Clinical impact and public health challenges of a PVL-MRSA bacteraemia outbreak amongst people who inject drugs in South Yorkshire, UK.

Background: Panton-Valentine leukocidin (PVL) Staphylococcus aureus (SA) is an emergent public health concern. PVL toxin has been mostly associated with methicillin-sensitive S. aureus (MSSA)-related skin and soft tissue infections occurring in high-risk groups such as people who inject drugs (PWID). The emergence of PVL methicillin-resistant S. aureus (MRSA) infection is causing severe and life-threatening disease in PWID. Clinical cases: We present an outbreak of eight PVL-MRSA bacteraemia cases at a UK teaching hospital between 2018 and 2022. An additional four patients developed bacteraemia with PVL-negative MRSA of the same multilocus sequence type (MLST). All patients were PWID and aged 33-51 years old. Four patients developed MRSA bacterial endocarditis. Three patients died. These cases represent the initial cases detected at Doncaster and Bassetlaw Teaching Hospitals of what is an ongoing and developing outbreak. Management: An outbreak investigation has been undertaken in association with the UK Health Security Agency. Epidemiological factors have been explored, including via direct contact at a local sheltered accommodation and the possibility of a contaminated drug supply. Whole-genome sequencing confirmed that all isolates were closely related and of the same MLST (sequence type 5). A community substance misuse group disseminated health education on the prevention of PVL-MRSA. Preventing infection in PWID presents a major challenge due to the impact of addiction on engagement with services and the significant barriers faced by our patients in observing infection prevention measures. Conclusion: PVL-MRSA is of major public health concern and outbreak investigation and mapping out local epidemiological patterns plays a vital role in preventing further spread throughout the community. Additionally, this work enables targeted and early treatment in patients in high-risk categories for disease. These cases of PVL-MRSA infection in PWID highlights the transmissibility, pathogenic potential and severe clinical disease spectrum within this population. Further work is required to tackle transmission and infection from this pathogenic strain.

Comprehensive evaluation of the implementation of episignatures for diagnosis of neurodevelopmental disorders (NDDs).

Episignatures are popular tools for the diagnosis of rare neurodevelopmental disorders. They are commonly based on a set of differentially methylated CpGs used in combination with a support vector machine model. DNA methylation (DNAm) data often include missing values due to changes in data generation technology and batch effects. While many normalization methods exist for DNAm data, their impact on episignature performance have never been assessed. In addition, technologies to quantify DNAm evolve quickly and this may lead to poor transposition of existing episignatures generated on deprecated array versions to new ones. Indeed, probe removal between array versions, technologies or during preprocessing leads to missing values. Thus, the effect of missing data on episignature performance must also be carefully evaluated and addressed through imputation or an innovative approach to episignatures design. In this paper, we used data from patients suffering from Kabuki and Sotos syndrome to evaluate the influence of normalization methods, classification models and missing data on the prediction performances of two existing episignatures. We compare how six popular normalization methods for methylarray data affect episignature classification performances in Kabuki and Sotos syndromes and provide best practice suggestions when building new episignatures. In this setting, we show that Illumina, Noob or Funnorm normalization methods achieved higher classification performances on the testing sets compared to Quantile, Raw and Swan normalization methods. We further show that penalized logistic regression and support vector machines perform best in the classification of Kabuki and Sotos syndrome patients. Then, we describe a new paradigm to build episignatures based on the detection of differentially methylated regions (DMRs) and evaluate their performance compared to classical differentially methylated cytosines (DMCs)-based episignatures in the presence of missing data. We show that the performance of classical DMC-based episignatures suffers from the presence of missing data more than the DMR-based approach. We present a comprehensive evaluation of how the normalization of DNA methylation data affects episignature performance, using three popular classification models. We further evaluate how missing data affect those models' predictions. Finally, we propose a novel methodology to develop episignatures based on differentially methylated regions identification and show how this method slightly outperforms classical episignatures in the presence of missing data.

Trends in coagulase-negative staphylococci (CoNS), England, 2010-2021.

Objective: To review the epidemiology of coagulase-negative staphylococci (CoNS) in England over the recent 12 year period. Methods: Laboratory-confirmed CoNS reported from sterile sites in patients in England to the UK Health Security Agency (UKHSA) between 2010 and 2021 were extracted from the national laboratory database and analysed. Results: Overall, 668 857 episodes of CoNS were reported. Unspeciated CoNS accounted for 56 % (374 228) of episodes, followed by Staphylococcus epidermidis (26 %; 174 050), S. hominis (6.5 %; 43 501) and S. capitis (3.9 %; 25 773). Unspeciated CoNS increased by 8.2 % (95 % CI, 7.1-9.3) annually between 2010 and 2016, then decreased annually by 6.4 % (95 % CI: -4.8 to -7.9) until 2021. Speciated CoNS increased by 47.6 % (95 % CI, 44.5-50.9) annually between 2010 and 2016 and increased annually by 8.9 % (95 % CI: 5.1 to 12.8) until 2021. Antimicrobial susceptibility profiles differed by species. Conclusions: Reports of CoNS from normally sterile body sites in patients in England increased between 2010 and 2016 and remained stable between 2017 and 2021. There has been a striking improvement in species-level identification of CoNS in recent years. Monitoring trends in CoNS epidemiology is crucial for development of observational and clinical intervention studies on individual species.

Soft Tissue Infection of Immunocompetent Man with Cat-Derived Globicatella Species.

We report a novel Globicatella species causing extensive soft tissue infection in a man bitten by a stray domestic cat in the United Kingdom. We identified this bacterium by 16S rRNA gene sequencing, whole-genome sequencing, and biochemical profiling and determined antimicrobial drug susceptibility.

Whole-genome sequencing reveals widespread presence of Staphylococcus capitis NRCS-A clone in neonatal units across the United Kingdom.

OBJECTIVE: Increased incidence of neonatal Staphylococcus capitis bacteraemia in summer 2020, London, raised suspicion of widespread multidrug-resistant clone NRCS-A. We set out to investigate the molecular epidemiology of this clone in neonatal units (NNUs) across the UK. METHODS: We conducted whole-genome sequencing (WGS) on presumptive S. capitis NRCS-A isolates collected from infants admitted to nationwide NNUs and from environmental sampling in two distinct NNUs in 2021. Previously published S. capitis genomes were added for comparison. Genetic clusters of NRCS-A isolates were defined based on core-genome single-nucleotide polymorphisms. RESULTS: We analysed WGS data of 838 S. capitis isolates and identified 750 NRCS-A isolates. We discovered a possible UK-specific NRCS-A lineage consisting of 611 isolates collected between 2005 and 2021. We determined 28 genetic clusters of NRCS-A isolates, which covered all geographical regions in the UK, and isolates of 19 genetic clusters were found in ≥2 regions, suggesting inter-regional spread. Within the NRCS-A clone, strong genetic relatedness was identified between contemporary clinical and incubator-associated fomite isolates and between clinical isolates associated with inter-hospital infant transfer. CONCLUSIONS: This WGS-based study confirms the dispersion of S. capitis NRCS-A clone amongst NNUs across the UK and urges research on improving clinical management of neonatal S. capitis infection.

Is Panton-Valentine leucocidin (PVL) toxin associated with poor clinical outcomes in patients with community-acquired Staphylococcus aureus bacteraemia?

Introduction. Panton-Valentine leucocidin (PVL) toxin is a potential determinant of virulence associated with S. aureus infection.Gap Statement. The contribution of PVL to S. aureus pathogenicity remains unclear.Aim. To compare clinical outcomes in hospitalized patients with PVL-positive and PVL-negative community-acquired (CA) S. aureus bacteraemia.Methods. Three national datasets were combined to provide clinical and mortality data for patients with CA S. aureus blood culture isolates sent to the UK reference laboratory for PVL testing, August 2018 to August 2021. Multivariable logistic regression models were built for the effect of PVL positivity on 30 day all-cause mortality and 90 day readmission.Results. In 2191 cases of CA S. aureus bacteraemia, there was no association between PVL and mortality (adjusted odds ratio, aOR: 0·90, 95 % confidence interval, CI: 0·50-1·35, P=0·602) and no difference in median LOS (14 versus 15 days, P=0.169). PVL-positive cases had lower odds of readmission (aOR 0·74, CI 0·55-0.98, P=0·038). There was no evidence that MRSA status modified this effect (P=0·207).Conclusions. In patients with CA S. aureus bacteraemia PVL toxin detection was not associated with worse outcomes.

An unusual familial Xp22.12 microduplication including EIF1AX: A novel candidate dosage-sensitive gene for premature ovarian insufficiency.

We report on the results of array-CGH and Whole exome sequencing (WES) studies carried out in a Tunisian family with 46,XX premature ovarian insufficiency (POI). This study has led to the identification of a familial Xp22.12 tandem duplication with a size of 559.4 kb, encompassing only three OMIM genes (RPS6KA3, SH3KBP1and EIF1AX), and a new heterozygous variant in SPIDR gene: NM_001080394.3:c.1845_1853delTATAATTGA (p.Ile616_Asp618del) segregating with POI. Increased mRNA expression levels were detected for SH3KBP1 and EIF1AX, while a normal transcript level for RPS6KA3 was detected in the three affected family members, explaining the absence of intellectual disability (ID). To the best of our knowledge, this is the first duplication involving the Xp22.12 region, reported in a family without ID, but rather with secondary amenorrhea (SA) and female infertility. As EIF1AX is a regulatory gene escaping X-inactivation, which has an extreme dosage sensitivity and highly expressed in the ovary, we suggest that this gene might be a candidate gene for ovarian function. Homozygous nonsense pathogenic variants of SPIDR gene have been reported in familial cases in POI. It has been suggested that chromosomal instability associated with SPIDR molecular defects supports the role of SPIDR protein in double-stranded DNA damage repair in vivo in humans and its causal role in POI. In this family, the variant (p.Ile616_Asp618del), present in a heterozygous state, is located in the domain that interacts with BLM and might disrupt the BLM binding ability of SPIDR protein. These findings strengthen the hypothesis that the additional effect of this variant could lead to POI in this family. Although the work represents the first evidence that EIF1AX duplication might be responsible for POI through its over-expression, further functional studies are needed to clarify and prove EIF1AX involvement in POI phenotype.

Emergence of methicillin resistance predates the clinical use of antibiotics.

The discovery of antibiotics more than 80 years ago has led to considerable improvements in human and animal health. Although antibiotic resistance in environmental bacteria is ancient, resistance in human pathogens is thought to be a modern phenomenon that is driven by the clinical use of antibiotics1. Here we show that particular lineages of methicillin-resistant Staphylococcus aureus-a notorious human pathogen-appeared in European hedgehogs in the pre-antibiotic era. Subsequently, these lineages spread within the local hedgehog populations and between hedgehogs and secondary hosts, including livestock and humans. We also demonstrate that the hedgehog dermatophyte Trichophyton erinacei produces two ß-lactam antibiotics that provide a natural selective environment in which methicillin-resistant S. aureus isolates have an advantage over susceptible isolates. Together, these results suggest that methicillin resistance emerged in the pre-antibiotic era as a co-evolutionary adaptation of S. aureus to the colonization of dermatophyte-infected hedgehogs. The evolution of clinically relevant antibiotic-resistance genes in wild animals and the connectivity of natural, agricultural and human ecosystems demonstrate that the use of a One Health approach is critical for our understanding and management of antibiotic resistance, which is one of the biggest threats to global health, food security and development.

Rarity of fetal cells in exocervical samples for noninvasive prenatal diagnosis.

OBJECTIVES: The possibility to isolate fetal cells from pregnant women cervical samples has been discussed for five decades but is not currently applied in clinical practice. This study aimed at offering prenatal genetic diagnosis from fetal cells obtained through noninvasive exocervical sampling and immuno-sorted based on expression of HLA-G. METHODS: We first developed and validated robust protocols for cell detection and isolation on control cell lines expressing (JEG-3) or not (JAR) the HLA-G antigen, a specific marker for extravillous trophoblasts. We then applied these protocols to noninvasive exocervical samples collected from pregnant women between 6 and 14 weeks of gestational age. Sampling was performed through insertion and rotation of a brush at the ectocervix close to the external os of the endocervical canal. Finally, we attempted to detect and quantify trophoblasts in exocervical samples from pregnant women by ddPCR targeting the male SRY locus. RESULTS: For immunohistochemistry, a strong specific signal for HLA-G was observed in the positive control cell line and for rare cells in exocervical samples, but only in non-fixative conditions. HLA-G positive cells diluted in HLA-G negative cells were isolated by flow cytometry or magnetic cell sorting. However, no HLA-G positive cells could be recovered from exocervical samples. SRY gene was detected by ddPCR in exocervical samples from male (50%) but also female (27%) pregnancies. CONCLUSIONS: Our data suggest that trophoblasts are too rarely and inconstantly present in noninvasive exocervical samples to be reliably retrieved by standard immunoisolation techniques and therefore cannot replace the current practice for prenatal screening and diagnosis.

MRSA surveillance programmes worldwide: moving towards a harmonised international approach.

Multinational surveillance programmes for methicillin-resistant Staphylococcus aureus (MRSA) are dependent on national structures for data collection. This study aimed to capture the diversity of national MRSA surveillance programmes and to propose a framework for harmonisation of MRSA surveillance. The International Society of Antimicrobial Chemotherapy (ISAC) MRSA Working Group conducted a structured survey on MRSA surveillance programmes and organised a webinar to discuss the programmes' strengths and challenges as well as guidelines for harmonisation. Completed surveys represented 24 MRSA surveillance programmes in 16 countries. Several countries reported separate epidemiological and microbiological surveillance. Informing clinicians and national policy-makers were the most common purposes of surveillance. Surveillance of bloodstream infections (BSIs) was present in all programmes. Other invasive infections were often included. Three countries reported active surveillance of MRSA carriage. Methodology and reporting of antimicrobial susceptibility, virulence factors, molecular genotyping and epidemiological metadata varied greatly. Current MRSA surveillance programmes rely upon heterogeneous data collection systems, which hampers international epidemiological monitoring and research. To harmonise MRSA surveillance, we suggest improving the integration of microbiological and epidemiological data, implementation of central biobanks for MRSA isolate collection, and inclusion of a representative sample of skin and soft-tissue infection cases in addition to all BSI cases.

Staphylococcus argenteus transmission among healthy Royal Marines: A molecular epidemiology case-study.

Objectives During a prospective study of S. aureus carriage in Royal Marines recruits, six S. argenteus strains were identified in four recruits. As S. argenteus sepsis leads to mortality similar to S. aureus, we determined the potential for within same troop transmission, to evaluate future outbreak risk. Methods We used whole-genome sequencing to characterise S. argenteus and investigate phylogenetic relationships between isolates. Results S. argenteus strains (t5078, ST2250) were detected in 4/40 recruits in the same troop (training cohort) in weeks 1, 6 or 15 of training. No mec, tsst or LukPV genes were detected. We identified differences of 1-17 core SNPs between S. argenteus from different recruits. In two recruits, two S. argenteus strains were isolated; these could be distinguished by 2 and 15 core SNPs. Conclusions The identification of S. argenteus within a single troop from the total recruit population suggests a common source for transmission, though high number of SNPs were identified, both within-host and within-cluster. The high number of SNPs between some isolates may indicate a common source of diverse isolates or a high level of S. argenteus mutation in carriage. S. argenteus is newly recognized species; and understanding of the frequency of genetic changes during transmission and transition from asymptomatic carriage to disease is required.

Performance and Diagnostic Value of Genome-Wide Noninvasive Prenatal Testing in Multiple Gestations.

OBJECTIVE: To evaluate the accuracy and diagnostic value of genome-wide noninvasive prenatal testing (NIPT) for the detection of fetal aneuploidies in multiple gestations, with a focus on dichorionic-diamniotic twin pregnancies. METHODS: We performed a retrospective cohort study including data from pregnant women with a twin or higher-order gestation who underwent genome-wide NIPT at one of the eight Belgian genetic centers between November 1, 2013, and March 1, 2020. Chorionicity and amnionicity were determined by ultrasonography. Follow-up invasive testing was carried out in the event of positive NIPT results. Sensitivity and specificity were calculated for the detection of trisomy 21, 18, and 13 in the dichorionic-diamniotic twin cohort. RESULTS: Unique NIPT analyses were performed for 4,150 pregnant women with a multiple gestation and an additional 767 with vanishing gestations. The failure rate in multiple gestations excluding vanishing gestations ranged from 0% to 11.7% among the different genetic centers. Overall, the failure rate was 4.8%, which could be reduced to 1.2% after single resampling. There were no common fetal trisomies detected among the 86 monochorionic-monoamniotic and 25 triplet cases. Two monochorionic-diamniotic twins had an NIPT result indicative of a trisomy 21, which was confirmed in both fetuses. Among 2,716 dichorionic-diamniotic twin gestations, a sensitivity of 100% (95% CI 74.12-100%) and a specificity of 100% (95% CI 99.86-100%) was reached for trisomy 21 (n=12). For trisomy 18 (n=3), the respective values were 75% (95% CI 30.06-95.44%) sensitivity and 100% (95% CI 99.86-100%) specificity, and for trisomy 13 (n=2), 100% (95% CI 20.65-100%) sensitivity and 99.96% (95% CI 99.79-99.99%) specificity. In the vanishing gestation group, 28 NIPT results were positive for trisomy 21, 18, or 13, with only five confirmed trisomies. CONCLUSION: Genome-wide NIPT performed accurately for detection of aneuploidy in dichorionic-diamniotic twin gestations.

Outcome of publicly funded nationwide first-tier noninvasive prenatal screening.

PURPOSE: Noninvasive prenatal screening (NIPS) using cell-free DNA has transformed prenatal care. Belgium was the first country to implement and fully reimburse NIPS as a first-tier screening test offered to all pregnant women. A consortium consisting of all Belgian genetic centers report the outcome of two years genome-wide NIPS implementation. METHODS: The performance for the common trisomies and for secondary findings was evaluated based on 153,575 genome-wide NIP tests. Furthermore, the evolution of the number of invasive tests and the incidence of Down syndrome live births was registered. RESULTS: Trisomies 21, 18, and 13 were detected in respectively 0.32%, 0.07%, and 0.06% of cases, with overall positive predictive values (PPVs) of 92.4%, 84.6%, and 43.9%. Rare autosomal trisomies and fetal segmental imbalances were detected in respectively 0.23% and 0.07% of cases with PPVs of 4.1% and 47%. The number of invasive obstetric procedures decreased by 52%. The number of trisomy 21 live births dropped to 0.04%. CONCLUSION: Expanding the scope of NIPS beyond trisomy 21 fetal screening allows the implementation of personalized genomic medicine for the obstetric population. This genome-wide NIPS approach has been embedded successfully in prenatal genetic care in Belgium and might serve as a framework for other countries offering NIPS.

Inherent colistin resistance in genogroups of the Enterobacter cloacae complex: epidemiological, genetic and biochemical analysis from the BSAC Resistance Surveillance Programme.

BACKGROUND: Polymyxins have re-entered use against problem Gram-negative bacteria. Resistance rates are uncertain, with estimates confounded by selective testing. METHODS: The BSAC Resistance Surveillance Programme has routinely tested colistin since 2010; we reviewed data up to 2017 for relevant Enterobacterales (n = 10 914). Unexpectedly frequent resistance was seen among the Enterobacter cloacae complex isolates (n = 1749); for these, we investigated relationships to species, genome, carbon source utilization and LPS structure. RESULTS: Annual colistin resistance rates among E. cloacae complex isolates were 4.4%-20%, with a rising trend among bloodstream organisms; in contrast, annual rates for Escherichia coli and Klebsiella spp. (including K. aerogenes) generally remained <2%. WGS split the E. cloacae complex isolates into seven genogroup clusters, designated A-G. Among isolates assigned to genogroups A-D, 47/50 sequenced were colistin resistant, and many of those belonging to genogroups A-C identified as E. asburiae. Isolates belonging to genogroups E-G consistently identified as E. cloacae and were rarely (only 3/45 representatives sequenced) colistin resistant. Genogroups F and G, the predominant colistin-susceptible clusters, were metabolically distinct from other clusters, notably regarding utilization or not of l-fucose, formic acid, d-serine, adonitol, myo-inositol, l-lyxose and polysorbates. LPS from resistant organisms grown without colistin pressure lacked substitutions with 4-amino-arabinose or ethanolamine but was more structurally complex, with more molecular species present. CONCLUSIONS: Colistin resistance is frequent in the E. cloacae complex and increasing among bloodstream isolates. It is associated with: (i) particular genomic and metabolic clusters; (ii) identification as E. asburiae; and (iii) with more complex LPS architectures.

Prenatally detected copy number variants in a national cohort: A postnatal follow-up study.

OBJECTIVE: Belgian genetic centers established a database containing data on all chromosomal microarrays performed in a prenatal context. A study was initiated to evaluate postnatal development in children diagnosed prenatally with a non-benign copy number variant (CNV). METHODS: All children diagnosed with a prenatally detected non-benign CNV in a Belgian genetic center between May 2013 and February 2015 were included in the patient population. The control population consisted of children who had undergone an invasive procedure during pregnancy, with no or only benign CNVs. Child development was evaluated at 36 months using three (3) questionnaires: Ages and Stages Questionnaire Third edition, Ages and Stages Questionnaire Social-Emotional Second Edition and a general questionnaire. RESULTS: A significant difference in communication and personal-social development was detected between children with a reported susceptibility CNV and both children with an unreported susceptibility CNV and the control population. The outcome of children with a particular CNV is discussed in a case-by-case manner. CONCLUSION: Our postnatal follow-up project of children with a prenatally detected non-benign CNV is the first nationwide project of its kind. A higher number of cases for each CNV category is however needed to confirm our findings.

Niche specialization and spread of Staphylococcus capitis involved in neonatal sepsis.

The multidrug-resistant Staphylococcus capitis NRCS-A clone is responsible for sepsis in preterm infants in neonatal intensive care units (NICUs) worldwide. Here, to retrace the spread of this clone and to identify drivers of its specific success, we investigated a representative collection of 250 S. capitis isolates from adults and newborns. Bayesian analyses confirmed the spread of the NRCS-A clone and enabled us to date its emergence in the late 1960s and its expansion during the 1980s, coinciding with the establishment of NICUs and the increasing use of vancomycin in these units, respectively. This dynamic was accompanied by the acquisition of mutations in antimicrobial resistance- and bacteriocin-encoding genes. Furthermore, combined statistical tools and a genome-wide association study convergently point to vancomycin resistance as a major driver of NRCS-A success. We also identified another S. capitis subclade (alpha clade) that emerged independently, showing parallel evolution towards NICU specialization and non-susceptibility to vancomycin, indicating convergent evolution in NICU-associated pathogens. These findings illustrate how the broad use of antibiotics can repeatedly lead initially commensal drug-susceptible bacteria to evolve into multidrug-resistant clones that are able to successfully spread worldwide and become pathogenic for highly vulnerable patients.

Duplication 2p16 is associated with perisylvian polymicrogyria.

Polymicrogyria (PMG) is a heterogeneous brain malformation that may result from prenatal vascular disruption or infection, or from numerous genetic causes that still remain difficult to identify. We identified three unrelated patients with polymicrogyria and duplications of chromosome 2p, defined the smallest region of overlap, and performed gene pathway analysis using Cytoscape. The smallest region of overlap in all three children involved 2p16.1-p16.3. All three children have bilateral perisylvian polymicrogyria (BPP), intrauterine and postnatal growth deficiency, similar dysmorphic features, and poor feeding. Two of the three children had documented intellectual disability. Gene pathway analysis suggested a number of developmentally relevant genes and gene clusters that were over-represented in the critical region. We narrowed a rare locus for polymicrogyria to a region of 2p16.1-p16.3 that contains 33-34 genes, 23 of which are expressed in cerebral cortex during human fetal development. Using pathway analysis, we showed that several of the duplicated genes contribute to neurodevelopmental pathways including morphogen, cytokine, hormonal and growth factor signaling, regulation of cell cycle progression, cell morphogenesis, axonal guidance, and neuronal migration. These findings strengthen the evidence for a novel locus associated with polymicrogyria on 2p16.1-p16.3, and comprise the first step in defining the underlying genetic etiology.

Toxic Shock Syndrome Toxin 1 Evaluation and Antibiotic Impact in a Transgenic Model of Staphylococcal Soft Tissue Infection.

Nonmenstrual toxic shock syndrome (nmTSS), linked to TSST-1-producing CC30 Staphylococcus aureus, is the leading manifestation of toxic shock syndrome (TSS). Due to case rarity and a lack of tractable animal models, TSS pathogenesis is poorly understood. We developed an S. aureus abscess model in HLA class II transgenic mice to investigate pathogenesis and treatment. TSST-1 sensitivity was established using murine spleen cell proliferation assays and cytokine assays following TSST-1 injection in vivo HLA-DQ8 mice were infected subcutaneously with a tst-positive CC30 methicillin-sensitive S. aureus clinical TSS-associated isolate. Mice received intraperitoneal flucloxacillin, clindamycin, flucloxacillin and clindamycin, or a control reagent. Abscess size, bacterial counts, TSST-1 expression, and TSST-1 bioactivity were measured in tissues. Antibiotic effects were compared with the effects of control reagent. Purified TSST-1 expanded HLA-DQ8 T-cell Vß subsets 3 and 13 in vitro and instigated cytokine release in vivo, confirming TSST-1 sensitivity. TSST-1 was detected in abscesses (0 to 8.0 µg/ml) and draining lymph nodes (0 to 0.2 µg/ml) of infected mice. Interleukin 6 (IL-6), gamma interferon (IFN-γ), KC (CXCL1), and MCP-1 were consistent markers of inflammation during infection. Clindamycin-containing antibiotic regimens reduced abscess size and TSST-1 production. Infection led to detectable TSST-1 in soft tissues, and TSST-1 was detected in draining lymph nodes, events which may be pivotal to TSS pathogenesis. The reduction in TSST-1 production and lesion size after a single dose of clindamycin underscores a potential role for adjunctive clindamycin at the start of treatment of patients suspected of having TSS to alter disease progression.IMPORTANCE Staphylococcal toxic shock syndrome (TSS) is a life-threatening illness causing fever, rash, and shock, attributed to toxins produced by the bacterium Staphylococcus aureus, mainly toxic shock syndrome toxin 1 (TSST-1). TSS was in the past commonly linked with menstruation and high-absorbency tampons; now, TSS is more frequently triggered by other staphylococcal infections, particularly of skin and soft tissue. Investigating the progress and treatment of TSS in patients is challenging, as TSS is rare; animal models do not mimic TSS adequately, as toxins interact best with human immune cells. We developed a new model of staphylococcal soft tissue infection in mice producing human immune cell proteins, rendering them TSST-1 sensitive, to investigate TSS. The significance of our research was that TSST-1 was found in soft tissues and immune organs of mice and that early treatment of mice with the antibiotic clindamycin altered TSST-1 production. Therefore, the early treatment of patients suspected of having TSS with clindamycin may influence their response to treatment.

Genomic and Epidemiological Evidence of a Dominant Panton-Valentine Leucocidin-Positive Methicillin Resistant Staphylococcus aureus Lineage in Sri Lanka and Presence Among Isolates From the United Kingdom and Australia.

Objective: To undertake the first detailed genomic analysis of methicillin-resistant Staphylococcus aureus (MRSA) isolated in Sri Lanka. Methods: A prospective observational study was performed on 94 MRSA isolates collected over a 4 months period from the Anuradhapura Teaching Hospital, Sri Lanka. Screening for mecA, mecC, and the Panton-Valentine leucocidin (PVL)-associated lukS-PV/lukF-PV genes and molecular characterization by spa typing was undertaken. Whole genome sequencing (WGS) and phylogenetic analysis was performed on selected multilocus sequence type (MLST) clonal complex 5 (CC5) isolates from Sri Lanka, England, Australia, and Argentina. Results: All 94 MRSA harbored the mecA gene. Nineteen spa types belonging to nine MLST clonal complexes were identified. Where origin of the sample was recorded, most isolates were from skin and soft tissue infections (70/91; 76.9%), with fewer causing bacteremia (16/91; 17.6%), empyema (3/91; 3.3%) and osteomyelitis (2/91; 2.2%). Sixty two (65.9%) isolates were PVL positive with the majority (56 isolates; 90.3%) belonging to a dominant CC5 lineage. This lineage, PVL-positive ST5-MRSA-IVc, was associated with both community and hospital-onset infections. Based on WGS, representative PVL-positive ST5-MRSA-IVc isolates from Sri Lanka, England and Australia formed a single phylogenetic clade, suggesting wide geographical circulation. Conclusions: We present the most detailed genomic analysis of MRSA isolated in Sri Lanka to date. The analysis identified a PVL-positive ST5-MRSA-IVc that is prevalent among MRSA causing clinical infections in Sri Lanka. Furthermore, this clone was also found among isolates from the United Kingdom and Australia.