ORIGINAL ARTICLE
Antibiotic Susceptibility of Lactobacilli Isolated from the Cervix of Healthy Women Gian Piero Testore1, Cesare Sarrecchia1, Edoardo Zupi2, Pasquale Sordillo1, Edoardo Valli2, Fernanda Bove3 and Massimo Andreoni1 From the 1Chair of Infectious Disease, Department of Public Health, 2Chair of Obstetrics and Gynaecology , Department of Surgery, University of Rome ‘Tor Vergata’, Rome, Italy, and 3Microbiology Service, S. Eugenio Hospital, Ple dell’Umanesimo 10, Rome, Italy Correspondence to: Prof. Massimo Andreoni, MD, Department of Public Health, University of Rome ‘Tor Vergata’, V. di Tor Vergata 135, 00133, Rome, Italy. Tel: »39 6 72596873; Fax: »39 6 2020799; E-mail:
[email protected]
Microbial Ecology in Health and Disease 2002; 14: 14 – 18 During antibiotic treatments normal vaginal micro ora can be replaced by resistant bacteria or fungi. The resulting infections are considered an unavoidable side effect. This study is aimed at identifying antibiotics with low activity against lactobacilli. Fifty isolates from 30 cervix specimens collected from 30 healthy women during colposcopy, typed by API 50CHL as lactobacilli, were tested for susceptibility to 41 antibiotics using the Bauer– Kirby test. The test was easy to perform and to interpret. Aztreonam, fusidic acid, ce xime, kanamycin and pe oxacin showed less activity against lactobacilli (72 – 86% of resistant isolates). All isolates were found to be susceptible to imipenem and piperacillin-tazobactam. Vancomycin showed higher antibacterial activity (22% of resistant isolates) than teicoplanin (54% resistance). Antibiotic treatments have to take into account the susceptibility of normal vaginal micro ora to prevent colonization by resistant pathogenic microorganisms. Key words : cervical micro ora, antibiotic susceptibility of lactobacilli, lactobacilli typing.
INTRODUCTION In a ‘post antibiotic era’ (1) a normal vaginal ecosystem could be the rst goal in the prevention of bacterial contamination of the vagina and, consequently, in the prevention of infectious diseases of the female genital tract (2, 3). In spite of this, the preservation of the vaginal micro ora during antibiotic treatment is not considered a goal of therapy. As a consequence, the use of chemo-antibiotics, active against lactobacilli, causes the destruction of normal micro ora and the resulting super-infections are, for clinicians, an unpleasant but unavoidable side effect of a systemic antibiotic therapy. The antibiotic susceptibility of the vaginal Gram-positive catalase-negative bacilli is usually not tested. The low pathogenicity of these bacteria (4, 5), their exigencies for growth, the absence of low-cost identi cation kits and, primarily, the absence of any standardized method to assess the susceptibility to antimicrobial drugs in vitro, can explain the dearth of information about the alteration on vaginal ora during antimicrobial treatment. During a study on normal cervical micro ora in healthy women, we typed Gram-positive catalase-negative bacilli by the commercial system API 50 CHL (BioMerieux, Marcy-l’Etoile, France) and performed the Bauer –Kirby susceptibility test © Taylor & Francis 2002. ISSN 0891-060 X
on Lactobacillus isolates with the aim of identifying drugs with low activity against vaginal lactobacilli, which can be used in women to minimize the alteration of the vaginal ecosystem. MATERIALS AND METHODS Specimens were collected from 30 healthy women during colposcopies. Patients gave informed consent to participate in the study. After removing cervical mucus, the cervical canal was brushed three times and the sample was dispersed into 1 ml of fresh brain-heart infusion broth maintained in an anaerobic atmosphere until use. The specimens were processed within 20 min from collection. One hundred ml of the inoculated broth was streaked onto Rogosa Agar (Oxoid Limited, Basingstoke, Hants, UK) and plates were incubated for 48 h in an anaerobic cabinet (atmosphere composition: N2 85%, H2 10%, CO2 5%) at 37°C. Growth colonies were sub-cultivated onto Columbia Blood Agar (Oxoid) as non-selective enrichment medium to evaluate the presence of b-haemolysis. The identi cation of lactobacilli was performed by the commercial system API 50 CHL (Bio-Merieux) according to the manufacturer’s instructions. The API system supplies a scale of identi cation quality that we utilized to group typing results in three levels: Microbial Ecology in Health and Disease
Antibiotic susceptibility of vaginal lactobacilli
Table I Susceptibility pattern of 50 lactobacillus strains to 41 chemotherapics obtained using the Bauer–Kirby test on Mueller–Hinton Agar supplemented by 5% of blood. Percentages are rounded for excess. A. Lincosamydes, penicillins, tetracycline, macrolides B. Cephalosporins, glycopeptides, carbapenems C. Monobattams, aminoglycosides, quinolones, others Drugs
Number of isolates
L. acidophylus (24 isolates)
Total
Susceptible
A Lincosamydes Lincomycin Clindamycin
50 50
21 29
(42) (58)
14 16
(58) (67)
2 2
(18) (18)
Penicillins Penicillin G Piperacillin Carbenicillin Methicillina Ampicillin
48 50 50 50 50
28 45 44 35 48
(58) (90) (88) (70) (96)
17 24 21 24 24
(88) (100) (88) (100) (100)
2 10 8 6 11
(18) (91) (73) (55) (100)
Tetracycline Doxiciclina
50
45
(90)
24
(100)
10
(91)
Macrolides Erythromycin
49
31
(62)
21
(88)
2
(18)
50 50 54 50 48
37 30 28 40 30
(74) (60) (52) (80) (62)
21 20 20 24 19
(88) (88) (88) (100) (79)
2 2 2 4 3
(18) (18) (18) (36) (28)
50 50 50
29 24 46
(58) (48) (92)
21 19 24
(88) (79) (100)
2 2 7
(18) (18) (64)
50 50 50 50 50
25 34 12 34 38
(50) (68) (24) (68) (76)
18 21 9 21 24
(75) (88) (37) (88) (100)
2 2 2 7 7
(18) (18) (18) (64) (64)
Glycopeptides Teicoplanin Vancomycina
50 50
23 39
(46) (78)
16 24
(67) (100)
2 7
(18) (64)
Carbapenem Imipenem Meropenem
50 50
50 50
(100) (100)
24 24
(100) (100)
11 11
(100) (100)
B Monobattams Aztreonam
49
7
(14)
8
(33)
3
(28)
Aminoglycosides Amikacin Gentamicin Kanamycin Netilmicin Tobramycin
50 50 50 50 50
38 35 13 43 16
(76) (70) (26) (86) (32)
18 19 3 24 5
(75) (79) (13) (100) (21)
8 4 2 4 4
(73) (36) (18) (36) (36)
B Cephalosporins (1st generation ) Cefaclor Cephadroxil Cephradine Cephalotin Cephalexin (2nd generation) Cefoxitin Cefuroxime Cefamandole (3rd generation) Ceftriaxone Cefoperazone Ce xime Cefotaxime Ceftazidime
(%)
Susceptible
L. paracasei (11 isolates) (%)
Susceptible
(%)
15
16
G. P. Testore et al.
Table I (Continued) Drugs
Number of isolates
Total
Susceptible
Quinolones Pe oxacin Nor oxacin Cipro oxacin
46 50 50
13 25 27
Vari Fusidic acid Rifampin Chloramphenicol Trimethoprim-Sulfa. Cefepime Piperacillin-tazobactam
50 50 50 50 50 50
9 44 39 16 41 50
L. acidophylus (24 isolates) (%)
L. paracasei (11 isolates)
Susceptible
(%)
(28) (50) (54)
3 11 24
(12) (46) (100)
3 5 6
(28) (45) (55)
(18) (88) (78) (32) (82) (100)
1 24 24 8 24 24
(4) (100) (100) (33) (100) (100)
4 6 7 4 6 11
(36) (55) (64) (36) (55) (100)
– 1° level (excellent, very good, good, acceptable identi cation); – 2° level (very good for the kind, good for the kind, good for the kind identi cation); – 3° level (doubtful, low discrimination, unacceptable identi cation). As regard the antibiotic susceptibility of lactobacilli, in the absence of a standard susceptibility test technique, we tested the isolates against 41 drugs using the Bauer –Kirby test (6) on Mueller – Hinton agar supplemented by 5% blood using the same method suggested for streptococci (7). The plates were incubated at 37°C in an anaerobic cabinet for 24 – 48 h. As a control, susceptibility to vancomycin and penicillin G was also evaluated for each isolate by the broth dilution method, using Mueller – Hinton broth. RESULTS One hundred and sixty strains were isolated from cervical specimens collected during colposcopy from 30 healthy women (a mean of 5.33 strains per specimen; range 1–7). Seventy-six isolates (47.5%) were Gram-positive catalasenegative bacteria. A pure culture of these isolates was typed using the API 50 CH-CHL commercial system. Sixty-two typings (79%) allowed us to identify bacteria belonging to Lactobacillus genus while the remaining 16 microorganisms were Pediococcus pentosaceus (n 6), Lacto coccus lactis subsp. lactis (n 6), Lactococcus raf nolactis (n 4). L. acidophilus and L. paracasei subsp. paracasei 24 (40%) and 11 (18%) isolates, respectively, represented 58.33% of all lactobacilli, while the remaining 41.67% consisted of 7 isolates of L. rhamnosus and L. brevis (11.66%), 3 isolates of L. delb subsp. delb and L. fermentum, 2 isolates of L. plantarum and L. curvatus, and 1 each of L. pentosi, L. crispatus and L. salivarius.
Susceptible
(%)
According to the quality identi cation scale, 50% of lactobacilli fall in the rst level (excellent, very good, good, acceptable identi cation), 23% in the second (very good for the kind, good for the kind, good for the kind identi cation) and 27% in the third (doubtful, low discrimination, unacceptable identi cation). Fifty lactobacilli were tested for their susceptibility to 41 chemotherapics (Table IA, B, C). In Table II, antibiotics are grouped according to the susceptibility of lactobacilli. Overall isolates showed a resistance level between 86% and 72% to 5 drugs (aztreonam, fusidic acid, ce xime, kanamycin, pe oxacin), between 68% and 50% to 7 drugs, and between 48% and 0% to 29 drugs. L. acidophilus and L. paracasei subsp. paracasei showed similar susceptibility to 13 drugs, a better susceptibility of L. paracasei subsp. paracasei was detected for 3 drugs but, for 25 antibiotics (61%) L. paracasei subsp. paracasei was more resistant compared with L. acidophilus. Eleven out of 50 (22%) lactobacilli were vancomycin resistant (4 L. paracasei subsp. paracasei, 2 L. brevis, 2 L. plantarum, and 1 each of L. fermentum, L. pentosaceus and L. rhamnosus ). Susceptibilities to vancomycin and penicillin G obtained using the broth dilution method overlap those obtained using the Bauer –Kirby test. DISCUSSION Despite the great importance of ecology in all aspects of human life, we know very little about the microecology of human body. The vagina, for its characteristics (brevity, accessibility, etc.), is ideal to study bacterial micro ora. Some aspects are quite clear: lactobacilli are the dominant genus (1), they adhere to the epithelial cells of the vagina (8– 12), inhibit the adherence of the Gardnerella genus (13) and interfere with the adherence to the vaginal cells of Candida albicans (14). Some lactobacilli produce H2O2 (15) and antibacterial substances (16). The quality of the bac-
Antibiotic susceptibility of vaginal lactobacilli
Table II Antibiotic subdivision according to lactobacilli antibiotic resistance. Drugs are divided in four classes from the less to the more active. Percentage of activity was obtained by dividing the number of resistant strains by the total number of tested strains and multiply ing results for 100 1st class (100–70%) Aztreonam Fusidic acid Ce xime Kanamycin Pe oxacin
86 82 76 74 72
2nd class (69–50%) Trimethoprim-Sulfa. Tobramycin Lincomycin Teicoplanin Cefuroxim Ceftriaxon Nor oxacin
68 66 58 54 52 50 50
3rd class (49–30%) Cephradine Cipro oxacin Cefoxitin Penicillin G Clindamycin Cefadroxil Cephalexin Erythromycin Cefoperazone Gentamycin Cefotaxime Methicillin
48 46 42 42 42 40 38 37 34 32 31 31
4th class (29–0%) Cefaclor Amikacin Ceftazidime Cephalotin Vancomycin Chloranphenicol Cefepime Netilmycin Carbenicillin Rinfapin Meropenem Piperacillin Doxicyclin Cephamandol Ampicillin Imipenem Tazobactam
26 24 24 22 22 22 19 14 12 12 10 10 10 8 4 0 0
teria of vaginal micro ora correlates with plasma level of estrogens (17, 18). Despite the importance of preserving micro ora for the health of a woman (19, 20), rapid tests for routine detection of vaginal micro ora have not yet been developed. In our study, using the API 50 CHL test (BioMerieux), we obtained acceptable typing only in 73% of isolates, whereas in 27% of isolates quality level typing scale was
17
doubtful or unacceptable, even when the typing was repeated. Moreover, our advice is that the method is too complex and expensive to be utilized routinely. This represents a major limit to lactobacilli identi cation, which contrasts with the good knowledge of the behaviour of these bacteria. Growth inhibition zones antibiotics were easily read using the Bauer –Kirby method, but presently this method is not standardized to evaluate lactobacilli susceptibility to antibiotics. A remarkable difference in antibiotic susceptibility among lactobacilli species was found. Particularly, L. paracasei subsp. paracasei showed a higher resistance than L. acidophilus with respect to more than 50% of tested drugs. These differences underline the importance of lactobacilli typing to asses a correct choice of antibiotic therapy. Grouping antibiotics according to the bacterial resistance in vitro permits us to de ne which drug is used to treat infections in women of fertile age, is less destructive to vaginal normal micro ora and, consequently, the best choice to preserve the vaginal environment. All drugs were active against lactobacilli and it is possible only to indicate which are less active or which have to be avoided because of being too active (e.g. imipenem and piperacillin-tazobactam). Moreover, also within antibiotic classes it is possible to choose between less or more active drugs. In our setting, with regard to amiglycosides class, for example, kanamycin (74% of resistant isolates) is less destructive then amikacin (24%), while tobramycin (68%) is more active in our isolates than previously reported elsewhere (18). Similarly, pe oxacin (72% of resistant isolates) can be utilized in women in place of cipro oxacin (46%), which is widely used in Italy. Fusidic acid (82% of resistant isolates) or teicoplanin (54%) can be preferable for infections caused by oxacillin-resistant staphylococci over vancomycin (22%). Lactobacilli are reported as being highly resistant to vancomycin. In this study a good level of activity of this drug was detected. It is possible to hypothesize about some differences in antibiotic susceptibility between lactobacilli isolated from the vagina and those derived from other environments. Moreover, we performed a phenotypic typing of Gram-positive catalase-negative bacteria, and the genotypic analysis could demonstrate that these vancomycin-susceptible isolates did not belong to Lactobacil lus genus ; however, this technique is not normally performed under routine laboratory conditions. The low percentage (22%) of lactobacilli that are resistant to vancomycin make the use of this drug for identi cation purposes useless in our setting (21). The high frequency of resistance to erythromycin and chlorampenicol among lactobacilli, especially among L. paracasei subsp. paracasei, can be related, only for erythromycin, to the frequent use of this drug in Italy. A cross-resistance between erythromycin and chlorampenicol mediated by plasmid was described in Bacteroides subsp. (22) and it is possible to
18
G. P. Testore et al.
hypothesize a similar mechanism for lactobacilli. The comparable activity of ceftazidime and cephalotin against lactobacilli, drugs with different bacterial targets, con rms that to generalize the drug spectrum of activity to genera not routinely studied can be unusable. Ampicillin, a lowcost antibiotic often utilized in respiratory and urinary tract infections, is strongly active against lactobacillus and should be avoided by women. Our data do not con rm a previous report about the low activity of doxycycline (23), which is normally prescribed to treat chlamydial infections. To use drugs not active against lactobacilli can be considered a health risk because the resistance could be transferred by resistant genes to other bacteria normally susceptible. Antibiotic treatment normally induces the development of resistant mutants that can substitute normal susceptible micro ora. It is our advice that it is more important to preserve the ecology of normal vaginal micro ora, even if resistant, than to alter the physiology of the reservoir with resistant bacteria belonging to faecal micro ora. To spare the normal human ecosystem, also when antibiotic treatment is needed, remains a goal for the microbiologist and clinician, and better knowledge of normal micro ora and its antibiotic susceptibility is needed.
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