Characterisation and transfer of antibiotic resistance genes from enterococci isolated from food

The genetic determinants responsible for the resistances against the antibiotics tetracycline [tet(M), tet(O), tet(S), tet(K) and tet(L)], erythromycin (ermA,B,C; mefA,E; msrA/B; and ereA,B) and chloramphenicol (cat) of 38 antibiotic-resistant Enterococcus faecium and Enterococcus faecalis strains from food were characterised. In addition, the transferability of resistance genes was also assessed using filter mating assays. The tet(L) determinant was the most commonly detected among tetracycline-resistant enterococci (94% of the strains), followed by the tet(M) gene, which occurred in 63.0% of the strains. Tet(K) occurred in 56.0% of the resistant strains, while genes for tet(O) and tet(S) could not be detected. The integrase gene of the Tn916-1545 family of transposons was present in 81.3% of the tetracycline resistant strains, indicating that resistance genes might be transferable by transposons. All chloramphenicol-resistant strains carried a cat gene. 81.8% of the erythromycin-resistant strains carried the ermB gene. Two (9.5%) of the 21 erythromycin-resistant strains, which did not contain ermA,B,C, ereA,B and mphA genes harboured the msrC gene encoding an erythromycin efflux pump, which was confirmed by sequencing the PCR amplicon. In addition, all E. faecium strains contained the msrC gene, but none of the E. faecalis strains. Transfer of the genetic determinants for antibiotic resistance could only be demonstrated in one filter mating experiment, where both the tet(M) and tet(L) genes were transferred from E. faecalis FAIR-E 315 to the E. faecalis OG1X recipient strain. Our results show the presence of various types of resistance genes as well as transposon integrase genes associated with transferable resistances in enterococci, indicating a potential for gene transfer in the food environment.     

Identification and characterization of antibiotic resistance genes in Lactobacillus reuteri and Lactobacillus plantarum

Aims:  The study aimed to identify the resistance genes mediating atypical minimum inhibitory concentrations (MICs) for tetracycline, erythromycin, clindamycin and chloramphenicol within two sets of representative strains of the species Lactobacillus reuteri and Lactobacillus plantarum and to characterize identified genes by means of gene location and sequencing of flanking regions.
Methods and Results:  A tet (W) gene was found in 24 of the 28 Lact. reuteri strains with atypical MIC for tetracycline, whereas four of the six strains with atypical MIC for erythromycin were positive for erm (B) and one strain each was positive for erm (C) and erm (T). The two Lact. plantarum strains with atypical MIC for tetracycline harboured a plasmid-encoded tet (M) gene. The majority of the tet (W)-positive Lact. reuteri strains and all erm -positive Lact. reuteri strains carried the genes on plasmids, as determined by Southern blot and a real-time PCR method developed in this study.
Conclusions:  Most of the antibiotic-resistant strains of Lact. reuteri and Lact. plantarum harboured known plasmid-encoded resistance genes. Examples of putative transfer machineries adjacent to both plasmid- and chromosome-located resistance genes were also demonstrated.
Significance and Impact of the Study:  These data provide some of the knowledge required for assessing the possible risk of using Lact. reuteri and Lact. plantarum strains carrying antibiotic resistance genes as starter cultures and probiotics.     

Plasmid profiling and curing of Lactobacillus strains isolated from the gastrointestinal tract of chicken

In this study, we assessed the susceptibility of 12 Lactobacillus strains, all of which had been isolated from the gastrointestinal tracts of chicken, to three antibiotics (chloramphenicol, erythromycin and tetracycline) used commonly as selective markers in transformation studies of lactic acid bacteria. Among these strains, 17%, 58%, and 25% were found to exhibit a high degree of resistance to 200 microg/ml of tetracycline, erythromycin, and chloramphenicol, respectively. Seven of the 12 Lactobacillus strains exhibiting resistance to at least 50 microg/ml of chloramphenicol or erythromycin, and five strains exhibiting resistance to at least 50 microg/ml of tetracycline, were subsequently subjected to plasmid curing with chemical curing agents, such as novobiocin, acriflavin, SDS, and ethidium bromide. In no cases did the antibiotic resistance of these strains prove to be curable, with the exception of the erythromycin resistance exhibited by five Lactobacillus strains (L. acidophilus I16 and I26, L. fermentum I24 and C17, and L. brevis C10). Analysis of the plasmid profiles of these five cured derivatives revealed that all of the derivatives, except for L. acidophilus I16, possessed profiles similar to those of wild-type strains. The curing of L. acidophilus I16 was accompanied by the loss of 4.4 kb, 6.1 kb, and 11.5 kb plasmids.     

Antimicrobial susceptibility of lactic acid bacteria isolated from a cheese environment

In the production of the Spanish traditional blue-veined Cabrales cheese, lactic acid bacteria strains free of antibiotic resistance that have a transferrable capacity are necessary as components of a specific starter. To select for these bacteria, the minimum inhibitory concentration (MIC) of 12 antibiotics and 2 mixtures (containing beta-lactamase inhibitor and penicillin) were determined by microbroth and agar dilution techniques in 146 strains belonging to the genera Lactococcus, Enterococcus, Lactobacillus, and Leuconostoc. The antibiotic-resistance profiles of Lactococcus and Enterococcus species were different from those of Lactobacillus and Leuconostoc, but clear genus- or species-associated patterns were not observed. Cefoxitin and metronidazole were not effective against bacteria of these genera. The MICs of beta-lactam antibiotics for lactobacilli and leuconostoc isolates were higher than those for lactococci and enterococci, but no strain was clinically resistant. All lactobacilli and leuconostoc isolates were resistant to high levels of vancomycin, a type of resistance not seen among the tested members of the genera Lactococcus and Enterococcus. The majority of the observed resistance appeared to be either intrinsic or nonspecific, although some strains of Lactococcus lactis, Enterococcus spp., and Lactobacillus spp. were resistant to antibiotics, such as chloramphenicol, erythromycin, clindamycin, or tetracycline.     

Antibiotic susceptibility patterns and resistance genes of starter cultures and probiotic bacteria used in food

A survey of starter and probiotic cultures was carried out to determine the current antibiotic resistance situation in microbial food additives in Switzerland. Two hundred isolates from 90 different sources were typed by molecular and other methods to belong to the genera Lactobacillus (74 samples), Staphylococcus (33 samples), Bifidobacterium (6 samples), Pediococcus (5 samples), or were categorized as lactococci or streptococci (82 samples). They were screened for phenotypic resistances to 20 antibiotics by the disk diffusion method. Twenty-seven isolates exhibiting resistances that are not an intrinsic feature of the respective genera were further analyzed by microarray hybridization as a tool to trace back phenotypic resistances to specific genetic determinants. Their presence was finally verified by PCR amplification or Southern hybridization. These studies resulted in the detection of the tetracycline resistance gene tet(K) in 5 Staphylococcus isolates used as meat starter cultures, the tetracycline resistance gene tet(W) in the probiotic cultures Bifidobacterium lactis DSM 10140 and Lactobacillus reuteri SD 2112 (residing on a plasmid), and the lincosamide resistance gene lnu(A) (formerly linA) in L. reuteri SD 2112.     

Evidence for extensive resistance gene transfer among Bacteroides spp. and among Bacteroides and other genera in the human colon

Transfer of antibiotic resistance genes by conjugation is thought to play an important role in the spread of resistance. Yet virtually no information is available about the extent to which such horizontal transfers occur in natural settings. In this paper, we show that conjugal gene transfer has made a major contribution to increased antibiotic resistance in Bacteroides species, a numerically predominant group of human colonic bacteria. Over the past 3 decades, carriage of the tetracycline resistance gene, tetQ, has increased from about 30% to more than 80% of strains. Alleles of tetQ in different Bacteroides species, with one exception, were 96 to 100% identical at the DNA sequence level, as expected if horizontal gene transfer was responsible for their spread. Southern blot analyses showed further that transfer of tetQ was mediated by a conjugative transposon (CTn) of the CTnDOT type. Carriage of two erythromycin resistance genes, ermF and ermG, rose from <2 to 23% and accounted for about 70% of the total erythromycin resistances observed. Carriage of tetQ and the erm genes was the same in isolates taken from healthy people with no recent history of antibiotic use as in isolates obtained from patients with Bacteroides infections. This finding indicates that resistance transfer is occurring in the community and not just in clinical environments. The high percentage of strains that are carrying these resistance genes in people who are not taking antibiotics is consistent with the hypothesis that once acquired, these resistance genes are stably maintained in the absence of antibiotic selection. Six recently isolated strains carried ermB genes. Two were identical to erm(B)-P from Clostridium perfringens, and the other four had only one to three mismatches. The nine strains with ermG genes had DNA sequences that were more than 99% identical to the ermG of Bacillus sphaericus. Evidently, there is a genetic conduit open between gram-positive bacteria, including bacteria that only pass through the human colon, and the gram-negative Bacteroides species. Our results support the hypothesis that extensive gene transfer occurs among bacteria in the human colon, both within the genus Bacteroides and among Bacteroides species and gram-positive bacteria.     

Antimicrobial susceptibility of some streptococci strains of anginosus group isolated from oral and maxillofacial infections

Streptococci strains of the anginosus group isolated from various oral and maxillofacial infections (OMF) were screened for their susceptibility to the following antimicrobial agents: benzylpenicillin, ampicillin, oxacillin, cephalothin, ceftazidime, cefotaxime, cefuroxime, erythromycin, clindamycin, tetracycline, chloramphenicol, vancomycin and trimethoprime-sulphamethoxazole. The isolates were susceptable to: clindamycin, chloramphenicol, vancomycin and all beta-lactam antibiotics, except ceftazidime to which 54.5% of the strains showed intermediate susceptibility. Intermediate susceptibility to tetracycline was found in 11.3% of the strains, whereas resistance to the same antibiotic was demonstrated in 61.4%. Resistance to erythromycin and trimethoprime-sulphamethoxazole was of 2.3% for both. In conclusion, penicillin is the drug of choice in infections caused by streptococci of the anginosus group.     

Antibiotic Susceptibility Profiles of Dairy Leuconostoc,Analysis of the Genetic Basis of Atypical Resistances and Transfer of Genes In Vitro and in a Food Matrix

In spite of a global concern on the transfer of antibiotic resistances (AR) via the food chain, limited information exists on this issue in species of Leuconostoc and Weissella, adjunct cultures used as aroma producers in fermented foods. In this work, the minimum inhibitory concentration was determined for 16 antibiotics in 34 strains of dairy origin, belonging to Leuconostoc mesenteroides (18), Leuconostoc citreum (11), Leuconostoc lactis (2), Weissella hellenica (2), and Leuconostoc carnosum (1). Atypical resistances were found for kanamycin (17 strains), tetracycline and chloramphenicol (two strains each), and erythromycin, clindamycin, virginiamycin, ciprofloxacin, and rifampicin (one strain each). Surprisingly, L. mesenteroides subsp. mesenteroides LbE16, showed resistance to four antibiotics, kanamycin, streptomycin, tetracycline and virginiamycin. PCR analysis identified tet(S) as responsible for tetracycline resistance in LbE16, but no gene was detected in a second tetracycline-resistant strain, L. mesenteroides subsp. cremoris LbT16. In Leuconostoc mesenteroides subsp. dextranicum LbE15, erythromycin and clindamycin resistant, an erm(B) gene was amplified. Hybridization experiments proved erm(B) and tet(S) to be associated to a plasmid of ≈35 kbp and to the chromosome of LbE15 and LbE16, respectively. The complete genome sequence of LbE15 and LbE16 was used to get further insights on the makeup and genetic organization of AR genes. Genome analysis confirmed the presence and location of erm(B) and tet(S), but genes providing tetracycline resistance in LbT16 were again not identified. In the genome of the multi-resistant strain LbE16, genes that might be involved in aminoglycoside (aadE, aphA-3, sat4) and virginiamycin [vat(E)] resistance were further found. The erm(B) gene but not tet(S) was transferred from Leuconostoc to Enterococcus faecalis both under laboratory conditions and in cheese. This study contributes to the characterization of AR in the Leuconostoc-Weissella group, provides evidence of the genetic basis of atypical resistances, and demonstrates the inter-species transfer of erythromycin resistance.     

Characterization and Transfer of Antibiotic Resistance in Lactic Acid Bacteria from Fermented Food Products

The study provides phenotypic and molecular analyses of the antibiotic resistance in lactic acid bacteria (LAB) from fermented foods in Xi'an, China. LAB strains (n = 84) belonging to 16 species of Lactobacillus (n = 73), and Streptococcus thermophilus (n = 11) were isolated and identified by sequencing their 16S rRNA gene. All strains were susceptible to ampicillin, bacitracin, and cefsulodin, and intrinsically resistant to nalidixic acid, kanamycin, and vancomycin (except L. bulgaricus, L. acidophilus, and S. thermophilus, which were susceptible to vancomycin). Some strains had acquired resistance for penicillin (n = 2), erythromycin (n = 9), clindamycin (n = 5), and tetracycline (n = 14), while resistance to gentamycin, ciprofloxacin, streptomycin, and chloramphenicol was species dependent. Minimum inhibitory concentrations presented in this study will help to review microbiological breakpoints for some of the species of Lactobacillus. The erm(B) gene was detected from two strains of each of L. fermentum and L. vaginalis, and one strain of each of L. plantarum, L. salivarius, L. acidophilus, L. animalis, and S. thermophilus. The tet genes were identified from 12 strains of lactobacilli from traditional foods. This is the first time, the authors identified tet(S) gene from L. brevis and L. kefiri. The erm(B) gene from L. fermentum NWL24 and L. salivarius NWL33, and tet(M) gene from L. plantarum NWL22 and L. brevis NWL59 were successfully transferred to Enterococcus faecalis 181 by filter mating. It was concluded that acquired antibiotic resistance is well dispersed in fermented food products in Xi'an, China and its transferability to other genera should be monitored closely.     

Removal of antibiotic resistance gene-carrying plasmids from Lactobacillus reuteri ATCC 55730 and characterization of the resulting daughter strain, L. reuteri DSM 17938

The spread of antibiotic resistance in pathogens is primarily a consequence of the indiscriminate use of antibiotics, but there is concern that food-borne lactic acid bacteria may act as reservoirs of antibiotic resistance genes when distributed in large doses to the gastrointestinal tract. Lactobacillus reuteri ATCC 55730 is a commercially available probiotic strain which has been found to harbor potentially transferable resistance genes. The aims of this study were to define the location and nature of beta-lactam, tetracycline, and lincosamide resistance determinants and, if they were found to be acquired, attempt to remove them from the strain by methods that do not genetically modify the organism before subsequently testing whether the probiotic characteristics were retained. No known beta-lactam resistance genes was found, but penicillin-binding proteins from ATCC 55730, two additional resistant strains, and three sensitive strains of L. reuteri were sequenced and comparatively analyzed. The beta-lactam resistance in ATCC 55730 is probably caused by a number of alterations in the corresponding genes and can be regarded as not transferable. The strain was found to harbor two plasmids carrying tet(W) tetracycline and lnu(A) lincosamide resistance genes, respectively. A new daughter strain, L. reuteri DSM 17938, was derived from ATCC 55730 by removal of the two plasmids, and it was shown to have lost the resistances associated with them. Direct comparison of the parent and daughter strains for a series of in vitro properties and in a human clinical trial confirmed the retained probiotic properties of the daughter strain.     

Molecular characterization, technological properties and safety aspects of enterococci from 'Hussuwa', an African fermented sorghum product

AIMS: To identify enterococci from Hussuwa, a Sudanese fermented sorghum product, and determine their technological properties and safety for possible inclusion in a starter culture preparation. METHODS AND RESULTS: Twenty-two Enterococcus isolates from Hussuwa were identified as Enterococcus faecium by using phenotypic and genotypic tests such as 16S rDNA gene sequencing, RAPD-PCR and restriction fragment length polymorphism of the 16S/23S intergenic spacer region fingerprinting. Genotyping revealed that strains were not clonally related and exhibited a considerable degree of genomic diversity. Some strains possessed useful technological properties such as production of bacteriocins and H2O2 or utilization of raffinose and stachyose. None produced alpha-amylase or tannase. A safety investigation revealed that all strains were susceptible to the antibiotics ampicillin, gentamicin, chloramphenicol, tetracycline and streptomycin, but some were resistant to ciprofloxacin, erythromycin, penicillin and vancomycin. Production of biogenic amines or presence of genes encoding virulence determinants occurred in some strains. CONCLUSIONS: Enterococcus faecium strains are associated with fermentation of Sudanese Hussuwa. Some strains exhibited useful technological properties such as production of antimicrobial agents and fermentation of indigestible sugars, which may aid in stabilizing and improving the digestibility of the product respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Enterococci were shown to play a role in the fermentation of African foods. While beneficial properties of these bacteria are indicated, their presence in this food may also imply a hygienic risk as a result of antimicrobial resistances or presence of virulence determinants.     

The sensitivity to 12 antibiotics of 125 strains of Streptococcus group B isolated in a maternity home

Sensitivity of 125 strains of group B streptococci isolated from newborns, their mothers and personnel in a maternity home was studied with respect to 12 antibiotics: benzylpenicillin, ampicillin, methicillin, cephalotin, erythromycin, lincomycin, levomycetin (chloramphenicol), oxacillin, tetracycline, streptomycin, gentamicin and ristomycin. The method of serial dilutions in a solid medium was applied. All the strains were sensitive to ristomycin and erythromycin. The predominating number of the strains were sensitive to lincomycin, levomycetin and the beta-lactam antibiotics. Strains resistant or moderately resistant to benzylpenicillin, ampicillin, oxacillin, methicillin and cephalotin were detected. The majority of the strains were resistant to streptomycin, tetracycline and gentamicin. Multiple antibiotic resistance with 2-7 determinants was revealed in 11.2 per cent of the strains. The antibiotic sensitivity of the strains isolated from the newborns, their mothers and the personnel in the maternity home was on the whole similar or insignificantly differed.     

Erythromycin- and tetracycline-resistant lactobacilli in Italian fermented dry sausages

Aims:  To assess the frequency of erythromycin- and tetracycline-resistant lactobacilli in Italian fermented dry sausages.
Methods and Results:  We isolated lactobacilli colonies from 20 salami from the north of Italy (Piacenza province) using selective medium supplemented with erythromycin or tetracycline; we determined the minimum inhibitory concentration and searched for selected erythromycin and tetracycline resistance genes. A total of 312 lactobacilli colonies were genetically ascribed to 60 different strains belonging to seven Lactobacillus species. Lactobacillus sakei , Lactobacillus curvatus and Lactobacillus plantarum were the most frequently found species. Thirty strains (50%) were phenotypically resistant to erythromycin, 45 (75%) to tetracycline and 27 (45%) were resistant to both. The most frequently detected resistance genes were tet (M) and erm (B).
Conclusions:  This study provides evidence of the presence of tetracycline- and, to a lesser extent, erythromycin-resistant lactobacilli in fermented dry sausages produced in northern Italy.
Significance and Impact of the Study:  Although these antibiotic-resistant lactobacilli could serve as reservoir organisms, in our study, 16 of 20 salami could be considered safe in regard to possible antibiotic resistance gene transfer to pathogens, whereas 4 of 20 could represent a borderline situation.     

Multiplex PCR detection of the antibiotic resistance genes in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> strains isolated from auricular infections

Thirty-five Staphylococcus aureus strains from auricular infections were isolated. The identification of strains was confirmed by Api ID 32 Staph strips, the antibiotic susceptibility test was performed using ATB Staph kit. PCR assay was used to detect the oxacillin resistance gene (mecA) and the erythromycin genes (ermA, ermB, ermC, msrA and mef). The susceptibility profile of all strains revealed a low resistance level to oxacillin and erythromycin. The PCR results show that 60 % of the strains are mecA positive. The frequency of erythromycin genes was: ermA (+) 22.8 %, ermB (+) 45.7, ermC (+) 17.1, msrA (+) 28.6. The mef gene was not detected in any strain. No correlations between genotypic and phenotypic methods for the determination of oxacillin and erythromycin resistance was found. However, multiplex PCR technique was shown to be a fast, practical and economic technique for the detection of methicillin-and erythromycin-resistant staphylococci.     

耐甲氧西林凝固酶阴性葡萄球菌中多种抗生素耐药基因的观察

探讨耐甲氧西林凝固酶阴性葡萄球菌(methicillin-resistant coagulase negative staphylococci,MRCNS)中多种抗生素耐药基因的分布情况,采取Kirby Bauer标准纸片扩散法(K-B纸片扩散法)对MRCNS开展药敏实验,并通过PCR检测菌种携带的抗生素耐药基因。药敏实验结果显示,96株MRCNS对庆大霉素、红霉素、莫匹罗星以及四环素的耐药性各有差异,但对氨苄西林的耐药性却达到100%,未发现对万古霉素具有耐药性的菌株;PCR检测结果显示,9种耐药基因merA、ermB、ermC、msrA、tetM、tetK、aac(6’)-aph(2")、aph(3’)-III、mupA的阳性检出率各有差异,没有扩增出vanA、ermA、vanB以及ant(6’)-I基因,但都扩增出mecA基因。结果表明,MRCNS可以同时携带多种抗生素耐药基因,属于一种多重耐药菌。     

The genetic diversity and phenotypic characterisation of Streptococcus agalactiae isolates from Rio de Janeiro, Brazil

Streptococcus agalactiae isolates are more common among pregnant women, neonates and nonpregnant adults with underlying diseases compared to other demographic groups. In this study, we evaluate the genetic and phenotypic diversity in S. agalactiae strains from Rio de Janeiro (RJ) that were isolated from asymptomatic carriers. We analysed these S. agalactiae strains using pulsed-field gel electrophoresis (PFGE), serotyping and antimicrobial susceptibility testing, as well as by determining the macrolide resistance phenotype, and detecting the presence of the ermA/B, mefA/E and lnuB genes. The serotypes Ia, II, III and V were the most prevalent serotypes observed. The 60 strains analysed were susceptible to penicillin, vancomycin and levofloxacin. Resistance to clindamycin, chloramphenicol, erythromycin, rifampin and tetracycline was observed. Among the erythromycin and/or clindamycin resistant strains, the ermA, ermB and mefA/E genes were detected and the constitutive macrolides, lincosamides and streptogramin B-type resistance was the most prevalent phenotype observed. The lnuB gene was not detected in any of the strains studied. We found 56 PFGE electrophoretic profiles and only 22 of them were allocated in polymorphism patterns. This work presents data on the genetic diversity and prevalent capsular serotypes among RJ isolates. Approximately 85% of these strains came from pregnant women; therefore, these data may be helpful in developing future prophylaxis and treatment strategies for neonatal syndromes in RJ.     

Erythromycin and penicillin resistance mechanisms among viridans group streptococci isolated from blood cultures of adult patients with underlying diseases

The aim of the study was to evaluate the species distribution, antimicrobial susceptibility and erythromycin-penicillin resistance mechanisms of viridans streptococci (VGS) isolates from blood cultures of adult patients with underlying diseases. Fifty VGS blood culture isolates were screened for their antibiotic susceptibilities against penicillin G, erythromycin and tetracycline by E-test. Clindamycin, cefotaxime, chloramphenicol, levofloxacin, linezolid and vancomycin susceptibility were performed by broth microdilution method. Erythromycin and penicillin resistance genotypes, ermB and mefA/E, pbp1a, pbp2b and pbp2x are amplified using PCR method. The clinical isolates included Streptococcus mitis (n. 19), S.oralis (n. 13), S.sanguinis, S.parasanguinis (n. 6, each), S.salivarius, S.vestibularis (n. 2, each), S.constellatus, S.sobrinus (n. 1, each). The percentage resistance against erythromycin and penicillin was 36% and 30%, respectively. The genotypic carriage rate of erythromycin resistance genes were: 56% ermB, 28% mefE, 8% ermB+mefE. Penicillin-resistant isolates carried pbp2b (33.3%) and pbp2x (20%) genes. Twenty-four VGS isolates were recovered from patients with cancer. S.mitis and S.oralis predominated among patients with cancer who had erythromycin and penicillin resistance isolates. The importance of classical antimicrobial agents like penicillin and erythromycin warrants the continuous surveillance of invasive VGS isolates and can guide better treatment options especially in patients with underlying diseases.     

Genetic determinants of antibiotic resistance in enterobacteria

Antibiotic resistance of enterobacterial strains from population isolated in Krasnodar region is rather often controlled by the "plasmid" genes. The conclusion is based on using the colony hybridization with [32P]-DNA fragments of plasmids, carrying the genetic determinants of antibiotic resistance, as a method for antibiotic resistance, genes screening. Kanamycin resistance in the majority of strains is coded by APH (3') II gene, streptomycin resistance by APH (3") gene, chloramphenicol resistance by CATI, sulphonilamide resistance by DHPS type II gene. Tetracycline resistance of the studied enterobacterial strains is not connected with the widespread genetic determinants of a new class tetracycline resistance.     

Antibiotic Susceptibility of Lactobacillus and Bifidobacterium Species from the Human Gastrointestinal Tract

One hundred and twenty-two strains of Bifidobacterium and Lactobacillus species have been tested against 12 antibiotics and two antibiotic mixtures by a commercial system (Sensititre Anaero3; Treck Diagnostic Systems). The upper limits of some minimum inhibitory concentrations (MICs) were completed on MRS agar plates by the NCCLS procedure. All strains were sensitive to chloramphenicol and imipenem and most of the strains were resistant to metronidazole. Bifidobacteria isolates were susceptible to cefoxitin, whereas about half of the lactobacilli were resistant. Approximately 30% of the Bifidobacterium isolates were resistant to tetracycline, as well as five Lactobacillus strains belonging to four different species. None of the tested Bifidobacterium isolates was resistant to vancomycin, whereas a species-dependent resistance was found among the lactobacilli. Single strains of Bifidobacterium longum, Bifidobacterium pseudocatenulatum, Lactobacillus acidophilus, Lactobacillus rhamnosus, and Lactobacillus brevis were resistant to erythromycin and/or clindamycin. Most of the observed resistances seemed to be intrinsic, but some others could be compatible with transmissible determinants.     

Molecular characterization of intrinsic and acquired antibiotic resistance in lactic acid bacteria and bifidobacteria

The minimum inhibitory concentrations (MICs) of 6 different antibiotics (chloramphenicol, clindamycin, erythromycin, streptomycin, tetracycline and vancomycin) were determined for 143 strains of lactic acid bacteria and bifidobacteria using the Etest. Different MICs were found for different species and strains. Based on the distribution of these MIC values, most of the strains were either susceptible or intrinsically resistant to these antibiotics. However, the MIC range of some of these antibiotics showed a bimodal distribution, which suggested that some of the tested strains possess acquired antibiotic resistance. Screening for resistance genes was performed by PCR using specific primers, or using a DNA microarray with around 300 nucleotide probes representing 7 classes of antibiotic resistance genes. The genes identified encoded resistance to tetracycline [tet(M), tet(W), tet(O) and tet(O/W)], erythromycin and clindamycin [erm(B)] and streptomycin [aph(E) and sat(3)]. Internal portions of some of these determinants were sequenced and found to be identical to genes described in other bacteria. All resistance determinants were located on the bacterial chromosome, except for tet(M), which was identified on plasmids in Lactococcus lactis. The contribution of intrinsic multidrug transporters to the antibiotic resistance was investigated by cloning and measuring the expression of Bifidobacterium breve genes in L. lactis.