Antibiotic susceptibility of different lactic acid bacteria strains

Beneficial Microbes, December 2011; 2(4): 335-339 Wageningen Academic P u b l i s h e r s Antibiotic susceptibility of different lactic acid bacteria strains N. Karapetkov, R. Georgieva, N. Rumyan and E. Karaivanova Lactina Ltd., 101 Sofia str., 1320 Bankya, Bulgaria; nikolay_karapetkov@lactina-ltd.com Abstract 1. Introduction Antibiotic resistance is a type of resistance at which a microorganism is able to survive exposure to an antibiotic. The ability of bacteria to resist antimicrobials could pose a threat to both human and animal health (Cocconcelli et al., 2004; Toomey et al., 2009). It is usually caused by the broad use of antimicrobials in primary health care and in veterinary medicine for growth promotion purposes in animal feed (Aarestrup, 2000). Resistance to an antimicrobial could be inherent to a bacterial species or genus (intrinsic resistance) (Danielsen and Wind, 2003) or acquired by mutation or through a gain of genes (Tenover, 2006). The antibiotic resistance of lactic acid bacteria (LAB) strains used for food, feed and probiotic applications is considered a major danger since this resistance could be transferred to pathogenic bacteria. For distinguishing between susceptible strains and cultures with acquired antimicrobial resistances, the Panel on Additives and Products or Substances used Received: 14 July 2011 / Accepted: 6 October 2011 2011 Wageningen Academic Publishers Five lactic acid bacteria (LAB) strains belonging to species Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus delbrueckii subsp. lactis and Streptococcus thermophilus were tested for their susceptibility to 27 antibiotics. The minimum inhibitory concentrations of each antimicrobial were determined using a microdilution test. Among the strains a high susceptibility was detected for most of the cell-wall synthesis inhibitors (penicillins, cefoxitin and vancomycin) and resistance toward inhibitors of DNA synthesis (trimethoprim/sulfonamides and fluoroquinolones). Generally, the Lactobacillus strains were inhibited by antibiotics such as chloramphenicol, erythromycin and tetracycline at breakpoint levels lower or equal to the levels defined by the European Food Safety Authority. Despite the very similar profile of S. thermophilus LC201 to lactobacilli, the detection of resistance toward erythromycin necessitates the performance of additional tests in order to prove the absence of transferable resistance genes. Keywords: antimicrobial resistance, LAB, minimum inhibitory concentration in Animal Feed (FEEDAP) of the European Food Safety Authority (EFSA) has defined microbiological breakpoints for a representative group of ten antibiotics, with the aim of identifying genotypes resistant to the most commonly used antimicrobials (EFSA, 2008). Microbiological breakpoints are determined by studying the distribution of minimum inhibitory concentrations (MICs) of chosen antimicrobials in bacterial populations belonging to the same species or genus. The European Commission has requested that bacterial strains harbouring transferable antibiotic resistance genes should not be used in animal feeds (EC, 2003). No legislation exists regarding microorganisms added to fermented food and probiotics for human use. However, based on the precautionary principle, it is recommended that these products follow similar requirements to feed additives (EFSA, 2007).The study aims to obtain data related to phenotypic susceptibility of four Lactobacillus and one Streptococcus thermophilus strains to a relevant range of antimicrobials with a view to their applicability as probiotic adjunct cultures. ISSN 1876-2833 print, ISSN 1876-2891 online, DOI 10.3920/BM2011.0016 335

N. Karapetkov et al. 2. Materials and methods Determination of the minimum inhibitory concentration LAB strains Lactobacillus acidophilus LC10, Lactobacillus helveticus LC3, Lactobacillus delbrueckii subsp. bulgaricus LC1, Lactobacillus delbrueckii subsp. lactis LC182, and S. thermophilus LC201 isolated from dairy products or fruits which are part of the laboratory collection of Lactina Ltd. (Bankya, Bulgaria) were selected for the present study. For long-term maintenance, the isolates were stored at -65 C in De Man, Rogose and Sharpe (MRS) or M17 supplemented with 20% (v/v) glycerol. Before analysis, the strains were routinely cultured at 37 C on MRS or M17 agar plates (Merck, Darmstadt, Germany) from which fresh cultures were prepared for inoculation of the final broth used for antimicrobial susceptibility testing. For selected LAB strains, the MICs of 27 antibiotics were determined using commercial Sensititre GPALL1F Grampositive MIC and EUST Staphylococci microbroth dilution tests (Trek Diagnostic Systems, East Grinstead, UK). The antibiotics used and their ranges of two-fold dilutions are indicated in Table 1. The tests were performed following the manufacturer s instructions. Colonies from solid media were picked, suspended and adjusted to 0.5 McFarland turbidity standard in sterile water. Ten ml aliquots of this suspension were transferred into 11 µl susceptibility test media. A standardised LSM broth consisting of a mixture of isosensitest (IST) broth (90% w/v; Oxoid, Basingstoke, UK), MRS broth (10% w/v), adjusted to ph 6.7 (Klare et al., Table 1. Antimicrobials included in the Sensititre GPALL1F and EUST and their range of dilutions used to determine the minimum inhibitory concentration. Antibiotic Type of antibiotic Range of dilutions (μg/ml) EUST GPALL1F Inhibitors of cell-wall synthesis Penicillin penicillin 0.12-2 0.06-8 Ampicillin penicillin - 0.12-8 Oxacillin+2%NaCl penicillin - 0.25-4 Cefoxitin cephalosporin 4-16 6 Vancomycin glycopeptide 1-16 0.25-32 Inhibitors of protein synthesis Chloramphenicol amphenicol 4-64 2-16 Gentamicin aminoglycoside 1-16 2-16 Streptomycin aminoglycoside 4-32 1000 Kanamycin aminoglycoside 4-64 - Quinupristin/dalfopristin streptogramins 1-4 0.5-4 Rifampin rifamycin 0.016-0.5 0.5-4 Tetracycline tetracycline 0.5-16 2-16 Erythromycin macrolide 0.25-8 0.25-4 Tigecycline glycylcycline - 0.03-0.5 Clindamycin lincosamide 0.12-4 0.5-2 Tiamulin pleuromutilin 0.5-4 - Linezolid oxazolidinone 1-8 1-8 Fusidate fusidan 0.5-4 - Mupirocin monoxycarbolic acid 0.5-256 - Inhibitors of DNA synthesis Trimethoprim/sulfamethoxazole trimethoprim/ sulfonamide - 0.5/9.5-4/76 Trimethoprim trimethropin 2-32 - Sulfamethoxazole sulfonamide 64-512 - Levofloxacin fluoroquinolone - 0.25-4 Moxifloxacin fluoroquinolone - 0.25-4 Ciprofloxacine fluoroquinolone 0.25-8 1-2 Nitrofurantoin nitrofuran - 32-64 Inhibitor of cell membrane function Daptomycin lipopeptide - 0.5-4 336 Beneficial Microbes 2(4)

Antibiotic susceptibility of different lactic acid bacteria strains 2007) and SSM consisting of IST broth (90%) and M17 broth (10%), supplemented with lactose (0.5% w/v) (Tosi et al., 2007) were used for the Lactobacillus strains and S. thermophilus, respectively. Microplates containing vacuum-dried antimicrobials were inoculated with 50 μl of the broth suspension. The final bacterial concentration was 1 10 5 to 5 10 5 cfu/ml. All plates were incubated at 37 C for 48 h and the MICs were visually read as recommended by the Clinical and Laboratory Standard Institute (Wayne, PA, USA). The lowest concentration of antibiotic preventing appearance of turbidity was considered to be the MIC. As positive control, a Enterococcus faecalis (ATCC 29212) strain was used. 3. Results and discussion In the present study, five LAB strains were tested for their susceptibility to 27 antimicrobials. The analyses were performed using two antibiotic susceptibility tests. The latter represent a microversion of the classic broth dilution method and can provide qualitative (susceptible or resistant) and quantitative (MIC) results in a dried plate format. Both tests covered the full list of ten antimicrobials that EFSA has set as a basic requirement for the group of LAB. Reference microbiological breakpoints for these antibiotics are presented in Table 2. A microorganism, inhibited at breakpoint level to a specific antimicrobial is defined as susceptible. When the MIC is higher than the breakpoint, it is considered resistant. Since, among lactic acid bacteria, the range of resistance to other antimicrobials Table 2. Distribution of minimum inhibitory concentrations (μg/ml) to antimicrobials for lactic acid bacterial strains using GPALL1F and EUST plates. 1 Antibiotic L. acidophilus LC10 L. lactis LC182 L. bulgaricus LC1 L. helveticus LC3 S. thermophilus LC201 EFSA 2 Sensititre EFSA Sensititre EFSA Sensititre EFSA Sensititre EFSA Sensititre Penicillin 0.25 0.06 0.06 0.06 0.06 Ampicillin 1 0.12 1 0.12 1 0.12 1 0.12 2 0.12 Oxacillin+2%NaCl 0.25 0.25 0.25 0.25 0.25 Cefoxitin 8 0.5 1 8 6 Vancomycin 2 1 2 1 2 1 2 1 4 0.5 Chloramphenicol 4 4 4 4 4 4 4 2 4 4 Gentamicin 16 8 16 >16 16 >16 16 8 32 16 Streptomycin 16 4 16 32 16 4 16 4 64 >32 Kanamycin 16 32 16 64 16 8 16 64 64 >64 Quinupristin/dalfopristin 4 1 4 0.5 4 0.5 4 0.5 4 0.5 Rifampin 4 0.12 0.06 >4 0.03 Tetracycline 4 2 4 0.5 4 0.5 4 1 4 0.5 Erythromycin 1 0.5 1 0.25 1 0.25 1 0.5 2 8 Tigecycline 0.12 0.12 0.12 >0.5 0.06 Clindamycin 1 1 1 0.12 1 0.12 1 1 2 0.25 Tiamulin 4 0.5 0.5 1 4 Linezolid 2 1 2 4 2 Fusidate >4 >4 >4 >4 1 Mupirocin 1 0.5 0.5 2 0.5 Trimethoprim/sulfamethoxazole >4/76 0.5/9.5 0.5/9.5 2/38 4/76 Trimethoprim >32 2 >32 >32 >32 Sulfamethoxazole >512 >512 >512 >512 >512 Levofloxacin >4 >4 >4 >4 4 Moxifloxacin >4 4 4 >4 4 Ciprofloxacine >8 >8 >8 >8 4 Nitrofurantoin 32 32 32 32 32 Daptomycin >4 >4 >4 >4 >4 1 Where the results of two assays differed, the upper minimum inhibitory concentration value is given. 2 Strains with minimum inhibitory concentrations higher than the breakpoints are considered as resistant (EFSA, 2008). Beneficial Microbes 2(4) 337

N. Karapetkov et al. can be broad with no clear breakpoint values, the MIC testing of these antibiotics is not considered relevant or the hazard of acquiring resistance is covered by testing for antimicrobials of the same group (EFSA, 2008). In general, a good correlation was observed between both tests. Strains defined as susceptible or resistant by EUST plates were defined as such by GPALL1F plates. In addition, the different range of concentrations for some antibiotics allows a more accurate determination of the MICs. For some strains different MICs to specific antibiotics between both tests was observed, which never exceeded 1 log 2 of dilution. In these cases, the higher MICs are presented in Table 2. The tested Lactobacillus strains and S. thermophilus did not show any significant differences in their antibioticresistance profile. High susceptibility was detected for most of the cell-wall synthesis inhibitors. Most of the strains displayed MICs to β-lactam antibiotics at the low end of the concentration ranges (Table 2). L. acidophilus LC10 and L. helveticus LC3 only resisted low concentrations of cefoxitin (MIC=8 µg/ml). Some LAB strains of Lactobacillus have been reported to be intrinsically resistant to vancomycin (Ruoff et al., 1988). In contrast, in our study, we found that all strains were inhibited at breakpoint values proposed by FEEDAP for this antibiotic and thus they were characterised as susceptible. Generally, lactobacilli are sensitive to the inhibitors of cell-wall synthesis, such as penicillins, but more resistant to cephalosporins (Ammor et al., 2007). A widespread sensitivity toward penicillins has already been observed in Lactobacillus strains used as probiotics or starter cultures (Belletti et al., 2009; Danielsen and Wind, 2003; Klare et al., 2007). Resistance to ampicillin and penicillin was not found among S. thermophilus or Lactobacillus strains used in yogurt production (Hummel et al., 2007). Species of the L. acidophilus group and L. delbrueckii were characterised as relatively more sensitive to penicillins and vancomycin in comparison with other LAB species (Klare et al., 2007). In contrast, high level of intrinsic resistance to glycopeptides was found in some probiotic lactobacilli such as Lactobacillus rhamnosus, Lactobacillus paracasei and Lactobacillus plantarum (Flòrez et al., 2005; Klare et al., 2007). The MICs of antibiotics affecting the synthesis of proteins showed the greatest variation in dependence on the antimicrobial used (Table 2). The tested Lactobacillus strains were characterised as susceptible toward chloramphenicol, streptomycin, tetracycline, erythromycin, quinupristin/dalfopristin and clindamycin, and resistant to at least one of the aminoglycoside antibiotics. Three of the strains showed growth in the presence of a high concentration of kanamycin. Gentamicin was less active against L. delbrueckii subsp. lactis LC182 and L. delbrueckii subsp. bulgaricus LC1 with MIC 16 μg/ml in comparison with 4 μg/ml for L. acidophilus LC10 and L. helveticus LC3. Only L. delbrueckii subsp. lactis LC182 was resistant to streptomycin. The high resistance of lactobacilli to kanamycin reported by Zhou et al. (2005) was confirmed in this study for L. acidophilus, L. delbrueckii subsp. lactis and L. helveticus. However, no resistance to tetracycline and erythromycin could be observed contrary to the results of Temmerman et al. (2003). The findings of the present study are essentially in accordance with previously reported results of Belleti et al. (2009), where most of the tested L. helveticus and L. delbrueckii subsp. lactis strains were susceptible to tetracycline, erythromycin and clindamycin and resistant to gentamicin. Lactobacilli are reported to be generally resistant to aminoglycosides (gentamicin, kanamycin and streptomycin) and susceptible to other protein synthesis inhibitors (Ammor et al., 2007; Zhou et al., 2005). Intrinsic resistance to aminoglycosides is attributed to the absence of cytochrome-mediated electron transport, enabling antibiotic uptake (Charteris et al., 2001). However, there are some discrepancies in the results obtained in this study regarding the sensitivity of lactobacilli to aminoglycosides and previously reported data from other authors. The L. delbrueckii subsp. lactis and L. delbrueckii subsp. bulgaricus strains gave expected results in terms of resistance to gentamicin and L. delbrueckii subsp. lactis to streptomycin, while for the rest of lactobacilli strains the aminoglycosides were effective inhibitors at levels lower than those set by FEEDAP, qualifying them as susceptible. S. thermophilus LC201 showed resistance to kanamycin, streptomycin and erythromycin and susceptibility toward the rest of the protein synthesis inhibitors. The incidence of resistance to erythromycin and tetracycline between S. thermophilus strains was generally low (Hummel et al., 2007). However, different S. thermophilus strains were previously described to have acquired resistance genes, which are responsible for their resistance to erythromycin, streptomycin and tetracycline (Tosi et al., 2007). Although S. thermophilus is non-pathogenic, it might transfer antibiotic resistance genes to pathogenic bacteria and hence represents a potential clinical risk that needs to be carefully evaluated. In this aspect, S. thermophilus LC201 should be subjected to additional genetic analysis in order to determine the nature of its antibiotic resistance, especially to erythromycin, by studying the presence of transferable resistance genes. Among the other inhibitors of the protein synthesis, the antibiotic linezolid showed antibacterial activity against most of the tested LAB with MIC in the range 2-4 μg/ ml. In addition, all lactobacilli were resistant to fusidate (MIC>4 µg/ml) and so the results reported by Zhou et al. (2005) could be confirmed. Within the genus Lactobacillus and dependent on the species a very different reaction toward fusidic acid was recorded (Klare et al., 2007). 338 Beneficial Microbes 2(4)

Antibiotic susceptibility of different lactic acid bacteria strains Most of the tested strains showed a high level of resistance towards the inhibitors of DNA synthesis (Table 2). Lactobacilli seem to be intrinsically resistant to fluoroquinolones, e.g. ciprofloxacin, levofloxacin by a currently unknown resistance mechanism (Hummel et al., 2007). Resistance to other inhibitors of nucleic acid synthesis such as trimethoprim and sulphonamides has also been reported as an intrinsic feature (Katla et al., 2001). 4. Conclusions The aim of the study was to evaluate LAB resistance to antibiotics, which could enhance their survival in environments containing antimicrobials. Differentiation of the bacterial strains into resistant or susceptible was made possible using the microdilution method. Our studies confirm the hypothesis that determination of the MICs to a relevant range of antimicrobials is a reliable way to predict the behaviour towards antibiotics of different LAB strains with respect to their use as probiotic adjunct cultures. All tested Lactobacillus strains were susceptible or intrinsically resistant to a recommended set of antibiotics. The presence of natural multi-resistance of L. delbrueckii subsp. lactis could be shown, however it is presumed to present a minimal potential for horizontal spread of resistance. As the genes in lactobacilli conferring resistance to specific antimicrobial compounds are probably not associated with mobile genetic elements, the risk of transfer to other organisms can be considered as minimal, thus making them safe for use in different probiotic products for human or animal consumption. 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