Front. Cell. Infect. Microbiol. Frontiers in Cellular and Infection Microbiology Front. Cell. Infect. Microbiol. 2235-2988 Frontiers Media S.A. 10.3389/fcimb.2021.789646 Cellular and Infection Microbiology Original Research Occurrence of NDM-1, VIM-1, and OXA-10 Co-Producing Providencia rettgeri Clinical Isolate in China Shen Siquan 1 2 † Huang Xiangning 3 † Shi Qingyu 1 2 Guo Yan 1 2 Yang Yang 1 2 Yin Dandan 1 2 Zhou Xun 1 2 Ding Li 1 2 Han Renru 1 2 Yu Hua 3 * Hu Fupin 1 2 * 1 Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, China 2 Key Laboratory of Clinical Pharmacology of Antibiotics, Ministry of Health, Shanghai, China 3 Department of Laboratory Medicine, Sichuan Provincial People’s Hospital, University of Electronic Science and Technology of China, Chengdu, China

Edited by: Guo-bao Tian, Sun Yat-sen University, China

Reviewed by: Abid Ali, University of Pittsburgh Medical Center, United States; Jian Sun, South China Agricultural University, China; Fangyou Yu, Tongji University, China

*Correspondence: Hua Yu, yuhua2002@163.com; Fupin Hu, hufupin@fudan.edu.cn

This article was submitted to Molecular Bacterial Pathogenesis, a section of the journal Frontiers in Cellular and Infection Microbiology

†These authors have contributed equally to this work, Author order was determined both alphabetically and in order of increasing seniority

 03 01 2022 2021 11 789646 05 10 2021 29 11 2021 Copyright © 2022 Shen, Huang, Shi, Guo, Yang, Yin, Zhou, Ding, Han, Yu and Hu 2022 Shen, Huang, Shi, Guo, Yang, Yin, Zhou, Ding, Han, Yu and Hu

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Providencia rettgeri is a nosocomial pathogen associated with urinary tract infections related to hospital-acquired Infections. In recent years, P. rettgeri clinical strains producing New Delhi Metallo-β-lactamase (NDM) and other β-lactamase which reduce the efficiency of antimicrobial therapy have been reported. However, there are few reports of P. rettgeri co-producing two metallo-β-lactamases in one isolate. Here, we first reported a P. rettgeri strain (P138) co-harboring bla NDM-1, bla VIM-1, and bla OXA-10. The specie were identified using MALDI-TOF MS. The results of antimicrobial susceptibility testing by broth microdilution method indicated that P. rettgeri P138 was resistant to meropenem (MIC = 64μg/ml), imipenem (MIC = 64μg/ml), and aztreonam (MIC = 32μg/ml). Conjugation experiments revealed that the bla NDM-1-carrying plasmid was transferrable. The carbapenemase genes were detected using PCR and confirmed by PCR-based sequencing. The complete genomic sequence of the P. rettgeri was identified using Illumina (Illumina, San Diego, CA, USA) short-read sequencing (150bp paired-end reads), and many common resistance genes had been identified, including bla NDM-1, bla VIM-1, bla OXA-10, aac(6’)-Il, aadA5, ant(2’’)-Ia, aadA1, aac(6’)-Ib3, aadA1, aph(3’)-Ia, aac(6’)-Ib-cr, qnrD1, qnrA1, and catA2. The bla NDM-1 gene was characterized by the following structure: IS110–TnpA–IntI1–aadB–IS91–GroEL–GroES–DsbD–PAI–ble–bla NDM-1–IS91–QnrS1–IS110. Blast comparison revealed that the bla NDM-1 gene structure shared >99% similarity with plasmid p5_SCLZS62 (99% nucleotide identity and query coverage). In summary, we isolated a P. rettgeri strain coproducing bla NDM-1, bla VIM-1, and blaOXA-10. To the best of our acknowledge, this was first reported in the world. The occurrence of the strain needs to be closely monitored.

 Providencia rettgeri bla NDM-1 bla VIM-1 bla OXA-10 Mobile gene elements Introduction

Providencia rettgeri is an opportunistic human pathogen, unlike other Enterobacterales, it is a little-known pathogen, which is mainly associated with hospital-acquired infections including catheter-related urinary tract infections, bacteremia, meningitis, diarrhea, and eye infections (Yoh et al., 2005; Tada et al., 2014). Treatment of these infections is challenging, as they are intrinsically resistant to multiple antibiotics including first-generation cephalosporins, amoxicillin-clavulanic acid, nitrofurantoin, tigecycline, and polymyxins. Imipenem, amikacin, and cefepime are effective against more than 90% of the isolates (Lee et al., 2007; Sharma et al., 2017). However, in recent years P. rettgeri has become increasingly carbapenemase producers carrying the carbapenem-resistant genes like bla NDM, bla VIM, and so on (Piza-Buitrago et al., 2020). The emergence of multidrug-resistant of P. rettgeri strains poses a serious threat to public health.

The widespread of metallo-β-lactamases (MBLs) remain a severe challenge in health care settings because the hydrolysis of β-lactams by MBL enzymes cannot be prevented by clinically available β-lactamase inhibitors, including avibactam, relebactam, and vaborbactam (Wu et al., 2019). New Delhi Metallo-β-lactamases were the most predominant MBL among Enterobacterales clinical isolates which were initially identified in Klebsiella pneumoniae in 2009 in a Swedish patient (Pillai et al., 2011). Currently, although bla NDM-1 was commonly related to K. pneumoniae (Han et al., 2020), E. coli, Enterobacter cloacae, and Citrobacter freundii strains in China (Yong et al., 2009; Zhang et al., 2021), reports on bla NDM-1 producing P. rettgeri are rare. The spread of plasmid-bearing MBL possess a great challenge for clinical treatment because these multidrug-resistant isolates will result in limitations on treatment options (Oteo et al., 2014; Piza-Buitrago et al., 2020). Here, we report the co-existence of the carbapenemase genes bla NDM-1, bla VIM-1, and bla OXA-10 in a P. rettgeri clinical isolate in China.

 Materials and Methods Species Identification, Antimicrobial Susceptibility Testing, and Confirmation of Carbapenemase Production

Species identification was performed using MALDI-TOF MS (bioMérieux, France). The minimal inhibitory concentration (MIC) was determined by the broth microdilution method according to the guidelines of the Clinical Laboratory Standards Institute (CLSI) (Clinical and Laboratory Standards Institute, 2021). The strains E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 were used as quality controls for antimicrobial susceptibility testing. Quality control and interpretation of the results were based on 2021 CLSI breakpoints (CLSI, 2021) for all the antimicrobial agents with the exception of cefepime-tazobactam, tigecycline, and polymyxin B. Cefepime- tazobactam MICs were interpreted using CLSI breakpoints for cefepime for comparison purposes only. Tigecycline and polymyxin B MICs were interpreted using the European Committee for Antimicrobial Susceptibility Testing (EUCAST) criteria (EUCAST, 2021). Carbapenemase production was phenotypically detected using imipenem- 3-aminobenzeneboronic acid/EDTA double disk synergy test. The existence of the carbapenemase genes (KPC, NDM, OXA, IMP, and VIM) was confirmed by NG-Test Carba-5 and PCR-based sequencing, as previously described (Poirel et al., 2011b; Weiß et al., 2017).

 Conjugation Assay and Plasmid Sequencing

Conjugation experiments were performed to explore the transferability of the plasmid using azide-resistant E. coli J53 as a recipient strain. The conjugants were selected on Mueller-Hinton (MH) agar supplemented with azide (100 mg/L) and ampicillin (50 mg/L). The conjugation frequency was calculated according to the number of conjugants per initial donor bacteria. The presence of the bla NDM-1, bla VIM-1, bla OXA-10 in conjugants was confirmed by PCR and PCR-based sequencing. The Qiagen Midi kit (Qiagen, Hilden, Germany) was used to extract the plasmid of the conjugant and the plasmid was sequenced using Illumina (Illumina, San Diego, CA, USA) short-read sequencing (150bp paired-end reads). SPAdes 3.12.0 was used to de novo assemble the sequencing reads, and the open reading frame prediction and annotation were done with RAST version 2.0 (https://rast.nmpdr.org) and BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The plasmid replicon was determined using the PCR-based replicon typing method (Carattoli et al., 2005). Plasmid comparisons were performed using BRIG (http://brig.sourceforge.net) (Alikhan et al., 2011) and Easyfig tools (http://mjsull.github.io/Easyfig) (Sullivan et al., 2011). Plasmids carrying bla NDM-1 were circularized using PCR and Sanger sequencing to fill in gaps between contigs. The conjugation elements were detected using oriTfinder, a web-based tool for the identification of origin of transfers in DNA sequences of bacterial mobile genetic elements (https://tool-mml.sjtu.edu.cn/oriTfinder/oriTfinder.html) (Li et al., 2018).

 Whole Genome Sequencing and Bioinformatics Analysis

The isolates’ genomic DNA was obtained by using one commercial kit, according to the manufacturer’s recommendation: Qiagen for P138. And the genomic DNA was sequenced using Illumina (Illumina, San Diego, CA, USA) short-read sequencing (150bp paired-end reads). Reads were trimmed with sickle (GitHub), subsequently, they were de novo assembled using SPAdes 3.12.0. Antimicrobial resistance genes analysis was performed using BacWGSTdb (http://bacdb.cn/BacWGSTdb/analysis_single.php) and the annotation process was done using RAST version 2.0 (https://rast.nmpdr.org).

 Results Overview of the <italic>P. rettgeri</italic> Clinical Isolate

The P. rettgeri strain P138 was isolated from a 51-year-old female patient that was admitted to a public hospital for the treatment of cervical cancer in 2019 in Sichuan Province in the southwest of China. A hysterectomy was performed for this patient. At the same time, due to the dense adhesion between the patient’s bilateral ureters and the paravaginal tissue, stents were put in the bilateral ureters. On the day before the operation, cefathiamidine (2g Q8h) was used for seven days for prophylaxis. On the ninth day after the operation, the patient developed a fever, an E. coli and the P. rettgeri strain P138 were isolated from urine culture, therapeutic regimen switched to levofloxacin (0.5g QD) and ceftizoxime (2g Q12h) for 2 days. Two days later, the patient’s body temperature returned to normal and the infection was controlled. Finally, the patient recovered and was discharged successfully.

The antimicrobial susceptibility profiles of P. rettgeri P138 are presented in Table 1 . The isolate was resistant to all tested antimicrobial agents including amikacin (MIC >128μg/ml), cefoperazone-sulbactam (MIC >128μg/ml), aztreonam (MIC = 32μg/ml), piperacillin-tazobactam (MIC ≥256μg/ml), meropenem (MIC =64μg/ml), imipenem (MIC =64μg/ml), ceftazidime-avibactam (MIC ≥64μg/ml), tigecycline (MIC = 2μg/ml), and polymyxin B (MIC > 16μg/ml).

Susceptibility of P. rettgeri clinical isolate, conjugant, and recipient to antimicrobial agents.

Strains β-Lactamase genes MIC (mg/liter)
CZA IPM MEM CAZ FEP TZP CSL ATM AMK FPT SXT LEV CIP TGC POL
P. rettgeri P138 blaNDM-1, blaVIM-1 and blaOXA-10 >32 64 64 >32 128 >256 >128 32 >128 >64 >32 >16 >8 2 >16
E. coli P138-C blaNDM-1 >32 2 8 >32 32 >256 >128 ≤1 2 32 0.25 0.5 0.5 0.125 0.25
E. coli J53 – 0.5 0.25 ≤0.03 0.5 ≤0.06 4 ≤1 ≤1 ≤1 ≤0.03 ≤0.25 0.125 ≤0.06 0.125 0.25

CZA, ceftazidime-avibactam; IPM, Imipenem; MEM, meropenem; CAZ, ceftazidime; FEP, cefepime; TZP, piperacillin-tazobactam; CSL, cefoperazone-sulbactam; ATM, aztreonam; AMK, amikacin; FPT, Cefepime-tazobactam; SXT, trimethoprim-sulfamethoxazole; LEV, levofloxacin; CIP, ciprofloxacin; TGC, tigecycline; POL, polymyxin B.

 Carbapenemase Genes and Conjugation Experiments

PCR-based sequencing demonstrated the presence of bla NDM-1, bla VIM-1, and bla OXA-10 in P. rettgeri strain P138. According to the results of Conjugation Experiments, conjugants were positive for bla NDM-1 but negative for bla VIM-1 and bla OXA-10, making the conjugants resistant to meropenem (MIC = 8μg/ml) and ceftazidime-avibactam (MIC = >32μg/ml), intermediate to imipenem (MIC = 2μg/ml). The meropenem, imipenem, and ceftazidime-avibactam MICs of conjugants increased at least 256, 8, 128-fold respectively, compared with the recipient E. coli J53 ( Table 1 ). The conjugation frequency is 2.47 × 10–5 (The conjugation frequency was calculated according to the number of conjugants per initial donor bacteria). Lots of modules associated with conjugation were detected in pP138-NDM, like the oriT gene (origin of transfer gene), relaxase, type IV coupling protein (TraD), and type IV secretion system (T4SS).

 WGS Analysis and Characterization of Plasmid Sequence Carrying <italic>bla</italic> <sub>NDM-1</sub> Gene

According to the whole-genome sequencing analysis, many common resistance genes had been identified, including the carbapenemase genes bla NDM-1, bla VIM-1 and bla OXA-10, the aminoglycoside resistance genes aac(6’)-Il, aadA5, ant(2’’)-Ia, aadA1, aac(6’)-Ib3, aadA1, aph(3’)-Ia and aac(6’)-Ib-cr, the fluoroquinolone resistance genes qnrD1 and qnrA1 and the phenicol resistance gene catA2. The sequencing of the conjugant’s plasmid localized bla NDM-1 on a plasmid of 120,528 bp, belonging to the IncC type. Four resistance genes were identified in the plasmid pP138-NDM, bla NDM-1, qnrA1, sul1, and ant(2’’)-Ia, conferring resistance to carbapenems, quinolones, sulphonamides, and aminoglycosides, respectively. BLAST comparison disclosed that the bla NDM−1 gene environment of the plasmid pP138-NDM shared >99% similarity with plasmid p5_SCLZS62 (99% nucleotide identity and query coverage), isolated from a Raoultella planticola strain from Sichuan, China (GenBank accession number CP082173). In both plasmids, bla NDM-1 and qnrA1 were located in an identical multidrug resistance region (MRR). The MRR was flanked by genes of IS110 family transposase on both sides, and also contained IS91. TnAs3, which belongs to the Tn3 family was also found in pP138-NDM. The full genetic environment surrounding bla NDM-1 is: IS110–TnpA–IntI1–aadB–IS91–GroEL–GroES–DsbD–PAI–ble–bla NDM-1–IS91–QnrS1–IS110.

In several plasmids with similar sequences ( Figure 1 ), pSAL-19-0623_NDM (99% nucleotide identity and query coverage), an IncA/C2-type bla NDM-1 carrying plasmid with 276,695 bp in a carbapenem-resistant Salmonella strain from Singapore (GenBank accession numbers NZ_CP020913) (Octavia et al., 2020). They all showed resistance to meropenem and ceftazidime/avibactam, the only difference is that the strain P138 in our study was resistant to aztreonam, and this is most likely mediated by bla OXA-10.

Circular comparison between plasmid pP138-NDM (MZ670000) and other similar plasmids. Plasmid pP138-NDM (the outer circle) was used by the BRIG software as a reference plasmid to perform the sequence alignment with BLASTN. The different colors indicate different plasmids and are listed in the color key.

 Discussion

The first isolate of NDM-1 producing P. rettgeri was reported in Israel in 2013 (Gefen-Halevi et al., 2013). Since then, NDM-1-producing P. rettgeri has been reported in various parts of the world (Pillai et al., 2011; Barrios et al., 2013; Carvalho-Assef et al., 2013; Mataseje et al., 2014; Pasteran et al., 2014; An et al., 2016). Reports in Nepal (Tada et al., 2014) as well as reports in Colombia (Marquez-Ortiz et al., 2017; Piza-Buitrago et al., 2020), and Korea (Shin et al., 2018), commonly associate P. rettgeri with high resistance rates to carbapenems. This resistance characteristic in P. rettgeri is commonly associated with the production of bla NDM-1. Recently, Piza-Buitrago et al. reported two NDM-1, VIM-2, and OXA-10 coproducing P. rettgeri strains GMR-RA257 and GMR-RA1153, similar to the drug resistance spectrum in our study, with resistance to the carbapenems imipenem and meropenem, and this was highly probable caused by the production of NDM-1 and VIM-1 (Piza-Buitrago et al., 2020). However, as to OXA-10, it seemed to have a limited effect on the hydrolysis of carbapenems, according to a study in Nigeria in 2011, a P. rettgeri isolate co-producing bla OXA-10, bla VEB-1, and bla CMY-4 genes with no presence of the MBL genes was susceptible to carbapenems (Aibinu et al., 2011).

The moving elements can aggregate and combine with resistance genes, resulting in multiple resistance transfer of plasmids (Partridge, 2011). Different from previous studies often associated bla NDM-1 with Tn125, especially ISAba125 (Nordmann et al., 2011; Poirel et al., 2011a; Nordmann et al., 2012), in our study, was TnAs3, which is relatively rare reported. As to insertion sequences, IS26 is widely distributed and it is often combined with Tn125 family transposons (Poirel et al., 2011a; Zheng et al., 2021), in P. rettgeri isolate P138, bla NDM-1 was associated at its 3’-end and 5’ -end with IS110 that is also relative rare reported. This further reflects the diversity of genetic elements, which leads to the wide spread of resistance genes among bacteria. Gene encoding small multidrug resistance (SMR) efflux transporter was also found in the MRR, such transmembrane proteins were frequently found in Gram-negative and Gram-positive bacteria where they were deduced to be associated with the efflux system (Kazama et al., 1998) ( Figure 2 ).

Multidrug resistance region (MRR) of bla NDM-1 in the plasmid pP138-NDM (MZ670000) and p5_SCLZS62 (CP082173). Resistance genes are indicated by blue symbols. Transposon-related genes and insertion sequences are indicated by yellow symbols. Other genes are indicated by violet symbols. Light gray shading indicated homologous regions (>99% DNA identity).

In our study, we reported a carbapenem resistant P. rettgeri isolate P138, co-harboring bla NDM-1, bla VIM-1, and bla OXA-10, combined with the previous results (Pillai et al., 2011; Barrios et al., 2013; Carvalho-Assef et al., 2013; Mataseje et al., 2014; Pasteran et al., 2014; Tada et al., 2014; An et al., 2016; Marquez-Ortiz et al., 2017; Piza-Buitrago et al., 2020), the presence of MBL genes as bla NDM-1, bla VIM-1, and bla VIM-2 contribute significantly to carbapenem resistance in P. rettgeri, while bla OXA-10 plays a relatively weak role. With the increasing number of such multi-drug resistant bacteria, especially these showed resistance to carbapenems like imipenem, meropenem, and new combination of antimicrobials like ceftazidime-avibactam, the clinical treatment options are limited, so the initial effective anti-infection treatment is important to reduce the mortality of infection caused by CRE. In the future, the laboratory should strengthen the monitoring of carbapenemase, and perform combined antimicrobial susceptibility tests to seek an effective therapeutic regime for the infection caused by CRE strain.

 Data Availability Statement

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/, MZ670000.

 Ethics Statement

The study protocol was approved by the Institutional Review Board of Huashan Hospital, Fudan University (Number: 2018-408).

 Author Contributions

FH and HY designed the study. SS and XH collected clinical samples and performed the experiments. SS, LD, YY, RH, QS, DY, YG, and XZ analyzed data. SS wrote the manuscript. All authors contributed to the article and approved the submitted version.

 Funding

This work was supported by the National Natural Science Foundation of China (81871690, and 81861138051), and Shanghai Public Health System Construction Three-Year Action Plan (2020-2022), Discipline leader Grant (GWV-10.2-XD02). The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.

 Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

 Publisher’s Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

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