Detection and epidemiology of plasmid-mediated AmpC ß-lactamase producing Escherichia coli in two Irish tertiary care hospitals.

This study determined the prevalence and distribution of plasmid-mediated AmpC (pAmpC) ß-lactamases in Irish Escherichia coli isolates. Clinical E. coli isolates (n=95) that were intermediate or resistant to cefoxitin and/or flagged by VITEK® 2 as potential AmpC-producers underwent confirmation using a MASTDISCS™ ESBL and AmpC Detection Kit. Multiplex PCR capable of detecting family-specific plasmid ampC genes was performed to detect the presence of these genes. Five PCR-negative isolates were selected for promoter analysis. PFGE and MLST were performed on E. coli isolates that harboured a plasmid ampC gene to determine their clonal relatedness. Plasmid ampC genes were detected in 19% (18/95) of phenotypic AmpC producing E. coli isolates. The CIT group was the most common plasmid family type (n=14); DHA (n=3) and ACC (n=1) groups were also detected. Promoter analysis showed that four isolates had multiple point mutations and one had a 1 bp insertion in the -10 box. PFGE demonstrated a polyclonal pattern for E. coli isolates. Furthermore, with the exception of two isolates with an identical sequence type (ST720), MLST analysis revealed that these isolates were not clonally related. This study revealed that there was a marked prevalence of pAmpC E. coli among phenotypic AmpC producing E. coli isolates but no evidence of cross-transmission of a single strain. Establishing the prevalence and clonality of these organisms is important in order to implement evidence-based infection control measures that reduce the spread of pAmpC ß-lactamase resistance in the hospital environment.

Evaluation of real-time polymerase chain reaction assays for the detection of herpes simplex virus in swab specimens.

An in-house herpes simplex virus (HSV) real-time polymerase chain reaction (PCR) hydrolysis probe assay and three commercial real-time HSV assays were evaluated for the detection of HSV from genital, oral, ocular and dermal clinical samples. Five hundred and sixty samples from patients displaying signs and symptoms of HSV infection were used to evaluate the in-house HSV assay. A representative 151 samples were processed using the artus HSV-1/2 TM PCR Kit, SmartCycler Non-typing and SmartCycler Typing ASR kits. The in-house PCR assay demonstrated an overall positivity rate of 38% (211/560), equating to a 14% increase in HSV detection compared to 24% for culture (135/560). All 76 culture-negative, in-house PCR-positive samples were confirmed using at least one HSV commercial kit. The in-house, SmartCycler Non-typing, artus and SmartCycler Typing assays showed improved sensitivity (100%, 100%, 98% and 99%, respectively) compared to culture (37%), and all real-time assays were highly specific (100%). The in-house and commercial real-time HSV PCR assays performed well and, combined with careful clinical interpretation, should improve the detection, differentiation and quantification of HSV from mucocutaneous swab samples.

Evaluation of the IDI-MRSA assay on the SmartCycler real-time PCR platform for rapid detection of MRSA from screening specimens.

Rapid accurate detection is a prerequisite for the successful control of meticillin-resistant Staphylococcus aureus (MRSA). The IDI-MRSA real-time polymerase chain reaction (PCR) assay was designed to provide rapid results from nasal specimens collected in Stuart's liquid transport medium. This study has evaluated the IDI-MRSA kit for use in a clinical laboratory by investigating the following parameters: (1) limits of detection (LoD), (2) performance with Amies' gel-based transport medium, (3) ability to detect strains of MRSA in a collection representative of MRSA in Ireland since 1974 (n=113) and (4) performance in a clinical trial with swabs from nose, throat and groin/perineum sites from 202 patients. LoDs (colony-forming units per ml) of the IDI-MRSA kit, direct culture on MRSA-Select chromogenic agar (CA) and salt-enrichment culture (with subculture onto CA) were 10(3), 10(3) and 10(2), respectively. LoDs with Stuart's and Amies' transport media were comparable. All except one of the 113 MRSA isolates were detected by the kit but, when six control strains carrying staphylococcal cassette chromosome mec (SCCmec) type IV element subtypes IVa-d and SCCmec types V and V(T) were tested, the kit failed to detect MRSA carrying SCCmec V. The sensitivity and specificity for detection of MRSA from nose, throat and groin/perineum specimens were comparable with slightly lower sensitivities from throat and groin/perineum specimens compared with nasal swabs (90%, 97%; 89%, 99%; 88%, 99%, respectively). Overall sensitivity, specificity and positive and negative predictive values for specimens from all sites were 88%, 99%, 94% and 97%, respectively. Further developments to improve the sensitivity of this highly worthwhile assay are required.

Rate of carriage of Serratia marcescens in patients with and without evidence of infection.

The epidemiology of Serratia marcescens is poorly understood. We designed a study to investigate carriage sites of the organism, and possible modes of transmission of infection. Using Sorbitol-MacConkey agar with colistin 200 IU/ml and MacConkey agar with a 10 microg colistin disc we performed cultures from various sites in patients already infected with S. marcescens. Over the same period of time we also investigated all patients in the intensive care unit (ICU) for colonization with the agent. Environmental screening was performed in the ICU only. Of 37 infected patients, 65% demonstrated carriage at a second site and 43% at multiple sites. Throat carriage was found in 59%, faecal carriage in 42%, nasal carriage in 31% and urinary carriage in 22%. Carriage over several weeks was found in 22%. Of 40 ICU patients, 10% demonstrated nasal and/or throat carriage. Environmental screening yielded 4 isolates. All ICU patient strains and a strain from the ICU bedpan macerator were O14:K14 with similar random amplified polymorphic DNA types. These results show that patients with S. marcescens infection are likely to carry the organism at multiple sites and that carriage may be prolonged. A significant level of carriage was also found in non-infected patients in a unit where the organism was prevalent.

An outbreak of an unusual strain of Serratia marcescens in two Dublin hospitals.

We describe a serious outbreak of infection caused by a strain of Serratia marcescens in two Dublin hospitals which occurred over an 11 week period and affected a total of 15 patients. A contaminated bed-pan macerator in the Intensive Care Unit of one hospital was identified as the possible source of infection and spread of the organism probably occurred via hand transmission by hospital personnel and via patient transfer to a second hospital. All isolates of S. marcescens involved in the outbreak had the same antimicrobial susceptibility pattern, with reduced susceptibility to gentamicin, cefotaxime and ciprofloxacin. Epidemiological typing revealed that the strains of S. marcescens isolated in the outbreak were of an uncommon serotype, O21:K14, and using pulsed-field gel electrophoresis, XbaI DNA macrorestriction profiles clustered at 90% similarity. The DNA patterns of the outbreak strain were also highly similar to S. marcescens isolates of the same serotype recovered from a separate Dublin hospital during the same time period as the outbreak described here. In addition, the isolates clustered at 82% similarity with strains of the same serotype from a retrospective collection of S. marcescens isolates from various hospitals in the Dublin area, indicating that these may be genetic variants of the same strain. Although the outbreak was brought under control following implementation of infection control measures, a significant number of similar O:21 isolates of S. marcescens have since been identified in four Dublin hospitals. These results suggest the unique spread of a single strain of S. marcescens in Dublin hospitals.

Increased expression of Fc gamma receptors on neutrophils and monocytes may reflect ongoing bacterial infection.

The Fc gammaR receptors for IgG, Fc gammaRI, Fc gammaRII and Fc gammaRIII were measured on neutrophils and monocytes from 36 patients suspected of systemic infection. These results were compared with 30 blood donor controls to assess the level of expression as an early indicator of bacterial infection. Fc gammaRI expression on neutrophils was found to be significantly increased from patients with systemic or localised infections, when compared to non-infected patient group, i.e., patients with no cultural evidence of bacterial infections, (p=0.02, p=0.04) or normal controls (p<0.0001, p=0.0005). Fc gammaRI expression on monocytes was also significantly increased in both of the infected groups compared to normal controls (p<0.0001, p=0.001); however, no significant difference could be seen when compared with the non-infected patients. Fc gammaRIII was found to be significantly increased on a subset of monocytes in patients with systemic or localised infections compared to the non-infected group (p=0.009, p=0.006) and compared to the normal controls (p=0.009, p=0.003). Infections caused by gram-negative bacilli induced a higher Fc gammaR response than infection with either streptococci or staphylococci. These data suggest that the measurement of Fc gammaRI on neutrophils and Fc gammaRIII on monocytes may be a useful rapid indicator of bacterial infection.

The in-vitro susceptibilities of vaginal lactobacilli to four broad-spectrum antibiotics, as determined by the agar dilution and E test methods.

The minimum inhibitory concentrations (MICs) of cefaclor, co-amoxiclav, clarithromycin and ciprofloxacin for 59 strains of vaginal lactobacilli were determined by both the reference agar dilution and E test methods. With the exception of clarithromycin, there was poor correlation between the results obtained by the two techniques. This was most apparent for the beta-lactams studied, the MICs of cefaclor as determined by the E test being particularly difficult to define. The stability of antimicrobial gradients in the E test may cause problems when testing slow-growing bacteria and/or organisms which grow only under anaerobic conditions. Accordingly, only those MICs determined by the agar dilution method are reported. The percentages of susceptible isolates were as follows: clarithromycin, 100; co-amoxiclav, 100; cefaclor, 20; and ciprofloxacin, 4. The administration of antimicrobials, such as ciprofloxacin and cefaclor, which have poor activities in vitro against lactobacilli, may therefore be advantageous to the host because it allows the protective effects of the normal vaginal flora to be preserved.