Molecular Evolutionary Pathways toward Two Successful Community-Associated but Multidrug-Resistant ST59 Methicillin-Resistant Staphylococcus aureus Lineages in Taiwan: Dynamic Modes of Mobile Genetic Element Salvages.
ABSTRACT: Clonal complex 59 (CC59) Staphylococcus aureus in Taiwan includes both methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA). As the most prominent community-associated MRSA (CA-MRSA) in Taiwan, CC59 has two major clones characterized as PVL-negative SCCmec IV (carrying the staphylococcal cassette chromosome mec IV but Panton-Valentine leukocidin-negative) and PVL-positive SCCmec V (5C2&5). We investigated the drug resistance, phylogeny and the distribution and sequence variation of SCCmec, staphylococcal bacteriophage ?SA3, genomic island ?Sa? and MES (an enterococcal mobile genetic element conferring multidrug resistance) in 195 CC59 S. aureus. Sequencing and PCR mapping revealed that all of the CC59/SCCmec V (5C2&5) MRSA strains had acquired MESPM1 or its segregants, and obtained a ?SA3-related fragment in ?Sa?. In contrast, MES6272-2 and MES4578, which showed gentamicin resistance that was not encoded by MESPM1, were dominant in SCCmec IVg MRSA. Translocation of a whole ?SA3 into ?Sa? instead of only a ?SA3-related fragment was common in SCCmec IVg MRSA. However, the non-subtype-g SCCmec IV MRSA (SCCmec IVa is the major) still carried MES and ?Sa? structures similar to those in SCCmec V (5C2&5) MRSA. A minimum spanning tree constructed by multiple-locus variable-number tandem repeat analysis revealed that SCCmec IVg MRSA and SCCmec V (5C2&5) MRSA grouped respectively in two major clades. The CC59 MSSA was equally distributed among the two clades, while the non-subtype-g SCCmec IV MRSA mostly clustered with SCCmec V (5C2&5) MRSA. Our findings strongly suggest that CC59 MSSA acquired divergent mobile genetic elements and evolved to SCCmec IVg MRSA and SCCmec V (5C2&5) MRSA/non-subtype-g SCCmec IV MRSA independently. The evolutionary history of CC59 S. aureus explains how mobile genetic elements increase the antimicrobial resistance and virulence and contribute to the success of CA-MRSA in Taiwan.
Project description:Clonal complex 59 (CC59) community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains were characterized using pulsed-field gel electrophoresis, spa typing, multilocus sequence typing, diagnostic DNA microarrays, and PCRs targeting staphylococcal cassette chromosome mec (SCCmec) elements and Panton-Valentine leukocidin (PVL). Six distinct groups within CC59 were characterized. At least seven different variants of SCCmec elements were identified (IVa [2B], IVb [2B], IVd [2B], IV variant [2B], IVa [2B&5], V variant [5C2], and V [5C2&5]). (The structural type is indicated by a Roman numeral, with a lowercase letter indicating the subtype, and the ccr complex and the mec complex are indicated by an Arabic numeral and an uppercase letter, respectively. Where there is an extra ccr element, this is indicated by "&" and an Arabic numeral designating the ccr type.) The first group is similar to the American sequence type 59 (ST59) MRSA-IV CA-MRSA strain USA1000. The second group includes a PVL-negative ST87 strain with an SCCmec element of subtype IVb (2B). The third group comprises PVL-variable ST59 MRSA-IV strains harboring multiple SCCmec IV subtypes. PVL-negative ST59 MRSA strains with multiple or composite SCCmec elements (IVa [2B&5]) form the fourth group. Group 5 corresponds to the internationally known "Taiwan clone," a PVL-positive strain with a variant SCCmec element (V [5C2&5]). This strain proved to be the most common CC59 MRSA strain isolated in Western Australia. Finally, group 6 encompasses the ST59 MRSA-V variant (5C2). The differentiation of CC59 into groups and strains indicates a rapid evolution and spread of SCCmec elements. Observed differences between groups of strains as well as intrastrain variability within a group facilitate the tracing of their spread.
Project description:Various types of the staphylococcal cassette chromosome mec (SCCmec) are known to confer methicillin resistance on the human pathogen Staphylococcus aureus. Such cassettes are not always stably maintained. The present studies were aimed at identifying the mechanism underlying the in vivo conversion of methicillin-resistant S. aureus (MRSA) to methicillin-susceptible S. aureus (MSSA) derivatives as encountered in two patients suffering from pneumonia and an umbilicus infection, respectively. All MRSA and MSSA isolates identified belong to multilocus sequence type (MLST) 398, have spa type t034, and are Panton-Valentine leukocidin positive. Sequencing of 27,616 nucleotides from the chromosomal SCCmec insertion site in orfX to the hsdR gene for a restriction enzyme revealed a type V (5C2&5) SCCmec. Sequence comparisons show that parts of the cassette are highly similar to sequences within SCCmec elements from coagulase-negative staphylococci, indicating a possible common origin. The cassette investigated contains ccrC-carrying units on either side of its class C2b mec gene complex. In vivo loss of the mec gene complex was caused by recombination between the recombinase genes ccrC1 allele 8 and ccrC1 allele 10. In vitro, the SCCmec was very stable, and low-frequency MRSA-to-MSSA conversion was only observed when MRSA isolates were cultivated at 41 degrees C for prolonged periods of time. In this case also, loss of the mec complex was due to ccrC gene recombination. Interestingly, the MRSA and MSSA isolates studied displayed no detectable differences in competitive growth and virulence, suggesting that the presence of the intact type V (5C2&5) SCCmec has no negative bearing on staphylococcal fitness under the conditions used.
Project description:This study aimed to investigate the clinical and molecular epidemiology and biofilm formation of Staphylococcus aureus (SA) isolated from pediatricians in China.SA strains were isolated from Beijing Children's hospital from February 2016 to January 2017. Isolates were typed by multilocus sequence typing (MLST), spa and SCCmec typing (for Methicillin-resistant SA [MRSA] only). Antimicrobial susceptibility testing was performed by agar dilution method except sulphamethoxazole/trimethoprim (E-test method). Biofilm formation and biofilm associated genes were detected.Totally 104 children (41 females and 63 males; median age, 5.2 months) were enrolled in this study, in which 60 patients suffered from MRSA infection. Among the 104 cases, 54.8% were categorized as community associated SA (CA-SA) infections. The children under 3 years were more likely to occur CA-SA infections compared with older ones (P?=?0.0131). ST59-SCCmec IV-t437 (61.7%) was the most prevalent genotype of MRSA, and ST22-t309 (18.2%), ST5-t002 (9.1%), ST6-t701 (9.1%), ST188-t189 (9.1%) were the top four genotypes of methicillin-sensitive SA (MSSA). All the present isolates were susceptible to linezolid, vancomycin, trimethoprim-sulfamethoxazole, mupirocin, tigecyclin, fusidic acid. No erythromycin-susceptible isolate was determined, and only a few isolates (3.8%) were identified as susceptible to penicillin. Multi-drug resistant isolates were reponsible for 83.8% of the ST59-SCCmec IV-t437 isolates. The isolates with strong biofilm formation were found in 85% of MRSA and 53.2% of MSSA, and in 88.7% of ST59-SCCmec IV-t437 isolates. Biofilm formation ability varied not only between MRSA and MSSA (P?= 0.0053), but also greatly among different genotypes (P?<?0.0001). The prevalence of the biofilm associated genes among ST59-SCCmec IV-t437 clone was: icaA (100.0%), icaD (97.3%), fnbpA (100.0%), fnbpB (0), clfA (100%), clfB (100%), cna (2.7%), bbp (0), ebpS (88.5%), sdrC (78.4%), sdrD (5.4%), and sdrE (94.5%).These results indicated strong homology of the MRSA stains isolated from Chinese children, which was caused by spread of multiresistant ST59-SCCmec IV-t437 clone with strong biofilm formation ability. The MSSA strains, in contrast, were very heterogeneity, half of which could produce biofilm strongly.
Project description:Molecular diagnostic tests can be used to provide rapid identification of staphylococcal species in blood culture bottles to help improve antimicrobial stewardship. However, alterations in the target nucleic acid sequences of the microorganisms or their antimicrobial resistance genes can lead to false-negative results. We determined the whole-genome sequences of 4 blood culture isolates of Staphylococcus aureus and 2 control organisms to understand the genetic basis of genotype-phenotype discrepancies when using the Xpert MRSA/SA BC test (in vitro diagnostic medical device [IVD]). Three methicillin-resistant S. aureus (MRSA) isolates each had a different insertion of a genetic element in the staphylococcal cassette chromosome (SCCmec)-orfX junction region that led to a misclassification as methicillin-susceptible S. aureus (MSSA). One strain contained a deletion in spa, which produced a false S. aureus-negative result. A control strain of S. aureus that harbored an SCCmec element but no mecA (an empty cassette) was correctly called MSSA by the Xpert test. The second control contained an SCCM1 insertion. The updated Xpert MRSA/SA BC test successfully detected both spa and SCCmec variants of MRSA and correctly identified empty-cassette strains of S. aureus as MSSA. Among a sample of 252 MSSA isolates from the United States and Europe, 3.9% contained empty SCCmec cassettes, 1.6% carried SCCM1, <1% had spa deletions, and <1% contained SCCmec variants other than those with SCCM1 These data suggest that genetic variations that may interfere with Xpert MRSA/SA BC test results remain rare. Results for all the isolates were correct when tested with the updated assay.
Project description:Staphylococcus aureus is known as an invasive human pathogen, resulting in significant morbidity and mortality worldwide; however, information on community-associated S. aureus (CA-SA) from bloodstream infections (BSI) in children in China remains scarce. This study aimed to investigate the molecular characteristics of 78 CA-SA isolates recovered from pediatric patients with BSI between 2012 and 2017 in Shanghai. All isolates including 51 (65.4%) methicillin-susceptible S. aureus (MSSA) and 27 (34.6%) methicillin-resistant S. aureus (MRSA) isolates were characterized based on antimicrobial resistance, virulence genes, multilocus sequence typing (MLST), spa, and SCCmec typing. A total of 18 distinct sequence types (STs) and 44 spa types were identified. ST188 and ST7 were the predominant MSSA clones and ST59-MRSA-SCCmecIV/V was the most common MRSA clone. Spa t189 (9.0%, 7/78) was the most common spa type. SCCmec types IV and V were observed at frequencies of 59.3 and 40.7%, respectively. Notably, 40 (51.3%) S. aureus BSI strains were multidrug resistant (MDR), and these were mostly resistant to penicillin, erythromycin, and clindamycin. MRSA strains were associated with substantially higher rates of resistance to multiple antibiotics than MSSA strains. Fifty (64.1%, 50/78) isolates, including 19 (70.3%) MRSA isolates, harbored ? 10 tested virulence genes, as evaluated in this study. Ten (37.0%) MRSA isolates and four (7.8%) MSSA isolates harbored the gene encoding Panton-Valentine leukocidin (PVL). Virulence genes analysis showed diversity in different clones; the seb-sek-seq genes were present in all ST59 strains, whereas the seg-sei-sem-sen-seo genes were present in different clones including ST5, ST20, ST22, ST25, ST26, ST30, ST121, and ST487 strains. In conclusion, this study revealed that community-associated S. aureus strains from BSI in children demonstrated considerable genetic diversity, and identified major genotypes of CA-MRSA and CA-MSSA, with a high prevalence of CA-MRSA. Furthermore, major genotypes were frequently associated with specific antimicrobial resistance and toxin gene profiles. Understanding the molecular characteristics of those strains might provide further insights regarding the spread of BSI S. aureus among children between communities in China.
Project description:Staphylococcus aureus colonies can spread on soft agar plates. We compared colony spreading of clinically isolated methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA). All MSSA strains showed colony spreading, but most MRSA strains (73%) carrying SCCmec type-II showed little colony spreading. Deletion of the entire SCCmec type-II region from these MRSA strains restored colony spreading. Introduction of a novel gene, fudoh, carried by SCCmec type-II into Newman strain suppressed colony spreading. MRSA strains with high spreading ability (27%) had no fudoh or a point-mutated fudoh that did not suppress colony spreading. The fudoh-transformed Newman strain had decreased exotoxin production and attenuated virulence in mice. Most community-acquired MRSA strains carried SCCmec type-IV, which does not include fudoh, and showed high colony spreading ability. These findings suggest that fudoh in the SCCmec type-II region suppresses colony spreading and exotoxin production, and is involved in S. aureus pathogenesis.
Project description:Genetic characteristics were analysed for recent clinical isolates of methicillin-resistant and -susceptible Staphylococcus aureus (MRSA and MSSA respectively) in Kathmandu, Nepal. MRSA isolates harbouring Panton-Valentine leukocidin (PVL) genes were classified into ST1, ST22 and ST88 with SCCmec-IV and ST772 with SCCmec-V (Bengal Bay clone), while PVL-positive MSSA into ST22, ST30 and ST772. ST22 isolates (PVL-positive MRSA and MSSA, PVL-negative MRSA) possessed a variant of elastin binding protein gene (ebpS) with an internal deletion of 180 bp, which was similar to that reported for ST121 S. aureus previously outside Nepal. Phylogenetic analysis indicated that the ebpS variant in ST22 might have occurred independently of ST121 strains. This is the first report of ST772 PVL-positive MRSA in Nepal and detection of the deletion variant of ebpS in ST22 S. aureus.
Project description:BACKGROUND:Staphylococcus aureus (S. aureus) with accessory gene regulator (agr) dysfunction occurs in health care settings. This study evaluated the prevalence and the molecular and drug resistance characteristics of S. aureus with dysfunctional agr in a pediatric population in Beijing, China. RESULTS:A total of 269 nonduplicate S. aureus clinical isolates were isolated from Beijing Children's Hospital, including 211 methicillin-resistant S. aureus (MRSA) from September 2010-2017 and 58 methicillin-sensitive S. aureus (MSSA) from February 2016-2017. Only 8 MRSA and 2 MSSA isolates were identified as agr dysfunction, and the overall prevalence rate was 3.7%. For MRSA isolates, ST59-SCCmec IV and ST239-SCCmec III were the most common clones, and the prevalence rate of agr dysfunction in ST239-SCCmec III isolates (17.39%) was significantly higher than in ST59-SCCmec IV (1.69%) and other genotype strains (P?=?0.006). Among the agr dysfunctional isolates, only one MRSA ST59 isolate and one MSSA ST22 isolate harbored pvl. No significant difference was detected between agr dysfunction and agr functional isolates regarding the biofilm formation ability (P?=?0.4972); however, 9/10 agr dysfunctional isolates could effectuate strong biofilm formation and multidrug resistance. Among MRSA, the non-susceptibility rates to ciprofloxacin, gentamicin, and trimethoprim-sulfamethoxazole were significantly higher in agr dysfunctional isolates than in isolates with functional agr (P?<?0.05). Two isolates belonging to ST239 had no mutations in agr locus, but a synonymous mutation was found in agrA in another ST239 isolate. The inactivating mutations were detected in other seven agr dysfunctional isolates. The variants were characterized by non-synonymous changes (n?=?5) and frameshift mutations (insertions, n?=?2), which mainly occurred in agrC and agrA. CONCLUSIONS:The results showed that agr dysfunctional S. aureus was not common in Chinese children, and ST59-SCCmec IV was associated with lower prevalence of agr dysfunction as compared to ST239-SCCmec III isolates. The agr dysfunctional isolates were healthcare-associated, multidrug resistant and form strong biofilm, which suggested that agr dysfunction might offer potential advantages for S. aureus to survive in a medical environment.
Project description:A comparative genomic microarray comprising 2,457 genes from two whole genomes of S. aureus was employed for the comparative genome hybridization analysis of 50 strains of divergent clonal lineages, including methicillin-resistant S. aureus (MRSA), methicillin-susceptible S. aureus (MSSA), and swine strains in China. Large-scale validation was confirmed via polymerase chain reaction in 160 representative clinical strains. All of the 50 strains were clustered into seven different complexes by phylogenetic tree analysis. Thirteen gene clusters were specific to different S. aureus clones. Ten gene clusters, including seven known (vSa3, vSa4, vSa?, vSa?, Tn5801, and phage ?Sa3) and three novel (C8, C9, and C10) gene clusters, were specific to human MRSA. Notably, two global regulators, sarH2 and sarH3, at cluster C9 were specific to human MRSA, and plasmid pUB110 at cluster C10 was specific to swine MRSA. Three clusters known to be part of SCCmec, vSa4 or Tn5801, and vSa? as well as one novel gene cluster C12 with homology with Tn554 of S. epidermidis were identified as MRSA-specific gene clusters. The replacement of ST239-spa t037 with ST239-spa t030 in Beijing may be a result of its acquisition of vSa4, phage ?Sa1, and ?Sa3. In summary, thirteen critical gene clusters were identified to be contributors to the evolution of host specificity and antibiotic resistance in Chinese S. aureus.
Project description:BACKGROUND:Staphylococcus aureus bloodstream infections (BSI) cause significant morbidity and mortality due to the frequent antibiotic resistance, toxin and adhesin production of the bacterium. These characteristics differ significantly in methicillin resistant (MRSA) and methicillin sensitive S. aureus (MSSA) and also among isolates of different MRSA clones, contributing to the outcome of S. aureus bacteraemia. METHODS:In this study, all MRSA BSI isolates from Semmelweis University, Budapest, Hungary, isolated between 2011-2016 and the same number of matched MSSA (overall 306 isolates) were characterised in terms of antibiotic susceptibility, virulence genes, clonality and their association with all-cause 30-day mortality. Effect of patient related variables, such as age, gender and comorbidities were also investigated. RESULTS:ST22-MRSA-IV and ST5-MRSA-II were the most prevalent clones in our study. SCCmec I isolates showed the highest resistance rates and SCCmec II carried most virulence genes. Infections caused by SCCmec IV isolates were associated with the highest mortality rate (42.2%), despite the similar comorbidity rates of the different patient groups. All-cause 30-day mortality was 39.9% in the MRSA and 30.7% in the MSSA group. Increased teicoplanin MIC was associated with high mortality rate. Resistance to ciprofloxacin, erythromycin and clindamycin was common in MRSA, whereas MSSA isolates were more sensitive to all antibiotics with the exception of doxycycline. All MRSA isolates were sensitive to glycopeptides and linezolid; resistance to rifampicin and sulfamethoxazole-trimethoprim was low. MRSA isolates carried more adhesion genes, superantigens were more frequent in MSSA. Panton-Valentine leukocidin was found in 2.3% of the isolates. CONCLUSION:This study provides insight into the clonal composition and associated mortality of BSI S. aureus isolates in Hungary. The results suggest that the outcome of the infection is determined by the antibiotic resistance, genotype of the bacterium, and patient-related factors; rather than the virulence factors carried by the bacteria.