Whole-genome sequencing of Salmonella serovars isolated from diarrheic and non-diarrheic foals

Diarrhea is one of the most relevant clinical conditions in foals, with up to 80% of foals experiencing one or more episodes of diarrhea during the first 6 mo of life. ¹⁷ Salmonella is a prevalent and important infectious agent associated with diarrhea in horses and is considered one of the most lethal pathogens in foals with diarrhea. ¹²

Our aim was to identify the serovar, the occurrence of genotypic multidrug resistance (MDR), and the presence of virulence and stress genes in Salmonella isolates obtained from fecal samples of foals with or without diarrhea via whole-genome sequencing (WGS). Our study was approved by the Committee on Ethics in the Use of Animals (CEUA), School of Veterinary Medicine and Animal Science, Sao Paulo State University (UNESP), Botucatu, SP, Brazil (protocol 0095/2020).

Thirty historical Salmonella isolates recovered from foals <12-mo-old (100 fecal samples from foals with diarrhea and 100 samples from healthy foals) were selected from a previous study ³ in which samples from 200 foals were tested for the presence of Salmonella spp. with 30 samples (15%) testing positive (21 from diarrheic and 9 from non-diarrheic fecal samples). Salmonella colonies obtained on XLD agar were transferred into lysogeny broth and incubated at 37°C for 2 d. The broth was then centrifuged (6,500 × g for 5 min), and the resulting pellet used for DNA extractions via a modified version of a published protocol (see Suppl. material). The DNA concentration was assessed (Qubit 2.0 fluorometer; Life Technologies). Library preparation (DNA Prep; Illumina) resulted in a normalized library with a mean fragment concentration of 4 nM and fragment size of 600–800 bp. WGS was performed (MiSeq platform; Illumina) with V3-600 cycle kits in a paired-read configuration (Suppl. Table 1).

The Mann–Whitney test was used to compare the difference in the number of resistance genes between diarrheal foals and healthy foals (Suppl. material). Statistical analyses were conducted via Prism v.6.01 (GraphPad; Dotmatics), and the significance level was set at p ≤ 0.05.

Of the 30 isolates, only 23 were recovered upon revival from storage, of which 16 isolates were from foals with diarrhea and 7 from foals without diarrhea, from 14 farms in the state of São Paulo, Brazil. The data from the whole-genome shotgun sequencing project were deposited in GenBank (BioProject PRJNA999707).

Salmonella Infantis was the predominant serovar detected, followed by Salmonella Minnesota (Suppl. Table 2). Notably, both serovars were isolated only from diarrheic foals. Salmonella Infantis was associated with a nosocomial Salmonella outbreak in horses at a veterinary teaching hospital. ¹⁶ A study in Brazil reported a Salmonella prevalence of 25% in foals with diarrhea, determined by fecal culture, compared to a prevalence of 7% in foals without diarrhea, with the predominant serovars identified being Infantis, Typhimurium, and Saintpaul. ¹² Serovar Infantis has also been recovered from a horse treated at a veterinary hospital in the Netherlands; although, the predominant serovar found across the study of 62 horses was Typhimurium. ¹⁸ Conversely, serovar Minnesota has not been reported previously from horses, and our isolates of this serovar had multidrug resistance. Serovar Newport was isolated from a foal with diarrhea in our study; this serovar has been described as the most prevalent serovar in horses with a variety of medical and surgical conditions in the United States^5,8 and Brazil. ¹³ We found serovars Braenderup and Give in diarrheic foals in our study; both serovars have been isolated previously from horses.^5,8 We also found serovar Cerro in a diarrheic foal and in a non-diarrheic foal in our study, a serovar that has been reported to be associated with salmonellosis. ⁵ The serovars Glostrup, Anatum, Javiana, 4,12:d:-, and Oranienburg were isolated only from foals without diarrhea in our study. Serovars Glostrup and Anatum have been isolated from horses with enteritis in Brazil.^7,13 Additionally, serovar Anatum was also shown to be associated with equine salmonellosis; serovar Javiana has been isolated from healthy horses in the United States. ⁵

The average number (and SD) of resistance genes was 9.93 (7.85) in the diarrheic foals and 4.00 (4.12) in the healthy foals (p = 0.08; Suppl. Table 2). Similarly, the average number of stress response genes was 11.2 (8.3) in the diarrheic foals and 5.5 (2.4) in the healthy foals (p = 0.24). The average number of virulence genes was 3.2 (0.57) in the diarrheic foals and 3.6 (0.53) in the healthy foals (p = 0.32).

Isolates are characterized as MDR when they exhibit resistance to ≥3 classes of antimicrobials in in vitro drug susceptibility tests. ⁹ We recovered 8 MDR Salmonella isolates from the diarrheic group, whereas only 1 was present in the non-diarrheic group. Interestingly, the healthy foal (17) from which an MDR isolate was recovered was from the same farm as the 2 other foals from which MDR isolates were obtained. Our results suggest that MDR isolates do not seem to occur commonly in the general equine population, although they have been found at a frequency of 11% in Salmonella species/serovars isolated from horses admitted to a veterinary teaching hospital in the United States. ⁸ All of our isolates had at least 2 resistance genes (Suppl. Tables 2, 3), and most of these genes were associated with a multidrug efflux pump complex.

Resistance to β-lactam antimicrobials was observed in 9 isolates from diarrheic foals, and all of these isolates had at least 1 antimicrobial resistance gene (ARG) related to the inactivation of these antimicrobials (bla_TEM-1, bla_CMY-2, bla_CTX-M-8). The isolates that were phenotypically resistant to the β-lactam class were consistently resistant to ampicillin, whereas the resistance to other β-lactams was variable. This result can be attributed to gene expression regulators or other undetected resistance mechanisms. An isolate from the diarrheic group had the bla_TEM-1 gene but remained sensitive to all of the β-lactams tested. CMY group genes (plasmid-carried genes encoding AmpC-like β-lactamase that hydrolyzes cephalosporins) in Salmonella spp. isolates from hospitalized or clinically ill horses have been reported. ⁸ Resistance to cephalosporins, which are often the first-choice drugs in the treatment of salmonellosis, is a relevant concern with regard to animal health.

Most isolates resistant to aminoglycosides were from diarrheic foals and had at least one described gene related to this class. Two isolates (1 from each of the 2 animal groups) carried aminoglycoside resistance genes but were phenotypically susceptible to drugs belonging to this class of antimicrobials. Similar results have been reported in Brazil, suggesting that the presence of known resistance genes does not predict phenotypic resistance due to unclear mechanisms. ² Isolates recovered from diarrheic foals 11 and 14 had an intermediate phenotypic profile for streptomycin despite having no resistance genes. Discrepancies in the genotypic/phenotypic profiles for gentamicin and streptomycin have been described previously. ¹⁰

The sul1, sul2, dfrA17, dfrA19, and dfrA27 genes were found mostly in Salmonella isolates from diarrheic foals. All of the antibiotic-resistant isolates in this class had at least one described resistance gene. However, 1 isolate from the diarrheic group had 2 resistance genes (sul1, dfrA17), but was not phenotypically resistant to sulfonamide or sulfamethoxazole–trimethoprim. Resistance to trimethoprim, an antimicrobial able to inhibit dihydrofolate reductase, occurs through the expression of genes in the dhfr and dfr classes, which encode enzymes resistant to the action of this antimicrobial. ¹

The resistance genes mph(A) and mph(E), which are responsible for the enzymatic inactivation of azithromycin, were found in 6 isolates from foals, 2 of which had phenotypic resistance to azithromycin. Eight isolates (7 from diarrheal foals and 1 from the non-diarrheic group) harbored the floR gene, which encodes an efflux pump responsible for resistance to amphenicols. All 8 of these isolates were phenotypically resistant to chloramphenicol, which is typically observed in MDR strains. ⁵ Isolates with phenotypic fluoroquinolone resistance but susceptible genotypes were observed. However, all of the isolates harboring the resistance genes qnrB6 and qnrB19 were resistant to at least one antimicrobial from the fluoroquinolone class.

Five of the 23 isolates had tetracycline resistance genes (tet(A), tet(B), tet(D)), which have been reported in Salmonella isolates obtained from food samples and humans. ² The tet(G) gene has been detected in Salmonella Typhimurium isolates from horses, ¹⁹ and the tet(A) and tet(E) genes have been detected in isolates from horses admitted to a referral hospital in the southern United States. ⁸

A total of 16 of the Salmonella isolates harbored at least one plasmid. The predominant type of plasmid was IncI1 (10 isolates; 7 from foals with diarrhea, 3 from foals without diarrhea). The IncA/C₂ plasmid was detected only in isolates from foals with diarrhea. The IncI1 and IncA/C₂ plasmids are responsible for carrying extended-spectrum β-lactamase genes and acquired AmpC. These plasmids are considered “epidemic resistance plasmids,” ⁴ and are detected worldwide in resistant Enterobacteriaceae isolates of different origins and sources. In general, IncI plasmids are associated with the spread of the bla_CMY-2, bla_CTX-M-1, and bla_TEM-52 genes. ⁴ Our results are somewhat consistent with the antimicrobials used in horses in Brazil (i.e., aminoglycoside and β-lactam antimicrobials). Other plasmids were found in the isolates in our study (Suppl. Table 3).

Five virulence genes were found in both the diarrheic and non-diarrheic groups of foals: iroB (100%/100%), sinH (100%/100%), cdtB (25%/43%), iroC (88%/100%), and sodC-1 (12%/14%). The sinH gene acts as a mediator of adhesion to host cells. The iroB and iroC genes play crucial roles in the synthesis, transport, and export of enterobactin, a siderophore produced by Salmonella species/serovars that is essential for facilitating iron uptake. The cdtB gene is related to the production of cytolethal distending toxin, a compound that hinders cellular growth, triggers apoptosis in infected host cells, and contributes to the prolonged persistence of pathogenic bacteria in host cells. ¹¹ The ability of the sodC-1 gene to reduce the effectiveness of reactive oxygen and nitrogen intermediate-mediated host defense mechanisms was experimentally demonstrated in Salmonella Choleraesuis. ¹⁴

We detected 20 stress genes from different operons with different actions against heavy metals and biocides. The asr gene, belonging to the arsenic operon, ¹⁵ and the fieF gene, which is responsible for encoding zinc exporters, were identified in all of the isolates. Genes present in the gold operon ¹⁵ (golS and golT) were detected in 22 of the 23 isolates. The mer genes, present in the mercury operon, ¹⁵ were found in 9 isolates, 8 of which originated from foals with diarrhea. The qacEdelta1 gene, which is responsible for biocide resistance, ¹⁵ was found in 3 isolates, all from foals with diarrhea. The pcoS gene (encoding a copper resistance–related membrane-spanning protein) was found in one isolate from a sick foal. A discernible relationship was found, indicating a significant association between the abundance of heavy metal operons and biocide resistance genes and a high occurrence of MDR genes per genome, similar to the findings of a previous study. ⁶

Our confirmation of the presence of MDR Salmonella isolates with a range of virulence and stress genes, particularly in foals with diarrhea, underscores the need to implement biosecurity measures at the farm level to reduce the risk of zoonotic outbreaks.

Supplemental Material

sj-pdf-1-vdi-10.1177_10406387251316314 – Supplemental material for Whole-genome sequencing of Salmonella serovars isolated from diarrheic and non-diarrheic foals

Supplemental material, sj-pdf-1-vdi-10.1177_10406387251316314 for Whole-genome sequencing of Salmonella serovars isolated from diarrheic and non-diarrheic foals by Roberta M. Basso, Fabrício M. Cerri, Fábio S. Possebon, Pollyana R. C. Braga, Monique R. T. Casas, José P. de Oliveira-Filho, João P. de Araújo Júnior, Márcio G. Ribeiro, Luis G. Arroyo and Alexandre S. Borges in Journal of Veterinary Diagnostic Investigation