Detection of KPC-producing Enterobacterales species in wastewater samples from the Gran Concepción Metropolitan area, Chile

Abstract

Background: Carbapenemase-mediated resistance to carbapenems is a significant public health concern due to its potential for widespread dissemination. The KPC family of carbapenemases, encoded by the bla_KPC gene and often associated with Tn4401-like transposons, is particularly important for its ability to be transferred through diverse plasmid types. In Chile, KPC-producing Gram-negative bacteria have been detected in clinical settings; however, their occurrence in wastewater (WW) remains unknown. This study addresses this gap by characterizing carbapenem-resistant Enterobacterales isolates from a wastewater treatment plant (WWTP) in the Gran Concepción Metropolitan Area, Chile.

Results: This study identifies three carbapenem-resistant Enterobacterales isolates, namely Klebsiella pasteurii M2/A/C/34, Klebsiella pneumoniae subsp. pneumoniae M3/A/M/3, and Citrobacter freundii sensu stricto. M4/A/C/32, all exhibiting multidrug-resistant profiles and carrying the bla_KPC-2 gene encoding KPC-like carbapenemases. These isolates also possessed genes for extended-spectrum β-lactamases (ESBLs) and aminoglycoside-modifying enzymes (AMEs). Sequence typing revealed that M2/A/C/34, M3/A/M/3, and M4/A/C/32 belonged to novel sequence types, specifically ST470, ST273, and ST214, respectively. All isolates carried plasmids belonging to groups commonly associated with ARGs, including IncF, IncP, and IncA. Both Klebsiella isolates (M2/A/C/34 and M3/A/M/3) carried the class 1 integron (intl1) gene. Phylogenomic analysis reveals that M2/A/C/34 is related to strains from China and Pakistan, while M3/A/M/3 shares similarities with a strain from Germany, indicating their potential dissemination.

Conclusions: This study represents the first detection of carbapenem-resistant Enterobacterales carrying bla_KPC-2 in Chilean WW, including the novel identification of K. pasteurii. These findings emphasize the critical role of genomic surveillance in WW under the One Health framework, enabling the monitoring of carbapenemase-producing bacteria and associated ARGs. Sustained surveillance efforts are essential to comprehend the dynamics of antibiotic resistance in environmental reservoirs and to develop strategies for its containment and mitigation.

Keywords: Antibiotic resistance; Carbapenem-resistant Enterobacterales; KPC-type carbapenemases; One health; Wastewater.

Fig. 1

Distribution of oxidase-negative glucose-fermenting Gram-negative bacilli (Ox[–]-F-GNB) obtained from wastewater samples (WW). MCC: MacConkey agar, MCC + M: MacConkey agar supplemented with meropenem (2 µ/mL)

Fig. 2

Carbapenemases production analyzed by Blue-Carba in influent (IF) strains growth in MCC + M plates. a K. pasteurii M2/A/C/34, b K. pneumoniae subsp. pneumoniae M3/A/M/3, c C. freundii s.str. M4/A/C/32, d K. pneumoniae UCO368 (KPC [+] control), e: K. pneumoniae UCO361 (NDM [ +] control), f K. pneumoniae UCO322 (OXA-48 [+] control), g E. coli ATCC25922 (negative control)

Fig. 3

Carbapenemases identification by the NG-Test® CARBA-5 immunochromatographic test in carbapenem-resistant strains (N = 3) from IF samples. a K. pasteurii M2/A/C/34, b K. pneumoniae subsp. pneumoniae M3/A/M/3, c C. freundii s.str. M4/A/C/32, d E. coli UCO327 (KPC [+] control). C: control; K: KPC-like; O: OXA-48-like; V: VIM-like; I: IMP-like; N: NDM-like

Fig. 4

Maximum likelihood phylogenomic tree depicting the relationship among K. pasteurii M2/A/C/34 and 38 other strains of the same species from around the world. The phylogeny was inferred in IQ-TREE v2.1.2 from core-genomes aligned by Roary v3.13.0 and filtered by SNP-sites v2.5.1. The values of the SH-aLRT, aBayes and UFboot are indicated in colors at the branches, which were performed based on 1000 replicates. The orange, yellow and green branches represent values between [70–80], [81–90] and ≥ 91 for SH-aLRT and UFBoot respectively, and [0.7–0.8], [0.81–0.9] and ≥ 0.91 for aBayes respectively. The colored ranges represent the strains STs. The strain M2/A/C/34 is labeled in bold blue and indicated by blue star while the close phylogenomically strains are in bold red. The tree was annotated and viewed in ITOL v6.8.2 and Klebsiella grimontii 4928STDY7071328 labeled in bold purple was used as outgroup. Branch lengths are not to scale, and the strains GenBank access numbers are found in the Table S2

Fig. 5

Maximum likelihood phylogenomic tree depicting the relationship among K. pneumoniae subsp. pneumoniae M3/A/M/3 ST273 and 138 other strains of the same species and sequence types (STs) from around the world. The phylogeny was inferred in IQ-TREE v2.1.2 from core-genomes aligned by Roary v3.13.0 and filtered by SNP-sites v2.5.1. The values of the SH-aLRT, aBayes and UFboot are indicated in colors at the branches, which were performed based on 1000 replicates. The orange, yellow and green branches represent values between [70–80], [81–90] and ≥ 91 for SH-aLRT and UFBoot respectively, and [0.7–0.8], [0.81–0.9] and ≥ 0.91 for aBayes respectively. The colored ranges represent the most frequent core-genomes sequence types (cgSTs) of the strains and the others are found in the Table S3. The strain M3/A/M/3 is labeled in bold blue and indicated by blue star while the close phylogenomically strain are in bold red. The tree was annotated and viewed in ITOL v6.8.2 and Klebsiella quasivariicola 08A119 labeled in bold purple was used as outgroup. Branch lengths are not to scale, and the strains GenBank access numbers are found in the Table S3