Spatial and temporal analysis of estuarine bacterioneuston and bacterioplankton using culture-dependent and culture-independent methodologies.

Bacterioneuston may play a key role in water-air exchange of gases and in processing organic matter and pollutants that accumulate at the sea-surface microlayer (SML). However, the phylogenetic diversity of bacterioneuston has been poorly characterized. We analyzed 24 samples each from the SML and underlying water (UW) at three sites in the Ria de Aveiro estuary, Portugal. Cultivation and culture-independent techniques were used to compare bacterioneuston and bacterioplankton. Culturable heterotrophic bacteria were enriched in the SML. The culturable community was dominated by Psychrobacter and Acinetobacter. The presence of high numbers of Psychrobacter was a notable result. Differences were confined to a few genera overrepresented in UW samples (Kocuria, Agrococcus and Vibrio). 16S rDNA DGGE profiles were highly stable in terms of number and position of bands between sampling sites and dates but cluster analysis revealed a slight tendency for grouping according to sampled layer. SML-specific DGGE bands affiliated with Actinobacteria, Cyanobacteria, Gammaproteobacteria and Bacteroidetes. Low similarity between nucleotide sequences of DGGE-bands and previously reported sequences suggest the occurrence of SML-specific populations. Enrichment of SML for Pseudomonas and Aeromonas was questioned and the diversity of both communities was analyzed. Consistent differences between SML and UW aeromonads communities were not identified. In terms of Pseudomonas, a culturable operational taxonomic unit was consistently overrepresented within SML samples. Taken together, our results indicate that the similarity between SML and UW communities depends on spatial and temporal factors.

Occurrence and diversity of integrons and beta-lactamase genes among ampicillin-resistant isolates from estuarine waters.

The aim of the present study was to assess the occurrence and molecular diversity of beta-lactamase genes and integrons among Gram-negative ampicillin-resistant bacteria from Ria de Aveiro. Ampicillin-resistant isolates were selected and subjected to genotyping using REP-PCR. Representatives from each REP pattern were affiliated with the following taxa by sequencing the 16S rRNA gene: Aeromonas caviae, A. hydrophila, A. media, A. molluscorum, A. veronii, A. salmonicida, Aeromonas sp., Pseudomonas putida, Pseudomonas sp., Escherichia coli, Escherichia sp., Shigella sonnei, Shigella sp., Klebsiella pneumoniae, K. oxytoca, Raoultella ornithinolytica, R. terrigena, R. planticola, Citrobacter freundii, Morganella morganii and Enterobacter sp. Isolates affiliated with genera Escherichia or Shigella were identified as Escherichia coli using phenotypic-based tests. PCR was used to assess beta-lactamase encoding sequences (bla(TEM), bla(SHV), bla(CARB), bla(CTX-M), bla(IMP), bla(VIM), bla(CphA/IMIS), bla(OXA-A), bla(OXA-B), bla(OXA-C)), class 1 and class 2 integrases, and integron variable regions. Sequence analysis of PCR products was performed. beta-Lactamase genes were detected in 77.8% of the Enterobacteriaceae and in 10.5% of the Aeromonas. The most frequently detected gene was bla(TEM), followed by bla(SHV,)bla(OXA-B), bla(CphA/IMIS) and bla(CARB). Retrieved sequences shared high homology with previously described beta-lactamases. The intI1 gene was present in 29.6% of the Enterobacteriaceae and in 21% of the Aeromonas isolates. The intI2 gene was present in 4 isolates. A total of 13 cassettes included in 12 different cassette arrays were identified. The most frequently found resistance gene cassettes were aadA variants. Previous investigations based on cultivation-independent approaches revealed higher molecular diversity among beta-lactamase-encoding sequences in this estuary. This fact reinforces the hypothesis that cultivation-dependent approaches may underestimate the prevalence of antibiotic resistance genes in environmental samples and may introduce bias in the recovery of their molecular variants.

Low Prevalence of Carbapenem-Resistant Bacteria in River Water: Resistance Is Mostly Related to Intrinsic Mechanisms.

Carbapenems are last-resort antibiotics to handle serious infections caused by multiresistant bacteria. The incidence of resistance to these antibiotics has been increasing and new resistance mechanisms have emerged. The dissemination of carbapenem resistance in the environment has been overlooked. The main goal of this research was to assess the prevalence and diversity of carbapenem-resistant bacteria in riverine ecosystems. The presence of frequently reported carbapenemase-encoding genes was inspected. The proportion of imipenem-resistant bacteria was on average 2.24 CFU/ml. Imipenem-resistant strains (n=110) were identified as Pseudomonas spp., Stenotrophomonas maltophilia, Aeromonas spp., Chromobacterium haemolyticum, Shewanella xiamenensis, and members of Enterobacteriaceae. Carbapenem-resistant bacteria were highly resistant to other beta-lactams such as quinolones, aminoglycosides, chloramphenicol, tetracyclines, and sulfamethoxazole/trimethoprim. Carbapenem resistance was mostly associated with intrinsically resistant bacteria. As intrinsic resistance mechanisms, we have identified the blaCphA gene in 77.3% of Aeromonas spp., blaL1 in all S. maltophilia, and blaOXA-48-like in all S. xiamenensis. As acquired resistance mechanisms, we have detected the blaVIM-2 gene in six Pseudomonas spp. (5.45%). Integrons with gene cassettes encoding resistance to aminoglycosides (aacA and aacC genes), trimethoprim (dfrB1b), and carbapenems (blaVIM-2) were found in Pseudomonas spp. Results suggest that carbapenem resistance dissemination in riverine ecosystems is still at an early stage. Nevertheless, monitoring these aquatic compartments for the presence of resistance genes and its host organisms is essential to outline strategies to minimize resistance dissemination.

Detection of HIV-1 subtype G using Cobas Ampliscreen test.

A nucleic acid amplification test for detection of HIV-1 RNA was designed for screening pools of human plasma. This test achieves a similar level of sensitivity for group M subtypes, but few samples of subtype G and none of its CRFs had been tested, which are the most relevant in Portugal. We found that the test is effective in detecting HIV-1 subtypes and has an analytical sensitivity similar to B subtype.

Molecular sequence analysis of prokaryotic diversity in the middle and outer sections of the Portuguese estuary Ria de Aveiro.

Construction of 16S rDNA libraries was undertaken to examine the structure of free-living bacterial communities in the estuarine system Ria de Aveiro, Portugal. Samples were collected in April 2002, from two sites representing marine (station N1) and brackish (station I6) water zones. Clones were characterized by RFLP patterns and sequence analysis of representative clones revealed that both libraries were clearly dominated by alpha-proteobacteria, followed by gamma-proteobacteria and beta-proteobacteria. Clones affiliated with the delta-proteobacteria, Verrucomicrobia, Bacteroidetes and Actinobacteria were exclusive of station I6 and sequences related to the Firmicutes were only found in station N1. Sequences retrieved are included in only a few major bacterial divisions and in general, shared a high degree of homology with sequences deposited in nucleotide databases, and recovered from aquatic environments of diverse geographic regions. Differences between the two sites may reflect adaptation to different environmental conditions, especially salinity. The pattern of prokaryotic diversity is comparable to other coastal and estuarine environments previously studied.

Gulls identified as major source of fecal pollution in coastal waters: a microbial source tracking study.

Gulls were reported as sources of fecal pollution in coastal environments and potential vectors of human infections. Microbial source tracking (MST) methods were rarely tested to identify this pollution origin. This study was conducted to ascertain the source of water fecal contamination in the Berlenga Island, Portugal. A total of 169 Escherichia coli isolates from human sewage, 423 isolates from gull feces and 334 water isolates were analyzed by BOX-PCR. An average correct classification of 79.3% was achieved. When an 85% similarity cutoff was applied 24% of water isolates were present in gull feces against 2.7% detected in sewage. Jackknifing resulted in 29.3% of water isolates classified as gull, and 10.8% classified as human. Results indicate that gulls constitute a major source of water contamination in the Berlenga Island. This study validated a methodology to differentiate human and gull fecal pollution sources in a real case of a contaminated beach.

Prevalence and diversity of carbapenem-resistant bacteria in untreated drinking water in Portugal.

We examined the prevalence and diversity of carbapenem-resistant bacteria (CRB) in untreated drinking water. Prevalence was estimated in plate count agar (PCA) and R2A media with or without antibiotics. Clonal relatedness of isolates was established by repetitive extragenic palindroitic (REP)-PCR. Phylogeny was based on the 16S rRNA gene. Antimicrobial susceptibility was assessed by disc diffusion methods. Genes encoding beta-lactamases and integrases were inspected by PCR. CRB ranged from 0.02% to 15.9% of cultivable bacteria, while ampicillin-resistant bacteria ranged from 1.5% to 31.4%. Carbapenem-resistant isolates affiliated with genera Stenotrophomonas, Pseudomonas, Janthinobacterium, Chryseobacterium, Sphingobacterium, Acidovorax, Caulobacter, Cupriavidus, and Sphingomonas. CRB were highly resistant to beta-lactams, but mostly susceptible to other classes. Transmissible beta-lactamase genes and integrase genes were not detected. The genus-specific bla(L1) was detected in 61% of the Stenotrophomonas isolates. Contrarily to what has been reported for extensively used antibiotics, low levels of carbapenem resistance were detected in untreated drinking water, often represented by intrinsically resistant genera. Production of chromosomal-encoded carbapenemases was the prevalent carbapenem resistance mechanism. Results suggest that the dissemination of anthropogenic-derived carbapenem resistance is at an early stage. This presents an opportunity to rationally develop monitoring strategies to identify dissemination routes and assess the impact of human actions in the environmental resistome.

Changes in the bacterial community structure in two-stage constructed wetlands with different plants for industrial wastewater treatment.

This study focused on the diversity of bacterial communities from two series of two-stage constructed wetlands (CWs) treating tannery wastewater, under different hydraulic conditions. Series were separately planted with Typha latifolia and Phragmites australis in expanded clay aggregates and operated for 31 months. The effect of plant species, hydraulic loading and unit stage on bacterial communities was addressed through bacterial enumeration and denaturating gradient gel electrophoresis (DGGE). Diverse and distinct bacterial communities were found in each system unit, which was related in part to the type of plant and stage position (first or second unit in the series). Numerical analysis of DGGE profiles showed high diversity in each unit with an even distribution of species. No clear relation was established between the sample collection time, hydraulic loading applied and the bacterial diversity. Isolates retrieved from plant roots and substrates of CWs were affiliated with gamma-Proteobacteria, Firmicutes, alpha-Proteobacteria, Sphingobacteria, Actinobacteria and Bacteroidetes. Both series were effective in removing organic matter from the inlet wastewater, however, based on batch degradation experiments it seems that biodegradation was limited by the recalcitrant properties of the wastewater.