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13. Endtz HP, Moutan RP, van der Reyden T, Ruijs GJ, Biever M: Fluoroquinoloneresistance in Campylobacter spp. isolated from human stools and poultry products. Lancet 1990, 335: 787. 14. Reina J, Bianco I, Catala A, Alomar P: Aislamiento de Campylobacterjejuni resistente al aeido nalidixico. Enfermedades Infeceiosas y Microbiologia Clinica 1989, 7: 288-289. 15. Reina J, Parras F, Alomar P: Deteceion de cepas de Carapylobacter jejuni resistentes a Ias quinotonas: resistencia post-tratamiento. Revista Espafiola de Mierobio|ogia Clinica 1989, 4: 318. i6. Aitwegg M, Burness A, Zollinger-Iten J, Penner JL: Problems in identificationof Campylobacterjejuni associated with acquisitionof resistance to nalidixicacid. Journal of Clinical Microbiology1987, 25: 1807-1808. 17. Adler-Mosca H, Liithy-Hottenstein J, Martinetti G, Burnens A, AItwegg M: Development of resistance to quinolonesin five patients with campylobacteriosis treated with norfloxacin or ciprofloxacin. European Journal of ClinicalMicrobiology& InfectiousDiseases 1991, 10: 953-95% 18. Douidar SM, Snodgrass WR: Potential role of fluoroquinolones in pediatric infections. Reviews of Infectious Diseases 1989, 11: 878--889.

In Vitro Antibacterial Activity of Antiseptics against Vaginal Lactobacilli C. Juliano 1, L. Piu l, E. Gavini 1, S. Z a n e t t i 2., G. F a d d a 2

The results of investigations carried out to evaluate the inhibitory activity in vitro of seven vaginal antiseptic douche solutions against several strains of vaginal lactobacilli isolated from asymptomatic women are reported. Some of the products examined showed marked antibacterial activity even at high dilutions and for short exposure times. The post-antibiotic effect of two of these antiseptics on vaginal lactobacilli was also evaluated. The results of these investigations suggest that uncontrolled use of antiseptic products could cause changes in the normal vaginal flora.

l Istituto di Tecnica Farmaceutica, Facolth di Farmacia, Universit~t di Sassari, Via Muroni 23/A, 07100 Sassari, Italy. 2Istituto di Microbiologiae Viro|ogia,Facolt~di Medicina e Chirurgia, Viale San Pietro 43/B, 07100 Sassari, Italy.

Eur, J. Clin. Microbiol. Infect. Dis.

The vaginal microbial flora in healthy adult women is a dynamic ecosystem subject to frequent shifts and consisting of both aerobic and anaerobic bacteria, anaerobes predominating (1, 2). In particular fermentative lactobacilli have frequently been isolated from the human vagina (3, 4), and their presence is considered crucial for maintaining the ecological balance of the vaginal flora. Lactobacilli ferment vaginal glycogen to lactic acid and maintain the pH between 4 and 5 (5). They thus cause local acidification, necessary for preserving the indigenous microflora, and are considered important in preventing growth of pathogenic microorganisms (6--8). Feminine hygiene measures, both as part of the daily routine and in relation to sexual intercourse, are carried out fairly frequently and often with specific products characterized by antiseptic properties (9). Hygiene is considered essential for preventing infections in the urogenital system (10). However, vaginal application of nonsuitable substances could negatively affect the flora, producing local physical and chemical alterations which favour the establishment of pathogenic microorganisms. In fact, a recent clinical study showed an association between vaginal douching and pelvic inflammation disease (11). In view of these observations, a study was carried out to evaluate the antibacterial activity in vitro of seven vaginal antiseptic douche solutions (12, 13) against strains of lactobacilli isolated from asymptomatic women.

Materials and Methods. Seven vaginal antiseptic douche solutions currently on the market were used in the study. Their characteristics and instructions for use, if present on the packages, have been reported in detail in a previous paper (12), Their chemical compositions are summarized in Table 1. Seven strains of lactobacilli were used, all isolated from vaginal secretions collected with sterile swabs from asymptomatic women. Four strains (PV14, PV15, PV16, PV22), kindly donated by Dr. G. Bo (Institute of Hygiene, Pavia, Italy), were homofermentative lactobacilli PV14 and PV15 were identified as Lactobacillus jensenii and PV16 and PV22 as Lactobacillus gasseri (3). The other three strains, designated SS1, SS2 and SS3, were isolated at the Institute of Microbiology and Virology of Sassari, and were identified as Lactobacillus species on the basis of cell and colony morphology, Gram stain and growth features.

Vo1.11,1992

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Table 1. Active constituents of the vaginal antiseptics tested. Antiseptic

Constituents

no.

1

Nonyl-phenoxyl-polyethoxyl ethanol (nonoxynol-9), alkylamido-betaine, lactic acid, dimethyldidecyl-ammonium chloride of pH 4.0 Amphoteric compound with aminoacid structure, non-ionic apolar, physiologic pH Germal1115 [urea-l,l-methylen-bis,3-hydroxymethyi-2,4-dioxo-5-imidazoline] 0.3 %, propylene glycol 200 mg, bergamotte oil 6 mg, water to 100 g Whey 0.045 %, glycolic salvia extract (1:0.5) 0.01%, tx-ketoglutaric acid 0.0025 %, anionic surface-active agent, lactic acid of pH 4.5 Arnphoteric surface active agents, hamamelis, lactic acid, distilled water [1,6-di(4-chlorophenyl biguanidine) exane] gluconate 0.08 g, menthol 0.500 g, polyoxyl (20) sorbitan monolaurate 15 g, N,N-dimethyl-N (3-alkylamido propyl) aminoacetate 3 g, chlorophyl 0.1 g, lavender oil 0.06 g, hydroglyceric solution 100 ml Salicaria hydroglycolic extract (0.5:1) 0.25 g, cx-ketoglutaric acid 0.0025 g, whey 0.045 g, sodium laurylcitrate 0.27 g, sterile water 100 ml, lactic acid of pH 4.5

F o r isolation of lactobacilli, material from the swabs was spread on SL Rogosa agar plates (Difco, U S A ) or de Man, Rogosa, Sharpe (MRS) agar plates (Oxoid, UK) and incubated for 48 h at 37 °C in an anaerobic jar using a Gas Generating Kit for microaerophilic organisms (Oxoid). Isolated coloni'es were taken and suspended in MRS b r o t h ( O x o i d ) . S t o c k c u l t u r e s w e r e s t o r e d at -20 °C in 10 % sterile skimmed milk (Oxoid). MICs of the douche solutions for lactobacilli strains were determined in liquid medium. Twofold serial dilutions (from 50 % to 0.39 %) of each solution were prepared in duplicate in MRS broth and inoculated with 1 x 10°cfu/ml of lactobacilli. After incubation at 37 °C for 24 h, testtubes were checked for growth of the bacteria. T h e lowest concentration at which no growth was detectable was defined as the MIC. T h e exposure time required to inhibit growth was determined as follows. Lactobacilli in the logarithmic phase of growth were centrifuged at 1200 x g for 15 min, washed in phosphate buffered saline (PBS) and resuspended in an appropriate volume of douche solution at a concentration of 5 x l06 bacteria/ml. A control tube (bacteria suspended in PBS at the same density) was included in each experiment. At time zero and after 1, 5, 10, 15, 20 and 30 min, 0.5 ml of suspension was removed, diluted with 6 ml of PBS and centrifuged, and the resulting pellet was resuspended in 6 ml of MRS broth. Testtubes

were read for bacterial growth after incubation for 24 h at 37 °C. The post-antibiotic effect (PAE) of some solutions was determined by a broth technique (14). Lactobacilli in the logarithmic phase of growth were washed in PBS and resuspended both in the douche solution and in PBS (control suspension) at a concentration of 5 x 106 cfu/ml. Testtubes were incubated at 37 °C for various periods. At the end of this incubation, suspensions were centrifuged at 1200 x g for 15 min. Bacterial pellets were then washed twice with PBS to remove douche solution and resuspended in fresh MRS broth. Colony counts (cfu/ml) were p e r f o r m e d at time zero and at prefixed times, at which 0.5 ml was removed from each sample. T h e 0.5 ml sample was subjected to serial tenfold dilutions in PBS and seeded on MRS agar plates. Plates were read after anaerobic incubation for 48 h at 37 °C. The PAE was calculated using the following formula: PAE = T - C where T is the time required for the colony count (cfu/ml) in the treated culture to increase 1 loglo above the n u m b e r observed at zero time, and C is the time required for the colony count in the control to increase 1 logl0 above the n u m b e r observed at zero time.

Results and Discussion. MICs of the douche solutions for the lactobacilli strains were in g e n e r a l high, ranging b e t w e e n 12.5 % and

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E u r . J. Clin. M i c r o b i o l . Infect. Dis.

Table 2: Minimum concentrations (expressed as a percentage) of the seven vaginal antiseptics required

to inhibit vaginal lactobacilli growth in vitro. MIC (expressed as percentage) of the vaginal antiseptics Strain

No. 1

No. 2

No. 3

No. 4

No. 5

No. 6

No. 7

PV14 PV15 PV16 PV22 SS1 SS2 SS3

1.56 1.56 1.56 1.56 1.56 ND 0.76

12.5 25 12.5 12.5 50 25 25

12.5 12.5 12.5 12.5 25 12.5 ND

12.5 12.5 25 25 25 12.5 12.5

12.5 25 12.5 12.5 25 ND 6.25

12.5 25 12.5 50 > 50 > 50 ND

12.5 12.5 12.5 12.5 25 25 25

ND = not determined.

Table 3: Minimum exposure times required by the seven vaginal antiseptics (undiluted and diluted 1:100 in PBS) to inhibit growth of vaginal lactobacilli in vitro. Exposure time (rain) Strain

No.1

PV14

No. 2

No. 3

No. 4

No. 5

No. 6

No. 7

1 1

1

15

1

1

15

1

1:100

1 1

1

20

1

1

> 30

5

1:100

1 5

I

> 30

1

1

> 30

1

1:100

5 5

> 30

> 30

1

> 30

ND

5

1:100

1 1

> 30

ND

1

> 30

> 30

10

I:100

1

> 30

> 30

1

ND

> 30

> 30

1:100

10 > 30

ND

ND

> 30

ND

ND

PV15 PV16 PV22 SS1 SS2 SS3

ND

ND = not determined.

> 5 0 %; h o w e v e r , s o l u t i o n no. 1 e x h i b i t e d m u c h l o w e r M I C v a l u e s ( T a b l e 2). Table 3 shows the minimum time required for each douche solution to inhibit lactobacilli g r o w t h . W h e r e a s with s o m e s o l u t i o n s (no. 3 a n d 6) this t i m e was p r o l o n g e d , in o t h e r c a s e s (no. 1 a n d 4) 1 m i n o f c o n t a c t was e n o u g h to t o t a l l y inh i b i t b a c t e r i a l g r o w t h . It is i n t e r e s t i n g to n o t e t h a t s o l u t i o n no. 1 still e x h i b i t e d n o t e w o r t h y a n t i b a c t e r i a l a c t i v i t y a f t e r 1:100 d i l u t i o n in P B S . T h e inh i b i t o r y a c t i v i t y o f s o l u t i o n s no. 2, 5 a n d 7 v a r i e d f r o m s t r a i n to strain. T h e P A E o f d o u c h e solutions no. 1 a n d no. 3 o n t h e P V 1 4 s t r a i n was also

evaluated. We chose these douche solutions bec a u s e t h e i r M I C v a l u e s a n d t h e i r a n t i b a c t e r i a l activity w e r e v e r y d i f f e r e n t ( T a b l e s 2 a n d 3). S o l u tion no. 1 was t e s t e d a f t e r a c o n t a c t t i m e o f 2 min a n d at 1:500 d i l u t i o n b e c a u s e l o w e r d i l u t i o n s d i d not allow bacterial growth after removal of the d o u c h e s o l u t i o n s , t h u s h i n d e r i n g m e a s u r e m e n t of t h e P A E . U n d e r o u r e x p e r i m e n t a l c o n d i t i o n s , the P A E v a l u e o f s o l u t i o n no. 1 was 5 h 40 rain. S o l u tion no. 3 was t e s t e d u n d i l u t e d ; a f t e r 5 m i n o f exp o s u r e n o s u p p r e s s i o n o f l a c t o b a c i l l i g r o w t h was observed, indicating there was no detectable PAE.

Vol. 11, 1992

In previous studies the inhibitory activity of antiseptic products against microorganisms responsible for urogenital diseases (Candida albicans,

Trichomonas vaginalis, Chlamydia trachomatis) has been investigated (12, 13). The findings of those studies indicated that improper use of vaginal antiseptics in the case of infectious diseases of the urogenital system could affect the results of diagnostic procedures for recovery of microorganisms from clinical specimens. Results obtained in the present study demonstrate that the same antiseptics previously examined show m a r k e d antibacterial activity in vitro against vaginal lactobacilli. This inhibition was often evident after very short exposure times, comparable with the times of actual presence of the antiseptics on the vaginal epithelium. A remarkable delay of bacterial growth was observed after very short exposure to a high dilution of douche solution no. 1. These findings suggest that the use of antiseptics, especially if frequent, could cause changes in the flora normally present in the vagina. They also indicate the need for in vivo investigations on the effects of antiseptic and contraceptive compounds on the vaginal ecosystem. References 1. Bartlett JG, Onderdonk AB, Drude E, Goldstein C, Anderka M, Alpert S, McCormack WM: Quantitative bacteriology of the vaginal flora. Journal of Infectious Disease 1977, 136: 271-277. 2. Goldacre MJ, Watt B, Loudon N, Milne LJ, Loudon JDO, Vessey MP: Vaginal microbial flora in normal young women. British Medical Journal 1979, i: 14501453. 3. Giorgi A, Torriani S, Deilaglio F, Bo G, Stola E, Bernuzzi L: Identification of vaginal lactobacilli from asymptomatic women. Microbiologica 1987, 10: 377384. 4. Magliano EM, Clerici P, Bestetti ML: Ricerche sulr isolamento, la quantizzazione e la biotipizzazione della flora lattobacillare di provenienza vaginale. Bollettino dell' Istituto Sieroterapico Milanese 1984, 63: 331-337. 5. Sharpe ME: The genus Lactobacillus.In: Starr MP, Stolp G, Trtiber C, Balows H, Schlegel K (ed): The prokaryotes. Volume 2. Springer-Verlag, Berlin, 1981. 6. Eschenbach D: Vaginal infection. Clinical Obstetrics and Gynecology 1983, 26: 186. 7. Hanna NF: The relation between vaginal pH and the microbiological status in vaginitis. British Journal of Obstetrics and Gynecology 1985, 92: 1267-1271. 8. Kiebanoff SJ, Hillier SL, Eschenbach DA, Wailersdorph AM: Control of the microbial flora of the vagina by H202 generating lactobacilli. Journal of Infectious Diseases 1991, 164: 94-100. 9. Vignini M, Pelfinl C: Feminine hygiene both daily and in relation to sexual intercourse. Journal of Applied Cosmetology 1987, 5: 102.

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10. Proto M, Cardinale F, Cortelazzi R: Controlli clinici e batteriologici dopo l'impiego di una lavanda vaginale monodose a base di salicaria. Giornale haliano di Ostetricia e Ginecologia 1989, 1: 60-64. 11. Wolner-Hanssen P, Eschenbach DA, Paavonen J, Stevens CE, Kiviat NB, Critchlow C, DeRouen T, Koutsky L, Holmes KK: Association between vaginal douching and acute pelvic inflammatory disease. Journal of the American Medical Association 1990, 263: 1936-1941. 12. Piu L, Juliano C: Requisiti tecnologici ed attivit~ "in vitro" di alcuni preparati antisettici per uso vaginale. L'Igiene Moderna 1990, 94: 688-700. 13. Juliano C, Piu L, Zanetti S: Requisiti tecnologici ed attivith "in vitro" di alcuni preparati antisettici per us vaginale. Nota II. L'Igiene Moderna 1991, 95: 672679. 14. Craig WA, Gudmundson S: The postantibiotic effect. In: Lorian V (ed): Antibiotics in laboratory medicine. William & Wilkins, Baltimore, 1986, p. 515-536.

Comparison of the E Test and a Reference Agar Dilution Method for Susceptibility Testing of Anaerobic Bacteria J.

Wrist*, U.

Hardegger

The susceptibility of 146 recent clinical isolates of gram-negative and gram-positive anaerobes was determined by the E test ( A B Biodisk) on both Wilkins-Chaigren and P D M A S M II ( A B Biodisk) agar. Results of the E test were compared with results obtained by the NCCLS agar dilution method using Wilkins-Chalgren agar. Incubation was for 20 hours and 44 hours in the E test and for 44 hours in the NCCLS method. In general, 44 hour results were more reliable; however, N C C L S readings were made only once after 44 hours. After two days of incubation, 9 1 % of E test results on Wiikins-Chaigren agar were within one dilution and 98 % within two dilutions of the corresponding N C C L S values; on P D M agar these values were 89 % and 98 %, respectively. Major and very major discrepancies combined were less than 1 % .

Department of Clinical Microbiology, University of Zurich, Gloriastr. 32, CH-8028 Ziirieh, Switzerland.

In vitro antibacterial activity of antiseptics against vaginal lactobacilli.

The results of investigations carried out to evaluate the inhibitory activity in vitro of seven vaginal antiseptic douche solutions against several st...
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