Streptococcal pyrogenic exotoxin B cleaves GSDMA and triggers pyroptosis.

Gasdermins, a family of five pore-forming proteins (GSDMA-GSDME) in humans expressed predominantly in the skin, mucosa and immune sentinel cells, are key executioners of inflammatory cell death (pyroptosis), which recruits immune cells to infection sites and promotes protective immunity1,2. Pore formation is triggered by gasdermin cleavage1,2. Although the proteases that activate GSDMB, C, D and E have been identified, how GSDMA-the dominant gasdermin in the skin-is activated, remains unknown. Streptococcus pyogenes, also known as group A Streptococcus (GAS), is a major skin pathogen that causes substantial morbidity and mortality worldwide3. Here we show that the GAS cysteine protease SpeB virulence factor triggers keratinocyte pyroptosis by cleaving GSDMA after Gln246, unleashing an active N-terminal fragment that triggers pyroptosis. Gsdma1 genetic deficiency blunts mouse immune responses to GAS, resulting in uncontrolled bacterial dissemination and death. GSDMA acts as both a sensor and substrate of GAS SpeB and as an effector to trigger pyroptosis, adding a simple one-molecule mechanism for host recognition and control of virulence of a dangerous microbial pathogen.

Virulence attributes of successful methicillin-resistant Staphylococcus aureus lineages.

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of severe and often fatal infections. MRSA epidemics have occurred in waves, whereby a previously successful lineage has been replaced by a more fit and better adapted lineage. Selection pressures in both hospital and community settings are not uniform across the globe, which has resulted in geographically distinct epidemiology. This review focuses on the mechanisms that trigger the establishment and maintenance of current, dominant MRSA lineages across the globe. While the important role of antibiotic resistance will be mentioned throughout, factors which influence the capacity of S. aureus to colonize and cause disease within a host will be the primary focus of this review. We show that while MRSA possesses a diverse arsenal of toxins including alpha-toxin, the success of a lineage involves more than just producing toxins that damage the host. Success is often attributed to the acquisition or loss of genetic elements involved in colonization and niche adaptation such as the arginine catabolic mobile element, as well as the activity of regulatory systems, and shift metabolism accordingly (e.g., the accessory genome regulator, agr). Understanding exactly how specific MRSA clones cause prolonged epidemics may reveal targets for therapies, whereby both core (e.g., the alpha toxin) and acquired virulence factors (e.g., the Panton-Valentine leukocidin) may be nullified using anti-virulence strategies.

Increased intracellular persulfide levels attenuate HlyU-mediated hemolysin transcriptional activation in Vibrio cholerae.

The vertebrate host's immune system and resident commensal bacteria deploy a range of highly reactive small molecules that provide a barrier against infections by microbial pathogens. Gut pathogens, such as Vibrio cholerae, sense and respond to these stressors by modulating the expression of exotoxins that are crucial for colonization. Here, we employ mass spectrometry-based profiling, metabolomics, expression assays, and biophysical approaches to show that transcriptional activation of the hemolysin gene hlyA in V. cholerae is regulated by intracellular forms of sulfur with sulfur-sulfur bonds, termed reactive sulfur species (RSS). We first present a comprehensive sequence similarity network analysis of the arsenic repressor superfamily of transcriptional regulators, where RSS and hydrogen peroxide sensors segregate into distinct clusters of sequences. We show that HlyU, transcriptional activator of hlyA in V. cholerae, belongs to the RSS-sensing cluster and readily reacts with organic persulfides, showing no reactivity or DNA dissociation following treatment with glutathione disulfide or hydrogen peroxide. Surprisingly, in V. cholerae cell cultures, both sulfide and peroxide treatment downregulate HlyU-dependent transcriptional activation of hlyA. However, RSS metabolite profiling shows that both sulfide and peroxide treatment raise the endogenous inorganic sulfide and disulfide levels to a similar extent, accounting for this crosstalk, and confirming that V. cholerae attenuates HlyU-mediated activation of hlyA in a specific response to intracellular RSS. These findings provide new evidence that gut pathogens may harness RSS-sensing as an evolutionary adaptation that allows them to overcome the gut inflammatory response by modulating the expression of exotoxins.

Panton-Valentine Leukocidin-Positive Staphylococcus aureus in Family and Pet Cat.

Continued detection of Panton-Valentine leukocidin-positive Staphylococcus aureus in samples from a family with severe repeated skin infections and their pet cat suggests transmission between the family and the cat. Decolonizing the pet led to successful elimination of the bacteria from the household. Clinicians should consider pet cats as possible reinfection sources.

Nasal carriage of virulent and multidrug resistant Staphylococcus aureus: a possible comorbidity of COVID-19.

BACKGROUND: Staphylococcus aureus (S. aureus) associated with COVID-19 has not been well documented. This cross-sectional study evaluated the association between nasal S. aureus carriage and COVID-19. METHODS AND RESULTS: Nasopharyngeal samples were collected from 391 participants presenting for COVID-19 test in Lagos, Nigeria, and S. aureus was isolated from the samples. Antimicrobial susceptibility test was done by disc diffusion method. All S. aureus isolates were screened for the presence of mecA, panton-valentine leucocidin (PVL) and toxic shock syndrome toxin (TSST) virulence genes by polymerase chain reaction. Staphylococcal protein A (spa) typing was conducted for all the isolates. Participants with COVID-19 had double the prevalence of S. aureus (42.86%) compared to those who tested negative (20.54%). A significant association was seen between S. aureus nasal carriage and COVID-19 (p = 0.004). Antimicrobial sensitivity results showed resistance to oxacillin (100%), cefoxitin (53%), and vancomycin (98.7%). However, only 41% of the isolates harbored the mecA gene, with SCCmecV being the most common SCCmec type. There was no association between the carriage of virulence genes and COVID-19. A total of 23 Spa types were detected, with t13249 and t095 being the two most common spa types. CONCLUSION: This study examined the association between nasal S. aureus carriage and SARS-COV-2 infection. Further research is required to fully explore the implications of S. aureus co-infection with COVID-19.

A case of refractory disseminated subcutaneous abscess with intrahousehold transmission by a USA300-LV-like strain of PVL-positive community-acquired MRSA clone.

A 44-year-old man with hypertension and dyslipidemia presented with pain in the buttocks. The patient was diagnosed with perianal ischiorectal fossa abscesses and cellulitis. He was subsequently diagnosed with a perineal subcutaneous abscess after a week, a right lower leg impetigo after a month, right periorchitis, a scrotal abscess, and Fournier's gangrene after two months. The patient was treated with various antimicrobials and underwent incisional drainage. Methicillin-resistant Staphylococcus aureus (MRSA) was detected in all draining specimens. Her daughter and son, who lived with the patient, presented with subcutaneous abscesses caused by MRSA. Suspecting repeated infections and household infections by virulent types of MRSA, such as PVL-positive strains, we performed genetic analyses of his and his son's strains. The results showed that the genotype and toxin gene profiles [ST8/t008/SCCmec type IVc/Panton-Valentine leucocidin (PVL) (+)/arginine catabolic mobile element (ACME) (-)] of both strains matched. single nucleotide polymorphism (SNP) analysis confirmed genetic homology between the two, concluding that home transmission by the same clone had occurred. In addition, the strain in this case differed from USA300 [ST8/t008/SCCmec type IVa/PVL (+) ACME (+)], which is a PVL-positive MRSA worldwide, including Japan, and its genetic profile matches that of USA300-LV, which is detected mainly in South America. Furthermore, SNP analysis showed that this strain is similar to USA300-LV/J (derived from USA300-LV) detected on Ishigaki Island, Okinawa Prefecture, Japan. This is the first report of refractory infections and household transmission of USA300-LV/J. Therefore, it is necessary to closely monitor both the USA300 and the USA300-LV.

Prevalence of the M1UK sublineage among emm1 Streptococcus pyogenes invasive strains isolated in the Czech Republic from December 2022 to May 2023. / Výskyt sub-linie M1 UK mezi invazivními kmeny Streptococcus pyogenes typ emm1 izolovanými od prosince 2022 do kvetna 2023 v Ceské republice.

AIMS: Since December 2022, an increase in invasive disease caused by Streptococcus pyogenes has been observed in the Czech Republic, with a shift in the clinical presentation and age of patients. Unlike in previous years, invasive disease is more common in children and adolescents under 18 years of age and in previously healthy middle-aged adults. An increase has been noticed in the number of S. pyogenes isolates from primarily sterile sites such as haemoculture, cerebrospinal fluid, pleural effusion fluid, joint fluid, and postmortem specimens. Routine emm gene typing revealed emm1 to be the predominant emm type of S. pyogenes. Between January 2023 and July 2023, 46% of all S. pyogenes isolates from invasive cases were assigned to the emm1 type. The globally spread M1UK sublineage is characterized by differences in the expression of seven genes, including the streptococcal pyrogenic toxin A (speA) gene, compared to historical emm1 iGAS strains. The aim of this study is to determine whether the more toxigenic M1UK sublineage is associated with the increase in invasive disease in the Czech Republic. METHODS: Whole genome sequencing of 41 S. pyogenes isolates from patients with invasive disease recovered in the Czech Republic in 2018 and 2019 and from December 2022 to May 2023 was performed using the MiSeq instrument (Illumina). Bioinformatics analysis was performed using freely available online tools the Bacterial and Viral Bioinformatics Resource Center. RESULTS: Based on whole genome sequencing data of 41 emm1 isolates of S. pyogenes from patients with invasive infectious disease recovered in 2018 and 2019 and from December 2022 to May 2023, the M1UK sublineage was found to be predominant from December 2022 to May 2023. CONCLUSION: The reason for the spread of the M1UK sublineage in the Czech Republic late in 2022 and in the first half of 2023 is not entirely clear, but it may be related to reduced immunity due to limited GAS transmission during lockdowns, especially in children. Another factor that may have contributed to the high incidence of invasive infectious diseases is the seasonal circulation of respiratory viruses.

SarS Is a Repressor of Staphylococcus aureus Bicomponent Pore-Forming Leukocidins.

Staphylococcus aureus is a successful pathogen that produces a wide range of virulence factors that it uses to subvert and suppress the immune system. These include the bicomponent pore-forming leukocidins. How the expression of these toxins is regulated is not completely understood. Here, we describe a screen to identify transcription factors involved in the regulation of leukocidins. The most prominent discovery from this screen is that SarS, a known transcription factor which had previously been described as a repressor of alpha-toxin expression, was found to be a potent repressor of leukocidins LukED and LukSF-PV. We found that inactivating sarS resulted in increased virulence both in an ex vivo model using primary human neutrophils and in an in vivo infection model in mice. Further experimentation revealed that SarS represses leukocidins by serving as an activator of Rot, a critical repressor of toxins, as well as by directly binding and repressing the leukocidin promoters. By studying contemporary clinical isolates, we identified naturally occurring mutations in the sarS promoter that resulted in overexpression of sarS and increased repression of leukocidins in USA300 bloodstream clinical isolates. Overall, these data establish SarS as an important repressor of leukocidins and expand our understanding of how these virulence factors are being regulated in vitro and in vivo by S. aureus.

Panton-Valentine Leukocidin-Positive CC398 MRSA in Urban Clinical Settings, the Netherlands.

We report detection of Panton-Valentine leukocidin-positive clonal complex 398 human-origin methicillin-resistant Staphylococcus aureus L2 in the Netherlands. This hypervirulent lineage originated in the Asia-Pacific Region and could become community-acquired in Europe after recurrent travel-related introductions. Genomic surveillance enables early detection to guide control measures and help limit spread of pathogens in urban settings.

The emerging threat of methicillin-resistant Staphylococcus aureus (MRSA) clone ST22-PT, carrying both Panton-Valentine leucocidin and toxic shock syndrome toxin 1 genes.

OBJECTIVES: Some MRSA strains produce Panton-Valentine leucocidin (PVL) and/or toxic shock syndrome toxin 1 (TSST-1), which are associated with severe infectious diseases. Although PVL- or TSST-1-positive strains have been isolated worldwide, strains carrying both PVL and TSST-1 genes are rare and sporadic. The objective of this study was to characterize these strains from Japan. METHODS: A total of 6433 MRSA strains isolated in Japan between 2015 and 2021 were analysed. Molecular epidemiological and comparative genomic analyses were conducted on PVL- and TSST-1-positive MRSA strains. RESULTS: A total of 26 strains from 12 healthcare facilities were PVL positive and TSST-1 positive, and all were classified as clonal complex (CC) 22. These strains exhibited similar genetic features to each other and were named as ST22-PT according to a previous report. Twelve and one of the ST22-PT strains were identified in patients with deep-seated skin infections and toxic shock syndrome-like symptoms, which are typical clinical features of PVL-positive and TSST-1-positive Staphylococcus aureus, respectively. Whole-genome comparative analysis revealed that the ST22-PT strains were highly similar to PVL- and TSST-1-positive CC22 strains isolated in several countries. Evaluation of the genome structure showed that ST22-PT possessed ΦSa2 harbouring PVL genes and a unique S. aureus pathogenicity island harbouring the TSST-1 gene. CONCLUSIONS: ST22-PT strains have recently emerged from several healthcare facilities in Japan, and ST22-PT-like strains have been identified in several countries. Our report highlights that the risk of international spread of PVL- and TSST-1-positive MRSA clone ST22-PT needs to be further investigated.

Panton-Valentine Leukocidin (PVL) genes may not be a reliable marker for community-acquired MRSA in the Dakahlia Governorate, Egypt.

BACKGROUND: Methicillin-resistant Staphylococcus aureus is linked to both nosocomial and community infections. One of the key virulence factors of S. aureus is Panton-Valentine leukocidin (PVL). The PVL genes are mostly associated with community-acquired MRSA (CA-MRSA). This study evaluates the prevalence of PVL genes as a marker for CA-MRSA at tertiary hospitals in Mansoura, Dakahlia, Egypt. S. aureus was isolated from clinical specimens obtained from different departments of tertiary hospitals, outpatient clinics, and hospital healthcare workers (HCWs). PCR was used to detect the mecA, PVL, and SCCmec genes among the recovered isolates. Standard broth microdilution method was used to determine the minimum inhibitory concentrations (MIC) of nine antibiotics against S. aureus. RESULTS: Two hundred S. aureus isolates were recovered and identified out of the total isolates (n = 320). The mecA gene was detected in 103 S. aureus isolates (51.5%). Among the MRSA isolates, 46.60% were PVL-positive. The incidence of the PVL genes of MRSA in nosocomial (HA), outpatient clinics (CA), and HCWs was 46.66%, 56.52%, and 42%, respectively. All MRSA isolates showed resistance to cefoxitin. The percentage of resistance to most tested antibiotics was high, except for ciprofloxacin (6.85%). Both antibiotic resistance and multidrug resistance among MRSA isolates were generally higher in PVL-positive isolates than in PVL-negative isolates in HA- and CA-MRSA isolates. While SCCmec type V was the most prevalent in PVL-positive MRSA stains, type I was the most prevalent in PVL-negative isolates. CONCLUSION: This study revealed that PVL genes are generally highly prevalent among mecA-positive MRSA isolates, whether they are CA-MRSA, HA-MRSA, or HCW isolates. Therefore, PVL is not a valid marker for CA-MRSA in Mansoura, Dakahlia Governorate, Egypt, as has been reported in other countries. Further epidemiologic studies are required to track the incidence of PVL in HA-MRSA, CA-MRSA, and HCW isolates in other Egyptian governorates.

Neutrophil-derived reactive agents induce a transient SpeB negative phenotype in Streptococcus pyogenes.

BACKGROUND: Streptococcus pyogenes (group A streptococci; GAS) is the main causative pathogen of monomicrobial necrotizing soft tissue infections (NSTIs). To resist immuno-clearance, GAS adapt their genetic information and/or phenotype to the surrounding environment. Hyper-virulent streptococcal pyrogenic exotoxin B (SpeB) negative variants caused by covRS mutations are enriched during infection. A key driving force for this process is the bacterial Sda1 DNase. METHODS: Bacterial infiltration, immune cell influx, tissue necrosis and inflammation in patient´s biopsies were determined using immunohistochemistry. SpeB secretion and activity by GAS post infections or challenges with reactive agents were determined via Western blot or casein agar and proteolytic activity assays, respectively. Proteome of GAS single colonies and neutrophil secretome were profiled, using mass spectrometry. RESULTS: Here, we identify another strategy resulting in SpeB-negative variants, namely reversible abrogation of SpeB secretion triggered by neutrophil effector molecules. Analysis of NSTI patient tissue biopsies revealed that tissue inflammation, neutrophil influx, and degranulation positively correlate with increasing frequency of SpeB-negative GAS clones. Using single colony proteomics, we show that GAS isolated directly from tissue express but do not secrete SpeB. Once the tissue pressure is lifted, GAS regain SpeB secreting function. Neutrophils were identified as the main immune cells responsible for the observed phenotype. Subsequent analyses identified hydrogen peroxide and hypochlorous acid as reactive agents driving this phenotypic GAS adaptation to the tissue environment. SpeB-negative GAS show improved survival within neutrophils and induce increased degranulation. CONCLUSIONS: Our findings provide new information about GAS fitness and heterogeneity in the soft tissue milieu and provide new potential targets for therapeutic intervention in NSTIs.

T-cell receptor Vß8 for detection of biologically active streptococcal pyrogenic exotoxin type C.

Streptococcus pyogenes is an important human pathogen, commonly spread by airborne droplets but also by ingestion of contaminated food. Apart from causing infection, this pathogen produces 13 distinct types of streptococcal pyrogenic exotoxins (SPE). The current method for detection cannot distinguish between the biologically active form of SPE that has been reported to cause foodborne outbreaks and the inactivated toxin that poses no health risk. To measure the biological activity of SPE type C (SPE-C), one such toxin that was linked to foodborne outbreaks associated with milk and milk products, we developed a cell-based assay that can discern between biologically active and inactive SPE-C. To the best of our knowledge, this is the first showing that SPE-C activates T-cells expressing Vß8. With this finding, we used a T-cell line natively expressing Vß8 that was genetically engineered to also express the luciferase reporter gene under the regulation of nuclear factor of activated T-cells response element in combination with a B-cell line to present the recombinant SPE-C (rSPE-C) toxin via major histocompatibility complex (MHC) class II to the Vß8 T-cell receptor (TCR) in an assay to detect and to discern between biologically active and inactive rSPE-C. By using this system, we demonstrated that SPE-C induced significant IL-2 secretion after 72 h and visible light emission after only 5 h, doubling by 24 h. We utilize this finding to assess the specificity of the assay and the effect of pasteurization on SPE-C activity. We observed no cross-reactivity with SPE-B and significant loss of SPE-C biological activity in spiked phosphate-buffered saline while SPE-C spiked into milk is heat stable. Once SPE-C has formed, it is infeasible to eliminate it from milk by thermal treatment.

Molecular epidemiology and change trend of methicillin-resistant Staphylococcus aureus from invasive infections of a hospital in Hangzhou from 2012 to 2018.

The disease caused by methicillin-resistant Staphylococcus aureus (MRSA) is a global public health challenge that threatens society and patients seriously. Therefore, the molecular epidemiology and change trend of MRSA is essential for the control and treatment of diseases caused by the pathogen in their regions. To explore molecular epidemiology of MRSA in Hangzhou, we collected 162 MRSA isolates from 2012 to 2018, conducted the antimicrobial susceptibility and used polymerase chain reaction(PCR) to test the molecular typing including multilocus sequence typing (MLST), staphylococcal chromosome cassette mec (SCCmec), staphylococcal protein A (spa A) and Panton-Valentine leucocidin (PVL). All the strains was divided into community-associated MRSA (CA-MRSA) or hospital-associated MRSA (HA-MRSA). 162 MRSA isolates were divided into 16 STs and 30 spa types. The major ST type was ST5 (96/162, 59.3%) and the predominant spa type was t311 (83/162, 51.2%). Five SCCmec types were found and the most common SCCmec type was type II (101/162, 61.7%). ST5-II-t311 was the predominant MRSA clone. And the prevalence of ST5 MRSA gradually declined from 2014 to 2018 but the prevalence of ST59 MRSA significantly increased. At the same time, livestock-associated methicillin-resistant Staphylococcus aureus(LA-MRSA) ST398 and ST9 were detected. Twenty-eight isolates were PVL gene positive (28/162, 17.3%). The most prevalent PVL-positive clone was ST59-IVa-t437. Comparing with HA-MRSA, CA-MRSA had a lower probability of ST5 (9.1% vs 67.1%, P=0.000) but a higher probability of ST59 (63.6% vs 11.4%, P=0.000), not only that, it was more likely to carrying PVL-positive gene (36.4% vs 14.3%, P=0.028). In summary, the molecular types of MRSA were getting complex over time. ST5-II-t311 was the predominant clone of MRSA isolate with a downward incidence from 2014 to 2018. ST59 MRSA strains, which is thought community related strain are spreading into hospitals and has an upward incidence from 2014 to 2018.

Genome-wide analysis of in vivo CcpA binding with and without its key co-factor HPr in the major human pathogen group A Streptococcus.

Catabolite control protein A (CcpA) is a master regulator of carbon source utilization and contributes to the virulence of numerous medically important Gram-positive bacteria. Most functional assessments of CcpA, including interaction with its key co-factor HPr, have been performed in nonpathogenic bacteria. In this study we aimed to identify the in vivo DNA binding profile of CcpA and assess the extent to which HPr is required for CcpA-mediated regulation and DNA binding in the major human pathogen group A Streptococcus (GAS). Using a combination RNAseq/ChIP-seq approach, we found that CcpA affects transcript levels of 514 of 1667 GAS genes (31%) whereas direct DNA binding was identified for 105 GAS genes. Three of the directly regulated genes encode the key GAS virulence factors Streptolysin S, PrtS (IL-8 degrading proteinase), and SpeB (cysteine protease). Mutating CcpA Val301 to Ala (strain 2221-CcpA-V301A) abolished interaction between CcpA and HPr and impacted the transcript levels of 205 genes (40%) in the total CcpA regulon. By ChIP-seq analysis, CcpAV301A bound to DNA from 74% of genes bound by wild-type CcpA, but generally with lower affinity. These data delineate the direct CcpA regulon and clarify the HPr-dependent and independent activities of CcpA in a key pathogenic bacterium.

A fatal case of pneumonia and sepsis caused by sequence type 398 methicillin-susceptible Staphylococcus aureus carrying Pantone-Valentine leukocidin in China.

Staphylococcus aureus (S. aureus) sequence type 398 (ST398) has aroused great concern for its spread throughout the world. ST398 community-acquired MRSA (CA-MRSA) has been given greater emphasis because of its high virulence and high probability of treatment failure. Herein, A 22-year-old male was admitted to our hospital with a history of fever, chest pain and dyspnea for 2 days. A chest CT scan showed infiltrative and nodular shadows. The sequence type of the isolates from blood culture was ST398, the virulence genes detected was PVL gene (lukS-PV and lukF-PV). Despite resuscitation efforts, he died of multiple organ failure on admission 3rd day. This is the first described case of severe pneumonia and sepsis due to hematogenous spread of scalp furuncles caused by Panton-Valentine leukocidin (PVL) positive community-acquired methicillin-susceptible S. aureus (CA-MSSA) ST398 strains in an immunocompentent adult in mainland China. This report highlight the emergence CA-PVL-MSSA ST398 infection and its association with life-threatening infections. Early decolonization and identification of ST398 is critical. Severe skin and soft tissue infections should be suspected for ST398 PVL-MSSA.

Invasive pectoral abscess and costal osteomyelitis with bloodstream infection caused by methicillin-resistant Staphylococcus aureus after nasal septoplasty in an immunocompetent adult patient.

There are few reports of multilocus sequence type (ST) 5/staphylococcal cassette chromosome (SCC) mec type IVc/toxic shock syndrome toxin (TSST)-1- positive methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infections. We report a case of community-onset MRSA (CO-MRSA) bloodstream infection in a healthy 41-year-old Japanese man after nasal septoplasty, followed by pectoral abscess and costal osteomyelitis. The patient presented with right anterior chest pain and fever. After admission, MRSA was isolated from two sets of blood cultures, and vancomycin was administered. On the fifth day, contrast-enhanced computed tomography (CT) scan and contrast-enhanced magnetic resonance imaging (MRI) scan showed an abscess in the right anterior chest to the right subpleural region. The dosage of vancomycin (4 g/day) did not reach the effective blood concentration; therefore, there was a switch to daptomycin. On the 23rd day, contrast-enhanced MRI revealed osteomyelitis of the right first rib, and as a result, linezolid was initiated. Two weeks later, contrast-enhanced CT of the chest showed improvement in the abscess. The patient was treated for 6 weeks during hospitalization and then switched to minocycline for 10 weeks. Molecular characterization of this isolate showed that it was ST5/SCCmec type IVc/TSST-1-positive/Panton-Valentine leucocidin (PVL)-negative. PVL-negative CO-MRSA can lead to hematogenous osteomyelitis and abscess even if the patient is immunocompetent, and if isolated from blood cultures, it is important to repeat imaging studies, even if the initial imaging studies were normal. It is possible that this strain contributes to the pathogenesis of invasive CO-MRSA, but further case accumulation is needed.

Methicillin-resistant Staphylococcus aureus transmission and hospital-acquired bacteremia in a neonatal intensive care unit in Greece.

BACKGROUND: Staphylococcus aureus is a common pathogen causing hospital acquired infections (HAIs) in neonates. In this study, the epidemiology of methicillin-resistant S. aureus (MRSA) colonization and infections in a 30-bed, level III university-affiliated neonatal intensive care unit (NICU) located in a children's hospital was retrospectively investigated for the period 2014-2018. METHODS: Genes encoding Panton-Valentine Leukocidin (lukS/lukF-PV, PVL), toxic shock syndrome toxin (tst), exfoliative toxins (eta, etb), and the resistance genes mecA, mecC and fusB, were defined in 46 representative strains by PCRs. Relatedness of strains was assessed by MLST. RESULTS: Of 1538 neonates, 77 (5%) had a positive culture for MRSA (23/77 were NICU-acquired and 54/77 imported cases). Four MRSA bacteremias occurred. Most isolates were multi-resistant. One major clone was identified, ST225, among 40 tested neonatal strains (23/40, 58%). Of these, 14/23 were imported from the same maternity hospital (MH). Another clone, ST217, was predominant (4/6) among health care workers (HCWs), found colonized. Four isolates classified as ST80 were PVL-positive. Additional four strains carried tst (10%), belonging to ST30 and ST225 (two strains each), and two etb. The implicated MH was notified for the problem, decolonization treatment was successfully performed in HCWs and neonates. Strengthening of infection control measures with emphasis on hand hygiene was applied. CONCLUSIONS: Uncovering reservoirs for on-going MRSA transmission in NICUs has proved challenging. Well known nosocomial MRSA clones are being constantly introduced and transmitted via MHs and HCWs. Effective infection prevention and control requires constant vigilance.

Aggregatibacter actinomycetemcomitans colonization and persistence in a primate model.

Aggregatibacter actinomycetemcomitans is associated with aggressive periodontitis resulting in premature tooth loss in adolescents. Tooth adherence and biofilm persistence are prerequisites for survival in the oral domain. Here, using a rhesus monkey model, 16S rRNA sequencing, and weighted network analysis, we assessed colonization of A. actinomycetemcomitans variants and ascertained microbial interactions in biofilm communities. Variants in A. actinomycetemcomitans leukotoxin (ltx) were created, labeled, inoculated, and compared with their progenitor strain for in vivo colonization. Samples of tooth-related plaque were assessed for colonization at baseline and after debridement and inoculation of labeled strains. Null, minimal, and hyper-Ltx-producing strains were created and assessed for hydroxyapatite binding and biofilm formation in vitro. Ltx-hyperproducing strains colonized with greater prevalence and at higher levels than wild type or ltx mutants (P = 0.05). Indigenous and inoculated A. actinomycetemcomitans strains that attached were associated with lactate-producing species (i.e., Leptotrichia, Abiotrophia, and Streptoccocci). A. actinomycetemcomitans was found at 0.13% of the total flora at baseline and at 0.05% 4 wk after inoculation. In vivo data were supported by in vitro results. We conclude that hyper-Ltx production affords these strains with an attachment advantage providing a foothold for competition with members of the indigenous microbiota. Increased attachment can be linked to ltx gene expression and up-regulation of adherence-associated genes. Growth of attached A. actinomycetemcomitans in vivo was enhanced by lactate availability due to consorting species. These associations provide A. actinomycetemcomitans with the constituents required for its colonization and survival in the complex and competitive oral environment.

Identification of a Thiol-Disulfide Oxidoreductase (SdbA) Catalyzing Disulfide Bond Formation in the Superantigen SpeA in Streptococcus pyogenes.

Mechanisms of disulfide bond formation in the human pathogen Streptococcus pyogenes are currently unknown. To date, no disulfide bond-forming thiol-disulfide oxidoreductase (TDOR) has been described and at least one disulfide bonded protein is known in S. pyogenes. This protein is the superantigen SpeA, which contains 3 cysteine residues (Cys 87, Cys90, and Cys98) and has a disulfide bond formed between Cys87 and Cys98. In this study, candidate TDORs were identified from the genome sequence of S. pyogenes MGAS8232. Using mutational and biochemical approaches, one of the candidate proteins, SpyM18_2037 (named here SdbA), was shown to be the catalyst that introduces the disulfide bond in SpeA. SpeA in the culture supernatant remained reduced when sdbA was inactivated and restored to the oxidized state when a functional copy of sdbA was returned to the sdbA-knockout mutant. SdbA has a typical C46XXC49 active site motif commonly found in TDORs. Site-directed mutagenesis experiments showed that the cysteines in the CXXC motif were required for the disulfide bond in SpeA to form. Interactions between SdbA and SpeA were examined using cysteine variant proteins. The results showed that SdbAC49A formed a mixed disulfide with SpeAC87A, suggesting that the N-terminal Cys46 of SdbA and the C-terminal Cys98 of SpeA participated in the initial reaction. SpeA oxidized by SdbA displayed biological activities suggesting that SpeA was properly folded following oxidation by SdbA. In conclusion, formation of the disulfide bond in SpeA is catalyzed by SdbA and the findings represent the first report of disulfide bond formation in S. pyogenes. IMPORTANCE Here, we reported the first example of disulfide bond formation in Streptococcus pyogenes. The results showed that a thiol-disulfide oxidoreductase, named SdbA, is responsible for introducing the disulfide bond in the superantigen SpeA. The cysteine residues in the CXXC motif of SdbA are needed for catalyzing the disulfide bond in SpeA. The disulfide bond in SpeA and neighboring amino acids form a disulfide loop that is conserved among many superantigens, including those from Staphylococcus aureus. SpeA and staphylococcal enterotoxins lacking the disulfide bond are biologically inactive. Thus, the discovery of the enzyme that catalyzes the disulfide bond in SpeA is important for understanding the biochemistry of SpeA production and presents a target for mitigating the virulence of S. pyogenes.