Streptothricin Resistance Gene Research Articles

Genomic characterization and virulence of Streptococcus suis serotype 4 clonal complex 94 recovered from human and swine samples.

Streptococcus suis is a zoonotic pathogen that causes invasive infections in humans and pigs. Herein, we performed genomic analysis of seven S. suis serotype 4 strains belonging to clonal complex (CC) 94 that were recovered from a human patient or from diseased and clinically healthy pigs. Genomic exploration and comparisons, as well as in vitro cytotoxicity tests, indicated that S. suis CC94 serotype 4 strains are potentially virulent. Genomic analysis revealed that all seven strains clustered within minimum core genome group 3 (MCG-3) and had a high number of virulence-associated genes similar to those of virulent serotype 2 strains. Cytotoxicity assays showed that both the human lung adenocarcinoma cell line and HeLa cells rapidly lost viability following incubation for 4 h with the strains at a concentration of 106 bacterial cells. The human serotype 4 strain (ID36054) decreased cell viability profoundly and similarly to the control serotype 2 strain P1/7. In addition, strain ST1689 (ID34572), isolated from a clinically healthy pig, presented similar behaviour in an adenocarcinoma cell line and HeLa cells. The antimicrobial resistance genes tet(O) and ermB that confer resistance to tetracyclines, macrolides, and lincosamides were commonly found in the strains. However, aminoglycoside and streptothricin resistance genes were found only in certain strains in this study. Our results indicate that S. suis CC94 serotype 4 strains are potentially pathogenic and virulent and should be monitored.

Species classification and novel plasmid identifications in Arcobacter cryaerophilus and Arcobacter cryaerophilus-like organisms.

The Arcobacter is a globally emerging foodborne and zoonotic pathogen that can cause diarrhea in humans. It is relatively homogenous and clearly distinguishes the group from other Epsilonproteobacteria. Arcobacter cryaerophilus (A. cryaerophilus) is a heterogeneous species and little is known about its genomic characterization in China. This study aims to determine the genetic and plasmid features of A. cryaerophilus based on whole-genome sequence (WGS). Average Nucleotide Identity (ANI) and in silico DNA–DNA hybridization (isDDH) were used for the species classification for 90 initially identified A. cryaerophilus strains. One complete genome and 42 draft genomes were obtained by whole genome sequencing. The genomic characteristics were determined using various bioinformatics software. The genomes of the strains examined were estimated to vary from 1.81 to 2.28 Mb in length, with a G + C content of around 27%. ANI and isDDH results indicated that 90 initially identified A. cryaerophilus strains should be reclassified into four new species (ANI > 96% or isDDH > 70%). Two clades (four subclades) were identified among 90 genomes with the phylogenetic analysis. The phylogenetic tree indicated these 90 genomes exhibited a high intra-species genomic diversity. No clustering was assorted with the host or geographic location among these genomes. Aminoglycoside resistance genes, such as aph(2’’)-Ih, AAC(6’)-Ie-APH(2’’)-Ia, aac(6’)-IIa, ant(6), and streptothricin resistance gene SAT-4 were detected in the chromosomes from a third of the Chinese strains. Virulence-related genes were identified in all the sequenced strains. A novel large multiple drug-resistant plasmid (named pCNAC48 with 161,992 bp in length) was identified in strain ICDCAC48. Two antibiotic-resistance islands were found in the plasmid with lengths of 7,950 and 25,137 bp and G + C content of 38.23 and 32.39%, respectively. The drug resistance genes and some transposable elements were cross-distributed among the islands in the plasmid. Antimicrobial susceptibility tests indicated these resistance genes in the plasmid were functional. Plasmid conjugation and curing experiments proved pCNAC48 was stable in strain ICDCAC48. It was the first identified multiple drug resistance plasmid in A. cryaerophilus-like.

Genomic Insights of First ermB-Positive ST338-SCCmecVT/CC59 Taiwan Clone of Community-Associated Methicillin-Resistant Staphylococcus aureus in Poland.

We report the first Polish representative of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA), lukS/F-PV-positive, encoding the ermB gene, as a genetic determinant of constitutive resistance to macrolides, lincosamides, and streptogramin B antibiotics, cMLS-B. This is the first detection of the CA-MRSA strain responsible for nosocomial infection in the Warsaw Clinical Hospital. Resistance to β-lactams associates with a composite genetic element, SCCmec cassette type VT (5C2&5). We assigned the strain to sequence type ST338 (single-locus variant of ST59), clonal complex CC59, spa-type t437, and agr-type I. Genomic-based comparison was designated SO574/12 as an international Taiwan clone, which has been so far described mainly in the Asia-Pacific region. The ermB gene locates on the chromosome within the 14,690 bp mobile element structure, i.e., the MESPM1-like structure, which also encodes aminoglycoside- and streptothricin-resistance genes. The MESPM1-like structure is a composite transposon containing Tn551, flanked by direct repeats of IS1216V insertion sequences, which probably originates from Enterococcus. The ermB is preceded by the 273 bp regulatory region that contains the regulatory 84 bp ermBL ORF, encoding the 27 amino acid leader peptides. The latest research suggests that a new leader peptide, ermBL2, also exists in the ermB regulatory region. Therefore, the detailed function of ermBL2 requires further investigations.

Detection of the florfenicol resistance gene floR in Chryseobacterium isolates from rainbow trout. Exception to the general rule?

Bacteria from the family Flavobacteriaceae often show low susceptibility to antibiotics. With the exception of two Chryseobacterium spp. isolates that were positive for the florfenicol resistance gene floR, no clinical resistance genes were identified by microarray in 36 Flavobacteriaceae isolates from salmonid fish that could grow in ≥ 4 mg/L florfenicol. Whole genome sequence analysis of the floR positive isolates revealed the presence of a region that contained the antimicrobial resistance genes floR, a tet(X) tetracycline resistance gene, a streptothricin resistance gene and a chloramphenicol acetyltransferase gene. In silico analysis of 377 published genomes for Flavobacteriaceae isolates from a range of sources confirmed that well-characterised resistance gene cassettes were not widely distributed in bacteria from this group. Efflux pump-mediated decreased susceptibility to a range of antimicrobials was confirmed in both floR positive isolates using an efflux pump inhibitor (phenylalanine-arginine β-naphthylamide) assay. The floR isolates possessed putative virulence factors, including production of siderophores and haemolysins, and were mildly pathogenic in rainbow trout. Results support the suggestion that, despite the detection of floR, susceptibility to antimicrobials in Flavobacteriaceae is mostly mediated via intrinsic mechanisms rather than the horizontally acquired resistance genes more normally associated with Gram-negative bacterial pathogens such as Enterobacteriaceae.

Construction of a shuttle vector for use in Riemerella anatipestifer

Riemerella anatipestifer causes epizootic infectious disease in poultry and serious economic losses, especially to the duck industry. Four complete genome sequences of R. anatipestifer strains are now available. However, functional studies have been limited by the lack of an effective shuttle vector. In this study, we constructed a shuttle vector, pRES, which was able to transfer plasmid DNA between Escherichia coli and R. anatipestifer strains. The vector contains the putative replication origin from R. anatipestifer plasmid pRA7026 and a ColE1 ori for replication in R. anatipestifer and E. coli respectively. In addition, it contains oriT for transferring the vector into R. anatipestifer by conjugation, and the putative promoter of the streptothricin resistance gene of plasmid pRA0726 for heterologous gene expression in R. anatipestifer. The vector pRES will be useful in the investigation of gene function in R. anatipestifer.

Analysis and distribution of class 1 and class 2 integrons and associated gene cassettes among Escherichia coli isolates from swine, horses, cats and dogs collected in the BfT-GermVet monitoring study

In the BfT-GermVet monitoring study, 417 Escherichia coli isolates collected during 2004-06 in Germany from various disease conditions of pigs (n = 87), horses (n = 102) or cats/dogs (n = 228) were investigated for their susceptibility to 24 antimicrobial agents. This study dealt with the identification of integron-associated resistance genes among these isolates. Class 1 and class 2 integrons were detected by PCR. The variable parts of the integrons were cloned and sequenced. Transformation and conjugation experiments were conducted to confirm a plasmid location of the integrons. Class 1 and/or class 2 integrons, alone or in different combinations, were detected in 79 of the 417 E. coli isolates. Four trimethoprim resistance genes (dfrA1/12/14/17), five streptomycin/spectinomycin resistance genes (aadA1/2/4/5/6), two streptothricin resistance genes (estX, sat2), one gentamicin/tobramycin/kanamycin resistance gene (aadB) and one chloramphenicol resistance gene (catB3) were detected. Seven different cassette arrangements were identified within class 1 integrons: aadA1 (21 isolates), dfrA1 + aadA1 (18 isolates), dfrA17 + aadA5 (9 isolates), dfrA12 + orfF + aadA2 (8 isolates), aadB + aadA1 (1 isolate), dfrA14 + recombined aadA6 (1 isolate) and dfrA1 + catB3 + aadA4 (1 isolate). Three different cassette arrangements in class 2 integrons, dfrA1 + sat2 + aadA1 (24 isolates), estX + sat2 + aadA1 (6 isolates) and estX + sat2 + DeltaaadA1 (1 isolate), were identified. The plasmid location of class 1 and/or class 2 integrons was confirmed in 37 isolates. Class 1 and/or class 2 integrons carrying resistance gene cassettes were detected in 18.9% of the isolates tested. This molecular analysis complements the phenotypic susceptibility testing conducted in the BfT-GermVet monitoring study and helps to explain the persistence of resistance genes even without direct selective pressure.

Construction of a Knockout Mutant of the Streptothricin-Resistance Gene in Streptomyces albulus by Electroporation

Streptothricins (STs) produced by Streptomyces strains are broad-spectrum antibiotics. All STs consist of a carbamoylated D-gulosamine to which the β-lysine homopolymer (1 to 7 residues) and the amide form of the unusual amino acid streptolidine (streptolidine lactam) are attached. In a previous study, we successfully isolated a novel ST-resistance gene (sttH) from Streptomyces albulus by shotgun cloning with a heterologous host. In vitro analysis of SttH demonstrated that this enzyme catalyzes the hydrolysis of the amide bond of streptolidine lactam, thereby conferring ST resistance. We suggested that the true role of SttH may not be its involvement in resistance against STs; instead, it may catalyze the hydrolysis of naturally occurring cyclic amide compounds in the metabolism of S. albulus. In this study, we therefore constructed an sttH gene-knockout mutant to better understand the true biological role of SttH in S. albulus and to clarify whether or not S. albulus possesses different ST-resistance gene(s). The minimum inhibitory concentration MIC of STs in the sttH gene-knockout mutant was determined to be 6.25 μg/ml, whereas the MIC of STs in the S. albulus wild strain was determined to be >400 μg/ml. This result clearly demonstrated that sttH is the sole gene responsible for ST resistance in the S. albulus strain. Moreover, we developed a transformation system for S. albulus by electroporation in this study.

Streptothricin resistance as a novel selectable marker for transgenic plant cells.

Streptothricins are known as antimicrobial agents produced by Streptomyces spp. Bacterial resistance to streptothricin is mediated by specific enzymes exhibiting an acetyltransferase activity which renders the drug non-toxic for bacteria. The nucleotide sequence of several streptothricin resistance genes from bacteria have been described. Certain cells of eukaryotic parasites (such as Ustilago maydis or Leishmania spp.) are sensitive to streptothricin and the introduction of the bacterial resistance gene sat2 renders them resistant. We show that numerous species of plants are sensitive to low concentrations of streptothricin. Moreover, introduction of the bacterial resistance gene sat3 under the control of the 35S cauliflower mosaic virus promoter protects these cells from the toxic action of streptothricin. Therefore, sat3-mediated streptothricin resistance appears to be a promising selective marker for genetic manipulation of plant cells.

Monitoring the spread of broad host and narrow host range plasmids in soil microcosms

Abstract Escherichia coli recipient and E. coli donor strains carrying streptothricin-resistance genes were inoculated together into different soil microcosms. These genes were localized on the narrow host range plasmids of incompatibility (Inc) groups FII, Il, and on the broad host range plasmids of IncP1, IncN, IncW3, and IncQ. The experiments were intended to study the transfer of these plasmids in sterile and non-sterile soil with and without antibiotic selective pressure and in planted soil microcosms. Transfer of all broad host range plasmids from the introduced E. coli donor into the recipient was observed in all microcosm experiments. These results indicate that broad host range plasmids encoding short and rigid pili might spread in soil environments by conjugative transfer. In contrast, transfer of the narrow host range plasmids of IncFII and IncI1, into E. coli recipients was not found in sterile or non-sterile soil. These plasmids encoded flexible pili or flexible and rigid pili, respectively. In all experiments highest numbers of transconjugants were detected for the IncP1-plasmid (pTH16). There was evidence with plasmids belonging to IncP group transferred by conjugation into a variety of indigenous soil bacteria at detectable frequencies. Significantly higher numbers of indigenous transconjugants were obtained for the IncP-plasmid under antibiotic selection pressure, and a greater diversity of transconjugants was detected. Availability of nutrients and rhizosphere exudates stimulated transfer in soil. Furthermore, transfer of the IncN-plasmid (pIE1037) into indigenous bacteria of the rhizosphere community could be detected. The transconjugants were determined by BIOLOG as Serratia liquefaciens . Despite the known broad host range of IncW3 and IncQ-plasmids, transfer into indigenous soil bacteria could not be detected.

Monitoring the spread of broad host and narrow host range plasmids in soil microcosms

Escherichia coli recipient and E. coli donor strains carrying streptothricin-resistance genes were inoculated together into different soil microcosms. These genes were localized on the narrow host range plasmids of incompatibility (Inc) groups FII, Il, and on the broad host range plasmids of IncP1, IncN, IncW3, and IncQ. The experiments were intended to study the transfer of these plasmids in sterile and non-sterile soil with and without antibiotic selective pressure and in planted soil microcosms. Transfer of all broad host range plasmids from the introduced E. coli donor into the recipient was observed in all microcosm experiments. These results indicate that broad host range plasmids encoding short and rigid pili might spread in soil environments by conjugative transfer. In contrast, transfer of the narrow host range plasmids of IncFII and IncI1, into E. coli recipients was not found in sterile or non-sterile soil. These plasmids encoded flexible pili or flexible and rigid pili, respectively. In all experiments highest numbers of transconjugants were detected for the IncP1-plasmid (pTH16). There was evidence with plasmids belonging to IncP group transferred by conjugation into a variety of indigenous soil bacteria at detectable frequencies. Significantly higher numbers of indigenous transconjugants were obtained for the IncP-plasmid under antibiotic selection pressure, and a greater diversity of transconjugants was detected. Availability of nutrients and rhizosphere exudates stimulated transfer in soil. Furthermore, transfer of the IncN-plasmid (pIE1037) into indigenous bacteria of the rhizosphere community could be detected. The transconjugants were determined by BIOLOG as Serratia liquefaciens. Despite the known broad host range of IncW3 and IncQ-plasmids, transfer into indigenous soil bacteria could not be detected.

Nucleotide sequence of the bacterial streptothricin resistance gene sat3

The nucleotide sequence of the sat3 gene which encodes resistance of enteric bacteria to the antibiotic streptothricin is reported. A protein with a molecular mass of about 23 kDa is expressed from this gene. The sat3 gene is not obviously related to any one of the streptothricin resistance determinants identified so far among Gram-negative or Gram-positive bacteria.

Characterization of the sat4 gene encoding a streptothricin acetyltransferase in Campylobacter coli BE/G4.

The sat4 streptothricin resistance gene from Campylobacter coli BE/G4 was cloned into pUC18, and its nucleotide sequence was determined. Streptothricin acetyltransferase activity was detected in Escherichia coli cells containing recombinant plasmid pAT132 which carries the sat4 gene as an insert. The deduced amino acid sequence displayed 21-27% amino acid identity with streptothricin acetyltransferases from E. coli and streptothricin producers Streptomyces lavendulae and Streptomyces noursei. The sat4 gene was detected by hybridization in clinical and environmental isolates of Campylobacter spp.

The trimethoprim resistance transposon Tn7 contains a cryptic streptothricin resistance gene

The transposon Tn7 codes for a trimethoprim resistance and for a streptomycin/spectinomycin resistance function of the bacterial host cells. Cloning of a restriction fragment of Tn7 into the vector plasmid pUC19 reveals the presence in Tn7 of an additional potential resistance determinant. A streptothricin resistance gene, which appears cryptic in the original Tn7 context becomes activated in the recombinant plasmid upon supplying the promoter function of the lacZ system of pUC19. These results together with previously published sequence data further disclose the modular character in the resistance gene regions of Tn7-like transposons.

Site-specific insertion of three structural gene cassettes in transposon Tn7

Transposon Tn7 has been known to carry genes for resistance to trimethoprim and spectinomycin. A poorly expressed streptothricin resistance gene, identical to the sat gene found in transposons Tn1825 and Tn1826, was localized between the two mentioned genes in Tn7. The surroundings of all three resistance genes indicated site-specific insertion of genetic cassettes.

DNA probes for studying streptothricin resistance evolution in enteric bacteria

Probes for the detection of streptothricin resistance genes have been derived from recombinant plasmids. These include the streptothricin resistance gene probe sat 1/2 derived from Tn 1826 and specific for both the sat-1 determinant of Tn 1825 and the sat-2 determinant of Tn 1826, and the probe sat D derived from and specific for the sat-1 determinant of transposon Tn 1825. A third streptothricin resistance gene probe, sat 3, represents the streptothricin resistance determinant sat-3 of the IncQ R plasmid pIE639. Hybridization studies did not reveal any sequence homology between sat-3 and the transposon-localized sat-1 and sat-2 determinants. Moreover, non of the different sat-determinants isolated from plasmids of gram negative bacteria hybridized with the analogous resistance determinant of Streptomyces noursei, which had been cloned and named nat by Krügel et al. (Gene, 1988, 62, 209-214). The sat 1/2 probe in combination with the sat D probe proved to be suitable for the identification and the differentiation of sat-1 and sat-2 determinants in different genetic environments. Streptothricin resistance genes related to those present on transposons Tn 1825 and Tn 1826 have been detected by hybridization with the probe sat 1/2 on plasmids isolated a long time ago before the application of streptothricins. The sat-3 determinant appears to be exclusively associated with the IncQ plasmid pIE639.

Purification and biochemical characterization of streptothricin acetyltransferase coded by the cloned streptothricin-resistance gene of Streptomyces lavendulae.

Streptothricin acetyltransferase was purified from Streptomyces lividans harboring a plasmid which carried the streptothricin-resistance gene cloned from a streptothricin-producing strain, Streptomyces lavendulae No. 1080. Some properties of the enzyme were determined and the reaction product was identified to be N beta-acetylstreptothricin by NMR spectroscopy.

Cloning and characterization of the streptothricin-resistance gene which encodes streptothricin acetyltransferase from Streptomyces lavendulae.

The streptothricin-resistance gene of Streptomyces lavendulae No. 1080 was cloned in S. lividans using pIJ41 as a vector. From subcloning experiments, the 1.6 kb BamH I fragment was determined to encode the structural gene. The cell extracts of S. lividans carrying the gene on the plasmid pKS7 had activity to inactivate streptothricin in the presence of S-acetyl coenzyme A, indicating that the gene product was streptothricin acetyltransferase.

Isolation and characterization of a pock-forming plasmid pTA4001 from Streptomyces lavendulae.

A covalently closed circular (ccc) DNA with a size of 5.9 kb was isolated from a strain producing streptothricins , Streptomyces lavendulae No. 1080, and its restriction map was determined. Colonies of the plasmid-carrying strain formed pocks on the lawn of the plasmid-free derivatives of the same organism. The pock-forming ability of pTA4001 was confirmed by transformation of S. lividans with the plasmid DNA. Insertion of the chromosomal streptothricin resistance gene of S. lavendulae No. 1080 into pTA4001 gave various composite plasmids with marked deletion in S. lividans as a host. By analyzing these derivatives, a 2.2 kb region essential for replication and a possible region for pock formation were determined in the map of pTA4001 . A cloning vector, pKST2 (4.3 kb) containing resistance genes to streptothricin and thiostrepton with several unique cloning sites was constructed from pTA4001 .