Chromosomal location of antibiotic resistance genes in Neisseria gonorrhoeae.

Transformation with purified plasmid and chromosomal deoxyribonucleic acid from a clinical isolate of Neisseria gonorrhoeae showed that each of seven loci affecting drug resistance (penA, penB, ery, str, tet, chl, and env) was chromosomal.     

Loss of low-level antibiotic resistance in Neisseria gonorrhoeae due to env mutations.

Mutations (env) which resulted in increased sensitivity of gonococci to diverse compounds were studied by transformation. Strains carrying an env mutation were more sensitive than wild-type strains to several antibiotics, dyes, and detergents. The env mutations resulted in complete phenotypic suppression of low-level resistance to these same drugs determined by mutation at ery. Recombination was observed in transformation crosses between various env mutants. The env locus was not linked to the cluster of antibiotic resistance genes near str and spc.     

Polygenes and modifier genes for tetracycline and penicillin resistance in Neisseria gonorrhoeae

The genetic basis for spontaneous resistance to tetracyline (Tet) and penicillin (Pen) in Neisseria gonorrhoeae was investigated. Tet and pen are polygenes which confer small but distinct levels of resistance to Tet and Pen, respectively. Mtr is a multiple-drug resistance polygene which increases resistance to Tet and Pen (as well as to other unrelated antibiotics). Tem is a modifier gene affecting resistance toTet and Pen. Pem is a modifier gene for Pen resistance. The following gene combinations code for resistance to five antibiotics: tet, mtr and tem for Tet; pen, mtr, pem and tem for Pen; tet, tem and mtr for doxycycline; pen and pem for ampicillin; pen, pem and mtr for nafcillin.     

Genetic analysis of antibiotic resistance in Streptococcus pyogenes.

The genetics of antibiotic resistance in mutant strains of Streptococcus pyrogenes was studied. Utilizing a type 6 strain (9440) primarily resistant to strepttomycin (Strr), classes of mutant strains were isolated that were resistant to one of the following antibiotics: rifampin (Rifr), erythromycin (Eryr), thiostrepton (Tstr), spiramycin (Sprr), fusidic acid (Fusr), gramicidin (Grcr), ethidium bromide (Ebrr), kanamycin (Kanr), neomycin (Neor), oleandomycin (Oler), gentamicin (Genr), and novobiocin (Novr). Transduction experiments separated antibiotic resistance markers into two distinct groups: transducible markers, including Fusr, Bacr, Ksg+, Spcr, Eryr, Sprr, Rifr, Stlr, and Tstr (Bacr, Ksgr, Spcr, and Stlr refer to resistance to bacitracin, kasugamycin, spectinomycin, and streptolydigan, respectively), and nontransducible markers, including Grcr, Ebrr, Kanr, Neor, Oler, Genr, and Novr. By means of two- and three-point crosses, transducible markers (excluding tst) were located in three separate linkage groups. spr was found to be linked with ery and spc in the order spc-ery-spr, whereas in a separate linkage group the order was determined to be str-fus-bac-ksg. The third linkage group contained the rif and stl markers.     

Novel approach for improving the productivity of antibiotic-producing strains by inducing combined resistant mutations

We developed a novel approach for improving the production of antibiotic from Streptomyces coelicolor A3(2) by inducing combined drug-resistant mutations. Mutants with enhanced (1.6- to 3-fold-higher) actinorhodin production were detected at a high frequency (5 to 10%) among isolates resistant to streptomycin (Str(r)), gentamicin (Gen(r)), or rifampin (Rif(r)), which developed spontaneously on agar plates which contained one of the three drugs. Construction of double mutants (str gen and str rif) by introducing gentamicin or rifampin resistance into an str mutant resulted in further increased (1.7- to 2.5-fold-higher) actinorhodin productivity. Likewise, triple mutants (str gen rif) thus constructed were found to have an even greater ability for producing the antibiotic, eventually generating a mutant able to produce 48 times more actinorhodin than the wild-type strain. Analysis of str mutants revealed that a point mutation occurred within the rpsL gene, which encodes the ribosomal protein S12. rif mutants were found to have a point mutation in the rpoB gene, which encodes the beta-subunit of RNA polymerase. Mutation points in gen mutants still remain unknown. These single, double, and triple mutants displayed in hierarchical order a remarkable increase in the production of ActII-ORF4, a pathway-specific regulatory protein, as determined by Western blotting analysis. This reflects the same hierarchical order observed for the increase in actinorhodin production. The superior ability of the triple mutants was demonstrated by physiological analyses under various cultural conditions. We conclude that by inducing combined drug-resistant mutations we can continuously increase the production of antibiotic in a stepwise manner. This new breeding approach could be especially effective for initially improving the production of antibiotics from wild-type strains.     

Inheritance of low-level resistance to penicillin, tetracycline, and chloramphenicol in Neisseria gonorrhoeae.

The genetics of low-level resistance to penicillin and other antibiotics in a clinical isolate and a multistep laboratory mutant of Neisseria gonorrhoea was studied by transformation. Mutations at three loci affected sensitivity to penicillin. Mutation at penA resulted in an eightfold increase in resistance to penicillin without affecting response to other antimicrobial agents. Mutation at ery resulted in a two- to fourfold increase in resistance to penicillin and similar increases in resistance to many other antibiotics, dyes, and detergents. Mutation at penB resulted in a fourfold increase in resistance to penicillin and tetracycline, the phenotypic expression of which was dependent on the presence of mutation at ery. The cumulative effect of mutations at penA, ery, and penB was an approximate 128-fold increase in penicillin resistance, to a minimum inhibitory concentration of 1.0 mug/ml. Low-level resistance to tetracycline or chloramphenicol was due to similar additive effects between mutations at the nonspecific ery and penB loci and a locus specific for resistance to each drug (tet and chl, respectively). No evidence was found for penicillinases or other drug-inactivating enzymes.     

Presence of specific antibiotic (tet) resistance genes in infant faecal microbiota

The widespread use of antibiotics for medical and veterinary purposes has led to an increase of microbial resistance. The antibiotic resistance of pathogenic bacteria has been studied extensively. However, antibiotics are not only selective for pathogens: they also affect all members of the gut microbiota. These microorganisms may constitute a reservoir of genes carrying resistance to specific antibiotics. This study was designed to characterize the gut microbiota with regard to the presence of genes encoding tetracycline resistance proteins (tet) in the gut of healthy exclusively breast-fed infants and their mothers. For this purpose we determined the prevalence of genes encoding ribosomal protection proteins (tet M, tet W, tet O, tet S, tet T and tet B) by PCR and characterized the gut microbiota by FISH in stools of infants and their mothers. The gene tet M was found in all the breast-fed infants and their mothers. tet O was found in all of the mothers' samples, whilst only 35% of the infants harboured this gene. tet W was less frequently found (85% of the mothers and 13% of the infants). None of the other genes analysed was found in any sample. Our results suggest that genes carrying antibiotic resistance are common in the environment, as even healthy breast-fed infants with no direct or indirect previous exposure to antibiotics harbour these genes.     

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

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

2010年广州地区淋球菌耐药监测结果分析

目的了解广州地区淋球菌对抗生素耐药性的变化及PPNG和TRNG的流行趋势。方法用琼脂稀释法测定头孢曲松、大观霉素、环丙沙星、阿奇霉素和四环素的最低抑菌浓度（MIC）;用纸片碘量法检测β-内酰胺酶。结果83株淋球菌检出PPNG24株（28．9％）、TRNG50株（60．2％）、环丙沙星耐药率高达98．8％,高度耐药株（MIC≥16mg／L）43株（51．8％）,而76株淋球菌中阿奇霉素耐药株11株（14．5％）,均未出现对头孢曲松、大观霉素耐药的菌株,抗菌活性强。结论合理规范使用抗生素及动态监测淋球菌耐药性变迁是临床减少淋球菌耐药菌株出现的有效办法。     

Double Infection,Recombination, and Segregation of Two R Factors,R 100 and Rts1, and Their Possible Bearing on the Genetic Structure of R 100

Applying the observation by Yokota et al (1969) that a cell doubly harboring an R factor (R100) and a temperature sensitive R factor (Rts1) produces segregant R factors with various resistance patterns, a total of 271 segregant R factors were obtained. There were 163 resistant to (sul, str, kan), 39 resistant to (sul, str, cml, kan), 62 resistant to (sul, str, tet, kan) and finally 7 resistant to (tet, kan). More than 90% of the former 3 segregants were fi⁺ and the remainder, including all of the (tet, kan) segregants, were fi^?. Some fi^? segregants with the former 3 resistance patterns and all of the (tet, kan) segregants were nontransmissible. All of these segregants were still temperature sensitive. Based upon the results of three experiments; (a) the growth at 43 C to observe linked loss of the kan gene and the genes derived from R 100, (b) a conjugal analysis of the relevant resistant markers, and (c) a transductional analysis of these same markers, several conclusions were made. The 2 R factors both consisting of a circle were supposed to have recombined to form a larger circle which then further resulted in the final formation of smaller circles. The possible bearing of these observations and conclusions on the genetic structure of R 100 was discussed.     

Genetic mapping of toxin regulatory mutations in Vibrio cholerae.

We have mapped a regulatory site mediating the hyperproduction of cholera toxin in mutants of Vibrio cholerae strain 569B. Mutations in this locus, called htx, result in the hypertoxinogenic phenotype, as measured by the ganglioside filter assay and immunoradial diffusion. Transposon-facilitated recombination was used to construct improved genetic donors in 569B parental and hypertoxinogenic mutant strains. Subsequent mapping by conjugation indicated that the htx locus was closely linked to the rif, str, and ilv loci of V. cholerae. Analysis of recombinants from these crosses suggested the following gene order: thy str htx rif ilv arg. The close genetic linkage of htx to rif (as high as 98%) resulted in a high comutation frequency of these two loci by nitrosoguanidine mutagenesis. Transfer of the htx mutant locus from a hypertoxinogenic donor to several unrelated Tox+ strains of V. cholerae caused a detectable elevation of toxin production in the recipients. These results suggest that toxin production in diverse strains of V. cholerae is controlled by a common regulatory mechanism in which the htx gene product plays a significant role.     

宁夏地区一株奶牛源耐甲氧西林金黄色葡萄球菌流行株的高通量测序分析

【背景】耐甲氧西林金黄色葡萄球菌(Methicillin-resistant Staphylococcus aureus, MRSA)是一种常见的条件性致病菌,由MRSA感染导致的奶牛乳腺炎给奶牛养殖户带来了重大的经济损失。【目的】了解宁夏地区奶牛源MRSA流行株基因组序列特征,为MRSA感染的防治提供理论依据。【方法】采用琼脂扩散法对分离株ld11进行抗菌药物敏感性试验,同时运用Illumina高通量测序平台对其基因组DNA进行高通量测序,使用网络数据库对获得的测序序列进行处理分析。【结果】药敏试验结果显示分离株ld11对头孢噻呋、磺胺异恶唑、氨苄西林、红霉素、庆大霉素、苯唑西林、克林霉素、四环素和多西环素耐药,数据分析显示分离株ld11携带耐药基因aadD、spc、str、blaZ、mecA、cat(pC194)、erm(A)、norA、tet(k)和tet(M),二者之间有很好的相关性;分离株ld11携带的耐药基因多于MRSA参考菌株,且分离株ld11和MRSA252的亲缘关系较近。COG功能分析和GO注释结果显示,与维持菌体基本功能和菌株生长增殖相关的基因占优势;KEGG通路分析结果显示,属于代谢通路的基因占比最多。从该菌株基因组序列上共检测到4个基因岛、9个疑似的CRISPR序列和1个完整的前噬菌体序列。【结论】揭示了宁夏地区奶牛源MRSA流行株的部分基因组序列信息,为MRSA菌株间基因组序列信息的比较分析及MRSA感染的防控提供参考依据。     

Drug resistance of enteric bacteria. VII. Recombination of R factors with tetracycline-sensitive mutants

Hashimoto, Hajime (Gunma University, Maebashi, Japan), and Susumu Mitsuhashi. Drug resistance of enteric bacteria. VII. Recombination of R factors with tetracycline-sensitive mutants. J. Bacteriol. 92:1351-1356. 1966.-The transmissible drug-resistance factor R is able to confer resistance to tetracycline (TC), chloramphenicol (CM), streptomycin (SM), and sulfonamide (SA) on a host bacterium when infected by cell-to-cell contact. Tetracycline-sensitive mutants were isolated from either CM- or SM-sensitive mutants of an R factor. Among 30 mutants isolated, 10 were point mutants which could recombine with each other, forming recombinant R factors able to grow on plates containing 50 mug/ml of TC. The recombination frequency of TC-resistant recombinants was 10(-2) to 10(-3) in bacterial cells carrying two types of TC-sensitive R factors by superinfection with both factors. Segregational patterns of the various markers on the R factor, i.e., chl, str, sul, and m, the locus determining R mating, and their linkage order, were investigated among TC-resistant recombinants of the R factor. When TC was used as the selective drug, the tet locus mapped on the R factor as an end marker. In view of the fact that these results are inconsistent with the linkage order of various markers reported previously, a circular genetic structure for the R factor which includes five tet-s and three chl-s loci is presented.     

Genetic analysis of drug resistance in Neisseria gonorrhoeae: production of increased resistance by the combination of two antibiotic resistance loci.

The studies reported here demonstrate that increased resistance of Neisseria gonorrhoeae to penicillin, tetracycline, and chloramphenicol results from the combined effect of two resistance loci. As shown by experiments with deoxyribonucleic acid from transformants carrying only a single resistance locus, transformants with an incresed level of resistance to penicillin result from the combination of a penicillin-specific locus, pen, and a multiple resistance locus, mtr. Similarly, transformants with an increased level of resistance to tetracycline result from the combination of mtr and a tetracycline-specific locus, tet. Transformants with an increased level of resistance to chloramphenicol result from the combination of mtr and a chloramphenicol-specific locus, cml. Deoxyribonucleic acid dilution experiments established that only a single dose of each of the two required resistance loci is necessary to give higher-level resistance. Higher-level-resistant transformants were not obtained when a double dose of one resistance locus or a combination of loci pairs other than mtr and pen, mtr and tet, or mtr and cml was introduced into a recipient. Combinations of the mtr and tet genes resulted in increased resistance to semisynthetic tetracyclines. The presence of the mtr and pen genes resulted in increased resistance to penicillinase-stable penicillins.     

Tetracycline residues and tetracycline resistance genes in groundwater impacted by swine production facilities

Antibiotics are used at therapeutic levels to treat disease; at slightly lower levels as prophylactics; and at low, subtherapeutic levels for growth promotion and improvement of feed efficiency. Over 88% of swine producers in the United States gave antimicrobials to grower/finisher pigs in feed as a growth promoter in 2000. It is estimated that ca. 75% of antibiotics are not absorbed by animals and are excreted in urine and feces. The extensive use of antibiotics in swine production has resulted in antibiotic resistance in many intestinal bacteria, which are also excreted in swine feces, resulting in dissemination of resistance genes into the environment. To assess the impact of manure management on groundwater quality, groundwater samples have been collected near two swine confinement facilities that use lagoons for manure storage and treatment. Several key contaminant indicators - including inorganic ions, antibiotics, and antibiotic resistance genes - were analyzed in groundwater collected from the monitoring wells. Chloride, ammonium, potassium, and sodium were predominant inorganic constituents in the manure samples and served as indicators of groundwater contamination. Based on these analyses, shallow groundwater has been impacted by lagoon seepage at both sites. Liquid chromatography-mass spectroscopy (LC-MS) was used to measure the dissolved concentrations of tetracycline, chlortetracycline, and oxytetracycline in groundwater and manure. Although tetracyclines were regularly used at both facilities, they were infrequently detected in manure samples and then at relatively trace concentrations. Concentrations of all tetracyclines and their breakdown products in the groundwater sampled were generally less than 0.5 microg/L. Bacterial tetracycline resistance genes served as distinct genotypic markers to indicate the dissemination and mobility of antibiotic resistance genes that originated from the lagoons. Applying PCR to genomic DNA extracted from the lagoon and groundwater samples, four commonly occurring tetracycline (tet) resistance genes - tet(M), tet(O), tet(Q), and tet(W) - were detected. The detection frequency of tet genes was much higher in wells located closer to and down-gradient from the lagoons than in wells more distant from the lagoons. These results suggested that in the groundwater underlying both facilities tetracycline resistance genes exist and are somewhat persistent, but that the distribution and potentially the flux for each tet gene varied throughout the study period.     

Isolation and characteristics of the biological and genetic properties of neamin-resistant mutants of Salmonella typhimurium and Salmonella dublin candidates for vaccine creation

Mutants, resistant to neamine and spectinomycin, have been isolated from S. typhimurium and S. dublin highly virulent strains. The neamine-resistant mutants can be divided into 3 classes in accordance with their sensitivity to streptomycin: sensitive, resistant to low and high concentrations of this antibiotic. The transduction analysis with the use of bacteriophage P 22 has revealed that the spectinomycin-resistant mutations under study are spc A mutations, while the mutations leading to resistance to neamine in class Near Strr 500 are nea B mutations. The mutation leading to resistance to spectinomycin (spc A) has been found to produce no changes in the virulence of salmonellae in the intraperitoneal infection of mice. The mutations leading to resistance to neamine and streptomycin (nea B and str A) have been found to decrease virulence.     

The prevalence of tetracycline resistance genes in clinical and commensal strains of Enterococcus faecalis isolated in Gdańsk

The common use of antibiotics is responsible for selecting of drug resistance not only in pathogenic, clinical bacteria but also in commensal, not pathogenic strains which could cause the rapid dissemination of the resistance to these antibacterial agents. However, information regarding the antibiotic resistance of commensal bacteria is very scarce, and the data is based mostly on phenotypical research. Therefore the use of genotyping methods for detection of tetracycline resistance genes, in commensal and medical isolates of bacteria, is essential, for understanding the spread of antibiotic resistance. In this study 24 commensal and 27 clinical isolates of Enterococcus faecalis has been screened by PCR methods for tet(M), tet(S) genes and Tn916 and Tn5397 transpozons. Subsequently, the tet(M) gene amplicones were sequenced and phylogenetic analysis was performed. We have found that the prevalence of tet(S) gene varied significantly between commensal and clinical strains. Moreover, the frequency of transpozons in clinical isolates was much higher comparing to strains isolated from healthy individuals. The phylogenetic analysis did not show significant differences between clinical and commensal strains but it could suggest that the genetic similarity between these two groups could be favourable factor for broad range spread of tet(M) gene.