Presence and antibiotic resistance of Porphyromonas gingivalis, Prevotella intermedia, and Prevotella nigrescens in children. - Related Documents




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241901.0000Presence and antibiotic resistance of Porphyromonas gingivalis, Prevotella intermedia, and Prevotella nigrescens in children. BACKGROUND/AIMS: Only limited information exists about the prevalence in children of pathogens associated with periodontitis. The aim of the present study was to determine by culture whether 8-11-year-old children carry Porphyromonas gingivalis, Prevotella intermedia, and/or P. nigrescens in samples from the gingiva and/or the buccal mucosa taken before, and after caries treatment and oral hygiene instruction. A second aim was to assess the proportion of subjects who had gram-negative anaerobes carrying the tet(Q) and erm(F) genes, suggesting antibiotic resistance to tetracycline or erythromycin. METHOD: A total of 150 children provided gingival and buccal swab bacterial samples that were cultured for P. gingivalis, P. intermedia, and P. nigrescens. The species was verified using DNA-DNA hybridization with species-specific probes made from the variable region of the 16S rRNA sequences. Antibiotic-resistant genes, tet(Q) and erm(F), were identified using specific DNA-DNA hybridization with specific DNA probes. RESULTS: A total of 116 isolates of black-pigmented bacteria were cultured from 47 (31%) of 150 children. Five isolates were identified as P. gingivalis, 29 as P. intermedia, 33 as P. nigrescens, and 49 as other species. In general, the bacteria were not culturable at more than one time period. We found that 55% of these 47 children harbored black pigmented bacteria that carried either one or both of the two antibiotic-resistant genes studied (tet(Q), and erm(F)). CONCLUSION: The present study demonstrated that children not exposed to regular dental treatment carry bacteria outside the gingival sulcus that have been associated with periodontitis, and that standard treatment procedures may not clear the presence of the putative pathogens. In addition, antibiotic-resistant genes are common in identifiable gram-negative anaerobes, including putative pathogens.200212445225
242010.9998Distribution of erm(F) and tet(Q) genes in 4 oral bacterial species and genotypic variation between resistant and susceptible isolates. BACKGROUND: Bacteroides forsythus, Porphyromonas gingivalis and Prevotella intermedia are Gram-negative anaerobic bacteria that are currently considered potential periopathogens. Prevotella nigrescens has recently been separated from P. intermedia and its rôle in periodontitis is unknown. The erm(F) gene codes for an rRNA methylase, conferring resistance to macrolides, lincosamides and streptogramin B (MLSB), and the tet(Q) gene for a ribosomal protection protein, conferring resistance to tetracycline. The presence of these resistance genes could impair the use of antibiotics for therapy. PURPOSE: The aim of this study was to determine the carriage of erm(F) and tet(Q), and genetic variability of 12 Porphyromonas gingivalis, 10 Prevotella intermedia, 25 Prevotella nigrescens and 17 Bacteroides forsythus isolates from 9 different patient samples. METHODS: We used polymerase chain reaction (PCR) for detecting antibiotic resistance genes, and pulsed-field gel electrophoresis (PFGE) for detecting genetic variability among the isolates. RESULTS: Thirty-one (48%) isolates were resistant to both erythromycin and tetracycline and carried the erm(F) and tet(Q) genes, eight (13%) were tetracycline resistant and carried the tet(Q) gene, 9 (14%) were erythromycin resistant and carried the erm(F) gene, and 12 (19%) isolates did not carry antibiotic resistance genes. PFGE was used to compare isolates from the same patient and isolates from different patient samples digested with XbaI. No association was found between antibiotic resistance gene carriage and PFGE patterns in any species examined. All isolates of the same species from the same patient had highly related or identical PFGE patterns. Isolates of same species from different patients had unique PFGE pattern for each species tested. CONCLUSION: All isolates of the same species from any one patient were genetically related to each other but distinct from isolates from other patients, and 66% of the patients carried antibiotic resistant isolates, which could impair antibiotic therapy.200211895543
593520.9997Antibiotic resistance genes in anaerobic bacteria isolated from primary dental root canal infections. Fourty-one bacterial strains isolated from infected dental root canals and identified by 16S rRNA gene sequence were screened for the presence of 14 genes encoding resistance to beta-lactams, tetracycline and macrolides. Thirteen isolates (32%) were positive for at least one of the target antibiotic resistance genes. These strains carrying at least one antibiotic resistance gene belonged to 11 of the 26 (42%) infected root canals sampled. Two of these positive cases had two strains carrying resistance genes. Six out of 7 Fusobacterium strains harbored at least one of the target resistance genes. One Dialister invisus strain was positive for 3 resistance genes, and 4 other strains carried two of the target genes. Of the 6 antibiotic resistance genes detected in root canal strains, the most prevalent were blaTEM (17% of the strains), tetW (10%), and ermC (10%). Some as-yet-uncharacterized Fusobacterium and Prevotella isolates were positive for blaTEM, cfxA and tetM. Findings demonstrated that an unexpectedly large proportion of dental root canal isolates, including as-yet-uncharacterized strains previously regarded as uncultivated phylotypes, can carry antibiotic resistance genes.201223108290
290430.9997The maintenance in the oral cavity of children of tetracycline-resistant bacteria and the genes encoding such resistance. OBJECTIVES: To investigate the maintenance of tetracycline-resistant oral bacteria and the genes encoding tetracycline resistance in these bacteria in children (aged 4--6 years) over a period of 12 months. METHODS: Plaque and saliva samples were taken from 26 children. Tetracycline-resistant bacteria were isolated and identified. The types of resistance genes and their genetic locations were also determined. RESULTS: Fifteen out of 18 children harboured tetracycline-resistant (defined as having a MIC>or=8 mg/L) oral bacteria at all three time points. The median percentage of tetracycline-resistant bacteria at 0, 6 and 12 months was 1.37, 1.37 and 0.85%, respectively; these were not significantly different. The MIC(50) of the group was 64 mg/L at all three time points compared with the MIC(90), which was 64 mg/L at 0 months, and 128 mg/L at 6 and 12 months. The most prevalent resistant species were streptococci (68%), which were isolated at all three time points in 13 children. The most prevalent gene encoding tetracycline resistance was tet(M) and this was found in different species at all three time points. For the first time, tet(32) was found in Streptococcus parasanguinis and Eubacterium saburreum. PCR and Southern-blot analysis (on isolates from three of the children) showed that the tet(M) gene was located on a Tn916-like element and could be detected at all three time points, in four different genera, Streptococcus, Granulicatella, Veillonella and Neisseria. CONCLUSIONS: The results of this study show that tetracycline-resistant bacteria and tet(M) are maintained within the indigenous oral microbiota of children, even though they are unlikely to have been directly exposed to tetracycline.200516027144
593740.9997Association of mutation patterns in GyrA and ParC genes with quinolone resistance levels in lactic acid bacteria. The quinolone resistance of 19 lactic acid bacterial strains belonging to the genera Enterococcus and Lactobacillus isolated from the natural fermented koumiss and yoghurt were investigated. The objective of this study was to determine the quinolone resistance levels and to explore the association of the resistance with the mutation patterns in gyrA and parC genes, as is currently recommended by the Food and Agriculture Organization/World Health Organization Joint Expert Committee in Guidelines for Evaluation of Probiotics in Food for probiotic lactic acid bacteria drug resistance in 2001. The Oxford Cup method and double-tube dilution method were used to determine the quinolone resistance levels of the isolated strains. Generally, all of the 19 strains showed resistance towards norfloxacin and ciprofloxacin when the Oxford cup method was used, whereas the incidence was lower (to norfloxacin 89.5% and to ciprofloxacin 68.4%) when minimum inhibitory concentration breakpoints (CLSI M100-S23) were tested. Furthermore, gene sequencing was conducted on gyrA and parC of topoisomerase II of these isolated strains. The genetic basis for quinolone resistance may be closely related to mutations in gyrA genes as there were 10 mutation sites in amino-acid sequences encoded by gyrA genes in 10 quinolone resistance strains and 14 mutation sites in Enterococcus durans HZ28, whereas no typical mutations were detected in parC genes.201525204345
241650.9997Detection of antibiotic resistance genes in samples from acute and chronic endodontic infections and after treatment. OBJECTIVE: The purpose of this study was twofold: survey samples from acute and chronic endodontic infections for the presence of genes encoding resistance to beta-lactams, tetracycline and erythromycin, and evaluate the ability of treatment to eliminate these genes from root canals. DESIGN: DNA extracts from samples of abscess aspirates (n=25) and root canals of teeth with asymptomatic apical periodontitis (n=24) were used as template for direct detection of the genes blaTEM, cfxA, tetM, tetQ, tetW, and ermC using real-time polymerase chain reaction (PCR). Bacterial presence was determined using PCR with universal bacterial primers. Root canals of the asymptomatic cases were also sampled and evaluated after chemomechanical procedures using NiTi instruments with 2.5% NaOCl irrigation. RESULTS: All abscess and initial root canal samples were positive for bacteria. At least one of the target resistance genes was found in 36% of the abscess samples and 67% of the asymptomatic cases. The most prevalent genes in abscesses were blaTEM (24%) and ermC (24%), while tetM (42%) and tetW (29%) prevailed in asymptomatic cases. The blaTEM gene was significantly associated with acute cases (p=0.02). Conversely, tetM was significantly more prevalent in asymptomatic cases (p=0.008). Treatment eliminated resistance genes from most cases. CONCLUSIONS: Acute and chronic endodontic infections harboured resistance genes for 3 classes of widely used antibiotics. In most cases, treatment was effective in eliminating these genes, but there were a few cases in which they persisted. The implications of persistence are unknown. Direct detection of resistance genes in abscesses may be a potential method for rapid diagnosis and establishment of proactive antimicrobial therapy.201323591127
599760.9997Resistance 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
592170.9997Prevalence of tetracycline resistance genes in oral bacteria. Tetracycline is a broad-spectrum antibiotic used in humans, animals, and aquaculture; therefore, many bacteria from different ecosystems are exposed to this antibiotic. In order to determine the genetic basis for resistance to tetracycline in bacteria from the oral cavity, saliva and dental plaque samples were obtained from 20 healthy adults who had not taken antibiotics during the previous 3 months. The samples were screened for the presence of bacteria resistant to tetracycline, and the tetracycline resistance genes in these isolates were identified by multiplex PCR and DNA sequencing. Tetracycline-resistant bacteria constituted an average of 11% of the total cultivable oral microflora. A representative 105 tetracycline-resistant isolates from the 20 samples were investigated; most of the isolates carried tetracycline resistance genes encoding a ribosomal protection protein. The most common tet gene identified was tet(M), which was found in 79% of all the isolates. The second most common gene identified was tet(W), which was found in 21% of all the isolates, followed by tet(O) and tet(Q) (10.5 and 9.5% of the isolates, respectively) and then tet(S) (2.8% of the isolates). Tetracycline resistance genes encoding an efflux protein were detected in 4.8% of all the tetracycline-resistant isolates; 2.8% of the isolates had tet(L) and 1% carried tet(A) and tet(K) each. The results have shown that a variety of tetracycline resistance genes are present in the oral microflora of healthy adults. This is the first report of tet(W) in oral bacteria and the first report to show that tet(O), tet(Q), tet(A), and tet(S) can be found in some oral species.200312604515
563880.9997PCR monitoring for tetracycline resistance genes in subgingival plaque following site-specific periodontal therapy. A preliminary report. BACKGROUND: The selection of antibiotic resistance genes during antibiotic therapy is a critical problem complicated by the transmission of resistance genes to previously sensitive strains via conjugative plasmids and transposons and by the transfer of resistance genes between gram-positive and gram-negative bacteria. The purpose of this investigation was to monitor the presence of selected tetracycline resistance genes in subgingival plaque during site specific tetracycline fiber therapy in 10 patients with adult periodontitis. METHOD: The polymerase chain reaction (PCR) was used in separate tests for the presence of 3 tetracycline resistance genes (tetM, tetO and tetQ) in DNA purified from subgingival plaque samples. Samples were collected at baseline, i.e., immediately prior to treatment, and at 2 weeks, and 1, 3, and 6 months post-fiber placement. The baseline and 6-month samples were also subjected to DNA hybridization tests for the presence of 8 putative periodontal pathogenic bacteria. RESULTS: PCR analysis for the tetM resistance gene showed little or no change in 5 patients and a decrease in detectability in the remaining 5 patients over the 6 months following tetracycline fiber placement. The results for tetO and tetQ were variable showing either no change in detectability from baseline through the 6-month sampling interval or a slight increase in detectability over time in 4 of the 10 patients. DNA hybridization analysis showed reductions to unmeasurable levels of the putative periodontal pathogenic bacteria in all but 2 of the 10 patients. CONCLUSIONS: These results complement earlier studies of tet resistance and demonstrate the efficacy of PCR monitoring for the appearance of specific resistance genes during and after antibiotic therapy.200010883874
290590.9997Scarce detection of mobile erm genes associated with tetQ in Bacteroides and Parabacteroides from Costa Rica. The frequency of finding of clindamycin-resistant anaerobic bacteria in clinical samples has doubled from 2008 to 2010 in Costa Rica. To determine whether this increase is due to dissemination of erm genes aided by tetQ elements, we analyzed 100 isolates of Bacteroides or Parabacteroides from a regional hospital, a national hospital, and the community. Antimicrobial susceptibilities were recorded with a broth micro-dilution method and erm genes were detected by PCR and Southern blotting. In addition, plasmid isolation and mating experiments were performed to clarify the location and mobility of the detected erm genes. Resistance to clindamycin was by far more frequent in the regional hospital (72%) than in the national hospital (29%) and the community (26%). Resistance to tetracycline was even more common, with the community (85%) outweighing the hospitals (71-72%). While MIC of clindamycin were higher in the hospitals than in the community (P < 0.05), the opposite was seen for tetracycline (P < 0.0001). Of the sought-after genes, only ermG (n = 2), ermA (n = 1), and ermF (n = 1) were detected in the hospitals and ermF in the community (n = 2). In opposition to the low frequency of finding of erm genes, 71% of the isolates were positive for tetQ. None of the detected genes were encoded on plasmids. Only three isolates from the hospitals transferred their erm genes laterally. By contrast, 13 hospital isolates and two community isolates transferred tetQ. Despite the widespread finding of tetracycline-resistant tetQ-positive bacteria, mobile erm genes were rare in our bacterial collection. We conclude that the detected erm genes are likely not included in typical conjugative transposons of Bacteroides and Parabacteroides.201323528984
5941100.9996Characterization of macrolide resistance genes in Haemophilus influenzae isolated from children with cystic fibrosis. OBJECTIVES: to determine the mechanism(s) of macrolide resistance in Haemophilus influenzae isolated from cystic fibrosis (CF) patients participating in a randomized placebo-controlled trial of azithromycin. METHODS: macrolide susceptibility, mutations and carriage of the macrolide resistance genes erm(A), erm(B), erm(C), erm(F) and mef(A) were determined using PCR assays and sequencing or hybridization of the PCR products. H. influenzae isolates were used as donors in conjugation studies with H. influenzae and Enterococcus faecalis recipients. Transconjugant susceptibility and the macrolide resistance genes carried were determined. RESULTS: of the 106 H. influenzae isolates, 27 were resistant and 78 intermediate resistant to azithromycin and/or erythromycin. All isolates carried one or more macrolide resistance gene(s), with the mef(A), erm(B) and erm(F) genes found in 74%, 31% and 29% of the isolates, respectively. None of the selected isolates had L4 or L22 mutations. Twenty-five donors, with various macrolide MICs, transferred macrolide resistance genes to H. influenzae Rd (3.5 × 10(-7)-1 × 10(-10)) and/or E. faecalis (1 × 10(-7)-1 × 10(-8)) recipients. The H. influenzae transconjugants were phenotypically resistant or intermediate to both macrolides while E. faecalis transconjugants were erythromycin resistant. CONCLUSIONS: this is the first identification of erm(A), erm(C) and erm(F) genes in H. influenzae or bacteria from CF patients and the first characterization of macrolide gene transfer from H. influenzae donors. The high level of H. influenzae macrolide gene carriage suggests that the use of azithromycin in the CF population may ultimately reduce the effectiveness of continued or repeated macrolide therapy.201121081549
5428110.9996Antimicrobial resistance and prevalence of resistance genes of obligate anaerobes isolated from periodontal abscesses. BACKGROUND: This study attempts to determine the antimicrobial resistance profiles of obligate anaerobic bacteria that were isolated from a periodontal abscess and to evaluate the prevalence of resistance genes in these bacteria. METHODS: Forty-one periodontal abscess samples were cultivated on selective and non-selective culture media to isolate the oral anaerobes. Their antibiotic susceptibilities to clindamycin, doxycycline, amoxicillin, imipenem, cefradine, cefixime, roxithromycin, and metronidazole were determined using the agar dilution method, and polymerase chain reaction assays were performed to detect the presence of the ermF, tetQ, nim, and cfxA drug resistance genes. RESULTS: A total of 60 different bacterial colonies was isolated and identified. All of the isolates were sensitive to imipenem. Of the strains, 6.7%, 13.3%, 16.7%, and 25% were resistant to doxycycline, metronidazole, cefixime, and amoxicillin, respectively. The resistance rate for both clindamycin and roxithromycin was 31.7%. Approximately 60.7% of the strains had the ermF gene, and 53.3% of the amoxicillin-resistant strains were found to have the cfxA gene. Two nim genes that were found in eight metronidazole-resistant strains were identified as nimB. CONCLUSIONS: In the present study, the Prevotella species are the most frequently isolated obligate anaerobes from periodontal abscesses. The current results show their alarmingly high resistance rate against clindamycin and roxithromycin; thus, the use of these antibiotics is unacceptable for the empirical therapy of periodontal abscesses. A brief prevalence of four resistance genes in the anaerobic bacteria that were isolated was also demonstrated.201423659425
2083120.9996A classification system for plasmids from enterococci and other Gram-positive bacteria. A classification system for plasmids isolated from enterococci and other Gram-positive bacteria was developed based on 111 published plasmid sequences from enterococci and other Gram-positive bacteria; mostly staphylococci. Based on PCR amplification of conserved areas of the replication initiating genes (rep), alignment of these sequences and using a cutoff value of 80% identity on both protein and DNA level, 19 replicon families (rep-families) were defined together with several unique sequences. The prevalence of these rep-families was tested on 79 enterococcal isolates from a collection of isolates of animal and human origin. Difference in prevalence of the designed rep-families were detected with rep(9) being most prevalent in Enterococcus faecalis and rep(2) in Enterococcus faecium. In 33% of the tested E. faecium and 32% of the tested E. faecalis no positive amplicons were detected. Furthermore, conjugation experiments were performed obtaining 30 transconjugants when selecting for antimicrobial resistance. Among them 19 gave no positive amplicons indicating presence of rep-families not tested for in this experimental setup.201019879906
5910130.9996Antimicrobial 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
5637140.9996Preparation and application of microarrays for the detection of antibiotic resistance genes in samples isolated from Changchun, China. The emergence of antibiotic-resistant bacteria, especially tetracycline- and beta-lactam-resistant bacteria, poses a great threat to human health. The purpose of this study was to develop and apply a suitable gene microarray for the detection of antibiotic resistance genes. We isolated 463 strains of bacteria from a hospital, a veterinary station, an animal nursery, and living environment of Changchun, China. After screening, it was found that 93.9% of these bacteria were resistant to tetracycline, 74.9% to ampicillin, 55.6% to deoxycycline, and 41.7% to ciprofloxacin. For amplification of antibiotic genes, we designed 28 pairs of primers. In addition, 28 hybridization probes for these genes were developed. The DNA microarray analysis was performed at 42 degrees C for 5 h. We were successful in detecting 12 resistance genes by microarray analysis. After detection, we also evaluated the sensitivity of the microarray analysis. The LDL (Lowest Detection Level) of the microarray was 1 x 10(6) copies/ml of template DNA. It is believed that such microarray-based determination of tetracycline and beta-lactam resistance genes can have a potential application in clinical studies in the future.201019642018
2909150.9996Determination of the prevalence of antimicrobial resistance genes in canine Clostridium perfringens isolates. Clostridium perfringens is a well documented cause of a mild self-limiting diarrhea and a potentially fatal acute hemorrhagic diarrheal syndrome in the dog. A recent study documented that 21% of canine C. perfringens isolates had MIC's indicative of resistance to tetracycline, an antimicrobial commonly recommended for treatment of C. perfringens-associated diarrhea. The objective of the present study was to further evaluate the antimicrobial susceptibility profiles of these isolates by determining the prevalence of specific resistance genes, their expression, and ability for transference between bacteria. One hundred and twenty-four canine C. perfringens isolates from 124 dogs were evaluated. Minimum inhibitory concentrations of tetracycline, erythromycin, tylosin, and metronidazole were determined using the CLSI Reference Agar Dilution Method. All isolates were screened for three tetracycline resistance genes: tetA(P), tetB(P) and tetM, and two macrolide resistance genes: ermB and ermQ, via PCR using primer sequences previously described. Ninety-six percent (119/124) of the isolates were positive for the tetA(P) gene, and 41% (51/124) were positive for both the tetA(P) and tetB(P) genes. No isolates were positive for the tetB(P) gene alone. Highly susceptible isolates (MIC< or = 4 microg/ml) were significantly more likely to lack the tetB(P) gene. One isolate (0.8%) was positive for the ermB gene, and one isolate was positive for the ermQ gene. The tetM gene was not found in any of the isolates tested. Two out of 15 tested isolates (13%) demonstrated transfer of tetracycline resistance via bacterial conjugation. Tetracycline should be avoided for the treatment of C. perfringens-associated diarrhea in dogs because of the relatively high prevalence of in vitro resistance, and the potential for conjugative transfer of antimicrobial resistance.200616330169
5909160.9996Antibiotic 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
2082170.9996Rapid screening technique for class 1 integrons in Enterobacteriaceae and nonfermenting gram-negative bacteria and its use in molecular epidemiology. A screening technique for integrons in members of the family Enterobacteriaceae and nonfermenting gram-negative bacteria by real-time PCR is reported. A total of 226 isolates of gram-negative bacteria obtained from a variety of clinical specimens were screened for class 1 integrons by real-time PCR performed on a LightCycler instrument. This technique used a primer pair specific for a 300-bp conserved region at the 5' ends of class 1 integrons. The screening assay was evaluated by comparison with results obtained by the conventional, thermal-block PCR (long PCR) by using established conditions and primers for the detection of class 1 integrons, and the real-time PCR technique was thus shown to be both sensitive and specific. DNA from 50 of 226 (22%) isolates screened was identified as containing an integron by the screening PCR, and sequence data were obtained across the integron for 34 of 50 (68%) of these isolates. In an attempt to study the molecular epidemiology of antimicrobial resistance genes carried within integrons, a comparison of the types of gene cassettes carried by isolates from different patients was made. Adenyltransferase genes conferring resistance to streptomycin and spectinomycin were the predominant gene cassettes amplified in the study. Resistance to trimethoprim was also frequently found to be encoded within integrons. Furthermore, multiple bacterial isolates obtained from one patient over a 5-month period were all shown to carry an integron containing the same single adenyltransferase gene cassette, suggesting that these elements were relatively stable in this case.200111257011
2240180.9996Evaluation of multiplex tandem PCR (MT-PCR) assays for the detection of bacterial resistance genes among Enterobacteriaceae in clinical urines. BACKGROUND: Increasing resistance drives empirical use of less potent and previously reserved antibiotics, including for urinary tract infections (UTIs). Molecular profiling, without culture, might better guide early therapy. OBJECTIVES: To explore the potential of AusDiagnostics multiplex tandem (MT) PCR UTI assays. METHODS: Two MT-PCR assays were developed successively, seeking 8 or 16 resistance genes. Amplification was tracked in real time, with melting temperatures used to confirm product identity. Assays were variously performed on: (i) extracted DNA; (ii) cultured bacteria; (iii) urine spiked with reference strains; and (iv) bacteria harvested from clinical urines. Results were compared with those from sequencing, real-time SybrGreen PCR or phenotypic susceptibility. RESULTS: Performance was similar irrespective of whether DNA, cultures or urines were used, with >90% sensitivity and specificity with respect to common β-lactamases, dfr genes and aminoglycoside resistance determinants except aadA1/A2/A3, for which carriage correlated poorly with streptomycin resistance. Fluoroquinolone-susceptible and -resistant Escherichia coli (but not other species) were distinguished by the melting temperatures of their gyrA PCR products. The time from urine to results was <3 h. CONCLUSIONS: The MT-PCR assays rapidly identified resistance genes from Gram-negative bacteria in urines as well as from cultivated bacteria. Used directly on urines, this assay has the potential to guide early therapy.201930476137
5534190.9996Antibiotic resistance in faecal microbiota of Greek healthy infants. Increasing use of antibiotics for the treatment of infectious diseases and also for non-therapeutic reasons (agriculture, animal husbandry and aquaculture) has led to the increasing incidence of antibiotic resistance and the ineffectiveness of antimicrobial treatment. Commensal intestinal bacteria are very often exposed to the selective pressure of antimicrobial agents and may constitute a reservoir of antibiotic resistance determinants that can be transferred to pathogens. The present study aimed to investigate the antibiotic susceptibility profile and the presence of selected resistance genes in cocci isolated from the faecal microbiota of 35 healthy, full-term infants at 4, 30 and 90 days after delivery. A total of 148 gram-positive, catalase-negative cocci were isolated and tested for susceptibility to 12 different antibiotics by disk-diffusion technique. Multiplex PCR analysis was performed for the identification of Enterococcus spp. isolates and the simultaneous detection of vancomycin-resistance genes. PCR-based methodology was used also for identification of tetracycline and erythromycin resistance determinants. Identification results indicated E. faecalis as the predominant species (81 strains), followed by E. faecium, E. casseliflavus/E. flavescens and E. gallinarum. High prevalence of resistance to tetracycline (39.9%), erythromycin (35.1%), vancomycin (19.6%) and to nucleic acid synthesis inhibitors was detected. PCR data revealed 24 out of 52 erythromycin-resistant isolates carrying the ermB gene and 32 out of 59 tetracycline-resistant strains carrying tet genes, with tet(L) determinant being the most frequently detected. Only intrinsic vancomycin resistance (vanC1 and vanC2/C3) was reported among tested isolates. In conclusion, erythromycin and tetracycline acquired resistant traits are widespread among faecal cocci isolates from Greek, healthy infants under no apparent antimicrobial selective pressure.201021831766