Lily Siew Yong Ng

Evaluation of Screening Methods To Detect Plasmid-Mediated AmpC in Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis

ABSTRACT There are currently no standardized phenotypic methods for the screening and detection of AmpC enzymes. This study aimed to evaluate different methods to detect AmpC enzymes in Escherichia coli, Klebsiella spp., and Proteus spp., comparing the results from two disk-based methods and an agar dilution method. AmpC activity was determined for 255 clinical isolates by use of a three-dimensional enzyme assay combined with a multiplex PCR assay for plasmid-borne ampC genes. These results were compared against a disk-based inhibitor assay using various combinations of cefpodoxime and cefoxitin as antibiotic substrates and boronic acid or cloxacillin as an AmpC inhibitor. The presence of enzyme induction by disk approximation was evaluated using imipenem, cefoxitin, and amoxicillin-clavulanate as inducing agents against ceftazidime. Finally, an agar dilution assay was performed, using cefoxitin with and without added cloxacillin. AmpC activity was present in 49.8% of test isolates, 93.7% of which were positive for plasmid-borne ampC genes. CIT-like enzymes were predominant in E. coli, and DHA-like enzymes were predominant in Klebsiella spp. The disk-based inhibitor tests performed better than the agar dilution assay, while detection of AmpC by disk induction had a poor sensitivity. The cefoxitin-cloxacillin disk combination provided the best overall performance, with a sensitivity and specificity of 95%. This study confirmed the accuracy of disk-based inhibitor screening for AmpC enzymes, which proved reliable at detecting CIT- and DHA-like plasmid-borne ampC genes. The methods are simple enough for introduction into clinical microbiology laboratories.

Automated Identification Systems and Burkholderia pseudomallei

Lowe et al. ([1][1]) recently reported the failure of the Vitek 2 system (bioMerieux) to correctly identify Burkholderia pseudomallei . They also warned that thoughtless reliance on automation runs the risk that incorrectly identified organisms may be reported without question. We recently had the

Evaluation of disc susceptibility tests performed directly from positive blood cultures

Aims: To evaluate the accuracy of direct disc susceptibility testing performed from positive BACTEC blood culture vials, using a predetermined dilution protocol. Methods: Direct susceptibility testing was performed from 432 positive blood culture vials, generating 3829 antibiotic-organism results. Results were compared with those obtained by standard disc susceptibility testing according to Clinical Laboratory Standards Institute (CLSI) methods. Results: When results were compared with the reference method, no very major errors were detected. One (0.03%) major error and 89 (2.3%) minor errors were found. Error rates by organism group ranged from 1.3% for Pseudomonas aeruginosa to 8.2% for β-haemolytic streptococci. Conclusions: Direct susceptibility testing provided accurate susceptibility results for most organism–antibiotic combinations, with the exception of the β-haemolytic streptococci.

The increased role of non-albicans species in candidaemia: results from a 3-year surveillance study.

Various studies have documented a shift in species distribution in Candida bloodstream infections (BSI), but there are little data from Southeast Asia. This study was performed to determine the species epidemiology and antifungal susceptibilities of Candida species BSI in Singapore. Candida spp. from BSI were collected from a tertiary and secondary referral hospital, and an obstetrics/paediatric hospital over a 3‐year period. The most common isolates were Candida albicans (36%), Candida tropicalis (27%), Candida glabrata (16%) and Candida parapsilosis (16%). Candida parapsilosis and C. albicans were predominant in the paediatric hospital, and C. albicans and C. tropicalis predominant in the other two institutions. Candida tropicalis temporarily replaced C. albicans as the predominant strain from BSI in 2006. Overall, 87.3% of Candida isolates were susceptible to fluconazole, and 10.4% classified as susceptible‐dose‐dependent. Fluconazole resistance was detected in C. tropicalis (3.6%), C. parapsilosis (2.1%) and C. glabrata (4.0%). Candida albicans is the predominant species isolated from BSI in Singapore. However, non‐albicans species accounted for nearly two‐thirds of all cases of candidaemia and the relative increase in C. tropicalis infections deserves further investigation. Resistance to fluconazole was uncommon.

CTX-M and ampC β-lactamases contributing to increased prevalence of ceftriaxone-resistant Escherichia coli in Changi General Hospital, Singapore☆☆☆

Data from an 800-bed hospital showed an increase in the incidence density of ceftriaxone-resistant Escherichia coli, from 13.1 to 21.7 per 100 000 inpatient days over a 4-year period. Detailed testing performed on 54 E. coli isolates showed bla(CTX-M) extended-spectrum beta-lactamase (ESBL) genes (n = 37) and bla(CMY-like)ampC genes (n = 15). Typing data showed only limited clonal transmission in isolates with ESBL.

Multidrug-resistant organisms in a routine ward environment: differential propensity for environmental dissemination and implications for infection control

Multidrug-resistant organisms (MDROs) pose significant infection-control challenges in settings with high prevalence and limited isolation facilities. This observational study in an 800-bed hospital determined the prevalence, bacterial density and genetic relatedness of MDROs isolated from ward surfaces, medical devices and the hands of healthcare professionals. The targeted MDROs were meticillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), Escherichia coli and Klebsiella pneumoniae resistant to extended-spectrum cephalosporins, and carbapenem-resistant (CR) Acinetobacter baumannii. During a 2-month period, microbiological sampling and molecular typing were performed on environment isolates, clinical isolates and isolates recovered from the hands of healthcare professionals. The target MDROs were recovered from 79% of sampled surfaces, predominantly MRSA (74% of all tested surfaces) and CR A. baumannii (29%) but also VRE (2%) and K. pneumoniae (1%). MRSA was recovered from most tested surfaces throughout the ward, whilst CR A. baumannii was significantly more likely to be recovered from near-patient surfaces. Hand sampling demonstrated infrequent recovery of MRSA (5%), CR A. baumannii (1%) and VRE (1%). Molecular typing of the study isolates identified seven MRSA and five Acinetobacter clonal clusters, respectively, and typing identified similar strains from the environment, patients and hands. Thus, in a healthcare setting with endemic circulation of MDROs, MRSA and CR A. baumannii were the predominant organisms recovered from ward surfaces, with MRSA in particular demonstrating widespread environmental dissemination. Molecular typing demonstrated the presence of related strains in patients, in the environment and on the hands of healthcare workers.

Clinical characteristics and antimicrobial susceptibilities of anaerobic bacteremia in an acute care hospital.

This study investigated the clinical features of anaerobic bacteraemia in an acute-care hospital, and evaluated the antimicrobial susceptibility of these isolates to commonly available antibiotics. Microbiological and epidemiological data from 2009 to 2011were extracted from the laboratory information system and electronic medical records. One hundred and eleven unique patient episodes consisting of 116 anaerobic isolates were selected for clinical review and antibiotic susceptibility testing. Susceptibilities to amoxicillin-clavulanate, clindamycin, imipenem, metronidazole, moxifloxacin, penicillin and piperacillin-tazobactam were performed using Etest strips with categorical interpretations according to current CLSI breakpoints. Metronidazole-resistant and carbapenem-resistant anaerobic Gram-negative bacilli were screened for the nim and cfiA genes. Clinical data was obtained retrospectively from electronic medical records. During the 3 year period, Bacteroides fragilis group (41%), Clostridium species (14%), Propionibacterium species (9%) and Fusobacterium species (6%) were the most commonly isolated anaerobes. Patients with anaerobic bacteraemia that were included in the study were predominantly above 60 years of age, with community-acquired infections. The most commonly used empiric antibiotic therapies were beta-lactam/beta-lactamase inhibitor combinations (44%) and metronidazole (10%). The crude mortality was 25%, and appropriate initial antibiotic therapy was not significantly associated with improved survival. Intra-abdominal infections (39%) and soft-tissue infections (33%) accounted for nearly three-quarters of all bacteraemia. Antibiotics with the best anaerobic activity were imipenem, piperacillin-tazobactam, amoxicillin-clavulanate and metronidazole, with in-vitro susceptibility rates of 95%, 95%, 94% and 92% respectively. Susceptibilities to penicillin (31%), clindamycin (60%) and moxifloxacin (84%) were more variable. Two multidrug-resistant isolates of Bacteroides species were positive for nim and cfiA genes respectively, while another two imipenem-resistant Fusobacterium species were negative for cfiA genes. This study demonstrated that anaerobic bacteraemia in our patient population was predominantly associated with intra-abdominal and soft-tissue infections. Overall antibiotic resistance was high for penicillin and clindamycin, and the presence of emerging resistance to carbapenems and metronidazole warrants further monitoring.

Performance characteristics and clinical predictive value of the string test for detection of hepato-virulent Klebsiella pneumoniae isolated from blood cultures

This study evaluated the phenotypic string test for identifying Klebsiella pneumoniae bacteraemic isolates associated with primary liver abscess. Test reproducibility and repeatability exceeded 95%, with varying sensitivity (66-90%) and specificity (64-67%) depending on cut-off values. The positive predictive value of a positive string test for identifying hepato-virulent isolates was <35%.

Evaluation of bacterial recovery and viability from three different swab transport systems

Summary This study evaluated three types of swab transport systems for organism recovery. Swabs with transport media were further assessed for organism viability over 24 hours over a range of different storage temperatures. Test organisms consisted of aerobes, fastidious aerobes and anaerobes. Swabs were tested according to the standardised quantitative elution method published by the Clinical Laboratory Standards Institute (CLSI; M-40A). There were substantial differences in primary organism recovery, with recovery rates from different swabs ranging from <0.1% to 78% for Streptococcus pyogenes. Similar differences were noted for other test organisms. In general, the flocked swab (ESwab) demonstrated highest rates of recovery for aerobic organisms, while higher rates of recovery of Fusobacterium nucleatum were demonstrated from a standard swab (Transwab). When considering organism viability, no single swab fulfilled all the criteria stipulated by the M-40A standard for all organism/temperature combinations. Organism viability was marginally better for the gel-based swab transport systems as compared to the liquid-media based ESwab. Significant differences between swab transport systems were demonstrated, including differences for primary organism recovery and viability. The ESwab showed the best recovery of organisms, while gel-based media demonstrated marginally better bacterial viability for most tested retention times and temperatures.

A cost-effective method for the presumptive identification of Enterobacteriaceae for diagnostic microbiology laboratories

Aims: This study evaluated the use of an abbreviated algorithm for the presumptive identification of Enterobacteriaceae isolated from clinical microbiology specimens. Methods: Identification was based on primary isolation of bacterial pathogens on blood, lactose fermentation based on colonial morphology on MacConkey agar, oxidase and indole tests, and a limited number of conventional biochemical tests. The accuracy of the study algorithm was prospectively evaluated against commercial bacterial identification kits, using clinical isolates from blood, urine and superficial wound and tissue sites. Results: Of 534 isolates, 518 (97%) were accurately identified to genus level. Identification of the study isolates was achieved with a 56% reduction in technologist time and 85% reduction in reagent costs, when compared to the use of a conventional biochemical identification panel. The main limitation of the protocol in the tested bacterial population was that indole‐negative Escherichia coli were likely to be misidentified as Enterobacter species. Conclusions: This protocol may be suitable for the presumptive identification of commonly isolated Enterobacteriaceae from non‐sterile sites by diagnostic laboratories in resource‐constrained settings.