In research, rhe horizon recedes as wt? advance ... the urgency of the pursurr grows more inrense. .. And reseurch i s always blcomplete

- Mark Panison

Limitations of the study

Every research project has its shortcomings. The present study is no exception.

,nough every effort was put in to overcome various obstacles, some were

insurmountable. Below are a few of the factors that render this study less than perfect.

I . The PCR-RFLP and envelope profiling studies were performed on a select

number of isolates due to constraints of reagent availability. A more

comprehensive picture could have been obtained with a larger sample size.

2 . Determination of the role of envelope proteins in fluoroquinolone resistance was

rcstricred to constitutive differences in the profiles on SDS-PAGE. Induction of

protein alterations following exposure to ciprofloxacin w;ls the next logical step in

thc investigation o f this Ilrpect but was not a p i u ~ of the study. Such studies

require more sensitive ins~rurnents than were available here, and so were not

pursued.

3 fTR-RRP ~tudies yielded valuable data regarding different genes that were

~nvcst igi~cd in connection with ciprofloxacin resistunce, Sequencing o f these

penes for the different isolates tested would have ceminly provided a wealth o f

~nformation. However, due to limited resources, sequencing o f the prA product

could not he pe r fo rn~d for all the i~olates. Neither W;F\ il p~ssible lo sequence the

mclr PCR prcxlucr\.

4 . :\ battcry o f tests, including both culture m d vrology uas employed for

confirming typhoid etiology in the ileal perforation cases. Using a DNA based

technique like PCR to detect specific DNA in the ileal biopsy mdy have provided

further evidence However, the PCR ficilities were not available till the latter half

of the study period.

5 R i d serum nnlplcs could hc obtained for only a few of the perfontion cases.

lmmunoblotting studies for the detection o f IgM antibodies were not a p;ur of this

work due to non. avi labi l i ty o f appropriate reagents.

Reagents/ Protocols

1. REAGENTS FOR PCR-RFLP: 0.5 M EDTA ph 8.0

Disodium EDTA 186.1 g

Double distilled waler 800 ml

The pH was adjusted to 8.0 wilh NaOH pclle~s (approx 20g)

5X TBE Bufler

Tris Base

0.5 M EDTA ph R . 0

Boric Acid

Ethidium bromide ( I0 mdnil. \lock)

Ethidium Brornrdc

Dirtilletl water

Composition of a g a m ~ l s

05X TBE Bufler

Agarosc 2.10 ntg 500 mg

Sample loading buller

Brorm~phcnol Blue 2.5mg

Xylene cyanol 2 .5 mg

50% Glyccml (in Mi l l~Q wd~er) I mL

Runnlng Bufler (05X TBE)

SX TEE buffer

Distilled water

Composition of 10X PCR buffer

(NH4)2S04

Tris HCI pH 8.8

MgClz

Tween 20

Composition of 10X RE buffer

Buffer Y Tango (for Hirfl, HpnlI)

Tris acetate

Magnesium acetalc

F'otxwium acetate

BS A

Buffer 11 pH 75 (for H ~ n f l , t i h u l )

T r ~ s t i c 1

XlgCI:

NaCl

D~[hltrrythr~ol

Hufler L pH 7 5 (for H p ~ r l l ~

T r ~ t HCI

SlgCI2

I)~th~oerythriol

Rufler E pH 7.9 (for Sl1u3r\l) ~lapnealum acetate

f'ota.wum actlate

'Tn\ setate

O~th~odur~rol

100 n ~ h l

100 mM

I OmM

t. REAGENTS FOR ANTIGEN EXTRACTION:

Nwmrl d i n e

NaCl 900 mg

Distilled water lOOmL

RoLeinaw K stock (2OmglmL)

Roleinate K 20 mg

MilliQ water I mL

Solution 1 ( IOmM Tris HCI, pH 8.0, also used for envelope extraction)

Tris Base 121 mg

Doublc distilled water 80 mL

The pH of h e solulion was adjusted to 8.0 with concentrated HCI and made up to

100 rnL

Sdution 2

lOmM Tris HCI pH 8.0

MgCI!

Triton-X 100

Solution 3

l h M Tria HCI pH 8.0

SDS

3. REAGENTS FOR SDS-PACE:

OJM Tris HCI pH 6.8

Tris base 6.lg

Distilled water 80 rnL

The pH of the solution was adjusted to 6.8 with concenmted HCI and the solution

made upto l 00 mL

15M Tris HCI pH 8.8

Tris b a ~ 18.15 g

Distilled water 80 mL

The pH of the solution was adjusted to 8.8 with concentrated HCI and made up to

100 rnl,

Solution A (Acrylarnide stock wlution)

Acrylarnidc 29.2g

Bisacrylamide 0.8g

Acrylamidc and bis-acrylamidc were dissolved in 80 mL of distilled water with

constun! s~irring. Thc solution w a filtered and made up to 100 mL, and stored in

dark hottles a1 4°C.

4X &prating gel elflet

I .5M Tris HCI pH 8.8

SDS

Distilled water

4X stacking gd Mer

0.SM Tris HCI pH 6.8

SDS

Distilled wilier

10% Ammonium pemlphate

Ammonium pcrsulphate

Distilled water

Electrophoresis buRer (pH 8.3)

Tris base 3g

Glycinc 14.4g

SDS I a bsti l l td water I000 mL

.Separating gel reagtnts

Stocking gel reagents

Ut.\tillcd water

Solution A

Solution C

10% APS

TEMED

Reagent

Sdution A

Sdutlon B

4X So)uWmtion buNer

I M Tris HTI pH 6.8

Glycerol

SDS

2 mercupfwthanol

Distilled wntcr

2.3 rnL

0.7 ml.

I mL

3opl

Spl

10% ge4

3 .4 mL

2.5 mL

0.6 ml.

2.5 mL

200 rng

0.5 mL

6.4 mL

125 % gel

4.1 rnL

2.5mL

Distilled water 3.4 mL P

10% APS

TEhlED -

.SO pl

5

-,

50 p1

5

4. SILVER STAINING REAGENTS:

Fixer

Methanol 50mL

Acetic acid 12 mL

Formaldehyde 18.5pL

Distilled water 38 rnL

Washing solution

Methanol

Distilled water

Pretreatment solution (freshly prepared prior to use.)

Sodium thiosulphate 10 mg

Distilled water 50 mL

Stainer

Silver nitnte

Formaldehyde

Disti llcd water

lkvelopcr

Stxltum carbonate

Formaldehyde

Distilled water

stop s d u h

Acetic acid

Distilled wuter

Sliver staining protocoi

Al l steps wen performed on a gel rocker. A l l glassware and plastic ware were

thoroughly cleaned prior to use, and gloves were used to handle the gels.

I . Following SDS-PAGE, the gel was place in the fixer for I hour (or overnight)

2. The fixer was poured off and the gel wa$ immersed in washing solution for 30

minutes. This was repeated with 2 changes of washing solution.

3. The gel was then immersed in freshly prepared pretreatment solution for I

minute.

4. This was followed by 3 washes in distilled water, each for 20 seconds

5 . The gel was then placed in staining solution for 60 minules in the dark.

6 , The gel waq then transferred to the developer solution for 2-10 minutes until

b i d s were visible (about 5 minutes for proteins, 10-15 minutes for LPS). When

the bands appeared, [he gels were tmmediately placed in distilled water.

7. The gels were wuhed with distilled water for 30 seconds.

8. The stop solution was then added and the gel shaken for 10 minutes.

0 , The gels were placed in wihhing solution for 20 minutes. The washing solution

was also used as a preservative.

5. IMMUNOBLO'ITING REAGENTS:

Tnlrsfer buffer

Tri s base

Glycine

Distilled water

Methanol

Phosphate Buffered Saline (PBS) pH7.2

NaCI

Na2HPO4

NaH2PO4

Distilled water

2% BSA

Bovine serum albumin

PBS pH 7.2

--- Tween i6

----.

Sample bufler (for diluting patien1 scm. conjugate)

0.05% Twter120 5rnL

2% BSA 5 rnL

Papers presented1 published Based on the work undertaken in this study, 4 papers have been published in peer

reviewed journals, and 4 papers have been presented at national level conferences.

Additionally, 2 papers were accepted for presentation at an international conference.

I. Nair S. Unnikrishnan M. Turner K, Parija SC, Churcher C, Wain J, Harish BN.

Molecular analysis of fluoroquinolone-resistant Salmonella Paratyphi A isolate.

India. Emerg Infect Dis 2006; 12(3):489-91

2. Madhulika U, Harish BN, Parija SC. Current pattern in antimicrobial

susceptibility of Salmonella Typhi isolates in Pondicherry. Indian J Med Res 2004

Aug; 120(2): l 11-4

3. Harish BN, Madhulika U. Parija SC. Isolated high-level cipmfloxacin resistance

in Salmonella cnterica suhsp, enterica serotype Paratyphi A. J Med Microbial

2004 Aug;53(PI 8):HIY

4. Harish BN, Madhulika [J, V~shnu Bhat B. Plrija SC. Neonatal typhoid fever.

Curr Pediatr Res !0(!4;8: 26-7

5 . Midhulika U. Hari\h BN. Parija SC. Analysis of sen from individuals with

uncomplicated typhoid fever and with perforation by immunoblotting. In:

Abskdcts of the 6'"ntern~tional Conference on Typhoid Fever and Other

Salmonellosis in Cullln China. November I?' -14', 2005

6. Harish BN. Midhulika IJ, Punla SC Multidrug resistance in Salmonella Typhi

iu)lates in Pondicherry lndia with special focus on reduced cipmfloxacin

susceptibility. I n Ahslnca of the 6' Interna~ional Conference on Typhoid Fever

and Other Salmonellosis in Guilin China. November 1 2 ~ -14" 2005

7 . Mdhulika 11, Hyish BN. W j a SC. Analysis of xra from individuals with

uncomplicated typhoid fever and with perforation by immunohlotting. In:

Abstracts of 29' Na~ional conference of he Indian Association of Medical

Microbiology (IAMM) in Chennai on Ocrohcr 21" -23"' 22005

8 . Harish BN, Madhulika U, Parija SC Multidrug resistance in Salmonella Typhi

isolotes in Pondicherry lndia with special focus on nduced ciprofloxacin

susceptibility. In: Abstracts of 29"ational conference of the Indian Association

of Medical Microbiology (IAMM) in Chennai on October 21" -23Id, 2005

9. Harish BN, Madhulika U, Parija SC Minimum inhibitory concentration of

ciprofloxacin of recent isolates of Salmonella Typhi at JIPMER Pondichmy. In:

Abstracts of 26"national conference of the IAMM in Bangalore on November

20"-2492002

10. Madhulika U, Harish BN, Parija SC C-reactive protein levels in cases of enteric

fever. In : Abstracts of 26Ih national conference of the IAMM in Bangalore on

November 20Ih-24Ih 2002

molecular basis of rcslstance and the genotype of thc Molecular Analysis rcslstant strains. High-level ciprofloxacin-resistant S. Paratyphi A (MIC 8 bg!mL) IS present in lndia (6) and of Fluoroquinolone- Japan (MlC~28u&mL.)(Q, However, becauc i,Typhi, the major cause of citeric fever in lndia. has not yet dcbei- resistant oped high-lcrcl resistance to fluoroquinolones, nteric Salmonella fevcrs are oftcn treated empirically with fluorcquinoloncs. I f this trcnd contlnucs. fluoroqutnolone-res~stant strains o f S. Paratyphi A are almost certain to become a major cause P a r a t ~ ~ h i A ofenteric fcver in many area.

Wc analyzrd, by DNA sequencing, the DNA gyrase and Isolate, India topo~somense I\' gencs o f the fiat reported highly fluoro- Satheesh Madhulika ,,nnikrlrhnan,t quimhinc-resistanl S Paratyphi A isolate (6). We looked at

Keith Turner,, Subash Chandra Parlja,t the full coding sequence, including the quinolonc resist.

Carol Churcher,' John Wain,* ancc- dctcrmininp rcgion (QRDR), o f both subunits o f

and Belgode Nararlmha Hariaht DNA eynsc and topoisomerase IV for mutations associal- cd with rcsistancc to lluoroqu~nolones. We also used mul-

Salmonella entenca serovar Paratyphi A ls Increasing. ~y a cause ol enterlc fever. Sequence analysis of an Indian !solate showed a unlque slrain wlth high-level resistance to c~ptofloxac~n associated with double mutations in the DNA gyrare subuntt gyrA (Ser834Phe and Asp87+Gly) and a mutatlon In lopoisomerase IV subunit parC (SerBO+Am).

5" Imrmull~r r n l r n c ~ serovar Paratyphi A 1s the sccond most common causc of enteric fcver after S. Typhi. \pproxlmalcly 0 25 S Paratyphi A ifections (parawhoid ic\crl occur for each S Typhl infection (typhoid fever) ( I ) . (!lien global cctlmates of ;21 million cascs of w h o ~ d Ic\cr In the ycar 2W0. ,5 million cases per year of S. P~ratyphl A probabl!, occur. I'aratyphoid fever is a major il lt~icai problem In Ind~a, but large outbreaks were not rcp(~ried un l~ l 1'4% (2). Elsewhere, in southern Ch~na for ~'r;m~ple. cstcnrlvc outbrcaks are also occurring (3).

SIIICC IO')X, plasm~d.niediatcd mullidntg resistance in S I'antyphl A, aasoc~atcd wilh chromosomally mcd~atud rcduccd suseeptlb~llty to clprofloxacin, h a caused concern 141. Rcduccd susceptibility to fluorcquinolones results In a pbor rcspt\sc of salmoncllosls pat~entr to treahnenl and may allou prolonged bactenai shedding ( 5 ) . Rlsing resir!- rncc lo Iluoroquinolones is likely lo be dnving an increase in caves of paratyphoid fcver in regions where fluoro- qu~nolunea are uscd Cmpincally to trcdl enteric fcvcr. We nlusl lnonitor lhc cmcrgence of rcsistancc in this enteric pathogen to dlfferent~ate between the acquisition of resisl- ancc durlng treatment (mutations occurring In different bacterial stralns) or clonal expansion o f a successful sadin hy person.10-person spread (identical mutallons associated ~ i t h a single straln). To do so, wc need to describe the

tilocus sequcncu typing (MLST) 10 confirm the identity of thc isolate.

The Study The stram described here (Pondl) was first isolated in

Pondlcherp. Indid. In Novcmber 2002 from the blcud of a 23-ycar-old man admitted w~th fcver and with no histor) of' having rcccl\cd antlmlcrobial chemotherapy (6). Thc ~soliltc was resistant to ciprofloxac~n and nalidixic acid. and the MIC of clprolloxacin was 8 mg/L. It was sensitive to all other antimicrobial drugs tested by disk diffusion: ampictllin, chloramphenicol, cohimoxazole, gentunicin, and cettnaxonc Repeat lesting showed that zones of i n h i bitlon lndicatlng susccptibllity were seen around both olloxactn (17 mm) and ciprofloxacin (21 mm) 5-kg disks; however. In light of the clproiloxacln MIC and resistance to nalidixic acid. Pond1 was consldcred resislant to fluoro. quinolones. h o slmilar isolates have becn seen in h s area slnce the ~nitlal repon, although the tolal number ofparaty- phoid fevcr cases has increased.

Polymerase chain reaction (PCR) amplification (Table I ) and direct DNA sequenc~ng of both strantis of the full lenyh of gyrase @r.4 and urB) and topoisomerase IV @urCandporEj subunit genes was performed with an ABI Prism dye terminator cycle sequencing k t (Perkii Elmer, Foster City. CA. USA) on an ABI 3730 automated scquencer. Results showed 3 mutations: 2 in g r A and I in parC. A comparison of hesc mutations with those prcr i- ously described in fluorcquinolonc-resistant S. Parawhi A is shown in Tablc 2. A rise of fluoroquinolone resistance over time IS apparent, and although the point mutations do not fully explain the MIC data, we noted general associa- tions: a sinale lnutation in mr.4 is always associated wlth

Wellme Tnrsl Ssngsr Influte, Csmbridgerh,re, Vn,lsd resistance to nalidix~c acid and reducrd sweptibrlity to K , ~ ~ ~ ~ ~ , and tJawehPrld Inslllule ol PoslgrPduate Medical ciprofloxacln and ofloxacin, and a double mutation i n u r A Educabm and Research. Pond~chern, Indm IS always accompanicd h) mutat~uns in lrurC and is asso-

Emarp~np Inlectlwa asaaoes w cdc pov/a!d. yoi 12 NO 3. March 20% 489

,#, 1 Primer W w m U la lmplLation d topplsmenm G Z Sequeme 5'4 Amplkon Geno (coding ssqumce length) ;A1 lo 5' GGGTCGACTOATTATGGTTTATGCCTCC 3' 31 89 2637 juA25 iR) 5' GAGACTTTCAGCGTAGHCG 3'

,. - . - ~ :trEB IR) 5' ATGCGCMGTGTCGCCATCAG 3' C

,:,ad with high-level (24 VgimL) resistance. Although thts s~nplc is limited, heterogeneity in mutations found at all lei suggests that this finding is not the result of a singlc jc,e\sful smin's sequentially acqutring mutations but a.Cr that resistance is arising in several diffcnnt strains.

:oncluslons Ihe QRDR within lopoisomcrascs containa hotspots

rniutations around the active site, which arc associatcd , I I ~ ril~sed MIC values for fluoroquinoloncs (10). For !,.I from nalld~xtc acid-resistant Salmonrllu iholatcs, 2 lula:lons are most frequently observed in clinical isolates: rrk3+Phe and SerR34Tyr (8,9,11). Thc association with :$:stance of mutations seen inparC, houever, is less clear ? I . Mutations inporCofgram-negative bacteria are usu- 11) utthln the QRDR at amino acids 80 and 84 (Scr ll4lc. Glu 8 4 4 l y , Lys). The first reported mutatton oThr57+Scr, and other mutations have been described. .ip!9+AIa, Gly 78-+Asp (9). Each mutation, in g r A or I#[' run give rise lo different MlCs in ditrcrcnt isolates, i.rh means that other factors must also influence the :~~uoncc phenotype in S. Paratyphl A. The most likely a1.e IS changes in expression levels of protein9 lnvolvcd :'I1 permeability barriers and efllua pumps. These langi could be ~ h c result of either polnt mutations In lnrcriptlon promoters and regulators or downstream kcla of mulations in topoisomerases. Known mecha- rmr of fluoroquinolone resistance that we were able ro rccn for include transmissible plasmidborne reristancc

- -- and efflux pumps. The qnr-containing plasmid was not detected with PCR using primers 5' GGG TAT GGA TAT TAT TGA TAA 3' and 5' CTA ATC CGG CAG CAC TAT TA 3' (13) in Pondl, and sensitiviv testing gave the same zone size around both tetracycline and chloramphenicol discs when compared with a nalidixic acid-susccptiblc ikolatc. This finding, combined with the ciprofloxacin MIC of 8 pgimL, argues against the presence of multiple antimicrobial drug rcsistancc efflux pumps. Thus tho high. lcvel resistance seen in Pondl appears to be associatcd direclly with 3 point mutations in the topoisomcrase genes.

'To confirm the identity of h e isolate as S. Paratyphi A, we used MLST, The primer sequences and MLST data are available from the MLST database at the Max-Planck. lnstitul Nr lnfektionsbiologie (htrp:!/web.mptb-berlin. mpg.de!mls~. Six of 7 sequenced loci matchcd exactly thc previously described scquenccs, and I was a unique allele. This finding mcans that the isolate described here is with- in (he clonal MLST group described as S. Paralphi A but IS a recognizable variant. This finding s u p p o ~ s prcvious typing data that show very linlc variation (14); typing S. Paratyphi A is problematic because genomic restriction analyses (pulsed-field gel elcchophoresis) of isolates from an oittbreak arc not always identical, and susceptiblc and resistant strains cannot be differentiated. For molecular rpidemiologic studies to be carded out, several methods need to be used (IJ), A broader study of single base-pair diffi.rences behveen strains of$. Paralphi Acould provide a usable typing scheme.

lble 2 Prmoffic of qnrtnd mutations In DNA g y m and topolaomrasr IV penaof Salmonrlla enterkc serovar Psratyphl A 'arm r r u r ; i a t ~ v ~ h durnaod ourcl~(lblIlty or r n s h r r e to clpmfaudn'

MIC ()lgcmL) auntry mar cp ~i sr gyrA PC Refareffie dla teOB 038 ~ 2 5 6 ND SeraSPhe ND iB) mgladest 1692 0.5 r256 ND Asp37+GIy N D (8) dla !OQg 0.5 >254 NO ScrOhPhb ND (s) xlpKcnp ~ W O 0 5 > 2 1 ND SerWTyr NM (9) Mn m L128 ,!& ND S d k P h o , Glu84-1Lyu (7)

As@7+Am Z.32 8 ,256 NP Ser8SPhe. Se fK~Ara t Thk 8hldy ---- -

A 7 4 b mudh m8 &eed ln rnd M cp, stmnorscln, Nal. MIMlllc ,Id:$, d M(IW NP. d p r M . NM, no mutsth Mh!n lhe

0 Emarplyl Intediout Ih8eases - wuw.cdc.govleld Vol. 12. No. 3. March 2WB

/

Reslslant Salmonella Paratyphi A Isolate, India

Reststancc in S. Paratyphi A populalions must be mon. ,lared because the acquisition o f rcsislance to fluoro.

gu~nolones, coupled wi th the reduction i n S. Typhi by the use o f typhoid-specif ic vaccination, may cause S. pantyphi A to become the main cause o f cnteric fever. DISC susceptibility testing does not always derccl resist- ance, and screening wi th nalidixtc acid and MIC testing

the method o f choice. The isolate dcscnbed here,

pond], contains a unique combinat~on o f mutations that

prov~des a way to track the spread o f this strain o f $ pantyphi A.

Acknow ldgm8n t l We thank the dtagnosttc m~crob~oluy~sts who ~wlated and

ihnraclerized the strnins described III th~s manuscnpl.

Sntheesh N a c John Wtin, and Kc~ t l i Turner ore funded by Ihr. Wellcome Trust u f Oreit Rritntn

Dr Nair 1s n rcseamh asroclate In trnpical bactcnology at Thc Wcllcome Trust Sanger Ittst~tute. The Wellcome Trust

(ienome Campus, Untlcd Kingdom HI^ research tnciudes the use d i molecular tools to detect genumic dtucrsity o f drug- r:i~nnn~~dmg.suwcpttbir Salmonriir~ spp lrom dillerrnt geo- ptaphtc and cp~dcrniolog~c backgrounds and ~ W c t s of drug r:

Indian J Med Re# 120, August 2004, pp I 11.1 14

Current pattern in antimicrobial susceptibility of Salmonella Typhi isolates in Pondicherry

I'. Madhuliku. B.N. Harish & S,C.P~rija

Dup~rrmetrr (4 M i r r n h i o l o ~ y , Jan'ahorlnl I r~sr irrrr~ (!I Por t~mduore Mrd i ra l Education & Research i'orldichrrn, India

Typhoid bvtr cmlinum to remsin a health p d t m us the cauntive o~anism SalmonrL Qphi hus dtrelaprd redstsnn to many 01 t k antibitin used This study was undertaken to ddennin the current p t k m d nslstmc lo anhiembid agrnls md phap typs dS.Typhi isolata Wwd in a h r t l l q M h care hmpilll in Pondkherry. Rllxd culture war done lor 12% suspatad r u ~ s 01 mttk Im and 151 strdna d S. Typhl wen irluled. Sensitivity to ompicillin, chlomphaid, wncrmidn, dprollox~eln and cellri~xone wsc dalerminrd by disc dinusion, and the minimum inNbitay cmmrntion (MIC) d a p d k m i n determid. There wen 61 mulYrug mistnnt (MDR) M s l n The MIC of cipmilaxpdn for 141 igolatn war 211.5 mp/l; of thac, 131 were resiatrnt to nd'ixic add. Phage typing WM dam for 123 isdates and 115 were found lo be d phqe type El, hiolypc I. A kelinc in the number of MDR isolalra WPR noled, Concumntlh then h r bnn an i- in lhc number u l bdslni rnsltive to all anlibiolics except nsliixic add, and ell theae irdDts s h e d ndud wceptlbility to cipmhuin. Ndidixk acid $usnptlbilily could be a udul z m n k I& l w thr detection d d c m d mseptihility d S. 'Typhi to ciprolloudn. The dinitism nhovld k dW (a wc nllriaxmr rh lve ly in csxn showing tion-renpwivmrrs 10 dprdlmtdn.

hr) w d Mull~drvy resalanl Snlmrrnrlin Typh~ . nal~dixic acid rcsislanl S. Dphi

Entcric fcvcr continues to he a major health problem increse in ciprofloxacin MIC has heen recorded in UKY Jv\pitc the use ol' ant~bicitics and the development nT us wcil as India""!, Given the variation i n the nener antihactcri~l drugs. Thc causative organism susceplhility pattemsrepned brS.Typhi,itisirnpttarit Si~lmtmtll~r Typhi has rapidly gained resistance to ~occ~nstmtiy monitoritsoas~oprovi&suitlblegui&lines ,~nlibicllics like ampicillin, chioramphenicol and for lrcatment. Recent rcpons on current sensitivily

:olr~moxulole, and also to previously efficacious paacrns of S.Qphi isolates in Pondicherry are lacking, J ~ g s like ciprofloxacinlJ, The incidence of muitidrug This study wasundertaken to nrsessmuilidrug resislance, rr.qscant (MDR) S. v p h i mpncd to he high susceptibility lo cipmfloxacin end predominant phage ill per cent while [here are reports noting a dccline"'. I)pt's of S,T~phi in 8 IeHiar~ health care facility in Kosurgence of resis~ant atrains has aim k e n reported! pundlcherl~.

US-haid siudy' ncied an increase in thc number 01' MDR strains and nalidixic acid resistant S. Typhi Blood cullure was done for 12% patients suspcltd NARST), although overall, the isolaks were sensitive to hilvc enteric fever at Jawaharlal Institute of

.[I cipmfloxacin and cefhax~ne, Another study from Postgraduate Medical Educatiun and Research

Bangladeshk repmd a deenae in MDR isol i l~s with (JIPMER) hospital and Government Hospital,

lo corresponding increase in sensitive strains. An Pondicherry during January 2002 to November 2003.

112 INDIAN I MED

Iiillates were identified biochemically" and confirned agglutination with 09 antiserum (Murex Biotech,

England). Antibiotic susceptibility testine was performed hy the Kirby Bauer disk diffusion method" to the h~llowingantibiotics (pg): ampicillin (lo), chlmphenicol (10), mtrimoxazole(2~),gentamicin (lo), nillidixic acid (301, cipmiloxacin ( 5 ) and ceftriaxone (30) (Hi.Media I.tboratoriesLtd, Mumbail. The disk strengths and zone rit,e interpretation was in accordance with National Committee for Clinical Laharatury Standards (h'CCLS)". MlCs of ciprofloxacin, amp~cillin and chluramphenicol were determined by apudilution, and oilnflrmed hy hroth microdilution prrfnrmcd in ;iccc~rdance with NCCLS slnndardil". Phagc typing was donc at the National Salmonella Phagc Typing Centre, I ~ d y Hardinge Medical College, New Dclhi.

A totul of 157 isolates of S.Typhi wcre t~hlrincd hy h11u1J culturc from suspected cases ol'c~ltcric fevcr. Of lhcse, 131 were nalidixic acid resistant and 129 ciprofloxucin sensitive (Tahlc). Al l iinlalcs werc \cnsit~ve lo ceftriwone. Sixty one isolates were found la k mullidrug resistant. Thcrc was n peak of ~stllat~on oh\crved between July-October 2002, and anuther in August 2003 and a corresponding increase in MDR lh~llalcs,

Of the 157 isol~tes, 10 showed MlCs ofciprufloxacin r i 0.25 mgl, 73 showed 0.5 mfl, 45 of I mpn and 29 of 2 mfl. Al l isola~es with MlCs of 0.25 m g md 2 with 0 5 mg/l wcre scnritlvc to nalidixic acid. The resl trcluding thc 14 thatcould ntlt he testcd, wcrc resirtant 1%) nalidixic acid (NARST).

Phagc typing was done for I23 iscllarcs. Thc majonty I l IS) were of phage type E l . The rest ol' the isolates

Tab!+. Anllb~lpam of S.Typh~ ~solald at Pu'undickrry (n=I57) p~ - -

Antih~olic Sensitive ln~cnned~aie Rrs~stanl

Ampicilhn 63 I0 84 Gvn~um~c~n 155 11 1 Chloramphenicol 72 3 .Q2 Cillrlmoxuolc 55 I1 102 Clprn~omin 129 28 0 Ccflriuonc 157 0 0 Nal~dixic r id* 12 0 111

' I4isoln1cscould not be lesled opsinsl nalidixic rcid

RES. AUGUST 2001

were of the A (4), 0 (2), U V M ( I ) phage types and one was Vi-negative. Among the E l type, 107, and of the rest, 4 were NARST. One each of 0 and A typc showcd aciprofloxacinMICof 2 mgl, the WS4 andVi-negative tsolatc had MICs of Img/l, The resl showed MlCs of 4 5 m g . Al l the MDR isolates were of the El phi~pe type.

First reported from Mexico, MDR isolatcs of S. Typhi rapidly appeared worldwide" In the present slody, we have found a decrease in the occurrence 01 MDR S.Typhi as compared to an earlier study". Previous reports from this region notcd m increase in MDR from l9ROonwards and in 1990, thc isolation was frcquent enough lo suggest an outhrcskl".

Nalidixic acid suscevtibilitv hias been vnlidntcd as u . . screening test for reduced susceptibility io ciprt~floxacin, and nalidixic acid resistance is associated with a high MIC of cipmfloxacin which in turn is associated whh treatmentfail~re~~~~~~~. In our study, all but2 ofthe isolates which showed nalidixic acid resistance hadcipmlloxacin MlCs of >0.5 m u . Any isolate that shows nalidixic acid resistance should be repnned as ~ntermediatcly su,ceptihle to cipmfloxacin and the clinician advibed to switch over to another antibiotic.

Threlfall et UP noted that 23 pcr cent of S. Typhi isolates in 1999inthe UKshoweddechused susceptibility to ciprofloxacin. In this region, MlCs of ciproiloxacin have k e n increasing over h e past few years. An earlier study from [hi# centre in 1998 showed that the MlCs of cipmnoxncin were between 0,0625.0.25 rngillx. In the prcsent study, MlCs ranged from 0.25.2 m@l.

Of the 147 isolates showing ciprofloxucin MlCs of 20.5 mg4,84 were sensitive toampicillin,chlomphenicol, andlor cotrimoxazole. While the MlCs in general, were higher than previously recorJed for his region, with a maximum recorded MIC of 2 mgll, what IS interesting to note was that a significont number of isolates showed high values of MICs forcipfloxacin alone. This probibly reflects the ovetua of cipmfloxacin in h e ueatmem of typhoid, as well as inotherumlatedinfenions, Incomplcle lreatment may also be a factorconvibuting todevelopment o f resistance. This provides a strong case for reconsidering the use of the first line of antibiotics for batrnent, espcially ampicillinand amoxicillin,

MADHULIKA tr dl: SALMONELL A TYPHl IN KINDICHERRY 113

A l l isolates were sensitive to ceftf laxone in contrast lo a study which recorded resistance to ctf ir iaxone", m i s underlines the impanance o f this d m g f o r treating

b(DR and ciprofloxacin resistant c a w in this region. Emphasis is la id o n the sparing useof th is drug. l tshould

used only i f the f irst and sectlnd l ine antihiodcs fa i l to a satisfactory respan% o r i f the isolate is resistant

lonalidixic acid.

A sh i l l in the predomtnam phage type I'rom 0 to E l ha\ k e n found i n this s~udy. In u sludy done earlicr, a l l ;he MDR S. Typhi isolates k l o n g e d ttr phagc t y p 0, Dtotype 11''. In conIrast, wc found a l l MDR i rolatcs belonged to phagc typ. E l , hiotype I. Other phage t y p s wcrc found l o he scnstttvc l o most antibiotics, wi th the

:xccplion ofna l id ix tc acid. This sh i l l i n the pmlominant nhrgc typc has h e n noted curlier". Ou r f inding is in :iincurrence w t l h a rccenl study which found thal most ~ i l c MDR S.Typhi isolatct wcrc o f phage lypc E lh .

I n conclusion. thc l tnd inps o f the present study ndtcated hiat thc f i n 1 i lno entihiolics may sti l l hake u rile to play i n the trealmcnl o f lyphoid fever. Also, the ~ re fu l ncss o f na l id ix tc ac i d as a screening lest f o r Ietecting reduced susceptibi l t ty to c i p ro f l ox r c i n is

:mph i i i ad . A simplc disk diffusion lcsl can rapidly

?rilvide i n f oma l i on t h a ~ can predict treatment failures. md obviates the need for the marc t imc ctlnsuming

l i lutlon tcsts. W i t h ceftr iaxane emerging ;IS !he sole

I e f cnx against N A R S T strains, physicians should he ~dvised against w i n g this dntgempirically, and tt should

r instituted on ly i n the event oI'ih~n.reaponsivencs~ to :iprolluxacin.

A c k n o w l e d g m e n t

The Authorrthank DrGltn Mehta. Nallonal SnlmunellaPhage lyplng Centre. Lndy Hvrdinkc Medical College. New Delhi for lhage typing the rlrainr, and I)r Sarangnpani. Deparlmenl of lilcrobiology, Govcrnmcnl Husp~tal. Dondtchcrry lor provldtne ~f S Typhi iwlatca.

References

kaudawn MV, Juhn TJ. Plnsmid medinled muhidrug r c~ i s l aw In Solnumelia Typhi, Indian 1 M d RPI 1992: VS : 6 7 ,

1, Butt T. Ahmsd RN. Mahmood A, Za~di S Cipn~floxacln trcatrnenl lailure i n ~yphoid Ievcr case, Poki~lan. EmrrsI&r~fDe2WJ: 9 : 1621.2

3. Sanghavl SK, Mane MP, Ntphadkur KB. Mullidrug reatstonce in Salmowlln scrntyar. lnrlian J Med Microbid IW9; 17 : 8R-90.

4. ChandeC.ShrikhaMeS.KapnIe5, Agrawal S. FuleRP.Change in antirn~nobial reslslanapmbn of SalmontilaTyphi in central India, Indian J,Mrd R P ~ 2002: 113 : 248.50

5. Saho hlR, Dulta Y, N ~ y u g i SK. Duua S. Mit ru U. Rnmamurlhy T. el 01. Uccreniing trcnd in the occurrence or Salmcmtlis enrrrr~,o icrotype Typhi amongst honpllali7ed children in Kolkatn. l n d ~ a during 1990.2000. Indian J M d R e r 21102; 1151 46.8.

6. Kumdr R. Anqu KK. K i~y P, Sharma M. Gupta R. Rarn S. Esalual~onolmin~mum tnhihls~ry ionvensuliun afquinoloncr and thlrd gcncratliln crphaluryorinr to Scirnonrila T j p h ~ isolaler lndron J MP~SIL !iK):: 56 : 1.8.

7. Ackerr hlL. h h r ND. Tauxc KV, hiin!?, Ei). Lnbordtlay.hercd iurreillancc of SnI!nonrllri seriilyv Typhi inkctioni in !he Un~led Sbttcs: antlmlcrnbiol lcilrttince on the r t rc .IAM,l 2003: 2R.I : 2ffiX.13.

8. KhtnanM,AhmadA.ShirmaS. ikulinetnrptdcm~cofmul~~ilrug reriatan1 Saimon~lle Typh~ is not ri

l I4 INDIAN J MED RES, AIIGUST 2W

1 , KoweH, WdLR,Th rdb l l El.Multidrugmsis~antSalmcmella 20. Hakanen A, Kotilainen P, l a l aa 1, Siitonen A, Huovinen P Typhl, u worldwide cpidsmic. Clin Infrrr [)is 1997; Drreclion o l dtcreosed fluoroqu~nolone suacop[ibility in 24 (Suppl I ) : SIM-9. Salmoncllar and ulidalion of nalid~xic acid scrcenlng ICSI.

J Clin Mi1robiel1999,~7: 8512.7. 11 Harish EN. Raahrnrh K, Mulidrugles~s~anl Salnu~nelh Typhi

Hilh lPCElal reference to i.irrn aclivity of cipronl,xaoin, 11. Kapil A, Kenuka I). Das 8. Nal~d~xic acid ruucp!ibility leal lo rcrcen c~prulloxacin realslance in S~l rnonr l l r~ Typhi.

HirimvD~inc 1998; 18: 62.6, lnilron J Mrd R P A 2002; /IS : 49.54. 10 Hao $1. Amamalh SKU Sulflfio S, An outbreak of ly~hold due 2 2 Saha SK. Talukder SY, Islam M, Saha S. A highly ceftriaxunr.

tu multldruf, resislanl Snlmcmrllo Typhl in Pondiuhcrry reialnnl Solmonrliu Typh~ in Aangladesh. Ptdilrrrlnfecr Db J T r~ rn~ R L. ln111 Mrd Hyg 1992: 86 : 201.5. IW, I # : 387.

~,l~rlrir rrclrrtrrl: Dr 11. Madhulikr, kpsnmcnl of Micmh~ologg. Jawahsrlal In~ l~ io lc of Ralgraduse Mcdical Wucaliirn & Research, Pondicherry 60iM6. ind~n email: m~ul ikagPrcd~f fmai l ,com

(solated high-level ciprofloxacin resistance in Salmonella enterica subsp, enterica serotype Paratyphi A

inlate of Salnron~II~ Parsryphi A horn a oii iif entert kver was found to have a ~~~rofiuxacin MIC of8 mgl-'.There have a,vt~ rupns of incrnring incidtnce of ) i r r l ~ h o i d lwer, along with a detreiv In , l lk~pt~b~lify to ciproIIoxac~n. This 18 the ~ b t repon of Salmattlla cntdfl l ~ r o t p e '~r r rphi A shuwing resistance to .lprotloxacin.

\!i.year.tiid malt war diagnovd as ... firing from enttric h e r 11 IIQMER. iun

Neonatal typhoid fever

0.11. HW, U. YndhuHlu, Vlrhnu Bhlt B', S.C. P~rl]l

Cq&& of Miaobsbey snd 'Pandialfa. Javaharlsl Nahru I m W e of Postgraduate Medi i Educat~on and Research iw&my, I n d i

A p n t n M l 1 m b o m l l 3 3 ~ t o 1 ~ r w l t h ~ I d l a b l l w . A 1 n 1 h o m d ~ v i d ~ n ~ ( o f Mtl dlctmc, bkod cultun m don. Sllmdh thlw W d fmm tha child. thouah not tmm thc m o t h n . ~ i d r l tal dw MI mothtr dmmk h lgh l i bn olmtlbodicc t b ~ . Ghl #O" and 'H" m. koW IypW h an nmmcly nn wm, md a hlph l n d u of tulplclon i8 n m u y l o dltca I.

~ ~ a ~ c # l m o f r m n P W m w t a b t y P n d ~ d ~ t y My fadaP mnmbuhng to l l s W heme hng a i d of ssrr(sbon snd hygene S lyphr is n unllely w s e ol wpWmta n n nlmt bb a k w cases have becn documented in chlidran 5 )am OM M less H o r w t n f W In children lsss han a pr P are raw We report one such c m in a n e h m

C r t Rlport

A p4m m l wghlng 2 4 kg ma h n at 33 weeks to a d!# wh p M l o n a i diabetes at JIPMER Hospnal The hhwy ma vqmd )HM h e appllCB(mn 01 hmel brmps Th

had evdenee ol felal dlslres and w a ~ shined lo h e nurwty Iw wen Blood cuiiure done (or the baby yielded S lyphr sen* to chbramphenlc~l gantamlun mtnmcwamk apdkm snd a h m reMan1 mly lo am@llln Tb Md i u w d mbtaxtme 120 mg IV and g tn lawn 6mg IV 12

IIhneabbnkin3daysPndw~sdpchugedwRhIhe r lnc4fiam~bsmnlmwdlor4mdap T k & n W ad a r ~ mnures done m w v e l y r r o n a g h Homw Wdrl alglubnahon lcsl was p b v e wrth ngndiwt btm d anbbcdhs g w s t bolh S whr '0' and 'H" antyns mpdwty (1 324 and 1 640 respcMy) She had no hlstwy of m b M hnpy just pior lo d0llMrf

T p M lewr a endemic in Indla. It uwdiy a m - a d u k and OM childre11 Mvgh h r e are repda el infecttons m chiidrsn

kiwn 2.5 years ol sgo. Repwls of ~nfeclloor in children less Vm 6 m l s of age KE rare, but h a n bacn know lo oaur 11. 31. Conveo~ty, inledions with nontyphodal salmonellae are h'gbd in h is p group, The data available pn this aspect of tvpho'id k m r is scanty, and that on the Indian pcenario, m e lo , in a M y done BI his instihrte on typhoid lever ~n children younger than 5 p a n , only me chiid vms less than 2 yea's of age 141, Anomclr dud7 hom Delhi pmfiied 18 children 2 yem old w bs and the youngesl was 9 months old IS]. lnlscl~ln in a 7. mfi old infanl has been reported from Mumbai 161

MnifesMuns of mhoid fever in mfants are reported to be miM, and d$nosis IJ olten by chance Mild lever and malaise have bewl k n m to m u r in inlank with cunure proven typhoid lever. Men, they may k asymplornalic, or may present !4th varying degrees of ga$tmenterilis 171 The ciassicai signs and symplwns of mid lewr are mnsp'cuous by lhetr absence Mher i n d i h n s tnclude meningism and convulsions, u th or mthout dianhsa. Enteric lewr is repotted lo be a cause ol abonion and premature delivery in Ye pregnancy It can also k Iransmltled varlically, manhsling &in 3 day, ol delivery Congenital I m s m b a n of enteric lever can m u r by hansplacenlal ~ n b l o n hom a bac$ramic mother to her fetus InIraparturn lransmission is dw poss~bb mur ing by a feco.oral mule from a carhr mlher (81 Transplacantal infection aems likely ~n lhis case, as tha disem mmbsted itself *in 3 days. B i d culture dona as a p t t s u m led to Ihe idation ol S. lyph,. Three dap of anlbiobc therapy led tothe baby becoming afebnle. The rothen stwl and u r i n r r e negetive for the organlam. Biood culture could not be don. However she had significant levels ol aniibddies lo '0' end

6, tamh SC, D& U, J& U( T@dd Fw h a 7. mOnmddWJ~MdlgIL5:41:1W169.

EM: MrM&@ha.eorn

ERRATA

As per the suggestion of Examiner 1, an abstract has been included in the revised thesis, following the Acknowledgments. The abstract reads as follows:-

Abstract Typhoid fever is a major cause of morbidity and mortality in the developing

world. The causative organism, Salmonella Typhi, has developed resistance to many of the antibiotics used in treatment. It is essential to monitor the development of antibiotic resistance in order to formulate effective treatment policies. Typhoid fever is alsa associated with life threatening complications like ileal perforations, the pathogenesis oi which is poorly understood. Aim and Objectives: The aims of this study were twofold-I) To look at antibiotic resistance in S. Typhi in general and fluoroquinolone resistance in particular, and 2) Tc investigate the antibody profile in typhoid perforation. Materials and M e w s : This study included isolates of S.Typhi and serum sampler from patients with signs and symptoms of typhoid fever, Isolation and identificatior followed standard protocols. Antibiotic susceptibility was determined by Kirby- Baue~ disc diffusion method for a panel of antibiotics. The MIC was determined by both aga~ dilution and broth microdilut~on for ampicillin, chloramphenicol, ciprofloxacin and ceftriaxone and interpreted in accordance with NCCLS guidelines. Envelope profiling and PCR-RFLP were done for 30 selected isolates. PCR was designed and performed f o ~ the amplification of 4 sequences located in the genes gyrA, marA, marB, and marR.

For antibody studies, 3 groups of samples were analysed- Group I, from patients with typhoid ileal perforations, Group 11, from patients with uncomplicated typhoid fevel and Group 111, consisting of healthy individuals or patients with non-enteric infections. Antibodies were detected in these sera by immunobloeing using Whole Cell Protein (WCP) extract, and also individually for the H, LPS ~ n d OMP antigens. Results and discussion: During the 35 months of this study, 177 isolates of S.Typhi were obtained. Multidrug resistance was seen to decline over this period from 56% to 7%. In contrast to multidrug resistance, reduced ciprofloxacin susceptibility was seen in a majority of the isolates- 159, of which 157 were also nalidixic acid resistant. There was a good correlation between nalidixic acid resistance and reduced ciprofloxacin susceptibility,

PCR-RFLP of the gyrA gene produced 3 patterns. One was exclusive to nalidixic acid sensitive isolates with ciprofloxacin MIC 5 0.25 pglmL. The second pattern was found in nalidixic acid resistant isolates, with raised ciprofloxacin MlCs between 0.5-2 pglmL. The third pattern was dso found in nalidixic acid resistant isolates with raised ciprofloxacin MICs, but suggested the existence of mixed genotypes. Amplification of the mar locus revealed that there were no mutations in this locus that affected fluoroquinolone susceptibility. Envelope protein profiling detected only minor changes, suggesting that constitutive changes in the envelopes were unlikely to play a major role.

Immunoblotting with WCP revealed that antibodies to S.Typhi antigens were present in the sera of patients with ileal perfomtions and were directed mostly against lower molecular weight proteins. In contrast, sera from typhoid fever patients contained

antibodies that also reacted with high molecular weight proteins. Antibodies to the H antigen were present in only 33% of Group I sere as compared to 70% in Group 11, suggesting that the lack of antibodies to the N antigen is a feature of typhoid ileal perforation, Unlike H antibodies, antibodies to LPS were detectable in both these groups. However, in Group 1, antibodies were detected to all components of LPS, while in Group 2, antibodies were directed against the 0 antigen. Immunoblotting for antibodies to OMPs revealed that these were more common in Group I ~han Group 11. Comparison with Group I11 suggested that immunoblotting for the detection of antibodies to H, LPS and OMP andgens can be of diagnostic value. Conclusions: In this part of India, multidrug resistance is being replaced by reduced susceptibility to ciprofloxacin and disc diffusion testing using nalidixic acid is a good indicator of this phenomenon, Of the different mechanisms that could mediate reduccd susceptibility to ciprofloxacin, a mutation in gyrA was the single most important factor, although hy no means the only one, PCR-RFLP of the gyrA gene also points to the existence of nixed genotypes, which mily be an indication of evolving resistance,

A comparison of antibodies in the sera of patients with uncomplicilted typhoid fever and with ileal perfordtion revealed ha t there are differences in the antibody profiles 01' the 2 groups and suggests that this differential response may contribute to the development of perforations. Finally, immunoblotting using the major antigens- H, LPS

, and OMP - may he of diagnostic and prognostic value. The observation of Examiner 2 that PCR-RFLP and envelope profiling should havc been done for more isolates h u been included in the "Limitations of the Study" in the Appendices section, It is pertinent to note that many of the isolates shared similar characteristics in terms of the selection criteria for PCR-RFLP and envelope profiling, ie, the antibiogram and phage types. Choosing representative isolates of each type was performed to cull the maimum possible data without repetition, 81 the same time minimizing reilgcnt usage