Necrotic activity of ExhC from Mammaliicoccus sciuri is mediated by specific amino acid residues.

Mammaliicoccus sciuri, a commensal and pathogenic bacterium of significant clinical and veterinary relevance, expresses exfoliative toxin C (ExhC), a specific glutamyl endopeptidase belonging to the chymotrypsin family as the principal virulence factor. However, unlike most members of this family, ETs are inactive against a wide range of substrates and possess exquisite specificity for desmoglein-1 (Dsg1), a cadherin-like adhesion molecule that is crucial to maintain tissue integrity, thereby preventing the separation of skin cells and the entry of pathogens. ExhC is of clinical importance since in addition to causing exfoliation in pigs and mice, it induces necrosis in multiple mammalian cell lines, a property not observed for other ETs. Previous experiments have implicated the ExhC79-128 fragment in causing necrosis. Site-directed mutagenesis of specific residues within this fragment were studied and led to the design of an ExhC variant containing four-point mutations (ExhCmut4) lacking necrotic potential but retaining nearly wild-type (wt) levels of enzymatic activity. Moreover, the determination of the ExhCwt and ExhCmut4 crystal structures identified the conformation in the necrosis-linked region. These results constitute an important step toward the understanding of the mechanisms underlying the necrotic and epidermolytic activity of ExhC.

Prevalence of Panton-Valentine leucocidin ( pvl) and exfoliative toxin A ( eta) gene within methicillin resistant and susceptible Staphylococcus aureus in an urban tertiary hospital: A molecular epidemiology pilot study.

Background: Staphylococcus aureus is well known to cause a multitude of clinical manifestations, from mild to severe bloodstream infections that could lead to death. Infections are common, either in community-acquired or hospital-acquired settings, and treatment remains a challenge due to methicillin-resistant Staphylococcus aureus (MRSA). The pathogenesis of S. aureus is mediated by several cell-surface and secreted virulence factors. The virulence factors discussed in this study are Panton-Valentine leucocidin ( pvl) and exfoliative toxin A ( eta). Identifying both pvl and eta gene may help in studying bacterial pathogenesis and biology thus creating possible therapeutic pathway or intervention.Our pilot study aimed to observe pvl and eta as virulence gene prevalence in a North Sumatera tertiary referral health center. Methods: Our study was a descriptive-analytical observational study with a cross-sectional design in which we collected isolates over a single time period. The frequency of genes is reported as a percentage comparison between MRSA and methicillin-susceptible S. aureus (MSSA). Qualitative gene prevalence analysis was carried out using the polymerase chain reaction (PCR). Results: Our results showed that from 38 MRSA sample isolates, 32 samples were found to be pvl-positive, or 84,3% of the total samples. From 40 MSSA sample isolates, one sample was found to be pvl-positive MSSA, or 97,5%. Regarding eta, from 38 MRSA sample isolates, 81,6% of the total sample did not have eta, while from 40 MSSA sample isolates, all samples were found to be positive for eta. We found that both pvl and eta were significantly more likely to be expressed in the MSSA strain. Conclusions: Our study shows that pvl and eta are more likely expressed in MSSA strains than in MRSA strains in Indonesia.

Antibiotic susceptibility of human-associated Staphylococcus aureus and its relation to agr typing, virulence genes, and biofilm formation.

BACKGROUND AND OBJECTIVE: Carriage of virulence factors confers some evolutionary benefit to bacteria, which favors the resistant strains. We aimed to analyze whether antibiotic susceptibility of Staphylococcus aureus strains is affected by agr typing, biofilm formation ability, and virulence profiles. METHODS: A total of 123 S. aureus clinical isolates were subjected to antimicrobial susceptibility testing by disk diffusion method, biofilm formation by microtiter plate method, as well as polymerase chain reaction screening to identify virulence genes and the accessory gene regulator (agr) types I-IV. A P value < 0.05 was considered significant. RESULTS: The most prevalent virulence gene was staphyloxanthin crtN, followed by hemolysin genes, capsular cap8H, toxic shock toxin tst, and enterotoxin sea, respectively. Resistant isolates were more commonly found in the agr-negative group than in the agr-positive group. Isolates of agr type III were more virulent than agr I isolates. Strong biofilm producers showed more antibiotic susceptibility and carried more virulence genes than non-strong biofilm producers. Associations were found between the presence of virulence genes and susceptibility to antibiotics. Carriage of the virulence genes and agr was higher in the inpatients; while, resistance and strong biofilms were more prevalent in the outpatients. CONCLUSION: These findings indicated the presence of several virulence factors, biofilm production capacity, agr types and resistance to antibiotics in clinical S. aureus isolates. Considering the importance of S. aureus for human medicine, an understanding of virulence and resistance relationships would help to reduce the impact of S. aureus infections.

Antimicrobial Resistance and Molecular Analysis of Staphylococcus aureus in Staphylococcal Scalded Skin Syndrome among Children in Korea.

BACKGROUND: Staphylococcal scalded skin syndrome (SSSS) is a skin disease characterized by blistering and desquamation caused by exfoliative toxins (ETs) of Staphylococcus aureus (S. aureus). Although many countries show predominance of methicillin-susceptible S. aureus (MSSA), cases of methicillin-resistant S. aureus (MRSA) have been reported. METHODS: Twenty-six children aged <15 years diagnosed with SSSS from January 2010 to December 2017 from three hospitals were included. S. aureus isolates from cases were analyzed for multilocus sequence types and ETs. Medical records were reviewed for clinical characteristics, treatment, and antimicrobial susceptibility patterns of S. aureus. RESULTS: Among the 26 cases, mean age was 2.3 years. According to skin manifestations patients were classified as generalized (n = 10, 38.5%), intermediate (n = 11, 42.3%), and abortive (n = 5, 19.2%). Among all cases, 96.2% (25/26) were due to MRSA and the macrolide-resistance rate was 92.3% (24/26). ST89 (n = 21, 80.8%) was the most prevalent clone, followed by single clones of ST1, ST5, ST72, ST121, and ST1507. The eta gene was detected in one (3.8%) isolate which was MSSA. The etb gene was detected in 14 (53.8%) isolates, all of which were ST89. Nafcillin or first-generation cephalosporin was most commonly prescribed (n=20, 76.9%). Vancomycin was administered in four patients (15.4%) and clindamycin in nine patients (34.6%). Among MRSA cases, there was no difference in duration of treatment when comparing the use of antimicrobials to which the causative bacteria were susceptible or non-susceptible (9.75 vs. 8.07 days, P > 0.05). CONCLUSION: S. aureus isolated from children with SSSS in Korea demonstrated a high prevalence of methicillin-resistant ST89 clones that harbored the etb gene. The predominance of MRSA suggests that antibiotics to which MRSA are susceptible may be considered for empirical antibiotic treatment in children with SSSS in Korea. Further studies on the role and effectiveness of systemic antibiotics in SSSS are warranted.

Synergistic antimicrobial activity of melittin with clindamycin on the expression of encoding exfoliative toxin in Staphylococcus aureus.

Staphylococcus aureus is an opportunistic human pathogens, with the ability to produce a series of virulence factors that contribute to the severity of infections. Exfoliative toxins (ETs) are one of the important virulence factors that participating in staphylococcal scalded skin syndrome. Melittin has different biological activities, comprising of antiviral, broad spectrum antibacterial, antiprotozoal, antifungal and anti-inflammatory effects. Twelve clinical isolates of methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) were obtained from wound infection in the burn patients. The MIC plus three sub-inhibitory concentrations (I, II and III) of clindamycin and melittin were tested. Next, the synergistic effects of melittin and clindamycin were evaluated using the broth microdilution checkerboard assay. The detection of exfoliative toxin A and B genes were examined by PCR method. Then the effects of sub-MIC melittin on the expression levels of eta and etb were assessed by quantitative real-time PCR (qRT-PCR) assay. Melittin MIC values against MRSA and MSSA planktonic cells were 0.25-0.5 and 0.25-1 µg/ml, respectively. The clindamycin MIC values against MRSA and MSSA were between 0.5 and 8 µg/ml and 0.5-2 µg/ml, respectively. The results of the time-kill kinetics assay (3.5log10 and 3log10) against MSSA and MRSA planktonic cells were determined within 24 h using melittin. The mean expression of eta in MRSA and MSSA was significantly downregulated to approximately 3.5 and 4 fold, respectively. Moreover, the mean expression of etb in MRSA and MSSA were significantly downregulated to approximately 2.5 and 3 fold, respectively. Hemolytic assay showed that the extracted melittin indicates a strong hemolytic activity (HD50 = 2 µg/ml). Melittin at 0.5 µg/ml induced cell lysis and stimulated the formation of vesicles in S. aureus strains. Melittin could reduce the expression of eta and etb as encoding exfoliative toxin A and B genes. This component appears to be a good candidate for the treatment of MRSA and MSSA strains. So, melittin in combination with clindamycin can be classified as a complementary treatment of wound infections in burn patients.

Frequency of superantigen encoding genes of Staphylococcus aureus isolates collected from multiple sclerosis (MS) patients and nasal carriers.

BACKGROUND: Bacterial superantigens are potent T cell activators that can have acute or chronic effects on the central nervous system. OBJECTIVES: In this study, the role of enterotoxins, exfoliative toxins and toxic shock syndrome toxin of Staphylococcus aureus was investigated in MS patients and healthy nasal carriers. METHODS: Three-hundred fifty nasal swabs were collected from healthy nasal carriers (n = 210) and MS (n = 140) patients. Staphylococcus aureus superantigens were detected by multiplex PCR. Antimicrobial susceptibility pattern was performed using disk diffusion method. RESULTS: The highest rates of nasal colonization were seen in MS patients (46.42%). The rates of nasal colonization in the healthcare workers were 30.95%. The most commonly detected superantigens were SEA (31.5%), SEB (17.7%) and ETA (16.9%). The Staphylococcus aureus isolates had the highest levels of resistance against erythromycin (57.7%), clindamycin (55.4%) and co-trimoxazole (43.1%). All isolates were susceptible to vancomycin, linezolid, and mupirocin. CONCLUSION: Our results revealed that the frequency of superantigen producing Staphylococcus aureus isolates is high in the MS patients. As well as these isolates are sensitive to mupirocin. Thus it is better to use of mupirocin for nasal decolonization of Staphylococcus aureus in the MS patients.

Two highly divergent lineages of exfoliative toxin B-encoding plasmids revealed in impetigo strains of Staphylococcus aureus.

Exfoliative toxin B (ETB) encoded by some large plasmids plays a crucial role in epidermolytic diseases caused by Staphylococcus aureus. We have found as yet unknown types of etb gene-positive plasmids isolated from a set of impetigo strains implicated in outbreaks of pemphigus neonatorum in Czech maternity hospitals. Plasmids from the strains of clonal complex CC121 were related to archetypal plasmid pETBTY4. Sharing a 33-kb core sequence including virulence genes for ETB, EDIN C, and lantibiotics, they were assigned to a stand-alone lineage, named pETBTY4-based plasmids. Differing from each other in the content of variable DNA regions, they formed four sequence types. In addition to them, a novel unique plasmid pETB608 isolated from a strain of ST130 was described. Carrying conjugative cluster genes, as well as new variants of etb and edinA genes, pETB608 could be regarded as a source of a new lineage of ETB plasmids. We have designed a helpful detection assay, which facilitates the precise identification of the all described types of ETB plasmids.

Staphylococci isolated from ready-to-eat meat - Identification, antibiotic resistance and toxin gene profile.

The aim of this study was to analyse the staphylococci isolated from ready-to-eat meat products, including pork ham, chicken cold cuts, pork sausage, salami and pork luncheon meat, sliced in the store to the consumer's specifications, along with species identification and determination of antibiotic resistance. Genes encoding staphylococcal enterotoxins, staphylococcal enterotoxin-like proteins, exfoliative toxins, and toxic shock syndrome toxin 1 were also investigated. From the 41 samples, 75 different staphylococcal isolates were obtained. Based on PCR-RFLP analysis of the gap gene using AluI and HpyCH4V restriction enzymes, the isolates were identified as Staphylococcus equorum (28%), S. vitulinus (16%), S. carnosus (14%), S. succinus (11%), S. xylosus (11%), S. saprophyticus (9%), S. warneri (9%), S. haemolyticus (1%) and S. pasteuri (1%). The incidence and number of resistances to antimicrobials was found to be species but not source of isolation dependent. All S. xylosus, S. saprophyticus, S. haemolyticus and S. pasteuri isolates showed antibiotic resistance. A lower percentage of resistance was recorded for S. warneri (71%) and S. vitulinus (58%), followed by S. equorum (57%), S. carnosus (50%) and S. succinus (50%). The most frequent resistance was observed to fusidic acid (43%). The mecA gene was amplified in 4% of the staphylococci. However, phenotypic resistance to methicillin was not confirmed in any of these isolates. On the other hand, the mecA gene was not detected in any of 9% of the isolates resistant to cefoxitin. It was also found that among 75 isolates, 60 (80%) harbored from 1 to 10 out of 21 analyzed superantigenic toxin genes. The most prevalent genes were: sei (36% isolates) among enterotoxins, seln (32% isolates) among enterotoxin-like proteins and eta encoding exfoliative toxin A (37% isolates). The findings of this study further extend previous observations that, when present in food, not only S. aureus but also other species of staphylococci could be of public health significance.

Antibiotic susceptibility and genomic variations in Staphylococcus aureus associated with Skin and Soft Tissue Infection (SSTI) disease groups.

BACKGROUND: Staphylococcus aureus is associated with human skin and soft tissue infections (SSTIs); however, the involvement of virulence factors in different clinical presentations is unclear. METHODS: We analyzed methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) strains from Taiwan to determine correlations among the clinical characteristics of SSTIs, antimicrobial susceptibility and virulence factors of S. aureus with specific genetic backgrounds. RESULTS: We identified 177 MRSA isolates and 130 MSSA isolates among the 307 SSTI-associated S. aureus isolates. Hospital-acquired (HA)- and community-acquired (CA)-MRSA isolates accounted for 61.6 % and 38.4 % of the isolates, respectively. Clinical presentations in SSTI patients differed significantly for the disease groups. Deep-seated MRSA infections presented with higher amputation rate than MSSA infections. MRSA isolates were all susceptible to linezolid, teicoplanin, and vancomycin, and >94 % of isolates were erythromycin- and clindamycin-resistant. Staphylococcal cassette chromosome (SCCmec) types IV, V, and VII were the most frequent in the CA-MRSA group (n = 68); types III, IV and V were the most frequent in the HA-MRSA group (n = 109). Panton-Valentine leukocidin (PVL) genes were significantly more frequent in CA-MRSA strains (75.0 %) than in HA-MRSA (33.0 %) and MSSA (24.6 %) and were found in 66.7 % (74/111) strains isolated from the abscess group. Exfoliatin A genes were more common in catheter-related exit-site MSSA infections (37.5 %) compared with other MSSA disease groups (P < 0.05). Exfoliatin B and superantigen exotoxin genes were uncommon in all SSTI disease types. Pulsotypes A (ST239), C, and D (ST59) were the predominant MRSA genotypes in deep-seated infections. CONCLUSIONS: If not treated appropriately, deep-seated MRSA-associated infections present with higher amputation rates than deep-seated MSSA-associated infections. PVL-positive MRSA strains caused more frequently pus-forming lesions and less bacteremia and invasive diseases. Methods for discriminating CA-MRSA from HA-MRSA strains are now unreliable due to circulation of both ST 239 and ST 59 strains in the community and nosocomial settings. Initial antibiotic treatments should consider MRSA for patients with SSTIs in areas where MRSA is prevalent.

Comparative genomics of toxigenic and non-toxigenic Staphylococcus hyicus.

The most common causative agent of exudative epidermitis (EE) in pigs is Staphylococcus hyicus. S. hyicus can be grouped into toxigenic and non-toxigenic strains based on their ability to cause EE in pigs and specific virulence genes have been identified. A genome wide comparison between non-toxigenic and toxigenic strains has never been performed. In this study, we sequenced eleven toxigenic and six non-toxigenic S. hyicus strains and performed comparative genomic and phylogenetic analysis. Our analyses revealed two genomic regions encoding genes that were predominantly found in toxigenic strains and are predicted to encode for virulence determinants for EE. All toxigenic strains encoded for one of the exfoliative toxins ExhA, ExhB, ExhC, or ExhD. In addition, one of these regions encoded for an ADP-ribosyltransferase (EDIN, epidermal cell differentiation inhibitor) and a novel putative RNase toxin (polymorphic toxin) and was associated with the gene encoding ExhA. A clear differentiation between toxigenic and non-toxigenic strains based on genomic and phylogenetic analyses was not apparent. The results of this study support the observation that exfoliative toxins of S. hyicus and S. aureus are located on genetic elements such as pathogenicity islands, phages, prophages and plasmids.

C55 bacteriocin produced by ETB-plasmid positive Staphylococcus aureus strains is a key factor for competition with S. aureus strains.

Exfoliative toxin (ET) produced by Staphylococcus aureus is closely associated with the onset of bullous impetigo. To date, three ETs (ETA, ETB and ETD) have been identified. The gene encoding ETB is located in a plasmid designated pETB. Bacteriocin synthesis genes are also located in this plasmid and pETB-positive strains reportedly produce the C55 bacteriocin. In this study, the antibacterial activity against S. aureus strains of the bacteriocin produced by the pETB-positive strain TY4 was investigated. This bacteriocin demonstrated antibacterial activity against all pETB-negative but not pETB-positive strains, including TY4. Additionally, a TY4- strain from which the pETB plasmid had been deleted exhibited susceptibility to the bacteriocin. Further experiments revealed that two immunity factors (orf 46-47 and orf 48) downstream of the bacteriocin synthesis genes in the pETB plasmid are associated with immunity against the bacteriocin produced by TY4. The TY4- with orf46-47 strain exhibited complete resistance to bacteriocin, whereas the TY4- with orf48 strain exhibited partial resistance. Whether bacteriocin affects the proportion of each strain when co-cultured with S. aureus strains was also investigated. When TY4 or TY4- was co-cultured with 209P strain, which is susceptible to the bacteriocin, the proportion of 209P co-cultured with TY4 was significantly less than when 209P was co-cultured with TY4-, whereas the proportion of TY4- with orf46-48 co-cultured with TY4 was greater than with TY4-. These results suggest that the C55 bacteriocin produced by pETB-positive strains affects the proportion of each strain when pETB-positive and -negative strains co-exist.

Complete genome analysis of two new bacteriophages isolated from impetigo strains of Staphylococcus aureus.

Exfoliative toxin A (ETA)-coding temperate bacteriophages are leading contributors to the toxic phenotype of impetigo strains of Staphylococcus aureus. Two distinct eta gene-positive bacteriophages isolated from S. aureus strains which recently caused massive outbreaks of pemphigus neonatorum in Czech maternity hospitals were characterized. The phages, designated ϕB166 and ϕB236, were able to transfer the eta gene into a prophageless S. aureus strain which afterwards converted into an ETA producer. Complete phage genome sequences were determined, and a comparative analysis of five designed genomic regions revealed major variances between them. They differed in the genome size, number of open reading frames, genome architecture, and virion protein patterns. Their high mutual sequence similarity was detected only in the terminal regions of the genome. When compared with the so far described eta phage genomes, noticeable differences were found. Thus, both phages represent two new lineages of as yet not characterized bacteriophages of the Siphoviridae family having impact on pathogenicity of impetigo strains of S. aureus.

The distribution of pathogenic and toxigenic genes among MRSA and MSSA clinical isolates.

Staphylococcus aureus (S. aureus) is considered as a notorious nosocomial pathogen among hospitalized patients and community-dwelling subjects. Its increasing morbidity and mortality is believed to be due to antibiotic resistance. However, the data concerning molecular properties of infecting strains are few. In this study, a total of 192 S. aureus strains, including 88 (45.8%) meticillin-sensitive S. aureus (MSSA) and 104 (54.2%) meticillin-resistant S. aureus (MRSA) were recovered from clinical samples. The prevalence of subtypes containing staphylococcal cassette chromosome mec (SSCmec), staphylococcal enterotoxins (SEs), toxic shock syndrome toxin (TSST) and exfoliative toxin was assessed by PCR. Antibiotic susceptibility pattern and vancomycin resistance of each isolate were evaluated by disk diffusion method and micro-dilution method, respectively. 9 (2.3%) strains required MIC > 2 mg/l of vancomycin, which significantly increased among multi drug resistant (MDR), MRSA and SCCmec type III strains (p < 0.05). 171 (89%), 140 (72.91%), 7 (3.6), 78 (48.6%), 5 (2.6%), 151 (78.64%), 129 (67.18%), 178 (92.7%) and 15 (7.8%) of 192 isolates harbored mecA, entA, entB, entC, entD, entE, eta, etb and tsst-1 genes, respectively. 31 (16.14%), 5 (2.6%), 95 (49.48%) and 7 (3.64%) of 192 isolates carried SCCmec type I, II, III and IV, respectively. We found a significantly higher rate of MRSA and resistance to all tested antibiotics, except to penicillin G, kanamycin and linezolide among the SCCmec type III class (p < 0.05). According to our findings, MSSA isolates should be taken as seriously as MRSA strains due to the potential presence of broad spectrum virulence factor genes.

PCR investigation of Panton-Valentine leukocidin, enterotoxin, exfoliative toxin, and agr genes in Staphylococcus aureus strains isolated from psoriasis patients.

BACKGROUND/AIM: Staphylococcus aureus colonization is a determiner of disease activation in psoriasis patients. Here we evaluate the presence of genes encoding Panton-Valentine leukocidin (PVL), enterotoxins, TSST-1, exfoliative toxins, and the accessory gene regulatory locus by polymerase chain reaction (PCR) in S. aureus isolates obtained from healthy and diseased skin regions and anterior nares of psoriasis patients and healthy controls. MATERIALS AND METHODS: The presence of PVL and toxin genes was investigated, and agr typing was performed by PCR. RESULTS: Eighteen of the isolated strains carried the sei, 1 carried the seb-sec, and 1 carried the seg enterotoxin gene. Eight of the strains carrying enterotoxin genes were isolated from nasal swabs, 6 from diseased skin swabs, and 4 from healthy skin swabs. None of the strains isolated from the control group carried the agr locus. On the other hand, 11 of the S. aureus strains isolated from the patients carried type 1, 7 carried type 1 + 3, 4 carried type 2, 4 carried type 3, and 1 carried type 1 + 2 agr loci. CONCLUSION: Enterotoxin production and the carried accessory gene regulatory locus may be important in the aggravation of psoriasis.

Prevalence of exfoliative toxin A and B genes in Staphylococcus aureus isolated from clinical specimens.

BACKGROUND: The pathogenicity of Staphylococcus aureus is based on the production of various virulence factors. The frequency of these factors can markedly differ according to the geographical region. MATERIALS AND METHODS: In this study, we investigated the prevalence of two frequent isoforms of exfoliative toxins and mecA genes using PCR in 197 clinical isolates obtained from clinical samples during the years 2011 and 2012. The samples were obtained from an educational hospital in northern Tehran, Iran. In addition, the antibiotic susceptibility pattern was studied for each isolate using the disc diffusion method. RESULTS: In this study, 186 (94.4%), 15 (7.6%) and 172 (86.3%) of the 197 isolates expressed the eta, etb and mecA genes, respectively. In addition, 164 (88.2%) and 12 (80%) strains, which harbored the eta and etb genes, respectively, were MRSA (methicillin resistant S. aureus). Furthermore, the prevalence of the mecA, eta and etb genes was higher among the wound samples (61.2%, 55.8% and 6.09%, respectively). We observed high rates of MDR (multi drug resistance) among our isolates. A significant correlation was detected between the presence of the mecA gene and the resistance to oxacillin, gentamicin, kanamycin, erythromycin, tetracycline, cotrimoxazole, clindamycin, and cephazolin as well as the multi-drug resistant property (P<0.05). In addition to penicillin, the MDR properties and resistances to the tested antibiotics in the etb-positive strains were significantly lower compared to the etb-negative strains (P<0.05). CONCLUSIONS: The prevalence of the eta, etb and mecA genes might be due to the specific geographic region.

Detection and genetic characterization of PVL-positive ST8-MRSA-IVa and exfoliative toxin D-positive European CA-MRSA-Like ST1931 (CC80) MRSA-IVa strains in Bangladesh.

Severe skin lesions caused by Staphylococcus aureus infection are associated with production from bacterial cells of Panton-Valentine leukocidin (PVL), a typical virulence factor of community-acquired methicillin-resistant S. aureus (CA-MRSA), as well as other toxins represented by exfoliative toxins. Through a retrospective study of 26 S. aureus strains isolated from skin lesions of diabetic patients admitted to a hospital in Bangladesh, 2 PVL-gene-positive MRSA-IVa strains and 8 PVL-negative, exfoliative toxin D (ETD) gene (etd)-positive MRSA-IVa strains were isolated. A PVL-positive MRSA-IVa strain had a type I arginine catabolic mobile element (ACME), belonged to ST8/agr-type I/spa-type t121 (a variant of t008), and harbored blaZ, tet(K), msrA, and aph(3')-IIIa, which are mostly typical characteristics found in USA300, a predominant CA-MRSA clone in the United States. Another PVL-positive MRSA strain, belonging to ST1929 (CC88)/agr-type III/spa-type t3341, was negative for ACME, but possessed blaZ and tet(K). The etd-positive MRSA-IVa strains possessed the epidermal cell differentiation inhibitor B (EDIN-B)-encoding gene (edinB) and belonged to ST1931 (CC80)/agr-type III/spa-type t11023 (a variant of t044), which was genetic trait similar to that of the European CA-MRSA ST80 clone. However, unlike the European ST80 strains, the etd-positive MRSA strains detected in the present study harbored seb, sek, and seq, while they were negative for tet(K), aph(3')-IIIa, and fusB, showing susceptibility to fusidic acid. These findings suggested that etd-positive ST1931 MRSA strains belong to the same lineage as the European ST80 MRSA clone, evolving from a common ancestral clone via acquisition of a different pathogenicity island. This is the first report of a USA300-like MRSA-IV strain, PVL-positive ST1929 (CC88) MRSA-IV, and European ST80 CA-MRSA-like etd-positive ST1931 (CC80) MRSA-IV strains isolated in Bangladesh.

Detection of genes for superantigen toxins in methicillin-resistant Staphylococcus aureus clinical isolates in Karachi.

OBJECTIVE: To detect genes for enterotoxins, exfoliative and toxic shock syndrome toxins in Staphylococcus aureus (S. aureus) strains isolated from clinical specimens. STUDY DESIGN: Cross-sectional observational study. PLACE AND DURATION OF STUDY: Department of Molecular Genetics, Dr. Ziauddin Hospital, Karachi, from January to December 2010. METHODOLOGY: Two hundred and ninety eight S. aureus clinical isolates were obtained from various clinical samples received at Dr. Ziauddin Hospital, Karachi. Out of these, 115 were detected as methicillin resistant (MRSA) by cefoxitin disk diffusion test showing a prevalence rate of 38.6%. Detection of individual toxin genes was performed by Polymerase Chain Reaction (PCR) by using only one primer pair for each tube. Uniplex primers were preferred as multiplex primers are longer in base pairs and have the potential for cross reaction due to non-specific binding and increase in optimization time. RESULTS: The possession of a single gene or more than a single gene in MRSA isolates was found in 61.73% of clinical samples; the highest number was found in pus swab, followed by sputum, blood, urethral swab, and urine. The prevalence of toxin genes was higher in MRSA as compared to methicillin sensitive (MSSA) isolates (19.12%). CONCLUSION: PCR detects strains possessing toxin genes independent of their expression. The possession of genes for super-antigens seems to be a frequent and habitual trait of S. aureus more so in MRSA.

Emergence of Staphylococcus aureus carrying multiple drug resistance genes on a plasmid encoding exfoliative toxin B.

We report the complete nucleotide sequence and analysis of pETBTY825, a Staphylococcus aureus TY825 plasmid encoding exfoliative toxin B (ETB). S. aureus TY825 is a clinical isolate obtained from an impetigo patient in 2002. The size of pETBTY825, 60.6 kbp, was unexpectedly larger than that of the archetype pETBTY4 (∼30 kbp). Genomic comparison of the plasmids shows that pETBTY825 has the archetype pETBTY4 as the backbone and has a single large extra DNA region of 22.4 kbp. The extra DNA region contains genes for resistance to aminoglycoside [aac(6')/aph(2″)], macrolide (msrA), and penicillin (blaZ). A plasmid deletion experiment indicated that these three resistance elements were functionally active. We retrospectively examined the resistance profile of the clinical ETB-producing S. aureus strains isolated in 1977 to 2007 using a MIC determination with gentamicin (GM), arbekacin (ABK), and erythromycin (EM) and by PCR analyses for aac(6')/aph(2″) and msrA using purified plasmid preparations. The ETB-producing S. aureus strains began to display high resistance to GM, which was parallel with the detection of aac(6')/aph(2″) and mecA, after 1990. Conversely, there was no significant change in the ABK MIC during the testing period, although it had a tendency to slightly increase. After 2001, isolates resistant to EM significantly increased; however, msrA was hardly detected in ETB-producing S. aureus strains, and only five isolates were positive for both aac(6')/aph(2″) and msrA. In this study, we report the emergence of a fusion plasmid carrying the toxin gene etb and drug resistance genes. Prevalence of the pETBTY825 carrier may further increase the clinical threat, since ETB-producing S. aureus is closely related to more severe impetigo or staphylococcal scalded-skin syndrome (SSSS), which requires a general antimicrobial treatment.

Characterization of colonizing Staphylococcus aureus isolated from surgical wards' patients in a Nigerian university hospital.

In contrast to developed countries, only limited data on the prevalence, resistance and clonal structure of Staphylococcus aureus are available for African countries. Since S. aureus carriage is a risk factor for postoperative wound infection, patients who had been hospitalized in surgical wards in a Nigerian University Teaching Hospital were screened for S. aureus carriage. All S. aureus isolates were genotyped (spa, agr) and assigned to multilocus sequence types (MLST). Species affiliation, methicillin-resistance, and the possession of pyrogenic toxin superantigens (PTSAg), exfoliative toxins (ETs) and Panton-Valentine Leukocidin (PVL) were analyzed. Of 192 patients screened, the S. aureus carrier rate was 31.8 % (n = 61). Of these isolates, 7 (11.5%) were methicillin-resistant (MRSA). The isolates comprised 24 spa types. The most frequent spa types were t064, t084, t311, and t1931, while the most prevalent MLST clonal complexes were CC5 and CC15. The most frequent PTSAg genes detected were seg/sei (41.0%) followed by seb (29.5%), sea (19.7%), seh (14.7%) and sec (11.5). The difference between the possession of classical and newly described PTSAg genes was not significant (63.9% versus 59.0% respectively; P = 0.602). PVL encoding genes were found in 39.3% isolates. All MRSA isolates were PVL negative, SCCmec types I and VI in MLST CC 5 and CC 30, respectively. Typing of the accessory gene regulator (agr) showed the following distribution: agr group 1 (n = 20), group II (n = 17), group III (n = 14) and group IV (n = 10). Compared to European data, enterotoxin gene seb and PVL-encoding genes were more prevalent in Nigerian methicillin-susceptible S. aureus isolates, which may therefore act as potential reservoir for PVL and PTSAg genes.