The Impact of Pipe Material on the Diversity of Microbial Communities in Drinking Water Distribution Systems.

As many cities around the world face the prospect of replacing aging drinking water distribution systems (DWDS), water utilities must make careful decisions on new pipe material (e.g., cement-lined or PVC) for these systems. These decisions are informed by cost, physical integrity, and impact on microbiological and physicochemical water quality. Indeed, pipe material can impact the development of biofilm in DWDS that can harbor pathogens and impact drinking water quality. Annular reactors (ARs) with cast iron and cement coupons fed with chloraminated water from a municipal DWDS were used to investigate the impact of pipe material on biofilm development and composition over 16 months. The ARs were plumbed as closely as possible to the water main in the basement of an academic building to simulate distribution system conditions. Biofilm communities on coupons were characterized using 16S rRNA sequencing. In the cast iron reactors, ß-proteobacteria, Actinobacteria, and α-proteobacteria were similarly relatively abundant (24.1, 22.5, and 22.4%, respectively) while in the cement reactors, α-proteobacteria and Actinobacteria were more relatively abundant (36.3 and 35.2%, respectively) compared to ß-proteobacteria (12.8%). Mean alpha diversity (estimated with Shannon H and Faith's Phylogenetic Difference indices) was greater in cast iron reactors (Shannon: 5.00 ± 0.41; Faith's PD: 15.40 ± 2.88) than in cement reactors (Shannon: 4.16 ± 0.78; Faith's PD: 13.00 ± 2.01). PCoA of Bray-Curtis dissimilarities indicated that communities in cast iron ARs, cement ARs, bulk distribution system water, and distribution system pipe biofilm were distinct. The mean relative abundance of Mycobacterium spp. was greater in the cement reactors (34.8 ± 18.6%) than in the cast iron reactors (21.7 ± 11.9%). In contrast, the mean relative abundance of Legionella spp. trended higher in biofilm from cast iron reactors (0.5 ± 0.7%) than biofilm in cement reactors (0.01 ± 0.01%). These results suggest that pipe material is associated with differences in the diversity, bacterial composition, and opportunistic pathogen prevalence in biofilm of DWDS.

Plant-Microbe Interactions Drive Denitrification Rates, Dissolved Nitrogen Removal, and the Abundance of Denitrification Genes in Stormwater Control Measures.

The microbial community and function along with nitrate/nitrite (NOx) removal rates, and nitrogen (N) partitioning into "uptake", "denitrification", and "remaining" via isotope tracers, were studied in soil bioretention mesocolumns (8 unique plant species). Total denitrification gene reads per million (rpm) were positively correlated with % denitrified ( r = 0.69) but negatively correlated with total NOx removal following simulated rain events ( r = -0.79). This is likely due to plant-microbe interactions. Plant species with greater root volume, plant and microbial assimilation %, and NOx removal % had lower denitrification genes and rates. This implies that although microorganisms have access to N, advantageous functions, like denitrification, may not increase. At the conclusion of the 1.5-year experiment, the microbial community was strongly influenced by plant species within the Top zone dominated by plant roots, and the presence or absence of a saturated zone influenced the microbial community within the Bottom zone. Leptospermum continentale was an outlier from the other plants and had much lower denitrification gene rpm (average 228) compared to the other species (range: 277 to 413). The antimicrobial properties and large root volume of Leptospermum continentale likely caused this denitrification gene depression.

What's the risk? Identifying potential human pathogens within grey-headed flying foxes faeces.

Pteropus poliocephalus (grey-headed flying foxes) are recognised vectors for a range of potentially fatal human pathogens. However, to date research has primarily focused on viral disease carriage, overlooking bacterial pathogens, which also represent a significant human disease risk. The current study applied 16S rRNA amplicon sequencing, community analysis and a multi-tiered database OTU picking approach to identify faecal-derived zoonotic bacteria within two colonies of P. poliocephalus from Victoria, Australia. Our data show that sequences associated with Enterobacteriaceae (62.8% ± 24.7%), Pasteurellaceae (19.9% ± 25.7%) and Moraxellaceae (9.4% ± 11.8%) dominate flying fox faeces. Further colony specific differences in bacterial faecal colonisation patterns were also identified. In total, 34 potential pathogens, representing 15 genera, were identified. However, species level definition was only possible for Clostridium perfringens, which likely represents a low infectious risk due to the low proportion observed within the faeces and high infectious dose required for transmission. In contrast, sequences associated with other pathogenic species clusters such as Haemophilus haemolyticus-H. influenzae and Salmonella bongori-S. enterica, were present at high proportions in the faeces, and due to their relatively low infectious doses and modes of transmissions, represent a greater potential human disease risk. These analyses of the microbial community composition of Pteropus poliocephalus have significantly advanced our understanding of the potential bacterial disease risk associated with flying foxes and should direct future epidemiological and quantitative microbial risk assessments to further define the health risks presented by these animals.

Evaluation of Techniques for Measuring Microbial Hazards in Bathing Waters: A Comparative Study.

Recreational water quality is commonly monitored by means of culture based faecal indicator organism (FIOs) assays. However, these methods are costly and time-consuming; a serious disadvantage when combined with issues such as non-specificity and user bias. New culture and molecular methods have been developed to counter these drawbacks. This study compared industry-standard IDEXX methods (Colilert and Enterolert) with three alternative approaches: 1) TECTA™ system for E. coli and enterococci; 2) US EPA's 1611 method (qPCR based enterococci enumeration); and 3) Next Generation Sequencing (NGS). Water samples (233) were collected from riverine, estuarine and marine environments over the 2014-2015 summer period and analysed by the four methods. The results demonstrated that E. coli and coliform densities, inferred by the IDEXX system, correlated strongly with the TECTA™ system. The TECTA™ system had further advantages in faster turnaround times (~12 hrs from sample receipt to result compared to 24 hrs); no staff time required for interpretation and less user bias (results are automatically calculated, compared to subjective colorimetric decisions). The US EPA Method 1611 qPCR method also showed significant correlation with the IDEXX enterococci method; but had significant disadvantages such as highly technical analysis and higher operational costs (330% of IDEXX). The NGS method demonstrated statistically significant correlations between IDEXX and the proportions of sequences belonging to FIOs, Enterobacteriaceae, and Enterococcaceae. While costs (3,000% of IDEXX) and analysis time (300% of IDEXX) were found to be significant drawbacks of NGS, rapid technological advances in this field will soon see it widely adopted.

Into the deep: Evaluation of SourceTracker for assessment of faecal contamination of coastal waters.

Faecal contamination of recreational waters is an increasing global health concern. Tracing the source of the contaminant is a vital step towards mitigation and disease prevention. Total 16S rRNA amplicon data for a specific environment (faeces, water, soil) and computational tools such as the Markov-Chain Monte Carlo based SourceTracker can be applied to microbial source tracking (MST) and attribution studies. The current study applied artificial and in-laboratory derived bacterial communities to define the potential and limitations associated with the use of SourceTracker, prior to its application for faecal source tracking at three recreational beaches near Port Phillip Bay (Victoria, Australia). The results demonstrated that at minimum multiple model runs of the SourceTracker modelling tool (i.e. technical replicates) were required to identify potential false positive predictions. The calculation of relative standard deviations (RSDs) for each attributed source improved overall predictive confidence in the results. In general, default parameter settings provided high sensitivity, specificity, accuracy and precision. Application of SourceTracker to recreational beach samples identified treated effluent as major source of human-derived faecal contamination, present in 69% of samples. Site-specific sources, such as raw sewage, stormwater and bacterial populations associated with the Yarra River estuary were also identified. Rainfall and associated sand resuspension at each location correlated with observed human faecal indicators. The results of the optimised SourceTracker analysis suggests that local sources of contamination have the greatest effect on recreational coastal water quality.

Colistin-resistant, lipopolysaccharide-deficient Acinetobacter baumannii responds to lipopolysaccharide loss through increased expression of genes involved in the synthesis and transport of lipoproteins, phospholipids, and poly-ß-1,6-N-acetylglucosamine.

We recently demonstrated that colistin resistance in Acinetobacter baumannii can result from mutational inactivation of genes essential for lipid A biosynthesis (Moffatt JH, et al., Antimicrob. Agents Chemother. 54:4971-4977). Consequently, strains harboring these mutations are unable to produce the major Gram-negative bacterial surface component, lipopolysaccharide (LPS). To understand how A. baumannii compensates for the lack of LPS, we compared the transcriptional profile of the A. baumannii type strain ATCC 19606 to that of an isogenic, LPS-deficient, lpxA mutant strain. The analysis of the expression profiles indicated that the LPS-deficient strain showed increased expression of many genes involved in cell envelope and membrane biogenesis. In particular, upregulated genes included those involved in the Lol lipoprotein transport system and the Mla-retrograde phospholipid transport system. In addition, genes involved in the synthesis and transport of poly-ß-1,6-N-acetylglucosamine (PNAG) also were upregulated, and a corresponding increase in PNAG production was observed. The LPS-deficient strain also exhibited the reduced expression of genes predicted to encode the fimbrial subunit FimA and a type VI secretion system (T6SS). The reduced expression of genes involved in T6SS correlated with the detection of the T6SS-effector protein AssC in culture supernatants of the A. baumannii wild-type strain but not in the LPS-deficient strain. Taken together, these data show that, in response to total LPS loss, A. baumannii alters the expression of critical transport and biosynthesis systems associated with modulating the composition and structure of the bacterial surface.

The major surface Vsp proteins of Brachyspira hyodysenteriae form antigenic protein complexes.

The Vsp proteins are the major outer membrane proteins of Brachyspira hyodysenteriae, the causative agent of swine dysentery. Eight vsp genes have been identified in B. hyodysenteriae strain B204, arranged into two four-gene loci, and at least two of the corresponding proteins are produced in vitro. The aims of this study were to characterise the vsp genes of the virulent Australian B. hyodysenteriae strain X576 and their corresponding proteins, Genomic sequence comparison with strains B204 and WA1 demonstrated that the number of vsp genes varies between B. hyodysenteriae strains, although the chromosomal locations of the vsp gene loci are consistent. We identified two additional vsp-like genes, designated vspI and vspJ, in each of the three strains. Double SDS-PAGE was used to demonstrate that Vsp proteins of B. hyodysenteriae strain X576 form multimeric protein complexes in the outer membrane that are stable in 6M urea but dissociate after boiling. The Vsp complexes primarily consisted of VspF but also contain VspE and VspI. VspD was also found in a series of complexes slightly larger than the more abundant VspF complexes. Vsp proteins are purported to be antigenic; however little direct data are available to support this claim. In this study convalescent pig sera did not bind denatured Vsp proteins by Western blotting, but did bind the Vsp complexes on Western blots, showing that conformational epitopes may be important in immune recognition of these major outer membrane proteins. This is the first definitive demonstration of the antigenicity of these proteins in swine dysentery.

Resequencing the Mycobacterium avium subsp. paratuberculosis K10 genome: improved annotation and revised genome sequence.

We report the resequencing and revised annotation of the Mycobacterium avium subsp. paratuberculosis K10 genome. A total of 90 single-nucleotide errors and a 51-bp indel in the original K10 genome were corrected, and the whole genome annotation was revised. Correction of these sequencing errors resulted in 28 frameshift alterations. The amended genome sequence is accessible via the supplemental section of study SRR060191 in the NCBI Sequence Read Archive and will serve as a valuable reference genome for future studies.

Complete genome sequence of Staphylococcus aureus strain JKD6159, a unique Australian clone of ST93-IV community methicillin-resistant Staphylococcus aureus.

Community methicillin-resistant Staphylococcus aureus (cMRSA) is an emerging issue that has resulted in multiple worldwide epidemics. We report the first complete genome sequence of an ST93-MRSA-IV clinical isolate that caused severe invasive infection and a familial outbreak of skin infection. This isolate is a representative of the most common Australian clone of cMRSA that is more distantly related to the previously sequenced genomes of S. aureus.

Differential expression of the Bhmp39 major outer membrane proteins of Brachyspira hyodysenteriae.

The enteric, anaerobic spirochete Brachyspira hyodysenteriae is the causative agent of swine dysentery, a severe mucohemorrhagic diarrheal disease of pigs that has economic significance in every major pork-producing country. Recent investigation into potential vaccine candidates has focused on the outer membrane proteins of B. hyodysenteriae. Bhmp39 (formerly Vsp39) is the most abundant surface-exposed outer membrane protein of B. hyodysenteriae; its predicted gene sequence has previously been shown to share sequence similarity to eight genes divided evenly between two paralogous loci. The peptide sequence suggested that Bhmp39 is encoded by one of these genes, bhmp39h. The biological significance of maintaining eight homologous bhmp39 genes is unclear, though it has been proposed that this may play a role in antigenic variation. In this study, real-time, reverse transcription-PCR was used to demonstrate that bhmp39f and bhmp39h were the transcripts most abundantly expressed by B. hyodysenteriae strain B204 cultured under in vitro growth conditions. Mass spectrometry data of the purified 39-kDa membrane protein showed that both Bhmp39f and Bhmp39h were present. Northern blot analysis across predicted Rho-independent terminators demonstrated that the genes of the bhmp39efgh locus result in monocistronic transcripts.

Characterization of a locus encoding four paralogous outer membrane lipoproteins of Brachyspira hyodysenteriae.

The identification of Brachyspira hyodysenteriae outer membrane proteins (OMPs) that may stimulate immunity to swine dysentery is important for vaccine development. We report here the analysis of a novel locus, blpGFEA, encoding four tandem paralogous proteins of approximately 30 kDa from B. hyodysenteriae. The four proteins share 31-39% sequence identity with lipoproteins from several species of bacterial pathogens, but the locus possesses a unique genetic organization. Using antisera raised to recombinant versions of each of these proteins, only BlpA and BlpE were found to be immunologically cross-reactive with the other proteins encoded by the locus. Northern hybridization indicated that only blpA was expressed under in vitro growth conditions. In addition, convalescent swine serum recognized recombinant BlpA in immunoblotting experiments, demonstrating that it is also expressed during infection. Analysis of the translated sequences of each of the genes revealed atypical spirochetal signal peptidase II recognition sites, and BlpA was shown to be a lipoprotein by incorporation of tritiated palmitic acid. Native BlpA was completely extracted by Triton X-114 (TX-114) and partitioned exclusively into the detergent phase during extraction of whole B. hyodysenteriae cells, implicating it as a component of the brachyspiral outer membrane. Consistent with the transcriptional and immunological data, analysis of the brachyspiral outer membrane proteome also revealed expression of only BlpA. Notably, inactivation of blpA homologs in Haemophilus influenzae and Salmonella enteritidis resulted in attenuation of virulence.