Lisa Louie

Rapid Detection of Methicillin-Resistant Staphylococci from Blood Culture Bottles by Using a Multiplex PCR Assay

ABSTRACT Rapid detection and accurate identification of methicillin-resistant staphylococci are critical for the effective management of infections caused by these organisms. We describe a multiplex PCR-based assay for the direct detection of methicillin-resistant staphylococci from blood culture bottles (BacT/Alert; Organon-Teknika, Durham, N.C.). A simple lysis method followed by a multiplex PCR assay designed to detect the nuc, mecA, and bacterial 16S rRNA genes was performed. A total of 306 blood culture specimens were collected over a period of 10 months from June 1998 to April 1999, consisting of 236 blood cultures growing staphylococci (including 124 methicillin-resistant Staphylococcus spp.), 50 positive blood cultures which grew organisms other than staphylococci, and 20 blood cultures that were negative for bacterial and fungal pathogens after 5 days of incubation and terminal subculture. DNA extraction, PCR, and detection could be completed in 2.5 h. Of the positive blood cultures with staphylococci, the multiplex PCR assay had a sensitivity and specificity of 99.2% and 100%, respectively. Our results show that rapid, direct detection of methicillin-resistant staphylococci is possible, allowing clinicians to make prompt and effective decisions for the management of patients with staphylococcal bacteremia.

Mupirocin-Resistant, Methicillin-Resistant Staphylococcus aureus Strains in Canadian Hospitals

ABSTRACT Mupirocin resistance in Staphylococcus aureus is increasingly being reported in many parts of the world. This study describes the epidemiology and laboratory characterization of mupirocin-resistant methicillin-resistant S. aureus (MRSA) strains in Canadian hospitals. Broth microdilution susceptibility testing of 4,980 MRSA isolates obtained between 1995 and 2004 from 32 Canadian hospitals was done in accordance with CLSI guidelines. The clinical and epidemiologic characteristics of strains with high-level mupirocin resistance (HLMupr) were compared with those of mupirocin-susceptible (Mups) strains. MRSA strains were characterized by pulsed-field gel electrophoresis (PFGE) and typing of the staphylococcal chromosomal cassette mec. PCR was done to detect the presence of the mupA gene. For strains with mupA, plasmid DNA was extracted and subjected to Southern blot hybridization. A total of 198 (4.0%) HLMupr MRSA isolates were identified. The proportion of MRSA strains with HLMupr increased from 1.6% in the first 5 years of surveillance (1995 to 1999) to 7.0% from 2000 to 2004 (P < 0.001). Patients with HLMupr MRSA strains were more likely to have been aboriginal (odds ratio [OR], 3.7; 95% confidence interval [CI], 1.5 to 9.4; P = 0.006), to have had community-associated MRSA (OR, 2.2; 95% CI, 1.0 to 5.0; P = 0.05), and to have been colonized with MRSA (OR, 1.7; 95% CI, 1.0 to 3.0; P = 0.04). HLMupr MRSA strains were also more likely to be resistant to fusidic acid (21% versus 4% for mupirocin-susceptible strains; P < 0.001). All HLMupr MRSA strains had a plasmid-associated mupA gene, most often associated with a 9-kb HindIII fragment. PFGE typing and analysis of the plasmid profiles indicate that both plasmid transmission and the clonal spread of HLMupr MRSA have occurred in Canadian hospitals. These results indicate that the incidence of HLMupr is increasing among Canadian strains of MRSA and that HLMupr MRSA is recovered from patients with distinct clinical and epidemiologic characteristics compared to the characteristics of patents with Mups MRSA strains.

Methicillin-Resistant Staphylococcus aureus Colonization or Infection in Canada: National Surveillance and Changing Epidemiology, 1995-2007

OBJECTIVE To determine the incidence and describe the changing epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) colonization or infection in Canadian hospitals from 1995-2007. SETTING Forty-eight hospitals participating in the Canadian Nosocomial Infection Surveillance Program. DESIGN Prospective, laboratory-based surveillance for incident cases of MRSA colonization or infection among hospitalized patients. METHODS Clinical and epidemiologic data were obtained by review of hospital records. Standard criteria were used to determine whether MRSA colonization or infection was present and whether the MRSA strain was healthcare associated or community associated. A representative subset of isolates was characterized by use of pulsed-field gel electrophoresis and staphylococcal cassette chromosome (SCC) mec typing. RESULTS From 1995 to 2007, a total of 37,169 hospitalized patients were newly identified as either infected or colonized with MRSA, and the overall incidence of both MRSA colonization and MRSA infection increased from 0.65 to 11.04 cases per 10,000 patient-days (P < .001). Of these 37,169 patients, 11,828 (32%) had an MRSA infection, and infection rate increased from 0.36 to 3.43 cases per 10,000 patient-days. The proportion of community-associated MRSA strains increased from 6% to 23% (P < .001). The most common genotype (47% of isolates) was CMRSA-2 (USA100/800); in 2007, CMRSA-10 (USA300) was the second most common strain (27% of isolates), associated with SCCmec type IV. Patients with CMRSA-10 were predominantly from western Canada and were more likely to be children (odds ratio [OR], 10.0 [95% confidence interval {CI}, 7.4-13.4]) and to have infection (OR, 2.3 [95% CI, 1.9-2.7]), especially skin and/or soft tissue infection (OR, 5.9 [95% CI, 5.0-6.9]). CONCLUSIONS The overall incidence of both MRSA colonization and MRSA infection increased 17-fold in Canadian hospitals from 1995 to 2007. There has also been a dramatic increase in cases of community-associated MRSA infection due to the CMRSA-10 (USA300) clone. Continued surveillance is needed to monitor the ongoing evolution of MRSA colonization or infection in Canada and globally.

MupB, a New High-Level Mupirocin Resistance Mechanism in Staphylococcus aureus

ABSTRACT Mupirocin is a topical antibiotic used for the treatment of skin infections and the eradication of methicillin-resistant Staphylococcus aureus carriage. It inhibits bacterial protein synthesis by interfering with isoleucyl-tRNA synthetase activity. High-level mupirocin resistance (MIC of ≥512 μg/ml) is mediated by the expression of mupA (ileS2), which encodes an alternate isoleucyl-tRNA synthetase. In this study, we describe high-level mupirocin resistance mediated by a novel locus, mupB. The mupB gene (3,102 bp) shares 65.5% sequence identity with mupA but only 45.5% identity with ileS. The deduced MupB protein shares 58.1% identity (72.3% similarity) and 25.4% identity (41.8% similarity) with MupA and IleS, respectively. Despite this limited homology, MupB contains conserved motifs found in class I tRNA synthetases. Attempts to transfer high-level mupirocin resistance via conjugation or transformation (using plasmid extracts from an mupB-containing strain) were unsuccessful. However, by cloning the mupB gene into a shuttle vector, it was possible to transfer the resistance phenotype to susceptible S. aureus by electroporation, proving that mupB was responsible for the high-level mupirocin resistance. Further studies need to be done to determine the prevalence of mupB and to understand risk factors and outcomes associated with resistance mediated by this gene.

Evaluation of a Latex Agglutination Test (MRSA-Screen) for Detection of Oxacillin Resistance in Coagulase-Negative Staphylococci

ABSTRACT The MRSA-Screen (Denka-Seiken, Tokyo, Japan) latex agglutination test was evaluated for its ability to detect PBP 2a from 200 clinical isolates of coagulase-negative staphylococci (CoNS; 84mecA-positive strains and 116mecA-negative strains) consisting of 108Staphylococcus epidermidis, 37 S. saprophyticus, 15 S. haemolyticus, 11 S. hominis, 10 S. capitis, 10 S. warneri, and 3 S. lugdunensis species as well as 6 other species of CoNS. The assay was compared with susceptibility testing with an agar screen plate with oxacillin at 6 μg/ml (OXA6), by oxacillin disk diffusion (DD), by broth microdilution (BMDIL), by the E test, and with Vitek GPS-SV and Vitek GPS-107 susceptibility cards. PCR for the detection of the mecA gene was used as the “gold standard.” The sensitivities and specificities for the methods evaluated were as follows: MRSA-Screen, 100 and 100%, respectively; OXA6, 100 and 99%, respectively; DD, 98 and 62%, respectively; BMDIL, 100 and 60%, respectively; E test, 100 and 51%, respectively; Vitek GPS-SV susceptibility card, 98 and 87%, respectively; and Vitek GPS-107 susceptibility card, 100 and 61%, respectively. The MRSA-Screen test accurately and rapidly detected oxacillin resistance in CoNS.

Evaluation of a Rapid PCR Assay for Diagnosis of Meningococcal Meningitis

ABSTRACT We compared the results of Gram staining and culture of cerebrospinal fluid to results obtained with a rapid PCR assay for the diagnosis of meningococcal meningitis in 281 cases of suspected bacterial meningitis. PCR had a sensitivity of 97% compared to a sensitivity of 55% for culture, and the PCR specificity was 99.6%. PCR results were available within 2 h of the start of the assay.

Multicenter comparison of nucleic acid extraction methods for detection of severe acute respiratory syndrome coronavirus RNA in stool specimens.

ABSTRACT The emergence of a novel coronavirus (CoV) as the cause of severe acute respiratory syndrome (SARS) catalyzed the development of rapid diagnostic tests. Stool samples have been shown to be appropriate for diagnostic testing for SARS CoV, although it has been recognized to be a heterogeneous and difficult sample that contains amplification inhibitors. Limited information on the efficiency of extraction methods for the purification and concentration of SARS CoV RNA from stool samples is available. Our study objectives were to determine the optimal extraction method for SARS CoV RNA detection and to examine the effect of increased specimen volume for the detection of SARS CoV RNA in stool specimens. We conducted a multicenter evaluation of four automated and four manual extraction methods using dilutions of viral lysate in replicate mock stool samples, followed by quantitation of SARS CoV RNA using real-time reverse transcriptase PCR. The sensitivities of the manual methods ranged from 50% to 100%, with the Cortex Biochem Magazorb method, a magnetic bead isolation method, allowing detection of all 12 positive samples. The sensitivities of the automated methods ranged from 75% to 100%. The bioMérieux NucliSens automated extractor and miniMag extraction methods each had a sensitivity of 100%. Examination of the copy numbers detected and the generation of 10-fold dilutions of the extracted material indicated that a number of extraction methods retained inhibitory substances that prevented optimal amplification. Increasing the volume of sample input did improve detection. This information could be useful for the extraction of other RNA viruses from stool samples and demonstrates the need to evaluate extraction methods for different specimen types.

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

ABSTRACT 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.

Detection and Characterization of Heterogeneous Vancomycin-Intermediate Staphylococcus aureus Isolates in Canada: Results from the Canadian Nosocomial Infection Surveillance Program, 1995-2006

ABSTRACT We describe the epidemiology of heterogeneously resistant Staphylococcus aureus (hVISA) identified in Canadian hospitals between 1995 and 2006. hVISA isolates were confirmed by the population analysis profiling-area under the curve method. Only 25 hVISA isolates (1.3% of all isolates) were detected. hVISA isolates were more likely to have been health care associated (odds ratio [OR], 5.1; 95% confidence interval [CI], 1.9 to 14.2) and to have been recovered from patients hospitalized in central Canada (OR, 3.0; 95% CI, 1.2 to 7.4). There has been no evidence of vancomycin “MIC creep” in Canadian strains of methicillin (meticillin)-resistant S. aureus, and hVISA strains are currently uncommon.

Characterization of Staphylococcus aureus Isolates with a Partial or Complete Absence of Staphylococcal Cassette Chromosome Elements

ABSTRACT Detection of methicillin-resistant Staphylococcus aureus (MRSA) by single-locus PCR assays that target the extremity of the staphylococcal cassette chromosome-mec (SCCmec) and part of the adjacent S. aureus-specific open reading frame gene (orfX) is a significant diagnostic advancement, since it provides real-time detection directly from screening specimens. However, isolates harboring mecA deletions within SCCmec may result in false-positive identification of MRSA in these assays. We characterized 24 methicillin-susceptible S. aureus (MSSA) isolates that tested positive in one such assay to investigate this phenomenon. Seven isolates resembled USA100 and carried SCCmec II elements with mecA deletions that spanned 20 to 46 kbp. The mecA excisions in USA100-resembling isolates appeared to be linked with IS431 transposable elements present in SCCmec II. For 17 isolates that resembled USA400 and/or MSSA476, the identity and possible excision of SCC elements could not be confirmed. The downstream common sequence (dcs) shared by SCCmec I, II, and IV elements was detected in these isolates. Sequence analysis of the chromosomal regions flanking the missing SCC element revealed an intact SCC integration site, a duplicate dcs, and the enterotoxin gene cluster downstream of orfX. An annealing sequence for one of the SCCmec-specific primers (mecii574) in the single-locus PCR assay was identified in the duplicate dcs. In the absence of SCC, a 176-bp amplicon can be generated from this mecii574 annealing sequence to yield a false-positive result. In conclusion, partial SCCmec II excisions via IS431 elements in strains that resembled USA100 and the presence of a duplicate mecii574 annealing sequence in strains that resembled USA400/MSSA476 were identified as causes for false-positive results in a single-locus PCR assay that targets the SCCmec/orfX junction.