Molecular Epidemiology of 16S rRNA Methyltransferase in Carbapenem-Resistant Enterobacterales: Insights from Nanjing Hospital

Introduction

Recent data from CHINET indicate that the isolation rate of carbapenem-resistant Enterobacterales (CRE) in China remains high. Notably, the resistance rate of Klebsiella to carbapenems is between 21.7% and 23.1%. This high resistance poses risks as CRE infections often result in severe clinical outcomes and increased mortality. CRE has been classified as one of the three most urgent antimicrobial resistance threats. However, Escherichia coli, Enterobacter cloacae, and other Enterobacteriaceae show high sensitivity to aztreonam-avibactam, amikacin, colistin, polycolistin B, and tigecycline, with sensitivity rates ranging from 87.1% to 95.5%.

Recommended treatments for CRE infection include aminoglycosides, tigecycline, colistin, and ceftazidime-avibactam (avycaz), used alone or in combination. Research indicates that combining aminoglycosides can enhance the effectiveness against CRE. Despite recent challenges from 16S rRNA methyltransferase (RMTase), which causes high-level aminoglycoside resistance, aminoglycosides still show effectiveness in clinical settings.

This study investigates the molecular epidemiology of 16S rRNA methyltransferase genes in CRE strains from a Nanjing hospital. The goal is to understand the prevalence of these genes and assist clinicians in using antibiotics effectively and safely.

Materials and Methods

Species Identification and Antimicrobial Susceptibility Testing

The study included 180 unique carbapenem-resistant Enterobacterales isolates collected from Nanjing Pukou People’s Hospital between January 2020 and December 2022. All isolates were identified using MALDI-TOF MS. The susceptibility of these CRE isolates to antibiotics was tested using the VITEK-2 COMPACT system. CRE was defined as strains resistant to imipenem or meropenem, with a minimum inhibitory concentration (MIC) of 4 µg/mL. The presence of carbapenemase genes (KPC, NDM, OXA, IMP, VIM) was confirmed through PCR testing.

Detection of Drug Resistance Genes

DNA was extracted by boiling bacterial colonies in sterile distilled water. Single PCR was used to analyze for carbapenemase genes (blaKPC, blaIMP, blaVIM, blaNDM, blaOXA-48) and 16S rRNA methylase genes (armA, rmtA, rmtB, npmA, rmtC, rmtD) using specific primers. The PCR products underwent agarose gel electrophoresis for analysis.

Statistical Analysis of Data

Data were analyzed using WHONET 5.6 and SPSS software (version 22.0). Chi-square tests assessed the relationship between different groups of CRE isolates.

Results

General Characteristics of CRE Isolates

A total of 180 CRE strains were identified. Among these, K. pneumoniae made up 50.1% (91/180), followed by E. coli (30.6%), E. cloacae (7.2%), P. mirabilis (5.6%), S. marcescens (3.3%), C. freundii (1.7%), and K. aerogenes (1.1%). The majority of strains were isolated from sputum (47.22%), followed by urine (30.00%) and bronchoalveolar lavage fluid (14.44%).

Prevalence of Genes in CRE

PCR analysis revealed that 63.3% (114/180) of CRE isolates carried at least one 16S rRNA methylase gene. The most common was rmtB, detected in 80 strains. The carbapenemase genes were found in 175 out of 180 CRE strains. blaKPC was present in 115 strains, while blaNDM appeared in 43 strains.

Antibiotic Susceptibilities of CRE

Of the 180 CRE isolates, 158 (87.8%) showed resistance to at least one aminoglycoside. Resistance rates were high for gentamicin (85.0%), tobramycin (82.8%), and amikacin (54.4%). CRE strains positive for 16S rRNA methylase genes were more resistant to certain drugs compared to negative isolates but showed increased sensitivity to trimethoprim-sulfamethoxazole, tetracycline, and minocycline.

Discussion

Carbapenems are essential for treating multi-drug-resistant gram-negative infections. Despite their importance, CRE strains are emerging globally. Patients with CRE infections have a higher risk of mortality. Combining tigecycline with aminoglycosides shows promise as an effective treatment strategy, although the presence of 16S rRNA methylases presents challenges due to their role in aminoglycoside resistance.

Conclusion

The positive rate of the 16S rRNA methyltransferase gene among CRE strains in this study was 63.3%, predominantly with the rmtB genotype. These strains often also carried the blaKPC carbapenemase genes. Monitoring the prevalence of 16S rRNA methyltransferase genes is crucial for understanding drug resistance patterns and guiding appropriate antibiotic use in clinical practice.