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Metallo-β-lactamase gene blaVIM was identified on the chromosome of four Pseudomonas sp. isolates from a chicken farm, including one Pseudomonas aeruginosa isolate from a swallow (Yanornis martini), one Pseudomonas putida isolate from a fly, and two P. putida isolates from chickens. The four isolates shared two variants of blaVIM-carrying genomic contexts that resemble the corresponding regions of clinical metallo-β-lactamase-producing Pseudomonas spp. Our study suggests that the surveillance of carbapenemase-producing bacteria in livestock and their surrounding environment is urgently needed.
Carbapenems are critically important antimicrobials as a last line of defense against multidrug-resistant Gram-negative bacterial infections (1, 2). As such, the increasing prevalence of carbapenemase-producing isolates in animal husbandry is of great concern. While the metallo-β-lactamase (MBL)-producing bacteria have been commonly identified from food animals (3,–7), blaMBL-carrying Pseudomonas spp. are rarely reported in animal husbandry or the surrounding environment. Although we have reported the high prevalence of NDM in Enterobacteriaceae isolates from poultry production in Shandong Province (7), carbapenemase-producing non-Enterobacteriaceae isolates have not been identified in the same region. Here, we report four chromosome-borne VIM-positive Pseudomonas isolates: one Pseudomonas aeruginosa isolate from a swallow (Yanornis martini), one Pseudomonas putida isolate from a fly, and two P. putida isolates from chickens. The blaVIM-2 gene was identified in the P. aeruginosa isolate, but 27 bp at the 3′-terminal region of blaVIM-2 was truncated by an IS6100 element in three P. putida isolates, resulting in a new variant of blaVIM gene, which was designated blaVIM-48; the blaVIM-carrying genomic regions in these Pseudomonas sp. isolates closely resembled the corresponding regions of clinical MBL-producing Pseudomonas spp.
Ninety-eight nonduplicated samples were randomly collected, with an informed consent form, from a commercial chicken farm in Shandong Province, China (chicken cloaca swabs, n = 30; flies, n = 30; dog anal swabs, n = 17; swallow fecal swabs, n = 10; farmer fecal swabs, n = 6; sewage, n = 5); the procedures for the collection of all samples were consistent with those previously reported (7). All samples were plated on CHROMagar Pseudomonas (CHROMagar, Paris, France) containing 8 μg/ml meropenem (Ouhe Technology Company, Beijing, China). Putative Pseudomonas colonies that turned blue when tested were recovered from 17 samples, and one colony from each sample was identified to the species level by 16S rRNA sequencing (8). Of these, four isolates were blaVIM positive, confirmed by PCR and sequencing (9). Further matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS; Bruker Daltonik GmbH, Bremen, Germany) analysis confirmed that the positive isolates consisted of one P. aeruginosa isolate from swallow fecal swabs, including DZ-B1; one P. putida isolate from a fly, DZ-F23; and two P. putida isolates, DZ-C20 and DZ-C18, from chicken cloaca swabs.
MIC analysis showed that the four Pseudomonas sp. isolates were almost resistant to all of the β-lactam antibiotics tested, including meropenem, imipenem, aztreonam, and ceftazidime, with only isolate DZ-B1 showing susceptibility to aztreonam (4 μg/ml) (Table 1) (10, 11). To further investigate the genetics background of the four isolates, whole-genome sequencing was conducted as described previously (7), and draft assembly sequences were searched against the antibiotic resistance gene database (https://cge.cbs.dtu.dk/services/ResFinder/), which confirmed the presence of blaVIM and other antibiotic resistance genes (Table 1). Additionally, to investigate the location of the resistance element, the genomic DNA of four VIM-producing isolates was digested with I-Ceu1 and separated by pulsed-field gel electrophoresis (PFGE). The result and Southern blot analysis showed that blaVIM was located on the chromosomes of all four Pseudomonas isolates (see Fig. S1 in the supplemental material). Moreover, the core genome phylogenetic analysis was conducted to reveal the relationship between the four isolates and other known Pseudomonas isolates carrying blaVIM in the NCBI database (see Table S1 in the supplemental material) using the Parsnp program (12). The phylogenetic tree revealed that three blaVIM-carrying P. putida isolates had distinct genomic heterogeneity, which is consistent with PFGE analysis of SpeI patterns (Fig. S1). The ST385 of the P. aeruginosa isolate DZ-B1 from our study was confirmed by whole-genome sequencing. This sequence type has been associated with clinical P. aeruginosa isolates from India (13). The genome context of DZ-B1 was closely related to that of seven blaVIM-2-carrying clinical P. aeruginosa isolates from GenBank (see Fig. S2 in the supplemental material).
Four blaVIM-carrying contigs were identified from four isolates and confirmed by PCR and sequencing (primers listed in Fig. S3 in the supplemental material). Analysis of the flanking regions of blaVIM-2 on the chromosome of DZ-B1 revealed that it was located in a Tn5090-like transposon bracketed by two 25-bp inverted repeats, IRi and IRt (Fig. 1A), suggesting that the whole region can be mobilized with the tni machinery (14, 15). This complete transposon shared 92.3% (7,296/7,907) nucleotide sequence identity to a P. aeruginosa Tn5090-like transposon, also containing blaVIM-2 gene, isolated from a Chinese patient (GenBank accession no. AM993098.1), and even greater sequence identity (99.4%, 4,147/4,171) was found within the corresponding tniC-blaVIM-2-aacA4-dhfr2 gene array located at the 3′ end of the Tn5090-like transposon. The Tn5090-like transposon contained a conserved segment with an integrase gene, intl1, at the 3′ end. Intl1 is associated with the integration of resistance gene cassette array blaVIM-2-aacA4-dhfr2, which is in the opposite orientation and confers resistance to carbapenems, some aminoglycosides, and trimethoprim, respectively. Another conserved gene cluster, tniA-tniB-hp-tniC, was located at the 5′ end of the transposon (14). tniA and tniB were predicted to be involved in the transposition, while tniC codes for a recombination protein that differs from those encoded by intl1, tniA, and tniB (Fig. 1A) (14). This gene arrangement, in combination with intl1, is involved in the transfer of a blaVIM-2-carrying cassette in clinical Pseudomonas spp. (16,–19).
A 9,875-bp fragment containing the blaVIM-2-like gene was observed in three P. putida strains that shared 99.9% sequence identity. Comparison analysis revealed that the blaVIM-2-like gene had a 27-bp deletion at the 3′-terminal region, including its original stop codon, versus the 801-bp blaVIM-2 in DZ-B1 and the previously reported P. aeruginosa isolates. Immediately downstream of the 27-bp deletion was an intact insertion element, IS6100, and the truncated blaVIM-2 gene appeared to be terminated at the TAG stop codon located in the right inverted repeat of IS6100. This resulted in an 810-bp open reading frame, designated VIM-48 (GenBank accession no. KY362199), that was 9 bp longer than the previously reported gene (Fig. 1B). Further analysis of the flanking regions revealed that it was very similar to the Tn6217 region in the blaIMP-9-carrying plasmid pOZ176 from a clinical P. aeruginosa isolate from Guangzhou, China (20). The upstream and downstream regions of blaVIM-48 contained the aacA4-intI1-Tn1403-like and sul1-hp-IS6100 gene clusters, respectively. These two regions shared 99.9% (2,792/2,794) and 99.9% (5,726/5,733) nucleotide sequence identity, respectively, to the corresponding regions of Tn6217; however, the downstream fragment was inverted, perhaps leading to the missing 27-bp segment at the 3′ end of blaVIM-2 (Fig. 1A).
Similar to the Tn5090-like transposon in P. aeruginosa isolate DZ-B1, a 3,801-bp segment harboring the IS6100-blaVIM-48-aacA4-intI1 gene cluster in the three P. putida isolates was bracketed by two 25-bp inverted repeats, IRi and IRt, suggesting that it reached its current location by transposition (Fig. 1A) (21). Moreover, compared to a 6,942-bp fragment in the 3′ region of the blaVIM-2-carrying segment in P. aeruginosa DZ-B1, only a 405-bp segment containing dhfr2 was absent from the P. putida strains. The remaining two nucleotide fragments were highly similar, sharing 100% (1,444/1,444) and 99.9% (5,091/5,097) identity, respectively (Fig. 1A).
The 810-bp blaVIM-48 gene was amplified by PCR and confirmed by sequencing using the primers listed in Fig. S3 and then cloned into vector pHSG398 (TaKaRa, Dalian, China), which resulted in a recombinant plasmid pHSG398-V01. The vector and recombinant plasmid were introduced into Escherichia coli DH5α (TaKaRa, Japan) by electroporation, resulting in two recombinant E. coli strains, designated HSG398-V01 and HSG398-DH5α. MIC analysis showed that HSG398-V01 was more active than HSG398-DH5α against imipenem and ceftazidime, with 4- and 8-fold increases, respectively (Table 1), which suggested that this blaVIM-48 gene is functional in the three P. putida isolates. Phenotypic detection of metallo-β-lactamases was performed on the three P. putida isolates according to the previous report (22), and synergy between imipenem and EDTA was observed for all three strains (data not shown).
To date, 47 variants of the blaVIM gene have been reported (23), and all of them were 801 bp long and differed in several amino acids, whereas the blaVIM48 gene in this study was 810 bp long, possibly due to the inverted insertion of a IS6100-contig-containing fragment, revealing a new generation mechanism for the variant of blaVIM gene. Similar to the spread of blaVIM-2 in bacteria of clinical origin, the mobile genetic elements, such as Tn5090, Tn1403, intl1, and IS6100, may play an important role in the dissemination of the blaVIM gene and its conserved flanking regions in bacteria among food animals (16, 17, 24). Our study has found MBL-producing Pseudomonas isolates not only in the livestock but also in their surrounding environment, suggesting that surveillance of carbapenemase-producing bacteria in livestock and their surrounding environment is urgently needed.
All genome assemblies of 4 strains were deposited in GenBank and are registered under BioProject accession number PRJNA381373, and the new VIM enzyme was designated VIM-48 and deposited in GenBank under accession number KY362199.
This work was supported in part by the National Natural Science Foundation of China (no. 31572568, 31422055, 31530076, and 81661138002), the National Key Basic Research Program of China (no. 2013CB127200).
T.R.W. was supported by MRC grant DETER-XDR-CHINA (MR/P007295/1).
Supplemental material for this article may be found at https://doi.org/10.1128/AAC.00167-17.