Quantitative Assessment of First Nations Drinking Water Distribution Systems for Detection and Prevalence of Thermophilic Campylobacter Species.

Water is considered a major route for transmitting human-associated pathogens. Although microbial water quality indicators are used to test for the presence of waterborne pathogens in drinking water, the two are poorly correlated. The current study investigates the prevalence of thermophilic DNA markers specific for Campylobacter spp. (C. jejuni and C. coli) in source water and throughout the water distribution systems of two First Nations communities in Manitoba, Canada. A total of 220 water samples were collected from various points of the drinking water distribution system (DWDS) between 2016 and 2018. Target Campylobacter spp. were always (100%) detected in a home with a fiberglass (CF) cistern, as well as the community standpipe (SP). The target bacteria were also frequently detected in treated water at the Water Treatment Plant (WTP) (78%), homes with polyethylene (CP) (60%) and concrete (CC) (58%) cisterns, homes with piped (P) water (43%) and water truck (T) samples (20%), with a maximum concentration of 1.9 × 103 cells 100 mL-1 (C. jejuni) and 5.6 × 105 cells 100 mL-1 (C. coli). Similarly, target bacteria were detected in 68% of the source water samples with a maximum concentration of 4.9 × 103 cells 100 mL-1 (C. jejuni) and 8.4 × 105 cells 100 mL-1 (C. coli). Neither target Campylobacter spp. was significantly associated with free and total chlorine concentrations in water. The study results indicate that there is an immediate need to monitor Campylobacter spp. in small communities of Canada and, particularly, to improve the DWDS in First Nations communities to minimize the risk of Campylobacter infection from drinking water sources. Further research is warranted in improving/developing processes and technologies to eliminate microbial contaminants from water.

Performance and recovery of nitrifying biofilm after exposure to prolonged starvation.

Achieving consistent ammonia removal in post-lagoon processes faces two major challenges impacting nitrifiers due to the unique seasonal variation of lagoon-based systems: summer to winter temperature drop and summer to fall ammonia starvation period while lagoon is removing ammonia. The objective of this study was to follow microbial diversity and define conditions that could overcome these challenges in a post-lagoon moving bed biofilm reactor (MBBR) operated at an initial surface area loading rate (SALR) of 0.3 g-NH4-N m-2d-1 from mesophilic (20 °C) to psychrophilic (4 °C). Initially the temperature was maintained at 20 °C and decreased to 10 °C until steady state was achieved. During starvation conditions (i.e., continuous, intermittent and no aeration without inflow; decanted media; and intermittent and continuous ammonia supplement) the temperature was decreased by 2 °C per week until 4 °C. The results indicated that operational procedures, such as intermittent ammonia supplement with SALR of 0.15 g-NH4-N m-2d-1 could improve performance with 80% ammonia removal achieved immediately after starvation period. Intermittent ammonia supplement had produced the greatest biofilm preservation comparable to the initial load with the highest specific and surface area removal rates. In the recovery phase (initial load restoration) 10 days were required to reestablish performance above 95% ammonia removal. When temperature was decreased from mesophilic to psychrophilic, the microbial diversity was found higher when starving biofilm compared to the control operated at the initial load while it converged to a similar population over recovery. The main actors associated to nitrification enriched at psychrophilic conditions were Proteobacteria and Bacteriodotes at phyla level. Ammonia oxidation to nitrite was mainly driven by the order Burkholderiales and nitrite oxidation to nitrate by Pseudomonadales. This procedure should be considered in the implementation of full-scale post-lagoon MBBR technologies to ensure reliable, robust, and consistent performance despite the inherent seasonal variability of lagoon-based processes.

Assessment of cleaning methods on bacterial burden of hospital privacy curtains: a pilot randomized controlled trial.

Healthcare-associated infections (HAIs) are an important global issue, leading to poor patient outcomes. A potential route of transmission of HAIs is through contact with hospital privacy curtains. The aim of this study is to evaluate cleaning on reduction of curtain bacterial burden. In this pilot cluster randomized controlled trial we compared the bacterial burden between three groups of 24 curtains on a regional burn/plastic surgery ward. A control group was not cleaned. Two groups were cleaned at 3-4 day intervals with either disinfectant spray or wipe. The primary outcome was the difference in mean CFU/cm2 between day 0 to day 21. The secondary outcome was the proportion of curtains contaminated with Methicillin-resistant Staphylococcus aureus (MRSA). By day 21, the control group was statistically higher (2.2 CFU/cm2) than spray (1.3 CFU/cm2) or wipe (1.5 CFU/cm2) (p < 0.05). After each cleaning at 3-4 day intervals, the bacterial burden on the curtains reduced to near day 0 levels; however, the level increased again over the intervening 3-4 days. By day 21, 64% of control curtains were contaminated with MRSA compared to 10% (spray) and 5% (wipe) (p < 0.05). This study show that curtains start clean and progressively become contaminated with bacteria. Regularly cleaning curtains with disinfectant spray or wipes reduces bacterial burden and MRSA contamination.

Effect of mesophilic anaerobic digestion on the resistome profile of dairy manure.

The effect of mesophilic anaerobic digestion (AD) on the resistome profile of manures from two different dairy farms was evaluated using a metagenomic approach. A total of 187 unique Antibiotic resistance genes (ARGs) for 17 different classes of antibiotics were detected in raw (undigested) manures. The results indicate that regardless of the origin of the dairy manure, mesophilic AD was capable of reducing or enriching the relative abundance of some ARGs. The main driver of these changes was strongly correlated with the evolution of the microbial community during the AD process. Putative ARG hosts were suggested by analyses of the co-occurrence of microbial groups and ARGs. Finally, network analyses revealed that mesophilic AD could also reduce the co-occurrence of different groups of ARGs potentially located in the same genetic elements. Our results provide valuable insights into the microbial mechanisms driving the diversity and abundance of ARGs during mesophilic AD.

A response regulator protein with antar domain, AvnR, in Acinetobacter baumannii ATCC 17978 impacts its virulence and amino acid metabolism.

Acinetobacter baumannii, a Gram-negative coccobacillus, is notorious for its involvement in opportunistic infections around the world. Its resistance to antibiotics makes treatment of infections challenging. In this study, we describe a novel response regulator protein, AvnR (A1S_2006) that regulates virulence-related traits in A. baumannii ATCC17978. Sequence analysis suggests that AvnR is a CheY-like response regulator and contains the RNA-binding ANTAR (AmiR and NasR transcription anti-termination regulators) domain. We show that AvnR plays a role in regulating biofilm formation (on glass and plastic surfaces), surface motility, adhesion to A549 cells as well as in nitrogen metabolism in A. baumannii. RNA-Seq analysis revealed that avnR deletion results in altered expression of more than 150 genes (116 upregulated and 42 downregulated). RNA-Seq data suggest that altered biofilm formation and surface motility observed in the avnR deletion mutant is likely mediated by previously unknown pathways. Of note, was the altered expression of genes predicted to be involved in amino acid transport and metabolism in avnR deletion mutant. Biolog phenotypic array showed that deletion of avnR hampered A. baumannii ATCC17978's ability to metabolize various nitrogen sources, particularly that of glutamic acid, serine, histidine, aspartic acid, isoleucine and arginine. Taken together our data show that AvnR, the first ANTAR protein described in A. baumannii, affects virulence phenotypes as well as its ability to metabolize nitrogen sources.

Effect of ceftiofur on mesophilic anaerobic digestion of dairy manure and the reduction of the cephalosporin-resistance gene cmy-2.

The effect of ceftiofur (CEF), a commonly used antibiotics on dairy farms, on the performance and stability of mesophilic batch anaerobic digestion (AD) of dairy manure was evaluated in terms of methane production, organic matter removal (COD, dCOD, TS, and VS), and synthesis of end-products (VFAs, CO2, and H2). The results indicated that only CEF concentrations of 10 mg/L or higher significantly affected the performance of the AD process, although the overall stability was not compromised. Biochemical analyses suggested that hydrolytic microorganisms were the most affected by the presence of CEF leading to lower COD removal, whereas acetogens were only temporarily slowed down. Methanogens, on the other hand, were not directly affected by any of the CEF concentrations tested (0.2-250 mg/L). Additionally, the presence of CEF was shown to alter the incidence of the cephalosporin-resistance marker, cmy-2, although an overall reduction was achieved in 15-day batch anaerobic digestion trials.

Two Component Regulatory Systems and Antibiotic Resistance in Gram-Negative Pathogens.

Gram-negative pathogens such as Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa are the leading cause of nosocomial infections throughout the world. One commonality shared among these pathogens is their ubiquitous presence, robust host-colonization and most importantly, resistance to antibiotics. A significant number of two-component systems (TCSs) exist in these pathogens, which are involved in regulation of gene expression in response to environmental signals such as antibiotic exposure. While the development of antimicrobial resistance is a complex phenomenon, it has been shown that TCSs are involved in sensing antibiotics and regulating genes associated with antibiotic resistance. In this review, we aim to interpret current knowledge about the signaling mechanisms of TCSs in these three pathogenic bacteria. We further attempt to answer questions about the role of TCSs in antimicrobial resistance. We will also briefly discuss how specific two-component systems present in K. pneumoniae, A. baumannii, and P. aeruginosa may serve as potential therapeutic targets.

Detection of fecal bacteria and antibiotic resistance genes in drinking water collected from three First Nations communities in Manitoba, Canada.

This study analyzed the microbiological quality of drinking and source water from three First Nations communities in Manitoba, Canada that vary with respect to the source, storage and distribution of drinking water. Community A relies on an aquifer and Community B on a lake as source water to their water treatment plants. Community C does not have a water treatment plant and uses well water. Quantification of free residual chlorine and fecal bacterial (E. coli and coliforms), as well as detection of antibiotic resistance genes (sul, ampC, tet(A), mecA, vanA, blaSHV, blaTEM, blaCTX-M, blaOXA-1, blaCYM-2, blaKPC, blaOXA-48, blaNDM, blaVIM, blaGES and blaIMP) was carried out. While water treatment plants were found to be working properly, as post-treatment water did not contain E. coli or coliforms, once water entered the distribution system, a decline in the chlorine concentration with a concomitant increase in bacterial counts was observed. In particular, water samples from cisterns not only contained high number of E. coli and coliforms, but were also found to contain antibiotic resistance genes. This work shows that proper maintenance of the distribution and storage systems in First Nations communities is essential in order to provide access to clean and safe drinking water.

Tn7-Based Single-Copy Insertion Vectors for Acinetobacter baumannii.

Acinetobacter baumannii is considered a problematic Gram-negative pathogen due to its widespread resistance to antibiotics. Understanding of resistance mechanisms in A. baumannii is critical for designing new and effective therapeutic options. However, this is hampered by the lack of tools to carry out genetic manipulations in A. baumannii. Here, we describe methods to use a chromosomal mini-Tn7-based single-copy gene expression system in A. baumannii. This system can be effectively used for performing genetic complementation studies, for tagging with fluorescent proteins, or for reporter fusion assays.

Rate of contamination of hospital privacy curtains in a burns/plastic ward: A longitudinal study.

BACKGROUND: Since hospital patient privacy curtains can harbor bacteria, are high-touch surfaces, and are cleaned infrequently, they may be involved in pathogen transmission. The aim of this longitudinal prospective study was to understand curtain contamination to inform curtain hygiene protocols, thereby minimizing the role of curtains in pathogen transmission. METHODS: Over 21 days, cultures of 10 freshly laundered curtains (8 test curtains surrounding patient beds and 2 controls in an unoccupied staff room) were taken in the Regional Burns/Plastics Unit. Contact plates were used to sample the curtains near the edge hem where they are most frequently touched. Microbial contamination and the presence of methicillin-resistant Staphylococcus aureus (MRSA) were determined. RESULTS: By day 3, test curtains showed increased microbial contamination (mean colony-forming units [CFU]/cm2 = 1.17) compared to control curtains (mean CFU/cm2 = 0.19). Test curtains became increasingly contaminated over time, with mean CFU/cm2 for days 17 and 21 of 1.86 and 5.11, respectively. By day 10, 1/8 test curtains tested positive for MRSA, and 5/8 were positive by day 14. CONCLUSIONS: Patient privacy curtains became progressively contaminated with bacteria, including MRSA. Between days 10 and 14 after being hung, curtains showed increased MRSA positivity. This may represent an opportune time to intervene, either by cleaning or replacing the curtains.

Isolation and Characterization of Acinetobacter baumannii Recovered from Campylobacter Selective Medium.

Acinetobacter baumannii, a Gram-negative opportunistic pathogen, is known to cause multidrug resistant infections. This organism has primarily been isolated from clinical environments and its environmental reservoirs remain largely unknown. In the present study, we recovered seven isolates of A. baumannii growing under conditions selective for Campylobacter spp. (microaerophilic at 42°C and in the presence of antibiotics) from dairy cattle manure storage tank or surface water impacted by livestock effluents. Antibiotic susceptibility tests revealed that all of these isolates were less susceptible to at least two different clinically relevant antibiotics, compared to the type strain A. baumannii ATCC17978. Expression of resistance-nodulation-division efflux pumps, an important mechanism of intrinsic resistance in these organisms, was analyzed, and adeB was found to be overexpressed in one and adeJ was overexpressed in three isolates. Comparison of these isolates using genomic DNA Macro-Restriction Fragment Pattern Analysis (MRFPA) revealed relatively low relatedness among themselves or with some of the clinical isolates from previous studies. This study suggests that A. baumannii isolates are capable of growing under selective conditions for Campylobacter spp. and that this organism can be present in manure and water.

Detection of Antibiotic Resistance Genes in Source and Drinking Water Samples from a First Nations Community in Canada.

UNLABELLED: Access to safe drinking water is now recognized as a human right by the United Nations. In developed countries like Canada, access to clean water is generally not a matter of concern. However, one in every five First Nations reserves is under a drinking water advisory, often due to unacceptable microbiological quality. In this study, we analyzed source and potable water from a First Nations community for the presence of coliform bacteria as well as various antibiotic resistance genes. Samples, including those from drinking water sources, were found to be positive for various antibiotic resistance genes, namely, ampC, tet(A), mecA, ß-lactamase genes (SHV-type, TEM-type, CTX-M-type, OXA-1, and CMY-2-type), and carbapenemase genes (KPC, IMP, VIM, NDM, GES, and OXA-48 genes). Not surprisingly, substantial numbers of total coliforms, including Escherichia coli, were recovered from these samples, and this result was also confirmed using Illumina sequencing of the 16S rRNA gene. These findings deserve further attention, as the presence of coliforms and antibiotic resistance genes potentially puts the health of the community members at risk. IMPORTANCE: In this study, we highlight the poor microbiological quality of drinking water in a First Nations community in Canada. We examined the coliform load as well as the presence of antibiotic resistance genes in these samples. This study examined the presence of antibiotic-resistant genes in drinking water samples from a First Nations Community in Canada. We believe that our findings are of considerable significance, since the issue of poor water quality in First Nations communities in Canada is often ignored, and our findings will help shed some light on this important issue.