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Currently, there is a great need for the development of three-dimensional (3D) in vitro lung models. Particularly, the production of a suitable 3D model of pulmonary epithelium for understanding the pathophysiology of diseases such as the COVID-19 must consider the tissue architecture and presence, for example, of the angiotensin-converting enzyme-2 (ACE-2) in the cells. Different polymeric membranes are being used to support cell culturing, especially of lung cells, however, there is still no information about the culture of these cells onto bacterial nanocellulose (BNC) matrices. We have used the BNC matrix CellFate® as a support for the assembly of a 3D in vitro model of lung epithelium, composed of human lung fibroblasts (HLF) and lung adenocarcinoma cells (CALU-3). CellFate® matrices were made from bacterial fermentation resulting in a natural and biocompatible biopolymer. Cells were cultured onto CellFate® and maintained in a 5% CO 2 humidified atmosphere at 37°C. Cell viability was assessed by the resazurin method The samples were, then, exposed to the air-liquid interface (ALI), and histologically analyzed. ACE-2 activity was verified on the hydrolyze of the fluorogenic substrate Mca-APK(Dnp)-OH, and its presence was evaluated by flow cytometry. The expression of the anionic transporter SLCO3A1 was evaluated by qPCR. Cell viability analysis indicates that CellFate® was not toxic to these cells. By flow cytometry, the presence of the ACE-2 was identified in the CALU-3 cells surface corroborating the results obtained from enzymatic activity analysis. The SLCO3A1 transporter expression was identified in cells cultured onto CellFate®, but not in cells cultured onto the transwell (control). CALU-3 cells cultivated onto CellFate® resulted in a pseudostratified organization, a typical morphology of the human respiratory tract epithelium. The current model opens perspectives for studies involving physiological characterization, improving its relevance for the understanding of the pathophysiology of diseases as well as the response to drugs., (© 2023 Wiley Periodicals LLC.)

Palmarosa essential oil (PEO) is an alternative to synthetic fungicides to control the contamination by food-deteriorating fungi, such as Aspergillus nomius . Nonetheless, the low long-term stability and volatility hamper its utilization. Thus, this study aimed to develop nanostructured lipid carriers (NLCs) containing PEO to improve its stability and consequently prolong the activity against A. nomius . A mixture design was applied to find the best preparation conditions for antifungal activity. The characterization analyses included size measurements, zeta potential (ζ-potential), entrapment efficiency (EE), and antifungal activity (by inhibition of mycelial growth (IMG) and/or in situ test (pre-contaminated Brazil nuts) tests). The nanocarriers presented particle sizes smaller than 300 nm, homogeneous size distribution, ζ-potential of -25.19 to -41.81 mV, and EE between 73.6 and 100%. The formulations F5 and F10 showed the highest IMG value (98.75%). Based on the regression model, three optimized formulations (OFs) were tested for antifungal activity (IMG and in situ test), which showed 100% of inhibition and prevented the deterioration of Brazil nuts by A. nomius . The preliminary stability test showed the maintenance of antifungal activity and physicochemical characteristics for 90 days. These results suggest a promising system as a biofungicide against A. nomius.

Bovine tuberculosis (bTB) is a zoonotic disease caused by Mycobacterium tuberculosis var. bovis, for which the definitive diagnosis is accomplished by bacterial isolation, which has biosafety issues and requires long time. Thus, diagnostic methods with potential to be faster and more efficient can represent an advance in bTB epidemiological knowledge and decrease exposure to M. tuberculosis var. bovis. This study aimed to validate a molecular test for bTB post-mortem diagnosis, as a strategy to reduce waste in bovine production. A total of 185 tissues from animals of infected herds or with suspected lesions at abattoir were evaluated through bacterial isolation, PCR and histopathology. PCR and histopathology showed sensitivities of 45.1% and 71.2%, respectively, and specificities of 83.3% and 83.0%, respectively, when compared to bacterial isolation. The combination of both tests resulted in enhanced specificity and positive predictive values.Therefore, PCR in conjunction with histopathology may be used as screening, in which concordant results can be considered conclusive, and discordant results may be submitted to bacterial isolation.

The present study aimed to investigate the frequency of pathogenic Leptospira spp. in Brazilian bats and to determine possible risk factors associated to it. Ninety two bats of 12 species were evaluated. Whole genomic DNA from kidneys was extracted and real-time PCR specific to pathogenic Leptospira spp. was applied. Association between the frequency of specimens positive for Leptospira spp. and sex, age, bat species or family, season of collection, geographic localization and feeding habits was evaluated. The results showed that 39.13% of analyzed bats were found positive for Leptospira spp. Nine bat species had at least one positive result. There was no association among the evaluated variables and frequency of pathogenic Leptospira spp. Although the limitations due to lack of Leptospira spp. isolation, leptospiral carriage was demonstrated in bats of different species from southern Brazil, which reinforces the need for surveillance of infectious agents in wild animals., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Foodborne diseases are a public health problem worldwide. The consumption of contaminated raw milk has been recognized as a major cause of transmission of bovine tuberculosis to humans. Other mycobacteria that may be present in raw milk and may cause diseases are those belonging to the Mycobacterium avium complex. In this study, molecular biology tools were applied to investigate raw milk contamination with Mycobacterium spp. in family dairy farms from Rio Grande do Sul, southern Brazil. Furthermore, different variables related to the source of the milk, herd characteristics, and management were evaluated for their effect on milk contamination. Five hundred and two samples were analyzed, of which 354 were from the Northwest region (102 farms with samples from 93 bulk tanks and 261 animals) and 148 from the South region of the state (22 farms with samples from 23 bulk tanks and 125 animals). Among them, 10 (1.99%) and 7 (1.39%) were positive for Mycobacterium tuberculosis (9 confirmed as Mycobacterium bovis) and M. avium complexes, respectively. There was no difference in the frequencies of positive samples between the regions or the sample sources. Of the positive samples, 4 were collected from a bulk tank (1 positive for M. avium and 3 for M. tuberculosis). Moreover, 1 sample was positive concomitantly for M. tuberculosis and M. avium complexes. On risk analysis, no variable was associated with raw milk contamination by M. tuberculosis complex species. However, washing the udders of all animals and drying them with paper towels were weakly classified as risk factors for M. avium contamination. Positive samples were obtained from both animals and bulk tanks, which emphasizes the importance of tuberculosis control programs and provides evidence that milk monitoring can be used as a control practice. Moreover, the findings of this study reinforce the need for awareness of the problems of raw milk consumption among the general population.

This study aimed at evaluating the bacteriological effects of the treatment of sheep meat contaminated with total coliforms, coliforms at 45 °C and Salmonella spp. by using irradiation at doses of 3 kGy and 5 kGy. Thirty sheep meat samples were collected from animals located in Rio de Janeiro State, Brazil, and then grouped in three lots including 10 samples: non-irradiated (control); irradiated with 3 kGy; and irradiated with 5 kGy. Exposure to gamma radiation in a (137)Cs source-driven irradiating facility was performed at the Nuclear Defense Section of the Brazilian Army Technological Center (CTEx) in Rio de Janeiro. The samples were kept under freezing temperature (-18 °C) until the analyses, which occurred in two and four months after irradiation. The results were interpreted by comparison with the standards of the current legislation and demonstrated that non-irradiated samples were outside the parameters established by law for all groups of bacteria studied. Gamma irradiation was effective in inactivating those microorganisms at both doses tested and the optimal dose was achieved at 3 kGy. The results have shown not only the need for sanitary conditions improvements in slaughter and processing of sheep meat but also the irradiation effectiveness to eliminate coliform bacteria and Salmonella spp.

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.

To investigate antimicrobial resistance, 96 Salmonella enterica subspecies enterica serovar Enteritidis strains isolated from salmonellosis outbreaks and poultry-related products obtained in southern Brazil were analyzed. Macrorestriction patterns, obtained by pulsed-field gel electrophoresis and phage types, were assessed. Although 43.75% of samples were sensitive to all drugs tested, resistance to sulfonamide (34.37%), trimethoprim-sulfamethoxazole (25.00%), nalidixic acid (14.58%), streptomycin (2.08%), gentamicin, and tetracycline (1.04%) was identified. Furthermore, 89.60% of strains belonged to phage type 4, and a predominant pulsed-field gel electrophoresis genotype represented by 82.29% of the strains was identified, suggesting that a clonal group was distributed in poultry, food, and human isolates. Although it was not possible to associate strains from different sources, the occurrence of antimicrobial-resistant Salmonella Enteritidis strains supports the need to establish monitoring programs to identify the emergence of potential resistance patterns and to direct policies for use of these drugs in food-producing animals.

Objectives: To determine the occurrence of antimicrobial resistance genes and role of integrons among 135 antimicrobial-resistant Salmonella enterica from Brazil., Methods: The presence of antimicrobial resistance genes, class 1 and 2 integrons and gene cassettes was analysed by PCR and sequencing. The genetic location of class 1 integrons was determined in 25 isolates by hybridization and plasmid transfer experiments., Results: Fifty-five of the isolates were positive for class 1 integrons. Integron-positive isolates represented 17 different serovars and were mainly from human (n=28) and animal (n=13) sources. The gene cassette arrangements could be determined in 51 of the positive isolates, which harboured one [dfrA22, aadA1 or orf3 (putative trimethoprim resistance)], two [aadA1-dfrA1, aac(6')-Ib-orf1 (unknown function) or aacA4-aadA1], three [dfrA15b-cmlA4-aadA2, orf2 (unknown function)-dfrA5-orfD] or four [orf4-aacA4-blaOXA-30 (interrupted by an IS1 element)-aadA1] cassettes in their variable region. Only one isolate harboured a class 2 integron with the gene cassette array dfrA1-sat-aadA1. Several integron unrelated resistance genes were also detected in the isolates. Sulphonamide resistance was primarily mediated by sul2 and sul3, tetracycline resistance by tet(B) and tet(A), chloramphenicol resistance by catA1, streptomycin resistance by strA and ampicillin resistance by blaTEM. blaCTX and blaCMY-2 were found in cephalosporin-resistant isolates. Mating and hybridization experiments demonstrated that a high-molecular-weight plasmid mediated the gene transfer of integrons and additional resistance determinants., Conclusions: The present study revealed that integron-mediated resistance genes contributed to the multiresistance phenotype observed in the isolates, but most resistance genes were located outside the integron structure, as independent genes. However, they might be located on the same conjugative plasmid.

272 isolates of Salmonella Enteritidis (111 isolated from frozen broiler chicken carcasses, 126 from human food and other biological materials involved in food poisoning outbreaks and 35 from different poultry materials) were selected for phage typing. From these, 111 were phage typed, 57.65% being classified as phage type 4, 32.43% as phage type 4a, 3.60% as phage type 6a and 0.90% as phage type 7, whereas 5.40% samples were not phage typeable. The predominance of phage type 4 is in agreement with the results published worldwide, and reinforces the need for studies related to the epidemiological meaning of these findings.

Of 7058 Vibrio cholerae strains recovered from patients suspected of cholera in the State of Ceará between December 1991 and September 1993, two were resistant to antimicrobials (Ampicillin, erythromycin, trimethoprim-sulfamethoxazole, tetracycline) and to vibriostatic agent O/129 (2,4-diamino-6,7-diisopropylpteridine). From the bacteriological standpoint, one strain was identified as V. cholerae serogroup O:1, biotype El Tor, serovar Inaba, and another as V. cholerae serogroup O:22, biochemically classified as Heiberg type II. It was shown that only in the serogroup O:1 strain, multiple resistance was encoded by a plasmid transferrable by conjugation to Escherichia coli K12 and a sensitive strains of V. cholerae O1 and non-O1, with at a frequency between 8 x 10(-2) and 5 x 10(-6). The plasmid, with a molecular weight of 147 Kb, encoded both multiple resistance to antimicrobials and the vibriostatic compound (O/129), compatible with descriptions reported in other parts of world.

The lysotypes, plasmidial profiles, and profiles of resistance to antimicrobial agents were determined in 111 Salmonella Typhimurium strains isolated from feces and blood of children treated in Rio de Janeiro and in Salvador. Six distinct lysotypes (19, 41, 97, 105, 120 and 193) were recognized, with a predominance of lysotype 193 (59.7%) in Rio de Janeiro and of phage type 105 (38.4) in Salvador. Approximately 86.7% of the lysotype 193 strains presented multiple resistance to more than six antimicrobial agents, whereas 93% of lysotype 105 strains were fully susceptible. More than 90% of the strains presented plasmids distributed into 36 different profiles in Rio de Janeiro and into 10 profiles in Salvador. A 40 MDa plasmid was the most frequent (47%) in the strains from Rio de Janeiro, whereas a 61 MDa plasmid predominated (14.5%) in Salvador. Combined analysis of plasmid profile and classification into lysotypes (especially those belonging to types 105 and 103, proved to be more discriminatory than the other methods applied).

The Salmonella serovars involved in 25 food poisoning episodes which occurred in the Southeast and South of Brazil from 1982 to 1991 were identified. The most frequently detected serotype was S. Typhimurium (13/25, 52%), and the food most frequently involved in the transmission of Salmonella was homemade mayonnaise. The need to set up a permanent program of epidemiologic alert for food poisoning is emphasized.

The in vitro susceptibilities of 703 clinical isolates of Salmonella to ciprofloxacin (CIP) and pefloxacin (PEF) were compared with those to trimethoprim-sulfamethoxazole (TMP-SMZ), chloramphenicol (CO) and ampicillin (AP). All isolates were susceptible to CIP, while PEF inhibited 90.7% of the strains. In contrast, resistance rates of 40, 29.2 and 27% were detected for AP, TMP-SMZ and CO, respectively. PEF resistance was detected in S. panama (1), S. typhi (3) and S. typhimurium (17), the latter representing the most frequently serovar isolated in our country. None of the S. typhi isolates was resistant to CO. Combined resistance was most frequently found among S. typhimurium isolates, with the patterns PEF-TMP-SMZ-AP (10) and PEF-TMP-SMZ-CO-AP (5) predominating.

The object of the investigation was the evaluation of the susceptibility to antibiotic and chemotherapeutic agents of 240 strains of Salmonella agona isolated from different sources (human, food and environment) obtained from five Brazilian states (Minas Gerais, São Paulo, Rio de Janeiro, Pernambuco and Rio Grande do Sul). The presence of R factors in 26 representative strains of the sample was also determined.

With the purpose of characterizing epidemiologic markers, 240 strains of S. agona isolated from differents sources (man, food and environment) and obtained from five Brazilian States (Minas Gerais, São Paulo, Rio de Janeiro, Pernambuco and Rio Grande do Sul) were analysed. The susceptibility to 15 antimicrobial agents and numeric codification of the resistance profiles allowed us to recognize 56 antibiotic resistance biotypes, while 40 strains were able to produce colicine, belonging to the types: Ia (55%); B (32.5%); Ib (10%) and untypable (2.5%). The application of these elements into intra-serotype differentiation is discussed.

326 samples of diarrheal feces obtained from children whose ages ranged from zero to 5 years, admitted in two rehydration hospitals in the city of Recife, Pernambuco, were analyzed. Feces were placed in Cary-Blair medium (4 degrees C) for shipment to the laboratory. There was no difference in the rate of bacteria isolation if the samples were analyzed within the period from 3 to 7 days of collection. 19.02% of the analyzed samples were positives for at least one of the searched bacteria, 26 Salmonella belonging to 3 species, 21 classic enteropathogenic E. coli, 1 invasive E. coli, 10 Shigella belonging to 3 serotypes and 1 Yersinia enterocolitica were found.