Introduction
Pseudomonas stutzeri is an aerobic, nonfermenting, active, Gram-negative oxidase-positive bacterium with unique colony morphology.1,2 Burri and Stutzer first described it in 1985,3 and the specific metabolic properties, such as denitrification, degradation of aromatic compounds, and nitrogen fixation, distinguish it from other pseudomonads species.2,4 Historically, P. stutzeri was not commonly isolated clinically and rarely caused human disease,5 exist mainly in environmental, occupying diverse ecological niches.2 However, the disease spectrum associated with its infections is broad, including endocarditis, bacteremia, pneumonia, osteomyelitis, arthritis, and ocular infections.6 P. stutzeri has been increasingly considered an opportunistic pathogen responsible for human infections.6–8
Carbapenem-resistant P. stutzeri was first isolated from Taiwan’s hospital environment in 2001, with three isolates carried IMP-1 and one harbored VIM-2.9 Subsequently, Netherlands identified a clinical P. stutzeri isolate with a single 70-kb plasmid carrying the blaDIM-1 gene in 2010,10 and Brazil reported blaIMP-16-carried P. stutzeri the same year.11 In 2017, class I integron containing blaVIM-2 emerged in P. stutzeri in Bangladesh.12 However, the carbapenemase resistance genes detected in P. stutzeri (except blaDIM-1) were identified as chromosomal located. The discovery of transferable plasmid carrying blaVIM-2 gene in P. stutzeri has not so far been reported.
The VIM-type Metallo-β-lactamases were first identified in Pseudomonas aeruginosa in Europe and have subsequently been reported worldwide in Enterobacteriaceae, Pseudomonas, and Acinetobacter.13,14 VIMs have a broad substrate hydrolysis profile, which can degrade almost all classes of β-lactams apart from the monobactams.13 The blaVIM is usually integrated into class I integron in the gene cassette and spreads among bacteria through mobile genetic elements.13 Besides, the coexistence of blaVIM and one or more aminoglycoside resistance genes, such as aacA4, aacA7, aadA1, aadA2, aadB, and aacC1, is very common.15 VIM-2 was first identified in southern France in 1996,16 which exhibits 93% amino acid identity to VIM-117 and sequence heterogeneity primarily observed in the NH2– and carboxy-terminal regions.13 Worldwide, VIM-2 is the most comprehensive distributed MBLs in Gram-negative bacteria.18
We first identified a clinical P. stutzeri isolate ZDHY95 with blaVIM-2-harboring plasmid and novel integron In1998 and implemented phylogenetic analysis in this study. Additionally, we elucidated the resistance mechanism of isolate and characterized the genetic environment and transfer mechanism of the plasmid.
Materials and Methods
Sample Collection and Bacterial Culture
Strains ZDHY95 and ZDHY372 were isolated from two cerebrospinal fluid specimens collected from the same patient with an interval of one month. The patient experienced intubation and drainage treatment after a cerebral hemorrhage in the Department of Neurosurgery of a teaching hospital of Zhengzhou University in April 2019. Subsequently, the patient developed a severe intracranial infection and persistent high fever. Therefore, the drained cerebrospinal fluid was collected for microbiological culture overnight at 37°C in 5% CO2 after inoculation on Columbia blood agar plates.
Phenotype Confirmation and Antimicrobial Susceptibility Testing
The species of the isolates ZDHY95 and ZDHY372 were confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) (Bruker, Bremen, Germany).19 Antimicrobial susceptibility testing (AST) was conducted by agar dilution, and standard strains of Escherichia coli (ATCC 25922) and P. aeruginosa (ATCC 27853) were used as quality controls. The results of AST were interpreted following the Clinical and Laboratory Standards Institute (CLSI) 2020 standards.20
Carbapenemase Gene Identification and Pulsed-Field Gel Electrophoresis
The carbapenemase-encoding gene was identified using PCR and DNA sequencing. The detailed technique for PCR amplification was as described previously.21 The homology analysis of the strains ZDHY95 and ZDHY372 was determined via pulsed-field gel electrophoresis (PFGE) as previously described, with a slight modification.22 Briefly, DNA plugs with whole-cell genomic DNA of culture-lysed cells were digested using the restriction enzyme SpeI (Takara Bio Inc., Japan). PFGE was undertaken on a CHEF-DR III system (Bio-Rad, Hercules, CA, USA), and the patterns were visually assessed and interpreted following published guidelines.23
Plasmid Characterization and Conjugation Assays
The number and sizes of the plasmids of the strains were characterized by S1-PFGE.24 The location of the blaVIM-2 gene was confirmed by Southern blotting and hybridization with a digoxigenin-labeled blaVIM probe using a DIG-High Prime DNA Labeling and Detection Starter Kit II (Roche Diagnostics). Conjugation transfer experiments were conducted to explore plasmids’ transferability with rifampin-resistant P. aeruginosa PAO1Ri as recipients, as described previously.25 Subsequently, Mueller-Hinton agar (OXOID, Hampshire, United Kingdom) plates that contained both 200 mg/L rifampicin and 2 mg/L meropenem were used to select transconjugants. The final identification of transconjugants includes MALDI-TOF/MS identification, blaVIM gene detection, and AST.
Whole-Genome Sequencing and Bioinformatics Analysis
The Bacterial DNA kit (Omega, Biotek, Norcross, USA) was utilized to extract the strains’ genomic DNA. Subsequently, the DNA was sequenced using Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA) and Oxford Nanopore (Oxford Nanopore Technologies, Oxford, UK) platforms. Assembly of sequencing fragments was performed via Unicycler v0.4.2, and finally obtained the strains’ complete genomic sequences. The genome sequences were annotated by RAST2.0 (rast.nmpdr.org/). Insertion sequence (IS) elements and integrons were detected via the ISfinder database (www-is.biotoul.fr/) and INTEGRALL (integrall.bio.ua.pt/). The identification of acquired antimicrobial resistance genes (ARGs) was carried out by Resfinder (cge.cbs.dtu.dk/services/ResFinder/). BLAST Ring Image Generator (BRIG) was used to generate a circular map to compare multiple plasmid genome sequences. The figures comparing the genetic context of the antibiotic resistance genes were developed with Easyfig 2.3.
Phylogenetic Analysis
The fasta files of all P. stutzeri strains were downloaded from the NCBI genome database and conducted ARGs analysis by Resfinder. The phylogenetic tree was constructed on core genome single nucleotide polymorphisms (SNPs) in WGS data via kSNP3.0 with P. stutzeri strain 40D2 as the reference. The maximum likelihood tree was visualized and modified using online tools iTOL (itol.embl.de/).
Accession Numbers and Ethical Approval
The complete nucleotide sequences of both the P. stutzeri ZDHY95 chromosome and plasmid pZDHY95-VIM-2 were deposited in GenBank with accession numbers CP063358 and CP063359. The ethical protocol was approved by the Ethics Committee of First Affiliated Hospital of Zhejiang University (no. 2018–752).
Results
Species Confirmation and Homology Analysis
Bacteriological culture of the cerebrospinal fluid was positive, Gram-negative bacilli ZDHY95 and ZDHY372 were isolated, and the species were confirmed as the opportunistic pathogen P. stutzeri. The PFGE results of the two strains are shown in Figure S1. Genetic relatedness based on the PFGE patterns and interpreted according to the criteria proposed by Tenover et al23 showed that ZDHY95 and ZDHY372 are of the same clone. Therefore, isolate ZDHY95 was selected for further detailed investigation.
AST of Pseudomonas Stutzeri ZDHY95
The results of AST of P. stutzeri ZDHY95 are shown in Table 1. According to the MIC breakpoints for other non-Enterobacterales in CLSI 2020 standards, isolate ZDHY95 exhibited resistance to piperacillin-tazobactam, ceftazidime, cefepime, cefotaxime, ceftriaxone, meropenem, imipenem, gentamicin, ciprofloxacin, and levofloxacin, intermediate resistance to aztreonam, with sensitivity only to amikacin.
Table 1 MIC Values of Antimicrobials for P. stutzeri ZDHY95, Recipient Strain PAO1Ri, and Transconjugant ZDHY95-PAO1Ri |
Location of blaVIM-2 and the AST of Transconjugant
S1-PFGE and Southern blot hybridization indicated that ZDHY95 contained an ~88 kb plasmid with blaVIM-2, designated as pZDHY95-VIM-2 (Figure S1). PCR and Sanger sequencing confirmed a transconjugant as blaVIM-2-encoding PAO1Ri. The comparison of AST of ZDHY95-PAO1Ri and PAO1Ri (Table 1) reveals that ZDHY95-PAO1Ri shows significant drug resistance to ceftazidime, ceftriaxone, cefotaxime, meropenem, imipenem, gentamicin, and ciprofloxacin; intermediate resistance to piperacillin-tazobactam, cefepime, levofloxacin. Although still sensitive to amikacin, its MIC value has increased. Only the MIC of aztreonam remains unchanged. These results indicate that the plasmid pZDHY95-VIM-2 has successfully transferred into recipient PAO1Ri and impacted bacterial resistance.
Characterization of the Genome of P. stutzeri ZDHY95 and In1998
The genome of ZDHY95 consisted of a circular chromosome of 4,500,524 bp and a plasmid of 88,056 bp. The genomic characteristics of P. stutzeri ZDHY95 are shown in Table S1. Fourteen drug resistance genes were identified by the Resfinder, of which six were plasmid located; the detailed results are shown in Table 2. Novel class I integron In1998 was identified in the chromosome of P. stutzeri ZDHY95 by INTEGRALL. Analysis of the sequence shows that In1998 has a sequenced size of 8183 bp and the gene cassette array includes 5ʹCS-aacA3 (aminoglycoside resistance)-aadA13 (aminoglycoside resistance)-cmlA8 (phenicol resistance)-blaOXA-246 (cephalosporin resistance)-arr3 (rifamycin resistance)-dfrA27 (trimethoprim resistance)-3ʹCS. NCBI BLAST analysis revealed that In1998 shared a high degree of genetic similarity (query coverage over 95% and identity 99%) with P. aeruginosa strain pae94323 (EU886980.1), P. aeruginosa strain RJ24624 (KU133339.1), P. aeruginosa strain NF811785 (HM175875.1), and P. aeruginosa strain PA26 (EU182575.1) from different regions of China. The gene cassette array and the genetic map of In1998 based on blastn and sequence analysis are shown in Figure 1.
Table 2 Antibiotic Resistance Genes of P. stutzeri ZDHY95 |
Structural Characterization of the Plasmid pZDHY95-VIM-2
The plasmid pZDHY95-VIM-2 has a sequence length of 88,056 bp and contains 91 protein-coding genes with a G+C content of 57% (Table S1). It consists of three central regions: the replication origin site repA and putative backbone genes downstream involved in maintaining stability (parB, arc, tonB, and pilL) and DNA metabolism (soj, dnaB, topB, rapA), conjugative transfer region, and multidrug resistance region. The plasmid could not be categorized into any of the known incompatibility groups by PlasmidFinder. NCBI BLAST analysis revealed that the complete sequence of pZDHY95-VIM-2 shared the highest degree of genetic similarity (query coverage over 17% and identity 99%) with p1160-VIM (MF144194.2) from P. aeruginosa 1160.25 The comparative plasmid circular map shows the genes and their locations (Figure 2A). The conjugative transfer region contains a large number of insertion sequences (ISPpu30, ISPst9, ISAzs36, IS481) and the putative genes associated with the conjugal transfer (traG, traI) and type IV secretion system (virB, virD4). The MDR region of plasmid pZDHY95-VIM-2 comprises In1722, the Tn402-like tni module, and transposon Tn5563. In1722 is an unreported integron carrying blaVIM-2 with the gene cassette array of 5ʹCS-aacA4ʹ–30–blaVIM-2–aacA4ʹ-3ʹCS. Adjoined to In1722 is the Tn402-like tni module, which has insertion mutations compared to the typical Tn402 tni module. The insertion of the gene fragment, including qnrVC1, catB11, and blaCARB-4, split tniA into two discontinuous parts, forming the Tn402-like tni module. Transposon Tn5563, with a sequenced length of 6253 bp, has the following structure: tnpR (resolvase)–orf2 (hypothetical protein)–pliT (pilT domain-containing protein)–tnpA (transposase)–mer (mercuric resistance gene locus). Further sequence alignment performed between pZDHY95-VIM-2 and p1160-VIM are shown in Figure 2B.
Phylogenetic Analysis
Twenty-two strains of P. stutzeri with drug resistance genes were screened out from a total of 283 in NCBI genome database and used for phylogenetic analysis together with P. stutzeri ZDHY95 (Figure 3). The results illustrated that the 23 strains aggregated into two clusters; P. stutzeri UBA4963 (GCA_002487265.1), P. stutzeri UBA6312 (GCA_002439185.1), P. stutzeri UBA4134 (GCA_002380885.1), P. stutzeri UBA3517 (GCA_002377205.1), P. stutzeri UBA6752 (GCA_002453575.1) composed a smaller cluster, and the remaining formed the other. P. stutzeri strains with drug resistance genes are mainly found in Pakistan and the USA, with a few scattered in China and Bangladesh. The strains isolated in Pakistan are all from accessible surfaces in the hospital ICU, such as bedside rail, washroom sink, and alcohol foam dispenser. They carry blaVIM-2, blaVIM-6, and blaIMP-34, respectively. The period of strain collection was six months. The strains isolated from the USA constitute the smaller cluster were all collected from natural environments, such as metal, plastic, and wood, and lacked carbapenemases. There are only two isolates from clinical samples, ZDHY95 and 40D2, both carrying blaVIM-2. P. stutzeri ZDHY95 is closely related to P. stutzeri T13 (GCA_000282955.1) isolated from China and P. stutzeri 40D2 (GCA_002027175.1) from Bangladesh.
Discussion
In recent years, the frequency of isolation of P. stutzeri from clinical material has increased remarkably. Nevertheless, these strains rarely exhibit pathogenicity1 and are considered more likely to represent opportunistic colonization or contamination of patients.5 However, P. stutzeri isolated from sterile sites should be given considerable attention.1Pseudomonas infection in the central nervous system is generally uncommon, and non-Pseudomonas aeruginosa infection is rare. To the best of our knowledge, central nervous system infections related to P. stutzeri are sporadic, with fewer than 10 cases reported to date. One case was an infection related to a brain abscess,26 and the remaining instances caused meningitis.11,27–31 To our knowledge, this is the first report of multidrug-resistant P. stutzeri with a blaVIM-2-carrying plasmid causing a nosocomially acquired central nervous system infection.
Low virulence and high antibiotic sensitivity are stereotypical characteristics of P. stutzeri compared with P. aeruginosa.1,2,32 Indeed, initial studies indicated that isolates of P. stutzeri are generally susceptible to third-generation cephalosporins, aminoglycosides, antipseudomonal penicillins, fluoroquinolones, monobactams, carbapenems, and trimethoprim/sulfamethoxazole and are variably vulnerable to ampicillin.1,5,32,33 However, this has changed since MBLs appeared in P. stutzeri. Currently, almost all of the reported P. stutzeri carrying carbapenemase are resistant to cefotaxime, ceftazidime, meropenem, and imipenem, and strain ZDHY95 in this study showed resistance to all antibiotics tested except aztreonam. These indicated that P. stutzeri is no longer the traditionally appreciated low-virulence, high-sensitivity colonizer or contaminant; instead, it may cause severe infection or mortality.6,11,34
Novel class I integron In1998 has three modules the same as classical class I integron: a 5ʹ conserved segment (5ʹCS), a variable region with resistance gene cassettes, and a 3ʹ conserved segment (3ʹCS).35 Sequence comparison of the strains (Figure 1) mentioned above indicated that aacA3-aadA13-cmlA8-blaOXA-246 might be transferred in bacteria as a resistance gene cassette. The presence of blaOXA-246 embedded in In1998 may result from the horizontal transfer of the resistance gene cassette. In addition to the similar genes shared by all sequences, In1998 carried two other resistance genes, arr3 and dfrA27, which inserted between blaOXA-246 and 3ʹCS. Although there are no mobile DNA elements in In1998, such as a Tn402 tni module, the insertion sequences and transposons upstream and downstream make the horizontal transfer possible (Table S2). To the best of our knowledge, In1998 is the second reported integron carrying blaOXA-246. OXA-246 is the OXA-10 family class D beta-lactamase, first detected in P. aeruginosa in China in 2014.36 OXA-246 has a nonsynonymous amino acid mutation at Lys141Asn compared with OXA-10 and was considered a non-ESBL β-lactamase for its narrow hydrolysis spectrum of ampicillin, penicillin G, carbenicillin, and ticarcillin.36 Subsequently, reports of blaOXA-246 only appeared in Vietnam37 and the Czech Republic,38 both in P. aeruginosa. Our study is the first report of blaOXA-246 in P. stutzeri to date.
In plasmid pZDHY95-VIM-2, blaVIM-2 is integrated into the class I integron In1722, designated by the INTEGRALL database, and the gene cassette array was 5ʹCS-aacA4ʹ-30–blaVIM-2–aacA4ʹ-3ʹCS. Unlike the typical class I integron, the 3ʹCS of In1722 was replaced by tniC/R, a functional transposon in Tn402 (Tn5090), instead of the qacEΔ1/sul1. The same phenomenon in class I integrons carrying blaVIM-2 has been described in 2007.39 It was considered an ancestral version of the class I integrons because Tn402 (Tn5090) was initially coupled to the typical 5ʹCS region of class I integron preceding the qac/sul region.40 In the structure of the MDR region, the Tn402-like tni module is connected downstream of In1722. Tn402, also known as Tn5090, is a transposon with a typical transposition module containing four genes: tniR/tniC, tniQ, tniB, and tniA.41 Previous studies predicted that the transposon encodes, in addition to the transposase TniA, two auxiliary proteins, TniB and TniQ, and the serine resolvase TniR/TniC.40,42 However, in pZDHY95-VIM-2, the tniA was truncated by an insertion segment containing qnrVC1, catB11, and blaCARB-4, suggesting that the Tn402-like transposon in pZDHY95-VIM-2 may lose the ability to undergo transposition. Unexpectedly, no mobile elements could be found around these three inserted genes. The same phenomenon was observed on the plasmid pVb1978, where resistance genes qnrVC1 and catB11 were inserted into tniA to cause truncation, but tniR/tniC and tniQ was deleted within the Tn402tni module of pVb1978.41 It is supposed that the insertion of resistance genes into tniA may be related to some particular recognition sequences between gene cassette intl1 and tniA. Tn5563 was initially characterized in Pseudomonas alcaligenes, which encoded transposase TnpA, the resolvase TnpR, and the mercuric ion transport proteins MerT, MerP. Previous research confirmed that Tn5563 is a functional Tn3 family transposon with mercury ion resistance.43 The presence of an MDR region and transfer region in pZDHY95-VIM-2 may cause the prevalence and spread of blaVIM-2. The ability of plasmid to capture exogenous resistance genes and be transmitted to pathogenic bacteria, such as P. aeruginosa, may lead to an epidemic of multidrug-resistant strains in hospitals.
Phylogenetic analysis showed that the isolate collection analyzed in this study was mainly divided into two clusters, the smaller one composed of strains isolated from the Uncultivated Bacteria and Archaea (UBA) project in the US, and the larger cluster mainly isolated from the ICU of a hospital in Pakistan. The continued isolation of P. stutzeri carrying MBLs from the ICU of a Pakistani hospital indicates that P. stutzeri with MBLs can be widely, continuously, and variably present on surfaces that medical workers and patients routinely touch. Once the patient has experienced long-term wound exposure or intubation, or doctors fail to disinfect relevant surfaces before an interventional operation thoroughly, the P. stutzeri hidden in the hospital settings will have the opportunity to enter the wound and cause unexpected infection. In fact, P. stutzeri isolated from the environment and surfaces, including medical equipment and ventilators, have played a role in nosocomial infections on multiple occasions.6,44,45 Infections caused by P. stutzeri vary geographically. It is reported that 62% of all globally reported cases were detected in the Mediterranean Basin.12 However, no strains from the Mediterranean region are found in Figure 3. Two different explanations are proposed for this phenomenon. One is that the P. stutzeri caused the infection has low virulence and high drug sensitivity and lacks drug resistance genes. Another is that because further WGS analysis was not performed, the sequence was unavailable in the public genome database, resulting in missing information.
P. stutzeri is ubiquitous in hospital settings, and research indicates that clinical strains come from the same populations as environmental isolates.2 Moreover, P. stutzeri is considered a potential reservoir of antibiotic resistance genes due to its genomic plasticity and capacity to capture genes from the environment.46 In recent years, populations of the genus Pseudomonas present in the hospital environment have frequently caused outbreaks of nosocomial infections and severe postoperative or posttraumatic infections of medical devices,6–8,22 and the isolated strains are generally multidrug-resistant and not susceptible to carbapenemase. Studies have revealed that environmental bacteria represent a reservoir for disseminating clinically relevant Metallo-β-lactamase genes,47 which poses a potential threat of outbreak and epidemic of MBLs in hospitals. However, very little is known about chromosomal and transferable MBLs in environmental Pseudomonas species, except Pseudomonas putida.48 The comprehensive description of the genetic characterization of P. stutzeri with a blaVIM-2-carrying plasmid and novel integron In1998 provides materials for further related research. Meanwhile, more vigilance and investigation should be provided for Pseudomonas in the hospital environment, and strict monitoring and thorough disinfection should be performed to control the spread of the strains.
Conclusion
In summary, we report the first identification of a clinical P. stutzeri isolate, ZDHY95, with a mobile plasmid coproducing VIM-2 and CARB-4 isolated from cerebrospinal fluid. We sequenced its complete genome and detected the novel class I integron In1998 in the chromosome of the strain. We performed a comprehensive phylogenetic analysis of P. stutzeri, determined the strain’s resistance mechanism, and characterized its genetic environment and plasmid transfer mechanism. Besides, Pseudomonas with MBLs in the hospital environment may cause infection in patients with long-term intubation or after interventional surgery and should be strictly monitored.
Data Sharing Statement
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.
Ethical Approval
The ethical protocol was approved by the Ethics Committee of First Affiliated Hospital of Zhejiang University (no. 2018-752).
Acknowledgments
The authors are grateful to Dr. Zhe Yin for providing the recipient bacteria PAO1Ri for the conjugation experiment in this paper.
Funding
We gratefully acknowledge the financial support of the National Natural Science Foundation of China (82072314), the National Key Research and Development Program of China (2016YFD0501105), the Mega-projects of Science Research of China (2018ZX10733402-004), and Henan Province Medical Science and Technology Research Project Joint Construction Project (No. LHGJ20190232).
Disclosure
The authors report no conflicts of interest in this work.
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