Violacein and its antifungal activity: comments and potentialities.

Violacein is an important natural antimicrobial pigment that is mainly produced by Chromobacterium violaceum and Janthinobacterium lividum. It presents a significant range of effects against phytopathogenic and human fungi, besides being featured as having low toxicity, and by its important ecological role in protecting amphibian species and applications in dyed medical fabric. The hypothesis about violacein's action mechanisms against mucormycosis (Rhizopus arrhizus) and candidiasis (Candida auris) is herein discussed based on data available in the scientific literature.

Classificação dos graus de lesões de aerossaculite em perus associadas com enterobactérias / [Classification of the levels of the airsacculitis injuries in turkeys associated with Enterobacteriaceae]

Foram padronizados os graus de lesões dos sacos aéreos em perus com aerossaculite, associadas com a presença de isolados de enterobactérias nesses órgãos. Um total de 110 amostras de sacos aéreos de perus machos com aerossaculite foi coletado para o estudo. Durante o processo de abate, as amostras foram coletadas por meio de swabs e submetidas a três métodos de armazenamento (imediato, congelado ou pré-incubado após congelamento) para posterior comparação das suas eficiências de isolamento. Os gêneros da família Enterobacteriaceae foram identificados pelas séries bioquímicas EPM, MILi e citrato de Simmons. O crescimento bacteriano ocorreu em 43,64% das amostras. Neste estudo, quatro padrões de lesões de aerossaculite foram identificados de acordo com as características patológicas dos sacos aéreos. Os principais gêneros de enterobactérias identificadas foram: Escherichia coli, Citrobacter, Proteus, Edwardsiella, Morganella, Kluyvera, Salmonella e Klebsiella. Foi observado que os graus padronizados como 3 e 4 apresentaram maior variedade de gêneros bacterianos. O armazenamento imediato apresentou maior porcentagem de positividade, 41,82%, no entanto o pré-incubado após congelamento se apresentou mais eficaz em relação à quantidade de colônias.(AU)

The degrees of air sac lesions in turkeys with airsacculitis were standardized, associated with the presence of Enterobacteriaceae isolated from these organs. A total of 110 samples of air sacs from male turkeys with airsacculitis were collected and analyzed. During the slaughtering process, the sample collection was done using swabs and submitted to three storage methods (immediate, frozen, or pre incubated after freezing) for further comparison of their isolated efficiency. The bacterial genera of the family Enterobacteriaceae were identified biochemical series EPM, MILi and Simmons citrate. Bacterial growth occurred in 43.64% of samples. In this study, four patterns of aerossaculitis lesions were identified according to the pathological characteristics of air sacs. The frequencies of the Enterobacteriaceae isolated identified in the samples were: Escherichia coli, Citrobacter, Proteus, Edwardsiella, Morganell, Kluyvera, Salmonella and Klebsiella. Otherwise, it was observed that the levels already standardized as level three and four showed higher variety of genus. The immediate storage showed higher percentage of positivity at 41.82%, however, the pre incubated after freezing showed more efficiency in relation to the quantity of colonies.(AU)

Antimicrobial resistance, diarrheagenic and avian pathogenic virulence genes in Escherichia coli from poultry feed and the ingredients / Resistência antimicrobiana, genes de virulência diarreiogênicos e patogênicos para aves em Escherichia coli isoladas de ingredientes e ração de frangos

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.(AU)

Escherichia coli diarreiogênicas (DEC) e patogênicas para aves (APEC) são associadas a infecções intestinais e extraintestinais (ExPEC), respectivamente. O objetivo do presente trabalho foi avaliar a sensibilidade antimicrobiana, a presença de genes que codificam os fatores de virulência relacionados à DEC e APEC, e a classificação filogenética em E. coli isoladas de 320 amostras de ração para frangos e ingredientes. A sensibilidade antimicrobiana foi determinada pelo método disco-difusão e calculou-se o índice de resistência múltipla aos antimicrobianos (IRMA) e a resistência a múltiplas drogas (MDR). Nas amostras que possuíam genes de virulência relacionados à DEC e/ou APEC, foi realizada a classificação filogenética. Foram isoladas 110 amostras de E. coli em 15% (49/320) dos insumos avaliados (n=13 farelos vegetais; n=33 farinhas de origem animal; n=3 rações). De forma geral, os isolados apresentaram as maiores frequências de resistência antimicrobiana à sulfonamida e à cefazolina e 18% (20/110) foram resistentes a múltiplas drogas. O IRMA das rações foi o mais alto (0,467). Os genes que codificam fatores de virulência associados à APEC e DEC foram detectados em seis e cinco isolados, respectivamente, pertencentes aos grupos filogenéticos A e B1. Os resultados demonstram a necessidade de rigoroso controle microbiológico durante o processo de produção desses alimentos.(AU)

New Approach For Simvastatin As An Antibacterial: Synergistic Effect With Bio-Synthesized Silver Nanoparticles Against Multidrug-Resistant Bacteria.

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.

Short communication: Detection of stx2 and elt genes in bovine milk by using a multiplex PCR system.

The aim of this study was to detect 2 important toxin genes from diarrheagenic Escherichia coli (DEC) in bovine milk using a new multiplex PCR. To standardize the multiplex PCR, the stx2 and elt genes were investigated for the detection of Shiga toxin-producing Escherichia coli (STEC) and enterotoxigenic E. coli (ETEC), respectively. The DNA template was prepared with a thermal procedure (boiling) and a commercial kit. Samples consisted of UHT and pasteurized milk, both skimmed, and STEC and ETEC were tested in concentrations between 101 and 109 cfu/mL. With the thermal procedure, the multiplex PCR system detected both pathotypes of E. coli at 109 cfu/mL in UHT and pasteurized milk. When the commercial kit was used for template preparation, STEC and ETEC could be detected at concentrations as low as 104 cfu/mL in UHT and pasteurized milk. Negative controls (Listeria monocytogenes, Salmonella Typhimurium, Salmonella Enteritidis, and Escherichia coli strain APEC 13) were not amplified with the multiplex PCR. These results indicate that the multiplex PCR was a rapid (less than 6 h) and efficient method to detect STEC and ETEC in milk using different methods for DNA preparation; however, the commercial kit was more sensitive than the thermal procedure.

Novel multiplex PCR for detection of diarrheagenic Escherichia coli strains isolated from stool and water samples.

Diarrhea is considered the second most common cause of infant mortality worldwide. The disease can be caused by many different pathogens, including diarrheagenic Escherichia coli (DEC), which includes the pathotypes enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAEC), Shiga toxin-producing E. coli (STEC), and enteropathogenic E. coli (EPEC). To develop a multiplex PCR system for the safe and accurate identification of the five main pathotypes of DEC, seven pairs of primers were determined for the following genes: aaiC, escV, bfpA, ipaH, elt, stx1, and stx2. To validate the system, 413 isolates from different sources (water and both animal and human stool) were analyzed that had been characterized previously. The sensitivity data were grouped by pathotype, in which 92.7% of the atypical EPEC were correlated, as were 92.8% of the STEC, 91.35% of the EAEC, and 100% of the typical EPEC, ETEC, and EIEC. These findings indicate that it is possible to detect the major five pathotypes of DEC from different sources, which can aid in determining the epidemiology of diarrhea with a low cost, high sensitivity and specificity, and the easy and safe viewing of the resulting PCR products.

Antibacterial synergic effect of honey from two stingless bees: Scaptotrigona bipunctata Lepeletier, 1836, and S. postica Latreille, 1807.

Several studies have tested antimicrobial activity of combinations of honey and various substances. In this study, we tested a combination of two stingless bee honeys against various bacterial strains. In particular: the antibacterial activity of honeys produced by Scaptotrigona bipunctata (SB) and Scaptotrigona postica (SP) was evaluated against Gram-positive and Gram-negative bacterial strains by agar well diffusion assays, minimum inhibitory concentration (MIC) assessment, construction of growth and viability curves and scanning electron microscopy (SEM). The interaction of the two honeys was also evaluated by the checkerboard assay. Inhibition zones ranged from 8 to 22 mm. The MIC values of the individual honeys ranged from 0.62 to 10% (v v(-1)) and decreased to 1/4 to 1/32 when the honeys were combined. SEM images showed division inhibition and cell wall disruption for the SB and SP honeys, respectively, and these alterations were observed in same field when the SB and SP honeys were combined. This study demonstrated that the natural honeys possess in vitro antimicrobial activity against Gram-positive and Gram-negative bacteria, including multidrug-resistant strains. Combination of the SB and SP honeys could lead to the development of new broad-spectrum antimicrobials that have the potential to prevent the emergence of resistant bacterial strains.

Action of phosphorylated mannanoligosaccharides on immune and hematological responses and fecal consistency of dogs experimentally infected with enteropathogenic Escherichia coli strains.

The therapeutic action of phosphorylated mannanoligosaccharides (MOS) was investigated regarding its prebiotic activity on enteropathogenic Escherichia coli (EPEC). Diarrhea was induced in dogs by experimental infection with EPEC strains. Then MOS was supplied once a day, in water for 20 days. Immunological (IgA and IgG), hematological (lymphocytes, neutrophils and monocytes) and bacteriological variables (PCR detection of the eae gene of EPEC recovered from stool culture), as well as occurrence of diarrhea were evaluated. All strains caused diarrhea at 24, 48 and 72 h after infection. PCR results indicated that E. coli isolated from stool culture of all infected animals had the eae gene. There was no significant difference among groups as to number of blood cells in the hemogram and IgA and IgG production. MOS was effective in recovering of EPEC-infected dogs since prebiotic-treated animals recovered more rapidly from infection than untreated ones (p < 0.05). This is an important finding since diarrhea causes intense dehydration and nutrient loss. The use of prebiotics for humans and other animals with diarrhea can be an alternative for the treatment and prophylaxis of EPEC infections.

Action of phosphorylated mannanoligosaccharides on immune and hematological responses and fecal consistency of dogs experimentally infected with enteropathogenic Escherichia coli strains

The therapeutic action of phosphorylated mannanoligosaccharides (MOS) was investigated regarding its prebiotic activity on enteropathogenic Escherichia coli (EPEC). Diarrhea was induced in dogs by experimental infection with EPEC strains. Then MOS was supplied once a day, in water for 20 days. Immunological (IgA and IgG), hematological (lymphocytes, neutrophils and monocytes) and bacteriological variables (PCR detection of the eae gene of EPEC recovered from stool culture), as well as occurrence of diarrhea were evaluated. All strains caused diarrhea at 24, 48 and 72 h after infection. PCR results indicated that E. coli isolated from stool culture of all infected animals had the eae gene. There was no significant difference among groups as to number of blood cells in the hemogram and IgA and IgG production. MOS was effective in recovering of EPEC-infected dogs since prebiotic-treated animals recovered more rapidly from infection than untreated ones (p < 0.05). This is an important finding since diarrhea causes intense dehydration and nutrient loss. The use of prebiotics for humans and other animals with diarrhea can be an alternative for the treatment and prophylaxis of EPEC infections.

Epidemiological characterization of resistance and PCR typing of Shigella flexneri and Shigella sonnei strains isolated from bacillary dysentery cases in Southeast Brazil.

Shigella spp are Gram-negative, anaerobic facultative, non-motile, and non-sporulated bacilli of the Enterobacteriaceae family responsible for "Shigellosis" or bacillary dysentery, an important cause of worldwide morbidity and mortality. However, despite this, there are very few epidemiological studies about this bacterium in Brazil. We studied the antibiotic resistance profiles and the clonal structure of 60 Shigella strains (30 S. flexneri and 30 S. sonnei) isolated from shigellosis cases in different cities within the metropolitan area of Campinas, State of São Paulo, Brazil. We used the following well-characterized molecular techniques: enterobacterial repetitive intergenic consensus, repetitive extragenic palindromic, and double-repetitive element-polymerase chain reaction to characterize the bacteria. Also, the antibiotic resistance of the strains was determined by the diffusion disk method. Many strains of S. flexneri and S. sonnei were found to be multi-resistant. S. flexneri strains were resistant to ampicillin in 83.3% of cases, chloramphenicol in 70.0%, streptomycin in 86.7%, sulfamethoxazole in 80.0%, and tetracycline in 80.0%, while a smaller number of strains were resistant to cephalothin (3.3%) and sulfazotrim (10.0%). S. sonnei strains were mainly resistant to sulfamethoxazole (100.0%) and tetracycline (96.7%) and, to a lesser extent, to ampicillin (6.7%) and streptomycin (26.7%). Polymerase chain reaction-based typing supported the existence of specific clones responsible for the shigellosis cases in the different cities and there was evidence of transmission between cities. This clonal structure would probably be the result of selection for virulence and resistance phenotypes. These data indicate that the human sanitary conditions of the cities investigated should be improved.

Epidemiological characterization of resistance and PCR typing of Shigella flexneri and Shigella sonnei strains isolated from bacillary dysentery cases in Southeast Brazil

Shigella spp are Gram-negative, anaerobic facultative, non-motile, and non-sporulated bacilli of the Enterobacteriaceae family responsible for "Shigellosis" or bacillary dysentery, an important cause of worldwide morbidity and mortality. However, despite this, there are very few epidemiological studies about this bacterium in Brazil. We studied the antibiotic resistance profiles and the clonal structure of 60 Shigella strains (30 S. flexneri and 30 S. sonnei) isolated from shigellosis cases in different cities within the metropolitan area of Campinas, State of São Paulo, Brazil. We used the following well-characterized molecular techniques: enterobacterial repetitive intergenic consensus, repetitive extragenic palindromic, and double-repetitive element-polymerase chain reaction to characterize the bacteria. Also, the antibiotic resistance of the strains was determined by the diffusion disk method. Many strains of S. flexneri and S. sonnei were found to be multi-resistant. S. flexneri strains were resistant to ampicillin in 83.3 percent of cases, chloramphenicol in 70.0 percent, streptomycin in 86.7 percent, sulfamethoxazole in 80.0 percent, and tetracycline in 80.0 percent, while a smaller number of strains were resistant to cephalothin (3.3 percent) and sulfazotrim (10.0 percent). S. sonnei strains were mainly resistant to sulfamethoxazole (100.0 percent) and tetracycline (96.7 percent) and, to a lesser extent, to ampicillin (6.7 percent) and streptomycin (26.7 percent). Polymerase chain reaction-based typing supported the existence of specific clones responsible for the shigellosis cases in the different cities and there was evidence of transmission between cities. This clonal structure would probably be the result of selection for virulence and resistance phenotypes. These data indicate that the human sanitary conditions of the cities investigated should be improved.

Attaching and effacing Escherichia coli isolated from dogs in Brazil: characteristics and serotypic relationship to human enteropathogenic E. coli (EPEC).

Escherichia coli isolates recovered from 182 fecal specimens from dogs up to five months old from the cities of São Paulo and Campinas, SP, Brazil, were examined by polymerase chain reaction (PCR) for several virulence factors and properties. The eae gene was found in 23 isolates of E. coli from 22 dogs, 19 of 146 (13%) from dogs with diarrhea and 3 of 36 (8.3%) from dogs with no diarrhea. Two different eae+ isolates were recovered from one dog with diarrhea. Isolates from two dogs with diarrhea harbored the bfpA gene, and none of the isolates possessed genes for enterotoxins, the EAF plasmid or Shiga toxins. PCR showed that, among the 23 isolates, eight were positive for beta intimin, six for gamma, two for, one for alpha, one for kappa, and five showed no amplification with any of the nine pairs of specific intimin primers used. PCR also showed that the LEE (locus of enterocyte effacement) was inserted in selC in four isolates, likely in pheU in seven isolates, and in undetermined sites in twelve isolates. Fifteen isolates adhered to HEp-2 cells and were fluorescence actin staining (FAS) positive. The predominant adherence pattern was the localized adherence-like (LAL) pattern. The eae-positive isolates belonged to a wide diversity of serotypes, including O111:H25, O119:H2 and O142:H6, which are serotypes that are common among human EPEC. These results confirmed the presence of EPEC in dogs (DEPEC) with and without diarrhea. The virulence factors found in these strains were similar to those in human EPEC, leading to the possibility that EPEC may move back and forth among human and canine populations.

Clonal distribution of an atypical MRP+, EF*, and suilysin+ phenotype of virulent Streptococcus suis serotype 2 strains in Brazil.

Streptococcus suis is considered one of the most important bacterial swine pathogens worldwide. The distribution of the 35 described serotypes in diseased animals may vary in different regions. Data regarding S. suis isolation from pigs in South America is not available. In the present study, 51 isolates of S. suis recovered in pure culture or as the predominant species from diseased animals in Brazil, were analyzed. These isolates were classified as serotypes 2 (58.8%), 3 (21.5%), 7 (13.7%), 1 (3.9%), and 14 (2%). Serotype 2 isolates were further studied for their production of virulence-related proteins muramidase-released protein (MRP), extracellular factor (EF), and suilysin. In addition, the genetic diversity was studied by randomly amplified polymorphic DNA. All but 1 of the serotype 2 isolates showed a clonal distribution of an atypical phenotype (MRP+, EF*, suilysin+), different from the known European (MRP+, EF+, suilysin+), and North American (MRPv, EF-, suilysin-), phenotypes.

Microbiological comparative study of isolates of Edwardsiella tarda isolated in different countries from fish and humans.

It is difficult to use tissue culture assays to investigate adherence and other properties of Edwardsiella tarda because the organism is invasive and produces a potent hemolysin. We therefore relied on polymerase chain reaction (PCR) to determine the occurrence of genes for enterotoxins (LT-I, EAST-1), Shiga toxin (Stx-1, Stx-2), cytotoxic necrotizing factors (CNF-1, CNF-2), aerobactin, invasion plasmid of enteroinvasive Escherichia coli, EPEC adherence factor (EAF), intimin (Eae), enterohemolysin (EntHly) and hemolysin (Hly) in 53 isolates of E. tarda from humans and fish from several countries. All isolates were negative for all genes investigated by PCR. Adhesion to and invasion of HeLa cells were determined by using the unusually short incubation time of 1h or 30 min. All isolates adhered and invaded in these tests. Finally, a random amplified polymorphic DNA (RAPD) test distinguished, with a few exceptions, isolates of human and fish origin.

[Studies on plaque formed on implants].

In vivo plaque formation on implant materials was studied. When different implant materials were set on the gingiva, the number of adhering viable bacteria depended on material surface properties 4 hours after setting, but not 48 hours after setting. The formation of pellicle-like thin layers and subsequent covering by lamellarly formed plaque were observed on the surfaces of all materials. Streptococcus species were predominant at the 4-hour setting time but anaerobes increased at the 48-hour setting time; this was common to all materials. The results indicate that surface properties of the implants influence early bacterial adherence, but do not influence bacterial flora or plaque maturation. The subgingival microflora at the neck of implants with clinically normal peri-implant tissues was compared with that at the neck of natural teeth. The bacterial isolates were classified based on their biochemical characteristics. For the spirochaetes, the number was counted directly under light microscopic observation. The most predominant bacterial species was Streptococcus, followed by Actinomyces, Neisseria and then Capnocytophaga at both sites. The ratio of spirochaetes in the microflora was extremely low for both the implant and natural tooth. Such a bacterial distribution pattern closely resembled the hitherto-reported distribution of bacteria existing in a healthy gingival crevicular. This suggested that the microflora in plaque at the neck of a normal implant is basically similar to that at the neck of a natural tooth. In conclusion, plaque formation on implant materials was not influenced by their surface properties in actual oral cavity.

[Study on air-powder abrasive system for prosthetic clinical application. 3. Bacterial adherence to the air-powder abraded metal surface].

The purpose of this study is to investigate the relation between the metal surface texture and bacterial adherence. The surface of Au-Ag-Pd alloy was treated with or without air-powder abrasion. Seven standard strains and two clinical isolates were used. The alloys were mixed with [6-3H]-thimidine labelled bacteria. The radioactivity was measured with a scintilation counter. Streptococcus sanguis and clinical isolated Bacteroides gingivalis were adhered in a larger amount to the air-powder abraded alloy surface than to nonabraded alloy surface. These results indicated that careful consideration is needed for application of air-powder abrasive system on metal crown with secondary caries and periodontal disease.

[Antibacterial actions of denture base resin on oral bacteria].

Antibacterial action of various denture base resins on thirteen species of bacteria were studied in vitro. Antibacterial effect of 5% tannin-fluoride preparation, 5% tannic acid and 5% chlorhexidine added to resins on these thirteen bacterial species were also investigated using heat-curing denture base resins. Fresh microwave-curing resin and pour-type resin each showed an antibacterial action on one bacterial strain. Fresh self-curing resins had antibacterial actions on several bacterial strains. However, after storage in water at 37 degrees C for one week, antibacterial action of microwave-curing and pour-type resin were diminished and self-curing resins partially lost their antibacterial actions. Denture base resin containing tannin-fluoride preparation or tannic acid showed an antibacterial effect on one bacterial strain. On the other hand, denture base resin containing chlorhexidine had an antibacterial action on eleven bacterial strains. However, color and mechanical properties of the drug-containing resins were not satisfactory.