Examining Antimicrobial Resistance in Escherichia coli: A Case Study in Central Virginia's Environment.

While environmental factors may contribute to antimicrobial resistance (AMR) in bacteria, many aspects of environmental antibiotic pollution and resistance remain unknown. Furthermore, the level of AMR in Escherichia coli is considered a reliable indicator of the selection pressure exerted by antimicrobial use in the environment. This study aimed to assess AMR variance in E. coli isolated from diverse environmental samples, such as animal feces and water from wastewater treatment plants (WWTPs) and drainage areas of different land use systems in Central Virginia. In total, 450 E. coli isolates obtained between August 2020 and February 2021 were subjected to susceptibility testing against 12 antimicrobial agents approved for clinical use by the U.S. Food and Drug Administration. Approximately 87.8% of the tested isolates were resistant to at least one antimicrobial agent, with 3.1% showing multi-drug resistance. Streptomycin resistance was the most common (73.1%), while susceptibility to chloramphenicol was the highest (97.6%). One isolate obtained from WWTPs exhibited resistance to seven antimicrobials. AMR prevalence was the highest in WWTP isolates, followed by isolates from drainage areas, wild avians, and livestock. Among livestock, horses had the highest AMR prevalence, while cattle had the lowest. No significant AMR difference was found across land use systems. This study identifies potential AMR hotspots, emphasizing the environmental risk for antimicrobial resistant E. coli. The findings will aid policymakers and researchers, highlighting knowledge gaps in AMR-environment links. This nationally relevant research offers a scalable AMR model for understanding E. coli ecology. Further large-scale research is crucial to confirm the environmental impacts on AMR prevalence in bacteria.

Unveiling the greenhouse gas emissions of drinking water treatment plant throughout the construction and operation stages based on life cycle assessment.

The carbon peaking and carbon neutrality targets proposed by the Chinese government have initiated a green transformation that is full of challenges and opportunities and endowed sustainable development strategy for combating global warming issue. It is essential to execute comprehensive identification and carbon reduction measures across all industries that produce greenhouse gas (GHG) emissions. Water supply system, as an energy-intensive sector, plays a crucial role in GHG reduction. This work conducted a life cycle assessment (LCA) to account the GHG emissions associated with the construction and operation phases of the drinking water treatment plant (DWTP). During the construction phase, the total GHG emissions were 19,525.762 t CO2-eq, with concrete work and rebar project being the dominant contributors (87.712%). The promotion of renewable or recyclable green building materials and low-carbon construction methods, such as the utilization of prefabricated components and on-site assembly, holds significant importance in reducing GHG emissions during the construction phase of DWTP. Regarding the operation stage, the DWTP possessed an average annual GHG emission of 37,660.160 t CO2-eq and an average GHG intensity of 0.202 kg CO2-eq/m3. Most emissions were attributed to electricity consumption (67.388%), chemicals utilization (12.893%), and heat consumption (10.414%). By increasing the use of clean energy and implementing strict control measures in the water supply pumps, energy consumption and GHG emissions can be effectively reduced. This study offers valuable insights into the mapping of GHG emissions in the DWTP, facilitating the identification of key areas for targeted implementation of energy-saving and carbon-reducing measures.

Escherichia cryptic clade II through clade VIII: Rapid detection and prevalence in feces and surface water.

Bacteria of the cryptic lineage of genus Escherichia, or Escherichia cryptic clades (cryptic clades), are phenotypically indistinguishable from Escherichia coli (E. coli) using standard biochemical tests. Except for clade I (C-I), cryptic clades were hypothetically believed to be environmental but not enteric. If so, they would hinder the interpretation of current E. coli-based water quality (fecal pollution) monitoring in the United States because environmental bacteria do not indicate the presence of harmful fecal material. This study was performed to develop a rapid method for the detection of cryptic clades and to investigate their potential impact on water quality monitoring. By whole-genome comparison, one gene, named ecc (Escherichiacryptic clades), was identified to be unique to C-II through C-VIII. An end-point polymerase chain reaction (PCR) method, eccPCR, was developed by targeting the ecc. The results of in-silico and wet tests demonstrated 100 % sensitivity and specificity of the eccPCR to detect C-II through C-VIII. Based on the EPA Method 1603, 519 presumptive E. coli isolates were obtained from the fecal samples of 13 different host species and 192 isolates from surface water samples taken at four locations in a watershed of mid-Missouri. As indicated by the eccPCR amplification, the overall prevalence of C-II through C-VIII in the presumptive E. coli isolates was estimated to be about 0.6 % in the fecal samples and about 1.6 % in the water samples. Therefore, the potential impact of cryptic clades on water quality monitoring may be limited if EPA Method 1603 is used. Furthermore, clades C-II through C-VIII in stream water samples were found repeatedly only at a single sampling site, but neither at the upstream sites nor five kilometers downstream of the site. The data do not support nor reject the environmental hypothesis about cryptic clades. Further study is needed to determine the implication of the observation.

Detection of viable Escherichia coli in environmental water using combined propidium monoazide staining and quantitative PCR.

The objectives of this study were to specifically detect viable Escherichia coli in environmental waters by targeting the ycjM gene in a propidium monoazide (PMA)-qPCR assay. PMA is a viability dye that can inhibit the amplification of DNA from dead cells, thus allowing for the detection and quantification of only viable cells. The ycjM primers were used to target E. coli that directly originated from the feces of warm blooded animals, and avoid false positive detection caused by "naturalized" E. coli that can exist in the environment. In this study, tap water and environmental waters were inoculated with E. coli isolated from animal feces. Following cell collection, samples were treated with PMA, followed by DNA isolation and qPCR detection. For pure cultures, 5 µM PMA with a 10-min light exposure was efficient at inhibiting the amplification of DNA from 105 CFU/mL dead E. coli cells, with a detection limit of 102 CFU/100 mL viable cells. For tap and environmental waters collected in the winter, a 10 µM PMA was required and as low as 103 CFU/100 mL viable cells could be detected in the presence of 105 CFU/100 mL dead cells. For water samples collected during the summer, 102 CFU/10 mL viable cells could be detected in the presence of 104 CFU/10 mL dead cells, after a 20 µM PMA treatment. No significant differences were found among the PMA-qPCR assay and two other standard culture-based methods for detection of viable E. coli in environmental water. In conclusion, with proper pretreatment of environmental water samples, this PMA-qPCR assay that targets the ycjM gene could quantify viable E. coli cells that directly come from the feces of warm-blooded animals, and therefore effectively and accurately indicate the quality of environmental water.

Biocontrol of the internalization of Salmonella enterica and Enterohaemorrhagic Escherichia coli in mung bean sprouts with an endophytic Bacillus subtilis.

Internalization of Salmonella enterica and enterohaemorrhagic Escherichia coli (EHEC) in seed sprouts poses a health risk to consumers, and the conventional sanitization methods are not always effective to reduce this risk. This study initiated a biocontrol approach to limit the internalization using endophytic Bacillus subtilis strains, which were isolated from the inner tissue of mung bean seeds or lettuce stems. By using the deferred agar method, 12 strains of B. subtilis out of 94 putative Bacillus isolates displayed inhibitory activity against at least one of the pathogenic indicators, S. enterica Typhimurium ATCC 14028 and E. coli O157:H7 505B. Two B. subtilis isolates (LCA1 and M24) showed a broad inhibitory spectrum against multiple strains of S. enterica and EHEC, Staphylococcus aureus sp., Klebsiella pneumoniae ATCC 700603, and Listeria monocytogenes Scott A, while the laboratory B. subtilis strain 168 was only moderately inhibitory against L. monocytogenes. To facilitate the tracking of the three B. subtilis strains (LCA1, M24, and 168) in the mung bean sprouts, the three strains were genetically engineered to carry the chloramphenicol acetyltransferase (cat), generating the strains LCA1-cat, M24-cat, and 168-cat, respectively. Data of the study using the cat-tagged strains demonstrated that both the two vegetable-associated and the laboratory B. subtilis strains could internalize in mung bean sprouts during the sprouting, but the latter displayed about 1.2 lg CFU/g of seeds lower in internalization. Overall, the presence of the three B. subtilis strains could significantly reduce the internalization of S. enterica or EHEC cocktail in mung bean sprouts during the sprouting. Among them, LCA1 showed the greatest inhibition against the EHEC cocktails with a reduction of about 2.0lg CFU/g of seeds by the end of sprouting (day 5), while 168 had the smallest reduction at about 0.6lg CFU/g of seeds. In addition, the three strains demonstrated a similar inhibition against the S. enterica cocktails by a reduction of about 1.1-1.4lg CFU/g of seeds by day 5. Results of this study suggest that the source (native vs. alien) of B. subtilis isolates may not affect the efficacy of the inhibition, but it might be affected by the production of antimicrobial substance and/or nutrition/space competition. The results also indicate that strain LCA1 may be useful as a biocontrol agent to reduce Salmonella and EHEC contamination in seed sprouts.

Evaluation of five microbial and four mitochondrial DNA markers for tracking human and pig fecal pollution in freshwater.

This study systematically evaluated five microbial and four mitochondrial DNA (mtDNA) markers, including sensitivities and specificities under PCR method, and fecal concentrations and decay rates in water under qPCR method. The microbial DNA markers were the three human-associated (BacH, HF183 and B.adolescentis) and two pig-associated (Pig-2-Bac and L.amylovorus), while the mtDNA ones were two human- (H-ND6 and H-ND5) and two pig-associated (P-CytB and P-ND5). All the mtDNA markers showed higher sensitivity (100%) than the microbial ones (84.0-88.8%) except Pig-2-Bac (100%). Specificities of the human mtDNA markers (99.1 and 98.1%) were higher than those of the human-associated microbial ones (57.0-88.8%). But this pattern was not observed in the pig-associated markers where Pig-2-Bac had 100% specificity. The reliability of H-ND6 and H-ND5 was further evidenced to identify locations of the most polluted within the Taihu Lake watershed of China. In general, the microbial DNA markers demonstrated a higher fecal concentration than the mtDNA ones; increasing temperature and sunlight exposure accelerated significantly the decay of all the DNA markers. Results of this study suggest that DNA markers H-ND6, H-ND5, and Pig-2-Bac may be among the best for fecal source tracking in water.

Uptake, translocation and metabolism of decabromodiphenyl ether (BDE-209) in seven aquatic plants.

Terrestrial plant uptake of PBDEs from contaminated soils has been widely reported recently. In this study the fate of deca-BDE within a plant/PBDEs/aquatic environment system was investigated through simulated pot experiments. Accumulations of the total PBDEs and deca-BDE were observed in tissues of seven test aquatic plant species, namely Phragmites australis, Cyperus papyrus, Alternanthera philoxeroides, Colocasia esculenta, Scirpus validus, Acorus calamus and Oryza sativa. In all seven plants, O. sativa leads the uptake and accumulation both in the total PBDEs (444.8 ng g(-1)) and deca-BDE (368.0 ng g(-1)) in roots. Among the six common phytoremediation aquatic plants, A. calamus leads the uptake (236.2 ng g(-1)), and P. australis leads the translocation (Cshoot/Croot = 0.35), while A. philoxeroides (43.4%) and P. australis (80.0%) lead in the metabolism efficiencies in the root and shoot, respectively. The detection of seventeen lesser brominated PBDE congeners provided the debromination evidence, and the specific PBDEs profiles in test plant species indicated there is no common metabolic pattern. Furthermore, a relative high proportion of lesser brominated PBDE congeners in shoots suggested the possible metabolic difference between roots and shoots. Finally, a noticeable percentage of penta- and octa-BDE derived from deca-BDE also hint the ecological risk in deca-BDE use. This comparative research on the aquatic plants provide a broad vision on the understanding of plant/PBDEs/aquatic environment interaction system, and may be applied to remediate PBDEs in contaminated waters and sediments.

Polymorphism of the glucosyltransferase gene (ycjM) in Escherichia coli and its use for tracking human fecal pollution in water.

This study examined polymorphism of the glucosyltransferase gene (ycjM) in fecal Escherichia coli isolates and evaluated the use of the sequence polymorphism for measuring human fecal pollution in water. Significant nucleotide variations were observed through comparative analysis of the ycjM sequences of 70 E. coli strains isolated from the feces of humans, domestic livestock, and wild animals. Three distinct types of ycjM sequences were found: universal-ycjM, human/chicken-ycjM, and human-ycjM. Using the human-ycjM sequences, both a polymerase chain reaction (PCR), Hycj-PCR and a quantitative PCR, Hycj-qPCR, were developed. As shown by the Hycj-PCR amplification, the human-ycjM marker appeared to be highly associated with the E. coli strains isolated from human feces, based on the analysis of 370 E. coli strains isolated from humans and seven other animal species. Similarly, the human-ycjM marker was highly linked with human feces, as demonstrated by the Hycj-PCR assay, when using 337 fecal DNA samples from 16 host animal sources, including both domestic and wild animals. Overall, the specificity and sensitivity of the human-ycjM marker for differentiating between the feces of humans and those of nonhuman groups were 99.7% and 100%, respectively; the prevalence of the marker appeared to be greater than 50% in the human-feces-associated E. coli population. In addition, our study showed that the quantification of human E. coli by the Hycj-qPCR was linearly correlated with the anthropogenic activity within a watershed. Our study suggests that this novel human-ycjM marker and the resulting PCR-based methods developed should be useful for measuring human-associated E. coli and human fecal pollution in water.

Differentiating enteric Escherichia coli from environmental bacteria through the putative glucosyltransferase gene (ycjM).

This study is to tackle the challenge posed by the "naturalized" Escherichia coli population against the worldwide practice of E. coli-based water quality monitoring. In the literature, the putative glucosyltransferase gene (ycjM) of E. coli has been identified in silico to be one of the 114 genes specific to enteric E. coli. Based on the sequence of E. coli K-12 MG1655, a PCR assay (ycjPCR) targeting ycjM was developed in this study. As demonstrated by the ycjPCR assay using 367 E. coli strains isolated from animal feces, 97.2% of the isolates carried the ycjM with variations from 93.9% to 100% among nine different host sources, but none of the 17 strains of non-E. coli bacteria and only 23.0% of the environment-isolated cryptic Escherichia strains contained the ycjM. These data experimentally confirmed ycjM to be enteric specific. Our study also showed that the ycjPCR assay was superior to the commonly used tuf- or uidA-based PCR methods in differentiating enteric E. coli from ß-D-glucuronidase-positive environmental bacteria. Furthermore, study on 190 E. coli isolates from water samples, using EPA Method 1603 followed by bacterial identification with Biolog MicroStation™ and ycjPCR assay, indicated that the prevalence of ycjM in the E. coli water isolates had a significant (p < 0.05, odds ratio ) spatial variation from 69.6% to 93.8%. These data suggest that E. coli profile using EPA Method 1603 or other ß-D-glucuronidase-activity-based methods may need further analysis using the ycjM profile to accurately determinate fecal pollution in water.

Using an intervening sequence of Faecalibacterium 16S rDNA to identify poultry feces.

This study was designed to identify poultry feces-specific marker(s) within sequences of Faecalibacterium 16S rDNA for detecting poultry fecal pollution in water. Bioinformatics tools were used in the comparative analysis of 7,458 sequences of Faecalibacterium 16S rDNA, reportedly associated with various poultry (chicken and turkey) and animal species. One intervening sequence (IVS) within between the hypervariable region 1 and the conserved region 2, designated as IVS-p, was found to be unique to poultry feces. Based on this sequence, a PCR assay (PCR-p) was developed. The PCR-p produced an amplicon of 132 bp only in the test when fecal or wastewater samples from poultry were used, but not when using fecal or wastewater samples from other sources. The non-poultry sources included feces of beef or dairy cattle, dog, horse, human, domestic or wild geese, seagull, sheep, swine, and wild turkey. These data indicate that IVS-p may prove to be a useful genetic marker for the specific identification of poultry fecal pollution in environmental waterways. Furthermore, results of data mining and PCR assay indicate that the IVS-p may have a broad geographic distribution. This report represents initial evidence of the potential utility of ribosomal intervening sequences as genetic markers for tracking host sources of fecal pollution in waterways.

Hormonal and nutritional regulation of muscle carnitine palmitoyltransferase I gene expression in vivo.

Transgenic mice carrying the human heart muscle carnitine palmitoyltransferase I (M-CPTI) gene fused to a CAT reporter gene were generated to study the regulation of M-CPTI gene expression. When the mice were fasted for 48 h, CAT activity and mRNA levels increased by more than 2-fold in heart and skeletal muscle, but not liver or kidney. In the diabetic transgenic mice, there was a 2- to 3-fold increase in CAT activity and CAT mRNA levels in heart and skeletal muscle which upon insulin administration reverted to that observed with the control insulin sufficient transgenic mice. Feeding a high fat diet increased CAT activity and mRNA levels by 2- to 4-fold in heart and skeletal muscle of the transgenic mice compared to the control transgenic mice on regular diet. Overall, the M-CPTI promoter was found to be necessary for the tissue-specific hormonal and dietary regulation of the gene expression.

Cysteine-scanning mutagenesis of muscle carnitine palmitoyltransferase I reveals a single cysteine residue (Cys-305) is important for catalysis.

Carnitine palmitoyltransferase (CPT) I catalyzes the conversion of long-chain fatty acyl-CoAs to acyl carnitines in the presence of l-carnitine, a rate-limiting step in the transport of long-chain fatty acids from the cytoplasm to the mitochondrial matrix. To determine the role of the 15 cysteine residues in the heart/skeletal muscle isoform of CPTI (M-CPTI) on catalytic activity and malonyl-CoA sensitivity, we constructed a 6-residue N-terminal, a 9-residue C-terminal, and a 15-residue cysteineless M-CPTI by cysteine-scanning mutagenesis. Both the 9-residue C-terminal mutant enzyme and the complete 15-residue cysteineless mutant enzyme are inactive but that the 6-residue N-terminal cysteineless mutant enzyme had activity and malonyl-CoA sensitivity similar to those of wild-type M-CPTI. Mutation of each of the 9 C-terminal cysteines to alanine or serine identified a single residue, Cys-305, to be important for catalysis. Substitution of Cys-305 with Ala in the wild-type enzyme inactivated M-CPTI, and a single change of Ala-305 to Cys in the 9-residue C-terminal cysteineless mutant resulted in an 8-residue C-terminal cysteineless mutant enzyme that had activity and malonyl-CoA sensitivity similar to those of the wild type, suggesting that Cys-305 is the residue involved in catalysis. Sequence alignments of CPTI with the acyltransferase family of enzymes in the GenBank led to the identification of a putative catalytic triad in CPTI consisting of residues Cys-305, Asp-454, and His-473. Based on the mutagenesis and substrate labeling studies, we propose a mechanism for the acyltransferase activity of CPTI that uses a catalytic triad composed of Cys-305, His-473, and Asp-454 with Cys-305 serving as a probable nucleophile, thus acting as a site for covalent attachment of the acyl molecule and formation of a stable acyl-enzyme intermediate. This would in turn allow carnitine to act as a second nucleophile and complete the acyl transfer reaction.

The F-ATPase operon promoter of Streptococcus mutans is transcriptionally regulated in response to external pH.

Streptococcus mutans F-ATPase, the major component of the acid-adaptive response of the organism, is transcriptionally upregulated at low pH. Fusions of the F-ATPase promoter to chloramphenicol acetyltransferase indicated that pH-dependent expression is still observed with a short promoter that contains a domain conserved between streptococcal ATPase operons.

Identification by mutagenesis of a conserved glutamate (Glu487) residue important for catalytic activity in rat liver carnitine palmitoyltransferase II.

Mammalian mitochondrial membranes express two active but distinct carnitine palmitoyltransferases: carnitine palmitoyltransferase I (CPTI), which is malonyl coA-sensitive and detergent-labile; and carnitine palmitoyltransferase II (CPTII), which is malonyl coA-insensitive and detergent-stable. To determine the role of the highly conserved C-terminal acidic residues glutamate 487 (Glu(487)) and glutamate 500 (Glu(500)) on catalytic activity in rat liver CPTII, we separately mutated these residues to alanine, aspartate, or lysine, and the effect of the mutations on CPTII activity was determined in the Escherichia coli-expressed mutants. Substitution of Glu(487) with alanine, aspartate, or lysine resulted in almost complete loss in CPTII activity. Because a conservative substitution mutation of this residue, Glu(487) with aspartate (E487D), resulted in a 97% loss in activity, we predicted that Glu(487) would be at the active-site pocket of CPTII. The substantial loss in CPTII activity observed with the E487K mutant, along with the previously reported loss in activity observed in a child with a CPTII deficiency disease, establishes that Glu(487) is crucial for maintaining the configuration of the liver isoform of the CPTII active site. Substitution of the conserved Glu(500) in CPTII with alanine or aspartate reduced the V(max) for both substrates, suggesting that Glu(500) may be important in stabilization of the enzyme-substrate complex. A conservative substitution of Glu(500) to aspartate resulted in a significant decrease in the V(max) for the substrates. Thus, Glu(500) may play a role in substrate binding and catalysis. Our site-directed mutagenesis studies demonstrate that Glu(487) in the liver isoform of CPTII is essential for catalysis.

Multiple pathways of Spx (YjbD) proteolysis in Bacillus subtilis.

ATP-dependent proteases degrade denatured or misfolded proteins and are recruited for the controlled removal of proteins that block activation of regulatory pathways. Among the ATP-dependent proteases, those of the Clp family are particularly important for the growth and development of Bacillus subtilis. Proteolytic subunit ClpP, together with regulatory ATPase subunit ClpC or ClpX, is required for the normal response to stress, for development of genetic competence, and for sporulation. The spx (formally yjbD) gene was previously identified as a site of mutations that suppress defects in competence conferred by clpP and clpX. The level of Spx in wild-type cells grown in competence medium is low, and that in clpP mutants is high. This suggests that the Spx protein is a substrate for ClpP-containing proteases and that accumulation of Spx might be partly responsible for the observed pleiotropic phenotype resulting from the clpP mutation. In this study we examined, both in vivo and in vitro, which ClpP protease is responsible for degradation of Spx. Western blot analysis showed that Spx accumulated in clpX mutant to the same level as that observed in the clpP mutant. In contrast, a very low concentration of Spx was detected in a clpC mutant. An in vitro proteolysis experiment using purified proteins demonstrated that Spx was degraded by ClpCP but only in the presence of one of the ClpC adapter proteins, MecA or YpbH. However, ClpXP, either in the presence or in the absence of MecA and YpbH, was unable to degrade Spx. Transcription of spx, as measured by expression of spx-lacZ, was slightly increased by the clpX mutation. To exclude a possible effect of clpX and clpP on spx transcription, the spx gene was placed under the control of the IPTG (isopropyl-beta-D-thiogalactopyranoside)-inducible Pspac promoter. In this strain, Spx accumulated when ClpX or ClpP was absent, suggesting that ClpX and ClpP are required for degradation of Spx. Taken together, these results suggest that Spx is degraded by both ClpCP and ClpXP. The putative proteolysis by ClpXP might require another adapter protein. Spx probably is degraded by ClpCP under as yet unidentified conditions. This study suggests that the level of Spx is tightly controlled by two different ClpP proteases.