Diversity and antibiotic resistance profiles of Pseudomonads from a hospital wastewater treatment plant.

AIMS: To evaluate the diversity of Pseudomonads and antibiotic resistance profiles of Pseudomonas aeruginosa in a hospital wastewater treatment plant (HWTP) located in Rio de Janeiro city, Brazil. Due its intrinsic multidrug resistance and its ability to colonize several environments, we selected Ps. aeruginosa isolates as indicator of antimicrobial resistance frequency. METHODS AND RESULTS: Twenty-seven Ps. aeruginosa strains isolated from five stages of HWTP identified by rrs 16S rDNA sequencing were submitted against 12 antimicrobials through disc diffusion method. Among these isolates, 62·9% showed aztreonam resistance, followed by ticarcillin/clavulanic acid (33·3%) and cefepime (22·2%). Of these isolates, 22·2% were classified as multidrug-resistant (MDR ≥ 3 classes). Five 16S rRNA gene libraries of Pseudomonas genus were constructed, one for each stages of the plant, yielding 93 sequences clustered in 41 Operational Taxonomic Units (OTUs). Each treatment step showed unique OTU's composition, suggesting changes in Pseudomonas spp. communities during the process. Several Pseudomonas species involved in biodegradation and bioremediation of xenobiotics were detected suggesting a positive impact in the wastewater treatment. CONCLUSIONS: Our strategy using metagenomics associated with the isolation of Ps. aeruginosa strains as bio-indicator allowed us to assess their antimicrobial susceptibility, the viability and diversity of Pseudomonas species in the hospital wastewater. SIGNIFICANCE AND IMPACT OF THE STUDY: The presence of MDR bacteria from treated effluents alerts for the need to improve these systems to avoid the spreading of resistance genes in aquatic ecosystems. This has special relevance in Brazil, where a significant portion of the population has no access to treated water.

Genotypic characteristics of multidrug-resistant Pseudomonas aeruginosa from hospital wastewater treatment plant in Rio de Janeiro, Brazil.

AIMS: To investigate Pseudomonas aeruginosa isolates from a hospital wastewater treatment plant (HWTP), focusing on enzyme-based mechanisms of ß-lactams resistance and the genetic relatedness among isolates. METHODS AND RESULTS: Forty-one Ps. aeruginosa strains recovered from a HWTP were identified by amplification of 16S rRNA gene. ß-lactamase production was screened by disc diffusion, CHROMagar extended-spectrum ß-lactamase (ESBL) and ß-lactamase strips. ß-lactamase and ESBL producing isolates were investigated by PCR for the presence of ESBL, metallo-ß-lactamase and Klebsiella pneumoniae carbapenemase encoding genes. Thirty-four isolates (83%) were resistant to at least one antibiotic belonging to three or more classes. Out of these 34 isolates, 28 (82%) were classified as multidrug-resistant (MDR) and 6 (18%) extensively drug-resistant (XDR). Genetic relatedness by Enterobacterial Repetitive Intergenic Consensus sequence-PCR and Multilocus sequence typing analysis showed 20 distinct profiles and 15 sequencing types respectively. Clonal Complex 244 (CC244) shows the pathogenic potential of this clone carrying MDR and XDR strains from clinical, environmental and hospital waste sources. CONCLUSIONS: Our results suggest that treatment facilities for hospital wastewater can stimulate the increase of antimicrobial resistance bacteria and genes. SIGNIFICANCE AND IMPACT OF THE STUDY: The great genetic diversity of Ps. aeruginosa recovered from HWTP constantly released into aquatic systems allow the spread of antimicrobial-resistant organisms and genes.

Prokaryotic diversity in one of the largest hypersaline coastal lagoons in the world.

Araruama Lagoon is an environment characterized by high salt concentrations. The low raining and high evaporation rates in this region favored the development of many salty ponds around the lagoon. In order to reveal the microbial composition of this system, we performed a 16S rRNA gene survey. Among archaea, most clones were related to uncultured environmental Euryarchaeota. In lagoon water, we found some clones related to Methanomicrobia and Methanothermococcus groups, while in the saline pond water members related to the genus Haloarcula were detected. Bacterial community was dominated by clones related to Gamma-proteobacteria, Actinobacteria, and Synechococcus in lagoon water, while Salinibacter ruber relatives dominated in saline pond. We also detected the presence of Alpha-proteobacteria, Pseudomonas-like bacteria and Verrucomicrobia. Only representatives of the genus Ralstonia were cosmopolitan, being observed in both systems. The detection of a substantial number of clones related to uncultured archaea and bacteria suggest that the hypersaline waters of Araruama harbor a pool of novel prokaryotic phylotypes, distinct from those observed in other similar systems. We also observed clones related to halophilic genera of cyanobacteria that are specific for each habitat studied. Additionally, two bacterioplankton molecular markers with ecological relevance were analyzed, one is linked to nitrogen fixation (nifH) and the other is linked to carbon fixation by bacterial photosynthesis, the protochlorophyllide genes, revealing a specific genetic distribution in this ecosystem. This is the first study of the biogeography and community structure of microbial assemblages in Brazilian tropical hypersaline environments. This work is directed towards a better understanding of the free-living prokaryotic diversity adapted to life in hypersaline waters.

Neisseria meningitidis PorA variable regions: rapid detection of P1.7 and P1.19 variants by PCR.

AIM: Rapid characterization of variable region (VR)1 variants of the porA gene among invasive strains is crucial for outbreak management and epidemiology studies. Recent sequence analysis studies in Brazil showed that the VR1 P1.7 and P1.19 variants are highly prevalent, accounting for 68%, of the total number of VR1 variants characterized. The aim of this work is to develop a rapid polymerase chain reaction (PCR)-based method for genosubtyping Neisseria meningitidis by detection of porA variable regions P1.7 and P1.19. METHODS AND RESULTS: PCR primers for the detection of porA VR1 P1.7 and P1.19 were designed and tested using 198 clinical N. meningitidis isolates that had been previously evaluated by porA sequencing. All 50 strains with VR1 P1.7 and all 65 strains with VR1 P1.19 were positively identified by the respective VR-specific PCR and no false-positive reactions occurred. CONCLUSIONS: VR-specific PCR amplification accurately identified VR P1.7 and P1.19 strains. SIGNIFICANCE AND IMPACT OF THE STUDY: To overcome the disadvantages of serosubtyping and sequencing for typing the porA VR1 segment of N. meningitidis, we developed a PCR-based method to rapidly and accurately detect VR1 P1.7 and P1.19 variants. This approach is highly specific and sensitive; moreover it may allow for genotype determination of culture-negative samples.

Exploring the biotechnologial applications in the archaeal domain

Archaea represent a considerable fraction of the prokaryotic world in marine and terrestrial ecosystems, indicating that organisms from this domain might have a large impact on global energy cycles. The extremophilic nature of many archaea has stimulated intense efforts to understand the physiological adaptations for living in extreme environments. Their unusual properties make them a potentially valuable resource in the development of novel biotechnological processes and industrial applications as new pharmaceuticals, cosmetics, nutritional supplements, molecular probes, enzymes, and fine chemicals. In the present mini-review, we show and discuss some exclusive characteristics of Archaea domain and the current knowledge about the biotechnological uses of the archaeal enzymes. The topics are: archaeal characteristics, phylogenetic division, biotechnological applications, isolation and cultivation of new microbes, achievements in genomics, and metagenomic.

As arqueas representam uma considerável fração dos procariotos nos ecossistemas marinhos e terrestes, indicando que estes organismos devem possuir um grande impacto nos ciclos energéticos. A natureza extremofílica de muitas arqueas tem estimulado intensos esforços para compreender sua adaptação fisiológica a ambientes extremos. Suas propriedades incomus as tornam uma fonte valiosa no desenvolvimento de novos processos biotecnológicos e aplicações industriais como novos fármacos, cosméticos, suplementos nutricionais, sondas moleculares, enzimas e reagentes. Na presente mini-revisão, mostramos e discutimos algumas de suas características exclusivas correlacionando-as com seu potencial biotecnológico e aplicação industrial. Os tópicos são: características das arqueas, divisão filogenética, aplicações biotecnológicas, isolamento e cultivo de novos microrganismos, genoma e metagenoma.

Archaeal diversity in naturally occurring and impacted environments from a tropical region.

AIMS: To evaluate archaeal diversity in natural and impacted habitats from Rio de Janeiro state, Brazil, a tropical region of South America. METHODS AND RESULTS: 16S rRNA gene was amplified directly by polymerase chain reaction (PCR) from genomic DNA, extracted from Guanabara Bay (GB) water, halomarine sediment (HS), municipal landfill leachate, agricultural soil and wastewater treatment (WT) system. Five archaeal 16S rDNA clone libraries were constructed. A total of 123 clones, within the five libraries analysed, were clustered into 29 operational taxonomic units, related to cultivated (24%) and uncultivated (76%) organisms. Rarefaction analysis showed that the libraries contained different levels of diversity. PCR-denaturing gradient gel electrophoresis (DGGE) of 16S-23S intergenic spacer regions confirmed the presence of a dominant phylotype, revealed by the WT system clone library. CONCLUSIONS: Archaeal communities of impacted environments seem to be confined to specific ecosystems with similar physicochemical properties, while communities from natural environments appear to be widely distributed. The presence of a high number of phylotypes related to uncultivated organisms suggests new archaeal lineages. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports, for the first time, the analysis of archaeal diversity in tropical environments from Brazil, and adds sequences from this region to the developing database of 16S rRNA clone libraries from environmental samples.

Seroprevalence and Toxoplasma gondii-IgG avidity in sheep from Lajes, Brazil.

Sheep are important domestic animals in the Northeast region of Brazil due to their minimal rearing and maintenance costs, and to their production of both meat and milk. In animals, Toxoplasma gondii infection results in significant reproductive and economic losses. The epidemiology of toxoplasmosis in sheep in the Northeast of Brazil has been little studied; particularly in Rio Grande do Norte State. Sera from 102 sheep intended for consumption in Lajes were subjected to the Toxoplasma-ELISA test to detect anti-T. gondii specific IgG confirming a past infection. Of the total tested, 30 (29.41%) sera were positive for IgG with an increasing number of positive animals with advancing age. We used IgG avidity ELISA in 30 positive samples and observed that 6 (20%) had low avidity antibodies and 24 (80%) had high avidity antibodies. Epidemiological studies are required in order to identify sources of infection for these hosts as well as their impact on animal breeding in the region and risk of transmission to humans.

PCR analyses of tRNA intergenic spacer, 16S-23S internal transcribed spacer, and randomly amplified polymorphic DNA reveal inter- and intraspecific relationships of Enterobacter cloacae strains.

PCR analysis of tRNA intergenic spacer (tDNA-PCR) and of the 16S-23S internal transcribed spacer (ITS-PCR) and random amplified polymorphic DNA (RAPD) analysis were evaluated for their usefulness in characterization of Enterobacter cloacae strains isolated from both clinical origins and vaccine microbial contamination. tDNA-PCR presented specific and reproducible patterns for Enterobacter sakazakii ATCC 29004, Enterobacter aerogenes ATCC 13048, and Enterobacter cloacae ATCC 13047 and 23355 that presented the same profile for all 16 E. cloacae isolates, offering an alternative tool for species-level identification. ITS-PCR and RAPD analysis yielded completely different banding patterns for the 20 strains studied, except for E. cloacae strains isolated from different batches of vaccine that exhibited a unique pattern, suggesting contamination by the same strain. The combined use of tDNA-PCR and ITS-PCR in a one-step protocol allows accurate identification and typing of E. cloacae strains a few hours after the colony has been isolated.