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ABSTRACT: Objectives: The draft genome sequence of Campylobacter jejuni (Cj26) was analysed to investigate genetic mechanisms of antimicrobial resistance, virulence-associated genes, and phylogenetic context. Methods: Antimicrobial resistance was assessed by agar dilution and disk diffusion. Cj26 was sequenced using NovaSeq 6000 technology. The genome was assembled and annotated. Resistance genes and chromosomal mutations were analysed using the Center for Genomic Epidemiology services, and the multilocus sequence type, SVR-flaA, and porA were determined. The virulome was determined using the Virulence Factor Database. Plasmid detection and assembly were performed using Unicycler v0.5.0 software. To infer the core genome phylogeny, prokka v1.14.5 was employed in conjunction with IQtree v2.0.3. Results: The Cj26 strain showed a high level of resistance to ciprofloxacin (32 µg/mL) and erythromycin (>128 µg/mL) and resistance to tetracycline and ampicillin. Multilocus sequence typing revealed that the strain belonged to sequence type 353. The substitutions Tre-86-Ile in gyrA and A2075G in 23s RNA were detected, along with the genes tetO, aph(3′)-III, ant(6)-Ia, and blaOXA 460. A consistent relationship among accessory and core genes was identified. When compared to other sequence type 353 genomes from Brazil, Cj26 clustered with strains that had more antimicrobial resistance genes than the other clusters. Conclusion: This report provides insight into the antimicrobial resistance determinants found in a C. jejuni strain and offers a valuable resource for further studies on Campylobacter genomics and antimicrobial resistance.

Campylobacter jejuni is the most frequent cause of bacterial gastroenteritis; therefore, the characteristics of its epidemiology must be continuously investigated to support possible mitigating measures. This is particularly important when evaluating representative strains from the world's leading chicken meat exporter, Brazil. We evaluated a panel of 14 virulence genes in 359 strains of C. jejuni isolated from chilled broiler carcasses in Brazil. The genes were classified into five virulence categories (B: biofilm/motility; SS: secretion/cytotoxicity system; CI: invasion/colonization; GB: Guillain-Barré; and AE: adaptation to stress). The percentage of strains with stress adaptation genes (86.07%) indicates the ability to survive in unfavorable environments; in addition, the strains showed a risk of causing infections in humans due to the frequency of the hcp gene (97.77%). Genes related to Guillain-Barre syndrome (GBS) in 77.44% of strains are an additional concern, which must be monitored. The gene panel showed the presence of 124 virulence profiles. Individual analyses by carcass, slaughter establishment, and municipalities in which they were located showed high index variabilities (I.Var.) of 0.82, 0.87, and 0.78, respectively. Georeferencing indicated the state of Paraná as a hotspot for virulent strains. Higher levels of isolation and multi-virulence were identified in the summer, which is hot and humid in Brazil. Together, our results showed that the studied strains are a potential danger to public health and that there is an urgent need for their surveillance and the adoption of control measures, especially in the state of Paraná.

Despite being considered fragile and fastidious, Campylobacter jejuni is the most prevalent cause of foodborne bacterial gastroenteritis, and chicken meat is considered the main vehicle of transmission to humans. This agent can survive adverse conditions in the form of biofilms, but extreme stress (nutritional, oxidative and thermal) promotes the acquisition of a state called viable but not culturable (VBNC). The emergence of this pathogen worldwide and the recent international requirements in its control instigated us to qualitatively and quantitatively estimate the time required for the acquisition of the VBNC form in 27 strains of C. jejuni, characterize morphological aspects, determine its adaptive and invasive potential and perform comparative metabolomic evaluation. Extreme stress promoted the complete acquisition of the VBNC form in a mean time of 26 days. Starting from an average initial count of 7.8 log CFU/mL, the first four days determined the greatest average reduction of the culturable form of 3.2 log CFU/mL. The scanning and transmission image analyses showed a transition from the typical viable form (VT) to the VBNC form, with initial acquisition of the straight rod shape, followed by loss of the flagella and subdivision into two to 11 imperfect cocci arranged in a chain and rich in cellular content, until their individual release. RT-PCR identified the presence of ciaB and p19 transcripts in the 27 cultivable C. jejuni strains, a character maintained in the VBNC form only for p19 and in 59.3% (16/27) of the VBNC strains for the ciaB gene. The average inoculation of 1.8 log CFU/mL of C. jejuni VBNC into primary chicken embryo hepatocyte cells promoted the occurrence of apoptosis processes significantly after 24 hours of contact by one of the strains tested. In C. jejuni VBNC, we detected higher expression of metabolites linked to protective and adaptation mechanisms and of volatile organic precursor compounds indicative of metabolism interruption. The oscillations in the time of acquisition of the VBNC form together with the presence of transcripts for ciaB and p19, the identification of cell lysis and metabolites that ensure the maintenance of the pathogen alert to the fact that C. jejuni VBNC remains virulent and adapted to stress, which makes evident the potential danger of this latent form, which is not detectable by official methodologies.

Horizontal gene transfer (HGT) in food matrices has been investigated under conditions that favor gene exchange. However, the major challenge lies in determining the specific conditions pertaining to the adapted microbial pairs associated with the food matrix. HGT is primarily responsible for enhancing the microbial repertoire for the evolution and spread of antimicrobial resistance and is a major target for controlling pathogens of public health concern in food ecosystems. In this study, we investigated Salmonella Heidelberg (SH) and Escherichia coli (EC) regarding gene exchange under conditions mimicking the industrial environment, with the coproducts whey (SL) and chicken juice (CJ). The S. Heidelberg strain was characterized by antibiotic susceptibility standards and PCR to detect the blaTEM gene. A concentration of 0.39 mg/mL was determined to evaluate the anti-conjugation activity of nanostructured lipid nanocarriers (NLCs) of essential oils to mitigate β-lactam resistance gene transfer. The results showed that the addition of these coproducts promoted an increase of more than 3.5 (whey) and 2.5 (chicken juice) orders of magnitude in the conjugation process (p < 0.01), and NLCs of sage essential oil significantly reduced the conjugation frequency (CF) by 74.90, 90.6, and 124.4 times when compared to the transfers in the absence of coproducts and the presence of SL and CJ, respectively. For NLCs from olibanum essential oil, the decrease was 4.46-fold for conjugations without inhibitors and 3.12- and 11.3-fold in the presence of SL and CJ. NLCs associated with sage and olibanum essential oils effectively control the transfer of antibiotic resistance genes and are a promising alternative for use at industrial levels.

This work describes the preparation, characterization and antimicrobial activity of four palladium(II) complexes, namely, [Pd(meg)(1,10-phen)] 1, [Pd(meg)(PPh3)2] 2, [Pd(og)(1,10-phen)] 3 and [Pd(og)(PPh3)2] 4, where meg = methyl gallate, og = octyl gallate, 1,10-phen = 1,10-phenanthroline and PPh3 = triphenylphosphine. As to the chemical structures, spectral and physicochemical studies of 1–4 indicated that methyl or octyl gallate coordinates a palladium(II) ion through two oxygen atoms upon deprotonation. A chelating bidentate phenanthroline or two triphenylphosphine molecules complete the coordination sphere of palladium(II) ion, depending on the complex. The metal complexes were tested against the Mycobacterium tuberculosis H37Rv strain and 2 exhibited high activity (MIC = 3.28 μg/mL). As to the tests with Campylobacter jejuni, complex 1 showed a significant effect in reducing bacterial population (greater than 7 log CFU) in planktonic forms, as well as in the biomass intensity (IBF: 0.87) when compared to peracetic acid (IBF: 1.11) at a concentration of 400 μg/mL. The effect provided by these complexes has specificity according to the target microorganism and represent a promising alternative for the control of microorganisms of public health importance.

ABSTRACT: Salmonella Infantis is frequently associated with human infections worldwide and is transmitted by consumption of contaminated foods, particularly those of animal origin, especially the chicken meat. We aimed to evaluate virulence characteristics, antimicrobial resistance and the genetic similarity of 51 strains of S. Infantis isolated from samples of poultry origin. The strains were isolated from 2009 to 2010 in a company with full cycle of broiler’s production in the state of São Paulo, Brazil. The antimicrobial susceptibility test was performed and, by PCR, we evaluated the presence of the genes lpfA (hem-adhesion), agfA (hem-biofilm) and sefA (hem-adhesion) and resistance genes to beta-lactams (blaTEM, blaSHV, bla CTX-M and blaAmpC ). The phylogenetic relationship was determined by RAPD-PCR method. Among the drugs tested, the highest percentages of resistance were to amoxicillin (35.3%) and to sulfonamide (15.7%). Eleven antimicrobial resistance patterns were identified (A1 to A11), none of them presented a multiresistance profile (> 3 antimicrobials classes). There was 100% of positivity for the agfA gene, 92.2% for the lpfA gene, and no strain presented the sefA gene. Most of the isolates showed similarities in virulence potential, since they were simultaneously positive for two studied genes, agfA and lpfA (92.2%, 47/51). Of the 18 (35.3%) strains resistant to antimicrobials of the β-lactam class, 10 (55.5%) were positive to blaAmpC gene, five (27.8%) for blaCTX-M , two (11.1%) to blaSHV and no strain presented the blaTEM gene. The phylogenetic evaluation has shown the presence of five clusters (A, B, C, D and E) with similarity greater than 80%, and three distinct strains which were not grouped in any cluster. Cluster B grouped 33 strains, all positive for lpfA and agfA genes, from both, the broiler farming facility and the slaughterhouse, persistent throughout all the study period. This cluster also grouped 18 strains clones with genetic similarity greater than 99%, all isolated in the slaughterhouse. The presence of virulence genes associated with persistent strains clones for a long period, warns to the possibility of S. Infantis to form biofilm, and should be constantly monitored in broilers’ production chain, in order to know the profile of the strains that may contaminate the final product and evaluate the hazards that represents to public health.

This work describes the synthesis and characterization of two metal complexes of the type [M(L1)2(phen)], where M = Pt2+ (complex I) or Pd2+ (complex II), L1 = 5-amino-1,3,4-thiadiazole-2(3H)-thiolate and phen = 1,10-phenanthroline. The in vitro antibacterial activity of these complexes was investigated in isolation and synergistically with ciprofloxacin (CIP) and erythromycin (ERY) in three strains of Campylobacter jejuni (MIC = 32 mg/L for CIP and ERY), selected from a bank of 235 strains representative of three poultry exporting states of the country (A, B and C), previously analyzed for epidemiology and resistance to CIP and ERY. A total of 53/235 (22.55%) strains showed co-resistance to CIP and ERY. Isolated resistance to CIP was higher than to ERY. Epidemiological analysis showed that resistance to CIP was more evident in state B (p < 0.0001), as well as a higher susceptibility to ERY in state C (p = 0.0028). Co-resistance was expressive in state A and in the spring and fall seasons. The evaluation of I alone and in synergy with CIP and ERY found values up to 0.25 mg/L not significant for ERY. Complex II did not show an antimicrobial effect on the three strains of tested C. jejuni. The effect provided by complex I represents a promising alternative for control of resistant strains of C. jejuni.

ABSTRACT: The aim was to determine the spread of genetically similar profiles of Campylobacter in chicken carcasses and evaluate their ability to produce transcripts for ciaB, dnaJ, p19 and sodB genes, before and after cultivation in Caco-2 cells. The strains used were isolated from 420 samples of chicken carcasses chilled and frozen ready for marketing. The species were identified by PCR-multiplex, the phylogeny was determined by RAPD-PCR and the presence of transcripts was performed by RT-PCR. We identified 74 (17.6%) of Campylobacter strains, being 55 (74.3%) C. jejuni and 19 (25.7%) C. coli. The phylogenetic relationship demonstrated heterogeneity between isolates of the same species, with absence of clones, indicating the high level of diversity of circulating genotypes. The gene transcription showed conflicting results before and after the culture in Caco-2 cell, so that before cultivation isolates showed greater capacity to transcribe genes related to survival and after the interaction with human cells, the strains showed higher potential to transcribe genes associated with virulence. The result of this study contributes to the understanding of how these seemingly fragile microorganisms are the most prevalent bacterial agents in human gastroenteritis.

The aim of the study was to evaluate the genotypic and phenotypic characteristics of 20 strains of S. Heidelberg (SH) isolated from broilers produced in southern Brazil. The similarity and presence of genetic determinants linked to virulence, antimicrobial resistance, biofilm formation, and in silico-predicted metabolic interactions revealed this serovar as a threat to public health. The presence of the ompC, invA, sodC, avrA, lpfA, and agfA genes was detected in 100% of the strains and the luxS gene in 70% of them. None of the strains carries the blaSHV, mcr-1, qnrA, qnrB, and qnrS genes. All strains showed a multidrug-resistant profile to at least three non-β-lactam drugs, which include colistin, sulfamethoxazole, and tetracycline. Resistance to penicillin, ceftriaxone (90%), meropenem (25%), and cefoxitin (25%) were associated with the presence of blaCTX–M and blaCMY–2 genes. Biofilm formation reached a mature stage at 25 and 37°C, especially with chicken juice (CJ) addition. The sodium hypochlorite 1% was the least efficient in controlling the sessile cells. Genomic analysis of two strains identified more than 100 virulence genes and the presence of resistance to 24 classes of antibiotics correlated to phenotypic tests. Protein-protein interaction (PPI) prediction shows two metabolic pathways correlation with biofilm formation. Virulence, resistance, and biofilm determinants must be constant monitoring in SH, due to the possibility of occurring infections extremely difficult to cure and due risk of the maintenance of the bacterium in production environments.

Campylobacter jejuni is the main pathogen identified in cases of foodborne gastroenteritis worldwide. Its importance in poultry production and public health is highlighted due to the growing antimicrobial resistance. Our study comparatively investigated the effect of five different classes of antimicrobials on the planktonic and biofilm forms of 35 strains of C. jejuni with high phylogenetic distinction in 30 of them. In the planktonic form, the existence of susceptible strains to colistin (7/35 – 20%) and resistance to meropenem (3/35 – 8.6%) represent a novelty in strains evaluated in Brazil. In biofilms formed with the addition of chicken juice, the number of resistant strains was significantly higher for colistin, erythromycin and meropenem (100%), but the susceptibility to tetracycline was shown as a control strategy for specific cases. High concentrations (1,060 ± 172.1mg/L) of antibiotics were necessary to control the biofilm structure in susceptible strains in the planktonic form, which is consistent with the high biomass produced in these strains. Stainless steel and polyurethane were the most (BFI=2.1) and least (BFI=1.6) favorable surfaces for the production of biomass treated with antimicrobials. It is concluded that the antimicrobial action was detected for all tested drugs in planktonic form. In sessile forms, the biomass production was intensified, except for tetracycline, which showed an antibiofilm effect.

The use of essential oils (EO) loaded with nanoparticles is the most promising alternative to increase food quality and safety. Interesting works describe the antimicrobial properties of EO for pathogen control in natural and processed foods for human health and animal production, also contributing to sustainability. Their association with different nanosystems allows novel developments in the micronutrition, health promotion, and pathogen control fields, preventing the aggravation of bacterial microevolution and combating antibiotic resistance. Benefits to the environment are also provided, as they are biodegradable and biocompatible. However, such compounds have some physicochemical properties that prevent commercial use. This review focuses on recent developments in antimicrobial EO-based nanoparticles and their application in different food matrices.

Campylobacter jejuni (CJ) is the most prevalent zoonotic pathogen of chicken meat and related products, which may lead to gastroenteritis and autoimmune diseases in humans. Although controlling this bacterium is important, CJ strains resistance against traditional antibiotic therapy has been increased. Vegetable oils and fats are natural biomaterials explored since the Ancient times, due to their therapeutic properties. Nanotechnology has promoted the miniaturization of materials, improving bioavailability and efficacy, while reducing the toxicity of loaded active molecules. In this work, a screening of 28 vegetable oils was firstly performed, in order to select anti-CJ candidates by the disc diffusion test. Thus, the selected liquid lipids were used as active molecules in nanostructured lipid carriers (NLC) formulations. The three resultant systems were characterized in terms of particle size (~200 nm), polydispersity index (~0.15), and zeta potential (~-35mV), and its physicochemical stability was confirmed for a year, at 25°C. The structural properties of NLC were assessed by infrared (FTIR-ATR) and differential scanning calorimetry (DSC) analyses. The spherical nanoparticle morphology and narrow size distribution was observed by transmission electron microscopy (TEM) and field emission scanning electron (FE-SEM) analyses, respectively. Then, the in vitro antimicrobial activity test determined the minimum inhibitory concentration (MIC) of each formulation against CJ strains, in both free (1–3 mg/ml−1) and sessile (0.78 mg/ml−1) forms. Finally, the in vitro biocompatibility of NLC was demonstrated through cell viability using VERO cell line, in which F6 was found twice less cytotoxic than pure olibanum oil. Considering the abovementioned achieved, F6 formulation is able to be evaluated in the in vivo anti-CJ efficacy assays.

Campylobacter spp. is an emerging pathogen that causes gastroenteritis in humans and the consumption of dairy food can characterize sources of infection. We aimed to verify the viability and a presence of transcripts associated with characteristics of virulence and adaptation of C. jejuni isolated from Minas Frescal cheeses, produced with contaminated milk and stored under refrigeration for up to ten days. The samples were analyzed for bioindicators, Campylobacter spp., pH, acidity, moisture and sodium chloride. Campylobacter spp. recovered were evaluated for the production of transcripts of: ciaB, dnaJ, p19 and sodB. The results were correlated with the viability of C. jejuni and changes in their transcriptome. Storage at low temperatures reduced C. jejuni from the first to the fourth day. The variations in humidity, pH and acidity influenced the decreasing of C. jejuni. There was a reduction in transcripts' production of the four genes, more pronounced on the fourth day, indicating the inability of the microorganism to perform its metabolic activities, due to the conditions of injury. Despite the presence of mechanisms of virulence and adaptation, C. jejuni could not remain viable four days after production. However, consumption of fresh cheese contaminated with Campylobacter jejuni can be a source of infection when consumed up to four days after production.

Salmonella spp. continues to figure prominently in world epidemiological registries as one of the leading causes of bacterial foodborne disease. We characterised 43 Brazilian lineages of Salmonella Typhimurium (ST) strains, characterized drug resistance patterns, tested copper (II) complex as control options, and proposed effective antimicrobial measures. The minimum inhibitory concentration was evaluated for seven antimicrobials, isolated and combined with the copper (II) complex [Cu(4-FH)(phen)(ClO4)2] (4-FH = 4-fluorophenoxyacetic acid hydrazide and phen = 1,10-phenanthroline), known as DRI-12, in planktonic and sessile ST. In parallel, 42 resistance genes were screened (PCR/microarray). All strains were multidrug resistant (MDR). Resistance to carbapenems and polymyxins (86 and 88%, respectively) have drawn attention to the emergence of the problem in Brazil, and resistance is observed also to CIP and CFT (42 and 67%, respectively), the drugs of choice in treatment. Resistance to beta-lactams was associated with the genes blaTEM/blaCTX-M in 39% of the strains. Lower concentrations of DRI-12 (62.7 mg/L, or 100 μM) controlled planktonic and sessile ST in relation to AMP/SUL/TET and AMP/SUL/TET/COL, respectively. The synergistic effect provided by DRI-12 was significant for COL/CFT and COL/AMP in planktonic and sessile ST, respectively, and represents promising alternatives for the control of MDR ST.

We aimed to investigate the occurrence, phylogeny, and virulence of E. coli in the uterine contents and urine of female dogs with pyometra, through the presence of virulence genes and their genetic similarity. Uterine secretions and urine samples from 52 female dogs with pyometra were collected and cultured. Strains identified as E. coli from 25 uterine and 7 urine samples were tested for virulence genes by PCR. Genetic similarity between the isolates was studied using RAPD-PCR. E. coli was observed in 48.07% uterine samples with pyometra and 20.0% urine samples. The strains showed high percentages for the presence of virulence genes: 96.9% had the gene sfa, 59.4% afa, 46.9% pap, 53.1% hly, and 68.75% cnf. Even with the high prevalence of virulence genes, the samples were not submitted to DNA sequencing to confirm the results. Analysis showed high genetic diversity in E. coli, however, strains isolated from the same animal indicate that cystitis and pyometra could be related. Our study indicated the association between E. coli in dogs with pyometra and cases of urinary tract infection and the pathogenic potential of strains increasing with animal age.

Objetivou-se descrever as condições higiênico-sanitárias de queijarias artesanais, determinar os parâmetros físico-químicos, bacteriológicos e o período mínimo de maturação que garanta a inocuidade de queijos minas artesanais produzidos em Uberlândia-MG. Foram analisados, até aos 26 dias de maturação, queijos de duas propriedades, antes e após treinamento em boas práticas, totalizando 10 lotes (fabricações) com 12 queijos cada. Foram realizadas análises físico-químicas e bacteriológicas nos queijos e análises bacteriológicas em amostras ambientais. Até 26 dias de maturação, o queijo foi classificado como gordo e de baixa umidade, mas, considerando a maturação até 10 dias (período de comercialização usual), são gordos com média umidade. O tempo de maturação influenciou nos índices físico-químicos, com exceção da gordura no extrato seco. Análises bacteriológicas ambientais demonstraram higiene deficiente e falhas ou ausência no tratamento da água. A maturação e o treinamento influenciaram na redução de coliformes totais e E. coli, mas não de Staphylococcus coagulase positiva, não sendo possível determinar um período de maturação mínimo que garantisse o atendimento à legislação. As análises físico-químicas demonstraram variações esperadas, como a redução do percentual de umidade, e as análises bacteriológicas refletiram o treinamento aplicado aos manipuladores, já que houve redução nas contagens de coliformes totais e E. coli. Contudo, para garantir a inocuidade do produto, é necessário um conjunto de ações que incluem reforço e repetição dos treinamentos, registro das atividades, rastreabilidade, associadas ao monitoramento e fiscalização regular.Palavras-chave: Maturação; Parâmetros físico-químicos; Qualidade bacteriológica; Queijaria

Este estudo avaliou a viabilidade e a produção de transcritos dos genes sodB, p19, ciaB e dnaJ em cepas de Campylobacter jejuni ATCC 33291, NCTC 11351 e 2383 IAL armazenadas em leite integral UHT e / ou neopeptona + 12% glicerol, submetidas ou não a pré-tratamentos a 4°C ou 10°C durante 30 minutos. As análises foram realizadas imediatamente após o congelamento em nitrogênio líquido (dia 0) e após a manutenção por 30, 60 e 90 dias à -20ºC. A viabilidade foi avaliada pelo método tradicional de cultura e a produção de transcritos, pela técnica de RT-PCR. A quantificação só foi possível no primeiro dia de análise (dia 0) e apresentou uma média de 3,0 x 107 UFC, e nos demais períodos de armazenamento as cepas apresentaram crescimento confluente, não permitindo sua enumeração. Os resultados indicaram que o leite integral UHT foi mais adequado para criopreservação em relação ao uso de neopeptona + 12% glicerol. A utilização de pré-tratamentos combinados com o uso de leite UHT como meio crioprotetor estabilizou as células para transcrever os genes ciaB, dnaJ, sodB e p19 nas cepas mantidas abaixo de -20° por 30 a 60 dias, indicando que são métodos mais adequados para a manutenção de cepas em laboratório.

This study simulated the contamination of two varieties of infant milk formulas (homemade and commercial) with 103 and 104 CFU/mL of Campylobacter jejuni, that were kept under refrigeration (4-7ºC) for up to 48 hours. The aim of this study was to verify the maintenance of the viability and ability of Campylobacter jejuni to produce transcripts of virulence and resistance to stress conditions during periods of 0 (after preparation), 24 and 48 hours. C. jejuni remained viable during all analyzed stages and the presence of coliforms was not detected. In general, the counts reduced 1 log cycle after 48 hours for all samples, except the 104 CFU/mL inoculum of commercial formula, which reduced 2 log cycles, indicating greater injury of C. jejuni in this food matrix. C. jejuni showed to be more adapted to homemade matrix, due to high transcription of the gene related to cell invasion, ciaB, and more susceptible in the commercial matrix, due to the high transcription of genes related to conditions of stress tolerance (dnaJ, p19, sodB). The low infective dose of C. jejuni coupled with greater vulnerability of children less than five years indicate the need for care in the preparation and maintenance of infant formulas, to prevent the use of contaminated raw material and cross-contamination, especially in homemade formulations.

Salmonella spp. is an important foodborne agent of salmonellosis, whose sources in humans often include products of avian origin. The control of this bacterium is difficult especially when Salmonella spp. is organized into biofilms. We hypothesized that the novel nanocomposites of ZnO nanocrystals doped with silver (Ag) and silver oxide (AgO) nanocrystals (ZnO:Ag-AgO) synthesized by the coprecipitation method could control or prevent the formation of Salmonella Enteritidis (SE) and Salmonella Heidelberg (SH) biofilm and its entry into turkey eggs. The diffraction characteristics of ZnO and AgO showed sizes of 28 and 30 nm, respectively. The Zn to Ag substitution into the ZnO crystalline structure was evidenced by the ionic radius of Ag+2 (1.26 Å), which is greater than Zn+2 (0.74 Å). For the SE analyses post-biofilm formation, the ZnO:Ag-AgO was not able to eliminate the biofilm, but the bacterial load was lower than that of the control group. Additionally, SE was able to infiltrate into the eggs and was found in both albumen and yolk. For the SH analyses applied onto the eggshells before biofilm formation, the ZnO:Ag-AgO treatment prevented biofilm formation, and although the bacterium infiltration into the eggs was observed in all treated groups, it was significantly smaller in ZnO:Ag-AgO pre-treated eggs, and SH could not reach the yolk. There was no difference in pore size between groups; therefore, the inhibition of biofilm formation and the prevention of bacterium entry into the egg were attributable to the use of ZnO:Ag-AgO, which was not influenced by the egg structure. Although the amount of Ag and Zn in the shell of the ZnO:Ag-AgO group was greater in relation to the control, this difference was not detected in the other egg components. In the search for new measures that are effective, safe and viable for controlling microorganisms in poultry farming, the application of a nanocomposite of Ag-doped ZnO and AgO nanocrystals appears as an alternative of great potential to prevent Salmonella sp biofilms in eggshells and other surfaces.

The presence of virulence genes, phylogenetic relationships, biofilm formation index (BFI), and ultrastructure in S. Minnesota at different temperatures (4, 25, and 36 °C) were analyzed. In addition, the ability of biocidal agents (chlorhexidine1%, sodium hypochlorite 1%, and peracetic acid 0.8%) to inhibit biofilms formed by 20 strains isolated from broiler slaughter plants from two Brazilian companies in 2009, 2010, and 2014 was determined. The presence of specific genes was evaluated by PCR and phylogeny between strains by pulsed-field gel electrophoresis. The BFI was determined using tryptone soy broth with 5% of chicken juice, and its structure was observed by scanning electron microscopy. The presence of specific genes indicated that S. Minnesota has the potential to cause disease in humans, adapting to adverse conditions. Temperatures of 25 and 36 °C favored biofilm formation, although at 4 °C, there was still biomass that could contaminate the final product. Tolerance to all biocides was identified in 12/20 (60%), representing a real risk of adaptation mechanisms development, especially regarding to resistance to sodium hypochlorite. Phylogenetic analysis indicated cross-contamination and spread among companies, which was probably related to biofilms formation. Results show the necessity of attention to this serovar considering its resistance to sodium hypochlorite, including the need for rigorous control, adopting low temperatures to prevent biofilms formation in the poultry industry.

Erysipelas is a disease caused by the Erysipelothrix genus, whose main species is the E. rhusiopathiae, the causative agent of animal erysipelas and human erysipeloid. We isolated Erysipelothrix sp. strain 2 (ES2) from turkey’s organs during an outbreak in Brazilian commercial and breeder flocks with sepsis and high mortality levels. We studied 18 flocks, accounting for 182 samples, being eight flocks (84 samples) as ES2 positive with individuals demonstrating clinical symptoms and high mortality. We obtained the genetic variability of 19 samples with PFGE and found two clones, both from the same flock but different samples, and two clusters. Interestingly, we found 15 strains with high genetic variability among and within flocks. We have found a positive association between the proximity of ES2 positive turkey flocks and commercial swine sites through epidemiological analysis. We infected Vero cells with two different isolates and three distinct concentrations of ES2. After performing the morphometry, we recorded enlargement of the nucleus and nucleolus. Moreover, we performed fluorescence assays that resulted in apoptotic and necrotic cells. We demonstrated that ES2 could multiply in the extracellular medium and invade and survive inside Vero cells. For the first time, our finds show that ES2 may have similar behavior as E. rhusiopathiae as a facultative intracellular microorganism, which may represent a hazard for humans.

This work describes the development of a gastroresistant antimicrobial formulation composed of two carriers, pectin and liposomes, intended to improve the efficiency of norfloxacin (NOR) against multi-resistant bacterial strains. The formulations showed physicochemical stability for 180 days (4 °C) in terms of size, polydispersity, and zeta potential of the vesicles, prolonging the in vitro release of NOR for 11 h. The hybrid nanocarriers improved the in vitro antimicrobial activity against different multidrug-resistant bacterial strains, such as Salmonella sp., Pseudomonasaeruginosa, E. coli and Campylobacterjejuni, in comparison to commercial NOR and liposomal suspensions. The in vivo toxicity assay in chicken embryos revealed that the hybrid systems were not toxic in any of the different parameters analyzed, a result also corroborated by the analyses of biochemical biomarkers of the chicken-embryos liver function.

Açaí berry, a Brazilian palm fruit widely distributed in northern South America, is acknowledged for its functional properties such as high antioxidant capacity and anti-inflammatory activities. Although the açaí juice is highly appreciated in Brazil and even worldwide, its commercialization is still limited. Microfiltration process is largely applied in juice processing, eliminating many of the traditional processing steps and reducing time, energy and addition of clarifying agents. Furthermore, microfiltration process may eliminate microorganisms and compounds responsible for turbidity in the juice. Current assay applies a microfiltration process to obtain a stabilized açaí permeate pulp. Microfiltrations of açaí pulp were carried out in a dead end configuration with a flat membrane of 0.22 μm pore size. Permeate pulp was characterized according to its turbidity, lipid concentration and microbiological analysis. Initial permeate flux was 103 kg m-2 h-1. After an initial flux decline during 30 min., due to membrane compaction and fouling occurrences, flux was stabilized at 20 kg m-2 h-1. The microfiltration process reduced the initial açaí pulp turbidity by 99.98% and lipids were not identified in the permeate. Microbiological analysis showed that the contamination by microorganism decreased in the permeate pulp when compared to that in raw açaí pulp.

Objetivou-se avaliar aspectos físico-químicos e microbiológicos do farelo úmido de glúten de milho (FUGM) em função do tempo de ensilagem. O FUGM foi avaliado em silos experimentais e, nos dias um, três, sete, 14, 21, 28 e 42, foram realizadas as avaliações físicas, químicas e microbiológicas da silagem. O delineamento experimental foi inteiramente casualizado com quatro repetições e obtidas equações de regressão em função do tempo. O tempo de ensilagem não afetou (P>0,05) o percentual de fibra em detergente neutro, fibra em detergente ácido, proteína bruta, matéria mineral, extrativos não-nitrogenados, nutrientes digestíveis totais e contagem de clostrídios. Os teores de matéria seca, extrato etéreo, perdas por efluentes, perdas por gases e população de bactérias láticas variaram entre 46,54 e 49,05%; 0,96 e 1,71%; 17,97 e 35,14g kg-1; 1,90 e 5,68% e 5,0 e 6,9log UFCg-1 de silagem, respectivamente, todos com equações com r2≤0,6.O pH (Y=4,12926-0,00894dias+0,00024126dias2,r2=0,75) e a temperatura (Y=19,82657+0,11734dias-0,00502dias2,r2=0,73) tiveram seus menores e maiores valores, respectivamente, em 18,5 e 11,7 dias. A contagem de bolores e leveduras sofreu redução em função do tempo de ensilagem (Y=6,8983-0,0363dias,r2=0,84). A ensilagem permite eficiente conservação dos aspectos químicos, microbiológicos e físicos do FUGM até 42 dias de ensilagem

To improve the understanding of implications of Campylobacter spp. infections in pets and children of different environments were analysed 160 faecal samples from children and 120 from pets (103 dogs and 17 cats). Campylobacter spp. were detected in 6.87% of the children and in 18.3% of the dogs and cats. From 33 stool samples positive for Campylobacter spp., 57.6% were identified as C. jejuni, and 33.4% were identified as C. coli. More than 50% of the isolates in pets were resistant to ceftiofur, sulphazotrim, norfloxacin and tetracycline. In humans, most of the isolates were resistant to amoxicillin, cefazolin, ceftiofur, erythromycin and norfloxacin. From 19 isolates of C. jejuni, 11 isolates from children and 5 from dogs contained two to four of the virulence genes flaA, pldA, cadF or ciaB. We found an association between the presence of virulence genes and diarrhoea. Furthermore, an association was observed between the presence of Campylobacter spp. and diarrhoea in dewormed pets with blood picture suggestive of bacterial infection, and the therapeutic use of antibiotics was associated with more positive detection of Campylobacter spp. in the faeces of pets. Our data indicate that virulent strains of Campylobacter spp. can be risk factor to diarrhoea in animals, and that high resistance to antimicrobial agents is common in pets.

Biofilm represents a way of life that allows greater survival of microorganisms in hostile habitats. Campylobacter jejuni is able to form biofilms in vitro and on surfaces at several points in the poultry production chain. Genetic determinants related to their formation are expressed differently between strains and external conditions are decisive in this respect. Our approach combines phylogenetic analysis and the presence of seven specific genes linked to biofilm formation in association with traditional microbiology techniques, using Mueller Hinton and chicken juice as substrates in order to quantify, classify, determine the composition and morphology of the biomass of simple and mixed biofilms of 30 C. jejuni strains. It also evaluates the inhibition of its formation by biocides commonly used in industry and also by zinc oxide nanoparticles. Genetic analysis showed high heterogeneity with the identification of 23 pulsotypes. Despite the diversity, the presence of flaA, cadF, luxS, dnaJ, htrA, cbrA, and sodB genes in all strains shows the high potential for biofilm formation. This ability was only expressed in chicken juice, where they presented phenotype of a strong biofilm producer, with a mean count of 7.37 log CFU/mL and an ultrastructure characteristic of mature biofilm. The composition of simple and mixed biofilms was predominantly composed by proteins. The exceptions were found in mixed biofilms with Pseudomonas aeruginosa, which includes a carbohydrate-rich matrix, lower ability to sessile form in chicken juice and compact architecture of the biofilm, this aspects are intrinsic to this species. Hypochlorite, chlorhexidine, and peracetic acid were more effective in controlling viable cells of C. jejuni in biofilm, but the existence of tolerant strains indicates exposure to sublethal concentrations and development of adaptation mechanisms. This study shows that in chicken juice C. jejuni presents greater potential in producing mature biofilms.

The aim of this study was to determine the most frequent Salmonella serovars in swine feces in the farm, piggery’s waiting slaughterhouse and after in their carcasses during slaughter, as well as their antimicrobial resistance profiles. Eighty six strains of Salmonella spp. were used, they were previously isolated from three different lots in different collections. The identification of serovars was done by serotyping and the resistance to antimicrobial agents was determined by disc diffusion. A variety of serovars was observed, and the 86 strains were serologically identified as: 28 (32.55%) S. Typhimurium, 20 (23.26%) S. Agona, 17 (19.77%) S. Infantis, 6 (6.98%) S. Panama, and 15 (17.44%) S. Minnesota. The diversity of serovars indicated that different factors influence the infection of finishing pigs and the persistence of microorganisms in the carcass after slaughter of these animals. More than 50% of the isolates were resistant to nine of the 11 tested antibiotics. The drugs that microorganisms had the highest percentages of sensitivity were sulphazotrim and norfloxacin, 22.1% and 14%, respectively. S. Typhimurium serovar was the most isolated, including in the carcasses, and also showed the largest difference in antimicrobial resistance when compared to other serovars. The profile of multidrug resistance observed in this study highlights to the necessity of a judicious observation of antimicrobial resistance in zoonotic foodborne bacteria.

O objetivo deste trabalho foi desenvolver uma bebida láctea sabor morango carbonatada e fermentada com bactérias potencialmente probióticas. Foram elaboradas quatro formulações de bebida láctea: Controle (BL), Fermentada (BLF), Carbonatada (BLC) e Fermentada Carbonatada (BLFC). Nas amostras submetidas à carbonatação, utilizou-se um carbonatador para injeção do gás dióxido de carbono (CO2 ) dissolvido em água potável e nas amostras fermentadas, adotou-se o cultivo Lactobacillus acidophilus-LA-5®, Bifidobacterium BB-12® e Streptococcus thermophilus (Biorich, Chr. Hansen). As amostras foram caracterizadas quanto a parâmetros físico-químicos, microbiológicos e sensoriais. A BLC apresentou leveduras e coliformes totais, mas as contagens indicaram que estava própria para o consumo no tempo 28 dias. A BL apresentou contagens médias de coliformes totais acima do limite estabelecido pela legislação após 21 dias de estocagem refrigerada. A presença das bactérias lácticas e CO2 exerceram efeitos em menores índices de proteólise, menores valores de pH e maiores valores de acidez e estiveram correlacionados com o efeito inibitório de micro-organismos contaminantes nas BLfe BLFC. A BLfapresentou melhores resultados nos testes sensoriais de aceitação e intenção de compra, mas as bebidas carbonatadas apresentaram resultados positivos, com médias superiores a 50% nos testes de aceitação com potencial inserção como diferencial em bebidas lácteas. Apenas a bebida BLFC foi considerada potencialmente probiótica, por apresentar contagens mínimas de Lactobacillus spp. durante o armazenamento. Novos estudos devem ser conduzidos com a tecnologia de carbonatação, uma vez que houve correlação da presença de CO2 com efeito inibitório de deteriorantes e menores alterações físico-químicas nas bebidas lácteas.

Salmonella spp. é um importante patógeno zoonótico que pode ser disseminado ao longo da cadeia produtiva de suínos. Objetivou-se avaliar a incidência de Salmonella spp. em fezes de suínos de terminação na granja, no pré-abate e amostras ambientais, identificar os sorovares e estabelecer a relação filogenética entre os isolados. Foram realizadas três coletas em lotes diferentes de suínos alojados na granja de terminação e nos mesmos animais após o transporte ao frigorífico totalizando 90 parcelas e 9 amostras ambientais. O transporte não influenciou na porcentagem de isolamento do microrganismo (p>0,05). Das 99 amostras, 50 (50,5%) foram identificados como Salmonella spp., sendo identificado uma multiplicidade de sorovares: Agona (30%), Typhimurium (26%), Minnesota (24%), Infantis (18%) e Panama (2%). Os dendrogramas demonstraram homologia entre isolados dos diferentes sorovares agrupados em clusters. A similaridade foi independente do local de isolamento indicando a presença de vários clones. As principais fontes de infecção determinadas foram a contaminação cruzada entre animais e ambiente e o consumo de ração contaminada. A diversidade de sorovares e a homologia entre eles indicam origem comum, demonstrando necessidade de monitoramento de bactérias zoonóticas e de implantação de medidas de controle mais eficazes para Salmonella spp. em suínos.

O queijo Minas artesanal, amplamente consumido no Brasil, é produzido com leite cru, e como tal, pode trazer prejuízos à saúde do consumidor pela presença de microrganismos patogênicos. Com o objetivo de verificar a qualidade de queijos artesanais comercializados em feiras livres de Uberlândia/MG foram analisadas 23 amostras deste produto. Realizou-se análi ses do teor de umidade (%), quanti ficação de coli formes totais e termotolerantes (NMP.g1), Staphylococcus coagulase positiva (UFC.g-1) e presença/ausência de Salmonella spp. Os resultados das contagens de coliformes e Staphylococcus foram correlacionados com os teores de umidade. Das amostras analisadas, 65,2% (15/23) e 100% apresentaram resultados acima dos permitidos pela legislação para o grupo coliforme e Staphylococcus coagulase positiva, respectivamente, sendo consideradas impróprias para o consumo. A umidade média dos queijos foi de 40,87%. O coeficiente de correlação entre umidade e contagens de coliformes totais e fecais foi de 0,87 e 0,82; e para Staphylococcus coagulase posi tiva foi de 0,57, indicando que quanto maior a umidade, maiores as contagens dos microrganismos. Não foi detectada a presença de Salmonella spp. em nenhuma amostra. Os resultados obtidos indicam que os queijos Minas artesanais comercializados nas feiras livres de Uberlândia/MG devem merecer atenção dos órgãos de saúde pública e dos consumidores por oferecerem riscos à saúde pública.

It was studied, in this work, the effects of supplementation of probiotics on the performance, on the pH of crop content, on the morphometry of the ileum and on the number of enterobacteria in the cecum of the broilers. Out of the 120 studied birds, 60 belonged to the test group and 60 to the control group. At 1, 7, 12, 18, 23 and 28 days of age, the chickens and rations were weighed and mortality and morbidity were determined. Then, the birds were sacrificed and the pH of the crop content was measured, ileum fragments were collected for morphometric analysis and the enterobacteria in the cecal content were quantified. The mean pH was lower in the group that was given probiotics at 1, 7 and 18 days of age. There were no differences on ration consumption, weight, mortality and morbidity rates. The counts of enterobacteria were lower in the supplemented animals at the ages of 7, 18 and 28 days. The measure of villus of the ileum was higher for the group treated with probiotics at all ages, except for 1 day of age in comparison to the control group. This work proves that supplementation with probiotics reduces the pH of the crop content at the ages of 1, 7 and 18 days and it can contribute to the reduction of pathogenic bacteria on the first days of life by reducing the quantity of enterobacteria in the cecum. In addition, probiotics increase the height of villi in the ileum of the birds.Neste trabalho foram estudados os efeitos da suplementação de probióticos sobre o desempenho, o pH do conteúdo do papo, a morfometria do íleo e o número de enterobactérias no ceco de frangos de corte. Das 120 aves estudadas, 60 eram pertencentes ao grupo teste e 60 ao grupo controle. Nas idades de 1, 7, 12, 18, 23 e 28 dias, os frangos e a ração foram pesados e a mortalidade e morbidade foram determinadas. As aves foram, então sacrificadas, o pH do papo foi medido, os fragmentos do íleo foram coletados para análise da morfometria e as enterobactérias do ceco foram quantificadas. A média do pH foi menor no grupo que recebeu probióticos nas idades de 1, 7 e 18 dias. Não houve diferença no consumo de ração, no peso das aves, e nas taxas de mortalidade e morbidade. A contagem de enterobactérias foi menor nos animais sob suplementação com probióticos nas idades de 7, 18 e 28 dias. A medida das vilosidades do íleo foi maior no grupo tratado com probióticos em todas as idades, à exceção de 1 dia em relação ao grupo controle. Este trabalho comprova que a suplementação com probióticos diminui o pH do conteúdo do papo nas idades de 1, 7 e 18 dias, e pode contribuir na redução de bactérias patogênicas nos primeiros dias de vida, diminuindo a quantidade de enterobactérias no ceco. Além disso, os probióticos aumentaram a altura dos vilos do íleo das aves.

A pós-acidificação e a viabilidade de bactérias láticas de iogurtes foram avaliadas durante 42 dias de estocagem refrigerada. Foi analisada a eficiência do Compact Dry® TC associado ao caldo De Man Rogosa Sharp (MRS) pH 5,4 para enumeração de bactérias láticas. Alíquotas foram diluídas e plaqueadas, simultaneamente, em placas contendo ágar MRS, incubadas em microaerofilia, e em placas de Compact Dry® TC, incubadas em duas condições de atmosfera distintas; em jarras hermeticamente fechadas com gerador de microaerofilia ou acondicionadas em filme plástico transparente (PVC). Para o cultivo no Compact Dry ® TC a última diluição foi realizada em caldo MRS pH 5,4. Após 48 horas, as colônias formadas foram enumeradas e as contagens foram comparadas por análise de variância (p

A granelização do leite contribuiu para a diminuição das contagens de bactérias mesófilas no mesmo. Todavia, microrganismos com capacidade de sobreviver em baixas temperaturas, como as bactérias psicrotróficas, passaram a ganhar importância. Estes microrganismos propiciam modificações nos produtos lácteos, interferindo nas suas características sensoriais e reológicas e diminuindo a vida de prateleira. O presente trabalho possuiu como objetivo quantificar microrganismos psicrotróficos em 19 amostras de leite cru provenientes de uma usina beneficiadora da cidade de Uberlândia - MG e analisar a aplicabilidade do método alternativo Compact Dry® TC na enumeração destes microrganismos. Das 19 amostras coletadas, 18 (94,7%) apresentaram contagem maior ou igual a 105 UFC.mL-1 e somente em 1 (5,3%) amostra a contagem foi inferior a 1,0 x 104 UFC.mL-1, para ambos os métodos. Os resultados evidenciam a necessidade de melhoria nas condições higiênico-sanitárias do manejo e armazenamento desta matéria-prima. A correlação entre os resultados obtidos nos dois métodos de análises utilizados foi positiva e significativa (r2>0,9). Esse coeficiente indica que os dois métodos são equivalentes, e que o Compact Dry® representa uma alternativa para análise de bactérias psicrotróficas.

Em produtos lácteos, como o queijo Parmesão, a presença de bactérias do grupo coliformes e do gênero Staphylococcus comprometem a qualidade higiênico-sanitária do produto. Esta pesquisa objetivou verificar a qualidade sanitária e viabilidade de bactérias láticas no leite fluido e queijo Parmesão ao longo do período de estocagem. Os queijos foram produzidos com a adição de diferentes culturas de Lactobacillus helveticus (A, Cc1, D1 e E5), submetidos à maturação a 18ºC durante 180 dias, e avaliados quanto a enumeração total de bactérias láticas, bactérias do grupo coliforme e Staphylococcus coagulase positiva. O leite empregado em cada processamento foi analisado quanto a incidência destes mesmos bioindicadores, incluindo a avaliação de mesófilos e Salmonella sp. Todas as amostras de leite e queijo Parmesão demonstraram conformidade aos padrões legais quanto aos bioindicadores de contaminação avaliados. Os queijos apresentaram qualidade sanitária adequada e presença de bactérias láticas, que registraram uma redução a partir de 90 dias de maturação.

The infection by Campylobacter spp. constitutes a zoonosis of worldwide distribution, with serious repercussions for public health and a significant socio-economic impact. The Campylobacter species that most often are implicated in outbreaks of gastrointestinal disease are C. jejuni and C. coli. Both are recognized as the most important and with greater pathogenic potential from the viewpoint of food safety. This review aims to clarify and discuss, based on literature, the epidemiology of Campylobacter spp. and the main challenges in their control, to assist in better understanding of the risk posed to human health. Due to the difficulty of isolating and cultivating of Campylobacter spp., the high cost and not mandatory, laboratory diagnosis by traditional cultivation techniques is not widely used in Brazil, resulting in lack of accurate data on the occurrence of the disease in the country. This factor has been identified as responsible for underdiagnosing and underreporting of the disease, which means that data on contamination of animal products or outbreaks are not statements of actual national situation. The epidemiology of Campylobacter spp. is considered complex presenting multiple determinants in the spread of this microorganism. Chickens are important reservoirs of Campylobacter sp. and the main target of control measures, however, other vehicles of transmission, such as raw cow milk, drinking water and other foods of animal origin must also be considered in the set of measures to be taken to control disease. The infection by Campylobacter spp. cause gastroenteritis in humans and occurred by oral contact with infected animals. In fact, are pets or production and food consumption, the most important pathways. The clinical manifestations of the disease can be from mild to severe symptoms, including watery diarrhea, sometimes with blood, abdominal pain, fever, headache and nausea. Sometimes the infection can get complications after the diarrhea stops and starts a progressive paralysis that can result in respiratory failure and severe neurological dysfunction and may even lead to death. The occurrence of campylobacteriosi's outbreaks should be mandatory to the authorities of surveillance, to provide an investigation of common sources and transmission control through preventive measures , thereby minimizing the potential risks to human health.