Antibiotic resistance

Proc Natl Acad Sci U S A, 2000 Jun 6, 97(12), 6640 - 5
One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products; Datsenko KA et al.; We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s) . In this procedure, recombination requires the phage lambda Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid . To demonstrate the utility of this approach, we generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites . By using the respective PCR products, we made 13 different disruptions of chromosomal genes . Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA . The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable . This procedure should be widely useful, especially in genome analysis of E . coli and other bacteria because the procedure can be done in wild-type cells.

FEMS Microbiol Lett, 2000 May 15, 186(2), 319 - 25
Conjugation and transformation of Streptomyces species by tylosin resistance; Fouces R et al.; The tlrB gene from Streptomyces fradiae has been cloned and used to construct bifunctional Streptomyces-Escherichia coli shuttle vectors carrying the antibiotic resistance genes to kanamycin-neomycin, thiostrepton and tylosin as selection markers . In the same way, the tlrB gene was subcloned in plasmids including the apramycin resistance gene and the oriT sequence from the plasmid pSET152 to facilitate conjugation of Streptomyces spores . The usefulness of the tlrB gene as tylosin resistance marker was ascertained in Streptomyces lividans, Streptomyces parvulus and Streptomyces coelicolor, but not in Streptomyces clavuligerus . The tlrB gene constitutes a useful selection marker when high-frequency of conjugation/transformation is not required or as secondary marker in recombinant Streptomyces species where thiostrepton and kanamycin have been utilized for primary selection.

Cochrane Database Syst Rev . 2000;(2):CD000245.
Antibiotics for acute bronchitis; Becker L et al.; BACKGROUND: Antibiotic treatment of acute bronchitis, which is one of the most common illnesses seen in primary care, is controversial . Most clinicians prescribe antibiotics in spite of expert recommendations against this practice . OBJECTIVES: People with acute bronchitis may show little evidence of bacterial infection . If effective, antibiotics could shorten the course of the disease . However if they are not effective, the risk of antibiotic resistance may be increased . The objective of this review was to assess the effects of antibiotic treatment for patients with a clinical diagnosis of acute bronchitis . SEARCH STRATEGY: We searched Medline, Embase, reference lists of articles and the authors' personal collections up to 1996, and Scisearch from 1989 to 1996 . SELECTION CRITERIA: Randomised trials comparing any antibiotic therapy with placebo in acute bronchitis . DATA COLLECTION AND ANALYSIS: At least two reviewers extracted data and assessed trial quality . MAIN RESULTS: Eight trials involving 750 patients aged eight to over 65 and including smokers and non-smokers were included . The quality of the trials was variable . A variety of outcome measures were assessed . In many cases, only outcomes that showed a statistically significant difference between groups were reported . Overall, patients receiving antibiotics had slightly better outcomes than did those receiving placebo . They were less likely to report feeling unwell at a follow up visit (odds ratio 0.42, 95% confidence interval 0.22 to 0.82), to show no improvement on physician assessment (odds ratio 0.43; 0.23 to 0.79), or to have abnormal lung findings (odds ratio 0.33, 95% confidence interval 0.13 to 0.86), and had a more rapid return to work or usual activities (weighted mean difference 0.7 days earlier, 95% confidence interval 0.2 to 1 . 3) . Antibiotic-treated patients reported significantly more adverse effects (odds ratio 1.64; 1.05 to 2.57) such as nausea, vomiting, headache, skin rash or vaginitis . REVIEWER'S CONCLUSIONS: Antibiotics appear to have a modest beneficial effect in the treatment of acute bronchitis, with a corresponding small risk of adverse effects . The benefits of antibiotics may be overestimated in this analysis because of the tendency of published reports to include complete data on only the outcomes found to be statistically significant.

J Dairy Sci, 2000 Apr, 83(4), 741 - 5
Efficacy of two modified nonantibiotic formulations (Victory) for treatment of papillomatous digital dermatitis in dairy cows; Shearer JK et al.; A field trial was conducted to compare the efficacy of the original and two modified formulations of Victory and oxytetracycline among dairy cows affected with papillomatous digital dermatitis . Seventy-eight cows with papillomatous digital dermatitis lesions were randomly allocated to one of four treatment groups (A, B, C, D) . Cows in group A (n = 19) were treated with an oxytetracycline solution; cows in group B (n = 22) were treated with the original formulation of Victory containing soluble copper, peroxide compound, and a cationic agent; cows in group C (n = 17) were treated with a modified formulation of Victory containing reduced soluble copper and peroxide compound but increased levels of cationic agent; and cows in group D (n = 20) were treated with a modified formulation of Victory containing levels of soluble copper and cationic agent equivalent to the original formulation but with reduced concentrations of peroxide compound . Cows were examined 7, 14 and 28 d after initial treatment; during each examination, pain and lesion scores were recorded . The modified nonantibiotic formulation used in cows in group C appeared to be the most effective for treatment of papillomatous digital dermatitis . Proportions of cows with signs of pain were significantly lower among cows in group C, compared with cows in group A . Similarly, pain scores were significantly lower among cows in treatment group C, compared to cows in group A . The 2 low efficacy of oxytetracycline was an unexpected result b and may have clinical implications associated with possible antibiotic resistance in dairy cows affected with papillomatous digital dermatitis.

Microbiology, 2000 Apr, 146 ( Pt 4), 1011 - 8
The gene encoding P27 lipoprotein and a putative antibiotic-resistance gene form an operon in Mycobacterium tuberculosis and Mycobacterium bovis; Bigi F et al.; P27 is an antigenic membrane lipoprotein synthesized by members of the Mycobacterium tuberculosis complex . Northern blotting and RT-PCR experiments indicated that the genes encoding P27 and a putative antibiotic transporter (P55) form an operon . A promoter region was identified and characterized by deletion analysis in Mycobacterium smegmatis . Two transcription initiation points were mapped in Mycobacterium bovis BCG by primer extension analysis to 76 bp and 87 bp upstream of the ATG initiation codon . Putative -10 and -35 promoter consensus sequences associated with these showed 66% similarity to previously identified mycobacterial promoters . These results suggest that the P27/P55 operon is transcribed from two promoters in M . bovis BCG.

Vaccine, 2000 Jun 1, 18(24), 2768 - 71
Development of a multi-specificity opsonophagocytic killing assay; Nahm MH et al.; The opsonophagocytic-killing assay (OPKA) is one of the primary surrogate assays for evaluating the pneumococcal capsular polysaccharide-protein conjugates under development as vaccines . Because each vaccine contains seven or more different conjugates, multiple OPKA must be performed on each serum . Moreover, the large number of assays can deplete serum samples from infants . To reduce the amount of serum and effort required to conduct OPKA we developed a multi-specificity OPKA using antibiotic resistant pneumococci . Equal numbers of optochin-resistant serotype 6B and streptomycin-resistant 19F pneumococci were used as the target bacteria . Surviving bacteria of each serotype were enumerated by plating on agar containing the appropriate antibiotic . In an examination of 25 immune sera the results obtained with this new assay correlated well with those obtained when bacterial targets were examined individually . By using additional antibiotic resistance markers, more than two specificities can be examined in a single assay.

Ann Pharmacother, 2000 Apr, 34(4), 459 - 64
Antibiotic use in a Canadian Province, 1995-1998; Carrie AG et al.; OBJECTIVE: Antibiotics are among the most commonly used classes of agents in community practice; yet, studies of antibiotic use in this setting are scarce . Data from developed countries suggest increasing use of newer broad-spectrum agents, which has implications for the development of antibiotic resistance as well as cost of therapy . In this study, we quantified changing patterns of antibiotic use in community practice in Manitoba, Canada, from 1995 to 1998 . DESIGN: A descriptive, population-based study of antibiotic use in Manitoba was facilitated by the Drug Programs Information Network (DPIN) of Manitoba Health; a data management system responsible for recording details of prescriptions dispensed for all Manitoba residents . Antibiotic use data, defined as numbers of prescriptions dispensed, were extracted from the DPIN from January 1, 1995, to March 31, 1998 . Antibiotic use is reported as prescriptions per 1000 persons per year (Rx/1000/Yr) based on quarterly use . RESULTS: Penicillins (48.3%), macrolides (16.0%), and sulfonamides (12.5%) accounted for 75% of total antibiotic use; total use decreased 19.1% between 1995 and 1998 . Use of the four most commonly prescribed agents decreased over the study period (amoxicillin, -17.4%; erythromycin, -29.0%; trimethoprim/sulfamethoxazole, -18.7%; penicillins G and V, -19.2%) . In contrast, use of newer and/or broad-spectrum agents increased (ciprofloxacin, 21.9%; cefuroxime, 30.7%; and azithromycin/clarithromycin, 29.5%) . Use of second-line agents as a percentage of total antibiotic use increased from 14.4% to 19.3% between January 1995 and March 1998 (p < 0.001) . CONCLUSIONS: Penicillins, macrolides, and sulfonamides accounted for 75% of antibiotic use . Total antibiotic use declined over the study period; however, use of newer, broad-spectrum agents increased while use of older, narrow-spectrum agents decreased.

Cell Growth Differ, 2000 Mar, 11(3), 173 - 83
Blocking HER-2/HER-3 function with a dominant negative form of HER-3 in cells stimulated by heregulin and in breast cancer cells with HER-2 gene amplification; Ram TG et al.; Amplification and overexpression of the HER-2 (neu/ erbB-2) gene in human breast cancer are clearly important events that lead to the transformation of mammary epithelial cells in approximately one-third of breast cancer patients . Heterodimer interactions between HER-2 and HER-3 (erbB-3) are activated by neu differentiation factor/heregulin (HRG), and HER-2/HER-3 heterodimers are constitutively activated in breast cancer cells with HER-2 gene amplification . This indicates that inhibition of HER-2/HER-3 heterodimer function may be an especially effective and unique strategy for blocking the HER-2-mediated transformation of breast cancer cells . Therefore, we constructed a bicistronic retroviral expression vector (pCMV-dn3) containing a dominant negative form of HER-3 in which most of the cytoplasmic domain was removed for introduction into cells . By using a bicistronic retroviral vector in which the antibiotic resistance gene and the gene of interest are driven by a single promoter, we attained 100% coordinate coexpression of antibiotic resistance with the gene of interest in target cell populations . Breast carcinoma cells with HER-2 gene amplification (21 MT-1 cells) and normal mammary epithelial cells without HER-2 gene amplification from the same patient (H16N-2 cells) were infected with pCMV-dn3 and assessed for HER-2/ HER-3 receptor tyrosine phosphorylation, p85PI 3-kinase and SHC protein activation, growth factor-dependent and -independent proliferation, and transformed growth in culture . Dominant negative HER-3 inhibited the HRG-induced activation of HER-2/HER-3 and signaling in H16N-2 and 21 MT-1 cells as well as the constitutive activation of HER-2/HER-3 and signaling in 21 MT-1 cells . Responses to exogenous HRG were strongly inhibited by dominant negative HER-3 . In contrast, the proliferation of cells stimulated by epidermal growth factor was not apparently affected by dominant negative HER-3 . The growth factor-independent proliferation and transformed growth of 21 MT-1 cells were also strongly inhibited by dominant negative HER-3 in anchorage-dependent and independent growth assays in culture . Furthermore, the HRG-induced or growth factor-independent proliferation of 21 MT-1 cells was inhibited by dominant negative HER-3, whereas the epidermal growth factor-induced proliferation of these cells was not: this indicates that dominant negative HER-3 preferentially inhibits proliferation induced by HER-2/HER-3.

Ear Nose Throat J, 2000 Mar, 79(3), 176 - 7
Description of an office technique for laser ventilation of the ears; Siegel GJ; The author presents a description of a technique he developed called laser office ventilation of ears (LOVE) . LOVE, an office-based procedure performed under local anesthesia, can provide intermediate-term ventilation for middle ear disease . This procedure has potential to change the standard of care for otitis media . Such changes might include a decrease in the need for antibiotics (and thus antibiotic resistance), a decrease in hearing loss associated with otitis media, and a decrease in the overall cost of treatment.

Int J Antimicrob Agents, 2000 Jan, 13(3), 143 - 53
Molecular epidemiology of antibiotic resistance; Stefani S et al.; Molecular typing methods based on the analysis of the genetic structure of bacteria, are used to address many different problems such as the study of genomic organisation and evolution, the identification of patterns of infection, the identification of sources of transmission, the epidemiological surveillance of infectious diseases and for investigations into outbreaks . Of particular interest is the application of these techniques for acquiring information on the spread of micro-organisms that have become resistant to many clinically important antibiotics . The emergence of antibiotic resistance is one of the most dangerous phenomena of the last 20 years and knowledge of the mechanisms of resistant-gene exchange means fully understanding their spread into all environments . Studies on the molecular epidemiology of antibiotic-resistance in micro-organisms should make it easier to distinguish clonality with respect to horizontal transfer of the determinants of resistance.

Antimicrob Agents Chemother, 2000 Apr, 44(4), 814 - 20
Non-target gene mutations in the development of fluoroquinolone resistance in Escherichia coli; Kern WV et al.; Mutations in loci other than genes for the target topoisomerases of fluoroquinolones, gyrA and parC, may play a role in the development of fluoroquinolone resistance in Escherichia coli . A series of mutants with increasing resistance to ofloxacin was obtained from an E . coli K-12 strain and five clinical isolates . First-step mutants acquired a gyrA mutation . Second-step mutants reproducibly acquired a phenotype of multiple antibiotic resistance (Mar) and organic solvent tolerance and showed enhanced fluoroquinolone efflux . None of the second-step mutants showed additional topoisomerase mutations . All second-step mutants showed constitutive expression of marA and/or overexpressed soxS . In some third-step mutants, fluoroquinolone efflux was further enhanced compared to that for second-step mutants, even when the mutant had acquired additional topoisomerase mutations . Attempts to circumvent the second-step Mar mutation by induction of the mar locus with sodium salicylate and thus to select for pure topoisomerase mutants at the second step were not successful . At least in vitro, non-target gene mutations accumulate in second- and third-step mutants upon exposure to a fluoroquinolone and typically include, but do not appear to be limited to, mutations in the mar or sox regulons with consequent increased drug efflux.

J Antimicrob Chemother, 2000 Mar, 45, 19 - 24
Optimizing economic outcomes in antibiotic therapy of patients with acute bacterial exacerbations of chronic bronchitis; Pechere JC et al.; The social, medical and economic effects of acute bacterial exacerbations of chronic bronchitis on individual patients and the resource implications of this disease for the healthcare sector are considerable . Optimizing the selection of patients who should receive antibiotics according to stringent clinical criteria is the first step in promoting good clinical practice and cost-effectiveness . Antibiotic efficacy is then the major driver of cost, especially when it reduces the need for hospitalization . Resistance to first-line antibiotics can be expected to increase the risk of treatment failure . Other drivers of cost include non-compliance, which predisposes to therapeutic failure, and the selection of resistant strains . Treatment regimens of short duration, once-daily dosing and good tolerability are determinants of good compliance and cost savings . The expenses of first-line antibiotics typically account for only a small proportion of the overall costs of healthcare and the cheapest antibiotics are not necessarily the most cost-effective . The clinical success rate of first-line therapy is the primary determinant of the overall expenditure on healthcare because of the high costs associated with treatment failure, especially if it leads to hospitalization . Factors such as poor patient compliance and high antibiotic resistance rates, which undermine the clinical efficacies of first-line therapy, will increase the overall costs of treatment.

Microbiology, 2000 Feb, 146 ( Pt 2), 345 - 52
A novel valanimycin-resistance determinant (vlmF) from Streptomyces viridifaciens MG456-hF10; Ma Y et al.; A novel valanimycin-resistance determinant (vImF) was isolated from a cosmid containing Streptomyces viridifaciens DNA that leads to valanimycin production in Streptomyces lividans . Expression of the vImF gene in both Escherichia coli and S . lividans provided valanimycin resistance . The nucleotide sequence of vImF consists of 1206 bp and the deduced amino acid sequence encodes a polypeptide with 12 putative transmembrane-spanning segments and a calculated pI of 10.1 . VImF shows significant similarities to other known or putative transmembrane efflux proteins that confer antibiotic resistance, but it appears to be specific for valanimycin . The sequence similarities suggest that VImF is a member of the DHA12 family within the major facilitator superfamily of transport proteins and that it is probably involved in active valanimycin efflux energized by a proton-dependent electrochemical gradient.

Annu Rev Genet, 1999, 33, 449 - 77
Toward an integrated genetic epidemiology of parasitic protozoa and other pathogens; Tibayrenc M; Due to the increase of human migrations, the appearance of emerging and reemerging endemies, growing antibiotic resistance, and climatic changes, infectious diseases most probably constitute the major challenge for medicine in the next century . The advent of molecular methods of pathogen characterization has considerably improved our knowledge of the epidemiology of these diseases . However, the use of concepts of evolutionary genetics for interpreting "molecular epidemiology" data remains limited, although the application of such methods would broaden considerably the scope of this field of research, and allow epidemiologic and taxonomic approaches to be ascertained on a much firmer basis . In turn, pathogens, hosts, and vectors provide fascinating models for basic research . The artificial character of the border between "basic" and "applied" research is especially apparent with regard to the "integrated genetic epidemiology of infectious diseases" concept . The goal of this chapter is to evaluate the respective impact, on the transmission and pathogenicity of infectious diseases, of the host's, the pathogen's, and the vector's (for vector-borne diseases) genetic diversity, and the interactions between these three parameters (coevolution phenomena).

Science, 2000 Feb 25, 287(5457), 1479 - 82
Effects of environment on compensatory mutations to ameliorate costs of antibiotic resistance; Bjorkman J et al.; Most types of antibiotic resistance impose a biological cost on bacterial fitness . These costs can be compensated, usually without loss of resistance, by second-site mutations during the evolution of the resistant bacteria in an experimental host or in a laboratory medium . Different fitness-compensating mutations were selected depending on whether the bacteria evolved through serial passage in mice or in a laboratory medium . This difference in mutation spectra was caused by either a growth condition-specific formation or selection of the compensated mutants . These results suggest that bacterial evolution to reduce the costs of antibiotic resistance can take different trajectories within and outside a host.

Int J Tuberc Lung Dis, 2000 Feb, 4(2 Suppl 1), S4 - 10
Molecular determinants of drug resistance in tuberculosis; Riska PF et al.; Rapid detection of drug-resistant tuberculosis (TB) has become increasingly important in the era of pandemic human immunodeficiency virus infection and antibiotic resistance . The identification of the molecular correlates of antibiotic resistance in Mycobacterium tuberculosis have engendered the development of DNA-based assays for the identification of drug-resistant TB . This review summarizes the recent discoveries concerning resistance to isoniazid, rifampin, pyrazinamide, ethambutol, streptomycin, amikacin, kanamycin and the quinolones.

Rev Med Interne, 2000 Jan, 21(1), 74 - 7
{Bronchiolitis obliterans with organized pneumonia: a rare complication of primary Gougerot-Sjögren syndrome}; Lambert M et al.; INTRODUCTION: Bronchiolitis obliterans organizing pneumonia (BOOP) is characterized by plugs of granulation tissue in bronchioles, alveolar ducts and alveoli . This pulmonary disorder has been reported in some cases in relation to drug consumption (D-penicillamine, amiodarone), with bacterial or viral infections (Mycoplasma pneumoniae, HIV), and with systemic diseases, such as rheumatoid arthritis . To our knowledge, only three cases of association BOOP-Sjogren's syndrome have been reported . EXEGESIS: We report three new cases of BOOP . These patients presented a primary Sjogren's syndrome without clinical or biological abnormalities suggestive of other autoimmune diseases . Initial presentation was an acute pulmonary disorder mimicking a bacterial pneumonia . Two patients had cutaneous vasculitis and the third vasculitic neuropathy . Corticosteroid therapy was begun and was quickly successful . None of the patients presented a relapse of BOOP . CONCLUSION: The incidence of BOOP is probably underestimated in patients with primary Sjogren's syndrome without cutaneous vasculitis . In case of pneumonia with antibiotic resistance, an immunological mechanism should be considered.

Zentralbl Chir, 1999, 124 Suppl 4, 19 - 22
{Treatment of septic complications od secondary peritonitis}; Winkeltau GJ et al.; The authors review the current management goals of surgical and antibiotic therapy in secondary peritonitis . Basic therapeutic regimen is the surgical elimination of the infectious source by means of a rational and risk-adapted operative procedure . Other technical procedures (such as intra- and postoperative lavage, drainage of the abdominal cavity, decompression, etc.) are critically reviewed and reduced to the scientific background . A calculated antibiotic therapy should be performed to eliminate the leading pathogens . The golden standard seems to be the short-term application of a third generation cephalosporin in combination with metronidazole . Antibiotic-induced endotoxinemia, bacterial shifting to grampositive species, and escalating antibiotic resistance require the examination of new therapeutic regimens (e.g . with quinolones).

J Antimicrob Chemother, 2000 Feb, 45(2), 205 - 11
Are routine sensitivity test data suitable for the surveillance of resistance? Resistance rates amongst Escherichia coli from blood and CSF from 1991-1997, as assessed by routine and centralized testing; Livermore DM et al.; Surveillance of antibiotic resistance can be undertaken by compilation of routine data or by central testing of isolates . Routine results can be obtained cheaply and in sufficient quantities for correlation with population and prescribing denominators but there is concern about their quality . As one of a series of ongoing studies to assess this quality, we compared the proportions of resistance amongst Escherichia coli from patients with bacteraemia or meningitis between 1991 and 1997 (i) as recorded in routine data reported to the PHLS and (ii) as found in tests performed at the PHLS Laboratory of Enteric Pathogens (LEP) . These two data sets both showed an overall upward trend in the proportion of isolates resistant to ampicillin, trimethoprim, gentamicin and ciprofloxacin . The average annual percentage increase in resistance was estimated in separate logistic regression models, and 95% confidence intervals (CI) were determined . The annual percentage increases in the proportions of isolates reported resistant were similar in the two data sets for trimethoprim, gentamicin and ciprofloxacin but differed for ampicillin . The upward trends were statistically significant except for gentamicin resistance in the LEP data set, where the 95% CI straddled zero . The proportions of resistant isolates for each antibiotic in the two data sets each year were in poorer agreement than the trends; however, the 95% CI of the difference of proportions resistant between the routine and LEP data sets straddled zero in 4 or 5 of the 7 years studied . Some discrepancies might be explained by geographical bias in the sampling or by differences in definitions of resistance . Thus (i) the proportion of resistant isolates tested at LEP almost always fell within the ranges bounded by the highest and lowest proportions for individual Regional Health Authorities, as recorded in the routine data, and (ii) the fact that LEP consistently recorded less gentamicin resistance but more ciprofloxacin resistance than the routine could be explained by breakpoint differences . We conclude that routine susceptibility data for ampicillin, ciprofloxacin, gentamicin and trimethoprim appear sound for E . coli and might be suitable for correlation with other data, e.g . for prescribing.

Mol Gen Genet, 2000 Jan, 262(6), 1070 - 80
The replication origin of Azotobacter vinelandii; Singh RA et al.; The putative replication origin of Azotobacter vinelandii was cloned as an autonomously replicating fragment after ligation to an antibiotic resistance cartridge . The resulting plasmids could be isolated and labelled by Southern hybridisation with the antibiotic resistance cartridge as probe and also visualised by electron microscopy . These plasmids integrated into the chromosome after a few generations, even in the recA mutant of A . vinelandii . The integrated copy of the plasmid was re-isolated from the chromosome and the DNA and its subfragments were cloned in the plasmid vector pBR322 . A 200-bp DNA fragment was sufficient to allow the replication of pBR322 in an Escherichia coli polA strain . Electron microscopic analysis of this plasmid showed that replication initiated mostly within the A . vinelandii DNA fragment . The nucleotide sequence of the putative replication origin and its flanking regions was determined . In the sequence of the 200-bp fragment many of the distinctive features found in other replication origins are lacking . A greater variation from the consensus DnaA binding sequence was observed in A . vinelandii . Direct sequencing of the relevant genomic fragment was also carried after amplifying it from A . vinelandii chromosomal DNA by PCR . This confirmed that no rearrangements had taken place while the cloned fragment was resident in E . coli . It was shown by hybridisation that the 200-bp chromosomal origin fragment of A . vinelandii was present in three other field strains of Azotobacter spp.

J Hosp Infect, 1999 Dec, 43 Suppl, S253 - 60
Stewardship of antibiotic use and resistance surveillance: the international scene; Gould IM; Audit is all about education and changing practice . It is well known that doctors are both difficult to target with educational initiatives and those who need educating are the most difficult to target! Changing established practice is even harder and maintaining change in practice is often considered just a dream . This emphasises the importance of the cycle of audit, feedback (education), re-audit etc . This process has been developed over a twelve-year period in Aberdeen while the antibiotic policy has evolved from a 15-page booklet on hospital antibiotic prescribing into a 90-page book combining both hospital and general practice prescribing . Both successes and failures of the audit process will be described in the context of Strategic Goals . The next phase is assessment of performance against other policies in Europe to establish the best methods of antibiotic stewardship . A European group has recently been formed to begin this process . Parameters that can be used to assess the comparative performance of policies will be discussed and include antibiotic resistance rates . Several international resistance surveillance and quality of antibiotic use programmes are highlighted.

Fam Med, 2000 Jan, 32(1), 22 - 9
An evaluation of statewide strategies to reduce antibiotic overuse; Mainous AG 3rd et al.; BACKGROUND: The rapid increase of antibiotic resistance poses a significant threat to human health . Overuse of antibiotics has been linked to rates of antibiotic resistance . This study assessed the utility of two common interventions--1) practice profiling and feedback and 2) patient education materials--implemented to decrease antibiotic prescribing for pediatric upper respiratory infections (URIs) . METHODS: Based on Medicaid regions in Kentucky, primary care physicians managing pediatric respiratory infections in Medicaid were randomized into four groups . Groups received either 1) performance feedback only, 2) patient education materials only, 3) both feedback and education materials, or 4) no intervention . Participating physicians had their antibiotic prescribing assessed for the period of July 1, 1996, to November 30, 1997, with an intervention in June 1997 . The study included 216 physicians and 124,092 episodes of care . RESULTS: All groups increased in proportion of episodes with antibiotics between the pre-intervention and post-intervention periods . Prescribing in the patient education group and the patient education and feedback group increased at a significantly lower rate than in the control group . Physicians did not change their coding of illness to justify antibiotics after the intervention, and there was no significant generalization of effect of the pediatric intervention on prescribing for adult URIs . CONCLUSIONS: These interventions demonstrate little if any impact on promoting appropriate antibiotic prescribing . Antibiotic prescribing for viral respiratory infections continues to increase, suggesting concomitant increases in antibiotic resistance.

Plasmid, 2000 Jan, 43(1), 35 - 48
Transcription of the transfer genes traY and traM of the antibiotic resistance plasmid R100-1 is linked; Stockwell D et al.; Three separate traY deletion mutants of R100-1 were prepared by allele replacement . These mutants retained the ability to transfer at a level 100 times greater than R100 and 1/50 that of the parental R100-1 . The mutants were complemented to normal R100-1 transfer levels by pDSP06, a multicopy traY clone . Comparison of transcripts initiated at the traY promoter, P(Y), by primer extension experiments showed that there was no detectable P(Y) activity in R100 and that the level of P(Y) activity in the traY deletion mutants was lower than that in R100-1 . Similar measurements performed on RNA from a set of previously described traM deletion mutants showed that those traM deletion mutants that produced more traM and finM (M) transcripts than the parental R100-1 also produced more traY transcripts than R100-1 and that those traM mutants that produced fewer M transcripts than R100-1 also produced fewer traY transcripts than R100-1 . We conclude that in R100, TraY regulates P(Y) activity and that transcripts originating in traM affect P(Y) activity.

Proc Natl Acad Sci U S A, 1999 Dec 21, 96(26), 15026 - 31
The mating-type and pathogenicity locus of the fungus Ustilago hordei spans a 500-kb region; Lee N et al.; The fungal pathogen Ustilago hordei causes the covered smut disease of barley and oats . Mating and pathogenicity in this fungus are controlled by the MAT locus, which contains two distinct gene complexes, a and b . In this study, we tagged the a and b regions with the recognition sequence for the restriction enzyme I-SceI and determined that the distance between the complexes is 500 kb in a MAT-1 strain and 430 kb in a MAT-2 strain . Characterization of the organization of the known genes within the a and b gene complexes provided evidence for nonhomology and sequence inversion between MAT-1 and MAT-2 . Antibiotic-resistance markers also were used to tag the a gene complex in MAT-1 strains (phleomycin) and the b gene complex in MAT-2 strains (hygromycin) . Crosses were performed with these strains and progeny resistant to both antibiotics were recovered at a very low frequency, suggesting that recombination is suppressed within the MAT region . Overall, the chromosome homologues carrying the MAT locus of U . hordei share features with primitive sex chromosomes, with the added twist that the MAT locus also controls pathogenicity.

Enferm Infecc Microbiol Clin, 1999, 17 Suppl 2, 27 - 31
{The other side of the coin: socioeconomic analysis of antibiotic resistance}; Garcia-Altes A et al.; BACKGROUND: The re-emergence of bacterial diseases and their negative consequences in terms of health and economic cost, have made this issue an important public health problem . The objective of this work is to review the economic literature about antibiotic resistance, and to suggest possible solutions in our health care context aimed to reduce their negative impact . METHODS: We made a bibliographic search in the main biomedical databases . Economic assessment studies published in Spanish, English, French or Italian and related to the appearance of antibiotic resistance were selected . Their main methodological characteristics and results were analysed . RESULTS: Two studies analysing the economic impact of the appearance of antibiotic resistance were identified . A minimum hospital cost of 1300 million dollars (in 1992), and a social cost between 100 and 30,000 million dollars (in 1989) were estimated . CONCLUSIONS: Economic analysis allows to quantify and assess the impact of several management strategies in relation with antibiotic administration, in terms of health and costs, and to choose the most cost-effective strategies . The identification of inappropriate consumption of antibiotics as a cause of a negative externality upon the rest of the society is basic to make health care professionals and public opinion conscious about this problem, and to develop strategies to reduce its negative impact.

Mol Cells, 1999 Oct 31, 9(5), 517 - 25
Isolation and identification of Fur binding genes in Escherichia coli; Oh M et al.; Fur (ferric uptake regulation) binding fragments were isolated by in vitro binding of purified Fur protein with Sau3AI-digested genomic DNA fragments . The Fur-bound DNA fragments were filtered on nitrocellulose paper, isolated, cloned, and sequenced . The protein binding was confirmed by gel retardation assay for five DNA fragments . The sequence data were used to identify the genes by comparison with the GenBank data . The proposed Fur binding regions lie on or near the putative promoter regions of marAB (multiple antibiotic resistance), pyrC (dihydroorotase), mreB (mecillinam resistance) and an unidentified gene (ecouw93) near argI and in the middle of the treBC (trehalose permease enzyme II) coding region . The proposed Fur binding sites of the known iron regulating operators including the genes of this work are AAT(pyrimidine) and A(purine)TT . The two conserved sequences are 10 bases apart and palindromic to each other, which might suggest the classical pattern of protein binding toward one side of the DNA in contrast to the concept of the Fur protein wrapping around the DNA.

Drugs, 1999, 58 Suppl 2, 71 - 7
Cost effectiveness of quinolones in hospitals and the community; Davey P; In hospitals, oral quinolone therapy has lower daily associated costs (acquisition and administration) than most intravenous regimens . In addition, oral switch therapy shortens the duration of hospital stay for most patients . However, randomised trials are required to measure the economic impact of the switch to oral therapy in terms of hospital care costs and the burden imposed on community health services, on relatives or carers and on the patient . Evidence about the reliability of absorption of quinolones in hospitalised patients is more likely to be obtained from large population kinetic studies than randomised effectiveness trials . The existing literature on cost effectiveness of quinolones in the community is disappointing . The principal problems are poor definition of diagnostic criteria, inclusion of irrelevant comparator drugs and a failure to include infections caused by bacteria that are resistant to the comparator . Consequently, there is little evidence to support the use of economic models to determine the consequences of antibiotic resistance in the community.

J Antimicrob Chemother, 1999 Nov, 44(5), 607 - 10
Characterization of expanded-spectrum cephalosporin resistance in E . coli isolates associated with bovine calf diarrhoeal disease; Bradford PA et al.; Antibiotic resistance among Escherichia coli isolates from diarrhoeal disease in cattle was studied . Many of the isolates were multiply resistant to beta-lactams, including expanded-spectrum cephalosporins, aminoglycosides, sulphonamides, tetracycline and fluoroquinolones . In many of the isolates, IEF revealed a strong beta-lactamase band compatible with overexpression of the AmpC beta-lactamase, either alone or in addition to TEM-type enzymes . Several of the isolates also possessed genes encoding virulence factors associated with animal and human diarrhoeal diseases . These results suggest that the use of antibiotics in animals could lead to a reservoir of antibiotic-resistant bacteria that could potentially infect humans.

Am J Infect Control, 1999 Dec, 27(6), S4 - 21
Impact of changing societal trends on the spread of infections in American and Canadian homes; Sattar SA et al.; Infectious diseases continue to exert a heavy toll on human health even in industrialized countries . Recent data from the World Health Organization suggests that infectious diseases are the leading cause of death in the world . Many changing trends in our society have a known or potential impact on infectious disease spread and may have an impact on the normal routine of home hygiene . Important amongst these societal trends are increasing population and life expectancy, changes in urbanization, grouping of susceptibles, increased ambulatory and home care, increased immunosuppression, increased and faster travel, changes in technology, increasing antibiotic resistance as a result of misuse of antibiotics, changes in food and water consumption, and changes in personal cleaning, washing, and laundry practices . This review will highlight these factors and their impact on home hygiene and steps that may be needed to reduce the risk from infections.

Biotechnol Prog, 1999 Nov-Dec, 15(6), 1046 - 52
Screening of transformed insect cell lines for recombinant protein production; Keith MB et al.; Nine insect cell lines were evaluated for their potential as host systems for recombinant protein production using a new expression vector permitting the continuous high-level expression of secreted glycoproteins by transformed insect cells (Farrell et al., 1998) . As a means of preliminary screening, all nine insect cell lines were transfected with the green fluorescence protein . Growth in static and suspension culture was then examined as a further method of screening . On the basis of their transfection efficiencies and cell growth characteristics, five insect cell lines, Bm5, High Five, IPLB-LdFB, IZD-MB-0503, and Sf-21, were selected for stable transformation to produce granulocyte-macrophage colony-stimulating factor (GM-CSF) . These five cell lines were stably transformed using an antibiotic resistance scheme and evaluated as a polyclonal population . Increasing the antibiotic concentration was found to cause not only a decrease in the specific growth rate but also an increase in the specific protein production rate and final GM-CSF concentration . The transformed High Five cells exhibited by far the greatest specific protein production rate of 5.1 x 10(-)(6) microgram/(cell.h), resulting in the highest final GM-CSF concentration of 22.8 mg/L when grown in static culture . One cloned High Five cell line produced a GM-CSF concentration of 46 mg/L in static culture and 27 mg/L in suspension culture.

Int J STD AIDS, 1999 Oct, 10(10), 677 - 9
HIV infection and sexually transmitted infections in Lithuania; Chaplinskas S et al.; Lithuania is a small country with a population of 3.7 million . It has recently been released from the yoke of Soviet rule . HIV infection was first identified in 1988 and while the numbers of cases are small, the incidence is beginning to rise precipitously . A National AIDS Centre has been established in the capital, Vilnius, and a nationwide epidemiological survey is underway . Efforts are being made to prevent HIV infection . Sixty one per cent of notified cases of HIV infection are in Klaipeda, a port city adjacent to the Kaliningrad region and the predominant mode of transmission is by intravenous drug use . The majority of cases of AIDS, however, are seen in Vilnius . Sexually transmitted infections (STIs) are poorly controlled and there is no national control strategy . While the incidence of gonorrhoea is declining, new cases of syphilis have been on the increase, reaching 101.4 cases per 100,000 of the population . Cases of congenital syphilis are still seenPIP: This paper examines the incidence of HIV infection and other sexually transmitted diseases in Lithuania . HIV infection was first identified in 1988, and while the numbers of cases are small, the incidence is beginning to rise rapidly . The National AIDS Center has been established in the capital, Vilnius, and a national epidemiological survey is underway . Cases of syphilis, gonorrhea, and HIV infection, as well as AIDS, are officially notified in Lithuania . There are about 120 male individuals infected with HIV, 21 of which have AIDS; about 8 have died . 45% of infections originated from intravenous drug users, 25% were acquired heterosexually, and 22% were acquired homosexually/bisexually . Disease counseling, conducted by the Lithuanian AIDS Center, has been set up to provide services to prostitutes . Other sexually transmitted infections in the country are syphilis and gonorrhea . It is concluded that laboratory services in Lithuania require further development, including the use of antibiotic resistance testing and more widespread introduction of molecular diagnostic techniques . Public education on STIs, including HIV infection, also needs to be further developed .

Am J Gastroenterol, 1999 Nov, 94(11), 3170 - 4
A survey of gastroenterologists' perceptions and practices related to Helicobacter pylori infection; Sharma VK et al.; OBJECTIVE: The aim of this study was to assess the current practice of gastroenterologists in the United States concerning Helicobacter pylori (H . pylori) infection . METHODS: We mailed a structured questionnaire to 1000 gastroenterologists chosen at random from a national database . We asked about personal and practice demographics and practices relating to testing for, and treating, H . pylori infection . RESULTS: A total of 922 questionnaires were delivered, from which we received 286 responses (31%) . Respondents used many different tests for H . pylori infection, but only 10% each had used either the 13C- or 14C-urea breath test . Testing for H . pylori infection was usually for appropriate reasons, although 21% indicated that they might not treat a patient with a positive test result . Different multiple treatment regimens were used; the most frequent were combinations of a proton pump inhibitor, clarithromycin, and either amoxicillin or metronidazole . Estimates of the prevalence of antibiotic resistance were highly variable and often inaccurate . Most respondents would not check asymptomatic individuals for the infection; however, in the absence of symptoms, 38% would personally undergo testing and treatment if positive . CONCLUSIONS: Gastroenterologists usually test for H . pylori infection in appropriate conditions, but may not always treat the infection based on a positive test result . Most use efficacious regimens to treat the infection although many have inaccurate information on resistance rates, which may adversely influence prescribing . Many would have testing and, if positive, treatment in the absence of symptoms or a specific diagnosis, but do not recommend this for their patients.

Eur J Gastroenterol Hepatol, 1999 Nov, 11(11), 1255 - 8
Reliability of biopsy-based diagnostic tests for Helicobacter pylori after treatment aimed at its eradication; van der Wouden EJ et al.; OBJECTIVE: Recurrence of Heliobacter pylori after apparently successful treatment mostly represents resurgence of the infection, rather than a new one . Therefore, the reliability of biopsy-based tests after treatment was investigated . METHODS: Four weeks or more after treatment, antral biopsy samples were taken for culture, histology, urease test and polymerase chain reaction (PCR), and a corpus specimen for culture . Treatment failure was defined as > or = 2 tests positive . If one test was positive, a 13C-urea breath test was performed and considered conclusive . RESULTS: One hundred and ninety-seven patients were evaluated . Endoscopy was performed 53 days (27-92 days) after treatment . Twenty-one patients with missing test results and 19 patients on acid-suppressive drugs were excluded . In 140 of 156 patients (89.7%), H . pylori was eradicated . Sensitivity and specificity of culture of antrum were, respectively, 100% and 100%; culture of corpus, 100% and 100%; rapid urease test, 87% and 99%; haematoxylin/eosin stain, 94% and 95%; Giemsa stain, 81% and 99%; and PCR, 88% and 100% . CONCLUSION: Although all biopsy-based tests are reliable after treatment, culture is the biopsy-based test of first choice as it is the most accurate and gives additional information on antibiotic resistance.

Proc Natl Acad Sci U S A, 1999 Nov 9, 96(23), 13276 - 81
Direct sequencing of bacterial and P1 artificial chromosome-nested deletions for identifying position-specific single-nucleotide polymorphisms; Chatterjee PK et al.; A loxP-transposon retrofitting strategy for generating large nested deletions from one end of the insert DNA in bacterial artificial chromosomes and P1 artificial chromosomes was described recently {Chatterjee, P . K . & Coren, J . S . (1997) Nucleic Acids Res . 25, 2205-2212} . In this report, we combine this procedure with direct sequencing of nested-deletion templates by using primers located in the transposon end to illustrate its value for position-specific single-nucleotide polymorphism (SNP) discovery from chosen regions of large insert clones . A simple ampicillin sensitivity screen was developed to facilitate identification and recovery of deletion clones free of transduced transposon plasmid . This directed approach requires minimal DNA sequencing, and no in vitro subclone library generation; positionally oriented SNPs are a consequence of the method . The procedure is used to discover new SNPs as well as physically map those identified from random subcloned libraries or sequence databases . The deletion templates, positioned SNPs, and markers are also used to orient large insert clones into a contig . The deletion clone can serve as a ready resource for future functional genomic studies because each carries a mammalian cell-specific antibiotic resistance gene from the transposon . Furthermore, the technique should be especially applicable to the analysis of genomes for which a full genome sequence or radiation hybrid cell lines are unavailable.

South Med J, 1999 Oct, 92(10), 971 - 6
Parental knowledge about common respiratory infections and antibiotic therapy in children; Collett CA et al.; BACKGROUND: Widespread antibiotic use has fostered the emergence of antibiotic-resistant bacteria . Parental expectations have been cited as one reason for physicians to overprescribe antibiotics . The objective of this study was to determine parental knowledge about antibiotics and their use for common respiratory tract infections . METHODS: A survey was administered to 100 adults at a rural pediatric office . RESULTS: Many respondents had misconceptions about the etiology of common respiratory tract infections and the effects of antibiotic therapy . Only 54% knew that a virus is the usual cause of the common cold, and 33% thought that a virus causes strep throat . Almost half (46%) believed that antibiotics kill viruses, while 17% were not sure whether antibiotics kill viruses . Most respondents (60%) had never heard about antibiotic resistance . CONCLUSION: Parental knowledge about common respiratory tract infections and about antibiotic therapy is often lacking . Improved parent education may alter parents' expectations concerning antibiotic therapy for their ill children.

Presse Med, 1999 Sep 25, 28(28), 1505 - 8
{Pneumococcal infections in intensive care . Retrospective 8 year study}; Landreau L et al.; OBJECTIVE: The aim of this study was to analyze the clinical presentations and severity of S . pneumoniae infections requiring hospitalization in an intensive care unit and evaluate the incidence and severity of infections caused by penicillin-resistant strains . PATIENTS AND METHODS: This retrospective study reviewed cases in our intensive care unit from January 1989 through December 1996 including all patients with pneumococcal infection . RESULTS: The study included 102 patients, mean age 59.6 years . Pneumonia was the most frequent (83 cases) followed by bacteriemia (31 cases) and meningitis (15 cases) . Mortality was high (43%) and influenced by age, simplified severity score, and presence of shock at admission . Antibiotic resistance appeared in 1991 and increased over the years reaching, in 1996: 24% for penicillin, 38% for macrolides, 20% for sulfamides, 19% for tetracyclins, and 14% for phenicols . Penicillin-resistance was not found to modify clinical expression nor severity of infection . Amoxicillin and third-generation cephalosporins were the most widely used antibiotics . CONCLUSION: Pneumococcal infections in intensive care patients are severe with high mortality . The emergence of more and more resistant strains has little clinical consequence on severity or treatment.

Clin Infect Dis, 1999 Nov, 29(5), 1189 - 96
Parenteral antibiotic use in acute-care hospitals: A standardized analysis of fourteen institutions; Carling PC et al.; Despite increasing concerns regarding the need to optimize appropriate antibiotic use in hospitals, a standardized method for evaluating interinstitutional antibiotic use has not been developed . To address this issue, antibiotic use was analyzed by means of a uniform methodology among 14 acute-care hospitals . Data were standardized by use of a defined daily dose for each antibiotic while adjusting for patient volume by calculating use per 1000 patient-days . Within the group, there was a 68% range in total parenteral antibiotic expenditures and wide variability in the use of individual agents . Analysis of these differences indicated that only the use of active antibiotic-management programs clearly correlated with antibiotic cost per 1000 patient-days (P<.001) . Given these results, we believe that wider comparative analysis of antibiotic use with a standardized methodology in conjunction with standardized analysis of nosocomial infection rates and antibiotic resistance data may enhance the stewardship of antibiotics in acute-care hospitals.

Can J Gastroenterol, 1999 Sep, 13(7), 581 - 3
Helicobacter pylori: novel therapies; Drouin E; The ideal therapy for Helicobacter pylori would cure the infection without resulting in the development of antibiotic resistance . Current therapies have variable cure rates; the reasons for treatment failure include bacterial resistance and poor compliance . Some antibiotics, such as furazolidone, may be affordable agents to treat this infection worldwide . New proton pump inhibitors, such as rabeprazole, can potentiate antibiotics . Nutriceuticals and probiotics demonstrate interesting in vitro activity against H pylori . Children rarely have symptoms to this infection and, therefore, are a suitable group in which to assess different nonaggressive therapies.

FEMS Microbiol Lett, 1999 Oct 15, 179(2), 361 - 6
Cloning and characterization of a replicon region of the IncHII plasmid pHH1457; Alonso G et al.; A replicative region of the large conjugative plasmid pHH1457 (incompatibility group HII (IncHII)) was cloned . A 1.4-kbp region, in a stable pSBII14 clone, containing a PolI-independent replicon and determinants for the HII incompatibility phenotype, was selected and characterized . High incompatibility with IncHII plasmids was corroborated . Independent replication of the insert was demonstrated by ligation to an antibiotic resistance cassette . pSBII14 was used as a probe to identify IncHII plasmids from other members of the H complex: IncHI (IncHI1, IncHI2 and IncHI3 subgroups) . Hybridization experiments revealed a high homology with the replication region of IncHII plasmids, but not with IncHI1 or IncHI3 plasmid prototypes . Homology with IncHI2 plasmids was observed, suggesting the presence of IncHII-like replicons among this subgroup of plasmids . This is the first report of the characterization of an IncHII plasmid maintenance region.

Proc Natl Acad Sci U S A, 1999 Oct 12, 96(21), 11740 - 5
A multiplasmid approach to preparing large libraries of polyketides; Xue Q et al.; A three-plasmid system for heterologous expression of 6-deoxyerythronolide B synthase (DEBS) has been developed to facilitate combinatorial biosynthesis of polyketides made by type I modular polyketide synthases (PKSs) . The eryA PKS genes encoding the three DEBS subunits were individually cloned into three compatible Streptomyces vectors carrying mutually selectable antibiotic resistance markers . A strain of Streptomyces lividans transformed with all three plasmids produced 6-deoxyerythronolide B at a level similar to that of a strain transformed with a single plasmid containing all three genes . The utility of this system in combinatorial biosynthesis was demonstrated through production of a library of modified polyketide macrolactones by using versions of each plasmid constructed to contain defined mutations . Combinations of these vector sets were introduced into S . lividans, resulting in strains producing a wide range of 6-deoxyerythronolide B analogs . This method can be extended to any modular PKS and has the potential to produce thousands of novel natural products, including ones derived from further modification of the PKS products by tailoring enzymes.

Curr Opin Microbiol, 1999 Oct, 2(5), 489 - 93
The biological cost of antibiotic resistance; Andersson DI et al.; The frequency and rates of ascent and dissemination of antibiotic resistance in bacterial populations are anticipated to be directly related to the volume of antibiotic use and inversely related to the cost that resistance imposes on the fitness of bacteria . The data available from recent laboratory studies suggest that most, but not all, resistance-determining mutations and accessory elements engender some fitness cost, but those costs are likely to be ameliorated by subsequent evolution.

J Med Chem, 1999 Sep 23, 42(19), 3852 - 9
Novel inhibitors of Erm methyltransferases from NMR and parallel synthesis; Hajduk PJ et al.; The Erm family of methyltransferases confers resistance to the macrolide-lincosamide-streptogramin type B (MLS) antibiotics through the methylation of 23S ribosomal RNA . Upon the methylation of RNA, the MLS antibiotics lose their ability to bind to the ribosome and exhibit their antibiotic activity . Using an NMR-based screen, we identified a series of triazine-containing compounds that bind weakly to ErmAM . These initial lead compounds were optimized by the parallel synthesis of a large number of analogues, resulting in compounds which inhibit the Erm-mediated methylation of rRNA in the low micromolar range . NMR and X-ray structures of enzyme/inhibitor complexes reveal that the inhibitors bind to the S-adenosylmethionine binding site on the Erm protein . These compounds represent novel methyltransferase inhibitors that serve as new leads for the reversal of Erm-mediated MLS antibiotic resistance.

Eur J Gastroenterol Hepatol, 1999 Aug, 11 Suppl 2, S39 - 42; discussion S43-5
What can be learnt from the new data about antibiotic resistance? Are there any practical clinical consequences of Helicobacter pylori antibiotic resistance?
Bazzoli F, Berretti D, De Luca L, Nicolini G, Pozzato P, Fossi S, Zagari M.
Effective treatment regimens are now available for the eradication of Helicobacter pylori, but one of the factors limiting their efficacy is antibiotic resistance . Omeprazole-based triple therapy (omeprazole plus two antibiotics) can, at present, be considered the treatment of choice for H . pylori infection; some of the best results have been achieved by combining omeprazole with either amoxycillin and clarithromycin or metronidazole and clarithromycin . However, the potential effectiveness of nitroimidazole derivatives and clarithromycin must be weighed against the possibility that resistance can develop to these agents . Eradication in metronidazole-resistant strains is lower than in sensitive strains, but is still about 75% (versus 97%) . However, clarithromycin resistance is thought to have more clinical significance, reducing the eradication rate of 95% in sensitive strains to 40% in resistant strains, although the overall importance of clarithromycin resistance for H . pylori eradication is still likely to be relatively low . Recent data on secondary resistance indicate that the rate is at least 50% for both metronidazole and clarithromycin in patients in whom eradication has failed . If, in the future, a large number of H . pylori-positive individuals undergo such treatment, treatment failures may become a major issue, and the problem of antibiotic resistance will have to be overcome.

Scand J Gastroenterol, 1999 Aug, 34(8), 750 - 6
Prevalence of resistance to clarithromycin and its clinical impact on the efficacy of Helicobacter pylori eradication; Ellenrieder V et al.; BACKGROUND: Triple therapy with a proton-pump inhibitor (PPI) in combination with metronidazole and clarithromycin is the method of choice for eradication of Helicobacter pylori . Failures have been primarily blamed on the development of resistance to clarithromycin . The present study investigated the prevalence and clinical significance of resistance to clarithromycin and metronidazole in determining therapeutic success of both triple therapy as a primary eradication method and high-dose dual therapy in non-responders . METHODS: On the basis of prior therapy, H . pylori-positive patients were assigned to one of two groups in the present prospective study . Group A (n = 93) included patients who had not undergone any prior eradication treatment, whereas group B (n = 15) consisted of patients who had received clarithromycin but in whom eradication had been unsuccessful . All patients underwent endoscopy with biopsy for bacterial culture and resistance studies . Patients in group A were treated with a 7-day regimen of pantoprazole (40 mg twice daily), metronidazole (500 mg twice daily), and clarithromycin (250 mg twice daily), whereas those in group B received omeprazole (40 mg three times a day) and amoxycillin (1000 mg three times a day ) for 14 days . Success of the eradication treatment was ascertained by means of the 13C urea breath test . RESULTS: In group A resistance to clarithromycin and metronidazole was identified in 3 patients (4.9%) and in 14 patients (22.9%), respectively . Eradication proved successful in 78 of 84 patients (92.6%) followed up . Two of the 3 patients with primary clarithromycin resistance and 1 of the 14 patients with metronidazole resistance did not respond to treatment . In group B isolated or combined resistance to clarithromycin was found in seven patients, whereas another four showed isolated resistance to metronidazole . Eradication proved successful in 10 of 13 controlled patients (76.9%) followed up, and only 2 patients reported severe side effects . CONCLUSION: Determination of antibiotic resistance before initiating therapy is not necessary, since primary resistance to clarithromycin is rare . The Italian triple therapy remains a highly effective primary therapeutic method . Further, routine determination of resistance in non-responders also seems to be superfluous because high-dose dual therapy is an effective and well-tolerated second-line therapy regardless of the patients' resistance status.

Int J Antimicrob Agents, 1999 Aug, 12(4), 275 - 8
Relationship between usage of antibiotics in food-producing animals and the appearance of antibiotic resistant bacteria; Shryock TR; Many studies and meeting reports have suggested that the use of some antibiotics in food animals can compromise the treatment of some infectious diseases in humans . Although the studies and reports are timely and important, it is difficult to assess the relative value of the conclusions in relationship to the overall situation concerning antibiotic resistant foodborne bacteria because the data used in the analyses are often of disparate origin . The studies have attempted to establish a cause and effect relationship between the use ('consumption') of antibiotics in food animals and treatment failures in human disease on the basis of {1} antibiotic usage data; {2} in vitro determinations of antibiotic susceptibility of animal and human isolates, {3} results obtained from controlled animal experiments or {4} epidemiological data . Each approach has sought to associate bacterial antibiotic resistance data with it's own immediate focus area of investigation . However, a true assessment of the degree of contribution to human antibiotic resistance problems from animal use can only be facilitated by comprehensively organizing these different approaches into a concerted, coordinated effort . Concurrently, the implementation of a multinational programme aimed at monitoring antibiotic usage in food animals and resistance in specific bacteria associated with those animals should be instituted . In parallel with this endeavour is the implementation of new prudent use guidelines for antibiotic use by veterinarians . Through the use of science-based approaches like these, the development and spread of antibiotic resistant bacteria associated with food animals could be minimized and contained.

Zentralbl Veterinarmed B, 1999 Aug, 46(6), 423 - 7
Prevalence of verotoxin-producing Escherichia coli (VTEC) in bovine coli mastitis and their antibiotic resistance patterns; Stephan R et al.; Between December 1996 and October 1997, milk samples from a total of 145 cows with coli mastitis were screened for the presence of verotoxin-producing E . coli (VTEC) . VTEC were found in four (2.8%) out of the 145 samples . The four isolated strains proved to be verotoxin (VT) 1-, VT2- or VT1- and VT2-positive . However, no strain contained all three virulence factors tested . Further strain characterization was carried out by serotyping as well as by resistance pattern analysis.

Nat Biotechnol, 1999 Sep, 17(9), 910 - 5
Fluorescent antibiotic resistance marker for tracking plastid transformation in higher plants; Khan MS et al.; Plastid transformation in higher plants is accomplished through a gradual process, during which all the 300-10,000 plastid genome copies are uniformly altered . Antibiotic resistance genes incorporated in the plastid genome facilitate maintenance of transplastomes during this process . Given the high number of plastid genome copies in a cell, transformation unavoidably yields chimeric tissues, which requires the identification of transplastomic cells in order to regenerate plants . In the chimeric tissue, however, antibiotic resistance is not cell autonomous: transplastomic and wild-type sectors both have a resistant phenotype because of phenotypic masking by the transgenic cells . We report a system of marker genes for plastid transformation, termed FLARE-S, which is obtained by translationally fusing aminoglycoside 3"-adenyltransferase with the Aequorea victoria green fluorescent protein . 3"-adenyltransferase (FLARE-S) confers resistance to both spectinomycin and streptomycin . The utility of FLARE-S is shown by tracking segregation of individual transformed and wild-type plastids in tobacco and rice plants after bombardment with FLARE-S vector DNA and selection for spectinomycin and streptomycin resistance, respectively . This method facilitates the extension of plastid transformation to nongreen plastids in embryogenic cells of cereal crops.

Vet Rec, 1999 Jul 10, 145(2), 50 - 3
The need for a veterinary antibiotic policy; Pedersen KB et al.; The international recognition of the 'stable to table' approach to food safety emphasises the need for appropriate and safe use of antibiotics in animal production . An appropriate use of antibiotics for food animals will preserve the long-term efficacy of existing antibiotics, support animal health and welfare and limit the risk of transfer of antibiotic resistance to humans . Furthermore, it may promote consumer confidence in the veterinary use of antibiotics . In advancing these arguments, the authors of this article argue that there is a need for a visible and operational policy for veterinary use of antibiotics, paying particular attention to the policies that are being developed in Denmark.

Trans R Soc Trop Med Hyg, 1999 Mar-Apr, 93(2), 171 - 3
Peptic ulcer disease in south Ethiopia is strongly associated with Helicobacter pylori; Henriksen TH et al.; Helicobacter pylori infection was detected in 93% of 174 patients with a peptic ulcer compared with 63% of 116 patients with normal findings (chi 2 = 37.3; P < 0.001) in a cohort of 834 consecutive patients examined by gastroscopy in Yirga Alem Hospital in south Ethiopia . Fourteen patients were given 14 days' treatment with metronidazole 500 mg t.i.d., doxycycline 100 mg b.i.d . and bismuth subnitrate mixture 150 mg q.i.d . Of 10 patients who returned for follow-up, only 2 patients were free from H . pylori and cured . Nineteen strains of H . pylori from 19 consecutive patients in the same hospital were tested for resistance in vitro against metronidazole, doxycycline and ampicillin . All but 1 were highly resistant to metronidazole; 2 were fully and 14 intermediate resistant against doxycycline . All strains were fully sensitive in vitro to ampicillin . Thus, peptic ulcer was strongly associated with H . pylori in south Ethiopia, but eradication of the infection was hampered by antibiotic resistance.

J Bacteriol, 1999 Aug, 181(16), 4842 - 7
The periplasmic murein peptide-binding protein MppA is a negative regulator of multiple antibiotic resistance in Escherichia coli; Li H et al.; MppA is a periplasmic binding protein in Escherichia coli essential for uptake of the cell wall murein tripeptide L-alanyl-gamma-D-glutamyl-meso-diaminopimelate . We have found serendipitously that E . coli K-12 strains carrying a null mutation in mppA exhibit increased resistance to a wide spectrum of antibiotics and to cyclohexane . Normal sensitivity of the mppA mutant to these agents is restored by mppA expressed from a plasmid . As is observed in the multiple antibiotic resistance phenotype in E . coli cells, the mppA null mutant overproduces the transcriptional activator, MarA, resulting in expression of the membrane-bound AcrAB proteins that function as a drug efflux pump . Reduced production of OmpF similar to that observed in the multiple antibiotic resistance phenotype is also seen in the mppA mutant . These and other data reported herein indicate that MppA functions upstream of MarA in a signal transduction pathway to negatively regulate the expression of marA and hence of the MarA-driven multiple antibiotic resistance . Overproduction of cytoplasmic GadA and GadB and of several unidentified cytoplasmic membrane proteins as well as reduction in the amount of the outer membrane protein, OmpP, in the mppA null mutant indicate that MppA regulates a number of genes in addition to those already known to be controlled by MarA.

Gene Ther, 1999 Feb, 6(2), 209 - 18
Minicircle: an improved DNA molecule for in vitro and in vivo gene transfer; Darquet AM et al.; Minicircles are a new form of supercoiled DNA molecule for nonviral gene transfer which have neither bacterial origin of replication nor antibiotic resistance marker . They are thus smaller and potentially safer than the standard plasmids currently used in gene therapy . They were obtained in E . coli by att site-specific recombination mediated by the phage lambda integrase, which was used to excise the expression cassette from the unwanted plasmid sequences . We produced two minicircles containing the luciferase or beta-galactosidase gene under the control of the strong human cytomegalovirus immediate-early enhancer/promoter . Comparing maximal differences, these minicircles gave 2.5 to 5.5 times more reporter gene activity than the unrecombined plasmid in the NIH3T3 cell line and rabbit smooth muscle cells . Moreover, injection in vivo into mouse cranial tibial muscle, or human head and neck carcinoma grafted in nude mice resulted in 13 to 50 times more reporter gene expression with minicircles than with the unrecombined plasmid or larger plasmids . Histological analysis in muscle showed there were more transfected myofibers with minicircles than with unrecombined plasmid.

Clin Infect Dis, 1999 Jul, 29(1), 155 - 60
Antibiotic prescribing for Canadian preschool children: evidence of overprescribing for viral respiratory infections; Wang EE et al.; Antibiotic resistance is associated with prior receipt of antibiotics . An analysis of linked computerized databases for physician visits and antibiotic prescriptions was used to examine antibiotic prescribing for different respiratory infections in preschool children in Canada . In 1995, 64% of 61,165 children aged <5 years made 140,892 visits (mean, 3.6 visits per child) for respiratory infections; 74% of children who made visits received antibiotic prescriptions . Antibiotics were prescribed to 49% of children with upper respiratory tract infection, 18% with nasopharyngitis, 78% with pharyngitis or tonsillitis, 32% with serous otitis media, 80% with acute otitis media, 61% with sinusitis, 44% with acute laryngitis or tracheitis, and 24% with influenza . Acute otitis media accounted for 33% of all visits and 39% of all antibiotic prescriptions . The estimated Canadian-dollar cost of overprescribing was $423,693, or 49% of the total cost of antibiotics ($859,893) used in this group . This population-based study confirms antibiotic overprescribing in Canada.

Microb Drug Resist, 1999 Summer, 5(2), 141 - 6
Effect of clarithromycin and omeprazole therapy on the diversity and stability of genotypes of Helicobacter pylori from duodenal ulcer patients; Owen RJ et al.; The genotypes of multiple isolates of Helicobacter pylori from 17 duodenal ulcer patients in the United Kingdom were compared to determine reasons for treatment failure . Isolates were from antrum and corpus biopsies taken before and after dual therapy with clarithromycin and omeprazole . All isolates were tested for antibiotic resistance and characterised by a novel scheme combining polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the ureA + ureB and 23S rRNA genes, vacA signal and midregion genotypes, and PCR detection of cagA . Combined genotypes of paired pre- and post-treatment isolates from 8 patients showed an infection with a single strain of H . pylori that had acquired resistance to clarithromycin . In 4 other patients, acquisition of clarithromycin resistance was associated with the presence of different strain types of H . pylori . The remaining 5 patients had clarithromycin-sensitive isolates . Overall, H . pylori from different patients had diverse genotypes, yet most (70%) were colonized by the same predominant and stable strain in both the antrum and corpus . There was no link between the emergence of in vitro clarithromycin resistance and a particular strain genotype for these UK isolates . It was concluded that colonization with a clarithromycin-resistant H . pylori was due to selection of a resistant strain or clonal variant within the infecting population . Present genomic markers had low predictive value for emergence of resistance.

FEMS Microbiol Lett, 1999 Jul 15, 176(2), 279 - 84
The effect of some antibiotic-resistance-conferring plasmids on the removal of the heat-aggregated proteins from Escherichia coli cells; Kedzierska S et al.; We found that the presence of plasmids expressing tetracycline resistance or chloramphenicol resistance genes, but not those expressing ampicillin resistance or kanamycin resistance genes, in Escherichia coli led to the retardation of the process of removal of the heat-aggregated proteins (i.e . the S fraction) from the bacterial cells . The presence of chloramphenicol acetyltransferase in the S fraction is demonstrated . Moreover, we observed that the expression of T7 RNA polymerase gene had an influence on S fraction removal . These results suggest that high level production of some heterologous proteins which are accumulated in the cytoplasm, but not proteins exported through the cell membranes, may cause overloading of the S fraction and delay in the removal of heat-aggregated proteins from bacterial cells.

Eur J Clin Pharmacol, 1999 Jun, 55(4), 325 - 32
"Doi moi" and private pharmacies: a case study on dispensing and financial issues in Hanoi, Vietnam; Chuc NT et al.; Vietnam, a developing country, has had comparatively good health and human survival at low cost . The economic reform changed the health care system, and private pharmacies during the last 5 years have taken over a majority of the drug distribution . Problems include weaknesses in drug regulation and reported increases in antibiotic resistance . This case study, a purposive sample of two private pharmacies in Hanoi, explored management, including dispensing, inventory and financing, using the concept of triangulation . Observations and interviews of customers were complemented by stock inventory and interviews of the pharmacy staff . Drugs were classified according to the ATC code and the essential drug list of Vietnam . Pretested protocols were used . In all 1833 encounters were studied during the 2 weeks, out of which 286 were children . Less than 1% of customers came with prescriptions and 94.9% decided by themselves which drugs to buy . Antibiotics represented 17%, of which 90% were broad spectrum . Ampicillin dominated, both in children and adults . Some 50% of the antibiotics were given for 2.5 days or less . Less than 50% of the drugs were essential drugs (ED) on dispensing and even less on inventory . Antibiotics and vitamins were the most commonly sold drugs and, overall, brand names dominated . Little if any drug information was observed . Antibiotics were said to represent the most profitable drugs, according to the pharmacy staff . More than 20% of all products were combination drugs, including irrational and popular products with antibiotics and corticosteroids and combinations of aspirin, phenacetin and caffeine . This study shows an unexpectedly high proportion of customers, being "Tu Lam Bac Sy" (their own doctors), deciding themselves which drugs to buy . Although the "Doi moi" renovation has led to much improved drug availability, at least in urban setting, our case study highlights major problems in need of urgent actions . In particular the prevailing practices regarding antibiotics and combination drugs need to be seriously scrutinized and drug regulatory mechanisms should be enforced.

Cas Lek Cesk, 1999 May 24, 138(11), 343 - 7
{Antibiotic resistance}; Blahova J et al.; The authors present a short review of the origin and evolution of antibiotic resistance from the beginning of antibiotic use . Transferable resistance, i.e . spread of resistance genes by mechanisms of the transduction, conjugation or transposition, plays an important role in the process of the development of resistance in susceptible bacterial strains . Equally, chromosomally coded resistance is recently becoming relevant . Large selective pressure of the antibiotics lends mutations of genes coding antibiotic resistance . So, bacterial strains produce a large amount of enzymes, which destroy antibiotic or lose the power for penetration of the antibiotics become completely resistant also to new antibiotics.

J Bacteriol, 1999 Aug, 181(15), 4669 - 72
Alteration of the repressor activity of MarR, the negative regulator of the Escherichia coli marRAB locus, by multiple chemicals in vitro; Alekshun MN et al.; MarR negatively regulates expression of the multiple antibiotic resistance operon (marRAB) in Escherichia coli . In this study, it was demonstrated that sodium salicylate, plumbagin, 2, 4-dinitrophenol, and menadione-inducers of the marRAB operon in whole cells-all interfered with the repressor activity of MarR in vitro . It is proposed that these compounds can interact directly with MarR to affect its repressor activity.

Am J Med, 1999 Jul, 107(1), 62 - 7
Antibiotics in acute bronchitis: a meta-analysis; Bent S et al.; PURPOSE: Most patients with acute bronchitis who seek medical care are treated with antibiotics, although the effectiveness of this intervention is uncertain . We performed a meta-analysis of randomized, controlled trials to estimate the effectiveness of antibiotics in the treatment of acute bronchitis . SUBJECTS AND METHODS: English-language studies published January 1966 to April 1998 were retrieved using MEDLINE, bibliographies, and consultation with experts . Only randomized trials that enrolled otherwise healthy patients with a diagnosis of acute bronchitis, used an antibiotic in the treatment group and a placebo in the control group, and provided sufficient data to calculate an effect size were included . RESULTS: We identified eight randomized controlled trials that satisfied all inclusion criteria . These studies used one of three antibiotics (erythromycin, doxycycline, trimethoprim/sulfamethoxazole) . The use of antibiotics decreased the duration of cough and sputum production by approximately one-half day (summary effect size 0.21; 95% CI, 0.05 to 0.36) . For specific symptoms, there were nonsignificant trends favoring the use of antibiotics: a decrease of 0.4 days of purulent sputum (95% CI, -0.1 to 0.8), a decrease of 0.5 days of cough (95% CI, -0.1 to 1.1), and a decrease of 0.3 days lost from work (95% CI, -0.6 to 1.1) . CONCLUSION: This meta-analysis suggests a small benefit from the use of the antibiotics erythromycin, doxycycline, or trimethoprim/sulfamethoxazole in the treatment of acute bronchitis in otherwise healthy patients . As this small benefit must be weighed against the risk of side effects and the societal cost of increasing antibiotic resistance, we believe that the use of antibiotics is not justified in these patients.

Int J Antimicrob Agents, 1999 Jun, 12(1), 19 - 26
Antibiotic resistance problems with Helicobacter pylori; Alarcon T et al.; Helicobacter pylori is very susceptible in vitro to most antibiotics, but when they are used in the clinical setting, eradication of the bacteria from the gastric mucosa is not obtained . Dual or triple therapy including two of the following antibiotics: amoxicillin, tetracycline, metronidazole or clarithromycin, plus a proton pump inhibitor, bismuth salt or ranitidine bismuth citrate is the most frequently used . Various in vitro susceptibility methods have been used: disk diffusion, agar dilution and Epsilometer test (E-test) . Metronidazole resistance among H . pylori strains is now found worldwide, and resistance rates vary according to the population studied . It is higher in developing than in developed countries and it could reach 80-90% in Africa . The prevalence on clarithromycin resistance is much lower, usually below 10%, although very high values are reported in Peru . Infection with metronidazole- or clarithromycin-resistant H . pylori strains is correlated with treatment failure when using regimens including these antibiotics.

Helicobacter, 1999 Jun, 4(2), 106 - 12
Pretreatment antibiotic resistance in Helicobacter pylori infection: results of three randomized controlled studies; Realdi G et al.; BACKGROUND: Although combinations of antibiotics and antisecretory drugs are useful for treatment of Helicobacter pylori infection, treatment failure is common . The aim of this study was to evaluate the relation between pretreatment antibiotic resistance and outcome by using six different treatment regimens for H . pylori infection . PATIENTS AND METHODS: Three hundred sixty-nine consecutive H . pylori-infected patients with dyspeptic symptoms were enrolled in three consecutive randomized, controlled, single-center clinical trials: trial A, 128 patients; trial B, 125 patients; trial C, 116 patients . Treatments consisted of (A) a 15-day course of dual therapy (omeprazole, 20 mg bid, and amoxicillin, 1 gm bid, or clarithromycin, 500 mg tid) (OA vs OC); (B) a 7-day triple therapy of omeprazole, 20 mg bid, plus metronidazole, 500 mg bid, and amoxicillin, 1,000 mg bid, or clarithromycin, 500 mg tid (OMA vs OMC); or (C) omeprazole, 20 mg bid, plus metronidazole, 500 mg bid, plus tetracycline, 500 mg qid, or doxycycline, 100 mg tid (OMT vs OMD) . Diagnostic endoscopy was made in all patients before and 5 to 6 weeks after therapy . Six biopsies were taken from each patient for histology, rapid urease test, and H . pylori culture; antibiotic susceptibility testing was performed using the E-test method . RESULTS: Overall cure rates were poor for both dual therapies OA and OC (38% and 37%, respectively) and for triple therapies OMA, OMC, and OMD (57%, 55%, and 58%, respectively) . The OMT combination was successful in 91% (95% confidence interval {CI}, 80.4%-97%) . Metronidazole resistance was present in 29.7% (95% CI, 24%-35%), amoxicillin resistance was present in 26% (95% CI, 21%-32%), clarithromycin resistance was present in 23.1% (95% CI, 18%-29%), tetracycline resistance was present in 14% (95% CI, 10%-20%), and doxycycline resistance was present in 33.3% (95% CI, 21%-47%) . Antibiotic resistance markedly reduced the cure rates and accounted for most of the poor results with the triple therapies: 89% versus 23%; 77% versus 26%; 100% versus 60%; and 67% versus 23% for OMC, OMA, OMT, and OMD, respectively . OMT appeared to be the best because of the high success rate with metronidazole-resistant H . pylori (71%) and in low-level tetracycline resistance . CONCLUSIONS: Pretreatment antibiotic-resistant H . pylori can, in part, explain the low cure rate of the infection and the variability in outcome in reported trials.

J Mol Biol, 1999 Jun 18, 289(4), 827 - 34
Erythromycin resistance mutations in ribosomal proteins L22 and L4 perturb the higher order structure of 23 S ribosomal RNA; Gregory ST et al.; We have used chemical modification to examine the conformation of 23 S rRNA in Escherichia coli ribosomes bearing erythromycin resistance mutations in ribosomal proteins L22 and L4 . Changes in reactivity to chemical probes were observed at several nucleotide positions scattered throughout 23 S rRNA . The L4 mutation affects the reactivity of G799 and U1255 in domain II and that of A2572 in domain V . The L22 mutation influences modification in domain II at positions m5U747, G748, and A1268, as well as at A1614 in domain III and G2351 in domain V . The reactivity of A789 is weakly enhanced by both the L22 and L4 mutations . None of these nucleotide positions has previously been associated with macrolide antibiotic resistance . Interestingly, neither of the ribosomal protein mutations produces any detectable effects at or within the vicinity of A2058 in domain V, the site most frequently shown to confer macrolide resistance when altered by methylation or mutation . Thus, while L22 and L4 bind primarily to domain I of 23 S rRNA, erythromycin resistance mutations in these ribosomal proteins perturb the conformation of residues in domains II, III and V and affect the action of antibiotics known to interact with nucleotide residues in the peptidyl transferase center of domain V . These results support the hypothesis that ribosomal proteins interact with rRNA at multiple sites to establish its functionally active three-dimensional structure, and suggest that these antibiotic resistance mutations act by perturbing the conformation of rRNA .

Chem Res Toxicol, 1999 Jun, 12(6), 501 - 7
Mutagenicity of site-specifically located 1,N2-ethenoguanine in Chinese hamster ovary cell chromosomal DNA; Akasaka S et al.; The adduct 1,N2-etheno(epsilon)-guanine (Gua) can be formed in DNA from exogenous or endogenous bifunctional electrophiles . Previous work with site-specifically modified oligonucleotides has shown all three possible base substitutions at the site of this residue in bacterial cells and in primer extension assays using purified polymerases (with the purified polymerases also showing deletions) . A 10-mer was synthesized containing 1,N2-epsilon-Gua at a specific position and ligated into a modified pCNheIA vector, which was used to insert the modified sequence into the chromosomes of AA8 (wild-type) and UV5 (nucleotide excision repair-deficient) Chinese hamster ovary cells . Transformants were selected by antibiotic resistance; DNA was amplified by polymerase chain reaction, and resistance to the restriction endonuclease NheI was used to estimate mutation frequency . In the AA8 cells, the apparent mutation frequency was elevated >10-fold due to the presence of 1, N2-epsilon-Gua (to 4.6%) . In UV5 cells, the mutation frequency was even higher (7.8%), but the estimate of the frequency in the control system (vector and unmodified sequence only) was 4.5% . Sequence analysis of 21 clones derived from the mutant fraction yielded five that correspond to base pair mutations directly at the 1, N2-epsilon-Gua site . The remainder of the mutants differed from those generated from the unmodified oligonucleotide and included deletions, rearrangements, double mutants, and base pair substitutions at sites nearby but not at the 1,N2-epsilon-Gua site.

J Mol Biol, 1999 Jun 4, 289(2), 277 - 91
The 2.2 A structure of the rRNA methyltransferase ErmC' and its complexes with cofactor and cofactor analogs: implications for the reaction mechanism; Schluckebier G et al.; The rRNA methyltransferase ErmC' transfers methyl groups from S -adenosyl-l-methionine to atom N6 of an adenine base within the peptidyltransferase loop of 23 S rRNA, thus conferring antibiotic resistance against a number of macrolide antibiotics . The crystal structures of ErmC' and of its complexes with the cofactor S -adenosyl-l-methionine, the reaction product S-adenosyl-l-homocysteine and the methyltransferase inhibitor Sinefungin, respectively, show that the enzyme undergoes small conformational changes upon ligand binding . Overall, the ligand molecules bind to the protein in a similar mode as observed for other methyltransferases . Small differences between the binding of the amino acid parts of the different ligands are correlated with differences in their chemical structure . A model for the transition-state based on the atomic details of the active site is consistent with a one-step methyl-transfer mechanism and might serve as a first step towards the design of potent Erm inhibitors .

J Eukaryot Microbiol, 1999 Mar-Apr, 46(2), 206 - 16
Characterization of the Euplotes crassus macronuclear rDNA and its potential as a DNA transformation vehicle; Erbeznik M et al.; We have cloned the macronuclear linear DNA molecule carrying the ribosomal RNA genes from the ciliated protozoan Euplotes crassus . DNA sequence analysis was carried out to locate coding regions and to determine whether sequences that have been mutated to confer antibiotic resistance are conserved in the E . crassus genes . The beginning and end of the primary transcript were mapped . In order to determine whether conserved sequences that might serve as replication origins were present, the 5' and 3' non-coding sequences from E . crassus were compared to the corresponding sequences from the macronuclear linear rDNA molecules from the following euplotid species: Euplotes vannus, Euplotes minuta, Euplotes raikovii and Euplotes rariseta . A DNA transformation construct was made by generating a putative anisomycin resistant mutation along with a mutation generating a restriction site polymorphism . Microinjection of the construct into the developing macronucleus of mated cells resulted in exconjugant cell lines with increased resistance to anisomycin . The injected rDNA with the restriction site polymorphism is detectable in the anisomycin resistant cells and appears to represent a minor fraction of the rDNA.

J Antimicrob Chemother, 1999 Apr, 43(4), 459 - 65
A review of the role of antibiotic policies in the control of antibiotic resistance; Gould IM; The optimal antibiotic control measures remain to be described and probably vary between institutions . Nevertheless, various control measures have been shown to be useful in reducing costs of therapy and total amounts of prescribing, while maintaining quality of care . More recently, interest has turned to whether antibiotic policies can reduce the spread of resistance and even reverse current high levels . Early studies indicated this was feasible, but mathematical models and the recent discovery of the role of transposons and integrons in multi-drug resistance have both cast doubt on likely future success in this area . Nevertheless, there have been some major successes in recent studies, both in the community and hospital . While cross-infection is a major impediment to control of resistance, there is little doubt that careful antibiotic prescribing can curtail the emergence and reduce the prevalence of resistance.

J Antibiot (Tokyo), 1999 Mar, 52(3), 288 - 96
Molecular analysis of tlrB, an antibiotic-resistance gene from tylosin-producing Streptomyces fradiae, and discovery of a novel resistance mechanism; Wilson VT et al.; The tlrB gene, which confers inducible resistance to a range of macrolide antibiotics including biosynthetic precursors of tylosin, was isolated and sequenced . In the genome of Streptomyces fradiae, it lies between pbp, which encodes a putative penicillin-binding protein, and tylN, encoding a glycosyltransferase involved in tylosin biosynthesis . The TlrB protein was produced in E . coli as a fusion to MalE . The fusion protein, but not MalE alone, inactivates macrolides in the presence of S-adenosyl-methionine (SAM) but the modified product(s) has not been characterised.

Braz J Med Biol Res, 1999 Feb, 32(2), 147 - 53
Main features of DNA-based immunization vectors; Azevedo V et al.; DNA-based immunization has initiated a new era of vaccine research . One of the main goals of gene vaccine development is the control of the levels of expression in vivo for efficient immunization . Modifying the vector to modulate expression or immunogenicity is of critical importance for the improvement of DNA vaccines . The most frequently used vectors for genetic immunization are plasmids . In this article, we review some of the main elements relevant to their design such as strong promoter/enhancer region, introns, genes encoding antigens of interest from the pathogen (how to choose and modify them), polyadenylation termination sequence, origin of replication for plasmid production in Escherichia coli, antibiotic resistance gene as selectable marker, convenient cloning sites, and the presence of immunostimulatory sequences (ISS) that can be added to the plasmid to enhance adjuvanticity and to activate the immune system . In this review, the specific modifications that can increase overall expression as well as the potential of DNA-based vaccination are also discussed.

Prescrire Int, 1998 Aug, 7(36), 101 - 2
Cefpodoxime: new dosage . A new, unproven, dose regimen; Penicillin-resistant pneumococci in southern Sweden et al.; Department of Medical Microbiology, Lund University Hospital, SwedenIn Malmohus County, Southern Sweden, the frequency of penicillin-resistant pneumococci in nasopharyngeal specimens of outpatients with respiratory tract infections increased from 3.1% in 1993 to 7.6% in 1995, and was thereafter rather stable . Over the period, 82-85% of the patients with penicillin-resistant strains were children 0-6 years of age . Ten groups/types constituted 96-100% of the penicillin-resistant isolates . Grouping/typing of 200 consecutive isolates in October and November each year indicated that the distribution of groups/types amongst patients with respiratory tract infections was rather constant over the period . The frequency of penicillin-resistant pneumococci of groups/types 6, 14, and 19 roughly corresponded to the occurrence of these groups/types amongst the consecutive isolates . Other groups/types 9, 15, 21, and 23 either showed a pronounced increase or decrease, which could not be related to the prevalence of these groups/types among the consecutive isolates or degree of antibiotic resistance . Penicillin-resistant group 9, introduced in the area in 1993, consisted of one single clone, 9V . The stabilized level of penicillin resistance since 1995 may be related to the preventive measures implemented in the area, including day-care interventions, and measures to reduce the prescription rate of antibiotics to outpatients with respiratory tract infections.

Nucleic Acids Res, 1999 Jun 1, 27(11), 2332 - 8
Demethylation and expression of methylated plasmid DNA stably transfected into HeLa cells; Qu GZ et al.; In vitro methylation at CG dinucleotides (CpGs) in a transfecting plasmid usually greatly inhibits gene expression in mammalian cells . However, we found that in vitro methylation of all CpGs in episomal or non-episomal plasmids containing the SV40 early promoter/enhancer (SV40 Pr/E) driving expression of an antibiotic-resistance gene decreased the formation of antibiotic-resistant colonies by only approximately 30-45% upon stable transfection of HeLa cells . In contrast, when expression of the antibiotic-resistance gene was driven by the Rous sarcoma virus long terminal repeat or the herpes simplex virus thymidine kinase promoter, this methylation decreased the yield of antibiotic-resistant HeLa transfectant colonies approximately 100-fold . The low sensitivity of the SV40 Pr/E to silencing by in vitro methylation was probably due to demethylation upon stable transfection . This demethylation may be targeted to the promoter and extend into the gene . By genomic sequencing, we showed that four out of six of the transfected SV40 Pr/E's adjacent Sp1 sites were hotspots for demethylation in the HeLa transfectants . High frequency demethylation at Sp1 sites was unexpected for a non-embryonal cell line and suggests that DNA demethylation targeted to certain aberrantly methylated regions may function as a repair system for epigenetic mistakes.

J Bacteriol, 1999 May, 181(10), 3303 - 6
Characterization of MarR superrepressor mutants; Alekshun MN et al.; MarR negatively regulates expression of the multiple antibiotic resistance (mar) locus in Escherichia coli . Superrepressor mutants, generated in order to study regions of MarR required for function, exhibited altered inducer recognition properties in whole cells and increased DNA binding to marO in vitro . Mutations occurred in three areas of the relatively small MarR protein (144 amino acids) . It is surmised that superrepression results from increased DNA binding activities of these mutant proteins.

J Bacteriol, 1999 May, 181(10), 3293 - 7
Identification and in vivo functional analysis of a virginiamycin S resistance gene (varS) from Streptomyces virginiae; Lee CK et al.; BarA of Streptomyces virginiae is a specific receptor protein for virginiae butanolide (VB), one of the gamma-butyrolactone autoregulators of the Streptomyces species, and acts as a transcriptional regulator controlling both virginiamycin production and VB biosynthesis . The downstream gene barB, the transcription of which is under the tight control of the VB-BarA system, was found to be transcribed as a polycistronic mRNA with its downstream region, and DNA sequencing revealed a 1,554-bp open reading frame (ORF) beginning at 161 bp downstream of the barB termination codon . The ORF product showed high homology (68 to 73%) to drug efflux proteins having 14 transmembrane segments and was named varS (for S . virginiae antibiotic resistance) . Heterologous expression of varS with S . lividans as a host resulted in virginiamycin S-specific resistance, suggesting that varS encoded a virginiamycin S-specific transport protein . Northern blot analysis indicated that the bicistronic transcript of barB-varS appeared 1 to 2 h before the onset of virginiamycin M1 and S production, at which time VB was produced, while exogenously added virginiamycin S apparently induced the monocistronic varS transcript.

Aliment Pharmacol Ther, 1999 May, 13(5), 667 - 73
The effect of antibiotic resistance on the outcome of three 1-week triple therapies against Helicobacter pylori; Pilotto A et al.; BACKGROUND: Resistance of Helicobacter pylori to antibiotics may be a major reason for treatment failure . AIM: To evaluate the effect of primary H . pylori resistance to antibiotics on the cure rates of three anti-H . pylori 1-week triple therapies . METHODS: One hundred and sixteen consecutive patients diagnosed H . pylori-positive by gastric histology, rapid urease test and culture were enrolled . Activity of tested antibiotics was determined by means of the E-test . Patients were treated for 7 days with: (i) pantoprazole 40 mg o.d . plus amoxycillin 1 g b.d . and metronidazole 250 mg q.d.s . (PAM); (ii) pantoprazole 40 mg o.d . plus clarithromycin 250 mg b.d . and metronidazole 250 mg q.d.s . (PCM); or (iii) pantoprazole 40 mg o.d . plus amoxycillin 1 g b.d . and clarithromycin 250 mg b.d . (PAC) . Two months after completion of therapy, endoscopy and gastric biopsies were repeated . RESULTS: Primary resistance rates to metronidazole, clarithromycin and amoxycillin were 17.2, 6.9 and 0%, respectively . Overall H . pylori cure rates expressed as intention-to-treat and per protocol analyses were, respectively, 79% and 86% with PAM, 82% and 89% with PCM, and 85% and 85% with PAC . Significantly lower cure rates were observed in metronidazole-resistant patients treated with PAM (56% vs . 96%, P = 0.01) or PCM (50% vs . 97%, P = 0.01) . A trend towards lower H . pylori cure rates was observed in clarithromycin-resistant patients treated with PCM (67% vs . 91%, P = 0.74) or PAC (50% vs . 87%, P = 0.68) . CONCLUSION: Primary resistance to metronidazole influences the H . pylori cure rate of anti-H . pylori proton pump inhibitor-based triple therapies which include this antibiotic . A similar trend exists for primary clarithromycin resistance.

Adv Enzymol Relat Areas Mol Biol, 1999, 73, 25 - 55, ix
Rethinking fundamentals of enzyme action; Northrop DB; Despite certain limitations, investigators continue to gainfully employ concepts rooted in steady-state kinetics in efforts to draw mechanistically relevant inferences about enzyme catalysis . By reconsidering steady-state enzyme kinetic behavior, this review develops ideas that allow one to arrive at the following new definitions: (a) V/K, the ratio of the maximal initial velocity divided by the Michaelis-Menten constant, is the apparent rate constant for the capture of substrate into enzyme complexes that are destined to yield product(s) at some later point in time; (b) the maximal velocity V is the apparent rate constant for the release of substrate from captured complexes in the form of free product(s); and (c) the Michaelis-Menten constant K is the ratio of the apparent rate constants for release and capture . The physiologic significance of V/K is also explored to illuminate aspects of antibiotic resistance, the concept of "perfection" in enzyme catalysis, and catalytic proficiency . The conceptual basis of congruent thermodynamic cycles is also considered in an attempt to achieve an unambiguous way for comparing an enzyme-catalyzed reaction with its uncatalyzed reference reaction . Such efforts promise a deeper understanding of the origins of catalytic power, as it relates to stabilization of the reactant ground state, stabilization of the transition state, and reciprocal stabilizations of ground and transition states.

Biochem Soc Symp, 1999, 64, 119 - 28
Transcriptional regulation via redox-sensitive iron-sulphur centres in an oxidative stress response; Demple B et al.; Genetic responses to oxidative stress are triggered by excessive levels of agents such as superoxide . The soxRS regulon of Escherichia coli includes at least a dozen oxidative-stress and antibiotic-resistance genes that are activated by the SoxS protein, the synthesis of which is controlled by the redox-sensing SoxR protein . SoxR is a homodimer of 17 kDa subunits, each of which contains a {2Fe-2S} cluster . Transcriptional activation by SoxR is controlled by the oxidation state of these metal centres . In the absence of oxidative stress, the {2Fe-2S} centres are in the reduced form and the protein is inactive, although it still binds the soxS promoter . Agents that generate superoxide in the cell (e.g . paraquat) cause rapid oxidation of the metal centres, which triggers the transcriptional activity of SoxR; removal of the oxidative stress is followed by rapid re-reduction of the {2Fe-2S} centres . This facile mechanism links oxidation state to control of protein activity and may be used widely to allow cells to respond to oxidative stress.

FEMS Microbiol Lett, 1999 Mar 15, 172(2), 255 - 60
Genetic basis of macrolide and lincosamide resistance in Brachyspira (Serpulina) hyodysenteriae; Karlsson M et al.; Macrolide antibiotic resistance is widespread among Brachyspira hyodysenteriae (formerly Serpulina hyodysenteriae) isolates . The genetic basis of macrolide and lincosamide resistance in B . hyodysenteriae was elucidated . Resistance to tylosin, erythromycin and clindamycin in B . hyodysenteriae was associated with an A-->T transversion mutation in the nucleotide position homologous with position 2058 of the Escherichia coli 23S rRNA gene . The nucleotide sequences of the peptidyl transferase region of the 23S rDNA from seven macrolide and lincosamide resistant and seven susceptible strains of Brachyspira spp . were determined . None of the susceptible strains were mutated whereas all the resistant strains had a mutation in position 2058 . Susceptible strains became resistant in vitro after subculturing on agar containing 4 micrograms ml-1 of tylosin . Sequencing of these strains revealed an A-->G transition mutation in position 2058.

Biotechnol Bioeng, 1998 Dec 20, 60(6), 656 - 63
High-level expression of secreted glycoproteins in transformed lepidopteran insect cells using a novel expression vector; Farrell PJ et al.; An expression cassette for continuous high-level expression of secreted glycoproteins by transformed lepidopteran insect cells has been developed as an alternative to baculovirus and mammalian cell expression systems . The expression cassette utilizes the promoter of the silkmoth cytoplasmic actin gene to drive expression from foreign gene sequences, and also contains the ie-1 transactivator gene and the HR3 enhancer region of BmNPV to stimulate gene expression . Using an antibiotic-resistance selection scheme, we have cloned a Bm5 (silkmoth) cell line overexpressing the secreted glycoprotein juvenile hormone esterase (JHE-KK) at levels of 190 mg/L in batch suspension cultures . A baculovirus (AcNPV) expressing the same gene under the control of the p10 promoter of AcNPV produced only 4 mg/L active JHE in static cultures of infected Sf21 cells . A cloned Bm5 cell line overexpressing a soluble isoform of the alpha-subunit of the granulocyte-macrophage colony stimulating factor receptor (solGMRalpha) was also generated and produced five times more solGMRalpha in static cultures than a cloned BHK cell line obtained by transformation with a recombinant expression cassette utilizing the human cytomegalovirus (CMV) enhancer-promoter system . Finally, we show that recombinant protein expression levels in transformed Bm5 cells remain high in serum-free media, that expression is stable even in the absence of antibiotic selection, and that lepidopteran cells other than Bm5 may be used equally efficiently with this new expression cassette for producing recombinant proteins .

Biotechnol Bioeng, 1998 Jan 20, 57(2), 238 - 44
Expression of the membrane protein glycophorin A as a fusion with the antibiotic resistance protein neomycin phosphotransferase II; Kromer WJ et al.; The gene for the integral membrane protein glycophorin A (GPA) was cloned in frame to the 5' end of the antibiotic resistance gene, neomycin phosphotransferase II (NPT) . Protein expression was achieved in Escherichia coli as well as in mammalian cells . In case of Chinese hamster ovary cells (CHO) the resistant populations were analyzed 2 weeks after transfection; the amount of GPA-NPT fusion protein produced was constant from experiment to experiment . Neomycin resistance was directly correlated with GPA expression, thus allowing the direct selection for a stable GPA-expressing cell population without the need of a cloning step . The amount of GPA-NPT produced was further increased by weakening the specific NPT enzymatic activity via site-directed mutagenesis . Detection was simplified by the fact that all different fusion proteins could be detected by the same anti-NPT antibody . This approach may be also applicable to other membrane proteins .

J Anim Sci, 1999 Feb, 77(2), 367 - 71
What is the pharmaceutical industry doing, and what does the pharmaceutical industry want from animal science departments?
Lauderdale JW.
Perceived contemporary issues are 1) food safety and food healthfulness, 2) environment, 3) sustainability, 4) biotechnology, 5) animal well-being, 6) animals as food, and 7) research funding . Food safety is the paramount contemporary issue, and environment and sustainability issues can be considered as a single issue . Biotechnology, animal well-being, and animals as food are addressed in this paper as separate issues, but they can be considered as components of food safety and healthfulness . The pharmaceutical industry addresses these issues by providing safe and effective products to the livestock industry . These products are used to treat and prevent disease and to increase livestock production efficiency . These products contribute to a safe food supply, enhance protection of the environment, and increase the sustainability of animal agriculture through increased efficiency of livestock production . The pharmaceutical industry wants the following from animal science departments: 1) students skilled in deductive and inductive thinking and communicating to peers and the public; 2) regional research on food safety, such as irradiation, steaming of carcasses, E . coli contamination, antibiotic resistance, production facilities, and carcass contamination; 3) improved research to identify the food values of animal products and effective communication of that research to the public; 4) research on topics having the greatest potential to increase efficiency of animal production consistent with a positive impact on the environment and sustainability of animal production; 5) leadership in developing and using technologies such as biotechnology, not only as descriptors of biological processes, but as technologies to test hypotheses leading to new understandings of biology; 6) research on animal well-being and production facilities that foster animal well-being; 7) research and education on ethical and moral aspects of animals as food through encouragement of one or more staff members to become effective animal science department spokespersons; and 8) active participation in activities such as FAIR 95, Federation of Animal Science Societies, and multidepartmental and(or) interdisciplinary programs.

Yonsei Med J, 1998 Dec, 39(6), 534 - 40
Molecular analysis of fluoroquinolone-resistance in Escherichia coli on the aspect of gyrase and multiple antibiotic resistance (mar) genes; Park YH et al.; We analyzed the fluoroquinolone resistance mechanism of 28 isolates of ciprofloxacin-resistant E . coli from patients who received ciprofloxacin as a regimen of a selective gut decontamination . Isolates distinctive by infrequent restriction site polymerase chain reaction (IRS-PCR) were subjected to Hinf I restriction fragment length polymorphism analysis, single-stranded conformation polymorphism (SSCP), and nucleotide sequencing of the quinolone resistance determining region (QRDR) in gyrA . Double mutations in QRDR of gyrA (Ser83 Leu and Asp87Asn) were found from most of the strains . Nucleotide sequencing of the marR locus showed that 18 out of 28 (64%) ciprofloxacin-resistant E . coli strains had three types of base change in marR loci: a double-base change at nucleotides 1628 and 1751, or 1629 and 1751: and a single-base change at 1751 . However, all the mutated strains showed no tolerance to cyclohexane test, suggesting the mutation in the marR region had no influence on overexpression of the MarA protein . In conclusion, mutation in gyrA was the main mechanism of ciporfloxacin resistance in E . coli from patients with selective gut decontamination . Therefore, mutation in the mar region did not influence the levels of ciprofloxacin resistance in our isolates.

J Bacteriol, 1999 Apr, 181(7), 2185 - 91
Transcriptional activation of ydeA, which encodes a member of the major facilitator superfamily, interferes with arabinose accumulation and induction of the Escherichia coli arabinose PBAD promoter; Bost S et al.; Induction of genes expressed from the arabinose PBAD promoter is very rapid and maximal at low arabinose concentrations . We describe here two mutations that interfere with the expression of genes cloned under arabinose control . Both mutations map to the ydeA promoter and stimulate ydeA transcription; overexpression of YdeA from a multicopy plasmid confers the same phenotype . One mutation is a large deletion that creates a more efficient -35 region (ATCACA changed to TTCACA), whereas the other affects the initiation site (TTTT changed to TGTT) . The ydeA gene is expressed at extremely low levels in exponentially growing wild-type cells and is not induced by arabinose . Disruption of ydeA has no detectable effect on cell growth . Thus, ydeA appears to be nonessential under usual laboratory growth conditions . The ydeA gene encodes a membrane protein with 12 putative transmembrane segments . YdeA belongs to the largest family of bacterial secondary active transporters, the major facilitator superfamily, which includes antibiotic resistance exporters, Lac permease, and the nonessential AraJ protein . Intracellular accumulation of arabinose is strongly decreased in mutant strains overexpressing YdeA, suggesting that YdeA facilitates arabinose export . Consistent with this interpretation, very high arabinose concentrations can compensate for the negative effect of ydeA transcriptional activation . Our studies (i) indicate that YdeA, when transcriptionally activated, contributes to the control of the arabinose regulon and (ii) demonstrate a new way to modulate the kinetics of induction of cloned genes.

Postgrad Med, 1999 Mar, 105(3), 137 - 40, 145-8
Practical advice on eradicating Helicobacter pylori infection; Graham DY et al.; Peptic ulcer disease associated with H pylori infection is curable . The important factors in selecting therapy are efficacy of eradication, prevention of resistance, avoidance or minimization of adverse effects, patient compliance, and cost . The most effective regimens include a bismuth preparation or antisecretory drug (proton pump inhibitor or H2 receptor antagonist) plus two antibiotics administered for 14 days . Dual-drug therapies are not recommended . Triple-drug regimens are more likely to eradicate H pylori and less likely to generate resistant strains among surviving organisms . In general, cure of the infection should be confirmed 4 weeks after completion of the treatment . Antibiotic resistance is an important consideration in choosing therapy, and patients should be taught the importance of compliance . When treatment fails, antibiotic combinations should not be repeated . Considerations for anti-H pylori treatment in a managed care environment mirror those for good medical practice in general, with special attention to stringent cost-control or outcomes-driven measures.

J Clin Gastroenterol, 1999 Mar, 28(2), 131 - 4
Efficacy of prolonged administration of intravenous erythromycin in an ambulatory setting as treatment of severe gastroparesis: one center's experience; DiBaise JK et al.; Intravenous erythromycin is a potent gastric prokinetic with demonstrated efficacy in the acute therapy of gastroparesis; long-term oral therapy has been limited by tolerance and modest efficacy . Our aim was to review our experience with prolonged administration of intravenous erythromycin in an ambulatory setting as therapy for severe gastroparesis, refractory to usual dietary and oral prokinetic regimens . We conducted a retrospective analysis of patients with gastroparesis treated with intravenous erythromycin for at least 1 month . Information on demographics; origin of gastroparesis; dosage, duration, and route of administration; clinical outcome in the short- and longer-term; and complications were determined . Eleven patients received a total of 14 courses of intravenous erythromycin for a median of 6.5 months (range, 1 to 19 months) at a median dosage of 300 mg/day (range, 150 to 1,000 mg/day) . One patient received no benefit, two had complete responses, and all others reported some benefit . Two had dramatic relapse on cessation of therapy and subsequently improved on its resumption . Parenteral nutrition could be discontinued in one of four patients . There were four episodes of line sepsis; two required catheter removal . A nonocclusive thrombus developed at the site of a central line in one patient . Secondary infections or antibiotic resistance were not encountered . Prolonged administration of intravenous erythromycin in an ambulatory setting is feasible, well tolerated, and effective in patients with severe gastroparesis.

Plant J, 1999 Jan, 17(1), 99 - 109
Genetic engineering of the multicellular green alga Volvox: a modified and multiplied bacterial antibiotic resistance gene as a dominant selectable marker; Hallmann A et al.; The green alga Volvox represents the simplest multicellular organism: Volvax is composed of only two cell types, somatic and reproductive . Volvox, therefore, is an attractive model system for studying various aspects of multicellularity . With the biolistic nuclear transformation of Volvox carteri, the powerful molecular genetic manipulation of this organism has been established, but applications have been restricted to an auxotrophic mutant serving as the DNA recipient . Therefore, a dominant selectable marker working in all strains and mutants of this organism is required . Among several gene constructs tested, the most advantageous results were obtained with a chimeric gene composed of the coding sequence of the bacterial ble gene, conferring resistance to the antibiotic zeocin, modified with insertions of two endogenous introns from the Volvox arylsulfatase gene and fused to 5' and 3' untranslated regions from the Volvox beta 2-tubulin gene . In the most suitable plasmid used, the gene dosage was increased 16-fold by a technique that allows exponential multiplication of a DNA fragment . Co-transformation of this plasmid and a non-selectable plasmid allowed the identification of zeocin resistant transformants with nuclear integration of both selectable and non-selectable plasmids . Stable expression of the ble gene and of genes from several non-selectable plasmids is demonstrated . The modified ble gene provides the first dominant marker for transformation of both wild-type and mutant strains of Volvox.

Vaccine, 1999 Feb 26, 17(7-8), 705 - 14
Immunogenicity of recombinant BCG producing the GRA1 antigen from Toxoplasma gondii; Supply P et al.; Toxoplasmosis is a major parasitic disease, responsible for foetopathy in humans and domestic animals, especially sheep . Toxoplasma gondii infection generally protects immunocompetent hosts against subsequent reinfection, suggesting that efficacious vaccines can be developed against this disease . Excreted/secreted T . gondii antigens have previously been shown to provide immunoprotection in small rodents, and protective immunity is thought to be cell-mediated . Mycobacterium bovis BCG is known to be a good inducer of cellular immunity . In this study, we have developed a BCG strain which produces and secretes GRA1, one of the major excreted/secreted T . gondii antigens . This strain does not carry antibiotic-resistance determinants and is therefore safe for the environment . The intraperitoneal immunisation of OF1 outbred mice with this BCG strain failed to induce GRA1-specific humoral or cellular immune responses and only conferred a very limited degree of protection against challenge with virulent T . gondii . However, in sheep immunised subcutaneously and boosted intravenously, this recombinant BCG strain induced GRA1-specific cell-mediated responses, as evidenced by the proliferation of peripheral blood mononuclear cells and by the production of IFN-gamma, although it failed to elicit GRA1-specific antibody responses . Following oocyst challenge infection, sheep immunised with recombinant BCG exhibited an abbreviated temperature response compared with controls, suggesting partial protection.

Lakartidningen, 1999 Feb 3, 96(5), 460 - 3
{Antibiotic resistance of Helicobacter pylori in Malmö . Therapeutic success in spite of antibiotic resistance}; Nilsson F et al.; Triple therapy (omeprazole, clarithromycin and metronidazole) is associated with a sensitive Helicobacter pylori (HP) eradication rate of over 90 per cent . As treatment failure is mainly due to poor patient compliance, satisfactory patient information is vital . Despite the high prevalence (29-40%) of metronidazole-resistant HP in Malmo, the eradication rate is over 90% . Clarithromycin-resistance among HP strains has increased in prevalence from 1 to 7% over the past four years, and always results in treatment failure . In order to avoid unnecessary antibiotic treatment, especially in cases of patients not responding to triple therapy, it is important to follow treatment up with, for example, the urea breath test.

Hepatology, 1999 Mar, 29(3), 834 - 8
The human p53 gene mutated at position 249 per se is not sufficient to immortalize human liver cells; Schleger C et al.; A particular point mutation of the tumor suppressor gene p53, namely a G-->T transversion at the third base of codon 249, is frequently detected in primary hepatocellular carcinomas from patients living in areas where the levels of dietary exposure to aflatoxin B1 and the rates of infection with the hepatitis B virus are very high . Very recently, a nontumorigenic liver epithelial cell line (HACL-1) with a finite life-span and expressing a number of hepatocyte-specific markers was established from a human hepatocellular adenoma in our laboratory . To analyze the role of mutated p53 in the immortalization of human liver cells, we transfected HACL-1 cells with an expression vector containing a human p53 complementary DNA mutated at the third base of codon 249 and analyzed the consequences of this gene transfer on the growth properties of this cell line . HACL-1 cells transfected with mutant p53 showed no increase in their life-span (when compared with HACL-1 cells transfected with the antibiotic resistance gene alone) and did not grow in soft agar, whereas transfection of wild-type p53 into HACL-1 cells led to a proliferation stop . Thus, these results strongly support the view that the mutation at codon 249 of the p53 gene may serve as a fingerprint for aflatoxin B1-induced hepatocellular carcinomas, but is not, by itself, sufficient to immortalize human liver cells.

J Bacteriol, 1999 Mar, 181(5), 1415 - 28
Ferritin mutants of Escherichia coli are iron deficient and growth impaired, and fur mutants are iron deficient; Abdul-Tehrani H et al.; Escherichia coli contains at least two iron storage proteins, a ferritin (FtnA) and a bacterioferritin (Bfr) . To investigate their specific functions, the corresponding genes (ftnA and bfr) were inactivated by replacing the chromosomal ftnA and bfr genes with disrupted derivatives containing antibiotic resistance cassettes in place of internal segments of the corresponding coding regions . Single mutants (ftnA::spc and bfr::kan) and a double mutant (ftnA::spc bfr::kan) were generated and confirmed by Western and Southern blot analyses . The iron contents of the parental strain (W3110) and the bfr mutant increased by 1.5- to 2-fold during the transition from logarithmic to stationary phase in iron-rich media, whereas the iron contents of the ftnA and ftnA bfr mutants remained unchanged . The ftnA and ftnA bfr mutants were growth impaired in iron-deficient media, but this was apparent only after the mutant and parental strains had been precultured in iron-rich media . Surprisingly, ferric iron uptake regulation (fur) mutants also had very low iron contents (2.5-fold less iron than Fur+ strains) despite constitutive expression of the iron acquisition systems . The iron deficiencies of the ftnA and fur mutants were confirmed by Mossbauer spectroscopy, which further showed that the low iron contents of ftnA mutants are due to a lack of magnetically ordered ferric iron clusters likely to correspond to FtnA iron cores . In combination with the fur mutation, ftnA and bfr mutations produced an enhanced sensitivity to hydroperoxides, presumably due to an increase in production of "reactive ferrous iron." It is concluded that FtnA acts as an iron store accommodating up to 50% of the cellular iron during postexponential growth in iron-rich media and providing a source of iron that partially compensates for iron deficiency during iron-restricted growth . In addition to repressing the iron acquisition systems, Fur appears to regulate the demand for iron, probably by controlling the expression of iron-containing proteins . The role of Bfr remains unclear.

Eur J Surg Suppl, 1998, (582), 11 - 5
H . pylori infection in non-ulcer patients--to treat or not to treat . The case against treatment; McCarthy DM; Heretofore regarded as a strict pathogen, recent identification of multiple mutants of H . pylori, varying in pathogenicity, genomic composition, antigenic structure and other characteristics, has led to speculation that not all strains of the organism merit elimination . Affecting half the world's population, H . pylori appears to cause clinically significant disease in <20% of cases . The costs of eradicating harmless infection in over 2 billion people are prohibitive, particularly in countries lacking resources, and are questionable even in advanced countries where infection, gastritis and related diseases are declining as social conditions improve . Major controversies surround empiric eradication of helicobacter infection in patients with asymptomatic gastritis or non-specific dyspepsia . Apart from cost, and feasibility, there are concerns that widespread campaigns to eradicate H . pylori might cause major increases in esophageal reflux disease and esophageal adenocarcinoma, while causing some serious iatrogenic illness and increasing antibiotic resistance, with uncertain consequences to affected populations.

Clin Exp Pharmacol Physiol, 1999 Jan, 26(1), 64 - 8
Radical ideas: genetic responses to oxidative stress; Demple B; 1 . Complex genetic systems counteract different types of 'oxidative stress' caused by reactive derivatives of oxygen . 2 . The bacterial oxyR system responds to peroxide stress and is governed by OxyR, a transcription factor activated by the formation of an intramolecular disulphide bond in H2O2-treated cells . Activated OxyR switches on several genes encoding antioxidant functions, such as catalase . During aerobic growth, oxyR acts homeostatically to regulate cellular H2O2 levels . 3 . The bacterial soxRS system responds to superoxide or nitric oxide (NO) stress and is activated in two transcriptional stages . The SoxR protein is activated by oxidation of its {2Fe-2S} centres in cells exposed to superoxide-generating agents, such as paraquat, or to No . Activated SoxR stimulates the soxS gene and SoxS protein then induces at least 15 genes encoding antioxidant functions, such as superoxide dismutase, metabolic functions, such as fumarase, and antibiotic resistance by activation of efflux pumps . The soxRS system may function in resistance to NO-generating immune cells and may contribute to clinical antibiotic resistance . 4 . Human cells respond to subtoxic levels of NO by inducing 12 proteins and down-regulating others . A key induced activity is haem oxygenase 1, which is controlled post-transcriptionally . 5 . Motor neurons exhibit adaptive resistance to NO, triggered by exposure to subtoxic NO levels, and providing resistance to usually cytotoxic levels of this agent or H2O2 . Adaptive resistance to NO depends strongly on the inducible heam oxygenase activity.

Proc Natl Acad Sci U S A, 1999 Feb 16, 96(4), 1645 - 50
In vivo transposition of mariner-based elements in enteric bacteria and mycobacteria; Rubin EJ et al.; mariner family transposons are widespread among eukaryotic organisms . These transposons are apparently horizontally transmitted among diverse eukaryotes and can also transpose in vitro in the absence of added cofactors . Here we show that transposons derived from the mariner element Himar1 can efficiently transpose in bacteria in vivo . We have developed simple transposition systems by using minitransposons, made up of short inverted repeats flanking antibiotic resistance markers . These elements can efficiently transpose after expression of transposase from an appropriate bacterial promoter . We found that transposition of mariner-based elements in Escherichia coli produces diverse insertion mutations in either a targeted plasmid or a chromosomal gene . With Himar1-derived transposons we were able to isolate phage-resistant mutants of both E . coli and Mycobacterium smegmatis . mariner-based transposons will provide valuable tools for mutagenesis and genetic manipulation of bacteria that currently lack well developed genetic systems.

Recenti Prog Med, 1998 Dec, 89(12), 625 - 9
{Bronchoalveolar lavage in lower respiratory tract infections in patients with bronchopulmonary carcinoma . Our experience}; Turitto G et al.; In order to evaluate the usefulness of the endoscopic procedures with BAL, we conducted a study on 92 patients with diagnosis of lung cancer, underwent to chemotherapy, identifying pathogenic species involved and the antibiotic sensibility and antibiotic resistance . Moreover, to evaluate possible modifications of alveolar cell population in neutropenic patients, we studied the specimens from BALs performed via fibreoptic bronchoscopy . METHODS: The cellular pattern of BALs in terms of concentration of total cells, concentrations of alveolar macrophages (AMs), of polymorphonucleates (PMNs) and lymphocytes (Ls) were compared in neutropenic and non-neutropenic patients . RESULTS: In the statistical analysis of our study we found a correlation, statistically significant, between patients with neutropenic episodes on the previous chemotherapy courses and incidence of LTRI . The mean concentrations x 100,000/ml of BAL fluid of total alveolar cells, AMs, PMNs and Ls were significantly lower, after chemotherapy, in group of neutropenic patients than in non neutropenic patients, respectively . CONCLUSIONS: In this study we found that during neutropenia the alveolar cell population was quantitatively deficient, confirming the hypothesis, proposed by other authors, of "lung aplasia" as a consequence or a concomitant event of chemotherapy-induced marrow toxicity.

Novartis Found Symp, 1998, 217, 195 - 205; discussion 205-8
Antibiotic resistance in mycobacteria; Davies J; Multiple drug resistance in mycobacteria compromises the use of antibiotics . Although the genetic and biochemical bases of antibiotic resistance in mycobacteria are largely understood, a number of questions remain to be addressed . This chapter discusses the potential roles of hypermutability and compensatory mutations in establishing stable resistant phenotypes in the pathogenic mycobacteria.

Bioorg Med Chem Lett, 1998 Dec 15, 8(24), 3489 - 94
The use of neamine as a molecular template: identification of active site residues in the bacterial antibiotic resistance enzyme aminoglycoside 3'-phosphotransferase type IIa by mass spectroscopy; Yang Y et al.; Four novel aminoglycoside-based affinity inactivators were shown to covalently modify the active site of aminoglycoside 3'-phosphotransferase type IIa (APH(3')-IIa), an important resistance factor in bacteria for aminoglycoside antibiotics . Standard peptide mapping techniques failed with this enzyme . A novel mass spectroscopic analysis which combines protease digestion on the instrument probe, followed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is described which permitted rapid identification of the sites of protein modification . By this new technique, Glu-3 and Asp-23 were identified as active-site residues, the side chains of which potentially may serve as counter ions for the ammonium functionalities at positions 6', and 1 and 3 of the antibiotic substrates, respectively . These findings contradict previous assertions that the C-terminal third of the enzyme should form the active site, by placing the active site clearly in the N-terminal portion of the enzyme.

Bioorg Med Chem Lett, 1998 Dec 15, 8(24), 3483 - 8
The use of neamine as a molecular template: inactivation of bacterial antibiotic resistance enzyme aminoglycoside 3'-phosphotransferase type IIa; Roestamadji J et al.; Aminoglycoside 3'-phosphotransferase type IIa {APH(3')-IIa} is a member of the family of bacterial aminoglycoside-modifying enzymes . Bacteria that harbor these enzymes are resistant to aminoglycoside antibiotics . Four aminoglycoside-based affinity inactivators were synthesized and were shown to be both substrates and inactivators for APH(3')-IIa . These affinity inactivators are N-bromoacetylated derivatives of neamine, an aminoglycoside antibiotic, where the bromoacetyl moiety in each was introduced regiospecifically at a different amine of the parent compound.

J Biol Chem, 1999 Feb 12, 274(7), 4180 - 8
Insertion of the N-terminal part of PsaF from Chlamydomonas reinhardtii into photosystem I from Synechococcus elongatus enables efficient binding of algal plastocyanin and cytochrome c6; Hippler M et al.; A strain of the cyanobacterium Synechococcus elongatus was generated that expresses a hybrid version of the photosystem I subunit PsaF consisting of the first 83 amino acids of PsaF from the green alga Chlamydomonas reinhardtii fused to the C-terminal portion of PsaF from S . elongatus . The corresponding modified gene was introduced into the genome of the psaF-deletion strain FK2 by cointegration with an antibiotic resistance gene . The transformants express a new PsaF subunit similar in size to PsaF from C . reinhardtii that is assembled into photosystem I (PSI) . Hybrid PSI complexes isolated from these strains show an increase by 2 or 3 orders of magnitude in the rate of P700(+) reduction by C . reinhardtii cytochrome c6 or plastocyanin in 30% of the complexes as compared with wild type cyanobacterial PSI . The corresponding optimum second-order rate constants (k2 = 4.0 and 1.7 x 10(7) M1 s1 for cytochrome c6 and plastocyanin) are similar to those of PSI from C . reinhardtii . The remaining complexes are reduced at a slow rate similar to that observed with wild type PSI from S . elongatus and the algal donors . At high concentrations of C . reinhardtii cytochrome c6, a fast first-order kinetic component (t(1)/(2) = 4 microseconds) is revealed, indicative of intramolecular electron transfer within a complex between the hybrid PSI and cytochrome c6 . This first-order phase is characteristic for P700(+) reduction by cytochrome c6 or plastocyanin in algae and higher plants . However, a similar fast phase is not detected for plastocyanin . Cross-linking studies show that, in contrast to PSI from wild type S . elongatus, the chimeric PsaF of PSI from the transformed strain cross-links to cytochrome c6 or plastocyanin with a similar efficiency as PsaF from C . reinhardtii PSI . Our data indicate that development of a eukaryotic type of reaction mechanism for binding and electron transfer between PSI and its electron donors required structural changes in both PSI and cytochrome c6 or plastocyanin.

Nat Biotechnol, 1999 Jan, 17(1), 67 - 72
Engineering a regulatable enzyme for homogeneous immunoassays; Legendre D et al.; We have engineered the phage displayed TEM-1 beta-lactamase to generate enzymes that can be used in homogeneous immunoassays because their activity can be modulated by binding to monoclonal antibodies (Mabs) raised against an unrelated protein . Random peptide libraries were genetically inserted into three loops to create hybrid enzymes with binding sites for Mabs . Insertion points were chosen to be close enough to the active site that complex formation could affect the activity . The antibiotic resistance provided by the beta-lactamase activity was used to select the clones encoding active enzymes . Biopanning of the active libraries on immobilized Mabs against the prostate specific antigen (PSA) or on streptavidin yielded enzymes with binding sites for these proteins . Their activity could be regulated by Mab or streptavidin binding . The dissociation constants of the complexes are in the 10(-9) to 10(-6) M range . In a competitive assay, PSA could be detected at a minimal concentration of 10(-9) M . The Mabs recognize mimotopes as no sequence similarity was found between inserts in regulated clones and fragments of the PSA sequence . The method can be developed to generate signaling molecules to be used for the detection of analytes in solution without identification of the epitope.

Aliment Pharmacol Ther, 1999 Jan, 13(1), 35 - 42
Novel therapies for Helicobacter pylori infection; Opekun AR et al.; BACKGROUND: Increasing antibiotic resistance has begun to impair our ability to cure Helicobacter pylori infection . AIM: To evaluate orally administered novel therapies for the treatment of H . pylori infection . METHODS: Healthy H . pylori infected volunteers received: (a) hyperimmune bovine colostral immune globulins, (b) an oligosaccharide containing an H . pylori adhesion target, Neu5Aca2-3Galb1-4Glc-(3'-sialyllactose), or (c) recombinant human lactoferrin . Outcome was assessed by urea breath test or histological assessment of the number of H . pylori present . RESULTS: None of the novel therapies appeared effective and no adverse events occurred . CONCLUSION: Although in vitro data appeared promising, in vivo results were disappointing . Higher doses, longer duration of therapy, adjunctive acid suppression, or a combination could possibly yield better results.

J Natl Cancer Inst, 1999 Jan 6, 91(1), 37 - 45
Telomerase repressor sequences on chromosome 3 and induction of permanent growth arrest in human breast cancer cells; Cuthbert AP et al.; BACKGROUND: Activation of the enzyme telomerase, which has been associated with cellular immortality, may constitute a key step in the development of human cancer . Telomerase is repressed in most normal human somatic cells . This study was conducted, using a genetic complementation approach, with the aim of identifying and mapping the genes responsible for repressing telomerase and, simultaneously, to establish the effect of experimentally induced telomerase repression on human tumor cell growth . METHODS: Individual human chromosomes isolated from normal diploid cells and tagged with bacterial antibiotic resistance genes (for later selection) were introduced into cells of the human breast carcinoma cell line 21NT by means of microcell transfer . Selected hybrid clones were screened for telomerase activity by use of the polymerase chain reaction-based telomere repeat amplification protocol (TRAP) assay, and the proliferative fate of the hybrid clones was determined . Regions of the introduced chromosomes associated with telomerase repression were mapped using segregant hybrids and a deletion analysis that employed microsatellite DNA markers . RESULTS: Strong repression of telomerase was observed following transfer of human chromosome 3 into 21NT cells but not after transfer of chromosomes 8, 12, or 20 . The vast majority of hybrid clones with repressed telomerase entered permanent growth arrest after 10-18 population doublings . Deletion analysis of nonrepressed segregant monochromosome 3 hybrids indicated two regions on the short arm of chromosome 3 (3p21.3-p22 and 3p12-21.1) where telomerase regulator genes may be located . CONCLUSIONS: Telomerase in human breast cancer cells is efficiently repressed by a gene or genes on normal human chromosome 3p, and this repression is associated with permanent growth arrest of the tumor cells.

J Bacteriol, 1999 Jan, 181(2), 642 - 7
The mithramycin gene cluster of Streptomyces argillaceus contains a positive regulatory gene and two repeated DNA sequences that are located at both ends of the cluster; Lombo F et al.; Sequencing of a 4.3-kb DNA region from the chromosome of Streptomyces argillaceus, a mithramycin producer, revealed the presence of two open reading frames (ORFs) . The first one (orfA) codes for a protein that resembles several transport proteins . The second one (mtmR) codes for a protein similar to positive regulators involved in antibiotic biosynthesis (DnrI, SnoA, ActII-orf4, CcaR, and RedD) belonging to the Streptomyces antibiotic regulatory protein (SARP) family . Both ORFs are separated by a 1.9-kb, apparently noncoding region . Replacement of the mtmR region by an antibiotic resistance cassette completely abolished mithramycin biosynthesis . Expression of mtmR in a high-copy-number vector in S . argillaceus caused a 16-fold increase in mithramycin production . The mtmR gene restored actinorhodin production in Streptomyces coelicolor JF1 mutant, in which the actinorhodin-specific activator ActII-orf4 is inactive, and also stimulated actinorhodin production by Streptomyces lividans TK21 . A 241-bp region located 1.9 kb upstream of mtmR was found to be repeated approximately 50 kb downstream of mtmR at the other end of the mithramycin gene cluster . A model to explain a possible route for the acquisition of the mithramycin gene cluster by S . argillaceus is proposed.

Front Biosci, 1999 Jan 01, 4, D9 - 21
Aminoglycoside phosphotransferases: proteins, structure, and mechanism; Wright GD et al.; Aminoglycoside antibiotics constitute an important class of clinically useful drugs which are imperiled by the emergence of resistant organisms . Aminoglycoside resistance in the clinics is primarily due to the presence of modifying enzymes which N-acetylate, O-adenylate or O-phosphorylate the antibiotics . The latter family of enzymes are termed the aminoglycoside phosphotransferases or kinases and are the subject of this review . There are seven classes of aminoglycoside phosphotransferases (APH(3'), APH(2''), APH(3'off'), APH(6), APH(9), APH(4), APH(7'')) and many isozymes in each class, and although there is very little overall general sequence homology among these enzymes, certain signature residues and sequences are common . The recent determination of the three-dimensional structure of the broad spectrum aminoglycoside kinase APH(3')-IIIa complexed with the product ADP, in addition to mechanistic and mutagenic studies on this and related enzymes, has added a great deal to our understanding of this class of antibiotic resistance enzyme . In particular, the revelation of structural and mechanistic similarities between APHs and Ser/Thr and Tyr kinases has set the stage for future inhibition studies which could prove important in reversing aminoglycoside resistance.

Ned Tijdschr Geneeskd, 1998 Oct 31, 142(44), 2388 - 90
{Surveillance of communicable diseases in Europe; more than just a means of communication among national institutes}; Verbrugh HA; The re-emergence of infectious diseases as a threat to the health of the public has led to the initiation of several surveillance programmes in the European community . The impact of these Europe-wide surveillance programmes, including the surveillance of antibiotic resistance, will depend on the ability of the epidemiologists involved to gather validated data that accurately reflect the trends in the occurrence of infectious diseases and of antibiotic resistances . The results of surveillance should be communicated not only between the national institutes but should also reach the medical communities and the public at large in the participating countries.

Mol Cell Biol, 1999 Jan, 19(1), 826 - 34
Ribosomal protein S14 of Saccharomyces cerevisiae regulates its expression by binding to RPS14B pre-mRNA and to 18S rRNA; Fewell SW et al.; Production of ribosomal protein S14 in Saccharomyces cerevisiae is coordinated with the rate of ribosome assembly by a feedback mechanism that represses expression of RPS14B . Three-hybrid assays in vivo and filter binding assays in vitro demonstrate that rpS14 directly binds to an RNA stem-loop structure in RPS14B pre-mRNA that is necessary for RPS14B regulation . Moreover, rpS14 binds to a conserved helix in 18S rRNA with approximately five- to sixfold-greater affinity . These results support the model that RPS14B regulation is mediated by direct binding of rpS14 either to its pre-mRNA or to rRNA . Investigation of these interactions with the three-hybrid system reveals two regions of rpS14 that are involved in RNA recognition . D52G and E55G mutations in rpS14 alter the specificity of rpS14 for RNA, as indicated by increased affinity for RPS14B RNA but reduced affinity for the rRNA target . Deletion of the C terminus of rpS14, where multiple antibiotic resistance mutations map, prevents binding of rpS14 to RNA and production of functional 40S subunits . The emetine-resistant protein, rpS14-EmRR, which contains two mutations near the C terminus of rpS14, does not bind either RNA target in the three-hybrid or in vitro assays . This is the first direct demonstration that an antibiotic resistance mutation alters binding of an r protein to rRNA and is consistent with the hypothesis that antibiotic resistance mutations can result from local alterations in rRNA structure.

Gene, 1998 Nov 26, 223(1-2), 213 - 9
pBRINT-Ts: a plasmid family with a temperature-sensitive replicon, designed for chromosomal integration into the lacZ gene of Escherichia coli; Le Borgne S et al.; A pBRINT-Ts family of integrative vectors for Escherichia coli was constructed by using a temperature-sensitive replicon derived from pSC101, a region of homology to the lacZ gene, and various antibiotic resistance markers (kanamycin, chloramphenicol and gentamycin) for selection of the integrants . The gene or group of genes to be integrated can be inserted into a multiple cloning site, flanked by an antibiotic resistance marker and lacZ sequences . A simple and rapid procedure was developed for the selection of cells where allelic exchange had occurred . With this procedure, the efficiency of integration of around 10-3 was observed, using several E . coli strains . From colonies with an integrated pBRINT-Ts plasmid, we detected an average allelic exchange event frequency of 7.5% . As a test for this system, we integrated the amy gene that codes for the alpha-amylase from B . stearothermophilus, into the lacZ gene of E . coli W3110 . Production of alpha-amylase was found to be proportional to copy number; at up to 10 copies per chromosome.

Gene, 1998 Nov 26, 223(1-2), 195 - 204
Regulatory implications of protein assemblies at the gamma origin of plasmid R6K - a review; Filutowicz M et al.; Recognition of the replication origin (ori) by initiator protein is a recurring theme for the regulated initiation of DNA replication in diverse biological systems . The objective of the work reviewed here is to understand the initiation process focusing specifically on the gamma-ori of the antibiotic-resistance plasmid R6K . The control of gamma-ori copy number is determined by both plasmid-encoded and host-encoded factors . The two central regulatory elements of the plasmid are a multifunctional initiator protein pi, and sequence-related DNA target sites, the inverted half-repeats (IRs) and the direct repeats (DRs) . The replication activator and inhibitor activities of pi seem to be at least partially distributed between two naturally occurring pi polypeptides (designated by their molecular weights pi35.0 and pi30.5) . Regulatory variants of pi with altered states of oligomerization in nucleoprotein complexes with DRs and IRs have been isolated . The properties of these mutants laid the foundation for our model of pi protein activity which proposes that different protein surfaces are required for the formation of functionally distinct complexes of pi with DRs and IRs . These mutants also suggest that pi polypeptides have a modular structure; the C-terminus contains the DNA-binding domain while the N-terminus controls protein oligomerization . Additionally, pi35.0 binds to a novel DNA sequence in the A+T-rich segment of gamma-ori . This binding site is at or near the site from which synthesis of the leading strand begins.

Gene, 1998 Nov 26, 223(1-2), 187 - 94
Observations on template switching during DNA replication through long inverted repeats; Ahmed A et al.; The bacterial Tn5 transposon consists of a pair of long inverted repeats flanking a central region that carries genes for antibiotic resistance . An analysis of DNA replication through Tn5 by two-dimensional gel electrophoresis has revealed two interesting features: (i) a spike representing X-shaped molecules, and (ii) a spot representing a barrier on the arc of Y-shaped replication intermediates . The electrophoretic behavior of various restriction fragments derived from this region indicates that the X molecules contain two linear Tn5 fragments joined together by a cross connection (Holliday junction) . However, their formation is recA independent . The junction seems to connect the right inverted repeat of one fragment to the left inverted repeat of the other . The structure of the X molecules suggests that they could be formed by template switching when synthesis of the leading strand enters the right inverted repeat . One possible mechanism is that switching occurs at the center of a transient cruciform structure . A similar switching event, occurring when synthesis of the leading strand enters the left inverted repeat, could give rise to the barrier . These results imply that the inviability of palindromic DNA, which has previously been attributed to the slowing down of replication, may actually be caused by frequent template switching.

Presse Med, 1998 Nov 14, 27(35), 1796 - 800
{Forum on bacterial resistance}; Bergogne-Berezin E et al.; Recently, in daily newspapers and on television, attention of the audience has been focused on the overuse of antibiotics and on the role it plays in the emergence and dissemination of resistance mechanisms in the human environment . The role of food from animal origin in relation to the use of antibiotic resistance, infectious diseases, medical practice and ENT infections have accepted to answer a series of questions concerning risks versus usefulness of antibiotic usage . From the answers, we may note convergent views and discrepancies: (i) there was agreement concerning the unnecessary prescription of antibiotics in rhinopharyngitis and few other common viral infections; (ii) the risk of misuse of antibiotics in patients with poor compliance and further risk of erroneous self prescription of the remaining tablets has been cited; (iii) in the problem of resistance resulting from growth promoting antibiotics in animals, it has been experimentally shown that from 2 bacteria of the same species introduced in the animal gut, one susceptible, the other resistant, the latter will be eliminated by means of the "barrier effect"; similarly in case of transfer of resistance from an exogenous bacteria to a "resident" organism of the gut, the latter will be eliminated by the homologous susceptible ones; only an antibiotic therapy may confer importance to the resistant bacteria . In this respect, care should be taken for resistance spread such as that concerning penicillin-resistant pneumococci and surveillance and control of resistance mechanisms has become necessary . However we should look with reluctance at the diffusion of inevitably simplified and truncated information from Media, showing the negative aspects of antibiotics only . Moreover, as underlined by the expert from the Institut Pasteur, there are new perspectives in the development of effective new agents based on the modern "genomic" research.

Rev Med Liege, 1998 Sep, 53(9), 529 - 31
{Bacterial resistance to antibiotics, an exemplary model of directed molecular evolution}; Ghuysen JM; The emergence and spread of antibiotic resistance among bacteria make use of the same strategies and obey the same laws as natural evolution but the process is oriented and accelerated.

Eur J Clin Microbiol Infect Dis, 1998 Sep, 17(9), 666 - 9
Antibiotic resistance pattern of enterotoxigenic Escherichia coli isolated from infants and young adults in Israel; Turner D et al.; The aim of this study was to describe antibiotic resistance rates of enterotoxigenic Escherichia coli in Israel in order to facilitate the empirical choice of antibiotic treatment or prophylaxis for traveler's diarrhea and infantile diarrhea in our region . A total of 281 enterotoxigenic Escherichia coli isolates were tested: 144 from Bedouin infants and 137 from Israeli soldiers . Antibiotic-resistant isolates were prevalent in both groups, but higher resistance rates were found in the pediatric group . Strains producing heat-labile toxin showed higher resistance rates than strains producing heat-stable toxin . The results obtained in Israel preclude the use of many commonly used antibiotics for the treatment of traveler's diarrhea . Quinolones, however, are still effective.

Infect Control Hosp Epidemiol, 1998 Nov, 19(11), 836 - 41
Management and outcome of tuberculosis in two St Louis hospitals, 1988 to 1994; L'Ecuyer PB et al.; OBJECTIVE: To describe management and outcome of tuberculosis (TB) and current practices for isolation in two urban hospitals in the Midwest . DESIGN: Retrospective cohort study . SETTING: Barnes Hospital and Jewish Hospital, tertiary-care and community hospitals affiliated with Washington University School of Medicine in St Louis, Missouri . PATIENTS: All adult patients with a positive culture for Mycobacterium tuberculosis from 1988 to 1994 . RESULTS: We identified 122 cases at Barnes and Jewish Hospitals (36.5/100,000 hospital discharges), median age was 59.0 years, 61.5% were non-Caucasian, and 54.9% resided within the city limits . Underlying risk conditions were common: substance abuse (25%), recent TB contact (24%), and foreign birth (13%) . Coexistent human immunodeficiency virus infection (8%) was uncommon . Of skin-tested cases, 22% were anergic; of the rest, 22% tested negative . Almost 20% of cases had prior positive skin tests, and thus were preventable, but had not received adequate prophylaxis . Of hospitalized patients with pulmonary TB, 70% received respiratory isolation . Antibiotic resistance was recognized in 16%; only 19% of cases initially received four-drug therapy . TB-related death occurred in 16% . CONCLUSIONS: In this area, TB cases primarily involve traditional risk groups without HIV coinfection . Current infection control practices, diagnostic strategies, and initial treatment regimens are suboptimal . Education about local disease epidemiology is needed to prevent nosocomial TB transmission.

J Food Prot, 1998 Nov, 61(11), 1511 - 4
Antibiotic resistance of Escherichia coli O157:H7 and O157:NM isolated from animals, food, and humans; Meng J et al.; Antibiotic resistance was determined for 118 E . coli O157:H7 and 7 O157:NM isolates from animals, foods, and humans . Among the 125 isolates, 30 (24%) were resistant to at least one antibiotic and 24 (19%) were resistant to three or more antibiotics . Cattle isolates had the highest rate (34%) of antibiotic resistance . The seven resistant food isolates were all from ground beef . The most frequent resistance type overall was streptomycin-sulfisoxazole-tetracycline, which accounted for over 70% of the resistant strains . Two E . coli O157:NM isolates from cattle were resistant to six antibiotics: ampicillin, kanamycin, sulfisoxazole, streptomycin, tetracycline, and ticarcillin . Streptomycin was the most common antibiotic to which E . coli O157:H7 and O157:NM were resistant (29 out of 30 isolates), followed by tetracycline (26 isolates) . This study suggests that E . coli O157:H7 and O157:NM have developed resistance to antibiotics . Research is needed to define mechanisms of antibiotic resistance in E . coli O157:H7 and to minimize the development of resistance.

Crit Rev Oral Biol Med, 1998, 9(4), 522 - 40
Antibiotic resistance in oral/respiratory bacteria; Roberts MC; In the last 20 years, changes in world technology have occurred which have allowed for the rapid transport of people, food, and goods . Unfortunately, antibiotic residues and antibiotic-resistant bacteria have been transported as well . Over the past 20 years, the rise in antibiotic-resistant gene carriage in virtually every species of bacteria, not just oral/respiratory bacteria, has been documented . In this review, the main mechanisms of resistance to the important antibiotics used for treatment of disease caused by oral/respiratory bacteria--including beta-lactams, tetracycline, and metronidazole--are discussed in detail . Mechanisms of resistance for macrolides, lincosamides, streptogramins, trimethoprim, sulfonamides, aminoglycosides, and chloramphenicol are also discussed, along with the possible role that mercury resistance may play in the bacterial ecology.

J Biotechnol, 1998 Sep 17, 64(1), 75 - 90
Horizontal gene transfer as a biosafety issue: a natural phenomenon of public concern; Droge M et al.; The transfer of genetic information between distantly or even unrelated organisms during evolution had been inferred from nucleotide sequence comparisons . These studies provided circumstantial evidence that in rare cases genes had been laterally transmitted amongst organisms of the domains bacteria, archaea and eukarya . Laboratory-based studies confirmed that the gene pools of the various domains of organisms are linked . Amongst the bacterial gene exchange mechanisms transduction, transformation and conjugation, the latter was identified as the mechanism with potentially the broadest host range of transfer . Previously, the issue of horizontal gene transfer has become important in the context of biosafety . Gene transfer studies carried out under more natural conditions such as in model ecosystems or in the environment established that all gene transfer mechanisms worked under these conditions . Moreover, environmental hot-spots were identified where favourable conditions such as nutrient enrichment increased the probability of genetic exchange among bacteria . In particular, the phytosphere was shown to provide conducive conditions for conjugative gene exchange . Concern has been expressed that transfer of recombinant DNA (e.g . antibiotic resistance genes) from genetically modified organisms (GMOs) such as transgenic plants to phytosphere bacteria may occur and thus contribute to the undesirable spread of antibiotic resistance determinants . Studies which were performed to address this issue clearly showed that such a transfer occurs, if at all, at extremely low frequency.

Berl Munch Tierarztl Wochenschr, 1998 Oct, 111(10), 374 - 8
{Phenotypic characterization of equine Dermatophilus congolensis field isolates}; Kruger B et al.; In 1993 and 1994 a highly increased occurrence of equine dermatophilosis was observed, and a study was initiated to determine phenotypic heterogeneity among 120 clinical isolates using biochemistry, antibiotic resistance profiles, membrane protein profiles and Western blotting . The biochemical examinations contained 1% equine serum in medium . Moreover, the API ZYM-test from bioMerieux was used . The biochemical reactions were suited to identify Dermatophilus congolensis but did not allow a differentiation among the various isolates . Antibiotic resistance in one or more isolates was observed against polymyxin B, enrofloxacin, oxacillin, neomycin and trimethoprim-sulfamethoxazol . All isolates were sensitive penicillin G, ampicillin, streptomycin, gentamicin, lincomycin, erythromycin, tetracycline, oxytetracycline, bacitracin and ceftiofur . The evaluation of silver-stained and immuno-stained membrane protein profiles showed minor differences among several isolates . In total, all isolates appeared to be closely related and the minor differences observed did not correlate with the geographic origin of the respective isolates.

Rev Prat, 1998 Sep 15, 48(14), 1530 - 4
{Structures and policy of nosocomial infection prevention: France is speeding up the process}; Carlet J; For the past 10 years France has tried to set up a programme to prevent and survey nosocomial infections and antibiotic resistance . A series of recommendations have been made available for hospitals . Hygiene, nosocomial infections, and resistance to antibiotics are recognized by medical doctors, nurses and administrative people as pertinent and logical quality indicators . Those indicators will probably be proposed by the French agency for accreditation and evaluation . We still need to define which events are preventable, which should be declared to the official agencies, and how to integrate infection control in a broader programme coordinating different systems of surveillance (drug, blood, ...) and risk management . We have also to make those informations available to the public in a more transparent mind.

J Bacteriol, 1998 Nov, 180(22), 5836 - 43
Molecular cloning and characterization of Tap, a putative multidrug efflux pump present in Mycobacterium fortuitum and Mycobacterium tuberculosis; Ainsa JA et al.; A recombinant plasmid isolated from a Mycobacterium fortuitum genomic library by selection for gentamicin and 2-N'-ethylnetilmicin resistance conferred low-level aminoglycoside and tetracycline resistance when introduced into M . smegmatis . Further characterization of this plasmid allowed the identification of the M . fortuitum tap gene . A homologous gene in the M . tuberculosis H37Rv genome has been identified . The M . tuberculosis tap gene (Rv1258 in the annotated sequence of the M . tuberculosis genome) was cloned and conferred low-level resistance to tetracycline when introduced into M . smegmatis . The sequences of the putative Tap proteins showed 20 to 30% amino acid identity to membrane efflux pumps of the major facilitator superfamily (MFS), mainly tetracycline and macrolide efflux pumps, and to other proteins of unknown function but with similar antibiotic resistance patterns . Approximately 12 transmembrane regions and different sequence motifs characteristic of the MFS proteins also were detected . In the presence of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), the levels of resistance to antibiotics conferred by plasmids containing the tap genes were decreased . When tetracycline accumulation experiments were carried out with the M . fortuitum tap gene, the level of tetracycline accumulation was lower than that in control cells but was independent of the presence of CCCP . We conclude that the Tap proteins of the opportunistic organism M . fortuitum and the important pathogen M . tuberculosis are probably proton-dependent efflux pumps, although we cannot exclude the possibility that they act as regulatory proteins.

Ann Allergy Asthma Immunol, 1998 Oct, 81(4), 293 - 302; quiz 302-3
Antibiotic resistance in community-acquired respiratory tract infections: current issues; Spach DH et al.; OBJECTIVE: In recent years, antibiotic resistance has emerged as an important global problem . The major goal of this review is to update important issues pertaining to antibiotic resistance, with an emphasis on antibiotic resistance involving community-acquired respiratory pathogens . In addition, this review examines potential reasons why antibiotic resistance has increased in recent years, how clinicians can better understand commonly used laboratory antibiotic resistance tests, and possible solutions to the increasing problem of antibiotic resistance . The article emphasizes the diagnosis, therapy, and prevention of antibiotic-resistant infections . DATA SOURCES: We identified relevant English-language articles through MEDLINE search (1966 to March 1998) . All articles related to antibiotic resistance and the scope of the articles included original investigative articles, reviews, letters, and editorials . In addition, we selected additional references from the bibliographies of the identified articles . STUDY SELECTION: We selected articles for detailed review if they provided direct insight into the cause of antibiotic resistance, testing for antibiotic resistance, or the treatment of antibiotic resistance . Most, but not all, of the articles selected pertained to antibiotic resistance and respiratory tract infections . We performed a detailed review on approximately 40% of the originally selected articles . RESULTS: Multiple factors that play a significant role in the development of antibiotic resistance include the overuse of antibiotics in both humans and animals, situations such as day care that enhance transmission via frequent close personal contact, and widespread dissemination of resistant strains via global travel . Most respiratory pathogens have developed resistance to commonly used antibiotics either by producing beta-lactamase or by altering binding site proteins . CONCLUSIONS: In many regions of the United States, the level of antibiotic resistance has impacted the clinical management of common respiratory pathogens . Future efforts to curtail antibiotic resistance will require a concerted effort in multiple areas, particularly enhanced epidemiologic surveillance to better detect resistance trends, judicious use of antibiotics, and new drug development.

J Immunol Methods, 1998 Aug 1, 217(1-2), 195 - 9
A vector for the expression of recombinant monoclonal Fab fragments in bacteria; Burioni R et al.; The availability of genes coding for monoclonal Fab fragments of a desired specificity permits their expression in bacteria and provides a simple method for the generation of good quality reagents . In this paper we describe a new phagemid vector for the production of recombinant Fabs from genes obtained from phage display combinatorial libraries . The phagemid features an antibiotic resistance cassette which, once inserted between the heavy chain fragment and the light chain genes, avoids unwanted recombination and preserves useful restriction sites not affecting the Fab production rate.

Lakartidningen, 1998 Sep 9, 95(37), 3940, 3943 - 4
{Antibiotic resistance here to stay? Compensatory mutations restore virulence of resistant bacteria}; Andersson DI et al.; Bacterial antibiotic resistance has increased alarmingly because of overuse of antibiotics both in humans and animals . One way of reversing this development is to reduce the use of antibiotics, thus promoting the disappearance of the resistant bacteria already present in humans and the environment . This approach is based on the assumption that resistance is conferred at the cost of impaired survival fitness in the absence of antibiotics, as compared with sensitive strains . It seems to be generally true that resistant bacteria are less fit than the respective sensitive strains, which suggests that resistance may be reversible . However, a complicating factor is the frequent finding in resistant strains of various types of compensatory mutations that restore fitness without concomitant loss of resistance . Thus, second-site compensatory mutations may allow resistant strains to persist and compete successfully with sensitive strains even in an environment depleted of antibiotics . It is concluded in the article that, if compensatory mutations are as common in clinical settings as they are in the laboratory, many types of resistance will be irreversible.

Proc Natl Acad Sci U S A, 1998 Oct 13, 95(21), 12468 - 73
Induction of microsatellite instability by oxidative DNA damage; Jackson AL et al.; Instability of repetitive sequences, both in intronic sequences and within coding regions, has been demonstrated to be a hallmark of genomic instability in human cancer . Understanding how these mutational events arise may provide an opportunity for prevention or early intervention in cancer development . To study the source of this instability, we have identified a region of the beta-lactamase gene that is tolerant to the insertion of fragments of exogenous DNA as large as 1,614 bp with minimal loss of enzyme activity, as determined by antibiotic resistance . Fragments inserted out-of-frame render Escherichia coli sensitive to antibiotic, and compensatory frameshift mutations that restore the reading frame of beta-lactamase can be selected on the basis of antibiotic resistance . We have utilized this site to insert a synthetic microsatellite sequence within the beta-lactamase gene and selected for mutations yielding frameshifts . This assay provides for detection of one frameshift mutation in a background of 10(6) wild-type sequences . Mismatch repair deficiency increased the observed frameshift frequency approximately 300-fold . Exposure of plasmid containing microsatellite sequences to hydrogen peroxide resulted in frameshift mutations that were localized exclusively to the microsatellite sequences, whereas DNA damage by UV or N-methyl-N'-nitro-N-nitrosoguanidine did not result in enhanced mutagenesis . We postulate that in tumor cells, endogenous production of oxygen free radicals may be a major factor in promoting instability of microsatellite sequences . This beta-lactamase assay may provide a sensitive methodology for the detection and quantitation of mutations associated with the development of cancer.

Aliment Pharmacol Ther, 1998 Sep, 12(9), 887 - 92
Low rate of emergence of clarithromycin-resistant Helicobacter pylori with amoxycillin co-therapy; Laine L et al.; BACKGROUND: Patients with persistent Helicobacter pylori infection following treatment with clarithromycin or omeprazole plus clarithromycin often develop clarithromycin resistance . AIM: To assess pre- and post-treatment antibiotic resistance in three double-blind trials of triple therapy with omeprazole, amoxycillin and clarithromycin . METHODS: Patients with H . pylori and duodenal ulcer (studies 1 and 2) or history of duodenal ulcer (study 3) were randomly assigned to 10 day courses of omeprazole 20 mg b.d., amoxycillin 1 g b.d . and clarithromycin 500 mg b.d . (OAC) or placebo, amoxycillin 1 g b.d . and clarithromycin 500 mg b.d . (AC) . Endoscopy was performed at baseline and 4 weeks after completion of therapy in studies 1 and 2, and at 4-6 weeks after therapy in study 3 . At baseline, H . pylori was diagnosed by CLO test with confirmation by histology, or by culture . Eradication was defined as no positive biopsy test and > or = 2 negative tests . Susceptibility testing was performed using the Etest . RESULTS: In the 91 patients with pre-treatment susceptible isolates who had persistent infection after AC, 10 developed resistance, eight had intermediate susceptibility and 73 continued to have clarithromycin-susceptible H . pylori isolates . In the 10 patients with pre-treatment susceptible isolates who had persistent infection after OAC, three developed clarithromycin resistance and seven still had susceptible isolates . CONCLUSIONS: Use of amoxycillin co-therapy results in a low rate of clarithromycin resistance developing in patients with persistent H . pylori infection following therapy with a clarithromycin-containing regimen.

J Bacteriol, 1998 Oct, 180(20), 5437 - 42
Point mutations in the integron integrase IntI1 that affect recombination and/or substrate recognition; Gravel A et al.; The site-specific recombinase IntI1 found in class 1 integrons catalyzes the excision and integration of mobile gene cassettes, especially antibiotic resistance gene cassettes, with a site-specific recombination system . The integron integrase belongs to the tyrosine recombinase (phage integrase) family . The members of this family, exemplified by the lambda integrase, do not share extensive amino acid identities, but three invariant residues are found within two regions, designated box I and box II . Two conserved residues are arginines, one located in box I and one in box II, while the other conserved residue is a tyrosine located at the C terminus of box II . We have analyzed the properties of IntI1 variants carrying point mutations at the three conserved residues of the family in in vivo recombination and in vitro substrate binding . We have made four proteins with mutations of the conserved box I arginine (R146) and three mutants with changes of the box II arginine (R280); of these, MBP-IntI1(R146K) and MBP-IntI1(R280K) bind to the attI1 site in vitro, but only MBP-IntI1(R280K) is able to excise cassettes in vivo . However, the efficiency of recombination and DNA binding for MBP-IntI1(R280K) is lower than that obtained with the wild-type MBP-IntI1 . We have also made two proteins with mutations of the tyrosine residue (Y312), and both mutant proteins are similar to the wild-type fusion protein in their DNA-binding capacity but are unable to catalyze in vivo recombination.

Gut, 1998 Jul, 43 Suppl 1, S10 - 3
Management of Helicobacter pylori infection in children; Oderda G; When trying to decide which children with Helicobacter pylori infection should be treated and at what stage they should be tested, we should take into account the fact that eradication of the infection may be useful both to induce symptom remission and to prevent later complications in adulthood . However, well designed studies to identify those infected children who are at risk of developing complications or have symptoms due to the infection are still lacking . Current literature only gives information on how to treat children with H pylori infection . Treatment regimens that include two drugs are usually more effective than in adults, and produce an eradication rate of 70-80%, but they should be given for at least two weeks, shorter treatments being less effective . Antibiotic resistance can impair eradication rate and the frequency of resistant strains in children should be studied . Combinations of antibiotics with antisecretory drugs are highly effective in adults, but triple therapy with two antibiotics and an antisecretory drug has been seldom tried in children; compliance is often poor so that the eradication rate is often similar to that produced by dual therapy . Compliance strongly influences eradication, and short simple treatment regimens that produce rapid symptom remission with few side effects are needed to optimise patient compliance . After treatment, eradication must be proved . Serological tests can help, provided that pretreatment serum is available and three to six months have passed since the treatment . A 13C-ureabreath test (13C-UBT) should be performed at least six weeks after treatment, but false negative results can occur and cut-off must be adjusted.

Infect Control Hosp Epidemiol, 1998 Aug, 19(8), 560 - 4
Preventing antibiotic resistance using rapid DNA-based diagnostic tests; Bergeron MG et al.; Improved diagnostic procedures should be an effective way to control infectious diseases and the spread of antibiotic resistance . To do so, diagnostics will need to be obtained within 1 hour of sampling and will require nucleic acid-based amplification tests directly on the clinical specimen.

J Biol Chem, 1998 Oct 9, 273(41), 26310 - 6
Involvement of waaY, waaQ, and waaP in the modification of Escherichia coli lipopolysaccharide and their role in the formation of a stable outer membrane; Yethon JA et al.; The waaY, waaQ, and waaP genes are located in the central operon of the waa (formerly rfa) locus on the chromosome of Escherichia coli . This locus contains genes whose products are involved in the assembly of the core region of the lipopolysaccharide molecule . In the R1 core prototype strain, E . coli F470, there are nine genes in this operon, and all but waaY, waaQ, and waaP have been assigned function . In this study, the waaY, waaQ, and waaP genes were independently mutated by insertion of a non-polar antibiotic resistance cassette, and the structures of the resulting mutant core oligosaccharides were determined by chemical analyses and phosphorus-nuclear magnetic resonance spectroscopy . All three of these mutations were shown to affect the modification of the heptose region of the core, a region whose structure is critical to outer membrane stability . Mutation of waaY resulted in a core oligosaccharide devoid of phosphate on HepII . Mutation of waaQ resulted in loss of the branch HepIII residue on HepII and impeded the activity of WaaY . Mutation of waaP resulted in loss of phosphoryl substituents on HepI and obviated WaaQ and WaaY activity . Only mutation of waaP resulted in hypersensitivity to novobiocin and sodium dodecyl sulfate, a characteristic of deep-rough mutations.

Arch Intern Med, 1998 Sep 14, 158(16), 1813 - 8
National trends in the use of antibiotics by primary care physicians for adult patients with cough; Metlay JP et al.; BACKGROUND: Increased antibiotic use for outpatient illnesses has been identified as an important determinant of the recent rise in antibiotic resistance among common respiratory pathogens . Efforts to reduce the inappropriate use will need to be evaluated against current trends in the outpatient use of antibiotics . OBJECTIVES: To examine national trends in the use of antibiotics by primary care physicians in the care of adult patients with cough and identify patient factors that may influence antibiotic use for these patients . METHODS: This study was based on a serial analysis of results from all National Ambulatory Medical Care Surveys beginning in 1980 (when therapeutic drug use was first recorded) to 1994 (the most recent survey year available) . These surveys are a random sampling of visits to US office-based physicians in 1980, 1981, 1985, and annually from 1989-1994 . Eligible visits included those by adults presenting to general internists, family practitioners, or general practitioners with a chief complaint of cough . A total of 3416 visits for cough were identified over the survey years . Survey results were extrapolated, based on sampling weights in each year, to project national rates of antibiotic use for patients with cough . Additional analyses examined the rates of antibiotic use stratified by patient age, race, and clinical diagnosis . RESULTS: Overall, an antibiotic was prescribed 66% of the time during office visits for patients with cough: 59% of patient visits in 1980 rising to 70% of visits in 1994 (P = .002 for trend) . In every study year, white, non-Hispanic patients and patients younger than 65 years were more likely to receive antibiotics compared with nonwhite patients and patients 65 years or older, respectively . CONCLUSIONS: The rate of antibiotic use by primary care physicians for patients with cough remained high from 1980 to 1994, and was influenced by nonclinical characteristics of patients.

J Gastroenterol Hepatol, 1998 Jan, 13(1), 1 - 12
Report of the 1997 Asia Pacific Consensus Conference on the management of Helicobacter pylori infection; Lam SK et al.; While European and United States guidelines for the management of Helicobacter pylori infection have been developed, there are no guidelines for the Asian Pacific . International experts and recognised local authorities met in Singapore in 1997 to develop appropriate guidelines, taking into account the high background prevalence of infection, high incidence rates of gastric cancer and resource limitations . Recommendations were made based on randomised controlled trials or where this was not possible, they were based on the current best available evidence or on good clinical practice . A number of acceptable diagnostic tests for infection are available throughout the region . The non-endoscopic methods of choice are the urea breath test or a locally validated antibody test . If endoscopy was to be performed, a biopsy urease test was recommended as the test of first choice, with histology recommended only if this was negative . Post treatment testing was not recommended for all patients; a urea breath test was considered the test of choice if available . All gastric and duodenal ulcer patients who are infected with H . pylori should be treated for H . pylori whether the ulcer is active or in remission . Patients requiring long term non-steroidal anti-inflammatory drug therapy who have a current or recent history of dyspepsia, patients with early gastric cancer or low grade gastric mucosa associated lymphoid tissue lymphoma, and patients with a family history of gastric cancer should be treated . However, it was concluded that there wasn't sufficient evidence that cure of H . pylori infection reduces the risk or prevents the development of gastric adenocarcinoma . Many patients with dyspepsia in the region will request or require early upper endoscopy because of an inherent fear of gastric cancer . However, where endoscopy is not available or is too costly, alternative acceptable approaches were recommended in high risk cancer regions . While evidence is inconclusive to support treatment of H . pylori infection in non-ulcer dyspepsia, it was agreed that treatment be offered to patients with documented infection on a case-by-case basis . Treatment regimens need to attain an eradication rate of 90% or greater by per protocol analysis and 80% or greater by intention-to-treat analysis . A number of 7-day regimens were recommended based on available evidence . These regimens were considered likely to maximize the chances of successful eradication with one course of treatment, thereby reducing the risk of acquired antibiotic resistance and leading to long term cost savings.

J Med Philos, 1998 Jun, 23(3), 297 - 302
Public health and bioethics; Lachmann PJ; Conduct that satisfies certain bioethical doctrines may come into conflict with the needs and ethics of public health . The growth of antibiotic resistance in bacteria and the spread of HIV both contribute to the difficulty of controlling infectious disease . These two sets of priorities need to be reconciled and this is likely to require a reassessment of prevailing ethical doctrines in the face of the needs of public health.

Mol Ecol, 1998 Sep, 7(9), 1205 - 16
Evidence for transfer of antibiotic-resistance genes in soil populations of streptomycetes; Wiener P et al.; Phylogenetic analysis was used to evaluate the hypothesis of gene transfer in streptomycetes, many of which are antibiotic producers . The diversity and possible origins of streptomycin-resistance genes was investigated for a population of Streptomyces strains isolated from a site in Brazil where antibiotic production had previously been implicated The analysis provides compelling evidence for the transfer of these genes . Examination of other Streptomyces-type strains also reveals a scattered distribution of streptomycin producers with respect to the overall phylogeny . These results suggest that horizontal gene transfer may be an important factor in the evolution of antibiotic genes in streptomycetes.

Int J Antimicrob Agents, 1998 May, 10(2), 161 - 4
Antibiotic utilisation for hospitalised paediatric patients; van Houten MA et al.; Antibiotics are among the most commonly prescribed drugs in paediatrics . Because of an overall rise in health care costs, lack of uniformity in drug prescribing and the emergence of antibiotic resistance, monitoring and control of antibiotic use is of growing concern and strict antibiotic policies are warranted . Before such policies can be implemented, detailed knowledge of antibiotic prescribing patterns is important . In this combined retrospective and prospective study the utilisation of antibiotics in a paediatric university hospital over three consecutive years has been analysed . Over an 8-week period (1 November-22 December) in 1994, 1995 and 1996 patient charts were reviewed with regard to antibiotic prescription (generic class, dose, duration and indication) . A total of 1120 patients were admitted during the study periods . Antibiotics were prescribed at least once for 36% of hospitalised children, although only 12.3% of the patients receiving antibiotics had a proven bacterial infection . During a single hospitalisation 13, 4.7, 2.6, and 2.7% of all children received 2, 3, 4 or more than four antibiotics, respectively . Infants less than 2 years received antibiotics more frequently than older children (25 and 11% respectively, P=0.0256) . More children admitted to the intensive care unit received antibiotics compared with patients admitted on medium care units (49.7 and 29.3% respectively, P < 0.0001) . They received more often several different antibiotic courses (2.6 courses per patient versus 1.9 courses per patient, P < 0.0001) . These children were also given more often intravenous rather than oral antibiotics (P < 0.0001) Significant differences could be found between the generic classes of antibiotics prescribed to children admitted to the intensive care unit and the medium care . However high variability in dose and duration of antibiotic therapy for the same clinical indication was shown . A high percentage of all hospitalised children receive antibiotics . In most cases antibiotics are started on an empirical basis, without proof of a bacterial infection, either before the start of therapy or afterwards . The fact that children admitted to intensive care units and patients of younger age groups are at special risk of receiving multiple courses of antibiotics, together with the knowledge that antibiotic resistance develops in this setting, suggest that strategies to control antibiotic use should focus on these patient populations.

Dermatol Clin, 1998 Jul, 16(3), 509 - 25
Antibiotics in the management of pediatric skin disease; Darmstadt GL; An increasingly large number of antibiotics are available for the treatment of uncomplicated skin and skin structure infections in children . Primary factors in the choice among these agents are the antibiotic resistance profile of the target pathogen(s), and the antibiotic's spectrum of activity, pharmacologic properties, potential adverse reactions and interactions and propensity to select for the emergence of resistant organisms . Based on a consideration of these principles, this article provides a practical guide to the use of antibiotics in the management of common cutaneous infections in the pediatric population.

Ann Trop Paediatr, 1998 Mar, 18(1), 17 - 21
Adherence to cotrimoxazole treatment for acute lower respiratory tract infections in rural Bangladeshi children; de Francisco A et al.; This study evaluates compliance with taking oral cotrimoxazole in an ALRI control programme in rural Bangladesh . Health workers administered the first dose to children with moderate disease and entrusted relatives to give the remaining doses . A team of medical assistants visited the families of cases 3 to 5 days after initiating treatment and counted the remaining tablets . Medical assistants undertook 367 visits to families of children under treatment at a mean (SD) of 4.4 (0.99) days after treatment began . All children appeared to have been given the antibiotic, but one-quarter were being under-dosed on the day of the visit . Under-dosing did not correlate with any of the socio-demographic variables studied, and seemed to be homogeneously distributed in the community . Under-dosed children did not seem to have a higher risk of subsequent ALRI episodes during the study period . There was no indication of progression to severe disease or death in home-managed cases of moderate pneumonia in this study . These findings raise a question about the need for 5 days of oral antibiotic in the management of moderate pneumonia . Careful studies of the effect on subsequent morbidity, mortality and antibiotic resistance of providing briefer treatment for moderate episodes of ALRI are required . If a briefer course proves effective, this would have important implications for funding programmes of control of ALRI in the community.

Vaccine, 1998 Jul, 16(11-12), 1116 - 21
Global progress in infectious disease control; Hinman AR; There is both good news and bad news concerning infectious disease control globally . The good news is that smallpox has been eradicated, eradication of poliomyelitis and guinea worm disease is on track, and many infectious diseases are under effective control in much of the world . The advances are primarily the result of improved sanitation, effective use of vaccines, and introduction and use of specific therapies (whose impact has primarily been on mortality, rather than incidence) . The bad news is that infectious diseases are still the leading cause of death world-wide, new diseases are emerging, old diseases are re-emerging, there are ominous interactions between diseases, and antibiotic resistance is emerging as a major problem . There are many promising developments for the future, including new and improved vaccines, new specific therapies, and new strategies to deal with infectious disease . However, unless eradicated, infectious diseases remain a threat and require continuous efforts to be kept under control . Given the ability of infectious agents to evolve, it is certain that the future will also hold new problems and new diseases.

Aliment Pharmacol Ther, 1998 Jun, 12(6), 545 - 50
Two-week course of pantoprazole combined with 1 week of amoxycillin and clarithromycin is effective in Helicobacter pylori eradication and duodenal ulcer healing; Louw JA et al.; BACKGROUND: Experience with proton pump inhibitor-based triple therapy is predominantly with omeprazole-containing regimens . AIM: To investigate the efficacy of a pantoprazole-based regimen, with either a 1 or 2-week course of antibiotic co-therapy, in eradicating H . pylori, healing duodenal ulcers and to assess the antibiotic sensitivity profiles of isolated H . pylori strains . METHODS: A single-blind, multicentre, parallel group comparison of patients with endoscopically proven, H . pylori associated, active duodenal ulceration . All patients received pantoprazole, 40 mg b.d . for 2 weeks . Patients were randomized to receive either 1 or 2 weeks of therapy with amoxycillin, 1 g b.d . and clarithromycin 500 mg b.d . Patients were endoscoped at entry, at 14 days and a minimum of 4 weeks after cessation of all therapy . H . pylori status was determined by urease reaction, histological assessment and culture from antral and body biopsies . Antibiotic sensitivity was determined using the agar dilution technique . RESULTS: Sixty-seven patients were randomized . One week co-therapy (n=33): eradication efficacy, ITT= 79% (95% CI: 61-91%); ulcer healing efficacy (at 6-week visit)=88% (95% CI: 72-97%) . Two-week co-therapy (n=34): eradication efficacy, ITT=91% (95% CI: 76-98%: ulcer healing efficacy= 88% (95% CI: 73-97%) . Both regimens were well tolerated and no primary antibiotic resistance was noted . CONCLUSION: Pantoprazole-based triple therapy, with either 1 or 2 weeks of co-therapy with amoxycillin and clarithromycin, is effective in eradicating H . pylori and healing duodenal ulceration.

Clin Infect Dis, 1998 Jul, 27(1), 84 - 9
Demonstration of unexpected antibiotic resistance of genotypically identical Helicobacter pylori isolates; Dore MP et al.; With use of multiple- and single-colony expansion procedures, the results of susceptibility testing of Helicobacter pylori isolates from patients with duodenal ulcer were assessed by Etest . The H . pylori genotype was assessed by repetitive extragenic palindrome-based polymerase chain reaction (REP-PCR) . There was a high degree of genotypic heterogeneity between different patients, but a single REP-PCR pattern was found for 92% of patients . In contrast, a high degree of phenotypic heterogeneity was shown among the isolated colonies . Antibiogram susceptibility patterns differed only with respect to metronidazole but not with respect to clarithromycin or amoxicillin . The 42% rate of resistance to metronidazole determined with use of the conventional multiple-strains expansion method was increased to 92% when the single-colony expansion method was used . Similarly, dual clarithromycin/metronidazole resistance was increased from 8% to 42% with single-colony expansion . Despite evidence of a single genotype in most patients, single-colony expansion shows that routine susceptibility testing may greatly underestimate the frequency of metronidazole resistance.

J Hepatol, 1998 Jun, 28(6), 1054 - 7
Use of granulocyte macrophage colony stimulating factor in children after orthotopic liver transplantation; Trindade E et al.; BACKGROUND/AIMS: Bacterial infections complicate the course of up to 80% of pediatric liver transplant recipients, and in some cases, neutropenia, surgical complications and/or antibiotic resistance prevent successful control of sepsis . The aim of the present study was to evaluate the safety and efficacy of granulocyte macrophage colony stimulating factors (GM-CSF) in treating neutropenia following pediatric orthotopic liver transplantation . METHODS: Among a cohort of 430 pediatric orthotopic liver transplantation recipients, 13 children (12 months to 15 years, median 2 years, 10 males) received 15 courses of GM-CSF, 5 microg x kg(-1) x d(-1) subcutaneously, during their post-transplant course . In nine cases, the initial neutrophil count was below 1000/mm3 . Ten patients were infected . Three received GM-CSF for severe sepsis without neutropenia . The mean duration of treatment was 16.3 days (range 4-49) . RESULTS: In all but one neutropenic patient the neutrophil count increased above 1500/mm3 and the mean neutrophil count increased from 1392+/-1912/mm3 (range 130-7170, median 640) to 4508+/-2459/mm3 (range 350-9630, median 4390) (p<0.01) . Only one neutropenic patient (FK506 related) failed to respond to treatment . No rejection episode was induced by treatment, no side effects were noted, and patients with sepsis were cured . CONCLUSION: In these patients, GM-CSF was safe, it achieved a significant increase in neutrophilic count, and was beneficial in patients with severe bacterial infections . This compound may prevent infectious complications in neutropenic patients and may benefit patients with severe sepsis with or without neutropenia.

Eur J Pediatr, 1998 Jun, 157(6), 479 - 81
Shift in antibiotic prescribing patterns in relation to antibiotic expenditure in paediatrics; van Houten MA et al.; In paediatrics, antibiotics are among the most commonly prescribed drugs . Because of an overall rise in health care costs, lack of uniformity in drug prescribing and the emergence of antibiotic resistance, monitoring and control of antibiotic use is of growing concern and strict antibiotic policies are warranted . Before such a policy can be implemented, detailed knowledge of antibiotic prescribing patterns and related costs is important . In this study a shift of antibiotic prescription patterns over time is described in relation to hospital antibiotic expenditure . CONCLUSIONS: A considerable shift in prescription patterns towards more expensive and broader spectrum antibiotics occurs in paediatrics, carrying a risk for the development of antibiotic resistance among the most prevalent micro-organisms in this age group.

Aliment Pharmacol Ther, 1998 May, 12(5), 439 - 45
Evaluation of short-term low-dose triple therapy for the eradication of Helicobacter pylori by factorial design in a randomized, double-blind, controlled study; Bazzoli F et al.; BACKGROUND: Studies demonstrating the efficacy of short-term low-dose triple therapies including omeprazole (O), clarithromycin (C) and a nitroimidazole (tinidazole, T) for Helicobacter pylori eradication have largely been open and uncontrolled, and have not assessed antibiotic sensitivity . Simpler regimens using the component drugs have not been evaluated . AIM: To evaluate the OCT regimen in a randomized, controlled trial, testing for pre- and post-treatment antibiotic resistance and comparing, in a factorial design, the OCT regimen with simpler combinations of its components . METHODS: One hundred and twenty-eight patients (68 males, 60 females, age 22-80 years, mean 53 years) with H . pylori gastritis were randomly assigned to one of the following four treatment groups: (C) clarithromycin 250 mg b.d.; (OC) omeprazole 20 mg o.d . + clarithromycin 250 mg b.d.; (CT) clarithromycin 250 mg b.d . + tinidazole 500 mg b.d.; (OCT) omeprazole 20 mg q.d.s . + clarithromycin 250 mg b.d . + tinidazole 500 mg b.d . The drugs were administered for 1 week . Medical interview, upper gastrointestinal endoscopy (with four antral and four corpus biopsies) and the 13C-urea breath test were carried out for all patients prior to and 4 weeks after treatment . Biopsy specimens were used for the urease test, histology, and culture and sensitivities . RESULTS: All but one patient completed treatment . Side-effects were rare and mild in all groups . The eradication rate was 93.8% in group OCT, 59.4% in group CT, 31.3% in group OC and 6.3% in group C . Pre-treatment metronidazole resistance was 12.8%, clarithromycin 1.1% and, to both antibiotics, 2.1% . In patients with pre-treatment metronidazole resistance, the eradication rate was 75% in group OCT and 33% in group CT . Post-treatment resistance to clarithromycin was induced in 28.5% of the failures in group C, but in none of group OC . Resistance to both antibiotics occurred in 22.2% of the failures in group CT and in none of group OCT . CONCLUSIONS: (i) The high efficacy of the OCT regimen is proved and each of the individual components of the regimen is essential to the result, possibly via a synergistic effect . (ii) Pre-treatment metronidazole resistance is scarcely relevant to the outcome . (iii) Acquired resistance is essentially nil if omeprazole is part of the regimen.

Cancer Res, 1998 Jun 15, 58(12), 2588 - 93
Preferential cytotoxicity of cells transduced with cytosine deaminase compared to bystander cells after treatment with 5-flucytosine; Lawrence TS et al.; In vitro experiments from our laboratory and others have suggested that herpes simplex virus thymidine kinase (HSV-TK)/ganciclovir (GCV) gene therapy depends on gap junctional intercellular communication (GJIC) to produce a strong bystander effect . Furthermore, we have shown that cells transduced with HSV-TK can be protected from GCV-mediated toxicity by GJIC with bystander cells . We wished to determine whether GJIC affected either the bystander or protective effect of the cytosine deaminase (CD)/5-flucytosine (5-FC) gene therapy approach, in which CD converts 5-FC to 5-fluorouracil (5-FU) . To test this, we designed a coculture system using communication-competent WB rat hepatocytes and a noncommunicating subclone (aB1), which were transduced with CD and with antibiotic resistance genes so that we could independently determine the survival of the CD-containing or bystander cells . We found that, compared to the HSV-TK/GCV strategy, bystander killing resulting from treatment with CD/5-FC does not depend on GJIC . However, our most striking finding was that both communication-competent and -incompetent CD-transduced cells were preferentially killed, by a factor of up to 500, compared to bystander cells . The lesser dependence of the CD/5-FC system on GJIC, combined with the finding that most cancer cells lack the capacity for GJIC, suggest that the CD/5-FC system may be superior to the HSV-TK/GCV approach for gene therapy . However, the premature death of the CD-transduced 5-FU "factory" suggests that other strategies may be necessary to produce a sufficient quantity of 5-FU for a duration long enough to produce permanent tumor regression.

J Mol Biol, 1998 Jun 19, 279(4), 873 - 88
Ribosomal proteins S5 and L6: high-resolution crystal structures and roles in protein synthesis and antibiotic resistance; Davies C et al.; Antibiotic resistance is rapidly becoming a major medical problem . Many antibiotics are directed against bacterial ribosomes, and mutations within both the RNA and protein components can render them ineffective . It is well known that the majority of these antibiotics act by binding to the ribosomal RNA, and it is of interest to understand how mutations in the ribosomal proteins can produce resistance . Translational accuracy is one important target of antibiotics, and a number of ribosomal protein mutations in Escherichia coli are known to modulate the proofreading mechanism of the ribosome . Here we describe the high-resolution structures of two such ribosomal proteins and characterize these mutations.The S5 protein, from the small ribosomal unit, is associated with two types of mutations: those that reduce translational fidelity and others that produce resistance to the antibiotic spectinomycin . The L6 protein, from the large subunit, has mutations that cause resistance to several aminoglycoside antibiotics, notably gentamicin . In both proteins, the mutations occur within their putative RNA-binding sites . The L6 mutations are particularly drastic because they result in large deletions of an RNA-binding region . These results support the hypothesis that the mutations create local distortions of the catalytic RNA component.When combined with a variety of structural and biochemical data, these mutations also become important probes of the architecture and function of the translational machinery . We propose that the C-terminal half of S5, which contains the accuracy mutations, organizes RNA structures associated with the decoding region, and the N-terminal half, which contains the spectinomycin-resistance mutations, directly interacts with an RNA helix that binds this antibiotic . As regards L6, we suggest that the mutations indirectly affect proofreading by locally distorting the EF-Tu.GTP.aminoacyl tRNA binding site on the large subunit .

FEMS Microbiol Rev, 1998 Apr, 22(1), 1 - 20
ATP-binding-cassette (ABC) transport systems: functional and structural aspects of the ATP-hydrolyzing subunits/domains; Schneider E et al.; Members of the superfamily of adenosine triphosphate (ATP)-binding-cassette (ABC) transport systems couple the hydrolysis of ATP to the translocation of solutes across a biological membrane . Recognized by their common modular organization and two sequence motifs that constitute a nucleotide binding fold, ABC transporters are widespread among all living organisms . They accomplish not only the uptake of nutrients in bacteria but are involved in diverse processes, such as signal transduction, protein secretion, drug and antibiotic resistance, antigen presentation, bacterial pathogenesis and sporulation . Moreover, some human inheritable diseases, like cystic fibrosis, adrenoleukodystrophy and Stargardt's disease are caused by defective ABC transport systems . Thus, albeit of major significance, details of the molecular mechanism by which these systems exert their functions are still poorly understood . In this review, recent data concerning the properties and putative role of the ATP-hydrolyzing subunits/domains are summarized and compared between bacterial and eukaryotic systems.

J Fam Pract, 1998 Jun, 46(6), 469 - 75
Treatment of acute bronchitis in adults . A national survey of family physicians; Oeffinger KC et al.; BACKGROUND: The purpose of this study was to determine how family physicians in the United States treat acute bronchitis in an otherwise healthy adult . METHODS: A 33-item questionnaire on the diagnosis and treatment of acute bronchitis was mailed to a random sample of 500 physicians who are members of the American Board of Family Practice . RESULTS: Thirty-two of the 500 sampled physicians could not be located by mail; 265 of those who received the questionnaire responded . The response rate was 57% (265/468) . Sixty-three percent of responding physicians indicated that antibiotics are their first choice of treatment for the otherwise healthy, nonsmoking adult with acute bronchitis . The decision to use antibiotics as the first choice of treatment did not vary by physician's sex, age, years in practice, practice location, practice type, or percentage of HMO patients . Only 6% of responding physicians reported using beta 2 agonist bronchodilators as their first choice of treatment . Physicians in this study stated that they prescribe an antibiotic 75% of the time in treating nonsmoking patients with acute bronchitis (first choice or otherwise) . If the patient is a smoker, physicians reported that they prescribe antibiotics 90% of the time (F = 110.25; df = 1; P > .0001) . Physicians reported that for patients who smoke it takes longer for coughs to totally resolve and longer for them to return to a normal activity level than for nonsmokers . CONCLUSIONS: Family physicians report that antibiotics are their most common treatment for acute bronchitis in the otherwise healthy adult . Previous clinical trials have shown only marginal improvement in symptoms when patients with this condition are treated with an antibiotic . With antibiotic resistance emerging as a major global health problem, it is essential that other methods of treatment be evaluated.

Nucleic Acids Res, 1998 May 1, 26(9), 2120 - 4
Repressor titration: a novel system for selection and stable maintenance of recombinant plasmids; Williams SG et al.; The propagation of recombinant plasmids in bacterial hosts, particularly in Escherichia coli, is essential for the amplification and manipulation of cloned DNA and the production of recombinant proteins . The isolation of bacterial transformants and subsequent stable plasmid maintenance have traditionally been accomplished using plasmid-borne selectable marker genes . Here we describe a novel system that employs plasmid-mediated repressor titration to activate a chromosomal selectable marker, removing the requirement for a plasmid-borne marker gene . A modified E.coli host strain containing a conditionally essential chromosomal gene (kan) under the control of the lac operator/promoter, lac O/P, has been constructed . In the absence of an inducer (allolactose or IPTG) this strain, DH1 lackan , cannot grow on kanamycin-containing media due to the repression of kan expression by LacI protein binding to lac O/P . Transformation with a high copy-number plasmid containing the lac operator, lac O, effectively induces kan expression by titrating LacI from the operator . This strain thus allows the selection of plasmids without antibiotic resistance genes (they need only contain lac O and an origin of replication) which have clear advantages for use as gene therapy vectors . Regulation in the same way of an essential, endogenous bacterial gene will allow the production of recombinant therapeutics devoid of residual antibiotic contamination.

Biotechniques, 1998 Jun, 24(6), 972 - 4, 976, 978 passim
Selection strategy for site-directed mutagenesis based on altered beta-lactamase specificity; Andrews CA et al.; Conventional approaches to oligonucleotide-directed mutagenesis rely upon the application of a selection strategy to maximize mutagenesis efficiencies . We have developed a mutagenesis procedure that incorporates a novel antibiotic resistance for selection . The selection involves altering the substrate specificity of TEM-1 beta-lactamase, the enzyme responsible for bacterial resistance to beta-lactam antibiotics such as ampicillin . The gene encoding beta-lactamase is commonly found on cloning and shuttle vectors used in molecular biology . Amino acid substitutions in several active site residues of beta-lactamase result in increased hydrolytic activity against extended-spectrum penicillins and cephalosporins . This increased activity confers a novel resistance specific to the mutant and thus provides the basis of the selection strategy . We describe a simple and efficient mutagenesis procedure and its application to creating a range of oligonucleotide-directed mutants.

Int J Antimicrob Agents, 1998 Apr, 10(1), 77 - 81
Antibiotic resistance of pneumococci in Norway; Bergan T et al.; A collection of 178 pneumococcal isolates found in Norway during the period 1987-1994 were tested for their susceptibility to benzylpenicillin, macrolides (azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, spiramycin), fluoroquinolones (ciprofloxacin, sparfloxacin), imipenem, chloramphenicol, and vancomycin by a standard agar dilution procedure . To benzylpenicillin, two strains (1%) showed resistance and 14 strains (8%) intermediate susceptibility . Towards erythromycin, eight strains (4%) showed resistance and four strains (2%) intermediate susceptibility . Cross-resistance was demonstrated among the macrolides . Among the fluoroquinolones, intermediate susceptibility occurred with 42% of the isolates for sparfioxacin and 90% for ciprofloxacin; to the latter 5.1% proved resistant . The sum of intermediate and highly resistant isolates was 53% for chloramphenicol . Both penicillin-resistant strains were isolated during the last 2 years of collection and came from patients of non-Norwegian ethnic background . Imported strains appeared over represented among the strains resistant to penicillin and macrolides . Only imipenem and vancomycin showed full susceptibility for all pneumococci tested . An over representation of serogroup 6 strains was apparent among the strains with intermediate susceptibility and high resistance to benzylpenicillin . It is apparent that high-level resistance has, not so far, become a difficult problem in Norway . Nevertheless, the situation requires monitoring of the resistance level, particularly in meningitis and septic patients, and certainly in patients who cntail a higher than usual possibility of acquiring pneumococci from pools of resistant strains outside Norway (visitors, immigrants and recent returness from abroad).

Antimicrob Agents Chemother, 1998 Jun, 42(6), 1334 - 5
Effect of metronidazole resistance on bacterial eradication of Helicobacter pylori in infected children; Raymond J et al.; A prospective study was performed with 23 Helicobacter pylori-infected children (mean age, 9.5 +/- 4.4 years) with clinical symptoms of gastritis and positive results of culture and histologic examination of gastric biopsy specimens to evaluate the influence of antibiotic resistance on eradication . Positive children were treated for 4 weeks with lansoprazole and for 2 weeks with either amoxicillin-metronidazole or spiramycin (a macrolide)-metronidazole . At endoscopy 1 month after the discontinuation of therapy, the eradication rate and improvement of histologically related gastritis were significantly dependent on the susceptibility or the resistance of the infecting organism to metronidazole (83 versus 17% and 88 versus 16.6%, respectively) . Pretreatment determination of the susceptibility is appropriate in any anti-H, pylori regimen, including one with metronidazole.

Genome Res, 1998 Apr, 8(4), 404 - 12
Modification of bacterial artificial chromosome clones using Cre recombinase: introduction of selectable markers for expression in eukaryotic cells; Kim SY et al.; Bacterial artificial chromosome clones (BACs) are widely used at present in human genome physical mapping projects . To extend the utility of these clones for functional genomic studies, we have devised a method to modify BACs using Cre recombinase to introduce a gene cassette into the loxP sequence, which is present in the vector portion of the BAC clone . Cre-mediated integration is site specific and thus maintains the integrity of the genomic insert sequences, while eliminating the steps that are involved in restriction digest-based DNA cloning strategies . The success of this method depends on the use of a DNA construct, RETRObac, which contains the reporter marker green fluorescent protein (GFP) and the selectable marker neomycin phosphotransferase (neo), but does not contain a bacterial origin of replication . BAC clones have been modified successfully using this method and the genomic insert shows no signs of deletions or rearrangements . Transfection efficiencies of the modified BACs into human or murine cell lines ranged from 1% to 6% . After culture in media containing G418 for 3 weeks, approximately 0 . 1% of cells previously sorted for GFP expression acquired stable antibiotic resistance . Introduction of a human BAC clone that contains genomic p53 sequences into murine NIH3T3 cells led to expression of human p53 mRNA as determined by RT-PCR, demonstrating that sequences contained on the BAC are expressed . We believe that GFP-neo modified BAC clones will be a valuable resource in efforts to study biological effects of known genes as well as in efforts to clone and analyze new genes and regulatory regions.

Eur J Gastroenterol Hepatol, 1998 May, 10(5), 367 - 70
Should antibiotic treatment of Helicobacter-positive patients with non-ulcer dyspepsia now be recommended?
Veldhuyzen van Zanten SJ, Talley NJ.
Whether cure of Helicobacter pylori infection leads to sustained improvement of non-ulcer dyspepsia (NUD) symptoms is one of the most important unresolved clinical questions in this area . H . pylori has not helped in explaining pathophysiological abnormalities which may cause dyspepsia symptoms . Currently there is insufficient evidence from randomized clinical trials to be sure that cure of H . pylori infection will improve symptoms . There are potential downsides to the widespread use of antibiotics, these include the problem of antibiotic resistance and the possibility of less adequate pH control with acid-suppressive therapy after the infection has been cured.

Theor Popul Biol, 1998 Apr, 53(2), 152 - 65
The population genetics of antibiotic resistance . II: Analytic theory for sustained populations of bacteria in a community of hosts; Stewart FM et al.; The phenomenon of antibiotic resistance is of practical importance and theoretical interest . As a foundation for further studies by simulation, experiment, and observation, we here develop a mathematical model for the dynamics of resistance among the bacteria resident in a population of hosts . The model incorporates the effects of natural selection within untreated hosts, colonization by bacteria from the environment, and the rapid increase of resistance in hosts who receive antibiotics . We derive explicit formulas for the distribution of resistance among hosts and for the rise or fall of resistance when the frequency of treatment is changed.

Cell Mol Life Sci, 1998 Apr, 54(4), 353 - 8
X-ray studies of enzymes that interact with penicillins; Kelly JA et al.; The technique of X-ray diffraction has been successfully applied to enzymes associated with peptidoglycan biosynthesis . The technique has taught us a great deal about the structures and catalytic mechanisms of penicillin-binding proteins and beta-lactamases . An insight into the structural basis for antibiotic resistance is given.

J Bacteriol, 1998 Jun, 180(11), 2901 - 5
Evidence for a conjugation-like mechanism of DNA transfer in Helicobacter pylori; Kuipers EJ et al.; Many strains of Helicobacter pylori are naturally competent for transformation in vitro . Since there is a high degree of genetic variation among H . pylori strains, we sought to determine whether mechanisms of DNA exchange other than transformation exist in these organisms . Studies were done with H . pylori cells that each were resistant to two different antibiotics; the procedure used involved mating of cells on plates or in broth, in the absence or presence of DNase . In each experiment, such matings produced progeny with the markers of both parents . Examination of the full resistance profile and random arbitrarily primed DNA PCR (RAPD-PCR) profiles of the progeny indicated that DNA transfer was bidirectional . DNase treatment reduced but did not eliminate transfer; only the presence of both DNase and a membrane separating the cells did so . For progeny derived from matings in the presence of DNase, antibiotic resistance and RAPD profiles indicated that transfer was unidirectional . DNase-treated cell-free supernatants also did not transform, ruling out transduction . These experiments indicate that both a DNase-sensitive mechanism (transformation) and a DNase-resistant conjugation-like mechanism involving cell-to-cell contact may contribute to DNA transfer between H . pylori cells.

Br Med Bull, 1998, 54(1), 207 - 16
Antibiotic resistance in Helicobacter pylori infection; Megraud F; Resistance to antibiotics is considered as the primary reason for failure of eradication therapies . Resistance to clarithromycin is due to a decrease in binding to the ribosomes associated with a point mutation on the 23S rRNA . Its rate in Europe varies from 0-15%, with 5% in the UK . The resistance influences dramatically the success of the treatments . Resistance to metronidazole is due to a lack of reduction of this compound whose genetic basis is still unknown . The resistance rate in Europe varies from 10-50%, with 25% in the UK . It influences the success of treatments to a lesser extent than clarithromycin resistance . The initial eradication treatment can be prescribed without testing for susceptibility and must include a combination of two antibiotics, while stressing the importance of compliance to the patient . In case of failure, susceptibility testing must be performed . Few data are currently available on alternative therapeutic strategies when H . pylori is resistant to both clarithromycin and metronidazole.

Plasmid, 1998, 39(3), 245 - 51
Eukaryotic expression vectors that replicate to low copy number in bacteria: transient expression of the Menkes protein; Fontaine SL et al.; A set of low copy number plasmid vectors for mammalian gene expression has been constructed . These vectors are derived from the previously described bacterial low copy number expression vectors, pWSK29 and pWKS30, which are present at six to eight copies per cell . The new plasmids also have the following useful properties: (1) they contain antibiotic resistance markers for the selection of stable mammalian cell lines; (2) they have either constitutive or inducible promoters; (3) a chimeric intron, for enhancing gene expression, is present; (4) they contain unique cloning sites; (5) they have an SV40 polyadenylation signal, and a subset of the vectors have an SV40 origin of replication for episomal replication and transient gene expression . A cDNA encoding the Menkes disease protein was cloned into two of these vectors, and transient expression studies in COS-7 cells showed that both constitutive and inducible expression was possible . This set of expression vectors will provide a useful tool for the manipulation, in Escherichia coli, of mammalian genes or cDNAs that are unstable in the high copy number vectors that are currently available .

Proc Natl Acad Sci U S A, 1998 Apr 14, 95(8), 4380 - 5
Translation of cytochrome f is autoregulated through the 5' untranslated region of petA mRNA in Chlamydomonas chloroplasts; Choquet Y et al.; A process that we refer to as control by epistasy of synthesis (CES process) occurs during chloroplast protein biogenesis in Chlamydomonas reinhardtii: the synthesis of some chloroplast-encoded subunits, the CES subunits, is strongly attenuated when some other subunits from the same complex, the dominant subunits, are missing . Herein we investigate the molecular basis of the CES process for the biogenesis of the cytochrome b6f complex and show that negative autoregulation of cytochrome f translation occurs in the absence of other complex subunits . This autoregulation is mediated by an interaction, either direct or indirect, between the 5' untranslated region of petA mRNA, which encodes cytochrome f, and the C-terminal domain of the unassembled protein . This model for the regulation of cytochrome f translation explains both the decreased rate of cytochrome f synthesis in vivo in the absence of its assembly partners and its increase in synthesis when significant accumulation of the C-terminal domain of the protein is prevented . When expressed from a chimeric mRNA containing the atpA 5' untranslated region, cytochrome f no longer showed an assembly-dependent regulation of translation . Conversely, the level of antibiotic resistance conferred by a chimeric petA-aadA-rbcL gene was shown to depend on the state of assembly of cytochrome b6f complexes and on the accumulation of the C-terminal domain of cytochrome f . We discuss the possible ubiquity of the CES process in organellar protein biogenesis.

An Esp Pediatr, 1997 Dec, 47(6), 621 - 6
{Preventive use of antibiotics in neonatal surgery}; de Alba Romero C et al.; OBJECTIVE: The objective of this study was to determine the incidence of surgical wound infection and the impact of this on the implementation of an antibiotic protocol according to the type of surgery and the prevailing endogenous flora in our neonatology unit . PATIENTS AND METHODS: Patients' charts were retrospectively reviewed in order to assess the effectiveness of a protocol of surgical prophylaxis, comparing the incidence of postsurgical wound infection in two periods of time . Surgical interventions were classified according to the period of the study in which they were performed, before (period A) and after (period B) the protocol was undertaken . In addition, if the antibiotics administered fit or not those indicated in the protocol, it was classified as correct or not . RESULTS: A total of 31 (37%) of the interventions were performed in period A, whereas 53 (63%) were carried out in period B . No statistically significant differences were found between periods with regard to the proportion of infections 925.8% vs 15%) . When prophylactic antibiotics were administered correctly, infection occurred in 10.2%, as compared to 31.4% when they did not fit the protocol (p < 0.05) . The duration of the antibiotics was longer in period A, in infected wounds and when prophylaxis was not correct . CONCLUSIONS: The correct adjustment to the protocol significantly decreases the incidence of wound infections, as well as the duration of antibiotic use . An appropriate policy of antibiotic prophylaxis in surgery is advantageous in terms of economic cost and might prevent antibiotic resistance and avoid unnecessary toxicity.

Int J Antimicrob Agents, 1998 Feb, 9(4), 255 - 67
Antibiotic resistance mechanisms in bacteria of oral and upper respiratory origin; Roberts MC; Over the past 20 years, antibiotic resistance has increased in virtually every species of bacteria examined . In this paper, the main mechanisms of antibiotic resistance currently known for antibiotics used for treatment of disease caused by oral and upper respiratory bacteria will be reviewed, with an emphasis on the most commonly used antibiotics . The possible role that mercury, which is released from silver amalgams, plays in the oral/respiratory bacterial ecology is also discussed, as it relates to possible selection of antibiotic resistant bacteria.

Int J Antimicrob Agents, 1998 Feb, 9(4), 235 - 8
Clinical use of antibiotics in dental practice; Fine DH et al.; Inappropriate use of antibiotics by clinicians leads to development of antibiotic resistance . For the most part, antibiotics are prescribed in dental practice for prophylactic and therapeutic reasons . Prophylactic antibiotics are prescribed to prevent diseases caused by members of the oral flora introduced to distant sites in a host at risk or introduced to a local compromised site in a host at risk . In most cases, prophylaxis is used for prevention of endocarditis . Therapeutic antibiotics are prescribed, in most cases, to treat diseases of hard and soft tissues in the oral cavity after local debridement has failed . Antibiotics used for prophylaxis must: (1) be active against the major pathogens; and (2) achieve a tissue loading dose before the bacteria are introduced . Antibiotics used for therapy are required in cases where the infection is already present and thus the agent must reach the site of infection at a high enough level for a long enough time to produce the desired effect . For an exogenous agent the goal is to eliminate the agent from the site of infection . In the case of an endogenous agent the antibiotic must suppress the organism at the site of infection . Recent evidence underscores the important role of antibiotics in the treatment and prevention of diseases initiated in the oral cavity that have the potential to spread to distant organs in the body.

Yeast, 1998 Mar 15, 14(4), 391 - 9
Construction of PCR-ligated long flanking homology cassettes for use in the functional analysis of six unknown open reading frames from the left and right arms of Saccharomyces cerevisiae chromosome XV; Pearson BM et al.; Six open reading frames (ORFs) of unknown function from Saccharomyces cerevisiae chromosome XV, three from the left and three from the right arm, were deleted in two diploid strains by the short flanking homology method (Wach et al., 1994) . Transformants were selected as Geneticin (G418)-resistant colonies and correct integration of the kanMX4 cassette was checked by colony PCR . Following sporulation of the diploids, tetrads were dissected and scored for the segregation of the G418-resistant marker . We have developed a widely applicable method for the construction of gap repair plasmids to obtain the cognate clones for each of the disrupted ORFs . The 5'- and 3'-flanks of the ORF in question are linked by a unique restriction endonuclease . When the plasmid is cut at this site it can be used to obtain, by selection for the appropriate antibiotic resistance, long flanking homology (LFH) cassettes containing the cognate clone or the disrupted allele . The LFH cassette containing the cognate clone or the disrupted allele can be released from the gap-repaired plasmid by cutting at the inserted flanking restriction sites . One of the six ORFs (YOR319w) corresponds to an essential gene whose product is part of the spliceosome complex . Haploid as well as homozygous and heterozygous diploid disruptant strains for each of the five non-essential ORFs were subjected to growth test on different media at 15 degrees C, 30 degrees C and 37 degrees C . Disruption of YOR322c causes osmotically sensitive growth on YEPD at 37 degrees C and the product of YOL091w appears to play a role in sporulation since the homozygous diploid disruptant has lost the ability to sporulate.

J Virol, 1998 May, 72(5), 4192 - 204
The envelope protein encoded by the A33R gene is required for formation of actin-containing microvilli and efficient cell-to-cell spread of vaccinia virus; Roper RL et al.; The vaccinia virus (VV) A33R gene encodes a highly conserved 23- to 28-kDa glycoprotein that is specifically incorporated into the viral outer envelope . The protein is expressed early and late after infection, consistent with putative early and late promoter sequences . To determine the role of the protein, two inducible A33R mutants were constructed, one with the late promoter and one with the early and late A33R promoter elements . Decreased A33R expression was associated with small plaques that formed comets in liquid medium . Using both an antibiotic resistance gene and a color marker, an A33R deletion mutant, vA33delta, was isolated, indicating that the A33R gene is not essential for VV replication . The plaques formed by vA33delta, however, were tiny, indicating that the A33R protein is necessary for efficient cell-to-cell spread . Rescue of the large-plaque phenotype was achieved by inserting a new copy of the A33R gene into the thymidine kinase locus, confirming the specific genetic basis of the phenotype . Although there was a reduction in intracellular virus formed in cells infected with vA33delta, the amount of infectious virus in the medium was increased . The virus particles in the medium had the buoyant density of extracellular enveloped viruses (EEV) . Additionally, amounts of vA33delta cell-associated extracellular enveloped viruses (CEV) were found to be normal . Immunogold electron microscopy of cells infected with vA33delta demonstrated the presence of the expected F13L and B5R proteins in wrapping membranes and EEV; however, fully wrapped vA33delta intracellular enveloped viruses (IEV) were rare compared to partially wrapped particles . Specialized actin tails that propel IEV particles to the periphery and virus-tipped microvilli (both common in wild-type-infected cells) were absent in cells infected with vA33delta . This is the first deletion mutant in a VV envelope gene that produces at least normal amounts of fully infectious EEV and CEV and yet has a small-plaque phenotype . These data support a new model for VV spread, emphasizing the importance of virus-tipped actin tails.

J Bacteriol, 1998 Apr, 180(7), 1887 - 94
A small protein (Ags1p) and the Pho80p-Pho85p kinase complex contribute to aminoglycoside antibiotic resistance of the yeast Saccharomyces cerevisiae; Wickert S et al.; We identified the AGS1 and AGS3 genes by their ability to partially complement an ags mutant (RC1707) which is supersensitive to various aminoglycoside antibiotics (J . F . Ernst and R . K . Chan, J . Bacteriol . 163:8-14, 1985) . AGS1 is located in proximity to the centromere of chromosome III and encodes a small protein of 88 amino acids . The size of the AGS1 transcript, which in wild-type cells is 1 kb, is reduced to 0.75 kb in mutant RC1707 . Disruption of AGS1 rendered strains supersensitive to hygromycin B and increased their resistance to vanadate . In addition, ags1delta strains underglycosylated invertase but had normal carboxypeptidase Y glycosylation, suggesting that Ags1p is required for the elaboration of outer N-glycosyl chains . AGS3 was found to be identical to PHO80 (TUP7), which encodes a cyclin activating the Pho85p protein kinase . Deletion of either PHO80 or PHO85 led to aminoglycoside supersensitivity; pho80delta ags1delta strains showed an enhanced-sensitivity phenotype compared to single mutants . pho80 and pho85 mutants were rendered resistant by deletion of PHO4, indicating that activation of the Pho4p transcription factor is required for increased aminoglycoside sensitivity . Thus, both the Pho80p-Pho85p kinase complex (by Pho4p phosphorylation) and a novel component of the N glycosylation pathway contribute to basal levels of aminoglycoside resistance in Saccharomyces cerevisiae.

J Bacteriol, 1998 Apr, 180(7), 1759 - 65
Genetic complementation and kinetic analyses of Rhodobacter capsulatus ORF1696 mutants indicate that the ORF1696 protein enhances assembly of the light-harvesting I complex; Young CS et al.; Rhodobacter capsulatus ORF1696 mutant strains were created by insertion of antibiotic resistance cartridges at different sites within the ORF1696 gene in a strain that lacks the light-harvesting II (LHII) complex . Steady-state absorption spectroscopy profiles and the kinetics of the light-harvesting I (LHI) complex assembly and decay were used to evaluate the function of the ORF1696 protein in various strains . All of the mutant strains were found to be deficient in the LHI complex, including one (deltaNae) with a disruption located 13 codons before the 3' end of the gene . A 5'-proximal disruption after the 31st codon of ORF1696 resulted in a mutant strain (deltaMun) with a novel absorption spectrum . The two strains with more 3' disruptions (deltaStu and deltaNae) were restored nearly to the parental strain phenotype when trans complemented with a plasmid expressing the ORF1696 gene, but deltaMun was not . The absorption spectrum of deltaMun resembled that of a strain which had a polar mutation in ORF1696 . We suggest that a rho-dependent transcription termination site exists between the MunI and proximal StuI sites of ORF1696 . A comparison of LHI complex assembly kinetics showed that assembly occurred 2.6-fold faster in the parental strain than in strain deltaStu . In contrast, LHI complex decay occurred 1.7-fold faster in the ORF1696 parental strain than in deltaStu . These results indicate that the ORF1696 protein has a major effect on LHI complex assembly, and models of ORF1696 function are proposed.

Am J Gastroenterol, 1998 Mar, 93(3), 386 - 9
Primary and acquired Helicobacter pylori resistance to clarithromycin, metronidazole, and amoxicillin--influence on treatment outcome; Adamek RJ et al.; OBJECTIVE: The aim of this study was to evaluate the primary and acquired resistance of H . pylori against clarithromycin, metronidazole, and amoxicillin, and to elucidate the consequential influence on H . pylori eradication . METHODS: A total of 195 patients with positive H . pylori status were consecutively included . In 172 patients, H . pylori could be cultured for evaluation of primary antibiotic resistance . Fifty patients received a 2-wk dual therapy with an acid inhibitor and amoxicillin 2,000 mg daily (A), the other 122 patients a 1-wk modified triple therapy with the acid inhibitor clarithromycin 500-1,000 mg daily, and metronidazole 1,000-1,500 mg daily (B: n = 78), or amoxicillin 2,000 mg daily and metronidazole 1,000 mg daily (C: n = 44), respectively . Acid inhibition was conducted with pantoprazole 40 mg b.i.d . (n = 62), omeprazole 20 mg b.i.d . (n = 50), lansoprazole 30 mg b.i.d . (n = 10), or ranitidine 150 mg t.i.d . (n = 50) . After therapy, 36 patients remained H . pylori-positive, 20 after dual therapy (A) and 16 after modified triple therapy (B: n = 7, C: n = 9) . In 32 of these patients, H . pylori could be recultured for evaluation of acquired resistance (A: n = 18, B: n = 7, C: n = 7) . RESULTS: Primary H . pylori resistance to metronidazole was observed in 36 of 172 patients (21%) and to clarithromycin in three of 172 (2%) . Acquired resistance was found in six of 14 (43%) and in two of seven (29%), respectively, whereas neither primary nor acquired H . pylori resistance to amoxicillin was noted . Patients infected with metronidazole resistant H . pylori strains were successfully treated in combination with clarithromycin (eight of nine vs 63 of 67 with sensitive strains, NS), but not with amoxicillin (one of eight vs 32 of 34 with sensitive strains, p < 0.0001) . In two patients with acquired combined clarithromycin and metronidazole resistance, modified triple therapy failed . CONCLUSION: The value of modified triple therapy with amoxicillin and metronidazole is significantly limited by metronidazole resistance . However, metronidazole resistance does not negatively influence treatment outcome in modified triple therapy including clarithromycin . H . pylori resistance to amoxicillin still is not present.

Microb Pathog, 1998 Jan, 24(1), 25 - 35
Isolation and characterization of transposon-induced mutants of Porphyromonas gingivalis deficient in fimbriation; Watanabe-Kato T et al.; Fimbriae are considered to be an important virulence factor of Porphyromonas gingivalis . In order to identify genes essential for fimbriation, other than fimA which encodes the major subunit protein of fimbriae, transposon mutagenesis and immunological screening techniques were used to isolate fimbria-deficient mutants . R751::*Omega4, a suicide vector that carries Tn4351, was transferred from Escherichia coli to P . gingivalis by conjugation . Twenty-two independent fimbria-deficient mutants were identified among the resulting transformants . Southern hybridization analysis with pBlue 4351, a transposon-specific probe, and R751 indicated that 45% of the mutants resulted from single transposon insertions and that the remaining 55% of the mutants resulted from cointegration of R751 sequences . Southern hybridization analysis with pUCBg12.1, a probe for the fimA region, indicated that nine of the mutants contained insertions within the 2.5 kb SacI DNA fragment of P . gingivalis that contains fimA, ORF1 (which encodes a 15 kDa protein), and the C-terminal portion of ORF5 (which encodes a 63 kDa protein) . Polymerase chain reaction (PCR) analysis and further Southern hybridization analysis indicated that the insertion site(s) for all nine of these mutants was within the fimA gene . Southern hybridization analysis also indicated that the remaining thirteen mutants contained insertions somewhere outside the 10 kb fimA region . Analysis by pulsed field gel electrophoresis (PFGE) revealed that insertions for most of the thirteen mutants mapped to a 300 kb NotI fragment and are located at least approximately 200 kb away from fimA . These results identify genetic loci other than fimA, that are required for fimbriation of P . gingivalis . Future cloning and characterization of these genetic loci should be straightforward since they are now marked by antibiotic resistance genes carried by the transposon .

Monaldi Arch Chest Dis, 1997 Oct, 52(5), 486 - 91
Management of lower respiratory tract infections in out-patients; Woodhead M; Lower respiratory tract infections (LRTIs) are one of the most frequent medical conditions seen in out-patients . They all cause morbidity, and although most are minor some may be life-threatening, thus appropriate disease management is important . Clinical features are usually used to classify LRTIs, but this approach may be inaccurate . It may therefore be simpler to describe a patient's symptoms without applying a label, such as "bronchitis", since the latter means different things to different people . Classification of LRTIs should aim to aid management . The two main management decisions are: whether to manage the patient at home; and whether to prescribe antibiotics . Investigations are carried out in the hospital environment to aid these decisions, however in the community investigation in only a minority of cases are done as they are costly and impractical . Markers of severity of LRTI have been identified in a number of studies and their value in clinical practice is now being assessed, however most studies suggest that severely ill patients are correctly identified and admitted to hospital . Currently, antibiotics are used liberally for lower respiratory tract infections . However most infections are not bacterial in origin and will not be affected by such therapy . The idea that antibiotics are harmless placebos for such illnesses is no longer tenable since the appearance and rapid spread of antibiotic resistance in bacteria . Only in community-acquired pneumonia and some patients with exacerbations of chronic bronchitis do antibiotics actually alter the course of the illness . In those groups antibiotics should be targetted at the casual pathogens and in other groups such therapy should be avoided . Much current research interest is focused on determining which (if any) is the best antibiotic in these situations.

New Horiz, 1998 Feb, 6(1), 75 - 83
Clinical practice guidelines for the management of pneumonia--do they work?
Wunderink RG.
Significant variation from physician to physician and from hospital to hospital occurs in the management of pneumonia, despite nearly identical patient populations and causative organisms . This situation seems ideal for the use of clinical practice guidelines (CPGs), and several have already been published . The underlying assumptions used to develop pneumonia CPGs need to be examined before further proliferation of pneumonia CPGs . Some issues with pneumonia CPGs are common to all CPGs including the need for validation, especially on a local basis, and the reluctance of practitioners to follow CPGs . Need to adjust antibiotic recommendations based on emerging antibiotic resistance is common to all CPGs for infectious problems . The ability of currently available pneumonia CPGs to affect outcome is suspect because most of the recommendations rely on data that were not outcome-based . Aspects of current pneumonia CPGs are reviewed based on subsequently available data which either validate or question the recommendations.

Gene Ther, 1997 Dec, 4(12), 1341 - 9
A new DNA vehicle for nonviral gene delivery: supercoiled minicircle; Darquet AM et al.; Plasmids currently used for nonviral gene transfer have the disadvantage of carrying a bacterial origin of replication and an antibiotic resistance gene . There is, therefore, a risk of uncontrolled dissemination of the therapeutic gene and the antibiotic resistance gene . Minicircles are new DNA delivery vehicles which do not have such elements and are consequently safer as they exhibit a high level of biological containment . They are obtained in E . coli by att site-specific recombination mediated by the phage lambda integrase . The desired eukaryotic expression cassette, bounded by the lambda attP and attB sites was cloned on a recombinant plasmid . The expression cassette was excised in vivo after thermoinduction of the integrase gene leading to the formation of two supercoiled molecules the minicircle and the starting plasmid lacking the expression cassette . In various cell lines, purified minicircles exhibited a two- to 10-fold higher luciferase reporter gene activity than the unrecombined plasmid . This could be due to either the removal of unnecessary plasmid sequences, which could affect gene expression or the smaller size of mini-circle which may confer better extracellular and intracellular bioavailability and result in improved gene delivery properties.

J Antimicrob Chemother, 1997 Dec, 40(6), 811 - 6
Characterization of antibiotic resistance plasmids from Bordetella bronchiseptica; Speakman AJ et al.; Of 52 antibiotic-resistant Bordetella bronchiseptica isolates from cats, ten carried plasmids . Only two of these plasmids, pLV1400 and pLV1401, were self-transmissible to Escherichia coli K12; both plasmids encoded resistance to ampicillin, tetracycline, sulphonamides, streptomycin and mercuric chloride, and were of incompatibility group P (IncP) . Transferable tetracycline resistance has not been reported in B . bronchiseptica previously . The plasmids were identical in size (c.51 kb), restriction endonuclease digestion pattern and gene sequences (trfA and korA) within the IncP replicon . The trfA and korA sequences differed from those of the archetypal IncP plasmids RP4 and R751 . Although the two B . bronchiseptica isolates were from epidemiologically and geographically separated cats, pulsed-field gel electrophoresis of their XbaI- or DraI-digested chromosomal DNA indicated that they were genotypically identical . The plasmid-encoded ampicillin resistance was mediated by a penicillinase of molecular weight 49,000, and pI 8.45 which was inhibited by clavulanate (IC50 = 0.1 mg/L) and tazobactam (IC50 = 0.42 mg/L) but not by parachloromercuribenzoate or EDTA . The high-level tetracycline resistance was mediated by a class C efflux mechanism that has not been described previously in this genus . The presence of transferable multi-drug resistance on a promiscuous plasmid may limit options for therapy of respiratory tract infection in companion and farm animals.

Helicobacter, 1997 Mar, 2(1), 21 - 6
The role of ranitidine bismuth citrate in significantly reducing the emergence of Helicobacter pylori strains resistant to antibiotics; McLaren A et al.; BACKGROUND: This study was carried out to investigate the clinical observation that the emergence of antibiotic resistance can be reduced in Helicobacter pylori if agents are administered in combination with bismuth salts . MATERIALS AND METHODS: Two H . pylori clinical isolates were grown on chocolate Columbia agar containing either ranitidine bismuth citrate (RBC) at one-half lethal concentration (8 micrograms/ml) or no drug control for 22 subcultures . After 1, 5, 8, 14, 17, and 22 subcultures, the emergence of antibiotic resistance to metronidazole, clarithromycin, and streptomycin was calculated and statistically analyzed . RESULTS: Acquisition of resistance to metronidazole was reduced significantly for both strains (p = .007 and 0.014) and for one strain against clarithromycin (p = .037) . However, spontaneous emergence of resistance to streptomycin was not altered significantly . In an analysis of the effect of long-term exposure of the isolates to RBC, susceptibility to bismuth was unaltered in one strain and had risen by only twofold in the other at experiment termination . CONCLUSION: RBC significantly decreased resistance acquisition in an inherently sensitive strain and also decreased resistance emergence in a strain that readily became metronidazole-resistant.

J Biol Chem, 1997 Dec 12, 272(50), 31265 - 71
Purification and characterization of recombinant catalase-peroxidase, which confers isoniazid sensitivity in Mycobacterium tuberculosis; Nagy JM et al.; The Mycobacterium tuberculosis katG gene encodes a dual-function enzyme called catalase-peroxidase, which confers sensitivity in M . tuberculosis to isonicotinic acid hydrazide . We have constructed a system for the high level expression of a recombinant form of this enzyme by amplifying the katG gene from the pYZ56 construct (1) and subcloning into a vector suitable for expression in Escherichia coli . The resulting plasmid, pTBCP, produced the catalase-peroxidase in large quantities, corresponding to 30% of total cell protein . The enzyme has been purified to homogeneity and appears to be a dimer in the native form . Using either hydrogen peroxide or t-butyl hydroperoxide and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) as substrates, kcat and Km values have been obtained for both catalatic and peroxidatic activities, respectively . The availability of significant quantities of an active, folded, recombinant form of M . tuberculosis catalase-peroxidase should thus facilitate future studies of its role in drug activation and antibiotic resistance.

Rev Chir Orthop Reparatrice Appar Mot, 1997, 84(5), 466 - 8
{Temporary antibiotic-loaded cemented prosthesis for two-stage septic hip arthroplasty}; Migaud H et al.; PURPOSE OF THE STUDY: During the excision period of a two-stage revision arthroplasty, the hip has a low function and an unacceptable leg length discrepancy . The goal of this study was to expose technical details in order to perform a simple articulated cement spacer which could be implanted during this period to improve hip function, to authorize partial weight bearing and to avoid leg length discrepancy . MATERIAL: This method was applied in three two-stage procedures justified because of particular immunodeficiency conditions: a 43 years old man who had bone marrow allograft and immunosuppressive therapy because of leukemia suffering of subacute septic hip arthritis; a 58 years old man suffering of diabetes and active C-hepatitis who had a septic loosening of a total hip arthroplasty (THA); a 76 years old woman suffering of diabetes who had a third septic loosening of THA . METHOD: The prosthesis was made of antibiotic-impregnated cement according to organisms antibiotic resistance . The prosthetic junction between head and diaphysis was reinforced with a tibial plate . Prosthetic shape was identical to the one of femoral broaches inserted in the femur after prosthetic and cement removal . The broach size was chosen when mechanical stability in the femur was obtained, and avoided leg length discrepancy after trials with cups . The tibial plate was bent in order to reinforce the junction with regard to the shape of the determined broach . Two doses of antibiotic-impregnated cement were mixed and molded with hands, then the plate was incorporated at the appropriate location, finally the broach was applied on this composite and cement in excess was removed before polymerisation . For prosthetic head, two options were available: to mold the cephalic zone of the cement at the patient acetabulum diameter with a soft aluminium cup previously molded in the acetabulum; to mold the cement cephalic zone with a trial cup in order to obtain a 22 or 28 ball . For this last option, a third dose of antibiotic-impregnated cement was prepared and placed in the acetabulum, a trial femoral head was applied in it to mold the location for the 22 or 28 prosthetic head . Before insertion, a collar was applied on the stem to prevent migration . Active mobilization was encouraged, and partial weight-bearing authorized . RESULTS: The mean range of hip flexion during period was 60 degrees . The patients were discharged approximatively 12 days after the first stage . Two patients had effective painless partial weight-bearing . The second stage was performed six weeks later on the average . The second procedure was easier than the second stage of a conventional two-stage procedure because of: easy and low hemorrhagic dissection authorized by the prosthesis; low difficulties with soft tissue tension as the prosthesis prevents leg length discrepancy; preservation of the articular space which prevents soft tissue sacrifice during the second stage . CONCLUSION: This simple technique is effective to prevent complications related to the excision period of a two-stage hip revision arthroplasty . Likewise, the economical aspect (short delay of hospitalisation, quick functional recovery) should be considered when compared with the excision period of a conventional two-stage procedure.

J Med Microbiol, 1998 Jan, 47(1), 85 - 90
Identification and antibiotic susceptibility of Nocardia farcinica and N . nova in the UK; Workman MR et al.; Nocardia asteroides has long been recognised as a heterogeneous group of organisms . The description and identification of two new subgroups, N . farcinica and N . nova, in other countries encouraged us to re-examine a collection of N . asteroides isolates from the UK . Of 73 clinical isolates identified as N . asteroides from different parts of England and Wales during 1991-1993, and now subjected to further differentiation tests by the Mycobacterial/Nocardial Reference Laboratory, 15 (20.5%) were identified as N . farcinica based on three out of four characteristics: growth property, acetamide production, rhamnose assimilation and a distinct antibiogram . No isolates were identified as N . nova . A revised identification and susceptibility system has significant clinical and taxonomic implications . Its introduction will improve speciation, identify antibiotic resistance and influence the choice of safer alternative therapy for patients infected with Nocardia spp . in the UK.

Mol Microbiol, 1997 Nov, 26(4), 779 - 87
Insertional mutagenesis of a peptide synthetase gene that is responsible for hepatotoxin production in the cyanobacterium Microcystis aeruginosa PCC 7806; Dittmann E et al.; Several bloom-forming cyanobacterial genera produce potent inhibitors of eukaryotic protein phosphatases called microcystins . Microcystins are hepatotoxic cyclic heptapeptides and are presumed to be synthesized non-ribosomally by peptide synthetases . We identified putative peptide synthetase genes in the microcystin-producing strain Microcystis aeruginosa PCC 7806 . Non-hepatotoxic strains of M . aeruginosa lack these genes . Strain PCC 7806 was transformed to chloramphenicol resistance . The antibiotic resistance cassette insertionally inactivated a peptide synthetase gene of strain PCC 7806 as revealed by Southern hybridization and DNA amplification . This is the first report of genetic transformation and mutation, by homologous recombination, of a bloom-forming cyanobacterium . Chemical and enzymatic analyses, including high-performance liquid chromatography (HPLC), mass spectrometry, amino acid activation, and protein phosphatase inhibition, revealed the inability of derived mutant cells to produce any variant of microcystin while maintaining their ability to synthesize other small peptides . The disrupted gene therefore encodes a peptide synthetase (microcystin synthetase) that is specifically involved in the biosynthesis of microcystins . Our results confirm that microcystins are synthesized non-ribosomally and that a basic difference between toxic and non-toxic strains of M . aeruginosa is the presence of one or more genes coding for microcystin synthetases.

Anal Biochem, 1997 Dec 15, 254(2), 157 - 78
Approaches to DNA mutagenesis: an overview; Ling MM et al.; In the last several years, the use of double-stranded DNA templates together with thermostable-polymerase PCR has essentially replaced the use single-stranded DNA templates using the thermolabile polymerase for in vitro mutagenesis . Numerous PCR methods are now available, such as overlap-extension PCR, megaprimer PCR, and inverse PCR . All these PCR methods are reliable, effective, and convenient, although they are more prone to high rates of spontaneous error in mutant DNAs than are methods using thermolabile polymerases . Some improvements, such as the introduction of methylated templates, have been employed to minimize PCR errors . On the other hand, because of the introduction of many selection measures (e.g., restoration of antibiotic resistance, restoration of replication origin and unique site elimination), both double-stranded and single-stranded DNAs can now be used as templates for mutagenesis using thermolabile polymerase methods . For PCR methods, selection measures such as nested PCR has developed . All these selection measures have greatly improved the efficiency of mutagenesis by removing wild-type templates prior to transformation . Many efficient methods are available for both SDM and REM . Mutations can be introduce in vitro or in vivo, either by mutagenic primers or by erroneous DNA synthesis . Thus, choices largely depend on the experimental needs and resources of the investigator.

Bull Soc Pathol Exot, 1997, 90(3), 156 - 9
{Molecular typing of Moroccan strains of Mycobacterium tuberculosis}; el Baghdadi J et al.; The insertion sequence IS 6110 was used to differentiate clinical Moroccan isolates of Mycobacterium tuberculosis by using two non radioactive probes . Among 16 strains isolated from patients clinically related, 10 had similar IS 6110 restriction fragment length polymorphism (RFLP) patterns, confirming that they were derived from a common source . Two strains were isolated from the same patient (sputum, lymph node) showed identical profiles hybridized with IS 6110 element . Four sequential strains isolated from the same patients before treatment and after one year had identical IS 6110 RFLP patterns suggesting relapse and not reinfection . Twenty-one strains with identical drug susceptibility showed different IS 6110 RFLP profiles confirming no correlation between antibiotic resistance profiles and IS 6110 RFLP patterns . Since RFLP analysis by using IS 6110 element is a useful tool for the epidemiological survey, of tuberculosis.

Pediatr Infect Dis J, 1997 Nov, 16(11), 1060 - 4
Reduction of pneumococcal nasopharyngeal carriage in early infancy after immunization with tetravalent pneumococcal vaccines conjugated to either tetanus toxoid or diphtheria toxoid; Dagan R et al.; BACKGROUND: Pneumococcal nasopharyngeal colonization is important for transmission of the organisms . We assessed the ability of two tetravalent conjugate vaccines administered in early infancy to prevent carriage of vaccine-related pneumococci . METHODS: A vaccine containing pneumococcal type 6B, 14, 19F and 23F polysaccharide conjugated to tetanus toxoid (Pnc-T) and a vaccine containing the same four polysaccharides conjugated to diphtheria toxoid (Pnc-D) were compared with placebo, in a double blinded study (25 infants per group) . Vaccines (or placebo) were injected at 2, 4 and 6 months of age . At 12 months of age a native (nonconjugate) polysaccharide vaccine was administered as a booster . Serum type-specific anticapsular antibody concentrations were measured and nasopharyngeal cultures were obtained at 2, 4, 6, 7, 12 and 13 months of age . RESULTS: In general carriage of all pneumococci (vaccine- and non-vaccine-related) was low at age 2 months and increased with age . However, for the vaccine-related serotypes (6A, 6B, 14, 19F and 23F) carriage was not increased with age in Pnc-D or Pnc-T recipients . Of all cultures obtained after the full primary series, 7 of 72 (10%), 3 of 62 (5%) and 19 or 70 (27%) were positive for the vaccine-related pneumococcal serotypes among the Pnc-D, Pnc-T and placebo recipients, respectively (P = 0.001 for Pnc-D vs . placebo; P = 0.014 for Pnc-T vs . placebo) . Most of the antibiotic-resistant isolates belonged to the vaccine-related serotypes . CONCLUSIONS: A significant reduction in the carriage of vaccine-related strains after administration of conjugate vaccines was observed . These preliminary results suggest that transmission of specific pneumococcal serotypes most often associated with disease and antibiotic resistance may at least partially be controlled by immunization.

Clin Infect Dis, 1997 Nov, 25(5), 983 - 9
Helicobacter pylori infection in Chile; Figueroa G et al.; This article summarizes studies designed to evaluate the role of Helicobacter pylori infection in Chile, described in 21 reports from nine centers in various Chilean regions published between 1985 and 1995 . According to their data, H . pylori infection is quite frequent among patients with a variety of gastric conditions, including adults (43%-92%) and children (6%-100%) . Levels of specific IgG antibodies to H . pylori are also elevated among patients with duodenal ulcers (100%) and gastritis (86%) as well as asymptomatic adults (75%) . Combination therapy with three (but not two) drugs has been proved effective, with clinical improvement, ulcer cure, and H . pylori eradication occurring in well-controlled studies . Available evidence suggests that antibiotic resistance is not a major problem in treatment . The H . pylori reinfection rate is low (4.2% per year), suggesting that combination therapy with three drugs constitutes a cost-effective alternative for treating colonized symptomatic patients . Concurrent preliminary studies revealed that antibodies to VacA but not CagA proteins correlate with disease severity in Chilean patients . It can be concluded that local research assists local administrators of health resources to implement adequate policies to prevent, control, and treat H . pylori-related pathologies.

Mol Microbiol, 1997 Nov, 26(3), 455 - 67
The finM promoter and the traM promoter are the principal promoters of the traM gene of the antibiotic resistance plasmid R100; Stockwell DT et al.; finP multicopy repression and traJ multicopy derepression indicate that the ratio of sense to antisense transcripts is important in the regulation of R100 conjugation . The extension of R100 traM transcripts into traJ shows that promoters in traM can affect this ratio, making the regulation of traM transcription important in the regulation of R100 conjugation . Since R100 traM, traY and tral proteins bind to the traM promoter region, we examined traM transcription in R100-1 traM, traY and tral mutants and compared it with traM transcription in both R100-1 and R100 . We verified that the traM and finM promoters provide virtually all the transcripts originating in the R100-1 traM gene . When either is deleted, as in VAR22 or VAR30, the remaining promoter is highly active . We show here that traY positively regulates R100-1 traM transcription, as has been found for F . We found that tral did not regulate R100-1 traM transcription . The measured activity of the native R100 traM promoter was 12% of that in R100-1, whereas the native R100 finM promoter was 45% of that in R100-1 . These data and data from the R100-1 traY and tral mutants show that the activities of the two promoters varied independently.

Mol Microbiol, 1997 Nov, 26(3), 441 - 53
Non-palindromic attl sites of integrons are capable of site-specific recombination with one another and with secondary targets; Hansson K et al.; Genes borne on cassettes are mobile owing to site-specific recombination systems called integrons, which have created various combinations of antibiotic resistance genes in R-plasmids . In these processes, the palindromic site, attC (59-base element), at cassette junctions has been proposed as being essential . Excised and circularized cassettes have been found to integrate with preference for an attl site at one end of the conserved sequence in integrons . In this work, we give evidence that recombination is possible in the absence of the highly organized attC sites between the more simply organized attl sites . Furthermore, at a very low frequency representing the background in our recombination assay, we observed cross-overs between attl and secondary sites . To characterize recombination excluding the attC sites, we have used naturally occurring attl variants and constructed mutants . The cross-over point was identified between a guanine and a thymine in attl using point mutations . Progressive deletions showed the extent of attl and identified two important regions in the conserved sequence 5' of the cross-over point . A region 27-36 bp 5' of attl influenced recombination with attC sites only, whereas a sequence 9-14 bp 5' of the cross-over point in attl was important for recombination with both attl and attC . Recombination between attl and secondary sites could allow fusion of the conserved sequence encoding the integron site-specific recombinase to new sequences.

Laryngoscope, 1997 Dec, 107(12 Pt 1), 1586 - 9
Role of middle meatus aspiration culture in the diagnosis of chronic sinusitis; Gold SM et al.; Although empiric antibiotic therapy is often used for sinusitis, the emergence of antibiotic resistance has increased the failure rate of this approach . Culture-directed therapy usually increases treatment success, but traditional antral puncture is often accompanied by poor patient and physician acceptance . Endoscopically directed middle meatal aspiration culture is increasingly used in this setting, but studies have not convincingly demonstrated the validity of this technique . Both endoscopic middle meatal and direct antral cultures were performed during endoscopic sinus surgery . Cytologic examination was performed to confirm the presence of inflammatory cells . When culture results were compared in 21 specimen pairs, exact correlation was found in 18 (85.7%) . Based on this study, endoscopically directed middle meatus aspiration culture appears to be a valuable alternative to antral puncture for guiding organism-specific antibiotic therapy in sinusitis.

J Bacteriol, 1997 Dec, 179(23), 7410 - 9
Fis, an accessorial factor for transcriptional activation of the mar (multiple antibiotic resistance) promoter of Escherichia coli in the presence of the activator MarA, SoxS, or Rob; Martin RG et al.; Transcription of the multiple antibiotic resistance marRAB operon increases when one of the sequence-related activators, MarA, SoxS, or Rob, binds to the "marbox" centered at -61.5 relative to the transcriptional start site . Previous deletion analyses showed that an adjacent upstream "accessory region" was needed to augment the marbox-dependent activation . To analyze the roles of the marbox and accessory regions on mar transcription, thirteen promoters, each with a different 5-bp transversion of the -96 to -32 sequence, were synthesized, fused to lacZ, and assayed for beta-galactosidase production in single-copy lysogens with appropriate genotypes . The accessory region is shown here to be a binding site for Fis centered at -81 and to bind Fis, a small DNA-binding and -bending protein, with a Kd of approximately 5 nM . The binding of MarA to the marbox and that of Fis to its site were independent of each other . MarA, SoxS, and Rob each activated the mar promoter 1.5-to 2-fold when it had a wild-type marbox but Fis was absent . In the presence of MarA, SoxS, or Rob, Fis further enhanced the activity of the promoter twofold provided the promoter was also capable of binding Fis . However, in the absence of MarA, SoxS, or Rob or in the absence of a wild-type marbox, Fis nonspecifically lowered the activity of the mar promoter about 25% whether or not a wild-type Fis site was present . Thus, Fis acts as an accessory transcriptional activator at the mar promoter.

Crit Care Nurs Q, 1997 Nov, 20(3), 1 - 11
Recognizing and preventing antibiotic-associated complications in the critical care setting; Foxworth J; Antibiotics are commonly used, even overused, in the intensive care unit (ICU) patient population . These agents, though useful and often lifesaving, are associated with many toxicities . These include infusion-related problems, allergic reactions, organ-system toxicities, drug accumulation in patients with renal disease, drug interactions, antibiotic resistance, diarrhea, interference with laboratory tests, and more . Informed vigilance by the ICU nurse can be helpful in increasing the benefit/toxicity ratio associated with these agents.

J Paediatr Child Health, 1997 Aug, 33(4), 287 - 95
Antibiotic management of pneumococcal infections in an era of increased resistance; Grimwood K et al.; Pneumococci are a leading cause of bacterial meningitis and bacteraemia, as well as pneumonia, otitis media and sinusitis in childhood . These organisms recently have shown a dramatic increase in antibiotic resistance . Penicillin-resistant pneumococci are of special concern as they are often resistant to other unrelated antibiotics . This is of particular significance to Aboriginal children who have among the highest rates of pneumococcal infection in the world . Laboratories should now test all invasive pneumococcal isolates for penicillin and third generation cephalosporin resistance . Local treatment guidelines are required for pneumococcal infections, especially for meningitis, taking into account the prevalence of resistant strains within the community . At present, penicillin and amoxycillin remain the drugs of choice for pneumococcal infections, with the exception of meningitis where initial empirical therapy must be with a third generation cephalosporin . Judicious antibiotic use, which avoids over-prescribing and unnecessary use of broad-spectrum agents, improved living standards in underprivileged communities and introduction of an effective conjugate vaccine, able to reduce the rates of pneumococcal infection and hopefully colonization, may limit the spread of resistant strains.

Microbiol Immunol, 1997, 41(9), 697 - 702
RobA-induced multiple antibiotic resistance largely depends on the activation of the AcrAB efflux; Tanaka T et al.; RobA is a member of the XylS/AraC subfamily of DNA binding proteins, and when overexpressed, it induces multiple antibiotic resistance in Escherichia coli . In this study, we introduced a multicopy robA plasmid (pMEP1) and its derivative into OmpF mutants and an AcrAB-deficient mutant . We found that a decrease in susceptibility to multiple antibiotics in these OmpF mutants when pMEP1 was introduced did not depend on OmpF porin expression . Interestingly, a delta ompF mutant (TK007) became more sensitive when pMEP1 was introduced . Moreover, no effect of RobA on the induction of multiple antibiotic resistance in an acrA1- mutant was observed . Therefore, we conclude that the multiple antibiotic resistance induced by the overexpression of RobA largely depends on the activation of the AcrAB efflux, as well as the activation of micF.

J Bacteriol, 1997 Oct, 179(19), 6122 - 6
Role of the acrAB locus in organic solvent tolerance mediated by expression of marA, soxS, or robA in Escherichia coli; White DG et al.; Escherichia coli K-12 strains are normally tolerant to n-hexane and susceptible to cyclohexane . Constitutive expression of marA of the multiple antibiotic resistance (mar) locus or of the soxS or robA gene product produced tolerance to cyclohexane . Inactivation of the mar locus or the robA locus, but not the soxRS locus, increased organic solvent susceptibility in the wild type and Mar mutants (to both n-hexane and cyclohexane) . The organic solvent hypersusceptibility is a newly described phenotype for a robA-inactivated strain . Multicopy expression of mar, soxS, or robA induced cyclohexane tolerance in strains with a deleted or inactivated chromosomal mar, soxRS, or robA locus; thus, each transcriptional activator acts independently of the others . However, in a strain with 39 kb of chromosomal DNA, including the mar locus, deleted, only the multicopy complete mar locus, consisting of its two operons, produced cyclohexane tolerance . Deletion of acrAB from either wild-type E . coli K-12 or a Mar mutant resulted in loss of tolerance to both n-hexane and cyclohexane . Organic solvent tolerance mediated by mar, soxS, or robA was not restored in strains with acrAB deleted . These findings strongly suggest that active efflux specified by the acrAB locus is linked to intrinsic organic solvent tolerance and to tolerance mediated by the marA, soxS, or robA gene product in E . coli.

Gene Ther, 1997 Aug, 4(8), 868 - 74
Enhancement of retroviral production from packaging cell lines expressing the human immunodeficiency type 1 VPU gene; Kobinger GP et al.; The HIV-1 Vpu protein stimulates virus production by enhancing the release of viral particles from infected cells . Interestingly, Vpu was also shown to enhance the release of capsids produced by gag gene contructs of other retroviruses that lack a Vpu-like activity . To investigate the effect of Vpu expression on viral particle production in retroviral packaging cell line, we developed the Damp-VpuP cell line in which vpu expression is under the control of the tetracycline-responsive promoter . Retroviral production was measured by dosage of virion-associated reverse transcriptase activity, by capsid protein immuno-detection in cell-free supernatants and by evaluating the transfer of antibiotic resistance to target cells . Induction of the Damp-VpuP cell line caused a 40-fold increase in the titer of infectious virus-like particles when compared with control cell lines . This increase in viral titer was not the result of a clonal effect nor was it a consequence of high selective pressure but rather the effect of a Vpu-mediated enhancement of viral particle production . Similar results using the third generation psi CRIP packaging cell line confirmed these findings . Constitutive expression of vpu caused a 13-fold increase in viral titer in this packaging cell line . These results indicate that the expression of HIV-1 vpu in retroviral packaging cell lines can significantly improve the titers of infectious retroviral particles.

Electrophoresis, 1997 Aug, 18(9), 1570 - 6
A study of the genetic diversity of Mycobacterium tuberculosis isolated from patients in the eastern province of South Africa using random amplified polymorphic DNA profiling; Richner SM et al.; This population genetics study was done to determine the degree of genetic diversity amongst clinical isolates of Mycobacterium tuberculosis in one of the largest provinces of South Africa . Three hundred and fifty-nine individual cultures were obtained from single patients over a nine-month period . Bacterial DNA was extracted and amplified with two arbitrary ten-mer primers, using the random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) technique . RAPD markers were separated by agarose gel electrophoresis, visualised with ethidium bromide, and then analysed for similarity using the GelCompar programme . The isolates were seen to fall into two major population groups . A high degree of genetic diversity was seen, with a total of 350 unique strains occurring amongst the 359 isolates . The majority of drug-resistant isolates grouped together in the smaller population group . The genetic data were also correlated with geographical locality . It was found that a slight preponderance of isolates that grouped together closely, vis . that were either genetically identical or very similar (similarity index of approximately 97%), were from areas that were considered to be geographically distant . Antibiotic resistance patterns were also examined, revealing a total of 16 different resistance profiles among the 68 drug-resistant isolates . RAPD-PCR has proved to be a cost- and time-effective technique, suitable for such a large-scale population genetics study . It has provided useful information on the genetic diversity of an organism which is responsible for an increasing amount of morbidity and mortality in South Africa's second-largest province.

J Biol Chem, 1997 Oct 3, 272(40), 24755 - 8
Inhibition of aminoglycoside antibiotic resistance enzymes by protein kinase inhibitors; Daigle DM et al.; Bacterial resistance to the aminoglycoside antibiotics is manifested primarily through the expression of enzymes which covalently modify these drugs . One important mechanism of aminoglycoside modification is through ATP-dependent O-phosphorylation, catalyzed by a family of aminoglycoside kinases . The structure of one of these kinases, APH(3')-IIIa has recently been determined by x-ray crystallography, and the general fold is strikingly similar to eukaryotic protein kinases (Hon, W . C., McKay, G . A., Thompson, P . R., Sweet, R . M., Yang, D . S . C., Wright, G . D., and Berghuis, A . M . (1997) Cell 89, 887-895) . Based on this similarity, we have examined the effect of known inhibitors of eukaryotic protein kinases on two aminoglycoside kinases, APH(3')-IIIa and the enzyme AAC(6')-APH(2") which also exhibits acetyl-CoA-dependent aminoglycoside modification activity . We report that several known protein kinase inhibitors are also good inhibitors of aminoglycoside kinases . Compounds belonging to the isoquinolinesulfonamide group are especially effective in this regard, giving competitive inhibition in the micromolar range with respect to ATP and noncompetitive inhibition versus the aminoglycoside substrate . This study provides the basis for future aminoglycoside kinase inhibitor design and for the development of compounds which could reverse antibiotic resistance in the clinic.

FEMS Microbiol Lett, 1997 Sep 15, 154(2), 385 - 8
Mercury induces multiple antibiotic resistance in Escherichia coli through activation of SoxR, a redox-sensing regulatory protein; Fuentes AM et al.; Sub-inhibitory mercury concentrations are capable of partially activating SoxR, as shown by the augmented expression of a soxS'::lacZ fusion, and a diminished sensitivity to antibiotics caused by mercury treatment . Mercury may elevate the intracellular concentration of superoxide or perhaps act as a putative metal ligand for SoxR.

J Bacteriol, 1997 Sep, 179(18), 5947 - 50
Acquisition of resistance genes by the IncQ plasmid R1162 is limited by its high copy number and lack of a partitioning mechanism; Becker EC et al.; R1162 is a representative member of the broad-host-range IncQ group of multicopy plasmids . Lower-copy-number derivatives of R1162 were constructed in vitro and shown to be unstable, indicating that partitioning of plasmid copies at cell division is due to random distribution and not to an active partitioning mechanism . However, the normal copy number of R1162 reduces cell fitness during growth in broth and favors the emergence of unstable, lower-copy-number variants . As a result, plasmid-borne antibiotic resistance genes active at a low copy number eventually result in plasmid loss during periods of no selection . We argue that the maintenance of R1162 in a population requires a gene that is selected only at high levels . This reduces the potential for acquiring genes from other R factors and could explain the limited variety of antibiotic resistance genes contained by naturally occurring IncQ plasmids.

Proc Natl Acad Sci U S A, 1997 Aug 5, 94(16), 8468 - 72
Phenotypic conversion of drug-resistant bacteria to drug sensitivity; Guerrier-Takada C et al.; Plasmids that contain synthetic genes coding for small oligoribonucleotides called external guide sequences (EGSs) have been introduced into strains of Escherichia coli harboring antibiotic resistance genes . The EGSs direct RNase P to cleave the mRNAs transcribed from these genes thereby converting the phenotype of drug-resistant cells to drug sensitivity . Increasing the EGS-to-target mRNA ratio by changing gene copy number or the number of EGSs complementary to different target sites enhances the efficiency of the conversion process . We demonstrate a general method for the efficient phenotypic conversion of drug-resistant bacterial cultures.

Geriatrics, 1997 Jul, 52(7), 34 - 6, 39-43
Antibiotic resistance: why is it increasing in nursing homes?
McCue JD.
Causes of the apparent increase in antibiotic resistance in the bacterial flora of nursing homes are multifactorial . Today's nursing home patients are older, in poorer health, and less able to function independently than has been true in the past . Infection and antibiotic use in this population may increase selective pressure for the emergence of resistant strains . The efficient transfer to nursing homes of patients from acute-care settings also contributes to the increase in colonization or infection with highly resistant bacteria . Prudent restraint in the use of antibiotics and better infection control in nursing homes may reduce or retard the increase or spread in resistant infections.

Curr Biol, 1997 Jul 1, 7(7), 530 - 3
Transposon-generated 'knock-out' and 'knock-in' gene-targeting constructs for use in mice; Westphal CH et al.; The conventional technique for targeted mutation of mouse genes entails placing a genomic DNA fragment containing the gene of interest into a vector for fine mapping, followed by cloning of two genomic arms around a selectable neomycin-resistance cassette in a vector containing thymidine kinase {1}; this generally requires 1-2 months of work for each construct . The single 'knock-out' construct is then transfected into mouse embryonic stem (ES) cells, which are subsequently subjected to positive selection (using G418 to select for neomycin-resistance) and negative selection (using FIAU to exclude cells lacking thymidine kinase), allowing the selection of cells which have undergone homologous recombination with the knockout vector . This approach leads to inactivation of the gene of interest {2} . Recently, an in vitro reaction was developed, on the basis of the yeast Ty transposon, as a useful technique in shotgun sequencing {3} . An artificial transposable element, integrase enzyme and the target plasmid are incubated together to engender transposition . The DNA is then purified, and subsequently electroporated into bacteria . The transposon and the target plasmid bear distinct antibiotic resistance markers (trimethoprim and ampicillin, respectively), allowing double selection for transposition events . In the present study, we have modified this system to allow the rapid, simultaneous generation of a palette of potential gene targeting constructs . Our approach led from genomic clone to completed construct ready for transfection in a matter of days . The results presented here indicate that this technique should also be applicable to the generation of gene fusion constructs {4-8}, simplifying this technically demanding method.

J Gastroenterol Hepatol, 1997 Jun, 12(6), S23 - 8
Peptic ulcer disease in the 1990s: an Asian perspective; Fock KM; Peptic ulcer disease is still a common disease in many parts of Asia, although it is less common today than it was 2-3 decades ago . Contrary to this general trend, peptic ulcers are on the rise in the elderly, particularly elderly females . Two important factors that could explain the observed changes in the trends of peptic ulcer disease are: Helicobacter pylori and NSAID . The seroprevalence of H . pylori, determined in three previous studies, would appear to have decreased over the last few decades, while NSAID and aspirin are used increasingly for arthritis, cerebrovascular disease and coronary artery disease . The major complication of peptic ulcer disease is gastrointestinal haemorrhage and in the 1990s endoscopic haemostatic therapy has replaced surgery as the treatment of choice . Treatment of peptic ulcer disease caused by H . pylori is directed at eradication of H . pylori itself; four classes of drug regimens are currently available for this . Antibiotic resistance, particularly metronidazole resistance, is an important factor that determines the outcome of therapy . Metronidazole resistance is reported to be present in 50% of all strains of H . pylori in Hong Kong and Singapore, and is present in 80-90% of all strains in India . Eradication rates in Asia, may for this reason, differ from those in the West, if the regimen contains metronidazole . Treatment of NSAID-associated ulcer consists of discontinuation of NSAID, if possible, and administration of anti-secretory drugs such as H2 blockers, proton pump inhibitors or mucosal protective agents . Co-prescription with misoprostol has been shown to reduce the risk of NSAID-induced ulcer . New NSAID or NO NSAID are being developed with few gastrointestinal side effects.

Pediatr Infect Dis J, 1997 Jun, 16(6), 560 - 4
Patterns and determinants of use of antibiotics for acute respiratory tract infection in children in China; Hui L et al.; BACKGROUND: Use of antibiotics for acute respiratory infection (ARI) of presumed viral etiology is a worldwide problem . The World Health Organization (WHO) has provided guidelines for diagnosis and treatment of ARI for developing countries . METHODS: Specially trained observers applied the WHO criteria to study the diagnosis and treatment of ARI given by 100 randomly selected health care workers (HCWs) in a rural county in China . A total of 750 cases of ARI were evaluated . RESULTS: Before the parents sought medical care, 47% of children in the county hospitals, 25% of those in the townships and 18% of those in the villages had already received antibiotics, available without prescription . Among the HCWs antibiotic abuse (antibiotics for presumably viral disease) was detected in the treatment of 97% of cases, and severe abuse (such as prescription of two incompatible antibiotics) was detected in 37% . Most (197 of 200) patients with bacterial disease received antibiotics, but inappropriate antibiotic treatment (dose or type) was observed in 63% of these cases . HCWs with university training and those with higher test scores on knowledge and attitude prescribed antibiotics more judiciously than those lacking those attributes . CONCLUSIONS: Abuse of antibiotics for ARI is a serious and costly problem in rural China, potentially leading to widespread antibiotic resistance . Educating HCWs in the management of ARI and proper use of antibiotics has high priority in China.

J Fam Pract, 1997 Jun, 44(6), 545 - 55
Evaluation of the dyspeptic patient: a cost-utility study; Ebell MH et al.; BACKGROUND: Traditionally, patients presenting with uncomplicated dyspepsia have been managed using empiric antisecretory therapy, followed by endoscopy in the event of persistent symptoms or complication . Since Helicobacter pylori is now accepted as an important and potentially reversible cause of ulcer disease, it is important to reevaluate the management of dyspepsia . The goal of this study is to evaluate seven outpatient strategies for the management of dyspeptic patients using a cost-utility analysis . METHODS: The study design was that of a cost-utility analysis . The model assumes that an adult patient with signs of dyspepsia but no signs of complication presents to the outpatient office of a primary care physician . Seven strategies are modeled: empiric antisecretory therapy; empiric H pylori eradication using oral omeprazole (20 mg {corrected} twice daily), clarithromycin (500 mg twice daily), and amoxicillin (1000 mg twice daily); use of either upper endoscopy, an upper gastrointestinal barium study (an upper GI), or the serum titer for H pylori as a diagnostic test to identify patients for H pylori eradication; or use of an initial diagnostic test followed by the serum titer for H pylori . The primary outcome was the cost per quality-adjusted life year (QALY) for each strategy for a 1-year period from presentation; secondary outcomes included the probability of symptomatic ulcer recurrence, cost per ulcer cure, and mortality . RESULTS: Three strategies were similarly cost-effective: empiric H pylori eradication ($1198 per QALY), use of a serum H pylori titer as an initial diagnostic test ($1214 per QALY), and empiric antisecretory therapy ($1288 per QALY) . Empiric antisecretory therapy, however, was associated with significantly more symptomatic ulcer recurrences and deaths than any other strategy . CONCLUSIONS: This cost-utility analysis suggests that two strategies are reasonable for patients presenting with dyspepsia: (1) empiric H pylori eradication and (2) use of a serum H pylori titer to identify patients who might benefit from H pylori eradication . The latter strategy may be preferable because it is less likely to lead to antibiotic resistance . Strategies utilizing an upper GI or upper endoscopy (either with or without serum H pylori titer) or empiric antisecretory therapy do not improve outcomes and are associated with greater cost, morbidity, and/or mortality.

Anal Biochem, 1997 Jun 1, 248(2), 297 - 9
Antibiotic resistance in Streptomyces lividans: fluorescence assay for streptogramin B lyase; Bateman KP et al.; A fluorescence assay for streptogramin B lyase, an enzyme that confers resistance to streptogramin B antibiotics, has been developed . The antibiotic substrates are fluorescent and the linear peptide products formed in the lyase-catalyzed reaction are relatively nonfluorescent . The assay has potential for assessing bacterial resistance to streptogramin B antibiotics and will be utilized to direct the purification of streptogramin B lyase from bacterial extracts.

Nucleic Acids Res, 1997 Jun 1, 25(11), 2205 - 12
Isolating large nested deletions in bacterial and P1 artificial chromosomes by in vivo P1 packaging of products of Cre-catalysed recombination between the endogenous and a transposed loxP site; Chatterjee PK et al.; A general approach for isolating large nested deletions in P1 artificial chromosomes (PACs) and bacterial artificial chromosomes (BACs) by retrofitting with a loxP site-containing Tn10 mini-transposon is described . Cre-mediated recombination between the loxP site existing in these clones and one introduced by transposition leads to deletions and inversions of the DNA between these sites . Large deletions are selectively recovered by transducing the retrofitted PAC or BAC clones with P1 phage . The requirement that both loxP sites in the cointegrate be packaged into a P1 head ensures that only large deletions are rescued . PCR analyses identified these deletions as products of legitimate recombination between loxP sites mediated by Cre protein . BACs produce deletions much more efficiently than PACs although the former cannot be induced to greater than unit copy in cells . Mammalian cell-responsive antibiotic resistance markers are introduced as part of the transposon into genomic clone deletions for subsequent functional analysis . Most importantly, the loxP site retrofitting and P1 transduction can be performed in the same bacterial host containing these clones directly isolated from PAC or BAC libraries . These procedures should facilitate physical and functional mapping of genes and regulatory elements in these large plasmids.

Gene, 1997 May 6, 190(2), 315 - 7
Antibiotic resistance gene cassettes derived from the omega interposon for use in E . coli and Streptomyces; Blondelet-Rouault MH et al.; Three antibiotic resistance gene cassettes, derived from the omega interposon (Prentki and Krisch (1984) Gene 29, 303-313) were constructed . These cassettes carry different antibiotic resistance genes, conferring resistance to geneticin, hygromycin or viomycin, flanked by short inverted repeats containing transcription and translation termination signals and synthetic polylinkers . These cassettes were designated omega aac, omega hyg and omega vph . Resistance phenotypes conferred by these constructions are selectable in E . coli and Streptomyces . These cassettes can be used for insertional mutagenesis or for vector construction.

Nippon Rinsho, 1997 May, 55(5), 1167 - 72
{Acquisition of antibiotic-resistance in bacteria by alteration of molecular target, or by the decreased permeability}; Hashimoto H; The target of an antibiotic is any essential enzyme for bacterial growth and quantitative or qualitative alteration of the enzyme or its substrate may cause resistance to the drug . Thus, the alteration of DNA gyrase and/or topoisomerase IV may result in resistance to fluoloquinolones, and alteration of beta-subunit of RNA polymerase may cause rifampicin-resistance . The cell wall synthesizing enzymes which cross-bridge peptidoglycan units are called PBPs and their alteration cause resistance to beta-lactams, and the alteration of the substrate D ala-D ala to D ala-D lactate causes vancomycin-resistance . In other drug-resistances, various instances of alteration of enzymes are known . Drug-resistances are also brought about by decreased permeability of the drug . Some instances are described.

J Biol Chem, 1997 Apr 25, 272(17), 11057 - 62
Reconstitution of mitogen-activated protein kinase phosphorylation cascades in bacteria . Efficient synthesis of active protein kinases; Khokhlatchev A et al.; Mitogen-activated protein (MAP) kinase pathways include a three-kinase cascade terminating in a MAP kinase family member . The middle kinase in the cascade is a MAP/extracellular signal-regulated kinase (ERK) kinase or MEK family member and is highly specific for its MAP kinase target . The first kinase in the cascade, a MEK kinase (MEKK), is characterized by its ability to activate one or more MEK family members . A two-plasmid bacterial expression system was employed to express active forms of the following MEK and MAP kinase family members: ERK1, ERK2, alpha-SAPK, and p38 and their upstream activators, MEK1, -2, -3, and -4 . In each kinase module, the upstream activator, a constitutively active mutant of MEK1 or MEKK1, was expressed from a low copy plasmid, while one or two downstream effector kinases were expressed from a high copy plasmid with different antibiotic resistance genes and origins of replication . Consistent with their high activity, ERK1 and ERK2 were doubly phosphorylated on Tyr and Thr, were recognized by an antibody specific to the doubly phosphorylated forms, and were inactivated by either phosphoprotein phosphatase 2A or phosphotyrosine phosphatase type 1 . Likewise, activated p38 and alpha-stress-activated protein kinase could also be inactivated by either phosphatase, and alpha-stress-activated protein kinase was recognized by an antibody specific to the doubly phosphorylated forms . These three purified, active MAP kinases have specific activities in the range of 0.6-2.3 micromol/min/mg . Coexpression of protein kinases with their substrates in bacteria is of great value in the preparation of numerous phosphoproteins, heretofore not possible in procaryotic expression systems.

Nucleic Acids Res, 1997 Apr 15, 25(8), 1469 - 75
Cysteine-to-alanine replacements in the Escherichia coli SoxR protein and the role of the {2Fe-2S} centers in transcriptional activation; Bradley TM et al.; The Escherichia coli soxRS regulon activates oxidative stress and antibiotic resistance genes in two transcriptional stages . SoxR protein becomes activated in cells exposed to excess superoxide or nitric oxide and then stimulates transcription of the soxS gene, whose product in turn activates>/=10 regulon promoters . Purified SoxR protein is a homodimer containing a pair of {2Fe-2S} centers essential for soxS transcription in vitro . The {2Fe-2S} centers are thought to be anchored by a C-terminal cluster of four cysteine residues in SoxR . Here we analyze mutant SoxR derivatives with individual cysteines replaced by alanine residues (Cys-->Ala) . The mutant proteins in cell-free extracts bound the soxS promoter with wild-type affinity, but upon purification lacked Fe or detectable transcriptional activity for soxS in vitro . Electron paramagnetic resonance measurements in vivo indicated that the Cys-->Ala proteins lacked the {2Fe-2S} centers seen for wild-type SoxR . The Cys-->Ala mutant proteins failed to activate soxS expression in vivo in response to paraquat, a superoxide- generating agent . However, when expressed to approximately 5% of the cell protein, the Cys-->Ala derivatives increased basal soxS transcription 2-4-fold . Overexpression of the Cys119-->Ala mutant protein strongly interfered with soxS activation by wild-type SoxR in response to paraquat . These studies demonstrate the essential role of the {2Fe-2S} centers for SoxR activation in vivo ; the data may also indicate oxidant-independent mechanisms of transcriptional activation by SoxR.

Mol Biotechnol, 1997 Apr, 7(2), 181 - 8
Site-directed mutagenesis using positive antibiotic selection; Bohnsack RN; Various mutagenesis protocols have been established that use the hybridization of a mismatched oligonucleotide to prime DNA synthesis on an M13 phagemid template . For efficient mutagenesis, all of these methods require a means to select for the mutant strand before or during amplification in an Escherichia coli host . In the Altered Sites II protocol, the mismatched oligonucleotide and an oligonucleotide that restores antibiotic resistance to the phagemid are simultaneously hybridized to the template and coupled by DNA synthesis and ligation . The restored antibiotic resistance is then used to select only those phagemids which incorporate the antibiotic repair oligonucleotide . Generally, between 60 and 90% of the phagemids recovered will incorporate both oligonucleotides . This method provides a simple an efficient technique for introducing specific mutations into DNA.

Appl Environ Microbiol, 1997 Apr, 63(4), 1288 - 97
Molecular detection of streptomycin-producing streptomycetes in Brazilian soils; Huddleston AS et al.; Actinomycetes were isolated from soybean rhizosphere soil collected as two field sites in Brazil . All the isolates were identified as Streptomyces species and were screened for streptomycin production and the presence of two genes, strA and strB1, known to be involved in streptomycin biosynthesis in Streptomyces griseus . Antibiotic resistance profiles were determined for 53 isolates from cultivated and uncultivated sites, and approximately half the strains were streptomycin resistance . Clustering by the unweighted pair group method with averages indicated the presence of two major clusters, with the majority of resistant strains from cultivated sites being placed in cluster 1 . Only representatives from this cluster contained strA . Streptomycetes containing strA and strB1 were phenotypically diverse, and only half could be assigned to known species . Sequence comparison of 16S rRNA and trpBA (tryptophan synthetase) genes revealed that streptomycin- producing streptomycetes were phylogenetically diverse . It appeared that a population of streptomycetes had colonized the rhizosphere and that a proportion of these were capable of streptomycin production.

Genetics, 1997 Apr, 145(4), 935 - 43
A dominant mutation in the Chlamydomonas reinhardtii nuclear gene SIM30 suppresses translational defects caused by initiation codon mutations in chloroplast genes; Chen X et al.; A suppressor of a translation initiation defect caused by an AUG to AUU mutation in the Chlamydomonas reinhardtii chloroplast petD gene was isolated, defining a nuclear locus that we have named SIM30 . A dominant mutant allele at this locus, sim30-1d, was found to increase the translation initiation rate of the mutant petD mRNA . sim30-1d was also able to suppress the translational defect caused by an AUG to AUC mutation in the petD gene, and an AUG to AUU mutation in the chloroplast petA gene . We therefore suggest that the SIM30 gene may encode a general chloroplast translation factor . The ability of sim30-1d to suppress the petD AUG to AUU mutation is diminished in the presence of one or more antibiotic resistance markers located within the 16S and 23S rRNAs, suggesting that the activity of the sim30-1d gene product in translation initiation may involve interaction with ribosomal subunits.

Gene, 1997 Mar 18, 187(2), 231 - 8
A method for construction of E . coli strains with multiple DNA insertions in the chromosome; Peredelchuk MY et al.; A system for construction of E . coli strains with multiple DNA insertions in the chromosome, based on elements of modules for site specific recombination of Tn1545 and phage lambda, has been developed . Circular non-replicating DNA fragments containing the transposon attachment site (attTn), an excisable cassette with a selectable marker, and a gene of interest integrate randomly into the chromosome of a host E . coli strain when provided with transposon integrase, Int-Tn (the host strain was obtained by insertion of the fragment containing transposon int-Tn gene coding for Int-Tn into the chromosome) . Integration of these fragments into the chromosome of int-Tn+ cells gives rise to a collection of antibiotic-resistant clones with single insertions at different locations in the chromosome . These insertions are transferred subsequently by P1 transduction into one strain and selected for antibiotic resistance provided by the cassette with the selectable marker . After transduction of each copy, a helper plasmid bearing phage lambda xis and int genes is introduced into the cells to excise the drug resistance gene flanked with the lambda attL and lambda attR sites from the chromosome . Cells cured of the helper plasmid can undergo the next cycle of P1 transduction/drug resistance gene excision . Each cycle adds another chromosomal copy of the foreign gene . To show the utility of the system, we constructed an E . coli strain bearing several chromosomal copies of lacZ at different locations.

EMBO J, 1997 Mar 3, 16(5), 1056 - 65
Spacing of promoter elements regulates the basal expression of the soxS gene and converts SoxR from a transcriptional activator into a repressor; Hidalgo E et al.; SoxR protein of Escherichia coli governs a global response against superoxide-generating agents (such as paraquat) or nitric oxide, and provides broad antibiotic resistance . A redox signal activates SoxR post-translationally to trigger transcription of a second regulatory gene, soxS . Activated and non-activated SoxR bind the soxS promoter with the same high affinity, but only the activated protein stimulates soxS transcription . SoxR acts by an unusual mechanism of positive control: the protein binds the soxS promoter between near-consensus -10 and -35 elements that are separated by an unusually long 19 bp (versus the optimal 17 bp) . We have constructed and analyzed site-specific deletions that alter the promoter element spacing . Reducing the spacer length to 16-18 bp dramatically elevated basal expression of soxS in vivo and in vitro, and nearly eliminated additional activation by SoxR in response to paraquat . More strikingly, shortening the spacer converted SoxR from an activator into a repressor regardless of paraquat treatment . Gel mobility-shift assays show that repression by SoxR of the promoters with 17 and 16 bp spacers is due to interference with binding by RNA polymerase . Thus, activated SoxR remodels the unusual configuration of the wild-type soxS promoter into a highly active form, probably by compensating for the suboptimal distance between the -10 and the -35 elements.

Trends Biotechnol, 1997 Mar, 15(3), 106 - 9
Vaccine strategies: selective elicitation of cellular or humoral immunity?
Shearer GM, Clerici M.
The discovery of immunoregulatory cytokines and the fact that they modulate the cellular and humoral arms of the immune system, generally in opposing directions raises fundamental questions concerning vaccine development . Because antibiotic-resistance infectious organisms are appearing at an increasingly rapid rate, more emphasis will need to be placed on prevention of infection/disease via immunization and less on post-infection antibiotics . To accomplish this task effectively, immune regulation should be integrated into vaccine design . Here we consider the opposing potentials of cytokine-regulated cellular and humoral immunity, and question whether the "best of both worlds' is possible or desirable.

Genetics, 1997 Mar, 145(3), 807 - 14
Fitness consequences of genetically engineered herbicide and antibiotic resistance in Arabidopsis thaliana; Purrington CB et al.; Researchers have often invoked the concept of metabolic drain to explain the lower growth rates of bacteria containing plasmids that confer antibiotic resistance . This idea posits that the energetic input needed to produce detoxifying enzymes diverts resources from clonal reproduction . In this paper we examine whether the concept of metabolic drain can be applied successfully to plants that differ from bacteria in several key aspects including their relative genome size and reproductive rate . We have conducted a field experiment using mutant and transgenic Arabidopsis thaliana that allows the comparison of genotypes differing by a single gene conferring resistance to either the herbicide chlorsulfuron or the antibiotic kanamycin . In addition to testing whether these traits reduce fitness, this experiment was conducted at two levels of resource availability to examine whether costs of resistance are sensitive to environmental quality . We found that herbicide-resistant individuals produced 26% fewer seeds than susceptible counterparts . However, contrasting published results in bacterial systems, the fecundity of individuals was completely unaffected by the expression of an introduced antibiotic resistance gene . The fitness cost associated with chlorsulfuron resistance was greater in nutrient-poor conditions relative to nutrient-rich conditions for comparisons involving mutant, but not transgenic, genotypes.

Mol Cell Biol, 1997 Feb, 17(2), 857 - 61
Positive selection of FLP-mediated unequal sister chromatid exchange products in mammalian cells; Aladjem MI et al.; Site-specific recombination provides a powerful tool for studying gene function at predetermined chromosomal sites . Here we describe the use of a blasticidin resistance system to select for recombination in mammalian cells using the yeast enzyme FLP . The vector is designed so that site-specific recombination reconstructs the antibiotic resistance marker within the sequences flanked by the FLP target sites . This approach allows the detection of DNA excised by FLP-mediated recombination and facilitates the recovery of recombination products that would not be detected by available screening strategies . We used this system to show that the molecules excised by intrachromosomal recombination between tandem FLP recombinase target sites do not reintegrate into the host genome at detectable frequencies . We further applied the direct selection approach to recover a rare FLP-mediated recombination event displaying the characteristics of an unequal sister chromatid exchange between FLP target sites . Implications of this approach for the generation of duplications to assess their effect on gene dosage and chromosome stability are discussed.

Nucleic Acids Res, 1997 Jan 15, 25(2), 447 - 8
Alanine-stretch scanning mutagenesis: a simple and efficient method to probe protein structure and function; Lefevre F et al.; We have developed a foolproof method to substitute a stretch of residues by alanines . After the introduction of aPstI site by IPCR, thus creating two alanine codons, additional codons are introduced at this site through the use of an 'alanine-stretch cartridge' . These cartridges comprise an antibiotic resistance gene flanked on both sides by alanine codons . Excision of the resistance gene byPvuII then yields the correct insertion of codons . The method is both highly reliable and flexible and should be of general use.

Curr Opin Ophthalmol, 1997 Feb, 8(1), 13 - 7
Anesthesia and preoperative and postoperative medications; Fichman RA; Retrobulbar blocks, although widely used, still have potentially serious complications . Topical anesthesia presents less risk of injury to the globe and less pain but requires careful usage and an experienced surgeon . New techniques, however, allow for an increase in the percentage of patients able to have topical anesthesia . Preoperatively, 2.5% phenylephrine is found to be just as effective as 10% phenylephrine, and, when compared with wound closure and surgeon's experience, the effect of prophylactic medications was found to be negated . Postoperatively, diclofenac is found to be as effective an anti-inflammatory agent as prednisolone . Also, the addition of 10% phenylephrine to 4% pilocarpine drops enhances the effectiveness of pharmacologic treatment of postoperative iridocorneal adhesions . In addition, ophthalmologists should be aware of emerging antibiotic resistance.

SAAS Bull Biochem Biotechnol, 1997, 10, 1 - 6
Construction of a psb C deletion strain in Synechocystis 6803; Goldfarb N et al.; Synechocystis 6803 is a cyanobacterium that carries out-oxygenic photosynthesis . We are interested in the introduction of mutations in the large extrinsic loop region of the CP43 protein of Photosystem II (PSII) . CP43 appears to be required for the stable assembly of the PSII complex and also appears to play a role in photosynthetic oxygen evolution . Deletion of short segments of the large extrinsic loop results in mutants incapable of evolving oxygen . Alterations in psbC, the gene encoding CP43, are introduced into Synechocystis 6803 by transformation and homologous recombination . Specifically, plasmid constructs bearing the site-directed mutations are introduced into a deletion strain where the portion of the gene encoding the area of mutation has been deleted and replaced by a gene conferring antibiotic resistance . We have constructed a deletion strain of Synechocystis appropriate for the introduction of mutations in the large extrinsic loop of CP43 and have used it successfully to produce site-directed mutants.

Ciba Found Symp, 1997, 207, 15 - 27; discussion 27-35
Origins, acquisition and dissemination of antibiotic resistance determinants; Davies JE; Since the introduction of antibiotics in the late 1940s there has been an inexorable propagation of antibiotic resistance genes in bacterial pathogens (and their relatives) . This survival phenomenon was first characterized as the appearance of point mutations that altered drug targets, but in the mid-1950s transmissible antibiotic resistance genes were reported in Japan . Since this time both resistance strategies have been used, often in concert . For some types of antibiotic, only resistance by mutation has been identified, for others only resistance by plasmid acquisition . There is conflicting evidence with respect to the presence of antibiotic resistance in bacterial pathogens in the 'pre-antibiotic' era; however, it is likely that the evolution of antibiotic resistance occurred over short periods . Thus, antibiotic resistance gene must be common in the environment, but their derivation remains to be established conclusively . This paper examines the proposals that antibiotic resistance genes originated in the bacterial population, either as bona fide resistance genes or genes encoding metabolic functions . In addition, the acquisition of heterologous resistance determinants by different genetic elements, their intergeneric exchange mechanisms, and the possible roles of antibiotics in the processes are discussed . Are there prospects for drug intervention that eliminate or retard these natural evolutionary processes?

Ciba Found Symp, 1997, 207, 1 - 9; discussion 9-14
Antibiotic resistance: an ecological imbalance; Levy SB; Antibiotic resistance thwarts the treatment of infectious diseases worldwide . Although a number of factors can be identified which contribute to the problem, clearly the antibiotic as a selective agent and the resistance gene as the vehicle of resistance are the two most important, making up a 'drug resistance equation' . Both are needed in order for a clinical problem to arise . Given sufficient time and quantity of antibiotic, drug resistance will eventually appear . But a public health problem is not inevitable if the two components of the drug resistance equation are kept in check . Enhancing the emergence of resistance is the case by which resistance determinants and resistant bacteria can spread locally and globally, selected by widespread use of the same antibiotics in people, animal husbandry and agriculture . Antibiotics are societal drugs . Each individual use contributes to the sum total of society's antibiotic exposure . In a broader sense, the resistance problem is ecological . In the framework of natural competition between susceptible and resistant bacteria, antibiotic use has encouraged growth of the resistant strains, leading to an imbalance in prior relationships between susceptible and resistant bacteria . To restore efficacy to earlier antibiotics and to maintain the success of new antibiotics that are introduced, we need to use antibiotics in a way which assures an ecological balance that favours the predominance of susceptible bacterial flora.

Annu Rev Neurosci, 1997, 20, 125 - 56
The role of vesicular transport proteins in synaptic transmission and neural degeneration; Liu Y et al.; Classical neurotransmitters are synthesized in the cytoplasm, so they require transport into secretory vesicles for regulated exocytotic release . Previous work has identified distinct vesicular transport activities for the different classical transmitters, and all depend on the H+-electrochemical gradient across the vesicle membrane but differ in the extent to which they rely on the chemical and electrical components of this gradient . Drugs that interfere with vesicular amine transport have implicated this activity in psychiatric disease . Selection for a cDNA encoding vesicular amine transport in the neurotoxin MPP+ also implicates the activity in Parkinson's disease . Molecular cloning of vesicular monoamine transporters shows sequence similarity to bacterial antibiotic resistance proteins, supporting a role for transport in detoxification and defining a novel mammalian gene family that now also includes a transporter for acetylcholine . Current work focuses on the mechanism of transport and the role that regulation of activity and its subcellular localization have in transmitter release, behavior, and neural degeneration.

Dermatol Clin, 1997 Jan, 15(1), 97 - 109
Acne . An overview of clinical research findings; Thiboutot DM; Although acne represents the most common chronic skin condition seen by dermatologists, there are still many unanswered questions regarding its pathophysiology, and patients are still in need of more effective therapies, particularly those aimed at the hormonal aspects of acne . Recent clinical research has led to advances in our understanding of factors such as cytokines in follicular hyperkeratinization and the role of androgens in acne, the emergence and significance of antibiotic resistance of P . acnes, the long-term safety and efficacy of isotretinoin, and the safety and efficacy of new topical retinoids, such as tazarotene and adapalene . Fruitful interactions between basic scientists and clinical researchers within medicine and the pharmaceutical industry will, it is hoped, provide for future advances in this area.

Clin Infect Dis, 1997 Jan, 24 Suppl 1, S151 - 3
Antibiotic resistance: a view from the pharmaceutical industry; Bax RP; The development of new antibiotics has been successful in significantly reducing morbidity and mortality . With increasing use there has occurred an increase in antibiotic resistance but not a parallel increase in new agents with significantly improved spectrum of activity . Without concerted action from the pharmaceutical industry, physicians, academia, health care providers, and governments, the prospects look gloomy.

Clin Infect Dis, 1997 Jan, 24 Suppl 1, S146 - 7
Perspectives on chemotherapeutic approaches to antibiotic-resistant bacteria; Kessler RE; This paper presents an overview of the potential chemotherapeutic approaches to antibiotic resistance, what those approaches have achieved, and future avenues of exploration . Chemical modifications of existing agent classes, interference with resistance mechanisms, searches for inhibitors of novel targets, and alternatives to chemotherapy with low-molecular-weight molecules are discussed . The research focusing on novel targets and on alternative approaches is most likely to yield breakthroughs against problem organisms in the future.

Appl Environ Microbiol, 1997 Jan, 63(1), 254 - 62
Isolation and molecular identification of planctomycete bacteria from postlarvae of the giant tiger prawn, Penaeus monodon; Fuerst JA et al.; Bacteria phenotypically resembling members of the phylogenetically distinct planctomycete group of the domain Bacteria were isolated from postlarvae of the giant tiger prawn, Penaeus monodon . A selective medium designed in the light of planctomycete antibiotic resistance characteristics was used for this isolation . Planctomycetes were isolated from both healthy and monodon baculovirus-infected prawn postlarvae . The predominant colony type recovered from postlarvae regardless of viral infection status was nonpigmented . Other, less commonly observed types were pink or orange pigmented . A planctomycete-specific 16S rRNA-directed probe was designed and used to screen the isolates for their identity as planctomycetes prior to molecular phylogenetic characterization . 16S rRNA genes from nine prawn isolates together with two planctomycete reference strains (Planctomyces brasiliensis and Gemmata obscuriglobus) were sequenced and compared with reference sequences from the planctomycetes and other members of the domain Bacteria . Phylogenetic analyses and sequence signatures of the 16S rRNA genes demonstrated that the prawn isolates were members of the planctomycete group . Five representatives of the predominant nonpigmented colony type were members of the Pirellula group within the planctomycetes, as were three pink-pigmented colony type representatives . Homology values and tree topology indicated that representatives of the nonpigmented and pink-pigmented colony types formed two discrete clusters within the Pirellula group, not identical to any known Pirellula species . A sole representative of the orange colony type was a member of the Planctomyces group, virtually identical in 16S rDNA sequence to P . brasiliensis, and exhibited distinctive morphology.

J Biol Chem, 1996 Dec 27, 271(52), 33173 - 5
The redox state of the {2Fe-2S} clusters in SoxR protein regulates its activity as a transcription factor; Ding H et al.; SoxR protein is a redox-responsive transcription factor that governs a regulon of oxidative stress and antibiotic resistance genes in Escherichia coli . Purified SoxR contains oxidized {2Fe-2S} clusters and stimulates in vitro transcription of its target gene soxS up to 100-fold . SoxR transcriptional activity, but not DNA binding, is completely dependent on the {2Fe-2S} clusters; apo-SoxR prepared in vitro binds the soxS promoter with unchanged affinity but does not have transcription activity . Thus, modulation of the SoxR {2Fe-2S} clusters was proposed to control the protein's function in transcription . Here, we provide evidence that SoxR with reduced {2Fe-2S} clusters is inactive . Redox titration of purified SoxR revealed a midpoint potential of -285 +/- 10 mV (pH 7.6) . In vitro transcription assays showed that SoxR was inactivated when the {2Fe-2S} cluster was reduced (-380 mV), and full activity was restored upon reoxidation (+100 mV) . The results suggest that one-electron oxidation and reduction of the {2Fe-2S} cluster regulate SoxR transcriptional activity.

Health Syst Rev, 1997 Jan-Feb, 30(1), 20 - 4
Resistance movement: the antibiotic crisis in hospitals; Goldmann DA; The near-miraculous healing power of antibiotic is being undermined by the rapid evolution of drug-resistant bacteria . The costs, in human and economic terms, could be staggering for all caregivers and patients, but especially for institutional providers like hospitals . In an article adapted from the Journal of the American Medical Association, Dr . Goldmann explains why antibiotic resistance is a crisis and how the danger can be mitigated.

EMBO J, 1996 Dec 2, 15(23), 6416 - 25
Targeted mutagenesis of acyl-lipid desaturases in Synechocystis: evidence for the important roles of polyunsaturated membrane lipids in growth, respiration and photosynthesis; Tasaka Y et al.; Acyl-lipid desaturases introduce double bonds (unsaturated bonds) at specifically defined positions in fatty acids that are esterified to the glycerol backbone of membrane glycerolipids . The desA, desB and desD genes of Synechocystis sp . PCC 6803 encode acyl-lipid desaturases that introduce double bonds at the delta12, omega3 and delta6 positions of C18 fatty acids respectively . The mutation of each of these genes by insertion of an antibiotic resistance gene cartridge completely eliminated the corresponding desaturation reaction . This system allowed us to manipulate the number of unsaturated bonds in membrane glycerolipids in this organism in a step-wise manner . Comparisons of the variously mutated cells revealed that the replacement of all polyunsaturated fatty acids by a monounsaturated fatty acid suppressed growth of the cells at low temperature and, moreover, it decreased the tolerance of the cells to photoinhibition of photosynthesis at low temperature by suppressing recovery of the photosystem II protein complex from photoinhibitory damage . However, the replacement of tri- and tetraunsaturated fatty acids by a diunsaturated fatty acid did not have such effects . These findings indicate that polyunsaturated fatty acids are important in protecting the photosynthetic machinery from photoinhibition at low temperatures.

Br J Biomed Sci, 1996 Dec, 53(4), 290 - 3
Drug efflux as a mechanism of resistance; Williams JB; A review of efflux as a mechanism of antibiotic resistance in both bacterial and eukaryotic cells is presented . This covers membrane transport, the evolution of efflux transporters, their genetic control and classification into families, and an appreciation of the widespread distribution and occurrence of these mechanisms in nature.

J Intern Med, 1996 Dec, 240(6), 319 - 32
Helicobacter pylori--can the mechanisms of pathogenesis guide us towards novel strategies for treatment and prevention?
Falk P.
Helicobacter pylori establishes a chronic infection in the stomach of humans . The infection is associated with a low grade inflammatory response in the epithelium that can develop into chronic active gastritis, peptic ulcer disease or neoplasia . Antibiotics have dramatically decreased the rate of recurrence of peptic ulcers . However, antibiotic resistance is already evident, casting doubts on the future efficacy of these strategies . The link between childhood infection and severe health problems, including increased risk for gastric tumours motivate efforts to develop vaccines . Characterization of the molecular mechanisms of pathogenesis will pave the way for novel strategies for treatment and prevention of H . pylori infection.

Vet Microbiol, 1996 Dec, 53(3-4), 349 - 54
Serogroups, toxins and antibiotic resistance of Escherichia coli strains isolated form diarrhoeic goat kids in Spain; Cid D et al.; Fifty-five Escherichia coli strains isolated from 55 diarrhoeic goat kids from 13 flocks in Spain were serotyped and investigated for production of enterotoxins (LT and STa), verotoxins (VT1 and VT2), cytotoxic necrotizing factors (CNF1 and CNF2), alpha-hemolysin (Hly) and enterohemolysin (EntHly), and for antibiotic resistance . Only 3 (5%) strains were toxigenic: 1 VT1+EntHly+ (serogroup O8) and 2 CNF2+ (both of serogroup O153) . The strains serotyped belonged to 19 serogroups . However, 31 (56%) were of one nine serogroups (O3, O8, O9, O10, O11, O21, O44, O103 and O153) and only three of them (O8, O9 and O11) accounted for 29% of the strains . The highest percentages of antibiotic resistance in order of frequency were: streptomycin (93%), sulfadiazine (89%), tetracyline (84%), kanamycin (82%), neomycin (82%) and ampicillin (69%) . We conclude that E . coli strains isolated from diarrhoeic goat kids are usually non-toxigenic and belong to a large number of serogroups.

Mil Med, 1996 Dec, 161(12), 728 - 31
The enemy within: diarrheal rates among British and Australian troops in Iraq; Rudland S et al.; British and Australian medical teams working in Northern Iraq in 1991 providing primary care to refugees and the war wounded were subjected to a descriptive retrospective survey, 5 weeks after arriving in Iraq . The aim was to document different rates of diarrhea in British and Australian troops . The British, who were not taking daily doxycycline and did not enforce a plate- and hand-washing routine, experienced higher rates of diarrhea (69% of British troops compared with 36% of Australian troops), which was more severe and of a longer duration (p < 0.001) and resulted in twice as many days being lost (p < 0.001) in spite of the British team being half the size of the Australian contingent, and the region having enteropathogens with a high rate of antibiotic resistance . Vigorous hand- and plate-washing routines along with doxycycline prophylaxis appear to significantly reduce incapacitation from diarrhea in this military setting and have an important implication for operational effectiveness.

Can J Microbiol, 1996 Dec, 42(12), 1274 - 6
Tn5431 arose by transposition of Tn3 into Tn1721; Zgur-Bertok D et al.; To further characterize Tn5431, which is composed of Tn3 and Tn1721, DNA sequencing was carried out . It was demonstrated that Tn5431 arose by transposition of Tn3 into Tn1721 . Multiple mutations in the Tn3 tnpA gene have occurred, rendering the Tn3 portion of Tn5431 transposition defective and ensuring the simultaneous transposition of the antibiotic resistance determinants on Tn3 and Tn1721.

Biochim Biophys Acta, 1996 Nov 11, 1309(1-2), 156 - 66
Mutations in yeast ribosomal proteins S28 and S4 affect the accuracy of translation and alter the sensitivity of the ribosomes to paromomycin; Synetos D et al.; Ribosomal proteins S12, S5 and S4 of Escherichia coli are essential for the control of translational accuracy . Their yeast equivalents, i.e., S28, S4 and S13, have also been implicated in this process . Using a poly(U)-dependent cell-free translation system, we determined the accuracy of translation and the sensitivity to antibiotic paromomycin of yeast ribosomes carrying mutant ribosomal proteins S28 and/or S4 . Our results confirm by quantitative biochemical methods previous genetic data showing that proteins S28 and S4 are involved in the decoding activity of the ribosome and interact to control translational accuracy . We find that the suppressor mutation SUP44 in yeast S4, decreased the accuracy of translation . To examine the effect of mutant S28, we disrupted RPS28B and introduced in RPS28A the same substitutions that cause hyperaccurate translation or antibiotic resistance in bacteria . Three of these substitutions (Lys-62-->Asn, Thr or Gln) similarly increased translational accuracy in vitro or antibiotic resistance . In the presence of the SUP44 mutation, these substitutions partially reversed the decrease of translational accuracy caused by SUP44 . However, the Lys-62-->Arg substitution decreased translational accuracy and caused antibiotic sensitivity both in nonsuppressor and in SUP44 haploids . These results establish the role of Lys-62 of S28 in optimizing translational accuracy and provide a more precise view of the functional role of two important ribosomal proteins.

Br J Hosp Med, 1996 Nov 6-19, 56(9), 481 - 5
Oral antibiotics for common infections in children: for and against; Payne D et al.; There are very real fears that the antibiotic era may soon be over . Increased use of antibiotics is a major factor in the widespread emergence of antibiotic resistance, which now exists in most of the common bacterial pathogens affecting children . Health-care workers must become aware that the implications are very serious and learn to use these valuable drugs with care.

Gene, 1996 Oct 17, 176(1-2), 23 - 6
Construction of a family of Mycobacterium/Escherichia coli shuttle vectors derived from pAL5000 and pACYC184: their use for cloning an antibiotic-resistance gene from Mycobacterium fortuitum; Ainsa JA et al.; The pSUM vectors, a family of Mycobacterium/Escherichia coli shuttle cloning vectors derived from the pSU series of vectors are described . They all contain the pACYC184 origin of replication enabling them to replicate in E . coli, the mycobacterial pAL5000 origin of replication and a kanamycin-resistance (KmR) gene which allows selection in both E . coli and mycobacteria . They carry a multiple cloning site (MCS) to facilitate cloning and the lacZ alpha reporter gene for screening inserts in E . coli . A library of total DNA from a strain of Mycobacterium fortuitum was constructed with one of these vectors, pSUM36, and a mycobacterial gentamicin-resistance (GmR) gene was cloned by selecting for GmR in Mycobacterium smegmatis.

Rev Prat, 1996 Oct 15, 46(16), 1940 - 7
{Epidemiology of sexually transmitted diseases, with the exception of AIDS}; Warszawski J et al.; Epidemiologic trends in sexually transmitted diseases strongly vary in the world . The situation is worrying in the developing countries . Both the incidences and prevalences are very high antibiotic resistance is expanding and interactions between sexually transmitted diseases and human immunodeficiency virus contribute to AIDS epidemic . In western Europe, the decline of some sexually transmitted diseases is likely to be linked with successful control programmes and gonorrhea and syphilis now are rare diseases . The USA situation is contrasted . Some inner-city minority population still have high level of gonorrhea and are concerned with resurgence of syphilis and chancroid . Chlamydia trachomatis infections, which are a major, cause of infertility and ectopic pregnancy, have become the most prevalent sexually transmitted disease in industrialized world.

Curr Biol, 1996 Oct 1, 6(10), 1219 - 21
Antibiotic resistance: counting the cost; Spratt BG; Acquisition of drug resistance should impose a cost on bacteria . Recent studies, however, suggest that natural selection acts to reduce, or eliminate, the growth disadvantage of resistant bacteria, making it difficult to reverse the high levels of antibiotic resistance currently found in hospitals and the community.

Chest, 1996 Oct, 110(4), 1025 - 34
Diagnosis and treatment of ventilator-associated pneumonia--impact on survival . A decision analysis; Sterling TR et al.; STUDY OBJECTIVE: To determine the impact of antibiotic treatment of ventilator-associated pneumonia (VAP) on survival . DESIGN: Decision analysis . PATIENTS: A hypothetical cohort of immunocompetent patients receiving mechanical ventilation who have suspected bacterial pneumonia . The analysis was performed separately for the following diagnostic techniques: clinical criteria, bronchoscopic protected specimen brush (PSB), and nonbronchoscopic protected BAL (pBAL) . Additional factors accounted for in the analysis included the presence or absence of prior antibiotic use, mortality of antibiotic-treated and untreated pneumonia, mortality attributable to VAP, development of antibiotic resistance, and mortality due to adverse drug reactions . MEASUREMENTS AND RESULTS: The overall survival of patients who receive antibiotic therapy was compared to survival if antibiotic therapy had been withheld . Antibiotic treatment of clinically diagnosed VAP was associated with lower overall survival than withholding treatment . Antibiotic treatment of VAP diagnosed by invasive (PSB) or semi-invasive (pBAL) techniques was associated with better survival than withholding treatment, although withholding antibiotic therapy was favored as the mortality rate of antibiotic-treated VAP approached 70% . CONCLUSIONS: Invasive or semi-invasive diagnostic techniques should be used to diagnose VAP, guide therapy, and thereby potentially improve survival . A prospective, randomized trial assessing outcome according to diagnostic technique is needed.

J Bacteriol, 1996 Oct, 178(20), 5946 - 53
Directed insertion of a selectable marker into a circular plasmid of Borrelia burgdorferi; Rosa P et al.; Studies of the biology of Borrelia burgdorferi and the pathogenesis of Lyme disease are severely limited by the current lack of genetic tools . As an initial step toward facile genetic manipulation of this pathogenic spirochete, we have investigated gene inactivation by allelic exchange using a mutated borrelial gyrB gene that confers resistance to the antibiotic coumermycin A1 as a selectable marker . We have transformed B . burgdorferi by electroporation with a linear fragment of DNA in which this selectable marker was flanked by sequences from a native borrelial 26-kb circular plasmid . We have identified coumermycin A1-resistant transformants in which gyrB had interrupted the targeted site on the 26-kb plasmid via homologous recombination with the flanking sequences . Antibiotic resistance conferred by the mutated gyrB gene on the plasmid is dominant, and transformed spirochetes carrying this plasmid do not contain any unaltered copies of the plasmid . Coumermycin A1 resistance can be transferred to naive B . burgdorferi by transformation with borrelial plasmid DNA from the initial transformants . This work represents the first example of a directed mutation in B . burgdorferi whereby a large segment of heterologous DNA (gyrB) has been inserted via homologous recombination with flanking sequences, thus demonstrating the feasibility of specific gene inactivation by allelic exchange.

Gene, 1996 Sep 26, 174(1), 103 - 10
Vector for IS element entrapment and functional characterization based on turning on expression of distal promoterless genes; Szeverenyi I et al.; We constructed and characterized a novel trap vector for rapid isolation of insertion sequences . The strategy used for the isolation of IS elements is based on the ability of many IS elements to turn on the expression of otherwise silent genes distal to some sites of insertion . The simple transposition of an IS element can sometimes cause the constitutive expression of promoterless antibiotic resistance genes resulting in selectable phenotypes . The trap vector pAW1326 is based on a pBR322 replicon, it carries ampicillin and streptomycin resistance genes, and also silenced genes that confer chloramphenicol and kanamycin resistance once activated . The trap vector pAW1326 proved to be efficient and 85 percent of all isolated mutations were insertions . The majority of IS elements resident in the studied Escherichia coli strains tested became trapped, namely IS2, IS3, IS5, IS150, IS186 and Tn1000 . We also encountered an insertion sequence, called IS10L/R-2, which is a hybrid of the two IS variants IS10L and IS10R . IS10L/R-2 is absent from most E . coli strains, but it is detectable in some strains such as JM109 which had been submitted to Tn10 mutagenesis . The distribution of the insertion sequences within the trap region was not random . Rather, the integration of chromosomal mobile genetic elements into the offered target sequence occurred in element-specific clusters . This is explained both by the target specificity and by the specific requirements for the activation of gene transcription by the DNA rearrangement . The employed trap vector pAW1326 proved to be useful for the isolation of mobile genetic elements, for a demonstration of their transposition activity as well as for the further characterization of some of the functional parameters of transposition.

Sex Transm Dis, 1996 Sep-Oct, 23(5), 407 - 12
Antibiotic prophylaxis among commercial sex workers in Cebu City, Philippines . Patterns of use and perceptions of efficacy; Abellanosa I et al.; BACKGROUND: This study describes the extent to which commercial sex workers (CSW) in Cebu City, Philippines perceive prophylactic antibiotic use to be an effective form of prevention for sexually transmitted diseases (STD) and human immunodeficiency virus (HIV), as well as the prevalence of this self-treatment practice . METHODS: A survey instrument was developed and pretested after 3 months of intensive ethnographic research on STD and acquired immune deficiency syndrome (AIDS) . A multistage sampling procedure was followed to ensure that a representative sample of CSW from four distinct work environments would be interviewed . Commercial sex workers registered at the Cebu City social hygiene clinic were sampled randomly from coded work establishment lists, and a convenience sample of unregistered freelance CSW was secured . In total, 200 CSW were interviewed . Of these 200 CSW, all were sexually active, but only 160 had been engaged actively in commercial sexual exchange the month before their interviews . RESULTS: Popular use of antibiotics as prophylaxis against STD is commonplace in the Philippines among CSW, with 38% reporting routine or occasional use and 31% reporting use in the last 2 weeks . Unregistered CSW are five times more likely to use prophylactic antibiotics than registered CSW, and they are seven times less likely to use condoms with 80% or more of their customers . They also have sex with three times as many customers . CONCLUSIONS: Use of prophylactic antibiotics by CSW offers them a false sense of security in a high-risk work environment . Self-treatment with low-dose prophylactic antibiotics provides no protection against STD, impedes STD screening efforts, and contributes to antibiotic resistance . An alarming percentage of CSW consider antibiotics a potential means of protecting themselves against AIDS . Public health interventions focusing on STD and AIDS in developing countries must address current patterns of prophylactic antibiotic usePIP: Some commercial sex workers (CSWs) believe that self-treatment with low-dose prophylactic antibiotics can protect them from contracting sexually transmitted diseases (STDs), including HIV and AIDS . Such practice, however, provides no protection against STD, impedes STD screening efforts, and contributes to antibiotic resistance . 200 sexually active female CSWs in Cebu City, Philippines, were interviewed to determine the extent to which they believe the prophylactic use of antibiotics can protect them from STD . 38% of the 160 CSWs involved in prostitution during the 2 weeks before the interviews reported routine or occasional use of antibiotics and 31% reported use in the past 2 weeks . CSWs not registered with the Cebu City social hygiene clinic were five times more likely to use prophylactic antibiotics than registered CSWs, seven times less likely to use condoms with 80% or more of their customers, and have sex with three times as many customers .

Biochemistry, 1996 Aug 20, 35(33), 10872 - 8
Biogenesis and topology of integral membrane proteins: characterization of lactose permease-chloramphenicol acetyltransferase hybrids; Zelazny A et al.; Use of beta-lactamase in gene fusions to study membrane protein topology permits exploitation of its biological activity to select for positive (external) hybrids on ampicillin agar plates . When the enzyme is attached to cytoplasmic loops of a membrane protein, it is not secreted and is therefore unable to confer ampicillin resistance . In this study, we examine the use of the cytoplasmic enzyme chloramphenicol acetyltransferase (Cat) as a complement to the use of periplasmic beta-lactamase, in gene fusion studies . This enzyme is responsible for chloramphenicol resistance in Escherichia coli . We show that Cat confers substantial antibiotic resistance when fused to cytoplasmic loops of lactose permease . As expected, periplasmically exposed Cat is enzymatically active in vitro but unable to confer significant chloramphenicol resistance, presumably because of the absence of acetylcoenzyme A in the periplasm . Therefore, Cat may serve as a topogenic sensor in gene fusion studies . The new Cat fusion approach is discussed with regard to its potential use for selecting E . coli mutants which are defective in the assembly of membrane proteins.

Biotechniques, 1996 Aug, 21(2), 255 - 9
Vectors encoding alternative antibiotic resistance for use in the yeast two-hybrid system; Watson MA et al.; We have altered the antibiotic resistance of the reporter plasmids and the pJG4-5 activation-domain and pEG202 DNA binding-domain plasmids used in the Brent interaction trap/two-hybrid system . These plasmids were each previously ampicillin-resistant, resulting in an inefficient purification of any one plasmid from a yeast strain containing all three plasmids that constitute the complete interaction trap . By creating derivatives of each of these plasmids expressing either kanamycin or chloramphenicol resistance, along with the parent plasmids, we now have the option to use the interaction trap in yeast with three E . coli differentially selectable vectors . This will allow isolation of any one plasmid by purifying all of the interaction trap plasmids from yeast simultaneously and plating E . coli transformed with the plasmids onto the appropriate antibiotic plate to select the particular plasmid of interest.

Epidemiol Infect, 1996 Aug, 117(1), 11 - 6
Enteroaggregative Escherichia coli associated with an outbreak of diarrhoea in a neonatal nursery ward; Cobeljic M et al.; Over a 9-day period in February 1995, 16 newborn babies (age range 2-11 days) and 3 infants (24, 47 and 180 days of age) in a neonatal nursery ward developed diarrhoea accompanied by pyrexia and weight loss . Known enteropathogens were not detected in their stools but Escherichia coli displaying aggregative adherence to HEp-2 cells (enteroaggregative E . coli) were found in 12 (63%) ill infants and in none of 5 well neonates (P = 0.02) . The illness lasted 3-9 days (mean 5.2) in 16 babies, whereas in 3 neonates it showed a protracted course of 18-20 days . The source of infection and the mode of transmission remained unclear . The outbreak isolates manifested properties common in this new group of diarrhoeagenic E . coli: mannose-resistant haemagglutination, haemolysis on blood agar, and clump formation in liquid culture medium . They belonged to the O4 E . coli serogroup and expressed multiple antibiotic resistance.

J Bacteriol, 1996 Aug, 178(16), 4965 - 74
Preponderance of Fis-binding sites in the R6K gamma origin and the curious effect of the penicillin resistance marker on replication of this origin in the absence of Fis; Wu F et al.; Fis protein is shown here to bind to 10 sites in the gamma origin of plasmid R6K . The Fis-binding sites overlap all the previously identified binding sites in the gamma origin for the plasmid-encoded pi initiator protein and three host-encoded proteins, DnaA, integration host factor, and RNA polymerase . However, the requirement of Fis for R6K replication depends on the use of copy-up pi-protein variants and, oddly, the antibiotic resistance marker on the plasmid . In Fis-deficient cells, copy-up pi variants cannot drive replication of R6K gamma-origin plasmids carrying the bla gene encoding resistance to penicillin (Penr) but can drive replication of plasmids with the same origin but carrying the chloramphenicol acetyltransferase gene encoding chloramphenicol resistance (Cmr) . In contrast, R6K replication driven by wild-type pi is unaffected by the antibiotic resistance marker in the absence of Fis protein . Individually, none of these elements (copy-up pi, Fis deficiency, or drug markers) prevents R6K replication . The replication defect is not caused by penicillin in the medium or runaway replication and is unaffected by the orientation of the bla gene relative to the origin . Replication remains inhibited when part of the bla coding segment is deleted but the bla promoter is left intact . However, replication is restored by insertion of transcriptional terminators on either side of the gamma origin, suggesting that excess transcription from the bla gene may inactivate replication driven by pi copy-up mutants in the absence of Fis . This study suggests that vector sequences such as drug markers may not be inconsequential in replication studies, as is generally assumed.

Cell Mol Biol (Noisy-le-grand), 1996 Jul, 42(5), 711 - 7
Effect of polyamines on plasmid-mediated kanamycin resistance and kanamycin phosphotransferase gene expression in Escherichia coli; Nastri HG et al.; The emergence of kanamycin resistance in a polyamine-deficient mutant of E . coli transformed with a plasmid encoding the kanamycin phosphotransferase gene has been studied . The initial inhibition of growth and protein synthesis caused by the addition of the antibiotic could be reversed earlier in polyamine-supplemented bacteria than in those depleted of the organic bases . Concomitantly, we have observed that the increase of kanamycin phosphotransferase activity evoking the antibiotic resistance was higher in bacteria cultivated in the presence of putrescine . This result seems to depend exclusively on the enhanced capacity of the translation process in bacteria grown with polyamines since the transcription of phosphotransferase gene was higher in cells subjected to polyamine starvation.

Science, 1996 Jun 14, 272(5268), 1659 - 62
Site-directed hydroxyl radical probing of the rRNA neighborhood of ribosomal protein S5; Heilek GM et al.; Cysteine residues were introduced into three different positions distributed on the surface of ribosomal protein S5, to serve as targets for derivatization with an Fe(II)-ethyl-enediaminetetraacetic acid linker . Hydroxyl radicals generated locally from the tethered Fe(II) in intermediate ribonucleoprotein particles or in 30S ribosomal subunits reconstituted from derivatized S5 caused cleavage of the RNA, resulting in characteristically different cleavage patterns for the three different tethering positions . These findings provide constraints for the three-dimensional folding of 16S ribosomal RNA (rRNA) and for the orientation of S5 in the 30S subunit, and they further suggest that antibiotic resistance and accuracy mutations in S5 may involve perturbation of 16S rRNA.

Mol Microbiol, 1996 Jun, 20(5), 1001 - 11
A glutamate/glutamine/aspartate/asparagine transport operon in Rhodobacter capsulatus; Zheng S et al.; A mutant of Rhodobacter capsulatus was identified in which an operon encoding a binding-protein-dependent transporter was interrupted by Tn5 transposition . Cloning and sequence analysis of the wild-type operon revealed a four-gene cluster with similarities to genes encoding periplasmic binding proteins (BztA), integral membrane proteins (BztB and BztC), and ATP-binding proteins (BztD) . To assess the function of this putative binding-protein-dependent transport system, a mutant was constructed in which most of the bztABCD operon was deleted and replaced by an antibiotic-resistance marker . The deletion mutant grew more slowly than the wild type in NH4(+)-free medium supplemented by glutamate, glutamine, aspartate or asparagine; it was resistant to toxic analogues of Glu, Asp, and Asn at concentrations that inhibited growth of the wild type; and it was defective in the uptake of Glu, Gln, and Asp . A complementing plasmid containing the wildtype copy of bztABCD was able to rescue all the mutant phenotypes . Taken together, these results indicate that the proteins encoded by bztABCD are active in the uptake of Glu, Gln, Asp, and Asn . In addition, competition experiments, in which the ability of each of the four amino acids to compete for the transport of one another was examined, demonstrated that all four substrates share at least one component of this transport system.

EMBO J, 1996 May 15, 15(10), 2593 - 603
Mutational analysis of mammalian poly(A) polymerase identifies a region for primer binding and catalytic domain, homologous to the family X polymerases, and to other nucleotidyltransferases; Martin G et al.; We have tested deletion and substitution mutants of bovine poly(A) polymerase, and have identified a small region that overlaps with a nuclear localization signal and binds to the RNA primer . Systematic mutagenesis of carboxylic amino acids led to the identification of three aspartates that are essential for catalysis . Sequence and secondary structure comparisons of regions surrounding these aspartates with sequences of other polymerases revealed a significant homology to the palm structure of DNA polymerase beta, terminal deoxynucleotidyltransferase and DNA polymerase IV of Saccharomyces cerevisiae, all members of the family X of polymerases . This homology extends as far as cca: tRNA nucleotidyltransferase and streptomycin adenylyltransferase, an antibiotic resistance factor.

Pediatr Infect Dis J, 1996 May, 15(5), 431 - 7
Pneumococcal disease among children in a rural area of west Africa; O'Dempsey TJ et al.; BACKGROUND . The pneumococcus is a frequent cause of pneumonia and other serious infections among young children in developing countries . Defining the pattern of pneumococcal infection in these countries is important so that, with the advent of pneumococcal conjugate vaccines, rational vaccination policies can be developed . METHODS . Children younger than 5 years of age who attended clinics in a rural area of The Gambia, West Africa, were screened by assistants during a 2-year period . Children with predefined features suggestive of a diagnosis of pneumonia, meningitis or septicemia were referred to the Medical Research Council Field Station at Basse for investigation . RESULTS . Of 2898 children investigated 103 cases of invasive pneumococcal disease (70 definite and 33 probable) were identified, suggesting that the incidence of this infection in the study community is at least 554/100,000/year in children younger than 1 year of age and 240/100,000/year in those younger than 5 years, rates many times higher than those found in industrialized societies . The mean age of presentation was 15 months; more boys than girls were affected . Cases of pneumonia were encountered 8 times more frequently than those of meningitis . Antibiotic resistance was rarely found and cases of pneumonia, but not meningitis, responded well to treatment . Case-fatality rates in children with pneumonia and meningitis were 1 and 55%, respectively . The most prevalent pneumococcal serotypes were types 6, 14, 19, 1 and 5 . CONCLUSION . About 60% of invasive pneumococcal infection in children in this community could potentially be prevented by a nine-valent pneumococcal conjugate vaccine (types 1, 4, 5, 6B, 9, 14, 18, 19F and 23) given at the ages of 2, 3 and 4 months.

Mt Sinai J Med, 1996 May-Sep, 63(3-4), 159 - 66
The emergence of "emerging diseases": a lesson in holistic epidemiology; Kilbourne ED; The term "emerging diseases" is a loosely defined category of entities comprising resurgent or recurrent old diseases (usually caused by "new" or mutated previously known agents), diseases truly new to man, but caused by preexisting ("old") zoonotic agents, and syndromes newly defined by the discovery of new agents through advances in biotechnology . Identification and solution of these problems depends, first, on recognition of their differences, and then upon tailoring appropriate strategies for their control . Thus, new influenza viruses appear each year to challenge immunity to their antecedents, but evoke the unchanged and centuries old symptom complex of influenza . Tuberculosis, is resurgent because of mycobacterial mutation to antibiotic resistance, immunosuppression by AIDS, and laxity in public health surveillance . Parvovirus B19 and herpesvirus 6 were revealed as cryptic infectors of white blood cells in studies of hepatitis B and AIDS, but since have been shown to be important causes of childhood rashes, aplastic anemia, and neurologic disease . The encroachment of human habitation on wilderness perimeters (ecosystem change) has increased contact with vectors of zoonotic viruses and bacteria, as evidenced by Lyme disease, Ebola virus infection, and the hemorrhagic fevers . The term "holistic epidemiology" embraces all these problems, from the molecular to the macroenvironmental level . Humans, parasites, and their environment will continue their ancient, fluctuating, dynamic relationship in the future, and new diseases will continue to emerge.

J Bacteriol, 1996 May, 178(9), 2507 - 13
Transcriptional activation of promoters of the superoxide and multiple antibiotic resistance regulons by Rob, a binding protein of the Escherichia coli origin of chromosomal replication; Jair KW et al.; The Rob protein, isolated on the basis of its ability to bind to the right arm of the Escherichia coli origin of chromosomal replication, is about 50% identical in amino acid sequence to SoxS and MarA, the direct regulators of the superoxide (soxRS) and multiple antibiotic resistance (mar) regulons, respectively . Having previously demonstrated that SoxS (as a MalE-SoxS fusion protein) and MarA are essentially identical in their abilities to activate in vitro transcription of genes of the sox-mar regulons, we investigated the properties of Rob as a transcriptional activator . We found that Rob (i) activates the transcription of zwf,fpr,fumC, micF, nfo, and sodA, (ii) requires a 21-bp soxbox-marbox-robbox sequence to activate zwf transcription, (iii) protects the soxbox/marbox/robbox from attack by DNase 1, (iv) is ambidextrous, i.e., requires the C-terminal domain of the alpha subunit of RNA polymerase for activation of zwf but not fumC or micF, (v) bends zwf and fumC DNA, and (vi) binds zwf and fumC DNA as a monomer . Since these transcription activation properties of Rob are virtually identical to those of MalE-SoxS and MarA, it appears as if the E . coli genome encodes three genes with the same functional capacity . However, in contrast to SoxS and MarA, whose syntheses are induced by specific environmental stimuli and elicit a clear defense response, Rob is expressed constitutively and its normal function is unknown.

Roum Arch Microbiol Immunol, 1996 Apr-Jun, 55(2), 133 - 43
Antibiotic resistance study of invasive S.pneumoniae; Mihalcu F et al.; 121 invasive pneumococci isolated from meningitis, septicemia, pneumopathies and other clinical infections (osteo-arthritis, peritonitis, sinusitis, otilis, conjunctivitis, plagues) were screened for susceptibility to penicillin G (P), erythromycim (E), chloramphenicol (Cl), tetracycline (T), ceftriaxone (Cro), and sulfametoxazole trimethoprim (SxT) by diffusimetric method, by MICs to P and Cro by agar dilutions and E-test . 48.8% were penicillin resistant (MICs > 0.12 microgram/ml), 22.7% being highly resistant (MICs 3-8 micrograms/ml) . Any strain was resistant to P only, but patterns with P included were frequent (55.8%) . Resistance to SxT was highest (monoresistance 25.6% as well as polyresistance 69.8%) . 72.9% were resistant strains to > or = 1 antibiotic with 12 patterns of resistance (1-5 antibiotics) . All of the strains were susceptible to Cro (MICs-0.003-0.5 microgram/ml) . Resistance was closely correlated to serotypes 6,9,14,19,23 to site of isolation and diagnosis . Pneumococci from meningitis were 2-3 times more susceptible to P,E,Cl,T and SxT than the isolates from pneumopathies or other infections.

Mol Microbiol, 1996 Apr, 20(1), 101 - 8
Dimerization of plasmid DNA accelerates selection for antibiotic resistance; Mazin AV et al.; Dimerization of multicopy plasmids is widely assumed to be disadvantageous both for plasmid maintenance and for the host cell . It is known that dimerization causes plasmid instability; dimer-containing cells grow slower than their monomer-containing counterparts . However, as we demonstrate here, under conditions of selective stress, dimers provide an advantage for bacteria . Dimers facilitate segregation of mutants from numerous copies of the parental plasmid . Accelerated segregation greatly increases the rate of accumulation of plasmids carrying mutations that are adaptive for bacteria . In contrast, resolution of dimers by site-specific recombination decreases, 10(3)-10(5)-fold, the efficiency of selection of spontaneous reversions in the tet gene of pBR327.

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