Genetic heterogeneity of Escherichia coli isolated from pasteurized milk in State of Paraná, Brazil

A contaminação de alimentos por patógenos entéricos é uma das principais causas de doenças diarréicas em todo o mundo, resultando em altas taxas de morbidade e mortalidade e perdas econômicas significativas. As bactérias são importantes agentes de doenças de origem alimentar, particularmente Escherichia coli diarreiogênicas. O presente estudo teve como objetivo avaliar a diversidade genética e a resistência a antimicrobianos de E. coli isoladas de leite pasteurizado, processados em 21 laticínios na região noroeste do Paraná - Brasil. Os 95 isolados de E. coli foram submetidos a testes de suscetibilidade aos antimicrobianos de acordo com as recomendações do Clinical and Laboratory Standards Institute e avaliados genotipicamente por ERIC-PCR (Enterobacterial Repetitive Intergenic Consensus - Polymerase Chain Reaction). O principal perfil de resistência encontrado entre os isolados foi resistência à cefalotina (55,78%). ERIC-PCR revelou alta diversidade genética, agrupando os 95 isolados bacterianos em 90 diferentes perfis genotípicos. Estes resultados mostraram uma população heterogênea de E. coli em amostras de leite produzido na região noroeste do Paraná e a necessidade de boas práticas na manipulação de todo o processamento de leite pasteurizado, a fim de reduzir o risco de doenças transmitidas por alimentos.Food contamination caused by enteric pathogens is a major cause of diarrheal disease worldwide, resulting in high morbidity and mortality and significant economic losses. Bacteria are important agents of foodborne diseases, particularly diarrheagenic Escherichia coli. The present study assessed the genetic diversity and antimicrobial resistance of E. coli isolates from pasteurized milk processed in 21 dairies in northwestern State of Parana, Brazil. The 95 E. coli isolates were subjected to antimicrobial susceptibility testing according to the recommendations of the Clinical and Laboratory Standards Institute and assessed genotypically by Enterobacterial Repetitive Intergenic Consensus-Polymerase Chain Reaction (ERIC-PCR). The highest rate of resistance was observed for cephalothin (55.78%). ERIC-PCR revealed high genetic diversity, clustering the 95 bacterial isolates into 90 different genotypic patterns. These results showed a heterogeneous population of E. coli in milk samples produced in the northwestern region of Paraná and the need for good manufacturing practices throughout the processing of pasteurized milk to reduce the risk of foodborne illnesses

Antibacterial and antibiofilm activity of carvacrol against Salmonella enterica serotype Typhimurium

The present study evaluated the antibacterial and antibiofilm activity of carvacrol against Salmonella Typhimurium. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined and the time-kill curve and scanning electron microscopy (SEM) were performed to evaluate antibacterial activity. Antibiofilm activity was evaluated by quantifying total biomass using crystal violet assay, and metabolic activity was determined using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The action of carvacrol against preformed biofilm on polypropylene and stainless steel was also evaluated by colony counting and SEM. The MIC and MBC was 312 µg mL-1. Carvacrol at MIC and 2 x MIC eliminated cells after 6 and 1 h of treatment, respectively, as exhibited in the time-kill curve. The greatest reduction in biofilm biomass and metabolic activity was 1,719 OD550 and 0,089 OD550 respectively, both at 4 x MIC of carvacrol. In carvacrol treated biofilms of S. Typhimurium on polypropylene, a reduction of 5.12 log was observed with 4 x MIC, while on stainless steel, carvacrol at 4 x MIC reduced bacterial counts by 5 log. The results showed that carvacrol exhibits antibacterial activity and can be used as an alternative for the control of S. Typhimurium biofilms

Genome of Herbaspirillum seropedicae Strain SmR1, a Specialized Diazotrophic Endophyte of Tropical Grasses

The molecular mechanisms of plant recognition, colonization, and nutrient exchange between diazotrophic endophytes and plants are scarcely known. Herbaspirillum seropedicae is an endophytic bacterium capable of colonizing intercellular spaces of grasses such as rice and sugar cane. The genome of H. seropedicae strain SmR1 was sequenced and annotated by The Paraná State Genome Programme—GENOPAR. The genome is composed of a circular chromosome of 5,513,887 bp and contains a total of 4,804 genes. The genome sequence revealed that H. seropedicae is a highly versatile microorganism with capacity to metabolize a wide range of carbon and nitrogen sources and with possession of four distinct terminal oxidases. The genome contains a multitude of protein secretion systems, including type I, type II, type III, type V, and type VI secretion systems, and type IV pili, suggesting a high potential to interact with host plants. H. seropedicae is able to synthesize indole acetic acid as reflected by the four IAA biosynthetic pathways present. A gene coding for ACC deaminase, which may be involved in modulating the associated plant ethylene-signaling pathway, is also present. Genes for hemagglutinins/hemolysins/adhesins were found and may play a role in plant cell surface adhesion. These features may endow H. seropedicae with the ability to establish an endophytic life-style in a large number of plant species

Partial characterization of an ATP / GTPase activity-enriched fraction obtained from rabbit testis

In this work we isolated and partially characterized a fraction of rabbit testis containing the ATPase/GTPase activity by using a phosphocellulose column. Testis was homogenated in 50mM Imidazole pH 8.5 with 10 mM EDTA/EGTA, 1.0 mM DTT, 0.3 mM PMSF, 1.0 mM benzamidine, and then centrifuged by 15.000g for 40 min. The supematant was loaded in DEAE-Sepharose column. Bound proteins were eluted with 50 mM Imidazole pH 6.5 and the eluate was directly loaded in a phosphocellulose column. The fraction containing ATPase activity was then eluated with 50 mM Imidazole pH 8.0. No proteins were retained in GTP-agarose column. The isolated fraction presented the following properties. 1) At high concentrations (mM) of ATP or GTP, GTPase larger than ATPase activity. At low substrate concentrations both nucleotides are indistinctly hidrolyzed. 2) Both activities were dependent on the presence of Mg++ or Ca++. In both cases, the Ca++-nucleotidase was larger of than Mg++-nucleotidase activity. 3) Ehfferenttly from the corresponding fraction isolated from rat brain (Coelho, 1988), Mg++ATPase activity presented no stimulation by Ca++-CaM. 4) ATPase activity is well preserved when stored at 4 to 10 «C by one week, but not at 75 »C by 5 minutes or in a freezer at -20 °C. 5) (K+-EDTA)ATPase activity was not detected. 6) Mg-ATPase activity was not sensitive to high salt concentrations (0.3 M KC1 or NaCl), 0.2 % Triton X-100, 1.0 mM azide w 250 nM NaVO3. However, it was 80% inhibited by A1F3 (2.4 mM NaF/ 0.1 mM AlClj) and 35 % in presence ofthe 1 mM NaVO3 or 5 mM NaF. . 1 4. tn GTP-agarose column were analyzed in Some polypeptides not bound to Utr agaiu , O0 J60 100 74 e 48 kDa bands. The column SDS-PAGE and caractenzed as 160, . ^tained just two polypeptides, with 32 and 16 kDa, that didn’t e i ite SMÍ.Í ilial ” lhe vesicle transport. The proteins here described may have a participation in mechanotransduction and signalization events, for what they still need more investigation.Dissertação (Mestrado)Neste trabalho foi isolado e caracterizado parcialmente uma fração de testículo de coelho em fosfocelulose enriquecida em atividade ATP/GTPase. Os testículos foram homogeneizados em tampão I (Imidazol-Cl 50 mM, EDTA 10 mM, EGTA 10 mM, DTT 1.0 mM, PMSF 0.3 mM e benzamidina 1 mM) pH 8.5 e centrifugado a 15.000g x 40 min. O sobrenadante foi aplicado em coluna de DEAE-Sepharose. Proteínas retidas foram eluídas com tampão Imidazol-Cl 50 mM, pH 6.5 e o eluato foi aplicado em coluna de fosfocelulose. A fração enriquecida em atividade ATPase foi obtida eluindo-se a coluna com tampão Imidazol-Cl, pH 8.0. Esta atividade não foi retida por uma coluna de GTP-aAga frroasçeã.o isolada de testículo de coelho possui as seguintes propriedades. 1) Em altas concentrações (mM) de substrato, a atividade GTPase é maior do que a ATPase. Em baixas concentrações os dois nucleotídeos são hidrolisados indistintamente. 2) Ambas as atividades ocorrem como de Ca++. Nos dois casos, a atividade Ca/Nucleotidase foi maior do que a atividade Mg/Nucleotidase. 3) Diferentemennte de uma fração correspondente obtida a partir de cérebro de rato (Coelho, 1988), a atividade MgATPase não Ca++/CaM. 4) A atividade ATPase é estável em baixa temperatura (4 «C/7 dias), mas é totalmente perdida quando aquecida (75 °C/ 5') ou congelada (-20 »C). 5) Não possui atividade (K-EDTA)ATPase 6) A atividade MgATPase não foi sensível a altas concentrações de sal (NaCl ou KC10 3 M) Triton X-100 0.2%, azida 1 mM ou NaVO, 250 pM. Contudo, foi inibida cerca de 80% por A1F3 (NaF 2.4 mM/ A1C1, 0.1 mM) e 35% na Presença de Na VO, 1 mM ou NaF 5 mM. No gel, observou-se que os polipeptídeos: 160, 100, 74 e 48 kDa apareceram no "flow through" da coiuna de GTP-agarose juntamente com a r • cAmplhante a atividade da fração PC. No eluato atividade MgATPase que foi semelhan r «ujpnq- 32 e 16 kDa, que não apresentaram detectou-se sómente dois polipeptídeos. apresentou estimulação poratividade MgGTPase. Estes dois polipeptídeos podem corresponder às pequenas proteínas G citoplasmáticas, que participam na organização do citoesqueleto e controle do transporte vesicular. Os resultados aqui apresentados mostram a caracterização parcial de uma Mg/Ca nucleotidase trifosfatásica de testículo de mamíferos e levanta a possibilidade de estudo de proteínas relacionadas as vias de mecanotransdução e transdução de sinal

Antibacterial and antibiofilm activity of carvacrol against Salmonella enterica serotype Typhimurium

<div><p>ABSTRACT The present study evaluated the antibacterial and antibiofilm activity of carvacrol against Salmonella Typhimurium. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined and the time-kill curve and scanning electron microscopy (SEM) were performed to evaluate antibacterial activity. Antibiofilm activity was evaluated by quantifying total biomass using crystal violet assay, and metabolic activity was determined using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The action of carvacrol against preformed biofilm on polypropylene and stainless steel was also evaluated by colony counting and SEM. The MIC and MBC was 312 µg mL-1. Carvacrol at MIC and 2 x MIC eliminated cells after 6 and 1 h of treatment, respectively, as exhibited in the time-kill curve. The greatest reduction in biofilm biomass and metabolic activity was 1,719 OD550 and 0,089 OD550 respectively, both at 4 x MIC of carvacrol. In carvacrol treated biofilms of S. Typhimurium on polypropylene, a reduction of 5.12 log was observed with 4 x MIC, while on stainless steel, carvacrol at 4 x MIC reduced bacterial counts by 5 log. The results showed that carvacrol exhibits antibacterial activity and can be used as an alternative for the control of S. Typhimurium biofilms.</p></div

Molecular mechanisms probably involved in plant colonization and plant growth promotion identified in the <i>H. seropedicae</i> SmR1 genome.

<p>Plant signals can modulate the expression of bacterial genes coding for adhesins, type IV <i>pili</i> and enzymes of lipopolysaccharide (LPS) synthesis, triggering bacterial attachment to root surfaces. The molecular communication involves bacterial protein secretion and phytohormones to stimulate plant growth and modulate plant defense response. In addition, modulation of plant ethylene levels by ACC deaminase may contribute to plant growth promotion. The success of the endophytic association depends on a compatible genetic background that leads to benefits for both organisms.</p

Proposed pathways for aromatic compounds metabolism in <i>H. seropedicae</i> SmR1.

<p>Proposed pathways for aromatic compounds metabolism in <i>H. seropedicae</i> SmR1.</p

General features of the genome of <i>Herbaspirillum seropedicae</i> SmR1.

<p>General features of the genome of <i>Herbaspirillum seropedicae</i> SmR1.</p

The type III secretion system gene cluster of <i>H. seropedicae</i> SmR1 and other organisms.

<p>Genes of the same color in different organisms are homologous. Genes colored in black have no counterpart in the genomic regions shown.</p

The genome of <i>Herbaspirillum seropedicae</i> SmR1.

<p>From inside to outside 1) G+C content; 2) GC skew; 3) genes color-coded according the COG functional categories; genes in the + strand and − strand are represented in the inside and outside circles respectively; 4) rRNAS operons; 5) putative horizontally transferred regions identified using IVOM: light red indicates low score and dark red indicates high score; 6) regions of <i>H. seropedicae</i> genome identical to castor bean (<i>Ricinus communis</i>) sequences (minimum of 200 bp in length and higher than 90% in identity).</p