Molecular characterisation of Staphylococcus aureus in school-age children in Guangzhou: associations among agr types, virulence genes, sequence types, and antibiotic resistant phenotypes.

BACKGROUND: Staphylococcus aureus, one of the most prevalent opportunistic pathogens, mainly colonizes the nasal cavity and is a risk factor for severe infections. Virulence factors and accessory gene regulator (agr) are key to the severity and diversity of staphylococcal infection. In this study, we aimed to characterise S. aureus agr-types and virulence genes and correlated them with genetic background and antibiotic-resistant phenotypes. RESULTS: Agr types were identified in 704 isolates (98.5%), with only 11 isolates were negative for agr type. Most of our isolates were classified as agr type I, followed by types III, II and IV. The enterotoxin c gene (sec) was detected in 48.6% of isolates, showing the highest prevalence among the five enterotoxin genes detected. The positivity rates for the lukS/F-PV and tsst genes were 4% and 2.2%, respectively, while neither sed nor SasX were detected. ST45, ST59, ST338, ST188, ST6, ST7, ST22, ST25, ST398, and ST944 belonged to agr I group, while ST5 and ST15 belonged to agr II group. ST30 and ST1 were classified into agr III group, and ST121 was assigned into agr IV group. The tsst gene was found exclusively within agr I and III types belonging to ST7 and ST30 isolates, while the lukS/F-PV was predominantly carried by agr I type isolates primarily within CC59 and CC22 clones. Among the methicillin-resistant S. aureus (MRSA) isolates, 89.7% belonged to agr I group, and 97.8% of rifampicin-resistant or intermediate isolates were assigned to agr I group. MRSA isolates harboured more tested virulence genes compared to methicillin-susceptible S. aureus isolates. CONCLUSIONS: We characterized the distributions of agr types and eight major virulence genes of 715 S. aureus isolates, and our findings revealed clear associations between agr types and STs, as well as virulence genes, and drug resistant phenotypes.

Methicillin-Resistant Staphylococcusaureus Strains Isolated from Burned Patients in a Tunisian Hospital: Molecular Typing, Virulence Genes, and Antimicrobial Resistance.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major causes of a variety of infections in hospitals and the community. Their spread poses a serious public health problem worldwide. Nevertheless, in Tunisia and other African countries, very little molecular typing data on MRSA strains is currently available. In our study, a total of 64 MRSA isolates were isolated from clinical samples collected from burned patients hospitalized in the Traumatology and Burns Center of Ben Arous in Tunisia. The identification of the collection was based on conventional methods (phenotypic and molecular characterization). The characterization of the genetic support for methicillin resistance was performed by amplification of the mecA gene by polymerase chain reaction (PCR), which revealed that 78.12% of S. aureus harbors the gene. The resistance of all the collection to different antibiotic families was studied. Indeed, the analysis of strain antibiotic susceptibility confirmed their multi-resistant phenotype, with high resistance to ciprofloxacin, gentamicin, penicillin, erythromycin, and tetracycline. The resistance to the last three antibiotics was conferred by the blaZ gene (73.43%), the erm(C) gene (1.56%), the msr(A) gene (6.25%), and tet(M) gene (7.81%), respectively. The clonal diversity of these strains was studied by molecular typing of the accessory gene regulator (agr) system, characterization of the SCCmec type, and spa-typing. The results revealed the prevalence of agr types II and III groups, the SCCmec type III and II cassettes, and the dominance of spa type t233. The characterization of the eight enterotoxins genes, the Panton-Valentine leukocidin and the toxic shock syndrome toxin, was determined by PCR. The percentage of virulence genes detected was for enterotoxins (55%), tst (71.88%), leukocidin E/D (79.69%), and pvl (1.56%) factors. Furthermore, our results revealed that the majority of the strains harbor IEC complex genes (94%) with different types. Our findings highlighted the emergence of MRSA strains with a wide variety of toxins, leukocidin associated with resistance genes, and specific genetic determinants, which could constitute a risk of their spread in hospitals and the environment and complicate infection treatment.

Molecular surveillance of methicillin-susceptible Staphylococcus aureus (MSSA) isolated over a one-year period from a Malaysian Teaching Hospital.

INTRODUCTION: We report the results of a molecular surveillance study carried out on methicillin-susceptible Staphylococcus aureus (MSSA) isolated in a one-year duration from Hospital Canselor Tuanku Muhriz (HCTM), a tertiary hospital located in Kuala Lumpur, Malaysia. METHODS: The first strain isolated from each MSSA infection in HCTM during the year 2009 was included into the study. Antimicrobial susceptibility testing and agr group typing were carried out for all strains; virulence gene (cna, seh, TSST-1 and PVL) typing results of the strains were obtained from a previous study. Pulsed-field gel electrophoresis (PFGE) was done on selected strains from the orthopedic ward. Relationship(s) between different typing methods used in the study was investigated, where a p value of <0.05 indicated significant association between typing methods. RESULTS: A total of 880 MSSA strains were included into the study. The strains were generally susceptible to most antibiotics, with most of them carrying cna and agr-I (51.6%, n=454; 39.8%, n=350, respectively). A total of 17 PFGE pulsotypes were identified using an 80% similarity cut-off value, where the main pulsotype (pulsotype E) consisted of 24 isolates (23.5%). agr-III strains were found to be usually positive for both cna and seh (p<0.05). In addition, some PFGE pulsotypes were also characteristic of certain virulence genes or agr groups. CONCLUSIONS: We did not identify a dominant MSSA clone circulating in HCTM in 2009. Nevertheless, results from this molecular surveillance will provide good baseline data for the hospital's second S. aureus surveillance planned for the year 2020.

Association Between agr Type, Virulence Factors, Biofilm Formation and Antibiotic Resistance of Staphylococcus aureus Isolates From Pork Production.

Livestock-associated Staphylococcus aureus colonization and/or infections exist in pigs and people in frequent contact with pigs. In this study, a total of 130 S. aureus isolates obtained from different stages of pork production were subjected to antimicrobial susceptibility, biofilm formation, as well as PCR screening to identify virulence genes, and the accessory gene regulator alleles (agr). Among all 130 S. aureus isolates, 109 (83.8%, 109/130) isolates were positive for agr. All swine farms isolates belonged to agr IV, whereas S. aureus isolated from slaughterhouse and retail indicated diverse agr types. All isolates exhibited biofilm formation ability, and raw meat isolates (belonging to agr I) exhibited a greater ability to form strong biofilms than swine farms isolates (belonging to agr IV). agr-positive isolates were associated with more virulence genes than agr-negative isolates. Most biofilm-producing isolates were positive for microbial surface component recognizing adhesive matrix molecule (MSCRAMM), capsule type and ica group genes. The results illustrate a significant association between the prevalence rate of MSCRAMM, capsule type and ica group genes among isolates producing weak, moderate and strong biofilms. The high prevalence of resistance to ciprofloxacin, gentamicin, tetracycline, clarithromycin, clindamycin, and trimethoprim-sulfamethoxazole were mainly observed in moderate and weak biofilm producers. Our findings indicate that S. aureus isolates from pork production displayed diverse molecular ecology.

Genotypic characterization of Staphylococcus aureus isolated from a burn centre by using agr, spa and SCCmec typing methods.

Infections caused by Staphylococcus aureus remain a major global healthcare problem. We aimed to find the common lineages of S. aureus strains circulating in a burn hospital in Tehran. A total of 167 isolates of S. aureus obtained from patients, healthcare workers (HCWs) and environment in Shahid Motahari burn hospital were genotyped by using spa, agr and staphylococcal cassette chromosome mec (SCCmec) typing methods. Antimicrobial susceptibility testing was performed by using the disc diffusion method. The frequency of methicillin-resistant S. aureus (MRSA) was 64.7% (n = 108), with distribution frequencies among patient, HCW and surface isolates of 64.2% (n = 79), 50% (n = 7) and 73.3% (n = 22), respectively. SCCmec type III (75%, n = 81) was found to be the most frequent SCCmec type among MRSA isolates, followed by SCCmec type I (20.4%, n = 22) and SCCmec type IV (1.8%, n = 2). The remaining MRSA isolates (2.8%, n = 3) were nontypeable by this method. About 78.4% (n = 131), 10.2% (n = 17) and 4.8% (n = 8) of all isolates were characterized as agr types I, II and III, respectively, and the other isolates (6.6%) were nontypeable. spa types t030 and t037 constituted the first and second most predominant spa types found in 56.4% (n = 57) and 25.6% (n = 26) of isolates, respectively. We also report here a novel spa type, t16471. The most prevalent genotypes of the isolates found among patient, surface and HCW samples were SCCmec type III/t030, t037/agr type I. Continuous tracking of epidemic isolates and better hospital infection control policies are recommended to efficiently prevent the spread of bacteria to inpatients.

Phenotypic and Molecular Characterization of Methicillin-Resistant Staphylococcus aureus Clones Carrying the Panton-Valentine Leukocidin Genes Disseminating in Iranian Hospitals.

The spread of Panton-Valentine leukocidin (PVL)-carrying Staphylococcus aureus strains in both hospital and the community is a significant worldwide problem. The aim of the study was to investigate the clonal dissemination pattern of PVL-producing S. aureus strains isolated from hospitalized patients in Tehran, Iran. In this cross-sectional study, 70 PVL-carrying S. aureus strains were recovered from 240 clinical specimens and characterized by antibiotic susceptibility testing, agr typing, SCCmec typing, spa typing, multilocus sequence typing, and virulence and adhesion gene profiling. All the PVL-carrying S. aureus strains were confirmed as methicillin-resistant S. aureus (MRSA) and recovered from wounds (48.6%), blood (25.7%), exudate/pus (11.4%), sputum (8.6%), and body fluid (5.7%) samples. Among the 70 PVL-carrying S. aureus strains tested, 38 (54.3%) were positive for ant (4')-Ia gene, 27 (38.6%) for aac (6')-Ie/aph (2″), 13 (18.6%) for msr(A), 13 (18.6%) for erm(C), 13 (18.6%) for tet(M), 11 (15.7%) for erm(A), 10(14.3%) for msr(B), 9 (12.9%) for aph (3')-IIIa, 5 (7.1%) for mupA, and 2 (2.9%) for erm(B) genes. Five clonal complexes (CC) and nine different clones were detected in this study. The most frequent CC was CC22 (ST22) (42.8%) followed by CC30 (ST30) (21.5%), CC8 (ST8) (17.2%), CC1 (ST772) (11.4%), and CC80 (ST80) (7.1%). In this study, ST22-SCCmec IV/t852 was the predominant PVL-positive MRSA clone (20%), followed by ST8-SCCmec IV/t008 (17.2%), ST30-SCCmec IV/t019 (12.9%), ST22-SCCmec IV/t790 (11.4%), ST22-SCCmec IV/t005 (11.4%), ST30-SCCmec IV/t021 (8.6%), ST80-SCCmec IV/t044 (7.1%), ST772-SCCmec V/t657 (7.1%), and ST772-SCCmec V/t10795 (4.3%). Diversity in clonal types of PVL-carrying MRSA strains in our study supports the need to perform a systematic surveillance of PVL-positive MRSA strains.

Survey of potential factors involved in the low frequency of CP5 and CP8 expression in Staphylococcus aureus isolates from mastitis of dairy cattle from Argentina, Chile, and Uruguay.

Staphylococcus aureus produces capsular polysaccharides (CPs) both in vivo and under defined culture conditions being serotypes 5 and 8 the most prevalent. S. aureus isolates that fail to produce CP5 or CP8 are defined as non-typeable (NT). Loss of capsule expression, however, may lead to S. aureus persistence in a chronically infected host. The prevalence of NT strains of S. aureus isolated from bovine mastitis varies according to the geographic origin of the strain. The aims of this work were to detect phenotypically and genotypically the capsular profile of 144 S. aureus isolated from bovine mastitis in Argentina, Chile, and Uruguay and explore the factors that are considered to be associated with capsule expression as presence of IS257, IScap, and agr typing of non-related collection. The detection of the IS257, IScap, cap genes, and agr typing was performed using PCR. The detection and quantification of capsular polysaccharide production were performed by ELISA assays. We found that 96% of the S. aureus isolates investigated carried cap5(8) genes but over 75% of strains do not express capsule in the three countries studied. However, only 6 isolates from Argentina carried the IScap element that totally suppressed the expression of the capsule, suggesting that other factors could influence on CP expression. Moreover, the agrI/NT association was statistically significant suggesting that this profile is a phenomenon observed not only in other parts of the world but also in our region.

Virulence characteristics and molecular relatedness of methicillin resistant Staphylococcus aureus harboring different staphylococcal cassette chromosome mec.

INTRODUCTION: Methicillin resistant Staphylococcus aureus (MRSA) is a versatile pathogen capable of causing multitude of human diseases. It is one of the most important nosocomial pathogen that implicated in community and healthcare associated infections. Therefore, this study aims to characterize different SCCmec elements found in MRSA isolates. Moreover, molecular typing was performed to investigate the genetic relatedness among MRSA isolates. METHODS: Phenotypic identification of MRSA was done by disc diffusion method. The MRSA isolates were typed based on the SCCmec, coa and agr genes. Phenotypic characterization included the detection of biofilm, lipase, protease, lecithinase, staphylokinase and hemagglutination. Also, hla, hlb, hlg, hld, tsst-1, psm-mec and mecI genes were detected genotypically. The correlation between the molecular types identified and the profile of virulence factors, clinical and geographical sources was determined for all isolates. RESULTS: Eighty five isolates were identified as MRSA. Eight types of SCCmec elements were detected among these isolates. Type V was the most observed type (56.47%). Regarding the correlation between SCCmec types and virulence factors, type V SCCmec exhibited a significant association with biofilm (p < 0.0001), staphylokinase (p = 0.0495) and tsst-1 (p = 0.0498). Molecular typing of coa gave an insight to the presence of specific types in specific hospital wards. Based on agr typing, agr I was the highest prevalent type in MRSA isolates (54.11%). CONCLUSION: There is an increase of MRSA infections particularly the community acquired with high variability in the distribution of virulence factors among different SCCmec types. The association between type III and V SCCmec with certain hospitals may be an evidence of nosocomial infection among these hospitals.

Characterization of virulence and antibiotic profile and agr typing of Staphylococcus aureus from milk of subclinical mastitis bovine in State of Rio de Janeiro / Caracterização do perfil de virulência e resistência antimicrobiana e tipagem agr de Staphylococcus aureus oriundos do leite bovino de mastite subclinica no estado do Rio de Janeiro

This study aims to detect the main virulence and antimicrobial resistance genes in Stapylococcus aureus from bovine mastitic milk as well as classifying them according to agr typing. A total of 55 strains from six dairy unities in the state of Rio de Janeiro were selected, of these 27.3% presented fbnA and 78,2% for fbnB genes, respectively. None of the strains tested were positive for cap5 gene, 3.6% were positive for cap8 gene. Additionally, 94.5% of strains had hlA gene and 89.1% had hlB gene while 67.3% of the strains had icaA gene and 87.3% had icaD gene. From these results it was possible to establish 12 different virulence profiles. Prevalence of agrII type was detected in 81.8% of the isolates. Concerning antimicrobial resistance evaluation, the studied strains were susceptible to all antibiotics tested except penicillin, 83.6% being resistant strains. None of the strains had mecA gene, however, 40% of the strains had blaZ gene. Associating virulence and resistance data made it possible to obtain 23 different profiles. This great diversity of strains shows wide array of bacterial strategies and the challenge of mastitis prevention in cattle. Despite antimicrobial susceptibility, these strains presented certain genes that allow its persistence in the herd.(AU)

O presente estudo teve como objetivo detectar os principais genes de virulência e resistência antimicrobiana em Staphylococcus aureus oriundos de leite bovino mastítico e classificá-los de acordo com a tipagem do gene agr. Foram selecionados 55 isolados de seis unidades produtores no estado do Rio de Janeiro. Destas, o gene fbnA foi encontrado em 27,3% das cepas e 78,2% possuíam o gene fbnB. Em nenhuma cepa foi encontrado o gene cap5 e 3,6% possuíam o gene cap8. O gene hlA foi encontrado em 94,5% das cepas e 89,1% possuíam o gene hlB. O gene icaA foi encontrado em 67,3% das cepas e 87,3% possuíam o gene icaD. Com base nesses resultados, foi possível estabelecer 12 diferentes perfis de virulência. Prevalência do agr tipo II foi detectada em 81,8% dos isolados. Considerando-se a avaliação da resistência antimicrobiana, as cepas estudadas foram suscetíveis a todos os antibióticos exceto penicilina, sendo detectado um percentual de 83,6% de cepas resistentes. Nenhuma das cepas apresentou o gene mecA, contudo 40% das cepas apresentaram o gene blaZ. Vinte e três perfis diferentes foram estabelecidos por associação de dados de virulência e resistência. Essa grande diversidade de cepas mostra a ampla gama de estratégias bacterianas e o desafio da prevenção à mastite no gado bovino, considerando-se que, a despeito da suscetibilidade antimicrobiana, essas cepas apresentam genes que permitem sua persistência no rebanho.(AU)

Molecular characteristics of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from patients with bacteremia based on MLST, SCCmec, spa, and agr locus types analysis.

The widespread emergence of methicillin resistant Staphylococcus aureus, as a common cause of nosocomial infections, is becoming a serious concern in global public health. The objective of the present study was to investigate antimicrobial susceptibility pattern, frequency of virulence genes and molecular characteristics of methicillin-resistant Staphylococcus aureus strains isolated from patients with bacteremia. A total of 128 methicillin-resistant Staphylococcus aureus isolates were collected during February 2015 to January 2016. In vitro antimicrobial susceptibility of the isolates was assessed using the disk diffusion method. Conventional PCR was performed for the detection of adhesion (can, bbp, ebp, fnbB, fnbA, clfB, clfA) and toxin (etb, eta, pvl, tst) encoding genes, determining the agr type, SCCmec, MLST and spa typing of the isolates. All the methicillin-resistant Staphylococcus aureus isolates were found to be sensitive to linezolid, teicoplanin, and vancomycin. Resistance to the tested antibiotics varied from 97.7% for penicillin to 24.2% for mupirocin. The rate of multi drug resistance (MDR) in the present study was 97.7%. The most commonly detected toxin and adhesion genes were tst (58.6%), and clfB (100%), respectively. The majority of SCCmec III isolates were found in agr group I while SCCmec IV and II isolates were distributed among agr group III. Multilocus Sequence Typing (MLST) of the MRSA isolates showed five different sequence types: ST239 (43%), ST22 (39.8%), ST585 (10.9%), ST45 (3.9%) and ST240 (2.3%). All of the pvl positive strains belonged to ST22-SCCmec IV/t790 clone and were MDR. Among different 7 spa types, the most common were t790 (27.3%), t037 (21.9%), and t030 (14.1%). spa types t016, t924 and spa type t383 were reported for the first time from Asia and Iran, respectively. It was shown that spa types circulating in the studied hospitals varied which support the need to perform future surveillance studies in order to understand methicillin-resistant Staphylococcus aureus distribution and thus more effective infection control.

Detection of biofilm related genes, classical enterotoxin genes and agr typing among Staphylococcus aureus isolated from bovine with subclinical mastitis in southwest of Iran.

Staphylococcus aureus by producing biofilm and facilitating chronic infection is a common cause of mastitis in cows and thereby can cause food poisoning by production of enterotoxins in milk. The agr typing method is an important tool for epidemiological investigation about S. aureus. The aims of the present study were to detect biofilm related genes, 5 classical enterotoxin genes and the agr types among S. aureus isolates. The ability of S. aureus isolates to produce biofilm was evaluated by modified CRA plate. Six biofilm related adhesion genes (icaD, icaA, fnbA, bap, clfA and cna), five classical enterotoxin genes (sea, seb, sec, sed and see) and tst-1 gene were detected by PCR methods. Multiplex-PCR was used to determination of the agr groups. 55 out of 80(68.8%) S. aureus isolates were biofilm producer. The icaD gene was detected in 70 (87.5%) of isolates. The prevalence rates of fnbA, icaA, clfA, cna and bap were 72.5, 56.25, 50, 22.5, and 5% respectively. The agr group I and III were detected in 57.5% 25% of studied isolates. The sea, sed and tst-1 genes were found in 10%, 7.5% and 1.25% of isolates respectively. The majority of S. aureus were able to produce biofilm. Significant associations were observed between presence of the icaD, icaA, fnbA, clfA and the cna genes as well as biofilm formation. The present study revealed that isolates with the agr type III are more potent for biofilm production. Our data supported a possible link between the agr types and certain SE genes.

Prevalence and molecular proifle of theStaphylococcus aureus strains harboring tstand/or pvl genes / 中国感染与化疗杂志

Objective To investigate the prevalence, accessory gene regulator (agr) and staphylococcal cassette chromosome mec (SCCmec, only for methicillin resistantS. aureus, MRSA) types of theS. aureus strains carrying toxic shock syndrome toxin-1 (tst) and/or panton-valentine leukocidin (pvl) genes.Methods Nine hundred and sixteen isolates ofS. aureus were collected from seven hospitals in Shanghai and Zhejiang Province and subjected to detection oftst,pvl,mecA andmecC genes by polymerase chain reaction (PCR). Theagr and SCCmec (only for MRSA) types were determined in thetst orpvl gene positive isolates.Results Of the 916 isolates, 208 carriedtst gene (22.7%), 35 harboredpvl gene (3.8%), and 665 weremecA positive (MRSA). No isolate was mecC positive. Out of the 665 MRSA isolates, 198 hosted thetst gene (29.8%). The most commonagr and SCCmec types were agr 2 (97.0%) and SCCmec II (94.4%), respectively. For thepvl gene, only 14 isolates were positive (2.1%). Theagr 1 (85.7%), SCCmecIII (42.9%) and SCCmec IVa (28.6%) were the most commonagr type and SCCmec type. In the 251 methicillin-sensitiveS. aureus (MSSA) isolates, 10 carriedtst gene (4.0%) and 21 carriedpvl gene (8.4%). The prevalence oftst gene in MRSA was higher than that in MSSA, while the prevalence ofpvlgene was just the opposite. However, the prevalence ofpvlgene in MRSA isolates from Zhejiang Province was higher than that in the MRSA isolates from Shanghai (P severeS. aureus infections.

Genetic diversity and virulence potential of Staphylococcus aureus isolates from raw and processed food commodities in Shanghai.

The risk of zoonotic transmission to humans highlights the need to understand the molecular ecology of Staphylococcus aureus in foods. In this study, 142 S. aureus isolates obtained from various raw and processed foods from Shanghai, China were characterized to determine their genetic diversity and virulence gene content. A total of 16 clonal complexes (CCs), 34 staphylococcal protein A (spa) types, and 6 accessory gene regulator (agr) allelic groups were identified and analyzed among the 142 S. aureus isolates. Among these, the genotype CC188-t189-agr Ι was the most prevalent, constituting 28.2% of all isolates. The presence of virulence genes encoding 20 staphylococcal enterotoxins (se), toxic shock syndrome toxin (tsst1), exfoliative toxins (eta, etb, and etd), Panton-Valentine leukocidin (lukS-PV and lukF-PV), as well as methicillin resistance gene (mecA), was determined by PCR. Of these S. aureus isolates, 72.5% harbored toxin genes, in which the most frequent toxin gene was sep (43.7%), followed by sej (26.1%) and pvl (21.1%). In contrast, see, ses, set, tsst1, etb, and etd were not found in any of the isolates tested. Eight S. aureus isolates (5.6%, 8/142), seven from raw milk and one from frozen food, were mecA positive and resistant to oxacillin, thus were MRSA. The 142 S. aureus isolates displayed 52 different toxin gene profiles. Although no direct association was found between toxin gene profile and the S. aureus genotype, the isolates belonging to CC5, CC9, CC20, CC50, and CC72 clonal lineages in general carried more toxin genes (>5) compared with the isolates in other CCs. It was also revealed that raw milk and raw meat were the major sources of isolates containing multiple toxin genes. S. aureus isolates from food that were genetically highly related, displayed diverse toxin gene profiles, implying the significant role of horizontal gene transfer in the emergence of highly toxigenic S. aureus isolates.