Genomic analysis of carbapenemase-encoding plasmids and antibiotic resistance in carbapenem-resistant Klebsiella pneumoniae isolates from Vietnam, 2021

Abstract

Carbapenem resistance in gram-negative rods is increasing in low- and middle-income countries. We conducted a single-center study to identify carbapenemase-encoding plasmids in carbapenem-resistant Klebsiella pneumoniae isolates causing human infections in Vietnam. The secondary objective was to investigate the prevalence of multidrug-resistant (MDR) and hypervirulent K. pneumoniae in this setting. Our genomic analysis study characterized 105 of 245 clinical K. pneumoniae isolates at the 108 Military Hospital in Hanoi, Vietnam, collected from intensive care unit and regular wards between 1 January 2021 and 31 December 2021. All isolates were characterized using long- and short-read sequencing, followed by hybrid assembly. Comprehensive genomic analysis was performed to identify carbapenemase-encoding plasmids, complemented by extended antibiotic susceptibility testing for commonly used and novel antibiotics. We observed a high prevalence of NDM-4-related carbapenem resistance (30.5%, 32/105) mostly carried by a specific 83-kb IncFII plasmid co-carrying the bla_TEM-1 (46.9%, 15/32). The genomic content of the bla_NDM-4-harboring plasmids is highly variable. While bla_OXA-181 and bla_OXA-48 were predominantly located on an IncX3 and an IncL plasmid, respectively, the majority of plasmids harboring bla_KPC-2 were not related to any named Inc-type. All isolates exhibited the MDR phenotype; however, the majority remained susceptible to the siderophore-cephalosporin cefiderocol (79%, 83/105). All isolates were susceptible to aztreonam/avibactam. In addition, we identified a hypervirulent, carbapenem-resistant K. pneumoniae ST23 strain, confirmed through both in vitro and in vivo experiments. Our study provides insights into plasmids harboring the carbapenemases New Delhi metallo-β-lactamase, oxacillinase-48 like, and K. pneumoniae carbapenemase-2 circulating in Vietnam.IMPORTANCECarbapenem resistance in Klebsiella pneumoniae is a major public health threat, especially in low- and middle-income countries. This study examined resistant strains from a hospital in Vietnam to understand how they spread and which antibiotics might still work. We found that a significant number of these bacteria carried resistance genes on different types of plasmids. Despite their resistance to many antibiotics, most strains remained susceptible to newer substances like cefiderocol and aztreonam/avibactam. Alarmingly, we also identified a hypervirulent strain that is carbapenem resistant, potentially posing an even greater risk to patients. This research provides insight into the epidemiology of the carbapenemase gene-harboring plasmids in a Vietnamese hospital. Understanding these resistance patterns can help guide antibiotic use and policy decisions to combat the growing threat of multidrug-resistant infections in Vietnam.

Keywords: KPC-2; NDM-4; OXA-48; Vietnam; carbapenemase-producing Klebsiella pneumoniae; hypervirulent Klebsiella pneumoniae.

Fig 1

ANI-based dendrogram and genomic content of the bla_NDM-4-carrying plasmids. Complete plasmids (n = 29) were compared using average nucleotide identity with ANIclustermap (v.1.1.0) and plasmid cluster. Four sections of the circle (separated by white gaps) represent, from inner to outer: (i) ST and primary cluster-ID color code as in the legend, (ii) replicon types (presence of the Inc-type in green, absence in gray), (iii) insertion sequence elements (presence of the IS in orange, absence in gray), and (iv) antimicrobial resistance genes present on the plasmids (presence of the AMR gene in black, absence in gray).

Fig 2

ANI-based dendrogram and genomic content of the bla_NDM-1-carrying plasmids. Complete plasmids (n = 9) were compared using average nucleotide identity with ANIclustermap (v.1.1.0) and plasmid cluster. The four sections of the circle (separated by white gaps) represent, from inner to outer: (i) ST and primary cluster-ID color code as in the legend, (ii) replicon types (presence of the Inc-type in green, absence in gray), (iii) insertion sequence elements (presence of the IS in orange, absence in gray), and (iv) antimicrobial resistance genes present on the plasmids (presence of the AMR gene in black, absence in gray).MLST, multi-locus sequence type.

Fig 3

ANI-based dendrogram and genomic content of the bla_OXA-48-carrying plasmids. Complete plasmids (n = 16) were compared using average nucleotide identity with ANIclustermap (v.1.1.0) and plasmid cluster. The four sections of the circle (separated by white gaps) represent, from inner to outer: (i) ST and primary cluster-ID color code as in the legend, (ii) replicon types (presence of the Inc-type in green, absence in gray), (iii) insertion sequence elements (presence of the IS in orange, absence in gray), and (iv) antimicrobial resistance genes present on the plasmids (presence of the AMR gene in black, absence in gray).

Fig 4

ANI-based dendrogram and genomic content of the bla_KPC-2-carrying plasmids. Complete plasmids (n = 36) were compared using average nucleotide identity with ANIclustermap (v.1.1.0) and plasmid cluster. The four sections of the circle (separated by white gaps) represent, from inner to outer: (i) ST and primary cluster-ID color code as in the legend, (ii) replicon types (presence of the Inc-type in green, absence in gray), (iii) insertion sequence elements (presence of the IS in orange, absence in gray), and (iv) antimicrobial resistance genes present on the plasmids (presence of the AMR gene in black, absence in gray). MLST, multi-locus sequence type.

Fig 5

Loci of virulence determinant conferring the hypervirulence phenotype in isolate Kp084. (a) The gene clusters conferring colibactin and yersiniabactin production are located within the chromosome. The colibactin operon was inserted by an IS5 family transposable element (IS102) upstream of the yersiniabactin operon. The aerobactin operon (iucABCD iutA genes) and the salmochelin operon (iroBCDN genes), indicated by red font, are located on an ~220-kb IncHI1b plasmid. There were no resistance genes detected on this plasmid. (b) The amount of siderophore secreted is expressed as the mean of the siderophore concentration in the supernatant of the bacterial culture and the standard error (n = 4). (c) Determination of hypermucoviscosity by sedimentation assay (n = 3). Results are expressed as the mean ratio of the OD₆₀₀ of the supernatant after centrifugation at 1,000 × g for 5 min to the total OD₆₀₀ and the standard error. (d) Survival in 50% human serum (n = 3). Results are expressed as mean and standard error of log2 fold change in CFU/mL after 4 h of incubation in the presence of human serum. (e) Resilience against 50 mg/mL bile salts (n = 3). Results are shown as mean percent survival rates and standard error. (f) Kaplan-Meier plot of mortality in the G. mellonella larvae infection model (n = 3). Results are expressed as mean percent mortality after the injection of 10⁵ CFU per larva. The well-characterized PBIO2030 and the E. coli K12 W3110 were used as hypervirulent K. pneumoniae reference and negative control (NC), respectively. For all results, Kp084 was statistically compared to PBIO2030 using the Kruskal-Wallis test, and the following indicates the level of significance (P value): ns, not significant (P ≥ 0.05); *, P < 0.05; ****, P < 0.0001.