Detection Of Methicillin-resistant Staphylococcus Aureus Research Articles

Prevalence of Methicillin-Resistant Staphylococcus aureus as an Etiology of Community-Acquired Pneumonia

Methicillin-resistant Staphylococcus aureus (MRSA) is a common cause of skin infections. Recent case series describe severe community-acquired pneumonia (CAP) caused by MRSA, but the prevalence and risk factors are unknown. We prospectively enrolled adults hospitalized with CAP from 12 university-affiliated emergency departments during the winter-spring of 2006 and 2007. Clinical information and culture results were collected, and factors associated with MRSA were assessed. Of 627 patients, 595 (95%) had respiratory (50%) and/or blood cultures (92%) performed. A pathogen was identified in 102 (17%); MRSA was identified in 14 (2.4%; range by site, 0%-5%) patients and in 5% of patients admitted to the intensive care unit. Two (14%) MRSA pneumonia patients died. All 9 MRSA isolates tested were pulsed-field type USA300. Features significantly associated with isolation of MRSA (as compared with any other or no pathogen) included patient history of MRSA; nursing home admission in the previous year; close contact in the previous month with someone with a skin infection; multiple infiltrates or cavities on chest radiograph; and comatose state, intubation, receipt of pressors, or death in the emergency department. Methicillin-resistant Staphylococcus aureus remains an uncommon cause of CAP. Detection of MRSA was associated with more severe clinical presentation.

Too close for comfort: Screening strategy to detect methicillin-resistant Staphylococcus aureus conversion in exposed roommates

All 899 roommates exposed to methicillin-resistant Staphylococcus aureus (MRSA) index cases were studied over 57 months. MRSA detection is better at approximately 3 days (50%-55%) or 7 days (56%) after contact has been broken than day 0 (30%). Polymerase chain reaction testing at day 3 performs similarly to culture at day 7. Nasal/rectal screening provides superior detection than nasal alone. Those exposed >48 hours are at significantly greater risk of colonization.

Genetic diversity of community-associated methicillin-resistant Staphylococcus aureus isolated from Tenerife Island, Spain

With the recent detection of MRSA (methicillin-resistant Staphylococcus aureus) infections in patients lacking health care-related risk factors, the term community-acquired MRSA (CA-MRSA) has been become widely recognised. Many cases of CA-MRSA spreading to the community have been described worldwide. The aim of this study was to determine the features of CA-MRSA isolates from Tenerife Island. Toward this end, one hundred MRSA isolates were collected from eight different health regions, and their molecular features were investigated. This study revealed a wide variety of MRSA clones, including an emergent ST: ST1434 (CC8) and two new spa types, t7575 (ST125) and t7678 (ST22). The PVL genes were found in only five isolates belonging to unrelated lineages, ST8, ST30 and ST22, which could indicate at least three independent introductions of PVL+ strains to Tenerife. Moreover, we detected that hospital MRSA clones, like EMRSA-15 and EMRSA-16, had spread to the community and are now circulating in both environments. Therefore, in our study, the CDC’s rules were not specific enough to distinguish CA-MRSA from HA-MRSA. Thus, we think that the current epidemiological information is not enough to discriminate between both MRSAs, and it is necessary for prevention guidelines to include the routine determination of at least the genetic background, the antimicrobial susceptibility profile, and the PVL genes of each MRSA isolate.

Role of MRSA strains in the pathogenesis of severe atopic dermatitis in childhood. The ways of remission achievement

This article presents results of examination of 63 patients with atopic dermatitis (AD) aged 3 to 18 years. It was demonstrated, that in 87.3% of cases their skin was contaminated by Staphylococcus aureus. In 58.2% of cases pathogenic strains of methicillin-resistant Staphylococcus aureus (MRSA) were detected. Direct correlation between the frequency of MRSA detection and severity of the disease (r = 0,68) as well as age of children (r = 0,58) was revealed. Authors revealed high frequency of multidrug-resistant strains to a variety of antibiotics, except mupirocin, vancomycin, clindamycin, and ciprofloxacin (in the total group of MRSA the prevalence of resistant strains to these antibiotics ranged from 2% in mupirocin to 18% in ciprofloxacin, depending on the severity of AD). Algorithm of the effective combined therapy with mupirocin containing topical drugs (Supirocin ointment, Supirocin B ointment) is presented. This algorithm allowed to achieve stable clinical remission in 100% of cases.

Detection of methicillin-resistant Staphylococcus aureus in Iberian pigs

Iberian pigs are bred in Spain for the production of high-value dry-cured products, whose export volumes are increasing. Animals are typically reared outdoors, although indoor farming is becoming popular. We compared carriage of methicillin-resistant Staphylococcus aureus (MRSA) in Iberian pigs, raised indoors and outdoors, with intensively farmed Standard White pigs. From June 2007 to February 2008, 106 skin swabs were taken from Iberian pigs and 157 samples from SWP at slaughterhouses in Spain. We found that Iberian pigs carried MRSA, although with a significantly lower prevalence (30/106; 28%) than SWP (130/157; 83%). A higher prevalence of indoor Iberian pigs compared with animals reared under outdoor conditions was not significant; however, all but one positive indoor Iberian pig samples were detected from one slaughterhouse. Overall, 16 different spa types were identified, with t011 predominating in all three animal populations. A subset of isolates was characterized by MLST. Most of these belonged to ST398. MRSA isolates from Iberian pigs presented a higher susceptibility to antibiotics than those isolated from SWP. Despite limited contact with humans, pigs raised outdoors are colonized by an MRSA population that genetically overlaps with that of intensively farmed pigs, although antimicrobial resistance is lower. To our knowledge, this is the first detection of MRSA in food animals raised in free-range conditions.

Which anatomical sites should be sampled for screening of methicillin-resistant Staphylococcus aureus carriage by culture or by rapid PCR test?

The nose is the anatomical site usually recommended for methicillin-resistant Staphylococcus aureus (MRSA) screening. Other sites are also recommended, but are more controversial. We showed that the sensitivities of MRSA detection from nasal swabs alone were 48% and 62% by culture or by rapid PCR test, respectively. These percentages increased to 79% and 92% with the addition of groin swabs, and to 96% and 99% with the addition of groin and throat swabs. In conclusion, neither by culture nor by rapid PCR test is nose sampling alone sufficient for MRSA detection. Additional anatomical sites should include at least the groin and throat.

Inclusion of 30-Day Postdischarge Detection Triples the Incidence of Hospital-Onset Methicillin-Resistant Staphylococcus aureus

Hospitalized patients are at increased risk for acquisition of methicillin-resistant Staphylococcus aureus (MRSA). As hospital length of stay shortens, hospital-acquired MRSA events may be more likely to be detected after discharge. We assessed the impact of attributing MRSA cases discovered within 30 days after discharge to the most recent hospitalization and identified patient characteristics associated with MRSA detection after discharge. Retrospective cohort study. Twenty-seven acute care hospitals in Orange County, California. Adult acute care admissions (2002-2007). Using a countywide hospital data set containing diagnostic codes with present-on-admission (POA) indicators, we identified the first admission with a MRSA code for each patient. This incident MRSA admission was defined as predischarge-detected (pre-DD) hospital-onset MRSA (HO-MRSA) when MRSA was not POA. If MRSA was POA and a prior admission occurred within 30 days, this prior admission was assigned postdischarge-detected (post-DD) HO-MRSA. We evaluated the impact of including post-DD HO-MRSA in the calculation of hospital HO-MRSA incidence using signed-rank tests and reviewed changes in hospital rankings. We conducted multivariate comparisons of patient characteristics of pre-DD versus post-DD HO-MRSA patients. Among 1,217,253 at-risk hospitalizations, the inclusion of post-DD HO-MRSA tripled the median hospital HO-MRSA incidence, from 12.2 to 35.7 cases per 10,000 at-risk admissions (P < .0001). Hospital ranking changed substantially when including post-DD HO-MRSA. Patients with shorter stays were more likely to have post-DD MRSA. On the basis of administrative claims data, the inclusion of post-DD HO-MRSA significantly increased the estimated HO-MRSA incidence and altered hospital rankings. This finding underscores the limitations of single-facility data when deriving HO-MRSA incidence and rank.

Rapid confirmation of suspected methicillin-resistant Staphylococcus aureus colonies on chromogenic agars by a new commercial PCR assay, the GenomEra MRSA/SA Diagnose

A new automated closed tube PCR assay, the GenomEra(™) MRSA/SA Diagnose (Abacus Diagnostica Oy, Finland) was evaluated for rapid confirmation of methicillin-resistant Staphylococcus aureus (MRSA) from cultured screening specimens. The ability of the assay to detect genotypically different MRSA strains was studied with a collection of 304 MRSA isolates covering 68 spa types. The specificity was investigated with a collection of 146 non-MRSA staphylococcus isolates. The usefulness of the assay for clinical purposes was assessed by a sequential combination of MRSA screening culture and confirmation of the colonies with the GenomEra MRSA/SA Diagnose assay. A total of 145 suspected MRSA colonies on chromogenic plates were analyzed this way. All MRSA isolates from the culture collection and from the clinical screening specimens were confirmed as MRSA with the GenomEra MRSA/SA Diagnose assay and none of the non-MRSA staphylococci caused false-positive results, which indicates both sensitivity and specificity of 100%. The combination of GenomEra MRSA/SA Diagnose with preceding culture on selective MRSA agar permitted MRSA confirmation within 24 h. This practice offers a reliable and quick detection of MRSA that is also suitable in areas where several strain types cause epidemics.

Isolation and Identification of Methicillin-Resistant Staphylococcus aureus from Keys of College Students Using Different Detection Methods

Aims: In this study, the methicillin-resistant Staphylococcus aureus (MRSA) were isolated and identified by using biochemical tests, antibiogram and polymerase chain reaction (PCR) to explore the circulation of MRSA among college students. Study design: Cross-sectional study. Place and Duration of Study: Department of Medical Microbiology, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia Research Article British Biotechnology Journal, 2(1): 13-25, 2012 14 between June 2010 and December 2010. Methodology: A total of 100 samples were collected from keys of college students. There were 39 isolates (39 %) Gram-positive cocci and Catalase positive. 29 (74.36%) were glucose oxidation and fermentation positive. From the 39 isolates, 16 (43.24%) were shown Mannitol Salt Agar (MSA) tests positive. The deoxyribonuclease (DNase) tests and tube coagulase tests with human and rabbit plasma were carried out to improve the efficiency of the MSA test. Results: 7 (43.75%) DNase positive and 2 (12.5%) tube coagulase positive. Both human and rabbit plasma showed similar sensitivity for the tube coagulase tests in this study. However, both isolates with tube coagulase positive were confirmed as S. aureus but not resistant to oxacillin, methicillin, erythromycin and cefoxitin. 2 (66.67%) of 3 (18.75%) isolates which is tube coagulase negative were resistant to erythromycin and 1 (33.33%) of them was resistant to methicillin. Rare strains of S. aureus can be coagulase negative. PCR assay was used. 1 (33.33%) of the coagulase negative isolate resistant to erythromycin was found to have nuc gene, mecA gene, ermC gene, msrA gene, linA gene, and femA gene. The isolate was confirmed as MRSA. Conclusion: In conclusion, PCR technique is more sensitive and reliable than tube coagulase test or antibiogram for the detection of MRSA. And keys were shown to be an important source of MRSA and other bacteria circulation in the community.

MRSA の院内伝播制御に有用な POT 法を用いた分子疫学解析

Surveillance is very important for preventing the nosocomial spread of methicillin-resistant Staphylococcus aureus (MRSA), and the pulsed-field gel electrophoresis (PFGE) method has long been used to identify the infection source and route as a molecular and epidemiological genotyping method. However, the use of the method in routine clinical laboratory measurements is difficult due to its complicated procedures. Since a molecular and epidemiological genotyping kit based on the POT (Phage Open-reading Frames Typing) method has been developed, we examined 192 MRSA isolates newly detected from inpatients in our hospital in 2010 in order to investigate the usefulness of POT for surveying outbreaks of MRSA. Among the 192 isolates 118 were suspected of nosocomial spread by the previous method, which defined a MRSA detection at more than 48 hours after admission as a nosocomial spread. The POT method was introduced at our laboratory in 2010, and we were able to recognize 38 patients as having strongly suspected nosocomial MRSA infection with the POT method taking into consideration the infection situation, such as places (wards and transfer) and time (date of admission and date of collected samples). Our Infection Control Division was confidently able to demonstrate the current condition of the nosocomial spread by providing the results to the clinical staff, who were also able to practice infection control confidently. We concluded that the POT method was very useful and convenient for investigating MRSA isolates and evaluating collected data because no particular analysis other than the digitizing electrophoretic pattern method was necessary.

Detection of methicillin-resistant Staphylococcus aureus (MRSA) from nasal samples by multiplex real-time PCR based on dual priming AT-rich primers

In this study, we reported on the design of a multiplex real-time PCR assay based on SYBR Green I, incorporating dual priming adenine-thymine (AT)-rich primers for direct detection of MRSA from nasal samples. The multiplex real-time polymerase chain reaction (RT-PCR) assay reported in this study is based on SYBR Green I with incorporation of six dual priming AT-rich primers designed from the SCCmec/orf junction. A string (4-6 bp) of low-melting bases, such as adenine and thymine, was incorporated into the primers, which virtually divided a single primer in two functional regions, thus decreasing non-specific PCR products. The analytical sensitivity and specificity of the RT-PCR assay was determined with genomic DNA of reference strains (MRSA, MSSA, and MRCoNS). RT-PCR assay was performed for analysis of 72 nasal swab specimens, and the results were confirmed by use of a culture method. Furthermore, the results of RT-PCR were compared with LightCycler MRSA advance test. The multiplex RT-PCR assay reproducibly detected a minimum of 1 pg genomic DNA (31.5 copy of genome) of MRSA reference strains and clinical isolates, with a specific melting peak at 83.5 ± 1.5°C, and neither fluorescence nor a melting peak was detected in non-target isolates. The concordance rate between RT-PCR assay and culture method was 87.5% with Cohen's kappa value (κ) 0.75, which showed good agreement between the two assays. The sensitivity, specificity, positive predictive value, and negative predictive value of the assay were 93.5%, 82.9%, 80.5%, and 94.4%, respectively. In a comparative study for the detection of 72 nasal samples, the sensitivity, specificity, positive predictive value, and negative predictive value of the multiplex RT-PCR assay with respect to LightCycler MRSA advance test was 84.2%, 88.2%, 89%, and, 83.3%, respectively. The results of RT-PCR assay demonstrated high specificity (88.2%) and positive predictive value (89%) for the direct detection of MRSA from nasal samples.

Clinical Usefulness of Multiplex PCR Lateral Flow in MRSA Detection: A Novel, Rapid Genetic Testing Method

Methicillin-resistant Staphylococcus aureus (MRSA) with exogenous cassette DNA containing the methicillin-resistant gene mecA (SCCmec) poses a problem as a drug-resistant bacterium responsible for hospital- and community-acquired infections. The frequency of MRSA detection has recently been increasing rapidly in Japan, and SCCmec has also been classified more diversely into types I–V. A rapid test is essential for early diagnosis and treatment of MRSA infections, but detection by conventional methods requires at least two days. The newly developed multiplex PCR lateral flow method allows specific amplification of femA to detect S. aureus, mecA to detect SCCmec, and kdpC to detect SCCmec type II; moreover, PCR products can be evaluated visually in about 3 h. In the present study, we developed a PCR lateral flow method for MRSA using this method and investigated its clinical usefulness in the detection of MRSA. The results showed a diagnostic concordance rate of 91.7% for MRSA and methicillin-susceptible S. aureus between bacteriological examination and PCR lateral flow, and a high level of specificity in PCR lateral flow. In addition, a higher detection rate for S. aureus using the same sample was observed for PCR lateral flow (70.2%) than for bacteriological tests (48.6%). The above results show that PCR lateral flow for MRSA detection has high sensitivity, specificity, and speed, and its clinical application as a method for early diagnosis of MRSA infections appears to be feasible.

Methicillin-Resistant Staphylococcus Aureus Screening in Total Joint Arthroplasty: A Worthwhile Endeavor

Infections with methicillin-resistant Staphylococcus aureus (MRSA) are an ever growing problem in the community, hospitals, and for orthopedic surgeons in particular. A conscious effort must be made to deal with this pathogen prior to total joint arthroplasty procedures. The drastic increase in prevalence of surgical-site infections (SSIs) after total joint replacement surgery has proved to be a major health care burden for both patients and surgeons from both a medical and financial standpoint. The development of screening techniques for detection of MRSA colonization in patients being admitted to hospitals is steadily increasing popularity. Particularly nasal swab rapid polymerase chain reaction detection of MRSA allows surgeons to identify patients at high risk for postoperative SSI. A variety of treatment regimens for eradication of MRSA colonization from the nares of surgical patients have surfaced, such as topical mupirocin prior to undergoing surgery. Decolonization of MRSA in patients undergoing joint arthroplasty procedures has demonstrated encouraging initial results in preventing SSIs and should be a serious focus of the future for orthopedic surgeons.

High prevalence of methicillin resistant Staphylococcus aureus in the surgical units of Mulago hospital in Kampala, Uganda

BackgroundThere is limited data on Methicillin resistant Staphylococcus aureus (MRSA) in Uganda where, as in most low income countries, the routine use of chromogenic agar for MRSA detection is not affordable. We aimed to determine MRSA prevalence among patients, healthcare workers (HCW) and the environment in the burns units at Mulago hospital, and compare the performance of CHROMagar with oxacillin for detection of MRSA.ResultsOne hundred samples (from 25 patients; 36 HCW; and 39 from the environment, one sample per person/item) were cultured for the isolation of Staphylococcus aureus. Forty one S. aureus isolates were recovered from 13 patients, 13 HCW and 15 from the environment, all of which were oxacillin resistant and mecA/femA/nuc-positive. MRSA prevalence was 46% (41/89) among patients, HCW and the environment, and 100% (41/41) among the isolates. For CHROMagar, MRSA prevalence was 29% (26/89) among patients, HCW and the environment, and 63% (26/41) among the isolates. There was high prevalence of multidrug resistant isolates, which concomitantly possessed virulence and antimicrobial resistance determinants, notably biofilms, hemolysins, toxin and ica genes. One isolate positive for all determinants possessed the bhp homologue which encodes the biofilm associated protein (BAP), a rare finding in human isolates. SCCmec type I was the most common at 54% prevalence (22/41), followed by SCCmec type V (15%, 6/41) and SCCmec type IV (7%, 3/41). SCCmec types II and III were not detected and 10 isolates (24%) were non-typeable.ConclusionsHyper-virulent methicillin resistant Staphylococcus aureus is prevalent in the burns unit of Mulago hospital.

Clinical Effectiveness and Cost Benefit of Universal versus Targeted Methicillin-Resistant Staphylococcus aureus Screening upon Admission in Hospitals

To conduct an exploratory study to evaluate the clinical effectiveness and cost benefit of universal versus targeted screening for methicillin-resistant Staphylococcus aureus (MRSA) to prevent hospital-acquired MRSA infections. Prospective, interventional study, using a case-control design, difference-in-differences, and cost-benefit analyses. Two community hospitals in Wisconsin. Consecutive sample of 15,049 adult admissions from April 2009 to July 2010. MRSA surveillance performed by polymerase chain reaction (PCR) on samples collected from all adult patients (aged over 18 years) within 30 days before or upon an admission to the hospital. During a 9-month baseline period, targeted screening was conducted at both hospitals. During the 5-month intervention period, all patients admitted to the intervention hospital were screened for MRSA. Infection control measures were consistent at both hospitals. Universal screening was associated with an increase in admission screening of 43.58 percentage points (P< .01), an increase in MRSA detection of 2.95 percentage points (P< .01), and a small, nonsignificant decline in hospital-acquired MRSA infections of 0.12 percentage points (P< .01). The benefit-to-cost ratio was 0.50, indicating that for every dollar spent on universal versus targeted screening, only $0.50 is recovered in avoided costs of hospital-acquired MRSA infection. Compared with targeted screening, universal screening increased the rate of detection of MRSA upon hospital admission but did not significantly reduce the rate of hospital-acquired MRSA infection. Universal screening was associated with higher costs of care and was not cost beneficial.

Evaluation of salt concentrations, chromogenic media and anatomical sampling sites for detection of methicillin-resistant Staphylococcus aureus in pigs

The performance of chromogenic media for the detection of methicillin-resistant Staphylococcus aureus (MRSA) in humans, has been evaluated in numerous studies. However, few comparative studies have been performed for the detection of MRSA in pigs. In this study two different salt concentrations (2.5% or 7.5% NaCl) were evaluated in the enrichment nutrient broth and three selective chromogenic media (chromID MRSA, BrillianceMRSA and MRSA Select) for their ability to detect MRSA in swabs from 29 pigs obtained from three different anatomical sampling sites (anterior nares, skin behind both ears and perineum). ChromID MRSA showed the highest relative sensitivity and specificity after enrichment in 7.5% NaCl, followed by MRSA Select and BrilianceMRSA. For all chromogenic media more MRSA-positive results were obtained for specimens collected from skin behind the ears than for specimens taken from both nares and perineum. The results with regard to the anatomical sampling sites were confirmed in a larger study on three different pig farms involving 60 pigs per farm. Skin behind the ears was the anatomical site with the highest relative sensitivity (91.4%) for MRSA detection compared to perineum and anterior nares, with a relative sensitivity of 76.5% and 75.3%, respectively. An increased relative sensitivity could be achieved when combining two anatomical sites. Sampling of anterior nares and skin behind the ears appeared to be the most sensitive combination with a relative sensitivity of 98.2%. These results show that sampling of only the anterior nares underestimates the real pig MRSA prevalence.

Screening of poultry-pig farms for methicillin-resistant Staphylococcus aureus: Sampling methodology and within herd prevalence in broiler flocks and pigs

Many reports described the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) in different livestock animals from one-species farms. However, in no published reports the prevalence on mixed poultry-pig farms was mentioned, nor the possible relation in MRSA colonization between those two species on one farm, and the possible role of the farmer in the dissemination of MRSA between those two species. Furthermore, no data is available on the optimal sampling site to detect MRSA in broilers. Therefore this study aimed to determine the most suitable sample location in broiler chickens for MRSA and the within flock prevalence of MRSA in various broiler flocks and compared this with the MRSA prevalence in pigs, the colonization of the farmer and the contamination in the barn environment in three mixed poultry-pig farms. MRSA was most frequently isolated from the cloaca and nose shell and to a lesser extent from the skin beneath the wing and the pharynx. The relative sensitivity of the different anatomical sites was, 44.4% for the cloaca, 33.3% for the nose shell, 16.7% for the skin beneath the wing and 5.6% for the pharynx. Based upon these relative sensitivities combining cloaca and nose shell would increase the chance of MRSA detection. A rather low within flock prevalence of MRSA varying between 0% and 28% was detected in broilers, whereas in pigs on the same farms the within herd prevalence varied between 82% and 92%. No MRSA contamination in the direct barn environment of the broilers was found, this in contrast to the environment of the pigs, indicating a relationship between MRSA prevalence and contamination in the environment. Two farmers were continuously colonized, while the third one was only once. In conclusion, a major difference was seen in MRSA occurrence between broilers and pigs from the same farm. This may suggest that broilers are naturally less susceptible to MRSA ST398 colonization than pigs. Conversely, short production time in broilers, vacancy of the barn environment during one week and the higher frequency of disinfection might also explain the lower prevalence in broilers. The farmer may play an important role in the dissemination of MRSA from pigs to poultry, especially in mixed farms where pigs are highly colonized and may act as a reservoir for MRSA ST398 carriage in humans.

Detection of novel chromosome-SCCmec variants in Methicillin Resistant Staphylococcus aureus and their inclusion in PCR based screening

FindingsTo facilitate automation, a novel DNA extraction method for MRSA was adopted. The MRSA specific chromosome-SCCmec PCR was adapted, additional primers were added, and the performance was validated. From various laboratories in The Netherlands we received a total of 86 MRSA clinical isolates, that were negative in commercially available tests. We identified 14 MRSA strains with new variant chromosome-SCCmec junctions by sequence analysis. These MRSA strains appeared to carry SCCmec sequences with a high degree of homology to SCC regions of S. epidermidis and S. haemolyticus. All were included for detection in chromosome-SCCmec based PCR.BackgroundEfficient management of Methicillin Resistant Staphylococcus aureus (MRSA) in the hospital is needed to prevent dissemination. It is important that MRSA can be rapidly identified, and effective infection control measures can be initiated. Equally important is a rapid MRSA negative report, especially for patients in isolation. For negative screening we implemented fully automated high through-put molecular screening for MRSA.ConclusionsFourteen variant chromosome-SCCmec junctions in MRSA, that are not detected in commercially available MRSA detection kits were added to our PCR to detect all currently known variant SCC-mec types of MRSA.

Methicillin‐Resistant Staphylococcus aureus (MRSA) in Three Dairy Herds in Southwest Germany

The objective of this study was to analyse the occurrence of methicillin-resistant Staphylococcus aureus (MRSA) in three dairy herds in the southwest of Germany that had experienced individual cases of clinical and subclinical mastitis associated with MRSA. The herds were identified by the detection of MRSA during routine resistance testing of mastitis pathogens. All quarters of all cows in the herds that were positive on California Mastitis Test were sampled for bacteriological analysis on two occasions. Bulk tank milk samples were also tested. Furthermore, nasal swabs were collected from people working on the farms and from cattle. Environmental samples were collected from associated pig holdings. Isolates were characterized using spa-typing and testing for antimicrobial resistance. Our results revealed a substantial spread of MRSA in the three dairy herds. In the first of the two investigations carried out on all cows in the three herds, milk samples of 5.1-16.7% of dairy cows were found positive for MRSA. The respective proportions in the second herd level investigation were 1.4-10.0%. Quarters harbouring MRSA had higher somatic cell counts than quarters that were negative on culture. Methicillin-resistant Staphylococcus aureus were also detected in nasal swabs of staff (7/9), cows (7/15) and calves (4/7), bulk tank milk samples (3/3) and environmental samples from pig premises (4/5) on the farm. Herds B and C had no contact to herd A. However, in all three herds MRSA of spa-type t011 were detected in milk samples. Results show that MRSA of spa-type t011 is a problem in dairy farms that needs urgent attention.

Genotypic and Phenotypic Characterization of Methicillin-Susceptible Staphylococcus aureus Isolates Misidentified as Methicillin-Resistant Staphylococcus aureus by the BD GeneOhm MRSA Assay

Twenty-three nasal swab samples that tested positive for methicillin-resistant Staphylococcus aureus (MRSA) on initial testing by the BD GeneOhm MRSA assay (BD-MRSA PCR; BD GeneOhm, San Diego, CA) were culture positive only for methicillin-susceptible S. aureus (MSSA) from an enrichment broth. The 23 recovered isolates were confirmed as MSSA by a variety of phenotypic methods, including the BD Phoenix automated microbiology system (BD Diagnostics, Sparks, MD), oxacillin screening agar (BD Diagnostics), BBL CHROMagar MRSA (BD Diagnostics), and a PBP2' assay (Denka Seiken Co., Tokyo, Japan); susceptibilities were determined by using Mueller-Hinton agar with oxacillin. All were positive by nuc PCR, specific for S. aureus, but negative for mecA with one exception. Isolates were characterized by using multiplex PCR methodology to determine structural types and variants (SCCmec typing); additional PCRs were performed for the detection of the ccr and mec complexes, the junkyard regions as well as the Panton-Valentine leukocidin. Pulsed-field gel electrophoresis was used to determine clonality. One phenotypic MSSA isolate contained an intact SCCmec. Twelve MSSA isolates tested positive for MRSA by the BD-MRSA PCR because of amplification of the mec priming site flanking the SCC insertion point, although these isolates lacked mecA. The 10 remaining isolates were not MRSA and tested as MSSA by phenotypic and genotypic assays. In our patient population, diagnostic and surveillance testing and subsequent infection control practices may be impacted by the frequency of these excision events when using the BD-MRSA PCR for MRSA detection.