Anaerobe 9 (2003) 277–280

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2 July 2002.

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doi:10.1016/j.an

Antimicrobial susceptibility

Antimicrobial susceptibility and strain prevalence ofKorean vaginal Lactobacillus spp.$

Soo Young Choia, Chung Eun Changa, Seung Cheol Kimb, Jae-Seong Soa,*aDepartment of Biological Engineering and Center for Advanced Bioseparation Technology, Inha University, Incheon 402-751, South Korea

bDepartment of Obstetrics and Gynecology, Ewha Womans University Hospital, Seoul 158-710, South Korea

Received 30 August 2002; received in revised form 30 July 2003; accepted 1 September 2003

Abstract

One hundred eight vaginal lactobacilli were isolated from Korean women and characterized in terms of their antibiotics

susceptibility and PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) pattern. The in vitro

antibiotics susceptibilities of 108 vaginal isolates to 13 antimicrobial agents were determined by broth dilution method based on

NCCLS reference protocol. High rates of resistance were demonstrated for gentamicin, kanamycin, metronidazole, and

streptomycin whereas all the isolates were susceptible to erythromycin. The concentrations of gentamicin, kanamycin,

metronidazole, streptomycin, and erythromycin at which 90% of the vaginal isolates were inhibited (MIC90) were 100, 200,

>200, 200 and 0.39mg/mL, respectively. For molecular identification, PCR-RFLP analysis was employed where the 16S rDNA was

amplified by PCR and the PCR products were digested with 8 different restriction endonucleases prior to being electrophoresed in

agarose gels. Based on PCR-RFLP results, approximately half of the isolates were identified as Lactobacillus crispatus. Several

isolates were further identified by DNA sequence analysis of their 16S rDNA.

r 2003 Published by Elsevier Ltd.

Keywords: Vaginal lactobacilli; Antimicrobial susceptibility; MIC; PCR-RFLP; Molecular identification

1. Introduction

It was first identified in 1894 by the German physicianA. Doderlein that Lactobacillus has been shown to bethe predominant bacterium in the vaginal microbialflora found in women of reproductive age [1]. Lactoba-cilli are facultative anaerobes that colonize the moistsurface of the vaginal epithelium, intestinal tract, andoral cavity of humans and non-human animals [1,2].Lactobacilli play an important role in maintaininghealthy vagina and they produce various inhibitorycompounds, which can prevent the growth of anaerobicpathogenic bacteria [3,4]. Lactobacilli metabolize glu-cose to a final end product of lactic acid, whichcontributes to the maintenance of a low vaginal pH(4.0–4.5) [2,3]. Many isolates of vaginal lactobacilli

Anaerobe Olympiad 2002. The 6th Biennial Congress

Society of the Americas, Park City, Utah, 29 June–

ng author. Tel.: +82-32-860-7516; fax: +82-32-875-

ss: sjaeseon@inha.ac.kr (J.-S. So).

front matter r 2003 Published by Elsevier Ltd.

aerobe.2003.09.001

produce hydrogen peroxide, a compound having broadantimicrobial activity [4,5]. However, in bacterialvaginosis (BV) patient, the number of lactobacillidecreases while the number of anaerobic bacteriaincreases for unknown reasons. In order to treat BVantimicrobial agents were used to eliminate the patho-genic bacteria but they can also eliminate vaginallactobacilli. While antimicrobial agents can be effectivefor eradication of urinary tract infections, they are oftenunable to cure BV or prevent complications that arisefrom it [6].Furthermore, antimicrobial agents have many side

effects such as multidrug-resistant microorganisms andsuch chemotherapeutic agents ideally should not be usedfor prophylaxis or health maintenance [7]. Therefore,the antimicrobial agent which can selectively inhibit thegrowth of anaerobic pathogenic bacteria while notinhibiting the growth of lactobacilli is needed foreffective treatment of BV. We isolated 108 vaginallactobacilli from healthy Korean women. Variouscharacteristics of 108 vaginal lactobacilli have beenstudied previously [8].

ARTICLE IN PRESSS.Y. Choi et al. / Anaerobe 9 (2003) 277–280278

In this study, first antimicrobial susceptibility ofKorean vaginal Lactobacillus spp. was determined.Furthermore, in order to find the strain prevalence ofKorean vaginal lactobacilli molecular identification wasperformed to the species level using PCR-RFLP.

2. Materials and methods

2.1. Antimicrobial susceptibility test

Thirteen antimicrobial agents were used for suscept-ibility test; ampicillin (Amp), chloramphenicol (Ch),erythromycin (Em), gentamicin (Gm), kanamycin (Km),metronidazole (Mtz), novobiocin (Nov), oxacillin(Oxa), penicillin G (PnG), streptomycin (Sm), tetracy-cline (Tc), ofloxacin (OF), cefotaxime (CF). Concentra-tions of antimicrobial agents used ranged from 0.01 to200 mg/mL. The antimicrobial susceptibility of 108vaginal isolates was determined by broth dilution testas described by NCCLS with slightly modification [9].

Lactobacillus spp. were cultured overnight anaerobi-cally in 5mL MRS broth. To prepare the culture broth,10% of overnight cultured cell were transferred to fresh5mL MRS broth and incubated for 3 h up to OD600=1.Serially two-fold diluted antimicrobial agents wereadded to each test tubes containing 5mL MRS broth.Then, the cultures were incubated for 18 h to measurethe optical density. Minimal inhibitory concentrations(MICs) were defined as the lowest drug concentrationthat gave only a slight growth corresponding approxi-mately to 10% of the control growth.

2.2. Molecular identification

For species-level identification, PCR-RFLP was used.Vaginal isolates and 10 ATCC type stains were culturedovernight in 5mL MRS broth and total genomic DNAs

Table 1

Antimicrobial susceptibility of the 108 Korean vaginal isolates

% of isolates

Amp Ch Em Gm Km Mtz

>200 (mg/mL) 5.97 84.21 93.97

200 (mg/mL) 25.37 11.84 6.02

100 (mg/mL) 46.26 3.94

50 (mg/mL) 14.92

25 (mg/mL) 5.97

12.5 (mg/mL) 1.49

6.25 (mg/mL) 5.81 46.91

3.125 (mg/mL) 45.34 53.08

1.5625 (mg/mL) 36.04

0.78125 (mg/mL) 6.97 7.89

0.39 (mg/ml) 5.81 28.94

0.1953 (mg/mL) 53.94

0.097 (mg/mL) 9.21

were prepared as previously reported [8]. PCR waspreformed with universial primer fD1(5AGAGTTT-GATCTGGCTCAG3) and rD1(5AAGGAGGTGAT-CCAGCC3) to amplify 1.5 kb of 16S rDNA [8,10]. PCRreaction mixture was as follows; template DNA 100 ng,10� buffer 5 mL, dNTP 2.5mM, primer 0.1 mM each,Taq(TaKaRa Ex Taqt) 1.25U, ddH2O 37.75 mL. PCRwas performed as follows: preheating at 95�C for 5min,denaturation at 94�C for 1min, primer annealing at60�C for 1min, polymerase extension at 72�C for 2min,final elongation at 72�C for 8min. PCR product wasdigested with 8 different restriction endonucleases;HinfI, AluI, MspI, Hae III (TaKaRa, Japan), RsaI,TaqI, DdeI and CfoI (Promega, USA). Enzyme digested16S rDNA was electrophoresed at 50mA for 10 h in 2%agarose gel. NTSYS-pc (numerical taxonomy system ofmultivariate statistical program) was used for PCR-RFLP data analysis [8].To sequence the 16S rDNA of several isolates,

polymerase chain reaction was performed using the 3primer sets; F1/R1, F2/R2, and F3/R3 [11]. Thesequence was determined using an ABI 377 automatedDNA sequencer (Perkin Elmer, Foster, USA) at theMolecular Core Facility of the University of Illinois,Chicago. The 16S rDNA sequence data were com-pared with known sequences registered at the NationalCenter for Biotechnological Information (http://www.ncbi.nlm.nih.gov//BLAST/) [9].

3. Results and discussion

3.1. Antimicrobial susceptibility test

High rates of resistance were demonstrated for genta-micin, kanamycin, metronidazole, and streptomycinwhereas all the isolates were susceptible to erythromycin.The distributions of MICs were presented in Table 1.

Nov Oxa PnG Sm Tc OF CF

11.66 24.19 30.5 2.17

6.66 20.96 23.72

15 46.77 16.94

1.03 11.66 4.83 13.55

4.47 1.03 15 6.77 2.17

1.49 10.3 18.33 1.61 5.08

7.46 36.08 13.33 1.61 8.97 3.38 8.69

5.97 22.68 5 61.53 39.13

14.9 14.43 3.33 29.48 45.65

31.34 4.12 2.17

29.85

2.98

1.49

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1Kb

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14

1Kb

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Fig. 2. Restriction pattern with RsaI of PCR-amplified product of 16S

rDNA genes of 9 ATCC type strains and 5 lab isolates. Lane M: 100 bp

DNA ladder, lanes 1–14: L. acidophilus ATCC 4356, L. casei subsp.

casei ATCC 393, L. crispatus ATCC 33820, L. delbrueckii subsp. lactis

ATCC 15808, L. fermentum ATCC 23271, L. gasseri ATCC 9857, L.

jensenii ATCC 25258, L. plantarum ATCC 14917, L. rhamnosus ATCC

7469, L. reuteri DSM 20016, Lactobacillus sp. KLB 1, Lactobacillus sp.

KLB 12, Lactobacillus sp. KLB 39, Lactobacillus spp. KLB 46.

S.Y. Choi et al. / Anaerobe 9 (2003) 277–280 279

The concentrations of gentamicin, kanamycin, metronida-zole, streptomycin, and erythromycin at which 90% of thevaginal isolates were inhibited (MIC90) were 100, 200,>200, 200 and 0.39mg/mL, respectively. The concentra-tions of metronidazole were ranged from 0.01 to 200mg/mL in our study and in these concentrations, OD of testculture was higher than the growth control. Similar resultswere previously reported in other paper [12]. Highconcentration of metronidazole, i.e. between 1000 and4000mg/mL, partially inhibited the growth of Lactobacil-

lus. Concentrations more or equal to 5000mg/mL com-pletely suppressed the growth of Lactobacillus.Concentrations between more or equal to 128 and lessor equal to 256mg/mL stimulated the growth of Lactoba-

cillus [12]. In vivo studies are required that may furtherelucidate the ideal antimicrobial agent for treatment of BVwithout lactobacilli susceptibility. Certainly our data couldbe used to design antimicrobial agent regimen for BVtreatment. Since clindamycin is popularly used for BVtreatment we did clindamycin susceptibility test and foundthat the concentration of clindamycin at which 90% of thevaginal isolates were inhibited (MIC90) was 12.5mg/mL(data not shown).

3.2. Molecular identification

Based on PCR-RFLP results, we have found that76% of Korean vaginal isolates belong to eitherLactobacillus crispatus or L. acidophilus (Fig. 1).Although in our RFLP experiments L. crispatus couldnot be separated from L. acidophilus DNA sequencingclearly distinguish between the two species [14]. It isnoteworthy that the predominant lactobacilli in Japa-nese women were found to be L. crispatus (52.7%)and L. gasseri (20.8%) by DNA–DNA hybridization

3% 1%1%4%4%

6%

5%

76%

L.acidophilus or

L.crispatus (76%)

L.fermentum (6%) L.gasseri (5%)

L.reuteri (4%) L.jensenii (4%) L.rhamnosus (3%)

L.plantarum (1%) L.casei (1%)

Fig. 1. Strain prevalence of vaginal isolates. Pie chart of the

percentage distribution of Lactobacillus spp. from the vaginas of

healthy Korean women.

analysis [13]. Most vaginal Lactobacillus strains fromwomen of geographically separated countries belongedto three species, L. crispatus, L. gasseri and L. jensenii

[14]. Overall strain prevalence of Korean vaginal isolateswere summurized in Fig. 1. Out of 108 isolates, 5 strainswere identified by 16S rDNA sequencing and theirsequence were deposited in GenBank as follows:Lactobacillus sp. KLB 46, L. crispatus (AF243145);Lactobacillus sp. KLB 58, L. paracasei (AF243173);Lactobacillus sp. KLB 79, L. crispatus (AF243144);Lactobacillus sp. KLB 12, L. fermentum (AF522394);Lactobacillus sp. KLB39, L. salivarius (AY112943). 16SrDNA sequencing results of these 5 strains were thesame as 16S rDNA PCR-RFLP data. The result showedthat PCR-RFLP is as effective as sequencing for specieslevel molecular identification.Interestingly, all lactobacilli strains including 108

vaginal isolates and 10 type strains showed thesame RFLP pattern when they were digested with RsaI(Fig. 2). This uniform RFLP pattern could be used todistinguish lactobacilli at the genus level from otherlactic acid bacteria such as bifidobacterium.

Acknowledgements

This study was supported by Center for AdvancedBioseparation Technology (ERC-BSEP), Inha Univer-sity.

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