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The impact of water application method on bacterial survival at or after the final irrigation was evaluated in bulb onions during commercially relevant field drying (curing). A three-strain rifampin-resistant cocktail of Escherichia coli was introduced to onions via a single overhead spray application in two separate trials (5.22 [trial 1] or 2.40 [trial 2] log CFU per onion) 2 to 3 days after the final irrigation. Onions were lifted from the soil 8 days after spray inoculation and, in some cases, foliage was removed (topping); onions remained in the field for an additional ca. 2 weeks (total ca. 3 weeks of curing). E. coli populations declined on the onions in the first 4 h after spray inoculation. E. coli was recovered from 38 (48%) or 28 (35%) of 80 whole-onion enrichments at the end of curing in trials 1 or 2, respectively. Topping did not significantly impact the percentage of E. coli-positive onions detected at the end of curing. From 8 h to 21 days, E. coli populations on positive onions ranged from 1 CFU per onion to 7 log CFU per onion in both trials, representing a potential risk of E. coli growth with overhead application of contaminated water at the end of onion production. In trial 2, additional rows of onions were inoculated via a 22-cm subsurface or surface drip irrigation line (1.94 log CFU/mL for 2.5 h). E. coli was detected in 0 (subsurface) and 4 (surface) of 50 whole-onion enrichments 3 h after the initiation of drip irrigation. Positive onions were detected at days 1 (4 of 50) and 7 (1 of 50) with subsurface drip inoculation, and at days 1 (7 of 50), 7 (2 of 50), and 14 (2 of 50) with surface drip inoculation. E. coli was not detected in whole-onion enrichments at the end of curing when inoculated by subsurface (0 of 50) or surface (0 of 50) drip irrigation. Application of contaminated water through drip irrigation, when coupled with field curing, results in low rates of contamination of bulb onions at the time of harvest.

Summary Background Hand washbasin U-bends have increasingly been associated with nosocomial outbreaks by Gram-negative bacteria, including Pseudomonas aeruginosa which is virtually ubiquitous in U-bends. Wastewater networks servicing U-bends are potential highways for trafficking pathogenic bacteria. Aim To use P. aeruginosa to investigate trafficking of bacteria between hospital washbasin U-bends. Methods Twenty-five washbasin U-bends in five locations in Dublin Dental University Hospital (DDUH) were investigated for trafficking of P. aeruginosa: 10 in Clinic 2 (C2), 10 in the Accident & Emergency Department (A&E) and five in three other locations. In addition, washbasin tap samples (N=80) and mains and tap water samples (N=72) were cultured for P. aeruginosa. Selected P. aeruginosa isolates recovered over 29 months underwent whole-genome sequencing, and relatedness was interpreted using whole-genome multi-locus sequence typing and pairwise single nucleotide polymorphism (SNP) analysis. Findings P. aeruginosa was recovered from all U-bends but not from taps or water. Eighty-three U-bend isolates yielded 10 sequence types (STs), with ST560 and ST179 from A&E, C2 and two other locations predominating (70%). ST560 was also recovered from a common downstream pipe. Isolates within ST560 and ST179 were highly related regardless of source. ST560 was divided into Cluster I (N=25) and Cluster II (N=2) with average allelic differences and SNPs of three and zero, and two and five, respectively. The 31 ST179 isolates exhibited an average allelic difference and SNPs of three and 12, respectively. Conclusion Highly related P. aeruginosa strains were identified in multiple U-bends in several DDUH locations, indicating trafficking via the wastewater network.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates (carrying the carbapenemase gene blaNDM-5) of sequence type 16 caused hospital-acquired bloodstream infection or gut colonization in two patients in an intensive care unit (ICU). It was hypothesized that handwashing sinks were the source, and all handwashing sinks in the ICU were sampled. Whole-genome sequencing and analysis revealed that one sink was the source of CRKP colonization/infection in both patients, instead of direct transmission of a common clone between the patients. This study highlights handwashing sinks as an important source of multi-drug-resistant organisms. Sink management, including prohibition of disposal of body fluids and daily disinfection with chlorine, curbed the transmission.

Cyanobacteria species are sensitive to many plant allelochemicals, such as pyrogallol. However, little attention has been paid to the relative effects of these xenobiotics on co-occurring toxigenic and non-toxigenic cyanobacterial strains, despite their co-existence in blooms. Hence, the responses of one toxigenic (TS2) and two non-toxigenic (NS1, NS2) Microcystis aeruginosa strains to pyrogallol were tested under three conditions: mono-culture and co-cultured either directly or separately by dialysis membrane. The study showed that the inhibitory effects of pyrogallol on the growth and photosynthetic yield (Fv/Fm) of either toxigenic or non-toxigenic M. aeruginosa strains were lower in direct and dialysis co-culture conditions than those in mono-culture conditions. This result indicated that chemical-mediated reciprocal effects occur between the co-existing toxigenic and non-toxigenic strains. The toxigenic M. aeruginosa strain was more sensitive to pyrogallol than the non-toxigenic strains in both mono- and co-culture systems, though whether this outcome is due to the former's toxigenic status is unclear. Intracellular microcystin-LR (MC-LR) concentrations of the toxigenic strain decreased after pyrogallol addition in both mono- and co-culture systems, whereas extracellular MC-LR concentrations increased. This finding may reflect the cell damage of M. aeruginosa because of the pyrogallol. At the same initial number of cells, the extracellular MC-LR concentration released from the same amount of TS2 cells in mono-culture was slightly higher than that in dialysis co-culture conditions. Overall, this study shows that plant allelochemicals may have the potential to reduce bloom toxicity by reducing the proportion of toxigenic cyanobacterial strains, and the effects of co-existing strains must be considered when assessing the effects of plant allelochemicals on target strains.

Escherichia coli (E. coli) is frequently used in assessment and regulation of recreational water quality, but it is a general fecal indicator that provides no information about fecal contamination source. Sewage-associated microorganisms and related marker genes have proven useful for microbial source tracking (MST) applications that link fecal contamination to host sources, but many MST marker genes are carried in taxa not used in regulatory contexts. A more direct connection with regulatory concerns, including human health risk and total maximum daily load (TMDL) assessments, could be accomplished with tools such as the human-associated marker genes of E. coli. We evaluated the performance of E. coli H8, H12, H14, and H24 marker genes for detection of domestic sewage at the E. coli isolate level in Florida. E. coli isolates (n = 1, 380) from reference fecal and wastewater samples were first tested by binary PCR for the presence of each H marker gene. H8 and H12 were >90% specific and sensitive for domestic sewage, while H14 and H24 were ≤86% specific. Therefore, quantitative PCR (qPCR) assays were used to quantify H8 and H12 marker genes at the sample level. Specificity values for the H8 and H12 qPCR assays were 96 and 93%, respectively, while both marker genes showed 100% sensitivity. H12 concentrations were tenfold lower in wastewater than H8 (~6–7 log10 gene copies (GC)/100 mL). H8 concentrations in wastewater and contaminated environmental water samples were correlated with the sewage-associated Bacteroides HF183 marker gene. This study suggests that E. coli H genes, and H8 in particular, can be useful for sewage contamination tracking and TMDL development in subtropical waters.

Water contamination by pathogenic bacteria is a global public health problem. Contamination of surface water utilized to irrigate food products, or for human consumption, causes outbreaks of foodborne and waterborne disease. Of these, those caused by diarrheagenic Escherichia coli (DEC) strains present substantial morbidity and mortality. The aim of this study was, therefore, to investigate the microbiological quality of surface water and the presence of DEC strains in different water bodies. A total of 472 water samples were collected from irrigation canal, dam, river, and dike water bodies from January through December 2015 in Sinaloa, a State located in Northwestern Mexico. Our studies demonstrated that 47.0% (222/472) of samples contained thermotolerant coliforms above permissive levels whereas E. coli strains were isolated from 43.6% (206/472). Among these E. coli isolates, DEC strains were identified in 14% (29/206) of samples including in irrigation canal (26/29) and river water (3/29) collected from the northern (83%) and central area (17%). Isolated DEC strains were classified as enteroaggregative E. coli (EAEC) 34.4% (10/29), enteropathogenic E. coli (EPEC) 31.0% (9/29), diffuse adherent E. coli (DAEC) 27.5% (8/29), and enterotoxigenic E. coli (ETEC) 6.8% (2/29). Moreover, 90% of isolated DEC strains exhibited resistance to at least one commonly prescribed antibiotic in Mexico whereas 17% were multi-drug resistant. In conclusion, the presence of DEC strains in surface water represents a potential source for human infection, and thus routine monitoring of DEC in surface water and other indirect affected areas should be considered at northwestern Mexico.

Background Mycobacterium leprae being an obligate intracellular parasite cannot be cultured in any artificial culture media but it has been shown to reside in wild armadillos in North America. Many studies suggested that M. leprae could be found in the environment and may have a role in continuing transmission of the disease. The exact role of the environment in the transmission dynamics is still speculative. The present study was undertaken to find out the presence of viable M. leprae around patients' environment like soil and water and association of free living pathogenic protozoa, Acanthamoeba which might play an important role in transmission of the disease. Methods Seven hundred soil and 400 water samples were collected from the surroundings of the houses of leprosy patients from endemic villages. Two hundred soil and 80 water samples were also collected from the surroundings of normal inhabitants from non-endemic villages as controls. These samples were screened for the presence of M. leprae and Acanthamoeba using DNA PCR. RNA was extracted from the PCR positive samples and Reverse Transcriptase - PCR targeting 16S rRNA gene region was performed for detection of viable M. leprae. Results We observed high PCR positivity in soil samples (218 out of 700; 31%) and water samples (73 out of 400; 18%). These samples when further screened for viability, it was observed that 106 soil samples (15% of total) and 34 water samples (8% of total) showed presence of 16S rRNA. We observed 18.3% of soil and 20.5% of water samples were PCR positive for Acanthamoeba. Soil samples from the control area, where no active leprosy case resided in the last 5 years, showed PCR positivity in 4 samples (2%) for M. leprae DNA in only soil samples with all water samples being negative. RT-PCR for all PCR positive soil samples was negative. Of the 106 soil samples positive for M. leprae RT-PCR, 30 samples were also positive for Acanthamoeba whereas out of 112 M. leprae RT-PCR negative but PCR positive samples only 10 samples were Acanthamoeba positive showing association of viability with presence of Acanthamoeba (p = .0021). Similarly, for water samples also, association of M. leprae viability with presence of Acanthamoeba was seen (p = .0009). Conclusion This study suggests that the surrounding environment (soil and water) of leprosy patients contain viable M. leprae and the viability has association with Acanthamoeba which may provide a protective niche for M. leprae. This could play an important role in the focal transmission of the disease.

We have critically assessed some of the dogmas in the microbiology of enhanced biological phosphorus removal (EBPR) and argue that the genus Tetrasphaera can be as important as Ca. Accumulibacter for phosphorus removal; and that proliferation of their competitors, the glycogen accumulating organisms, does not appear to be a practical problem for EBPR efficiency even under tropical conditions. An increasing number of EBPR-related genomes are changing our understanding of their physiology, for example, their potential to participate in denitrification. Rather than trying to identify organisms that adhere to strict phenotype metabolic models, we advocate for broader analyses of the whole microbial communities in EBPR plants by iterative studies with isolates, lab enrichments, and full-scale systems.

Prolific growth of pathogenic Legionella pneumophila within engineered water systems and premise plumbing, and human exposure to aerosols containing this bacterium results in the leading health burden of any water-related pathogen in developed regions. Ecologically, free-living amoebae (FLA) are an important group of the microbial community that influence biofilm bacterial diversity in the piped-water environment. Using fluorescent microscopy, we studied in-situ the colonization of L. pneumophila in the presence of two water-related FLA species, Willaertia magna and Acanthamoeba polyphaga in drinking water biofilms. During water flow as well as after periods of long-stagnation, the attachment and colonization of L. pneumophila to predeveloped water-biofilm was limited. Furthermore, W. magna and A. polyphaga showed no immediate interactions with L. pneumophila when introduced to the same natural biofilm environment. A. polyphaga encysted within 5–7 d after introduction to the tap-water biofilms and mostly persisted in cysts till the end of the study period (850 d). W. magna trophozoites, however, exhibited a time delay in feeding on Legionella and were observed with internalized L. pneumophila cells after 3 weeks from their introduction to the end of the study period and supported putative (yet limited) intracellular growth. The culturable L. pneumophila in the bulk water was reduced by 2-log over 2 years at room temperature but increased (without a change in mip gene copies by qPCR) when the temperature was elevated to 40 °C within the same closed-loop tap-water system without the addition of nutrients or fresh water. The overall results suggest that L. pneumophila maintains an ecological balance with FLA within the biofilm environment, and higher temperature improve the viability of L. pneumophila cells, and intracellular growth of Legionella is possibly cell-concentration dependent. Observing the preferential feeding behavior, we hypothesize that an initial increase of FLA numbers through feeding on a range of other available bacteria could lead to an enrichment of L. pneumophila, and later force predation of Legionella by the amoeba trophozoites results in rapid intracellular replication, leading to problematic concentration of L. pneumophila in water. In order to find sustainable control options for legionellae and various other saprozoic, amoeba-resisting bacterial pathogens, this work emphasizes the need for better understanding of the FLA feeding behavior and the range of ecological interactions impacting microbial population dynamics within engineered water systems.

Ultrafiltration concentration of microorganisms in large volume water samples containing high levels of particulate matter was evaluated in a proof of concept study. The organisms tested were Bacillus atrophaeus subspecies globigii spores and MS2 bacteriophage. To produce the large volume samples, fresh water sediment of a known particle size was added to 51 liters of tap water. Five different concentrations of particulate matter were studied: 0, 50, 100, 150 and 750 mg solids/L. The concentration procedure used a dialysis filter as the ultrafilter configured for axial flow, either with or without recirculation. The target number of organisms spiked was 1 × 10(5) of either spores or bacteriophage per 51 liters. After concentration, the filters were dissected to retrieve the fibers which were then washed using surfactant solution which was then analyzed for the target organisms. Two washes of the filter fibers were carried out sequentially. For axial flow with recirculation, the first wash produced statistically greater recovery of B. globigii spores (26 – 40 % of spike) compared to the second wash (8 – 13% of spike). Total recovery (the sum of the recoveries for the first and second washes) ranged from 35 to 53 %. Recovery increased as the solids level increased from 0 to 150 mg solids/L. Recovery at the 100 and 150 mg solids/L loadings was statistically higher at the P < 0.05 level than recovery at 0 mg/L solids. At 150 mg solids/L, axial flow without recirculation (dead end) yielded lower recovery than axial flow with recirculation, however the difference was not significant at the P < 0.05 level. Recovery of B. globigii at 750 mg solids/L averaged 38% using dead end axial flow. The average recovery of MS2 bacteriophage was 45 % at a solids concentration of 150 mg/L using axial flow with recirculation. PhiX174 and Phi8 were also studied, however these bacteriophage appeared to be inactivated in the matrix of concentrated wash water. One hundred liters of water containing 750 mg solids/L was concentrated using dead end axial flow, and only minimal problems with filter clogging were observed. Results described herein suggest axial flow ultrafiltration is an effective concentration method for microorganisms in water containing high levels of particulate matter.

We investigated inflow of a wastewater treatment plant and sediment of an urban lake for the presence of Clostridioides difficile by cultivation and PCR. Among seven colonies we sequenced the complete genomes of three: two non-toxigenic isolates from wastewater and one toxigenic isolate from the urban lake. For all obtained isolates, a close genomic relationship with human-derived isolates was observed. (C) 2019 Elsevier Ltd. All rights reserved.

In this study, the spatial and seasonal bacterioplankton community dynamics were investigated in the main channel of the Middle Route of the South-to-North Water Diversion Project (MRP) using Illumina HiSeq sequencing. Water samples were collected in spring and summer from south to north at eight water quality monitoring stations, respectively. The results showed that seasonal changes had a more pronounced effect on the bacterioplankton community compositions (BCCs) than spatial variation. The diversity analysis results indicated that samples of summer have more operational taxonomic units (OTUs), higher richness and diversity than those in spring. The main phyla, Proteobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria and Chloroflexi, displayed significant differences (P 0.05) between spring and summer in the main channel. The Redundancy analysis (RDA) targeting all samples indicated that specific conductivity (SPC), dissolved oxygen (DO), pH and temperature (T) might be key factors in driving BCCs, while trophic status showed no significant correlation (P 0.05). The present study provides important insights into the potential ecological roles of specific taxa in the new artificial ecosystem and it offers reference for studies on ecosystem succession of other giant interbasin water diversion project in the world.

Current metagenomic tools allow the recovery of microbial genomes directly from the environment. This can be accomplished by binning metagenomic contigs according to their coverage and tetranucleotide frequency, followed by an estimation of the bin quality. The public availability of bioinformatics tools, together with the decreasing cost of next generation sequencing, are democratizing this powerful approach that is spreading from specialized research groups to the general public. Using metagenomes from hypersaline environments, as well as mock metagenomes composed of Archaea and Bacteria frequently found in these systems, we have analyzed the advantages and difficulties of the binning process in these extreme environments to tackle microbial population diversity. These extreme systems harbor relatively low species diversity but high intraspecific diversity, which can compromise metagenome assembly and therefore the whole binning process. The main goal is to compare the output of the binning process with what is previously known from the analyzed samples, based on years of study using different approaches. Several scenarios have been analyzed in detail: (i) a good quality bin from a species highly abundant in the environment; (ii) an intermediate quality bin with incongruences that can be solved by further analyses and manual curation, and (iii) a low-quality bin to investigate the failure to recover a very abundant microbial genome as well as some possible solutions. The latter can be considered the “great metagenomics anomaly” and is mainly due to assembly problems derived from the microdiversity of naturally co-existing populations in nature., The help of the owners of the Salines d'es Trenc and Bras del Port and the Fundación Valle Salado de Añana/Añanako Gatz Harana is greatly appreciated. This research was supported by the Spanish Ministry of Economy project CLG2015_66686-C3-3 (to JA), which also included European Regional Development Fund (FEDER) funds.

Made available in DSpace on 2019-10-04T12:35:30Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) USDA-ARS CRIS Project Sensitive detection of Cryptosporidium oocysts is important because the protozoan can cause clinical infection in humans at extremely low numbers. In the present study, 1.5 x 10(2), 1.0 x 10(3), or 1.0 x 10(4)C. parvum oocysts were spiked into 101 of source or finished water in triplicate followed by recovery using Envirochek HV sampling capsules. One subsample of the recovered oocysts was analyzed by commercial immuno fluorescence assay (IFA), while a second subsample was subjected to DNA-RNA extraction, followed by RT-PCR using primers directed to the gene encoding Cryspovirus capsid. IFA analysis of Envirochek filter eluates of finished water detected oocysts at all 3 C. parvum oocyst doses, but only at the 1.0 x 10(3) and 1.0 x 10(4) doses in source water. Cryspovirus RT-PCR appeared to offer greater sensitivity than IFA because C. parvum oocysts were detected using this molecular technique in both source and finished water concentrates at all 3 spiking levels. A linear relationship was observed between log oocysts spiking dose and the relative intensity of the Cryspovirus RT-PCR signal for finished water, but not for source water. These data indicate that Cryspovirus RT-PCR is a sensitive method for detecting C. parvum oocysts in source and finished water. Univ Estadual Paulista, Coll Vet Med, Dept Clin Surg & Anim Reprod, Clovis Pestana 793, BR-16050680 Sao Paulo, Brazil ARS, Environm Microbial & Food Safety Lab, USDA, Beltsville, MD 20705 USA Univ Estadual Paulista, Coll Vet Med, Dept Clin Surg & Anim Reprod, Clovis Pestana 793, BR-16050680 Sao Paulo, Brazil FAPESP: 2015/07147-2 USDA-ARS CRIS Project: 8042-32000-100-00-D

We evaluated the diagnostic performances of two media (BCYE, MWY) on 951 Legionella-positive hospital water samples. MWY allowed detecting Legionella in 89.2% of samples, but in 10.8% (103/951) Legionella was found on BCYE plates only. In samples where Legionella was isolated with other micro-organisms (663/951), MWY was essential to detect the majority of positive samples (349/663, 52.6%), as fewer plates resulted unreadable; however, in those containing Legionella only, a higher frequency of positive samples was recorded with BCYE (94.8%, 273/288) compared to MWY (85.1%, 245/288). Considering the 484 concordant positive samples, overall Legionella counts were significantly higher on BCYE (P=0.0029), with 47% of samples showing higher counts on BCYE compared to MWY plates. Furthermore, discordant samples (positive on only one medium) showed different relative proportions between Legionella pneumophila and non-pneumophila, the latter being found more frequently on BCYE only (P=0.0296).Our findings confirm the appropriateness of the ISO 11731:2017 update.

The worldwide spread of toxin-producing and multi-drug resistant bacteria in water, food and the environment is considered a major threat to human health. Drinking water quality is controlled by inspection of fecal indicators presence whereby viable contaminants will be efficiently reduced by chlorination which is a common process for disinfection. However, the all-out efficiency is arguable, because bacterial regrowth has been documented after disinfection. In this study, we investigated the stability of Shiga toxin producing Escherichia coli (STEC) and β-lactamase expressing E. coli and Pseudomonas aeruginosa isolates, both equipped with multiple or single β-lactamase resistance genes. The aim of the study was to analyze the efficiency of chlorine (Cl2) disinfection against shigatoxigenic or β-lactamase producing bacteria. Cl2 reacts with the bacterial cells after first contact. Counts of antibiotic resistant E. coli were lower after short than upon extended Cl2 treatment. P. aeruginosa counts decreased moderately upon 15–60 min treatment with 1.2 mg Cl2/l, while cells adapted to tap water were not cultivable anymore. We assume that the bacterial physiology changed to a temporary non-cultivatable state at first Cl2 contact followed by resuscitation of some cells at later stages. STEC viability went down continuously at low Cl2 concentrations and these toxigenic E. coli isolates exhibited slightly increased stability to Cl2 treatment compared with non-toxigenic E. coli. Controlling the efficiency of disinfection, realistic counts of cultivatable cells are achieved after extended Cl2 action.

Increasing numbers of outbreaks caused by enterohemorrhagic Escherichia coli (EHEC) are associated with the consumption of contaminated fresh produce. The contamination of the plants may occur directly on the field via irrigation water, surface water, manure or fecal contamination. Suggesting a low infectious dose of 10 to 102 cells, internalization of EHEC into plant tissue presents a serious public health threat. Therefore, the ability of EHEC O157:H7 strain Sakai to adhere to and internalize into root tissues of the lamb's lettuce Valerianella locusta was investigated under the environmental conditions of a greenhouse. Moreover, the influence of the two adherence and colonization associated genes hcpA and iha was surveyed regarding their role for attachment and invasion. Upon soil contamination, the number of root-internalized cells of EHEC O157:H7 strain Sakai exceeded 102 cfu/g roots. Deletion of one or both of the adherence factor genes did not alter the overall attachment of EHEC O157:H7 strain Sakai to the roots, but significantly reduced the numbers of internalized bacteria by a factor of between 10 and 30, indicating their importance for invasion of EHEC O157:H7 strain Sakai into plant roots. This study identified intrinsic bacterial factors that play a crucial role during the internalization of EHEC O157:H7 strain Sakai into the roots of Valerianella locusta grown under the growth conditions in a greenhouse.

Bacteria adapt to their living environment forming organised biofilms. The survival strategy makes them more resistant to disinfectants, which results in acute biofilm-caused infections, secondary water pollution by biofilm metabolites and bio-corrosion. New, efficient and environmentally friendly strategies must be developed to solve this problem. Water soluble N-acetyl derivative of L-cysteine (NAC) is a non-toxic compound of mucolytic and bacteriostatic properties that can interfere with the formation of biofilms. However, it can also be a source of C and N for undesired microorganisms, as well as a reason for some adverse human health effects. Consequently, novel procedures are required, that would decrease the take-up of NAC but not reduce its antibacterial properties. We have grafted N-acetyl-l-cysteine onto linear poly(vinylsilsesquioxanes) and poly(methylvinylsiloxanes) via thiol-ene addition. Antibacterial activity of the obtained hybrid materials (respectively, NAC-Si-1 and NAC-Si-2) was determined against Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus strains. Native NAC inhibited growth of planktonic cells for the tested bacteria at concentration 0.25% w/v. Inhibition with equivalent solutions of the polymer derivatives was less effective due to the lack of SH groups. However, the tested polymers proved to be quite effective in eradication of mature biofilms. Treatment with 1% w/v emulsions of the hybrid polymers resulted in a significant reduction of viable cells in biofilm matrix despite the absence of thiol moieties. The effect was most pronounced for mature biofilms of S. aureus eradicated with NAC-Si-2.

Objectives Recent research has demonstrated that natural populations of bacteria carry large numbers of mobile genetic elements that may harbour antibiotic resistance determinants. This study aimed to investigate carbapenem resistance in Gram-negative bacteria isolated from natural environments in Bejaia (Algeria) and to determine the horizontal gene transfer potential of a subset of these antibiotic resistance genes (ARGs). Methods Antibiotic-resistant bacteria were isolated and the host was identified using MALDI-TOF/MS and 16S rRNA sequencing. ARG carriage was investigated by the double-disk synergy test, metallo-β-lactamase (MBL) production test and PCR screening for carbapenemase genes. Conjugation experiments were performed to determine potential ARG mobility. To identify ARGs, genomic libraries were constructed and functionally screened and inserts were sequenced. Results A total of 62 antibiotic-resistant strains isolated from soil and water samples were classified as belonging to the Enterobacteriaceae, Pseudomonadaceae, Xanthomonadaceae and Aeromonadaceae families. Four highly imipenem-resistant (MIC > 64 μg/mL) and cefotaxime-resistant (MIC > 8 μg/mL) clinically-relevant strains were selected for further characterisation. All four strains produced extended-spectrum β-lactamases, but MBL production was not confirmed. Imipenem and cefotaxime resistance was transferable to Escherichia coli but was not conferred by blaAmpC, blaIMP, blaNDM, blaKPC, blaOXA-48 or blaGES genes. Novel putative resistance mechanisms were identified, including a novel DHA β-lactamase conferring clinical resistance to cefotaxime. Conclusions The environment is a reservoir of carbapenem-resistant bacteria. Further investigation of the evolution and dissemination of antibiotic resistance in environmental bacteria is required in order to understand and prevent the emergence of resistance in the clinical environment.

Deep Sea sediment cores were collected from the surrounding of active volcanic Barren Island, Andaman & Nicobar Islands. A total of 24 halophilic eubacteria were isolated and identified based on their biochemical and 16S rDNA sequences. Three major classes ( Gamma-Proteobacteria , Alpha - Proteobacteria and Bacilli ) of bacteria were detected in the deep sea sediments of active volcanic Barren Island. Among those, 37% of isolates exhibited antimicrobial activity against all tested Gram positive and Gram negative clinical pathogens. 60% of isolates revealed the presence of either PKS or NRPS genes and 65% isolates disclosed medium to higher level of cytotoxicity in MB-231 breast cancer cell line. Majority of the isolates revealed excellent potential for bioprospecting of novel byproducts with industrial and pharmaceutical importance.

Waterborne pathogens are a global concern for public health worldwide. Despite continuing efforts to maintain water safety, water quality is still affected by deterioration and pollution. Legionella pneumophila colonizes man-made water systems and can infect humans causing Legionnaire's disease (LD), pneumonia. The prevention of LD is a public health issue and requires specific systems to control and detect these microorganisms. Culture plate is the only technique currently approved, but requires more than 10 days to obtain results. A rapid test that inform in hours about the presence of Legionella pneumophila in water samples will improve the control of this pathogen colonization. In order to control colonization by L. pneumophila we developed a membrane filter method to capture and immunodetect this microorganism in water samples. This membrane filter is used to retain the bacteria using a nitrocellulose disc inside a home-made cartridge. Subsequently we perform the immunodetection of the bacteria retained in the nitrocellulose (blocking, antibody incubation, washings and developing). On comparing our test with the gold-standard, the most important finding is the considerably reduction in time maintaining the same detection limit. This rapid test is easily automated for L. pneumophila detection allowing a comprehensive surveillance of L. pneumophila in water facilities and reducing the variability in the analyses due to the low need for manipulation. Moreover, corrective measures may be applied the same day of the analysis. This method considerably reduces the detection time compared with the conventional, gold-standard detection culture method that requires more than 10 days, being decisive to prevent outbreaks.

Objectives The aim of this study was to monitor the spread, persistence and antibiotic resistance patterns of Legionella spp. strains found in a hospital water distribution system. These environmental studies are intended to help detect the presence of antibiotic resistant strains before they infect patients. Methods: Antimicrobial surveillance tests were performed at 27 different sampling points of the water network of a large Spanish hospital over two years. Water samples were screened for Legionella according to ISO 11731:2007. Legionella spp. isolates were identified by serotyping and by mass spectrometry (MALDI-ToF). Epidemiological molecular typing was done by Pulse-Field Gel Electrophoresis (PFGE) and by Sequence-Based Typing (SBT). Antibiotic susceptibility tests were performed using disk diffusion and ETEST®. Results Legionella spp. were recurrently isolated for 2 years. All isolates belonged the same group, L. pneumophila serogroups 2–14. Isolates were all attributed by SBT to sequence type (ST) ST328, although PFGE revealed 5 different patterns. No significant change in antibiotic susceptibility could be observed for this study period, irrespectively of the method used. Conclusion Colonization of water systems by Legionella spp. is still occurring, although all the prevention rules were strictly followed. Antibiotic resistance monitoring may help us to find resistance in bacteria with environmental reservoirs but difficult to isolate from patients. The knowledge of the antibiotic susceptibility in environmental strains may help us to predict changes in clinical strains. This study might also help reconsidering Legionnaires’ disease (LD) diagnostic methods. L. pneumophila serogroups 2–14 present all along the time of the investigation in the water distribution system can cause LD. However, they may not be detected by routine urine tests run on patients, thereby missing an ongoing LD infection.

The emergence of antibiotic resistance represents a major threat to human health. In this work we investigated the elimination of antibiotic resistant bacteria (ARB) by solar light and solar photo-Fenton processes. As such, we have designed an experimental plan in which several bacterial strains (Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae) possessing different drug-susceptible and -resistant patterns and structures (Gram-positive and Gram-negative) were subjected to solar light and the photo-Fenton oxidative treatment in water. We showed that both solar light and solar photo-Fenton processes were effective in the elimination of ARB in water and that the time necessary for solar light disinfection and solar photo-Fenton disinfection were similar for antibiotic-susceptible and antibiotic-resistant strains (mostly 180-240 and 90-120 min, respectively). Moreover, the bacterial structure did not significantly affect the effectiveness of the treatment. Similar regrowth pattern was observed (compared to the susceptible strain) and no development of bacteria with higher drug-resistance values was found in waters after any treatment. Finally, both processes were effective to reduce AR genes (ARGs), although solar photo-Fenton was more rapid than solar light. In conclusion, the solar photo-Fenton process ensured effective disinfection of ARB and elimination of ARGs in water (or wastewater) and is a potential mean to ensure limitation of ARB and ARG spread in nature. (C) 2018 Elsevier Ltd. All rights reserved.

Viral contamination can compromise the safety of water utilized for direct consumption, produce irrigation, and postharvest washing of produce. Zero-valent iron (ZVI) is used commercially for chemical remediation of water and has been demonstrated to remove some biological contaminants from water in laboratory and field studies. This study investigated the efficacy of ZVI to remove human norovirus surrogates, Tulane virus (TV) and murine norovirus (MNV), from water and to characterize the reversibility and nature of viral association with ZVI. Genomic material of TV and MNV recovered from the effluent of inoculated water treatment columns containing a 1:1 mixture of ZVI and sand was 2 and 3 log, respectively, less than that recovered from the effluent of treatment columns containing only sand. Elution buffers (citrate buffers, pH 4 and 7, and virus elution buffer, pH 9.5, with and without added 1 M NaCl) did not increase recovery of infectious TV and MNV from ZVI as compared with elution with water alone. TV-inoculated lettuce washed with water in the presence of ZVI yielded 1.5 to 2 log fewer infectious TV from washwater as compared with lettuce washed with water alone or in the presence of sand. These data demonstrate the enhanced removal of human norovirus surrogates, TV and MNV, from water by ZVI and provide indications that unrecovered viruses are not readily disassociated from ZVI by buffers of various pH and ionic strength. These findings warrant further investigation into larger-scale simulations of water remediation of viral contaminants for potential application in the treatment of water used for drinking, irrigation, and food processing.

Algicidal bacteria associated with Karenia mikimotoi have been isolated, yet the distribution of the algicidal bacteria has been rarely studied. Here, we postulated and demonstrated that terrestrial environment harbors diverse algicidal bacteria, which can survive in seawater along water flowing into marine and suppress Karenia mikimotoi. In summary, 9 and 5 bacteria with algicidal activity on Karenia mikimotoi were isolated from seawater and estuarine soil, respectively. Similar with the marine bacteria (Alteromonas sp., Halomonas sp., Marinobacter sp., Paracoccus sp., Rhodobacteraceae, Idiomarina sp.), the soil strains (Pseudoalteromonas sp. and Flavobaterium sp.) showed high mortality in Karenia mikimotoi with the inhibitory rate of 87% and 93.5%, respectively, after two days co-cultivation. Algicidal activity of the two strains was detected in the cell-free filtrate not in bacterial cells. The results suggest that algicidal bacteria associated with Karenia mikimotoi widely exist in terrestrial and marine environments, and have application potential on controlling Karenia mikimotoi.

Bacteriophages are among the most abundant and diverse biological units in the biosphere. They have contributed to our understanding of the central dogma of biology and have been instrumental in the evolutionary success of bacterial pathogens. In contrast to our current understanding of marine viral communities, the soil virome and its function in terrestrial ecosystems has remained relatively understudied. Here, we examine, in a comparative fashion, the knowledge gathered from studies performed in soil versus marine settings. We address the information with respect to the abundance, diversity, ecological significance, and effects of, in particular, bacteriophages on their host's evolutionary trajectories. We also identify the main challenges that soil virology faces and the studies that are required to accompany the current developments in marine settings.

International audience; A novel anaerobic methylotrophic halophilic methanogen strain SLHTYRO T was isolated from a deep hypersaline anoxic basin called "Tyro" located in the Eastern Mediterranean Sea. Cells of SLHTYRO T were motile cocci. The strain SLHTYRO T grew between 12 and 37 °C (optimum 30 °C), at pH between 6.5 and 8.2 (optimum pH 7.5) and salinity from 45 to 240 g L −1 NaCl (optimum 135 g L −1). Strain SLHTYRO T was methylotrophic methanogen able to use methylated compounds (trimethylamine, dimethylamine, monomethylamine and methanol). Strain SLHTYRO T was able to grow at in situ hydrostatic pressure and temperature conditions (35 MPa, 14 °C). Phylogenetic analysis based on 16S rRNA gene and mcrA gene sequences indicated that strain SLHTYRO T was affiliated to genus Methanohalophilus within the order Methanosarcinales. It shared >99.16% of the 16S rRNA gene sequence similarity with strains of other Methanohalophilus species. Based on ANIb, AAI and dDDH measurements, and the physiological properties of the novel isolate, we propose that strain SLHTYRO T should be classified as a representative of a novel species, for which the name Methanohalophilus profundi sp. nov. is proposed; the type strain is SLHTYRO T (=DSM 108854 = JCM 32768 = UBOCC-M-3308).

Fecal contamination of surface water is commonly evaluated by quantification of bacterial or viral indicators such as Escherichia coli and coliphages, or by direct testing for pathogens such as enteric viruses. Retention of fecally derived organisms in biofilms and sediments is less frequently considered. In this study, we assessed the distribution of E. coli, somatic coliphages, and enteric viruses including human adenovirus (HAdV), enterovirus (EV), norovirus genogroup GII (NoV GII) and group A rotavirus (RoV) in an urban river environment in Germany. 24 samples each of water, epilithic biofilms and sediments were examined. E. coli and somatic coliphages were prevalent not only in the flowing water, but also in epilithic biofilms and sediments, where they were accumulated compared to the overlying water. During enhanced rainfall, E. coli and coliphage concentrations increased by approximately 2.5 and 1 log unit, respectively, in the flowing water, whereas concentrations did not change significantly in epilithic biofilms and sediments. The occurrence of human enteric viruses detected by qPCR was higher in water than in biofilms and sediments. 87.5% of all water samples were positive for HAdV. Enteric viruses found less frequently were EV, RoV and NoV GII in 20.8%, 16.7% and 8.3% of the water samples, respectively. In epilithic biofilms and sediments, HAdV was found in 54.2% and 50.0% of the samples, respectively, and EV was found in 4.2% of both biofilm and sediment samples. RoV and NoV GII were not detected in any of the biofilms and sediments. Overall, the prevalence of enteric viruses was in the order of HAdV > EV > RoV ≥ NoV GII. In conclusion, epilithic biofilms and sediments can be reservoirs for fecal indicators and enteric viruses and thus should be taken into consideration when assessing microbial pollution of surface water environments.

Currently the gold standard for validating the efficiency of UV-C disinfection of water is biodosimetry. This method allows the assessment of operating conditions under which a UV reactor can be operated safely, but relies on the addition of a live reference organism into the water matrix. Whereas biodosimetry enables off-line testing prior to installation of UV reactors into water treatment plants, it is not compatible with on-site tests. To measure disinfection efficiency, we lent a principle for the assessment of genotoxicity originally developed for cancer research. The approach utilizes the amplification of gene sequences of different lengths by quantitative PCR. The principle is based on the fact that UV-C-induced DNA damage suppresses the PCR amplification of long DNA sequences stronger than it does in case of short DNA sequences. Comparison of threshold cycles of samples taken before and after UV disinfection allows the calculation of the number of PCR-effective DNA lesions. A clear dose-response relationship was obtained with laboratory-grown and -irradiated E. coli suspensions. The extent of DNA damage was less pronounced in bacteria naturally contained in drinking water from works applying UV-C, which was presumably due to distinct UV susceptibilities of different bacterial species. Indeed, DNA sequencing of bacterial populations before and after reactor passage revealed a strong shift of amplifiable sequences from proteobacteria towards highly UV-resistant actinobacteria . Interestingly, the original water microbiome was quickly re-established after passage through a clean water tank suggesting that UV exposure only perturbed the bacterial community for a short time.

The aim of the present study was to verify the antimicrobial susceptibility profile and virulence factors of Vibrio parahaemolyticus isolated from water and bivalve mollusks. A high percentage of V. parahaemolyticus was isolated in natura, processed bivalves tissues, and surrounding water (75%, 20%, and 59%, respectively). The most potential virulence phenotype in V. parahaemolyticus isolates was amylase production (97%) followed by DNase (83%), phospholipase (70%), β-hemolytic activity (57%). The tdh and trh genes were not detected. Besides, a high antimicrobial resistance was observed for ampicillin (97%), minimum inhibitory concentration [MIC] = 400 μg and cephalothin (93%, MIC ≤ 100 μg). The absence of expression of tdh and trh virulence genes excluded the toxigenic potential of V. parahaemolyticus isolates; however, the high prevalence of antimicrobial resistance among the environmental strains is a risk to human health.

The application of tandem MALDI-TOF MS screening with 16S rRNA gene sequencing of selected isolates has been demonstrated to be an excellent approach for retrieving novelty from large-scale culturing. The application of such methodologies in different hypersaline samples allowed the isolation of the culture-recalcitrant Salinibacter ruber second phylotype (EHB-2) for the first time, as well as a new species recently isolated from the Argentinian Altiplano hypersaline lakes. In this study, the genome sequences of the different species of the phylum Rhodothermaeota were compared and the genetic repertoire along the evolutionary gradient was analyzed together with each intraspecific variability. Altogether, the results indicated an open pan-genome for the family Salinibacteraceae, as well as the codification of relevant traits such as diverse rhodopsin genes, CRISPR-Cas systems and spacers, and one T6SS secretion system that could give ecological advantages to an EHB-2 isolate. For the new Salinibacter species, we propose the name Salinibacter altiplanensis sp. nov. (the designated type strain is AN15T = CECT 9105T = IBRC-M 11031T). Fil: Viver, Tomeu. Consejo Superior de Investigaciones Cientificas. Instituto Mediterraneo de Estudios Avanzados; España Fil: Orellana, Luis. Instituto Tecnológico de Georgia; Estados Unidos. Georgia Institute of Techology; Estados Unidos Fil: González Torres, Pedro. Universidad de Alicante; España Fil: Díaz, Sara. Consejo Superior de Investigaciones Cientificas. Instituto Mediterraneo de Estudios Avanzados; España Fil: Urdiain, Mercedes. Consejo Superior de Investigaciones Cientificas. Instituto Mediterraneo de Estudios Avanzados; España Fil: Farias, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina Fil: Benes, Vladimir. European Molecular Biology Laboratory Heidelberg; Fil: Kaempfer, Peter. Justus Liebig University Giessen; Fil: Shahinpei, Azadeh. University Of Tehran; Irán Fil: Ali Amoozegar, Mohammad. University Of Tehran; Irán Fil: Amann, Rudolf. Max Planck Institut Für Marine Mikrobiologie; Fil: Antón, Josefa. Universidad de Alicante; España Fil: Konstantinidis, Konstantinos T.. Georgia Institute of Techology; Estados Unidos. Instituto Tecnológico de Georgia; Estados Unidos Fil: Rosselló Móra, Ramon. Consejo Superior de Investigaciones Cientificas. Instituto Mediterraneo de Estudios Avanzados; España

The current study was conducted to isolate the marine bioluminescent bacteria from the blue swimmer crab Portunus pelagicus along the Thondi Coast. Morphological, biochemical and molecular characterization techniques including 16S rRNA gene sequencing and phylogenetic analyses confirmed that the isolated strain was Vibrio harveyi. Experiments were further carried out at different temperatures and various time intervals and the results revealed a significant effects of high temperature and extended time duration on elimination of V. harveyi. Hence, high temperature treatments could facilitate the suppression of V. harveyi from sea food and thereby, preventing food borne infections during human consumption.

Background Pseudomonas aeruginosa is an important nosocomial pathogen that commonly colonizes hospital water supplies, including in taps and sinks. We report the transmission of P. aeruginosa from water to patients in a clinical hematology setting. Methods P. aeruginosa from water samples were compared to clinical isolates from hematology ward patients, via molecular typing (pulsed field gel electrophoresis). Results P. aeruginosa cultured from blood cultures from 3 patients was indistinguishable from water strains, by molecular typing. Based on infection control inspections, the transmission event was surmised to be due to cleaning of equipment, specifically an infusion therapy procedure tray used to transport intravenous drugs to patients, with water from an outlet colonized by P. aeruginosa. Conclusion We show the importance of holistic factors, such as disposal of patient waste water, cleaning of tap outlets, and cleaning of medical equipment, in the transmission of P. aeruginosa, and demonstrate that the role of waterborne transmission of this organism in a hematology setting cannot be overlooked. We suggest that appropriate management of water, including both holistic and engineering interventions, is needed to stop transmission of P. aeruginosa from water to patients.

The Produce Safety Rule of the U.S. Food Safety Modernization Act includes restrictions on the use of agricultural water of poor microbiological quality. Mitigation options for poor water quality include the application of an irrigation-to-harvest interval of

Microorganisms are globally distributed but new evidence shows that the microbial structure of their communities can vary due to geographical location and environmental parameters. In this study, 50 samples including brines and sediments from Europe, Spanish-Atlantic and South America were analysed by applying the operational phylogenetic unit (OPU) approach in order to understand whether microbial community structures in hypersaline environments exhibited biogeographical patterns. The fine-tuned identification of approximately 1000 OPUs (almost equivalent to ?species?) using multivariate analysis revealed regionally distinct taxa compositions. This segregation was more diffuse at the genus level and pointed to a phylogenetic and metabolic redundancy at the higher taxa level, where their different species acquired distinct advantages related to the regional physicochemical idiosyncrasies. The presence of previously undescribed groups was also shown in these environments, such as Parcubacteria, or members of Nanohaloarchaeota in anaerobic hypersaline sediments. Finally, an important OPU overlap was observed between anoxic sediments and their overlaying brines, indicating versatile metabolism for the pelagic organisms. Fil: Mora Ruiz, M.del R.. Consejo Superior de Investigaciones Cientificas. Instituto Mediterraneo de Estudios Avanzados; España Fil: Cifuentes, Ana. Consejo Superior de Investigaciones Cientificas. Instituto Mediterraneo de Estudios Avanzados; España Fil: Font Verdera, Francisca. Consejo Superior de Investigaciones Cientificas. Instituto Mediterraneo de Estudios Avanzados; España Fil: Pérez Fernández, César A.. Universidad de Puerto Rico; Puerto Rico Fil: Farias, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; Argentina Fil: González, Bernardo V.. Universidad Adolfo Ibanez; Chile Fil: Orfila, Alejandro. Consejo Superior de Investigaciones Cientificas. Instituto Mediterraneo de Estudios Avanzados; España Fil: Rosselló Móra, Ramón. Consejo Superior de Investigaciones Cientificas. Instituto Mediterraneo de Estudios Avanzados; España

Effects of sulfadiazine and ciprofloxacin on the occurrence of free-living and particle-associated opportunistic pathogens in bulk water of simulated drinking water distribution systems (DWDSs) were investigated. It was found that sulfadiazine and ciprofloxacin greatly promoted the occurrence of opportunistic pathogens including Pseudomonas aeruginosa, Legionella pneumophila, Mycobacterium avium and its broader genus Mycobacterium spp., as well as the amoebae Acanthamoeba spp. and Hartmanella vermiformis, in bulk water of DWDSs. Moreover, sulfadiazine and ciprofloxacin exhibited much stronger combined effects on the increase of these opportunistic pathogens. Based on the analysis of the antibiotic resistance genes (ARGs) and extracellular polymeric substances (EPS), it was verified that EPS production was increased by the antibiotic resistant bacteria arising from the effects of sulfadiazine/ciprofloxacin. The combined effects of sulfadiazine and ciprofloxacin induced the greatest increase of EPS production in DWDSs. Furthermore, the increased EPS with higher contents of proteins and secondary structure β-sheet led to greater bacterial aggregation and adsorption. Meanwhile, large numbers of suspended particles were formed, increasing the chlorine-resistance capability, which was responsible for the enhancement of the particle-associated opportunistic pathogens in bulk water of DWDSs with sulfadiazine/ciprofloxacin. Therefore, sulfadiazine and ciprofloxacin promoted the occurrence of particle-associated opportunistic pathogens in bulk water of DWDSs due to the role of EPS produced by the bacteria with ARGs.

It is well known that the pathogenic multidrug resistant microbes are highly distributed and transferred to human through contaminated food and water. Advanced technologies have been developed for controlling these microbes using synthesized nanoparticles. In this study, biosynthesized silver nanoparticles were prepared, characterized and integrated with two synthetic and natural polymers. The polymers-silver nanoparticles were characterized using SEM, FTIR and mechanical properties of the membrane synthesized from either polymers with/without nanosilver. Both of pathogenic bacteria and yeast were tested for their resistance against 10 different antibiotics. The pathogens showed high resistance against 9 antibiotics and only one was recorded as potent. The cytotoxicity of nanosilver integrated polymers were tested against Hamster kidney cells and Human skin fibroblast cells, and the non-cytotoxic dose was checked for its antimicrobial activity against the selected pathogens. The obtained results in this study confirm that the using of the nanomaterials in safe doses could be a good substitution for biogenic antibiotics and chemicals used in water treatment. Moreover, the beads which were used in this study could be lasts for long period in water treatment station with high antibacterial capacity, in addition, it can be gathered easily at the end of the run.

The wide use of antibiotics in aquaculture for prophylactic and therapeutic purposes can potentially lead to the prevalence of antibiotic resistance genes (ARGs). This study reports for the first time the profile of ARGs from effluents of coastal aquaculture located in South Jeolla province and Jeju Island, South Korea. Using quantitative PCR (qPCR), twenty-two ARGs encoding tetracycline resistance ( tetA , tetB , tetD , tetE , tetG , tetH , tetM , tetQ , tetX , tetZ , tetBP ), sulfonamide resistance ( sul1 , sul2 ), quinolone resistance ( qnrD , qnrS , aac(6′)-Ib-cr ), β-lactams resistance ( bla TEM , bla CTX , bla SHV ), macrolide resistance ( ermC ), florfenicol resistance ( floR ) and multidrug resistance ( oqxA ) and a class 1 integrons-integrase gene ( intI1 ) were quantified. In addition, Illumina Miseq sequencing was applied to investigate microbial community differences across fish farm effluents. Results from qPCR showed that the total number of detected ARGs ranged from 4.24 × 10 −3 to 1.46 × 10 −2 copies/16S rRNA gene. Among them, tetB and tetD were predominant, accounting for 74.8%–98.0% of the total ARGs. Furthermore, intI1 gene showed positive correlation with tetB , tetD , tetE , tetH , tetX , tetZ tetQ and sul1 . Microbial community analysis revealed potential host bacteria for ARGs and intI1 . Two genera, Vibrio and Marinomonas belonging to Gammaproteobacteria , showed significant correlation with tetB and tetD , the most dominant ARGs in all samples. Also, operational taxonomic units (OTUs)-based network analysis revealed that ten OTUs, classified into the phyla Proteobacteria , Cyanobacteria/Chloroplast , Bacteroidetes , Verrucomicrobia and an unclassified phylum, were potential hosts of tetracycline resistance genes (i.e., tetA , tetG , tetH , tetM , tetQ and tetZ ). Further systematic monitoring of ARGs is warranted for risk assessment and management of antibacterial resistance from fish farm effluents.

The risk of pathogen exposure in recreational water is a concern worldwide. Moreover, suspended particles, as ideal shelters for pathogens, in these waters also need attention. However, the risk caused by the pathogen-particle attachment is largely unknown. Accordingly, water samples in three recreational lakes in Beijing were collected and separated into free-floating (FL, 0.22–5 μm) and particle-attached (PA, > 5 μm) fractions. Next-generation sequencing (NGS) was employed to determine the diversity of genera containing pathogens, and quantitative PCR (qPCR) was used to assess the presence of genes from Escherichia coli ( uidA ), Salmonella enterica ( invA ), Aeromonas spp. ( aerA ), Mycobacterium avium ( 16S ) and Pseudomonas aeruginosa ( oaa ). The NGS results showed stable pathogen genera composition distinctions between the PA and FL fractions. Some genera, such as Aeromonas and Mycobacterium , exhibited higher abundances in the PA fractions. qPCR revealed that most of the gene concentrations were higher within particles than were FL fractions. Some gene levels showed correlations with the particle concentrations and lake nutrient levels. Further quantitative microbial risk assessment (QMRA) of selected strains ( S . enterica and M . avium ) indicated a higher health risk during secondary contact activities in lakes with more nutrients and particles. We concluded that suspended particles (mainly composed of algae) in urban recreational water might influence the pathogen distribution and could serve as reservoirs for pathogen contamination, with important management implications.

One of the most important resistance mechanisms in Gram-negative bacteria today is the production of enzymes causing resistance to cephalosporin and carbapenem antibiotics. The spread of extended-spectrum β-lactamases (ESBL)- and carbapenemase- producing Gram-negative bacteria is an emerging global public health problem. The aim of the present study was to (i) assess the prevalence of carbapenem-resistant bacteria (CRB) and ESBL-producing strains in sewage effluents from two major hospitals in Singapore, (ii) characterize the isolated strains and (iii) identify some of the ESBL and carbapenemase genes responsible for the resistance. CHROMagar ESBL and KPC plates were used to rapidly screen for ESBL-producing bacteria and those expressing reduced susceptibility to carbapenems, respectively. The abundance of ESBL-producers and CRB in hospital wastewater ranged between 10 3 and 10 6 CFU/mL. Out of the 66 isolates picked from ESBL and KPC plates, 95%, 82%, 82% and 76% were resistant to ceftriaxone, ceftazidime (3rd generation cephalosporin family), ertapenem and meropenem (carbapenem family), respectively. Among the resistant isolates, the most predominant taxa identified were Pseudomonas spp. (28.2%), Klebsiella spp. (28.2%), Enterobacter spp. (18.3%) and Citrobacter spp. (11.3%). PCR and sequencing analysis showed that the predominant β-lactamase genes were bla SHV (41.1%) followed by bla NDM-1 (35.6%), bla CTX (35.6%) and bla KPC (28.8%). The results of this study show a high prevalence of bacteria resistant to modern extended-spectrum cephalosporins and carbapenems and the presence of ESBL- and carbapenemase producers in hospital effluents. These findings support the need to improve management of hospital wastewater in order to minimize the spread of antimicrobial resistant microorganisms from this source.

Legionellae, i.e. Legionella pneumophila, are human bacterial hydrophilic facultative pathogens causing pneumonia (Legionnaires' disease). Free-living amoebae (FLA) can serve as natural hosts and thus as reservoirs of many amoebae-resistant bacteria. An encysted amoeba can contribute to the resistance of intracellular L. pneumophila to various chemical and physical treatments. Humans can be infected by droplets containing bacteria from an environmental source or human-made devices such as shower heads, bathtubs, air-conditioning units or whirlpools. In this study, we were investigating the presence of FLA and L. pneumophila in plumbing systems of healthcare facilities in Bratislava (Slovakia) by standard diagnostic methods, while the presence of L. pneumophila was verified also by MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) analysis. The results showed the occurrence of L. pneumophila and FLA in 62.26% and 66.4% of samples taken from four paediatric clinics, respectively. Both standard methods and MALDI-TOF MS showed comparable results and they can be successfully applied for the identification of L. pneumophila strains in environmental samples. Our approach could be useful for further monitoring, prevention and decreasing risk of Legionella infection also in other hospitals.

To establish the association between microbial indicators and the presence of foodborne pathogens in irrigation water, Escherichia coli was enumerated using two quantification methods (plate counts and PMA-qPCR) and presence/absence of pathogenic microorganisms, including five strains from the Shiga toxigenic E. coli (O157:H7, O26, O103, O111 and O145) and Salmonella spp. were evaluated. The results confirmed that surface water can be considered a microbial hazard when used for irrigation. The levels of viable E. coli were very similar to those of cultivable E. coli, except for irrigation water obtained from water reservoirs. Comparison between the E. coli counts in samples positive and negative for the presence of pathogenic bacteria for the evaluated water sources identified E. coli level of 2.35 log cfu/100 mL as a cut-off able to correctly predict positive and negative samples with 93% sensitivity and 66% specificity, respectively. Thus, for the samples with levels of E. coli under 2.35 log cfu/100 mL (e.g., 2.24 log cfu/100 mL) there was a 90% probability that the samples were not contaminated with pathogenic microorganism in locations with similar prevalence. E. coli levels in irrigation water were affected by the ambient temperature confirming that water source and climate conditions should be taken into account by growers when designing a sampling program and the frequency of the monitoring to make a better and more efficient use of their resources.

International audience; Water networks in hospitals are frequently contaminated by opportunistic premise plumbing pathogens (OPPPs) leading to installation of antimicrobial filters on water points-of-use (POU) in order to limit patients' exposure.AIM:To assess the spread of OPPPs through secondary water routes (outside the plumbing system) in an adult haematology unit in which 52 out of 73 water POU were high risk for patients and protected by antimicrobial filters.METHODS:An observational audit identified six secondary water routes for which bacteria tracking and typing were performed in 315 surface samplings. Bacterial isolates were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and compared to the infra-species level by multiplex repetitive element sequence-based polymerase chain reaction and/or by restriction fragment length polymorphism in pulse-field gel electrophoresis.FINDINGS:Pseudomonas aeruginosa and Stenotrophomonas maltophilia, as well as non-pathogenic OPPP indicators, were detected in water collected upstream of antimicrobial filters. P. aeruginosa was the sole OPPP retrieved from tested surfaces (5.1%). The same clone of P. aeruginosa spread from water source to dry surfaces in the same room and cross-contaminated two sinks in different rooms. Three clones of non-pathogenic OPPP indicators spread more widely in different rooms.CONCLUSION:A strategy based on filtration of most (but not all) water POU in a haematology unit could be sufficient to limit the spread of OPPPs to the environment, provided a functional mapping of 'high-risk' POU has been undertaken. The residual spread of OPPPs and OPPP indicators linked to non-filtered water POU argues for careful monitoring of non-filtered water use.

Drinking water quality typically degrades after treatment during conveyance through the distribution system. Potential causes include biofilm growth in distribution pipes which may result in pathogen retention, inhibited disinfectant diffusion, and proliferation of bad tastes and odors. However, there is no standard method for direct measurement of biofilms or quantification of biofilm cells in drinking water distribution systems. Three methods are compared here for quantification of biofilm cells grown in pipe loops samplers: biofilm heterotrophic plate count (HPC), biofilm biovolume by confocal laser scanning microscopy (CLSM) and biofilm total cell count by flow cytometry (FCM) paired with Syto 9. Both biofilm biovolume by CLSM and biofilm total cell count by FCM were evaluated for quantification of the whole biofilms (including non-viable cells and viable but not culturable cells). Signal-to-background ratios and overall performance of biofilm biovolume by CLSM and biofilm total cell count by FCM were found to vary with the pipe material. Biofilm total cell count by FCM had a low signal-to-background ratio on all materials, indicating that further development is recommended before application in drinking water environments. Biofilm biovolume by CLSM showed the highest signal-to-background ratio for cement and cast iron, which suggests promise for wider application in full-scale systems. Biofilm biovolume by CLSM and Syto 9 staining allowed in-situ biofilm cell quantification thus elimination variable associated with cell detachment for quantification but had limitations associated with non-specific staining of cement and, to a lesser degree, auto-fluorescence of both cement and polyvinyl chloride materials. Due to variability in results obtained from each method, multiple methods are recommended to assess biofilm growth in drinking water distribution systems. Of the methods investigated here, HPC and CLSM and recommended for further development towards application in full-scale systems. HPC is a sample and widely applied method that quantifies viable culturable cells. CLSM analysis allows the elimination of experimental variables associated with cell detachment and affords the opportunity to evaluate biofilm components such as extracellular polymeric substances through the addition of specific probes. These two methods can be applied together to assess biofilms known to degrade treated water quality during conveyance in full-scale drinking water treatment systems. The significance of improved biofilm assessment methods for drinking water distribution systems lies in advancing understanding of biofilm growth and control mechanisms that may lead to improved water quality during conveyance and at the tap for greater public health protection.

A rapid method that provides information on the viability of organisms is needed to protect public health and ensure that remediation efforts following a release of a biological agent are effective. The rapid viability-polymerase chain reaction (RV-PCR) method combines broth culture and molecular methods to provide results on whether viable organisms are present in less than 15 h. In this study, a modified RV-PCR (mRV-PCR) method was compared to a membrane-filtration culture method for the detection of viable Bacillus spores in water matrices. Samples included small and large volumes of chlorine and non‑chlorine treated tap water. Large volume water samples (up to 100 L), were processed by ultrafiltration using a semi-automated waterborne pathogen concentrator, followed by centrifugation as a secondary concentration technique. The concentrated samples were analyzed by mRV-PCR and culture methods. The overall agreement between the mRV-PCR and culture methods when seed concentrations were greater than 10 spores per sample volume analyzed was 96%. The total time from the start of sample processing to the final sample result for the mRV-PCR method was decreased by approximately 2 h, in comparison to the previously published RV-PCR method because of the incorporation of shorter, more efficient primary and secondary concentration steps and a shorter DNA extraction technique. Overall, this study confirmed that RV-PCR is a promising approach for identifying viable Bacillus spores in small- and large-volume water samples and for producing results in less time than traditional culture methods.

Four strains of Planctomycetes, (JC636, JC649, JC650T, JC657T) which are all salt and alkali tolerant, pink coloured, with spherical to oval shaped, Gram-stain-negative, non-motile cells were isolated from different regions of Chilika lagoon, India. All strains have obligate requirement for N-acetylglucosamine (NAG) and share highest 16S rRNA gene sequence identity with members of the genus Tautonia ( 99.5%. Respiratory quinone for all the strains was MK6. Major fatty acids of all the strains were C18:1ω9c, C16:0 and C18:0. Major polar lipid of the strain JC650T was phosphatidylethanolamine, while, phosphatidylcholine and phosphatidylglycerol for strain JC657T. Spermidine was the only common polyamine for all the four strains. Strains JC657T, JC636 and JC649 shared highest phenotypic similarity along with 100% 16S rRNA gene sequence identity. Strains JC657T, JC636 and JC649 differed from strain JC650T phenotypically, chemotaxonomically and genotypically, thus belong to a different species. The genomic size of strain JC650T and JC657T are 7.06 Mb and 6.96 Mb with DNA G + C content of 63.9 and 62.7 mol%, respectively. Based on phylogenetic, genomic (ANI, AAI, POCP, dDDH), chemotaxonomic, physiological and biochemical characteristics, we conclude that strains JC650T and JC657T (together with strains JC636, JC649) belong to the genus Tautonia and constitute two novel species for which we propose the names Tautonia marina sp. nov., and Tautonia rosea sp. nov., respectively. These two novel species are represented by the type strains JC650T (=KCTC 72177T = NBRC 113885T) and JC657T (=KCTC 72597T = NBRC 113883T) respectively.

The abundance, phosphorus solubilizing ability and community composition of phosphorus solubilizing bacteria (PSB) attached on two bloom-forming cyanobacteria, Microcystis and Anabeana, were investigated in Guanqiao ponds in 2014 and Lake Chaohu in 2015 and 2016. Thirty organic phosphate-mineralizing bacteria (OPB) and eighteen inorganic phosphate-solubilizing bacteria (IPB) isolated from Guanqiao ponds and Lake Chaohu were identified. The community compositions of PSB attached on Microcystis and Anabeana were found to be entirely different. Some PSB were found to be shared by OPB and IPB, especially the species attached on Microcystis, such as Rhizobium sp. Compared to the PSB attached on Anabeana, the PSB attached on Microcystis showed the lower numbers, higher phosphorus solubilizing ability and extensive substrate adaptability. This indicated that the PSB were important for the growth of Microcystis through meeting soluble reactive phosphorus (SRP) demand, which was further supported by the data from Guanqiao ponds where succession process from Anabeana to Microcystis was recorded. All these facts can explain why the succession from Anabeana to Microcystis frequently occurred in shallow eutrophic lakes. Therefore, the attached PSB should be considered as a crucial contributor on algal growth, succession and collapse, depending on algal species.

The development of an intensive aquaculture industry has been accompanied by increasing environmental impacts, especially nitrogen pollution. In this study, a novel halophilic bacterium capable of heterotrophic nitrification and aerobic denitrification was isolated from mariculture water and identified as Bacillus litoralis N31. The efficiency of ammonium, nitrite and nitrate removal by N31 were 86.3%, 89.3% and 89.4%, respectively, after a 48-h cultivation in sole N-source medium with initial nitrogen approximately 20 mg/L. However, ammonium was removed preferentially, and no obvious nitrite accumulated during the simultaneous nitrification and denitrification process in mixed N-source media. The existence of hao, napA and nirS genes further proved the heterotrophic nitrification–aerobic denitrification capability of N31. The optimal conditions for ammonium removal were 30°C, initial pH 7.5–8.5, C/N ratio 5–20 and salinity 30–40‰, and the nitrification rate of N31 increased with increasing initial NH 4 + – N from 10 to 250 mg/L. Biosecurity assessment with shrimp indicated that strain N31 could be applied in the marine aquaculture industry safely for culture water remediation and effluent treatment.

Biofilm formation, loose deposit accumulation and water quality deterioration in drinking water distribution systems have been widely reported. However, the accumulation and distribution of harbored elements and microbes in the different niches (loose deposits, PVC-U biofilm, and HDPE biofilm) and their corresponding potential contribution to water quality deterioration remain unknown. This precludes an in-depth understanding of water quality deterioration and the development of proactive management strategies. The present study quantitatively evaluated the distribution of elements, ATP, Aeromonas spp., and bacterial communities in distribution pipes (PVC-U, D = 110 mm, loose deposit and biofilm niches) and household connection pipes (HDPE, D = 32 mm, HDPE biofilm niches) at ten locations in an unchlorinated distribution system. The results show that loose deposits in PVC-U pipes, acting as sinks, constitute a hotspot (highest total amount per meter pipe) for elements, ATP, and target bacteria groups (e.g., Aeromonas spp., Mycobacterium spp., and Legionella spp.). When drinking water distribution system niches with harbored elements and microbes become sources in the event of disturbances, the highest quality deterioration potential (QDP) is that of HDPE biofilm; this can be attributed to its high surface-to-volume ratio. 16s rRNA analysis demonstrates that, at the genus level, the bacterial communities in the water, loose deposits, PVC-U biofilm, and HDPE biofilm were dominated, respectively, by Polaromonas spp. (2–23%), Nitrosipra spp. (1–47%), Flavobacterium spp. (1–36%), and Flavobacterium spp. (5–67%). The combined results of elemental composition and bacterial community analyses indicate that different dominant bio-chemical processes might occur within the different niches—for example, iron-arsenic oxidizing in loose deposits, bio-calumniation in PVC-U biofilm, and methane oxidizing in HDPE biofilm. The release of 20% loose deposits, 20% PVC-U biofilm and 10% HDPE biofilm will cause significant changes of water bacterial community.

Background Viability quantitative Polymerase Chain Reaction (v-qPCR) is a recent analytical approach for only detecting live microorganisms by DNA amplification-based methods This approach is based on the use of a reagent that irreversibly fixes dead cells DNA. In this study, we evaluate the utility of v-qPCR versus culture method for Legionellosis risk management. Methods The present study was performed using 116 real samples. Water samples were simultaneously analysed by culture, v-qPCR and qPCR methods. Results were compared by means of a non-parametric test. Results In 11.6% of samples using both methods (culture method and v-qPCR) results were positive, in 50.0% of samples both methods gave rise to negative results. As expected, equivalence between methods was not observed in all cases, as in 32.1% of samples positive results were obtained by v-qPCR and all of them gave rise to negative results by culture. Only in 6.3% of samples, with very low Legionella levels, was culture positive and v-qPCR negative. In 3.5% of samples, overgrowth of other bacteria did not allow performing the culture. When comparing both methods, significant differences between culture and v-qPCR were in the samples belonging to the cooling towers-evaporative condensers group. The v-qPCR method detected greater presence and obtained higher concentrations of Legionella spp. (p < 0.001). Otherwise, no significant differences between methods were found in the rest of the groups. Conclusions The v-qPCR method can be used as a quick tool to evaluate Legionellosis risk, especially in cooling towers-evaporative condensers, where this technique can detect higher levels than culture. The combined interpretation of PCR results along with the ratio of live cells is proposed as a tool for understanding the sample context and estimating the Legionellosis risk potential according to 4 levels of hierarchy.