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Infectious Diseases in Obstetrics and Gynecology 5:36-44 (I 997)(C) 1997 Wiley-Liss, Inc.

Phage Infection in Vaginal Lactobacilli" An InVitro Study

Sylvia I. Pavlova, Ali O. Kilig,2 Susan M. Mou,1 and Lin Tao1,2.

1Department of Obstetrics and Gynecology, School of Medicine, University ofMissouri-Kansas City,Kansas City, MO

eDepartment of Oral Biology, School of Dentistry, University ofMissouri-Kansas City,Kansas City, MO

ABSTRACT

Objective: During bacterial vaginosis, an unexplained decrease of vaginal lactobacilli occurs. Toidentify whether these lactobacilli could be infected by phages, we isolated phages from vaginallactobacilli and analyzed their potential virulence in attacking vaginal lactobacilli in vitro.

Methods: Vaginal samples were obtained from 39 reproductive-aged women. The selectiveRogosa SL agar was used to isolate lactobacilli, from which phages induced by mitomycin C orreleased spontaneoulsy were analyzed by the agar spot method.

Results: Of 20 samples from women with vaginal infections, 12 did not have lactobacilli. Fromthe remaining 8 infection samples and the 19 samples from healthy women, 37 Lactobacillus strainswere isolated, from which 7 temperate phages were identified. Upon analysis, all 7 phages infectedvaginal lactobacilli from the same and/or different women in vitro. Two phages, (bkc005 and(bkc007, had a broad host range, infecting 7 of 8 species tested. A control intestinal Lactobacillusphage also lysed several vaginal strains. One vaginal phage, (bkc039, was apparently lytic againstvaginal lactobacilli from 7 other women. This phage was characterized as follows: plaque morphol-ogy, small and clear; burst size, 300 phages per cell; spontaneous induction rate, 1 per 106 cells;DNA, double-stranded and linear, 41 kb; and shape, a hexogonal head and a non-contractile tail.

Conclusions: Bacteriophages were isolated from vaginal lactobacilli of some women and wereshown in vitro to lyse vaginal Lactobacillus strains from the same and/or different women. It wassuggested that vaginal lactobacilli might be suppressed by phages. Infect. Dis. Obstet. Gynecol.5:36-44, 1997. (C) 1997 Wiley-Liss, Inc.

KEY WORDS

Lactobacillus phage; 1ysogen; bacteriolysis; bacterial vaginosis

ndigenous vaginal lactobacilli play an importantrole in maintaining vaginal health. However, an

unexplained decrease of vaginal lactobacilli occurs

during bacterial vaginosis (BV).z-4 Women whosuffer from BV may have an increased, milk-likedischarge that has an unpleasant odor.s AlthoughBV itself may be a mild disease, its sequelae in-

clude pelvic infections, premature labor, low birth

weight, and mid-trimester pregnancy loss.6 BV can

thus be a serious disease state. The cause of BV is

unknown, but recently BV has been defined as a

condition in which the normal Lactobacillus-

predominant vaginal flora is replaced with anaero-

bic bacteria, Gardnerella vaginalis, and Mycoplasma

Contract grant sponsor: Concerned Parents for AIDS Research/American Foundation for AIDS Research; Contract grantnumber: 02069-15-RG; Contract grant sponsor: University of Missouri Research Board; Contract grant number: K-3-40532.Dr. Kili is now at the Department of Microbiology and Clinical Microbiology, School of Medicine, Karadeniz TechnicalUniversity, 61080 Trabzon, Turkey.*Correspondence to: Dr. Lin Tao, Department of Oral Biology, School of Dentistry, University of Missouri-Kansas City, 650East 25th Street, Kansas City, MO 64108. E-mail: [email protected].

Received 16 October 1996Clinical Symposium Accepted 31 March 1997

PHAGES IN VAGINAL LACTOBACILL1 PAVLOVA ET AL.

hominis.6 Several possible mechanisms by whichvaginal lactobacilli decrease have been proposed.These include douching,7 the use of spermicidesuch as Nonoxynol-9,8 and treatment with antibi-otics for other infections. However, it is unknownwhether vaginal lactobacilli can decrease by naturalcauses.

Lactobacilli produce lactic acid and hydrogenperoxide (HzOz), which can prevent the over-

growth of other microorganisms in the vagina,z--4

including Escherichia coli,9,1 G. vaginalis, 11 Chla-mydia trachomatis, lz and Neisseria gonorrhoeae. 13

However, during BV, the number of HzOz-producing lactobacilli decreases drastically, whilemany anaerobic bacteria, such as G. vaginalis, M.hominis, Ureaplasma urealyticum, and Mobiluncusspecies, overgrow, z-4 It is currently unknownwhether vaginal lactobacilli decrease first, allowingthe BV-associated anaerobes to overgrow, or theanaerobes overgrow first, suppressing vaginal lac-tobacilli. Because these anaerobes are sensitive to

lactic acid and HzOz produced by vaginal lactoba-cilli, theoretically they should not outgrow lactoba-cilli. Therefore, it is possible that lactobacilli de-crease first in the vagina before the anaerobes over-

grow. If so, other yet unidentified factors mightinhibit vaginal lactobacilli.A natural inhibitor of bacteria is bacteriophage

or phage, the virus that infects bacteria. 14 Phagescan exist as free viruses in the environment or as

parasites in the host bacteria. A bacterium thathosts a latent phage (prophage) is called lysogen,which can release a small number of phages spon-taneously or a large number of phages upon induc-tion. Phages can be lyric (virulent) or temperate.The lyric phages can lyse bacteria completely, re-

leasing many new phages into the environment.The temperate phages do not lyse all the infectedbacterial cells; they convert some of them into ly-sogens and coexist with them. However, some tem-

perate phages may become lyric due to mutationsof the phage or changes of the bacterial host back-ground.4,s

Lactobadllus phages have been isolated fromvarious sources, including dairy products,6 sau-

sage,7 human intestines, 18 and sewage.9 To date,studies on Lactobacillus phages have mainly focusedon the food fermentation industry that uses lacto-bacilli as starter cultures.6- These foods include

yogurts and fermented meats, such as sausages. A

study of the production of salami dry sausageshowed that phage infection can delay acid produc-tion by the lactobadllus starter culture and drasti-cally reduce the number of lactobacilli in the sau-

sage and disrupt the ripening process of the sau-

sage.7 Likewise, phages may also infect vaginallactobacilli, delay their acid production, and reducetheir numbers. However, whether phages can in-fect human vaginal lactobacilli has not been re-

ported. Therefore, the purposes of this study were

to isolate phages from vaginal lactobacilli, to deter-mine their infectivity and virulence by cross-

infecting these phages against a collection of vagi-nal Lactobacillus strains, and to characterize one ofthe vaginal Lactobacillus phages.

SUBJECTS AND METHODSIsolation of Vaginal Lactobacilli

The clinical research was approved by the TrumanMedical Center Institutional Review Board in Kan-sas City, MO. Vaginal samples were obtained from39 reproductive-aged, non-pregnant women. Allsubjects gave written informed consent. First, a

sample was obtained to diagnose BV. As previouslydescribed,s the diagnosis of BV was based upon thepresence of a vaginal discharge with three of thefour following characteristics: a homogenous ap-pearance; a pH > 4.5; the presence of a fishy amineodor upon the addition of 10% potassium hydrox-ide (KOH); and the presence of clue cells. Thevaginal discharge of healthy women had no more

than one of these four characteristics, and none

contained clue cells. Then, a vaginal sample was

taken for isolating lactobacilli. A BBL CulturetteCollection and Transport System (Becton Dickin-son Microbiological Systems, Cockeysville, MD)was used. The Culturette tube contains a rayonswab attached to a cap by a plastic stick and an

ampule with 0.5 ml of modified Stuart’s transportmedium. A swab was inserted into the vagina, ro-

tated a few turns along the vaginal sidewall, andallowed to absorb for a few seconds before with-

drawing. The swab was then placed back into thetube containing the transport medium and sent to

the laboratory for analysis. The samples were in-oculated onto the selective Rogosa SL agar plates(Difco, Detroit, MI) for isolating vaginal lactoba-cilli. The agar plates were incubated in a candle jarat 37C for 48 h. Lactobacilli were identified on thebasis of growth on the selective Rogosa SL agar

INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 37

PHAGES IN VAGINAL LACTOBACILLI PAVLOVA ET AL.

(pH 5.2), rod cell morphology, positive Gram stain,and negative catalase reaction. Further identifica-tion of the species of these lactobacilli was basedon carbohydrate fermentation patterns and gas pro-duction in the lactobacilli AIRS broth (Difco, De-troit, MI) described in Bergey’s Manual ofSystematicBacteriology.e Purifed cultures were maintained at

-70C in MRS broth with 10% glycerol. Two vagi-nal Lactobacillus type strains, L. gasseri ATCC 9857and L. jensenii ATCC 25258, were obtained fromthe American Type Culture Collection (Rockville,MD) to be used as controls. Additional controlstrains included a human intestinal strain, L. gasseriADH and its phage qbadh (from Dr. Klaenhammer,University of North Carolina), and a dairy typestrain, L. delbrueckii subsp, lactis ATCC 15808.

Phage Induction

Mitomycin C (Sigma, St. Louis, MO) was used to

induce phages from vaginal lactobacilli as previ-ously described. 1(’ The induction of Lactobacillusprophages was indicated by the lysis of a Lactoba-cillus culture 4-7 h after the addition of mitomycinC. These lysates were then centrifuged, filteredthrough a 0.2 pm pore-size filter, and maintained at

4C with a drop of chloroform.

Phage Infectivity AssayPhage infectivity was determined by the agar spotmethod as previously described. 16 All positive re-

suits were verified by single plaque formation,phage DNA isolation, and observation of phagesunder an electron microscope to rule out possiblebacterial inhibitory effects due to bacteriocin,

HeOe, or organic acids.

Phage Titration

The temperate (non-virulent or non-lytic) phage,kc039, from a vaginal Lactobadllus isolate (L. del-brueckii subsp, delbruecbii KC039), was found to belytic (virulent) against another vaginal isolate, L.delbruecbii subsp, delbruecbii KC013. Thus, KC013was used as an indicator strain to analyze the titerof bkc039. Plaques were counted after 24 h of in-cubation at 37C on MRS agar with 10 mM CaC1z.

One-Step Growth Curve

KC013 was grown at 37C in MRS broth until mid-

exponential phase. To ml of this culture, 0.1 mlof bkc039 stock [10s plaque forming unit (PFU)/

ml; multiplicity of infection, 1:10] was added andmixed. The mixture of cells was added into 4 ml ofmelted soft 0.6% MRS agar with 10 mM CaC1e,

rapidly mixed, poured onto MRS plates, and incu-bated at 37C. Plaques were numerated after 24 hof incubation.

Spontaneous Phage Induction

KC039 was grown in 2 ml of MRS broth to mid-

exponential phase. One milliliter of the culture wasdiluted and plated on MRS agar plates for cellcount. Another ml was centrifuged to harvest thesupernatant, which was filtered through a sterile

0.2 pm pore-size filter. The supernatant was di-luted and used to infect the indicator strain KC013by the soft agar overlay method as described above.Plaques were numerated after 24 h of incubation at

37C.

Phage DNA AnalysisThe bacteriophage qbkc039 was purifed from ofmitomycin-induced lysate by a standard method,el

The phage DNA was extracted with the QIAGEN(Chatsworth, CA) lambda phage DNA isolation kit.Restriction enzyme digests of qbkc039 DNA were

subjected to gel electrophoresis on a 0.8% agarosegel at 40 V for 3 h. The gel was stained with ethid-ium bromide and photographed under an ultravio-let light.

Electron Microscopy

One drop of the purifed phage dkc039 in 0.1 Mammonium acetate (pH 7.0) was spotted on gridswith a carbon-coated Formvar film (Ladd Research

Industry, Burlington, VT). After drying for 30 sec,the sample was negatively stained with 2% uranylacetate (pH 4.2). Electron microscopy was per-formed with the CM12 transmission electron mi-

croscope (Philips Electronic Instruments, Inc.,Mahwah, NJ) at 80 kV.

RESULTSIsolation and Identification of

Vaginal Lactobacilli

Vaginal samples were obtained from 39 reproduc-tive-aged women (1 Native American, 5 Asians, 6

Blacks, and 27 Caucasians; age range: 19-52 years).Evaluation of the vaginal secretions revealed 19

normal women, 16 women with BV, and 4 womenwith vaginal candidiasis (VC). All women with the

38 INFECTIOUS DISEASES IN OBSrl-’ETRICS AND GYNECOLOGY


clinical diagnosis of BV, the presence of at leastthree of four Amsel’s criteria,s had a reduction or

absence in vaginal lactobacilli. VC was confirmedby the growth of Candida cells in the culture test.

Lactobacilli were isolated from all of the 19 healthywomen and 4 VC patients, but from only 4 of 16women who had BV. Some vaginal samples hadmultiple Lactobacillus strains/species; therefore, a

total of 37 Lactobacillus strains were isolated fromthe 27 women. Based on sugar fermentation pat-terns, the species of the 37 vaginal Lactobacillusisolates were tentatively identified as follows: L.acidophilus, 10 strains; L. delbrueckii subsp, deLbruecleii, 19 strains; L. fermentum, 3 strains; L. jensenii,4 strains; and L. plantarum, strain.

Phage Isolation and Cross-Infection

Phage induction was performed with the mitomy-cin C method in 37 clinically isolated vaginalstrains, 2 control vaginal type strains (L. gasseriATCC 9857 and L. jensenii ATCC 25258), and 2other control strains (L. gasseri ADH and L. del-bruecbii subsp, lactis ATCC 15808). The lysateswere used to interact with the 41 Lactobacillusstrains to screen for phage-sensitive indicatorstrains. Seven lysates were confirmed to containphages because they formed single plaques on the

agar plates of sensitive strains. Among the 7phages, 3 were from 29 Lactobacillus strains of 19healthy women, 2 from 4 strains of 4 VC patients,and 2 from 4 strains of 4 BV patients. Although therate of phage isolation was apparently lower fromlactobacilli in healthy women than in women withBV or VC, them was no apparent difference in

phage sensitivity among vaginal Lactobacillusstrains isolated from these women. The sources

and host ranges of these phages and the sensitivityof various vaginal Lactobacillus isolates to thesephages are shown in Table 1. Strains from all Lac-tobacillus species studied, except L. fermentum, were

infected by phages. The phage bkc005 isolatedfrom a vaginal L. acidophilus strain of a women with

VC had a broad host range and was very virulent.Unlike other phages, qbkc005 could continue to

spread its infection on a soft agar plate by enlargingits lytic zones with time (Fig. 1). Interestingly, thecontrol phage isolated from the intestinal strain L.gasseri ADH also lysed 6 of these vaginal strains.

In Vitro Study of Vaginal LactobacillusPhage Infection

All 7 temperate phages isolated from vaginal lacto-bacilli lysed vaginal lactobacilli from the same and/or different women in vitro (Table 1). Among thesephages, the temperate phage, (bkc039, isolatedfrom a vaginal Lactobacillus strain, KC039, was

found to be lytic to 7 other vaginal Lactobacillusclinical strains and vaginal type strain. Among the8 strains, KC013 was the most sensitive one to

(bkc039 infection. A bacterial strain that is sensitiveto a phage by showing plaques on agar plate uponinfection can be used to analyze the phage as an

indicator strain. Thus, KC013 was selected as theindicator strain for analyzing bkc039. Normally,temperate phages only partially lyse their bacterialindicator strains by showing turbid plaques on agarplates, such as the case of dkc005 (Fig. 1). How-ever, qbkc039 showed clear plaques after the phageinfected Lactobacillus strain KC013 on agar plates(data not shown). The complete lysis of KC013 bydkc039 allowed isolation of a large amount ofphage particles and gcnomic DNA. This greatlyfacilitated characterizations of this phage. More-over, the phage bkc039 and its indicator strainKC013 formed an ideal in vitro model to show thata temperate phage released from a vaginal Lactoba-cillus stain of one woman could become a lyticphage against a different vaginal Lactobacillus strainfrom another woman.

Characterization of bkc039The infection cycle of bkc039 against its new hoststrain KC013 in MRS broth with 10 mM CaC1e was

characterized by its one-step growth kinetics. Fig-ure 2 graphically represents the increase of PFU/mlas a function of time during phage infection. Theevents in bacteriophage development were deter-mined: the time between adsorption and lysis (la-tent period) was 30 min; the rise period was 90 min;and the average number of phage particles releasedper infected host cell (burst size) was about 300

phages per cell. The plaque morphology of bkc039was small and clear, with a diameter ranging from0.5 to mm. The spontaneous induction rate ofKC039 was about in 106 cells. The restriction

pattern of bkc039 DNA is shown in Figure 3. The

genome size of bkc039 was estimated to be 41 kb

INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY 39


TABLE I. Sensitivity of vaginal lactobacilli to phages released from different lactobacilli

Lactobacillus strain (N 41)

Vaginal Lactobacillus phages

L. acidophilus L. delbrueckiid Intestinal phage:L. gasseri

qbkc005b qbkc007 dpkc012b bkc021 bkc023 bkc031 kc039 badhVaginal isolates (N 37)

L. acidophilus KC001 a + + + +KC007a + +KC012 + + +KC021 + +KC026 + + + +

L. delbrueckii KC003 + +KC004a + + +KC004b + + + +KC006a + + + +KC006b + +KC010 + + +KC013 + + + + +KC023 + + + +KC027 + + +KC029 + + + + +KC031 + + + +KC032 + + +KC033 + +KC034 + + + + +KC036b + + + + +KC038 + +KC039 + +

L. jensenii KC008 + + +KC009 + +KC035 + + + +

L. plantarum KC020 + + +

Control strains (N 4)L. gasseri ATCC 9857 + + + + +L. jensenii ATCC 25258 + + + + +L. lactis ATCC 15808 + + + + +

Only phage-sensitive strains are shown; vaginal strains and control strain, ADH, were not infected by these phages. To multiple strains isolatedfrom the same woman, a lowercase letter is added to their strain designations.blsolated from women with VC.Clsolated from women with BV.dSubspecies of L. delbrueckii.

by an average of the sums of the molecular sizes ofthese restriction fragments. The agarose gel elec-trophoresis, which allows discrimination among co-

valently closed supercoiled circular, nick-relaxedcircular, and linear DNA molecules showed onlyone distinct band of undigested phage qbkc039DNA (data not shown). This indicated that thephage genome was a double-stranded linear DNA.The electron micrograph illustrates that the phagebkc039 has a hexagonal head, about 67 nm in di-

ameter, and a flexible but non-contractile tail about250 nm in length with about 60 discs (Fig. 4). Thisphage morphology apparently belongs to Bradleytype B phages,zz

DISCUSSION

BV is a common vaginal infection that may result in

serious sequelae, such as increased risk of pretermdelivery, low birth weight, and mid-trimester loss.6

Although the exact cause of BV is unknown, it hasbeen well documented that during BV, the normal,Lactobacillus-predominant vaginal flora is replacedwith anaerobic bacteria,z-6,z3 However, becausevaginal anaerobic bacteria are normally suppressedby lactic acid and HzOz produced by lactobacilli,z-4

theoretically the anaerobes cannot remove lactoba-cilli unless the latter diminishes or disappears first.

Phages are natural inhibitors of bacteria. If

40 INFECTIOUS DISEASES IN OBSTETRICS AND GYNECOLOGY


Fig. I. Lactobacillus phage infections on a soft agar plate. The larger circle of bacteriolysis on the left was cause by a drop of

I1 of bkc005. The four smaller circles of bacteriolysis on the right were caused by drops of lal each of dkc012. Theindicator strain was L. delbrueckii subsp, lactis ATCC 15808. Normally, the lysis of Lactobacillus cells would stop after 2 daysof phage infection, with a lysis zone similar to the four smaller zones caused by dkc012. However, the infection of bkc005continued as shown by the enlargement of the lytic zone with time. The result on this plate was 7 days after infection.

10 9

10 8

lO 7

106

1050 30 60 90 120 150 180

Time (min)

Fig. 2. One-step growth curve of vaginal Lactobacillusphage dpkc039.

phages can infect vaginal lactobacilli, they may bea natural cause for the reduction of vaginal lacto-bacilli. Although phages have been isolated fromlactobacilli of various sources, 1"s-19 phage infectionsin vaginal lactobacilli have not been reported. Thisstudy observed that some vaginal lactobacilli were

lysogens that released phages spontaneously or

upon induction. The rate of phage isolation fromvaginal lactobacilli was apparently lower in healthywomen than in women with BV or VC. As to phagesensitivity among various Lactobacillus speciestested, L. fermentum did not release any phages, nor

were they sensitive to phages released by otherLactobacillus species. Moreover, the phage adh re-

leased from a human intestinal L. gasseri strain

ADH also lysed 6 of 37 vaginal Lactobacillus strains

tested, suggesting that infectious phages in the va-

gina may come from the fecal-urogenital route.

However, an increased number of cases will beneeded to confirm these observations.

Since it is difficult to test phage infections in

vaginal lactobacilli in vivo in humans, in vitro stud-ies were performed to show that phages releasedfrom some women’s vaginal lactobacilli could lysevaginal lactobacilli isolated from the same and/ordifferent women (Table 1). This observation sug-gested that vaginal lactobacilli could be suppressedby phages in vivo in humans.The analysis of these phages revealed some un-

INFECTIOUS DISEASES IN OBSTE,TRICS AND GYNECOLOGY 41


123456

(kb)23.1

Fig. 3. Restriction analysis of vaginal Lactobacillus phagedpkc039 DNA. Bacteriophage +kc039 DNA was digestedwith Aval (lane I), BamHI (Lane 2), Bglll (lane 3), EcoRI(lane 4), and Sail (lane 5). A Hindlll digest of lambda DNAwas used as the molecular weight standard (lane 6).

usual and interesting characteristics. First, most

phages have a specific or narrow host range, andtheir infection process stops when the host bacteriaare not actively growing. 14,1s However, two of thevaginal Lactobacillus phages, +kc005 and qbkc007,had a very broad host range, infecting 7 of 8 Lac-tobacillus species tested (Table 1). The phageqbkc005 could also continue its infection after thehost cell had stopped active growth (Fig. 1).

Second, a bacterium that carries a prophagc,namely a lysogcn, is normally immune from infec-tion by other phages of the same type. This char-acteristic is called "super-infection immunity,"which is controlled by a phage repressor proteinencoded by the prophagc genes already in the hostbacterial chromosome or by other mechanisms. 14

This may explain why the phages released by a

vaginal Lactobacillus strain normally would not lysc

Fig. 4. Electron micrograph of the vaginal Lactobacillusphage bkc039. Stained with 2% uranyl acetate, x240,000.Bar 1,000 A (100 nm).

its own bacterial host. However, the "super-infection immunity" may not protect vaginal lacto-bacilli from infections by different types of phages.The observation that phages released from one

Lactobacillus strain can lyse other lysogenic strains

suggests that different types of phages may exist inthese vaginal lactobacilli. But exceptions exist.Two phages, qbkc007 and qbkc012, lysed their ownhost strains, suggesting that these phages might bedefective in their super-infection immunity.

Third, truly lytic or virulent phages are usuallyshort-lived. Once they appear, the virulent phagescan rapidly eliminate their host bacteria; as a result,they lose their shelter that enables them to live andto reproduce themselves. Therefore, phages thatare temperate to some bacteria but lytic to othersare of concern. All 7 vaginal Lactobacillus phagesisolated in this study were temperate phages fromlysogenic vaginal Lactobacillus strains. It is wellknown that some temperate phages can become

42 INFt,;C’I’IOUS DISEASIS IN OBS’IT{7’RICS AN/) GYNI,JCOLOGY

PHAGES IN VAGINAL LACTOBACILLI

virulent due to genetic mutations, is However, it isunknown why some temperate phages, such as

+kc039, can become lyric against other vaginal Lac-tobacillus strains. Probably certain differences inbacterial host backgrounds prohibited these phagesfrom integrating their DNA into the chromosomeof their new hosts to form lysogens,z4 Among manycharacteristics of these phages, the spontaneous re-

lease of a small number of phages by lysogeniclactobacilli is noteworthy. This suggests that lyso-gcnic vaginal lactobacilli may be a source of poten-tially infectious phages.

In summary, phages were isolated from vaginallactobacilli. In vitro studies showed that phages iso-lated from vaginal lactobacilli of some women

could infect vaginal lactobacilli of the same and/ordifferent women. Also, a phage isolated from a hu-man intestinal Lactobacil/us strain lysed some ofthese vaginal lactobacilli, suggesting that infectiousphages in the vagina may come from the fecal-urogenital route. Although the phage infection invaginal lactobacilli observed in vitro may not nec-

essarily represent that a similar situation could hap-pen in vivo, the results imply that vaginal lactoba-cilli may be inhibited by phages. This implicationmay be important for studying the etiology of BVthat is associated with the decrease of vaginal lac-tobacilli. However, further studies with an in-creased number of clinical cases will be needed to

associate phage infections in vaginal lactobacilliwith women’s vaginal health status.

ACKNOWLEDGMENTSWe thank Dr. T. Klaenhammer for sending us L.gasseri ADH strain and phage qbadh, Dr. C. Lee, S.Robinson, and D. Sackuvich for assisting with elec-tron microscopy, D. Coats for photography, and P.Puntenney for editing the manuscript.

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