Molecular phylogeny of the salmonellae: relationships among Salmonella species and subspecies determined from four housekeeping genes and evidence of lateral gene transfer events

The salmonellae are a diverse group of bacteria within the family Enterobacteriaceae that includes two species, Salmonella enterica and Salmonella bongori. In order to characterize the phylogenetic relationships of the species and subspecies of Salmonella, we analyzed four housekeeping genes, gapA, phoP, mdh and recA, comprising 3,459 bp of nucleotide sequence data for each isolate sequenced. Sixty-one isolates representing the most common serotypes of the seven subspecies of Salmonella enterica and six isolates of Salmonella bongori were included in this study. We present a robust phylogeny of the Salmonella species and subspecies that clearly defines the lineages comprising diphasic and monophasic subspecies. Evidence of intersubspecies lateral gene transfer of the housekeeping gene recA, which has not previously been reported, was obtained.     

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus. However, there are two species, six subspecies, and over 2,500 different Salmonella serovars, and not all are equal in their significance to public health. For example, finding S. enterica subspecies IIIa Arizona on a table egg layer farm is insignificant compared to the isolation of S. enterica subspecies I serovar Enteritidis, the leading cause of salmonellosis linked to the consumption of table eggs. Serovars are identified based on antigenic differences in lipopolysaccharide (LPS)(O antigen) and flagellin (H1 and H2 antigens). These antigenic differences are the outward appearance of the diversity of genes and gene alleles associated with this phenotype.We have developed an allelotyping, multiplex PCR that keys on genetic differences between four major S. enterica subspecies I serovars found in poultry and associated with significant human disease in the US. The PCR primer pairs were targeted to key genes or sequences unique to a specific Salmonella serovar and designed to produce an amplicon with size specific for that gene or allele. Salmonella serovar is assigned to an isolate based on the combination of PCR test results for specific LPS and flagellin gene alleles. The multiplex PCRs described in this article are specific for the detection of S. enterica subspecies I serovars Enteritidis, Hadar, Heidelberg, and Typhimurium.Here we demonstrate how to use the multiplex PCRs to identify serovar for a Salmonella isolate.     

Detection of Salmonella spp. Using a Generic and Differential FRET-PCR

To facilitate the detection of Salmonella and to be able to rapidly and conveniently determine the species/subspecies present, we developed and tested a generic and differential FRET-PCR targeting their tetrathionate reductase response regulator gene. The differential pan-Salmonella FRET-PCR we developed successfully detected seven plasmids that contained partial sequences of S. bongori and the six S. enterica subspecies. The detection limit varied from ∼5 copies of target gene/per PCR reaction for S. enterica enterica to ∼200 for S. bongori. Melting curve analysis demonstrated a T _m of ∼68°C for S. enterica enterica, ∼62.5°C for S. enterica houtenae and S. enterica diarizonae, ∼57°C for S. enterica indica, and ∼54°C for S. bongori, S. enterica salamae and S. enterica arizonae. The differential pan-Salmonella FRET-PCR also detected and determined the subspecies of 4 reference strains and 47 Salmonella isolated from clinically ill birds or pigs. Finally, we found it could directly detect and differentiate Salmonella in feline (5/50 positive; 10%; one S. enterica salamae and 4 S. enterica enterica) and canine feces (15/114 positive; 13.2%; all S. enterica enterica). The differential pan-Salmonella FRET-PCR failed to react with 96 non-Salmonella bacterial strains. Our experiments show the differential pan-Salmonella FRET-PCR we developed is a rapid, sensitive and specific method to detect and differentiate Salmonella.     

Comparison of Salmonella enterica Serovar Typhimurium LT2 and Non-LT2 Salmonella Genomic Sequences, and Genotyping of Salmonellae by Using PCR

Genes of Salmonella enterica serovar Typhimurium LT2 expected to be specifically present in Salmonella were selected using the Basic Local Alignment Search Tool (BLAST) program. The 152 selected genes were compared with 11 genomic sequences of Salmonella serovars, including Salmonella enterica subsp. I and IIIb and Salmonella bongori (V), and were clustered into 17 groups by their comparison patterns. A total of 38 primer pairs were constructed to represent each of the 17 groups, and PCR was performed with various Salmonella subspecies including Salmonella enterica subsp. I, II, IIIa, IIIb, IV, VI, and V to evaluate a comprehensive DNA-based scheme for identification of Salmonella subspecies and the major disease-causing Salmonella serovars. Analysis of PCR results showed that Salmonella enterica subsp. I was critically divided from other subspecies, and Salmonella strains belonging to S. enterica subsp. I were clustered based on their serovars. In addition, genotypic relationships within S. enterica subsp. I by PCR results were investigated. Also, Salmonella signature genes, Salmonella enterica serovar Typhimurium signature genes, and Salmonella enterica subsp. I signature genes were demonstrated based on their PCR results. The described PCR method suggests a rapid and convenient method for identification of Salmonella serovars that can be used by nonspecialized laboratories. Genome sequence comparison can be a useful tool in epidemiologic and taxonomic studies of Salmonella.     

Development of an oligonucleotide microarray method for Salmonella serotyping

Adequate identification of Salmonella enterica serovars is a prerequisite for any epidemiological investigation. This is traditionally obtained via a combination of biochemical and serological typing. However, primary strain isolation and traditional serotyping is time‐consuming and faster methods would be desirable. A microarray, based on two housekeeping and two virulence marker genes (atpD, gyrB, fliC and fljB), has been developed for the detection and identification of the two species of Salmonella (S. enterica and S. bongori), the five subspecies of S. enterica (II, IIIa, IIIb, IV, VI) and 43 S. enterica ssp. enterica serovars (covering the most prevalent ones in Austria and the UK). A comprehensive set of probes (n = 240), forming 119 probe units, was developed based on the corresponding sequences of 148 Salmonella strains, successfully validated with 57 Salmonella strains and subsequently evaluated with 35 blind samples including isolated serotypes and mixtures of different serotypes. Results demonstrated a strong discriminatory ability of the microarray among Salmonella serovars. Threshold for detection was 1 colony forming unit per 25 g of food sample following overnight (14 h) enrichment.     

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.     

Rapid classification and identification of salmonellae at the species and subspecies levels by whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry

Variations in the mass spectral profiles of multiple housekeeping proteins of 126 strains representing Salmonella enterica subsp. enterica (subspecies I), S. enterica subsp. salamae (subspecies II), S. enterica subsp. arizonae (subspecies IIIa), S. enterica subsp. diarizonae (subspecies IIIb), S. enterica subsp. houtenae (subspecies IV), and S. enterica subsp. indica (subspecies VI), and Salmonella bongori were analyzed to obtain a phylogenetic classification of salmonellae based on whole-cell matrix-assisted laser desorption ionization-time of flight mass spectrometric bacterial typing. Sinapinic acid produced highly informative spectra containing a large number of biomarkers and covering a wide molecular mass range (2,000 to 40,000 Da). Genus-, species-, and subspecies-identifying biomarker ions were assigned on the basis of available genome sequence data for Salmonella, and more than 200 biomarker peaks, which corresponded mainly to abundant and highly basic ribosomal or nucleic acid binding proteins, were selected. A detailed comparative analysis of the biomarker profiles of Salmonella strains revealed sequence variations corresponding to single or multiple amino acid changes in multiple housekeeping proteins. The resulting mass spectrometry-based bacterial classification was very comparable to the results of DNA sequence-based methods. A rapid protocol that allowed identification of Salmonella subspecies in minutes was established.     

Inhibition of Salmonella enterica Cells in Deli-Type Salad by Enterocin AS-48 in Combination with Other Antimicrobials

The cyclic antibacterial peptide enterocin AS-48 acted synergistically with p-hydroxybenzoic acid methyl ester (PHBME) and with 2-nitropropanol against Salmonella enterica serovar Enteritidis CECT 4300 in Russian salad. In challenge tests on a cocktail of Salmonella strains (S. enterica ssp. enterica serotype Typhi CECT 409, S. enterica ssp. enterica serovar Choleraesuis CECT 915, S. enterica ssp. enterica serovar Enteritidis CECT 4300, S. enterica ssp. arizonae serovar Arizonae CECT 4395, and S. enterica ssp. salamae CECT 4000) in salad at 10°C, the combinations of PHBME and AS-48 (80 μg/g) or 2-nitropropanol and AS-48 (40 μg/g) reduced the concentrations of viable Salmonella from 4.27 to 4.75 log CFU/g down to the limit of detection for 7 days. Salmonella populations did not increase in control samples (without any antimicrobials or in the presence of AS-48 alone) probably due to the low pH of this type of salad and low temperature of incubation, but retained over 85% viability after 1 week. This work opens new possibilities to expand the spectrum of inhibition of enterocin AS-48 against Salmonella enterica.     

The Salmonella In Silico Typing Resource (SISTR): An Open Web-Accessible Tool for Rapidly Typing and Subtyping Draft Salmonella Genome Assemblies

For nearly 100 years serotyping has been the gold standard for the identification of Salmonella serovars. Despite the increasing adoption of DNA-based subtyping approaches, serotype information remains a cornerstone in food safety and public health activities aimed at reducing the burden of salmonellosis. At the same time, recent advances in whole-genome sequencing (WGS) promise to revolutionize our ability to perform advanced pathogen characterization in support of improved source attribution and outbreak analysis. We present the Salmonella In Silico Typing Resource (SISTR), a bioinformatics platform for rapidly performing simultaneous in silico analyses for several leading subtyping methods on draft Salmonella genome assemblies. In addition to performing serovar prediction by genoserotyping, this resource integrates sequence-based typing analyses for: Multi-Locus Sequence Typing (MLST), ribosomal MLST (rMLST), and core genome MLST (cgMLST). We show how phylogenetic context from cgMLST analysis can supplement the genoserotyping analysis and increase the accuracy of in silico serovar prediction to over 94.6% on a dataset comprised of 4,188 finished genomes and WGS draft assemblies. In addition to allowing analysis of user-uploaded whole-genome assemblies, the SISTR platform incorporates a database comprising over 4,000 publicly available genomes, allowing users to place their isolates in a broader phylogenetic and epidemiological context. The resource incorporates several metadata driven visualizations to examine the phylogenetic, geospatial and temporal distribution of genome-sequenced isolates. As sequencing of Salmonella isolates at public health laboratories around the world becomes increasingly common, rapid in silico analysis of minimally processed draft genome assemblies provides a powerful approach for molecular epidemiology in support of public health investigations. Moreover, this type of integrated analysis using multiple sequence-based methods of sub-typing allows for continuity with historical serotyping data as we transition towards the increasing adoption of genomic analyses in epidemiology. The SISTR platform is freely available on the web at https://lfz.corefacility.ca/sistr-app/.     

Variation of the Ribosomal Operon 16S-23S Gene Spacer Region in Representatives of Salmonella enterica Subspecies

The 16S-23S spacer regions of two ribosomal operons (rrnA and rrnE) have been sequenced in seven representatives of the Salmonella enterica subspecies. Isolated nucleotide substitutions were found at the same sites as in Escherichia coli but the number of polymorphic sites was much larger, as could be expected for a more heterogeneous species. Still, as in E. coli, most of the variation found was due to insertions and/or deletions affecting blocks of nucleotides generally located at equivalent regions of the putative secondary structure for both species. Isolated polymorphic sites generated phylogenetic trees generally consistent with the subspecies structure and the accepted relationships among the subspecies. However, the sequences of rrnE put subspecies I closer to E. coli K-12 than to the other S. enterica subspecies. The distribution of polymorphisms affecting blocks of nucleotides was much more random, and the presence of equivalent sequences in distantly related subspecies, and even in E. coli, could reflect relatively frequent horizontal transfer. The smallest 16S-23S spacers in other genera of the family Enterobacteriaceae were also sequenced. As expected, the level of variation was much larger. Still, the phylogenetic tree inferred is consistent with those of 16S rRNA or housekeeping genes.     

Comparison of a semi-automated rep-PCR system and multilocus sequence typing for differentiation of Salmonella enterica isolates

The accurate sub-typing of Salmonella enterica isolates is essential for epidemiological investigations and surveillance of Salmonella infections. Salmonella isolates are currently identified using the Kauffman-White serotyping scheme. Multilocus sequence typing (MLST) schemes have been developed for the major bacterial pathogens including Salmonella and have assisted in understanding the molecular epidemiology and population biology of these organisms. Recently, the DiversiLab rep-PCR system has been developed using micro-fluidic chips to provide standardized, semi-automated fingerprinting for pathogens including S. enterica. In the current study, 71 isolates of S. enterica, representing 21 serovars, were analyzed using MLST and the DiversiLab rep-PCR system. MLST was able to identify 31 sequence types (STs), while the DiversiLab system revealed 38 DiversiLab types (DTs). The rep-PCR distinguished isolates of different serovars and showed greater discriminatory power (0.95) than MLST typing (0.89). Rep-PCR exhibited 92% concordance with MLST and 90% with serotyping, while the concordance level of MLST typing with serotyping was 96%, representing a strong association. Comparison of rep-PCR profiles with those held in an online library database led to the accurate prediction of serovar in 63% of cases and resulted in inaccurate predictions for 10% of profiles. MLST and the rep-PCR system may provide useful additional informative techniques for the molecular identification of S. enterica. We conclude that the DiversiLab rep-PCR system may provide a rapid (less than 4 h) and standardized method for sub-typing isolates of S. enterica.     

Phylogenomic Analysis Identifies Gene Gains That Define Salmonella enterica Subspecies I

Comparative methods for analyzing whole genome sequence (WGS) data enable us to assess the genetic information available for reconstructing the evolutionary history of pathogens. We used the comparative approach to determine diagnostic genes for Salmonella enterica subspecies I. S. enterica subsp. I strains are known to infect warm-blooded organisms regularly while its close relatives tend to infect only cold-blooded organisms. We found 71 genes gained by the common ancestor of Salmonella enterica subspecies I and not subsequently lost by any member of this subspecies sequenced to date. These genes included many putative functional phenotypes. Twenty-seven of these genes are found only in Salmonella enterica subspecies I; we designed primers to test these genes for use as diagnostic sequence targets and data mined the NCBI Sequence Read Archive (SRA) database for draft genomes which carried these genes. We found that the sequence specificity and variability of these amplicons can be used to detect and discriminate among 317 different serovars and strains of Salmonella enterica subspecies I.     

Characterization of Salmonella occurring at high prevalence in a population of the land iguana Conolophus subcristatus in Galápagos Islands, Ecuador

The aim of the study was to elucidate the association between the zoonotic pathogen Salmonella and a population of land iguana, Colonophus subcristatus, endemic to Galápagos Islands in Ecuador. We assessed the presence of Salmonella subspecies and serovars and estimated the prevalence of the pathogen in that population. Additionally, we investigated the genetic relatedness among isolates and serovars utilising pulsed field gel electrophoresis (PFGE) on XbaI-digested DNA and determined the antimicrobial susceptibility to a panel of antimicrobials. The study was carried out by sampling cloacal swabs from animals (n = 63) in their natural environment on in the island of Santa Cruz. A high prevalence (62/63, 98.4%) was observed with heterogeneity of Salmonella subspecies and serovars, all known to be associated with reptiles and with reptile-associated salomonellosis in humans. Serotyping revealed 14 different serovars among four Salmonella enterica subspecies: S. enterica subsp. enterica (n = 48), S. enterica subsp. salamae (n = 2), S. enterica subsp. diarizonae (n = 1), and S. enterica subsp. houtenae (n = 7). Four serovars were predominant: S. Poona (n = 18), S. Pomona (n = 10), S. Abaetetuba (n = 8), and S.Newport (n = 5). The S. Poona isolates revealed nine unique XbaI PFGE patterns, with 15 isolates showing a similarity of 70%. Nine S. Pomona isolates had a similarity of 84%. One main cluster with seven (88%) indistinguishable isolates of S. Abaetetuba was observed. All the Salmonella isolates were pan-susceptible to antimicrobials representative of the most relevant therapeutic classes. The high prevalence and absence of clinical signs suggest a natural interaction of the different Salmonella serovars with the host species. The interaction may have been established before any possible exposure of the iguanas and the biocenosis to direct or indirect environmental factors influenced by the use of antimicrobials in agriculture, in human medicine or in veterinary medicine.     

TRS-based PCR as a potential tool for inter-serovar discrimination of Salmonella Enteritidis,S. Typhimurium,S. Infantis,S. Virchow,S. Hadar,S. Newport and S. Anatum

Salmonella enterica subsp. enterica comprises a number of serovars, many of which pose an epidemiological threat to humans and are a worldwide cause of morbidity and mortality. Most reported food infection outbreaks involve the serovars Salmonella Enteritidis and Salmonella Typhimurium. Rapid identification to determine the primary sources of the bacterial contamination is important to the improvement of public health. In recent years, many DNA-based techniques have been applied to genotype Salmonella. Herein, we report the use of a manual TRS-PCR approach for the differentiation of the Salmonella enterica subspecies enterica serovars in a single-tube assay. One hundred seventy Salmonella strains were examined in this work. These consisted of serovars S. Enteritidis, S. Typhimurium, S. Infantis, S. Virchow, S. Hadar, S. Newport and S. Anatum. Five of the TRS-primers, N₆(GTG)₄, N₆(CAC)₄, N₆(CGG)₄, N₆(CCG)₄ and N₆(CTG)₄, perfectly distinguished the S. Enteritidis and S. Typhimurium serovars, and the N₆(GTG)₄ primer additionally grouped the other five frequently isolated serovars. In our opinion, the TRS-PCR methodology could be recommended for a quick and simple DNA-based test for inter-serovar discrimination of Salmonella strains.     

Analysis of the Type 1 Pilin Gene Cluster fim in Salmonella: Its Distinct Evolutionary Histories in the 5′ and 3′ Regions

The type 1 pilin encoded by fim is present in both Escherichia coli and Salmonella natural isolates, but several lines of evidence indicate that similarities at the fim locus may be an example of independent acquisition rather than common ancestry. For example, the fim gene cluster is found at different chromosomal locations and with distinct gene orders in these closely related species. In this work we examined the fim gene cluster of Salmonella, the genes of which show high nucleotide sequence divergence from their E. coli counterparts, as well as a different G+C content and codon usage. DNA hybridization analysis revealed that, among the salmonellae, the fim gene cluster is present in all isolates of S. enterica but is absent from S. bongori. Molecular phylogenetic analyses of the fimA and fimI genes yield an estimate of phylogeny that is in satisfactory congruence with housekeeping and other virulence genes examined in this species. In contrast, phylogenetic analyses of the fimZ, fimY, and fimW genes indicate that horizontal transfer of this region has occurred more than once. There is also size variation in the fimZ, fimY, and fimW intergenic regions in the 3′ region, and these genes are absent in isolate S2983 of subspecies IIIa. Interestingly, the G+C contents of the fimZ, fimY, and fimW genes are less than 46%, which is considerably lower than those of the other six genes of the fim cluster. This study demonstrates that horizontal transmission of all or part of the same gene cluster can occur repeatedly, with the result that different regions of a single gene cluster may have different evolutionary histories.     

Ferrioxamine-Mediated Iron(III) Utilization by Salmonella enterica

Utilization of ferrioxamines as sole sources of iron distinguishes Salmonella enterica serotypes Typhimurium and Enteritidis from a number of related species, including Escherichia coli. Ferrioxamine supplements have therefore been used in preenrichment and selection media to increase the bacterial growth rate while selectivity is maintained. We characterized the determinants involved in utilization of ferrioxamines B, E, and G by S. enterica serotype Typhimurium by performing siderophore cross-feeding bioassays. Transport of all three ferric siderophores across the outer membrane was dependent on the FoxA receptor encoded by the Fur-repressible foxA gene. However, only the transport of ferrioxamine G was dependent on the energy-transducing protein TonB, since growth stimulation of a tonB strain by ferrioxamines B and E was observed, albeit at lower efficiencies than in the parental strain. Transport across the inner membrane was dependent on the periplasmic binding protein-dependent ABC transporter complex comprising FhuBCD, as has been reported for other hydroxamate siderophores of enteric bacteria. The distribution of the foxA gene in the genus Salmonella, as indicated by DNA hybridization studies and correlated with the ability to utilize ferrioxamine E, was restricted to subspecies I, II, and IIIb, and this gene was absent from subspecies IIIa, IV, VI, and VII (formerly subspecies IV) and Salmonella bongori (formerly subspecies V). S. enterica serotype Typhimurium mutants with either a transposon insertion or a defined nonpolar frameshift (+2) mutation in the foxA gene were not able to utilize any of the three ferrioxamines tested. A strain carrying the nonpolar foxA mutation exhibited a significantly reduced ability to colonize rabbit ileal loops compared to the foxA⁺ parent. In addition, a foxA mutant was markedly attenuated in mice inoculated by either the intragastric or intravenous route. Mice inoculated with the foxA mutant were protected against subsequent challenge by the foxA⁺ parent strain.     

Salmonella Serovars in the Herpetofauna of Indiana County, Pennsylvania

Herpetofaunal Salmonella enterica serovars have not been fully examined in any U.S. region. Thirty-three Salmonella serovars were isolated from 156 samples from 34 species, all within Indiana County, Pennsylvania. Results suggest that herpetofaunas could potentially pose a threat to humans. Further understanding of Salmonella in herpetofaunas may prevent future human cases.     

Salmonella enterica Diversity in Central Californian Coastal Waterways

Salmonella enterica is one of the most important bacterial enteric pathogens worldwide. However, little is known about its distribution and diversity in the environment. The present study explored the diversity of 104 strains of Salmonella enterica isolated over 2 years from 12 coastal waterways in central California. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing were used to probe species diversity. Seventy-four PFGE patterns and 38 sequence types (STs) were found, including 18 newly described STs. Nineteen of 25 PFGE patterns were indistinguishable from those of clinical isolates in PulseNet. The most common ST was consistent with S. enterica serovar Typhimurium, and other frequently detected STs were associated with the serovars Heidelberg and Enteritidis; all of these serovars are important etiologies of salmonellosis. An investigation into S. enterica biogeography was conducted at the level of ST and subspecies. At the ST and subspecies level, we found a taxon-time relationship but no taxon-area or taxon-environmental distance relationships. STs collected during wet versus dry conditions tended to be more similar; however, STs collected from waterways adjacent to watersheds with similar land covers did not tend to be similar. The results suggest that the lack of dispersal limitation may be an important factor affecting the diversity of S. enterica in the region.     

Wild Griffon Vultures (Gyps fulvus) as a Source of Salmonella and Campylobacter in Eastern Spain

The existence of Campylobacter and Salmonella reservoirs in wildlife is a potential hazard to animal and human health; however, the prevalence of these species is largely unknown. Until now, only a few studies have evaluated the presence of Campylobacter and Salmonella in wild griffon vultures and based on a small number of birds. The aim of this study was to evaluate the presence of Campylobacter and Salmonella in wild griffon vultures (n = 97) during the normal ringing programme at the Cinctorres Observatory in Eastern Spain. In addition, the effect of ages of individuals (juveniles, subadult and adult) on the presence were compared. Campylobacter was isolated from 1 of 97 (1.0%) griffon vultures and identified as C. jejuni. Salmonella was isolated from 51 of 97 (52.6%) griffon vultures. No significant differences were found between the ages of individuals for the presence of Salmonella. Serotyping revealed 6 different serovars among two Salmonella enterica subspecies; S. enterica subsp. enterica (n = 49, 96.1%) and S. enterica subsp. salamae (n = 2, 3.9%). No more than one serovar was isolated per individual. The serovars isolated were S. Typhimurium (n = 42, 82.3%), S. Rissen (n = 4, 7.8%), S. Senftenberg (n = 3, 5.9%) and S. 4,12:b[-] (n = 2, 3.9%). Our results imply that wild griffon vultures are a risk factor for Salmonella transmission, but do not seem to be a reservoir for Campylobacter. We therefore rule out vultures as a risk factor for human campylobacteriosis. Nevertheless, further studies should be undertaken in other countries to confirm these results.