Shigatoxin encoding Bacteriophage ϕ24B modulates bacterial metabolism to raise antimicrobial tolerance.

How temperate bacteriophages play a role in microbial infection and disease progression is not fully understood. They do this in part by carrying genes that promote positive evolutionary selection for the lysogen. Using Biolog phenotype microarrays and comparative metabolite profiling we demonstrate the impact of the well-characterised Shiga toxin-prophage ϕ24B on its Escherichia coli host MC1061. As a lysogen, the prophage alters the bacterial physiology by increasing the rates of respiration and cell proliferation. This is the first reported study detailing phage-mediated control of the E. coli biotin and fatty acid synthesis that is rate limiting to cell growth. Through ϕ24B conversion the lysogen also gains increased antimicrobial tolerance to chloroxylenol and 8-hydroxyquinoline. Distinct metabolite profiles discriminate between MC1061 and the ϕ24B lysogen in standard culture, and when treated with 2 antimicrobials. This is also the first reported use of metabolite profiling to characterise the physiological impact of lysogeny under antimicrobial pressure. We propose that temperate phages do not need to carry antimicrobial resistance genes to play a significant role in tolerance to antimicrobials.

Evaluation of a new chromogenic medium, chromID OXA-48, for recovery of carbapenemase-producing Enterobacteriaceae from patients at a university hospital in Turkey.

The purpose of this study was to evaluate a new chromogenic medium, chromID OXA-48, for the isolation of carbapenemase-producing Enterobacteriaceae (CPE) directly from rectal swabs. chromID CARBA and chromID OXA-48 are two chromogenic media that have been commercialized for the isolation of CPE directly from clinical samples. Both media were evaluated alongside a broth enrichment method recommended by the CDC for isolation of CPE, with rectal swabs from 302 unique hospitalized patients at the Hacettepe University Hospital, Ankara, Turkey. A total of 33 patients (11 %) were found to be colonized with CPE using a combination of all methods, and all CPE produced OXA-48 carbapenemase. Klebsiella pneumoniae was by far the most dominant species of CPE and was isolated from 31 patients. Culture on chromID OXA-48 offered the highest sensitivity (75.8 %) for detection of CPE compared with the other two methods (sensitivity for both other methods was 57.6 %) and also offered the highest specificity (99.3 %). However, a combination of methods (either chromID OXA-48 plus CDC method or chromID OXA-48 plus chromID CARBA) was necessary to achieve an acceptable sensitivity (90.9 %). For isolation of CPE, in a setting where OXA-48 carbapenemase is the dominant type of carbapenemase, chromID OXA-48 is a highly useful medium but using a combination of methods is optimal for adequate detection. The combined use of two chromogenic media offered acceptable sensitivity (90.9 %) and the highest specificity (98.5 %) and also allowed for isolation of CPE within 18-20 h.

Prevalence of NDM-1 carbapenemase in patients with diarrhoea in Pakistan and evaluation of two chromogenic culture media.

AIMS: To evaluate two chromogenic media, Brilliance CRE and chromID CARBA, with stool samples referred to the Public Health Laboratories Division of the National Institute of Health in Islamabad, and assess the prevalence of carbapenemase-producing Enterobacteriaceae (CPE) in this population. METHODS AND RESULTS: One hundred and fifty-two stool samples from patients with diarrhoea were referred to the Microbiology Department and were investigated for the presence of CPE using two chromogenic culture media, Brilliance CRE and chromID CARBA. Thirteen patients (8·6%) were found to be colonized with CPE and all produced NDM-1 carbapenemase. Twelve of these patients (92%) were found to be colonized by culture on chromID CARBA compared with seven (54%) using Brilliance CRE. CONCLUSIONS: If only coloured colonies were considered as presumptive CPE, the sensitivity, specificity and positive predictive value were 54, 23 and 6% for Brilliance CRE and 85, 85 and 36% for chromID CARBA, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: We conclude that Enterobacteriaceae that produce NDM-1 carbapenemase can be found in patients from all major provinces of Pakistan and that chromID CARBA was the most effective of the two chromogenic media in this setting.

The preterm gut microbiota: changes associated with necrotizing enterocolitis and infection.

AIM: To describe gut colonization in preterm infants using standard culture and 16S gene rRNA profiling, exploring differences in healthy infants and those who developed NEC/late onset sepsis (LOS). METHODS: Ninety-nine stools from 38 infants of median 27-week gestation were cultured; 44 stools from 27 infants had their microbial profiles determined by 16S. Ordination analyses explored effects of patient variables on gut communities. RESULTS: Standard microbiological culture identified a mean of two organisms (range 0-7), DGGE 12 (range 3-18) per patient. Enterococcus faecalis and coagulase negative staphylococci (CONS) were most common by culture (40% and 39% of specimens). Meconium was not sterile. No fungi were cultured. Bacterial community structures in infants with NEC and LOS differed from healthy infants. Infants who developed NEC carried more CONS (45% vs 30%) and less Enterococcus faecalis (31% vs 57%). 16S identified Enterobacter and Staphylococcus presence associated with NEC/LOS, respectively. CONCLUSIONS: Important differences were found in the gut microbiota of preterm infants who develop NEC/LOS. The relationship of these changes to current practices in neonatal intensive care requires further exploration.

The transmission of nosocomial pathogens in an intensive care unit: a space-time clustering and structural equation modelling approach.

We investigated the incidence of cases of nosocomial pathogens and risk factors in an intensive treatment unit ward to determine if the number of cases is dependent on location of patients and the colonization/infection history of the ward. A clustering approach method was developed to investigate the patterns of spread of cases through time for five microorganisms [methicillin-resistant Staphylococcus aureus (MRSA), Acinetobacter spp., Klebsiella spp., Candida spp., and Pseudomonas aeruginosa] using hospital microbiological monitoring data and ward records of patient-bed use. Cases of colonization/infection by MRSA, Candida and Pseudomonas were clustered in beds and through time while cases of Klebsiella and Acinetobacter were not. We used structural equation modelling to analyse interacting risk factors and the potential pathways of transmission in the ward. Prior nurse contact with colonized/infected patients, mediated by the number of patient-bed movements, were important predictors for all cases, except for those of Pseudomonas. General health and invasive surgery were significant predictors of cases of Candida and Klebsiella. We suggest that isolation and bed movement as a strategy to manage MRSA infections is likely to impact upon the incidence of cases of other opportunist pathogens.

The molecular diversity of the methanogenic community in a hypereutrophic freshwater lake determined by PCR-RFLP.

AIMS: To combine database-held sequence information with a programme of experimental molecular ecology to define the methanogenic community of a hypereutrophic lake by a PCR-restriction fragment length polymorphism (RFLP) analysis. METHODS AND RESULTS: Methanogen diversity in a hypereutrophic freshwater lake was analysed using 16S rDNA PCR-RFLP. Database-held 16S rRNA gene sequences for 76 diverse methanogens were analysed for specific restriction sites that permitted unequivocal differentiation of methanogens. Restriction digestion and agarose gel electrophoresis of the 16S rDNA from selected methanogen pure cultures generated observed restriction profiles that corroborated the expected patterns. This method was then tested by analysing methanogen diversity in samples obtained over 1 year from sediment and water samples taken from the same sampling site. CONCLUSIONS: Restriction analysis of the 16S rRNA gene sequences from 157 methanogen clones generated from lakewater and sediment samples showed that over 50% were similar to Methanoculleus spp. Furthermore, a total of 16 RFLP types (1-16) were identified, eight of which contained no cultured representative archaeal 16S rRNA gene sequences. SIGNIFICANCE AND IMPACT OF THE STUDY: This RFLP strategy provides a robust and reliable means to rapidly identify methanogens in the environment.