Investigating the influence of Food Safety Management Systems (FSMS) on microbial diversity of Canastra cheeses and their processing environments.

Canastra Cheese is one of the most commercialized artisanal cheeses in Brazil and intrinsic characteristics of its production, such as the use of raw milk and natural whey starter cultures as well short ripening time on wooden shelves, offer risk of contamination by a plethora of microorganisms. Here, we used 16 S rRNA gene amplicon sequencing approach to characterize the bacterial communities from Canastra cheese processing environments and final products, accessing cheesemaking facilities with distinct profiles of Food Safety Management Systems (FSMS), in order to estimate whether differences in microbial composition and diversity could also be observed between the two sampled groups of facilities. Our results revealed that the diversity of bacterial communities in the processing environments was much higher than that observed for cheeses, with greater discrepancy for facilities with inadequate FSMS. Additionally, in facilities with inadequate FSMS the bacterial communities from environments, especially hand surfaces and ripening wooden shelves, were similar to those during processing and finished cheese. These evidences highlight the importance of implementing and maintaining FSMS in the facilities, in order to assure quality and safety of Canastra cheese, but also the stability and economic viability of the Canastra cheese production chain.

Microbial composition of a hydropower cooling water system reveals thermophilic bacteria with a possible role in primary biofilm formation.

Microfouling, ie biofilm formation on surfaces, can have an economic impact and requires costly maintenance in water-powered energy generation systems. In this study, the microbiota of a cooling system (filter and heat exchanger) in the Irapé hydroelectric power plant in Brazil was examined. The goal was to identify bacteria that could be targeted to more efficiently reduce biofilm formation. Two sampling campaigns were made corresponding to two well-defined seasons of the Brazilian Cerrado biome: the dry (campaign 1) and the wet (campaign 2). Microfouling communities varied considerably over time in samples obtained at different times after the last clearance of the heat exchanger. The thermophilic bacteria Meiothermus, Thermomonas and Symbiobacterium were exclusive and abundant in the microfouling of the heat exchanger in campaign 2, while methanotrophs and iron-reducing bacteria were abundant only in filter sediments. These findings could help to guide strategies for ecofriendly measures to reduce biofilm fouling in hydroelectric power plants, minimizing environmental and economic losses.

Nitrogen removal from food waste digestate using partial nitritation-anammox process: Effect of different aeration strategies on performance and microbial community dynamics.

The feasibility of employing anammox and partial nitritation-anammox (PN/A) processes for nitrogen removal from food waste (FW) digestate was investigated in this study. The effects of different aeration strategies on the microbial community were also investigated. To achieve this, after anammox enrichment (Phase 1), the reactor was fed with digestate supplemented with nitrite (Phase 2), and subsequently different aeration strategies were evaluated to establish PN/A. Aeration strategies with high anoxic periods (30 and 45 min) in relation to aerobic periods (15 min) coupled with low air flow rates (0.026 L  min-1. Lreator-1) were found to be better for establishing PN/A, as coefficients of produced nitrate/removed ammonium were closer to those reported previously (0.17 and 0.21). Aeration conditions considerably altered the microbial community. Candidatus Brocadia was replaced by Candidatus Jettenia, after the first aeration strategies. These results support the feasibility of FW digestate treatment using anammox and PN/A processes and provide a better understanding of the effect of aeration on microbial dynamics in PN/A reactors.

Porcine stomachs with and without gastric ulcer differ in Lactobacillus load and strain characteristics.

Although Lactobacillus species are recognized as normal inhabitants of porcine gastric mucosa, the association of these bacteria with health status or gastric ulcer disease has never been considered. We investigated the bacterial load of Lactobacillus isolated from the antrum, corpus, and pars esophagea of stomachs with (n = 13) and without (n = 10) ulcer of the pars esophagea of slaughtered pigs. We also evaluated in vitro antagonistic properties against typical pathogens of strains isolated from stomachs without ulcer. To quantify Lactobacillus, gastric mucosa samples obtained with 5 mm biopsy punches were smeared on MRS agar and colonies were counted after 48 h of incubation under anaerobic conditions. The score of Lactobacillus was significantly greater in the antrum and corpus of stomachs without ulcer (P < 0.001 for both) when compared with stomachs with ulcer. Fingerprint profiles, obtained by repetitive sequence-based PCR using (GTG)5 primers, showed that the isolates were highly diverse. The reduction of Lactobacillus load in porcine stomachs may be a contributing factor for gastric ulcer. Strains isolated from healthy stomachs, which showed a wide spectrum of antagonistic activity against pathogens, may be viewed as an untapped source of bacteria with potential beneficial properties that deserve to be further investigated.

Antibiotic resistance profile and occurrence of AmpC between Pseudomonas aeruginosa isolated from a domestic full-scale WWTP in southeast Brazil.

Wastewater treatment plants (WWTPs) represent an important reservoir of antibiotic resistance determinants. Although many studies have been conducted to evaluate resistance profiles in Enterobacteriaceae isolates from this setting, the dynamics of this phenomenon are poorly known to the bacterium Pseudomonas aeruginosa. Here we aimed to evaluate the resistance profiles and the production of AmpC ß-lactamase in P. aeruginosa isolates from a domestic full-scale WWTP. Samples of the raw sewage and effluent were collected and the bacterium P. aeruginosa was isolated on cetrimide agar. Susceptibility to ß-lactams, fluoroquinolones and aminoglycosides was evaluated by the disc diffusion method, and the presence of AmpC ß-lactamase was investigated phenotypically and by molecular method. We recovered 27 isolates of P. aeruginosa. Of these, 81.5% were susceptible to all antimicrobials tested. However, a considerable rate of resistance to carbapenems (11%) was found among the isolates. Twenty-two isolates were positive in the phenotypic test for inducible AmpC ß-lactamase but the blaampc gene was only identified in four isolates, suggesting the presence of other independent resistance mechanisms besides this ß-lactamase. In summary, we have shown that P. aeruginosa isolates from a domestic WWTP represents a potential reservoir of blaampC genes and other resistance determinants, including those that result in low susceptibility to carbapenems and aminoglycosides.

Diversity and distribution of iron-oxidising bacteria belonging to Gallionellaceae in different sites of a hydroelectric power plant.

Iron (Fe) is the fourth most abundant element on the planet, and iron-oxidising bacteria (FeOB) play an important role in the biogeochemical cycle of this metal in nature. FeOB stands out as Fe oxidisers in microaerophilic environments, and new members of this group have been increasingly discussed in the literature, even though their isolation can still be challenging. Among these bacteria is the Gallionellaceae family, mainly composed of neutrophilic FeOB, highlighting Gallionella ferruginea, and nitrite-oxidiser genera. In the previous metagenomic study of the biofilm and sediments of the cooling system from the Irapé hydroelectric power plant (HPP-Irapé), 5% of the total bacteria sequences were related to Gallionellaceae, being 99% unclassified at genus level. Thus, in the present study, a phylogenetic tree based on this family was constructed, in order to search for shared and unique Gallionellaceae signatures in a deep phylogenetic level affiliation and correlated them with geomorphologic characteristics. The results revealed that Gallionella and Ferrigenium were ubiquitous reflecting their ability to adapt to various locations in the power plant. The cave was considered a hotspot for neutrophilic FeOB since it harboured most of the Gallionellaceae diversity. Microscopic biosignatures were detected only in the CS1 sample, which presented abundance of the stalk-forming Ferriphaselus and of the sheath-forming Crenothrix. Further studies are required to provide more detailed insights on Gallionellaceae distribution and diversity patterns in hydroelectric power plants, particularly its biotechnological potential in this industry.

The first report of the qnrB19, qnrS1 and aac(6´)-Ib-cr genes in urinary isolates of ciprofloxacin-resistant Escherichia coli in Brazil.

In this study, we investigated the presence of plasmid-mediated quinolone resistance (PMQR) genes among 101 ciprofloxacin-resistant urinary Escherichia coli isolates and searched for mutations in the quinolone-resistance-determining regions (QRDRs) of the DNA gyrase and topoisomerase IV genes in PMQR-carrying isolates. Eight isolates harboured the qnr and aac(6')-Ib-cr genes (3 qnrS1, 1 qnrB19 and 4 aac(6')-Ib-cr). A mutational analysis of the QRDRs in qnr and aac(6')-Ib-cr-positive isolates revealed mutations in gyrA, parC and parE that might be associated with high levels of resistance to quinolones. No mutation was detected in gyrB. Rare gyrA, parC and parE mutations were detected outside of the QRDRs. This is the first report of qnrB19, qnrS1 and aac(6')-Ib-cr -carrying E. coli isolates in Brazil.

Spatiotemporal dynamics of the resistome and virulome of riverine microbiomes disturbed by a mining mud tsunami.

Aquatic ecosystems are highly vulnerable to anthropogenic activities. However, it remains unclear how the microbiome responds to press disturbance events in these ecosystems. We examined the impact of the world's largest mining disaster (Brazil, 2015) on sediment microbiomes in two disturbed rivers compared to an undisturbed river during 390 days post-disturbance. The diversity and structure of the virulome and microbiome, and of antibiotic and metal resistomes, consistently differed between the disturbed and undisturbed rivers, particularly at day 7 post-disturbance. 684 different ARGs were predicted, 38% were exclusive to the disturbed rivers. Critical antibiotic resistance genes (ARGs), e.g., mcr and ereA2, were significantly more common in the disturbed microbiomes. 401 different ARGs were associated with mobile genetic elements (MGEs), 95% occurred in the disturbed rivers. While plasmids were the most common MGEs with a broad spectrum of ARGs, spanning 16 antibiotic classes, integrative conjugative elements (ICEs) and integrons disseminated ARGs associated with aminoglycoside and tetracycline, and aminoglycoside and beta-lactam, respectively. A significant increase in the relative abundance of class 1 integrons, ICEs, and pathogens was identified at day 7 in the disturbed microbiomes, 72-, 14- and 3- fold higher, respectively, compared with the undisturbed river. Mobile ARGs associated with ESKAPEE group pathogens, while metal resistance genes and virulence factor genes in nonpathogenic hosts predominated in all microbiomes. Network analysis showed highly interconnected ARGs in the disturbed communities, including genes targeting antibiotics of last resort. Interactions between copper and beta-lactam/aminoglycoside/macrolide resistance genes, mostly mobile and critical, were also uncovered. We conclude that the mud tsunami resulted in resistome expansion, enrichment of pathogens, and increases in promiscuous and mobile ARGs. From a One Health perspective, mining companies need to move toward more environmentally friendly and socially responsible mining practices to reduce risks associated with pathogens and critical and mobile ARGs.

Exploring antibiotic resistance in environmental integron-cassettes through intI-attC amplicons deep sequencing.

INTRODUCTION: Freshwater ecosystems provide propitious conditions for the acquisition and spread of antibiotic resistance genes (ARGs), and integrons play an important role in this process. MATERIAL AND METHODS: In the present study, the diversity of putative environmental integron-cassettes, as well as their potential bacterial hosts in the Velhas River (Brazil), was explored through intI-attC and 16S rRNA amplicons deep sequencing. RESULTS AND DISCUSSION: ORFs related to different biological processes were observed, from DNA integration to oxidation-reduction. ARGs-cassettes were mainly associated with class 1 mobile integrons carried by pathogenic Gammaproteobacteria, and possibly sedentary chromosomal integrons hosted by Proteobacteria and Actinobacteria. Two putative novel ARG-cassettes homologs to fosB3 and novA were detected. Regarding 16SrRNA gene analysis, taxonomic and functional profiles unveiled Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria as dominant phyla. Betaproteobacteria, Alphaproteobacteria, and Actinobacteria classes were the main contributors for KEGG orthologs associated with resistance. CONCLUSIONS: Overall, these results provide new information about environmental integrons as a source of resistance determinants outside clinical settings and the bacterial community in the Velhas River.

Dynamics of microbial contaminants is driven by selection during ethanol production.

Brazil is the second largest ethanol producer in the world and largest using sugarcane feedstock. Bacteria contamination is one of the most important issues faced by ethanol producers that seek to increase production efficiency. Each step of production is a selection event due to the environmental and biological changes that occur. Therefore, we evaluated the influence of the selection arising from the ethanol production process on diversity and composition of bacteria. Our objectives were to test two hypotheses, (1) that species richness will decrease during the production process and (2) that lactic acid bacteria will become dominant with the advance of ethanol production. Bacterial community assemblage was accessed using 16S rRNA gene sequencing from 19 sequential samples. Temperature is of great importance in shaping microbial communities. Species richness increased between the decanter and must steps of the process. Low Simpson index values were recorded at the fermentation step, indicating a high dominance of Lactobacillus. Interactions between Lactobacillus and yeast may be impairing the efficiency of industrial ethanol production.

Insights into the Genome Sequence of Chromobacterium amazonense Isolated from a Tropical Freshwater Lake.

Members of the genus Chromobacterium have been isolated from geographically diverse ecosystems and exhibit considerable metabolic flexibility, as well as biotechnological and pathogenic properties in some species. This study reports the draft assembly and detailed sequence analysis of Chromobacterium amazonense strain 56AF. The de novo-assembled genome is 4,556,707 bp in size and contains 4294 protein-coding and 95 RNA genes, including 88 tRNA, six rRNA, and one tmRNA operon. A repertoire of genes implicated in virulence, for example, hemolysin, hemolytic enterotoxins, colicin V, lytic proteins, and Nudix hydrolases, is present. The genome also contains a collection of genes of biotechnological interest, including esterases, lipase, auxins, chitinases, phytoene synthase and phytoene desaturase, polyhydroxyalkanoates, violacein, plastocyanin/azurin, and detoxifying compounds. Importantly, unlike other Chromobacterium species, the 56AF genome contains genes for pore-forming toxin alpha-hemolysin, a type IV secretion system, among others. The analysis of the C. amazonense strain 56AF genome reveals the versatility, adaptability, and biotechnological potential of this bacterium. This study provides molecular information that may pave the way for further comparative genomics and functional studies involving Chromobacterium-related isolates and improves our understanding of the global genomic diversity of Chromobacterium species.

Synthesis, characterization, and antibacterial activity of three palladium(II) complexes of tetracyclines.

Pd(II) complexes with three antibiotics of the tetracycline family (tetracycline, doxycycline and chlortetracycline) were synthesized and characterized by elemental, thermogravimetric, and conductivity analyses, and infrared spectroscopy. The interactions between Pd(II) ions and tetracycline were investigated in aqueous solution by (1)H NMR. All the tetracyclines studied form 1:1 complexes with Pd(II) via the oxygen of the hydroxyl group at ring A and that of the amide group. The effect of the three complexes on the growth of bacterial strains sensitive and resistant to tetracycline was studied. The Pd(II) complex of tetracycline is practically as efficient as tetracycline in inhibiting the growth of two Escherichia coli (E. coli) sensitive bacterial strains and 16 times more potent against E. coli HB101/pBR322, a bacterial strain resistant to tetracycline. Pd(II) coordination to doxycycline also increased its activity in the resistant strain by a factor of 2.

Antibacterial activity of different types of snake venom from the Viperidae family against Staphylococcus aureus / Atividade antibacteriana de diferentes tipos de veneno de serpentes da família Viperidae contra Staphylococcus aureus

Toxins and venoms produced by living organisms have exhibited a variety of biological activities against microorganisms. In this study, we tested seven snake venoms from the family Viperidae for antibacterial activity and the activities of reversal of antibiotic resistance and inhibition of biofilm formation against 22 clinical isolates of Staphylococcus aureus. Bothrops moojeni venom exhibited anti staphylococcal activity with the lowest mean value of minimum inhibitory concentration (MIC). Moreover, reversal of antibiotic resistance was observed for combinations of B. moojeni venom (½ x MIC) and norfloxacin or ampicillin (both ½ x MIC) for 86.4% and 50% of the isolates, respectively. B. moojeni venom alone at ½ MIC inhibited 90% of biofilm formation, whereas in combination with ciprofloxacin, both at ½ MIC, a reduction on the NorA efflux pump activity was observed. The detection of in vitro mutants colonies of S. aureus resistant to B. moojeni venom was low and they did not survive. A phospholipase A2 was purified from the venom of B. moojeni and displayed anti-staphylococcal activity when tested alone or in combination with ciprofloxacin. The results presented here will contribute to the search for new antimicrobial agents against resistant S. aureus.

Toxinas e venenos exibem uma variedade de atividades biológicas contra micro-organismos. Neste estudo, investigou-se a atividade de sete venenos de serpentes, da família Viperidae, sobre o crescimento de Staphylococcus aureus, na reversão fenotípica da resistência a antibióticos e inibição de formação de biofilme contra 22 isolados clínicos de S. aureus. O veneno de Bothrops moojeni apresentou a menor média de concentração inibitória mínima (CIM). Além disso, observou-se reversão da resistência a antibióticos para combinações do veneno de B. moojeni (½ x CIM) e norfloxacina ou ampicilina (ambos ½ x CIM) para 86,4% e 50% dos isolados, respectivamente. O veneno de B. moojeni na concentração de ½ CIM inibiu 90% de formação de biofilme, enquanto ele em combinação com ciprofloxacina, ambos na concentração de ½ CIM, diminuiu a atividade da bomba de efluxo NorA. A detecção in vitro de colônias mutantes de S. aureus resistente ao veneno de B. moojeni foi baixa e eles não sobreviveram. Uma fosfolipase A2 purificada a partir do veneno de B. moojeni exibiu atividade antibacteriana quando testada sozinha ou em combinação com ciprofloxacina. Os dados obtidos poderão contribuir para a pesquisa de novos agentes antimicrobianos contra S. aureus.

Phenotypic and genetic analysis of Enterobacter spp. from a Brazilian oligotrophic freshwater lake.

We characterized a population of Enterobacter spp. of the Enterobacter cloacae complex isolated from an oligotrophic lake; most isolates were identified as E. cloacae. Fingerprinting polymerase chain reaction (PCR), along with morphological, biochemical, physiological, and plasmid profiles analyses, including antimicrobial susceptibility testing, were performed on 22 environmental isolates. Misidentification occurred when using the API 20E identification system. Analysis of 16S rDNA sequences confirmed the close relatedness between species of the E. cloacae complex. The tDNA PCR allowed the differentiation and identification of the E. cloacae isolates. Evaluation of genetic diversity by 16S rDNA sequence, tDNA, internal transcribed spacers, and enterobacterial repetitive intergenic concensus profiles revealed nearly identical isolates, although they exhibited different physiological and antimicrobial resistance profiles. Among the Enterobacter isolates, 96% were resistant to at least one antimicrobial; multiple resistance was also found at a high frequency (86%). The antimicrobials against which resistance was found most frequently were beta-lactams, chloramphenicol, and streptomycin. Plasmids were found in 21 of the 22 Enterobacter isolates. This confirms the conception that antibiotic resistance can occur in oligotrophic freshwater lake bacteria, which has important implications for public health.

The first report of the qnrB19, qnrS1 and aac(6´)-Ib-cr genes in urinary isolates of ciprofloxacin-resistant Escherichia coli in Brazil

In this study, we investigated the presence of plasmid-mediated quinolone resistance (PMQR) genes among 101 ciprofloxacin-resistant urinary Escherichia coli isolates and searched for mutations in the quinolone-resistance-determining regions (QRDRs) of the DNA gyrase and topoisomerase IV genes in PMQR-carrying isolates. Eight isolates harboured the qnr and aac(6')-Ib-cr genes (3 qnrS1, 1 qnrB19 and 4 aac(6')-Ib-cr). A mutational analysis of the QRDRs in qnr and aac(6')-Ib-cr-positive isolates revealed mutations in gyrA, parC and parE that might be associated with high levels of resistance to quinolones. No mutation was detected in gyrB. Rare gyrA, parC and parE mutations were detected outside of the QRDRs. This is the first report of qnrB19, qnrS1 and aac(6')-Ib-cr -carrying E. coli isolates in Brazil.

Synergic interaction between ascorbic acid and antibiotics against Pseudomonas aeruginosa

Investigou-se in vitro o efeito da combinação do ácido ascórbico (AA) com seis antibióticos frente a 12 isolados multirresistentes de Pseudomonas aeruginosa. As concentrações inibitórias mínimas (CIM) foram determinadas pelo método de diluição em caldo. Foi estudado o efeito do AA nas CIM pelo cálculo das concentrações inibitórias fracionais (CIF). Para quase todas as combinações AA-antibiótico foi detectado efeito sinérgico, exceto para ampicilina e tobramicina. Indiferença foi observada na interação com todos os antibióticos, porém antagonismo foi somente observado para cloranfenicol. Os resultados deste estudo indicam que o sinergismo contra P. aeruginosa resistentes pode ocorrer entre AA e cloranfenicol, canamicina, estreptomicina e tetraciclina, ainda que as linhagens sejam resistentes aos antibióticos individualmente. Além disso, estes resultados encorajam futuros trabalhos in vivo a respeito da interação AA-antimicrobianos na incessante busca de novas alternativas para o controle de linhagens multirresistentes de P.aeruginosa.

Clonagem do operon de resistência ao íon mercúrio do plasmidio pBH100 em escherichia coli 5K, usando o vetor pAT 153 / Cloning of the mercuric ion-resistance operon of pBH100 into escherichia coli 5K using pAT153 as vector

Resumo: Inicialmente, o plasmidio pBH100 (95 Kb) que codifica resistência, canamicina, cloranfenicol, estreptomicina e mercúrio inorgânico, foi transferido, por conjugaçäo, da linhagem selvagem Escherichia coli BH100 para a receptora. E. coli 5K. Para clonar o operon Hg plasmideos recombinantes foram construidos in vitro a partira ligaçäo (ligase) de fragmentos de pBH100, contendo os marcadores de resistência ao mercúrio, com o veiculo de clonagem pAT153, ambos digeridos coma endonuclease BamH I. Após transformaçäo de Escherihia coli 5K análises genéticas e eletroforética permitiram a detecçäo dos plasmídios recombinantes pATHg 1, pATHg2, pATHg3. Análise de restriçäo do plasmídio pATHg1, digerido com BamHI e Hind III, evidenciou três bandas de 24, 4 e 1 Kb e duas bandas de 21 e 8 Kb, respectivamente. O pATHg3 apresentou menor tamanho (12 Kb) e menor estabilidade 37 (por cento)(au)