Thirty clinical K. pneumoniae strains were recovered from diseased dogs and cats during July, August, and September in 2010 and from July to October in 2012 at one animal hospital in Guangdong Province, China. Further information about the antimicrobial treatments of these pets were unfortunately not available. Samples were collected from the exudates of the infection areas (urinary tract infections, skin infections, or intra-abdominal infections) or feces, then seeded on MacConkey agar at 37°C. Each isolate was from a single animal. All bacterial species were identified with classical biochemical methods and confirmed using a matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) method (Shimadzu, Japan). As 16S-RMTase can confer high-level resistance to both GEN and amikacin (AMK), all isolates were subcultured in MacConkey agar containing 64 mg/L GEN and 64 mg/L AMK to screen for GEN/AMK-resistant isolates.

All the strains exhibiting aminoglycoside resistance were screened for 16S-RMTase genes including rmtA, rmtB, rmtC, rmtD, rmtE, rmtF, rmtG, rmtH, armA, and npmA (Hidalgo et al., 2013b; Xia et al., 2016). Aminoglycoside-resistant isolates were further analyzed for the presence of extended spectrum β-lactamase (ESBL) genes (bla_TEM, bla_CTX-M, and bla_SHV) using previously described primers (Liao et al., 2015).

The minimal inhibitory concentration (MIC) of ampicillin (AMP), cefotaxime (CTX), meropenem, ciprofloxacin (CIP), norfloxacin (NOR), GEN, kanamycin (KAN), AMK, streptomycin (STR), neomycin (NEO), apramycin (APR), chloramphenicol (CHL), florfenicol (FFC), tetracycline (TET), sulfamethoxazole/trimethoprim (SXT) were determined by the agar dilution method according to Clinical and Laboratory Standards Institute guidelines (Clinical and Laboratory Standards Institute, 2015). E. coli ATCC 25922 was used as a quality control strain.

Chromosomal DNA digested with XbaI restriction enzyme was used for pulsed-field gel electrophoresis (PFGE) to analyze the genetic relatedness of all isolates containing 16S-RMTase genes (CHEF Mapper1, Bio-Rad Laboratories, Hercules, CA, USA) as previously described (Fang et al., 2015). PFGE patterns were analyzed with the Dice coefficient and the unweighted pair group method with average linkages (UPGMA) clustering method using BioNumerics software (Applied Maths, Ghent, Belgium). PFGE types were defined with >90% similarity between clusters. Multilocus sequence typing (MLST) of 16S-RMTase genes containing K. pneumoniae was performed according to the protocols described on the K. pneumoniae database website¹. Seven chromosomal genes were PCR amplified and sequenced. Then the sequences were compared with those reference sequences, submitted to the MLST database to determine allele numbers and STs (Belbel et al., 2014).

Isolates positive for 16S-RMTase genes were selected for conjugation experiments by the broth-mating method using STR-resistant E. coli C600 as the recipient to determine the transferability of 16S-RMTase genes. Transconjugants were selected on MacConkey agar plates supplemented with STR (1000 μg/mL) and AMK (64 μg/mL). Transconjugants harboring 16S-RMTase genes were confirmed by PCR and antimicrobial susceptibility testing as described above (Fang et al., 2015). Antimicrobial susceptibility testing of the transconjugants and co-transfer of other resistance genes as mentioned above were also determined.

Plasmids were preliminarily classified according to their incompatibility group by using the PCR-based replicon typing (PBRT) scheme described previously (Yao et al., 2011). To better characterize IncF plasmids, replicon sequence typing of IncF plasmids was performed according to the protocol previously described (Yang Q.E. et al., 2015). The A- and B- symbols indicate the absence of the FIA and FIB replicons, respectively.

PFGE with S1 nuclease (TakaRa Biotechnology, Dalian, China) digestion of genomic DNA was performed for all the transconjugants as described previously (Xia et al., 2015). After Southern blotting, fix the DNA to a Hybond-N⁺ membrane (GE Healthcare, Little Chalfont, UK), the plasmids were probed with the 16S-RMTase genes and bla_CTX-M-1G/9G gene (DIG High Prime DNA Labeling and Detection Starter Kit I, Roche Applied Science, Mannheim, Germany). All the transconjugants were subjected to restriction enzyme digestion (EcoRI) analysis to clarify whether a specific plasmid had been disseminated among the isolates (Deng et al., 2011b). EcoRI-RFLP types were defined with >80% similarity between clusters using the method described as PFGE analysis.

Reports on the genetic contexts of the 16S-RMTase genes indicated that IS26, Tn3, ISCR1, and ISCR3 played an important role in the dissemination of these genes. These transfer events enabled horizontal spread among different bacterial lineages (Deng et al., 2011b). Here we focused on the link between 16S-RMTase genes and mobile elements by PCR mapping. The primers using in PCR mapping were listed in Supplementary Table S1.

In this study, the owners of the companion animals from which fecal swabs and the exudates of the infection areas were taken gave permission for their animals to be used in this study. Written informed consent was obtained, and the study protocol in our research was approved by the South China Agriculture University Animal ethics committee and carried out in accordance with relevant guidelines.

Among 30 K. pneumoniae isolates from the hospital, 12 were resistant to AMK and GEN. However, all K. pneumoniae strains contained the 16S-RMTase genes rmtB (11/30) and/or armA (2/30) and a single isolate carried both genes (1/30). The AMK/GEN-resistant K. pneumoniae isolates showed an MDR phenotype (resistant to three or more classes of antimicrobials). Apart from aminoglycoside antibiotics, there was a very high frequency (>90.0%) of resistance to AMP, CTX, SXT, TET, CIP, NOR, CHL, and FFC. The most frequently observed pattern of MDR was GEN-CTX-SXT-AMP-CIP-TET-STR-KAN-AMK-CHL-NEO-NOR (Table 1).

We examined the prevalence of ESBL and AME genes in the 16S-RMTase gene-positive isolates. Among these, nine isolates contained bla_CTX-M that were distributed as bla_CTX-M-55 (6), bla_CTX-M-55 and bla_CTX-M-27 (2), and bla_CTX-M-55 and bla_CTX-M-14 (1). Two other ESBLs were also present in these isolates as bla_TEM-1 (10/12) and bla_{SHV -1} (10/12) (Table 1).

PFGE was successfully performed in all twelve 16S-RMTase genes positive K. pneumoniae isolates and were grouped into five clonal patterns designated PFGE types A to E (Figure 1). The predominant PFGE type was type C that accounted for 67% (8/12) of the isolates, suggesting that one small clonal outbreak had occurred. The MLST results revealed that one ST was the most prevalent in this collection: ST37 (9/12). ST2018, ST395, and ST147 each had one isolate (Table 1). The results obtained by MLST were consistent with those obtained by PFGE; eight rmtB-positive K. pneumoniae strains showing similar PFGE patterns were belonged to ST37.

Plasmids carrying rmtB from 10 isolates were successfully transferred to recipients by conjugation. All the transconjugants were highly resistant to AMK, GEN, and KAN (MICs ≥ 512 μg/mL). PBRT of the plasmid incompatibility groups showed that nine rmtB-bearing plasmids from K. pneumoniae isolates carried two replicons. The two replicons were confirmed to be IncFII in combination with IncN, and the other rmtB-bearing plasmid was non-typeable. All the IncFII plasmids were further determined to be F33:A-:B-.

Nine of the 10 transconjugants carried one approximately 97 kb plasmid. The other one harbored both ∼60 kb (rmtB positive) and ∼200 kb (armA positive) plasmids at the same time. Moreover, rmtB and bla_CTX-M-55 genes were located on the same 97 kb plasmid. The RFLP analysis indicated that 7 out of the 10 transconjugants had identical plasmid restriction patterns. The two remaining plasmids shared the same RFLP type (Figure 2).

The genetic profiles were successfully obtained in the 10 rmtB-positive transconjugants. We found four new genetic profiles in the 10 rmtB-positive transconjugants and all possessed Tn3 and bla_TEM-1 directly upstream of rmtB. Tn3 was truncated by IS26 in 9 of the 10 transconjugants, which were inserted at the same site (3573 of Tn3). The other isolate KP01 harbored the intact tnpA–tnpR genetic structure of Tn3, with a Na⁺/H⁺-exchanging protein gene and ISCR1 downstream rmtB. IS26 was also found directly downstream of rmtB in seven plasmids, which was located in the same orientation to the 5′ IS26 elements. Moreover, the Na⁺/H⁺-exchanging protein gene located between rmtB and IS26 (in the opposite orientation) occurred in strain KP30 plasmid (Figure 3). The four unique sequences were deposited in GenBank under accession numbers KY402260, KY402261, KY402262, and KY402263.