Chromogenic media for MRSA diagnostics

For the past decade, a number of chromogenic media for methicillin-resistance Staphylococcus aureus (MRSA) detection have been developed and applied, including Oxoid Brilliance? MRSA, CHROMagar? MRSA, BBL? CHROMagar? MRSA, MRSASelect and chromID MRSA. The advantages of these chromogenic media offers direct detection of visible staphylococcal colonies, coupled with the use of chromogenic enzymatic substrates that can be hydrolyzed by S. aureus to confirm species or strain identification. BBL? CHROMagar? MRSA and MRSASelect are designed for detection of nasal colonization by MRSA, while CHROMagar? MRSA, Oxoid Brilliance? MRSA and chromID MRSA are readily applied in bacterial screening. This review summarizes the characteristics, principles and capacities of these selective media, and focuses on comparison of different chromogenic media.     

Evaluation of chromogenic media for the isolation of vancomycin-resistant enterococci from stool samples

Aims:  This study sought to evaluate the performance of two chromogenic media designed for the isolation of vancomycin-resistant enterococci (VRE) and compare them with a traditional bile-esculin medium for the isolation of VRE from stool samples.
Methods and Results:  A total of 285 stool samples were inoculated onto Chromogenic VRE Agar (AES VRE agar; AES Chemunex), chromID VRE (bioMérieux) and VRE Agar (Oxoid) both directly and also following broth enrichment. In total 18 strains of vancomycin-resistant Enterococcus faecium were recovered, including 17 harbouring the vanA gene and one with vanB . On direct culture, the sensitivity of the three media was 66·7%, 77·8% and 44·4% and after broth enrichment 66·7%, 83·3% and 77·8% using AES VRE Agar, chromID VRE and Oxoid VRE Agar respectively.
Conclusions:  All three media are useful tools for the isolation of VRE from stool samples. AES VRE Agar and bioMérieux chromID VRE are easier to use than Oxoid VRE Agar due to diffusion of black coloration from the latter.
Significance and Impact of the Study:  This is the first study to evaluate the performance of AES VRE Agar and the first to compare two media containing synthetic chromogens for the isolation of VRE.     

Comparative Evaluation of Five Culture Media with Triplex PCR Assay for Detection of Methicillin-Resistant Staphylococcus aureus

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) is responsible for nosocomial and community-acquired infections. Hence, rapid and accurate laboratory diagnosis of MRSA is a vital constituent of control measures. The present study evaluated five different methods for the identification of MRSA. A total of 207 S. aureus clinical isolates that consisted of 89 MRSA and 118 methicillin-susceptible S. aureus (MSSA) strains confirmed by PCR were tested. MRSA strains were evaluated by five different methods: chromogenic MRSA agar (CMRSA), oxacillin resistance screening agar base (ORSAB), mannitol salt oxacillin agar (MSO), mannitol salt cefoxitin agar with two different concentrations of cefoxitin [4 μg/ml (MSC-4) and 6 μg/ml (MSC-6)]. The results of the different methods were compared to mecA PCR as the gold standard. MSC-6 showed only six false-positive MRSA in comparison with PCR. The sensitivities and specificities of MSC-6, MSC-4, MSO-4, ORSAB, and CMRSA were as follows: 98.9/94.9%, 100/83.1%, 89.9/87.3%, 97.8/96.6%, and 95.5/94.9%, respectively. In comparison with PCR, it was found that both MSC-6 and ORSAB were relatively the least expensive screening tests (0.70 and0.70 and 1.00, respectively). In conclusion, all methods were comparable, but MSC-6 was the least expensive medium for MRSA screening.     

The molecular epidemiology of Stenotrophomonas maltophilia bacteraemia in a tertiary referral hospital in the United Arab Emirates 2000–2004

Background

Methicillin-resistant Staphylococcus aureus (MRSA) has become increasingly prevalent worldwide since it was first reported in a British hospital. The prevalence however, varies markedly in hospitals in the same country, and from one country to another. We therefore sought to document comprehensively the prevalence and antimicrobial susceptibility pattern of MRSA isolates in Trinidad and Tobago.

Methods

All Staphylococcus aureus isolates encountered in routine clinical specimens received at major hospitals in the country between 2000 and 2001 were identified morphologically and biochemically by standard laboratory procedures including latex agglutination test (Staphaurex Plus; Murex Diagnostics Ltd; Dartford, England); tube coagulase test with rabbit plasma (Becton, Dickinson & Co; Sparks, MD, USA), and DNase test using DNase agar (Oxoid Ltd; Basingstoke, Hampshire, England). MRSA screening was performed using Mueller-Hinton agar containing 6 μg oxacillin and 4% NaCl, latex agglutination test (Denka Seiken Co. Ltd, Tokyo, Japan) and E-test system (AB Biodisk, Solna, Sweden). Susceptibility to antimicrobial agents was determined by the modified Kirby Bauer disc diffusion method while methicillin MICs were determined with E-test system.

Results

Of 1,912 S. aureus isolates received, 12.8% were methicillin (oxacillin) resistant. Majority of the isolates were recovered from wound swabs (86.9%) and the least in urine (0.4%) specimens. Highest number of isolates was encountered in the surgical (62.3%) and the least from obstetrics and gynaecology (1.6%) facilities respectively. Large proportions of methicillin sensitive isolates are >85% sensitive to commonly used and available antimicrobials in the country. All MRSA isolates were resistant to ceftriaxone, erythromycin, gentamicin and penicillin but were 100% sensitive to vancomycin, rifampin and chloramphenicol.

Conclusion

There is a progressive increase in MRSA prevalence in the country but the present rate is still low in comparison to values in some other countries. Vancomycin is still the drug of choice for treating multidrug resistant MRSA infections. Further use of molecular studies to monitor the epidemiology of MRSA in these hospitals in the country is highly recommended too.     

Rapid detection of methicillin resistance in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolates; evaluation of colorimetric Quicolor ES agar and determination of breakpoint inhibition zone diameters of cefoxitin

In this study, it was aimed to evaluate colorimetric Quicolor ES agar for the rapid detection of methicillin resistance and to determine susceptibility and resistance breakpoint zone diameters for cefoxitin by using 51 methicillin susceptible Staphylococcus aureus (MSSA) and 63 methicillin resistant S. aureus (MRSA) isolates. In the study, while oxacillin and cefoxitin results were obtained within 4–7 h (5.5 h in average) for MSSA isolates, the results of MRSA isolates were obtained within 5.5–9 h (6.6 h in average) for both antibiotics on QC ES agar. QC ES agar is an inexpensive medium for rapid detection (4–9 h) of methicillin resistance by disc diffusion method using oxacillin or cefoxitin. Additional studies for further evaluation of the efficiency of QC-ES agar in rapid determination of methicillin resistance in S. aureus may be beneficial.     

Comparison of methods for the detection of methicillin resistance in Staphylococcus aureus isolates from food products

AIMS: To compare several methods for detection of methicillin resistance in Staphylococcus aureus isolates from food. METHODS AND RESULTS: Two hundred S. aureus isolates from food of animal origin were screened for methicillin resistance by a PCR assay specific for the mecA gene, an oxacillin agar screen test and a cefoxitin disk diffusion test. Six out of 200 strains (3%) were found to be methicillin-resistant Staphylococcus aureus (MRSA) by PCR. The oxacillin agar screen test detected only one of the MRSA isolates (sensitivity of 16.7%) and mischaracterized three additional strains as MRSA (specificity of 98.45%). None of the MRSA strains was detected by the cefoxitin test (sensitivity of 0%), while 15 methicillin-susceptible S. aureus (MSSA) strains were misclassified as resistant (specificity of 92.3%). Fifteen MSSA strains displayed a beta-lactamase hyperproducer-like phenotype. The six MRSA (mecA-positive) strains resembled the characteristics of heteroresistant strains. CONCLUSIONS: As MRSA of animal origin may display atypical phenotypes, PCR appears to be more reliable for detection of methicillin resistance in animal strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The study stresses the need for implementing the methods of screening S. aureus from food of animal origin for methicillin resistance.     

Evaluation of a rapid culture-based screening test for detection of methicillin resistant Staphylococcus aureus

The performance of a culture based assay, BacLite Rapid MRSA for the rapid detection (5 hours) of methicillin resistant Staphylococcus aureus (MRSA) from specimens (n = 377) obtained from nares, throat, wounds and perineum was investigated. Compared to culture based reference methods (chromogenic MRSA ID (bioMerieux)), selective enrichment broth, PBP2' latex agglutination (Oxoid) and VITEK 2 identification (bioMerieux), an overall sensitivity of 71% with a 82% specificity and a negative predictive value (NPV) of 95% was provided. The Baclite test is rapid and easy to use and has the advantage of a culture-based detection method for MRSA.     

Differential performance of marubakaido apple rootstock shoots grown in culture media containing different agar brands: dynamic rheological analysis

Shoots of the marubakaido apple rootstock grown in culture medium containing BBL agar presented significantly lower multiplication rate (MR) compared to MRs found for shoots grown in medium containing A-7002, A-7921, Select, and Phytagar as gelling agents. In addition, significant hyperhydricity was found for shoots grown in Phytagar and A-7921 agar-containing media. Analysis of elastic (G′) and viscous (G″) modulus showed that for all of the five agar brands used in this study, G′ was always much higher, i.e., typically one order of magnitude higher than G″, which characterizes a strong gel. G′ changed randomly with time for all of the agar brands studied, except for BBL, which presented progressive decline in G′ throughout the culture cycle. Examination of G′, within the same week, showed that Select agar always had the smallest G′, while Phytagar always had the highest G′. Analysis of the loss tangent (tan δ = G″/G′), a better indicator for gel behavior compared to G′ isolated, showed that tan δ for Select and Phytagar were always between tan δ values found for A-7002 and BBL. In addition, analysis of tan δ also indicated that BBL and Select agars showed a significantly weaker gel network, compared to Phytagar, A-7002 and A-7921 agars after the third week of culture. When seen together, these results indicate that shoot performance for the marubakaido rootstock is not related to agar gel strength. In addition, the high hyperhydricity rate found for shoots grown on agars A-7921 and Phytagar could not be related to agar gel strength, as well. Analysis of HPSEC profiles indicated that the best performance, i.e., multiplication rate, of marubakaido shoots in agars A-7002 and A-7921 is likely to be related to their lower polydispersity and/or smaller amount of high molecular weight fractions, compared to BBL, Phytagar, and Select agars.     

Detection of methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> using double duplex real-time PCR and dye Syto 9

A screening method for methicillin-resistant Staphylococcus aureus (MRSA) using real-time polymerase chain reaction (PCR) and dye Syto 9 was developed and evaluated. The assay was based on the two duplex reactions run simultaneously. The detection reaction amplified staphylococcal cassette chromosome mec (SCCmec) right extremity sequences and S. aureus-specific 442-bp DNA (Sa442). The control reaction amplified S. aureus-specific nuclease gene nuc and a marker of methicillin resistance, mecA. The method was evaluated by analyzing 214 clinical S. aureus isolates yielding 98.7 % sensitivity, 100 % specificity, 100 % positive predictive value and 96.6 % negative predictive value for detection of MRSA. The detection limit was determined to be 15–80 genome copies per real-time PCR. It was able to discriminate between MRSA, methicillin resistant coagulase negative staphylococci and methicillin susceptible S. aureus (MSSA) isolates containing only small fragments of the right extremity of the SCCmec (MSSA revertants).     

Prevalence of NDM‐1 carbapenemase in patients with diarrhoea in Pakistan and evaluation of two chromogenic culture media

Aims

To evaluate two chromogenic media, Brilliance CRE and chromID CARBA, with stool samples referred to the Public Health Laboratories Division of the National Institute of Health in Islamabad, and assess the prevalence of carbapenemase‐producing Enterobacteriaceae (CPE) in this population.

Methods and Results

One hundred and fifty‐two stool samples from patients with diarrhoea were referred to the Microbiology Department and were investigated for the presence of CPE using two chromogenic culture media, Brilliance CRE and chromID CARBA. Thirteen patients (8·6%) were found to be colonized with CPE and all produced NDM‐1 carbapenemase. Twelve of these patients (92%) were found to be colonized by culture on chromID CARBA compared with seven (54%) using Brilliance CRE.

Conclusions

If only coloured colonies were considered as presumptive CPE, the sensitivity, specificity and positive predictive value were 54, 23 and 6% for Brilliance CRE and 85, 85 and 36% for chromID CARBA, respectively.

Significance and Impact of the Study

We conclude that Enterobacteriaceae that produce NDM‐1 carbapenemase can be found in patients from all major provinces of Pakistan and that chromID CARBA was the most effective of the two chromogenic media in this setting.     

Evaluation of BBL CHROMagar Listeria agar for the isolation and identification of Listeria monocytogenes from food and environmental samples

The performance of BBL CHROMagar Listeria chromogenic agar for the detection of Listeria monocytogenes was evaluated for its ability to isolate and identify L. monocytogenes from food and environmental samples. The medium was compared to non-chromogenic selective agars commonly used for Listeria isolation: Oxford, Modified Oxford, and PALCAM. BBL CHROMagar Listeria had a sensitivity of 99% and 100% for the detection of L. monocytogenes from 200 natural and artificially inoculated food samples, respectively, with a colony confirmation rate of 100%. The sensitivity of non-chromogenic selective media for the detection of L. monocytogenes from these same samples was 97-99% with colony confirmation rates of 65-67.5%. From 93 environmental samples, BBL CHROMagar Listeria agar results correlated 100% with a Listeria spp. visual immunoassay (TECRA) performed on these same samples and the USDA-FSIS standard culture method for the isolation of L. monocytogenes. From environmental samples, the L. monocytogenes confirmation rate was 100% for BBL CHROMagar Listeria as compared to 50% for conventional agars tested. On BBL CHROMagar Listeria, L. monocytogenes forms a translucent white precipitation zone (halo) surrounding blue-pigmented colonies of 2-3 mm in diameter, with an entire border. BBL CHROMagar Listeria offers a high degree of specificity for the confirmation of suspect L. monocytogenes colonies, whereas non-chromogenic selective agars evaluated were not differential for L. monocytogenes from other Listeria species.     

Identification and Discrimination of Methicillin Resistant Staphylococcus aureus Strains Isolated from Burn Wound Sites Using PCR and Authentication with MALDI-TOF–MS

The present study demonstrates isolation and identification of methicillin resistance Staphylococcus aureus (MRSA) strains in the samples collected from burn patients. About 106 swab samples were collected from burn patients of >40% burn injury and were subjected to isolation using nutrient agar followed by screening using Me Re Sa selective medium agar. A total of 10 isolates with identity to S. aureus were obtained and further authenticated using Polymerase Chain Reaction and matrix assisted laser desorption/ionization time of flight mass spectrometry analysis. Presence of mec A gene and the peak pattern observations suggested seven of the 10 isolates are MRSA. Thus, the present study emphasizes the process of identification of MRSA using two different bio-analytical techniques, which authenticate the presence of MRSA.     

Evaluation of BacLiteRapidMRSA, a rapid culture based screening test for the detection of ciprofloxacin and methicillin resistantS. aureus(MRSA) from screening swabs

Background  

Methicillin-resistantStaphylococcus aureus(MRSA) is a major nosocomial pathogen worldwide. The need for accurate and rapid screening methods to detect MRSA carriers has been clearly established. The performance of a novel assay, BacLiteRapidMRSA (Acolyte Biomedica, UK) for the rapid detection (5 h) and identification of hospital associated ciprofloxacin resistant strains of MRSA directly from nasal swab specimens was compared to that obtained by culture on Mannitol salt agar containing Oxacillin (MSAO) after 48 h incubation.     

Efficiency of the Cepheid Xpert vanA/vanB assay for screening of colonization with vancomycin-resistant enterococci during hospital outbreak

This study aimed to assess the efficiency of the Cepheid Xpert vanA/vanB test for detecting vancomycin-resistant enterococci (VRE) colonization during a VanA Enterococcus faecium outbreak and to compare the Cepheid Xpert vanA/vanB (Cepheid, Sunnyvale, USA) test to a culture method with chromogenic medium chromID VRE agar (bioMérieux). The Cepheid Xpert vanA/vanB assay showed sensitivity 61.5%, specificity 79.2%, positive predictive value 61.5% and negative predictive value 79.2%. The results obtained in this study demonstrate that a positive result in the Cepheid Xpert vanA/vanB test for vanA enables the rapid (less than 1 h) presumptive, prior to culture, recognition of patients colonized with VRE. However, the Cepheid Xpert vanA/vanB assay cannot be the only test used to screen patients during an ongoing VRE outbreak, because additional culturing of all samples negative for both vanA and vanB or positive for vanB should be performed in order to confirm the carrier status of the patient.     

Comparison of <Emphasis Type="Italic">mecA</Emphasis> Polymerase Chain Reaction With Phenotypic Methods for the Detection of Methicillin-Resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

In the present study, several conventional methods to detect methicillin-resistant Staphylococcus aureus (MRSA) were compared with polymerase chain reaction (PCR) detection of mecA gene–positive isolates. Cefoxitin E-test was also evaluated as a possible phenotypic method of MRSA detection. Oxacillin agar screen and PBP2′ latex agglutination methods were found to be more sensitive than oxacillin and cefoxitin disk-diffusion methods. Cefoxitin disk diffusion was found to be the most specific. A combination of oxacillin agar screening with cefoxitin disk diffusion, or oxacillin disk diffusion with PBP2′, improved sensitivity and specificity. Cefoxitin E-test with the current break points had low sensitivity and specificity (33.3% and 75%, respectively) for the detection of MRSA. However, changing the break points to ≤ 4 μg/ml and to ≥ 6 μg/ml for sensitive and resistant, respectively, greatly improved both. Changing the 30-μg cefoxitin disk-diffusion break points to ≤ 21 mm for resistant slightly improved sensitivity but had no effect on specificity. It was therefore concluded that the use of more than one screening method is necessary to detect all MRSA isolates in clinical settings.     

New categories designated as healthcare‐associated and community‐associated methicillin‐resistant Staphylococcus pseudintermedius in dogs

The incidence of MRSP has been increasing, and treatment options in veterinary medicine are limited. Few previous studies of MRSP have described the relationships between the genotypes, phenotypes, and clinical backgrounds of the isolates. To gain insight into the associations between the microbiological and clinical characteristics of MRSP, we analyzed 282 Staphylococcus pseudintermedius isolates from dogs. A total of 195 (69.1%) strains were identified as mecA‐positive MRSP and were classified into mainly two genotypes: SCCmec types III (II‐III) (52.8%) and V (37.4%). SCCmec type III MRSP strains were significantly correlated with hospital admission and antimicrobial therapy of the dogs, and exhibited a homogeneous genotype similar to sequence type 71‐MRSP, which is a globally endemic clone in dogs. In contrast, SCCmec type V MRSP strains were not highly correlated with hospital admission and antimicrobial therapy and exhibited genotypic and phenotypic heterogeneity. Properties of MRSP strains SCCmec types III and V were similar to those of HA‐ and CA‐MRSA, respectively. Therefore, we designated these isolates carrying SCCmec types III and V as HA‐MRSP and CA‐MRSP, respectively. Discrimination between HA‐ and CA‐MRSP by oxacillin MIC will provide useful information for treatment and infection control measures for canine MRSP infections.     

Comparison of Real-Time PCR with Disk Diffusion, Agar Screen and E-test Methods for Detection of Methicillin-Resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is a nosocomial pathogen. Our main objective was to compare oxacillin disk test, oxacillin E-test, and oxacillin agar screen for detection of methicillin resistance in S. aureus, using real-time PCR for mecA as the “gold standard” comparison assay. 196 S. aureus isolates were identified out of 284 Staphylococcus isolates. These isolates were screened for MRSA with several methods: disk diffusion, agar screen (6.0 μg/ml), oxacillin E-test, and real-time PCR for detection of mecA gene. Of the 196 S. aureus isolates tested, 96 isolates (49%) were mecA-positive and 100 isolates (51%) mecA-negative. All methods tested had a statistically significant agreement with real-time PCR. E-test was 100% sensitive and specific for mecA presence. The sensitivity and specificity of oxacillin agar screen method were 98 and 99%, respectively and sensitivity and specificity of oxacillin disk diffusion method were 95 and 93%, respectively. In the present study, oxacillin E-test is proposed as the best phenotypic method. For economic reasons, the oxacillin agar screen method (6.0 μg/ml), which is suitable for the detection of MRSA, is recommended due to its accuracy and low cost.     

Methicillin-resistant Staphylococcus pseudintermedius isolated from canine pyoderma in North China

Aims: To determine the prevalence of carriage of methicillin‐resistant Staphylococcus pseudintermedius (MRSP) among dogs with pyoderma from two small animal hospitals in North China during a 21‐month period and to characterize these isolates. Methods and Results: Swabs were taken from 260 dogs with pyoderma, and the staphylococcal species isolated and methicillin resistance were confirmed phenotypically and genotypically. The identified MRSP isolates were characterized by multilocus sequence typing (MLST), spa typing, staphylococcal cassette chromosome (SCC) mec typing, testing for susceptibility to nine antimicrobial agents and SmaI‐digested pulsed‐field gel electrophoresis. Thirty‐three (12·7%) dogs were positive for MRSP. The most prevalent genotypes detected among MRSP were ST71(MLST)‐t06(spa)‐II‐III(SCCmec) (n = 22, 66·7%), followed by ST5‐t19 (n = 8, 24·2%), ST126‐III(n = 2, 6·1%) and ST6‐t02‐V (3·0%). All MRSP isolates showed extended resistance to tested antimicrobial agents. Eight different SmaI patterns were observed in 21 typeable MRSP isolates. Conclusions: Clinical isolates of MSRP isolated from dogs in North China belonged to two major clonal lineages ST71 and ST5. Significance and Impact of the Study: This study is the first report on MRSP from canine pyoderma in China. Further surveillance study is needed to gain more detailed data concerning this major clinical challenge in veterinary medicine.     

两种纸片检测耐甲氧西林葡萄球菌结果比较

目的以苯唑西林琼脂筛选法为标准,比较苯唑西林和头孢西丁2种药敏纸片检测耐甲氧西林葡萄球菌(MRS)结果的准确性和一致性。方法分别用苯唑西林琼脂筛选法,头孢西丁和苯唑西林纸片扩散法检测临床分离的595株葡萄球菌。以琼脂筛选法为标准,计算2种纸片法的敏感性和特异性,比较二者的检测准确率差异是否存在统计学意义。结果金黄色葡萄球菌中,MRSA的检出率为55.97%,两药敏纸片的敏感性和特异性均在97%以上;二者在筛选MRSA的准确率上差异无统计学意义(P>0.05)。凝固酶阴性葡萄球菌中,MRSCN的检出率为69.68%,两纸片的敏感性均为100.00%,特异性分别为92.86%(头孢西丁)和77.38%(苯唑西林)。二者在检测MRSCN的准确率上差异存在统计学意义(P(0.05)。结论常规工作中,头孢西丁可准确检测所有的MRS,但苯唑西林只能用于检测MRSA,不能检测MRSCN。     

Molecular epidemiology of methicillin-resistant Staphylococcus aureus isolates from clinical specimens of patients with nosocomial infection: are there unnoticed silent outbreaks?

Bacteriological and epidemiological studies were carried out on 90 isolates of methicillin-resistant Staphylococcus aureus (MRSA) at Turgut Ozal Medical Center of In?nü University, (Malatya/Turkey). MRSA isolates were obtained from patients with nosocomial infections. Staphylococcus aureus clinical isolates were collected between May 2004-May 2005. Isolates were tested for resistance to methicillin. Antimicrobial susceptibility testing and slime production evaluation was performed. Genotype studies were carried out by arbitrarily primed polymerase chain reaction (AP-PCR) and consequent cluster analysis. All of the isolates were mecA-positive in a PCR-based assay; all exhibited resistance to oxacillin, by agar dilution (MICs > or = 4 mg/L) and disc diffusion methods, and multiple antibiotics. Most MRSA isolates were collected in intensive care units. Of 90 samples, 53 were found to be unrelated to the others while the remaining 37 strains were either identical or closely related. Dendrogram analysis identified nine major clusters. These data support the opinion that MRSA are significant nosocomial pathogens in intensive care units and that resistant clones may be transmitted between patients. Molecular epidemiological tools are helpful for understanding transmission patterns and sources of infection, and are useful for measuring outcomes of intervention strategies implemented to reduce nosocomial MRSA.