Abstract
Salmonella is one of the most important foodborne pathogens associated with severe diseases in animals and humans. Meat samples are considered as one of the main sources of Salmonella infections. Consequently, the survey of Salmonella contamination in meat samples is of outmost importance for the control and prevention of severe diseases. In this study, 250 meat samples were selected for surveys of Salmonella contaminations. Results indicated that 12% (n=30) of samples tested were positive to Salmonella. The genetic characterization of 30 Salmonella was studied by enterobacterial repetitive intergenic consensus–polymerase chain reaction (ERIC-PCR), and 22 of ERIC-PCR types were found with D of 94.8%. In addition, the resistant characterization was also carried out using nine antibiotics test, and nine resistant patterns were observed with D of 88.7%. A good correlation was also observed between ERIC-PCR fingerprinting and resistant patterns in some Salmonella such as SAL 6 and SAL 7.
Introduction
S
Traditionally, epidemiological investigations for Salmonella have been relied on the phenotypic characteristics. However, phenotypic analyses present some limitations. For this reason, DNA fingerprinting has been applied as a useful tool to facilitate surveillance, tracing routes from a source to an infected individual (Drudy et al., 2006). Enterobacterial repetitive intergenic consensus–polymerase chain reaction (ERIC-PCR) fingerprinting could generate species- or strain-specific patterns to differentiate closely related strains (Versalovic et al., 1991). In addition, ERIC elements have been discovered in the noncoding intergenic regions of Salmonella species (Hulton et al., 1991).
In this study, 30 Salmonella strains have been isolated from 250 meat samples. In addition, ERIC-PCR and antibiotics test were used to characterize Salmonella isolates.
Materials and Methods
Isolation of Salmonella from meat samples
Two hundred fifty meat samples were collected from different farmer's markets of Guangdong Province during November 2006 to December 2007. The isolation protocols of Salmonella are as follows: 25 g sample was mixed into 25 mL normal saline for homogenate. Then, half of the homogenate was incubated with 100 mL tetrathionate broth (Huankai) containing iodine solution and brilliant green at 42°C for 18–24 h. Using a sterile loop, the enrichment samples were streaked onto both Salmonella–Shigella (Huankai) agar at 37°C for 40–48 h and bismuth sulfite (Huankai) agar at 37°C for 18–24 h. Bacterial colonies showing black colonies on bismuth sulfite and colorless transparent colonies with central black dot on Salmonella–Shigella agar were further confirmed by biochemical tests including triple sugar iron (Huankai), indole test, urea test (pH=7.0), and KCN test. Salmonella typhi CMCC 50071 and Escherichia coli ATCC 25922 were used as positive and negative controls, respectively.
Confirmation of Salmonella isolates by specific PCR
Bacterial colonies showing KCN negative, urea negative, H2S positive, and indole negative or positive for Salmonella were considered as presumptive Salmonella and were then confirmed by specific PCR methods (Xu et al., 2008). The positive band was purified using a commercial kit (Sangon) for sequencing. The sequences were aligned with expected fragments by BLAST in GenBank of NCBI (
Resistance patterns of Salmonella isolates
To test the antibiotic resistance of the isolated strains, disk diffusion assay was carried out according to the instructions of the National Committee for Clinical Standards (CLSI, 2005). Antibiotic disks were represented by ampicillin (10 μg), cephalotin (30 μg), gentamicin (10 μg), kanamycin (30 μg), vancomycin (30 μg), erythromycin (15 μg), norfloxacin (10 μg), tetracycline (30 μg), and chloromycetin (30 μg). Colonies of Salmonella isolates were incubated on Luria-Bertani agar at 37°C for 12–16 h, and then the colonies were selected and suspended in 2 mL of Mueller-Hinton broth (Oxoid) to obtain suspensions with an absorbance of 0.08–0.10 at 625 nm. The suspension was spread onto the Mueller-Hinton agar plates and the inoculated plates were dried for 5–10 min. Then, four or five antibiotic disks were placed on each plate, which were incubated at 37°C for 12–16 h under aerobic conditions. The diameters of the inhibition zones were measured and interpreted according to CLSI guidelines for Enterobacteriaceae (Staphylococcus spp. for erythromycin) (CLSI, 2005; Kim et al., 2008). The index of discriminatory ability (D) was calculated as described by Ye et al. (2010).
ERIC-PCR for Salmonella isolates
For ERIC-PCR, the primers ERIC-1R (5′-ATGTAAGCTCCTGGGGATTCAC-3′) and ERIC2 (5′-AAGTAAGTGACTGGGGTGAGCG-3′) (Versalovic et al., 1991) were used. The PCR was performed in a 25 μL solution containing 1 μM of each primer, 2.5 μL of 10× PCR buffer, 200 μm dNTPs, 2.5 mM MgCl2, and 3.0 U of Taq DNA polymerase (Sangon). The PCR conditions were one cycle at 95°C for 5 min, followed by 30 cycles of 1 min at 94°C, 1 min at 46°C, and 4 min at 72°C, and the last extension at 72°C for 8 min. The ERIC-PCR fingerprinting was analyzed as described by Ye et al. (2010), using Bionumerics 4.0 (Applied Maths).
Results and Discussion
In this study, 250 meat samples were collected for the surveys of Salmonella contamination. Results indicated that 12% (n=30) of samples tested were positive to Salmonella, as seen in Table 1, by a combination of traditional method and PCR with sequencing (Xu et al., 2008).
To determine the genetic diversity and resistant patterns, ERIC-PCR and antibiotics tests were used to characterize Salmonella isolated from meat samples. Thirty Salmonella isolates were grouped into four clusters (A, B, C, D) with D of 94.8%, as seen in Figure 1. Isolate 12 from chicken and isolate 15 from pork in Jiangmen city have 83.3% similarity within cluster A, showing closely related ERIC-PCR types. Likewise, three isolates (16, 17, and 19) have shown related types having 80.1%–92.3% similarity. Two strains (6 and 7) grouped into D cluster came from the same city having 100% similarity, which indicated that two isolates may be the same strain or from the same origin. All strains grouped into C cluster were from the same city except SAL 24. In addition, the isolates 20 and 25, 24 and 26, and 21 and 29 have 93.3%, 85.7%, and 88.9% similarity, respectively, which indicated their genetic relationship in phylogeny.
Dendrogram obtained from cluster analysis (Dice similarity coefficient; unweighted pair-group average method) of enterobacterial repetitive intergenic consensus–polymerase chain reaction patterns of Salmonella isolates.
The nine resistant patterns were observed using nine antibiotics test with D of 88.7% as seen in Figure 2. Each isolate was resistant to at least one antibiotic. Twenty-three strains were resistant to two or more antibiotics. Only four strains in H pattern were sensitive to erythromycin, and other strains were resistant to it. Interestingly, the correlation between ERIC-PCR fingerprint patterns and resistant patterns was found in some isolates. In particular, isolates 6 and 7 from the same city have same ERIC-PCR and resistant patterns. These data further indicated that they may be related in evolution. Correlation of antibiotic test and ERIC-PCR patterns indicated that a combination of two or more methods for typing of pathogens will be accurate or valid, yielding important insight into the epidemiology of infectious diseases.
Antibiotic resistance patterns (a to i) for Salmonella isolates from meat samples. 1, Ampicillin; 2, tetracycline; 3, vancomycin; 4, chloromycetin; 5, norfloxacin; 6, cephalotin; 7, gentamicin; 8, kanamycin; 9, erythromycin. S, susceptible; R, resistant; I, intermediate.
Footnotes
Acknowledgments
We acknowledge the financing support of the start-up funding of Doctor of Hefei University of Technology (No. GDBJ-036) and Guangdong Project of Science and Technology (2009B040500001) and The Cooperation Project in Industry, Education, and Research of Guangdong Province and Ministry of Education of China (2006D90504009).
Disclosure Statement
No competing financial interests exist.