Divisions of 1Clinical Bacteriology, F82, and 2 Infectious Diseases, Karolinska University Hospital Huddinge, SE-141 86 Stockholm, Sweden; 3 Division of Infectious Diseases, Karolinska University Hospital Solna, Karolinska Institutet, Stockholm, Sweden
Received 16 March 2004; returned 13 June 2004; revised 30 June 2004; accepted 20 July 2004
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Abstract |
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Methods: Twenty patients admitted to hospital due for treatment with penicillin and 16 patients due for treatment with ciprofloxacin or norfloxacin were included in the study. In either group, the patients were randomized into two groups, receiving placebo or an active probiotic product. Faecal samples were collected before treatment, on day 10 and 1 month after the start of the treatment. Isolates of enterococci, enterobacteria and Bacteroides fragilis species were screened for resistance to penicillin and ciprofloxacin, respectively.
Results: Administration of penicillin did not influence resistance in enterococci while quinolone resistance increased during quinolone treatment. Susceptibility to ampicillin and piperacillin/tazobactam decreased in enterobacteria during penicillin treatment and ciprofloxacin resistance increased in the quinolone group. Penicillin and quinolones did not influence the resistance rates of Bacteroides isolates. No major differences were observed between the probiotic- and placebo-supplemented groups.
Conclusions: There was a limited effect of Lactobacillus F19 on the emergence of resistant isolates during treatment with penicillin and quinolones.
Keywords: probiotics , resistance , antimicrobial agents
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Introduction |
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Among the aerobic microorganisms, enterococci are regarded as one of the most common causes of nosocomial infection. The majority of enterococcal infections are caused by Enterococcus faecalis followed by Enterococcus faecium.3 Enterococci are intrinsically resistant to several antibacterial agents, i.e. low-level resistance to cell-wall-active agents and aminoglycosides.4 However, in the Nordic countries the level of resistance in enterococci is still low.5
Other aerobic microorganisms frequently encountered among clinical isolates are members of the family Enterobacteriaceae.6 It has been shown that long-term hospitalized elderly patients are at increased risk of being infected with Gram-negative bacilli resistant to ampicillin.7 A problem with regard to Enterobacteriaceae, since the introduction of the third-generation cephalosporins, is the increasing number of strains expressing extended-spectrum ß-lactamases (ESBLs). Almost 17% of Klebsiella pneumoniae strains in a European multi-centre study were found to be potential carriers of ESBL enzymes.6
Anaerobic bacteria outnumber aerobic bacteria in the gastrointestinal tract, and among the anaerobic microorganisms, Bacteroides spp. are the most frequent pathogenic bacteria isolated.8 Clinically important species are the Bacteroides fragilis group. The majority of the B. fragilis group is resistant to penicillins and cephalosporins. Resistance to clindamycin is increasing while resistance to carbapenems and nitroimidazoles is still rare.8
The gastrointestinal microbial ecosystem is relatively stable but quantitative and qualitative disturbances are seen after oral administration of antibiotics.9 The normal flora limits the concentration of potentially pathogenic microorganisms, which can reach high numbers in connection with intake of antimicrobial agents.10 The use of probiotics for prevention of gastrointestinal diseases is well established.11 However, no studies have been performed on the role of probiotics in the prevention of emergence of resistant isolates. By competition for receptors and nutrition and by production of lactic acid and antimicrobial substances, probiotic strains could be a means by which colonization with resistant strains could be prevented.
The aim of the present study was to examine whether administration of Lactobacillus F19 in conjunction with treatment with penicillin or quinolones (ciprofloxacin or norfloxacin) prevents the emergence of resistant isolates of enterococci, enterobacteria and B. fragilis spp. in the intestinal tract.
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Material and methods |
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Forty-one patients admitted to the Division of Infectious Diseases, Karolinska University Hospital Huddinge, Stockholm, Sweden receiving treatment with penicillin G followed by penicillin V, and 23 patients receiving treatment with ciprofloxacin or norfloxacin were included in the study. None of the patients had any history of gastrointestinal, liver or kidney diseases, and had not been treated with antimicrobial agents within 3 months preceding the study. The two treatment groups were randomized into one placebo and one active group regarding the probiotic supplement in a double-blind fashion. The study was approved by the local ethics committee of Karolinska University Hospital, Karolinska Institutet, Stockholm, Sweden.
Administration of antimicrobial agents
Patients in the penicillin group received penicillin G (Bensylpenicillin; Astra Läkemedel, Södertälje, Sweden) 1 or 3 g three times daily followed by penicillin V (Kåvepenin; Astra Läkemedel) 1 g three times daily and patients in the quinolone group received either ciprofloxacin (Ciproxin; Bayer AB, Göteborg, Sweden) 500 or 750 mg twice per day or norfloxacin (Lexinor; Astra Läkemedel) 400 mg twice daily.
Administration of probiotic and placebo products
The probiotic product consisted of powdered milk (10 g) and 1010 colony forming units (cfu) of freeze-dried Lactobacillus paracasei ssp. paracasei F19 (Arla Foods, Stockholm, Sweden). A similar product was given to patients in the placebo groups but with no added microorganisms. The products were prepared in connection with the start of the study and were stored at 70°C. After inclusion of a patient, the product was stored at 8°C. The products were dissolved in 100 mL water immediately before use. The products were administered twice daily for 14 days. The cell viability was checked after storage under similar conditions.
Compliance
Signed diary cards that were returned at the end of the study checked compliance in respect of the probiotic product. Assay of the probiotic microorganism in faecal samples was a further verification of compliance.
Sampling procedures
Stool specimens were collected before the administration of antimicrobial agents and probiotic/placebo products (day 1), on day 10 and 30 days after the start of administration. The samples were collected in sterile plastic containers, which were frozen at 70°C until processed.
Microbiological procedures
Stool specimens were suspended in pre-reduced peptoneyeast extract medium, diluted to 107 and inoculated on the following selective media: Enterococcosel agar (BBL, Cockeysville, MD, USA) for detection of enterococci, CLED agar (Merck, Darmstadt, Germany) for detection of Enterobacteriaceae, BKV (kanamycinvancomycinblood) agar for detection of B. fragilis group, Rogosa agar (Difco, Detroit, MI, USA) for cultivation of lactobacilli and TCCFA agar (Pepton from Casein/Proteose PeptonIII 40 mg/mL, sodium hydrogen phosphate 5 mg/mL, potassium dihydrogen phosphate 1 mg/L, sodium chloride 2 mg/mL, magnesium sulphate 0.2 mg/mL, Bacto agar/agaragar 20 mg/L, taurocholic acid 1 mg/mL, neutral red 0.03 mg/mL, fructose 15%, Clostridium difficile-supplemented D-cycloserine, cefoxitin) for cultivation of C. difficile. Blood agar (LabM Kemila, Bury, UK) was used for total counts of aerobic and anaerobic bacteria. ASM agar (PDM; Biodisk, Solna, Sweden) supplemented with 5% blood and 8 mg/L penicillin G for aerobic microorganisms and 2 mg/L penicillin G for anaerobic microorganisms or ciprofloxacin 4 mg/L (used for aerobic and anaerobic bacteria) was used for assessing the total number of penicillin- or quinolone-resistant microorganisms, respectively. The aerobic agar plates were incubated for 24 h at 37°C and the anaerobic plates for 48 h at 37°C in anaerobic jars (GasPak; BBL). After incubation different colony types were counted, isolated in pure cultures and identified to genus level. All isolates were analysed according to Gram's stain and colony morphology, followed by biochemical tests. The API 20E kit (bioMérieux SA, Marcy l'Étoile, France) was used for identification of Enterobacteriaceae. Anaerobic microorganisms were identified to genus level by gasliquid chromatography of metabolites from glucose. The lower limit of detection was 102 microorganisms per gram faeces.
Isolated strains of C. difficile were tested for cytotoxin production on McCoy cells with an antitoxin kit for detection of toxin B (TechLab, Blacksburg, VA, USA). C. difficile CCUG 19126 was used as the reference strain.
Antibiotic susceptibility tests
Up to five colonies of enterococci, Enterobacteriaceae and B. fragilis group spp. were isolated from each patient on each sampling occasion. Depending on which antimicrobial agent had been used as treatment, the MIC of penicillin or ciprofloxacin was determined by the agar dilution method, according to the NCCLS M7-A6 document for aerobic bacteria12 and the NCCLS M11-A5 document for anaerobic bacteria.13 Species intrinsically resistant and those found to be resistant on susceptibility testing were further tested for susceptibility to several different antimicrobial agents, i.e. isolates of enterococci were tested against ampicillin, gentamicin, linezolid and vancomycin, Enterobacteriaceae were tested against ampicillin, piperacillin/tazobactam, cefotaxime and gentamicin, and B. fragilis group isolates were tested against ampicillin/clavulanic acid, cefoperazone, cefoxitin, imipenem, clindamycin and metronidazole.
The control strains used were E. faecalis ATCC 29212, Escherichia coli ATCC 25922, B. fragilis ATCC 25285 and Bacteroides thetaiotaomicron ATCC 29741. The final inoculum for aerobic bacteria was 1 x 104 cfu/spot and for anaerobic bacteria 1 x 105 cfu/spot. Inoculated plates were incubated for 18 h (aerobic bacteria) and 48 h (anaerobic bacteria) at 37°C. The breakpoint values used were according to NCCLS recommendations.
Detection of ESBLs
Isolates of Enterobacteriaceae intermediately resistant or resistant to cefotaxime were tested for production of ESBLs using the Etest (AB Biodisk). K. pneumoniae ATCC 700603 and E. coli ATCC 35218 were used as positive and negative controls, respectively.
Identification of Lactobacillus F19 strains
Colony forming units exhibiting colony and Gram's stain morphology similar to the probiotic strain Lactobacillus F19 were analysed by randomly amplified polymorphic DNA-PCR (RAPD-PCR) to verify the identification. The primer Lbc-19 (5'-AGTAGCCAC-3') was used in screening and OPA-02 (5'-TGCCGAGCTC-3') for identification of Lactobacillus F19.14
Statistical analysis
The quantitative alterations were compared within groups between pretreatment (day 1) and day 10 and between pretreatment and after treatment (day 30) and were analysed by Wilcoxon's signed rank test for paired samples. The MIC values for each species were compared within groups between pretreatment and day 10 and between pretreatment and day 30 by the MannWhitney U test in order to detect significant decreases in susceptibility during and after the administration period. Differences in MIC value between the groups were likewise analysed by the MannWhitney U test. P values <0.05 were considered statistically significant and were adjusted for the multiple analysis.
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Results |
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Of 41 randomized patients, 20 (49%) completed the study. Seventeen patients were treated for pneumonia and three for streptococcal infections (tonsillitis and erysipelas). The mean age was 59 years (range 1889 years), the mean length of hospital stay was 5 days (range 211 days), the mean amount of penicillin G was 11 g (range 330 g) and the mean total length of antimicrobial treatment (penicillin G followed by penicillin V) was 14 days (range 1117 days). There were no statistically significant differences between the patients regarding the above parameters in the active and placebo groups. Twenty-one patients did not complete the study, of whom 13 received new antimicrobial agents, three patients were nauseated by the ingestion of the probiotic/placebo product, four patients felt too ill to be able to complete the study, and in, one case, faecal samples were not received.
Patients: quinolone group
Of 23 randomized patients, 16 (70%) completed the study. Fourteen patients had urinary tract infections and two patients had fever of unknown origin and cholecystitis. Nine patients were treated with ciprofloxacin 500 mg twice daily and one patient received 750 mg twice daily for 3 days followed by norfloxacin 400 mg twice daily for 14 days and 200 mg twice daily for 14 days. The remaining six patients were treated with norfloxacin 400 mg twice daily. The mean age was 61 years (range 2886 years), the mean duration of hospital stay was 4 days (range 28 days), and the total length of antimicrobial treatment was 12 days (range 731 days) (15 patients 15 days). There were no statistically significant differences between the patients concerning these parameters in the active and placebo groups. Seven patients did not complete the study. In three cases there was a change of medication or the medication was withdrawn, one patient felt nauseous when drinking the supplement and three subjects failed to produce faecal samples.
Compliance
Four patients in the penicillin group receiving the active probiotic product returned completed diary cards. In six cases, information was lacking concerning the intake of the probiotic product. In the placebo group, there were six completed diary cards and four incomplete. In the quinolone-treated patients in the active and placebo groups, there were six and seven completed cards and two and one uncompleted cards, respectively.
Impact of antimicrobial agents and Lactobacillus F19 on numbers of microorganisms
The numbers of enterococci, enterobacteria and Bacteroides species in patients receiving penicillin showed no major changes apart from increased numbers of enterococci in the active group (increase from day 1 to day 30 P < 0.05). The total numbers of penicillin-resistant microorganisms on the penicillin-containing non-selective agar remained stable during the study period.
In the patients treated with quinolones, there was a statistically significant increase in the numbers of resistant isolates on the ciprofloxacin-containing non-selective agar (P < 0.05) between day 1 and 10 and numbers of Enterobacteriaceae (P < 0.05) between day 1 and day 30 in the microflora of patients in the placebo group. In the placebo group, the numbers of B. fragilis spp. (P < 0.05), the total number of anaerobic microorganisms (P < 0.001) as well as the total numbers of resistant strains (P < 0.05) increased from day 1 to day 30. No statistically significant changes were seen in the intestinal microflora in the active group.
C. difficile was isolated in four samples of four patients treated with quinolones, in one patient on day 10 and in three patients on day 30. Two patients belonged to the placebo and two to the active group. None of these isolates produced toxins.
Prevalence of Lactobacillus F19
Lactobacillus F19 was recovered in three samples from 10 patients in the active group of penicillin-treated individuals (log10 cfu range 3.34.7) on day 10. In the active group of quinolone-treated patients, Lactobacillus F19 was isolated in three samples of eight patients (log10 cfu 3.35.2) on day 10, and one patient still harboured the probiotic strain on day 30 (log10 cfu 2.3). Five of the six patients positive for Lactobacillus F19 returned their diary cards completed.
In vitro activity of antimicrobial agents against enterococci
One isolate from one patient in the active penicillin group was resistant to penicillin, ampicillin and ciprofloxacin on day 10, while the same isolate was sensitive to vancomycin, linezolid and gentamicin.
The numbers of ciprofloxacin-resistant enterococci in quinolone-treated patients increased in the placebo group from three of 32 (9%) isolates on day 1 to 21 of 25 (84%) isolates on day 10. Thirty days after the start of treatment, six of 46 (13%) isolates were resistant to ciprofloxacin. In the active group, the corresponding figures were seven of 26 (27%) isolates on day 1, 12 of 20 (60%) isolates on day 10 and 17 of 39 (44%) isolates on day 30. Isolates resistant to ampicillin were detected in one patient in the placebo group and in one patient in the active group in samples from days 10 and 30. Gentamicin-resistant isolates were also detected in all samples from one patient in the active group. All isolates were sensitive to vancomycin and linezolid.
In vitro activity of antimicrobial agents against Enterobacteriaceae
Before administration of antimicrobial agents, isolates of enterobacteria with decreased susceptibility to ampicillin and piperacillin/tazobactam were detected in patients treated with penicillin (Table 1). The MIC values increased for isolates from sample 2 and decreased again on day 30 in both the placebo and active groups. There was a statistically significant difference between the groups concerning the susceptibility to piperacillin/tazobactam also existing before the start of the study. Exclusion of patients with piperacillin/tazobactam-resistant isolates before treatment revealed the emergence of resistant or intermediately resistant isolates in four patients in the placebo group and one in the active group. In 23 of 135 (17%) isolates in the placebo group, reduced susceptibility to both ampicillin and piperacillin/tazobactam was observed. The corresponding figure for the active group was nine of 130 (7%) isolates. In both the active and placebo groups, all isolates were susceptible to ciprofloxacin. In the placebo group, four isolates from two patients with decreased susceptibility to cefotaxime were detected on days 10 and 30. Two isolates were intermediately resistant to gentamicin on day 10.
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ESBLs
One patient treated with penicillin was found to harbour ESBL-positive strains of Klebsiella oxytoca on day 10. The patient belonged to the placebo group. Amongst the quinolone-treated patients, ESBL-positive strains of E. coli was identified in two subjects, one from the placebo and one from the active group. In both cases the strains were found before treatment and on day 10 and in one patient also on day 30.
In vitro activity of antimicrobial agents against B. fragilis spp
In the penicillin-treated group, isolates of B. fragilis spp. had reduced susceptibility to ampicillin/clavulanic acid, cefoxitin, cefoperazone and clindamycin before, during and after the administration of penicillin (Table 2). There were no differences between the active and placebo groups. Over 90% of all isolates were resistant to penicillin. Two isolates from two patients in the placebo group were resistant to ampicillin/clavulanic acid, cefoxitin, cefoperazone, imipenem and metronidazole on day 10. All other isolates were susceptible to imipenem and metronidazole.
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No adverse events were reported. Eight patients treated with penicillin reported looser stools, four in the active and four in the placebo group. In the quinolone-treated patients there were two individuals who experienced looser stools, one from each of the placebo and active groups. One of the patients also complained of nausea during treatment.
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Discussion |
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In the quinolone-treated group the numbers of enterobacteria increased in the placebo group. The increased numbers of enterobacteria partly explain the increase in total numbers of quinolone-resistant aerobic isolates in this group but also reflect the increased numbers of resistant enterococci. Use of fluoroquinolone and length of hospital stay have been shown to be associated with ampicillin resistance in enterococci.15 Only two patients harboured enterococci resistant to ampicillin and also to ciprofloxacin. Four patients were already colonized with resistant enterobacteria before the treatment. Intake of quinolones during the month preceding admission to hospital has been shown to be a risk factor for the emergence of quinolone-resistant intestinal enterobacteria.16 Selection of quinolone-resistant E. coli has also been shown to occur after short exposure to quinolones in the intestinal microflora of cancer patients.17 None of the patients in this study had received antimicrobial agents or been admitted to hospital within the 3 months preceding the study. The resistance rates in B. fragilis group spp. in quinolone-treated patients followed the same pattern as for penicillin-treated patients.
The overall difference between placebo and active supplemented groups was rather limited in this study. A plausible explanation could be poor compliance, which is indicated by the low rate of completed diary cards, in particular in the penicillin group. Some patients were old and weak and several subjects complained that they did not enjoy the taste and the consistency of the products. The recovery rate of the probiotic strain was also limited. Lactobacillus F19 was only detected in three of 10 in the penicillin-treated group and in three of eight of the quinolone-treated patients. The antimicrobial agents may also have influenced the probiotic strain: the MICs of penicillin and ciprofloxacin are 0.5 and 1.5 mg/L, respectively. Furthermore, the proportions of the patient groups may have been another factor of importance. The exclusion criteria used restricted the selection, and in particular the quinolone-treated group was rather small. The effect of probiotics is strain dependent and the strain Lactobacillus F19 in combination with Lactobacillus acidophilus and Bifidobacterium lactis has been shown to have a normalizing effect on the microflora of healthy subjects during administration of clindamycin.18 In that study, which was commenced after the present one, the probiotic was delivered in a fermented milk product that might have been more agreeable to the patients.
In conclusion, the effect of Lactobacillus F19 supplement on the emergence of resistance in enterococci, enterobacteria and B. fragilis group during treatment with penicillin or quinolones was limited.
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Footnotes |
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References |
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Sullivan, A., Barkholt, L. & Nord, C. E. (2003). Lactobacillus acidophilus, Bifidobacterium lactis and Lactobacillus F19 prevent antibiotic-associated ecological disturbances of Bacteroides fragilis in the intestine. Journal of Antimicrobial Chemotherapy 52, 30811.
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