Adapting spa typing for national laboratory-based surveillance of methicillin-resistant Staphylococcus aureus

Laboratory-based surveillance of methicillin-resistant Staphylococcus aureus (MRSA) monitors the baseline occurrence of different genotypes and identifies strains and transmission chains responsible for outbreaks. The consequences of substituting pulsed-field gel electrophoresis (PFGE) with spa typing as a first-line typing method were analyzed by typing 589 strains isolated between 1997 and 2006, with a focus on both short- and long-term correspondence between the PFGE and spa typing results. The study, covering these ten years, included all Finnish MRSA blood isolates and representatives of the two most prevalent MRSA strains (PFGE types FIN-4 and FIN-16) in Finland. In addition, all sporadic isolates from 2006 were included. spa typing was more expensive but approximately four times faster to perform than PFGE. Nearly 90% of FIN-4 and FIN-16 isolates showed consistent spa types, t172 and t067, respectively. spa typing predicted the PFGE result of the blood isolates by a Wallace coefficient of 0.9009, recognized internationally successful strains (t041, t067) to be common also in Finland, and identified a separate cluster of isolates, also related in time and place among the FIN-4 strains. Additional typing by another method was needed to provide adequate discrimination or to characterize isolates with a newly recognized spa type in Finland.     

Molecular characterization of Staphylococcus aureus from outpatients in the Caribbean reveals the presence of pandemic clones

Staphylococcus aureus infections continue to pose a global public health problem. Frequently, this epidemic is driven by the successful spread of single S. aureus clones within a geographic region, but international travel has been recognized as a potential risk factor for S. aureus infections. To study the molecular epidemiology of S. aureus infections in the Caribbean, a major international tourist destination, we collected methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) isolates from community-onset infections in the Dominican Republic (n = 112) and Martinique (n = 143). Isolates were characterized by a combination of pulsed-field gel electrophoresis (PFGE), spa typing, and multilocus sequence typing (MLST) typing. In Martinique, MRSA infections (n = 56) were mainly caused by t304-ST8 strains (n = 44), whereas MSSA isolates were derived from genetically diverse backgrounds. Among MRSA strains (n = 22) from the Dominican Republic, ST5, ST30, and ST72 predominated, while ST30 t665-PVL+ (30/90) accounted for a substantial number of MSSA infections. Despite epidemiological differences in sample collections from both countries, a considerable number of MSSA infections (~10%) were caused by ST5 and ST398 isolates at each site. Further phylogenetic analysis suggests the presence of lineages shared by the two countries, followed by recent genetic diversification unique to each site. Our findings also imply the frequent import and exchange of international S. aureus strains in the Caribbean.     

Population structure analyses of Staphylococcus aureus at Tygerberg Hospital,South Africa,reveals a diverse population,a high prevalence of Panton–Valentine leukocidin genes,and unique local methicillin-resistant S. aureus clones

Studies reporting on the population structure of Staphylococcus aureus in South Africa have focused only on methicillin-resistant S. aureus (MRSA). This study describes the population structure of S. aureus, including methicillin-susceptible S. aureus (MSSA) isolated from patients at Tygerberg Academic Hospital, Western Cape province. Pulsed-field gel electrophoresis (PFGE), detection of Panton–Valentine leukocidin (PVL), spa typing, multilocus sequence typing (MLST), agr typing and SCCmec typing were used to characterize strains. Of 367 non-repetitive S. aureus isolates collected over a period of 1 year, 56 (15.3%) were MRSA. Skin and soft tissue infections were the most frequent source (54.8%), followed by bone and joint (15.3%) and respiratory tract infections (7.7%). For strain typing, PFGE was the most discriminative method, and resulted in 31 pulsotypes (n = 345, 94.0%), as compared with 16 spa clonal complexes (CCs) (n = 344, 93.4%). Four MLST CCs were identified after eBURST of sequence types (STs) of selected isolates. One hundred and sixty isolates (MSSA, n = 155, 42.2%) were PVL-positive, and agr types I–IV and SCCmec types I–V were identified. Our S. aureus population consisted of genotypically diverse strains, with PVL being a common characteristic of MSSA. MSSA and MRSA isolates clustered in different clones. However, the dominant MRSA clone (ST612) also contained an MSSA isolate, and had a unique genotype. Common global epidemic MRSA clones, such as ST239-MRSA-III and ST36-MRSA-II, were identified. A local clone, ST612-MRSA-IV, was found to be the dominant MRSA clone.     

False-negative test results in the Slidex Staph Plus (bioMérieux) agglutination test are mainly caused by spa-type t001 and t001-related strains

The relative sensitivity of commercial agglutination kits for fast identification of S. aureus is usually given to be about 98%. This reported sensitivity has sometimes been questioned. In this study, three collections of molecularly defined, single-copy strains of S. aureus were used to compare the sensitivities of agglutination-based identification and the MALDI-TOF mass spectrometry-based identification using the Biotyper 2.0 database to a molecularly defined reference method. Clinical isolates (n = 363) of methicillin-susceptible S. aureus (MSSA) and 240 clinical isolates of methicillin-resistant S. aureus (MRSA) were included. In order to rule out a predominance of local MRSA-strains, a collection of 104 pulsed-field-gel electrophoresis divergent MRSA strains were also tested. MALDI-TOF MS using Biotyper database (Bruker) identified all isolates, whereas the Slidex Staph Plus (bioMérieux) detected only 98.0% of the MSSA, 94.5% of the MRSA and only 70.1% of the MRSA of the molecularly divergent strain collection. Interestingly, strains with a false-negative test result in the agglutination methods were mostly spa-type t001 and t001 related. The MALDI-TOF MS based identification can thus be used as an alternative identification method for suspected false-negative results from the agglutination tests, especially if the local prevalence of t001 and t001 related strains is high.     

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.     

A comparison of methicillin-resistant and methicillin-susceptible Staphylococcus aureus reveals no clinical and epidemiological but molecular differences

Most studies on Staphylococcus aureus have focused on the molecular epidemiology of methicillin-resistant S. aureus (MRSA) infections. In contrast, little information is available regarding the molecular epidemiology of currently circulating methicillin-susceptible S. aureus (MSSA) isolates in hospital settings, an epoch when the epidemiology of S. aureus has undergone significant changes. We conducted a cross-sectional study to compare the clinical, epidemiological, and genetic characteristics of MSSA and MRSA isolates at 3 tertiary-care hospitals in Medellín, Colombia, from February 2008 to June 2010. The infections were classified according to the Centers for Disease Control and Prevention (CDC) definitions. Genotypic analysis included spa typing, multilocus sequence typing (MLST) and staphylococcal cassette chromosome (mec) (SCCmec) typing. A total of 810 patients was enrolled. One hundred infections (12.3%) were classified as community-associated (31 CA-MSSA, 69 CA-MRSA), 379 (46.8%) as healthcare-associated community-onset (136 HACO-MSSA, 243 HACO-MRSA), and 331 (40.9%) as healthcare-associated hospital-onset (104 HAHO-MSSA, 227 HAHO-MRSA). Genotype analyses showed a higher diversity and a more varied spa type repertoire in MSSA than in MRSA strains. Most of the clinical-epidemiological characteristics and risk factors evaluated did not allow for discriminating MRSA- from MSSA-infected patients. The lack of equivalence among the genetic backgrounds of the major MSSA and MRSA clones would suggest that the MRSA clones are imported instead of arising from successful MSSA clones. This study emphasizes the importance of local surveillance to create public awareness on the changing S. aureus epidemiology.     

Molecular Characterization of Methicillin-Susceptible Staphylococcus aureus Clinical Isolates in the United States, 2004 to 2010

While much is known about the geographic distribution of different clonal types of methicillin-resistant Staphylococcus aureus (MRSA), few studies have assessed the molecular epidemiology of methicillin-susceptible S. aureus (MSSA), despite its continued clinical importance. In each U.S. Census region, reference laboratories collected successive MSSA isolates from patients with invasive or superficial staphylococcal infections for use in the Tigecycline Evaluation and Surveillance Trial. All isolates from the periods of 2004 to 2005 and 2009 to 2010 underwent antimicrobial susceptibility testing and characterization of their staphylococcal protein A (spa) type. Of the 708 isolates analyzed, 274 spa types were identified and divided into 15 genetic clusters. The most common clones were spa t002 (n = 63, 8.9%) and t008 (n = 56, 7.9%). While the distribution of the predominant spa types did not differ by U.S. Census region or time period, spa t008 was nearly twice as common in community skin and soft tissue infections than in nosocomial bloodstream infections (11.1% versus 5.6%, respectively; P = 0.008). Despite such differences, both community and nosocomial settings had diverse staphylococcal clonal types representing all major spa clusters. In contrast to those of MRSA, MSSA infectious isolates show wide genetic diversity without clear geographical or temporal clustering. Notably, the prevalent MSSA strains (spa t002 and spa t008) are analogous to the predominant MRSA clones, further demonstrating the importance of both lineages.     

Typing of Panton-Valentine leukocidin-encoding phages carried by methicillin-susceptible and methicillin-resistant Staphylococcus aureus from Italy

Panton-Valentine leukocidin (PVL) is the hallmark of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) but can also be found in methicillin-susceptible S. aureus (MSSA) sharing pathogenic and epidemiological characteristics of CA-MRSA. PVL is encoded by two co-transcribed genes that are carried by different staphylococcal bacteriophages. We applied an extended PCR-based typing scheme for the identification of two morphological groups (elongated-head group and icosahedral-head group I phages) and specific PVL phage types in S. aureus isolates recovered in Italy. We examined 48 PVL-positive isolates (25 MSSA and 23 MRSA) collected from different hospital laboratories from April 2005 to May 2011. spa typing, multilocus sequence typing and staphylococcal cassette chromosome mec typing were applied to categorize the isolates. Phage typeability was 48.0% in MSSA and 91.3% in MRSA, highlighting the limitation of the PCR typing scheme when applied to PVL-positive MSSA. Five different PVL phages and two variants of a known phage were detected, the most prevalent being ΦSa2usa, recovered in 15 out of 48 (31.2%) isolates, and carried by both MSSA and MRSA belonging to CC8 and CC5. The recently described ΦTCH60 was recovered in four isolates. A PVL phage (ΦSa119) from an ST772 MRSA, that was not detected using the previous typing scheme, was sequenced, and new primers were designed for the identification of the icosahedral-head group II PVL phages present in ST772 and ST59 MRSA. A comprehensive PVL-phage typing can contribute to the understanding of the epidemiology and evolution of PVL-positive MSSA and MRSA.     

Characterisation of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolates from bloodstream infections,Democratic Republic of the Congo

Staphylococcus aureus is known worldwide as an invasive pathogen, but information on S. aureus from bloodstream infections in Central Africa remains scarce. A collection of S. aureus blood culture isolates recovered from hospitals in four provinces in the Democratic Republic of the Congo (2009–2013) was assessed. A total of 27/108 isolates were methicillin-resistant S. aureus (MRSA), of which >70% were co-resistant to aminoglycosides, tetracyclines, macrolides and lincosamides. For MRSA and methicillin-susceptible S. aureus (MSSA) isolates, resistance to chloramphenicol and trimethoprim–sulphamethoxazole (TMP-SMX) was <10%. However, 66.7% (72/108) of all isolates harboured the trimethoprim resistance gene dfrG. More than three-quarters (84/108, 77.8%) of isolates belonged to CC5, CC8, CC121 or CC152. Genetic diversity was higher among MSSA (31 spa types) compared to MRSA (four spa types). Most MRSA (23/27, 85.2%) belonged to CC8-spa t1476-SCCmec V and 17/23 (73.9%) MRSA ST8 were oxacillin susceptible but cefoxitin resistant. Among MRSA and MSSA combined, 49.1% (53/108) and 19.4% (21/108) contained the genes encoding for Panton–Valentine leucocidin (lukS-lukF PV, PVL) and toxic shock syndrome toxin-1 (tst, TSST-1), respectively. PVL was mainly detected among MSSA (51/53 isolates harbouring PVL were MSSA, 96.2%) and associated with CC121, CC152, CC1 and CC5. TSST-1 was associated with CC8-spa t1476-SCCmec V. The immune evasion cluster (IEC) genes scn, sak and chp were detected in 81.5% of isolates (88/108, equally represented among MSSA and MRSA). The present study confirms the occurrence of MRSA with high levels of multidrug co-resistance and PVL-positive MSSA among invasive S. aureus isolates in Central Africa.     

High frequency of multidrug‐resistant Staphylococcus aureus with SCCmec type III and Spa types t037 and t631 isolated from burn patients in southwest of Iran

Methicilin resistance Staphylococcus aureus (MRSA) infections are the major challenges in hospitals, especially in the burn units. The use of molecular typing methods is essential for tracking the spread of S. aureus infection and epidemiological investigations. The aim of this study was to find the profile of the spa types and also the prevalence of each SCCmec type of S. aureus strains in a central burn hospital in southwest of Iran. A total of 81 non‐duplicate S. aureus were isolated from burn patients between April 2011 and February 2012. The susceptibility of the isolates against 13 different antibiotics was tested by disk agar diffusion (DAD) method. MRSA strains were identified by amplification of mecA gene. Multiplex‐polymerase chain reaction (PCR) technique was used to determine the SCCmec types of MRSA strains and all the S. aureus isolates were typed by spa typing method. Detection of mecA gene showed that 70 (86.4%) of the isolates were MRSA. The highest rate of resistance was observed for penicillin (97.5%) and erythromycin (77.8%). None of the isolates were resistant to vancomycin. Sixty‐seven of the 70 MRSA isolates harbored only SCCmec type III and three untypeable isolates. Five different spa types were detected. The most common spa types were t037 (42.5%) and t631 (34.5%) and were only found in MRSA isolates. Only SCCmec type III was found in burn patients which emphasizes the HA‐MRSA origin of these strains. Only five different spa types identified in this study are in accordance with one SCCmec type which indicates that a limited number of bacterial colons are circulated in the burn unit in this hospital.     

Molecular typing of methicillin-resistantStaphylococcus aureus on the basis of protein A gene polymorphism

The polymorphic X-region of the protein A gene (spa) was used for molecular typing of methicillin-resistantStaphylococcus aureus (MRSA) strains. The X-region is characterized by a variable number (between 3 and 15) of small repeats. DNA sequencing of MRSA strains revealed 25 distinct repeats. Analysis of MRSA strains grown in vitro and in vivo revealed that the X-region was sufficiently stable for epidemiologic typing of MRSA strains.Spa typing of MRSA strains was compared to phage typing and, in general, concordance was found between the two methods. However,spa typing was more sensitive, allowing differentiation of strains within a particular phage type. Results obtained withspa typing suggest that hospital outbreaks may be caused by two or more MRSA strains.Spa typing may be an important tool in unravelling the spread of MRSA strains within and between hospitals.     

Molecular characterization and susceptibility of methicillin-resistant and methicillin-susceptible Staphylococcus aureus isolates from hospitals and the community in Vladivostok,Russia

A prospective study was conducted during an 8-month period, from August 2006 to April 2007, to describe the epidemiology of Staphylococcus aureus-associated infections. In addition, the molecular characteristics, antimicrobial susceptibilities and antibiotic resistance determinants were identified in S. aureus isolates from hospitals and the community in Vladivostok, Russia. Among the 63 S. aureus isolates eligible for this study, methicillin resistance was observed in 48% (n = 30). Hospital-acquired strains accounted for 93% (28/30) of all methicillin-resistant S. aureus (MRSA) isolates. The major MRSA clone (sequence type (ST) 239, staphylococcal cassette chromosome mec (SCCmec) type III, Panton--Valentine leukocidin (PVL)-negative, with two related staphylococcal protein A gene (spa) types (types 3 and 351)) represented 90% of all of the MRSA isolates. This clone was multidrug-resistant, and 41% of isolates showed resistance to rifampicin. Community-acquired MRSA isolates (n = 2) were categorized as ST30, SCCmecIV, spa type 19, and PVL--positive, and as ST8, SCCmecIV, of a novel spa type 826, and PVL-negative. Eight different STs were detected among methicillin-susceptible S. aureus (MSSA) isolates, of which 55% were PVL--positive. One MSSA clone, which was categorized as ST121, spa type 273, and PVL--positive, caused fatal community-acquired pneumonia infections. The strains predominantly isolated in hospitals in Russia belonged to the multidrug-resistant Brazilian/Hungarian ST239 MRSA clone; however, this clone has new antibiotic susceptibilities. Additionally, the emergence of PVL--positive MSSA strains with enhanced virulence was observed, warranting continued surveillance.     

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.     

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.     

Combinations of cefoxitin plus other β-lactams are synergistic in vitro against community associated methicillin-resistant Staphylococcus aureus

In vitro studies demonstrate that oxacillin minimal inhibitory concentrations (MICs) of methicillin-resistant S. aureus (MRSA) strains USA300 and 400 decrease in the presence of cefoxitin. The aim of this study was to characterize the activity of cefoxitin plus β-lactams against a collection of MRSA isolates. We assessed the in vitro antimicrobial activity of a selection of β-lactams alone and together with subinhibitory concentrations of cefoxitin against a collection of MRSA, methicillin-susceptible S. aureus (MSSA), and vancomycin-intermediate S. aureus (VISA) isolates using MICs and time kill assays. For community-associated (CA) MRSA strains USA300 and USA400, MICs of nafcillin, cefazolin, cephalexin, cefuroxime, ceftriaxone and cefotaxime decreased by 8- to 64-times in the presence of 10 μg/ml cefoxitin. In contrast, for hospital-associated (HA) strains COLn, N315, and Mu50, there was no change in any β-lactam MIC in the presence of cefoxitin. When combined with cefoxitin, the cephalexin MIC decreased for eight CA-MRSA and five MSSA sequence types but did not change for seven HA-MRSA sequence types. β-lactam/cefoxitin combinations were synergistic against CA- but not HA-MRSA strains in time kill assays. Cefoxitin combined with a variety of β-lactams enhances their activity against CA-MRSA strains in vitro. Further studies of combination β-lactam therapy may provide insight into β-lactam biology, penicillin binding protein cooperativity, and novel therapeutic strategies against MRSA.     

Analysis of typing methods for epidemiological surveillance of both methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains

Sequence-based methods for typing Staphylococcus aureus, such as multilocus sequence typing (MLST) and spa typing, have increased interlaboratory reproducibility, portability, and speed in obtaining results, but pulsed-field gel electrophoresis (PFGE), remains the method of choice in many laboratories due to the extensive experience with this methodology and the large body of data accumulated using the technique. Comparisons between typing methods have been overwhelmingly based on a qualitative assessment of the overall agreement of results and the relative discriminatory indexes. In this study, we quantitatively assess the congruence of the major typing methods for S. aureus, using a diverse collection of 198 S. aureus strains previously characterized by PFGE, spa typing, MLST, and, in the case of methicillin-resistant S. aureus (MRSA), SCCmec typing in order to establish the quantitative congruence between the typing methods. The results of most typing methods agree in that MRSA and methicillin-susceptible S. aureus (MSSA) differ in terms of diversity of genetic backgrounds, with MSSA being more diverse. Our results show that spa typing has a very good predictive power over the clonal relationships defined by eBURST, while PFGE is less accurate for that purpose but nevertheless provides better typeability and discriminatory power. The combination of PFGE and spa typing provided even better results. Based on these observations, we suggest the use of the conjugation of spa typing and PFGE typing for epidemiological surveillance studies, since this combination provides the ability to infer long-term relationships while maintaining the discriminatory power and typeability needed in short-term studies.     

Prevalence and accessory gene regulator (agr) analysis of vancomycin-intermediate Staphylococcus aureus among methicillin-resistant isolates in Taiwan—SMART program, 2003

The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in nosocomial staphylococcal infections in Taiwan has exceeded 50% since 2000. However, little relevant data has been available concerning vancomycin-intermediate S. aureus (VISA) and heteroresistant VISA (hVISA). We collected 1,000 MRSA isolates from ten medical center hospitals in Taiwan during 2003. All were initially screened for reduced susceptibility to vancomycin on brain heart infusion (BHI) agar containing 5 mg/L vancomycin. Among 34 MRSA isolates that grew on the screening plates, two VISA isolates (0.2%) and seven hVISA isolates (0.7%) were evident. Vancomycin-resistant S. aureus was not detected. The accessory gene regulator (agr) typing of all 1,000 MRSA strains were typed by multiplex polymerase chain reaction (PCR); 919 strains (91.9%) including the VISA and hVISA isolates belonged to agr group I, 78 strains (7.8%) were agr group II, two strains (0.2%) were agr group III, and one isolate (0.1%) was agr group IV. There was no relationship between sample sites and agr typing. In 2003, the incidence of hVISA and VISA in Taiwan was low. Continued surveillance is recommended, given the implementation of new Clinical and Laboratory Standards Institute (CLSI) criteria for S. aureus and the increasing clinical use of glycopeptides.     

Characterization of methicillin-resistant Staphylococcus aureus from healthy carrier chickens

Methicillin-resistant Staphylococcus aureus (MRSA) has long been recognized as an important pathogen in human medicine leading to hospital and community-acquired infections. However, it is now also considered a growing problem in veterinary medicine, although causing little or no disease. Although MRSA has already been detected in livestock including poultry, little is known about the epidemiology of MRSA in broiler and layer chickens. We therefore investigated 372 poultry farms in Belgium. We also compared the isolation method recommended by the European Food Safety Authority using two enrichment steps with an isolation method using only one enrichment step. Isolated MRSA was characterized by means of antimicrobial resistance profiling, spa typing, multi-locus sequence typing, and SCCmec typing. MRSA prevalence was 0.8% using the double broth enrichment method, while using the single broth enrichment method it was 1.8%. Five MRSA strains belonged to the livestock-associated (LA) MRSA ST398 (four with spa type t011 and one with t899), and three to the hospital-acquired MRSA ST239 spa type t037. The ST239 strains carried SCCmec type III while those belonging to ST398 carried SCCmec type IV or V. All isolates showed additional resistance to erythromycin and tetracycline apart from the expected resistance to cefoxitin and penicillin. All strains were susceptible to linezolid, mupirocin and vancomycin. In conclusion, a higher sensitivity for the isolation of LA-MRSA was obtained using only one enrichment step. While the typical LA-MRSA ST398 was present at low prevalence in poultry, human-associated strains have also been found.     

What Is the Origin of Livestock-Associated Methicillin-Resistant Staphylococcus aureus Clonal Complex 398 Isolates from Humans without Livestock Contact? An Epidemiological and Genetic Analysis

Fifteen percent of all methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 398 (CC398) human carriers detected in The Netherlands had not been in direct contact with pigs or veal calves. To ensure low MRSA prevalence, it is important to investigate the likely origin of this MRSA of unknown origin (MUO). Recently, it was shown that CC398 strains originating from humans and animals differ in the presence of specific mobile genetic elements (MGEs). We hypothesized that determining these specific MGEs in MUO isolates and comparing them with a set of CC398 isolates of various known origin might provide clues to their origin. MUO CC398 isolates were compared to MRSA CC398 isolates obtained from humans with known risk factors, a MRSA CC398 outbreak isolate, livestock associated (LA) MRSA CC398 isolates from pigs, horses, chickens, and veal calves, and five methicillin-susceptible Staphylococcus aureus (MSSA) CC398 isolates of known human origin. All strains were spa typed, and the presence or absence of, scn, chp, φ3 int, φ6 int, φ7 int, rep7, rep27, and cadDX was determined by PCRs. The MRSA CC398 in humans, MUO, or MRSA of known origin (MKO) resembled MRSA CC398 as found in pigs and not MSSA CC398 as found in humans. The distinct human MSSA CC398 spa type, t571, was not present among our MRSA CC398 strains; MRSA CC398 was tetracycline resistant and carried no φ3 bacteriophage with scn and chp. We showed by simple PCR means that human MUO CC398 carriers carried MRSA from livestock origin, suggestive of indirect transmission. Although the exact transmission route remains unknown, direct human-to-human transmission remains a possibility as well.     

Genotyping and antimicrobial resistance of Staphylococcus aureus isolates from diseased turkeys

Staphylococcus aureus is a highly versatile pathogen in a large number of domestic animals, including avian species. To gain deeper insight into the epidemiology and diversity of S. aureus associated with articular disease in domestic turkeys, isolates were collected from infected foot joints of turkeys in Brittany (France). A total of 34 isolates were recovered and characterized by means of antimicrobial resistance, staphylococcal protein A typing, macrorestriction pulsed-field gel electrophoresis and micro-array analysis. Thirty isolates were identified as clonal complex (CC) 398 and methicillin-susceptible S. aureus (MSSA), one was identified as a methicillin-resistant S. aureus (MRSA) CC398 isolate, and the remaining were also MSSA and belonged to CC5, CC101, and CC121. Eleven different antimicrobial resistance patterns were detected, with most isolates resistant to penicillin and tetracycline. Based on all typing methods used, the 34 isolates could be divided into 22 different strains. Results on selected isolates, genotyped using microarrays, indicated a high homogeneity among pathogenic MSSA isolates from turkeys. Moreover, all isolates, except the unique MRSA isolate, carried specific φAvβ prophage avian-niche-specific genes, demonstrating the versatility of S. aureus to adapt to the specific ecological poultry niche.