Two genes involved in clindamycin resistance of Bacillus licheniformis and Bacillus paralicheniformis identified by comparative genomic analysis. - Related Documents




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522501.0000Two genes involved in clindamycin resistance of Bacillus licheniformis and Bacillus paralicheniformis identified by comparative genomic analysis. We evaluated the minimum inhibitory concentrations of clindamycin and erythromycin toward 98 Bacillus licheniformis strains isolated from several types of fermented soybean foods manufactured in several districts of Korea. First, based on recent taxonomic standards for bacteria, the 98 strains were separated into 74 B. licheniformis strains and 24 B. paralicheniformis strains. Both species exhibited profiles of erythromycin resistance as an acquired characteristic. B. licheniformis strains exhibited acquired clindamycin resistance, while B. paralicheniformis strains showed unimodal clindamycin resistance, indicating an intrinsic characteristic. Comparative genomic analysis of five strains showing three different patterns of clindamycin and erythromycin resistance identified 23S rRNA (adenine 2058-N6)-dimethyltransferase gene ermC and spermidine acetyltransferase gene speG as candidates potentially involved in clindamycin resistance. Functional analysis of these genes using B. subtilis as a host showed that ermC contributes to cross-resistance to clindamycin and erythromycin, and speG confers resistance to clindamycin. ermC is located in the chromosomes of strains showing clindamycin and erythromycin resistance and no transposable element was identified in its flanking regions. The acquisition of ermC might be attributable to a homologous recombination. speG was identified in not only the five genome-analyzed strains but also eight strains randomly selected from the 98 test strains, and deletions in the structural gene or putative promoter region caused clindamycin sensitivity, which supports the finding that the clindamycin resistance of Bacillus species is an intrinsic property.202032271828
599610.9998Molecular characterization of intrinsic and acquired antibiotic resistance in lactic acid bacteria and bifidobacteria. The minimum inhibitory concentrations (MICs) of 6 different antibiotics (chloramphenicol, clindamycin, erythromycin, streptomycin, tetracycline and vancomycin) were determined for 143 strains of lactic acid bacteria and bifidobacteria using the Etest. Different MICs were found for different species and strains. Based on the distribution of these MIC values, most of the strains were either susceptible or intrinsically resistant to these antibiotics. However, the MIC range of some of these antibiotics showed a bimodal distribution, which suggested that some of the tested strains possess acquired antibiotic resistance. Screening for resistance genes was performed by PCR using specific primers, or using a DNA microarray with around 300 nucleotide probes representing 7 classes of antibiotic resistance genes. The genes identified encoded resistance to tetracycline [tet(M), tet(W), tet(O) and tet(O/W)], erythromycin and clindamycin [erm(B)] and streptomycin [aph(E) and sat(3)]. Internal portions of some of these determinants were sequenced and found to be identical to genes described in other bacteria. All resistance determinants were located on the bacterial chromosome, except for tet(M), which was identified on plasmids in Lactococcus lactis. The contribution of intrinsic multidrug transporters to the antibiotic resistance was investigated by cloning and measuring the expression of Bifidobacterium breve genes in L. lactis.200817957105
584920.9997Characterisation and molecular cloning of the novel macrolide-streptogramin B resistance determinant from Staphylococcus epidermidis. A total of 110 staphylococcal isolates from human skin were found to express a novel type of erythromycin resistance. The bacteria were resistant to 14-membered ring macrolides (MIC 32-128 mg/l) but were sensitive to 16-membered ring macrolides and lincosamides. Resistance to type B streptogramins was inducible by erythromycin. A similar phenotype, designated MS resistance, was previously described in clinical isolates of coagulase-negative staphylococci from the USA. In the UK, MS resistance is widely distributed in coagulase-negative staphylococci but was not detected in 100 erythromycin resistant clinical isolates of Staphylococcus aureus. Tests for susceptibility to a further 16 antibiotics failed to reveal any other selectable marker associated with the MS phenotype. Plasmid pattern analysis of 48 MS isolates showed considerable variability between strains and no common locus for the resistance determinant. In one strain of S. epidermidis co-resistance to tetracycline, penicillin and erythromycin (MS) was associated with a 31.5 kb plasmid, pUL5050 which replicated and expressed all three resistances when transformed into S. aureus RN4220. The MS resistance determinant was localised to a 1.9 kb fragment which was cloned on to the high-copy-number vector, pSK265. A constitutive mutant of S. aureus RN4220 containing the 1.9 kb fragment remained sensitive to clindamycin. This observation, together with the concentration-dependent induction (optimum 5 mg/l of erythromycin) of virginiamycin S resistance suggests that the MS phenotype is not due to altered expression of MLS resistance determinants (erm genes) but probably occurs via a different mechanism.19892559912
522630.9997Human vaginal Lactobacillus rhamnosus harbor mutation in 23S rRNA associated with erythromycin resistance. Little is known about the diversity and distribution of resistance determinants in human commensal bacteria. The aim of this study was to determine the molecular mechanism responsible for high-level erythromycin resistance among five human vaginal Lactobacillus rhamnosus isolates. PCR screening for the presence of ermA, ermB and ermC methylase genes revealed no determinants responsible for detected erythromycin resistance. Therefore, sequences of 23S rRNA genes from L. rhamnosus strains were studied by PCR-RFLP analysis and sequencing of 23S rRNA genes. According to the results, in all erythromycin-resistant L. rhamnosus strains, the presence of a A-->G transition mutation at position 2058 was discovered. Additionally, the isolates exhibited heterozygosity for the A2058/G2058 mutation among 23S rRNA gene copies. Presumably, the greatest number of mutated 23S rRNA operons was observed for the L. rhamnosus BGHV1' strain that also had the highest MIC for erythromycin (MIC=2048 microg mL(-1)). This study reports the presence of transition mutations in the V region of 23S rRNA genes that most probably account for high-level erythromycin resistance observed for the first time in human vaginal lactobacilli.200919619642
549940.9997Antibiotic Resistance/Susceptibility Profiles of Staphylococcus equorum Strains from Cheese, and Genome Analysis for Antibiotic Resistance Genes. In food, bacteria carrying antibiotic resistance genes could play a prominent role in the spread of resistance. Staphylococcus equorum populations can become large in a number of fermented foods, yet the antibiotic resistance properties of this species have been little studied. In this work, the resistance/susceptibility (R/S) profile of S. equorum strains (n = 30) from cheese to 16 antibiotics was determined by broth microdilution. The minimum inhibitory concentration (MIC) for all antibiotics was low in most strains, although higher MICs compatible with acquired genes were also noted. Genome analysis of 13 strains showed the S. equorum resistome to be composed of intrinsic mechanisms, acquired mutations, and acquired genes. As such, a plasmidic cat gene providing resistance to chloramphenicol was found in one strain; this was able to provide resistance to Staphylococcus aureus after electroporation. An msr(A) polymorphic gene was identified in five strains. The Mrs(A) variants were associated with variable resistance to erythromycin. However, the genetic data did not always correlate with the phenotype. As such, all strains harbored a polymorphic fosB/fosD gene, although only one acquired copy was associated with strong resistance to fosfomycin. Similarly, a plasmid-associated blaR1-blaZI operon encoding a penicillinase system was identified in five ampicillin- and penicillin G-susceptible strains. Identified genes not associated with phenotypic resistance further included mph(C) in two strains and norA in all strains. The antibiotic R/S status and gene content of S. equorum strains intended to be employed in food systems should be carefully determined.202337511416
522750.9997Mutation at the position 2058 of the 23S rRNA as a cause of macrolide resistance in Streptococcus pyogenes. BACKGROUND: In streptococci, three macrolide resistance determinants (erm(B), erm(TR) and mef(A)) have been found. In addition, certain mutations at the ribosomal 23S RNA can cause resistance to macrolides. Mutation at the position 2058 of the 23S rRNA of the Streptococcus pyogenes as a cause of macrolide resistance has not been described before. METHODS: Antibiotic resistance determinations for the clinical S. pyogenes strain ni4277 were done using the agar dilution technique. Macrolide resistance mechanisms were studied by PCR and sequencing. All six rRNA operons were amplified using operon-specific PCR. The PCR products were partially sequenced in order to resolve the sequences of different 23S rRNA genes. RESULTS: One clinical isolate of S. pyogenes carrying an adenine to guanine mutation at the position 2058 of the 23S rRNA in five of the six possible rRNA genes but having no other known macrolide resistance determinants is described. The strain was highly resistant to macrolides and azalides, having erythromycin and azithromycin MICs > 256 microgram/ml. It was resistant to lincosamides (clindamycin MIC 16 microgram/ml) and also MIC values for ketolides were clearly elevated. The MIC for telithromycin was 16 microgram/ml. CONCLUSION: In this clinical S. pyogenes strain, a mutation at the position 2058 was detected. No other macrolide resistance-causing determinants were detected. This mutation is known to cause macrolide resistance in other bacteria. We can conclude that this mutation was the most probable cause of macrolide, lincosamide and ketolide resistance in this strain.200415128458
357360.9996Antibiotic Susceptibility Profiles of Lactic Acid Bacteria from the Human Vagina and Genetic Basis of Acquired Resistances. Lactic acid bacteria can act as reservoirs of antibiotic resistance genes that can be ultimately transferred to pathogens. The present work reports on the minimum inhibitory concentration (MIC) of 16 antibiotics to 25 LAB isolates of five Lactobacillus and one Bifidobacterium species from the human vagina. Acquired resistances were detected to kanamycin, streptomycin, chloramphenicol, gentamicin, and ampicillin. A PCR analysis of lactobacilli failed to identify genetic determinants involved in any of these resistances. Surprisingly, a tet(W) gene was detected by PCR in two Bifidobacterium bifidum strains, although they proved to be tetracycline-susceptible. In agreement with the PCR results, no acquired genes were identified in the genome of any of the Lactobacillus spp. strains sequenced. A genome analysis of B. bifidum VA07-1AN showed an insertion of two guanines in the middle of tet(W) interrupting the open reading frame. By growing the strain in the presence of tetracycline, stable tetracycline-resistant variants were obtained. An amino acid substitution in the ribosomal protein S12 (K43R) was further identified as the most likely cause of VA07-1AN being streptomycin resistance. The results of this work expand our knowledge of the resistance profiles of vaginal LAB and provide evidence for the genetic basis of some acquired resistances.202032276519
357970.9996The Tetracycline Resistance Gene, tet(W) in Bifidobacterium animalis subsp. lactis Follows Phylogeny and Differs From tet(W) in Other Species. The tetracycline resistance gene tet(W) encodes a ribosomal protection protein that confers a low level of tetracycline resistance in the probiotic bacterium Bifidobacterium animalis subsp. lactis. With the aim of assessing its phylogenetic origin and potential mobility, we have performed phylogenetic and in silico genome analysis of tet(W) and its flanking genes. tet(W) was found in 41 out of 44 examined B. animalis subsp. lactis strains. In 38 strains, tet(W) was flanked by an IS5-like element and an open reading frame encoding a hypothetical protein, which exhibited a similar GC content (51-53%). These genes were positioned in the same genomic context within the examined genomes. Phylogenetically, the B. animalis subsp. lactis tet(W) cluster in a clade separate from tet(W) of other species and genera. This is not the case for tet(W) encoded by other bifidobacteria and other species where tet(W) is often found in association with transferable elements or in different genomic regions. An IS5-like element identical to the one flanking the B. animalis subsp. lactis tet(W) has been found in a human gut related bacterium, but it was not associated with any tet(W) genes. This suggests that the IS5-like element is not associated with genetic mobility. tet(W) and the IS5 element have previously been shown to be co-transcribed, indicating that co-localization may be associated with tet(W) expression. Here, we present a method where phylogenetic and in silico genome analysis can be used to determine whether antibiotic resistance genes should be considered innate (intrinsic) or acquired. We find that B. animalis subsp. lactis encoded tet(W) is part of the ancient resistome and thereby possess a negligible risk of transfer.202134335493
606580.9996Screening for enterocins and detection of hemolysin and vancomycin resistance in enterococci of different origins. The inhibitory activity of 122 out of 426 Enterococcus strains of geographically widespread origin and from different sources (food and feed, animal isolates, clinical and nonclinical human isolates) was tested against a wide range of indicator bacteria. Seventy-two strains, mainly belonging to the species Enterococcus faecium and Enterococcus faecalis were bacteriocinogenic. A remarkable variation of inhibitory spectra occurred among the strains tested, including inhibition of, for instance, only closely related enterococci, other lactic acid bacteria (LAB), food spoilage and pathogenic bacteria. No correlation could be found between the origin of the strains and the type of inhibitory spectrum, although a clustering of human isolates from both fecal and clinical origin was observed in the group of strains inhibiting lactic acid bacteria, Listeria, and either Staphylococcus or Clostridium. No relationship could be established between the presence of enterocin structural genes and the origin of the strain either, and hence no correlation seemed to exist between the presence of known enterocin genes and the activity spectra of these enterococci. The structural gene of enterocin A was widely distributed among E. faecium strains, whereas that of enterocin B only occurred in the presence of enterocin A. The vancomycin resistance phenotype as well as the presence of vancomycin resistance genes was also investigated. The vanA gene only occurred among E. faecium strains. The incidence of beta-hemolysis was not restricted to E. faecalis strains, but among the E. faecium strains the structural genes of cytolysin were not detected. beta-Hemolysis occurred in strains both from food and nonfood origin. It has been concluded that bacteriocin-producing E. faecium strains lacking hemolytic activity and not carrying cytolysin nor vancomycin resistance genes may be useful as starter cultures, cocultures, or probiotics.200312810293
243990.9996Differences in distribution of MLS antibiotics resistance genes in clinical isolates of staphylococci belonging to species: S. epidermidis, S. hominis, S. haemolyticus, S. simulans and S. warneri. BACKGROUND: Macrolides and lincosamides are two leading types of antibiotics commonly used in therapies. The study examines the differences in resistance to these antibiotics and their molecular bases in S. epidermidis as well as in rarely isolated species of coagulase-negative staphylococci such as S. hominis, S. haemolyticus, S. warneri and S. simulans. The isolates were tested for the presence of the erm(A), erm(B), erm(C), lnu(A), msr(A), msr(B), mph(C), ere(A) and ere(B) genes. Phenotypic resistance to methicillin and mecA presence were also determined. RESULTS: The MLS(B) resistance mechanism was phenotypically found in isolates of species included in the study. The most prevalent MLS(B) resistance mechanism was observed in S. hominis, S. haemolyticus and S. epidermidis isolates mainly of the MLS(B) resistance constitutive type. Macrolide, lincosamide and streptogramin B resistance genes were rarely detected in isolates individually. The erm(B), ere(A) and ere(B) genes were not found in any of the strains. The erm(A) gene was determined only in four strains of S. epidermidis and S. hominis while lnu(A) was seen in eight strains (mainly in S. hominis). The erm(C) gene was present in most of S. epidermidis strains and predominant in S. hominis and S. simulans isolates. The examined species clearly differed between one another in the repertoire of accumulated genes. CONCLUSIONS: The presence of genes encoding the MLS(B) resistance among CoNS strains demonstrates these genes' widespread prevalence and accumulation in opportunistic pathogens that might become gene reservoir for bacteria with superior pathogenic potential.201931182020
5909100.9996Antibiotic susceptibility profiles of Lactobacillus reuteri and Lactobacillus fermentum. Lactobacillus reuteri and Lactobacillus fermentum, which are commonly used as food processing aids and probiotics, can potentially act as reservoirs of antibiotic resistance genes. Acquired resistance genes may be transferred via the food chain or in the gastrointestinal tract to pathogenic bacteria. Knowledge of the distributions of antibiotic MICs for a species is needed when using a phenotypic method to assess the presence of acquired resistance genes. In the present study, 56 L. reuteri and 56 L. fermentum strains that differed by source and spatial and temporal origin were assessed for antibiotic susceptibility using an Etest kit and a broth microdilution protocol. L. fermentum strains displayed a uniform distribution of MICs for all six antibiotics tested. L. reuteri strains had a bimodal distribution of MICs or a distribution with MICs above the test range for 7 of the 14 antibiotics tested. Genetic relatedness was observed among L. reuteri strains with high MICs for both ampicillin and tetracycline and among strains with high MICs for both erythromycin and clindamycin. Results obtained with the Etest and the broth microdilution method corresponded well with each other. Thus, further research may make it possible to define microbiological breakpoints for distinguishing between strains with and without acquired resistance genes.200717340877
5992110.9996Emergence of Enterococcus gallinarum carrying vanA gene cluster displaying atypical phenotypes. Motile enterococci such as Enterococcus gallinarum has the ability to acquire and transfer antibiotic resistance genes to other enterococci. Even though infections caused by E. gallinarum are rare, the discovery of this bacteria in food sources and in clinical environments is disturbing. Here, we report the isolation and identification of E. gallinarum from the wound of a hospital in-patient. The isolate was identified using 16S rDNA sequencing. Isolate 146 harboured the vanA and vanC1 gene clusters, was vancomycin-susceptible, and displayed resistance to ampicillin, penicillin, erythromycin and teicoplanin. This isolate also showed intermediate resistance to linezolid and sequencing of the 23S rRNA peptidyl transferase region did not unveil any known mutations associated to the conferment of linezolid resistance. The presence of vanA did not confer resistance to vancomycin. Structural analyses into the Tn1546 transposon carrying the vanA gene revealed distinct genetic variations in the vanS, vanY and vanS-vanH intergenic region that could be associated to the atypical antibiotic resistance phenotypes of isolate 146. Finding from this study are suggestive of the occurrence of interspecies horizontal gene transfer and that similarities in genotypic characteristic may not necessarily correlate with actual antibiotic resistance pattern of E. gallinarum.201633579083
5997120.9996Resistance of potential probiotic lactic acid bacteria and bifidobacteria of African and European origin to antimicrobials: determination and transferability of the resistance genes to other bacteria. Probiotic bacteria and starter cultures of Lactobacillus, Weissella and Bifidobacterium of African and European origins were studied and compared for their susceptibility to antimicrobials. The study included, for all isolates, determination of MICs (Minimal Inhibitory Concentration) for 24 antimicrobials, detection of resistance genes by PCR reactions using specific primers and sequencing of positive amplicons. The ability of Lb. reuteri from Africa to transfer the erythromycin resistance gene erm(B) to closely related bacteria was investigated by conjugation. Variations were observed and high levels of intrinsic resistance were found among the tested species. Positive amplicons were obtained for resistance genes encoding aminoglycoside (aph(3')-III, aadA, aadE) and tetracycline (tet(S)) from isolates from Europe and macrolide (erm(B)) from an isolate from Africa. However, only the erm(B) gene found in Lb. reuteri L4: 12002 from Africa contained a homologous sequence to previously published sequences. This gene could be transferred in vitro to enterococci. Higher prevalence of phenotypic resistance for aminoglycoside was found in isolates from Europe.200818063151
2442130.9996Macrolide, lincosamide, and streptogramin B resistance in lipophilic Corynebacteria inhabiting healthy human skin. Corynebacteria exist as part of human skin microbiota. However, under some circumstances, they can cause opportunistic infections. The subject of the study was to examine the macrolide-lincosamide-streptogramin B (MLSB) antibiotic resistance in 99 lipophilic strains of Corynebacterium genus isolated from the skin of healthy men. Over 70% of the tested strains were resistant to erythromycin and clindamycin. All of which demonstrated a constitutive type of MLSB resistance mechanism. In all strains, there were being investigated the erm(A), erm(B), erm(C), erm(X), lin(A), msr(A), and mph(C) genes that could be responsible for the different types of resistance to marcolides, lincosamides, and streptogramin B. In all strains with the MLSB resistance phenotype, the erm(X) gene was detected. None of the other tested genes were discovered. Strains harboring the erm(X) gene were identified using a phenotypic method based on numerous biological and biochemical tests. Identification of the chosen strains was compared with the results of API Coryne, MALDI-TOF MS, and 16S rDNA sequencing methods. Only 7 out of the 23 investigated resistant strains provided successful results in all the used methods, showing that identification of this group of bacteria is still a great challenge. The MLSB resistance mechanism was common in most frequently isolated from healthy human skin Corynebacterium tuberculostearicum and Corynebacterium jeikeium strains. This represents a threat as these species are also commonly described as etiological factors of opportunistic infections.201424735183
5848140.9996Plasmid and chromosomal basis of tolerance to cadmium and resistance to antibiotics in normal bovine duodenal bacterial flora. Cadmium (Cd) tolerance and antibiotic resistance was studied in duodenal flora of 20 normal bovine samples. Twelve bacterial isolates (5 Staphylococcus spp, 4 Enterococcus faecalis, 2 Bacillus spp, and a Pseudomonas sp) were grown in Luria broth containing 0.05 to 0.8 mM of cadmium chloride (CdCl). All isolates displayed multiple antibiotic resistance, with 2 Enterococcus strains and Pseudomonas pickettii demonstrating resistance to 12/17 antibiotics tested. With the exception of Staphylococcus sp, all contained plasmid DNA. Curing to remove plasmid DNA determined if Cd tolerance and/or antibiotic resistance was plasmid or chromosomally mediated. None of the bacteria became sensitive to CdCl after curing, suggesting that tolerance was not plasmid-mediated. Six bacteria became sensitive to antibiotics after curing indicating that antibiotic2 resistance was plasmid mediated. Two of these bacteria became sensitive to multiple antibiotics; a Staphylococcus sp became sensitive to ampicillin, ceftiofur and cephalothin, and a Enterococcus strain became sensitive to neomycin, oxacillin, and tiamulin. All of the isolates were probed for the presence of known Cd-resistance genes (cadA, cadC, and cadD). DNA-DNA hybridization revealed cadA- and cadC-related sequences in chromosomal DNA of a Staphylococcus sp, an Enterococcus strain, and in plasmid DNA of another Staphylococcus sp. No cadD-related sequences were detected in any of the 12 isolates even under reduced stringency of hybridization.200111383651
5863150.9996Molecular characterization of tet(M) genes in Lactobacillus isolates from different types of fermented dry sausage. 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.200312571056
5982160.9996Genetic diversity of penicillin-binding protein 2B and 2X genes from Streptococcus pneumoniae in South Africa. Streptococcus pneumoniae (the pneumococcus) is believed to have developed resistance to penicillin by the production of altered forms of penicillin-binding proteins (PBPs) that have decreased affinity for penicillin. Sixty-eight clinical isolates of serogroup 6 and 19 pneumococci (MICs, < 0.015 to 8 micrograms/ml) were randomly selected from hospitals across South Africa which are at substantial geographic distance from each other. The polymerase chain reaction was used to isolate the penicillin-binding domain of PBPs 2B and 2X from the chromosomal DNAs of the bacteria; the purified PBP DNA was digested with restriction enzymes, the fragments were end-labelled and separated on polyacrylamide gels, and the DNA fingerprints were visualized following autoradiography. Fingerprint analysis revealed that at least 19 PBP 2B gene variants occur in the serogroup 6 and 19 pneumococci. The PBP 2B gene revealed a uniform profile among penicillin-susceptible isolates, with variation from this profile occurring only in isolates for which MICs were > or = 0.06 micrograms/ml. Analysis of the PBP 2X gene revealed a greater diversity in the population with 26 variant genes, including some diversity among susceptible isolates. Discrete profiles of both genes were found only within narrow bands of the penicillin MIC, so that the gene pattern predicted the MIC. PBP 2X gene variation and the lack of variability among PBP 2B genes in pneumococci inhibited at low MICs confirm that PBP 2X alteration may be responsible for low-level penicillin resistance, while alterations in both PBP 2B and PBP 2X are required for high-level resistance. The extensive diversity of PBP genes in South African serogroup 6 and 19 strains suggests that altered PBP genes have arisen frequently in this population.19938239609
6064170.9996Evaluation of marine bacteriocinogenic enterococci strains with inhibitory activity against fish-pathogenic Gram-negative bacteria. Use of lactic acid bacteria (LAB) as probiotics may provide an alternative to the use of antibiotics in aquaculture. LAB strains isolated from wild fish viscera and skin were evaluated for bacteriocin production and safety aspects (lack of antibiotic resistance, production of virulence factors). 16S rRNA gene sequences revealed the presence of Enterococcus faecium (13 isolates) and Lactococcus lactis (3 isolates) from fish samples. Pulsed-field gel electrophoresis analyses of the 13 enterococci isolates showed that they were all clustered, with greater than 95% similarity. However, RAPD analysis revealed significant molecular diversity between enterococci strains. Six enterococci strains were chosen and evaluated for their antibacterial activities. These strains produced a bacteriocin-like substance and exhibited a broad spectrum of inhibition against pathogenic bacteria isolated from diseased fish, including Streptococcus parauberis, Vagococcus spp., and Carnobacterium maltaromaticum, and in particular against the Gram-negative bacteria Flavobacterium frigidarium, Vibrio pectenicida, V. penaeicida, and Photobacterium damselae. The inhibition activity towards bacterial indicator strains was at a maximum when bacteria were grown at 37°C. However, bacteriocin production was observed at 15°C after 12 h of incubation. Only structural genes of enterocins A and B were detected by PCR in the 6 enterococci strains, suggesting the production of these enterocins. In addition, these strains did not harbor any virulence factors or any significant antibiotic resistance, and they tolerated bile. Our results suggest that enterococci are an important part of the bacterial flora of fish and that some strains have the potential to be used as probiotics.201626865233
3574180.9996Antibiotic resistances of starter and probiotic strains of lactic acid bacteria. The antibiotic resistances of 45 lactic acid bacteria strains belonging to the genera Lactobacillus, Streptococcus, Lactococcus, Pediococcus, and Leuconostoc were investigated. The objective was to determine antibiotic resistances and to verify these at the genetic level, as is currently suggested by the European "qualified presumption of safety" safety evaluation system for industrial starter strains. In addition, we sought to pinpoint possible problems in resistance determinations. Primers were used to PCR amplify genes involved in beta-lactam antibiotic, chloramphenicol, tetracycline, and erythromycin resistance. The presence of ribosomal protection protein genes and the ermB gene was also determined by using a gene probe. Generally, the incidences of erythromycin, chloramphenicol, tetracycline, or beta-lactam resistances in this study were low (<7%). In contrast, aminoglycoside (gentamicin and streptomycin) and ciprofloxacin resistances were higher than 70%, indicating that these may constitute intrinsic resistances. The genetic basis for ciprofloxacin resistance could not be verified, since no mutations typical of quinolone resistances were detected in the quinolone determining regions of the parC and gyrA genes. Some starter strains showed low-level ampicillin, penicillin, chloramphenicol, and tetracycline resistances, but no known resistance genes could be detected. Although some strains possessed the cat gene, none of these were phenotypically resistant to chloramphenicol. Using reverse transcription-PCR, these cat genes were shown to be silent under both inducing and noninducing conditions. Only Lactobacillus salivarius BFE 7441 possessed an ermB gene, which was encoded on the chromosome and which could not be transferred in filter-mating experiments. This study clearly demonstrates problems encountered with resistance testing, in that the breakpoint values are often inadequately identified, resistance genes may be present but silent, and the genetic basis and associated resistance mechanisms toward some antibiotics are still unknown.200717122388
5910190.9996Antimicrobial Susceptibility Testing and Tentative Epidemiological Cutoff Values for Five Bacillus Species Relevant for Use as Animal Feed Additives or for Plant Protection. Bacillus megaterium (n = 29), Bacillus velezensis (n = 26), Bacillus amyloliquefaciens (n = 6), Bacillus paralicheniformis (n = 28), and Bacillus licheniformis (n = 35) strains from different sources, origins, and time periods were tested for the MICs for nine antimicrobial agents by the CLSI-recommended method (Mueller-Hinton broth, 35°C, for 18 to 20 h), as well as with a modified CLSI method (Iso-Sensitest [IST] broth, 37°C [35°C for B. megaterium], 24 h). This allows a proposal of species-specific epidemiological cutoff values (ECOFFs) for the interpretation of antimicrobial resistance in these species. MICs determined by the modified CLSI method were 2- to 16-fold higher than with the CLSI-recommended method for several antimicrobials. The MIC distributions differed between species for five of the nine antimicrobials. Consequently, use of the modified CLSI method and interpretation of resistance by use of species-specific ECOFFs is recommended. The genome sequences of all strains were determined and used for screening for resistance genes against the ResFinder database and for multilocus sequence typing. A putative chloramphenicol acetyltransferase (cat) gene was found in one B. megaterium strain with an elevated chloramphenicol MIC compared to the other B. megaterium strains. In B. velezensis and B. amyloliquefaciens, a putative tetracycline efflux gene, tet(L), was found in all strains (n = 27) with reduced tetracycline susceptibility but was absent in susceptible strains. All B. paralicheniformis and 23% of B. licheniformis strains had elevated MICs for erythromycin and harbored ermD The presence of these resistance genes follows taxonomy suggesting they may be intrinsic rather than horizontally acquired. Reduced susceptibility to chloramphenicol, streptomycin, and clindamycin could not be explained in all species.IMPORTANCE When commercializing bacterial strains, like Bacillus spp., for feed applications or plant bioprotection, it is required that the strains are free of acquired antimicrobial resistance genes that could potentially spread to pathogenic bacteria, thereby adding to the pool of resistance genes that may cause treatment failures in humans or animals. Conversely, if antimicrobial resistance is intrinsic to a bacterial species, the risk of spreading horizontally to other bacteria is considered very low. Reliable susceptibility test methods and interpretation criteria at the species level are needed to accurately assess antimicrobial resistance levels. In the present study, tentative ECOFFs for five Bacillus species were determined, and the results showed that the variation in MICs followed the respective species. Moreover, putative resistance genes, which were detected by whole-genome sequencing and suggested to be intrinsic rather that acquired, could explain the resistance phenotypes in most cases.201830030233