Anammox bacteria enrichment and characterization from municipal activated sludge.

A sustainable option for nitrogen removal is the anaerobic ammonium-oxidizing (anammox) process in which ammonium is oxidized to nitrogen gas with nitrite as electron acceptor. Application of this process, however, is limited by the availability of anammox biomass. In this study, two Brocadia-like anammox phylotypes were successfully enriched, detected and identified from an activated sludge taken from a domestic wastewater treatment plant (Minas Gerais, Brazil) employing a Sequencing Batch Reactor (SBR). The dominant phylotype was closely related to 'Candidatus Brocadia sinica', but one clone seemed to represent a novel species for which we propose the name 'Candidatus Brocadia brasiliensis'. Based on Fluorescence in situ hybridization (FISH) analysis, this enrichment led to a relative population size of 52.7% (±15.6) anammox bacteria after 6 months of cultivation. The cultivation process can be divided into three phases: phase 1 (approximately 25 days) was characterized by heterotrophic denitrification metabolism, phase 2 was the propagation phase and phase 3 (from the 87th day onwards), in which significant anammox activity was detected. A long-term performance of the SBR showed a near perfect removal of nitrite based on the influent NO(2)(-)-N concentration of 61-95 mg L(-1). The average ammonia removal efficiency was 90% with the influent NH(4)(+)-N concentration of 55-82 mg L(-1). Therefore, anammox cultivation and enrichment from activated sludge was possible under a controlled environment within 3 months.

Dynamics of Aeromonas species isolated from wastewater treatment system.

Aeromonas are widely distributed in the aquatic environment, and are considered to be emerging organisms that can produce a series of virulence factors. The present study was carried out in a sanitary sewage stabilization pond treatment system, located in Lins, State of São Paulo, Brazil. Most probable number was applied for estimation of the genus Aeromonas. Colony isolation was carried out on blood agar ampicillin and confirmed by biochemical characterization. Aeromonas species were isolated in 72.4% of influent samples, and in 55.2 and 48.3% of effluent from anaerobic and facultative lagoons, respectively. Thirteen Aeromonas species were isolated, representing most of the recognized species of these organisms. Even though it was possible to observe a tendency of decrease, total elimination of these organisms from the studied system was not achieved. Understanding of the pathogenic organism's dynamics in wastewater treatment systems with a reuse potential is especially important because of the risk it represents.

Emergence of Klebsiella pneumoniae carrying the novel extended-spectrum beta-lactamase gene variants bla(SHV-40), bla(TEM-116) and the class 1 integron-associated bla(GES-7) in Brazil.

A clinical Klebsiella pneumoniae isolate carrying the extended-spectrum beta-lactamase gene variants bla(SHV-40), bla(TEM-116) and bla(GES-7) was recovered. Cefoxitin and ceftazidime activity was most affected by the presence of these genes and an additional resistance to trimethoprim-sulphamethoxazole was observed. The bla(GES-7) gene was found to be inserted into a class 1 integron. These results show the emergence of novel bla(TEM) and bla(SHV) genes in Brazil. Moreover, the presence of class 1 integrons suggests a great potential for dissemination of bla(GES) genes into diverse nosocomial pathogens. Indeed, the bla(GES-7) gene was originally discovered in Enterobacter cloacae in Greece and, to our knowledge, has not been reported elsewhere.

Molecular detection of enterotoxins in environmental strains of Aeromonas hydrophila and Aeromonas jandaei.

Aeromonas species are widely distributed in aquatic environments and recent studies include the genus in the emergent pathogens group because of its frequent association with local and systemic infections in immunocompetent humans. Aiming to search for virulence genes in environmental strains of Aeromonas hydrophila and Aeromonas jandaei, we designed specific primers to detect act/hlyA/aer complex and alt genes. Primers described elsewhere were used to detect ast. Eighty-seven strains previously identified using phenotypic and genotypic tests as A. hydrophila (41) and A. jandaei (46) were analysed for the presence of the virulence genes using PCR. DNA fragments of expected size were purified and directly sequenced. Among the 41 strains of A. hydrophila 70.7% (29), 97.6% (40) and 26.8% (11) possessed act/hlyA/aer complex, ast and alt genes, respectively. Among the 46 strains of A. jandaei, 4.4% (2), 0% (0) and 32.6% (15) were positive for act/hly A/aer complex, ast and alt genes, respectively. Sequencing allowed for the confirmation of amplified products using BLAST. The present work proposes a specific and rapid diagnostic method to detect the main virulence determinants of Aeromonas, a genus potentially pathogenic to humans.

Detection of metallo-beta-lactamases-encoding genes in environmental isolates of Aeromonas hydrophila and Aeromonas jandaei.

AIMS: To determine the prevalence and expression of metallo-beta-lactamases (MBL)-encoding genes in Aeromonas species recovered from natural water reservoirs in southeastern Brazil. METHODS AND RESULTS: Eighty-seven Aeromonas isolates belonging to Aeromonas hydrophila (n = 41) and Aer. jandaei (n = 46) species were tested for MBL production by the combined disk test using imipenem and meropenem disks as substrates and EDTA or thioglycolic acid as inhibitors. The presence of MBL genes was investigated by PCR and sequencing using new consensus primer pairs designed in this study. The cphA gene was found in 97.6% and 100% of Aer. hydrophila and Aer. jandaei isolates, respectively, whereas the acquired MBL genes bla(IMP), bla(VIM) and bla(SPM-1) were not detected. On the other hand, production of MBL activity was detectable in 87.8% and 10.9% of the cphA-positive Aer. hydrophila and Aer. jandaei isolates respectively. CONCLUSIONS: Our results indicate that cphA seems to be intrinsic in the environmental isolates of Aer. hydrophila and Aer. jandaei in southeastern Brazil, although, based on the combined disk test, not all of them are apparently able to express the enzymatic activity. SIGNIFICANCE AND IMPACT OF THE STUDY: These data confirm the presence of MBL-producing Aeromonas species in natural water reservoirs. Risk of waterborne diseases owing to domestic and industrial uses of freshwater should be re-examined from the increase of bacterial resistance point of view.

Risk factors for colonisation of newborn infants during an outbreak of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae in an intermediate-risk neonatal unit.

We describe a cross-sectional survey to identify risk factors for colonisation of neonates by extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae. This occurred following exposure to a colonised healthcare worker during an outbreak in an intermediate-risk neonatal unit. In total, 120 neonates admitted consecutively during a three-month period were screened for ESBL-producing K. pneumoniae by rectal swabbing and 27 were identified as colonised. Multivariate analysis showed colonisation to be independently associated with use of antibiotics and absence of breastfeeding. Previous use of antibiotics presented an odds ratio (OR) of 12.3 [95% confidence interval (CI): 3.66-41.2, P<0.001]. The most commonly used antibiotics were penicillin and amikacin. Breastfeeding was associated with reduced risk for colonisation (OR: 0.22; 95% CI: 0.05-0.99; P=0.049). Nine isolates recovered during the first stage of the outbreak and 27 isolates from surveillance cultures were typed thereafter by pulsed-field gel electrophoresis, revealing six different profiles (A-F). Clones A, C, and E were implicated in the first stage of the outbreak, whereas among the 27 strains recovered from surveillance cultures, all six clones were identified. Clone A was also found on the hand of a nursing auxiliary with onychomycosis. We concluded that prior antimicrobial use predisposed to colonisation. The possible role of breastfeeding as a protective factor needs to be further elucidated. Detection of different genotypes of ESBL-producing K. pneumoniae suggests that dissemination of mobile genetic elements bearing the ESBL gene may have been superimposed on the simple dissemination of a clone during the outbreak.

Sanitation assessment of wastewater treated by stabilization ponds for potential reuse in agricultural irrigation sanitation assessment.

Wastewater reuse has become an important alternative to agricultural irrigation; on the other hand, it poses concern with regard to public health. Total coliform and Escherichia coli concentration, presence of helminth eggs and Salmonella, and physical-chemical parameters were evaluated in raw and treated wastewater. Chemical and biochemical oxygen demand removal efficiency was 74.6 and 77.9%, respectively. As for organic nitrogen, total phosphorus, and total suspended solids, total efficiency removal was 17.4, 12.5, and 32.9%, respectively. The average density of total coliforms and E. coli was 3.5 x 10(9) and 1.8 x 10(8) MPN/100 mL and 1.1 x 10(7) MPN/100 mL and 3.9 x 10(5) MPN/100 mL for raw and treated wastewater, respectively. Ascaris eggs were observed in 80.8% of the samples collected, and viable eggs in 42.3% of the samples. Salmonella was detected in 36.4% of the samples. The values observed in treated wastewater did not show the adequate bacteriological quality, as recommended by World Health Organization (Geneva, Switzerland). Therefore, additional measures should be taken to achieve an improved microbiological and parasitological quality.

Retrieval of the species Alteromonas tetraodonis Simidu et al. 1990 as Pseudoalteromonas tetraodonis comb. nov. and emendation of description.

A polyphasic taxonomy study was undertaken of three strains of Pseudoalteromonas haloplanktis subsp. tetraodonis (Simidu et al. 1990) Gauthier et al. 1995. DNA was prepared from each of the strains and genomic relatedness was measured by DNA-DNA hybridization. Strains KMM 458T and IAM 14160T shared 99% genetic relatedness, but were only 48-49% related to the type strain of Pseudoalteromonas haloplanktis subsp. haloplanktis, IAM 12915T. The third strain, P. haloplanktis subsp. tetraodonis A-M, showed 83% genetic similarity with P. haloplanktis subsp. haloplanktis IAM 12915T and 32% with KMM 458T. From these results, it is concluded that strains KMM 458T and IAM 14160T comprise a separate species, originally described as Alteromonas tetraodonis, whereas strain A-M belongs to the species Pseudoalteromonas haloplanktis. Based on phenotypic and chemotaxonomic data, genomic fingerprint patterns, DNA-DNA hybridization data and phylogenetic analysis of 16S rRNA, it is proposed that the species Alteromonas tetraodonis be retrieved and recognized as Pseudoalteromonas tetraodonis comb. nov. (type strain IAM 14160T = KMM 458T).

Molecular analysis of Vibrio cholerae O1, O139, non-O1, and non-O139 strains: clonal relationships between clinical and environmental isolates.

A total of 26 strains of Vibrio cholerae, including members of the O1, O139, and non-O1, non-O139 serogroups from both clinical and environmental sources, were examined for the presence of genes encoding cholera toxin (ctxA), zonula occludens toxin (zot), accessory cholera enterotoxin (ace), hemolysin (hlyA), NAG-specific heat-stable toxin (st), toxin-coregulated pilus (tcpA), and outer membrane protein (ompU), for genomic organization, and for the presence of the regulatory protein genes tcpI and toxR in order to determine relationships between epidemic serotypes and sources of isolation. While 22 of the 26 strains were hemolytic on 5% sheep blood nutrient agar, all strains were PCR positive for hlyA, the hemolysin gene. When multiplex PCR was used, all serogroup O1 and O139 strains were positive for tcpA, ompU, and tcpI. All O1 and O139 strains except one O1 strain and one O139 strain were positive for the ctxA, zot, and ace genes. Also, O1 strain VO3 was negative for the zot gene. All of the non-O1, non-O139 strains were negative for the ctxA, zot, ace, tcpA, and tcpI genes, and all of the non-O1, non-O139 strains except strain VO26 were negative for ompU. All of the strains except non-O1, non-O139 strain VO22 were PCR positive for the gene encoding the central regulatory protein, toxR. All V. cholerae strains were negative for the NAG-specific st gene. Of the nine non-ctx-producing strains of V. cholerae, only one, non-O1, non-O139 strain VO24, caused fluid accumulation in the rabbit ileal loop assay. The other eight strains, including an O1 strain, an O139 strain, and six non-O1, non-O139 strains, regardless of the source of isolation, caused fluid accumulation after two to five serial passages through the rabbit gut. Culture filtrates of all non-cholera-toxigenic strains grown in AKI media also caused fluid accumulation, suggesting that a new toxin was produced in AKI medium by these strains. Studies of clonality performed by using enterobacterial repetitive intergenic consensus sequence PCR, Box element PCR, amplified fragment length polymorphism (AFLP), and pulsed-field gel electrophoresis (PFGE) collectively indicated that the V. cholerae O1 and O139 strains had a clonal origin, whereas the non-O1, non-O139 strains belonged to different clones. The clinical isolates closely resembled environmental isolates in their genomic patterns. Overall, there was an excellent correlation among the results of the PCR, AFLP, and PFGE analyses, and individual strains derived from clinical and environmental sources produced similar fingerprint patterns. From the results of this study, we concluded that the non-cholera-toxin-producing strains of V. cholerae, whether of clinical or environmental origin, possess the ability to produce a new secretogenic toxin that is entirely different from the toxin produced by toxigenic V. cholerae O1 and O139 strains. We also concluded that the aquatic environment is a reservoir for V. cholerae O1, O139, non-O1, and non-O139 serogroup strains.

Idiomarina gen. nov., comprising novel indigenous deep-sea bacteria from the Pacific Ocean, including descriptions of two species, Idiomarina abyssalis sp. nov. and Idiomarina zobellii sp. nov.

Two bacterial strains, KMM 227T and 231T, were isolated from seawater samples collected from the north-western Pacific Ocean at a depth of 4000-5000 m and were characterized using polyphasic taxonomy. Both were Gram-negative, psychrotolerant, heterotrophic, aerobic and required NaCl for growth (0.6-15.0%). The temperature for growth was 4-30 degrees C. Both strains were rod-shaped, with a single flagellum. However, strain KMM 231T revealed a single long fimbrium. Cellular fatty acids detected in the isolates were predominantly odd-numbered and iso-branched, with 15 and 17 carbons (ca. 70%). Also present were saturated and monounsaturated straight-chain fatty acids. Results of phylogenetic analyses, employing three tree-making methods, strongly indicated that the two strains formed a distinct lineage within a clade containing the genera Alteromonas, Colwellia and Pseudoalteromonas, in the gamma-Proteobacteria. The two strains shared 16S rDNA sequence similarity of 96.9% and genomic DNA relatedness of 27%; the latter was determined by dot-blot hybridization. The strains were differentiated by the presence of fimbria, production of chitinase, ability to grow on 15% NaCl and BIOLOG profiles. Given the polyphasic evidence accumulated in this study, it is proposed that the two deep-sea isolates be classified in the genus Idiomarina gen. nov., as Idiomarina abyssalis sp. nov. (type strain is KMM 227T) and Idiomarina zobellii sp. nov. (type strain is KMM 231T).

Potentially pathogenic vibrios associated with mussels from a tropical region on the Atlantic coast of Brazil.

Mussels (Perna perna) harvested on the coast of Ubatuba, in three different stations in the State of São Paulo, Brazil, were examined for Vibrio spp. over a 1 year period. The ranges of most probable number (MPN 100 g-1) were: Vibrio alginolyticus (< 3-24,000), V. parahaemolyticus (< 3-24,000), V. fluvialis (< 3-1100), V. cholerae non-O1 (< 3-23), V. furnissii (< 3-30), V. mimicus (< 3-9) and V. vulnificus (< 3-3). The highest incidence was observed for V. alginolyticus (92-100%), followed by V. parahaemolyticus (67-92%), V. fluvialis (34-67%), V. vulnificus (8-17%), V. furnissii (0-17%), V. mimicus (0-17%) and V. cholerae non-O1 (0-8%). Tests for virulence factors were positive in 34.1% of the vibrios in the rabbit ileal loop and 31.7% in the Dean test. Positive results in the Kanagawa test were obtained with 0.51% of V. parahaemolyticus strains. The mean values (MPN 100 g-1) of faecal coliforms in mussels from the three regions varied from 1100 to 44,000, and seawater collected at the same stations gave average values for faecal coliforms in the range 18-3300 MPN 100 ml-1. These results highlight the potential risks of food poisoning associated with raw or undercooked seafood.