Methicillin-resistant Staphylococcus aureus detection using chromogenic media: the Sheffield experience

Methicillin-resistant Staphylococcus aureus (MRSA) continues to cause major problems, both in hospitals and the community. Microbiology departments need to review their methodology regularly to ensure that they are contributing in the most appropriate manner to the battle against MRSA. Media employing chromogenic enzymes to aid the isolation and identification of MRSA is a relatively new approach. In this study, 192 swabs from 112 different patients were inoculated on two chromogen-containing media and four other commonly used solid MRSA media to determine which gave the appropriate combination of sensititivity, specificity and speed of result. Methicillin-resistant S. aureus was isolated on at least one of the six media from 102 of the 192 swabs. Both chromogenic media proved to be statistically significantly more sensitive than the other media after overnight incubation and had a sensitivity of 96% after 48 hours' incubation. The recent introduction of chromogen-containing MRSA media offers microbiology laboratories the opportunity to isolate and confirm the majority of MRSA infections/colonisations in 24 hours, which should result in better patient care. The possible slight increase in costs should not provide a valid excuse for using inferior methodologies.     

Evaluation of different chromogenic media for the detection of methicillin-resistant Staphylococcus aureus CC398 in broilers

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has emerged in a wide variety of animal species, including poultry. The objective of this study was to evaluate three different chromogenic media for MRSA clonal complex (CC) 398 detection in broilers. On three Belgian poultry farms, 50 broiler chickens were sampled per farm from both nose shell and cloaca. All swab specimens were enriched and inoculated the following day on three chromogenic media: chromID MRSA (bioMérieux), Brilliance MRSA 2 Agar (Oxoid) and MRSASelect (Bio-Rad). ChromID had the highest isolation rates, yet, Brilliance MRSA 2 Agar demonstrated the highest relative sensitivity, while MRSASelect and Brilliance MRSA 2 Agar showed the highest relative specificity. A subset of MRSA isolates was confirmed to be CC398 by the polymerase chain reaction (PCR) targeting sau1-hsdS1. In conclusion, Brilliance MRSA 2 Agar outperformed MRSASelect and chromID MRSA for the detection of MRSA in broilers.     

Comparison of Methicillin-Resistant Staphylococcus aureus Community-Acquired and Healthcare-Associated Pneumonia

Purpose

Methicillin-resistant Staphylococcus aureus (MRSA) is recognized as an important cause of not only healthcare-associated pneumonia (HCAP) but also community-acquired pneumonia (CAP). We determined the impact of MRSA on differences in clinical characteristics, courses, and outcomes between CAP and HCAP.

Materials and Methods

We conducted a retrospective observational study on 78 adult patients admitted with MRSA pneumonia at a university-affiliated tertiary hospital between January 2008 and December 2011. We compared baseline characteristics, chest radiographs, treatment outcomes, and drug resistance patterns between the CAP and HCAP groups.

Results

Of the 78 patients with MRSA pneumonia, 57 (73.1%) were HCAP and 21 (26.9%) were CAP. MRSA infection history in the previous year (29.8% vs. 14.3%, p=0.244) tended to be more common in HCAP than in CAP. Despite similar Pneumonia Severity Index scores (151 in CAP vs. 142 in HCAP), intubation rates (38.1% vs. 17.5%; p=0.072) and intensive care unit admission (42.9% vs. 22.8%; p=0.095) tended to be higher in the CAP group, while 28-day mortality was higher in the HCAP group (14.3% vs. 26.3%; p=0.368), although without statistical significance. All patients showed sensitivity to vancomycin and linezolid; meanwhile, HCAP patients showed greater resistance to gentamicin than CAP patients (58.3% vs. 16.6%; p=0.037). The median total hospital charges were 6899 American dollars for CAP and 5715 American dollars for HCAP (p=0.161).

Conclusion

MRSA pneumonia showed significantly differences in baseline characteristics, chest radiographs, treatment outcomes, and medical expenses between HCAP and CAP groups.     

Community-Associated Methicillin-Resistant Staphylococcus aureus Mediastinitis

Community-associated methicillin (meticillin)-resistant Staphylococcus aureus (CA-MRSA) continues to emerge as a cause of serious infections, chiefly of the skin and soft tissues. We present the first documented case of CA-MRSA mediastinitis in an adult. Blood and mediastinal isolates were characterized as CA-MRSA by pulsed-field gel electrophoresis and susceptibility testing.     

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.     

New chromogenic identification and detection of Staphylococcus aureus and methicillin-resistant S. aureus

This paper describes a new chromogenic plate medium, CHROMagar Staph aureus (CHROMagar, Paris, France), for the identification of Staphylococcus aureus on the basis of colony pigmentation. The abilities of CHROMagar Staph aureus, thermostable nuclease (DNase), and mannitol salt agar (MSA) to identify S. aureus isolates (n = 114) and discriminate between S. aureus and coagulase-negative staphylococci (CoNS; n = 22) were compared. CHROMagar Staph aureus proved to be more sensitive and specific than DNase and MSA, allowing a reliable, simple, and rapid method for the identification of S. aureus isolates. All CoNS encountered in this study with the exception of S. chromogenes could be easily differentiated from S. aureus on this medium. The supplementation with 4 microgram of oxacillin or methicillin per ml allowed simple identification of methicillin resistance in hospital-acquired S. aureus strains which show multiple-drug resistance profiles. Community-acquired methicillin-resistant S. aureus strains showing non-multi-drug resistance profiles require further evaluation on this new chromogenic medium. Methicillin or oxacillin resistance of all S. aureus isolates was confirmed by the detection of penicillin-binding protein 2a, encoded by the mecA gene, using the latex slide agglutination MRSA-Screen test (PBP 2' Test, DR900M; Oxoid).     

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.     

Evaluation of two chromogenic agar media for recovery and identification of Staphylococcus aureus small-colony variants

To identify the most rapid and reliable technique for recovery and identification of Staphylococcus aureus small-colony variants (SCVs), the colonial appearance of 106 isolates representing SCVs and the normal phenotype were evaluated on two newly described chromogenic agar media. Although almost all of the SCVs grew on the chromogenic agar media, they did not exhibit a change of color. In comparison with conventional media, S. aureus ID agar (SAID; bioMerieux, La Balme Les Grottes, France) showed the most reliable results, with 49 of 53 SCVs tested growing either as an SCV colony or with a normal phenotype after only 24 h of incubation. Growth of SCVs was often not detected before 72 h of incubation on some of the media tested. In conclusion, the most accurate and rapid method to detect both the species S. aureus and the SCV phenotype is to inoculate specimens onto both Columbia blood agar and SAID.     

Evaluation of S. aureus ID, a new chromogenic agar medium for detection of Staphylococcus aureus

S. aureus ID (bioMérieux, La Balme Les Grottes, France) is a new chromogenic agar medium designed to enable the isolation of staphylococci and the specific identification of Staphylococcus aureus. S. aureus produces green colonies on this medium due to production of α-glucosidase. To evaluate this medium, a total of 350 wound swabs were cultured onto S. aureus ID, CHROMagar Staph. aureus, and conventional media routinely used in our laboratory. After 18 to 20 h of incubation, 96.8% of strains formed green colonies on S. aureus ID compared with 91.1% of strains forming mauve colonies on CHROMagar Staph. aureus. A total of 94.3% of strains were recovered within 18 to 20 h with conventional media. The sensitivity was increased after 48 h of incubation to 98.7, 96.2, and 95.6% with S. aureus ID, CHROMagar Staph. aureus, and conventional media, respectively. A total of 97.4% of green colonies on S. aureus ID were confirmed as S. aureus compared with 94.4% of mauve colonies on CHROMagar Staph. aureus. We conclude that S. aureus ID is a highly sensitive and specific medium for the isolation and identification of S. aureus from wound swabs.     

A chromogenic method for rapid identification of Staphylococcus aureus

Staphylococcus aureus is responsible for septicaemia and serious nosocomial infections. A rapid and specific identification of this species is of great importance in clinical microbiology. Current methods for S. aureus identification require a 18 to 24 h-incubation. We describe a two hour-identification method based on the detection of the staphylocoagulase, using human prothrombin and a chromogenic substrate. 242 staphylococcal strains (160 S. aureus, 82 coagulase-negative staphylococci (CNS)) were collected from 4 French hospitals. They have been identified by the following methods: (i) clotting of citrated rabbit plasma, which is considered as reference method; (ii) biochemical tests (Rapidec Staph and Api Staph or ID 32 Staph); (iii) and agglutination test (Pastorex Staph or Pastorex Staph-plus). A strain of S. intermedius was provided by the Collection of the Pasteur Institute (Paris). An adapted culture medium is inoculated with staphylococci and adjusted to 2 Mac Farland unities. This medium is then mixed to an equal volume with a human prothrombin solution and the chromogenic substrate. After 1 to 2 hours incubation at 37 degrees C, the strength of the yellow colour of the mixture is observed to the naked eye, or measured at 405 nm with a spectrophotometer. Fifteen chromogenic tripeptides having a thrombin-like affinity and paranitroanilin as leaving group were compared. With the substrate which has the higher hydrolysis velocity and enzymatic affinity (SQ149), all S. aureus strains gave a positive result: 94.7% of the methicillin-susceptible S. aureus were detected after 1 hour incubation, but only 52.3% of the methicillin-resistant S. aureus. 98.4% of the methicillin-resistant S. aureus were detected after 2 hours. No false positive result was observed for the 82 CNS strains. The chromogenic method shows good within-run and day-to-day precision tests. It doesn't need any complementary test. The sensitivity and the specificity are 99.4% and 100% respectively.     

Detection of nasal colonization methicillin-resistant Staphylococcus aureus: a prospective study comparing real-time genic amplification assay vs selective chromogenic media

In contrast to "classical" genic amplification, real-time genic amplification can be performed in every laboratory without the need of sophisticated isolation procedures. Moreover, real-time genic amplification allows an early detection of meticillin resistant Staphylococcus aureus colonization, 2 hours compared to 1 or 2 days for culture. OBJECTIVE: In order to assess the feasibility on Smartcycler of the IDI-MRSA real-time genic amplification assay in comparison with chromogenic media. METHODS: A prospective study has been initiated in July 2004: nasal swabs were taken from patients entering the ICU, vascular surgery, diabetology and geriatry wards. During a 4 months period, 682 specimens have been obtained from 508 patients. RESULTS: Sixty-four (9.3%) patients were positive by genic amplification and selective agar culture (CHROMagar MRSA, MRSASelect and/or ORSAB), 19 (2.9%) were positive by genic amplification only (3 of these patients were under antibiotic treatment); 572 specimens remained negative by both methods. The sensitivity and specificity of this assay were 100% and 96% respectively with a positive predictive value of 70% and negative predictive value of 100%. Initially 82 nasal specimens were unresolved (12%). 38 were resolved following a freeze-thaw cycle. Thus, 44 (6.4%) were unresolved specimens. Comparison between CHROMagar MRSA and MRSASelect showed a good correlation for the detection at 24 hours (5.5% and 5.6% respectively). These two chromogenic media allowed a much better detection of MRSA than ORSAB medium within 24H. CONCLUSION: The results obtained by the early real-time genic amplification for the detection of meticillin resistant Staphylococcus aureus are promising. Despite 6.4% amplification failure, we consider that IDI-MRSA real-time genic amplification assay represents a significant breakthrough in the detection of colonization.     

Evaluation of three chromogenic media (MRSA-ID, MRSA-Select and CHROMagar MRSA) and ORSAB for surveillance cultures of methicillin-resistant Staphylococcus aureus

Screening specimens were homogenised in saline 0.9% w/v before either direct inoculation or following enrichment in broth on three chromogenic media (MRSA-ID, CHROMagar MRSA and MRSA Select) and ORSAB medium for the detection of methicillin-resistant Staphylococcus aureus (MRSA). In total, 102 of 466 specimens yielded MRSA on at least one medium. After incubation for 16-18 h, the sensitivity was 51%, 59%, 47% and 65% on MRSA-ID, CHROMagar MRSA, ORSAB and MRSA Select, respectively, compared with 82%, 75%, 67% and 80%, respectively, after 42 h, and 93%, 95%, 79% and not tested, respectively, following broth enrichment. There were significantly more MRSA colonies on MRSA-Select after 16-18 h than on ORSAB or MRSA ID (p 0.001 and 0.0022, respectively), whereas there were more MRSA colonies after 42 h on MRSA-ID and MRSA-Select than on ORSAB (p 0.0004 and 0.012, respectively). The specificity of the media for identifying MRSA based on the colour of colonies after incubation for 16-18 h was 100%, 99%, 99% and 100%, respectively, compared with 98%, 97%, 98% and 98%, respectively, after 42 h, and 100%, 99%, 100% and not tested, respectively, following broth enrichment. The speed of detection (mean time to report a positive result) was 1.65, 1.72, 2.31 and 1.35 days, respectively. For each of the three media tested following enrichment, the use of an enrichment broth increased the detection rate of MRSA by 16-24%.     

Evaluation of Chromogenic Media for Detection of Methicillin-Resistant Staphylococcus aureus

Rapid laboratory diagnosis is critical for treating, managing, and preventing methicillin-resistant Staphylococcus aureus (MRSA) infections. We evaluated and compared the potential for MRSA detection of five chromogenic media, Brilliance MRSA agar (Oxoid), ChromID (bioMérieux), MRSASelect (Bio-Rad), CHROMagar (CHROMagar Microbiology), and BBL-CHROMagar (BD Diagnostics). Media were tested with log serial dilutions (10⁰ to 10⁶ CFU) of pure isolates of MRSA (n = 60), non-MRSA (n = 27), and defined mixtures thereof simulating clinical samples (n = 84). Further evaluations were done on pre-enriched nasal and groin screening swabs (n = 213) from 165 hospitalized patients. Randomized samples were spiral plated on each medium and independently scored by five investigators for characteristic colonies at 24 and 48 h of incubation. Confirmatory testing of up to five putative MRSA colonies recovered from each medium was done. The cumulative average sensitivity with isolates, mixtures, and clinical samples was the highest for Brilliance MRSA agar (97%) and similar for the other four media (≥92%). The cumulative average specificity was the highest for BBL-CHROMagar (99%), followed by MRSASelect (98%), CHROMagar (97%), ChromID (89%), and Brilliance MRSA agar (86%). All of the media detected MRSA at 10 and 1 CFU, although at these low loads, few MRSA samples harboring SCCmec type III or IV were misinterpreted as non-MRSA by investigators. False-positive results were mainly due to methicillin-resistant S. epidermidis. For an arbitrary MRSA prevalence of 5% and based on patient sample evaluations, the positive predictive values for BBL-CHROMagar and CHROMagar (∼84%) were the highest. The negative predictive values of all of the media were ≥92% for MRSA prevalences ranging from 5% to 30%. In conclusion, BBL-CHROMagar and CHROMagar gave the best overall results for detection of MRSA, irrespective of the sample concentration, investigator, or incubation period.Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a major nosocomial pathogen in the last decade. Patients colonized with MRSA serve as reservoirs of self-infection or dissemination to other patients and to the hospital environment (6, 12, 22). Hence, screening for MRSA carriage and contact isolation of MRSA carriers are crucial for effective hospital infection control (9). Employing rapid and sensitive screening assays for MRSA detection could help to further improve infection control, as well as decrease costs (10, 13).In recent years, the use of chromogenic media has become a key method for the rapid identification of microorganisms in clinical samples (20). These media detect key microbial enzymes as diagnostic markers for pathogens through the use of “chromogenic” substrates incorporated into a solid-agar-based matrix (20). In contrast to conventional culture media, chromogenic media allow direct colony color-based identification of the pathogen from the primary culture. This reduces the need for subculture for further biochemical testing and hence the time until a result is obtained. Currently available chromogenic media for MRSA detection incorporate chromogens to differentiate S. aureus from other pathogens and antibiotics for selective growth of MRSA. These media differ in their chromogenic substrates, antibiotic formulations, and/or concentrations, factors that impact their sensitivity and specificity for MRSA detection (reviewed in reference 13). We compared the potential of five of the most commonly used commercial chromogenic media for MRSA detection using pure MRSA isolates, non-MRSA isolates, and mixtures thereof at defined concentrations simulating clinical samples. Further evaluations of the media were carried out with nasal and groin screening samples from hospitalized patients.(Part of this work was presented at the 48th Interscience Conference on Antimicrobial Agents and Chemotherapy, Washington, DC, 24 to 28 October 2008.)     

Prospective Two-Center Comparison of Three Chromogenic Agars for Methicillin-Resistant Staphylococcus aureus Screening in Hospitalized Patients

Three chromogenic media, chromID MRSA SMART (SMART), chromID MRSA first generation (chromID), and Brilliance MRSA (OX2), were evaluated for methicillin-resistant Staphylococcus aureus (MRSA) screening using 1,220 samples. The sensitivity at 24 h was significantly better with the SMART agar (66.4%) than that with chromID agar (50.5%). Enrichment and incubation until 48 h are still needed for an optimal yield.     

Performance of MRSA ID, a new chromogenic medium for detection of methicillin-resistant Staphylococcus aureus

MRSA ID was evaluated for its ability to identify methicillin-resistant Staphylococcus aureus. A well-defined collection of staphylococci was used (n = 998). The sensitivity after 24 h was 96.4%, increasing to 98.8% after 48 h. The specificity was 98.2% after 24 h and decreased to 89.7% after 48 h.     

Comparison of the BBL CHROMagar Staph aureus agar medium to conventional media for detection of Staphylococcus aureus in respiratory samples

Screening for Staphylococcus aureus has become routine in certain patient populations. This study is the first clinical evaluation of the BBL CHROMagar Staph aureus agar (CSA) medium (BD Diagnostics, Sparks, Md.) for detection of S. aureus in nasal surveillance cultures and in respiratory samples from cystic fibrosis (CF) patients. S. aureus colonies appear mauve on CSA. Other organisms are inhibited or produce a distinctly different colony color. S. aureus was identified from all media by slide coagulase, exogenous DNase, and mannitol fermentation assays. Susceptibility testing was performed using the agar dilution method. A total of 679 samples were evaluated. All samples were inoculated onto CSA. Nasal surveillance cultures were inoculated onto sheep blood agar (SBA) (BD Diagnostics), and samples from CF patients were inoculated onto mannitol salt agar (MSA) (BD Diagnostics). Of the 679 samples cultured, 200 organisms produced a mauve color on CSA (suspicious for S. aureus) and 180 were positive for S. aureus on SBA or MSA. Of 200 CSA-positive samples 191 were identified as S. aureus. Nine mauve colonies were slide coagulase negative and were subsequently identified as Staphylococcus lugdunensis (one), Staphylococcus epidermidis (three), Staphylococcus haemolyticus (one), and Corynebacterium species (four). CSA improved the ability to detect S. aureus by recovering 12 S. aureus isolates missed by conventional media. Of the 192 S. aureus isolates recovered, 122 were methicillin susceptible and 70 were methicillin resistant. Overall, the sensitivity and specificity of CSA in this study were 99.5 and 98%, respectively. There was no difference in the performance of the slide coagulase test or in susceptibility testing performed on S. aureus recovered from CSA compared to SBA or MSA. Our data support the use of CSA in place of standard culture media for detection of S. aureus in heavily contaminated respiratory samples.