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Antimicrobial susceptibility of Enterococcus strains used in clinical practice as probiotics
J Infect Chemother DOI 10.1007/s10156-013-0633-6
ORIGINAL ARTICLE
Antimicrobial susceptibility of Enterococcus strains used in clinical practice as probiotics Tetsuo Yamaguchi • Yuri Miura • Tetsuya Matsumoto
Received: 22 July 2012 / Accepted: 2 June 2013 Ó Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2013
Abstract The aim of this study was to evaluate the antimicrobial susceptibilities of probiotic strains that are suggested to be effective for preventing antibiotics-associated diarrhea (AAD). The minimum inhibitory concentrations (MICs) of 17 antibiotics against probiotic strains were tested by the agar plate dilution method or broth microdilution method. In all, eight probiotic strains containing Enterococcus faecalis, Bifidobacterium spp., Clostridium butyricum, and Lactobacillus acidophilus were tested. Although the MIC range was wide, from less than 0.0625 to more than 1,024 lg/ml, the MICs of 11 betalactams were high for three of four enterococci, with a range of 32 to more than 1,024 lg/ml. In contrast, fluoroquinolones and vancomycin showed potent activities against all enterococci, of which MICs were 0.25–8 lg/ml. Two Bifidobacterium strains and one Lactobacillus strain showed low MICs against many of the beta-lactams, fluoroquinolones, macrolides, and vancomycin, with MICs of 8 lg/ml or less. Fosfomycin showed generally mild activity against enterococci (MIC, 8–32 lg/ml) and anaerobic strains (MIC, 32 to [1,024 lg/ml), respectively. The probiotics strains with high MIC values may survive in the intestinal tract, even if the patient was concomitantly using the antibiotics in clinical practice. Therefore, our results suggest that adequate combinations of probiotics strains and antibiotics should be important for preventing AAD. Further study is needed to determine the efficacy of probiotics in clinical practice.
T. Yamaguchi Y. Miura T. Matsumoto (&) Department of Microbiology, Tokyo Medical University, 6-1-1, Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan e-mail: [email protected]
Keywords Probiotics Enterococci Antimicrobial susceptibility Antibiotics-associated diarrhea
Introduction Antibiotics-associated diarrhea (AAD) is the most common adverse effect during antibiotic therapy, occurring in from 5 % to more than 30 % of the patients during the course of therapy [1]. AAD is associated with alterations in intestinal microflora, mucosal integrity, vitamin and nutrient metabolism and absorption, and other systems or processes [2]. In severe cases, inflammation of the digestive organs and intestinal bleeding may occur and the condition may become life threatening [3]. Moreover, infectious diseases such as Clostridium difficile infection (CDI) complicate AAD further by infecting the intestinal lumen damaged both by the drug itself and by the toxins produced from the organism [4]. Incidences are higher among children, the elderly, and immunocompromised patients, and are frequently reported as nosocomial infections [5–7]. Antibiotics are believed to complicate AAD further by reducing the ‘colonization resistance’ of the bowel, allowing subsequent colonization and infection with pathological bacteria [8]. Therefore, even in milder cases of AAD, antibiotic therapies often need to be either switched or modified, adding burdens on patients and medical systems. The guideline for enteric infection issued by the Japanese Society for Clinical Microbiology in 2010 indicated AAD as having three mechanisms in disease development. First is the functional disorder caused by the decrease of intestinal flora. Second, it is also an intestinal mucosal disorder directly caused by the antibiotics themselves. Finally, CDI further influences the severity of disease.
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Among the various measures for the prevention of AAD, the use of probiotic products intended to decrease the frequency and the magnitude of AAD has been attracting attention from both healthcare professionals and the general public [9, 10]. Probiotic products of natural or traditional food forms produced in controlled facilities with known colony-forming units are used in some studies, and some reports indicated the use of probiotics as risk free [11]. However, controversies remain among healthcare professionals whether probiotic preparations are efficacious in preventing AAD, calling for scientific evaluation [12– 14]. As one of the reasons, many types of bacterial strains found in those probiotic products showed sensitivity toward routinely used antibiotics. Therefore, among major issues to be investigated is the ability of the bacteria from probiotic preparations to survive in the intestine, if they were to prevent AAD with a proposed mechanism such as ‘‘colonization resistance’’ toward worsening of AAD, by decreased pH and protection of intestinal epithelium by encouraging mucin production in the colon [15]. We think that a suitable combination of antibiotics and probiotic products should be scientifically and comprehensively judged from three major types of evidence, such as (1) in vitro MIC evaluation data, (2) pharmacokinetics (PK) evaluation data estimating antibiotics drug concentrations in the intestinal tract, and (3) clinical efficacy data evaluating beneficial outcomes including AAD prevention effects, etc., when these agents are concomitantly used. As one of the first steps to scientifically investigate the effectiveness of bacterial strains from probiotics, we aimed to study the organism’s in vitro sensitivities toward various antibiotics used in modern clinical practice. In this study, we determined the minimum inhibitory concentrations (MICs) of the antimicrobial agents against each bacterial strain used in the probiotics by the agar plate dilution method and broth microdilution method.
Materials and methods
infantis SMR of Lebenin (Wakamoto Pharmaceutical, Tokyo, Japan), Bifidobacterium bifidum G9-1 of Biofermin Tablet (Biofermin Pharmacuticals), and Clostridium butyricum MIYAIRI 588 of MiyaBM (Miyarisan Pharmaceuticals, Tokyo, Japan). Antibiotics The antibiotics commonly used in clinical practice, such as beta-lactams including penicillins, cephems, and carbapenems, fluoroquinolones, macrolides, glycopeptide, and fosfomycin (FOM), were evaluated. Penicillins used were ampicillin (ABPC), sulbactam/ampicillin (SBT/ABPC), and tazobactam/piperacillin (TAZ/PIPC); cephems used were cefepime (CFPM), cefozopran (CZOP), ceftazidime (CAZ), and sulbactam/cefoperazone (SBT/CPZ); carbapenems used were meropenem (MEPM), doripenem (DRPM), panipenem (PAPM), and biapenem (BIPM); fluoroquinolones used were garenoxacin (GRNX), levofloxacin (LVFX), and tosufloxacin (TFLX); and macrolides and glycopeptides used were clarithromycin (CAM) and vancomycin (VCM), respectively. Determination of MIC The MICs (lg/ml) of the antibiotics were determined by the agar plate dilution method and broth microdilution method, in accordance with the ‘‘M07-A8. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard, Eighth Edition’’ by the Clinical and Laboratory Standards Institute (CLSI) [16] for enterococci, and ‘‘M11-A7. Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria; Approved Standard, Seventh Edition’’ by CLSI [17] for anaerobic bacteria. The agar plate dilution method was conducted using LSM-C agar plates and commercially available discs (Oxoid, Basingstoke, UK) containing each drug. The broth microdilution method used commercially available microtiter plates (original plate: custom order, Eiken Chemical, Tokyo Japan) containing each drug.
Bacterial strains We selected the probiotics commonly used for colitis care and readily available. Bacterial strains tested were enterococci and anaerobic bacteria as used in the probiotics. Strains were provided by the manufacturers of the probiotics: Enterococcus faecalis 129 BIO 3B of Biofermin (Biofermin Pharmaceuticals, Kobe, Japan), Enterococcus faecalis 129 BIO 3B-R of Biofermin R (Biofermin Pharmacuticals), Enterococcus faecalis BIO-4R (the strain is registered as Streptococcus faecalis BIO-4R) of Entenoron R (Meguro Institute, Osaka, Japan), Enterococcus faecalis PCR, Lactobacillus acidophilus 4AR, and Bifidobacterium
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Results Susceptibilities of enterococci to beta-lactams Tables 1 and 2 show antibiotic MICs against enterococci by the agar plate dilution method and broth microdilution method, respectively. Overall, results showed a wide range of MICs of the antibiotics evaluated in this study, from less than 0.25 to more than 1,024 lg/ml. The beta-lactams had high MICs, 128 to more than 1,024 lg/ml, against three of four enterococci, i.e., Enterococcus faecalis 129 BIO 3B-
ABPC ampicillin, SBT/ABPC sulbactam/ampicillin, TAZ/PIPC tazobactam/piperacillin, CFPM cefepime, CZOP cefozopran, CAZ ceftazidime, SBT/CPZ sulbactam/cefoperazone, MEPM meropenem, DRPM doripenem, PAPM panipenem, BIPM biapenem, GRNX garenoxacin, LVFX levofloxacin, TFLX tosufloxacin, CAM clarithromycin, VCM vancomycin, FOM fosfomycin
Minimum inhibitory concentration (MIC) values of beta-lactams, fluoroquinolones, macrolides, glycopeptides, and FOM against four enterococci used in probiotics were evaluated by agar plate dilution method with LSM-C agar plates and discs
8
0.5
16
0.5
2
4
1
2 4
1
256 1,024
[1,024 512 1,024
[1,024 [1,024 [1,024 [1,024 256 128 Enterococcus faecalis PCR
512
[1,024 [1,024 [1,024 [1,024 1,024
[1,024
256 256 Enterococcus faecalis BIO-4R
[1,024
256
0.25
[512
8 0.5 [512 1 4 4 512 256 [1,024 [1,024 [1,024 [1,024 1,024 256 256 Enterococcus faecalis 129 BIO 3B-R
512
512
32 1 4 4 4 8 8 16 8 Enterococcus faecalis 129 BIO 3B
4
[1,024
512 [1,024
64
16
16
16
LVFX GRNX BIPM PAPM DRPM MEPM SBT/ CPZ CAZ CZOP CFPM TAZ/ PIPC SBT/ ABPC ABPC
MICs (lg/ml) Enterococcus strains
Table 1 Susceptibilities of enterococci to beta-lactams, fluoroquinolones, macrolide, glycopeptide, and fosfomycin (FOM) (agar plate dilution method)
TFLX
CAM
VCM
FOM
J Infect Chemother
R, E. faecalis BIO-4R, and E. faecalis PCR, by agar plate dilution method (Table 1), and 32 to more than 512 lg/ml by broth microdilution method (Table 2). Susceptibilities of anaerobic bacteria to beta-lactams Tables 3 and 4 show antibiotic MICs against anaerobic bacteria by the agar plate dilution method and broth microdilution method, respectively. Overall, results showed a wide range of MICs of the antibiotics evaluated in this study, from less than 0.0625 to more than 1,024 lg/ml. The beta-lactams had an MIC range of 0.0625–128 lg/ml against anaerobic bacteria by agar plate dilution method (Table 3) and less than 0.5–64 lg/ml by broth microdilution method (Table 4). The antibiotics MICs of beta-lactams against anaerobic bacteria were generally lower than those against enterococci (Tables 1 vs. 3, and Tables 2 vs. 4). Comparison of MIC values between beta-lactams and other antibiotics The MIC values against enterococci were compared between beta-lactams and other antibiotics such as fluoroquinolones, macrolide, glycopeptide, and FOM using the agar plate dilution method (Tables 1, 3). The MIC values of beta-lactams were generally higher than those of other antibiotics. Fluoroquinolones and VCM showed potent activity against all enterococci, with MICs of 0.25–8 lg/ml, and FOM showed generally mild activity against enterococci with MICs of 8–32 lg/ml (Table 1). As with beta-lactams, the MICs of other antibiotics against anaerobic bacteria were low. Two Bifidobacterium strains and one Lactobacillus strain showed low MICs against many of the beta-lactams, fluoroquinolones, macrolides, and VCM with MICs of 8 lg/ml or less. FOM showed mild activity against anaerobic strains with MICs of 32 to more than 1,024 lg/ml (Table 3). Comparison of MIC values among beta-lactams The MIC values of cephems against enterococci were higher than those of penicillins or carbapenems (Tables 1, 2). For penicillins, the MIC values of TAZ/PIPC against enterococci tended to be higher than those of ABPC or SBT/ABPC, but no notable difference between antibiotics was observed in cephems and carbapenems (Tables 1, 2). The MIC values of penicillins, cephems, and carbapenems against anaerobic bacteria were generally similar, and no notable difference between antibiotics was observed in each group (Tables 3, 4).
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J Infect Chemother Table 2 Susceptibilities of enterococci to beta-lactams (broth microdilution method) Enterococcus strains
MICs (lg/ml) ABPC
SBT/ ABPC
TAZ/ PIPC
CFPM
CZOP
CAZ
SBT/CPZ
MEPM
DRPM
PAPM
BIPM
Enterococcus faecalis 129 BIO 3B
\8
\2/4
4/16
[512
128
[64
16
8
8
16
Enterococcus faecalis 129 BIO 3B-R
128
64/128
[4/512
[512
[128
[64
[512/512 [128
[64
128
[128
Enterococcus faecalis BIO-4R
128
32/64
[4/512
256
[128
[64
[512/512 [128
[64
256
[128
Enterococcus faecalis PCR
128
32/64
[4/512
[512
[128
[64
[64
32
128
32/32
256/256
128
The MIC values of beta-lactams against four enterococci used in probiotics were evaluated by broth microdilution method with microtiter plates
Comparison of MIC values evaluated by agar plate dilution method and broth microdilution method The MIC values of beta-lactams against enterococci obtained from the agar plate dilution assay were tended to be higher than those from the broth microdilution assay. However, the tendencies of differences in MICs among beta-lactams were similar between the two methods (Tables 1, 2). In penicillins and cephems, there was no major difference between the MIC values against anaerobic bacteria obtained from both assay methods, but in carbapenems the MIC values obtained from the agar plate dilution assay tended to be higher than those from the broth microdilution assay (Tables 3, 4).
Discussion Our data indicated that there was an extremely wide range of MICs, and the bacterial strains used in probiotics had various levels of sensitivities toward the broad-spectrum antibiotics used in modern medicine. Therefore, we think it is important to select the appropriate bacterial strain when the probiotics are used as prophylactic for AAD, considering the MIC values of the antibiotics. Probiotics using enterococci such as E. faecalis 129 BIO 3B-R, E. faecalis BIO-4R, and E. faecalis PCR may be effective when concomitantly administered with beta-lactams, because these had high MICs for this type of antibiotic and a good chance of surviving in the patient’s intestine during antibiotic treatment. We also compared the MIC values of beta-lactams against these enterococci with other antibiotics such as fluoroquinolones, macrolides, glycopeptides, and FOM. The MIC values of beta-lactams were generally higher than those of other antibiotics, and these enterococci are suggested to be more effective when combined with beta-lactams than with other antibiotics. On the other hand, anaerobic bacteria resulting in microorganisms with low MIC values may not survive
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during antibiotic therapy. Iwata et al. [18, 19] previously reported that after administration of MEPM or BIPM to children, these antibiotics decreased the number of fecal anaerobic bacteria including Bifidobacterium, which had low MIC values of these antibiotics, in the range of 0.125–4 lg/ml, in this study. The group also investigated the fecal concentrations of MEPM or BIPM after administration, which was 0.35–66.0 or 0.24–2.22 lg/g feces, respectively. The decrease of the number of fecal flora was transient or recoverable. However, they concluded that caution is required for the long-term administration of MEPM or BIPM to patients with high risk for AAD. It is difficult to correctly know the real effect of antibiotics on the viabilities of probiotic strains in the gut. Especially, the exact measurements of intestinal concentrations of antibiotics are difficult, because the concentrations differ with the site of the intestinal tract and at different sampling times after administration. In fact, the intestinal or fecal concentrations differed substantially among the previous reports [18–25]. However, MIC values of most beta-lactams against the three enterococci obtained from our study were higher than the intestinal concentrations of beta-lactams in previous reports. Therefore, we think that the probiotics strains with high MICs may not be killed by concomitantly administered beta-lactams. The probiotics using the enterococci may be most effective when concomitantly administered with cephems among beta-lactams, because the MIC values of cephems against enterococci were higher than those of penicillins or carbapenems. All the cephems evaluated in this study are either third or fourth generation, but there was no distinguishable difference between the drug generations. Previous reports indicated that the newer generations, third and fourth, of cephems were the possible factors for AAD and suggested the higher incidence of AAD [26, 27]. Our results suggest the possibility of combining probiotics including enterococci and cephems for preventing AAD. Among penicillins, TAZ/PIPC had higher MICs against enterococci, and these organisms may be effective toward prevention of AAD by TAZ/PIPC, possibly by
The MIC values of beta-lactams, fluoroquinolones, macrolide, glycopeptide, and FOM against four anaerobic bacteria strains used in probiotics were evaluated by agar plate dilution method with LSM-C agar plates and discs
32 0.5 0.25 Bifidobacterium infantis SMR
4
4
8
8
32
16
1
8
0.5
4
1
4
4
\0.0625
[1024 1 128 4 64 0.5 16 8 4 2 4 16 4 8 1 1 Lactobacillus acidophilus 4AR
1
32 0.5 \0.0625
64 1 \0.0625
0.25 1
4 1
0.125 64
2 1
16 4
0.25 0.125
1 1
0.25 1
128 64
0.125 0.0625
64 1
0.25
0.5 Clostridium butyricum MIYAIRI 588
0.0625 0.25 Bifidobacterium bifidum G9-1
0.5
4
VCM LVFX GRNX BIPM PAPM DRPM MEPM SBT/ CPZ CAZ CZOP CFPM TAZ/ PIPC SBT/ ABPC ABPC
MICs (lg/ml) Anaerobic bacteria strains
Table 3 Susceptibilities of anaerobic bacteria to beta-lactams, fluoroquinolones, macrolides, glycopeptides, and FOM (agar plate dilution method)
TFLX
CAM
FOM
J Infect Chemother
compensating for the adverse effects of antibiotics that decrease or disrupt the normal intestinal flora. However, in two previous studies [21, 22], TAZ/PIPC was reported to decrease the number of intestinal microflora slightly and transiently after administration of TAZ/PIPC to human adults. One of the two studies also indicated that TAZ/ PIPC was detectable in the intestinal lumen in two of seven healthy volunteers, and decrease of fecal flora was only observed in these two cases [22]. This finding indicates that the concentration of the antibiotics in the intestine is very important when the probiotic supplement is used as a prophylactic for AAD. Therefore, the effective combinations of the antibiotics and the probiotics have to be carefully considered based on the MICs and the drug concentration in the intestinal lumen. As the limitation of this in vitro study, the results may not reflect efficacy when probiotics are administered to actual patients. The antibiotics evaluated in this study include injectable and/or oral formulations, and the drug concentration of antibiotics in the gastrointestinal tract would be much higher when administered orally. Therefore, in addition to the in vitro MIC values, the administration route of the antibiotics is an important point to determine the effectiveness of a combination of antibiotics with probiotics. Also, PK/pharmacodynamics of the antibiotics after administration need to be accounted for determining the actual effects. Moreover, the marketed probiotic products often have combinations of bacterial strains, which may have synergistic effects in the clinical setting; however, our experimental design did not cover such aspects. Another point we should notice is the potential for spread of resistance genes. Most bacterial strains used for the probiotics are established to be clinically safe, but the resistance genes of these safe strains may be carried by a mobile genetic element into pathogenic bacterial strains such as a self-transferable plasmid or transposon [28]. Especially, drug-resistant enterococci are often detected in clinical practice, and vanA gene coding the vancomycin resistance locates in the plasmid DNA of the vancomycinresistant enterococci [29]. Although there were no vancomycin-resistant strains in our study, some enterococci were highly resistant against beta-lactams. The drug resistance mechanism for each strain of enterococci in this study remains unclear; enterococci tend to be resistant against beta-lactams because penicillin-binding proteins of the enterococci originally have low binding affinity to betalactams [30]. Usually, the potential for horizontal gene transfer of this resistance gene is low, because this gene generally locates in the chromosomal DNA. However, we should carefully consider this point because an acquired form of this resistance mechanism through transposons or plasmids is also reported.
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J Infect Chemother Table 4 Susceptibilities of anaerobic bacteria to beta-lactams (broth microdilution method) Anaerobic bacteria strains
MICs (lg/ml) ABPC
SBT/ ABPC
TAZ/ PIPC
CFPM
CZOP
CAZ
SBT/ CPZ
MEPM
DRPM
PAPM
BIPM
Bifidobacterium bifidum G9-1
\8
\2/4
\4/4
\4
Clostridium butyricum MIYAIRI 588
\8
\2/4
\4/4
64
\1
2
\4/4
\1
\0.5
\4
\1
16
64
\2/4
\1
\0.5
\4
\1
Lactobacillus acidophilus 4AR
\8
2/4
\4/4
\4
\1
1
\4/4
\1
\0.5
\4
\1
Bifidobacterium infantis SMR
\8
\2/4
\4/4
8
2
32
8/8
\1
1
\4
\1
The MIC values of beta-lactams against four anaerobic bacteria strains used in probiotics were evaluated by broth microdilution method with microtiter plates
Together with in vitro MIC evaluation data and PK evaluation data, we also think that a clinical study should be finally conducted to evaluate the beneficial outcomes including AAD prevention effects when antibiotics and probiotics are concomitantly used. The Yale Workshop on Probiotics published their updated recommendations for the clinical use of probiotics in 2011. They recommended some specific strains of organisms for clinical use to prevent AAD, given as A ratings, and indicated their analysis references [31]. A clinical study with the probiotics evaluated in our work should be conducted, consulting these recommendations and referred literature. Furthermore, in this in vitro study, we selected commonly used probiotics and antibiotics in the present Japanese clinical practice. We believe that our findings will be clinically useful evidence, together with PK data and clinical efficacy data, in the near future. In this study, we tested the antimicrobial susceptibility of strains in probiotics that were commonly used in clinical settings. Probiotics that contain each strain tested in this study have a 69.1 % share of the probiotics used in clinical settings in Japan [32]. Therefore, we think that the results will provide helpful suggestions to many patients with therapeutic adaptation of probiotics. In conclusion, the probiotics using these three enterococci are suggested to have a high chance of surviving in the patient’s intestine during antibiotic treatment and may be effective in preventing AAD for beta-lactams. Our study may provide background information on preventing the decrease of intestinal flora that may contribute to the initial step of preventing AAD. We further intended to provide data for clinical studies and subsequent applications when the combinations of probiotics and antibiotics are considered. Acknowledgments We appreciate Hideyuki Sasaoka and Takahiro Morishita (Department of Microbiology, Bunkyo Gakuin University), and Toshie Fukui (Department of Microbiology, Tokyo Medical University) for their helpful expertise in our experiments. Conflict of interest
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None.
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ORIGINAL ARTICLE
Antimicrobial susceptibility of Enterococcus strains used in clinical practice as probiotics Tetsuo Yamaguchi • Yuri Miura • Tetsuya Matsumoto
Received: 22 July 2012 / Accepted: 2 June 2013 Ó Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2013
Abstract The aim of this study was to evaluate the antimicrobial susceptibilities of probiotic strains that are suggested to be effective for preventing antibiotics-associated diarrhea (AAD). The minimum inhibitory concentrations (MICs) of 17 antibiotics against probiotic strains were tested by the agar plate dilution method or broth microdilution method. In all, eight probiotic strains containing Enterococcus faecalis, Bifidobacterium spp., Clostridium butyricum, and Lactobacillus acidophilus were tested. Although the MIC range was wide, from less than 0.0625 to more than 1,024 lg/ml, the MICs of 11 betalactams were high for three of four enterococci, with a range of 32 to more than 1,024 lg/ml. In contrast, fluoroquinolones and vancomycin showed potent activities against all enterococci, of which MICs were 0.25–8 lg/ml. Two Bifidobacterium strains and one Lactobacillus strain showed low MICs against many of the beta-lactams, fluoroquinolones, macrolides, and vancomycin, with MICs of 8 lg/ml or less. Fosfomycin showed generally mild activity against enterococci (MIC, 8–32 lg/ml) and anaerobic strains (MIC, 32 to [1,024 lg/ml), respectively. The probiotics strains with high MIC values may survive in the intestinal tract, even if the patient was concomitantly using the antibiotics in clinical practice. Therefore, our results suggest that adequate combinations of probiotics strains and antibiotics should be important for preventing AAD. Further study is needed to determine the efficacy of probiotics in clinical practice.
T. Yamaguchi Y. Miura T. Matsumoto (&) Department of Microbiology, Tokyo Medical University, 6-1-1, Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan e-mail: [email protected]
Keywords Probiotics Enterococci Antimicrobial susceptibility Antibiotics-associated diarrhea
Introduction Antibiotics-associated diarrhea (AAD) is the most common adverse effect during antibiotic therapy, occurring in from 5 % to more than 30 % of the patients during the course of therapy [1]. AAD is associated with alterations in intestinal microflora, mucosal integrity, vitamin and nutrient metabolism and absorption, and other systems or processes [2]. In severe cases, inflammation of the digestive organs and intestinal bleeding may occur and the condition may become life threatening [3]. Moreover, infectious diseases such as Clostridium difficile infection (CDI) complicate AAD further by infecting the intestinal lumen damaged both by the drug itself and by the toxins produced from the organism [4]. Incidences are higher among children, the elderly, and immunocompromised patients, and are frequently reported as nosocomial infections [5–7]. Antibiotics are believed to complicate AAD further by reducing the ‘colonization resistance’ of the bowel, allowing subsequent colonization and infection with pathological bacteria [8]. Therefore, even in milder cases of AAD, antibiotic therapies often need to be either switched or modified, adding burdens on patients and medical systems. The guideline for enteric infection issued by the Japanese Society for Clinical Microbiology in 2010 indicated AAD as having three mechanisms in disease development. First is the functional disorder caused by the decrease of intestinal flora. Second, it is also an intestinal mucosal disorder directly caused by the antibiotics themselves. Finally, CDI further influences the severity of disease.
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Among the various measures for the prevention of AAD, the use of probiotic products intended to decrease the frequency and the magnitude of AAD has been attracting attention from both healthcare professionals and the general public [9, 10]. Probiotic products of natural or traditional food forms produced in controlled facilities with known colony-forming units are used in some studies, and some reports indicated the use of probiotics as risk free [11]. However, controversies remain among healthcare professionals whether probiotic preparations are efficacious in preventing AAD, calling for scientific evaluation [12– 14]. As one of the reasons, many types of bacterial strains found in those probiotic products showed sensitivity toward routinely used antibiotics. Therefore, among major issues to be investigated is the ability of the bacteria from probiotic preparations to survive in the intestine, if they were to prevent AAD with a proposed mechanism such as ‘‘colonization resistance’’ toward worsening of AAD, by decreased pH and protection of intestinal epithelium by encouraging mucin production in the colon [15]. We think that a suitable combination of antibiotics and probiotic products should be scientifically and comprehensively judged from three major types of evidence, such as (1) in vitro MIC evaluation data, (2) pharmacokinetics (PK) evaluation data estimating antibiotics drug concentrations in the intestinal tract, and (3) clinical efficacy data evaluating beneficial outcomes including AAD prevention effects, etc., when these agents are concomitantly used. As one of the first steps to scientifically investigate the effectiveness of bacterial strains from probiotics, we aimed to study the organism’s in vitro sensitivities toward various antibiotics used in modern clinical practice. In this study, we determined the minimum inhibitory concentrations (MICs) of the antimicrobial agents against each bacterial strain used in the probiotics by the agar plate dilution method and broth microdilution method.
Materials and methods
infantis SMR of Lebenin (Wakamoto Pharmaceutical, Tokyo, Japan), Bifidobacterium bifidum G9-1 of Biofermin Tablet (Biofermin Pharmacuticals), and Clostridium butyricum MIYAIRI 588 of MiyaBM (Miyarisan Pharmaceuticals, Tokyo, Japan). Antibiotics The antibiotics commonly used in clinical practice, such as beta-lactams including penicillins, cephems, and carbapenems, fluoroquinolones, macrolides, glycopeptide, and fosfomycin (FOM), were evaluated. Penicillins used were ampicillin (ABPC), sulbactam/ampicillin (SBT/ABPC), and tazobactam/piperacillin (TAZ/PIPC); cephems used were cefepime (CFPM), cefozopran (CZOP), ceftazidime (CAZ), and sulbactam/cefoperazone (SBT/CPZ); carbapenems used were meropenem (MEPM), doripenem (DRPM), panipenem (PAPM), and biapenem (BIPM); fluoroquinolones used were garenoxacin (GRNX), levofloxacin (LVFX), and tosufloxacin (TFLX); and macrolides and glycopeptides used were clarithromycin (CAM) and vancomycin (VCM), respectively. Determination of MIC The MICs (lg/ml) of the antibiotics were determined by the agar plate dilution method and broth microdilution method, in accordance with the ‘‘M07-A8. Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard, Eighth Edition’’ by the Clinical and Laboratory Standards Institute (CLSI) [16] for enterococci, and ‘‘M11-A7. Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria; Approved Standard, Seventh Edition’’ by CLSI [17] for anaerobic bacteria. The agar plate dilution method was conducted using LSM-C agar plates and commercially available discs (Oxoid, Basingstoke, UK) containing each drug. The broth microdilution method used commercially available microtiter plates (original plate: custom order, Eiken Chemical, Tokyo Japan) containing each drug.
Bacterial strains We selected the probiotics commonly used for colitis care and readily available. Bacterial strains tested were enterococci and anaerobic bacteria as used in the probiotics. Strains were provided by the manufacturers of the probiotics: Enterococcus faecalis 129 BIO 3B of Biofermin (Biofermin Pharmaceuticals, Kobe, Japan), Enterococcus faecalis 129 BIO 3B-R of Biofermin R (Biofermin Pharmacuticals), Enterococcus faecalis BIO-4R (the strain is registered as Streptococcus faecalis BIO-4R) of Entenoron R (Meguro Institute, Osaka, Japan), Enterococcus faecalis PCR, Lactobacillus acidophilus 4AR, and Bifidobacterium
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Results Susceptibilities of enterococci to beta-lactams Tables 1 and 2 show antibiotic MICs against enterococci by the agar plate dilution method and broth microdilution method, respectively. Overall, results showed a wide range of MICs of the antibiotics evaluated in this study, from less than 0.25 to more than 1,024 lg/ml. The beta-lactams had high MICs, 128 to more than 1,024 lg/ml, against three of four enterococci, i.e., Enterococcus faecalis 129 BIO 3B-
ABPC ampicillin, SBT/ABPC sulbactam/ampicillin, TAZ/PIPC tazobactam/piperacillin, CFPM cefepime, CZOP cefozopran, CAZ ceftazidime, SBT/CPZ sulbactam/cefoperazone, MEPM meropenem, DRPM doripenem, PAPM panipenem, BIPM biapenem, GRNX garenoxacin, LVFX levofloxacin, TFLX tosufloxacin, CAM clarithromycin, VCM vancomycin, FOM fosfomycin
Minimum inhibitory concentration (MIC) values of beta-lactams, fluoroquinolones, macrolides, glycopeptides, and FOM against four enterococci used in probiotics were evaluated by agar plate dilution method with LSM-C agar plates and discs
8
0.5
16
0.5
2
4
1
2 4
1
256 1,024
[1,024 512 1,024
[1,024 [1,024 [1,024 [1,024 256 128 Enterococcus faecalis PCR
512
[1,024 [1,024 [1,024 [1,024 1,024
[1,024
256 256 Enterococcus faecalis BIO-4R
[1,024
256
0.25
[512
8 0.5 [512 1 4 4 512 256 [1,024 [1,024 [1,024 [1,024 1,024 256 256 Enterococcus faecalis 129 BIO 3B-R
512
512
32 1 4 4 4 8 8 16 8 Enterococcus faecalis 129 BIO 3B
4
[1,024
512 [1,024
64
16
16
16
LVFX GRNX BIPM PAPM DRPM MEPM SBT/ CPZ CAZ CZOP CFPM TAZ/ PIPC SBT/ ABPC ABPC
MICs (lg/ml) Enterococcus strains
Table 1 Susceptibilities of enterococci to beta-lactams, fluoroquinolones, macrolide, glycopeptide, and fosfomycin (FOM) (agar plate dilution method)
TFLX
CAM
VCM
FOM
J Infect Chemother
R, E. faecalis BIO-4R, and E. faecalis PCR, by agar plate dilution method (Table 1), and 32 to more than 512 lg/ml by broth microdilution method (Table 2). Susceptibilities of anaerobic bacteria to beta-lactams Tables 3 and 4 show antibiotic MICs against anaerobic bacteria by the agar plate dilution method and broth microdilution method, respectively. Overall, results showed a wide range of MICs of the antibiotics evaluated in this study, from less than 0.0625 to more than 1,024 lg/ml. The beta-lactams had an MIC range of 0.0625–128 lg/ml against anaerobic bacteria by agar plate dilution method (Table 3) and less than 0.5–64 lg/ml by broth microdilution method (Table 4). The antibiotics MICs of beta-lactams against anaerobic bacteria were generally lower than those against enterococci (Tables 1 vs. 3, and Tables 2 vs. 4). Comparison of MIC values between beta-lactams and other antibiotics The MIC values against enterococci were compared between beta-lactams and other antibiotics such as fluoroquinolones, macrolide, glycopeptide, and FOM using the agar plate dilution method (Tables 1, 3). The MIC values of beta-lactams were generally higher than those of other antibiotics. Fluoroquinolones and VCM showed potent activity against all enterococci, with MICs of 0.25–8 lg/ml, and FOM showed generally mild activity against enterococci with MICs of 8–32 lg/ml (Table 1). As with beta-lactams, the MICs of other antibiotics against anaerobic bacteria were low. Two Bifidobacterium strains and one Lactobacillus strain showed low MICs against many of the beta-lactams, fluoroquinolones, macrolides, and VCM with MICs of 8 lg/ml or less. FOM showed mild activity against anaerobic strains with MICs of 32 to more than 1,024 lg/ml (Table 3). Comparison of MIC values among beta-lactams The MIC values of cephems against enterococci were higher than those of penicillins or carbapenems (Tables 1, 2). For penicillins, the MIC values of TAZ/PIPC against enterococci tended to be higher than those of ABPC or SBT/ABPC, but no notable difference between antibiotics was observed in cephems and carbapenems (Tables 1, 2). The MIC values of penicillins, cephems, and carbapenems against anaerobic bacteria were generally similar, and no notable difference between antibiotics was observed in each group (Tables 3, 4).
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J Infect Chemother Table 2 Susceptibilities of enterococci to beta-lactams (broth microdilution method) Enterococcus strains
MICs (lg/ml) ABPC
SBT/ ABPC
TAZ/ PIPC
CFPM
CZOP
CAZ
SBT/CPZ
MEPM
DRPM
PAPM
BIPM
Enterococcus faecalis 129 BIO 3B
\8
\2/4
4/16
[512
128
[64
16
8
8
16
Enterococcus faecalis 129 BIO 3B-R
128
64/128
[4/512
[512
[128
[64
[512/512 [128
[64
128
[128
Enterococcus faecalis BIO-4R
128
32/64
[4/512
256
[128
[64
[512/512 [128
[64
256
[128
Enterococcus faecalis PCR
128
32/64
[4/512
[512
[128
[64
[64
32
128
32/32
256/256
128
The MIC values of beta-lactams against four enterococci used in probiotics were evaluated by broth microdilution method with microtiter plates
Comparison of MIC values evaluated by agar plate dilution method and broth microdilution method The MIC values of beta-lactams against enterococci obtained from the agar plate dilution assay were tended to be higher than those from the broth microdilution assay. However, the tendencies of differences in MICs among beta-lactams were similar between the two methods (Tables 1, 2). In penicillins and cephems, there was no major difference between the MIC values against anaerobic bacteria obtained from both assay methods, but in carbapenems the MIC values obtained from the agar plate dilution assay tended to be higher than those from the broth microdilution assay (Tables 3, 4).
Discussion Our data indicated that there was an extremely wide range of MICs, and the bacterial strains used in probiotics had various levels of sensitivities toward the broad-spectrum antibiotics used in modern medicine. Therefore, we think it is important to select the appropriate bacterial strain when the probiotics are used as prophylactic for AAD, considering the MIC values of the antibiotics. Probiotics using enterococci such as E. faecalis 129 BIO 3B-R, E. faecalis BIO-4R, and E. faecalis PCR may be effective when concomitantly administered with beta-lactams, because these had high MICs for this type of antibiotic and a good chance of surviving in the patient’s intestine during antibiotic treatment. We also compared the MIC values of beta-lactams against these enterococci with other antibiotics such as fluoroquinolones, macrolides, glycopeptides, and FOM. The MIC values of beta-lactams were generally higher than those of other antibiotics, and these enterococci are suggested to be more effective when combined with beta-lactams than with other antibiotics. On the other hand, anaerobic bacteria resulting in microorganisms with low MIC values may not survive
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during antibiotic therapy. Iwata et al. [18, 19] previously reported that after administration of MEPM or BIPM to children, these antibiotics decreased the number of fecal anaerobic bacteria including Bifidobacterium, which had low MIC values of these antibiotics, in the range of 0.125–4 lg/ml, in this study. The group also investigated the fecal concentrations of MEPM or BIPM after administration, which was 0.35–66.0 or 0.24–2.22 lg/g feces, respectively. The decrease of the number of fecal flora was transient or recoverable. However, they concluded that caution is required for the long-term administration of MEPM or BIPM to patients with high risk for AAD. It is difficult to correctly know the real effect of antibiotics on the viabilities of probiotic strains in the gut. Especially, the exact measurements of intestinal concentrations of antibiotics are difficult, because the concentrations differ with the site of the intestinal tract and at different sampling times after administration. In fact, the intestinal or fecal concentrations differed substantially among the previous reports [18–25]. However, MIC values of most beta-lactams against the three enterococci obtained from our study were higher than the intestinal concentrations of beta-lactams in previous reports. Therefore, we think that the probiotics strains with high MICs may not be killed by concomitantly administered beta-lactams. The probiotics using the enterococci may be most effective when concomitantly administered with cephems among beta-lactams, because the MIC values of cephems against enterococci were higher than those of penicillins or carbapenems. All the cephems evaluated in this study are either third or fourth generation, but there was no distinguishable difference between the drug generations. Previous reports indicated that the newer generations, third and fourth, of cephems were the possible factors for AAD and suggested the higher incidence of AAD [26, 27]. Our results suggest the possibility of combining probiotics including enterococci and cephems for preventing AAD. Among penicillins, TAZ/PIPC had higher MICs against enterococci, and these organisms may be effective toward prevention of AAD by TAZ/PIPC, possibly by
The MIC values of beta-lactams, fluoroquinolones, macrolide, glycopeptide, and FOM against four anaerobic bacteria strains used in probiotics were evaluated by agar plate dilution method with LSM-C agar plates and discs
32 0.5 0.25 Bifidobacterium infantis SMR
4
4
8
8
32
16
1
8
0.5
4
1
4
4
\0.0625
[1024 1 128 4 64 0.5 16 8 4 2 4 16 4 8 1 1 Lactobacillus acidophilus 4AR
1
32 0.5 \0.0625
64 1 \0.0625
0.25 1
4 1
0.125 64
2 1
16 4
0.25 0.125
1 1
0.25 1
128 64
0.125 0.0625
64 1
0.25
0.5 Clostridium butyricum MIYAIRI 588
0.0625 0.25 Bifidobacterium bifidum G9-1
0.5
4
VCM LVFX GRNX BIPM PAPM DRPM MEPM SBT/ CPZ CAZ CZOP CFPM TAZ/ PIPC SBT/ ABPC ABPC
MICs (lg/ml) Anaerobic bacteria strains
Table 3 Susceptibilities of anaerobic bacteria to beta-lactams, fluoroquinolones, macrolides, glycopeptides, and FOM (agar plate dilution method)
TFLX
CAM
FOM
J Infect Chemother
compensating for the adverse effects of antibiotics that decrease or disrupt the normal intestinal flora. However, in two previous studies [21, 22], TAZ/PIPC was reported to decrease the number of intestinal microflora slightly and transiently after administration of TAZ/PIPC to human adults. One of the two studies also indicated that TAZ/ PIPC was detectable in the intestinal lumen in two of seven healthy volunteers, and decrease of fecal flora was only observed in these two cases [22]. This finding indicates that the concentration of the antibiotics in the intestine is very important when the probiotic supplement is used as a prophylactic for AAD. Therefore, the effective combinations of the antibiotics and the probiotics have to be carefully considered based on the MICs and the drug concentration in the intestinal lumen. As the limitation of this in vitro study, the results may not reflect efficacy when probiotics are administered to actual patients. The antibiotics evaluated in this study include injectable and/or oral formulations, and the drug concentration of antibiotics in the gastrointestinal tract would be much higher when administered orally. Therefore, in addition to the in vitro MIC values, the administration route of the antibiotics is an important point to determine the effectiveness of a combination of antibiotics with probiotics. Also, PK/pharmacodynamics of the antibiotics after administration need to be accounted for determining the actual effects. Moreover, the marketed probiotic products often have combinations of bacterial strains, which may have synergistic effects in the clinical setting; however, our experimental design did not cover such aspects. Another point we should notice is the potential for spread of resistance genes. Most bacterial strains used for the probiotics are established to be clinically safe, but the resistance genes of these safe strains may be carried by a mobile genetic element into pathogenic bacterial strains such as a self-transferable plasmid or transposon [28]. Especially, drug-resistant enterococci are often detected in clinical practice, and vanA gene coding the vancomycin resistance locates in the plasmid DNA of the vancomycinresistant enterococci [29]. Although there were no vancomycin-resistant strains in our study, some enterococci were highly resistant against beta-lactams. The drug resistance mechanism for each strain of enterococci in this study remains unclear; enterococci tend to be resistant against beta-lactams because penicillin-binding proteins of the enterococci originally have low binding affinity to betalactams [30]. Usually, the potential for horizontal gene transfer of this resistance gene is low, because this gene generally locates in the chromosomal DNA. However, we should carefully consider this point because an acquired form of this resistance mechanism through transposons or plasmids is also reported.
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J Infect Chemother Table 4 Susceptibilities of anaerobic bacteria to beta-lactams (broth microdilution method) Anaerobic bacteria strains
MICs (lg/ml) ABPC
SBT/ ABPC
TAZ/ PIPC
CFPM
CZOP
CAZ
SBT/ CPZ
MEPM
DRPM
PAPM
BIPM
Bifidobacterium bifidum G9-1
\8
\2/4
\4/4
\4
Clostridium butyricum MIYAIRI 588
\8
\2/4
\4/4
64
\1
2
\4/4
\1
\0.5
\4
\1
16
64
\2/4
\1
\0.5
\4
\1
Lactobacillus acidophilus 4AR
\8
2/4
\4/4
\4
\1
1
\4/4
\1
\0.5
\4
\1
Bifidobacterium infantis SMR
\8
\2/4
\4/4
8
2
32
8/8
\1
1
\4
\1
The MIC values of beta-lactams against four anaerobic bacteria strains used in probiotics were evaluated by broth microdilution method with microtiter plates
Together with in vitro MIC evaluation data and PK evaluation data, we also think that a clinical study should be finally conducted to evaluate the beneficial outcomes including AAD prevention effects when antibiotics and probiotics are concomitantly used. The Yale Workshop on Probiotics published their updated recommendations for the clinical use of probiotics in 2011. They recommended some specific strains of organisms for clinical use to prevent AAD, given as A ratings, and indicated their analysis references [31]. A clinical study with the probiotics evaluated in our work should be conducted, consulting these recommendations and referred literature. Furthermore, in this in vitro study, we selected commonly used probiotics and antibiotics in the present Japanese clinical practice. We believe that our findings will be clinically useful evidence, together with PK data and clinical efficacy data, in the near future. In this study, we tested the antimicrobial susceptibility of strains in probiotics that were commonly used in clinical settings. Probiotics that contain each strain tested in this study have a 69.1 % share of the probiotics used in clinical settings in Japan [32]. Therefore, we think that the results will provide helpful suggestions to many patients with therapeutic adaptation of probiotics. In conclusion, the probiotics using these three enterococci are suggested to have a high chance of surviving in the patient’s intestine during antibiotic treatment and may be effective in preventing AAD for beta-lactams. Our study may provide background information on preventing the decrease of intestinal flora that may contribute to the initial step of preventing AAD. We further intended to provide data for clinical studies and subsequent applications when the combinations of probiotics and antibiotics are considered. Acknowledgments We appreciate Hideyuki Sasaoka and Takahiro Morishita (Department of Microbiology, Bunkyo Gakuin University), and Toshie Fukui (Department of Microbiology, Tokyo Medical University) for their helpful expertise in our experiments. Conflict of interest
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None.
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