Emergence of an SGI 1-bearing Salmonella enterica serotype Kentucky isolated from septic poultry in Nigeria

Introduction: Salmonella enterica serotype Kentucky was isolated from septic poultry in Nigeria. The objective of this study was to characterize this isolate by screening for SGI1 and hyper-virulence. Methodology: The strain was characterized by identification of Salmonella genomic island 1 (SGI1) through a PCR assay and we used a tissue culture invasion assay to assess protozoa-mediated hyper-invasion. Results: Salmonella genomic island 1 (SGI1) was identified in the strain along with an SGI1 gene (SO13) implicated in hyper-virulence. Protozoa-mediated hyper-invasiveness was also documented in the strain. Conclusion: The hyper-invasion is concordant with this emerging strain’s ability to cause fowl paratyphoid.

DT104 bearing SGI1 were found to be hyperinvasive following exposure to rumen protozoa (RPz) that are normal flora of cattle [5].In a subsequent study, an SGI1 gene (designated as SO13) gene was identified to coordinately up-regulate the observed hyper-invasion together with an unknown SGI1 gene(s) [6].
These studies accounted for the increased mortality of calves infected with DT104 when compared to calves infected with antibioticsensitive S. enterica serovar Typhimurium [7].This finding is also true of other Salmonella serotypes, such as Agona and Infantis, possessing SGI1 [5].
The current study characterizes the causative agent of the outbreak by screening for SGI1 and hyper-invasion.Our results identify a potentially emerging multiresistant strain of S. Kentucky capable of causing significant systemic disease in poultry through a hyper-virulence mechanism.

Methodology
The Salmonella isolate and the study The Salmonella isolate used for this study was recently obtained from organs of septic poultry in commercial poultry farms in Nigeria.Birds exhibited clinical signs of septicemia characterized by depression, prostration, anorexia, pyrexia, and death [14].

Serotyping of the isolate
The multidrug resistant poultry isolates were subcultured into tripticase soy agar (TSA) and submitted to National Veterinary Service Laboratories in Ames, Iowa, USA, for serotyping.Serotyping was performed per the Kauffman-White Scheme.

Determination of the minimum inhibitory concentrations of antibiotics
Ampicillin, chloramphenicol, streptomycin, sulfamethoxazole and tetracycline (all obtained from Sigma-Aldrich (Jos, Nigeria).Minimum inhibitory concentrations (MICs) were determined using the two-fold micro-broth dilution method per Clinical and Laboratory Standards Institute (CLSI) standards [15].MICs were ascribed to the lowest concentration of the antibiotics that inhibited visible growth of the isolate while DT104 was used as the positive control.

SGI1 screening and invasion assay
Since the multi-resistance and putative hypervirulence of the isolate were suggestive of the presence of SGI1, we examined the isolate for the presence of SGI1 elements such as SO13.The latter gene is involved in protozoal-mediated hypervirulence of Salmonella through hyper expression of HilA while the former contains the following antibiotic resistance gene markers: floR (chloramphenicol/florfenicol resistance), tetR (regulator of tetracycline resistance), and aadA2 (streptomycin/spectinomycin resistance) [6].We used a forward primer specific to floR and a reverse primer specific to tetR for the antibiotic resistance genes floR (chloramphenicol/florfenicol resistance), tetR (regulator of tetracycline resistance), and aadA2 (streptomycin/spectinomycin resistance) in a PCR assay as earlier described [6,16].A PCR product corresponding to aadA2 was purified and sequenced at the Iowa State University DNA Sequencing Facility (Ames, IA, USA).
As earlier described [6,16], approximately 10 9 colony forming units of bacteria were added to approximately 10 5 Rumen protozoa (RPz).The Salmonella-RPz mixture was then gently rolled for 16 hours at 37°C in a sealed 5-ml glass tube.Extracellular bacteria were then killed using 300 µg/ml florfenicol (Schering-Plough).For in vitro studies, RPz were lysed for 60 seconds at 4,800 rpm using 2.5 mM glass beads and a Mini-Bead beater (Biospec Products, Bartlesville, OK, USA).The lysate was centrifuged at 15,000 rpm for 2 minutes and then resuspended in 350 µl Lennox L broth.The lysate was used in a tissue culture invasion assay to assess protozoa-mediated hyper-invasion by allowing free-living protozoa (Tetrahymena) to engulf the strain prior to the HEp-2 cell tissue culture invasion.Percent invasion was calculated by dividing CFU recovered by CFU added [5,6].

Results
Gross and histopathological lesions observed in the carcasses from which the strain was isolated included hepatomegaly, bronze discoloration of the liver (Figure 1), fibrinonecrotic typhlitis (Figure 2), and multifocal hepatic degeneration suggestive of a paratyphoid nodule (Figure 3).
As shown in Figure 4, the S. Kentucky isolate contains SO13 and exhibits hyper-invasion by this gene in a manner similar to that observed with DT104.PCR studies revealed that floR and tetR are present and adjacent in the genome of the S. Kentucky isolate.Additionally, PCR revealed the presence of aadA2 despite streptomycin susceptibility in the S. Kentucky isolate.As shown in Figure 5, the S. Kentucky aadA2 contains a deletion of a thymidine at nucleotide 198 that is concordant with streptomycin susceptibility via a truncated AadA2 protein lacking the ability to adenylate and inactivate streptomycin (Genbank accession number: JN119849).Arrows outline an area of hepatonecrosis and hyperplasia that is associated with systemic salmonellosis [18].DT104 was used as a positive control while SGI1-free Salmonella was used as a negative control.Invasion was measured in a tissue culture invasion assay following engulfment by and egress from Tetrahymena [5,6].The gel picture insert above the invasion data illustrates the SO13 amplicons derived by PCR using primers described previously [6].

Discussion
Our study describes the characterization of Salmonella enterica serotype Kentucky isolated from septic poultry in Nigeria.This isolate contains SGI1 or an SGI1-like integron based on the presence of the SO13, floR, tetR, and aadA2.This isolate bears an aadA2 isoform that does not confer streptomycin resistance due to an inactivating mutation in which the nucleotide 200 is missing and a different amino acid sequence starting at 66 (C instead of L) with a premature stop codon at amino acid 147 when compared with DT104.This observation is responsible for the sensitivity of this particular isolate to streptomycin, whereas DT104 is resistant.
Our previous studies have implicated protozoa as instigators of hyper-virulence in SGI1-bearing nontyphoidal [5,6] and paratyphoidal Salmonella [17].DT104 may be more virulent than other Salmonella strains, and this putative phenomenon has been attributed to the presence of SGI1 [5].Humans infected with DT104 are known to be two to three times more likely to be hospitalized than those infected with other strains [13], and calves infected with DT104 are 13 times more likely to die than calves infected with antibiotic-sensitive S. enterica serovar Typhimurium [7].This finding is also extended to other Salmonella serotypes, e.g., Agona and Infantis, which possess SGI1 [5].(RPz, microbiota that natively inhabit the major forestomach of ruminants such as cattle, were identified as mediators of DT104 hyper-invasion.In a model study, RPz engulf DT104 and then hyperactivate the enteroinvasive phenotype.The RPz/DT104 bacteria are then moved to the abomasum, the "true" stomach of ruminants, where the RPz are digested and DT104 is released.DT104 then moves to the small intestine where invasion, or in this case hyper-invasion, ensues.Hyper-invasion leads to a faster onset of clinical signs, a greater recovery rate of the pathogen, and a poorer prognosis.This phenomenon was not observed in the absence of RPz or SGI1 [5].Thus RPz and SGI1 appear to co-contribute to the enhancement of invasion.
In this study, S. Kentucky strain possesses DT104 like characteristics such as SGI1, therefore we assessed protozoa-mediated hyper-invasion in the isolate.Tetrahymena, a common free-living waterborne non-pathogenic protozoan, induced a significant increase in tissue culture invasion for S. Kentucky.This finding is thus consistent with the systemic nature of this isolate.Future studies will identify the specific protozoan from the affected poultry farms that possibly contributed to the hyper virulence / hyperinvasiveseness of this isolate.Nonetheless, this is the first report of an SGI1bearing Salmonella isolate capable of causing avian paratyphoid from Nigeria.

Figure 1 .
Figure 1.Gross pathology of septic chickens infected with SGI1-bearing S. Kentucky.Moderate enlargement of liver with an arrow highlighting marked bronze degeneration.

Figure 2 .
Figure 2. Gross pathology of septic chickens infected with SGI1-bearing S. Kentucky.The arrow indicates inspissation of fibrinonecrotic debris within the cecum.

Figure 3 .
Figure 3. Histopathologic lesion of an apparent paratyphoid nodule in a septic chicken.

Figure 4 .
Figure 4. Hyperinvasion and the presence of SO13 in the S. Kentucky isolated from septic poultry.