Stn Gene Research Articles

Detection of virulent and ESBL-resistant Salmonella species from cattle and associated farm environment

The aim of this study was to explore the virulence characteristics and antibiotic resistance in Salmonella species isolated from cattle farms. During the study, 10 cattle farms were selected and from each farm, 35 samples consisting of faeces, milk, udder swabs, hand swabs, floor swabs, animal drinking water, pit water, composite fodder sample, and dairy utensil swabs were collected. Accordingly, 350 samples were collected from 10 farms and processed for the detection of Salmonella spp. Presumptive Salmonella isolates were confirmed by PCR and characterized for virulence and antibiotic resistance genes. From 350 samples, 19 (5.40%) Salmonellae were recovered, and out of 19 Salmonellae, 12 were ESBL producers. Similarly, out of 19 isolates, spvC was the predominant gene detected (42.10%), followed by stn (31.5%), and spvR (26.31%), whereas, invA, and iroB genes were detected in all 19 Salmonella isolates. Most of the Salmonella isolates were multi-drug resistant strains exhibiting resistance to nalidixic acid (94.73%), tetracycline (36.84%), and ceftazidime (42.10%). Out of 12 ESBL-positive Salmonellae, 9 (75%) isolates harboured ESBL genes, viz. blaSHV and blaTEM (25% each) and blaCTX-M and blaOXA (16.66% each). Six Salmonella isolates showed the presence of tetA gene. The current study indicated healthy cattle and the associated farm environments could be a source of virulent and drug-resistant Salmonellae. The current study also advocates phenotypic and genotypic antibiotic resistance monitoring at farm animal premises and agricultural ecosystems.

Rapid and simultaneous detection of Salmonella Sp. and Staphylococcus aureus in seafood by Multiplex PCR (mPCR)

aims: The present study is to investigate the prevalence of Salmonella sp. and Staphylococcus in seafood sample using multiplex polymerase chain reaction (mPCR). background: The rapid detection of food borne pathogen is increasing to ensure safety to consumers as major food borne illness is caused by pathogenic bacteria. Salmonellosis caused by Salmonella sp. is one of the primary concerns in many countries. Staphylococcus aureus is capable of generating toxins which can produce food poisoning in human body. objective: The thermostable nuclease (nuc) gene of Staphylococcus aureus and enterotoxin (stn) gene of Salmonella were used as target genes for mPCR detection. method: Totally, 10 seafood which includes fishes, crab and prawns which are generally available in Indian fish markets were selected for the present study. Samples that carried both the strains Salmonella and Staphylococcus were selected for mPCR by targeting the stn and nuc gene. result: Among 10 seafood samples collected, 7 of them carried Salmonella strain and 5 of them carried Staphylococcus strains. The results showed that 75% of the salmonella strains carried stn gene and 75% of the Staphylococcus strains carried nuc gene. conclusion: This study suggests that mPCR can be used for simultaneous detection by targeting the stn gene and nuc gene of salmonella and Staphylococcus food borne pathogens in seafood. other: Not applicable

Colistin-, cefepime-, and levofloxacin-resistant Salmonella enterica serovars isolated from Egyptian chicken carcasses

ObjectivesThe emergence of multidrug-resistant (MDR) Salmonella strains, especially resistant ones toward critically important antimicrobial classes such as fluoroquinolones and third- and fourth-generation cephalosporins, is a growing public health concern. The current study, therefore, aimed to determine the prevalence, and existence of virulence genes (invA, stn, and spvC genes), antimicrobial resistance profiles, and the presence of β-lactamase resistance genes (blaOXA, blaCTX-M1, blaSHV, and blaTEM) in Salmonella strains isolated from native chicken carcasses in Egypt marketed in Mansoura, Egypt, as well as spotlight the risk of isolated MDR, colistin-, cefepime-, and levofloxacin-resistant Salmonella enterica serovars to public health.MethodsOne hundred fifty freshly dressed native chicken carcasses were collected from different poultry shops in Mansoura City, Egypt between July 2022 and November 2022. Salmonella isolation was performed using standard bacteriological techniques, including pre-enrichment in buffered peptone water (BPW), selective enrichment in Rappaport Vassiliadis broth (RVS), and cultivating on the surface of xylose-lysine-desoxycholate (XLD) agar. All suspected Salmonella colonies were subjected to biochemical tests, serological identification using slide agglutination test, and Polymerase Chain Reaction (PCR) targeting the invasion A gene (invA; Salmonella marker gene). Afterward, all molecularly verified isolates were screened for the presence of virulence genes (stn and spvC). The antimicrobial susceptibility testing for isolated Salmonella strains towards the 16 antimicrobial agents tested was analyzed by Kirby–Bauer disc diffusion method, except for colistin, in which the minimum inhibition concentration (MIC) was determined by broth microdilution technique. Furthermore, 82 cefotaxime-resistant Salmonella isolates were tested using multiplex PCR targeting the β-lactamase resistance genes, including blaOXA, blaCTX-M1, blaSHV, and blaTEM genes.ResultsSalmonella enterica species were molecularly confirmed via the invA Salmonella marker gene in 18% (27/150) of the freshly dressed native chicken carcasses. Twelve Salmonella serotypes were identified among 129 confirmed Salmonella isolates with the most predominant serotypes were S. Kentucky, S. Enteritidis, S. Typhimurium, and S. Molade with an incidence of 19.4% (25/129), 17.1% (22/129), 17.1% (22/129), and 10.9% (14/129), respectively. All the identified Salmonella isolates (n = 129) were positive for both invA and stn genes, while only 31.8% (41/129) of isolates were positive for the spvC gene. One hundred twenty-one (93.8%) of the 129 Salmonella-verified isolates were resistant to at least three antibiotics. Interestingly, 3.9%, 14.7%, and 75.2% of isolates were categorized into pan-drug-resistant, extensively drug-resistant, and multidrug-resistant, respectively. The average MAR index for the 129 isolates tested was 0.505. Exactly, 82.2%, 82.2%, 63.6%, 51.9%, 50.4%, 48.8%, 11.6%, and 10.1% of isolated Salmonella strains were resistant to cefepime, colistin, cefotaxime, ceftazidime/clavulanic acid, levofloxacin, ciprofloxacin, azithromycin, and meropenem, respectively. Thirty-one out (37.8%) of the 82 cefotaxime-resistant Salmonella isolates were β-lactamase producers with the blaTEM as the most predominant β-lactamase resistance gene, followed by blaCTX-M1 and blaOXA genes, which were detected in 21, 16, and 14 isolates respectively).ConclusionThe high prevalence of MDR-, colistin-, cefepime-, and levofloxacin-resistant Salmonella serovars among Salmonella isolates from native chicken is alarming as these antimicrobials are critically important in treating severe salmonellosis cases and boost the urgent need for controlling antibiotic usage in veterinary and human medicine to protect public health.Graphical

Escherichia coli and Salmonella enterica isolated from Egyptian dairy cattle herds: The prevalence and molecular characteristics.

The pathogens Escherichia coli and Salmonella enterica that caused substantial health problems and financial losses were believed to have originated primarily from Egypt's dairy farms. The purpose of this study was to ascertain the occurrence of E. coli and S. enterica in three large dairy farms located in the Egyptian governorate of Sharkia. Furthermore, biochemical and serological characteristics of the isolated isolates were described. Further analysis revealed that several E. coli serovars had the genes stx1, stx2, eaeA, and hylA, while invA, stn, and hilA genes were found in several S. enterica serotypes using a multi-plex PCR. A total of 540 samples of fresh raw cow milk, water, feedstuffs, feces, (108 each), as well as swabs from feeders, milker hands and cattle crushes (36 each ), were gathered and analyzed. The recovery of E. coli from various sampling sources was shown to have an overall prevalence of 62.2% (336/540) in the results. Fecal samples had isolated S. enterica, with a frequency of 0.74% (4/540). The existence of various groups of serovars, such as O26, O44, O55, O78 and O111 for E. coli and Salmonella enteritidis, Salmonella typhimurium and Salmonella inganda for S. enterica was revealed by serological identification of the two species. However, it was discovered that a number of E. coli serovars had much higher percentages of the eaeA and hylA genes as well as shiga-toxin types 1 and 2 (stx1 and stx2). The presence of the invA gene, a diagnostic marker for S. enterica was 100% across all serovars. Salmonella enteritidis possessed both the enterotoxin gene (stn) and the hyper-invasive locus gene (hilA). Salmonella typhimurium had the hilA gene, whereas S. inganda had the stn gene. Escherichia coli and S. enterica recovered in this study have significant genetic risk factors for high pathogenicity and virulence, posing a real threat to dairy population productivity and health, which could spread to the general public through milk.

Chapter 6 - Childhood-inherited white matter disorders with calcification

Intracranial calcification (ICC) occurs in many neurologic disorders both acquired and genetic. In some inherited white matter disorders, it is a common or even invariable feature where the presence and pattern of calcification provides an important pointer to the specific diagnosis. This is particularly the case for the Aicardi-Goutières syndrome (AGS) and for Coats plus (CP) and leukoencephalopathy with calcifications and cysts (LCC), which are discussed in detail in this chapter. AGS is a genetic disorder of type 1 interferon regulation, caused by mutations in any of the nine genes identified to date. In its classic form, AGS has very characteristic clinical and neuroimaging features which will be discussed here. LCC is a purely neurologic disorder caused by mutations in the SNORD118 gene, whereas CP is a multisystem disorder of telomere function that may result from mutations in the CTC1, POT1, or STN genes. In spite of the different pathogenetic basis for LCC and CP, they share remarkably similar neuroimaging and neuropathologic features. Cockayne syndrome, in which ICC is usually present, is discussed elsewhere in this volume. ICC may occur as an occasional feature of many other white matter diseases, including Alexander disease, Krabbe disease, X-ALD, and occulodentodigital dysplasia.

Antibacterial efficiency of apple vinegar marination on beef-borne Salmonella.

Salmonella-related foodborne illnesses are a significant public health concern. Naturally, antibacterial food components have been shown to limit microbial growth proliferation with various degrees of efficacy. To examine the occurrence, microbial load, and effect of apple vinegar on Salmonella serovars in beef and beef products. 150 beef and beef products were collected between March and May 2022. Total viable count (TVC), Enterobacteriaceae count (ENT), isolation and identification of Salmonella, and their virulence factors detection by multiplex PCR were determined, and an experimental study of the effect of natural apple vinegar marination on Salmonella spp. TVC was higher in meatballs (3.32 × 106 ± 1.07 × 106) while beef burgers (4.22 × 103 ± 0.71 × 103) had the highest ENT. Concerning the prevalence of Salmonella spp., meatball (46.7%) and beef burger (25.3%) samples were the highest contamination rate. The common serovars detected were Salmonella typhimurium (6%), Salmonella enteritidis (6%), and Salmonella infantis (4%). Based on the results of PCR, 12, 11, and 11 out of 18 samples of Salmonella isolates possess hila, stn, and invA genes. By immersing the inoculated steak meat in apple vinegar at different concentrations (50%, 70%, and 100%), the initial populations of the Salmonella strains after 12 hours were reduced to 0.38 × 102 ± 0.05 × 102 log CFU/ml; however, after 48 hours become the most reduction (0.31 × 102 ± 0.07 × 102 log CFU/ml) at a concentration of 100% apple vinegar. An enhancement in the sensory attributes was noted across all concentrations. The consumed beef and beef products are contaminated with pathogenic bacteria such as Salmonella spp. Marinades made using apple vinegar concentrations of 50%, 75%, and 100% effectively minimized the prevalence of artificially inoculated Salmonella and extended the shelf life of preserved refrigerated beef products to 48 hours.

Bacteriological Examination of Deboned Turkey Meat in Gharbia Province

The present work was carried out to evaluate the microbiological quality of deboned turkey meat from the retail outlets in Gharbia province via determination of Aerobic bacterial count (ABC), coliforms count (CC) and isolation and identification of Salmonella, Staphylococcus aureus, Escherichia coli So, this study was carried out on a total of 100 sample of deboned turkey meat including Breast muscle and Thigh muscle of turkey samples (50 of each) that, were collected from local markets of Tanta University Garbia province –Egypt. All samples were transported without delay in a frozen state to the laboratory of animal health research institute – Tanta. Samples were subjected to bacteriological investigations. it was recorded that total bacterial counts showed a higher level of aerobic plate counts showed a higher level of total aerobic plate count observed in Thigh muscle (5.9×109) than the Breast muscle (2.5×106), While, the coliforms count showed a higher mean level observed in Thigh muscle (9.9×106) than the Breast muscle (2.8×105),The Salmonella counts showed a higher incidences observed in Breast muscle (18 %) than the Thigh muscle (12 %), The Escherichia coli counts showed the higher incidences of Escherichia coli count observed in thigh muscle (16 %) than the breast muscle (12 %). While, the higher level of Staphylococcus aureus counts observed in Thigh muscle (16 %) than the Breast muscle (12 %). While present results on the PCR results cleared that, the PCR for detection of Escherichia coli the Photograph of agarose gel electrophoresis of multiplex PCR Gave positive results for stx1 at (614 bp), stx2 at (779 bp) and for eaeA (890 bp) genes for characterization of Enteropathogenic Escherichiacoli. The higher incidences of Enteroppathogenic Escherichia Coli genes observed in stx1 (4 %) followed by stx2 (5 %) and eae A gene (1 %), While, the PCR results of Salmonella Spp cleared that, the agarose gel electrophoresis of multiplex PCR cleared that, the genes for salmonellae includes stn (260 bp), hilA (497 bp) and fimH (1008 bp) virulence genes for characterization of Salmonella species. The genes responsible for pathogenicity of salmonellae includes stn observed in (3 %) of examined samples, followed by hilA (4 %) and fimH 85 %) of examined samples. While, our results on the Staphylococcus aureus cleared that, the Photograph of agarose gel electrophoresis of multiplex PCR of genes responsible for pathogenicity of Escherichia coli includes sea (120 bp), seb (478 bp), sec (257 bp) and sed (317 bp) enterotoxin genes for characterization of S. aureus. The lanes of positive Staphylococcus aureus strains gave positive results for sea gene, seb gene, sec and sed genes. This study concluded that turkey meat possess a higher number of microorganisms like Salmonella, Staphylococcus aureus, Escherichia coli. Also, the presence of Salmonella, Staphylococcus aureus, Escherichia coli in turkey meat products may be due to unsanitary conditions, cross-contamination and post-processing contamination during handling, packaging, storage, distribution and selling on the other hand application of Hazard Analysis and Critical Control Point System (HACCP) which control the technological line of the production at all points from the farm to table to produce a safe product fit for human consumption.

Molecular Identification and Phylogenetic Analysis of Salmonella‎ species ‎Isolated from Diarrheal Children and Dogs in Baghdad Governorate, ‎Iraq‎

This work aimed to use conventional PCR to identify Salmonella‎ spp. that ‎were isolated from diarrheal children and healthy and diarrheic dogs based on four ‎virulence genes, hilA, stn, spvR‎, and marT. Sixteen Salmonella‎ isolates including: 9 ‎isolated from children's diarrhea from three species (S. Typhimurium, S. Enteritidis, S. ‎Typhi) and seven isolated from dogs including (S. Typhimurium, S. Enteritidis, S. ‎Muenchen), were identified primarily by several methods. The PCR products of the 16S ‎rRNA gene were sequenced and examined using BLAST analysis to find differences and ‎similarities between these Iraqi isolates and already-known global strains in order to ‎construct the phylogenetic tree of S. Muenchen which was detected for the first time in ‎dogs in Iraq. The results of the study revealed that all isolates of Salmonella‎ obtained ‎from children possess the hilA and stn genes. The marT gene was detected in 88% of the ‎Salmonella‎ serovars, and the spvR‎ gene was carried in 55% of the isolates. Among dog ‎Salmonella‎ isolates, the hilA gene was detected at 100%, the stn gene was at 85.7%, the ‎marT gene was present at 71.4%, while the spvR‎ gene was found at 57.1%. The result of ‎DNA sequencing and phylogenetic tree indicated that the local Iraqi S. Muenchen was ‎extremely close to the national strain and share the same ‎16S rRNA gene sequence, the ‎isolate was registered at NCBI and became a global reference with the accession number OQ999043.1. In conclusion, the presence of these important virulence genes among ‎Salmonella‎ serovars isolated from children and dogs alerted on the potential risk of ‎contamination of the environment and may lead to a community health crisis‎.

Extensively drug-, ciprofloxacin-, cefotaxime-, and azithromycin-resistant Salmonella enterica serovars isolated from camel meat in Egypt

Given the great importance of Salmonella as a leading foodborne pathogen of global concern and the few available data regarding its prevalence in camel meat, the present study aimed to determine the prevalence, antimicrobial resistance (AMR) profile, virulence genes, β-lactamase genes of Salmonella enterica serovars isolated from camel meat marketed in Egypt. Forty-five (29.6 %) of the 152 camel meat samples examined were positive for Salmonella spp. Among the 432 Salmonella presumptive colonies isolated, 128 were molecularly verified as Salmonella after confirmation by PCR targeting the Salmonella marker (invA) gene. Virulence genes, encompassing stn, spvC, and hilA genes, were detected in 91.4 % (117/128), 20.3 % (26/128), and 80.5 % (103/128) of the isolates, respectively. S. Enteritidis, S. Typhimurium, S. Cerro, and S. Montevideo were the most prevalent serovars with incidences of 25 % (32/128), 15.6 % (20/128), 15.6 % (20/128), and 12.5 % (16/128), respectively. Interestingly, 56.3 %, 53.1 %, 37.5 %, 28.1 %, 21.9 %, 18.8 %, 12.5 %, and 3.1 % of the isolates tested showed resistance to cefepime, ciprofloxacin, levofloxacin, cefotaxime, gentamicin, colistin, meropenem, and azithromycin, respectively. Salmonella isolates showed resistance to at least one antibiotic, with a mean multiple antibiotic resistance (MAR) index of 0.472. Interestingly, 59.4 %, 15.6 %, and 3.1 % of the isolates were categorized into multidrug-resistant, extensively drug-resistant, and pan-drug-resistant, respectively. Only 23 (25 %) of the 92 ampicillin-resistant isolates were proven to be Extended Spectrum Beta-Lactamase (ESBL)-producing Salmonella, in which β-lactamase (bla) genes were detected. The blaOXA-2 was the most existing gene where it was detected lonely in 10 of the ampicillin-resistant isolates and coexisted with blaCMY-1 in 4 isolates and with blaCMY-2 in a single isolate. The blaCMY-1, however, existed in 11 isolates, whereas the blaCMY-2 gene was only detected in 3 isolates tested. The present findings affirm that camel meat could be a leading reservoir for multi- and extensively-drug-resistance β-lactamase-producing Salmonella, representing a global public health challenge. Therefore, further research is necessary to detect the prevalence and AMR of Salmonella serovars from camel meat in Egypt and other countries to put camel meat as a source of Salmonella in foods of animal origin.

Assessment of stn, sipB and sopB Virulence Genes in Various Salmonella Serovars.

Salmonella is a zoonotic bacterium that is considered to be one of the most common causes of foodborne infections worldwide. Bearing in mind the genes involved in its virulence, identifying these genes can enable experts to better understand bacterial pathogenicity, which could subsequently help develop more efficient means to control and prevent infections. This study aimed to analyze stn, sipB, and sopB genes in various Salmonella serovars. To carry out this study, 103 Salmonella serovars were extracted from livestock, poultry, and humans from existing samples at the Department of Microbiology of the Razi Serum and Vaccine Research Institute in Karaj, Iran. These samples were cultured in selection and differential media, and their serovars were identified using specific antibodies based on Kaufman-White Tables. Utilizing PCR and specific primers, stn, sopB, and sipB genes were detected among these serovars. In this investigation, the most common human serovars were Salmonella paratyphi A, Salmonella paratyphi B, and Salmonella enteritidis; the most common serovars among livestock consisted of Salmonella dublin and Salmonella typhimurium and the most common Salmonella serovars among poultry consisted of Salmonella infantis and Salmonella enteritidis. The results of PCR on stn, sipB, and sopB genes demonstrated segments with 617bp, 875 bp, and 220 bp on agar gel, respectively. Based on the obtained findings, stn, sipB, and sopB genes were detected in 96.11%, 99.02%, and 98.05% of Salmonella serovars, respectively. Considering the fact that the aforementioned genes play significant roles in bacterial virulence, they can be used to develop diagnostic ELISA kits and recombinant vaccines.

Virulence genes identification in Salmonella enterica isolates from humans, crocodiles, and poultry farms from two regions in Colombia.

Salmonella spp. is frequently found in the digestive tract of birds and reptiles and transmitted to humans through food. Salmonellosis is a public health problem because of pathogenicity variability in strains for virulence factors. This study aimed to identify the virulence genes in Salmonella isolates from humans, crocodiles, broiler cloacas, and broiler carcasses from two departments of Colombia. This study was conducted on 31 Salmonella enterica strains from humans with gastroenteritis (seven), crocodiles (seven), broiler cloacas (six), and broiler carcasses (12) from Tolima and Santander departments of Colombia, belonging to 21 serotypes. All samples were tested for Salmonella spp. using culture method on selective and non-selective mediums. Extraction of genomic DNA was performed from fresh colonies, DNA quality was verified by spectrophotometry and confirmed by amplification of InvA gene using conventional polymerase chain reaction (PCR). bapA, fimA, icmF, IroB, marT, mgtC, nlpI, oafA, pagN, siiD, spvC, spvR, spvB, Stn, and vexA genes were amplified by PCR. The most prevalent gene was bapA (100%), followed by marT (96.77%), mgtC (93.55%), and fimA (83.87%). Likewise, IroB (70.97%), Stn (67.74%), spvR (61.29%), pagN (54.84%), icmF (54.8%), and SiiD (45.16%) were positive for more than 50% of the strains. Furthermore, none of the isolates tested positive for the vexA gene. Salmonella isolates presented 26 virulence profiles. This study reported 14 virulence genes in Salmonella spp. isolates from humans with gastroenteritis, crocodiles, and broiler cloacas and carcasses. The distribution of virulence genes differed among sources. This study could help in decision-making by health and sanitary authorities.

Paratyphoid Salmonella serovars in chickens: molecular detection of virulence and antimicrobial resistance genes

Paratyphoid salmonellosis is a serious disease threatens the poultry industry worldwide, besides its public health hazard. The aims of this study were characterization of paratyphoid Salmonella spp. in chicken flocks of some Egyptian governorates, demonstration of the antimicrobial susceptibility of the isolated Salmonella spp., and detection of some virulence genes and antibiotic resistance genes using recent molecular techniques. A total of 238 organ samples were collected from 52 broiler, layer, and breeder chicken flocks, representing 9 Egyptian governorates. Conventional characterization of Salmonella isolates revealed a total isolation rate of 56.3% (134/238). Moreover, the isolation rates of Salmonella spp. were (49/79; 62%), (47/81; 58%), (10/18; 55.5%), (9/20; 45%), (2/6; 33.3%), (2/3; 66.7%), and (15/82; 53.6%) from liver, yolk sac, heart, spleen, caecum, ovary, and dead-in-shell embryos, respectively. A total of 32/238 (13.44%) isolates of Salmonella were found. Serological identification revealed presence of S. enteritidis (21.9%), S. kentucky (15.6%), S. typhimurium (12.5%), S. molade (12.5%), S. takoradi (9.4%), S. wingrove (6.3%), S. infantis (6.3%), S. tsevie (6.3), S. shangani (3.1%), S. bargny (3.1%), and S. papuana (3.1%). All Salmonella strains (32/32; 100%) were resistant to streptomycin, while almost all of them (31/32; 96.9%) were susceptible to meropenem. The amplification of 16S rRNA gene of Salmonella isolates using uniplex polymerase chain reaction (PCR) generated a specific Salmonella product of approximate 550 base pair. The multiplex PCR revealed presence of invA (100%), stn (65.6%), and sopB (40.6 %) virulence-associated genes as well as aadA1 (100%), blaTEM (59.4%), aadB (18.75%), and sul1 (28.1%) antibiotic resistance genes. In conclusion, virulent paratyphoid Salmonella spp. are circulating in the Egyptian flocks, causing economic loses. Additionally, they became resistant to the most commonly used field antibiotics. Therefore, regular molecular surveillance studies on the circulating Salmonella spp. and their resistance to the used antibiotics are of significant importance.

Antibiotic Resistance Pattern and Distribution of Resistance Genes in Salmonella Isolated from Chicken and Duck Eggs in Chhattisgarh, India

Background: Salmonella is recognized as the most prevalent bacterial cause of foodborne diseases worldwide and animal-sourced foods have been reported as a common source of Salmonella infections among humans. Methods: The commercial chicken eggs, backyard chicken eggs, and duck eggs samples, 60 each, were processed for isolation and identification of Salmonella. All Salmonella isolates were further tested for resistance against six different antibiotics. The prevalence of virulence and antimicrobial resistance genes in the Salmonella isolates was determined by PCR. Result: A total of 28 Salmonella isolates were recovered with an overall prevalence of 15.6% and out of them, 11.1% and 4.4% were from eggshell and egg content, respectively. All the isolates were found sensitive to Gentamicin however maximum resistance was observed against Cefotaxime. PCR results revealed that 100% of the isolates were carrying the invA gene however stn gene was detected in 78.6% of isolates. Among presumptively identified β-lactam-resistant Salmonella isolates, 100% and 50% isolates harbored blaTEM and blaCTX-M genes, respectively whereas none of the isolates contained the blaSHV gene. All tetracycline-resistant isolates harbored the tetA gene whereas none of the isolates carried the tetB gene. 100% of fluoroquinolone-resistant isolates were carrying the gyrA gene however parC gene was present only in 60% of isolates. These results indicate that drug-resistant Salmonella spp. were prevalent in eggs sold in the study area which can pose a serious public health problem.

Assessment of stn, sipB and sopB virulence genes in various Salmonella serovars

Salmonella is a zoonotic bacterium that is considered to be one of the most common causes of foodborne infections worldwide. Bearing in mind the genes involved in its virulence, identifying these genes can enable experts to better understand bacterial pathogenicity, which could subsequently help develop more efficient means to control and prevent infections. This study aimed to analyze stn, sipB, and sopB genes in various Salmonella serovars. To carry out this study, 103 Salmonella serovars were extracted from livestock, poultry, and humans from existing samples at the Department of Microbiology of the Razi Serum and Vaccine Research Institute in Karaj, Iran. These samples were cultured in selection and differential media, and their serovars were identified using specific antibodies based on Kaufman-White Tables. Utilizing PCR and specific primers, stn, sopB, and sipB genes were detected among these serovars. In this investigation, the most common human serovars were Salmonella paratyphi A, Salmonella paratyphi B, and Salmonella enteritidis; the most common serovars among livestock consisted of Salmonella dublin and Salmonella typhimurium and the most common Salmonella serovars among poultry consisted of Salmonella infantis and Salmonella enteritidis. The results of PCR on stn, sipB, and sopB genes demonstrated segments with 617bp, 875 bp, and 220 bp on agar gel, respectively. Based on the obtained findings, stn, sipB, and sopB genes were detected in 96.11%, 99.02%, and 98.05% of Salmonella serovars, respectively. Considering the fact that the aforementioned genes play significant roles in bacterial virulence, they can be used to develop diagnostic ELISA kits and recombinant vaccines.

Detection of Virulence and β-lactamase resistance genes of non-typhoidal Salmonella isolates from human and animal origin in Egypt "one health concern"

BackgroundNon-typhoidal Salmonella (NTS) is a major foodborne zoonotic pathogen worldwide. In the current study, Various NTS strains were isolated from (cows, milk and dairy products in addition to humans) in New Valley and Assiut Governorate, Egypt. NTS were firstly serotyped and tested by antibiotic sensitivity test. Secondly, some virulence genes and Antibiotic resistance genes have been identified by using PCR. Finally, Phylogenesis was performed depending on the invA gene, for two S. typhimurium isolates (one of animal origin and the other of human origin for evaluating zoonotic potential).ResultsOut of 800 examined samples, the total number of isolates was 87 (10.88%), which were classified into 13 serotypes, with the most prevalent being S. Typhimurium and S. enteritidis. Both bovine and human isolates showed the highest resistance to clindamycin and streptomycin, with 90.80% of the tested isolates exhibiting MDR. The occurrence of the invA gene was 100%, while 72.22%, 30.56%, and 94.44% of the examined strains were positive for stn, spvC, and hilA genes, respectively. Additionally, blaOXA-2 was detected in 16.67% (6/ 36) of the tested isolates, while blaCMY-1 was detected in 30.56% (11of 36) of the tested isolates. Phylogenesis revealed a high degree of similarity between the two isolates.ConclusionsThe high occurrence of MDR strains of NTS in both human and animal samples with high degree of genetic similarity, shows that cows, milk and milk product may be a valuable source of human infection with NTS and interfere with treatment procedures.

Cas12a-based primer production enables isothermal amplification for nucleic acid detection

Clustered regularly interspaced short palindromic repeats (CRISPR) and the associated Cas proteins have been employed as innovative tools for biomolecule detection. However, the functionality of the Cas protein is limited by the RNA-guided nuclease that cleaves pre-amplified products to generate fluorescence signals. As such, we proposed a strategy to use the Cas12a protein for active primer production and as an initiator for subsequent isothermal amplification. Target nucleic acids activate Cas12a to cleave the DNA component of RNA-DNA chimera primers with a 3′ phosphate group, converting inactive RNA-DNA chimera primers into active ones. We then used two isothermal amplification methods based on Klenow DNA polymerase/T7 RNA polymerase and terminal deoxynucleotidyl transferase to generate fluorescence and colorimetric signals, respectively. The Salmonella enterotoxin (stn) gene was effectively amplified with satisfactory sensitivity and selectivity even in real samples such as drinking water and human serum, paving the way for Cas12a-derived active primers that may be useful in diverse detection platforms.

Identification and Characterisation of Salmonella spp. Isolated from Stool Samples of a Food Poisoning Outbreak in Chite, Mizoram, India due to Consumption of Bekang-Um (Fermented Soybean)

Introduction: Traditional fermented soybean (“bekang-um”) is widely consumed by the indigenous Mizo tribe in Mizoram and plays a significant part of the local staple food. They are commonly prepared with herbs like culantro and often with just locally dried chilli flakes, implying it is mostly consumed raw without further cooking. Though it plays a staple part of the diet because of its unique taste, awareness on proper and regulated hygienic conditions during its preparation and fermentation processes is quite negligent. The fermentation process often warrants for the growth of not only commensal lactic acid fermenter bacteria like Acinetobacter spp., Lactobacillus spp., but also provides ample growth conditions for harmful bacteria like Salmonella spp., Clostridium spp., Intestinal Pathogenic E.Coli (IPEC) and Bacillus cereus. Aim: Phenotypic and molecular characterisation of Salmonella spp. isolated from the fermented food product bekang-um. Materials and Methods: The cross-sectional study was conducted within three months (October to December, 2020) in Multidisciplinary Research Unit (MRU), Zoram Medical College in collaboration with the Department of Veterinary Microbiology, CVSc and AH, Central Agricultural University, Selesih, Mizoram. A total of 18 stool samples were collected from patients (living in Chite, a small locality in Aizawl) who were still exhibiting symptoms of food poisoning (diarrhoea, vomiting and nausea) after consumption of fermented soybean all bought from their local neighbourhood fermenter and seller of the food item. The data was tabulated and statistically analysed using Microsoft Excel and sensitivity/resistance patterns were expressed in percentages. Results: Salmonella spp. was isolated from 17 out of 18 stool samples collected. Out of 17 samples, 15 exhibited Multi-Drug Resistance (MDR) phenotypically to clinically relevant and widely used antibiotics like cefotaxime, ceftriaxone, ciprofloxacin, amoxicillin-clavulanic acid. Alarmingly, resistance genes like blaCTX-M1 (70.6%), blaCTX-M2 (35.3%), blaSHV (70.6%), blaTEM (52.9%), blaNDM (5.8%), blaAmpC (100%), blaKPC (29.4%), blaVIM (23.5%), blaIMP (11.8%) were commonly detected. Moreover, Salmonella virulence determinants like invA (41.2%), stn (41.2%), and biofilm-associated virulence genes like csgA (11.8%), csgD (70.6%) and adrA (5.9%) were also identified. Conclusion: The identification and characterisation of Salmonella spp. isolated from a widely consumed food product “bekangum” (fermented soybean) with co-occurring crucial virulence, biofilm-producing and resistance gene determinants is quite alarming. The findings suggest the urgent need of implementing food safety conditions and proper preparation standards of the exotic food item, and the seriousness of the potential threat it can cause in cases of unhygienic fermentation processes.

Identification of Salmonella carriers by amplification of FimA, Stn and InvA genes and bacterial culture methods in fecal samples of buffalo.

Salmonellosis is one of the most important bacterial diseases in human and animals. Rapid diagnosis and sub sequence accurate treatment of Salmonella carriers help reduce the salmonellosis in human and livestock animals. In this study, 420 fecal samples were taken during year 2019 from buffalo in the Urmia, Khoy and Piranshahr regions in west Azerbaijan province, Iran. Samplings were carried out in different seasons. Presence of Salmonella invasion genes (FimA, Stn and InvA) were evaluated by polymerase chain reaction. The bacterial culture and biochemical tests were performed on feces samples for isolation of bacterium Salmonella; however, all samples were negative in culture method. PCR findings showed that, 50 (11.90%) fecal samples were positive to the genes. The analysis of results showed that frequency of salmonellosis outbreak in different parts of west Azerbaijan province followed a similar pattern and the incidence of salmonellosis according to forecast in the warm seasons (spring and summer) was more than in cold seasons (autumn and winter). The prevalence of Salmonella in buffalo's feces based on warm and cold seasons were 32 (64.00%) and 18 (36.00%), respectively. The results showed significant difference between cold and warm season in the prevalence of salmonellosis. Therefore, the application of molecular technics is essential for the prevention and treatment of salmonellosis. The results also showed that specificity of PCR method was better than culture method for detection of Salmonella in feces sample.

Determination of MIC, MPC, and MSW of Ilex paraguariensis against non-typhoidal Salmonella with identification of the mechanisms of resistance and pathogenicity factors.

This study investigated the antibacterial activity of the aqueous extract of Ilex paraguariensis against 32 different strains of nontyphoidal Salmonella (NTS) through the determination of the minimum inhibitory concentration (MIC), mutant prevention concentration (MPC), and mutant selection window (MSW) and the detection of virulence genes by multiplex PCR assays. The MIC values of Ilex paraguariensis against Salmonella spp. strains varied between 0.78mg/ml and 6.25mg/ml with a MIC90 of 3.12mg/ml. The highest MPC in this study was 48mg/ml yielding a mutant selection window of 41.75mg/ml. The MSW values of the remaining strains varied between 1.56 and 8.87mg/ml. Genes of pathogenicity detected in Salmonella spp. isolates were most commonly the stn, sdiA, invA, sopB, invH, and sopE genes. The antibacterial activity of yerba mate extract was not affected by the antimicrobial resistance patterns or pathogenicity genes expressed. More work is needed to identify the active antibacterial compound(s) responsible for the antibacterial activity.

Detection and Molecular Identification of Salmonella Pathogenic Islands and Virulence Plasmid Genes of Salmonella in Xuzhou Raw Meat Products

Virulence genes expressed in Salmonella are a primary contributing factor leading to the high morbidity and mortality of salmonellosis in humans. The pathogenicity of Salmonella is mainly determined by the specific virulence factors that it carries. These factors also confer greater virulence and play a role in infection of a host and transmission of disease, and most Salmonella enterica can cause cross-infections between humans and animals. In this study, 265 samples in total were collected from a farmer's market and two supermarkets in Xuzhou, Jiangsu province, China, including 205 pork samples and 60 chicken samples. The suspected Salmonella isolates were isolated and identified using microbiological and molecular methods, and the confirmed isolates were used for serovar analysis and antimicrobial susceptibility testing. The virulence genes of Salmonella pathogenic islands (SPIs) and Salmonella virulence plasmids (Spv) in Salmonella-positive isolates were subsequently detected. Salmonella was isolated from 29.0% of samples, and all isolates were confirmed by PCR targeting the stn gene. Among the Salmonella isolates, resistance was most frequently observed against ciprofloxacin (84.4%), followed by tetracycline (71.4%) and streptomycin (68.8%). Resistance to amoxicillin-clavulanic acid (6.3%) and aztreonam (5%) was less commonly detected. The presence of the following virulence genes was determined by specific PCRs: hilA (SPI-1), sifA (SPI-2), misL (SPI-3), siiE (SPI-4), sopB (SPI-5), and spvC. The detection rate for SPI-1 to SPI-5 was 93.5, 87.0, 97.4, 97.4, and 97.4%, respectively. In addition, the detection rate of the spvC gene was 96.1%. Except for sopB (94.7%), all isolates of the dominant serovar S. enterica subsp.. enterica serovar Enteritidis contained all virulence genes from SPI-1 to SPI-5. This study demonstrated the epidemiological status of Salmonella in raw meat products in Xuzhou, and the complex antibiotic resistance and high isolation rate of virulence genes observed reveal many potential risks of which the findings presented herein will provide orientation to improve public health safeguards.