This article was downloaded by: [University of Nebraska, Lincoln] On: 01 September 2015, At: 11:57 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: 5 Howick Place, London, SW1P 1WG
British Poultry Science Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cbps20
Antibiotic susceptibility of Lactobacillus strains isolated from domestic geese a
a
a
a
M. Dec , A. Wernicki , A. Puchalski & R. Urban-Chmiel a
Sub-Department of Veterinary Prevention and Avian Diseases, Institute of Biological Bases of Animal Diseases, Faculty of Veterinary Medicine, University of Life Sciences, 20-033 Lublin, Poland Accepted author version posted online: 24 Jun 2015.Published online: 26 Jun 2015.
Click for updates To cite this article: M. Dec, A. Wernicki, A. Puchalski & R. Urban-Chmiel (2015) Antibiotic susceptibility of Lactobacillus strains isolated from domestic geese, British Poultry Science, 56:4, 416-424, DOI: 10.1080/00071668.2015.1058919 To link to this article: http://dx.doi.org/10.1080/00071668.2015.1058919
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
British Poultry Science, 2015 Vol. 56, No. 4, 416–424, http://dx.doi.org/10.1080/00071668.2015.1058919
Antibiotic susceptibility of Lactobacillus strains isolated from domestic geese M. DEC, A. WERNICKI, A. PUCHALSKI
AND
R. URBAN-CHMIEL
Downloaded by [University of Nebraska, Lincoln] at 11:57 01 September 2015
Sub-Department of Veterinary Prevention and Avian Diseases, Institute of Biological Bases of Animal Diseases, Faculty of Veterinary Medicine, University of Life Sciences, 20-033 Lublin, Poland
Abstract 1. The aim of this study was to determine the antibiotic susceptibility of 93 Lactobacillus strains isolated from domestic geese raised on Polish farms. The minimal inhibitory concentration (MIC) of 13 antimicrobial substances was determined by the broth microdilution method. 2. All strains were sensitive to the cell wall inhibitors ampicillin and amoxicillin (MIC ≤ 8 μg/ml). Resistance to inhibitors of protein synthesis and to fluoroquinolone inhibitors of replication was found in 44.1% and 60.2% of isolates, respectively; 26.9% strains were resistant to neomycin (MIC ≥ 64 μg/ml), 23.6% to tetracycline (MIC ≥ 32 μg/ml), 15% to lincomycin (MIC ≥ 64 μg/ml), 18.3% to doxycycline (MIC ≥ 32 μg/ml), 9.7% to tylosin (MIC ≥ 32 μg/ml), 56% to flumequine (MIC ≥ 256 μg/ml) and 22.6% to enrofloxacin (MIC ≥ 64 μg/ml). 3. Bimodal distribution of MICs indicative of acquired resistance and unimodal distribution of the high MIC values indicative of intrinsic resistance were correlated with Lactobacillus species. Eleven (11.8%) strains displayed multiple resistance for at least three classes of antibiotics. 4. Data derived from this study can be used as a basis for reviewing current microbiological breakpoints for categorisation of susceptible and resistant strains of Lactobacillus genus and help to assess the hazards associated with the occurrence of drug resistance among natural intestinal microflora.
INTRODUCTION Lactobacilli are gram-positive, non-spore-forming, catalase-negative, aerotolerant or anaerobic and acid-tolerant rod-shaped bacteria. They belong to the broadly defined group of lactic acid bacteria (LAB) characterised by the formation of lactic acid as the sole or main end product of carbohydrate metabolism (Claesson et al., 2007). At the time of writing (November 2014), the genus Lactobacillus appeared to be one of the most species-rich genera within the Firmicutes phylum, with 196 recognised species (NCBI taxonomy database). They can be found in plants or material of plant origin and in fermented foods, as well as in the oral cavities, gastrointestinal tracts (GIT) and vaginas of humans and animals. Lactobacilli play an important role in the physiology of their host, as they maintain the microbial balance around mucous membranes. They create a hostile environment for pathogenic bacteria, which they
constantly compete with for food and living space. Moreover, they improve digestion and assimilation of nutrients, remove toxic substances and enhance immunity (Lebeer et al., 2008; Dec et al., 2014a). The Lactobacillus species found in the GIT have received particular attention due to their health-promoting properties. They are commonly used as probiotics, which are defined by the FAO/WHO (2001) as live microorganisms that when administered in adequate amounts confer a health benefit on the host. Excessive, often unjustified use of antimicrobial substances in animal production and veterinary medicine has contributed to the development of resistance not only among pathogenic strains but also among the commensal microflora, such as lactobacilli. Antibiotic-resistant strains not only pose a danger to animals, but, as they spread via the food chain, also contribute to problems in humans. Despite attempts to stop the spread of antibiotic resistance, the level of resistant bacteria
Correspondence to: Marta Dec, Sub-Department of Veterinary Prevention and Avian Diseases, Institute of Biological Bases of Animal Diseases, Faculty of Veterinary Medicine, University of Life Sciences, Akademicka 12, 20-033 Lublin, Poland. E-mail: [email protected] Accepted for publication 12 May 2015.
© 2015 British Poultry Science Ltd
Downloaded by [University of Nebraska, Lincoln] at 11:57 01 September 2015
ANTIBIOTIC SUSCEPTIBILITY OF GOOSE LACTOBACILLI
is on the rise and the hypothesis that the GIT acts as a reservoir of antibiotic resistance genes is widely accepted (Schjørring and Krogfelt, 2011; Hu et al., 2013). Several studies have confirmed the presence of genes encoding antibiotic resistance in Lactobacillus (Darsanaki et al., 2013). Some of these genes are localised in mobile genetic elements, such as plasmids, insertion sequences (IS), transposons and introns, which could be transferred horizontally between lactobacilli and other species of the intestinal microbiota, significantly increasing their pathogenic potential (Morelli et al., 2004; Devirgiliis et al., 2013). The determination of antibiotic susceptibility of a bacterial strain is an important prerequisite to recognising it as safe for human and animal consumption. Natural antibiotic resistance, generally linked to the presence of low-affinity targets, the absence of targets or decreased uptake, accumulation or efflux of the drug, is desirable in the case of probiotics as it enables them to survive in the GIT when they are co-administered with antibiotics. However, microorganisms intended for use as probiotics should not carry transmissible antibiotic resistance genes acquired by the bacteria via acquisition of exogenous DNA. Ingestion of bacteria carrying such genes is undesirable, as horizontal gene transfer to recipient bacteria in the gut could lead to the development of new antibiotic-resistant pathogens and subsequent failure of antibiotic treatment (Zhou et al., 2005). According to the EFSA’s FEEDAP Panel (European Food Safety Authority Panel on Additives and Products or Substances used in Animal Feed) (EFSA, 2008), strains carrying acquired resistance are unacceptable for use as animal feed additives unless it can be shown that the genetic basis of the acquired resistance is a chromosomal mutation that presents a low risk of horizontal dissemination. In phenotypic methods, FEEDAP (2008) requires determination of the minimal inhibitory concentrations (MICs) of the most relevant antibiotics in order to distinguish susceptible and resistant strains. When resistance to an antimicrobial is inherent for a bacterial species, it is generally referred to as “intrinsic resistance” (natural resistance), and is typical of all strains of that species. In contrast, when a strain of a typically susceptible species is resistant to a given antimicrobial drug, this is considered to be “acquired resistance”. The aim of this study was to determine the antibiotic susceptibility of cloacal Lactobacillus strains isolated from domestic geese. There are few data available in the current literature on the antibiotic sensitivity of poultry lactobacilli, and we have found no publication describing this phenomenon in LAB originating in geese. A total of 93 Lactobacillus isolates of various species
417
were collected from different locations and environments in order to achieve the most diverse possible set of strains. The bacteria were tested for susceptibility in order to determine wild-type MIC distributions. The results obtained will be helpful in assessing the potential risk of poultry lactobacilli acting as a pool of resistance genes. Furthermore, the study is one step in the selection of probiotic strains, which will eliminate from further study isolates considered to have acquired resistance.
MATERIALS AND METHODS Tested bacteria A total of 93 Lactobacillus strains isolated from fresh faeces or cloacae of 50 White Koluda geese from 14 poultry farms located in south-east Poland were included in this study. They belonged to the species L. salivarius (n = 35), L. johnsonii (n = 17), L. ingluviei (n = 10), L. agilis (n = 8), L. reuteri (n = 6), L. plantarum (n = 5), L. paracasei (n = 5), L. crispatus (n = 3), L. amylovorus (n = 2) and L. oris (n = 2). The identification of isolates to species level using MALDI-TOF mass spectrometry and analysis of 16 S-23 S regions in rDNA were previously described by Dec et al. (2014b). The strains were maintained in deMan Rogosa Sharpe broth (MRS, BTL, Poland) containing 20% glycerol at −80°C. Prior to antimicrobial susceptibility testing, cultures were streaked on LSM agar (Klare et al., 2005) consisting of 90% IsoSensitest broth (Oxoid) and 10% MRS broth and incubated for about 20 h at 37°C in 5% CO2. Determination of minimal inhibitory concentration Antibiotic susceptibility of all bacterial isolates was determined by the broth microdilution procedure (Mayrhofer et al., 2010) using the LAB susceptibility test medium (LSM) recommended by the International Organization of Standardization (ISO)/International Dairy Federation (IDF) (ISO 10932/IDF 223 standard). Briefly, 96-well flat-bottomed microtitre plates (Medlab, Raszyn, Poland) were used to determine the MICs of 9 antibiotic and chemotherapeutic agents: ampicillin and amoxicillin as inhibitors of cell wall synthesis; tetracycline, doxycycline, neomycin sulphate, tylosin and lincomycin as inhibitors of protein synthesis; and enrofloxacin and flumequine as inhibitors of DNA replication. The antibiotic substances tested, except for ampicillin and tetracycline, are currently widely used in the treatment of poultry disease in Poland. All antimicrobial agent powders were obtained from Sigma-Aldrich (Poznań, Poland). Enrofloxacin (Enrocin, 50 mg/ml) was
418
M. DEC ET AL.
Table 1.
Antimicrobial substances and their dilutions used to determine MICs Antimicrobial substance
Mechanism of action
Group of antimicrobial agent
Diluent
Inhibitors of cell wall synthesis
Ampicillin Amoxycilin
β-Lactams
BR II1 0.1 M NH4OH
Inhibitors of protein synthesis
Tetracycline Doxycycline Neomycin sulphate Lincomycin Tylosin
Tetracyclines
0.1
Aminoglycosides Lincosamides Macrolides
BR II1 H2O
Fluoroquinolones
– 0.1 M NH4OH
Inhibitors of DNA synthesis
Enrofloxacin Flumequine
M
HCl
Range of concentration (µg/ml) 0.125–64 0.125–64 0.5–512 0.25–256 1–512 0.25–512 0.06–256 0.5–512 4–2048
1
Downloaded by [University of Nebraska, Lincoln] at 11:57 01 September 2015
BR II buffer: 16.73 g K2HPO4 and 0.523 g KH2PO4 diluted in 1000 ml of distilled H2O, pH 7.9.
purchased from Vet-Agro (Lublin, Poland). Each of the antibiotic powders was carefully weighed, and dissolved in appropriate diluents and filtersterilised before being added to LSM. For each antimicrobial substance, serial dilutions of the antibiotics were prepared (Table 1). Inocula were prepared by suspending strains of bacteria grown on the LSM in 5 ml 0.9% NaCl so that the optical density (OD) of the suspension at 600 nm was 0.5. Subsequently, adjusted inocula were diluted 1:500 in LSM broth. Microdilution plates were inoculated with 50 μl of a 1:500diluted inoculum and 50 μl of the appropriate antibiotic concentration (stock solution dissolved previously in LSM), resulting in the final range of concentrations shown in Table 1 (ISO standard 10932/IDF 223). Wells containing the bacterial inocula without the addition of an antibiotic or chemotherapeutic agent were used as positive controls and LSM was used as a negative control. After the plates were incubated at 37°C in 5% CO2 for 48 h, MIC values were read as the lowest concentration of an antimicrobial agent at which visible growth was inhibited. The experiments were done in two replications to verify the reproducibility of the method under the conditions described.
RESULTS The distribution of MIC values for inhibitors of cell wall, protein and DNA synthesis is presented in Table 1. For all tested strains the range of MIC for ampicillin and amoxicillin was relatively narrow: 0.25–8 μg/ml. The MICs for the other antimicrobial substances varied widely and were as follows: for tetracycline
British Poultry Science Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cbps20
Antibiotic susceptibility of Lactobacillus strains isolated from domestic geese a
a
a
a
M. Dec , A. Wernicki , A. Puchalski & R. Urban-Chmiel a
Sub-Department of Veterinary Prevention and Avian Diseases, Institute of Biological Bases of Animal Diseases, Faculty of Veterinary Medicine, University of Life Sciences, 20-033 Lublin, Poland Accepted author version posted online: 24 Jun 2015.Published online: 26 Jun 2015.
Click for updates To cite this article: M. Dec, A. Wernicki, A. Puchalski & R. Urban-Chmiel (2015) Antibiotic susceptibility of Lactobacillus strains isolated from domestic geese, British Poultry Science, 56:4, 416-424, DOI: 10.1080/00071668.2015.1058919 To link to this article: http://dx.doi.org/10.1080/00071668.2015.1058919
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
British Poultry Science, 2015 Vol. 56, No. 4, 416–424, http://dx.doi.org/10.1080/00071668.2015.1058919
Antibiotic susceptibility of Lactobacillus strains isolated from domestic geese M. DEC, A. WERNICKI, A. PUCHALSKI
AND
R. URBAN-CHMIEL
Downloaded by [University of Nebraska, Lincoln] at 11:57 01 September 2015
Sub-Department of Veterinary Prevention and Avian Diseases, Institute of Biological Bases of Animal Diseases, Faculty of Veterinary Medicine, University of Life Sciences, 20-033 Lublin, Poland
Abstract 1. The aim of this study was to determine the antibiotic susceptibility of 93 Lactobacillus strains isolated from domestic geese raised on Polish farms. The minimal inhibitory concentration (MIC) of 13 antimicrobial substances was determined by the broth microdilution method. 2. All strains were sensitive to the cell wall inhibitors ampicillin and amoxicillin (MIC ≤ 8 μg/ml). Resistance to inhibitors of protein synthesis and to fluoroquinolone inhibitors of replication was found in 44.1% and 60.2% of isolates, respectively; 26.9% strains were resistant to neomycin (MIC ≥ 64 μg/ml), 23.6% to tetracycline (MIC ≥ 32 μg/ml), 15% to lincomycin (MIC ≥ 64 μg/ml), 18.3% to doxycycline (MIC ≥ 32 μg/ml), 9.7% to tylosin (MIC ≥ 32 μg/ml), 56% to flumequine (MIC ≥ 256 μg/ml) and 22.6% to enrofloxacin (MIC ≥ 64 μg/ml). 3. Bimodal distribution of MICs indicative of acquired resistance and unimodal distribution of the high MIC values indicative of intrinsic resistance were correlated with Lactobacillus species. Eleven (11.8%) strains displayed multiple resistance for at least three classes of antibiotics. 4. Data derived from this study can be used as a basis for reviewing current microbiological breakpoints for categorisation of susceptible and resistant strains of Lactobacillus genus and help to assess the hazards associated with the occurrence of drug resistance among natural intestinal microflora.
INTRODUCTION Lactobacilli are gram-positive, non-spore-forming, catalase-negative, aerotolerant or anaerobic and acid-tolerant rod-shaped bacteria. They belong to the broadly defined group of lactic acid bacteria (LAB) characterised by the formation of lactic acid as the sole or main end product of carbohydrate metabolism (Claesson et al., 2007). At the time of writing (November 2014), the genus Lactobacillus appeared to be one of the most species-rich genera within the Firmicutes phylum, with 196 recognised species (NCBI taxonomy database). They can be found in plants or material of plant origin and in fermented foods, as well as in the oral cavities, gastrointestinal tracts (GIT) and vaginas of humans and animals. Lactobacilli play an important role in the physiology of their host, as they maintain the microbial balance around mucous membranes. They create a hostile environment for pathogenic bacteria, which they
constantly compete with for food and living space. Moreover, they improve digestion and assimilation of nutrients, remove toxic substances and enhance immunity (Lebeer et al., 2008; Dec et al., 2014a). The Lactobacillus species found in the GIT have received particular attention due to their health-promoting properties. They are commonly used as probiotics, which are defined by the FAO/WHO (2001) as live microorganisms that when administered in adequate amounts confer a health benefit on the host. Excessive, often unjustified use of antimicrobial substances in animal production and veterinary medicine has contributed to the development of resistance not only among pathogenic strains but also among the commensal microflora, such as lactobacilli. Antibiotic-resistant strains not only pose a danger to animals, but, as they spread via the food chain, also contribute to problems in humans. Despite attempts to stop the spread of antibiotic resistance, the level of resistant bacteria
Correspondence to: Marta Dec, Sub-Department of Veterinary Prevention and Avian Diseases, Institute of Biological Bases of Animal Diseases, Faculty of Veterinary Medicine, University of Life Sciences, Akademicka 12, 20-033 Lublin, Poland. E-mail: [email protected] Accepted for publication 12 May 2015.
© 2015 British Poultry Science Ltd
Downloaded by [University of Nebraska, Lincoln] at 11:57 01 September 2015
ANTIBIOTIC SUSCEPTIBILITY OF GOOSE LACTOBACILLI
is on the rise and the hypothesis that the GIT acts as a reservoir of antibiotic resistance genes is widely accepted (Schjørring and Krogfelt, 2011; Hu et al., 2013). Several studies have confirmed the presence of genes encoding antibiotic resistance in Lactobacillus (Darsanaki et al., 2013). Some of these genes are localised in mobile genetic elements, such as plasmids, insertion sequences (IS), transposons and introns, which could be transferred horizontally between lactobacilli and other species of the intestinal microbiota, significantly increasing their pathogenic potential (Morelli et al., 2004; Devirgiliis et al., 2013). The determination of antibiotic susceptibility of a bacterial strain is an important prerequisite to recognising it as safe for human and animal consumption. Natural antibiotic resistance, generally linked to the presence of low-affinity targets, the absence of targets or decreased uptake, accumulation or efflux of the drug, is desirable in the case of probiotics as it enables them to survive in the GIT when they are co-administered with antibiotics. However, microorganisms intended for use as probiotics should not carry transmissible antibiotic resistance genes acquired by the bacteria via acquisition of exogenous DNA. Ingestion of bacteria carrying such genes is undesirable, as horizontal gene transfer to recipient bacteria in the gut could lead to the development of new antibiotic-resistant pathogens and subsequent failure of antibiotic treatment (Zhou et al., 2005). According to the EFSA’s FEEDAP Panel (European Food Safety Authority Panel on Additives and Products or Substances used in Animal Feed) (EFSA, 2008), strains carrying acquired resistance are unacceptable for use as animal feed additives unless it can be shown that the genetic basis of the acquired resistance is a chromosomal mutation that presents a low risk of horizontal dissemination. In phenotypic methods, FEEDAP (2008) requires determination of the minimal inhibitory concentrations (MICs) of the most relevant antibiotics in order to distinguish susceptible and resistant strains. When resistance to an antimicrobial is inherent for a bacterial species, it is generally referred to as “intrinsic resistance” (natural resistance), and is typical of all strains of that species. In contrast, when a strain of a typically susceptible species is resistant to a given antimicrobial drug, this is considered to be “acquired resistance”. The aim of this study was to determine the antibiotic susceptibility of cloacal Lactobacillus strains isolated from domestic geese. There are few data available in the current literature on the antibiotic sensitivity of poultry lactobacilli, and we have found no publication describing this phenomenon in LAB originating in geese. A total of 93 Lactobacillus isolates of various species
417
were collected from different locations and environments in order to achieve the most diverse possible set of strains. The bacteria were tested for susceptibility in order to determine wild-type MIC distributions. The results obtained will be helpful in assessing the potential risk of poultry lactobacilli acting as a pool of resistance genes. Furthermore, the study is one step in the selection of probiotic strains, which will eliminate from further study isolates considered to have acquired resistance.
MATERIALS AND METHODS Tested bacteria A total of 93 Lactobacillus strains isolated from fresh faeces or cloacae of 50 White Koluda geese from 14 poultry farms located in south-east Poland were included in this study. They belonged to the species L. salivarius (n = 35), L. johnsonii (n = 17), L. ingluviei (n = 10), L. agilis (n = 8), L. reuteri (n = 6), L. plantarum (n = 5), L. paracasei (n = 5), L. crispatus (n = 3), L. amylovorus (n = 2) and L. oris (n = 2). The identification of isolates to species level using MALDI-TOF mass spectrometry and analysis of 16 S-23 S regions in rDNA were previously described by Dec et al. (2014b). The strains were maintained in deMan Rogosa Sharpe broth (MRS, BTL, Poland) containing 20% glycerol at −80°C. Prior to antimicrobial susceptibility testing, cultures were streaked on LSM agar (Klare et al., 2005) consisting of 90% IsoSensitest broth (Oxoid) and 10% MRS broth and incubated for about 20 h at 37°C in 5% CO2. Determination of minimal inhibitory concentration Antibiotic susceptibility of all bacterial isolates was determined by the broth microdilution procedure (Mayrhofer et al., 2010) using the LAB susceptibility test medium (LSM) recommended by the International Organization of Standardization (ISO)/International Dairy Federation (IDF) (ISO 10932/IDF 223 standard). Briefly, 96-well flat-bottomed microtitre plates (Medlab, Raszyn, Poland) were used to determine the MICs of 9 antibiotic and chemotherapeutic agents: ampicillin and amoxicillin as inhibitors of cell wall synthesis; tetracycline, doxycycline, neomycin sulphate, tylosin and lincomycin as inhibitors of protein synthesis; and enrofloxacin and flumequine as inhibitors of DNA replication. The antibiotic substances tested, except for ampicillin and tetracycline, are currently widely used in the treatment of poultry disease in Poland. All antimicrobial agent powders were obtained from Sigma-Aldrich (Poznań, Poland). Enrofloxacin (Enrocin, 50 mg/ml) was
418
M. DEC ET AL.
Table 1.
Antimicrobial substances and their dilutions used to determine MICs Antimicrobial substance
Mechanism of action
Group of antimicrobial agent
Diluent
Inhibitors of cell wall synthesis
Ampicillin Amoxycilin
β-Lactams
BR II1 0.1 M NH4OH
Inhibitors of protein synthesis
Tetracycline Doxycycline Neomycin sulphate Lincomycin Tylosin
Tetracyclines
0.1
Aminoglycosides Lincosamides Macrolides
BR II1 H2O
Fluoroquinolones
– 0.1 M NH4OH
Inhibitors of DNA synthesis
Enrofloxacin Flumequine
M
HCl
Range of concentration (µg/ml) 0.125–64 0.125–64 0.5–512 0.25–256 1–512 0.25–512 0.06–256 0.5–512 4–2048
1
Downloaded by [University of Nebraska, Lincoln] at 11:57 01 September 2015
BR II buffer: 16.73 g K2HPO4 and 0.523 g KH2PO4 diluted in 1000 ml of distilled H2O, pH 7.9.
purchased from Vet-Agro (Lublin, Poland). Each of the antibiotic powders was carefully weighed, and dissolved in appropriate diluents and filtersterilised before being added to LSM. For each antimicrobial substance, serial dilutions of the antibiotics were prepared (Table 1). Inocula were prepared by suspending strains of bacteria grown on the LSM in 5 ml 0.9% NaCl so that the optical density (OD) of the suspension at 600 nm was 0.5. Subsequently, adjusted inocula were diluted 1:500 in LSM broth. Microdilution plates were inoculated with 50 μl of a 1:500diluted inoculum and 50 μl of the appropriate antibiotic concentration (stock solution dissolved previously in LSM), resulting in the final range of concentrations shown in Table 1 (ISO standard 10932/IDF 223). Wells containing the bacterial inocula without the addition of an antibiotic or chemotherapeutic agent were used as positive controls and LSM was used as a negative control. After the plates were incubated at 37°C in 5% CO2 for 48 h, MIC values were read as the lowest concentration of an antimicrobial agent at which visible growth was inhibited. The experiments were done in two replications to verify the reproducibility of the method under the conditions described.
RESULTS The distribution of MIC values for inhibitors of cell wall, protein and DNA synthesis is presented in Table 1. For all tested strains the range of MIC for ampicillin and amoxicillin was relatively narrow: 0.25–8 μg/ml. The MICs for the other antimicrobial substances varied widely and were as follows: for tetracycline
