Comparison of Fingerprinting Methods for Typing Methicillin-Resistant Staphylococcus aureus Sequence Type 398

This study evaluates the multiple-locus variable-number tandem-repeat assay (MLVA) and pulsed-field gel electrophoresis (PFGE) when using restriction enzymes BstZI, SacII, and ApaI to fingerprint a diverse collection of methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) sequence type 398 (ST398) isolates. These isolates had been characterized previously by multilocus sequence typing, spa typing, and staphylococcal cassette chromosome mec (SCCmec) typing. Typeability and discriminatory power were analyzed, and the concordance between the various methods was determined. All MRSA ST398 isolates were typeable by the MLVA and PFGE using BstZI, SacII, and ApaI. With each method, the MRSA ST398 isolates formed a separate group from the two non-ST398 MRSA strains. PFGE, performed with any of the three restriction enzymes, had the most discriminatory power, followed by MLVA, spa typing, and SCCmec typing. The MLVA showed the highest concordance with PFGE using ApaI and spa typing. As further expressed by the Wallace coefficient, the MLVA type was poorly predicted by spa typing, whereas the spa type was well predicted by MLVA. PFGE, using a combination of all three restriction enzymes, had the highest concordance with the MLVA but had a low probability of being predicted by MLVA. PFGE, using a combination of all three restriction enzymes, was able to predict SCCmec type and MLVA type completely and had a high probability of predicting spa type. Both the MLVA and PFGE could be used to discriminate among the MRSA ST398 isolates. Although the MLVA is a faster technique, PFGE had more discriminatory power than the MLVA, especially when a combination of restriction enzymes was used.Infections caused by methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) have been a problem in hospitals and nursing homes for many decades. These MRSA isolates are therefore called health care-associated MRSA (HA-MRSA) isolates (1). Since the early 1990s, MRSA has emerged in healthy persons without risk factors for MRSA infections. These isolates are the so-called community-associated MRSA (CA-MRSA) isolates (20). In the last few years, MRSA has been isolated from livestock animals (pigs in particular) and pig farmers (5, 6, 30). These MRSA strains are called animal-associated MRSA (AA-MRSA) strains. It seems that (livestock) animals form a new, separate reservoir. These AA-MRSA strains all appear to belong to the new clonal complex 398 (CC398), with sequence type 398 (ST398) as the basic type, as determined by multilocus sequence typing (MLST) (29). MRSA ST398 has already been isolated in Europe, Asia, and North America (32). Considering the worldwide spread of MRSA, epidemiological questions arise about its transmission within farms, among farms, and from farms to the population. Fast and inexpensive typing methods with good discriminatory power are necessary to conduct large-scale epidemiological studies.Traditionally, human MRSA isolates have been typed by pulsed-field gel electrophoresis (PFGE), using SmaI as the restriction enzyme (19). The advantages of using PFGE are good discriminatory power and good reproducibility at the interlaboratory level when standardized protocols are used. However, AA-MRSA is not typeable by this method, as the activity of SmaI is blocked due to methylation of the restriction site (2).More recently, methods based on DNA sequencing, such as MLST and spa typing, are increasingly being used to discriminate among different MRSA strains. Given their excellent interlaboratory reproducibility, online databases have been made to collate and harmonize data from various geographic regions. The drawback of MLST, which measures sequence variation at seven housekeeping loci, is its limited use with epidemiological studies due to its weak discriminatory power, time-consuming protocols, and high costs. spa typing, based on the variation in repeats present in the X-region of staphylococcal protein A, has a discriminatory power that lies between those of PFGE and MLST. Within ST398, several spa types have been distinguished, although the number of spa types seems rather limited in most countries.One promising method is the multiple-locus variable-number tandem-repeat assay (MLVA), a PCR-based method, based on the analysis of the number of repeats in the variable-number tandem-repeat regions of various individual genes. This method has proven to be useful for typing both Staphylococcus aureus and clinical MRSA isolates with good reproducibility and good discriminatory power. Because the MLVA is also simple, inexpensive, and easy to interpret, it is useful as a typing method for large-scale epidemiological studies (10, 11, 12, 15, 16, 17, 23, 24, 27).This study aimed to investigate various methods for typing MRSA ST398 isolates. An MLVA, consisting of a selection of primers from three existing MLVA systems, was tested with a collection of MRSA ST398 isolates. In addition, PFGE with restriction enzymes other than SmaI was performed with this set of isolates. These isolates had been previously characterized by MLST, spa typing, and staphylococcal cassette chromosome mec (SCCmec) typing. Typeability and discriminatory power were analyzed for all methods, and the concordance among the different methods was determined.     

Methicillin-Resistant Staphylococcus aureus in Spain: Molecular Epidemiology and Utility of Different Typing Methods

In a point-prevalence study performed in 145 Spanish hospitals in 2006, we collected 463 isolates of Staphylococcus aureus in a single day. Of these, 135 (29.2%) were methicillin (meticillin)-resistant S. aureus (MRSA) isolates. Susceptibility testing was performed by a microdilution method, and mecA was detected by PCR. The isolates were analyzed by pulsed-field gel electrophoresis (PFGE) after SmaI digestion, staphylococcal chromosomal cassette mec (SCCmec) typing, agr typing, spa typing with BURP (based-upon-repeat-pattern) analysis, and multilocus sequence typing (MLST). The 135 MRSA isolates showed resistance to ciprofloxacin (93.3%), tobramycin (72.6%), gentamicin (20.0%), erythromycin (66.7%), and clindamycin (39.3%). Among the isolates resistant to erythromycin, 27.4% showed the M phenotype. All of the isolates were susceptible to glycopeptides. Twelve resistance patterns were found, of which four accounted for 65% of the isolates. PFGE revealed 36 different patterns, with 13 major clones (including 2 predominant clones with various antibiotypes that accounted for 52.5% of the MRSA isolates) and 23 sporadic profiles. Two genotypes were observed for the first time in Spain. SCCmec type IV accounted for 6.7% of the isolates (70.1% were type IVa, 23.9% were type IVc, 0.9% were type IVd, and 5.1% were type IVh), and SCCmec type I and SCCmec type II accounted for 7.4% and 5.2% of the isolates, respectively. One isolate was nontypeable. Only one of the isolates produced the Panton-Valentine leukocidin. The isolates presented agr type 2 (82.2%), type 1 (14.8%), and type 3 (3.0%). spa typing revealed 32 different types, the predominant ones being t067 (48.9%) and t002 (14.8%), as well as clonal complex 067 (78%) by BURP analysis. The MRSA clone of sequence type 125 and SCCmec type IV was the most prevalent throughout Spain. In our experience, PFGE, spa typing, SCCmec typing, and MLST presented good correlations for the majority of the MRSA strains; we suggest the use of spa typing and PFGE typing for epidemiological surveillance, since this combination is useful for both long-term and short-term studies.Methicillin (meticillin)-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-acquired infections worldwide (5, 25). The appearance of MRSA in the community and the potential risk of it entering hospitals are also matters of concern (29, 44). Moreover, the increasing prevalence of multidrug resistance and the emergence of isolates with intermediate or high-level vancomycin resistance emphasize the importance of the use of infection control measures (2, 49, 50). Although the rates of isolation of MRSA have been increasing throughout the world for the last few decades and in some areas the rates reach >50%, there are considerable variations in the prevalence of MRSA according to geographic area (3, 18, 21, 39, 44). In Spain, the prevalence of MRSA increased from 1.5% in 1986 to 29.2% in 2006, although it seems to have stabilized (13). Despite the worldwide increase in isolation rates, only a limited number of clones of MRSA have spread in most countries (20).Historically, the dissemination of epidemic clones such as EMRSA type 15 (EMRSA-15), EMRSA-16, the Iberian clone, and the Brazilian clone, as well as the high incidence of the community-acquired MRSA USA300 clone, has led to the increased use of molecular typing methods (11, 38, 42, 47, 53).In recent years, a variety of molecular techniques have been used for the typing of MRSA isolates. Of these, SmaI macrorestriction analysis is the “gold standard” for the analysis of the local epidemiology in the short term, spa typing in combination with BURP (based-upon-repeat-pattern) analysis has become a frontline tool for routine epidemiological typing, and multilocus sequence typing (MLST)-staphylococcal chromosomal cassette mec (SCCmec) typing is the reference method for the definition of MRSA clones (10, 34, 37, 46).The aim of the present study was to determine which clones are circulating in Spain and whether the strains have spread between hospitals by analyzing a representative sample of isolates collected in a point-prevalence study. Isolates were grouped by using pulsed-field gel electrophoresis (PFGE) and spa typing and were assigned to MRSA clones on the basis of MLST and SCCmec typing. The congruence between the different grouping methods was assessed.(This study was presented in part at the 47th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 2007 [O. Cuevas, C. Marcos, P. Trincados, T. Boquete, E. Cercenado, E. Bouza, and A. Vindel; abstr. C2-148].)     

Comparison of Typing Results Obtained for Methicillin-Resistant Staphylococcus aureus Isolates with the DiversiLab System and Pulsed-Field Gel Electrophoresis

We compared the results of typing methicillin-resistant Staphylococcus aureus (MRSA) isolates using the DiversiLab system (DL) to the results obtained using pulsed-field gel electrophoresis (PFGE). One hundred five MRSA isolates of PFGE types USA100 to USA1100 and the Brazilian clone, from the Centers for Disease Control and Prevention (CDC) and Project ICARE strain collections, were typed using DL. In addition, four unique sets of MRSA isolates from purported MRSA outbreaks that had been previously typed by DL, each consisting of six isolates (where five isolates were classified as indistinguishable by DL and one was an unrelated DL type) were typed by PFGE. DL separated the 105 MRSA isolates of known USA types into 11 clusters and six unique banding patterns. DL grouped most of the USA100, USA200, and USA1100 isolates into unique clusters. Multilocus sequence type 8 isolates (i.e., USA300 and USA500) often clustered together at >95% similarity in DL dendrograms. Nevertheless, USA300 and USA500 DL patterns could be distinguished using the pattern overlay function of the DL software. Among the hospital outbreak clusters, PFGE and DL identified the same “unrelated” organism in three of four sets. However, PFGE showed more pattern diversity than did DL, suggesting that two of the sets were less likely to represent true outbreaks. In summary, DL is useful for screening MRSA isolates to rule out potential outbreaks of MRSA in hospitals, but PFGE provides better discrimination of potential outbreak strains and is more useful for confirming strain relatedness and specific USA types.Although pulsed-field gel electrophoresis (PFGE) is often considered the gold standard for typing methicillin-resistant Staphylococcus aureus (MRSA) isolates for epidemiologic studies (8, 12, 13), PFGE requires several days to complete and the results are often difficult for inexperienced users to interpret. On the other hand, DNA sequence-based methods, such as spa typing, which has also been shown to be useful for epidemiologic studies of MRSA (3), are not practical for many clinical laboratories in the United States, which lack access to DNA sequencing facilities. An alternative strain typing method, which is available commercially, is the DiversiLab typing system (DL) (bioMérieux, Inc., Durham, NC), which uses the presence of DNA repetitive elements present in the organism''s genome to determine the genetic relatedness of bacterial and fungal isolates (4-6, 9, 18). DL has been used successfully in several MRSA typing studies to distinguish sporadic from outbreak-related isolates and is noted to be more rapid to perform and easier to learn than PFGE (14, 15). Agreement between DL clusters of organisms and USA PFGE types, as defined by McDougal et al. (12), was reported for five well-defined U.S. outbreaks, although specific data were not shown (14). However, a recent study of representative MRSA strains from the Harmony collection in Europe concluded that while DL, PFGE, and multilocus sequence typing (MLST) provided concordant classification of strains, PFGE showed a higher level of strain discrimination than either DL or MLST (17). Thus, whether DL can differentiate accurately among USA types remains an open question.The goal of this study was to use DL to characterize a series of MRSA isolates of known PFGE types from U.S. hospitals to determine whether DL could (i) differentiate among PFGE types USA100 through USA1100, (ii) identify DL banding patterns that correlated with specific USA types, and (iii) differentiate contemporary outbreak-related MRSA isolates from sporadic isolates collected from U.S. hospitals.     

Improved Methods for Detection of Methicillin-Resistant Staphylococcus aureus

 In order to assess the performance of two detection methods, a set of 93 recent clinical isolates of Staphylococcus aureus, including a large number of strains that demonstrated low-level methicillin-resistance were evaluated using the MRSA-Screen (Denka Seiken, Japan), a commercial latex agglutination test to detect penicillin-binding protein 2′ (PBP2′), and a polymerase chain reaction assay using the LightCycler Instrument (Roche Diagnostics, Switzerland). The results show that the latex agglutination test is highly sensitive if performed after induction by cefoxitin. Inconclusive results can be rapidly confirmed on the same day by real-time polymerase chain reaction used to detect mecA and femA genes.     

Prospective Genotyping of Hospital-Acquired Methicillin-Resistant Staphylococcus aureus Isolates by Use of a Novel,Highly Discriminatory Binary Typing System

In settings of high methicillin-resistant Staphylococcus aureus (MRSA) prevalence, detection of nosocomial transmission events can be difficult without strain typing. Prospective typing of all MRSA isolates could potentially identify transmission in a timely fashion, making infection control responses to outbreaks more effective. We describe the development and evaluation of a novel 19-target binary typing system for MRSA using the multiplex-PCR/reverse line blot hybridization platform. Pulse-field gel electrophoresis (PFGE), spa typing, and phage-derived open reading frame (PDORF) typing were performed for comparison. The system was utilized to identify transmission events in three general surgical wards over a 12-month period. Initial MRSA isolates from 273 patients were differentiated into 55 unique binary types. One or more potential contacts colonized with the same MRSA strain were identified in 69 of 87 cases (79%) in which definite or possible nosocomial MRSA acquisition had occurred. The discriminatory power of the typing system was similar to that of PFGE (Simpson''s index of diversity [D] = 0.994, versus 0.987) and higher than that of spa typing (D = 0.926). Strain typing reduced the total number of potential MRSA-colonized source contacts from 859 to 212 and revealed temporal clustering of transmission events. Prospective MRSA typing using this novel binary typing method can rapidly identify nosocomial transmission events, even in high-prevalence settings, which allows timely infection control interventions. The system is rapid, inexpensive, discriminatory, and suitable for routine, high-throughput use in the hospital microbiology laboratory.     

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.     

Community Strain of Methicillin-Resistant Staphylococcus aureus Involved in a Hospital Outbreak

Western Australia (WA) has been able to prevent methicillin-resistant Staphylococcus aureus (MRSA) strains from outside of the state from becoming established in its hospitals. Recently, a single-strain outbreak of MRSA occurred in a WA metropolitan teaching hospital following admission of an infected patient from a remote community. The strain responsible for the outbreak was unrelated to any imported strains and spread rapidly in the hospital. Screening of two remote communities in the region from which the index case came revealed that 42% of the people in one community and 24% in the other carried MRSA. Isolates were typed by resistance pattern, plasmid analysis, contour-clamped homogeneous electric field electrophoresis, bacteriophage pattern, and coagulase gene restriction fragment length polymorphism. It was found that of the people carrying MRSA, 39% in the former community and 17% in the latter community were carrying an MRSA strain which was indistinguishable from the strain that caused the hospital outbreak.     

Comparison of Two Multilocus Variable-Number Tandem-Repeat Methods and Pulsed-Field Gel Electrophoresis for Differentiating Highly Clonal Methicillin-Resistant Staphylococcus aureus Isolates

In the United Kingdom, EMRSA-15 and EMRSA-16 account for the majority (∼90%) of nosocomial methicillin-resistant Staphylococcus aureus (MRSA) infections. Currently, the standard typing technique, pulsed-field gel electrophoresis (PFGE), is laborious and insufficient for discriminating between closely related subtypes of EMRSA-15 and -16. The objective of the present study was to compare the usefulness of multilocus variable-number tandem-repeat fingerprinting (MLVF) and multilocus variable-number tandem-repeat analysis (MLVA) with PFGE for subtyping these highly clonal MRSA lineages. A panel of 85 MRSA isolates (41 EMRSA-15, 20 EMRSA-16, and 24 MRSA isolates with diverse PFGE patterns) was investigated. In addition, a further 29 EMRSA-15s with identical PFGE patterns from two geographically linked but epidemiologically distinct outbreaks and several sporadic cases were analyzed. PFGE, MLVF, and MLVA resolved 66 (Simpson''s index of diversity [SID] = 0.984), 51 (SID = 0.95), and 42 (SID = 0.881) types, respectively, among the 85 MRSA isolates. MLVF was more discriminatory than MLVA for EMRSA-15 and -16 strains, but both methods had comparable discriminatory powers for distinguishing isolates in the group containing diverse PFGE types. MLVF was comparable to PFGE for resolving the EMRSA-15s but had a lower discriminatory power for the EMRSA-16s. MLVF and MLVA resolved the 29 isolates with identical PFGE patterns into seven and six subtypes, respectively. Importantly, both assays indicated that the two geographically related outbreaks were caused by distinct subtypes of EMRSA-15. Taken together, the data suggest that both methods are suitable for identifying and tracking specific subtypes of otherwise-indistinguishable MRSA. However, due to its greater discriminatory power, MLVF would be the most suitable alternative to PFGE for hospital outbreak investigations.Methicillin-resistant Staphylococcus aureus (MRSA) is a major human pathogen causing considerable morbidity and mortality worldwide. In Scotland and the United Kingdom in general, the incidence of MRSA is high (∼35%) with two epidemic clones, EMRSA-15 (ST22) and EMRSA-16 (ST36/ST30), accounting for 70 and 20%, respectively, of isolates referred to the Scottish MRSA Reference Laboratory (SMRSARL). Molecular typing of clinical isolates is important to inform decisions regarding effective control measures. For over a decade, pulsed-field gel electrophoresis (PFGE) of SmaI-digested genomic DNA has been used in United Kingdom reference laboratories for outbreak investigations and epidemiological surveillance, owing to its high discriminatory power and validated interpretation scheme (18). However, it is laborious and time-consuming, with poor interlaboratory comparability, and has no common international nomenclature. Furthermore, ca. 50% of EMRSA-15 strains and 35% of EMRSA-16 strains are indistinguishable by PFGE (4). Accordingly, the method is unsuitable for the tracing of many epidemics caused by EMRSA-15 and EMRSA-16 strains.Various techniques have been developed to address some of the limitations of PFGE, including spa typing and the variable-number tandem-repeat (VNTR)-based methods, multilocus VNTR fingerprinting (MLVF) (15) and multilocus VNTR analysis (MLVA) (6, 13, 16). spa typing involves DNA sequencing of the polymorphic VNTR in the 3′ coding region of the S. aureus-specific staphylococcal protein A (spa) gene (7). The method is more rapid and less laborious than PFGE, and the output is a digital profile, which is easily comparable between laboratories (1). However, it is less discriminatory than PFGE (10, 17) and is unsuitable for investigating the transmission of MRSA in hospitals dominated by EMRSA-15 and EMRSA-16 (8).MLVF analyzes the variation in the number of tandem repeats in seven genes (clfA, clfB, sdrC, sdrD, sdrE, spa, and sspA) by multiplex PCR and has been reported to be highly discriminatory and reproducible (9, 10, 15, 19). Previously, Tenover et al. (19) and Moser et al. (11) demonstrated that MLVF can distinguish between strains with identical PFGE patterns.MLVA differs from MLVF in that the number of repeats at each locus is determined to produce a numerical profile that can be incorporated into electronic databases and easily shared between laboratories. Although several MLVA schemes have been described, which differ in the loci and PCR protocol used, the MLVA described by Schouls et al. (16) benefits from automated fragment sizing on a DNA sequencer.To date, the effectiveness of MLVF and MLVA for tracing hospital outbreaks has not been compared. The aim of the present study was to investigate the usefulness of MLVF and MLVA compared to PFGE for subtyping highly clonal EMRSA-15s (ST22) and EMRSA-16S (ST36/ST30) and for tracing hospital outbreaks of infection.     

Molecular Characterization and Panton-Valentine Leucocidin Typing of Community-Acquired Methicillin-Sensitive Staphylococcus aureus Clinical Isolates

Limited comprehensive molecular typing data exist currently for Panton-Valentine leucocidin (PVL)-positive, methicillin-sensitive Staphylococcus aureus (PVL-MSSA) clinical isolates. Characterization of PVL-MSSA isolates by multilocus sequence typing (MLST) and spa typing in this study showed a genetic similarity to PVL-positive, methicillin-resistant S. aureus (PVL-MRSA) strains, although three novel spa types and a novel MLST (ST1518) were detected. Furthermore, the detection of PVL phages and haplotypes in PVL-MSSA identical to those previously found in PVL-MRSA isolates highlights the role these strains may play as precursors of emerging lineages of clinical significance.     

Prevalence and Characterization of Methicillin-Resistant Staphylococcus aureus Isolates from Retail Meat and Humans in Georgia

There is increasing interest in the presence of Staphylococcus aureus, specifically methicillin-resistant S. aureus (MRSA), on retail meat products. In this study, staphylococci were isolated from retail pork and retail beef in Georgia, and MRSA from the products was compared to human MRSA from the same geographic area using broth microdilution antimicrobial susceptibility testing, multilocus sequence typing (MLST), spa typing, SCCmec typing, and pulsed-field gel electrophoresis (PFGE). S. aureus was isolated from 45% (45/100) of pork products and 63% (63/100) of beef products; mecA was detected in S. aureus from both pork (3/100; 3%) and beef (4/100; 4%). Fifty percent (50/100) of human S. aureus also contained mecA. Multidrug resistance was detected among MRSA from all sources. All MRSA (n = 57) was SCCmec type IV, and nine different spa types were present among the isolates (t002, t008, t012, t024, t179, t337, t548, t681, and t1062). Four sequence types (ST5, ST8, ST9, and ST30) were detected using MLST; the majority of MRSA isolates belonged to ST8, followed by ST5. One retail beef MRSA isolate belonged to ST8, while the remaining three were ST5. In retail pork MRSA, ST5, ST9, and ST30 were observed. The majority of human MRSA isolates belonged to ST8. Thirty-seven MRSA isolates, one of which was a retail beef MRSA isolate, were pvl⁺. Using PFGE, MLST, and spa typing, three retail beef MRSA isolates were found to be identical in PFGE pattern, ST, and spa type to two human clonal MRSA isolates (USA100 and USA300). One additional retail beef MRSA isolate had a PFGE pattern similar to that of a human MRSA isolate, whereas none of the retail pork MRSA isolates had PFGE patterns similar to those of human MRSA isolates. These data suggest that the retail beef samples were contaminated by a human source, possibly during processing of the meat, and may present a source of MRSA for consumers and others who handle raw meat.     

Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus Isolates from Patients Newly Identified as Nasal Carriers

We aimed to determine whether additional molecular and microbiological evaluations of methicillin-resistant Staphylococcus aureus (MRSA) isolated from patients newly identified as nasal carriers were useful for control strategies and whether longitudinal testing during the same or repeat hospitalization changed MRSA status. Nasal swabs from patients positive by Xpert MRSA PCR and not known to be colonized in the previous year were cultured for S. aureus. Isolates were tested for resistance to a variety of antibiotics, including high-level mupirocin resistance (HLMR) and low-level mupirocin resistance (LLMR) and the presence of genes mecA and mupA and those for Panton-Valentine leukocidin (PVL), USA300, and USA400. Repeat nasal screens during the 6-month study were tested for continued presence of MRSA. Among 130 patients, cultures revealed MRSA in 85 (65.4%), methicillin-susceptible S. aureus in 19 (14.6%), and no growth in 26 (20%). MRSA isolates were USA300 positive in 13/85 (15.3%) and LLMR in 8/85 (9.4%) patients. No isolates were HLMR or mupA positive. mecA dropout was detected in 9/130 (6.9%) patients. The rate of subsequent MRSA infections in USA300-positive versus -negative patients was not different. MRSA nasal status remained concordant in 69/70 (98.6%) patients who had follow-up testing. The findings do not support expanding MRSA surveillance to include routine detection of genes for USA300, PVL, or mupA, all of which were either of low frequency or not significantly associated with MRSA infection risk in our population of newly identified nasal carriers. Repeat nasal screening for MRSA during the same or subsequent hospitalizations over 6 months could also be deferred, reducing costs associated with screening.     

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.     

Molecular and Phenotypic Characteristics of Methicillin-Resistant and Vancomycin-Intermediate Staphylococcus aureus Isolates from Patients with Septic Arthritis

Staphylococcus aureus is one of the most common pathogens in community- and hospital-associated infections and frequently causes severe and intractable infections in osteoarticular tissues. S. aureus isolates that are resistant to methicillin (meticillin) (MRSA isolates) and intermediately resistant to vancomycin (VISA isolates) have emerged. In this report, we described two patients, one female and one male, diagnosed with septic arthritis due to S. aureus infections. A total of 13 MRSA isolates were obtained from these two patients. All but one isolate belonged to the VISA group. All seven isolates from the female patient were determined to be community associated, multilocus sequence type (MLST) 59, and staphylococcal cassette chromosome mecA (SCCmec) type IV; had direct repeat units (DRUs) of nine repeats; were spa type t437; and were susceptible to sulfa and quinolone antibiotics. The other six isolates, from the male patient, were determined to be hospital associated, MLST 239, and SCCmec type III; had DRUs of 14 repeats; were spa type t037; and were resistant to sulfa and quinolone antibiotics. All 13 MRSA isolates were in agr group I, were pvl negative, and showed no evidence of any association between vancomycin resistance and autolysis.Staphylococcus aureus, which causes severe infections in the skin and soft tissue and in the cardiovascular, osteoarticular, and respiratory systems, is one of the most common and important pathogens in both community-associated and hospital-associated infections (7, 24, 31). Methicillin (meticillin)-resistant S. aureus (MRSA) emerged in the 1960s and has maintained a prevalence of 60 to 80% since the 1980s (15, 31). Vancomycin-intermediate S. aureus (VISA) strains have also emerged in the past 10 years due to frequent use of glycopeptide antibiotics for treatment of MRSA infections (20).Hematogenous and surgery-related septic arthritides are severe infections of the osteoarticular system, and S. aureus is the most common causative pathogen (1, 10). Although glycopeptides, including vancomycin and teicoplanin, are commonly used to treat MRSA septic arthritis (44), poor penetration of antibiotics into the periarticular space and biofilm formation on prosthesis make it very difficult to treat septic arthritis caused by S. aureus (17, 36).We had reported a patient with septic arthritis caused by VISA isolates which were resistant to autolysis in the Triton X-100 lysis assay due to increased thickness of the cell wall (32). Although several molecular typing methods, such as pulsed-field gel electrophoresis (PFGE) (35, 47), staphylococcal cassette chromosome mecA (SCCmec) typing (23, 27), and multilocus sequence typing (MLST) (12), have been developed for typing, few molecular epidemiology studies of S. aureus strains from patients with osteoarticular infections have been reported. In this study, we applied these methods to delineate the molecular types and phenotypes of S. aureus isolates from two patients with septic arthritis of distinct sources.     

Clinical Outcomes with Rapid Detection of Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus Isolates from Routine Blood Cultures

Staphylococcus aureus is a common cause of bacteremia, with a substantial impact on morbidity and mortality. Because of increasing rates of methicillin-resistant Staphylococcus aureus, vancomycin has become the standard empirical therapy. However, beta-lactam antibiotics remain the best treatment choice for methicillin-susceptible strains. Placing patients quickly on the optimal therapy is one goal of antimicrobial stewardship. This retrospective, observational, single-center study compared 33 control patients utilizing only traditional full-susceptibility methodology to 22 case patients utilizing rapid methodology with CHROMagar medium to detect and differentiate methicillin-resistant and methicillin-susceptible Staphylococcus aureus strains hours before full susceptibilities were reported. The time to targeted therapy was statistically significantly different between control patients (mean, 56.5 ± 13.6 h) and case patients (44.3 ± 17.9 h) (P = 0.006). Intensive care unit status, time of day results emerged, and patient age did not make a difference in time to targeted therapy, either singly or in combination. Neither length of stay (P = 0.61) nor survival (P = 1.0) was statistically significantly different. Rapid testing yielded a significant result, with a difference of 12.2 h to targeted therapy. However, there is still room for improvement, as the difference in time to susceptibility test result between the full traditional methodology and CHROMagar was even larger (26.5 h). This study supports the hypothesis that rapid testing plays a role in antimicrobial stewardship by getting patients on targeted therapy faster.     

Genotyping of Epidemic Methicillin-Resistant Staphylococcus aureus Phage Type 15 Isolates by Fluorescent Amplified-Fragment Length Polymorphism Analysis

Fluorescent amplified-fragment length polymorphism (FAFLP) analysis was investigated for its ability to identify and subtype isolates of an epidemic methicillin-resistant phage type of Staphylococcus aureus, EMRSA-15. These isolates were also characterized by PCR-restriction fragment length polymorphism (PCR-RFLP) of the coagulase gene and pulsed-field gel electrophoresis (PFGE). For FAFLP, DNA was double digested with restriction enzymes ApaI plus TaqI or EcoRI plus MseI. Site-specific adaptors were ligated to one or the other set of restriction fragments, and PCR amplification was carried out with adaptor-specific primers. Amplified fragments separated on an ABI 377 automated sequencer and analyzed with Genescan version 2.1 software generated FAFLP profiles for all the isolates. The presence or absence of fragments was scored, similarity coefficients were calculated, and UPGMA (unweighted pair group method using arithmatic averages) cluster analysis was performed. Either enzyme-primer combination readily differentiated EMRSA-15 from other methicillin-resistant S. aureus (MRSA) isolates and also revealed heterogeneity within the phage type. The discriminatory power of FAFLP was high. By combining both enzyme-primer data sets, 24 isolates were divided into 11 profiles. PCR-RFLP did not discriminate among these EMRSA-15 isolates. PFGE could discriminate well between isolates but was not as reproducible as FAFLP. All S. aureus and MRSA isolates in this study were typeable by FAFLP, which was easy to perform, robust, and reproducible, with evident potential to subtype MRSA for purposes of hospital infection control.