Direct detection of iro B, stn and hil A virulence genes in Salmonella enterica serovar typhimurium from non-ripened cheese
Bulletin of the National Research Centre volume 46, Article number: 175 (2022)
Microorganisms' survival is based on the importance of growth factors found in the microbial environment, as well as their overwhelming appetite for survival which is controlled by their genetic material. This study was designed to investigate the virulence patterns of Salmonella enterica serovar typhimurium associated with soft cheese. Two hundred and sixty (260) soft cheese samples were collected from vendors at Akure, Ikare and Owo in Ondo State, Nigeria. Molecular characterization of six (6) Salmonella enterica isolates for the possession of iro B, stn, hil A and spv C gene were revealed through multiplex polymerase chain reaction (PCR) analysis with DNA extraction, polymerase chain reaction (PCR) amplification, sequencing and genome blasting, respectively.
Four (4) possess only iro B; two (2) possess only stn gene; and another three (3) possess hil A gene as none of the isolates possess spv C gene. This study revealed antibiotic-resistant Salmonella enterica serovar typhimurium is present in locally made soft cheese samples. It also revealed that virulence genes comprising hil A, stn and iro B as well as plasmids-mediating antibiotic resistance are present in S. enterica serovar typhimurium found in locally produced cheese.
The presence of molecularly elucidated virulence genes comprising Iro B, Stn and Hil A makes the soft cheese potentially viable for pathogenicity. This study recommends food-borne salmonellosis should be constantly monitored with appropriate cleaning of preparatory paraphernalia in cheese production. Constant sensitization of nomads to improve their awareness on milk-borne zoonosis and its associated risk factors is needed.
The presence of Salmonella enterica serovar typhimurium poses serious threat to human health and food antimicrobial sensitivity akin to drastically reducing the antimicrobial importance of food (Amiri and Moradli 2016). The bacterium is a primary enteric pathogen infecting both humans and animals. This infection begins with the ingestion of contaminated food or water by the ease passage of Salmonellae into intestinal epithelium of the human system to trigger gastrointestinal disease (Amiri and Moradli 2016). Also, this organism is among the most common etiological agent of cases and outbreak of food-borne diarrheal illnesses. For Salmonella enterica serovar typhimurium to cause any infection, the presence of O and H antigens is essential which indicate the somatic and the flagella strains of genes, respectively, i.e. the invasion and the spread of the pathogen (Larrisa et al. 2016). Mostly, Salmonella enterica serovars are found worldwide in warm-blooded animals. They are intracellular pathogens which insinuate they reside inside the body of living host (Larrisa et al. 2016).
Salmonellosis is associated with severe morbidity and even mortality in farm animals representing a major economic productivity loss in the food and animal industries (Leekitcharoenphon et al. 2016). Hence, it is systematic infection caused by Salmonella species and serotypes. The most common clinical symptoms range from headache, fever, abdominal cramps and vomiting. Young and old individuals especially those with weakened immune system, i.e. either they are immune-compromised or immune-suppressed, are bound to develop severe form of salmonellosis like reactive arthritis, irritable bowel syndrome (Leekitcharoenphon et al. 2016). Salmonella enterica serovars has major public health burden worldwide, causing over 1 billion human infections annually with its impact being responsible for significant morbidity and mortality (Leekitcharoenphon et al. 2016). An estimated 94% of salmonellosis is transmitted by food (Luvsansharav et al. 2020). As a result, implicated foods are often of animal origin such as beef, poultry, milk, and eggs. Any food, however, can become contaminated through cross-contamination, environmental contamination, or by the unwashed hands of food workers (Leekitcharoenphon et al. 2016).
The emergence of multidrug resistant strains limits the possibilities of effective treatment, and new antimicrobial targets are required (Akinyemi et al. 2018). Salmonella enterica serovar typhimurium is broadly adapted to the host with the respect of being the major cause of food-associated nontyphoidal gastrointestinal disease, with asymptomatic carriage in farm animals presenting a common reservoir for transmission to humans (Rivero-Calle et al. 2021). However, the prevalence of enteritis and its accompanying diarrheal and other health challenges linked to infections with Salmonella has continuously plagued sub-Saharan Africa (Akinyemi et al. 2018; Rivero-Calle et al. 2021). In Nigeria, typhoid fever is among the major widespread diseases affecting both young and old as a result of many interrelated factors such as inadequate sanitation, indiscriminate use of antibiotics and faecal contamination of water sources (Akinyemi et al. 2018). Morbidity associated with illness due to Salmonella continues to increase with untold fatal consequences, often resulting in death. An accurate figure of cases is difficult to arrive at because only large outbreaks are mostly investigated whereas sporadic cases are under-reported (Rivero-Calle et al. 2021).
In Africa, S. enteritidis and S. typhimurium were the two most common serotypes reported, occurring in 26% and 25% of the isolates, respectively (Jajere 2019). Salmonella enterica serovar typhimurium (29%) was most frequently reported in clinical isolates in North America, followed by S. enteritidis (21%). In spite of improvements in hygiene and sanitation, the incidence of nontyphoidal Salmonella (NTS) infections continue to increase, creating a burden in both industrialized and underdeveloped countries (Majowicz et al. 2010).
Salmonella is of great concern for dairy industry. It causes morbidity and mortality throughout the world with the host immune response which can either be infection is acute (limited) or chronic (Systemic) (Pui et al. 2011; Ruszel et al. 2021). The nature of pathogenicity of an organism lies in the virulence genes or virulence factors. The majority of virulence genes of Salmonella are clustered in a region distributed over the chromosome, called Salmonella Pathogenicity Islands (SPI) (Eng et al. 2015; Dos Santos et al. 2021). Hil A gene, an invasive operon of salmonella, is found to trigger the internalization of deeper tissue of host systems as revealed by Yanestria et al. (2019). Salmonella virulence factors were also detected in virulence plasmids in certain Salmonella serovars like Salmonella enterica serovar typhimurium as juxtaposed by Yanestria et al. (2019).
Cheese is a nutritional versatile dairy food. A wide variety of cheese types are available to meet specific consumer requirements and allow convenience use (Aldrete-Tapia et al. 2018). The microbiota present in local soft cheese production across seasons is a crucial role as it shows the diversity in growth of microorganisms which is based on milk composition and environmental conditions like temperature, humidity, etc. (Aldrete-Tapia et al. 2018). However, bacterial species that were found at the start of production of cheese reduces, as there were subsequent processes involved in the production of cheese that can inhibit or destroy the presence of resident organisms. A preceding study has shown that these organisms are potentially antagonistic in nature (Aldrete-Tapia et al. 2018). Apart from Salmonella enterica, several organisms such as Listeria monocytogenes, Escherichia coli, Enterococcus faecalis, and Shigella dysenteriae are quite present in unsafe cheese production as detailed by Sangoyomi et al. (2010); Al-Gamal et al. (2019).
The production of diverse dairy products comprising soft cheese from raw milk improved its microbial quality by increasing the salinity of the manufactured dairy products (Al-Ashmawy et al. 2016). Conversely, the primordial method of manufacturing under unrestrained sanitized conditions in developing countries is a barrier to good soft cheese with sound microbiological quality (Omar et al. 2018). Satisfactory milk pasteurization application in non-ripened cheese could inactivate pathogenic derivatives during soft cheese production and storage (Fusco et al. 2020). Hence, this study set out to investigate the virulence patterns of Salmonella enterica serovar typhimurium associated with soft cheese in South-West, Nigeria.
Colony counter (J-2 PEC MEDICAL, New Jersey, USA), Salmonella-shigella agar (SSA), sterile distilled water, 70% ethanol, sodium acetate, magnesium chloride (MgCl2), BLAST-Basic local alignment search tool, soft cheese, microtube, and refrigerated universal centrifuge.
This is an experimental study conducted between January and late April 2019. It was hypothesized that there are complex genetic dynamics of mobile genetic elements that codes for the establishment of pathogens in host cells. As a rider to this, soft cheese samples were randomly collected from nomads to checkmate the hygienic practices of sellers and potential pathogenicity for susceptible consumers.
Description of Study location
The study location was accessed through experimental design. Areas of study are Ikare, Owo and Akure which are located at the Northern (Ikare and Owo), and central part (Akure) of Ondo State, respectively (Fig. 1). Ikare lies between latitudes 070.27′N; 070.31′N and longitudes 0050.43′E; 0050.47′E. Akure lies between latitudes 7.25′ N and longitude, 5.195′ E. Owo, on the other hand, is located between latitude 7.2° N and longitude, 5.59° E. These areas are moderately populated with moderate standard of living. Residents living in these areas are majorly farmers, teachers, traders, civil servants, legal and medical practitioners.
Collection of samples
Two hundred and sixty (260) cheese samples were collected from the suburbs of Ilepa at Ikare Akoko, Federal institutions and residential Estates at Akure as well as parks at Owo from January to late April 2019. The samples were stored in a cooler averagely filled with ice packs (4 °C) for three (3) hours because of the proximity between the area of collection and transfer to the laboratory. However, fresh milk of a healthy cow was the positive control experiment, while water source which was used for the cooking of cheese product was the negative control experiment.
Isolation and enumeration of bacteria
Colony counting was carried out visually by counting the number of visible colonies that appeared on the Salmonella-shigella agar (SSA) plates using rapid plating method; plates that had distinct colonies were used. Calculation of colony forming unit (CFU) per millilitre (ml) for the bacteria were recorded in standard form (Bayode et al. 2021). The number of colonies on each plate was recorded utilizing a colony counter (J-2 PEC MEDICAL, New Jersey, USA) (McHugh et al. 2017). Identification of the Salmonella enterica serovar Typhimurium was divulged via to the white–Kauffmann–Le minor protocol (Issenhuth-Jeanjean et al. 2014). Bacterial organisms were distinctively identified culturally and biochemically following the method of Onifade and Oladapo (2020). Nutrient agar and salmonella-shigella agar media were sterilized according to manufacturers’ specifications. One (1) gram of each cheese sample was aspirated into 9 ml of sterile 0.1% peptone water, shaken vigorously for a minute and serially diluted up to 10–4. One millilitre (1 m) of diluent 10–2 and 10−4were aseptically transferred into two (2) sterile Petri dishes, respectively. Fifteen millimetres (15 ml) of each growth medium (NA and SSA) were aseptically poured into Petri plates containing the diluents and were homogenized gently and allowed to gel. The labelled inoculated plates were incubated at 37 °C aerobically at 24 h. Pure culture of Salmonella enterica were stored in sterile double strength nutrient agar slant bottles and kept at 4 °C for further studies.
Extraction of bacterial deoxyribonucleic acid (DNA)
DNA Extraction method used and DNA concentration were evaluated through the steps described by Jena bioscience Kit™ (Jena GmbH, Loebstedter, Jena, Germany). The steps were classified into three (3) which are lysis, precipitation and hybridization (Munch et al. 2019). The lysis of bacteria cells was carried out, as 1 ml of each pure isolate was transferred into a 1.5-ml microtube, centrifuged at 14,850 revolutions per minute (rpm) for 1 min using refrigerated universal centrifuge (BR Biochem Life Sciences Private Limited, New Delhi, India). The supernatant obtained was discarded, leaving the residue (pellet) in the microtube. The pellet contains peptidoglycan particles that encapsulate the chromosome (DNA or RNA). The pellets were re-suspended in cell resuspension solution. 1.5 µl of RNase A solution was added to lyse the ribonucleic acid (RNA) genome, mixed by inversion, incubated at 37 °C for 15–30 min and cooled on ice for 1 min (Vaidya et al. 2018).
Polymerase chain reaction (PCR) assay
Reconstitution of primers
Stock primers were stored in lyophilized form in order to maintain the efficacy of the primer and to avoid contamination. These were virulence primers of S. enterica serovar typhimurium which include hil A, stn, iro B, and spv B which were in forward and reverse (Table 1). However, to work with these primers, there is a need to reconstitute these primers to a working solution, i.e. it has to be diluted. The lyophilized stock primers were transported in microtubes and were diluted by adding some amount of PCR-grade water (nucleic water) into the microtubes containing the primer (Tassinari et al. 2020).
The tubes were spun down because pellet can often be dislodged during shipping and may be in the cap. A 100 μM master stock was prepared by adding specific amount of nucleic water to each of the lyophilized primer in the tube. The amount of nucleic water needed was measured according to manufacturer’ specification, i.e. multiplying number of ‘nmol’ of primer in the tube by 10. The original primer tubes were used for this 100-μM stock. Newly suspended master stock primers were allowed to sit at room temperature for 10 min before they were used for working stock dilutions. Afterwards, the master stock primer was evenly homogenized by vortex mixer and diluted further with sterile molecular grade water in the ratio 1:10 to obtain a working stock (Shome et al. 2020).
PCR analysis of stn, hil A, iro B and spv C gene embedded in Salmonella enterica serovar typhimurium
PCR analysis was run with the working stock primers uniquely produced for the detection of Salmonella enterica serovar typhimurium virulence genes. The PCR mix comprises of 1 µl of 10X buffer, 0.4 µl of 50 mM MgCl2, 0.5 µl of 25 m MdNTPs, 0.5 µl of 5 mM selected working primers, 0.05 µl of 5 µl Taq polymerase with 2 µl of template DNA and 5.05 µl of sterile distilled water to make up 10 µl reaction mix (Kebede et al. 2016). The PCR profile used was initial denaturation temperature of 94 °C which lasted for 3 min, followed by 30 cycles of 60 s, 56 °C for 60 s, 72 °C for 120 s and final extension temperature of 72 °C for 5 min and the 10 °C on hold for few hours (Kebede et al. 2016).
Purification of PCR products
The amplicon was further purified before the sequencing using 2 M sodium acetate wash techniques. To about 10 µl of the PCR product, 1 µl of 2 M NaAct pH 5.2 was added. 20 µl absolute ethanol was also added, stored at -20 °C for 1 h, centrifuged at 10,000 rpm for 10 min, washed with 70% ethanol and then air-dried. The mixture was re-suspended in 5-µl sterile distilled water and kept at 4 °C for sequencing (Tassinari et al. 2020).
Preparation of samples for Gene Sequencer (ABI 3130 × 1 machine)
The cocktail mix was a combination of 9 µl of Hi Di formamide with 1 µl of purified sequence, making a total of 10 µl. The purified PCR products were loaded in the ABI 3130xl machine (genetic analyser system) CA, USA, and the nucleotide sequences were obtained (Shome et al. 2020).
The PCR mix used comprises 0.5 µl of Big Dye Terminator™ mix, 1 µl of 5X sequencing buffer, 1 µl of forward working stock primers with 6.5 µl distilled water and 1 µl of the PCR product which make up a total of 10 µl. The PCR profile for sequencing was rapid profile, the initial rapid thermal ramp to 96 °C for 1 minute, followed by 25 cycles of rapid thermal ramp to 96 °C for 10 s rapid thermal ramp to 50 °C for 5 s and rapid thermal ramp to 60 °C for 4 min. This was followed by rapid thermal ramp to 4 °C and placed on hold for few hours (Costessi et al. 2018).
Purification of PCR sequencing products
The PCR sequence product was also purified before the sequencing running using 2 M sodium acetate wash technique (Costessi et al. 2018). To 10 µl of the PCR product, 1 µl of 2 M NaAct pH 5.2 was added, followed by adding 20 µl absolute ethanol. The mixture was thereafter kept at -20 °C for 1 h, spun at 10,000 rpm for 10 min, washed with 70% ethanol, after which it was air-dried. It was re-suspended in 5-µl sterile distilled water and kept at 4 °C for sequencing running (Costessi et al. 2018). These reaction products were then re-suspended and analysed for nucleotide sequence determination using capillary electrophoresis (Applied Biosystems (ABI) prism 310 Genetic Analyser) (Shome et al. 2020). The sequence obtained was translated using gene sequential software and were compared with online genomic database using BLAST programme (http://www.ncbi.nlm.nih.gov/BLAST/blast) by submitting the sequence obtained to Gen Bank of National Centre for Biotechnology Information (NCBI) website.
Statistical analysis of data
Data obtained were subjected to one-way analysis of variance (ANOVA) and treatment means were separated using Duncan’s new multiple range test (DNMRT) at p ≤ 0.05 level of significance with the aid of Statistical Package for Social Sciences (SPSS) version 23.
Frequency of bacterial occurrence in cheese samples from selected cities
Table 2 shows that Akure metropolis had the highest plate count of enteric pathogens with 18.50 ± 0.71 × 105 CFU/ml on nutrient agar as well as 5.79 ± 0.45 × 104 CFU/ml on Salmonella-shigella agar. The frequency of bacterial occurrence isolated from cheese samples obtained from three (3) senatorial districts of Ondo State (Akure, Ikare and Owo) is shown in Table 3. A total of two hundred and sixty (260) cheese samples were studied for the presence and the virulence patterns of Salmonella enterica serovar typhimurium as illustrated in Table 3. Fifty (50) cheese samples were vended in Ikare province with varying locations ranging from transit garages, and market square. At Akure, one hundred and thirty (130) vended cheese samples were obtained from bus stops, car parks, suburbs, as well as market square. The remaining 80 cheese samples were vended across towns in Owo province. The highest percentage frequency of bacterial occurrence in local soft cheese obtained was accounted from Ikare Akoko, (100%) while the lowest frequency of bacterial occurrence was detected in samples obtained from vendors in Owo town (51.3%).
Presumptive identification of bacterial isolates obtained from cheese samples
Twenty-nine (29) bacterial isolates were obtained from cheese samples and analysed for morphological and biochemical characteristics (Table 4). These isolates were presumptively identified as Salmonella enterica, Shigella spp, Escherichia coli, Listeria monocytogenes, Yersinia enterocolitica, and Enterococcus faecalis all of which are gram negative.
Molecular identity of virulence genes in Salmonella enterica serovar typhimurium
Six (6) Salmonella enterica isolates were screened for the possessions of four (4) virulence genes, namely iro B, stn, hil A and spv C. Figure 2 shows the gel electrophoretic image of iro gene fragments of Salmonella enterica isolates with 1.1 kilo base pairs. Isolate code, A6, IK2, IK3, W2 were found to possess iro B genes which were molecularly identified as glycosol transferase iro B with accession numbers, CQ108275.1, CQA90090.1, AFD59678.1, and AVU70088.1, respectively, as shown in Table 5.
The electrophoretic gel image of stn fragments shown in Fig. 3 reveals that Salmonella isolates, A8 and IK3, possess stn genes which are molecularly identified as domain containing protein and accession numbers, ECY4622154.1, BTY1142154.1, respectively as shown in Table 6. Isolates, A8, IK2, IK3 were molecularly confirmed to have hyper-invasion hil A (Fig. 4) with accession numbers, AOY22154.1, CT_33212.5, and ECN5074153.1 as shown in Table 7. spvC virulence gene was found to be negative in all six Salmonella enterica isolates as no visible bands were observed as shown in Fig. 5.
Nutrient agar (NA) was used to quantify the total viable bacterial count of the cheese samples collected, while salmonella-shigella agar (SSA) was used to identify the specific Salmonella enterica and other species of Salmonella from the collected cheese samples. The high total bacterial count, 18.50 ± 0.71 × 105 CFU/ml, recorded in this study (Table 2) corroborates with the work of Omemu et al. (2014) who observed the bacterial count of soft white cheese at 20.0 × 107 CFU/ml vended within Abeokuta metropolis in Ogun State, Nigeria. This high count might be attributed to the use of low milk quality. According to the outcome of their study, the microbial quality of raw milk was crucial for the production of any high quality dairy food. It could also be as a result of unsanitary conditions by the vendors during processing and handling of the cheese. Mishandling and disregard of hygienic measures on the part of the food vendors may enable pathogens to come into contact with foods and in some cases, survive and multiply in sufficient numbers to cause gastrointestinal illness in the consumers after ingestion (Birke and Zawide 2019).
The bacterial occurrence revolves around the water used in the process of production. This claim was supported by the research carried out by Saka et al. (2013) whose team worked on the morphological and chemical assessment of water from different rural settings in Ikare Akoko. The team quantified that the mean count of faecal coliforms to be 1.04 × 106 CFU/ml. The highest frequency of bacterial occurrence obtained in Table 3 was accounted for soft cheese samples vended at the rural settings of Ikare, Ondo State, Nigeria. Ikare town is known to be a recreational centre for tourist and in yesteryears has friendly people whose hospitality knows no bound for all tribes of Nigeria. The Fulani are the main vendors of soft cheese in Ondo State and 90% are young girls with piece of personal hygiene.
Hence, the highest frequency of occurrence is a rider to the behavioural pattern of the vendors and the water used at the final stage of production, i.e. cleaning of utensils, washing of hands and packaging baskets. Also, the source of water supply from polluted wells and streams whose locations are at sewer channels and tanks utilized for cheese processing could be considered as a rationale for contamination.
The virulence patterns of plasmid-mediated multidrug resistant Salmonella enterica serovar typhimurium was detected in a similar study (Onifade and Oladapo 2020) associated with soft cheese in South-West, Nigeria. Apart from Salmonella enterica identified in Table 4, the centre of the study, five (5) other enteric organisms were identified in cheese samples. These are E. coli, Shigella dysenteriae, Enterococcus faecalis and Listeria monocytogenes. The biochemistry of S. enterica serovar typhimurium observed in this study is in line with the outcome of Sangoyomi et al. (2010) who performed biochemical characterization on bacteria isolated from cheese, a claim also supported by Al-Gamal et al. (2019).
The presence of Salmonella enterica in soft cheese samples recorded in this study (Table 4) is in line with the observation made by Frasson et al. (2016) who accounted for the presence of Salmonella enterica serovars in dairy foods. However, the findings from this study have shown that Salmonella enterica isolated from cheese is relatively small. This is due to the in-processes involved in the cheese-making which affects the growth of the bacterium. Nonetheless, they were susceptible pathogens associated with cheese. In fact, the earlier observations made by Frasson et al. (2016) showed that the prevalence of food-borne Salmonella enterica was reported to be 81%, indicating the ease of contamination involved in cheese production if not carefully monitored.
Findings from the study also revealed the virulence genes of Salmonella enterica serovar typhimurium through the PCR amplification of iro B, stn, and hil A. The primer pairs targeting the enterotoxin, stn and the histidine transport operon met all of our criteria for application to cheese samples which is in agreement with the evaluation of primers as demonstrated by Ziemer and Steadham (2003), Heymans et al. (2018). The criteria for accepting a primer set was based on PCR amplification of all Salmonella strains tested (Heymans et al. 2018).
The molecular determination of ferric uptake regulator in Salmonella enterica serovar typhimurium has revealed the presence of iro B gene in cheese samples in Fig. 2. This is in agreement with the study carried out by Zishiri et al. (2016) who worked on the prevalence of virulence genes of Salmonella in South Africa. They revealed that out of the overall 146 Salmonella-positive samples that were screened for the iro B gene, most of them were confirmed to be Salmonella enterica with high prevalence rates of iro B ranging between 69–85%, a gene responsible for the shortage of blood in host. In fact, findings from Bearson et al. (2008) demonstrated the influence of iro B gene in S. enterica serovar typhimurium. They claimed that there is notable competition of iron acquisition between the host and S. enterica serovar typhimurium. This confirmed the travail of Salmonella enterica serovar typhimurium in host system, once consumed.
The hil A (hyper-invasion locus A) confirmed positive in this study (Fig. 3) is the grand orator of invasion in Salmonella enterica serovar typhimurium which were distributed in all sources of cheese samples. This corroborates with the research conducted by Yanestria et al. (2019). They opined that 12.5% of hil A gene exhibited by Salmonella were distributed in Milkfish sources. This clearly questions the potential location and the routes of Salmonella transmission in cheese making. The First step in the intracellular pathogenicity cycle of Salmonella is the invasion of intestinal epithelial cells, and this step is controlled by the hil A gene, in a review confirmed by Sharma (2016). Therefore, intake of cheese samples contaminated with Salmonella enterica serovar typhimurium may result to inflammation, liver cirrhosis and some other chronic gastro intestinal infections.
However, stn gene operon with molecular size of 260 bp was found in 33.3% of the isolates analysed for virulence (Fig. 4). The two (2) pure isolates of S. enterica serovar typhimurium had enterotoxic and cytotoxic effects on host systems. The photographic plates of Salmonella enterica serovar typhimurium showed the differences in the bands, as a band appears blurred and the other, distinct, which is a clear indication that there is difference of phenotypes of gene and regulation in the membrane composition and integrity. Moreover, Masayuki et al. (2012); ElSheikh et al. (2019) analysed the importance of Stn gene and how it regulates membrane composition and Salmonella’ integrity. These were done through in vitro and in vivo using wild-type and Δstn mutant Salmonella. From their research, the team opined that differences of virulence phenotypes between the wild type and Δstn led to the production of R-S-EXE triad motif, which is essential for the ADP-ribosyltransferase of CT and LT, a virulence factor of Salmonella as reported by Masayuki et al. (2012) and ElSheikh et al. (2019).
From this, it is possible that stn might play a role in unique function, a known operon capable of causing severe acute gastroenteritis. Spv operon, on the other hand, has not been detected (Fig. 5), as the Salmonella plasmid gene is noted for growth regulation which is being activated during the interaction with the host cell framework. However, the deprivation of nutrient by the phagosome present in host phagocytic cells can be the environmental signal activating Salmonella plasmid virulence gene expression. In fact, a study confirmed that analyses of reporter gene expression, mRNA synthesis, deletion mutagenesis, and gene complementation have demonstrated a novel ‘regulatory system’. This is a consequence of the selective transcription of spv during the stationary phase of the bacterial growth (Guiney and Fierer 2011; Lv et al. 2019).
Therefore, locally produced soft cheese used for this study was found to be unsafe for human consumption due to the low microbiological quality made evident in the microbiological profile of the vended soft cheese. The presence of molecularly elucidated virulence genes comprising Iro B, Stn and Hil A makes the soft cheese potentially viable for pathogenicity. This study recommends that food-borne salmonellosis should be constantly monitored with appropriate cleaning of preparatory paraphernalia in cheese production encouraged. Constant sensitization of nomads to improve their awareness on milk-borne zoonosis and its associated risk factors is needed.
Availability of data and materials
There is availability of data and materials in supplementary data files.
Deoxyribose nucleic acid
Ribose nucleic acid
- MgCl2 :
Modified dynamic needle tip positioning
Basic local alignment search tool
Akinyemi KO, Ajoseh SO, Iwalokun BA, Oyefolu AOB, Fakorede CO, Abegunrin RO, Adunmo O (2018) Antimicrobial Resistance and Plasmid Profiles of Salmonella enterica Serovars from Different Sources in Lagos. Nigeria Health 10(06):758. https://doi.org/10.4236/health.2018.106058
Al-Ashmawy MA, Sallam KI, Abd-Elghany SM, Elhadidy M, Tamura T (2016) Prevalence, molecular characterization, and antimicrobial susceptibility of methicillin-resistant Staphylococcus aureus Isolated from Milk and Dairy Products. Foodborne Pat and Dis 13(3):156–162. https://doi.org/10.1089/fpd.2015.2038
Aldrete-Tapia JA, Escobar-Ramírez CM, Tamplin ML, Hernández-Iturriaga M (2018) Characterization of bacterial communities in Mexican Artisanal Raw Milk “Bola de Ocosingo” cheese by high-throughput sequencing. Front Microbiol 9:2598
Al-Gamal MS, Ibrahim GA, Sharaf OM, Radwan AA, Dabiza NM, Youssef AM, El-Ssayad MF (2019) The protective potential of selected lactic acid bacteria against the most common contaminants in various types of cheese in Egypt. Heliyon 5(3):e01362. https://doi.org/10.1016/j.heliyon.2019.e01362
Amiri S, Moradli GH (2016) Molecular identification of virulence genes (agfA and mgtC) in Salmonella typhimurium strains isolated from children with gastroenteritis using multiplex PCR method. J Babol Univ Med Sci 18(10):40–45
Bayode MT, Olalemi AO, Oladejo BO (2021) Multiple antibiotic resistant index and detection of qnrS and qnrB genes in bacterial consortium of urine samples from clinical settings. Euro J Biol Res 11(1):45–56. https://doi.org/10.5281/zenodo.4304311
Bearson BL, Bearson SM, Uthe JJ, Dowd SE, Houghton JO, Lee I, Lay DC Jr (2008) Iron-regulated genes of Salmonella enterica serovar Typhimurium in response to norepinephrine and the requirement of fepDGC for norepinephrine-enhanced growth. Microb Infect 10(7):807–816
Birke W, Zawide F (2019) Transforming research results in food safety to community actions: A call for action to advance food safety in Ethiopia. Environ Ecol Res 7:153–170. https://doi.org/10.13189/eer.2019.070305
Costessi A, van den Bogert B, May A, Loren V, van Themaat E, Roubos JA, Kolkman MA, Pirovano W (2018) Novel sequencing technologies to support industrial biotechnology. FEMS Microbiol Lett 365(16):103. https://doi.org/10.1093/femsle/fny103
Dos Santos AM, Ferrari RG, Panzenhagen P, Rodrigues GL, Conte-Junior CA (2021) Virulence genes identification and characterization revealed the presence of the Yersinia High Pathogenicity Island (HPI) in Salmonella from Brazil. Gene 787:145646. https://doi.org/10.1016/j.gene.2021.145646
ElSheikh M, Abdeen E, Ammar A (2019) Molecular Detection of some virulence genes of salmonella serotypes isolated from poultry in Egypt. J Curr Vet Res 1(1):86–93. https://doi.org/10.21608/jcvr.2019.36570
Eng SK, Pusparajah P, Ab-Mutalib NS, Ser HL, Chan KG, Lee LH (2015) Salmonella: a review on pathogenesis, epidemiology and antibiotic resistance. Front Life Sci 8(3):284–293. https://doi.org/10.1080/21553769.2015.1051243
Frasson I, Bettanello S, De Canale E, Richter SN, Palù G (2016) Serotype epidemiology and multidrug resistance patterns of Salmonella enterica infecting humans in Italy. Gut Pat 8(1):26. https://doi.org/10.1186/s13099-016-0110-8
Fusco V, Chieffi D, Fanelli F, Logrieco AF, Cho GS, Kabisch J, Böhnlein C, Franz CMAP (2020) Microbial quality and safety of milk and milk products in the 21st century. Compr Rev Food Sci Food Saf 2020(19):2013–2049. https://doi.org/10.1111/1541-4337.12568
Guiney DG, Fierer J (2011) The role of the spv genes in Salmonella pathogenesis. Front Microbiol 2:129. https://doi.org/10.3389/fmicb.2011.00129
Heymans R, Vila A, van Heerwaarden CA, Jansen CC, Castelijn GA, van der Voort M, Biesta-Peters EG (2018) Rapid detection and differentiation of Salmonella species, Salmonella Typhimurium and Salmonella Enteritidis by multiplex quantitative PCR. PLoS ONE 13(10):e0206316. https://doi.org/10.1371/journal.pone.0206316
Issenhuth-Jeanjean S, Roggentin P, Mikoleit M, Guibourdenche M, de Pinna E, Nair S (2014) Supplement 2008–2010 (no. 48) to the white–Kauffmann–Le minor scheme. Res Microbiol 165:526–530. https://doi.org/10.1016/j.resmic.2014.07.004
Jajere SM (2019) A review of Salmonella enterica with particular focus on the pathogenicity and virulence factors, host specificity and antimicrobial resistance including multidrug resistance. Vet World 12(4):504. https://doi.org/10.14202/vetworld.2019.504-521
Kebede A, Kemal J, Alemayehu H, Habte MS (2016) Isolation, identification, and antibiotic susceptibility testing of Salmonella from slaughtered bovines and ovines in Addis Ababa Abattoir Enterprise, Ethiopia: a cross-sectional study. Intl J Bacteriol 2016:1–8. https://doi.org/10.3389/fmicb.2011.00129
Larrisa B, Moonjely S, Behie SW, Bidochka MJ (2016) Fungi with multifunctional lifestyles: endophytic insect pathogenic fungi. Plant Mol Biol 90(6):657–664
Leekitcharoenphon P, Hendriksen RS, Le Hello S, Weill FX, Baggesen DL, Jun SR, Aarestrup FM (2016) Global genomic epidemiology of Salmonella enterica serovar Typhimurium DT104. Appl Environ Microbiol 82(8):2516–2526. https://doi.org/10.1128/aem.03821-15
Luvsansharav UO, Vieira A, Bennett S, Huang J, Healy JM, Hoekstra RM, Cole D (2020) Salmonella serotypes: A novel measure of association with foodborne transmission. Foodborne Pat Dis 17(2):151–155. https://doi.org/10.1089/fpd.2019.2641
Lv M, Hu M, Li P, Jiang Z, Zhang LH, Zhou J (2019) A two-component regulatory system VfmIH modulates multiple virulence traits in Dickeya zeae. Mol Microbiol 111(6):1493–1509. https://doi.org/10.1111/mmi.14233
Majowicz SE, Musto J, Scallan E, Angulo FJ, Kirk M, O'Brien SJ and International Collaboration on Enteric Disease “Burden of Illness” Studies (2010) The global burden of non-typhoidal Salmonella gastroenteritis. Clin Infect Dis 50(6):882–889. https://doi.org/10.1086/650733
Masayuki N, Yamasaki E, Ichinose A, Shimohata T, Takahashi A, Akada JK, Kurazono H (2012) Salmonella enterotoxin (Stn) regulates membrane composition and integrity. Dis Models Mech 5(4):515–521
McHugh AJ, Feehily C, Hill C, Cotter PD (2017) Detection and enumeration of spore-forming bacteria in powdered dairy products. Front Microbiol 8:109. https://doi.org/10.3389/fmicb.2017.00109
Munch MM, Chambers LC, Manhart LE, Domogala D, Lopez A, Fredricks DN (2019) Srinivasan S (2019) Optimizing bacterial DNA extraction in urine. PLoS ONE 14(9):e0222962
Omar D, Al-Ashmawy M, Ramadan H, El-Sherbiny M (2018) Occurrence and PCR identification of Salmonella spp. from milk and dairy products in Mansoura, Egypt. Int Food Res J 25(1):446–452
Omemu A, Taiwo G, Obuotor T (2014) Microbiological assessment and prevalence of food borne pathogens in street vended Wara-Nigerian white cheese. Am J Food Nut 2(4):59–62
Onifade AK, Oladapo DM (2020) Prevalence and antibiotic sensitivity patterns of Salmonella enterica serovar typhimurium isolated from Local Cheese in Ondo State, Nigeria. South Asian J Res Microbiol 5(3):1–10. https://doi.org/10.9734/SAJRM/2019/v5i330133
Pui CF, Wong WC, Chai LC, Robin T, Ponniah J (2011) Salmonella: a foodborne pathogen. Int Food Res J 18:465–473. https://doi.org/10.1016/j.foodcont.2010.05.021
Rivero-Calle I, Gómez-Rial J, Bont L, Gessner BD, Kohn M, Dagan R, Martinón-Torres F (2021) TIPICO X: report of the 10th interactive infectious disease workshop on infectious diseases and vaccines. https://doi.org/10.1080/21645515.2020.1788301
Ruszel K, Dubel R, Chodun W, Nieradko-Iwanicka B (2021) Salmonella–still a threat? Epidemiological analysis of infecion. J Educ Health Sport 11(8):38–43. https://doi.org/10.12775/jehs.2021.11.08.004
Saka BA, Ejelonu BC, Lasisi AA, Adeogun AI (2013) Microbiological and chemical assessment of spring water from a rural setting in Ondo State Southwest, Nigeria. Afr J Environ Sci Technol 7(6):555–559. https://doi.org/10.5897/ajest13.1517
Sangoyomi TE, Owoseni AA, Okerokun O (2010) Prevalence of enteropathogenic and lactic acid bacteria species in wara: a local cheese from Nigeria. Afr J Microbiol Res 4(15):1624–1630
Sharma I (2016) Detection of invA gene in isolated Salmonella from marketed Poultry Meat by PCR Assay. J Food Proc Technol 7(1):3. https://doi.org/10.4172/2157-7110.1000564
Shome A, Kumawat M, Pesingi PK, Bhure SK, Mahawar M (2020) Isolation and identification of periplasmic proteins in Salmonella Typhimurium. Intl J Curr Microbiol Appl Sci 9(6):1923–1936. https://doi.org/10.20546/ijcmas.2020.906.238
Tassinari E, Bawn M, Thilliez G, Charity O, Acton L, Kirkwood M (2020) Kingsley RA (2020) Whole-genome epidemiology links phage-mediated acquisition of a virulence gene to the clonal expansion of a pandemic Salmonella enterica serovar Typhimurium clone. Microb Genom. https://doi.org/10.1099/mgen.0.000456
Vaidya JD, van den Bogert B, Edwards JE, Boekhorst J, Van Gastelen S, Saccenti E, Smidt H (2018) The effect of DNA extraction methods on observed microbial communities from fibrous and liquid rumen fractions of dairy cows. Front Microbiol 9:92. https://doi.org/10.3389/fmicb.2018.00092
Yanestria SM, Rahmaniar RP, Wibisono FJ, Effendi MH (2019) Detection of invA gene of Salmonella from milkfish (Chanos chanos) at Sidoarjo wet fish market, Indonesia, using polymerase chain reaction technique. Vet World 12(1):170. https://doi.org/10.14202/vetworld.2019.170-175
Ziemer CJ, Steadham SR (2003) Evaluation of the specificity of Salmonella PCR primers using various intestinal bacterial species. Lett Appl Microbiol 37(6):463–469. https://doi.org/10.1046/j.1472-765x.2003.01430.x
Zishiri OT, Mkhize N, Mukaratirwa S (2016) Prevalence of virulence and antimicrobial resistance genes in Salmonella spp. isolated from commercial chickens and human clinical isolates from South Africa and Brazil. Onderstepoort J Vet Res 83(1):1–11. https://doi.org/10.4102/ojvr.v83i1.1067
Authors are grateful to Erevna laboratories, Adebajo Street, New Bodija, Ibadan, Oyo State, Nigeria, for their technical input in the virulence analysis performed in this study. Authors also acknowledge the efforts of Inqaba Biotech West Africa Ltd, located at the Bioscience Unit, International Institute for Tropical Agriculture (IITA), Ibadan, Nigeria, for the virulence primer stocks utilized in this study.
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Oladapo, O.D., Onifade, A.K. & Bayode, M.T. Direct detection of iro B, stn and hil A virulence genes in Salmonella enterica serovar typhimurium from non-ripened cheese. Bull Natl Res Cent 46, 175 (2022). https://doi.org/10.1186/s42269-022-00857-2
- Antibiotic resistance
- Soft cheese
- Salmonella enterica serovar typhimurium
- Virulence gene