Introduction C.difficile is a Gram-positive anaerobe that can cause life-threatening diarrhoea. There is no current method with which to identify pathogenic C.difficile colonisation (PCDC) quickly. In order to be pathogenic, C.difficile must have the ability to make toxin B. We have designed and validated an rtPCR assay targeting the toxin B with which to diagnose PCDC quickly using a perianal swab. Methods Perianal swabs were taken prospectively from 99 patients with proven stool culture-positive C.difficile infection (CDI) within 24 hours of diagnosis. DNA from swab tips was extracted using the automated QIAsymphony platform. Half of the DNA extracts from patients with CDI along with control C.difficile DNA were used to optimise an rtPCR assay using primers targeting the C.difficile toxin B gene. PCRs were run as 10μL reactions on 96 well plates in duplicate and the volume of extract per reaction was varied (1–3μL). PCR positive was defined as: amplification curve crossing the threshold at <40 cycles, <0.5 CT difference between duplicates, melting points to be ± 1.25 OC of that of C.difficile DNA and no amplification in negative controls. PCR positivity was compared to culture result as the gold standard. Once optimised, the assay was validated using the remaining 48 DNA extracts from CDI patients and extracts from swabs taken from 11 control patients with C.difficile-negative diarrhoea. Results In optimisation, assay sensitivity increased as more DNA extract was added (40%, 62% and 78% for 1μL, 2μL and 3μL respectively, p = 0.0042). The 3μL assay showed 92% efficiency and linearity of 0.997 was selected for the validation study. For validation using perianal swabs from 48 patients with proven CDI and 11 C.difficile stool culture-negative patients, assay sensitivity was 69%, specificity 93%, positive predictive value (PPV) 97% and negative predictive value (NPV) 48%. Conclusion We designed an rtPCR assay to identify C.difficile toxin B in DNA extracted from perianal swabs taken from patients with diarrhoea. Although the sensitivity in the validation experiments was low (69%), the high specificity and PPV mean that the assay may have clinical applicability. As almost all patients testing positive with the PCR swab test will be colonised with pathogenic C.difficile, they could be isolated on hospital admission to reduce the chance of nosocomial spread. Patients testing positive could also be prospectively followed to see if colonisation with pathogenic C.difficile on admission is a risk factor for subsequent C.difficile-induced diarrhoea (CDI), poorer outcome if diagnosed with CDI, or relapsing disease. Disclosure of Interest None Declared

OBJECTIVES The objectives of this study were to characterize ESBL-producing Enterobacteriaceae present in 24 neonatal units (NNUs) in eight networks participating in a multicentre probiotic study and to test the hypothesis that specific strains would cluster within individual units and networks. METHODS We performed analysis of stool samples for the presence of ESBL-producing Enterobacteriaceae at 2 weeks post-natal age and 36 weeks post-menstrual age. ESBL-producing Enterobacteriaceae were characterized and typed using molecular methods. RESULTS ESBL-producing Enterobacteriaceae (n = 71) were isolated from 67/1229 (5.5%) infants from whom we received a sample at either sampling time or both sampling times, and from infants in 18 (75%) of the 24 recruiting NNUs. Thirty-three Escherichia coli, 23 Klebsiella spp. and 6 Enterobacter spp. strains were characterized. ESBL-producing E. coli were all distinguishable within individual NNUs by antibiotic resistance genotype, serogroup (O25b), phenotype, phylotype or ST. Ten of the 33 were ST131 and 9 of the 10 ST131 isolates were ciprofloxacin resistant. Seven of the 10 ST131 isolates carried genes encoding CTX-M group 1 enzymes. ST131 isolates were isolated from centres within five of the eight NNU networks. There were clusters of indistinguishable ESBL-producing Klebsiella and Enterobacter isolates associated with specific NNUs. CONCLUSIONS Strains of E. coli ST131 were distributed across neonatal networks in the south of England. There was no evidence of clustering of clonally related ESBL-producing E. coli strains, by contrast with Klebsiella spp. and Enterobacter spp., which did cluster within units. The possibility that ESBL-producing E. coli strains are spread by vertical transmission requires further investigation.

Australian avocado orchards are currently planted on seedling rootstocks, which are genetically diverse encompassing genotypes from the three botanical races of Persea americana. This diversity increases the difficulty of getting a uniform outcome from standard management practices. For example, over a 6-year period a 400% difference in yield was measured between ‘Hass’ trees in the same orchard under identical management. Additionally, large differences have been recorded between trees in the susceptibility of fruit developing post harvest rots which negatively impacts on consumers. These differences have been attributed to different rootstocks exerting changes on scion physiology/chemistry. Phytophthora cinnamomi was present in eastern Australia before avocados were introduced in the late 19

Colonization of the anterior nares with Staphylococcus aureus has been shown to be a risk factor for infection. The purpose of this study was to test the hypothesis that methicillin-resistant strains of S. aureus (MRSA) compete with methicillin-sensitive (MSSA) strains for colonization of the anterior nares. As part of the local National Health Service trust MRSA infection control strategy, patients who have been in a healthcare institution in the last year are routinely sampled and tested for MRSA colonization at the time of hospital admission. The sampling and testing methods were modified for the six-month period of this study to allow the detection of both MSSA and MRSA/MSSA co-colonization. MRSA alone was carried by 56 (8%) of 680 patients, MSSA alone by 115 patients (17%), 505 patients (74.3%) carried neither, and four patients (0.6%) carried both MRSA and MSSA. The deviance between the observed number of co-colonized swabs and that expected under the null hypothesis of no competition between MSSA and MRSA was significant (P=0.02, Fisher's exact test). The statistical approach is unaffected by the confounding effect of factors that affect the relative frequencies of MRSA or MSSA colonization. When logistic regression was used to estimate the extent of competition, controlling for effects of age and sex, we estimated a protective efficacy of MSSA colonization in the prevention of MRSA colonization of 78% (95% CI 29-99%). Results from this cross-sectional study support the hypothesis that MRSA and MSSA compete for colonization space, and provides an estimate of the extent to which MSSA interferes with MRSA colonization.

ABSTRACT Lactobacilli, principally the strains that are hydrogen peroxide (H2O2) producing, may have a protective effect against vaginal colonization by pathogenic species such as those that cause bacterial vaginosis. Previous reports have also suggested that H2O2-producing lactobacilli in the vagina may protect pregnant women against ascending infection of the chorioamniotic membranes and uterine cavity. We report the identification and H2O2 production of lactobacilli isolated from vaginal swabs collected at 20 weeks' gestation from a population of pregnant women at high risk of preterm birth. We also report the correlation between identification and H2O2 production in relation to the outcomes of chorioamnionitis and preterm birth. Lactobacilli were identified by partial 16S rRNA gene sequencing. H2O2 production by isolates was determined by a semiquantitative method. The most commonly isolated species were L. crispatus, L. gasseri, L. vaginalis and L. jensenii. Amounts of H2O2 produced by lactobacilli varied widely. The presence of lactobacilli producing high levels of H2O2 in the vagina of this population of pregnant women was associated with a reduced risk of bacterial vaginosis at 20 weeks' gestation and subsequent chorioamnionitis. L. jensenii and L. vaginalis produced the highest levels of H2O2. We postulate that H2O2-producing lactobacilli are able to reduce the incidence of ascending infections of the uterus and the subsequent production of proinflammatory molecules which are important in the pathogenesis of chorioamnionitis and preterm birth.

Background: Microbial antibiotic resistance is a major public health concern. The bowel flora of babies nursed on neonatal units is characterised by few normal commensals and may be dominated by hospital acquired organisms. In a pilot study 18 of 22 (82%) babies who had been on NICU >7days were found to have stools colonised with antibiotic resistant Gram negative bacilli (rGNB) at times between discharge and 357days. The objective of the present study was to investigate the rate and clinical associates of colonisation with rGNB in a population of VLBW babies on a second neonatal unit. Methods: Babies <1500g birthweight and >23 weeks gestational age were recruited into a study involving randomisation to receive Bifidobacterium breve BBG for 28 days starting within 48h of birth. Quantitative microbiological analysis was performed on the available stool closest to 28 days. Results: 40 babies (median BWt 864g, IQR 777–1144g; GA 26.8w, IQR 25.4–29.0w) were recruited of whom 38 survived to 28d. Stools were available for 34 of these. 25(74%) were colonised with GNB resistant to one class of antibiotics and 10(29%) had multi-resistant strains. Of 16 babies born to women on antibiotics at the time of delivery, 8 (50%) were colonised with multi-resistant GNB (mrGNB) compared with only 2 of 18(11%) babies whose mothers were not on antibiotics, p=0.023. There were no other statistically significant associations between gestational age, birthweight, birhweight Z-score, sex, prolonged membrane rupture, antenatal steroids, caesarian section, whether or not the baby was colonised with Bifidebacterium breve BBG at 28d, the number of days the baby was on antibiotics or whether the baby had received a cephalosporin in the first 28d and colonisation with either rGNB or mrGNB. Conclusion: Rates of intestinal colonisation of babies cared for on our NICUs with antibiotic resistant Gram negative bacilli appear to be high and posibly to be associated with the use of maternal antibiotics around the time of birth. Further work is needed to explore this phenomenon further and to determine whether the graduates of our nurseries represent a public health risk in the community following discharge.