Derek J. Brown

Meticillin-resistant Staphylococcus aureus with a novel mecA homologue in human and bovine populations in the UK and Denmark: a descriptive study

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 (mecALGA251) was discovered in the LGA251 genome located in a novel staphylococcal cassette chromosome mec element, designated type-XI SCCmec. The mecALGA251 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 mecALGA251 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 mecALGA251. Funding Department for Environment, Food and Rural Affairs, Higher Education Funding Council for England, Isaac Newton Trust (University of Cambridge), and the Wellcome Trust.

Emergence and global spread of epidemic healthcare-associated Clostridium difficile

Epidemic C. difficile (027/BI/NAP1) has rapidly emerged in the past decade as the leading cause of antibiotic-associated diarrhea worldwide. However, the key events in evolutionary history leading to its emergence and the subsequent patterns of global spread remain unknown. Here, we define the global population structure of C. difficile 027/BI/NAP1 using whole-genome sequencing and phylogenetic analysis. We show that two distinct epidemic lineages, FQR1 and FQR2, not one as previously thought, emerged in North America within a relatively short period after acquiring the same fluoroquinolone resistance–conferring mutation and a highly related conjugative transposon. The two epidemic lineages showed distinct patterns of global spread, and the FQR2 lineage spread more widely, leading to healthcare-associated outbreaks in the UK, continental Europe and Australia. Our analysis identifies key genetic changes linked to the rapid transcontinental dissemination of epidemic C. difficile 027/BI/NAP1 and highlights the routes by which it spreads through the global healthcare system.

Host adapted serotypes of Salmonella enterica.

Salmonella constitutes a genus of zoonotic bacteria of worldwide economic and health importance. The current view of salmonella taxonomy assigns the members of this genus to two species: S. enterica and S. bongori. S. enterica itself is divided into six subspecies, enterica, salamae, arizonae, diarizonae, indica, and houtenae, also known as subspecies I, II, IIIa, IIIb, IV, and VI, respectively [1]. Members of Salmonella enterica subspecies enterica are mainly associated with warm-blooded vertebrates and are usually transmitted by ingestion of food or water contaminated by infected faeces. The pathogenicity of most of the distinct serotypes remains undefined, and even within the most common serotypes, many questions remain to be answered regarding the interactions between the organism and the infected host.Salmonellosis manifests itself in three major forms: enteritis, septicaemia, and abortion, each of which may be present singly or in combination, depending on both the serotype and the host involved. Although currently over 2300 serovars of Salmonella are recognized, only about 50 serotypes are isolated in any significant numbers as human or animal pathogens [2, 3] and they all belong to subspecies enterica. Of these, most cause acute gastroenteritis characterized by a short incubation period and a severe systemic disease in man or animals, characterized by septicaemia, fever and/or abortion, and such serotypes are often associated with one or few host species [4–6].It is the intention of this review to present a summary of current knowledge of these host-adapted serotypes of S. enterica. The taxonomic relationships between the serotypes will be discussed together with a comparison of the pathology and pathogenesis of the disease that they cause in their natural host(s). Since much of our knowledge on salmonellosis is based on the results of work on Typhimurium, this serotype will often be used as the baseline in discussion. It is hoped that an appreciation of the differences that exist in the way these serotypes interact with the host will lead to a greater understanding of the complex host–parasite relationship that characterizes salmonella infections.

EUCAST Expert Rules in Antimicrobial Susceptibility Testing

EUCAST expert rules have been developed to assist clinical microbiologists and describe actions to be taken in response to specific antimicrobial susceptibility test results. They include recommendations on reporting, such as inferring susceptibility to other agents from results with one, suppression of results that may be inappropriate, and editing of results from susceptible to intermediate or resistant or from intermediate to resistant on the basis of an inferred resistance mechanism. They are based on current clinical and/or microbiological evidence. EUCAST expert rules also include intrinsic resistance phenotypes and exceptional resistance phenotypes, which have not yet been reported or are very rare. The applicability of EUCAST expert rules depends on the MIC breakpoints used to define the rules. Setting appropriate clinical breakpoints, based on treating patients and not on the detection of resistance mechanisms, may lead to modification of some expert rules in the future.

Clonal lines of Salmonella enterica serotype enteritidis documented by IS200-, ribo-, pulsed-field gel electrophoresis and RFLP typing

Sixty-two selected strains of Salmonella serotype Enteritidis of 33 phage types (PTs), and one strain classified as RDNC, were characterised by four different chromosomally based typing methods to elucidate genetic relationships among strains of different phage types. Based on IS200-hybridisation patterns, two major groups, containing strains of the most commonly encountered phage types, and six minor groups (seven with the RDNC strain included) were observed. IS200 pattern was a stable epidemiological marker in strains of all phage types except PT 6a and 14b. Ribotyping separated strains of the phage types into one major and five minor groups; the pattern of the RDNC strain was not seen with other strains. More than one ribotype was observed among strains of Enteritidis PTs 6, 7, 14b and 21. By pulsed-field gel electrophoresis, strains of 21 of the 33 phage types formed one large cluster when bands > 125 kb were used as the criterion for separation. Among strains belonging to PTs 1, 6, 7 and 14b, more than one pattern was observed by this method. By probing with five random cloned fragments of the Enteritidis chromosome, strains from 27 of 31 phage types examined showed the same hybridisation pattern. With the combined use of four genotypic methods, two groups of strains, representing eight and seven of 33 Enteritidis phage types, were formed; these two groups may be considered as the main evolutionary lines of Enteritidis. Strains of the remaining phage types, and the RDNC strain, belonged to separate groups.

Distinguishable epidemics of multidrug-resistant Salmonella Typhimurium DT104 in different hosts.

Sourcing Antibiotic Resistance It is widely assumed that antibiotic resistance in farm animals contributes in a major way to antibiotic resistance in humans. Mather et al. (p. 1514, published online 12 September; see the Perspective by Woolhouse and Ward) analyzed hundreds of genome sequences from Salmonella isolates collected from both livestock and patients in Scotland between 1990 and 2004. The relative contributions of animal-derived and human-derived sources of infection were quantified and the phylogenetic diversity of resistance profiles was matched with bacterial phylogenies. The results suggest that most human infections are caught from other humans rather than from livestock and that humans harbor a greater diversity of antibiotic resistance. Antibiotic resistance travels in independent epidemics in humans and their livestock. [Also see Perspective by Woolhouse and Ward] The global epidemic of multidrug-resistant Salmonella Typhimurium DT104 provides an important example, both in terms of the agent and its resistance, of a widely disseminated zoonotic pathogen. Here, with an unprecedented national collection of isolates collected contemporaneously from humans and animals and including a sample of internationally derived isolates, we have used whole-genome sequencing to dissect the phylogenetic associations of the bacterium and its antimicrobial resistance genes through the course of an epidemic. Contrary to current tenets supporting a single homogeneous epidemic, we demonstrate that the bacterium and its resistance genes were largely maintained within animal and human populations separately and that there was limited transmission, in either direction. We also show considerable variation in the resistance profiles, in contrast to the largely stable bacterial core genome, which emphasizes the critical importance of integrated genotypic data sets in understanding the ecology of bacterial zoonoses and antimicrobial resistance.

Intrinsic resistance to β-lactam antibiotics in staphylococcus aureus

Methicillin-resistant strains of Stu@rylococcus aureus were first described in 1961 [1,2]. These strains demonstrate cross-resistance to /3-lactam antibiotics other than methicillin [3] and resistance is intrinsic rather than being due to penicillinase activity, as penicillinase-negative variants retain resistance to fl-lactam antibiotics [4]. The resistance is unusual in that cell populations are heterogeneous with respect to the level of resistance expressed [5] although all cells in the population retain the ability to express resistance. The degree of resistance is affected dramatically by alterations in the environmental conditions. In particular, resistance is increased by growing cells at <37”C [6] or by incorporating 5% NaCl into the medium [7]. Intrinsic resistance to /3-lactam antibiotics has been shown to be due to alteration either in the amounts [8] or the affinities for @-lactam antibiotics [9-l l] of the penicillin-binding proteins (PBPs) of certain bacteria other than S. aureus. Here we demonstrate that intrinsic resistance to /I-lactam antibiotics in S. cureus results from an increase in the amount of modified PBP, or the presence of a new PBP, which has a lower affinity for fl-lactam antibiotics than PBPs in sensitive strains.

Shigella sonnei genome sequencing and phylogenetic analysis indicate recent global dissemination from Europe.

Shigella are human-adapted Escherichia coli that have gained the ability to invade the human gut mucosa and cause dysentery, spreading efficiently via low-dose fecal-oral transmission. Historically, S. sonnei has been predominantly responsible for dysentery in developed countries but is now emerging as a problem in the developing world, seeming to replace the more diverse Shigella flexneri in areas undergoing economic development and improvements in water quality. Classical approaches have shown that S. sonnei is genetically conserved and clonal. We report here whole-genome sequencing of 132 globally distributed isolates. Our phylogenetic analysis shows that the current S. sonnei population descends from a common ancestor that existed less than 500 years ago and that diversified into several distinct lineages with unique characteristics. Our analysis suggests that the majority of this diversification occurred in Europe and was followed by more recent establishment of local pathogen populations on other continents, predominantly due to the pandemic spread of a single, rapidly evolving, multidrug-resistant lineage.

Penicillin-binding proteins of β-lactam-resistant strains of Staphylococcus aureus: Effect of growth conditions

Methidllin‐resistant clinical isolates of Staphylococcus aureus are intrinsically resistant to β‐lactam antibiotics in that the resistance mechanism is unrelated to the possession of β‐lactamases. We have demonstrated that a new, high‐molecular‐mass penicillin‐binding protein (PBP) is present in these strains with a low affinity for β‐lactams and that its amount is regulated by the growth conditions. The new PBP from all strains that have been examined has an identical mobility on SDS gel electrophoresis and is the only PBP still present in an uncomplexed state with β‐lactams (and therefore the only functional PBP) when these strains are grown in media containing concentrations of β‐lactam antibiotics sufficient to kill sensitive strains.

Anti-MRSA Agent Discovery Using Diversity-Oriented Synthesis†

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/