Molecular genotyping of Salmonella spp. isolated from cheese samples of local stores in Al-Diwaniyah city, Iraq
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ABSTRACT:
SUBMITTER: Faja O
PROVIDER: S-EPMC10658018 | biostudies-literature | 2023 Oct
REPOSITORIES: biostudies-literature
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Project description:The methods currently used for detecting Salmonella in environmental samples require 2 days to produce results and have limited sensitivity. Here, we describe the development and validation of a real-time PCR Salmonella screening method that produces results in 18 to 24 h. Primers and probes specific to the gene invA, group D, and Salmonella enterica serovar Enteritidis organisms were designed and evaluated for inclusivity and exclusivity using a panel of 329 Salmonella isolates representing 126 serovars and 22 non-Salmonella organisms. The invA- and group D-specific sets identified all the isolates accurately. The PCR method had 100% inclusivity and detected 1 to 2 copies of Salmonella DNA per reaction. Primers specific for Salmonella-differentiating fragment 1 (Sdf-1) in conjunction with the group D set had 100% inclusivity for 32 S Enteritidis isolates and 100% exclusivity for the 297 non-Enteritidis Salmonella isolates. Single-laboratory validation performed on 1,741 environmental samples demonstrated that the PCR method detected 55% more positives than the Vitek immunodiagnostic assay system (VIDAS) method. The PCR results correlated well with the culture results, and the method did not report any false-negative results. The receiver operating characteristic (ROC) analysis documented excellent agreement between the results from the culture and PCR methods (area under the curve, 0.90; 95% confidence interval of 0.76 to 1.0) confirming the validity of the PCR method.IMPORTANCE This validated PCR method detects 55% more positives for Salmonella in half the time required for the reference method, VIDAS. The validated PCR method will help to strengthen public health efforts through rapid screening of Salmonella spp. in environmental samples.
| S-EPMC5494621 | biostudies-literature
Project description:The potential role of environmental Mycobacterium tuberculosis in the epidemiology of TB remains unknown. We investigated the transmission of M tuberculosis from humans to the environment and the possible transmission of M tuberculosis from the environment to humans.A total of 1,500 samples were collected from three counties of the Tehran, Iran metropolitan area from February 2012 to January 2014. A total of 700 water samples (47%) and 800 soil samples (53%) were collected. Spoligotyping and the mycobacterial interspersed repetitive units-variable number of tandem repeats typing method were performed on DNA extracted from single colonies. Genotypes of M tuberculosis strains isolated from the environment were compared with the genotypes obtained from 55 patients with confirmed pulmonary TB diagnosed during the study period in the same three counties.M tuberculosis was isolated from 11 of 800 soil samples (1%) and 71 of 700 water samples (10%). T family (56 of 82, 68%) followed by Delhi/CAS (11 of 82, 13.4%) were the most frequent M tuberculosis superfamilies in both water and soil samples. Overall, 27.7% of isolates in clusters were related. No related typing patterns were detected between soil, water, and clinical isolates. The most frequent superfamily of M tuberculosis in clinical isolates was Delhi/CAS (142, 30.3%) followed by NEW-1 (127, 27%). The bacilli in contaminated soil (36%) and damp water (8.4%) remained reculturable in some samples up to 9 months.Although the dominant M tuberculosis superfamilies in soil and water did not correspond to the dominant M tuberculosis family in patients, the presence of circulating genotypes of M tuberculosis in soil and water highlight the risk of transmission.
| S-EPMC4388118 | biostudies-other
Project description:Aim:This study was directed during an outbreak of suspected foot-and-mouth disease (FMD) in cattle in Al-Qadisiyah province, Iraq 2016. The disease has made a huge economic loss in livestock. It was suspected that the vaccination has failed to protect the animals from the infection because of the difference in the strains. Consequently, we designed the study to make the diagnosis and detect the strain of the causative virus. Materials and Methods:The extraction of the DNA was done on 73 samples and Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) was used in the detection of FMD virus (FMDV) for primary diagnosis, and serotype-specific diagnosis was done with universal primer sets 1F/1R, A-1C612, and O-ARS4 with the expected band of 329, 865, and 1301 bp, respectively. Results:Universal primer pair 1F/1R detected FMD in 55 of 73 (75.3%); of these, 37 (67.3%) were females and 18 (32.7%) were males, with high significance (p<0.01) between males and females in the PCR positivity ratio. The tested samples with positive universal primer were amplified with specific primers A-IC612 with no reaction for serotype O-ARS4. Conclusion:The products of RT-PCR were sent for RNA sequencing, and the results were 100% positive to serotype A which means that it is the predominant type in Iraq. It may help in the importing or production of the vaccine to make a preventive plan for the disease. The virus of FMD is contagious and dangerous due to its role in the huge economic loses. The detection of this virus is widely explained in lots of articles, but it is more specific and sensitive in RT-PCR and sequencing. Consequently, the authorities responsible for importing and/or production vaccines have to avoid the importing of other serotypes because it will be losing money and more outbreaks will explode.
| S-EPMC6200567 | biostudies-literature
Project description:The growing concern over antibiotic resistance in foodborne pathogens necessitates comprehensive assessments of its prevalence and associated risks in various food products. The present study aimed to assess the occurrence of Enterococcus spp. in samples of fish purchased at various points of sale in the Tricity region. The selection of products (n = 74) was based on their availability and included both fish caught in the Baltic region and products imported from, Vietnam, China, Norway, and European Union (EU) countries. For bacterial isolation, samples were inoculated into selective broth, and the growth of enterococci was assessed based on turbidity. Positive cultures were confirmed by a change in color in bromocresol purple broth and were isolated on Slanetz-Bartley agar. Bacteria were present in all tested samples regardless of the degree of raw material processing as follows: frozen (F)- 55%, fresh/raw (FS)- 70.6%, thawed (DF)- 30%, smoked (S)- 50%, and the packaging methods, modified atmosphere packaging (MAP)- 34.4%, unit packaging (UP)- 75%, and sold in bulk (SB)- 76.9%, with an overall frequency of occurrence of 58.1%. The number of bacteria ranged from not detected to 4.28-log cfu/g, with the lowest mean values for thawed fish and those packed in MAP. Tests conducted on 24 strains isolated from samples showed their varied sensitivity to tetracyclines. Single cases of multidrug resistance of the tested strains were also observed. The conducted statistical analysis did not show statistically significant differences in the count of enterococci based on the origin, degree of processing, or packaging (p < 0.05). Moreover, differences in strain sensitivity to ampicillin were observed. Detected cases of resistance, especially to tetracycline, require careful monitoring and action to limit the health risks associated with resistant bacterial strains in food products.
| S-EPMC11232986 | biostudies-literature
Project description:Cutaneous leishmaniasis (CL) is highly prevalent in southern Iraq and neighboring countries, but is non-endemic to the Kurdistan Region of Iraq, particularly in the Garmian area. This study aimed to investigate the causative agent of CL at the molecular level by amplifying the small subunit (18S) rRNA and internal transcribed spacer 1 (ITS1) region. The present study was conducted from December 2019 to December 2020 at Kalar General Hospital, Kalar, Kurdistan Region, Iraq. Eighty-five clinical specimens were collected selectively from patients with suspected CL lesions via fine needle aspiration. After parasitic genomic DNA was extracted from the removed fluid, PCR and DNA sequencing targeting the 18S rRNA and ITS1 region were performed for molecular detection and species identification. Additionally, for 14 samples, the target bands of amplified DNA fragments for both 18S rRNA and ITS1 were extracted and sequenced via Sanger method using both the directional primers employed in the PCR. Seventy-one (83.53%) of the 85 suspected patients had CL, based on amplification of 18S rRNA and ITS1 via PCR. The sequence analysis revealed that all samples were Leishmania major. Phylogenetic analysis based on ITS1 was also performed. Our study revealed that our molecular method was an efficient technique for detecting CL and a valuable method for identifying Leishmania species in clinical samples. Sequence analysis indicated that the causative agent of CL in the Garmian area was L. major and the disease was rural in origin.
| S-EPMC8810569 | biostudies-literature
Project description:Salmonellosis, caused by Salmonella spp., is a widely reported foodborne zoonosis frequently associated with ingestion of poultry products. Salmonella vaccination of chickens can be used to reduce bacterial shedding and risk of human infection. To determine Salmonella burden in chicken farms, culture methods of environmental samples that require a turn-around time of 5-7 days are usually used. Rapid screening using molecular assays such as PCR of pre-enriched broth has been reported for Salmonella spp. detection in feed, floor dust, and drag swabs within 2-3 days. Here we report an adaptation of the method for detection of Salmonella in poultry dust samples collected using a settle plate method under experimental conditions. Key features:•Passive dust sample collection using dry settle plates without media suspended from dropper lines of drinkers.•Small amount of sample required for the pre-enrichment process.•Quantification of Salmonella DNA with high sensitivity using an inexpensive extraction protocol.
| S-EPMC8374386 | biostudies-literature
Project description:To the best of our knowledge, this is the first Iraqi study to detect C. felis in cattle by the polymerase chain reaction (PCR) assay and to confirm the local isolates in the National Center for Biotechnology Information (NCBI). Overall, 130 diarrheic calves of different ages and sexes were selected randomly from rural and sub-urban areas in Wasit province (Iraq) from February to April (2021) and subjected to direct collection of fresh fecal samples for DNA extraction and PCR examination. Targeting the heat shock protein 70 (HSP70) gene of C. felis showed that 17.69% of the animals were positive. Findings from clinical examination revealed no significant differences in values of temperature, pulse, or respiratory rates between the positive and negative calves. The association between the positive results and demographic risk factors showed no significant differences in the prevalence rate of infection and risk of exposure to C. felis between the rural and sub-urban areas; however, higher significant values were reported in calves aged 6 months than in calves aged 12 months, as well as in females than in males. Five of the local C. felis isolates were documented under the accession numbers MZ964600.1, MZ964601.1, MZ964602.1, MZ964603.1, and MZ964604.1. Finally, the data presented here provide epidemiological and molecular evidence that the range of C. felis in cattle is wider than expected globally, with a high probability of infection transmission from cattle to humans.
| S-EPMC10237573 | biostudies-literature
Project description:Culture-based detection is still considered as the standard way for detection of Salmonella in foods, although molecular methods, such as viability PCR (vPCR), have been introduced to overcome some disadvantages of traditional culture methods. Despite the success of the vPCR methodology, the problem of false-positive results is a major drawback, especially when applied to environmental samples, hindering the interpretation of the results. To improve the efficiency of vPCR, many approaches have been introduced by several authors during the last years. In the present work, the combination of PEMAX dye, double tube change, and double photo-activation step was established as a strategy to improve vPCR protocol. By combining these approaches, we developed an improved sample treatment protocol able to neutralize DNA signals of up to 5.0×107 dead cells/sample from both pure culture and artificially contaminated food samples. Our results indicate that vPCR can work reliable and has a potential for high throughput detection of live Salmonella cells in food samples, minimizing false-positive signals.
| S-EPMC5726647 | biostudies-literature
Project description:The transmission of Salmonella enterica within a vertically integrated poultry operation was investigated longitudinally over an 18-month period (2013-2014). Thirty six percent of all samples collected (1503 of 4219) were positive for salmonellae with seven Salmonella enterica subsp. enterica serovars, and one Salmonella enterica subsp. salamae serovar detected. Both Salmonella enterica subsp. enterica serovars Infantis and Typhimurium were detected in all locations sampled. Salmonella Typhimurium was the most frequently detected serovar (63% of serotyped samples) with 8 phage types (PT) and 41 multiple-locus variable-number tandem-repeats analysis (MLVA) profiles identified. The most frequently identified phage types were PT135a and DT135. A total of 62 PT/MLVA combinations were observed. MLVA profiles 03-14-10-09-525 and 03-15-11-11-525 were the most frequently identified and 83% of the isolates shared at least one MLVA profile with an isolate from another phage type. The use of phage typing and MLVA profiling, on their own or in combination, were insufficient to understand the complexity of the epidemiological relationships between locations within this production system. Despite the high level of apparent diversity, cluster analysis was unable to differentiate the transmission pathways of all S. Typhimurium variants detected within the integrated enterprise. Using additional epidemiological information, the parent breeder rearing site was identified as the most likely point of introduction of two S. Typhimurium isolates into the production system with subsequent dissemination to the broiler flocks via the hatchery. This complexity is unable to be resolved in the absence of intensive sampling programs at all generations of the production system.
| S-EPMC6053207 | biostudies-literature
Project description:Infectious bronchitis virus (IBV) is one of the most critical pathogens in the poultry industry, causing serious economic losses in all countries including Iraq. IBV has many genotypes that do not confer any cross-protection. This virus has been genotyped by sequence analysis of the S1 glycoprotein gene. A total of 100 tracheal and kidney tissue specimens from different commercial broiler flocks in the middle and south of Iraq were collected from September 2013 to September 2014. Thirty-two IBV-positive samples were selected from among the total and were further characterized by nested PCR. Phylogenetic analysis revealed that isolates belong to four groups (group I, variant 2 [IS/1494-like]; group II, 793/B-like; group III, QX-like; group IV, DY12-2-like). Sequence analysis revealed nucleotide sequence identities within groups I, II, and III of 99.68 %-100 %, 99.36 %-100 %, and 96.42 %-100 %, respectively. Group I (variant 2) was the dominant IBV genotype. One Chinese-like recombinant virus (DY12-2-like) that had not been reported in the Middle East was detected. In addition, the presence of QX on broiler chicken farms in the area studied was confirmed. This is the first comprehensive study on the genotyping of IBV in Iraq with useful information regarding the molecular epidemiology of IBV. The phylogenetic relationship of the strains with respect to different time sequences and geographical regions displayed complexity and diversity. Further studies are needed and should include the isolation and full-length molecular characterization of IBV in this region.
| S-EPMC7086866 | biostudies-literature