Methicillin-Resistant Staphylococcus aureus (MRSA) Nasal Real-Time PCR: A Predictive Tool for Contamination of the Hospital Environment

OBJECTIVE We sought to determine whether the bacterial burden in the nares, as determined by the cycle threshold (CT) value from real-time MRSA PCR, is predictive of environmental contamination with MRSA. METHODS Patients identified as MRSA nasal carriers per hospital protocol were enrolled within 72 hours of room admission. Patients were excluded if (1) nasal mupirocin or chlorhexidine body wash was used within the past month or (2) an active MRSA infection was suspected. Four environmental sites, 6 body sites and a wound, if present, were cultured with premoistened swabs. All nasal swabs were submitted for both a quantitative culture and real-time PCR (Roche Lightcycler, Indianapolis, IN). RESULTS At study enrollment, 82 patients had a positive MRSA-PCR. A negative correlation of moderate strength was observed between the CT value and the number of MRSA colonies in the nares (r=−0.61; P<0.01). Current antibiotic use was associated with lower levels of MRSA nasal colonization (CT value, 30.2 vs 27.7; P<0.01). Patients with concomitant environmental contamination had a higher median log MRSA nares count (3.9 vs 2.5, P=0.01) and lower CT values (28.0 vs 30.2; P<0.01). However, a ROC curve was unable to identify a threshold MRSA nares count that reliably excluded environmental contamination. CONCLUSIONS Patients with a higher burden of MRSA in their nares, based on the CT value, were more likely to contaminate their environment with MRSA. However, contamination of the environment cannot be predicted solely by the degree of MRSA nasal colonization

sodC-Based Real-Time PCR for Detection of Neisseria meningitidis

Real-time PCR (rt-PCR) is a widely used molecular method for detection of Neisseria meningitidis (Nm). Several rt-PCR assays for Nm target the capsule transport gene, ctrA. However, over 16% of meningococcal carriage isolates lack ctrA, rendering this target gene ineffective at identification of this sub-population of meningococcal isolates. The Cu-Zn superoxide dismutase gene, sodC, is found in Nm but not in other Neisseria species. To better identify Nm, regardless of capsule genotype or expression status, a sodC-based TaqMan rt-PCR assay was developed and validated. Standard curves revealed an average lower limit of detection of 73 genomes per reaction at cycle threshold (Ct) value of 35, with 100% average reaction efficiency and an average R2 of 0.9925. 99.7% (624/626) of Nm isolates tested were sodC-positive, with a range of average Ct values from 13.0 to 29.5. The mean sodC Ct value of these Nm isolates was 17.6±2.2 (±SD). Of the 626 Nm tested, 178 were nongroupable (NG) ctrA-negative Nm isolates, and 98.9% (176/178) of these were detected by sodC rt-PCR. The assay was 100% specific, with all 244 non-Nm isolates testing negative. Of 157 clinical specimens tested, sodC detected 25/157 Nm or 4 additional specimens compared to ctrA and 24 more than culture. Among 582 carriage specimens, sodC detected Nm in 1 more than ctrA and in 4 more than culture. This sodC rt-PCR assay is a highly sensitive and specific method for detection of Nm, especially in carriage studies where many meningococcal isolates lack capsule genes

Genetic Analysis of the Capsule Locus of Haemophilus influenzae Serotype f

A 19-kb DNA region containing genes involved in the biosynthesis of the capsule of Haemophilus influenzae serotype f (Hif) has been cloned and characterized. The Hif cap locus organization is typical of group II capsule biosynthetic loci found in other H. influenzae serotype b bacteria and other gram-negative bacteria. However, the Hif cap locus was not associated with an IS1016 element. Three new open reading frames, Fcs1, Fcs2, and Fcs3, were identified in the Hif capsule-specific region II. The chromosomal location of the Hif cap locus and the organization of the flanking sequences differed significantly from previously described division I H. influenzae serotypes

Complete Sequence of the cap Locus of Haemophilus influenzae Serotype b and Nonencapsulated b Capsule-Negative Variants

The complete capsule (cap) loci from three Haemophilus influenzae strains, one serotype b (Hib) and two nonencapsulated b capsule-negative variants, were sequenced. Two new open reading frames, hcsA and hcsB, were identified in region III and thought to be involved in postpolymerization modification of the capsule. The location of the cap locus in the Haemophilus influenzae chromosome was identified within section 97 of the Rd genome (chromosomal coordinates 1074542 to 1086327) and found to be the same for the Hib and two Hib(−) strains as well as some other encapsulated division I H. influenzae strains

Altered Growth, Pigmentation, and Antimicrobial Susceptibility Properties of Staphylococcus aureus Due to Loss of the Major Cold Shock Gene cspB▿

An insertional mutation made in the major cold shock gene cspB in Staphylococcus aureus strain COL, a methicillin-resistant clinical isolate, yielded a mutant that displayed a reduced capacity to respond to cold shock and many phenotypic characteristics of S. aureus small-colony variants: a growth defect at 37°C, a reduction in pigmentation, and altered levels of susceptibility to many antimicrobials. In particular, a cspB null mutant displayed increased resistance to aminoglycosides, trimethoprim-sulfamethoxazole, and paraquat and increased susceptibility to daptomycin, teicoplanin, and methicillin. With the exception of the increased susceptibility to methicillin, which was due to a complete loss of the type I staphylococcal cassette chromosome mec element, these properties were restored to wild-type levels by complementation when cspB was expressed in trans. Taken together, our results link a stress response protein (CspB) of S. aureus to important phenotypic properties that include resistance to certain antimicrobials

Capsule Gene Analysis of Invasive Haemophilus influenzae: Accuracy of Serotyping and Prevalence of IS1016 among Nontypeable Isolates▿

We evaluated the accuracy of serologic capsule typing by analyzing capsule genes and related markers among invasive Haemophilus influenzae isolates before and after the introduction of H. influenzae serotype b (Hib) conjugate vaccines. Three hundred and sixty invasive H. influenzae isolates were collected as part of Active Bacterial Core surveillance within the Georgia Emerging Infections Program between 1 January 1989 and 31 July 1998. All isolates were biotyped, serotyped by slide agglutination serotyping (SAST), and evaluated using PCR capsule typing. Nontypeable H. influenzae (NTHi) isolates were probed with Hib cap-gene-containing plasmid pUO38 and with IS1016; a subset was examined with phosphoglucose isomerase (pgi) genotyping and pulsed-field gel electrophoresis (PFGE). Discrepancies between SAST and PCR capsule typing were found for 64/360 (17.5%) of the isolates; 48 encapsulated by SAST were NTHi by PCR, 8 NTHi by SAST were encapsulated by PCR, 6 encapsulated by SAST were a different capsule type by PCR, and 2 encapsulated by SAST were capsule-deficient Hib variants (Hib-minus). None of the PCR-confirmed NTHi isolates demonstrated homology with residual capsule gene sequences; 19/201 (9.5%) had evidence of IS1016, an insertion element associated with division I H. influenzae capsule serotypes. The majority of IS1016-positive NTHi were biotypes I and V and showed some genetic relatedness by PFGE. In conclusion, PCR capsule typing was more accurate than SAST and Hib-minus variants were rare. IS1016 was present in 9.5% of NTHi isolates, suggesting that this subset may be more closely related to encapsulated organisms. A better understanding of NTHi may contribute to vaccine development