Abstract
Salmonella Typhimurium is an important serovar causing non-typhoidal salmonellosis in humans. Poultry products are a common source of infection. In the present study, we used 46 isolates of S. Typhimurium, which were isolated from poultry eggs. Isolates were screened for their virulence, virulence factors (genes and plasmids), presence of ampicillin-resistant bla PSE-1 gene and Minimum Inhibitory Concentration (MIC) for ampicillin. Out of 46 isolates, bla PSE-1 gene was present in all 46 isolates and MIC ranged from 2.0 to 64 µg/ml. In mice inoculation test, out of 46 isolates, three isolates were found to be avirulent. Among virulence genes, stn, inv A, pef and spv C genes were detected in 45 (97.82%), 44(95.65%),13(28.26%), 4 (8.6%) isolates, respectively. Plasmids were detected in 37 isolates (80.43%). Plasmids, stn and inv A gene were more closely associated with virulence as compared to pef and spv C genes. As these isolates were virulent and exhibited resistance to ampicillin, the spread of such isolates may result in a serious threat to human and animal health.
Keywords: Poultry eggs, S. Typhimurium, bla PSE-1 gene, Virulence genes, Plasmids, Minimum Inhibitory Concentration (MIC)
Introduction
Salmonella is one of the most common pathogenic bacteria which have been detected in various food products and water using various molecular and microbiological tools [1]. Salmonella causes two types of diseases in humans, Typhoid and Non-typhoidal salmonellosis. S. Typhimurium is a major serovar associated with non-typhoidal salmonellosis [2]. The virulence of S. Typhimurium is attributed to the genes located on the chromosomes and plasmids [3]. Four chromosomal genes, inv A, stn, pef and spv C are responsible for invasion, diarrheal enterotoxin production, attachment to intestinal epithelium and replication of Salmonella in target organs and apoptosis [4–7]. Also, the presence of large size plasmid correlates positively with virulence [4]. Non-typhoidal salmonellosis causes 150 million illness and 60,000 deaths annually in the World [8]. Poultry products like poultry eggs are the common source of Salmonella infection. Punjab, Haryana, Uttarakhand and nearby areas of Delhi are having many commercial poultry farms and were selected for screening of Salmonella in poultry eggs as per the objective of DBT funded project entitled “Molecular Typing and Virulence gene analysis of Salmonella in North India”. Ampicillin is a very commonly used antibiotic in poultry industry and resistance for ampicillin has been reported [9]. Therefore, the isolates were screened for ampicillin resistance along with virulence and virulence contributing genes. This work will facilitate evaluation of threats to human health and establish a correlation between virulence contributing markers (genes/plasmids) with in vivo virulence in mice model.
Material and Methods
Bacterial Culture
All the 46 field isolates of Salmonella Typhimurium belonging to poultry eggs and collected from different locations ( one isolate from Delhi, three isolates from Punjab, 13 isolates from Haryana and 29 isolates from Uttarakhand) (Table-1) were obtained from Animal Biotechnology Center, College of Veterinary and Animal Sciences, G.B. Pant University of Agriculture and Technology, Pantnagar. The isolates were revived on Brilliant Green Agar (Hi-media) and a single colony was tested by Salmonella specific PCR targeting his gene [10]. The isolates were maintained in Luria Bertani Agar (LB Agar) (Himedia). The reference strain E.coli (MTCCV517) containing the plasmids of different molecular weights was procured from the Institute of Microbial Technology, Chandigarh, India and maintained in LB agar.
Isolation of Plasmid(s)
Plasmid (s) of the field isolates and reference culture were isolated by Midi preparation [11]. The plasmids were subjected to electrophoresis in a horizontal agarose 0.8% w/v gel, calibrated with plasmids of E. coli reference strain MTCCV517.
Screening of Virulence Genes by PCR
Genomic DNA was isolated using CTAB method [12] and used for detection of virulence genes by PCR.
Salmonella Plasmid Virulence ( Spv C) Gene Detection
Following primers were used for detection of spv C gene by PCR [13].
Primer 1: 5’ TTG TAG CTG CTT ATG GGG CGG 3’.
Primer 2: 5’ TGG AGA AAC GAC GCA TAC TGC 3’.
For 25 µl of PCR reaction mixture, 5 µl of genomic DNA (40 ng/µl), 1 µl of dNTPs mix (200 µm each), 1 µl of primers (20 pmol each), 1 µl of Taq DNA polymerase (3U/ µl) (Bangalore GeNei™), 2.5 µl of 10X PCR buffer (1.5 M MgCl2) and 13.5 µl of sterilized triple distilled water were used. The PCR mixture was subjected to initial denaturation at 94 °C for 5 min, 30 cycles of denaturation ( 94 °C for 1 min), annealing (60 °C for 30 s),extension (72 °C for 1 min each) and a final extension at 72 °C for 5 min. Each sample was tested thrice. The molecular weight of the amplicon was calculated by comparison with a standard molecular weight marker (100 bp-1 Kb ladder, Bangalore GeNei™) on 1.5% agarose gel.
Enterotoxin (stn) Gene Detection
Following primers were used for detection of stn gene by PCR [14]
Primer 1: 5’ TTG TGT CGC TAT CAC TGG CAA CC 3’.
Primer 2: 5’ ATT CGT AAC CCG CTC TCG TCC 3’.
All the conditions of PCR reaction mix and PCR programme were similar as described earlier except annealing temperature was kept at 59 °C for 30 s. The molecular weight of the amplicon was calculated by comparison with a standard molecular weight marker (100 bp-1 Kb ladder, Bangalore GeNei™) on 1.5% agarose gel.
Invasion (invA) Gene Detection
Following primers were used for detection of invA gene by PCR described by [15].
Primer 1: 5’ ACA GTG CTC GTT TAC GAC CTG AAT 3’.
Primer 2: 5’ AGA CGA CTG GTA CTG ATC GAT AAT 3’.
All the conditions of PCR reaction mix and PCR programme were similar except annealing temperature was kept at 56 °C for 30 s. The molecular weight of the amplicon was calculated by comparison with a standard molecular weight marker (100 bp-1 Kb ladder, Bangalore GeNei™) on 1.5% agarose gel.
Plasmid Encoded Fimbriae (pef) Gene Detection
Following primers were used for detection of pef gene by PCR [14].
Primer 1: 5’ TGT TTC CGG GCT TGT GCT 3’
Primer 2: 5’ CAG GGC ATT TGC TGA TTC TTC C 3’
All the conditions of PCR reaction mix and PCR programme were similar except annealing temperature was kept at 56 °C for 55 s. The molecular weight of the amplicon was calculated by comparison with a standard molecular weight marker (100 bp-1 Kb ladder, Bangalore GeNei™) on 1.5% agarose gel.
In vivo Virulence Test
In vivo virulence of the isolates was conducted by mice inoculation test [16]. Swiss albino mice (N = 230) of either sex and 3–4 weeks of age were procured from Laboratory Animal Unit, Division of Animal Genetics, Indian Veterinary Research Institute, Izatnagar. The mice were reared in animal houses as per the guidelines of the Institutional Animal Ethics Committee (IAEC, approval number: IAEC/ Vet Biochemistry / DBT 05). Absence of Salmonella in feces was tested using PCR. Each isolate of S. Typhimurium was injected into a group of five mice. A volume of 100 µl (108 CFU) of culture was injected intraperitoneally. The mice of each isolated group were kept separately and observed for two weeks for mortality. Salmonella was isolated from the spleen and intestine of dead mice and confirmed using PCR. The mice that survived after the 2 weeks were sacrificed and disposed ethically as per the guidelines of IAEC.
Determination of Minimum Inhibitory Concentration (MIC)
Minimum inhibitory concentration (MIC) was calculated using Hi Comb strips{ (strip A (2 µg to 256 µg) and (strip B(0.016 µg to 2.048 µg)} (Himedia™ India). Briefly, a single colony was inoculated in 2 ml LB broth and cultured at 37 °C. The turbidity was matched with the 0.5 McFarland standard. The lawn of bacterial culture was prepared on the Mueller Hinton agar plate and onto which Hi Comb strips were placed. The plates were incubated at 37 °C for 18–24 h. The zone of inhibition in the form of the ellipse was examined and MIC was calculated.
Detection of bla PSE-1 gene
Isolates were tested for the presence of bla PSE-1 gene (ampicillin resistant gene) by PCR [17]. A volume of 5 µl of bacterial cell lysate was used instead of genomic DNA. The annealing temperature was kept at 60 °C for 30 s. Remaining conditions were same as in virulence genes PCR. The molecular weight of the amplicon was calculated by comparing it with a standard molecular weight marker on 1.5% agarose gel.
Statistical Analysis for the Presence of Virulence Contributing Genes, Plasmids and in vivo Virulence in Mice
Chi-square test (with Yate’s correction) was performed to test the association between the presence of virulence genes, the presence of plasmid and In vivo virulence in mice.
Results and Discussion
All the cultures were revived and confirmed as Salmonella by PCR.
Plasmid Profiling
Of the 46 isolates, only 37 showed the presence of plasmid. Thirty three isolates exhibited a single plasmid of approximately 35Mda. Three isolates (PM 32, PM 37 and V-7) showed the presence of a 37Mda plasmid. However, isolate A-833 possessed both the plasmids (35 Mda and 37 Mda) (Fig. 1). Two types of plasmids, small size and large size have been reported in Salmonella. Small size plasmid showed their association with drug resistance [18] while large size plasmids exhibit their role in virulence [18, 19]. We could isolate the large size plasmids from most of the virulent isolates, similar to the findings of other workers [19–22].
Fig. 1.
Plasmid isolated from poultry isolates of Salmonella Typhimurium. M- Marker (E. coli MTCC V517) Lane 1-A 40 Lane 2-A 193 Lane 3-A 201 Lane 4-A 285 Lane 5-A 833 Lane 6-A 840 Lane 7-A 853 Lane 8-A 973 Lane 9-L 9 Lane 10-A 972 Lane 11-L 1089 Lane 12-ANG
Detection of Virulence Contributing Genes
Out of 46 isolates, spv C gene was present in only four isolates (8.6%) and exhibited spvC gene amplicon of 472 base pairs ( Fig. 2). Salmonella enterotoxin, coded by stn gene, was present in 45 (97.82%) isolates (617 bp amplicon) except one (A-973) (Fig. 3).Only 13 (28.26%) isolates amplified pef gene (700 bp amplicon) responsible for the biosynthesis of fimbriae of Salmonella (Fig. 4) and the invA gene (571 bp amplicon) was amplified in 44 isolates ( 95.65%) except two isolates ( A-833 and V-3) (Fig. 5) and (Table 1). The Spv region of Salmonella plasmid is composed of five genes spv R, A,B,C,D and is reported to increase the rate of replication of Salmonella in the liver and spleen [23].The spv C gene encodes Spv C protein (28 Kda), which has phospho-threonine lyase activity. Phospho-threonine lyase activity of Spv C protein inactivates MAPkinase system of the host and by inhibiting p38 gene, it triggers the apoptosis of the host cell [7]. In our study, spv C gene could be detected in very few numbers of isolates (8.5%). In earlier reports also, spv C gene was found uncommon among the field isolates of Salmonella [24–27]. Maximum isolates (97.82%) possessed stn gene which is responsible for diarrhea and also in regulating the membrane composition and integrity of the membrane of Salmonella during the infection [28]. High presence of stn in Salmonella has also been observed earlier [24, 28]. The pef gene was present in only 28.26% of isolates, the frequency of pef gene was found to be less which is similar to findings of earlier workers [25, 26, and 27]. Inv operon, composed of four genes inv A, B, C, D play an important role in the invasion of Salmonella in the host cell. Mutational analysis depicted loss of invasion ability of Salmonella on the mutation of inv A gene [32]. Therefore, inv A may be considered as an important virulence contributing gene of Salmonella. In our study, inv A gene was possessed in majority of the isolates (95.65%) as also reported earlier [33–36].
Fig. 2.
Amplification of spv C gene, stn genes, pef gene and invA gene of Salmonella Typhimurium. spv C gene amplicons. Lane M: DNA Marker (100-3000 bp) (Low range DNA ruler) (Bangalore GeNei, India) Lane 1, 2, 5, 6 : Salmonella Typhimurium isolates PM28, PM32, V7, 4 Lane 3: Negative Salmonella Typhimurium isolate 15 Lane 4: Negative control
Fig. 3.
Enterotoxin (stn) gene amplicons. Lane M-DNA Marker (100 bp Ladder) (Bangalore GeNei, India) Lane 1-24 Salmonella Typhimurium isolates A50, A40, A193, A201, A285, V3, A840, A853, A972, A823,A999, L9, L973, L1089, ANG, D349, PM21, PM22, PM23, PM24, PM25, PM26, PM28, PM29
Fig. 4.
pef gene amplicons. Lane M: DNA Marker (100-3000 bp) (Low range DNA ruler) (Bangalore GeNei, India) Lane 1-13: Salmonella Typhimurium isolates A193, A285, PM25, PM28, PM31, PM32, PM34, PMX1, V7, 13, 4, 11, 12. Lane 14 Negative control
Fig. 5.
Invasion (invA) gene amplicons. Lane M-DNA Marker (100 bp Ladder) (Bangalore GeNei, India) Lane 1-24 Salmonella Typhimurium isolates A50, A40, A193, A201, A285, A823, A840, A853, A972, A973, A999, L9, L973, L1089, ANG, D349, PM21, PM22, PM23, PM24, PM25, PM26, PM28, PM29
Table 1.
Comparative analysis of the presence of spvC, stn, invA, pef genes and in vivo virulence in mice (− indicates gene or plasmid absent, + indicates gene or plasmid present, a = 35 MDa Plasmid, b = 37 MDa Plasmid)
| Isolate No | Source Location | Plasmid (MDa) |
spvC (472 bp) |
invA (571 bp) |
pef (700 bp) |
Stn (617 bp) |
Mortality in mice (%) |
MIC for ampicillin (µg/ml) |
|---|---|---|---|---|---|---|---|---|
| A-50 | Haryana | – | – | + | – | + | 100 | 4.0 |
| A-40 | Haryana | a | – | + | – | + | 100 | 4.0 |
| A-193 | Haryana | a | – | + | + | + | 100 | 4.0 |
| A-201 | Haryana | a | – | + | – | + | 100 | 4.0 |
| A-285 | Haryana | a | – | + | + | + | 100 | 8.0 |
| A-823 | Haryana | – | – | + | – | + | 100 | 8.0 |
| A-833 | Haryana | a and b | – | - | – | + | 80 | 64.0 |
| A-840 | Haryana | a | – | + | – | + | 100 | 32.0 |
| A-853 | Haryana | a | – | + | – | + | 100 | 32.0 |
| A-972 | Haryana | a | – | + | – | + | 100 | 8.0 |
| A-973 | Haryana | – | – | + | – | - | 80 | 32.0 |
| A-999 | Haryana | – | – | + | – | + | 80 | 16.0 |
| L-9 | Punjab | a | – | + | – | + | 100 | 4.0 |
| L-973 | Punjab | a | – | + | – | + | 100 | 16.0 |
| L-1089 | Punjab | a | – | + | – | + | 100 | 8.0 |
| ANG | Haryana | a | – | + | – | + | 100 | 4.0 |
| D-349 | Delhi | – | – | + | – | + | 100 | 2.0 |
| PM-21 | Uttarakhand | a | – | + | – | + | 60 | 4.0 |
| PM-22 | Uttarakhand | a | – | + | – | + | 100 | 2.0 |
| PM-23 | Uttarakhand | – | – | + | – | + | 100 | 4.0 |
| PM-24 | Uttarakhand | a | – | + | – | + | 80 | 2.0 |
| PM-25 | Uttarakhand | a | – | + | + | + | 100 | 2.0 |
| PM-26 | Uttarakhand | a | – | + | – | + | 100 | 2.0 |
| PM-28 | Uttarakhand | a | + | + | + | + | 100 | 4.0 |
| PM-29 | Uttarakhand | – | – | + | – | + | 0 | 4.0 |
| PM-30 | Uttarakhand | a | – | + | – | + | 100 | 4.0 |
| PM-31 | Uttarakhand | a | – | + | + | + | 100 | 2.0 |
| PM-32 | Uttarakhand | b | + | + | + | + | 100 | 8.0 |
| PM-33 | Uttarakhand | a | – | + | – | + | 80 | 4.0 |
| PM-34 | Uttarakhand | a | – | + | + | + | 100 | 2.0 |
| PM-35 | Uttarakhand | a | – | + | – | + | 60 | 4.0 |
| PM-37 | Uttarakhand | b | – | + | – | + | 100 | 4.0 |
| 20 | Uttarakhand | a | – | + | – | + | 0 | 8.0 |
| PMX-1 | Uttarakhand | a | – | + | + | + | 100 | 2.0 |
| PMX-2 | Uttarakhand | a | – | + | – | + | 80 | 4.0 |
| V-3 | Uttarakhand | – | – | – | – | + | 60 | 2.0 |
| V-6 | Uttarakhand | – | – | + | – | + | 80 | 2.0 |
| V-7 | Uttarakhand | b | + | + | + | + | 100 | 2.0 |
| 13 | Uttarakhand | a | – | + | + | + | 100 | 8.0 |
| 14 | Uttarakhand | a | – | + | – | + | 100 | 8.0 |
| 15 | Uttarakhand | a | – | + | – | + | 0 | 8.0 |
| 4 | Uttarakhand | a | + | + | + | + | 100 | 4.0 |
| 5 | Uttarakhand | a | – | + | – | + | 100 | 8.0 |
| 6 | Uttarakhand | a | – | + | – | + | 100 | 4.0 |
| 11 | Uttarakhand | a | – | + | + | + | 100 | 4.0 |
| 12 | Uttarakhand | a | – | + | + | + | 100 | 8.0 |
Testing of Virulence by Mice Inoculation Test
Mice inoculation test performed on 46 isolates revealed 43 to be virulent in nature as they caused mortality in mice of different groups. The mortality percentage varied from 60–100% (Table 1). Salmonella organisms were isolated from the spleen and liver of dead mice. Three isolates (PM 29, 20 and 15) were found to be avirulent (Table 1). Lab animals have been used for the assessment of the virulence of pathogenic bacteria and their mutants [27]. Variation in the degree of virulence was exhibited as also reported earlier by various authors, who used mice model for the determination of virulence of the isolates [37].
Analysis of Minimum Inhibitory Concentration (MIC)
All the isolates (46) used in present study were found resistant for ampicillin. MIC value for ampicillin among the isolates ranged from 2.0 to 64 µg/ml. Isolate number A833 exhibited the highest MIC of 64 µg/ml followed by A840, A853 and A973 having MIC values of 32 µg/ml(Table 2). Though in a few earlier reports, resistance for ampicillin was correlated with the presence of drug-resistant plasmid [38, 39] but in our study, we could not establish any such correlation rather the resistance was found to be chromosomal in nature as bla PSE-1 gene was present in all the isolates (Table 1). Resistance for the ampicillin in field isolates of Salmonella has been reported earlier also and few reports are available on MIC of ampicillin also [9, 38]. Our findings are in agreement with them.
Table 2.
In vivo virulence in mice and statistical analysis of virulence genes (spvC, stn, pef and invA), plasmid invA), plasmid
| SI. No | Test of independence between two variables | P-value | Significance |
|---|---|---|---|
| 1 | stn Vs invA | 0.000313 | Highly significant |
| 2 | pef Vs spvC | 0.003 | Highly significant |
| 3 | spvC Vs Plasmid (37 MDa) | 0.0031 | Highly Significant |
| 4 | Plasmid (35 MDa) Vs in vivo Virulence | 0.009 | Highly Significant |
| 5 | invA Vs in vivo Virulence | 0.050 | Significant |
| 6 | pef Vs Plasmid (35 MDa) | 0.050 | Significant |
| 7 | stn Vs in vivo Virulence | 0.0118 | Significant |
| 8 | stn Vs Plasmid(35 MDa) | 0.0578 | Non-significant |
| 9 | stn Vs pef | 0.717 | Non-significant |
| 10 | stn Vs spvC | 0.557 | Non-significant |
| 11 | invA Vs spvC | 0.675 | Non-significant |
| 12 | invA Vs Plasmid (35 MDa) | 0.0556 | Non-significant |
| 13 | invA Vs pef | 0.534 | Non-significant |
| 14 | pef Vs in vivo Virulence | 0.402 | Non-significant |
| 15 | spvC Vs Plasmid (35 MDa) | 0.588 | Non-significant |
| 16 | spvC Vs in vivo Virulence | 0.85 | Non-significant |
Detection of bla PSE-1 gene
Out of 46 isolates, bla PSE-1 gene was found to be present in all 46 isolates (amplicon size 256 bp) (Table 1).
Statistical Analysis of the Presence of Virulence Gene (spv C, stn, pef, and inv A), Plasmid and In vivo Virulence in Mice
The chi-square test with Yate’s correction was performed to test the independence of different variables. The association between stn and invA, pef and spvC, spv C and plasmid of 37 Mda, in vivo virulence and plasmid (35 Mda) were found to be highly significant (p value ≤ 0.001). The association between invA and in vivo virulence, pef gene and 35 MDa plasmid and stn gene and in vivo virulence were found to be significant (p value ≤ 0.05).Rest of the associations were found to be non-significant(p value ≥ 0.05) (Table 2).
Conclusion
In India, vaccination against Salmonella in poultry is not a very common practice, hence, the commercially available poultry eggs were found contaminated with Salmonella Typhimurium. Most isolates were virulent in mice model, thereby posing a threat to humans. Two genes, stn and inv A were found closely associated with the virulence as compared to pef and spv C. Large size plasmids were also present in virulent isolates. Ampicillin, commonly used therapeutic drug in poultry industry was resistant in all the isolates. Therefore, the study warrants necessary steps in controlling the occurrence of Salmonella in poultry industry.
Acknowledgements
The authors highly acknowledge the Director of Experiment Station and Dean, College of Veterinary and Animal Sciences G.B. Pant University of Agriculture & Technology for providing necessary facilities for this study. The financial support of Department of Biotechnology, Government of India is highly acknowledged.
Funding
Department of Biotechnology, Ministry of Science and Technology, India, Grant No. BT/PR7699/AAQ/298, Mumtesh Kumar Saxena
Declarations
Competing interest
There is no conflict of interest among the authors.
Footnotes
Publisher's Note
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