Detection of Campylobacter jejuni and Salmonella typhimurium in chicken using PCR for virulence factor hipO and invA genes (Saudi Arabia)

Campylobacter jejuni and Salmonella typhimurium are the leading causes of bacterial food contamination in chicken carcasses. Contamination is particularly associated with the slaughtering process. The present study isolated C. jejuni and S. typhimurim from fifty chicken carcass samples, all of which were acquired from different companies in Riyadh, Saudi Arabia. The identification of C. jejuni was performed phenotypically by using a hippurate test and genetically using a polymerase chain reaction with primers for 16S rRNA and hippurate hydrolase (hipO gene). For the dentification of S. typhimurim, a serological Widal test was carried out using serum anti-S. typhimurium antibodies. Strains were genetically detected using invA gene primers. The positive isolates for C. jejuni showed a specific molecular size of 1448 bp for 16S rRNA and 1148 bp for hipO genes. However, the positive isolates of the invA gene exhibited a specific molecular size at 244 bp using polymerase chain reaction (PCR). Comparing sequencing was performed with respect to the invA gene and the BLAST nucleotide isolates that were identified as Salmonella enterica subsp. enterica serovar typhimurium strain ST45, thereby producing a similarity of 100%. The testing identified C.jejuni for hippuricase, GenBank: Z36940.1. While many isolates of Salmonella spp. that contained the invA gene were not necessarily identified as S. typhimurim, the limiting factor for the Widal test used antiS. typhimurum antibodies. The multidrug resistance (MDR) of C. jejuni isolates in chickens was compared with the standard C. jejuni strain ATCC 22931. Similarly, S. typhimurium isolates were compared with the standard S. typhimurium strain ATCC 14028.

A rapid, sensitive, specific and visual detection of Salmonella in retail meats using the loop-mediated isothermal amplification (LAMP), targeting the invA gene

Salmonella is one of the major pathogenic bacteria causing food-borne diseases. The rapid detection of Salmonella in food is of great significance to food safety. In this study, the loop-mediated isothermal amplification (LAMP) method was developed and the primers were designed targeting the invA gene of Salmonella. Then, the standard samples of recombinant invA-plasmid and 100 retail meat samples were tested by LAMP and compared with the results tested by the conventional PCR and the routine China National Food Safety Standard Methods for Food Microbiological Examination-Salmonella (GB/T4789.4-2016), respectively. The results showed that Salmonella strains of 8 different serotypes were amplified successfully by the developed LAMP assay. And, it was 1000-fold more sensitive than the conventional PCR with the analytical sensitivity of 8×102 copies/μL of the standard sample of invA-plasmid. The results were visualized directly by adding Calcein/MnCl2 into the LAMP reaction tube and the positively amplified products turned green after an incubation of 2 min. In the parallel detection, the positive rate of Salmonella by the LAMP assay was highly correlated with the routine China national standard method. The results of the study demonstrated that the developed LAMP assay is a simple, rapid, strongly specific, highly sensitive and visual detection method for Salmonella.

Antibiotic Susceptibility of Salmonella spp Isolated from Fresh Leafy Vegetables Samples by Using Culture and Polymerase Chain Reaction Methods

Background: Microbial contamination continues to be one of the leading risks to food safety. Contaminated leafy green vegetables are the primary cause of infection among children, elderly, and immunocompromised people. The purposes of this work were to isolate and identify of Salmonella spp. in fresh leafy vegetables collected from Jeddah Central Market, Jeddah district, western area, kingdom of Saudi Arabia, estimated of the number and percentage of isolated Salmonella spp and determined the antimicrobial susceptibility of the isolated Salmonella spp. Methods: Five-hundred samples were examined for the presence of Salmonella spp, by using standard microbiological and biochemical tests. Further, detection of Salmonella spp. was done by PCR with the primers targeting invA gene, a key factor for entry of Salmonella into epithelial cells. Susceptibility of the isolated Salmonella spp was done toward thirteen different antibiotics. Results: The percentage of isolation of Salmonella spp was 1.2 % (06/500). It was isolated as (0.40%, 02/500) from Basil, (0.20%, 01/500) from Spinach, Rocket, Parsley and Chards. Two isolates (2/6, 33.3%) showed positive Salmonella invA gene (244 bp). All isolated Salmonella showed resistance to Cephalexin (30 µg/disc), Metronidazole (5 µg/disc) and Methicillin (5 µg/disc).

Molecular Serotyping by Phylogenetic Analyses of a 1498bp Segment of the invA Gene of Salmonella

The current gold standard for Salmonella serotyping is costly, labor-intensive and time-consuming. However, proper identification is key to monitor Salmonella transmission and implementation of necessary control measures. The onset of advanced molecular techniques has lessened resource and labor requirements; however, it still remains complex, unestablished and plagued with insufficiencies. Hence, a simpler serotyping method with sufficient resolution is needed. In this study, the invA virulence gene, associated with Salmonella invasion into host cells and is considered as a marker for Salmonella detection, was amplified and sequenced among isolates from meat samples in Metro Manila, Philippines. This was followed by sequence alignments with reference sequences (Refseqs), oversaturation and model tests, phylogenetic tree analyses and signal detections. Unfortunately, alignment of a 229bp amplified and sequenced invA gene segment with Refseqs generated little to no base variations and consequently provided insufficient phylogenetic resolution for molecular serotyping (0 of the 17 serotypes tested). However, another segment of 1498bp, outside the amplified region, showed considerable base variation in alignment and consequently resolved a maximum of 13 out of 17 (76.47%) serotypes tested, all generated trees considered. These suggest the potential of the invA virulence gene as a single-gene marker for molecular serotyping of Salmonella through phylogenetic analyses.

Detection of INVA Gene and Cytotoxin of Salmonella entertidis in Food Samples Using Molecular Methods

Salmonella entertidis is a foodborne pathogen that causes various diseases in human beings worldwide. The toxin of Salmonella can cause infectious diseases. In this research project, Salmonella was detected through various microbial, biochemical and molecular tests in diverse food samples collected from highly populated, moderately populated and less populated areas of Lahore, Pakistan. Enriched cultures of all food samples such as apples, tomatoes, yogurt and mayonnaise was streaked on violet-red bile glucose agar, Simmon’s citrate agar and eosin-methylene blue agar (EMB). Salmonella isolates were screened for the presence of toxin encoding gene through PCR. 27% apples, 19% tomatoes, 5% mayonnaise and 7% yogurt were found to be positive for INVA genes (invasion protein genes). In medical and pharmaceutical point of views the INVA gene can also help to develop specific medicines against salmonella. The cytotoxin that is protein in nature was confirmed by SDS PAGE in mayonnaise samples. This study illustrates that foods of highly populated areas are reservoir for Salmonella entertidis in Pakistan. There is need to develop specific drugs, precautionary measures to control salmonella and its disease.

Molecular Characterization of Salmonella Species Isolates from Some Hospitals in Jos, Nigeria

The conventional methods of identification of Salmonella involving microbiological enrichment and successive identification mostly are tedious, time consuming and not specific. Therefore, the aim of this study was to utilize molecular techniques to characterize Salmonella species isolates from some Hospitals in Jos, Nigeria. The 10 isolates collected from some Hospitals in Jos, Nigeria were screened for Salmonella using conventional biochemical methods. The positive isolates were identified using polymerase chain reaction (PCR) for discernment of invasion A (invA) gene at explicit molecular size (284 bp) utilizing explicit primers (forward and reverse). Sequencing of the invA gene was performed and the similarities and differences between our invA gene and published sequences on GenBank were assessed. Seven out of ten confirmed Salmonella species isolates were positive to the invA gene while the remaining three were negative. The homology level of nucleotide sequence (97.746%) demonstrated high similitude between the local isolates and the other sequences on GenBank. Molecular characterization of the Salmonella isolates provides data about the virulence of the pathogen just as its relatedness to different organisms which offer data about the genome of the organisms and are helpful for epidemiological examinations. Therefore, Molecular methods which enable the detection of virulent genes are extremely important surveillance tools that are required to assist in curbing the escalation of infections caused by Salmonella.

Experimental Study of the Potential Role of Salmonella enterica subsp. diarizonae in the Diarrhoeic Syndrome of Lambs

The objectives of this experimental work were the evaluation of the potential role of Salmonella enterica subsp. diarizonae in diarrhoeic syndrome in lambs and the investigation of facets of the pathogenesis of the infection. In total, 12 lambs were challenged orally on the first day of life, with a S. enterica subsp. diarizonae isolate from a clinical case of diarrhoeic syndrome. Sequential blood, faecal and buccal samples were collected from lambs and faecal and milk samples were taken from their dams. Lambs were euthanised 1, 2, 4, 7, 10, 14 and 21 days after challenge. Samples were processed for recovery of the challenge organism; they were also subjected to examination by PCR for detection of the invA gene. Tissue samples from lambs were also examined as above and histopathologically. S. enterica subsp. diarizonae was recovered from faecal samples of all lambs, in total, from 45/77 samples (median duration: 2.4 days post-inoculation). It was also recovered from buccal samples (10/77) from seven lambs (median duration: 0.8 days), and from tissue samples (small intestine, abomasum, liver, gallbladder) of nine lambs. It was recovered from two consecutive milk samples from the same ewe, but not from any faecal sample from ewes. The invA gene was detected in samples from all lambs (median duration: 5.5 days in faecal and 1.3 days in buccal samples), as well as in milk samples from three ewes. Histopathological findings included abomasitis with subepithelial presence of eosinophils, lymphocytes and plasma cells, consistently observed in all lambs. In the small intestine, salient lesions initially included distension and oedema of intestinal villi, leucocytic infiltration and hyperplasia of lymphoid nodules with apparent germinal centres; this was followed at later stages by atrophy and/or degeneration of the lymphoid tissue of the intestine with marked subepithelial infiltration of lymphocytes, plasma cells and eosinophils.

Detection of invA and blaCTM-genes in Salmonella spp isolated from febrile patients in Lagos hospitals, Nigeria

Salmonella infections remain a global challenge. The culture method is the gold standard for the detection of genus Salmonella. Application of Polymerase Chain Reaction (PCR) has become an effective tool for the detection of virulence and antimicrobial resistance genes. This study investigated the prevalence of Salmonella by culture and detection of invA gene and blaCTX-M and blaCTX-M-3 gene markers by PCR. A total of 612 blood samples were collected from hospitalized febrile patients between March 2020 and April 2021. The samples were cultured, isolates identified by standard method with Analytical Profile Index (API 20-E) kits and were subjected to in-vitro antimicrobial susceptibility test (AST) using disk diffusion method. Extended-spectrum beta-lactamase (ESBL) detection was carried out by double-disc synergy test. Detection of invA gene and antibiotic-resistant genes makers was done by qPCR. A total of 24 Salmonella isolates were identified given a prevalence of 3.9% Salmonella-associated bacteraemia. Children within 1-10 years with persistent pyrexia of unknown origin (PUO) accounted for 50% of the Salmonella isolated with a mean age of 5.299 years. Specifically, 75% (18/24) Salmonella isolates and their corresponding samples of positive Salmonella culture were positive for the invA gene. The AST results indicated 100% Salmonella isolates developed resistance to ceftazidime, cefotaxime , augmentin, ampicillin, ertapenem, and doripenem. None of drug resistant-Salmonella isolates expressed ESBL enzyme phenotypically. Seven resistance patterns were observed, and the pattern CAZ-CTX-OFL-AUG-NIT-AMP-ETR-DOR was the most encountered pattern. Twelve (50%) Salmonella isolates harbored the blaCTX-M and blaCTX-M-3 genes and were mostly from children. The study has added to the growing knowledge on the suitability of the invA gene primer set as a PCR target for the detection of Salmonella. It also revealed a paradigm shift in the occurrence of invasive Salmonella harboring blaCTX-M and blaCTX-M-3 genes in PUO cases. There is a need for judicious use of cephalosporin and carbapenem antibiotics to preserve their efficacies.

Contribution of InvA Gene PCR to Recovery of Salmonella spp. Strains

Aims: Salmonella infection remains a major public health concern worldwide, contributing the economic burden of both industrialized and developing countries through the costs associated with surveillance, prevention, and treatment of disease. This zoonosis has a harmful health and economic impact in terms of death, hospitalization, and destruction of livestock on farms. To adapts the means of control and prevention against this threat, the phenotypical characterization of Salmonella strains, both those recently identified and those which have been conserved for a long time, is necessary. So, the aim of this study was to check quality of the salmonella strains first stored in storage tubes in NRC of salmonella of Institute Pasteur of Côte d'Ivoire. Place and Duration of Study: This study was done in Institute Pasteur of Côte d’Ivoire between July 2019 and October 2019. Methodology: A total of 56 tubes used to store salmonella strains with few or no agar were analyzed to assess presence of Salmonella. The strains were first cultured in broth and then on selective agar (Hecktoen) medium and nutrient agar. Then, the Salmonella-specific InvA gene was directly detected in the tubes. Results: The results obtained showed that the quality of the Salmonella strains initially conserved for at least 10 years had clearly deteriorated because none of them had been cultured after enrichment and culture on agar media. However, 73.21% of tubes not containing storage agar have traces of Salmonella DNA followed by molecular identification. The preponderant contaminating bacterial flora was represented by Gram positive. Conclusion: These results should encourage all laboratories to proceed immediately with the quality control of their strain collections for excellent biobanking.