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1.
Aims:  The focus of this work was to compare the survival of Escherichia coli introduced into streambed sediments from goose, deer and bovine faeces vs indigenous E. coli. Methods and Results:  The survival experiments were conducted in flow‐through chambers for 32 days using two sediments (mineral and organic) obtained from a first‐order creek in Maryland. Bovine, goose and deer faeces were collected fresh and diluted or enriched so that added E. coli and indigenous populations were equivalent. Escherichia coli and total coliforms were enumerated using the Colilert‐18 Quanti‐Tray system. Patterns of E. coli survival and inactivation rates were virtually identical for indigenous strains in both mineral and organic sediments. The addition of E. coli strains from bovine, goose or deer faeces had relatively little impact on final E. coli concentrations, with the exception of deer‐borne E. coli populations in the organic sediment. Conclusion:  These results indicate that indigenous sediment‐borne E. coli strains are generally, or more, persistent than those deposited into sediments, including wildlife. Significance and Impact of the Study:  This is the first study on the survival of E. coli originating from wildlife faeces, in sediments, as opposed to bovine faeces or laboratory‐cultured strains. As wildlife are likely to be the primary source of E. coli in most non agricultural watersheds, an understanding of the persistence of these strains is important to understanding microbial water quality.  相似文献   

2.
Aims: Developing a rapid method for detection of faecal pollution is among the critical goals set forth by the Environmental Protection Agency in its revision of water quality criteria. The purpose of this study is to devise and test covalently linked antibody–bead complexes for faecal indicator bacteria (FIB), specifically Escherichia coli or Enterococcus spp., in measuring water quality in freshwater and marine systems. Methods and Results: Covalently linked complexes were 58–89% more robust than antibody–bead complexes used in previous studies. Freshwater and marine water samples analysed using covalently linked immunomagnetic separation/adenosine triphosphate quantification technique (Cov‐IMS/ATP) and culture‐based methods yielded good correlations for E. coli (R = 0·87) and Enterococcus spp. (R = 0·94), with method detection limits below EPA recreational water quality health standards for single standard exceedances (E. coli– 38 cells per 100 ml; Enterococcus spp. – 25 cells per 100 ml). Cov‐IMS/ATP correctly classified 87% of E. coli and 94% of Enterococcus spp. samples based on these water quality standards. Cov‐IMS/ATP was also used as a field method to rapidly distinguish differential loading of E. coli between two stream channels to their confluence. Conclusions: Cov‐IMS/ATP is a robust, in‐field detection method for determining water quality of both fresh and marine water systems as well as differential loading of FIB from two converging channels. Significance and Impact of the Study: To our knowledge, this is the first work to present a viable rapid, in‐field assay for measuring FIB concentrations in marine water environments. Cov‐IMS/ATP is a potential alternative detection method, particularly in areas with limited laboratory support and resources, because of its increased economy and portability.  相似文献   

3.
The aim of this study was to report the antimicrobial resistance, the molecular mechanisms associated and the detection of virulence determinants within faecal Enterococcus spp. and Escherichia coli isolates of Iberian wolf. Enterococci (= 227) and E. coli (= 195) isolates were obtained from faecal samples of Iberian wolf (Canis lupus signatus). High rates of resistance were detected for tetracycline and erythromycin among the enterococci isolates, and most of resistant isolates harboured the tet(M) and/or tet(L) and erm(B) genes, respectively. The blaTEM, tet(A) and/or tet(B), and aadA or strAstrB genes were detected among most ampicillin‐, tetracycline‐ or streptomycin‐resistant E. coli isolates, respectively. E. coli isolates were ascribed to phylogroups A (= 56), B1 (91), B2 (13) and D (35). The occurrence of resistant enterococci and E. coli isolates in the faecal flora of Iberian wolf, including the presence of resistant genes in integrons, and virulence determinants was showed in this study. Iberian wolf might act as reservoir of certain resistance genes that could be spread throughout the environment.

Significance and Impact of the Study

This study shows antimicrobial resistance in commensal bacteria from the free‐range, Portuguese, Iberian wolf population. The results indicate that the Iberian wolf could contribute to the spread of resistant bacteria throughout the environment. Additionally, in case of infection, an increased risk of therapeutic failure due to the presence of multiresistant bacteria may represent a health problem for this endangered species. Future studies must be performed to analyse the possible contamination of these animals through the environment and/or the food chain.  相似文献   

4.
Aims: We developed an improved Fluorescent In Situ Hybridization FISH‐based method to detect viable Escherichia coli cells by solid phase cytometry (SPC), and results were compared to those obtained by the standard culture method. Methods and Results: The method includes a direct viable count (DVC) assay, multi‐probes labelled and unlabelled (helpers) to detect specifically viable E. coli cells and to enhance SPC cell counts. We demonstrate that helpers increase the fluorescence intensity of hybridized E. coli cells as detected by SPC and assess the high specificity of the DVC–FISH procedure on a large panel of cultured strains. Application to seawater, freshwater and wastewater samples showed a good correlation between SPC cells counts and standard plate counts. Conclusion: The high specificity of the procedure was demonstrated as well as its accuracy for detecting and counting viable E. coli cells in environmental samples. Significance and Impact of the Study: The developed approach may be used to monitor faecal contamination sources and to investigate the occurrence of viable E. coli in natural environments.  相似文献   

5.
The reported fate of Escherichia coli in the environment ranges from extended persistence to rapid decline. Incomplete understanding of factors that influence survival hinders risk assessment and modeling of the fate of fecal indicator bacteria (FIB) and pathogens. FIB persistence in subtropical aquatic environments was explored in outdoor mesocosms inoculated with five E. coli strains. The manipulated environmental factors were (i) presence or absence of indigenous microbiota (attained by natural, disinfected, and cycloheximide treatments), (ii) freshwater versus seawater, and (iii) water column versus sediment matrices. When indigenous microbes were removed (disinfected), E. coli concentrations decreased little despite exposure to sunlight. Conversely, under conditions that included the indigenous microbiota (natural), significantly greater declines in E. coli occurred regardless of the habitat. The presence of indigenous microbiota and matrix significantly influenced E. coli decline, but their relative importance differed in freshwater versus seawater. Cycloheximide, which inhibits protein synthesis in eukaryotes, significantly diminished the magnitude of E. coli decline in water but not in sediments. The inactivation of protozoa and bacterial competitors (disinfected) caused a greater decline in E. coli than cycloheximide alone in water and sediments. These results indicate that the autochthonous microbiota are an important contributor to the decline of E. coli in fresh and seawater subtropical systems, but their relative contribution is habitat dependent. This work advances our understanding of how interactions with autochthonous microbiota influence the fate of E. coli in aquatic environments and provides the framework for studies of the ecology of enteric pathogens and other allochthonous bacteria in similar environments.  相似文献   

6.
The history of colibacteria The E. coli bacterium discovered by Theodor Escherich 125 years ago has influenced the development of molecular‐biological research and medicinal and industrial biotechnology like no other bacterium. In particular, the characteristics of the K12‐strain with respect to apathogenicity, culturability and transformability made E. coli the “workhorse” of geneticists and molecular biologists. The easiness with which genetically modified E. coli can be made let this bacterium become a popular production organism of modern biotechnology for the making of drugs and fine chemicals. As a physiological inhabitant of the intestine of humans and animals, E. coli is used as an indicator organism of faecal pollution of ground and drinking water. Alongside its micro‐ecological role in the gastrointestinal tract, the E. coli bacterium, in terms of pathogenic strains, also has significance as a causative agent of diarrhoeal diseases.  相似文献   

7.
Microbial water quality monitoring for the presence of faecal indicator bacteria (FIB) is a mandatory activity in many countries and is key in public health protection. Despite technological advances and a need for methodological improvements, chromogenic and fluorogenic enzymatic techniques remain the mainstays of water quality monitoring for both public health agencies and regulated utilities. We demonstrated that bioelectroanalytical approaches to FIB enumeration are possible and can be achieved using commercially available enzyme‐specific resorufin glycosides, although these are expensive, not widely available or designed for purpose. Following this, we designed two naphthoquinone glycosides which performed better, achieving Escherichia coli detection in the range 5.0 × 102 to 5.0 × 105 CFU ml?1 22–54% quicker than commercially available resorufin glycosides. The molecular design of the naphthoquinone glycosides requires fewer synthetic steps allowing them to be produced for as little as US$50 per kg. Tests with environmental samples demonstrated the low tendency for abiotic interference and that, despite specificity being maintained between β‐glucuronidase and β‐galactosidase, accurate enumeration of E. coli in environmental samples necessitates development of a selective medium. In comparison to a commercially available detection method, which has U.S. Environmental Protection Agency (EPA) approval, our approach performed better at high organism concentrations, detecting 500 organisms in 9 h compared with 13.5 h for the commercial method. Bioelectroanalytical detection is comparable to current approved methods and with further development could result in improved detection times. A recent trend for low‐cost open‐source hardware means that automated, potentiostatically controlled E. coli detection systems could be constructed for less than US$100 per channel.  相似文献   

8.
The presence of coliform bacteria, faecal coliforms, Escherichia coli, diarrhoeagenic E. coli pathotypes (DEP) and Salmonella were determined in ready‐to‐eat cooked vegetable salads (RECS) from restaurants in Pachuca city, Mexico. The RECS were purchased from three types of restaurants: national chain restaurants (A), local restaurants (B) and small restaurants (C). Two restaurants for each A and B, and three for C, were included. Forty RECS samples were purchased at each A and B restaurant and 20 at each C restaurant. Of the overall total of 220 analysed samples, 100, 98·2, 72·3, 4·1 and 4·1% had coliform bacteria, faecal coliforms, E. coli, DEP and Salmonella, respectively. Identified DEP included enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC) and Shiga toxin‐producing E. coli (STEC). The EPEC, ETEC and STEC were isolated each from 1·4% of samples. No E. coli O157:H7 were detected in any STEC‐positive samples. The analysis of Kruskal–Wallis anova and median test of microbiological data showed that the microbiological quality of RECS did not differ between the different restaurants (P > 0·05).

Significance and Impact of the Study

This is the first report regarding microbiological quality and Salmonella, enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC) and Shiga toxin‐producing E. coli (STEC) isolation from ready‐to‐eat cooked vegetable salads from Mexican restaurants. Ready‐to‐eat cooked vegetable salads could be an important factor contributing to the endemicity of EPEC, ETEC and STEC, and Salmonella caused gastroenteritis in Mexico.  相似文献   

9.
Aims: This study aims to investigate the ecology of coliphages, an important microbial pollution indicator. Specifically, our experiments address (i) the ability of environmental Escherichia coli (E. coli) to serve as hosts for coliphage replication, and (ii) the temporal and spatial distribution of coliphages in coastal waters. Methods and Results: Water samples from three locations in California’s Newport Bay watershed were tested for the presence of coliphages every 2 weeks for an entire year. A total of nine E. coli strains isolated from various sources served as hosts for coliphage detection. Coliphage occurrence was significantly different between freshwater, estuarine and coastal locations and correlated with water temperature, salinity and rainfall in the watershed. The coliphages isolated on the environmental hosts had a broad host‐range relative to the coliphages isolated on an E. coli strain from sewage and a US EPA recommended strain for coliphage detection. Conclusions: Coliphage occurrence was related to the temperature, rainfall and salinity within the bay. The adaptation to a broad host‐range may enable the proliferation of coliphages in the aquatic environment. Significance and Impact of the Study: Understanding the seasonal variation of phages is useful for establishing a background level of coliphage presence in coastal waters. The broad host‐range of coliphages isolated on the environmental E. coli host calls for investigation of coliphage replication in the aquatic environment.  相似文献   

10.

Aims

To determine the herd prevalence of Enterobacteriaceae producing CTX‐M‐type extended‐spectrum β‐lactamases (ESBLs) among 381 dairy farms in Japan.

Methods and Results

Between 2007 and 2009, we screened 897 faecal samples using BTB lactose agar plates containing cefotaxime (2 μg ml?1). Positive isolates were tested using ESBL confirmatory tests, PCR and sequencing for CTX‐M, AmpC, TEM and SHV. The incidence of Enterobacteriaceae producing CTX‐M‐15 (= 7), CTX‐M‐2 (= 12), CTX‐M‐14 (= 3), CMY‐2 (= 2) or CTX‐M‐15/2/14 and CMY‐2 (= 4) in bovine faeces was 28/897 (3·1%) faecal samples. These genes had spread to Escherichia coli (= 23) and three genera of Enterobacteriaceae (= 5). Herd prevalence was found to be 20/381 (5·2%) dairy farms. The 23 E. coli isolates showed clonal diversity, as assessed by multilocus sequence typing and pulsed‐field gel electrophoresis. The pandemic E. coli strain ST131 producing CTX‐M‐15 or CTX‐M‐27 was not detected.

Conclusions

Three clusters of CTX‐M (CTX‐M‐15, CTX‐M‐2, CTX‐M‐14) had spread among Japanese dairy farms.

Significance and Impact of the Study

This is the first report on the prevalence of multidrug‐resistant CTX‐M‐15–producing E. coli among Japanese dairy farms.  相似文献   

11.
The specific processes that facilitate pathogen transmission are poorly understood, particularly for wild animal populations. A major impediment for investigating transmission pathways is the need for simultaneous information on host contacts and pathogen transfer. In this study, we used commensal Escherichia coli strains as a model system for gastrointestinal pathogens. We combined strain‐sharing information with detailed host contact data to investigate transmission routes in mountain brushtail possums. Despite E. coli being transmitted via the faecal‐oral route, we revealed that, strain‐sharing among possums was better explained by host contacts than spatial proximity. Furthermore, and unexpectedly, strain‐sharing was more strongly associated with the duration of brief nocturnal associations than day‐long den‐sharing. Thus, the most cryptic and difficult associations to measure were the most relevant connections for the transmission of this symbiont. We predict that future studies that employ similar approaches will reveal the importance of previously overlooked associations as key transmission pathways.  相似文献   

12.
Aims: This study evaluated the applicability of standard faecal indicator bacteria (SFIB) for alpine mountainous water resources monitoring. Methods and Results: Escherichia coli, enterococci (ENTC) and Clostridium perfringens were investigated by standard or frequently applied phenotypic and genotypic methods in a broad range of animal and human faecal sources in a large alpine mountainous area. Clostridium perfringens occurred only in human, livestock and carnivorous source groups in relevant average concentrations (log 4·7–7·0 CFU g?1) but not in herbivorous wildlife sources. Escherichia coli proved to be distributed in all faecal source groups with remarkably balanced average concentrations (log 7·0–8·4 CFU g?1). Except for single faecal samples from the cattle source group, prevalence rates for ENTC source groups were generally >87% with average concentrations of log 5·3–7·7 CFU g?1. To test the faecal indication capacity in the environment, faecal prevalence data were comparatively analysed with results from the concurrently performed multi‐parametric microbial source tracking effort on karst spring water quality from the investigated alpine mountainous catchment ( Reischer et al. 2008 ; Environ Microbiol 10:2598–2608). Conclusion: Escherichia coli and enterococci are reliable faecal indicators for alpine mountainous water resources monitoring, although E. coli is the more sensitive one. Clostridium perfringens did not prove to be an indicator of general faecal pollution but is suggested a conservative microbial source tracking marker for anthropogenic faecal influence. Significance and Impact of the Study: Applicability of SFIB is currently hotly debated. This is the first study providing comprehensive information on the applicability of SFIB at alpine mountainous habitats.  相似文献   

13.
There is a growing interest in the use of microalgae as low‐cost hosts for the synthesis of recombinant products such as therapeutic proteins and bioactive metabolites. In particular, the chloroplast, with its small, genetically tractable genome (plastome) and elaborate metabolism, represents an attractive platform for genetic engineering. In Chlamydomonas reinhardtii, none of the 69 protein‐coding genes in the plastome uses the stop codon UGA, therefore this spare codon can be exploited as a useful synthetic biology tool. Here, we report the assignment of the codon to one for tryptophan and show that this can be used as an effective strategy for addressing a key problem in chloroplast engineering: namely, the assembly of expression cassettes in Escherichia coli when the gene product is toxic to the bacterium. This problem arises because the prokaryotic nature of chloroplast promoters and ribosome‐binding sites used in such cassettes often results in transgene expression in E. coli, and is a potential issue when cloning genes for metabolic enzymes, antibacterial proteins and integral membrane proteins. We show that replacement of tryptophan codons with the spare codon (UGG→UGA) within a transgene prevents functional expression in E. coli and in the chloroplast, and that co‐introduction of a plastidial trnW gene carrying a modified anticodon restores function only in the latter by allowing UGA readthrough. We demonstrate the utility of this system by expressing two genes known to be highly toxic to E. coli and discuss its value in providing an enhanced level of biocontainment for transplastomic microalgae.  相似文献   

14.
15.
Aims: To determine the presence of antibiotic‐resistant faecal Escherichia coli and Enterococcus spp. in feral pigeons (Columba livia forma domestica) in the Czech Republic. Methods and Results: Cloacal swabs of feral pigeons collected in the city of Brno in 2006 were cultivated for antibiotic‐resistant E. coli. Resistance genes, class 1 and 2 integrons, and gene cassettes were detected in resistant isolates by polymerase chain reaction (PCR). The samples were also cultivated for enterococci. Species status of enterococci isolates was determined using repetitive extragenic palindromic‐PCR. Resistance genes were detected in resistant enterococci by PCR. E. coli isolates were found in 203 of 247 pigeon samples. Antibiotic resistance was recorded in three (1·5%, nE. coli = 203) isolates. Using agar containing ciprofloxacin, 12 (5%, nsamples = 247) E. coli strains resistant to ciprofloxacin were isolated. No ESBL‐producing E. coli isolates were detected. A total of 143 enterococci were isolated: Ent. faecalis (36 isolates), Ent. faecium (27), Ent. durans (19), Ent. hirae (17), Ent. mundtii (17), Ent. gallinarum (12), Ent. casseliflavus (12) and Ent. columbae (3). Resistance to one to four antibiotics was detected in 45 (31%) isolates. Resistances were determined by tetK, tetL, tetM, tetO, aac(6′)aph(2′′), ant(4′)‐Ia, aph(3′)‐IIIa, ermB, pbp5, vanA and vanC1 genes. Conclusions: Antibiotic‐resistant E. coli and Enterococcus spp. occurred in feral pigeons in various prevalences. Significance and Impact of the Study: Feral pigeon should be considered a risk species for spreading in the environment antimicrobial resistant E. coli and enterococci.  相似文献   

16.
Under stress conditions, such as nutrient deprivation, bacteria enter into a hibernation stage, which is characterized by the appearance of 100S ribosomal particles. In Escherichia coli, dimerization of 70S ribosomes into 100S requires the action of the ribosome modulation factor (RMF) and the hibernation‐promoting factor (HPF). Most other bacteria lack RMF and instead contain a long form HPF (LHPF), which is necessary and sufficient for 100S formation. While some structural information exists as to how RMF and HPF mediate formation of E. coli 100S (Ec100S), structural insight into 100S formation by LHPF has so far been lacking. Here we present a cryo‐EM structure of the Bacillus subtilis hibernating 100S (Bs100S), revealing that the C‐terminal domain (CTD) of the LHPF occupies a site on the 30S platform distinct from RMF. Moreover, unlike RMF, the BsHPF‐CTD is directly involved in forming the dimer interface, thereby illustrating the divergent mechanisms by which 100S formation is mediated in the majority of bacteria that contain LHPF, compared to some γ‐proteobacteria, such as E. coli.  相似文献   

17.
Aims: Escherichia coli has emerged as a viable heterologous host for the production of complex, polyketide natural compounds. In this study, polyketide biosynthesis was compared between different E. coli strains for the purpose of better understanding and improving heterologous production. Methods and Results: Both B and K‐12 E. coli strains were genetically modified to support heterologous polyketide biosynthesis [specifically, 6‐deoxyerythronolide B (6dEB)]. Polyketide production was analysed using a helper plasmid designed to overcome rare codon usage within E. coli. Each strain was analysed for recombinant protein production, precursor consumption, by‐product production, and 6dEB biosynthesis. Of the strains tested for biosynthesis, 6dEB production was greatest for E. coli B strains. When comparing biosynthetic improvements as a function of mRNA stability vs codon bias, increased 6dEB titres were observed when additional rare codon tRNA molecules were provided. Conclusions: Escherichia coli B strains and the use of tRNA supplementation led to improved 6dEB polyketide titres. Significance and Impact of the Study: Given the medicinal potential and growing field of polyketide heterologous biosynthesis, the current study provides insight into host‐specific genetic backgrounds and gene expression parameters aiding polyketide production through E. coli.  相似文献   

18.
In vitro experimental evolution has taught us many lessons on the molecular bases of adaptation. To move towards more natural settings, evolution in the mice gut has been successfully performed. Yet, these experiments suffered from the use of laboratory strains as well as the use of axenic or streptomycin‐treated mice to maintain the inoculated strains. To circumvent these limitations, we conducted a one‐year experimental evolution in vivo using a natural isolate of E. coli, strain 536, in conditions mimicking as much as possible natural environment with mother‐to‐offspring microbiota transmission. Mice were then distributed in 24 independent cages and separated into two different diets: a regular one (chow diet, CD) and high‐fat and high‐sugar one (Western Diet, WD). Genome sequences revealed an early and rapid selection during the breastfeeding period that selected the constitutive expression of the well‐characterized lactose operon. E. coli was lost significantly more in CD than WD; however, we could not detect any genomic signature of selection, nor any diet specificities during the later part of the experiments. The apparently neutral evolution presumably due to low population size maintained nevertheless at high frequency the early selected mutations affecting lactose regulation. The rapid loss of lactose operon regulation challenges the idea that plastic gene expression is both optimal and stable in the wild.  相似文献   

19.
Converting renewable feedstocks to aromatic compounds using engineered microbes offers a robust approach for sustainable, environment‐friendly, and cost‐effective production of these value‐added products without the reliance on petroleum. In this study, rationally designed E. coli–E. coli co‐culture systems were established for converting glycerol to 3‐hydroxybenzoic acid (3HB). Specifically, the 3HB pathway was modularized and accommodated by two metabolically engineered E. coli strains. The co‐culture biosynthesis was optimized by using different cultivation temperatures, varying the inoculum ratio between the co‐culture strains, recruitment of a key pathway intermediate transporter, strengthening the critical pathway enzyme expression, and adjusting the timing for inducing pathway gene expression. Compared with the E. coli mono‐culture, the optimized co‐culture showed 5.3‐fold improvement for 3HB biosynthesis. This study demonstrated the applicability of modular co‐culture engineering for addressing the challenges of aromatic compound biosynthesis.  相似文献   

20.
Hybridization between distantly related organisms can facilitate rapid adaptation to novel environments, but is potentially constrained by epistatic fitness interactions among cell components. The zoonotic pathogens Campylobacter coli and C. jejuni differ from each other by around 15% at the nucleotide level, corresponding to an average of nearly 40 amino acids per protein‐coding gene. Using whole genome sequencing, we show that a single C. coli lineage, which has successfully colonized an agricultural niche, has been progressively accumulating C. jejuni DNA. Members of this lineage belong to two groups, the ST‐828 and ST‐1150 clonal complexes. The ST‐1150 complex is less frequently isolated and has undergone a substantially greater amount of introgression leading to replacement of up to 23% of the C. coli core genome as well as import of novel DNA. By contrast, the more commonly isolated ST‐828 complex bacteria have 10–11% introgressed DNA, and C. jejuni and nonagricultural C. coli lineages each have <2%. Thus, the C. coli that colonize agriculture, and consequently cause most human disease, have hybrid origin, but this cross‐species exchange has so far not had a substantial impact on the gene pools of either C. jejuni or nonagricultural C. coli. These findings also indicate remarkable interchangeability of basic cellular machinery after a prolonged period of independent evolution.  相似文献   

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