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Monitoring and Identifying Antibiotic Resistance Mechanisms in Bacteria M. T. Roe and S. D. Pillai Food Safety & Environmental Microbiology Program, Poultry Science Department, Texas A&M University, College Station, Texas 77843-2472 ABSTRACT Sub-therapeutic administration of antibiotics to animals is under intense scrutiny because they contribute to the dissemination of antibiotic-resistant bacteria into the food chain. Studies suggest that there is a link between the agricultural use of antibiotics and antibiotic-resistant human infections. Antibiotic-resistant organisms from animal and human wastes reenter the human and animal populations through a number of pathways including natural waters, irrigation water, drinking water, and vegetables and foods. Antibiotic usage in the United States for animal production (disease prevention and growth promotion) is estimated to be 18 million pounds annually. As much as 25 to 75% of the antibiotics administered to feedlot animals are excreted unaltered in feces. Because about 180 million dry tons of livestock and poultry waste is generated annually in the United States, it is not surprising that animal-derived antibioticresistant organisms are found contaminating groundwater, surface water, and food crops. It is extremely important to clearly understand the molecular mechanisms that could potentially cause lateral or horizontal gene transfer of antibiotic resistance genes among bacteria. Once the mechanisms and magnitude of resistance gene transfer are clearly understood and quantified, strategies can be instituted to reduce the potential for dissemination of these genes. (Key words: integrons, antimicrobial resistance, bacteria, environment, characterization) 2003 Poultry Science 82:622–626 This paper provides an overview of integrons, which are considered to be one of the primary mechanisms by which antimicrobial resistance genes are transferred between bacteria. The paper focuses on integrons in poultry production and processing and in natural ecosystems. INTRODUCTION The use of antimicrobials as feed supplements is a common practice in livestock production. Studies suggest that there may be links between this practice and the development of antimicrobial resistance among the microflora of these animals, especially the enteric bacterial pathogens (Aarestrup, 1995; Witte, 1998; Smith et al., 1999; Wegener et al., 1999; Endtz et al., 1990; McEwen and Fedorka-Cray, 2002). Antimicrobial resistance in poultry-related pathogens is of concern because many of these compounds are no longer useful for treating bacterial infections in poultry (Bass et al., 1999; Schwarz and Chaslus-Dancla, 2001). Even though animal growers argue that the use of antimicrobials in animal feed has beneficial effects such as promoting growth and reducing cost, regulatory agencies such as the United States Food and Drug Administration, the Centers for Disease Control and Prevention, and the World Health Organization all argue that sub-therapuetic use of antimicrobials in animal feed should be restricted (Feinman, 1998; Harrison and Lederberg, 1998). ANTIMICROBIAL RESISTANCE MECHANISMS Microbial populations develop resistance to antimicrobials through several mechanisms. The rate at which an individual gene mutates to express an antimicrobial resistance phenotype is a complex phenomenon in which environment, cell physiology, bacterial genetics, and population dynamics all play roles (Martinez and Baquero, 2000). In addition, for full resistance to occur, mutations must develop within multiple genes because of genetic redundancy in the antimicrobial targets. A primary example of this is the fluoroquinolone target gene gyrA, gyrB, parA, and parC that are all targets for the fluoroquinolone antimicrobial, and all must have mutations for full resistance phenotypes to develop (Martinez and Baquero, 2000). Bacteria can also acquire antimicrobial resistance genes through horizontal or lateral gene transfer. Bacterial cells can acquire genetic sequences from other organisms through several processes. First, there is the take up of naked DNA from their immediate sur- 2003 Poultry Science Association, Inc. Received for publication October 21, 2002. Accepted for publication December 18, 2002. 1 To whom correspondence should be addressed: spillai@poultry. tamu.edu. 622 SYMPOSIUM: USE OF ANTIMICROBIALS IN PRODUCTION roundings by a process termed transformation. The frequency with which bacteria acquire DNA from the environment depends on several factors including cell wall structure and bacterial species with transfer frequencies being as low as 10−7. Bacterial cells have to be “competent” to acquire extraneous DNA by transformation. Bacteria such as Campylobacter are thought to be naturally competent. The process of transformation is complex, and there are differences in the process among gram-positive and gram-negative bacteria. The process involves the specific recognition sequences in order for the new DNA to be taken up by the bacteria (Elkins et al., 1991). Bacteria can also exchange and acquire genetic material through conjugation of self-replicating extra chromosomal DNA or plasmids. This requires physical contact between cells, which allows the plasmids to be exchanged between donor and recipient cells. An example of this is the floR gene encoding florofenicol resistance in Escherichia coli found in cattle (Cloeckaert et al., 2000). The floR antimicrobial resistance gene encodes a 1,122 amino acid protein that confers resistance to florofenicol through an unknown mechanism (Arcangioli et al., 1999). A third mechanism of horizontal gene transfer is the introduction of genetic material into a bacterium by a bacteriophage, or transduction. In this method, the virus attaches and injects its own nucleic acids to the bacterial cell, which in some cases facilitates the introduction of new genes into the bacterial genome. This concept has been used in molecular cloning by introducing vector DNA, including antimicrobial resistance, through bacteriophage λ for some time (Sambrook et al., 1989). Transposons, which are genetic elements conferring a selectable phenotype flanked by two insertion sequences, are involved in horizontal gene transfer events between bacteria. Transposons are unique in that they have the ability of excising themselves from one genetic locus and moving to another, whether it is within the same bacteria or bacteria in another taxa (Ochman et al., 2000). Transposons can be transferred through all of the methods mentioned above namely, conjugation, transformation, and transduction. Transposons play a significant role in antimicrobial resistance development because they often contain gene sequences mediating antimicrobial resistance called integron gene sequences. Integrons are thought to play a significant role in the rapid dissemination of antimicrobial resistance among bacteria (Ochman et al., 2000). INTEGRONS AND ANTIMICROBIAL RESISTANCE First identified by Stokes and Hall (1989), the integron gene sequence has become identified as a primary method by which bacteria acquire antimicrobial resistance. The integron gene sequence is unique in that it acts as a site-specific recombination system capable of capturing or excising novel genetic elements called gene 623 cassettes. These gene cassettes are promoterless genes with a recombination site known as a 59-base element located downstream of the gene. The gene cassette codes for a wide range of antimicrobial resistant determinants (Recchia and Hall, 1995). Integrons are also thought to play a role in the transfer of pathogenic elements (Mazel et al., 1998). These circular gene cassettes are exchanged between bacteria and are linearized by the integrase enzyme before being inserted at the integration site. Multiple gene cassettes can be inserted into the integron gene sequence to confer a multiple antimicrobial resistant phenotype to the bacteria (Hall and Collis, 1995; Rowe-Magnus and Mazel, 1999; Ochman et al., 2000). The integron has several unique genetic components that allow them to insert gene cassettes. One is the 5′ intI gene, which codes for the site-specific recombinase (integrase) enzyme. The second is the Pant promoter site that acts as the promoter governing inserted gene cassettes. The third unique element is the attI, which is an inverted repeated sequence, that is the recombination site for the lineraized gene cassettes (Stokes and Hall, 1989; Hall and Collis, 1995; Rowe-Magnus and Mazel, 1999). Multiple gene cassettes can be integrated at one locus on the chromosome or on a plasmid to form the integron gene sequence array (Recchia and Hall, 1995, 1997). Four different classes of integrons, namely class 1, class 2, class 3, and class 4, have been described to date, each of which has several distinctive traits (Hall and Collis, 1995; Mazel et al., 1998). The four classes of integrons differ primarily by the sequence of their integrase gene. The integrase genes of class 2 and class 3 integrons (intI2 and intI3) share only 45% and 60% amino acid similarity respectively with the class 1 integrase, intI1 (Hall and Collis, 1995). The class 1 integron designated In2 is most commonly associated with the Tn21 transposon (Liebert et al., 1999) and contains a 3′ conserved sequence composed of the qacE∆1 and sul1 resistance genes with an unknown open reading frame, orf5, located further downstream. In the class 1 integron In16 located on transposon Tn402, however, the 3′ conserved segment genes qacE∆1, sul1, and orf5 are replaced by the tni genes (Partridge et al., 2001). Class 2 integrons are commonly associated with the Tn7 transposon and its derivatives all of which contain a 3′ conserved segment composed of the tns gene module which contain five genes that encode products involved in the movement of the transposon. The class 3 integron has only been identified in Serratia marcescens and is not commonly associated with a transposon carrier (Arakawa et al., 1995). The class 4 integron gene sequence has been classified only in Vibrio cholerae genome and is associated with the Vibrio cholerae repeated sequences (VCR) but not a transposon sequence (Mazel et al., 1998). INTEGRONS IN POULTRY PROCESSING AND RETAIL CHICKEN PRODUCTS Hofacre et al. (2001) have linked multiple antimicrobial resistance phenotype in bacteria isolated from ani- 624 ROE AND PILLAI mal feed to integron sequences. They isolated gramnegative bacterial isolates from 165 rendered animal products (meat, bone, fish, and feather meal), which were used as protein sources for animal feed and determined their resistance patterns. The colonies were probed with DNA probes specific to the intI1 gene coding for the class 1 integrase. Resistance profiles showed that multiple antimicrobial resistance (resistance to five or more antimicrobials tested) was observed in three of the 36 isolates tested (8%) among the gram-negative isolates. Resistance was more prevalent in meat and bone meal than in feather meal. The class 1 integrase gene was found in nine of the 24 isolates (37%). Integrons have also been shown to be associated with multiple antimicrobial resistance in isolates taken from on-farm poultry (Bass et al., 1999). This study utilized 100 bacterial isolates of pathogenic E. coli of poultry origin, which were screened for antimicrobial resistance to the four major families of antimicrobials, namely βLactams, chloramphenicols, tetracyclines, and aminoglycosides. They reported that class 1 integrons were found in 63% of multiple antimicrobial-resistant E. coli of poultry origin. The isolates were also screened for the presence of the genes intI1 and qacE∆1 using DNAspecific probes. Furthermore, they identified the presence of the aadA1 (aminoglycoside resistance) and merA (mercury resistance) gene cassettes in 50% of the isolates. They acknowledged that the antimicrobial resistance phenotypes of the isolates could not be explained by the class 1 integron gene sequences solely. Because merA is harbored on the transposon Tn21, the authors suggested that the class 1 integrons are actually components on a Tn21 like transposon placing the class 1 integrons in the In2 family. Goldstein et al. (2001) examined the prevalence of class 1 and class 2 integrons in 588 bacterial isolates from clinical and poultry isolates using specific DNA probes. Multiple drug resistance phenotype (resistant to five or more antimicrobials tested) was evident in 56% (64/115) of the isolates. Of the poultry isolates tested, 73% possessed the class 1 integrase gene intI1. The class 2 integrase gene intI2 was found in 8% of the isolates, and the intI1 and intI2 genes were found together in 7% of the isolates. The class 3 integrase gene was only found in a total of 12% of all isolates that contained an integrase gene. To confirm the class 3 integrase presence, the authors utilized DNA PCR to amplify the intI3 gene; however, the isolates did not amplify. Class 4 integron genes were not found in any of the isolates tested. Hudson et al. (2000) examined the antimicrobial resistance profiles and the prevalence of the class 1 integron genes intI1, merA, and aadA1 within 22 isolates of Salmonella enterica serovar Typhimurium DT104 in non-domesticated birds (birds of the passerine species such as cowbirds, and English sparrows, which are not grown as companion animals or for food). By using probes specific for the class 1 integron associated genes, namely intI1, aadA1, and merA, they found that these genes were present in various combinations in 41% (9/22) of isolates examined. White et al. (2001) examined the prevalence of antimicrobial-resistant Salmonella spp. from retail ground meats. Out of 200 meat samples that were tested, 22% contained Salmonella spp., and 53% of these isolates were resistant to three or more antimicrobials. When the sulfamethoxazole-resistant isolates were screened for the class 1 integron gene sequence (because class 1 integrons typically contain the sul1 gene mediating sulfamethoxazole resistance), most them had integrons with genes mediating resistance to aminoglycosides, sulfonamides, trimethoprim, and β-lactams. Lucey et al. (2000) studied the contribution of class 1 integron in Campylobacter spp. by using isolates from poultry and porcine carcass samples collected at slaughter. Most (62.3%) of the isolates were resistant to sulfonamide. Using PCR analysis, they found that 40% (40/ 55) of the isolates tested contained the class 1 integron. Individual amplicons ranging in size from 230 bp to 1.47 kb were detected with a 350-bp fragment common to all but one of the isolates tested. Several amplified ‘gene cassette patterns’ or variable regions were identified from each isolate, and the sul1 and qacE∆1 were also identified using PCR amplification to confirm their presence in these isolates. INTEGRONS IN NATURAL ECOSYSTEMS The occurrence of integrons in natural ecosystems is important because the environment apparently serves as a source and sink for agriculture. However, studies on the prevalence of the integron gene sequence within natural environments have been limited to date. Rosser and Young (1999) examined the prevalence and characterization of the class 1 integron gene sequence in bacteria from an estuary of the Tay and Earn River west of Dundee, United Kingdom. In this study, 3,000 gramnegative isolates were collected over 2 mo. The colonies were analyzed using colony blot analysis with DNA probes targeted against the class 1 integrase gene intI1 and the quaternary ammonium compound resistance gene qacE∆1, both of which are linked to the class 1 integron. Of the 3,000 isolates screened for the class 1 integron gene sequence, 3.6% of the isolates hybridized to the intI1 probe using the colony blot hybridization technique. Eighty-five isolates containing the class 1 integron were examined for other gene cassettes using PCR amplification utilizing primers for gene cassettes commonly found in class 1 integron variable regions. Amplified cassettes were cloned and sequenced to map the variable region. The authors found broad diversity of gene cassettes present in the variable regions studied including the aadA1 and aadA2 gene cassettes, which encode resistance to streptomycin and spectinomycin antimicrobials. Of the 85 integron-positive isolates that were analyzed, 26 were Pseudomonas spp., 28 were Vibrio spp., and 31 were coliforms. This study illustrated that SYMPOSIUM: USE OF ANTIMICROBIALS IN PRODUCTION class 1 integrons are present in natural surface waters and are distributed among a variety of bacterial genera. Integron sequences have also been isolated from soils (Nield et al., 2001). Using culture-independent methods, they detected several novel integrase genes. However, complete gene cassettes were not identified. In studies in our laboratory, out of 322 E. coli isolates originally isolated from irrigation water and associated sediments, 32 isolates (10%) were resistant to two or more antimicrobial compounds. Of these 32 multidrugresistant isolates, four isolates (13%) contained class 1specific integron sequences (class 1 integron variable region and the class 1 integrase genes). Only one isolate contained class 2 integron-specific sequences (class II integron variable and class II integrase genes). Nucleotide sequence analysis showed that the class I integron bearing strains contained a conserved 780-bp aadA gene cassette, whereas the class II integron bearing strain contained two distinct gene cassettes, sat-1 and aadA. Even though all integron-bearing strains harbored the aadA sequence, none of the strains were resistant to streptomycin. The results suggest that irrigation water and sediments contaminated with fecal waste can be sources of class 1 and class 2 integron-bearing Escherichia coli and that environmental factors can cause a lack of phenotypic expression in these strains (Roe, unpublished data). Pillai and Pepper (1990) in their work with transposable elements had also reported on this similar lack of phenotypic expression in bacterial cells in cells, even when the genotypic potential exists. In conclusion, studies suggest that poultry products at the farm, processing, and retail levels harbor antibioticresistant organisms and the genetic determinants that could potentially disseminate these genes. Relying solely on the phenotypic characterization of antibiotic resistance may result in underestimation of the true genetic potential of bacteria to resist antimicrobial compounds. Culture-independent assays such as analysis of total microbial community nucleic acids for resistancecoding genes will provide a better picture of the genetic potential of a microbial community to acquire or transmit resistance genes. Nucleotide sequence analysis of the antimicrobial resistance gene determinants will help identify whether certain agronomic or processing methods are selecting for specific genotypes. It is critical for the poultry, beef, and porcine industries to understand the underlying mechanistic processes of the dissemination of antimicrobial resistance during preharvest and processing of their products. Possible research areas include the role of quorum sensing on exposure to sub-therapeutic antibiotic concentrations and acquisition of antibiotic resistance and the impacts(s) that specific poultry processing steps on retarding the transfer or resistance determinants (Lu et al., 2003). 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