Pathogenic analysis of Vibrio alginolyticus infectionin a mouse model

Xiao-Fei Liu & Helin Zhang & Xingshan Liu &Yanwen Gong & Yingjian Chen & Yuan Cao & Chengjin Hu

Received: 16 April 2013 /Accepted: 13 September 2013 /Published online: 25 September 2013# Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2013

Abstract Vibrio alginolyticus is a Gram-negative halophilicbacterium and has been recognized as an opportunistic pathogento both humans and marine animals. So far, most studies havebeen focused on marine animals and few reports have beenaimed at mammals, including human. In this study, we firstestablished a mouse model to understand the pathogenic mech-anism of V. alginolyticus infection. After infection via intraper-itoneal injection, hematological and liver function indicatorswere evaluated and serum interleukin (IL)-1 and IL-6 expres-sion were detected by ELISA. Furthermore, we compared thevirulence of two V. alginolyticus strains, ATCC17749T andE0666. The results demonstrated that V. alginolyticus infectioncauses robust lung and liver damage and induces changes in IL-1, IL-6, hematological, and liver indicators. In addition, theATCC17749T strain appeared to be more virulent than theE0666 strain. Better understanding of the pathogenicmechanismof V. alginolyticus infection should guide effective preventionand therapy for V. alginolyticus infection.

Introduction

Vibrio alginolyticus is a Gram-negative, halophilic bacteriumfound in many marine floras. On the coast of Southern China,

V. alginolyticus is the dominant Vibrio species found both inseawater and in farmedmarine animals (Xie et al. 2005). It hasbeen recognized as an opportunistic pathogen to both humansand marine animals (Zhao et al. 2010), such as shrimp(George et al. 2005), sea bream (Balebona et al. 1998), clam(Gomez-Leon et al. 2005), and seahorse (Balcazar et al. 2010).The bacterium has been reported to cause wound infections(Horii et al. 2005; Matsiota-Bernard and Nauciel 1993;Sganga et al. 2009; Spark et al. 1979), ear (Ardic and Ozyurt2004; Feingold and Kumar 2004; Reina Prieto and HervasPalazon 1993), and ocular (Lessner et al. 1985; Li et al. 2009)and gastrointestinal diseases (Levine and Griffin 1993;Libinzon et al. 1975) in humans. Although rare, deep-seatedor invasive infections can develop. Cases of invasive V.alginolyticus infections defined as bloodstream infection ordeep-seated or necrotizing soft tissue infection (Gomez et al.2003; Ho et al. 1998; Howard et al. 1985) have been reported.The route of these infections is direct contact with contami-nated seawater or ingestion of raw seafood, which is the sameas that of other Vibrio infections.

V. alginolyticus infection is most common during summerseason. As global warming increases ocean temperature, theincidence of out-of-season infections caused by V. alginolyticushas been escalating (Sganga et al. 2009). Thus, it is urgent tostudy the pathogenic mechanism and therapeutic strategies totarget V. alginolyticus, especially related to human infection. Todate, most of the studies of V. alginolyticus pathogenic mecha-nisms has been focused on marine animals, such as Epinephelusawoara (Chen et al. 2012), shrimp (George et al. 2005), Pagrusmajor (Ye et al. 2008),Pseudosciaena crocea (Chen et al. 2008),Epinephelus bruneus (Harikrishnan et al. 2012), Haliotisdiversicolor supertexta (Wu et al. 2011), Lutjanus sanguineus(Liang et al. 2010), and Lutjanus erythropterus (Cai et al. 2010).Few reports have been focused on mammals, including humans.

X.

In this study, we established a V. alginolyticus-infected mousemodel to study the pathogenic mechanism of V. alginolyticusinfection.

Materials and methods

Bacterial strains and growth conditions

Two strains of V. alginolyticus were used in this study. StrainATCC17749T was originally isolated from spoiled horsemackerel , which caused food poisoning (Soto-Rodriguezet al. 2003), and strain E0666 was isolated from ascites ofEpinephelus coioides . Both strains were kindly provided byMarine culture collection of China (Xiamen, Fujian Province,China). The strains were confirmed in our laboratory using16S rDNA sequencing and a VITEK 2 system (bioMrieuxVitek, Durham, NC, USA). The strains were selected onthiosulfate citrate bile salt sucrose (TCBS) agar and grownwith agitation at 37 C for 16 to 20 h in alkaline peptone water(APW) supplemented with 2%(w /v ) NaCl. The bacterial cellswere centrifuged at 2,500 g for 5 min and then resuspended inAPW. The concentration of the bacterial suspension wasconfirmed by plate count and was adjusted to 1109 CFU/mL prior to inoculation.

Infection of mice with V. alginolyticus strains

Six to 8-week-old female BALB/c mice weighing approxi-mately 20 g were purchased from the Center of ExperimentalAnimals in Shandong University (Jinan, China). These ani-mals were certified as specific-pathogen-free (SPF). All ani-mal experiments were approved by the Animal Ethical andExperimental Committee of the General Hospital of JinanMilitary Region of PLA. The mice were infected with V.alginolyticus by intraperitoneal injection, and age-matchedcontrol mice were mock-inoculated with APW. For reasonsrelated to biosecurity, the infected mice were disposed of byautoclaving at the end of experiments.

Determination of the median lethal dose (LD50) for mice

Determination of the LD50 was conducted in accordance witha previous study (Kim et al. 2005). Briefly, 6 to 8-week-oldSPF female BALB/c mice were administered with fivefoldserially diluted bacterial suspensions ranging from 5107

CFU to 5109 CFU. The number of dead mice within 24 hand their survival time were recorded, and the LD50 valueswere calculated as the minimal dose of bacteria required for50 % lethality within 24 h. Six mice were used for each dose,and the experiment was repeated twice.

Culture and identification of bacteria isolated from tissuesand blood

The mice were anatomized after death or sacrificed after V.alginolyticus infection for 24 h. The heart and lung werecollected and homogenized in 1 ml sterile phosphate bufferedsaline using a sterilized homogenizer. The tissue homogenateswere inoculated in TCBS plates and incubated at 37 C for 16to 20 h. Blood collected from the eyeball was inoculated inAPW and grown with agitation at 37 C for 16 to 20 h. Thebacteria were identified by their morphology after Gram stain-ing and the VITEK 2 system using a VITEK 2 Gram-negativeidentification card (VITEK 2 GN Test Kit).

Table 1 Effect of the intraperitoneal injection on the LD50 of V.alginolyticus

Strain Body/organmass (g)

LD50 (CFU) Mean survivaltime (h)

ATCC17749T 21.81.1 1108 11.8

E0666 21.61.5 1109 18.3

BALB/c mice were injected with either the ATCC17749Tor E0666 strainintraperitoneally, and LD50 and mean survival time were calculated

Fig. 1 Histopathology sections of liver and lung. The mice were infectedwith V. alginolyticus ATCC17749T or E0666 for 12 h. Liver and lungsections were stained with H & E

168 Folia Microbiol (2014) 59:167171

Histopathology

The mice were sacrificed 12 h after infection with V. alginolyticusat 1108 CFU. The heart, liver, spleen, lung, kidney, duodenum,and greater curvature of the stomach were collected for hematox-ylin and eosin (H & E) staining. Histopathologic evaluation wasperformed by two pathologists in a blinded fashion.

Hematological and biochemical indicator testing

Blood samples were collected by peeling off eyeballs 12 hafter infection withV. alginolyticus and euthanized. Red bloodcell (RBC), white blood cell (WBC), hemoglobin (Hb), andplatelet (PLT) counts were performed using a Sysmex KX-21analyzer (Sysmex Corporation, Kobe, Japan). Serum sampleswere separated and alanine amid-transaminase (ALT), asparticacid amid-transaminase (AST), and total bilirubin level(TBIL) were measured using a Beckman DXC800 analyzer(Beckman Coulter, Brea, CA, USA).

ELISA for IL-1 and IL-6

The mice were sacrificed at the indicated times and bloodsamples were collected from the eyeballs. Serum concentrationsof interleukin (IL)-1 and IL-6 were measured using commer-cial ELISA kits (R&D Systems, Minneapolis, MN, USA)

according to the manufacturers instruction. Assays wereperformed in duplicates.

Statistical analysis

Data were expressed as means standard error. The differ-ences between groups were determined using the two-tailedstudent t test in SPSS software, version 13.0 (SPSS). A pvalue of

Isolation of bacteria from tissues and blood of deadand survival mice

Clinical data have revealed that V. alginolyticus infection resultsin bacteremia and is associated with high mortality (English andLindberg 1977; Janda et al. 1986; Lee et al. 2008). In order tobetter understand the lethal mechanism, we examined the heart,lung, and eyeball blood from animals that survived or died afterthe bacterial injection. We were unable to isolate bacteria fromthe surviving mice; however, bacteria were isolated from theheart, lung, and eyeball blood of the dead mice (data not shown).These bacteria were identified asV. alginolyticus , suggesting thatthey were from the intraperitoneal injection. Based on theseresults, it is reasonable to speculate that when a large amountof V. alginolyticus is injected into the peritoneal cavity, theperitoneum absorbs bacteria directly into the blood. In addition,rapid propagation promotes V. alginolyticus spreading to organsthrough blood circulation and causes multiple organ damage,which finally leads to lethality.

Histopathology

It has been reported that V. alginolyticus infection causes septicshock or necrotizing fasciitis in patients with cirrhosis (Ho et al.1998; Lee et al. 2008). Infection with another species, Vibriovulnificus, also results in necrotizing fasciitis and sepsis, andmost patients infected with this microbe have liver dysfunctionas an underlying disease (Matsumoto et al. 2010). To explore thepathogenicity of V. alginolyticus in the mouse model, mice wereinfected by intraperitoneal injection for 12 h with eitherATCC17749T or E0666 at 1108 CFU. The heart, liver, spleen,lung, kidney, duodenum, and stomach were sectioned to performH&E staining. Light microscopy observation revealed that afterinfection, hepatic cell suffered porosity, hydrops, and sinushepaticus suffered hemangiectasia and congestion, and the pul-monary tissue was subjected to congestion, hydrops, and pulmo-nary alveoli collapse (Fig. 1). The pathology sections revealedthat ATCC17749T caused more severe damage than E0666.

Hematological and biochemical indicators

Elevated serum ALT and AST levels are indications of a highspecificity and reasonable sensitivity of liver injury. Testing forASTandALTas a primary screening parameter for liver diseasesis part of many routine screening approaches (Wedemeyer et al.2010). Serum TBIL level was also used as an indicator forevaluating liver function (Lu et al. 2007). To further evaluatethe pathogenicity ofV. alginolyticus , eyeball bloodwas collectedand hematological and biochemical indicators were detected.Figure 2a, b shows that WBC and platelet numbers were signif-icantly decreased and RBC number was significantly increasedafter V. alginolyticus infection. The decrease or increase of theATCC17749T treated group was significantly higher that of the

E0666 treated group. The hemoglobin content of theATCC17749T treated group was also significantly higher com-pared with the E0666 treated or the control group. We thendetected biochemical indicators of liver function. Figure 2c, dshowed that ALT, AST, and TBIL were significantly increasedafter V. alginolyticus infection. These data showed that V.alginolyticus infection induced liver injury. The ALT and ASTexpression after infection with ATCC17749T were increasedsignificantly compared to that of E0666. These results suggestthat V. alginolyticus infection could induce changes of hemato-logical and liver indicators. The ATCC17749T strain inducedmore changes than the E0666 strain.

Inflammatory factor levels in serum

Previous clinical cases have reported that V. alginolyticus infec-tion could cause tissue inflammation, such as otitis (Ardic andOzyurt 2004; Feingold and Kumar 2004) or endophthalmitis (Liet al. 2009). The published data also showed that IL-1 expres-sion increased after injection with V. alginolyticus in fish (Weiet al. 2011). To see if V. alginolyticus infection could induceinflammation in mice, we examined inflammatory factor levelsin serum 12 h after V. alginolyticus infection. The serum IL-1and IL-6 expression was detected by ELISA. The results showedthat IL-1 and IL-6 level was significantly higher in serum of theV. alginolyticus-infected group compared to those in the controlgroup (p

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Folia Microbiol (2014) 59:167171 171

Pathogenic analysis of Vibrio alginolyticus infection in a mouse modelAbstractIntroductionMaterials and methodsBacterial strains and growth conditionsInfection of mice with V. alginolyticus strainsDetermination of the median lethal dose (LD50) for miceCulture and identification of bacteria isolated from tissues and bloodHistopathologyHematological and biochemical indicator testingELISA for IL-1 and IL-6Statistical analysis

Results and discussionIntraperitoneal LD50 in miceIsolation of bacteria from tissues and blood of dead and survival miceHistopathologyHematological and biochemical indicatorsInflammatory factor levels in serum

References