Antimicrobianos

Journal of Applied Microbiology ISSN 1364-5072
ORIGINAL ARTICLE
Towards a compatible probioticantibiotic combination therapy: assessment of antimicrobial resistance in the Japanese probiotics
A.M. Hammad and T. Shimamoto
Laboratory of Food Microbiology and Hygiene, Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan
Keywords antibiotic compatibility, Japanese probiotics. Correspondence Tadashi Shimamoto, Laboratory of Food Microbiology and Hygiene, Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima 739-8528, Japan. E-mail: tadashis@hiroshima-u.ac.jp
Abstract Aim: To determine the antimicrobial resistance of the Japanese probiotics available in the market without a pharmacists supervision. Methods and Results: A total of 43 isolates were obtained from 40 samples of probiotics (30 dairy products and 10 products in tablet form). Isolates were identied using 16S rRNA gene sequencing and tested for their susceptibility to 14 antimicrobials. They were screened using PCR for some antibiotic resistance genes. Inactivation of cefepime, clarithromycin and vancomycin by different inocula of 11 strains was evaluated using the antibiotic inactivation bioassay. None of the dairy probiotics showed a level of constitutive resistance or carried inducible resistance genes, making them suitable to be administrated with macrolides. Among the probiotics in tablet form only Enterococcus faecium strains carrying the msrC gene showed an MIC90 of 4 lg ml)1. Extended-spectrum b-lactams, tetracyclines and ampicillin exhibited powerful germicidal activity against the vast majority of the probiotic strains. Conclusions: There is a limited choice of the Japanese probiotics that can be administered with clinically used antibiotics. Signicance and Impact of the Study: Japanese probiotics are widely distributed all over the world. Through the ndings of our study, we have attempted to provide guidance for clinicians interested in using the Japanese probiotics in combination with antibiotics.
2009 2213: received 4 January 2010, revised 15 April 2010 and accepted 15 April 2010
doi:10.1111/j.1365-2672.2010.04762.x
Introduction Whereas the discovery and use of antimicrobial agents has been one of medicines greatest achievements, frequent and lengthy use of antibiotics usually results in alteration in the complexity of the intestinal commensal ora. This enhances the ability of otherwise silent intestinal microbes to cause disease. How antibiotic-mediated elimination of commensal bacteria promotes infection by other bacteria is still a fertile area for speculation, with few dened mechanisms (Pamer 2007; Brandl et al. 2008). Moreover, preventive therapy for diseases and side effects emerging during treatment with antibiotics, such as antibiotic-associated diarrhoea (AAD), Clostridium difcile disease and infection with antibiotic-resistant bacteria (Pamer 2007; Brandl et al. 2008), is still not available.
Medical professionals in this era have found that prescribing probiotics with antibiotics is an effective approach in counteracting the adverse effects of the antibiotics. In addition, some viable probiotics have shown strong germicidal activity against certain pathogens, such as Helicobacter pylori (Zou et al. 2009). As a consequence, they are coprescribed with antibiotics. This raises an important question: What are the rationales for the choice of probiotics to be coadministered with antibiotics? We believe that a deeper understanding of their antibiotic resistance mechanisms will lead to the development of more effective coadministration protocols. The conclusion that probiotics are unlikely to offer a protective effect in vivo, when they are themselves susceptible to the same antibiotics considered to be at high risk of causing AAD (Gould and Short 2008) may underestimate some
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2010 The Authors Journal compilation 2010 The Society for Applied Microbiology, Journal of Applied Microbiology 109 (2010) 13491360
Antibiotic resistance of probiotics
valuable probiotic strains. A bacterial strain carrying an inducible antibiotic resistance gene may appear susceptible to some antibiotics in in vitro tests, but have the potential to develop resistance to these antibiotics upon in vivo selection by the appropriate antibiotic (Schmitz et al. 2002). On the other hand, should maintaining the potency of the antibiotic in the probioticantibiotic combination therapy be considered a safety criterion in this therapeutic approach? It has been known that the relative potency of vancomycin declines with the density of bacteria exposed (Udekwu et al. 2009). Reduction in the effective concentration of the antibiotic (potency) in the medium may be because a number of factors that include antibioticinactivating or denaturing enzymes, binding of the antibiotic to cell structures and the DNA or RNA of killed as well as viable bacteria (Hunt et al. 1987; Davies 1994; Udekwu et al. 2009). This may lead to the hypothesis that probiotics that are usually ingested in large numbers, during probioticantibiotic combination therapy, may interact antagonistically with antibiotics. Antibiotic inactivation bioassay is a simple and reliable technique for studying this theory. In recent years, Japan has undergone regulatory reform. In addition to the large variety of dairy probiotics available in the Japanese market, some of the over-the-counter drugs, including probiotics with well established safety records, have been reclassied into the category of quasi drugs or as functional foods to reduce health care costs (Amagase 2008). These products are no longer limited to being sold at drug stores and can now be sold more widely in the marketplace. It is unclear whether the probiotics available in the Japanese market are compatible for use in probioticantibiotic combination therapy. Phenotypic and molecular assessment of their antimicrobial resistance is needed to elucidate this issue. Therefore, the objective of this study was to determine the spectrum of antibiotic resistance of the probiotics available in the Japanese market and to verify their genetic mechanisms of resistance. Material and methods Collection of samples and isolation of bacterial strains A total of 40 samples of probiotic products, comprising 30 dairy products and 10 products in tablet form produced by different companies, were collected from Japanese supermarkets. Bacterial isolation was performed as follows. Briey, after vigorous shaking of the product (probiotic or probio-yogurt) in its closed package, 1 ml was mixed with 9 ml buffered peptone water (Difco, Becton Dickinson and Co., Sparks, MD, USA) and
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incubated for 3 h at 37C. From the incubated dairy products, loopfuls were streaked on to De Man Rogosa and Sharpe Agar medium (Merck, Darmstadt, Germany) supplemented with 03 g l)1 cysteine hydrochloride (SigmaAldrich, Tokyo, Japan) for the isolation of Lactobacillus and Bidobacterium strains and on to M17 medium (Merck) for isolation of Streptococcus strains. In the case of products in tablet form, one tablet was dissolved in 9 ml buffered peptone water and incubated for 3 h at 37C and then, according to the strain names declared on the product labels, loopfuls were streaked on different media. In addition to the two previously mentioned media, Kanamycin esculin azide agar (Merck) was used for the isolation of Enterococcus spp. All plates with different media were incubated under standard conditions. DNA extraction Total genomic DNA was extracted using the DNeasy Tissue kit (Qiagen, Tokyo, Japan) following the manufacturers protocol for Gram-positive bacteria. Identication of isolates The 16S rRNA gene was amplied by PCR using primers as described by Frank et al. (2008). Another set of primers consisting of rrs (forward) (Kariyama et al. 2000) and 1492r (reverse) (Frank et al. 2008) were used to amplify 16S rRNA gene in DNA templates that could not be amplied by the rst set of primers. For species identication of enterococcal isolates, a pair of degenerate primers was used to characterize a 438-bp-long DNA fragment internal to the sodA gene encoding the manganesedependent superoxide dismutase (Poyart et al. 2000). The PCR products were puried using a QIAquick PCR purication kit (Qiagen) according to the manufacturers instructions and subsequently commercially sequenced using an ABI 3730 XL automated sequencer (Applied Biosystems, Foster City, CA, USA). Antimicrobial susceptibility tests Characterized strains were tested for their susceptibility to 14 antimicrobial agents by the disc diffusion method. They were as follows (lg): ampicillin (10), cefepime (30), cefotaxime (30), cefpirome (30), ceftriaxone (30), ciprooxacin (5), clarithromycin (15), erythromycin (15), gentamicin (10), kanamycin (30), metronidazole (50), streptomycin (10), tetracycline (30) and vancomycin (30). They were tested according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) [formerly, the National Committee for Clinical
Laboratory Standards (NCCLS)] (CLSI 2005) but with the following modications: plates of lactic acid bacteria (LAB) susceptibility test medium supplemented with cysteine (LSM + cysteine) [i.e. 90% Iso-Sensitest broth (Oxoid, Wesel, Germany), 10% MRS broth (Merck) and 15 g l)1 agar supplemented with 03 g l)1 l-cysteine HCl (Sigma)] were used for all LAB and bidobacteria (Klare et al. 2005). Streptococcus thermophilus sensitive medium (90% Iso-Sensitest broth, 10% M17 broth, 05% lactose and 15 g l)1 agar) was used for Strep. thermophilus strains (Tosi et al. 2007). Muller Hinton agar (Oxoid) was used for non-LAB. The antimicrobial discs (Table 1) were purchased from Nissui Pharmaceutical Co. (Tokyo, Japan), with the exception of metronidazole, which was purchased from Oxoid. The minimal inhibitory concentrations (MICs), expressed in lg ml)1, of the same antibiotics purchased from different companies (Bristol-Myers Squibb, Tokyo Japan; Nacalai Tesque, Kyoto, Japan; Sigma-Aldrich; Wako Chemical Co., Osaka, Japan) were determined using the broth microdilution method, using the same media without agar, according to the guidelines of CLSI (CLSI 2005). Antibiotic inactivation bioassays Inactivation of clarithromycin, cefepime and vancomycin by one strain representing each probiotic species (total 11 strains) was tested as follows: three tubes of LSM broth supplemented with cysteine and containing either 10 lg ml)1 of clarithromycin or cefepime or 500 lg ml)1 vancomycin were inoculated with exponential cultures of these strains until the concentrations were equal to 05 and 1 McFarland standard (for Enterococcus faecium strains, MullerHinton broth was used instead of LSM broth). Then, they were incubated for 12 h at 37C. After centrifugation, sample aliquots of these cultures were ltered (022 lm) to remove the cells after 6 and 12 h. Thirty microlitres of the supernatants was deposited on sterile discs over MullerHinton agar plates, previously seeded with Kocuria rhizophila ATCC 9341 as an indicator organism for clarithromycin and cefepime, and with a vancomycin sensitive Bacillus subtilis laboratory strain as indicator for vancomycin bioassay. The plates were incubated for 18 h and the zone sizes around the discs, which indicate the antibiotic remaining in the culture medium, were measured. The zone sizes were compared with those produced when the antibiotic was incubated with medium alone. The broth microdilution assay for the detection of effective residual concentration of antibiotics in the culture medium, which is thought to be more sensitive than the disc method, was carried out as described by Udekwu
et al. (2009) to conrm the negative results. Briey, probiotic cultures were challenged with each antibiotic and sample aliquots of these cultures were ltered as mentioned earlier. Then, overnight cultures of K. rhizophila ATCC 9341 (in case of clarithromycin and cefepime) or B. subtilis laboratory strain (in case of vancomycin) were diluted to c. 5 105 CFU ml)1, and the MICs of the ltered media were estimated by broth microdilution assay modied from the CLSI protocol. Detection of antibiotic resistance genes The presence of antibiotic resistance genes was investigated in all strains isolated in this work. PCR amplications of genes associated with resistance to macrolides (ermA, ermB, ermC, msrA B, ereA, ereB, mphA and mefA E genes) (Sutcliffe et al. 1996), aminoglycosides (aph[3], ant[6], aadE and aacA-aphD) (Van de Klundert and Vliegenthart 1993; Clark et al. 1999; Leelaporn et al. 2008), vancomycin (vanA, vanB, vanC1, and vanC2 or vanC3) (Bell et al. 1998), tetracycline (the ribosomal protection proteins degenerate primer pairs DI DII (Clermont et al. 1997), Ribo-2-FW Ribo-2-RV and tet(W) (Aminov et al. 2001) were carried out. Amplicons were puried and sequenced as previously described. Results Identication of isolates 16S rRNA sequence analyses using primers described by Frank et al. (2008) allowed species identication of nearly all strains. The alternative set of primers was needed for the identication of Lactobacillus delbrueckii ssp. bulgaricus, Lactobacillus helveticus and Lactobacillus acidophilus strains that did not have amplicons suitable for sequencing with the rst set. From the dairy probiotics, the lactobacilli were Lactobacillus casei (n = 11), Lact. acidophilus (n = 5), Lactobacillus gasseri (n = 2), Lactobacillus plantarum (n = 2), Lact. delbrueckii ssp. bulgaricus (n = 6), Lactobacillus brevis (n = 1) and Lact. helveticus (n = 1). The streptococci were Strep. thermophilus (n = 3). The bidobacteria were Bidobacterium animalis ssp. lactis (n = 3) and Bidobacterium longum (n = 2). From probiotics in tablet form, one strain each of Lact. casei, Lact. acidophilus and Lact. gasseri were identied from three different products. In addition, there were four Ent. faecium strains. In the case of dairy products, identied species were always in agreement with the strain names declared on the product label (when written), whereas in the case of products in tablet form, Ent. faecium strains were mislabelled as Enterococcus faecalis in all the four products containing this species.
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Table 1 Susceptibility of probiotic bacteria to antimicrobial agents using the disc diffusion method
Inhibition zone diameter range (mm) CLR 15 lg 2935 3137 3135 3037 2225 34* 29 2830 3040 2930 1416 CIP 5 lg Lact. casei (12) Lact. acidophilus (6) Lact. gasseri (3) Lact. delbrueckii ssp. bulgaricus (6) Lact. plantarum (2) Lact. helveticus (1) Lact. brevis (1) Strep. thermophilus (3) Bif. animalis ssp. lactis (3) Bif. longum (2) Ent. faecium (4) 1417 6 6 1113 6 6 6 1920 1112 17 1216 ERY 15 lg 2530 3033 2930 2838 1921 30* 26 2930 3032 3132 1314 MTR 50 lg 6 6 6 6 6 6 6 6 3941* 3739 6 VAN 30 lg 6 2930 2223 2325 6 29 6 2225 2527 25 2122 CTX 30 lg 2529 2933 2931 2537 3032 40 17 3032 3032 3235 2426 TET 30 lg 3033 3033 2728 2933 15 33 15 2832 2022 3035 2226 CRO 30 lg 1519 2527 2326 2836 2931 35 17 3033 3036 2933 2023 STR 10 lg 810 1226 1415 1322 6 26 11 1112 6 6 612 FEP 30 lg 615 2530 2730 1537 3032 28 14 3031 3638 3136 2529 GEN 10 lg 1924 1425 1015 1126 1316 23 20 1415 69 6 1314 CFR 30 lg 2227 3034 2937 2535 3033 35 16 3436 3436 3537 2428 KAN 30 lg 1416 620 6 1428 1113 25 14 1517 6 6 6 AMP 10 lg 2326 3236 2730 3238 2025 42 24 3435 3038 3235 2729
Species (number of strains) Lactobacillus casei (12) Lactobacillus acidophilus (6) Lactobacillus gasseri (3) Lactobacillus delbrueckii ssp. bulgaricus (6) Lactobacillus plantarum (2) Lactobacillus helveticus (1) Lactobacillus brevis (1) Streptococcus thermophilus (3) Bidobacterium animalis ssp. lactis (3) Bidobacterium longum (2) Enterococcus faecium (4)
AMP, ampicillin; CFR, cefpirome; CIP, ciprooxacin; CLR, clarithromycin; CRO, ceftriaxone; CTX, cefotaxime; ERY, erythromycin; FEP, cefepime; GEN, gentamicin; KAN, kanamycin; MTR, metronidazole; STR, streptomycin; TET, tetracycline; VAN, vancomycin. *Pin point colonies often observed within inhibition zone. Haze of growth observed within inhibition zone.
Antimicrobial susceptibility tests The results of the antimicrobial disc diffusion susceptibility tests on 43 strains for 14 antibiotics are summarized in Table 1. Their MICs are listed in terms of MIC50 [MIC (lg ml)1) that inhibited 50% of the number of isolates tested], MIC90 [MIC (lg ml)1) that inhibited 90% of the number of isolates tested] and the MIC range (Table 2). Because no cut-off values have been ofcially dened for LAB or bidobacterial species, the breakpoints established by the FEEDAP Panel of the European Food Safety Authority (EC 2008) were employed as a reference for most of the antibiotics tested. Using these cut-offs, none of the dairy probiotics showed a level of constitutive resistance for macrolides (MIC breakpoint 4 lg ml)1). Among the probiotics in tablet form, only Ent. faecium strains showed the highest natural resistance to erythromycin (MIC90 of 4 lg ml)1). High resistance levels to vancomycin were observed in Lact. casei, Lact. brevis and Lact. planIndeed, tarum strains (MIC 128 lg ml)1).
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heterofermentative lactobacilli are naturally resistant to glycopeptides and their MICs for vancomycin are not reliable for the differentiation of intrinsic and acquired resistance mechanisms (EC 2005). On the other hand, the MICs of vancomycin shown by obligate homofermentative lactobacilli including Lact. gasseri, Lact. delbrueckii ssp. bulgaricus and Lact. helveticus were lower than the MIC breakpoint of 4 lg ml)1 for vancomycin. Tetracycline and ampicillin resistant strains appeared to be rare in the Japanese market. Only Bif. animalis ssp. lactis (n = 3), Lact. brevis (n = 1) and Lact. plantarum (n = 2) showed moderate resistance levels to tetracycline (MIC range 816 lg ml)1). In contrast, both naturally resistant aminoglycosides and metronidazole strains were abundant. It should be noted that there was a tendency towards lower MICs for gentamicin when compared with kanamycin and streptomycin. This nding was inconsistent with the results obtained by Danielsen and Wind (2003). Pinpoint colonies were observed within the inhibition zone of metronidazole using the disc diffusion test for all bidobacterial strains.
Table 2 Susceptibility of probiotic bacteria to antimicrobial agents using broth microdilution method
MIC (lg ml)1) Antibiotics Clarithromycin Species (number of strains) Lactobacillus casei (12) Lactobacillus acidophilus (6) Lactobacillus gasseri (3) Lactobacillus delbrueckii ssp. bulgaricus (6) Lactobacillus plantarum (2) Lactobacillus helveticus (1) Lactobacillus brevis (1) Streptococcus thermophilus (3) Bidobacterium animalis ssp. lactis (3) Bidobacterium longum (2) Enterococcus faecium (4) Lact. casei (12) Lact. acidophilus (6) Lact. gasseri (3) Lact. delbrueckii ssp. bulgaricus (6) Lact. plantarum (2) Lact. helveticus (1) Lact. brevis (1) Strep. thermophilus (3) Bif. animalis ssp. lactis (3) Bif. longum (2) Ent. faecium (4) Lact. casei (12) Lact. acidophilus (6) Lact. gasseri (3) Lact. delbrueckii ssp. bulgaricus (6) Lact. plantarum (2) Lact. helveticus (1) Lact. brevis (1) Strep. thermophilus (3) Bif. animalis ssp. lactis (3) Bif. longum (2) Ent. faecium (4) Lact. casei (12) Lact. acidophilus (6) Lact. gasseri (3) Lact. delbrueckii ssp. bulgaricus (6) Lact. plantarum (2) Lact. helveticus (1) Lact. brevis (1) Strep. thermophilus (3) Bif. animalis ssp. lactis (3) Bif. longum (2) Ent. faecium (4) Lact. casei (12) Lact. acidophilus (6) Lact. gasseri (3) Lact. delbrueckii ssp. bulgaricus (6) Lact. plantarum (2) Lact. helveticus (1) Lact. brevis (1) Strep. thermophilus (3) Bif. animalis ssp. lactis (3) MIC range 0125025 0125 0125 0125 025 0125 025 0125 0125 0125 24 0125025 0125 0125 0125 02505 0125 025 0125 0125 0125 4 128 05 1 05 128 025 128 0125025 05 025 1 1 12 12 051 8 1 16 0251 8 012505 052 816 058 432 058 1632 1 8 8 32 MIC50 0125 0125 0125 0125 025 0125 0125 0125 4 0125 0125 0125 0125 02505 0125 0125 0125 4 128 05 1 05 025 05 025 1 1 1 2 1 8 1 8 0125 05 8 4 4 8 1632 8 32 MIC90 0125 0125 0125 0125 0 0125 0125 4 0125 0125 0125 0125 0125 0125 4 128 05 1 05 025 05 1 1 1 2 1 1 8 05 8 4 4 8 8 32
Erythromycin
Vancomycin
Tetracycline
Streptomycin
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Table 2 (Continued)
MIC (lg ml)1) Antibiotics Species (number of strains) Bif. longum (2) Ent. faecium (4) Lact. casei (12) Lact. acidophilus (6) Lact. gasseri (3) Lact. delbrueckii ssp. bulgaricus Lact. plantarum (2) Lact. helveticus (1) Lact. brevis (1) Strep. thermophilus (3) Bif. animalis ssp. lactis (3) Bif. longum (2) Ent. faecium (4) Lact. casei (12) Lact. acidophilus (6) Lact. gasseri (3) Lact. delbrueckii ssp. bulgaricus Lact. plantarum (2) Lact. helveticus (1) Lact. brevis (1) Strep. thermophilus (3) Bif. animalis ssp. lactis (3) Bif. longum (2) Ent. faecium (4) Lact. casei (12) Lact. acidophilus (6) Lact. gasseri (3) Lact. delbrueckii ssp. bulgaricus Lact. plantarum (2) Lact. helveticus (1) Lact. brevis (1) Strep. thermophilus (3) Bif. animalis ssp. lactis (3) Bif. longum (2) Ent. faecium (4) Lact. casei (12) Lact. acidophilus (6) Lact. gasseri (3) Lact. delbrueckii ssp. bulgaricus Lact. plantarum (2) Lact. helveticus (1) Lact. brevis (1) Strep. thermophilus (3) Bif. animalis ssp. lactis (3) Bif. longum (2) Ent. faecium (4) Lact. casei (12) Lact. acidophilus (6) Lact. gasseri (3) Lact. delbrueckii ssp. bulgaricus Lact. plantarum (2) Lact. helveticus (1) Lact. brevis (1) MIC range 16 832 12 0254 48 0258 28 05 1 4 816 16 8 16 432 3264 116 16 2 16 16 64 32 128128 12 832 64 4 816 64 32 12 4 2 24 128 64128 128 128 128 128 128 128 4 1 128 24 02505 051 01251 025 05 16 MIC50 16 32 2 4 4 2 28 4 16 16 8 16 16 32 16 16 16 64 32 128 2 32 64 4 8 1 4 2 4 128 128 128 128 128 128 4 1 128 4 05 05 1 025 MIC90 32 2 4 4 4 4 16 8 16 16 32 16 16 64 128 2 32 64 4 1 4 4 128 128 128 128 128 128 4 128 4 05 1 1
Gentamicin
(6)
Kanamycin
(6)
Ciprooxacin
(6)
Metronidazole
(6)
Cefotaxime
(6)
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Table 2 (Continued)
MIC (lg ml)1) Antibiotics Species (number of strains) Strep. thermophilus (3) Bif. animalis ssp. lactis (3) Bif. longum (2) Ent. faecium (4) Lact. casei (12) Lact. acidophilus (6) Lact. gasseri (3) Lact. delbrueckii ssp. bulgaricus Lact. plantarum (2) Lact. helveticus (1) Lact. brevis (1) Strep. thermophilus (3) Bif. animalis ssp. lactis (3) Bif. longum (2) Ent. faecium (4) Lact. casei (12) Lact. acidophilus (6) Lact. gasseri (3) Lact. delbrueckii ssp. bulgaricus Lact. plantarum (2) Lact. helveticus (1) Lact. brevis (1) Strep. thermophilus (3) Bif. animalis ssp. lactis (3) Bif. longum (2) Ent. faecium (4) Lact. casei (12) Lact. acidophilus (6) Lact. gasseri (3) Lact. delbrueckii ssp. bulgaricus Lact. plantarum (2) Lact. helveticus (1) Lact. brevis (1) Strep. thermophilus (3) Bif. animalis ssp. lactis (3) Bif. longum (2) Ent. faecium (4) Lact. casei (12) Lact. acidophilus (6) Lact. gasseri (3) Lact. delbrueckii ssp. bulgaricus Lact. plantarum (2) Lact. helveticus (1) Lact. brevis (1) Strep. thermophilus (3) Bif. animalis ssp. lactis (3) Bif. longum (2) Ent. faecium (4) MIC range 0125 05 02505 32* 816 051 02505 01251 0125025 1 16 0125 051 12 1632* 1632 051 0125025 01258 0125 2 16 0125 0125 0125 128 24 01250025 0125025 012505 0125 1 8 0125 0125 0125 24* 051 025 025 0125 0125025 025 1 0125 0125 0125025 051 MIC50 0125 05 02505 32* 16 1 05 1 0125025 0125 05 12 32* 16 05 025 1 0125 0125 0125 0125 128 2 025 025 05 0125 0125 0125 0125 4* 1 025 025 0125 0125025 0125 0125 0125025 05 MIC90 0125 05 32* 16 1 1 0125 05 32* 16 05 0 1 0125 0125 128 2 025 025 05 0125 0125 4* 1 025 025 0125 0125 0125 1
Ceftriaxone
(6)
Cefepime
(6)
Cefpirome
(6)
Ampicillin
(6)
MIC, minimal inhibitory concentrations. MIC50 and MIC90, MICs (lg ml)1) that inhibited 50 and 90% of the number of isolates tested, respectively. *All enterococcal strains were able to give a haze of growth in presence of cefotaxime, ceftriaxone and cefpirome till higher concentrations (cefotaxime and ceftriaxone MIC 128 and cefpirome MIC at 128).
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Extended-spectrum b-lactams including cefpirome, cefepime, cefotaxime and ceftriaxone showed high germicidal activity against the vast majority of tested strains. Lactobacillus casei strains exhibited a different behaviour for this group. They showed high levels of resistance to cefepime and ceftriaxone but appeared almost susceptible to cefpirome and cefotaxime. On the other hand, all enterococcal strains showed zones of partial inhibition in the disc diffusion test for this group. Also, in the MIC test, all enterococcal strains were able to give a haze of growth in the presence of these antibiotics until higher concentrations were reached (cefotaxime and ceftriaxone MIC 128 lg ml)1 and cefpirome MIC 128 lg ml)1). Ciprooxacin exhibited different patterns of response in the tested strains. A wide range of MICs was observed in Lactobacillus species. However, according to the criteria suggested by Danielsen and Wind (2003) for lactobacilli (MIC breakpoint of 32 lg ml)1), nearly all strains, with the exception of Lact. gasseri and Lact. helveticus, appeared to possess intrinsic mechanisms of resistance. Antibiotic inactivation bioassays No decline in the effective concentrations of vancomycin, clarithromycin and cefepime in the ltrates was observed, after the incubation of heavy inoculums of the 11 tested strains with these three antibiotics. Antibiotic resistance genes Even though strains of the same species identied in this study appeared phenotypically resistant to antibiotics, they did not carry antibiotic resistance genes. This may be attributed to intrinsic mechanisms of resistance or the presence of antibiotic resistance genes that were not screened in this study. Positive amplicons were obtained using msrA B primers from all enterococcal strains. Sequence analysis revealed the msrC gene. All Bif. animalis ssp. lactis with an MIC90 of 8 lg ml)1 for tetracycline were found to have positive amplicons using degenerate primers that identify all known ribosome-protection-type Tcr genes. All had the tet(W) gene as revealed from the sequencing of the positive amplicons using tet(W) specic primers. Discussion There has been a signicant worldwide rise in the sales and consumption of probiotic products being taken together with antibiotics. It is important that such probiotic products are correctly prescribed with antibiotics. In other words, antibiotic and probiotic combination therapy can only be considered to be compatible if the
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potency of the antibiotic and the viability of the probiotic are maintained. This study is the rst to provide collective data on antibiotic compatibility of the Japanese probiotics. Through the ndings of our study, we have attempted to provide guidance for clinicians interested in using the Japanese probiotics in combination with antibiotics and to improve the knowledge of the general public with regard to the safety aspects of the ingestion of probiotics during antibiotic therapy. Probiotics are dened as live micro-organisms administered in adequate amounts which confer a benecial health effect on the host (FAO WHO 2001). Starting from this vague denition and in the absence of approved standards for the phenotypic or genotypic evaluation of the antibiotic resistance of probiotics, moving towards a therapeutic approach with probiotics is complicated. This denition is based on the benecial effects of viable bacteria and should not be confused with the probiotic effects achieved by nonviable bacteria or even by bacterial DNA (Szajewska et al. 2006). We believe that two important concepts should be considered when choosing this therapeutic approach. First, viable probiotics should reach their target site in the gastrointestinal tract in large enough numbers to achieve their effects. Therefore, they should have high natural constitutive resistance levels or nontransferable inducible antibiotic resistance genes enabling them to survive in the actual concentration of antibiotics that can be present in the human gastrointestinal tract. Second, when susceptible bacteria are exposed to bactericidal concentrations of an antibiotic, most of the population is killed within 12 h and some bacteria survive even in the presence of active antibiotic (Wiuff and Andersson 2007). Thus, in randomized double-blind placebo-controlled trials, the benecial effects of probiotics isolated after the end of therapy should not always be attributed to viable normal probiotics. Probiotic strains have been tested for their clinical efcacy against H. pylori infection (Zou et al. 2009). This infection is one of the most important established risk factors for the development of gastric cancer, with more than 100 000 new cases each year in Japan (Ferlay et al. 2002). The Lact. gasseri, Lact. acidophilus, Lact. casei and Bidobacterium lactis Bb12 strains were found to amplify the eradication of this pathogen (Zou et al. 2009). However, physicians usually prescribe probiotics in combination with antibiotic therapy to achieve complete eradication. Helicobacter pylori eradication regimen, proton pump inhibitor + amoxicillin (AMPC) + clarithromycin (CAM), or PPI AC is widely used all over the world and is the only currently approved H. pylori eradication regimen for insurance coverage in Japan. Surprisingly, according to our ndings, none of the dairy probiotic strains investigated could be appropriately combined with
this regime, as they were highly susceptible to clarithromycin (MIC90 of 0125 lg ml)1). Indeed, the gastric concentration of clarithromycin (lg ml)1) was recorded to be 13 and 43 after 6 h of low and high dose regimes, respectively (Nakamura et al. 2003). Thus, it is not expected that these probiotics will remain viable if coadministered with clarithromycin. Additionally, these probiotics did not carry any inducible antibiotic resistance genes that could have enabled them to survive in vivo. Moreover, the same mentioned species isolated from probiotic products in tablet form were also very susceptible to clarithromycin. It should be noted that the MIC90 for macrolides obtained in this study for Lactobacillus species was lower than the MIC90 obtained in some previous studies (Danielsen and Wind 2003). However, it was inconsistent with the erythromycin MIC90 for the large number of Lactobacillus species tested in a European project intended to propose tentative epidemiological cut-off values for recognizing intrinsic and acquired antimicrobial resistances (Klare et al. 2007). On the other hand, the expected concentration of metronidazole, the second line of antimicrobial treatment, in the gastric juice was around 20 lg ml)1 at 1 h after administration (Calafatti 2000). This was higher than the MIC of Bif. animalis ssp. lactis strains. The appearance of pinpoint colonies around the metronidazole discs (50 lg) using the disc diffusion test indicated that the emergence of mutants is more likely to occur in vivo. However, the number of these mutants is not expected to reach the proposed number of probiotics that can deliver benecial effects. Cephalosporins are rapidly losing their usefulness as frontline antimicrobial agents, because of their potential to cause Cl. difcile-associated diarrhoea (Billyard 2007). The mode of excretion of these antibiotics results in an MIC concentration in the lower part of the intestinal tract that is reported to be higher than the MIC that can be tolerated by all of the intestinal microora, leading to their partial elimination (Brismar et al. 1990). Using the currently proposed MIC breakpoint for ciprooxacin (Danielsen and Wind 2003), our Lactobacillus strains appeared to be susceptible. Therefore, their survival during therapy is a cause for suspicion. Our in vitro results were inconsistent with the outcomes of previous clinical trials using similar Lactobacillus strains (Sullivan et al. 2003). Sullivan et al. (2003) could detect only low number of probiotic bacteria in stools of few treated patients after 10 days of quinolone therapy. This indicated the emergence of a few phenotypically resistant probiotics that could only be detected after this period. In addition, the vast majority of the strains detected in this study were clearly susceptible to tetracycline and extended-spectrum b-lactams, making the choice of probiotics that remain viable with these antibiotics limited.
A Bif. animalis ssp. lactis strain carrying the tet(W) gene, which had a similar MIC to the strains identied in this study, has been shown to survive during tetracycline therapy (Saarela et al. 2007). Its survival depended on the evolution of a genetic mechanism of resistance, rather than its natural ability to withstand the intestinal concentration of tetracycline. This strain had tetracycline MICs of >256, 256 and 32 lg ml)1 during therapy. More importantly, it did not revert to the susceptible state after growing in antibiotic-free media. This means that a genetic mechanism of resistance, rather than phenotypic resistance, was responsible for the acquired phenotype (Balaban et al. 2004). Interestingly, all enterococcal strains identied in this study showed two unique characters. First, they carried msrC gene that is thought to confer low-level protection against the 14-membered-ring macrolides and type B streptogramins. This is the rst report of msrC gene in Ent. faecium strains used as probiotics. It has been reported that msrC is prevalent, but not intrinsic, in Ent. faecium isolates and encodes an efux pump that is likely to be of an inducible nature (Singh et al. 2001; Werner et al. 2001). From a clinical perspective, induction of this gene during macrolide therapy may enable these strains to survive in vivo. The second unique character of the isolated enterococcal strains appeared in antibiotic susceptibility tests of the extended-spectrum b-lactams (cefpirome, cefepime and cefotaxime). No clear-cut results were detected in both disc diffusion and MIC tests. This phenomenon has not been recorded in the guidelines for the susceptibility testing of Enterococcus spp. (CLSI 2005), but a similar phenomenon has been reported for anaerobes (National Committee for Clinical Laboratory Standards, NCCLS) (CLSI 2001). In such cases, MIC was determined as the lowest concentration of antimicrobial agent that resulted in a signicant drop-off in the amount of growth. It should be noted that in the evaluation of the antibiotic resistance levels of probiotics, we should consider the survival of the proposed number of probiotics that are expected to give benecial effects. Further clinical studies are still needed to clarify this point in these probiotic strains. Taking into consideration the high cell density of probiotics in tablet form and in dairy products on the one hand and the traditional habitat of eating large amounts of probiotics in Asian countries on the other, we tried in this study to investigate the impact of 11 strains from different species with different cell components and genomes on the effective concentrations of three different classes of antibiotics. It was found that an increase in inoculum size of LAB resulted in elevation of their MICs to some antibiotics, including b-lactam (ampicillin) and macrolide rn et al. 2007). On the (erythromycin) antibiotics (Egerva other hand, vancomycin was proved to be inactivated by
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heavy inoculums of vancomycin susceptible Staphylococcus aureus strains (Udekwu et al. 2009). It is unclear whether the heavy inoculum of probiotic bacteria has the ability to inactivate certain classes of antibiotics. Therefore, the interactions of cefepime, clarithromycin and vancomycin, antibiotics widely used in clinic, with probiotic bacteria were investigated in this study using antibiotic inactivation bioassay. The effective residual concentrations of the three antibiotics in the ltrate of culture media were not affected by heavy inoculums of the probiotic strains. Interestingly, LAB including certain species belonging to the genus Lactobacillus synthesize low afnity peptidoglycan precursors ending exclusively in D-Lac that lack the ability to bind with vancomycin (Handwerger et al. 1994; Deghorain et al. 2007). In agreement with this view, the heavy inoculums of resistant strains in this study showed no effect on the potency of vancomycin. Our in vitro nding clearly showed that bacterial components of probiotics had no antagonistic effect on the potency of antibiotics tested. Further studies are still needed to investigate whether the high cell density of probiotics adversely affects the rate and extent of antibiotic-mediated killing (the efcacy of antibiotic) during probioticantibiotic combination therapy. With regard to a general concern about the safety of probiotics, such as potential transferability of resistance determinants, the vast majority of Japanese probiotics, with their low natural resistance to antibiotics tested, appear risk-free. The previous published data (Portillo rez et al. 2006) concerning the antibiotic et al. 2000; Flo resistance genes identied in this study revealed that they were always chromosomally encoded. Moreover, given that transferable resistance genes might be present in all strains of a given species, further studies are also still needed to elucidate the mechanisms of resistance of some strains exhibiting high MICs. In conclusion, according to the molecular and phenotypic characteristics of the Japanese probiotics detected in this study, probioticantibiotic combination therapy in many cases may not achieve the same positive outcomes as those obtained in clinical trials using viable probiotics alone. This is because many probiotic strains are susceptible to antibiotics used in clinic. In view of the similarity between our in vitro susceptibility prole of Japanese probiotics and the viability of probiotics demonstrated in previous clinical studies, clinicians are advised to use only those probiotic strains shown to be viable in the presence of antibiotics. Acknowledgement The authors gratefully acknowledge funding from a Grant-in-Aid for Scientic Research to T.S. from the
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Ministry of Education, Culture, Sports, Science and Technology of Japan. References

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Journal of Applied Microbiology ISSN 1364-5072

Antibiotic resistance of probiotics

A.M. Hammad and T. Shimamoto

A.M. Hammad and T. Shimamoto

Antibiotic resistance of probiotics

Antibiotic resistance of probiotics

A.M. Hammad and T. Shimamoto

A.M. Hammad and T. Shimamoto

Antibiotic resistance of probiotics

Antibiotic resistance of probiotics

A.M. Hammad and T. Shimamoto

A.M. Hammad and T. Shimamoto

Antibiotic resistance of probiotics

Antibiotic resistance of probiotics

A.M. Hammad and T. Shimamoto

A.M. Hammad and T. Shimamoto

Antibiotic resistance of probiotics

Antibiotic resistance of probiotics

A.M. Hammad and T. Shimamoto

Ministry of Education, Culture, Sports, Science and Technology of Japan. References

A.M. Hammad and T. Shimamoto

Antibiotic resistance of probiotics

Antibiotic resistance of probiotics

A.M. Hammad and T. Shimamoto

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