First Experimental Evidence for the Presence of Potentially Virulent Klebsiella oxytoca in 14 Species of Commonly Consumed Aquatic Animals, and Phenotyping and Genotyping of K. oxytoca Isolates

Abstract

Klebsiella oxytoca is a recently emerging pathogen that can cause necrotizing enterocolitis, hemorrhagic colitis, sepsis-associated purpura fulminans, and infective endocarditis in humans. The bacterium is ubiquitous in water and soil environments. Nevertheless, current literature on K. oxytoca in aquatic products is rare. In this study, we surveyed K. oxytoca contamination in 41 species of consumable aquatic animals sold in July, August, and September of 2018 and 2019 in Shanghai, China, 40 of which had no history of carrying this bacterium. K. oxytoca was for the first time isolated from 14 species with high abundance in benthic animals. None of the K. oxytoca isolates (n = 125) harbored toxin genes mviM, tisB, and yqgB. However, a high occurrence of virulence-associated genes was observed, including brkB (73.6%), cdcB (66.4%), pduV (64.8%), and virk (63.2%). Resistance to sulphamethoxazole-trimethoprim (56.0%) was the most predominant among the isolates, followed by chloramphenicol (6.4%), tetracycline (5.6%), and kanamycin (3.2%). Approximately 8.0% of the isolates displayed multidrug resistant phenotypes. Meanwhile, high percentages of the isolates tolerated the heavy metals Cu2+ (84.8%), Pb2+ (80.8%), Cr3+ (66.4%), Zn2+ (66.4%), and Hg2+ (49.6%). Different virulence and resistance profiles were observed among K. oxytoca isolates in 3 types and 14 species of aquatic animals. The ERIC-PCR-based genome fingerprinting of the 125 K. oxytoca isolates revealed 108 ERIC genotypes with 79 singletons, which demonstrated the genetic diversity of the isolates. The results of this study fill gaps for policy and research in the risk assessment of K. oxytoca in consumable aquatic animals.

First Report of Potentially Pathogenic Klebsiella pneumoniae from Serotype K2 in Mollusk Tegillarca granosa and Genetic Diversity of Klebsiella pneumoniae in 14 Species of Edible Aquatic Animals

“…The samples were pretreated as described in our recent research [ 33 ]. Briefly, an aliquot (25 g) of each sample was mixed with 225 mL of 1× phosphate-buffered saline (PBS, pH 7.4–7.6, Shanghai Sangon Biological Engineering Technology and Services Co., Ltd., Shanghai, China), homogenized, serial diluted, and spread onto the MacConkey Inositol Adonitol Carbenicillin (MIAC, Beijing Land Bridge Technology Co., Ltd., Beijing, China) agar plates [ 34 ]. The red colonies grown on the MIAC agar plates at 37 °C were randomly picked out for further identification.…”

“…The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) platform (Microflex LT/SH, MALDI Biotyper, Bruker, Germany) was used to identify presumptive K. pneumoniae colonies; the platform was run under the same conditions and parameters in our recent studies [ 34 , 35 ]. Mass spectra were collected and analyzed by using the Microflex LT Mass Spectrometer (Bruker 422 Daltonics, Bremen, Germany) [ 34 , 35 ].…”

“…K. pneumoniae strains were also confirmed using the polymerase chain reaction (PCR) assay to amplify the 16S rRNA gene with the universal bacterial primers ( Table S2 ) and DNA sequencing analysis [ 33 , 34 ].…”

“…The 20 μL PCR reaction mixture contained 8 μL of DNase/RNase-free deionized water (Tiangen Biotech Co., Ltd., Beijing, China), 10 μL of 2× Taq Master Mix (Novoprotein Technology Co., Ltd., Shanghai, China), 0.5 μL of each primer (5 μM), and 1 μL of DNA template. PCR amplification was performed as described in our previous research [ 33 , 34 , 42 ], but with different annealing temperatures and elongation times, based on the melting temperatures of the primer pairs and the predicted sizes of the PCR products ( Table S2 ). The obtained PCR products were analyzed as previously described [ 33 , 34 , 42 ].…”

“…PCR amplification was performed as described in our previous research [ 33 , 34 , 42 ], but with different annealing temperatures and elongation times, based on the melting temperatures of the primer pairs and the predicted sizes of the PCR products ( Table S2 ). The obtained PCR products were analyzed as previously described [ 33 , 34 , 42 ]. DNA sequencing was carried out by Suzhou Jinweizhi Biotechnology Co., Ltd., Suzhou, China.…”