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Water is the main resource for maintaining life. Anthropic activities influence the microbial epidemiological chain in watersheds, which can act as ways of disseminating microorganisms resistant to antimicrobial drugs, with impacts on human, animal, and environmental health. Here, we characterized aquatic microbial communities and their resistomes in samples collected along Rio das Ostras watershed during two seasons. Surface water samples were collected at eleven sites from the Jundia, Iriry, and Rio das Ostras rivers in two seasons (dry and wet season). Microbial DNA was extracted, high-throughput sequenced and screened for antimicrobial resistance genetic (ARG) markers. The physicochemical characteristics and the microbiota data confirmed that Rio das Ostras watershed can be divided into three well defined portions: rural, urban, and marine. Rural areas were enriched by bacteria typically found in limnic environments and Patescibacteria phyla. The urban portion was characterized by sites with low pH and groups associated with iron oxidation. Some genera of clinical relevance were also identified, though in relatively low abundance. The marine site was enriched mainly by Cyanobacteria and bacteria that showed strong correlation with conductivity, salinity, and chloride. Twenty-six ARG markers were identified on the resistome, being found most frequently in the urban area, despite being present in rural sites. Among them were some related to classes of great clinical concern, such as genes coding for extended-spectrum beta-lactamase (blaCTX-M and blaTEM), resistance to carbapenems (blaKPC) and to methicillin by Staphylococcus aureus (mecA). These results broaden our understanding of the microbial community of a watershed impacted by anthropogenic actions. The large number of ARGs detected along the Rio das Ostras watershed contrasts with the small number of microorganisms of clinical relevance observed, suggesting that antimicrobial resistance has arisen from non-clinical environments and microbes. Our results corroborate that freshwater acts as a reservoir of antimicrobial resistance genes.

Vaginose Bacteriana (VB) e uma sindrome polimicrobiana, caracterizada pelo desequilibrio da microbiota vaginal, associada a substituicao da populacao bacteriana vaginal predominantemente aerobia ( Lactobacillus spp. ) por uma populacao anaerobia (principalmente Gardnerella vaginalis ), gerando descarga vaginal anormal em mulheres em idade reprodutiva. Este trabalho tem como objetivo determinar a presenca de VB entre as pacientes atendidas nos servicos de ginecologia da rede publica (SUS) e rede privada (RP), de Juiz de Fora, MG, utilizando o escore de Nugent, e estabelecer a validade e a confiabilidade do encontro de clue-cells como indicador da presenca da sindrome, correlacionando aos resultados obtidos no exame preventivo (Papanicolaou). Amostras de secrecao vaginal foram coletadas e processadas para isolamento seletivo. A presenca de VB foi determinada por meio do escore de Nugent. A concordância entre a presenca de clue-cells e o resultado positivo de Papanicolaou foi de 75,0% na RP e 57,1% no SUS. Os dados sugerem que a bacterioscopia pelo metodo de Gram pode ser uma tecnica incorporada aos criterios diagnosticos de forma complementar ao Papanicolaou, auxiliando e agilizando o diagnostico de VB.

An experimental infection based on a tissue cage model was reproduced to evaluate the interference subinhibitory concentration (SIC) of metronidazole in Bacteroides fragilis OMV production patterns and immunological and histological characteristics of the host facing the experimental challenge.A tissue cage model was reproduced for B. fragilis experimental challenge in three Wistar rats groups: negative control group (NC) without bacterial inoculation; positive control group (PC) infected with parental strain; and experimental group (EG) infected with the parental strain and treated with metronidazole SIC. Tissue cage sections and histological preparations were evaluated under optical and transmission electron microscope. Observations included OMV identification and count and cellular envelope evaluation. Transcriptional analyses were performed to evaluate cytokines expression levels.Total counts of leukocytes and neutrophils were higher for EG, and slight increase in PC group. It was observed an exacerbated inflammatory infiltrate after 8 days on infection. The expression of TNF-α was increased during the experiments, along with IL-1α and IL-6. MCP-1 levels were suppressed in almost every evaluated time-point. The IL-10 was exacerbated in EG group. A massive production and release of OMV and cell wall thickening were observed especially the EG group.Despite literature data suggest positive association between OMV and antimicrobial stress for Gram negatives, no correlations are made for B. fragilis and drug-response during experimental model of infection. Results corroborate observations in which OMV may be involved in bacterial pathogenicity once the phenomenon was observed along histological evidence of exacerbated inflammation and cytokines modulation.

Introduction: Bacterial vaginosis (BV) is characterized by the depletion of Lactobacillus spp. population and increase of other species, especially Gardnerella vaginalis and Atopobium vaginae. This study aimed to investigate the vaginal microbiota structure of Brazilian women with and without BV according to Nugent Score and to assess the correlation among Nugent score and the quantification of BV-associated bacteria. Methodology: Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) assay was employed to characterize the vaginal microbiota structure. Quantification of Lactobacillus spp., G. vaginalis, A. vaginae, Mobiluncus sp. and M. hominis were determined by quantitative real-time PCR (qPCR). Results: Clustering by PCR-DGGE revealed differences in microbial structure of the different patient groups. Gardnerella vaginalis, A. vaginae, M. hominis and Mobiluncus sp. were detected at high loads in BV-associated microbiota. Quantification of Lactobacillus spp. showed an inverse correlation with Nugent score while the loads of G. vaginalis, A. vaginae, M. hominis and Mobiluncus sp. indicated a direct correlation with this method. Conclusions: Despite Nugent score is considered the gold standard for BV diagnosis, qPCR stands out as a useful tool for bacteria quantification and an alternative for BV diagnosis. Vaginal microbiota is a complex microbial community although there is a common core among BV and non-BV women. Investigation of vaginal microbiota structure may contribute to the development of tools for diagnosis improvement and therapeutic regimen optimization.

Fungi have a complex role in the intestinal tract, influencing health and disease, with dysbiosis contributing to obesity. Our objectives were to investigate fungal diversity in human gut microbiota among eutrophic, overweight, and obese. Epidemiological and nutritional information were collected from adult individuals, as well as stool samples processed for selective fungi isolation and identification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (yeasts) or microculture (filamentous fungi). Further 18S rDNA sequencing was performed to confirm identification. The mean count of fungi was 241 CFU/g of feces. Differences in the population level of the filamentous fungi were observed within eutrophic and obese groups. Overall, 34 genera were identified. The predominant phylum was Ascomycota with 20 different genera, followed by Basidiomycota and Zygomycota. As for Ascomycota, the most prevalent species were Paecilomyces sp., Penicillium sp., Candida sp., Aspergillus sp., Fonsecaea sp., and Geotrichum sp. (76.39, 65.28, 59.72, 58.33, 12.50, and 9.72%, respectively). As for Basidiomycota, Trichosporon sp. and Rhodotorula sp. were the most prevalent (30.56 and 15.28%, respectively), and for Zygomycota, Rhizopus sp. and Mucor sp. were the most numerous (15.28 and 9.72%, respectively). As expected there is a mycobiota shift towards obesity, with slightly higher diversity associated to eutrophic individuals. This mycobiota shift seems also to be related to the nutritional behavior of the individuals, as observed that the macronutrients intake may be positively related to the different fungi occurrences. Other studies are needed to better understand relationships between mycobiota and obesity, which could be used in future obesity treatments.

Although most Brazilian dairy products meet high technological standards, there are quality issues regarding milk production, which may reduce the final product quality. Several microbial species may contaminate milk during manufacture and handling. If antimicrobial usage remains uncontrolled in dairy cattle, the horizontal transfer of antimicrobial resistance genes in foodstuffs may be of particular concern for both food producers and dairy industry. This study focused on the evaluation of putative Gram positive cocci in Minas cheese and of antimicrobial and biocide resistance genes among the isolated bacteria. Representative samples of 7 different industrially trademarked Minas cheeses (n = 35) were processed for selective culture and isolation of Gram positive cocci. All isolated bacteria were identified by DNA sequencing of the 16S rRNA gene. Antimicrobial resistance genes were screened by PCR. Overall, 208 strains were isolated and identified as follows: Enterococcus faecalis (47.6%), Macrococcus caseolyticus (18.3%), Enterococcus faecium (11.5%), Enterococcus caseliflavus (7.7%), Staphylococcus haemolyticus (7.2%), Staphylococcus aureus (4.3%), Staphylococcus epidermidis (2.9%), and Enterococcus hirae (0.5%). The genetic markers mecA (78.0%) and smr (71.4%) were the most prevalent, but others were also detected, such as blaZ (65.2%), msrA (60.9%), msrB (46.6%), linA (54.7%), and aacA-aphD (47.6%). The occurrence of opportunist pathogenic bacteria harboring antimicrobial resistance markers in the cheese samples are of special concern, since these bacteria are not considered harmful contaminating agents according to the Brazilian sanitary regulations. However, they are potentially pathogenic bacteria and the cheese may be considered a reservoir for antimicrobial resistance genes available for horizontal transfer through the food chain, manufacturing personnel and consumers.

Although lifestyle and physiology in obese individuals are accepted to lead to changes in the intestinal microbiota, uncertainty remains about microbiota dysbiosis, and xenobiotics intake, as a source of selective pressure, independent of antimicrobial chemotherapy. The aim of this study was to compare the occurrence of antimicrobial resistance genetic markers (ARG) in faecal specimens of eutrophic, overweight and obese individuals, and their correlation with xenobiotic intake and gut bacteria density. Methods: This was a cross-sectional case-controlled study including 72 adult participants with no record of intestinal or systemic diseases, or recent use of antimicrobials, grouped as eutrophic, overweight, or obese. Anthropometric profile, eating habits and oral xenobiotics intake were recorded. Faecal metagenomic DNA was used to screen for ARG by PCR, and to measure bacterial groups by fluorescence in situ hybridization (FISH). Student&rsquo, s t and Wilcoxon tests were used to compare means and differences in ARG detection (95% confidence intervals). Correlation analyses (odds ratio) and relationships between bacteria density and ARG were determined. Results: Increase in abdominal circumference, waist circumference, hip, waist-hip ratio, BMI, carbohydrate, fibres, and total calorie intakes were different from eutrophic to obese participants. Habitual use of antihypertensive and anti-inflammatory drugs, antacids, and artificial sweeteners were associated mainly with obesity and overweight. Nutritional supplements were associated to the eutrophic group. ARG screening showed differences being more frequent among obese, and positive for 27 genetic markers related to &beta, lactams, tetracyclines, the macrolide lincosamide and streptogramin group, quinolones, sulfonamides, aminoglycosides, and efflux pump. Positive correlation between ARG and BMI, caloric intake, and intake of xenobiotics, was observed for obese individuals. Relationships among ARG detection and bacteria densities were also different. Conclusions: This study reinforces the hypothesis that obese individuals may harbour an altered gut microbiota, if compared to eutrophic. The overweight individuals display a transitional gut microbiota which seems to be between eutrophic and obese. Furthermore, the increased xenobiotic intake associated to obesity may play an important role in the antimicrobial resistance phenomenon.

The knowledge about microbiota from cheeses plays a key role in safety improvement and quality enhancement of the final product. We used a high throughput sequencing methodology to provide insights into the microbial composition of Minas Frescal Cheese (MFC). The results obtained from the analysis of 28 cheese samples showed that cheeses were brand-clustered, exhibiting unique bacterial profiles which varied in diversity and abundance of taxa. Proteobacteria dominated in all samples, corresponding to a mean of 88.7% from all sequences. Groups associated with contamination, as Pseudomonas, Acinetobacter and Vibrio were highly frequent in most of brands. Low abundance of lactic acid bacteria was also observed. The core microbiome shared by 100% of the samples indicated predominance of Enterobacteriaceae family. Our data point to a concern regarding the occurrence of potential spoilage microbiota and opportunistic pathogens in cheeses. This metataxonomic study suggests correlations between putative bacteria and antimicrobial resistance phenomenon in foodstuff, as already reported for MFC. Together these data highlight that productive chain of MFC might be related to spread of medically important bacteria, which are underestimated for their possible impact on human health. The observations may also reinforce the importance of food safety regulations facing the One Health point of view.

Three series of d-galactose derivatives linked to a lipophilic aminoalcohol moiety were synthesized and their antibacterial activity was evaluated against Mycobacterium tuberculosis and representative species of Gram positive and Gram negative bacteria. Five out of the thirteen tested compounds displayed activity against M. tuberculosis, with a minimal inhibitory concentration (MIC) of 12.5 μg/mL and seven compounds were active against the four bacterial strains tested. The best results were obtained for amino alcohols 10 and 11 against Staphylococcus epidermidis (MIC = 2 μg/mL). The antitumor activity was evaluated against three tumor cell lines (MCF-7, HeLa and MO59J) and compared to the normal cell line GM07492A. The results showed that the lowest IC50 values were observed for the amino alcohol 16 against MCF-7 (11.9 μM) and MO59J (10.0 μM).

To characterize methicillin-resistant Staphylococcus aureus isolates from an intensive care unit of a tertiary-care teaching hospital, between 2005 and 2010. A total of 45 isolates were recovered from patients admitted to the intensive care unit in the study period. Resistance rates higher than 80% were found for clindamycin (100%), erythromycin (100%), levofloxacin (100%), azithromycin (97.7%), rifampin (88.8%), and gentamycin (86.6%). The SCCmec typing revealed that the isolates harbored the types III (66.7%), II (17.8%), IV (4.4%), and I (2.2%). Four (8.9%) isolates carried non-typeable cassettes. Most (66.7%) of the isolates were related to the Brazilian endemic clone from CC8/SCCmec III, which was prevalent (89.3%) between 2005 and 2007, while the USA100/CC5/SCCmec II lineage emerged in 2007 and was more frequent in the last few years. The study showed high rates of antimicrobial resistance among methicillin-resistant S. aureus isolates and the replacement of Brazilian clone, a well-established hospital lineage, by the USA100 in the late 2000s, at the intensive care unit under study. Keywords: Clonal complex, MRSA, PFGE lineages, SCCmec

Different series of N-alkylated diamines and their derivatives condensed to quinic acid were synthesized and tested for antibacterial properties against Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Mycobacterium tuberculosis. The lipophilic chain and carbohydrate moiety modulate the antibacterial activity and the compounds showed a structure-activity relationship. Overall, 11 compounds displayed better activity than chloramphenicol against Gram-positive and Gram-negative bacteria. Monoalkylated amines 2a-h displayed an activity similar to that of ethambutol against Mycobacterium tuberculosis.

Novel lipophilic gold(I) complexes containing 1,3,4-oxadiazol-2-thione or 1,3-thiazolidine-2-thione derivatives were synthesized and characterized by IR, high resolution mass spectrometry, and 1H, 13C 31P NMR. The cytotoxicity of the compounds was evaluated considering cisplatin and/or auranofin as reference in different tumor cell lines: colon cancer (CT26WT), metastatic skin melanoma (B16F10), breast adenocarcinoma (MCF-7), cervical carcinoma (HeLa), glioblastoma (M059 J). Normal human lung fibroblasts (GM07492-A) and kidney normal cell (BHK-21) were also evaluated. The gold(I) complexes were more active than their respective free ligands and cisplatin. Furthermore, antibacterial activity was evaluated against Gram-positive bacteria Staphylococcus aureus ATCC 25213, Staphylococcus epidermidis ATCC 12228 and Gram-negative bacteria Escherichia coli ATCC 11229 and Pseudomonas aeruginosa ATCC 27853 and expressed as the minimum inhibitory concentration (MIC). The complexes exhibited lower MIC values when compared to the ligands and chloramphenicol against Gram-positive bacteria and Gram-negative bacteria. Escherichia coli was sensitive one to the action of gold(I) complexes.

Although most Brazilian dairy products meet high technological standards, there are quality issues regarding milk production, which may reduce the final product quality. Several microbial species may contaminate milk during manufacture and handling. If antimicrobial usage remains uncontrolled in dairy cattle, the horizontal transfer of antimicrobial resistance genes in foodstuffs may be of particular concern for both food producers and dairy industry. This study focused on the evaluation of putative Gram positive cocci in Minas cheese and of antimicrobial and biocide resistance genes among the isolated bacteria. Representative samples of 7 different industrially trademarked Minas cheeses (n = 35) were processed for selective culture and isolation of Gram positive cocci. All isolated bacteria were identified by DNA sequencing of the 16S rRNA gene. Antimicrobial resistance genes were screened by PCR. Overall, 208 strains were isolated and identified as follows: Enterococcus faecalis (47.6%), Macrococcus caseolyticus (18.3%), Enterococcus faecium (11.5%), Enterococcus caseliflavus (7.7%), Staphylococcus haemolyticus (7.2%), Staphylococcus aureus (4.3%), Staphylococcus epidermidis (2.9%), and Enterococcus hirae (0.5%). The genetic markers mecA (78.0%) and smr (71.4%) were the most prevalent, but others were also detected, such as blaZ (65.2%), msrA (60.9%), msrB (46.6%), linA (54.7%), and aacA-aphD (47.6%). The occurrence of opportunist pathogenic bacteria harboring antimicrobial resistance markers in the cheese samples are of special concern, since these bacteria are not considered harmful contaminating agents according to the Brazilian sanitary regulations. However, they are potentially pathogenic bacteria and the cheese may be considered a reservoir for antimicrobial resistance genes available for horizontal transfer through the food chain, manufacturing personnel and consumers.

In continuation of our efforts to find new antimicrobial compounds, series of fatty N -acyldiamines were prepared from fatty methyl esters and 1,2-ethylenediamine, 1,3-propanediamine or 1,4-butanediamine. The synthesized compounds were screened for their antibacterial activity against Gram-positive bacteria ( Staphylococcus aureus , Staphylococcus epidermidis ), Gram-negative bacteria ( Escherichia coli , Pseudomonas aeruginosa ) and for their antifungal activity against four species of Candida ( C. albicans , C. tropicalis , C. glabrata and C. parapsilosis ). Compounds 5a ( N -(2-aminoethyl)dodecanamide), 5b ( N -(2-aminoethyl)tetracanamide) and 6d ( N -(3-aminopropyl)oleamide) were the most active against Gram-positive bacteria, with MIC values ranging from 1 to 16 μg/mL and were evaluated for their activity against 21 clinical isolates of methicillin-resistant S. aureus . All the compounds exhibited good to moderate antifungal activity. Compared to chloramphenicol, compound 6b displayed a similar activity (MIC 50 = 16 μg/mL). A positive correlation could be established between lipophilicity and biological activity.

Beta-lactamases enzymes such as extended-spectrum beta-lactamases (ESBL) and carbapenemase type beta-lactamases (KPC) confer resistance to beta-lactam drugs among Gram-negative rods, mainly Enterobacteriaceae, as those frequently related to urinary tract infections (UTI). The aim of this study was to evaluate ESBL and KPC among enterobacteria isolated from monomicrobial UTI and to establish correlations between the presence of genetic markers and the phenotypic resistance to beta-lactam antibiotics. Out of 12 304 urine samples collected during 2009, 93 enterobacteria showing an ESBL phenotype were recovered. Imipenem was used for KPC screening and modified disk approximation assay was used for detection of ESBL phenotype. Polymerase chain reaction was used for screening of bla(SHV), bla(TEM), bla(CTX-M), and bla(KPC). Considering the isolated bacteria showing ESBL phenotype 56% of the isolates were positive for two genes. The bla(TEM) was the most frequent (87·1%). Neither KPC phenotype nor bla(KPC)-harboring bacteria were observed. Monitoring the antimicrobial resistance is extremely important to sustain empirical therapy of community-acquired urinary tract infections (Co-UTI).

We report in this work the preparation and in vitro antimicrobial evaluation of novel amphiphilic aromatic amino alcohols synthesized by reductive amination of 4-alkyloxybenzaldehyde with 2-amino-2-hydroxymethyl-propane-1,3-diol. The antibacterial activity was determined against four standard strains (Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa) and 21 clinical isolates of methicillin-resistant Staphylococcus aureus. The antifungal activity was evaluated against four yeast (Candida albicans, Candida tropicalis, Candida glabrata and Candida parapsilosis). The results obtained showed a strong positive correlation between the lipophilicity and the antibiotic activity of the tested compounds. The best activities were obtained against the Gram-positive bacteria (MIC = 2–16 μg ml−1) for the five compounds bearing longer alkyl chains (4c–g; 8–14 carbons), which were also the most active against Candida (MIC = 2–64 μg ml−1). Compound 4e exhibited the highest levels of inhibitory activity (MIC = 2–16 μg ml−1) against clinical isolates of MRSA. A concentration of twice the MIC resulted in bactericidal activity of 4d against 19 of the 21 clinical isolates.

The Fluorescent in situ Hybridization (FISH) technique was employed to enumerate potential probiotic and putative pathogenic bacteria in the gut of tilapia ( Oreochromis niloticus ). Bacteria used in the study were isolated from water, sediment and intestines of tilapia ( Oreochromis niloticus ) raised in an aquaculture system. These isolates were tested in vitro on antagonism tests against putative pathogenic bacteria ( Aeromonas hydrofila , Enterococcus faecalis , Edwardsiella tarda , Pseudomonas fluorescens and Pseudomonas putida ), also isolated from the same aquaculture system. Two isolates that inhibited largest number of pathogenic bacteria were identified by sequencing as Bacillus sp. and Enterococcus sp. and were added to the commercial feed (10 6 cells g − 1 ) for in vivo tests. Treatments of the in vivo experiment were: 1) Control — fish fed with no added bacteria, 2) Bacil. — fish fed diets containing Bacillus sp.; 3) Enter. — fish fed diets containing Enterococcus sp., and 4) Bacil. + Enter. — fish fed diets containing Bacillus sp. and Enterococcus sp. (1:1). Each treatment consisted of four replicates with 15 juveniles of tilapia ( O. niloticus — 16.74 ± 4.35 g e 9.82 ± 0.85 cm). The experiment lasted for 30 days and at the end of this period, three fish from each tank were killed, and the intestines were taken for microbiological analysis by FISH technique, where Bacillus and Enterococcus , as well as two putative pathogenic bacteria ( Aeromonas and Pseudomonas sp.) were quantified. Enterococcus sp. and Bacillus sp. were present in high number in the gut microbiota of fish. However, Bacillus sp., showed an increase in its abundance, indicating a successful incorporation of this potential probiotic bacteria into the tilapia gut microbiota. Furthermore, in the Bacil. treatment it was observed a significant reduction of Aeromonas and Pseudomonas sp. abundances compared with the other treatments. These results indicate that the FISH technique is a potential tool to characterize the dynamics of potential probiotic bacteria and their efficiency in the control of pathogenic bacteria.

Coagulase-negative staphylococci (CoNS), which are generally neglected as foodborne bacteria, are emerging as significant opportunistic pathogens that may be highly resistant to available antimicrobial drugs. In this study, antimicrobial susceptibility patterns, mecA gene occurrence, and virulence-associated characteristics were evaluated in CoNS isolated from soft cheese in Brazil. A total of 227 bacterial isolates were recovered from 35 cheese samples belonging to 5 batches with 7 different trademarks. The CoNS counts ranged from 10(6) to 10(7) CFU/g. High antimicrobial resistance percentages were observed for oxacillin (76.2%), penicillin (78.5%), erythromycin (67.8%), gentamicin (47.2%), clindamycin (35.7%), rifampicin (26.8%), azithromycin (14.7%), tetracycline (14.7%), levofloxacin (14.2%), and sulfamethoxazole-trimethoprim (11.9%). A low antimicrobial resistance percentage was observed for chloramphenicol (2.3%), and all of the tested bacteria were susceptible to vancomycin and linezolid. In total, a multiple antibiotic resistance (MAR) index of >0.2 was observed for 80.6% of the isolated CoNS. However, the MAR index ranged from 50% to 92.6% when only bacterial cheese isolates belonging to the same trademark were considered. Regarding to the prevalence of CoNS carrying mecA gene, 81.5% of the isolated strains were mecA(+) , and 76.2% of these were phenotypically resistant to oxacillin. Three isolates carried the enterotoxin A gene (sea), 29.5% produced biofilm in a laboratory test, and α- or s-hemolysis were observed for 3% and 5.2%, respectively. This study highlights the extent of the antimicrobial resistance phenomenon in neglected foodborne microorganisms and the potential public health risks that are related to the consumption of CoNS-contaminated soft cheese.

Rivers and streams are important reservoirs of freshwater for human consumption. These ecosystems are threatened by increasing urbanization, because raw sewage discharged into them alters their nutrient content and may affect the composition of their microbial community. In the present study, we investigate the taxonomic and functional profile of the microbial community in an urban lotic environment. Samples of running water were collected at two points in the São Pedro stream: an upstream preserved and non-urbanized area, and a polluted urbanized area with discharged sewage. The metagenomic DNA was sequenced by pyrosequencing. Differences were observed in the community composition at the two sites. The non-urbanized area was overrepresented by genera of ubiquitous microbes that act in the maintenance of environments. In contrast, the urbanized metagenome was rich in genera pathogenic to humans. The functional profile indicated that the microbes act on the metabolism of methane, nitrogen and sulfur, especially in the urbanized area. It was also found that virulence/defense (antibiotic resistance and metal resistance) and stress response-related genes were disseminated in the urbanized environment. The structure of the microbial community was altered by uncontrolled anthropic interference, highlighting the selective pressure imposed by high loads of urban sewage discharged into freshwater environments.

The use of antimicrobials and toxic metals should be considered carefully in aquaculture and surrounding environments. We aimed to evaluate medically relevant bacteria in an aquaculture system and their susceptibility to antimicrobials and toxic metals. Selective cultures for enterobacteria (ENT), non-fermenting Gram-negative rods (NFR) and Gram-positive cocci (GPC) were obtained from water samples collected in two different year seasons. The isolated bacteria were biochemically identified and antimicrobial and toxic metal susceptibility patterns were determined. Overall, 407 representative strains were recovered. In general, bacteria isolated from fish ponds showed higher multiple antibiotic resistance indices when compared to those isolated from a water-fed canal. Resistance to penicillin and azithromycin was observed more frequently in the GPC group, whereas resistance to ampicillin and ampicillin/sulbactam or gentamicin was observed more frequently in the ENT and NFR groups, respectively. All the isolated bacteria were tolerant to nickel, zinc, chromium and copper at high levels (≥1,024 μg mL(-1)), whereas tolerance to cadmium and mercury varied among the isolated bacteria (2-1,024 μg mL(-1)). Multidrug-resistant bacteria were more frequent and diverse in fish ponds than in the water-fed canal. A positive correlation was observed between antimicrobial resistance and metal tolerance. The data point out the need for water treatment associated with the aquaculture system.

Prevotella intermedia is a rod-shaped, Gram-negative anaerobic bacterium found in human indigenous microbiota that plays an important role in opportunistic infections. The successful colonization depends on the ability of anaerobes to respond to oxidative stress (OS) in oxygenated tissues as well as to resist oxidative events from the host immune system until anaerobic conditions are present at the infection site. As knowledge of the mechanisms of protection against OS in Prevotella is limited, studies are needed to clarify aspects of molecular biology, physiology and ecology of this bacterium. The aim of this study was to access the proteins differentially regulated in P. intermedia after exposure to molecular oxygen by using two-dimensional gel electrophoresis (2DE) associated with the approach of MALDI-TOF/TOF Tandem Mass Spectrometry. The identity of the protein was evaluated by database search for homologous genomic sequences of P. intermedia strain 17 (TIGR). Twenty five out of 72 proteins found were identified as up-regulated (17) or down-regulated (9). These proteins were related to a variety of metabolic process, some of which could be associated to antioxidant and redox regulatory roles. Our data indicate that OS may stimulate an adaptive response in P. intermedia whose effect on its biology may be evidenced by the increase in aerotolerance and changes in protein abundance in the oxygen adapted cells.

We describe the synthesis and the antibacterial evaluation 2’,N3-cyclonucleoside 3 analogue of MTA that is characterized by the presence of an additional linkage between the heterocyclic ring and the sugar moiety.

Two series of aromatic and heteroaromatic amino alcohols were synthesized from alcohols and aldehydes and evaluated for their antibacterial activities. All the octylated compounds displayed a better activity against the four bacteria tested when evaluated by the agar diffusion method and were selected for the evaluation of minimal inhibitory concentration. The best results were obtained for p-octyloxybenzyl derivatives against Staphylococcus epidermidis (minimal inhibitory concentrations = 32 μm).

Objective In this study of patients with chronic periodontitis (CP), the severity of the disease and the main periodontal pathogens identified in patients with chronic kidney disease (CKD) were compared with those detected in individuals without systemic disease. Design Nineteen patients with CP without evidence of systemic disease (control group), 25 patients with CP and CKD who were in the pre-dialysis stages (pre-dialysis group), and 22 patients with CP and CKD who were on renal replacement therapy (RRT group) were examined. The severity of CP was based on the investigation of probing depth (PD) and clinical attachment level (CAL). The definition and stage of CKD were based on the criteria proposed by the Kidney Disease Outcomes Quality Initiative of the National Kidney Foundation. Glomerular filtration rate (GFR) was estimated using the equation of Modification of Diet in Renal Disease and the identification of microorganisms in subgingival plaque was performed using polymerase chain reaction (PCR). Results Candida albicans, Porphyromonas gingivalis, Tannerella forsythia, and Treponema denticola were more common in patients who were on RRT and pre-dialysis than in control subjects. CP was more severe in patients with CKD. A strong association was observed between the frequency of C. albicans (P = 0.056), P.gingivalis (P = 0.008), T. denticola (P = 0.013) and CAL, when CKD patients were compared with the control group. Conclusion CP is more severe and is associated with increased frequency of C. albicans, P. gingivalis, T. forsythia, and T. denticola in patients with CKD.

Urban pigeons (Columba livia) come into close contact with humans and animals, and may contribute to the spread of infectious agents. These may include human pathogens such as diarrheagenic Escherichia coli strains, which are able to survive in pigeon feces, thus creating potential for human exposure and infection. Our objectives were to determine the occurrence of diarrheagenic E. coli strains in fresh feces from urban pigeons and their drug susceptibility patterns. E. coli strains were isolated from 100 fresh feces samples and presumptive phenotypic species identification was carried out, confirmed by amplification of specific 16S ribosomal RNA encoding DNA. Multiplex PCR was performed to characterize pathogenic strains. Drug susceptibility patterns were determined by the agar dilution method. Enteroinvasive E. coli, Shiga toxin-producing E. coli, enteropathogenic E. coli, and enterotoxigenic E. coli were detected at an overall rate of 12.1%. Among the isolated E. coli strains, 62.1% were susceptible to all tested drugs, whereas 37.9% were resistant to at least one of the antimicrobials tested. Amikacin was the less effective drug (36.8% resistance), followed by ampicillin (7.8%). No resistance was detected to gentamicin, ceftriaxone, and ceftazidime and almost all the isolates were susceptible to ampicillin-sulbactam (98.4%), levofloxacin (97.8%), and trimethoprim-sulfamethoxazole (96.1%). Since these pigeons may harbor multidrug-resistant pathogens, their presence in an urban environment could be an important component of infection spread, with impact on public health.

The protein profile of the outer membrane of Leptospira interrogans serovar Hardjoprajitno was determined by two-dimensional gel electrophoresis. The outer membrane was extracted with Triton x 114 and the proteins were precipitated with acetone. The images were analyzed for the determination of the molecular weight of the detected proteins. Thirty-five spots for the proteins that are predominant in the outer membrane of this Leptospira were observed and five proteins were found in higher quantities: 22.54KDa (LipL22), 30/26KDa (LipL32), 34.41KDa (PME34) (2), 42.75KDa (LipL41), and 58.59KDa (LipL63).

Summary Background and objectives This study aims to determine antibacterial activities of Cocos nucifera (husk fiber), Ziziphus joazeiro (inner bark), Caesalpinia pyramidalis (leaves), aqueous extracts and Aristolochia cymbifera (rhizomes) alcoholic extract against Prevotella intermedia, Porphyromonas gingivalis, Fusobacterium nucleatum, Streptococcus mutans and Lactobacillus casei. The antioxidant activity and acute toxicity of these extracts were also evaluated. Material and methods The plant extracts antibacterial activity was evaluated in vitro and the minimal inhibitory concentration (MIC) was determined by the broth micro-dilution assay. The bacterial killing kinetic was also evaluated for all extracts. In addition, the antibacterial effect of the extracts was tested in vitro on artificial oral biofilms. The acute toxicity of each extract was determined in according to Lorke [Lorke D. A new approach to practical acute toxicity testing. Arch Toxicol 1983;54:275–87] and the antioxidant activity was evaluated by DPPH photometric assay [Mensor LL, Menezes FS, Leitao GG, Reis AS, Santos TC, Coube CS, et al. Screening of Brazilian plants extract for antioxidant activity by the use of DPPH free radical method. Phytother Res 2001;15:127–30]. Results MIC and the bactericidal concentrations were identical, for each evaluated extract. However, microbes of artificial biofilms were less sensitive to the extracts than the planktonic strains. A. cymbifera extract induced the highest bactericidal effect against all tested bacteria, followed by C. nucifera, Z. joazeiro and C. pyramidalis extracts, respectively. All extracts showed good antioxidant potential, being C. nucifera and C. pyramidalis aqueous extracts the most active ones. Conclusion In conclusion, all oral bacteria tested (planktonic or in artificial biofilms) were more susceptible to, and rapidly killed in presence of A. cymbifera, C. pyramidalis and C. nucifera than Z. joazeiro extracts, respectively. Thus, these extracts may be of great interest for future studies about treatment of oral diseases, considering their potent antioxidant activity and low toxicity.

Anaerobic digestion is an alternative method for the treatment of animal manure and wastewater. The anaerobic bioconversion of biomass requires a multi-step biological process, including microorganisms with distinct roles. The diversity and composition of microbial structure in pilot-scale anaerobic digestion operating at ambient temperature in Brazil were studied. Influence of the seasonal and temporal patterns on bacterial and archaeal communities were assessed by studying the variations in density, dynamic and diversity and structure. The average daily biogas produced in the summer and winter months was 18.7 and 16 L day(-1), respectively, and there was no difference in the average methane yield. Quantitative PCR analysis revealed that no differences in abundances and dynamics were found for bacterial communities and the total number of Archaea in different seasons. Analysis of bacterial clone libraries revealed a predominance of Firmicutes (54.5 %/summer and 46.7 %/winter) and Bacteroidetes (31.4 %/summer and 44.4 %/winter). Within the Archaea, the phylum Euryarchaeota was predominant in both digesters. Phylogenetic distribution showed changes in percentage between the phyla identified, but no alterations were recorded in the quality and amount of produced methane or community dynamics. The results may suggest that redundancy of microbial groups may have occurred, pointing to a more complex microbial community in the ecosystem related to this ambient temperature system.

Ertapenem and piperacillin/tazobactam are β-lactam antibiotics with a broad spectrum of activity used for the treatment of mixed infections in which Bacteroides fragilis and Escherichia coli play an important aetiological role. In this study, the activities of piperacillin/tazobactam and ertapenem (MIC and time–kill kinetics) against these bacteria were compared. MICs were determined by the agar dilution method, and the time and slope of time–kill curves were analysed. In the in vitro pharmacodynamic assays, pure and mixed cultures of E. coli and B. fragilis were exposed to peak concentrations of ertapenem (8.0 μg ml−1) and piperacillin/tazobactam (64.0/8.0 μg ml−1) for 48 h. Treatment with ertapenem reduced the viability of E. coli and/or B. fragilis by 3 logs in all experiments, whereas piperacillin/tazobactam only affected the viability of B. fragilis. Both drugs exhibited their fastest rates of killing when bacteria were grown in mixed cultures. According to the results, ertapenem exhibited activity similar to that of piperacillin/tazobactam against B. fragilis alone or in mixed culture. However, ertapenem exhibited a markedly higher activity against E. coli alone or in combination with B. fragilis relative to piperacillin/tazobactam.

Introduction: Bacterial vaginosis (BV) is a syndrome related to Gardnerella vaginalis and is characterized by an imbalance in the vaginal microbiota. This work focused on the evaluation of antimicrobial susceptibility patterns and the occurrence of the vaginolysin ( vly ) gene in G. vaginalis isolated from BV and non-BV patients. Methodology: The vaginal secretions were collected randomly and processed for G. vaginalis isolation. The isolates were presumptively identified by β-hemolysis and oxidase and catalase tests. Polymerase chain reaction (PCR) was performed to confirm bacterial identity and to detect the vly gene. Antimicrobial susceptibility patterns were determined. Results: Of 89 patients, G. vaginalis was isolated from 42 (37 BV and 5 non-BV), and 204 isolates were selected (179 from BV and 25 non-BV). The vly gene was detected in all G. vaginalis isolated from non-BV women and in 98.3% of the bacteria from BV patients. High resistance was observed for ampicillin (54.4%), metronidazole (59.8%), tinidazole (60.3%) and secnidazole (71.6%). Conclusions: Further studies are needed to better address the role of G. vaginalis and the vly gene in BV pathogenesis.

Introduction: Bacterial resistance is a growing concern in the nosocomial environment in which Klebsiella pneumoniae and Enterobacter aerogenes play an important role due to their opportunism and carbapenemase-production. This work aimed to evaluate physiological and molecular characteristics of carbapenem-resistant K. pneumoniae and E. aerogenes isolated in a Brazilian tertiary hospital. Methodology: In total, 42 carbapenem-resistant bacteria isolated from clinical specimens were included (21 K. pneumoniae and 21 E. aerogenes). Drug-sensitive K. pneumoniae (n = 27) were also included. Antimicrobial susceptibility and biocide tolerance patterns, hemolytic activity, tolerance to oxidative stress, and aggregative ability were assessed. Genetic markers related to carbapenem resistance, or ESBL-production were screened by PCR. Results: Compared to drug-sensitive strains, carbapenem-resistant K. pneumoniae were more tolerant to biocides and to oxidative stress, and they displayed an increase in biofilm formation. The genetic markers blaKPC (95.2%) and blaTEM (90.5%) were the most frequent. Among the carbapenem-resistant E. aerogenes strains, blaKPC, and blaTEM were detected in all bacteria. Drug-sensitive E. aerogenes were not isolated in the same period. blaSHV, blaVIM, and blaCTX markers were also observed among carbapenem-resistant bacteria. Conclusions: Results suggest that carbapenemase-producing enterobacteria might show peculiar characteristics regarding their physiology associated with their environmental persistency, virulence, and multidrug resistance. The observed phenomenon may have implications not only for antimicrobial chemotherapy, but also for the prognosis of infectious diseases and infection control.

Introduction: Non-fermenting Gram-negative bacteria such as Pseudomonas aeruginosa and Acinetobacter baumannii are widespread in the environment and are increasingly associated with nosocomial infections. Extensive and indiscriminate use of antibiotics in hospitals has contributed to an increased number of infections caused by these microorganisms, that are resistant to a wide variety of antimicrobials, including β-lactams. This study aimed to isolate and identify carbapenem-resistant Acinetobacter spp. and P. aeruginosa from hospitalized patients, to determine their antimicrobial susceptibility patterns and to screen for bla OXA-23 , bla OXA-24 , bla OXA-51 , bla OXA-58 , and bla OXA-143 genes among the isolated bacteria . Methodology: Antimicrobial resistance patterns were performed using the disk-diffusion method. Genetic markers related to carbapenem resistance were screened by polymerase chain reaction. Results: Carbapenem-resistant Acinetobacter spp. (n = 44) and P. aeruginosa (n = 28) samples were isolated from patients admitted to a tertiary hospital. Polymyxin B was the only effective drug for all isolates. Considering the oxacillinase gene screening, genetic markers were observed only in Acinetobacter isolates. The most frequent genotype observed was bla OXA-23 + / bla OXA-51 + (45.5%), followed by bla OXA-51 + / bla OXA-143 + (41%). The oxacillinase genes bla OXA-24 and bla OXA-58 were not detected. High mortality rates (> 70%) were observed. Conclusions: The data suggest the need for rational use of antimicrobials associated with early diagnosis of multidrug-resistant bacteria, especially considering non-fermenting Gram-negative rods, which are widespread in hospitals. The findings of bla oxa-51 ‑ strains suggest the occurrence and spread of non- A. baumannii species throughout our hospitals. Effective implementation of surveillance programs in hospitals is needed to reduce infectious and resistant intra- and inter-species bacteria.