Isolation, identification, molecular detection and sensitivity to antibiotics of Salmonella from cattle faeces

2021 ◽  
Vol 24 (1) ◽  
pp. 57-66
Author(s):  
M. N. K. Khan ◽  
M. R. Das ◽  
M. A. Sabur ◽  
M. M. Rahman ◽  
M. B. Uddin ◽  
...  
Keyword(s):  
Antibiotic Sensitivity ◽  
Culture Method ◽  
Sensitivity Study ◽  
Culture Methods ◽  
Isolation And Identification ◽  
Conventional Culture ◽  
Faecal Samples ◽  
Antibiotic Sensitivity Test ◽  
Cattle Faeces ◽  
Polymerase Chain

The present study was designed with the aim of isolation and identification of Salmonella by con-ventional culture method and their confirmation by polymerase chain reaction (PCR). Antibacte-rial sensitivity study of isolated Salmonella from cattle faeces was also performed. During the study period of July 2017 to June 2018, a total of 200 faecal samples were collected from different government and private farms in Sylhet district of Bangladesh. Out of 200 samples, 24 (12%) were found to be positive for Salmonella by conventional culture methods. Among the twenty four suspected colonies of Salmonella, seventeen were confirmed by biochemical test and same number was detected by PCR estimating a prevalence of 8.5% (17/200). The prevalence was high-er in calves under 1 year of age (16%) compared with older animals (11.25% of 1–2 years; 10% of above 2 years of age) but without statistically significant differences (χ2=4.835, P=0.089). Moreo-ver, in diarrhoeic animals the prevalence was significantly higher (32.14%, χ2=49.414, P<0.01) than in apparently healthy animals (8.72%). The antibiotic sensitivity test showed that highest number of Salmonella isolates were sensitive to ciprofloxacin (100%), gentamicin (100%) and neomycin (100%). On the other hand, significantly high resistance of Salmonella isolates was detected to erythromycin (100%), amoxicillin (100%), cotrimoxazole (81.48%), streptomycin (62.96%) followed by tetracycline (55.56%).

scholarly journals Molecular Detection and Antibiotic Sensitivity of Salmonella Species Isolated from Goat Feces in Sylhet District of Bangladesh

2021 ◽  
Vol 11 (3) ◽  
pp. 395-401
Author(s):  
Md. Abdus Sabur ◽  
Mouri Rani Das ◽  
Md Bashir Uddin ◽  
Md. Mahfujur Rahman ◽  
Md. Rafiqu Islam ◽  
...  
Keyword(s):  
Molecular Detection ◽  
Antibiotic Sensitivity ◽  
Culture Methods ◽  
Specific Primers ◽  
Antimicrobial Sensitivity ◽  
Conventional Culture ◽  
Antibiotic Sensitivity Test ◽  
One Year ◽  
Pcr Techniques

The present study aimed at the molecular detection of Salmonella species from feces of goats and the characterization of the isolated Salmonella by biochemical and antimicrobial sensitivity techniques. A total of 220 goat feces samples were collected, of which 27 (12.27%) were positive for Salmonella by conventional culture methods and 20 (9.09%) by biochemical and PCR techniques. The prevalence was higher in goats under one year of age (20%), compared to older animals aged one to two years (7.8%) and more than two years of age (4.7%), respectively. Moreover, the prevalence of diarrheic goats was significantly higher (38.46%) than healthy animals (2.76%). DNA was extracted from Salmonella strains and amplified by PCR using the specific primers of Salmonella invasion gene (invA gene). The antibiotic sensitivity test indicated that Ciprofloxacin (100 percent sensitivity), Gentamycin (100 percent sensitivity), and Neomycin (100 percent sensitivity) were the most effective antibiotics for the majority of Salmonella isolates. On the other hand, Salmonella isolates were found to have substantially high resistance to Erythromycin (100%), Amoxicillin (100%), Trimethoprim-Sulfamethoxazole (81.48%), Streptomycin (62.96%), and Tetracycline (55.56 percent). Since the rate of Salmonella carriers was relatively high, eating goat meat could increase the risk of foodborne salmonellosis.

Evaluation of TaqMan PCR Assay for Detecting Salmonella in Raw Meat and Shrimp

1999 ◽  
Vol 62 (4) ◽  
pp. 329-335 ◽  
Author(s):  
BON KIMURA ◽  
SUSUMU KAWASAKI ◽  
TATEO FUJII ◽  
JUN KUSUNOKI ◽  
TAKESHI ITOH ◽  
...  
Keyword(s):  
False Negative ◽  
Culture Method ◽  
Nuclease Activity ◽  
Food Samples ◽  
Pcr Assay ◽  
Culture Methods ◽  
Conventional Culture ◽  
Fluorogenic Probe ◽  
Taqman Pcr ◽  
Polymerase Chain

We evaluated the TaqMan Salmonella amplification/detection kit from PE Applied Biosystems, which uses a polymerase chain reaction (PCR) assay for rapid detection of Salmonella in food samples. This system uses the 5′ nuclease activity of Taq DNA polymerase, which digests an internal fluorogenic probe to monitor the amplification of the target gene. The system's sensitivity and specificity were evaluated using 42 serotypes of 68 Salmonella strains isolated from fecal samples from patients in Tokyo, Japan, and 39 non-Salmonella strains in 22 genera. There were no false-negative or false-positive results. This PCR assay can detect 3 CFU per PCR tube of Salmonella in pure culture (120 CFU/ml of TSB culture). PCR signals were attenuated with artificially contaminated shrimp, but a similar detection limit was obtained. TaqMan's performance was tested with 100 meat and chicken samples purchased from stores in Tokyo. Overall, two of the DNA extraction protocols (the Chelex and EnviroAmp methods) worked equally well, with some exceptions. Of the 100 samples analyzed, 10 were positive for Salmonella with both conventional culture methods and the kit and 89 were negative with both. One sample was negative by the culture method but positive by the kit assay. These results indicate that TaqMan is a reliable and rapid method for Salmonella analysis in the food industry. With this system, food samples can be analyzed for Salmonella in less than 20 h.

Comparison of a Real-Time PCR Method with a Culture Method for the Detection of Salmonella enterica Serotype Enteritidis in Naturally Contaminated Environmental Samples from Integrated Poultry Houses

2012 ◽  
Vol 75 (4) ◽  
pp. 743-747 ◽  
Author(s):  
BWALYA LUNGU ◽  
W. DOUGLAS WALTMAN ◽  
ROY D. BERGHAUS ◽  
CHARLES L. HOFACRE
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Salmonella Enteritidis ◽  
Culture Method ◽  
Pcr Assay ◽  
Culture Methods ◽  
The Real ◽  
Selective Enrichment ◽  
Conventional Culture ◽  
Field Samples

Conventional culture methods have traditionally been considered the “gold standard” for the isolation and identification of foodborne bacterial pathogens. However, culture methods are labor-intensive and time-consuming. A Salmonella enterica serotype Enteritidis–specific real-time PCR assay that recently received interim approval by the National Poultry Improvement Plan for the detection of Salmonella Enteritidis was evaluated against a culture method that had also received interim National Poultry Improvement Plan approval for the analysis of environmental samples from integrated poultry houses. The method was validated with 422 field samples collected by either the boot sock or drag swab method. The samples were cultured by selective enrichment in tetrathionate broth followed by transfer onto a modified semisolid Rappaport-Vassiliadis medium and then plating onto brilliant green with novobiocin and xylose lysine brilliant Tergitol 4 plates. One-milliliter aliquots of the selective enrichment broths from each sample were collected for DNA extraction by the commercial PrepSEQ nucleic acid extraction assay and analysis by the Salmonella Enteritidis–specific real-time PCR assay. The real-time PCR assay detected no significant differences between the boot sock and drag swab samples. In contrast, the culture method detected a significantly higher number of positive samples from boot socks. The diagnostic sensitivity of the real-time PCR assay for the field samples was significantly higher than that of the culture method. The kappa value obtained was 0.46, indicating moderate agreement between the real-time PCR assay and the culture method. In addition, the real-time PCR method had a turnaround time of 2 days compared with 4 to 8 days for the culture method. The higher sensitivity as well as the reduction in time and labor makes this real-time PCR assay an excellent alternative to conventional culture methods for diagnostic purposes, surveillance, and research studies to improve food safety.

A Real-Time PCR Assay for the Detection of Salmonella in a Wide Variety of Food and Food-Animal Matrices†

2007 ◽  
Vol 70 (5) ◽  
pp. 1080-1087 ◽  
Author(s):  
V. M. BOHAYCHUK ◽  
G. E. GENSLER ◽  
M. E. McFALL ◽  
R. K. KING ◽  
D. G. RENTER
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Culture Method ◽  
Pcr Assay ◽  
Culture Methods ◽  
The Real ◽  
Conventional Culture ◽  
Food Animal ◽  
Highly Sensitive ◽  
Field Samples

Conventional culture methods have traditionally been considered the “gold standards” for the isolation and identification of foodborne pathogens. However, culture methods are labor-intensive and time-consuming. We have developed a real-time PCR assay for the detection of Salmonella in a variety of food and food-animal matrices. The real-time PCR assay incorporates both primers and hybridization probes based on the sequence of the Salmonella invA gene and uses fluorescent resonance energy transfer technology to ensure highly sensitive and specific results. This method correctly classified 51 laboratory isolates of Salmonella and 28 non-Salmonella strains. The method was also validated with a large number of field samples that consisted of porcine feces and cecal contents, pork carcasses, bovine feces and beef carcasses, poultry cecal contents and carcasses, equine feces, animal feeds, and various food products. The samples (3,388) were preenriched in buffered peptone water and then selectively enriched in tetrathionate and Rappaport-Vassiliadis broths. Aliquots of the selective enrichment broths were combined for DNA extraction and analysis by the real-time PCR assay. When compared with the culture method, the diagnostic sensitivity of the PCR assay for the various matrices ranged from 97.1 to 100.0%, and the diagnostic specificity ranged from 91.3 to 100.0%. Kappa values ranged from 0.87 to 1.00, indicating excellent agreement of the real-time PCR assay to the culture method. The reduction in time and labor makes this highly sensitive and specific real-time PCR assay an excellent alternative to conventional culture methods for surveillance and research studies to improve food safety.

scholarly journals Analysis of River Water Along Klang Valley to Evaluate the Prevalence of Antibiotics Resistant Strains within Urbanized Areas of Selangor, Malaysia

2020 ◽  
Vol 2 (1) ◽  
pp. 59
Keyword(s):  
River Water ◽  
Antibiotic Sensitivity ◽  
Most Probable Number ◽  
Resistant Bacteria ◽  
Minimal Bactericidal Concentration ◽  
Isolation And Identification ◽  
Antibiotic Resistant ◽  
Probable Number ◽  
Antibiotic Sensitivity Test ◽  
Klang Valley

The determination of antibiotic-resistant bacteria in Klang river water in Klang valley is performed as the river exposed to various environments. The analysis is performed through enumeration, isolation, and identification process. The water samples were obtained from the origin of the river, housing region, and hospital region. The coliforms obtained through enumeration and identification was then used to determine antibiotic sensitivity, minimum inhibitory concentration (MIC), and minimal bactericidal concentration (MBC). The level of coliforms was indicated through the most probable number (MPN), which 70 MPN per 100 ml of river water in the origin of the river while housing and hospital regions showed more than 1600 MPN per 100 ml of river water. The results obtained from the antibiotic sensitivity test showed that the degree of resistance of coliforms is varied in different regions. The zone of inhibition to ampicillin and tetracyclin for coliforms in housing regions is 20 mm, while the coliforms in the hospital region are 6 mm and 7 mm, respectively. The overall results showed that the level of coliforms and the antibiotic sensitivity of coliforms are different in various regions. The coliforms in the hospital region are more resistant to antibiotics compared to the housing region.

scholarly journals High Prevalence of yadA-Positive Yersinia enterocolitica in Pig Tongues and Minced Meat at the Retail Level in Finland

1999 ◽  
Vol 62 (2) ◽  
pp. 123-127 ◽  
Author(s):  
MARIA FREDRIKSSON-AHOMAA ◽  
SEBASTIAN HIELM ◽  
HANNU KORKEALA
Keyword(s):  
Yersinia Enterocolitica ◽  
Culture Method ◽  
Selective Enrichment ◽  
Conventional Culture ◽  
High Prevalence ◽  
Retail Outlets ◽  
Polymerase Chain ◽  
Meat Samples ◽  
Minced Meat ◽  
Conventional Culture Method

Polymerase chain reaction (PCR) was used to determine the prevalence of yadA-positive Yersinia enterocolitica in pig tongues and minced meat at the retail level in Finland and to confirm the yadA-positive Y. enterocolitica isolates recovered from the same samples using the conventional culture method. A total of 51 pig tongues purchased at 12 retail outlets and 255 minced meat samples purchased at 40 retail outlets in the Helsinki area were studied. The prevalence of Y. enterocolitica carrying the yadA gene was 92% in pig tongues and 25% in minced meat using PCR and 78% in tongues and 2% in minced meat with the culture method. The prevalence of yadA-positive tongues was higher (98%) when both PCR- and culture-positive results were included because Y. enterocolitica carrying the yadA gene could also be isolated in three PCR-negative tongue samples. In the minced meat samples, all PCR-negative samples were also culture-negative. With the culture method, 66 of 80 yadA-positive isolates in 38 tongues and all yadA-positive isolates (4) in four minced meat samples were recovered after selective enrichment. A total of 92 isolates of Y. enterocolitica bioserotype 4/O:3 in tongues and 5 isolates in minced meat were found, of which 13% in tongues and 20% in minced meat did not carry the yadA gene.

scholarly journals Bovine tuberculosis: Diagnosis by PCR technique in bovine milk samples

2013 ◽  
Vol 37 (2) ◽  
pp. 156-160
Author(s):  
Mohammed J. Alwan
Keyword(s):  
Bovine Milk ◽  
Culture Method ◽  
Bacterial Isolation ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Culture Methods ◽  
Milk Samples ◽  
Tuberculosis Diagnosis ◽  
Pcr Method ◽  
Polymerase Chain

     The aim of this study was to determine the percentage of borne tuberculin infection in milk sample by using PCR.  (102) milk samples were collected from  cows , AL-Dejella (39) samples,  AL-Suara (20) samples cow station, AL-Fthalia (20) samples,  AL-Azezia (11) samples and AL-Twarege (12) samples (Iraq) during the period  July 10th   2010 to  Nov.30th   2010. The samples were examined by direct smear stained by Ziehle-Neelson stain, culture methods and confirmed the isolates by Polymerase Chain Reaction (PCR) assay. The results showed that  5, 102 (4.9%) milk samples were M. bovis positive that were detected by direct milk smear method and 10 out 102(9.8%) M.bovis +ve were detected by culture method and PCR assay. The results also showed that high percentage of bacterial isolates from milk samples AL-Dejella city show (12.8%) by culturing and PCR method followed by AL-Suara (10%), AL-Fthelia (10%), Al-Twarege (8.3%) but no bacterial isolation was recorded in AL-Azezia milk samples. This study concluded that M.bovis infection was spreading in dairy cow within the mentioned areas and PCR was more sensitive, rapid, and accurate technique for M.bovis infection diagnosis.

scholarly journals Evaluation of a manual identification system for detection of Mycobacterium tuberculosis in a primary tuberculosis laboratory in China

2019 ◽  
Vol 47 (6) ◽  
pp. 2666-2673 ◽  
Author(s):  
Ping Zhao ◽  
Qin Yu ◽  
Yu Zhang
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Diagnostic Performance ◽  
Culture Method ◽  
Cross Sectional Study ◽  
Identification System ◽  
Culture Methods ◽  
Cross Sectional ◽  
Indicator Tube ◽  
Conventional Culture ◽  
Time To Detection

Objective To compare the diagnostic performance of the manual BACTEC™ Mycobacteria Growth Indicator Tube (MGIT™) system (M-MGIT) with the automated BACTEC™ MGIT™ 960 system (A-MGIT) and Löwenstein-Jensen (L-J) culture method in detecting mycobacteria in sputum specimens from patients with suspected pulmonary tuberculosis (TB). Methods For this cross-sectional study, sputum samples were taken from patients aged ≥18 years attending a TB clinic in Beijing, China between July 2015 and October 2016. Processed sputum samples were inoculated into the MGIT systems and L-J medium for up to 6 and 8 weeks, respectively. Results The M-MGIT and A-MGIT methods detected significantly more Mycobacterium tuberculosis complex (MTC) isolates than L-J culture from the 565 sputum samples (39%, 40% and 32%, respectively). Using a positive result from any of the three culture systems as reference, the sensitivity of M-MGIT, A-MGIT and L-J methods were 92%, 94%, and 74%, respectively. The time-to-detection of mycobacteria was 12.9±4.2 days for M-MGIT, 11.8±5.2 days for A-MGIT and 24.2±8.7 days for L-J. Conclusions M-MGIT has a similar diagnostic performance to A-MGIT, and is a fast and reliable alternative to conventional culture methods in the diagnosis of pulmonary TB in a developing country.

scholarly journals Direct detection of Mycobacterium avium in environmental water and scale samples by loop-mediated isothermal amplification

2013 ◽  
Vol 12 (2) ◽  
pp. 211-219 ◽  
Author(s):  
Yukiko Nishiuchi ◽  
Aki Tamaru ◽  
Yasuhiko Suzuki ◽  
Seigo Kitada ◽  
Ryoji Maekura ◽  
...  
Keyword(s):  
Mycobacterium Avium ◽  
Environmental Samples ◽  
Direct Detection ◽  
Culture Method ◽  
Environmental Water ◽  
Isothermal Amplification ◽  
Lamp Method ◽  
Culture Methods ◽  
Loop Mediated Isothermal Amplification ◽  
Conventional Culture

We previously demonstrated the colonization of Mycobacterium avium complex in bathrooms by the conventional culture method. In the present study, we aimed to directly detect M. avium organisms in the environment using loop-mediated isothermal amplification (LAMP), and to demonstrate the efficacy of LAMP by comparing the results with those obtained by culture. Our data showed that LAMP analysis has detection limits of 100 fg DNA/reaction for M. avium. Using an FTA® elute card, DNA templates were extracted from environmental samples from bathrooms in the residences of 29 patients with pulmonary M. avium disease. Of the 162 environmental samples examined, 143 (88%) showed identical results by both methods; 20 (12%) and 123 (76%) samples were positive and negative, respectively, for M. avium. Of the remaining 19 samples (12%), seven (5%) and 12 (7%) samples were positive by the LAMP and culture methods, respectively. All samples that contained over 20 colony forming units/primary isolation plate, as measured by the culture method, were also positive by the LAMP method. Our data demonstrate that the combination of the FTA elute card and LAMP can facilitate prompt detection of M. avium in the environment.

Isolation and Identification of Campylobacter spp. from Poultry and Poultry By-Products in Tunisia by Conventional Culture Method and Multiplex Real-Time PCR

2017 ◽  
Vol 80 (10) ◽  
pp. 1623-1627 ◽  
Author(s):  
Hela Jribi ◽  
Hanen Sellami ◽  
Siala Mariam ◽  
Salma Smaoui ◽  
Asma Ghorbel ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Poultry Meat ◽  
Culture Methods ◽  
Isolation And Identification ◽  
Conventional Culture ◽  
Multiplex Pcr Assay ◽  
Thermophilic Campylobacter ◽  
By Products ◽  
Campylobacter Spp

ABSTRACT Thermophilic Campylobacter spp. are one of the primary causes of bacterial human diarrhea. The consumption of poultry meats, by-products, or both is suspected to be a major cause of human campylobacteriosis. The aims of this study were to determine the prevalence of thermophilic Campylobacter spp. in fresh poultry meat and poultry by-products by conventional culture methods and to confirm Campylobacter jejuni and Campylobacter coli isolates by using the multiplex PCR assay. Two hundred fifty fresh poultry samples were collected from a variety of supermarkets and slaughterhouses located in Sfax, Tunisia, including chicken (n =149) and turkey (n =101). The samples were analyzed using conventional microbiological examinations according to the 2006 International Organization for Standardization method (ISO 10272-1) for Campylobacter spp. Concurrently, a real-time PCR was used for identification of C. jejuni and C. coli. Of the 250 samples of poultry meat and poultry by-products, 25.6% (n = 64) were contaminated with Campylobacter spp. The highest prevalence of Campylobacter spp. was found in chicken meat (26.8%) followed by turkey meat (23.7%). Among the different products, poultry breasts showed the highest contamination (36.6%) followed by poultry by-products (30%), poultry wings (28%) and poultry legs (26%) showed the lowest contamination, and no contamination was found on neck skin. Of the 64 thermophilic Campylobacter isolates, C. jejuni (59.7%) was the most frequently isolated species and 10.9% of the isolates were identified as C. coli. All of the 64 Campylobacter isolates identified by the conventional culture methods were further confirmed by PCR. The seasonal peak of Campylobacter spp. contamination was in the warm seasons (spring and summer). The study concluded that high proportions of poultry meat and poultry by-products marketed in Tunisia are contaminated by Campylobacter spp. Furthermore, to ensure food safety, poultry meats must be properly cooked before consuming.

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