Research Article Characterization of Staphylococcus aureus Isolates ...downloads.hindawi.com/journals/ijmicro/2015/358489.pdf · Research Article Characterization of Staphylococcus

Research ArticleCharacterization of Staphylococcus aureusIsolates That Colonize Medical Students in a Hospitalof the City of Cali Colombia

Luis Fernando Collazos Mariacuten1 Gina Estupintildean Arciniegas1 and Monica Chavez Vivas2

1Programa de Medicina Facultad de Medicina Universidad Santiago de Cali Hospital San Juan de Dios de CaliCarrera 4 No 17-67 Cali Colombia2Centro de Estudios e Investigacion en Salud (CEIS) Facultad de Salud Universidad Santiago de CaliCampus Pampalinda Calle 5 No 62-00 Cali Colombia

Correspondence should be addressed to Monica Chavez Vivas monikchavezgmailcom

Received 1 April 2015 Revised 4 June 2015 Accepted 9 July 2015

Academic Editor Sung-Pin Tseng

Copyright copy 2015 Luis Fernando Collazos Marın et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Introduction Nasal carriage of methicillin-resistant Staphylococcus aureus (MRSA) represents a risk for the spread of bacteria Thisstudy characterized the S aureus isolated frommedical students who were in their clinical rotation at a hospital in the city of CaliMaterials and Methods 216 students participated in the study and 63 isolates of S aureus were evaluated for susceptibility and PCRamplification of agr andmecA genesThe origin of MRSA isolates was established by analyzing agr polymorphisms Results A totalof 292 of students were colonized by S aureus and nasal carriage rate was 236 and 143 MRSA Three agr groups (agr IIand agr III) were identified the agr I group was the most common with a 35 prevalence this group is from community originConclusion The present study demonstrates that medical students carry S aureus strains with the threat of spreading them bothto community and hospital environments

1 Introduction

Staphylococcus aureus resistant to methicillin (MRSA) iscurrently among the bacteria of global concern [1 2] Itis responsible for a broad range of community-associatedMRSA infections especially in young individuals soft tissuewithout risk factors [3ndash5] and at hospitals specifically inintensive care units (ICU) [1 6] The most serious casesof MRSA resistance are those mediated by the mecA genethese strains can simultaneously acquire pathogenicity genesthat make them able to develop more aggressive infections[1]

Asymptomatic carriage of S aureus in healthy individualshas been shown to have a high prevalence especially in chil-dren young adults and healthcare workers [7ndash10] Medicalstudents represent an important portion of the healthcarestaff whom are in frequent contact with community in gen-eral or healthcare environment and may spread the bacteria

to other community members or to susceptible patientsrespectively [10ndash16]

Epidemiological studies conducted in Latin Americanhospitals report a prevalence of S aureus colonization from20 to 60 in health area students [10ndash13] with an importantpresence of MRSA strains in higher percentage than inEurope [14ndash16]

Epidemiological knowledge of the status ofMRSA strainscirculating in our environment helps to establish controlmeasures to prevent the transmission of the pathogenthrough adequate use of biosafety barriers by the healthcarestaff including medical students In this sense two of theaspects that must be considered are whether the S aureuscarried by the healthcare staff is of intrahospital origin orfrom the community and how it is disseminated in hospitalwards

Molecular approaches like analysis of the polymorphismin agr genes have permitted determining the variability and

Hindawi Publishing CorporationInternational Journal of MicrobiologyVolume 2015 Article ID 358489 6 pageshttpdxdoiorg1011552015358489

2 International Journal of Microbiology

origin of isolates generating epidemic outbreaks Accordingto this genersquos variability pattern to date four variants havebeen defined denominated as groups I to IV [17] Isolatesbelonging to group III are related to infections associatedwiththe community while group II is detected predominantly instrains isolated at intrahospital level [18 19]

The objective of this study was to establish the geneticdiversity of isolates of S aureus and detect the presence ofthemecA gene in strains isolated from asymptomaticmedicalstudents whowere in their clinical rotation phase in a hospitalin the city of Cali

2 Materials and Methods

21 Study Population During the study 481medical studentswere in their preclinical and clinical rotation cycle 216students signed the informed consent and fulfilled with theinclusion and exclusion criteria which included not havingreceived antibiotic treatment within the last three monthsand not presenting respiratory or skin diseases A total of119 participants were women and 97 were men who were inhospital practices from October to December 2010 at the SanJuan de Dios Hospital in the city of Cali A cross-sectionaldescriptive study was carried out and it was evaluated andapproved by the Ethics and Bioethics Committee of theFaculty of Health at the university

22 Culture Conditions Samples were taken with sterile cot-ton swabs through smears of the nasal mucosa and of the skinin each student The samples were processed immediately inthe microbiology laboratory The isolates were managed bycodes thus respecting student confidentiality

To isolate species of the Staphylococcus genus the sampleswere seeded in mannitol salt phenol red agar (Oxoid LtdHampshire United Kingdom) and incubated for 24 to 48hours at 37∘C Identification of S aureus was accomplishedthrough fermentation of mannitol in selective agar (mediumyellow coloration) the positive reaction of coagulase test andmicroscope observation of Gram positive cocci in clustersfrom a direct extended Gram stain The S aureus wasdifferentiated from coagulase-negative staphylococci (CoNS)by using the DNase test

23 Antibiotic Susceptibility Tests Antimicrobial susceptibil-ity testing was conducted on paired samples using the agardisc diffusion method following the recommendations of theClinical and Laboratory Standards Institute (CLSI) [20]

To conduct this test a standardized amount of S aureuswas inoculated (standard 05 by Mc Farland) in Mueller-Hinton agar medium (Scharlau Chemie SA) and thenthe sensi-disks were placed oxacillin (OXA 1120583g) cefoxitin(FOX 30 120583g) cephalexin (LEX 30 120583g) gentamicin (GEN10 120583g) ciprofloxacin (CIP 5 120583g) erythromycin (ERY 15 120583g)clindamycin (CLI 2 120583g) trimethoprimsulfamethoxazole(SXT 1252375 120583g) tetracycline (TET 30 120583g) chlorampheni-col (CHL 30 120583g) vancomycin (VAN 30120583g) imipenem (IPM10 120583g) and penicillin (PEN 10U) (Oxoid) To verify the

action of the sensi-disks during susceptibility analysis theATCC 25923 strain of S aureus was used as control

Determination of MRSA isolates was performed accord-ing to the results obtained in the evaluation of oxacillin andcefoxitin and by detecting themecA gene [21 22]

24 Molecular Analyses of Isolates TheDNA of the referencestrains and of the bacterial isolates was extracted by usingthe modified protocol by Cheng and Jiang based on bacteriallysis using 25 sucrose 10mgmL of lysozyme and 1mgmLof Proteinase K at 56∘C [23]

To establish resistance to methicillin the mecA genewas amplified following the protocol reported by Lee andcolleagues using primers MR1 51015840 (478)-GTG GAA TTGGCC AAT ACA GG-(497) 31015840 and MR2 51015840 (1816)-TGA GTTCTG CAG TAC CGG AT-(1797) 31015840 [22]

To determine the four agr groups fragments of 440 bp572 bp 406 bp and 588 pb respectively were amplified inde-pendently using a set of primers made up of the universalsense primer with the agr gene sequence 51015840-ATG CAC ATGGTGCACATGC-31015840 and one of the specific primers for eachgroup in antisense agr I 51015840-GTC ACA AGT ACT ATA AGCTGCGAT-31015840 agr II 51015840-GTA TTA CTAATTGAAAAG TGCCATAGC-3 agr III 51015840-CTGTTGAAAAAGTCAACTAAAAGC TC-31015840 and agr IV 51015840-CGA TAA TGC CGT AAT ACCCG-31015840 [18]

The PCR reactions were conducted in 50 120583L volume ofa reaction mixture composed of MgCl

225mM 200120583M of

the four dNTPs 05U of Taq DNA polymerase (Invitrogen)10 pmol of each primer and 1 120583L of DNA solution in athermocycler GeneAmp PCR system 2400 In this study Saureus ATCC 25923 strain was used for the positive control

25 Statistical Analysis The unit of analysis was the bacte-rial isolate obtained from the nasal swab from which themicrobiological andmolecular characteristicswere registeredand were related with the sociodemographic characteristicsof the carriers like gender and academic semester Themicrobiological variables were the presence of S aureus andthe degree of susceptibility or resistance to each antibioticevaluated categorized into different degrees thus resistance(1) intermediate susceptibility (2) and susceptibility (3)according to the standards for each antibiotic established forS aureus The molecular variables were presence of mecAgenes and the variants of the agr gene A database wasconstructed with the variables of interest by using the Excelprogram

The presence of S aureus was determined as percentageof time of permanence and gender In the group colonizedby S aureus an association analysis was performed amongthe different variables like resistance phenotype to differentantibiotics presence of mecA gene and the agr geneticvariant bearing in mind the academic semester and thenumber of hours per day of practice three hours for third-year students five hours for fourth-year students 8 hoursfor fifth-year students and 12 hours for sixth-year studentsPrevalence of the different agr gene variants was determinedin MRSA and MSSA phenotypes

International Journal of Microbiology 3

Table 1 Frequency of isolation of S aureus from medical students

Academic degreeStudents Students colonizedTotal S aureus OR CI 95 119875

119873 119899 () Nose 119899 () Skin 119899 () Min Max3 31 144 9 (143) 7 (159) 2 (105) 1608 0302 8569 05754 69 319 31 (492) 18 (409) 13 (684) 0322 0102 0998 00455 55 255 11 (175) 10 (273) 1 (53) 5294 0627 44707 00946 61 282 12 (19) 9 (205) 3 (158) 1371 0327 5755 0665Total 216 100 63 (292) 44 (204) 19 (89) 2870 1658 4969 0000OR odds ratio CI confidence intervals statistical significance was based on 119875 le 005

The findings were statistically analyzed using descriptivestatistics Chi-square test (1205942) and 119875 value (119875 lt 005statistical significant) The risk factor analysis of MRSAcolonization was performed using SPSS statistical package(version 200 SPSS Inc Chicago IL USA)

3 Results

A total of 292 (63) of the students who were in the differentservices at the hospital were colonized by S aureus Table 1shows the isolates according to the studentsrsquo academic yearit can be noted that fourth-year students had the highestnumber of isolates (45) (119875 lt 005) The frequency of nasalcarriage of S aureus was significantly higher than on skin(204 versus 89 OR = 2870 119875 lt 005) and fifth-yearstudents presented the highest frequency of S aureus in thenose than on the skin (182 versus 18 OR = 5294 119875 =0094) (Table 1)

31 Antibiotic Susceptibility Analysis Isolates with resistanceto 120573-lactam antibiotics were distributed in a high numberof students from all the academic grades with a variationbetween 90 and 22 (Table 2) Isolates resistant to antibioticsinhibiting protein synthesis were registered between 18and 90 and to antibiotics inhibiting nucleic acid synthesisregistered between 11 and 44

32 Molecular Analysis of Isolates Amplification of themecAgene was detected in the five isolates presenting simultaneousresistance to oxacillin-cefoxitin and in four isolates resistantonly to cefoxitin (143)Themultidrug resistance phenotypewas the distinctive characteristic in these MRSA isolates withsimultaneous resistance to 120573-lactam macrolide aminoglyco-side and quinolone antibiotics (Table 3)

A total of 54 isolates were positive to agr gene amplifi-cation with a difference in prevalence of specific agr groupsbetween MRSA and methicillin sensitive S aureus (MSSA)isolates Thirty-five bacterial isolates (556) were classifiedinto agr group I 31 (492) strains were MSSA and 4(63) strains were MRSA the differences were statisticallysignificant (119875 lt 005)

FiveMRSA isolates (93) were identified in agr group IIthe mecA gene was detected in all isolates and agr group IIIwas found only in 14 MSSA isolates (222) (Table 4)

The majority of S aureus strains isolated from studentsbelonged to agr group I followed by agr group III (41 strains)and finally agr group II (9 strains) was distributed amongthe isolates of fourth- fifth- and sixth-year students The agrgroups I to III occurred in the major percentages (259 and185 resp) in fourth-year students (data not shown)

4 Discussion

The present study evidenced the presence of S aureus with292 of prevalence in medical students in clinical rotationand nasal carriage was a risk factor The tendency is similarin Latin American countries with percentages between 50and 20 [10ndash13] In Europe a prevalence of nasal carriers ofS aureus is reported at 25 [14ndash16] In Asia prevalence ofcolonization by S aureus in Chinese Malaysian and Indianmedical students is registered in 31 of the cases [24]

Although some studies report increased frequency ofnasal carriers asmedical students have greater exposure to thehospital environment during clinical rotations [25 26] ourresults did not show significant differences among the nasalcarriers and the time of permanence in the hospital envi-ronment However a significant number of isolates resistantto imipenem were determined in third-year students and ahigher number of isolates with resistance to aminoglycosideserythromycins and quinolones in students with greater timeof permanence in the hospital (Table 2)

Our study determined a high frequency of S aureusisolates resistant to penicillin cephalexin imipenem ery-thromycin chloramphenicol clindamycin tetracycline gen-tamicin trimethoprim-sulfamethoxazole and ciprofloxacingiving way to the presence of multidrug resistant isolatesThese isolates can evolve in response to selective pressureexerted on microflora residing in patients which are thenpassed on to students or can be directly generated in themicroflora of students

Similar results were obtained in studies conducted by Leeand colleagues [22] all isolates with resistance to cefoxitinpresented themecA gene In their study Datta and colleaguesdemonstrated that cefoxitin induces the expression of thePBP2a protein encoded in the mecA gene the cefoxitin disk


Table2Antibiotic

susceptib

ilitypatte

rnso

fSaureusisolates

Academ

icdegree

(119873)

Stud

ents

Total

RT Hou

rsOXA119899(

)PE

N119899(

)FO

X119899(

)LE

X119899(

)IPM119899(

)VA

N119899(

)CI

P119899(

)SX

T119899(

)TC

Y119899(

)GEN119899(

)CL

I119899(

)ER

Y119899(

)CH

L119899(

)119899

3

31144

30(0)

9(100)

0(0)

8(889)5lowast

(56)

6(667)

1(111)

4(444)

5(222)

2(222)

6(667)

8(333)

7(556)

469

319

52(65)

27(871)

4(129)

29(935)

5(161)

11(355)

12(387)

10(233)

15(233)

9(30)13lowast

(233)

22(50)

22(467)

555

255

82(182)

11(100)

3(273

)10

(909)

2(182)10lowast

(909)

2(182)

6(545)

7(633)

2(182)

8(727)

10(909)

8(727)

661

282

121(83)

11(917

)2(167)

10(833)

2(167)

3(25)

4(333)

6(50)

7(583)

5(455)

9(75)

10(833)5lowast

(417

)To

tal

216

100

5(79)

58(921)

9(14

3)

57(905)

14(222)

30(476

)19

(302)

26(413

)34

(54)

18(286)

36(365)

50(571)

42(667)

RTresidence

timeo

fmedicalstu

dentsinho

spita

llowast

119875le005

NoteO

XAo

xacillin

PENp

enicillinF

OX

cefoxitin

LEX

cephalexin

IPMimipenem

VANvancomycinC

IPciproflo

xacin

SXT

trim

etho

prim

-sulfametho

xazoleT

ETtetracyclineG

ENgentamicinC

LI

clindamycinE

RYerythromycinC

HLchloramph

enicol


Table 3 Frequency of MRSA and MSSA strains according of antibiotic susceptibility patterns

Isolate AntibioticPEN FOX IPM VAN CLI ERI CHL GEN TET SXT CIP

MRSA(119899 = 9) 100 (9) 100 (9) 778 (7) 778 (7) 889 (8) 100 (9) 778 (7) 778 (7) 100 (9) 666 (6) 666 (6)

MSSA(119899 = 54) 907 (49) 889 (48) 13 (7) 0 (0) 519 (28) 76 (41) 648 (35) 204 (11) 463 (25) 37 (20) 241 (13)

MRSA methicillin-resistant S aureus MSSA methicillin-sensitive S aureus

Table 4 Genetic polymorphism of the agr locus amongMRSA andMSSA strains carried

agr I119899 ()

agr II119899 ()

agr III119899 ()

agr (minus)119899 ()

Total119899

MRSA 4 (63) 5 (79) 0 0 9MSSA 31 (492) 0 14 (222) 9 (143) 54Total 35 (556) 5 (79) 14 (222) 9 (143) 63

diffusion test had the best diagnostic performance among thephenotypic methods for detection of MRSA [21]

One of the factors that permit the presence of MRSAstrains in asymptomatic carriers is due to antibiotic intakethat strengthens overgrowth of organisms on the skin and onmucosa surface [2 6] It has been demonstrated that use ofpenicillin is associated with acquisition of MRSA likewiseuse of fluoroquinolones macrolides and cephalosporinsincreases the risk of having these strains [27]

Although in Colombia it is reported that the majority ofMRSA strains are not of nosocomial origin [2] an increasingnumber of reports indicate an epidemiological change withthe prevalence of strains of community origin [5 28]

This study determined that strains of hospital origin (AH-MRSA) presented resistance to 120573-lactam antibiotics and tomultiple antibiotics (Table 3) and according to the agr geneanalysis these isolates were located in agr group II a groupcompatible with hospital origin [17 18]

The presence of AH-MRSA strains in asymptomaticcarriers is a potential risk of disseminating in the communityadditionally these strains are resistant to multidrug with fewtherapeutic alternatives

However we also found isolates with characteristicsof community origin (AC-MRSA) in four MRSA isolatesgrouped in agr I Nasal carriage of MRSA becomes animportant reservoir and source of pathogen propagationamong the personnel the community and the patient

As observed in Table 3 MSSA isolates also presentedresistance to multidrug characteristics Studies conductedin Spain reveal that MSSA isolates susceptibility also showsresistance to erythromycin (135) clindamycin (115) andciprofloxacin (19) [16]

In addition 492 and 222 of MSSA isolates weregrouped in agr I and agr III respectively associating themwith a molecular community origin

Several studies have found that the MRSA and MSSAcirculating in Colombia have genetic similarities to clone

USA300 [29 30] This clone usually (ST8-MRSA-IVa) har-bors the sek seq bsaB lukF-PV and lukS-PV genes whichencode the staphylococcal enterotoxins K andQ bacteriocinand Panton-Valentine leukocidin (PVL) respectively Addi-tionally the clone typically contains a type I agr operon Thespread of MRSA could also be caused by the acquisition ofSCCmec among asymptomatic carriers of S aureus in thecommunity [30] Thus the asymptomatic carriage of MSSAin medical students may contribute to the spread of MRSAbetween the community and hospitals

Although some of the risk factors for colonization by Saureus are age gender hospitalizations use of antibioticsand some diseases and habitat [3 12] among the populationanalyzed these variables were not evaluated which becamea limitation of the study The results of this study howeverevidence the important presence of asymptomatic carriers Saureus in medical students from the time they begin theirclinical practice Additionally increased MRSA colonizationwas found in students as they spentmore time in the hospitalas it is seen in fifth-year students showing the highest numberof carriers

Conflict of Interests

The authors have no conflict of interests to declare

Acknowledgments

The authors thank the General Research Direction (DGI) atUniversidad Santiago de Cali for the economic support toconduct this study gratitude is also expressed to BellazminArenas coordinator of the Center of Research and Studies ofthe Faculty of Health (CEIS) for the support granted

References

[1] R Valaperta M R Tejada M Frigerio et al ldquoStaphylococcusaureus nosocomial infections the role of a rapid and low-costcharacterization for the establishment of a surveillance systemrdquoNew Microbiologica vol 33 no 3 pp 223ndash232 2010

[2] R H Deurenberg and E E Stobberingh ldquoThe evolution ofStaphylococcus aureusrdquo Infection Genetics and Evolution vol 8no 6 pp 747ndash763 2008

[3] J R Mediavilla L Chen B Mathema and B N KreiswirthldquoGlobal epidemiology of community-associated methicillinresistant Staphylococcus aureus (CA-MRSA)rdquo Current Opinionin Microbiology vol 15 no 5 pp 588ndash595 2012


[4] X X Ma A Galiana W Pedreira et al ldquoCommunity-acquiredmethicillin-resistant Staphylococcus aureus UruguayrdquoEmergingInfectious Diseases vol 11 no 6 pp 973ndash976 2005

[5] C A Alvarez N Yomayusa A L Leal et al ldquoNosoco-mial infections caused by community-associated methicillin-resistant Staphylococcus aureus in Colombiardquo American Journalof Infection Control vol 38 no 4 pp 315ndash318 2010

[6] T S Naimi K H LeDell K Como-Sabetti et al ldquoComparisonof community- and health care-associated methicillin-resistantStaphylococcus aureus infectionrdquo Journal of the American Medi-cal Association vol 290 no 22 pp 2976ndash2984 2003

[7] U Seybold S Schubert J R Bogner and M HogardtldquoStaphylococcus aureus infection following nasal colonizationan approach to rapid risk stratification in a university healthcaresystemrdquo Journal of Hospital Infection vol 79 no 4 pp 297ndash3012011

[8] S Cesur and F Cokca ldquoNasal carriage of methicillin-resistantStaphylococcus aureus among hospital staff and outpatientsrdquoInfection Control and Hospital Epidemiology vol 25 no 2 pp169ndash171 2004

[9] M Dulon C Peters A Schablon and A Nienhaus ldquoMRSAcarriage among healthcare workers in non-outbreak settingsin Europe and the United States a systematic reviewrdquo BMCInfectious Diseases vol 14 article 363 2014

[10] A Bettin C Causil andN Reyes ldquoMolecular identification andantimicrobial susceptibility of Staphylococcus aureus nasal iso-lates from medical students in Cartagena Colombiardquo BrazilianJournal of Infectious Diseases vol 16 no 4 pp 329ndash334 2012

[11] I A Mendez D F Holguın-Riano D P Pachon-Barinas et alldquoPrevalence and antimicrobial susceptibility of Staphylococcusaureusmethicilin resistant isolated frommedical studentsrdquoCESMedicina vol 27 no 1 pp 21ndash30 2013

[12] S Lopez-Aguilera M M Goni-Yeste L Barrado C MGonzalez-Rodrıguez-Salinas J R Otero and F ChavesldquoStaphylococcus aureus nasal colonization in medical studentsimportance in nosocomial transmissionrdquo Enfermedades Infec-ciosas y Microbiologıa Clınica vol 31 no 8 pp 500ndash505 2013

[13] K A Prates A M Torres L B Garcia S F Y Ogatta C LCardoso and M C B Tognim ldquoNasal carriage of methicillin-resistant Staphylococcus aureus in university studentsrdquoBrazilianJournal of Infectious Diseases vol 14 no 3 pp 316ndash318 2010

[14] S Baliga R Bansil U Suchitra B Bharati KVidyalakshmi andS Shenoy ldquoNasal carriage of meticillin-resistant Staphylococcusaureus in medical studentsrdquo Journal of Hospital Infection vol68 no 1 pp 91ndash92 2008

[15] JMarques J Barbosa and I Alves ldquoStaphylococcus aureusnasaland hand carriage among students from a Portuguese healthschoolrdquo British Journal of Biomedical Science vol 67 no 1 pp5ndash8 2010

[16] C Rodrıguez-Avial A Alvarez-Novoa A Losa and J J PicazoldquoAumento significativo de la colonizacion por Staphylococcusaureus entre los estudiantes de medicina durante la realizacionde las practicas en el hospitalrdquo Enfermedades Infecciosas yMicrobiologıa Clınica vol 31 no 8 pp 516ndash519 2013

[17] S Jarraud C Mougel J Thioulouse et al ldquoRelationshipsbetween Staphylococcus aureus genetic background virulencefactors agr groups (alleles) and human diseaserdquo Infection andImmunity vol 70 no 2 pp 631ndash641 2002

[18] G Sakoulas G M Eliopoulos R C Moellering Jr et alldquoAccessory gene regulator (agr) locus in geographically diverseStaphylococcus aureus isolates with reduced susceptibility to

vancomycinrdquo Antimicrobial Agents and Chemotherapy vol 46no 5 pp 1492ndash1502 2002

[19] A Azimian S Najar-Pirayeh S Mirab-Samiee and M NaderildquoOccurrence of methicillin resistant Staphylococcus aureus(MRSA) among clinical samples in TehranmdashIran and its corre-lation with polymorphism of specific accessory gene regulator(AGR) groupsrdquo Brazilian Journal of Microbiology vol 43 no 2pp 779ndash785 2012

[20] Clinical and Laboratory Standards Institute Performance Stan-dards for Antimicrobial Susceptibility Testing Twenty-ThirdInformational Supplement M100-S23 Clinical and LaboratoryStandards Institute (CLSI) Wayne Pa USA 2013

[21] P Datta N Gulati N Singla et al ldquoEvaluation of variousmethods for the detection of meticillin-resistant Staphylococcusaureus strains and susceptibility patternsrdquo Journal of MedicalMicrobiology vol 60 no 11 pp 1613ndash1616 2011

[22] Y Lee C K Kim M Kim D Yong K Lee and Y ChongldquoDetection of mecA in strains with oxacillin and cefoxitin disktests for detection of methicillin-resistant Staphylococcusrdquo TheKorean Journal of Laboratory Medicine vol 27 no 4 pp 276ndash280 2007

[23] H-R Cheng and N Jiang ldquoExtremely rapid extraction of DNAfrom bacteria and yeastsrdquo Biotechnology Letters vol 28 no 1pp 55ndash59 2006

[24] N Vasanthakumari A S D Alshrari E G Rad et al ldquoHighlydynamic transient colonization by Staphylococcus aureus inhealthy Malaysian studentsrdquo Journal of Medical Microbiologyvol 58 no 11 pp 1531ndash1532 2009

[25] E Guclu T Yavuz A Tokmak et al ldquoNasal carriage ofpathogenic bacteria in medical students effects of clinic expo-sure on prevalence and antibiotic susceptibilityrdquo EuropeanArchives of Oto-Rhino-Laryngology vol 264 no 1 pp 85ndash882007

[26] M A Gaona D I Rıos M C Pena M I Pinilla and G RGutierrez ldquoVariacion del estado de portador de Staphylococcusaureus en una poblacion de estudiantes de medicinardquo RevistaCiencia y Salud vol 7 no 1 pp 37ndash46 2009

[27] R C Moellering Jr ldquoCurrent treatment options for comm-unity-acquired methicillin-resistant Staphylococcus aureusinfectionrdquo Clinical Infectious Diseases vol 46 no 7 pp1032ndash1037 2008

[28] C A Arias J Reyes M Zuniga et al ldquoMulticentre surveillanceof antimicrobial resistance in Enterococci and Staphylococcifrom Colombian hospitals 2001-2002rdquo Journal of AntimicrobialChemotherapy vol 51 no 1 pp 59ndash68 2003

[29] J A Escobar-Perez B E Castro and R A Marquez-OrtizldquoAislamientos de Staphylococcus aureus sensibles a meticilinarelacionados geneticamente con el clon USA300 origen de losaislamientos SARM de genotipo comunitario en ColombiardquoBiomedica vol 34 no 1 pp 124ndash136 2014

[30] S S Huang and R Platt ldquoRisk of methicillin-resistant Staphylo-coccus aureus infection after previous infection or colonizationrdquoClinical Infectious Diseases vol 36 no 3 pp 281ndash285 2003

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origin of isolates generating epidemic outbreaks Accordingto this genersquos variability pattern to date four variants havebeen defined denominated as groups I to IV [17] Isolatesbelonging to group III are related to infections associatedwiththe community while group II is detected predominantly instrains isolated at intrahospital level [18 19]

The objective of this study was to establish the geneticdiversity of isolates of S aureus and detect the presence ofthemecA gene in strains isolated from asymptomaticmedicalstudents whowere in their clinical rotation phase in a hospitalin the city of Cali

2 Materials and Methods

21 Study Population During the study 481medical studentswere in their preclinical and clinical rotation cycle 216students signed the informed consent and fulfilled with theinclusion and exclusion criteria which included not havingreceived antibiotic treatment within the last three monthsand not presenting respiratory or skin diseases A total of119 participants were women and 97 were men who were inhospital practices from October to December 2010 at the SanJuan de Dios Hospital in the city of Cali A cross-sectionaldescriptive study was carried out and it was evaluated andapproved by the Ethics and Bioethics Committee of theFaculty of Health at the university

22 Culture Conditions Samples were taken with sterile cot-ton swabs through smears of the nasal mucosa and of the skinin each student The samples were processed immediately inthe microbiology laboratory The isolates were managed bycodes thus respecting student confidentiality

To isolate species of the Staphylococcus genus the sampleswere seeded in mannitol salt phenol red agar (Oxoid LtdHampshire United Kingdom) and incubated for 24 to 48hours at 37∘C Identification of S aureus was accomplishedthrough fermentation of mannitol in selective agar (mediumyellow coloration) the positive reaction of coagulase test andmicroscope observation of Gram positive cocci in clustersfrom a direct extended Gram stain The S aureus wasdifferentiated from coagulase-negative staphylococci (CoNS)by using the DNase test

23 Antibiotic Susceptibility Tests Antimicrobial susceptibil-ity testing was conducted on paired samples using the agardisc diffusion method following the recommendations of theClinical and Laboratory Standards Institute (CLSI) [20]

To conduct this test a standardized amount of S aureuswas inoculated (standard 05 by Mc Farland) in Mueller-Hinton agar medium (Scharlau Chemie SA) and thenthe sensi-disks were placed oxacillin (OXA 1120583g) cefoxitin(FOX 30 120583g) cephalexin (LEX 30 120583g) gentamicin (GEN10 120583g) ciprofloxacin (CIP 5 120583g) erythromycin (ERY 15 120583g)clindamycin (CLI 2 120583g) trimethoprimsulfamethoxazole(SXT 1252375 120583g) tetracycline (TET 30 120583g) chlorampheni-col (CHL 30 120583g) vancomycin (VAN 30120583g) imipenem (IPM10 120583g) and penicillin (PEN 10U) (Oxoid) To verify the

action of the sensi-disks during susceptibility analysis theATCC 25923 strain of S aureus was used as control

Determination of MRSA isolates was performed accord-ing to the results obtained in the evaluation of oxacillin andcefoxitin and by detecting themecA gene [21 22]

24 Molecular Analyses of Isolates TheDNA of the referencestrains and of the bacterial isolates was extracted by usingthe modified protocol by Cheng and Jiang based on bacteriallysis using 25 sucrose 10mgmL of lysozyme and 1mgmLof Proteinase K at 56∘C [23]

To establish resistance to methicillin the mecA genewas amplified following the protocol reported by Lee andcolleagues using primers MR1 51015840 (478)-GTG GAA TTGGCC AAT ACA GG-(497) 31015840 and MR2 51015840 (1816)-TGA GTTCTG CAG TAC CGG AT-(1797) 31015840 [22]

To determine the four agr groups fragments of 440 bp572 bp 406 bp and 588 pb respectively were amplified inde-pendently using a set of primers made up of the universalsense primer with the agr gene sequence 51015840-ATG CAC ATGGTGCACATGC-31015840 and one of the specific primers for eachgroup in antisense agr I 51015840-GTC ACA AGT ACT ATA AGCTGCGAT-31015840 agr II 51015840-GTA TTA CTAATTGAAAAG TGCCATAGC-3 agr III 51015840-CTGTTGAAAAAGTCAACTAAAAGC TC-31015840 and agr IV 51015840-CGA TAA TGC CGT AAT ACCCG-31015840 [18]

The PCR reactions were conducted in 50 120583L volume ofa reaction mixture composed of MgCl

225mM 200120583M of

the four dNTPs 05U of Taq DNA polymerase (Invitrogen)10 pmol of each primer and 1 120583L of DNA solution in athermocycler GeneAmp PCR system 2400 In this study Saureus ATCC 25923 strain was used for the positive control

25 Statistical Analysis The unit of analysis was the bacte-rial isolate obtained from the nasal swab from which themicrobiological andmolecular characteristicswere registeredand were related with the sociodemographic characteristicsof the carriers like gender and academic semester Themicrobiological variables were the presence of S aureus andthe degree of susceptibility or resistance to each antibioticevaluated categorized into different degrees thus resistance(1) intermediate susceptibility (2) and susceptibility (3)according to the standards for each antibiotic established forS aureus The molecular variables were presence of mecAgenes and the variants of the agr gene A database wasconstructed with the variables of interest by using the Excelprogram

The presence of S aureus was determined as percentageof time of permanence and gender In the group colonizedby S aureus an association analysis was performed amongthe different variables like resistance phenotype to differentantibiotics presence of mecA gene and the agr geneticvariant bearing in mind the academic semester and thenumber of hours per day of practice three hours for third-year students five hours for fourth-year students 8 hoursfor fifth-year students and 12 hours for sixth-year studentsPrevalence of the different agr gene variants was determinedin MRSA and MSSA phenotypes






3 Results







4 Discussion






Table2Antibiotic

susceptib

ilitypatte

rnso

fSaureusisolates

Academ

icdegree

(119873)

Stud

ents

Total

RT Hou

rsOXA119899(

)PE

N119899(

)FO

X119899(

)LE

X119899(

)IPM119899(

)VA

N119899(

)CI

P119899(

)SX

T119899(

)TC

Y119899(

)GEN119899(

)CL

I119899(

)ER

Y119899(

)CH

L119899(

)119899

3

31144

30(0)

9(100)

0(0)

8(889)5lowast

(56)

6(667)

1(111)

4(444)

5(222)

2(222)

6(667)

8(333)

7(556)

469

319

52(65)

27(871)

4(129)

29(935)

5(161)

11(355)

12(387)

10(233)

15(233)

9(30)13lowast

(233)

22(50)

22(467)

555

255

82(182)

11(100)

3(273

)10

(909)

2(182)10lowast

(909)

2(182)

6(545)

7(633)

2(182)

8(727)

10(909)

8(727)

661

282

121(83)

11(917

)2(167)

10(833)

2(167)

3(25)

4(333)

6(50)

7(583)

5(455)

9(75)

10(833)5lowast

(417

)To

tal

216

100

5(79)

58(921)

9(14

3)

57(905)

14(222)

30(476

)19

(302)

26(413

)34

(54)

18(286)

36(365)

50(571)

42(667)

RTresidence

timeo

fmedicalstu

dentsinho

spita

llowast

119875le005

NoteO

XAo

xacillin

PENp

enicillinF

OX

cefoxitin

LEX

cephalexin

IPMimipenem

VANvancomycinC

IPciproflo

xacin

SXT

trim

etho

prim

-sulfametho

xazoleT

ETtetracyclineG

ENgentamicinC

LI

clindamycinE

RYerythromycinC

HLchloramph

enicol




MRSA(119899 = 9) 100 (9) 100 (9) 778 (7) 778 (7) 889 (8) 100 (9) 778 (7) 778 (7) 100 (9) 666 (6) 666 (6)

MSSA(119899 = 54) 907 (49) 889 (48) 13 (7) 0 (0) 519 (28) 76 (41) 648 (35) 204 (11) 463 (25) 37 (20) 241 (13)



agr I119899 ()

agr II119899 ()

agr III119899 ()


Total119899

MRSA 4 (63) 5 (79) 0 0 9MSSA 31 (492) 0 14 (222) 9 (143) 54Total 35 (556) 5 (79) 14 (222) 9 (143) 63














Acknowledgments


References








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






3 Results







4 Discussion






Table2Antibiotic

susceptib

ilitypatte

rnso

fSaureusisolates

Academ

icdegree

(119873)

Stud

ents

Total

RT Hou

rsOXA119899(

)PE

N119899(

)FO

X119899(

)LE

X119899(

)IPM119899(

)VA

N119899(

)CI

P119899(

)SX

T119899(

)TC

Y119899(

)GEN119899(

)CL

I119899(

)ER

Y119899(

)CH

L119899(

)119899

3

31144

30(0)

9(100)

0(0)

8(889)5lowast

(56)

6(667)

1(111)

4(444)

5(222)

2(222)

6(667)

8(333)

7(556)

469

319

52(65)

27(871)

4(129)

29(935)

5(161)

11(355)

12(387)

10(233)

15(233)

9(30)13lowast

(233)

22(50)

22(467)

555

255

82(182)

11(100)

3(273

)10

(909)

2(182)10lowast

(909)

2(182)

6(545)

7(633)

2(182)

8(727)

10(909)

8(727)

661

282

121(83)

11(917

)2(167)

10(833)

2(167)

3(25)

4(333)

6(50)

7(583)

5(455)

9(75)

10(833)5lowast

(417

)To

tal

216

100

5(79)

58(921)

9(14

3)

57(905)

14(222)

30(476

)19

(302)

26(413

)34

(54)

18(286)

36(365)

50(571)

42(667)

RTresidence

timeo

fmedicalstu

dentsinho

spita

llowast

119875le005

NoteO

XAo

xacillin

PENp

enicillinF

OX

cefoxitin

LEX

cephalexin

IPMimipenem

VANvancomycinC

IPciproflo

xacin

SXT

trim

etho

prim

-sulfametho

xazoleT

ETtetracyclineG

ENgentamicinC

LI

clindamycinE

RYerythromycinC

HLchloramph

enicol




MRSA(119899 = 9) 100 (9) 100 (9) 778 (7) 778 (7) 889 (8) 100 (9) 778 (7) 778 (7) 100 (9) 666 (6) 666 (6)

MSSA(119899 = 54) 907 (49) 889 (48) 13 (7) 0 (0) 519 (28) 76 (41) 648 (35) 204 (11) 463 (25) 37 (20) 241 (13)



agr I119899 ()

agr II119899 ()

agr III119899 ()


Total119899

MRSA 4 (63) 5 (79) 0 0 9MSSA 31 (492) 0 14 (222) 9 (143) 54Total 35 (556) 5 (79) 14 (222) 9 (143) 63














Acknowledgments


References








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table2Antibiotic

susceptib

ilitypatte

rnso

fSaureusisolates

Academ

icdegree

(119873)

Stud

ents

Total

RT Hou

rsOXA119899(

)PE

N119899(

)FO

X119899(

)LE

X119899(

)IPM119899(

)VA

N119899(

)CI

P119899(

)SX

T119899(

)TC

Y119899(

)GEN119899(

)CL

I119899(

)ER

Y119899(

)CH

L119899(

)119899

3

31144

30(0)

9(100)

0(0)

8(889)5lowast

(56)

6(667)

1(111)

4(444)

5(222)

2(222)

6(667)

8(333)

7(556)

469

319

52(65)

27(871)

4(129)

29(935)

5(161)

11(355)

12(387)

10(233)

15(233)

9(30)13lowast

(233)

22(50)

22(467)

555

255

82(182)

11(100)

3(273

)10

(909)

2(182)10lowast

(909)

2(182)

6(545)

7(633)

2(182)

8(727)

10(909)

8(727)

661

282

121(83)

11(917

)2(167)

10(833)

2(167)

3(25)

4(333)

6(50)

7(583)

5(455)

9(75)

10(833)5lowast

(417

)To

tal

216

100

5(79)

58(921)

9(14

3)

57(905)

14(222)

30(476

)19

(302)

26(413

)34

(54)

18(286)

36(365)

50(571)

42(667)

RTresidence

timeo

fmedicalstu

dentsinho

spita

llowast

119875le005

NoteO

XAo

xacillin

PENp

enicillinF

OX

cefoxitin

LEX

cephalexin

IPMimipenem

VANvancomycinC

IPciproflo

xacin

SXT

trim

etho

prim

-sulfametho

xazoleT

ETtetracyclineG

ENgentamicinC

LI

clindamycinE

RYerythromycinC

HLchloramph

enicol




MRSA(119899 = 9) 100 (9) 100 (9) 778 (7) 778 (7) 889 (8) 100 (9) 778 (7) 778 (7) 100 (9) 666 (6) 666 (6)

MSSA(119899 = 54) 907 (49) 889 (48) 13 (7) 0 (0) 519 (28) 76 (41) 648 (35) 204 (11) 463 (25) 37 (20) 241 (13)



agr I119899 ()

agr II119899 ()

agr III119899 ()


Total119899

MRSA 4 (63) 5 (79) 0 0 9MSSA 31 (492) 0 14 (222) 9 (143) 54Total 35 (556) 5 (79) 14 (222) 9 (143) 63














Acknowledgments


References








































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




MRSA(119899 = 9) 100 (9) 100 (9) 778 (7) 778 (7) 889 (8) 100 (9) 778 (7) 778 (7) 100 (9) 666 (6) 666 (6)

MSSA(119899 = 54) 907 (49) 889 (48) 13 (7) 0 (0) 519 (28) 76 (41) 648 (35) 204 (11) 463 (25) 37 (20) 241 (13)



agr I119899 ()

agr II119899 ()

agr III119899 ()


Total119899

MRSA 4 (63) 5 (79) 0 0 9MSSA 31 (492) 0 14 (222) 9 (143) 54Total 35 (556) 5 (79) 14 (222) 9 (143) 63














Acknowledgments


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








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Characterization of Staphylococcus aureus Isolates ...downloads.hindawi.com/journals/ijmicro/2015/358489.pdf · Research Article Characterization of Staphylococcus