Background
Members of the genus
Vibrio are abundant in marine environments [
1] and in inland rivers where seawater intrusion occurs [
2]. The levels of
Vibrio spp. in various seafood commodities have been reported to be significantly higher in the summer than in other seasons [
3]. Under this genus,
V. furnissii is a motile, oxidase-positive, gram-negative, halophilic bacterium with phenotypic characteristics highly similar to those of
V. fluvialis [
4]. Although
V. furnissii is closely related to
V. fluvialis, it differs in its ability to produce gas through carbohydrate fermentation [
4].
V. furnissii is a potential pathogen of European eel (
Anguilla anguilla) [
5] and also one of the non-cholera
Vibrio species pathogenic in humans that can spread through the consumption of contaminated seafood products or exposure to coastal waters [
6,
7].
V. furnissii has been associated with outbreaks or sporadic cases of gastroenteritis with cholera-like symptoms including diarrhoea, abdominal cramps, nausea and vomiting [
7,
8]. Cases of
V. furnissii bacteraemia associated with skin lesions or cellulitis have also been reported [
9,
10].
V. furnissii infection has been reported to be rarer than
V. fluvialis infection. The US Centers for Disease Control and Prevention (CDC) reported only 10 isolates of
V. furnissii from 1997 to 2008: three from blood, two from a wound and five from stool [
9]. However, since 2006, 24 sequences of
V. furnissii have been submitted to GenBank in succession, which means that
V. furnissii infection is increasingly reported.
V. fluvialis infection has been reported worldwide [
11‐
13] and shows features of multi-drug resistance [
12,
14] including resistance to fluoroquinolones and β-lactam antimicrobials,
blaNDM−1-mediated carbapenem resistance and azithromycin resistance [
15‐
17].
V. fluvialis has the ability to cause epidemics, so the rapid increase in and spread of antibiotic resistance in this pathogen during the past 20 years has become a major cause of concern [
18].
V. furnissii is phylogenetically close to
V. fluvialis, and recently, a
mph(A)- and
blaOXA−1-bearing conjugative plasmid, which mediates resistance to cephalosporins and azithromycin, was also discovered in a
V. furnissii strain isolated from hospital sewage in Zhuhai, a coastal city in China [
19].
Genome analysis of
V. furnissii NCTC11218 isolated from estuaries [
20] has shown that this species has a dynamic and fluid genome that can quickly adapt to environmental perturbation and has a series of virulence-related genes, such as quorum sensing-related genes
cqsA,
cqsS,
luxS,
luxU/O and
luxP/Q; biofilm formation-related genes
hapR and
vpsT; major pilin subunit-encoding gene
tcpA; and haemolysis-related genes
rtx and
hlyA. These genes are also widely distributed in other pathogenic
Vibrio spp.
In this study, we isolated seven V. furnissii strains from the stool samples of patients with diarrhoea; surveyed the clinical characteristics of the sampled patients; and analysed the antibiotic resistance and virulence phenotypes of, and related genes in, the isolated strains by using whole-genome sequencing.
Discussion
Although
V. furnissii and
V. fluvialis are two closely related species,
V. furnissii differs from
V. fluvialis in its ability to produce gas through glucose fermentation. However, gas production from glucose fermentation has been reported be variable and thus not reliable for differentiating between the two species [
46]. In our study, VITEK MS identified
V. furnissii strains as accurately as did whole-genome sequencing, and other studies also used MS to confirm
V. furnissii colonies [
7,
10,
46]. These results indicate that MS could be used in clinical laboratories to identify
V. furnissii quickly and easily.
Many selective-differential media have been developed for isolation of
Vibrio spp., however, none of the media developed to date combines the sensitivity to low numbers with the specificity necessary to inhibit growth of other organisms [
47]. AMP is frequently added to isolation medium as a selective agent when culturing
Aeromonas [
48]. In this study, as the isolated
V. furnissii strains caused strong beta haemolysis on blood agar and had intermediate resistance or high resistance to AMP, blood agar plates with 20 µg/mL AMP also helped to isolate more
V. furnissii compared with only using gentamicin selective medium.
V. furnissii has previously been isolated in inland cities such as Beijing, and its total isolation rate in our study was 0.4% (7/1985), which is close to the rate (0.5%) reported in Recife, Brazil [
49], an Atlantic seaport city. The reason for such similar rates might be that seafood products are widely consumed worldwide and bacteria of the
Vibrio genus can contaminate seafood [
22]. Although the patients in our study reported no exposure to seawater or seafood, we inferred that seafood might have polluted the food they ate.
Phylogenetic analysis showed that the V. furnissii strains were clustered into three main clades, which were not directly related to the isolation location or host source. Our seven V. furnissii isolates were in different monophyletic clades in the phylogenetic tree, suggesting that these strains were not associated with outbreaks but were seven independent cases of gastroenteritis.
V. furnissii strains express many putative virulence factors, such as a series of virulence factors described in the genome analysis of
V. furnissii NCTC11218 [
20]. In addition, the virulence genes
vfh,
vfp and
hupO, which occur widely in
V. fluvialis, were also found in
V. furnissii. VFH expression is associated with strong beta-haemolysis on blood agar [
39] and can induce interleukin-1β secretion through the activation of the NLRP3 inflammasome [
42]. Given the high similarity of
vfh between
V. fluvialis and
V. furnissii, the haemolysin characterisation of
V. furnissii was also likely to be related to
vfh. VFP is a metalloprotease that exhibits haemagglutinating, permeability-enhancing, haemorrhagic and proteolytic activities [
43]. HupO, an iron-regulated hemin-binding outer membrane protein, stimulates haemolysin production and resistance to oxidative stress [
44].
V. furnissii also showed the presence of IlpA, a potent immunogenic lipoprotein that triggers cytokine production in human monocytes by activating Toll-like receptor 2 in
V. vulnificus [
41]. T6SS was first identified in
V. cholerae [
50] and
Pseudomonas aeruginosa [
51]. To date, it has been shown to be widely distributed in approximately 25% of all gram-negative bacteria [
51]. T6SS can mediate
V. cholerae infection in humans [
50], and the T6SS of
V. fluvialis is important for interbacterial competition [
52]. In our study,
V. furnissii was found to express functional T6SS.
V. furnissii strains were resistant to ampicillin (85.7%) and cephalothin (21.4%), a first-generation cephalosporin, in a Peru survey in 1995 [
8]. Some case reports have also reported that their
V. furnissii isolates were sensitive to most antibiotics except ampicillin [
7,
9,
10]. In comparison, the
V. furnissii strains in our study showed higher resistance to streptomycin, tetracycline and cefazolin (a first-generation cephalosporin), as well as high rates of intermediate resistance to ampicillin/sulbactam and imipenem. VFBJ02 was also resistant to imipenem and meropenem, both of which are carbapenems, although carbapenem resistance-related genes were not detected. This suggests the presence of an undiscovered carbapenem-resistant mechanism in
V. furnissii. Imipenem is thus not recommended to treat
V. furnissii infections. The CDC recommends combination therapy with doxycycline and ceftazidime to treat
V. vulnificus infection [
53]. Although antimicrobial therapy for
V. furnissii infection has not yet been established, our results suggest that fluoroquinolones and third-generation cephalosporins, such as ceftazidime and doxycycline, are effective at treating
V. furnissii infection.
Except for the streptomycin resistance shown by VFBJ01, VFBJ05 and VFBJ07 being completely consistent with the presence of
strA,
strB,
aph(3’’)-Ib and
aph(6)-Id genes, the antibiotic susceptibility patterns of the seven
V. furnissii strains were partly consistent with the presence of other antibiotic resistance genes. For example, although VFBJ01, VFBJ05 and VFBJ07 carried
sul genes, their sensitivity to sulphonamides only slightly declined. Similarly, prior studies have shown that resistance genes do not completely correlate with phenotypic resistance [
54,
56]. In the present study, the tetracycline resistance of VFBJ05 and VFBJ07 was consistent with the positive detection of
tetA,
tetB and
tetR; however, VFBJ01 and VFBJ02 were also resistant to tetracycline despite carrying no
tet genes. This finding suggests that other unknown genes influence the relationship between the genotype and phenotype of tetracycline resistance. In addition, although VFBJ07 carried
aac(6’)-IIa, its fluoroquinolone resistance level was only slightly raised and it remained sensitive to fluoroquinolones. The reason may be that several resistance mechanisms together contribute to fluoroquinolone resistance, including mutations in the quinolone resistance-determining genetic regions and plasmid-mediated quinolone resistance [
57]. Ultimately, although the detection and bioinformatic analysis of antibiotic resistance genes cannot completely replace antibiotic susceptibility tests, the existence of antibiotic resistance genes might mean that the strains’ sensitivity to the corresponding antibiotics is declining, thus providing useful guidance for infectious disease treatment.
Several mobile genetic elements, such as insertion sequences, transposons and gene cassettes/integrons, can move within or between DNA molecules and transfer between bacterial cells [
58]. GCA_021249365 was isolated from hospital sewage in Zhuhai, Guangdong province, China [
19], and GCF_024220035 [
40] was isolated from the stool sample of a 63-year-old man in Zhongshan, Guangdong province. These two strains were quite close on the phylogenetic tree, indicating that they might have originated from the same colony. The two strains also possessed the same antimicrobial resistance gene-bearing conjugative plasmid. In our samples, antibiotic resistance genes occurred on transposon islands in VFBJ05 and VFBJ07. The existence of transposon islands carrying antimicrobial resistance indicates that these strains may greatly increase the spread of drug resistance among clinical isolates through the process of infection, especially VFBJ05, in which transposon islands are located on a plasmid.
In conclusion, we found that diarrhoea associated with V. furnissii infection occurred sporadically and was more common than expected in the summer in Beijing, China. Fluoroquinolones and third-generation cephalosporins, such as ceftazidime and doxycycline, may be effective at treating V. furnissii infection. Two strains – VFBJ05 and VFBJ07 – were found to carry transposon islands containing antibiotic resistance genes. V. furnissii had unique virulence characteristics indicated mainly by the presence of T6SS and haemolysis. Overall, the results contribute to our understanding of the bioinformatic and clinical features of V. furnissii infections. Future academic and clinical efforts should focus on continual and improved laboratory-based surveillance to prevent and control V. furnissii infections and antibiotic resistance gene dissemination.
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