Detection of Salmonella Spp . in Feed and Their Antibiotic Susceptibility for Alternative Therapy

Article history: Received on: 05/02/2016 Revised on: 08/03/2016 Accepted on: 11/04/2016 Available online: 28/05/2016 In the study, about 695 samples comprising (fish powder: 320 samples, blood meal: 41 samples, bone meal: 123 samples), finished feed (pellets of pig feed: 213 samples) were collected and detected by polymerase chain reaction (PCR). Isolation prevalence in fish powder, blood meal, bone meal, finished feed was 23(7.19 %), 9 (21.95%), 48 (39.67%), 2 (0.94%) respectively. These Salmonella showed different antibiotic sensitivities to erythromycin, ampicillin, penicillin, ciprofloxacin. However, all these strains were inhibited with plantaricin produced by Lactobacillus plantarum PN05 isolated in Coryandrium sativum. Our findings highlighted a potential public health hazard and warned human the outbreaks of human salmonellosis with high resistance due to the consumption of contaminated feed and also suggested the prevention by plantaricin of Lactobacillus plantarum PN05.


INTRODUCTION
Salmonella causes a health problem in the world.In United States, Salmonella infected in eggs was detected (Braden, 2006).Salmonella that occurred in developing countries commonly affect feed.Detection of Salmonella in feed is necessary in the processing chain guarantees.The identification, typing and fingerprinting of Salmonella were performed in the old days (Threlfall and Frost, 1990).Currently, polymerase chain reaction (PCR) is becoming the most utilized rapid method to detect Salmonella in food.In this context, several PCR-based assays have already been described (Iun-Fan et al., 2008).However, only some of these assays are applicable as diagnostic tools.Although there are numerous alternative methods for Salmonella detection, their application in feed is still narrow because of the presence of PCR inhibitors in the sample.Moreover, antibiotic resistance in Salmonella is high, leading the difficulties in treatment of salmonellosis (Hald et al., 2007).Rapid detection will contribute to the study on the risk of Salmonella to human because there was an association of phylogeny and virulence (Litrup et al., 2010).
Moreover, the antibiotic susceptibility of Salmonella would be exploited soon so that a treatment would be alternative (Hendriksen et al., 2007).Nowadays, bacteriocins isolated from Lactobacillus are potential in pathogen prevention.However, each bacteriocin shows their significant effects on different pathogens.With the rapid detection, a rapid collection of pathogens obtained for solving many important problems caused by pathogens.
Therefore, the study established the polymerase chain reaction (PCR) method for Salmonella detection and then the antibiotic susceptibility was done.Then, an alternative therapy by bacteriocins of Lactobacillus plantarum was also tested.

Sample collection and bac-terial cultivation
The entire sample derived from analytical service at Center of analytical service and experimentation (CASE) in Hochiminh city.Three feed ingredients (fish powder: 320 samples, blood meal: 41 samples, bone meal: 123 samples), finished feed (pellets of pig feed: 213 samples).All the samples were cultured in Luria broth.

DNA extraction and PCR
DNA was extracted from above samples using a modified PowerPrep TM DNA extraction from food and feed kit (Kogenebiotech).One mL buffered peptone water (BPW) aliquot of each frozen sample was diluted with 400 µL of lysis buffer A and 40 µL of lysis buffer B in a microtube and mixed for 10 sec; 10 µL of proteinase K and 10 µL RNase A were added before incubation at 65 o C for 1 hour; then, 400 µL of chloroform was added and the samples were centrifuged for 15 min at 12,000 rpm.200 µL of supernatant was used to extract the DNA according to the manufacturer's instructions.For the PCR assay, concentrations of reagents for PCR reaction were used according to AccuLite Salmonella spp.Detection Kit (KT Biotech) with a primer pair (F: 5'-TAC TTA ACA GTG CTC GTT TAC-3') and (R: 5'-ATA AAC TTC ATC GCA CCG TCA-3') (Iun-Fan et al., 2008), targeting the invA gene of Salmonella spp. to obtain an amplicon with 570 base pairs.5 µL sample was used for PCR.The total volume for PCR was 25 µL.The temperature program was 94 o C for 15 min in the first cycle.The conditions for next 40 cycles were 94 o C (30 s), 60 o C (30 s) and 70 o C (30 s).Then, one next cycle was 72 o C for 5 min.Finally, the reaction was cooled to 4 o C. The PCR products were analyzed with gel electrophoresis using 1 % agarose gels containing ethidium bromide (0.5 mg/mL) in TBE buffer (89 mM Tris-HCl pH 8.3, 89 mM boric acid, 2.5 mM EDTA).The DNA bands were observed by irradiating the pre stained gel under UV illuminator at 302 nm and photographed.

Antibiotic susceptibility test
To test the antibiotic susceptibility of isolated Salmonella, minimum inhibition concentration (MIC) should be determined.In this study, the antibiotics (ampicillin, penicillin, erythromycin, ciprofloxaxin) were diluted from 1024 to 1 µg/mL in 1mL broth volume in standard test tubes.One inoculum of Salmonella (10 6 cfu) was mixed in test tubes and incubated in 12h.For the suspect tubes, they were checked for the bacterial survival on agar to make sure the definite inhibition.The tests were performed by triplicate.The lowest concentration of antibiotics in which microorganism was not survival is the minimal inhibitory concentrations.

Plantaricin preparation and its anti-Salmonella activity test
Lactobacillus plantarum PN05 isolated in Coryandrum sativum (Le et al., 2015) was cultured in De Man-Rogosa-Sharpe (MRS) (Biokar Diagnostics, Beauvais, India) and incubated at 37 o C under aerobic conditions.Cultures were collected at different phase of incubation and centrifuged at 10000 rpm for 30 min to separate the cell from the broth.The cell-free supernatant was precipitated with 40% ammonium sulphate.The supernatant was precipitated continuously with 60% ammonium sulphate.The pellet was collected and solubilized in water.This solution was dialyzed in dialysis tube (cut-off: 1KDa) to eliminated ammonium sulphate.This final solution contained plantaricin.Plantaricin concentration was determined by spectrophometer.Plantaricin was used for its anti-Salmonella activity, using agar diffusion test according to Tagg (Tagg et al., 1971).In this study, plantaricin were used 20 µg/mL to applied 6 mm wells on plates inoculated with one inoculum of Salmonella (10 6 cfu) and then incubated in 12h.The diameter of inhibition zones were measured next day.The tests were performed by triplicate.

Statistical analyses
The SPSS 16.0 software (SPSS Inc., Chicago, IL, USA) was used to calculate the means and standard deviations in any experiments involving triplicate analyses of any samples.The statistical significance of any observed difference was elauated by oneway analysis of variance (One way ANOVA).Difference at P < 0.05 were considered to be significant.

Detection of Salmonella
Salmonella detection was presented in table 1.The ratio of Salmonella spp.detected by the PCR method was 11.80%.The study showed that PCR could be applied to detect Salmonella spp. in feed.The results in table 1 also showed that feed had a high risk to Salmonella infection, especially the blood meal (21.95%) and bone meal (36.97%).Although there was a lower percent of Salmonella detected in fish powder (7.19%), it was meant that there was a big problem of fish sources and food processing.
There were detected 2 strains (Sal 5 and Sal 8) showing ampicillin resistance of microorganisms (Table 2).Interestingly, Sal 7 showed strong resistance to penicillin (Table 3).For ciprofloxacin resistance, Sal 7 and Sal 10 showed strong resistance (Table 4).All 11 strains showed resistance to erythromycin (Table 5).The antibiotic susceptibility tests pointed that there are many kinds of Salmonella showing the significant resistance to antibiotics when human contacts to their feed.Seriously, one hundred percent of Salmonella strains were resistant to erythromycin.It was meant that this is a warning that erythromycin should not be used for Salmonella treatment.
Interestingly, these strains was strongly inhibited by plantaricin, a bacteriocin of Lactobacillus plantarum PN05 isolated in Coryandrium sativum (Table 1, Figure 1).Consequently, plantaricin can be used in preservation of food as well as in alternative treatment of Salmonella.

DISCUSSION
As seeing in table 1, the detection ability was high (11.8%)using PowerPrep TM DNA extraction from food and feed kit (Kogenebiotech), pointing the potency of this kit while there were many affordable DNA-extraction method from only Salmonella enterica for PCR experiments (Karimnasab et al., 2013).
The resistance of Salmonella to amphicillin and penicillin due to Salmonella may contain beta-lactam gene (bla).However, there was the difference in resistance to ampicillin from penicillin in same Salmonella strains (Table 2 and 3), for example Salmonella strain (Sal 7).It was meant that there was the structural changes in penicillin binding protein in Sal 7, leading to resistance to penicillin not ampicillin.
As presented in table 4, Sal 7 and Sal 8 were resistant to ciprofloxacin due to multi-drug resistant (MDR) gene (Franco et al., 2015).Commonly, minimum inhibitory concentration (MIC) of Salmonella containing MDR was 0.25 µg/mL (Franco et al., 2015).The MICs of Sal 7 and Sal 8 were over 1024 µg/mL.The results in this study suggested Sal 7 and Sal 8 containing resistant markers other MDR.In Sal 1, Sal 2, Sal 3, Sal 4, Sal 5, Sal 6, Sal 9, Sal 10, Sal 11, the MICs were 128 µg/mL that was higher than 0.25 µg/mL, pointing that these strains probably contained resistant markers.Further study will be done to understand well the resistance of Salmonella isolated in feed.
Interestingly, all tested Salmonella strains (1-11) in table 5 were resistant to erythromycin that claimed us that Salmonella appearing in Vietnam was high resistant to this antibiotic.Therefore, using this antibiotic for Salmonella treatment should be checked carefully.These strains might have antibiotic efflux pump, leading low drug accumulation.However, the resistant mechanism should be more clarified.
Although Salmonella isolates were resistant to current antibiotics, Salmonella could be inhibited well with plantaricin of Lactobacillus plantarum AD1 (Table 6, Figure 1).It was meant that multi drug resistance (MDR) of Salmonella would not necessary to recognize plantaricin.The study indicated that plantaricin could be used in food preservation and alternative therapy for Salmonella infection.
With the diversity of Salmonella from different food sources due to a rapid, reliable PCR method, the information of drug susceptibility of Salmonella, the prevention in Salmonella infection will be effective.The factors relating to drug susceptibility will be announced soon.

CONCLUSION
The study supplied information for detection of Salmonella by PCR and the preliminary prevention of Salmonella.Moreover, the dug susceptibility of Salmonella isolated from feed warned us to use antibiotic carefully because of the high resistance in Salmonella.
Consequently, using this alternative antibiotic to treat Salmonella should have further research due to these strains might have antibiotic efflux pump, leading low drug accumulation.On the other hand, the resistant mechanism should be further explained.
Although Salmonella isolates were resistant to existing antibiotics, Salmonella could be inhibited acceptably with plantaricin of Lactobacillus plantarum AD1 as presented on this paper.It was expected that MRD of Salmonella recognize plantaricin.The results on this research indicate that plantaricin could be used in food preservation and alternative therapy for Salmonella infection.

Table 1 :
Detection Salmonella spp. in the samples.

Table 2 :
The antibiotic sensitivity of strains with ampicillin.

Table 3 :
The antibiotic sensitivity of strains with penicillin

Table 4 :
The antibiotic sensitivity of strains with ciprofloxacin

Table 5 :
The antibiotic sensitivity of strains with erythromycin.

Table 6 :
The antibiotic inhibition of bacterocin.