Analysis of Integrons and Antimicrobial Resistances of Multidrug Resistant Escherichia coli Isolated in Korea

Antibacterial drugs are one of the most important therapeutic agents of bacterial infections but multidrug resistant Escherichia coli (MDREC) is an increasing problem worldwide. Major resistance mechanism of MDREC is horizontal gene transfer of R plasmids harboring integrons, which the integron integrase (IntI) catalyzes gene cassette insertion and excision through site specific recombination. In this study, resistance profiles of integron harboring E. coli isolated in Korea and the genetic environments of integron gene cassettes were analyzed by PCR and direct sequencing to clarify the mechanisms of spread of integron harboring E. coli. Resistance rates of integron harboring E. coli, including β-lactams, aminoglycosides, and fluoroquinolones and MDR frequencies were significantly higher than that of E. coli without integron (p < 0.01). Majority (80%) of integron harboring E. coli showed resistance transfer by conjugation. Most (80%) of E. coli had dfrA17-aadA5 cassette array and PcH1 hybrid promoter; 16.7% of E. coli had dfrA12-orfF-aadA2 cassette array and PcW promoter. The higher prevalence of weak Pc variants among most (96.7%) of integron harboring MDREC suggests that a flexible cassette array is more important than enhanced expression. All the integrons had LexA binding motif suggests that SOS responses control the expression of these integrons. In conclusion, the genetic bases of integrons were diverse, and the spread and the expression of prevalent gene cassette arrays may be deeply related with strengths of Pc promoters in integrons. These informations will provide important knowledge to control the increase of integron harboring MDREC.

Caracterization of the SOS response in Leptospira interrogans sorovar Copenhageni / Caracterização da resposta SOS em Leptospira interrogans sorovar Copenhageni

Leptospira is a basal genus in an ancient group of bacteria, the spirochetes. The pathogenic species are responsible for leptospirosis, a disease with worldwide distribution and of public health importance in developed tropical countries. L. interrogans serovar Copenhageni is the agent for the majority of human leptospirosis in Brazil. In this work, we used a great variety of experimental approaches to characterize the SOS system in this serovar, to identify its impact in general DNA damage response, as well as to assess the DNA repair toolbox owned by pathogenic and saprophytic leptospires. We identified an additional repressor LexA, acquired by lateral gene transfer, exclusively in serovar Copenhageni. We also observed that UV-C irradiation led to massive death of cells and blockage of cell division in the survivors. Both repressors were active and we identified the sequences responsible for binding to promoters. However, the LexA1 SOS box was redefined after a de novo motif search on LexA1 ChIP-seq enriched sequences. This regulator was able to bind to at least 25 loci in the genome. DNA damage also caused a massive rearrangement of metabolism: increase in expression was observed in transposon and prophage genes, in addition to DNA repair pathways and mutagenesis inducers; on the other hand, motility, general metabolism and almost all virulence genes were repressed. Two induced prophages provided several proteins with useful functions. We also assessed the DNA repair-related genes presented by the three species of Leptospira: the saprophytic L. biflexa, the facultative pathogen L. interrogans and the obligatory pathogen L. borgpetersenii. There are more diversity and redundancy of repair genes in L. interrogans in comparison with the other species. Lateral gene transfer seems to be an important supplier of DNA repair functions. In addition, leptospires share characteristics of both Gram-positives and Gram-negatives bacteria. Representative genes from several different pathways were induced during infection of susceptible mice kidneys, suggesting DNA repair genes are active while causing disease. All these data suggest mobile genetic elements are the major forces in leptospiral evolution. Moreover, during DNA damage response, several SOS-dependent and independent mechanisms are employed to decrease cell growth and virulence in favor of controlled induction of mechanisms involved in genetic variability

Leptospira é um gênero basal em um grupo já considerado um dos mais ancestrais, as espiroquetas. As espécies patogênicas são responsáveis pela leptospirose, uma doença presente em todo o mundo e de principal importância em países tropicais em desenvolvimento. L. interrogans sorovar Copenhageni é o agente da maior parte dos casos no Brasil. Nesse trabalho, utilizamos diversas abordagens experimentais para caracterizar o sistema SOS nesse sorovar, identificar seu impacto na resposta geral a danos no DNA, assim como avaliar as funções de reparo de DNA disponíveis em leptospiras patogênicas e saprofíticas. Identificamos um repressor LexA adicional, adquirido por transferência horizontal e exclusivo do sorovar Copenhageni. Observamos também que irradiação por UV-C causou significativa morte celular e bloqueio da divisão celular dos sobreviventes. Ambos os repressores são ativos e identificamos as sequências que utilizam para se ligar aos promotores dos genes regulados. Entretanto, o SOS box de LexA1 foi redefinido após uma busca de novo por motivos enriquecidos nas sequências recuperadas por ChIP-seq. Esse regulador ligou-se ao menos a 25 locais do genoma. A maioria desses alvos teve aumento de expressão após UV-C. Danos no DNA também causaram um importante rearranjo metabólico: houve aumento de expressão em transposons e profagos, além de indutores de mutagênese e vias de reparo; por outro lado, mobilidade, crescimento celular e quase todos os fatores de virulência foram reprimidos. Dois profagos induzidos durante essa resposta, possivelmente proporcionam algumas proteínas de funções importantes. Nós também avaliamos a presença de genes envolvidos no reparo de DNA em três espécies de leptospira: L. biflexa, L. interrogans e L. borgpetersenii. L. interrogans é a espécie com maior diversidade e redundância de genes de reparo. Além disso, transferência horizontal parece ser um importante fornecedor de funções de reparo nesse gênero. Leptospiras também apresentam genes característicos tanto de bactérias Gram-positivas quanto Gram-negativas. Genes representando diferentes vias de reparo foram induzidos durante infecção em modelo animal, sugerindo que essas vias estão ativas no curso da doença. Todos esses dados, em conjunto, sugerem que elementos genéticos móveis são de extrema importância na evolução do gênero e das vias de reparo. Assim, durante a resposta a danos no DNA, diversos mecanismos dependentes e independentes de SOS são empregados para frear o crescimento celular e virulência em favor da indução controlada de mecanismos para aumentar variabilidade genética

The molecular physiological and genetic mechanisms underlying the superb efficacy of quinolones / 药学学报

The fluoroquinolones are the most widely used broad-spectrum antibiotics, accounting for 18% of global antibacterial market share. They can kill bacteria rapidly with variety of derivatives available. Different quinolones vary significantly in rate and spectrum of killing, oxygen requirement for metabolism and reliance upon protein synthesis. Further understanding the sophisticated mechanisms of action of this important antibiotic family based on the molecular genetic response of bacteria can facilitate the discovery of better quinolone derivatives. Factors such as SOS response, bacterial toxin-antitoxin system, programmed death, chromosome fragmentation and reactive oxygen have been implicated in the action to some extent. "Two steps characteristic" of quinolones killing is also emphasized, which might inspire future better quinolones modification.

Efeitos genotóxicos e indução do SOS em mutantes derivados de Escherichia coli K-12 durante o processo de interação com superfícies bióticas e abióticas / Genotoxic and SOS induction in mutants derived from Escherichia coli K-12 during the process of interaction with biotic and abiotic surfaces

A célula epitelial é o primeiro contato entre os micro-organismos e o hospedeiro. Essa interação pode levar a produção de diversas citocinas, quimiocinas, moléculas inflamatórias e também estimular a geração de espécies reativas de oxigênio (ERO). Neste trabalho avaliamos se a interação com as células HEp-2 poderia ser genotóxica para os mutantes derivados de Escherichia coli K-12 deficientes em algumas enzimas que fazem parte do sistema de reparo por excisão de base (BER). Além disto, avaliamos a expressão do sistema SOS, que é induzido pela presença de danos no genoma bacteriano. Os resultados obtidos mostraram a presença de filamentos, na interação com células HEp-2, principalmente, no mutante xthA (BW9091) e no triplo mutante xthA nfo nth (BW535). Quando a interação foi quantificada na ausência da D-manose, observamos um aumento das bactérias aderidas. Além disto, a quantidade e o tamanho dos filamentos também aumentaram, mostrando que as adesinas manose-sensíveis estavam envolvidas na filamentação bacteriana. Para comprovar se o aumento da filamentação observada neste ensaio foram uma consequência da indução do sistema SOS, desencadeada pela interação com as células HEp-2, quantificamos a expressão do SOS, na presença e na ausência da D-manose. De fato, observamos que a indução do SOS na ausência da D-manose foi maior, quando comparada, com o ensaio realizado na presença de D-manose. Além disto, observamos que a ausência de xthA foi importante para o aumento da filamentação observada na ausência de D-manose. Diante destes resultados, verificamos se a resposta de filamentação ocorreria quando as bactérias interagiam com uma superfície abiótica como o vidro. Observamos também inúmeros filamentos nos mutantes BER, BW9091 e BW535, quando comparados a cepa selvagem AB1157. Essa filamentação foi associada à indução do SOS, em resposta a interação das bactérias com o vidro...

The epithelial cell is the first contact between microorganisms and host. This interaction results in production of several cytokines, chemokines, and inflammatory molecules by epithelial cells and also stimulate the generation of reactive oxygen species (ROS). In the present study, we have evaluated whether the interaction to HEp-2 cells causes genotoxicity to mutants derived from Escherichia coli K-12 deficient in some enzymes that are part of the system of base excision repair (BER). Moreover, we measured the expression of SOS system, which is induced by the presence of damage to the bacterial genome. Our results showed mainly presence of filamentous bacterial growth in xthA mutant (BW9091) and triple xthA nfo nth mutant (BW535) when submitted to HEp-2 cells interaction assays. When experiments were performed in the absence of mannose, data showed enhanced interaction of viable bacteria to HEp-2 cells for all strains tested. Furthermore, the removal of D-mannose resulted in an increase in both number and size of bacterial filamentous forms, indicating the involvement of mannose-sensitive adhesins in the filamentation of these strains. In order to verify whether the increased filamentation growth in this assay was a consequence of SOS induction, triggered by interaction to HEp-2 cells, we measured expression of SOS in the presence and absence of D-mannose. Indeed, we observed higher expression of SOS response in the absence of mannose than in experiments performed in the presence of D-mannose. Moreover, we observed that the absence of xthA was important to filamentation increasing in absence of D-mannose. Based on these results, we verified if interaction to abiotic surfaces, like glass, could lead to filamentation of these strains. We also observed numerous filaments in BER mutants, BW9091 and BW535, when compared to wild-type strain AB1157. The filamentation observed was a consequence of SOS induction, triggered by attachment to the glass surface...

Antigenotoxic and antimutagenic potential of an annatto pigment (norbixin) against oxidative stress

Carotenoids are 40-carbon molecules with conjugated double bonds, making them particularly effective for quenching free radicals. They have always been believed to possess anticancer properties, which could be due to their antioxidant potential. Norbixin is an unusual dicarboxylic water-soluble carotenoid present as a component in the pericarp of the seeds of Bixa orellana L. (from the Bixaceae family), a tropical shrub commonly found in Brazil. The main carotenoids present in these seeds, bixin and norbixin, form a coloring material, known as annatto, which is mainly used in the food industry. As annatto is only used as a coloring material, most studies of annatto pigments have focused on the determination of annatto levels in food. However, little attention has been given to the biological properties of bixin and norbixin. We evaluated the effect of norbixin on the response of Escherichia coli cells to DNA damage induced by UV radiation, hydrogen peroxide (H2O2) and superoxide anions (O2*-)) and found that norbixin protects the cells against these agents. Norbixin enhanced survival at least 10 times. The SOS induction by UVC was inhibited 2.3 times more when cells were grown in the presence of norbixin. We also found that norbixin has antimutagenic properties, with a maximum inhibition of H2O2-induced mutagenic activity of 87%, based on the Salmonella mutagenicity test

Efecto de la radiación gamma sobre la supervivencia y la inducción de la respuesta SOS en células de Escherichia coli deficientes en la reparación por escisión de nucleótidos y por recombinación / Effect of gamma radiation on the survival and the induction of the SOS response in Eschericia coli cells deficient in the repair by excision of neuclotides and recombination

Se estudiaron los daños primarios producidos por la radiación gamma en el ADN de cepas de Escherichia coli portadoras de la fusión génica sulA::lacZ y de mutaciones en diferentes genes de reparación del ADN. Además, se determinó su radiosensibilidad. Las cepas con mutaciones en los genes recN y uvrA fueron más sensibles que la tipo salvaje. Esto concuerda con estudios previos donde se demostró que los genes rec y uvr participaban en la reparación del daño en el ADN producido por los rayos gamma. En las cepas mutantes se encontraron diferencias significativas en los niveles de expresión del gen sulA en relación con el tipo salvaje. Se discute la utilidad de las cepas estudiadas como biosensores de genotoxicidad así como en estudios de radioprotección

DNA repair in Chromobacterium violaceum

Chromobacterium violaceum is a Gram-negative beta-proteobacterium that inhabits a variety of ecosystems in tropical and subtropical regions, including the water and banks of the Negro River in the Brazilian Amazon. This bacterium has been the subject of extensive study over the last three decades, due to its biotechnological properties, including the characteristic violacein pigment, which has antimicrobial and anti-tumoral activities. C. violaceum promotes the solubilization of gold in a mercury-free process, and has been used in the synthesis of homopolyesters suitable for the production of biodegradable polymers. The complete genome sequence of this organism has been completed by the Brazilian National Genome Project Consortium. The aim of our group was to study the DNA repair genes in this organism, due to their importance in the maintenance of genomic integrity. We identified DNA repair genes involved in different pathways in C. violaceum through a similarity search against known sequences deposited in databases. The phylogenetic analyses were done using programs of the PHILYP package. This analysis revealed various metabolic pathways, including photoreactivation, base excision repair, nucleotide excision repair, mismatch repair, recombinational repair, and the SOS system. The similarity between the C. violaceum sequences and those of Neisserie miningitidis and Ralstonia solanacearum was greater than that between the C. violaceum and Escherichia coli sequences. The peculiarities found in the C. violaceum genome were the absence of LexA, some horizontal transfer events and a large number of repair genes involved with alkyl and oxidative DNA damage

Site-Specific Mutagenesis in Escherichia coli by Bulky Exocyclic Amino-Substituted Guanine and Adenine Derivatives in Double-Stranded or Gapped Plasmids / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: 7-Bromomethylbenz[alpha]anthracene is a known mutagen and carcinogen. The mutagenic potency of its two major DNA adducts, i.e., N2-(benz[alpha]anthracen-7-ylmethyl)-2'-deoxyguanosine (b[alpha]a2G) and N6-(benz[alpha]anthracen-7-ylmethyl)-2'-deoxyadenosine (b[alpha]a6A), as well as the simpler benzylated analogs, N2-benzyl-2'-deoxyguanosine (bn2G) and N6-benzyl-2'-deoxyadenosine (bn6A), were determined in E. coli. MATERIALS AND METHODS: Double-stranded and gapped plasmid vectors were used to determine the mutagenicity of b[alpha]a2G, b[alpha]a6A, bn2G and bn6A in E. coli. The four, suitably protected, bulky exocyclic amino-substituted adducts were incorporated into 16-base oligodeoxyribonucleotides, in place of normal guanine or adenine residues, which form part of the ATG initiation codon for the lacZ' alpha-complementation gene. The site-specifically modified oligodeoxyribonucleotides were then incorporated into double-stranded plasmids, which contained uracil residues in the complementary strand in the vicinity of the initiation codon. The uracil residues lead to the creation of a gap in the complementary strand due to the actions of E. coli uracil-DNA glycosylase and AP endonuclease. Following the transfection of these plasmid vectors into E. coli strain GP102, a lacZ alpha complementing version of the parent strain AB1157, their propensity to induce mutation was investigated. RESULTS: The percentages of mutant colonies produced by the four modified nucleosides, in both the double-stranded and gapped plasmid vectors, were not significantly different from those produced by the unmodified plasmids. The mutagenicities of the b[alpha]a2G and b[alpha]a6A were extremely low, and a totally unexpected result, whereas, those of the bn2G and bn6A were undetectable. CONCLUSION: In this E. coli site-specific mutagenesis system, these bulky aralkylated adducts exhibited no significant mutagenicities, either with or without SOS induction.

Genotoxic evaluation of the organophosphorous pesticide temephos

The chemical compound temephos (0,0,0',0'-tetrametyl-0,0'-thiodi-p-phenylene phosphorothioate) is an organophosphorous pesticide that has been used in Brazil since 1967 in control campaigns against the mosquito Aedes aegypti, the vector of dengue and yellow fever. We used single cell gel electrophoresis (SCGE), SOS/umu and Ames/Salmonella assays to test the toxicity and mutagenicity of temephos. Temephos was genotoxic in the SCGE assay, inducing severe DNA lesions (type IV lesions) at doses above 1.34 micro M. It was mutagenic, but not toxic, in the SOS/umu assay to Escherichia coli strain PQ37, but not to PQ35, at concentrations above 1.33 micro M, particularly when the S9 mixture was not used in the assay. Temephos was not mutagenic in the Ames assay with S. typhimurium strains TA97, TA98, TA100 and TA102, both with and without metabolic activation. However, temephos at concentrations above 3.33 micro M was mutagenic to TA98NR, YG7104 and YG7108, both with and without metabolic activation. In conclusion, temephos was genotoxic and mutagenic in all the three tests used, and in two of them at concentrations similar to those routinely used to combat Aedes aegypti

Genotoxic evaluation of the effect of Thuya occidentalys tinctures

Three tinctures samples from extracts of the popular medicinal plant Thuya occidentalis were tested in vitro through two short term tests for measuring the activity of genotoxic chemicals. Using the Salmonella/mammalian-microsome (Mutatest) assay and the SOS-chromotest (induction of beta-galactosidase in Escherichia coli), none of the extract was effective in inducing mutagenesis or beta-galactosidase synthesis (as an indicator of general and early sign of DNA damage), even with metabolization

Respuesta SOS en E Coli: test de inducción de genes SOS como ensayo de mutagenicidad / SOS response in E Coli: induction test of SOS genes as a mutagenecity assay

Se presentan las particularidades de la respuesta SOS en células de E. coli y se realiza una descripción de los tipos de daños que inducen dicha respuesta. Se tratan además los aspectos relacionados con la regulación del circuito RecA/Lex A, así como de la señal inductora de la respuesta. Se resumen, de acuerdo con la bibliografía actualizada del tema, las funciones de los principales productos génicos de esta respuesta, en particular, RecA y UmuC y D, durante la restauración de la replicación y se discute un modelo que explica el fenómeno de la mutagénesis SOS en E. coli. Se hacen algunas consideraciones evolutivas de la mutagénesis SOS en bacterias de acuerdo con el modelo cairsiano de evolución. Se explican las particularidades de los tests de inducción de genes SOS, así como su utilidad, tanto en la evaluación de efectos genotóxicos como en la prospección y estudio de mecanismos de acción de sustancias antimutagénicas, radioprotectoras, o ambas.

Membrane permeability and sensitivity to lethal heat are affected by lexA and recA mutations in Escherichia coli K12

Membrane permeability was evaluated in several SOS-deficient strains. Great heat sensitivity was observed in all the lexA (Ind-) strains, which was associated to an increase in membrane permeability (up to 120 per cent increase above the wild-type control), as assayed by the crystal violet (CV) growth inhibition. After irradiation with a single UV dose (75 J.m-2 delivered to wild-type and 2 J.m-2 to the lex A3 strain), survival was followed by plating cells in both nutrient and membrane permeability-selective (nutrient + CV) media and a great lethality due to CV was observed in a lexA mutant, which appeared to be about 100 times more sensitive to CV compared to its wild-type parent stain. The decreased membrane integrity found in the lex A-deficient strains suggests that LexA protein and/or LexA-repressed genes may interact with the bacterial membrane, which could be the location of SOS events

Genotoxicity of plant extracts

Aqueous extracts of seven species used in Brazilian popular medicine (Achyrocline satureoides, Iodina rhombifolia, Desmodium incanum, Baccharis anomala, Tibouchina asperior, Luehea divaricata, Maytenus ilicifolia) were screened to the presence of mutagenic activity in the Ames test (Salmonella/microsome). Positive results were obtained for A. satureoides, B anomala and L. divaricata with microsomal activation. As shown elsewhere (Vargas et al., 1990) the metabolites of A. satureoides extract also show the capacity to induce prophage and/or SOS response in microscreen phage induction assay and SOS spot chromotest.