Emergence of methicillin-resistant,vancomycin-intermediate Staphylococcus aureus among patients associated with group A Streptococcal pharyngitis infection in southern India

Beyond Staphylococcus aureus being an etiological agent for several serious clinical complications, the foot prints of S. aureus in pharyngitis infection has also been recently recognized. With due response to the fact, a prospective study was conducted between 2009 and 2010 to describe the molecular epidemiology of S. aureus in throat swabs of pharyngitis patients. A total of 63 methicillin-resistant S. aureus (MRSA) and 102 methicillin-susceptible S. aureus (MSSA) isolates were recovered from 265 throat swabs, representing a community-acquired outpatient population from Tamil Nadu, India. Molecular characterization of MRSA was done by two conventional multiplex PCR assays including Panton-Valentine leukocidin (PVL), mecA and nuc genes, and staphylococcal cassette chromosome mec (SCCmec) typing. Among 165 S. aureus isolates, methicillin resistance was observed in 38.2% (n = 63), in which 69.8% (n = 44/63) of the MRSA along with 55.9% (n = 57/102) of MSSA harbored PVL toxin genes. SCCmec typing showed 50.8% of isolates as SCCmec V (n = 32), 44.4% as SCCmec III (n = 28), and 1.6% as SCCmec types I, II and IVa (n = 1). Multilocus sequence typing performed for 26 selected MRSA isolates resulted in 12 different sequence types (ST), including a novel ST2129/SCCmec III, PVL-positive. Ten MRSA isolates were categorized as ST772 (38.5%)/SCCmec V, PVL-positive, and three isolates as ST368 (11.5%)/SCCmec III, PVL-negative. Though the prominent clones of ST772/SCCmec V were multidrug-susceptible worldwide, they were highly multidrug-resistant in the current study, including four clones intermediate to vancomycin. Totally, 10 (15.9%) out of 63 MRSA isolates were documented as vancomycin-intermediate S. aureus (VISA). Collectively, the present study for the first time portrayed the high prevalence of active MRSA pharyngitis infection and also emphasizes an alarming need for discrimination of pharyngeal-asymptomatic carriers of S. aureus from those with an active S. aureus pharyngitis infection.     

Bovine mastitis Staphylococcus aureus: Antibiotic susceptibility profile,resistance genes and molecular typing of methicillin-resistant and methicillin-sensitive strains in China

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) infection in dairy animals is of great concern for livestock and public health. The aim of present study was to detect new trends of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA) towards antibiotic susceptibility, resistance genes and molecular typing by methods of disc diffusion, multiplex PCR assay and multilocus sequence typing (MLST). A total of 219 S. aureus strains were isolated from bovine mastitis cases from six provinces of China, including 34 MRSA strains. The results revealed that more than 70% isolated strains showed resistance to various antibiotics, and multiple-drugs resistance to more than five categories of antibiotics was found more common. The ermC was the most prevalent resistance gene, followed by other genes; however, ermA was the least frequently detected gene. Twenty-eight mecA-negative MRSA and six mecA-positive MRSA strains were detected, and in which three strains were ST97-MRSA-IV, others were ST965-MRSA-IV, ST6-MRSA-IV and ST9-MRSA-SCCmec-NT. The mecA-negative MRSA strains were found resistant to most of the antibiotics, and harbored aac(6′)/aph(2′′), aph(3′)-III and tetM genes higher than MSSA strains. The resistance to most of the antibiotics was significantly higher in MRSA than in MSSA strains. The MLST profiles showed that these strains mainly belonged to CC5, CC398, CC121 and CC50 lineage, especially within ST97 and ST398, while some novel sequence types (ST2154, ST2165 and ST2166) were identified and deposited in the MLST database. This indicates that the resistance of S. aureus is becoming more complicated by changes in multi-drug resistance mechanism and appearance of mecA-negative MRSA isolates, and importantly, MRSA-IV strains in different MLST types are emerging.     

Molecular analysis and the toxin,MSCRAMM, and biofilm genes of methicillin-resistant Staphylococcus aureus strains isolated from pemphigus wounds: A study based on SCCmec and dru typing

IntroductionPemphigus is a chronic autoimmune blistering disease. Pemphigus blisters can damage the natural skin barrier and increase the risk of life-threatening conditions. Colonization of pemphigus wounds with methicillin-resistant Staphylococcus aureus (MRSA) prolongs wound healing and increases mortality rate. Assessing MRSA prevalence, types, and toxin and adhesion genes can facilitate the detection of MRSA strains which cause infections, selection of appropriate treatments, and healing of pemphigus wounds. This study aimed to determine the SCCmec, the direct repeat unit (dru) types (dts), and the toxin, MSCRAMM, and biofilm genes of MRSA strains isolated from pemphigus wounds.MethodsIn this cross-sectional study, 118 S. aureus isolates were gathered from 118 patients with pemphigus. MRSA detection was performed using the mecA gene. Using the polymerase chain reaction method, all MRSA isolates were assessed for the presence of the sea, seb, sec, tst, eta, pvl, hla, hlb, MSCRAMM, and ica genes. Typing and subtyping were performed through respectively SCCmec typing and dru typing methods. The Bionumerics software was used for analyzing the data and drawing the minimum spanning tree.FindingsFrom 118 S. aureus isolates, 51 were MRSA. SCCmec typing revealed the prevalence of SCCmec II with a prevalence of 64.7% (33 out of 51 isolates) and SCCmec III with a prevalence of 35.3% (18 out of 51 isolates). Dru typing indicated seven dts, namely dts 10a, 10g, 10m, 13i, 8h, 8i, and 9ca in two main clusters. The dt9ca was a new dru type and was registered in the dru-typing database (www.dru-typing.org). The prevalence rates of the hla, sea, and sec genes in MRSA isolates were respectively 54.9%, 27.4%, and 1.9%, while the hlb, seb, eta, and pvl genes were not detected at all. Only one MRSA with SCCmec III and dt10a carried the tst encoding gene. MSCRAMM gene analysis revealed the high prevalence of the eno (31.3%) and the fib (21.5%) genes. The prevalence rates of the icaA and icaD biofilm formation genes were 3.9% and 5.8%, respectively. There were no significant differences between the two detected SCCmec types and between the two detected dts clusters respecting the prevalence of the encoding genes of virulence factors and MSCRAMMs.ConclusionThe toxin genes hla and sea are prevalent among MRSA strains with SCCmec II and III isolated from pemphigus wounds. The most prevalent dts are dt10a and dt10g among MRSA with SCCmec III and dt8h and dt8i among MRSA with SCCmec II.     

Clinical and molecular characteristics of nosocomial meticillin-resistant Staphylococcus aureus skin and soft tissue isolates from three Indian hospitals

We analysed risk factors for nosocomial meticillin-resistant Staphylococcus aureus (MRSA) skin and soft tissue infections (SSTIs) in three Indian hospitals. We also determined antimicrobial resistance patterns and genotypic characteristics of MRSA isolates using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and staphylococcal cassette chromosome (SCCmec) typing. Medical records of 709 patients admitted to three tertiary hospitals with nosocomial S. aureus SSTIs were clinically evaluated. Antimicrobial susceptibility testing of patient isolates was performed in accordance with Clinical and Laboratory Standards Institute guidelines, with meticillin and mupirocin resistance confirmed by multiplex polymerase chain reaction. PFGE analysis of 220 MRSA isolates was performed, followed by MLST and SCCmec typing of a selected number of isolates. MRSA was associated with 41%, 31% and 7.5% of infections at the three hospitals, respectively. Multiple logistic regression analysis identified longer duration of hospitalisation [odds ratio (OR): 1.78; OR: 2.83 for ≥20 days], intra-hospital transfer (OR: 1.91), non-infectious skin conditions (3.64), osteomyelitis (2.9), neurological disorders (2.22), aminoglycoside therapy (1.74) and clindamycin therapy (4.73) as independent predictors for MRSA SSTIs. MRSA isolates from all three hospitals were multidrug resistant, with fifteen clones (I–XV) recognised. A majority of the strains possessed type III cassette. The common sequence type (ST) 239 was considered the signature MLST sequence for PFGE clone III. This major MRSA clone III was closely related to the UK EMRSA-1 and was significantly more resistant to antibiotics. Dissemination of multidrug-resistant MRSA clones warrants continuous tracking of resistant genotypes in the Indian subcontinent.     

Genotyping of community-associated methicillin resistant Staphylococcus aureus (CA-MRSA) in a tertiary care centre in Mysore,South India: ST2371-SCCmec IV emerges as the major clone

The burden of community-associated methicillin resistant Staphylococcus aureus (CA-MRSA) is on the rise in population and clinical settings on account of the adaptability and virulence traits of this pathogen. We characterized 45 non-duplicate CA-MRSA strains implicated mainly in skin and soft tissue infections (SSTIs) in a tertiary care hospital in Mysore, South India. All the isolates were genotyped by staphylococcal cassette chromosome mec (SCCmec) typing, staphylococcal protein A (spa) typing, accessory gene regulator (agr) typing, and multi-locus sequence typing (MLST). Four sequence types (STs) belonging to three major clonal complexes (CCs) were identified among the isolates: CC22 (ST2371 and ST22), CC1 (ST772) and CC8 (ST8). The majority (53.3%) of the isolates was of the genotype ST2371-t852-SCCmec IV [sequence type-spa type-SCCmec type], followed by ST22-t852-SCCmec IV (22.2%), ST772-t657-SCCmec V (13.3%) and ST8-t008-SCCmec IV (11.1%). ST237I, a single locus variant of ST22 (EMRSA-15 clone), has not been reported previously from any of the Asian countries. Our study also documents for the first time, the appearance of ST8-SCCmec IV (USA300) strains in India. Representative strains of the STs were further analyzed by pulsed field gel electrophoresis (PFGE). agr typing detected type I or II alleles in the majority of the isolates. All the isolates were positive for the leukotoxin gene, pvl (Panton–Valentine leukocidin) and the staphylococcal enterotoxin gene cluster, egc. Interestingly, multidrug resistance (resistance to ⩾3 classes of non-beta-lactam antibiotics) was observed in 77.8% (n = 35) of the isolates. The highest (75.5%) resistance was recorded for ciprofloxacin, followed by erythromycin (53.3%), and quinupristin–dalfopristin (51.1%). Inducible clindamycin-resistance was identified in 37.7% of the isolates and it was attributed to the presence of erm(A), erm(C) and a combination of erm(A) and erm(C) genes. Isolates which showed a phenotypic pattern of M^R/L^S (macrolide-resistance/lincosamide-sensitivity) harbored the msr(A) gene. In conclusion, we report a high rate of multidrug resistance among Indian strains of CA-MRSA and the emergence of the lineages ST2371 and ST8 in India.     

Molecular characterization of invasive Staphylococcus aureus strains isolated from patients with diabetes in Iran: USA300 emerges as the major type

There have been few studies focused on the molecular characterization of invasive Staphylococcus aureus strains in patients with diabetes in Iran. In the present study, 20 invasive S. aureus strains recovered from the patients with diabetes characterized by the virulence and resistance analysis, biofilm formation, staphylocoagulase (SC) typing, S. aureus protein A locus (spa) typing staphylococcal cassette chromosome mec (SCCmec) typing, and multilocus sequence typing (MLST). Virulence gene detection indicated a high prevalence of strains encoding the pvl genes (50%), a low prevalence of the tst and seg gene (each of them was 5%) and a markedly high prevalence of fnbB (95%), fnbA (85%), icaD (75%), icaA (65%). A total of 3 coagulase types (III, 85%; II, 10%; V, 5%), 2 agr types (I, 90%; II 10%) and 2 SCCmec types (IV, 65%; III, 35%) and four different clones namely ST8-MRSA-IV/t008 (50%) (USA300), ST239-MRSA-III/t030 (35%), ST5-MRSA-IV/t002 (10%), and ST45-MRSA-IV/t038 (5%) were detected in this study. Eighty-five percent of the isolates were biofilm producers. All the 4 high-level mupirocin resistance (HLMUPR) strains belonged to CC/ST8-MRSA-IV/t008 clone and carried mupA gene. Fusidic acid-resistant isolate belonged to ST239-SCCmec III/t030 clone. One vancomycin-intermediate resistance isolates was detected in our study, which belonged to ST5-MRSA-IVt002. Circulating clone in MRSA strains (USA300) isolated from the patients with diabetes highlighting the possibility of transmission of these microorganisms' clones between hospital, community, and environments. However, further studies require providing critical insights into the importance of continued controlling and treatment of S. aureus infections in patients with diabetes.     

The evolution of Staphylococcus aureus

A broad variety of infections, ranging from minor infections of the skin to post-operative wound infections can be caused by Staphylococcus aureus. The adaptive power of S. aureus to antibiotics leaded, in the early 1960s, to the emergence of methicillin-resistant S. aureus (MRSA). The cause of resistance to methicillin and all other β-lactam antibiotics is the mecA gene, which is situated on a mobile genetic element, the staphylococcal cassette chromosome mec (SCCmec). Seven major variants of SCCmec, type I to VII, are distinguished. The most important techniques used to investigate the molecular epidemiology of S. aureus are pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), S. aureus protein A (spa) typing and SCCmec typing (only for MRSA). These techniques have been used to study the evolution of the MRSA clones that have emerged since the early 1960s, and to study their subsequent worldwide dissemination. The early MRSA clones were hospital-associated (HA-MRSA). However, from the late 1990s, community-associated MRSA (CA-MRSA) clones emerged worldwide. CA-MRSA harbors SCCmec type IV, V or VII, the majority belong to other S. aureus lineages compared to HA-MRSA, and CA-MRSA is often associated with the presence of the toxin Panton-Valentine leukocidin (PVL). However, during recent years, the distinction between HA-MRSA and CA-MRSA has started to disappear, and CA-MRSA is now endemic in many US hospitals. MRSA probably originated trough the transfer of SCCmec into a limited number of methicillin-sensitive S. aureus (MSSA) lineages. This review describes the latest observations about the structure of SCCmec, the techniques used to study the molecular epidemiology and evolution of S. aureus as well as some challenges that researchers face in the future.     

Development of a multilocus sequence typing (MLST) scheme for Mannheimia haemolytica and assessment of the population structure of isolates obtained from cattle and sheep

Mannheimia haemolytica is an important veterinary pathogen affecting cattle and sheep. Previous typing methods, including restriction enzyme analysis, pulsed-field gel electrophoresis and multilocus enzyme electrophoresis (MLEE) have indicated a clonal population structure. Multilocus sequence typing (MLST) has now almost replaced MLEE and is a definitive and portable typing method, allowing global data exchange. The purpose of this study was to develop a MLST scheme for M. haemolytica. A collection of isolates from 10 countries, including the type strain and reference strains for all recognized serotypes were included in the study. Partial sequences of the housekeeping genes adk, aroE, deoD, gapDH, gnd, mdh and zwf were used to define the MLST scheme. The 95 isolates demonstrated 34 different sequence types (ST) of which 19 were connected in three clonal complexes (CC). ST1 constituted more than one-third of the isolates and was most frequently demonstrated among isolates from bovine sources. The analysis indicated a common evolutionary origin of 33 isolates from the French alps, collected from domestic and wild animals and demonstrating several related STs. An analysis of 17 isolates from the USA demonstrated the same ST in 14 of the isolates. In conclusion, an unambiguous typing scheme is presented for M. haemolytica and results obtained confirm previous observations of a clonal population of the organism.     

Multiple-locus variable-number tandem-repeat analysis of meticillin-resistant Staphylococcus aureus discriminates within USA pulsed-field gel electrophoresis types

Many isolates of meticillin-resistant Staphylococcus aureus (MRSA) are indistinguishable when compared using the standard pulsed-field gel electrophoresis (PFGE) typing method. This may present a problem when investigating local outbreaks of MRSA transmission in a healthcare setting. It also impedes investigation of the widely disseminated community-acquired MRSA (USA 300-0114) in the inpatient setting, which is displacing other traditional hospital-acquired PFGE types. Combination of methods, including multiple-locus sequence typing (MLST), spa typing and staphylococcal cassette chromosome mec (SCCmec) typing, have been used with, or in place of, PFGE to characterise MRSA for epidemiological purposes. These methods are technically challenging, time-consuming and expensive and are rarely feasible except in large laboratories in tertiary care medical centres. Another method, which is simpler and with faster turnaround time, is multiple-locus variable-number tandem-repeat analysis (MLVA). We investigated the utility of MLVA to distinguish common PFGE types. The results suggest that MLVA can be used to identify unrelated strains with identical PFGE patterns or confirm close genetic composition of linked isolates. MLVA could potentially be used in conjunction with PFGE to validate relationships, but further prospective evaluation of these relationships will be required in order to define the proper role, if any, for use of this method in hospital epidemiology.     

Reassessment of MLST schemes for Leptospira spp. typing worldwide

Leptospirosis is a neglected zoonosis of global importance. Several multilocus sequence typing (MLST) methods have been developed for Leptospira spp., the causative agent of leptospirosis.In this study we reassessed the most commonly used MLST schemes in a set of worldwide isolates, in order to select the loci that achieve the maximum power of discrimination for typing Leptospira spp. Global eBURST algorithm was used to detect clonal complexes among STs and phylogenetic relationships among concatenated and individual sequences were inferred through maximum likelihood (ML) analysis. The evaluation of 12 loci combined to type a subset of strains rendered 57 different STs. Seven of these loci were selected into a final scheme upon studying the number of alleles and polymorphisms, the typing efficiency, the discriminatory power and the ratio dN/dS per nucleotide site for each locus. This new 7-locus scheme was applied to a wider collection of worldwide strains. The ML tree constructed from concatenated sequences of the 7 loci identified 6 major clusters corresponding to 6 Leptospira species. Global eBURST established 8 CCs, which showed that genotypes were clearly related by geographic origin and host. ST52 and ST47, represented mostly by Argentinian isolates, grouped the higher number of isolates. These isolates were serotyped as serogroups Pomona and Icterohaemorrhagiae, showing a unidirectional correlation in which the isolates with the same ST belong to the same serogroup.In summary, this scheme combines the best loci from the most widely used MLST schemes for Leptospira spp. and supports worldwide strains classification. The Argentinian isolates exhibited congruence between allelic profile and serogroup, providing an alternative to serological methods.     

Isolation and clinical sample typing of human leptospirosis cases in Argentina

Leptospira typing is carried out using isolated strains. Because of difficulties in obtaining them, direct identification of infective Leptospira in clinical samples is a high priority. Multilocus sequence typing (MLST) proved highly discriminatory for seven pathogenic species of Leptospira, allowing isolate characterization and robust assignment to species, in addition to phylogenetic evidence for the relatedness between species. In this study we characterized Leptospira strains circulating in Argentina, using typing methods applied to human clinical samples and isolates. Phylogenetic studies based on 16S ribosomal RNA gene sequences enabled typing of 8 isolates (6 Leptospira interrogans, one Leptospira wolffii and one Leptospira broomii) and 58 out of 85 (68.2%) clinical samples (55 L. interrogans, 2 Leptospira meyeri, and one Leptospira kirschneri). MLST results for the L. interrogans isolates indicated that five were probably Canicola serogroup (ST37) and one was probably Icterohaemorrhagiae serogroup (ST17). Eleven clinical samples (21.6%), provided MLST interpretable data: five were probably Pyrogenes serogroup (ST13), four Sejroe (ST20), one Autumnalis (ST22) and one Canicola (ST37). To the best of our knowledge this study is the first report of the use of an MLST typing scheme with seven loci to identify Leptospira directly from clinical samples in Argentina. The use of clinical samples presents the advantage of the possibility of knowing the infecting strain without resorting to isolates. This study also allowed, for the first time, the characterization of isolates of intermediate pathogenicity species (L. wolffii and L. broomii) from symptomatic patients.     

All methicillin-resistant Staphylococcus auveus (MRSA) are not equal

New typing techniques are giving a greater understanding of the evolution and epidemiology of methicillin-resistant Staphylococcus aureus (MRSA). A comparison of the overall genetic background of isolates is now possible using MultiLocus sequence typing (MLST). However, even isolates with the same genetic background can be different from each other because of the acquisition of antibiotic-resistance and pathogenicity determinants. Sequencing of the region involved with methicillin resistance, the mec region or staphylococcal cassette chromosome mec (SCCmec), has demonstrated that there are different SCCmecs. It has been found that strains with similar genetic backgrounds can have different SCCmecs and that strains with different genetic background can have similar SCCmecs. This, and molecular evidence, indicates that SCCmecs are mobile elements that are able to move between cells by an as yet unidentified mechanism.Typing has also demonstrated that some strains, known as epidemic MRSA (EMRSA), spread more readily in hospitals and health care institutions. However, it is not known why they have this ability. Whereas in the past MRSAs were regarded as being associated with health care institutions, strains have emerged in the community that are quite different from those associated with health care institutions. These strains are not multiply resistant to antimicrobials like most of the strains associated with health care institutions; however, there is concern that they will acquire additional resistance determinants and present problems for the treatment of staphylococcal infections. Some EMRSA are not multiply resistant and, on their resistance profiles, could be mistaken for community MRSA. This highlights the importance of typing in identifying and tracing MRSA for infection control.     

Multilocus sequence typing (MLST) of leptospiral strains isolated from two geographic locations of Tamil Nadu,India

Here the rodent carrier status for the transmission of human leptospirosis in Tiruchirappalli, district, Tamil Nadu, India was assessed. The predominantly circulating leptospiral STs were recognized by multilocus sequence typing (MLST). A total of 113 rodents were trapped from different provinces of the Tiruchirappalli district. The most prevalent rodent was Bandicota bengalensis (37.2%), and of the total, 52.2% (n = 59) rodents were found to be positive for leptospiral 16S rRNA. These results were validated with a leptospiral culture positivity of 45.8% (n = 27). Three isolates from Chennai (2 rodents and 1 human) and 1 human isolate from Tiruchirappalli were included to understand the spatial variations and to track the source of human leptospirosis. The serogroup, serovar, and species level identification of all 31 isolates identified 28 to be Leptospira borgpetersenii serovar Javanica and three as Leptospira interrogans serovar Autumnalis. MLST analysis defined all isolates to the existing ST profiles (ST145 and ST27) with the exception of 6 L. borgpetersenii (ST DR) isolates that showed variations in the sucA and pfkB loci. The DR ST was locally confined to Chatram province of Tiruchirappalli suggesting an epidemiological link. The predominant STs, ST145 and ST-DR form a group, indicating the presence of original strain that subsequently diverged evolutionarily into two STs. The variations between L. borgpetersenii in sucA and pfkB loci may be an indication that evolutionary changes transpired in Tiruchirappalli.     

A reliable combination method to identification and typing of epidemic and endemic clones among clinical isolates of Acinetobacter baumannii

The multi-drug resistant (MDR) Acinetobacter baumannii as an important nosocomial pathogen has emerged a global health concern in recent years. In this study, we applied three easier, faster, and cost-effective methods including PCR-based open reading frames (ORFs) typing, sequence typing of bla_OXA-51-like and RAPD-PCR method to rapid typing of A. baumannii strains. Taken together in the present study the results of ORFs typing, PCR-sequencing of bla_OXA-51-like genes and MLST sequence typing revealed there was a high prevalence (62%, 35/57) of ST2 as international and successful clone which detected among clinical isolates of multi-drug resistant A. baumannii with ORF pattern B and bla_OXA-66 gene. Only 7% (4/57) of MDR isolates belonged to ST1 with ORF pattern A and bla_OXA-69 gene. Interestingly, we detected singleton ST513 (32%, 18/57) that encoded bla_OXA-90 and showed the ORF pattern H as previously isolated in Middle East. Moreover, our data showed RAPD-PCR method can detect divergent strains of the STs. The Cl-1, Cl-2, Cl-3, Cl-4, Cl-10, Cl-11, Cl-12, Cl-13 and Cl-14 belonged to ST2. While the Cl-6, Cl-7, Cl-8 and Cl-9 belonged to ST513. Only Cl-5 belonged to ST1. It seems that the combination of these methods have more discriminatory than any method separately and could be effectively applied to rapid detection of the clonal complex (CC) of A. baumannii strains without performing of MLST or PFGE.     

Molecular epidemiology and antibiotic resistance of methicillin-resistant Staphylococcus aureus circulating in the Russian Federation

The aim of this study was to investigate the patterns of antimicrobial resistance and molecular features of methicillin-resistant Staphylococcus aureus (MRSA) isolates in Russia. Isolates recovered from hospital patients (n = 480), healthy medical personnel (n = 25), and healthy carriers (n = 13) were included in the study. Hospital-acquired MRSA (HA-MRSA) demonstrated high resistance to ciprofloxacin, gentamicin, and chloramphenicol (76%–92%), moderate – to tetracycline, erythromycin, clindamycin, and rifampicin (38%–54%), and low – to fusidic acid, co-trimoxazole, mupirocin, and daptomycin (2%–7%). Elevated MIC (2.0 μg/ml) of vancomycin was detected in 26% of isolates. All isolates were susceptible to linezolid and tigecycline. Multilocus sequence typing (MLST) revealed that CC8 isolates (ST8 + ST239) constituted 83.1% of HA-MRSA and that this genetic lineage dominated in all regions from Krasnoyarsk to Saint Petersburg. A local ST239 variant harboring the tst gene (ST239_Kras) was detected in Krasnoyarsk. The other HA-MRSA isolates belonged to clonal complex 5 (CC5) (21 isolates, 12.2%) and CC22 (2, 1.2%). The majority of CC5 isolates were affiliated with sequence type 228 (ST228) and were characterized with decreased susceptibility to ceftaroline (MIC = 2 μg/ml). We also detected, for the first time in Russia, livestock-associated MRSA (LA-MRSA) from clusters CC398 and CC97 in humans. Among the 2053 healthy persons screened for nasal carriage of S. aureus, the bacteria were isolated from 426 (21%); among them, 13 carried isolates identified as community-associated MRSA (CA-MRSA). Eleven of 13 CA-MRSA isolates belonged to ST22 (spa types t223, t3243, and t3689; SCCmec types IVa and IVc, agr type I, tst-positive) and were similar to the EMRSA-15/Middle Eastern variant (Gaza strain).     

Population deviation of piggery-associated methicillin-resistant Staphylococcus aureus based on mec-associated direct repeat unit analysis

Piggery-associated methicillin-resistant Staphylococcus aureus (MRSA) is a potential zoonotic pathogen. We constructed the population structure and dynamics of staphylococcal chromosome cassette mec (SCCmec) in MRSA ST9 isolates from different geographical areas of Taiwan. A total of 140 MRSA (135 piggery and 5 human clinical) isolates from three populations located in western Taiwan (n = 96 including the 5 clinical isolates), central eastern Taiwan (n = 22), and Penghu Island (n = 22) were collected and characterized by analysis of the mec-associated direct repeat unit (dru). Twenty-eight dru types (with 24 novel) and 15 dru-Clonal Complexes (CCs) were identified. The predominant novel dt12w type (48.6%) was widespread in all populations and may have a superior ability to transmit among populations. The minimum spanning network showed that at least two ancestral dru types (dt11a and dt12w) were identified, and the genetics between different populations could be differentiated. Temporal distributions of clone population dynamics estimated through the Bayesian skyline plot indicated a stable population with a long evolutionary history for MRSA ST9 in Taiwan. Findings indicating that some dru types are shared between piggery-associated and human-clinical MRSA ST9 suggest the occurrence of cross-species horizontal transmission of SCCmec.     

马鞍山地区耐甲氧西林金黄色葡萄球菌的分子特征研究

目的 了解马鞍山地区耐甲氧西林金黄色葡萄球菌(金葡菌)(MRSA)肠毒素、溶血素分布情况、菌株克隆群关系及其耐药性。方法 采用全自动酶联荧光免疫系统和PCR技术分别检测肠毒素和溶血素基因分布;选择金葡菌的7个管家基因作为目的基因,对34株MRSA和3株甲氧西林敏感金葡菌(MSSA)进行多位点序列分型(MLST),然后与网上数据库比对,获得序列型(ST),根据eBURST的ST进行亲缘性分析;采用琼脂稀释法检测MRSA对12种抗生素的耐药情况。结果 210株金葡菌肠毒素阳性率为50.9%,溶血素基因携带率为97.1%,其中51株MRSA全部含有溶血素基因。34株MRSA有10个ST,以ST239为主(47.1%,16/34),其次为ST5(17.6%,6/34);3株MSSA的ST为ST188、ST1281和ST7。17株患者来源菌株分为6个ST,以ST239为主(35.3%,6/17),其次为ST5(29.4%,5/17);20株食品来源菌株有9个ST别,以ST239为主(45.0%,9/20),其次为ST7(15.0%,3/20)。ST585、ST630以及ST239的亲缘关系较近,其他ST之间亲缘关系较远。除万古霉素外,所有菌株对10种抗生素有不同程度的耐药。结论 金葡菌溶血素普遍存在;ST239为马鞍山地区MRSA的主要优势菌株,各ST间亲缘关系较远。     

Livestock-associated Methicillin-Resistant Staphylococcus aureus Sequence Type 398 in Humans, Canada

Rates of colonization with livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) sequence type 398 have been high for pigs and pig farmers in Canada, but prevalence rates for the general human population are unknown. In this study, 5 LA-MRSA isolates, 4 of which were obtained from skin and soft tissue infections, were identified from 3,687 tested MRSA isolates from persons in Manitoba and Saskatchewan, Canada. Further molecular characterization determined that these isolates all contained staphylococcal cassette chromosome (SCC) mecV, were negative for Panton-Valentine leukocidin, and were closely related by macrorestriction analysis with the restriction enzyme Cfr91. The complete DNA sequence of the SCCmec region from the isolate showed a novel subtype of SCCmecV harboring clustered regularly interspaced short palindromic repeats and associated genes. Although prevalence of livestock-associated MRSA seems to be low for the general population in Canada, recent emergence of infections resulting from this strain is of public health concern.     

Identification of a dominant Chlamydia trachomatis strain in patients attending sexual transmitted infection clinic and female sex workers in Tunisia using a high resolution typing method

BackgroundThe distribution of Chlamydia trachomatis genotypes in Tunisia was previously studied using the reverse hybridization method. In this study, we used multilocus sequence typing (MLST) to describe Chlamydia trachomatis genetic diversity among heterosexual populations in Tunisia. The obtained sequence types (STs) were compared with those from a heterosexual population from Amsterdam, the Netherlands.MethodsClinical Tunisian patients and female sex workers provided 107 Chlamydia trachomatis positive samples that were used for MLST. Samples from 256 heterosexuals visiting the Amsterdam STI clinic were included as a reference group. Six highly variable genetic regions including the ompA gene were amplified and sequenced. The ST numbers were derived from a Chlamydia typing database (http://mlstdb.uu.se) and used to draw minimum spanning trees.ResultsompA sequencing detected 7 genotypes among the Tunisian populations of which genotype E was the most prevalent (66.3%). This genotype E resolved into 23 different STs and among these the ST3 was predominant (53.5%). MLST displayed 43 STs, of which 28 (65%) were new in the database. Minimum spanning tree analysis of all Tunisian samples identified 4 clusters of which one formed a clonal cluster with samples presenting the most prevalent ST3. When comparing samples from the Tunisian and Dutch populations in one minimum spanning tree, there was little overlap between the Chlamydia trachomatis samples.ConclusionThe CT-hrMLST scheme allowed us to identify that the Tunisian distribution was dominated by one genotype E (ST3) strain which is also highly prevalent in many other countries worldwide.     

纹带棒状杆菌多位点序列分型及应用

目的 建立纹带棒状杆菌的多位点序列分型（MLST）方法,探讨临床分离纹带棒状杆菌的种群结构和遗传进化关系。方法 筛选出7个管家基因（gyrA、gyrB、hsp65、sodA、secA1、rpoB、16S rRNA）,设计引物并进行PCR扩增和测序,测序所得序列通过SeqMan软件进行拼接。采用DnaSP 5.10.01软件、Splits tree 4.14.2软件对管家基因的多样性及基因重组特征进行评价;采用MEGA 7.0.14软件基于序列型别（ST）采用M-L法构建系统发育树,采用BioNumerics软件基于ST特征值构建最小生成树,并用eBURST软件分析ST间遗传进化关系。结果 所选的7个位点在所有试验菌株中均获得了预期的扩增产物;Splits tree表明所有纹带棒状杆菌的聚类一致,提示基因重组是推动纹带棒状杆菌进化的潜在动力;MLST将344株纹带棒状杆菌分成72个STs,85.7%的菌株形成克隆复合体（CC）结构,CC19形成了优势克隆复合体,但包含菌株数最多的ST为该克隆复合体中的ST16。ST具有一定的地域聚集性且与分离年份具有一定的相关性。结论 我国纹带棒状杆菌呈现高度的遗传多样性,CC19为优势克隆复合体。本研究建立的MLST分型方案可用于纹带棒状杆菌的分型,但尚需优化改进。