Characteristics of antimicrobial susceptibility of the mcr-positive K. pneumoniae strain
We collected fecal samples from chicken manure, chicken farm workers, and healthy workers in chicken farms and residents nearby villages around Hangzhou neighborhood. A total of 112 strains of Enterobacteriaceae were identified, among which 60 strains were isolated from chicken, 5 strains were from chicken farm workers, and 47 strains were from healthy residents. Colony PCR was conducted to screen mcr genes using primers as described in materials and methods. Two K. pneumoniae KP26 and KP29 from chicken manure were found to harbor mcr-8 gene. Antimicrobial susceptibility analysis of KP26 and KP29 were performed and interpreted according to CLSI guidelines (Table 1). The result showed that KP26 and KP29 exhibited low-level resistance to polymyxin B (KP26, MIC = 4 mg/L; KP29, MIC = 4 mg/L) and colistin (KP26, MIC = 8 mg/L; KP29, MIC = 4 mg/L). Besides that, KP26 and KP29 also displayed resistance to tigecycline (KP26, MIC = 8 mg/L; KP29, MIC = 8 mg/L). MIC data revealed that KP26 and KP29 exhibited resistance to cefazolin (> 128 mg/L), levofloxacin (> 32 mg/L), aztreonam (64 mg/L), ceftriaxone (> 64 mg/L), cefepime (> 64 mg/L), amikacin (> 128 mg/L), ceftazidime (64 mg/L), cefepime-sulbactam (64/32 mg/L), and ciprofloxacin (> 32 mg/L), but is susceptible to carbapenem antibiotics, including ertapenem (0.25 mg/L), imipenem (0.125 mg/L), and meropenem (0.03 mg/L) (Table 1).
Conjugation experiments showed that the colistin resistance gene mcr-8 of KP26 and KP29 could be successfully transferred to the recipient E. coli J53, making the conjugants acquire colistin resistance phenotype. Results of S1-PFGE and Southern hybridization showed that KP26 carried 4 plasmids and KP29 carried 5 plasmids, with mcr-8 gene located at plasmid 101.2 kb (Fig. 1).
S1-PFGE profile (left) and Southern hybridization (right) analysis using mcr-8-specific probes to detect mcr-8-harboring isolates. Salmonella strain H9812 was used as a control strain and size marker. The names of the isolates are shown in the first line. The arrows indicated the locations of mcr-8 harboring plasmids according to the Southern hybridization experiment
Genomic analysis and molecular type of K. pneumonia KP26 and KP29
The complete genomes of isolates KP26 and KP29 were sequenced by WGS. KP26 consists of a 5,252,651 bp chromosome and four plasmids (pKP26-tmexCD1, pKP26-mcr8, pKP26-3, pKP26-4) (Table 2). KP29 consists of a 5,253,463 bp chromosome and five plasmids (pKP29-tmexCD1, pKP29-2, pKP29–mcr8, pKP29-4, pKP29-5) (Table 2). MLST analysis showed that the KP26 and KP29 strains belong to ST11.
Antibiotic resistance determinants blaSHV-182, fosA, oqxA and oqxB were both detected on chromosomes of KP26 and KP29. Most of the resistance genes are located on plasmids, especially on the large plasmids pKP26-tmexCD1 and pKP29-tmexCD1, which are 271,379 bp. They include aph(6)-Id, aph(3′)-Ia, aph(3”)-Ib, aadA1, aadA2b, armA, blaDHA-1, msr(E), mph(E), cmlA1, qnrB4, sul1, sul3, tmexD1, toprJ1, and tmexC1. Only one resistance gene, mcr-8, has been identified in pKP26-mcr8 and pKP29-mcr8. Other resistance genes were identified on two other plasmids of KP26 and three other plasmids of KP29.
Characterization of mcr-8-harboring plasmids
Plasmid pKP26-mcr8 was of type IncFIA/IncFII with a total length of 101,185 bp and contained only one antimicrobial resistance gene, mcr-8.1. It was 100% identical to plasmid pKP29-mcr8. Therefore, we only describe the characterization of pKP26-mcr8. The mcr-8.1 gene on pKP26-mcr8 was associated with the genetic context of IS903B-orf-dgkA–baeS–copR–mcr-8.1-orf-IS903B. The pKP26-mcr8 was shown to be conjugative under laboratory conditions. We performed comparative genomic analysis based on DNA sequences of pKP26-mcr8 and five plasmids with different sources in NCBI (Fig. 2). The results showed that pKP26-mcr8, pKP46-mcr8 and pKP91 [13] isolated from animals had similar genetic background around the mcr-8 gene IS903B-orf-dgkA–baeS–copR–mcr-8.1-orf-IS903B. In clinical plasmid pMCR8_095845, p18-29mcr-8.2 and environmental plasmid pZZW20-88 K, ISKpn26 took the position of IS903B, which was located in the downstream region of mcr-8 gene.
Genetic features of the mcr-8-carrying plasmid in K. pneumoniae strain KP26 and KP29. The linear genetic environment surrounding mcr-8 is depicted. Arrows indicate the direction of transcription for each gene, and different colors represent different genes. The mcr-8 gene is shown in red, genes associated with mobile elements are shown in green, the three genes in the figure are shown in black, and hp and other functional genes are shown in yellow. Regions with high homology are represented by gray shading
Evolutionary analysis of isolates carrying mcr-8
We retrieved genome sequences of 138 mcr-8 positive K. pneumoniae from the NCBI database. Among these isolates, 122 strains had definite source. There are 63 strains isolated from animals, 56 strains from human, and 3 from environment (Fig. 3). Twenty strains did not have complete genome, so we constructed a phylogenetic tree consisting of 118 K. pneumoniae isolates, K. pneumoniae KP26 and KP 29 based on single nucleotide polymorphisms (SNPs) of core genomes. The 120 K. pneumoniae isolates were clustered in 3 clonal groups (Fig. 4). These isolates were identified in North America, Asia and Africa. Most of them were detected in China, followed by Thailand and Vietnam, indicating that Asia is the main prevalent continent for mcr-8 positive K. pneumoniae. The most common MLST type among the 114 strains was ST43 (17/114), followed by ST11 (16/114), which is also the dominant epidemic strain in China [20]. K. pneumoniae KP26 and KP29 were belong to cluster II and closed to KP_SAMN23139063. It is worth noting that isolates in cluster III were from different countries around world isolated from humans, animals and environment. This implies that mcr-8 positive K. pneumoniae are undergoing evolution during the process of dissemination.
Among all 138 isolates containing mcr-8 in the NCBI database, 131 of them had geographical distribution with well-defined locations. Different hosts of the isolates are marked with different colors, with red, yellow, blue and black indicating human, animal, environmental and unknown origin respectively. The number of isolates harboring mcr-8 in each country is plotted under the country name on a world map. And the world map was created using the corresponding map data of the R package ggplot2 v3.3.5
Phylogenetic tree generated from the core genome sequences of the mcr-8-harboring isolates identified in this study and other mcr-8-harboring K. pneumoniae isolates in the NCBI database. Each isolate has a sample identifier (ID), location, MLST, and host. The figure shows important antimicrobial resistance genes (ARGs) and major virulence-associated genes (VGs)
In order to investigate the virulence genes in mcr-8-positive K. pneumoniae, the whole genome sequence of mcr-8-positive K. pneumoniae were compared with virulence factor database VFDB. The results showed that the virulence genes included iron-absorption virulence gene (entA, entB, fepC), invasive virulence genes (ompA), adhesive virulence gene (yagV/ecpE, yagW/ecpD, yagX/ecpC, yagY/ecpB, yagZ/ecpA, ykgK/ecpR), and enterotoxin gene (astA). Among the 120 strains of K. pneumoniae, only 1 strain was found to carry both iroB and iucA.
Characterization of the tmexCD1-toprJ1-harboring plasmid
The plasmid type of pKP26-tmexCD1 was IncFIB(Mar)/IncHI1B, with a length of 271,379 bp. Resistance genes include aph(6)-Id, aph(3’)-Ia, aph(3’)-Ib, aadA1, aadA2b, armA, blaDHA-1, msr(E), mph(E), cmlA1, qnrB4, sul1, and sul3. The sequence of pKP29-tmexCD1 was 100% identical to pKP26-tmexCD1. To investigate the core genetic environment of tmexCD1-toprJ1, we analyzed the tmexCD1-toprJ1-containing regions from four tmexCD1-toprJ1-bearing plasmids in Klebsiella spp. isolated from animals, humans and environment. The core genetic structures of tmexCD1-toprJ1 in Klebsiella spp. were divided into two types (Fig. 5). The type I genetic context is IS903B–strB–strA–tnpR–∆tnpA–toprJ1–tmexD1–tmexC1–tnfxB1–∆hp-IS26. Four tmexCD1-toprJ1-bearing plasmids (pHKU57_1, pKP26-tmexCD1, pKP46-3, and pKPT698-tmexCD) were belong to type I, and slightly different is that the pHKU57_1 lacks an IS903B upstream. They possessed two insertion sequence (IS) elements IS26 located on the flank of tmexCD1-toprJ1, which conferred the ability to acquire and mobilize tmexCD1-toprJ1, resulting the transmission in Klebsiella. The core genetic environment of type II was strB–strA–tnpR–ΔtnpA–toprJ1–tmexD1–tmexC1–tnfxB1–hp in plasmid pHNAH8I-1[16]. It lacked the flanked IS26 element compared with type I, which indicates that the mobilization of tmexCD1-toprJ1 might be driven by IS26 from the plasmid pHNAH8I-1[21]. The IS elements play an important role in the spreading of tmexCD1-toprJ1.
Genetic features of the tmexCD1-toprJ1-carrying plasmid in K. pneumoniae strain KP26 and KP29. The linear genetic environment surrounding tmexCD1-toprJ1 is depicted. Arrows indicate the direction of transcription of each gene, and different genes are shown in different colors. The tmexCD1-toprJ1 gene is shown in red, genes associated with mobile elements are shown in green, the three genes in the figure are shown in black, hp and other functional genes are shown in yellow, and int genes are shown in purple
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