1. Background
Salmonella is a common causative agent of food-borne illnesses in humans and a growing worldwide public health problem (1, 2). In Thailand, epidemiological reports indicate that Salmonella is encountered as the most common cause of diarrhea, which is widespread in all parts of the country (3). Salmonella is able to colonize in animal intestinal tracts, mainly swine and chicken, and shed in the feces (4-6). Therefore, Salmonella contamination in pork or chicken is unavoidable in the human food supply chain. Additionally, probably due to the use of manure fertilizer in their cultivation, Salmonella contamination can also be found in various types of vegetables (7). The illness is usually transmitted through food contaminated by feces. Furthermore, the resistance of Salmonella to a range of antimicrobial agents has become a serious global concern in public health.
An increasing number of antimicrobial resistant Salmonella has been reported in both developed and developing countries. Recently, a study in Denmark reported that the frequency of quinolone-resistant S. Enteritidis has increased from 0.8% in 1995 to 8.5% in 2000 (8). According to an investigation in England and Wales from 2000 to 2004, antimicrobial resistant S. Enteritidis has increased from 19% in 2000 to 35% in 2004, and antimicrobial resistant S. Typhimurium has increased from 82% in 2000 to 90% in 2002 (9). Moreover, a consequence of multidrug resistant (MDR) Salmonella infection has been previously reported, in which patients who were infected by MDR strain of S. typhimurium had an approximately 10 times higher mortality rate than general population (10). To ensure adequate consumer protection, especially in urban areas, several methods for decreasing contamination have been implemented in animal production and slaughtering system. In the countryside of Thailand, most animal and vegetable production is supplied for local consumption and therefore, the issue of food safety and Salmonella contamination in food, animal products, and vegetables may receive insufficient attention in these areas.
2. Objectives
To reduce contamination and illness caused by Salmonella infection, the prevalence of Salmonella contamination and antimicrobial susceptibility data in meat and vegetable products must be monitored. Hence, the aims of this study were to investigate the prevalence and antimicrobial resistance profiles of Salmonella found in contaminated pork, chicken meat, and vegetables in Phatthalung Province, Southern Thailand.
3. Materials and Methods
3.1. Samples
Forty-one samples of pork, 40 samples of chicken meat, and 80 samples of fresh vegetables were randomly selected from retail markets in Phatthalung Province during the period of October to December 2010. Samples were kept in separate sterile plastic bags, stored in cool boxes, and transported to laboratory on the same day of sample collection for isolation and identification of Salmonella.
3.2. Salmonella Isolation and Identification
Approximately 25 Grams of surface area of pork, chicken meat, and fresh vegetables were cut into small pieces by sterile scissors before being added to a stomach bag containing 225 mL of buffered peptone water (BPW) and incubated at 37 °C for 24 hours. Subsequently, 100 µL and 5 mL of pre-enriched cultures were transferred to Modified Semi-solid Rappaport-Vassiliadis (MSRV) medium (Criterion, U.S.A) and Rappaport Vassiliadis (RV) broth, respectively and incubated at 42 °C for 24 hours. Then, one loopful from each of the enriched broths were streaked onto Xylose Lysine Desoxycholate (XLD) agar (Himedia, India) plates and incubated at 37 °C for 18 to 24 hours. At least 5 single typical colonies of Salmonella were randomly picked up and stabbed into Triple Sugar Iron (TSI) agar (Criterion, U.S.A) and Lysine Indole Motile (LIM) medium (Himedia, India) and incubated at 37 °C for 18 to 24 hours. Typical characteristics of Salmonella exhibited on TSI agar were selected to subculture on Tryptic Soy (TSA) agar (Himedia, India). Suspected Salmonella colonies were confirmed by biochemical reactions (motility, indole production, lysine decarboxylase and carbohydrate fermentation) and slide agglutination with Salmonella O antigen antiserum. Final confirmation test was obtained through Salmonella serotyping performed by slide agglutination with O and H Salmonella antisera (S&A reagent, Thailand).
3.3. Antimicrobial Susceptibility Testing
All isolates were tested for 10 antimicrobial drugs (oxoids); tetracycline (30 µg), ciprofloxacin (5µg), chloramphenicol (30 µg), ampicillin (10 µg), norfloxacin (10 µg), nalidixic acid (30 µg), streptomycin (10 µg), trimethoprim/sulfamethoxazole (25 µg), cephalothin (30 µg), and gentamicin (10 µg). Susceptibility testing was performed according to recommendations of Clinical and Laboratory Standards Institute (CLSI) (11). To standardize bacterial suspension (in 0.8% NaCl), the density of suspension was adjusted to 0.5 McFarland and spread over the entire surface of Mueller Hinton agar (MHA) plates using a sterile cotton swab. Antimicrobial discs were placed on the agar surface followed by incubation of the plates at 37 °C for 24 hours. Inhibition zones were measured by Venire Caliper and interpreted accordingly by CLSI recommendations. The results were analyzed by WHONET 5 program.
4. Results
4.1. Prevalence of Salmonella Serotypes in Samples
Of 161 samples, 41 samples were prepared from retail pork, 40 samples from chicken meat, and 80 samples from fresh vegetables. The prevalence of Salmonella contamination in retail pork, chicken meat, and fresh vegetables were 82% (34/41), 67.5% (27/40), and 46% (37/80), respectively. Among all positive samples, 45 isolates, 38 isolates, and 29 isolates of Salmonella were segregated from pork, chicken meat, and vegetables, respectively. The most predominant serotypes isolated from pork, chicken meat, and fresh vegetables were S. Rissen (28.8%), S. albany (44.7%), and S. Typhimurium (33.3%), respectively. Frequency of isolated serotypes from pork, chicken meat, and vegetables is presented in Table 1.
Table 1.Salmonella serotypes Isolated From Pork, Chicken Meat, and Vegetable Samples at Phatthalung Province, Thailand.
| Pork, No. (%) | Chicken Meat, No. (%) | Vegetables, No. (%) | |
|---|---|---|---|
| Rissen | 13 (28.8) | 3 (7.8) | |
| Anatum | 3 (6.6) | ||
| Weltevreden | 12 (26.6) | 1 (2.6) | 6 (20) |
| Typhimurium | 2 (4.4) | 7 (18.4) | 10 (33.3) |
| Give | 4 (8.8) | 5 (13.15) | |
| Dirby | 1 (2.2) | ||
| Kentucky | 1 (2.2) | 1 (2.6) | |
| Bredeney | 9 (20) | ||
| Albany | 17 (44.7) | ||
| Hvittingfoss | 1 (2.6) | 7 (23.3) | |
| Kalamu | 3 (7.8) | ||
| Eastbourne | 3 (10) | ||
| Paratyphi B | 2 (6.6) | ||
| Kotu | 1 (3.3) | ||
| Total | 45 | 38 | 29 |
4.2. Antimicrobial Susceptibility Testing and Multi Drug Resistance Profiles
Antimicrobial susceptibility testing was performed for all isolates. The results revealed that Salmonella isolated from pork were mostly resistant to tetracycline (77%), streptomycin (71%), and ampicillin (51%). For Salmonella isolated from chicken samples, highest resistance was observed against streptomycin (92%) followed by nalidixic acid (76%), ampicillin (68%), and chloramphenicol (68%). Despite of meat samples, fewer antimicrobial resistant Salmonella were observed in vegetable samples, mostly resistant to tetracycline (33%) followed by ampicillin (20%) and streptomycin (20%). All antimicrobial resistance profiles in Salmonella isolated from each sample are presented in Table 2.
Table 2.Antimicrobial Resistance of Salmonella serotypes Isolated From Pork, Chicken Meat, and Vegetables
| Antimicrobial Resistance of Salmonella Serovars Isolated From Pork, Chicken Meat and Vegetables | ||||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| TCY | CHL | CEP | AMP | CIP | NAL | STR | NOR | GEN | SXT | |||||||||||||||||||||
| P | C | V | P | C | V | P | C | V | P | C | V | P | C | V | P | C | V | P | C | V | P | C | V | P | C | V | P | C | V | |
| Rissen | 6 | 3 | 0 | 0 | 3 | 0 | 1 | 0 | 0 | 1 | 3 | 0 | 0 | 0 | 0 | 0 | 3 | 0 | 6 | 3 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 6 | 0 | 0 |
| Anatum | 3 | - | - | 0 | - | - | 1 | - | - | 1 | - | - | 0 | - | - | 0 | - | - | 1 | - | - | 0 | - | - | 0 | - | - | 0 | - | - |
| Weltevreden | 11 | 1 | 1 | 3 | 1 | 0 | 5 | 0 | 0 | 9 | 1 | 1 | 0 | 0 | 0 | 0 | 1 | 0 | 10 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 1 |
| Typhimurium | 1 | 4 | 2 | 0 | 4 | 0 | 0 | 1 | 0 | 2 | 4 | 0 | 0 | 0 | 0 | 0 | 6 | 1 | 2 | 7 | 2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 2 |
| Give | 4 | 2 | - | 1 | 1 | - | 0 | 0 | - | 3 | 1 | - | 0 | 0 | - | 0 | 3 | - | 3 | 3 | - | 0 | 0 | - | 0 | 0 | - | 0 | 1 | - |
| Dirby | 0 | - | - | 0 | - | - | 0 | - | - | 0 | - | - | 0 | - | - | 0 | - | - | 1 | - | - | 0 | - | - | 0 | - | - | 0 | - | - |
| Kentucky | 1 | 0 | - | 0 | 1 | - | 0 | 0 | - | 1 | 1 | - | 0 | 0 | - | 0 | 1 | - | 1 | 1 | - | 0 | 0 | - | 0 | 0 | - | 0 | 0 | - |
| Bredeney | 9 | - | - | 2 | - | - | 6 | - | - | 6 | - | - | 0 | - | - | 0 | - | - | 8 | - | - | 0 | - | - | 0 | - | - | 1 | - | - |
| Albany | - | 11 | - | - | 12 | - | - | 1 | - | - | 13 | - | - | 0 | - | - | 11 | - | - | 16 | - | - | 1 | - | - | 0 | - | - | 1 | - |
| Hvitingfoss | - | 1 | 4 | - | 1 | 0 | - | 0 | 2 | - | 1 | 3 | - | 0 | 2 | - | 1 | 1 | - | 1 | 3 | - | 0 | 0 | - | 0 | 0 | - | 0 | 0 |
| Kalamu | - | 1 | - | - | 3 | - | - | 0 | - | - | 2 | - | - | 0 | - | - | 3 | - | - | 3 | - | - | 0 | - | - | 0 | - | - | 0 | - |
| Eastboume | - | - | 3 | - | - | 0 | - | - | 0 | - | - | 2 | - | - | 1 | - | - | 0 | - | - | 0 | - | - | 0 | - | - | 0 | - | - | 0 |
| Paratyphi B | - | - | 0 | - | - | 0 | - | - | 0 | - | - | 0 | - | - | 0 | - | - | 0 | - | - | 0 | - | - | 0 | - | - | 0 | - | - | 0 |
| Kotu | - | - | 0 | - | - | 0 | - | - | 0 | - | - | 0 | - | - | 0 | - | - | 0 | - | - | 0 | - | - | 0 | - | - | 0 | - | - | 0 |
| Total (%) | 35 (77) | 23 (60) | 10 (33) | 5 (11) | 26 (68) | 0 (0) | 13 (28) | 2 (5) | 2 (6) | 23 (51) | 26 (68) | 6 (20) | 0 (0) | 0 (0) | 3 (10) | 0 (0) | 29 (76) | 2 (6) | 32 (71) | 35 (92) | 6 (20) | 0 (0) | 1 (2) | 0 (0) | 0 (0) | 0 (0) | 0 (0) | 8 (17) | 2 (5) | 3 (10) |
In addition, all Salmonella isolated were analyzed for multiple drugs resistance profiles, and it was revealed that in thirty-one pork samples (68%) several isolates were of MDR strains, mostly S. Weltevreden. Thirty-two samples (84%) isolated from chicken were of the MDR strains, mostly S. Albany. However, only 7 samples (29%) isolated from vegetables exhibited resistance to two or more antimicrobial drugs. Distribution of serotypes and multiple antimicrobial resistance patterns pertaining to pork, chicken meat, and vegetables are presented in Table 3.
Table 3.Multiple Antimicrobial Resistances of Salmonella Isolated From Pork, Chicken Meat, and Vegetables
| Pork Isolates | Chicken Isolates | Vegetables isolates | ||||||
|---|---|---|---|---|---|---|---|---|
| Serovar | Antibiogram | Number | Serovar | Antibiogram | Number | Serovar | Antibiogram | Number |
| Anatum | ||||||||
| TCY CEP AMP STR | 1 | Albany | TCY STR | 1 | Eastbourne | TCY CIP | 1 | |
| Bredeny | ||||||||
| TCY AMP STR | 1 | NAL STR | 1 | TCY AMP | 2 | |||
| TCY CEP STR | 1 | AMP NAL STR | 2 | Hvittingfoss | AMP CIP STR | 1 | ||
| TCY CHL CEP | 1 | CHL NAL STR NOR | 1 | CEP AMP NAL STR | 1 | |||
| TCY CHL AMP STR | 1 | CHL CEP AMP NAL STR | 1 | CEP AMP CIP STR | 1 | |||
| TCY CEP AMP STR | 4 | TCY CHL AMP STR | 5 | Typhimurium | TCY NAL | 1 | ||
| Give | ||||||||
| TCY AMP STR | 3 | TCY CHL AMP NAL STR | 5 | |||||
| Kentucky | ||||||||
| TCY AMP STR | 1 | Give | AMP NAL STR | 1 | ||||
| Rissen | ||||||||
| TCY STR | 5 | TCY NAL STR | 2 | |||||
| TCY STR AMP | 1 | Hvittingfoss | TCY CHL AMP NAL STR | 1 | ||||
| Typhimurium | ||||||||
| TCY AMP STR | 1 | kalamu | CHL AMP NAL STR | 2 | ||||
| Weltevreden | ||||||||
| TCY STR | 1 | TCY CHL NAL STR | 1 | |||||
| TCY CEP | 1 | Kentucky | CHL AMP NAL STR | 1 | ||||
| TCY AMP STR | 3 | Rissen | TCY CHL AMP NAL STR | 3 | ||||
| TCY CEP AMP STR | 2 | Typhimurium | NAL STR | 3 | ||||
| TCY CHL AMP STR | 1 | TCY CHL AMP NALSTR | 3 | |||||
| TCY CHL CEP AMP STR | 1 | TCY CHL CEP AMP STR | 1 | |||||
| TCY CEP AMP NAL STR | 1 | Weltevreden | TCY CHL AMP NAL STR | 1 | ||||
| TCY CHL AMP CIP NAL STR | 1 | |||||||
| Total | 31 (68%) | Total | 32 (84%) | Total | 7 (29%) | |||
5. Discussion
The problems attributed to Salmonella infection have increased significantly, in terms of both incidence and severity. Furthermore, an increase of antimicrobial resistance in this pathogen makes the treatment of infection more difficult that probably results in death. Therefore, epidemiological information and monitoring systems are necessary to control Salmonella infection in public health sector. In this study, we observed prevalence and antimicrobial susceptibility patterns of Salmonella isolated from pork, chicken meat, and vegetables. The study showed that the retail pork was highly contaminated by Salmonella, followed by chicken meat and fresh vegetables. These products, according to many reports, are the resources of Salmonella contamination (1, 2, 12, 13).
Serotyping of Salmonella isolated from pork indicated that S. Rissen was the most predominant serotype followed by S. Weltevreden, whereas S. albany and S. typhimurium were the most predominant serotyping found in chicken meat and vegetables, respectively. Our results are inconsistent with previous findings in Thailand which indicated that S. Albany and S. give were predominantly found in swine and chicken farms, respectively (14). The inconsistency in observed serotypes may be attributed to different areas studied and sample types used in this investigation. Although serotyping were not the same as in other studies, those that were observed were included within 25 most common serotyping isolated from human and other sources in Thailand (15).
High contamination of Salmonella implies that hygienic performance in carcass production processes , especially in slaughters of Phattalung Province area, probably are insufficiently attended. Antimicrobial susceptibility testing revealed high resistance rates against several antimicrobial drugs used in both medical and agricultural fields. Salmonella isolated from pork samples were commonly resistant to tetracycline, ampicillin, and streptomycin, as was in those isolated from chicken, which were additionally resistant to nalidixic acid. High resistance to those antimicrobial drugs were consistent to previous observations from various countries (16-18), which implies that the wide consumption of such antimicrobials as feed additives in livestocks contributes to emergence and dissemination of resistance in Salmonella. In addition, it had also been reported the sub-therapeutic doses of antimicrobial drugs in animal husbandry as a responsible factor in emergence and maintenance of multiple antimicrobial resistant pathogenic bacteria (19-21).
According to several reports, our results demonstrated that the fluoroquinolone groups such as ciprofloxacin and norfloxacin are still the most effective drugs to treat Salmonella infection (22-24). In recent years, evidence for decreasing susceptibility to fluoroquinolones in Salmonella has been reported. Increasing resistance to fluoroquinolones is growing as an issue receiving special attention, since fluoroquinolones are effective drugs against Salmonella in clinical performance and are usually considered as treatment of choice in life threatening cases (25). Our study found that Salmonella isolated from fresh vegetables were different from meat samples in both serotype distributions and antimicrobial resistance patterns. This imply that contamination in vegetable samples may originate from environmental sources that are different from animal contamination. However, this study indicated that retail meat and animals can serve as a source for MDR strains of Salmonella that may transfer to vegetables. Association or cross contamination between meat and vegetable samples is subject to further evaluations.
