Antimicrobial Resistance in Nontyphoidal Salmonella

Transcription

1 780 Journal of Food Protection, Vol. 70, No. 3, 2007, Pages Copyright, International Association for Food Protection Review Antimicrobial Resistance in Nontyphoidal Salmonella SAMUEL D. ALCAINE, 1 LORIN D. WARNICK, 2 AND MARTIN WIEDMANN 1 * 1 Department of Food Science and 2 Department of Population Medicine and Diagnostic Sciences, Cornell University, Ithaca, New York 14853, USA MS : Received 17 May 2006/Accepted 20 October 2006 ABSTRACT Salmonella is one of the leading causes of foodborne illness in countries around the world. Treatment of Salmonella infections, in both animals and humans has become more difficult with the emergence of multidrug-resistant (MDR) Salmonella strains. Foodborne infections and outbreaks with MDR Salmonella are also increasingly reported. To better monitor and control the spread of MDR Salmonella, it is important to understand the mechanisms responsible for drug resistance and how drug resistance is transmitted to and between Salmonella strains. This review summarizes current knowledge on antimicrobial drugs used to treat Salmonella infections and provides an overview of MDR Salmonella in the United States and a discussion of the genetics of Salmonella drug resistance, including the mechanisms responsible for the transmission of drug-resistance genes in Salmonella, using data from the United States and other countries. Salmonella is a gram-negative bacillus that causes infections in a large number of avian, mammalian, and reptilian species. The genus Salmonella includes the species Salmonella bongori and Salmonella enterica, which is divided into six subspecies: I, II, IIIa, IIIb, IV, and VI (30). Subspecies I represents roughly 99% of all reported human isolates in the United States (30). Salmonella isolates are traditionally classified by serotype, which is based on the O and H antigen immunoreactivity of an isolate. More than 2,500 Salmonella serotypes have been identified, and the 20 most common human serotypes represented 78% of all human Salmonella isolates reported in the United States in 2003 (30). Salmonella infections are typically contracted through the consumption of contaminated food, water, or through contact with an infected host. Salmonella is one of the leading causes of foodborne illness in the United States and the European Union (EU) (46, 79), with estimated incidences of 15.1 cases per 100,000 persons in the United States (8) and 42.2 cases per 100,000 persons in the EU (46). Most Salmonella infections do not require treatment and result in temporary gastroenteritis (92). In invasive life-threatening infections, the use of antimicrobial drugs is required (108), but the efficacy of many of these drugs is decreasing as antimicrobial-resistant Salmonella subtypes emerge (6, 116, 125). The emergence and spread of antimicrobial-resistant Salmonella strains, particularly those that are resistant to multiple antimicrobial drugs (i.e., multidrug-resistant [MDR] Salmonella), is a major public health concern. The goal of this review is to provide an overview of the importance of MDR Salmonella in the United States, with a focus on the mechanisms of drug resistance and the occur- * Author for correspondence. Tel: ; Fax: ; mw16@cornell.edu. rence and transmission of drug resistance genes among members of the genus Salmonella, based on data from the United States and other countries. ANTIMICROBIAL RESISTANCE DEFINITIONS AND OVERVIEW Resistance to an antimicrobial drug implies that the drug is not efficacious for treatment of clinical disease caused by a particular bacterial pathogen. Our focus in this review is on acquired resistance, where genetic changes in a bacterium result in resistance to a previously effective drug. Laboratory measurements of antimicrobial drug susceptibility are used for prediction of clinical efficacy and for phenotypic characterization of bacterial isolates. Susceptibility most often is measured by either broth dilution methods or inhibition zones from disk diffusion assays. Interpretive criteria typically are used to categorize isolates as susceptible, intermediate, or resistant based on broth dilution MICs or inhibition zone diameters. Interpretive criteria are established using available pharmacokinetic, pharmacodynamic, and clinical efficacy data and population MIC distributions. Resistance should be used to designate isolates that are not inhibited by usually achievable systemic concentration of the agent with normal dosage schedules and/or fall in the range where specific antimicrobial resistance mechanisms are likely (e.g. beta-lactamases) and clinical efficacy has not been reliable in treatment studies (89). Validated interpretive criteria are not available for all clinically important bacteria, drug, and host combinations. Data are particularly scarce for certain bacteria and drugs important in veterinary medicine. Veterinary interpretive criteria for Salmonella are extrapolated for most drugs from human data (90). For some antimicrobial drugs (e.g., ceftiofur), breakpoints established for other pathogens are

2 J. Food Prot., Vol. 70, No. 3 ANTIMICROBIAL RESISTANCE IN SALMONELLA 781 widely used in reporting Salmonella susceptibility results, even though breakpoints have not been validated specifically for Salmonella (29). Literature related to Salmonella antimicrobial susceptibility often does not clearly define what is meant by resistance. In our review, we rely on the authors interpretation of resistance, recognizing that the term may encompass a range of meanings, including (i) a relative decrease in susceptibility to a drug compared with population distributions, (ii) identification of a known resistance mechanism, (iii) exceeding a widely used, though not validated, interpretive criterion, or (iv) classification of resistance based on clinically validated breakpoints. HUMAN SALMONELLA INFECTIONS WITH DRUG-RESISTANT STRAINS The rise of MDR Salmonella poses an increasing health risk to human populations (116). Of the 10 serotypes most commonly reported by the Centers for Disease Control and Prevention (CDC) as isolated from human infections in the United States (30), 8 include at least some isolates that showed resistance to five or more antimicrobial drugs (29). Salmonella serotypes Typhimurium, Heidelberg, and Newport most commonly exhibited multidrug resistance (29). MDR Salmonella Typhimurium isolates commonly had one of two resistance patterns, including (i) resistance to ampicillin, kanamycin, streptomycin, sulfamethoxazole, and tetracycline or (ii) resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline, the resistance type typically associated with Salmonella Typhimurium DT104 (29). Salmonella Heidelberg isolates most commonly displayed resistance to ampicillin, amoxicillin clavulanic acid, ceftiofur, and cephalothin (29). Among Salmonella Newport isolates, the most commonly encountered resistance type is referred to as MDR-AmpC, which indicates resistance to ampicillin, chloramphenicol, amoxicillin clavulanic acid, ceftiofur, cephalothin, streptomycin, sulfamethoxazole, and tetracycline and reduced susceptibility to ceftriaxone (29). From 1993 to 2003, the CDC recorded a 160% increase in the number of Salmonella Newport isolates reported (30). In 2002, 22% of these isolates tested by the National Antimicrobial Resistance Monitoring System displayed multidrug resistance (29); 27% of Salmonella Typhimurium isolates and 8% of Salmonella Heidelberg isolates also displayed multidrug resistance (29). MDR strains have also been identified among other Salmonella serotypes of epidemiological importance, such as Agona, Dublin, Hadar, and Senftenberg (29). Although the overall incidence of human clinical Salmonella isolates that are resistant to one or more drug classes declined from 1996 to 2003 in the United States (30), infections by drugresistant Salmonella strains pose potential treatment difficulties, and recent evidence indicates that human infections with drug-resistant Salmonella strains may be more severe than those caused by sensitive strains (62). The human health impact of drug-resistant Salmonella strains is also evident in the high number of human salmonellosis outbreaks caused by MDR Salmonella strains (Table 1) (7, 9, , 116, 128). These outbreaks have been caused by the consumption of contaminated foods or have been linked to direct contact with infected animals. Isolates obtained during a 2000 Salmonella Typhimurium outbreak in New Jersey, which was linked to consumption of contaminated milk, were resistant to five antimicrobial drugs: ampicillin, kanamycin, streptomycin, sulfamethoxazole, and tetracycline (95). In 2002, an outbreak of Salmonella Newport MDR-AmpC infection related to the consumption of contaminated beef products affected 47 people in five U.S. states (7). The three isolates from this outbreak that were tested were resistant to nine antimicrobial drugs: amoxicillin-clavulanate, ampicillin, cefoxitin, ceftiofur, cephalothin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline; two isolates also displayed kanamycin resistance and reduced sensitivity to ceftriaxone. A series of outbreaks of Salmonella Typhimurium DT120 infection that occurred from December 2003 to late 2004 was linked to handling of pet rodents (9). These isolates were resistant to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline. MDR Salmonella strains thus clearly represent a health risk to the U.S. population. New methods for surveillance and control of MDR Salmonella strains are necessary to prevent future outbreaks or at least to minimize their impact. ANTIMICROBIAL RESISTANCE MECHANISMS IN SALMONELLA The following portion of this review is separated into sections that cover the main classes of antimicrobial drugs to which Salmonella isolates have shown resistance. Within each section, we describe the mode of action of the antimicrobial drug, the mechanisms of resistance found in Salmonella, and the occurrence of resistance to a given antimicrobial drug class in strains of Salmonella. Table 2 contains a summary, organized by antimicrobial drug classes, of genes that commonly encode antimicrobial resistance and Salmonella serotypes in which resistance genes commonly have been found. Aminoglycosides. Aminoglycosides were first discovered in 1943, when streptomycin was isolated from Streptomyces griseus (55). Other aminoglycosides are neomycin, amikacin, and gentamicin (55). Based on their chemical structure, aminoglycosides are divided into four classes: (i) streptidines, a class containing streptomycin and dihydrostreptomycin; (ii) streptamines, whose only clinically significant member is spectinomycin; (iii) 4,5-distributed 2- deoxystreptamines, whose clinical efficacy is limited by their toxicity and that are represented in clinical use only by neomycin; and (iv) 4,6-disubstituted 2-deoxystreptamines, represented by kanamycin, gentamicin, amikacin, and tobramycin (78). Aminoglycosides are effective for treating infections caused by gram-negative bacilli and usually are used in combination with other antimicrobial drugs to ensure a broad spectrum of action (55). These drugs bind to conserved sequences within the 16S rrna of the 30S ribosomal subunit (78), and this binding leads to codon misreading and translation inhibition. Most aminoglycosides are bactericidal, with the exception of spectinomycin, which has a bacteriostatic mode of action (78). Although

3 782 ALCAINE ET AL. J. Food Prot., Vol. 70, No. 3 TABLE 1. Selected human outbreaks caused by multidrug-resistant Salmonella strains Salmonella Implicated serotype Resistance a vehicle Location Year Reference Blockley CKST and KSTN Unknown Greece Heidelberg KST Stuffed ham Maryland Newport ACKSSuT Ground beef California Newport MDR-AmpC Farm exposure Massachusetts Newport MDR-AmpC Ground beef Connecticut, New York, Michigan, Pennsylvania, Ohio Stanley KTB Alfalfa sprouts Arizona Typhimurium AckSSuT Raw milk Arizona Typhimurium ACKSSuT Contact with infected Idaho animals Typhimurium ACSSuT Cheese Washington Typhimurium ACSSuT Raw milk Vermont Typhimurium ACSSuT Handling of rodents Georgia, Illinois, Kentucky, Michigan, Minnesota, Missouri, New Jersey, North Carolina, Pennsylvania, South Carolina Typhimurium AKSSuT Milk Illinois Typhimuium AKSSuT Farm exposure Ohio Typhimurium AKSSuT Milk Pennsylvania, New Jersey Typhimurium TS Salad bar Oregon Typhimurium DT104 ACSSuT quinolone Pork Denmark (reduced susceptibility) Typhimurium DT104 ACSSuT Contact with infected Minnesota, Washington animals Typhimurium DT104 ACSSuT Prepared lunch boxes Japan a A, ampicillin; C, chloramphenicol; K, kanamycin; N, nalidixic acid; S, streptomycin; Su, sulfamethoxazole; T, tetracycline; B, trimethoprim-sulfamethoxazole. MDR-AmpC, ACSSuT amoxicillin clavulanic acid, cephalothin, cefoxitin, and ceftiofur resistance and reduced susceptibility to ceftriaxone. Salmonella isolates may appear susceptible to aminoglycosides in vitro, the NCCLS (now the Clinical and Laboratory Standards Institute [CLSI]) has cautioned that this antimicrobial drug class is not clinically effective against Salmonella (88). Primary mechanisms for bacterial resistance to aminoglycosides are (i) decreased drug uptake, (ii) drug modification, and (iii) modification of the ribosomal target of the drug (108). Although efflux pumps, which remove antimicrobial drugs from the bacterial cell, play a role in aminoglycoside resistance in other enterobacteria such as Escherichia coli (2, 104) and play a role in the resistance of Salmonella to other antimicrobial drugs, this mechanism has yet to be associated with aminoglycoside resistance in Salmonella. Ribosomal modification also has not been reported as a mechanism in naturally occurring Salmonella aminoglycoside resistance but has been identified after selection experiments in the laboratory (18). Aminoglycoside resistance in Salmonella is generally associated with the expression of aminoglycoside-modifying enzymes (52, 58). These enzymes fall into three groups that are named according to the types of reactions they catalyze: acetyltransferases, phosphotransferases, and nucleotidyltransferases. A comprehensive review of aminoglycoside resistance mechanisms was provided by Shaw et al. (108). Aminoglycoside acetyltransferases are enzymes that primarily modify aminoglycoside amino groups (78, 108). These enzymes are subdivided into four groups, AAC(1), AAC(2 ), AAC(3), and AAC(6 ), based on the location in the aminoglycoside molecule they modify (78). Genes encoding these enzymes are typically designated aac (114); these genes have been found as part of Salmonella genomic islands (43), integrons (59, 71, 97), and plasmids (59). Many aac variants have been identified in a wide range of Salmonella serotypes, including Newport (43), Agona (86), Typhimurium (32), Typhimurium var. Copenhagen (49), Kentucky (71), and 4,5,12:i: (59). Aminoglycoside acetyltransferases provide resistance to gentamicin, tobramycin, and kanamycin (78). Aminoglycoside phosphotransferases are enzymes that catalyze ATP-dependent phosphorylation of specific aminoglycoside hydroxyl groups (78, 108). This enzyme group is divided into subgroups whose classification depends on the specific site of phosphorylation. Genes encoding subgroup APH(3 ) and APH(6) enzymes, both of which provide resistance to streptomycin (78), have been found on plasmids harbored by Salmonella isolates (52). Most genes encoding these enzymes are designated as aph (114), although the genes aph(3 )-Ib and aph(6)-id are commonly referred in the literature as stra and strb, respectively (75, 108). Genes from both families have been found in Salmonella serotypes Agona, Anatum, Blockely, Bredeney, Derby, Give, Hadar, Heidelberg, London, Saintpaul, and Typhimurium (75, 98). Genes encoding enzymes of the

4 J. Food Prot., Vol. 70, No. 3 ANTIMICROBIAL RESISTANCE IN SALMONELLA 783 TABLE 2. Characteristics of resistance to different antimicrobial drugs in Salmonella Antimicrobial drug class Mode of action Common resistance genes Salmonella serotypes References Aminoglycosides Inhibits protein synthesis aac(3)-iv, aac(3)-iva, aacc2, stra, strb, aph(3 )-IIA, aada1, aada2, aadb Beta-lactams Inhibits cell wall formation bla CMY-2, bla CTX-M9, bla TEM-1, bla TEM-53, bla CARB2, bla OXA-30 4,5,12:i:, Agona, Anatum, Blockley, Bredeney, Derby, Give, Hadar, Heidelberg, Kentucky, London, Infantis, Saintpaul, Newport, Typhimurium Anatum, Agona, Blockley, Dublin, Enteritidis, Haardt, Muenchen, Newport, Stanley, Typhimurium, Virchow Chloramphenicol Inhibits protein synthesis cat1, cat2, cmla, flor Albany, Agona, Derby, Enteritidis, Haardy, Kiambo, Newport, Typhimurium 30, 42, 47, 57, 68, 71, 80, 93 6, 9, 12, 30, 40, 51, 56, 106, 111, 113, 120 6, 17, 23, 30, 40, 41, 56, 76 Quinolones Inhibits topoisomerase gyra, gyrb, parc a Enteritidis, Typhimurium 13, 27, 59, 69 Tetracyclines Inhibits protein synthesis tet(a), tet(b) Agona, Anatum, Blockley, Bredeney, Colorado, Derby, Dublin, Enteritidis, Give, Haardt, Hadar, Heidelberg, Infantis, Orion, Senftenberg, Typhimurium 31, 48, 56, 93 Sulfonamides Inhibits dihydropteroate synthetase sul1, sul2, sul3 a Resistance is mediated by point mutations in these genes. 4,5,12:i:, Agona, Albany, Anatum, Brandenburg, Derby, Djugu, Enteritidis, Hadar, Heidelberg, Orion, Rissen, Typhimurium 10, 30, 41, 42, 54 APH(3 ) subgroup provide resistance to kanamycin and neomycin (78) and have been found in several Salmonella serotypes such as Derby (32), Haardt (32), Enteritidis (32), Typhimurium (52), and Typhimurium var. Copenhagen (49). The final group of enzymes providing aminoglycoside resistance are nucleotidyltransferases (78, 108). These enzymes also target the hydroxyl groups and are divided into several subgroups based on the site of modification. Genes encoding nucleotidyltransferases are usually designated aad (for aminoglycoside adenylyltransferase) (114), although some are also designated as ant (for aminoglycoside nucleotidyltransferase). The aada gene, which has also been referred to as ant(3 ) (108), provides streptomycin resistance in Salmonella isolates (78). There are several variants of this gene, and they have been found in Salmonella serotypes Agona, Anatum, Bredeney, Derby, Enteritidis, Give, Heidelberg, Saint Paul, and Typhimurium (32, 75, 98). The aadb gene, also known as ant(2 )-Ia (108), confers resistance to gentamicin and tobramycin (78). It has been found in Salmonella serotypes Typhimurium and Typhimurium var. Copenhagen (27, 48, 49). Both aada and aadb have been found in integronborne gene cassettes (27, 99, 126). Beta-lactams. There are three major groups of betalactams: penicillins, cephalosporins, and carbapenems. The antimicrobial effects of these drugs are mediated by their ability to interfere with a group of proteins known as penicillin-binding proteins. These proteins are involved in the synthesis of peptidoglycan, an essential component of the bacterial cell wall. Beta-lactams are generally considered bacteriocidal; however, the activity varies among beta-lactams, organisms, and target penicillin-binding proteins. In Salmonella and E. coli, inhibition of the essential penicillinbinding proteins (1 through 3) leads to bacteriocidal activity (96, 106). Possibly because of the widespread clinical use of penicillins, resistance to drugs such as ampicillin and methicillin has become common (6). In response to this problem, a second class of beta-lactams, the cephalosporins, was developed. Penicillins have a five-member thiazolidine ring fused to the beta-lactam ring, whereas cephalosporins have a six-member ring (a dihydrothiazine ring) fused to the beta-lactam ring (63). These changes provide cephalosporins with a broader range of activity and greater stability in the presence of beta-lactamases. There are four generations of cephalosporins, and each progressive generation is effective against a broader range of organisms (63). Although Salmonella isolates may appear susceptible to firstand second-generation cephalosporins in vitro, the CLSI cautions that this antimicrobial drug class may not be clinically effective against Salmonella (91). Cephalosporins have become popular antimicrobial drugs, but with increased use has come increased resistance. The latest group of beta-lactams to be discovered is the carbapenems, which have a five-member ring that does not contain sulfur fused to the four-member beta-lactam ring. These beta-lactams

5 784 ALCAINE ET AL. J. Food Prot., Vol. 70, No. 3 are sometimes paired with beta-lactamase inhibitors (78). Carbapenems have a much broader range of activity against both gram-negative and gram-positive bacteria than do other beta-lactams and are more stable against beta-lactamases (78). Their use is generally reserved for severe infections by MDR bacteria. Nevertheless, Salmonella isolates that possess resistance to carbapenems such as imipenem already have been reported (12, 83). A detailed account of the structure and function of beta-lactams was provided by Mascaretti (78). For beta-lactams to reach their penicillin-binding protein targets, they must first traverse the bacterial outer membrane. This passage is facilitated by two porins, OmpC and OmpF (65). Although porin loss or modification is an uncommon mechanism for beta-lactam resistance in Salmonella, cases have been documented where decreases in either OmpF (17) or OmpC (80) porin concentrations resulted in observable increases in resistance to beta-lactams such as ampicillin, cefoxitin, and other cephalosporins (cephalothin, ceftriaxone, and cefazolin). In one study on Salmonella envb mutants, which have reduced porin content, the reduction in OmpF and OmpD porin expression actually led to decreased resistance to most beta-lactams other than mecillinam and imipenem, although this decrease also may have been due to effects of the envb mutation on expression of proteins other than porins (96). The most common mechanism of resistance to betalactams in Salmonella is the secretion of beta-lactamases into the environment. These enzymes work by hydrolyzing the beta-lactam ring structure, yielding beta amino acids with no antimicrobial activity. The genes encoding for betalactamases produced by Salmonella are typically carried on plasmids, although most of these genes are chromosomally encoded in other bacterial species (16, 33, 41, 58, 66, 78, 125, 126). There are several different classification schemes for the characterization of beta-lactamases, but for the purposes of this review Ambler s classification scheme, which is based on primary structure and amino acid sequence identity, is used (5). In general, class A beta-lactamases are the most commonly reported class of beta-lactamases in Salmonella. They are plasmid encoded and provide a range of resistance against penicillins, early generation cephalosporins, and carbapenems. There are several different gene families encoding for enzymes in this class, and TEM is the most prevalent among Salmonella isolates. The gene bla TEM-1 has been found in isolates of Salmonella serotypes Enteritidis, Dublin, Haardt, Muenchen, and Typhimurium (32, 53). A variant, bla TEM-52, has been found in Salmonella serotypes Enteritidis, Blockley, Panama, and Typhimurium (113, 121, 129). Other class A beta-lactamase genes such as bla PSE-1, which is also known in the literature as bla CARB-2 (74), also have been found in a number of Salmonella serotypes (74, 122). Recently, a class A beta-lactamase gene, bla KPC-2, which provides resistance to imipenem, was found in a Salmonella Cubana isolate (83). The emergence of cefotaximases (CTX-M), which are class A beta-lactamases conferring resistance to ampicillin and cephalosporins, is an important trend to watch (13). Variants of bla CTX-M have been identified in isolates of Salmonella serotypes Anatum, Enteritidis, Stanley, Typhimurium, and Virchow from Europe (13, 123) and other continents, including Asia and Africa (20, 64). Class C beta-lactamases are another commonly reported class of beta-lactamases. These are typically encoded by chromosomal ampc genes and provide resistance against cephalosporins such as cefoxitin and ceftiofur. Salmonella has no chromosomal ampc gene; instead, these genes are harbored in plasmids (85, 125). Current research is primarily focused on the presence of bla CMY-2, which has been associated with resistance to ceftiofur (3, 125), an extended spectrum cephalosporin approved for use in domestic animals in the United States. The spread of bla CMY-2 is a public health concern because the presence of this gene appears to mediate resistance or at least reduced susceptibility to ceftriaxone, another extended-spectrum cephalosporin that is the drug of choice for the treatment of Salmonella infections in children (125). Several Salmonella serotypes such as Typhimurium, Agona, and Newport (3, 41) carry this gene. Other class C genes and CMY variants, such as bla CMY-4 (12) and bla CMY-7 (60), have been found but have not been as commonly reported in Salmonella. Class B corresponds to metallobeta-lactamases. These enzymes provide resistance to all beta-lactam antibiotics, including carbapenems such as imipenem (78). Metallobeta-lactamases are usually chromosomally encoded, although plasmid-mediated class B beta-lactamases, such as IMP-1 and VIM-1, exist (78). Class B beta-lactamases are not commonly found in Salmonella. Class D beta-lactamases appear to be uncommon among Salmonella isolates. This class of enzymes provides resistance to lactams closely related to oxacillin, such as cloxacillin and methicillin. The gene bla OXA-1 was found in a Salmonella Paratyphi isolate (25) and bla OXA-30 has been found in isolates of Salmonella serotypes Muenchen and Typhimurium (10, 53, 60). Phenicols. Phenicols include chloramphenicol and florfenicol. Chloramphenicol was once the drug of choice for the treatment of typhoid fever (78). Production of chloramphenicols by Streptomyces venezuelae was discovered in Chloramphenicol works by binding to the peptidyltransferase center of the 50S ribosomal unit, thus preventing formation of peptide bonds (78). Chloramphenicol s broad range activity against gram-positive and gram-negative bacteria and its ability to cross the blood-brain barrier make it a powerful choice for the treatment of systemic infections. Its toxicity, which can lead to bone marrow damage and aplastic anemia (78, 130), and widespread resistance have generally limited chloramphenicol use to occasions where the risk of the infection, such as bacterial meningitis, is greater than the risk of adverse effects from the drug (78). Chloramphenicol is still widely used in developing countries because of its low cost (44). Chloramphenicol resistance in Salmonella isolates is conferred through two mechanisms: (i) the enzymatic inactivation of the antibiotic via chloramphenicol O-acetyltransferase (CAT) and (ii) the removal of the antibiotic via

6 J. Food Prot., Vol. 70, No. 3 ANTIMICROBIAL RESISTANCE IN SALMONELLA 785 an efflux pump. The genes encoding for CAT are plasmidborne (58) and commonly found in Salmonella Typhi isolates (78, 107). CAT genes, such as cat1 and cat2, have also been found in nontyphoidal Salmonella serotypes, including Derby, Enteritidis, Haardt, and Typhimurium (32, 58). Chloramphenicol efflux pumps in Salmonella isolates have been reported to be encoded by two closely related genes, cmla (25) and flor (124). The flor gene appears to be widespread in Salmonella isolates, whereas cmla is less widely distributed. Salmonella serotypes Albany, Agona, Kiambo, Newport, Typhimurium, and Typhimurium var. Copenhagen have been found to carry flor (3, 21, 25, 41, 42, 81). This gene is highly mobile and has been found in Salmonella genomic islands (21, 42, 122) and in many different plasmids (35, 41, 81). It appears to be associated with multidrug resistance (3, 41). For example, in Salmonella Typhimurium DT104 and other MDR Salmonella Typhimurium phage types it is located chromosomally between two integrons (118). Quinolones and fluoroquinolones. Quinolones and fluoroquinolones are synthetic bacteriocidal drugs. In 1962, nalidixic acid became the first quinolone approved for medical use (78). Several generations of quinolones have been developed, with each new generation having improved action against bacterial infections. The early generation quinolones target DNA gyrase, and the late-generation quinolones target DNA gyrase and DNA topoisomerase IV (78, 127). The mode of action for quinolones is quite complex and not completely understood (78). Although quinolones target topoisomerases, they do not actually bind to the topoisomerase but to the double-stranded DNA in the topoisomerase complex (109). Further information on quinolones and their mode of action has been provided in comprehensive reviews (78, 127). Although there are documented cases of Salmonella isolates with resistance to nalidixic acid and low-level resistance to other quinolones (22, 84), high-level resistance to quinolones is still rare (28, 94). Quinolone resistance of Salmonella isolates has been linked to two mechanisms. The first mechanism is mediated by target mutations in the quinolone resistance determining region of gyra and gyrb, the two genes that encode the subunits of DNA gyrase, and in the parc subunit of topoisomerase IV (14, 28, 36, 61, 72). The second mechanism involves changes in the expression of the AcrAB-TolC efflux system, mostly due to mutations in the genes encoding regulators of this system (e.g., marrab) that result in overexpression of this efflux system (4, 14, 15, 36, 67, 71, 93) and consequently decreased quinolone sensitivity. No single mutation confers high-level resistance to quinolones; resistance results from the accumulation of mutations (61). The facts that Salmonella isolates must acquire multiple unlinked mutations and that some of those mutations reduce fitness, particularly those involved in the regulation of the efflux pump (54), may explain why this kind of resistance is so infrequent. In some bacterial species, such as E. coli (120) and Klebsiella pneumoniae (119), quinolone resistance also has been linked to the expression of the plasmid-mediated qnr gene (73). This gene codes for a protein that appears to bind to DNA gyrase and protect it from quinolone inhibition (73). Research conducted on plasmids harboring qnr revealed that this gene could be transferred from other bacterial species to Salmonella via conjugation (77). Although documented cases of plasmid-mediated quinolone resistance in Salmonella isolates are rare, a recent study indicated that the spread of such plasmids to Salmonella isolates has occurred (33). The appearance of plasmid-mediated quinolone resistance in Salmonella isolates is a very important emerging public health concern. Plasmids harboring qnr also can harbor other resistance genes (73), suggesting that the treatment of infections with Salmonella strains containing this plasmid may be increasingly difficult. In a recent study, reduced susceptibility to ciprofloxacin was conferred by a variant of the gene encoding aminoglycoside acetyltransferase AAC(6 )-Ib. The presence of this gene has been reported in E. coli but not yet in Salmonella (103). Tetracyclines. Tetracyclines were discovered in the 1940s. The first tetracycline, chlorotetracycline, was isolated from Streptomyces aureofaciens (78). Tetracyclines were popular because of their minimal adverse effects and broad spectrum of activity. They were effective against most bacteria, including chlamydias and mycoplasms, and even some protozoa (34, 78). Tetracyclines act by preventing the binding of trna to the A site of the 30S ribosomal subunit, thus inhibiting protein synthesis (78). Unfortunately, the rise of resistant bacteria has severely limited the use of tetracyclines. A more in-depth review of tetracycline structure and function was provided by Chopra and Roberts (34). Tetracycline resistance of Salmonella isolates is attributed to production of an energy-dependent efflux pump, which removes this antimicrobial drug from the bacterial cell. Other mechanism of resistance, such as modification of the ribosomal target and enzymatic inactivation of tetracycline, have been documented in other bacterial species but have yet to be reported in Salmonella isolates (34, 78). Deletion or inactivation of the marrab operon also has been linked to reduced susceptibility to tetracycline (4). There are at least 32 different genes that confer resistance to tetracycline and oxytetracycline (34, 78). Of these, tet(a), tet(b), tet(c), tet(d), tet(g), and tet(h) have been found in Salmonella isolates (26, 34, 49). The most commonly reported of these genes is tet(a). It has been found in Salmonella genomic island 1 (26), on integrons (23), and on transferable plasmids (50, 53, 98). The tet(a) gene has been detected in isolates of Salmonella serotypes Agona, Anatum, Blockley, Bredeney, Colorado, Derby, Enteritidis, Give, Haardt, Hadar, Heidelberg, Infantis, Orion, Senftenberg, and Typhimurium (32, 98). The tet(b) gene is also relatively common and has been found in isolates of Salmonella serotypes Agona, Dublin, Choleraesuis, Heidelberg, and Typhimurium (32, 51, 58). Like tet(a), tet(b) also has been located on transferable plasmids (58). These genes appear to be easily transferred and widespread among Salmonella isolates. They also tend to be found in isolates that

7 786 ALCAINE ET AL. J. Food Prot., Vol. 70, No. 3 display multidrug resistance (26, 32, 98), making them an important marker in identifying potentially serious Salmonella infections. Sulfonamides and trimethoprim. Although initially prescribed separately, sulfonamides and trimethoprim have been used in combination for the treatment of bacterial infection since the late 1960s (78). These compounds are bacteriostatic antimicrobial drugs that act by competitively inhibiting enzymes involved in the synthesis of tetrahydrofolic acid. Sulfonamides inhibit dihyrdropteroate synthetase (DHPS), and trimethoprim inhibits dihydrofolate reductase (DHFR) (78). The combination of a sulfonamide and trimethoprim has been a popular form of treatment for decades, and although resistance among Salmonella isolates has emerged (87), this resistance does not appear to be common (19, 31, 131). Sulfonamide resistance in Salmonella isolates has been attributed to the presence of an extra sul gene, which expresses an insensitive form of DHPS (11, 78). Three main sul genes have been identified: sul1, sul2, and sul3. The sul1 gene has been found in a wide range of Salmonella serotypes such as Agona, Albany, Derby, Djugu, Enteritidis, Hadar, Heidelberg, Orion, Rissen, and Typhimurium (11, 32, 42, 43). This gene is often associated with class I integrons that contain other resistance genes (57, 69, 105). These integronborne gene cassettes have been found on transferable plasmids (58) and as part of Salmonella genomic island variants (21, 42, 43). Although sometimes found in isolates also harboring sul1 (11, 32), sul2 appears to associated with plasmids but not with class I integrons (11). Isolates of Salmonella serotypes Agona, Enteritidis, and Typhimurium have been reported to carry sul2 (32). The sul3 gene has been identified only recently in Salmonella, and it has been associated with plasmids (56) and class I integrons (11) in Salmonella, suggesting that there may be further dissemination of this gene within Salmonella populations. The sul3 gene already has been found in Salmonella serotypes 4,5,12:i:, Agona, Anatum, Brandenburg, Heidelberg, Rissen, and Typhimurium (11, 56). Deletion or inactivation of the marrab operon also has been linked to reduced sulfonamide susceptibility (4). Similar to sulfonamide resistance, trimethoprim resistance is attributed to the expression of DHFR that does not bind trimethoprim (78). There are many variants of the dhfr and dfr genes that encode this resistance (1, 78), such as dhfr1, dfra1, and dhfr12 (32, 42, 70). These genes have been found as part of integronborne gene cassettes also associated with sul1 and sul3 (11), on transferable plasmids carrying other resistance genes (59, 117), and on Salmonella genomic islands (42, 43). Salmonella serotypes known to carry trimethoprim resistance genes are 4,5,12:i:, Agona, Albany, Derby, Djugu, Hadar, Newport, Rissen, and Typhimurium (11, 40, 42, 43, 59, 76). TRANSMISSION OF ANTIMICROBIAL RESISTANCE IN SALMONELLA The two mechanisms critical for the spread of antimicrobial resistance in Salmonella populations are (i) horizontal transfer of antibiotic resistance genes and (ii) clonal spread of antimicrobial drug resistant Salmonella isolates (24, 82). Horizontal transfer of resistance genes can occur between Salmonella strains or from other bacterial species to Salmonella. These other species can be a source of antibiotic resistance genes that might not be found in the Salmonella genetic pool at a given time. In Salmonella, plasmids and class I integrons are primarily responsible for such transfers. Genes conferring resistance to aminoglycosides (86, 108), beta-lactams (16, 41, 66), chloramphenicols (35, 41), tetracyclines (59, 98), sulfonamides (11, 56), and trimethoprim (1, 117) all have been found on several different plasmid types. Many of these plasmids carry multiple antibiotic resistance genes that are transferable to other Salmonella strains and other bacterial species (41, 58, 59, 117). Class I integrons are composed of (i) a conserved 5 region consisting of the integrase gene int and a promoter, (ii) a conserved 3 region consisting of the ethidium bromide quaternary ammonium compound resistance locus qace 1 and the sulfonamide resistance gene sul1, and (iii) a gene cassette containing multiple antibiotic resistance genes in various combinations (69, 105). Integrons have been found as part of plasmids (58, 59) and transposons (98, 117) carried by Salmonella. Class I integrons are primary components of Salmonella genomic islands, which are the genetic elements responsible for multidrug resistance in isolates of Salmonella serotypes Agona, Albany, Newport, and Typhimurium DT104 (21, 40, 42, 43, 70). Recent research has indicated that these genomic islands are complex class I integrons (39). They can be horizontally transferred in the presence of a conjugative helper plasmid and have been found in multiple Salmonella subtypes (39), indicating that further dissemination of this multidrug resistance element among Salmonella strains is likely. Although horizontal gene transfer provides a mechanism for emergence of drug-resistant Salmonella subtypes, factors that govern subsequent clonal spread and dispersal of these subtypes are not as well understood. Even though a number of drug-resistant and MDR Salmonella subtypes have been identified, some appear to have been particularly successful in spreading geographically and through different host species. The most well known MDR clonal group is Salmonella serotype Typhimurium DT104. This group has been found worldwide (45, 100, 102, 115) and has been responsible for numerous outbreaks (68, 84). MDR Salmonella Newport is another drug-resistant clonal group that is widely distributed, including in the United States (29, 47, 101). Although widespread use of antimicrobial drugs in human and animal populations has been suggested as providing critical selective pressure for clonal expansion and spread of MDR Salmonella (7), some evidence indicates that drug-resistant clones may emerge and spread independently of antimicrobial drug use on individual farms (37). Some evidence also suggests that exposure to sanitizers (e.g., chlorine) or food preservatives may select for Salmonella with reduced sensitivity to commonly used antibiotics (e.g., tetracycline and chloramphenicol) apparently because of mutations in marr, which may be associated with a decreasing influx and increasing efflux of toxic

8 J. Food Prot., Vol. 70, No. 3 ANTIMICROBIAL RESISTANCE IN SALMONELLA 787 agents (4, 99). The importance of naturally occurring antibiotics, which are produced by other bacteria and fungi, as a factor contributing to the emergence and spread of MDR Salmonella strains is poorly understood. Recent research revealed that many nonpathogenic soil and environmental microorganisms are resistant to multiple antibiotic classes, supporting the presence of considerable selective pressure for maintenance of antimicrobial resistance phenotypes outside mammalian hosts (38). Antimicrobial resistance genes are typically found on mobile genetic elements, such as integrons and plasmids, which are readily transferred among Salmonella strains and between other bacterial species and Salmonella. MDR Salmonella strains resulting from acquisition of these genetic elements have been found worldwide and are a growing concern for public health and food safety. Although future research efforts on the ecology, epidemiology, and evolution of drug-resistant Salmonella, in conjunction with technological advances to allow rapid identification and characterization of antimicrobial-resistant Salmonella isolates, are needed to address this food safety issue, new knowledge and tools will be effective only if applied in the framework of a comprehensive farm-to-table approach that also takes into consideration the role of non host-associated environments in the emergence and spread of antibiotic-resistant bacteria. ACKNOWLEDGMENTS Research on Salmonella in the authors laboratories is supported by a number of sources, including an International Life Sciences Institute North America Future Leader Award (to M. Wiedmann), a U.S. Department of Agriculture Special Research Grant ( to M. Wiedmann), and federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under contract no. N01-AI (to L. Warnick). The opinions expressed herein are those of the authors and do not necessarily represent the views of the International Life Sciences Institute North America or the U.S. Department of Agriculture. The authors thank Wan-Lin Su for help with collecting information used for this review. REFERENCES 1. Agodi, A., M. Marranzano, C. S. Jones, and E. J. Threlfall Molecular characterization of trimethoprim resistance in Salmonellas isolated in Sicily, Eur. J. Epidemiol. 11: Aires, J. R., and H. Nikaido Aminoglycosides are captured from both periplasm and cytoplasm by the AcrD multidrug efflux transporter of Escherichia coli. J. Bacteriol. 187: Alcaine, S. D., S. S. Sukhnanand, L. D. Warnick, W. L. Su, P. McGann, P. McDonough, and M. Wiedmann Ceftiofur-resistant Salmonella strains isolated from dairy farms represent multiple widely distributed subtypes that evolved by independent horizontal gene transfer. Antimicrob. Agents Chemother. 49: Alekshun, M. N., and S. B. Levy Regulation of chromosomally mediated multiple antibiotic resistance: the mar regulon. Antimicrob. Agents Chemother. 41: Ambler, R. P The structure of beta-lactamases. Philos. Trans. R. Soc. Lond. B Biol. Sci. 289: Angulo, F. J., K. R. Johnson, R. V. Tauxe, and M. L. Cohen Origins and consequences of antimicrobial-resistant nontyphoidal Salmonella: implications for the use of fluoroquinolones in food animals. Microb. Drug Resist. 6: Anonymous Outbreak of multidrug-resistant Salmonella Newport United States, January April Morb. Mortal. Wkly. Rep. 51: Anonymous Preliminary FoodNet data on the incidence of foodborne illnesses selected sites, United States, Morb. Mortal. Wkly. Rep. 51: Anonymous Outbreak of multidrug-resistant Salmonella Typhimurium associated with rodents purchased at retail pet stores United States, December 2003 October Morb. Mortal. Wkly. Rep. 54: Antunes, P., J. Machado, J. C. Sousa, and L. Peixe Dissemination amongst humans and food products of animal origin of a Salmonella Typhimurium clone expressing an integron-borne OXA-30 beta-lactamase. J. Antimicrob. Chemother. 54: Antunes, P., J. Machado, J. C. Sousa, and L. Peixe Dissemination of sulfonamide resistance genes (sul1, sul2, and sul3) in Portuguese Salmonella enterica strains and relation with integrons. Antimicrob. Agents Chemother. 49: Armand-Lefevre, L., V. Leflon-Guibout, J. Bredin, F. Barguellil, A. Amor, J. M. Pages, and M. H. Nicolas-Chanoine Imipenem resistance in Salmonella enterica serovar Wien related to porin loss and CMY-4 beta-lactamase production. Antimicrob. Agents Chemother. 47: Batchelor, M., K. Hopkins, E. J. Threlfall, F. A. Clifton-Hadley, A. D. Stallwood, R. H. Davies, and E. Liebana bla(ctx-m) genes in clinical Salmonella isolates recovered from humans in England and Wales from 1992 to Antimicrob. Agents Chemother. 49: Baucheron, S., E. Chaslus-Dancla, and A. Cloeckaert Role of TolC and parc mutation in high-level fluoroquinolone resistance in Salmonella enterica serotype Typhimurium DT204. J. Antimicrob. Chemother. 53: Baucheron, S., H. Imberechts, E. Chaslus-Dancla, and A. Cloeckaert The AcrB multidrug transporter plays a major role in high-level fluoroquinolone resistance in Salmonella enterica serovar Typhimurium phage type DT204. Microb. Drug Resist. 8: Bauernfeind, A., I. Stemplinger, R. Jungwirth, and H. Giamarellou Characterization of the plasmidic beta-lactamase CMY-2, which is responsible for cephamycin resistance. Antimicrob. Agents Chemother. 40: Bellido, F., I. R. Vladoianu, R. Auckenthaler, S. Suter, P. Wacker, R. L. Then, and J. C. Pechere Permeability and penicillinbinding protein alterations in Salmonella Muenchen: stepwise resistance acquired during beta-lactam therapy. Antimicrob. Agents Chemother. 33: Bjorkman, J., P. Samuelsson, D. I. Andersson, and D. Hughes Novel ribosomal mutations affecting translational accuracy, antibiotic resistance and virulence of Salmonella typhimurium. Mol. Microbiol. 31: Blau, D. M., B. J. McCluskey, S. R. Ladely, D. A. Dargatz, P. J. Fedorka-Cray, K. E. Ferris, and M. L. Headrick Salmonella in dairy operations in the United States: prevalence and antimicrobial drug susceptibility. J. Food. Prot. 68: Blomberg, B., R. Jureen, K. P. Manji, B. S. Tamim, D. S. Mwakagile, W. K. Urassa, M. Fataki, V. Msangi, M. G. Tellevik, S. Y. Maselle, and N. Langeland High rate of fatal cases of pediatric septicemia caused by gram-negative bacteria with extendedspectrum beta-lactamases in Dar es Salaam, Tanzania. J. Clin. Microbiol. 43: Boyd, D., A. Cloeckaert, E. Chaslus-Dancla, and M. R. Mulvey Characterization of variant Salmonella genomic island 1 multidrug resistance regions from serovars Typhimurium DT104 and Agona. Antimicrob. Agents Chemother. 46: Breuil, J., A. Brisabois, I. Casin, L. Armand-Lefevre, S. Fremy, and E. Collatz Antibiotic resistance in salmonellae isolated from humans and animals in France: comparative data from 1994 and J. Antimicrob. Chemother. 46: Briggs, C. E., and P. M. Fratamico Molecular characterization of an antibiotic resistance gene cluster of Salmonella Typhimurium DT104. Antimicrob. Agents Chemother. 43:

9 788 ALCAINE ET AL. J. Food Prot., Vol. 70, No Butaye, P., G. B. Michael, S. Schwarz, T. J. Barrett, A. Brisabois, and D. G. White The clonal spread of multidrug-resistant non-typhi Salmonella serotypes. Microbes Infect. 8: Cabrera, R., J. Ruiz, F. Marco, I. Oliveira, M. Arroyo, A. Aladuena, M. A. Usera, M. T. Jimenez De Anta, J. Gascon, and J. Vila Mechanism of resistance to several antimicrobial agents in Salmonella clinical isolates causing traveler s diarrhea. Antimicrob. Agents Chemother. 48: Carattoli, A., E. Filetici, L. Villa, A. M. Dionisi, A. Ricci, and I. Luzzi Antibiotic resistance genes and Salmonella genomic island 1 in Salmonella enterica serovar Typhimurium isolated in Italy. Antimicrob. Agents Chemother. 46: Carattoli, A., L. Villa, C. Pezzella, E. Bordi, and P. Visca Expanding drug resistance through integron acquisition by IncFI plasmids of Salmonella enterica Typhimurium. Emerg. Infect. Dis. 7: Casin, I., J. Breuil, J. P. Darchis, C. Guelpa, and E. Collatz Fluoroquinolone resistance linked to GyrA, GyrB, and ParC mutations in Salmonella enterica Typhimurium isolates in humans. Emerg. Infect. Dis. 9: Centers for Disease Control and Prevention National Antimicrobial Resistance Monitoring System for enteric bacteria (NARMS): 2002 human isolates final report. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, Atlanta. 30. Centers for Disease Control and Prevention Salmonella surveillance annual summary, U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, Atlanta. 31. Centers for Disease Control and Prevention National Antimicrobial Resistance Monitoring System for enteric bacteria (NARMS): 2003 human isolates final report. U.S. Department of Health and Human Services, Centers for Disease Control and Prevention, Atlanta. 32. Chen, S., S. Zhao, D. G. White, C. M. Schroeder, R. Lu, H. Yang, P. F. McDermott, S. Ayers, and J. Meng Characterization of multiple-antimicrobial-resistant Salmonella serovars isolated from retail meats. Appl. Environ. Microbiol. 70: Cheung, T. K., Y. W. Chu, M. Y. Chu, C. H. Ma, R. W. Yung, and K. M. Kam Plasmid-mediated resistance to ciprofloxacin and cefotaxime in clinical isolates of Salmonella enterica serotype Enteritidis in Hong Kong. J. Antimicrob. Chemother. 56: Chopra, I., and M. Roberts Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiol. Mol. Biol. Rev. 65: Cloeckaert, A., S. Baucheron, and E. Chaslus-Dancla Nonenzymatic chloramphenicol resistance mediated by IncC plasmid R55 is encoded by a flor gene variant. Antimicrob. Agents Chemother. 45: Cloeckaert, A., and E. Chaslus-Dancla Mechanisms of quinolone resistance in Salmonella. Vet. Res. 32: Davis, M. A., D. D. Hancock, and T. E. Besser Multiresistant clones of Salmonella enterica: the importance of dissemination. J. Lab. Clin. Med. 140: D Costa, V. M., K. M. McGrann, D. W. Hughes, and G. D. Wright Sampling the antibiotic resistome. Science 311: Doublet, B., D. Boyd, M. R. Mulvey, and A. Cloeckaert The Salmonella genomic island 1 is an integrative mobilizable element. Mol. Microbiol. 55: Doublet, B., P. Butaye, H. Imberechts, D. Boyd, M. R. Mulvey, E. Chaslus-Dancla, and A. Cloeckaert Salmonella genomic island 1 multidrug resistance gene clusters in Salmonella enterica serovar Agona isolated in Belgium in 1992 to Antimicrob. Agents Chemother. 48: Doublet, B., A. Carattoli, J. M. Whichard, D. G. White, S. Baucheron, E. Chaslus-Dancla, and A. Cloeckaert Plasmid-mediated florfenicol and ceftriaxone resistance encoded by the flor and bla CMY-2 genes in Salmonella enterica serovars Typhimurium and Newport isolated in the United States. FEMS Microbiol. Lett. 233: Doublet, B., R. Lailler, D. Meunier, A. Brisabois, D. Boyd, M. R. Mulvey, E. Chaslus-Dancla, and A. Cloeckaert Variant Salmonella genomic island 1 antibiotic resistance gene cluster in Salmonella enterica serovar Albany. Emerg. Infect. Dis. 9: Doublet, B., F. X. Weill, L. Fabre, E. Chaslus-Dancla, and A. Cloeckaert Variant Salmonella genomic island 1 antibiotic resistance gene cluster containing a novel 3 -N-aminoglycoside acetyltransferase gene cassette, aac(3)-id, in Salmonella enterica serovar Newport. Antimicrob. Agents Chemother. 48: Duke, T., M. Michael, D. Mokela, T. Wal, and J. Reeder Chloramphenicol or ceftriaxone, or both, as treatment for meningitis in developing countries? Arch. Dis. Child. 88: Esaki, H., A. Morioka, A. Kojima, K. Ishihara, T. Asai, Y. Tamura, H. Izumiya, J. Terajima, H. Watanabe, and T. Takahashi Epidemiological characterization of Salmonella Typhimurium DT104 prevalent among food-producing animals in the Japanese veterinary antimicrobial resistance monitoring program ( ). Microbiol. Immunol. 48: European Food Safety Authority The community summary report on trends and sources of zoonoses, zoonotic agents and antimicrobial resistance in the European Union in European Food Safety Authority, Brussels. 47. Fontana, J., A. Stout, B. Bolstorff, and R. Timperi Automated ribotyping and pulsed-field gel electrophoresis for rapid identification of multidrug-resistant Salmonella serotype Newport. Emerg. Infect. Dis. 9: Frana, T. S., S. A. Carlson, and R. W. Griffith Relative distribution and conservation of genes encoding aminoglycoside-modifying enzymes in Salmonella enterica serotype Typhimurium phage type DT104. Appl. Environ. Microbiol. 67: Frech, G., C. Kehrenberg, and S. Schwarz Resistance phenotypes and genotypes of multiresistant Salmonella enterica subsp. enterica serovar Typhimurium var. Copenhagen isolates from animal sources. J. Antimicrob. Chemother. 51: Frech, G., and S. Schwarz Plasmid-encoded tetracycline resistance in Salmonella enterica subsp. enterica serovars Choleraesuis and Typhimurium: identification of complete and truncated Tn1721 elements. FEMS Microbiol. Lett. 176: Frech, G., and S. Schwarz Molecular analysis of tetracycline resistance in Salmonella enterica subsp. enterica serovars Typhimurium, Enteritidis, Dublin, Choleraesuis, Hadar and Saintpaul: construction and application of specific gene probes. J. Appl. Microbiol. 89: Gebreyes, W. A., and C. Altier Molecular characterization of multidrug-resistant Salmonella enterica subsp. enterica serovar Typhimurium isolates from swine. J. Clin. Microbiol. 40: Gebreyes, W. A., and S. Thakur Multidrug-resistant Salmonella enterica serovar Muenchen from pigs and humans and potential interserovar transfer of antimicrobial resistance. Antimicrob. Agents Chemother. 49: Giraud, E., A. Cloeckaert, S. Baucheron, C. Mouline, and E. Chaslus-Dancla Fitness cost of fluoroquinolone resistance in Salmonella enterica serovar Typhimurium. J. Med. Microbiol. 52: Gonzalez, L. S., III, and J. P. Spencer Aminoglycosides: a practical review. Am. Fam. Physician 58: Guerra, B., E. Junker, and R. Helmuth Incidence of the recently described sulfonamide resistance gene sul3 among German Salmonella enterica strains isolated from livestock and food. Antimicrob. Agents Chemother. 48: Guerra, B., S. Soto, S. Cal, and M. C. Mendoza Antimicrobial resistance and spread of class 1 integrons among Salmonella serotypes. Antimicrob. Agents Chemother. 44: Guerra, B., S. Soto, R. Helmuth, and M. C. Mendoza Characterization of a self-transferable plasmid from Salmonella enterica serotype Typhimurium clinical isolates carrying two integron-borne gene cassettes together with virulence and drug resistance genes. Antimicrob. Agents Chemother. 46: Guerra, B., S. M. Soto, J. M. Arguelles, and M. C. Mendoza