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Journal: Applied and environmental microbiology

81

Extreme habitats are not only limited to natural environments, but also apply to man-made systems, for instance household appliances such as dishwashers. Limiting factors, such as high temperatures, high and low pH, high NaCl concentrations, presence of detergents and shear force from water during washing cycles define the microbial survival in this extreme system. Fungal and bacterial diversity in biofilms isolated from rubber seals of 24 different household dishwashers were investigated using next generation sequencing. Bacterial genera such as Pseudomonas, Escherichia and Acinetobacter, known to include opportunistic pathogens, were represented in most samples. The most frequently encountered fungal genera in these samples belonged to Candida, Cryptococcus and Rhodotorula, also known to include opportunistic pathogenic representatives. This study showed how specific conditions of the dishwashers impact the abundance of microbial groups, and investigated on the inter- and intra-kingdom interactions that shape these biofilms. The age, the usage frequency and hardness of incoming tap water of dishwashers had significant impact on bacterial and fungal composition. Representatives of Candida spp. were found at highest prevalence (100%) in all dishwashers and are assumingly one of the first colonizers in recent dishwashers. Pairwise correlations in tested microbiome showed that certain bacterial groups co-occur and so did the fungal groups. In mixed bacterial-fungal biofilms, early adhesion, contact and interactions were vital in the process of biofilm formation, where mixed complexes of the two, bacteria and fungi, could provide a preliminary biogenic structure for the establishment of these biofilms.IMPORTANCE Worldwide demand for household appliances, such as dishwashers and washing machines, is increasing, as well as the number of immune-compromised individuals. The harsh conditions in household dishwashers should prevent growth of most microorganisms. However, our research shows that persisting poly-extremotolerant groups of microorganisms in household appliances are well established under these unfavourable conditions, supported by the biofilm mode of growth. The significance of our research is in identifying the microbial composition of biofilms formed on dishwasher rubber seals, how diverse abiotic conditions affects microbiota and which key members were represented in early colonisation and contamination of dishwashers, as these appliances can present a source of domestic cross-contamination leading to broader medical impacts.

Concepts: Archaea, Bacteria, Microbiology, Eukaryote, Fungus, Pseudomonas aeruginosa, Pathogen, Biofilm

64

The development and continuous improvement of high-throughput sequencing platforms has stimulated interest in the study of complex microbial communities. Currently, the most popular sequencing approach to study microbial community composition and dynamics is targeted 16S rRNA gene metabarcoding. To prepare samples for sequencing, there are a variety of processing steps, each with the potential to introduce bias at the data analysis stage. In this short review, key information from the literature pertaining to each processing step is described and consequently, general recommendations for future 16S rRNA gene metabarcoding experiments are made.

Concepts: Archaea, Microbiology, Species, Ribosomal RNA, Biotechnology, 16S ribosomal RNA, 30S

46

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

42

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

41

The skin is the first line of defense between an animal and its environment, and disruptions in skin-associated microorganisms can be linked to an animal’s health and nutritional state. To better understand the skin microbiome of large whales, high-throughput sequencing of partial small subunit ribosomal RNA genes was used to study the skin-associated bacteria of 89 seemingly healthy humpback whales (Megaptera novaeangliae) sampled along the Western Antarctic Peninsula (WAP) during early (2010) and late (2013) austral summers. Six core genera of bacteria were present in 93% or more of all humpback skin samples. A shift was observed in the average relative abundance of these core genera over time, with the emergence of four additional core genera corresponding to a decrease in water temperature, possibly caused by seasonal or foraging related changes in skin biochemistry that influenced microbial growth, or other temporal-related factors. The skin microbiome differed between whales sampled at several regional locations along the WAP, suggesting that environmental factors or population may also influence the whale skin microbiome. Overall, the skin microbiome of humpback whales appears to provide insight into animal and environmental-related factors and may serve as a useful indicator for animal health or ecosystem alterations.IMPORTANCE The microbiomes of wild animals are currently understudied, but may provide information about animal health and/or animal-environmental interactions. In the largest sampling of any marine mammal microbiome, this study demonstrates conservation in the skin microbiome of 89 seemingly healthy humpback whales sampled in the Western Antarctic Peninsula, with shifts in the bacterial community composition related to temporal and regional variability. This study is important because it suggests that the skin microbiome of humpback whales could provide insight into animal nutritional or seasonal/environmental-related factors, which are becoming increasingly important to recognize due to unprecedented rates of climate change and anthropogenic impact on ocean ecosystems.

Concepts: Archaea, Bacteria, RNA, Ribosomal RNA, Ribosome, Whale, Humpback whale, Whale surfacing behaviour

35

In this study differences in the placental microbiota of term and preterm deliveries from a large UK pregnancy cohort were studied using 16S targeted amplicon sequencing. The impact of contamination from DNA extraction, PCR reagents, as well as those from delivery itself were also examined. A total of 400 placental samples from 256 singleton pregnancies were analysed and differences investigated between spontaneous preterm, non-spontaneous preterm, and term delivered placenta. DNA from recently delivered placenta was extracted, and screening for bacterial DNA was carried out using targeted sequencing of the 16S rRNA gene on the Illumina MiSeq platform. Sequenced reads were analysed for presence of contaminating operational taxonomic units (OTUs) identified via sequencing of negative extraction and PCR blank samples. Differential abundance and between sample (beta) diversity metrics were then compared. A large proportion of the reads sequenced from the extracted placental samples mapped to OTUs that were also found in negative extractions. Striking differences in the composition of samples were also observed, according to whether the placenta was delivered abdominally or vaginally, providing strong circumstantial evidence for delivery contamination as an important contributor to observed microbial profiles. When OTU and genus level abundances were compared between the groups of interest, a number of organisms were enriched in the spontaneous preterm cohort, including organisms that have been previously associated with adverse pregnancy outcomes, specifically Mycoplasma spp., and Ureaplasma spp.. However, analyses of overall community structure did not reveal convincing evidence for the existence of a reproducible ‘preterm placental microbiome’.IMPORTANCE Preterm birth is associated with both psychological and physical disabilities and is the leading cause of infant morbidity and mortality worldwide. Infection is known to be an important cause of spontaneous preterm birth, and recent research has implicated variation in the ‘placental microbiome’ with preterm birth risk. Consistent with previous studies, the abundance of certain clinically relevant species differed between spontaneous preterm and non-spontaneous preterm or term delivered placenta. These results support the view that a proportion of spontaneous preterm births have an intra-uterine infection component. However, an additional observation from this study was that a substantial proportion of reads sequenced were contaminating reads, rather than DNA from endogenous, clinically relevant species. This observation warrants caution in the interpretation of sequencing output from such low biomass samples as the placenta.

33

This study aimed at assessing the dynamics of lactic acid bacteria and other Firmicutes associated with durum wheat organs and processed products. 16S rRNA gene-based high-throughput sequencing approaches and culture-independent analyses showed that Lactobacillus, Streptococcus, Enterococcus and Lactococcus were the main epiphytic and endophytic genera among lactic acid bacteria. Bacillus, Exiguobacterium, Paenibacillus and Staphylococcus completed the picture of the core genera microbiome. The relative abundance of each lactic acid bacteria genus was affected by cultivars, phenological stages, other Firmicutes genera, environmental temperature and water activity (aw) of plant organs. Lactobacilli, showing the highest sensitivity to aw, markedly decreased during milk development (Odisseo) and physiological maturity (Saragolla). At these stages, Lactobacillus was mainly replaced by Streptococcus, Lactococcus and Enterococcus. However, a key sourdough species such as Lactobacillus plantarum was associated to plant organs during the life cycle of Odisseo and Saragolla wheat. The composition of the sourdough microbiota and the overall quality of leavened baked goods is also determined throughout the phenological stages of wheat cultivation, with variations depending on environmental and agronomic factors. Based on the adaptability of lactic acid bacteria on wheat plant, future research has to assess the potential of these bacteria for biocontrol and plant growth promotion.

Concepts: Bacteria, Microbiology, Streptococcus, Wheat, Lactobacillus, Firmicutes, Lactic acid bacteria, Lactobacillales

29

The food-borne pathogen Listeria (L) monocytogenes is able to survive a variety of stress conditions leading to the colonization of different niches like the food processing environment. This study focuses on the hypervariable genetic hotspot lmo0443-lmo0449 haboring three inserts: the stress survival islet 1 (SSI-1), the single-gene insert LMOf2365_0481 and two homologous genes of the non-pathogenic species L. innocua: lin0464, a putative transcriptional regulator and lin0465, an intracellular PfpI protease. Our prevalence study revealed a different distribution of the inserts between human and food-associated isolates. The lin0464-lin0465 insert was predominantly found in food-associated strains of sequence type (ST) 121. Functional characterization of this insert showed that the putative PfpI protease Lin0465 is involved in alkaline and oxidative stress response, but not in acidic, gastric, heat, cold, osmotic and antibiotic stress. In parallel, deletion of lin0464 decreased the survival under alkaline and oxidative stress. The expression of both genes increased significantly under oxidative stress conditions independently of the alternative sigma factor σ(B) Furthermore, we showed that the expression of the protease lin0465 is regulated by the transcription factor lin0464 under stress conditions, suggesting that lin0464 and lin0465 form a functional unit.In conclusion, we identified a novel stress survival islet 2 (SSI-2), predominantly present in L. monocytogenes ST121 strains, beneficial for survival under alkaline and oxidative stress, potentially supporting adaptation and persistence of L. monocytogenes in food processing environments.IMPORTANCEListeria (L.) monocytogenes strains of ST121 are known to persist for months and even years in food processing environments, thereby increasing the risk of food contamination and listeriosis. However, the molecular mechanism underlying this remarkable niche-specific adaptation is still unknown. Here, we demonstrate that the genomic islet SSI-2, predominantly present in L. monocytogenes ST121 strains, is beneficial for survival under alkaline and oxidative stress conditions, which are routinely encountered in food processing environments. Our findings suggest that SSI-2 is part of a diverse set of molecular determinants contributing to niche-specific adaptation and persistence of L. monocytogenes ST121 strains in food processing environments.

Concepts: DNA, Genetics, Gene expression, Transcription, Oxidative stress, Listeria monocytogenes, Listeria, Listeriosis

28

The South China Sea (SCS), the largest marginal sea in the Western Pacific Ocean, is a huge oligotrophic water body with very limited influx of nitrogenous nutrients. This suggests that sediment microbial N(2) fixation plays an important role in the production of bioavailable nitrogen. To test the molecular underpinning of this hypothesis, the diversity, abundance, biogeographical distribution, and community structure of the sediment diazotrophic microbiota were investigated at 12 sampling sites, including estuarine, coastal, offshore, deep-sea, and methane hydrate reservoirs or their prospective areas by targeting nifH and some other functional biomarker genes. Diverse and novel nifH sequences were obtained, significantly extending the evolutionary complexity of extant nifH genes. Statistical analyses indicate that sediment in situ temperature is the most significant environmental factor influencing the abundance, community structure, and spatial distribution of the sediment nifH-harboring microbial assemblages in the northern SCS (nSCS). The significantly positive correlation of the sediment pore water NH(4)(+) concentration with the nifH gene abundance suggests that the nSCS sediment nifH-harboring microbiota is active in N(2) fixation and NH(4)(+) production. Several other environmental factors, including sediment pore water PO(4)(3-) concentration, sediment organic carbon, nitrogen and phosphorus levels, etc., are also important in influencing the community structure, spatial distribution, or abundance of the nifH-harboring microbial assemblages. We also confirmed that the nifH genes encoded by archaeal diazotrophs in the ANME-2c subgroup occur exclusively in the deep-sea methane seep areas, providing for the possibility to develop ANME-2c nifH genes as a diagnostic tool for deep-sea methane hydrate reservoir discovery.

Concepts: Archaea, Bacteria, Water, Hydrogen, Pacific Ocean, Ocean, South China Sea, East China Sea

28

Evaluating different swabbing materials for spore recovery efficiency (RE) from steel surfaces, we recorded the maximum RE (71%) of 10(7) Bacillus subtilis spores with Tulips cotton buds, followed by Johnson’s cotton buds and standard Hi-Media cotton, polyester, nylon, and foam (23%) swabs. Among cotton swabs, instant water-absorbing capacity or the hydrophilicity index appeared to be the major indicator of RE, as determined by testing three more brands. Tulips swabs worked efficiently across diverse nonporous surfaces and on different Bacillus spp., registering 65 to 77% RE.

Concepts: Cotton, Spore, Bacillus, Asexual reproduction, Bacillus subtilis, Endospore, American inventions, Cotton swab