Copper nanoparticles effectively reduce Salmonella Enteritidis in broiler chicken diet and water.

RESEARCH HIGHLIGHTS: Supplementation with CuNP in feed and water reduced Salmonella Enteritidis count.Supplementation with CuNP did not affect intestinal integrity of broilers.CuNP did not affect weight gain or total lactic acid bacterial counts.The results demonstrate the potential of CuNP as alternative antimicrobials.

Development of Predictive Modeling for Removal of Multispecies Biofilms of Salmonella Enteritidis, Escherichia coli, and Campylobacter jejuni from Poultry Slaughterhouse Surfaces.

Salmonella Enteritidis, Escherichia coli, and Campylobacter jejuni are among the most common foodborne pathogens worldwide, and poultry products are strongly associated with foodborne pathogen outbreaks. These pathogens are capable of producing biofilms on several surfaces used in the food processing industry, including polyethylene and stainless steel. However, studies on multi-species biofilms are rare. Therefore, this study aimed to develop predictive mathematical models to simulate the adhesion and removal of multispecies biofilms. All combinations of microorganisms resulted in biofilm formation with differences in bacterial counts. E. coli showed the greatest ability to adhere to both surfaces, followed by S. Enteritidis and C. jejuni. The incubation time and temperature did not influence adhesion. Biofilm removal was effective with citric acid and benzalkonium chloride but not with rhamnolipid. Among the generated models, 46 presented a significant coefficient of determination (R2), with the highest R2 being 0.88. These results provide support for the poultry industry in creating biofilm control and eradication programs to avoid the risk of contamination of poultry meat.

Galleria mellonella larvae as an alternative model to determine the pathogenicity of avian pathogenic Escherichia coli.

RESEARCH HIGHLIGHTS: Galleria mellonella larvae are a viable model for determining APEC pathogenicity.Larval disease score is the main variable for determining APEC pathogenicity.Response variables should be evaluated up to 24 h post-inoculation.

Antilisterial activity of cinnamon essential oil, pomegranate extract, or strawberry tree extract against Listeria monocytogenes in slices of dry-cured ham and pork loin.

Owing to concerns about the antimicrobial resistance of agents that can prevent the growth of Listeria monocytogenes in meat, researchers have investigated natural preservatives with antilisterial effects. However, in vivo application of essential oils and plant extracts usually results in reduced antimicrobial activity in meat products when compared to in vitro studies. This study aimed to evaluate the in vivo antimicrobial activity of cinnamon essential oil, pomegranate, and strawberry tree extracts in slices of dry-cured ham and pork loin against L. monocytogenes. Fragments of sterile dry-cured ham were inoculated with 100 µL cinnamon oil 0.5%, pomegranate, or strawberry crude extract. After 10 min, 100 µL of L. monocytogenes serotype 4b (104 colony-forming unit [CFU]/mL) was inoculated, and samples were incubated at 7 °C for 7 d to simulate the processing and storage temperature conditions of dry-cured meat products. L. monocytogenes was detected and quantified. Only strawberry extract presented significant differences (P < 0.05) from the control; thus, it was selected for the assay with 2% and 4% salt-treated pork loin. The strawberry tree extract significantly (P < 0.05) reduced the growth of L. monocytogenes in dry-cured ham. However, it could not reduce L. monocytogenes growth in pork loin, regardless of the salt concentration. This is the first report on the antimicrobial effect of strawberry tree leaf extract against L. monocytogenes in dry-cured ham.

Adhesion capacity of Salmonella Enteritidis, Escherichia coli and Campylobacter jejuni on polystyrene, stainless steel, and polyethylene surfaces.

Poultry products are recognized as the main source of Salmonella and Campylobacter jejuni infections in humans, while avian pathogenic Escherichia coli may have zoonotic potential and can be transmitted from chicken meat to humans. Biofilm formation contributes to their spread through the food chain. This study aimed to compare the adhesion of Salmonella Enteritidis, E. coli, and C. jejuni strains isolated from poultry, food implicated in outbreaks, and poultry slaughterhouses on three surfaces widely used in poultry production (polystyrene, stainless steel, and polyethylene). S. Enteritidis and E. coli adhesion on the three surfaces tested were not significantly different (p > 0.05). Interestingly, the number of C. jejuni cells on stainless steel (4.51-4.67 log10 CFU/cm.-2) was significantly higher (p = 0.0004) than that on polystyrene (3.80-4.25 log10 CFU/cm.-2), but similar (p > 0.05) to that on polyethylene (4.03-4.36 log10 CFU/cm.-2). However, C. jejuni adhesion was significantly lower (p < 0.05) than S. Enteritidis and E. coli adhesion, regardless of the surface evaluated. In addition, scanning electron microscopy analyses have shown an increased irregularity of the stainless steel surface when compared to polyethylene and polystyrene. These irregularities form small spaces ideal for microbial adhesion.

Characterization of avian pathogenic Escherichia coli isolates based on biofilm formation, ESBL production, virulence-associated genes, and phylogenetic groups.

Escherichia coli is a part of both animal and human commensal microbiota. Avian pathogenic E. coli (APEC) is responsible for colibacillosis in poultry, an economically important disease. However, the close similarities among APEC isolates make it difficult to differentiate between pathogenic and commensal bacteria. The aim of this study was to determine phenotypic and molecular characteristics of APEC isolates and to compare them with their in vivo pathogenicity indices. A total of 198 APEC isolates were evaluated for their biofilm-producing ability and extended-spectrum ß-lactamase (ESBL) production phenotypes. In addition, 36 virulence-associated genes were detected, and the isolates were classified into seven phylogenetic groups using polymerase chain reaction. The sources of the isolates were not associated with biofilms, ESBL, genes, or phylogroups. Biofilm and ESBL production were not associated with pathogenicity. Group B2 had the highest pathogenicity index. Groups B2 and E were positively associated with high-pathogenicity isolates and negatively associated with low-pathogenicity isolates. In contrast, groups A and C were positively associated with apathogenic isolates, and group B1 was positively associated with low-pathogenicity isolates. Some virulence-associated genes showed positive or negative associations with specific phylogenetic groups. None of the individual techniques produced results that correlated with the in vivo pathogenicity index. However, the combination of two techniques, namely, detection of virulence-associated genes and the phylogenetic groups, could help the classification of the isolates as pathogenic or commensal.

Antibiofilm activity of electrochemically activated water (ECAW) in the control of Salmonella Heidelberg biofilms on industrial surfaces.

Owing to its antimicrobial activity, electrochemically activated water (ECAW) is a potential alternative to chemical disinfectants for eliminating foodborne pathogens, including Salmonella Heidelberg, from food processing facilities. However, their antibiofilm activity remains unclear. This study aimed to evaluate the antibiofilm activity of ECAW against S. Heidelberg biofilms formed on stainless steel and polyethylene and to determine its corrosive capacity. ECAW (200 ppm) and a broad-spectrum disinfectant (0.2%) were tested for their antibiofilm activity against S. Heidelberg at 25 °C and 37 °C after 10 and 20 min of contact with stainless steel and polyethylene. Potentiostatic polarization tests were performed to compare the corrosive capacity of both compounds. Both compounds were effective in removing S. Heidelberg biofilms. Bacterial counts were significantly lower with ECAW than with disinfectant in polyethylene, regardless the time of contact. The time of contact and the surface significantly influenced the bacterial counts of S. Heidelberg. Temperature was not an important factor affecting the antibiofilm activities of the compounds. ECAW was less corrosive than the disinfectant. ECAW demonstrated a similar or even superior effect in the control of S. Heidelberg biofilms, when compared to disinfectants, reducing bacterial counts by up to 5 log10 CFU cm-2. The corrosion of stainless steel with ECAW was similar to that of commercial disinfectants. This technology is a possible alternative for controlling S. Heidelberg in the food production chain.

Bacteriophages as an alternative for biological control of biofilm-forming Salmonella enterica.

Salmonellosis is one of the most common foodborne diseases worldwide. Surface adherence and biofilm formation are among the main strategies evolved by Salmonella to survive under harsh conditions and are risk factors for its spread through the food chain. Owing to the increase in antimicrobial resistance, there is a growing need to develop other methods to control foodborne pathogens, and bacteriophages have been suggested as a potential alternative for this purpose. The aim of this study was to evaluate bacteriophages as a biological control of Salmonella enterica serotypes to inhibit and remove bacterial biofilms. A total of 12 S. enterica isolates were selected for this study, all of which were biofilm producers. Seven bacteriophages were tested, individually and in a cocktail, for their host range and efficiency of plating (EOP). The phage cocktail was evaluated for its antibiofilm effect against the Salmonella biofilms. Phages UPF_BP1, UPF_BP2, UPF_BP3, UPF_BP6, and 10:2 possessed a broad lytic spectrum and could infect all S. enterica strains. Phages 10:2, UPF_BP6, and UPF_BP3 had high EOP in 10, 9, and 9 out of the 12 S. enterica strains, respectively. The cocktail was able to infect all S. enterica strains and had a high EOP in 10 out of 12 S. enterica isolates, presenting a broader host range than any of the tested single phages. A wide variation of inhibition among strains was observed, ranging from 14.72% to 88.53%. Multidrug-resistant and strong biofilm producer strains showed high biofilm inhibition levels by phage cocktail. Our findings demonstrate the ability of the cocktail to prevent biofilm formation and remove formed biofilms of Salmonella. These results indicate that the phage cocktail is a promising candidate to be used as an alternative for the control of Salmonella biofilms through surface conditioning.

Different Multidrug-Resistant Salmonella spp. Serovars Isolated from Slaughter Calves in Southern Brazil.

Bovines are carriers of Salmonella spp., a relevant foodborne pathogen, acting as contamination sources in slaughterhouses. Calves are prone to infection, and antimicrobial resistance may occur in such bacteria. This study aimed to determine the prevalence and virulence determinants of Salmonella spp. recovered from calves in the Rio Grande do Sul state, Brazil. Eighty-five calves' carcasses were evaluated (leather and veal meat). Thirteen Salmonella spp. isolates (8%) from 11 animals (13%) were obtained only from leather, indicating that contamination occurred before slaughter and that the meat was safe regarding this aspect. The serotypes S. Minnesota, S. Abony, S. Cerro, and S. Gafsa were identified, and all isolates were multidrug-resistant. The isolates had at least 19 virulence-related genes, and the blaOXA-48 resistance gene was detected in three (23%). The data suggest that treating infections caused by these bacteria may be difficult in animals from these farms and can also be an extended human health problem.

Antibiofilm activity of the biosurfactant and organic acids against foodborne pathogens at different temperatures, times of contact, and concentrations.

Biofilm formation has been suggested to play a significant role in the survival of pathogens in food production. Interest in evaluating alternative products of natural origin for disinfectant use has increased. However, there is a lack of information regarding the effects of biosurfactants and organic acids on Salmonella enterica serotype Enteritidis, Escherichia coli, and Campylobacter jejuni biofilms, mainly considering temperatures found in environments of poultry processing, as well as simulating the contact times used for disinfection. The aim of this study was to evaluate the antibiofilm activity of rhamnolipid, malic acid, and citric acid on the adhesion of S. Enteritidis, E. coli, and C. jejuni on polystyrene surfaces at different temperatures (4, 12, and 25 °C), compound concentrations, and times of contact (5 and 10 min), and to analyze the potential use of these compounds to disrupt formed biofilms. All three compounds exhibited antibiofilm activity under all analyzed conditions, both in the prevention and removal of formed biofilms. Contact time was less important than temperature and concentration. The antibiofilm activity of the compounds also varied according to the pathogens involved. In the food industry, compound selection must consider the temperature found in each stage of product processing and the target pathogens to be controlled.

Bacterial community identification in poultry carcasses using high-throughput next generation sequencing.

Poultry products are susceptible to contamination by pathogenic and spoilage bacteria during the slaughtering process. Molecular techniques have been used to assist in the identification of microorganisms in various microbiomes. The aim of this study was to identify bacterial components of the microbiome in poultry carcasses during the slaughter process, using high-throughput next generation sequencing (HT-NGS). Samples were collected from three slaughterhouses (A, B, and C) located in southern Brazil and included those taken from three points (initial, middle, and end) in the chiller tanks and two carcass pools (at the entrance to the clean area and after the final carcass packaging) at each establishment. A total of 104 carcasses were collected from each slaughterhouse. For this study, HT-NGS allows for a precise, quantitative and culture-independent microbiome assessment in poultry products. Three phyla (Firmicutes, Bacteroidetes, and Proteobacteria) were found in all establishments, and one phylum (Verrucomicrobia) was found only in Establishment A. Common set of genera (Anaerotruncus, Bacteroides, Campylobacter, Erysipelatoclostridium, Faecalibacterium, Lachnoclostridium, and Subdoligranulum) was identified in processing establishments along with the groups unique to a particular site. Pathogenic and spoilage bacteria, as well as other microorganisms that were not expected in poultry products, were detected by HT-NGS technique. The Shannon diversity index was the highest in Establishment B (2.40), followed by establishments C (1.98) and A (1.43). As we progressed through sample analysis, from the entrance of the clean area to the final carcass packaging area, we found significant reductions (p < 0.05) in the quantities of sequences of all phyla in establishments A and B. Significant differences (p < 0.05) in the quantities of sequences of all phyla were found between different stages in the slaughtering process. More stringent control procedures in establishments A and B were associated with reduced contamination even though all establishments followed the official sanitary standards. Our findings provide new insight into the chicken meat microbiome, and can be used in future studies to help ensure food safety in slaughterhouses.

Antimicrobial activity of copper surfaces against biofilm formation by Salmonella Enteritidis and its potential application in the poultry industry.

As a consequence of developing antimicrobial resistance to disinfectants, copper, which exhibits antimicrobial activity, has been studied as a possible alternative to the use of stainless steel surfaces. The aim was to evaluate the antimicrobial activity of copper surfaces in preventing biofilm formation by Salmonella Enteritidis and to determine their corrosive capacity. Strains of S. Enteritidis were incubated at 4 °C, 12 °C, and 25 °C with 1 cm2 coupons of electrolytic copper (99.9% Cu), brass (70% Cu), copper coated with tin, and stainless steel (control). A planktonic cell-suspension assay was used, followed by serial dilutions and bacterial counts. The corrosion test was performed with two disinfectants: benzalkonium chloride and sodium hypochlorite (100, 200, and 400 ppm). There was a significant reduction in biofilm production (log10 CFU cm-2) on the copper (2.64 at 4 °C, 4.20 at 12 °C, 4.56 at 25 °C) and brass (2.79 at 4 °C, 3.49 at 12 °C, 4.55 at 25 °C) surfaces compared to the control (5.68 at 4 °C, 5.89 at 12 °C, 6.01 at 25 °C). The antimicrobial surfaces showed uniform corrosion similar to that of surfaces generally used. These results demonstrated the effectiveness of copper surfaces in reducing S. Enteritidis and suggest they can be used as a complementary antimicrobial to control for this pathogen.

Antimicrobial susceptibility and detection of virulence-associated genes of Escherichia coli and Salmonella spp. isolated from domestic pigeons (Columba livia) in Brazil.

Overpopulation of domestic pigeons is considered to be one of the major problems of urban centers, as these birds are responsible for the dissemination of relevant pathogens to animal and human health. The aim of this study was to detect potentially pathogenic Escherichia coli and Salmonella spp. in domestic pigeons captured in areas near silos used for grain and feed storage, analyzing the antimicrobial sensitivity and the presence of virulence-associated genes. We evaluated 41 pigeons. From each bird, cecal contents and a pool of viscera (heart, spleen, and liver) were collected. Fifty strains of E. coli and three strains of S. Typhimurium were isolated. The antimicrobial susceptibility assay showed that 2% of the isolates of E. coli were resistant to chloramphenicol and the combination of sulfamethoxazole + trimethoprim and 4% to tetracycline, doxycycline, and sulfonamide. The three S. Typhimurium strains were sensitive to all antimicrobials tested. The pathogenicity profile demonstrated that no E. coli isolates showed a STEC compatible profile. Regarding the APEC pathotype, all genes were observed in 8% of E. coli, 6% had only the iss gene and 4% presented ompT, hlyF, and iutA genes. invA, hilA, avrA, and lpfA genes were detected in 100% of Salmonella isolates. The sitC and pefA genes were only present in one strain and the remaining genes were detected in two. In conclusion, it was found that pigeons living in the vicinity of silos are carriers of important pathogens, and control measures should be taken to minimize animal and human health risks.

Detection of virulence genes in Salmonella Heidelberg isolated from chicken carcasses.

During the last years, Brazilian government control programs have detected an increase of Salmonella Heidelberg in poultry slaughterhouses a condition that poses a threat to human health However, the reasons remain unclear. Differences in genetic virulence profiles may be a possible justification. In addition, effective control of Salmonella is related to an efficient epidemiological surveillance system through genotyping techniques. In this context, the aim of this study was the detection of 24 virulence-associated genes in 126 S. Heidelberg isolates. We classified the isolates into 56 different genetic profiles. None of the isolates presented all the virulence genes. The prevalence of these genes was high in all tested samples as the lowest number of genes detected in one isolate was 10/24. The lpfA and csgA (fimbriae), invA and sivH (TTSS), and msgA and tolC (intracellular survival) genes were present in 100% of the isolates analyzed. Genes encoding effector proteins were detected in the majority of SH isolates. No single isolate had the sefA gene. The pefA gene was found in only four isolates. We have also performed a screening of genes associated with iron metabolism: 88.9% of isolates had the iroN geneand 79.4% the sitC gene . Although all the isolates belong to the same serotype, several genotypic profiles were observed. These findings suggest that there is a diversity of S. Heidelberg isolates in poultry products. The fact that a single predominant profile was not found in this study indicates the presence of variable sources of contamination caused by SH. The detection of genetic profiles of Salmonella strains can be used to determine the virulence patterns of SH isolates.

Influence of catecholamines on biofilm formation by Salmonella Enteritidis.

Salmonella spp. are the main pathogens responsible for foodborne disease worldwide. Bacterial communities use the quorum sensing system to control biofilm formation. These systems function through the secretion of substances, called auto-inducers (AI), into the environment. AI-3 is structurally similar to epinephrine (EPI) and norepinephrine (NOR) -catecholamines secreted by eukaryotic cells to communicate with each other. In this context, this work aimed to evaluate the effect of EPI and NOR on biofilm formation by S. Enteritidis at 12 °C and 25 °C. Also, we detected the presence of the csgD, adrA, and fimA genes in these strains. Biofilm formation was investigated at two temperatures (12 °C and 25 °C) using a microtiter plate assay, under four different treatments (50 mM EPI, 100 mM EPI, 50 mM NOR; 100 mM NOR) and a control group. PCR was used to detect the virulence genes associated with biofilm production. A greater number of biofilm producer isolates were observed at 25 °C than at 12 °C, regardless of the treatment. The number of biofilms forming strains at 12 °C was significantly higher in the treatment with norepinephrine at 100 µM. The proportion of non-producer and biofilm producer strains at 25 °C did not differ significantly among the treatments. All strains presented the three genes (csgD, adrA, and fimA). The approach carried out in this work is a precursor in veterinary medicine, focusing on both public and poultry health, and evaluates the influence of catecholamines on the formation of biofilms with S. Enteritidis, an important pathogen with zoonotic potential. Norepinephrine seems to be more efficient at stimulating biofilm formation by S. Enteritidis strains at 12 °C. csgD, fimA, and adrA were detected in all strains.

Detection and quantification of Salmonella spp. in poultry slaughterhouses of southern Brazil.

INTRODUCTION: Salmonella is a major cause of foodborne illness throughout the world. The use of quantitative techniques is important for assessing the risk and determining the capacity of each step of the slaughtering process to decrease or increase bacterial contamination. We aimed to detect and to quantify the presence of Salmonella in Brazilian processing plants by real-time quantitative polymerase chain reaction (qPCR). METHODOLOGY: A total of 139 poultry slaughterhouses samples were collected in order to detect to and quantify Salmonella by qPCR. RESULTS: Almost all collection points (3/18), except water from pre-chiller tank, carcasses after pre-chiller, and carcasses frozen at -12ºC for 60 days, and 49% (68/139) of samples were positive for Salmonella. Quantification means varied equally among all of the tested sources, and we could not establish any pattern of variation. A large proportion (52.6%) of cloacal swabs was Salmonella-positive. Also, contamination in transport cages was increased after the cleaning process, indicating that the process was ineffective. The overall prevalence in samples obtained during the slaughtering process was 48.9%, and on the whole rinsed carcasses, this proportion was 50%. The detection of Salmonella in frozen carcasses, even after long periods of storage, indicates that the carcasses are a potential source of infection for consumers. CONCLUSIONS: We found that contamination levels remain similar throughout the slaughtering. qPCR proved to be an efficient method for the detection of Salmonella.

Biofilm formation by Salmonella Enteritidis and Salmonella Typhimurium isolated from avian sources is partially related with their in vivo pathogenicity.

Salmonella Enteritidis and Salmonella Typhimurium are among the most prevalent serotypes isolated from salmonellosis outbreaks and poultry. Salmonella spp. have the capacity to form biofilms on several surfaces, which can favour survival in hostile environments, such as slaughterhouses. Salmonella strains present differences in pathogenicity. However, there is little information regarding the pathogenicity of S. Enteritidis and S. Typhimurium isolated from avian sources and their relationship to biofilm production. The aim of this study was to use a novel pathogenicity index and a biofilm production assay to evaluate their relationships within these serotypes. In addition, we detected the presence of the spiA and agfA genes in these strains. Biofilm formation was investigated at two temperatures (37 °C and 28 °C) using microtiter plate assay, and the results were compared with the individual pathogenicity index of each strain. PCR was used to detect spiA and agfA, virulence genes associated with biofilm production. S. Enteritidis and S. Typhimurium strains were capable of producing biofilm at 37 °C and 28 °C. Sixty-two percent and 59.5% of S. Enteritidis and 73.8% and 46.2% of S. Typhimurium produced biofilm at 37 °C and 28 °C, respectively. Biofilm production at 37 °C was significantly higher in both serotypes. Only S. Enteritidis was capable of adhering strongly at both temperatures. Biofilm production was related to pathogenicity index only at 28 °C for S. Enteritidis. spiA and agfA were found in almost all strains and were not statistically associated with biofilm production.

Phenotypic and Molecular Characterization of Salmonella Enteritidis SE86 Isolated from Poultry and Salmonellosis Outbreaks.

Salmonella Enteritidis remains a standout among the leading causes of foodborne diseases worldwide. Previous studies have demonstrated that a unique clonal group of Salmonella Enteritidis, named SE86, is involved in foodborne outbreaks in southern Brazil and is frequently identified among strains isolated from poultry. The aim of this study was to determine the influence of the isolation source (food products involved in salmonellosis outbreaks and poultry sources) on the phenotypic and molecular characteristics of Salmonella Enteritidis SE86. A biofilm formation assay, antimicrobial susceptibility test, polymerase chain reaction identification of virulence-associated genes, and phage type 4 (PT4) assessment were performed to characterize Salmonella Enteritidis SE86. The human strains presented less antimicrobial resistance than the poultry strains. Resistance to some substances was related to the isolation source of the strain. Strains of the same clonal group presented different biofilm production abilities. Biofilm formation was independent of the isolation source at all temperatures. Temperature influenced biofilm formation only by the poultry strains. Most of the investigated genes presented a high frequency and a regular distribution, regardless of the isolation source. The spvB, spiA, pagC, sipB, prgH, spaN, sitC, and lpfC genes were associated with the avian strains, whereas iroN was associated with the strains isolated from food products involved in salmonellosis outbreaks. Most strains belonged to PT4. No relationship was found between biofilm production and antimicrobial resistance or between the virulence profile and biofilm production or antimicrobial resistance.

Spread of a Major Clone of Salmonella enterica Serotype Enteritidis in Poultry and in Salmonellosis Outbreaks in Southern Brazil.

Salmonella spp. are among the most important agents of foodborne diseases all over the world. Human Salmonella outbreaks are often associated with the consumption of poultry products (meat and eggs), and one of the most prevalent serotypes associated with these products is Salmonella Enteritidis. Brazil is one of the most important poultry exporters in the world. In southern Brazil, three closely related clones of Salmonella Enteritidis have been responsible for the majority of foodborne Salmonella outbreaks over the past decade. However, until now, there has been little information regarding the clonal relationship among the Brazilian Salmonella strains of avian origin and those involved in foodborne outbreaks. Therefore, the aim of the present study was to complete the molecular characterization of Salmonella Enteritidis strains isolated from poultry and food sources involved in Salmonella outbreaks. PCR ribotyping was performed to discriminate the strains into different ribotype profiles according to the banding pattern amplification. This technique was able to differentiate the Salmonella Enteritidis strains into two banding patterns: R2 and R4. R2 accounted for 98.7% of the strains. DNA sequencing of the 600-bp fragment, present in all ribotypes, was applied to confirm this result. The sequences generated showed high levels of similarity, ranging from 99.7 to 100%, and were grouped into a single cluster. These results suggest that there is a clonal relationship among the Salmonella Enteritidis strains responsible for several salmonellosis outbreaks and the strains collected from poultry sources.

Use of Molecular Pathogenicity Indices to Identify Pathogenic Strains of Pasteurella multocida.

In addition to being the causative agent of fowl cholera (FC), Pasteurella multocida is also one of the most prevalent opportunistic pathogens associated with respiratory diseases in various hosts. However, understanding of the traits that distinguish the virulent isolates that cause FC is still limited. The objective of this study was to characterize P. multocida isolates of Brazil by PCR-restriction fragment length polymorphism (PCR-RFLP) analysis in order to determine if strain-type correlates with virulence or with 22 previously studied virulence genes. The PCR-RFLP was used to classify the isolates into seven strain types, and the isolates in Profile II had a higher pathogenicity index (P < 0.05) than did those in Profiles I, V, and VI. The overall identity among the nucleotide sequences of the ompH was 89.8%. Furthermore, strains available in GenBank showed a high level of homology of the different bacterial serotypes with the groupings resulting from the PCR-RFLP. Strain Types I and II showed the highest identity with Serotypes 3 (100%) and 3-4 (99.1%), respectively. Detection of the pfhA gene indicated the presence of strains that are highly pathogenic. The screening detection of 22 virulence genes and inference through the decision tree models comparing the results of pathogenicity indices permitted the identification of the most highly pathogenic strains of P. multocida .