Antibiotic production improvement in the rare actinomycete Planobispora rosea by selection of mutants resistant to the Aminoglycosides Streptomycin and Gentamycin and to Rifamycin

During a strain improvement program, spontaneous mutants with single or combined resistance to streptomycin (Str^r), gentamycin (Gen^r) or rifamycin (Rif^r) were selected from the industrial strain of Planobispora rosea, which is the producer of thiazolylpeptide GE2270. Among the mutants resistant to each single antibiotic, higher producers occurred more frequently (60%) among Gen^r than in Rif^r (10%) and Str^r (24%) populations. Two Gen^r mutants showed up to 1.5-fold improvement in GE2270 production while single resistant mutants Str^r and Rif^r produced slightly more than the parental strains. The combination of Str^r and Rif^r in the same strain improved GE2270 yield up to 1.7-fold. Finally, a higher GE2270 producing strain (1.8-fold improvement with respect to the parental strain) was selected among those mutants with triple resistance to streptomycin, rifamycin and gentamycin. A hierarchical increase in aerial mycelium and spore formation was observed which paralleled GE2270 production improvement.     

Genetic improvement of technological characteristics of starters for fermented milk products

Possibility for improvement of technological characteristics of lactobacilli using mutations of resistance to rifampicin (rif ^r ) and streptomycin (str ^r ) was studied. Using starter model of Narine Lactobacillus acidophilus INMIA-9602 Armenian diet milk product, it was showed that a possibility for selecting strains with increased rate of milk fermentation and acid production is higher in Rif^r and Str^r mutants induced by nitrosoguanidine than in cultures sensitive to antibiotics. The milk products obtained using Rif^r and Str^r strains had high viscosity, improved texture, increased amount of alive cells and good organoleptic features.     

Contraselectable Streptomycin Susceptibility Determinant for Genetic Manipulation and Analysis of Helicobacter pylori

Many Helicobacter pylori genetic studies would benefit from an ability to move DNA sequences easily between strains by transformation and homologous recombination, without needing to leave a conventional drug resistance determinant at the targeted locus. Presented here is a two-gene cassette that can be selected both (i) against, due to a Campylobacter jejuni rpsL gene (dominant streptomycin susceptibility in cells also carrying an rpsL-str^r allele), and (ii) for, due to an erm gene (erythromycin resistance). This rpsL,erm cassette's utility was assessed by using it to replace four gene loci (mdaB, frxA, fur, and nikR) in four streptomycin-resistant [Str^r] strain backgrounds (derivatives of 26695, SS1, X47, and G27MA). The resultant 16 strains (phenotypically erythromycin resistant [Erm^r] and Str^s) were each transformed with wild-type genomic DNAs, and Str^r derivatives were selected. The desired Erm^s Str^r isolates were obtained at frequencies that ranged from 17 to 96% among Str^r transformants, with the Erm^s yield apparently depending on the strain background and genome location of the targeted locus. The ease of isolating unmarked transformants described here should be valuable for many H. pylori molecular genetic and evolutionary analyses.     

The dependence of UV-induced reversions to prototrophy on the streptomycin resistance allele in Escherichia coli

UV-induced mutability to prototrophy depended on the alleles of the strA locus. The yield of UV-induced suppressor mutations was decreased ten-fold in a Str^r strain compared with an isogenic Str^s strain. This decrease was due to the inability of the majority of suppressors to be expressed phenotypically in the Str^r strain in the course of selection. The addition of streptomycin to the selective media raised the number of selected suppressor revertants, by making such expression possible. The probable relation of streptomycin resistance and UV-induced suppressors is discussed.     

Antibacterial and Antifungal Spectra of Antibiotics Produced by Different Strains of Erwinia herbicola (= Pantoea agglomerans)

Nine antibiotic producer strains of Erwinia herbicola (=Pantoea agglomerans), belonging to different groups, strongly inhibited growth of 21 streptomycin sensitive strains and 6 streptomycin resistant strains of E. amylovora. The antibacterial spectra of antibiotics produced by the tested strains of E. herbicola were mainly limited to E. amylovora and related tested species. The tested strains of E. amylovora that are resistant to streptomycin did not show cross-resistance to the different types of antibiotics produced by the tested strains of E. herbicola. The antibiotics produced by the different tested strains of E. herbicola did not exert any activity on tested fungi with the exception that strains Eh 153 and Eh 351 slightly inhibited the growth of Verticillium dahliae.     

Innovative Approach for Improvement of an Antibiotic-Overproducing Industrial Strain of Streptomyces albus

Working with a Streptomyces albus strain that had previously been bred to produce industrial amounts (10 mg/ml) of salinomycin, we demonstrated the efficacy of introducing drug resistance-producing mutations for further strain improvement. Mutants with enhanced salinomycin production were detected at a high incidence (7 to 12%) among spontaneous isolates resistant to streptomycin (Str^r), gentamicin, or rifampin (Rif^r). Finally, we successfully demonstrated improvement of the salinomycin productivity of the industrial strain by 2.3-fold by introducing a triple mutation. The Str^r mutant was shown to have a point mutation within the rpsL gene (encoding ribosomal protein S12). Likewise, the Rif^r mutant possessed a mutation in the rpoB gene (encoding the RNA polymerase β subunit). Increased productivity of salinomycin in the Str^r mutant (containing the K88R mutation in the S12 protein) may be a result of an aberrant protein synthesis mechanism. This aberration may manifest itself as enhanced translation activity in stationary-phase cells, as we have observed with the poly(U)-directed cell-free translation system. The K88R mutant ribosome was characterized by increased 70S complex stability in low Mg²⁺ concentrations. We conclude that this aberrant protein synthesis ability in the Str^r mutant, which is a result of increased stability of the 70S complex, is responsible for the remarkable salinomycin production enhancement obtained.     

Molecular Epidemiology of Antimicrobial-Resistant Commensal Escherichia coli Strains in a Cohort of Newborn Calves

Pulsed-field gel electrophoresis (PFGE) was used to investigate the dissemination and diversity of ampicillin-resistant (Amp^r) and nalidixic acid-resistant (Nal^r) commensal Escherichia coli strains in a cohort of 48 newborn calves. Calves were sampled weekly from birth for up to 21 weeks and a single resistant isolate selected from positive samples for genotyping and further phenotypic characterization. The Amp^r population showed the greatest diversity, with a total of 56 different genotype patterns identified, of which 5 predominated, while the Nal^r population appeared to be largely clonal, with over 97% of isolates belonging to just two different PFGE patterns. Distinct temporal trends were identified in the distribution of several Amp^r genotypes across the cohort, with certain patterns predominating at different points in the study. Cumulative recognition of new Amp^r genotypes within the cohort was biphasic, with a turning point coinciding with the housing of the cohort midway through the study, suggesting that colonizing strains were from an environmental source on the farm. Multiply resistant isolates dominated the collection, with >95% of isolates showing resistance to at least two additional antimicrobials. Carriage of resistance to streptomycin, sulfamethoxazole, and tetracycline was the most common combination, found across several different genotypes, suggesting the possible spread of a common resistance element across multiple strains. The proportion of Amp^r isolates carrying sulfamethoxazole resistance increased significantly over the study period (P < 0.05), coinciding with a decline in the most common genotype pattern. These data indicate that calves were colonized by a succession of multiply resistant strains, with a probable environmental source, that disseminated through the cohort over time.     

Revertants of a streptomycin-resistant, oligosporogenous mutant ofBacillus subtilis

Summary Revertants of a streptomycin-resistant (Str^R), oligosporogenous (Spo^-) mutant ofBacillus subtilis were selected for the ability to sporulate. The revertants obtained fell into two phenotypic classes: Str^S Spo⁺ (streptomycin-sensitive, sporeforming), which arose by reversion of the streptomycin resistance mutations of the parent strain; and Str^R Spo⁺, which arose by the acquisition of additional mutations, some of which were shown to affect ribosomal proteins. Alterations of ribosomal proteins S4 and S16 in the 30S subunit and L18 in the 50S subunit were detected in Str^R Spo⁺ revertants by polyacrylamide gel electrophoresis. Streptomycin resistance of the parental strain and the Str^R revertants was demonstrated to reside in the 30S ribosomal subunit. The second site mutations of the revertants depressed the level of streptomycin resistance in vivo and in the in vitro translation of phage SP01 messenger ribonucleic acid (mRNA) relative to the resistance exhibited by the Str^R parental strain. The Str^R parent grew slowly and sporulated at approximately 1% of the wild type level. The Str^S revertants closely resembled the wild type strain with regard to growth and sporulation. The Str^R revertants grew at rates intermediate between those of the Str^R parent and wild type, and sporulated at wild type levels.     

Status of Streptomycin Resistance Development in Xanthomonas oryzae pv. oryzae and Xanthomonas oryzae pv. oryzicola in China and their Resistance Characters

Rice leaves with bacterial blight or bacterial leaf streak symptoms were collected in southern China in 2007 and 2008. Five hundred and thirty‐four single‐colony isolates of Xanthomonas oryzae pv. oryzae and 827 single‐colony isolates of Xanthomonas oryzae pv. oryzicola were obtained and tested on plates for sensitivity to streptomycin. Four strains (0.75%) of X. oryzae pv. oryzae isolated from the same county of Province Yunnan were resistant to streptomycin, and the resistance factor (the ratio of the mean median effective concentration inhibiting growth of resistant isolates to that of sensitive isolates) was approximately 226. The resistant isolate also showed streptomycin resistance in vivo. In addition to resistant isolates, isolates of less sensitivity were also present in the population of X. oryzae pv. oryzae from Province Yunnan. However, no isolates with decreased streptomycin‐sensitivity were obtained from the population of X. oryzae pv. oryzicola. Mutations in the rpsL (encoding S12 protein) and rrs genes (encoding 16S rRNA) and the presence of the strA gene accounting for streptomycin resistance in other phytopathogens or animal and human pathogenic bacteria were examined on sensitive and resistant strains of X. oryzae pv. oryzae by polymerase chain reaction amplification and sequencing. Neither the presence of the strA gene nor mutations in the rpsL or rrs were found, suggesting that different resistance mechanisms are involved in the resistant isolates of X. oryzae pv. oryzae.     

Interactions between viomycin resistance and streptomycin resistance on ribosomes of Mycobacterium smegmatis.

We have investigated the mechanism of the expression of resistance to high levels of viomycin and coresistance to streptomycin in a mutant strain of Mycobacterium smegmatis ATCC 14468 (AC-13) which was obtained by serial transfers of parental cells to media containing increasing concentrations of viomycin. It was shown previously that resistance to viomycin by strain AC-13 was due to an alteration in the 50 S ribosomal subunit (20). However, genetic analysis has shown that mutation in 50 S subunits alone gave only low level resistance to viomycin. When a streptomycin resistant mutation (caused by an alteration in the 30 S subunit) was introduced into the low level viomycin resistant recombinant strains, most of them were highly resistant to viomycin. Some recombinants were resistant to intermediate levels of viomycin, and the remainder were not affected by the introduction of the str^r allele. Studies with in vitro cell-free systems have shown that streptomycin resistant 30 S ribosomal subunits obtained from a high level viomycin resistant recombinant were able to modify the levels of resistance to viomycin expressed by the 50 S ribosomal subunit. These findings provide additional evidence concerning the functional relationship between 30 S and 50 S ribosomal components in ribosomes.     

Conjugal Transfer of Genetic Information in Group N Streptococci

Streptococcus lactis strains ML3 and C₂O and S. lactis subsp. diacetylactis strains DRC3, 11007, and WM₄ were found to transfer lactose-fermenting ability to LM0230, an S. lactis C2 lactose-negative (Lac⁻) derivative which is devoid of plasmid deoxyribonucleic acid (DNA). Lactose-positive streptomycin-resistant (Lac⁺ Str^r) recombinants were found when the Lac⁺ Str^s donor was mixed with Lac⁻ Str^r LM0230 in solid-surface matings. Transduction and transformation were ruled out as the mechanism of genetic exchange in strains ML3, DRC3, 11007, and WM₄, nor was reversion responsible for the high number of Lac⁺ Str^r recombinants. Furthermore, chloroform treatment of the donor prevented the appearance of recombinants, indicating that transfer of lactose-fermenting ability required viable cell-to-cell contact. Strain C₂O demonstrated transduction as well as conjugation. Transfer of plasmid DNA during conjugation for all strains was confirmed by demonstrating the presence of plasmid DNA in the transconjugants by using agarose gel electrophoresis. In some instances, a cryptic plasmid was transferred in conjunction with the lactose plasmid by using strains DRC3, 11007, and WM₄. In S. lactis C2 × LM0230 matings, the Str^r marker was transferred from LM0230 to C2, suggesting conjugal transfer of chromosomal DNA. The results confirm conjugation as another mechanism of genetic exchange occurring in dairy starter cultures.     

Campylobacter jejuni Strains Compete for Colonization in Broiler Chicks

Campylobacter jejuni isolates possess multiple adhesive proteins termed adhesins, which promote the organism's attachment to epithelial cells. Based on the proposal that one or more adhesins are shared among C. jejuni isolates, we hypothesized that C. jejuni strains would compete for intestinal and cecal colonization in broiler chicks. To test this hypothesis, we selected two C. jejuni strains with unique SmaI pulsed-field gel electrophoresis macrorestriction profiles and generated one nalidixic acid-resistant strain (the F38011 Nal^r strain) and one streptomycin-resistant strain (the 02-833L Str^r strain). In vitro binding assays revealed that the C. jejuni F38011 Nal^r and 02-833L Str^r strains adhered to LMH chicken hepatocellular carcinoma epithelial cells and that neither strain influenced the binding potential of the other strain at low inoculation doses. However, an increase in the dose of the C. jejuni 02-833L Str^r strain relative to that of the C. jejuni F38011 Nal^r strain competitively inhibited the binding of the C. jejuni F38011 Nal^r strain to LMH cells in a dose-dependent fashion. Similarly, the C. jejuni 02-833L Str^r strain was found to significantly reduce the efficiency of intestinal and cecal colonization by the C. jejuni F38011 Nal^r strain in broiler chickens. Based on the number of bacteria recovered from the ceca, the maximum number of bacteria that can colonize the digestive tracts of chickens may be limited by host constraints. Collectively, these data support the hypothesis that C. jejuni strains compete for colonization in chicks and suggest that it may be possible to design novel intervention strategies for reducing the level at which C. jejuni colonizes the cecum.     

Relation between resistance to β-lactam antibiotics and cadmium in salmonellae isolated from pigs

Of 50Salmonella species isolated from pigs, 30 were resistant to cadmium and 18 of these also to azlocillin. The azlocillin-resistant isolates were resistant to cadmium at 80–500, mg/L CdSO₄. A broader spectrum of resistance to azlocillin, ampicillin and cephazollin was found in strains resistant to <200 mg/L CdSO₄. Resistance to silver, mercury, chloramphenicol and streptomycin was independent of the resistance to β-lactam antibiotics and Cd²⁺. Production and levels of β-lactamase do not correlate with the spectrum of resistance.     

Phenotypic and genetic diversity of Erwinia amylovora: the causal agent of fire blight

Erwinia amylovora is a polyphagous bacterium causing fire blight on apple, pear and over 130 other plant species belonging mainly to the Rosaceae family. Although E. amylovora is regarded as a very homogenous species, the particular strains can differ in pathogenic ability as far as their host range is concerned (e.g. those originating from Rubus or Maloidae plants) as well as by the extent of the disease they cause. It was found that strains originating from North America are generally more genetically heterogeneous than those from Europe. Diversity of E. amylovora is also related to streptomycin resistance as a result of its application to control of fire blight. The level of genetic heterogeneity of E. amylovora is so low (comparative genome analysis revealed a similarity of over 99% for the two genomes tested) that standard DNA-based techniques fail in detection of intra-species variability. Amplified fragment length polymorphism was found to be most useful for differentiation of strains of fire blight causal agent as well as techniques ensuing release of pan-genome sequences of two E. amylovora strains: multi-locus variable number of tandem repeats analysis and clustered regularly interspaced short palindrome repeats.     

相对定量分析异烟肼、链霉素单耐药与多耐药结核分枝杆菌临床分离株的蛋白质组差异

【目的】探讨异烟肼(isoniazid,INH)、链霉素(streptomycin,SM)单耐药结核分枝杆菌(Mycobacterium tuberculosis,MTB)与INH/SM多耐药MTB蛋白质组差异。【方法】应用i TRAQ结合Nano LC-MS/MS定量蛋白质组学技术,分析临床分离INH、SM或INH/SM耐药MTB与H37Rv标准株间均表达差异蛋白;并以INH/SM耐药MTB与H37Rv比值为对照,相对定量分析单耐药与多耐药MTB蛋白表达差异倍数;运用DAVID 6.7分析差异蛋白生物功能;STITCH 5.0分析差异蛋白与INH和SM相互作用。【结果】与H37Rv标准株比较,58个蛋白在INH、SM耐药与INH/SM耐药MTB间均有表达差异,共同差异蛋白生物功能主要为氧化还原酶活性和转移酶活性;主要参与丙酸代谢信号通路。共同差异蛋白中,与INH/SM耐药MTB比较,Rv2986c和Rv1908c在INH、SM耐药MTB均表达上调1.25倍;Rv3133c和Rv0577则均表达下调0.7倍;生物信息学预测发现以上4种蛋白可直接或间接与INH、SM进行相互作用。【结论】INH、SM单耐药和INH/SM多耐药MTB蛋白表达谱有较大差异,蛋白Rv2986c、Rv1908c、Rv3133c和Rv0577表达水平及相互作用可能与INH和SM耐药有关。     

Development of a direct test of baculovirus resistance in wild codling moth populations

The occurrence of resistance of codling moth (CM, Cydia pomonella L.) to Cydia pomonella granulovirus (CpGV) used as biological control agent revealed the need for fast and reliable resistance monitoring methods. Here, we describe the development of a laboratory resistance test that is directly performed on larvae extracted from infested apples. This test is based on a 14‐day bioassay at a discriminative CpGV concentration of 2 × 10⁵ occlusion bodies/ml diet and can be applied to L1–L4 larvae. Information on virus resistance can be obtained within <4 weeks. In a survey, CM larvae were isolated and tested from 6698 apples from 10 different orchards in Germany, Austria, Switzerland, Italy and the Netherlands. We identified seven CM populations resistant or partly resistant to CpGV isolates. Although some of the orchards were treated with commercial CpGV products, this method allowed us to obtain reliable information about the resistance status of the examined populations.     

In vivo transfer of genes from Escherichia coli to Bacillus subtilis

Summary Escherichia coli cells, carrying plasmid pRD1 with (a) drug resistance markers from Pseudomonas (km^r, carb^r, tc^r) and (b) the nif-gene group from Klebsiella, were incubated together with Bacillus subtilis cells (str^r), whose cell wall had been disintegrated with lysozyme. Upon plating the cell mixtures onto appropriately supplemented selective medium, multiple drug resistant Bacillus subtilis cells were obtained. Their nature was verified by suitable biochemical tests and checking for the presence of additional genetic markers. The majority of the isolates was unstable. Some however retained multiple drug resistance for longer periods of time, and several produced nitrogenase activity. The data are interpreted as evidence not only for the transfer of the respective genes but also for their expression in the gram-positive recipient cells.Abbreviations pRD1 a hybrid plasmid, renamed by Ray Dixon - pRP4 plasmid from Pseudomonas, originally described by Datta et al., J. Bacteriol 108, 1244 (1971) - km ^r, carb ^r, tc ^r, str ^r resistance against kanamycin, carbenicillin, tetracyclin and streptomycin, respectively - r restriction negative. For other bacterial markers refer to Bachmann, B.J. et al., Bacteriological Reviews 40, 116 (1976)     

Identification of a globally distributed clinical streptomycin-resistance plasmid and other resistance determinants in a coastal bay of China

Aims: To study streptomycin‐resistant bacteria isolated from Jiaozhou Bay and their molecular determinants of resistance. Methods and Results: Twenty‐seven tetracycline‐resistant and 49 chloramphenicol‐resistant bacterial isolates from surface seawater of Jiaozhou Bay were selected for investigation. More than 88% of these isolates were resistant to streptomycin. Half of the streptomycin‐resistant bacteria harboured the strA–strB gene pair, and six isolates carried Tn5393‐like transposons by PCR detection. The p9123‐related plasmids containing the sul2–strA–strB gene cluster were characterized in two environmental Escherichia coli isolates. Transposon Tn5393 was first identified on a Klebsiella pneumoniae plasmid, which also carried Tn1721, estP and umu genes responsible for antimicrobial and insecticide resistance. Conclusions: Coresistance to streptomycin and tetracycline or chloramphenicol was found with high frequency. p9123‐related plasmid and Tn5393 transposon may contribute to the wide distribution and spread of the strA–strB gene pair in Jiaozhou Bay. The detection of streptomycin‐resistance plasmid pQ1‐1 from Jiaozhou Bay seawater bacteria and human bacterial pathogens from USA indicates its global dissemination and transmission, across different components of the microbiota on earth. Significance and Impact of the Study: Streptomycin resistance can be recognized as an important bioindicator of environmental quality, owing to its association with anthropogenic pollution and the multidrug‐resistant microbiota.     

分离自母婴肠道的假小链双歧杆菌的耐药性分析

【背景】由于滥用抗生素导致细菌耐药性日益严重。对于双歧杆菌,人们往往注重其益生功能的挖掘而忽视了对其耐药性的研究,存在一定的安全隐患。【目的】检测母婴肠道中假小链双歧杆菌的耐药性,探究婴儿肠道中假小链双歧杆菌耐药性的来源。【方法】利用微量肉汤稀释法测定48株分离自母婴肠道的假小链双歧杆菌对14种抗生素的耐药性,比较分离自不同家庭母婴肠道中假小链双歧杆菌的耐药性。【结果】48株母婴肠道分离株对四环素、氯霉素、新霉素、环丙沙星100%耐药,对其余10种抗生素耐药率依次为：卡那霉素98%、利福平80%、克林霉素78%、甲氧苄啶63%、红霉素59%、庆大霉素43%、链霉素16%、万古霉素14%、氨苄西林6%、利奈唑胺2%。母婴肠道分离株的耐药性无显著差异,分离自同一家庭母婴肠道的菌株具有相似的耐药表型。【结论】分离自母婴肠道的假小链双歧杆菌对多种抗生素具有耐药性,婴儿肠道中假小链双歧杆菌的耐药性可能是由母亲肠道垂直传递而来。     

Incidence of antimicrobial resistance among Enterobacteriaceae isolated in Riyadh

The antimicrobial resistance of 1,018 isolates of Enterobacteriaceae isolated from fecal specimens of the urban population of Riyadh, Saudi Arabia, was studied. Resistance to 1 or more of 10 antimicrobial agents was encountered in 50.2% of the isolates. Of the isolates tested,Escherichia coli (0.8%) andKlebsiella species (1.6%) were found resistant to seven antimicrobial agents simultaneously: ampicillin, chloramphenicol, kanamycin, colistin, streptomycin, tetracycline, and carbenicillin. Resistance to nalidixic acid was encountered in only 0.68% of theE. coli isolates. No isolate was found to be resistant to gentamicin. Eighty-six of the resistant strains were tested for their ability to transfer their resistance. Forty percent were able to do so withE. coli K-12.