Coagulase gene-based typing of Staphylococcus aureus from mastitic cattle and goats from arid region in India

Staphylococcus aureus is an important pathogen and causes mastitis and many other infections in animals as well as in humans. In the present study, S. aureus isolates were investigated for any variations based on coa gene polymorphism and AluI restriction fragment length polymorphism of coa amplicons. Thirty S. aureus isolates obtained from cattle and goats with clinical mastitis were identified by their phenotypic properties and then confirmed genotypically by PCR where all the isolates produced species-specific amplicon of 1,250?bp. Amplification of the coa gene produced products of three different sizes (600, 680, or 850?bp), one specific to each isolate. The strains of S. aureus were common to both cattle and goat, but the predominance of S. aureus isolates with coa gene amplicon of 680?bp was observed in cattle and 600?bp coa gene amplicon in goats. No difference in the restriction fragment length polymorphism (RFLP) patterns of coa amplicons with AluI endonuclease was obtained in isolates from cattle or goats, and there was limited strain variations in the region under study. The coa amplicons and RFLP patterns of isolates were similar to those recorded elsewhere.     

Molecular subtyping of Staphylococcus aureus isolated from bovine mastitis in Pernambuco State,Brazil

The present study describes genotypic variations of Staphylococcus aureus strains isolated from bovine mastitis in Pernambuco State, Brazil. Twenty-four isolates of S. aureus from individual animals from 14 different herds were including on study. The isolates were analyzed by three different molecular typing methods: PCR amplification of the X region of the spa gene, the 3′ end of the coa gene, and the spacer region between the 16S and 23S rRNA. All isolates could be differentiated from each other with PCR products ranging the X region of the spa gene of 290 to 380 bp, the sizes of the coa gene PCR products of 700 to 1,200 bp, and PCR amplification of the 16S–23S rRNA variable region of 700 to 1,000 bp. The S. aureus isolates can be classified into five distinct clusters; the cluster IV has the higher number of isolates (54.17 %) and has representation of different herds. PCR amplification of the X region of the spa gene, the 3′ end of the coa gene, and the spacer region between 16S and 23S rRNA was technically simple with high reproducibility.     

Discrimination of Staphylococcus aureus isolates on the basis of gene coding protein A using PCR-restriction enzyme analysis

In a comparative study, isolates of Staphylococcus aureus from bovine mastitis cases with known coa and aroA types were analyzed by molecular typing based on polymerase chain reaction and restriction enzyme analysis (REA) of protein A-encoding gene (spa) for assessment of its utility over coa and aroA typing in discrimination of the isolates. Fifty-eight isolates of S. aureus from nine dairy herds in two Iranian provinces were typed based on polymorphism characterizing the gene encoding for the X region of protein A (spa). Five differently sized amplicons of approximately 1,200?bp to 1,410?bp were observed. Spa gene REAs produced a total of eight distinct patterns, designated as S1–S8, after digestion with restriction endonuclease Hin6I. For the spa gene, the lack of amplification was also considered a distinct genotype (S9). The majority of isolates were classified into spa types S2 (24.14%) and S6 (24.14%). This study also showed that genetic analysis of the repeat region of protein A might help to understand the distribution of prevalent S. aureus clones among bovine mastitis isolates. Interestingly, based on spa, coa, and aroA typing, some isolates which were identified as dominant types by one method were classified as rare types by another and vice versa. Finally, spa typing has 62.1% concordance with coa typing from the standpoint of the assignment of the isolates to predominant and rare lineages. This study also demonstrates the importance of spa genotyping in the discrimination of S. aureus isolates, which were otherwise indistinguishable by coa and aroA gene typing.     

coa types and antimicrobial resistance profile of Staphylococcus aureus isolates from cases of bovine mastitis

The objective of the study was to determine the interrelationship between coa types of Staphylococcus aureus isolates from bovine mastitis cases and their susceptibility patterns to commonly used antimicrobial agents. In this study, a total of 63 S. aureus isolates obtained from 387 clinical and subclinical bovine mastitis milk specimens were examined for antimicrobial susceptibility against various classes of antibiotics and coa typing according to the size of the PCR product. Overall, five resistance profiles, nomenclatured PA, PAAc, PAE, PAAcE, and PAAcT according to their resistance and five different coa types, numbered I to V, were noted among the isolates studied. The most coa types were type I (38.1% of the 63 isolates) and type III (36.51%) and indicates that a few coagulase gene types of S. aureus are responsible for the majority of bovine mastitis cases. Resistance to penicillin, ampicillin, and amoxicillin/clavulanic acid were most common, respectively, with 63 (100%), 63 (100%), and 41 (65.1%) isolates and appeared to be higher than those usually reported. No resistance was detected for trimethoprim and gentamicin. Furthermore, a good relationship between phenotypic antibiogram and the polymorphism of coagulase gene was found in this study. Predominant coa types I and III were significantly associated with resistance profiles PAAcT and PAAcE, respectively. These two predominant types also showed high antibiotic resistance characteristic in comparison with rare types. As a result, this study illustrated a tendency for isolates from different coa types to have specific antimicrobic susceptibility profiles, presumably because of genetic diversity between isolates and/or selection force through the use of drugs.     

Antibiotic resistance, population structure and spread of Staphylococcus aureus in nursing homes in the Euregion Meuse-Rhine

To determine the spread of Staphylococcus aureus within and between nursing home (NH) residents in the Euregion Meuse-Rhine, a cross-border region of the Netherlands and Germany, we investigated the prevalence of antibiotic resistance, genetic background and population structure of both methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolates. A total of 245 S. aureus isolates were collected from NH residents. Susceptibility testing was performed with microbroth dilution. The genetic background was determined using spa typing, SCCmec typing, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Differences in the prevalence of resistance between the German and Dutch MSSA isolates were observed for the macrolides (15 % vs. 2 %, p?=?0.003), clindamycin (15 % vs. 0 %, p?=?0.003) and ciprofloxacin (34 % vs. 25 %). The macrolide and ciprofloxacin resistance varied between the NHs, while trimethoprim–sulfamethoxazole resistance was low in all residents. The MRSA prevalence was 3.5 % and <1 % among the German and Dutch NH residents, respectively (p?=?0.005). The German MRSAs, isolated in 7 out of 10 NHs, belonged to ST22-MRSA-IV or ST225-MRSA-II. spa clonal complexes (spa-CCs) 015 and 002 were prevalent among the German MSSA isolates and spa-CCs 024 and 1716 were prevalent among the Dutch MSSA isolates. The antibiotic resistance of MSSA and the MRSA prevalence were significantly higher among the German NH residents. The spread of two MRSA clones was observed within and between the German NHs, but not between the Dutch and German NHs. Differences in the prevalence of resistance and the prevalence of MRSA between NHs on both sides of the border warrant the continuation of surveillance at a local level.     

Genotyping of Staphylococcus aureus isolated from bovine mastitis based on PCR-RFLP analysis of the aroA gene

The aim of this study was the genotypical characterization of 58 Staphylococcus aureus isolates from nine dairy herds in the Tabriz and Urmia regions of east and west Azerbaijan provinces, Iran. In this study, 58 S. aureus isolates from 370 milk samples from cows with clinical and subclinical mastitis were analyzed by polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) analysis of the aroA gene. Amplification of the aroA gene resulted in a single amplicon with a size of approximately 1,153 bp from all 58 isolates of S. aureus. To obtain the RFLP patterns of the isolates, the PCR products were digested with TaqI restriction enzyme and the fragments separated by gel electrophoresis. Four distinct RFLP patterns were observed among the studied isolates. Three out of four detected genotypes had the same RFLP patterns (A, B, and N) as reported by previous studies. The fourth newly detected genotype in this study was named H. Genotypes A and B were the most frequent, being observed in 24 (41.38%) and 29 (50%) isolates, respectively. Genotypes N and H comprised 1.7% and 6.9% of all isolates, respectively. With the exception of the RFLP pattern N, which was observed only in the Tabriz region, all other patterns were found in both Tabriz and Urmia regions. The results demonstrate that strain variations of S. aureus could occur within and between herds and also between different regions, although a few genotypes of S. aureus were predominant in bovine mastitis. This study also indicated that PCR amplification of the aroA gene is specific for S. aureus identification.     

MOLECULAR TYPING OF METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS STRAINS BY PCR-RFLP OF SPA GENE: A REFERENCE LABORATORY PERSPECTIVE

Purpose: To characterize methicillin-resistant Staphylococcus aureus (MRSA) strains by molecular typing based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of spa gene and to assess the utility of spa genotyping over bacteriophage typing in the discrimination of the strains. Materials and Methods: Studies were undertaken on 125 MRSA strains representing the most predominant phage types and the non phage typeable strains. Strains were typed by bacteriophage typing and PCR-RFLP of spa gene. DNA sequence analysis of the amplified spa gene fragment of the representative RFLP patterns was performed using standard protocols. Results: All the strains resistant to oxacillin were found to contain mec A gene. Fifty-two per cent of these strains were typeable by the international basic set of 23 phages. Five different PCR-RFLP patterns were observed among 125 MRSA strains. Non phage typeable strains were differentiated into four PCR-RFLP patterns. Sequencing of the spa gene from the representative strains of each RFLP pattern confirmed the length of these restriction fragments due to variation in the 24 bp and the 174 bp tandem repeats. It also revealed the presence of three new spa repeat patterns. Conclusion: The study demonstrates the importance of spa genotyping in the discrimination of MRSA strains, which were otherwise indistinguishable by bacteriophage typing. spa genotyping allowed differentiation of strains within a particular phage type. Nucleotide sequencing of isolates of different PCR-RFLP patterns indicated a correlation between the RFLP patterns of a variable number of tandem repeats and the phage type. The study provides valuable information on the epidemiological characterization of MRSA strains.     

Molecular Characterization of Endocarditis-Associated Staphylococcus aureus

Infective endocarditis (IE) is a life-threatening infection of the heart endothelium and valves. Staphylococcus aureus is a predominant cause of severe IE and is frequently associated with infections in health care settings and device-related infections. Multilocus sequence typing (MLST), spa typing, and virulence gene microarrays are frequently used to classify S. aureus clinical isolates. This study examined the utility of these typing tools to investigate S. aureus epidemiology associated with IE. Ninety-seven S. aureus isolates were collected from patients diagnosed with (i) IE, (ii) bloodstream infection related to medical devices, (iii) bloodstream infection not related to medical devices, and (iv) skin or soft-tissue infections. The MLST clonal complex (CC) for each isolate was determined and compared to the CCs of members of the S. aureus population by eBURST analysis. The spa type of all isolates was also determined. A null model was used to determine correlations of IE with CC and spa type. DNA microarray analysis was performed, and a permutational analysis of multivariate variance (PERMANOVA) and principal coordinates analysis were conducted to identify genotypic differences between IE and non-IE strains. CC12, CC20, and spa type t160 were significantly associated with IE S. aureus. A subset of virulence-associated genes and alleles, including genes encoding staphylococcal superantigen-like proteins, fibrinogen-binding protein, and a leukocidin subunit, also significantly correlated with IE isolates. MLST, spa typing, and microarray analysis are promising tools for monitoring S. aureus epidemiology associated with IE. Further research to determine a role for the S. aureus IE-associated virulence genes identified in this study is warranted.     

Molecular typing of Staphylococcus aureus isolated from food samples in Iran

Staphylococcus aureus is the third most common cause of confirmed food poisoning in the world and is the predominant species involved in staphylococcal food poisoning outbreaks. Considerable genetic heterogeneity has been shown in natural populations of S. aureus isolates. Coagulase gene typing is one of the numerous molecular techniques to identify and compare S. aureus genotypes. The present study was conducted to type the coagulase gene in 25 S. aureus isolates isolated from food samples. All isolates were identified by routine biochemical tests and then confirmed by species-specific PCR and yielded products with the expected molecular size of 1.3 kb. PCR amplification of DNA with the primers COAG2 and COAG3 yielded single-banded PCR products in 24 isolates with the molecular size of approximately 500 bp (n?=?2, 8 %), 750 bp (n?=?1, 4 %), 850 bp (n?=?12, 48 %), and 950 bp (n?=?9, 36 %), while one isolate produced no band in PCR amplification of coagulase gene. Since human and bovine reservoirs of S. aureus represent two subpopulations that rarely cross-infect, detection of single bands by coagulase PCR in S. aureus isolates suggests that these isolates may be of bovine origin not human one, and contamination of food samples may initiate from the animal source not the food handlers. Digestion of coagulase PCR products with restriction endonuclease enzymes AluI and Hin6I yielded four different restriction profiles that indicate presence of heterogeneity in the coagulase gene of the isolates. This work showed that restriction analysis of the coagulase gene can be considered as a reliable and fast method for determining the origin of S. aureus in food samples.     

Distribution of collagen adhesin gene among various types of Staphylococcus aureus strains associated with bovine mammary gland

A subset of 58 bovine mastitis-associated Staphylococcus aureus isolates with known coa types were investigated for the presence of collagen adhesin gene, cna. According to the enzyme restriction pattern of gene encoding coagulase, strains were divided into nine genotypes. All isolates were investigated by PCR for the presence of gene encoding Cna, which was considered as an important virulence factor associated with bacteria adhesion. Interestingly, 49 (84.5%) strains were found to be cna ⁺ with significant variations across the predominant and rare genotypes. According to the results of this study, it might be emphasized that cna is significantly more common in bovine mastitis-associated S. aureus isolates and distributed dependently between genotypes. The finding that collagen–adhesin is present in the majority of the bovine mastitis-associated S. aureus isolates encourages the development of new strategies to prevent mastitis, based on antagonist ligands able to interact with surface adhesin and block its specific binding with matrix collagen.     

Molecular Characterisation of Staphylococcus aureus Using spa Typing as a Diagnostic Tool in Haryana,India

Background: Staphylococcus aureus is one of the top six most common etiologic agents of nosocomial, community and livestock acquired bacterial infections. These infections although initially were described as a major problem in hospitals have now also become a serious threat in community not only in India but also worldwide. Its prevalence varies depending on the health-care setting, country or a particular region. Thus to better understand the epidemiology of methicillin-resistant S. aureus (MRSA) in a particular geographical location, it is important to study the variations in the population using molecular tools. Methods: This prospective study was carried out in the Department of Microbiology of Shree Guru Gobind Singh Tricentenary (SGT) Medical College. Staphylococcal protein A (spa) typing was done on 250 S. aureus isolates obtained from various clinical specimens including pus, wound swabs, urine, catheters, blood and cerebrosspinal fluid from both indoor and outdoor patients of SGT Hospital, Budhera, Gurgaon. Results: The selected region of the spa gene of all 250 isolates which includes 87 MRSA and 163 methicillin-susceptible S. aureus were amplified. The spa gene was detected in 248 out of 250 isolates (99.2%), whereas in 2 isolates (0.8%), it remained undetected and referred as non-typable isolates. The 248 S. aureus isolates were typed into 39 spa types, which clustered into six different spa clonal clusters and eight singletons. Conclusion: High diversity observed within S. aureus isolates indicated that many different strains circulate in the study region or in the hospital. The results would contribute in the understanding of epidemiology related to S. aureus spread and prevention.     

Molecular Typing of Staphylococcus aureus Based on PCR Restriction Fragment Length Polymorphism and DNA Sequence Analysis of the Coagulase Gene

A typing procedure for Staphylococcus aureus was developed based on improved PCR amplification of the coagulase gene and restriction fragment length polymorphism (RFLP) analysis of the product. All coagulase-positive staphylococci produced a single PCR amplification product of either 875, 660, 603, or 547 bp. Those strains of epidemic methicillin-resistant S. aureus 16 (EMRSA-16) studied all gave a product of 547 bp. PCR products were digested with AluI and CfoI, and the fragments were separated by gel electrophoresis. Ten distinct RFLP patterns were found among 85 isolates of methicillin-resistant S. aureus (MRSA) and 10 propagating strains (PS) of methicillin-sensitive S. aureus (MSSA) examined. RFLP patterns 1, 2, and 3 were specific to strains of EMRSA-3, -15, and -16, respectively. By contrast, RFLP patterns 4 and 5 were seen with a heterogeneous collection of strains, together with drug-resistant forms of S. aureus isolated in Europe and four propagating strains used for the international phage set. RFLP pattern 6 was given by the Airedale isolate and PS 95. RFLP pattern 7 encompassed EMRSA-2 (isolate 331), PS 94, and PS 96. An isolate from Germany gave RFLP pattern 8. Eight strains of MSSA gave patterns similar to those of methicillin-resistant strains (RFLP patterns 3, 4, 5, 6, and 7), but two, PS 42E and PS 71, gave unique RFLP patterns 9 and 10, respectively. The coagulase gene PCR products for 24 isolates of MRSA and two isolates of MSSA were sequenced for both strands. The sequences were aligned, and evolutionary lineages were inferred based on pairwise distances between isolates.     

Biofilm Forming Ability and <Emphasis Type="Italic">Spa</Emphasis> Gene Polymorphism in Methicillin Resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> Clinical Isolates in North of Iran

Spa typing has been shown to function as a genetic marker for Staphylococcus aureus outbreak investigations and epidemiological studies. This study was aimed to investigate biofilm formation capacity and spa gene polymorphism in methicillin resistant S. aureus (MRSA) strains isolated from clinical samples. A total of 102 S. aureus isolated during 2016, were analyzed for methicillin resistance and biofilm formation using phenotypic assays and PCR-based detection of associated genes. The polymorphic region of the spa gene was amplified by PCR using specific primers and subsequently in MRSA strains the amplified products were sequenced and spa types determined by using the spa database website. Out of 102 S. aureus, 41 isolates (40.2%) recognized as MRSA in phenotypic and genotypic investigations. In phenotypic assay, biofilm forming ability was detected in 71 isolates. The frequency of icaA and fnbA in test isolate were 53.9 and 65.7% respectively. Amplification of polymorphic region of the spa gene in all 102 tested isolates resulted in eight size fragments ranged between 168–336 bp. In MRSA strains thirteen distinct spa types with 5–12 repeats were observed. The most frequent types of spa were t030, t037, t325, t421, t937, t1814 and t084. spa types t2421, t1814, t359 and t2617 identified for the first time in Iran. The present results showed high biofilm formation capacity and great diversity of variable region of spa gene in MRSA strains and confirmed that, spa typing provides valuable information on the epidemiologic features and discrimination of this bacterium.     

Is living in a border region a risk for a high prevalence of resistance?

This study assessed the antimicrobial resistance and population structure of Staphylococcus aureus isolated from general practice (GP) patients and nursing home (NH) residents in the province of Limburg (near the border with Germany and Belgium) in comparison with those obtained in the remaining provinces of the Netherlands. A total of 617 and 418 S. aureus isolates were isolated from 2,691 to 1,351 nasal swabs from GP patients and NH residents, respectively. Quantitative antibiotic susceptibility testing was performed using a microbroth dilution method. Putative methicillin-resistant S. aureus (MRSA) isolates were tested for the presence of the mecA gene and spa typing was performed on all S. aureus isolates. No significant differences in the prevalence of resistance were found between the two groups of GP isolates, but the isolates from the NH residents showed a lower resistance for trimethoprim–sulfamethoxazole (p = 0.003) in Limburg province compared with the remaining provinces in the Netherlands. Among the isolates from NH residents in Limburg province, the prevalence of spa-CC 084 was higher (p = 0.003) and that of spa-CC 002 was lower (p = 0.01) compared with isolates from NHs in the remaining provinces of the Netherlands. We observed no differences in resistance and population structure between S. aureus isolates from GP patients in Limburg and the remaining provinces of the Netherlands, and only a few differences were observed between the NH populations. There was no higher prevalence of resistance among the GP and NH isolates from Limburg compared with the remaining provinces.     

A recommendation to perform a blood culture before the administration of intravenous antibiotics increased the detection of Staphylococcus aureus bacteremia

In 2004, the Surviving Sepsis Campaign was launched to increase awareness and improve the outcome of severe sepsis. Accordingly, in Jönköping County, Sweden, a strong recommendation to perform a blood culture before the start of intravenous antibiotic treatment was introduced in 2007. Moreover, a reminder was included in the laboratory report to consult an infectious disease specialist when Staphylococcus aureus was isolated from a blood culture. Retrospectively, patients with at least one blood culture growing S. aureus during 2002 through 2003 (pre intervention n?=?58) or during 2008 through 2009 (post intervention n?=?100) were included. Medical records were evaluated regarding clinical data and outcome. Blood culture isolates were characterized by antibiotic susceptibility testing (AST) and S. aureus protein A (spa) gene typing. The annual incidence of S. aureus bacteremia (SAB) increased from 28 per 100,000 inhabitants at the pre intervention period to 45 per 100,000 at the post intervention period (p?=?0.046). During post intervention, the SAB incidence was significantly higher in men (p?=?0.009). The mortality rate during hospital stay was 14 % during pre intervention and 18 % during post intervention (p?=?0.47). The most common spa types were t012 and t084. The Surviving Sepsis Campaign resulted in an increased number of detected cases of SAB. The mortality rate was the same before and after the intervention, and no spa type correlated to certain clinical manifestations or mortality.     

Comparing Whole-Genome Sequencing with Sanger Sequencing for spa Typing of Methicillin-Resistant Staphylococcus aureus

spa typing of methicillin-resistant Staphylococcus aureus (MRSA) has traditionally been done by PCR amplification and Sanger sequencing of the spa repeat region. At Hvidovre Hospital, Denmark, whole-genome sequencing (WGS) of all MRSA isolates has been performed routinely since January 2013, and an in-house analysis pipeline determines the spa types. Due to national surveillance, all MRSA isolates are sent to Statens Serum Institut, where the spa type is determined by PCR and Sanger sequencing. The purpose of this study was to evaluate the reliability of the spa types obtained by 150-bp paired-end Illumina WGS. MRSA isolates from new MRSA patients in 2013 (n = 699) in the capital region of Denmark were included. We found a 97% agreement between spa types obtained by the two methods. All isolates achieved a spa type by both methods. Nineteen isolates differed in spa types by the two methods, in most cases due to the lack of 24-bp repeats in the whole-genome-sequenced isolates. These related but incorrect spa types should have no consequence in outbreak investigations, since all epidemiologically linked isolates, regardless of spa type, will be included in the single nucleotide polymorphism (SNP) analysis. This will reveal the close relatedness of the spa types. In conclusion, our data show that WGS is a reliable method to determine the spa type of MRSA.     

Genetic Variation among Staphylococcus aureus Strains from Bovine Milk and Their Relevance to Methicillin-Resistant Isolates from Humans

In genetic analysis of bovine Staphylococcus aureus isolates that are recognized as an important pathogenic bacterium in bovine mastitis, multilocus sequence typing (MLST) showed strong correlation to the results of pulsed-field gel electrophoresis, coa PCR-restriction fragment length polymorphism (RFLP), spa typing, and the coagulase serotyping method. According to MLST results, strains derived from sequence type 97 (ST97) and ST705 were suggested as not only dominant bovine S. aureus lineages in Japan but also pandemic bovine S. aureus lineages. Although both lineages seem to be distantly related to each other by phylogenetic analysis, both had common characteristics, i.e., lukM/lukF′-PV and coagulase serotype VI. These characteristics were very rare among minor bovine strains and human strains and may contribute to the host specificity of these lineages. Four methicillin-resistant S. aureus (MRSA) isolates were first confirmed from bovine milk in Japan; these isolates showed geno- and serotypes that were identical or similar to those of human MRSA isolates in Japan (ST5, staphylococcal cassette chromosome mec type II [SCCmec II], Spa type t002 or t375, and coagulase serotype II, and ST89, SCCmec IIIa, Spa type t5266, and coagulase serotype I). ST5 and ST89 are uncommon among bovine isolates in the world, whereas these STs are common among human MRSA isolates in Japan.Staphylococcus aureus is a major causal bacterium in contagious bovine mastitis. Although S. aureus has been isolated from heifer body sites and in the dairy farm environment, the lactating mammary gland is the primary reservoir of S. aureus involved in bovine mastitis (35, 36). Analyzing the genetic variation among S. aureus isolates from bovine milk is essential in bovine mastitis studies such as the identification of protective antigens for vaccine development and elucidation of the mechanism of pathogenesis. Furthermore, investigation of the relationship with isolates from environmental and other origins in dairy farms will lead to the identification of the sources of contagion on dairy farms and ways to prevent contagion from occurring.There have been reports of the isolation of methicillin-resistant S. aureus (MRSA) from bovine milk for a long time (7). The emergence of MRSA infection on dairy farms is of great concern for animal and public hygiene. MRSA-contaminated livestock products, including bovine milk, may become causal agents for human MRSA infection (27). In addition, the spread of MRSA in herds will cause a delay in the treatment of not only mastitis but also other bovine diseases, because most MRSA strains in Japan exhibit multidrug resistance (37). Therefore, screening for MRSA from bovine milk and genetic analysis of isolates are indispensable, and these analyses will lead to elucidation of the mechanisms of MRSA emergence or invasion in dairy farms.Many different typing methods have been developed for epidemiological studies or the analysis of genetic characteristics and relationships, but each method has drawbacks and advantages, so it is important that an optimal typing method be selected depending on the purpose. The correlation among different genotyping results, the relations with the phenotype, and the discriminatory power become reference points to select a genotyping method. Pulsed-field gel electrophoresis (PFGE), which shows high discriminatory power, is considered “the gold standard” for the typing of S. aureus isolates, but it is difficult to establish a precise database with PFGE (3). However, multilocus sequence typing (MLST) and X region of protein A gene (spa) typing are established and have websites with very useful resources (www.mlst.net and www.spaserver.ridom.de) for sharing and analyzing substantial databases of genotypes (9, 14). Other than these methods, multiple-locus variable-number tandem-repeat analysis (MLVA), which is based on the number of direct repeats of staphylococcal interspersed repeat units (SIRUs), has demonstrated high discriminatory power for human S. aureus isolates (19). Furthermore, staphylococcal cassette chromosome mec (SCCmec) typing is indispensable for the full characterization of MRSA (31).This study analyzed the genetic variation among S. aureus isolates from bovine milk in Japan using different typing methods. Consequently, the genetic characteristics of bovine milk isolates were evaluated along with their relationship with foreign bovine milk isolates and human MRSA isolates. Furthermore, the discriminatory powers of the different typing methods for bovine S. aureus isolates were evaluated and compared.     

Rapid and high-resolution distinction of community-acquired and nosocomial Staphylococcus aureus isolates with identical pulsed-field gel electrophoresis patterns and spa types

Methicillin-resistant Staphylococcus aureus (MRSA) represent a serious threat for public health worldwide. Of particular concern is the emergence of community-acquired MRSA, which is often difficult to distinguish from nosocomial MRSA due to a lack of suitable typing methods for early detection. For example, the USA300 pulsed-field gel electrophoresis (PFGE) pattern includes both the ‘classical’ community-acquired USA300 clone with spa type t008 and an epidemiologically unrelated nosocomial clone with spa type t024. Likewise, spa typing cannot distinguish the classic USA300 from nosocomial MRSA with the spa type t008. Since the fast and high-resolution distinction of these S. aureus types is important for infection prevention and surveillance, we investigated whether multiple-locus variable number tandem repeat fingerprinting (MLVF) can be applied to overcome these limitations. Indeed, MLVF correctly grouped 91 MRSA isolates belonging to the classic USA300 lineage, nosocomial MRSA isolates with the USA300 PFGE profile and spa type t024, and nosocomial MRSA isolates with spa type t008 into 3 distinct clusters. Importantly, several sub-clusters were also identified, reflecting epidemiological relationships between the respective isolates. We conclude that MLVF has the discriminatory power needed to rapidly distinguish very similar community-acquired and nosocomial MRSA isolates and that MLVF-based sub-clustering of isolates is highly useful for epidemiological investigations, outbreak prevention, and control.     

Clonality and Antimicrobial Susceptibility of Staphylococcus aureus and Methicillin-Resistant S. aureus Isolates from Food Animals and Other Animals

Out of 3,081 animals studied, 24.9% of pigs, 4.7% of chickens, 6.3% of dogs, 10.5% of cats, and 7.1% of rodents were Staphylococcus aureus positive. Prevalence of methicillin-resistant S. aureus (MRSA) was high in pigs (animals, 21.3%; batches, 46.5%), with all MRSA isolates and most methicillin-sensitive S. aureus isolates belonging to clonal complex 9 (CC9) and being multidrug resistant. The predominant S. aureus CCs among dog and cat isolates were similar. Among rodent isolates, CC398 predominated, with spa t034 the most frequent spa type detected.     

Rapid detection,differentiation and typing of methicillin-resistant Staphylococcus aureus harbouring either mecA or the new mecA homologue mecALGA251

The recent finding of a new mecA homologue, mecA_LGA251, with only 70% nucleotide homology to the conventional mecA gene has brought the routine testing for mecA as a confirmatory test for methicillin-resistant Staphylococcus aureus (MRSA) into question. A multiplex PCR was designed to differentiate mecA_LGA251 from the known mecA together with detection of lukF-PV and the spa gene fragments, enabling direct spa typing by sequencing of the PCR amplicons. The PCR analysis and subsequent spa typing were validated on a large collection (n = 185) of contemporary MRSA and methicillin-sensitive S. aureus isolates, including 127 isolates carrying mecA_LGA251. The mecA_LGA251 gene was situated in staphylococcal cassette chromosome mec type XI elements, and sequence variation within a 631-bp fragment of mecA_LGA251 in 79 isolates indicated a very conserved gene sequence. Following a successful validation, the multiplex PCR strategy was implemented in the routine testing of MRSA for national surveillance. Over a 2-month period, among 203 samples tested, 12 new MRSA cases caused by isolates carrying mecA_LGA251 were identified, emphasizing the clinical importance of testing for these new MRSA isolates.