changing trends in antibiogram and molecular analysis of quinolone resistant...

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2017Vol. 3 No. 1: 2

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Research Article

Journal of Infectious Diseases and Treatment ISSN 2472-1093

DOI: 10.21767/2472-1093.100029

© Under License of Creative Commons Attribution 3.0 License | This article is available in: http://infectious-diseases-and-treatment.imedpub.com/archive.php

Muhammad Zubair Saleem1, Abida Arshad2,Mazhar Qayyum2,Muhammad Imran Shabbir3, Aamir Ali4, Ishfaq Ahmad5 and Muhammad Arshad3

1 College of Basic Medical Sciences, Dalian Medical University Dalian, Liaoning, PR China

2 Department of Zoology, Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi, Pakistan

3 DepartmentofBioinformaticsandBiotechnology,InternationalIslamicUniversity, Islamabad, Pakistan

4 HealthBiotechnologyDivision,NationalInstituteforBiotechnologyandGeneticEngineering(NIBGE),Faisalabad,Pakistan

5 DepartmentofMathematicsandStatistics,InternationalIslamicUniversity, Islamabad, Pakistan

.

Corresponding author: Muhammad Arshad

[email protected]

InternationalIslamicUniversity,Islamabad,Pakistan.

Tel: 00923338228084

Citation: Saleem MZ, Arshad A, Qayyum M, etal.ChangingTrendsinAntibiogramandMolecular Analysis of Quinolone Resistant Salmonella typhi Isolates in Pakistan. J Infec Dis Treat. 2017, 3:1.

BackgroundTyphoid fever is one of life threatening illnesses. Salmonella enterica serovar Typhi commonly known as S. typhi is the causative agent of sporadic outbreak of typhoid fever. Whenleftuntreated,itresultsinseriousinfectionamongchildrenandadults triggering bacteremia and inflammatory obliteration oforgansmainlyintestine[1].

Typhoid is most prevalent disease with more than 100 cases per 100,000 persons per year in South central Asia and Southeast Asia [2]. Salmonella is responsible for 3 million worldwide deaths, 16 millionannualtyphoidcasesand1.3billiongastroenteritiscases[3]. Asian regions are at the hit list of Salmonella with almost 80%deathsandcaseswhilerestinAfricaandLatinAmerica.Theproportion, epidemics and fatality of typhoid fever is larger in

Changing Trends in Antibiogram and Molecular Analysis of Quinolone Resistant Salmonella

typhi Isolates in Pakistan

Received: February 13, 2017; Accepted: February 25, 2017;Published: March 09, 2017

Abstract Multidrug resistance is the ongoing burning issue in Salmonella typhi around the whole globe. This study was conducted to investigate changing trends inantibioticsresistanceandmechanismofresistanceagainstNalidixicacidamongmultidrug resistant Salmonella typhi strains isolated from Islamabad Capital Territory, Pakistan.

Methodology: Prior to blood sampling, demographic data of patients wasrecorded. A total of 103 clinical isolates from blood of typhoid patients wereidentified usingmicrobiological techniques such as colonymorphology, Gram’sstaining and confirmed using standard biochemical techniques. For molecularconfirmation,hypervariableregionVIofflagellinfliC gene was targeted using PCR. AntibiogramoftheisolateswastestedbyKirbyBauerdiscdiffusionmethodusingfifteenregularlyusedantibiotics.Efficacyofantibioticswasevaluatedinrespecttovariousseasons,agegroupsandgenderofpatients.RelevantgenesgyrA and gyrBweretargetedandquinoloneresistancedeterminingregion(QRDR)ofthesegeneswassequencedtoanalyzemutations.

Results: Antibiogram study demonstrated that 90.3% isolates were multidrugresistant.75.7%isolatesweresensitivetocefipimewhile80.58%wereresistanttonalidixicacid.66.02%isolateswerefoundtobeciprofloxacinresistantexposingreducedsusceptibilityofsalmonella typhi tofluoroquinolones.Molecularstudiesrevealedasinglepointmutationwithsubstitutionofserine-83byphenyl-alanine.Thissinglepointmutationseemstoberesponsibleforresistanceagainstnalidixicacid in S. typhi.

Conclusion: The incidence of typhoid is high in Islamabad with significantresistance of S. typhiisolatesagainstcurrentlyadministratedantibioticsduetothepresenceofonepointmutation.Therefore,itmustbemandatoryforthehealthcare professionals to test for the antibiogram before prescribing appropriateantibiotics.

Keywords: Antibiogram;Salmonella typhi;Resistance,Susceptible;Antibiotics

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differentpartsofSoutheastAsia,includingPakistan,India,China,Bangladesh,Indonesia,Malaysia,VietnamandTajikistan[4,5].

In Pakistan, typhoid fever is 4thmajorkillerdisease.Intheareaofhigher transmission and inappropriate treatment, fatality rate of typhoidpatientstouches30%insteadof5%commonmortality.Byusingsuitabledrugs,thisexaggerationcanbereducedtolessthan0.5%[6]butinappropriateuseoftheantibioticshaveledtothe advent ofmultidrug resistantS. typhi (MDRST) throughout Pakistan.Eventually,itappearsasaleadingcauseoflong-lastingstayinhospitals,additionalcostoffitnesscareandsomeseveredifficulties triggering morbidity and mortality [7,8]. In somecases,thesedrug-resistantorganismsspreaduptoriskylevelduetocrossinfectionsofhospitalizedpatients[9].

Mechanism developed antibiotics resistance is due to geneticdiversity and adaptability of S. typhi. This adaptation is causedbyeitherplasmidacquisitionorchromosomalmutations[10,11].InMDRST, plasmidmediated resistance ismore common [12].Theseplasmids belong to incompatibility complex group IncHI. These are responsible for resistant gene transfer in bacteria and theseareoriginatedfromSoutheastAsia[13,14].

Due to these MDRST, fluoroquinolone such as ciprofloxacinbecamefirst lineofdrugforaction[15-17]. Inacute infections,thesedrugsarepositivelyusedtoshortendurationofsymptomsandfecaleliminationofsalmonellae.Overtheyears,resistancehasbeenincreasedeventoquinolonesindifferentpartsoftheworldparticularlyinAsia[18].Nalidixicacid(NA)isaquinoloneused as a sample for in vitro screening tests. Unfortunately, NA resistant strains are displaying reduced susceptibility tociprofloxacinacrosstheglobe[19]. In thiscase,azithromycin issetasanalternativedrugfortreatmentoftyphoidfeverbutnowithaslostitstributeduetoresistantstrainsonsets[20].

Therefore, it is important to study the resistance pattern of S. typhi isolatesfromthepatients.Atthesametime,alotofeffortisneededtofindgeneticvariationscausingresistance.Keepingin view the above stated reasons, the basic aim of this research work was to find resistance patterns along with their geneticreasoninginordertolayoutfuturerecommendationsforproperuseofsuitableantibiotics.

Materials and MethodsSample collectionBlood sampleswerecollected fromsuspected typhoidpatientsin different diagnostic centers and hospitals. All sampleswere collected using standard sterile measures. Prior to the blood sampling, age, gender and different clinical/laboratoryexaminationfindingswererecorded.

Isolation of coloniesTwomlofbloodwasinoculatedintoabottlecontaining16.0mltrypticasesoybroth(TSB)andkeptat37˚Cfor72hours.OnemlofTSB(BD211768)fromprimarycultivatedsamplewaspouredon both blood and MacConkey agar (Oxoid, USA) plates. Theplates were kept at 37°C for 24 hours. The plates which indicated growth were separated. Colonies were taken from these plates andre-cultivatedbyprimary,secondaryandtertiarystreakingon

freshplatesofMacConkeyagar.Freshstreakedplateswerekeptovernightat37˚C.

Microbiological and biochemical identificationAllisolateswereidentifiedbystandardmicrobiologicalproceduresincludingcolonymorphology,Gramstainingandwereverifiedbyvariousbiochemicalreactionsspecificallytriplesugarirontests.ForfurtherconfirmationofS. typhi, API 20E strips were used.

Molecular identification of isolatesFor molecular identification, DNA was extracted by standardPhenol-chloroform method. The reliability of extracted DNAwas mediated by agarose gel electrophoresis in which 1% agarose gel was used. Eagle Eye UV trans-illuminator wasused to photograph bands showing purity of DNA samples. Furthermore, these samples were analyzed for quality usingspectrophotometer. Hyper variable region VI of flagellin fliC gene was targeted using two sets of primers (Figure 1a). These primers were ST 1 (5'-ACTGCTAAAACCACTACT-3') and ST2(5'-TGGAGACTTCGGTCGCGTAG-3')asreportedbySongetal.[21]andFrankel[22].

PCR reagents comprised template DNA (5 ng/µL), dNTPs (0.7nmol/µL),PCRbufferwithoutMgCl2(10X),MgCl2 (25mM),forwardprimer(25pmol/µl),reverseprimer(25pmol/µl),TaqPolymerase(5U/µL)andTrisbufferofpH:8.3 (10mM).50µlvolumewasattained by adding distilled water to reagents. PCR conditionswereused inwayof initialdenaturing temperatureof94°C for2 minutes followed by 30 cycles of denaturing temperature of 94°C,annealingtemperatureof50°Candextensiontemperatureof 72°C for aminute each and a final extension of 72°C for 5minutes.

Sensitivity testingForsensitivitytesting,Kirby-Bauerdiscdiffusionmethod[23]wasperformed according to the guidelines of Clinical and Laboratory Standards Institute, formerly National Committee for ClinicalLaboratoryStandards(NCCLS)[24,25].Inthismethod,antibioticsability was tested to inhibit growth of bacteria by forming a zone of inhibition around the disk. Penicillin, carbapenum,cephalosporin, aminoglycosides, macrolides/lincosamides,quinolones, fluoroquinolones and some other miscellaneousgroups of antibiotics were applied to petri plates of S. typhi. The illustrative drugs among above mentioned antibioticsgroupwereampicillin (10µg), imepenum/meropenum(10µg),cefixime(5µg),ceftrixone(30µg),ceftazidime(30µg),cefipime(30µg),gentamicin(10µg),erythromycin(15µg),azithromycin(15µg), nalidixic acid (30µg), ciprofloxacin (5µg), levofloxacin(5 µg), ofloxacin (5 µg), co-trimaxazole (1.25/23.75 µg) andchloramphenicol(30µg).Isolateswereconsideredasmultidrugresistantwhenresistanttomorethantwoclassesofantibioticsat least.

Genes for antimicrobial resistanceThestrainsfoundresistanttociprofloxacinandnalidixicacidwereselectedformolecularcharacterization.Alargenumberofgenesare reported previously for phenotypic resistance but in this study, gyrA and gyrBwereselected.Primersused for targeting

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gyrA (Figure 1b)genewereStgyrA-1(TGTCCGAGATGGCCTGAAGC)and StgyrA-2 (TACCGTCATASGTTATCCACG) as mentioned byGriggs et al. [26]. In the same way, primers for gyrB (Figure 1c) were StgyrB-1(CAAACTGGCGGACTGTCAGG) and StgyrB-2(TTCCGGCATCTGACGATAGA)asmentionedbyLingetal. [27].

These genes were amplified by using PCR. 50 µl of reactionmixture contained similar reagents asmentioned forfliC gene. PCR conditions were followed in way of initial denaturingtemperature of 92°C for 2 minute followed by 30 cycles of denaturing temperature of 92°C and annealing temperature of 62°Cforaminuteeachwhileextensiontemperatureof74°Cfor2minutes.Finalextensiontemperaturewasof74°Cforaminute.

Obtained amplified products were sequenced fromMacrogen,Korea. The nucleotide sequence for these genes was blast atNCBI.Proteomic serverwasused for comparisonofnucleotidesequenceswithreferenceS. typhi str. CT 18.

Statistical analysisStatisticalpackageforsocialscience(SPSSversion16)wasusedforstatisticalanalysis inwhichanalysisofvariancewithinteractionwasappliedtodistinguish the impactofantibioticsgroupsandtheir representative drugs. Meanwhile, resistance patterns ofisolates in terms of different environmental and demographicvariables like season, age and gender were also tested.

ResultsEpidemiology In this study, suspected typhoid patients were categorized indifferentagegroupsfrom≤2yearsupto80asshowninTable 1. These were divided into infants, children, sub-adults/adultsandpostadults.Thesepatientsweresynchronizedforresearchpurposeonthebasisofinvestigatedpersistenceofsymptoms.

Isolation strengthAmong103isolates,55wereobtainedfrommalepatientswhileremaining48werefemalepatients.Inthisstudy,59.2%patientsbelonged to urban area while remaining 40.8% were rural which revealed typhoid fever to be more common in urban areas of Rawalpindi and Islamabad Capital Territory (Table 1).

Drug sensitivitySixtimeperiodswere framed to regulate the consequencesofdiscdiffusionmethod,performed,throughouttheyear.Eachtimeconstraintcomprisedaphaseoftwomonths.Thesetimeframesweredesignedonthebaseofseasonalvariationsofstudyareato reveal seasonal outbreaks of S. typhi strains. These seasons included summer, monsoon, autumn, winter and spring.

Afterapplyingallselectedantibiotics,itwasfoundthatcefipime,afifthgenerationcephalosporin,wasmosteffectiveandsensitivedrug to87 (84.47%) isolates. 78 (75.73%) isolateswere foundsensitivetoceftriaxonewhichhadnearlysimilaroutcomeof77(74.76%) to cefixime. As far as resistance is concerned, 90.3%isolateswereconfirmedtobeMDRST.83(80.58%)isolateswereresistanttonalidixicacidwhile68(66.02%)outof103werefoundto be resistant to ciprofloxacin and imepenum/meropenum.

Nalidixicacidandciprofloxacinwerenotsuitabledrugsbecauseofhighestresistancepercentage.Cephalosporinswereconfirmedto be suitable drugs as shown in Figure 2.

Statistical analysis for drug sensitivityByapplyingstatistics, itwasproved that resistance todifferentantibiotics groups was independent of seasons. There was asignificant difference (P ≤ 0.05) between antibiotics group’sresistance and seasons (Figure 3).Ahighnon-significantdifference(P=0.302 or P ≥ 0.05)wasprofessedbetweenantibioticsgroup’sresistance and age groups (Figure 4).Inthesameway,highnon-significantdifference (P=0.976 or P ≥ 0.05)wasalso confirmedbetweenantibioticsgroup’sresistanceandgender(Figure 4).

LSD was applied to weigh peculiar effectiveness and multiplecomparisons of drugswith specificity to impact of season andage categories. Cephalosporin was unique group with a highsignificant difference (P=0.000) from other applied groups.Fluoroquinoloneswereadjac

ent in their actions to penicillin, carbapenum, macrolidesand miscellaneous antibiotics (P ≥ 0.05) while diverse fromcephalosporin and aminoglycosides (P ≤ 0.05). Cefipime,among cephalosporin, was effective drug having significantdifference (P=0.000) in action fromother applieddrugs exceptthird generation cephalosporins like cefixime, ceftriaxone andceftazidimeusedinKirby-Bauerdiskdiffusionmethod.Monsoonand autumn were the seasons of typhoid outbreak. Results for summer, winter and spring season were similar while monsoon andfallweredifferent fromotherseasonsof thestudyarea inrespecttoantibiogramsofS. typhi (Figure 3).Excessiveantibioticsresistancewithhighsignificantdifference(P = 0.000)wasfoundinpatientshavingagebetween10-20years(Figure 5).

Genes for drug resistanceIsolates resistant to nalidixic acid were selected for theirmolecularanalysis tofindgeneticmechanismof their resistantbehavior. The reason behind selection of isolates was theirhighest resistance to nalidixic acid in the study region. Foranalysisofquinoloneresistance,gyrA and gyrB was checked and thenamplifiedbyusing regularPCR.AmplificationofgyrA was indicatedbysingleproductof347bps.Ontheotherhand,gyrB wasamplifiedshowingasingleproductof345bps.

Analysis of sequencingIn this study, a common single point mutation was detectedamong all selected isolates which was substituting Serine-83(TCC) with Phenyl-alanine (TTC). This single point mutation isassociatedwithresistanceagainstnalidixicacidinS. typhi[28].

DiscussionTyphoidfeverremainsamajorcommunalhealthprobleminmostdeveloping countries such as Pakistan. It is due to factors blend includingdeprivedcleannessandhealthcareset-up.Antimicrobialresistance is a major public health problem generated inSalmonella typhi. Some of the reasons of antibiotic resistanceincludeimproperuseofantibioticsaswellaspooraccesstothedoctors. These reasons ultimately boost the intolerable sellingpracticeofantibiotics[29].

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1a fliC gene.Figureshowsmolecular identificationofS. typhi.Lane1-8:AmpliconsoffliCgene.Lane10:Generuler(Invitrogen). 1b) gyrA gene. PCR results for gyrAgene.Lane1-8:AmpliconsofgyrAgene.Lane9:Generuler(Invitrogen).1c) gyrB gene. PCR results for gyrBgene.Lane1-9:AmpliconsofgyrBgene.Lane10:Generuler(Invitrogen).

Figure 1

AntibiogramstudiesofS. typhi throughout the year from May, 2014 to April, 2015.Figure 2

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Antibioticsresistantisolatesrelativetoseasons(P ≤ 0.05).Figure 3

Antibioticsresistantisolatesrelativetodifferentagegroupsofpatients(P=0.302 or P ≥ 0.05).Figure 4

ThereishighburdenofantibioticsresistanceinAsia.InBangladesh,resistancetociprofloxacinandofloxacinwasonly8%in2000[12] but71%resistancepatternswereobservedin2005.Resistancetofluoroquinolonesreachedto90%in2009[30]. In 2014, it was found thatnalidixicacidismostinaptantibioticsfollowedbycefixime,levofloxacinandciprofloxacininBengladesh.AntibiogramstudyatGulbargaUniversity,Indiarevealedcompletesensitivityofallisolates to imipenum. Highest resistance was against nalidixicacid, ampicillin and chloramphenicol i.e., 31.57%, 29.47% and 28.42%,respectively.10% isolatesweremultidrugresistant [2]. InNepal,26.4% isolateswereconfirmed tobeMDR ina studyfrom year 2000 to 2005. High resistance of 77% was found against nalidixic acid. 30% isolates were resistant to ampicillin

while resistance to chloramphenicol and co-trimaxazole was27%.Noisolatewasresistanttociprofloxacinduringthefiveyearstudy [31]. Ina studyconducted inChina in2006-2007,ahighfrequencyofantibioticsresistancewasrecordedagainstdifferentantibiotics. Trimethoprim resistance was among 96% isolates,similarly,resistancetosulfamethoxazole(95%),ampicillin(95%),gentamicin (95%), streptomycin (91%), ciprofloxacin (96%),nalidixicacid(95%)andchloramphenicol(84%)wasrecorded[18].

In 2011, a study was conducted in NIBGE, Pakistan to checkantibiograms of S. typhi. 30% and 10% isolates were resistant to nalidixic acid and ciprofloxacin respectively. Meanwhile,resistance to cefiximeand ceftriaxonewereobserved in13.3%and3.3%isolatesrespectively[32]. Previously,severaldramatic

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changingtrendsinantibioticsresistancearediscussedfortyphoidsalmonellaeinRawalpindi/Islamabad.Fromyear1996to2000,no isolateswere found tobe resistant tofluoroquinolones likeciprofloxacin in a study conducted in Armed Forces Instituteof Pathology, Rawalpindi but in session 2001-2003, some ofthe ciprofloxacin resistant isolates were attained from nativepopulation[33].

This study was conducted to evaluate quantitative relationof antimicrobials resistance and their use as well as to assessfluctuatingtendenciesofantibioticsresistanceinS. typhi. Among 15 different antibiotics applied, quinolone/fluoroquinolonesi.e., nalidixic acid and ciprofloxacin and cephalosporin likecefixime, ceftrixone, ceftazidime and peculiarly cefipime weremonitoredadequatelyduetotheircurrentalarmingstatus.Otherconventional antimicrobials comprised ampicillin, imepenum/meropenum, gentamicin, erythromycin, azithromycin,levofloxacin,ofloxacin,co-trimaxazoleandchloramphenicol.

At culmination of this comprehensive study, nalidixic acid,ciprofloxacin, imepenum/ meropenum, chloramphenicol, co-trimaxazole, ampicillin, erythromycin and levofloxacin weredrugs against which more than half of isolates were resistant. Nalidixicacidandciprofloxacinwereobservantdrugswithhighestresistance percentage of 80.58% and 66.02% respectively. Anintroducedfifthgenerationcephalosporin, cefipime,was foundreliable drug against 87 S. typhi isolates.Otherthirdgenerationcephalosporins were also dependable drugs with less number of resistant isolates. Ceftrixone, cefixime and ceftazidimeweresuitable drugs for 78, 77 and 75 isolates, respectively. Among103 samples, 93 were confirmed to beMDR exhibiting 90.2%MDRST isolates. These outcomes have exposed astonishingintensification inMDRSTaswellasfluoroquinolone resistantS. typhi isolates for last ten years in the Islamabad capital territory.

Point mutations in quinolone resistance determining regions(QRDR)oftargetgenes i.e.,gyrA and gyrB is responsible factor

Antibioticsresistantisolatesrelativetogenderofpatients(P=0.976).Figure 5

Age Isolates Gender No. of Patients Socio-economic Status(Year) No. % Male Female Rural Urban Lower Middle Upper

Infants≤2 9 8.74 5 4 2 7 1 8 -

Children02-Oct 24 23.3 14 10 14 10 10 12 2

Sub adults & adultsOct-20 21 20.39 15 6 13 8 12 8 120-30 10 9.7 7 3 4 6 3 6 130-40 14 13.6 3 11 2 12 4 6 440-50 15 14.56 6 9 5 10 4 9 2

Post adults50-60 6 5.83 2 4 2 4 2 3 160-70 3 2.91 3 - - 3 - 3 -70-80 1 0.97 - 1 - 1 - 1 -Total 103 100 55 48 42 61 36 56 11

Table 1Gender,age,localityandsocio-economicstatusofpatients.

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for resistance in quinolone and reduced susceptibility tofluoroquinolones[34].IngyrA gene, the most common reported pointmutationsaresubstitutionsatSer-83positiontoAla,TyrorPheandsubstitutionsatAsp-87toAsn,TyrorGly[35].

Inpresentstudy,thebasiccausefornalidixicacidresistancewasconfirmedtobeassociatedwithsinglepointmutationwithaminoacid alteration fromSer83- position to Phe, a similarmutationfoundatNIBGE,inyear2011.Likewise,itwasconcludedthatMICofciprofloxacinneedstobeimproved.

Conclusions Onthebasisofcurrentstudy, it isconcludedthatcefipimeandthird generation cephalosporin like ceftriaxone are suitabledrugstobeprescribedinsteadoffluoroquinolonesinIslamabad,the capital territory of Pakistan. It is understandable that the occurrence of infection is apparently high. Blood isolates of S. typhi were more resistant to most of the commonly prescribed antibiotics like ciprofloxacin. Therefore it must be compulsoryupon the committed medical doctors, clinical microbiologistsand public health officials to familiarize themselves with theinformationoflocalantimicrobialresistancepatternsofS. typhi.

Itwouldhelpfordirectingexperimentalandpathogen-specifictherapy. The proper health education like antibiotic sensitivitytestingaswellastheaccurateremedyanddosageofantibioticswill help to restrict the expansion of antibiotics resistance bySalmonella.

AcknowledgementsAuthors would like to thank Higher Education Commission,Pakistan for research support. We also thank IslamabadDiagnosticCenterandKulsoomInternationalHospital,Islamabadfor providing support to make this

research effort possible. Meanwhile, authors pay gratitude toNIBGE,Faisalabadforprovidingtheirtechnicalsupport.

Conflict of InterestTheauthorsdeclarethattheyhavenoconflictofinterest.

Ethical ApprovalForthistypeofstudyformalconsentisnotrequired.Thisarticledoesnotcontainanystudieswithhumanparticipantsoranimalsperformed by any of the authors.

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