USA300 Clone Research Articles

Characterization of methicillin resistant Staphylococcus Aureus in municipal wastewater in Finland

Wastewater-based surveillance (WBS) of multidrug-resistant bacteria could complement clinical data, serving as a population-level early warning tool. This study evaluated WBS as a pandemic preparedness tool, by selectively isolating and culturing methicillin-resistant Staphylococcus aureus (MRSA) with CHROMagar MRSA. Some 24-h composite wastewater samples (n = 80) were collected from ten treatment plants across Finland between February 2021 and January 2022. MRSA prevalence in wastewater samples was 27.5% (n = 22/80), showing seasonal and temporal variations. Phenotypic antimicrobial susceptibility testing (AST) with microdilution showed that over 80% of isolates were drug-resistant to clindamycin, sulfamethoxazole/trimethoprim, tetracycline, fusidic acid, and erythromycin. Four isolates (18.2%) were vancomycin-resistant. WGS revealed that 31.8% (n = 7) of the isolates belonged to the ST8-t008 and ST6-t304 spa types, respectively. In addition, two spa types (t011 and t034) belong to the CC398 complex. The mecA gene was found in all isolates (n = 22) and three tetracycline resistance determinants (tet38, tetK, and tetM) were detected with tet38 being the most abundant (81.8%, n = 18/22). Three isolates harboured the plasmid-mediated sat4 gene that confers resistance to Streptothricin. In addition, resistance determinants to macrolide antibiotics (mph (C)/msr (A) and fosfomycin (fosB) were detected in the seven isolates that belonged to spa type t008. All isolates except one harboured the SCCmec_type_IVa(2B). Six ST8 isolates harboured the LukS/F-PV genes encoding the Panton–Valentine leukocidin (PVL) and were also positive for the Arginine Catabolic Mobile Element (ACME), suggesting they belong to the USA300 clone. The Inc18 plasmid was the most abundant as it was detected in 72.7% (n = 16/22) of the isolates. Other plasmid replicons detected were the rep_trans and repA_N which were detected in 45.4% (n = 10/22) and 40.9% (n = 9/22) of the isolates respectively. Ten isolates harboured at least three plasmid replicons and no plasmid replicons were detected in four isolates (ST6/t304). The cgMLST revealed that some isolates aggregated into two genomically indistinguishable clusters: ST6/t304 belonging to cluster type CT12405 (≤20 allelic differences) and ST8/t008 belonging to cluster type CT1925 (<8 allelic differences). Overall, we found a high genotypic concordance with the national clinical bacterial resistance data. Our study demonstrates the sensitivity of culture-based wastewater surveillance for MRSA using clinical media following pre-enrichment, reliably predicting pathogen occurrence at the population level.

Genetic characterization of methicillin-resistant / susceptible Staphylococcus aureus (MRSA/MSSA) and Staphylococcus argenteus clinical isolates in Bangladesh: Dominance of ST6-MRSA-IV/t304 and detection of cfr/fexA in ST8-MSSA/t008

ObjectivesCoagulase-positive staphylococcus (CoPS), represented by Staphylococcus aureus, is a major cause of infections in humans. This study aimed to investigate molecular epidemiological characteristics, antimicrobial resistance, and their trends of CoPS in Bangladesh. MethodsClinical isolates of CoPS were collected from two medical institutions in Bangladesh for a 2-year period and analyzed for their species, genotypes, virulence factors, antimicrobial susceptibility, and resistance determinants. Results172 CoPS isolates collected were identified as S. aureus or S. argenteus (170 and two, respectively). Methicillin-resistant S. aureus (MRSA) accounted for 36% (n = 61), having Staphylococcal cassette chromosome mec (SCCmec)-IV (82%) or V (18%). Panton-Valentine leukocidin (PVL) genes were detected at higher rate in methicillin-susceptible S. aureus (MSSA) (62%) than MRSA (26%). MRSA comprised 11 STs, including a dominant type ST6 (46%) associated with mostly SCCmec-IVa/spa-t304, and one isolate had genetic features of the USA300 clone (ST8/SCCmec-IVa/coa-IIIa/spa-t008/ACME-I/ΦSa2USA). STs of CC1, CC88, and CC398 were common in MSSA, with CC88 showing the highest PVL-positive rate. One MSSA isolate (ST8/spa-t008) harbored fexA and cfr showing susceptibility to linezolid. S. argenteus was methicillin-susceptible and belonged to ST2250/coa-XId. ConclusionsGenetic characteristics of current MRSA/MSSA in Bangladesh were revealed, with first identification of S. argenteus at low prevalence.

Evolution of Invasive and Colonizing Pediatric Staphylococcus aureus Isolates

Abstract Background Staphylococcus aureus is the most common bacterial pathogen isolated in children with invasive bloodstream and musculoskeletal infections. A major barrier to vaccine and novel therapeutic development against S. aureus is its continuous evolution. Many recent and current vaccine candidates target antigens that may have appeared crucial to its pathogenesis, only to see these factors recede from circulating invasive strains. Characterization of clinically relevant S. aureus isolates over time is necessary to find targets for novel therapeutics. Efforts toward new interventions should target factors that remain prevalent in invasive isolates over time. Methods We obtained 131 invasive S. aureus isolates from children admitted to the Monroe Carell Jr. Children's Hospital at Vanderbilt from 2010 to 2022, and 246 colonizing isolates. Whole genome sequences were obtained using the Illumina sequencing platform. Virulence factors and multi-locus sequence type (MLST) were determined using Geneious software and through PubMLST, an open-access curated database. Results There were 10 unique clonal complexes (CC) identified among 320 isolates. CC8 remains the most common invasive clonal complex, though CC8 is significantly less frequent now than in 2010-2012. Diversity of invasive strains has increased substantially, with the emergence of CC5 and CC121 lineages causing invasive disease in children. CC1 was found exclusively in colonization isolates. Virulence factor prevalence has changed significantly over time. Panton-Valentine leucocidin (PVL), enterotoxins K and Q (SEK/SEQ), and the pathogenic arginine catabolic mobile element (ACME), have decreased significantly in invasive S. aureus isolates since 2010, from 53-70% to 21-35% of current isolates. Leukocidin ED (LukED) and surface protein staphylokinase (Sak) are strongly associated with invasive isolates compared to colonization isolates. Conversely, toxic shock toxin (TST) and Staphylococcal enterotoxin B (SEB) were seen more commonly in colonization isolates (12-15%) compared to invasive isolates (5-7%). The genes encoding for gamma hemolysins (HlgA-C), leukocidin AB (LukAB), iron-regulated surface proteins (IsdA, IsdB), staphylococcal binding immunoglobulin protein (sbi), extracellular fibrinogen-binding protein (Efb), and clumping factors A and B (ClfA, ClfB) were identified in all isolates tested. Figure 1 Figure 2 Conclusions Significant shifts have occurred over the past decade in the predominant circulating S. aureus strains and their virulence factor repertoires. The once-dominant CC8 (USA300 clone) has receded, and with that has come a significant reduction in some factors once thought crucial for pathogenesis. These findings also have implications for infection prevention and control practices, as eradication of colonizing strains with low potential for pathogenicity may inadvertently allow for replacement by more virulent strains.

Comparative analysis of genomic characteristics, virulence and fitness of community-associated Staphylococcus aureus ST121 clone causing fatal diseases in China and other CA-MRSA clones

ABSTRACT The increasing rate of community-associated Staphylococcus aureus (CA-SA) worldwide has aroused global public concern for decades. Although ST121 clone is one of the prevalent CA-SA in China, there is still limited knowledge about it. In this study, we conducted a genomic analysis of 28 CA-SA ST121 isolates from severe bloodstream infection cases and 175 ST121 isolates from the public database. Phylogenetic analysis revealed the consistency and the complexity of global ST121 lineages, and suggested potential cross-country even cross-continental transmission of ST121 isolates. By investigating the virulence and fitness between ST121-CA-methicillin-resistant SA (CA-MRSA) and other CA-MRSA clones, we found that ST121-MRSA exhibits virulence comparable to the highly virulent USA300 clone, exceeding that of the predominant CA-MRSA lineage ST59 in China and the other American CA-MRSA clone MW2. Notably, based on analyses of virulence genes, eta, etb, edin-C and egc were only found in ST121, suggesting that the high virulence of ST121 may be attributed to the combination of these virulence factors encoded by mobile genetic elements. However, results of experiments in mice nasal and human alveolar epithelial cells showed that the colonization capacity of ST121 is much lower than that of other clones. Moreover, ST121-MRSA displayed much lower acid tolerance, suggesting that ST121-MRSA may not have such capacity to achieve the epidemiological success of other CA-MRSA clones and become the dominant lineage. Our findings expand current understanding of the epidemiology and pathogenicity of the hypervirulent ST121 clone, and highlight the importance of colonization capacity and environmental adaption in MRSA epidemiological success.

Clonal Complexes Distribution of Staphylococcus aureus Isolates from Clinical Samples from the Caribbean Islands.

The aim of this study was to comprehensively characterise S. aureus from the Caribbean Islands of Trinidad and Tobago, and Jamaica. A total of 101 S. aureus/argenteus isolates were collected in 2020, mainly from patients with skin and soft tissue infections. They were characterised by DNA microarray allowing the detection of ca. 170 target genes and assignment to clonal complexes (CC)s and strains. In addition, the in vitro production of Panton-Valentine leukocidin (PVL) was examined by an experimental lateral flow assay. Two isolates were identified as S. argenteus, CC2596. The remaining S. aureus isolates were assigned to 21 CCs. The PVL rate among methicillin-susceptible S. aureus (MSSA) isolates was high (38/101), and 37 of the 38 genotypically positive isolates also yielded positive lateral flow results. The isolate that did not produce PVL was genome-sequenced, and it was shown to have a frameshift mutation in agrC. The high rate of PVL genes can be attributed to the presence of a known local CC8-MSSA clone in Trinidad and Tobago (n = 12) and to CC152-MSSA (n = 15). In contrast to earlier surveys, the USA300 clone was not found, although one MSSA isolate carried the ACME element, probably being a mecA-deficient derivative of this strain. Ten isolates, all from Trinidad and Tobago, were identified as MRSA. The pandemic ST239-MRSA-III strain was still common (n = 7), but five isolates showed a composite SCCmec element not observed elsewhere. Three isolates were sequenced. That showed a group of genes (among others, speG, crzC, and ccrA/B-4) to be linked to its SCC element, as previously found in some CC5- and CC8-MRSA, as well as in S. epidermidis. The other three MRSA belonged to CC22, CC72, and CC88, indicating epidemiological connections to Africa and the Middle East.

Emergence of community-associated methicillin-resistant Staphylococcus aureus ΨUSA300 among Japanese people with HIV, resulted from stepwise mutations in 2010s

Although infection with the methicillin-resistant Staphylococcus aureus (MRSA) clone USA300 is extremely rare in Japan, the uniquely evolved clone ΨUSA300 has been reported in Japan. An outbreak of a distinct USA300 clone was recently reported in an HIV/AIDS referral hospital in Tokyo. The present study investigated the evolutionary origin and genetic diversity of USA300-related clones causing regional outbreaks among people living with HIV (PLWHIV) in Tokyo. MRSA isolates collected from PLWHIV in an HIV/AIDS referral center in Tokyo were subjected to whole-genome sequencing and their genetic features were compared with those of previously described USA300 MRSA genomes. Of the 28 MRSAs isolated in 2016–2019, 23 (82.1%) were identified as USA300, with 22 (95.6%) of the latter identified as ΨUSA300. Although the genomic structure of ΨUSA300 was identical to the structures of reference USA300 strains, one clade (cluster A) was found to have acquired 29 previously identified lineage-specific mutations in a stepwise manner. The estimated divergence dates of ΨUSA300 and Cluster A were 2009 and 2012, respectively. These findings suggested that the ΨUSA300 clone had spread among PLWHIVs in Tokyo in the early 2010s, with stepwise acquisition of lineage-specific nonsynonymous mutations.

Clinical Impact of Staphylococcus aureus Skin and Soft Tissue Infections.

The pathogenic bacterium Staphylococcus aureus is the most common pathogen isolated in skin-and-soft-tissue infections (SSTIs) in the United States. Most S. aureus SSTIs are caused by the epidemic clone USA300 in the USA. These infections can be serious; in 2019, SSTIs with S. aureus were associated with an all-cause, age-standardized mortality rate of 0.5 globally. Clinical presentations of S. aureus SSTIs vary from superficial infections with local symptoms to monomicrobial necrotizing fasciitis, which can cause systemic manifestations and may lead to serious complications or death. In order to cause skin infections, S. aureus employs a host of virulence factors including cytolytic proteins, superantigenic factors, cell wall-anchored proteins, and molecules used for immune evasion. The immune response to S. aureus SSTIs involves initial responders such as keratinocytes and neutrophils, which are supported by dendritic cells and T-lymphocytes later during infection. Treatment for S. aureus SSTIs is usually oral therapy, with parenteral therapy reserved for severe presentations; it ranges from cephalosporins and penicillin agents such as oxacillin, which is generally used for methicillin-sensitive S. aureus (MSSA), to vancomycin for methicillin-resistant S. aureus (MRSA). Treatment challenges include adverse effects, risk for Clostridioides difficile infection, and potential for antibiotic resistance.

Time-Course Transcriptome Analysis of the Lungs of Mice Challenged with Aerosols of Methicillin-Resistant Staphylococcus aureus USA300 Clone Reveals Inflammatory Balance.

USA300, a dominant clone of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA), is circulating globally and can cause necrotizing pneumonia with high morbidity and mortality. To further reveal the host anti-MRSA infection immune response, we established a mouse model of acute primary MRSA pneumonia challenged with aerosols of the USA300 clone. A time-course transcriptome analysis of the lungs collected at 0, 12, 24, 48 and 96 h post-infection (hpi) was conducted using RNA sequencing (RNA-seq) and multiple bioinformatic analysis methods. The change trend of histopathology and five innate immune cell (neutrophils, mononuclear cells, eosinophils, macrophages, DC cells) proportions in the lungs after infection was also examined. We observed a distinct acute pulmonary recovery process. A rapid initiation period of inflammation was present at 12 hpi, during which the IL-17 pathway dominantly mediated inflammation and immune defense. The main stages of host inflammatory response occurred at 24 and 48 hpi, and the regulation of interferon activation and macrophage polarization played an important role in the control of inflammatory balance at this stage. At 96 hpi, cellular proliferation processes associated with host repair were observed, as well as adaptive immunity and complement system responses involving C1q molecules. More importantly, the data provide new insight into and identify potential functional genes involved in the checks and balances occurring between host anti-inflammatory and proinflammatory responses. To the best of our knowledge, this is the first study to investigate transcriptional responses throughout the inflammatory recovery process in the lungs after MRSA infection. Our study uncovers valuable research targets for key regulatory mechanisms underlying the pathogenesis of MRSA lung infections, which may help to develop novel treatment strategies for MRSA pneumonia.

Severity and intractableness of skin infections caused by Panton–Valentine leukocidin‐positive methicillin‐resistant Staphylococcus aureus

AbstractCases of skin infections caused by Panton–Valentine leukocidin (PVL)‐positive methicillin‐resistant Staphylococcus aureus (MRSA), particularly USA300 clone, have been increasing in Japan. We report that clinical findings of 5 patients with PVL‐positive MRSA and compared to those of four patients with PVL‐negative MRSA. Severities of patients with PVL‐positive MRSA were significantly higher than those of patients with PVL‐negative MRSA. Average durations of antimicrobial therapy for patients with PVL‐positive MRSA were 3.4‐fold longer than those for patients with PVL‐negative MRSA. Our data suggest that PVL‐positive MRSA should be deal with a causative agent for intractable skin infections in Japan likewise other countries.

Pandemic clone USA300 in a Brazilian hospital: detection of an emergent lineage among methicillin-resistant Staphylococcus aureus isolates from bloodstream infections

BackgroundStaphylococcus aureus is one of the leading causes of bloodstream infections (BSI) worldwide. In Brazil, the hospital-acquired methicillin-resistant S. aureus USA100/SCCmecII lineage replaced the previously well-established clones. However, the emergence of community-associated (CA) MRSA lineages among hospitalized patients is an increasing issue.MethodsConsecutive S. aureus isolates recovered from BSI episodes of patients admitted between January 2016 and December 2018 in a Brazilian teaching hospital were tested for antimicrobial resistance, their genotypic features were characterized, and the clinical characteristics of the patients were evaluated.ResultsA total of 123 S. aureus isolates were recovered from 113 patients. All isolates were susceptible to linezolid, teicoplanin and vancomycin and 13.8% were not susceptible to daptomycin. Vancomycin MIC50 and MIC90 of 2 mg/L were found for both MRSA and MSSA isolates. The MRSA isolation rate was 30.1% (37/123), and 51.4% of them carried the SCCmec type II, followed by SCCmecIV (40.5%). Among the 37 MRSA isolates, the main lineages found were USA100/SCCmecII/ST5 and ST105 (53.7%) and USA800/ST5/SCCmecIV (18.9%). Surprisingly, six (16%) CA-MRSA isolates, belonging to USA300/ST8/SCCmecIVa that carried PVL genes and the ACME cassette type I, were detected. These six patients with USA300 BSI had severe comorbidities, including cancer, and most had a Charlson score ≥ 5; furthermore, they were in wards attended by the same health professionals. MRSA isolates were associated with hospital acquired infections (p = 0.02) in more elderly patients (p = 0.03) and those diagnosed with hematologic cancer (p = 0.04). Among patients diagnosed with MRSA BSI, 19 (54.3%) died.ConclusionsThe pandemic MRSA USA300 was detected for the first time in the Brazilian teaching hospital under study, and its cross-transmission most probably occurred between patients with BSI. This lineage may already be circulating among other Brazilian hospitals, which highlights the importance of carrying out surveillance programs to fight multidrug resistant and hypervirulent isolates.

Clinical features and antimicrobial treatment of skin infections caused by Panton‐Valentine leukocidin‐positive methicillin‐resistant Staphylococcus aureus

Skin infections caused by Panton-Valentine leukocidin (PVL)-positive methicillin-resistant Staphylococcus aureus (MRSA), especially the USA300 clone, have been increasing in Japan. To prevent an epidemic of PVL-positive MRSA, rapid diagnosis and effective antimicrobial therapy are essential. However, the clinical features of, and antimicrobial efficacy against, these skin infections are not well understood in Japan. Here, we report 10 cases of skin infections caused by PVL-positive MRSA that presented over a two-year period in our clinic. Genetic analyses revealed that 90% of the PVL-positive MRSA strains were identified as USA300 and its related clones. Notably, 70% of the patients had atopic dermatitis (AD) as an underlying disease. Average durations of antimicrobial therapy for AD patients (10.6 weeks) were 2.9-fold longer than those for non-AD patients (3.7 weeks). However, all cases were improved by a long-term course of fosfomycin, minocycline, doxycycline, and/or rifampicin. Our data suggest that AD may be an important risk factor for intractable skin infections caused by PVL-positive MRSA.

Genetic Characterization of Staphylococcus aureus, Staphylococcus argenteus, and Coagulase-Negative Staphylococci Colonizing Oral Cavity and Hand of Healthy Adults in Northern Japan.

The spread of methicillin resistance and virulence among staphylococci in the community poses a public health concern. In this study, we investigated the prevalence of Staphylococcus species colonizing the oral cavity and hand (skin) of healthy university students and their phenotypic and genetic characteristics in northern Japan. Among a total of 332 subjects, 6 and 110 methicillin-resistant and susceptible Staphylococcus aureus (MRSA and MSSA, respectively) isolates were recovered from 105 subjects. MRSA isolates were genotyped as CC5, CC8, CC45, and CC59 with SCCmec-IIa or IV, among which an isolate of ST6562 (single-locus variant of ST8) harbored SCCmec-IVa, PVL genes and ACME-I, which are the same traits as the USA300 clone. ST1223 S. argenteus was isolated from the oral cavity and hand of a single student. Coagulase-negative Staphylococcus (CoNS) was recovered from 154 subjects (172 isolates), and classified into 17 species, with S. capitis being the most common (38%), followed by S. warneri (24%) and S. epidermidis (15%), including nine mecA-positive isolates. S. capitis was differentiated into seven clusters/subclusters, and genetic factors associated with the NRCS-A clone (nsr, tarJ, ebh) were detected in 10–21% of isolates. The colonization of the USA300-like MRSA variant and S. capitis with the traits of the NRCS-A clone in healthy individuals was noteworthy.

The trend of environmental and clinical methicillin-resistant Staphylococcus aureus in a hospital in Taiwan: Impact of USA300.

The environment may facilitate transmission of health care-associated methicillin-resistant Staphylococcus aureus (MRSA) and the pathogen is frequently shed by patients. However, the molecular characteristics and genetic relatedness between clinical and environmental MRSA isolates remain largely unclear in the clinical setting. A total of 100 hospitalized patients with MRSA infection and 25 hospitalized patients without MRSA infection were enrolled in a medical center, Taiwan in 2019. Environmental and clinical MRSA isolates were characterized by antibiotic susceptibility testing and molecular methods. In the study, we detected 17 MRSA isolates in the environment that surrounded 15 MRSA-infected patients and one environmental MRSA isolate from one patient without MRSA infection. The molecular analysis revealed a high genetic diversity within either environmental or clinical MRSA isolates, while the USA300 clone (pulsotype AI, SCCmec IV, ST8, PVL-positive) accounts for 39% (7/18) of environmental and 33% (7/21) of clinical MRSA isolates. Moreover, 13 of the 15 MRSA-infected patients had identical paired clinical-environmental MRSA isolates, which exhibited indistinguishable genetic relatedness and highly similar antibiotic susceptibility phenotype, suggesting a possible transmission cycle of MRSA in the hospital. The environmental MRSA was closely linked to MRSA isolated from patients, suggesting that the environment may act as a reservoir of MRSA. Besides, the USA300 MRSA has become a major clone in the hospital setting. An effective and rigorous approach to environmental cleaning and decontamination is suggested to eradicate MRSA in the hospital.

Environmental Surveillance and Characterization of Antibiotic Resistant Staphylococcus aureus at Coastal Beaches and Rivers on the Island of Hawai'i.

Staphylococcus aureus are human facultative pathogenic bacteria and can be found as contaminants in the environment. The aim of our study was to determine whether methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) isolated from coastal beach and river waters, anchialine pools, sand, and wastewater on the island of Hawaiʻi, Hawaiʻi, are a potential health risk. Samples were collected from three regions on Hawaiʻi Island from July to December 2020 during the COVID-19 pandemic and were characterized using whole-genome sequencing (WGS). From WGS data, multilocus sequence typing (MLST), SCCmec type, antimicrobial resistance genes, virulence factors, and plasmids were identified. Of the 361 samples, 98.1% were positive for Staphylococcus spp. and 7.2% were S. aureus positive (n = 26); nine MRSA and 27 MSSA strains were characterized; multiple isolates were chosen from the same sample in two sand and seven coastal beach water samples. The nine MRSA isolates were multi-drug resistant (6–9 genes) sequence type (ST) 8, clonal complex (CC) 8, SCCmec type IVa (USA300 clone), and were clonally related (0–16 SNP differences), and carried 16–19 virulence factors. The 27 MSSA isolates were grouped into eight CCs and 12 STs. Seventy-eight percent of the MSSA isolates carried 1–5 different antibiotic resistance genes and carried 5–19 virulence factors. We found S. aureus in coastal beach and river waters, anchialine pools, and sand at locations with limited human activity on the island of Hawaiʻi. This may be a public health hazard.

Staphylococcus aureus Bacteremia Among Patients Receiving Maintenance Hemodialysis: Trends in Clinical Characteristics and Outcomes

Staphylococcus aureus (Saureus) bacteremia (SAB) is associated with morbidity and mortality in patients receiving maintenance hemodialysis (HD). We evaluated changes in clinical and bacterial characteristics, and their associations with clinical outcomes with SAB in this population over a 21-year period. Prospective cohort study. 453 hospitalized, non-neutropenic adults receiving maintenance HD who developed monomicrobial SAB between 1995 and2015. Clinical characteristics and bacterial genotype. All-cause and SAB-attributable mortality, persistent bacteremia, and metastatic complications. Proportions of participants experiencing each outcome were calculated overall and by calendar year. Secular trends were estimated using binomial risk regression, a generalized linear model with the log link function for a binomial outcome. Associations with outcomes were estimated using logistic regression. Over the 21-year study period, patients receiving maintenance HD experienced significant increases in age- and diabetes-adjusted SAB-attributable mortality (0.45% [95% CI, 0.36%-0.46%] per year), persistent bacteremia (0.86% [95% CI, 0.14%-1.55%] per year), metastatic complications (0.84% [95% CI, 0.11%-1.56%] per year), and infection with the virulent Saureus clone USA300 (1.47% [95% CI, 0.33%-2.52%] per year). Over time, the suspected source of SAB was less likely to be a central venous catheter (-1.32% [95% CI,-2.05 to-0.56%] per year) or arteriovenous graft (-1.08% [95% CI,-1.54 to-0.56] per year), and more likely to be a nonvascular access source (1.89% [95% CI, 1.29%-2.43%] per year). Patients with a nonvascular access suspected source of infection were more likely to die as a result of their S aureus infection (OR, 3.20 [95% CI, 1.36-7.55]). The increase in USA300 infections may have contributed to the observed increase in persistent bacteremia (OR, 2.96 [95% CI, 1.12-7.83]) but did not explain the observed increases in SAB-attributable mortality (OR, 0.83 [95% CI, 0.19-3.61]) or metastatic complications (OR, 1.34 [95% CI, 0.53-3.41]). Single-center, inpatient cohort. The clinical and molecular epidemiology of SAB in patients receiving maintenance HD has changed over time, with an increase in SAB-attributable mortality and morbidity despite a decline in catheter-related infections.

#31: Children with Invasive S. aureus Infection Produce Broadly Neutralizing Antibodies Against Distantly Related Variants of the Cytotoxin LukAB

Abstract Background Staphylococcus aureus is the most common invasive bacterial pathogen in children, and novel targets of intervention are urgently needed. The two-component leukotoxin, LukAB, is critical for S. aureus targeting and killing of human neutrophils and is abundantly produced in the setting of invasive human infection. LukAB is unique among S. aureus cytotoxins in that it exists in variant form across distantly related strains of clinically relevant S. aureus. The broad diversity of circulating clinical S. aureus strains must be taken into account to successfully develop new anti-staphylococcal preventives or therapeutics. This diversity and ongoing S. aureus evolution may explain previous unsuccessful attempts to intervene against S. aureus at the virulence factor level. We have previously shown that LukAB is ubiquitous among clinical isolates and that children with invasive disease mount a high-titer neutralizing response, but the breadth of function of this response has not previously been explored. Methods B-cells were isolated from children with invasive S. aureus infections (e.g. bacteremia or acute hematogenous osteomyelitis). Following EBV-transformation, cell supernatants were screened for LukAB binding and selected for generation of monoclonal hybridomas. MAbs were assessed for binding by ELISA and neutralizing function by in vitro cytotoxicity assays, where neutrophil-like HL-60 cells were cultured in the presence of human mAbs and diverse allelic variants of LukAB. The infecting S. aureus isolates were typed using multilocus sequence typing (MLST) to determine clonal complexes. Results 34 distinct human anti-LukAB mAbs were generated from 3 children with invasive S. aureus disease. Of these, 22% were isolated following infection with a strain belonging to clonal complex 8 (CC8), consistent with the epidemic USA300 clone, and 78% were generated against CC5 strains. Within this panel, all mAbs potently neutralized LukAB from the same clonal complex as the corresponding infecting isolate, but 23 also demonstrated neutralization against other allelic variants of LukAB. 7 mAbs were capable of broad, potent neutralization against LukAB variants from all clinically relevant clonal complexes tested (CC8, CC30, CC45, CC75, CC1, CC5, and CC398). Conclusions A subset of human mAbs isolated from children with invasive S. aureus disease were capable of broad neutralization against distantly related variants of the important toxin LukAB. This has two important implications: First, we found strong evidence of a conserved target (or targets) for antibody-mediated toxin neutralization across diverse strains of S. aureus. Second, this provides additional support for this toxin as a target of intervention, as some previous vaccine attempts were likely unsuccessful due to activity against a narrow subset of circulating S. aureus strains.

Staphylococcus aureus secreted lipases do not inhibit innate immune killing mechanisms.

Background: Staphylococcus aureus causes an array of diseases in both humans and livestock. Pathogenesis is mediated by a plethora of proteins secreted by S. aureus, many of which remain incompletely characterised. For example, S. aureus abundantly secretes two isoforms of the enzyme lipase into the extracellular milieu, where they scavenge upon polymeric triglycerides. It has previously been suggested that lipases may interfere with the function of innate immune cells, such as macrophages and neutrophils, but the impact of lipases on phagocytic killing mechanisms remains unknown. Methods: We employed the epidemic S. aureus clone USA300 strain LAC and its lipase deficient isogenic mutant, along with recombinant lipase proteins, in in vitro experimental infection assays. To determine if lipases can inhibit innate immune killing mechanisms, the bactericidal activity of whole blood, human neutrophils, and macrophages was analysed. In addition, gentamycin protection assays were carried out to examine the influence of lipases on S. aureus innate immune cell escape. Results: There were no differences in the survival of S. aureus USA300 LAC wild type and its lipase-deficient isogenic mutant after incubation with human whole blood or neutrophils. Furthermore, there was no detectable lipase-dependent effect on phagocytosis, intracellular survival, or escape from both human primary and immortalised cell line macrophages, even upon supplementation with exogenous recombinant lipases. Conclusions: S. aureus lipases do not inhibit bacterial killing mechanisms of human macrophages, neutrophils, or whole blood. These findings broaden our understanding of the interaction of S. aureus with the innate immune system.

Epidemiology of the Staphylococcus aureus CA-MRSA USA300 in Belgium.

The methicillin-resistant Staphylococcus aureus (MRSA) sequence type (ST) 8 Panton-Valentine toxin (PVL)-positive USA300 clone has a worldwide distribution. The USA300 North American (NA) variant, harbouring the arginine catabolic mobile element (ACME), is predominant in the USA while the Latin American (LV) variant is predominant in Northern South America. Both variants have failed to become endemic in Europe. We examined here the epidemiology of the USA300 clone in Belgium from 2006 to 2019. A total of 399 clonal complex 8 PVL-positive MRSA isolates received between 2006 and 2019 by the Belgian National Reference Laboratory for S. aureus were investigated for the presence of ACME. Selected ACME-positive (n=102) and ACME-negative (n=16) isolates were sequenced, characterized for the presence of several resistance and virulence molecular markers and subjected to phylogenetic analysis. A total of 300 isolates were USA300-NA (ACME-positive), while only 99 were ACME-negative. Most USA300-NA interspersed in the phylogeny analysis with isolates from other countries, suggesting multiple introductions. However, two big clades were maintained and spread over a decade, peaking between 2010 and 2017 to finally decrease. Few ACME-negative isolates, mainly related to trips to South America, were identified as USA300-LV. The remaining ACME-negative isolates were ST8 SCCmec IVb or ST923 SCCmec IVa (COL923). Two clades of the USA300-NA clone have successfully spread in Belgium, but seem to currently decrease. Related South American variants have been detected for the first time in Belgium, including the emerging COL923 clone.

IBT-V02: A Multicomponent Toxoid Vaccine Protects Against Primary and Secondary Skin Infections Caused by Staphylococcus aureus.

Staphylococcus aureus causes a wide range of diseases from skin infections to life threatening invasive diseases such as bacteremia, endocarditis, pneumonia, surgical site infections, and osteomyelitis. Skin infections such as furuncles, carbuncles, folliculitis, erysipelas, and cellulitis constitute a large majority of infections caused by S. aureus (SA). These infections cause significant morbidity, healthcare costs, and represent a breeding ground for antimicrobial resistance. Furthermore, skin infection with SA is a major risk factor for invasive disease. Here we describe the pre-clinical efficacy of a multicomponent toxoid vaccine (IBT-V02) for prevention of S. aureus acute skin infections and recurrence. IBT-V02 targets six SA toxins including the pore-forming toxins alpha hemolysin (Hla), Panton-Valentine leukocidin (PVL), leukocidin AB (LukAB), and the superantigens toxic shock syndrome toxin-1 and staphylococcal enterotoxins A and B. Immunization of mice and rabbits with IBT-V02 generated antibodies with strong neutralizing activity against toxins included in the vaccine, as well as cross-neutralizing activity against multiple related toxins, and protected against skin infections by several clinically relevant SA strains of USA100, USA300, and USA1000 clones. Efficacy of the vaccine was also shown in non-naïve mice pre-exposed to S. aureus. Furthermore, vaccination with IBT-V02 not only protected mice from a primary infection but also demonstrated lasting efficacy against a secondary infection, while prior challenge with the bacteria alone was unable to protect against recurrence. Serum transfer studies in a primary infection model showed that antibodies are primarily responsible for the protective response.

Predominance of PVL-negative community-associated methicillin-resistant Staphylococcus aureus sequence type 8 in newly diagnosed HIV-infected adults, Tanzania

Difficult-to-treat infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are of concern in people living with HIV infection as they are more vulnerable to infection. We aimed to identify molecular characteristics of MRSA colonizing newly diagnosed HIV-infected adults in Tanzania. Individuals newly diagnosed with HIV infection were recruited in Dar es Salaam, Tanzania, from April 2017 to May 2018, as part of the randomized clinical trial CoTrimResist (ClinicalTrials.gov identifier: NCT03087890). Nasal/nasopharyngeal isolates of Staphylococcus aureus were susceptibility tested by disk diffusion method, and cefoxitin-resistant isolates were characterized by short-reads whole genome sequencing. Four percent (22/537) of patients carried MRSA in the nose/nasopharynx. MRSA isolates were frequently resistant towards gentamicin (95%), ciprofloxacin (91%), and erythromycin (82%) but less often towards trimethoprim-sulfamethoxazole (9%). Seventy-three percent had inducible clindamycin resistance. Erythromycin-resistant isolates harbored ermC (15/18) and LmrS (3/18) resistance genes. Ciprofloxacin resistance was mediated by mutations of the quinolone resistance-determining region (QRDR) sequence in the gyrA (S84L) and parC (S80Y) genes. All isolates belonged to the CC8 and ST8-SCCmecIV MRSA clone. Ninety-five percent of the MRSA isolates were spa-type t1476, and one exhibited spa-type t064. All isolates were negative for Panton-Valentine leucocidin (PVL) and arginine catabolic mobile element (ACME) type 1. All ST8-SCCmecIV-spa-t1476 MRSA clones from Tanzania were unrelated to the globally successful USA300 clone. Carriage of ST8 MRSA (non-USA300) was common among newly diagnosed HIV-infected adults in Tanzania. Frequent co-resistance to non-beta lactam antibiotics limits therapeutic options when infection occurs.