Comprehensive study reveals phenotypic heterogeneity in Klebsiella pneumoniae species complex isolates.

Here, we conducted a comprehensive analysis of 356 Klebsiella pneumoniae species complex (KpSC) isolates that were classified as classical (cl), presumptive hypervirulent (p-hv) and hypermucoviscous-like (hmv-like). Overall, K. pneumoniae (82.3%), K. variicola (2.5%) and K. quasipneumoniae (2.5%) were identified. These isolates comprised 321 cl-KpSC, 7 p-hv-KpSC and 18 hmv-like-KpSC. A large proportion of cl-KpSC isolates were extended-spectrum-ß-lactamases (ESBLs)-producers (64.4%) and 3.4% of isolates were colistin-resistant carrying carbapenemase and ESBL genes. All p-hv-KpSC showed an antibiotic susceptible phenotype and hmv-like isolates were found to be ESBL-producers (8/18). Assays for capsule production and capsule-dependent virulence phenotypes and whole-genome sequencing (WGS) were performed in a subset of isolates. Capsule amount differed in all p-hv strains and hmv-like produced higher capsule amounts than cl strains; these variations had important implications in phagocytosis and virulence. Murine sepsis model showed that most cl strains were nonlethal and the hmv-like caused 100% mortality with 3 × 108 CFUs. Unexpectedly, 3/7 (42.9%) of p-hv strains required 108 CFUs to cause 100% mortality (atypical hypervirulent), and 4/7 (57.1%) strains were considered truly hypervirulent (hv). Genomic analyses confirmed the diverse population, including isolates belonging to hv clonal groups (CG) CG23, CG86, CG380 and CG25 (this corresponded to the ST3999 a novel hv clone) and MDR clones such as CG258 and CG147 (ST392) among others. We noted that the hmv-like and hv-ST3999 isolates showed a close phylogenetic relationship with cl-MDR K. pneumoniae. The information collected here is important to understand the evolution of clinically important phenotypes such as hypervirulent and ESBL-producing-hypermucoviscous-like amongst the KpSC in Mexican healthcare settings. Likewise, this study shows that mgrB inactivation is the main mechanism of colistin resistance in K. pneumoniae isolates from Mexico.

Carbapenemase-Encoding Genes and Colistin Resistance in Gram-Negative Bacteria During the COVID-19 Pandemic in Mexico: Results from the Invifar Network.

In this study, we report the carbapenemase-encoding genes and colistin resistance in Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa in the second year of the COVID-19 pandemic. Clinical isolates included carbapenem-resistant K. pneumoniae, carbapenem-resistant E. coli, carbapenem-resistant A. baumannii, and carbapenem-resistant P. aeruginosa. Carbapenemase-encoding genes were detected by PCR. Carbapenem-resistant K. pneumoniae and carbapenem-resistant E. coli isolates were analyzed using the Rapid Polymyxin NP assay. mcr genes were screened by PCR. Pulsed-field gel electrophoresis and whole-genome sequencing were performed on representative isolates. A total of 80 carbapenem-resistant E. coli, 103 carbapenem-resistant K. pneumoniae, 284 carbapenem-resistant A. baumannii, and 129 carbapenem-resistant P. aeruginosa isolates were recovered. All carbapenem-resistant E. coli and carbapenem-resistant K. pneumoniae isolates were included for further analysis. A selection of carbapenem-resistant A. baumannii and carbapenem-resistant P. aeruginosa strains was further analyzed (86 carbapenem-resistant A. baumannii and 82 carbapenem-resistant P. aeruginosa). Among carbapenem-resistant K. pneumoniae and carbapenem-resistant E. coli isolates, the most frequent gene was blaNDM (86/103 [83.5%] and 72/80 [90%], respectively). For carbapenem-resistant A. baumannii, the most frequently detected gene was blaOXA-40 (52/86, 60.5%), and for carbapenem-resistant P. aeruginosa, was blaVIM (19/82, 23.2%). For carbapenem-resistant A. baumannii, five indistinguishable pulsotypes were detected. Circulation of K. pneumoniae New Delhi metallo-ß-lactamase (NDM) and E. coli NDM was detected in Mexico. High virulence sequence types (STs), such as K. pneumoniae ST307, E. coli ST167, P. aeruginosa ST111, and A. baumannii ST2, were detected. Among K. pneumoniae isolates, 18/101 (17.8%) were positive for the Polymyxin NP test (two, 11.0% positive for the mcr-1 gene, and one, 5.6% with disruption of the mgrB gene). All E. coli isolates were negative for the Polymyxin NP test. In conclusion, K. pneumoniae NDM and E. coli NDM were detected in Mexico, with the circulation of highly virulent STs. These results are relevant in clinical practice to guide antibiotic therapies considering the molecular mechanisms of resistance to carbapenems.

Molecular and Genomic Insights of mcr-1-Producing Escherichia coli Isolates from Piglets.

The use of colistin in food-producing animals favors the emergence and spread of colistin-resistant strains. Here, we investigated the occurrence and molecular mechanisms of colistin resistance among E. coli isolates from a Mexican piglet farm. A collection of 175 cephalosporin-resistant colonies from swine fecal samples were recovered. The colistin resistance phenotype was identified by rapid polymyxin test and the mcr-type genes were screened by PCR. We assessed the colistin-resistant strains by antimicrobial susceptibility test, pulse-field gel electrophoresis, plasmid profile, and mating experiments. Whole-Genome Sequencing data was used to explore the resistome, virulome, and mobilome of colistin-resistant strains. A total of four colistin-resistant E. coli were identified from the cefotaxime-resistant colonies. All harbored the plasmid-borne mcr-1 gene, which was located on conjugative 170-kb IncHI-2 plasmid co-carrying ESBLs genes. Thus, high antimicrobial resistance rates were observed for several antibiotic families. In the RC2-007 strain, the mcr-1 gene was located as part of a prophage carried on non-conjugative 100-kb-plasmid, which upon being transformed into K. variicola strain increased the polymyxin resistance 2-fold. The genomic analysis showed a broad resistome and virulome. Our findings suggest that colistin resistance followed independent acquisition pathways as clonal and non-genetically related mcr-1-harboring strains were identified. These E. coli isolates represent a reservoir of antibiotic resistance and virulence genes in animals for human consumption which could be potentially propagated into other interfaces.

Molecular and genome characterization of colistin-resistant Escherichia coli isolates from wild sea lions (Zalophus californianus).

Using molecular and whole-genome sequencing tools, we investigated colistin-resistant Escherichia coli isolates from wild sea lions. Two unrelated E. coli colistin-resistant isolates, ST8259 and ST4218, were identified, both belonging to the B2 phylogroup and different serotypes. Polymorphisms in PmrA, PmrB, and PhoQ proteins were identified, and the role of PmrB and PhoQ in contributing to colistin resistance was determined by complementation assays. However, the mutations characterized in the present study are not involved in colistin resistance, which have been described in E. coli isolates from clinical settings. Therefore, the acquired mutations in pmrB and phoQ genes in resistance to colistin in bacteria related to marine environment animals are different. This work contributes to the surveillance and characterization of colistin resistance in Escherichia coli obtained from animals from aquatic environments.