The emergence and diversification of a zoonotic pathogen from within the microbiota of intensively farmed pigs.

The expansion and intensification of livestock production is predicted to promote the emergence of pathogens. As pathogens sometimes jump between species, this can affect the health of humans as well as livestock. Here, we investigate how livestock microbiota can act as a source of these emerging pathogens through analysis of Streptococcus suis, a ubiquitous component of the respiratory microbiota of pigs that is also a major cause of disease on pig farms and an important zoonotic pathogen. Combining molecular dating, phylogeography, and comparative genomic analyses of a large collection of isolates, we find that several pathogenic lineages of S. suis emerged in the 19th and 20th centuries, during an early period of growth in pig farming. These lineages have since spread between countries and continents, mirroring trade in live pigs. They are distinguished by the presence of three genomic islands with putative roles in metabolism and cell adhesion, and an ongoing reduction in genome size, which may reflect their recent shift to a more pathogenic ecology. Reconstructions of the evolutionary histories of these islands reveal constraints on pathogen emergence that could inform control strategies, with pathogenic lineages consistently emerging from one subpopulation of S. suis and acquiring genes through horizontal transfer from other pathogenic lineages. These results shed light on the capacity of the microbiota to rapidly evolve to exploit changes in their host population and suggest that the impact of changes in farming on the pathogenicity and zoonotic potential of S. suis is yet to be fully realized.

HAM-ART: An optimised culture-free Hi-C metagenomics pipeline for tracking antimicrobial resistance genes in complex microbial communities.

Shotgun metagenomics is a powerful tool to identify antimicrobial resistance (AMR) genes in microbiomes but has the limitation that extrachromosomal DNA, such as plasmids, cannot be linked with the host bacterial chromosome. Here we present a comprehensive laboratory and bioinformatics pipeline HAM-ART (Hi-C Assisted Metagenomics for Antimicrobial Resistance Tracking) optimised for the generation of metagenome-assembled genomes including both chromosomal and extrachromosomal AMR genes. We demonstrate the performance of the pipeline in a study comparing 100 pig faecal microbiomes from low- and high-antimicrobial use pig farms (organic and conventional farms). We found significant differences in the distribution of AMR genes between low- and high-antimicrobial use farms including a plasmid-borne lincosamide resistance gene exclusive to high-antimicrobial use farms in three species of Lactobacilli. The bioinformatics pipeline code is available at https://github.com/lkalmar/HAM-ART.

Mutation rate dynamics reflect ecological change in an emerging zoonotic pathogen.

Mutation rates vary both within and between bacterial species, and understanding what drives this variation is essential for understanding the evolutionary dynamics of bacterial populations. In this study, we investigate two factors that are predicted to influence the mutation rate: ecology and genome size. We conducted mutation accumulation experiments on eight strains of the emerging zoonotic pathogen Streptococcus suis. Natural variation within this species allows us to compare tonsil carriage and invasive disease isolates, from both more and less pathogenic populations, with a wide range of genome sizes. We find that invasive disease isolates have repeatedly evolved mutation rates that are higher than those of closely related carriage isolates, regardless of variation in genome size. Independent of this variation in overall rate, we also observe a stronger bias towards G/C to A/T mutations in isolates from more pathogenic populations, whose genomes tend to be smaller and more AT-rich. Our results suggest that ecology is a stronger correlate of mutation rate than genome size over these timescales, and that transitions to invasive disease are consistently accompanied by rapid increases in mutation rate. These results shed light on the impact that ecology can have on the adaptive potential of bacterial pathogens.

Large-scale genomic analysis of antimicrobial resistance in the zoonotic pathogen Streptococcus suis.

BACKGROUND: Antimicrobial resistance (AMR) is among the gravest threats to human health and food security worldwide. The use of antimicrobials in livestock production can lead to emergence of AMR, which can have direct effects on humans through spread of zoonotic disease. Pigs pose a particular risk as they are a source of zoonotic diseases and receive more antimicrobials than most other livestock. Here we use a large-scale genomic approach to characterise AMR in Streptococcus suis, a commensal found in most pigs, but which can also cause serious disease in both pigs and humans. RESULTS: We obtained replicated measures of Minimum Inhibitory Concentration (MIC) for 16 antibiotics, across a panel of 678 isolates, from the major pig-producing regions of the world. For several drugs, there was no natural separation into 'resistant' and 'susceptible', highlighting the need to treat MIC as a quantitative trait. We found differences in MICs between countries, consistent with their patterns of antimicrobial usage. AMR levels were high even for drugs not used to treat S. suis, with many multidrug-resistant isolates. Similar levels of resistance were found in pigs and humans from regions associated with zoonotic transmission. We next used whole genome sequences for each isolate to identify 43 candidate resistance determinants, 22 of which were novel in S. suis. The presence of these determinants explained most of the variation in MIC. But there were also interesting complications, including epistatic interactions, where known resistance alleles had no effect in some genetic backgrounds. Beta-lactam resistance involved many core genome variants of small effect, appearing in a characteristic order. CONCLUSIONS: We present a large dataset allowing the analysis of the multiple contributing factors to AMR in S. suis. The high levels of AMR in S. suis that we observe are reflected by antibiotic usage patterns but our results confirm the potential for genomic data to aid in the fight against AMR.

A One Health Study of the Genetic Relatedness of Klebsiella pneumoniae and Their Mobile Elements in the East of England.

BACKGROUND: Klebsiella pneumoniae is a human, animal, and environmental commensal and a leading cause of nosocomial infections, which are often caused by multiresistant strains. We evaluate putative sources of K. pneumoniae that are carried by and infect hospital patients. METHODS: We conducted a 6-month survey on 2 hematology wards at Addenbrooke's Hospital, Cambridge, United Kingdom, in 2015 to isolate K. pneumoniae from stool, blood, and the environment. We conducted cross-sectional surveys of K. pneumoniae from 29 livestock farms, 97 meat products, the hospital sewer, and 20 municipal wastewater treatment plants in the East of England between 2014 and 2015. Isolates were sequenced and their genomes compared. RESULTS: Klebsiella pneumoniae was isolated from stool of 17/149 (11%) patients and 18/922 swabs of their environment, together with 1 bloodstream infection during the study and 4 others over a 24-month period. Each patient carried 1 or more lineages that was unique to them, but 2 broad environmental contamination events and patient-environment transmission were identified. Klebsiella pneumoniae was isolated from cattle, poultry, hospital sewage, and 12/20 wastewater treatment plants. There was low genetic relatedness between isolates from patients/their hospital environment vs isolates from elsewhere. Identical genes encoding cephalosporin resistance were carried by isolates from humans/environment and elsewhere but were carried on different plasmids. CONCLUSION: We identified no patient-to-patient transmission and no evidence for livestock as a source of K. pneumoniae infecting humans. However, our findings reaffirm the importance of the hospital environment as a source of K. pneumoniae associated with serious human infection.

Streptococcus suis contains multiple phase-variable methyltransferases that show a discrete lineage distribution.

Streptococcus suis is a major pathogen of swine, responsible for a number of chronic and acute infections, and is also emerging as a major zoonotic pathogen, particularly in South-East Asia. Our study of a diverse population of S. suis shows that this organism contains both Type I and Type III phase-variable methyltransferases. In all previous examples, phase-variation of methyltransferases results in genome wide methylation differences, and results in differential regulation of multiple genes, a system known as the phasevarion (phase-variable regulon). We hypothesized that each variant in the Type I and Type III systems encoded a methyltransferase with a unique specificity, and could therefore control a distinct phasevarion, either by recombination-driven shuffling between different specificities (Type I) or by biphasic on-off switching via simple sequence repeats (Type III). Here, we present the identification of the target specificities for each Type III allelic variant from S. suis using single-molecule, real-time methylome analysis. We demonstrate phase-variation is occurring in both Type I and Type III methyltransferases, and show a distinct association between methyltransferase type and presence, and population clades. In addition, we show that the phase-variable Type I methyltransferase was likely acquired at the origin of a highly virulent zoonotic sub-population.

Truncation of GdpP mediates ß-lactam resistance in clinical isolates of Staphylococcus aureus.

OBJECTIVES: High-level ß-lactam resistance in MRSA is mediated in the majority of strains by a mecA or mecC gene. In this study, we identified 10 mec gene-negative MRSA human isolates from Austria and 11 bovine isolates from the UK showing high levels of ß-lactam resistance and sought to understand the molecular basis of the resistance observed. METHODS: Different antimicrobial resistance testing methods (disc diffusion, Etest and VITEK® 2) were used to establish the ß-lactam resistance profiles for the isolates and the isolates were further investigated by WGS. RESULTS: A number of mutations (including novel ones) in PBPs, AcrB, YjbH and the pbp4 promoter were identified in the resistant isolates, but not in closely related susceptible isolates. Importantly, a truncation in the cyclic diadenosine monophosphate phosphodiesterase enzyme, GdpP, was identified in 7 of the 10 Austrian isolates and 10 of the 11 UK isolates. Complementation of four representative isolates with an intact copy of the gdpP gene restored susceptibility to penicillins and abolished the growth defects caused by the truncation. CONCLUSIONS: This study reports naturally occurring inactivation of GdpP protein in Staphylococcus aureus of both human origin and animal origin, and demonstrates clinical relevance to a previously reported association between this truncation and increased ß-lactam resistance and impaired bacterial growth in laboratory-generated mutants. It also highlights possible limitations of genomic determination of antibiotic susceptibility based on single gene presence or absence when choosing the appropriate antimicrobial treatment for patients.

Streptococcus bovimastitidis sp. nov., isolated from a dairy cow with mastitis.

Here we describe a new species of the genus Streptococcus that was isolated from a dairy cow with mastitis in New Zealand. Strain NZ1587T was Gram-positive, coccus-shaped and arranged as chains, catalase and coagulase negative, γ-haemolytic and negative for Lancefield carbohydrates (A-D, F and G). The 16S rRNA sequence did not match sequences in the NCBI 16S rRNA or GreenGenes databases. Taxonomic classification of strain NZ1587T was investigated using 16S rRNA and core genome phylogeny, genome-wide average nucleotide identity (ANI) and predicted DNA-DNA hybridisation (DDH) analyses. Phylogeny based on 16S rRNA was unable to resolve the taxonomic position of strain NZ1587T, however NZ1587T shared 99.4 % identity at the 16S rRNA level with a distinct branch of S. pseudoporcinus. Importantly, core genome phylogeny demonstrated that NZ1587T grouped amongst the 'pyogenic' streptococcal species and formed a distinct branch supported by a 100 % bootstrap value. In addition, average nucleotide identity and inferred DNA-DNA hybridisation analyses showed that NZ1587T represents a novel species. Biochemical profiling using the rapid ID 32 strep identification test enabled differentiation of strain NZ1587T from closely related streptococcal species. In conclusion, strain NZ1587T can be classified as a novel species, and we propose a novel taxon named Streptococcus bovimastitidis sp. nov.; the type strain is NZ1587T. NZ1587T has been deposited in the Culture Collection University of Gothenburg (CCUG 69277T) and the Belgian Co-ordinated Collections of Micro-organisms/LMG (LMG 29747).

Atmin mediates kidney morphogenesis by modulating Wnt signaling.

The DNA damage protein and transcription factor Atmin (Asciz) is required for both lung tubulogenesis and ciliogenesis. Like the lungs, kidneys contain a tubular network that is critical for their function and in addition, renal ciliary dysfunction has been implicated in the pathogenesis of cystic kidney disease. Using the Atmin mouse mutant Gasping6 (Gpg6), we investigated kidney development and found it severely disrupted with reduced branching morphogenesis, resulting in fewer epithelial structures being formed. Unexpectedly, transcriptional levels of key cilia associated genes were not altered in Atmin(Gpg6/Gpg6) kidneys. Instead, Gpg6 homozygous kidneys exhibited altered cytoskeletal organization and modulation of Wnt signaling pathway molecules, including ß-catenin and non-canonical Wnt/planar cell polarity (PCP) pathway factors, such as Daam2 and Vangl2. Wnt signaling is important for kidney development and perturbation of Wnt signaling pathways can result in cystic, and other, renal abnormalities. In common with other PCP pathway mutants, Atmin(Gpg6/Gpg6) mice displayed a shortened rostral-caudal axis and mis-oriented cell division. Moreover, intercrosses between Atmin(Gpg6/+) and Vangl2(Lp/+) mice revealed a genetic interaction between Atmin and Vangl2. Thus we show for the first time that Atmin is critical for normal kidney development and we present evidence that mechanistically, Atmin modifies Wnt signaling pathways, specifically placing it as a novel effector molecule in the non-canonical Wnt/PCP pathway. The identification of a novel modulator of Wnt signaling has important implications for understanding the pathobiology of renal disease.

Dissemination of carbapenemase-producing Enterobacterales in Ireland from 2012 to 2017: a retrospective genomic surveillance study.

The spread of carbapenemase-producing Enterobacterales (CPE) is of major public health concern. The transmission dynamics of CPE in hospitals, particularly at the national level, are not well understood. Here, we describe a retrospective nationwide genomic surveillance study of CPE in Ireland between 2012 and 2017. We sequenced 746 national surveillance CPE samples obtained between 2012 and 2017. After clustering the sequences, we used thresholds based on pairwise SNPs, and reported within-host diversity along with epidemiological data to infer recent putative transmissions. All clusters in circulating clones, derived from high-resolution phylogenies, of a species (Klebsiella pneumoniae, Escherichia coli, Klebsiella oxytoca, Enterobacter cloacae, Enterobacter hormaechei and Citrobacter freundii) were individually examined for evidence of transmission. Antimicrobial resistance trends over time were also assessed. We identified 352 putative transmission events in six species including widespread and frequent transmissions in three species. We detected putative outbreaks in 4/6 species with three hospitals experiencing prolonged outbreaks. The bla OXA-48 gene was the main cause of carbapenem resistance in Ireland in almost all species. An expansion in the number of sequence types carrying bla OXA-48 was an additional cause of the increasing prevalence of carbapenemase-producing K. pneumoniae and E. coli.

Simultaneous Nasal Carriage by Methicillin-Resistant and Methicillin Susceptible Staphylococcus aureus of Lineage ST398 in a Live Pig Transporter.

Methicillin-resistant Staphylococcus aureus (MRSA) sequence type (ST)398 is a livestock associated (LA) lineage with zoonotic potential, especially in humans with live pig contact. The objective of this study was to characterize two S. aureus strains of lineage ST398 (one methicillin-resistant (MRSA), one methicillin-susceptible (MSSA)) isolated from the same nasal sample of a patient admitted in the Intensive-Care Unit of a Spanish Hospital, and with previous occupational exposure to live pigs, by whole-genome-sequencing (WGS). The sample was obtained during routine surveillance for MRSA colonization. Purified genomic DNA was sequenced using Illumina HiSeq 2000 and processed using conventional bioinformatics software. The two isolates recovered were both S. aureus t011/ST398 and showed similar resistance-phenotypes, other than methicillin susceptibility. The possession of antibiotic resistance genes was the same, except for the mecA-gene located in SCCmecV in the MRSA isolate. The MSSA isolate harbored remnants of a SCCmec following the deletion of 17342bp from a recombination between two putative primases. Both isolates belonged to the livestock-associated clade as defined by three canonical single-nucleotide-polymorphisms, and neither possessed the human immune evasion cluster genes, chp, scn, or sak. The core genome alignment showed a similarity of 99.6%, and both isolates harbored the same mobile genetic elements. The two nasal ST398 isolates recovered from the patient with previous occupational exposure to pigs appeared to have a livestock origin and could represent different evolutionary steps of animal-human interface lineage. The MSSA strain was formed as a result of the loss of the mecA gene from the livestock-associated-MRSA lineage.

Whole-genome sequence profiling of antibiotic-resistant Staphylococcus aureus isolates from livestock and farm attendants in Ghana.

OBJECTIVE: Epidemic methicillin-resistant Staphylococcus aureus (MRSA) clones have been described in Ghana, but so far, no typical livestock-associated MRSA isolates (CC398) have been found. In this study we provide baseline information on antimicrobial resistance, population structure, and virulence gene content of S. aureus isolates from livestock and farm attendants. METHODS: Nasal samples were collected from cattle, pigs, goats, sheep, and farm attendants from three farms. Staphylococcus aureus was identified by matrix-assisted laser desorption/ionisation time-of-flight and antimicrobial susceptibility testing was performed using VITEK II (Biomerieux, Marcy l'Etoile, France) and interpreted according to EUCAST guidelines. Whole-genome sequencing was performed using the Illumina, San Diego, CA, USA MiSeq Platform. RESULTS: In total, 401 nasal swab samples were obtained from 57 farm attendants, 208 pigs, 30 goats, 26 sheep, and 80 cattle. The S. aureus isolates (n = 25) recovered (farm attendants: n = 10; pigs: n = 8; and goats: n = 7) were frequently resistant to penicillin (68%), tetracycline (44%), and ciprofloxacin (32%); two human isolates were MRSA. Twelve isolates (48%) were multidrug resistant (MDR) (>3 classes). Genome sequencing of the isolates revealed ST152-t355, ST9-t1430, and ST133-t8662 as dominant clones among farm attendants, pigs, and goats, respectively. The two MRSA isolates detected belonged to ST8-t334 and ST152-t355. The scn and sak genes associated with human-adaption were detected in 10 isolates; 9 from humans and 1 from a goat. Typing results provided evidence of a single potential transmission event (t861, PVL-, scn+). CONCLUSION: No MRSA was detected among livestock, perhaps because of low intensive farming; however, the relatively high prevalence of MDR isolates may be a result of inappropriate antibiotic usage in Ghanaian livestock production.

Mechanisms of ß-lactam resistance of Streptococcus uberis isolated from bovine mastitis cases.

A number of veterinary clinical pathology laboratories in New Zealand have been reporting emergence of increased minimum in inhibitory concentrations for ß-lactams in the common clinical bovine mastitis pathogen Streptococcus uberis. The objective of this study was to determine the genetic basis of this increase in MIC for ß-lactams amongst S. uberis. Illumina sequencing and determination of oxacillin MIC was performed on 265 clinical isolates. Published sequences of the five penicillin binding proteins pbp1a, pbp1b, pbp2a, pbp2b, and pbp2x were used to identify, extract and align these sequences from the study isolates. Amino acid substitutions resulting from single nucleotide polymorphisms (SNP) within these genes were analysed for associations with elevated (≥ 0.5 mg/L) oxacillin MIC together with a genome wide association study. The population structure of the study isolates was approximated using a phylogenetic tree generated from an alignment of the core genome. A total of 53 % of isolates had MIC ≥ 0.5 mg/L for oxacillin. A total of 101 substitutions within the five pbp were identified, of which 11 were statistically associated with an MIC ≥ 0.5 mg/L. All 140 isolates which exhibited an increased ß-lactam MIC had SNPs leading to pbp2x E381K and Q554E substitutions. The phylogenetic tree indicated that the genotype and phenotype associated with the increased MIC for oxacillin were present in several different lineages suggesting that acquisition of this increased ß-lactam MIC had occurred in multiple geographically distinct regions. Reanalysis of the data from the intervention studies from which the isolates were originally drawn found a tendency for the pbp2x E381K substitution to be associated with lower cure rates. It is concluded that there is geographically and genetically widespread presence of pbp substitutions associated with reduced susceptibility to ß-lactam antimicrobials. Additionally, presence of pbp substitutions tended to be associated with poorer cure rate outcomes following antimicrobial therapy for clinical mastitis.

One Health Genomic Surveillance of Escherichia coli Demonstrates Distinct Lineages and Mobile Genetic Elements in Isolates from Humans versus Livestock.

Livestock have been proposed as a reservoir for drug-resistant Escherichia coli that infect humans. We isolated and sequenced 431 E. coli isolates (including 155 extended-spectrum ß-lactamase [ESBL]-producing isolates) from cross-sectional surveys of livestock farms and retail meat in the East of England. These were compared with the genomes of 1,517 E. coli bacteria associated with bloodstream infection in the United Kingdom. Phylogenetic core genome comparisons demonstrated that livestock and patient isolates were genetically distinct, suggesting that E. coli causing serious human infection had not directly originated from livestock. In contrast, we observed highly related isolates from the same animal species on different farms. Screening all 1,948 isolates for accessory genes encoding antibiotic resistance revealed 41 different genes present in variable proportions in human and livestock isolates. Overall, we identified a low prevalence of shared antimicrobial resistance genes between livestock and humans based on analysis of mobile genetic elements and long-read sequencing. We conclude that within the confines of our sampling framework, there was limited evidence that antimicrobial-resistant pathogens associated with serious human infection had originated from livestock in our region.IMPORTANCE The increasing prevalence of E. coli bloodstream infections is a serious public health problem. We used genomic epidemiology in a One Health study conducted in the East of England to examine putative sources of E. coli associated with serious human disease. E. coli from 1,517 patients with bloodstream infections were compared with 431 isolates from livestock farms and meat. Livestock-associated and bloodstream isolates were genetically distinct populations based on core genome and accessory genome analyses. Identical antimicrobial resistance genes were found in livestock and human isolates, but there was limited overlap in the mobile elements carrying these genes. Within the limitations of sampling, our findings do not support the idea that E. coli causing invasive disease or their resistance genes are commonly acquired from livestock in our region.

Genomic survey of Clostridium difficile reservoirs in the East of England implicates environmental contamination of wastewater treatment plants by clinical lineages.

There is growing evidence that patients with Clostridiumdifficile-associated diarrhoea often acquire their infecting strain before hospital admission. Wastewater is known to be a potential source of surface water that is contaminated with C. difficile spores. Here, we describe a study that used genome sequencing to compare C. difficile isolated from multiple wastewater treatment plants across the East of England and from patients with clinical disease at a major hospital in the same region. We confirmed that C. difficile from 65 patients were highly diverse and that most cases were not linked to other active cases in the hospital. In total, 186 C. difficile isolates were isolated from effluent water obtained from 18 municipal treatment plants at the point of release into the environment. Whole genome comparisons of clinical and environmental isolates demonstrated highly related populations, and confirmed extensive release of toxigenic C. difficile into surface waters. An analysis based on multilocus sequence types (STs) identified 19 distinct STs in the clinical collection and 38 STs in the wastewater collection, with 13 of 44 STs common to both clinical and wastewater collections. Furthermore, we identified five pairs of highly similar isolates (≤2 SNPs different in the core genome) in clinical and wastewater collections. Strategies to control community acquisition should consider the need for bacterial control of treated wastewater.

Genomic Surveillance of Enterococcus faecium Reveals Limited Sharing of Strains and Resistance Genes between Livestock and Humans in the United Kingdom.

Vancomycin-resistant Enterococcus faecium (VREfm) is a major cause of nosocomial infection and is categorized as high priority by the World Health Organization global priority list of antibiotic-resistant bacteria. In the past, livestock have been proposed as a putative reservoir for drug-resistant E. faecium strains that infect humans, and isolates of the same lineage have been found in both reservoirs. We undertook cross-sectional surveys to isolate E. faecium (including VREfm) from livestock farms, retail meat, and wastewater treatment plants in the United Kingdom. More than 600 isolates from these sources were sequenced, and their relatedness and antibiotic resistance genes were compared with genomes of almost 800 E. faecium isolates from patients with bloodstream infection in the United Kingdom and Ireland. E. faecium was isolated from 28/29 farms; none of these isolates were VREfm, suggesting a decrease in VREfm prevalence since the last UK livestock survey in 2003. However, VREfm was isolated from 1% to 2% of retail meat products and was ubiquitous in wastewater treatment plants. Phylogenetic comparison demonstrated that the majority of human and livestock-related isolates were genetically distinct, although pig isolates from three farms were more genetically related to human isolates from 2001 to 2004 (minimum of 50 single-nucleotide polymorphisms [SNPs]). Analysis of accessory (variable) genes added further evidence for distinct niche adaptation. An analysis of acquired antibiotic resistance genes and their variants revealed limited sharing between humans and livestock. Our findings indicate that the majority of E. faecium strains infecting patients are largely distinct from those from livestock in this setting, with limited sharing of strains and resistance genes.IMPORTANCE The rise in rates of human infection caused by vancomycin-resistant Enterococcus faecium (VREfm) strains between 1988 to the 2000s in Europe was suggested to be associated with acquisition from livestock. As a result, the European Union banned the use of the glycopeptide drug avoparcin as a growth promoter in livestock feed. While some studies reported a decrease in VREfm in livestock, others reported no reduction. Here, we report the first livestock VREfm prevalence survey in the UK since 2003 and the first large-scale study using whole-genome sequencing to investigate the relationship between E. faecium strains in livestock and humans. We found a low prevalence of VREfm in retail meat and limited evidence for recent sharing of strains between livestock and humans with bloodstream infection. There was evidence for limited sharing of genes encoding antibiotic resistance between these reservoirs, a finding which requires further research.

Identification of LukPQ, a novel, equid-adapted leukocidin of Staphylococcus aureus.

Bicomponent pore-forming leukocidins are a family of potent toxins secreted by Staphylococcus aureus, which target white blood cells preferentially and consist of an S- and an F-component. The S-component recognizes a receptor on the host cell, enabling high-affinity binding to the cell surface, after which the toxins form a pore that penetrates the cell lipid bilayer. Until now, six different leukocidins have been described, some of which are host and cell specific. Here, we identify and characterise a novel S. aureus leukocidin; LukPQ. LukPQ is encoded on a 45 kb prophage (ΦSaeq1) found in six different clonal lineages, almost exclusively in strains cultured from equids. We show that LukPQ is a potent and specific killer of equine neutrophils and identify equine-CXCRA and CXCR2 as its target receptors. Although the S-component (LukP) is highly similar to the S-component of LukED, the species specificity of LukPQ and LukED differs. By forming non-canonical toxin pairs, we identify that the F-component contributes to the observed host tropism of LukPQ, thereby challenging the current paradigm that leukocidin specificity is driven solely by the S-component.

Comparison of two chromogenic media for the detection of vancomycin-resistant enterococcal carriage by nursing home residents.

We compared ChromID VRE and Brilliance VRE media for the detection of vancomycin-resistant enterococci (VRE). Using a panel of 28 enterococcal isolates, 10 vanA Enterococcus faecium and three vanA Enterococcus faecalis isolates grew as per manufacturers' instructions whilst growth of two vanC and eight vancomycin-susceptible enterococci was inhibited on both media. Important differences were noted in the selectivity and chromogenic properties of the two media for vanA Enterococcus raffinosus and vanB E. faecium. The two media were further evaluated using 295 stool samples from nursing home residents, 34 of which grew VRE (11.5%). ChromID and Brilliance had comparable sensitivity, which was increased markedly by prolonging incubation to 48 hours (from 29% to 82%, and from 41% to 85%, respectively) and by a pre-enrichment step (to 97% and 100%, respectively). Brilliance VRE agar had higher selectivity at 48 hours, and after pre-enrichment.