A Wohlfahrtiimonas chitiniclastica with a novel type of blaVEB–1-carrying plasmid isolated from a zebra in China

Background Wohlfahrtiimonas chitiniclastica is an emerging fly-borne zoonotic pathogen, which causes infections in immunocompromised patients and some animals. Herein, we reported a W. chitiniclastica BM-Y from a dead zebra in China. Methods The complete genome sequencing of BM-Y showed that this isolate carried one chromosome and one novel type of blaVEB–1-carrying plasmid. Detailed genetic dissection was applied to this plasmid to display the genetic environment of blaVEB–1. Results Three novel insertion sequence (IS) elements, namely ISWoch1, ISWoch2, and ISWoch3, were found in this plasmid. aadB, aacA1, and gcuG were located downstream of blaVEB–1, composing a gene cassette array blaVEB–1–aadB–aacA1–gcuG bracketed by an intact ISWoch1 and a truncated one, which was named the blaVEB–1 region. The 5′-RACE experiments revealed that the transcription start site of the blaVEB–1 region was located in the intact ISWoch1 and this IS provided a strong promoter for the blaVEB–1 region. Conclusion The spread of the blaVEB–1-carrying plasmid might enhance the ability of W. chitiniclastica to survive under drug selection pressure and aggravate the difficulty in treating infections caused by blaVEB–1-carrying W. chitiniclastica. To the best of our knowledge, this is the first report of the genetic characterization of a novel blaVEB–1-carrying plasmid with new ISs from W. chitiniclastica.

It is speculated that W. chitiniclastica is intrinsically resistant to fosfomycin due to the fosfomycin efflux proteins, the gene homolog encoding for transferases, and the gene homologous to mdtG that related to fosfomycin resistance (Kopf et al., 2021).In addition, research has revealed that W. chitiniclastica shows resistance to amikacin (Kopf et al., 2021), tetracycline (Snyder et al., 2020), and tobramycin (Kopf et al., 2021), and it is intermediate to ampicillin (Qi et al., 2016).However, W. chitiniclastica is susceptible to the majority of the known antibiotics including β-lactams, quinolones, and trimethoprim/sulfamethoxazole (Kopf et al., 2022).Therefore, these three categories of antimicrobials may be the best options for treating W. chitiniclastica infections (Schröttner et al., 2017).
This study showed the complete genome sequences of a W. chitiniclastica from the pancreas of a dead zebra in China, which carried one chromosome and one novel type of bla VEB−1carrying plasmid.Detailed genetic dissection was applied to this plasmid to display the genetic environment of bla VEB−1 .The transcription start site and the promoter of the bla VEB−1 region were identified.The data presented here provided a deeper genomics and bioinformatics understanding of W. chitiniclastica for clinical treatment and pathogenesis research.To the best of our knowledge, this is the first report of the genetic characterization of a novel bla VEB−1 -carrying plasmid from W. chitiniclastica.

Bacterial isolation
The blood, heart, liver, spleen, lung, kidney, and pancreas specimens from a dead zebra were collected from Shenzhen Safari Park in 2013 and transferred to our laboratory in Changchun under sterile conditions for bacterial isolation.For each specimen, the tissue was plated onto brain heart infusion agar medium, 5% sheep blood agar medium, and MacConkey agar medium.The sample was then incubated at 37 • C for 18 h under aerobic conditions.
The W. chitiniclastica strain DSM 18708 (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH, Germany), isolated from artificial culture of W. magnifica in Hungary, was used as the reference strain in this study (Kõljalg et al., 2015).

Bacterial identification and phenotypic characteristics
Gram staining, BD Phoenix TM -100 Automated Microbiology System (Becton, Dickinson and Company, USA) detection, PCR amplification of the 16S rRNA, rpoB, and gyrB genes followed by sequencing, and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS, Bruker Daltonics, USA) were conducted for bacterial identification.
A transmission electron microscopy investigation was carried out.The motility of the bacteria was tested on motility agar (Tittsler and Sandholzer, 1936).To determine the bacterial growth curves, the optical density at 600 nm (OD 600 ) was monitored for each culture with a 1-h interval during the first 12 h and a 2-h interval during the last 12 h using the NanoPhotometer N60 (Implen, Germany).
The biochemical characteristics were tested using the BD Phoenix TM -100 Automated Microbiology System, and the cellular fatty acid composition was detected using gas chromatography (Hewlett Packard 6890, USA) and identified using the Sherlock Microbial Identification System (MIDI, USA).

Sequencing and sequence assembly
The bacterial genomic DNA was isolated using the UltraClean microbial kit (Qiagen, Germany) and sequenced with a PacBio RS II sequencer (Pacific Biosciences, USA) (Fu et al., 2022;Li et al., 2022).The reads were assembled de novo using SMARTdenovo.1

Bacterial precise species identification
Bacterial precise species identification was conducted using pairwise average nucleotide identity (ANI) analysis between the bacterial genomic DNA sequenced in this study and the reference genome.2A cutoff of ≥ 95% ANI was used to define a bacterial species (Richter and Rosselló-Móra, 2009).

Phylogenetic analysis
The core genes and specific genes of the bacterial genomes were analyzed using the CD-HIT rapid clustering of similar proteins software with a threshold of 50% pairwise identity and 0.7 length difference cutoff in amino acid (Li et al., 2001(Li et al., , 2002;;Li and Godzik, 2006).The core amino acid sequences were extracted and aligned using MUSCLE (Edgar, 2004).Based on the core amino acid sequences, a maximum-likelihood phylogenetic tree was constructed using TreeBeST3 with a bootstrap iteration of 1,000.

Identification of the transcription start site and the promoter of the bla VEB−1 region
The total RNA of the bacterial isolates was extracted using the RNeasy maxi kit (Qiagen, Germany).Rapid amplification of cDNA ends (RACE) was performed using 5 -RACE system version 2.0 (Invitrogen, USA) according to the manufacturer's instructions.Three gene-specific primers GSP1 (5 -ATCCTTCTCATTGCTG-3 ), GSP2 (5 -CTCCTATTCTGGCATTTTTTG-3 ), and GSP3 primer (5 -AAGTTGTCAGTTTGAGCATTT-3 ) were used.The final PCR products were cloned into the pMD18-T vector, and then, the positive clones were identified and sequenced.The 5 -RACE experiment was repeated three times.Five clones were selected randomly and sequenced each time.The transcription start site was determined according to the sequence comparison between the positive clones and the bla VEB−1 region.The online database BPROM (Cassiano and Silva-Rocha, 2020) was used for promoter prediction.

Conjugation and electroporation experiments
Conjugal transfer and electroporation were performed as described previously (Liang et al., 2018;Guan et al., 2022).

Data availability statement
The complete sequences of the chromosome of BM-Y and the plasmid pBM-Y were submitted to GenBank under the accession numbers CP115969 and CP115970, respectively.

Identification of W. chitiniclastica BM-Y and its phenotypic characteristics
BM-Y was isolated from the pancreas specimens of a dead zebra.According to Gram staining and the sequences of the amplicon of the 16S rRNA, rpoB, and gyrB genes, BM-Y was identified as the Gram-negative bacteria W. chitiniclastica.The MALDI-TOF MS score of BM-Y was 2.418, confirming W. chitiniclastica.Scores above 2.300 represent a highly probable species identification.No other bacterial isolates were discovered in this study.BM-Y has a 97.19% ANI value with the reference genome DSM 18708 (accession number AQXD01000000).BM-Y contained one chromosome (2.0Mb in length) and one plasmid pBM-Y (42.3 Kb in length), and they were fully sequenced herein.The chromosome carried no resistance genes.The plasmid pBM-Y harbored bla VEB−1 , aadB, aacA1, and tetA(H).
Colonies of BM-Y were small (colony diameter 0.8-1.0mm), entire, convex, smooth, and glistening and were composed of short, straight, and non-motile rods (0.1-0.2 × 1.0-1.5 µm, Figure 1), which did not produce hemolysin on 5% sheep blood agar medium.The MICs of ceftazidime, cefotaxime, cefepime, aztreonam, gentamicin, and tetracycline of BM-Y were higher than that of DSM 18708.The MICs of 14 antimicrobial drugs are shown in Table 1.

Antibiotics
Minimum inhibitory concentration (MIC, µg/mL) Trimethoprim/ sulfamethoxazole < 0.5/9.5 < 0.5/9.5 < 0.5/9.5 The biochemical test results of BM-Y and DSM 18708 are shown in Table 2.A varied reaction between these two strains was observed for acetate, polymyxin B, and glycine.The cellular fatty acid profile (Table 3) of BM-Y revealed a proportionally high level of C 18:1 ω7c, C 14:0 , and C 16:0 , which was consistent with DSM 18708.

Phylogenetic analysis
Based on the bacterial core/pan-genome analysis, we conducted the phylogenetic analysis of 27 sequenced W. chitiniclastica isolates (Supplementary Table 1), including one complete genome sequence from this study and 26 draft genome sequences from GenBank (last accessed March 17, 2023).Construction of a phylogenetic tree (Figure 3) based on core genes (1602/3597, 44.53%) of 27 W. chitiniclastica genomes revealed that all strains clustered in one subclade with the reference genome DSM 18708, and BM-Y shared the closest genetic relationship with MUWRP0946 from Uganda.This result indicated a surprisingly   conserved core/pan genome without clear host or geographical clustering, suggesting a potential spread and transmission.This is in line with Kopf 's report (Kopf et al., 2022).

Organization of the bla VEB−1 -carrying plasmid pBM-Y
The modular structure of pBM-Y (Figure 4) was separated into the backbone and the accessory modules.The backbone contained three different rep (replication) with no similar DNA sequences (nucleotide identity < 95%) in GenBank, two parA (partition) with different sequences, three groups of relBE (a toxin-antitoxin system) with low nucleotide identities, one group of yefM/yoeB (a toxin-antitoxin system), and one group of hsdMSR (a type I restriction-modification system).No conjugal transfer genes were identified in the backbone.The accessory modules were composed of a bla VEB−1 region, a tetA(H) region, and intact or truncated insertion sequences (ISs) ISWoch2 and ISWoch3.
bla VEB−1 was originally found in Tn2000 in the plasmid pNLT1 from E. coli MG-1 (Poirel et al., 1999;Naas et al., 2001).Tn2000 was an IS26-composite transposon composed of In53 and was used as a reference herein.A detailed sequence comparison (Figure 5) was applied to the bla VEB−1 -carrying In53 from Tn2000 and the bla VEB−1 region from pBM-Y.Both In53 and the bla VEB−1 region harbored the bla VEB−1 cassette (with a 133-bp attachment site of the cassette, attC), the aadB cassette (with a 60-bp attC), and the aacA1/gcuG fusion gene cassette (with a 118-bp attC) (Naas et al., 2001), but they showed three major modular variations: (i) an intact attC_aacA1/gcuG in In53, while an interrupted one in the bla VEB−1 region; (ii) a gene cassette array (GCA) qacL-aadB-aacA1-gcuG-bla VEB−1 -aadB-arr-2-cmlA5-bla OXA−10 -aadA1 in In53, while a GCA bla VEB−1 -aadB-aacA1-gcuG in the bla VEB−1 region; and (iii) a truncated 5 -conserved segment (5 -CS) and a 3 -CS were upstream and downstream, respectively, of the GCA in In53, while a novel IS element ISWoch1 and a truncated one were upstream and downstream, respectively, of the GCA in the bla VEB−1 region.No direct repeats (DRs) were identified at the ends of the bla VEB−1 region from pBM-Y.
ISWoch1, ISWoch2, and ISWoch3 were named based on the first four letters of the species in which they were first discovered (Wohlfahrtiimonas chitiniclastica) and had IR of 37, 45, and 21 bp, respectively (Figure 4).ISWoch1 and ISWoch2 encoded two transposases, while ISWoch3 carried a single one.No DNA sequences similar (nucleotide identity < 95%) to these transposases were found using blastn analysis.The promoter of ISWoch1 was determined using 5 -RACE experiments.Fourteen of 15 positive clones were successfully sequenced.GACCT (Figure 6), the transcription start site of the bla VEB−1 region, was identified through multiple sequence alignments.This site was found in ISWoch1 and located 173 bp upstream of the initiation codon of bla VEB−1 .A promoter with the −10/−35 region (TATAAT/TTAGCA) was located upstream of this site (Figure 5).The spacing between the −10/−35 region was 18 bp.Based on the results of the promoter prediction, ISWoch2 contained a −10/−35 region (TATCAT/ATACCA, with a 21-bp spacer), and ISWoch3 carried a −10/−35 region (TAAAAT/TTATCA, with a 19-bp spacer).ISWoch1-bla VEB−1 -aadB-aacA1-gcuG-ISWoch1 was composed of the bla VEB−1 region (Figure 5).ISWoch2 was located upstream of the truncated ISWoch3 and downstream of the repA.Three intact ISWoch3 and three truncated ones were found in pBM-Y.ISWoch3-orf405-tetR(H)-tetA(H)-orf423 formed the tetA(H) region.

Conjugation and electroporation experiments
The bla VEB−1 -carrying transconjugants could not be obtained, regardless of the number of times the conjugation experiments were performed.This result might be due to the absence of the essential conjugal transfer genes, including rlx (relaxase), oriT (origin of conjugative replication), pri (DNA primase), and cpl (coupling protein), and the type IV secretion system (T4SS) in pBM-Y.
The bla VEB−1 -carrying electroporants were not obtained, no matter how many times the electroporation experiments were conducted.Because we failed to extract the plasmid pBM-Y from strain BM-Y.

Discussion
Wohlfahrtiimonas chitiniclastica is an emerging fly-borne zoonotic pathogen, which is often carried by different species of flies, and it causes infections in immunocompromised patients and some animals (Almuzara et al., 2011;Thaiwong et al., 2014), leading to bacteremia, sepsis, and other infections.β-lactams, quinolones, and trimethoprim/sulfamethoxazole are used to treat patients and animals infected by W. chitiniclastica (Schröttner et al., 2017).
Herein, BM-Y was isolated from the pancreas of a dead zebra in Shenzhen Safari Park in China in 2013.We speculated that W. chitiniclastica was possibly transferred to this park through either or both internal and international transport routes.Notably, W. chitiniclastica was first isolated and identified in China from C. megacephala captured from the Pudong International Airport in 2011 (Cao et al., 2013).This fly is one of the most common species found in South China (Liu et al., 2009), and it may have become the depositor of W. chitiniclastica in China.However, BM-Y shares the closest genetic relationship with MUWRP0946 from Uganda.W. chitiniclastica is more likely to be transferred from other countries to this park through international food (animal-and plant-based) trade or travel.We failed to isolate W. chitiniclastica from the flies collected in Shenzhen Safari Park, although we made several attempts.
We are not sure whether BM-Y was associated with the death of the zebra because we failed to find the maggots from the zebra.Generally, flies transmit W. chitiniclastica to the host by laying eggs that subsequently hatch into larvae inside an open wound (Schröttner et al., 2017).In this study, neither an open wound nor a maggot was found, but the flies might have carried W. chitiniclastica to deposit the eggs on the mucosal surfaces of the zebra (Schröttner et al., 2017).It is necessary to continuously monitor the spread of W. chitiniclastica, especially the ones that carry antimicrobial resistance genes, in the future.pBM-Y is a very specific plasmid.It carries three different and novel rep genes.Currently, no other similar genes can be found in GenBank.pBM-Y encodes two toxin-antitoxin systems: RelBE and YefM/YoeB, which have been shown to increase plasmid maintenance (Gotfredsen and Gerdes, 1998).The HsdMSR type I restriction-modification system is also identified in pBM-Y.This system can stabilize plasmids by degrading the unmethylated incoming DNA (Oliveira et al., 2014).It is speculated herein that the loss of the toxin-antitoxin or restriction-modification system might lead to the instability of pBM-Y.No conjugative genes were identified; so, pBM-Y is putatively mobilized by the conjugative plasmids (Xu et al., 2021).
The MICs of ceftazidime, cefotaxime, cefepime, aztreonam, gentamicin, and tetracycline of BM-Y were higher than that of DSM 18708, which was related to the resistance genes bla VEB−1 , aadB, aacA1, and tetA(H).These genes were identified in pBM-Y using the CARD and ResFinder online databases.This indicates that these genes can express effectively and are involved in the increase in MICs.The bla VEB−1 gene cassette, the aadB gene cassette, and the aacA1/gcuG fusion cassette are individual mobile units and are usually found inserted into an integron (Partridge et al., 2018).
Two copies of ISWoch1 participated in the movement of bla VEB−1 -aadB-aacA1-gcuG.A single IS can also move an adjacent region that includes one or more resistance genes by forming a TU (Partridge et al., 2018).Herein, ISWoch3 was located upstream of orf405-tetR(H)-tetA(H)-orf423, indicating that it might be involved in the mobilization of tetA(H), and this tetA(H) region might preferentially insert next to an existing copy of ISWoch3 in the recipient molecule, generating a structure of ISWoch3-orf405-tetR(H)-tetA(H)-orf423-ISWoch3 (Partridge et al., 2018).It should also be noted that six intact or truncated copies of ISWoch3 were presented in pBM-Y, demonstrating that they participated in complex homologous recombination events and promoted the assembly of complex structures as observed in pBM-Y.A promoter was found or predicted in each of ISWoch1, ISWoch2, and ISWoch3, meaning these three ISs might provide a promoter for captured genes.Taken together, ISWoch1, ISWoch2, and ISWoch3 might promote the dissemination of drug resistance genes and affect the expression of these genes to regulate antimicrobial resistance (Noel et al., 2022).Surveillance studies for these three ISs are necessary.
The promoter region (containing the −10 and −35 elements) of the bla VEB−1 region was located in ISWoch1.The optimal spacing between the −10 and 35 elements is 16-18 bp (Aoyama et al., 1983), and in this study, it was 18 bp.This indicates that ISWoch1 provides a strong promoter for the bla VEB−1 region.Therefore, BM-Y can express β-lactamase and show resistance to ceftazidime, cefepime, and aztreonam.This means that some β-lactams are not suitable for treating infections caused by bla VEB−1 -carrying W. chitiniclastica, while quinolones and trimethoprim/sulfamethoxazole can still be the first choices.
To the best of our knowledge, this is the first report of the genetic characterization of a novel bla VEB−1 -carrying plasmid with three new ISs from W. chitiniclastica.The acquisition of this plasmid can give bacteria the fitness advantage for adapting to mammals and enable bacteria to acquire new antimicrobial resistance genes.The resistance of W. chitiniclastica to ceftazidime, cefepime, aztreonam, and tetracycline might enhance its ability to survive under drug selection pressure and aggravate the difficulty in treating infections caused by W. chitiniclastica.It is necessary to continuously monitor the spread of the bla VEB−1 -carrying W. chitiniclastica and the possibility of acquiring other drug resistance genes in W. chitiniclastica.

FIGURE 1
FIGURE 1Transmission electron micrographs of Wohlfahrtiimonas chitiniclastica BM-Y.BM-Y was incubated on a brain heart infusion agar medium at 37 • C for 18 h.

FIGURE 2
FIGURE 2Bacterial growth curves of W. chitiniclastica BM-Y and DSM 18708.

FIGURE 3
FIGURE 3Maximum-likelihood phylogenetic tree of 27 W. chitiniclastica isolates.The numbers above branches indicate bootstrap values of 1,000 times.The bar corresponds to a scale of sequence divergence.

FIGURE 4
FIGURE 4 Organization of the plasmid pBM-Y.(A) Schematic map of pBM-Y.Genes are denoted by arrows, and the backbone and accessory module regions are highlighted in black and color, respectively.The innermost circle presents the GC-skew [(G-C)/(G+C)], with a window size of 500 bp and a step size of 20 bp.The next-to-innermost circle presents the GC content.(B) Linear structure of pBM-Y.Genes are denoted by arrows.The genes, accessory genetic elements (AGEs), and other features are colored based on their functional classification.The numbers in brackets indicate nucleotide positions within pBM-Y.

FIGURE 5
FIGURE 5Comparison of the bla VEB−1 region from pBM-Y and bla VEB−1 -carrying Tn2000.Genes are denoted by arrows.The genes, AGEs, and other features are colored based on their functional classification.The shading in light blue denotes regions of homology (nucleotide identity ≥ 95%).The 3 fragment of attC_aacA1/gcuG is upstream of bla VEB−1 , while the 5 fragment is downstream of gcuG.The numbers in the brackets indicate the nucleotide positions within pBM-Y.Tn2000(Naas et al., 2001) (accession number AF205943) was used as a reference.

FIGURE 6
FIGURE 6Identification of the transcription start site of the bla VEB−1 region.Multiple sequence alignments of 14 positive clones and partial pBM-Y.

TABLE 1
Minimum inhibitory concentrations of 14 antimicrobial drugs.

TABLE 2
Biochemical characteristics of BM-Y and DSM 18708.