A chromosomal chloramphenicol acetyltransferase determinant from a probiotic strain of Bacillus clausii

The mechanism of resistance to chloramphenicol was studied in four strains of Bacillus clausii included in a probiotic mixture, which is administered to humans for prevention of gastrointestinal side effects due to oral antibiotic therapy. By cloning experiments, a chloramphenicol acetyltransferase (CAT) gene, cat _Bcl , coding for a putative 228-amino acid CAT protein was identified in B. clausii SIN. The deduced amino acid sequence displayed from 31% to 85% identity with 56 CAT proteins from other Gram-positive bacterial strains. The cat _Bcl gene was also detected by PCR in the three other B. clausii strains resistant to chloramphenicol, whereas it was absent in the three control strains susceptible to chloramphenicol. Pulse-field gel electrophoresis of total DNA digested by I-CeuI followed by hybridization with a cat -specific probe as well as unsuccessful repeated attempts of in vitro transfer of chloramphenicol resistance to various recipient cells indicated that cat _Bcl was chromosomally located in all four resistant B. clausii strains.     

籼稻品种地谷抗稻瘟病基因的遗传

籼稻地谷是我国杂效裟育种上重要的稻瘟病抗源之一。利用我国稻区的稻瘟病菌系ＺＢ１３和ＺＢ１５对地谷与感病品种江南香糯的杂效Ｆ１、Ｆ２和Ｂ１Ｆ１群体,以及地谷与感病品种丽江新团黑谷、冈４６Ｂ和８９８７的Ｆ２群体进行接种鉴定,根据抗病性的分离确认地谷对ＺＢ１３和ＺＢ１５的抗性受显性基因控制。利用菌系ＺＢ１３接种地谷和１０个具有已知抗病基因的鉴别品种及其杂交Ｆ１和Ｆ２群体,进一步证明了地谷的抗温性受１对显     

Characterisation and transfer of antibiotic resistance genes from enterococci isolated from food

The genetic determinants responsible for the resistances against the antibiotics tetracycline [tet(M), tet(O), tet(S), tet(K) and tet(L)], erythromycin (ermA,B,C; mefA,E; msrA/B; and ereA,B) and chloramphenicol (cat) of 38 antibiotic-resistant Enterococcus faecium and Enterococcus faecalis strains from food were characterised. In addition, the transferability of resistance genes was also assessed using filter mating assays. The tet(L) determinant was the most commonly detected among tetracycline-resistant enterococci (94% of the strains), followed by the tet(M) gene, which occurred in 63.0% of the strains. Tet(K) occurred in 56.0% of the resistant strains, while genes for tet(O) and tet(S) could not be detected. The integrase gene of the Tn916-1545 family of transposons was present in 81.3% of the tetracycline resistant strains, indicating that resistance genes might be transferable by transposons. All chloramphenicol-resistant strains carried a cat gene. 81.8% of the erythromycin-resistant strains carried the ermB gene. Two (9.5%) of the 21 erythromycin-resistant strains, which did not contain ermA,B,C, ereA,B and mphA genes harboured the msrC gene encoding an erythromycin efflux pump, which was confirmed by sequencing the PCR amplicon. In addition, all E. faecium strains contained the msrC gene, but none of the E. faecalis strains. Transfer of the genetic determinants for antibiotic resistance could only be demonstrated in one filter mating experiment, where both the tet(M) and tet(L) genes were transferred from E. faecalis FAIR-E 315 to the E. faecalis OG1X recipient strain. Our results show the presence of various types of resistance genes as well as transposon integrase genes associated with transferable resistances in enterococci, indicating a potential for gene transfer in the food environment.     

Spoligotype analysis of Mycobacterium bovis isolates from Northern México

Bovine tuberculosis is still rife in Latin America, producing huge economic losses. There are very few studies of the way this disease is spread through this geographical region, particularly in countries that border those that are almost free of Mycobacterium bovis. In this work, we have analyzed the spacer oligonucleotide typing (spoligotype) patterns of M. bovis isolates from cattle at Ciudad Juárez, a Mexican city close to El Paso, Texas. Fifty-eight M. bovis isolates collected from a herd in Northern Mexico were studied by spoligotyping. Nine spoligotype patterns were observed in total. Two were predominant (SB0121 and SB0140) and accounted for 50% and 14% of the isolates, respectively. Six patterns were found to be already described in an international M. bovis spoligotype database, while the other three (SB0985, SB0986, and SB0987) were novel. Interestingly, none of the isolates corresponded to any other Mexican pattern previously reported. This is the first spoligotype analysis of M. bovis strains from a border city between Mexico and the United States. The necessity for further studies to formulate a better identification of M. bovis strains within, and its dissemination between, the two countries is discussed.     

Ecotypes of the Mycobacterium tuberculosis complex

A phylogeny of the Mycobacterium tuberculosis complex has recently shown that the animal-adapted strains are found in a single lineage marked by the deletion of chromosomal region 9 (RD9) [Brosch et al., 2002. A new evolutionary scenario for the Mycobacterium tuberculosis complex. Proc. Natl Acad. Sci. USA 99 (6), 3684-3689]. We have obtained the spoligotype patterns of the RD9 deleted strains used to generate this new evolutionary scenario and we show that the presence of spoligotype spacers 3, 9, 16, 39, and 40-43 is phylogenetically informative in this lineage. We have used the phylogenetically informative spoligotype spacers to screen a database of spoligotype patterns and have identified further members of a group of strains apparently host-adapted to antelopes. The presence of the spoligotype spacers is congruent with the phylogeny generated by chromosomal deletions, suggesting that recombination is rare or absent between strains of this lineage. The phylogenetically informative spacers, in concert with the previously identified single nucleotide mutations and chromosomal deletions, can be used to identify a series of clades in the RD9 deleted lineage each with a separate host preference. Finally, we discuss the application of the ecotype concept to this series of clades and suggest that the M. tuberculosis complex may best be described as a series of host-adapted ecotypes.     

Genetic typing of Mycobacterium tuberculosis strains by spoligotyping and genome fingerprinting techniques

A total of 122 M. tuberculosis clinical drug-resistant strains isolated in Central Russia were studied by spoligotyping and genome fingerprinting techniques. According to spoligotyping results 77% of M. tuberculosis strains were distributed to 13 oligotypes, while 23% of these strains were found to form unique patterns. Most of them belonged to the families Beijing and Haarlem (43.4% and 13.9% respectively). The patterns of the strains of oligotype 12 (7F-7F-7E-0E-78-3E) were identical to those of the strains isolated in Brazil, France and the Netherlands. The strains of the spoligotype 22 (7F-1E-7F-7F-07-3E) had the patterns identical to those of the strains of group S13, also isolated in Brazil. According to genome fingerprinting 31.4% of the strains were found to belong to clusters with the similarity coefficient equal to 1. The strains belonging to genotypes W and A1 were found to prevail in the analyzed group.     

Use of restriction fragment length polymorphism to detect dihydropteroate synthase gene mutations in strains of Pneumocystis jirovecii isolated in Poland

Point mutation of the dihydropteroate synthase gene causes Pneumocystis jirovecii resistance to sulfa drugs. The RFLP method was used to search for DHPS polymorphism in P. jirovecii strains isolated in Poland. Positive results of DHPS gene fragment amplification using nested PCR were achieved in 25 out of the 30 examined Pneumocystis DNA samples. Two (8%) of the 25 isolates had point mutation at codon 55 (Thr55Ala). The mutation-positive strains were obtained from HIV positive (1/15) and HIV negative (1/10) patients. The observed DHPS gene polymorphism may point to the appearance of P. jirovecii strains resistant to sulfa drugs in Poland.     

Exclusion of vanA, vanB and vanC type glycopeptide resistance in strains of Lactobacillus reuteri and Lactobacillus rhamnosus used as probiotics by polymerase chain reaction and hybridization methods

Strains of Lactobacillus reuteri and Lact. rhamnosus are used as probiotics in man and animal. The aim of this study was to determine whether the glycopeptide resistance in these lactobacilli has a similar genetic basis as in enterococci. Five Lact. reuteri strains and one Lact. rhamnosus, as well as four Enterococcus control strains, were probed for the vanA gene cluster, the vanB gene and the vanC gene by PCR and Southern hybridization, and DNA/DNA hybridization. Their resistance and plasmid patterns were also investigated. All Lactobacillus strains were resistant to vancomycin but susceptible to a broad range of antibiotics. Four of the Lactobacillus strains (including the Lact. rhamnosus strain) did not harbour any plasmid and two of them contained five and 6 plasmid bands respectively. None of the Lactobacillus strains possessed the vanA, vanB or vanC gene. These findings indicate that the glycopeptide resistance of the Lactobacillus strains analysed is different from the enterococcal type. The study provides reassurance on the safety of the Lactobacillus strains used as probiotics with regard to their vancomycin resistance.     

Characterization of a new erm-related macrolide resistance gene present in probiotic strains of Bacillus clausii

The mechanism of resistance to macrolides, lincosamides, and streptogramins B was studied in four Bacillus clausii strains that are mixed in a probiotic administered to humans for prevention of gastrointestinal side effects due to oral antibiotic chemotherapy and in three reference strains of B. clausii, DSM8716, ATCC 21536, and ATCC 21537. An 846-bp gene called erm(34), which is related to the erm genes conferring resistance to these antibiotics by ribosomal methylation, was cloned from total DNA of B. clausii DSM8716 into Escherichia coli. The deduced amino acid sequence presented 61% identity with that of Erm(D) from B. licheniformis, B. halodurans, and B. anthracis. Pulsed-field gel electrophoresis of total DNA digested by I-CeuI, followed by hybridization with an erm(34)-specific probe, indicated a chromosomal location of the gene in all B. clausii strains. Repeated attempts to transfer resistance to macrolides by conjugation from B. clausii strains to Enterococcus faecalis JH2-2, E. faecium HM1070, and B. subtilis UCN19 were unsuccessful.     

Nisin Resistance in Clostridium botulinum Spores and Vegetative Cells

The frequencies at which vegetative cells and spores of Clostridium botulinum strains 56A, 62A, 17409A, 25763A, 213B, B-aphis, and 169B formed colonies on agar media containing 0, 10(sup2), 10(sup3), and 10(sup4) IU of nisin per ml at 30(deg)C were determined. Strain 56A had the highest frequencies of nisin resistance, while strains 62A, 169B, and B-aphis had the lowest. For most strains, spores were more resistant than vegetative cells. One exposure to nisin was sufficient to generate stable nisin-resistant isolates in some strains. Stepwise exposure to increasing concentrations of nisin generated stable resistant isolates from all strains. Spores produced from nisin-resistant isolates maintained their nisin resistance. The frequency of spontaneous nisin resistance was reduced considerably by lowering the pH of the media and adding 3% NaCl. Nisin-resistant isolates of strains 56A and 169B also had increased resistance to pediocin PA1, bavaricin MN, plantaricin BN, and leuconocin S.     

多重耐药鲍曼不动杆菌的耐药性及其耐药基因分析

目的:探讨多重耐药鲍曼不动杆菌(MDR-Ab)的耐药性及其耐药基因,为临床合理选择抗菌药物提供依据。方法:回顾性分析2018年1月至2018年12月鲍曼不动杆菌感染的住院患者信息。使用VITEK-32微生物分析仪/梅里埃药敏卡片GN13鉴定MDR-Ab 95株。采用聚合酶链式反应(多重PCR)检测MDR-Ab携带相关耐药基因。结果:95株MDR-Ab对头孢类抗菌药物耐药率为100%。对氨苄西林-舒巴坦和头孢哌酮-舒巴坦耐药率分别为95.79%和81.05%,对美罗培南和亚胺培南耐药率分别为56.84%和57.89%,对庆大霉素和阿米卡星耐药率均为88.42%,对环丙沙星和左氧氟沙星耐药率分别为100%和88.42%,对四环素、米诺环素、替加环素耐药率分别为87.37%、16.84%和9.47%,对多粘菌素B耐药率为1.05%。95株MDR-Ab中携带β-内酰胺酶中A类酶耐药基因TEM、PER分别95株和25株,D类酶耐药基因OXA-51、carO和adeB各95株,OXA-23基因90株。携带消毒剂耐药基因qacE 60株。携带16S r RNA甲基化酶耐药基因armA 75株。每株MDR-Ab除携带TEM+carO+adeB+OXA-51四种基因外,另同时携带四种基因20株(21.05%),三种基因38株(40.00%)。结论:MDR-Ab对多种抗菌药物的耐药率较高,携带的耐药基因型主要为TEM、carO、adeB及OXA-51。携带多种耐药基因是MDR-Ab耐药重要原因。加强医院感染防控、合理应用抗菌药物对于延缓泛鲍曼不动杆菌耐药性发展具有重要的临床意义。     

Molecular characterisation of <Emphasis Type="Italic">Mycobacterium bovis</Emphasis>isolated from cattle slaughtered at the Bamako abattoir in Mali

Background

Mali is one of the most important livestock producers of the Sahel region of Africa. A high frequency of bovine tuberculosis (BTB) has been reported but surveillance and control schemes are restricted to abattoir inspections only. The objective of this study was to conduct, for the first time, molecular characterisation of Mycobacterium bovis strains isolated from cattle slaughtered at the Bamako abattoir. Of 3330 animals screened only 60 exhibited gross visible lesions. From these animals, twenty strains of M. bovis were isolated and characterised by spoligotyping.

Results

Organ lesions typical of BTB were most often detected in the liver, followed by the lung and the peritoneum. M. bovis was isolated from 20 animals and 7 different spoligotypes were observed among these 20 strains; three of the patterns had not been previously reported. Spoligotype patterns from thirteen of the strains lacked spacer 30, a characteristic common in strains of M. bovis found in Chad, Cameroon and Nigeria. However, unlike the other three Central African countries, the majority of spoligotype patterns observed in Mali also lacked spacer 6. Of the remaining seven strains, six had spoligotype patterns identical to strains commonly isolated in France and Spain.

Conclusion

Two groups of M. bovis were detected in cattle slaughtered at the Bamako abattoir. The spoligotype pattern of the first group has similarities to strains previously observed in Chad, Cameroon and Nigeria. The additional absence of spacer 6 in the majority of these strains suggests a Mali specific clone. The spoligotype patterns of the remaining strains suggest that they may have been of European origin.

     

印第安纳沙门氏菌对氯霉素类药物耐药性分析

[目的]调查鸡源沙门氏菌对氯霉素类药物的耐药特征和相关耐药基因的流行情况.[方法]从山东省部分地区鸡孵化场、养殖场、屠宰场分离样品进行沙门氏菌鉴定和药物敏感性试验;设计引物,对氯霉素类药物的耐药基因进行PCR扩增和序列分析.[结果]试验分离到印第安纳沙门氏菌,分离率为23.28％.孵化场、养殖场、屠宰场印第安纳沙门氏菌对氯霉素耐药率分别为50.00％、83.33％和93.30％,对氟苯尼考耐药率为83.33％、100％和100％,对甲砜霉素耐药率为63％、65％和77％.在80株氯霉素类耐药菌株中,54株检测到catA1基因,74株检测到floR基因,5株检测到cmlA基因.[结论]不同来源印第安纳沙门氏菌对氯霉素类药物耐药率存在差异,catA1和floR基因广泛存在于耐药菌株中.     

A recombinase-mediated system for elimination of antibiotic resistance gene markers from genetically engineered Bacillus thuringiensis strains.

A TnpI-mediated site-specific recombination system to construct genetically modified Bacillus thuringiensis strains was developed. Recombinant B. thuringiensis strains from which antibiotic resistance genes can be selectively eliminated were obtained in vivo with a new vector based on the specific resolution site of transposon Tn4430. For example, a cryIC gene, whose product is active against Spodoptera littoralis, was introduced into B. thuringiensis Kto harboring a cryIA(c) gene active against Ostrinia nubilalis. The resulting strain had a broader activity spectrum than that of the parental strain. It contained only B. thuringiensis DNA and was free of antibiotic resistance genes. This should facilitate regulatory approval for its development as a commercial biopesticide.     

Occurrence of macrolide-lincosamide-streptogramin B resistant isolates of Staphylococcus aureus in clinical specimens in 2003-2004

The aim of the study was the analysis of frequency of macrolide-lincosamide-streptogramin B (MLS(B)) resistance among MSSA (n=1682) and MRSA (n=272) strains which were isolated in 2002-2004 from various clinical materials from patients hospitalized in the University Hospital at the L. Rydygier Collegium Medicum in Bydgoszcz, University of Nicolaus Copernicus in Toruń. Susceptibility testing and examination of methicillin-resistant strains were performed by the disc diffusion techniques according to recommendation of NCCLS. Resistance to the MLS(B) antimicrobials agents was higher among MRSA compared to MSSA isolates. The MLS, constitutive phenotype was more prevalent than the MLS(B) inducible phenotype among investigated MRSA (65.4%) and MSSA (7.6%) isolates. Inducible resistance had only 2.5% of the MSSA and 2.6% of the MRSA strains. Moreover in 2004 there were found increasing frequency of inducible MLS(B) resistance from 1.1% to 5.7% and decreasing frequency of constitutive MLS(B) resistance from 9.2% to 4.7% among MSSA strains, in comparison to 2003. The investigated MSSA MLS(B)-, MLS(B)- and MRSAMLS(B)+, MLS(B)- strains were the most frequently isolated from pus (adequately 5.2%, 28.8% and 30.5%, 10.7%) and also from nosopharynx swabs (1.7% MSSA MLS(B)+ and 22.9% MSSA MLS(B)-) and biomaterials (15,1% MRSA MLS(B)+ and 9.6% MRSA MLS(B)-). They mainly came from patients of the outpatient clinic (2,4% MSSA MLS(B)+ and 19.9% MSSA MLS(B)-) and patients treated at the neurosurgical ward (20.6% MRSA MLS(B)+ and 12.1% MRSA MLS(B)-).     

欧文氏菌2-酮基醛糖还原酶基因敲除的研究

根据已知基因序列,利用ＰＣＲ技术,从克隆质粒和欧文氏菌（Ｅｒｗｉｎｉａ ｓｐ．）ＳＣＢ１２５染色体中重新扩增得到含有２－酮基醛糖还原酶Ａ和Ｂ（２－ＫＲＡ和Ｂ）基因的片段,分别用于基因表达和敲除。用于表达的片段定向连接到表达载体ｐＢＬ并转化大肠杆菌ＢＨ５α后,获得高酶活表达。在证实发生了突变的基因表达产物仍具有酶活的基础上,对其进行基因敲除的研究。在体外将链霉素抗性基因插入到ｔｋｒＡ内部使其突变失活     

利用荧光差异显示技术分离的家蚕抗NPV相关基因s3a

通过荧光差异显示技术,分析了家蚕Bombyx mori对BmNPV抗性品系NB、感性品系306和近等基因系306NNZZ添毒和未添毒处理区的基因表达的差异。根据差异显示的结果克隆了一条702 bp长度的cDNA片段,并用Northern blot进行了验证。该序列经过NCBI EST库的同源性比较获得了电子延伸。延伸后的序列用特异引物进行RT-PCR扩增获得了一条782 bp的序列,拼接后基因cDNA序列全长为827 bp,推导的氨基酸序列与草地夜蛾Spodoptera frugiperda S3A同源性最高达97.7％;其次是烟芽夜蛾Heliothis virescens S3A,同源性为94.0％;与黑腹果蝇Drosophila melanogaster S3A同源性为75.3%。比较结果显示这是一个新的家蚕基因,定名为家蚕s3a基因。本实验获得的s3a基因在家蚕感性和抗性品系以及添毒处理和未添毒处理中都具有差异表达,其中在抗性品系和近等基因系中的表达高于感性品系,在添毒处理中的表达高于未添毒组。因此推测它是一个与家蚕抗BmNPV相关的新基因。     

Antibiotic 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.     

The genetic diversity of Mycobacterium tuberculosis and an assessment of risk factors of tuberculosis spread in Russia's Siberian region by molecular epidemiological methods

Molecular epidemiology approaches provided for a new interpretation of the TB infection transmission dynamics, contributed to changing the focuses of attention and updated the monitoring practice. On the basis of 101 cases of isolates of Mycobacterium tuberculosis (MBT) complex sampled from 84 patients with pulmonary tuberculosis in the Siberian region, we proved that the independent methods of IS6110 RFLP genetic typing and VNTR-typing by five accurate repeat tandems of ETR A, B, C, D, and E bring about similar results and can be used in studying the MTB clonal structure population in the Siberian region for the purpose of defining the TB infection transmission dynamics. The most widespread genetic types were detected, i.e. Beeijing family strains, the S42 spoligotype, and the 31323 VNTR type, which account for 52.3% of all samples. The general parameters describing the epidemic process intensity were evaluated, i.e. those characterizing the strains (91.6%) and the transmission activity factor (72%). Consequently, each three of the four analyzed TB cases resulted from a recent transmission. However, there is a trend, within the analyzed samples, towards a higher percentage of clusterization in the age group ranging from 40 to 60. Such trend is typical of a prevalence of TB reactivation cases caused by MBT complex strains spread intensively in the discussed territory. As for the clusterized isolates, which are endemic for the territory, such data should be interpreted as a recent transmission only cautiously. 28.5% of the studied isolates are resistant to anti-TB drugs used in medical practice; and 35.7% of them are resistant to izoniazide and rifampicin, therefore, according to the WHO classification they are considered to be poly-antibiotics-resistant (PAR). No strict associations were found between the spectrum of antibiotics-resistance and any of genotypes, however, 30% of PAR strains are 32525 and 42525 types VNTR (spoligotype S1 or Beejing type).     

Microarray analysis of erythromycin resistance determinants

AIMS: To develop a DNA microarray for analysis of genes encoding resistance determinants to erythromycin and the related macrolide, lincosamide and streptogramin B (MLS) compounds. METHODS AND RESULTS: We developed an oligonucleotide microarray containing seven oligonucleotide probes (oligoprobes) for each of the six genes (ermA, ermB, ermC, ereA, ereB and msrA/B) that account for more than 98% of MLS resistance in Staphylococcus aureus clinical isolates. The microarray was used to test reference and clinical S. aureus and Streptococcus pyrogenes strains. Target genes from clinical strains were amplified and fluorescently labelled using multiplex PCR target amplification. The microarray assay correctly identified the MLS resistance genes in the reference strains and clinical isolates of S. aureus, and the results were confirmed by direct DNA sequence analysis. Of 18 S. aureus clinical strains tested, 11 isolates carry MLS determinants. One gene (ermC) was found in all 11 clinical isolates tested, and two others, ermA and msrA/B, were found in five or more isolates. Indeed, eight (72%) of 11 clinical isolate strains contained two or three MLS resistance genes, in one of the three combinations (ermA with ermC, ermC with msrA/B, ermA with ermC and msrA/B). CONCLUSIONS: Oligonucleotide microarray can detect and identify the six MLS resistance determinants analysed in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results suggest that microarray-based detection of microbial antibiotic resistance genes might be a useful tool for identifying antibiotic resistance determinants in a wide range of bacterial strains, given the high homology among microbial MLS resistance genes.