Abstract
Thirty-five of Staphylococcus aureus isolated from food raw materials and workers' hands in a noshery were characterized using antibiotic susceptibility testing, enterobacterial repetitive intergenic consensus–polymerase chain reaction (ERIC-PCR), and random amplified polymorphic DNA (RAPD)–PCR. As a similarity threshold of 90%, ERIC-PCR classified 35 S. aureus isolates into 28 ERIC types with discrimination indexes (D) of 0.984, while RAPD-PCR grouped 35 S. aureus isolates into five clusters (A–E) showing 19 RAPD types with D of 0.949. Four resistance patterns were observed with D of 0.826. A comparison of characterization of S. aureus indicated a clear correlation between ERIC-PCR and RAPD-PCR or resistance patterns in some strains. It was concluded that ERIC-PCR method could be used for genetic diversity of S. aureus and tracing the sources of it from the food chain.
Introduction
S
To date, no special studies have focused on the genetic characterization of S. aureus by ERIC-PCR. In this study, ERIC-PCR and RAPD-PCR were used to characterize S. aureus and determine the possible correlation between DNA fingerprinting and antibiotic resistance patterns.
Materials and Methods
Isolation of S. aureus strains
Thirty-five S. aureus strains were isolated from food materials and workers' hands in a noshery by following the method described by Bennett and Lancette (2001). Both Escherichia coli ATCC25922 and S. aureus ATCC25923 were used as negative and positive controls, respectively. Four isolates (SA1–SA4) were from the tegument of Jiaozi; five each from meat stuffing of Jiaozi (SA6–SA10) and workers' hands (SA5, SA11, SA24, SA34, SA15); three each from celery stuffing (SA22, SA23, SA19); five each from mushroom stuffing (SA16–SA18, SA20, SA21) and sesame stuffing of Tangyuan (SA30–SA33, SA35); four isolates from surface of Tangyuan (SA12–SA14, SA25) and greengrocery stuffing of Jiaozi (SA26–SA29).
Antibiotic susceptibility testing
The susceptibility of all isolates was tested by the disk-agar diffusion method according to the Clinical and Laboratory Standards Institute (CLSI, 2005). Antibiotic disks were represented by oxacillin (1.0 μg), azithromycin (15 μg), clindamycin (2.0 μg), tetracycline (30 μg), vancomycin (30 μg), and penicillin (10 units). Plates were inoculated, and zone sizes were interpreted by following the instructions of CLSI (2005). The type of strain S. aureus ATCC25923 was used as the positive control. The numerical index of discriminatory ability was calculated as described by Hunter and Gaston (1988).
ERIC-PCR for S. aureus
Genomic DNA of S. aureus was extracted with lysostaphin (100 μg/mL) and 1% sodium dodecyl sulfate as described by Bannerman et al. (1995). For ERIC-PCR, a 50 μL mixture consisted of 50 ng DNA template; 10× buffer at 5 μL; 4.0 U Taq polymerase (Fermentas); 200 μM of dNTP; and 0.5 μM for each primer of ERIC1R(5′-ATGTAAGCTCCTGGGGATTCAC-3′) and ERIC2(5′-AAGTAAGTG ACTGGGGTGAGCG-3′) (Versalovic et al., 1991); adding water to 50 μL. ERIC-PCR was performed under the following conditions: 95°C, 5 min; 36°C, 1 min; 72°C, 4 min; followed by 35 cycles at 94°C, 1 min; 36°C, 1 min; and 72°C for 4 min; 72°C, 8 min for last extension. The products (8 μL) were subjected to electrophoresis. The index of discriminatory (D) ability was calculated as described by Hunter and Gaston (1988). The ERIC-PCR fingerprinting was analyzed as described by Ye et al. (2010), using Bionumerics 4.0 (Applied Maths). The fingerprinting of S. aureus with a similarity index of ≥90% was considered to have the same ERIC type Rodriguez-Calleja et al., 2006).
RAPD-PCR for S. aureus
The PCR reaction system (50 μL) consisted of 10 mM Tris-HCl, 10 mM KCl, 2.5 mM MgCl2; 200 μM dNTP; 0.5 μM each primer of primer B (5′-AGG GAA CGA G-3′) (Martin et al., 2004) and primer C (5′-AAGACGCCG T-3′) (Rodriguez-Calleja et al., 2006); 50 ng DNA template; and 4.0 U Taq polymerase (Fermentas). The thermal conditions, electrophoresis procedure, dendrogram creation, and the cluster analysis were described in “ERIC-PCR for S. aureus” section.
Results and Discussion
In the present study, 35 S. aureus isolates were characterized by ERIC-PCR fingerprinting for the first time, as well as antibiotics susceptibility testing, and RAPD-PCR. Four resistant types were observed with D of 0.826 (Fig. 1A, B). Antibiotics susceptibility testing indicated that five isolates (SA1, SA2, SA3, SA4, and SA5) were resistant to oxacillin and penicillin, six isolates (SA19, SA23, SA26, SA29, SA30, and SA32) were found to be resistant to tetracycline.
ERIC-PCR fingerprinting and resistance patterns of 35 S. aureus isolates
ERIC-PCR assigned 35 S. aureus isolates into 28 ERIC types with discrimination indexes (D) of 0.984 (Fig. 1A). Four isolates (SA1, SA2, SA3, and SA4) from skin of boiled dumping and SA34 from a worker's hand were grouped into V cluster. Cluster III collected seven isolates (SA6–SA12), two of which (SA11 and SA12) were from workers' hands and rice glue balls. Meanwhile, two isolates from mushroom stuffing were assigned to cluster I. Four isolates resistant to oxacillin and penicillin were grouped into the same cluster, while six isolates exhibiting tetracycline resistance were assigned to cluster IV by ERIC-PCR, which was supported in Salmonella strains resistant to the same antibiotic showing similar ERIC-PCR types (Ye et al., 2011). Compared with ERIC-PCR, RAPD-PCR has relatively low discriminatory ability and assigned 19 different patterns into five clusters (A–E) with D value of 0.949 (Fig. 1B). Four S. aureus isolates (SA1–SA4) from skin of boiled dumping and SA5 from a worker's hand were found to have identical patterns. Likewise, five S. aureus isolates from mushroom stuffing (SA16, SA17, SA18, SA20, and SA21) were allocated to cluster B. It is notable that SA9 and SA10 (cluster B) from meat stuffing have an identical pattern exhibited in SA11 and SA12 isolates (B) as well as in SA13 and SA14 (D). In the previous publication, some Salmonella strains from the same location have similar molecular patterns described by Ye et al. (2011). On the other hand, four of five isolates from gingili stuffing were grouped into cluster D, five isolates (SA1, SA2, SA3, SA4, and SA5) resistant to oxacillin and penicillin were categorized into cluster A, and the other six isolates resistant to tetracycline were distributed to different clusters by RAPD-PCR. In addition, a clear correlation between ERIC-PCR and RAPD-PCR or resistance patterns in some strains such as SA1–SA4 indicate that their combination might provide useful and reliable information of S. aureus strains for tracing origins of S. aureus from food chain.
Conclusion
Thirty-five of S. aureus were characterized using antibiotic susceptibility testing, ERIC-PCR, and RAPD-PCR, which show diversity of S. aureus with D index of 0.826, 0.984, and 0.949, respectively. A comparison of characterization from these three methods indicated that ERIC-PCR method could be used for genetic diversity of S. aureus.
Footnotes
Acknowledgments
We acknowledge the financing support by Dr. start-up funding of Hefei University of Technology (No. GDBJ-036407) and Guangdong Project of Science and Technology (2008A040101001, 2009B040500001).
Disclosure Statement
No competing financial interests exist.