Isolation Procedure for CP E. coli from Caeca Samples under Review Towards an Increased Sensitivity

Due to the increasing reports of carbapenemase-producing Enterobacteriaceae (CPE) from livestock in recent years, the European Reference Laboratory for Antimicrobial Resistances (EURL-AR) provided a protocol for their recovery from caecum and meat samples. This procedure exhibited limitations for the detection of CPE with low carbapenem MIC values. Therefore, it was modified by a second, selective enrichment in lysogeny broth with cefotaxime (CTX 1 mg/L) and with meropenem (MEM 0.125 mg/L) at 37 °C under microaerophilic conditions. By Real-time PCR, these enrichments are pre-screened for the most common carbapenemase genes. Another adaptation was the use of in-house prepared MacConkey agar with MEM and MEM+CTX instead of commercial selective agar. According to the EURL-method, we achieved 100% sensitivity and specificity using the in-house media instead of commercial agar, which decreased the sensitivity to ~75%. Comparing the method with and without the second enrichment, no substantial influence on sensitivity and specificity was detected. Nevertheless, this enrichment has simplified the CPE-isolation regarding the accompanying microbiota and the separation of putative colonies. In conclusion, the sensitivity of the method can be increased with slight modifications.

Download Full-text

Selective enrichment of R−segregants as the main mechanism of ‘curing’ of the R factor by acridine dyes

Genetics Research ◽

10.1017/s001667230001199x ◽

1971 ◽

Vol 17 (1) ◽

pp. 9-16 ◽

Cited By ~ 4

Author(s):

Masanosuke Yoshikawa

Keyword(s):

Shigella Flexneri ◽

Host Cells ◽

Bacterial Strains ◽

Initial Inoculum ◽

Selective Enrichment ◽

E Coli ◽

R Factor ◽

Acridine Dyes ◽

Increased Sensitivity ◽

R Factors

SUMMARYBy the use of appropriate strains ofEscherichia coli, Shigella flexneriandSalmonella typhimuriumwith and without an R factor, R100, the mechanism of ‘curing’ of R factor by acridine dyes was examined. This R factor was shown to confer increased sensitivity to acriflavine upon the host cells.E. colistrain W-3630, once infected with R100, has never been observed to segregate R−cells. When mixtures of R+and R−cells of this strain were grown in acriflavine broth, the proportion of R−cells increased and was also correlated with the proportion in the initial inoculum. Other bacterial strains carrying R100segregate R~ cells spontaneously. Growth tests starting with varying proportion of R+and R−cells of these strains in acriflavine broth also gave a marked correlation between the initial and final proportions of R−cells, and indicated that the main cause of ‘curing’ the R factor was the selective enrichment of R−segregants present in the initial inocula or arising spontaneously during growth of the R+culture. These results suggest that the mechanisms underlying the ‘curing’ of F and R factors are different. Tests with several acridine dyes gave results similar to those with acriflavine.

Download Full-text

Prevalence and Antibiotic Resistance Profiles of Escherichia coli O157:H7 in Beef Products from Retail Outlets in Gaborone, Botswana

Journal of Food Protection ◽

10.4315/0362-028x-68.2.403 ◽

2005 ◽

Vol 68 (2) ◽

pp. 403-406 ◽

Cited By ~ 17

Author(s):

CLIFF A. MAGWIRA ◽

BERHANU A. GASHE ◽

ERNEST K. COLLISON

Keyword(s):

Escherichia Coli ◽

Antibiotic Resistance ◽

Escherichia Coli O157 ◽

Potassium Tellurite ◽

Selective Enrichment ◽

E Coli ◽

Retail Outlets ◽

Beef Products ◽

Meat Samples ◽

Minced Meat

Four hundred meat samples (134 meat cubes, 133 minced meat, 133 fresh sausages) were collected from 15 supermarkets and butcheries in Gaborone, Botswana, between the summer months of October 2002 and March 2003. Samples were assayed for Escherichia coli O157 by selective enrichment in modified E. coli broth containing novobiocin, followed by immuno-magnetic separation and plating onto sorbitol MacConkey agar supplemented with potassium tellurite. The isolates were biochemically and serologically confirmed by API 20E and O157 antisera, respectively. The prevalence rates for E. coli O157 were 5.22% in meat cube samples, 3.76% in minced meat samples, and 2.26% in fresh sausages. The isolates showed single, double, and triple antibiotic resistance. Fifty-three percent of them were resistant to cephalothin. Resistance was also recorded for sulphatriad (33%), colistin sulphate (26%), streptomycin (0.7%), and tetracycline (26%). It is recommended that the cause for antibiotic resistance be investigated using a larger number of samples from cattle, especially from ranching areas of the country.

Download Full-text

Occurrence of Shiga Toxin–Producing Escherichia coli O157 in Selected Dairy and Meat Products Marketed in the City of Rabat, Morocco

Journal of Food Protection ◽

10.4315/0362-028x-67.6.1234 ◽

2004 ◽

Vol 67 (6) ◽

pp. 1234-1237 ◽

Cited By ~ 13

Author(s):

N. BENKERROUM ◽

Y. BOUHLAL ◽

A. EL ATTAR ◽

A. MARHABEN

Keyword(s):

Escherichia Coli ◽

Meat Products ◽

Latex Agglutination ◽

Agglutination Test ◽

Escherichia Coli O157 ◽

Selective Enrichment ◽

E Coli ◽

Meat Samples ◽

Antigen Antibody ◽

The City

Samples of meat and dairy products taken from the city of Rabat, Morocco, were examined for the presence of Escherichia coli O157 by the selective enrichment procedure followed by plating on cefixime–tellurite–sorbitol MacConkey agar and a latex agglutination test. The ability of isolates to produce Shiga toxins (ST1 or ST2) was also tested by an agglutination test using sensitized latex. Dairy samples (n = 44) included different products commonly consumed in the country. Meat samples (n = 36) were taken from traditional butchers because these products are generally marketed in this way. Random samples were taken from each product during the period of January through May. Of the 80 samples tested, 8 (10%) harbored E. coli O157. Four dairy and four meat samples were contaminated (9.1 and 11.1%, respectively). Of 10 E. coli O157 isolates from contaminated samples demonstrating true antigen-antibody agglutination, 5 (50%) produced either ST2 alone or ST2 plus ST1. Four of the five strains (80%) were meat isolates and produced ST2 with or without ST1, and the fifth was a dairy isolate producing ST2.

Download Full-text

MOLECULAR DETECTION OF ENTEROHAEMORRHAGIC ESCHERICHIA COLI IN RAW BEEF MEAT FROM ABEOKUTA, OGUN STATE, NIGERIA

African Journal of Science and Nature ◽

10.46881/ajsn.v10i0.189 ◽

2020 ◽

Vol 10 ◽

pp. 164

Author(s):

Anotu Mopelola Deji-Agboola ◽

Olubunmi Adetokunbo Osinupebi ◽

Rotimi Tope Akinlalu

Keyword(s):

Escherichia Coli ◽

Shiga Toxin ◽

Serological Test ◽

Polymerase Chain Reaction Method ◽

Open Market ◽

Macconkey Agar ◽

Public Health Importance ◽

E Coli ◽

Enterohaemorrhagic Escherichia Coli ◽

Meat Samples

The presence of Enterohaemorrhagic Escherichia coli in beef and beef products is of public health importance. Therefore, the current study was carried out to evaluate the prevalence of E. coli serotype O157:H7 from raw beef collected from Abeokuta. Meat samples from selected abattoir and open beef omarket in Abeokuta were aseptically collected into sterile peptone water, incubate at 37C for 24 hours and subcultured on MacConkey agar. The bacteria isolated were identified using standard method and owere cultured on Sorbitol MacConkey agar, incubated at 37C for 24hrs. Serological test was performed using O157:H7 polyvalent and monovalent anti-E. coli O and H sera. The presence of Shiga toxin 1 and 2 was detected by Polymerase Chain Reaction method. Antibiotic susceptibility testing was carried out using disk diffusion technique. Out of the 100 samples collected 60% yielded lactose fermenting colonies identified biochemically as E. coli, 43.3% from the abattoirs and 56.7% were from the open market. Only 3.3% of the Escherichia coli were non-Sorbitol fermenter and were confirmed as Enterohaemorrhagic Escherichia coli O157:H7 serologically, 1.7% carries the Stx 1 gene. The E. coli were resistant to Augmentin 95% and Cefuroxime 83.3%, Gentamycin 20%, Ofloxacin 21.7%, Ciprofloxacin 26.7%, Cefixime 38.3% and Ceftazidime 43.3%. The isolates that possessed Stx 1 gene were resistant to all the antibiotics tested. The meat samples in the abattoir and open market were contaminated with E coli which contain enterohaemorrhagic strains producing shiga toxin 1 (Stx 1) and were highly resistant to antibiotics.

Download Full-text

Use of gene probes for the detection of quiescent enteric bacteria in marine and fresh waters

Water Science & Technology ◽

10.2166/wst.1995.0628 ◽

1995 ◽

Vol 31 (5-6) ◽

pp. 291-298

Author(s):

Sally A. Anderson ◽

Gillian D. Lewis ◽

Michael N. Pearson

Keyword(s):

Membrane Filtration ◽

Enteric Bacteria ◽

Probe Method ◽

Detection Methods ◽

23S Rrna ◽

Specific Gene ◽

Gene Probe ◽

Selective Media ◽

Selective Enrichment ◽

E Coli

Specific gene probe detection methods that utilise a non-selective culturing step were tested for the ability to recognise the presence of quiescent enteric bacteria (Escherichia coli and Enterococcus faecalis ) within illuminated freshwater and seawater microcosms. An E. coli specific uidA gene probe and a 23S rRNA oligonucleotide probe for Enterococci were compared with recoveries using membrane filtration and incubation on selective media (mTEC and mE respectively). From these microcosm experiments a greater initial detection (from 4 hours to 1 day) of E. coli and Ent. faecalis using gene probe methods was observed. Additionally, a comparison of E. coli direct viable counts (DVC) in sunlight exposed microcosms with recoveries by selective media and gene probe methods revealed a large number of viable non-culturable cells. This suggests that enumeration of E. coli by a gene probe method is limited by the replication of the bacteria during the initial non-selective enrichment step. The detection of stressed Ent. faecalis by the oligonucleotide gene probe method was significantly greater than recovery on selective mE agar, indicating an Enterococci non-growth phase.

Download Full-text

Quality of beef diaphragm meat in naturally occurring Sarcocystic infection in cattle

Czech Journal of Food Sciences ◽

10.17221/89/2017-cjfs ◽

2018 ◽

Vol 36 (No. 5) ◽

pp. 378-385

Author(s):

Vytautas Januskevicius ◽

Grazina Januskeviciene ◽

Gintare Zaborskiene

Keyword(s):

Meat Quality ◽

Harmful Effect ◽

Coliform Bacteria ◽

E Coli ◽

Noticeable Increase ◽

Physico Chemical ◽

Infected Meat ◽

Technological Quality ◽

Meat Samples

The aim of this study was to investigate the possible harmful effect of Sarcocystis parasites on bovine diaphragm meat quality. Meat samples were collected from 120 bulls aged 20–24 months. Meat quality was investigated using microbiological and physico-chemical (RP-HPLC, GC) methods 48 hours after slaughter. Sarcocystis infection was associated with increased fat content, lightness L* and drip loss, and decreased ash and protein percentages. Infection also had a significant effect on the amount of amino acids (AAs), which slowly decreased as the number of sarcocysts increased. The total amount of AAs correlated with glutamic acid content (R = 0.966, P < 0.05). Heavily infected samples contained significantly lower amounts of putrescine, histamine, spermine and spermidine (P < 0.05) and<br /> a noticeable increase in the total count of aerobic microorganisms, but no change in the numbers of E. coli and coliform bacteria in comparison with no infected samples. Sarcocysts in beef diaphragms did not cause serious changes in the technological quality of the meat, but the biological quality of infected meat was reduced.

Download Full-text

Comparison of colony and stool blots for detection of enteropathogens by DNA probes

Canadian Journal of Microbiology ◽

10.1139/m91-066 ◽

1991 ◽

Vol 37 (5) ◽

pp. 407-410

Author(s):

Mônica A. M. Vieira ◽

Beatriz E. C. Guth ◽

Tânia A. T. Gomes

Keyword(s):

Escherichia Coli ◽

Key Words ◽

Sensitivity And Specificity ◽

Dna Probes ◽

Type I ◽

Enteropathogenic Escherichia Coli ◽

Key Words Dna ◽

E Coli ◽

Heat Stable

DNA probes that identify genes coding for heat-labile type I (LT-I) and heat-stable type 1 (ST-I) enterotoxins, enteropathogenic Escherichia coli adherence factor (EAF), and Shigella-like, invasiveness (INV) are used to evaluate the sensitivity and specificity of stool blots in comparison with the sensitivity and specificity of colony blots in detecting enteropathoghens. The sensitivities of the probes in stool blots are 91.7% for the LT-I probe, 76.9% for the ST-I probes, 78.9% for the EAF probe, and 45.5% for the INV probe. The specificity of all probes is higher than 95%. In general, the stool blot method identifies as many if not more LT-I-, ST-I-, and EAF-producing E. coli infections than the colony blots. Key words: DNA probes, stool blots, enteropathogens, diagnosis.

Download Full-text

Rapid Detection, Identification, and Enumeration ofEscherichia coli Cells in Municipal Water by Chemiluminescent In Situ Hybridization

Applied and Environmental Microbiology ◽

10.1128/aem.67.1.142-147.2001 ◽

2001 ◽

Vol 67 (1) ◽

pp. 142-147 ◽

Cited By ~ 29

Author(s):

Henrik Stender ◽

Adam J. Broomer ◽

Kenneth Oliveira ◽

Heather Perry-O'Keefe ◽

Jens J. Hyldig-Nielsen ◽

...

Keyword(s):

In Situ Hybridization ◽

16S Rrna ◽

Sensitivity And Specificity ◽

Bacterial Species ◽

Rdna Sequence ◽

Sequence Information ◽

Sequence Alignments ◽

E Coli ◽

Municipal Water

ABSTRACT A new chemiluminescent in situ hybridization (CISH) method provides simultaneous detection, identification, and enumeration of culturableEscherichia coli cells in 100 ml of municipal water within one working day. Following filtration and 5 h of growth on tryptic soy agar at 35°C, individual microcolonies of E. coliwere detected directly on a 47-mm-diameter membrane filter using soybean peroxidase-labeled peptide nucleic acid (PNA) probes targeting a species-specific sequence in E. coli 16S rRNA. Within each microcolony, hybridized, peroxidase-labeled PNA probe and chemiluminescent substrate generated light which was subsequently captured on film. Thus, each spot of light represented one microcolony of E. coli. Following probe selection based on 16S ribosomal DNA (rDNA) sequence alignments and sample matrix interference, the sensitivity and specificity of the probe Eco16S07C were determined by dot hybridization to RNA of eight bacterial species. Only the rRNA of E. coli and Pseudomonas aeruginosa were detected by Eco16S07C with the latter mismatch hybridization being eliminated by a PNA blocker probe targetingP. aeruginosa 16S rRNA. The sensitivity and specificity for the detection of E. coli by PNA CISH were then determined using 8 E. coli strains and 17 other bacterial species, including closely related species. No bacterial strains other thanE. coli and Shigella spp. were detected, which is in accordance with 16S rDNA sequence information. Furthermore, the enumeration of microcolonies of E. coli represented by spots of light correlated 92 to 95% with visible colonies following overnight incubation. PNA CISH employs traditional membrane filtration and culturing techniques while providing the added sensitivity and specificity of PNA probes in order to yield faster and more definitive results.

Download Full-text

Prevalence of non-O157 Shiga Toxin-producing E. Coli in Children and Calves in Al- Muthanna Province, Iraq

Al-Anbar Journal of Veterinary Sciences ◽

10.37940/ajvs.2021.14.2.10 ◽

2021 ◽

Vol 14 (2) ◽

pp. 78-90

Author(s):

Ahmed Jarad ◽

Kh. Al- Jeboori

Keyword(s):

Shiga Toxin ◽

Latex Agglutination ◽

Macconkey Agar ◽

Biochemical Tests ◽

Bacteriological Study ◽

E Coli ◽

Additional Information ◽

Single Colony ◽

Big Six ◽

Reliable Isolation

The present study focus on non-O157 Shiga toxin-producing E. Coli (STEC), included a bacteriological study was subjected to provide additional information for non-O157 STEC prevalence in children and calves. Isolation by using selective culturing media (CHROMagar STEC and CHROMagar O157) from 127 children suffering from diarrhea and 133 calves in Al- Muthanna province. Characterization depends on culturing positive colony on MacConkey agar and Levin’s Eosin Methylene blue agar, staining single colony from the growth by gram stain, biochemical tests; Indole, the Methyl Red, Voges-Proskauer, Citrate test, Oxidase, Catalase, Urease, Motility, Kligler Iron and Api-20E, were done to confirm a diagnosis of non-O157 STEC, The reliable isolation as non-O157 STEC serotyping by specific latex agglutination test for the target non-O157 STEC (big six) serogroup (O26, O45, O103, O111, O121 and O145). The current study showed the prevalence of non-O157 STEC was 20 of out 127 (15.73%) in samples collected from children and 27 / 133 (20.30%) in calves samples in conclusion the Non-O157 STEC is an important cause of diarrhea in children, and calves; finally, the calves play an important reservoir for Non-O157 STEC.

Download Full-text

Evaluation of a novel and rapid screening method for the detection of contaminated colistin-resistant bacteria in food

10.21203/rs.3.rs-118889/v1 ◽

2020 ◽

Author(s):

Hanh Vu ◽

Cornelia Appiah-Kwarteng ◽

Kaori Tanaka ◽

Ryuji Kawahara ◽

Diep Thi Khong ◽

...

Keyword(s):

Real Time ◽

Rapid Method ◽

High Speed ◽

Screening Method ◽

Rapid Screening ◽

Resistant Bacteria ◽

Practical Utility ◽

E Coli ◽

Retail Meat ◽

Meat Samples

Abstract Background: The dissemination of colistin-resistant bacteria carrying the colistin-resistant mobile gene, mcr-1 threatens medical care worldwide. In particular, contamination of food with colistin-resistant bacteria accelerates the community dissemination of colistin-resistant bacteria. Therefore, monitoring of colistin-resistant bacteria in food is important for controlling resistant bacteria. Unfortunately, the conventional culture methods for detecting colistin-resistant bacteria are not practical for monitoring food saftey. Therefore, development of a simple and rapid method to detect food contamination with colistin-resistant bacteria is desirable as an effective means for preventing the dissemination of resistant bacteria, particularly colistin-resistant bacteria.Findings: We developed a simple and rapid method for detecting Escherichia coli harboring the mcr-1 colistin resistance gene using a high-speed real-time polymerase chain reaction (PCR). The entire procedure, from sample processing to finals results, was performed within one hour. The practical utility of this method was verified by analyzing 27 retail meat samples for the presence of colistin-resistant bacteria. The results of the developed method were in agreement with the results of culturing colistin-resistant E. coli from the meat samples, demonstrating its efficacy and usefulness.Conclusions: A simple and rapid real-time PCR-based screening method was developed for detecting E. coli harboring mcr-1 in food samples. The practical utility of the procedure was confirmed using retail meat samples, indicating its potential as a convenient and rapid method to detect bacterial contamination of food items, especially in developing communities.

Download Full-text