California's experience with SMA newborn screening: A successful path to early intervention.

BACKGROUND: Universal spinal muscular atrophy (SMA) newborn screening was implemented in California on June 24, 2020. OBJECTIVE: We describe California's experience with the first 18 months of SMA newborn screening, including our assay methodology, timeliness of screening and follow-up milestones, and clinical and epidemiological outcomes observed. METHODS: Dried blood spots are screened for SMA using multiplex real time polymerase chain reaction (RT-PCR) to detect deletions of exon 7 in the survival of motor neuron 1 (SMN1) gene. Short-term follow-up data is collected from clinical staff via an online data collection tool. RESULTS: In the first 18 months, 628,791 newborns from California's diverse population were tested for SMA. Thirty-four screened positive and were confirmed to have the disorder. Infants were referred, diagnosed, and treated at a median of 8, 12, and 33 days of life, respectively. Nearly all infants received the desired treatment modality, and 62% received treatment while still asymptomatic. CONCLUSIONS: SMA newborn screening is a highly sensitive and specific test which identifies infants with SMA early when treatment is most effective. Even with newborn screening's success in facilitating early intervention, there is still work to be done to expedite treatment, especially for infants with the most severe form of the disease.

Draft Genome Sequences of 171 Listeria monocytogenes Isolates from Food-Related Listeriosis Outbreaks in California from 2007 to 2017.

This publication reports the availability of draft genome sequences of 171 Listeria monocytogenes strains isolated from various food-related sources from California between 2007 and 2017. All isolates contain at least two antimicrobial resistance genes.

Multi-Laboratory Validation of a Loop-Mediated Isothermal Amplification Method for Screening Salmonella in Animal Food.

Loop-mediated isothermal amplification (LAMP) has gained wide popularity in the detection of Salmonella in foods owing to its simplicity, rapidity, and robustness. This multi-laboratory validation (MLV) study aimed to validate a Salmonella LAMP-based method against the United States Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Chapter 5 Salmonella reference method in a representative animal food matrix (dry dog food). Fourteen independent collaborators from seven laboratories in the United States and Canada participated in the study. Each collaborator received two sets of 24 blind-coded dry dog food samples (eight uninoculated; eight inoculated at a low level, 0.65 MPN/25 g; and eight inoculated at a high level, 3.01 MPN/25 g) and initiated the testing on the same day. The MLV study used an unpaired design where different test portions were analyzed by the LAMP and BAM methods using different preenrichment protocols (buffered peptone water for LAMP and lactose broth for BAM). All LAMP samples were confirmed by culture using the BAM method. BAM samples were also tested by LAMP following lactose broth preenrichment (paired samples). Statistical analysis was carried out by the probability of detection (POD) per AOAC guidelines and by a random intercept logistic regression model. Overall, no significant differences in POD between the Salmonella LAMP and BAM methods were observed with either unpaired or paired samples, indicating the methods were comparable. LAMP testing following preenrichment in buffered peptone water or lactose broth also resulted in insignificant POD differences (P > 0.05). The MLV study strongly supports the utility and applicability of this rapid and reliable LAMP method in routine regulatory screening of Salmonella in animal food.

Draft Genome Sequence of an ortho-Nitrophenyl-ß-d-Galactoside (ONPG)-Negative Strain of Vibrio cholerae, Isolated from Drakes Bay, California.

We present the draft whole-genome sequence of a Vibrio cholerae strain (Vc25-3) isolated from Drakes Bay, California. This environmental isolate has an atypical morphology and is ortho-nitrophenyl-ß-d-galactoside (ONPG)-negative.

Draft Whole-Genome Sequences of Three Shiga Toxin-Producing Escherichia coli O91:H21 Isolates, Two from Hemolytic Uremic Syndrome Patients and One of Porcine Origin.

This study presents three genomes of O91:H21 isolates, two from hemolytic uremic syndrome patients and one of porcine origin. Genome analyses reveal that one of the human isolates contains both Shiga toxin-encoding genes (stx1 and stx2), and all three isolates contain putative adhesin (iha and eaeH) and antibiotic resistance (ampC) genes.

Characterization of wastewater treatment plant microbial communities and the effects of carbon sources on diversity in laboratory models.

We are developing a laboratory-scale model to improve our understanding and capacity to assess the biological risks of genetically engineered bacteria and their genetic elements in the natural environment. Our hypothetical scenario concerns an industrial bioreactor failure resulting in the introduction of genetically engineered bacteria to a downstream municipal wastewater treatment plant (MWWTP). As the first step towards developing a model for this scenario, we sampled microbial communities from the aeration basin of a MWWTP at three seasonal time points. Having established a baseline for community composition, we investigated how the community changed when propagated in the laboratory, including cell culture media conditions that could provide selective pressure in future studies. Specifically, using PhyloChip 16S-rRNA-gene targeting microarrays, we compared the compositions of sampled communities to those of inocula propagated in the laboratory in simulated wastewater conditionally amended with various carbon sources (glucose, chloroacetate, D-threonine) or the ionic liquid 1-ethyl-3-methylimidazolium chloride ([C2mim]Cl). Proteobacteria, Bacteroidetes, and Actinobacteria were predominant in both aeration basin and laboratory-cultured communities. Laboratory-cultured communities were enriched in γ-Proteobacteria. Enterobacteriaceae, and Aeromonadaceae were enriched by glucose, Pseudomonadaceae by chloroacetate and D-threonine, and Burkholderiacea by high (50 mM) concentrations of chloroacetate. Microbial communities cultured with chloroacetate and D-threonine were more similar to sampled field communities than those cultured with glucose or [C2mim]Cl. Although observed relative richness in operational taxonomic units (OTUs) was lower for laboratory cultures than for field communities, both flask and reactor systems supported phylogenetically diverse communities. These results importantly provide a foundation for laboratory models of industrial bioreactor failure scenarios.

Microbial community responses to organophosphate substrate additions in contaminated subsurface sediments.

BACKGROUND: Radionuclide- and heavy metal-contaminated subsurface sediments remain a legacy of Cold War nuclear weapons research and recent nuclear power plant failures. Within such contaminated sediments, remediation activities are necessary to mitigate groundwater contamination. A promising approach makes use of extant microbial communities capable of hydrolyzing organophosphate substrates to promote mineralization of soluble contaminants within deep subsurface environments. METHODOLOGY/PRINCIPAL FINDINGS: Uranium-contaminated sediments from the U.S. Department of Energy Oak Ridge Field Research Center (ORFRC) Area 2 site were used in slurry experiments to identify microbial communities involved in hydrolysis of 10 mM organophosphate amendments [i.e., glycerol-2-phosphate (G2P) or glycerol-3-phosphate (G3P)] in synthetic groundwater at pH 5.5 and pH 6.8. Following 36 day (G2P) and 20 day (G3P) amended treatments, maximum phosphate (PO4(3-)) concentrations of 4.8 mM and 8.9 mM were measured, respectively. Use of the PhyloChip 16S rRNA microarray identified 2,120 archaeal and bacterial taxa representing 46 phyla, 66 classes, 110 orders, and 186 families among all treatments. Measures of archaeal and bacterial richness were lowest under G2P (pH 5.5) treatments and greatest with G3P (pH 6.8) treatments. Members of the phyla Crenarchaeota, Euryarchaeota, Bacteroidetes, and Proteobacteria demonstrated the greatest enrichment in response to organophosphate amendments and the OTUs that increased in relative abundance by 2-fold or greater accounted for 9%-50% and 3%-17% of total detected Archaea and Bacteria, respectively. CONCLUSIONS/SIGNIFICANCE: This work provided a characterization of the distinct ORFRC subsurface microbial communities that contributed to increased concentrations of extracellular phosphate via hydrolysis of organophosphate substrate amendments. Within subsurface environments that are not ideal for reductive precipitation of uranium, strategies that harness microbial phosphate metabolism to promote uranium phosphate precipitation could offer an alternative approach for in situ sequestration.

Microbial biogeography across a full-scale wastewater treatment plant transect: evidence for immigration between coupled processes.

Wastewater treatment plants use a variety of bioreactor types and configurations to remove organic matter and nutrients. Little is known regarding the effects of different configurations and within-plant immigration on microbial community dynamics. Previously, we found that the structure of ammonia-oxidizing bacterial (AOB) communities in a full-scale dispersed growth activated sludge bioreactor correlated strongly with levels of NO2 (-) entering the reactor from an upstream trickling filter. Here, to further examine this puzzling association, we profile within-plant microbial biogeography (spatial variation) and test the hypothesis that substantial microbial immigration occurs along a transect (raw influent, trickling filter biofilm, trickling filter effluent, and activated sludge) at the same full-scale wastewater treatment plant. AOB amoA gene abundance increased >30-fold between influent and trickling filter effluent concomitant with NO2 (-) production, indicating unexpected growth and activity of AOB within the trickling filter. Nitrosomonas europaea was the dominant AOB phylotype in trickling filter biofilm and effluent, while a distinct "Nitrosomonas-like" lineage dominated in activated sludge. Prior time series indicated that this "Nitrosomonas-like" lineage was dominant when NO2 (-) levels in the trickling filter effluent (i.e., activated sludge influent) were low, while N. europaea became dominant in the activated sludge when NO2 (-) levels were high. This is consistent with the hypothesis that NO2 (-) production may cooccur with biofilm sloughing, releasing N. europaea from the trickling filter into the activated sludge bioreactor. Phylogenetic microarray (PhyloChip) analyses revealed significant spatial variation in taxonomic diversity, including a large excess of methanogens in the trickling filter relative to activated sludge and attenuation of Enterobacteriaceae across the transect, and demonstrated transport of a highly diverse microbial community via the trickling filter effluent to the activated sludge bioreactor. Our results provide compelling evidence that substantial immigration between coupled process units occurs and may exert significant influence over microbial community dynamics within staged bioreactors.

Dissecting a complex chemical stress: chemogenomic profiling of plant hydrolysates.

The efficient production of biofuels from cellulosic feedstocks will require the efficient fermentation of the sugars in hydrolyzed plant material. Unfortunately, plant hydrolysates also contain many compounds that inhibit microbial growth and fermentation. We used DNA-barcoded mutant libraries to identify genes that are important for hydrolysate tolerance in both Zymomonas mobilis (44 genes) and Saccharomyces cerevisiae (99 genes). Overexpression of a Z. mobilis tolerance gene of unknown function (ZMO1875) improved its specific ethanol productivity 2.4-fold in the presence of miscanthus hydrolysate. However, a mixture of 37 hydrolysate-derived inhibitors was not sufficient to explain the fitness profile of plant hydrolysate. To deconstruct the fitness profile of hydrolysate, we profiled the 37 inhibitors against a library of Z. mobilis mutants and we modeled fitness in hydrolysate as a mixture of fitness in its components. By examining outliers in this model, we identified methylglyoxal as a previously unknown component of hydrolysate. Our work provides a general strategy to dissect how microbes respond to a complex chemical stress and should enable further engineering of hydrolysate tolerance.

The influence of in situ chemical oxidation on microbial community composition in groundwater contaminated with chlorinated solvents.

In situ chemical oxidation with permanganate has become an accepted remedial treatment for groundwater contaminated with chlorinated solvents. This study focuses on the immediate and short-term effects of sodium permanganate (NaMnO(4)) on the indigenous subsurface microbial community composition in groundwater impacted by trichloroethylene (TCE). Planktonic and biofilm microbial communities were studied using groundwater grab samples and reticulated vitreous carbon passive samplers, respectively. Microbial community composition was analyzed by terminal restriction fragment length polymorphism and a high-density phylogenetic microarray (PhyloChip). Significant reductions in microbial diversity and biomass were shown during NaMnO(4) exposure, followed by recovery within several weeks after the oxidant concentrations decreased to <1 mg/L. Bray-Curtis similarities and nonmetric multidimensional scaling showed that microbial community composition before and after NaMnO(4) was similar, when taking into account the natural variation of the microbial communities. Also, 16S rRNA genes of two reductive dechlorinators (Desulfuromonas spp. and Sulfurospirillum spp.) and diverse taxa capable of cometabolic TCE oxidation were detected in similar quantities by PhyloChip across all monitoring wells, irrespective of NaMnO(4) exposure and TCE concentrations. However, minimal biodegradation of TCE was observed in this study, based on oxidized conditions, concentration patterns of chlorinated and nonchlorinated hydrocarbons, geochemistry, and spatiotemporal distribution of TCE-degrading bacteria.

Systems biology approach to bioremediation.

Bioremediation has historically been approached as a 'black box' in terms of our fundamental understanding. Thus it succeeds and fails, seldom without a complete understanding of why. Systems biology is an integrated research approach to study complex biological systems, by investigating interactions and networks at the molecular, cellular, community, and ecosystem level. The knowledge of these interactions within individual components is fundamental to understanding the dynamics of the ecosystem under investigation. Understanding and modeling functional microbial community structure and stress responses in environments at all levels have tremendous implications for our fundamental understanding of hydrobiogeochemical processes and the potential for making bioremediation breakthroughs and illuminating the 'black box'.

PCR amplification-independent methods for detection of microbial communities by the high-density microarray PhyloChip.

Environmental microbial community analysis typically involves amplification by PCR, despite well-documented biases. We have developed two methods of PCR-independent microbial community analysis using the high-density microarray PhyloChip: direct hybridization of 16S rRNA (dirRNA) or rRNA converted to double-stranded cDNA (dscDNA). We compared dirRNA and dscDNA communities to PCR-amplified DNA communities using a mock community of eight taxa, as well as experiments derived from three environmental sample types: chromium-contaminated aquifer groundwater, tropical forest soil, and secondary sewage in seawater. Community profiles by both direct hybridization methods showed differences that were expected based on accompanying data but that were missing in PCR-amplified communities. Taxon richness decreased in RNA compared to that in DNA communities, suggesting a subset of 20% in soil and 60% in groundwater that is active; secondary sewage showed no difference between active and inactive populations. Direct hybridization of dscDNA and RNA is thus a viable alternative to PCR-amplified microbial community analysis, providing identification of the active populations within microbial communities that attenuate pollutants, drive global biogeochemical cycles, or proliferate disease states.

Characterization of coastal urban watershed bacterial communities leads to alternative community-based indicators.

BACKGROUND: Microbial communities in aquatic environments are spatially and temporally dynamic due to environmental fluctuations and varied external input sources. A large percentage of the urban watersheds in the United States are affected by fecal pollution, including human pathogens, thus warranting comprehensive monitoring. METHODOLOGY/PRINCIPAL FINDINGS: Using a high-density microarray (PhyloChip), we examined water column bacterial community DNA extracted from two connecting urban watersheds, elucidating variable and stable bacterial subpopulations over a 3-day period and community composition profiles that were distinct to fecal and non-fecal sources. Two approaches were used for indication of fecal influence. The first approach utilized similarity of 503 operational taxonomic units (OTUs) common to all fecal samples analyzed in this study with the watershed samples as an index of fecal pollution. A majority of the 503 OTUs were found in the phyla Firmicutes, Proteobacteria, Bacteroidetes, and Actinobacteria. The second approach incorporated relative richness of 4 bacterial classes (Bacilli, Bacteroidetes, Clostridia and alpha-proteobacteria) found to have the highest variance in fecal and non-fecal samples. The ratio of these 4 classes (BBC:A) from the watershed samples demonstrated a trend where bacterial communities from gut and sewage sources had higher ratios than from sources not impacted by fecal material. This trend was also observed in the 124 bacterial communities from previously published and unpublished sequencing or PhyloChip- analyzed studies. CONCLUSIONS/SIGNIFICANCE: This study provided a detailed characterization of bacterial community variability during dry weather across a 3-day period in two urban watersheds. The comparative analysis of watershed community composition resulted in alternative community-based indicators that could be useful for assessing ecosystem health.

Optimization of a whole-cell cadmium sensor with a toggle gene circuit.

This work demonstrates improvement of a whole-cell cadmium detection sensor through construction of a gene circuit. A cadmium (II) specific regulatory promoter, P(cadR,) from Psuedomonas putida 06909, is used in the assembly of a toggle circuit. The circuit contains the cadR promoter fused to lacIq and gfp, and a divergently transcribed tac promoter and cadR. The toggle sensor exhibits lower background fluorescence, and a 20-fold lower detection limit in comparison to a nontoggle gene circuit. The detection limit of the toggle sensor is 0.01 microM (1.12 ppb) cadmium chloride, and tunable with the addition of isopropyl-b-D-thiogalactopyranoside (IPTG). The toggle sensor is highly specific to cadmium (II), and no response is elicited from zinc, lead, manganese, nickel, copper, and mercury.

Developing microbe-plant interactions for applications in plant-growth promotion and disease control, production of useful compounds, remediation and carbon sequestration.

Interactions between plants and microbes are an integral part of our terrestrial ecosystem. Microbe-plant interactions are being applied in many areas. In this review, we present recent reports of applications in the areas of plant-growth promotion, biocontrol, bioactive compound and biomaterial production, remediation and carbon sequestration. Challenges, limitations and future outlook for each field are discussed.

Detection of recombinant Pseudomonas putida in the wheat rhizosphere by fluorescence in situ hybridization targeting mRNA and rRNA.

A method was developed to detect a specific strain of bacteria in wheat root rhizoplane using fluorescence in situ hybridization and confocal microscopy. Probes targeting both 23S rRNA and messenger RNA were used simultaneously to achieve detection of recombinant Pseudomonas putida (TOM20) expressing toluene o-monooxygenase (tom) genes and synthetic phytochelatin (EC20). The probe specific to P. putida 23S rRNA sequences was labeled with Cy3 fluor, and the probe specific to the tom genes was labeled with Alexa647 fluor. Probe specificity was first determined, and hybridization temperature was optimized using three rhizosphere bacteria pure cultures as controls, along with the P. putida TOM20 strain. The probes were highly specific to the respective targets, with minimal non-specific binding. The recombinant strain was inoculated into wheat seedling rhizosphere. Colonization of P. putida TOM20 was confirmed by extraction of root biofilm and growth of colonies on selective agar medium. Confocal microscopy of hybridized root biofilm detected P. putida TOM20 cells emitting both Cy3 and Alexa647 fluorescence signals.

Versatile microbial surface-display for environmental remediation and biofuels production.

Surface display is a powerful technique that uses natural microbial functional components to express proteins or peptides on the cell exterior. Since the reporting of the first surface-display system in the mid-1980s, a variety of new systems have been reported for yeast, Gram-positive and Gram-negative bacteria. Non-conventional display methods are emerging, eliminating the generation of genetically modified microorganisms. Cells with surface display are used as biocatalysts, biosorbents and biostimulants. Microbial cell-surface display has proven to be extremely important for numerous applications, ranging from combinatorial library screening and protein engineering to bioremediation and biofuels production.

Engineering plant-microbe symbiosis for rhizoremediation of heavy metals.

The use of plants for rehabilitation of heavy-metal-contaminated environments is an emerging area of interest because it provides an ecologically sound and safe method for restoration and remediation. Although a number of plant species are capable of hyperaccumulation of heavy metals, the technology is not applicable for remediating sites with multiple contaminants. A clever solution is to combine the advantages of microbe-plant symbiosis within the plant rhizosphere into an effective cleanup technology. We demonstrated that expression of a metal-binding peptide (EC20) in a rhizobacterium, Pseudomonas putida 06909, not only improved cadmium binding but also alleviated the cellular toxicity of cadmium. More importantly, inoculation of sunflower roots with the engineered rhizobacterium resulted in a marked decrease in cadmium phytotoxicity and a 40% increase in cadmium accumulation in the plant root. Owing to the significantly improved growth characteristics of both the rhizobacterium and plant, the use of EC20-expressing P. putida endowed with organic-degrading capabilities may be a promising strategy to remediate mixed organic-metal-contaminated sites.

Enhanced arsenic accumulation in engineered bacterial cells expressing ArsR.

The metalloregulatory protein ArsR, which offers high affinity and selectivity toward arsenite, was overexpressed in Escherichia coli in an attempt to increase the bioaccumulation of arsenic. Overproduction of ArsR resulted in elevated levels of arsenite bioaccumulation but also a severe reduction in cell growth. Incorporation of an elastin-like polypeptide as the fusion partner to ArsR (ELP153AR) improved cell growth by twofold without compromising the ability to accumulate arsenite. Resting cells overexpressing ELP153AR accumulated 5- and 60-fold-higher levels of arsenate and arsenite than control cells without ArsR overexpression. Conversely, no significant improvement in Cd(2+) or Zn(2+) accumulation was observed, validating the specificity of ArsR. The high affinity of ArsR allowed 100% removal of 50 ppb of arsenite from contaminated water with these engineered cells, providing a technology useful to comply with the newly approved U.S. Environmental Protection Agency limit of 10 ppb. These results open up the possibility of using cells overexpressing ArsR as an inexpensive, high-affinity ligand for arsenic removal from contaminated drinking and ground water.

Enhanced mercury biosorption by bacterial cells with surface-displayed MerR.

The metalloregulatory protein MerR, which exhibits high affinity and selectivity toward mercury, was exploited for the construction of microbial biosorbents specific for mercury removal. Whole-cell sorbents were constructed with MerR genetically engineered onto the surface of Escherichia coli cells by using an ice nucleation protein anchor. The presence of surface-exposed MerR on the engineered strains enabled sixfold-higher Hg(2+) biosorption than that found in the wild-type JM109 cells. Hg(2+) binding via MerR was very specific, with no observable decline even in the presence of 100-fold excess Cd(2+) and Zn(2+). The Hg(2+) binding property of the whole-cell sorbents was also insensitive to different ionic strengths, pHs, and the presence of metal chelators. Since metalloregulatory proteins are currently available for a wide variety of toxic heavy metals, our results suggest that microbial biosorbents overexpressing metalloregulatory proteins may be used similarly for the cleanup of other important heavy metals.