Research ArticleComparative Analysis of Quinolone Resistance inClinical Isolates of Klebsiella pneumoniae and Escherichia colifrom Chinese Children and Adults
Ying Huang12 James O Ogutu1 Jiarui Gu1 Fengshu Ding3 Yuhong You4
Yan Huo1 Hong Zhao1 Wenjing Li1 Zhiwei Zhang1 Wenli Zhang1 Xiaobei Chen2
Yingmei Fu12 and Fengmin Zhang12
1Department of Microbiology Pathogenic Biology Harbin Medical University Harbin Heilongjiang 150081 China2Heilongjiang Provincial Key Laboratory of Immunity and Infection Harbin Heilongjiang 150081 China3Harbin Childrenrsquos Hospital Harbin Heilongjiang 150081 China4Department of Laboratory Diagnosis Heilongjiang Provincial Hospital Harbin Heilongjiang 150081 China
Correspondence should be addressed to Yingmei Fu yingmeifufoxmailcomand Fengmin Zhang fengminzhangemshrbmueducn
Received 7 October 2014 Revised 19 December 2014 Accepted 27 January 2015
Academic Editor Teresa M Coque
Copyright copy 2015 Ying Huang et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
The objective of this study was to compare quinolone resistance and gyrA mutations in clinical isolates of Klebsiella pneumoniaeand Escherichia coli from Chinese adults who used quinolone in the preceding month and children without any known historyof quinolone administration The antimicrobial susceptibilities of 61 isolates from children and 79 isolates from adults weredetermined The mutations in the quinolone resistance-determining regions in gyrA gene were detected by PCR and DNAsequencing Fluoroquinolone resistance and types of gyrA mutations in isolates from children and adults were compared andstatistically analyzed No significant differences were detected in the resistance rates of ciprofloxacin and levofloxacin betweenchildren and adults among isolates of the two species (all 119875 gt 005) The double mutation Ser83rarr Leu + Asp87rarrAsn in theciprofloxacin-resistant isolates occurred in 737 isolates from the children and 679 from the adults respectively (119875 = 05444)Children with no known history of quinolone administration were found to carry fluoroquinolone-resistant Enterobacteriaceaeisolates The occurrence of ciprofloxacin resistance and the major types of gyrA mutations in the isolates from the childrenwere similar to those from adults The results indicate that precautions should be taken on environmental issues resulting fromwidespread transmission of quinolone resistance
1 Introduction
It is widely accepted that overuse of antibiotics in hospitalsand communities is one of the major contributing factorsto bacterial resistance [1] However it is worth noting thatdecreasing antibiotic use does not necessarily directly corre-spond to complete eradication of antimicrobial resistance inbacterial populations [2 3] It has been reported that drugresistance and its determinants could persist in the bacteriumeven in the absence of the corresponding antibiotics [1]Furthermore it has recently been shown that quinoloneresistance also develops in the absence of antibiotic selective
pressure [4 5]Therefore wemay postulate that the existenceof antimicrobial-resistant isolates from clinical cases may notnecessarily result from antimicrobial use
Fluoroquinolones have been widely used in the treatmentof a variety of infections due to their broader antimi-crobial spectra and improved pharmacokinetic propertiesHowever the problem of quinolone resistance is becomingincreasingly serious with the extensive use of the agents[6 7] Klebsiella pneumoniae and Escherichia coli are thetwo most common quinolone-resistant EnterobacteriaceaeResistance to quinolones in enterobacteria is mainly due tomutations in the quinolone target enzymes (DNA gyrase and
Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 168292 6 pageshttpdxdoiorg1011552015168292
2 BioMed Research International
topoisomerase IV) encoded by gyrA and parC [8 9] Besidesplasmid-mediated quinolone resistance genes also confer lowlevels of quinolone resistance [10]
Due to their adverse effects on childrenrsquos cartilage theprescription of quinolones has been restricted from pediatriccases [11] Thus children can be considered as a populationlacking exposure to quinolones With the purpose of inves-tigating drug resistance among people with and without thehistory of fluoroquinolone administration within the samearea we collected the clinical isolates of K pneumoniae andE coli from both preschool children who have no knownhistory of quinolone administration and adults who usedquinolone in the preceding month of the sample collectionin Harbin Heilongjiang China Fluoroquinolone resistanceand types of gyrA mutation in the clinical isolates weredetected and compared between the two groups
2 Materials and Methods
21 Bacterial Collection During the period of March toDecember 2009 77 K pneumoniae and 63 E coli non-repetitive isolates were consecutively collected from threetertiary hospitals in Harbin city the provincial headquartersof Heilongjiang province Northeast China Seventy-nineisolates came from adult patients while sixty-one isolatesfrom children comprising 21 infants (lt1 year) 20 toddlers(1ndash3 years) and 20 preschoolers (3ndash6 years) The strains wereisolated from stool nasopharyngeal swabs and sputum sam-ples of in-patients in surgical wards internal medicine wardsintensive care units and pediatric wards A questionnairesurveyed information about quinolone usage in childrenand adults The clinical strains were obtained only fromchildren without the history of quinolone administrationand adults who had taken quinolones in the four weekspreceding specimen collection All the specimen providers orthe childrenrsquos guardians agreed to participate in the researchprojects and signed the informed consent The InstituteResearch Board of Harbin Medical University checked andapproved the study (Report number HMUIRB20140009)
Identification of the bacteria to species level was donethrough the API 20E system (BioMerieux Marcy-IrsquoEtoileFrance) All isolates were confirmed as nonreplicate strains byRandomly Amplified Polymorphism DNA (RAPD) analysis(data not shown)
22 Antimicrobial Susceptibility Test Minimum InhibitoryConcentrations (MICs) of ciprofloxacin were determinedby the microdilution method according to Clinical andLaboratory Standards Institute (CLSI) guidelines [12] Thesusceptibility to other antimicrobial agents was determinedusing Kirby-Bauer disk diffusion test with antimicrobialagent discs (OXOID UK) following the CLSI guidelines [13]E coli ATCC 25922 and K pneumoniae ATCC 10031 wereused as means of quality control
Isolates with reduced susceptibility to third generationcephalosporins were subjected to phenotypic detection ofESBL production by using cefotaxime (CTX) and ceftazidime(CAZ) discs and double-disk synergy (DDS) method as
recommended by the Clinical and Laboratory StandardsInstitute [14] AmpC production was detected using cefoxitinalone and in combination with boronic acid and confirma-tion was done by three-dimensional disk method Isolateswith reduced susceptibility to cefoxitin were subjected to amodified three-dimensional extract test to confirm AmpCproduction [15]
23 Amplification and Sequencing of the gyrAGene FragmentsAll isolates of K pneumoniae and E coli were used to deter-mine the DNA sequence of QRDR of gyrA Oligonucleotideprimers used for amplification of fragments encompassingthe QRDR were gyrA-F (51015840-TGCGAGAGAAATTACACC-31015840) and gyrA-R (51015840-AATATGTTCCATCAGCCC-31015840) ThegyrA gene fragments were amplified from crude cell lysatesPCR products were purified and then subjected to bidirec-tional sequencing using an automated DNA sequencer (ABIPRISM 373 Applied Biosystems Foster City CA) with thesame primers used in the PCR amplification The nucleotidesequences and the deduced amino acids were compared tothat of K pneumoniae ATCC 13883 (GenBank DQ673325)and E coli K-12 (GenBank NC 0104731) using the onlineClustalW2 multiple sequence alignment program All theexperiments were performed in accordance with relevantguidelines and regulations The partial sequences of thevariant gyrA genes in the clinical isolates were submittedto GenBank under accession numbers EU430280 throughEU430289 and JQ694717 through JQ694722
24 Statistical Methods Resistance rates among the isolateswere analyzed with paired chi-square test Comparisons offluoroquinolone resistance and individual GyrA alterationsbetween adults and children of all age groups were analyzedby Fisherrsquos exact test or Pearson chi-square test (IBM SPSS160 statistical package) 119875 lt 005 was considered as statisti-cally significant
3 Results
31 Antimicrobial Resistance in K pneumoniae and E coliIsolates A comparison of antimicrobial agent susceptibilitybetween isolates from children and adults in K pneumoniaeand E coli was shown in Table 1 Among isolates of the twospecies there were no significant differences in the resistancerates of ciprofloxacin and levofloxacin between children andadults (all 119875 gt 005) In K pneumoniae the MIC
50 MIC
90
andMIC range for ciprofloxacin in isolates from children andadults were 8mgL versus 2mgL 256mgL versus 256mgLand 05ndash256mgL versus 05ndash512mgL respectively In Ecoli isolates from children and adults the ciprofloxacinMIC50
was 64mgL versus 64mgL MIC90
was 128mgLversus 256mgL and MIC range was 05ndash512mgL versus1ndash512mgL The prevalence of ciprofloxacin resistance inchildren varied with the age Resistance to ciprofloxacin wasfound in 375 of the isolates from infants aged under 6months which was lower than the percentage observed in theother age groups (6923 of 6-month to 1-year infants 700
BioMed Research International 3
Table 1 Resistance of 140 K pneumoniae and E coli isolates to antimicrobial agents in children and adults
of 1- to 3-year toddlers and 600 of 3- to 6-year preschoolchildren)
As shown in Table 1 resistance rates to other classes ofantimicrobial agents in E coli isolated from children andadults were quite similar (all 119875 gt 005) However in Kpneumoniae the resistance rates for cefotaxime ceftriaxoneand ceftazidime in children were statistically higher thanthose in adults (all 119875 lt 005) Occurrences of the productionof extended-spectrum beta-lactamases (ESBLs) isolates fromchildren and adults were 579 and 385 respectively Incontrast cefoxitin resistance rate was significantly lower inthe isolates from children (119875 = 00085) Of note theoccurrence of AmpC in children was 79 which is similarto that in adults (154 the Fisher exact test 119875 = 04806)Theamikacin resistance rate of K pneumoniae in children is alsohigher than that of adults (119875 lt 005) Among the resistantisolates from adults 9 (231) were found an absence of
inhibition zone around the amikacin disc (diameter lt6mm)while 25 (658) of the isolates from children were observedto have this phenomenon (1205942 = 142398119875 = 00002 Table 1)
32 Resistance to Ciprofloxacin and gyrAMutations in Isolatesfrom Children and Adults All the 140 isolates were inves-tigated for QRDR mutations of gyrA 46 of the 61 isolates(754) from children were found to contain gyrAmutationsA similar prevalence pattern of the gyrA mutation was alsoobserved in the isolates of adult origin (6479 810 119875 =043) Among the 38 isolates resistant to ciprofloxacin fromchildren 37 (974 3738) were found to contain the gyrAmutation which is quite similar to the prevalence in isolatesfrom adults (5556 982 119875 = 100)
As shown in Table 2 the double mutation Ser83rarr Leu +Asp87rarrAsn was the most prevalent type of GyrA changesin antimicrobial-resistant isolates from both children and
4 BioMed Research International
adults (737 versus 679 119875 = 05444) The single mutationSer83rarr Ile was not detected in the isolates from childrenbut was detected in 196 of the isolates from adults (119875 =00026) The single mutation Ser83rarr Leu was detected in184 of the isolates from children but was not found inthose from adults (119875 = 00012 Table 2) GryA mutationwas also found in 18 isolates nonresistant to ciprofloxacinincluding 9 isolates from children and 9 isolates from adultsThe mutation patterns were listed in Table 2
33 GyrA Mutations in Children with Different Age GroupsThe 61 isolates from children were assigned into 4 groupsaccording to physiological development and participation insocial activities namely young infants (lt6 months) infants(6ndash12 months) toddlers (1ndash3 years) and preschoolers (age of3ndash6 years) It was found that the mutations Ser83rarr Leu +Asp87rarrAsn Ser83rarr Leu Ser83rarrThr and Asp87rarrAsnwere the common types of mutations in GyrA and Ser83rarrLeu + Asp87rarrAsn was the most prevalent double mutationIn addition there was no significant difference in terms ofindividual types of mutation in GyrA in children amongdifferent age groups (119875 gt 005)
4 Discussion
There is a growing concern over antibiotics resistance as aresult of antibiotics overuse in hospital and communitiesThus restriction on the use of antibiotics has become oneof the most important measures for control emergenceand spread of drug-resistant bacteria This study involvesclinical isolates obtained from children in the absence offluoroquinolone usage history as quinolones are not rec-ommended for pediatric use due to the adverse effects onjoints However our findings showed that children har-bored quinolone-resistant isolates being similarly resistantto ciprofloxacin and levofloxacin compared with the isolatesfrom adults Moreover isolates from children were found tohave similarly high ciprofloxacin MIC levels with those fromadults By sequencing the QRDRs of gyrA we found that thedouble mutation Ser83rarr Leu + Asp87rarrAsn was similarlypredominant among the quinolone-resistant isolates fromboth children and adults
A previous study conducted in Spain found 5 resistancerate of ciprofloxacin in E coli isolates from healthy children[16] Other studies have also recorded quinolone-resistantEnterobacteriaceae from children in recent years [17 18]GyrA mutations involved in quinolone resistance in isolatesfrom infants were most recently discovered in Spain [19]The resistance rate to ciprofloxacin in children was up to22 in these regions [17] Compared to these regions theciprofloxacin resistance rate and the resistant level (MIC)found in the present study are notably higher In spiteof the difference in resistance rate and resistant level thefindings in the present study and others demonstrated thequinolone resistance and the involved mutation mechanismsin nonexposed population under high antibiotic pressureIt indicates that drug resistance may be transmitted andpersist in people who are not exposed to the correspondingantibiotics
Various reports on the different aspects of resistance haverevealed that persistence of resistance in human or animalpopulations relies more on the transmission of resistance inthe public environment than on drug pressure [5 20] Drug-resistant isolates of bacteria were detected even in remotemountainous areas of Amazon where antibiotics have neverbeen used [4] Presence of drug-resistant isolates in childrenmight be attributed to their increased contact with adultsand playmates in families daycare or school settings [2122] Among the environmental factors food supply chainmay be the major source of antibiotic resistance in ldquonaıverdquohumans as an indirect consequence of overuse of antibioticsfor curative and preventative purpose in stock farming andagricultural products In 2000 the use of antibiotics inraising animals was questioned [23] Shortly after antibiotic-resistant E coli was detected in poultry products [24] Thesituation of antibiotic-resistant bacteria from food chainsources has become a notable societal issue which shouldbe worthy of attention [25ndash29] Furthermore pollution byantibiotic resistance genes from stock farming and antibioticsin manure and metabolites could increase the chances ofresistance acquisition by human pathogens [30]
In summary our data showed similar prevalence of quin-olone resistance and type of gyrA genemutation in E coli andK pneumoniae among children and adults The conclusionthat there exists similarity in fluoroquinolone resistance ratesbetween children and adults in a region under high commu-nity antibiotic pressure constrains us to pay close attentionto the environmental and community reservoirs of resistanceand take precautions to prevent perpetuating antibiotic-resistant isolates and their transmission and dissemination
Conflict of Interests
All authors declare that they have no conflict of interests
Authorsrsquo Contribution
Ying Huang Yingmei Fu and Fengmin Zhang participatedin the design conduction analysis and interpretation of thestudy Ying Huang James O Ogutu and Jiarui Gu wereinvolved in all phases of the experiment Fengshu DingYuhong You and YanHuo were involved in themanipulationof clinical isolates Hong Zhao andWenjing Li were involvedin the DNA sequencing Zhiwei Zhang Wenli Zhang andXiaobei Chen conducted the statistical analysis Ying Huangwrote the paper Yingmei Fu and Fengmin Zhang reviewedthe initial and final drafts of the paper All authors read andapproved the final paper
Acknowledgments
This work was supported by grants from National NaturalScience Foundation of China (NSFC) (81101300 31370164J1103609 and J1210062) Heilongjiang Educational Agency(1155G34) Heilongjiang Provincial Science and TechnologyInnovation Team inHigher Education Institutes for Infectionand Immunity and Harbin Medical University
BioMed Research International 5
References
[1] P J Johnsen J P Townsend T Boslashhn G S Simonsen ASundsfjord and K M Nielsen ldquoFactors affecting the reversal ofantimicrobial-drug resistancerdquo The Lancet Infectious Diseasesvol 9 no 6 pp 357ndash364 2009
[2] T W Hennessy K M Petersen D Bruden et al ldquoChangesin antibiotic-prescribing practices and carriage of penicillin-resistant Streptococcus pneumoniae a controlled interventiontrial in rural Alaskardquo Clinical Infectious Diseases vol 34 no 12pp 1543ndash1550 2002
[3] G Barkai D Greenberg N Givon-Lavi E Dreifuss D Vardyand R Dagan ldquoCommunity prescribing and resistant Strepto-coccus pneumoniaerdquo Emerging Infectious Diseases vol 11 no 6pp 829ndash837 2005
[4] L Pallecchi A Bartoloni E Riccobono et al ldquoQuinoloneresistance in absence of selective pressure the experience of avery remote community in the Amazon forestrdquo PLoS NeglectedTropical Diseases vol 6 no 8 Article ID e1790 2012
[5] M P Quintana-Hayashi and S Thakur ldquoPhylogenetic analysisreveals common antimicrobial resistant Campylobacter colipopulation in antimicrobial-free (ABF) and commercial swinesystemsrdquo PLoS ONE vol 7 no 9 Article ID e44662 2012
[6] LMartınez-Martınez A Pascual andG A Jacoby ldquoQuinoloneresistance from a transferable plasmidrdquoThe Lancet vol 351 no9105 pp 797ndash799 1998
[7] M Montes E Tamayo B Orden J Larruskain and E Perez-Trallero ldquoPrevalence and clonal characterization of Streptococ-cus pyogenes clinical isolates with reduced fluoroquinolone sus-ceptibility in Spainrdquo Antimicrobial Agents and Chemotherapyvol 54 no 1 pp 93ndash97 2010
[8] K L Hopkins R H Davies and E J Threlfall ldquoMechanisms ofquinolone resistance in Escherichia coli and Salmonella recentdevelopmentsrdquo International Journal of Antimicrobial Agentsvol 25 no 5 pp 358ndash373 2005
[9] L MWeigel C D Steward and F C Tenover ldquogyrAmutationsassociated with fluoroquinolone resistance in eight species ofEnterobacteriaceaerdquo Antimicrobial Agents and Chemotherapyvol 42 no 10 pp 2661ndash2667 1998
[10] A Robicsek G A Jacoby and D C Hooper ldquoThe worldwideemergence of plasmid-mediated quinolone resistancerdquo LancetInfectious Diseases vol 6 no 10 pp 629ndash640 2006
[11] Committee on Infectious Diseases ldquoThe use of systemic fluoro-quinolonesrdquo Pediatrics vol 118 no 3 pp 1287ndash1292 2006
[12] Clinical and Laboratory Standards Institute Methods for Dilu-tion Antimicrobial Susceptibility Tests for Bacteria That GrowAerobically (M07-A8) CLSI Wayne Pa USA 8th edition2009
[13] M Pfaller L Boyken R Hollis et al ldquoComparison of the brothmicrodilutionmethods of the EuropeanCommittee onAntimi-crobial Susceptibility Testing and the Clinical and LaboratoryStandards Institute for testing itraconazole posaconazole andvoriconazole against Aspergillus isolatesrdquo Journal of ClinicalMicrobiology vol 49 no 3 pp 1110ndash1112 2011
[14] Clinical and Laboratory Standards Institute ldquoPerformancestandards for antimicrobial susceptibility testing Twentiethinformational supplementrdquo M100-S20 Wayne Pa USA CLSI2010
[15] Clinical and Laboratory Standards Institute ldquoPerformancestandards for antimicrobial susceptibility testing Eighteenthinformational supplementrdquo 2008
[16] E Domınguez M Zarazaga Y Saenz L Brinas and C TorresldquoMechanisms of antibiotic resistance in Escherichia coli isolatesobtained from healthy children in Spainrdquo Microbial DrugResistance vol 8 no 4 pp 321ndash327 2002
[17] J Ayatollahi H Shahcheraghi R Akhondi and S S SolutildquoAntibiotic resistance patterns of Escherichia coli isolated fromchildren in shahid sadoughi hospital of Yazdrdquo Iranian Journalof Pediatric Hematology and Oncology vol 3 no 2 pp 78ndash822013
[18] AGarraffoCMarguet AChecoury et al ldquoUrinary tract infec-tions in hospital pediatrics many previous antibiotherapy andantibiotics resistance including fluoroquinolonesrdquoMedecine etMaladies Infectieuses vol 44 no 2 pp 63ndash68 2014
[19] M J Pons S Mosquito C Gomesa L J del Valle T J Ochoaand J Ruiz ldquoAnalysis of quinolone-resistance in commensaland diarrheagenic Escherichia coli isolates from infants in limaPerurdquo Transactions of the Royal Society of Tropical Medicine andHygiene vol 108 no 1 pp 22ndash28 2014
[20] M P Quintana-Hayashi and S Thakur ldquoLongitudinal study ofthe persistence of antimicrobial-resistantCampylobacter strainsin distinct swine production systems on farms at slaughter andin the environmentrdquo Applied and Environmental Microbiologyvol 78 no 8 pp 2698ndash2705 2012
[21] J Isendahl A Turlej-Rogacka C Manjuba A Rodrigues C GGiske and P Naucler ldquoFecal carriage of ESBL-producing E coliand K pneumoniae in children in Guinea-Bissau a hospital-based cross-sectional studyrdquo PLoS ONE vol 7 no 12 ArticleID e51981 2012
[22] R Pires C Ardanuy D Rolo et al ldquoEmergence of cipro-floxacin-nonsusceptible Streptococcus pyogenes isolates fromhealthy children and pediatric patients in Portugalrdquo Antimicro-bial Agents and Chemotherapy vol 54 no 6 pp 2677ndash26802010
[23] R J Bywater and M W Casewell ldquoAn assessment of theimpact of antibiotic resistance in different bacterial species andof the contribution of animal sources to resistance in humaninfectionsrdquo Journal of Antimicrobial Chemotherapy vol 46 no6 p 1052 2000
[24] J R Johnson M R Sannes C Croy et al ldquoAntimicrobial drug-resistant Escherichia coli from humans and poultry productsMinnesota and Wisconsin 2002ndash2004rdquo Emerging InfectiousDiseases vol 13 no 6 pp 838ndash846 2007
[25] M Bagheri R Ghanbarpour and H Alizade ldquoShiga toxin andbeta-lactamases genes in Escherichia coli phylotypes isolatedfrom carcasses of broiler chickens slaughtered in Iranrdquo Interna-tional Journal of Food Microbiology vol 177 pp 16ndash20 2014
[26] M V C Cossi R C K Burin D A Lopes et al ldquoAntimicrobialresistance and virulence profiles of Salmonella isolated frombutcher shops in minas gerais Brazilrdquo Journal of Food Protec-tion vol 76 no 9 pp 1633ndash1637 2013
[27] F Reich V Atanassova and G Klein ldquoExtended-spectrumbeta-lactamase- and ampc-producing enterobacteria in healthybroiler chickens Germanyrdquo Emerging Infectious Diseases vol19 no 8 pp 1253ndash1259 2013
[28] M Mellata ldquoHuman and avian extraintestinal pathogenicEscherichia coli infections zoonotic risks and antibiotic resis-tance trendsrdquo Foodborne Pathogens and Disease vol 10 no 11pp 916ndash932 2013
6 BioMed Research International
[29] K Holvoet I Sampers B Callens J Dewulf and M Uyt-tendaele ldquoModerate prevalence of antimicrobial resistance inEscherichia coli isolates from lettuce irrigation water and soilrdquoApplied and Environmental Microbiology vol 79 no 21 pp6677ndash6683 2013
[30] J L Martinez ldquoEnvironmental pollution by antibiotics andby antibiotic resistance determinantsrdquo Environmental Pollutionvol 157 no 11 pp 2893ndash2902 2009
topoisomerase IV) encoded by gyrA and parC [8 9] Besidesplasmid-mediated quinolone resistance genes also confer lowlevels of quinolone resistance [10]
Due to their adverse effects on childrenrsquos cartilage theprescription of quinolones has been restricted from pediatriccases [11] Thus children can be considered as a populationlacking exposure to quinolones With the purpose of inves-tigating drug resistance among people with and without thehistory of fluoroquinolone administration within the samearea we collected the clinical isolates of K pneumoniae andE coli from both preschool children who have no knownhistory of quinolone administration and adults who usedquinolone in the preceding month of the sample collectionin Harbin Heilongjiang China Fluoroquinolone resistanceand types of gyrA mutation in the clinical isolates weredetected and compared between the two groups
2 Materials and Methods
21 Bacterial Collection During the period of March toDecember 2009 77 K pneumoniae and 63 E coli non-repetitive isolates were consecutively collected from threetertiary hospitals in Harbin city the provincial headquartersof Heilongjiang province Northeast China Seventy-nineisolates came from adult patients while sixty-one isolatesfrom children comprising 21 infants (lt1 year) 20 toddlers(1ndash3 years) and 20 preschoolers (3ndash6 years) The strains wereisolated from stool nasopharyngeal swabs and sputum sam-ples of in-patients in surgical wards internal medicine wardsintensive care units and pediatric wards A questionnairesurveyed information about quinolone usage in childrenand adults The clinical strains were obtained only fromchildren without the history of quinolone administrationand adults who had taken quinolones in the four weekspreceding specimen collection All the specimen providers orthe childrenrsquos guardians agreed to participate in the researchprojects and signed the informed consent The InstituteResearch Board of Harbin Medical University checked andapproved the study (Report number HMUIRB20140009)
Identification of the bacteria to species level was donethrough the API 20E system (BioMerieux Marcy-IrsquoEtoileFrance) All isolates were confirmed as nonreplicate strains byRandomly Amplified Polymorphism DNA (RAPD) analysis(data not shown)
22 Antimicrobial Susceptibility Test Minimum InhibitoryConcentrations (MICs) of ciprofloxacin were determinedby the microdilution method according to Clinical andLaboratory Standards Institute (CLSI) guidelines [12] Thesusceptibility to other antimicrobial agents was determinedusing Kirby-Bauer disk diffusion test with antimicrobialagent discs (OXOID UK) following the CLSI guidelines [13]E coli ATCC 25922 and K pneumoniae ATCC 10031 wereused as means of quality control
Isolates with reduced susceptibility to third generationcephalosporins were subjected to phenotypic detection ofESBL production by using cefotaxime (CTX) and ceftazidime(CAZ) discs and double-disk synergy (DDS) method as
recommended by the Clinical and Laboratory StandardsInstitute [14] AmpC production was detected using cefoxitinalone and in combination with boronic acid and confirma-tion was done by three-dimensional disk method Isolateswith reduced susceptibility to cefoxitin were subjected to amodified three-dimensional extract test to confirm AmpCproduction [15]
23 Amplification and Sequencing of the gyrAGene FragmentsAll isolates of K pneumoniae and E coli were used to deter-mine the DNA sequence of QRDR of gyrA Oligonucleotideprimers used for amplification of fragments encompassingthe QRDR were gyrA-F (51015840-TGCGAGAGAAATTACACC-31015840) and gyrA-R (51015840-AATATGTTCCATCAGCCC-31015840) ThegyrA gene fragments were amplified from crude cell lysatesPCR products were purified and then subjected to bidirec-tional sequencing using an automated DNA sequencer (ABIPRISM 373 Applied Biosystems Foster City CA) with thesame primers used in the PCR amplification The nucleotidesequences and the deduced amino acids were compared tothat of K pneumoniae ATCC 13883 (GenBank DQ673325)and E coli K-12 (GenBank NC 0104731) using the onlineClustalW2 multiple sequence alignment program All theexperiments were performed in accordance with relevantguidelines and regulations The partial sequences of thevariant gyrA genes in the clinical isolates were submittedto GenBank under accession numbers EU430280 throughEU430289 and JQ694717 through JQ694722
24 Statistical Methods Resistance rates among the isolateswere analyzed with paired chi-square test Comparisons offluoroquinolone resistance and individual GyrA alterationsbetween adults and children of all age groups were analyzedby Fisherrsquos exact test or Pearson chi-square test (IBM SPSS160 statistical package) 119875 lt 005 was considered as statisti-cally significant
3 Results
31 Antimicrobial Resistance in K pneumoniae and E coliIsolates A comparison of antimicrobial agent susceptibilitybetween isolates from children and adults in K pneumoniaeand E coli was shown in Table 1 Among isolates of the twospecies there were no significant differences in the resistancerates of ciprofloxacin and levofloxacin between children andadults (all 119875 gt 005) In K pneumoniae the MIC
50 MIC
90
andMIC range for ciprofloxacin in isolates from children andadults were 8mgL versus 2mgL 256mgL versus 256mgLand 05ndash256mgL versus 05ndash512mgL respectively In Ecoli isolates from children and adults the ciprofloxacinMIC50
was 64mgL versus 64mgL MIC90
was 128mgLversus 256mgL and MIC range was 05ndash512mgL versus1ndash512mgL The prevalence of ciprofloxacin resistance inchildren varied with the age Resistance to ciprofloxacin wasfound in 375 of the isolates from infants aged under 6months which was lower than the percentage observed in theother age groups (6923 of 6-month to 1-year infants 700
BioMed Research International 3
Table 1 Resistance of 140 K pneumoniae and E coli isolates to antimicrobial agents in children and adults
of 1- to 3-year toddlers and 600 of 3- to 6-year preschoolchildren)
As shown in Table 1 resistance rates to other classes ofantimicrobial agents in E coli isolated from children andadults were quite similar (all 119875 gt 005) However in Kpneumoniae the resistance rates for cefotaxime ceftriaxoneand ceftazidime in children were statistically higher thanthose in adults (all 119875 lt 005) Occurrences of the productionof extended-spectrum beta-lactamases (ESBLs) isolates fromchildren and adults were 579 and 385 respectively Incontrast cefoxitin resistance rate was significantly lower inthe isolates from children (119875 = 00085) Of note theoccurrence of AmpC in children was 79 which is similarto that in adults (154 the Fisher exact test 119875 = 04806)Theamikacin resistance rate of K pneumoniae in children is alsohigher than that of adults (119875 lt 005) Among the resistantisolates from adults 9 (231) were found an absence of
inhibition zone around the amikacin disc (diameter lt6mm)while 25 (658) of the isolates from children were observedto have this phenomenon (1205942 = 142398119875 = 00002 Table 1)
32 Resistance to Ciprofloxacin and gyrAMutations in Isolatesfrom Children and Adults All the 140 isolates were inves-tigated for QRDR mutations of gyrA 46 of the 61 isolates(754) from children were found to contain gyrAmutationsA similar prevalence pattern of the gyrA mutation was alsoobserved in the isolates of adult origin (6479 810 119875 =043) Among the 38 isolates resistant to ciprofloxacin fromchildren 37 (974 3738) were found to contain the gyrAmutation which is quite similar to the prevalence in isolatesfrom adults (5556 982 119875 = 100)
As shown in Table 2 the double mutation Ser83rarr Leu +Asp87rarrAsn was the most prevalent type of GyrA changesin antimicrobial-resistant isolates from both children and
4 BioMed Research International
adults (737 versus 679 119875 = 05444) The single mutationSer83rarr Ile was not detected in the isolates from childrenbut was detected in 196 of the isolates from adults (119875 =00026) The single mutation Ser83rarr Leu was detected in184 of the isolates from children but was not found inthose from adults (119875 = 00012 Table 2) GryA mutationwas also found in 18 isolates nonresistant to ciprofloxacinincluding 9 isolates from children and 9 isolates from adultsThe mutation patterns were listed in Table 2
33 GyrA Mutations in Children with Different Age GroupsThe 61 isolates from children were assigned into 4 groupsaccording to physiological development and participation insocial activities namely young infants (lt6 months) infants(6ndash12 months) toddlers (1ndash3 years) and preschoolers (age of3ndash6 years) It was found that the mutations Ser83rarr Leu +Asp87rarrAsn Ser83rarr Leu Ser83rarrThr and Asp87rarrAsnwere the common types of mutations in GyrA and Ser83rarrLeu + Asp87rarrAsn was the most prevalent double mutationIn addition there was no significant difference in terms ofindividual types of mutation in GyrA in children amongdifferent age groups (119875 gt 005)
4 Discussion
There is a growing concern over antibiotics resistance as aresult of antibiotics overuse in hospital and communitiesThus restriction on the use of antibiotics has become oneof the most important measures for control emergenceand spread of drug-resistant bacteria This study involvesclinical isolates obtained from children in the absence offluoroquinolone usage history as quinolones are not rec-ommended for pediatric use due to the adverse effects onjoints However our findings showed that children har-bored quinolone-resistant isolates being similarly resistantto ciprofloxacin and levofloxacin compared with the isolatesfrom adults Moreover isolates from children were found tohave similarly high ciprofloxacin MIC levels with those fromadults By sequencing the QRDRs of gyrA we found that thedouble mutation Ser83rarr Leu + Asp87rarrAsn was similarlypredominant among the quinolone-resistant isolates fromboth children and adults
A previous study conducted in Spain found 5 resistancerate of ciprofloxacin in E coli isolates from healthy children[16] Other studies have also recorded quinolone-resistantEnterobacteriaceae from children in recent years [17 18]GyrA mutations involved in quinolone resistance in isolatesfrom infants were most recently discovered in Spain [19]The resistance rate to ciprofloxacin in children was up to22 in these regions [17] Compared to these regions theciprofloxacin resistance rate and the resistant level (MIC)found in the present study are notably higher In spiteof the difference in resistance rate and resistant level thefindings in the present study and others demonstrated thequinolone resistance and the involved mutation mechanismsin nonexposed population under high antibiotic pressureIt indicates that drug resistance may be transmitted andpersist in people who are not exposed to the correspondingantibiotics
Various reports on the different aspects of resistance haverevealed that persistence of resistance in human or animalpopulations relies more on the transmission of resistance inthe public environment than on drug pressure [5 20] Drug-resistant isolates of bacteria were detected even in remotemountainous areas of Amazon where antibiotics have neverbeen used [4] Presence of drug-resistant isolates in childrenmight be attributed to their increased contact with adultsand playmates in families daycare or school settings [2122] Among the environmental factors food supply chainmay be the major source of antibiotic resistance in ldquonaıverdquohumans as an indirect consequence of overuse of antibioticsfor curative and preventative purpose in stock farming andagricultural products In 2000 the use of antibiotics inraising animals was questioned [23] Shortly after antibiotic-resistant E coli was detected in poultry products [24] Thesituation of antibiotic-resistant bacteria from food chainsources has become a notable societal issue which shouldbe worthy of attention [25ndash29] Furthermore pollution byantibiotic resistance genes from stock farming and antibioticsin manure and metabolites could increase the chances ofresistance acquisition by human pathogens [30]
In summary our data showed similar prevalence of quin-olone resistance and type of gyrA genemutation in E coli andK pneumoniae among children and adults The conclusionthat there exists similarity in fluoroquinolone resistance ratesbetween children and adults in a region under high commu-nity antibiotic pressure constrains us to pay close attentionto the environmental and community reservoirs of resistanceand take precautions to prevent perpetuating antibiotic-resistant isolates and their transmission and dissemination
Conflict of Interests
All authors declare that they have no conflict of interests
Authorsrsquo Contribution
Ying Huang Yingmei Fu and Fengmin Zhang participatedin the design conduction analysis and interpretation of thestudy Ying Huang James O Ogutu and Jiarui Gu wereinvolved in all phases of the experiment Fengshu DingYuhong You and YanHuo were involved in themanipulationof clinical isolates Hong Zhao andWenjing Li were involvedin the DNA sequencing Zhiwei Zhang Wenli Zhang andXiaobei Chen conducted the statistical analysis Ying Huangwrote the paper Yingmei Fu and Fengmin Zhang reviewedthe initial and final drafts of the paper All authors read andapproved the final paper
Acknowledgments
This work was supported by grants from National NaturalScience Foundation of China (NSFC) (81101300 31370164J1103609 and J1210062) Heilongjiang Educational Agency(1155G34) Heilongjiang Provincial Science and TechnologyInnovation Team inHigher Education Institutes for Infectionand Immunity and Harbin Medical University
BioMed Research International 5
References
[1] P J Johnsen J P Townsend T Boslashhn G S Simonsen ASundsfjord and K M Nielsen ldquoFactors affecting the reversal ofantimicrobial-drug resistancerdquo The Lancet Infectious Diseasesvol 9 no 6 pp 357ndash364 2009
[2] T W Hennessy K M Petersen D Bruden et al ldquoChangesin antibiotic-prescribing practices and carriage of penicillin-resistant Streptococcus pneumoniae a controlled interventiontrial in rural Alaskardquo Clinical Infectious Diseases vol 34 no 12pp 1543ndash1550 2002
[3] G Barkai D Greenberg N Givon-Lavi E Dreifuss D Vardyand R Dagan ldquoCommunity prescribing and resistant Strepto-coccus pneumoniaerdquo Emerging Infectious Diseases vol 11 no 6pp 829ndash837 2005
[4] L Pallecchi A Bartoloni E Riccobono et al ldquoQuinoloneresistance in absence of selective pressure the experience of avery remote community in the Amazon forestrdquo PLoS NeglectedTropical Diseases vol 6 no 8 Article ID e1790 2012
[5] M P Quintana-Hayashi and S Thakur ldquoPhylogenetic analysisreveals common antimicrobial resistant Campylobacter colipopulation in antimicrobial-free (ABF) and commercial swinesystemsrdquo PLoS ONE vol 7 no 9 Article ID e44662 2012
[6] LMartınez-Martınez A Pascual andG A Jacoby ldquoQuinoloneresistance from a transferable plasmidrdquoThe Lancet vol 351 no9105 pp 797ndash799 1998
[7] M Montes E Tamayo B Orden J Larruskain and E Perez-Trallero ldquoPrevalence and clonal characterization of Streptococ-cus pyogenes clinical isolates with reduced fluoroquinolone sus-ceptibility in Spainrdquo Antimicrobial Agents and Chemotherapyvol 54 no 1 pp 93ndash97 2010
[8] K L Hopkins R H Davies and E J Threlfall ldquoMechanisms ofquinolone resistance in Escherichia coli and Salmonella recentdevelopmentsrdquo International Journal of Antimicrobial Agentsvol 25 no 5 pp 358ndash373 2005
[9] L MWeigel C D Steward and F C Tenover ldquogyrAmutationsassociated with fluoroquinolone resistance in eight species ofEnterobacteriaceaerdquo Antimicrobial Agents and Chemotherapyvol 42 no 10 pp 2661ndash2667 1998
[10] A Robicsek G A Jacoby and D C Hooper ldquoThe worldwideemergence of plasmid-mediated quinolone resistancerdquo LancetInfectious Diseases vol 6 no 10 pp 629ndash640 2006
[11] Committee on Infectious Diseases ldquoThe use of systemic fluoro-quinolonesrdquo Pediatrics vol 118 no 3 pp 1287ndash1292 2006
[12] Clinical and Laboratory Standards Institute Methods for Dilu-tion Antimicrobial Susceptibility Tests for Bacteria That GrowAerobically (M07-A8) CLSI Wayne Pa USA 8th edition2009
[13] M Pfaller L Boyken R Hollis et al ldquoComparison of the brothmicrodilutionmethods of the EuropeanCommittee onAntimi-crobial Susceptibility Testing and the Clinical and LaboratoryStandards Institute for testing itraconazole posaconazole andvoriconazole against Aspergillus isolatesrdquo Journal of ClinicalMicrobiology vol 49 no 3 pp 1110ndash1112 2011
[14] Clinical and Laboratory Standards Institute ldquoPerformancestandards for antimicrobial susceptibility testing Twentiethinformational supplementrdquo M100-S20 Wayne Pa USA CLSI2010
[15] Clinical and Laboratory Standards Institute ldquoPerformancestandards for antimicrobial susceptibility testing Eighteenthinformational supplementrdquo 2008
[16] E Domınguez M Zarazaga Y Saenz L Brinas and C TorresldquoMechanisms of antibiotic resistance in Escherichia coli isolatesobtained from healthy children in Spainrdquo Microbial DrugResistance vol 8 no 4 pp 321ndash327 2002
[17] J Ayatollahi H Shahcheraghi R Akhondi and S S SolutildquoAntibiotic resistance patterns of Escherichia coli isolated fromchildren in shahid sadoughi hospital of Yazdrdquo Iranian Journalof Pediatric Hematology and Oncology vol 3 no 2 pp 78ndash822013
[18] AGarraffoCMarguet AChecoury et al ldquoUrinary tract infec-tions in hospital pediatrics many previous antibiotherapy andantibiotics resistance including fluoroquinolonesrdquoMedecine etMaladies Infectieuses vol 44 no 2 pp 63ndash68 2014
[19] M J Pons S Mosquito C Gomesa L J del Valle T J Ochoaand J Ruiz ldquoAnalysis of quinolone-resistance in commensaland diarrheagenic Escherichia coli isolates from infants in limaPerurdquo Transactions of the Royal Society of Tropical Medicine andHygiene vol 108 no 1 pp 22ndash28 2014
[20] M P Quintana-Hayashi and S Thakur ldquoLongitudinal study ofthe persistence of antimicrobial-resistantCampylobacter strainsin distinct swine production systems on farms at slaughter andin the environmentrdquo Applied and Environmental Microbiologyvol 78 no 8 pp 2698ndash2705 2012
[21] J Isendahl A Turlej-Rogacka C Manjuba A Rodrigues C GGiske and P Naucler ldquoFecal carriage of ESBL-producing E coliand K pneumoniae in children in Guinea-Bissau a hospital-based cross-sectional studyrdquo PLoS ONE vol 7 no 12 ArticleID e51981 2012
[22] R Pires C Ardanuy D Rolo et al ldquoEmergence of cipro-floxacin-nonsusceptible Streptococcus pyogenes isolates fromhealthy children and pediatric patients in Portugalrdquo Antimicro-bial Agents and Chemotherapy vol 54 no 6 pp 2677ndash26802010
[23] R J Bywater and M W Casewell ldquoAn assessment of theimpact of antibiotic resistance in different bacterial species andof the contribution of animal sources to resistance in humaninfectionsrdquo Journal of Antimicrobial Chemotherapy vol 46 no6 p 1052 2000
[24] J R Johnson M R Sannes C Croy et al ldquoAntimicrobial drug-resistant Escherichia coli from humans and poultry productsMinnesota and Wisconsin 2002ndash2004rdquo Emerging InfectiousDiseases vol 13 no 6 pp 838ndash846 2007
[25] M Bagheri R Ghanbarpour and H Alizade ldquoShiga toxin andbeta-lactamases genes in Escherichia coli phylotypes isolatedfrom carcasses of broiler chickens slaughtered in Iranrdquo Interna-tional Journal of Food Microbiology vol 177 pp 16ndash20 2014
[26] M V C Cossi R C K Burin D A Lopes et al ldquoAntimicrobialresistance and virulence profiles of Salmonella isolated frombutcher shops in minas gerais Brazilrdquo Journal of Food Protec-tion vol 76 no 9 pp 1633ndash1637 2013
[27] F Reich V Atanassova and G Klein ldquoExtended-spectrumbeta-lactamase- and ampc-producing enterobacteria in healthybroiler chickens Germanyrdquo Emerging Infectious Diseases vol19 no 8 pp 1253ndash1259 2013
[28] M Mellata ldquoHuman and avian extraintestinal pathogenicEscherichia coli infections zoonotic risks and antibiotic resis-tance trendsrdquo Foodborne Pathogens and Disease vol 10 no 11pp 916ndash932 2013
6 BioMed Research International
[29] K Holvoet I Sampers B Callens J Dewulf and M Uyt-tendaele ldquoModerate prevalence of antimicrobial resistance inEscherichia coli isolates from lettuce irrigation water and soilrdquoApplied and Environmental Microbiology vol 79 no 21 pp6677ndash6683 2013
[30] J L Martinez ldquoEnvironmental pollution by antibiotics andby antibiotic resistance determinantsrdquo Environmental Pollutionvol 157 no 11 pp 2893ndash2902 2009
of 1- to 3-year toddlers and 600 of 3- to 6-year preschoolchildren)
As shown in Table 1 resistance rates to other classes ofantimicrobial agents in E coli isolated from children andadults were quite similar (all 119875 gt 005) However in Kpneumoniae the resistance rates for cefotaxime ceftriaxoneand ceftazidime in children were statistically higher thanthose in adults (all 119875 lt 005) Occurrences of the productionof extended-spectrum beta-lactamases (ESBLs) isolates fromchildren and adults were 579 and 385 respectively Incontrast cefoxitin resistance rate was significantly lower inthe isolates from children (119875 = 00085) Of note theoccurrence of AmpC in children was 79 which is similarto that in adults (154 the Fisher exact test 119875 = 04806)Theamikacin resistance rate of K pneumoniae in children is alsohigher than that of adults (119875 lt 005) Among the resistantisolates from adults 9 (231) were found an absence of
inhibition zone around the amikacin disc (diameter lt6mm)while 25 (658) of the isolates from children were observedto have this phenomenon (1205942 = 142398119875 = 00002 Table 1)
32 Resistance to Ciprofloxacin and gyrAMutations in Isolatesfrom Children and Adults All the 140 isolates were inves-tigated for QRDR mutations of gyrA 46 of the 61 isolates(754) from children were found to contain gyrAmutationsA similar prevalence pattern of the gyrA mutation was alsoobserved in the isolates of adult origin (6479 810 119875 =043) Among the 38 isolates resistant to ciprofloxacin fromchildren 37 (974 3738) were found to contain the gyrAmutation which is quite similar to the prevalence in isolatesfrom adults (5556 982 119875 = 100)
As shown in Table 2 the double mutation Ser83rarr Leu +Asp87rarrAsn was the most prevalent type of GyrA changesin antimicrobial-resistant isolates from both children and
4 BioMed Research International
adults (737 versus 679 119875 = 05444) The single mutationSer83rarr Ile was not detected in the isolates from childrenbut was detected in 196 of the isolates from adults (119875 =00026) The single mutation Ser83rarr Leu was detected in184 of the isolates from children but was not found inthose from adults (119875 = 00012 Table 2) GryA mutationwas also found in 18 isolates nonresistant to ciprofloxacinincluding 9 isolates from children and 9 isolates from adultsThe mutation patterns were listed in Table 2
33 GyrA Mutations in Children with Different Age GroupsThe 61 isolates from children were assigned into 4 groupsaccording to physiological development and participation insocial activities namely young infants (lt6 months) infants(6ndash12 months) toddlers (1ndash3 years) and preschoolers (age of3ndash6 years) It was found that the mutations Ser83rarr Leu +Asp87rarrAsn Ser83rarr Leu Ser83rarrThr and Asp87rarrAsnwere the common types of mutations in GyrA and Ser83rarrLeu + Asp87rarrAsn was the most prevalent double mutationIn addition there was no significant difference in terms ofindividual types of mutation in GyrA in children amongdifferent age groups (119875 gt 005)
4 Discussion
There is a growing concern over antibiotics resistance as aresult of antibiotics overuse in hospital and communitiesThus restriction on the use of antibiotics has become oneof the most important measures for control emergenceand spread of drug-resistant bacteria This study involvesclinical isolates obtained from children in the absence offluoroquinolone usage history as quinolones are not rec-ommended for pediatric use due to the adverse effects onjoints However our findings showed that children har-bored quinolone-resistant isolates being similarly resistantto ciprofloxacin and levofloxacin compared with the isolatesfrom adults Moreover isolates from children were found tohave similarly high ciprofloxacin MIC levels with those fromadults By sequencing the QRDRs of gyrA we found that thedouble mutation Ser83rarr Leu + Asp87rarrAsn was similarlypredominant among the quinolone-resistant isolates fromboth children and adults
A previous study conducted in Spain found 5 resistancerate of ciprofloxacin in E coli isolates from healthy children[16] Other studies have also recorded quinolone-resistantEnterobacteriaceae from children in recent years [17 18]GyrA mutations involved in quinolone resistance in isolatesfrom infants were most recently discovered in Spain [19]The resistance rate to ciprofloxacin in children was up to22 in these regions [17] Compared to these regions theciprofloxacin resistance rate and the resistant level (MIC)found in the present study are notably higher In spiteof the difference in resistance rate and resistant level thefindings in the present study and others demonstrated thequinolone resistance and the involved mutation mechanismsin nonexposed population under high antibiotic pressureIt indicates that drug resistance may be transmitted andpersist in people who are not exposed to the correspondingantibiotics
Various reports on the different aspects of resistance haverevealed that persistence of resistance in human or animalpopulations relies more on the transmission of resistance inthe public environment than on drug pressure [5 20] Drug-resistant isolates of bacteria were detected even in remotemountainous areas of Amazon where antibiotics have neverbeen used [4] Presence of drug-resistant isolates in childrenmight be attributed to their increased contact with adultsand playmates in families daycare or school settings [2122] Among the environmental factors food supply chainmay be the major source of antibiotic resistance in ldquonaıverdquohumans as an indirect consequence of overuse of antibioticsfor curative and preventative purpose in stock farming andagricultural products In 2000 the use of antibiotics inraising animals was questioned [23] Shortly after antibiotic-resistant E coli was detected in poultry products [24] Thesituation of antibiotic-resistant bacteria from food chainsources has become a notable societal issue which shouldbe worthy of attention [25ndash29] Furthermore pollution byantibiotic resistance genes from stock farming and antibioticsin manure and metabolites could increase the chances ofresistance acquisition by human pathogens [30]
In summary our data showed similar prevalence of quin-olone resistance and type of gyrA genemutation in E coli andK pneumoniae among children and adults The conclusionthat there exists similarity in fluoroquinolone resistance ratesbetween children and adults in a region under high commu-nity antibiotic pressure constrains us to pay close attentionto the environmental and community reservoirs of resistanceand take precautions to prevent perpetuating antibiotic-resistant isolates and their transmission and dissemination
Conflict of Interests
All authors declare that they have no conflict of interests
Authorsrsquo Contribution
Ying Huang Yingmei Fu and Fengmin Zhang participatedin the design conduction analysis and interpretation of thestudy Ying Huang James O Ogutu and Jiarui Gu wereinvolved in all phases of the experiment Fengshu DingYuhong You and YanHuo were involved in themanipulationof clinical isolates Hong Zhao andWenjing Li were involvedin the DNA sequencing Zhiwei Zhang Wenli Zhang andXiaobei Chen conducted the statistical analysis Ying Huangwrote the paper Yingmei Fu and Fengmin Zhang reviewedthe initial and final drafts of the paper All authors read andapproved the final paper
Acknowledgments
This work was supported by grants from National NaturalScience Foundation of China (NSFC) (81101300 31370164J1103609 and J1210062) Heilongjiang Educational Agency(1155G34) Heilongjiang Provincial Science and TechnologyInnovation Team inHigher Education Institutes for Infectionand Immunity and Harbin Medical University
BioMed Research International 5
References
[1] P J Johnsen J P Townsend T Boslashhn G S Simonsen ASundsfjord and K M Nielsen ldquoFactors affecting the reversal ofantimicrobial-drug resistancerdquo The Lancet Infectious Diseasesvol 9 no 6 pp 357ndash364 2009
[2] T W Hennessy K M Petersen D Bruden et al ldquoChangesin antibiotic-prescribing practices and carriage of penicillin-resistant Streptococcus pneumoniae a controlled interventiontrial in rural Alaskardquo Clinical Infectious Diseases vol 34 no 12pp 1543ndash1550 2002
[3] G Barkai D Greenberg N Givon-Lavi E Dreifuss D Vardyand R Dagan ldquoCommunity prescribing and resistant Strepto-coccus pneumoniaerdquo Emerging Infectious Diseases vol 11 no 6pp 829ndash837 2005
[4] L Pallecchi A Bartoloni E Riccobono et al ldquoQuinoloneresistance in absence of selective pressure the experience of avery remote community in the Amazon forestrdquo PLoS NeglectedTropical Diseases vol 6 no 8 Article ID e1790 2012
[5] M P Quintana-Hayashi and S Thakur ldquoPhylogenetic analysisreveals common antimicrobial resistant Campylobacter colipopulation in antimicrobial-free (ABF) and commercial swinesystemsrdquo PLoS ONE vol 7 no 9 Article ID e44662 2012
[6] LMartınez-Martınez A Pascual andG A Jacoby ldquoQuinoloneresistance from a transferable plasmidrdquoThe Lancet vol 351 no9105 pp 797ndash799 1998
[7] M Montes E Tamayo B Orden J Larruskain and E Perez-Trallero ldquoPrevalence and clonal characterization of Streptococ-cus pyogenes clinical isolates with reduced fluoroquinolone sus-ceptibility in Spainrdquo Antimicrobial Agents and Chemotherapyvol 54 no 1 pp 93ndash97 2010
[8] K L Hopkins R H Davies and E J Threlfall ldquoMechanisms ofquinolone resistance in Escherichia coli and Salmonella recentdevelopmentsrdquo International Journal of Antimicrobial Agentsvol 25 no 5 pp 358ndash373 2005
[9] L MWeigel C D Steward and F C Tenover ldquogyrAmutationsassociated with fluoroquinolone resistance in eight species ofEnterobacteriaceaerdquo Antimicrobial Agents and Chemotherapyvol 42 no 10 pp 2661ndash2667 1998
[10] A Robicsek G A Jacoby and D C Hooper ldquoThe worldwideemergence of plasmid-mediated quinolone resistancerdquo LancetInfectious Diseases vol 6 no 10 pp 629ndash640 2006
[11] Committee on Infectious Diseases ldquoThe use of systemic fluoro-quinolonesrdquo Pediatrics vol 118 no 3 pp 1287ndash1292 2006
[12] Clinical and Laboratory Standards Institute Methods for Dilu-tion Antimicrobial Susceptibility Tests for Bacteria That GrowAerobically (M07-A8) CLSI Wayne Pa USA 8th edition2009
[13] M Pfaller L Boyken R Hollis et al ldquoComparison of the brothmicrodilutionmethods of the EuropeanCommittee onAntimi-crobial Susceptibility Testing and the Clinical and LaboratoryStandards Institute for testing itraconazole posaconazole andvoriconazole against Aspergillus isolatesrdquo Journal of ClinicalMicrobiology vol 49 no 3 pp 1110ndash1112 2011
[14] Clinical and Laboratory Standards Institute ldquoPerformancestandards for antimicrobial susceptibility testing Twentiethinformational supplementrdquo M100-S20 Wayne Pa USA CLSI2010
[15] Clinical and Laboratory Standards Institute ldquoPerformancestandards for antimicrobial susceptibility testing Eighteenthinformational supplementrdquo 2008
[16] E Domınguez M Zarazaga Y Saenz L Brinas and C TorresldquoMechanisms of antibiotic resistance in Escherichia coli isolatesobtained from healthy children in Spainrdquo Microbial DrugResistance vol 8 no 4 pp 321ndash327 2002
[17] J Ayatollahi H Shahcheraghi R Akhondi and S S SolutildquoAntibiotic resistance patterns of Escherichia coli isolated fromchildren in shahid sadoughi hospital of Yazdrdquo Iranian Journalof Pediatric Hematology and Oncology vol 3 no 2 pp 78ndash822013
[18] AGarraffoCMarguet AChecoury et al ldquoUrinary tract infec-tions in hospital pediatrics many previous antibiotherapy andantibiotics resistance including fluoroquinolonesrdquoMedecine etMaladies Infectieuses vol 44 no 2 pp 63ndash68 2014
[19] M J Pons S Mosquito C Gomesa L J del Valle T J Ochoaand J Ruiz ldquoAnalysis of quinolone-resistance in commensaland diarrheagenic Escherichia coli isolates from infants in limaPerurdquo Transactions of the Royal Society of Tropical Medicine andHygiene vol 108 no 1 pp 22ndash28 2014
[20] M P Quintana-Hayashi and S Thakur ldquoLongitudinal study ofthe persistence of antimicrobial-resistantCampylobacter strainsin distinct swine production systems on farms at slaughter andin the environmentrdquo Applied and Environmental Microbiologyvol 78 no 8 pp 2698ndash2705 2012
[21] J Isendahl A Turlej-Rogacka C Manjuba A Rodrigues C GGiske and P Naucler ldquoFecal carriage of ESBL-producing E coliand K pneumoniae in children in Guinea-Bissau a hospital-based cross-sectional studyrdquo PLoS ONE vol 7 no 12 ArticleID e51981 2012
[22] R Pires C Ardanuy D Rolo et al ldquoEmergence of cipro-floxacin-nonsusceptible Streptococcus pyogenes isolates fromhealthy children and pediatric patients in Portugalrdquo Antimicro-bial Agents and Chemotherapy vol 54 no 6 pp 2677ndash26802010
[23] R J Bywater and M W Casewell ldquoAn assessment of theimpact of antibiotic resistance in different bacterial species andof the contribution of animal sources to resistance in humaninfectionsrdquo Journal of Antimicrobial Chemotherapy vol 46 no6 p 1052 2000
[24] J R Johnson M R Sannes C Croy et al ldquoAntimicrobial drug-resistant Escherichia coli from humans and poultry productsMinnesota and Wisconsin 2002ndash2004rdquo Emerging InfectiousDiseases vol 13 no 6 pp 838ndash846 2007
[25] M Bagheri R Ghanbarpour and H Alizade ldquoShiga toxin andbeta-lactamases genes in Escherichia coli phylotypes isolatedfrom carcasses of broiler chickens slaughtered in Iranrdquo Interna-tional Journal of Food Microbiology vol 177 pp 16ndash20 2014
[26] M V C Cossi R C K Burin D A Lopes et al ldquoAntimicrobialresistance and virulence profiles of Salmonella isolated frombutcher shops in minas gerais Brazilrdquo Journal of Food Protec-tion vol 76 no 9 pp 1633ndash1637 2013
[27] F Reich V Atanassova and G Klein ldquoExtended-spectrumbeta-lactamase- and ampc-producing enterobacteria in healthybroiler chickens Germanyrdquo Emerging Infectious Diseases vol19 no 8 pp 1253ndash1259 2013
[28] M Mellata ldquoHuman and avian extraintestinal pathogenicEscherichia coli infections zoonotic risks and antibiotic resis-tance trendsrdquo Foodborne Pathogens and Disease vol 10 no 11pp 916ndash932 2013
6 BioMed Research International
[29] K Holvoet I Sampers B Callens J Dewulf and M Uyt-tendaele ldquoModerate prevalence of antimicrobial resistance inEscherichia coli isolates from lettuce irrigation water and soilrdquoApplied and Environmental Microbiology vol 79 no 21 pp6677ndash6683 2013
[30] J L Martinez ldquoEnvironmental pollution by antibiotics andby antibiotic resistance determinantsrdquo Environmental Pollutionvol 157 no 11 pp 2893ndash2902 2009
adults (737 versus 679 119875 = 05444) The single mutationSer83rarr Ile was not detected in the isolates from childrenbut was detected in 196 of the isolates from adults (119875 =00026) The single mutation Ser83rarr Leu was detected in184 of the isolates from children but was not found inthose from adults (119875 = 00012 Table 2) GryA mutationwas also found in 18 isolates nonresistant to ciprofloxacinincluding 9 isolates from children and 9 isolates from adultsThe mutation patterns were listed in Table 2
33 GyrA Mutations in Children with Different Age GroupsThe 61 isolates from children were assigned into 4 groupsaccording to physiological development and participation insocial activities namely young infants (lt6 months) infants(6ndash12 months) toddlers (1ndash3 years) and preschoolers (age of3ndash6 years) It was found that the mutations Ser83rarr Leu +Asp87rarrAsn Ser83rarr Leu Ser83rarrThr and Asp87rarrAsnwere the common types of mutations in GyrA and Ser83rarrLeu + Asp87rarrAsn was the most prevalent double mutationIn addition there was no significant difference in terms ofindividual types of mutation in GyrA in children amongdifferent age groups (119875 gt 005)
4 Discussion
There is a growing concern over antibiotics resistance as aresult of antibiotics overuse in hospital and communitiesThus restriction on the use of antibiotics has become oneof the most important measures for control emergenceand spread of drug-resistant bacteria This study involvesclinical isolates obtained from children in the absence offluoroquinolone usage history as quinolones are not rec-ommended for pediatric use due to the adverse effects onjoints However our findings showed that children har-bored quinolone-resistant isolates being similarly resistantto ciprofloxacin and levofloxacin compared with the isolatesfrom adults Moreover isolates from children were found tohave similarly high ciprofloxacin MIC levels with those fromadults By sequencing the QRDRs of gyrA we found that thedouble mutation Ser83rarr Leu + Asp87rarrAsn was similarlypredominant among the quinolone-resistant isolates fromboth children and adults
A previous study conducted in Spain found 5 resistancerate of ciprofloxacin in E coli isolates from healthy children[16] Other studies have also recorded quinolone-resistantEnterobacteriaceae from children in recent years [17 18]GyrA mutations involved in quinolone resistance in isolatesfrom infants were most recently discovered in Spain [19]The resistance rate to ciprofloxacin in children was up to22 in these regions [17] Compared to these regions theciprofloxacin resistance rate and the resistant level (MIC)found in the present study are notably higher In spiteof the difference in resistance rate and resistant level thefindings in the present study and others demonstrated thequinolone resistance and the involved mutation mechanismsin nonexposed population under high antibiotic pressureIt indicates that drug resistance may be transmitted andpersist in people who are not exposed to the correspondingantibiotics
Various reports on the different aspects of resistance haverevealed that persistence of resistance in human or animalpopulations relies more on the transmission of resistance inthe public environment than on drug pressure [5 20] Drug-resistant isolates of bacteria were detected even in remotemountainous areas of Amazon where antibiotics have neverbeen used [4] Presence of drug-resistant isolates in childrenmight be attributed to their increased contact with adultsand playmates in families daycare or school settings [2122] Among the environmental factors food supply chainmay be the major source of antibiotic resistance in ldquonaıverdquohumans as an indirect consequence of overuse of antibioticsfor curative and preventative purpose in stock farming andagricultural products In 2000 the use of antibiotics inraising animals was questioned [23] Shortly after antibiotic-resistant E coli was detected in poultry products [24] Thesituation of antibiotic-resistant bacteria from food chainsources has become a notable societal issue which shouldbe worthy of attention [25ndash29] Furthermore pollution byantibiotic resistance genes from stock farming and antibioticsin manure and metabolites could increase the chances ofresistance acquisition by human pathogens [30]
In summary our data showed similar prevalence of quin-olone resistance and type of gyrA genemutation in E coli andK pneumoniae among children and adults The conclusionthat there exists similarity in fluoroquinolone resistance ratesbetween children and adults in a region under high commu-nity antibiotic pressure constrains us to pay close attentionto the environmental and community reservoirs of resistanceand take precautions to prevent perpetuating antibiotic-resistant isolates and their transmission and dissemination
Conflict of Interests
All authors declare that they have no conflict of interests
Authorsrsquo Contribution
Ying Huang Yingmei Fu and Fengmin Zhang participatedin the design conduction analysis and interpretation of thestudy Ying Huang James O Ogutu and Jiarui Gu wereinvolved in all phases of the experiment Fengshu DingYuhong You and YanHuo were involved in themanipulationof clinical isolates Hong Zhao andWenjing Li were involvedin the DNA sequencing Zhiwei Zhang Wenli Zhang andXiaobei Chen conducted the statistical analysis Ying Huangwrote the paper Yingmei Fu and Fengmin Zhang reviewedthe initial and final drafts of the paper All authors read andapproved the final paper
Acknowledgments
This work was supported by grants from National NaturalScience Foundation of China (NSFC) (81101300 31370164J1103609 and J1210062) Heilongjiang Educational Agency(1155G34) Heilongjiang Provincial Science and TechnologyInnovation Team inHigher Education Institutes for Infectionand Immunity and Harbin Medical University
BioMed Research International 5
References
[1] P J Johnsen J P Townsend T Boslashhn G S Simonsen ASundsfjord and K M Nielsen ldquoFactors affecting the reversal ofantimicrobial-drug resistancerdquo The Lancet Infectious Diseasesvol 9 no 6 pp 357ndash364 2009
[2] T W Hennessy K M Petersen D Bruden et al ldquoChangesin antibiotic-prescribing practices and carriage of penicillin-resistant Streptococcus pneumoniae a controlled interventiontrial in rural Alaskardquo Clinical Infectious Diseases vol 34 no 12pp 1543ndash1550 2002
[3] G Barkai D Greenberg N Givon-Lavi E Dreifuss D Vardyand R Dagan ldquoCommunity prescribing and resistant Strepto-coccus pneumoniaerdquo Emerging Infectious Diseases vol 11 no 6pp 829ndash837 2005
[4] L Pallecchi A Bartoloni E Riccobono et al ldquoQuinoloneresistance in absence of selective pressure the experience of avery remote community in the Amazon forestrdquo PLoS NeglectedTropical Diseases vol 6 no 8 Article ID e1790 2012
[5] M P Quintana-Hayashi and S Thakur ldquoPhylogenetic analysisreveals common antimicrobial resistant Campylobacter colipopulation in antimicrobial-free (ABF) and commercial swinesystemsrdquo PLoS ONE vol 7 no 9 Article ID e44662 2012
[6] LMartınez-Martınez A Pascual andG A Jacoby ldquoQuinoloneresistance from a transferable plasmidrdquoThe Lancet vol 351 no9105 pp 797ndash799 1998
[7] M Montes E Tamayo B Orden J Larruskain and E Perez-Trallero ldquoPrevalence and clonal characterization of Streptococ-cus pyogenes clinical isolates with reduced fluoroquinolone sus-ceptibility in Spainrdquo Antimicrobial Agents and Chemotherapyvol 54 no 1 pp 93ndash97 2010
[8] K L Hopkins R H Davies and E J Threlfall ldquoMechanisms ofquinolone resistance in Escherichia coli and Salmonella recentdevelopmentsrdquo International Journal of Antimicrobial Agentsvol 25 no 5 pp 358ndash373 2005
[9] L MWeigel C D Steward and F C Tenover ldquogyrAmutationsassociated with fluoroquinolone resistance in eight species ofEnterobacteriaceaerdquo Antimicrobial Agents and Chemotherapyvol 42 no 10 pp 2661ndash2667 1998
[10] A Robicsek G A Jacoby and D C Hooper ldquoThe worldwideemergence of plasmid-mediated quinolone resistancerdquo LancetInfectious Diseases vol 6 no 10 pp 629ndash640 2006
[11] Committee on Infectious Diseases ldquoThe use of systemic fluoro-quinolonesrdquo Pediatrics vol 118 no 3 pp 1287ndash1292 2006
[12] Clinical and Laboratory Standards Institute Methods for Dilu-tion Antimicrobial Susceptibility Tests for Bacteria That GrowAerobically (M07-A8) CLSI Wayne Pa USA 8th edition2009
[13] M Pfaller L Boyken R Hollis et al ldquoComparison of the brothmicrodilutionmethods of the EuropeanCommittee onAntimi-crobial Susceptibility Testing and the Clinical and LaboratoryStandards Institute for testing itraconazole posaconazole andvoriconazole against Aspergillus isolatesrdquo Journal of ClinicalMicrobiology vol 49 no 3 pp 1110ndash1112 2011
[14] Clinical and Laboratory Standards Institute ldquoPerformancestandards for antimicrobial susceptibility testing Twentiethinformational supplementrdquo M100-S20 Wayne Pa USA CLSI2010
[15] Clinical and Laboratory Standards Institute ldquoPerformancestandards for antimicrobial susceptibility testing Eighteenthinformational supplementrdquo 2008
[16] E Domınguez M Zarazaga Y Saenz L Brinas and C TorresldquoMechanisms of antibiotic resistance in Escherichia coli isolatesobtained from healthy children in Spainrdquo Microbial DrugResistance vol 8 no 4 pp 321ndash327 2002
[17] J Ayatollahi H Shahcheraghi R Akhondi and S S SolutildquoAntibiotic resistance patterns of Escherichia coli isolated fromchildren in shahid sadoughi hospital of Yazdrdquo Iranian Journalof Pediatric Hematology and Oncology vol 3 no 2 pp 78ndash822013
[18] AGarraffoCMarguet AChecoury et al ldquoUrinary tract infec-tions in hospital pediatrics many previous antibiotherapy andantibiotics resistance including fluoroquinolonesrdquoMedecine etMaladies Infectieuses vol 44 no 2 pp 63ndash68 2014
[19] M J Pons S Mosquito C Gomesa L J del Valle T J Ochoaand J Ruiz ldquoAnalysis of quinolone-resistance in commensaland diarrheagenic Escherichia coli isolates from infants in limaPerurdquo Transactions of the Royal Society of Tropical Medicine andHygiene vol 108 no 1 pp 22ndash28 2014
[20] M P Quintana-Hayashi and S Thakur ldquoLongitudinal study ofthe persistence of antimicrobial-resistantCampylobacter strainsin distinct swine production systems on farms at slaughter andin the environmentrdquo Applied and Environmental Microbiologyvol 78 no 8 pp 2698ndash2705 2012
[21] J Isendahl A Turlej-Rogacka C Manjuba A Rodrigues C GGiske and P Naucler ldquoFecal carriage of ESBL-producing E coliand K pneumoniae in children in Guinea-Bissau a hospital-based cross-sectional studyrdquo PLoS ONE vol 7 no 12 ArticleID e51981 2012
[22] R Pires C Ardanuy D Rolo et al ldquoEmergence of cipro-floxacin-nonsusceptible Streptococcus pyogenes isolates fromhealthy children and pediatric patients in Portugalrdquo Antimicro-bial Agents and Chemotherapy vol 54 no 6 pp 2677ndash26802010
[23] R J Bywater and M W Casewell ldquoAn assessment of theimpact of antibiotic resistance in different bacterial species andof the contribution of animal sources to resistance in humaninfectionsrdquo Journal of Antimicrobial Chemotherapy vol 46 no6 p 1052 2000
[24] J R Johnson M R Sannes C Croy et al ldquoAntimicrobial drug-resistant Escherichia coli from humans and poultry productsMinnesota and Wisconsin 2002ndash2004rdquo Emerging InfectiousDiseases vol 13 no 6 pp 838ndash846 2007
[25] M Bagheri R Ghanbarpour and H Alizade ldquoShiga toxin andbeta-lactamases genes in Escherichia coli phylotypes isolatedfrom carcasses of broiler chickens slaughtered in Iranrdquo Interna-tional Journal of Food Microbiology vol 177 pp 16ndash20 2014
[26] M V C Cossi R C K Burin D A Lopes et al ldquoAntimicrobialresistance and virulence profiles of Salmonella isolated frombutcher shops in minas gerais Brazilrdquo Journal of Food Protec-tion vol 76 no 9 pp 1633ndash1637 2013
[27] F Reich V Atanassova and G Klein ldquoExtended-spectrumbeta-lactamase- and ampc-producing enterobacteria in healthybroiler chickens Germanyrdquo Emerging Infectious Diseases vol19 no 8 pp 1253ndash1259 2013
[28] M Mellata ldquoHuman and avian extraintestinal pathogenicEscherichia coli infections zoonotic risks and antibiotic resis-tance trendsrdquo Foodborne Pathogens and Disease vol 10 no 11pp 916ndash932 2013
6 BioMed Research International
[29] K Holvoet I Sampers B Callens J Dewulf and M Uyt-tendaele ldquoModerate prevalence of antimicrobial resistance inEscherichia coli isolates from lettuce irrigation water and soilrdquoApplied and Environmental Microbiology vol 79 no 21 pp6677ndash6683 2013
[30] J L Martinez ldquoEnvironmental pollution by antibiotics andby antibiotic resistance determinantsrdquo Environmental Pollutionvol 157 no 11 pp 2893ndash2902 2009
[1] P J Johnsen J P Townsend T Boslashhn G S Simonsen ASundsfjord and K M Nielsen ldquoFactors affecting the reversal ofantimicrobial-drug resistancerdquo The Lancet Infectious Diseasesvol 9 no 6 pp 357ndash364 2009
[2] T W Hennessy K M Petersen D Bruden et al ldquoChangesin antibiotic-prescribing practices and carriage of penicillin-resistant Streptococcus pneumoniae a controlled interventiontrial in rural Alaskardquo Clinical Infectious Diseases vol 34 no 12pp 1543ndash1550 2002
[3] G Barkai D Greenberg N Givon-Lavi E Dreifuss D Vardyand R Dagan ldquoCommunity prescribing and resistant Strepto-coccus pneumoniaerdquo Emerging Infectious Diseases vol 11 no 6pp 829ndash837 2005
[4] L Pallecchi A Bartoloni E Riccobono et al ldquoQuinoloneresistance in absence of selective pressure the experience of avery remote community in the Amazon forestrdquo PLoS NeglectedTropical Diseases vol 6 no 8 Article ID e1790 2012
[5] M P Quintana-Hayashi and S Thakur ldquoPhylogenetic analysisreveals common antimicrobial resistant Campylobacter colipopulation in antimicrobial-free (ABF) and commercial swinesystemsrdquo PLoS ONE vol 7 no 9 Article ID e44662 2012
[6] LMartınez-Martınez A Pascual andG A Jacoby ldquoQuinoloneresistance from a transferable plasmidrdquoThe Lancet vol 351 no9105 pp 797ndash799 1998
[7] M Montes E Tamayo B Orden J Larruskain and E Perez-Trallero ldquoPrevalence and clonal characterization of Streptococ-cus pyogenes clinical isolates with reduced fluoroquinolone sus-ceptibility in Spainrdquo Antimicrobial Agents and Chemotherapyvol 54 no 1 pp 93ndash97 2010
[8] K L Hopkins R H Davies and E J Threlfall ldquoMechanisms ofquinolone resistance in Escherichia coli and Salmonella recentdevelopmentsrdquo International Journal of Antimicrobial Agentsvol 25 no 5 pp 358ndash373 2005
[9] L MWeigel C D Steward and F C Tenover ldquogyrAmutationsassociated with fluoroquinolone resistance in eight species ofEnterobacteriaceaerdquo Antimicrobial Agents and Chemotherapyvol 42 no 10 pp 2661ndash2667 1998
[10] A Robicsek G A Jacoby and D C Hooper ldquoThe worldwideemergence of plasmid-mediated quinolone resistancerdquo LancetInfectious Diseases vol 6 no 10 pp 629ndash640 2006
[11] Committee on Infectious Diseases ldquoThe use of systemic fluoro-quinolonesrdquo Pediatrics vol 118 no 3 pp 1287ndash1292 2006
[12] Clinical and Laboratory Standards Institute Methods for Dilu-tion Antimicrobial Susceptibility Tests for Bacteria That GrowAerobically (M07-A8) CLSI Wayne Pa USA 8th edition2009
[13] M Pfaller L Boyken R Hollis et al ldquoComparison of the brothmicrodilutionmethods of the EuropeanCommittee onAntimi-crobial Susceptibility Testing and the Clinical and LaboratoryStandards Institute for testing itraconazole posaconazole andvoriconazole against Aspergillus isolatesrdquo Journal of ClinicalMicrobiology vol 49 no 3 pp 1110ndash1112 2011
[14] Clinical and Laboratory Standards Institute ldquoPerformancestandards for antimicrobial susceptibility testing Twentiethinformational supplementrdquo M100-S20 Wayne Pa USA CLSI2010
[15] Clinical and Laboratory Standards Institute ldquoPerformancestandards for antimicrobial susceptibility testing Eighteenthinformational supplementrdquo 2008
[16] E Domınguez M Zarazaga Y Saenz L Brinas and C TorresldquoMechanisms of antibiotic resistance in Escherichia coli isolatesobtained from healthy children in Spainrdquo Microbial DrugResistance vol 8 no 4 pp 321ndash327 2002
[17] J Ayatollahi H Shahcheraghi R Akhondi and S S SolutildquoAntibiotic resistance patterns of Escherichia coli isolated fromchildren in shahid sadoughi hospital of Yazdrdquo Iranian Journalof Pediatric Hematology and Oncology vol 3 no 2 pp 78ndash822013
[18] AGarraffoCMarguet AChecoury et al ldquoUrinary tract infec-tions in hospital pediatrics many previous antibiotherapy andantibiotics resistance including fluoroquinolonesrdquoMedecine etMaladies Infectieuses vol 44 no 2 pp 63ndash68 2014
[19] M J Pons S Mosquito C Gomesa L J del Valle T J Ochoaand J Ruiz ldquoAnalysis of quinolone-resistance in commensaland diarrheagenic Escherichia coli isolates from infants in limaPerurdquo Transactions of the Royal Society of Tropical Medicine andHygiene vol 108 no 1 pp 22ndash28 2014
[20] M P Quintana-Hayashi and S Thakur ldquoLongitudinal study ofthe persistence of antimicrobial-resistantCampylobacter strainsin distinct swine production systems on farms at slaughter andin the environmentrdquo Applied and Environmental Microbiologyvol 78 no 8 pp 2698ndash2705 2012
[21] J Isendahl A Turlej-Rogacka C Manjuba A Rodrigues C GGiske and P Naucler ldquoFecal carriage of ESBL-producing E coliand K pneumoniae in children in Guinea-Bissau a hospital-based cross-sectional studyrdquo PLoS ONE vol 7 no 12 ArticleID e51981 2012
[22] R Pires C Ardanuy D Rolo et al ldquoEmergence of cipro-floxacin-nonsusceptible Streptococcus pyogenes isolates fromhealthy children and pediatric patients in Portugalrdquo Antimicro-bial Agents and Chemotherapy vol 54 no 6 pp 2677ndash26802010
[23] R J Bywater and M W Casewell ldquoAn assessment of theimpact of antibiotic resistance in different bacterial species andof the contribution of animal sources to resistance in humaninfectionsrdquo Journal of Antimicrobial Chemotherapy vol 46 no6 p 1052 2000
[24] J R Johnson M R Sannes C Croy et al ldquoAntimicrobial drug-resistant Escherichia coli from humans and poultry productsMinnesota and Wisconsin 2002ndash2004rdquo Emerging InfectiousDiseases vol 13 no 6 pp 838ndash846 2007
[25] M Bagheri R Ghanbarpour and H Alizade ldquoShiga toxin andbeta-lactamases genes in Escherichia coli phylotypes isolatedfrom carcasses of broiler chickens slaughtered in Iranrdquo Interna-tional Journal of Food Microbiology vol 177 pp 16ndash20 2014
[26] M V C Cossi R C K Burin D A Lopes et al ldquoAntimicrobialresistance and virulence profiles of Salmonella isolated frombutcher shops in minas gerais Brazilrdquo Journal of Food Protec-tion vol 76 no 9 pp 1633ndash1637 2013
[27] F Reich V Atanassova and G Klein ldquoExtended-spectrumbeta-lactamase- and ampc-producing enterobacteria in healthybroiler chickens Germanyrdquo Emerging Infectious Diseases vol19 no 8 pp 1253ndash1259 2013
[28] M Mellata ldquoHuman and avian extraintestinal pathogenicEscherichia coli infections zoonotic risks and antibiotic resis-tance trendsrdquo Foodborne Pathogens and Disease vol 10 no 11pp 916ndash932 2013
6 BioMed Research International
[29] K Holvoet I Sampers B Callens J Dewulf and M Uyt-tendaele ldquoModerate prevalence of antimicrobial resistance inEscherichia coli isolates from lettuce irrigation water and soilrdquoApplied and Environmental Microbiology vol 79 no 21 pp6677ndash6683 2013
[30] J L Martinez ldquoEnvironmental pollution by antibiotics andby antibiotic resistance determinantsrdquo Environmental Pollutionvol 157 no 11 pp 2893ndash2902 2009
[29] K Holvoet I Sampers B Callens J Dewulf and M Uyt-tendaele ldquoModerate prevalence of antimicrobial resistance inEscherichia coli isolates from lettuce irrigation water and soilrdquoApplied and Environmental Microbiology vol 79 no 21 pp6677ndash6683 2013
[30] J L Martinez ldquoEnvironmental pollution by antibiotics andby antibiotic resistance determinantsrdquo Environmental Pollutionvol 157 no 11 pp 2893ndash2902 2009
