Presence of class I integrons in multidrug-resistant, low-prevalence Salmonella serotypes, Italy.

In 1997 to 1999, we detected class I integrons in multidrug-resistant isolates of Salmonella enterica serovars Anatum, Blockley, Brandenburg, Bredeney, Derby, Heidelberg, Livingstone, Newport, Ohio, Panama, Paratyphi B, Saintpaul, Sandiego, and Stanley.


The Study
Seventy-four strains of Salmonella (of serotypes other than S. Enteritidis and S. Typhimurium) resistant to three or more antibacterial drugs were identified from January 1997 to December 1999. Isolates were from human and nonhuman sources. Sixty-two isolates were available for further investigation. Identification was performed by the API 20E system (Biomerieux, Marcy l'Etoile, France) and serotyping (11) by commercially obtained antisera (Sanofi Diagnostics Pasteur, Marnes-La Coquette, France).
The rifampin-resistant strain of Escherichia coli K12J5 Rif r was used as the recipient in conjugation experiments (13). Transconjugants were selected on Luria-Bertani agar containing 250 µg/mL of rifampin plus 50 µg/mL of ampicillin or sulfonamides or 30 µg/mL of chloramphenicol, streptomycin, tetracycline, or trimethoprim.
Screening of isolates for presence of class I integrons was performed by a high-stringency protocol with oligonucleotide primers specific for the sequence of the published 5'-CS and 3'-CS regions adjacent to the site-specific recombinational insertion sequence (15). Primer sequences were: 5'-CS, GGCATCCAAG-CAGCAAG and 3'-CS, AAGCAGACTTGACCTGA (15).
Further polymerase chain reaction (PCR) analysis was performed on the 26 isolates harboring class I integrons to better characterize the antibiotic resistance genes associated with the integron structure. This was done by using primers located at the beginning extremities of the inserted resistance genes in combination with that specific for the 5'-CS conserved segment. The following sequences were tested: sulfonamide resistance gene sulI; beta-lactam resistance genes oxa2, pse1, and tem; aminoglycoside resistance genes aac(3)-Ia, aac(3)-IIa, aac(6')-Ib, ant(3")-Ia, aadA2 [also named ant(3")-Ib]; and trimethoprim resistance gene dhfrI (15). The presence of the pasppflo-like (flor) and tetG genes, conferring resistance to chloramphenicol, florfenicol, and tetracycline in MDR-DT104, was also investigated by using PCR primers specific for these sequences (16).
From 1997 to 1999, 18 Salmonella serotypes were identified, including isolates resistant to three or more antibacterial drugs: Anatum, Blockley, Brandenburg, Bredeney, Derby, Hadar, Heidelberg, Livingstone, Muenchen, Newport, Ohio, Panama, Paratyphi B, Saintpaul, Sandiego, Stanley, Thompson, and Virchow. Seventy-four multidrugresistant isolates were identified, which accounted for 10.0% of the strains belonging to the serotypes under study. The proportion of isolates with a pattern of resistance to three or more drugs is summarized (Table 1); 26 isolates belonging to 14 serotypes contained class I integrons (Table 2). Screening for the presence of plasmid DNA detected no plasmids in 14 strains and plasmids, between 35 and 140 megadaltons in size, in the remaining 12. Three isolates of serotype Brandenburg clustered as an epidemic, according to epidemiologic data and shared identical plasmid DNA and integron profiles. Transfer of plasmids was associated with transmission to E. coli of the complete or partial resistance pattern ( Table 2). In all but one case, PCR analysis with the 5'CS and 3'CS primers confirmed the presence of integrons in the recipient cells.
Heterogeneous integron-associated resistance genes were present in the isolates under study, despite the extensive similarities of the antibiotic resistance phenotypes (Table 3). Strains belonging to serotypes Ohio, Panama, and Saintpaul carried the ant(3")-Ia and pseI gene cassettes previously described in two different chromosome-located integrons in MDR-DT104 but inserted in a single integron transferable by conjugation. The integron-associated aminoglycoside resistance genes aac(3)-IIa and aac(6')-Ib were not detected in the strains tested.
Both tetG and flor resistance determinants, known to characterize MDR-DT104 strains (16), were found in one strain of Paratyphi B isolated from tropical fish imported from Singapore.

Conclusions
The emergence of multidrug resistance in Salmonella serotypes is causing growing concern because of the high potential of human involvement through food and animal contact. We have detected integrons in MDR-resistant isolates of Salmonella identified in southern Italy in the last 3 years. Our findings confirmed not only that integrons are not confined to S. Typhimurium DT104 but also that they can be found in many less-prevalent serotypes with extensive reservoirs, encompassing animal species (swine, poultry, domestic pets) and environmental sites (rivers, sewage effluents). A further concern is the presumed location of integrons on the chromosome, detected in isolates of nine different serotypes. This resistance gene location has proved to be very efficient in acquiring and establishing resistance traits and in supporting spread of S. Typhimurium DT104 through the food chain in western countries (17). We also recognized in different serotypes a pattern of resistance similar to the five-drug pattern typical of DT104, a phenomenon reported by Glynn et al. (10). The heterogeneous distribution and organization of resistance genes within several low-prevalence serotypes of Salmonella suggest the possible emergence of MDR-DT104-like patterns in serotypes other than S. Typhimurium that share a similar selective pressure because of intensive use of antimicrobial agents in farming. Moreover, tetG and flor resistance sequences in one S. Paratyphi B isolate from Singapore tropical fish suggest that the use of antimicrobial agents in aquaculture in Asia is contributing to the emergence and spread of multidrug resistance within fish pathogens and, subsequently, MDR-DT 104 strains (18).
The association between emergence of MDR Salmonella strains and excessive use of antibiotics in animal husbandry (as growth promoters and for disease prevention and therapy) is receiving increasing attention in developed countries. The presence of integrons in zoonotic serotypes such as Blockley, Brandenburg, Derby, or Saintpaul, which in southern Italy are epidemiologically linked to farming practices, underscores the public health problem of antibiotic resistance diffusion.
Surveillance and monitoring of antimicrobial-drug resistance, including screening for class I integrons as likely indicators of evolution of drug resistance mechanisms and acquisition of new resistance traits, are necessary steps in planning effective strategies for containing this phenomenon within foodborne infectious organisms.
Dr. Nastasi is professor of hygiene at the Department of Public Health of the University of Florence, Italy. His research interests include epidemiology and surveillance of infectious diseases.
Dr. Mammina is a physician at the Department of Hygiene and Microbiology of the University of Palermo, Italy. Her work focuses on epidemiologic investigation of infectious diseases by molecular typing.