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Journal: Applied and environmental microbiology

261

There have been an increasing number of reports implicating Gammaproteobacteria often carrying genes of drug resistance from colonized sink traps to vulnerable hospitalized patients. However, the mechanism of transmission from the wastewater of the sink P-trap to patients remains poorly understood. Herein we report the use of a designated hand washing sink lab gallery to model dispersion of green fluorescent protein (GFP)- expressing Escherichia coli from sink wastewater to the surrounding environment. We found no dispersion of GFP-E.coli directly from the P-trap to the sink basin or surrounding countertop with coincident water flow from a faucet. However, when the GFP-E.coli were allowed to mature in the P-trap under conditions similar to a hospital environment a GFP-E.coli containing putative biofilm extended upward over seven days to reach the strainer. This subsequently resulted in droplet dispersion to the surrounding areas (<30 inches) during faucet operation. We also demonstrated that P-trap colonization could occur by retrograde transmission along a common pipe. We postulate that the organisms mobilize up to the strainer from the P-trap resulting in droplet dispersion rather than directly from the P-trap. This work helps to further define the mode of transmission of bacteria from a P-trap reservoir to a vulnerable hospitalized patient.Importance Many recent reports demonstrate that sink drain pipes become colonized with highly consequential multidrug resistant bacteria, which then result in hospital acquired infections. However, the mechanism of dispersal of bacteria from the sink to patients has not been fully elucidated. Through establishment of a unique sink gallery this work found that a staged mode of transmission involving biofilm growth from the lower pipe to the sink strainer and subsequent splatter to the bowl and surrounding area occurs rather than splatter directly from the water in the lower pipe. We have also demonstrated that bacterial transmission can occur via connections in wastewater plumbing to neighboring sinks. This work helps to more clearly define the mechanism and risk of transmission from a wastewater source to hospitalized patients in a world with increasingly antibiotic resistant bacteria which can thrive in wastewater environments and cause infections in vulnerable patients.

Concepts: Protein, Gene, Bacteria, Antibiotic resistance, Escherichia coli, Pseudomonas aeruginosa, Plumbing, Sink

170

Comparative analysis of ospC genes from 127 Borrelia burgdorferi sensu stricto strains collected in Lyme disease endemic and non-endemic European and North American regions revealed close relatedness of geographically distinct populations. OspC alleles A, B and L were detected on both continents in vectors and hosts including humans. Six ospC alleles, A, B, L, Q, R and V, were prevalent in Europe; 4 of them were detected in samples of human origin. Ten ospC alleles, A, B, D, E3, F, G, H, H3, I3 and M, were identified in the far-western U.S.A. Four ospC alleles, B, G, H and L, were abundant in the southeastern U.S.A. Here we present the first expanded analysis of ospC alleles of B. burgdorferi strains from the southeastern U.S.A with respect to their relatedness to strains from other North American and European localities. We demonstrate that ospC genotypes commonly associated with human Lyme disease in endemic European and North American regions were detected in B. burgdorferi strains isolated from non-human biting tick Ixodes affinis and rodent hosts in southeastern U.S.A. We discovered that some ospC alleles previously known only from Europe are widely distributed in the southeastern U.S.A., a finding that confirms the hypothesis of trans-oceanic migration of Borrelia species.

Concepts: Bacteria, Lyme disease, Ixodes scapularis, North America, Borrelia burgdorferi, Borrelia, Allen Steere, Willy Burgdorfer

162

In recent decades, fresh and minimally processed produce items have been associated with an increasing proportion of foodborne illnesses. Most pathogens associated with fresh produce are enteric (fecal) in origin, and contamination can occur anywhere along the farm-to-fork chain. Microbial source tracking (MST) is a tool developed in the environmental microbiology field to identify and quantify the dominant source(s) of fecal contamination. This study investigated the utility of an MST method based on Bacteroidales 16S rDNA sequences as a means of identifying potential fecal contamination, and its source, in the fresh produce production environment. The method was applied to rinses of fresh produce, source and irrigation waters, and harvester hand rinses collected over the course of one year from nine farms (growing tomatoes, jalapeño peppers, and cantaloupe) in Northern Mexico. Of 174 samples, 39% were positive for a universal Bacteroidales marker (AllBac), including 66% of samples from cantaloupe farms (3.6 log10 genome equivalence copies [GEC]/100 ml), 31% of samples from tomato farms (1.7 log10 GEC/100 ml), and 18% of samples from jalapeño farms (1.5 log10 GEC/100 ml). Of 68 AllBac-positive samples, 46% were positive for one of 3 human-specific markers (BFD, HF183, BVulg), and none were positive for a bovine-specific marker (BoBac). There was no statistically significant correlation between Bacteroidales and generic E. coli across all samples. This study provides evidence that Bacteroidales markers may serve as alternative indicators for fecal contamination in fresh produce production, allowing for determination of both general contamination and that derived from the human host.

Concepts: Bacteria, Microbiology, Escherichia coli, Marker, Tomato, Identification, Mexico, Marker pen

150

There is still an important interest in controlling bacterial endospores. The use of chemical disinfectants and notably oxidising agents to sterilize medical devices is increasing. With this in mind hydrogen peroxide (H2O2) and peracetic acid (PAA) have been used in combination but until now there has been no explanation for the observed increased in sporicidal activity. This study provides information on the mechanism of synergistic interaction of PAA and H2O2 against bacterial spores. The investigations of the efficacy of different combinations, including pre-treatments with the two oxidisers were performed against wild-type and a range of spore mutants deficient in their spore coat or small acid-soluble spore proteins. The concentrations of the two biocides were also measured in the reaction vessels enabling the assessment of any shift from H2O2 to PAA formation. This study confirmed the synergistic activity of H2O2 and PAA combination. However, we observed that the sporicidal activity of the combination is largely due to PAA and not H2O2. Furthermore, we observed that the synergistic combination was based on H2O2 compromising the spore coat, which was the main spore resistance factor, likely allowing better penetration of PAA, resulting in the increased sporicidal activity.

Concepts: Oxygen, Hydrogen peroxide, Chlorine, Disinfectant, Peroxide, Disinfectants, Peracetic acid, Peroxy acid

139

We show in this report that traces of juices released from salad leaves as they became damaged can significantly enhance Salmonella enterica salad leaf colonisation. Salad juices in water increased Salmonella growth by 110% over the un-supplemented control, and in host-like serum based media by more than 2400-fold over controls. In serum based media salad juices induced growth of Salmonella via provision of Fe from transferrin, and siderophore production was found to be integral to the growth induction process. Other aspects relevant to salad leaf colonisation and retention were enhanced, such as motility and biofilm formation, which increased over controls by >220% and 250% respectively; direct attachment to salad leaves increased by >350% when a salad leaf juice was present. In terms of growth and biofilm formation the endogenous salad leaf microbiota was largely unresponsive to leaf juice, suggesting that Salmonella gains a marked advantage from fluids released from salad leaf damage. Salad leaf juices also enhanced pathogen attachment to the salad bag plastic. Over 5 days refrigeration (a typical storage time for bagged salad leaves) even traces of juice within the salad bag fluids increased Salmonella growth in water by up to 280-fold over control cultures, as well as enhancing salad bag colonisation, which could be an unappreciated factor in pathogen fresh produce retention. Collectively, this study shows that exposure to salad leaf juice may contribute to the persistence of Salmonella on salad leaves, and strongly emphasizes the importance of ensuring the microbiological safety of fresh produce.

Concepts: Bacteria, Iron, Microbiology, Salmonella enterica, Salmonella, Leaf, Lettuce, Leaf vegetable

120

Bacterial cross-contamination from surfaces to food can contribute to foodborne disease. The cross-contamination rate of Enterobacter aerogenes was evaluated on household surfaces using scenarios that differed by surface type, food type, contact time (<1, 5, 30 and 300 s), and inoculum matrix (tryptic soy broth or peptone buffer). The surfaces used were stainless steel, tile, wood and carpet. The food types were watermelon, bread, bread with butter and gummy candy. Surfaces (25 cm(2)) were spot inoculated with 1 ml of inoculum and allowed to dry for 5 h, yielding an approximate concentration of 10(7) CFU/surface. Foods (with 16 cm(2) contact area) were dropped on the surfaces from a height of 12.5 cm and left to rest as appropriate. Post transfer surfaces and foods were placed in sterile filter bags and homogenized or massaged, diluted and plated on tryptic soy agar. The transfer rate was quantified as the log % transfer from the surface to the food. Contact time, food and surface type all had a highly significant effect (P<0.000001) on log % transfer of bacteria. The inoculum matrix (TSB or peptone buffer) also had a significant effect on transfer (P = 0.013), and most interaction terms were significant. More bacteria transferred to watermelon (∼0.2-97%) relative to other foods, while fewer bacteria transferred to gummy candy (∼0.1-62%). Transfer of bacteria to bread (∼0.02-94%) and bread with butter (∼0.02-82%) were similar, and transfer rates under a given set of condition were more variable compared with watermelon and gummy candy.

Concepts: Bacteria, Food, Bread, Foodborne illness, Food safety, Enterobacter, Enterobacter aerogenes, Gummi bear

104

In the United States, 1 in 8 women will be diagnosed with breast cancer in her lifetime. Along with genetics, the environment also contributes to disease development but what these exact environmental factors are remain unknown. We have previously shown that breast tissue is not sterile but contains a diverse population of bacteria. We thus believe that the host’s local microbiome could be modulating the risk of breast cancer development. Using 16S rRNA amplicon sequencing we show that bacterial profiles differ between normal adjacent tissue from women with breast cancer and tissue from healthy controls. Women with breast cancer had higher relative abundances of Bacillus, Enterobacteriaceae and Staphylococcus Escherichia coli (member of the Enterobacteriaceae family) and Staphylococcus epidermidis, isolated from breast cancer patients, were shown to induce DNA double stranded breaks in HeLa cells using the γH2AX assay. We also found that microbial profiles are similar between normal adjacent tissue and tissue sampled directly from the tumour. This novel study raises important questions as to what role the breast microbiome plays in disease development or progression and how we can manipulate this microbiome for possible therapeutics or prevention.

Concepts: DNA, Genetics, Cancer, Breast cancer, Bacteria, Gut flora, Oncology, Escherichia coli

84

Staphylococcus aureus is an important pathogen of humans and animals. We genome-sequenced ninety S. aureus isolates from the Gambia: 46 isolates from invasive disease in humans, 13 human carriage isolates and 31 monkey carriage isolates. We inferred multiple anthroponotic transmissions of S. aureus from humans to green monkeys, Chlorocebus sabaeus in The Gambia over different timescales. We report a novel monkey-associated clade of S. aureus that emerged from a human-to-monkey switch estimated to have occurred 2,700 years ago. Adaptation of this lineage to the monkey host is accompanied by loss of phage-encoded genes that are known to play an important role in human colonisation. We also report recent anthroponotic transmission of the well-characterised human lineages, ST6 and ST15, to monkeys, probably because of steadily increasing encroachment of humans into the monkeys' habitat. Although we have found no evidence of transmission of S. aureus from monkeys to humans, as the two species come into ever-closer contact there might be an increased risk of additional inter-species exchanges of potential pathogens.

Concepts: Bacteria, Microbiology, Africa, Pneumonia, Staphylococcus aureus, Staphylococcus, Primate, Coagulase

42

In recent years, a greater appreciation for the microbes inhabiting human body sites has emerged. In the female mammary gland, milk has been shown to contain bacterial species, ostensibly from the skin. We decided to investigate whether there is a microbiome within the mammary tissue. Using 16S rRNA sequencing and culture, we analysed breast tissue from 81 women with and without cancer in Canada and Ireland. A diverse population of bacteria were detected within tissue collected from sites all around the breast in women aged 18 to 90, not all of whom had a history of lactating. The principal phylum was Proteobacteria. The most abundant taxa in the Canadian samples were Bacillus (11.4%), Acinetobacter (10.0%), unclassified Enterobacteriaceae (8.3%), Pseudomonas (6.5%), Staphylococcus (6.5%), Propionibacterium (5.8%), unclassified Comamonadaceae (5.7%), unclassified Gammaproteobacteria (5.0%) and Prevotella (5.0%). In the Irish samples the most abundant taxa were unclassified Enterobacteriaceae (30.8%), Staphylococcus (12.7%), Listeria welshimeri (12.1%), Propionibacterium (10.1%) and Pseudomonas (5.3%). None of the subjects had signs or symptoms of infection, but the presence of viable bacteria was confirmed in some samples by culture. The extent to which these organisms play a role in health or disease remains to be determined.

Concepts: Bacteria, Prolactin, Milk, Lactation, Breast, Mammary gland

41

This paper introduces a novel method for sampling pathogens in natural environments. It uses fabric boot socks worn over walkers' shoes allowing collection of composite samples over large areas. Wide area sampling is better suited to studies focussing upon human exposure to pathogens (e.g. recreational walking). This sampling method is implemented using a Citizen Science approach: groups of three walkers wearing boot socks undertook one of six routes, 40 times over 16 months in the North West (NW) and East Anglian (EA) regions of England. To validate this methodology we report the successful implementation of this Citizen Science approach, the observation that Campylobacter was detected on 47% of boot socks, and the observation that multiple boot socks from individual walks produced consistent results. Findings indicate elevated Campylobacter presence in the livestock dominated NW in comparison to EA (55.8% vs 38.6%). Seasonal variation in Campylobacter presence was found between regions, with indications of winter peaks in both regions, but a spring peak in NW. Campylobacter presence on boot socks was negatively associated with ambient temperature (p=0.011) and positively associated with precipitation (p<0.001), results which are consistent with our understanding of Campylobacter survival and the probability of material adhering to boot socks. C. jejuni was the predominant species found, with C. coli largely restricted to the livestock dominated NW. Source attribution analysis indicated that the potential source of C. jejuni was predominantly sheep in NW and wild birds in EA but did not vary between peak and non-peak periods of human incidence.Importance There is debate in the literature on the pathways through which pathogens transfer from the environment to humans. We report on the success of a novel method for sampling human-pathogen interactions using boot socks and citizen science techniques, which enable us to sample human-pathogen interactions that may occur through visits to natural environments. This contrasts with traditional environmental sampling, which is based upon spot sampling techniques and does not sample human-pathogen interactions. Our methods are of practical value to scientists trying to understand transmission of pathogens from the environment to people. Our findings provide insight into the risk of Campylobacter from recreational visits and an understanding of how these risks vary seasonally and the factors behind these patterns. We highlight the Campylobacter species predominantly encountered and the potential sources of the C. jejuni.

Concepts: Sample, Human, Sample size, Sampling, Environment, Natural environment, Nature, Campylobacter