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Sulfide-Induced Dissimilatory Nitrate Reduction to Ammonium Supports Anammox in an Open-Water Unit Process Wetland.
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Sulfide-Induced Dissimilatory Nitrate Reduction to Ammonium Supports Anammox in an Open-Water Unit Process Wetland.

Appl Environ Microbiol. 2017 May 19;:

Authors: Jones ZL, Jasper JT, Sedlak DL, Sharp JO

Abstract
Open-water unit process wetlands host a benthic diatomaceous and bacterial assemblage capable of nitrate removal from treated municipal wastewater with unexpected contributions from anammox processes. In exploring mechanistic drivers of anammox, 16S rRNA gene sequencing profiles of the biomat revealed significant microbial community shifts along the flow path and with depth. Notably there was an increasing abundance of sulfate reducers (Desulfococcus and other Deltaproteobacteria) and anammox microorganisms (Brocadiaceae) with depth. Pore water profiles demonstrated that nitrate and sulfate concentrations exhibited a commensurate decrease with biomat depth accompanied by the accumulation of ammonium. Quantitative PCR targeting the anammox hydrazine synthase gene, hzsA, revealed a 3-fold increase in abundance with biomat depth as well as a 2-fold increase in the sulfate reductase gene, dsrA These microbial and geochemical trends were most pronounced in proximity to the influent region of the wetland where the biomat was thickest and influent nitrate concentrations were highest. While direct genetic queries for dissimilatory nitrate reduction to ammonium (DNRA) microorganisms proved unsuccessful, an increasing depth-dependent dominance of Gammaproteobacteria and diatoms that have previously been functionally linked to DNRA was observed. To further explore this potential, a series of microcosms containing field-derived biomat material confirmed the ability of the community to produce sulfide and reduce nitrate; however, significant ammonium production was only observed in the presence of hydrogen sulfide. Collectively, these results suggest that biogenic sulfide induces DNRA, which in turn can explain the requisite coproduction of ammonium and nitrite from nitrified effluent necessary to sustain the anammox community.Importance This study aims to increase understanding of why and how anammox is occurring in an engineered wetland with limited exogenous contributions of ammonium and nitrite. In doing so, it has implications for how geochemical parameters could potentially be leveraged to impact nutrient cycling and attenuation during the operation of treatment wetlands. The work also contributes to ongoing discussions about biogeochemical signatures surrounding anammox processes and enhances our understanding of the contributions of anammox processes in freshwater environments.

PMID: 28526796 [PubMed - as supplied by publisher]




Exploring a possible link between the intestinal microbiota and feed efficiency in pigs.
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Exploring a possible link between the intestinal microbiota and feed efficiency in pigs.

Appl Environ Microbiol. 2017 May 19;:

Authors: McCormack UM, Curião T, Buzoianu SG, Prieto ML, Ryan T, Varley P, Crispie F, Magowan E, Metzler-Zebeli BU, Berry D, O'Sullivan O, Cotter PD, Gardiner GE, Lawlor PG

Abstract
Feed efficiency (FE) is critical in pig production for both economic and environmental reasons. As the intestinal microbiota plays an important role in energy harvest, it is likely to influence FE. Our aim was therefore to characterize the intestinal microbiota of pigs ranked as low, medium and high residual feed intake (RFI; a metric for FE), where genetic, nutritional and management effects were minimized, in order to explore a possible link between the intestinal microbiota and FE. Eighty one pigs were ranked on RFI between weaning and day 126 post-weaning, and 32 were selected as the extremes in RFI (12 low, 10 medium, 10 high). Intestinal microbiota diversity, composition and predicted functionality were assessed by 16S rRNA gene sequencing. Although no differences in microbial diversity were found, some RFI-associated compositional differences were revealed, principally among members of Firmicutes, and predominantly in faeces at slaughter (albeit mainly for low abundance taxa). In particular, microbes associated with a leaner and healthier host (e.g. Christensenellaceae, Oscillibacter, Cellulosilyticum) were enriched in low RFI (more feed efficient) pigs. Differences were also observed in the ileum of low RFI pigs; most notably Nocardiaceae (Rhodococcus) were less abundant. Predictive functional analysis suggested improved metabolic capabilities in these animals, especially within the ileal microbiota. Higher ileal isobutyric acid concentrations were also found in more efficient pigs. Overall, differences observed within the intestinal microbiota of low RFI pigs compared to their high RFI counterparts, albeit relatively subtle, suggest a possible link between the intestinal microbiota and FE in pigs.IMPORTANCE This study is one of the first to show that differences in intestinal microbiota composition, albeit subtle, may, at least in part, explain improved FE in low RFI pigs. One of the main findings is that, although microbial diversity did not differ among animals of varying FE, specific intestinal microbes could potentially be linked with porcine FE. However, as the factors impacting FE are still not fully understood, intestinal microbiota composition may not be a major factor determining differences in FE. Nonetheless, this work has provided a potential set of microbial biomarkers for FE in pigs. Although culturability could be a limiting factor, and intervention studies are required, these taxa could potentially be targeted in the future, in order to manipulate the intestinal microbiome so as to improve FE in pigs. If successful, this has the potential to reduce both production costs and the environmental impact of pig production.

PMID: 28526795 [PubMed - as supplied by publisher]




Raffinose induces biofilm formation by Streptococcus mutans in low concentrations of sucrose by increasing production of extracellular DNA and fructan.
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Raffinose induces biofilm formation by Streptococcus mutans in low concentrations of sucrose by increasing production of extracellular DNA and fructan.

Appl Environ Microbiol. 2017 May 19;:

Authors: Nagasawa R, Sato T, Senpuku H

Abstract
Streptococcus mutans is the primary etiological agent of dental caries and causes tooth decay by forming a firmly attached biofilm on tooth surfaces. Biofilm formation is induced by the presence of sucrose, which is a substrate for the synthesis of extracellular polysaccharides, but not in the presence of oligosaccharides. Nonetheless, in this study, we found that raffinose, which is an oligosaccharide with an intestinal regulatory function and antiallergic effect, induced biofilm formation by S. mutans in a mixed culture with sucrose which was at concentrations less than those required to induce biofilm formation directly. We analyzed the possible mechanism behind the small requirement for sucrose for biofilm formation in the presence of raffinose. Our results suggested that sucrose contributed to an increase in bacterial cell surface hydrophobicity and biofilm formation. Next, we examined how the effects of raffinose interacted with the effects of sucrose for biofilm formation. We showed that the presence of raffinose induced fructan synthesis by fructosyltransferase and aggregated extracellular DNA (eDNA, which is probably genomic DNA released from dead cells) into the biofilm. eDNA seemed to be important for biofilm formation, because the degradation of DNA by DNase I resulted in a significant reduction in biofilm formation. When assessing the role of fructan in biofilm formation, we found that fructan enhanced eDNA-dependent cell aggregation. Therefore, our results show that raffinose and sucrose have cooperative effects and that this induction of biofilm formation depends on supportive elements that mainly consist of eDNA and fructan.IMPORTANCE The sucrose-dependent mechanism of biofilm formation in Streptococcus mutans has been studied extensively. Nonetheless, the effects of carbohydrates other than sucrose are inadequately understood. Our findings concerning raffinose advance the understanding of the mechanism underlying the joint effects of sucrose and other carbohydrates on biofilm formation. Since raffinose has been reported to have positive effects on enterobacterial flora, research on the effects of raffinose on the oral flora are required prior to its use as a beneficial sugar for human health. Here, we showed that raffinose induced biofilm formation by S. mutans in low concentrations of sucrose. The induction of biofilm formation generally generates negative effects on the oral flora. Therefore, we believe that this finding will aid in the development of more effective oral care techniques to maintain oral flora health.

PMID: 28526794 [PubMed - as supplied by publisher]




Genomic analysis of Calderihabitans maritimus KKC1, a thermophilic hydrogenogenic carboxydotrophic bacterium isolated from marine sediment.
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Genomic analysis of Calderihabitans maritimus KKC1, a thermophilic hydrogenogenic carboxydotrophic bacterium isolated from marine sediment.

Appl Environ Microbiol. 2017 May 19;:

Authors: Omae K, Yoneda Y, Fukuyama Y, Yoshida T, Sako Y

Abstract
Calderihabitans maritimus KKC1 is a thermophilic hydrogenogenic carboxydotroph isolated from a submerged marine caldera. Here, we describe the de novo sequencing and feature analysis of the C. maritimus KKC1 genome. Genome-based phylogenetic analysis confirmed that C. maritimus KKC1 was most closely related to the genus Moorella, which includes well-studied acetogenic members. Comparative genomic analysis revealed that, like Moorella, C. maritimus KKC1 retained both the CO2-reducing Wood-Ljungdahl pathway and energy-converting hydrogenase-based module activated by reduced ferredoxin, but lacked the HydABC and NfnAB electron bifurcating enzymes and pyruvate:ferredoxin oxidoreductase required for ferredoxin reduction during acetogenic growth. Furthermore, C. maritimus KKC1 harbored six genes encoding CooS, a catalytic subunit of the anaerobic CO dehydrogenase that can reduce ferredoxin via CO oxidation, whereas Moorella possessed only two CooS genes. Our analysis revealed that three cooS formed known gene clusters in other microorganisms: cooS-acetyl-CoA synthase (which contained a frameshift mutation), cooS-energy-converting hydrogenase, and cooF-cooS-FAD-NAD oxidoreductase, while the other three had novel genomic contexts. Sequence composition analysis indicated that these cooS genes likely evolved from a common ancestor. Collectively, these data suggest that C. maritimus KKC1 may be highly dependent on CO as a low potential electron donor to directly reduce ferredoxin and more suited to carboxydotrophic growth compared to the acetogenic growth observed in Moorella, which show adaptation at a thermodynamic limit.IMPORTANCECalderihabitans maritimus KKC1 and members of the genus Moorella are phylogenetically related, but physiologically distinct. The former is a hydrogenogenic carboxydotroph that can grow on carbon monoxide (CO) with H2 production, whereas the latter includes acetogenic bacteria that grow on H2 + CO2 with acetate production. Both species may require reduced ferredoxin as an actual 'energy equivalent,' but ferredoxin is a low potential electron carrier and requires a high-energy substrate as an electron donor for reduction. Comparative genomic analysis revealed that C. maritimus KKC1 lacked specific electron bifurcating enzymes and possessed six CO dehydrogenases, unlike Moorella species. This suggests that C. maritimus KKC1 may be more dependent on CO-a strong electron donor that can directly reduce ferredoxin via CO dehydrogenase-and exhibit a survival strategy different from that of acetogenic Moorella that solve the energetic barrier associated with endergonic reduction of ferredoxin with hydrogen.

PMID: 28526793 [PubMed - as supplied by publisher]




Environmental spread of NDM-1-producing multi-drug resistant bacteria in Dhaka, Bangladesh.
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Environmental spread of NDM-1-producing multi-drug resistant bacteria in Dhaka, Bangladesh.

Appl Environ Microbiol. 2017 May 19;:

Authors: Islam MA, Islam M, Hasan R, Hossain MI, Nabi A, Rahman M, Goessens WHF, Endtz HP, Boehm AB, Faruque SM

Abstract
Resistance to carbapenem antibiotics through the production of New Delhi Metallo-β-lactamase-1 (NDM-1) constitutes an emerging challenge in the treatment of bacterial infections. To monitor possible source of spread of these organisms in Dhaka, Bangladesh, we conducted comparative analysis of waste water samples from hospital adjacent areas (HAR) and from community areas (COM) as well as public tap water samples for the occurrence and characteristics of NDM-1 producing bacteria. Of 72 HAR samples tested 51 (71%) were positive for NDM-1-producing bacteria as evidenced by phenotypic tests and the presence of blaNDM-1 gene, compared to 5 of 41 (12.1%) from COM samples (P<0⋅001). All tap water samples were negative for NDM-1-producing bacteria. Klebsiella pneumoniae (44%) was the predominant bacterial species among blaNDM-1 positive isolates followed by E. coli (29%), Acinetobacter sp., (15%) and Enterobacter spp. (9%). These bacteria were also positive for one or more other antibiotic resistance genes including blaCTX-M-1 (80%),blaCTX-M-15 (63%), blaTEM (76%), blaSHV (33%), blaCMY-2 (16%), blaOXA-48-like (2%), blaOXA-1 (53%) and blaOXA-47-like (60%). Around 40% of the isolates contained qnr gene while 50% had 16s-rRNA methylase genes. The majority of isolates hosted multiple plasmids, and plasmids of 30-50 MDa carrying blaNDM-1 were self-transmissible. Our results highlight a number of issues related to the characteristics and source of spread of multiple drug resistant bacteria as a potential public health threat. In view of existing practice of discharging untreated liquid wastes into the environment, hospitals in Dhaka city contribute to potential dissemination of NDM-1-producing bacteria into the community.IMPORTANCE. Infections caused by carbapenemase-producing Enterobacteriaceae are extremely difficult to manage due to their marked resistance to a wide range of antibiotics. NDM-1 is the most recently-described carbapenemase, and the blaNDM-1 gene encoding NDM-1 is located on self-transmissible plasmids that also carry a considerable number of other antibiotic resistance genes. The present study shows high prevalence of NDM-1-producing organisms in the wastewater samples from hospital adjacent areas as a potential source for spread of these organisms to community areas in Dhaka, Bangladesh. The study also examines characteristics of the isolates and their potential to horizontally transmit the resistance determinants. The significance of our research is in identifying the mode of spread of multiple antibiotic resistant organisms, which will allow the development of containment measures, leading to broader impacts in reducing their spread to the community.

PMID: 28526792 [PubMed - as supplied by publisher]




Pseudomonas chlororaphis produces two distinct R-tailocins that contribute to bacterial competition in biofilms and on roots.
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Pseudomonas chlororaphis produces two distinct R-tailocins that contribute to bacterial competition in biofilms and on roots.

Appl Environ Microbiol. 2017 May 19;:

Authors: Dorosky RJ, Yu JM, Pierson LS, Pierson EA

Abstract
R-type tailocins are high molecular weight bacteriocins that resemble bacteriophage tails and are encoded within the genomes of many Pseudomonas species. In this study, analysis of the P. chlororaphis 30-84 R-tailocin gene cluster revealed that it encodes the structural components to produce two R-tailocins of different ancestral origin. Two distinct R-tailocin populations differing in length were observed in UV induced lysates of P. chlororaphis 30-84 via transmission electron microscopy. Mutants defective in the production of one or both R-tailocins demonstrated that the killing spectrum of each tailocin is limited to Pseudomonas species. The spectrum of pseudomonads killed by the two R-tailocins differed, although a few Pseudomonas species were either killed by or insusceptible to both tailocins. Tailocin release was disrupted by deletion of the holin gene within the tailocin gene cluster, demonstrating that the lysis cassette is required for the release of both R-tailocins. The loss of functional tailocin production reduced the ability of P. chlororaphis 30-84 to compete with an R-tailocin sensitive strain within biofilms and rhizosphere communities. Our study demonstrates that Pseudomonas species can produce more than one functional R-tailocin particle sharing the same lysis cassette, but differing in their killing spectrum. This study provides evidence for the role of R-tailocins as determinants of bacterial competition among plant-associated Pseudomonas in biofilms and the rhizosphere.IMPORTANCE Recent studies have identified R-tailocin gene clusters potentially encoding more than one R-tailocin within the genomes of plant-associated Pseudomonas, but have not demonstrated that more than one particle is produced or the ecological significance of the production of multiple R-tailocins. This study demonstrates for the first time that Pseudomonas strains can produce two distinct R-tailocins with different killing spectra, both of which contribute to bacterial competition between rhizosphere-associated bacteria. These results provide new insight into the previously uncharacterized role of R-tailocin production by plant-associated Pseudomonas species in bacterial population dynamics within surface attached biofilms and on roots.

PMID: 28526791 [PubMed - as supplied by publisher]




Post epizootic persistence of asymptomatic Mycoplasma conjunctivae infection in Iberian ibex.
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Post epizootic persistence of asymptomatic Mycoplasma conjunctivae infection in Iberian ibex.

Appl Environ Microbiol. 2017 May 19;:

Authors: Fernández-Aguilar X, Cabezón O, Granados JE, Frey J, Serrano E, Velarde R, Cano-Manuel FJ, Mentaberre G, Ráez-Bravo A, Fandos P, López-Olvera JR

Abstract
The susceptibility of Iberian ibex (Capra pyrenaica) to Mycoplasma conjunctivae ocular infection and the changes in their interaction over time were studied in terms of clinical outcome, molecular detection and IgG immune response in a captive population that underwent a severe infectious keratoconjunctivitis (IKC) outbreak. Mycoplasma conjunctivae was detected for the first time in the Iberian ibexes coinciding with the IKC outbreak. Its prevalence had a decreasing trend in 2013 that was consistent with the clinical resolution (August 35.4%; September 8.7%; November 4.3%). Infections without clinical outcome were however still detected in the last handling in November. Sequencing and cluster analyses of the M. conjunctivae strains found one year later in the ibex population confirmed the persistence of the same strain lineage that caused the IKC outbreak, but with a high prevalence (75.3%) of mostly asymptomatic infections and with lower DNA load of M. conjunctivae in the eyes (qPCR Ct mean 36.1 vs. 20.3 in severe IKC). Significant age related differences of M. conjunctivae prevalence were only observed in IKC epizootic conditions. No substantial effect of systemic IgG on M. conjunctivae DNA in the eye was evidenced with a linear mixed models selection, which indicated that systemic IgG do not necessarily drive the resolution of M. conjunctivae infection and do not explain the epidemiological changes observed. Results show how both epidemiological scenarios, severe IKC outbreak and mostly asymptomatic infections, can consecutively occur by entailing the mycoplasma persistence.ImportanceMycoplasma infections are reported in a wide range of epidemiological scenarios that involve from severe disease to asymptomatic infections. This study allows a better understanding of the transition between two different Mycoplasma conjunctivae epidemiological scenarios described in wild hosts populations and highlights the ability of M. conjunctivae to adapt, persist and establish diverse interactions with its hosts. The proportion of asymptomatic and clinical M. conjunctivae infections in a host population may not be regarded only in response to intrinsic host-species traits (i.e. susceptibility) but also to a specific host-pathogen interaction, which in turn has influences on the infection dynamics. Both epidemic infectious keratoconjunctivitis and high prevalence of asymptomatic M. conjunctivae infections may occur in the same host population depending on the circulation of M. conjunctivae, its maintenance and the progression of the host-pathogen interactions.

PMID: 28526790 [PubMed - as supplied by publisher]




A novel, iron-dependent enzyme that catalyzes the initial step in the biodegradation of N-nitroglycine by Variovorax sp. strain JS1663.
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A novel, iron-dependent enzyme that catalyzes the initial step in the biodegradation of N-nitroglycine by Variovorax sp. strain JS1663.

Appl Environ Microbiol. 2017 May 19;:

Authors: Mahan KM, Zheng H, Fida TT, Parry RJ, Graham DE, Spain JC

Abstract
Nitramines are key constituents of most of the explosives in current use and consequently contaminate soil and groundwater at many military facilities around the world. Toxicity from nitramine contamination poses a health risk to plants and animals, thus understanding how nitramines are biodegraded is critical to environmental remediation. The biodegradation of synthetic nitramine compounds such as hexahydro-1,3,5-trinitro-1,3,5- triazine (RDX) has been studied for decades, but little is known about the catabolism of naturally produced nitramine compounds. In this study, we report the isolation of a soil bacterium Variovorax sp. JS1663, that degrades N-nitroglycine (NNG), a naturally produced nitramine, and the key enzyme involved in its catabolism. Variovorax sp. JS1663 is a Gram-negative, non-spore-forming, motile bacterium, isolated from activated sludge based on its ability to use NNG as a sole growth substrate under aerobic conditions. A single gene (nnlA) encodes an iron-dependent enzyme that releases nitrite from NNG through a proposed β-elimination reaction. Bioinformatics analysis of the amino acid sequence of NNG lyase identified a PAS (Per Arnt Sim) domain. PAS domains can be associated with heme cofactors and function as signal sensors in signaling proteins. This is the first instance of a PAS domain present in a denitration enzyme. The NNG biodegradation pathway should provide the basis for identification of other enzymes that cleave the N-N bond and facilitate the development of enzymes to cleave similar bonds in RDX, nitroguanidine, and other nitramine explosives.Importance Production of antibiotics and other allelopathic chemicals is a major aspect of chemical ecology. Biodegradation of such chemicals can play an important ecological role in mitigating or eliminating the effects of such compounds. N-Nitroglycine (NNG) is produced by the Gram-positive filamentous soil bacterium Streptomyces noursei This study reports the isolation of a Gram-negative soil bacterium, Variovorax sp. JS1663, that is able to use NNG as a sole growth substrate. The proposed degradation pathway occurs via a β-elimination reaction that releases nitrite from NNG. The novel NNG lyase requires iron(II) for activity. The identification of a novel enzyme and catabolic pathway provides evidence of a substantial and underappreciated flux of the antibiotic in natural ecosystems. Understanding the NNG biodegradation pathway will help in the identification of other enzymes that cleave the N-N bond and facilitate the development of enzymes to cleave similar bonds in synthetic nitramine explosives.

PMID: 28526789 [PubMed - as supplied by publisher]




Zinc Resistance within Swine Associated Methicillin Resistant Staphylococcus aureus (MRSA) Isolates in the USA is Associated with MLST Lineage.
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Zinc Resistance within Swine Associated Methicillin Resistant Staphylococcus aureus (MRSA) Isolates in the USA is Associated with MLST Lineage.

Appl Environ Microbiol. 2017 May 19;:

Authors: Hau SJ, Frana T, Sun J, Davies PR, Nicholson TL

Abstract
Zinc resistance in livestock-associated methicillin resistant Staphylococcus aureus (LA-MRSA) sequence type (ST) 398 is primarily mediated by the czrC gene co-located with the mecA gene, encoding methicillin resistance, within the type V SCCmec element. Because czrC and mecA are located within the same mobile genetic element, it has been suggested that the use of in feed zinc as an antidiarrheal agent has the potential to contribute to the emergence and spread of MRSA in swine through increased selection pressure to maintain the SCCmec element in isolates obtained from pigs. In this study we report the prevalence of the czrC gene and phenotypic zinc resistance in US swine associated LA-MRSA ST5 isolates, MRSA ST5 isolates from humans with no swine contact, and US swine associated LA-MRSA ST398 isolates. We demonstrate that the prevalence of zinc resistance in US swine associated LA-MRSA ST5 isolates was significantly lower than the prevalence of zinc resistance in MRSA ST5 isolates from humans with no swine contact, swine associated LA-MRSA ST398 isolates, and previous reports describing zinc resistance in other LA-MRSA ST398 isolates. Collectively our data suggest that selection pressure associated with zinc supplementation in feed is unlikely to have played a significant role in the emergence of LA-MRSA ST5 in the US swine population. Additionally, our data indicate that zinc resistance is associated with MLST lineage suggesting a potential link between genetic lineage and carriage of resistance determinants.Importance Our data suggest that coselection thought to be associated with the use of zinc in feed as an antimicrobial agent is not playing a role in the emergence of livestock-associated methicillin resistant Staphylococcus aureus (LA-MRSA) ST5 in the US swine population. Additionally, our data indicate that zinc resistance is more associated with multi locus sequence type (MLST) lineage suggesting a potential link between genetic lineage and carriage of resistance markers. This information is important to public health professionals, veterinarians, producers, and consumers.

PMID: 28526788 [PubMed - as supplied by publisher]




Development of multi-well plate methods using pure cultures of methanogens to identify new inhibitors for suppressing ruminant methane emissions.
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Development of multi-well plate methods using pure cultures of methanogens to identify new inhibitors for suppressing ruminant methane emissions.

Appl Environ Microbiol. 2017 May 19;:

Authors: Weimar MR, Cheung J, Dey D, McSweeney C, Morrison M, Kobayashi Y, Whitman WB, Carbone V, Schofield LR, Ronimus RS, Cook GM

Abstract
Hydrogenotrophic methanogens typically require strictly anaerobic culturing conditions in glass tubes with overpressures of H2 and CO2 that are both time-consuming and costly. To increase the throughput for screening chemical compound libraries, 96-well microtiter plate methods for the growth of a marine (environmental) methanogen Methanococcus maripaludis strain S2 and the rumen methanogen Methanobrevibacter species AbM4 were developed. A number of key parameters (inoculum size, reducing agents for media preparation, duration of assay, inhibitor solvents and culture volume) were optimised to achieve robust and reproducible growth in a high-throughput microtiter plate format. The method was validated using published methanogen inhibitors and statistically assessed for sensitivity and reproducibility. The Sigma-Aldrich LOPAC library containing 1280 pharmacologically active compounds and an in-house natural product library (120 compounds) were screened against M. maripaludis as a proof of utility. This screen identified a number of bioactive compounds, and minimum inhibitory concentration (MIC) values were confirmed for some of them against M. maripaludis and AbM4. The developed method provides for a significant increase in throughput for screening compound libraries and can now be used to screen larger compound libraries for the discovery of novel methanogen-specific inhibitors for mitigation of ruminant methane emissions.IMPORTANCE Methane emissions from ruminants are a significant contributor to global greenhouse gas emissions and new technologies are required to control emissions in the Agritech sector. The discovery of small-molecule inhibitors of methanogens using high-throughput phenotypic (growth) screening against compound libraries (synthetic and natural products) is an attractive avenue. However, phenotypic inhibitor screening is currently hindered by our inability to grow methanogens in a high-throughput format. We have developed, optimised and validated a high-throughput 96-well microtiter plate assay for the growth of environmental and rumen methanogens. Using this platform, we identified several new inhibitors of methanogen growth, demonstrating the utility of this approach to fast track the development of methanogen-specific inhibitors for controlling ruminant methane emissions.

PMID: 28526787 [PubMed - as supplied by publisher]




Impact of Acanthamoeba cysts on stress resistance of Salmonella enterica serovar Typhimurium, Yersinia enterocolitica 4/O:3, Listeria monocytogenes 1/2a and Escherichia coli O:26.
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Impact of Acanthamoeba cysts on stress resistance of Salmonella enterica serovar Typhimurium, Yersinia enterocolitica 4/O:3, Listeria monocytogenes 1/2a and Escherichia coli O:26.

Appl Environ Microbiol. 2017 May 19;:

Authors: Lambrecht E, Baré J, Sabbe K, Houf K

Abstract
The formation of robust resting cysts enables Acanthamoeba to resist harsh environmental conditions. This study investigated to what extent these cysts are resistant to physical and chemical stresses as applied in food industry cleaning and disinfection procedures. Moreover it was assessed whether certain intracystic meatborne bacterial pathogens are more stress-resistant than free-living bacterial monocultures and if intracystic passage and subsequent association with trophozoites induces cross-tolerance towards other stressors.Several physical and chemical stressors [NaCl, H2O2, benzalkoniumchloride, 55°C, heating until boiling, ethanol, dishwashing detergent and sodium hypochlorite[rsqb] frequently used in domestic and industrial food related environments, were tested against (i) A. castellanii cysts, (ii) single strains of bacterial monocultures, (iii) intracystic bacteria and (iv) bacteria after intracystic passage (cyst-primed bacteria). Only heating until boiling and hypochlorite treatment were cysticidal. After boiling, no viable trophozoites could be recovered from the cysts and hypochlorite treatment caused a 1.34 to 4.72 log10 cells/mL reduction in cyst viability. All treatments were effective in reducing or even eliminating the tested bacterial monocultures, whereas bacteria residing inside cyst were more tolerant towards these stressors. All cyst-primed bacteria exhibited an increased tolerance towards subsequent H2O2 (> 92 % decrease in median Log10 cfu/ml reduction) and 70% ethanol treatment (>99 %). Moreover, intracystic passage significantly increased the survival of Y. enterocolitica (74% decrease in median Log10 reduction), E. coli (58 %) and S. enterica (48%) after NaCl treatment and of E. coli (96%), S. enterica (99%) and L. monocytogenes (99%) after sodium hypochlorite treatment compared to non-primed bacteria.IMPORTANCE This study demonstrated that both viable and non-viable amoebal cysts can protect internalized bacteria against stressful conditions. Moreover, cyst passage can induce cross-tolerance in bacteria, increasing their survival when exposed to selected stressors. These findings underscore the potential importance of free-living amoebae in food related environments and their impact on the persistence of meatborne bacterial pathogens.

PMID: 28526786 [PubMed - as supplied by publisher]




Effects of Arginine on Growth, Virulence Gene Expression, and Stress Tolerance by Streptococcus mutans.
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Effects of Arginine on Growth, Virulence Gene Expression, and Stress Tolerance by Streptococcus mutans.

Appl Environ Microbiol. 2017 May 19;:

Authors: Chakraborty B, Burne RA

Abstract
Streptococcus mutans is a common constituent of oral biofilms and a primary etiologic agent of human dental caries. The bacteria associated with dental caries have a potent ability to produce organic acids from dietary carbohydrates and to grow and metabolize in acidic conditions. In contrast, many commensal bacteria produce ammonia through the arginine deiminase system (ADS), which moderates the pH of oral biofilms. Arginine metabolism by the ADS is a significant deterrent to the initiation and progression of dental caries. In this study, we observed how exogenously provided L-arginine affects growth, virulence properties, and tolerance of environmental stresses of S. mutans Supplementation with 1.5% arginine (final concentration) had inhibitory effects on the growth of S. mutans in complex and chemically defined media, particularly when cells were exposed to acid or oxidative stress. Genes for virulence factors required for attachment/accumulation (gtfB, spaP), for bacteriocins (nlmA, nlmB, nlmD, cipB), and for the sigma factor required for competence development (comX) were down regulated during growth with 1.5% arginine. Deep-sequencing of RNA (RNA-Seq) comparing the transcriptomes of S. mutans growing in a chemically defined medium with and without 1.5% arginine revealed differential expression of genes for ATP-binding cassette transporters, metal transporters, and constituents required for survival, metabolism and biofilm formation. Therefore, the mechanisms of action by which arginine inhibits dental caries include direct adverse effects on multiple virulence-related properties of the most common human dental caries pathogen.Importance Metabolism of the amino acid arginine by the arginine deiminase system (ADS) of certain oral bacteria raises the pH of dental plaque and provides a selective advantage to health-associated bacteria, thereby protecting the host from dental caries (cavities). Here, we examine the effects of arginine on the cavity-causing bacterium Streptococcus mutans We find that arginine negatively impacts the growth, the pathogenic potential, and the tolerance of environmental stresses in a way that is likely to compromise the ability of S. mutans to cause disease. Using genetic and genomic techniques, multiple mechanisms by which arginine exerts its influence on virulence-related properties of S. mutans are discovered. This is the first report demonstrating that a primary mechanism of action by which arginine inhibits the initiation and progression of dental caries may be by reducing the pathogenic potential of S. mutans.

PMID: 28526785 [PubMed - as supplied by publisher]