Rappaport Vassiliadis Research Articles

Characterization of Diarrheagenic Escherichia coli and Salmonella enterica from Produce in the Chobe District of Botswana

Diarrheal disease is a leading cause of death in children in low- and moderate-income countries. Fresh produce, including fruits and vegetables, may harbor diarrheal disease-causing bacteria including strains of Salmonella enterica and Escherichia coli. This study aimed to determine the prevalence and antibiotic resistance profiles of S. enterica and E. coli isolated from produce samples (n = 207) obtained from retail markets in northern Botswana in Chobe District of Botswana in 2022. Samples were enriched in the appropriate selective media: Brilliant Green Bile Broth for E. coli and Rappaport Vassiliadis Broth for S. enterica. E. coli were confirmed by PCR detecting the phoA gene, and classified as potentially pathogenic through screening for the eae, stx, and stx2 and estIb genes. S. enterica isolates were confirmed using invA primers. Isolates were evaluated for resistance to ampicillin, amoxicillin-clavulanic acid, chloramphenicol, cefotaxime, doxycycline, streptomycin, sulfamethoxazole, and tetracycline antibiotic using the Kirby-Bauer Disk Diffusion method. E. coli was isolated from 15.5% of produce samples (n = 207). The gene eae was detected from 1.5% of samples, while stx1, stx2, and estIb were not detected. Resistance to one or more antibiotics was common (72%) with the majority of the resistant E. coli (n = 32) isolated from fruits (22%) and greens (18%) compared to other types of vegetables. Multidrug resistance (MDR, resistant to 3 or more antibiotics) was identified in 18% of samples. S. enterica was isolated from 3.4% of produce samples (7, n = 207). Resistance was uncommon among the S. enterica isolates (1/7). Overall prevalence of diarrheagenic S. enterica and E. coli was low; however, their presence and that of MDR E. coli in foods commonly consumed raw increases the risk to vulnerable populations. Strategies to reduce contamination of fresh produce and public education on washing and cooking some types of produce may be useful to reduce disease.

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

Evaluation of Salmonella Enteritidis Isolated from Layer Hens and Murine Fecal Pellets in Poultry Farms of Libya

The rodents play a significant role in the transmission of Salmonella between farms and regions. The present study aimed to compare the virulence of Salmonella enteritidis isolated from fecal samples of laying hens and murine within the same poultry house but different regions in Libya using Vivo-quantitative measurement of invasiveness (chicken intestinal loop model). A total of 540 cloacal swabs from laying hens (Hy-line brown chickens) aged 36 weeks and 200 batches of murine fecal pellets were collected from the same poultry house at Gaser Bin Gisher and Furnag regions in Libya. The samples were passed on pre-enrichment broth (Buffered Peptone Water) and enrichment broths (Rappaport Vassiliadis, Selenite broth, and tetrathionate), then the samples were cultured onto Xylose Lysine Deoxycholate agar, brilliant green agar, Salmonella Shigella agar, and Hektoen enteric agar. Single colonies were selected and stained by gram stain and tested biochemically using analytical profile index (API) 20 tests. Salmonella enteritidis was isolated from all the collected samples. The invasion of Salmonella enteritidis isolated from laying hens and murine feces was significantly higher in the anterior inoculation position compared to the posterior position of jejunum in both regions. The account of Salmonella enteritidis isolated from laying feces of hens and murine at Gaser Bengasher region was significantly higher than that isolated from the AlFurnge region. In the present study, the rodents act only as mechanical transmitters without affecting Salmonella invasiveness capacity. Furthermore, the invasion of Salmonella enteritidis depends on the inoculation position in the jejunum. Moreover, the invasiveness variation of Salmonella enteritidis isolated from the Gaser Bengasher and AlFurnge regions could be attributed to the presence of different Salmonella strains in the studied area. Salmonella enteritidis isolated from poultry and murine in the current study was sensitive to gentamicin, ciprofloxacin, and enrofloxacin and resistant to doxycycline, chloramphenicol, sulfafurazol, and ampicillin.

Distinctive Culture Expressions of Enterobacteria Interfering with Isolation of Salmonella spp. during the Application of the Recommended ISO 6579-1:2017

The objective of the present report is the dissemination of information acquired during the application of ISO 6579-1:2017 for the isolation of Salmonella spp. from swine samples. ISO 6579-1:2017 is the protocol officially recommended by the EU for the isolation of Salmonella spp., aiming for the harmonization of effective control of Salmonella infection in food-producing animals. Successful control of animal salmonellosis is highly dependent on the sensitivity and reliability of the biochemical methods used to detect the presence of the pathogen in various stages of food production. Application of ISO 6579-1:2017 resulted in the isolation of twelve Salmonella spp. and eighteen other members of the family of Enterobacteriaceae biochemically and other bacteria antigenically resembling salmonellae. An evaluation of the culture media was conducted, including xylose-lysine-desoxycholate agar (XLD), Salmonella–Shigella Agar (SS), Brilliant Green Agar (BGA), Salmonella Chromogenic Agar (SCA), Triple Sugar Iron (TSI), and modified semi-solid Rappaport–Vassiliadis (MSRV) agar. The evaluation showed that these culture-selective media differed significantly in their performance for the isolation of Salmonella from swine samples. It was concluded that the presence of atypical Salmonella strains negatively affects the prevalence of Salmonella, thus affecting the identification of carrier pigs and eventually affecting the efficiency of control programs. Thus, doubtful results require additional biochemical testing to confirm the accuracy of such universally recommended isolation methods.

Growth assessment of Salmonella enterica multi-serovar populations in poultry rinsates with commonly used enrichment and plating media

Isolation of Salmonella from enrichment cultures of food or environmental samples is a complicated process. Numerous factors including fitness in various selective enrichment media, relative starting concentrations in pre-enrichment, and competition among multi-serovar populations and associated natural microflora, come together to determine which serovars are identified from a given sample. A recently developed approach for assessing the relative abundance (RA) of multi-serovar Salmonella populations (CRISPR-SeroSeq or Deep Serotyping, DST) is providing new insight into how these factors impact the serovars observed, especially when different selective enrichment methods are used to identify Salmonella from a primary enrichment sample. To illustrate this, we examined Salmonella-positive poultry pre-enrichment samples through the selective enrichment process in Tetrathionate (TT) and Rappaport Vassiliadis (RVS) broths and assessed recovery of serovars with each medium. We observed the RA of serovars detected post selective enrichment varied depending on the medium used, initial concentration, and competitive fitness factors, all which could result in minority serovars in pre-enrichment becoming dominant serovars post selective enrichment. The data presented provide a greater understanding of culture biases and lays the groundwork for investigations into robust enrichment and plating media combinations for detecting Salmonella serovars of greater concern for human health.

Simultaneous Salmonella and bacteriophage isolation on Modified Semisolid Rappaport Vassiliadis media

Salmonella represents a food safety concern worldwide. Despite the application of National Control Programs (NCP) against Salmonella, regulated by the European Union, every year the European Food Safety Authority reports new cases. On the look for new alternatives to antibiotics, bacteriophages, or phages, rise as a promising alternative to treat multidrug resistance infections. Although they are known to be ubiquitous in the environment, their high specificity to host cells hinders their isolation and usage for phage therapy. The ISO 6579-1:2017 is performed as a reference method in the NCP and uses an unspecific media to enrich the sample the same way most phage isolation protocols do. Later, the protocol uses a more selective media to isolate the Salmonella, Modified Semisolid Rappaport Vassiliadis (MSRV). This paper aims to find out whether, due to the similarity between phage isolation protocols and the ISO 6579-1:2017, this last one could be used as a protocol to also isolate phages against the same bacterium that is being simultaneously isolated. To do so, 2 experiments were performed to assess phage isolation from MSRV media in in-vivo conditions. The results from experiments 1 and 2 proved that the MSRV media was usable for simultaneous phage and pathogen isolation through a single procedure. Additionally, there is a correlation between the antigenic formulae from the bacteria and the phage's host range, seeming to be effective against bacteria with similar antigenic formulae.

Detection of Atypical Salmonella Infantis Phenotypes in Broiler Environmental Samples.

In numerous countries, strict and targeted measures concerning Salmonella monitoring and control are implemented and high quality of surveillance is ensured by obligatory investigation of samples from the primary production level of animals according to EN/ISO standards. Here, 2 phenotypic characteristics of Salmonella exhibited on compulsory media are crucial, namely, motility demonstrated on modified semisolid Rappaport Vassiliadis agar (MSRV), and production of hydrogen sulfide (H2S) on xylose lysine deoxycholate agar (XLD). In the present study, we describe the detection of Salmonella Infantis variants found in broiler environmental samples with major alterations in their growth characteristics on MSRV, XLD, and brilliant green-phenol red-agar (BPLS). The variants proved to be non-motile on MSRV and displayed non-confirming colony appearances on the previously mentioned selective agars. The growth spectrum comprised pinhead sized yellow colonies with small black centers, but also pinpoint sized colorless colonies, both colony types of regular shape. Our work contributes to highlight the finding of S. Infantis variants which possess more than one phenotypic deviation from the "typical" growth characteristics and by this limit the detection power of the actual obligatory used media. IMPORTANCE Salmonellosis caused by non-typhoidal Salmonella serovars is the second most frequently reported zoonotic disease in humans in the EU. The transmission of these agents is mainly via contaminated food of animal origin. In this context, poultry products are the main source of infection. Therefore, continuous and standardized surveillance of the prevalence of such Salmonella serovars at the primary production level is essential. Our findings show the phenotypic heterogeneity of the serovar Infantis and provide growth characteristics of atypical variants. Such variants pass unnoticed official screening methods, resulting in incorrect identification and being underrepresented in epidemiological surveillance programs.

Isolation and antimicrobial sensitivity test of Salmonella isolates from selected intensive dairy farms in Bishoftu town, Central Ethiopia

Salmonella is one of persisting foodborne pathogenic infection particularly food of animal origin including dairy cattle products. Accoringly a cross-sectional study was conducted from July to September 2021 to determine the prevalence and antibiotics resistant of Salmonella in Bishoftu town. To conduct the study 61 samples were collected randomly from the individual lactating cows. The Salmonella was recovered from the samples; conventional culture characterization of Salmonella was conducted by pre-enrichment on the buffered peptone water broth and enrichment in Rappaport- Vassiliadis (RV) broth, subsequently isolation was done on selective XLD and biochemical confirmation of Salmonella isolates. Finally, antibiotic sensitivity tests against the identified isolate were done by using eight drugs. Salmonella was isolated from 13/61 (21.3%, 95% CI: 11.3-31.3) of the total samples. Out of the 13 Salmonella isolates, 11/49 (22.4% 95% CI: 11.4-33.4) and 2/121 (16.7% 95% CI: 4.77-38.17) were isolated from milk cows and milk container respectively with no significance (P-value = 0.362). The isolates were susceptible to Ciprofloxacin, Penicillin and Vancomycin) were susceptible to the isolates, while, Streptomycin, Ampicillin, Tetracycline Rifampicillin and Cloxacillin) were resistant to the isolates. The effectiveness of such drugs like ciprofloxacin may be because they are not widely used in countries like Ethiopia and other African countries for animal’s treatment. 100% rate of resistant was recorded to Ampicillin, Chloramphenicol, Cloxacillin, Streptomycin and Amoxicillin. The study indicated the prevalence of Salmonella with high proportion of multiple antimicrobial resistance to the commercial used in the study area. Therefore, the government, farmers and other stakeholders should give concern for dairy farm hygiene and antibiotics uses in dairy farms.

Escherichia coli O157:H7 and Salmonella species Isolated from Ducks and Indigenous chickens in live-bird Markets in Ibadan, Oyo State, Nigeria.

This study investigated the occurrence of Escherichia coli O157:H7 and Salmonella spp. and their antimicrobial susceptibility from ducks and indigenous chickens in major live-bird markets at Ibadan, Oyo State, Nigeria. Thirty-one cloacae samples were each collected from both ducks and indigenous chickens in three different sample locations for a total of 186 cloacae swab samples. Isolation of Escherichia coli (E. coli) was done using MacConkey agar and Sorbitol MacConkey agar selective for E. coli O157:H7, while serological latex agglutination test kit was used to confirm isolates. Rappaport Vassiliadis and Xylose Lysine Deoxycholate agar were used for Salmonella spp. Antibiotic susceptibility was determined using the disc diffusion method and interpreted using the CLSI 2020 standards. Data were analyzed using descriptive statistics and Fisher's exact test (p ≤ 0.05). Escherichia coli O157:H7 was confirmed in 31 samples (16.7%). E. coli isolates showed high resistance (90.3-93.5%) to cefuroxime, cefixime, ceftazidime, and amoxicillin, while they were highly susceptible to ofloxacin (96.8%) and gentamycin (80.7%). Salmonella was confirmed in 24 samples (12.9%). Salmonella showed 100% resistance to cefuroxime, cefixime ceftazidime, and amoxicillin, but was highly susceptible to gentamycin (91.7%) and nitrofurantoin (66.7%). No statistically significant association (p<0.05) was observed between the occurrence of E. coli O157 and Salmonella within the three live-bird markets. This study reveals that E. coli and Salmonella spp. occur in ducks and indigenous chickens from major live bird markets in Ibadan, Oyo state with antimicrobial susceptibility. Findings from this study underscores the need for further studies on these pathogenic organisms from ducks in Nigeria because there is paucity of data on this species of poultry that may serve as reservoir for these zoonotic organisms.

Animal feed contains diverse populations of Salmonella.

In food animals, Salmonella can exist as multiserovar populations, and the goal of this study was to determine whether Salmonella-positive animal feed samples also consist of multiserovar populations. In all, 50 Salmonella-positive samples, collected from 10 countries, were cultured using three different media for Salmonella isolation: universal pre-enrichment broth, Rappaport-Vassiliadis (RV) broth and tetrathionate (TT) broth. The samples included 25 samples from feed ingredients, 13 from complete feed and 12 feed mill dust samples. Samples from pelleted overnight cultures were analysed by CRISPR-SeroSeq to examine serovar populations in individual samples. Serovars Anatum and Mbandaka were the most commonly identified and were found in feed, feed ingredients and feed environments. Serovars commonly associated with human illness were also identified, and included serovars Enteritidis, Typhimurium and Infantis. Overall, we detected 12 different serogroups (37 different serovars), with eight serovars belonging to the O:7 serogroup (C1 ). Over half (56%) of the samples contained two or more serovars, with 11 serovars found in one sample. Feed ingredients exhibited higher serovar diversity, with an average of three serovars. Across paired samples of pre-enriched and enriched populations, the Bray-Curtis dissimilarity metric showed that 83% of serovar populations were a strong match. The data presented show that serovars belonging to the O:7 serogroup are commonly found in feed, and that feed can contain multiple serovars. The serovar populations across different Salmonella media were largely concordant. The presence of Salmonella in animal feed is considered a transmission route into meat and poultry products and this study demonstrates that animal feed can contain multiple Salmonella serovars.

Mixed Salmonella cultures reveal competitive advantages between strains during pre‐enrichment and selective enrichment

AbstractCulture‐based Salmonella isolation consists of nonselective pre‐enrichment, followed by selective enrichment in Rappaport–Vassiliadis (RV) or tetrathionate (TT) broths, and subsequent plating on selective indicator agar. This study aimed to assess the recovery of two strains belonging to serovars Montevideo (strain ATCC‐8387) and Typhimurium (strain ATCC‐14028) when grown together in different media. The two strains were co‐inoculated 1:1, 10:1, 100:1, and the reciprocal, and CRISPR‐SeroSeq was used to assess the relative frequency of both strains after each culture step. Individually, there was no growth difference between both strains in universal pre‐enrichment (UP) or RV broths, though both strains had a higher growth in the former. In TT, both strains had reduced growth, especially ser. Montevideo‐8387. When the strains were combined, the growth of ser. Typhimurium‐14028 strain was higher in UP and TT broths, while the ser. Montevideo strain was higher in RV broth. The ser. Typhimurium strain also grew better on xylose lysine tergitol‐4 (XLT‐4) agar. Using media that are commonly used for Salmonella isolation, this work reveals strain‐to‐strain differences in growth and that in three conditions (UP, RV, XLT‐4), these differences were only manifested when the strains were in competition with each other. These findings illustrate the importance of dual selective enrichments to be able to capture all Salmonella present in a sample.

Comparison of selective enrichment and plating media for Salmonella isolation from broiler carcasses

AbstractSalmonella detection and isolation rely on different selective enrichment media, which can influence which serovars are detected. The objective of this study was to compare Salmonella recovery from broiler carcass rinses using three different selective enrichment protocols and three differential plating agars. Eight prechill broiler carcasses were collected at a commercial slaughter facility. Each carcass was subjected to whole carcass rinse procedure in buffered peptone water (BPW). An aliquot of the rinse and whole carcasses in the remaining rinse were incubated as a pre‐enrichment before subculturing in selective enrichment broths (Rappaport Vassiliadis [RV], Tetrathionate Hajna [TT], and TT to RV in series [TT‐to‐RV]). Enriched samples were streaked on the three differential agars (Hektoen Enteric [HE], Brilliant Green Sulfa [BGS], and Xylose‐Lysine‐Tergitol‐4 [XLT‐4]). Salmonella was isolated from all eight carcasses. Considering all sample preparations as independent subsamples, Salmonella was detected in 88% (128/144) of subsamples with a 100% recovery from the TT‐to‐RV enrichment, and 92 and 71% from RV and TT broths, individually. A high concordance in recovery on BGS versus XLT‐4 agar plates was observed compared to HE versus BGS and HE versus XLT‐4 plates. These data suggest that choice of pre‐enrichment method, selective enrichment medium, and differential agar can influence the recovery of Salmonella from poultry samples.

CONTAMINATION OF CHICKEN EGGSHELLS AND EGG CONTENTS WITH SALMONELLA SPECIES FROM SELECTED FARMS IN KOSGAMA, COLOMBO DISTRICT

Salmonellosis is a common, widely distributed foodborne disease. Consumption of raw or undercooked chicken eggs infected with Salmonella has been reported in association with salmonellosis cases; however, minimum attention has been paid to regulate the quality of eggs released for consumption. This study aimed to investigate the presence of Salmonella in eggs collected from selected farms from Kosgama area and to compare the egg quality of backyard and commercial farms. Randomly purchased eggs from selected chicken farms were analyzed for the presence of Salmonella. Egg content was mixed thoroughly, and 25.0mL was inoculated into 225.0mL of 1% Buffered Peptone Water (BPW) and incubated at 350C (24h). From the pre-enriched specimen, 0.1ml was added to 10.0ml of Rappaport Vassiliadis broth and incubated at 420C (24h). The same procedure was followed for shells. Isolated cultures were streaked on Brilliant Green Agar (BGA) and Xylose Lysine Deoxycholate Agar (XLD) and incubated at 350C for 24h. Colonies were investigated with Gram staining, biochemical tests and serotyping was carried out to identify the species. Of 78 eggs, 35 were from backyard and 43 from commercial farms. Six specimens (4 from shell and 2 from content) yielded Salmonella (7.69 %). Four of the positive specimens were from backyard farms (4/35, 8.91%) and remaining two (2/43, 3.62%) were from commercial farms. Isolates were identified as S. Typhimurium and S. Enteriditis. The prevalence of Salmonella was 7.69 % (n=6). The proportion of Salmonella showed no significant difference (p=0.782) between backyard and commercial farms.

Prevalence and Antibiotic Susceptibility Pattern of Salmonella Species

Aim: This research was carried out to determine the prevalence of Salmonella species from smoked fish sold in the three major markets in Tamale Metropolis and examined the isolates resistance patterns to various antibiotics. &#x0D; Study Design: The study was in two parts which include administering questionnaires and collecting samples. The second part was the laboratory analysis to detect Salmonella species from the collected samples.&#x0D; Place and Duration of Study: The study was carried out in the Spanish laboratory (microbiology section) of the Department of Biotechnology, Faculty of Biosciences, of the University for Development Studies.&#x0D; Methodology: A total of one hundred samples were examined. The samples were enriched on buffered peptone water and inoculated on Modified Semi-Solid Rappaport Vassiliadis. Xylose Lysine Deoxycholate was used to identify the bacteria, and Simons Citrate agar was used for the biochemical test. The antibiotic test was done by using the Kirby-Bauer disc diffusion test.&#x0D; Results: After the laboratory analysis, 67 (67%) out of the 100 samples were confirmed to be positive for Salmonella species of which Cut fish (11.9%), Redfish (11.9%), Mudfish (11.9%), and Chale fish (11.9%) recorded the highest contamination, and among the three markets, Central market had the highest fish contamination. The resistance patterns of the isolates to the various antibiotics used were; Ciprofloxacin (2.98%), Ceftriaxone (34.32%), Ampicillin (83.58%), Doxycycline (88.05%), Amoxicillin/Clavulanic acid (91.04%), and Oxytetracycline (92.53%).&#x0D; Conclusion: This study revealed that smoked fish sold in the Tamale Metropolis contained Salmonella positive that can cause food poisoning and other gastrointestinal problems.

Selective pre-enrichment method to lessen time needed to recover Salmonella from commercial poultry processing samples

Conventional Salmonella detection is time consuming, often employing a 24-h pre-enrichment step in buffered peptone water (BPW), followed by a 24-h selective enrichment in either Rappaport Vassiliadis (RV) or tetrathionate (TT) broths before streaking onto selective indicator agar. To reduce this time, we sought to optimize pre-enrichment for Salmonella recovery by evaluating the addition of selective chemicals to BPW. Duplicate samples each representative of 500 carcasses were collected by catching processing water drip under moving carcass shackle lines immediately after feather removal in each of nine commercial processing plants. Carcass drip samples were cultured under selective pre-enrichment conditions in parallel with BPW pre-enrichment followed by RV and TT selective enrichment. Addition of bile salts (1 g/L) and novobiocin (0.015 g/L) resulted in Salmonella recovery from 89% samples when plated directly after pre-enrichment compared to 67% recovery in non-selective BPW alone. Salmonella serovar identities were determined using CRISPR-SeroSeq. Overall, serovars matched between selective pre-enrichment and traditional enrichment methods. These data suggest that increasing the selectivity of Salmonella pre-enrichment step may lessen the need for a separate selective enrichment step thereby reducing time required for Salmonella isolation by 24 h.

Potential Zoonotic Faecal Bacteria from Sunda Porcupine (Hystrix javanica ) and Their Antimicrobial Resistance Profiles

Sunda porcupine (Hystrix javanica) is one of the Indonesian endemic species which is often sought after for their meat. Although it is becoming increasingly popular for extreme culinary, information regarding biological risks arising from this wildlife is very limited. This study aimed to assess potential zoonotic faecal bacteria carried by Sunda porcupine with culture-dependant approach and to investigate whether antimicrobial resistant isolates can be found in wildlife. A total 22 faecal samples were collected from captive Sunda porcupine and tested for the presence of pathogens in selective media for Salmonella and Listeria. After inoculating the samples in Rappaport-Vassiliadis (RV) Salmonella enrichment broth, two samples (9%) were regarded as positive for Salmonella in this presumptive test which indicated by growth black colonies on xylose lysine deoxycholate (XLD) agar. Meanwhile, the presence for Listeria was presumptively positive in all samples (100%), indicated by black colour appearance in Listeria isolation transwab. In total, 38 bacterial isolates were successfully purified, preserved and subjected for antimicrobial susceptibility testing (AST) by disk diffusion method. Resistance to ceftriaxone (3rd generation cephalosporins) was not detected while resistance to one or two antimicrobials was observed in seven isolates. Further, 16S rRNA bacterial identification was performed for selected isolates and based on sequence similarity on GenBank® databases and phylogenetic tree construction, those isolates were denoted as Pseudomonas xinjiangensis (XG4.4), Shigella sonnei (XD8.2 and G11.3), Proteus mirabilis (XH3.3, H4.2, and E1.2) and Klebsiella quasipneumoniae subsp. similipneumoniae (XF4.2). All identified isolates were sensitive to amikacin, amoxicillin-clavulanic acid, cefoxitin and ceftriaxone, except for one isolate Shigella sonnei (XD8.2) which was resistant to cefoxitin. Further research to confirm the pathogenicity of the isolates is still needed but based on these results, we support the hypothesies that Sunda porcupine is potential as a reservoir pathogenic bacteria and preventive measures are crucial to prevent transmission when processing this bushmeat.

Potential Zoonotic Faecal Bacteria and Antimicrobial Resistance from Sunda Porcupine (Hystrix javanica): A Preliminary Study

Sunda porcupine (Hystrix javanica) is one of the Indonesian endemic species which is often sought after for their meat. Although it is becoming increasingly popular for extreme culinary, information regarding biological risks arising from this wildlife is very limited. This study aimed to assess potential zoonotic faecal bacteria carried by Sunda porcupine with culture-dependant approach and to investigate whether antimicrobial resistant isolates can be found in wildlife. A total 22 faecal samples were collected from captive Sunda porcupine and tested for the presence of pathogens in selective media for Salmonella and Listeria. After inoculating the samples in Rappaport-Vassiliadis (RV) Salmonella enrichment broth, two samples (9%) were regarded as positive for Salmonella in this presumptive test which indicated by growth black colonies on xylose lysine deoxycholate (XLD) agar. Meanwhile, the presence for Listeria was presumptively positive in all samples (100%), indicated by black colour appearance in Listeria isolation transwab. In total, 38 bacterial isolates were successfully purified, preserved and subjected for antimicrobial susceptibility testing (AST) by disk diffusion method. Resistance to ceftriaxone (3rd generation cephalosporins) was not detected while resistance to one or two antimicrobials was observed in seven isolates. Further, 16S rRNA bacterial identification was performed for selected isolates and based on sequence similarity on GenBank® databases and phylogenetic tree construction, those isolates were denoted as Pseudomonas xinjiangensis (XG4.4), Shigella sonnei (XD8.2 and G11.3), Proteus mirabilis (XH3.3, H4.2, and E1.2) and Klebsiella quasipneumoniae subsp. similipneumoniae (XF4.2). All identified isolates were sensitive to amikacin, amoxicillin-clavulanic acid, cefoxitin and ceftriaxone, except for one isolate Shigella sonnei (XD8.2) which was resistant to cefoxitin. Further research to confirm the pathogenicity of the isolates is still needed but based on these results, we support the hypothesies that Sunda porcupine is potential as a reservoir pathogenic bacteria and preventive measures are crucial to prevent transmission when processing this bushmeat.

Development of a genomic DNA reference material for Salmonella enteritidis detection using polymerase chain reaction

Several rapid methods based on nucleic acids can detect foodborne pathogens, such as Salmonella spp. However, a common reference that enables metrological traceability among measurement results is not available. Reference materials (RM) are thus key to guarantee methodological comparability. This study developed a candidate genomic DNA reference material for Salmonella enteritidis quantification to establish performance conditions and reference values for normalized RM production. The growth of Salmonella enteritidis ATCC® 13076 in Rappaport Vassiliadis selective medium was characterized, and we optimized a method of DNA extraction using cetrimonium bromide (CTAB) and LiCl.In a first stage six concentrations of DNA were prepared with and without yeast RNA (40 ng/μL) to evaluate its effect as a stabilizer in terms of homogeneity and short-term stability. Based on the findings, in a second stage two DNA concentrations were prepared and a reference value with its uncertainty was assigned based on the results of characterization, homogeneity, and stability studies using digital polymerase chain reaction and the gene targets, invA, ttr, and hilA. The material was stable for 9 months at 4 °C, with a expanded uncertainty contribution range of 11%–14%. The novel candidate RM is the first to be developed nationwide and will improve the quality of measurements in the area of food safety.

Performance evaluation of a selective-enrichment-isolation protocol for Salmonella enterica from seafood

In this study using 57 finfish samples of marine origin, selective enrichment in Rappaport-Vassiliadis (RV) broth followed by isolation on the Hektoen enteric agar (HEA) yielded 50 (53.2%) of 94 isolates. The results suggest RV-HEA as the most suitable media combination for the recovery of Salmonella from tropical seafood.

Screening of Salmonella enterica Serovars, Typhi, Typhimurium, and Enteritidis in Raw Milk and Dairy Products in South-Khorasan, Iran: Conventional versus Molecular Method

Background and Objective: Food-borne Salmonellosis has been reported as the second most common bacterial infection. Salmonella enterica subsp. enterica serotype enteritidis (S. Enteritidis) and S. enterica subsp. enterica serotype typhimurium (S. Typhimurium) are the most common serotypes worldwide as salmonellosis agents. Salmonella yyphi is the causative agent of typhoid fever worldwide. The purpose of the present study was to determine the contamination rate of raw milk and dairy products to Salmonella typhi, S. typhimurium and S. Enteritidis in South-Khorasan, Iran. It is very important in food safety risk assessment and human health. Methods: A total of 260 raw milk and 181 dairy products were obtained from South-Khorasan, Iran. Dairy samples were pre-enriched in buffered peptone water and enriched in Rappaport Vassiliadis (RV). Raw milk was enriched in RV. Plating of the enriched samples was carried out on Xylose Lysine Desoxycholate (XLD) agar and Brilliant Green agar (BGA). All of the enriched samples were also tested by M-PCR for detection of S. typhi, S. typhimurium and S. Enteritidis. Results: Among the 441 tested samples only 4 samples were contaminated with Salmonella spp. in culture method. PCR assay, didn’t find any positive sample regarding Salmonella spp. In chi-square test, the difference of two methods of isolation was significant (P&lt; 0.05). Conclusions: In conclusion, the results of the present study showed a good hygienic state of raw milk and dairy products. Enrichment based PCR assay is more economical than time-consuming culture method for Salmonella detection.