Enferm Infecc Microbiol Clin, 2002 Aug-Sep, 20(7), 341 - 5 {Structure and function of integrons}; Sabate M et al.; Integrons are genetic elements known for their role in the acquisition and expression of genes conferring antibiotic resistance . Integrons have an integrase gene (intI), an attachment site (attI), into which individual resistance genes are inserted and a promotor sequence (Pant), allowing expression of resistance genes (cassette-associated genes), which do not have promotors . Integrase recognizes 59-be, a specific sequence in certain resistance genes, which is captured by recombination at the attI attachment site . The fragment intI - attI is highly conserved in all integrons and is called 59 -CS.Integrons have been classified according to the sequence of their integrase and the ones most frequently detected in isolated clinical strains belong to Class I . Class I integrons contain the 59 -CS region followed by gene cassettes in a variable region and finally, a conserved region known as 39 -CS containing two genes, the quaternary ammonium resistance gene (qacEDI) and the sulphonamide resistance gene (sul1); both genes are fixed in this structure . Accordingly, the structure of a Class 1 integron would be IntI - attI {R11 R21.} - qacED1 - sul1.Integrons are probably not mobile, but they are often found in transposons within conjunctive plasmids, which assures their mobility, as can be seen by their wide diffusion among bacteria. J Perinat Neonatal Nurs, 2002 Sep, 16(2), 54 - 72 Vancomycin and gentamicin in neonates: hindsight, current controversies, and forethought; Dawson PM; The use of both vancomycin and gentamicin in the treatment of suspected or documented neonatal infections, while routine, is a challenge for bedside and advanced practice nurses caring for neonates in intensive care units . A review of the background information surrounding neonatal infections as well as the history, intended use, and the pharmacokinetic and pharmacodynamic properties of vancomycin and gentamicin is presented with the goal of aiding in proper treatment with these two medications . Specific attention is given to doses in special situations, means of drug monitoring, strategies for avoiding antibiotic resistance, alternative medication choices, and areas for future investigation. Semin Respir Infect, 2002 Sep, 17(3), 184 - 8 Resistance mechanisms; Acar JF; During the past decade, there has been extensive investigation of bacterial resistance mechanisms.The interplay of genetic and antibiotic factors on antibiotic resistance are multiple and complex . Antibiotic resistance is the clinical expression of these factors . This article reviews the most studied and understood mechanisms of resistance in bacterial species in which resistance problems have clinical significance . Besides plasmid mediated resistance some resistance problems among respiratory pathogens are clonal resistance . The precise cause of this actual genetic event causing bacterial mutation recombination and selection is not always clear . Although resistance mechanisms may be identical within an antibiotic class, expression of resistance often varies among antibiotics of the same class . Med J Aust, 2002 Sep 16, 177(6), 325 - 9 11: Antibiotic resistance; Collignon PJ; Antibiotic resistance is a consequence of antibiotic use - we need to use antibiotics less and to use them prudently . Plans to combat antibiotic resistance were recently proposed by the World Health Organization, a United States interagency taskforce and the Australian Joint Expert Technical Advisory Committee on Antibiotic Resistance . Prudent antibiotic use includes not using antibiotics when benefit is minimal (eg, in many respiratory tract infections), using narrow-spectrum antibiotics whenever possible and using optimal dosages and regimens . The need for antibiotic therapy can be reduced by preventing infections through vaccination, infection control measures and improved sanitation . Surveillance of antibiotic resistance is needed to target interventions for minimising antibiotic use . More research is needed into new antibiotics and regimens and into improving medical devices and protocols to prevent infection . Some simple changes to practice could reduce development and spread of antibiotic resistance Vaccine, 2002 Sep 10, 20(27-28), 3379 - 88 PCR-generated linear DNA fragments utilized as a hantavirus DNA vaccine; Johansson P et al.; The field of DNA vaccines has grown rapidly, and since most such vaccines involve the inoculation of large circular DNA molecules previously propagated in bacteria, several inconveniences (e.g . the presence of antibiotic resistance genes, impurities from bacterial cultures or inefficient uptake of the large and bulky plasmid DNA molecules to the nucleus) are debated . In this study, we have explored the possibility of using smaller and more flexible PCR-generated linear DNA fragments instead . Phosphorothioate (PTO)-modified primers were used successfully to protect the PCR-generated DNA fragments from exonuclease degradation, and by using a nuclear localization signal-peptide to target the linear DNA to the nucleus the immune response against the encoded antigen was further improved . This approach was tested in cell culture using a sensitive reporter system and in vivo with DNA encoding the amino-terminus of the Puumala hantavirus nucleocapsid protein . Our results indicate that linear DNA fragments have a great potential as a genetic vaccine and phosphorothioate modification in combination with a nuclear localization signal peptide increase the stability and targets the linear DNA molecules to the nucleus resulting in an improved biological response examined both in vitro and in vivo. J Mol Recognit, 2002 May-Jun, 15(3), 113 - 25 Conformational analysis of bacterial cell wall peptides indicates how particular conformations have influenced the evolution of penicillin-binding proteins, beta-lactam antibiotics and antibiotic resistance mechanisms; Grail BM et al.; Our aim was to use a conformational analysis technique developed for peptides to identify structural relationships between bacterial cell wall peptides and beta-lactam antibiotics that might help to explain their different actions as substrates and inhibitors of penicillin binding proteins (PBPs) . The conformational forms of the model cell wall peptide Ac-L-Lys(Ac)-D-Ala-D-Ala are described by just a few backbone torsion combinations: three C-terminal carboxylate regions, with Tor8 (psi(i+1)) ranges of D3 region (50 degrees to 70 degrees ), D6 region (140 degrees to 170 degrees ) and D9 region (-50 degrees to -70 degrees ) are combined with either of two Tor6 (phi(i))-Tor4 (psi(i)) combinations, C4 region (-50 degrees to -80 degrees ) with B8 region (-40 degrees to -70 degrees ) or C11 region (30 degrees to 50 degrees ) with B2 region (30 degrees to 70 degrees ) . From these results, and comparisons with conformational analyses of various beta-lactams and Ac-L-Lys(Ac)-D-Ala-D-Lac, it is concluded that molecular recognition of cell wall peptide substrates by PBPs requires conformers with backbone torsion angles of D3C4B8 . beta-Lactam antibiotics are constrained compounds with fewer conformational forms; these match well the backbone torsions of cell wall peptides at D3C4, allowing their recognition and acylation by PBPs, whereas their unique Tor4 produces differently orientated CO and N atoms that appear to prevent subsequent deacylation, leading to their action as suicide substrates . The results are also related to the selective pressures involved in evolution of beta-lactamases from PBPs . From analysis of conformers of Ac-L-Lys(Ac)-D-Ala-D-Ala and the vancomycin-resistant analogue Ac-L-Lys(Ac)-D-Ala-D-Lac, it is concluded that vancomycin may recognise D6C11B2 conformers, giving it complementary substrate specificity to PBPs . This approach could have applications in the rational design of antibiotics targeted against PBPs and their substrates . Helicobacter, 2002, 7 Suppl 1, 56 - 62 Helicobacter pylori infection in geriatrics; Pilotto A et al.; The prevalence of Helicobacter pylori infection increases with age worldwide . However, the percentage of H . pylori-positive elderly patients who are tested and treated for their infection remains very low . We now have data that demonstrate the benefit of curing H . pylori infection in elderly patients with H . pylori-associated peptic ulcer disease and severe chronic gastritis . Furthermore, the cure of H . pylori may prevent progression of intestinal metaplasia and gastric atrophy . Studies are needed to clarify the role of eradication for elderly patients who have nonulcer dyspepsia, gastroesophageal reflux disease and who use nonsteroidal anti-inflammatory drugs . H . pylori infection may be easily diagnosed by histological evaluation, rapid urease test or culture performed on gastric biopsies taken during endoscopy . However, the biopsy site must be carefully selected in elderly patients . For noninvasive monitoring of H . pylori infection after treatment, the 13C-urea breath test has significantly higher accuracy than serology in the elderly . The role of the H . pylori stool antigen test in old age still needs to be clarified . One-week PPI-based triple therapy regimens including clarithromycin, amoxycillin and/or nitroimidazoles are highly effective and well tolerated in elderly patients . Low doses of both PPIs and clarithromycin (in combination with standard doses of amoxycillin or nitroimidazoles) are sufficient . Antibiotic resistance and low compliance are the main factors related to treatment failure at any age. Biochemistry, 2002 Sep 3, 41(35), 10764 - 70 Cloning, overexpression, and purification of aminoglycoside antibiotic 3-acetyltransferase-IIIb: conformational studies with bound substrates; Owston MA et al.; Aminoglycoside 3-acetyltransferase-IIIb (AAC3), which acetylates N3 amine of aminoglycoside antibiotics, was cloned from P . Aeruginosa and purified from overexpressing E . coli BL21 (DE3) cells . Bound conformations of kanamycin A and ribostamycin, in the active site of the enzyme that modifies the essential N3B of aminoglycoside antibiotics, were determined by NMR spectroscopy . Experimentally determined interproton distances were used in a simulated annealing protocol to determine enzyme-bound conformations of both antibiotics . Two conformations, consistent with the NOE restraints, were determined for ribostamycin . The only difference between the two conformers was the orientation of the A ring with respect to the rest of the molecule . The average glycosidic dihedral angles were Phi(1A) = -22 degrees +/- 3 and Psi(1A) = -42 degrees +/- 1 (conformer 1) and Phi(1A) = -67 degrees +/- 0.7 and Phi(1A) = -59 degrees +/- 0.8 (conformer 2) . Three conformers were determined for the enzyme-bound kanamycin A . Two conformers of kanamycin A were matched well with the two conformers of ribostamycin when the A and the B rings of the antibiotics were superimposed . Conformations of kanamycin A and ribostamycin were compared to those of other aminoglycosides that are bound to different enzymes and RNA . The results lend further support to our earlier hypothesis that the A and B rings of aminoglycosides adopt a conformation that is recognized not only by the aminoglycoside-modifying enzymes but also by RNA (Serpersu, E . H., Cox, J . R., Digiammarino, E . L., Mohler, M . L., Akal, A., Ekman, D . R., and Owston, M . (2000) Cell Biochem . Biophys . 33, 309-321) . These results may be useful in designing new antibiotics to combat the antibiotic resistance against infectious diseases. J Mol Evol, 2002 Sep, 55(3), 314 - 21 Phylogenetic analysis shows that the OXA beta-lactamase genes have been on plasmids for millions of years; Barlow M et al.; The OXA genes encode a class of beta-lactamases that confer resistance to a wide range of beta-lactam antibiotics . To determine whether the diversity of the OXA beta-lactamases is the result of recent or ancient events, and to determine whether mobilization of the OXA genes from chromosomes to plasmids occurred recently or long ago, we have constructed a Bayesian phylogeny of the OXA beta-lactamase genes . Analysis of that phylogeny shows that much of the diversity is the result of ancient events and that the OXA genes were mobilized from chromosomes to plasmids on at least two independent occasions that occurred millions of years ago . That observation contradicts the commonly held impression that mobilization of antibiotic resistance genes is strictly the result of modern use of antibiotics. J Med Microbiol, 2002 Aug, 51(8), 705 - 9 Antibiotic susceptibility of Helicobacter pylori in Germany: stable primary resistance from 1995 to 2000; Wolle K et al.; The issue of antibiotic resistance in Helicobacter pylori is of particular concern and has become an important factor leading to eradication failure . This paper reports the prevalence of primary resistance to clarithromycin, amoxicillin, metronidazole and tetracycline among H . pylori isolates in the north-eastern part of Germany . A total of 1644 clinical H . pylori isolates was investigated over a period of 6 years from 1995 to 2000 . The MICs were determined by the Etest . The overall rate of primary resistance was 26.2% for metronidazole and 2.2% for clarithromycin . No significant changes in the resistance rates during the period of investigation were observed . No isolate was resistant to amoxicillin or tetracycline . PCR-RFLP analysis for the detection of point mutations associated with clarithromycin resistance was performed with 36 H . pylori isolates . The A --> G transition mutation at position 2143 was detected in 19 H . pylori isolates (52.8%), whereas the mutation at position 2142 was found in 13 isolates (36.1%). J Am Chem Soc, 2002 Aug 14, 124(32), 9422 - 30 Molecular dynamics at the root of expansion of function in the M69L inhibitor-resistant TEM beta-lactamase from Escherichia coli; Meroueh SO et al.; Clavulanate, an inhibitor for beta-lactamases, was the very first inhibitor for an antibiotic resistance enzyme that found clinical utility in 1985 . The clinical use of clavulanate and that of sulbactam and tazobactam, which were introduced to the clinic subsequently, has facilitated evolution of a set of beta-lactamases that not only retain their original function as resistance enzymes but also are refractory to inhibition by the inhibitors . This article characterizes the properties of the clinically identified M69L mutant variant of the TEM-1 beta-lactamase from Escherichia coli, an inhibitor-resistant beta-lactamase, and compares it to the wild-type enzyme . The enzyme is as active as the wild-type in turnover of typical beta-lactam antibiotics . Furthermore, many of the parameters for interactions of the inhibitors with the mutant enzyme are largely unaffected . The significant effect of the inhibitor-resistant trait was a relatively modest elevation of the dissociation constant for the formation of the pre-acylation complex . The high-resolution X-ray crystal structure for the M69L mutant variant revealed essentially no alteration of the three-dimensional structure, both for the protein backbone and for the positions of the side chains of the amino acids . It was surmised that the difference in the two enzymes must reside with the dynamic motions of the two proteins . Molecular dynamics simulations of the mutant and wild-type proteins were carried out for 2 ns each . Dynamic cross-correlated maps revealed the collective motions of the two proteins to be very similar, yet the two proteins did not behave identically . Differences in behavior of the two proteins existed in the regions between residues 145-179 and 155-162 . Additional calculations revealed that kinetic effects measured experimentally for the dissociation constant for the pre-acylation complex could be mostly attributed to the electrostatic and van der Waals components of the binding free energy . The effects of the mutation on the behavior of the beta-lactamase were subtle, including the differences in the measured dissociation constants that account for the inhibitor-resistant trait . It would appear that nature has selected for incorporation of the most benign alteration in the structure of the wild-type TEM-1 beta-lactamase that is sufficient to give the inhibitor-resistant trait. Crit Care Med, 2002 Aug, 30(8), 1877 - 82 Pilot study of antibiotic cycling in a pediatric intensive care unit; Moss WJ et al.; OBJECTIVE: This pilot study was performed to determine the safety and size of effect of antibiotic cycling to reduce colonization and infection with antibiotic-resistant bacteria . DESIGN: Open, observational study . SETTING: The study was performed in a 16-bed pediatric medical-surgical intensive care unit . PATIENTS: Critically ill children requiring antibiotic therapy . INTERVENTIONS: Three antibiotic classes were systematically cycled for 3-month intervals over 18 months . Antibiotic regimens were used for all empirical therapy and continued if the bacterial isolate was susceptible . MEASUREMENTS: The primary outcome was colonization with antibiotic-resistant bacteria, determined by surveillance cultures obtained twice monthly from all patients in the unit . Rates of antibiotic-resistant, nosocomial blood stream infections, and risks of colonization over calendar time in the intensive care unit were also evaluated . MAIN RESULTS: The cycling of broad-spectrum, empirical antibiotics was safe and did not generate increased antibiotic resistance nor select for new organisms . Over the study period, the trend in prevalence of children colonized with antibiotic-resistant bacteria was from 29% to 24% (p =.41) . The effect on prevalence of resistant blood stream infections was similar (p =.29) . Changes in individual risks of colonization with resistant bacteria over calendar time were consistent with the ecologic effect in size and direction . CONCLUSIONS: Results of this pilot intervention suggest that cycling antibiotics may be a safe and viable strategy to minimize the emergence of antibiotic resistance in intensive care units . A definitive study will require a randomized and controlled trial of only four pediatric intensive care units over an 18-month period. J Ind Microbiol Biotechnol, 2002 Aug, 29(2), 60 - 9 The detection of diverse aminoglycoside phosphotransferases within natural populations of actinomycetes; Anderson AS et al.; The conserved nature of the genes that code for actinomycete secondary metabolite biosynthetic pathways suggests a common evolutionary ancestor and incidences of lateral gene transfer . Resistance genes associated with these biosynthetic pathways also display a high degree of similarity . Actinomycete aminoglycoside phosphotransferase antibiotic resistance enzymes (APH) are coded for by such genes and are therefore good targets for evaluating the bioactive potential of actinomycetes . A set of universal PCR primers for APH encoding genes was used to probe genomic DNA from three collections of actinomycetes to determine the utility of molecular screening . An additional monitoring of populations for the predominance of specific classes of enzymes to predict the potential of environmental sites for providing isolates with interesting metabolic profiles . Approximately one-fifth of all isolates screened gave a positive result by PCR . The PCR products obtained were sequenced and compared to existing APH family members . Sequence analysis resolved the family into nine groups of which six had recognizable phenotypes: 6'-phosphotransferase (APH(6)), 3'-phosphotransferase (APH(3)), hydroxyurea phosphotransferase (HUR), peptide phosphotransferase, hygromycin B phosphotransferase (APH(7")) and oxidoreductase . The actinomycetes screened fell into seven groups, including three novel groups with unknown phenotypes . The strains clustered according to the environmental site from where they were obtained, providing evidence for the movement of these genes within populations . The value of this as a method for obtaining novel compounds and the significance to the ecology of antibiotic biosynthesis are discussed. Expert Opin Investig Drugs, 2002 Aug, 11(8), 1127 - 38 New strategies for the prevention and treatment of Helicobacter pylori infection; Ruggiero P et al.; Helicobacter pylori infects the stomach of > 50% of the human population worldwide, with higher prevalence in the developing countries . A strict correlation between H . pylori infection and gastroduodenal diseases has been demonstrated, including gastritis, peptic ulcer and gastric cancer . Current therapies against H . pylori consist of an antisecretory plus antibiotics . These therapies are effective in 80 - 90% of the cases; presently, no alternative therapies have been shown to give comparable or better results . There are two main reasons for therapy failure: poor compliance, which results in cure discontinuation, and antibiotic resistance . To overcome the drawbacks inherent to any antibiotic therapy, a prophylactic vaccine seems to be the most reasonable approach . Vaccines have been developed based on data obtained in animal models, a number of which are currently in Phase I clinical trials, in some cases giving encouraging data for safety and immunogenicity . In the absence of any immunological correlate of protection against H . pylori, it will be possible to evaluate the efficacy of these vaccines only in large Phase III clinical trials. Expert Opin Investig Drugs, 2002 Aug, 11(8), 1023 - 32 Reversing antibiotic resistance; Hodgkinson R et al.; Over the past decade many well-tried chemotherapeutic agents have lost their effectiveness . This is due to a phenomenon referred to as multi-drug resistance . The most likely cause of multi-drug resistance is an increase in the activity of an efflux pump mediated through the actions of a P-glycoprotein . There is a continuing search, not only for new chemotherapeutic agents, but also for agents that can reverse the acquired resistance to existing agents. Bull Acad Natl Med, 2001, 185(9), 1683 - 97 {Towards non-viral gene therapy}; Scherman D; In vivo gene transfer might be useful for gene therapy application such as cancer or therapeutic neoangiogenesis . Non-viral gene therapy, which uses plasmid DNA as expression vector, presents several advantages . In particular, plasmids are more simple to develop than viral vectors, and they are not immunogenic . We have improved the safety and efficiency of non viral gene therapy by optimizing plasmid backbone and developing new gene delivery technologies . Backbone optimized minicircles are gene expression vectors of minimal size, which are devoid of bacterial origin of replication and of antibiotic resistance gene . Triple helix affinity chromatography allows rapid obtention of highly purified plasmid preparation, with minimal contamination by endotoxins and bacterial chromosomal linear DNA . Electrotransfer is a physical gene delivery technique with leads to high transgene expression in muscle and tumors after direct intratissular injection . In skeletal muscle, plasmid DNA electrotransfer leads to sustained protein production, for more than 18 months, and the transgene products can be released in the circulation . Preclinical evidence suggests that this technology might be useful for the treatment of hemophilia, beta-thalassemia, rheumatoid arthritis, or metabolic disorders . Finally, chemical DNA delivery vectors might prove useful for the targeting of disseminated metastasis. Bull Soc Pathol Exot, 2002 Jun, 95(2), 66 - 70 {Antibiotic resistance of Helicobacter pylori in Reunion Island: therapeutic consequences}; Picot S et al.; The aims of this paper were to assess resistance of Helicobacter pylori to antibiotics included in the so-called French triple regimens and to identify the possible causes of therapeutic failure in Reunion island . Antibiotic resistance was determined for 109 strains . All the strains were sensitive to amoxicillin and tetracycline, 93.6% were sensitive to ciprofloxacin, 92.7% to erythromycin and 60.6% to metronidazole . Fifty three patients who had previously tested positive for H . pylori received for one week regimen of amoxicillin (1 g bd), clarithromycin (0.5 g bd) and omeprazole (20 mg bd) . Eradication rate after therapy was of 73.6% . Therapeutic failure was analysed for 9 patients using random amplified polymorphic DNA and the presence or not of antibiotic resistance . One cause of failure is clarithromycin resistance . These data show that triple therapy can be used in Reunion Island . In case of failure, sensitivity must be detected because the rate of resistance to metronidazole is over 30%. Drug Resist Updat, 2002 Apr, 5(2), 73 - 87 Emerging resistance to antibiotics against respiratory bacteria: impact on therapy of community-acquired pneumonia in children; Esposito S et al.; Perhaps because of its etiologic complexity, community-acquired pneumonia (CAP) in infants and children remains a significant problem worldwide . Over the last few years, difficulties related to CAP treatment in children have greatly increased because of the emergence of resistance to the most widely used antibiotics against some of the bacterial pathogens involved in the development of the disease . There are few data describing the impact of antibiotic resistance on clinical outcomes in CAP, but many experts believe that the clinical impact is limited . We here discuss the prevalence of different etiologic agents in CAP of children, the diagnostic criteria, problems related to antibiotic resistance, therapeutic strategies, and future implications . Curr Opin Infect Dis, 2002 Aug, 15(4), 401 - 5 Infection in the intensive care unit: prevention strategies; Bonten MJ; PURPOSE OF THE REVIEW: Nosocomial infections remain among the most common treatment complications, particularly in intensive care unit patients . In many countries antibiotic resistance is increasingly hampering treatment of these infections . Preventive strategies have therefore become more important and have been directed both against the development of specific infections and against the spread of antibiotic-resistant pathogens . The present review addresses recent data on the latter issue . In particular, we discuss the first approaches to use mathematical modelling as a tool to analyse and guide strategies to prevent infection, and the effects of antibiotic cycling . RECENT FINDINGS: Several mathematical models to address the dynamics of pathogen transmission in hospital settings have been developed . One of the models may allow quantification of the effects of different strategies to prevent infection in intensive care units, and another may be used to determine the relative importance of different colonization routes, without the need for expensive genotyping methods . The results of the first prospective studies on antibiotic cycling are inconclusive, and again mathematical modelling may help to provide testable hypotheses for such interventions . Finally, recent studies have shown that alcohol-based hand rubs are better than hand washing with soap and water for most hand disinfection purposes . SUMMARY: The first results of use of mathematical modelling to guide infection control strategies should be subjected to prospective, empirical testing in order to determine their clinical usefulness . More rigorously designed studies are needed to determine the benefits of antibiotic cycling strategies . Hands should be disinfected with alcohol-based hand rubs, which should be available at each bedside. Curr Opin Infect Dis, 2002 Aug, 15(4), 395 - 400 Antibiotic policies and control of resistance; Gould IM; PURPOSE OF THE REVIEW: The current worldwide pandemic of antibiotic resistance shows no signs of abating . It is clear that it is driven mainly by heavy and often inappropriate antibiotic use . Although control measures are widely practised, it is important that we assess their efficacy critically in order to concentrate expensive control efforts where they will be most effective . The past year has seen much activity in this area, with evidence-based assessments of the literature according to strict guidelines, as well as progress in basic science studies of mechanisms of resistance, and their causes and relations to pathogenicity and adaptability . RECENT FINDINGS: The present review summarizes current developments in the causes of antibiotic resistance, the classification of antibiotic stewardship and control measures, the evidence base for their efficacy, current problems in hospital practice, the adaptability of bacteria, the content of antibiotic policies and anticipated activities . SUMMARY: The conclusions from the published literature are that much of it that pertains to changing prescribing practices does not stand up to modern evidence-based analysis concepts . Nevertheless, we can learn from experience in changing other areas of medical practice . We must be pragmatic and must not expect to change the world, but rather take it step by step, recognizing barriers and measuring outcomes and quality indicators . Studies into the molecular basis of resistance confirm the superb genetic adaptability of micro-organisms . They will always be several steps ahead of us . Nevertheless, we are learning how to modify our prescribing habits to minimize resistance, not only by using antibiotics less frequently but also by altering dosing schedules in various ways. Curr Opin Infect Dis, 2002 Aug, 15(4), 387 - 94 Nosocomial pneumonia; Mehta RM et al.; PURPOSE OF REVIEW: Despite abundant literature on the management of nosocomial pneumonia, a number of aspects, from diagnosis to the therapy of nosocomial pneumonia, are still controversial . This review focuses on recent advances that can aid in the day-to-day care of these critically ill patients . RECENT FINDINGS: The risk factors for nosocomial pneumonia in specific subsets of trauma, postoperative and burn injury patients have been identified, with emphasis on the type of pneumonia developing in these populations - early or late onset nosocomial pneumonia . Resolution of nosocomial pneumonia, in terms of improvement of clinical parameters such as oxygenation, fever, leukocytosis and bacterial eradication, has been reported, and these data can lead to a better understanding of the natural course of the disease . The importance of initial, accurate empiric therapy in improving mortality in nosocomial pneumonia has been reinforced by multiple studies . Newer techniques to study colonization and the routes of spread of pathogenic organisms in the intensive care unit are adding to our understanding of how pneumonia develops, the role of infection control measures and the types of strategies that are needed for prevention . Oral decontamination is showing promise as a technique to prevent ventilator-associated pneumonia, and noninvasive ventilation has been shown to be useful in various etiologies of respiratory failure, with the beneficial effect of reducing the incidence of ventilator-associated pneumonia and its associated mortality . The implementation of protocolized treatment guidelines and antibiotic rotation policies are emerging as useful tools for reducing the frequency of antibiotic resistance and the impact of nosocomial pneumonia . SUMMARY: There is a better understanding of nosocomial pneumonia risk factors, mechanisms of bacterial colonization, and resolution of illness, with exciting developments in prevention and treatment emerging, and these data can help us achieve more effective management of this complex illness. Semin Pediatr Infect Dis, 2002 Apr, 13(2), 129 - 33 Influenza virus vaccines in children and their impact on the incidence of otitis media; Rafei K; Otitis media has been identified as the most frequent reason for outpatient antibiotic therapy . Several studies have linked viral respiratory infections with bacterial otitis media . In light of rising concerns about antibiotic resistance, the possibility of reducing the incidence of otitis media through vaccination against respiratory viruses has received increasing attention . This article reviews inactivated and live attenuated influenza virus vaccines and their possible impact on the incidence of otitis media . Inactivated and live attenuated influenza virus vaccines are safe and immunogenic in children older than 6 months and are linked to a decrease in the incidence of otitis media . Influenza vaccination of infants younger than 6 months has resulted in less predictable immunogenicity and deserves further investigation. Antimicrob Agents Chemother, 2002 Aug, 46(8), 2636 - 9 Salicylate reduces susceptibility of Mycobacterium tuberculosis to multiple antituberculosis drugs; Schaller A et al.; Salicylate induces multiple antibiotic resistance in various bacterial species . Here we investigated the effect of salicylate on the susceptibility of Mycobacterium tuberculosis to a range of antituberculosis (anti-TB) drugs . In the presence of salicylate, the killing effects of isoniazid (INH), rifampin (RMP), ethambutol (EMB), streptomycin (STR), and p-aminosalicylate (PAS) were reduced, as shown with a tetrazolium redox dye viability assay and a bacterial survival assay . Salicylate-induced resistance was more pronounced for PAS, STR, and EMB but was not apparent for INH and RMP when salicylate and the anti-TB agents were incorporated into 7H11 plates . The significance of these findings for TB treatment needs to be further evaluated in vivo. Curr Biol . 2002 Jul 9;12(13):R441. Flaws bug television gene drama; Dixon B; MEDIAWATCH: Rejection of scientific advice by the writers of a BBC thriller based on the consequences of genetic modification of wheat with an antibiotic-resistance gene renders the plot absurd, writes Bernard Dixon. J Am Geriatr Soc, 2002 Jul, 50(7 Suppl), S242 - 6 Management of antibiotic-resistant bacteria; Norman DC; Optimizing the management of antibiotic resistance is an important strategy in improving outcomes for infectious diseases in older persons . Strategies that manage antibiotic resistance must take into account all clinical settings, because resistant pathogens previously seen only in acute care facilities are becoming increasingly common in long-term care facilities . Recently, modest improvement in therapeutic options for the treatment of infections due to resistant pathogens has become available because of the development of newer antibiotics . Some of these drugs are briefly discussed in this review, but the best strategy is to limit the potential for the development of resistance and transmission of these pathogens . This can best be accomplished by minimizing misuse of antibiotics and maximizing adherence to basic hygiene standards. Clin Infect Dis, 2002 Aug 1, 35(3), 298 - 304 Epub 2002 Jul 08. Helicobacter pylori: consensus and controversy; Passaro DJ et al.; Helicobacter pylori is uniquely adapted to colonize the human stomach . Infection leads to a range of subclinical and clinical outcomes that depend on properties of the infecting strain, the host, and the environment . Eradication therapy is indicated for infected persons who develop peptic ulcer disease or gastric lymphoma or who are beginning long-term treatment with nonsteroidal anti-inflammatory drugs . However, treatment may worsen gastroesophageal reflux disease and increase the risk of esophageal cancer . H . pylori infections can be diagnosed noninvasively and can be eradicated with approximately 85% success by a variety of multidrug, 7-14-day regimens . Unfortunately, antibiotic resistance is affecting treatment effectiveness in the United States and abroad . A more complete understanding of the variation in H . pylori pathogenesis should lead to clearer recommendations about treatment for infected persons who have neither peptic ulcer disease nor gastric lymphoma. Transgenic Res, 2002 Jun, 11(3), 315 - 9 Selection of transgenic Xenopus laevis using antibiotic resistance; Moritz OL et al.; We previously established lines of transgenic Xenopus laevis expressing green fluorescent protein (GFP) or GFP fusion proteins in the rod photoreceptors of their retinas under control of the X . laevis opsin promoter, which permits easy identification of transgenic animals by fluorescence microscopy . However, GFP tags can alter the properties of fusion partners, and in many circumstances a second selectable marker would be useful . The transgene constructs we used also encode a gene that confers resistance to the antibiotic G418 in cultured mammalian cells . In this study, we show that F2 transgenic offspring of these animals are more resistant to G418 toxicity than their non-transgenic siblings, as are primary transgenic X . laevis . G418 resistance can be used as a selectable marker in transgenic X . laevis, and possibly other aquatic transgenic animals. J Infect Chemother, 2002 Jun, 8(2), 155 - 62 Characterization of cefotaxime-resistant Escherichia coli isolates from a nosocomial outbreak at three geriatric hospitals; Ma L et al.; Over a 22-month period, there was an unusual upsurge in the incidence of cefotaxime-resistant Escherichia coli among hospitalized patients in three geriatric hospitals in the same district . Sixteen highly cefotaxime-resistant strains were obtained from clinical specimens during the period January 1996 through October 1997 . All strains were characterized by antibiotic resistance pattern analysis, the detection of the TEM- and Toho-type beta-lactamase or CTX-M-type beta-lactamase gene by polymerase chain reaction (PCR), plasmid profiling, Southern hybridization analysis, and pulsed-field gel electrophoresis (PFGE) . Antibiotic resistance analysis showed that all strains were highly resistant to ampicillin, piperacillin, carbenicillin, cephaloridine, and cefotaxime; intermediately resistant to cefoxitin; moderately susceptible to moxalactam and ceftazidime; and susceptible to imipenem . Detailed analysis of beta-lactamase content revealed that all cefotaxime-resistant strains harbored a plasmid that mediated an extended-spectrum beta-lactamase of the Toho-type or CTX-M-type by PCR and Southern hybridization analysis . PCR detection showed that all the E . colistrains, except for strains TUM1023, TUM1101, TUM1227, and TUM1229, also possessed bla(TEM) genes . Furthermore, Southern hybridization analysis showed that all strains, except for TUM1102, gave a similar signal with the Toho probe . The PFGE profiles of the E . colistrains obtained with XbaI showed four patterns that correlated well with the plasmid profiles . The Dice value of 15 strains, including Toho-2 producer (TUM1083), for their PFGE patterns indicated a similarity of 80% or more . Our results suggest that 15 of the 17 Toho type beta-lactamase-producing E . coli strains (including strain TUM1083) studied belong to a single epidemic strain, while the other two strains are different from them, and the Toho-type or CTX-M-type beta-lactamase encoding gene may be acquired by plasmid conjugation or a mobile element. Curr Drug Targets, 2002 Aug, 3(4), 345 - 9 Mutation and evolution of antibiotic resistance: antibiotics as promoters of antibiotic resistance? Blazquez J, Oliver A, Gomez-Gomez JM. Antibiotic resistance appearance and spread have been classically considered the result of a process of natural selection, directed by the use of antibiotics . Bacteria, that have to face the antibiotic challenge, evolve to acquire resistance and, under this strong selective pressure, only the fittest survive, leading to the spread of resistance mechanisms and resistant clones . Horizontal transference of resistance mechanisms seems to be the main way of antibiotic resistance acquisition . Nevertheless, recent findings on hypermutability and antibiotic-induced hypermutation in bacteria have modified the landscape . Here, we present a review of the last data on molecular mechanisms of hypermutability in bacteria and their relationship with the acquisition of antibiotic resistance . Finally, we discuss the possibility that antibiotics may act not only as selectors for antibiotic resistant bacteria but also as resistance promoters. Protein Eng, 2002 Jun, 15(6), 463 - 70 Improving a circularly permuted TEM-1 beta-lactamase by directed evolution; Osuna J et al.; Circular permutation of proteins is a powerful technique to explore the importance of the polypeptide secondary structure order for attaining the final three-dimensional structure . Here, we designed a circular permutation of the TEM beta-lactamase in order to produce a new domain-forming amino acid arrangement in the polypeptide sequence . Closing the normal N- and C-termini with the connecting peptide GGS and creating new N- and C-termini at position 216, produces a severely impaired permuted protein . Introduction of a connector with random components allows the isolation of enzymes with better activities and indicates a selection for a potential helix-stop signal at the new super-secondary motif . We applied several directed-evolution cycles, starting from permuted enzymes with each of the two different connecting peptides, and selecting for antibiotic resistance and isolated several mutants with resistance levels close to those of the wild-type enzyme . We also analyze some of the data collected on the outcomes and paths of these evolutionary experiments . A purified sixth cycle variant with connector peptide GGS showed catalytic efficiency values approximately 8% of the natural enzyme. J Mol Biol, 2002 Jun 28, 320(1), 85 - 95 Evolution of an antibiotic resistance enzyme constrained by stability and activity trade-offs; Wang X et al.; Pressured by antibiotic use, resistance enzymes have been evolving new activities . Does such evolution have a cost? To investigate this question at the molecular level, clinically isolated mutants of the beta-lactamase TEM-1 were studied . When purified, mutant enzymes had increased activity against cephalosporin antibiotics but lost both thermodynamic stability and kinetic activity against their ancestral targets, penicillins . The X-ray crystallographic structures of three mutant enzymes were determined . These structures suggest that activity gain and stability loss is related to an enlarged active site cavity in the mutant enzymes . In several clinically isolated mutant enzymes, a secondary substitution is observed far from the active site (Met182-->Thr) . This substitution had little effect on enzyme activity but restored stability lost by substitutions near the active site . This regained stability conferred an advantage in vivo . This pattern of stability loss and restoration may be common in the evolution of new enzyme activity . (c) 2002 Elsevier Science Ltd. J Am Coll Nutr, 2002 Jun, 21(3 Suppl), 166S - 173S Food safety evaluation of crops produced through biotechnology; Chassy BM; Agricultural biotechnology has been widely adopted in agriculture but is also the focus of controversy . Questions have arisen regarding food and environmental safety . In the US, responsibility for ensuring agricultural and environmental safety is delegated to the USDA and EPA, respectively . The FDA has primary responsibility for food safety, with the exception that the EPA has responsibility for the safety of proteins in plants associated with insect defense mechanisms . The food safety assessment, whether performed by the FDA or the EPA, requires evaluation of the safety of 1) the newly added DNA, 2) the safety of the newly introduced gene product and 3) the overall safety of the balance of the food . A paradigm called "Substantial Equivalence" guides the assessment . The principal food safety issues for new varieties crops are 1) potential toxicity of the newly introduced protein(s), 2) potential changes in allergenicity, 3) changes in nutrient composition, 4) unintended effects giving rise to allergenicity or toxicity and 5) the safety of antibiotic resistance marker-encoded proteins included with the transgene . All of these must be taken in the context of the predicted range of dietary exposures . The evaluation seeks to establish that there is a "reasonable likelihood of safety" and that new varieties are as safe as or safer than crops produced by traditional methods . Indeed, after extensive safety testing and some five years of experience with such crops in the marketplace, there is not a single report that would lead an expert food scientist to question the safety of such transgenic crops now in use. Commun Dis Public Health, 2002 Mar, 5(1), 7 - 12 Impact of infections on primary care--greater than expected; Fleming DM et al.; Though infection has declined as a cause of death it consumes a major proportion of primary care resources, and because of antibiotic resistance is increasingly important . We examined the burden of illness attributable to infection in primary care by analysing data from the fourth practice-based national morbidity survey which monitored all consultations by diagnosis in 470,000 persons over a 12 month period from September 1991 to August 1992 . Rates of persons consulting, new episodes of illness and consultations were calculated according to the list of infections published by Wilson and Bhopal (W&B list), and as amended by the Communicable Disease Surveillance Centre (CDSC list); selected comparisons were made with national data on hospital derived finished consultant episodes and deaths . Forty-one percent of all registered persons consulted on at least one occasion during the year for infection (estimated by either list) . Infections accounted for 40% of all new episodes of illness and 29% of all consultations; respiratory infections accounted for approximately half the infection total . New episode rates were highest in children aged less than 1 year and lowest in males 25-54 years and females 55-64 years . Except in infancy, rates were higher in females . There were 734 episodes of infection annually per 1,000 population reported in general practice compared with 20 per 1,000 finished consultant episodes . The average episode of infection prompted 1.2 general practice consultations . Improved management through more precise diagnosis by near patient tests is desirable, but is unlikely to be obtained cost effectively if consultation numbers or the time spent is substantially increased . These results emphasise the importance of adequate training for general practitioners in the natural history, epidemiology, diagnosis and treatment (pharmacology) of infection. Res Microbiol, 2002 May, 153(4), 199 - 204 Structure and role of coupling proteins in conjugal DNA transfer; Gomis-Ruth FX et al.; Type IV secretory systems are transmembrane bacterial multiprotein complexes . They are pivotal for conjugation, bacterial-induced plant tumour formation, toxin secretion and mammalian pathogen intracellular activity . These systems are involved in the spread of antibiotic resistance genes among bacteria by enabling conjugative DNA transfer . When such translocons transport DNA, they require the assistance of multimeric integral inner membrane proteins, the type IV coupling proteins . Its structural prototype is plasmid R388 TrwB protein, responsible for coupling the relaxosome with the DNA transport apparatus during bacterial conjugation . Its monomeric molecular structure is reminiscent of ring helicases and AAA ATPases . The quaternary structure is made up by six equivalent protomers featuring a flattened sphere resembling F1-ATPase, with a central channel traversing the particle, thus connecting cytoplasm and periplasm. Physiol Plant, 2002 Mar, 114(3), 429 - 438 Genetic transformation of cauliflower (Brassica oleracea var . botrytis) by direct DNA uptake into mesophyll protoplasts; Radchuk VV et al.; Mesophyll protoplasts of Brassica oleracea var . botrytis were successfully transformed using polyethylene glycol (PEG) . The success of plant transformation depended on both gene transfer and plant regeneration . Parameters, such as PEG and vector concentrations and heat shock conditions were tested in experiments on transient expression of the beta-glucuronidase (EC 3.2.1.31) gene and the most suitable conditions for DNA uptake were determined . Two antibiotic resistance marker genes for neomycin phosphotransferase (EC 2.7.1.95) and hygromycin phosphotransferase (EC 2.7.1.104), and three vector plasmids with different lengths were used to obtain stable transformants. J Biol Chem, 2002 Aug 30, 277(35), 32149 - 56 Epub 2002 Jun 10. The structural bases of antibiotic resistance in the clinically derived mutant beta-lactamases TEM-30, TEM-32, and TEM-34; Wang X et al.; Widespread use of beta-lactam antibiotics has promoted the evolution of beta-lactamase mutant enzymes that can hydrolyze ever newer classes of these drugs . Among the most pernicious mutants are the inhibitor-resistant TEM beta-lactamases (IRTs), which elude mechanism-based inhibitors, such as clavulanate . Despite much research on these IRTs, little is known about the structural bases of their action . This has made it difficult to understand how many of the resistance substitutions act as they often occur far from Ser-130 . Here, three IRT structures, TEM-30 (R244S), TEM-32 (M69I/M182T), and TEM-34 (M69V), are determined by x-ray crystallography at 2.00, 1.61, and 1.52 A, respectively . In TEM-30, the Arg-244 --> Ser substitution (7.8 A from Ser-130) displaces a conserved water molecule that usually interacts with the beta-lactam C3 carboxylate . In TEM-32, the substitution Met-69 --> Ile (10 A from Ser-130) appears to distort Ser-70, which in turn causes Ser-130 to adopt a new conformation, moving its O gamma further away, 2.3 A from where the inhibitor would bind . This substitution also destabilizes the enzyme by 1.3 kcal/mol . The Met-182 --> Thr substitution (20 A from Ser-130) has no effect on enzyme activity but rather restabilizes the enzyme by 2.9 kcal/mol . In TEM-34, the Met-69 --> Val substitution similarly leads to a conformational change in Ser-130, this time causing it to hydrogen bond with Lys-73 and Lys-234 . This masks the lone pair electrons of Ser-130 O gamma, reducing its nucleophilicity for cross-linking . In these three structures, distant substitutions result in accommodations that converge on the same point of action, the local environment of Ser-130. Toxicol Lett, 2002 Feb 28, 127(1-3), 337 - 40 Clinical risk assessment of GM foods; Lack G; The main concerns about adverse effects of genetically modified (GM) foods on health are the transfer of antibiotic resistance, toxicity and allergenicity . There are two issues from an allergic standpoint . First, the transfer of a known allergen may occur from a crop into a non-allergenic target crop . The second scenario is the creation of a neo-allergen where de novo sensitisation occurs in the population . The first scenario occurred in 1996 when the 2S albumen protein from Brazil nut was transferred into soy bean (N . Engl . J . Med . 334 (1996) 688) . 2S albumen was found to be a major Brazil nut allergen and the newly expressed protein in transgenic soy retained its allergenicity . Patients allergic to Brazil nuts and not to soy bean now showed an IgE mediated response towards GM soy bean . We argue that it is possible to prevent such occurrences by doing IgE-binding studies and taking into account physico-chemical characteristics of proteins and referring to known allergen databases . The second possible scenario of de novo sensitisation does not easily lend itself to risk assessment . We compare GM technology to traditional plant breeding and food processing methods . There is no evidence that the technology used for the production of GM foods poses an allergic threat per se compared to other methodologies widely accepted in the food industry . We need to proceed cautiously in the future, assessing individual GM foods on the basis of their individual merits and risks prior to introducing them into the market. Aliment Pharmacol Ther, 2002 Jul, 16 Suppl 4, 105 - 13 Review article: Helicobacter pylori infection from pathogenesis to treatment--a critical reappraisal; Vaira D et al.; The main areas of this review are Helicobacter pylori and disease pathogenesis; the relationship of H . pylori to lower gastrointestinal diseases, liver disease and extra-gastrointestinal conditions; the relationship of H . pylori to gastro-oesophageal reflux disease; infection in the very young and very old; diagnostic techniques; and management of H . pylori infections with particular emphasis on eradication regimens and antibiotic resistance. Am Fam Physician, 2002 May 15, 65(10), 2039 - 44 Diagnosis and management of acute bronchitis; Knutson D et al.; Acute bronchitis is one of the top 10 conditions for which patients seek medical care . Physicians show considerable variability in describing the signs and symptoms necessary to its diagnosis . Because acute bronchitis most often has a viral cause, symptomatic treatment with protussives, antitussives, or bronchodilators is appropriate . However, studies indicate that many physicians treat bronchitis with antibiotics . These drugs have generally been shown to be ineffective in patients with uncomplicated acute bronchitis . Furthermore, antibiotics often have detrimental side effects, and their overuse contributes to the increasing problem of antibiotic resistance . Patient satisfaction with the treatment of acute bronchitis is related to the quality of the physician-patient interaction rather than to prescription of an antibiotic. Hum Reprod, 2002 Jun, 17(6), 1433 - 6 Chlamydia trachomatis in subfertile women undergoing uterine instrumentation . The clinician's role; Macmillan S; Most women attending a fertility clinic will undergo uterine instrumentation either diagnostically and/or therapeutically . This places them at potential risk of chlamydial pelvic inflammatory disease . Clinicians remain unclear about the roles of screening, serology and prophylactic antibiotics . A review of the evidence suggests that women <25 years and those older with risk factors, men with risk factors and gamete donors should have their lower genital tract screened for Chlamydia trachomatis by a sensitive test . More information is required before screening men by age can be recommended . Serology in its present form cannot be advocated as a screening tool . Those women with a past history of chlamydial morbidity or a diagnosis of tubal pathology should, in addition to screening, be covered with prophylactic antibiotics when undergoing uterine instrumentation . The partner should be screened for sexually transmitted infections . Non-selective use of prophylactic antibiotics serves only to increase the problem of antibiotic resistance and maintain the bacterial load of chlamydia in the community. Gene Ther, 2002 Jun, 9(11), 679 - 82 Extrachromosomal genes: a powerful tool in gene targeting approaches; Colosimo A et al.; Several studies, some of which have been updated during the recent workshop entitled Genome Medicine: Gene Therapy for the Millennium (Rome, 30 September-3 October 2001), have highlighted the usefulness of extrachromosomal or episomal genes in gene targeting strategies . Due to the selectable nature of antibiotic resistance and reporter genes, targeted correction of mutated versions of these extrachromosomal genes allows an accurate quantification of correction frequency . In addition, these model systems facilitate and speed up the optimization of critical parameters for the successful application of gene targeting approaches . In fact, type of cell line, gene delivery system, molar ratio of episomal target/therapeutic constructs, nature and design of therapeutic complexes and different recombinative proteins may be critical for the actual feasibility of each method . Although virus-based approaches are now being investigated as well, this article is focusing on the targeted correction of extrachromosomal genes by the use of small DNA fragments (SDF), chimeric RNA/DNA oligonucleotides (RDO) and triplex-forming oligonucleotides (TFO). Prof Nurse, 2001 Sep, 17(1), 56 - 9 Addressing the problem of increased antibiotic resistance; Kelly J; Growing bacterial resistance to antibiotics is causing grave concern in both community and hospital settings . Mismanagement has been identified as the main cause . The problem can be addressed at ground level by careful use of antibiotics, through meticulous personal and environmental hygiene and preventive infection-control measures. J Pediatr Nurs, 2002 Apr, 17(2), 126 - 32 Prescribing practices that promote antibiotic resistance: strategies for change; Larrabee T; Approximately 90% of all upper respiratory infections are caused by viruses, yet antibiotics are prescribed for 50% to 70% of patients who seek medical care for these conditions (Neiderman, Skerrett, & Yamauchi, 1998) . Prescribing antibiotics for conditions for which there is no proven benefit is not a harmless practice; it contributes to the development of antibiotic resistance (Ware, 2000) . This article will discuss the magnitude of the problem of antibiotic resistance as it relates to pediatric outpatient upper respiratory tract infection and otitis media, analyze practitioner prescribing practices that contribute to widespread antibiotic use, and suggest provider and patient intervention to improve the rational use of antibiotics . Philos Trans R Soc Lond B Biol Sci, 2002 Apr 29, 357(1420), 505 - 19 The evolution of drug-resistant malaria: the role of drug elimination half-life; Hastings IM et al.; This paper seeks to define and quantify the influence of drug elimination half-life on the evolution of antimalarial drug resistance . There are assumed to be three general classes of susceptibility of the malaria parasite Plasmodium falciparum to a drug: Res0, the original, susceptible wildtype; Res1, a group of intermediate levels of susceptibility that are more tolerant of the drug but still cleared by treatment; and Res2, which is completely resistant to the drug . Res1 and Res2 resistance both evolve much faster if the antimalarial drug has a long half-life . We show that previous models have significantly underestimated the rate of evolution of Res2 resistance by omitting the effects of drug half-life . The methodology has been extended to investigate (i) the effects of using drugs in combination, particularly when the components have differing half-lives, and (ii) the specific example of the development of resistance to the antimalarial pyrimethamine-sulphadoxine . An important detail of the model is the development of drug resistance in two separate phases . In phase A, Res1 is spreading and replacing the original sensitive forms while Res2 remains at a low level . Phase B starts once parasites are selected that can escape drug action (Res1 genotypes with borderline chemosensitivity, and Res2): these parasites are rapidly selected, a process that leads to widespread clinical failure . Drug treatment is clinically successful during phase A, and health workers may be unaware of the substantial changes in parasite population genetic structure that predicate the onset of phase B . Surveillance programs are essential, following the introduction of a new drug, to monitor effectively changes in treatment efficacy and thus provide advance warning of drug failure . The model is also applicable to the evolution of antibiotic resistance in bacteria: in particular, the need for these models to incorporate drug pharmacokinetics to avoid potentially large errors in their predictions. Hindustan Antibiot Bull, 1999 Feb-Nov, 41(1-4), 25 - 31 Antibiotic resistance--a close look; Bhattacharyya BK; The discovery of antibiotic greatly improved the human health care system . But today we are facing the problem of antibiotic resistance due to uncontrolled use of the compound . This can be by passed by controlled use of antibiotic and formulation of newer ones. J Dairy Sci, 2002 Apr, 85(4), 992 - 1001 Farmers' choice of medical treatment of mastitis in Danish dairy herds based on qualitative research interviews; Vaarst M et al.; A qualitative research study was conducted to describe and analyze farmers' perspectives on their own choices regarding decisions to have cows treated for mastitis . Through qualitative research interviews of 16 Danish dairy farmers, four levels of the decision-making process used by farmers to decide whether or not to treat a cow with antibiotics were identified . Those levels were: 1) symptom level (seriousness of the mastitis case), 2) cow level (to the extent a cow fulfilled goals of the farmer and the herd), 3) herd level (the situation of the herd, e.g., in relation to milk quota), and 4) level of alternatives (whether the farmer regards such practices as blinding of teats or homoeopathy as serious alternatives to antibiotic treatment) . All four levels could be recognized in all herds, but with differing weights and relative importance across herds . Directions of different possibilities within each level also varied among farmers . By identifying those four levels, a model for understanding the farmers' choices is provided . This provides background for dialogue with each farmer about choices in the context of each specific herd . It also provides insight into implications of mastitis treatments for effective treatment versus issues of antibiotic resistance when discussing choices on a more general level . Communication and understanding between farmers and their veterinarians and cattle-oriented advisors is essential . Farmers were shown to be coherent in their choices of treatment, but their decisions often seemed to differ from normal veterinary recommendations . Such differences have to be understood and implemented into effective decisions for the whole farm. J Appl Microbiol, 2002, 92(6), 1116 - 22 Novel intercellular communication system in Escherichia coli that confers antibiotic resistance between physically separated populations; Heal RD et al.; AIMS: To determine whether intercellular signalling can occur between physically separated populations of Escherichia coli . METHODS AND RESULTS: Intercellular signalling between physically discrete populations of E . coli BL21 was analysed in bi-partite Petri dishes . Transfer of a growth-promoting signal resulted in induction of resistance to the antibiotic ampicillin . Optimal expression of the signal occurred when the signalling population was established as a bacterial lawn for 24 h . This represented an entry into the stationary phase of growth, as indicated by the expression profile of the RNA polymerase subunit sigma38 (sigmaS; sigma S) . The growth-promoting effect was also observed when E . coli DH5alpha (luxS-) was used as the signalling population . Preventing passage of air between the two populations resulted in a complete cessation of the growth-promoting effect . CONCLUSIONS: A growth-promoting signal occurs between physically separated cultures of E . coli . The exact nature of the signal remains to be determined but does not involve the production of autoinducer-2 from the luxS gene . Signal transmission is likely to involve airborne transfer of a signal species . SIGNIFICANCE AND IMPACT OF THE STUDY: Intercellular signalling systems exist in bacteria that enable antibiotic resistance to be conferred between physically separated populations. Am J Gastroenterol, 2002 Apr, 97(4), 857 - 60 Colloidal bismuth subcitrate-based twice-a-day quadruple therapy as primary or salvage therapy for Helicobacter pylori infection; Dore MP et al.; OBJECTIVE: Helicobacter pylori (H . pylori) eradication rates in northern Sardinia using standard 1-wk triple therapies (i.e., a proton pump inhibitor and two antibiotics) are typically <60%, primarily because of antibiotic resistance . The aim of this study was to test b.i.d . quadruple therapy as primary and as salvage therapy in this population . METHODS: This was a prospective, single center study of consecutive dyspeptic H . pylori-infected patients . Therapy consisted of omeprazole 20 mg, tetracycline 500 mg, metronidazole 500 mg, and bismuth subcitrate caplets 240 mg, all b.i.d . with the midday and evening meals for 14 days . H . pylori status was evaluated by 13C-urea breath test and histology before and 4-6 wk after therapy . Eradication was defined as no positive test . RESULTS: We enrolled 118 consecutive dyspeptic patients (mean age 46 yr; 73 men, including 15 with peptic ulcer disease) . Of the patients, 42 (38%) had failed prior therapy: twice in 21 cases, three times in 12, and four or more times in nine . The intention-to-treat cure rate was 95% (110 of 116) (95% CI = 90-98%) overall, and 98% per protocol, irrespective of diagnosis, age, prior treatment failure, or smoking status . Moderate or severe side effects were experienced by only 5% of patients . CONCLUSIONS: Bismuth subcitrate-based b.i.d . quadruple therapy was an excellent primary and salvage therapy and should be considered as first line therapy. Wien Med Wochenschr, 2002, 152(5-6), 141 - 7 {Therapy of Helicobacter pylori infection: "Who to treat, how to treat?"}; Sebesta Ch et al.; Seventeen years after detection of Helicobacter pylori as the pathogenetic factor in a variety of gastroduodenal diseases, current treatment options are about 90% effective in eradicating the bacteria and thereby curing the affiliated disorders . In 1987 successful therapy has been shown to reduce relapse rates of duodenal and gastric ulcers dramatically and clinicians all over the world started to perform a still ongoing vast number of trials searching for the most effective first--and in case of failure, second line-treatment options . The results of these trials led to strictly evidence based and generally accepted guidelines formulated by Consensus Reports . The main questions "who to treat" and "how to treat" can be answered by using the categories "strongly recommended indications", "advisable indications" that leave some space for individual decisions, and consideration of first and second time eradication therapies together as a package . Treatment has become simple: three drugs, twice daily for one week . All available protone pump inhibitors (PPI) have been shown to be effective and presently there is only a few antibiotic agents used in combination and defined dose, proven to ensure treatment success . The future will encounter clinicians with problems concerning patient compliance, antibiotic resistance and more effective second and even third line therapies . This paper will focus on two subjects: indications ("who to treat") and evidence-based eradication regimens ("how to treat") in a reasonable setting close to everyday clinical practice. Emerg Infect Dis, 2002 May, 8(5), 496 - 502 Sentinel surveillance: a reliable way to track antibiotic resistance in communities? Schrag SJ, Zell ER, Schuchat A, Whitney CG. We used population-based data to evaluate how often groups of randomly selected clinical laboratories accurately estimated the prevalence of resistant pneumococci and captured trends in resistance over time . Surveillance for invasive pneumococcal disease was conducted in eight states from 1996 to 1998 . Within each surveillance area, we evaluated the proportion of all groups of three, four, and five laboratories that estimated the prevalence of penicillin-nonsusceptible pneumococci (%PNSP) and the change in %PNSP over time . We assessed whether sentinel groups detected emerging fluoroquinolone resistance . Groups of five performed best . Sentinel groups accurately predicted %PNSP in five states; states where they performed poorly had high between-laboratory variation in %PNSP . Sentinel groups detected large changes in prevalence of nonsusceptibility over time but rarely detected emerging fluoroquinolone resistance . Characteristics of hospital-affiliated laboratories were not useful predictors of a laboratory's %PNSP . Sentinel surveillance for resistant pneumococci can detect important trends over time but rarely detects newly emerging resistance profiles. J Am Chem Soc, 2002 May 15, 124(19), 5333 - 40 An ultrahigh resolution structure of TEM-1 beta-lactamase suggests a role for Glu166 as the general base in acylation; Minasov G et al.; Although TEM-1 beta-lactamase is among the best studied enzymes, its acylation mechanism remains controversial . To investigate this problem, the structure of TEM-1 in complex with an acylation transition-state analogue was determined at ultrahigh resolution (0.85 A) by X-ray crystallography . The quality of the data was such as to allow for refinement to an R-factor of 9.1% and an R(free) of 11.2% . In the resulting structure, the electron density features were clear enough to differentiate between single and double bonds in carboxylate groups, to identify multiple conformations that are occupied by residues and loops, and to assign 70% of the protons in the protein . Unexpectedly, even at pH 8.0 where the protein was crystallized, the active site residue Glu166 is clearly protonated . This supports the hypothesis that Glu166 is the general base in the acylation half of the reaction cycle . This structure suggests that Glu166 acts through the catalytic water to activate Ser70 for nucleophilic attack on the beta-lactam ring of the substrate . The hydrolytic mechanism of class A beta-lactamases, such as TEM-1, appears to be symmetrical, as are the serine proteases . Apart from its mechanistic implications, this atomic resolution structure affords an unusually detailed view of the structure, dynamics, and hydrogen-bonding networks of TEM-1, which may be useful for the design of inhibitors against this key antibiotic resistance target. Ir J Med Sci, 2002 Jan-Mar, 171(1), 42 - 3 Vancomycin-resistant peritonitis associated with peritoneal dialysis: a cause for concern; O'Riordan A et al.; BACKGROUND: Peritonitis resulting from peritoneal dialysis (PD) remains a serious cause of morbidity and even mortality among dialysis patients . AIM: To highlight the danger of antibiotic resistance in patients on dialysis who have received multiple courses of antibiotics . METHODS: Two cases are reported in which the patients developed peritonitis resistant to vancomycin . CONCLUSIONS: Multi-drug resistance is a growing danger . It is imperative to use the most appropriate antibiotics in the proper dosage . If infections persist, early removal of the catheters is essential . The use of antibiotics in PD patients needs to be limited . Sensitivity patterns of the cultured organisms must be monitored regularly as the lack of vigilance may help accelerate the development of the so-called 'super bug' resistant to all antibiotics. Vaccine, 2002 May 6, 20(15), 1999 - 2003 Issues for improving multiplasmid DNA vaccines for HIV-1; Muthumani K et al.; Since the first reports of plasmid vaccines, there have been substantial changes made to the design of plasmid backbones, as well as to the antibiotic resistance markers chosen for clinical vectors compared with first generation vectors . These changes aid manufacturing, production and scale up and at the same time aid conceptual safety by limiting the ability of the vaccines to transfer useful genetic selection genes to other bacterial infectious agents . In contrast, there has been little change to the original promoters or polyadenlyation tracts in the last decade.We have learned that these first generation plasmid vaccines for HIV-1 appear very well tolerated in humans . However, while safe and immunogenic, improving the immune potency of DNA vaccines is a critical goal for this technology . The combination of antigens used should be carefully examined for possible immune interference . Such interference may only become apparent when each component of the vaccine is tested individually . This interference also suggests one mechanism of immune pathogenesis possibly by HIV-1 . Optimization of the immune response can come through manipulation of the transfection efficiency, expression or through the use of various T cell and B cell plasmid adjuvants . It is likely that the combination of such advancements will significantly improve the clinical phenotype of this important vaccine modality. Plasmid, 2002 Mar, 47(2), 129 - 37 Construction of mini-Tn4001tet and its use in Mycoplasma gallisepticum; Pour-El I et al.; The Mollicutes are a group of cell-wall-less bacteria and are important plant and animal pathogens . Progress toward analyzing their pathogenic mechanisms has been hampered by the few available genetic tools . Of the two transposons shown to function in mycoplasmas, only Tn4001 is readily amenable to modification and development . One disadvantage of using Tn4001 in mycoplasmas has been independent insertion of the insertion sequence, IS256, probably as a result of inadequate control of the transposase expression in mycoplasmas . In this study, we describe the construction of a mini-Tn4001 containing the tetM antibiotic resistance gene from Tn916 . The transposase gene was placed outside the inverted repeats to lower the frequency of independent transposition events . Transposition of mini-Tn4001tet in Mycoplasma gallisepticum occurred at a frequency of 1-8 x 10(-6), a frequency similar to that of the parent transposon . Insertions of mini-Tn4001tet were random and only single insertions were observed . Several unique restriction sites between the inverted repeat sequences provide for further development of mini-Tn4001 . Curr Opin Biotechnol, 2002 Apr, 13(2), 173 - 80 Marker-free transformation: increasing transformation frequency by the use of regeneration-promoting genes; Zuo J et al.; The generation of transgenic plants free of antibiotic resistance markers is a major challenge to plant biologists and plant breeders . Currently, there are two main strategies to achieve this goal: one approach is to excise or segregate marker genes from the host genome after regeneration of transgenic plants, and the second is based on so-called 'marker-free' transformation . Marker-free transformation has been successfully demonstrated by the use of several plant and non-plant genes that are capable of promoting explant regeneration . This approach appears not only to be effective for the generation of marker-free transgenic plants, but also has great potential to improve the transformation frequency of recalcitrant species. Proc Natl Acad Sci U S A, 2002 Apr 16, 99(8), 5601 - 5 Epub 2002 Apr 09. How to assess the relative importance of different colonization routes of pathogens within hospital settings; Pelupessy I et al.; The emergence of antibiotic resistance among nosocomial pathogens has reemphasized the need for effective infection control strategies . The spread of resistant pathogens within hospital settings proceeds along various routes of transmission and is characterized by large fluctuations in prevalence, which are typical for small populations . Identification of the most important route of colonization (exogenous by cross-transmission or endogenous caused by the selective pressure of antibiotics) is important for the design of optimal infection control strategies . Such identification can be based on a combination of epidemiological surveillance and costly and laborious as well as time-consuming methods of genotyping . Furthermore, analysis of the effects of interventions is hampered by the natural fluctuations in prevalence . To overcome these problems, we introduce a mathematical algorithm based on a Markov chain description . The input is longitudinal prevalence data only . The output is estimates of the key parameters characterizing the two colonization routes . The algorithm is tested on two longitudinal surveillance data sets of intensive care patients . The quality of the estimates is determined by comparing them to accurate estimates based on additional information obtained by genotyping . The results warrant optimism that this algorithm may help to quantify transmission dynamics and can be used to evaluate the effects of infection control interventions more carefully. J Clin Pharmacol, 2002 Apr, 42(4), 461 - 6 Lack of bioequivalence of ciprofloxacin when administered with calcium-fortified orange juice: a new twist on an old interaction; Neuhofel AL et al.; Fluoroquinolones are known to interact with drugs containing multivalent ions . Current Food and Drug Administration (FDA) labeling states that ciprofloxacin and most other fluoroquinolones are safe to be given with food and dietary calcium but not calcium supplements . Although many of the currently marketed calcium fortified foods have calcium contents that usually exceed those in dietary calcium sources, it is unclear whether they represent a risk for less than optimal absorption of fluoroquinolones, which may result in subsequent clinical failures due to lack of bacterial eradication and antibiotic resistance . The purpose of this three-way, randomized, crossover study was to characterize and compare the bioequivalence of single doses of oral ciprofloxacin in 15 healthy volunteers when administered with water, concurrently with orange juice, and concurrently with calcium-fortified orange juice . Compared to the control arm, the Cmax of ciprofloxacin significantly decreased when it was given with orange juice (23%, p = 0.001) and with calcium-fortified orange juice (41%, p < 0.001) . Twenty-four-hour ciprofloxacin AUCs were also decreased for both forms of the orange juice (22% {p < 0.001} and 38% {p < 0.001}, respectively) . When compared to each other, neither of the orange juice regimens were bioequivalent to each other, with the Cmax and AUC for the fortified form being 22% (p = 0.005) and 21% (p = 0.015) lower than those of the nonfortified form . By FDA standards, although ciprofloxacin is marginally bioequivalent when administered with orange juice, it is not when it is administered with calcium-fortified orange juice . The changes in Cmax and AUC have the potential to significantly decrease clinical efficacy and promote antibiotic resistance . Not warning patients about potential food-drug interactions with fortified foods may be a major unrealized and unstudied inadvertent source of clinical failures and resistance trends with fluoroquinolones. J Chemother, 2001 Nov, 13 Spec No 1(1), 108 - 11 Antibiotic prophylaxis in clean surgery: breast surgery and hernia repair; D'Amico DF et al.; Use of prophylactic antibiotics in clean surgery is still controversial . We reviewed the literature of the last 10 years to identify the best way to approach clean surgery . The question is more important for patients undergoing breast surgery . The presence of an infected breast wound delays the beginning of postoperative adjuvant anticancer therapy: there is good evidence to suggest that delayed adjuvant therapy compromises the outcome for patients in terms of both local control and survival . There are several clinical trials that have addressed the efficacy of prophylactic antibiotics for patients undergoing breast surgery and hernia repair . Platt et al assessed the efficacy of preoperative antibiotic prophylaxis in a clinical trial of 1218 patients undergoing clean surgery with an absolute reduction rate of 39% in wound infections . Gupta et al reported no influence on the incidence of infective complications by antibiotic prophylaxis in 357 patients undergoing elective breast surgery . Like breast surgery, use of prophylaxis in hernia repair is not clear: a prospective, randomized, double-blind, multicenter study of 619 patients assessed no benefit of antibiotic prophylaxis . On the other hand Lewis et al reported a 75% reduction of infections in low-risk patients when a single dose of cefotaxime was used in clean operations . A particularly interesting point is the use of prosthetic mesh in hernia repair and primary reconstructive surgery in breast surgery . Amland et al reported a significant reduction of the incidence of wound infections in a group of patients undergoing reconstructive breast surgery, receiving azithromycin vs placebo (5% vs 20%) . In hernia repair we stress the need to prevent wound infections: currently Liechtestein's technique is widely performed all over the world . Mesh infection is an unpleasant event that requires prosthesis removal . The lack of conclusive studies about antibiotic prophylaxis in clean surgery suggests that a single-dose of cephalosporin at the induction of anesthesia may be prudent . This procedure is certainly inexpensive and safe and, more importantly, probably does not have an impact on antibiotic resistance. Aliment Pharmacol Ther, 2002 Apr, 16(4), 683 - 91 Review article: an approach to Helicobacter pylori infection in the elderly; Pilotto A et al.; The prevalence of Helicobacter pylori infection increases with age world-wide, reaching levels of 40-60% in asymptomatic elderly subjects and over 70% in elderly patients with gastroduodenal diseases . However, the percentage of H . pylori-positive elderly patients who are treated for their infection remains very low . Data are now available that demonstrate the benefit of curing H . pylori infection in elderly patients with H . pylori-associated peptic ulcer disease and severe chronic gastritis . Furthermore, the cure of H . pylori may prevent the progression of intestinal metaplasia and gastric atrophy . New studies are needed to clarify the role of eradication in elderly patients with non-ulcer dyspepsia and gastro-oesophageal reflux disease and in those who use non-steroidal anti-inflammatory drugs . H . pylori infection may be easily diagnosed by histological evaluation, rapid urease test or culture performed on gastric biopsies taken during endoscopy . However, the biopsy site must be carefully selected in elderly patients . For non-invasive monitoring of H . pylori infection after treatment, the 13C-urea breath test has significantly higher accuracy than serology in the elderly; further studies are needed to clarify the role of the H . pylori stool antigen test in old age . One-week proton pump inhibitor-based triple therapy regimens, including clarithromycin, amoxicillin and/or nitroimidazoles, are highly effective and well tolerated in elderly patients . Low doses of both proton pump inhibitors and clarithromycin (in combination with standard doses of amoxicillin or nitroimidazoles) are sufficient . Low compliance and antibiotic resistance are the main factors related to treatment failure in old age. Avian Dis, 2002 Jan-Mar, 46(1), 143 - 52 Pathotypes of avian Escherichia coli as related to tsh-, pap-, pil-, and iuc-DNA sequences, and antibiotic sensitivity of isolates from internal tissues and the cloacae of broilers; Ngeleka M et al.; One hundred four Escherichia coli isolates were collected from internal tissues and the cloacae of broilers with colibacillosis or from the cloacae of healthy birds . The isolates were tested for the presence of DNA sequences for temperature-sensitive hemagglutinin (tsh), for P (pap) and F1 (pil) fimbriae, and for aerobactin synthesis (iuc) by DNA/DNA hybridization . The isolates were also tested for O1, O2, and O78 serogroups, serum and antibiotic resistance, and virulence in day-old chickens . The Tsh/Pil/Iuc was the major pathotype detected in 53.8% of isolates from internal tissues, as compared with only 28.8% of isolates from the cloacae . The Tsh/Pap/Iuc pathotype was detected at a lower frequency (15.4%) but only in isolates from internal tissues . Among the virulence-associated marker genes, tsh and iuc were detected in most of the isolates from internal tissues (90.4% and 92.3%), as compared with only 51.9% and 63.5% of isolates from the cloacae, respectively, pap was detected to a lesser extent, in 25% of isolates but only from internal tissues . In contrast to the pil gene, the tsh-, pap-, and iuc-DNA sequences were more frequently detected in isolates from internal tissues than in isolates from the cloacae . O-antigen typing revealed that 25% of isolates belonged to serogroups O1 (4.8%), O2 (9.6%), and O78 (10.6%) . Although most isolates appeared to be resistant to serum, only isolates from internal tissues were virulent in day-old chickens in contrast to isolates from the cloacae . More than 10% of isolates were resistant to most of the antibiotics used for the study . However, less resistance to enrofloxacin and norfloxacin was observed . Our data suggest that the Tsh/Pil/Iuc and Tsh/Pap/Iuc pathotypes and Tsh and Iuc virulence-associated markers are important factors of avian pathogenic E . coli . Enrofloxacin appeared to be the best choice for treatment of the infection. J Paediatr Child Health, 2001 Dec, 37(6), 529 - 30 Perspectives on infective ear disease in indigenous Australian children; Leach AJ et al.; Australian Aboriginal children experience the highest rates of bacterial respiratory diseases reported in the literature . Neonatal acquisition of multiple bacterial pathogenic species and strains predicts persistent and severe disease throughout childhood, particularly infective ear disease . The dynamics of bacterial nasopharyngeal colonization and transmission are poorly understood . The importance of host factors, bacterial competition and co-operation in the transition from asymptomatic carriage to disease are also uncertain . Treatment outcomes are poor, possibly due to the high density of bacterial infection following early age exposure, poor compliance and increasing levels of antibiotic resistance . The relationship between antibiotic use, clinical outcomes and bacterial resistance needs to be better understood in high-risk populations if the benefits associated with treatment are to be maximized . While there is an urgent need for vaccines, the early age of infection and the high rates of transmission and bacterial antigenic diversity mean these may also be less effective than predicted from studies in low-risk populations. Prev Med, 2002 Mar, 34(3), 346 - 52 Antibiotic use and upper respiratory infections: a survey of knowledge, attitudes, and experience in Wisconsin and Minnesota; Belongia EA et al.; BACKGROUND: Public attitudes and expectations contribute to inappropriate antibiotic prescribing and antibiotic resistance . This study assessed knowledge, attitudes, and experiences regarding antibiotic use for respiratory infection or illness . METHODS: Random-digit-dialing telephone surveys of adults and parents of children <5 years old were conducted in Wisconsin and Minnesota during 1999 . RESULTS: The survey was completed by 405 adults and 275 parents of children <5 years old . The median age was 32 years for parents and 50 years for adults . Seven percent of parents and 17% of adults believed that antibiotics are never or almost never necessary for bronchitis . More than 70% in each group believed that antibiotics are needed for green or yellow nasal drainage, and nearly half of respondents believed that they knew whether an antibiotic was needed before seeing a physician . Exposure to multiple information sources on antibiotic resistance in the past 6 months was independently associated with a knowledge score greater than or equal to the median for nine questions . CONCLUSIONS: The general public has misconceptions regarding indications for antibiotic use, and this may contribute to inappropriate prescribing . Providing multiple and varied antibiotic-related informational messages may increase knowledge of appropriate antibiotic prescribing and decrease patient demand for antibiotics . (C)2002 American Health Foundation and Elsevier Science (USA). Mol Biochem Parasitol, 2002 Apr 9, 120(2), 195 - 204 A new expression vector for Crithidia fasciculata and Leishmania; Tetaud E et al.; Crithidia fasciculata is a monogenetic parasite of insects . It grows in fully defined media without requiring serum, which facilitates biochemical analysis . We have constructed a series of expression systems that allows expression of transfected genes in the kinetoplastid protozoa Crithidia and Leishmania . These cells can be readily transfected with plasmid DNA by electroporation and transformants selected with various antibiotic resistance markers . 5'-Trans-splicing signals and poorly defined regions within the 3'-untranslated regions of genes are required for optimal expression of genes in trypanosomatids . We, therefore, inserted the intergenic region of the C . fasciculata phosphoglycerate kinase (PGK) genes A and B, which allows polyadenylation of the target gene and spliced leader addition to the selectable marker gene . Part of the intergenic region of the PGK locus was added upstream of the target gene to permit its trans-splicing . A 3'-untranslated sequence from the Crithidia glutathionylspermidine synthetase (GSPS) was also added to allow the polyadenylation of the selectable marker gene . Genes can be readily inserted using a multiple cloning site and can be expressed as a fusion protein with a poly-histidine sequence at either the N or C-terminus or fused with green fluorescent protein . Biologically active proteins can be expressed in C . fasciculata or L . amazonensis promastigotes and purified by affinity chromatography using a metal chelating column. Semin Respir Infect, 2002 Mar, 17(1), 57 - 64 Old and new antibiotics for pediatric pneumonia; Bradley JS; A variety of antibiotics, both parenteral and oral, are available to the clinician caring for a child with pneumonia . Although viral pathogens are the common etiologic agents causing community-acquired pneumonia, significant morbidity and mortality exists from disease caused by bacteria and atypical pneumonia agents . Treatment of nosocomial bacterial pneumonia has become particularly difficult with ever-increasing resistance documented in hospital-acquired organisms . This article discusses antibiotic therapy based on clinical presentation and based on identified pathogens, with a discussion of mechanisms of antibiotic resistance and the newer agents that have been designed to meet this continually evolving challenge. J Biol Chem, 2002 May 17, 277(20), 18118 - 26 Epub 2002 Mar 07. Investigation of the roles of catalytic residues in serotonin N-acetyltransferase; Scheibner KA et al.; Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase (AANAT)) is a critical enzyme in the light-mediated regulation of melatonin production and circadian rhythm . It is a member of the GNAT (GCN-5-related N-acetyltransferase) superfamily of enzymes, which catalyze a diverse array of biologically important acetyl transfer reactions from antibiotic resistance to chromatin remodeling . In this study, we probed the functional properties of two histidines (His-120 and His-122) and a tyrosine (Tyr-168) postulated to be important in the mechanism of AANAT based on prior x-ray structural and biochemical studies . Using a combination of steady-state kinetic measurements of microviscosity effects and pH dependence on the H122Q, H120Q, and H120Q/H122Q AANAT mutants, we show that His-122 (with an apparent pK(a) of 7.3) contributes approximately 6-fold to the acetyltransferase chemical step as either a remote catalytic base or hydrogen bond donor . Furthermore, His-120 and His-122 appear to contribute redundantly to this function . By analysis of the Y168F AANAT mutant, it was demonstrated that Tyr-168 contributes approximately 150-fold to the acetyltransferase chemical step and is responsible for the basic limb of the pH-rate profile with an apparent (subnormal) pK(a) of 8.5 . Paradoxically, Y168F AANAT showed 10-fold enhanced apparent affinity for acetyl-CoA despite the loss of a hydrogen bond between the Tyr phenol and the CoA sulfur atom . The X-ray crystal structure of Y168F AANAT bound to a bisubstrate analog inhibitor showed no significant structural perturbation of the enzyme compared with the wild-type complex, but revealed the loss of dual inhibitor conformations present in the wild-type complex . Taken together with kinetic measurements, these crystallographic studies allow us to propose the relevant structural conformations related to the distinct alkyltransferase and acetyltransferase reactions catalyzed by AANAT . These findings have significant implications for understanding GNAT catalysis and the design of potent and selective inhibitors. J Am Chem Soc, 2002 Mar 13, 124(10), 2318 - 26 EPR study of substrate binding to the Mn(II) active site of the bacterial antibiotic resistance enzyme FosA: a better way to examine Mn(II); Smoukov SK et al.; FosA is a manganese metalloglutathione transferase that confers resistance to the broad-spectrum antibiotic fosfomycin, (1R,2S)-epoxypropylphosphonic acid . The reaction catalyzed by FosA involves the attack by glutathione on fosfomycin to yield the product 1-(S-glutathionyl)-2-hydroxypropylphosphonic acid . The enzyme is a dimer of 16 kDa subunits, each of which harbors one mononuclear Mn(II) site . The coordination environment of the Mn(II) in the FosA x Mn(2+) complex is composed of a glutamate and two histidine ligands and three water molecules . Here we report EPR spectroscopic studies on FosA, in which EPR spectra were obtained at 35 GHz and 2 K using dispersion-detection rapid-passage techniques . This approach provides an absorption envelope line shape, in contrast to the conventional (slow-passage) derivative line shape, and is a more reliable way to collect spectra from Mn(II) centers with large zero-field splitting . We obtain excellent spectra of FosA bound with substrate, substrate analogue phosphate ion, and product, whereas these states cannot be studied by X-band, slow-passage methods . Simulation of the EPR spectra shows that binding of substrate or analogue causes a profound change in the electronic parameters of the Mn(II) ion . The axial zero-field splitting changes from {D} = 0.06 cm(-1) for substrate-free enzyme to 0.23 cm(-1) for fosfomycin-bound enzyme, 0.28 (1) cm(-1) for FosA with phosphate, and 0.27 (1) cm(-1) with product . Such a large zero-field splitting is uncommon for Mn(II) . A simple ligand field analysis of this change indicates that binding of the phosphonate/phosphate group of substrate or analogue changes the electronic energy levels of the Mn(II) 3d orbitals by several thousand cm(-1), indicative of a significant change in the Mn(II) coordination sphere . Comparison with related enzymes (glyoxalase I and MnSOD) suggests that the change in the coordination environment on substrate binding may correspond to loss of the glutamate ligand. BMC Biochem . 2002;3(1):4 . Epub 2002 Feb 13. Probing substrate binding to metallo-beta-lactamase L1 from Stenotrophomonas maltophilia by using site-directed mutagenesis; Carenbauer AL et al.; BACKGROUND: The metallo-beta-lactamases are Zn(II)-containing enzymes that hydrolyze the beta-lactam bond in penicillins, cephalosporins, and carbapenems and are involved in bacterial antibiotic resistance . There are at least 20 distinct organisms that produce a metallo-beta-lactamase, and these enzymes have been extensively studied using X-ray crystallographic, computational, kinetic, and inhibition studies; however, much is still unknown about how substrates bind and the catalytic mechanism . In an effort to probe substrate binding to metallo-beta-lactamase L1 from Stenotrophomonas maltophilia, nine site-directed mutants of L1 were prepared and characterized using metal analyses, CD spectroscopy, and pre-steady state and steady state kinetics . RESULTS: Site-directed mutations were generated of amino acids previously predicted to be important in substrate binding . Steady-state kinetic studies using the mutant enzymes and 9 different substrates demonstrated varying Km and kcat values for the different enzymes and substrates and that no direct correlation between Km and the effect of the mutation on substrate binding could be drawn . Stopped-flow fluorescence studies using nitrocefin as the substrate showed that only the S224D and Y228A mutants exhibited weaker nitrocefin binding . CONCLUSIONS: The data presented herein indicate that Ser224, Ile164, Phe158, Tyr228, and Asn233 are not essential for tight binding of substrate to metallo-beta-lactamase L1 . The results in this work also show that Km values are not reliable for showing substrate binding, and there is no correlation between substrate binding and the amount of reaction intermediate formed during the reaction . This work represents the first experimental testing of one of the computational models of the metallo-beta-lactamases. Aliment Pharmacol Ther, 2002 Mar, 16(3), 569 - 75 Effectiveness of two quadruple, tetracycline- or clarithromycin-containing, second-line, Helicobacter pylori eradication therapies; Georgopoulos SD et al.; BACKGROUND: There are no guidelines on second-line therapies for Helicobacter pylori eradication failures of omeprazole-clarithromycin-amoxicillin triple therapy . AIM: To compare the efficacy of two second-line therapies for persistent H . pylori infection . METHODS: Over a 6-year period, patients with persistent H . pylori infection following omeprazole-clarithromycin-amoxicillin eradication therapy were randomized to receive omeprazole, 20 mg twice daily, bismuth, 120 mg four times daily, metronidazole, 500 mg twice daily, and either tetracycline, 500 mg four times daily, or clarithromycin, 500 mg twice daily, given for 7 days . Before therapy, patients underwent endoscopy with biopsies for histology, culture and antibiotic susceptibility tests . H . pylori infection was confirmed by histology . RESULTS: Of the 95 randomized patients, 88 (93%) completed the study . Age, sex, smoking, ulcer/non-ulcer dyspepsia ratio and antibiotic resistance were not significantly different between the treatment groups . On intention-to-treat analysis, eradication was achieved in 41 of the 49 patients (84%; 95% confidence interval, 70.4-92.7%) and 27 of the 46 patients (59%; 95% confidence interval, 43.3-73.0%) of the tetracycline- and clarithromycin-containing groups, respectively (P=0.007) . On multivariate regression analysis, the sensitivity of H . pylori to metronidazole had a likelihood ratio of 5.2 (P=0.022), followed by the type of quadruple therapy (likelihood ratio, 4.4; P=0.036) . CONCLUSIONS: Tetracycline-containing quadruple rescue therapy is highly effective in treating H . pylori eradication failures of the omeprazole-amoxicillin-clarithromycin regimen. J Bacteriol, 2002 Mar, 184(6), 1712 - 21 IntI2 integron integrase in Tn7; Hansson K et al.; Integrons can insert and excise antibiotic resistance genes on plasmids in bacteria by site-specific recombination . Class 1 integrons code for an integrase, IntI1 (337 amino acids in length), and are generally borne on elements derived from Tn5090, such as that found in the central part of Tn21 . A second class of integron is found on transposon Tn7 and its relatives . We have completed the sequence of the Tn7 integrase gene, intI2, which contains an internal stop codon . This codon was found to be conserved among intI2 genes on three other Tn7-like transposons harboring different cassettes . The predicted peptide sequence (IntI2*) is 325 amino acids long and is 46% identical to IntI1 . In order to detect recombination activity, the internal stop codon at position 179 in the parental allele was changed to a triplet coding for glutamic acid . The sequences flanking the cassette arrays in the class 1 and 2 integrons are not closely related, but a common pool of mobile cassettes is used by the different integron classes; two of the three antibiotic resistance cassettes on Tn7 and its close relatives are also found in various class 1 integrons . We also observed a fourth excisable cassette downstream of those described previously in Tn7 . The fourth cassette encodes a 165-amino-acid protein of unknown function with 6.5 contiguous repeats of a sequence coding for 7 amino acids . IntI2*179E promoted site-specific excision of each of the cassettes in Tn7 at different frequencies . The integrases from Tn21 and Tn7 showed limited cross-specificity in that IntI1 could excise all cassettes from both Tn21 and Tn7 . However, we did not observe a corresponding excision of the aadA1 cassette from Tn21 by IntI2*179E. J Antimicrob Chemother, 2002 Mar, 49(3), 541 - 3 In vitro activity of rifampicin against Helicobacter pylori isolated from children and adults; Fujimura S et al.; Antibiotic resistance in Helicobacter pylori has been a growing problem with current treatment regimens . However, second-line therapy for cases with eradication failure remains to be established . The aim of this study was to evaluate (in vitro) the potential use of rifampicin for H . pylori eradication . The susceptibility of 52 H . pylori strains isolated from children and adults to amoxicillin, clarithromycin, metronidazole and rifampicin was determined . Forty H . pylori isolates susceptible to these antibiotics were investigated for in vitro inducement of resistance . All isolates were susceptible to rifampicin . None of the H . pylori isolates tested acquired rifampicin resistance during prolonged exposure in in vitro inducement experiments . It is suggested that rifampicin has stable activity against H . pylori. Clin Microbiol Infect, 1998 Apr, 4(4), 213 - 219 Isolation and characterization of enteroaggregative Escherichia coli (EAggEC) by genotypic and phenotypic markers, isolated from diarrheal children in Congo; Jalaluddin S et al.; OBJECTIVE: To determine the prevalence of enteroaggregative Escherichia coli (EAggEC) in African diarrheal children in Lwiro, Congo, to characterize EAggEC isolates by possible genotypic and phenotypic markers, and to evaluate the EAggEC probe pCVD432 in identifying EAggEC . METHODS: The Hep-2 cell adhesion assay and colony-blot hybridization assays were carried out for the identification of EAggEC . O:H serotyping, biotyping, antibiogram and plasmid-profile analysis were done . To detect the E . coli LT and ST, ELISA tests were used and, for VT, a vero cell assay was used . RESULTS: EAggEC strains were isolated from 56 out of 115 diarrheal children (48.7%): the organism was present alone and presumed to cause diarrhea in 22 (19.1%) cases . The rest of the cases were associated with two or more diarrheagenic E . coli strains . EAggEC strains were isolated from 25% of total diarrheal children (first day of isolation) and 8.86% of age-matched healthy individuals (p<0.03) . This isolation rate was significantly higher than the one found for other diarrheagenic E . coli strains . In parallel, we evaluated the sensitivity and specificity of EAggEC probe pCVD432, and found that it had 56% sensitivity with 100% specificity compared with the Hep-2 cell test . EAggEC isolates were characterized by serotyping, biotyping, antibiotic resistance pattern, plasmid profiling and toxin production analysis . They did not produce any one of these classical toxins and nor did they relate to any particular serotypes . Plasmid analysis of the 79 EAggEC isolates (n=315) showed seven different profiles . Ten resistance patterns were identified and 34 strains were sensitive to all drugs . There was no association between plasmid profiles and antibiotic resistance patterns . All 16 classical E . coli biotypes were found in this small EAggEC population . CONCLUSIONS: EAggEC has been emerging as a cause of childhood diarrhea in African children in Congo . From the accumulated data it was found that there is a great heterogeneity in EAggEC populations. Methods, 2001 Nov, 25(3), 358 - 64 In vivo selection of functional variations in essential sites of ribosomal RNA; Moine H et al.; The technique of "in vivo selection of functional ribosomes" is a genetic approach to dissecting the link between the structure and function of critical sites of rRNA . This method proceeds through selection of functional variants among cells that express ribosomes from a pool of rRNA-containing randomized sites . The selection of bacterial clones with functional ribosomes is based on the use of a plasmid carrying a rRNA operon in which a site of interest has been randomized and a point mutation conferring an antibiotic resistance has been introduced . Cells expressing functional ribosomes are then selected on medium containing the antibiotic . With this approach one can isolate at once all the possible variations at a given rRNA site that are able to sustain normal ribosome function . The identification of covariations in between several nucleotides that maintain wild-type ribosome activity can thus help demonstrate the function of specific interactions in rRNA . Virology, 2002 Feb 1, 293(1), 26 - 30 Generation of E3-deleted canine adenoviruses expressing canine parvovirus capsid by homologous recombination in bacteria; Morrison MD et al.; E3-deleted canine adenovirus type 1 (CAV-1) was generated by homologous recombination in bacterial cells, using an antibiotic resistance marker to facilitate the recovery of recombinants . This marker was flanked by unique restriction endonuclease sites, which allowed its subsequent removal and the insertion of cassettes expressing the canine parvovirus capsid at the E3 locus . Infectious virus was recovered following transfection of canine cells and capsid expression was observed by RT-PCR from one of the virus constructs . A second construct, containing a different promoter, showed delayed growth and genome instability which, based on the size difference between these inserts, suggests a maximum packaging size of 106 to 109% wild-type genome size for CAV-1. Ther Umsch, 2002 Jan, 59(1), 51 - 5 {Successful strategies against increasing antibiotic resistance}; Sax H; The development of antibiotic resistance, known since the early days of antibiotic therapy, has accelerated in recent years and makes a postantibiotic era a realistic future scenario . The battle against increasingly resistant strains has become a major concern for infectious disease and infection control specialists worldwide . The issue has now gained heightened awareness among physicians in all specialties and the public, and has become a challenge to hospital leadership . Detailed evidence about the causes and conditions of antibiotic resistance is accumulating, but the main axes have been known for some time: overuse and misuse of antibiotic therapy and transmission of multiresistant pathogens or genetic material between humans . Selection pressure by the systematic use of antibiotics as growth promoters in animal husbandry and the resulting transmission by the food chain is an additional important cause . This overview presents the most promising strategies to contain further resistance spread and hopefully inverse the current trend. Ther Umsch, 2002 Jan, 59(1), 5 - 10 {Mechanisms of antibiotic resistance}; Muhlemann K; Antibiotics interfere with structural and regulatory elements of bacterial cells leading to growth arrest or cell death . Bacteria have evolved a variety of strategies to overcome the effects of antibiotics . Examples are enzymatic destruction, alteration of the target, efflux and permeability changes . Resistance towards the same substance can be mediated by several mechanisms . Efflux pumps can probably act as mediators of higher resistance development . Alteration of common targets can lead to cross-resistance against several classes of antibiotics . Genetic events, such as point mutations, transfer of plasmids and gen regulation, can mediate a rapid emergence of resistance . Therefore, substances like rifampicin should be only used in combination with other drugs . Accumulation of resistance genes under common regulatory control in integrons induces co-resistance against substances of different specificity . Detailed knowledge of resistance mechanisms, their evolution and dynamics is important for a rational use of antibiotics and other strategies against antibiotic resistance. J Appl Microbiol, 2002, 92(1), 109 - 17 Comparison of molecular and antibiotic resistance profile methods for the population analysis of Bradyrhizobium spp . (TGx) isolates that nodulate the new TGx soybean cultivars in Africa; Abaidoo RC et al.; AIMS: Comparison of molecular and antibiotic resistance profile methods to identify an easy method that can differentiate between strains of introduced Bradyrhizobium japonicum and the indigenous Bradyrhizobium spp . (TGx) isolates which nodulate the newly developed TGx soybean cultivars in Africa . METHODS AND RESULTS: Restriction fragment length polymorphism (RFLP) of 16S rDNA generated by five restriction enzymes, banding patterns in Southern hybridization using nod and nif genes as probes, and resistance patterns of the isolates to nine antibiotics, were used to group 26 Bradyrhizobium spp . (TGx) isolates and four other Bradyrhizobium strains . The clusters of isolates obtained from the four grouping methods were all different, although all methods revealed large genetic diversity among the isolates . CONCLUSIONS: Results indicate that the antibiotic resistance profile method is as good as the three molecular methods used in this study for phylogenetic grouping of the Bradyrhizobium spp . (TGx) isolates, which may serve as a basis for further characterization of selected isolates from each group . SIGNIFICANCE AND IMPACT OF THE STUDY: The antibiotic resistance profile method can be used as a simple means of assessing genetic variability and grouping of a large number of Bradyrhizobium spp . (TGx) isolates . Representative isolates from each group can then be selected for further characterization. Aliment Pharmacol Ther, 2002 Mar, 16 Suppl 1, 24 - 30 Review article: treatment of Helicobacter pylori infection and factors influencing eradication; Qasim A et al.; Currently available Helicobacter pylori eradication therapies are considered very effective and safe . The most recent eradication guidelines proposed in the Maastricht 2-2000 Consensus Report recommend the use of proton pump inhibitors (standard b.d.) along with clarithromycin (500 mg b.d.) and amoxycillin (1000 mg b.d.) or metronidazole (500 mg b.d.) for a minimum of 7 days . The combination of amoxycillin and clarithromycin is preferred because it may favour best results with a second-line proton pump inhibitor quadruple therapy . The recommended second-line therapy includes a combination of a proton pump inhibitor (standard b.d.) with bismuth salt (subsalicylate/subcitrate 120 mg q.d.s.), metronidazole (500 mg t.d.s.), and tetracycline (500 mg q.d.s.) for a minimum of 7 days . Extended proton pump inhibitor-based triple therapy can be used if bismuth is not available . Specialists should manage subsequent failures . Based on direct and indirect evidence from well-designed studies and clinical experience, eradication is recommended in gastric and duodenal ulcers, MALToma, atrophic gastritis, postgastric cancer resection, and in first-degree relatives of gastric cancer patients . The most common reason for treatment failure is poor compliance with eradication guidelines . Antibiotic resistance may be a significant factor in certain geographical areas . Proton pump inhibitors are an integral part of the eradication regimens as proved by meta-analyses of clinical trials . Novel agents used in secondary failure are few and depend on the use of new antibiotics . The role of H . pylori-specific antibiotics, probiotics, and vaccines is not established as yet . Widespread acceptance of the eradication guidelines should be regarded as the single most important factor in eradication success. Biotechniques, 2002 Feb, 32(2), 300, 302 - 4 Rapid construction of sequencing templates by random insertion of antibiotic resistance genes; Nabirochkina EN et al.; We describe a novel and handy method for generating a population of templates for sequencing . The method is based on the random insertion of antibiotic resistance gene in plasmid DNA digested by DNase I . The advantages of this approach are the small quantity of DNA necessary for mutagenesis and the complete independence from the restriction map of the plasmid . DNase I digestion provides a random distribution of the insertions, while antibiotic selection provides low background . We also present a convenient PCR-based procedure for the analysis and ordering of obtained insertion mutants. J Gen Intern Med, 2002 Feb, 17(2), 87 - 94 Tensions in antibiotic prescribing: pitting social concerns against the interests of individual patients; Metlay JP et a