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Appl Environ Microbiol. Apr 2012; 78(8): 3028–3032.
PMCID: PMC3318844

Functional Cloning and Characterization of Antibiotic Resistance Genes from the Chicken Gut Microbiome

Abstract

Culture-independent sampling in conjunction with a functional cloning approach identified diverse antibiotic resistance genes for different classes of antibiotics in gut microbiomes from both conventionally raised and free-range chickens. Many of the genes are phylogenetically distant from known resistance genes. Two unique genes that conferred ampicillin and spectinomycin resistance were also functional in Campylobacter, a distant relative of the Escherichia coli host used to generate the genomic libraries.

TEXT

Zoonotic bacterial pathogens cause diseases in animals and/or humans, consequently threatening animal production, food safety, and public health (22, 27, 29). Increasing evidence indicates that antimicrobial usage in animals promotes the emergence of resistant zoonotic pathogens, greatly compromising the effectiveness of antibiotic treatments (7, 24). It has been proposed that normal intestinal microbiotas play a critical role in antibiotic resistance (AR) development and transmission via horizontal gene transfer (HGT) (13, 26). However, the majority of intestinal microbiotas cannot be cultivated using traditional culturing methods, greatly impeding our understanding of the AR reservoir in gut microbiotas. Recently, Sommer et al. (21) functionally characterized the AR reservoir in the human gut microbiota; many AR genes identified using a culture-independent approach are evolutionarily distant from known resistance genes, first demonstrating the immense diversity of AR genes in the human microbiome. Based on this recent human study, the gut microbiota in food animals may also contain diverse and novel AR genes that could be accessible to clinically relevant pathogens. However, the reservoirs of mobile AR genes in the gut microbiomes from food animals are still largely unknown. In addition, it is still not clear if the novel AR genes identified in the gut microbiome can function normally in a new host or a distant relative when HGT successfully occurs, a key issue for the emergence of AR in bacterial pathogens.

In this study, we examined AR genes in gut microbiomes of chickens from different production systems and determined the functional compatibility of the identified novel AR genes. The major bacterial strains and plasmids used in this study are listed in Table 1. We first isolated total genomic DNA directly from cecal contents of two free-range chickens that have never been treated with antimicrobials (kindly provided by a free-range farm in Dandridge, TN) and two conventionally raised chickens that have received antibiotic-containing feed (from Koch Foods, Inc., Morristown, TN). The sheared DNA fragments were cloned into expression vector pZE21-MCS (12) and transformed into Escherichia coli One Shot TOP10 electrocompetent cells (Epicentre) for library construction as described by Sommer et al. (21). The total sizes of the 4 expression libraries ranged from 0.4 × 108 bp to 2.4 × 108 bp. Using the same screening protocol as Sommer et al. (21), we selected AR clones from each library by plating culture on Luria broth agar plates containing the antibiotic of interest at the appropriate concentration (50 μg/ml of ampicillin, 10 μg/ml of tetracycline, 25 μg/ml of chloramphenicol, 75 μg/ml of spectinomycin, 12.5 μg/ml of norfloxacin, or 10 μg/ml of ciprofloxacin). Fifty inserts conferring resistance were sequenced by using primers pZE-F and pZE-R (Table 2); the sequences were annotated and analyzed as described by Sommer et al. (21).

Table 1
Bacterial strains and plasmids used in this study
Table 2
PCR primers used in this study

As shown in Table 3, 3 AR genes that conferred resistance to ampicillin or spectinomycin were identified in the gut microbiomes of two free-range chickens, while a total of 11 AR genes were identified from conventionally raised chickens (Table 3). The novel AR genes that shared low sequence similarity (58% to 76% at the amino acid level) with the known resistance genes constitute a substantial proportion of the AR genes identified in this study (6 of 14) (Table 3). Notably, among the 6 novel genes, 3 were unique and even shared low sequence similarities (<68% at the amino acid level) with the novel AR genes recently identified from the human gut microbiome using the same functional cloning approach (21); these 3 genes are FRAmp1.1, FRSpe1.1, and CRChl2.2 (Table 3). Discovery of the 3 AR genes from free-range chickens in this study strongly supports a recent theory that environmental bacteria harbor the readily available and diverse pool of AR genes regardless of antibiotic exposure (2). This finding is also consistent with a recent work directly showing that antibiotic resistance is a natural phenomenon that predates the modern selective pressure of clinical antibiotic use (4). It is not surprising that more AR genes, in terms of number and diversity, were identified in the conventionally raised chickens than in the free-range chickens. The extensive antimicrobial usage in conventionally raised chickens may enrich AR genes in the gut microbiome, consequently increasing likelihood and ease of identification of AR genes using the functional cloning approach. However, given the limitation of the small sample size of this study (2 birds for each production system), this finding needs to be confirmed in future large-scale experiments using animals from diverse sources.

Table 3
Antibiotic resistance genes identified in the cecal contents of free-range and conventionally raised chickensa

To better understand the uniqueness of the identified novel AR genes, we further analyzed the phylogenetic relationship of FRAmp1.1, a novel β-lactamase identified in the gut microbiome of a free-range chicken (Table 3), to 25 presently identified β-lactamases that represent 21 different families. As shown in Fig. 1, FRAmp1.1 and the β-lactamases recently identified in the human gut microbiome (21) were phylogenetically distant from most known β-lactamases that were characterized in cultivated organisms. In particular, FRAmp1.1 was very distantly related to the β-lactamases (Cj0229, Cam1, CJS_0285, and CJE0344) (Fig. 1) that have been identified in Campylobacter, an important food-borne bacterial pathogen with chicken as a major animal host (10, 11) and a distant relative of the E. coli host used here to generate genomic libraries. This finding prompted us to explore another significant issue of antibiotic resistance by taking advantage of the identified novel AR genes: can novel AR genes function normally in different bacterial species if the HGT barrier is successfully overcome? To address this issue, we first constructed an E. coli-Campylobacter shuttle vector (pZW) (Table 1) carrying a Campylobacter promoter according to the procedure described by Larsen et al. (8). The two selected novel AR gene products (FRAmp1.1, conferring ampicillin resistance, and FRSpe1.1, conferring spectinomycin resistance) shared low sequence similarity to the major determinants of ampicillin resistance (Cj0299) and spectinomycin resistance (AadA) in Campylobacter (6, 17). FRAmp1.1 and FRSpe1.1 were PCR amplified using proper primers pairs (Table 2), and the PCR fragments were cloned into pZW, creating pZW1 and pZW2, respectively. The promoterless kanamycin resistance gene KAN-2 from a Campylobacter jejuni strain (11) was also cloned into pZW to generate pZW3 (Table 1). All plasmids described above were transferred into C. jejuni 81-176 (JL242) (Table 1) using triparental conjugation as described previously (32). All the C. jejuni constructs were subjected to MIC tests for different antibiotics as determined by a standard microtiter broth dilution method (10). The JL857 strain (C. jejuni 81-176 containing pZW3) displayed a dramatically enhanced resistance to kanamycin (MIC > 128 μg/ml) compared to that of the control strain JL854 (MIC = 8 μg/ml), indicating that pZW was an effective expression vector for Campylobacter. The constructs harboring the novel AR genes (JL855 and JL856) (Table 1) also showed significantly higher levels of resistance to the corresponding antibiotics. Specifically, the JL856 strain containing the FRSpe1.1 gene displayed dramatically enhanced resistance to spectinomycin (MIC > 256 μg/ml) compared to that of the control strain JL854 (MIC = 8 μg/ml). Expression of FRAmp1.1 in JL855 also led to enhanced ampicillin resistance compared to that of the control JL854 strain (8-fold increase) (Table 4). The JL855 strain also displayed significantly increased resistance to a variety of β-lactams in C. jejuni, including those (e.g., cefotaxime) to which Cj0299 fails to confer high resistance (6) (Table 4). Together, these findings further support a previous hypothesis that if a barrier to gene transfer exists between the constituents of gut microbiomes and pathogens, it must be attributed to processes other than functional compatibility (21).

Fig 1
Phylogenetic relationship of β-lactamases from different sources. Unrooted phylogenetic trees were generated from ClustalW2 alignments using the neighbor-joining algorithm. FRAmp1.1, discovered in this study, was analyzed together with 25 identified ...
Table 4
MICs of different β-lactam antibiotics for the C. jejuni JL865 strain that carries resistance gene FRAmp1.1 and for the control strain JL854

Characterization of novel AR genes from animal gut microbiomes is important in several ways. First, the genes that are divergent in sequence can help to assemble a more complete image of the evolutionary history of AR genes (2). Second, further functional and structural study of novel AR genes will improve our understanding of the relationship between sequence diversity and the resistance spectrum/level (28). In particular, identification of novel AR genes using the functional cloning approach would greatly complement modern high-throughput pyrosequencing of the metagenome by improving annotation power. Third, since the novel AR genes may function effectively in zoonotic pathogens, thorough examination of novel AR gene pools in food animals and even other agriculture ecosystems (e.g., manure) will greatly facilitate development of more-powerful molecular diagnostic tools to monitor AR in zoonotic pathogens and examine the development and transmission of AR (20). Finally, considering the fact that genes for antibiotic biosynthesis were sometimes clustered with AR genes, identification of novel AR genes may lead to the discovery of potentially novel antibiotics (19).

Although the novel gut AR genes have been successfully identified in both conventionally raised and free-range chickens in this study, the following limitations and issues should be addressed for future large-scale investigation of the AR reservoir. First, the sizes of expression libraries are a limiting factor of the likelihood of identifying more novel AR genes. The sizes of the libraries used in both a recent human study (~109 bp per library) (21) and this study (~108 bp per library) cover only a proportion of the gut microbiome (18). Thus, increasing library size, for instance by cloning larger insertions into the vector, will provide a greater chance to identify more novel AR genes. Second, we used chickens as a model here to analyze the gut AR reservoir in food animals. The functional cloning method can be extended into the investigation of the AR gene reservoir in other habitats, including the guts of other food animals as well as specific niches of agricultural ecosystems (e.g., manure, soil, water, lagoon). Recent studies for the identification of specific AR genes in soils also showed the feasibility of the similar methodology in revealing a natural AR gene reservoir, which exhibited a higher level of diversity than previously expected (1, 5, 19). Finally, the complex mechanisms of HGT should be elucidated in parallel to the effort for identification of novel AR gene reservoirs. The novel AR genes need to overcome multiple HGT barriers (23) before being able to confer resistance to the pathogens. AR gene reservoirs and HGT are equally important issues to be addressed in the future to improve our understanding of the development, transmission, and evolution of AR genes.

Nucleotide sequence accession numbers.

The full coding sequences of the 14 AR genes identified in this study were deposited in the GenBank database under accession numbers JN625754, JN625755, JN625756, JN625757, JN625758, JN625759, JN625760, JN625761, JN625762, JN625763, JN625764, JN625765, JN625766, and JN625767.

ACKNOWLEDGMENTS

We thank Andree A. Hunkapiller for technical support.

This study was supported by Microbiology across Campuses Educational and Research Venture (M-CERV) and AgResearch at The University of Tennessee.

Footnotes

Published ahead of print 27 January 2012

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