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Article

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

by
Ling Ni
1,2,†,
Yingwei Xu
1,† and
Lanming Chen
1,*
1
Key Laboratory of Quality and Safety Risk Assessment for Aquatic Products on Storage and Preservation (Shanghai), Ministry of Agriculture and Rural Affairs of China, College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China
2
Laboratory of Food Quality and Safety Testing, Shanghai Ocean University, Shanghai 201306, China
*
Author to whom correspondence should be addressed.
†
These authors have contributed equally to this work.
Antibiotics 2021, 10(10), 1235; https://doi.org/10.3390/antibiotics10101235
Submission received: 29 August 2021 / Revised: 28 September 2021 / Accepted: 30 September 2021 / Published: 11 October 2021
(This article belongs to the Special Issue Antimicrobials and Antimicrobial Resistance in Foodborne Pathogens)
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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.

1. Introduction

Klebsiella oxytoca is a Gram-negative bacterium that ubiquitously resides in water and soil environments [1,2]. The bacterium is also found in the nasopharynx and intestine of healthy individuals in humans [3]. K. oxytoca was originally isolated from a yogurt specimen in 1886 and named Bacillus oxytoca. In 1963, this organism was taxonomically classified as a member of the genus Klebsiella [4]. Recently, it has been reported that K. oxytoca is the causative agent of necrotizing enterocolitis [5], hemorrhagic colitis [6], sepsis-associated purpura fulminans [7], and infective endocarditis in humans [8], arguing that K. oxytoca is an emerging pathogen [1].
Outbreak of K. oxytoca in hospitals involved strains with extended-spectrum beta-lactamases and carbapenemases, which consequently lead to therapeutic problems [9]. Multidrug resistant (MDR) K. oxytoca isolates of clinical origins have been reported, particularly in developing nations. For instance, Gunduz et al. isolated K. oxytoca strains (n = 33) from different clinical samples collected in the Department of Pediatrics of Turgut Ozal University in Ankara, Turkey from September 2014 to April 2016. High percentages of these K. oxytoca strains were resistant to ciprofloxacin (CIP) (81%), ceftazidime (CAZ) (72%), amikacin (AK) (71%), gentamicin (GEN) (71%), and imipenem (IPM) (59%) [10]. Recently, Alemayehu et al. reported that K. oxytoca strains (n = 9) isolated from clinical specimens in Sidama, Ethiopia from February 13 to June 7 of 2020 displayed resistance to ampicillin (AMP) (100%), CAZ (100%), CIP (100%), cefuroxime (CRX) (100%), cefotaxime (CTX) (100%), meropenem (MER) (11.1%), GEN (77.8%), cotrimoxazole (COT) (71.4%), nitrofurantoin (NIT) (66.7%), and piperacillin-tazobactam (PIT) (42.9%) [11]. Nevertheless, the available literature on K. oxytoca isolates of environmental origins (e.g., aquatic environments and animals) is rare. To our knowledge, few studies have been conducted in this research field. Mann et al. reported on one K. oxytoca strain, isolated from a wastewater treatment plant in the North West Province of South Africa, showing resistance to oxytetracycline (OT) [12]. Recently, Håkonsholm et al. collected 476 batches of marine bivalve mollusks along the Norwegian coast from September 2019 to March 2020, including Mytilus edulis, Crassostrea gigas, Pecten maximus, Modiolus modiolus, Arctica islandica, Cerastoderma edule, Politapes rhomboides, Mya arenaria, and Strongylocentrotus droebachiensis. A total of 41 K. oxytoca strains were isolated from M. edulis and C. gigas, 39 of which were resistant to AMP [2]. The presence of K. oxytoca in aquatic products poses an emergent threat to food safety systems and the public health.
The pollution of aquatic environments by toxic heavy metals has also given rise to one of the most important ecological and organismic problems [13,14]. The heavy metals accumulated in consumable aquatic animals may lead to serious hazards to humans through the food chain include cadmium (Cd), chromium (Cr), mercury (Hg), nickel (Ni), and lead (Pb) [15]. They increase the selection of antibiotic resistance of bacteria to a certain extent, and vice versa [16]. Heavy metals in the water, in the sediments of rivers, lakes, and oceans, and in fish farming environments worldwide have been reported, particularly in developing countries [17]. For example, Ni et al. recently reported higher positive sample rates (PSR) of the heavy metals copper (Cu) (100%), Hg (100%), Pb (77.4%), and Cd (34.0%) in aquatic product samples (n = 108) collected in Shanghai, China in the summer of 2018 and 2019, none of which exceeded individual maximum residue limits (MRLs) [18]. Notably, Alboghobeish et al. reported one K. oxytoca strain, ATHA6, isolated from industrial wastewater of Isfahan in Iran, showing resistance to Ni2+ (24 mM) [19].
China is the largest producer, consumer, and exporter of aquatic products in the world, accounting for about 60% (65,490,200 tons) of the global amount in 2020 (National Bureau of Statistics, http://www.stats.gov.cn/, accessed on 20 August 2021). In the present study, we conducted a survey on a larger scale to investigate K. oxytoca contamination in 41 species of commonly consumed aquatic animals, including 21 species of mollusks, 17 species of fish, and 3 species of crustaceans (Supplementary Material: Table S1). The samples were collected from two large fish markets in July, August, and September of 2018 and 2019 in Shanghai [18], the largest city for aquatic product transportation and consumption in China. To our knowledge, K. oxytoca has not ever been detected in these 40 species of aquatic animals. The results in this study provide data to fill gaps for the risk assessment of K. oxytoca in aquatic products.

2. Results

2.1. Prevalence of K. oxytoca in 41 Species of Consumable Aquatic Animals

A total of 567 red, viscous, and single colonies grown on selective MacConkey Inositol Adonitol Carbenicillin (MIAC) agar plates were randomly picked out, which were recovered from 14 of the 41 species of consumable aquatic animals. Approximately 22.0% (125 of the 567 colonies) were identified as K. oxytoca by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF/MS) analysis. Moreover, the 125 K. oxytoca strains were detected positive in the capsular staining but negative in Gram’s staining and the dynamic tests. The results were confirmed by 16S rRNA gene sequencing and analysis. Among the 125 K. oxytoca isolates, approximately 88.0% (n = 110), 11.2% (n = 14), and 0.80% (n = 1) were recovered from the mollusk, fish, and crustacean samples, respectively (Table S1). Approximately 77.6% (n = 97) and 22.4% (n = 28) of the isolates were derived from 11 and 3 species of seawater and freshwater animal samples, respectively. Remarkably, most (96.8%, n = 121) of the K. oxytoca isolates were recovered from benthic aquatic animals.
K. oxytoca was present in 14 species of aquatic animals, including 10 species of mollusks: Anodonta woodiana (n = 21), Babylonia areolata (n = 5), Cipangopaludina cahayensis (n = 1), Haliotis rubra (n = 10), Mactra antiquata (n = 1), Mytilus edulis (n = 2), Neptunea cumingi Crosse (n = 31), Scapharca subcrenata (n = 5), Sinonovacula constricta (n = 6), Tegillarca granosa (n = 28); 3 species of fish: Blotchy rock cod, Carassius auratus (Crucian), and Carassius auratus (Ditrema temmincki Bleeker); and one species of crustacean: Procambarus clarkii. Additionally, the other 27 species of aquatic animals were absent from K. oxytoca (Table S1).
The 125 K. oxytoca isolates were recovered from aquatic animal products originating from four provinces and one city located along the East China Sea, one of the major fishing grounds along China’s coast, which encompasses the Fujian, Jiangsu, Shandong, and Zhejiang provinces, as well as Shanghai City in China. The highest abundance of K. oxytoca isolates were observed in the samples originating from Zhejiang Province (32.8%, 41 of the 125 isolates), followed by Fujian Province (25.6%, 32/125), Shandong Province (20.8%, 26/125), and Shanghai City (19.2, 24/125). Only a few isolates (1.6%, 2/125) originated from Jiangsu Province. Approximately 91.2% of the 125 K. oxytoca were isolated from the samples collected in the Luchao Port Aquatic Market, and 8.8% from the Jiangyang Aquatic Market in Shanghai, China.

2.2. Virulence Associated-Genes in the K. oxytoca Isolates

All of the 125 K. oxytoca isolates were detected negative for virulence-associated genes mviM, tisB, and yqgB by PCR assay. Nevertheless, higher incidence of the genes encoding virulence determinants brkB (73.6%), cdcB (66.4%), pduV (64.8%), and virk (63.2%) were observed among the isolates. Moreover, relE (43.2%), symE (37.6%), and vagC (23.2%) genes were also detected positive among the isolates. These amplified genes were confirmed by DNA sequencing and analysis, and the obtained sequences were deposited in the GenBank database under the accession numbers MW380322 to MW380328 (Table S2).
The K. oxytoca isolates recovered from the three types of aquatic products had different virulence-associated gene profiles. Notably, high percentages of the brkB, cdcB, pduV genes were observed in the K. oxytoca isolates recovered from the mollusk (72.7–64.5%) and fish samples (78.6–57.1%). Meanwhile, relatively higher detection frequencies of the relE, and symE genes were found in the mollusks (44.5%, and 40.9%) than in the fish (28.6%, and 7.1%). The vagC and virk genes were only present in the isolates recovered from these two types of aquatic products, but with higher incidence from the mollusks (23.6%, 70.0%) than in the fish (21.4%, 14.3%). Additionally, the isolate (K. o- P. clarkii 8-1-12-7) from the crustacean (P. clarkia) carried the brkB, cdcB, relE, pduV, and symE genes (Figure not shown).
Based on the limited numbers of K. oxytoca isolates recovered from the 14 species of aquatic animals, different virulence-associated gene profiles were also observed (Figure 1A–C). For example, six isolates originated from A. woodiana, N. cumingi Crosse, S. subcrenata, and T. granosa had the maximum number (n = 7, brkB+/cdcB+/pduV+/relE+/symE+/vagC+/virk+) of the virulence-associated genes tested. Conversely, 17 isolates from the 14 species of aquatic animals carried the smallest number of these genes (n = 1, brkB+ or virk+).
The 125 K. oxytoca isolates harbored 41 different virulence gene profiles (Table S3). Among these, the cdcB+/pduV+/relE+/symE+/virk+ profile was the most predominant (11.2%, n = 14), followed by BrkB+/Virk+ (10.4%, n = 13), and BrkB+ (9.6%, n = 12). In contrast, one isolate (K. o- A. woodiana 8-1-8-11) did not harbor any of the virulence-associated genes tested (Table S3).

2.3. Antibiotic Resistance of the K. oxytoca Isolates

The susceptibility of the 125 K. oxytoca isolates to nine commonly used antibiotics was determined, and the resulting data are illustrated in Figure 2. Approximately 28% of the K. oxytoca isolates were susceptible to all the antibiotics evaluated. Moreover, all the isolates were sensitive to imipenem (IPM) (100%) and meropenem (MEM) (100%), and most isolates were also sensitive to norfloxacin (NOR) (98.4%), GEN (96.8%), chloramphenicol (CHL) (93.6%), tetracycline (TET) (92.0%), CIP (87.2%) and kanamycin (KAN) (56.0%). Conversely, sulphamethoxazole-trimethoprim (SXT) resistance was the most predominant (56.0%) among the K. oxytoca isolates, followed by CHL (6.4%), TET (5.6%), and KAN (3.2%). Approximately 12.0% and 40.8% of the isolates also exhibited intermediate susceptibility to CIP and KAN, respectively. The resistance trend of the 125 K. oxytoca was SXT > CHL > TET > KAN > CIP = GEN = NOR > MEM = IPM (Figure 2).
The K. oxytoca isolates recovered from the 3 types of aquatic products had different antibiotic resistance profiles (Figure not shown). Higher rates of resistance to SXT were observed in the isolates recovered from the mollusks (60.9%, 67/110) than those from the fish (21.4%, 3/14) and crustacean (0%, n = 1). The CHL, TET, KAN, and GEN resistance were solely present in the isolates from the mollusks (7.3%, 6.4%, 3.6%, 0.9%), whereas CIP and NOR resistance were only found in the isolates derived from the fish (7.1%, and 7.1%) (Figure not shown).
Additionally, different antibiotic resistance profiles were also observed among the K. oxytoca isolates recovered from the 14 species of aquatic animals (Figure 3A,B). Remarkably, resistance to SXT was prevalent among the isolates from 10 of the 14 species, except C. cahayensis, C. auratus (Ditrema temmincki Bleeker), M. antiquate, and P. clarkii. For example, all isolates (n = 2) recovered from M. edulis were resistant to SXT, and more than half of the isolates from A. woodiana (61.9%, 13/21), B. areolate (60%, 3/5), and S. constricta (50%, 3/6) showed resistance to SXT. Resistance to CHL was present in a few isolates originating from H. rubra (1 of 10 isolates), M. edulis (2 of 2 isolates), N. cumingi Crosse (4 of 31 isolates), and T. granosa (1 of 28 isolates). One isolate (K. o- C. auratus 8-11-1) from C. auratus (Crucian) showed resistance to CIP and NOR, while GEN resistance was solely observed in one isolate (K. o- S. subcrenata 8-2-11) from S. subcrenata. Additionally, none of the isolates from C. cahayensis (n = 1), C. auratus (Ditrema temmincki Bleeker) (n = 2), M. antiquata (n = 1), and the crustacean P. clarkii (n = 1) was resistant to any of the nine antibiotics evaluated in this study.

2.4. MDR Phenotypes of the K. oxytoca Isolates

Approximately 8.0% (n = 10) of the K. oxytoca isolates had MDR phenotypes showing resistance to two or more antimicrobial agents. The isolates originating from the mollusks had the highest occurrence of MDR phenotypes (8.2%, 9/110), followed by the fish (7.1%, 1/14), and the crustacean isolates (0.0%, 0/1). Moreover, the MDR isolates were recovered from 6 of the 14 species of aquatic animals, including M. edulis (n = 2), C. auratus (Crucian) (n =1), S. subcrenata (n = 1), N. cumingi Crosse (n = 4), H. rubra (n = 1), and T. granosa (n = 1). Conversely, no MDR strain was found in the other 8 species of aquatic animals.
The multiple antimicrobial resistance index (MARI) values of the 125 K. oxytoca isolates ranged from 0.44 to 0.00, which indicated varying degrees of exposure to the nine antibiotics evaluated. The mean MARI values of the isolates originating from the mollusks, fish, and crustacean were 0.21, 0.15, and 0, respectively. Among the 14 species of aquatic animals, the maximum MARI value was found from the isolates recovered from N. cumingi Crosse (0.44), and H. rubra (0.44), followed by C. auratus (Crucian) (0.33), M. edulis (0.33), T. granosa (0.33), and S. subcrenata (0.22), whereas the isolates from the other 8 species had smaller MARI values (≤0.11). Notably, two isolates (K. o- H. rubra 8-2-2-11 and K. o- N. cumingi Crosse 8-6-19), originating from H. rubra and N. cumingi Crosse, respectively, had the largest MARI value of 0.44, showing resistance to four of the nine antibiotics tested.

2.5. Heavy Metal Tolerance of the K. oxytoca Isolates

Tolerance of the 125 K. oxytoca isolates to 8 heavy metals was determined (Table S4). The maximum observed values of minimal inhibitory concentrations (MICs) among the K. oxytoca isolates were 3200 μg/mL for Cr3+, Pb2+, Mn2+, and Cu2+; 1600 μg/mL for Ni2+, and Zn2+; 800 μg/mL for Cd2+; and 50 μg/mL for Hg2+, when compared with the quality control strain E. coli K12. Tolerance to Cu2+ and Pb2+ were most prevalent among the isolates (84.8%, 80.8%), followed by Cr3+ (66.4%), Zn2+ (66.4%), Hg2+ (49.6%), Mn2+ (11.2%), Cd2+ (9.6%), and Ni2+ (0.8%). The tolerance trend of the 125 K. oxytoca isolates to heavy metals was Cu2+ > Pb2+ > Cr3+ = Zn2+ > Hg2+ > Cd2+ > Mn2+ > Ni2+.
K. oxytoca isolates recovered from the three types of aquatic products had different heavy metal tolerance profiles. The majority of the isolates from all three types of aquatic products were tolerant to Cu2+ (100% to 84.5%), Hg2+ (100% to 48.2%), Pb2+ (100% to 79.1%) and Zn2+ (100% to 61.8%). Tolerance to Cr3+ was prevalent among the isolates from the fish (100%) and mollusks (62.7%), whereas lower percentages of Cd2+ tolerance were found in the isolates from the mollusks (10.0%) and fish (7.1%). Tolerance to Mn2+ (12.7%) and Ni2+ (0.9%) were solely observed in the isolate of the mollusks (Figure not shown).
Different heavy metal tolerance profiles were also found among the K. oxytoca isolates in the 14 species of aquatic animals (Figure 4A–C). The K. oxytoca isolates recovered from S. constricta were tolerant to all eight of the heavy metals tested, followed by T. granosa, and H. rubra (7 heavy metals); S. subcrenata, and C. auratus (Crucian) (6 heavy metals); C. auratus (Ditrema temmincki Bleeker), B. areolata, B. rock cod, and N. cumingi Crosse (5 heavy metals). Tolerance to Ni2+ was only found in one isolate from S. constricta (16.7%, 1/6).

2.6. Genetic Diversity of the K. oxytoca Isolates

The enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) was used to analyze the genetic diversity of the 125 K. oxytoca isolates recovered from the 14 species of aquatic animals. The obtained genome fingerprinting profiles comprised various numbers of DNA bands mainly ranging from 100 to 1000 bp (Figure 5a,b). Based on the fingerprinting profiles, all the isolates were classified into 108 different ERIC-genotypes, 73.2% of which were assigned as singletons (n = 79). Approximately 36.7% (n = 29), 30.4% (n = 24), and 25.3% (n = 20) of these singletons were derived from the mollusks N. cumingi Crosse, T. granosa, and A. woodiana, respectively. The UPGMA algorithm grouped all the 108 ERIC genotypes into 14 distinct clusters (clusters I to XIV) at a 32.0% similarity cut-off level (Figure 5). Approximately 14.4% (n = 18) of the K. oxytoca isolates were classified into the largest cluster, II, followed by 11.2% (n = 14), 9.6% (n = 12), and 8.0% (n = 10) into clusters VIII, III, and I, respectively. The remaining isolates (56.8%) fell into clusters III–V, VII, and IX–XIV (7.2% to 0.8%). Most isolates had a similarity coefficient of 30.0–85.0%, and a Simpson’s diversity index of 0.8485. These results demonstrate the considerable genetic diversity of the 125 K. oxytoca isolates recovered from the 14 species of aquatic animals.
Approximately 23.2% (n = 29) of the 125 K. oxytoca isolates shared 12 ERIC-genotypes. For example, four isolates with the ERIC-genotype K. o- 00044 were derived from S. constricta, suggesting near-present relatives or clonal relatedness. Additionally, there were four ERIC-genotypes (K. o- 00011, K. o- 00071, K. o- 00083, and K. o- 00108) containing the isolates derived from different species of aquatic animals. For example, the two isolates K. o-H. rubra 8-2-2-16, and K. o- S. constricta 8-2-3-1, which share the same ERIC-genotype K. o- 00071, were recovered from H. rubra and S. constricta, respectively.

2.7. Comparison of the MDR and Heavy Metal Tolerance

To get insights into co-selection between antibiotics and heavy metals in K. oxytoca, we further analyzed the 10 isolates with MDR phenotypes by phylogenetic analysis (Figure 6). The resulting data revealed the genetic diversity of the MDR K. oxytoca isolates with a Simpson’s index of 0.8556. Moreover, the MDR isolates belonging to 8 ERIC-genotypes were classified into 5 distinct clusters (clusters A–E) (Figure 6).
Most of the MDR K. oxytoca isolates (90.0%, n = 9) were tolerant to two or more heavy metals. Specifically, one K. oxytoca isolate (K. o- N. cumingi crosse 8-1-6-19) displayed resistance to 4 antibiotics (CHL/SXT/KAN/TET), and was also tolerant to 3 heavy metals (Pb2+/Hg2+/Zn2+). Another K. oxytoca isolate (K. o- H. rubra 8-2-2-11) was resistant to 4 antibiotics (CHL/SXT/KAN/TET) and 6 heavy metals (Cr3+/Cu2+/Hg2/Mn2+/Pb2+/Zn2+). These results provided direct evidence for the cross-resistance between the MDR and heavy metals in the K. oxytoca isolates originating from consumable aquatic animals.

3. Discussion

K. oxytoca is the second most common Klebsiella species after K. pneumoniae [5]. Nevertheless, systematic research on K. oxytoca is still in its infancy, and current literature on K. oxytoca in aquatic products is rare [1,20]. Recently, Håkonsholm et al. reported 40 K. oxytoca strains isolated from M. edulis and one from C. gigas [2]. In this study, we surveyed K. oxytoca contamination in 41 species of commonly consumed aquatic animal products sold in Shanghai in July, August, and September of 2018 and 2019. K. oxytoca was for the first time isolated from 14 species of aquatic animals, including 10 species of mollusks: A. woodiana, B. areolata, C. cahayensis, H. rubra, M. antiquata, M. edulis, N. cumingi Crosse, S. subcrenata, S. constricta and T. granosa; 3 species of fish: B. rock cod, C. auratus (Crucian), and C. auratus (Ditrema temmincki Bleeker); and one species of crustacean: P. clarkii. Our data also provided the first experimental evidence for high detection frequencies of K. oxytoca in benthic aquatic animals, such as N. cumingi crosse, T. granosa, and A. woodiana. These results suggested a potential health risk of the bacterial transmission to communities through the aquatic animals.
The cytotoxin-producing K. oxytoca has recently been identified as a new candidate etiologic agent in the pathogenesis of necrotizing enterocolitis [5]. In fact, information in genes associated with the virulence of K. oxytoca still remains to be discovered. Previous studies have indicated that the genes encoding adhesins, siderophores, and invasins of the genus Klebsiella were associated with deleterious traits [21]. In this study, virulence-related genes (brkB, cdcB, pduV, relE, symE, vagC, and virK) in the 125 K. oxytoca isolates recovered from 14 species of aquatic animals were detected simultaneously. High incidence of the brkB (73.6%), cdcB (66.4%), pduV (64.8%), and virk (63.2%) genes was observed. The brkB gene encodes the YihY/virulence factor BrkB family protein, while cdcB, pduV, and virK encode a cytotoxin, a type I toxin-antitoxin system hok family toxin, and the virulence factor VirK, respectively [22,23]. The RelE toxin in Escherichia coli was a global inhibitor of translation [24]. SymE was toxic in the inhibition of protein synthesis and RNA degradation [25]. The virulence factor VagC was involved in the bacteriocin secretion system and the type II toxin-antitoxin system in Turkish Salmonella serovar Infantis isolates [26]. In this study, the virulence-associated genes mviM, tisB, and yqgB were absent from the K. oxytoca isolates. As a virulence gene, mviM was associated with the exercise, biofilm formation and antimicrobial resistance of Cronobacter sakazakii [27]. TisB is a component of the TisB/IstR-1 toxin-antitoxin system in E. coli [28]. The adaptive factor YqgB in Bacillus thuringiensis facilitated the bacterial colonization of the host [29]. In this study, the K. oxytoca isolates recovered from the 3 types and 14 species of aquatic animals harbored different virulence-associated gene profiles. Remarkably, the isolates originating from A. woodiana, N. cumingi crosse, S. subcrenata, and T. granosa harbored the maximum number (n = 7) of the virulence-associated genes tested, which suggests a health risk from the potentially virulent K. oxytoca in these consumable aquatic animals.
The propagation and spread of resistant pathogenic bacteria pose serious threats to the public heath for humans and animals [30,31]. It is estimated that antibiotic resistance may lead to 10 million deaths per year by 2050 [31]. Misuse of antimicrobial agents is the main cause of antibiotic resistance in pathogenic bacteria, particularly in developing nations [32,33,34]. In this study, our results indicate that SXT resistance was the most predominant (56.0%) among the 125 K. oxytoca isolates of aquatic animal origins. SXT-resistant K. oxytoca isolates in humans has also been reported. For example, Maharjan et al. recently reported that 9.4% of K. oxytoca strains (n = 48) isolated from stool specimens of healthy adult volunteers (n = 510) in Kathmandu in Nepal showed the highest resistance toward SXT (45.8%) [35]. In this study, incidences of intermediate susceptibility to KAN (40.8%), and CIP (12.0%) were observed, which suggests a potential resistance trend of K. oxytoca in aquaculture environments.
MARI is commonly used to determine the health risk associated with antibiotic resistance [17]. In this study, the mean MARI values for K. oxytoca isolates derived from the mollusks, fish, and crustacean samples were 0.21, 0.15 and 0, respectively. Among the 14 species of aquatic animals, the maximum MARI value was derived from the isolates in H. rubra (0.44), and N. cumingi Crosse (0.44). These results suggested likely extended exposure of the mollusks, particularly H. rubra, and N. cumingi Crosse, to the antibiotic drugs evaluated in this study. Recently, Ni et al. reported that 10 antibiotics (AMP, CHL, CIP, ENR, OFX, OT, pefloxacin (PEF), sulfadiazine (SDZ), sulfisomidine (SIM), and sulfapyridine (SP)) were detected positive in the 41 species of aquatic animal samples (n = 108) with an overall detection frequency of 61.3%. The residual CHL (47.2%), AMP (31.1%), and SP (1.9%) exceeded their MRLs [18], which provided direct evidence for the antibiotic-resistant phenotypes of K. oxytoca isolates observed in this study.
A high level of the bioaccumulation of toxic heavy metals (e.g., Cd2+, Cr3+, Hg2+, Ni2+, and Pb2+) through the food chain is a grave threat to human health, due to their non-degradable nature [36]. Numerous studies have reported heavy metal residues in various aquatic environments and species of food animals (shellfish, fish, crustaceans, and crabs) sampled worldwide, particularly in developing nations [13,14,15,18,37]. In the recent surveys by our research group [17,38,39], the heavy metal-tolerant waterborne pathogen Vibrio cholerae has been discovered in many species of aquatic animals. In this study, the results indicated that the K. oxytoca isolates originated from the 3 types and 14 species of aquatic animals had different heavy metal tolerance profiles. Tolerance to Cu2+ and Pb2+ was the most prevalent among the isolates (84.8% and 80.8%). Cu is an essential element for various biological functions in many organisms, and it is also a cofactor for hemocyanin in aquatic arthropods and mollusks. However, excessive levels of Cu can be toxic or disrupting metabolic processes [40]. In this study, our data also revealed a higher detection frequency of Cr3+ tolerance in the K. oxytoca isolates from the fish samples than that from the mollusks. Moreover, the isolates recovered from S. constricta were tolerant to all 8 heavy metals, followed by H. rubra, and T. granosa (7 heavy metals); C. auratus (Crucian), and S. subcrenata (6 heavy metals); B. areolata, B. rock cod, C. auratus (Ditrema temmincki Bleeker), and N. cumingi Crosse (5 heavy metals). Tolerance to Ni2+ was only detected in the isolates from S. constricta. These data suggest serious heavy metal pollution likely occurred in the aquaculture environments, consistent with previous reports [18,38,39]. Recently, Ni et al. reported that the heavy metals Cu, Hg, Pb, and Cd were observed in the 41 species of aquatic animals with PSRs of 100%, 100%, 77.4%, and 34.0%, respectively, none of which exceeded their MRLs [18], which provided direct evidence for the high incidence of K. oxytoca tolerance to heavy metals in this study. Additional attention should be paid to the potential health risk of heavy metal pollution in consumable aquatic animals sold in Shanghai, China.
In this study, ten K. oxytoca isolates had MDR phenotypes, nine of which also exhibited tolerance to two or more than two heavy metals. For example, the K. oxytoca isolate K. o- H. rubra 8-2-2-11, showing resistance to the four antibiotics CHL, SXT, KAN, and TET, also tolerated the 6 heavy metals Cr3+, Cu2+, Hg2+, Mn2+, Pb2+, and Zn2+. These results suggested that heavy metal pollution likely co-selected for antibiotic resistance in K. oxytoca, and vice versa. In our previous research, antibiotic resistance correlating positively with heavy metal resistance was also observed in the V. cholerae isolates recovered from the 41 species of aquatic animals [18]. Cross-resistance due to co-selection can be inferred as the most likely mechanism of the rising antibiotic-resistant pathogens [41].

4. Materials and Methods

4.1. Sample Collection

Forty-one species of fresh aquatic animals, including 21 species of mollusks, 17 species of fish, and 3 species of crustaceans (Table S1), were sampled from Shanghai Jiangyang Aquatic Market (31°21′25.90″ N, 121°26′50.68″ E), and Shanghai Luchao Port Aquatic Market (30°51′40.21″ N, 121°50′48.40″ E) located in Shanghai, China in July, August, and September of 2018 and 2019 [18]. The samples (n = 108) were collected in sterile plastic bags (Nanjing Maojie Microbial Technology Co., Ltd., Nanjing, China), stored in iceboxes (700 × 440 × 390 mm), and immediately transported to the laboratory at Shanghai Ocean University, Shanghai, China for the analyses described below.

4.2. Isolation and Identification of K. oxytoca

K. oxytoca was isolated and identified in agreement with the instructions of the Chinese Government Standard (SN/T 1962–2007). Briefly, 25 g of each fish intestine sample or whole-tissue mollusk or crustacean sample was mixed with 225 mL sterile 1 × phosphate-buffered saline (PBS, pH 7.4–7.6, Shanghai Sangon Biological Engineering Technology and Services Co., Ltd., Shanghai, China), and then homogenized for 10 min using the Stomacher (Bagmixer 400 W, Interscience, Saint Nom la Bretèche, France). Ten-fold serial dilutions were prepared, and 100 μL of each dilution (to 1:104) was spread onto the selective MIAC (Beijing Land Bridge Technology Co., Ltd., Beijing, China) agar medium, and then incubated at 37 °C for 18 h. The red, wet, and sticky colonies that were 2 to 3 mm in diameter grown on the MIAC agar plates were picked out for further analysis.
Presumptive K. oxytoca colonies were identified using the MALDI-TOF/MS platform (Microflex LT/SH, MALDI Biotyper, Bruker, Germany) [42]. Briefly, 1.2 mL of 75% ethanol (Sangon Biotech Co., Ltd., Shanghai, China) and 50 μL of 70% formic acid (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) were added to the bacterial culture of each strain, according to the protocol recommended by the manufacturer (Bruker Daltonics, Bremen, Germany). The mixture was centrifuged at 14,000 rpm for 2 min and the supernatant was used for MALDI identification. The sample was spotted on the MBT Biotarget 96 target plates. After air-drying, 1 mL of saturated α-cyano-4-hydroxycinnamic acid (HCCA) (Bruker Daltonics, Bremen, Germany) matrix solution in 50% of acetonitrile and 2.5% of trifluoroacetic acid (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) was added. Mass spectra were acquired using a Microflex LT Mass Spectrometer (Bruker Daltonics, Bremen, Germany) with default parameters, including detection in linear positive mode, laser frequency of 60 Hz, ion source voltages of 2.0 and 1.8 kV, and lens voltage of 6 kV, within the m/z of 2000–20,000. For each strain, a total of 24 spectra from eight independent spots were acquired. External calibration of the mass spectra was performed using the Bruker bacterial test standard (BTS, Bruker Daltonics, Bremen, Germany) [42].
K. oxytoca strains were further identified by biochemical tests, including the capsular staining, Gram′s staining, and dynamic tests, using biochemical identification reagents (Qingdao haibo Bio Co., Ltd., Qingdao, China) following the instructions of the manufacturer and the Chinese Government Standard (SN/T 1962–2007). K. oxytoca strains were also identified by 16S rRNA gene amplification and sequencing using the universal bacterial primers 27F and 1492R (Table S5) as described previously [43]. Standard strain K. oxytoca ATCC43165 (Guangdong Culture Collection Center, Guangzhou, China) was used as a positive control strain in this study.

4.3. Detection of Virulence Genes in K. oxytoca

Virulence-associated genes (brkB, cdcB, mviM, pduV, relE, symE, tisB, vagC, virK, yqgB) were detected by PCR assay. All oligonucleotide primers (Table S5) were synthesized by Shanghai Sangon Biological Engineering Technology and Services Co., Ltd., Shanghai, China. PCR products were purified using Takara MiniBEST DNA Fragment Purification Kit Ver. 4.0 (Takara Biomedical Technology (Beijing) Co., Ltd., Beijing, China). Genomic DNA was extracted using TaKaRa MiniBEST Bacterial Genomic DNA Extraction Kit Ver.3.0 (TaKaRa, Beijing, China) following the manufacturer’s instructions. 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 reactions were performed as described previously [17,38,39], 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 S5). The extracted DNA samples and amplified PCR products were analyzed by agarose gel electrophoresis analysis, visualized, and recorded as described previously [17,38,39]. The PCR products were purified and sequenced by Suzhou Jinweizhi Biotechnology Co., Ltd., Suzhou, China. DNA sequences were analyzed using the basic local alignment search tool (BLAST) of the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/, accessed on 19 August 2021).

4.4. ERIC-PCR Assay

Genotyping was conducted by ERIC-PCR with the primers ERIC1R and ERIC2 (Table S5) [39,44]. The 20 μL reaction mixture contained 6 μL of DNase/RNase-free deionized water (Tiangen, China), 10 μL of 2 × Taq Master Mix (Novoprotein Technology, Shanghai, China), 1 μL of each primer (5 μM), and 2 μL of a DNA template. ERIC-PCR reactions were performed at 95 °C for 30 s, 52 °C for 1 min, and at 65 °C for 8 min for 32 reaction cycles. Amplified DNA fragments were visualized, recorded, and analyzed using BioNumerics 7.6 software, as described earlier [17,38,39]. Cluster analysis was performed by the unweighted pair group with arithmetic averages (UPGMA), and the Simpson index was calculated for the biodiversity of K. oxytoca isolates [17,38,39].

4.5. Antibiotic Susceptibility and Heavy Metal Tolerance Assays

The K. oxytoca isolates were measured for susceptibility to nine antimicrobial agents (Oxoid, UK) according to the method issued by the Clinical and Laboratory Standards Institute (2018, CLSI M100-S28), for CHL (30 μg), CIP (5 μg), GEN (10 μg), IPM (10 μg), KAN (30 μg), MEM (10 μg), NOR (10 μg), SXT (23.75 μg and 1.25 μg), and TET (30 μg). K. oxytoca ATCC43165 was used as a quality control strain. Broth dilution testing (microdilution) was used to measure the MICs of 8 heavy metals, including HgCl2, NiCl2, CrCl3, CdCl2, PbCl2, CuCl2, ZnCl2, and MnCl2 (Analytical Reagent, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) [17,38,39]. E. coli K12 (Institute of Industrial Microbiology, Shanghai, China) was used as a quality control strain. All tests were performed in triplicate in this study.

4.6. Statistical Analysis

Data analysis was performed using the SPSS statistical analysis software, version 17.0 (SPSS Inc., Chicago, IL, USA). The MARI of an isolate was defined as a/b, where a represents the number of antibiotics to which the isolate was resistant, while b represents the number of antibiotics for which the isolate was examined [17,38,39,45].

5. Conclusions

In the present 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, including 21 species of mollusks, 17 species of fish, and 3 species of crustaceans. Among these, 40 species of the bacterium had not been detected previously.. K. oxytoca was for the first time isolated from 14 species, including 10 species of mollusks: A. woodiana, B. areolata, C. cahayensis, H. rubra, M. antiquata, M. edulis, N. cumingi Crosse, S. subcrenata, S. constricta, and T. granosa; 3 species of fish: B. rock cod, C. auratus (Crucian), and C. auratus (Ditrema temmincki Bleeker); and one species of crustacean: P. clarkii. The other 27 species of aquatic animals were without K. oxytoca. Remarkably, K. oxytoca was found in high abundance in benthic animals, such as N. cumingi crosse, T. granosa, and A. woodiana.
None of K. oxytoca isolates (n = 125) harbored the 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 SXT (56.0%) was the most predominant among the isolates, followed by CHL (6.4%), TET (5.6%), and KAN (3.2%). Incidence of intermediate susceptibility to KAN (40.8%), and CIP (12.0%) were also observed. Approximately 8.0% of the isolates displayed MDR phenotypes. Meanwhile, high percentages of the isolates were tolerant to 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 the 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 considerable genetic diversity of the isolates.
Overall, the results of this study provided first experimental evidence for the presence of potentially virulent K. oxytoca in 14 species of commonly consumed aquatic animals, which poses a potential threat to food safety systems and the public health. In future research, the molecular mechanisms underlying the cross-resistance between antibiotics and heavy metals should be further examined in the emerging pathogen K. oxytoca.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/antibiotics10101235/s1, Table S1. The 41 species of aquatic animal products and the recovered K. oxytoca isolates, Table S2. Virulence-associated genes in representative K. oxytoca isolates, Table S3. Virulence-associated gene profiles of the 125 K. oxytoca isolates, Table S4. Tolerance of the 125 K. oxytoca isolates to eight heavy metals, Table S5. Oligonucleotide primers used in this study.

Author Contributions

L.N. and Y.X. major experiments, data analysis, and writing—original draft; L.C. funding acquisition, conceptualization, and writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by Shanghai Municipal Science and Technology Commission, grant number 17050502200, and the National Natural Science Foundation of China, grant number 31671946.

Data Availability Statement

The raw sequences generated in this study are available from the NCBI GenBank under the accession numbers MZ380322 to MZ380328.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Virulence-related genes of the 125 K. oxytoca isolates recovered from 14 species of aquatic animals. (A) mollusks; (B) crustacean; (C) fish.
Figure 1. Virulence-related genes of the 125 K. oxytoca isolates recovered from 14 species of aquatic animals. (A) mollusks; (B) crustacean; (C) fish.
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Figure 2. Antimicrobial susceptibility of the 125 K. oxytoca isolates.
Figure 2. Antimicrobial susceptibility of the 125 K. oxytoca isolates.
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Figure 3. Antibiotic resistance of the 125 K. oxytoca isolates in the 13 species of aquatic animals. (A) mollusks; (B) fish.
Figure 3. Antibiotic resistance of the 125 K. oxytoca isolates in the 13 species of aquatic animals. (A) mollusks; (B) fish.
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Figure 4. Heavy metal tolerance profiles of the 125 K. oxytoca isolates recovered from the14 species of aquatic animals. (A) mollusks; (B) crustacean; (C) fish.
Figure 4. Heavy metal tolerance profiles of the 125 K. oxytoca isolates recovered from the14 species of aquatic animals. (A) mollusks; (B) crustacean; (C) fish.
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Figure 5. ERIC-PCR-based genotyping of the 125 K. oxytoca isolates.
Figure 5. ERIC-PCR-based genotyping of the 125 K. oxytoca isolates.
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Figure 6. ERIC-PCR-based genotyping of the 10 K. oxytoca isolates with MDR phenotypes.
Figure 6. ERIC-PCR-based genotyping of the 10 K. oxytoca isolates with MDR phenotypes.
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Ni, L.; Xu, Y.; Chen, L. 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. Antibiotics 2021, 10, 1235. https://doi.org/10.3390/antibiotics10101235

AMA Style

Ni L, Xu Y, Chen L. 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. Antibiotics. 2021; 10(10):1235. https://doi.org/10.3390/antibiotics10101235

Chicago/Turabian Style

Ni, Ling, Yingwei Xu, and Lanming Chen. 2021. "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" Antibiotics 10, no. 10: 1235. https://doi.org/10.3390/antibiotics10101235

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