Showing papers by "Gerson Nakazato published in 2019"

Distribution of ExPEC Virulence Factors, blaCTX-M, fosA3, and mcr-1 in Escherichia coli Isolated From Commercialized Chicken Carcasses

Abstract: Pathogenic Escherichia coli found in humans and poultry carcasses harbor similar virulence and resistance genes. The present study aimed to analyze the distribution of extraintestinal pathogenic E. coli (ExPEC) virulence factors (VF), bla CTX-M groups, fosA3, and mcr-1 genes in E. coli isolated from commercialized chicken carcasses in southern Brazil and to evaluate their pathogenic risk. A total of 409 E. coli strains were isolated and characterized for genes encoding virulence factors described in ExPEC. Results of antimicrobial susceptibility testing confirmed that the strains were resistant to β-lactams, fosfomycin, colistin, and others resistance groups. The highest prevalence of VFs was observed in isolates belonging to the CTX-M groups, especially the CTX-M-2 group, when compared to those in other susceptible strains or strains with different mechanisms of resistance. Furthermore, ESBL strains were found to be 1.40 times more likely to contain three to five ExPEC virulence genes than non-ESBL strains. Our findings revealed the successful conjugation between ESBL-producing E. coli isolated from chicken carcass and the E. coli recipient strain J53, which suggested that genetic determinants encoding CTX-M enzymes may have originated from animals and could be transmitted to humans via food chain. In summary, chicken meat is a potential reservoir of MDR E. coli strains harboring resistance and virulence genes that could pose serious risks to human public health.

Characterization of CMY-2-type beta-lactamase-producing Escherichia coli isolated from chicken carcasses and human infection in a city of South Brazil.

Abstract: Food-producing animals, mainly poultry, have been associated with the maintenance and dissemination of antibiotic-resistant bacteria, such as plasmid-mediated AmpC (pAmpC)-producing Enterobacteriaceae, to humans, thus impacting food safety. Many studies have shown that Escherichia coli strains isolated from poultry and humans infections share identical cephalosporin resistance, suggesting that transmission of resistance from poultry meat to humans may occur. The aim of this study was to characterize pAmpC-producing E. coli strains isolated from chicken carcasses and human infection in a restrict area and to determine their antimicrobial resistance profiles, and molecular type by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). A total of 14 pAmpC-producing E. coli strains were isolated, including eight strains from chicken carcasses and six strains from human infections (from urine, tissue and secretion). The blaCMY-2 gene was identified in all pAmpC-producing E. coli strains by polymerase chain reaction (PCR) and DNA sequencing. High percentages of strains resistant to tetracycline, nalidixic acid and sulfamethoxazole-trimethoprim (78–92%) were detected, all of which were considered multidrug-resistant. Among the non-beta-lactam resistance genes, the majority of the strains showed tetA, tetB, sulI and sulII. No strain was considered an extended-spectrum beta-lactamases (ESBL) producer, and the blaTEM-1 gene was found in 2 strains isolated from human infection. Six strains from chicken carcasses and four strains from humans infections were linked to an ISEcp1-like element. Through MLST, 11 sequence types were found. Three strains isolated from human infection and one strain isolated from chicken carcasses belonged to the same sequence type (ST354). However, considerable heterogeneity between the strains from chicken carcasses and humans was confirmed by PFGE analysis. This study showed the prevalence of E. coli strains producing blaCMY-2 linked to ISEcp1 that were present in both chickens and humans in a restricted area. Our results also suggest the presence of a highly diverse strains that harbor pAmpC, indicating no clonal dissemination. Therefore, continuous monitoring and comparative analyses of resistant bacteria from humans and food-producing animals are needed.

New approach for simvastatin as an antibacterial: synergistic effect with bio-synthesized silver nanoparticles against multidrug-resistant bacteria

Abstract: Background Multidrug-resistant bacteria such as extended-spectrum beta-lactamase (ESBL), Enterobacteriaceae, and methicillin-resistant Staphylococcus aureus (MRSA) pose a challenge to the human health care system. MRSA is among the major causes of hospital-acquired and community infections. Methods Therefore, in the present study, we evaluated the antibacterial activity of silver nanoparticles synthesized by Fusarium oxysporum (AgNPbio) in combination with simvastatin against reference and multidrug-resistant bacterial strains. Results Simvastatin showed a minimal inhibitory concentration (MIC) ranging from 0.062 to 0.25 mg mL-1 against MRSA. AgNPbio with a size of 77.68± 33.95 nm and zeta potential -34.6 ± 12.7 mV showed an MIC of 0.212 mg mL-1 against S. aureus including MRSA strains. The checkerboard assay and time-kill curves exhibited a synergistic effect of the simvastatin-AgNPbio combination on antibacterial activity against MRSA strains. The combination of simvastatin and AgNPbio demonstrated antibacterial activity against Escherichia coli producing ESBL. Scanning electron microscopy showed the formation of cell surface protrusions after treatment with AgNPbio and the formation of a large amorphous mass after treatment with simvastatin, both in MRSA. Conclusion Our results indicate that the combination of AgNPbio and simvastatin could be a great future alternative in the control of bacterial infections, where, when combined with simvastatin, smaller doses of AgNPbio are required, with the same antibacterial activity.

In vitro anti-Pythium insidiosum activity of biogenic silver nanoparticles.

Abstract: Pythium insidiosum belongs to the phylum Oomycota. It is capable of infecting mammals causing a serious condition called pythiosis, which affects mainly horses in Brazil and humans in Thailand. The objective of the present study was to verify the in vitro anti-P. insidiosum activity of a biogenic silver nanoparticle (bio-AgNP) formulation. The in vitro assays were evaluated on P. insidiosum isolates (n = 38) following the M38-A2 protocol. Damage to the P. insidiosum hyphae ultrastructure was verified by means of scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Bio-AgNP inhibition concentrations on P. insidiosum isolates ranged from 0.06 to 0.47 μg/ml. It was observed through SEM that P. insidiosum hyphae treated showed surface roughness, as well as cell walls with multiple retraction areas, loss of continuity, and rupture in some areas. The TEM of treated hyphae did not differentiate organelle structures; also, the cellular wall was rarefied, showing wrinkled and partly ruptured borders. The bio-AgNP evaluated has excellent in vitro anti-P. insidiosum activity. However, further studies on its in vivo action are necessary as so to determine the possibility of its use in the treatment of the disease in affected hosts.

Molecular characterization of multidrug-resistant Shiga toxin-producing Escherichia coli harboring antimicrobial resistance genes obtained from a farmhouse.

Abstract: Shiga toxin-producing Escherichia coli (STEC) colonize the gastrointestinal tract of animals; however, STEC may also cause severe diarrheal diseases. Food-producing animals have been acting as reservoirs and disseminators of multidrug-resistant (MDR) bacteria and antimicrobial resistance genes (ARGs); however, there are few studies characterizing molecularly bacterial isolates from sheep. Therefore, this study aimed to characterize E. coli isolates obtained from feces of sheep in a Brazilian farmhouse. A total of 14 MDR E. coli isolates were obtained from 100 feces samples, six of which were classified as non-O157 STEC (stx1, stx2 and ehxA). MDR E. coli isolates presented different ARGs [blaCTX-M-Gp9, blaCMY, blaSHV, qnrS, oqxB, aac(6')-Ib, tet(A), tet(B), tet(C), sul1, sul2, and cmlA] and plasmids (IncI1, IncFrepB, IncFIB, IncFIA, IncHI1, IncK, and ColE-like). In addition, mutations in the quinolone-resistance determining region of GyrA (Ser83Leu; Asp87Asn) and ParC (Glu84Asp) were detected. PFGE showed a high genetic diversity (30.9 to 83.9%) and thirteen STs were detected (ST25, ST48, ST155, ST162, ST642, ST1247, ST1518, ST1725, ST2107, ST2522, ST3270, ST5036, and ST7100). Subtyping of the fimH gene showed seven fimH-type (25, 32, 38, 41, 54, 61, and 366). The results found in the present study showed high genetic diversity among MDR ARGs-producing E. coli obtained from a farmhouse. This study reports for the first time, the presence of MDR STEC and non-STEC belonging to ST25, ST162, ST642, ST1247, ST1518, ST1725, ST2107, ST3270, ST5036, and ST7100 in sheep, and contributes to the surveillance studies associated with One Health concept.

Metallic nanoparticles as a potential antimicrobial for catheters and prostheses

Abstract: Infections caused by catheters and prostheses are serious problems in hospitals and the community alike, mainly due to biofilms being produced in these materials. So composites with antimicrobial action are important to prevent infections in these patients. Nanoparticle use has been applied in catheters and prostheses. The metallic nanoparticles showed different antimicrobial activity properties. In this chapter, we will discuss specific types of metallic nanoparticles (silver, copper, zinc, gold, aluminum, titanium, and iron) and their antimicrobial action. Three aspects are correlated with this activity: direct action, inhibition of biofilm formation, and drug delivery. Different types of nanoparticles and their characteristics (synthesis, size, and shape) showed different antimicrobial effects (types of microorganisms and action levels). The use of metallic nanoparticles is interesting in terms of the synergic effect on antimicrobial activity and impeding bacterial resistance. Our research group has studied antimicrobial (fungi and bacterial) and antibiofilm activity using metallic nanoparticles with different approaches in the biomedical field. This chapter is designed to provide information and to make a comparison between metallic nanoparticles applied in catheters and prostheses.

Violacein@Biogenic Ag system: synergistic antibacterial activity against Staphylococcus aureus.

Abstract: To examine the synergistic antibacterial activity of violacein and silver nanoparticles (AgNPs) against ATCC bacteria, Staphylococcus aureus, Escherichia coli and two bacteria isolated from bovine mastitis. Violacein from Chromobacterium violaceum and biogenic AgNPs from Fusarium oxysporum were evaluated in antimicrobial tests. E. coli isolates were not inhibited by violacein at concentrations up to 400 μM and they showed sensitivity for AgNPs between 62.5 and 250 μM. Staphylococcus aureus showed sensitivity to violacein with MIC of 200 μM, and the MIC with AgNPs between 250 μM and 125 μM. It was also tested the association between the two compounds through a concentration gradient and was observed the reduction of the MIC in the combination for both strains. The bactericidal effect of violacein against S. aureus was better when combined with AgNPs (synergistic).

Análise comparativa dos fatores de virulência dos isolados clínicos e ambientais de pseudomonas aeruginosa

Abstract: Pseudomonas aeruginosa is a very important bacteria for public health because it is present in the environment and clinical infections. The aim of this study was to evaluate the virulence factors such as motility, protease and rhamnolipids in clinical and environmental P. aeruginosa isolates. Twenty-five clinical isolates and ten environmental isolates were analyzed by phenotypic assays and categorized into non-mobile, weakly, moderately and highly mobile strains; and producers of protease and rhamnolipids. The isolates were tested in triplicate on three different days. Environmental isolates produced virulence factors such as motility (Swimming and Twitching), and Ramnolipids significantly higher than clinical isolates. This study alerts us to the high level of pathogenicity of P. aeruginosa strains, mainly environmental strains. For a better understanding of motility and rhamnolipids, virulence factors that are directly associated with the biofilms formation, may favor studies that complement the research aimed at the control of pathogenic bacteria.

Antimicrobial resistance, diarrheagenic and avian pathogenic virulence genes in Escherichia coli from poultry feed and the ingredients

Abstract: Diarrheagenic (DEC) and avian pathogenic Escherichia coli (APEC) are associated with intestinal and extra-intestinal infections (ExPEC), respectively. We aimed to analyze the antimicrobial susceptibility, gene encoding virulence factors associated to DEC and APEC, and phylogenetic classification in E. coli isolated from 320 samples of feed and ingredients. Antimicrobial susceptibility was performed using the disk diffusion method and Multiple Antibiotic Resistance (MAR) Index and Multi-Drug Resistance (MDR) were calculated. Phylogenetic classification was performed on samples harboring DEC and/or APEC virulence-associated genes. A total of 110 E. coli strains were isolated in 15% (49/320) of the evaluated inputs (n=13 vegetable meal; n=33 animal meal, n=3 feed). In general, the isolates showed the highest rates of antimicrobial resistance to sulfonamide and cefazolin and 18% (20/110) were multi-drug resistant. MAR index of feed samples was the highest (0.467). Six and five strains had APEC and DEC virulence-associated genes, respectively, and belonging to phylogenetic groups A and B1. These findings point to the need for strict microbiological control during the production process of these foods.

Characterization of non-O157 Shiga toxin-producing Escherichia coli (STEC) obtained from feces of sheep in Brazil.

Abstract: Shiga toxin-producing Escherichia coli (STEC) are zoonotic pathogens and may induce severe diarrheagenic diseases in humans and other animals. Non-O157 STEC have been emerging as important pathogens causing outbreaks worldwide. Bacterial resistance to antimicrobials has become a global public health problem, which involves different ecological spheres, including animals. This study aimed to characterize the resistance to antimicrobials, plasmids and virulence, as well as the serotypes and phylogenetic groups in E. coli isolated from sheep in Brazil. A total of 57 isolates were obtained and showed different antimicrobial resistance profiles. Nineteen isolates presented acquired antimicrobial resistance genes (ARGs) (blaCTX-M-Gp9, qnrB, qnrS, oqxB, oqxA, tetA, tetB, tetC, sul1 and sul2) and plasmid families (F, FIA, FIB, I1, K, HI1 and ColE-like). The stx1, stx2 and ehxA virulence genes were detected by PCR, being 50 isolates (87.7%) classified as STEC. A great diversity of serotypes was detected, being O176:HNM the most predominant. Phylogenetic group E was the most prevalent, followed by B1, A and B2. To the best of our knowledge, this is the first report in the world of blaCTX-M-Gp9 (O75, O114, O100, O128ac and O176 serogroups), qnrB and oqxB genes in non-O157 STEC in healthy sheep. The results obtained in the present study call attention to the monitoring of antimicrobial-resistant non-O157 STEC harboring acquired ARGs worldwide and indicate a zoonotic risk due to the profile of virulence, resistance and serotype found.

As Ciências Biológicas nas Dimensões Humanista, Crítica e Reflexiva 2

Abstract: Bioprospection of microorganisms at unexplored sites, such as insect microbiota, is important for obtaining both microorganisms and enzymes, with potential biotechnological applications. Glucosidases are enzymes that catalyze the hydrolysis of glycosidic bonds of oligosaccharides and glycoconjugates, used in the production of biofuels and the food industry. The objective of this work was the evaluation of extracellular αand β-glucosidases produced by endosymbiont yeasts from the gut of Broca-da-Andiroba larvae (Hypsipyla spp.). The isolates were screened for the production of extracellular glucosidase on solid medium in the presence Esculin 0.1%. The effect of pH (4.0-9.0) and temperature (25oC-70oC) on glucosidase activity were analyzed using p-nitrophenyl-glucopyranoside as substrate. All yeasts had extracellular enzymatic activity: β-glucosidases showed an alkaline pH optimum and temperature ~40oC, highlighting the strain Candida jaroonii (M2). α-glucosidases presented a pH optimum rangeing from acid to alkaline and temperature >40oC, highlighting the strains Candida tropicalis (M4) and Candida jaroonii (M2) with activity at 70°C, 76 mU.ml-1 and 91 mU.ml-1, respectively. Higher activities in alkaline pH can provide a broader application of this class of enzymes. The present work is pioneer in the study of yeast glucosidases of Hypsipyla spp. microbiota.