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360300.9994Diversity of tet resistance genes in tetracycline-resistant bacteria isolated from a swine lagoon with low antibiotic impact. Tetracycline resistance has been extensively studied and shown to be widespread. A number of previous studies have clearly demonstrated that a variety of tetracycline resistance genes are present in swine fecal material, treatment lagoons, and the environments surrounding concentrated animal feeding operations (CAFOs). The diversity of tetracycline resistance within a swine lagoon located at a CAFO that used only bacitricin methylene disalicylate as an antibiotic was evaluated by screening 85 tetracycline-resistant isolates for the presence of 18 different genes by performing PCR with primers that target tetracycline efflux genes of Gram-negative bacteria and ribosomal protection proteins. In addition, partial 16S rRNA sequences from each of these isolates were sequenced to determine the identity of these isolates. Of the 85 isolates examined, 17 may represent potential novel species based on BLAST results. Greater than 50% of the isolates (48 out of 85) were found to not contain targeted tet efflux genes. Though minimum inhibitory concentrations ranged widely (16 - >256 mg/L), these values did not give an indication of the tet genes present. Ten new genera were identified that contain at least one tet efflux gene. Five other genera possessed tet efflux genes that were not found in these organisms previously. Interestingly, none of the isolates possessed any of the selected ribosomal protection protein genes. Though tetracycline resistance was found in bacteria isolated from a swine CAFO lagoon, it appears that the limited antibiotic use at this CAFO might have impacted the presence and diversity of tetracycline resistance genes.200718059563
563410.9993Effects of antibiotic use in sows on resistance of E. coli and Salmonella enterica Typhimurium in their offspring. To determine effects of exposure of parental animals to antibiotics on antibiotic resistance in bacteria of offspring, sows were either treated or not treated with oxytetracycline prior to farrowing and their pigs were challenged with Salmonella enterica Typhimurium and treated or not treated with oxytetracycline and apramycin. Fecal Escherichia coli were obtained from sows, and E. coli and salmonella were recovered from pigs. Antibiotic resistance patterns of isolates were determined using a minimum inhibitory concentration (MIC) analysis. Polymerase chain reaction (PCR) and electroporation were used to characterize the genetic basis for the resistance and to determine the location of resistance genes. Treatments had little effect on resistance of the salmonella challenge organism. The greatest resistance to apramycin occurred in E. coli from pigs treated with apramycin and whose sows had earlier exposure to oxytetracycline. Resistance to oxytetracycline was consistently high throughout the study in isolates from all pigs and sows; however, greater resistance was noted in pigs nursing sows that had previous exposure to that drug. The aac(3)-IV gene, responsible for apramycin resistance, was found in approximately 90% of apramycin-resistant isolates and its location was determined to be on plasmids. Several resistant E. coli bio-types were found to contain the resistance gene. These results indicate that resistance to apramycin and oxytetracycline in E. coli of pigs is affected by previous use of oxytetracycline in sows.200516156702
352920.9993High dietary zinc supplementation increases the occurrence of tetracycline and sulfonamide resistance genes in the intestine of weaned pigs. BACKGROUND: Dietary zinc oxide is used in pig nutrition to combat post weaning diarrhoea. Recent data suggests that high doses (2.5 g/kg feed) increase the bacterial antibiotic resistance development in weaned pigs. Therefore, the aim of this study was to investigate the development of enterobacterial antibiotic resistance genes in the intestinal tract of weaned pigs. FINDINGS: Weaned pigs were fed diets for 4 weeks containing 57 (low), 164 (intermediate) or 2425 (high) mg kg(-1) analytical grade ZnO. DNA extracts from stomach, mid-jejunum, terminal ileum and colon ascendens were amplified by qPCR assays to quantify copy numbers for the tetracycline (tetA) and sulfonamide (sul1) resistance genes in Gram-negative bacteria. Overall, the combined data (n = 336) showed that copy numbers for tetracycline and sulfonamide resistance genes were significantly increased in the high zinc treatment compared to the low (tetA: p value < 10(-6); sul1: p value = 1 × 10(-5)) or intermediate (tetA: P < 1.6 × 10(-4); sul1: P = 3.2 × 10(-4)) zinc treatment. Regarding the time dependent development, no treatment effects were seen 1 week after weaning, but significant differences between high and low/intermediate zinc treatments evolved 2 weeks after weaning. The increased number of tetA and sul1 copies was not confined to the hind gut, but was already present in stomach contents. CONCLUSIONS: The results of this study suggest that the use of high doses of dietary zinc beyond 2 weeks after weaning should be avoided in pigs due to the possible increase of antibiotic resistance in Gram-negative bacteria.201526322131
279630.9993Distribution of specific tetracycline and erythromycin resistance genes in environmental samples assessed by macroarray detection. A macroarray system was developed to screen environmental samples for the presence of specific tetracycline (Tc(R)) and erythromycin (erm(R)) resistance genes. The macroarray was loaded with polymerase chain reaction (PCR) amplicons of 23 Tc(R) genes and 10 erm(R) genes. Total bacterial genomic DNA was extracted from soil and animal faecal samples collected from different European countries. Macroarray hybridization was performed under stringent conditions and the results were analysed by fluorescence scanning. Pig herds in Norway, reared without antibiotic use, had a significantly lower incidence of antibiotic resistant bacteria than those reared in other European countries, and organic herds contained lower numbers of resistant bacteria than intensively farmed animals. The relative proportions of the different genes were constant across the different countries. Ribosome protection type Tc(R) genes were the most common resistance genes in animal faecal samples, with the tet(W) gene the most abundant, followed by tet(O) and tet(Q). Different resistance genes were present in soil samples, where erm(V) and erm(E) were the most prevalent followed by the efflux type Tc(R) genes. The macroarray proved a powerful tool to screen DNA extracted from environmental samples to identify the most abundant Tc(R) and erm(R) genes within those tested, avoiding the need for culturing and biased PCR amplification steps.200717298370
353040.9993Occurrence of the transferable copper resistance gene tcrB among fecal enterococci of U.S. feedlot cattle fed copper-supplemented diets. Copper, an essential micronutrient, is supplemented in the diet at elevated levels to reduce morbidity and mortality and to promote growth in feedlot cattle. Gut bacteria exposed to copper can acquire resistance, which among enterococci is conferred by a transferable copper resistance gene (tcrB) borne on a plasmid. The present study was undertaken to investigate whether the feeding of copper at levels sufficient to promote growth increases the prevalence of the tcrB gene among the fecal enterococci of feedlot cattle. The study was performed with 261 crossbred yearling heifers housed in 24 pens, with pens assigned randomly to a 2×2 factorial arrangement of treatments consisting of dietary copper and a commercial linseed meal-based energy protein supplement. A total of 22 isolates, each identified as Enterococcus faecium, were positive for tcrB with an overall prevalence of 3.8% (22/576). The prevalence was higher among the cattle fed diets supplemented with copper (6.9%) compared to normal copper levels (0.7%). The tcrB-positive isolates always contained both erm(B) and tet(M) genes. Median copper MICs for tcrB-positive and tcrB-negative enterococci were 22 and 4 mM, respectively. The transferability of the tcrB gene was demonstrated via a filter-mating assay. Multilocus variable number tandem repeat analysis revealed a genetically diverse population of enterococci. The finding of a strong association between the copper resistance gene and other antibiotic (tetracycline and tylosin) resistance determinants is significant because enterococci remain potential pathogens and have the propensity to transfer resistance genes to other bacteria in the gut.201323666328
358950.9993Prevalence and diversity of tetracycline resistant lactic acid bacteria and their tet genes along the process line of fermented dry sausages. 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.200312866855
281260.9993Antibiotic resistance in faecal bacteria isolated from horses receiving virginiamycin for the prevention of pasture-associated laminitis. Enterococcus faecium, a major cause of potentially life-threatening hospital-acquired human infections, can be resistant to several antimicrobials, such that streptogramin quinupristin-dalfopristin (Q/D) is one of the few antibiotics still effective. Consequently use of the streptogramin virginiamycin as an animal growth promoter was banned in the EU in 1999 as some believed this contributed to the emergence of Q/D resistant E. faecium. Virginiamycin is advocated for preventing equine pasture-associated laminitis, but its effect on equine faecal bacterial Q/D resistance has not been determined. Faecal samples were obtained from horses receiving virginiamycin, horses co-grazing and horses not exposed to virginiamycin. Streptogramin resistant E. faecium were cultured from 70% (21/30) of animals treated with virginiamycin, 75% (18/24) of co-grazing animals and 69% (11/16) of animals not exposed. ermB and vatD genes were detected using real time PCR in 63% and 66% of animals treated with virginiamycin, 75% and 71% of co-grazing animals and 63% and 69% of animals not exposed. Antimicrobial resistance genes were present only in samples which had cultured Q/D resistant E. faecium. There was no significant difference between groups with respect to antimicrobial resistance. The gene load of vatD was significantly (p=0.04) greater in unexposed animals compared to those treated with virginiamycin. The use of virginiamycin to prevent pasture-associated laminitis does not appear to be related to an increased Q/D resistance frequency. However, in view of the high frequency of resistance within all groups, the horse is a reservoir of Q/D resistant genes and clones that potentially could be transferred transiently to humans.201121676560
361470.9993Structure and diversity of arsenic resistant bacteria in an old tin mine area of Thailand. The microbial community structure in Thailand soils contaminated with low and high levels of arsenic was determined by denaturing gradient gel electrophoresis (DGGE). Band pattern analysis indicated that the bacterial community was not significantly different in the two soils. Phylogenetic analysis obtained by excising and sequencing six bands indicated that the soils were dominated by Arthobacter koreensis and proteobacteria. Two hundred and sixty-two bacterial isolates were obtained from arsenic contaminated soils. The majority of the As resistant isolates were gram-negative bacteria. MIC studies indicated that all of the tested bacteria had greater resistance to arsenate than arsenite. Some strains were capable of growing in medium containing up to 1,500 mg/l arsenite and arsenate. Correlations analysis of resistance patterns of arsenite resistance indicated that the isolated bacteria could be categorized into 13 groups, with a maximum similarity value of 100%. All strains were also evaluated for resistance to eight antibiotics. The antibiotic resistance patterns divided the strains into 100 unique groups, indicating that the strains were very diverse. Isolates from each antibiotic resistance group were characterized in more detail by using the repetitive extragenic palindromic-PCR (rep-PCR) DNA fingerprinting technique with ERIC primers. PCR products were analyzed by agarose gel electrophoresis. The genetic relatedness of 100 bacterial fingerprints, determined by using Pearson product moment similarity coefficient, showed that the isolates could be divided into four clusters, with similarity values ranging from 5-99%. While many isolates were genetically diverse, others were clonal in nature Additionally, the arsenic-resistant isolates were examined for the presence of arsenic resistance (ars) genes by using PCR, and 30% of the isolates were found to carry an arsenate reductase encoded by the arsC gene.201020134249
563580.9993Antimicrobial resistance characteristics and fitness of Gram-negative fecal bacteria from volunteers treated with minocycline or amoxicillin. A yearlong study was performed to examine the effect of antibiotic administration on the bacterial gut flora. Gram-negative facultative anaerobic bacteria were recovered from the feces of healthy adult volunteers administered amoxicillin, minocycline or placebo, and changes determined in antimicrobial resistance (AMR) gene carriage. Seventy percent of the 1039 facultative anaerobic isolates recovered were identified by MALDI-TOF as Escherichia coli. A microarray used to determine virulence and resistance gene carriage demonstrated that AMR genes were widespread in all administration groups, with the most common resistance genes being bla TEM, dfr, strB, tet(A), and tet(B). Following amoxicillin administration, an increase in the proportion of amoxicillin resistant E. coli and a three-fold increase in the levels of bla TEM gene carriage was observed, an effect not observed in the other two treatment groups. Detection of virulence genes, including stx1A, indicated not all E. coli were innocuous commensals. Approximately 150 E. coli collected from 6 participants were selected for pulse field gel electrophoresis (PFGE), and a subset used for characterisation of plasmids and Phenotypic Microarrays (PM). PFGE indicated some E. coli clones had persisted in volunteers for up to 1 year, while others were transient. Although there were no unique characteristics associated with plasmids from persistent or transient isolates, PM assays showed transient isolates had greater adaptability to a range of antiseptic biocides and tetracycline; characteristics which were lost in some, but not all persistent isolates. This study indicates healthy individuals carry bacteria harboring resistance to a variety of antibiotics and biocides in their intestinal tract. Antibiotic administration can have a temporary effect of selecting bacteria, showing co-resistance to multiple antibiotics, some of which can persist within the gut for up to 1 year.201425566232
357490.9993Antibiotic 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
5636100.9993Impact of Ciprofloxacin and Clindamycin Administration on Gram-Negative Bacteria Isolated from Healthy Volunteers and Characterization of the Resistance Genes They Harbor. The aim of this study was to assess the impact of ciprofloxacin, clindamycin, and placebo administration on culturable Gram-negative isolates and the antibiotic resistance genes they harbor. Saliva and fecal samples were collected from healthy human volunteers before and at intervals, up to 1 year after antibiotic administration. Samples were plated on selective and nonselective media to monitor changes in different colony types or bacterial species. Following ciprofloxacin administration, there was a decrease of Escherichia coli in feces and after clindamycin administration a decrease of Bacteroides in feces and Leptotrichia in saliva, which all returned to pretreatment levels within 1 to 4 months. Ciprofloxacin administration also resulted in an increase in ciprofloxacin-resistant Veillonella in saliva, which persisted for 12 months. Additionally, 949 aerobic and anaerobic isolates purified from ciprofloxacin- and clindamycin-containing plates were screened for the presence of resistance genes. Resistance gene carriage was widespread in isolates from all three treatment groups, and no association was observed between genes and antibiotic administration. Although the anaerobic component of the microbiota was not a major reservoir of aerobe-associated antimicrobial resistance (AMR) genes, we detected the sulfonamide resistance gene sul2 in anaerobic isolates. The longitudinal nature of the study allowed identification of distinct Escherichia coli clones harboring multiple resistance genes, including one carrying an extended-spectrum β-lactamase blaCTX-M group 9 gene, which persisted in the gut for up to 4 months. This study provided insight into the effects of antibiotic administration on healthy microbiota and the diversity of resistance genes harbored therein.201525987611
5633110.9993Effect of the growth promoter avilamycin on emergence and persistence of antimicrobial resistance in enteric bacteria in the pig. AIM: To assess the effect of the growth promoter avilamycin on emergence and persistence of resistance in enteric bacteria in the pig. METHODS AND RESULTS: Pigs (treated with avilamycin for 3 months and controls) were challenged with multi-resistant Salmonella Typhimurium DT104 and faecal counts were performed for enterococci, Escherichia coli, S. Typhimurium and Campylobacter (before, during and 5 weeks post-treatment). Representative isolates were tested for antibiotic resistance and for the presence of resistance genes. Avilamycin-resistant Enterococci faecalis (speciated by PCR) were isolated from the treated pigs and continued to be detected for the first week after treatment had ceased. The avilamycin-resistance gene was characterized by PCR as the emtA gene and speciation by PCR. MIC profiling confirmed that more than one strain of Ent. faecalis carried this gene. There was no evidence of increased antimicrobial resistance in the E. coli, Salmonella and Campylobacter populations, although there was a higher incidence of tetB positive E. coli in the treated pigs than the controls. CONCLUSION: Although avilamycin selects for resistance in the native enterococci population of the pig, no resistant isolates were detected beyond 1 week post-treatment. This suggests that resistant isolates were unable to persist once selective pressure was removed and were out-competed by the sensitive microflora. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data suggest the risk of resistant isolates becoming carcass contaminants and infecting humans could be minimized by introducing a withdrawal period after using avilamycin and prior to slaughter.200515715858
6360120.9992Antimicrobial Resistance in Vaginal Bacteria in Inseminated Mares. Antimicrobials are added to semen extenders to inhibit the growth of bacteria that are transferred to the semen during collection. However, this non-therapeutic use of antimicrobials could contribute to the development of antimicrobial resistance. The objective of this study was to determine changes in the antibiotic susceptibility of vaginal microbiota after artificial insemination. Swabs were taken from the vagina of 26 mares immediately before artificial insemination and again 3 days later. Bacteria isolated from the vagina at both time points were subjected to antibiotic susceptibility testing and whole-genome sequencing. In total, 32 bacterial species were identified. There were increases in the resistance of Escherichia coli to trimethoprim (p = 0.0006), chloramphenicol and (p = 0.012) tetracycline (p = 0.03) between day 0 and day 3. However, there was no significant effect of exposure to antibiotics in semen extenders with respect to the resistance of Staphylococcus simulans and Streptococcus equisimilis (p > 0.05). Whole-genome sequencing indicated that most phenotypic resistance was associated with genes for resistance. These results indicate that the resistance patterns of vaginal bacteria may be affected by exposure to antibiotics; therefore, it would be prudent to minimize, or preferably, avoid using antibiotics in semen extenders.202336986297
2799130.9992Genetic and physiological characterization of oxytetracycline-resistant bacteria from giant prawn farms. Four hundred and thirteen oxytetracycline-resistant bacteria were recovered from six freshwater giant prawn farms with a history of oxytetracycline use. Most oxytetracyclineresistant isolates were Gram-negative bacteria. Six groups of oxytetracycline-resistant bacteria were classified using cluster analysis based on a comparison of levels of oxytetracycline resistance. Complex fingerprint patterns were obtained for 71 isolates studied. In general, the band patterns of isolates from different ponds were very similar, and the data indicated that the isolates were closely related. The exploration for crossresistance found that most of the 71 oxytetracycline-resistant isolates were also resistant to tetracycline and chlortetracycline, but had a relatively low resistance to doxycycline. Many isolates showed higher chlortetracycline resistance than oxytetracycline resistance. Additionally, the oxytetracyclineresistant isolates were examined for the presence of tetracycline resistance (tet) genes. Fifty percent of the isolates carried one of the 14 known tet genes examined. The most common determinants were TetA and TetD. However, TetB, TetC, TetE, TetK, TetL, and TetM were also found with various frequencies.200818309262
2813140.9992Quantity of the tetracycline resistance gene tet(M) differs substantially between meat at slaughterhouses and at retail. Concentrations of the tetracycline resistance gene tet(M) per square centimeter were assessed in meat from the slaughterhouse (n = 100) and from retail (n = 100) by real-time quantitative PCR. The study revealed a substantial contamination of retail meat with the tetracycline resistance gene tet(M), with a mean of 4.34 log copies per cm² fasces in chicken and 5.58 log copies per cm² fasces in pork. Quantitative resistance gene analysis provides an interesting tool for risk assessment and is becoming increasingly important. For both chicken and pork, tet(M) concentrations were significantly higher in meat at retail, compared to meat at slaughter. Cultural investigations revealed substantial differences in the prevalence of listeria and enterococci, and of E. coli and coliforms, between meat at slaughter (n = 500) and at retail (n = 500). However, the differences in the prevalence of 2 investigated groups of potential tet(M)-carriers (enterococci, listeria) could not sufficiently explain the differences in tet(M) concentrations, since increasing concentrations of tet(M) were accompanied by decreasing prevalences of these potential tet(M)-carriers. The percentage of tetracycline susceptible indicator bacteria (E. faecalis, E. coli) did not differ between meat at slaughter and meat at retail. Higher concentrations of tet(M) at retail might correlate with the proliferation of other genera than enterococci and listeria, but there is also a reason to discuss whether secondary contaminants might carry tet(M) more often than the primary flora of meat. PRACTICAL APPLICATION: We successfully applied the direct quantitative monitoring of resistance genes in meat, which generally might aid as a useful and rapid additional tool for risk assessment. We know that bacteria provide a large pool of resistance genes, which are widely shared between each other-the larger the pool is, the more genes might be exchanged. Thus, in terms of resistance gene monitoring, we should sometimes overcome the restricted view on single bacteria and look at the gene pool, instead.201121729069
5651150.9992Class 1 integron causes vulnerability to formaldehyde in Escherichia coli. In this study, the relationships of integron 1 element, formaldehyde dehydrogenase, and orfF genes with the level of formaldehyde resistance of isolated E. coli were investigated. E. coli bacteria were isolated from apparently healthy and colibacillosis-affected broilers of Fars Province, Iran. Formaldehyde resistance level and the presence of genetic markers were measured using MIC, and PCR tests, respectively. The prevalence of integron 1 element, orfF, and formaldehyde dehydrogenase genes in E. coli isolates were 61%, 8%, and 94%, respectively. In addition, according to our cut off definition, 15% and 85% of isolates were resistant and sensitive to formaldehyde, respectively. None of the genes had a statistically significant relationship with the formaldehyde resistance; however, the isolates containing integron 1 were significantly more sensitive to formaldehyde in the MIC test than those without integron 1. Integron 1 gene cassette could carry some bacterial surface proteins and porins with different roles in bacterial cells. Formaldehyde could also interfere with the protein functions by alkylating and cross-linking, and this compound would affect bacterial cell surface proteins in advance. Through an increase in the cell surface proteins, the presence of integron 1 gene cassette might make E. coli more sensitive to formaldehyde. As integron 1 was always involved in increasing bacterial resistance to antibiotics and disinfectants such as QACs, this is the first report of bacterial induction of sensitivity to a disinfectant through integron 1. Finally, integron 1 does not always add an advantage to E. coli bacteria, and it could be assumed as a cause of vulnerability to formaldehyde.202134148112
5638160.9992PCR 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
5910170.9992Antimicrobial 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
2419180.9992Presence 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
3569190.9992Identification of a new ribosomal protection type of tetracycline resistance gene, tet(36), from swine manure pits. Previously, only one ribosome protection type of a tetracycline resistance gene, tetQ, had been identified in Bacteroides spp. During an investigation of anaerobic bacteria present in swine feces and manure storage pits, a tetracycline-resistant Bacteroides strain was isolated. Subsequent analysis showed that this new Bacteroides strain, Bacteroides sp. strain 139, did not contain tetQ but contained a previously unidentified tetracycline resistance gene. Sequence analysis showed that the tetracycline resistance gene from Bacteroides sp. strain 139 encoded a protein (designated Tet 36) that defines a new class of ribosome protection types of tetracycline resistance. Tet 36 has 60% amino acid identity over 640 aa to TetQ and between 31 and 49% amino acid identity to the nine other ribosome protection types of tetracycline resistance genes. The tet(36) region was not observed to transfer from Bacteroides sp. strain 139 to another Bacteroides sp. under laboratory conditions. Yet tet(36) was found in other genera of bacteria isolated from the same swine manure pits and from swine feces. Phylogenetic analysis of the tet(36)-containing isolates indicated that tet(36) was present not only in the Cytophaga-Flavobacter-Bacteroides group to which Bacteroides sp. strain 139 belongs but also in gram-positive genera and gram-negative proteobacteria, indicating that horizontal transfer of tet(36) is occurring between these divergent phylogenetic groups in the farm environment.200312839793