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