Mutagenesis and chromosome mobilization in Hyphomicrobium facilis B-522

1992 ◽  
Vol 38 (11) ◽  
pp. 1167-1174 ◽  
Author(s):  
Christian G. Gliesche ◽  
Peter Hirsch
Keyword(s):  
High Frequency ◽  
Tetracycline Resistance ◽  
Transposon Mutagenesis ◽  
Chemical Mutagenesis ◽  
Direct Selection ◽  
Broad Host Range ◽  
Antibiotic Resistant ◽  
Auxotrophic Mutants ◽  
Restricted Facultative Methylotroph ◽  
Insertion Mutants

Spontaneously derived antibiotic-resistant mutants of Hyphomicrobium facilis B-522, a restricted facultative methylotroph, occurred at a high frequency on agar plates with low antibiotic concentrations. Mutants specifically defective in methanol oxidation have been obtained using an allyl alcohol direct selection technique. By chemical mutagenesis with N-methyl-N′ -nitro-N-nitrosoguanidine in the presence of chloramphenicol several stable auxotrophic mutants could be isolated: three leucine auxotrophs, two threonine auxotrophs, and two leucine–methionine double auxotrophic mutants. Optimal conditions for transposon mutagenesis have been developed by comparing several transposon delivery vectors. With the suicide plasmid pRK2013 as a vector, the tetracycline resistance conferring transposon Tn5-132 was introduced into the genome of H. facilis B-522. The following insertion mutants have been obtained: leu-3::Tn5-132, ilv-1::Tn5-132, and pur-1::Tn5-132. Broad host range IncP-1 plasmids could be successfully transferred by interspecific matings. Chromosome mobilization was demonstrated with the conjugative IncP-1 plasmids RP1, R68.45, pMO60, and H. facilis 2189 (leu-2, met-1, mox-1, nfs-1, str-12) as recipient strain. Transconjugants occurred at frequencies ranging from 10−6 to 10−8 for each marker. Key words: methylotrophs, Hyphomicrobium, N-methyl-N′-nitro-N-nitrosoguanidine mutagenesis, transposon mutagenesis, promiscuous plasmids, chromosome mobilization.

scholarly journals Direct Selection of IS903 Transposon Insertions by Use of a Broad-Host-Range Vector: Isolation of Catalase-Deficient Mutants of Actinobacillus actinomycetemcomitans

1999 ◽  
Vol 181 (23) ◽  
pp. 7298-7307 ◽  
Author(s):  
Valeri J. Thomson ◽  
Mrinal K. Bhattacharjee ◽  
Daniel H. Fine ◽  
Keith M. Derbyshire ◽  
David H. Figurski
Keyword(s):  
Host Range ◽  
Transposon Mutagenesis ◽  
Actinobacillus Actinomycetemcomitans ◽  
Direct Selection ◽  
Nucleotide Sequence Analysis ◽  
Broad Host Range ◽  
Dna Uptake ◽  
Transposase Gene ◽  
Insertion Mutants ◽  
Selection Of

ABSTRACT Transposon mutagenesis in bacteria generally requires efficient delivery of a transposon suicide vector to allow the selection of relatively infrequent transposition events. We have developed an IS903-based transposon mutagenesis system for diverse gram-negative bacteria that is not limited by transfer efficiency. The transposon, IS903φkan, carries a cryptic kangene, which can be expressed only after successful transposition. This allows the stable introduction of the transposon delivery vector into the host. Generation of insertion mutants is then limited only by the frequency of transposition. IS903φkan was placed on an IncQ plasmid vector with the transposase gene located outside the transposon and expressed from isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible promoters. After transposase induction, IS903φkaninsertion mutants were readily selected in Escherichia coliby their resistance to kanamycin. We used IS903φkan to isolate three catalase-deficient mutants of the periodontal pathogenActinobacillus actinomycetemcomitans from a library of random insertions. The mutants display increased sensitivity to hydrogen peroxide, and all have IS903φkan insertions within an open reading frame whose predicted product is closely related to other bacterial catalases. Nucleotide sequence analysis of the catalase gene (designated katA) and flanking intergenic regions also revealed several occurrences of an 11-bp sequence that is closely related to the core DNA uptake signal sequence for natural transformation of Haemophilus influenzae. Our results demonstrate the utility of the IS903φkan mutagenesis system for the study of A. actinomycetemcomitans. Because IS903φkan is carried on a mobilizable, broad-host-range IncQ plasmid, this system is potentially useful in a variety of bacterial species.

scholarly journals Construction of a Derivative of Agrobacterium tumefaciens C58 That Does Not Mutate to Tetracycline Resistance

2001 ◽  
Vol 14 (1) ◽  
pp. 98-103 ◽  
Author(s):  
Zhao-Qing Luo ◽  
Thomas E. Clemente ◽  
Stephen K. Farrand
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Host Range ◽  
High Frequency ◽  
Tetracycline Resistance ◽  
Physiological Traits ◽  
Selection Marker ◽  
Detectable Effect ◽  
Broad Host Range ◽  
Binary Vectors ◽  
Agrobacterium Tumefaciens C58

Agrobacterium tumefaciens C58 mutates to tetracycline resistance at high frequency, complicating the use of many broad-host-range cloning and binary vectors that code for resistance to this antibiotic as the selection marker. Such mutations are associated with a resistant gene unit, tetC58, that is present in the genome of this strain. By deleting the tetC58 locus, we constructed NTL4, a derivative of C58 that no longer mutates to tetracycline resistance. The deletion had no detectable effect on genetic or physiological traits of NTL4 or on the ability of this strain to transform plants.

Transferable tetracycline resistance in Clostridium perfringens strains of porcine origin

1983 ◽  
Vol 29 (10) ◽  
pp. 1241-1246 ◽  
Author(s):  
Julian I. Rood
Keyword(s):  
Antibiotic Resistance ◽  
Clostridium Perfringens ◽  
High Frequency ◽  
Tetracycline Resistance ◽  
Donor Cell ◽  
Genetic Studies ◽  
Antibiotic Resistant ◽  
Resistant Strains ◽  
Derivatives Of ◽  
Porcine Origin

These studies represent the first systematic survey of the incidence of conjugative antibiotic resistance in Clostridium perfringens. Ninety-two antibiotic-resistant porcine strains were examined to see if they could donate their antibiotic-resistance determinants to sensitive recipient strains. Fifteen of the 89 tetracycline-resistant strains transferred their tetracycline resistance in mixed-plate mating experiments but no transfer of macrolide–lincosamide resistance was detected. The efficiencies of transfer of tetracycline resistance varied from 1.3 × 10−3 to 1.9 × 10−6 transconjugants per donor cell. Significantly higher transfer efficiencies were observed when both the donor and recipient strains were derivatives of strain CW 362. These values ranged from 3.7 × 10−1 to 4.6 × 10−2 transconjugants per donor cell. This high frequency transfer system should prove invaluable for further genetic studies on this microorganism.

scholarly journals Occurrence and Antimicrobial Resistance of Arcobacter spp. Recovered from Aquatic Environments

Antibiotics ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 288
Author(s):  
Sonia Sciortino ◽  
Pietro Arculeo ◽  
Vincenzina Alio ◽  
Cinzia Cardamone ◽  
Luisa Nicastro ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Tetracycline Resistance ◽  
Multi Drug Resistance ◽  
Genetic Determinants ◽  
Antibiotic Resistant ◽  
Resistance Profile ◽  
Antimicrobial Resistance Profile ◽  
Drug Resistance Profile ◽  
Arcobacter Butzleri ◽  
Seawater Samples

Arcobacter spp. are emerging waterborne and foodborne zoonotic pathogens responsible for gastroenteritis in humans. In this work, we evaluated the occurrence and the antimicrobial resistance profile of Arcobacter isolates recovered from different aquatic sources. Besides, we searched for Arcobacter spp. in seaweeds and the corresponding seawater samples. Bacteriological and molecular methods applied to 100 samples led to the isolation of 28 Arcobacter isolates from 27 samples. The highest prevalence was detected in rivers followed by artificial ponds, streams, well waters, and spring waters. Seaweeds contained a higher percentage of Arcobacter than the corresponding seawater samples. The isolates were identified as Arcobacter butzleri (96.4%) and Arcobacter cryaerophilus (3.6%). All the isolates showed a multi-drug resistance profile, being resistant to at least three different classes of antibiotics. Molecular analysis of genetic determinants responsible for tetracycline resistance in nine randomly chosen isolates revealed the presence of tetO and/or tetW. This work confirms the occurrence and the continuous emergence of antibiotic-resistant Arcobacter strains in environmental samples; also, the presence of quinolone-resistant Arcobacter spp. in aquatic sources used for water supply and irrigation represents a potential risk for human health.

Presence of antibiotic-resistant commensal bacteria in samples from agricultural, city, and national park environments evaluated by standard culture and real-time PCR methods

2010 ◽  
Vol 56 (9) ◽  
pp. 761-770 ◽  
Author(s):  
Hua Yang ◽  
Oleksandr A. Byelashov ◽  
Ifigenia Geornaras ◽  
Lawrence D. Goodridge ◽  
Kendra K. Nightingale ◽  
...  
Keyword(s):  
Real Time ◽  
Resistance Gene ◽  
Water Samples ◽  
Real Time Pcr ◽  
National Park ◽  
Tetracycline Resistance ◽  
Commensal Bacteria ◽  
Tetracycline Resistance Gene ◽  
Antibiotic Resistant ◽  
Standard Culture

This study examined the presence of antibiotic-resistant commensal bacteria among cattle operations representing areas heavily affected by agriculture, city locations representing areas affected by urban activities and indirectly affected by agriculture, and a national park representing an area not affected by agriculture. A total of 288 soil, fecal floor, and water samples were collected from cattle operations, from the city of Fort Collins, and from Rocky Mountain National Park (RMNP) in Colorado. In addition, a total of 42 new and unused feed, unused bedding, compost, and manure samples were obtained from the cattle operations. Total, tetracycline-resistant, and ceftiofur-resistant bacterial populations were enumerated by both standard culture plating and real-time PCR methods. Only wastewater samples from the cattle operations demonstrated both higher tetracycline-resistant bacterial counts (enumerated by the culture plating method) and tetracycline resistance gene copies (quantified by real-time PCR) compared to water samples collected from non-farm environments. The ceftiofur resistance gene, blaCMY-2, was not detectable in any of the samples, while the tetracycline resistance genes examined in this study, tet(B), tet(C), tet(W), and tet(O), were detected in all types of tested samples, except soil samples from RMNP. Tetracycline resistance gene pools quantified from the tet(O) and tet(W) genes were bigger than those from the tet(B) and tet(C) genes in fecal and water samples. Although only limited resistance genes, instead of a full set, were selected for real-time PCR quantification in this study, our results point to the need for further studies to determine natural and urban impacts on antibiotic resistance.

scholarly journals Alterations of Salmonella enterica Serovar Typhimurium Antibiotic Resistance under Environmental Pressure

2018 ◽  
Vol 84 (19) ◽  
Author(s):  
Mengfei Peng ◽  
Serajus Salaheen ◽  
Robert L. Buchanan ◽  
Debabrata Biswas
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Salmonella Enterica ◽  
Tetracycline Resistance ◽  
Multidrug Resistant ◽  
Genetic Alterations ◽  
Antibiotic Resistant ◽  
Content Type ◽  
Farm Systems ◽  
Serovar Typhimurium

ABSTRACT Microbial horizontal gene transfer is a continuous process that shapes bacterial genomic adaptation to the environment and the composition of concurrent microbial ecology. This includes the potential impact of synthetic antibiotic utilization in farm animal production on overall antibiotic resistance issues; however, the mechanisms behind the evolution of microbial communities are not fully understood. We explored potential mechanisms by experimentally examining the relatedness of phylogenetic inference between multidrug-resistant Salmonella enterica serovar Typhimurium isolates and pathogenic Salmonella Typhimurium strains based on genome-wide single-nucleotide polymorphism (SNP) comparisons. Antibiotic-resistant S. Typhimurium isolates in a simulated farm environment barely lost their resistance, whereas sensitive S. Typhimurium isolates in soils gradually acquired higher tetracycline resistance under antibiotic pressure and manipulated differential expression of antibiotic-resistant genes. The expeditious development of antibiotic resistance and the ensuing genetic alterations in antimicrobial resistance genes in S. Typhimurium warrant effective actions to control the dissemination of Salmonella antibiotic resistance. IMPORTANCE Antibiotic resistance is attributed to the misuse or overuse of antibiotics in agriculture, and antibiotic resistance genes can also be transferred to bacteria under environmental stress. In this study, we report a unidirectional alteration in antibiotic resistance from susceptibility to increased resistance. Highly sensitive Salmonella enterica serovar Typhimurium isolates from organic farm systems quickly acquired tetracycline resistance under antibiotic pressure in simulated farm soil environments within 2 weeks, with expression of antibiotic resistance-related genes that was significantly upregulated. Conversely, originally resistant S. Typhimurium isolates from conventional farm systems lost little of their resistance when transferred to environments without antibiotic pressure. Additionally, multidrug-resistant S. Typhimurium isolates genetically shared relevancy with pathogenic S. Typhimurium isolates, whereas susceptible isolates clustered with nonpathogenic strains. These results provide detailed discussion and explanation about the genetic alterations and simultaneous acquisition of antibiotic resistance in S. Typhimurium in agricultural environments.

scholarly journals Complete Nucleotide Sequence of the Conjugative Tetracycline Resistance Plasmid pFBAOT6, a Member of a Group of IncU Plasmids with Global Ubiquity

2004 ◽  
Vol 70 (12) ◽  
pp. 7497-7510 ◽  
Author(s):  
Glenn Rhodes ◽  
Julian Parkhill ◽  
Christine Bird ◽  
Kerrie Ambrose ◽  
Matthew C. Jones ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Transfer Functions ◽  
Genetic Load ◽  
Tetracycline Resistance ◽  
Complete Sequence ◽  
Pair Formation ◽  
Broad Host Range ◽  
Mating Pair ◽  
Associated Bacteria ◽  
Type Iv

ABSTRACT This study presents the first complete sequence of an IncU plasmid, pFBAOT6. This plasmid was originally isolated from a strain of Aeromonas caviae from hospital effluent (Westmorland General Hospital, Kendal, United Kingdom) in September 1997 (G. Rhodes, G. Huys, J. Swings, P. McGann, M. Hiney, P. Smith, and R. W. Pickup, Appl. Environ. Microbiol. 66:3883-3890, 2000) and belongs to a group of related plasmids with global ubiquity. pFBAOT6 is 84,748 bp long and has 94 predicted coding sequences, only 12 of which do not have a possible function that has been attributed. Putative replication, maintenance, and transfer functions have been identified and are located in a region in the first 31 kb of the plasmid. The replication region is poorly understood but exhibits some identity at the protein level with replication proteins from the gram-positive bacteria Bacillus and Clostridium. The mating pair formation system is a virB homologue, type IV secretory pathway that is similar in its structural organization to the mating pair formation systems of the related broad-host-range (BHR) environmental plasmids pIPO2, pXF51, and pSB102 from plant-associated bacteria. Partitioning and maintenance genes are homologues of genes in IncP plasmids. The DNA transfer genes and the putative oriT site also exhibit high levels of similarity with those of plasmids pIPO2, pXF51, and pSB102. The genetic load region encompasses 54 kb, comprises the resistance genes, and includes a class I integron, an IS630 relative, and other transposable elements in a 43-kb region that may be a novel Tn1721-flanked composite transposon. This region also contains 24 genes that exhibit the highest levels of identity to chromosomal genes of several plant-associated bacteria. The features of the backbone of pFBAOT6 that are shared with this newly defined group of environmental BHR plasmids suggest that pFBAOT6 may be a relative of this group, but a relative that was isolated from a clinical bacterial environment rather than a plant-associated bacterial environment.

Deletion and Transposon Mutagenesis and Sequence Analysis of the pRO1600 OriR Region Found in the Broad-Host-Range Plasmids of the pQF Series

Plasmid ◽  
1994 ◽  
Vol 31 (3) ◽  
pp. 265-274 ◽  
Author(s):  
Ingrid Jansons ◽  
Gail Touchie ◽  
Robert Sharp ◽  
Kurt Almquist ◽  
Mark A. Farinha ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Host Range ◽  
Transposon Mutagenesis ◽  
Broad Host Range ◽  
Broad Host Range Plasmids

scholarly journals Joint antibiotic and phage therapy: addressing the limitations of a seemingly ideal phage for treating Staphylococcus aureus infections

2020 ◽  
Author(s):  
Brandon A. Berryhill ◽  
Douglas L. Huseby ◽  
Ingrid C. McCall ◽  
Diarmaid Hughes ◽  
Bruce R. Levin
Keyword(s):  
Staphylococcus Aureus ◽  
Bacterial Infections ◽  
Phage Therapy ◽  
Receptor Site ◽  
Clinical Isolates ◽  
Broad Host Range ◽  
Antibiotic Resistant ◽  
Small Colony Variants ◽  
Small Colony

AbstractIn response to increasing frequencies of antibiotic-resistant pathogens, there has been a resurrection of interest in the use of bacteriophage to treat bacterial infections: phage therapy. Here we explore the potential of a seemingly ideal phage, PYOSa, for combination phage and antibiotic treatment of Staphylococcus aureus infections. (i) This K-like phage has a broad host range; all 83 tested clinical isolates of S.aureus tested were susceptible to PYOSa. (ii) Because of the mode of action of PYOSaS. aureus is unlikely to generate classical receptor-site mutants resistant to PYOSa; none were observed in the 13 clinical isolates tested. (iii) PYOSa kills S. aureus at high rates. On the downside, the results of our experiments and tests of the joint action of PYOSa and antibiotics raise issues that must be addressed before PYOSa is employed clinically. Despite the maintenance of the phage, PYOSa does not clear the populations of S. aureus. Due to the ascent of a phenotypically diverse array of small colony variants following an initial demise, the bacterial populations return to densities similar to that of phage-free controls. Using a combination of mathematical modeling and in vitro experiments, we postulate and present evidence for a mechanism to account for the demise–resurrection dynamics of PYOSa and S. aureus. Critically for phage therapy, our experimental results suggest that treatment with PYOSa followed by bactericidal antibiotics can clear populations of S. aureus more effectively than the antibiotics alone.Significance StatementThe increasing frequency of antibiotic-resistant pathogens has fostered a quest for alternative means to treat bacterial infections. Prominent in this quest is a therapy that predates antibiotics: bacteriophage. This study explores the potential of a phage, PYOSa, for treating Staphylococcus aureus infections in combination with antibiotics. On first consideration, this phage, isolated from a commercial therapeutic cocktail, seems ideal for this purpose. The results of this population dynamic and genomic analysis study identify a potential liability of using PYOSa for therapy. Due to the production of potentially pathogenic atypical small colony variants, PYOSa alone cannot eliminate S. aureus populations. However, we demonstrate that by following the administration of PYOSa with bactericidal antibiotics, this limitation and potential liability can be addressed.

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