1: Lett Appl Microbiol. 2004;38(3):206-10. Use of folic acid casei medium reveals trimethoprim susceptibility of Lactobacillus species. Danielsen M, Andersen HS, Wind A. Applied Biotechnology, Identification section, Horsholm, Denmark. morten.danielsen@dk.chr-hansen.com AIM: Lactobacilli have been reported to have intrinsic resistance to trimethoprim. The susceptibility of lactobacilli to trimethoprim on different media was investigated in order to search for a phenotypic test method that could indicate the presence of acquired resistance genes. METHODS AND RESULTS: Strains of Lactobacillus acidophilus, Lact. paracasei, Lact. rhamnosus and Lact. plantarum were susceptibility tested with E-tests on folic acid casei medium (FACM), MRS and defined medium 1. The effects of addition or removal of nucleosides and thymidine phosphorylase were investigated. E-tests on FACM yielded reproducible minimal inhibitory concentrations (MICs) for trimethoprim but addition of nucleosides was necessary for growth of Lact. acidophilus. MICs for the tested strains were 0.125-0.19, 0.25-3 and 0.064-0.19 microg ml(-1) for Lact. paracasei, Lact. rhamnosus and Lact. plantarum, respectively. With the addition of deoxyuridine and deoxyadenosine to FACM the MICs of Lact. acidophilus were 0.064-1 microg ml(-1). CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacilli do not have intrinsic resistance to trimethoprim. The results show that trimethoprim susceptibility testing of the tested Lactobacillus species is possible and indicate that transferable resistance genes are absent in all the tested strains. PMID: 14962041 [PubMed - indexed for MEDLINE] 2: Appl Environ Microbiol. 2003 Dec;69(12):7173-80. Heat shock treatment increases the frequency of loss of an erythromycin resistance-encoding transposable element from the chromosome of Lactobacillus crispatus CHCC3692. Stroman P, Muller CC, Sorensen KI. Department of Genomics and Strain Development, Chr. Hansen A/S, DK-2970 Horsholm, Denmark. per.stroeman@dk.chr-hansen.com A 3,165-bp chromosomally integrated transposon, designatedTn3692, of the gram-positive strain Lactobacillus crispatus CHCC3692 contains an erm(B) gene conferring resistance to erythromycin at concentrations of up to 250 micrograms/ml. Loss of this resistance can occur spontaneously, but the rate is substantially increased by heat shock treatment. Heat shock treatment at 60 degrees C resulted in an almost 40-fold increase in the frequency of erythromycin-sensitive cells (erythromycin MIC, 0.047 micrograms/ml). The phenotypic change was followed by a dramatic increase in transcription of the transposase gene and the concomitant loss of an approximately 2-kb DNA fragment carrying the erm(B) gene from the 3,165-bp erm transposon. In cells that were not subjected to heat shock, transcription of the transposase gene was not detectable. The upstream sequence of the transposase gene did not show any homology to known heat shock promoters in the gene data bank. Significant homology (>99%) was observed between the erythromycin resistance-encoding gene from L. crispatus CHCC3692 and the erm(B) genes from other gram-positive bacteria, such as Streptococcus agalactiae, Streptococcus pyogenes, Enterococcus faecium, and Lactobacillus reuteri, which strongly indicates a common origin of the erm(B) gene for these species. The transposed DNA element was not translocated to other parts of the genome of CHCC3692, as determining by Southern blotting, PCR analysis, and DNA sequencing. No other major aberrations were observed, as judged by colony morphology, growth performance of the strain, and pulsed-field gel electrophoresis. These observations suggest that heat shock treatment could be used as a tool for the removal of unwanted antibiotic resistance genes harbored in transposons flanked by insertion sequence elements or transposases in lactic acid bacteria used for animal and human food production. PMID: 14660363 [PubMed - indexed for MEDLINE] 3: Plasmid. 2003 Nov;50(3):190-201. Sequence and genetic organization of the 19.3-kb erythromycin- and dalfopristin-resistance plasmid pLME300 from Lactobacillus fermentum ROT1. Gfeller KY, Roth M, Meile L, Teuber M. Laboratory of Food Microbiology, Institute of Food Science and Nutrition, Swiss Federal Institute of Technology Zurich, ETH-Zentrum, Zurich CH-8092, Switzerland. Lactobacillus fermentum ROT1 was isolated from a raw milk dairy product. It is resistant to novobiocin, tetracycline, erythromycin and dalfopristin. A chromosomal tetracycline-resistance determinant was identified as tetM. A 19,398-bp plasmid (pLME300), present in several erythromycin-resistant strains of Lb. fermentum, was isolated from strain ROT1 and completely sequenced. Based on putative open reading frames, pLME300 contains at least four different functional regions. In region I, ORF1 shows high homologies to replication proteins of different theta-replicating plasmids. In addition, a tandem repeat of a 22-bp sequence appears 4.5 times. In region II, ORF3 may code for a methylase, and ORF4 has homologies to Mrr restriction system proteins of Deinococcus radiodurans and Escherichia coli suggesting a restriction-modification system. Region III harbours antibiotic-resistance genes, coding for a macrolide-lincosamide-streptogramin B (MLS) methylase Erm(LF) and the streptogramin A acetyltransferase Vat(E), which is identical to Vat(E) from Enterococcus faecium. Furthermore, region III shows a 91% nucleotide sequence identity to an erm-vat linkage of E. faecium. Region IV carries ORFs that appear to be involved in plasmid mobilization as characterized by a putative origin of transfer and a mobilization protein. pLME300 is the largest completely sequenced multi-resistance plasmid isolated from any Lactobacillus strain so far. PMID: 14597008 [PubMed - indexed for MEDLINE] 4: FEMS Microbiol Lett. 2003 Aug 8;225(1):125-30. In vitro conjugal transfer of tetracycline resistance from Lactobacillus isolates to other Gram-positive bacteria. Gevers D, Huys G, Swings J. Laboratory of Microbiology, Faculty of Sciences, Ghent University, B-9000 Ghent, Belgium. dirk.gevers@ugent.be The ability of 14 Lactobacillus strains, isolated from fermented dry sausages, to transfer tetracycline resistance encoded by tet(M) through conjugation was examined using filter mating experiments. Seven out of 14 tetracycline-resistant Lactobacillus isolates were able to transfer in vitro this resistance to Enterococcus faecalis at frequencies ranging from 10(-4) to 10(-6) transconjugants per recipient. Two of these strains could also transfer their resistance to Lactococcus lactis subsp. lactis, whereas no conjugal transfer to a Staphylococcus aureus recipient was found. These data suggest that meat lactobacilli might be reservoir organisms for acquired resistance genes that can be spread to other lactic acid bacteria. In order to assess the risk of this potential hazard, the magnitude of transfer along the food chain merits further research. PMID: 12900030 [PubMed - indexed for MEDLINE] 5: Syst Appl Microbiol. 2003 Jun;26(2):277-83. Prevalence and diversity of tetracycline resistant lactic acid bacteria and their tet genes along the process line of fermented dry sausages. Gevers D, Masco L, Baert L, Huys G, Debevere J, Swings J. Laboratory of Microbiology, Faculty of Sciences, Ghent University, Gent, Belgium. dirk.gevers@rug.ac.be In order to study the prevalence and diversity of tetracycline resistant lactic acid bacteria (Tc(r) LAB) along the process line of two different fermented dry sausage (FDS) types, samples from the raw meat, the meat batter and the fermented end product were analysed quantitatively and qualitatively by using a culture-dependent approach. Both the diversity of the tet genes and their bacterial hosts in the different stages of FDS production were determined. Quantitative analysis showed that all raw meat components of both FDS types (FDS-01 and FDS-08) contained a subpopulation of Tc(r) LAB, and that for FDS-01 no Tc(r) LAB could be recovered from the samples after fermentation. Qualitative analysis of the Tc(r) LAB subpopulation in FDS-08 included identification and typing of Tc(r) LAB isolates by (GTG)5-PCR fingerprinting, plasmid profiling, protein profiling and a characterization of the resistance by PCR detection of tet genes. Two remarks can be made when the results of this analysis for the different samples are compared. (i) The taxonomic diversity of Tc(r) LAB varies along the process line, with a higher diversity in the raw meat (lactococci, lactobacilli, streptococci, and enterococci), and a decrease after fermentation (only lactobacilli). (ii) Also the genetic diversity of the tet genes varies along the process line. Both tet(M) and tet(S) were found in the raw meat, whereas only tet(M) was found after fermentation. A possible relationship was found between the disappearing of species other than lactobacilli and tet(S), because tet(S) was only found in lacotocci, enterococci, and streptococci. These data suggest that fermented dry sausages are among those food products that can serve as vehicles for Tc(r) LAB and that the raw meat already contains a subpopulation of these bacteria. Whether these results reflect the transfer of resistant bacteria or of bacterial resistance genes from animals to man via the food chain is difficult to ascertain and may require a combination of cultivation-dependent and cultivation-independent approaches. PMID: 12866855 [PubMed - indexed for MEDLINE] 6: Appl Environ Microbiol. 2003 Feb;69(2):1270-5. Molecular characterization of tet(M) genes in Lactobacillus isolates from different types of fermented dry sausage. Gevers D, Danielsen M, Huys G, Swings J. Laboratory of Microbiology, Faculty of Sciences, Ghent University, B-9000 Ghent, Belgium. dirk.gevers@rug.ac.be The likelihood that products prepared from raw meat and milk may act as vehicles for antibiotic-resistant bacteria is currently of great concern in food safety issues. In this study, a collection of 94 tetracycline-resistant (Tc(r)) lactic acid bacteria recovered from nine different fermented dry sausage types were subjected to a polyphasic molecular study with the aim of characterizing the host organisms and the tet genes, conferring tetracycline resistance, that they carry. With the (GTG)(5)-PCR DNA fingerprinting technique, the Tc(r) lactic acid bacterial isolates were identified as Lactobacillus plantarum, L. sakei subsp. carnosus, L. sakei subsp. sakei, L. curvatus, and L. alimentarius and typed to the intraspecies level. For a selection of 24 Tc(r) lactic acid bacterial isolates displaying unique (GTG)(5)-PCR fingerprints, tet genes were determined by means of PCR, and only tet(M) was detected. Restriction enzyme analysis with AccI and ScaI revealed two different tet(M) allele types. This grouping was confirmed by partial sequencing of the tet(M) open reading frame, which indicated that the two allele types displayed high sequence similarities (>99.6%) with tet(M) genes previously reported in Staphylococcus aureus MRSA 101 and in Neisseria meningitidis, respectively. Southern hybridization with plasmid profiles revealed that the isolates contained tet(M)-carrying plasmids. In addition to the tet(M) gene, one isolate also contained an erm(B) gene on a different plasmid from the one encoding the tetracycline resistance. Furthermore, it was also shown by PCR that the tet(M) genes were not located on transposons of the Tn916/Tn1545 family. To our knowledge, this is the first detailed molecular study demonstrating that taxonomically and genotypically diverse Lactobacillus strains from different types of fermented meat products can be a host for plasmid-borne tet genes. PMID: 12571056 [PubMed - indexed for MEDLINE] 7: Int J Food Microbiol. 2003 Jan 26;82(1):1-11. Susceptibility of Lactobacillus spp. to antimicrobial agents. Danielsen M, Wind A. Identification Section, Applied Biotechnology, Chr. Hansen A/S, Boge Alle 10-12, 2970 Horsholm, Denmark. morten.danielsen@dk.chr-hansen.com Bacteria used as probiotics or in starter cultures may serve as hosts of antibiotic resistance genes, which can be transferred to pathogenic bacteria. Before launching a starter culture or a probiotic product into the market, it is therefore important to verify that the single bacterial isolates (strains) do not contain transferable resistance genes. A study has been undertaken to establish the levels of susceptibility of Lactobacillus spp. to various antimicrobial agents. This is a prerequisite for differentiating putative transferable resistance from natural resistance. A selection of 62 strains has been screened with the use of the Etest (ABBiodisk, Stockholm, Sweden) for their susceptibility to 25 antimicrobial agents. The strains belonged to the following species: Lactobacillus plantarum/pentosus, L. rhamnosus, L. paracasei, L. sakei, L. curvatus and species of the L. acidophilus group: L. johnsonii, L. crispatus, L. gasseri, and L. acidophilus.The results from the Etests have shown that the level of susceptibility to the antimicrobial agents is species-dependent. For the following antimicrobial agents, susceptibility varied several folds between species: vancomycin, teicoplanin, tetracycline, norfloxacin, ciprofloxacin, fusidic acid, and clindamycin. The differences between the species were more subtle for the rest of the tested antimicrobial agents. On the basis of the result, it was possible to suggest minimal inhibition concentrations (MICs) for the individual Lactobacillus species to be used as a microbiological breakpoint when screening strains for transferable resistance genes. PMID: 12505455 [PubMed - indexed for MEDLINE] 8: Microb Drug Resist. 2003 Fall;9(3):293-7. Macrolide and lincosamide resistance in the gram-positive nasal and tonsillar flora of pigs. Martel A, Meulenaere V, Devriese LA, Decostere A, Haesebrouck F. Department of Pathology, Bacteriology, and Poultry Diseases, Faculty of Veterinary Medicine, Ghent University, B-9820 Merelbeke, Belgium. An.Martel@rug.ac.be Macrolide and lincosamide resistance phenotypes and the presence of the erm(A), erm(B), erm(C), and mef(A) genes were determined in 344 bacterial strains belonging to 34 species and nine genera, isolated from the tonsils and nasal cavities of 2-week- and 6-week-old piglets, derived from four different farms. These piglets had never before been treated with macrolides or lincosamides. Macrolide and lincosamide resistance was most frequently present in Streptococcus and Enterococcus strains, of which over two-thirds were resistant. These genera were followed in decreasing order of resistance frequency by Lactobacillus, Rothia, Staphylococcus, Arcanobacterium, Actinomyces, Pediococcus strains. Only five infrequently occurring species did not show resistance. This high frequency of resistance in nontreated piglets indicates that resistant strains circulate in the herds. In streptococci, enterococci, and Lactobacillus strains, resistance was most often encoded by the erm(B) gene and in staphylococci by erm(A) or erm(C). The erm(B) gene was sporadically detected in other bacterial genera (Actinomyces, Rothia, Aerococcus, Pediococcus). The sequence of the erm(B) gene of 29 strains of 11 pigs originating from the four different farms was determined. This sequence was identical in 12 strains and only differed by 1-6 nucleotides in the other strains, indicating that exchanges of resistance genes might occur between bacterial species and genera belonging to the nasal or tonsillar flora of piglets. PMID: 12959408 [PubMed - indexed for MEDLINE] 9: Scand J Infect Dis. 2003;35(6-7):404-8. Six cases of Lactobacillus bacteraemia: identification of organisms and antibiotic susceptibility and therapy. Arpi M, Vancanneyt M, Swings J, Leisner JJ. Arhus University Hospital, Skejby, Arhus, Denmark. Six cases of bacteraemia in hospitalized patients, 5 with a depressed immune status, were caused by lactobacilli. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell proteins and API 50 CH carbohydrate patterns assigned the causative agents to the species Lactobacillus rhamnosus, Lactobacillus curvatus, Lactobacillus delbrueckii subsp. lactis and Lactobacillus paracasei subsp. paracasei. Publication Types: Case Reports PMID: 12953954 [PubMed - indexed for MEDLINE] 10: Plasmid. 2002 Sep;48(2):98-103. Characterization of the tetracycline resistance plasmid pMD5057 from Lactobacillus plantarum 5057 reveals a composite structure. Danielsen M. Applied Biotechnology, Identification Section, Chr. Hansen A/S, Boge Alle 10-12, 2970 Horsholm, Denmark. morten.danielsen@dk.chr-hansen.com The 10,877bp tetracycline resistance plasmid pMD5057 from Lactobacillus plantarum 5057 was completely sequenced. The sequence revealed a composite structure containing DNA from up to four different sources. The replication region had homology to other plasmids of lactic acid bacteria while the tetracycline resistance region, containing a tet(M) gene, had high homology to sequences from Clostridium perfringens and Staphylococcus aureus. Within the tetracycline resistance region a Lactobacillus IS-element was found. The remaining part of the plasmid contained three open reading frames with unknown functions. The composite structure with several truncated genes suggests a recent assembly of the plasmid. This is the first sequence of an antibiotic resistance plasmid isolated from L. plantarum. PMID: 12383727 [PubMed - indexed for MEDLINE] 11: Appl Environ Microbiol. 2002 Mar;68(3):1088-95. Effects of pressure-induced membrane phase transitions on inactivation of HorA, an ATP-dependent multidrug resistance transporter, in Lactobacillus plantarum. Ulmer HM, Herberhold H, Fahsel S, Ganzle MG, Winter R, Vogel RF. Lehrstuhl fur Technische Mikrobiologie, Weihenstephaner Steig 16, TU Munchen, D-85350 Freising, Germany. The effects of pressure on cultures of Lactobacillus plantarum were characterized by determination of the viability and activity of HorA, an ATP-binding cassette multidrug resistance transporter. Changes in the membrane composition of L. plantarum induced by different growth temperatures were determined. Furthermore, the effect of the growth temperature of a culture on pressure inactivation at 200 MPa was determined. Cells were characterized by plate counts on selective and nonselective agar after pressure treatment, and HorA activity was measured by ethidium bromide efflux. Fourier transform-infrared spectroscopy and Laurdan fluorescence spectroscopy provided information about the thermodynamic phase state of the cytoplasmic membrane during pressure treatment. A pressure-temperature diagram for cell membranes was established. Cells grown at 37 degrees C and pressure treated at 15 degrees C lost >99% of HorA activity and viable cell counts within 36 and 120 min, respectively. The membranes of these cells were in the gel phase region at ambient pressure. In contrast, cells grown at 15 degrees C and pressure treated at 37 degrees C lost >99% of HorA activity and viable cell counts within 4 and 8 min, respectively. The membranes of these cells were in the liquid crystalline phase region at ambient pressure. The kinetic analysis of inactivation of L. plantarum provided further evidence that inactivation of HorA is a crucial step during pressure-induced cell death. Comparison of the biological findings and the membrane state during pressure treatment led to the conclusion that the inactivation of cells and membrane enzymes strongly depends on the thermodynamic properties of the membrane. Pressure treatment of cells with a liquid crystalline membrane at 0.1 MPa resulted in HorA inactivation and cell death more rapid than those of cells with a gel phase membrane at 0.1 MPa. PMID: 11872454 [PubMed - indexed for MEDLINE] 12: J Antimicrob Chemother. 2002 Mar;49(3):535-9. BAL 9141, a new broad-spectrum pyrrolidinone cephalosporin: activity against clinically significant anaerobes in comparison with 10 other antimicrobials. Wootton M, Bowker KE, Holt HA, MacGowan AP. Bristol Centre for Antimicrobial Research and Evaluation, North Bristol NHS Trust/University of Bristol, Department of Medical Microbiology, Southmead Hospital, Westbury-on-Trym, Bristol BS10 5NB, UK. The in vitro potency of BAL 9141, a new pyrrolidinone cephalosporin, was tested against non-duplicate strains of anaerobic bacteria. The MIC(50) was 1 mg/L against Actinomyces species, Clostridium species, Gram-positive anaerobic cocci, Porphyromonas species, Fusobacterium species, Lactobacillus species, Prevotella species and Veillonella species. The MIC(50) was 16 mg/L for Bacteroides fragilis and other Bacteroides species. BAL 9141 was not active against cefoxitin-resistant Bacteroides fragilis. PMID: 11864955 [PubMed - indexed for MEDLINE] 13: Lett Appl Microbiol. 2002;34(6):402-6. Influence of the culture medium on antibiotic susceptibility testing of food-associated lactic acid bacteria with the agar overlay disc diffusion method. Huys G, D'Haene K, Swings J. Laboratory of Microbiology, Ghent University, Ghent, Belgium. geert.huys@rug.ac.be AIMS: To investigate the influence of the culture medium on antibiotic susceptibility testing of food-associated lactic acid bacteria (LAB) with the agar overlay disc diffusion (DD) method. METHOD: The antibiotic resistance profile of 39 food-associated lactobacilli and enterococci was determined with the agar overlay DD method using a defined medium (i.e. Iso-sensitest agar; ISA) or an undefined medium (i.e. de Man, Rogosa, Sharpe or MRS agar). RESULTS: The study revealed that ampicillin discs and, although to a lesser extent, also tetracycline discs consistently produced larger zones on MRS medium compared to ISA medium. For the antibiotics gentamicin, bacitracin and erythromycin, the radius of the inhibition zones produced on MRS medium was significantly smaller in relation to ISA. For categorizing LAB isolates into resistant, intermediate and susceptible groups, it was demonstrated that major errors can occur in determining bacitracin and gentamicin resistance if MRS medium instead of ISA medium is used. On the other hand, the performance of both media was found to be equivalent for testing tetracycline resistance. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Despite the fact that MRS medium generally supports the growth of lactic acid bacteria much better than the nutrient-poor ISA medium, the present study clearly demonstrates that both media are not compatible in susceptibility testing against various classes of antibiotics. These results may stimulate future discussions on a generally recommended DD method for susceptibility testing of food LAB strains. PMID: 12028419 [PubMed - indexed for MEDLINE] 14: J Food Prot. 2001 Dec;64(12):2007-14. Gradient diffusion antibiotic susceptibility testing of potentially probiotic lactobacilli. Charteris WP, Kelly PM, Morelli L, Collins JK. SET Consultants Ltd., Douglas, Cork, Ireland. bcharteris@glanbia.ie Minimum inhibitory contentrations (MICs) of selected inhibitors of cell wall synthesis (benzylpenicillin, ampicillin, and vancomycin), protein synthesis (gentamicin, streptomycin, tetracycline, chloramphenicol, and erythromycin), and nucleic acid synthesis (co-trimoxazole, rifampicin, and metronidazole) were determined by gradient diffusion (E test; AB Biodisk, Solna, Sweden) on deMan, Rogosa, Sharpe (MRS) agar for Lactobacillus strain GG and 11 closely related, rapidly growing, facultatively anaerobic, potentially probiotic Lactobacillus rhamnosus strains. All strains were resistant to vancomycin (MIC90 > or = 256 microg/ml), co-trimoxazole (MIC90 > or = 32 microg/ml), metronidazole (MIC90 > or = 32 microg/ml), gentamicin (MIC90 > or = 128 microg/ml), and streptomycin (MIC90 > or = 256 microg/ml), and sensitive to pencillin G (MIC90 > 0.375 microg/ml), ampicillin (MIC90 > 0.750 microg/ml), rifampicin (MIC90 > 0.375 microg/ml), tetracycline (MIC90 > 1.5 microg/ml), chloramphenicol (MIC90 > 8 microg/ml), and erythromycin (MIC90 > 2 microg/ml). E test MICs were also determined for L. acidophilus National Collection of Food Bacteria (NCFB) 1748 and L. reuteri Deutsche Sammlung von Mikroorganismen 20016T by the inoculum application method recommended by the manufacturer (swabbing), with and without antibiotic prediffusion for 1 h at room temperature, and by an alternative inoculum application (agar overlay) method, without antibiotic prediffusion. Antibiotic prediffusion increased the MICs for penicillin G, ampicillin, tetracycline, and chloramphenicol by up to 2 log2 MIC dilutions without changing antibiotic susceptibility category. Agar overlay application also increased the MICs for these antibiotics as well as for gentamicin by up to 3 log2 MIC dilutions without changing antibiotic susceptibility category. Exact agreement between MICs determined by swab and agar overlay application without antibiotic prediffusion was strain dependent: 54.5% for strain DSM 20016T and 72.7% for strain NCFB 1748. The swab and agar overlay gradient diffusion methods provide a reliable basis for antibiotic susceptibility testing of rapidly growing, facultatively anaerobic lactobacilli, using MRS agar as test medium and are readily applicable for testing individual isolates as needed. PMID: 11770631 [PubMed - indexed for MEDLINE] 15: J Bacteriol. 2001 Sep;183(18):5371-5. Hop resistance in the beer spoilage bacterium Lactobacillus brevis is mediated by the ATP-binding cassette multidrug transporter HorA. Sakamoto K, Margolles A, van Veen HW, Konings WN. Brewing Research & Development Laboratory, Asahi Breweries, Ltd., Moriya-machi, Kitasoma-gun, Ibaraki 302-0106, Japan. Lactobacillus brevis is a major contaminant of spoiled beer. The organism can grow in beer in spite of the presence of antibacterial hop compounds that give the beer a bitter taste. The hop resistance in L. brevis is, at least in part, dependent on the expression of the horA gene. The deduced amino acid sequence of HorA is 53% identical to that of LmrA, an ATP-binding cassette multidrug transporter in Lactococcus lactis. To study the role of HorA in hop resistance, HorA was functionally expressed in L. lactis as a hexa-histidine-tagged protein using the nisin-controlled gene expression system. HorA expression increased the resistance of L. lactis to hop compounds and cytotoxic drugs. Drug transport studies with L. lactis cells and membrane vesicles and with proteoliposomes containing purified HorA protein identified HorA as a new member of the ABC family of multidrug transporters. PMID: 11514522 [PubMed - indexed for MEDLINE] 16: Int J Food Microbiol. 2001 Jul 20;67(1-2):147-52. Antimicrobial susceptibility of starter culture bacteria used in Norwegian dairy products. Katla AK, Kruse H, Johnsen G, Herikstad H. Regional Food Control Authority of Midt-Rogaland, Stavanger, Norway. katla@nmt-mrog.rl.no Commercial starter culture bacteria are widely used in the production of dairy products and could represent a potential source for spread of genes encoding resistance to antimicrobial agents. To learn more about the antimicrobial susceptibility of starter culture bacteria used in Norwegian dairy products, a total of 189 isolates of lactic acid bacteria were examined for susceptibility to ampicillin, penicillin G, cephalothin, vancomycin, bacitracin, gentamicin, streptomycin, erythromycin, tetracycline, chloramphenicol, quinupristin/dalfopristin, ciprofloxacin, trimethoprim and sulphadiazine using Etest for MIC determination. Most of the isolates (140) originated from 39 dairy products (yoghurt, sour cream, fermented milk and cheese), while 49 were isolated directly from nine commercial cultures. The bacteria belonged to the genera Lactobacillus, Lactococcus, Leuconostoc and Streptococcus. Only one of the 189 isolates was classified as resistant to an antimicrobial agent included in the study. This isolate, a lactobacillus, was classified as high level resistant to streptomycin. The remaining isolates were not classified as resistant to the antimicrobial agents included other than to those they are known to have a natural reduced susceptibility to. Thus, starter culture bacteria in Norwegian dairy products do not seem to represent a source for spread of genes encoding resistance to antimicrobial agents. PMID: 11482563 [PubMed - indexed for MEDLINE] 17: Curr Microbiol. 2001 Jul;43(1):17-20. Sequence analyses of a broad host-range plasmid containing ermT from a tylosin-resistant Lactobacillus sp. Isolated from swine feces. Whitehead TR, Cotta MA. Fermentation Biochemistry Research Unit, National Center for Agricultural Utilization Research, USDA, Agricultural Research Service, 1815 N. University Street, Peoria, IL 61604, USA. whitehtr@mail.ncaur.usda.gov Anaerobic bacteria resistant to the macrolide antibiotics tylosin and erythromycin were isolated from the feces of swine. One of the strains, 121B, was initially identified by 16S rDNA sequence analysis as an unknown Lactobacillus sp. The strain was found to contain at least two plasmids, one of which was capable of replicating and providing erythromycin and tylosin resistance to Bacillus subtilis, Streptococcus gordonii, and Escherichia coli. DNA sequence analyses of the 4,232-bp plasmid, p121BS, identified one open reading frame encoding a methylase gene highly similar (> 98% amino acid identity, > 99% DNA sequence identity) to the ermT gene from the Lactobacillus reuteri plasmid pGT633. This is only the second ermT gene to be reported. p121BS also contains two additional open reading frames with significant amino acid similarities to replication proteins from Lactobacillus and other Gram-positive bacteria. PMID: 11375658 [PubMed - indexed for MEDLINE] 18: Syst Appl Microbiol. 2001 Apr;24(1):116-21. Antimicrobial susceptibility of intestinal bacteria from Swiss poultry flocks before the ban of antimicrobial growth promoters. Frei A, Goldenberger D, Teuber M. Department of Food Science, Swiss Federal Institute of Technology, Zurich. From the crop and the caecum of Swiss broilers slaughtered between November 1997 and January 1998, Escherichia coli, enterococci, staphylococci, lactobacilli and Campylobacter species were isolated. After identification to the genus or species level, their minimal inhibitory concentrations (MIC's) for several clinically used antimicrobial agents were determined with the E-Test stripes and compared to those from studies in other European countries. All strains of Enterococcus faecalis (n = 38), E. faecium (27), staphylococci (n = 39) and lactobacilli (n = 14) showed a hundred percent resistance against bacitracin which was included in the feed of the mother animals, but not in the feed of the investigated animals. E.coli strains (n = 60) showed higher resistance incidences than in comparable studies from Finland and Denmark, but lower than those in studies from Italy and Germany. In staphylococci, low resistance rates were observed. A high susceptibility of the 13 Campylobacter jejuni strains was found against therapeutically used antimicrobials. These data can be used as a baseline to determine antibiotic resistance rates after implementation of the growth promotor ban in 1999 in Switzerland. PMID: 11403390 [PubMed - indexed for MEDLINE] 19: Scand J Infect Dis. 2001;33(5):344-9. Antibacterial susceptibility of intestinal lactobacilli of healthy children. Mandar R, Lijvukene K, Huftt P, Karki T, Mikelsaar M. Department of Microbiology, University of Tartu, Estonia. reetm@med.ut.ee We investigated the antibacterial susceptibility of intestinal lactobacilli of Estonian and Swedish children aged 1-2 y. Sixty isolates (10 species) of lactobacilli (29 Estonian and 31 Swedish strains) were tested against ampicillin, cefuroxime, cefoxitin, gentamicin, ciprofloxacin, tetracycline, vancomycin, metronidazole and erythromycin. We observed that intestinal lactobacilli do not display uniform susceptibility to antibiotics. None of the tested lactobacilli was resistant to ampicillin, gentamicin and erythromycin. Single strains were resistant to cefuroxime and tetracycline, about half of the strains to cefoxitin and ciprofloxacin and 73% of the strains to vancomycin. All studied strains were resistant to metronidazole. Most of the strains investigated were resistant to two or three antibiotics out of nine. Some differences in susceptibility were noted between strains belonging to different fermentation types. No differences in susceptibility were found between Estonian and Swedish isolates. Metronidazole, cefoxitin, vancomycin and ciprofloxacin seem to be safer for gastrointestinal lactoflora than other tested antibiotics in both countries. PMID: 11440219 [PubMed - indexed for MEDLINE] 20: J Appl Microbiol. 2000 Nov;89(5):815-24. Exclusion of vanA, vanB and vanC type glycopeptide resistance in strains of Lactobacillus reuteri and Lactobacillus rhamnosus used as probiotics by polymerase chain reaction and hybridization methods. Klein G, Hallmann C, Casas IA, Abad J, Louwers J, Reuter G. Institute of Meat Hygiene and Technology, Veterinary Faculty, Free University of Berlin, Germany. guenter.klein@bgvv.de Strains of Lactobacillus reuteri and Lact. rhamnosus are used as probiotics in man and animal. The aim of this study was to determine whether the glycopeptide resistance in these lactobacilli has a similar genetic basis as in enterococci. Five Lact. reuteri strains and one Lact. rhamnosus, as well as four Enterococcus control strains, were probed for the vanA gene cluster, the vanB gene and the vanC gene by PCR and Southern hybridization, and DNA/DNA hybridization. Their resistance and plasmid patterns were also investigated. All Lactobacillus strains were resistant to vancomycin but susceptible to a broad range of antibiotics. Four of the Lactobacillus strains (including the Lact. rhamnosus strain) did not harbour any plasmid and two of them contained five and 6 plasmid bands respectively. None of the Lactobacillus strains possessed the vanA, vanB or vanC gene. These findings indicate that the glycopeptide resistance of the Lactobacillus strains analysed is different from the enterococcal type. The study provides reassurance on the safety of the Lactobacillus strains used as probiotics with regard to their vancomycin resistance. PMID: 11119156 [PubMed - indexed for MEDLINE] 21: J Food Prot. 2000 Oct;63(10):1369-76. Effect of conjugated bile salts on antibiotic susceptibility of bile salt-tolerant Lactobacillus and Bifidobacterium isolates. Charteris WP, Kelly PM, Morelli L, Collins JK. SET Consultants Ltd., Douglas, Cork, Ireland. bcharteris@glanbia.ie Virtually every antibiotic may cause in vivo alterations in the number, level, and composition of the indigenous microbiotae. The degree to which the microbiotae are disturbed depends on many factors. Although bile may augment antibiotic activity, studies on the effect of bile on the antibiotic susceptibility of indigenous and exogenous probiotic microorganisms are lacking. It was against this background that the antibiotic susceptibility of 37 bile salt-tolerant Lactobacillus and 11 Bifidobacterium isolates from human and other sources was determined in the presence of 0.5% wt/wt oxgall (conjugated bile salts). Oxgall did not affect the intrinsic resistance of lactobacilli to metronidazole (5 microg), vancomycin (30 microg), and cotrimoxazole (25 microg), whereas it resulted in a complete loss of resistance to polymyxin B (300 microg) and the aminoglycosides gentamicin (10 microg), kanamycin (30 microg), and streptomycin (10 microg) for most strains studied (P < 0.001). Oxgall did not affect the intrinsic resistance of bifidobacteria to metronidazole and vancomycin, whereas polymyxin B and co-trimoxazole resistance was diminished (P < 0.05) and aminoglycoside resistance was lost (P < 0.001). Seven lactobacilli, but no bifidobacteria strain, showed unaltered intrinsic antibiotic resistance profiles in the presence of oxgall. Oxgall affected the extrinsic susceptibility of lactobacilli and bifidobacteria to penicillin G (10 microg), ampicillin (10 microg), tetracycline (30 microg), chloramphenicol (30 microg), erythromycin (15 microg), and rifampicin (5 microg) in a source- and strain-dependent manner. Human strain-drug combinations of lactobacilli (P < 0.05) and bifidobacteria (P < 0.01) were more likely to show no change or decreased susceptibility compared with other strain-drug combinations. The antimicrobial activity spectra of polymyxin B and the aminoglycosides should not be considered limited to gram-negative bacteria but extended to include gram-positive genera of the indigenous and transiting microbiotae in the presence of conjugated bile salts. Those lactobacilli (7 of 37) that show unaltered intrinsic and diminished extrinsic antibiotic susceptibility in the presence of oxgall may possess greater upper gastrointestinal tract transit tolerance in the presence of antibiotics. PMID: 11041136 [PubMed - indexed for MEDLINE] 22: Lett Appl Microbiol. 2000 Jul;31(1):57-62. Antimicrobial susceptibility of bifidobacteria. Yazid AM, Ali AM, Shuhaimi M, Kalaivaani V, Rokiah MY, Reezal A. Department of Food Technology, Universiti Putra Malaysia, Serdang, Selangor, Malaysia. myazid@fsb.upm.edu.my Eighteen Bifidobacterium strains were tested for their susceptibility to a range of antimicrobial agents. All the strains tested, including the reference culture Lactobacillus acidophilus CH2, were susceptible to several groups of antimicrobial agents, they were cephalosporin (cefamandole, cefazolin, cefaperazone, cefoxitin), polypeptide (bacitracin), macrolide (erythromycin), penicillin (amoxicillin), phenicol (chloramphenicol) and beta-lactam (imipenem). Fourteen strains were resistant to more than 10 antibiotics. The reference culture was resistant to only three antibiotics. The results showed that bifidobacteria are resistant to a wide range of antimicrobial agents. PMID: 10886616 [PubMed - indexed for MEDLINE] 23: Syst Appl Microbiol. 2000 Jun;23(2):279-84. Isolation and identification of tetracycline resistant lactic acid bacteria from pre-packed sliced meat products. Gevers D, Huys G, Devlieghere F, Uyttendaele M, Debevere J, Swings J. Department of Biochemistry, Physiology and Microbiology, University of Gent, Belgium. dirk.gevers@rug.ac.be In recent years, the food chain has been recognised as one of the main routes for transmission of antibiotic resistant bacteria between the animal and human population. In this regard, the current study aimed to investigate if tetracycline resistant (tetR) lactic acid bacteria (LAB) are present in ready-to-eat modified atmosphere packed (MAP) sliced meat products including fermented dry sausage, cooked chicken breast meat and cooked ham. From population graphs based on doubling tetracycline concentrations between 0 and 256 microg ml(-1), only fermented dry sausage was shown to contain a high-level retR LAB population (5.10(1) - 2,23.10(4) CFU/g), and this in four out of ten examined sausages. From these four positive sausages, a total of 100 strains were isolated on de Man, Rogosa and Sharpe-sorbic acid (MRS-S) agar without tetracycline (n = 45) and on MRS-S agar supplemented with a tetracycline breakpoint concentration of 64 microg ml(-1) (n = 55). Using resistance histograms derived from the disc diffusion method, all these strains were grouped as sensitive to rifampicin, erythromycin and ampicillin. All strains from the tetracycline-containing MRS-S plates were resistant to tetracycline. Identification with whole-cell protein profiling revealed that the total strain set represented four different species: Pediococcus pentosaceus, Lactobacillus plantarum, Lactobacillus sakei subsp. carnosus and Lactobacillus curvatus. All species are commonly associated with fermented dry sausage, either as starter culture or as natural contaminants. The latter three species were found to comprise all tetracycline resistant strains. To our knowledge, this is the first report providing evidence for the presence of tetR LAB in final ready-to-eat pre-packed fermented dry sausages. PMID: 10930081 [PubMed - indexed for MEDLINE] 24: FEMS Microbiol Lett. 2000 Apr 1;185(1):1-7. Enterococcal-type glycopeptide resistance genes in non-enterococcal organisms. Patel R. Division of Infectious Diseases and Infectious Diseases Research Laboratory, Mayo Clinic and Foundation, Rochester, MN, USA. patel.robin@mayo.edu Although the emergence of vancomycin-resistant enterococci can be attributed, in part, to the increasing use of vancomycin in clinical practice, and glycopeptide use in animal husbandry, the origins of the enterococcal vancomycin resistance genes are not clear. The vancomycin resistance-associated genes in Enterococcus gallinarum, Enterococcus casseliflavus/flavescens, Lactobacillus spp., Leuconostoc spp., Pediococcus spp., and Erysipelothrix rhusiopathiae, are not the source of the high-level vancomycin resistance-associated genes in enterococci. There are, however, environmental organisms which have been found to have gene clusters homologous to the enterococcal vanA, vanB and vanC gene clusters; these include the biopesticide Paenibacillus popilliae, and, to a lesser extent, the glycopeptide-producing organisms Amycolatopsis orientalis and Streptomyces toyocaensis. Still, the exact sources of the enterococcal vancomycin resistance genes remain a mystery. Publication Types: Review Review, Tutorial PMID: 10731599 [PubMed - indexed for MEDLINE] 25: J Hosp Infect. 1999 Aug;42(4):275-82. Intrinsically vancomycin-resistant gram-positive organisms: clinical relevance and implications for infection control. Nelson RR. Department of Clinical Microbiology, Western Infirmary, Glasgow. Intrinsic resistance to vancomycin in gram-positive bacteria presumably predates acquired vancomycin resistance in enterococci but it has only recently generated interest. Intrinsically resistant enterococci possessing the vanC gene and the non-enterococcal genera Leuconostoc, Lactobacillus, Pediococcus and Erysipelothrix are known to cause human infection. This review examines the available data on their identification, resistance mechanisms, epidemiology, clinical infections and antimicrobial susceptibility. Intrinsically vancomycin-resistant gram-positives are usually opportunistic pathogens. Although serious infections may occur, treatment options remain available. No additional infection control measures for the intrinsically resistant genera appear justified with currently available evidence, although vigilance should be maintained to detect future changes in susceptibility patterns. Publication Types: Review Review, Tutorial PMID: 10467540 [PubMed - indexed for MEDLINE] 26: J Clin Microbiol. 1999 Mar;37(3):729-33. Lactobacillus species identification, H2O2 production, and antibiotic resistance and correlation with human clinical status. Felten A, Barreau C, Bizet C, Lagrange PH, Philippon A. Service de Bacteriologie-Virologie-Hygiene, Hopital St-Louis, 75475 Paris Cedex 10, France. Lactobacilli recovered from the blood, cerebrospinal fluid, respiratory tract, and gut of 20 hospitalized immunocompromised septic patients were analyzed. Biochemical carbohydrate fermentation and total soluble cell protein profiles were used to identify the species. Hydrogen peroxide production was measured. Susceptibility to 19 antibiotics was tested by a diffusion method, and the MICs of benzylpenicillin, amoxicillin, imipenem, erythromycin, vancomycin, gentamicin, and levofloxacin were determined. A small number of species produced H2O2, and antibiotic susceptibilities were species related. Eighteen (90%) of the isolates were L. rhamnosus, one was L. paracasei subsp. paracasei, and one was L. crispatus. L. rhamnosus, L. paracasei subsp. paracasei isolates, and the type strains were neither H2O2 producers nor vancomycin susceptible (MICs, >/=256 microgram/ml). L. crispatus, as well as most of the type strains of lactobacilli which belong to the L. acidophilus group, was an H2O2 producer and vancomycin susceptible (MICs, <4 microgram/ml). PMID: 9986841 [PubMed - indexed for MEDLINE] 27: J Food Prot. 1998 Dec;61(12):1636-43. Antibiotic susceptibility of potentially probiotic Lactobacillus species. Charteris WP, Kelly PM, Morelli L, Collins JK. SET Consultants Ltd., Douglas, Cork, Ireland. bcharteris@awg.ie In recent years, the time-honored reputation of lactobacilli as promoters of gastrointestinal and female urogenital health has been qualified. This has occurred due to a rare association with human infection in the presence of certain predisposing factors and their potential to act as a source of undesirable antibiotic resistance determinants to other members of the indigenous microbiota. This necessitates greater caution in their selection for use in microbial adjunct nutrition and disease management (prophylaxis and therapy). It was against this background that 46 Lactobacillus strains from human and dairy sources were assayed for susceptibility to 44 antibiotics. All strains were resistant to a group of 14 antibiotics, which included inhibitors of cell wall synthesis (cefoxitin [30 microg] and aztreonam [30 microg]), protein synthesis (amikacin [30 microg], gentamicin [10 microg], kanamycin [30 microg], and streptomycin [10 microg]), nucleic acid synthesis (norfloxacin [10 microg], nalidixic acid [30 microg], sulphamethoxazole [100 microg], trimethoprim [5 microg], co-trimoxazole [25 microg], and metronidazole [5 microg]), and cytoplasmic membrane function (polymyxin B [300 microg] and colistin sulphate [10 microg]). All strains were susceptible to tetracycline (30 microg), chloramphenicol (30 microg), and rifampicin (5 microg). Four human strains and one dairy strain exhibited atypical resistance to a penicillin, bacitracin (10 microg), and/or nitrofurantoin (300 microg). One human strain was also resistant to erythromycin (15 microg) and clindamycin (2 microg). These resistances may have been acquired due to antibiotic exposure in vivo, but conclusive evidence is lacking in this regard. Seven microorganism-drug combinations were evaluated for beta-lactamase activity using synergy and nitrocefin tests. The absence of activity suggested that cell wall impermeability appeared responsible for beta-lactam resistance. The occurrence of a minority of lactobacilli with undesirable, atypical resistance to certain antibiotics demonstrates that not all strains are suitable for use as probiotics or bacteriotherapeutic agents. The natural resistance of lactobacilli to a wide range of clinically important antibiotics may enable the development of antibiotic/probiotic combination therapies for such conditions as diarrhea, female urogenital tract infection, and infective endocarditis. PMID: 9874341 [PubMed - indexed for MEDLINE] 28: J Antimicrob Chemother. 1998 Sep;42(3):297-301. In-vitro activity of the new ketolide antibiotic HMR 3647 against gram-positive bacteria. Schulin T, Wennersten CB, Moellering RC Jr, Eliopoulos GM. Department of Medicine, Beth Israel Deaconess Medical Center, and Harvard Medical School, Boston, MA 02115, USA. The comparative in-vitro activity of HMR 3647, a new ketolide antibiotic, was investigated against 492 clinical isolates of gram-positive bacteria, including multiply resistant strains, by an agar-dilution technique. All streptococci tested were inhibited by the new ketolide at concentrations < or = 0.5 mg/L. HMR 3647 was more potent than erythromycin against staphylococci. For enterococci the new compound yielded an MIC90 of 8 mg/L. Erysipelothrix spp., Pediococcus spp., Leuconostoc spp., Lactobacillus spp., JK diphtheroids and Listeria monocytogenes were also susceptible to the new ketolide. PMID: 9786468 [PubMed - indexed for MEDLINE] 29: Int J Food Microbiol. 1998 Jun 16;41(3):195-204. Vancomycin resistance factor of Lactobacillus rhamnosus GG in relation to enterococcal vancomycin resistance (van) genes. Tynkkynen S, Singh KV, Varmanen P. Valio Ltd, R&D, Finland. soile.tynkkynen@valio.fi Lactobacillus rhamnosus GG (ATCC 53103) is a probiotic strain used in fermented dairy products in many countries and is also used as a food supplement in the form of freeze-dried powder. The relationship of the vancomycin resistance factor in L. rhamnosus GG and the vancomycin resistance (van) genes of Enterococcus faecalis and E. faecium were studied using polymerase chain reaction (PCR), Southern hybridization and conjugation methods. Our results show that the vancomycin resistance determinant in L. rhamnosus GG is not closely related to enterococcal van genes, since no PCR product was amplified in L. rhamnosus GG with any of the three sets of vanA primers used, and enterococcal vanA, vanB, vnH, vanX, vanZ, vanY, vanS and vanR genes did not hybridize with DNA of L. rhamnosus GG. This strain does not contain plasmids and transfer of chromosomal vancomycin resistance determinant from L. rhamnosus GG to enterococcal species was not detected. Our results are in accordance with previous findings of intrinsically vancomycin-resistant lactic acid bacteria. PMID: 9706787 [PubMed - indexed for MEDLINE] 30: J Bacteriol. 1997 Oct;179(19):6208-12. Peptidoglycan structure of Lactobacillus casei, a species highly resistant to glycopeptide antibiotics. Billot-Klein D, Legrand R, Schoot B, van Heijenoort J, Gutmann L. L.R.M.A., Universite Paris VI, France. The structure of the peptidoglycan of Lactobacillus casei ATCC 393, a species highly resistant to glycopeptide antibiotics, was examined. After digestion, 23 muropeptides were identified; monomers represented 44.7% of all muropeptides, with monomer tetrapeptides being the major ones. Fifty-nine percent of the peptidoglycan was O-acetylated. The cross-bridge between D-alanine and L-lysine consisted of one asparagine, although aspartate could be found in minor quantities. Since UDP-MurNAc-tetrapeptide-D-lactate is the normal cytoplasmic precursor found in this species, monomer tetrapeptide-lactate was expected to be found. However, such a monomer was found only after exposure to penicillin, suggesting that penicillin-sensitive D,D-carboxypeptidases were very active in normal growing cells. PMID: 9324275 [PubMed - indexed for MEDLINE] 31: Mol Cell Probes. 1997 Oct;11(5):317-22. Detection of erythromycin resistant methylase gene by the polymerase chain reaction. Nawaz MS, Khan AA, Cerniglia CE. Division of Microbiology, National Center for Toxicological Research, Jefferson, AR 72079, USA. A polymerase chain reaction (PCR) protocol was developed that could specifically amplify a 520-bp region of the erythromycin resistant methylase (ermC) gene sequence. The identity of the PCR-amplified 520-bp DNA was confirmed by HinCII endonuclease restriction digestion, which produced the predicted 440-bp and 80-bp DNA fragments. A 20-mer (alpha-32P) oligonucleotide probe specifically hybridized with these amplified products confirming the specificity and reliability of this diagnostic assay. The assay could detect the ermC gene in bacterial suspensions containing as few as 10(3) cells ml-1. The assay was used to detect the presence of the ermC gene in several Gram-positive bacterial strains identified as Streptococcus sp., Staphylococcus sp., Micrococcus sp., Lactobacillus sp. and Enterococcus sp., isolated from water samples maintained in experimental animal cages and clinical sources. Only bacteria identified as Staphylococcus sp. were resistant to the antibiotic. Although 17 strains of Staphylococcus sp. isolated from clinical samples were resistant to erythromycin, only seven of these isolates tested positive for the presence of the ermC gene. Of these strains, five were identified as coagulase-positive S. aureus and the rest were identified as coagulase-negative S. epidermidis. The erythromycin resistance in all seven ermC positive isolates was constitutive. The entire diagnostic assay, including template preparation, amplification and electrophoresis can be completed within 6 h. PMID: 9375290 [PubMed - indexed for MEDLINE] 32: Appl Environ Microbiol. 1997 Jun;63(6):2117-23. Single-crossover integration in the Lactobacillus sake chromosome and insertional inactivation of the ptsI and lacL genes. Leloup L, Ehrlich SD, Zagorec M, Morel-Deville F. Laboratoire de Recherches sur la Viande, INRA, Jouy-en-Josas, France. Single-crossover homologous integration in Lactobacillus sake was studied. Integration was conducted with nonreplicative delivery vector pRV300. This vector is composed of a pBluescript SK- replicon for propagation in Escherichia coli and an erythromycin resistance marker. Random chromosomal DNA fragments of L. sake 23K ranging between 0.3 and 3.4 kb were inserted into pRV300. The resulting plasmids were able to integrate into the chromosome by homologous recombination as single copies and were maintained stably. The single cross-over integration frequency was logarithmically proportional to the extent of homology between 0.3 and 1.2 kb and reached a maximum value of 1.4 x 10(3) integrants/micrograms of DNA. We used this integration strategy to inactivate the ptsI gene, encoding enzyme I of the phosphoenolpyruvate:carbohydrate phosphotransferase system, and the lacL gene, which is one of the two genes required for the synthesis of a functional beta-galactosidase. The results indicated that our method facilitates genetic analysis of L. sake. PMID: 9172327 [PubMed - indexed for MEDLINE] 33: Antonie Van Leeuwenhoek. 1997 Feb;71(1-2):117-28. The role of transport processes in survival of lactic acid bacteria. Energy transduction and multidrug resistance. Konings WN, Lolkema JS, Bolhuis H, van Veen HW, Poolman B, Driessen AJ. Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands. Lactic acid bacteria play an essential role in many food fermentation processes. They are anaerobic organisms which obtain their metabolic energy by substrate phosphorylation. In addition three secondary energy transducing processes can contribute to the generation of a proton motive force: proton/substrate symport as in lactic acid excretion, electrogenic precursor/product exchange as in malolactic and citrolactic fermentation and histidine/histamine exchange, and electrogenic uniport as in malate and citrate uptake in Leuconostoc oenos. In several of these processes additional H+ consumption occurs during metabolism leading to the generation of a pH gradient, internally alkaline. Lactic acid bacteria have also developed multidrug resistance systems. In Lactococcus lactis three toxin excretion systems have been characterized: cationic toxins can be excreted by a toxin/proton antiport system and by an ABC-transporter. This cationic ABC-transporter has surprisingly high structural and functional analogy with the human MDR1-(P-glycoprotein). For anions an ATP-driven ABC-like excretion systems exist. Publication Types: Review Review, Tutorial PMID: 9049023 [PubMed - indexed for MEDLINE] 34: Chin Med J (Engl). 1997 Feb;110(2):157-9. Multiple antibiotic-resistant lactic acid bacteria preparation eliminated MRSA from the decubitus of a bed-ridden elderly patient. Shigeru K, Yumiko T, Hiroko T, Shigehiko M, Haruki K, Motoharu K. First Department of Internal Medicine, Kyoto Prefectural University of Medicine, Japan. Publication Types: Case Reports PMID: 9594292 [PubMed - indexed for MEDLINE] 35: Arch Tierernahr. 1997;50(1):25-9. Effect of maduramicin and monensin on survival of Lactobacillus salivarius 51R administered in the crop and caeca of young chickens. Rada V, Marounek M. Czech University of Agriculture Prague, Czech Republic. A rifampicin-resistant Lactobacillus salivarius 51R was administered orally to newly hatched broiler chickens. The resistance to rifampicin enabled us to differentiate the organism administered from indigenous strains. One day after inoculation, Lactobacillus salivarius 51R dominated among lactobacilli in the crop and caeca of all inoculated chickens, even in those ones receiving maduramicin and monensin at 5 and 100 mg per kg of feed mixture, respectively. Coliform counts in both crop and caeca of inoculated chickens were significantly lowered on the first day after treatment. Also, counts of the crop enterococci were decreased in inoculated chickens. Rifampicin-resistant lactobacilli were still present in high numbers in the crop and caecal contents of inoculated chickens sampled 5 days after inoculation. Differences in counts of total lactobacilli, coliform bacteria, and enterococci were mostly nonsignificant in these samples. Our results demonstrate that (i) bacterial counts in the chicken gut were influenced by probiotic Lactobacillus administration, and (ii) chicken lactobacilli are resistant to ionophore coccidiostats under in vivo conditions. PMID: 9205734 [PubMed - indexed for MEDLINE] 36: Int J Antimicrob Agents. 1997 Jan;9(3):153-63. Quality assessment of glycopeptide susceptibility tests: a European collaborative study. European Glycopeptide Resistance Group. Brown DF, Courvalin P. Public Health and Clinical Microbiology Laboratory, Addenbrooke's Hospital, Cambridge, UK. The ability of seventy clinical laboratories in nine European countries to detect glycopeptide resistance in Gram-positive bacteria was investigated. Results of routine tests were compared with those on the same strains by a reference method in national co-ordinating laboratories. In addition, control strains were tested by some of the participants. Errors in reporting susceptibility of Staphylococcus aureus to teicoplanin and vancomycin and coagulase-negative staphylococci to vancomycin were < 1%. With coagulase-negative staphylococci however, 44 (3.4%) teicoplanin susceptible isolates were reported intermediate and six (0.4%) resistant; 18 (58.1%) of 31 teicoplanin intermediate isolates were reported susceptible and five (16.1%) resistant; and six of nine teicoplanin resistant isolates were reported susceptible and two intermediate. All seven isolates of enterococci intermediate to vancomycin were reported susceptible. Distribution of a known vancomycin intermediate strain of E. gallinarum indicated problems with vancomycin susceptibility testing (44.4% reported susceptible, 32.7% intermediate, 32.1% resistant) and identification (only 34.1% correct) of this organism. Two of 28 teicoplanin resistant enterococci and three of 30 vancomycin resistant isolates were reported susceptible. Among other organisms, one resistant Lactobacillus sp. was reported susceptible to teicoplanin and vancomycin. In reporting teicoplanin susceptible organisms, there were fewer errors with comparative/Stokes methods than with most other methods and more errors with the ATB and Sceptor methods than most other methods. None of the methods used were reliable for testing teicoplanin intermediate and resistant coagulase-negative staphylococci or low-level vancomycin resistant enterococci. Alternative methods, such as breakpoint screening, should be considered for detecting glycopeptide resistance. PMID: 9552711 [PubMed - indexed for MEDLINE] 37: Plasmid. 1997;37(3):199-203. Isolation and characterization of a plasmid from Lactobacillus fermentum conferring erythromycin resistance. Fons M, Hege T, Ladire M, Raibaud P, Ducluzeau R, Maguin E. Unite d'Ecologie et de Physiologie du Systeme Digestif, Institut National de la Recherche Agronomique, Jouy-en-Josas, France. Lactobacillus fermentum is a lactic acid bacterial species commonly found in the digestive tracts of pigs and rodents and also present in man. We characterized a 5.7-kb plasmid, pLEM3, conferring erythromycin resistance, which was isolated from a porcine strain of L. fermentum. Plasmid pLEM3 established efficiently in L. fermentum, conferred high-level erythromycin resistance (MIC > 1 mg/ml), and was segregationally stable. A deletion derivative of pLEM3, called pLEM5, was constructed and found to be as genetically stable as the parent. A multiple cloning site was inserted into pLEM5, generating plasmid pLEM7. Nucleotide sequence determination of pLEM5 revealed similarities with known genes. The replicon itself is a member of the pC194 family of rolling circle plasmids. The region responsible for erythromycin resistance was 98.2% identical to the erm gene of conjugative transposon Tn1545. PMID: 9200223 [PubMed - indexed for MEDLINE] 38: Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):10668-72. Multidrug resistance mediated by a bacterial homolog of the human multidrug transporter MDR1. van Veen HW, Venema K, Bolhuis H, Oussenko I, Kok J, Poolman B, Driessen AJ, Konings WN. Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Haren, The Netherlands. Resistance of Lactococcus lactis to cytotoxic compounds shares features with the multidrug resistance phenotype of mammalian tumor cells. Here, we report the gene cloning and functional characterization in Escherichia coli of LmrA, a lactococcal structural and functional homolog of the human multidrug resistance P-glycoprotein MDR1. LmrA is a 590-aa polypeptide that has a putative topology of six alpha-helical transmembrane segments in the N-terminal hydrophobic domain, followed by a hydrophilic domain containing the ATP-binding site. LmrA is similar to each of the two halves of MDR1 and may function as a homodimer. The sequence conservation between LmrA and MDR1 includes particular regions in the transmembrane domains and connecting loops, which, in MDR1 and the MDR1 homologs in other mammalian species, have been implicated as determinants of drug recognition and binding. LmrA and MDR1 extrude a similar spectrum of amphiphilic cationic compounds, and the activity of both systems is reversed by reserpine and verapamil. As LmrA can be functionally expressed in E. coli, it offers a useful prokaryotic model for future studies on the molecular mechanism of MDR1-like multidrug transporters. PMID: 8855237 [PubMed - indexed for MEDLINE] 39: Plasmid. 1996 Sep;36(2):116-24. Molecular characterization of a plasmid-borne (pTC82) chloramphenicol resistance determinant (cat-TC) from Lactobacillus reuteri G4. Lin CF, Fung ZF, Wu CL, Chung TC. Department of Veterinary Medicine, National Chung-Hsing University, Taichung, Taiwan, Republic of China. Lactobacillus reuteri G4 contains a 7.0-kb plasmid (pTC82) encoding resistance to chloramphenicol (Cm). Determination of the nucleotide sequence of the genetic determinant (cat-TC) encoding resistance to Cm on pTC82 revealed an open reading frame for a 238-amino-acid Cm acetyltransferase (CAT) monomer. This structural cat gene, 714 bp in length, was highly related (ca. 95% nucleotide and ca. 81% amino acid identity) to the 648-bp cat gene from Staphylococcus aureus plasmid pC194. A total of 6 bp transversions and 4 bp deletions was observed along the whole DNA sequence of cat-TC compared to that of cat-pC194. To determine the activity of the putative cat-TC gene, recombinant plasmid pUC8217 containing the cat determinant from pTC82 was subjected to a maxicell analysis. The observed molecular mass of the synthesized protein, based on electrophoretic mobility, was in reasonable agreement with the 27.3 kDa predicted from the DNA sequence. This is the first reported nucleotide sequence of a Cm-resistance determinant from L. reuteri and also the first evidence of adding Lactobacillus to the list of versatile bacterial genera which naturally acquire the cat-pC194 gene in the microbial ecological system. PMID: 8954883 [PubMed - indexed for MEDLINE] 40: Eur J Pediatr. 1996 May;155(5):421. Resistance of Bifidobacteria and Lactobacilli to tobramycin. Heine W, Mohr C, Ullrich S, Plath C, Uhlemann M. Publication Types: Letter PMID: 8741044 [PubMed - indexed for MEDLINE] 41: Gene. 1995 Jan 11;152(1):79-83. Erratum in: Gene 1995 Aug 8;161(1):139. Analysis of genes encoding D-alanine:D-alanine ligase-related enzymes in Leuconostoc mesenteroides and Lactobacillus spp. Elisha BG, Courvalin P. Unite des Agents Antibacteriens, Centre National de la Recherche Scientifique EP J0058, Institut Pasteur, Paris, France. Degenerate oligodeoxyribonucleotides complementary to sequences encoding conserved amino acid (aa) motifs in D-alanine:D-alanine ligases (Ddl) were used to amplify approx. 600-bp fragments from glycopeptide-resistant strains of Leuconostoc mesenteroides (Lm), Lactobacillus plantarum, La. salivarius and La. confusus, and from a susceptible strain of La. leichmannii. Comparison of the deduced aa sequences of the PCR products revealed that the Ddl-related enzymes of resistant Lm and Lactobacillus spp. are more akin to each other (47-63% aa identity) than to that of susceptible La. leichmannii (33-37% aa identity), indicating that the Ddl-related enzymes in these intrinsically resistant species of Gram+ bacteria exhibit structural differences with those in susceptible species. The Ddl-related enzymes, VanA and VanB, implicated in acquired resistance to glycopeptides in enterococci, were not closely related to their counterparts in Lm and Lactobacillus spp., as they displayed only 26-32% aa identity. PMID: 7828933 [PubMed - indexed for MEDLINE] 42: J Bacteriol. 1994 Apr;176(8):2398-405. Modification of peptidoglycan precursors is a common feature of the low-level vancomycin-resistant VANB-type Enterococcus D366 and of the naturally glycopeptide-resistant species Lactobacillus casei, Pediococcus pentosaceus, Leuconostoc mesenteroides, and Enterococcus gallinarum. Billot-Klein D, Gutmann L, Sable S, Guittet E, van Heijenoort J. Laboratoire de RMN, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France. The biochemical basis for the acquired or natural resistance of various gram-positive organisms to glycopeptides was studied by high-pressure liquid chromatographic analysis of their peptidoglycan UDP-MurNAc-peptide precursors. In all cases, resistance was correlated with partial or complete replacement of the C-terminal D-Ala-D-Ala-containing UDP-MurNAc-pentapeptide by a new precursor with a modified C terminus. Nuclear magnetic resonance analysis by sequential assignment showed that the new precursor encountered in Enterococcus faecium D366, a strain belonging to the VANB class, which expresses low-level resistance to vancomycin, was UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-lactate, identical to that previously found in the VANA class, which expresses high-level resistance to vancomycin. High-pressure liquid chromatographic analyses, composition determinations, and digestion by R39 D,D-carboxypeptidase demonstrated the exclusive presence of the new precursor in Lactobacillus casei and Pediococcus pentosaceus, which are naturally highly resistant to glycopeptides. The low-level natural resistance of Enterococcus gallinarum to vancomycin was found to be associated with the synthesis of a new precursor identified as a UDP-MurNAc-pentapeptide containing a C-terminal D-serine. The distinction between low and high levels of resistance to glycopeptides appeared also to depend on the presence or absence of a substantial residual pool of a D-Ala-D-Ala-containing UDP-MurNAc-pentapeptide. PMID: 8157610 [PubMed - indexed for MEDLINE] 43: J Bacteriol. 1994 Jan;176(1):260-4. Vancomycin-resistant Leuconostoc mesenteroides and Lactobacillus casei synthesize cytoplasmic peptidoglycan precursors that terminate in lactate. Handwerger S, Pucci MJ, Volk KJ, Liu J, Lee MS. Laboratory of Microbiology, Rockefeller University, New York, New York 10021. The emergence of acquired high-level resistance among Enterococcus species has renewed interest in mechanisms of resistance to glycopeptide antibiotics in gram-positive bacteria. In Enterococcus faecalis and Enterococcus faecium, resistance is encoded by the van gene cluster and is due to the production of a peptidoglycan precursor terminating in D-alanyl-D-lactate, to which vancomycin does not bind. Most Leuconostoc and many Lactobacillus species are intrinsically resistant to high levels of glycopeptide antibiotics, but the mechanism of resistance has not been elucidated. To determine whether the mechanisms of resistance are similar in intrinsically resistant bacteria, cytoplasmic peptidoglycan precursors were isolated from Leuconostoc mesenteroides and Lactobacillus casei and analyzed by mass spectrometry, revealing structures consistent with UDP-N-acetylmuramyl-L-Ala-D-Glu-L-Lys-(L-Ala)-D-Ala-D-lactate and UDP-N-acetylmuramyl-L-Ala-D-Glu-L-Lys-D-Ala-D-lactate, respectively. PMID: 8282706 [PubMed - indexed for MEDLINE] 44: Plasmid. 1994 Jan;31(1):60-71. Molecular characterization of a plasmid-borne (pGT633) erythromycin resistance determinant (ermGT) from Lactobacillus reuteri 100-63. Tannock GW, Luchansky JB, Miller L, Connell H, Thode-Andersen S, Mercer AA, Klaenhammer TR. Department of Microbiology, University of Otago, Dunedin, New Zealand. Lactobacillus reuteri 100-63 contains a 9.8 kb plasmid (pGT633) encoding resistance to the macrolide-lincosamide-streptogramin (MLS) type B antibiotics. The restriction endonucleases AccI, BamHI, BclI, EcoRI, EcoRV, HhaI, HindII, HpaI, KpnI, SalI, and SspI were employed to establish a physical map of pGT633. Next, genetic transfer experiments were conducted to establish the host range of pGT633. The results revealed that pGT633 replicated autonomously and encoded constitutive resistance to erythromycin in a variety of bacterial hosts, including Bacillus subtilis BD170, Streptococcus sanguis DL1, Staphylococcus aureus RN4220, and Enterococcus faecalis 19433, as well as several Lactobacillus spp. A 2.2 kb BamHI fragment of pGT633 containing the genetic determinant (ermGT) encoding resistance to erythromycin was cloned into Escherichia coli. Determination of the nucleotide sequence of ermGT revealed a methylase gene 731 bp in length that was highly related (ca. 81% nucleotide and ca. 78% amino acid identity) to the ermC gene from S. aureus plasmid pE194. The leader sequences of ermGT and ermC were identical except for a single base change at nt 51. PMID: 8171126 [PubMed - indexed for MEDLINE] 45: J Clin Microbiol. 1993 Sep;31(9):2499-501. Identification of vancomycin-resistant lactic bacteria isolated from humans. Mackey T, Lejeune V, Janssens M, Wauters G. Microbiology Unit, UCL 5490, University of Louvain, Brussels, Belgium. By using cell morphology, arginine dihydrolase, and gas production in de Man, Sharp, Rogosa broth, 122 isolates of vancomycin-resistant lactic bacteria from humans were assigned to five profiles, allowing us to distinguish Pediococcus, homofermentative and heterofermentative Lactobacillus, and Leuconostoc species. The absence of L-(+)-lactic acid, as detected spectrophotometrically, was confirmatory for Leuconostoc species. API 50 CHL panels were useful for the identification of Lactobacillus species. PMID: 8408575 [PubMed - indexed for MEDLINE] 46: Antimicrob Agents Chemother. 1993 Jun;37(6):1364-6. In vitro activity of ramoplanin against vancomycin-resistant gram-positive organisms. Collins LA, Eliopoulos GM, Wennersten CB, Ferraro MJ, Moellering RC Jr. Department of Medicine, New England Deaconess Hospital, Boston, Massachusetts 02215. In vitro activity of ramoplanin, a cyclic lipoglycopeptide, against 92 vancomycin-resistant gram-positive organisms was evaluated. Ramoplanin demonstrated potent activity against many highly vancomycin-resistant organisms including enterococci (MICs for 90% of strains tested of 0.5 micrograms/ml) and against Lactobacillus spp., Leuconostoc spp., and Pediococcus spp., all of which were inhibited at concentrations of < or = 0.25 micrograms/ml. This drug or a derivative compound merits further investigation as a potential therapeutic agent for infections due to vancomycin-resistant enterococci. PMID: 8328787 [PubMed - indexed for MEDLINE] 47: Diagn Microbiol Infect Dis. 1993 Feb;16(2):111-8. Antimicrobial activity and spectrum of rifaximin, a new topical rifamycin derivative. Hoover WW, Gerlach EH, Hoban DJ, Eliopoulos GM, Pfaller MA, Jones RN. Department of Pathology, University of Iowa College of Medicine, Iowa City. Rifaximin, a rifamycin derivative, was evaluated in vitro to assess its spectrum and potency against a wide variety of bacteria, yeasts, viruses, and parasites. High concentrations of rifaximin were often used to reflect topically achieved levels since this compound is poorly absorbed by oral route. Like rifampin, rifaximin possessed best activity against Staphylococcus spp. (MIC50 < or = 0.015 microgram/ml), Streptococcus spp. (MIC50s, < or = 0.03-0.12 microgram/ml), Enterococcus spp. (MIC50s, 0.25-2 micrograms/ml), Bacillus cereus (MIC50, 0.06 microgram/ml), Moraxella catarrhalis (MIC50, < or = 0.03 microgram/ml), and Haemophilus influenzae (MIC50, 0.25 microgram/ml). Rifaximin demonstrated potential use as a topical agent for bacterial vaginosis by inhibiting Bacteroides bivius-disiens, Gardnerella vaginalis, Lactobacillus spp., and Mobiluncus spp. strains (all MICs < or = 1 microgram/ml). Strains of Haemophilus ducreyi and Neisseria gonorrhoeae (MIC50s, 0.25 microgram/ml) were also inhibited. However, some organisms associated with genital tract infections were rifaximin resistant, for example, Candida spp., herpes virus, mycoplasmas, Trichomonas vaginalis, and Ureaplasma urealyticum. Clinical trials appear warranted using rifaximin topical concentrations that will minimize mutations to rifamycin resistance. PMID: 8385592 [PubMed - indexed for MEDLINE] 48: FEMS Microbiol Lett. 1992 Jul 1;73(1-2):101-4. Correlation between hydrophobicity and resistance to nonoxynol-9 and vancomycin for urogenital isolates of lactobacilli. Tomeczek L, Reid G, Cuperus PL, McGroarty JA, van der Mei HC, Bruce AW, Khoury AE, Busscher HJ. Department of Microbiology, University of Toronto, Canada. Seven clinical isolates of lactobacilli were found to be relatively hydrophobic with a mean water-contact angle of 66 +/- 15 degrees, and to be susceptible to 1% nonoxynol-9 and vancomycin. However, seven other strains were relatively hydrophilic with a mean water-contact angle of 32 +/- 13 degrees, and found to be resistant to 25% nonoxynol-9 and vancomycin. Thus, the surface properties of lactobacilli that influence susceptibility to antimicrobial agents may involve surface hydrophobicity. Possibly the penetration barrier posed by the cell surface towards these two nonionic antimicrobials is lower for hydrophobic cells than for hydrophilic cells. PMID: 1325935 [PubMed - indexed for MEDLINE] 49: APMIS. 1992 Jun;100(6):543-52. In vitro activity of teicoplanin and vancomycin against gram-positive bacteria from human clinical and veterinary sources. Jensen KT, Schonheyder H, Pers C, Thomsen VF. Department of Clinical Microbiology, Statens Seruminstitut, Copenhagen, Denmark. The minimum inhibitory concentration (MIC) of teicoplanin and vancomycin was determined by the agar dilution method for 186 Gram-positive bacteria from human clinical and veterinary sources. Teicoplanin MIC values were less than or equal to 4 micrograms/ml for 94% of staphylococci (group A, n = 52) and less than or equal to 2 micrograms/ml for all streptococci, enterococci, aerococci and pediococci (group B, n = 75). Seventy-eight percent of Gram-positive rods, Rhodococcus and Leuconostoc spp. (group C, n = 59) were inhibited by 4 micrograms/ml. Teicoplanin resistance (MIC greater than or equal to 16 micrograms/ml) was demonstrated for all Nocardia strains and for some strains of Lactobacillus, E. rhusiopathiae, Leuconostoc, and S. haemolyticus. Cross-resistance between teicoplanin and vancomycin was observed for all Nocardia strains and for some strains of Lactobacillus, E. rhusiopathiae, and Leuconostoc. Three methicillin-resistant S. haemolyticus strains were either resistant or intermediately susceptible to teicoplanin and susceptible to vancomycin. Eight strains (motile enterococci four, E. rhusiopathiae three and Leuconostoc sp. one) were susceptible to teicoplanin and resistant to vancomycin. Teicoplanin disc diffusion on Danish Blood Agar with NeoSensitabs (Rosco), PDM AB Biodisc and locally prepared discs revealed a wide range of zone diameters in groups B and C. The relation between MIC values and zone diameters for teicoplanin was analysed by the error-rate bounded method. Zone size interpretive criteria as suggested by the manufacturers (greater than or equal to 15 mm) produced 2.7% (95% confidence limits 0.9-6.2%) and 1.6% (95% confidence limits 0.3-4.6%) very major errors for NeoSensitabs and PDM-disc, respectively. Using a zone size breakpoint for susceptibility of greater than or equal to 25 mm for NeoSensitabs and greater than or equal to 20 mm for PDM-disc, the proportions of very major errors were 0.5% (95% confidence limits 0.0-3.0%) at the expense of 5.9% (95% confidence limits 3.0-10.3%) indeterminate strains that belonged to E. rhusiopathiae, Leuconostoc, Lactobacillus and S. haemolyticus. However, using these zone size breakpoints five major errors (beta-haemolytic streptococci, group B three, S. aureus one, Leuconostoc sp. one) were observed for NeoSensitabs and two major errors (beta-haemolytic streptococcus, group B one, Leuconostoc sp. one) were observed for PDM-disc. Susceptibility testing against teicoplanin among these taxa should therefore include a determination of MIC. PMID: 1535201 [PubMed - indexed for MEDLINE] 50: Plasmid. 1992 May;27(3):169-76. Mobilization and location of the genetic determinant of chloramphenicol resistance from Lactobacillus plantarum caTC2R. Ahn C, Collins-Thompson D, Duncan C, Stiles ME. Department of Food Science, University of Alberta, Edmonton, Canada. The mobilization of a nonconjugative plasmid (pCaT) that mediates chloramphenicol resistance in Lactobacillus plantarum caTC2R was achieved by comobilization with the conjugative plasmid pAM beta 1. The conjugation studies confirmed that the 8.5-kb pCaT in L. plantarum caTC2R contains the gene responsible for chloramphenicol resistance and that the plasmid has several unique restriction sites which make it useful for genetic studies in Carnobacterium spp. Cloning studies showed that the gene responsible for chloramphenicol resistance is located in the 2.6-kb EcoRV-SalI region of pCaT. This was confirmed by probing the 3.0-kb BglII fragment of pCaT with a biotin-labeled 1.6-kb BstEII-HpaII fragment from the streptococcal-derived plasmid pVA797(Cmr). Expression of chloramphenicol resistance in Carnobacterium as well as in other Lactobacillus species was achieved by electrotransformation using donor DNA from pCaT. PMID: 1513874 [PubMed - indexed for MEDLINE] 51: Antimicrob Agents Chemother. 1990 Apr;34(4):543-9. Antimicrobial susceptibility of vancomycin-resistant Leuconostoc, Pediococcus, and Lactobacillus species. Swenson JM, Facklam RR, Thornsberry C. Antimicrobics Investigation Branch, Centers for Disease Control, Atlanta, Georgia 30333. Eighty-five strains of vancomycin-resistant gram-positive bacteria from three genera, Leuconostoc, Pediococcus, and Lactobacillus, were tested to determine susceptibility to 24 antimicrobial agents by broth microdilution and to 10 agents by disk diffusion. The MICs of vancomycin and teicoplanin ranged from 64 to greater than 512 micrograms/ml; however, the MICs of daptomycin, a new lipopeptide, were all less than or equal to 0.25 micrograms/ml. None of the organisms were resistant to imipenem, minocycline, chloramphenicol, gentamicin, or daptomycin. The MICs of penicillin were in the moderately susceptible range for all but three strains. Susceptibility to the other agents varied by genus and, in some cases, by species. When disk diffusion results were compared with MICs for drugs recommended for streptococci by the National Committee for Clinical Laboratory Standards, Villanova, Pa., few very major or major errors were obtained, but the number of minor errors was 19.3%. Therefore, we recommended that MIC testing be used instead of disk diffusion testing for these organisms. PMID: 2344161 [PubMed - indexed for MEDLINE] 52: Plasmid. 1990 Mar;23(2):119-25. Characterization of plasmids and plasmid-borne macrolide resistance from Lactobacillus sp. strain 100-33. Rinckel LA, Savage DC. Department of Microbiology, University of Tennessee, Knoxville 37996. Lactobacillus sp. strain 100-33 is resistant to macrolides, lincosamides, and streptogramin B-type antibiotics (MLSR) and appears to contain several major and minor plasmids. One of these plasmids, pLAR33, is approximately 18 kbp in size. When cells of strain 100-33 were protoplasted and regenerated, an MLSS isolate was derived. The derivative, designated strain ES1, contained a unique plasmid complement in which it had apparently lost the major plasmids of the parental strain, including pLAR33, and retained only a minor plasmid seen in low concentrations in strain 100-33. The MLSR determinant was cloned from plasmid DNA of strain 100-33 on a 3-kbp EcoRV fragment into pBR322 and localized to pLAR33. The determinant expressed macrolide and lincosamide resistance in Escherichia coli HB101, was localized to approximately 1 kbp on the cloned sequence, and is apparently under the control of its own promoter. MLSR electroporants were derived from strain ES1 electroporated with plasmid DNA from strain 100-33; these MLSR isolates had acquired a plasmid complement similar to that of strain 100-33, including pLAR33. Endonuclease digestion and Southern analysis of plasmid DNA from both strains indicated that the major plasmids are multimeric and deleted forms of one archetypal extrachromosomal element. PMID: 2163536 [PubMed - indexed for MEDLINE] 53: Antimicrob Agents Chemother. 1990 Feb;34(2):261-4. Genetic basis of tetracycline resistance in urogenital bacteria. Roberts MC, Hillier SL. Department of Pathobiology, University of Washington, Seattle 98195. The distributions of the nucleotide sequences related to the tetracycline resistance determinants Tet K, Tet L, Tet M, and Tet O were studied by dot blot hybridization with randomly chosen clinical urogenital tract isolates of viridans group streptococci, Streptococcus agalactiae, Enterococcus faecalis, Gardnerella vaginalis, Lactobacillus spp., Fusobacterium nucleatum, Peptostreptococcus spp., and Veillonella parvula. Among the Peptostreptococcus spp., 79% of the isolates hybridized with one (64%) or more (36%) of the probes for Tet K (27%), Tet L (30%), Tet M (75%) and Tet O (13%). Of the viridans group streptococci, 82% of the strains hybridized with one (34%) or more (66%) of the four probes. The distribution of the four determinants in this group was as follows: Tet K, 36%; Tet L, 31%; Tet M, 43%; Tet O, 61%. Twenty-nine percent of the enterococci and forty-six percent of the group B streptococci hybridized with the probes; however, the Tet K, Tet L, and Tet O determinants were found in only a few strains, while the Tet M determinant predominated. A total of 29% of the F. nucleatum isolates, 55% of the G. vaginalis isolates, and 26% of the V. parvula isolates hybridized with the Tet M determinant. In contrast, 43% of the Lactobacillus spp. hybridized with the Tet O determinant. The data indicate that tetracycline resistance determinants are common to many of the microorganisms isolated from the urogenital tract. PMID: 2327774 [PubMed - indexed for MEDLINE] 54: Antimicrob Agents Chemother. 1989 Sep;33(9):1477-81. Activity of glycopeptides against vancomycin-resistant gram-positive bacteria. Nicas TI, Cole CT, Preston DA, Schabel AA, Nagarajan R. Lilly Research Laboratories, Eli Lilly & Co., Indianapolis, Indiana 46285-0438. Gram-positive bacteria resistant to vancomycin are rare; but they include members of the genera Leuconostoc, Lactobacillus, and Pediococcus, as well as recently emerging vancomycin-resistant strains of Enterococcus faecium and Enterococcus faecalis. Vancomycin, teicoplanin, and several vancomycin derivatives were tested for their activities against vancomycin-resistant gram-positive bacteria. Vancomycin-resistant E. faecium and E. faecalis were generally cross-resistant to other glycopeptides, but some N-substituted vancomycin derivatives were active against the resistant strains, with MICs of 2 to 32 micrograms/ml. These vancomycin derivatives also had significant levels of activity against intrinsically vancomycin-resistant organisms such as Leuconostoc sp. While vancomycin resistance in E. faecium and E. faecalis was inducible, resistance in members of the genera Leuconostoc, Lactobacillus, and Pediococcus appeared to be expressed constitutively. Antibody to a vancomycin-induced membrane protein found in membranes of resistant enterococci did not detect a cross-reacting protein in other vancomycin-resistant species. PMID: 2817848 [PubMed - indexed for MEDLINE] 55: Antimicrob Agents Chemother. 1989 Aug;33(8):1383-4. In vitro activities of daptomycin and other antimicrobial agents against vancomycin-resistant gram-positive bacteria. de la Maza L, Ruoff KL, Ferraro MJ. Francis Blake Bacteriology Laboratories, Massachusetts General Hospital, Boston 02114. A comparative evaluation of daptomycin and eight other antimicrobial agents was performed by the agar dilution technique with 56 strains of vancomycin-resistant gram-positive bacteria, including Leuconostoc, Lactobacillus, and Pediococcus spp. Erythromycin, deptomycin, clindamycin, and gentamicin exhibited the greatest activities, whereas penicillin, ampicillin, and cefotaxime showed moderate activities. The organisms were all highly resistant to vancomycin and cefoxitin. PMID: 2552910 [PubMed - indexed for MEDLINE] 56: FEMS Microbiol Lett. 1989 Jul 15;51(1):149-52. Conjugal transfer of plasmid pIP501 from Lactococcus lactis to Lactobacillus delbruckii subsp. bulgaricus and Lactobacillus helveticus. Langella P, Chopin A. Laboratoire de Genetique Microbienne, INRA-Domaine de Vilvert, Jouy-en-Josas, France. Plasmid pIP501 was transferred by conjugation from Lactococcus lactis to Lactobacillus delbruckii subsp. bulgaricus and Lactobacillus helveticus. Only Lb. delbruckii subsp. bulgaricus transconjugants could act as a donor in crosses with Lc. lactis. No Lactobacillus transconjugants were detected after inter- or intra-species Lactobacillus crosses. Plasmid pIP501 has undergone no detectable deletion or rearrangement during transfer from Lc. lactis to Lactobacillus strains. PMID: 2506107 [PubMed - indexed for MEDLINE] 57: J Dent Res. 1988 Dec;67(12):1518-22. Antibiotic resistance and production of inhibitory substances among bacterial strains isolated from the oral cavities of BALB/c mice. St-Amand L, Lavoie MC. Departement de biochimie, Faculte des sciences et de genie, Universite Laval, Quebec, Canada. In the oral cavities of BALB/c mice, microbial population levels are regulated by multifactorial processes. Factors include the production of inhibitory substances and the exchange of genetic material. In this work, 371 isolates from different sites (saliva, tongue, teeth, and mucosa) of the oral cavities of BALB/c mice were screened for resistance to antibiotics and antimicrobial activity. Antibiotic-resistant strains represented 25% of the total flora. Among the predominant species, all the S. faecalis isolates showed multiresistance, and 23% of the Lactobacillus murinus isolates and 15% of the Staphylococcus aureus were resistant to at least one antibiotic. Resistance to aminoglycosides (neomycin, streptomycin, kanamycin, and gentamicin) was most frequently encountered. In S. faecalis, high levels of resistance were recorded to neomycin and streptomycin but not to gentamicin or kanamycin. Macrolides (M), lincosamides (L), streptogramin B (S), tetracycline (Tc), and chloramphenicol (Cm) resistance was also present in multiresistance patterns, especially among S. faecalis isolates. Hemolytic (Hly+) streptococci were less resistant to MLS, Tc, and Cm than were non-hemolytic (Hly-) isolates. Resistance to beta-lactam antibiotics was detected only among staphylococci and with a low prevalence (4%). The frequencies of strains producing antimicrobial substances against the indicator strains (S. mutans LG-1, S. sanguis Ny 101, and A. viscosus Ny 1) were high for L. murinus (76%) and S. faecalis (57% for Hly- and 90% for Hly+), but low for S. aureus (7%). These results indicate that the indigenous oral flora could interfere with colonization by allochthonous micro-organisms and that resistance patterns should be taken into account for the elimination of the oral indigenous flora by antibiotic treatment.(ABSTRACT TRUNCATED AT 250 WORDS) PMID: 3143751 [PubMed - indexed for MEDLINE] 58: J Appl Bacteriol. 1988 Nov;65(5):371-5. In vivo transfer of pAM beta 1 from Lactobacillus reuteri to Enterococcus faecalis. Morelli L, Sarra PG, Bottazzi V. Istituto di Microbiologia, Universita Cattolica del Sacro Cuore, Piacenza, Italy. Trials were conducted to determine the in vivo transferability of plasmid-mediated antibiotic resistance between two strains of enteric Gram-positive bacteria. Germ-free mice were associated with the donor Lactobacillus reuteri DSM 20016 strain, carrying the broad host range pAM beta 1 plasmid, and with the Enterococcus faecalis JH2SS recipient strain. Analysis of faecal content of associated mice demonstrated that the in vivo transfer of this plasmid did occur and that frequencies of conjugation were affected by the presence of subtherapeutic levels of antibiotic in the diet. PMID: 2976754 [PubMed - indexed for MEDLINE] 59: J Clin Microbiol. 1988 Oct;26(10):2064-8. Vancomycin-resistant gram-positive bacteria isolated from human sources. Ruoff KL, Kuritzkes DR, Wolfson JS, Ferraro MJ. Francis Blake Bacteriology Laboratories, Massachusetts General Hospital, Boston 02114. Recent reports of infections with vancomycin-resistant gram-positive bacteria prompted us to study vancomycin-resistant isolates from human sources to characterize the types of bacteria displaying this phenotype. Thirty-six vancomycin-resistant gram-positive isolates, 14 from clinical specimens and 22 from stool samples, were identified. These isolates were tentatively identified as Lactobacillus spp. (25 strains), Leuconostoc spp. (6 strains), and Pediococcus spp. (3 strains) on the basis of morphology and physiological tests. Two isolates of indeterminate morphology could not be unambiguously assigned to a genus. Four isolates of vancomycin-resistant lactobacilli from normally sterile body sites were considered to be clinically significant. Vancomycin-resistant gram-positive bacteria may represent an emerging class of nosocomial pathogens. Better methods for distinguishing the various genera in the clinical microbiology laboratory are needed. PMID: 3182995 [PubMed - indexed for MEDLINE] 60: Plasmid. 1988 Sep;20(2):171-4. Identification and cloning of a plasmid-encoded erythromycin resistance determinant from Lactobacillus reuteri. Axelsson LT, Ahrne SE, Andersson MC, Stahl SR. Department of Microbiology, Swedish University of Agricultural Sciences, Uppsala. Plasmid analysis, plasmid curing, cloning, and hybridization experiments were used to study four Lactobacillus reuteri strains showing high resistance to erythromycin. Plasmid curing with acriflavine resulted in a loss of erythromycin resistance in a frequency of 1-10%. For three of the strains this was accompanied by a loss of a 6.9-MDa plasmid, which was shown to be identical for the different strains and designated pLUL631. The erythromycin (erm) gene was located on a 5.5-MDa plasmid in the fourth strain. A restriction map of pLUL631 was constructed and the location of the erm gene on the plasmid was identified by cloning in Escherichia coli. By using a Streptococcus lactis-E. coli shuttle vector, the erm gene was also transformed to S. lactis and expressed. The erm gene from L. reuteri was shown to be related to the erm gene from pIP501 (Streptococcus agalactiae) by DNA-DNA hybridization. PMID: 3237864 [PubMed - indexed for MEDLINE] 61: J Appl Bacteriol. 1988 Jul;65(1):43-7. Metabolism and some characteristics of lactobacilli isolated from the rumen of young calves. Marounek M, Jehlickova K, Kmet V. Institute of Animal Physiology and Genetics, Prague, Czechoslovakia. Fourteen strains of lactobacilli isolated from the rumen of young calves were studied to determine their biochemical characteristics, growth parameters, metabolism on lactose and sensitivity to 28 antimicrobial agents. Thirteen homofermentative strains belonged to Lactobacillus acidophilus and one heterofermentative strain resembled Lact. fermentum. The relevance of rumen lactobacilli to the nutrition of calves is discussed. PMID: 3209515 [PubMed - indexed for MEDLINE] 62: J Infect. 1988 May;16(3):279-83. Vancomycin resistance of clinical isolates of lactobacilli. Holliman RE, Bone GP. Public Health Laboratory, St George's Hospital, London, U.K. Strains of lactobacilli were cultured from two patients with clinical evidence of infection. Each isolate was found to be highly resistant to vancomycin but inactivation of the antibiotic could not be demonstrated. Lactobacillus isolates should be regarded as potential pathogens. Reliable vancomycin susceptibility testing is required since susceptibility among Gram-positive bacteria in general should not be assumed. Publication Types: Case Reports PMID: 3135337 [PubMed - indexed for MEDLINE] 63: J Hosp Infect. 1988 Apr;11(3):292. Vancomycin resistant lactobacilli. Golledge C. Publication Types: Letter PMID: 2899115 [PubMed - indexed for MEDLINE] 64: Appl Environ Microbiol. 1988 Mar;54(3):824-6. Conjugal transfer of group B streptococcal plasmids and comobilization of Escherichia coli-Streptococcus shuttle plasmids to Lactobacillus plantarum. Shrago AW, Dobrogosz WJ. Department of Microbiology, North Carolina State University, Raleigh 27695. The antibiotic resistance group B streptococcal plasmids, pIP501 and pVA797, were conjugally transferred from Streptococcus faecalis to Lactobacillus plantarum. The Escherichia coli-Streptococcus shuttle plasmids, pVA838 and pSA3, were mobilized from S. sanguis to L. plantarum by pVA797 via cointegrate formation. pVA838 readily resolved from pVA797 and was present in L. plantarum as deletion derivatives. The pVA797::pSA3 cointegrate failed to resolve in L. plantarum. PMID: 3132101 [PubMed - indexed for MEDLINE] 65: Appl Environ Microbiol. 1987 Jul;53(7):1620-5. Susceptibility and resistance of ruminal bacteria to antimicrobial feed additives. Nagaraja TG, Taylor MB. Department of Animal Sciences and Industry, Kansas State University, Manhattan 66506. Susceptibility and resistance of ruminal bacterial species to avoparcin, narasin, salinomycin, thiopeptin, tylosin, virginiamycin, and two new ionophore antibiotics, RO22-6924/004 and RO21-6447/009, were determined. Generally, antimicrobial compounds were inhibitory to gram-positive bacteria and those bacteria that have gram-positive-like cell wall structure. MICs ranged from 0.09 to 24.0 micrograms/ml. Gram-negative bacteria were resistant at the highest concentration tested (48.0 micrograms/ml). On the basis of their fermentation products, ruminal bacteria that produce lactic acid, butyric acid, formic acid, or hydrogen were susceptible and bacteria that produce succinic acid or ferment lactic acid were resistant to the antimicrobial compounds. Selenomonas ruminantium was the only major lactic acid-producing bacteria resistant to all the antimicrobial compounds tested. Avoparcin and tylosin appeared to be less inhibitory (MIC greater than 6.0 micrograms/ml) than the other compounds to the two major lactic acid-producing bacteria, Streptococcus bovis and Lactobacillus sp. Ionophore compounds seemed to be more inhibitory (MIC, 0.09 to 1.50 micrograms/ml) than nonionophore compounds (MIC, 0.75 to 12.0 micrograms/ml) to the major butyric acid-producing bacteria. Treponema bryantii, an anaerobic rumen spirochete, was less sensitive to virginiamycin than to the other antimicrobial compounds. Ionophore compounds were generally bacteriostatic, and nonionophore compounds were bactericidal. The specific growth rate of Bacteroides ruminicola was reduced by all the antimicrobial compounds except avoparcin. The antibacterial spectra of the feed additives were remarkably similar, and it appears that MICs may not be good indicators of the potency of the compounds in altering ruminal fermentation characteristics. PMID: 3116929 [PubMed - indexed for MEDLINE] 66: J Hosp Infect. 1987 Jul;10(1):1-3. Vancomycin-resistant leuconostocs, lactobacilli and now pediococci. Colman G, Efstratiou A. Division of Hospital Infection, Central Public Health Laboratory, London. Publication Types: Editorial PMID: 2888805 [PubMed - indexed for MEDLINE] 67: Appl Environ Microbiol. 1985 Nov;50(5):1319-21. Plasmid profiles and transfer of plasmid-encoded antibiotic resistance in Lactobacillus plantarum. West CA, Warner PJ. Plasmids were visualized in strains of Lactobacillus plantarum by use of a rapid method. Plasmids pIP501 and pAM beta 1 were transferred by conjugation from Streptococcus strains to Lactobacillus plantarum, and recipient strains were shown to act as donors in crosses to S. lactis. Attempts to transfer these plasmids between strains of L. plantarum were not successful. PMID: 4091561 [PubMed - indexed for MEDLINE] 68: J Antimicrob Chemother. 1985 Jul;16 Suppl A:91-100. Multiplicity of macrolide-lincosamide-streptogramin antibiotic resistance determinants. Courvalin P, Ounissi H, Arthur M. Bacteria can resist macrolide, lincosamide, and streptogramin (MLS) antibiotics enzymically by alteration of the target site or detoxification of the antibiotic. N6-dimethylation of adenine in 23 S ribosomal RNA confers resistance to M, L, and S B-type (MLSB) antibiotics. Investigation, by DNA annealing, of the relationship between the genes specifying this resistance mechanism from Streptococcus (groups A,B,D, and H, and pneumoniae), Staphylococcus aureus, Bacillus licheniformis, Bacteroides fragilis, Lactobacillus casei, and Streptomyces erythreus indicated substantial sequence diversity among the MLSB resistance (R) determinants. A minimum of four distinct classes of MLSB R determinants could be defined: classes A and B for the Gram-positive cocci (streptococci pmeumococci and staphylococci), class C for B. licheniformis, and class D for Bact. fragilis. These data do not support the hypothesis that the R determinants were acquired recently from a single common origin and suggest an easy exchange of genetic information among the Gram-positive cocci. The genetic classes do not correlate with differences in phenotypic expression or in regulation (inducibility or constitutivity) of resistance towards MLSB antibiotics. Inactivation of the drug confers resistance to M and/or L and/or S or SA or SB antibiotics and has been detected in strains of Streptococcus, Staph. aureus, Lactobacillus, C. perfringens, Streptomyces, and recently in the Gram-negative organism Escherichia coli. We have cloned and sequenced a DNA fragment conferring high level resistance (MIC greater than 2 g/l) to erythromycin by hydrolysis of the antibiotic. The distribution of this 'new' character in enterobacteria isolated from human faeces was studied by colony hybridization using an intragenic probe. The gene for the erythromycin esterase was detected in numerous strains of E. coli belonging to various biotypes, in Klebsiella pneumoniae, Enterobacter agglomerans, and in one 'coliform'. Moreover, our results indicated the existence of at least two classes of genes specifying resistance to erythromycin by inactivation of the antibiotic in enterobacteria. PMID: 3932312 [PubMed - indexed for MEDLINE] 69: Microbiologica. 1983 Apr;6(2):145-54. Plasmids and antibiotic resistances in Lactobacillus helveticus and Lactobacillus bulgaricus isolated from natural whey culture. Morelli L, Vescovo M, Bottazzi V. PMID: 6688117 [PubMed - indexed for MEDLINE] 70: Ann Microbiol (Paris). 1981 Jan-Feb;132A(1):51-7. Degradation of macrolide-lincosamide-streptogramin antibiotics by Lactobacillus strains from animals. Dutta GN, Devriese LA. Ability to degrade macrolide, lincosamide and streptogramin antibiotics was noted in Lactobacillus strains isolated from the caeca of pigs, cattle and poultry. Some of the strains degraded all the three classes of antibiotics while some degraded either macrolides and lincosamides or macrolidese and streptogramins. There were also strains which degraded only macrolide or streptogramin type A antibiotics. Strains with any of these degradation abilities were always resistant to the antibiotics concerned, but strains with resistance to these antibiotics were often, but not always able to degrade them. PMID: 6789742 [PubMed - indexed for MEDLINE] 71: Antimicrob Agents Chemother. 1980 Mar;17(3):359-63. Bactericidal synergy between penicillin or ampicillin and aminoglycosides against antibiotic-tolerant lactobacilli. Bayer AS, Chow AW, Morrison JO, Guze LB. The bactericidal activities of penicillin G and ampicillin alone were compared with those of their combinations with streptomycin or gentamicin against 17 strains of lactobacilli classified as tolerant to various beta-lactam antibiotics. The penicillin G combinations with streptomycin and gentamicin were synergistic against 17 and 16 of these strains, respectively, whereas the corresponding ampicillin-aminoglycoside combinations were synergistic against 12 and 15 strains, respectively. Importantly, synergy was manifested at concentrations of these antibiotics that are attained in serum after their administration in conventional dose regimens. In no instances were combinations antagonistic. These in vitro observations provide a partial explanation for the favorable results obtained in preliminary clinical evaluations of the benefits of combination regimens in the treatment of lactobacillus infections refractory to single-drug therapy. PMID: 6903434 [PubMed - indexed for MEDLINE] 72: J Bacteriol. 1979 Jan;137(1):614-9. Transfer of plasmid-mediated antibiotic resistance from streptococci to lactobacilli. Gibson EM, Chace NM, London SB, London J. The transmissible plasmid pAMbeta1, which codes for erythromycin and lincomycin resistance, was conjugally transferred from a Lancefield group F Streptococcus to a strain of Streptococcus avium. Both organisms served as pAMbeta1 donors for three strains of Lactobacillus casei. Introduction of pAMbeta1 into one of the L. casei strains caused the organism to lose its native 6.7 X 10(6)-dalton plasmid. Loss of the native plasmid produced no alterations in the organism's growth characteristics or fermentation pattern. PMID: 104973 [PubMed - indexed for MEDLINE]