Plasmids of incompatibility group A/C (IncA/C) are becoming increasingly prevalent within pathogenic Enterobacteriaceae They are associated with the dissemination of multiple clinically relevant resistance genes, including blaCMY and blaNDM Current typing methods for IncA/C plasmids offer limited resolution. In this study, we present the complete sequence of a blaNDM-1-positive IncA/C plasmid, pMS6198A, isolated from a multidrug-resistant uropathogenic Escherichia coli strain. Hypersaturated transposon mutagenesis, coupled with transposon-directed insertion site sequencing (TraDIS), was employed to identify conserved genetic elements required for replication and maintenance of pMS6198A. Our analysis of TraDIS data identified roles for the replicon, including repA, a toxin-antitoxin system; two putative partitioning genes, parAB; and a putative gene, 053 Construction of mini-IncA/C plasmids and examination of their stability within E. coli confirmed that the region encompassing 053 contributes to the stable maintenance of IncA/C plasmids. Subsequently, the four major maintenance genes (repA, parAB, and 053) were used to construct a new plasmid multilocus sequence typing (PMLST) scheme for IncA/C plasmids. Application of this scheme to a database of 82 IncA/C plasmids identified 11 unique sequence types (STs), with two dominant STs. The majority of blaNDM-positive plasmids examined (15/17; 88%) fall into ST1, suggesting acquisition and subsequent expansion of this blaNDM-containing plasmid lineage. The IncA/C PMLST scheme represents a standardized tool to identify, track, and analyze the dissemination of important IncA/C plasmid lineages, particularly in the context of epidemiological studies.
Local Thai and imported Malaysian beef in southern Thailand area carry several Shiga toxin-producing Escherichia coli (STEC) serotypes. STEC O104 is an important pathogen capable of causing outbreaks with considerable morbidity and mortality. This study investigated the presence of E. coli O104 from local Thai and imported Malaysian beef obtained from markets in Hat Yai City, Songkhla Province during August 2015 - February 2016. Thirty-one E. coli O104 strains were isolated from 12 beef samples (16% and 23% Thai and imported Malaysian, respectively). Thirty strains possessed aggA (coding for a major component of AAF/I fimbriae), a gene associated with enteroaggregative E. coli (EAEC) pathotype, and all strains carried fimH (encoding Type 1 fimbriae). Thirty strains belonged to phylogenetic group B1 and one strain (from Malaysian beef) to group A. Agglutination of yeast cells was observed among 29 E. coli O104 strains. Investigation of stx2 phage occupancy loci demonstrated that sbcB was occupied in 12 strains. Antimicrobial susceptibility assay revealed that 7 strains were resistant to at least one antimicrobial agent and two were multi-drug resistant. One strain carried extended spectrum β-lactamase gene blaCTX-M and three carried blaTEM. PFGE-generated DNA profiling showed identical DNA pattern between that of one EAEC O104 strain from Thai beef and another from Malaysian beef, indicating that these two strains originated from the same clone. This is the first report in Thailand describing the presence of EAEC O104 from both Thai and imported Malaysian beef and their transfer between both countries. Thorough surveillance of this pathogen in fresh meats and vegetables should help to prevent any possible outbreak of E. coli O104.
This study aimed at determining the presence and characterization of Escherichia coli and Shiga toxin-producing E. coli (STEC) from imported frozen beef meats. Seventy-four (74) frozen imported beef meat samples from two countries, India (42 samples) and Australia (32 samples), were collected and tested for E. coli. These samples were purchased from the frozen meat sections of five different supermarkets in different locations in Selangor, Malaysia, from April 2012 to October 2014. A total of 222 E. coli strains were isolated from the meat samples; 126 strains were isolated from country A (India), and 96 E. coli strains were from country of origin B (Australia), respectively. A total of 70 E. coli strains were identified and characterized. All E. coli strains were isolated into Fluorocult medium and identified using API 20E kit. All selected E. coli strains were characterized for Shiga toxin genes (stx1 and stx2). All biochemically identified E. coli in this study were further subjected to molecular detection through polymerase chain reaction (PCR) amplification and characterization using 16S ribosomal RNA (rRNA) gene of Shiga toxin-producing E. coli. Of the 70 E. coli strains, 11 strains were positive for both Shiga toxin genes (stx1 and stx2) and 11 (11/70) strains were positive for stx1 gene, while 25 (25/70) strains were positive for stx2 gene. The analysis of 16S rRNA gene of all the E. coli isolates in this study was successfully sequenced and analyzed, and based on sequence data obtained, a phylogenetic tree of the 16S rRNA gene was performed using Clustal W programme in MEGA 6.06 software. Phylogenetic tree showed that the E. coli isolates in our study cluster with the strain of E. coli isolated in other countries, which further confirm that the isolates of E. coli in this study are similar to those obtained in other studies. As a result, all the strains obtained in this study proved to be a strain of pathogenic E. coli, which may cause a serious outbreak of food-borne disease. The isolation of pathogenic E. coli strains from the imported meat samples calls for prudent management of imported meats by the relevant authorities.
In view of the epidemiological success of CTX-M-15-producing lineages of Escherichia coli and particularly of sequence type 131 (ST131), it is of significant interest to explore its prevalence in countries such as India and to determine if antibiotic resistance, virulence, metabolic potential, and/or the genetic architecture of the ST131 isolates differ from those of non-ST131 isolates. A collection of 126 E. coli isolates comprising 43 ST131 E. coli, 40 non-ST131 E. coli, and 43 fecal E. coli isolates collected from a tertiary care hospital in India was analyzed. These isolates were subjected to enterobacterial repetitive intergenic consensus (ERIC)-based fingerprinting, O typing, phylogenetic grouping, antibiotic sensitivity testing, and virulence and antimicrobial resistance gene (VAG) detection. Representative isolates from this collection were also analyzed by multilocus sequence typing (MLST), conjugation, metabolic profiling, biofilm production assay, and zebra fish lethality assay. All of the 43 ST131 E. coli isolates were exclusively associated with phylogenetic group B2 (100%), while most of the clinical non-ST131 and stool non-ST131 E. coli isolates were affiliated with the B2 (38%) and A (58%) phylogenetic groups, respectively. Significantly greater proportions of ST131 isolates (58%) than non-ST131 isolates (clinical and stool E. coli isolates, 5% each) were technically identified to be extraintestinal pathogenic E. coli (ExPEC). The clinical ST131, clinical non-ST131, and stool non-ST131 E. coli isolates exhibited high rates of multidrug resistance (95%, 91%, and 91%, respectively), extended-spectrum-β-lactamase (ESBL) production (86%, 83%, and 91%, respectively), and metallo-β-lactamase (MBL) production (28%, 33%, and 0%, respectively). CTX-M-15 was strongly linked with ESBL production in ST131 isolates (93%), whereas CTX-M-15 plus TEM were present in clinical and stool non-ST131 E. coli isolates. Using MLST, we confirmed the presence of two NDM-1-positive ST131 E. coli isolates. The aggregate bioscores (metabolite utilization) for ST131, clinical non-ST131, and stool non-ST131 E. coli isolates were 53%, 52%, and 49%, respectively. The ST131 isolates were moderate biofilm producers and were more highly virulent in zebra fish than non-ST131 isolates. According to ERIC-based fingerprinting, the ST131 strains were more genetically similar, and this was subsequently followed by the genetic similarity of clinical non-ST131 and stool non-ST131 E. coli strains. In conclusion, our data provide novel insights into aspects of the fitness advantage of E. coli lineage ST131 and suggest that a number of factors are likely involved in the worldwide dissemination of and infections due to ST131 E. coli isolates.
Maintenance of recombinant plasmid vectors in host bacteria relies on the presence of selection antibiotics in the growth media to suppress plasmid -free segregants. However, presence of antibiotic resistance genes and antibiotics themselves is not acceptable in several applications of biotechnology. Previously, we have shown that FabV-Triclosan selection system can be used to select high and medium copy number plasmid vectors in E. coli. Here, we have extended our previous work and demonstrated that expression vectors containing FabV can be used efficiently to express heterologous recombinant proteins in similar or better amounts in E. coli host when compared with expression vectors containing β-lactamase. Use of small amount of non-antibiotic Triclosan as selection agent in growth medium, enhanced plasmid stability, applicability in various culture media, and compatibility with other selection systems for multiple plasmid maintenance are noteworthy features of FabV-Triclosan selection system.
Escherichia coli ST131 is now recognised as a leading contributor to urinary tract and bloodstream infections in both community and clinical settings. Here we present the complete, annotated genome of E. coli EC958, which was isolated from the urine of a patient presenting with a urinary tract infection in the Northwest region of England and represents the most well characterised ST131 strain. Sequencing was carried out using the Pacific Biosciences platform, which provided sufficient depth and read-length to produce a complete genome without the need for other technologies. The discovery of spurious contigs within the assembly that correspond to site-specific inversions in the tail fibre regions of prophages demonstrates the potential for this technology to reveal dynamic evolutionary mechanisms. E. coli EC958 belongs to the major subgroup of ST131 strains that produce the CTX-M-15 extended spectrum β-lactamase, are fluoroquinolone resistant and encode the fimH30 type 1 fimbrial adhesin. This subgroup includes the Indian strain NA114 and the North American strain JJ1886. A comparison of the genomes of EC958, JJ1886 and NA114 revealed that differences in the arrangement of genomic islands, prophages and other repetitive elements in the NA114 genome are not biologically relevant and are due to misassembly. The availability of a high quality uropathogenic E. coli ST131 genome provides a reference for understanding this multidrug resistant pathogen and will facilitate novel functional, comparative and clinical studies of the E. coli ST131 clonal lineage.
Escherichia coli strains isolated from patients with diarrhea or hemolytic uremic syndrome (HUS) at Pusan University Hospital, South Korea, between 1990 and 1996 were examined for traits of the O157:H7 serogroup. One strain isolated from a patient with HUS belonged to the O157:H7 serotype, possessed a 60-MDa plasmid, the eae gene, and ability to produce Shiga toxin 1 but not Shiga toxin 2. Arbitrarily primed PCR analysis suggested that this strain is genetically very close to a O157:H7 strain isolated in Japan.
Amongst 107 diarrheal cases studied a bacterial agent was isolated from 71 (66%) cases of which 60 (85%) were due to a single agent and the remaining 11 (15%) were of mixed infections. Enterotoxigenic Escherichia coli (ETEC) was isolated from 65 cases. Other pathogens isolated included Salmonella spp, Shigella spp and rotavirus. There was a higher isolation rate of ETEC from females and rotavirus from males. The infection rate was found to higher for the 0-2 year age group as compared to the 3-5 year age group. Amongst the ETEC isolated the STa 2 toxotype was the predominant type.
To explore new approaches of phage-based bio-process of specifically pathogenic Escherichia coli bacteria in food products within a short period. One hundred and forty highly lytic designed coliphages were used. Escherichia coli naturally contaminated and Enterohemorrhagic Escherichia coli experimentally inoculated samples of lettuce, cabbage, meat, and egg were used. In addition, experimentally produced biofilms of E. coli were tested. A phage concentration of 10(3) PFU/ml was used for food products immersion, and for spraying of food products, 10(5) PFU/ml of a phage cocktail was used by applying a 20-s optimal dipping time in a phage cocktail. Food samples were cut into pieces and were either sprayed with or held in a bag immersed in lambda buffer containing a cocktail of 140 phages. Phage bio-processing was successful in eliminating completely E. coli in all processed samples after 48 h storage at 4°C. Partial elimination of E. coli was observed in earlier storage periods (7 and 18 h) at 24° and 37°C. Moreover, E. coli biofilms were reduced >3 log cycles upon using the current phage bio-processing. The use of a phage cocktail of 140 highly lytic designed phages proved highly effective in suppressing E. coli contaminating food products. Proper decontamination/prevention methods of pathogenic E. coli achieved in this study can replace the current chemically less effective decontamination methods.
We report a case of neonatal Bartter syndrome in a 31 weeks premature baby girl with antenatal unexplained polyhydramnios requiring amnioreduction. She presented with early onset E. coli septicaemia and severe dehydration leading to pre-renal renal impairment which obscure the typical biochemical changes of hypokalaemic hypochloraemic metabolic alkalosis.
The emergence of Escherichia coli resistant to extended-spectrum cephalosporins (ESCs) is of concern as ESC is often used to treat infections by Gram-negative bacteria. One-hundred and ten E. coli strains isolated in 2009-2010 from children warded in a Malaysian tertiary hospital were analyzed for their antibiograms, carriage of extended-spectrum beta-lactamase (ESBL) and AmpC genes, possible inclusion of the beta-lactamase genes on an integron platform, and their genetic relatedness. All E. coli strains were sensitive to carbapenems. About 46% of strains were multidrug resistant (MDR; i.e., resistant to ≥3 antibiotic classes) and almost half (45%) were nonsusceptible to ESCs. Among the MDR strains, high resistance rates were observed for ampicillin (98%), tetracycline (75%), and trimethoprim/sulfamethoxazole (73%). Out of 110 strains, bla(TEM-1) (49.1%), bla(CTX-M) (11.8%), and bla(CMY-2) (6.4%) were detected. Twenty-one strains were ESBL producers. CTX-M-15 was the predominant CTX-M variant found and this is the first report of a CTX-M-27-producing E. coli strain from Malaysia. Majority (3.1%) of the strains harbored class 1 integron-encoded integrases with a predominance of aadA and dfr genes within the integron variable region. No gene cassette encoding ESBL genes was found and integrons were not significantly associated with ESBL or non-ESBL producers. Possible clonal expansion was observed for few CTX-M-15-positive strains but the O25-ST131 E. coli clone known to harbor CTX-M-15 was not detected while CMY-2-positive strains were genetically diverse.
A baseline study was carried out to assess the metal concentrations and microbial contamination at selected Lake waters in and around Miri City, East Malaysia. Sixteen surface water samples were collected at specific Lakes in the environs of major settlement areas and recreational centers in Miri City. The Physico-chemical parameters [pH, Electrical Conductivity (EC) and Dissolved Oxygen (DO)], metals (Fe, Mn, Cu, Cd, Ni and Zn) and Escherichia coli (E. coli) were analysed. The concentrations of Fe, Mn and Ni have been found to be above the permissible limits of drinking water quality standards. The metals data have also been used for the calculation of heavy metal pollution index. Higher values of E. coli indicate microbial contamination in the Lake waters.
The aim of this study was to explore the utilization of polymeric membrane for bio-sensing application in most efficient and rapid way. Customization of membrane formulation via phase separation study to modify its morphologies and properties enable the detection of different pathogens in a specific manner. Experimental findings (FESEM, through-pore distribution, porosity, capillary flow test and protein binding test) verified the predictions of faster capillary flow time and higher membrane's protein binding by the addition of cellulose acetate and nitrocellulose to the membrane casting dope, respectively. Throughout the phase separation study, the potential phase behavior was investigated, which was correlating various membrane structures to its performances for potential pathogens detection in water.
The efficacy of bacteriophage EC1, a lytic bacteriophage, against Escherichia coli O78:K80, which causes colibacillosis in poultry, was determined in the present study. A total of 480 one-day-old birds were randomly assigned to 4 treatments groups, each with 4 pens of 30 birds. Birds from the control groups (groups I and II) received PBS (pH 7.4) or 10(10) pfu of bacteriophage EC1, respectively. Group III consisted of birds challenged with 10(8) cfu of E. coli O78:K80 and treated with 10(10) pfu of bacteriophage EC1 at 2 h postinfection, whereas birds from group IV were challenged with 10(8) cfu of E. coli O78:K80 only. All the materials were introduced into the birds by intratracheal inoculation. Based on the results of the present study, the infection was found to be less severe in the treated E. coli-challenged group. Mean total viable cell counts of E. coli identified on eosin methylene blue agar (designated EMB + E. coli) in the lungs were significantly lower in treated, E. coli-challenged birds than in untreated, E. coli-challenged birds on d 1 and 2 postinfection. The EMB + E. coli isolation frequency was also lower in treated birds; no E. coli was detectable in blood samples on any sampling day, and E. coli were isolated only in the liver, heart, and spleen of treated chickens at a ratio of 2/6, 1/6, and 3/6, respectively, at d 1 postinfection. The BW of birds from the E. coli-challenged group treated with bacteriophage EC1 were not significantly different from those of birds from both control groups but were 15.4% higher than those of the untreated, E. coli-challenged group on d 21 postinfection. The total mortality rate of birds during the 3-wk experimental period decreased from 83.3% in the untreated, E. coli-challenged birds (group IV) to 13.3% in birds treated with bacteriophage EC1 (group III). These results suggest that bacteriophage EC1 is effective in vivo and could be used to treat colibacillosis in chickens.
Consumption of probiotics has been associated with decreased risk of colon cancer and reported to have antimutagenic/ anti-carcinogenic properties. One possible mechanism for this effect involves physical binding of the mutagenic compounds, such as heterocyclic amines (HCAs), to the bacteria. Therefore, the objective of this study was to examine the binding capacity of bifidobacterial strains of human origin on mutagenic heterocyclic amines which are suspected to play a role in human cancers. In vitro binding of the mutagens Trp-p-2, IQ, MeIQx, 7,8DiMeIQx and PhIP by three bacterial strains in two media of different pH was analysed using high performance liquid chromatography. Bifidobacterium pseudocatenulatum G4 showed the highest decrease in the total HCAs content, followed by Bifidobacterium longum, and Escherichia coli. pH affects binding capacity; the highest binding was obtained at pH 6.8. Gram-positive tested strains were found to be consistently more effective than the gram-negative strain. There were significant decreases in the amount of HCAs in the presence of different cell concentrations of B. pseudocatenulatum G4; the highest decrease was detected at the concentration of 10(10) cfu/ml. The results showed that HCAs were able to bind with all bacterial strains tested in vitro, thus it may be possible to decrease their absorption by human intestine and increase their elimination via faeces.
Green fluorescent protein (GFP) is a versatile reporter protein and has been widely used in biological research. However, its quantitation requires expensive equipment such as a spectrofluorometer. In the current study, a gel documentation imaging system using a native polyacrylamide gel for the quantitation of GFP was developed. The assay was evaluated for its precision, linearity, reproducibility, and sensitivity in the presence of Escherichia coli cells and was compared with the spectrofluorometric method. Using this newly established, gel-based imaging technique; the amount of GFP can be quantified accurately.
With advances in point-of-care testing (POCT), lateral flow assays (LFAs) have been explored for nucleic acid detection. However, biological samples generally contain complex compositions and low amounts of target nucleic acids, and currently require laborious off-chip nucleic acid extraction and amplification processes (e.g., tube-based extraction and polymerase chain reaction (PCR)) prior to detection. To the best of our knowledge, even though the integration of DNA extraction and amplification into a paper-based biosensor has been reported, a combination of LFA with the aforementioned steps for simple colorimetric readout has not yet been demonstrated. Here, we demonstrate for the first time an integrated paper-based biosensor incorporating nucleic acid extraction, amplification and visual detection or quantification using a smartphone. A handheld battery-powered heating device was specially developed for nucleic acid amplification in POC settings, which is coupled with this simple assay for rapid target detection. The biosensor can successfully detect Escherichia coli (as a model analyte) in spiked drinking water, milk, blood, and spinach with a detection limit of as low as 10-1000 CFU mL(-1), and Streptococcus pneumonia in clinical blood samples, highlighting its potential use in medical diagnostics, food safety analysis and environmental monitoring. As compared to the lengthy conventional assay, which requires more than 5 hours for the entire sample-to-answer process, it takes about 1 hour for our integrated biosensor. The integrated biosensor holds great potential for detection of various target analytes for wide applications in the near future.
Microbial strain optimisation for the overproduction of a desired phenotype has been a popular topic in recent years. Gene knockout is a genetic engineering technique that can modify the metabolism of microbial cells to obtain desirable phenotypes. Optimisation algorithms have been developed to identify the effects of gene knockout. However, the complexities of metabolic networks have made the process of identifying the effects of genetic modification on desirable phenotypes challenging. Furthermore, a vast number of reactions in cellular metabolism often lead to a combinatorial problem in obtaining optimal gene knockout. The computational time increases exponentially as the size of the problem increases. This work reports an extension of Bees Hill Flux Balance Analysis (BHFBA) to identify optimal gene knockouts to maximise the production yield of desired phenotypes while sustaining the growth rate. This proposed method functions by integrating OptKnock into BHFBA for validating the results automatically. The results show that the extension of BHFBA is suitable, reliable, and applicable in predicting gene knockout. Through several experiments conducted on Escherichia coli, Bacillus subtilis, and Clostridium thermocellum as model organisms, extension of BHFBA has shown better performance in terms of computational time, stability, growth rate, and production yield of desired phenotypes.