lnternatitmal Journal of Ft~d Mic~ "obiolog% 16 (1992) 349-352 349 © 1992 Elsevier Science Publishers B.V. All rights reserced 0168-161)5/92/$05.~)

FOOD 00531

Short communication

Preliminary examinations on the enterotoxigenicity of isolates of Klebsiella pneumoniae from seafoods

Bhoj Raj Singh and S.B. Kulshreshtha WHO C~dlal~)rating Centre for Vt'lcrinao' Pt~blit" fh'allh, hnlian Veterinary Research Institute. Izatnagar.

BareiUy. India

(Received 4 March ITS}2; revision received Ifi June 1~2: accepted 24 June ITS2)

One hundred and eighty-five seati,~gl samples, consisting of 96 freshwater fish, 37 marine fish, 13 freshwater prawn° 13 marine prawn and 26 molluscs were screened for presence of Kleb,w'ella. Out of these, 12 isolates of Klebsiella were identified, Four K. tmeum~miae var. ozaenae were im*lated from marine fish samples and eight h: pnenmtmiae vat. pneumoniue, six from freshwater fish and two from freshwater prawns. All 12 isolates were tested for enterotoxigenicity by the va~lmrmeability factor test in rabbits, the mouse f(~)t pad test, the latex agglutination test and the coagglutinathm test. One isolate of K. pneumoniae var pneumoniae, isolated from fresh water prawn was found enterotoxigenic.

Key words: Kleb.~iella paeumoniae vat. imetmnmiae; Kh'b.~iella pm'omoniae var. ozaenae; Enterotoxin

Introduction

Klcbsi¢lla arc common on scafoods and fish because thcy inhabitat¢ most of the natural waters. The load of Klebsiella increases with pollution of natural waters with excreta and these are cycled again to humans via consumption of fish and o ther seafoods, Klebsiella has been reported from seafoods, at a number of t imes as par t of the normal microflora of fish and seafoods (Koburger and Wahlquist, 1979; Okfer and Ncaako, 1985; Saxena, 1985; Boutin et al., 1986).

Klebsiella is a wcU known opportunistic pa thogen and can cause bacteraemia, pneumonia and urinary tract infections, but its enteropathogenici ty came into light in 1976 when Wads t roem ct al. (1976) isolatcd enterotoxigcnic strains of Klebsiella from clinical cases of diarrhoea. Klipstein and Engert (1977) isolated and purified heat stable and heat labile enterotoxins from K. p n e u m o n i a e and established their role in enterotoxicity.

However, environmental isolates of K. p n e u m o n i a e from oysters and waters f rom various localities did not show any enterotoxigenicity ei ther in vitro or in vivo

Correspondence address: Bhoj Raj Singh, Division of Veterinary Public Health, Indian Veterina~ Research Institute, Izamagar, Bareilly, India.

351)

T A B L E I

Results of enlerotoxigenieity of Kh'hsMla i~lales from fish and scafo<~Is

Area/market Type of ~afta~d Type of Klebsielht Enterotoxigenicity (No. of samples) isolated (No. of isolates) with MFPT. VPF'I',

LAT. COT

Bijnor Freshwater fish (18) None Freshwater prawn (3) K. pneamoniae vat. + + + +

I~tletO~tot#a# (1)

C a l c u t t a M a r i n e f ish (13) K. pm,mnoniae var . - - - - o.zae,,lae ( 4 )

Marine prawn (6) None

Delhi Marine fish (111) None Marine prawn (3) None

Bareilly Freshwater fish (78) K pneummaiae var. - - - - pnelttJlt~ae ( 6 )

F r e s h w a t e r p r a w n ( l i t ) K pnelonotlia~, v a t . - - - -

ptn,ultloniae ( | ) Marine fish (I) None Molluscs (26) None

Bombay Marine fish ( 13 ) None Marine prawns (4) None

M F P T . m o u s e l~t~ot p a d tes t : V P F T , v a s o p e r m e a b i l i t y f a c t o r tes t : L A T , l a tex a g g l u t i n a t i o n tes t ; C O T .

coagglntination lest,

(Boutin ct al., 1986). Hence enterotoxigenici ty appears to bc quest ionable for environmental isolates.

Mater ia l s and Methods

With steri le swabs, slime from the gills or surface of 96 freshwater fish, 37 marine fish, 13 freshwater prawns, 13 marine prawns and 26 freshwater molluscs was swabbed from fish markets of Bombay, Calcutta, Delhi, Bareiily and Bijnor (Table I). The swabs were immediate ly t ransferred to Cary Blair t ransport media (Difco) vials stored under ice and brought to laboratory for isolation of Klebsiella. Specimens were resuscitated in 0.1% peptone water for 6-8 h at 37°C. After resuscitation 0.1 ml of cul ture was transferred to MacConkey (Difco) broth tubes, incubated overnight at 37°C and la ter s t reaked on MacConkey (Difc'O agar plates. The plates were incubated at 37°C for 24 h. Lactose ferment ing pink, large, mucoid colonies were tested for their morphology and biochemical characterist ics to confirm their identity as Klebsiella (Edwards and Ewing, 1972).

For preparat ion of cell free culture fi l trate (CFCF), Klebsiella isolatcs were inoculated into 20 ml Brain Hear t lnfusion (BHI) broth (DIFCO) tubes. These tubes were incubated at 37°C in a shaking water ba th (100 rpm) for 18 h. The broth cultures were centrifuged at 4000 rpm for 30 min at 4°C. The supernatant was

351

filtered through 0.45-p,m poresize membrane filter and cell-free culture filtrate (CFCF) stored at 4°C until testing for cnterotoxicity by different methods.

The Vase permeability factor test (VPFT) was done according to Evans et al. (t9731.

The mouse foot pad test (MFPT) (Singh, 19891 was conducted on the test CFCF and control BHI broth, injecting 0.05 ml intradermally in the centre of the hind foot pad of mice along with suitable positive (E.scherichia coil LT) and negative (normal saline) controls. The mice were caged for 18 h and then observed for swelling in the foot pads and recorded as positive or negative by comparison with positive or negative controls respectively.

Mouse foot pads reacting positively in the MFPT with Klebsiella enterotoxin preparations (CFCF) were removed (after humane killing) along with suitable negative controls. The foot pads were perfuscd in vials containing a sufficient amount of saline with 1(1% (v/v) formaldehyde for 3 days, After this they were cut into thin pieces and washed for about 12 h in tap water to remove formaldehyde from the tissues. The tissues were then dehydrated in increasing concentrations of ethyl alcohol, cleared in turpentine oil and embedded in paraffin (melting point 60°C). Sections were cut at 4 -6 /xm thickness. The sections were stained as a routine with haematoxylin and eosin (Luna, 19681.

The Latex agglutination test (I_,AT) was performed as described by Finkelstein and Yang (1983) for E. coil

Slide coagglutination test was performed on a glass slide by putting one drop of test CFCF and mixing it with one drop of coloured (tetrazolium salt) cell suspen- sion of Staphyloc'occus aureus sensitized with optimal dose of anticholera entero- toxin serum (Kapoor, 1989).

Results and Discussion

The results obtained with 12 isolates of Klebsiella from fish and seafoods are presented in Table I. All 4 KI. pneumoniae var. ozaenae strain were isolated from marine fish while 6 K. pneumoniae var. pneumoniae were isolated from fresh-water fish and 2 from freshwater prawns. One isolate of K. pneumoniae vat. pneumoniae from prawns of Bijnor market was enterotoxigenic when tested with VPFT, MFPT, LAT and coagglutination test.

There appears to be some immunological relation of this enterotoxin of K. pneumoniae with the Cholerae toxin of V. cholerae as it gave positive LAT and coagglutination test antisera of heat labile E, coli enterotoxin and cholera toxin (results not shown). Our findings confirm the previous observations of Klipstein and Engert (19771.

In the negative control group, mouse foot pads did not reveal any significant change, while there was massive swelling in foot pads injected with enterotoxic CFCF of Klebsiella (results not shown).

In the foot pad sections of the negative control group the epidermis and dermis layers were apparently normal, while in foot pad sections where the enterotoxic

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culture filtrate of Klebsiella had been injected, marked oedema in the epidermal and dermal layers with massive infiltration of polymorphs and a few mononuclear cells was observed; m u ~ u l a r layers were invaded by a few polymorphs (results not shown).

Though this is a preliminary work and needs more investigations to identify the nature of the enterotoxin and its effect on various cell lines, this isolation of enterotoxigenic K. p n e u m o n i a e is of special importance, because the enterotoxi- gt:nie glebsiel la has been isolated from the environment or foods.

Acknowledgements

The authors are thankful to the Director, IVR! and Head of Division of Veterinary Public Heal th, IVRI , Izatnagar , Bareilly, UP for providing facilities to conduct the study.

References

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