Priscila Guerra | University of Copenhagen (original) (raw)

Papers by Priscila Guerra

In a previous study, a novel virulence gene, bstA, identified in a Salmonella enterica serovar Ty... more In a previous study, a novel virulence gene, bstA, identified in a Salmonella enterica serovar Typhimurium sequence type 313 (ST313) strain was found to be conserved in all published Salmonella enterica serovar Dublin genomes. In order to analyze the role of this gene in the host-pathogen interaction in S. Dublin, a mutant where this gene was deleted (S. Dublin ΔbstA) and a mutant which was further genetically complemented with bstA (S. Dublin 3246-C) were constructed and tested in models of in vitro and in vivo infection as well as during growth competition assays in M9 medium, Luria-Bertani broth, and cattle blood. In contrast to the results obtained for a strain of S. Typhimurium ST313, the lack of bstA was found to be associated with increased virulence in S. Dublin. Thus, S. Dublin ΔbstA showed higher levels of uptake than the wild-type strain during infection of mouse and cattle mac-rophages and higher net replication within human THP-1 cells. Furthermore, during mouse infections, S. Dublin ΔbstA was more virulent than the wild type following a single intraperitoneal infection and showed an increased competitive index during competitive infection assays. Deletion of bstA did not affect either the amount of cyto-kines released by THP-1 macrophages or the cytotoxicity toward these cells. The histol-ogy of the livers and spleens of mice infected with the wild-type strain and the S. Dub-lin ΔbstA mutant revealed similar levels of inflammation between the two groups. The gene was not important for adherence to or invasion of human epithelial cells and did not influence bacterial growth in rich medium, minimal medium, or cattle blood. In conclusion , a lack of bstA affects the pathogenicity of S. Dublin by decreasing its virulence. Therefore, it might be regarded as an antivirulence gene in this serovar.

Polyamines (putrescine and spermidine) are small-cationic amines ubiquitous in nature and present... more Polyamines (putrescine and spermidine) are small-cationic amines ubiquitous in nature and present in most living cells. In recent years they have been linked to virulence of several human pathogens including Shigella spp and Salmonella enterica serovar Typhimurium (S. Typhimurium). Central to S. Typhimurium virulence is the ability to survive and replicate inside macrophages and resisting the antimicrobial attacks in the form of oxidative and nitrosative stress elicited from these cells. In the present study, we have investigated the role of polyamines in intracellular survival and systemic infections of mice. Using a S. Typhimurium mutant defective for putrescine and spermidine biosynthesis, we show that polyamines are essential for coping with reactive nitrogen species, possibly linking polyamines to increased intracellular stress resistance. However, using a mouse model defective for nitric oxide production, we find that polyamines are required for systemic infections independently of host-produced reactive nitrogen species. To distinguish between the physiological roles of putrescine and spermidine, we constructed a strain deficient for spermidine biosynthesis and uptake, but with retained ability to produce and import putrescine. Interestingly, in this mutant we observe a strong attenuation of virulence during infection of mice proficient and deficient for nitric oxide production suggesting that spermidine, specifically, is essential for virulence of S. Typhimurium.

A B S T R A C T Over the last few years, polyamines have been described as key-signal of virulenc... more A B S T R A C T Over the last few years, polyamines have been described as key-signal of virulence in pathogenic bacteria. In the current study, we investigated whether the knockout of genes related to polyamine biosynthesis and putrescine transport affected the virulence of an avian pathogenic E. coli (APEC) strain. One-week-old White Leghorn chickens were infected intratracheally with mutants in polyamine biosynthesis (ΔspeB/C and ΔspeD/E) and transport genes (ΔpotE) of a well-characterized APEC strain of ST117 (O83: H4). All polyamine mutants and the wild-type strain were able to infect chicken; however, we observed significantly fewer lesions in the lungs of the chickens infected with the polyamine mutants in comparison with chicken infected with the wild-type. Results derived from histology of infected lungs detected significantly fewer lesions in the lung of birds infected within particular the putrescine transport mutant (ΔpotE). A decrease in colonization levels was observed in the liver and spleen of birds infected with the putrescine biosynthesis mutant ΔspeB/C, and likewise, a decrease of the colonization levels of all organs from birds infected with the ΔpotE was detected. Together, our data demonstrate that the deletion of polyamine genes, and in particular the PotE membrane protein, attenuates the virulence of APEC during infection of chickens.

Pesquisa Veterinária Brasileira, 2014

Veterinaria Em Foco, Aug 21, 2014

As bactérias do gênero Helicobacter são bacilos Gram negativos, helicoidais, que requerem uma atm... more As bactérias do gênero Helicobacter são bacilos Gram negativos, helicoidais, que requerem uma atmosfera de microaerofi lia para seu crescimento. Podem habitar tanto a mucosa gástrica quanto o interior das glândulas gástricas de animais assintomáticos ou com sinais clínicos de gastropatia. Esses pacientes podem apresentar quadros de dispepsia, gastrite crônica, erosões, ulcerações e até adenocarcinoma, ocasionado dor e desconforto abdominal. Devido à elevada prevalência dessa bactéria em diferentes espécies animais, torna-se importante conhecer as principais espécies envolvidas na infecção, bem como os principais métodos diagnósticos utilizados na medicina veterinária. Os métodos invasivos, os quais necessitam de endoscopia digestiva alta, são os mais empregados em medicina veterinária, incluem-se a histologia, o teste rápido da urease, a cultura bacteriana e os métodos moleculares de diagnóstico. Para fi ns de pesquisa, recomenda-se a associação de múltiplas técnicas para alcançar um diagnóstico mais preciso. O objetivo do trabalho foi revisar os principais métodos diagnósticos invasivos empregados em medicina veterinária, bem como as principais espécies de Helicobacter que acometem os animais domésticos.

Acta Scientiae Veterinariae, 2012

Polyamines (putrescine and spermidine) are small-cationic amines ubiquitous in nature and present... more Polyamines (putrescine and spermidine) are small-cationic amines ubiquitous in nature and present in most living cells. In recent years they have been linked to virulence of several human pathogens including Shigella spp and Salmonella enterica serovar Typhimurium (S. Typhimurium). Central to S. Typhimurium virulence is the ability to survive and replicate inside macrophages and resisting the antimicrobial attacks in the form of oxidative and nitrosative stress elicited from these cells. In the present study, we have investigated the role of polyamines in intracellular survival and systemic infections of mice. Using a S. Typhimurium mutant defective for putrescine and spermidine biosynthesis, we show that polyamines are essential for coping with reactive nitrogen species, possibly linking polyamines to increased intracellular stress resistance. However, using a mouse model defective for nitric oxide production, we find that polyamines are required for systemic infections independently of hostproduced reactive nitrogen species. To distinguish between the physiological roles of putrescine and spermidine, we constructed a strain deficient for spermidine biosynthesis and uptake, but with retained ability to produce and import putrescine. Interestingly, in this mutant we observe a strong attenuation of virulence during infection of mice proficient and deficient for nitric oxide production suggesting that spermidine, specifically, is essential for virulence of S. Typhimurium.

Journal of Zoo and Wildlife Medicine, 2014

After demonstrating chronic weight loss, prostration, and muscle flaccidness, a captive-bred 9-mo... more After demonstrating chronic weight loss, prostration, and muscle flaccidness, a captive-bred 9-mo-old boa constrictor (Boa constrictor constrictor) died and was submitted for necropsy. Along the spinal column there were multiple, yellowish white, macroscopic nodules of 1-5 mm in diameter in the ventral side of the vertebral body and in the intervertebral spaces. Severe multifocal necrotizing osteomyelitis associated with granulomatous inflammation was the main histologic finding in the vertebral column. In the liver, there was discrete but similar granulomatous changes. Positive anti-Salmonella immunostaining was observed in the spinal column and in the liver. Salmonella enterica serovar Derby was isolated from fragments of the spinal column. These bacteria are important cause of disease in captive reptiles.

Archives of Veterinary Science, 2012

RESUMO: O objetivo deste estudo foi determinar a presença e distribuição de perfis clonais de iso... more RESUMO: O objetivo deste estudo foi determinar a presença e distribuição de perfis clonais de isolados de Listeria monocytogenes, agrupados após a sorotipificação e macrorestrição de DNA seguida de eletroforese em campo pulsado (PFGE), em carcaças de suínos amostradas em quatro pontos da linha de abate e no ambiente de matadouros frigoríficos. Suabes de superfície foram coletados no ambiente (n=30) e na superfície de carcaças (n=240) após a escaldagem, chamuscamento, evisceração e na entrada da câmara fria, em dois matadouros frigoríficos localizados no sul do Brasil. Dos 270 suabes analisados, 21 (7,7%) foram positivos para o gênero Listeria, distribuídas nas espécies, L. innocua (10), L. monocytogenes (9), L. ivanovii (1) e L. seeligeri (1). L. monocytogenes foi isolada apenas de amostras coletadas de carcaça após a evisceração e do ambiente da câmara fria em um dos matadouros frigoríficos. Os isolados de L. monocytogenes foram classificados em três sorotipos (1/2a, 1/2b e 1/2c) e dois pulsotipos. A combinação dos sorotipos e pulsotipos resultaram na formação de quatro perfis clonais (I-IV). A identificação de um perfil clonal (I) comum em cinco carcaças amostradas após a etapa de evisceração demonstra que esta etapa é critica para contaminação cruzada durante o processo de abate. Palavras-chave: abate de suínos; PFGE; Listeria ABSTRACT: The aim of this study was to evaluate the isolation and distribution of Listeria monocytogenes clonal groups originated from serotyping and pulsed-field gel eletrophoresis (PFGE) analysis of isolates from environment and from pork carcasses sampled at four different points of the slaughter line. Swabs were taken from the environment (n=30) and from the surface of carcasses (n=240) after scalding, flaming, evisceration and before cooling in two slaughterhouses of Southern Brazil. From 270 performed swabs, 21 (7.7%) were positive for Listeria spp., identified as follow: L. innocua (10), L. monocytogenes (9), L. ivanovii (1) e L. seeligeri (1). L. monocytogenes was isolated from carcass samples taken after evisceration and before cooling. L. monocytogenes strains were classified in three serotypes (1/2a, 1/2b and 1/2c) and two pulsotypes. The association of serotypes and pulsotypes originated four clonal groups (I-IV) among L. monocytogenes strains. The identification of a single clonal group (I) of L. monocytogenes in five carcasses after the evisceration demonstrates that this is a critical step for cross-contaminating during the slaughter process.

Pesquisa Veterinária Brasileira, 2014

Journal of Food Protection, 2012

This study was conducted to assess the dissemination of Salmonella clonal groups in slaughterhous... more This study was conducted to assess the dissemination of Salmonella clonal groups in slaughterhouses that received batches of Salmonella-positive pigs and used different routine processing procedures. Eight serial sampling sessions were conducted in three slaughterhouses (A, B, and C). Blood was collected randomly (n~25) from each batch of pigs and processed for serology. Carcasses (n~12) were identified and sampled after dehairing, after singeing, after evisceration, and before chilling. A section of cecum also was collected. Salmonella isolates were submitted to pulsed-field gel electrophoresis. The overall seroprevalence of Salmonella was 80.6% (316 of 392 samples), and cecal contents were positive for Salmonella in 23.8% (26 of 109) of the pigs sampled. Carcasses after dehairing had a significantly higher prevalence of Salmonella (P~0.004) and the highest Salmonella levels (median~0.26 log CFU/300 cm 2 ). The singeing step significantly affected the Salmonella status of the carcasses (P , 0.001); however, the efficacy of singeing differed among slaughterhouses. In the prechilling step, 14.7% (16 of 109) of the carcasses were positive for Salmonella. Salmonella pulsotypes found on the prechill carcasses were also found in the lairage, in the cecal contents, and on carcasses after dehairing, suggesting that the main source of contamination was the slaughter process before singeing. Slaughterhouse C was the most likely (odds ration [OR]~6.51) to have pigs carrying Salmonella in the gut, and slaughterhouse B was the most likely (OR~14.66) to have contaminated carcasses at the prechilling step. These findings indicate that the procedures adopted in slaughterhouse B contributed to the spread of Salmonella strains. In contrast, in slaughterhouse C the Salmonella strains carried by the pigs or found in the lairage were not recovered from prechilled carcasses, validating the effectiveness of the slaughterhouse interventions. These results indicate that an effective slaughter process can help decrease the number of Salmonella-positive carcasses in slaughterhouses that receive Salmonella-positive pig batches.

In a previous study, a novel virulence gene, bstA, identified in a Salmonella enterica serovar Ty... more In a previous study, a novel virulence gene, bstA, identified in a Salmonella enterica serovar Typhimurium sequence type 313 (ST313) strain was found to be conserved in all published Salmonella enterica serovar Dublin genomes. In order to analyze the role of this gene in the host-pathogen interaction in S. Dublin, a mutant where this gene was deleted (S. Dublin ΔbstA) and a mutant which was further genetically complemented with bstA (S. Dublin 3246-C) were constructed and tested in models of in vitro and in vivo infection as well as during growth competition assays in M9 medium, Luria-Bertani broth, and cattle blood. In contrast to the results obtained for a strain of S. Typhimurium ST313, the lack of bstA was found to be associated with increased virulence in S. Dublin. Thus, S. Dublin ΔbstA showed higher levels of uptake than the wild-type strain during infection of mouse and cattle mac-rophages and higher net replication within human THP-1 cells. Furthermore, during mouse infections, S. Dublin ΔbstA was more virulent than the wild type following a single intraperitoneal infection and showed an increased competitive index during competitive infection assays. Deletion of bstA did not affect either the amount of cyto-kines released by THP-1 macrophages or the cytotoxicity toward these cells. The histol-ogy of the livers and spleens of mice infected with the wild-type strain and the S. Dub-lin ΔbstA mutant revealed similar levels of inflammation between the two groups. The gene was not important for adherence to or invasion of human epithelial cells and did not influence bacterial growth in rich medium, minimal medium, or cattle blood. In conclusion , a lack of bstA affects the pathogenicity of S. Dublin by decreasing its virulence. Therefore, it might be regarded as an antivirulence gene in this serovar.

Polyamines (putrescine and spermidine) are small-cationic amines ubiquitous in nature and present... more Polyamines (putrescine and spermidine) are small-cationic amines ubiquitous in nature and present in most living cells. In recent years they have been linked to virulence of several human pathogens including Shigella spp and Salmonella enterica serovar Typhimurium (S. Typhimurium). Central to S. Typhimurium virulence is the ability to survive and replicate inside macrophages and resisting the antimicrobial attacks in the form of oxidative and nitrosative stress elicited from these cells. In the present study, we have investigated the role of polyamines in intracellular survival and systemic infections of mice. Using a S. Typhimurium mutant defective for putrescine and spermidine biosynthesis, we show that polyamines are essential for coping with reactive nitrogen species, possibly linking polyamines to increased intracellular stress resistance. However, using a mouse model defective for nitric oxide production, we find that polyamines are required for systemic infections independently of host-produced reactive nitrogen species. To distinguish between the physiological roles of putrescine and spermidine, we constructed a strain deficient for spermidine biosynthesis and uptake, but with retained ability to produce and import putrescine. Interestingly, in this mutant we observe a strong attenuation of virulence during infection of mice proficient and deficient for nitric oxide production suggesting that spermidine, specifically, is essential for virulence of S. Typhimurium.

A B S T R A C T Over the last few years, polyamines have been described as key-signal of virulenc... more A B S T R A C T Over the last few years, polyamines have been described as key-signal of virulence in pathogenic bacteria. In the current study, we investigated whether the knockout of genes related to polyamine biosynthesis and putrescine transport affected the virulence of an avian pathogenic E. coli (APEC) strain. One-week-old White Leghorn chickens were infected intratracheally with mutants in polyamine biosynthesis (ΔspeB/C and ΔspeD/E) and transport genes (ΔpotE) of a well-characterized APEC strain of ST117 (O83: H4). All polyamine mutants and the wild-type strain were able to infect chicken; however, we observed significantly fewer lesions in the lungs of the chickens infected with the polyamine mutants in comparison with chicken infected with the wild-type. Results derived from histology of infected lungs detected significantly fewer lesions in the lung of birds infected within particular the putrescine transport mutant (ΔpotE). A decrease in colonization levels was observed in the liver and spleen of birds infected with the putrescine biosynthesis mutant ΔspeB/C, and likewise, a decrease of the colonization levels of all organs from birds infected with the ΔpotE was detected. Together, our data demonstrate that the deletion of polyamine genes, and in particular the PotE membrane protein, attenuates the virulence of APEC during infection of chickens.

Pesquisa Veterinária Brasileira, 2014

Veterinaria Em Foco, Aug 21, 2014

As bactérias do gênero Helicobacter são bacilos Gram negativos, helicoidais, que requerem uma atm... more As bactérias do gênero Helicobacter são bacilos Gram negativos, helicoidais, que requerem uma atmosfera de microaerofi lia para seu crescimento. Podem habitar tanto a mucosa gástrica quanto o interior das glândulas gástricas de animais assintomáticos ou com sinais clínicos de gastropatia. Esses pacientes podem apresentar quadros de dispepsia, gastrite crônica, erosões, ulcerações e até adenocarcinoma, ocasionado dor e desconforto abdominal. Devido à elevada prevalência dessa bactéria em diferentes espécies animais, torna-se importante conhecer as principais espécies envolvidas na infecção, bem como os principais métodos diagnósticos utilizados na medicina veterinária. Os métodos invasivos, os quais necessitam de endoscopia digestiva alta, são os mais empregados em medicina veterinária, incluem-se a histologia, o teste rápido da urease, a cultura bacteriana e os métodos moleculares de diagnóstico. Para fi ns de pesquisa, recomenda-se a associação de múltiplas técnicas para alcançar um diagnóstico mais preciso. O objetivo do trabalho foi revisar os principais métodos diagnósticos invasivos empregados em medicina veterinária, bem como as principais espécies de Helicobacter que acometem os animais domésticos.

Acta Scientiae Veterinariae, 2012

Polyamines (putrescine and spermidine) are small-cationic amines ubiquitous in nature and present... more Polyamines (putrescine and spermidine) are small-cationic amines ubiquitous in nature and present in most living cells. In recent years they have been linked to virulence of several human pathogens including Shigella spp and Salmonella enterica serovar Typhimurium (S. Typhimurium). Central to S. Typhimurium virulence is the ability to survive and replicate inside macrophages and resisting the antimicrobial attacks in the form of oxidative and nitrosative stress elicited from these cells. In the present study, we have investigated the role of polyamines in intracellular survival and systemic infections of mice. Using a S. Typhimurium mutant defective for putrescine and spermidine biosynthesis, we show that polyamines are essential for coping with reactive nitrogen species, possibly linking polyamines to increased intracellular stress resistance. However, using a mouse model defective for nitric oxide production, we find that polyamines are required for systemic infections independently of hostproduced reactive nitrogen species. To distinguish between the physiological roles of putrescine and spermidine, we constructed a strain deficient for spermidine biosynthesis and uptake, but with retained ability to produce and import putrescine. Interestingly, in this mutant we observe a strong attenuation of virulence during infection of mice proficient and deficient for nitric oxide production suggesting that spermidine, specifically, is essential for virulence of S. Typhimurium.

Journal of Zoo and Wildlife Medicine, 2014

After demonstrating chronic weight loss, prostration, and muscle flaccidness, a captive-bred 9-mo... more After demonstrating chronic weight loss, prostration, and muscle flaccidness, a captive-bred 9-mo-old boa constrictor (Boa constrictor constrictor) died and was submitted for necropsy. Along the spinal column there were multiple, yellowish white, macroscopic nodules of 1-5 mm in diameter in the ventral side of the vertebral body and in the intervertebral spaces. Severe multifocal necrotizing osteomyelitis associated with granulomatous inflammation was the main histologic finding in the vertebral column. In the liver, there was discrete but similar granulomatous changes. Positive anti-Salmonella immunostaining was observed in the spinal column and in the liver. Salmonella enterica serovar Derby was isolated from fragments of the spinal column. These bacteria are important cause of disease in captive reptiles.

Archives of Veterinary Science, 2012

RESUMO: O objetivo deste estudo foi determinar a presença e distribuição de perfis clonais de iso... more RESUMO: O objetivo deste estudo foi determinar a presença e distribuição de perfis clonais de isolados de Listeria monocytogenes, agrupados após a sorotipificação e macrorestrição de DNA seguida de eletroforese em campo pulsado (PFGE), em carcaças de suínos amostradas em quatro pontos da linha de abate e no ambiente de matadouros frigoríficos. Suabes de superfície foram coletados no ambiente (n=30) e na superfície de carcaças (n=240) após a escaldagem, chamuscamento, evisceração e na entrada da câmara fria, em dois matadouros frigoríficos localizados no sul do Brasil. Dos 270 suabes analisados, 21 (7,7%) foram positivos para o gênero Listeria, distribuídas nas espécies, L. innocua (10), L. monocytogenes (9), L. ivanovii (1) e L. seeligeri (1). L. monocytogenes foi isolada apenas de amostras coletadas de carcaça após a evisceração e do ambiente da câmara fria em um dos matadouros frigoríficos. Os isolados de L. monocytogenes foram classificados em três sorotipos (1/2a, 1/2b e 1/2c) e dois pulsotipos. A combinação dos sorotipos e pulsotipos resultaram na formação de quatro perfis clonais (I-IV). A identificação de um perfil clonal (I) comum em cinco carcaças amostradas após a etapa de evisceração demonstra que esta etapa é critica para contaminação cruzada durante o processo de abate. Palavras-chave: abate de suínos; PFGE; Listeria ABSTRACT: The aim of this study was to evaluate the isolation and distribution of Listeria monocytogenes clonal groups originated from serotyping and pulsed-field gel eletrophoresis (PFGE) analysis of isolates from environment and from pork carcasses sampled at four different points of the slaughter line. Swabs were taken from the environment (n=30) and from the surface of carcasses (n=240) after scalding, flaming, evisceration and before cooling in two slaughterhouses of Southern Brazil. From 270 performed swabs, 21 (7.7%) were positive for Listeria spp., identified as follow: L. innocua (10), L. monocytogenes (9), L. ivanovii (1) e L. seeligeri (1). L. monocytogenes was isolated from carcass samples taken after evisceration and before cooling. L. monocytogenes strains were classified in three serotypes (1/2a, 1/2b and 1/2c) and two pulsotypes. The association of serotypes and pulsotypes originated four clonal groups (I-IV) among L. monocytogenes strains. The identification of a single clonal group (I) of L. monocytogenes in five carcasses after the evisceration demonstrates that this is a critical step for cross-contaminating during the slaughter process.

Pesquisa Veterinária Brasileira, 2014

Journal of Food Protection, 2012

This study was conducted to assess the dissemination of Salmonella clonal groups in slaughterhous... more This study was conducted to assess the dissemination of Salmonella clonal groups in slaughterhouses that received batches of Salmonella-positive pigs and used different routine processing procedures. Eight serial sampling sessions were conducted in three slaughterhouses (A, B, and C). Blood was collected randomly (n~25) from each batch of pigs and processed for serology. Carcasses (n~12) were identified and sampled after dehairing, after singeing, after evisceration, and before chilling. A section of cecum also was collected. Salmonella isolates were submitted to pulsed-field gel electrophoresis. The overall seroprevalence of Salmonella was 80.6% (316 of 392 samples), and cecal contents were positive for Salmonella in 23.8% (26 of 109) of the pigs sampled. Carcasses after dehairing had a significantly higher prevalence of Salmonella (P~0.004) and the highest Salmonella levels (median~0.26 log CFU/300 cm 2 ). The singeing step significantly affected the Salmonella status of the carcasses (P , 0.001); however, the efficacy of singeing differed among slaughterhouses. In the prechilling step, 14.7% (16 of 109) of the carcasses were positive for Salmonella. Salmonella pulsotypes found on the prechill carcasses were also found in the lairage, in the cecal contents, and on carcasses after dehairing, suggesting that the main source of contamination was the slaughter process before singeing. Slaughterhouse C was the most likely (odds ration [OR]~6.51) to have pigs carrying Salmonella in the gut, and slaughterhouse B was the most likely (OR~14.66) to have contaminated carcasses at the prechilling step. These findings indicate that the procedures adopted in slaughterhouse B contributed to the spread of Salmonella strains. In contrast, in slaughterhouse C the Salmonella strains carried by the pigs or found in the lairage were not recovered from prechilled carcasses, validating the effectiveness of the slaughterhouse interventions. These results indicate that an effective slaughter process can help decrease the number of Salmonella-positive carcasses in slaughterhouses that receive Salmonella-positive pig batches.