first report of blactx-m-28 in enterobacteriaceae isolates in the united...
TRANSCRIPT
Research ArticleFirst Report of blaCTX-M-28 in Enterobacteriaceae Isolates inthe United Arab Emirates
Mubarak Alfaresi1 Garwin Kim Sing2 and Abiola Senok 3
1College of Medicine University of Sharjah Sharjah UAE2Department of Microbiology and Immunology College of Medicine Alfaisal University Riyadh Saudi Arabia3Department of Basic Medical Sciences College of Medicine Mohammed Bin Rashid University ofMedicine and Health Sciences Dubai UAE
Correspondence should be addressed to Abiola Senok abiolasenokmbruacae
Received 12 November 2017 Accepted 20 December 2017 Published 18 January 2018
Academic Editor Alfizah Hanafiah
Copyright copy 2018 Mubarak Alfaresi et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited
Background The CTX-M family of extended-spectrum beta lactamase (ESBL) enzymes is comprised of over 60 119887119897119886CTX-M genevariants with the predominance of 119887119897119886CTX-M-15 in many regions In this report we present the first description of 119887119897119886CTX-M-28 inthe United Arab EmiratesMethods Forty-five non-duplicate ESBL producing isolates identified in a secondary care facility in theUnited Arab Emirates from June to July 2016 were studied Gene sequencing was performed and DNA sequences were annotatedusing theBLASTprogram to identify the gene subtypesResultsThemajority of the ESBLpositive isolateswereE coli (119899119873 = 3945866) followed by K pneumoniae (119899 = 5) and K oxytoca (119899 = 1) All isolates harboured 119887119897119886CTX-M and 119887119897119886TEM genes 18 had 119887119897119886SHVand 2 were 119887119897119886VIM positive Thirty-seven isolates (822) were positive for 119887119897119886CTX-M-28 Other 119887119897119886CTX-M genes identified include119887119897119886CTX-M-167 (119899 = 2 isolates 1 and 26) and one each for 119887119897119886CTX-M-38 119887119897119886CTX-M-163 and 119887119897119886CTX-M-198 No 119887119897119886CTX-M-15 was identifiedThe predominant 119887119897119886TEM subtype was 119887119897119886TEM-171 (119899 = 8) followed by one of each of 119887119897119886TEM-120 119887119897119886TEM-163 and 119887119897119886TEM-206 The 119887119897119886SHVsubtypes were 119887119897119886SHV-148 and 119887119897119886SHV-187 Conclusion The findings indicate the first description of 119887119897119886CTX-M-28 in a setting where119887119897119886CTX-M-15 was previously predominant
1 Introduction
Extended-spectrum beta lactamase (ESBL) producing Enter-obacteriaceae are major agents of nosocomial and com-munity acquired infections In contrast to ESBL enzymessuch as TEM SHV and OXA types which arose from pointmutations the CTX-M family of enzymes are derived fromKluyvera spp chromosomal beta lactamase genes Since thefirst description in 1992 the CTX-M family has now becomethe predominant ESBL enzymes in Enterobacteriaceae [1 2]The CTX-M family comprises over 60 119887119897119886CTX-M variantswhich have been classified into five major phylogeneticclusters [3] The rapid dissemination of CTX-M-positiveESBL producing bacteria has hitherto been largely driven bythe predominance of CTX-M-15 across geographical regionsand in diverse bacterial isolates [2] The 119887119897119886CTX-M-28 geneis closely related to the 119887119897119886CTX-M-15 gene differing only bya single amino acid at the C-terminus and both belong to
CTX-M-Group 1 In the Arabian Gulf region one of thehighest rates of ESBL prevalence has been from the UnitedArab Emirates (UAE) [4] Previously reportedwork indicateda predominance of 119887119897119886CTX-M-15 [5 6] In this study we presentthe first report of 119887119897119886CTX-M-28 in the UAE and its predomi-nance among the Enterobacteriaceae isolates studied
2 Methods
Bacterial Isolates The study was carried out at a secondarycare general hospital in the UAE A total of 45 nonduplicateconsecutive phenotypically confirmed ESBL isolates identi-fied in theMicrobiology Laboratory from June-July 2016werestudied Only the first isolate per patient was included in thestudy Isolates were obtained from clinical specimens sub-mitted for routine diagnostic investigation Bacterial iden-tification and antibiotic susceptibility testing were carried
HindawiJournal of PathogensVolume 2018 Article ID 1304793 5 pageshttpsdoiorg10115520181304793
2 Journal of Pathogens
out using the Vitek2 automated system (BioMerieux Marcy-lrsquoEtoile France) and the N204 VITEK susceptibility card wasused for ESBL confirmation Isolates were stored at minus80∘Cformolecular characterization Ethical approval was obtainedfrom hospital review board (Medical Executive Committee)
21 Genotypic Testing for ESBL PCR was performed usingpreviously described primers and protocol [7] Each 25120583Lreaction mixture was made up of 125 120583L of GoTaq GreenMasterMix (PromegaCoMadisonWI) 04 120583Mprimer and3 120583L of sample DNA The PCR protocol followed was initialdenaturation at 95∘C for 5min cyclic denaturation at 95∘Cfor 50 s annealing at 56∘C 50∘C or 60∘C for 40 s elongationat 72∘C for 1min for 35 cycles and final extension at 72∘Cfor 5min Detection of PCR products was on 1 agarose gelPositive amplicons were purified by Promega Wizard SV Geland PCR Clean-up System (Promega Co Madison WI) andsequenced at Macrogen Laboratories (Seoul South Korea)DNA sequenceswere annotated by using the BLASTprogram(httpsblastncbinlmnihgov) to identify the gene subtypesBriefly raw AB1 sequence trace files were translated intoFASTQ format and individual sequences quality-trimmedPairwise alignments of quality-filtered paired forward andreverse reads ge200 nt were produced Reference sequencesof complete bacterial bla and mcr-1 genes were retrievedfrom the NCBI nucleotide database A reference BLASTnucleotide databasewas compiled fromgene features (FASTAformat) with makeblastdb v260+ F The FASTA sequenceswere queried against the blamcr-1 subtypes BLAST databaseThe query hits were checked for gene specificity (all hitshad to be from the same gene to be valid) Sequences witha single highest bit score were assigned to the respectivegene subtype Query sequences with multiple highest bitscores were assigned to the respective gene type withoutspecification of a subtype
3 Results
Of the 208 Enterobacteriaceae identified during the studyperiod 45 (216) were ESBL positive The majority of theESBL positive isolates were E coli (119899119873 = 3945 866) fol-lowed byK pneumoniae (119899 = 5) andK oxytoca (119899 = 1) Mostwere from inpatients (119899119873 = 3445 755)The isolates wereobtained from urine (119899 = 36) sputum (119899 = 4) pus (119899 = 4)and blood (119899 = 1) All the isolates harboured 119887119897119886CTX-M and119887119897119886TEM genes 18 had 119887119897119886SHV gene and 2 were positive for119887119897119886VIM gene Table 1 shows the distribution of the isolates andthe ESBL genes identified in each isolate Over half of theisolates (119899 = 25) harboured a combination of three genesand 19 had two genes (Table 1) One isolate harboured acombination of four genes namely 119887119897119886CTX-M-167 119887119897119886CTX-M-28119887119897119886SHV 119887119897119886TEM (isolate 26 Table 1) Thirty-seven isolates(822) were positive for 119887119897119886CTX-M-28 Other 119887119897119886CTX-M genesidentified include 119887119897119886CTX-M-167 (119899 = 2 isolates 1 amp 26)The other CTX-M subtypes identified include 119887119897119886CTX-M-167(119899 = 2) and one each for 119887119897119886CTX-M-38 119887119897119886CTX-M-163 and119887119897119886CTX-M-198With the exception of the 119887119897119886CTX-M-163 isolate allothers were also positive for 119887119897119886CTX-M-28 No 119887119897119886CTX-M-15 wasidentified For seven isolates (9 14 22 25 28 38 and 45 see
Table 1) the CTX-M subtype could not be resolved as querysequences with multiple highest hit scores were identified
The two 119887119897119886SHV subtypes identified were 119887119897119886SHV-148 (iso-late 4) and 119887119897119886SHV-187 (isolate 8) The predominant 119887119897119886TEMsubtype was 119887119897119886TEM-171 (isolates 10 13 20 28 30 32 39 and40 Table 1) The other 119887119897119886TEM subtypes were 119887119897119886TEM-120 (iso-late 42) 119887119897119886TEM-163 (isolate 41) and 119887119897119886TEM-206 (isolate 31)A total of 18 isolates were identified as harbouring 119887119897119886SHV butassignment into subtypes could not be done because querysequences with multiple highest hit scores were identifiedSimilarly there were 33 isolates identified with 119887119897119886TEM butwithout subtype classification Two isolates harboured 119887119897119886VIM(isolates 44 and 45) None of the isolates harboured the119887119897119886OXA-48 119887119897119886IMP 119887119897119886NDM or mcr-1 genes The antibiotic sus-ceptibility profile of each isolate is shown in the supplemen-tary file (available here) There was no evidence of carbape-nem resistance as all isolates were sensitive to imipenemmeropenem and ertapenem In addition fosfomycin sensi-tivity was seen in all isolates 18 were sensitive to ciprofloxacinand trimethoprim-sulphamethoxazole
4 Discussion
During the study period 216 of Enterobacteriaceae iden-tified in this healthcare facility were ESBL positive which islower than the 36ndash41 previously reported in studies fromthe UAE [4 5] However these previous studies were carriedout over a one-year period and inmultiple care facilitiesThisrepresents the first study from the UAE carried out in a singlesecondary care facility where cases coming straight from thecommunity are seen Thus the finding of a relatively highESBL prevalence suggests that ESBL dissemination remainsan ongoing occurrence in the community in the UAE Thisis particularly pertinent as the majority of the isolates wereobtained from urine
Our findings identified several CTX-M SHV and TEMgenes which had not been previously reported in the UAEThis is largely because of the sequencing approach which wasused in this studyThe predominant ESBL gene identified was119887119897119886CTX-M which is in keeping with the previously reportedwork from the UAE and neighboring countries [5 6 8ndash10]Indeed CTX-M group one genes and specifically 119887119897119886CTX-M-15had been identified as the predominant CTX-M type inESBL producers in the Arabian Gulf region [5 10ndash12]Previous work done in the UAE by Alfaresi et al identified119887119897119886CTX-M-15 in majority of ESBL isolates [5] The findings inthis study indicate a predominance of 119887119897119886CTX-M-28 Unlike119887119897119886CTX-M-15 which had been frequently been described inthe literature 119887119897119886CTX-M-28 has been sparsely documentedSporadic identification has been documented in reports fromIndia Brazil Bosnia and Herzegovina China and Tunisia[13ndash18] In the Arabian Gulf region 119887119897119886CTX-M-28 has only beenidentified in one E coli isolate in Kuwait [19] In contrast tothese sporadic reports 621 of the ESBL producers reportedin SouthKorea byYoo et al were 119887119897119886CTX-M-28 whichwas a shiftfrom the predominance of 119887119897119886CTX-M-15 [17]The findings fromthis study represent the first report of similar predominanceof 119887119897119886CTX-M-28 in the Arabian Gulf region Similar to thefindings in South Korea the majority of our isolates were
Journal of Pathogens 3
Table 1 Distribution of isolates and ESBL genes
Collection date Isolate Site Source ESBL genes(1) 6162016 E coli Outpatient Urine 119887119897119886CTX-M-167 119887119897119886CTX-M-28 119887119897119886TEM(2) 6162016 E coli Outpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM(3) 6192016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM(4) 6192016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV-148 119887119897119886TEM(5) 6192016 K pneumoniae Outpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM(6) 6252014 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV-187 119887119897119886TEM(7) 6262016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM(8) 6262016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM(9) 6292016 E coli Inpatient Sputum 119887119897119886CTX-M 119887119897119886SHV 119887119897119886TEM(10) 6292016 E coli Inpatient Blood 119887119897119886CTX-M-28 119887119897119886TEM-171
(11) 6292016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM(12) 6302016 K pneumoniae Inpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM(13) 6302016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM-171
(14) 6302016 E coli Inpatient Urine 119887119897119886CTX-M 119887119897119886SHV 119887119897119886TEM(15) 742016 E coli Inpatient Urine 119887119897119886CTX-M28 119887119897119886SHV 119887119897119886TEM(16) 752016 K oxytoca Inpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM(17) 752016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM(18) 762016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM(19) 762016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM(20) 782016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM-171
(21) 782016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM(22) 7102016 E coli NR Sputum 119887119897119886CTX-M 119887119897119886SHV 119887119897119886TEM(23) 7112016 E coli Inpatient Pus 119887119897119886CTX-M-198 119887119897119886CTX-M-28 119887119897119886TEM(24) 7122016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM(25) 7122016 K pneumoniae Outpatient Urine 119887119897119886CTX-M 119887119897119886SHV 119887119897119886TEM(26) 7122016 E coli Outpatient Urine 119887119897119886CTX-M-167 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM(27) 7132016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM(28) 7132016 E coli Inpatient Urine 119887119897119886CTX-M 119887119897119886TEM-171
(29) 7132016 K pneumoniae Inpatient Pus 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM(30) 7142016 E coli Outpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM-171
(31) 7152016 E coli Inpatient Urine 119887119897119886CTX-M-163 119887119897119886TEM-206
(32) 7182016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM-171
(33) 7182016 E coli Inpatient Sputum 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM(34) 7192016 E coli Outpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM(35) 7232016 E coli Outpatient Urine 119887119897119886CTX-M-28 119887119897119886CTX-M-38 119887119897119886TEM(36) 7232016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM(37) 7242016 K pneumoniae Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM(38) 7252016 E coli Inpatient Pus 119887119897119886CTX-M 119887119897119886TEM-206
(39) 7252016 E coli Inpatient Sputum 119887119897119886CTX-M-28 119887119897119886TEM-171
(40) 7252016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM-171
(41) 7262016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM-163
(42) 7262016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM-120
(43) 7272016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM(44) 7302016 E coli Outpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM 119887119897119886VIM(45) 7302017 E coli Outpatient Pus 119887119897119886CTX-M 119887119897119886TEM 119887119897119886VIMNotes NR not recorded
E coli [17] Both 119887119897119886CTX-M-28 and 119887119897119886CTX-M-15 belong to CTX-M group 1 and differ by just a single amino acid at the 31015840-end of the CTX-M gene It has been suggested that becauseof this close similarity and the proximity of the sequence
differences to the 51015840-end (position 21) and 31015840-end (position865) of the CTX-M gene the primers designed for detectionof the CTX-M-1 group might misidentify these two CTX-M genes This might explain why 119887119897119886CTX-M-28 has hitherto
4 Journal of Pathogens
been underreported Previous work done by Alfaresi et al[5] using a sequencing methodology similar to that usedin the present study found predominance of 119887119897119886CTX-M-15Additionally the primers used in this study were designedto ensure adequate differentiation of these two CTX-M genestaking into cognizance the recommendations by Menezes etal [20] Hence these findings are therefore indicative of thefirst report of 119887119897119886CTX-M-28 in the UAE It was interesting tofind that the two isolates harbouring the 119887119897119886VIM gene werecarbapenem sensitive It is possible that this might be dueto nonexpression of this metallo-beta-lactamase gene andfurther work is needed to confirm this
A limitation of our work is the short study duration andnumber of isolates reported However the findings indicatethe need for multicenter studies to determine if there is anemerging shift in ESBL epidemiology and the clinical rele-vance of ESBL genes which had not been previously reportedin our setting but were found in this study Furthermoresuch large studies will enable further understanding of theevolutionary trend and clinical implications of 119887119897119886CTX-M-28As 119887119897119886CTX-M-15 was notorious for rapid dissemination glob-ally close surveillance and monitoring is needed for earlydetection of the spread of 119887119897119886CTX-M-28
Conflicts of Interest
None of the authors has any financial or other relationshipsthat may constitute conflicts of interest
Authorsrsquo Contributions
Mubarak Alfaresi conceived the study and collected the dataAbiola Senok analyzed and interpreted the data Abiola Senokprepared the manuscript with contributions from MubarakAlfaresi and Garwin Kim Sing All authors approved the finalversion of the manuscript
Acknowledgments
Strategic Research Grant no 307191502153 from AlfaisalUniversity Riyadh Saudi Arabia (Garwin Kim Sing andAbiola Senok) is acknowledged
Supplementary Materials
Specimen collection datesourcehospital unit and Antibioticsusceptibility profile for each isolate (Supplementary Materi-als)
References
[1] D M Livermore R Canton M Gniadkowski et al ldquoCTX-Mchanging the face of ESBLs in Europerdquo Journal of AntimicrobialChemotherapy vol 59 no 2 pp 165ndash174 2007
[2] E R Bevan A M Jones and P M Hawkey ldquoGlobal epidemiol-ogy of CTX-M 120573-lactamases temporal and geographical shiftsin genotyperdquo Journal of Antimicrobial Chemotherapy vol 72 no8 pp 2145ndash2155 2017
[3] R Bonnet ldquoGrowing group of extended-spectrum 120573-lactamas-es The CTX-M Enzymesrdquo Antimicrobial Agents and Chemo-therapy vol 48 no 1 pp 1ndash14 2004
[4] M Al-Zarouni A Senok F Rashid S M Al-Jesmi and D Pan-igrahi ldquoPrevalence and antimicrobial susceptibility pattern ofextended-spectrum beta-lactamase-producing enterobacteri-aceae in the United Arab Emiratesrdquo Medical Principles andPractice vol 17 no 1 pp 32ndash36 2008
[5] M S Alfaresi A A ElkoushHMAlshehhi andA I Abdulsa-lam ldquoMolecular characterization and epidemiology of extend-ed-spectrum beta-lactamase-producing escherichia coli andklebsiella pneumoniae isolates in the United Arab EmiratesrdquoMedical Principles and Practice vol 20 no 2 pp 177ndash180 2011
[6] M Al-Zarouni A Senok N Al-Zarooni F Al-Nassay and DPanigrahi ldquoExtended-spectrum 120573-lactamase-producing enter-obacteriaceae in vitro susceptibility to fosfomycin nitrofuran-toin and tigecyclinerdquoMedical Principles and Practice vol 21 no6 pp 543ndash547 2012
[7] J Du P Li H Liu D Lu H Liang and Y Dou ldquoPhenotypic andmolecular characterization of multidrug resistant Klebsiellapneumoniae isolated from a university teaching hospitalChinardquo PLoS ONE vol 9 no 4 Article ID e95181 2014
[8] K M Bindayna H S Khanfar A C Senok and G A BottaldquoPredominance of CTX-Mgenotype among extended spectrumbeta lactamase isolates in a tertiary hospital in Saudi ArabiardquoSaudi Medical Journal vol 31 no 8 pp 859ndash863 2010
[9] KM Bindayna A C Senok andA E Jamsheer ldquoPrevalence ofextended-spectrum beta-lactamase-producing Enterobacteri-aceae in Bahrainrdquo Journal of Infection and Public Health vol 2no 3 pp 129ndash135 2009
[10] A M Somily M Z Arshad G A Garaween and A C SenokldquoPhenotypic and genotypic characterization of extended-spec-trum 120573-lactamases producing Escherichia coli and Klebsiellapneumoniae in a tertiary care hospital in Riyadh Saudi ArabiardquoAnnals of Saudi Medicine vol 35 no 6 pp 435ndash439 2015
[11] V O Rotimi W Jamal T Pal A Sovenned and M J AlbertldquoEmergence of CTX-M-15 type extended-spectrum 120573-lacta-mase-producing Salmonella spp inKuwait and theUnitedArabEmiratesrdquo Journal ofMedicalMicrobiology vol 57 no 7 pp 881ndash886 2008
[12] HM ZowawiHH Balkhy T RWalsh andD L Paterson ldquo120573-Lactamase production in key gram-negative pathogen isolatesfrom theArabian PeninsulardquoClinicalMicrobiology Reviews vol26 no 3 pp 361ndash380 2013
[13] N Ben Achour P S Mercuri P Power et al ldquoFirst detection ofCTX-M-28 in a Tunisian hospital from a cefotaxime-resistantKlebsiella pneumoniae strainrdquo Pathologie Biologie vol 57 no 5pp 343ndash348 2009
[14] A Ibrahimagic B Bedenic F Kamberovic and S UzunovicldquoHigh prevalence of CTX-M-15 and first report of CTX-M-3 CTX-M-22 CTX-M-28 and plasmid-mediated AmpCbeta-lactamase producing Enterobacteriaceae causing urinarytract infections in Bosnia and Herzegovina in hospital andcommunity settingsrdquo Journal of Infection and Chemotherapyvol 21 no 5 pp 363ndash369 2015
[15] J Kingsley and S Verghese ldquoSequence analysis of blaCTX-M-28 an ESBL responsible for third-generation cephalosporinresistance in Enterobacteriaceae for the first time in IndiardquoIndian Journal of Pathology and Microbiology vol 51 no 2 pp218ndash221 2008
[16] A C S Lopes D L Veras A M S Lima R D C A Melo andJ Ayala ldquo 119887119897119886CTX-M-2 and 119887119897119886CTX-M-28 extended-spectrum
Journal of Pathogens 5
120573-lactamase genes and class 1 integrons in clinical isolatesof Klebsiella pneumoniae from Brazilrdquo Memorias do InstitutoOswaldo Cruz vol 105 no 2 pp 163ndash167 2010
[17] J S Yoo J Byeon J Yang J I Yoo G T Chung and Y S LeeldquoHigh prevalence of extended-spectrum 120573-lactamases and plas-mid-mediated AmpC 120573-lactamases in Enterobacteriaceae iso-lated from long-term care facilities in KoreardquoDiagnostic Micro-biology and Infectious Disease vol 67 no 3 pp 261ndash265 2010
[18] Y Yu S Ji Y Chen et al ldquoResistance of strains producing ex-tended-spectrum 120573-lactamases and genotype distribution inChinardquo Infection vol 54 no 1 pp 53ndash57 2007
[19] N Almaraghi Characterisation of CTX-M-120573-lactamases in En-terobacteriaceaeae in hospitals in Kuwait DissertationPhDthesis University of Edinburgh Edinburgh UK 2013
[20] G A Menezes M A Khan and J P Hays ldquoImportant method-ological considerations with respect to differentiation of CTX-M-15 and CTX-M-28 extended-spectrum beta-lactamasesrdquoIndian Journal of Medical Microbiology vol 28 no 1 pp 81-822010
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2 Journal of Pathogens
out using the Vitek2 automated system (BioMerieux Marcy-lrsquoEtoile France) and the N204 VITEK susceptibility card wasused for ESBL confirmation Isolates were stored at minus80∘Cformolecular characterization Ethical approval was obtainedfrom hospital review board (Medical Executive Committee)
21 Genotypic Testing for ESBL PCR was performed usingpreviously described primers and protocol [7] Each 25120583Lreaction mixture was made up of 125 120583L of GoTaq GreenMasterMix (PromegaCoMadisonWI) 04 120583Mprimer and3 120583L of sample DNA The PCR protocol followed was initialdenaturation at 95∘C for 5min cyclic denaturation at 95∘Cfor 50 s annealing at 56∘C 50∘C or 60∘C for 40 s elongationat 72∘C for 1min for 35 cycles and final extension at 72∘Cfor 5min Detection of PCR products was on 1 agarose gelPositive amplicons were purified by Promega Wizard SV Geland PCR Clean-up System (Promega Co Madison WI) andsequenced at Macrogen Laboratories (Seoul South Korea)DNA sequenceswere annotated by using the BLASTprogram(httpsblastncbinlmnihgov) to identify the gene subtypesBriefly raw AB1 sequence trace files were translated intoFASTQ format and individual sequences quality-trimmedPairwise alignments of quality-filtered paired forward andreverse reads ge200 nt were produced Reference sequencesof complete bacterial bla and mcr-1 genes were retrievedfrom the NCBI nucleotide database A reference BLASTnucleotide databasewas compiled fromgene features (FASTAformat) with makeblastdb v260+ F The FASTA sequenceswere queried against the blamcr-1 subtypes BLAST databaseThe query hits were checked for gene specificity (all hitshad to be from the same gene to be valid) Sequences witha single highest bit score were assigned to the respectivegene subtype Query sequences with multiple highest bitscores were assigned to the respective gene type withoutspecification of a subtype
3 Results
Of the 208 Enterobacteriaceae identified during the studyperiod 45 (216) were ESBL positive The majority of theESBL positive isolates were E coli (119899119873 = 3945 866) fol-lowed byK pneumoniae (119899 = 5) andK oxytoca (119899 = 1) Mostwere from inpatients (119899119873 = 3445 755)The isolates wereobtained from urine (119899 = 36) sputum (119899 = 4) pus (119899 = 4)and blood (119899 = 1) All the isolates harboured 119887119897119886CTX-M and119887119897119886TEM genes 18 had 119887119897119886SHV gene and 2 were positive for119887119897119886VIM gene Table 1 shows the distribution of the isolates andthe ESBL genes identified in each isolate Over half of theisolates (119899 = 25) harboured a combination of three genesand 19 had two genes (Table 1) One isolate harboured acombination of four genes namely 119887119897119886CTX-M-167 119887119897119886CTX-M-28119887119897119886SHV 119887119897119886TEM (isolate 26 Table 1) Thirty-seven isolates(822) were positive for 119887119897119886CTX-M-28 Other 119887119897119886CTX-M genesidentified include 119887119897119886CTX-M-167 (119899 = 2 isolates 1 amp 26)The other CTX-M subtypes identified include 119887119897119886CTX-M-167(119899 = 2) and one each for 119887119897119886CTX-M-38 119887119897119886CTX-M-163 and119887119897119886CTX-M-198With the exception of the 119887119897119886CTX-M-163 isolate allothers were also positive for 119887119897119886CTX-M-28 No 119887119897119886CTX-M-15 wasidentified For seven isolates (9 14 22 25 28 38 and 45 see
Table 1) the CTX-M subtype could not be resolved as querysequences with multiple highest hit scores were identified
The two 119887119897119886SHV subtypes identified were 119887119897119886SHV-148 (iso-late 4) and 119887119897119886SHV-187 (isolate 8) The predominant 119887119897119886TEMsubtype was 119887119897119886TEM-171 (isolates 10 13 20 28 30 32 39 and40 Table 1) The other 119887119897119886TEM subtypes were 119887119897119886TEM-120 (iso-late 42) 119887119897119886TEM-163 (isolate 41) and 119887119897119886TEM-206 (isolate 31)A total of 18 isolates were identified as harbouring 119887119897119886SHV butassignment into subtypes could not be done because querysequences with multiple highest hit scores were identifiedSimilarly there were 33 isolates identified with 119887119897119886TEM butwithout subtype classification Two isolates harboured 119887119897119886VIM(isolates 44 and 45) None of the isolates harboured the119887119897119886OXA-48 119887119897119886IMP 119887119897119886NDM or mcr-1 genes The antibiotic sus-ceptibility profile of each isolate is shown in the supplemen-tary file (available here) There was no evidence of carbape-nem resistance as all isolates were sensitive to imipenemmeropenem and ertapenem In addition fosfomycin sensi-tivity was seen in all isolates 18 were sensitive to ciprofloxacinand trimethoprim-sulphamethoxazole
4 Discussion
During the study period 216 of Enterobacteriaceae iden-tified in this healthcare facility were ESBL positive which islower than the 36ndash41 previously reported in studies fromthe UAE [4 5] However these previous studies were carriedout over a one-year period and inmultiple care facilitiesThisrepresents the first study from the UAE carried out in a singlesecondary care facility where cases coming straight from thecommunity are seen Thus the finding of a relatively highESBL prevalence suggests that ESBL dissemination remainsan ongoing occurrence in the community in the UAE Thisis particularly pertinent as the majority of the isolates wereobtained from urine
Our findings identified several CTX-M SHV and TEMgenes which had not been previously reported in the UAEThis is largely because of the sequencing approach which wasused in this studyThe predominant ESBL gene identified was119887119897119886CTX-M which is in keeping with the previously reportedwork from the UAE and neighboring countries [5 6 8ndash10]Indeed CTX-M group one genes and specifically 119887119897119886CTX-M-15had been identified as the predominant CTX-M type inESBL producers in the Arabian Gulf region [5 10ndash12]Previous work done in the UAE by Alfaresi et al identified119887119897119886CTX-M-15 in majority of ESBL isolates [5] The findings inthis study indicate a predominance of 119887119897119886CTX-M-28 Unlike119887119897119886CTX-M-15 which had been frequently been described inthe literature 119887119897119886CTX-M-28 has been sparsely documentedSporadic identification has been documented in reports fromIndia Brazil Bosnia and Herzegovina China and Tunisia[13ndash18] In the Arabian Gulf region 119887119897119886CTX-M-28 has only beenidentified in one E coli isolate in Kuwait [19] In contrast tothese sporadic reports 621 of the ESBL producers reportedin SouthKorea byYoo et al were 119887119897119886CTX-M-28 whichwas a shiftfrom the predominance of 119887119897119886CTX-M-15 [17]The findings fromthis study represent the first report of similar predominanceof 119887119897119886CTX-M-28 in the Arabian Gulf region Similar to thefindings in South Korea the majority of our isolates were
Journal of Pathogens 3
Table 1 Distribution of isolates and ESBL genes
Collection date Isolate Site Source ESBL genes(1) 6162016 E coli Outpatient Urine 119887119897119886CTX-M-167 119887119897119886CTX-M-28 119887119897119886TEM(2) 6162016 E coli Outpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM(3) 6192016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM(4) 6192016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV-148 119887119897119886TEM(5) 6192016 K pneumoniae Outpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM(6) 6252014 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV-187 119887119897119886TEM(7) 6262016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM(8) 6262016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM(9) 6292016 E coli Inpatient Sputum 119887119897119886CTX-M 119887119897119886SHV 119887119897119886TEM(10) 6292016 E coli Inpatient Blood 119887119897119886CTX-M-28 119887119897119886TEM-171
(11) 6292016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM(12) 6302016 K pneumoniae Inpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM(13) 6302016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM-171
(14) 6302016 E coli Inpatient Urine 119887119897119886CTX-M 119887119897119886SHV 119887119897119886TEM(15) 742016 E coli Inpatient Urine 119887119897119886CTX-M28 119887119897119886SHV 119887119897119886TEM(16) 752016 K oxytoca Inpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM(17) 752016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM(18) 762016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM(19) 762016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM(20) 782016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM-171
(21) 782016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM(22) 7102016 E coli NR Sputum 119887119897119886CTX-M 119887119897119886SHV 119887119897119886TEM(23) 7112016 E coli Inpatient Pus 119887119897119886CTX-M-198 119887119897119886CTX-M-28 119887119897119886TEM(24) 7122016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM(25) 7122016 K pneumoniae Outpatient Urine 119887119897119886CTX-M 119887119897119886SHV 119887119897119886TEM(26) 7122016 E coli Outpatient Urine 119887119897119886CTX-M-167 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM(27) 7132016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM(28) 7132016 E coli Inpatient Urine 119887119897119886CTX-M 119887119897119886TEM-171
(29) 7132016 K pneumoniae Inpatient Pus 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM(30) 7142016 E coli Outpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM-171
(31) 7152016 E coli Inpatient Urine 119887119897119886CTX-M-163 119887119897119886TEM-206
(32) 7182016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM-171
(33) 7182016 E coli Inpatient Sputum 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM(34) 7192016 E coli Outpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM(35) 7232016 E coli Outpatient Urine 119887119897119886CTX-M-28 119887119897119886CTX-M-38 119887119897119886TEM(36) 7232016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM(37) 7242016 K pneumoniae Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM(38) 7252016 E coli Inpatient Pus 119887119897119886CTX-M 119887119897119886TEM-206
(39) 7252016 E coli Inpatient Sputum 119887119897119886CTX-M-28 119887119897119886TEM-171
(40) 7252016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM-171
(41) 7262016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM-163
(42) 7262016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM-120
(43) 7272016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM(44) 7302016 E coli Outpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM 119887119897119886VIM(45) 7302017 E coli Outpatient Pus 119887119897119886CTX-M 119887119897119886TEM 119887119897119886VIMNotes NR not recorded
E coli [17] Both 119887119897119886CTX-M-28 and 119887119897119886CTX-M-15 belong to CTX-M group 1 and differ by just a single amino acid at the 31015840-end of the CTX-M gene It has been suggested that becauseof this close similarity and the proximity of the sequence
differences to the 51015840-end (position 21) and 31015840-end (position865) of the CTX-M gene the primers designed for detectionof the CTX-M-1 group might misidentify these two CTX-M genes This might explain why 119887119897119886CTX-M-28 has hitherto
4 Journal of Pathogens
been underreported Previous work done by Alfaresi et al[5] using a sequencing methodology similar to that usedin the present study found predominance of 119887119897119886CTX-M-15Additionally the primers used in this study were designedto ensure adequate differentiation of these two CTX-M genestaking into cognizance the recommendations by Menezes etal [20] Hence these findings are therefore indicative of thefirst report of 119887119897119886CTX-M-28 in the UAE It was interesting tofind that the two isolates harbouring the 119887119897119886VIM gene werecarbapenem sensitive It is possible that this might be dueto nonexpression of this metallo-beta-lactamase gene andfurther work is needed to confirm this
A limitation of our work is the short study duration andnumber of isolates reported However the findings indicatethe need for multicenter studies to determine if there is anemerging shift in ESBL epidemiology and the clinical rele-vance of ESBL genes which had not been previously reportedin our setting but were found in this study Furthermoresuch large studies will enable further understanding of theevolutionary trend and clinical implications of 119887119897119886CTX-M-28As 119887119897119886CTX-M-15 was notorious for rapid dissemination glob-ally close surveillance and monitoring is needed for earlydetection of the spread of 119887119897119886CTX-M-28
Conflicts of Interest
None of the authors has any financial or other relationshipsthat may constitute conflicts of interest
Authorsrsquo Contributions
Mubarak Alfaresi conceived the study and collected the dataAbiola Senok analyzed and interpreted the data Abiola Senokprepared the manuscript with contributions from MubarakAlfaresi and Garwin Kim Sing All authors approved the finalversion of the manuscript
Acknowledgments
Strategic Research Grant no 307191502153 from AlfaisalUniversity Riyadh Saudi Arabia (Garwin Kim Sing andAbiola Senok) is acknowledged
Supplementary Materials
Specimen collection datesourcehospital unit and Antibioticsusceptibility profile for each isolate (Supplementary Materi-als)
References
[1] D M Livermore R Canton M Gniadkowski et al ldquoCTX-Mchanging the face of ESBLs in Europerdquo Journal of AntimicrobialChemotherapy vol 59 no 2 pp 165ndash174 2007
[2] E R Bevan A M Jones and P M Hawkey ldquoGlobal epidemiol-ogy of CTX-M 120573-lactamases temporal and geographical shiftsin genotyperdquo Journal of Antimicrobial Chemotherapy vol 72 no8 pp 2145ndash2155 2017
[3] R Bonnet ldquoGrowing group of extended-spectrum 120573-lactamas-es The CTX-M Enzymesrdquo Antimicrobial Agents and Chemo-therapy vol 48 no 1 pp 1ndash14 2004
[4] M Al-Zarouni A Senok F Rashid S M Al-Jesmi and D Pan-igrahi ldquoPrevalence and antimicrobial susceptibility pattern ofextended-spectrum beta-lactamase-producing enterobacteri-aceae in the United Arab Emiratesrdquo Medical Principles andPractice vol 17 no 1 pp 32ndash36 2008
[5] M S Alfaresi A A ElkoushHMAlshehhi andA I Abdulsa-lam ldquoMolecular characterization and epidemiology of extend-ed-spectrum beta-lactamase-producing escherichia coli andklebsiella pneumoniae isolates in the United Arab EmiratesrdquoMedical Principles and Practice vol 20 no 2 pp 177ndash180 2011
[6] M Al-Zarouni A Senok N Al-Zarooni F Al-Nassay and DPanigrahi ldquoExtended-spectrum 120573-lactamase-producing enter-obacteriaceae in vitro susceptibility to fosfomycin nitrofuran-toin and tigecyclinerdquoMedical Principles and Practice vol 21 no6 pp 543ndash547 2012
[7] J Du P Li H Liu D Lu H Liang and Y Dou ldquoPhenotypic andmolecular characterization of multidrug resistant Klebsiellapneumoniae isolated from a university teaching hospitalChinardquo PLoS ONE vol 9 no 4 Article ID e95181 2014
[8] K M Bindayna H S Khanfar A C Senok and G A BottaldquoPredominance of CTX-Mgenotype among extended spectrumbeta lactamase isolates in a tertiary hospital in Saudi ArabiardquoSaudi Medical Journal vol 31 no 8 pp 859ndash863 2010
[9] KM Bindayna A C Senok andA E Jamsheer ldquoPrevalence ofextended-spectrum beta-lactamase-producing Enterobacteri-aceae in Bahrainrdquo Journal of Infection and Public Health vol 2no 3 pp 129ndash135 2009
[10] A M Somily M Z Arshad G A Garaween and A C SenokldquoPhenotypic and genotypic characterization of extended-spec-trum 120573-lactamases producing Escherichia coli and Klebsiellapneumoniae in a tertiary care hospital in Riyadh Saudi ArabiardquoAnnals of Saudi Medicine vol 35 no 6 pp 435ndash439 2015
[11] V O Rotimi W Jamal T Pal A Sovenned and M J AlbertldquoEmergence of CTX-M-15 type extended-spectrum 120573-lacta-mase-producing Salmonella spp inKuwait and theUnitedArabEmiratesrdquo Journal ofMedicalMicrobiology vol 57 no 7 pp 881ndash886 2008
[12] HM ZowawiHH Balkhy T RWalsh andD L Paterson ldquo120573-Lactamase production in key gram-negative pathogen isolatesfrom theArabian PeninsulardquoClinicalMicrobiology Reviews vol26 no 3 pp 361ndash380 2013
[13] N Ben Achour P S Mercuri P Power et al ldquoFirst detection ofCTX-M-28 in a Tunisian hospital from a cefotaxime-resistantKlebsiella pneumoniae strainrdquo Pathologie Biologie vol 57 no 5pp 343ndash348 2009
[14] A Ibrahimagic B Bedenic F Kamberovic and S UzunovicldquoHigh prevalence of CTX-M-15 and first report of CTX-M-3 CTX-M-22 CTX-M-28 and plasmid-mediated AmpCbeta-lactamase producing Enterobacteriaceae causing urinarytract infections in Bosnia and Herzegovina in hospital andcommunity settingsrdquo Journal of Infection and Chemotherapyvol 21 no 5 pp 363ndash369 2015
[15] J Kingsley and S Verghese ldquoSequence analysis of blaCTX-M-28 an ESBL responsible for third-generation cephalosporinresistance in Enterobacteriaceae for the first time in IndiardquoIndian Journal of Pathology and Microbiology vol 51 no 2 pp218ndash221 2008
[16] A C S Lopes D L Veras A M S Lima R D C A Melo andJ Ayala ldquo 119887119897119886CTX-M-2 and 119887119897119886CTX-M-28 extended-spectrum
Journal of Pathogens 5
120573-lactamase genes and class 1 integrons in clinical isolatesof Klebsiella pneumoniae from Brazilrdquo Memorias do InstitutoOswaldo Cruz vol 105 no 2 pp 163ndash167 2010
[17] J S Yoo J Byeon J Yang J I Yoo G T Chung and Y S LeeldquoHigh prevalence of extended-spectrum 120573-lactamases and plas-mid-mediated AmpC 120573-lactamases in Enterobacteriaceae iso-lated from long-term care facilities in KoreardquoDiagnostic Micro-biology and Infectious Disease vol 67 no 3 pp 261ndash265 2010
[18] Y Yu S Ji Y Chen et al ldquoResistance of strains producing ex-tended-spectrum 120573-lactamases and genotype distribution inChinardquo Infection vol 54 no 1 pp 53ndash57 2007
[19] N Almaraghi Characterisation of CTX-M-120573-lactamases in En-terobacteriaceaeae in hospitals in Kuwait DissertationPhDthesis University of Edinburgh Edinburgh UK 2013
[20] G A Menezes M A Khan and J P Hays ldquoImportant method-ological considerations with respect to differentiation of CTX-M-15 and CTX-M-28 extended-spectrum beta-lactamasesrdquoIndian Journal of Medical Microbiology vol 28 no 1 pp 81-822010
Stem Cells International
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Submit your manuscripts atwwwhindawicom
Journal of Pathogens 3
Table 1 Distribution of isolates and ESBL genes
Collection date Isolate Site Source ESBL genes(1) 6162016 E coli Outpatient Urine 119887119897119886CTX-M-167 119887119897119886CTX-M-28 119887119897119886TEM(2) 6162016 E coli Outpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM(3) 6192016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM(4) 6192016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV-148 119887119897119886TEM(5) 6192016 K pneumoniae Outpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM(6) 6252014 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV-187 119887119897119886TEM(7) 6262016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM(8) 6262016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM(9) 6292016 E coli Inpatient Sputum 119887119897119886CTX-M 119887119897119886SHV 119887119897119886TEM(10) 6292016 E coli Inpatient Blood 119887119897119886CTX-M-28 119887119897119886TEM-171
(11) 6292016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM(12) 6302016 K pneumoniae Inpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM(13) 6302016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM-171
(14) 6302016 E coli Inpatient Urine 119887119897119886CTX-M 119887119897119886SHV 119887119897119886TEM(15) 742016 E coli Inpatient Urine 119887119897119886CTX-M28 119887119897119886SHV 119887119897119886TEM(16) 752016 K oxytoca Inpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM(17) 752016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM(18) 762016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM(19) 762016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM(20) 782016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM-171
(21) 782016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM(22) 7102016 E coli NR Sputum 119887119897119886CTX-M 119887119897119886SHV 119887119897119886TEM(23) 7112016 E coli Inpatient Pus 119887119897119886CTX-M-198 119887119897119886CTX-M-28 119887119897119886TEM(24) 7122016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM(25) 7122016 K pneumoniae Outpatient Urine 119887119897119886CTX-M 119887119897119886SHV 119887119897119886TEM(26) 7122016 E coli Outpatient Urine 119887119897119886CTX-M-167 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM(27) 7132016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM(28) 7132016 E coli Inpatient Urine 119887119897119886CTX-M 119887119897119886TEM-171
(29) 7132016 K pneumoniae Inpatient Pus 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM(30) 7142016 E coli Outpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM-171
(31) 7152016 E coli Inpatient Urine 119887119897119886CTX-M-163 119887119897119886TEM-206
(32) 7182016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM-171
(33) 7182016 E coli Inpatient Sputum 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM(34) 7192016 E coli Outpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM(35) 7232016 E coli Outpatient Urine 119887119897119886CTX-M-28 119887119897119886CTX-M-38 119887119897119886TEM(36) 7232016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM(37) 7242016 K pneumoniae Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM(38) 7252016 E coli Inpatient Pus 119887119897119886CTX-M 119887119897119886TEM-206
(39) 7252016 E coli Inpatient Sputum 119887119897119886CTX-M-28 119887119897119886TEM-171
(40) 7252016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM-171
(41) 7262016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM-163
(42) 7262016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM-120
(43) 7272016 E coli Inpatient Urine 119887119897119886CTX-M-28 119887119897119886SHV 119887119897119886TEM(44) 7302016 E coli Outpatient Urine 119887119897119886CTX-M-28 119887119897119886TEM 119887119897119886VIM(45) 7302017 E coli Outpatient Pus 119887119897119886CTX-M 119887119897119886TEM 119887119897119886VIMNotes NR not recorded
E coli [17] Both 119887119897119886CTX-M-28 and 119887119897119886CTX-M-15 belong to CTX-M group 1 and differ by just a single amino acid at the 31015840-end of the CTX-M gene It has been suggested that becauseof this close similarity and the proximity of the sequence
differences to the 51015840-end (position 21) and 31015840-end (position865) of the CTX-M gene the primers designed for detectionof the CTX-M-1 group might misidentify these two CTX-M genes This might explain why 119887119897119886CTX-M-28 has hitherto
4 Journal of Pathogens
been underreported Previous work done by Alfaresi et al[5] using a sequencing methodology similar to that usedin the present study found predominance of 119887119897119886CTX-M-15Additionally the primers used in this study were designedto ensure adequate differentiation of these two CTX-M genestaking into cognizance the recommendations by Menezes etal [20] Hence these findings are therefore indicative of thefirst report of 119887119897119886CTX-M-28 in the UAE It was interesting tofind that the two isolates harbouring the 119887119897119886VIM gene werecarbapenem sensitive It is possible that this might be dueto nonexpression of this metallo-beta-lactamase gene andfurther work is needed to confirm this
A limitation of our work is the short study duration andnumber of isolates reported However the findings indicatethe need for multicenter studies to determine if there is anemerging shift in ESBL epidemiology and the clinical rele-vance of ESBL genes which had not been previously reportedin our setting but were found in this study Furthermoresuch large studies will enable further understanding of theevolutionary trend and clinical implications of 119887119897119886CTX-M-28As 119887119897119886CTX-M-15 was notorious for rapid dissemination glob-ally close surveillance and monitoring is needed for earlydetection of the spread of 119887119897119886CTX-M-28
Conflicts of Interest
None of the authors has any financial or other relationshipsthat may constitute conflicts of interest
Authorsrsquo Contributions
Mubarak Alfaresi conceived the study and collected the dataAbiola Senok analyzed and interpreted the data Abiola Senokprepared the manuscript with contributions from MubarakAlfaresi and Garwin Kim Sing All authors approved the finalversion of the manuscript
Acknowledgments
Strategic Research Grant no 307191502153 from AlfaisalUniversity Riyadh Saudi Arabia (Garwin Kim Sing andAbiola Senok) is acknowledged
Supplementary Materials
Specimen collection datesourcehospital unit and Antibioticsusceptibility profile for each isolate (Supplementary Materi-als)
References
[1] D M Livermore R Canton M Gniadkowski et al ldquoCTX-Mchanging the face of ESBLs in Europerdquo Journal of AntimicrobialChemotherapy vol 59 no 2 pp 165ndash174 2007
[2] E R Bevan A M Jones and P M Hawkey ldquoGlobal epidemiol-ogy of CTX-M 120573-lactamases temporal and geographical shiftsin genotyperdquo Journal of Antimicrobial Chemotherapy vol 72 no8 pp 2145ndash2155 2017
[3] R Bonnet ldquoGrowing group of extended-spectrum 120573-lactamas-es The CTX-M Enzymesrdquo Antimicrobial Agents and Chemo-therapy vol 48 no 1 pp 1ndash14 2004
[4] M Al-Zarouni A Senok F Rashid S M Al-Jesmi and D Pan-igrahi ldquoPrevalence and antimicrobial susceptibility pattern ofextended-spectrum beta-lactamase-producing enterobacteri-aceae in the United Arab Emiratesrdquo Medical Principles andPractice vol 17 no 1 pp 32ndash36 2008
[5] M S Alfaresi A A ElkoushHMAlshehhi andA I Abdulsa-lam ldquoMolecular characterization and epidemiology of extend-ed-spectrum beta-lactamase-producing escherichia coli andklebsiella pneumoniae isolates in the United Arab EmiratesrdquoMedical Principles and Practice vol 20 no 2 pp 177ndash180 2011
[6] M Al-Zarouni A Senok N Al-Zarooni F Al-Nassay and DPanigrahi ldquoExtended-spectrum 120573-lactamase-producing enter-obacteriaceae in vitro susceptibility to fosfomycin nitrofuran-toin and tigecyclinerdquoMedical Principles and Practice vol 21 no6 pp 543ndash547 2012
[7] J Du P Li H Liu D Lu H Liang and Y Dou ldquoPhenotypic andmolecular characterization of multidrug resistant Klebsiellapneumoniae isolated from a university teaching hospitalChinardquo PLoS ONE vol 9 no 4 Article ID e95181 2014
[8] K M Bindayna H S Khanfar A C Senok and G A BottaldquoPredominance of CTX-Mgenotype among extended spectrumbeta lactamase isolates in a tertiary hospital in Saudi ArabiardquoSaudi Medical Journal vol 31 no 8 pp 859ndash863 2010
[9] KM Bindayna A C Senok andA E Jamsheer ldquoPrevalence ofextended-spectrum beta-lactamase-producing Enterobacteri-aceae in Bahrainrdquo Journal of Infection and Public Health vol 2no 3 pp 129ndash135 2009
[10] A M Somily M Z Arshad G A Garaween and A C SenokldquoPhenotypic and genotypic characterization of extended-spec-trum 120573-lactamases producing Escherichia coli and Klebsiellapneumoniae in a tertiary care hospital in Riyadh Saudi ArabiardquoAnnals of Saudi Medicine vol 35 no 6 pp 435ndash439 2015
[11] V O Rotimi W Jamal T Pal A Sovenned and M J AlbertldquoEmergence of CTX-M-15 type extended-spectrum 120573-lacta-mase-producing Salmonella spp inKuwait and theUnitedArabEmiratesrdquo Journal ofMedicalMicrobiology vol 57 no 7 pp 881ndash886 2008
[12] HM ZowawiHH Balkhy T RWalsh andD L Paterson ldquo120573-Lactamase production in key gram-negative pathogen isolatesfrom theArabian PeninsulardquoClinicalMicrobiology Reviews vol26 no 3 pp 361ndash380 2013
[13] N Ben Achour P S Mercuri P Power et al ldquoFirst detection ofCTX-M-28 in a Tunisian hospital from a cefotaxime-resistantKlebsiella pneumoniae strainrdquo Pathologie Biologie vol 57 no 5pp 343ndash348 2009
[14] A Ibrahimagic B Bedenic F Kamberovic and S UzunovicldquoHigh prevalence of CTX-M-15 and first report of CTX-M-3 CTX-M-22 CTX-M-28 and plasmid-mediated AmpCbeta-lactamase producing Enterobacteriaceae causing urinarytract infections in Bosnia and Herzegovina in hospital andcommunity settingsrdquo Journal of Infection and Chemotherapyvol 21 no 5 pp 363ndash369 2015
[15] J Kingsley and S Verghese ldquoSequence analysis of blaCTX-M-28 an ESBL responsible for third-generation cephalosporinresistance in Enterobacteriaceae for the first time in IndiardquoIndian Journal of Pathology and Microbiology vol 51 no 2 pp218ndash221 2008
[16] A C S Lopes D L Veras A M S Lima R D C A Melo andJ Ayala ldquo 119887119897119886CTX-M-2 and 119887119897119886CTX-M-28 extended-spectrum
Journal of Pathogens 5
120573-lactamase genes and class 1 integrons in clinical isolatesof Klebsiella pneumoniae from Brazilrdquo Memorias do InstitutoOswaldo Cruz vol 105 no 2 pp 163ndash167 2010
[17] J S Yoo J Byeon J Yang J I Yoo G T Chung and Y S LeeldquoHigh prevalence of extended-spectrum 120573-lactamases and plas-mid-mediated AmpC 120573-lactamases in Enterobacteriaceae iso-lated from long-term care facilities in KoreardquoDiagnostic Micro-biology and Infectious Disease vol 67 no 3 pp 261ndash265 2010
[18] Y Yu S Ji Y Chen et al ldquoResistance of strains producing ex-tended-spectrum 120573-lactamases and genotype distribution inChinardquo Infection vol 54 no 1 pp 53ndash57 2007
[19] N Almaraghi Characterisation of CTX-M-120573-lactamases in En-terobacteriaceaeae in hospitals in Kuwait DissertationPhDthesis University of Edinburgh Edinburgh UK 2013
[20] G A Menezes M A Khan and J P Hays ldquoImportant method-ological considerations with respect to differentiation of CTX-M-15 and CTX-M-28 extended-spectrum beta-lactamasesrdquoIndian Journal of Medical Microbiology vol 28 no 1 pp 81-822010
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
4 Journal of Pathogens
been underreported Previous work done by Alfaresi et al[5] using a sequencing methodology similar to that usedin the present study found predominance of 119887119897119886CTX-M-15Additionally the primers used in this study were designedto ensure adequate differentiation of these two CTX-M genestaking into cognizance the recommendations by Menezes etal [20] Hence these findings are therefore indicative of thefirst report of 119887119897119886CTX-M-28 in the UAE It was interesting tofind that the two isolates harbouring the 119887119897119886VIM gene werecarbapenem sensitive It is possible that this might be dueto nonexpression of this metallo-beta-lactamase gene andfurther work is needed to confirm this
A limitation of our work is the short study duration andnumber of isolates reported However the findings indicatethe need for multicenter studies to determine if there is anemerging shift in ESBL epidemiology and the clinical rele-vance of ESBL genes which had not been previously reportedin our setting but were found in this study Furthermoresuch large studies will enable further understanding of theevolutionary trend and clinical implications of 119887119897119886CTX-M-28As 119887119897119886CTX-M-15 was notorious for rapid dissemination glob-ally close surveillance and monitoring is needed for earlydetection of the spread of 119887119897119886CTX-M-28
Conflicts of Interest
None of the authors has any financial or other relationshipsthat may constitute conflicts of interest
Authorsrsquo Contributions
Mubarak Alfaresi conceived the study and collected the dataAbiola Senok analyzed and interpreted the data Abiola Senokprepared the manuscript with contributions from MubarakAlfaresi and Garwin Kim Sing All authors approved the finalversion of the manuscript
Acknowledgments
Strategic Research Grant no 307191502153 from AlfaisalUniversity Riyadh Saudi Arabia (Garwin Kim Sing andAbiola Senok) is acknowledged
Supplementary Materials
Specimen collection datesourcehospital unit and Antibioticsusceptibility profile for each isolate (Supplementary Materi-als)
References
[1] D M Livermore R Canton M Gniadkowski et al ldquoCTX-Mchanging the face of ESBLs in Europerdquo Journal of AntimicrobialChemotherapy vol 59 no 2 pp 165ndash174 2007
[2] E R Bevan A M Jones and P M Hawkey ldquoGlobal epidemiol-ogy of CTX-M 120573-lactamases temporal and geographical shiftsin genotyperdquo Journal of Antimicrobial Chemotherapy vol 72 no8 pp 2145ndash2155 2017
[3] R Bonnet ldquoGrowing group of extended-spectrum 120573-lactamas-es The CTX-M Enzymesrdquo Antimicrobial Agents and Chemo-therapy vol 48 no 1 pp 1ndash14 2004
[4] M Al-Zarouni A Senok F Rashid S M Al-Jesmi and D Pan-igrahi ldquoPrevalence and antimicrobial susceptibility pattern ofextended-spectrum beta-lactamase-producing enterobacteri-aceae in the United Arab Emiratesrdquo Medical Principles andPractice vol 17 no 1 pp 32ndash36 2008
[5] M S Alfaresi A A ElkoushHMAlshehhi andA I Abdulsa-lam ldquoMolecular characterization and epidemiology of extend-ed-spectrum beta-lactamase-producing escherichia coli andklebsiella pneumoniae isolates in the United Arab EmiratesrdquoMedical Principles and Practice vol 20 no 2 pp 177ndash180 2011
[6] M Al-Zarouni A Senok N Al-Zarooni F Al-Nassay and DPanigrahi ldquoExtended-spectrum 120573-lactamase-producing enter-obacteriaceae in vitro susceptibility to fosfomycin nitrofuran-toin and tigecyclinerdquoMedical Principles and Practice vol 21 no6 pp 543ndash547 2012
[7] J Du P Li H Liu D Lu H Liang and Y Dou ldquoPhenotypic andmolecular characterization of multidrug resistant Klebsiellapneumoniae isolated from a university teaching hospitalChinardquo PLoS ONE vol 9 no 4 Article ID e95181 2014
[8] K M Bindayna H S Khanfar A C Senok and G A BottaldquoPredominance of CTX-Mgenotype among extended spectrumbeta lactamase isolates in a tertiary hospital in Saudi ArabiardquoSaudi Medical Journal vol 31 no 8 pp 859ndash863 2010
[9] KM Bindayna A C Senok andA E Jamsheer ldquoPrevalence ofextended-spectrum beta-lactamase-producing Enterobacteri-aceae in Bahrainrdquo Journal of Infection and Public Health vol 2no 3 pp 129ndash135 2009
[10] A M Somily M Z Arshad G A Garaween and A C SenokldquoPhenotypic and genotypic characterization of extended-spec-trum 120573-lactamases producing Escherichia coli and Klebsiellapneumoniae in a tertiary care hospital in Riyadh Saudi ArabiardquoAnnals of Saudi Medicine vol 35 no 6 pp 435ndash439 2015
[11] V O Rotimi W Jamal T Pal A Sovenned and M J AlbertldquoEmergence of CTX-M-15 type extended-spectrum 120573-lacta-mase-producing Salmonella spp inKuwait and theUnitedArabEmiratesrdquo Journal ofMedicalMicrobiology vol 57 no 7 pp 881ndash886 2008
[12] HM ZowawiHH Balkhy T RWalsh andD L Paterson ldquo120573-Lactamase production in key gram-negative pathogen isolatesfrom theArabian PeninsulardquoClinicalMicrobiology Reviews vol26 no 3 pp 361ndash380 2013
[13] N Ben Achour P S Mercuri P Power et al ldquoFirst detection ofCTX-M-28 in a Tunisian hospital from a cefotaxime-resistantKlebsiella pneumoniae strainrdquo Pathologie Biologie vol 57 no 5pp 343ndash348 2009
[14] A Ibrahimagic B Bedenic F Kamberovic and S UzunovicldquoHigh prevalence of CTX-M-15 and first report of CTX-M-3 CTX-M-22 CTX-M-28 and plasmid-mediated AmpCbeta-lactamase producing Enterobacteriaceae causing urinarytract infections in Bosnia and Herzegovina in hospital andcommunity settingsrdquo Journal of Infection and Chemotherapyvol 21 no 5 pp 363ndash369 2015
[15] J Kingsley and S Verghese ldquoSequence analysis of blaCTX-M-28 an ESBL responsible for third-generation cephalosporinresistance in Enterobacteriaceae for the first time in IndiardquoIndian Journal of Pathology and Microbiology vol 51 no 2 pp218ndash221 2008
[16] A C S Lopes D L Veras A M S Lima R D C A Melo andJ Ayala ldquo 119887119897119886CTX-M-2 and 119887119897119886CTX-M-28 extended-spectrum
Journal of Pathogens 5
120573-lactamase genes and class 1 integrons in clinical isolatesof Klebsiella pneumoniae from Brazilrdquo Memorias do InstitutoOswaldo Cruz vol 105 no 2 pp 163ndash167 2010
[17] J S Yoo J Byeon J Yang J I Yoo G T Chung and Y S LeeldquoHigh prevalence of extended-spectrum 120573-lactamases and plas-mid-mediated AmpC 120573-lactamases in Enterobacteriaceae iso-lated from long-term care facilities in KoreardquoDiagnostic Micro-biology and Infectious Disease vol 67 no 3 pp 261ndash265 2010
[18] Y Yu S Ji Y Chen et al ldquoResistance of strains producing ex-tended-spectrum 120573-lactamases and genotype distribution inChinardquo Infection vol 54 no 1 pp 53ndash57 2007
[19] N Almaraghi Characterisation of CTX-M-120573-lactamases in En-terobacteriaceaeae in hospitals in Kuwait DissertationPhDthesis University of Edinburgh Edinburgh UK 2013
[20] G A Menezes M A Khan and J P Hays ldquoImportant method-ological considerations with respect to differentiation of CTX-M-15 and CTX-M-28 extended-spectrum beta-lactamasesrdquoIndian Journal of Medical Microbiology vol 28 no 1 pp 81-822010
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Journal of Pathogens 5
120573-lactamase genes and class 1 integrons in clinical isolatesof Klebsiella pneumoniae from Brazilrdquo Memorias do InstitutoOswaldo Cruz vol 105 no 2 pp 163ndash167 2010
[17] J S Yoo J Byeon J Yang J I Yoo G T Chung and Y S LeeldquoHigh prevalence of extended-spectrum 120573-lactamases and plas-mid-mediated AmpC 120573-lactamases in Enterobacteriaceae iso-lated from long-term care facilities in KoreardquoDiagnostic Micro-biology and Infectious Disease vol 67 no 3 pp 261ndash265 2010
[18] Y Yu S Ji Y Chen et al ldquoResistance of strains producing ex-tended-spectrum 120573-lactamases and genotype distribution inChinardquo Infection vol 54 no 1 pp 53ndash57 2007
[19] N Almaraghi Characterisation of CTX-M-120573-lactamases in En-terobacteriaceaeae in hospitals in Kuwait DissertationPhDthesis University of Edinburgh Edinburgh UK 2013
[20] G A Menezes M A Khan and J P Hays ldquoImportant method-ological considerations with respect to differentiation of CTX-M-15 and CTX-M-28 extended-spectrum beta-lactamasesrdquoIndian Journal of Medical Microbiology vol 28 no 1 pp 81-822010
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom
Stem Cells International
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
MEDIATORSINFLAMMATION
of
EndocrinologyInternational Journal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Disease Markers
Hindawiwwwhindawicom Volume 2018
BioMed Research International
OncologyJournal of
Hindawiwwwhindawicom Volume 2013
Hindawiwwwhindawicom Volume 2018
Oxidative Medicine and Cellular Longevity
Hindawiwwwhindawicom Volume 2018
PPAR Research
Hindawi Publishing Corporation httpwwwhindawicom Volume 2013Hindawiwwwhindawicom
The Scientific World Journal
Volume 2018
Immunology ResearchHindawiwwwhindawicom Volume 2018
Journal of
ObesityJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Computational and Mathematical Methods in Medicine
Hindawiwwwhindawicom Volume 2018
Behavioural Neurology
OphthalmologyJournal of
Hindawiwwwhindawicom Volume 2018
Diabetes ResearchJournal of
Hindawiwwwhindawicom Volume 2018
Hindawiwwwhindawicom Volume 2018
Research and TreatmentAIDS
Hindawiwwwhindawicom Volume 2018
Gastroenterology Research and Practice
Hindawiwwwhindawicom Volume 2018
Parkinsonrsquos Disease
Evidence-Based Complementary andAlternative Medicine
Volume 2018Hindawiwwwhindawicom
Submit your manuscripts atwwwhindawicom