Epidemiological and Genetic Characteristics of Clinical Carbapenem-Resistant Pseudomonas aeruginosa Strains in Guangdong Province, China

Abstract

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is a bacterial pathogen that may cause serious drug-resistant infections that are potentially fatal. To investigate the genetic characteristics of these organisms, we tested 416 P. aeruginosa strains recovered from 12 types of clinical samples collected in 29 different hospital wards in 10 hospitals in Guangdong Province, China, from 2017 to 2020. These strains were found to belong to 149 known sequence types (STs) and 72 novel STs, indicating that transmission of these strains involved multiple routes. A high rate of resistance to imipenem (89.4%) and meropenem (79.4%) and a high prevalence of pathogenic serotypes (76.4%) were observed among these strains. Six STs of global high-risk clones (HiRiCs) and a novel HiRiC strains, ST1971, which exhibited extensive drug resistance, were identified. Importantly, ST1971 HiRiC, which was unique in China, also exhibited high virulence, which alarmed the further surveillance on this highly virulent and highly resistant clone. Inactivation of the oprD gene and overexpression of efflux systems were found to be mainly responsible for carbapenem resistance in these strains; carriage of metallo-β-lactamase (MBL)-encoding genes was less common. Interestingly, frameshift mutations (49.0%) and introduction of a stop codon (22.4%) into the oprD genes were the major mechanisms of imipenem resistance. On the other hand, expression of the MexAB-OprM efflux pump and MBL-encoding genes were mechanisms of resistance in >70% of meropenem-resistant strains. The findings presented here provide insights into the development of effective strategies for control of worldwide dissemination of CRPA. IMPORTANCE Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is a major concern in clinical settings worldwide, yet few genetic and epidemiological studies on CRPA strains have been performed in China. Here, we sequence and analyze the genomes of 416 P. aeruginosa strains from hospitals in China to elucidate the genetic, phenotypic, and transmission characteristics of CRPA strains and to identify the molecular signatures responsible for the observed increase in the prevalence of CRPA infections in China. These findings may provide new insight into the development of effective strategies for worldwide control of CRPA and minimize the occurrence of untreatable infections in clinical settings.

Keywords: Pseudomonas aeruginosa; carbapenem resistance; efflux-pump-encoding genes; molecular epidemiology; oprD.

FIG 1

Phenotypic and epidemiological characteristics of 416 clinical P. aeruginosa strains collected from 10 tertiary hospitals in Guangdong, China, from 2017 to 2020. (a) Distribution of age of patients from whom the P. aeruginosa strains were recovered. (b) Rate of recovery of P. aeruginosa strains from different clinical departments. (c) Rate of recovery of P. aeruginosa strains from different clinical specimens. (d) Antimicrobial susceptibility of the 416 clinical P. aeruginosa strains. IMP, imipenem; MEM, meropenem; LEV, levofloxacin; AZT, aztreonam; CIP, ciprofloxacin; CAZ, ceftazidime; TZP, piperacillin-tazobactam; FEP, Cefepime; TOB, tobramycin; AMK, amikacin; CST, colistin. Interpretation of resistance phenotypes adheres to the CLSI M100-S26. (e and f) Distribution of the top 10 prevalent ST-types of strains (e) and serotypes (f). The five-pointed star indicates HiRiCs ST-types of strains. (g) Accumulation of pan genes. (h) Number of core genes (99% ≤ strains ≤ 100%) in the 416 P. aeruginosa strains.

FIG 2

Geographical distribution of MLST types among 416 clinical P. aeruginosa strains collected from 10 tertiary hospitals in Guangdong, China, from 2017 to 2020. A total of 149 known STs and 72 new STs of strains were identified among these strains. The most prevalent STs were ST244 (n = 17), ST274 (n = 13), ST1971 (n = 12), ST313 (n = 11), ST357 (n = 11), ST179 (n = 9), ST532 (n = 8), ST773 (n = 7), ST253 (n = 7), ST316 (n = 6), ST277 (n = 5), and ST606 (n = 4). The high-risk STs are depicted in red text. The shading represents the observed clonal complex.

FIG 3

Phylogenetic tree of 416 P. aeruginosa strains collected from 10 tertiary hospitals in Guangdong, China, from 2017 to 2020. Circle 1 depicts strains isolated from five municipal cities in Guangdong, China. Circle 2 denotes 10 different investigating hospitals. Circle 3 depicts the distribution of O-antigen serotypes. Circle 4 depicts the distribution of high-risk clone strains. Circles 5 to 10 depict the antibiotic susceptibilities to currently used antibiotics.

FIG 4

Virulence potential of the HiRiC ST1971 strains. Virulence potential of six strains, including the HiRiC ST1971 strains b164, R530, and 19C16, ST381 types of strain Q410, and control strain PAO1, were evaluated in a zebrafish larva infection model (n = 100). The survival rates of zebrafish larvae infected by corresponding strains (400 CFU) are presented in various colors. Statistical analysis was performed by using a log-rank (Mantel-Cox) test and GraphPad Prism 8.0. ****, P < 0.0001; ***, P = 0.0002; **, P = 0.0058; *, P = 0.0428; ns, P = 0.6681.

FIG 5

AMR gene analysis of 416 clinical P. aeruginosa strains collected in 10 tertiary hospitals in Guangdong, China, from 2017 to 2020. Heatmaps were obtained by aligning the draft genome sequence of each strain to the AMR gene database. P. aeruginosa strains are clustered using a maximum-likelihood tree. Red and cyan blue depict the presence and absence, respectively, of AMR genes in the test strains. The right side denotes the category of AMR genes. Almost all strains contained intrinsic AMR genes, including fosA, armA, aph, bla_OXA-50, and rsmA. Only 17.8% (74/416) of the strains had acquired exogenous AMR genes. Strain names are depicted at the bottom of the chart.