Infection of Diabetes Foot Caused by Carbapenem-Resistant Proteus penneri Mediated by a Novel Plasmid Containing bla_NDM

Introduction

Over the last decades, Proteus species infections have received special attention because of the emergence of some species resistant to various antimicrobial agents, particularly β-lactams.¹ Proteus penneri, first recognized in 1982, is a member of the Enterobacteriaceae family, which is salicin- and esculin-negative and chloramphenicol-resistant.^1,2 P. penneri is primarily associated with nosocomial acquired infections and is mainly isolated from urine samples (50%), wound and soft tissue exudates (25%), and blood cultures (15%).^3,4 P. penneri, same as P. mirabilis, is naturally resistant to penicillin G, oxacillin, all tested macrolides, lincosamides, streptogramins, glycopeptides, rifampicin, and fusidic acid.¹ However, P. penneri is sporadically reported carbapenem-resistant from human and environmental sources.⁴ Antimicrobial resistance (AMR) has become a pressing issue that poses a significant risk to global public health.⁵ The increasing spread of carbapenemase-encoding genes has increased the carbapenem-resistant Enterobacteriaceae (CRE) infection threat.^6,7

In this retrospective study, a strain of P. penneri with carbapenem resistance was found in patients with diabetic foot infections. Therefore, antimicrobial susceptibility testing and whole-genome sequencing (WGS) were implemented to understand the drug resistance and genome of carbapenemase-resistant P. penneri (CR-PPE). The homology of CR-PPE was analyzed according to the National Center for Biotechnology Information (NCBI) database to provide data support for clinical prevention and treatment of infections caused by CR-PPE.

Materials and Methods

Bacterial Strains

CR-PPE strain was isolated from a hospitalized patient from July 16, 2016, to July 23, 2016, in a tertiary hospital in China. CR-PPE was isolated through bacterial culture from purulence and identified using a Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) Vitek-MS (Sysmex-bioMerieux, Marcy l’Etoile, France). Escherichia coli (ATCC 25922) was used as a quality control strain.

Antimicrobial Susceptibility Testing

The antimicrobials used included the following: ceftriaxone (CRO), amikacin (AMK), gentamicin (GEN), ampicillin (AMP), aztreonam (ATM), ceftazidime (CAZ), ciprofloxacin (CIP), levofloxacin (LVX), cefepime (FEP), trimethoprim-sulfamethoxazole (SXT), tobramycin (TOB), cefotetan (CTT), piperacillin-tazobactam (TZP), ampicillin-sulbactam (SAM), ertapenem (ETP), and piperacillin (PIP), and were analyzed using the VITEK 2 Compact (GN13) (Sysmex-bioMerieux, Marcy l’Etoile, France). The drugs not included in the GN13 card were tested using the Kirby–Bauer (K-B) diffusion method (Oxoid, Hampshire, UK). The antimicrobial drugs detected using the K-B method included meropenem (MEM), cefuroxime (CXM), cefazolin (CZO), cefoperazone/sulbactam (CSL), cefoxitin (FOX) and imipenem (IPM). The results of susceptibility testing were interpreted according to Clinical and Laboratory Standards Institute (CLSI) guidelines. E. coli ATCC 25922 was used as the quality control strain.

Bacterial Genome Extraction

Conservation strains were first incubated overnight in an enriched broth and then streaked onto Colombian blood plates (AutoBio, Zhengzhou, China). Species identified with Vitek MS were inoculated overnight in 3 mL of Luria-Bertani (L.B.) broth medium (Oxoid, Hampshire, UK). Bacterial genomic DNA was extracted using an Omega Bio-Tek Bacterial DNA Kit (Doraville, GA, USA), according to the manufacturer’s instructions, and the genomic DNA concentration and quality were checked using a NanoDrop spectrophotometer 2000 (Waltham, MA, USA). The obtained DNA was stored at −80 °C until further analysis.

Sequencing and Sequence Assembly

Bacterial genomic DNA was sequenced from a paired-end library with an average insert size of 350 bp using a NovaSeq sequencer (Illumina, CA, USA). FASTQ format files for each sample set were assembled independently using the de novo assembler SPAdes 3.13.0.^8,9 with K-values of 21, 33, 55, 77, and 99, and careful mode to reduce the number of mismatches. Plasmid sequences were assembled using SPAdes.⁷

WGS Analysis

Antibiotic resistance genes (ARGs) and virulence factor (VF) were identified using NCBI and VF databases with Abricate 0.8 (https://github.com/tseemann/abricate).¹⁰ The plasmid type (Inc type) of contigs harboring carbapenemase genes was determined using PlasmidFinder.¹¹ The comparison of different plasmids harboring carbapenemase genes was performed using the BLAST Ring Image Generator.¹² The phylogeny was used for single nucleotide polymorphism (SNP) analysis.¹³ Unrooted maximum-likelihood phylogenetic trees were further generated using MEGAX 10.1.8 with a bootstrap iteration of 1000.¹⁴ The phylogenetic tree was visualized with iTOL.¹⁵ The strain data for constructing the phylogenetic tree, except for CR-PPE strains in the study, were obtained from NCBI database. There were nine strains of P. penneri in NCBI database (up to Nov 8, 2022) that were used to build the phylogenetic tree and were named after their accession numbers. The sequence data in this study were deposited in the BioProject database under PRJNA901201.

Results and Discussion

CR-PPE was isolated from the purulence of an elderly man with serious basic diseases. The exact isolation time was July 17, 2016, during hand and foot surgery. The clinical characteristics of patients are summarized in Table 1.

Table 1 The Characteristics of Patient

Analysis of Medical Record Characteristics

The patient had diabetes for four years and took metformin at home, but the blood glucose level was unstable The patient had a history of left-limb paralysis caused by cerebral thrombosis 20 years prior. The patient presented with a left foot infection for 20 days. Because the treatment effect in the local hospital was poor, he visited a tertiary hospital in East China for further therapy. On admission, a dry gangrenous wound approximately 3×3 cm in size was observed on the left dorsum of the foot, with the bottom of the wound reaching the bone. The surrounding skin was dark, and the local epidermis could be seen to fall off. An ulcer approximately 2×1 cm in size was observed on the dorsum of the right foot. Pulsation of the right dorsalis pedis artery was weakened, and the skin temperature of the right foot was reduced. After admission, the patient received a dressing change and antibacterial and symptomatic treatment, such as hypoglycemic therapy, invigorating blood circulation, and relieving swelling. After treatment, the blood glucose level improved, and the right foot wound healed, but infection control was poor. The dry gangrene wound on the left dorsum of the foot did not improve. Tissue debridement of wound skin and subcutaneous or amputation were recommended by doctors. The patient and his family refused surgery and asked for conservative treatment, changing the dressing at home after discharge. An integrated view of the medical records is depicted in Figure 1.

Figure 1 Integrated view of the medical record. Abbreviations: CZO, Cefazolin; LVX, Levofloxacin. Notes: The date marked by arrow was the date of isolation of the P. penneri strains.

On July 17, 2016, CR-PPE was isolated through bacterial culture of the purulence collected, while CZO sodium pentahydrate antibacterial (2 g/24 h) (CLSI recommends 1 g/12 h) was administered on the same day for three days. The infection did not improve after three days. The wound was characterized by necrotic substances and exudation with a peculiar smell. LVX lactate (0.2 g/24 h) (CLSI recommend 0.75 g/24 h) antibacterial treatment was administered on July 21, 2016, until the discharge on July 23. At discharge, the infection was poorly controlled. The size of the dry gangrenous wound (3×3 cm) on the left dorsum of the foot did not decrease, and exudation and smell persisted.

Antimicrobial Susceptibility Phenotype and Genotype

CR-PPE is resistant to two carbapenems (IPM and ETP), GEN, CRO, CZO, CXM, and FOX and sensitive to ATM, TZP, and CTT. Tet(B), sul2, bla_OXA-1, and other resistance genes have been detected to exist in CR-PPE. The resistant phenotype of CR-PPE was consistent with the genotype, and no virulence gene was detected. For plasmid prediction, Col3M_1 is forecasted. The characteristics of patient are summarized in Table 1. Specific antimicrobial susceptibility testing results and predicted resistance genes are displayed in Table 2.

Table 2 Outcomes of Antimicrobial Susceptibility Testing, Predicted Resistance Genes and Plasmid Type of CR-PPE

Shaded parts represent sensitive results of antimicrobial susceptibility testing.

WGS Analysis and Plasmid Profile Analysis of pWF127-NDM

The results of WGS analysis demonstrated that the drug resistance phenotype of CR-PPE was consistent with the genotype; thus, CR-PPE demonstrated multidrug resistance (MDR) to common clinical drugs. Moreover, WGS analysis indicated that the strain was bla_NDM positive, and sequencing confirmed it to be the bla_NMD-1 type. No virulence genes were detected. No virulence gene was detected in this bacterium, indicating that CR-PPE is a low-virulence strain and generally does not cause serious infection or even death. The clinical manifestations also confirmed the above findings.

However, bla_NDM does not belong to the plasmid Col3M_1. Therefore, we speculated that the plasmid containing bla_NDM is a new type of plasmid, pWF127-NDM. By predicting the sequence of second-generation sequencing, the plasmid pWF127-NDM was 78, 874 bp in size, with 104 open reading frames. The partial structures of Tn6588, Tn125, and Tn6727 were located in the exogenous insertion region of the plasmid. bla_NDM-1, whose cluster is arranged sequentially as ISAba125, bla_NDM-1, and ble_MBL elements, was embedded in the composite transposon Tn125. The specific plasmid structure is illustrated in Figure 2. There have been few reports of CR-PPE infection cases in China.¹⁶ However, the resistance genes and plasmids of CR-PPE infection cases have not been studied. To the best of our knowledge, pWF127-NDM is the first report of a carbapenem-resistant plasmid containing bla_NDM-1 in P. penneri in China. The blad_NDM-1 originates from an unknown environmental bacterial ancestor and is integrated into the chromosome of Acinetobacter. The Tn125 transposon carrying bla_NDM-1 is likely to be subsequently constructed by this Acinetobacter, which is then transferred to various host plasmids and then horizontally transferred to Enterobacteriaceae, such as Proteus.¹⁷

Figure 2 Plasmid map of the plasmid pWF127-NDM harboring blaNDM-1.

Environment of Carbapenem Resistance Gene

We found that the multidrug-resistance-encoding region of pWF127-NDM contained Tn125, Tn6588, and Tn6806. bla_NDM-1, which is located on Tn125, is situated in the second variable area of the class I integron. The upstream region of bla_NDM-1 is ble_MBL, a component of Tn125, and transposase tnpA is carried by ISCR1. ISAba125 and aphA6 are downstream of bla_NDM-1. The specific gene structure is displayed in Figure 3. Tn125 promoted bla_NDM-1 expression and enhanced the hydrolysis activity of the strain toward carbapenems. The ble_MBL gene upstream of bla_NDM-1 could stabilize bla_NDM-1.¹⁸ In addition, consistent with its ability to transpose, ISAba125 was described in association with increased resistance levels to β-lactams in Enterobacteriaceae.¹⁵ Compared with the reference plasmid pHFK418-NDM (Accession: MH491967), there is an approximately 60k fragment deletion in the plasmid skeleton region, while other skeleton regions are the same. The differences in the exogenous insertion region were mainly concentrated in the Tn6588 and Tn125 regions. Only two genes, trpF and dsbD, in Tn125 of pWF127-NDM swap positions compared to pHFK418-NDM. The other parts of Tn125 were the same. bla_NDM-1 was transmitted by the plasmid. In the conjugation experiments, the plasmid pHFK418-NDM from P. mirabilis HFK418 was transferred to E. coli, thereby generating bla_NDM-1 positive E. coli.¹⁹ Therefore, we speculate that bla_NDM-1 in this CR-PPE may be transmitted from other Enterobacteriaceae-resistant plasmids. Moreover, the plasmid pWF127-NDM may spread bla_NDM-1 to other bacteria, leading to carbapenem resistance. CR-PPE infection, spread of transmission of drug-resistant plasmids, and carbapenem resistance should be given extensive attention.

Figure 3 Genetic environment of blaNDM-1. Adapted from Wang P, Jiang X, Mu K, et al. DANMEL: a manually curated reference database for analyzing mobile genetic elements associated with bacterial drug resistance. mLife. 2022; 1: 460-64. © 2022 The Authors. mLife published by John Wiley & Sons Australia, Ltd. on behalf of Institute of Microbiology, Chinese Academy of Sciences.20

Homology Analysis

The phylogenetic tree consisted of P. penneri from this study and nine P. penneri strains. The P. penneri of this study has the closest evolutionary relationship with GCF 024129515.1, which was found in Gallus gallus in the Czech Republic in 2019. Second, P. penneri is also closely related to GCF 019375415.1 and GCF 022369495.1, both from China, in terms of evolutionary relationships. Therefore, we speculated that homologous transmission of P. penneri may be more common. Meanwhile, among the eight P. penneri strains in a clinical observation study biochemically, the strains were homologous except for a few reactions.²¹ In addition, from the phylogenetic tree, the strains were isolated from humans and animals, such as Gallus gallus, swine, and Manis javanica. Several strains have been identified since 2014. A specific evolutionary tree is illustrated in Figure 4.

Figure 4 Evolutionary tree of 10 strains of Proteus penneri.

Limitation

The conclusion of this study was based on only one patient, and further research is required.

Conclusion

Proteus penneri has been overlooked and understudied, and there are few reports on the isolation of P. penneri from patients with infection.^22,23 In this retrospective study, a strain of CR-PPE was found in the gangrenous purulence of a patient with diabetes. CR-PPE is resistant to two carbapenems (IPM and ETP) and is sensitive to ATM, TZP, and CTT. The major carbapenem resistance mechanism of this strain was determined by the plasmid pWF127-NDM, which contains bla_NDM-1 located on Tn125. This plasmid has never been reported in P. penneri. CR-PPE has the closest evolutionary relationship with GCF 024129515.1, which was found in Gallus gallus in the Czech Republic in 2019. Second, according to the evolutionary tree, CR-PPE has high homology with the two P. penneri found in China. P. penneri usually infects the urinary tract, blood, and abdominal wounds.^4,24 In hospitals, P. penneri infections often occur because of multiple-drug resistance, especially carbapenem resistance, with the potential to spread drug-resistant plasmids of nosocomial pathogens. Therefore, patients with underlying diseases, such as diabetes and weak immunity, should be carefully monitored for CR-PPE infection, as well as those undergoing abdominal or urogenital surgery.