Isolation of Salmonella typhi from apparently healthy liver

Isolation of Salmonella typhi from apparently healthy liver
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  Short communication Isolation of   Salmonella typhi  from apparently healthy liver Gopal Nath a, ⇑ , Chandra Bhan Pratap a , Saurabh K. Patel a , Anil K. Gulati a , Sunil Kumar Tripathi b a Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India b Forensic Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India a r t i c l e i n f o  Article history: Received 9 June 2011Received in revised form 24 August 2011Accepted 30 August 2011Available online 6 September 2011 Keywords:S. typhi Chronic typhoid carriersNested PCR LiverGallbladder a b s t r a c t It is suggested that  Salmonella typhi  resides mostly in hepatobiliary system especially in gallbladder inchronic typhoid carriers. It is not very clear whether in gallbladder lumen or on its wall or in liver. How-ever, we had been successful in detecting  S. typhi  in liver by PCR targeting flagellin gene sequences.Therefore, in the present study, we tried to isolate the bacterium from liver tissue collected from deadbodiesbrought forpostmortemexamination. Wecouldisolate S.typhi  in2of 20suchlivertissues exam-inedbyusingconventional isolationtechniques.Theisolateswereidentifiedbyroutinephenotypicchar-acters and were confirmed by amplification and sequencing of two conserved genes i.e. 16S rDNA andflagellin (  fliC  ) gene followed by blasting on www.ncbi.nlm.nih.gov.   2011 Elsevier B.V. All rights reserved. 1. Introduction Chronic carriage of   Salmonella enterica  subspecies  enterica  sero-type typhi ( Salmonella typhi ) with focus usually in the gallbladderis considered far more common than any other site. Chronictyphoid carriage may develop either as a sequel of acute typhoidfever or subclinical infection by the bacterium. Subclinical typhoidinfections have been predicted five times more than the acutecases in an endemic area. About 10% of the apparently healthypopulation has been found to be chronic typhoid carrier based onVi serology in endemic area (Mohan et al., 2006). In agreement with the results of Vi serology, nested PCR based amplification of   fliC   gene sequences of   S. typhi  in hepatobiliary system of deadbodies of accidental deaths have shown almost similar prevalence(Nathet al., 2010a,b). Further, inthisstudy, wecouldprovethat  S.typhi  primarily resides in liver rather than gallbladder. Ourobservation has been supported by the demonstration of   S. typhi bacteria by immunohistochemistry (Ramachandran et al., 1974) and evidences of hepatic dysfunction in 21–60% of the acutetyphoid cases (Khosla et al., 1988). Therefore, it became prudent to isolate  S. typhi  fromliver to confirmthe speculation of presenceofthisbacteriuminliver.Therefore,thepresentstudywasplannedto isolate  S. typhi  from liver tissue collected from dead bodieswhich were apparently free from gallbladder diseases.A total of 20 corpses were included in the present study. Theproposal was cleared by Institute Ethics Committee of BanarasHindu University, Varanasi. They were victims of unnatural deathsbrought to the Centre for Advanced Studies, Department of Foren-sic Medicine of the University Hospital within 18h of death forpost mortem examinations. The study period extended from JanuarytoFebruary, 2010. Livertissuewascollectedandsubjectedfor isolation by inoculating directly on MacConkey agar andDesoxycholate citrate agar and Selenite F enrichment broth within1h of collection and subsequent subculture on MA agar and DCA. 2. Identification of the isolates  2.1. Phenotypic identification The suspected colonies were confirmed by Grams’ staining,motility testing and inoculating a battery of test substrates.  2.2. Genotypic identification 2.2.1. Isolation of bacterial DNA The bacterial DNAwas extracted following the standard phenolchloroform method (Sambrook and Russell, 2001).  2.2.2. Amplification and sequencing of S. typhi specific sequences The DNA extracts from each of the isolates in the quantity of 100ng was subjectedto PCRusing primer specific to flagellin(  fliC  )gene of   S. typhi  following the method described by Song et al.(1993) which was further modified by Frankel (1994). While 16S rDNAwasamplifiedbyusinguniversalprimersspecifictoeubacte-ria (Carrol et al., 2000) (Table 1). 1567-1348/$ - see front matter   2011 Elsevier B.V. All rights reserved.doi:10.1016/j.meegid.2011.08.029 ⇑ Corresponding author. E-mail address:  gopalnath@gmail.com (G. Nath).Infection, Genetics and Evolution 11 (2011) 2103–2105 Contents lists available at SciVerse ScienceDirect Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid  Although, all the essential precautions were taken to avoid lab-oratory contamination. Known positive (DNA from reference strainof   S. typhi , MTCC 3216) andnegativecontrols(distilledwater) wereput to detect contamination. Amplicon generated for  fliC   and 16SrDNA were sent for partial sequencingto Bangalore Genei, India. Se-quences were analyzed using BLAST N (http://www.ncbi.nlm.nih.-gov/BLAST/) to verify identity of the sequences as  S. typhi .Two of the 20 specimens of hepatic srcin yielded positive for S. typhi . One of the deceased was a female of age 35 years, diedinstantaneously due to injury to brain and other vital organs in aroad traffic accident. The other dead body was a case of drown-ing, a male of 36 years age, had injury to lungs and chest walland died due to shock and haemorrhages. Isolates from liver of both dead bodies gave similar biochemical reactions. These iso-lates were observed growing on simple medium, were able tobreak down glucose both by oxidative and fermentative path-ways, did not produced cytochrome oxidase but nitrate was re-duced. They did not produce phenyl pyruvic deaminase andurease enzymes. There was alkali/acid reaction with very smallamount of H 2 S production in triple sugar iron but indole wasnot produced. Glucose and mannitol were fermented withoutproduction of gas. Lactose and sucrose were not fermented andlysine was decarboxylated. The isolates were further confirmedby serological agglutination using poly O, poly H, factors O9,H-d and Vi antisera.Fig. 1A and B shows the gel photograph of amplification for  fliC  and 16S rDNA gene respectively .  The sequencing data on blastingshowed >99% similarity with serotype typhi.Both of the above isolates were from the victims of accidentaldeath with the remote possibility of their being cases of acute ty-phoid fever. The speculation that the  S. typhi  might have migratedfrom gallbladder or from intestine seems to be untrue because wehave already observed that  S. typhi  were exclusively present in 55%of the liver specimens (Nath et al., 2010a,b). Isolation of   S. typhi fromlivertissue fromsuspected casesof chroniccarriersis thefirstreport of its kind. However, in the light of precise history of thesecases, we cannot claim that both cases were definitely chronic car-riers. They might be convalescent excreters or with asymptomaticinfection caused by  S. typhi . Previous reports of isolation of   S. typhi areavailable,butmostlyfromcasesofliverabscessduetoserotypetyphi with or without  Entamoeba histolytica  (Kabra and Wadhwa,2006; Soni et al., 1994). Occurrence of hepatomegaly, jaundice,raised serum enzymes and bilirubin level in acute typhoid fevercases has already been reported (Khosla et al., 1988). Moreover, frequent failure of cholecystectomy to eradicate the carrier statealso supports the view that gallbladder is not the sole niche of the bacterium in chronic typhoid carriers. To assess the involve-ment of liver in chronic carriage, liver function test may be sug-gested. We should improvise modalities which can deal withintracellular abode of the bacterium in phagocytic cells which isusuallydifficulttotreat.Presently,afewof theavailableantibioticsi.e. chloramphenicol, macrolides, aminoglycosides, rifampicin etc.have been used for many weeks duration but without definite curefrom chronic typhoid carriage. The other treatment modalities forinfectious diseases caused by intracellular pathogens may be inthe form of vaccines inducing cell mediated immune response,newer chemotherapeutic agents, bacteriophage therapy, inductionof autophagy by chemical agents etc. may be explored (Finlay andHancock, 2004; Schwegmann and Brobacher, 2008; Chiu et al.,2009). Although, simple removal of gallbladder is not going to helpin eradication of typhoid carriage state, it will definitely help inpreventing development of gallbladder cancer and other nonmalignant inflammatory conditions might be occurring due topresence of bacterium in liver.  Table 1 Primers used for amplification of 16S rDNA and flagellin (  fliC  ) gene of   Salmonella typhi . S. No. Gene Primer sequence Product size Annealing1 16S rDNA 16S F 5 0 -TTG GAG AGT TTG ATC CTG GCT C-3 0 16S R 5 0 -ACG TCA TCC CCA CCT TCC TC-3 0  1194 bp 59   C2 Flagellin (  fliC  ) gene(i) Primary amplification(ii) Nested amplificationST1 – 5 0 -ACT GCT AAA ACC ACT ACT-3 0 ST2 – 5 0 -TTA ACG CAG TAA AGA CAG-3 0 ST3 – 5 0 -AGA TGG TAC TGG CGT TGC TC-3 0 ST4 – 5 0 -TGG AGA CTT CGG TCG CGT AG-3 0 495 bp364 bp57   C63   C Fig. 1.  (A) Gel Picture showing amplified product of flagellin (H1 d ) gene of   Salmonella typhi  by using specific nested primer: lane 1 – molecular marker (100 bp); lane 2 –negative control; lane 3 – positive control ( Salmonella typhi  MTCC 3216); lane 4 – RL-130 isolate; lane 5 – RL-131 isolate. (B) Gel-Picture showing 16S rDNA amplification of  Salmonella typhi : lane 1 – molecular marker (100 bp); lane 2 – negative control; lane 3 – positive control ( Salmonella typhi  MTCC 3216); lane 4 – RL-130 isolate; lane 5 – RL-131 isolate; lane 6 – molecular marker (1 kb).2104  G. Nath et al./Infection, Genetics and Evolution 11 (2011) 2103–2105  References Carrol, N.M., Jaeger, E.F., Choudhury, S., Dunlop, A.A., Matheson, M.M., Adamson, P.,Okhravi, N., Lightman, S., 2000. Detection of and discrimination between gram-positive and gram-negative bacteria in intraocular samples by using nestedPCR. J. Clin. Microbiol. 38, 1753–1757.Chiu, H.C., Kulp, S.K., Soni, S., Wang, D., Gunn, J.S., Schlesinge, L.S., Chen, C.S., 2009.Eradication of Intracellular  Salmonella enterica  Serovar Typhimurium with aSmall-Mole. Antimicrob. Agents Chemother. 53, 5236–5244.Finlay, B.B., Hancock, R.E., 2004. Can innate immunity be enhanced to treatmicrobial infections? Nat. Rev. Microbiol. 2, 497–504.Frankel, G., 1994. Detection of   Salmonella typhi  by PCR. J Clin Microbiol. 32, 1415.Kabra, S., Wadhwa, V., 2006. Hepatic abscess caused by  Salmonella typhi . Ind. Paed.43, 81–82.Khosla, S.N., Singh, R., Singh, G.P., Trehan, V.K., 1988. The spectrum of hepatic injuryin enteric fever. Am. J. Gastroenterol. 83, 413–416.Mohan, U., Mohan, V., Raj, K., 2006. A study of carrier state of   S. typhi , intestinalparasites and personal hygiene amongst food handlers in Amritsar city. Ind. J.Comm. Med. 31, 60–61.Nath, G., Maurya, P., Gulati, A.K., Singh, T.B., Srivastava, R.C., Kumar, M., Tripathi,S.K., 2010a. Comparison of Vi Serology and nested PCR in diagnosis of chronictyphoid carriers in two different study populations in typhoid endemic area of India. Southeast Asian J. Trop. Med. Pub. Health 41, 636–640.Nath, G., Singh, Y.K., Maurya, P., Gulati, A.K., Srivastava, R.C., Tripathi, S.K., 2010b.Does  Salmonella typhi  primarily reside in the liver of chronic typhoid carriers? J.Infect. Dev. Ctries. 4, 259–261.Ramachandran, S., Godfrey, J.J., Perera, M.V., 1974. Typhoid hepatitis. JAMA 230,236–240.Sambrook, J., Russell, D.W., 2001. Molecular cloning: a laboratory manual 3rd ed..Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, p. 1.31..Schwegmann, A., Brobacher, F., 2008. Host-directed drug targeting of factorshijacked by pathogens. Sci. Signal 1, 8.Song, J.H., Cho, H., Park, M.Y., Na, D.S., Moon, H.B., Pail, C.H., 1993. Detection of   S.typhi  in the blood of patients with typhoid fever by PCR. J. Clin. Microbiol. 31,1439–1443.Soni, P.N., Hoosen, A.A., Pillay, D.G., 1994. Hepatic abscess caused by  Salmonellatyphi . A case report and review of the literature. Dig. Dis. Sci. 39,1694–1696. G. 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