Microbiome and Resistome Profiles along a Sewage-Effluent-Reservoir Trajectory Underline the Role of Natural Attenuation in Wastewater Stabilization Reservoirs.

Antibiotic-resistant bacteria and antibiotic resistance gene (ARGs) loads dissipate through sewage treatment plants to receiving aquatic environments, but the mechanisms that mitigate the spread of these ARGs are not well understood due to the complexity of full-scale systems and the difficulty of source tracking in downstream environments. To overcome this problem, we targeted a controlled experimental system comprising a semicommercial membrane-aerated bioreactor (MABR), whose effluents fed a 4,500-L polypropylene basin that mimicked effluent stabilization reservoirs and receiving aquatic ecosystems. We analyzed a large set of physicochemical measurements, concomitant with the cultivation of total and cefotaxime-resistant Escherichia coli, microbial community analyses, and quantitative PCR (qPCR)/digital droplet PCR (ddPCR) quantification of selected ARGs and mobile genetic elements (MGEs). The MABR removed most of the sewage-derived organic carbon and nitrogen, and simultaneously, E. coli, ARG, and MGE levels dropped by approximately 1.5- and 1.0-log unit mL-1, respectively. Similar levels of E. coli, ARGs, and MGEs were removed in the reservoir, but interestingly, unlike in the MABR, the relative abundance (normalized to 16S rRNA gene-inferred total bacterial abundance) of these genes also decreased. Microbial community analyses revealed the substantial shifts in bacterial and eukaryotic community composition in the reservoir relative to the MABR. Collectively, our observations lead us to conclude that the removal of ARGs in the MABR is mainly a consequence of treatment-facilitated biomass removal, whereas in the stabilization reservoir, mitigation is linked to natural attenuation associated with ecosystem functioning, which includes abiotic parameters, and the development of native microbiomes that prevent the establishment of wastewater-derived bacteria and associated ARGs. IMPORTANCE Wastewater treatment plants are sources of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs), which can contaminate receiving aquatic environments and contribute to antibiotic resistance. We focused on a controlled experimental system comprising a semicommercial membrane-aerated bioreactor (MABR) that treated raw sewage, whose effluents fed a 4,500-L polypropylene basin that mimicked effluent stabilization reservoirs. We evaluated ARB and ARG dynamics across the raw-sewage-MABR-effluent trajectory, concomitant with evaluation of microbial community composition and physicochemical parameters, in an attempt to identify mechanisms associated with ARB and ARG dissipation. We found that removal of ARB and ARGs in the MABR was primarily associated with bacterial death or sludge removal, whereas in the reservoir it was attributed to the inability of ARBs and associated ARGs to colonize the reservoir due to a dynamic and persistent microbial community. The study demonstrates the importance of ecosystem functioning in removing microbial contaminants from wastewater.

Survival of clinical and environmental carbapenem-resistant Klebsiella pneumoniae ST147 in surface water.

Carbapenem-resistant Klebsiella pneumoniae represents a healthcare threat, already disseminated in the environment. This study aimed to compare the behaviour of a clinical and an environmental K. pneumoniae strain (multilocus sequence type ST147) harbouring the gene blaKPC-3 in water. The abundance of the genes phoE (specific for K. pneumoniae) and blaKPC-3 was monitored by quantitative PCR in urban runoff water and sterile ultra-pure water microcosms, aiming to assess survival, blaKPC-3 persistence, and the effect of the native water microbiota. In sterile ultra-pure water, the abundance of cultivable K. pneumoniae and blaKPC-3 gene did not change over the incubation period (8 days). In contrast, in urban runoff, the K. pneumoniae and the genes phoE and blaKPC genes decreased by up to 3 log-units. These results suggest that K. pneumoniae were outcompeted by the native microbiota of the urban runoff water and that the decay of blaKPC-3 gene was due to host death, rather than to gene loss. The study highlights that although native microbiota is essential to hamper the persistence of non-native bacteria, carbapenemase producing K. pneumoniae can survive in urban runoff water for at least one week.

Dissemination of Metallo-ß-Lactamase-Producing Pseudomonas aeruginosa in Serbian Hospital Settings: Expansion of ST235 and ST654 Clones.

This nationwide study aimed to investigate the molecular characteristics of metallo-ß-lactamase (MBL)-producing Pseudomonas aeruginosa in Serbia, underlying resistance mechanisms, the genetic context of detected MBL genes, and the clonal relationship between isolates harboring genes-encoding MBL. Overall, 320/5334 isolates collected from 2018 to 2021 were identified as P. aeruginosa. Carbapenem-resistant P. aeruginosa (CRPA) were screened for the presence of blaVIM, blaIMP, and blaNDM, genes whereas MBL-positive isolates were tested for the presence of the blaCTX-M-2, blaPER, blaTEM, blaSHV, blaVEB, and blaGES. Multilocus sequence typing and phylogenomic analysis were performed for P. aeruginosa-producing MBL. The majority of the P. aeruginosa isolates were recovered from the lower respiratory tract (n = 120; 37.5%) and wound specimens (n = 108; 33.75%). CRPA isolates accounted for 43.1% (n = 138) of the tested isolates, 31 out of them being blaNDM-1-positive (22.5%). The colistin resistance rate was 0.3%. MLST analysis revealed the occurrence of ST235 (n = 25) and ST654 (n = 6), mostly confined to Serbia. The distribution of beta-lactamase-encoding genes in these isolates suggested clonal dissemination and possible recombination: ST235/blaNDM-1, ST235/blaNDM-1/blaPER-1, ST654/blaNDM-1, ST654/blaNDM-1/blaPER-1, and ST654/blaNDM-1/blaGES-5. High-risk clones ST235 and ST654 identified for the first time in Serbia, are important vectors of acquired MBL and ESBL and their associated multidrug resistance phenotypes represent a cause for considerable concern.

Betaproteobacteria are predominant in drinking water: are there reasons for concern?

Betaproteobacteria include some of the most abundant and ubiquitous bacterial genera that can be found in drinking water, including mineral water. The combination of physiology and ecology traits place some Betaproteobacteria in the list of potential, yet sometimes neglected, opportunistic pathogens that can be transmitted by water or aqueous solutions. Indeed, some drinking water Betaproteobacteria with intrinsic and sometimes acquired antibiotic resistance, harbouring virulence factors and often found in biofilm structures, can persist after water disinfection and reach the consumer. This literature review summarises and discusses the current knowledge about the occurrence and implications of Betaproteobacteria in drinking water. Although the sparse knowledge on the ecology and physiology of Betaproteobacteria thriving in tap or bottled natural mineral/spring drinking water (DW) is an evidence of this review, it is demonstrated that DW holds a high diversity of Betaproteobacteria, whose presence may not be innocuous. Frequently belonging to genera also found in humans, DW Betaproteobacteria are ubiquitous in different habitats, have the potential to resist antibiotics either due to intrinsic or acquired mechanisms, and hold different virulence factors. The combination of these factors places DW Betaproteobacteria in the list of candidates of emerging opportunistic pathogens. Improved bacterial identification of clinical isolates associated with opportunistic infections and additional genomic and physiological studies may contribute to elucidate the potential impact of these bacteria.

Oryzisolibacter propanilivorax gen. nov., sp. nov., a propanil-degrading bacterium.

Strain EPL6T, a Gram-negative, motile, short rod was isolated from a propanil and 3,4-dichloroaniline enrichment culture produced from rice paddy soil. Based on the analyses of the 16S rRNA gene sequence, strain EPL6T was observed to be a member of the family Comamonadaceae, sharing the highest pairwise identity with type strains of the species Alicycliphilus denitrificans K601T (96.8 %) and Melaminivora alkalimesophila CY1T (96.8 %). Strain EPL6T was able to grow in a temperature range of 15-37 °C, pH 6-9 and in the presence of up to 4 % (w/v) NaCl and tested positive for catalase and oxidase reactions. The major respiratory quinone was Q8. The genomic DNA had a G+C content of 69.4±0.9 mol%. The major polar lipids were phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol, and the major fatty acid methyl esters comprised C16 : 0, C18 : 1ω7c and summed feature 3 (C16 : 1ω7c/iso-C15 : 0 2-OH). Comparison of the genome sequence of strain EPL6T and of its closest neighbours, Melaminivora alkalimesophila CY1T and Alicycliphilus denitrificans K601T, yielded values of ANI ≤84.1 % and of AAI ≤80.3 %. Therefore, the genetic, phylogenetic, phenotypic and chemotaxonomic characteristics support the classification of this organism into a new taxon. Considering the genetic divergence of strain EPL6T from the type strains of the closest species, which belong to distinct genera, we propose a new genus within the family Comamonadaceae, named Oryzisolibacter propanilivorax gen. nov., sp. nov., represented by the isolate EPL6T as the type strain of the species (=LMG 28427T=CECT 8927T).

Hydromonas duriensis gen. nov., sp. nov., isolated from freshwater.

An aerobic, Gram-stain-negative rod, designated strain A2P5T, was isolated from the Douro river, in Porto, Portugal. Cells were catalase- and oxidase-positive. Growth occurred at 15-30 °C, at pH 6-8 and in the presence of 1 % (w/v) NaCl. The major respiratory quinone was Q8, the genomic DNA had a G+C content of 47 ± 1 mol%, and phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol were amongst the major polar lipids. On the basis of 16S rRNA gene sequence analysis, strain A2P5T was observed to be a member of the family Burkholderiaceae, but could not be identified as a member of any validly named genus. The low levels of 16S rRNA gene sequence similarity to other recognized taxa ( < 91 %), together with the comparative analysis of phenotypic and chemotaxonomic characteristics, supported the proposal of a novel species of a new genus within the family Burkholderiaceae. The name Hydromonas duriensis gen. nov., sp. nov. is proposed. The type strain of Hydromonas duriensis is A2P5T ( = LMG 28428T = CCUG 66137T).

Towards the definition of an antibiotic resistome signature in wastewater and downstream environments.

Domestic wastewater is a significant reservoir of antibiotic resistance genes, which pose environmental and public health risks. We aimed to define an antibiotic resistome signature, represented by core genes, i.e., shared by ≥90% of the metagenomes of each of three conceptual environmental compartments - wastewater (influent, sludge, effluent), freshwater, and agricultural soil. The definition of resistome signatures would support the proposal of a framework for monitoring treatment efficacy and assessing the impact of treated wastewater discharge into the environment, such as freshwater and agricultural soil. Metagenomic data from 163 samples originating from wastewater (n=81), freshwater (n=58), and agricultural soils (n=24) across different regions (29 countries, 5 continents), were analysed regarding antibiotic resistance diversity, based on annotation against a database that merged CARD and ResFinder databases. The relative abundance of the total antibiotic resistance genes (corresponding to the ratio between the antibiotic resistance genes and total reads number) was not statistically different between raw and treated wastewater, being significantly higher than in freshwater or agricultural soils. The latter had the significantly lowest relative abundance of antibiotic resistance genes. Genes conferring resistance to aminoglycosides, beta-lactams, and tetracyclines were among the most abundant in wastewater environments, while multidrug resistance was equally distributed across all environments. The wastewater resistome signature included 27 antibiotic resistance genes that were detected in at least 90% of the wastewater resistomes, and that were not frequent in freshwater or agricultural soil resistomes. Among these were genes responsible for resistance to tetracyclines (n=8), macrolide-lincosamide-streptogramin B (n=7), aminoglycosides (n=4), beta-lactams (n=3), multidrug (n=2), sulphonamides (n=2), and polypeptides (n=1). This comprehensive assessment provides valuable insights into the dynamics of antibiotic resistance in urban wastewater systems and their potential ecological implications in diverse environmental settings. Furthermore, provides guidance for the implementation of One Health monitoring approaches.

Making waves: The NORMAN antibiotic resistant bacteria and resistance genes database (NORMAN ARB&ARG)-An invitation for collaboration to tackle antibiotic resistance.

With the global concerns on antibiotic resistance (AR) as a public health issue, it is pivotal to have data exchange platforms for studies on antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in the environment. For this purpose, the NORMAN Association is hosting the NORMAN ARB&ARG database, which was developed within the European project ANSWER. The present article provides an overview on the database functionalities, the extraction and the contribution of data to the database. In this study, AR data from three studies from China and Nepal were extracted and imported into the NORMAN ARB&ARG in addition to the existing AR data from 11 studies (mainly European studies) on the database. This feasibility study demonstrates how the scientific community can share their data on AR to generate an international evidence base to inform AR mitigation strategies. The open and FAIR data are of high potential relevance for regulatory applications, including the development of emission limit values / environmental quality standards in relation to AR. The growth in sharing of data and analytical methods will foster collaboration on risk management of AR worldwide, and facilitate the harmonization in the effort for identification and surveillance of critical hotspots of AR. The NORMAN ARB&ARG database is publicly available at: https://www.norman-network.com/nds/bacteria/.

Patulibacter medicamentivorans sp. nov., isolated from activated sludge of a wastewater treatment plant.

A Gram-positive, aerobic, non-motile, non-endospore-forming rod-shaped bacterium with ibuprofen-degrading capacity, designated strain I11(T), was isolated from activated sludge from a wastewater treatment plant. The major respiratory quinone was demethylmenaquinone DMK-7, C18 : 1 cis9 was the predominant fatty acid, phosphatidylglycerol was the predominant polar lipid, the cell wall contained meso-diaminopimelic acid as the diagnostic diamino acid and the G+C content of the genomic DNA was 74.1 mol%. On the basis of 16S rRNA gene sequence analysis, the closest phylogenetic neighbours of strain I11(T) were Patulibacter ginsengiterrae CECT 7603(T) (96.8 % similarity), Patulibacter minatonensis DSM 18081(T) (96.6 %) and Patulibacter americanus DSM 16676(T) (96.6 %). Phenotypic characterization supports the inclusion of strain I11(T) within the genus Patulibacter (phylum Actinobacteria). However, distinctive features and 16S rRNA gene sequence analysis suggest that is represents a novel species, for which the name Patulibacter medicamentivorans sp. nov. is proposed. The type strain is I11(T) ( = DSM 25962(T) = CECT 8141(T)).

Diversity and antibiotic resistance of Acinetobacter spp. in water from the source to the tap.

Acinetobacter spp. are ubiquitous bacteria in the environment. Acinetobacter spp. isolated from a municipal drinking water treatment plant and from connected tap water were identified to the species level on the basis of rpoB gene partial sequence analysis. Intraspecies variation was assessed based on the analysis of partial sequences of housekeeping genes (rpoB, gyrB, and recA). Antibiotic resistance was characterized using the disk diffusion method and isolates were classified as wild or non-wild type (non-WT), according to the observed phenotype. The strains of Acinetobacter spp. were related to 11 different validly published species, although three groups of isolates, presenting low rpoB sequence similarities with previously described species, may represent new species. Most of the isolates were related to the species A. johnsonii and A. lwoffii. These two groups, as well as others related to the species A. parvus and A. tjernbergiae, were detected in the water treatment plant and in tap water. Other strains, related to the species A. pittii and A. beijerinckii, were isolated only from tap water. Most of the isolates (80 %) demonstrated wild type (WT) to all of the 12 antibiotics tested. Non-WT for tetracycline, meropenem, and ceftazidime, among others, were observed in water treatment plant or in tap water samples. Although, in general, this study suggests a low prevalence of acquired antibiotic resistance in water Acinetobacter spp., the potential of some species to acquire and disseminate resistance via drinking water is suggested.

Insight into phylogenomic bias of blaVIM-2 or blaNDM-1 dissemination amongst carbapenem-resistant Pseudomonas aeruginosa.

OBJECTIVES: Pseudomonas aeruginosa (P. aeruginosa) are ubiquitous opportunistic pathogens that combine intrinsic and acquired multidrug resistance phenotypes. Due to different types of acquired genes, carbapenem resistance has been expanding in this species. This study hypothesised that the spread of carbapenem resistance among P. aeruginosa is influenced by phylogenomic features, being distinct for different genes. METHODS: To test this hypothesis, the genomes of P. aeruginosa harbouring blaVIM-2 or blaNDM-1 genes were compared. The blaVIM-2 gene was selected because, although frequent, it is almost restricted to this species and blaNDM-1 gene due to its wide interspecies distribution. A group of genomes harbouring the genes blaVIM-2 (n = 116) or blaNDM-1 (n = 27), available in GenBank, was characterised based on core phylogenomic analysis, functional categories in the accessory genome and mobile genetic elements flanking the selected genes. RESULTS: Most blaVIM-2 gene hosts belonged to multilocus sequence types (ST) ST111 (n = 32 of 116) and ST233 (n = 27 of 116) and were reported in Europe (n = 75 of 116). The blaNDM-1 gene hosts were distributed by different STs (ST38, ST773, ST235, ST357 and ST654), frequently from Asia (n = 11 of 27). Significant differences in the prevalence of functional protein/enzyme annotations per number of accessory genomes were observed between blaVIM-2+ and blaNDM-1+. The blaVIM-2 gene was frequently inserted in the Tn402-like and Tn21 transposons family and rarely in IS6100, while blaNDM-1 gene was preferentially flanked by ISAba125 and bleMBL genes or associated with IS91 insertion sequence. CONCLUSION: The hypothesis that carbapenem resistance gene acquisition is not random among phylogenomic lineages was confirmed, suggesting the importance of phylogeny in the dissemination of antibiotic resistance genes.

Candidate biomarkers of antibiotic resistance for the monitoring of wastewater and the downstream environment.

Urban wastewater treatment plants (UWTPs) are essential for reducing the pollutants load and protecting water bodies. However, wastewater catchment areas and UWTPs emit continuously antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs), with recognized impacts on the downstream environments. Recently, the European Commission recommended to monitor antibiotic resistance in UWTPs serving more than 100 000 population equivalents. Antibiotic resistance monitoring in environmental samples can be challenging. The expected complexity of these systems can jeopardize the interpretation capacity regarding, for instance, wastewater treatment efficiency, impacts of environmental contamination, or risks due to human exposure. Simplified monitoring frameworks will be essential for the successful implementation of analytical procedures, data analysis, and data sharing. This study aimed to test a set of biomarkers representative of ARG contamination, selected based on their frequent human association and, simultaneously, rare presence in pristine environments. In addition to the 16S rRNA gene, ten potential biomarkers (intI1, sul1, ermB, ermF, aph(3'')-Ib, qacEΔ1, uidA, mefC, tetX, and crAssphage) were monitored in DNA extracts (n = 116) from raw wastewater, activated sludge, treated wastewater, and surface water (upstream and downstream of UWTPs) samples collected in the Czech Republic, Denmark, Israel, the Netherlands, and Portugal. Each biomarker was sensitive enough to measure decreases (on average by up to 2.5 log-units gene copy/mL) from raw wastewater to surface water, with variations in the same order of magnitude as for the 16S rRNA gene. The use of the 10 biomarkers allowed the typing of water samples whose origin or quality could be predicted in a blind test. The results show that, based on appropriate biomarkers, qPCR can be used for a cost-effective and technically accessible approach to monitoring wastewater and the downstream environment.

Bacillus purgationiresistans sp. nov., isolated from a drinking-water treatment plant.

A Gram-positive, aerobic, non-motile, endospore-forming rod, designated DS22(T), was isolated from a drinking-water treatment plant. Cells were catalase- and oxidase-positive. Growth occurred at 15-37 °C, at pH 7-10 and with <8% (w/v) NaCl (optimum growth: 30 °C, pH 7-8 and 1-3% NaCl). The major respiratory quinone was menaquinone 7, the G+C content of the genomic DNA was 36.5 mol% and the cell wall contained meso-diaminopimelic acid. On the basis of 16S rRNA gene sequence analysis, strain DS22(T) was a member of the genus Bacillus. Its closest phylogenetic neighbours were Bacillus horneckiae NRRL B-59162(T) (98.5% 16S rRNA gene sequence similarity), Bacillus oceanisediminis H2(T) (97.9%), Bacillus infantis SMC 4352-1(T) (97.4%), Bacillus firmus IAM 12464(T) (96.8%) and Bacillus muralis LMG 20238(T) (96.8%). DNA-DNA hybridization, and biochemical and physiological characterization allowed the differentiation of strain DS22(T) from its closest phylogenetic neighbours. The data supports the proposal of a novel species, Bacillus purgationiresistans sp. nov.; the type strain is DS22(T) (=DSM 23494(T)=NRRL B-59432(T)=LMG 25783(T)).

Comparison of ubiquitous antibiotic-resistant Enterobacteriaceae populations isolated from wastewaters, surface waters and drinking waters.

This study aimed at assessing the role of ubiquitous (non-Escherichia coli) Enterobacteriaceae in the dissemination of antimicrobial resistance through the urban water cycle. Enterobacteriaceae isolated from a municipal wastewater treatment plant (111 isolates), urban water streams (33 isolates) and drinking water (123 isolates) were compared in terms of: (i) genera distribution, (ii) resistance to 12 antibiotics, and (iii) class 1 and class 2 integrons. The predominant bacterial genera were the same in the different types of water, although with a distinct pattern of species. The most prevalent resistance phenotypes were observed for amoxicillin, ticarcillin, cephalothin and sulphamethoxazole (24-59% in the three types of water). No resistance against ceftazidime or meropenem was observed. Resistance to cephalothin, amoxicillin and sulphamethoxazole was significantly more prevalent in drinking water, water streams and wastewater, respectively, than in the other types of water. It was possible to recognize antibiotic-resistance associations, namely for the pairs streptomycin-tetracycline (positive) and ticarcillin-cephalotin (negative). Class 1 and/or class 2 integrons with similar gene cassettes were detected in the three types of water. This study demonstrated that Enterobacteriaceae are important vehicles of antibiotic resistance, namely in drinking water.

Quality Control of Drinking Water in the City of Ilave, Region of Puno, Peru.

The region of Puno, in Peru, is described as a region with some health conditions that may be associated with the water quality, such as a high index of anemia or cases of acute diarrhea in children. This study aimed at monitoring the drinking water quality of the city of Ilave, in Peru, and determining possible correlations between physical-chemical and microbiological parameters, and the water distribution conditions, such as the period of water availability. Physical-chemical parameters (turbidity, residual chlorine, temperature, conductivity, and pH), microbiological parameters (presence of coliforms), and heavy metals (Zn, Mn, Ni, Fe, and Cu) were determined. All the parameters quantified were within the maximum permissible limits according to Peruvian regulations, except for residual chlorine, which was, for all the treated water samples, below the recommended value of 0.5 mg/L. Coliforms that should be absent from drinking water were detected in all the household samples. These results demonstrate the need for the inclusion of additional steps of re-chlorination along the distribution system to guarantee the maintenance of residual levels of chlorine that assure the microbiological quality of water. The quality of the drinking water was not observed to correlate with the period of water availability.

Monitoring antibiotic resistance genes in wastewater environments: The challenges of filling a gap in the One-Health cycle.

Antibiotic resistance (AR) is a global problem requiring international cooperation and coordinated action. Global monitoring must rely on methods available and comparable across nations to quantify AR occurrence and identify sources and reservoirs, as well as paths of AR dissemination. Numerous analytical tools that are gaining relevance in microbiology, have the potential to be applied to AR research. This review summarizes the state of the art of AR monitoring methods, considering distinct needs, objectives and available resources. Based on the overview of distinct approaches that are used or can be adapted to monitor AR, it is discussed the potential to establish reliable and useful monitoring schemes that can be implemented in distinct contexts. This discussion places the environmental monitoring within the One-Health approach, where two types of risk, dissemination across distinct environmental compartments, and transmission to humans, must be considered. The plethora of methodological approaches to monitor AR and the variable features of the monitored sites challenge the capacity of the scientific community and policy makers to reach a common understanding. However, the dialogue between different methods and the production of action-oriented data is a priority. The review aims to warm up this discussion.

Diversity and antibiotic resistance patterns of Sphingomonadaceae isolates from drinking water.

Sphingomonadaceae (n = 86) were isolated from a drinking water treatment plant (n = 6), tap water (n = 55), cup fillers for dental chairs (n = 21), and a water demineralization filter (n = 4). The bacterial isolates were identified based on analysis of the 16S rRNA gene sequence, and intraspecies variation was assessed on the basis of atpD gene sequence analysis. The isolates were identified as members of the genera Sphingomonas (n = 27), Sphingobium (n = 28), Novosphingobium (n = 12), Sphingopyxis (n = 7), and Blastomonas (n = 12). The patterns of susceptibility to five classes of antibiotics were analyzed and compared for the different sites of isolation and taxonomic groups. Colistin resistance was observed to be intrinsic (92%). The highest antibiotic resistance prevalence values were observed in members of the genera Sphingomonas and Sphingobium and for beta-lactams, ciprofloxacin, and cotrimoxazole. In tap water and in water from dental chairs, antibiotic resistance was more prevalent than in the other samples, mainly due to the predominance of isolates of the genera Sphingomonas and Sphingobium. These two genera presented distinct patterns of association with antibiotic resistance, suggesting different paths of resistance development. Antibiotic resistance patterns were often related to the species rather than to the site or strain, suggesting the importance of vertical resistance transmission in these bacteria. This is the first study demonstrating that members of the family Sphingomonadaceae are potential reservoirs of antibiotic resistance in drinking water.

Candidimonas nitroreducens gen. nov., sp. nov. and Candidimonas humi sp. nov., isolated from sewage sludge compost.

Two bacterial strains (SC-089(T) and SC-092(T)) isolated from sewage sludge compost were characterized by using a polyphasic approach. The isolates were Gram-negative short rods, catalase- and oxidase-positive, and showed good growth at 30 °C, at pH 7 and with 1 % (w/v) NaCl. Ubiquinone 8 was the major respiratory quinone, and phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol were amongst the major polar lipids. On the basis of 16S rRNA gene sequence analysis, the strains were observed to be members of the family Alcaligenaceae, but could not be identified as members of any validly described genus. The low levels of 16S rRNA gene sequence similarity to other recognized taxa, together with comparative analysis of phenotypic traits and chemotaxonomic markers, supported the proposal of a new genus within the family Alcaligenaceae, for which the name Candidimonas gen. nov. is proposed. Strains SC-089(T) and SC-092(T), which shared 99.1 % 16S rRNA gene sequence similarity, could be differentiated at the phenotypic level, and DNA-DNA hybridization results supported their identification as representing distinct species. The names proposed for these novel species are Candidimonas nitroreducens sp. nov. (type strain, SC-089(T) = LMG 24812(T) = CCUG 55806(T)) and Candidimonas humi sp. nov. (type strain, SC-092(T) = LMG 24813(T) = CCUG 55807(T)).

Acinetobacter rudis sp. nov., isolated from raw milk and raw wastewater.

Two bacterial strains, G30(T) and A1PC16, isolated respectively from raw milk and raw wastewater, were characterized using a polyphasic approach. Chemotaxonomic characterization supported the inclusion of these strains in the genus Acinetobacter, with Q-8 and Q-9 as the major respiratory quinones, genomic DNA G+C contents within the range observed for this genus (38-47 mol%) and C(16:0), C(18:1)ω9c and C(16:1)ω7c/iso-C(15:0) 2-OH as the predominant fatty acids. The observation of 16S rRNA gene sequence similarity lower than 97% with other Acinetobacter species with validly published names led to the hypothesis that these isolates could represent a novel species. This hypothesis was supported by comparative analysis of partial sequences of the genes rpoB and gyrB, which showed that strains G30(T) and A1PC16 did not cluster with any species with validly published names, forming a distinct lineage. DNA-DNA hybridizations confirmed that the two strains were members of the same species, which could be distinguished from their congeners by several phenotypic characteristics. On the basis of these arguments, it is proposed that strains G30(T) and A1PC16 represent a novel species, for which the name Acinetobacter rudis sp. nov. is proposed. The type strain is strain G30(T) (=LMG 26107(T) =CCUG 57889(T) =DSM 24031(T) =CECT 7818(T)).

Culture-dependent and culture-independent diversity surveys target different bacteria: a case study in a freshwater sample.

Compared with culture-independent approaches, traditionally used culture-dependent methods have a limited capacity to characterize water microbiota. Nevertheless, for almost a century the latter have been optimized to detect and quantify relevant bacteria. A pertinent question is if culture-independent diversity surveys give merely an extended perspective of the bacterial diversity or if, even with a higher coverage, focus on a different set of organisms. We compared the diversity and phylogeny of bacteria in a freshwater sample recovered by currently used culture-dependent and culture-independent methods (DGGE and 454 pyrosequencing). The culture-dependent diversity survey presented lower coverage than the other methods. However, it allowed bacterial identifications to the species level, in contrast with the other procedures that rarely produced identifications below the order. Although the predominant bacterial phyla detected by both approaches were the same (Proteobacteria, Actinobacteria, Bacteroidetes), sequence similarity analysis showed that, in general, different operational taxonomical units were targeted by each method. The observation that culture-dependent and independent approaches target different organisms has implications for the use of the latter for studies in which taxonomic identification has a predictive value. In comparison to DGGE, 454 pyrosequencing method had a higher capacity to explore the bacterial richness and to detect cultured organisms, being also less laborious.