Aeromonas allosaccharophila Strain AE59-TE2 Is Highly Antagonistic towards Multidrug-Resistant Human Pathogens, What Does Its Genome Tell Us?

Multidrug-resistant bacteria are of critical importance and a problem for human health and food preservation; the discovery of new antimicrobial substances to control their proliferation is part of the solution. This work reports on 57 antagonistic Aeromonas strains, of which 38 strains were antagonistic towards problematic human pathogens. The genome of the most antagonistic strain was sequenced and identified as Aeromonas allosaccharophila. Its genome was fully annotated and mined for genes that might explain that activity. Strain AE59-TE was antagonistic toward clinically relevant gram-negative and gram-positive multidrug-resistant bacteria, including Klebsiella pneumoniae KPC, Escherichia coli ESBL, Salmonella typhimurium, and Staphylococcus aureus MRSA. Strain AE59-TE2 was identified by multilocus sequence analysis. Genome mining identified four genes homologous to the bacteriocin, zoocin A from Streptococcus equi and a gene 98% similar to cvpA linked to colicin V production. A. allosaccharophila strain AE59-TE2 produced antimicrobial activity against a broad range of bacteria, including important gram-negative bacteria, not typically targeted by bacteriocins. Herewere described novel zoocin genes that are promising for industrial applications in the food and health sectors. Interesting and important antagonistic activity is described combined with the first detailed genomic analysis of the species Aeromonas allosaccharophila.

Draft Genome Sequence of a Wohlfahrtiimonas chitiniclastica Strain Isolated from Frozen Chicken in Rio De Janeiro, Brazil.

Here, we report the draft genome sequence of Wohlfahrtiimonas chitiniclastica strain 20, isolated from a chicken carcass originated from indoor broiler farming and identified using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry followed by sequencing of the 16S rRNA gene.

Antimicrobial Susceptibility and Enterotoxin-Encoding Genes in Staphylococcus spp. Recovered from Kitchen Equipment from a University Hospital in Rio de Janeiro, Brazil.

This study was conducted to determine the occurrence of antimicrobial resistance and enterotoxin-encoding genes (EEGs) in Staphylococcus spp. recovered from equipment used to prepare hospital meals, in a university hospital in Rio de Janeiro, Brazil. Sixty samples were collected from semi-industrial equipment (one blender and one mixer) in the hospital's kitchen. Resistance genes and SCCmec types were detected by PCR. From the 40 isolates of Staphylococcus spp. identified, 8 were Staphylococcus aureus. Thirty-two (80%) Staphylococcus spp. isolates were resistant to at least one antimicrobial agent. Resistance genetic determinants were detected: erm gene (Staphylococcus epidermidis [n = 2]; Staphylococcus hominis [n = 1]), mecA gene (S. epidermidis [n = 2]), and aa(6')-aph(2'') gene (Staphylococcus caprae [n = 1], S. epidermidis [n = 2], S. hominis [n = 1], Staphylococcus pausteri [n = 1], Staphylococcus simulans [n = 1], and Staphylococcus warneri [n = 1]). The presence of at least one EEG in 83% (n = 33) of the isolates was identified. Two strains of S. epidermidis were methicillin-resistant S. epidermidis (MRSE) and harboring SCCmec type IV. Staphylococcus spp. contaminated some hospital kitchen's equipment, indicating that hygiene procedures should be improved. Results also indicate that meals can be a vehicle to disseminate multiresistant Staphylococcus spp., including MRSE, and Staphylococcus with EEGs.

Ex vivo model of rabbit intestinal epithelium applied to the study of colonization by enteroaggregative escherichia coli / Modelo ex vivo de epitélio intestinal de coelho aplicado ao estudo de colonização por Escherichia coli enteroagregativa

ABSTRACT BACKGROUND The diarrheal syndrome is considered a serious public health problem all over the world and is considered a major cause of morbidity and mortality in developing countries. The high incidence of enteroaggregative Escherichia coli in diarrheal syndromes classified as an emerging pathogen of gastrointestinal infections. After decades of study, your pathogenesis remains uncertain and has been investigated mainly using in vitro models of adhesion in cellular lines. OBJECTIVE The present study investigated the interaction of enteroaggregative Escherichia coli strains isolated from childhood diarrhea with rabbit ileal and colonic mucosa ex vivo, using the in vitro organ culture model. METHODS The in vitro adhesion assays using cultured tissue were performed with the strains co-incubated with intestinal fragments of ileum and colon over a period of 6 hours. Each strain was tested with three intestinal fragments for each region. The fragments were analysed by scanning electron microscopy. RESULTS Through scanning electron microscopy we observed that all strains adhered to rabbit ileal and colonic mucosa, with the typical aggregative adherence pattern of “stacked bricks” on the epithelium. However, the highest degree of adherence was observed on colonic mucosa. Threadlike structures were found in greater numbers in the ileum compared to the colon. CONCLUSION These data showed that enteroaggregative Escherichia coli may have a high tropism for the human colon, which was ratified by the higher degree of adherence on the rabbit colonic mucosa. Finally, data indicated that in vitro organ culture of intestinal mucosa from rabbit may be used to elucidate the enteroaggregative Escherichia coli pathogenesis.

RESUMO CONTEXTO A síndrome diarréica é considerada um grave problema de saúde pública em todo o mundo e é considerada uma das principais causas de morbidade e mortalidade nos países em desenvolvimento. A elevada incidência de Escherichia coli enteroagregativa nas síndromes diarreicas a classificou como um patógeno emergente de infecções gastrintestinais. Depois de décadas de estudo, sua patogênese ainda é incerta e tem sido investigada usando principalmente modelos in vitro de adesão em linhagens celulares. OBJETIVO O presente estudo investigou a interação de cepas de Escherichia coli enteroagregativa isoladas de diarreia infantil com mucosa ileal e colônica de coelho ex vivo, utilizando o modelo de cultura de órgão in vitro. MÉTODOS Os ensaios de adesão in vitro utilizando tecido cultivado foram realizados com as cepas co-incubadas com fragmentos intestinais de íleo e de cólon durante um período de 6 horas. Cada cepa foi testada em três fragmentos intestinais para cada região. Os fragmentos foram analisados por microscopia eletrônica de varredura. RESULTADOS Através da microscopia eletrônica de varredura observamos que todas as cepas aderiram a mucosa ileal e colônica de coelho, com o padrão de aderência agregativo típico de “tijolos empilhados” no epitélio. Entretanto, o maior grau de adesão foi observado na mucosa do cólon. Estruturas filiformes foram encontradas em maior número no íleo em comparação com o cólon. CONCLUSÃO Esses dados mostraram que Escherichia coli enteroagregativa pode ter um maior tropismo para o cólon humano, o que foi ratificado pelo maior grau de aderência na mucosa do cólon de coelho. Finalmente, os dados indicaram que a cultura de órgão in vitro da mucosa intestinal de coelho pode ser utilizado para elucidar a patogênese de Escherichia coli enteroagregativa.

EX VIVO MODEL OF RABBIT INTESTINAL EPITHELIUM APPLIED TO THE STUDY OF COLONIZATION BY ENTEROAGGREGATIVE ESCHERICHIA COLI.

BACKGROUND: The diarrheal syndrome is considered a serious public health problem all over the world and is considered a major cause of morbidity and mortality in developing countries. The high incidence of enteroaggregative Escherichia coli in diarrheal syndromes classified as an emerging pathogen of gastrointestinal infections. After decades of study, your pathogenesis remains uncertain and has been investigated mainly using in vitro models of adhesion in cellular lines. OBJECTIVE: The present study investigated the interaction of enteroaggregative Escherichia coli strains isolated from childhood diarrhea with rabbit ileal and colonic mucosa ex vivo, using the in vitro organ culture model. METHODS: The in vitro adhesion assays using cultured tissue were performed with the strains co-incubated with intestinal fragments of ileum and colon over a period of 6 hours. Each strain was tested with three intestinal fragments for each region. The fragments were analysed by scanning electron microscopy. RESULTS: Through scanning electron microscopy we observed that all strains adhered to rabbit ileal and colonic mucosa, with the typical aggregative adherence pattern of "stacked bricks" on the epithelium. However, the highest degree of adherence was observed on colonic mucosa. Threadlike structures were found in greater numbers in the ileum compared to the colon. CONCLUSION: These data showed that enteroaggregative Escherichia coli may have a high tropism for the human colon, which was ratified by the higher degree of adherence on the rabbit colonic mucosa. Finally, data indicated that in vitro organ culture of intestinal mucosa from rabbit may be used to elucidate the enteroaggregative Escherichia coli pathogenesis.

Adhesion and cytotoxicity of Aeromonas caviae to rabbit intestinal epithelium ex vivo.

UNLABELLED: Aeromonads are considered potential pathogens for humans and animals and are responsible for the etiology of intestinal and extraintestinal diseases. The presence of Aeromonas spp. in food and water shows that it is an important vehicle of infection in humans. The pathology caused by these bacteria involves several virulence factors, such as the ability to produce toxins, adhesion and invasion. The present study investigated the interaction of five Aeromonas caviae strains isolated from human diarrheic faeces with rabbit ileal and colonic mucosa ex vivo, using in vitro organ culture model. The in vitro adhesion assays using cultured tissue were performed with A. caviae strains co-incubated with intestinal fragments of ileum and colon over a period of 6 h. The fragments were analyzed by light and electron microscopy. All strains adhered to rabbit ileal and colonic mucosa ex vivo, with higher degree of adherence presented on colonic mucosa. The typical aggregative adherence pattern was observed among strains studied. Through electron and light microscopy, we observed extensive colonization of ileal and colonic mucosa, large mucus production, biofilm formation and morphological alterations such as intense vacuolization, structural disorganization, cell extrusion and destruction of the villi. These results demonstrate that in vitro organ culture of intestinal mucosa from rabbit may be used to investigate Aeromonas spp. PATHOGENESIS: Finally, our results support the pathogenic potential of Aeromonas emphasising their importance in public health.

First report of the emerging zoonotic agent Wohlfahrtiimonas chitiniclastica isolated from a retail frozen chicken in Rio de Janeiro, Brazil.

Wohlfahrtiimonas chitiniclastica is an emerging zoonotic bacterium commensally living in larvae of particular flies. It has been associated with human and animal infections but never isolated from food. In the present study, a whole chicken carcass was rinsed in buffered peptone water which was then inoculated into BHI and the growth plated onto selective medium. Species identification was performed by MALDI-TOF MS. Those bacteria identified as W. chitiniclastica were subjected to 16S rRNA sequencing for confirmation and MEGA software was used to obtain their phylogenetic position. The findings of this study raise concerns regarding the abattoir, transport and stock practices of frozen meat carcasses and should be of interest with regard to microbiology, entomology and food production.

Adhesion, invasion, intracellular survival and cytotoxic activity of strains of Aeromonas spp. in HEp-2, Caco-2 and T-84 cell lines.

The genus Aeromonas contains important pathogen for both humans and other animals, being responsible for the etiology of intestinal and extraintestinal diseases. The pathology caused by these bacteria involves several virulence factors, such as the ability to produce toxins, adhesion and invasion. The properties conferred by these factors have been extensively studied in experiments of interaction between bacterial strains and cell culture. We evaluate the interaction of eight Aeromonas spp. strains, previously isolated from human faeces, food and water with HEp-2, Caco-2 and T-84 cell lines. Cytotoxic effects, the pattern of adhesion, invasive capacity and intracellular survival were analyzed. The results showed that Aeromonas strains were adherent to three cells lines in 6 h of incubation, displaying the aggregative adherence pattern. Among eight strains studied, 50% produced cytotoxic effects on HEp-2 cells, while none of the strains produced cytotoxic effects on Caco-2 and T-84 cells at 48 h. This study demonstrated that subsets of Aeromonas isolated from different sources were able to invade intestinal (T-84, Caco-2) and epithelial (HEp-2) cell lines cultivated in vitro surviving in intracellular environments up to 72 h. Finally, our results support the pathogenic potential of Aeromonas, especially those of food and clinical sources.

Prevalence of R-type ACSSuT in strains of Salmonella serovar Typhimurium DT193 isolated from human infections in Brazil.

OBJECTIVE: To determine the prevalence of resistance to ampicillin, chloramphenicol, streptomycin, sulphonamides, and tetracyclines (ACSSuT) in Salmonella serovar Typhimurium definitive [phage] type (DT) 193 strains isolated from human sources over the last four decades. METHODS: From 2008 to 2010, 553 DT193 isolates out of 810 human-origin Salmonella ser. Typhimurium phage-typed strains isolated from the 1970s through 2008 were selected and tested for ACSSuT resistance: 91 strains isolated during the 1970s, 65 from the 1980s, 70 from the 1990s, and 327 from 2000-2008. Resistance profiles were determined using the disk diffusion method. RESULTS: An antimicrobial susceptibility assay indicated 20.9%, or 116, of all isolates tested were ACSSuT-resistant, 52.0% (287) were resistant to one or more drugs in the ACSSuT profile, and 27.1% (150) were nonresistant (susceptible to antimicrobials). Based on the assay, overall antimicrobial resistance was extremely high in the 1970s (affecting 99.0% of isolates from that period) and remained high during the 1980s, when 95.4% of isolates had some type of antimicrobial resistance and incidence of Salmonella ser. Typhimurium DT193 R-type ACSSuT increased to 73.8%. R-type ACSSuT dropped to 27.1% (19 isolates) during the 1990s, and to 5.2% (17) during 2000-2008, despite a substantial increase in the number of isolates tested (397 versus 204, 111, and 98, respectively, for the previous three decades). CONCLUSIONS: Although prevalence of Salmonella ser. Typhimurium DT193 R-type ACSSuT in Brazil has decreased since the 1970s, ACSSuT resistance markers continue to circulate. Therefore, continuous surveillance should be conducted to evaluate the occurrence of Salmonella ser. Typhimurium DT193 and its antimicrobial resistance.

Prevalence of R-type ACSSuT in strains of Salmonella serovar Typhimurium DT193 isolated from human infections in Brazil / Prevalencia de resistencia de tipo ACSSuT en cepas de Salmonella serovariedad Typhimurium DT193 aisladas a partir de infecciones humanas en el Brasil

OBJECTIVE: To determine the prevalence of resistance to ampicillin, chloramphenicol, streptomycin, sulphonamides, and tetracyclines (ACSSuT) in Salmonella serovar Typhimurium definitive [phage] type (DT) 193 strains isolated from human sources over the last four decades. METHODS: From 2008 to 2010, 553 DT193 isolates out of 810 human-origin Salmonella ser. Typhimurium phage-typed strains isolated from the 1970s through 2008 were selected and tested for ACSSuT resistance: 91 strains isolated during the 1970s, 65 from the 1980s, 70 from the 1990s, and 327 from 2000-2008. Resistance profiles were determined using the disk diffusion method. RESULTS: †An antimicrobial susceptibility assay indicated 20.9 percent, or 116, of all isolates tested were ACSSuT-resistant, 52.0 percent (287) were resistant to one or more drugs in the ACSSuT profile, and 27.1 percent (150) were nonresistant (susceptible to antimicrobials). Based on the assay, overall antimicrobial resistance was extremely high in the 1970s (affecting 99.0 percent of isolates from that period) and remained high during the 1980s, when 95.4 percent of isolates had some type of antimicrobial resistance and incidence of Salmonella ser. Typhimurium DT193 R-type ACSSuT increased to 73.8 percent. R-type ACSSuT dropped to 27.1 percent (19 isolates) during the 1990s, and to 5.2 percent (17) during 2000-2008, despite a substantial increase in the number of isolates tested (397 versus 204, 111, and 98, respectively, for the previous three decades). CONCLUSIONS: †Although prevalence of Salmonella ser. Typhimurium DT193 R-type ACSSuT in Brazil has decreased since the 1970s, ACSSuT resistance markers continue to circulate. Therefore, continuous surveillance should be conducted to evaluate the occurrence of Salmonella ser. Typhimurium DT193 and its antimicrobial resistance.

OBJETIVO: Determinar la prevalencia de resistencia a la ampicilina, el cloranfenicol, la estreptomicina, las sulfonamidas y las tetraciclinas (ACSSuT) en cepas de Salmonella serovariedad Typhimurium fagotipo definitivo (DT) 193 aisladas de fuentes de origen humano durante las cuatro últimas décadas. MÉTODOS: Entre el 2008 y el 2010 se seleccionaron 553 aislados de DT193 entre 810 cepas de Salmonella serovariedad Typhimurium fagotipificadas aisladas desde la década de 1970 hasta el 2008, y en ellos se analizó la resistencia a ACSSuT: se estudiaron 91 cepas aisladas durante la década de 1970, 65 aisladas durante la década de 1980, 70 aisladas durante la década de 1990, y 327 aisladas entre el 2000 y el 2008, respectivamente. Los perfiles de resistencia a los antibióticos se determinaron mediante el método de difusión en disco. RESULTADOS: El antibiograma indicó que 20,9 por ciento (116) de todos los aislados que se analizaron fueron resistentes a ACSSuT, 52,0 por ciento (287) fueron resistentes a uno o más antibióticos del grupo ACSSuT y 27,1 por ciento (150) no fueron resistentes (es decir, fueron sensibles a dichos antibióticos). Según el análisis, la resistencia general a los antibióticos fue muy alta en la década de 1970 (y comprendió a 99,0 por ciento de los aislados de ese período) y continuó siendo alta durante la década de 1980, cuando 95,4 por ciento de los aislados presentó algún tipo de resistencia a los antibióticos y la incidencia de Salmonella serovariedad Typhimurium DT193 con resistencia de tipo ACSSuT aumentó hasta 73,8 por ciento. La resistencia de tipo ACSSuT descendió a 27,1 por ciento (31 aislados) durante la década de 1990, y a 5,2 por ciento (17 aislados) entre el 2000 y el 2008, a pesar del aumento importante en el número de aislados que se evaluaron (397 frente a 204, 111 y 98 en las tres décadas anteriores, respectivamente). CONCLUSIONES: Aunque la prevalencia de Salmonella serovariedad Typhimurium DT193 con resistencia de tipo ACSSuT en el Brasil ha disminuido desde la década de 1970, los marcadores de resistencia a ACSSuT continúan en circulación. Por consiguiente, debe llevarse a cabo una vigilancia permanente para evaluar la aparición de infecciones por Salmonella serovariedad Typhimurium DT193 y su resistencia a los antibióticos.

The involvement of tetA and tetE tetracycline resistance genes in plasmid and chromosomal resistance of Aeromonas in Brazilian strains.

This study analyzed the involvement of tetA and tetE genes in the tetracycline resistance of 16 strains of genus Aeromonas, isolated from clinical and food sources. Polymerase chain reactions revealed that 37.5% of the samples were positive for tetA, and also 37.5% were tetE positive. One isolate was positive for both genes. Only the isolate A. caviae 5.2 had its resistance associated to the presence of a plasmid, pSS2. The molecular characterization of pSS2 involved the construction of its restriction map and the determination of its size. The digestion of pSS2 with HindIII originated two fragments (A and B) that were cloned separately into the pUC18 vector. The tetA gene was shown to be located on the HindIII-A fragment by PCR. After transforming a tetracycline-sensitive strain with pSS2, the transformants expressed the resistance phenotype and harbored a plasmid whose size was identical to that of pSS2. The results confirmed the association between pSS2 and the tetracycline resistance phenotype, and suggest a feasible dissemination of tetA and tetE among strains of Aeromonas. This study suggests the spreading tetA and tetE genes in Aeromonas in Brazil and describes a resistance plasmid that probably contributes to the dissemination of the resistance.

The involvement of tetA and tetE tetracycline resistance genes in plasmid and chromosomal resistance of Aeromonas in Brazilian strains

This study analyzed the involvement of tetA and tetE genes in the tetracycline resistance of 16 strains of genus Aeromonas, isolated from clinical and food sources. Polymerase chain reactions revealed that 37.5 percent of the samples were positive for tetA, and also 37.5 percent were tetE positive. One isolate was positive for both genes. Only the isolate A. caviae 5.2 had its resistance associated to the presence of a plasmid, pSS2. The molecular characterization of pSS2 involved the construction of its restriction map and the determination of its size. The digestion of pSS2 with HindIII originated two fragments (A and B) that were cloned separately into the pUC18 vector. The tetA gene was shown to be located on the HindIII-A fragment by PCR. After transforming a tetracycline-sensitive strain with pSS2, the transformants expressed the resistance phenotype and harbored a plasmid whose size was identical to that of pSS2. The results confirmed the association between pSS2 and the tetracycline resistance phenotype, and suggest a feasible dissemination of tetA and tetE among strains of Aeromonas. This study suggests the spreading tetA and tetE genes in Aeromonas in Brazil and describes a resistance plasmid that probably contributes to the dissemination of the resistance.

Antimicrobial resistance in food and clinical Aeromonas isolates.

This study highlights the incidence of resistance and the presence of plasmids in human and food isolates of Aeromonas in Brazil. A total of 83 Aeromonas spp. strains (28 isolated from human and 55 from fresh lettuce) were studied. Thirty-five were identified as A. hydrophila complex and 48 as A. caviae complex. All strains were shown to be susceptible to imipenem, amikacin, gentamicin, tobramycin and ciprofloxacin by the disk diffusion method. Resistance to antimicrobial agents was observed in strains of both food and clinical origin. The food strains were resistant to ampicillin/sulbactam, cefoxitin and tetracycline, while the clinical strains presented resistance to ampicillin/sulbactam, cefotaxime, ceftazidime, cefoxitin, sulfamethoxazole/trimethoprim, chloramphenicol and tetracycline. The minimal inhibitory concentrations of chloramphenicol, tetracycline and sulfamethoxazole/trimethoprim were tested by agar dilution. Thirteen strains isolated from vegetables were resistant to tetracycline (MIC 16 microg ml-1). Two A. hydrophila strains and one A. caviae strain presented extracromosomal DNA (3 and 15 kb plasmids, respectively). The tetracycline resistance phenotype determinant was related to the 15 kb plasmid according to cure and transformation experiments.