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Summary Background Animals can act as a reservoir and source for the emergence of novel meticillin-resistant Staphylococcus aureus (MRSA) clones in human beings. Here, we report the discovery of a strain of S aureus (LGA251) isolated from bulk milk that was phenotypically resistant to meticillin but tested negative for the mecA gene and a preliminary investigation of the extent to which such strains are present in bovine and human populations. Methods Isolates of bovine MRSA were obtained from the Veterinary Laboratories Agency in the UK, and isolates of human MRSA were obtained from diagnostic or reference laboratories (two in the UK and one in Denmark). From these collections, we searched for mecA PCR-negative bovine and human S aureus isolates showing phenotypic meticillin resistance. We used whole-genome sequencing to establish the genetic basis for the observed antibiotic resistance. Findings A divergent mecA homologue ( mecA LGA251 ) was discovered in the LGA251 genome located in a novel staphylococcal cassette chromosome mec element, designated type-XI SCC mec . The mecA LGA251 was 70% identical to S aureus mecA homologues and was initially detected in 15 S aureus isolates from dairy cattle in England. These isolates were from three different multilocus sequence type lineages (CC130, CC705, and ST425); spa type t843 (associated with CC130) was identified in 60% of bovine isolates. When human mecA -negative MRSA isolates were tested, the mecA LGA251 homologue was identified in 12 of 16 isolates from Scotland, 15 of 26 from England, and 24 of 32 from Denmark. As in cows, t843 was the most common spa type detected in human beings. Interpretation Although routine culture and antimicrobial susceptibility testing will identify S aureus isolates with this novel mecA homologue as meticillin resistant, present confirmatory methods will not identify them as MRSA. New diagnostic guidelines for the detection of MRSA should consider the inclusion of tests for mecA LGA251 . Funding Department for Environment, Food and Rural Affairs, Higher Education Funding Council for England, Isaac Newton Trust (University of Cambridge), and the Wellcome Trust.

Antibacterial drugs have played an essential role in the global increase in quality of life and life expectancy. However, these gains are at serious risk owing to bacterial drug resistance by so-called “superbugs”, such as methicillin-resistant Staphylococcus aureus (MRSA). The discovery of new antibiotics with novel modes of action is vital to tackle the threat of multidrug-resistant bacteria. Traditionally, antibiotics have been discovered from natural sources; however, there are many disadvantages to using extracts (e.g. limited availability, bioactive constituent identification, and complex analogue synthesis). These problems have led to a complementary approach of synthesizing structurally diverse, natural-product-like small molecules directly and efficiently, an approach known as diversity-oriented synthesis (DOS). Whereas compound collections of a common scaffold decorated with diverse building blocks have been synthesized efficiently, there are limited examples of the synthesis of small molecules with a high degree of skeletal diversity (usually by a build–couple–pair strategy). Previously, we have used a diazoacetate starting unit to mimic nature8s divergent synthetic strategy with acetyl CoA (by a pluripotent functional-group strategy) to synthesize compounds with natural-product scaffolds (e.g. cocaine and warfarin). Herein, we report the use of a solid-supported phosphonate unit to synthesize 242 drug-like compounds based on 18 natural-product-like scaffolds in two to five steps and their use in discovering a new structural class of antibiotic with anti-MRSA activity. The solid-supported phosphonate 1 (Scheme 1) was identified as an attractive DOS starting unit for three key reasons. First, the reactive phosphonate functionality permits the stereoselective formation of a,b-unsaturated acyl imidazolidinones (2) that could be used to generate enantioselectively a wide range of scaffolds that can be diversified further. Second, the imidazolidinone linker not only enables twopoint binding of chiral catalysts but also permits divergent cleavage of the exocyclic acyl group (hydrolysis, reduction, esterification, and amide formation). Thirdly, immobilization of 1 on a silyl polystyrene support simplified reaction optimization and work-up procedures in the multistep parallel synthesis (total of over 1000 individual steps), thereby allowing the efficient production of milligram quantities of 242 compounds without the requirement for automation equipment. In the first step of the diversity-oriented synthesis, 1 was treated with aldehyde building blocks (aryl, heteroaryl, and alkyl; see the Supporting Information) to deliver twelve a,bunsaturated acyl imidazolidinones (2). The second steps of the solid-supported synthesis exploited three catalytic, enantioselective, divergent reaction pathways (Scheme 1): 1) [2+3] cycloaddition (reaction b, ee 60–65%, de 7899%), 2) dihydroxylation (reaction c, ee 88–91%), and 3) [4+2] cycloaddition (reaction d, ee 89–98%, de 74– 74%). Similar selectivities were observed when repeating the reactions in solution with a triisopropylsilyl-protected linker (as opposed to the diisopropylpolystyrene group; see the Supporting Information). The reactions were also conducted with achiral catalysts to give racemic products, which were used for the later steps of the synthesis. This procedure enabled the diversity-oriented synthesis to be streamlined to half the size, yet permitted the enantioselective synthesis of hits during the structure–activity relationship stages of this [*] Dr. G. L. Thomas, R. J. Spandl, F. G. Glansdorp, Dr. M. Ladlow, Dr. D. R. Spring Department of Chemistry, University of Cambridge Lensfield Road, Cambridge, CB2 1EW (UK) Fax: (+44) 1223-336362 E-mail: drspring@ch.cam.ac.uk Homepage: http://www-spring.ch.cam.ac.uk/

The prevalence of CTX-M-producing members of the Enterobacteriaceae is increasing worldwide. A novel, multiplex, real-time TaqMan PCR assay to detect and type bla CTX-M genes is described which is an improvement on previously described techniques with respect to reduced assay time, elimination of the need for protracted post-PCR processing and the convenience of a single reaction vessel. Based on β-lactam antibiogram and MIC data, 478 of 1279 Enterobacteriaceae isolates from clinical blood and urine culture specimens were selected and tested for extended-spectrum β-lactamase (ESBL) production using phenotypic methods. The new TaqMan assay detected and typed bla CTX-M genes in 21 of 28 ESBL-producing isolates.

Summary 1.From September 2003, acute National Health Service trusts in England have been required by the Department of Health to undertake mandatory surveillance of bacteraemias caused by glycopeptide-resistant enterococci (GRE). 2.In total, 4855 enterococcal bacteraemias (including 256 reports of ‘Group D streptococci') were reported through the voluntary reporting system to the Communicable Disease Surveillance Centre (CDSC) in England and Wales in 2002, and 8.9% of the reports that contained information on vancomycin susceptibility indicated that the isolate was resistant to vancomycin. Most hospitals reported fewer than five GRE bacteraemias per year to the CDSC, with only 13 hospitals reporting more than five episodes per year, although there is undoubtedly an element of under-reporting. 3.This Working Group was established to review the methods used to identify and test the susceptibility of enterococci to glycopeptides, and to make recommendations on the reporting of GRE bacteraemias, as there were indications that current methods would not provide a suitable basis for this surveillance. 4.A review of methods was undertaken, including a national questionnaire on methods, approaches to testing and risk factors for GRE bacteraemia. This, together with data from routine laboratory reports and special surveys, revealed the following. –A significant proportion of enterococci are not identified to species level. –There are problems with identification of enterococci. Commercially available kits do not identify some enterococcal species reliably. –Not all laboratories test vancomycin susceptibility of enterococci from bacteraemia. This does not allow a robust assessment of GRE bacteraemia rates because the prevalence of glycopeptide resistance will be underestimated if laboratories test teicoplanin alone or no glycopeptides against enterococci. –There are technical difficulties in detecting non-VanA resistance in enterococci and this will also contribute to under-reporting of glycopeptide resistance. 5.Clinically significant GRE infection is associated with a worse prognosis than infection with susceptible strains, but this factor has not been clearly separated from the possible influence of co-morbidities and antibiotic usage. 6.GRE bacteraemia occurs mainly on specialist units, particularly transplantation, renal, haematological malignancy and intensive care units. Hospitals with such units will inevitably have a higher risk of featuring prominently in surveillance studies. Hence, meaningful denominators for surveillance of GRE bacteraemia are required. Recommendations for GRE bacteraemia surveillance 1.As the mandatory surveillance of glycopeptide-resistant enterococci (GRE) bacteraemias has already started, this report recommends a phased approach to some of the developments, such as using meaningful denominators for the bacteraemia rates. 2.Rates of GRE bacteraemias in trusts should not be used as a performance indicator because the numbers are too small for valid analysis in this way. 3.GRE bacteraemias as a proportion of all clinically significant bacteraemias in the trust should be measured as an indicator of changing trends. 4.Since most GRE are likely to be associated with specialist units, reports of GRE bacteraemia should indicate the specialty in which the patient acquired the infection. 5.Data on clinical activity in the special units where patients acquired GRE bacteraemias should be obtained from trusts with more than five GRE bacteraemias in a year. 6.The significance of blood cultures containing GRE should be assessed clinically. If the bacteraemia that includes GRE is deemed to be clinically significant (according to the case definitions for bacteraemia), whether mono- or polymicrobial, it should be reported as a GRE bacteraemia. 7.Enterococci from blood cultures should be identified to species level to provide data on resistance rates in different species. 8.Biochemical identification should be augmented by antimicrobial susceptibility patterns. The identification is suspect if Enterococcus faecalis appears to be ampicillin resistant and/or quinupristin/dalfopristin susceptible, or if Enterococcus faecium appears to be ampicillin susceptible and/or quinupristin/dalfopristin resistant. The identification and susceptibility of such isolates should be checked and, if confirmed, the isolate should be sent to a reference laboratory for further investigation. 9.Enterococci from blood cultures should be tested for susceptibility to vancomycin. Teicoplanin is not an acceptable alternative to vancomycin for these purposes. 10.Standardized methods should be used for glycopeptide susceptibility testing of enterococci, and the technical requirements of the methods must be adhered to. 11.The situation should be reviewed after a year and the methodology should be amended as appropriate. Recommendations for further research 1.A group should be established to explore methods of data collection for global and individual risk factors for GRE bacteraemia. 2.Further studies on the performance of commercial identification kits with a range of enterococcal species are necessary. 3.Further studies to improve the reliability of susceptibility testing methods for detection of GRE are necessary.

The increase since the mid 1980s in glycopepetide resistant enterococci (GRE) raised concerns about the limited options for antimicrobial therapy, the implications for ever-increasing numbers of immunocompromised hospitalised patients, and fuelled fears, now realised, for the transfer of glycopeptide resistance to more pathogenic bacteria, such as Staphylococcus aureus. These issues underlined the need for guidelines for the emergence and control of GRE in the hospital setting. This Hospital Infection Society (HIS) and Infection Control Nurses Association (ICNA) working party report reviews the literature relating to GRE prevention and control. It provides guidance on microbiological investigation, treatment and management, including antimicrobial prescribing and infection control measures. Evidence identified to support recommendations has been categorized. A risk assessment approach is recommended and areas for research and development identified.

These evidence-based guidelines have been produced after a literature review of the laboratory diagnosis and susceptibility testing of methicillin-resistant Staphylococcus aureus (MRSA). We have considered the detection of MRSA in screening samples and the detection of reduced susceptibility to glycopeptides in S. aureus. Recommendations are given for the identification of S. aureus and for suitable methods of susceptibility testing and screening for MRSA and for S. aureus with reduced susceptibility to glycopeptides. These guidelines indicate what tests should be used but not when the tests are applicable, as aspects of this are dealt with in guidelines on control of MRSA. There are currently several developments in screening media and molecular methods. It is likely that some of our recommendations will require modification as the new methods become available.

To describe the current patterns and trends in antimicrobial susceptibility in enterococci and streptococci (excepting pneumococci) from bacteraemia in the UK and Ireland from 2001 to 2006.In each year 2001-06, blood culture isolates were collected by 25 laboratories distributed across the UK and Ireland. In total, there were 1408 isolates of enterococci, 1332 of beta-haemolytic streptococci and 1012 of alpha- and non-haemolytic streptococci. A single central laboratory re-identified the isolates and measured MICs by the BSAC agar dilution method.The prevalence of reduced susceptibility in streptococci and enterococci did not change significantly for most antibiotics, but trends were noted to increased ampicillin, imipenem and vancomycin resistance in Enterococcus faecium. The prevalence of reduced susceptibility to macrolides and tetracycline in streptococci, to tetracycline and gentamicin (high level) in enterococci and to beta-lactams and glycopeptides in E. faecium were all high, with some differences in the prevalence among species or groups.Reduced susceptibility to some antimicrobial agents among streptococci and enterococci remains common and continued surveillance is warranted.

BackgroundClostridium difficile is a leading infectious cause of health care associated diarrhoea. Several industrialised countries have reported increased C. difficile infections and outbreaks, which have been attributed to the emergent PCR ribotype 027 strain.Methods and findingsWe conducted a case-case study to compare severity of C. difficile disease for patients with 027 versus non-027 ribotypes. We retrospectively collected clinical information about 123/136 patients with C. difficile infections admitted to hospitals in the East of England region in 2006 and from whom stool isolates were cultured and ribotyped as part of an earlier national survey. We defined severe C. difficile disease as having one or more of shock, paralytic ileus, pseudo membranous colitis or toxic megacolon. Patient median age was 83 years old (range 3 to 98, interquartile range 75 to 89), 86% were prescribed antibiotics in the eight weeks before illness onset, 41% had ribotype 027 and 30-day all cause mortality during hospital admission was 21%. Severe disease occurred in 24% (95%CI 13% to 37%) and 17% (95%CI 9% to 27%) of patients with PCR ribotype 027 and non-027 ribotypes respectively. In a multivariable model, ribotype 027 was not associated with severe disease after adjusting for sex, discharge from hospital prior to 60 days of current admission, gastroenteritis on admission, number of initiator antibiotics for C. difficile disease, and hospital where the patient was admitted.ConclusionsOur study found no evidence to support previous assertions that ribotype 027 is more virulent than other PCR ribotypes. This finding raises questions about the contribution of this strain to the recent increase in C. difficile disease throughout North America and Europe.

Sir, Organisms producing extended-spectrum -lactamases (ESBLs) characteristically have reduced susceptibility to newer cephalosporins and/or aztreonam. In vitro resistance, however, as indicated by routine disc diffusion or dilution methods may be low or moderate. In addition, routine disc diffusion or dilution methods do not distinguish -lactam resistance mediated by ESBLs from resistance mediated by other mechanisms unless an extended range of different -lactam agents is tested. As outbreaks of infection with ESBL-producing strains have been reported worldwide, the identification of such strains is desirable so that infection control measures can be instigated. Tests proposed for discrimination of ESBLs include the double-disc potentiation method, the three-dimensional test and the Etest. These tests are based on antagonism by clavulanic acid of the action of ESBLs on newer cephalosporins, particularly ceftazidime, and/or aztreonam. The three-dimensional test is technically difficult and is not widely used. The double-disc potentiation test is technically simple but depends on subjective reading of interactions between zones of inhibition and is affected by spacing of discs. In addition ESBL-producing Proteus mirabilis and organisms hyper-producing chromosomal cephalosporinase in addition to ESBLs may cause falsenegative results and inhibition of some hyper-produced enzymes by clavulanic acid may lead to false-positive results. The double-disc potentiation method is, however, generally reliable for the detection of most ESBLs in Escherichia coli and Klebsiella spp., the organisms that most commonly produce ESBLs. The Etest is less subjective than double-disc potentiation as interpretation is based on quantitative reading of the MIC of ceftazidime in the presence and absence of clavulanic acid. A ceftazidime MIC greater than four-fold lower in the presence of clavulanic acid indicates ESBL production. We tested the performance of the Etest and double-disc potentiation method for detection of a range of ESBLs and investigated reproducibility by testing the same isolates in four laboratories. Eighteen ESBL-producing strains tested were E. coli with TEM-3, TEM-4, TEM-5, TEM-6, TEM-7, TEM-8, TEM-9, TEM-10, TEM-15, TEM-24, TEM-101, SHV-2, SHV-3, SHV-4 (two strains), SHV-5 and BIL-1 enzymes and Klebsiella sp. with the K1 enzyme. Ten strains with -lactamases other than ESBLs were E. coli with TEM-1 (two strains), TEM-2 (two strains), SHV-l, OXA-2 and hyper-produced AmpC (two strains), and Citrobacter freundii with inducible and hyper-produced AmpC enzymes. Seven strains of E. coli with no -lactamase were also included. The strains were distributed by one centre with the identity of the strains blinded. Strains were tested in the four laboratories by the Etest method with ceftazidime ESBL strips (Cambridge Diagnostic Services, Cambridge, UK) and the double-disc potentiation method with ceftazidime 30 g and co-amoxiclav 30 g discs. Results were returned to one laboratory for decoding of identity and analysis of results. The seven strains with no -lactamase were all correctly reported with both methods in all four laboratories (Table). Fifteen of the 18 ESBL-producing strains were correctly reported with both methods by all four laboratories. The strains with the K1 and BIL-1 enzymes were correctly reported resistant by all laboratories but these enzymes are not inhibited by clavulanic acid and were not reported as ESBL-producing strains. Similarly, clavulanic acidresistant TEM enzymes would not be distinguished by these methods, but these enzymes are not widely distributed at present. One strain with a TEM-24 enzyme was reported as an ESBL producer by both methods in all laboratories except one, which reported the strain resistant, but not an ESBL producer with the Etest method. By the Etest this strain had a ceftazidime MIC of 32 mg/L in all four laboratories. In the presence of clavulanic acid the MIC was 0.75 mg/L (with colonies inside the zone edge) 1 mg/L, 2 mg/L and 8 mg/L in the four laboratories, resulting in reports of ESBL production in all except the Correspondence Journal of Antimicrobial Chemotherapy (2000) 46, 323–342