Molecular Characterization of a Multidrug-Resistant Klebsiella pneumoniae Strain R46 Isolated from a Rabbit

To investigate the mechanisms of multiple resistance and the horizontal transfer of resistance genes in animal pathogens, we characterized the molecular structures of the resistance gene-related sequences in a multidrug-resistant Klebsiella pneumoniae strain R46 isolated from a rabbit. Molecular cloning was performed to clone the resistance genes, and minimum inhibitory concentrations (MICs) were measured to determine the resistance characteristics of the cloned genes and related strains. A conjugation experiment was conducted to assess the transferability of the resistance plasmids. Sequencing and comparative genomic methods were used to analyze the structures of the resistance gene-related sequences. The K. pneumoniae R46 genome consisted of a chromosome and three resistance plasmids named pR46-27, pR46-42, and pR46-270, respectively. The whole genome encoded 34 antibiotic resistance genes including a newly identified chromosome-encoded florfenicol resistance gene named mdfA2. pR46-270, besides encoding 26 antibiotic resistance genes, carried four clusters of heavy metal resistance genes and several virulence-related genes or gene clusters. The plasmid-encoded resistance genes were mostly associated with mobile genetic elements. The plasmid with the most similarity to the floR gene-harboring plasmid pR46-27 was pCTXM-2271, a plasmid from Escherichia coli. The results of this work demonstrated that the plasmids with multidrug resistance genes were present in animal-derived bacteria and more florfenicol resistance genes such as mdfA2 could be present in bacterial populations. The resistance genes encoded on the plasmids may spread between the bacteria of different species or genera and cause the resistance dissemination.

The antibiotic resistance, especially multiple-drug resistance bacteria of different species in both human and animal pathogens, becomes an increasing global problem. The multidrug resistance in the microbial organisms, in particular the Enterobacteriaceae, is often due to the acquisition of resistance genes from a shared pool [11]. The resistance genes in this pool appeared to have been captured from the chromosomes of various species by the mobile genetic elements, such as insertion sequences (IS) [12], transposons [13], and integrons [14]. A great number of genotypes (subgenotypes) of the antibiotic resistance genes have been found encoded on the mobile genetic elements. These mobile genetic elements can translocate between different bacterial DNA molecules, such as chromosomes and plasmids, especially the conjugative plasmids, and can be transferred between different bacterial species or genera through horizontal gene transfer, causing resistance dissemination [15].
With the widespread use of antimicrobial agents and detergents without effective supervision and the greater incidence of animal-caused foodborne illnesses, animals, especially those in agricultural practice, may become potential reservoirs for the dissemination of antimicrobial resistance. In this work, to investigate the resistance characteristics of K. pneumoniae isolated from animals, we used molecular cloning and comparative genomic methods to identify resistance gene profiles and to assess the horizontal transfer of resistance genes in a K. pneumoniae strain isolated from a rabbit in a farm in Wenzhou, South China.

Materials and Methods
2.1. Bacterial Isolation and Identification. K. pneumoniae R46 was isolated on a MacConkey agar (MCA) plate with florfenicol (8 mg/L) from an anal swab sample of a rabbit from a farm in Wenzhou, South China. Species identification was conducted with the Vitek-60 microorganism auto-analysis system (BioMerieux Corporate, Lyon, France). Further verification was performed by using homology comparisons of the 16S ribosomal RNA gene and the whole genome sequence from NCBI nucleotide database (http://www.ncbi. nlm.nih.gov) via BLASTN software. Multilocus sequence typing (MLST) was performed by analyzing the housekeeping genes gapA, infB, mdh, pgi, phoE, rpoB, and tonB [16] via the online service MLST-1.8 (https://cge.cbs.dtu.dk/ services/MLST/) [17].

Antimicrobial Susceptibility
Testing. The agar dilution method was used to determine the minimum inhibitory concentrations (MICs) of the antibiotics against bacteria in accordance with the guidelines of the Clinical and Laboratory Standards Institute (CLSI document M100-S28, 2017) and European Committee on Antimicrobial Susceptibility Testing (EUCAST 2017). All tests were carried out in triplicate. Briefly, each isolate was diluted in the logarithmic growth period with 0.9% saline to achieve a 0.5 McFarland standard suspension, equivalent to an inoculum with 5 * 10 5 CFU/mL. Suspensions of approximately 5 μL were plated on Mueller-Hinton (MH) agar plates (with a series of antibiotic concentrations between 0.0625 and 1,024 mg/L). The inoculated plates were then incubated for 18 hours at 37°C in a constant-temperature incubator. The MIC was defined as the lowest antibiotic concentration showing no colony growth. The commercially available E. coli strain ATCC 25922 served to ensure the quality of the plates, and E. coli DH5α carrying an empty cloning vector was used for comparison.
2.3. Cloning of K. pneumoniae R46 Resistance Genes. The genomic DNA of K. pneumoniae R46 was extracted using an AxyPrep Bacterial Genomic DNA Miniprep Kit (Axygen Scientific, Union City, CA, USA). To clone resistance genes, the primers of the ORFs with their upstream promotor regions were designed by using Primer Premier 5.0 and were synthesized by Shanghai Sunny Biotechnology Co. Ltd. (Shanghai, China) ( Table 1). A standard protocol for PCR with PrimeSTAR HS DNA Polymerase (TaKaRa, Dalian, China) was performed under the following conditions: an initial cycle of 94°C for 1 min, followed by 35 cycles of 10 s at 94°C, 15 s at a specific annealing temperature, and then an extension step of 1 min/kb at 72°C, with a final extension step of 10 min at 72°C (Table 1). The PCR products were cloned into the pMD™19-T vector (TaKaRa, Dalian, China) ( Table 2). The resulting recombinant plasmids were transformed into E. coli DH5α using the calcium chloride method, and bacterial colonies were grown on Luria-Bertani agar plates supplemented with ampicillin (100 mg/L). The recombinant plasmids were isolated and PCR-amplified to confirm the sizes of the inserted fragments. The cloned fragments from the transformants were further characterized by Sanger sequencing (ABI3730 Analyzer, USA) and compared to reference resistance genes using the BLASTN program.

Conjugation Experiment.
A conjugation experiment was carried out by biparental mating on sterile nitrocellulose filters, and rifampin-resistant E. coli C600 (EC600) was used as the recipient. Overnight cultures of the donor strain K. pneumoniae R46 and the recipient strain EC600 were mixed together in 3 mL of LB broth, harvested, and resuspended in 80 μL of LB. The mixture was spotted on a 1 cm 2 filter membrane, placed on an LB plate, and then incubated for mating at 37°C for 12-18 hours. The transconjugants were selected on two kinds of Mueller-Hinton agar plates: one containing 1,024 mg/L rifampin and 32 mg/L florfenicol and the other containing 1,024 mg/L rifampin and 32 mg/L chloramphenicol. The plasmid DNA of the transconjugant was extracted and analyzed by agarose gel electrophoresis and compared to the plasmid profile of K. pneumoniae R46. PCR analysis of the genes encoded in the plasmid was performed to further verify the plasmid in the transconjugants. The primers for ORFs with the predicted promoter regions. b The underlines present the restriction enzyme sites and their protective bases.
2.5. Sequencing and Annotations of the K. pneumoniae R46 Genome. Genomic DNA was extracted from K. pneumoniae R46 as mentioned above and sequenced with Pacific Biosciences sequencers at Annoroad Gene Technology Co. LTD (Beijing, China). In addition, a paired-end library with 300 bp insert sizes was constructed and sequenced from both ends with Illumina HiSeq-2500. Pacific Biosciences sequencing reads of approximately 10-20 kb in length were assembled by Canu v1.2 [18]. The Illumina reads were then mapped onto the assembled contigs to correct the primary assembly by using bwa0.7.13, samtools1.3, and GenomeAna-lysisTK2.3.9 [19,20]. Glimmer3.02 software with default parameters was used to predict potential open reading frames (ORFs) [21]. ORF annotations were determined by performing BLASTX comparisons with the NCBI nonredundant protein database. Comparisons of nucleotide sequences and amino acid sequences were performed by BLASTN and BLASTP, respectively [22]. BLASTP was applied to compare amino acid sequences with those in the Antibiotic Resistance Genes Database (ARDB). A map of the plasmid with GC content and GC skew was drawn with the online CGView Server (http://stothard.afns.ualberta.ca/cgview_server/) and local GView 1.7 with a visual interface [23]. The rRNA gene sequences were annotated by the online tool RNAmmer (http://www.cbs.dtu.dk/services/RNAmmer/) [24], and the tRNA sequences were annotated by the online tool tRNAscan-SE 2.0 (http://lowelab.ucsc.edu/tRNAscan-SE/) [25]. Promoter sites were determined by using Soft Berry BPROM software (http://linux1.softberry.com/berry.phtml? topic=bprom&group=programs&subgroup=gfindb). The plasmid sequences used in this study were downloaded from the NCBI nucleotide database (http://www.ncbi. nlm.nih.gov).
2.6. Comparative Genomic Analysis. Sequences containing resistance genes were obtained from the NCBI nucleotide database by the BLASTN program using the resistance gene sequences of K. pneumoniae R46 as the query. The resulting sequences were filtered, and only sequences containing resistance genes were retained. CD-HIT was used to cluster the retained sequences using the genome sequence of K. pneumoniae R46 as the reference with identity of 90% and coverage of 85% [26]. The sequence sharing the greatest similarity to the other sequences in each cluster was chosen as the candidate for ortholog analysis. Orthologous groups of the genes from the candidate sequences were identified using BLASTP [22]. Sequence retrieval, statistical analysis, and other bioinformatic tools used in this study were applied with Perl and Bioperl scripts (http:// www.perl.org/).

Nucleotide Accession Numbers.
The sequences are now available in the NCBI nucleotide database. The accession numbers for the chromosome and the plasmids are CP035777, CP035774, CP035775, and CP035776, respectively.

Results
3.1. Isolation and Identification of K. pneumoniae R46. K. pneumoniae R46 is a gram-negative bacillus with small, nonhemolytic, gray, and mucoid colonies after overnight culture on blood agar plates. Whole-genome comparative analysis combined with 16S ribosomal RNA gene homology analysis showed that the most closely related species were three Klebsiella pneumoniae strains: Klebsiella pneumoniae strain INF042 (CP024542), Klebsiella pneumoniae strain INF059 (CP024545), and Klebsiella pneumoniae strain KSB1_7J (CP024548) all with coverages of 99% and identities of 99%. MLST of seven housekeeping genes from the K. pneumoniae R46 genome showed optimum matching to the same housekeeping genes of the K. pneumoniae ST37 genome, with identities of 100.00%, 100.00%, 100.00%, 99.77%, 100.00%, 100.00%, and 100.00%, respectively. Finally, we classified the strain into the genus K. pneumoniae and named it K. pneumoniae R46 with MLST type ST37.

The Conjugative Plasmid pR46-270 Carries Multiple
Resistance and Virulence Genes. Sequence annotation revealed that a conjugative system consisting of 24 tra genes, 5 trb genes, and a finO gene was encoded in a region approximately 36.5 kb in length in pR46-270 (Figure 1(b)). The conjugation experiment confirmed the transferability of pR46-270, facilitating the mobilization of pR46-27 (Table 5). All 26 resistance genes carried by pR46-270 were clustered in a region 47.5 kb in length (Figure 1(b), Figure 3). Interestingly, all three known sulfonamide genes (sul1 (orf00311 and orf00319), sul2 (orf00327), and sul3 (orf00284)) were found to be encoded in one K. pneumoniae plasmid for the first time in this study. Comparative genomic analysis demonstrated that the MDR region could be divided into six fragments, and all of the resistance genes were associated with the mobile genetic elements, including two class 1 integrons. The first fragment was a transposon mediated by Tn1721 (5,749 bp in length, between 177.11 and 171.36 kb) encoding two resistance genes, tetA and tetR, which showed >99% similarity to a fragment in pK1HV from a K. pneumoniae strain isolated from a healthy neonate in Vietnam. The second was a class 1 integron (11,770   insertion sequences (IS, IS26, IS6100, and ISCR1). This fragment shared the highest identity (99%) with a sequence in plasmid pJIE137 from a K. pneumoniae strain isolated from a patient in Australia. The fourth fragment was a 7,634 bp sequence (between 204.11 and 211.74 kb) that could be divided into two parts. One part contained a truncated Tn6029 (containing repA and repC of the repABC operon) and three resistance genes (sul2 and strAB). The other part was a Tn4352 containing two copies of IS26 and aphA1a. Both parts were similar (>99% identity) to a region in plasmid p123 from an E. coli strain isolated from a canine. The fifth fragment, containing IS26, aac3, and tmrB, was a 1,934 bp sequence (between 211.97 and 223.91 kb) that showed >99% similarity to a fragment in plasmid p12181-KPC from a K. pneumoniae strain isolated from a patient in China. The last fragment was a 3,374 bp sequence (between 214.38 and 217.75 kb) composed of a class 1 integron with three resistance gene cassettes (aacA4, bla OXA-1 , and catB3) that showed >99% sequence similarity to a fragment in plasmid pAUSMDU8141-1 from a Citrobacter farmeri strain isolated from a patient. In addition to the multiantibiotic resistance genes, four clusters (operons) of heavy metal resistance genes, including those for silver (silA, silB, silC, silE, silF, silR, and silS), copper (copA, copB, copC, copD, pcoE, copG, pcoR, and pcoS), arsenic (arsR, arsD, arsA, arsB, and arsC), and mercury (merA, merC, merP, merE, merD, merT, and merR), were identified in the plasmid. Moreover, several gene clusters or genes related to virulence were identified in the plasmid, such as two iron uptake systems (fecABCDEIR and iucABCDiutA), the genes of the lac operon (lacIYZ), and the fimbrial protein gene fimK.

Discussion
In this work, we analyzed the MDR strain K. pneumoniae R46 of ST37 isolated from a rabbit in south China. According to MLST typing, there are more than 3,000 ST types of K. pneumoniae identified worldwide (http://bigsdb. pasteur.fr), of which ST37 strains have mostly been reported to be MDR clones. The K. pneumoniae ST37 bacterium has largely been isolated from human clinical samples [27,28]. Recently, ST37 was reported to have been isolated from animals, including companion animals [29] and chickens [30]. K. pneumoniae R46 carried 34 resistance genes and showed resistance to a variety of antibiotics, including β-lactams, amphenicols, aminoglycosides, and quinolones. In addition, this strain harbored three resistance plasmids, and 76.47% (26/34) of its resistance genes were encoded in a conjugative plasmid, all of the resistance genes were associated with mobile genetic elements. It demonstrated that horizontal gene transfer played an important role in the development of the multidrug resistance of K. pneumoniae R46.
To date, all mdfA variants have been found to be encoded in chromosomes, and no mobile genetic elements have been identified in their neighboring regions, indicating that mdfA genes are relatively conserved and might not be carried and   transferred by mobile genetic elements. Our comparative genomic analysis demonstrated that the sequences next to the mdfA genes in different bacterial genera had different structures, while sequences of the same species or genera, to some extent, showed conserved structures. These observations indicated that sequence rearrangement rarely occurs in the mdfA-encoding region.
In addition to antibiotic agents used for human and animal therapy, a large number of other chemical substances with antibacterial activities, such as heavy metals and detergents, are used in human health care and agricultural practices. In addition to encoding 26 antibiotic resistance genes, the pR46-270 plasmid harbored 4 clusters of heavy metal resistance genes (including silver, copper, arsenic, and mercury, a total of 29 genes) and a number of other metal (iron) and virulence-related genes or gene clusters. Associations between heavy metal and antibiotic resistance have been reported previously [34]. Resistance to metals and to antibiotics might be subject to coselection [34]. Metal contamination represents a long-standing, widespread, and recalcitrant selective pressure with both environmental and clinical importance, and it potentially contributes to the maintenance and spread of antibiotic resistance factors.

Conclusion
Whole-genome sequencing of an animal-derived K. pneumoniae isolate harboring three resistance plasmids revealed that in addition to floR encoded in the pR46-27 plasmid, a chromosome-encoded mdfA variant, named mdfA2, also conferred florfenicol resistance. The genome encoded 34 drug resistance genes, of which 26 were encoded in the conjugative plasmid pR46-270. These 26 resistance genes were all associated with mobile genetic elements that shared homologous sequences in different bacterial genera of different origins. Four clusters of heavy metal (silver, copper, arsenic, and mercury) resistance genes and a number of virulence-related genes or gene clusters were also identified in the pR46-270 plasmid. The results of this work demonstrated that the plasmids with multidrug resistance genes were present in animal-derived bacteria and more florfenicol resistance genes such as mdfA2 could be present in bacterial populations. Active surveillance efforts are imperative for monitoring the prevalence of antibiotic-resistant bacteria, especially florfenicol-resistant strains, in animals around the world.

Abbreviations
BLAST: Basic local alignment search tool.

Data Availability
The data related to this paper are deposited in the NCBI database.