Desulfohalophilus alkaliarsenatis gen. nov., sp. nov., an extremely halophilic sulfate- and arsenate-respiring bacterium from Searles Lake, California.

A haloalkaliphilic sulfate-respiring bacterium, strain SLSR-1, was isolated from a lactate-fed stable enrichment culture originally obtained from the extreme environment of Searles Lake, California. The isolate proved capable of growth via sulfate-reduction over a broad range of salinities (125-330 g/L), although growth was slowest at salt-saturation. Strain SLSR-1 was also capable of growth via dissimilatory arsenate-reduction and displayed an even broader range of salinity tolerance (50-330 g/L) when grown under these conditions. Strain SLSR-1 could also grow via dissimilatory nitrate reduction to ammonia. Growth experiments in the presence of high borate concentrations indicated a greater sensitivity of sulfate-reduction than arsenate-respiration to this naturally abundant anion in Searles Lake. Strain SLSR-1 contained genes involved in both sulfate-reduction (dsrAB) and arsenate respiration (arrA). Amplicons of 16S rRNA gene sequences obtained from DNA extracted from Searles Lake sediment revealed the presence of close relatives of strain SLSR-1 as part of the flora of this ecosystem despite the fact that sulfate-reduction activity could not be detected in situ. We conclude that strain SLSR-1 can only achieve growth via arsenate-reduction under the current chemical conditions prevalent at Searles Lake. Strain SLSR-1 is a deltaproteobacterium in the family Desulfohalobiacea of anaerobic, haloalkaliphilic bacteria, for which we propose the name Desulfohalophilus alkaliarsenatis gen. nov., sp. nov.

Genome sequence of the methanotrophic alphaproteobacterium Methylocystis sp. strain Rockwell (ATCC 49242).

Methylocystis sp. strain Rockwell (ATCC 49242) is an aerobic methane-oxidizing alphaproteobacterium isolated from an aquifer in southern California. Unlike most methanotrophs in the Methylocystaceae family, this strain has a single pmo operon encoding particulate methane monooxygenase but no evidence of the genes encoding soluble methane monooxygenase. This is the first reported genome sequence of a member of the Methylocystis species of the Methylocystaceae family in the order Rhizobiales.

Validation of the AtlasTMCampylobacter Detection Assay: AOAC Performance Tested MethodSM 032101.

BACKGROUND: Campylobacter spp. are a major causal agent for diarrheal illness in humans. Detection of Campylobacter spp. in food is critical to reduce foodborne illness, and to provide safe foods. OBJECTIVE: The aim was to evaluate the Atlas Campylobacter Detection Assay for AOAC Performance Tested MethodsSM certification for detecting C. jejuni, C. coli, and C. lari in foods after 12 h enrichment. METHOD: The Atlas Campylobacter Detection Assay was compared to the ISO 10272-1:2017 reference culture method for chicken carcass rinse, turkey carcass sponge, raw ground poultry, raw ground pork, and ready-to-eat (RTE) meats. Inclusivity, exclusivity, product consistency, product stability, and robustness studies were also performed. An independent laboratory evaluated the performance of the Atlas Campylobacter Detection Assay method on chicken carcass rinse. RESULTS: No significant differences were observed between the Atlas Campylobacter Detection Assay and the reference ISO method in spiked food matrixes. The Atlas Campylobacter Detection Assay detected all 50 inclusive organisms and none of the 30 exclusive organisms. Product consistency and stability studies showed no statistical differences between lots or over the term of the shelf-life using accelerated method study. Finally, the robustness study showed no statistical difference between different sample volumes, enrichment times, and storage time after sample transfer. CONCLUSIONS: The results of this study indicate that the Atlas Campylobacter Detection Assay is comparable to ISO 10272-1:2017 for detecting Campylobacter in chicken carcass rinse, turkey carcass sponge, raw ground poultry, raw ground pork, and RTE meats. HIGHLIGHTS: The Atlas Campylobacter Detection Assay is a rapid, accurate molecular method able to detect C. jejuni, C. coli, and C. lari in in chicken carcass rinse, turkey carcass sponge, raw ground poultry, raw ground pork, and RTE meats within 12-18 h.

Prevalence of SARS-CoV-2 Contamination on Food Plant Surfaces as Determined by Environmental Monitoring.

ABSTRACT: The SARS-CoV-2 pandemic has presented new challenges to food manufacturers. During the early phase of the pandemic, several large outbreaks of coronavirus disease 2019 (COVID-19) occurred in food manufacturing plants resulting in deaths and economic loss, with approximately 15% of personnel diagnosed as asymptomatic for COVID-19. Spread by asymptomatic and presymptomatic individuals has been implicated in large outbreaks of COVID-19. In March 2020, we assisted in implementation of environmental monitoring programs for SARS-CoV-2 in zones 3 and 4 of 116 food production facilities. All participating facilities had already implemented measures to prevent symptomatic personnel from coming to work. During the study period, from 17 March to 3 September 2020, 1.23% of the 22,643 environmental samples tested positive for SARS-CoV-2, suggesting that infected individuals were actively shedding virus. Virus contamination was commonly found on frequently touched surfaces such as doorknobs, handles, table surfaces, and sanitizer dispensers. Most processing plants managed to control their environmental contamination when they became aware of the positive findings. Comparisons of positive test results for plant personnel and environmental surfaces in one plant revealed a close correlation. Our work illustrates that environmental monitoring for SARS-CoV-2 can be used as a surrogate for identifying the presence of asymptomatic and presymptomatic personnel in workplaces and may aid in controlling infection spread.

Effects of ammonium and nitrite on growth and competitive fitness of cultivated methanotrophic bacteria.

The effects of nitrite and ammonium on cultivated methanotrophic bacteria were investigated. Methylomicrobium album ATCC 33003 outcompeted Methylocystis sp. strain ATCC 49242 in cultures with high nitrite levels, whereas cultures with high ammonium levels allowed Methylocystis sp. to compete more easily. M. album pure cultures and cocultures consumed nitrite and produced nitrous oxide, suggesting a connection between denitrification and nitrite tolerance.

Coupled arsenotrophy in a hot spring photosynthetic biofilm at Mono Lake, California.

Red-pigmented biofilms grow on rock and cobble surfaces present in anoxic hot springs located on Paoha Island in Mono Lake. The bacterial community was dominated ( approximately 85% of 16S rRNA gene clones) by sequences from the photosynthetic Ectothiorhodospira genus. Scraped biofilm materials incubated under anoxic conditions rapidly oxidized As(III) to As(V) in the light via anoxygenic photosynthesis but could also readily reduce As(V) to As(III) in the dark at comparable rates. Back-labeling experiments with (73)As(V) demonstrated that reduction to (73)As(III) also occurred in the light, thereby illustrating the cooccurrence of these two anaerobic processes as an example of closely coupled arsenotrophy. Oxic biofilms also oxidized As(III) to As(V). Biofilms incubated with [(14)C]acetate oxidized the radiolabel to (14)CO(2) in the light but not the dark, indicating a capacity for photoheterotrophy but not chemoheterotrophy. Anoxic, dark-incubated samples demonstrated As(V) reduction linked to additions of hydrogen or sulfide but not acetate. Chemoautotrophy linked to As(V) as measured by dark fixation of [(14)C]bicarbonate into cell material was stimulated by either H(2) or HS(-). Functional genes for the arsenate respiratory reductase (arrA) and arsenic resistance (arsB) were detected in sequenced amplicons of extracted DNA, with about half of the arrA sequences closely related ( approximately 98% translated amino acid identity) to those from the family Ectothiorhodospiraceae. Surprisingly, no authentic PCR products for arsenite oxidase (aoxB) were obtained, despite observing aerobic arsenite oxidation activity. Collectively, these results demonstrate close linkages of these arsenic redox processes occurring within these biofilms.

Bacteria beneath the West Antarctic ice sheet.

Subglacial environments, particularly those that lie beneath polar ice sheets, are beginning to be recognized as an important part of Earth's biosphere. However, except for indirect indications of microbial assemblages in subglacial Lake Vostok, Antarctica, no sub-ice sheet environments have been shown to support microbial ecosystems. Here we report 16S rRNA gene and isolate diversity in sediments collected from beneath the Kamb Ice Stream, West Antarctic Ice Sheet and stored for 15 months at 4 degrees C. This is the first report of microbes in samples from the sediment environment beneath the Antarctic Ice Sheet. The cells were abundant ( approximately 10(7) cells g(-1)) but displayed low diversity (only five phylotypes), likely as a result of enrichment during storage. Isolates were cold tolerant and the 16S rRNA gene diversity was a simplified version of that found in subglacial alpine and Arctic sediments and water. Although in situ cell abundance and the extent of wet sediments beneath the Antarctic ice sheet can only be roughly extrapolated on the basis of this sample, it is clear that the subglacial ecosystem contains a significant and previously unrecognized pool of microbial cells and associated organic carbon that could potentially have significant implications for global geochemical processes.

Ecophysiology of "Halarsenatibacter silvermanii" strain SLAS-1T, gen. nov., sp. nov., a facultative chemoautotrophic arsenate respirer from salt-saturated Searles Lake, California.

Searles Lake occupies a closed basin harboring salt-saturated, alkaline brines that have exceptionally high concentrations of arsenic oxyanions. Strain SLAS-1(T) was previously isolated from Searles Lake (R. S. Oremland, T. R. Kulp, J. Switzer Blum, S. E. Hoeft, S. Baesman, L. G. Miller, and J. F. Stolz, Science 308:1305-1308, 2005). We now describe this extremophile with regard to its substrate affinities, its unusual mode of motility, sequenced arrABD gene cluster, cell envelope lipids, and its phylogenetic alignment within the order Halanaerobacteriales, assigning it the name "Halarsenatibacter silvermanii" strain SLAS-1(T). We also report on the substrate dynamics of an anaerobic enrichment culture obtained from Searles Lake that grows under conditions of salt saturation and whose members include a novel sulfate reducer of the order Desulfovibriales, the archaeon Halorhabdus utahensis, as well as a close homolog of strain SLAS-1(T).

Comparison of ELISA and DNA Lateral Flow Assays for Detection of Pork, Horse, Beef, Chicken, Turkey, and Goat Contamination in Meat Products.

Background: Concerns about the contamination of meat products with undeclared meats and new regulations for the declaration of meat adulterants have established the need for a sensitive test to detect meat adulteration. To address this need, Microbiologique, Inc. has developed ELISA assays that can detect the presence of pork, horse, beef, chicken, turkey, and goat meat adulterants to 0.1% (w/w) and a deoxyribonucleic acid (DNA) lateral flow assay for pork, horse, beef, chicken, turkey, goat, and lamb adulterants to 0.1% (w/w). Objective: We compared the results of the DNA lateral flow assay to the ELISA assays. Methods: ELISA and DNA lateral flow assays were performed on the same spiked meat samples, prepared meats, and pet foods. Results: Both the DNA lateral flow and the ELISA assays were sensitive to 0.1% meat adulterant, and the agreement between the DNA lateral flow and ELISA assays for spiked samples, prepared meat, and pet foods was 100%. Conclusions: Based on the 100% concordance between the two assay formats, the choice between the two is dependent on whether quantitation is desired, which assay is more familiar to the particular laboratory, availability of the required equipment, and time restrictions. Highlights: The ELISA assays are less time consuming, taking about 1.5 h, compared with about 2.5 h for the DNA lateral flow assay. Because the DNA lateral flow test detects seven species in one test, it can be more cost effective when the potential adulterant is not known, while the ELISA may be better for quantification.

Rapid Detection of Listeria in Ice Cream in 13 Hours Using the Roka Listeria Detection Assay.

Background: Listeria contamination is a major concern in the ice cream industry; therefore, early and accurate detection is vital. Current detection methods require about a 24 h enrichment period for detection. Objective: Enhance the early detection of Listeria in ice cream using the highly sensitive isothermal ribosomal RNA-based Roka/Atlas Listeria Detection Assay. Methods: The R2 Medium was developed for Listeria enrichment by Molecular Epidemiology, Inc. (Seattle, WA). Comparative growth curve studies were performed on the new R2 Medium for Listeria and the currently validated media for the Roka Listeria Detection Assay. Subsequently, a method comparison between the Roka Listeria Detection Assay and the U.S. Food and Drug Administration's (FDA) Bacteriological Analytical Manual (BAM) Chapter 10 reference method on ice cream was carried out. Results: The R2 Medium supports the growth of L. monocytogenes better than Buffered Listeria Enrichment Broth, Demi-Fraser broth, and Modified University of Vermont Broth, as indicated by the faster growth rate of the organism. When used as an enrichment medium in a method comparison study of ice cream, the results showed that R2 Medium-enriched samples tested with the Roka Listeria Detection Assay gave an equivalent performance compared with the 24 h FDA-BAM reference method at 10 and 18 h post-enrichment for Listeria. Conclusions: The results from this study indicate that the new R2 Medium and the highly sensitive Roka Listeria Detection Assay allowed for the rapid detection of Listeria species in ice cream in 13 h. Highlights: The Roka Listeria Detection Assay, in conjunction with a new media formulation (R2 Medium), allowed for the early detection of Listeria in ice cream and may be applied in other food matrixes and environmental samples.

New Delhi metallo-ß-lactamase-1 (NDM-1) Escherichia coli isolated from household vacuum cleaner-Oregon, 2013.

The first Oregon case of New Delhi metallo-ß-lactamase-1 (NDM-1)-producing Escherichia coli was reported during November 2013. Epidemiologic investigation revealed only local outpatient medical care and no travel outside Oregon for both the patient and his household contact. Environmental sampling discovered a matching isolate from the patient's household vacuum cleaner, suggesting environmental persistence.

Molecular ecological analysis of planktonic bacterial communities in constructed wetlands invaded by Culex (Diptera: Culicidae) mosquitoes.

The succession of the planktonic bacterial community during the colonization by Culex (Diptera: Culicidae) mosquitoes of 0.1-ha treatment wetlands was studied using denaturing gradient gel electrophoresis (DGGE) methodology. Relationships between apparent bacterial diversity and ecological factors (water quality, total bacterial counts, and immature mosquito abundance) were determined during a 1-mo flooding period. Analysis of DGGE banding patterns indicated that days postflooding and temporal changes in water quality were the primary and secondary determinants, respectively, of diversity in bacterial communities. Lower levels of diversity were associated with later postflood stages and increases in ammoniacal nitrogen concentration and total bacterial counts. Diversity was therefore most similar for bacteria present on the same sampling date at wetland locations with similar flooding regimes and water quality, suggesting that wastewater input was the driving force shaping bacterial communities. Comparatively small changes in bacterial diversity were connected to natural processes as water flowed through the wetlands. Greater immature mosquito abundance coincided with less diverse communities composed of greater total numbers of bacteria. Five individual DGGE bands were directly associated with fluctuations in mosquito production, and an additional 16 bands were associated with hydrological aspects of the environment during the rise and fall of mosquito populations. A marked decline in mosquito numbers 21 d after inundation may have masked associations of bacterial communities and mosquito recruitment into the sparsely vegetated wetlands. DGGE was an effective tool for the characterization of bacteria in mosquito habitat in our study, and its potential application in mosquito ecology is discussed.

Draft Genome Sequence of the Beer Spoilage Bacterium Megasphaera cerevisiae Strain PAT 1T.

The genus Megasphaera harbors important spoilage organisms that cause beer spoilage by producing off flavors, undesirable aroma, and turbidity. Megasphaera cerevisiae is mainly found in nonpasteurized low-alcohol beer. In this study, we report the draft genome of the type strain of the genus, M. cerevisiae strain PAT 1(T).

A viable microbial community in a subglacial volcanic crater lake, Iceland.

We describe a viable microbial community in a subglacial lake within the Grímsvötn volcanic caldera, Iceland. We used a hot water drill to penetrate the 300-m ice shelf and retrieved lake water and volcanic tephra sediments. We also acquired samples of borehole water before and after penetration to the lake, overlying glacial ice and snow, and water from a nearby subaerial geothermal lake for comparative analyses. Lake water is at the freezing point and fresh (total dissolved solids = 260 mg L(-1)). Detectable numbers of cells were found in samples of the lake water column and tephra sediments: 2 x 10(4) ml(-1) and 4 x 10(7) g(-1), respectively. Plate counts document abundant cold-adapted cultivable organisms in the lake water, but not in the borehole (before penetration) or glacial ice. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments amplified from genomic DNA extracted from Grímsvötn samples indicates that the lake community is distinct from the assemblages of organisms in borehole water (before penetration) and the overlying ice and snow. Sequencing of selected DGGE bands revealed that many sequences are highly similar to known psychrophilic organisms or cloned DNA from other cold environments. Significant uptake of 14C-labeled bicarbonate occurred in dark, low-temperature incubations of lake water samples, indicating the presence of autotrophs. Acetylene reduction assays under similar incubation conditions showed no significant nitrogen fixation potential by lake water samples. This may be a consequence of the inhibition of diazotrophy by nitrogen in the lake.

Draft Genome Sequence of blaNDM-1-Positive Escherichia coli O25b-ST131 Clone Isolated from an Environmental Sample.

A multidrug-resistant NDM-1 carbapenamase-producing Escherichia coli sequence type 131 (ST131) organism was obtained from vacuum cleaner dust collected from the home of a case patient. Here, we report the assembly and annotation of its genome.

Composition, diversity, and stability of microbial assemblages in seasonal lake ice, miquelon lake, central alberta.

The most familiar icy environments, seasonal lake and stream ice, have received little microbiological study. Bacteria and Eukarya dominated the microbial assemblage within the seasonal ice of Miquelon Lake, a shallow saline lake in Alberta, Canada. The bacterial assemblages were moderately diverse and did not vary with either ice depth or time. The closest relatives of the bacterial sequences from the ice included Actinobacteria, Bacteroidetes, Proteobacteria, Verrucomicrobia, and Cyanobacteria. The eukaryotic assemblages were less conserved and had very low diversity. Green algae relatives dominated the eukaryotic gene sequences; however, a copepod and cercozoan were also identified, possibly indicating the presence of complete microbial loop. The persistence of a chlorophyll a peak at 25-30 cm below the ice surface, despite ice migration and brine flushing, indicated possible biological activity within the ice. This is the first study of the composition, diversity, and stability of seasonal lake ice.

Enhancing nitrification at low temperature with zeolite in a mining operations retention pond.

Ammonium nitrate explosives are used in mining operations at Diavik Diamond Mines Inc. in the Northwest Territories, Canada. Residual nitrogen is washed into the mine pit and piped to a nearby retention pond where its removal is accomplished by microbial activity prior to a final water treatment step and release into the sub-Arctic lake, Lac de Gras. Microbial removal of ammonium in the retention pond is rapid during the brief ice-free summer, but often slows under ice cover that persists up to 9 months of the year. The aluminosilicate mineral zeolite was tested as an additive to retention pond water to increase rates of ammonium removal at 4°C. Water samples were collected across the length of the retention pond monthly over a year. The structure of the microbial community (bacteria, archaea, and eukarya), as determined by denaturing gradient gel electrophoresis of PCR-amplified small subunit ribosomal RNA genes, was more stable during cold months than during July-September, when there was a marked phytoplankton bloom. Of the ammonia-oxidizing community, only bacterial amoA genes were consistently detected. Zeolite (10 g) was added to retention pond water (100 mL) amended with 5 mM ammonium and incubated at 12°C to encourage development of a nitrifying biofilm. The biofilm community was composed of different amoA phylotypes from those identified in gene clone libraries of native water samples. Zeolite biofilm was added to fresh water samples collected at different times of the year, resulting in a significant increase in laboratory measurements of potential nitrification activity at 4°C. A significant positive correlation between the amount of zeolite biofilm and potential nitrification activity was observed; rates were unaffected in incubations containing 1-20 mM ammonium. Addition of zeolite to retention ponds in cold environments could effectively increase nitrification rates year-round by concentrating active nitrifying biomass.

An oligarchic microbial assemblage in the anoxic bottom waters of a volcanic subglacial lake.

In 2006, we sampled the anoxic bottom waters of a volcanic lake beneath the Vatnajökull ice cap (Iceland). The sample contained 5 x 10(5) cells per ml, and whole-cell fluorescent in situ hybridization (FISH) and PCR with domain-specific probes showed these to be essentially all bacteria, with no detectable archaea. Pyrosequencing of the V6 hypervariable region of the 16S ribosomal RNA gene, Sanger sequencing of a clone library and FISH-based enumeration of four major phylotypes revealed that the assemblage was dominated by a few groups of putative chemotrophic bacteria whose closest cultivated relatives use sulfide, sulfur or hydrogen as electron donors, and oxygen, sulfate or CO(2) as electron acceptors. Hundreds of other phylotypes are present at lower abundance in our V6 tag libraries and a rarefaction analysis indicates that sampling did not reach saturation, but FISH data limit the remaining biome to <10-20% of all cells. The composition of this oligarchy can be understood in the context of the chemical disequilibrium created by the mixing of sulfidic lake water and oxygenated glacial meltwater.

Rapid screening for freshwater bacterial groups by using reverse line blot hybridization.

The identification of phylogenetic clusters of bacteria that are common in freshwater has provided a basis for probe design to target important freshwater groups. We present a set of 16S ribosomal RNA gene-based oligonucleotide probes specific for 15 of these freshwater clusters. The probes were applied in reverse line blot hybridization, a simple method that enables the rapid screening of PCR products from many samples against an array of probes. The optimized assay was made stringent to discriminate at approximately the single-mismatch level. This made 10 of the probes highly specific, with at least two mismatches to the closest noncluster member in the global database. Screening of PCR products from bacterioplankton of 81 diverse lakes from Belgium, The Netherlands, Denmark, Sweden, and Norway showed that the respective probes were reactive against 5 to 100% of the lake samples. Positive reactivity of six highly specific probes showed that bacteria from actinobacterial clusters ACK-M1 and Sta2-30 and from verrucomicrobial cluster CLO-14 occurred in at least 90% of the investigated lakes. Furthermore, bacteria from alpha-proteobacterial cluster LD12 (closely related to the marine SAR11 cluster), beta-proteobacterial cluster LD28 and cyanobacterial cluster Synechococcus 6b occurred in more than 70% of the lakes. Reverse line blot hybridization is a new tool in microbial ecology that will facilitate research on distribution and habitat specificity of target species at relatively low costs.