Evaluation of the Potential Antimicrobial Resistance Transfer from a Multi-Drug Resistant Escherichia coli to Salmonella in Dairy Calves

Previous research conducted in our laboratory found a significant prevalence of multi-drug resistant (MDR) Salmonella and MDR Escherichia coli (MDR EC) in dairy calves and suggests that the MDR EC population may be an important reservoir for resistance elements that could potentially transfer to Salmonella. Therefore, the objective of the current research was to determine if resistance transfers from MDR EC to susceptible strains of inoculated Salmonella. The experiment utilized Holstein calves (approximately 3 weeks old) naturally colonized with MDR EC and fecal culture negative for Salmonella. Fecal samples were collected for culture of Salmonella and MDR EC throughout the experiment following experimental inoculation with the susceptible Salmonella strains. Results initially suggested that resistance did transfer from the MDR E. coli to the inoculated strains of Salmonella, with these stains demonstrating resistance to multiple antibiotics following in vivo exposure to MDR EC. However, serogrouping and serotyping results from a portion of the Salmonella isolates recovered from the calves post-challenge, identified two new strains of Salmonella; therefore transfer of resistance was not demonstrated under these experimental conditions.     

Biofilm Formation by Salmonella Enterica Serovar 1,4,[5],12:i:- Portuguese Isolates: A Phenotypic,Genotypic, and Socio-geographic Analysis

Biofilm-forming ability is well established as an important virulence factor. However, there are no studies available regarding biofilm formation of Salmonella Typhimurium 1,4,[5],12:i:-, the new pandemic serovar in Europe. To address this problem, biofilm expression by Salmonella 1,4,[5],12:i:- was evaluated using 133 isolates from clinical, environmental and animal origins, collected in Portugal from 2006 to 2011. Biofilm detection was performed by phenotypic and genotypic methods, such growth characterization in agar and broth medium, optical density determination by microtiter assays and direct observation by fluorescent in situ hybridization. Biofilm-related genes adrA, csgD and gcpA were detected by PCR. A socio-geographic characterization of strains as biofilm producers was also performed. Results showed that biofilm formation in monophasic Salmonella is widely distributed in Portuguese isolates and could be one of the reasons for its dissemination in this country. Biofilm expression varies between locations, showing that isolates from some regions like Lisboa or Ponta Delgada have an increased ability to persist in the environment due to an enhanced biofilm production. Biofilm formation also varies between risk groups, with a higher prevalence in isolates from salmonellosis infections in women. Therefore, the analysis of the socio-geographic distribution of biofilm-forming bacteria should be considered for the establishment of more adequate regulatory measures or therapeutics regimens, especially important due to the continuous increase of infections caused by antimicrobial resistant microorganisms.     

Anti-salmonella properties of kefir yeast isolates: An in vitro screening for potential infection control

The rise of antibiotic resistance has increased the need for alternative ways of preventing and treating enteropathogenic bacterial infection. Various probiotic bacteria have been used in animal and human. However, Saccharomyces boulardii is the only yeast currently used in humans as probiotic. There is scarce research conducted on yeast species commonly found in kefir despite its claimed potential preventative and curative effects. This work focused on adhesion properties, and antibacterial metabolites produced by Kluyveromyces lactis and Saccharomyces unisporus isolated from traditional kefir grains compared to Saccharomyces boulardii strains. Adhesion and sedimentation assay, slide agglutination, microscopy and turbidimetry assay were used to analyze adhesion of Salmonella Arizonae and Salmonella Typhimurium onto yeast cells. Salmonella growth inhibition due to the antimicrobial metabolites produced by yeasts in killer toxin medium was analyzed by slab on the lawn, turbidimetry, tube dilution and solid agar plating assays. Alcohol and antimicrobial proteins production by yeasts in killer toxin medium were analyzed using gas chromatography and shotgun proteomics, respectively. Salmonella adhered onto viable and non-viable yeast isolates cell wall. Adhesion was visualized using scanning electron microscope. Yeasts-fermented killer toxin medium showed Salmonella growth inhibition. The highest alcohol concentration detected was 1.55%, and proteins with known antimicrobial properties including cathelicidin, xanthine dehydrogenase, mucin-1, lactadherin, lactoperoxidase, serum amyloid A protein and lactotransferrin were detected in yeasts fermented killer medium. These proteins are suggested to be responsible for the observed growth inhibition effect of yeasts-fermented killer toxin medium. Kluyveromyces lactis and Saccharomyces unisporus have anti-salmonella effect comparable to Saccharomyces boulardii strains, and therefore have potential to control Salmonella infection.     

Simple and Rapid Detection of Salmonella by Direct PCR Amplification of Gene fimW

This study established a simple method of specifically detecting Salmonella species by amplifying fimW gene, which was involved in regulating Salmonella type I fimbriae expression. A pair of primers was designed to target and discriminate the 68 Salmonella strains of 23 Salmonella serovars available to us from 12 non-Salmonella strains of five different kinds of bacteria by polymerase chain reaction (PCR) amplification. Results showed that specific DNA fragment with an expected size of 477 bp was successfully amplified from all Salmonella serovars, while no target band was detected in non-Salmonella species. The sensitivity of this PCR-amplifying system reached to 1 pg DNA chromosome and 10² cfu of Salmonella enteritis strain CMCC(B) 50336. The above results demonstrated the method as a simple, sensitive, and specific way for Salmonella detection.     

Quantification of acid-base interactions based on contact angle measurement allows XDLVO predictions to attachment of Campylobacter jejuni but not Salmonella

Acid-base (AB) interactions play the most important role in bacterial attachment to surfaces and can be quantified based on electron donor/electron acceptor data from contact angle measurement (CAM) according to the extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory. It follows that the XDLVO theory could fail to explain attachment numbers if differences in AB interactions between strains are not apparent by CAM. This study aimed to investigate the validity of the above assumptions by comparing empirical data on attachment of six bacterial strains (three strains of Campylobacter jejuni and three strains of Salmonella) to stainless steel and XDLVO theory predictions. A significant difference (P < 0.05) in AB interactions, apparent by CAM, between C. jejuni strains allowed prediction of attachment of this species by the XDLVO theory. However, the theory failed to explain the attachment numbers for Salmonella due to similar AB interactions, as established by CAM, between the three Salmonella strains. Qualitative analysis of AB interactions by microbial adhesion to solvents (MATS) revealed a significant difference (P < 0.05) in electron donor property between the three Salmonella strains suggesting that these strains may differ with respect to AB interactions. No significant correlation with respect to electron donor property (P = 0.502, r² = 12%) was apparent between CAM and MATS. These data suggest that CAM may not always reflect exactly AB interactions and that the difference in the outcomes from MATS and CAM should be considered when the XDLVO theory is used to predict bacterial attachment to surfaces.     

Independent Bottlenecks Characterize Colonization of Systemic Compartments and Gut Lymphoid Tissue by Salmonella

Vaccination represents an important instrument to control typhoid fever in humans and protects mice from lethal infection with mouse pathogenic serovars of Salmonella species. Mixed infections with tagged Salmonella can be used in combination with probabilistic models to describe the dynamics of the infection process. Here we used mixed oral infections with tagged Salmonella strains to identify bottlenecks in the infection process in naïve and vaccinated mice. We established a next generation sequencing based method to characterize the composition of tagged Salmonella strains which offers a fast and reliable method to characterise the composition of genome-tagged Salmonella strains. We show that initial colonization of Salmonella was distinguished by a non-Darwinian selection of few bacteria setting up the infection independently in gut associated lymphoid tissue and systemic compartments. Colonization of Peyer''s patches fuels the sustained spread of bacteria into mesenteric lymph nodes via dendritic cells. In contrast, infection of liver and spleen originated from an independent pool of bacteria. Vaccination only moderately reduced invasion of Peyer''s patches but potently uncoupled bacterial populations present in different systemic compartments. Our data indicate that vaccination differentially skews the capacity of Salmonella to colonize systemic and gut immune compartments and provide a framework for the further dissection of infection dynamics.     

The Early Innate Response of Chickens to Salmonella enterica Is Dependent on the Presence of O-Antigen but Not on Serovar Classification

Salmonella vaccines used in poultry in the EU are based on attenuated strains of either Salmonella serovar Enteritidis or Typhimurium which results in a decrease in S. Enteritidis and S. Typhimurium but may allow other Salmonella serovars to fill an empty ecological niche. In this study we were therefore interested in the early interactions of chicken immune system with S. Infantis compared to S. Enteritidis and S. Typhimurium, and a role of O-antigen in these interactions. To reach this aim, we orally infected newly hatched chickens with 7 wild type strains of Salmonella serovars Enteritidis, Typhimurium and Infantis as well as with their rfaL mutants and characterized the early Salmonella-chicken interactions. Inflammation was characterized in the cecum 4 days post-infection by measuring expression of 43 different genes. All wild type strains stimulated a greater inflammatory response than any of the rfaL mutants. However, there were large differences in chicken responses to different wild type strains not reflecting their serovar classification. The initial interaction between newly-hatched chickens and Salmonella was found to be dependent on the presence of O-antigen but not on its structure, i.e. not on serovar classification. In addition, we observed that the expression of calbindin or aquaporin 8 in the cecum did not change if inflammatory gene expression remained within a 10 fold fluctuation, indicating the buffering capacity of the cecum, preserving normal gut functions even in the presence of minor inflammatory stimuli.     

Detection of Salmonella enterica in Naturally Contaminated Liquid Eggs by Loop-Mediated Isothermal Amplification, and Characterization of Salmonella Isolates

Loop-mediated isothermal amplification (LAMP) assay was effective in detecting Salmonella enterica in naturally contaminated liquid egg samples. Salmonella was detected in 110 samples taken from four egg-breaking plants. The egg samples were pre-enriched in buffered peptone water (BPW) at 37°C for 20 h. The selective enrichment was done in Rappaport-Vassiliadis or tetrathionate broth and plated onto xylose lysine deoxycholate agar and brilliant green agar, modified. In addition, the PCR assay was used to detect Salmonella after pre-enrichment in BPW at 37°C for 20 h. The culture method and PCR assay were compared to the LAMP assay, which was also performed after pre-enrichment in BPW. PCR failed to detect Salmonella in 10% of 110 samples, whereas the culture method and LAMP assay successfully identified Salmonella in all samples. However, the LAMP assay was found to be much more rapid than the culture method and as sensitive in detecting Salmonella from liquid eggs. In all of the egg-breaking plants studied, Salmonella was isolated on most tested days. The positive samples showed that more than 75% of the Salmonella strains had identical genetic patterns when analyzed by pulsed-field gel electrophoresis. This suggests that the same Salmonella strains having survived long periods of time in the plants were contaminating the production line. The LAMP assay is rapid, specific, and sensitive for Salmonella detection in liquid eggs and is able to monitor Salmonella contamination in egg-handling plants more reliably.     

Analysis of hopanoids in bacteria involved in food technology and food contamination

Hopanoids are pentacyclic triterpenoids which are believed to act as reinforcers of membranes in certain prokaryotic microorganisms. A rapid and sensitive method for their screening in bacteria was elaborated, involving extraction of non-saponifiable lipids and the analysis by gas chromatography-mass spectrometry, selectively monitoring the ion of m/z=191. Using the method, hopanoids were detected in strains of Acetobacter pasteurianus, but were found to be absent in lactic acid bacteria (Lactobacillus spp., Lactococcus spp.) and in food-contaminating bacteria (Salmonella spp., Listeria spp., Yersinia spp. and others).     

In vitro virulence characteristics of rare serovars of <Emphasis Type="Italic">Salmonella enterica</Emphasis> isolated from sand lizards (<Emphasis Type="Italic">Lacerta agilis</Emphasis> L.)

The aim of this study was to estimate virulence potential of Salmonella enterica strains colonizing the gut of free-living sand lizards (Lacerta agilis L.). The strains belonged to three Salmonella serovars: Abony, Schleissheim, and Telhashomer. Adhesion and invasion abilities of the strains were determined in quantitative assays using the gentamicin protection method. Induction of apoptosis was assessed using HeLa cell monolayers. PCR assays were used for detection of 26 virulence genes localised within mobile elements: pathogenicity islands, virulence plasmids, and prophage sequences. In vitro studies revealed that all strains had adhesion and invasion abilities to human epithelial cells. The isolates were cytotoxic and induced apoptosis of the cells. The serovars differed in the number of virulence-associated genes: up to 18 genes were present in Salmonella Schleissheim, 17 in Salmonella Abony, whereas as few as six genes were found in Salmonella Telhashomer. Generally, Salmonella Abony and Salmonella Schleissheim did not differ much in gene content connected with the presence SPI-1 to -5. All of the strains lacked genes localised within bacteriophages and plasmids. The presence of virulence-associated genes and in vitro pathogenicity assays suggest that Salmonella sp. strains originating from autochthonous, free-living lizards can potentially infect and cause disease in humans.     

Immunogenicity of Salmonella enterica serovar Enteritidis virulence protein,InvH, and cross-reactivity of its antisera with Salmonella strains

Acellular vaccines containing bacterial immunodominant components such as surface proteins may be potent alternatives to live attenuated vaccines in order to reduce salmonellosis risk to human health. invH gene, an important part of needle complex in type three secretion system (TTSS) plays important role in efficient bacterial adherence and entry into epithelial cells. In this work we hypothesize that use of a 15 kDa recombinant InvH as Salmonella enterica serovar Enteritidis surface protein could provoke antibody production in mouse and would help us study feasibility of its potential for diagnosis and/or a recombinant vaccine. The purified InvH provoked significant rise of IgG in mice. Active protection induced by immunization with InvH against variable doses of S. enterica serovar Enteritidis, indicated that the immunized mice were completely protected against challenge with 10⁴ LD₅₀. The immunoreaction of sera from immunized mice with other Salmonella strains or cross reaction with sera of Salmonella strains inoculated mice is indicative of possessing by Salmonella strains of the surface protein, InvH, that can be employed in both prophylactic and diagnostic measures against S. enterica. Bacteria free spleen and ileum of the immunized mice in this study indicate that the invH gene affects bacterial invasion. Efficacy of the virulence protein, InvH, in shuttling into host cells in injectisome of S. enterica serovar Enteritidis and inhibition of this phenomenon by active immunization was shown in this study. In conclusion immunization with InvH protein can develop protection against S. enterica serovar Enteritidis infections. InvH in Salmonella strains can be exploited in protective measures as well as a diagnostic tool in Salmonella infections.     

Calcium-dependent antibacterial activity of donkey’s milk against <Emphasis Type="Italic">Salmonella</Emphasis>

The aim of this study was to examine the antibacterial activity of raw donkey’s milk (DM) against Salmonella Enteritidis and Salmonella Typhimurium as well as to determine the dependence of this antibacterial activity on calcium, lysozyme and lactoferrin content. Antibacterial assays were conducted in DM artificially contaminated with these Salmonella serotypes and then incubated at 38 °C for 8 hours. Calcium concentration was found to have a strong influence on the antibacterial activity of the contaminated DM samples supplemented with CaCl₂ and EDTA. The antibacterial activity of DM against the tested Salmonella strains was observed to be strongly calcium dependent, with the addition of CaCl₂ to DM samples improving its antibacterial potential against both pathogens. Salmonella Enteritidis appeared to be less sensitive to the antimicrobial agents in DM than S. Typhimurium. One explanation is the calcium-dependant antibacterial activity of DM is attributable to the calcium-binding ability of its lysozyme. Lysozyme may be the main antibacterial agent, most probably via a nonenzymatic mode of action against tested Salmonella strains.     

Characterization of the RpoS Status of Clinical Isolates of Salmonella enterica

The stationary-phase-inducible sigma factor, σ^S (RpoS), is the master regulator of the general stress response in Salmonella and is required for virulence in mice. rpoS mutants can frequently be isolated from highly passaged laboratory strains of Salmonella. We examined the rpoS status of 116 human clinical isolates of Salmonella, including 41 Salmonella enterica serotype Typhi strains isolated from blood, 38 S. enterica serotype Typhimurium strains isolated from blood, and 37 Salmonella serotype Typhimurium strains isolated from feces. We examined the abilities of these strains to produce the σ^S protein, to express RpoS-dependent catalase activity, and to resist to oxidative stress in the stationary phase of growth. We also carried out complementation experiments with a cloned wild-type rpoS gene. Our results showed that 15 of the 41 Salmonella serotype Typhi isolates were defective in RpoS. We sequenced the rpoS allele of 12 strains. This led to identification of small insertions, deletions, and point mutations resulting in premature stop codons or affecting regions 1 and 2 of σ^S, showing that the rpoS mutations are not clonal. Thus, mutant rpoS alleles can be found in freshly isolated clinical strains of Salmonella serotype Typhi, and they may affect virulence properties. Interestingly however, no rpoS mutants were found among the 75 Salmonella serotype Typhimurium isolates. Strains that differed in catalase activity and resistance to hydrogen peroxide were found, but the differences were not linked to the rpoS status. This suggests that Salmonella serotype Typhimurium rpoS mutants are counterselected because rpoS plays a role in the pathogenesis of Salmonella serotype Typhimurium in humans or in the transmission cycle of the disease.     

Comparative Phenotypic and Genotypic Characterization of Salmonella spp. in Pig Farms and Slaughterhouses in Two Provinces in Northern Thailand

Salmonella spp. are an important group of bacterial zoonotic pathogens which can cause acute food-borne diseases in humans. Pork products are the main source of salmonellosis, but the origins and transmission routes of the disease have not been clearly determined. The purpose of this study was to characterize Salmonella spp. isolated in pig production lines both from pig farms and from slaughterhouses in Chiang Mai and Lamphun provinces in northern Thailand. The study focuses on the association among serotypes, antimicrobial resistance patterns and Pulse Field Gel Electrophoresis (PFGE) patterns to investigate possible sources of infection and to provide information which could help strengthen salmonellosis control programs in the region. A total of 86 strains of Salmonella comprising five majority serotypes were identified. Antibiotic resistance to tetracycline was found to be the most prevalent (82.56%) followed by ampicillin (81.40%) and streptomycin (63.95%). Seven clusters and 28 fingerprint-patterns generated by PFGE were identified among strains recovered from various locations and at different times, providing information on associations among the strains as well as evidence of the existence of persistent strains in some areas. Study results suggest that Salmonella control programs should be implemented at slaughterhouse production lines, including surveillance to insure good hygiene practices, in addition to regular monitoring of large populations of farm animals.     

Diversity of Salmonella Strains Isolated from the Aquatic Environment as Determined by Serotyping and Amplification of the Ribosomal DNA Spacer Regions

Salmonella species are pathogenic bacteria often detected in sewage, freshwater, marine coastal water, and groundwater. Salmonella spp. can survive for long periods in natural waters, and the persistence of specific and epidemic strains is of great concern in public health. However, the diversity of species found in the natural environment remains unknown. The aim of this study was to investigate the diversity of Salmonella strains isolated from different natural aquatic systems within a Mediterranean coastal watershed (river, wastewater, and marine coastal areas). A total of 574 strains isolated from these natural environments were identified by both conventional serotyping and the ribosomal spacer-heteroduplex polymorphism (RS-HP) method (M. A. Jensen and N. Straus, PCR Methods Appl. 3:186–194, 1993). More than 40 different serotypes were found, and some serotypes probably mobilized from widespread animal-rearing activities were detected only during storm events. These serotypes may be good indicators of specific contamination sources. Furthermore, the RS-HP method based on the PCR amplification of the intergenic spacer region between the 16S and 23S rRNA genes can produce amplicon profiles allowing the discrimination of species at both serotype and intraserotype levels. This method represents a powerful tool that could be used for rapid typing of Salmonella isolates.     

Oral Delivery of a Novel Attenuated Salmonella Vaccine Expressing Influenza A Virus Proteins Protects Mice against H5N1 and H1N1 Viral Infection

Attenuated strains of invasive enteric bacteria, such as Salmonella, represent promising gene delivery agents for nucleic acid-based vaccines as they can be administrated orally. In this study, we constructed a novel attenuated strain of Salmonella for the delivery and expression of the hemagglutinin (HA) and neuraminidase (NA) of a highly pathogenic H5N1 influenza virus. We showed that the constructed Salmonella strain exhibited efficient gene transfer activity for HA and NA expression and little cytotoxicity and pathogenicity in mice. Using BALB/c mice as the model, we evaluated the immune responses and protection induced by the constructed Salmonella-based vaccine. Our study showed that the Salmonella-based vaccine induced significant production of anti-HA serum IgG and mucosal IgA, and of anti-HA interferon-γ producing T cells in orally vaccinated mice. Furthermore, mice orally vaccinated with the Salmonella vaccine expressing viral HA and NA proteins were completely protected from lethal challenge of highly pathogenic H5N1 as well as H1N1 influenza viruses while none of the animals treated with the Salmonella vaccine carrying the empty expression vector with no viral antigen expression was protected. These results suggest that the Salmonella-based vaccine elicits strong antigen-specific humoral and cellular immune responses and provides effective immune protection against multiple strains of influenza viruses. Furthermore, our study demonstrates the feasibility of developing novel attenuated Salmonella strains as new oral vaccine vectors against influenza viruses.     

Plasmid Profile and Pulsed–Field Gel Electrophoresis Analysis of Salmonella enterica Isolates from Humans in Turkey

This study was conducted for typing Salmonella enterica subspecies enterica strains in Turkey using pulsed–field gel electrophoresis (PFGE) and plasmid DNA profile analysis. Fourty-two strains were isolated from clinical samples obtained from unrelated patients with acute diarrhea. The samples were collected from state hospitals and public health laboratories located at seven provinces in different regions of Turkey at different times between 2004 and 2010. The strains were determined to belong to 4 different serovars. The Salmonella enterica strains belonged to the serovars Salmonella Enteritidis (n = 23), Salmonella Infantis (n = 14), Salmonella Munchen (n = 2), and Salmonella Typhi (n = 3). Forty-two Salmonella enterica strains were typed with PFGE methods using XbaI restriction enzyme and plasmid analysis. At the end of typing, 11 different PFGE band profiles were obtained. Four different PFGE profiles (type 1, 4, 9, and 10) were found among serotype S. Enteritidis species, 3 different PFGE profiles (type 3, 5, 6) were found among S. Infantis species, 2 different PFGE profiles were found among S. Typhi species (type 2 and 11), and 2 different PFGE profiles were found among S. Munchen species (type 7, 8). The UPGMA dendrogram was built on the PFGE profiles. In this study, it was determined that 4 strains of 42 Salmonella enterica strains possess no plasmid, while the isolates have 1–3 plasmids ranging from 5.0 to 150 kb and making 12 different plasmid profiles (P1–P12). In this study, we have applied the analysis of the PFGE patterns and used bioinformatics methods to identify both inter and intra serotype relationships of 4 frequently encountered serotypes for the first time in Turkey.     

Substantial within-Animal Diversity of Salmonella Isolates from Lymph Nodes,Feces, and Hides of Cattle at Slaughter

Lymph nodes (mandibular, mesenteric, mediastinal, and subiliac; n = 68) and fecal (n = 68) and hide (n = 35) samples were collected from beef carcasses harvested in an abattoir in Mexico. Samples were analyzed for Salmonella, and presumptive colonies were subjected to latex agglutination. Of the isolates recovered, a subset of 91 was characterized by serotyping, pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility phenotyping. Salmonella was isolated from 100% (hide), 94.1% (feces), 91.2% (mesenteric), 76.5% (subiliac), 55.9% (mandibular), and 7.4% (mediastinal) of samples. From the 87 typeable isolates, eight Salmonella enterica serotypes, including Kentucky (32.2%), Anatum (29.9%), Reading (17.2%), Meleagridis (12.6%), Cerro (4.6%), Muenster (1.1%), Give (1.1%), and Mbandaka (1.1%), were identified. S. Meleagridis was more likely (P = 0.03) to be recovered from lymph nodes than from feces or hides, whereas S. Kentucky was more likely (P = 0.02) to be recovered from feces and hides than from lymph nodes. The majority (59.3%) of the Salmonella isolates were pansusceptible; however, multidrug resistance was observed in 13.2% of isolates. Typing by PFGE revealed that Salmonella strains generally clustered by serotype, but some serotypes (Anatum, Kentucky, Meleagridis, and Reading) were comprised of multiple PFGE subtypes. Indistinguishable PFGE subtypes and, therefore, serotypes were isolated from multiple sample types, and multiple PFGE subtypes were commonly observed within an animal. Given the overrepresentation of some serotypes within lymph nodes, we hypothesize that certain Salmonella strains may be better at entering the bovine host than other Salmonella strains or that some may be better adapted for survival within lymph nodes. Our data provide insight into the ecology of Salmonella within cohorts of cattle and offer direction for intervention opportunities.     

Identification by PCR of Non-typhoidal Salmonella enterica Serovars Associated with Invasive Infections among Febrile Patients in Mali

Background

In sub-Saharan Africa, non-typhoidal Salmonella (NTS) are emerging as a prominent cause of invasive disease (bacteremia and focal infections such as meningitis) in infants and young children. Importantly, including data from Mali, three serovars, Salmonella enterica serovar Typhimurium, Salmonella Enteritidis and Salmonella Dublin, account for the majority of non-typhoidal Salmonella isolated from these patients.

Methods

We have extended a previously developed series of polymerase chain reactions (PCRs) based on O serogrouping and H typing to identify Salmonella Typhimurium and variants (mostly I 4,[5],12:i:-), Salmonella Enteritidis and Salmonella Dublin. We also designed primers to detect Salmonella Stanleyville, a serovar found in West Africa. Another PCR was used to differentiate diphasic Salmonella Typhimurium and monophasic Salmonella Typhimurium from other O serogroup B, H:i serovars. We used these PCRs to blind-test 327 Salmonella serogroup B and D isolates that were obtained from the blood cultures of febrile patients in Bamako, Mali.

Principal Findings

We have shown that when used in conjunction with our previously described O-serogrouping PCR, our PCRs are 100% sensitive and specific in identifying Salmonella Typhimurium and variants, Salmonella Enteritidis, Salmonella Dublin and Salmonella Stanleyville. When we attempted to differentiate 171 Salmonella Typhimurium (I 4,[ 5],12:i:1,2) strains from 52 monophasic Salmonella Typhimurium (I 4,[5],12:i:-) strains, we were able to correctly identify 170 of the Salmonella Typhimurium and 51 of the Salmonella I 4,[5],12:i:- strains.

Conclusion

We have described a simple yet effective PCR method to support surveillance of the incidence of invasive disease caused by NTS in developing countries.     

Fast DNA Serotyping and Antimicrobial Resistance Gene Determination of Salmonella enterica with an Oligonucleotide Microarray-Based Assay

Salmonellosis caused by Salmonella (S.) belongs to the most prevalent food-borne zoonotic diseases throughout the world. Therefore, serotype identification for all culture-confirmed cases of Salmonella infection is important for epidemiological purposes. As a standard, the traditional culture method (ISO 6579:2002) is used to identify Salmonella. Classical serotyping takes 4–5 days to be completed, it is labor-intensive, expensive and more than 250 non-standardized sera are necessary to characterize more than 2,500 Salmonella serovars currently known. These technical difficulties could be overcome with modern molecular methods. We developed a microarray based serogenotyping assay for the most prevalent Salmonella serovars in Europe and North America. The current assay version could theoretically discriminate 28 O-antigens and 86 H-antigens. Additionally, we included 77 targets analyzing antimicrobial resistance genes. The Salmonella assay was evaluated with a set of 168 reference strains representing 132 serovars previously serotyped by conventional agglutination through various reference centers. 117 of 132 (81%) tested serovars showed an unique microarray pattern. 15 of 132 serovars generated a pattern which was shared by multiple serovars (e.g., S. ser. Enteritidis and S. ser. Nitra). These shared patterns mainly resulted from the high similarity of the genotypes of serogroup A and D1. Using patterns of the known reference strains, a database was build which represents the basis of a new PatternMatch software that can serotype unknown Salmonella isolates automatically. After assay verification, the Salmonella serogenotyping assay was used to identify a field panel of 105 Salmonella isolates. All were identified as Salmonella and 93 of 105 isolates (88.6%) were typed in full concordance with conventional serotyping. This microarray based assay is a powerful tool for serogenotyping.