Phenotypic and molecular characterization of Klebsiella spp. isolates causing community-acquired infections

Abstract

Klebsiella spp. isolates from community-acquired infections were characterized. A total of 39 Klebsiella spp. isolates were obtained from outpatients at four rural hospitals in Mexico (2013-2014). The biochemical tests identified all as being K. pneumoniae. The molecular multiplex-PCR test identified 36 (92.4%) K. pneumoniae isolates and one (2.5%) K. variicola isolate, and phylogenetic analysis of the rpoB gene identified two isolates (5.1%) belonging to K. quasipneumoniae subsp. quasipneumoniae and K. quasivariicola. The last one was confirmed by phylogenetic analysis of six-loci concatenated genes. Mostly the isolates were multidrug resistant; however, a minority were extended-spectrum β-lactamase producing (10.2%). The extended-spectrum β-lactamase CTX-M-15 gene was identified in these isolates. Analysis of biofilm production and the hypermucoviscosity phenotype showed a total of 35 (92.3%) and seven (17.9%) of the isolates were positive for these phenotypes respectively. The K2 (4/39, 10.2%), K5 (2/39, 5.1%) and K54 (1/39, 2.5%) serotypes were identified in seven (17.9%) of the isolates, and only 28.5% (2/7) hypermucoviscous isolates were positive for the K2 and K5 serotypes. In general, the sequence type (ST) analysis and phylogenetic analysis of seven multilocus sequence typing loci were heterogeneous; however, ST29 was the most prevalent ST in the analysed isolates, accounting for 19% (4/21) of the total isolates. Two of the four ST29 isolates had the hypermucoviscosity phenotype. The virulence factors for fimbriae were the most prevalent, followed by siderophores. Community-acquired infections are caused by various species from Klebsiella genus, with different profiles of antibiotic resistance and heterogeneous virulence factors.

Keywords: Antimicrobial susceptibility; Bacterial resistance; Cephalosporin resistance; Community infection; ESBL; Hypermucoviscosity.

Fig. 1

Phylogenetic analysis of rpoB genes determined of Klebsiella pneumoniae 10471 (MH003688) and 10457 (MH003687); Klebsiella quasipneumoniae 10441 (MH003686); Klebsiella variicola 10447 (MH003689); and Klebsiella quasivariicola 10446 (MH003680) isolates. Nucleotide sequence of rpoB gene was obtained from K. pneumoniae MGH78578 (CP000647.1), Klebsiella quasipneumoniae subsp. quasipneumoniae 18A069 (KpII-A) (CBZM010000001.1), Klebsiella quasipneumoniae subsp. similipneumoniae 07A044 (KpII-B) (CBZR010000001.1), Klebsiella variicola At-22 (CP001891.1), Klebsiella quasivariicola KPN1705 (CP022823.1), Klebsiella sp. 10982 (NZ_AKYX00000000), Escherichia coli K-12 MG1655 (NC_000913.3) and Salmonella enterica Ty21a (NC_021176.1) used as reference bacterial species.

Fig. 2

Identification of clonal groups by PFGE and dendrogram analysis of Klebsiella pneumoniae isolates with different phenotypic and molecular characteristics. Hospital, PFGE pattern, ESBL production ability, presence or absences of hypermucoviscous phenotype, serotype and ST are indicated. ESBL, extended-spectrum β-lactamase; HMV, hypermucoviscous phenotype; PFGE, pulsed-field gel electrophoresis; ST, sequence type.

Fig. 3

Maximum-likelihood phylogeny tree from seven-loci multilocus sequence typing of Klebsiella pneumoniae clinical isolates. Bootstrapping of gene tree was implemented to evaluate support of groups. Numbers adjacent to nodes are bootstrap values. Clade I: isolates 10454, 10483, 10455, 10445, 10457, 10456, 10450, 10474, 10476, 10481, 10479 and 10482. Clade II: isolates 10471, 10444, 10440, 10449, 10443, 10453, 10458, 10472 and 10460.

Fig. 4

Jaccard index of phenotypic and molecular characteristics identified using microbiologic and molecular tests in Klebsiella pneumoniae, Klebsiella quasipneumoniae, Klebsiella variicola and Klebsiella quasivariicola isolates.