Antibiogram, adhesive characteristics, and incidence of class 1 integron in Aeromonas species...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 127570 8 pageshttpdxdoiorg1011552013127570

Research ArticleAntibiogram Adhesive Characteristics and Incidence ofClass 1 Integron in Aeromonas Species Isolated from Two SouthAfrican Rivers

Isoken H Igbinosa Vincent N Chigor Etinosa O IgbinosaLawrence C Obi and Anthony I Okoh

Applied and Environmental Microbiology Research Group (AEMREG) Department of Biochemistry and MicrobiologyUniversity of Fort Hare Private Bag X1314 Alice 5700 South Africa

Correspondence should be addressed to Isoken H Igbinosa ladyisokengmailcom

Received 29 May 2013 Revised 25 August 2013 Accepted 27 August 2013

Academic Editor Yanmin Hu

Copyright copy 2013 Isoken H Igbinosa 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

Aeromonas species are well distributed in freshwater environments and their natural susceptibility to antimicrobials rendersthem interesting candidates for the survey of antimicrobial resistance in freshwater milieu Water samples were collected fromKat and Tyume rivers in the Eastern Cape province of South Africa and a total of 45 isolates identified as Aeromonas specieswere recovered from the two rivers All Aeromonas isolates were resistant to oxacillin penicillin clindamycin cephalothinvancomycin and rifamycin while appreciable susceptibilities (893 941 821 941 857 882 and 929 882)were observedagainst ciprofloxacin chloramphenicol nitrofurantoin and gentamicin fromKat andTyume rivers respectivelyMultiple antibioticresistance (MAR) indices ranged from0016 to 0044 for the two rivers Class 1 integronwas detected in about 20of the isolates andall the isolates except one showed ability to produce biofilm in vitro as weak producers (5333) moderate producers (1556) andstrong producers (289) This investigation provides a baseline data on antibiotic resistance as well as the adhesive characteristicsof Aeromonas isolates from Tyume and Kat rivers in the Eastern Cape province of South Africa

1 Introduction

Aeromonas species are Gram-negative rod-shaped non-spore-forming facultatively anaerobic bacteria that occurubiquitously and autochthonously in aquatic environmentsTheAeromonas genus has undergone a number of taxonomicand nomenclature revisions in the past two decades [1]Initially Aeromonas was placed in the family Vibrionaceaebut successive phylogenetic analyses revealed thatAeromonasis not closely related to Vibrios and resulted in movingAeromonas to a new family the Aeromonadaceae [2 3]Aeromonads share in common many biochemical character-istics with members of the Enterobacteriaceae however theyare easily differentiated by being oxidase positive

Aeromonas species are agents of infection in fish [4] andare associated with human diarrheal diseases and woundinfections which may result due to contact with contami-nated water [5 6] Wound infections may become severe

and systemic Aeromonas is also associated with sepsisrespiratory tract eye and other systemic infections [7] Infish Aeromonas causes bacterial infections which may poserelatively high resistance to antibiotics including clinicallyrelevant cases and diseases [8] Treatment of Aeromonasinfection is usually with the use of antibiotics howeverantimicrobial resistance canmake these infections difficult totreat

The ubiquity of Aeromonas species in aquatic ecosystemsand their natural susceptibility to antimicrobials renderthem interesting candidates for the survey of antimicrobialresistance in freshwater environments [9 10] Freshwaterstreams are usually receptors of many industrial domesticand agricultural wastes which could contain antimicrobialagents and antimicrobial-resistant bacteria [10 11] Due todiverse microbial population in such ecosystems freshwaterenvironment provides favourable conditions for the spread ofantimicrobial resistance

2 BioMed Research International

Aquatic bacteria such as Aeromonas may become reser-voirs of antibiotic resistance determinants as a result ofinfluents from diverse sources entering into the river and assuch may transfer these antibiotic resistance determinants toother aquatic organisms including pathogenic bacteria [10]In the United States a study has shown that several rivers arebecoming major reservoirs of antibiotic resistance microor-ganisms [12] With widespread commerce and global travelantibiotic resistant organisms can spread across the globeThe occurrence and distribution of Aeromonas in aquaticecosystems its emerging significance as a contaminant ofwater and the pathogenic potential mediated by mesophilicAeromonas species are all of public health concern [13 14]The ability of bacteria to develop multiple-drug resistanceresult in part from their ability to acquire new antibioticresistance genes Mobile elements called integrons determinea site-specific recombination system that is responsible for theacquisition of many antibiotic resistance determinants [1516]

Bacterial adherence to surfaces is one of the initial stepsleading to biofilm formation and is therefore a significantmicrobiological event in medicine and the environment [1718] Aeromonas hydrophila can attach to and form biofilmson polystyrene glass surface stainless steel and polyvinylchloride[19] Aeromonas can also attach to solid surfacesand form biofilms in aquatic environments The presenceof Aeromonas in biofilm samples from water distributionsystems in South Africa has been documented [20] Biofilmis an irreversible growth of a combination of bacterial micro-colonies on surfaces entrenched in extracellular polysaccha-ride matrix [21 22] The formation of biofilm results inresistance of bacteria to antimicrobial drugs and persistentinfections [21] which can lead to the severity of severalbacterial diseases affecting both human [23] and animalhealth [24]

AlthoughAeromonas species are well distributed in fresh-water habitat and have been assigned as an emerging threatto human health [3 25] no data exists about the antibioticsusceptibility profiles and adhesive properties of aeromonadsfrom Kat and Tyume rivers in the Eastern Cape province ofSouth AfricaThe Food and Agriculture OrganizationWorldHealthOrganization (FAOWHO) commission recommendsthat to prevent waterborne diseases in developing countriesaquatic environments having direct impact on human pop-ulations should be characterized physically chemically andmicrobiologically In view of this recommendation and aspart of our surveillance of reservoir of antibiotic resistantcommensal bacteria this present study aimed to (i) evaluatethe levels of antimicrobial resistance in aeromonads isolatesfrom Kat and Tyume rivers (ii) determine the presenceof class 1 and 2 integron associated gene cassette and (iii)evaluate their biofilm forming capabilities

2 Materials and Methods

21 Study Area Sampling and Processing of Samples Katriver is located in a semiurban location at geographicalcoordinates S32∘465471015840 E026∘384561015840 while Tyume river is

located in a rural community at geographical coordinatesS32∘472791015840 E026∘ 505201015840 in the Eastern Cape province ofSouth Africa Water samples were collected four times atrandom between April 2011 and March 2012 Water sampleswere collected in duplicates using 2 L bottles placed on ice andtransported to the laboratory for analysisHundredmicroliter(100 120583L) was spread on several Glutamate phenol(GSP) agar(biolab merck SA) on the other hand 500 120583L was inoculatedinto 145 mL sterile nutrient broth and incubated in a rotaryincubator at 150 rpm overnight at 36∘C At the end of theincubation period a loopful of the culture was spread andorstreaked on GSP agar and all agar plates were incubatedat 36∘C for 24 h Typical yellow colonies on GSP agar werepurified using the samemedia Pure colonieswere transferredunto nutrient agar plates and subjected to oxidase andcatalase tests Oxidase and catalase positive isolates werefurther screened for biochemical characteristics using API20NE kit The strips were then read and final identificationwas made using API lab plus software (bioMerieux MarcylrsquoEtoile France)

22 Antimicrobial Susceptibility Testing Isolates were sub-cultured on nutrient agar plates incubated for 24 h at 36∘CColonies were picked from the agar plates and suspendedin normal saline (085 wv) and adjusted to an A

560

value of 012 plusmn 002 (05 McFarland standard) The bacterialsuspension was spread on the Mueller Hinton agar platesusing a sterile swab stick allowed to dry and impregnatedwith antibiotic disk The antibiotics used were as followsciprofloxacin (5120583g) trimethoprim (5120583g) chloramphenicol(3 120583g) penicillins (10 120583g) clindamycins (2120583g) ofloxacin(5 120583g) ampicillin-sulbactam (20120583g) oxacillin (1 120583g) ampi-cillin (25 120583g) gentamicin (10 120583g) nalidixic acid (30 120583g) cefo-taxime (30 120583g) nitrofurantoin (300 120583g) sulfamethoxazole(25 120583g) cephalothin (30 120583g) erythromycin (15 120583g) tetracy-cline (10 120583g) minocycline (30 120583g) vancomycin (30120583g) andrifampicin (5120583g) Disks were purchased from Mast Diag-nostics (Mast Group Merseyside UK) Plates were incubatedat 36∘C for 24 h Diameters of the zones of inhibitionwere measured and interpreted as susceptible intermedi-ate or resistant according to the Clinical Laboratory Stan-dard Guidelines [26] The frequency of antibiotic-resistantAeromonas isolates was calculated by the following equation119860119861 times 100 where 119860 is the number of isolates resistant toan antibiotic and 119861 is the total number of isolates from thesample The multiple antibiotic resistance (MAR) index ofeach samples was estimated by the following equation 119886(119887times119888) where 119886 represents the aggregate antibiotic resistance scoreof all isolates from the sample 119887 represents the number ofantibiotics and 119888 represents the number of isolates from thesample as outlined in [27ndash29]

23 PCR Detection of Integrons DNA was extracted fol-lowing the method described elsewhere [30 31] TemplateDNA was stored at minus20∘C until it was ready for use Theprimer used for the detection of class 1 and class 2 integronis shown in Table 1 The PCR conditions were as followsinitial denaturation at 94∘C for 2min followed by 30 cycles

BioMed Research International 3

Table 1 Primer set for the detection of class 1 and 2 integron

Target gene Primer sequence 51015840 rarr 31015840 Size Reference

Class 1 integron GGC ATC CAA GCA GCA AG Variable [55]GGC ATC CAA GCA GCA AG

Class 2 integron CGG GAT CCC CGG CAT GCA CGA TTT GTA Variable [55]GAT GCC ATC GCA AGT ACG AG

of denaturation at (95∘C for 45 s) annealing (56∘C for 1min)extension (72∘C for 90 s) and a final extension at 72∘C for10min

24 Biofilm Formation Assay Quantitatively biofilm forma-tion among Aeromonas isolates was assessed using microtitreplatemethod described by Stepanovic et al [32] andOdeyemiet al [22] with modification Wells of 96 flat bottomedmicrotiter plates were filled with 200120583L of Tryptone SoyBroth (TSB) and inoculated with 20120583L ofAeromonas isolatesgrown overnight and standardized to 05 McFarland stan-dard Plates were incubated at 37∘C for 24 h Positive controlwells contained Aeromonas hydrophila ATCC 7699 and thenegative control wells contained uninoculated Tryptone SoyBroth Contents of each well were aspirated and washed threetimes with sterile phosphate-buffered saline (PBS) After air-drying wells were stained with 200 120583L of 1 crystal violet for30min The wells were carefully washed with distilled waterto remove the excess stain Plates were allowed to dry at roomtemperature Dye bound to adherent cells was resolubilizedwith 150 120583L of absolute ethanol Microplate reader (Synergymx Biotek USA) was used to read the plates at 570 nmwavelength Average optical density (OD) of each duplicateresult was taken including positive and negative controlsIsolates were categorized as nonbiofilm producer (ODi ltODc) weak (ODc lt ODi lt 01) moderate (ODi = 01 lt 012)and strong (ODi gt 012) producers according to the modifiedmethods of [33 34]

3 Statistical Analysis

Susceptibility data were compared by using a Chi-square testwith SPSS software for Windows version 170 Both suscepti-bility and resistance were calculated as percentages with 95confidence intervals A 119875 value lt 005 was considered to bestatistically significant

4 Results

A total of 45 isolates (17 from Kat river and 28 from Tyumeriver) were identified as Aeromonas hydrophilacaviae Theisolates were evaluated for their antibiotic susceptibilitiesGenerally all isolates were resistant to oxacillin penicillinclindamycin cephalothin vancomycin and rifamycin whileover 80 of the isolates were susceptible to ciprofloxacinchloramphenicol gentamicin and nitrofurantoin (Table 2)Isolates from both rivers showed a diversified resistancetrend to the same antibiotics For example all the Aeromonasisolates from Tyume river were resistant to ofloxacin while

100

90

80

70

60

50

40

30

20

10

0

CIP

TMC PG CD O

FXSA

M OX AP

GM NA

CTX NI

SMX KF E T

MN VA RP

Kat riverTyume river

AntibioticRe

sista

nce f

requ

ency

()

Figure 1 The Frequency of antibiotic-resistant Aeromonas isolatesfrom Kat and Tyume rivers CIP ciprofloxacin TM trimetho-prim C chloramphenicol PG penicillin CD clindamycin OFXofloxacin SAM ampicillin-sulbactam OX oxacillin AP ampi-cillin GM gentamicin NA nalidixic acid CTX cefotaxime NInitrofurantoin SMX sulfamethoxazole KF cephalothin E ery-thromycin T tetracycline MN minocycline VA vancomycin RPrifamycin

893 of isolates from Kat river were susceptible to ofloxacin(Table 2) Also 706 of the isolates from Tyume riverwere resistant against tetracycline while 821 of the isolatesfrom Kat river were susceptible to the antibiotic SimilarlyAeromonas isolates showed 941 resistance and 607 sus-ceptibility against trimethoprim and 823 resistance and786 susceptibility against cefotaxime from Tyume and Katrivers respectively as shown in Table 2

The frequency of antibiotic resistance among theAeromonas isolates from Kat and Tyume rivers is shown inFigure 1 The MAR index of Kat river ranged from 0026to 0044 with a mean value of 0034 The highest MARindex was observed against only one isolate (MAR 0044)while six isolates exhibited MAR index of 0029 Aeromonasisolates from Tyume river demonstrated MAR index whichranged from 0016 to 0029 with a mean value of 00214Twelve isolates exhibited the lowest MAR index of 0016PCR amplification of class 1 and class 2 integron detectedclass 1 integron in 20 of Aeromonas isolates while class 2integron was not detected

Aeromonas in this study was able to form biofilms invitro on a polystyrene microtitre plate Optical densities andbiofilm production status of Aeromonas isolates from Katand Tyume rivers are as shown in Table 3 Isolates were


Table 2 Antibiogram of Aeromonas isolates recovered from Kat river and Tyume river

Antibiotics Kat river () Tyume river ()119878 119868 119877 119878 119868 119877

Ciprofloxacin 893 0 107 941 59 0Trimethoprim 607 321 714 59 0 941Chloramphenicol 821 179 0 941 59 0Penicillin 0 0 100 0 0 100Clindamycin 0 0 100 0 0 100Ofloxacin 893 714 36 0 0 100Ampicillin-sulbactam 36 0 964 0 0 100Oxacillin 0 0 100 0 0 100Ampicillin 0 0 100 59 0 941Gentamicin 929 0 714 882 0 118Nalidixic acid 143 36 821 706 0 294Cefotaxime 786 143 714 118 59 823Nitrofurantoin 857 36 107 882 118 0Sulfamethoxazole 0 36 964 0 0 100Cephalothin 0 0 100 0 0 100Erythromycin 714 571 357 588 59 352Tetracycline 821 214 714 177 118 706Minocycline 429 571 0 235 118 647Vancomycin 0 0 100 0 0 100Rifampicin 0 0 100 0 0 100

30

25

20

15

10

5

0

Num

ber o

f org

anism

s

Non biofilmproducers producers producers producers

Weak biofilm Moderate biofilm Strong biofilm

Figure 2 Occurrence of biofilm production of Aeromonas isolatesin microtitre plates

categorized into four groups according to the criteria asdescribed above Weak producers of biofilm which makeup 5333 of the isolates were highest among the groupsclassified Thirteen isolates (289) were strong producerswhile 7 (1556) demonstrated moderate capabilities forbiofilm production (Figure 2) Table 4 shows the adherentcharacteristics of Aeromonas isolates from Tyume and Katrivers

5 Discussion

The presence of multidrug resistant bacteria in surfacewater is a major public health burden as drug resistant

bacteria could be transferred to humans by means of drink-ing contaminated water which subsequently contributes tothe spread and persistence of antibiotic resistance bacteriain general population and environment [29] Aeromonasspecies are well distributed in freshwater ecosystems andtheir potential to enter distribution systems increases asa result of ineffective water treatment [35] The result ofthis investigation demonstrates the presence of multidrugresistant aeromonads in the two rivers assayed Absoluteresistance was observed against several antibiotics with nocomplete susceptibility to any however appreciable suscep-tibility was observed against ciprofloxacin chloramphenicoland nitrofurantoin Considerable susceptibility was observedfor the aminoglycosides tested (gentamicin) with highersusceptibility occurring with isolates from Kat river Ourfindings corroborate with the study carried out by Abulhamd[1] which reports susceptibility ofAeromonas species isolatedfrom environmental water source to gentamicin Similarfindings of aminoglycosides susceptibility fromwater sourceshave been observed [36]

Resistance to the penicillin group of antibiotics wasobserved which can be attributed to Aeromonas instinc-tive resistance to penicillins especially to ampicillin due tothe production of 120573-lactamases Among the cephalosporinstested 100 resistance was observed against cephalothinrelative susceptibility was observed against cefotaxime withisolates from Kat river and insignificant susceptibility withAeromonas isolates from Tyume river Bizani and Brandelli[36] reported 100 resistance of Aeromonas species isolatedfrom water distribution system used in a bovine abattoirhowever in contrast to our observation other studies have


Table 3 Optical densities and biofilm production status ofAeromonas species isolated from both Kat and Tyume rivers

Isolate code Isolate source(river)

Mean ODi plusmn SD Biofilmproducing status

Control (TSB) 0069 plusmn 0003

ISKAE001 Kat 0084 plusmn 0006 WeakISKAE002 Kat 00725 plusmn 0015 WeakISKAE003 Kat 0092 plusmn 0021 WeakISKAE004 Kat 0095 plusmn 0013 WeakISKAE005 Kat 0102 plusmn 0024 ModerateISKAE006 Kat 0073 plusmn 0004 WeakISKAE007 Kat 0076 plusmn 0037 WeakISKAE008 Kat 0128 plusmn 0056 StrongISKAE009 Kat 0123 plusmn 0035 StrongISKAE010 Kat 0098 plusmn 0009 WeakISKAE011 Kat 0085 plusmn 0004 WeakISKAE012 Kat 0119 plusmn 0028 StrongISKAE020 Kat 0083 plusmn 0016 WeakISKWA003 Kat 0097 plusmn 0006 WeakISKWA007 Kat 0073 plusmn 0027 WeakISKWA013 Kat 0092 plusmn 0045 WeakISKWA034 Kat 0096 plusmn 003 WeakISKJA001 Tyume 0105 plusmn 0019 ModerateISKJA006 Tyume 0137 plusmn 0018 StrongISKJA008 Tyume 0080 plusmn 0009 WeakISKJA011 Tyume 0132 plusmn 004 StrongISKJA014 Tyume 0074 plusmn 0015 WeakISKJA015 Tyume 014 plusmn 0002 StrongISKJA016 Tyume 0092 plusmn 0021 WeakISKJA017 Tyume 0093 plusmn 0005 WeakISKJA018 Tyume 0152 plusmn 0007 StrongISKJA019 Tyume 0134 plusmn 0012 StrongISKJA 021 Tyume 0142 plusmn 0009 StrongISKJA 030 Tyume 0065 plusmn 0003 Non-producerISKJA 031 Tyume 0093 plusmn 0015 WeakISKJA 032 Tyume 0188 plusmn 0002 StrongISKJA 033 Tyume 0163 plusmn 0007 StrongISKJA 038 Tyume 0078 plusmn 0006 WeakISKJA 053 Tyume 0082 plusmn 0007 WeakISKJA054 Tyume 0092 plusmn 0004 WeakISKJA 055 Tyume 0119 plusmn 0013 ModerateISKJA 056 Tyume 0089 plusmn 0002 WeakISKJA 062 Tyume 0098 plusmn 0008 WeakISKJA 064 Tyume 0104 plusmn 0009 ModerateISKWA 033 Tyume 012 plusmn 0002 ModerateISKWA 035 Tyume 0117 plusmn 0011 ModerateISKWA 060 Tyume 013 plusmn 0007 StrongISKXA 063 Tyume 0095 plusmn 0004 WeakISKXA 074 Tyume 0149 plusmn 0003 StrongISKXA 076 Tyume 0114 plusmn 0017 Moderate

Table 4 Adherence capability of Aeromonas isolates from Kat andTyume rivers

River No of isolates Adherence propertiesNA WA MA SA

Kat 17 0 13 1 3Tyume 28 1 11 6 10NA nonadherent WA weakly adherent MA moderately adherent and SAstrongly adherent

documented a rare incidence of cephalosporin resistancefrom Aeromonas isolated from water sources [37 38]

Aeromonas isolates from both sources showed similarbehaviours to some antibiotics for instance isolates fromboth sources showed high level of susceptibility against chlo-ramphenicol and none was resistant Similarly none of theisolates were susceptible to sulfamethoxazole or cephalothinwhile the isolates demonstrated a diversified variation intheir susceptibility and resistance against trimethoprim andnalidixic acids These differences in resistance patterns toantibiotics of the isolates from Kat and Tyume rivers couldbe as a result of sampling locations (rural and semiurban)anthropogenic activities and other environmental factors

Variable susceptibility against the quinolones wasobserved for Aeromonas isolates from both sources butofloxacin showed a distinct pattern as it showed 89susceptibility with isolates from Kat river and absoluteresistance against isolates from Tyume river The reason forthese differences may be attributed to enzymatic conductionand selective environmental pressure of Aeromonas isolatesfrom these two settings The presence of multidrug resistantAeromonas hydrophila from aquatic animals including fish[39] eel and catfish [40 41] could be as a result of widespreaddistribution of multidrug resistance among A hydrophila inaquatic (freshwater) habitat hence the need for continuoussurveillance of emerging and resistance pool of antibioticresistance determinant

Integrons are elements that encode a site-specific recom-bination system that identifies and captures mobile genecassettes and are closely related to multiple resistances ofenvironmental microorganisms [42] The role of class 1 inte-grons in conferring antibiotic resistance to clinical isolatesof different bacterial strains is well documented [43ndash45]Studies have also shown link between incidence of class 1integron and antibiotic resistance in bacterial pathogens suchas Escherichia coli and Aeromonas salmonicida [46 47] Theincidences of integron-bearing Aeromonas isolates in ourstudy suggest that these isolates are potential contaminantsand that have the possibility for horizontal gene transferexits Lukkana et al [41] has documented the presence ofclass 1 integron in Aeromonas hydrophilia isolated from NileTilapia in Thailand Rosser and Young [48] documented theincidence of class 1 integrons in 36 of bacteria isolated fromthe Tay estuary and Lin and Biyela [49] reported the presenceof class 1 integron in 58 of Enterobacteriaceae isolated fromMhlathuze river in KwaZulu-Natal Republic of South Africaand this may imply a wide distribution and persistence ofclass 1 integron in South African aquatic milieuThe presence


of integron in a wide variety of bacteria and in differenthabitat substantiates the horizontal mobility and stability ofthis gene capture system [50]

Biofilm-producing bacteria have been shown to be associ-ated with numerous human diseases and capable of coloniz-ing a wide range of environments In aquatic environmentmicrobial adhesion initiates biofilm formation exacerbatescontamination reduces the aesthetic quality of the waterbody and reduces microbiological safety through augmentedsurvival of pathogens [18 51]The result obtained in this studyis consistent with the observation of Saidi et al [52] whofound high percentage (95) of biofilm forming Aeromonasspecies isolated from a river near the seacoast of MonastirTunisia Also our findings are also similar to the result docu-mented by Odeyemi et al [22] who foundAeromonas isolatesfrom estuary in Malaysia to be biofilm formers with highpercentage of weak biofilm producers and strong producerstrailing as the second in occurrence This is the first carriedout study on the adhesive properties of environmental Ahydrophila strains isolated from aquatic source in the EasternCape province of South Africa Ability of Aeromonas toform biofilm in aquatic environment enhances the recyclingof nutrients and minerals in aquatic environment therebypromoting the growth of potential pathogen in the aquaticmilieu Motility and flagella play a vital role in adhesionbiofilm formation and colonization of several pathogenicbacteria such asAeromonas hydrophila [52 53] Our previousstudy has shown that Aeromonas isolates from the studiedmicrohabitat possess some virulent potential [31] and theability of these virulent strains to form biofilm in aquaticenvironment may enhance an elevated waterborne dispersalcapacity an attribute that has been related to an elevated riskof bacterial transmission and infectivity [54] Biofilm forma-tion and development by microorganisms play a vital rolein the pathogenesis of a disease hence the biofilm formingcapability demonstrated in vitro by Aeromonas isolates fromTyume and Kat rivers further suggests enhanced pathogenicstatus of these isolates

6 Conclusion

This study provides a baseline data on the antibiotic resistanceprofile of Aeromonas species isolated from Kat and Tyumerivers in the EasternCape province of SouthAfricaThe resultof this study reveals that the antibiotic resistance patterns ofAeromonas species isolated from the two rivers and the inci-dence of class 1 integron suggest the possibility of horizontalgene transfer of antibiotic resistance determinants in theseisolatesThe result of this study shows thatAeromonas strainsfromKat andTyume rivers have ability to bind to surfaces andform biofilms which is of public health significance as biofilmformation results in resistance of bacteria to conventionalantimicrobial agents and persistent infections

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

The authors are grateful to the University of Fort Hare for thefinancial support

References

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[2] R R Colwell M T MacDonell and J De Ley ldquoProposal torecognize the family Aeromonadaceae fam novrdquo InternationalJournal of Systematic Bacteriology vol 36 no 3 pp 473ndash4771986

[3] I H Igbinosa E U Igumbor F Aghdasi M Tom and AI Okoh ldquoEmerging Aeromonas species infections and theirsignificance in public healthrdquo The Scientific World Journal vol2012 Article ID 625023 13 pages 2012

[4] M J Saavedra S Guedes-Novais A Alves et al ldquoResistanceto 120573-lactam antibiotics in Aeromonas hydrophila isolated fromrainbow trout (Oncorhynchus mykiss)rdquo International Microbiol-ogy vol 7 no 3 pp 207ndash211 2004

[5] I Kuhn G Allestam G Huys et al ldquoDiversity persistence andvirulence of Aeromonas strains isolated from drinking waterdistribution systems in Swedenrdquo Applied and EnvironmentalMicrobiology vol 63 no 7 pp 2708ndash2715 1997

[6] S W Joseph ldquoAeromonas gastrointestinal disease a case studyin causationrdquo inTheGenus Aeromonas B Austin M AltweggP J Gosling and S W Joseph Eds pp 311ndash326 John Wiley ampSons Chichester UK 1996

[7] J M Janda and P S Duffey ldquoMesophilic aeromonads inhuman disease current taxonomy laboratory identificationand infectious disease spectrumrdquo Reviews of Infectious Diseasesvol 10 no 5 pp 980ndash997 1988

[8] M J Figueras A AlperiM J Saavedra et al ldquoClinical relevanceof the recently described species Aeromonas aquariorumrdquoJournal of Clinical Microbiology vol 47 no 11 pp 3742ndash37462009

[9] J R Huddleston J C Zak and R M Jeter ldquoAntimicrobialsusceptibilities of Aeromonas spp isolated from environmentalsourcesrdquo Applied and Environmental Microbiology vol 72 no11 pp 7036ndash7042 2006

[10] L Gordon A Cloeckaert B Doublet et al ldquoComplete sequenceof the floR-carrying multiresistance plasmid pAB5S9 fromfreshwater Aeromonas bestiarumrdquo Journal of AntimicrobialChemotherapy vol 62 no 1 pp 65ndash71 2008

[11] B Halling-Soslashrensen S Nors Nielsen P F Lanzky F IngerslevH C Holten Lutzhoslashft and S E Joslashrgensen ldquoOccurrence fateand effects of pharmaceutical substances in the environment areviewrdquo Chemosphere vol 36 no 2 pp 357ndash393 1998

[12] R J Ash B Mauck and M Morgan ldquoAntibiotic resistanceof gram-negative bacteria in rivers United Statesrdquo EmergingInfectious Diseases vol 8 no 7 pp 713ndash716 2002

[13] N Borrell M J Figueras and J Guarro ldquoPhenotypic identifi-cation of Aeromonas genomospecies from clinical and environ-mental sourcesrdquo Canadian Journal of Microbiology vol 44 no2 pp 103ndash108 1998

[14] S W Joseph and A M Carnahan ldquoUpdate on the genusAeromonasrdquo ASM News vol 66 pp 218ndash223 2000


[15] R M Hall and H W Stokes ldquoIntegrons novel DNA elementswhich capture genes by site-specific recombinationrdquo Geneticavol 90 no 2-3 pp 115ndash132 1993

[16] R M Hall and C M Collis ldquoMobile gene cassettes andintegrons capture and spread of genes by site-specific recom-binationrdquo Molecular Microbiology vol 15 no 4 pp 593ndash6001995

[17] S Bayoudh L Ponsonnet H B Ouada A Bakhrouf andA Othmane ldquoBacterial detachment from hydrophilic andhydrophobic surfaces using a microjet impingementrdquo Colloidsand Surfaces A vol 266 no 1ndash3 pp 160ndash167 2005

[18] L C Simoes N Azevedo A Pacheco C W Keevil and M JVieira ldquoDrinking water biofilm assessment of total and cultur-able bacteria under different operating conditionsrdquo Biofoulingvol 22 no 2 pp 91ndash99 2006

[19] S M Kirov M Castrisios and J G Shaw ldquoAeromonas flagella(polar and lateral) are enterocyte adhesins that contribute tobiofilm formation on surfacesrdquo Infection and Immunity vol 72no 4 pp 1939ndash1945 2004

[20] S M September F A Els S N Venter and V S BrozelldquoPrevalence of bacterial pathogens in biofilms of drinking waterdistribution systemsrdquo Journal of Water and Health vol 5 no 2pp 219ndash227 2007

[21] RM Donlan ldquoBiofilm formation a clinically relevant microbi-ological processrdquo Clinical Infectious Diseases vol 33 no 8 pp1387ndash1392 2001

[22] O A Odeyemi A Asmat and G Usup ldquoAntibiotics resistanceand putative virulence factors ofAeromonas hydrophila isolatedfrom estuaryrdquo Journal of Microbiology Biotechnology and FoodSciences vol 1 no 6 pp 1339ndash1357 2012

[23] H M Probert and G R Gibson ldquoBacterial biofilms in thehuman gastrointestinal tractrdquoCurrent Issues in Intestinal Micro-biology vol 3 no 2 pp 23ndash27 2002

[24] M BMelchior H Vaarkamp and J Fink-Gremmels ldquoBiofilmsa role in recurrent mastitis infectionsrdquo Veterinary Journal vol171 no 3 pp 398ndash407 2006

[25] M J Figueras A Suarez-Franquet M R Chacon et al ldquoFirstrecord of the rare species Aeromonas culicicola from a drinkingwater supplyrdquo Applied and Environmental Microbiology vol 71no 1 pp 538ndash541 2005

[26] Clinical and Laboratory Standards Institute (CLSI) Methodsfor Dilution of Antimicrobial Susceptibility Tests for Bacteriathat Grow Aerobically Approved Standard M7-A7 Clinical andLaboratory Standards Institute Wayne Pa USA 7th edition2006

[27] P H Krumperman ldquoMultiple antibiotic resistance indexing ofEscherichia coli to identify high-risk sources of fecal contami-nation of foodsrdquo Applied and Environmental Microbiology vol46 no 1 pp 165ndash170 1983

[28] C D Miranda and R Zemelman ldquoAntimicrobial multiresis-tance in bacteria isolated from freshwater Chilean salmonfarmsrdquo Science of the Total Environment vol 293 no 1ndash3 pp207ndash218 2002

[29] R Tao G G Ying H C Su H W Zhou and J P S SidhuldquoDetection of antibiotic resistance and tetracycline resistancegenes in Enterobacteriaceae isolated from the Pearl rivers inSouth Chinardquo Environmental Pollution vol 158 no 6 pp 2101ndash2109 2010

[30] J Sambrook andDW RussellMolecular Cloning A LaboratoryManual Cold Spring Harbor Laboratory Press Cold SpringHarbor NY USA 3rd edition 2001

[31] I H Igbinosa and A I Okoh ldquoDetection and distribution ofputative virulence associated genes in Aeromonas species fromfreshwater and wastewater treatment plantrdquo Journal of BasicMicrobiology 2013

[32] S Stepanovic D Vukovic I Davic B Savic and M Svabic-Vlahovic ldquoA modified microtiter-plate test for quantificationof staphylococcal biofilm formationrdquo Journal of MicrobiologicalMethods vol 40 no 2 pp 175ndash179 2000

[33] N Cevahir M Demir I Kaleli M Gurbuz and S TikveslildquoEvaluation of biofilm production gelatinase activity andmannose-resistant hemagglutination in Acinetobacter bau-mannii strainsrdquo Journal of Microbiology Immunology and Infec-tion vol 41 no 6 pp 513ndash518 2008

[34] A Hassan J Usman F Kaleem M Omair A Khalid andM Iqbal ldquoEvaluation of different detection methods of biofilmformation in the clinical isolatesrdquo Brazilian Journal of InfectiousDiseases vol 15 no 4 pp 305ndash311 2011

[35] P Holmes L M Niccolls and D P Sartory ldquoThe ecologyof mesophilic Aeromonas in the aquatic environmentrdquo in TheGenus Aeromonas B Austin M Altwegg P J Gosling and SJoseph Eds pp 127ndash150 Wiley New York NY USA 1996

[36] D Bizani andA Brandelli ldquoAntimicrobial susceptibility hemol-ysis and hemagglutination among Aeromonas spp isolatedfrom water of a bovine abattoirrdquo Brazilian Journal of Microbi-ology vol 32 no 4 pp 334ndash339 2001

[37] C D Miranda and G Castillo ldquoResistance to antibiotic andheavy metals of motile aeromonads from Chilean freshwaterrdquoScience of the Total Environment vol 224 no 1ndash3 pp 167ndash1761998

[38] M Goni-Urriza M Capdepuy C Arpin N Raymond and CQ Pierre Caumette ldquoImpact of an urban effluent on antibioticresistance of riverine Enterobacteriaceae and Aeromonas spprdquoApplied and Environmental Microbiology vol 66 no 1 pp 125ndash132 2000

[39] M Hatha A A Vivekanandhan G Julie Joice and C ChristolldquoAntibiotic resistance pattern of motile aeromonads from farmraised fresh water fishrdquo International Journal of Food Microbiol-ogy vol 98 no 2 pp 131ndash134 2005

[40] J Penders and E E Stobberingh ldquoAntibiotic resistance ofmotileaeromonads in indoor catfish and eel farms in the southernpart ofThe Netherlandsrdquo International Journal of AntimicrobialAgents vol 31 no 3 pp 261ndash265 2008

[41] M Lukkana J Wongtavatchai and R Chuanchuen ldquoClass 1integrons in Aeromonas hydrophila isolates from farmed NileTilapia (Oreochromis nilotica)rdquo Journal of Veterinary MedicalSciences vol 74 no 4 pp 435ndash440 2012

[42] L Ma X X Zhang S Cheng et al ldquoOccurrence abundanceand elimination of class 1 integrons in one municipal sewagetreatment plantrdquo Ecotoxicology vol 20 no 5 pp 968ndash973 2011

[43] D Kucken H H Feucht and P M Kaulfers ldquoAssociation ofqacE and qacEΔ1 with multiple resistance to antibiotics andantiseptics in clinical isolates of Gram-negative bacteriardquo FEMSMicrobiology Letters vol 183 no 1 pp 95ndash98 2000

[44] PAWhite andWDRawlinson ldquoCurrent status of the aadAdfrgene cassette familiesrdquo Journal of Antimicrobial Chemotherapyvol 47 no 4 pp 495ndash496 2001

[45] M A Leverstein-Van Hall H E M Blok A R T Donders APaauw A C Fluit and J Verhoe ldquoMultidrug resistance amongenterobacteriaceae is strongly associated with the presenceof integrons and is independent of species or isolate originrdquoJournal of Infectious Diseases vol 187 no 2 pp 251ndash259 2003


[46] H Soslashrum T M LrsquoAbee-Lund A Solberg and A WoldldquoIntegron-containing IncU R plasmids pRAS1 and pAr-32 fromthe fish pathogenAeromonas salmonicidardquoAntimicrobial Agentsand Chemotherapy vol 47 no 4 pp 1285ndash1290 2003

[47] W H Gaze N Abdouslam P M Hawkey and E M HWellington ldquoIncidence of class 1 integrons in a quaternaryammonium compound-polluted environmentrdquo AntimicrobialAgents and Chemotherapy vol 49 no 5 pp 1802ndash1807 2005

[48] S J Rosser and H K Young ldquoIdentification and charac-terization of class 1 integrons in bacteria from an aquaticenvironmentrdquo Journal of Antimicrobial Chemotherapy vol 44no 1 pp 11ndash18 1999

[49] J Lin and P T Biyela ldquoConvergent acquisition of antibioticresistance determinants amongst the Enterobacteriaceae iso-lates of the Mhlathuze River KwaZulu-Natal (RSA)rdquoWater SAvol 31 no 2 pp 257ndash260 2005

[50] V Dubois M P Parizano C Arpin L Coulange M C Bezianand C Quentin ldquoHigh genetic stability of integrons in clinicalisolates of Shigella spp of worldwide originrdquo AntimicrobialAgents and Chemotherapy vol 51 no 4 pp 1333ndash1340 2007

[51] Y P Tsai ldquoSimulation of biofilm formation at different assim-ilable organic carbon concentrations under lower flow velocityconditionrdquo Journal of Basic Microbiology vol 45 no 6 pp 475ndash485 2005

[52] N Saidi M Snoussi D Usai S Zanetti and A BakhroufldquoAdhesive properties of Aeromonas hydrophila strains isolatedfromTunisian aquatic biotopesrdquoAfrican Journal ofMicrobiologyResearch vol 5 no 31 pp 5644ndash5655 2011

[53] A A Rabaan I Gryllos J M Tomas and J G Shaw ldquoMotilityand the polar flagellum are required for Aeromonas caviaeadherence to HEp-2 cellsrdquo Infection and Immunity vol 69 no7 pp 4257ndash4267 2001

[54] C A Fux J W Costerton P S Stewart and P Stoodley ldquoSur-vival strategies of infectious biofilmsrdquo Trends in Microbiologyvol 13 no 1 pp 34ndash40 2005

[55] M Nawaz S A Khan A A Khan et al ldquoDetection andcharacterization of virulence genes and integrons inAeromonasveronii isolated from catfishrdquo Food Microbiology vol 27 no 3pp 327ndash331 2010

Submit your manuscripts athttpwwwhindawicom

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Microbiology


Aquatic bacteria such as Aeromonas may become reser-voirs of antibiotic resistance determinants as a result ofinfluents from diverse sources entering into the river and assuch may transfer these antibiotic resistance determinants toother aquatic organisms including pathogenic bacteria [10]In the United States a study has shown that several rivers arebecoming major reservoirs of antibiotic resistance microor-ganisms [12] With widespread commerce and global travelantibiotic resistant organisms can spread across the globeThe occurrence and distribution of Aeromonas in aquaticecosystems its emerging significance as a contaminant ofwater and the pathogenic potential mediated by mesophilicAeromonas species are all of public health concern [13 14]The ability of bacteria to develop multiple-drug resistanceresult in part from their ability to acquire new antibioticresistance genes Mobile elements called integrons determinea site-specific recombination system that is responsible for theacquisition of many antibiotic resistance determinants [1516]

Bacterial adherence to surfaces is one of the initial stepsleading to biofilm formation and is therefore a significantmicrobiological event in medicine and the environment [1718] Aeromonas hydrophila can attach to and form biofilmson polystyrene glass surface stainless steel and polyvinylchloride[19] Aeromonas can also attach to solid surfacesand form biofilms in aquatic environments The presenceof Aeromonas in biofilm samples from water distributionsystems in South Africa has been documented [20] Biofilmis an irreversible growth of a combination of bacterial micro-colonies on surfaces entrenched in extracellular polysaccha-ride matrix [21 22] The formation of biofilm results inresistance of bacteria to antimicrobial drugs and persistentinfections [21] which can lead to the severity of severalbacterial diseases affecting both human [23] and animalhealth [24]

AlthoughAeromonas species are well distributed in fresh-water habitat and have been assigned as an emerging threatto human health [3 25] no data exists about the antibioticsusceptibility profiles and adhesive properties of aeromonadsfrom Kat and Tyume rivers in the Eastern Cape province ofSouth AfricaThe Food and Agriculture OrganizationWorldHealthOrganization (FAOWHO) commission recommendsthat to prevent waterborne diseases in developing countriesaquatic environments having direct impact on human pop-ulations should be characterized physically chemically andmicrobiologically In view of this recommendation and aspart of our surveillance of reservoir of antibiotic resistantcommensal bacteria this present study aimed to (i) evaluatethe levels of antimicrobial resistance in aeromonads isolatesfrom Kat and Tyume rivers (ii) determine the presenceof class 1 and 2 integron associated gene cassette and (iii)evaluate their biofilm forming capabilities

2 Materials and Methods

21 Study Area Sampling and Processing of Samples Katriver is located in a semiurban location at geographicalcoordinates S32∘465471015840 E026∘384561015840 while Tyume river is

located in a rural community at geographical coordinatesS32∘472791015840 E026∘ 505201015840 in the Eastern Cape province ofSouth Africa Water samples were collected four times atrandom between April 2011 and March 2012 Water sampleswere collected in duplicates using 2 L bottles placed on ice andtransported to the laboratory for analysisHundredmicroliter(100 120583L) was spread on several Glutamate phenol(GSP) agar(biolab merck SA) on the other hand 500 120583L was inoculatedinto 145 mL sterile nutrient broth and incubated in a rotaryincubator at 150 rpm overnight at 36∘C At the end of theincubation period a loopful of the culture was spread andorstreaked on GSP agar and all agar plates were incubatedat 36∘C for 24 h Typical yellow colonies on GSP agar werepurified using the samemedia Pure colonieswere transferredunto nutrient agar plates and subjected to oxidase andcatalase tests Oxidase and catalase positive isolates werefurther screened for biochemical characteristics using API20NE kit The strips were then read and final identificationwas made using API lab plus software (bioMerieux MarcylrsquoEtoile France)

22 Antimicrobial Susceptibility Testing Isolates were sub-cultured on nutrient agar plates incubated for 24 h at 36∘CColonies were picked from the agar plates and suspendedin normal saline (085 wv) and adjusted to an A

560

value of 012 plusmn 002 (05 McFarland standard) The bacterialsuspension was spread on the Mueller Hinton agar platesusing a sterile swab stick allowed to dry and impregnatedwith antibiotic disk The antibiotics used were as followsciprofloxacin (5120583g) trimethoprim (5120583g) chloramphenicol(3 120583g) penicillins (10 120583g) clindamycins (2120583g) ofloxacin(5 120583g) ampicillin-sulbactam (20120583g) oxacillin (1 120583g) ampi-cillin (25 120583g) gentamicin (10 120583g) nalidixic acid (30 120583g) cefo-taxime (30 120583g) nitrofurantoin (300 120583g) sulfamethoxazole(25 120583g) cephalothin (30 120583g) erythromycin (15 120583g) tetracy-cline (10 120583g) minocycline (30 120583g) vancomycin (30120583g) andrifampicin (5120583g) Disks were purchased from Mast Diag-nostics (Mast Group Merseyside UK) Plates were incubatedat 36∘C for 24 h Diameters of the zones of inhibitionwere measured and interpreted as susceptible intermedi-ate or resistant according to the Clinical Laboratory Stan-dard Guidelines [26] The frequency of antibiotic-resistantAeromonas isolates was calculated by the following equation119860119861 times 100 where 119860 is the number of isolates resistant toan antibiotic and 119861 is the total number of isolates from thesample The multiple antibiotic resistance (MAR) index ofeach samples was estimated by the following equation 119886(119887times119888) where 119886 represents the aggregate antibiotic resistance scoreof all isolates from the sample 119887 represents the number ofantibiotics and 119888 represents the number of isolates from thesample as outlined in [27ndash29]

23 PCR Detection of Integrons DNA was extracted fol-lowing the method described elsewhere [30 31] TemplateDNA was stored at minus20∘C until it was ready for use Theprimer used for the detection of class 1 and class 2 integronis shown in Table 1 The PCR conditions were as followsinitial denaturation at 94∘C for 2min followed by 30 cycles










4 Results


100

90

80

70

60

50

40

30

20

10

0

CIP

TMC PG CD O

FXSA

M OX AP

GM NA

CTX NI

SMX KF E T

MN VA RP


AntibioticRe

sista

nce f

requ

ency

()









30

25

20

15

10

5

0

Num

ber o

f org

anism

s





5 Discussion




















6 Conclusion




Acknowledgment


References

































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology










4 Results


100

90

80

70

60

50

40

30

20

10

0

CIP

TMC PG CD O

FXSA

M OX AP

GM NA

CTX NI

SMX KF E T

MN VA RP


AntibioticRe

sista

nce f

requ

ency

()









30

25

20

15

10

5

0

Num

ber o

f org

anism

s





5 Discussion




















6 Conclusion




Acknowledgment


References

































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





30

25

20

15

10

5

0

Num

ber o

f org

anism

s





5 Discussion




















6 Conclusion




Acknowledgment


References

































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

















6 Conclusion




Acknowledgment


References

































































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Antibiogram, adhesive characteristics, and incidence of class 1 integron in Aeromonas species...

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Transcript of Antibiogram, adhesive characteristics, and incidence of class 1 integron in Aeromonas species...