Evaluation of the microbiological efficacy of cleaning agents for tracheostomy inner cannulas.

PURPOSE: Biofilms are a significant cause of morbidity in patients with indwelling medical devices. Biofilms pose a potential risk with reusable inner cannulas by increasing the risk of infections. Effective decontamination is thus vital in decreasing bioburden. The current guidelines for cleaning inner cannulas are varied, with multiple techniques being recommended, which are not supported by strong evidence. This randomized, controlled, cross-over study attempted to enumerate the bacterial count of inner cannulas used in tracheostomy patients (n = 60) pre-and post-decontamination with detergent (A) or sterile water (B). MATERIALS AND METHODS: The patients were randomly allocated to sequence A > B or B > A in 1:1 fashion. The saline flushing of the inner cannulas was plated on trypticase soy agar with 5 % sheep blood to enumerate the bacterial count. RESULTS: The mean ratio [Log (CFU)post/Log (CFU)pre]A/[Log (CFU)post/Log (CFU)pre]B based on 53 samples was 0.918 ± 0.470, two-sided 90 % confidence interval (CI) 0.812, 1.024. The equivalence criterion was met as the mean ratio after cleaning fell within the equivalence region of 0.8 and 1.25. CONCLUSION: This study demonstrated the microbiological efficacy of both detergent and sterile water in the decontamination of inner cannulas, and that sterile water was not less effective than detergent in reducing the bacterial load for safe re-use of inner cannulas. This has the potential to promote cost savings for patients with tracheostomy, both in the hospital and the community. The study findings may also be relevant in formulating tracheostomy care policies.

Evaluation of the clinical sensitivity and specificity of the BD Max™ Enteric Bacterial Panel for molecular detection of pathogens for acute gastroenteritis in the Singaporean population.

PURPOSE: Acute gastroenteritis (AGE) is caused by a wide range of pathogens. Culture methods for the detection of bacterial pathogens is time consuming and labour intensive. This study compared a same-day-to-result commercial molecular method using BD Max™ Enteric Bacterial Panel against conventional culture and laboratory-developed PCR assays (LDTs), and characterised the epidemiology of bacterial AGE in Singapore. METHODOLOGY: PCRs for Campylobacter spp., Salmonella spp., Shigella spp./Enteroinvasive Escherichia coli (EIEC) and Shiga toxin-producing E. coli (STEC)/Shigella dysenteriae were performed on the BD Max™ platform. Concurrent routine bacterial culture ("reference standard") was performed for Campylobacter, Salmonella, Shigella, Vibrio and Aeromonas spp. In the event of a discrepancy, an "expanded reference standard" (bacterial culture with LDT) was used. RESULTS: There were 299 stool specimens in the study, with no bacterial pathogens detected in 190 samples (63.5%). The positive samples (n = 109,36.5%) were detected with Salmonella (n = 57,19.1%), Campylobacter (n = 28,9.4%), Vibrio parahaemolyticus (n = 6,2.0%), Shigella/EIEC (n = 6,2.0%), ETEC (n = 4,1.3%), STEC (n = 2,0.7%), Aeromonas (n = 2,0.7%), Plesiomonas shigelloides (n = 1,0.3%) and 3(1.0%) co-infections. Compared to the "expanded reference standard", conventional culture missed 38/112 (33.9%) pathogens. Conversely, testing by BD Max™ alone failed to detect 17 pathogens. BD Max™ reported seven (2.3%) false-positive results. CONCLUSIONS: BD Max™ increased the detection rate of bacterial AGE pathogens in the panel, but was limited by the absence of detection capability for Vibrio and Aeromonas spp.

Hypermucoviscosity, rmpA, and aerobactin are associated with community-acquired Klebsiella pneumoniae bacteremic isolates causing liver abscess in Singapore.

INTRODUCTION: This retrospective study investigated the clinical etiology of community-acquired bacteremic Klebsiella pneumoniae infections, and characterized laboratory and genetic markers which may be associated with primary liver abscess (PLA). METHODS: Community-onset K. pneumoniae bacteremic episodes from 2010 to 2011 were identified from the laboratory information system. Isolates were retrieved for susceptibility testing, hypermucoviscosity testing, PCR-based serotyping (K1, K2 and K5) and PCR detection of virulence genes (rmpA, alls, kfu and aerobactin). Clinical data collected from electronic medical records included primary and secondary diagnoses, co-existing morbidities, antibiotic therapy, and in-patient mortality. RESULTS: 129 bacteremic episodes were identified. The most common primary infections were pneumonia (n = 24, 18.6%), primary liver abscess (n = 21, 16.3%) and urinary tract infections (n = 21, 16.3%). Hypermucoviscosity was present in 55 isolates (42.6%). The most commonly detected virulence genes were aerobactin (n = 63, 48.8%) and rmpA (n = 59, 45.7%). Isolates causing liver abscess were significantly associated with a positive string test, rmpA, aerobactin gene, and capsular serotype K1 (all p < 0.01), but not with capsular serotype K2, K5, kfu, or allS genes. The absence of a positive string test, rmpA, or aerobactin genes had a 97.3%-100% negative predictive value for PLA. The positive predictive values of the string test, rmpA, aerobactin genes, and serotype K1 for PLA ranged from 31.7% to 35.6%. CONCLUSION: In our study population, pneumonia and PLA were the most common sources of community-acquired bacteremia. Hypermucoviscosity, rmpA, aerobactin, and serotype K1 could be useful laboratory markers to alert clinicians to arrange abdominal imaging to detect liver abscess.

Development of a real-time assay to determine the frequency of qac genes in methicillin resistant Staphylococcus aureus.

PURPOSE: The emergence of antiseptic resistance and/or antiseptic-resistance genes in methicillin-resistant Staphylococcus aureus (MRSA) may result in failure of decolonization treatments. Plasmid-encoded efflux pump genes qacA/B and qacC (smr) confer tolerance to chlorhexidine and quaternary ammonium compounds. The objective of this study was to develop and validate a multiplex real time-PCR assay for detection of antiseptic-resistance genes, apply the assay on 200 MRSA isolates and explore if carriage of these genes was associated with resistance to topical antibiotics. METHODOLOGY: A SYBR-Green based multiplex real time-PCR assay was developed to detect qacA/B, qacC, and mecA (internal control) simultaneously. The multiplex assay was compared against conventional single-plex PCR followed by agarose gel electrophoresis, using DNA from the first 73 MRSA isolates, followed by multiplex testing of the remaining 127 MRSA isolates. All 200 MRSA isolates were tested for susceptibility to mupirocin, retapamulin, neomycin, bacitracin and octenidine. The genetic diversity of the isolates was investigated by spa-typing. RESULTS: The concordance between multiplex and conventional PCR, in assignments of qacA/B and qacC status were 99%(72/73) and 100%(73/73) respectively. Among 200 MRSA isolates, 48(24%) and 44(23%) were found to harbour qacA/B and qacC genes, respectively. These isolates remained susceptible to many common decolonization agents, except mupirocin. The predominant spa-types were t020 and t1081 (41 and 32 isolates respectively). CONCLUSION: The real-time assay performed acceptably for the detection of qac genes. A high prevalence of antiseptic-resistance genes were detected in the MRSA isolates in our population and appeared to be associated with spa-type t1081.

Faster and economical screening for vancomycin-resistant enterococci by sequential use of chromogenic agar and real-time polymerase chain reaction.

BACKGROUND/PURPOSE: Screening for vancomycin-resistant enterococci (VRE) by culture takes days to generate results, while polymerase chain reaction (PCR) testing directly from clinical specimens lacks specificity. The aims of this study were to develop a real-time PCR to detect and identify Enterococcus faecium, Enterococcus faecalis, and vanA and vanB genes, and to evaluate the impact of this PCR on test-reporting times when performing it directly from suspect VRE isolates present on screening chromogenic media. METHODS: The tetraplex PCR primers were designed to amplify E. faecium, E. faecalis, and vanA and vanB genes, with melt-curve analysis of PCR products. Following analytical and clinical validation of the molecular assay, PCR testing was performed for target colonies present on VRE chromogenic media. PCR results were evaluated against conventional phenotypic identification and susceptibility testing, with the time to result being monitored for both modalities. RESULTS: A total of 519 colonies from clinical specimens were tested concurrently by real-time PCR and phenotypic methods. In all, 223 isolates were identified with phenotypic vancomycin resistance (vanA, n = 108; vanB, n = 105; non-vanA/vanB = 10), with complete agreement between PCR and phenotypic testing for vancomycin-resistant E. faecium and E. faecalis. The majority (88.6%) of PCR results were reported, on average, 24.8 hours earlier than those of phenotypic testing, with 68% reduction in total costs. CONCLUSION: The use of culture on selective media, followed by direct colony PCR confirmation allows faster and economical VRE screening.

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.

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

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.

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%.

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.

Five clinical cases of Necropsobacter rosorum bacteremia.

Five cases of bacteremia with Necropsobacter rosorum are described, originating from intra-abdominal infections or localized soft tissue infections in the pelvic region. N. rosorum is consistently misidentified by commercial identification systems, which may delay recognition of this organism as a human pathogen.

Characterisation of significant Gram positive bacilli from soft tissue infections.

INTRODUCTION: Gram positive bacilli (GPB) isolated from soft tissue infections are often neglected or ignored due to their fastidious nature and the lack of reliable phenotypic identification methods. This study was done to characterise clinically significant aero-tolerant GPB isolated from surgically obtained samples in patients with soft tissue infections. METHODS: Forty-six GPB isolates collected during a 2 year study period were identified using partial 16s rRNA sequencing and API Coryne. Antibiotic susceptibility testing to penicillin, amoxycillin/clavulanate, moxifloxacin and erythromycin was performed on these isolates using Etest. Clinical data were gathered from patients' medical records. RESULTS: The most common isolates identified by 16s rRNA sequencing were Actinomyces species (n = 30, 65%) and Corynebacterium species (n = 9, 20%). The majority of the Actinomyces species infections were located below the waist, in particular the perianal region. There was poor agreement between API Coryne and genotypic identification, with only one-third of the isolates being correctly identified to species level. Actinomyces species were uniformly susceptible to penicillin and amoxicillin/clavulanate. Antibiotic susceptibilities were more varied for the other genera isolated. CONCLUSION: Actinomyces species comprised two-thirds of aerobically growing GPB isolates and may represent an under-reported cause of bacterial soft tissue infections. Penicillin and amoxycillin/clavulanate may be the empiric antibiotics of choice for Actinomyces species as all isolates were susceptible.

Influence of different Mueller-Hinton agars and media age on Etest susceptibility testing of tigecycline.

This study investigated the effect of different Mueller-Hinton agars and media age on tigecycline MICs, obtained by Etest. Variations in MIC values on different Mueller-Hinton were noted, which may result in changes in categoric susceptibility. The use of stored Mueller-Hinton media had minimal effect on MIC values.

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.

CTX-M and ampC beta-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.

Evaluation of screening methods to detect plasmid-mediated AmpC in Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis.

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.

In vitro effect of minocycline and colistin combinations on imipenem-resistant Acinetobacter baumannii clinical isolates.

OBJECTIVES: The study investigated the effect of colistin and minocycline when tested singly and in combination against Acinetobacter baumannii. METHODS: Thirteen unrelated imipenem-resistant A. baumannii clinical isolates were included in the study. MICs of colistin sulphate and minocycline were determined by broth macrodilution and Etest. Organisms were also tested against the two antibiotics singly and in combination using time-kill methods and an Etest-based method. RESULTS: Neither colistin nor minocycline when tested alone demonstrated bactericidal activity. However, the combination of colistin and minocycline demonstrated bactericidal activity against most of the isolates tested. At 24 h, the combination of antibiotics demonstrated synergy in 12 of the 13 isolates by time-kill methods. None of the isolates demonstrated synergy by Etest methods. CONCLUSIONS: The combination of colistin and minocycline was found to be bactericidal and synergistic against A. baumannii by time-kill methods. There was no agreement between time-kill and Etest methods for synergy testing.

Susceptibility testing of unconventional antibiotics against multiresistant Acinetobacter spp. by agar dilution and Vitek 2.

Treatment options are increasingly limited for carbapenem-resistant Acinetobacter baumannii. This study set out to determine the in vitro susceptibility of multiresistant Acinetobacter spp. to colistin, minocycline, and rifampicin using agar dilution, and to compare the accuracy of testing results obtained from an automated susceptibility testing system (Vitek 2). MICs for colistin, minocycline, and rifampicin were obtained by agar dilution and Vitek 2 for 44 unrelated strains of multiresistant Acinetobacter spp. All tested strains were susceptible to colistin, whereas 61% were susceptible to minocycline and 52% were susceptible to rifampicin. Results for colistin testing showed categoric agreement between Vitek 2 and agar dilution, with no false-resistance reported by Vitek 2. However, there was a poor agreement for minocycline and rifampicin when results obtained from Vitek 2 testing were compared with those obtained by agar dilution.

Comparison of three standardized disc susceptibility testing methods for colistin.

OBJECTIVES: With increasing antibiotic resistance in Gram-negative bacteria, the use of the polymyxins has increased in recent years. Antibiotic disc susceptibility testing remains the most widely used method in clinical laboratories, but there is very little data on the accuracy of disc testing methods for colistin. In this study, the accuracy of three standardized methods of disc susceptibility testing for colistin was compared with agar dilution. METHODS: A total of 228 clinical isolates of Acinetobacter spp., Pseudomonas aeruginosa and Enterobacteriaceae were included in the study. Isolates were tested by agar dilution for susceptibility to colistin, and results were compared with those obtained by three disc susceptibility testing methods (product insert based on CLSI methodology, British BSAC and French SFM). RESULTS: Colistin displayed good activity against Acinetobacter spp., Klebsiella spp. and Escherichia coli (MIC(90) 2 mg/L) but was less active against P. aeruginosa (MIC(90) 4 mg/L) and Enterobacter spp. (MIC(90) >or= 128 mg/L). Totally, 81%, 79% and 89% of colistin-resistant isolates were falsely reported as susceptible when tested by the product insert, BSAC and SFM testing methods, respectively. There were no false-resistant results. CONCLUSIONS: Disc susceptibility testing methods are unreliable at detecting colistin resistance. Dilution methods should be the method of choice for susceptibility testing of colistin.