Assessment of Internalin A Gene Sequences and Cell Adhesion and Invasion Capacity of Listeria monocytogenes Strains Isolated from Foods of Animal and Related Origins.

Listeria monocytogenes is a foodborne pathogen of global relevance that causes outbreaks and sporadic cases of listeriosis, acquired through the consumption of contaminated products, including milk or meat products and ready-to-eat meat products subjected to intensive handling. The objective of the present study was to classify L. monocytogenes isolated from various food-related sources in the Federal District of Brazil and surrounding areas to sequence internalin A (inlA) genes from these isolates and assess their adhesion and invasion capacity using Caco-2 cells. In addition, 15 were classified as group I, 3 as group II, and 7 classified as group IV. Premature stop codons (PMSCs) at the nucleotide position 976 (GAA→TAA) of the inlA gene were identified in 5 of the 25 isolates. Adhesion and invasion tests in Caco-2 cells showed that all the isolates were capable of adhesion and cellular invasion, with isolates containing PMSCs exhibiting on average higher invasion capacity than those without PMSCs (p = 0.041) and a median of adhesion very distinctive from those without stop codons. These results are the first report of PMSCs in the inlA gene of L. monocytogenes from the Federal District of Brazil and Brazil.

Molecular characterization and biofilm-formation analysis of Listeria monocytogenes, Salmonella spp., and Escherichia coli isolated from Brazilian swine slaughterhouses.

This study aimed to verify the presence of Listeria monocytogenes, Salmonella spp., and Escherichia coli in two Brazilian swine slaughterhouses, as well as to perform antibiograms, detect virulence and antimicrobial resistance genes, and evaluate the in vitro biofilm-forming capability of bacterial isolates from these environments. One Salmonella Typhi isolate and 21 E. coli isolates were detected, while L. monocytogenes was not detected. S. Typhi was isolated from the carcass cooling chamber's floor, resistant to several antimicrobials, including nalidixic acid, cefazolin, chloramphenicol, doxycycline, streptomycin, gentamicin, tetracycline, and sulfonamide, and contained resistance genes, such as tet(B), tet(C), tet(M), and ampC. It also showed moderate biofilm-forming capacity at 37°C after incubating for 72 h. The prevalence of the 21 E. coli isolates was also the highest on the carcass cooling chamber floor (three of the four samplings [75%]). The E. coli isolates were resistant to 12 of the 13 tested antimicrobials, and none showed sensitivity to chloramphenicol, an antimicrobial prohibited in animal feed since 2003 in Brazil. The resistance genes MCR-1, MCR-3, sul1, ampC, clmA, cat1, tet(A), tet(B), and blaSHV, as well as the virulence genes stx-1, hlyA, eae, tir α, tir ß, tir γ, and saa were detected in the E. coli isolates. Moreover, 5 (23.8%) and 15 (71.4%) E. coli isolates presented strong and moderate biofilm-forming capacity, respectively. In general, the biofilm-forming capacity increased after incubating for 72 h at 10°C. The biofilm-forming capacity was the lowest after incubating for 24 h at 37°C. Due to the presence of resistance and virulence genes, multi-antimicrobial resistance, and biofilm-forming capacity, the results of this study suggest a risk to the public health as these pathogens are associated with foodborne diseases, which emphasizes the hazard of resistance gene propagation in the environment.

WHO Critical Priority Escherichia coli as One Health Challenge for a Post-Pandemic Scenario: Genomic Surveillance and Analysis of Current Trends in Brazil.

The dissemination of carbapenem-resistant and third generation cephalosporin-resistant pathogens is a critical issue that is no longer restricted to hospital settings. The rapid spread of critical priority pathogens in Brazil is notably worrying, considering its continental dimension, the diversity of international trade, livestock production, and human travel. We conducted a nationwide genomic investigation under a One Health perspective that included Escherichia coli strains isolated from humans and nonhuman sources, over 45 years (1974-2019). One hundred sixty-seven genomes were analyzed extracting clinically relevant information (i.e., resistome, virulome, mobilome, sequence types [STs], and phylogenomic). The endemic status of extended-spectrum ß-lactamase (ESBL)-positive strains carrying a wide diversity of blaCTX-M variants, and the growing number of colistin-resistant isolates carrying mcr-type genes was associated with the successful expansion of international ST10, ST38, ST115, ST131, ST354, ST410, ST648, ST517, and ST711 clones; phylogenetically related and shared between human and nonhuman hosts, and polluted aquatic environments. Otherwise, carbapenem-resistant ST48, ST90, ST155, ST167, ST224, ST349, ST457, ST648, ST707, ST744, ST774, and ST2509 clones from human host harbored blaKPC-2 and blaNDM-1 genes. A broad resistome to other clinically relevant antibiotics, hazardous heavy metals, disinfectants, and pesticides was further predicted. Wide virulome associated with invasion/adherence, exotoxin and siderophore production was related to phylogroup B2. The convergence of wide resistome and virulome has contributed to the persistence and rapid spread of international high-risk clones of critical priority E. coli at the human-animal-environmental interface, which must be considered a One Health challenge for a post-pandemic scenario. IMPORTANCE A One Health approach for antimicrobial resistance must integrate whole-genome sequencing surveillance data of critical priority pathogens from human, animal and environmental sources to track hot spots and routes of transmission and developing effective prevention and control strategies. As part of the Grand Challenges Explorations: New Approaches to Characterize the Global Burden of Antimicrobial Resistance Program, we present genomic data of WHO critical priority carbapenemase-resistant, ESBL-producing, and/or colistin-resistant Escherichia coli strains isolated from humans and nonhuman sources in Brazil, a country with continental proportions and high levels of antimicrobial resistance. The present study provided evidence of epidemiological and clinical interest, highlighting that the convergence of wide virulome and resistome has contributed to the persistence and rapid spread of international high-risk clones of E. coli at the human-animal-environmental interface, which must be considered a One Health threat that requires coordinated actions to reduce its incidence in humans and nonhuman hosts.

Molecular characterization of Salmonella spp. and Listeria monocytogenes strains from biofilms in cattle and poultry slaughterhouses located in the federal District and State of Goiás, Brazil.

Listeria monocytogenes and Salmonella spp. are considered important foodborne pathogens that are commonly associated with foods of animal origin. The aim of this study was to perform molecular characterization of L. monocytogenes and Salmonella spp. isolated from biofilms of cattle and poultry slaughterhouses located in the Federal District and State of Goiás, Brazil. Fourteen L. monocytogenes isolates and one Salmonella sp. were detected in poultry slaughterhouses. No isolates were detected in cattle slaughterhouses. All L. monocytogenes isolates belonged to lineage II, and 11 different pulsotypes were detected. Pulsed-field gel electrophoresis analysis revealed the dissemination of two strains within one plant, in addition to the regional dissemination of one of them. The Salmonella isolate was identified via whole genome sequencing as Salmonella enterica serovar Minnesota ST548. In the sequence analysis, no premature stop codons were detected in the inlA gene of Listeria. All isolates demonstrated the ability to adhere to Caco-2 cells, while 50% were capable of invading them. Antimicrobial resistance was detected in 57.1% of the L. monocytogenes isolates, and resistance to sulfonamide was the most common feature. The tetC, ermB, and tetM genes were detected, and four isolates were classified as multidrug-resistant. Salmonella sp. was resistant to nine antimicrobials and was classified as multidrug-resistant. Resistance genes qnrB19, blaCMY-2, aac(6')-Iaa, sul2, and tetA, and a mutation in the parC gene were detected. The majority (78.5%) of the L. monocytogenes isolates were capable of forming biofilms after incubation at 37°C for 24 h, and 64.3% were capable of forming biofilms after incubation at 12°C for 168 h. There was no statistical difference in the biofilm-forming capacity under the different evaluated conditions. Salmonella sp. was capable of forming biofilms at both tested temperatures. Biofilm characterization was confirmed by collecting the samples consistently, at the same sampling points, and by assessing biofilm formation in vitro. These results highlight the potential risk of cross-contamination in poultry slaughterhouses and the importance of surveillance and pathogen control maintenance programs within the meat production industry.

Protein kinase C beta controls nuclear factor kappaB activation in B cells through selective regulation of the IkappaB kinase alpha.

Activation of the nuclear factor (NF)-kappaB transcription complex by signals derived from the surface expressed B cell antigen receptor controls B cell development, survival, and antigenic responses. Activation of NF-kappaB is critically dependent on serine phosphorylation of the IkappaB protein by the multi-component IkappaB kinase (IKK) containing two catalytic subunits (IKKalpha and IKKbeta) and one regulatory subunit (IKKgamma). Using mice deficient for protein kinase C beta (PKCbeta) we show an essential role of PKCbeta in the phosphorylation of IKKalpha and the subsequent activation of NF-kappaB in B cells. Defective IKKalpha phosphorylation correlates with impaired B cell antigen receptor-mediated induction of the pro-survival protein Bcl-xL. Lack of IKKalpha phosphorylation and defective NF-kappaB induction in the absence of PKCbeta explains the similarity in immunodeficiencies caused by PKCbeta or IKKalpha ablation in B cells. Furthermore, the well established functional cooperation between the protein tyrosine kinase Bruton's tyrosine kinase (Btk), which regulates the activity of NF-kappaB and PKCbeta, suggests PKCbeta as a likely serine/threonine kinase component of the Btk-dependent NF-kappaB activating signal transduction chain downstream of the BCR.

Conservation of the internal quality of eggs using a biodegradable coating.

This study aimed to evaluate the effect of a pectin biofilm on the preservation of refrigerated and unrefrigerated eggs during 5 wk of storage based on egg weight loss, albumen height, Haugh unit (HU), and the yolk index (YI). A total of 1,200 nonfertile eggs from GLK Bankiva laying hens (40 wk of age), which were freshly laid and came from a single collection, were obtained from a model poultry rearing system (Planaltina, Federal District, Brazil) that meets all animal welfare criteria. The experimental outline was entirely randomized, with 20 treatments in a factorial scheme of 2 × 2 × 5, with 2 biofilm treatments (with and without) × 2 storage temperatures (refrigeration: 5°C and ambient: 25°C) × 5 storage periods (7, 14, 21, 28, and 35 d), with 12 repetitions per treatment. Starting from the third storage week, increased weight loss (%) was observed in noncoated eggs (4.46 ± 1.06; 5.61 ± 1.37; 6.93 ± 1.66%) compared with biofilm-coated eggs (3.57 ± 1.26; 4.74 ± 1.8; 6.05 ± 2.21%), respectively. The HU variation in the pectin-coated eggs (86.84-78.02) was smaller than that in the noncoated eggs (83.01-64.36) between the beginning (7 d) and the end (35 d) of the experimental period. Eggs with and without biofilm stored in the refrigerator presented average HU values of 91.26 ± 6.27 and 88.35 ± 6.96, respectively. In contrast, when kept at room temperature, eggs with the coating presented higher HU values (71.27 ± 10.78) than eggs without the coating (59.11 ± 15.97). Coated eggs (0.37 ± 0.16) showed higher YI values than noncoated eggs (0.35 ± 0.16). A pectin-based biofilm effectively maintained egg quality during the 35 d of storage.

Effect of transportation distances, seasons and crate microclimate on broiler chicken production losses.

The goal of this research was to evaluate the microclimate (temperature, relative humidity and ECI-enthalpy comfort index) of commercial loads of broiler chickens at different transport distances: Dist15 (15 km on average) and Dist90 (90 km on average) in the summer and winter seasons and their effects on the production parameters body weight difference (BWD), mortality (%) and bruising prevalence (%). Twelve broiler loads were monitored using dataloggers to record temperature and humidity, with a total of 24 target crates per load. The experiment followed a factorial design [2 seasons (rainy and dry) × 2 distances (Dist15 and Dist90)] with a randomized complete block arrangement, 3 sexes (all males, all females, or mixed shipments) and one shipment per combination. BWD had a heterogeneous distribution throughout the load, and this distribution was not significantly correlated with the mean ECI measured during transport at 12 positions along the load. In terms of comfort, summer is the most critical period for broiler transport. In the interaction between rainy season and Dist90, the highest ECI was scored in the lethal zone (where physiological mechanisms are not enough to control body temperature). Mortality during the rainy season was not significantly different between distances. However, during the dry season, mortality was twice as high as broilers that travelled for 15 km. The prevalence of bruising on carcasses was not affected by the interaction between season and distance. As we know, broiler chicken performance, during transport, can be also related to road conditions, being hard to evaluate the real impact of seasons and distances on animal welfare. Load microclimate can compromise broiler chicken welfare during transport and it does not necessary reflect significant losses pre and post-slaughter.

Isolamento de Listeria monocytogenes em salsichas comercializadas no Distrito Federal / Isolation of Listeria monocytogenesin sausages commercialized in the Distrito Federal

A bactéria Listeria monocytogenes é o patógeno causador da listeriose, importante doença transmitida por alimentos que acomete humanos e animais podendo causar morte. Foram coletadas 100 amostras de salsicha de mercados do Distrito Federal. A metodologia usada para o isolamento foi a descrita na Instrução Normativa Nº 40 do Ministério da Agricultura, Pecuária e Abastecimento. As amostras que apresentaram motilidade característica de Listeria sp p. foram submetidas a técnica de PCR para confirmação da espécie L. monocytogenes. Os resultados positivos para Listeria sp p. foram vistos em 14 amostras e em 5 confirmou-se a presença de L. monocytogenes. Através dos resultados desse experimento conclui-se que salsichas podem estar contaminadas com bactérias do gênero Listeria sp p. e L. monocytogenes, oferecendo risco de listeriose aos consumidores.

Antimicrobial resistance of Campylobacter jejuni isolated from chicken carcasses in the Federal District, Brazil.

The aim of the present study was to perform microbiological isolation of Campylobacter jejuni from chilled chicken carcasses marketed in the Federal District of Brazil and to subject the strains to an antibiogram. A total of 92 samples from chilled chicken carcasses were acquired, 18 of which (19.56%) tested positive for C. jejuni. A total of 16 strains were tested for susceptibility to eight antimicrobial drugs. All 16 strains were resistant to ciprofloxacin, 15 strains to nalidixic acid, streptomycin, tetracycline, and gentamycin, 14 strains to amoxicillin, 11 strains to erythromycin, and 6 strains to chloramphenicol. The present study is the first to report on the presence of C. jejuni in chilled chicken carcasses marketed in the Federal District region of Brazil. These results may indicate flaws in certain steps of this food processing and highlight a possible public health problem due to the high level of resistance exhibited by the isolated strains.

Pesquisa de Clostridium perfringens em carnes bovinas embaladas a vácuo comercializadas no Distrito Federal e entorno / Clostridium perfringens research on vacuum packed beef commercialized on Distrito Federal and in the surrounding area

O objetivo deste trabalho foi detectar a presença de Clostridium perfringens em 54 amostras de carne bovina embaladas a vácuo comercializadas na região do Distrito Federal, bem como detectar a presença da toxina cpe por PCR, ainda avaliar os meios de cultivo ágar SPS® e ágar TSC®, com e sem etapa de pré-enriquecimento das amostras em caldo infusão de cérebro e coração (BHI) na câmara de anaerobiose, e posterior incubação das placas de SPS® e TSC® tanto em jarra de anaerobiose, como em câmara de anaerobiose. Na análise da incubação das placas em ágar SPS® e TSC®, sem a etapa de pré-enriquecimento em caldo BHI na câmara de anaerobiose, observou-se o crescimento em apenas uma (1,85%) das 54 amostras analisadas, em ambos os meios de cultivo e formas de incubação. Com a etapa de pré-enriquecimento com caldo BHI em câmara de anaerobiose, observou-se crescimento em todas as 54 amostras (100%), em ambos os meios de cultivo e formas de incubação. Na reação em cadeia de polimerase (PCR) nenhuma das cepas oriundas das amostras analisadas apresentaram a amplificação de fragmento do gene da toxina cpe. Os resultados evidenciam a presença de C. perfringens em carnes embaladas a vácuo comercializadas no Distrito Federal e Entorno, porém não foi detectada a toxina cpe em nenhuma cepa isolada analisada. Na comparação estatística aplicando o teste qui-quadrado de McNemar, observou-se que houve diferença significativa (p<0,001) entre as análises sem e com a etapa de pré-enriquecimento em caldo BHI, verificando-se a influencia positiva do meio na recuperação de esporos, destacando desta forma a importância do enriquecimento prévio em meio BHI e a incubação em câmara de anaerobiose, na recuperação de esporos deste microrganismo.

The aim of this work was to detect Clostridium perfringens in 54 samples of vacuum packed beef sold at the Federal District area, and to detect the presence of the cpe toxin gene by Polymerase chain reaction (PCR), also evaluate the culture medium SPS® agar and TSC® agar, with and without pre-enrichment step of the samples in Brain Heart Infusion (BHI) broth in the anaerobic chamber, and to promote the incubation of the plates in anaerobic jar and anaerobic chamber. The results of the incubation on SPS® agar and TSC® agar, without the pre-enrichment step in BHI, growth was observed in only one (1,85%) of the 54 analyzed samples, in both culture media and incubation methods. With the pre-enrichment step with BHI broth in the anaerobic chamber, growth was observed in all 54 samples (100%), in both culture media and incubation methods. At the PCR, In the polymerase chain reaction (PCR), none of the strains from the analyzed samples showed fragment amplification of the toxin cpe gene. The results showed the presence of C. perfringens in vacuum packed beef samples commercialized at the Distrito Federal area and surroundings, however the cpe toxin was not detected in any isolated strain analyzed. In the statistical test using the McNemar chi-square test, a significant difference (p<0,001) was observed between the analysis with and without pre-enrichment step in BHI broth, verifying the positive influence of the medium in spore recovery, therefore to enhance the importance of the pre-enrichment stage in BHI broth and the incubation in anaerobic chamber in spore recovery for this microorganism.