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Horizontal transfer of antibiotic resistance has been drawing more and more attention due to its critical role in the emergence and dissemination of antibiotic-resistant bacteria in hospitals and the food animal production environment. However, most studies on horizontal gene transfer in nature have been focused on conjugation and transformation, whereas much less data are available on phage-mediated transfer.
Phage genomes and/or phage remnants are common in the genome of most Salmonella (Porwollik, Microbes and Infection, 2003, 5: 977). About 95% of strains of S. Typhimurium examined to date contain complete inducible prophage genomes and 99% of these phages were capable of generalized transduction of chromosomal host markers and plasmids (Schicklmaier, Antonie van Leeuwenhoek, 1998, 73: 49). Schmieger et al. (Schmieger, FEMS Microbiology Letters, 1999, 170: 251) has reported that antibiotic resistance genes of S. Typhimurium DT104 isolates can be efficiently transduced by phage released from the DT104 isolates. However, the extent to which transduction contributes to the dissemination of antibiotic resistance among Salmonella serovars other than Typhimurium is unknown.
This problem is further complicated by the fact that treatment with antibiotics, such as those used for treatment of sick animals or enhance growth promotion may actually induce phage, causing lysogenized prophage to be released from their chromosomal location, start a lytic cycle of growth and potentially enhance the probability of the dissemination of antibiotic resistance markers.
The stated objectives for this work were:
A total of 46 Salmonella isolates of bovine origin were selected from the Salmonella isolate collection in the LeJeune’s Laboratory. Salmonella serovars were determined at the U.S. Department of Agriculture National Veterinary Service Laboratories in Ames, Iowa.
Phage Induction and Host Range Determination
Phages were induced from their host bacteria with 2μg/ml of mitomycin C as an inducing agent and then tested for their host range by spot lysis assay. A 10μl-aliquot of phage lysate was dropped on Tryptic Soy Agar which was seeded by top agar (TSB with 0.5% of agar) with the following indicator strains: ATCC 13311, ATCC BAA-712, MZ1260, MZ1261, MZ1262, and MZ1263. Those isolates that did not release detectable phages on the 6 indicator strains were further screened on 10 additional Salmonella, individually, for inducible phages.
A volume of 200μl of the induced phage was mixed with 200μl of the overnight culture of recipient strain (S. Typhimurium MZ1262, susceptible to all antibiotics tested) followed by 15min incubation. The mixture was added to 3ml of top agar and poured onto a membrane filter placed on an antibiotic-free TSA plate and incubated for 2h to allow the antibiotic resistance phenotype to develop. The membrane was then transferred to the TSA supplemented with ampicillin (32μg/ml) or tetracycline (16μg/ml). Transduction frequency was calculated as the number of transductants per input PFU.
Antibiotic Susceptibility Testing
Antibiotic MICs for the 46 Salmonella isolates and transductants were determined by using the Sensititre automated antimicrobial susceptibility system (Trek Diagnostic Systems, Westlake, Ohio) and interpreted according to Clinical and Laboratory Standards Institute (CLSI) standards for broth microdilution methods. E. coli ATCC 25922 and E. coli ATCC 35218 were used as quality control organisms.
Determination of antibiotic resistance mechanisms
PCR was performed to determine the molecular basis of ampicillin and tetracycline resistance. DNA template for PCR was made by boiling overnight culture of the Salmonella isolates and transductants. The target genes were blaCMY-2, blaTEM-1, tet-A, and tet-B. The temperature profile for each pair of primers was: 95°C for 10 min, followed by 30 cycles of 95°C for 30 s, 55°C for 1 min, and 72°C for 1 min, and a final step of 72°C for 7 min (blaCMY-2 and blaTEM-1); 95°C for 5 min, followed by 40 cycles of 95°C for 1min, 55°C for 30sec, and 72°C for 30sec, and a final step of 72°C for 7 min (tet-A, and tet-B).
Twelve serovars were identified among 46 Salmonella of bovine-origin. Transducing phages were detected in 28 of 46 (61%) isolates using 6 standard indicator strains of Salmonella and 10 additional Salmonella from the strain collection. The findings that all 14 S. Typhimurium released phages that could be detected in a plaque assay and that an overall 61% of the isolates tested liberated phages suggest a high percentage of natural Salmonella of bovine origin contained inducible phages on their chromosomal backbone. Considering that horizontal gene transfer is of great importance in the spread of antibiotic resistance genes and virulence, it is reasonable to assume that phage transduction may play an important role in antibiotic resistance transfer among natural Salmonella. This assumption is also partly supported by the observation that inducible phages were detected in 84% of antimicrobial-resistant isolates versus 44% of non-resistant ones, which suggests a potential linkage between lysogenic bacteria and antibiotic resistance phenotype in Salmonella.
A total of 11 patterns of phage host range profiles on the 6 standard indicator strains were identified among the 24 phages. Eighteen of 24 phage samples could multiply in two or more standard indicator strains. Four more phage-releasing Salmonella were detected when additional 10 indicator strains were included for detection of lysogenic Salmonella among the 22 phages that failed to form detectable plaques on the standard indicator strains. One phage released from S. Montevideo showed a host range of seven Salmonlla, including serovars Cerro, Hartford, Heidelberg, Kentucky, Senftenberg, and Typhimurium. Despite that phages are always restricted to the same bacterial species or even strains that they are isolated from, our study identified 21 of 28 phage samples that were able to grow on two or more Salmonella of various serovars, demonstrating a broad host range of these isolated phages, which may, therefore, facilitate the genetic exchange among Salmonella in the environment.
Since all the six standard indicator strains used were susceptible to all the antibiotics tested, they were not appropriate to serve as transduction donor strains for the purpose of antibiotic resistance transfer. Thus, the 24 released phages detected by the standard indicator strains were tested for propagation on the antibiotic-resistant Salmonella in the collection to screen for a potential donor for transduction assay. The phage titre should reach 1012 phage performing units (PFU)/ml suitable for transduction. One phage released from isolate S32 (S. Typhimurium) was shown to be able to transduce antiobiotic resistance markers from an MDR isolate S33 (S. Heidelberg) to a pan-susceptible standard strain of MZ1262 (S. Typhimurium). The transduction resulted in independent acquisition of blaCMY-2, tet-A and tet-B and the transduction frequency was about 10-9 Transductants/PFU for both ampicillin and tetracycline resistance.
Since phage is highly specific to its bacterial host, what causes concern is the cross-serovar transfer of antibiotic resistance from S. Heidelberg to S. Typhimurium via transduction. Food animal production environment is the reservoir of diverse microorganisms, including Salmonella of various serovars, which determines an important role played by phage in the exchange of genetic materials among different strains of Salmonella serovars. The cross-serovar event may also explain the relatively lower transduction frequency of phage P24 as there may be a recombination barrier caused by slight differences between the two recombining DNA sequences in S. Heidelberg and S. Typhimurium and the mismatch repair system from the recipient may inhibit the integration of the donor DNA into the recipient chromosome.
Wild-type phage is common among Salmonella of bovine origin. Many phages have broad host ranges and are able to multiply in various serovars of Salmonella, which may facilitate the genetic exchange among Salmonella in the environment. β-lactam resistance and tetracycline resistance can be transduced from S. Heidelberg to S. Typhimurium via wild-type phage transduction. Phage-mediated antibiotic resistance transfer may contribute to the emergence and dissemination of antibiotic-resistant Salmonella in the food production environment.