Relative Distribution and Conservation of Genes Encoding Aminoglycoside-Modifying Enzymes in Salmonella enterica Serotype Typhimurium Phage Type DT104

ABSTRACT PCR was used to identify genes encoding aminoglycoside-modifying enzymes in 422 veterinary isolates of Salmonella entericaserotype Typhimurium. The identities of extra-integron genes encoding resistance to streptomycin, gentamicin, kanamycin, and apramycin were evaluated. Gentamicin resistance was conferred by theaadB gene. Kanamycin resistance was encoded by either theaphA1-Iab gene or the Kn gene. Apramycin resistance was determined by the aacC4 gene. Analysis of gene distribution did not reveal significant differences with regard to phage type, host species, or region except for theKn gene, which was found mostly in nonclinical isolates. The data from this study indicate that pentaresistant DT104 does not acquire extra-integron genes in species- or geography-related foci, which supports the hypothesis that clonal expansion is the method of spread of this organism.

a The eastern, middle, and western regions are described in the text. b A total of 315, 54, 33, 13, and 10 isolates belonged to phage types DT104, U302, DT193, DT208, and DT120, respectively. c The states from which the isolates were obtained were not identified; 18 strains were from the FSIS, two strains were from foreign sources, and the origin of one strain was not recorded.
The isolates used in the survey were obtained from ruminant species, swine, avian species, companion animals (including horses), and nonclinical settings (Food Safety and Inspection Service [FSIS]). The phage types included DT104 (n ϭ 312), U302 (n ϭ 54), DT193 (n ϭ 33), DT208 (n ϭ 13), and DT120 (n ϭ 10). The isolates were submitted from 32 different states, and the isolate pool is summarized in Table 1. The resistance breakpoints for the antibiotics used were those established by the National Antimicrobial Susceptibility Monitoring Program (6) and were determined as previously described (3,4). DNA isolation was performed as previously described (5). PCR was performed with an automated thermocycler (Hybaid, Teddington, United Kingdom) as previously described (3) by using the primers shown in Table 2. All of the gentamicin-resistant isolates harbored the aadB gene that codes for 2-aminoglycoside acetyltransferase (accession number AF078527). Similarly, a single gene, aacC4, which encodes for the 6-N-aminoglycoside acetyltransferase (accession number AJ009820), conferred all of the apramycin resistance. Kanamycin resistance was con-ferred by either of two genes, aphA1-Iab (111 isolates) (accession number AF093572) or Kn (11 isolates) (accession number U66885); both of these genes code for an aminoglycoside 3Ј phosphotransferase product, although there is only 54% identity between the two genes.
The results of a chi-square analysis and multivariate logistic regression are shown in Table 3. No significant gene differences were identified with regard to host, geographic region, or phage type, except for the Kn gene. Of the 11 isolates found to contain the Kn gene, 8 were nonclinical in origin. Streptomycin resistance is explained in phage type DT104 by the presence of an integron containing the aadA2 gene (2). All non-DT104 phage types were tested for the presence of the DT104-specific integron. Only phage type DT208 isolates lacked this element, as determined by the cmlA-tetR amplicon (5), but all DT208 isolates contained the aadA2 gene ( Fig. 1). Geographic regions of the United States were arbitrarily classified as eastern (including Alabama, Connecticut, Delaware, Florida, Georgia, Indiana, Kentucky, Massachusett Maryland, Maine, Michigan,  The results of this study indicate that single genes are responsible for conferring gentamicin resistance and apramycin resistance, and no host, geographic (Fig. 2), or phage type differences were noted. These findings suggest two possibilities regarding gentamicin and apramycin resistance genes in DT104. First, although many genes can confer gentamicin or apramycin resistance, only a small subset of gentamicin or apramycin resistance genes or enzymes are compatible with vitality of pathogenic microorganisms. For example, the GϩC content or enzyme kinetics may dictate, and thus limit, potential acquisition candidates. Second, DT104 is abundantly exposed to microbes that possess and transfer these two specific genes. This seems less likely since intestinal microfloras are diverse and since the isolates were obtained from a wide array of hosts. We favor the former possibility since DT104 resistance genes can be found in fish pathogens (1,2,8,13), suggesting that certain resistance genes have a predilection for moving from bacterium to bacterium.
Two separate genes, however, conferred kanamycin resistance, although a single gene conferred kanamycin resistance in the clinical isolates. The Kn gene was much more likely to be found in nonclinical isolates, suggesting that its presence and/or expression is not compatible with overt virulence. This is consistent with our recent finding that certain kanamycinresistant DT104 isolates are less invasive and less pathogenic than the rest of the group (3). Alternatively, it is possible that the Kn gene is abundant in slaughterhouse environments and that its presence in the nonclinical isolates represents a postharvest transfer or acquisition event.
Recently, we reported that a DT104-related phage type (U302) and a phage type not related to DT104 (DT120) are capable of possessing the integrons found in DT104 (4). In this study we demonstrated that another phage type not related to DT104 (DT193) also has this capability. However, it appears that multiresistant DT208 does not contain the genetic array characterized for the ACSSuT pattern but does have the aadA2 gene typically associated with the DT104-specific inte-  gron. This suggests that DT208 may contain an uncharacterized integron harboring this gene and perhaps other antibiotic resistance genes. Our study, which was aimed at detecting integron-independent genes encoding aminoglycoside-modifying enzymes, did not reveal significant differences in the genes conferring gentamicin, kanamycin, and apramycin resistance in clinical isolates. This suggests that DT104 did not acquire these genes in a geography-or species-specific manner. Our data support the theory of clonal dissemination of DT104, although it is possible that the overwhelming prevalence of single aminoglycoside resistance genes is due to physiochemical compatibilities between DT104 metabolic properties and aminoglycoside-modifying enzymes. Our results should be compared to the results of similar studies performed in the future and similar studies performed with human isolates. The results should provide an understanding of gene acquisition and the mode of spread of multiresistant DT104 in order to provide insight into the emergence of DT104 as an important health concern worldwide.