Staphylococcus aureus colonization and acquisition of skin and soft tissue infection among Royal Marines recruits: a prospective cohort study.

OBJECTIVES: Skin and soft tissue infections (SSTIs) are a serious health issue for military personnel. Of particular importance are those caused by methicillin-resistant Staphylococcus aureus and Panton-Valentine leucocidin (PVL)-positive S. aureus (PVL-SA), as they have been associated with outbreaks of SSTIs. A prospective observational study was conducted in Royal Marine (RM) recruits to investigate the prevalence of PVL-SA carriage and any association with SSTIs. METHODS: A total of 1012 RM recruits were followed through a 32-week training programme, with nose and throat swabs obtained at weeks 1, 6, 15 and 32. S. aureus isolates were characterized by antibiotic susceptibility testing, spa typing, presence of mecA/C and PVL genes. Retrospective review of the clinical notes for SSTI acquisition was conducted. RESULTS: S. aureus colonization decreased from Week 1 to Week 32 (41% to 26%, p < 0.0001). Of 1168 S. aureus isolates, three out of 1168 (0.3%) were MRSA and ten out of 1168 (0.9%) PVL-positive (all MSSA) and 169 out of 1168 (14.5%) were resistant to clindamycin. Isolates showed genetic diversity with 238 different spa types associated with 25 multi-locus sequence type (MLST) clonal complexes. SSTIs were seen in 35% (351/989) of recruits with 3 training days lost per recruit. SSTI acquisition rate was reduced amongst persistent carriers (p < 0.0283). CONCLUSIONS: Nose and throat carriage of MRSA and PVL-SA was low among recruits, despite a high incidence of SSTIs being reported, particularly cellulitis. Carriage strains were predominantly MSSA with a marked diversity of genotypes. Persistent nose and/or throat carriage was not associated with SSTI acquisition. Putative person-to-person transmission within troops was identified based on spa typing requiring further research to confirm and explore potential transmission routes.

Outbreak report of investigation and control of an outbreak of Panton-Valentine Leukocidin-positive methicillin-sensitive Staphylococcus aureus (PVL-MSSA) infection in neonates and mothers.

BACKGROUND: In January 2011, there was an outbreak of Panton-Valentine Leukocidin-positive methicillin-sensitive Staphylococcus aureus (PVL-MSSA) infection in a neonatal unit (NNU). We describe the investigation and control of an outbreak of PVL-MSSA infection in neonates. SETTING: Neonatal unit in West London. METHODS: We performed descriptive and analytical (case-control study) epidemiological investigations. Microbiological investigations including screening of MSSA isolates by PCR for the presence of the luk-PV, mecA and mecC genes and comparison of isolate with Pulsed field gel electrophoresis (PFGE). Control measures were also introduced. RESULTS: Sixteen babies were infected/colonised with the outbreak strain. Of these, one baby developed blood stream infection, 12 developed skin pustules and four babies were colonised. Four mothers developed breast abscesses. Eighty-seven babies in the unit were screened and 16 were found to have same PVL-MSSA strain (spa type t005, belonging to MLST clonal complex 22). Multivariate analysis showed gestational age was significantly lower in cases compared to controls (mean gestational age: 31.7 weeks v 35.6 weeks; P = 0.006). Length of stay was significantly greater for cases, with a median of 25 days, compared to only 6 days for controls (P = 0.01). Most (88%) cases were born through caesarean section, compared to less than half of controls. (P = 0.002). No healthcare worker carriers and environmental source was identified. The outbreak was controlled by stopping new admissions to unit and reinforcing infection control precautions. The outbreak lasted for seven weeks. No further cases were reported in the following year. CONCLUSIONS: Infection control teams have to be vigilant for rising prevalence of particular S. aureus clones in their local community as they may cause outbreaks in vulnerable populations in healthcare settings such as NNUs.

Whole-Genome Sequencing Reveals the Contribution of Long-Term Carriers in Staphylococcus aureus Outbreak Investigation.

Whole-genome sequencing (WGS) makes it possible to determine the relatedness of bacterial isolates at a high resolution, thereby helping to characterize outbreaks. However, for Staphylococcus aureus, the accumulation of within-host diversity during carriage might limit the interpretation of sequencing data. In this study, we hypothesized the converse, namely, that within-host diversity can in fact be exploited to reveal the involvement of long-term carriers (LTCs) in outbreaks. We analyzed WGS data from 20 historical outbreaks and applied phylogenetic methods to assess genetic relatedness and to estimate the time to most recent common ancestor (TMRCA). The findings were compared with the routine investigation results and epidemiological evidence. Outbreaks with epidemiological evidence for an LTC source had a mean estimated TMRCA (adjusted for outbreak duration) of 243 days (95% highest posterior density interval [HPD], 143 to 343 days) compared with 55 days (95% HPD, 28 to 81 days) for outbreaks lacking epidemiological evidence for an LTC (P = 0.004). A threshold of 156 days predicted LTC involvement with a sensitivity of 0.875 and a specificity of 1. We also found 6/20 outbreaks included isolates with differing antimicrobial susceptibility profiles; however, these had only modestly increased pairwise diversity (mean 17.5 single nucleotide variants [SNVs] [95% confidence interval {CI}, 17.3 to 17.8]) compared with isolates with identical antibiograms (12.7 SNVs [95% CI, 12.5 to 12.8]) (P < 0.0001). Additionally, for 2 outbreaks, WGS identified 1 or more isolates that were genetically distinct despite having the outbreak pulsed-field gel electrophoresis (PFGE) pulsotype. The duration-adjusted TMRCA allowed the involvement of LTCs in outbreaks to be identified and could be used to decide whether screening for long-term carriage (e.g., in health care workers) is warranted. Requiring identical antibiograms to trigger investigation could miss important contributors to outbreaks.

Detection and molecular characterization of Livestock-Associated MRSA in raw meat on retail sale in North West England.

Limited data are available on the prevalence of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) in the UK. We tested 124 raw meat samples for MRSA including pork (n = 63), chicken (n = 50) and turkey (n = 11) collected from retail outlets in North West England between March and July 2015. MRSA was recovered from nine (7·3%) samples (four chicken, three pork and two turkey) from different butchers and supermarkets. Four were labelled of UK origin, three were from continental Europe; the origin was not specified for two samples. Whole-genome sequencing (WGS), spa typing and the presence of lineage-specific canonical single nucleotide polymorphisms confirmed that they belonged to the livestock-associated clade of clonal complex (CC) 398. Seven (77·8%) isolates were multi-drug resistant. Phylogenetic analyses showed the isolates were diverse, suggesting multiple silent introductions of LA-MRSA into the UK food chain. Two chicken meat isolates belonged to a sub-clade recently reported from human cases in Europe where poultry meat was the probable source. The low levels of MRSA identified (<20 CFU per g) and absence of enterotoxin genes suggest the risk of acquisition of, or food-poisoning due to, LA-MRSA is low. Nevertheless, the MRSA contamination rate is higher than previously estimated; further evaluation of the public health impacts of LA-MRSA is warranted. SIGNIFICANCE AND IMPACT OF THE STUDY: Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) is a public health concern worldwide, but has only been reported sporadically in the UK. In the largest UK study to date, samples of raw meat at retail sale were examined for both the presence and levels of MRSA. We report the first isolations of CC398 LA-MRSA from poultry meat in the UK including representatives of a particular sub-clade associated with cases of human infection/colonization in Europe. Although levels were low (<20 CFU per g), the contamination rate was higher than previous UK studies. Moreover, whole-genome sequencing revealed multiple independent introductions of LA-MRSA into the UK food chain.

First report of identification of livestock-associated MRSA ST9 in retail meat in England.

Sixty percent of all meat consumed in the UK is imported from European countries where there have been increasing reports of methicillin-resistant Staphylococcus aureus (MRSA) identified in food-producing animals, but rarely from such animals in the UK. Thirty samples each of raw chicken, pork and beef, sourced in England, were collected from retail outlets in Greater Manchester. MRSA was recovered from three chicken samples and one each of pork and beef, all from prepackaged supermarket meat. Four isolates were identified as representatives of the most common human healthcare-associated MRSA clone in the UK [EMRSA-15, spa type t032, belonging to multilocus sequence type clonal complex 22 (MLST-CC22)], suggesting contamination from human source(s) during meat processing. The fifth isolate (from chicken) was multiply-resistant (including oxacillin, ciprofloxacin, erythromycin, clindamycin and tetracycline), identified as ST9-SCCmecIV, spa type t1939 and lacked the immune evasion cluster, a characteristic of livestock-associated strains. This lineage has been identified previously from animals and meat products in Asia and mainland Europe but not the UK.

Prediction of Staphylococcus aureus antimicrobial resistance by whole-genome sequencing.

Whole-genome sequencing (WGS) could potentially provide a single platform for extracting all the information required to predict an organism's phenotype. However, its ability to provide accurate predictions has not yet been demonstrated in large independent studies of specific organisms. In this study, we aimed to develop a genotypic prediction method for antimicrobial susceptibilities. The whole genomes of 501 unrelated Staphylococcus aureus isolates were sequenced, and the assembled genomes were interrogated using BLASTn for a panel of known resistance determinants (chromosomal mutations and genes carried on plasmids). Results were compared with phenotypic susceptibility testing for 12 commonly used antimicrobial agents (penicillin, methicillin, erythromycin, clindamycin, tetracycline, ciprofloxacin, vancomycin, trimethoprim, gentamicin, fusidic acid, rifampin, and mupirocin) performed by the routine clinical laboratory. We investigated discrepancies by repeat susceptibility testing and manual inspection of the sequences and used this information to optimize the resistance determinant panel and BLASTn algorithm. We then tested performance of the optimized tool in an independent validation set of 491 unrelated isolates, with phenotypic results obtained in duplicate by automated broth dilution (BD Phoenix) and disc diffusion. In the validation set, the overall sensitivity and specificity of the genomic prediction method were 0.97 (95% confidence interval [95% CI], 0.95 to 0.98) and 0.99 (95% CI, 0.99 to 1), respectively, compared to standard susceptibility testing methods. The very major error rate was 0.5%, and the major error rate was 0.7%. WGS was as sensitive and specific as routine antimicrobial susceptibility testing methods. WGS is a promising alternative to culture methods for resistance prediction in S. aureus and ultimately other major bacterial pathogens.

Prolonged outbreak of meticillin-resistant Staphylococcus aureus in a cardiac surgery unit linked to a single colonized healthcare worker.

BACKGROUND: In low- as well as in high-prevalence settings, healthcare workers (HCWs) may be a substantial, under-recognized, reservoir of meticillin-resistant Staphylococcus aureus (MRSA) and an important potential source of transmission to patients. AIM: To report an outbreak of MRSA in a cardiac surgery unit in England over a 10-month period. METHODS: Cases were defined as patients and staff on the cardiac surgery unit from whom the outbreak strain was newly isolated between 20 May 2011 and 16 March 2012. Representative isolates from all cases were characterized by spa-typing, pulsed-field gel electrophoresis and multi-locus variable-number tandem-repeat analysis (MLVA). FINDINGS: Four patients appeared to acquire MRSA during their inpatient stay on the cardiac surgery unit. All four patients and one HCW were found to be carrying an identical epidemic (E)MRSA-15 strain (spa t032, pulsotype A, MLVA profile 16-6-3-1-1-17-1-4). No other members of staff were found to be colonized with MRSA. The colonized HCW was thought to be the source of the outbreak and was decolonized using a combination of nasal mupirocin, chlorhexidine body wash and oral rifampicin and doxycycline. CONCLUSIONS: This report highlights recent changes in the epidemiology of MRSA in England and suggests an important role for colonized HCWs in the transmission of MRSA to patients. Screening HCWs may provide an increasingly valuable strategy in managing linked hospital acquisitions and well-defined outbreaks where initial investigation does not reveal a source.

Association of a lukM-positive clone of Staphylococcus aureus with fatal exudative dermatitis in red squirrels (Sciurus vulgaris).

Fatal exudative dermatitis (FED) is a recently described condition affecting red squirrels (Sciurus vulgaris) on the Isle of Wight and Jersey (Simpson et al., 2010a). Staphylococcus aureus strains isolated from skin lesions in cases of FED were characterised by molecular and phenotypic approaches. The strains were found to belong to a single MLST clonal complex (CC49) representing either ST49 or a novel single locus variant thereof (ST1957), were closely related by other molecular typing approaches, and all possessed the leukotoxin M encoding gene (lukM). In contrast S. aureus was either not isolated from none-FED cases or belonged to distinct and diverse molecular types that, with one exception, did not encode lukM. All isolates from FED cases were susceptible to all antimicrobials tested, including penicillin, and all proved negative for mecA and mecC as well as 14 other staphylococcal toxin genes. As all squirrels affected by FED were infected with S. aureus of the same lineage and encoded the lukM gene, it is possible that strains of this lineage may be involved in the pathogenesis of the dermatitis.

Putative link between Staphylococcus aureus bacteriophage serotype and community association.

Methicillin-resistant Staphylococcus aureus (MRSA) from humans can be broadly separated into 3 groups: healthcare-associated (HA), community-associated (CA), and livestock-associated (LA) MRSA. Initially based on epidemiological features, division into these classes is becoming increasingly problematic. The sequencing of S. aureus genomes has highlighted variations in their accessory components, which likely account for differences in pathogenicity and epidemicity. In particular, temperate bacteriophages have been regarded as key players in bacterial pathogenesis. Bacteriophage-associated Panton-Valentine leukocidin genes (luk-PV) are regarded as epidemiological markers of the CA-MRSA due to their high prevalence in CA strains. This paper describes the development and application of a partial composite S. aureus virulence-associated gene microarray. Epidemic, pandemic, and sporadic lineages of UK HA and CA S. aureus were compared. Phage structural genes linked with CA isolates were identified and in silico analysis revealed these to be correlated with phage serogroup. CA strains predominantly carried a PVL-associated phage either of the A or Fb serogroup, whilst HA strains predominantly carried serogroup Fa or B phages. We speculate that carriage of a serogroup A/Fb PVL-associated phage rather than the luk-PV genes specifically is correlated with CA status.

Outbreak of a South West Pacific clone Panton-Valentine leucocidin-positive meticillin-resistant Staphylococcus aureus infection in a UK neonatal intensive care unit.

BACKGROUND: Panton-Valentine leucocidin-positive meticillin-resistant Staphylococcus aureus (PVL-MRSA) has become a globally common cause of community-acquired infections. AIM: We report an outbreak of PVL-MRSA in a regional neonatal unit in the UK involving three babies and three staff members. METHODS: Quinolone susceptibility was helpful in identifying potential PVL-MRSA but toxin gene profiling and sequence-based typing were required to distinguish between two PVL-MRSA strains present in the unit. FINDINGS: All three symptomatic babies and two staff carriers, one of whom was symptomatic, were found to be carrying the South West Pacific (SWP) clone of PVL-MRSA (ST30). One of the staff carriers had recently visited the Philippines and was thought to be the source of the outbreak. Control was established using standard infection control procedures but one baby with relapsing MRSA colonization has required more than 100 days in isolation. CONCLUSION: This is the first reported neonatal outbreak associated with the SWP clone in the UK. Our study highlights the potential risk of further introductions of this organism by healthcare staff or patients epidemiologically linked with the Philippines.

Decline and fall of epidemic meticillin-resistant Staphylococcus aureus-16.

Epidemic meticillin-resistant Staphylococcus aureus-16, which was widespread throughout the UK and the rest of the world, has declined markedly in recent years. The reasons for this are not clear.

Molecular diversity within clonal complex 22 methicillin-resistant Staphylococcus aureus encoding Panton-Valentine leukocidin in England and Wales.

Panton-Valentine leukocidin (PVL)-positive methicillin-resistant Staphylococcus aureus (MRSA) that are multi-locus sequence type clonal complex 22 (CC22) comprise a significant public health problem in the UK. In the present study we sought to determine the genetic diversity, and the respective patient demographics, among 47 PVL-MRSA with a CC22 pulsotype that occurred sporadically or in clusters in community and healthcare settings in eight of nine geographic regions in England and Wales between January 2005 and September 2007. Patient demographics and disease presentations were typical for PVL-S. aureus infections (mostly skin and soft tissue infections in individuals <40 years old); one patient with community-acquired pneumonia died. Although the isolates were closely genotypically related by spa typing and pulsed field gel electrophoresis, at least two variant groups were suggested. PCR detections demonstrated that the majority of the CC22 PVL-MRSA identified (n = 42; 89%) harboured SCCmecIVc, three had SCCmecIVd, one had SCCmecIV but was non-subtypeable, and one isolate harboured SCCmecV. At least three different PVL-encoding phages were detected: ФPVL, Ф108PVL and an unidentified icosahedral phage. Agar dilution MIC determinations showed that the CC22 PVL-MRSA identified were typically resistant to gentamicin and trimethoprim (43 of 47 isolates) and ciprofloxacin resistance was also noted in six isolates. In conclusion, the CC22 PVL-MRSA tested were geographically disseminated but highly genetically related. The observed variances in acquired elements (most notably SCCmec and PVL-encoding phages) suggested that CC22 PVL-MRSA in England and Wales have evolved on multiple occasions.

Distinct bacteriophages encoding Panton-Valentine leukocidin (PVL) among international methicillin-resistant Staphylococcus aureus clones harboring PVL.

Genetically diverse community-associated methicillin resistant Staphylococcus aureus (CA-MRSA) can harbor a bacteriophage encoding Panton-Valentine leukocidin (PVL) lysogenized into its chromosome (prophage). Six PVL phages (ΦPVL, Φ108PVL, ΦSLT, ΦSa2MW, ΦSa2USA, and ΦSa2958) are known, and single-nucleotide polymorphisms (SNPs) in the PVL genes have been reported. We sought to determine the distribution of lysogenized PVL phages among MRSA strains with PVL (PVL-MRSA strains), the PVL gene sequences, and the chromosomal phage insertion sites in 114 isolates comprising nine clones of PVL-MRSA that were selected for maximal underlying genetic diversity. The six PVL phages were identified by PCR; ΦSa2USA was present in the highest number of different lineages (multilocus sequence type clonal complex 1 [CC1], CC5, CC8, and sequence type 93 [ST93]) (n = 37 isolates). Analysis of 92 isolates confirmed that PVL phages inserted into the same chromosomal insertion locus in CC22, -30, and -80 but in a different locus in isolates of CC1, -5, -8, -59, and -88 and ST93 (and CC22 in two isolates). Within the two different loci, specific attachment motifs were found in all cases, although some limited inter- and intralineage sequence variation occurred. Overall, lineage-specific relationships between the PVL phage, the genes that encode the toxin, and the position at which the phage inserts into the host chromosome were identified. These analyses provide important insights into the microepidemiology of PVL-MRSA, will prove a valuable adjunct in outbreak investigation, and may help predict the emergence of new strains.

Community and nosocomial transmission of Panton-Valentine leucocidin-positive community-associated meticillin-resistant Staphylococcus aureus: implications for healthcare.

In the UK, infections due to Panton-Valentine leucocidin-positive community-associated meticillin-resistant Staphylococcus aureus (PVL-MRSA) have been reported sporadically. In September 2006, a fatal PVL-MRSA infection occurred in a Filipino healthcare worker (HCW) after she underwent caesarean section. Throat and nasal swabs were obtained from contacts of cases in community and hospital. MRSA with an antibiogram similar to the PVL-MRSA strain were characterised including toxin gene profiling, polymerase chain reaction- and sequence-based typing. Carriers underwent decolonisation treatment, and HCWs were restricted from patient care until they and their household members were considered negative for PVL-MRSA. The PVL-MRSA belonged to ST30, was protein A gene (spa) type t019, SCCmec IVc, agr 3, and resistant only to beta-lactam antibiotics. Representatives of the same lineage were identified among a further 16 individuals in community and hospital. Infections likely to be caused by PVL-MRSA had occurred in 12 cases, and were likely to be hospital-acquired in two patients (one fatal) and occupationally acquired in one HCW. Nine cases worked as nursing staff in the hospital. Eight of these had emigrated from the Philippines in the previous five years and were linked socially. Thus, PVL-MRSA-ST30 was detected in a HCW community in the UK. This is the first report of nosocomial transmission of this pandemic clone in the UK associated with a fatality. Increased vigilance in healthcare and community is needed in response to this emerging threat.

Enhanced surveillance of methicillin-resistant Staphylococcus aureus (MRSA) bacteraemia in children in the UK and Ireland.

OBJECTIVE: To determine the incidence and demographic features of methicillin-resistant Staphylococcus aureus (MRSA) bacteraemia in children in the UK and Ireland and to characterise MRSA isolated from cases. DESIGN: Prospective surveillance study. SETTING: Children aged <16 years hospitalised with bacteraemia due to MRSA. METHODS: Cases were ascertained by active surveillance involving paediatricians reporting to the British Paediatric Surveillance Unit and by routine laboratory surveillance. Patient characteristics were obtained using questionnaires sent to reporting paediatricians. MRSA isolates were characterised using molecular and phenotypic techniques including antimicrobial susceptibility testing. RESULTS: 265 episodes of MRSA bacteraemia were ascertained, involving 252 children. The overall incidence rate was 1.1 per 100 000 child population per year (95% CI 0.9 to 1.2): 61% of the children were aged <1 year (a rate of 9.7 cases per 100 000 population per year (95% CI 8.2 to 11.4)) and 35% were <1 month. Clinical data were obtained from 115 cases. The clinical presentation varied, with fever present in only 16% of neonates compared with 72% of older children. A history of invasive procedure was common, with 32% having had intravascular lines and 13% having undergone surgery. 62% of patients for whom data were available were receiving high-dependency care (46% in SCBU/NICU and 16% in PICU). Of 93 MRSA isolates studied, 73% belonged to epidemic strains widely associated with nosocomial infection in the UK and Ireland. CONCLUSIONS: MRSA bacteraemia in children was relatively uncommon and was predominantly seen in very young children, often those receiving neonatal or paediatric intensive care. Bacteraemia predominantly involved well-documented epidemic strains of MRSA associated with nosocomial infection.

Patient segregation and aggressive antibiotic eradication therapy can control methicillin-resistant Staphylococcus aureus at large cystic fibrosis centres.

BACKGROUND: The prevalence of MRSA in patients with CF has risen in recent years. We adhere to a policy of segregation and barrier nursing to manage patients with MRSA, and we actively pursue eradication of MRSA. We have evaluated our experiences of MRSA infection in our large adult CF centre. METHOD: A retrospective review of all MRSA-positive patients from 1998 to 2008 was undertaken. Isolates were subjected to molecular identification to elucidate possible patient-to-patient transmission events. Eradication attempts were scrutinised. RESULTS: We have maintained a low incidence and prevalence (below 3%) of MRSA within this large cohort. A total of 15 pulsotypes of MRSA were identified among the 24 isolates examined, epidemiological data suggested no patient-patient transmission. Based on 6 month follow-up data, successful eradication was achieved in 81% patients. This includes those who had harboured infection for some time. Twenty-one (80.8%) required only one course of treatment, 3 (11.6%) patients required two different regimes and 2 (7.5%) required three courses to fully eradicate the organism. CONCLUSION: Strict infection control procedures can control MRSA infection and keep the prevalence low in CF clinics. Eradication is achievable in the majority of patients even when significant time has lapsed from initial isolation. In some instances, up to 3 courses of antibiotics were required to achieve eradication.

Clinical, molecular and epidemiological description of a cluster of community-associated methicillin-resistant Staphylococcus aureus isolates from injecting drug users with bacteraemia.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) is an increasing problem, predominantly in previously healthy individuals including notable risk groups such as the homeless, those who play close-contact sports, military personnel, men who have sex with men (MSM) and injecting drug users (IDUs). Over a 5-month period, four IDUs were admitted to Addenbrooke's Hospital, Cambridge, UK, with MRSA bacteraemia. All four patients presented with complex clinical features, with more than one focus of infection, and were linked epidemiologically. The atypical antibiogram of the MRSA isolates (ciprofloxacin-susceptible) prompted further characterization, both phenotypically (antibiotic resistance typing; phage typing) and genotypically (detection of toxin genes by PCR; pulsed-field gel electrophoresis (PFGE); Staphylococcal chromosome cassette (SCC) mec typing; multi-locus sequence typing (MLST)). All four isolates had similar antibiograms, were Panton-Valentine Leucocidin (PVL) toxin gene-negative, harboured SCCmec type IV and were closely related as shown by phage typing and PFGE. These isolates were representatives of a community-associated clone, ST1-MRSA-IV, known to be circulating in IDUs in the UK since 2001. This paper presents a detailed description of the clinical, microbiological and epidemiological features of a series of CA-MRSA bacteraemias in IDUs in the UK.