Emergence and dissemination of epidemic-causing OXA-244 carbapenemase-producing Escherichia coli ST38 through hospital sewage in Norway, 2020-2022.

BACKGROUND: Population-based sewage surveillance has emerged as a promising approach for studying the prevalence of antibiotic resistance in pathogens. AIM: To determine the temporal prevalence of cefotaxime-resistant Escherichia coli in sewage from five sewage treatment plants located in Bergen city, to determine whether ESBL- and carbapenemase-producing E. coli are consistently disseminated in the receiving environment through sewage. METHOD: A total of 569 cefotaxime-resistant E. coli were isolated over a period of 19 months (August 2020 to February 2022) using ECC CHROMagar™ plates from 82 samples, antibiotic sensitivity profiles were determined, using Sensititre™ plates. The draft genome sequences were determined, using Illumina MiSeq-based sequencing. Complete genome sequences were determined, using Oxford Nanopore-based sequencing. FINDINGS: All 569 strains obtained from influent (N=461) and effluent (N=108) were multi-drug resistant. Most of the sequenced strains (52 of 61) carried blaCTX-M-15 (38.5%) and blaCTX-M-27 (34.6%). The most prevalent sequence types (STs) for ESBL-carrying strains were ST131 (32.8%) and ST38 (21.3%). All CTX-M-27-carrying ST131 strains belonged to clade A or C1, while CTX-M-15-harbouring strains were present in all the clades. Five OXA-244-producing ST38 strains, genetically similar to epidemic-causing strains from Western Norway, France and the Netherlands, were isolated only from raw and treated sewage of the treatment plant receiving hospital sewage. CONCLUSION: This is the first study showing persistent dissemination of OXA-244-producing ST38 clones through sewage in Norway, demonstrating that hospital sewage is the likely source of OXA-244-producing ST38 clones reaching the receiving environment.

Observational multi-centre, prospective study to characterize novel pathogen-and host-related factors in hospitalized patients with lower respiratory tract infections and/or sepsis - the "TAILORED-Treatment" study.

BACKGROUND: The emergence and spread of antibiotic resistant micro-organisms is a global concern, which is largely attributable to inaccurate prescribing of antibiotics to patients presenting with non-bacterial infections. The use of 'omics' technologies for discovery of novel infection related biomarkers combined with novel treatment algorithms offers possibilities for rapidly distinguishing between bacterial and viral infections. This distinction can be particularly important for patients suffering from lower respiratory tract infections (LRTI) and/or sepsis as they represent a significant burden to healthcare systems. Here we present the study details of the TAILORED-Treatment study, an observational, prospective, multi-centre study aiming to generate a multi-parametric model, combining host and pathogen data, for distinguishing between bacterial and viral aetiologies in children and adults with LRTI and/or sepsis. METHODS: A total number of 1200 paediatric and adult patients aged 1 month and older with LRTI and/or sepsis or a non-infectious disease are recruited from Emergency Departments and hospital wards of seven Dutch and Israeli medical centres. A panel of three experienced physicians adjudicate a reference standard diagnosis for all patients (i.e., bacterial or viral infection) using all available clinical and laboratory information, including a 28-day follow-up assessment. Nasal swabs and blood samples are collected for multi-omics investigations including host RNA and protein biomarkers, nasal microbiota profiling, host genomic profiling and bacterial proteomics. Simplified data is entered into a custom-built database in order to develop a multi-parametric model and diagnostic tools for differentiating between bacterial and viral infections. The predictions from the model will be compared with the consensus diagnosis in order to determine its accuracy. DISCUSSION: The TAILORED-Treatment study will provide new insights into the interplay between the host and micro-organisms. New host- or pathogen-related biomarkers will be used to generate a multi-parametric model for distinguishing between bacterial and viral infections. This model will be helpful to better guide antimicrobial therapy for patients with LRTI and sepsis. This study has the potential to improve patient care, reduce unnecessary antibiotic prescribing and will contribute positively to institutional, national and international healthcare economics. TRIAL REGISTRATION: NCT02025699 . Registration Date: January, 1, 2014.

Complete genome sequence of the marine Rhodococcus sp. H-CA8f isolated from Comau fjord in Northern Patagonia, Chile.

Rhodococcus sp. H-CA8f was isolated from marine sediments obtained from the Comau fjord, located in Northern Chilean Patagonia. Whole-genome sequencing was achieved using PacBio RS II platform, comprising one closed, complete chromosome of 6,19 Mbp with a 62.45% G + C content. The chromosome harbours several metabolic pathways providing a wide catabolic potential, where the upper biphenyl route is described. Also, Rhodococcus sp. H-CA8f bears one linear mega-plasmid of 301 Kbp and 62.34% of G + C content, where genomic analyses demonstrated that it is constituted mostly by putative ORFs with unknown functions, representing a novel genetic feature. These genetic characteristics provide relevant insights regarding Chilean marine actinobacterial strains.

Uncommonly isolated clinical Pseudomonas: identification and phylogenetic assignation.

Fifty-two Pseudomonas strains that were difficult to identify at the species level in the phenotypic routine characterizations employed by clinical microbiology laboratories were selected for genotypic-based analysis. Species level identifications were done initially by partial sequencing of the DNA dependent RNA polymerase sub-unit D gene (rpoD). Two other gene sequences, for the small sub-unit ribosonal RNA (16S rRNA) and for DNA gyrase sub-unit B (gyrB) were added in a multilocus sequence analysis (MLSA) study to confirm the species identifications. These sequences were analyzed with a collection of reference sequences from the type strains of 161 Pseudomonas species within an in-house multi-locus sequence analysis database. Whole-cell matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analyses of these strains complemented the DNA sequenced-based phylogenetic analyses and were observed to be in accordance with the results of the sequence data. Twenty-three out of 52 strains were assigned to 12 recognized species not commonly detected in clinical specimens and 29 (56 %) were considered representatives of at least ten putative new species. Most strains were distributed within the P. fluorescens and P. aeruginosa lineages. The value of rpoD sequences in species-level identifications for Pseudomonas is emphasized. The correct species identifications of clinical strains is essential for establishing the intrinsic antibiotic resistance patterns and improved treatment plans.

Flavobacterium tructae sp. nov. and Flavobacterium piscis sp. nov., isolated from farmed rainbow trout (Oncorhynchus mykiss).

Four Gram-staining-negative, catalase- and oxidase-positive, pale-orange pigmented bacterial strains (435-08(T), 47B-3-09, 412R-09(T) and 60B-3-09) were isolated from diseased rainbow trout. Analysis of their 16S rRNA gene sequences suggested their adscription to the genus Flavobacterium. Strains formed two phylogenetic groups represented by strains 435-08(T) and 47B-3-09 (group A), and strains 412R-09(T) and 60B-3-09 (group B) displaying 16S rRNA sequence similarities greater than 99.8-99.9% within their respective groups. Strain 435-08(T) exhibited the highest levels of similarity with Flavobacterium aquidurense WB-1.1.56(T) (98.6% sequence similarity) and strain 412R-09(T) with Flavobacterium frigidimaris KUC-1(T) and Flavobacterium aquidurense WB-1.1.56(T) (98.9% and 98.6% sequence similarity, respectively). DNA-DNA hybridization studies showed low levels of relatedness between strain 435-08(T) and strain 412R-09(T) and between both strains and the most closely related species of the genus Flavobacterium. The genomic DNA G+C contents of strains 435-08(T) and 412R-09(T) were 36.2 and 34.3 mol%, respectively. The predominant respiratory quinone of both strains was MK-6 and the major fatty acids were iso-C(15 : 0), C(16 : 1)ω7c and C(15 : 0). The two groups of strains could be distinguished from each other and from related species of the genus Flavobacterium by a number of phenotypic properties. Phylogenetic, genotypic and phenotypic evidence indicated that strains of groups A and B represent two novel species of the genus Flavobacterium, for which the names Flavobacterium tructae sp. nov. (type strain 435-08(T) = CECT 7791(T) = CCUG 60100(T)) and Flavobacterium piscis sp. nov. (type strain 412R-09(T) = CECT 7911(T) = CCUG 60099(T)) are proposed.

Shift of CTX-M genotypes has determined the increased prevalence of extended-spectrum ß-lactamase-producing Escherichia coli in south-western Sweden.

The prevalence of Escherichia coli producing extended-spectrum ß-lactamases (ESBLs) markedly increased during 2004-2008 in south-western Sweden, with a greater increase in urinary isolates in hospitals (0.2-2.5%) than in the community (0.2-1.6%). ESBLs of genotype CTX-M predominated, with a significant (p <0.02) shift from the CTX-M-9 to CTX-M-1 phylogroup occurring among urinary ESBL-producing E. coli isolated early (n = 41) as compared with late (n = 221) in the study period. The increase in ESBL-producing E. coli was polyclonal, and only partly attributable to an increase (0-24%) in the number of O25b-ST131 isolates carrying CTX-M-15. The increase was prominent in men and in elderly patients, and warrants continued surveillance.

Chryseobacterium tructae sp. nov., isolated from rainbow trout (Oncorhynchus mykiss).

Three pale-orange bacteria (strains 1083-08, 1084-08(T) and 1095B-08) were isolated from diseased rainbow trout. The isolates were Gram-staining-negative, catalase- and oxidase-positive, rod-shaped cells. Analyses of their 16S rRNA gene sequences confirmed their adscription to the genus Chryseobacterium. The three isolates shared 100% 16S rRNA gene sequence similarity and 98.5% similarity with Chryseobacterium indologenes CCUG 14556(T), being the closest phylogenetically related species. Genomic DNA-DNA hybridization similarity values between the three isolates were 94-100% and 2-39% between strain 1084-08(T) and the type strains of other related Chryseobacterium species, confirming that the isolates represent a novel species within the genus Chryseobacterium. The DNA G+C content of the species was 33.6-36.1mol%. The predominant respiratory quinone of strain 1084-08(T) was MK-6 and the major fatty acids were iso-C(15:0), iso-C(17:1)ω9c, iso-C(17:0) 3-OH and C(16:1)ω6c. The isolates were distinguished from related Chryseobacterium species by a number of phenotypic properties. Based on the phenotypic, genotypic and phylogenetic findings, it is proposed that the new isolates from rainbow trout be classified as a new species of the genus Chryseobacterium, with the name of Chryseobacterium tructae sp. nov. The type strain is 1084-08(T) (=CECT 7798(T)=CCUG 60111(T)).

Flavobacterium oncorhynchi sp. nov., a new species isolated from rainbow trout (Oncorhynchus mykiss).

Eighteen isolates of a Gram-negative, catalase and oxidase-positive, rod-shaped bacterium, recovered from diseased rainbow trout (Oncorhynchus mykiss), were characterized, using a polyphasic taxonomic approach. Studies based on comparative 16S rRNA gene sequence analysis showed that that the eighteen new isolates shared 99.2-100% sequence similarities. Phylogenetic analysis revealed that isolates from trout belonged to the genus Flavobacterium, showing the highest sequence similarities to F. chungangense (98.6%), F. frigidimaris (98.1%), F. hercynium (97.9%) and F. aquidurense (97.8%). DNA-DNA reassociation values between the trout isolates (exemplified by strain 631-08(T)) and five type strains of the most closely related Flavobacterium species exhibited less than 27% similarity. The G+C content of the genomic DNA was 33.0 mol%. The major respiratory quinone was observed to be menaquinone 6 (MK-6) and iso-C(15:0), C(15:0) and C(16:1) ω7c the predominant fatty acids. The polar lipid profile of strain 631-08(T) consisted of phosphatidylethanolamine, unknown aminolipids AL1 and AL3, lipids L1, L2, L3 and L4 and phospholipid PL1. The novel isolates were differentiated from related Flavobacterium species by physiological and biochemical tests. On the basis of the evidence from this polyphasic study, it is proposed that the isolates from rainbow trout be classified as a new species of the genus Flavobacterium, Flavobacterium oncorhynchi sp. nov. The type strain is 631-08(T) (= CECT 7678(T) = CCUG 59446(T)).

Chryseobacterium viscerum sp. nov., isolated from diseased fish.

A taxonomic study was carried out on five Gram-staining-negative, catalase- and oxidase-positive, rod-shaped bacteria isolated from the gills and livers of five diseased rainbow trout. The five novel isolates were designated strains 687B-08(T), 445-08, 452-08, 453B-08 and 967B-08. In phylogenetic analyses based on 16S rRNA gene sequences, the five novel strains appeared almost identical (99.0-100 % sequence similarity) and to belong to the genus Chryseobacterium. Strain 687B-08(T) (the strain selected to represent the five novel isolates) was found to be most closely related to Chryseobacterium oncorhynchi 701B-08(T) (98.9% sequence similarity), Chryseobacterium ureilyticum F-Fue-04IIIaaaa(T) (98.6%), Chryseobacterium indologenes ATCC 29897(T) (98.3%), Chryseobacterium jejuense JS17-8(T) (98.1%) and Chryseobacterium gleum ATCC 35910(T) (98.1%). In DNA-DNA hybridizations, DNA-DNA relatedness values of 99-100% were recorded between the five novel strains. Lower DNA-DNA relatedness values (21-57%) were recorded between strain 687B-08(T) and C. oncorhynchi 701B-08(T), C. ureilyticum F-Fue-04IIIaaaa(T) and the type strains of other closely related, established species of the genus Chryseobacterium. The predominant respiratory quinone of strain 687B-08(T) was MK-6 and the major cellular fatty acids were iso-C(15:0), iso-C(17:1)ω9c, iso-C(17:0) 3-OH and C(16:1)ω6c. The G+C content of the genomic DNA of strain 687B-08(T) was 38.6 mol%. Based on the phenotypic and genotypic evidence, the five novel strains isolated from rainbow trout represent a single, novel species of the genus Chryseobacterium, for which the name Chryseobacterium viscerum sp. nov. is proposed. The type strain is 687B-08(T) ( = CECT 7793(T)  = CCUG 60103(T)).

Chryseobacterium oncorhynchi sp. nov., isolated from rainbow trout (Oncorhynchus mykiss).

Genotypic and phenotypic analyses were performed on five Gram-negative, catalase and oxidase-positive, rod-shaped bacteria isolated from the gill and liver of four rainbow trout. Studies based on comparative 16S rRNA gene sequence analysis showed that the five new isolates shared 99.8-100% sequence similarity and that they belong to the genus Chryseobacterium. The nearest phylogenetic neighbours of the strain 701B-08(T) were Chryseobacterium ureilyticum F-Fue-04IIIaaaa(T) (99.1% 16S rRNA gene sequence similarity) and Chryseobacterium joosteii LMG 18212(T) (98.6%). DNA-DNA hybridization values between the five isolates were 91-99% and ranged from 2 to 53% between strain 701B-08(T) and the type strains of phylogenetically closely related species of Chryseobacterium. Strain 701B-08(T) had a DNA G+C content of 36.3 mol%, the major fatty acids were iso-C(15:0), iso-C(17:1)ω9c, C(16:1)ω6c and iso-C(17:0) 3-OH and the predominant respiratory quinone was MK-6. The novel isolates were distinguished from related Chryseobacterium species by physiological and biochemical tests. The genotypic and phenotypic properties of the isolates from rainbow trout suggest their classification as representatives of a novel species of the genus Chryseobacterium, for which the name Chryseobacterium oncorhynchi sp. nov. is proposed. The type strain is 701B-08(T) (=CECT 7794(T)=CCUG 60105(T)).

Taxonomic characterisation of ceftazidime-resistant Brevundimonas isolates and description of Brevundimonas faecalis sp. nov.

Three ceftazidime-resistant strains isolated from the sewage water of a municipal hospital in Palma de Mallorca, Spain, were analysed phenotypically and genotypically to clarify their taxonomic positions. Sequence determinations and phylogenetic analyses of the 16S rRNA genes indicated that strains CS20.3(T), CS39 and CS41 were affiliated with the species of the alphaproteobacterial genus Brevundimonas, most closely related to B. bullata, B. diminuta, B. naejangsanensis and B. terrae. Additional sequences analyses of the ITS1 region of the rRNA operon and the genes for the housekeeping enzymes DNA gyrase ß-subunit and RNA polymerase ß-subunit, genomic DNA-DNA hybridisation similarities, cell fatty acid profiles and physiological and biochemical characterizations supported the recognition of CS20.3(T) (CCUG 58127(T)=CECT 7729(T)) as a distinct and novel species, for which the name Brevundimonas faecalis sp. nov. is proposed. Strains CS39 and CS41 were ascribed to the species B. diminuta.

Differentiation of species of the Streptococcus bovis/equinus-complex by MALDI-TOF Mass Spectrometry in comparison to sodA sequence analyses.

The Streptococcus bovis/equinus complex is a heterogeneous group within the group D streptococci with important clinical relevance regarding infective endocarditis, sepsis and colon carcinoma. The taxonomic identification of species and sub-species of this complex, by the standard methods remains difficult. In the present study, we compared the cluster analysis of 88 strains of species of the S. bovis/equinus complex by sequence analysis of the manganese-dependent superoxide dismutase gene (sodA) and by Matrix Assisted Laser Desorption/Ionization-Time Of Flight Mass Spectrometry (MALDI-TOF MS). We observed a high congruence of strain grouping by MALDI-TOF MS in comparison with sodA sequence analyses, demonstrating the accuracy and reliability of MALDI-TOF MS in comparison to DNA sequence-based method. By generating mass spectra for each species and sub-species, we were able to discriminate all members of the S. bovis/equinus complex. Furthermore, we demonstrated reliable identifications to the species level by MALDI-TOF MS, independently of cultivation conditions.

Bacterial community dynamics during in-situ bioremediation of petroleum waste sludge in landfarming sites.

In-situ bioremediation of petroleum waste sludge in landfarming sites of Motor Oil Hellas (petroleum refinery) was studied by monitoring the changes of the petroleum composition of the waste sludge, as well as the changes in the structure of the microbial community, for a time period of 14 months. The analyses indicated an enhanced degradation of the petroleum hydrocarbons in the landfarming areas. A depletion of n-alkanes of approximately 75-100% was obtained. Marked changes of the microbial communities of the landfarms occurred concomitantly with the degradation of the petroleum hydrocarbons. The results obtained from terminal restriction fragment length polymorphism (T-RFLP) analysis of polymerase chain reaction (PCR) amplified 16S rRNA genes demonstrated that bacteria originating from the refinery waste sludge and newly selected bacteria dominated the soil bacterial community during the period of the highest degradation activity. However, the diversity of the microbial community was decreased with increased degradation of the petroleum hydrocarbons contained in the landfarms. T-RFLP fingerprints of bacteria of the genera Enterobacter and Ochrobactrum were detected in the landfarmed soil over the entire treatment period of 14 months. In contrast, the genus Alcaligenes appeared in significant numbers only within the 10 month old landfarmed soil. Genes encoding catechol 2,3-dioxygenase (subfamily I.2.A) were detected only in DNA of the untreated refinery waste sludge. However, none of the genes known to encode the enzymes alkane hydroxylase AlkB, catechol 2,3-dioxygenase (subfamily I.2.A) and naphthalene dioxygenase nahAc could be detected in DNA of the landfarmed soils.

Insights into the genomic basis of niche specificity of Pseudomonas putida KT2440.

A major challenge in microbiology is the elucidation of the genetic and ecophysiological basis of habitat specificity of microbes. Pseudomonas putida is a paradigm of a ubiquitous metabolically versatile soil bacterium. Strain KT2440, a safety strain that has become a laboratory workhorse worldwide, has been recently sequenced and its genome annotated. By drawing on both published information and on original in silico analysis of its genome, we address here the question of what genomic features of KT2440 could explain or are consistent with its ubiquity, metabolic versatility and adaptability. The genome of KT2440 exhibits combinations of features characteristic of terrestrial, rhizosphere and aquatic bacteria, which thrive in either copiotrophic or oligotrophic habitats, and suggests that P. putida has evolved and acquired functions that equip it to thrive in diverse, often inhospitable environments, either free-living, or in close association with plants. The high diversity of protein families encoded by its genome, the large number and variety of small aralogous families, insertion elements, repetitive extragenic palindromic sequences, as well as the mosaic structure of the genome (with many regions of 'atypical' composition) and the multiplicity of mobile elements, reflect a high functional diversity in P. putida and are indicative of its evolutionary trajectory and adaptation to the diverse habitats in which it thrives. The unusual wealth of determinants for high affinity nutrient acquisition systems, mono- and di-oxygenases, oxido-reductases, ferredoxins and cytochromes, dehydrogenases, sulfur metabolism proteins, for efflux pumps and glutathione-S-transfereases, and for the extensive array of extracytoplasmatic function sigma factors, regulators, and stress response systems, constitute the genomic basis for the exceptional nutritional versatility and opportunism of P. putida , its ubiquity in diverse soil, rhizosphere and aquatic systems, and its renowned tolerance of natural and anthropogenic stresses. This metabolic diversity is also the basis of the impressive evolutionary potential of KT2440, and its utility for the experimental design of novel pathways for the catabolism of organic, particularly aromatic, pollutants, and its potential for bioremediation of soils contaminated with such compounds as well as for its application in the production of high-added value compounds.

Thermomonas haemolytica gen. nov., sp. nov., a gamma-proteobacterium from kaolin slurry.

Four aerobic, gram-negative bacterial strains isolated from kaolin slurry used in the production of paper were subjected to a polyphasic analysis and characterization to determine their taxonomic position. Analysis of the 16S rDNA sequences of the four strains revealed that they represent a new lineage within the gamma-Proteobacteria, related to the genera Xanthomonas, Pseudoxanthomonas, Stenotrophomonas, Luteimonas, Xylella and Rhodanobacter. Analysis of the quinone system, the polyamines, the fatty acids and the polar lipids revealed a combination of characteristics that is unique and not described for the phylogenetic relatives. The four strains contain a ubiquinone Q-8, spermidine as the major polyamine, diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine as the predominant polar lipids, and a fatty acid profile with predominantly iso-branched fatty acids. The G+C content of the genomic DNA was determined to be within the narrow range 67.1-68.7 mol%. Determination of DNA relatedness, as well as riboprint band patterns and amplified fragment length polymorphism profiles, clearly demonstrated that the four strains are members of a single species. Antibiotic-susceptibility patterns were identical for the four strains. Although showing a high degree of similarites in physiological and biochemical patterns, each of the four strains could be distinguished from the others on the basis of a few biochemical characteristics. On the basis of the estimates of phylogenetic relationships derived from the 16S rDNA sequence analyses, the observed chemotaxonomic characteristics and other phenotypic traits, a new genus, Thermomonas gen. nov., and species, Thermomonas haemolytica sp. nov., are proposed for the strains A50-7-3T (= DSM 13605T = LMG 19653T), B 50-7-1 (= DSM 13598 = LMG 19655), D50-7-1 (= DSM 13610 = LMG 19656) and B50-8-1 (= DSM 13599 = LMG 19654), with strain A50-7-3T as the type strain.

Phylogeny and Abundance of Novel Denitrifying Bacteria Isolated from the Water Column of the Central Baltic Sea.

The Baltic Sea is an estuarine ecosystem where denitrification in the low oxic and anoxic parts of the deep water contributes significantly to the nitrogen budget. Seventy-six heterotrophic, denitrifying, strains have been isolated by four cultivation procedures from the water column of the Gotland Deep, the main anoxic basin of the Central Baltic. Phylogenetic positions of representative strains of 10 different genotypes, grouped beforehand by low molecular weight (LMW) RNA profiling, were estimated by 16S rRNA sequence analysis. The 10 genotypes consisted of two members of the alpha subclass of the Proteobacteria and eight members of the gamma subclass. The major fraction of the genotypes was considered to be novel species or even genera. The gamma-Proteobacteria were the most abundant of the denitrifying isolates (96% of the total isolates) with a predominance of Shewanella baltica (77%), whereas the alpha-Proteobacteria were represented by single isolates. The diversity spectrum of Baltic sea denitrifying isolates was rather distinct from that previously described for marine and freshwater environments. Denitrifying bacteria could be isolated from all depths of the water column with the highest diversity and abundance of genotypes detected in samples of the oxic-anoxic interface, the layer of high in situ denitrification. For success of isolation of phylogenetically divers denitrifiers, both sample origin and cultivation procedure were observed to have an impact.

Role of Microcolony Formation in the Protistan Grazing Defense of the Aquatic Bacterium Pseudomonas sp. MWH1.

A BSTRACTThe defense strategy of the aquatic bacterium Pseudomonas sp. MWH1 against flagellate grazing was investigated in chemostat and batch experiments. The influence of predation on the Pseudomonas population was studied in the absence and presence of a potential competitor ( Vibrio sp. CB5), as well as under starvation conditions and in a situation of unlimited growth. In the competition experiment the two bacterial strains were distinguished by immunofluorescence microscopy. When the Pseudomonas strain was cultured in the absence of the predator Ochromonas sp. DS, only mobile single cells were detectable. Grazing by this bacterivorous flagellate resulted in all experiments in the occurrence of a Pseudomonas subpopulation, which grew as floclike, suspended microcolonies. These microcolonies consisted of up to approximately 1,000 cells and were, because of their large size, protected against flagellate grazing. The microcolony subpopulation dominated the total Pseudomonas population in situations of high grazing pressure at a wide range of bacterial growth conditions. Thus, the formation of the microcolonies is interpreted as a successful grazing-defense strategy, which is effective under several growth conditions, allowing for the survival of the strain even when substrate depletion is combined with strong grazing pressure. Batch culture experiments demonstrated that the change in morphology of Pseudomonas sp. MWH1 is not controlled by growth rate, although no formation of microcolonies was observed after the addition of 0.2-&mgr;m-filtered flagellate cultures to Pseudomonas cultures, indicating that a chemical trigger released by the flagellate is not involved in the control of this defense mechanism.

Interaction of Sphingomonas and Pseudomonas strains in the degradation of chlorinated dibenzofurans.

We have studied the concerted degradation of two monochlorodibenzofurans by a bacterial consortium, consisting of the chlorodibenzofurans-cometabolizing and chlorosalicylates-excreting strain Sphingomonas sp RW16, and Pseudomonas sp RW10, which mineralized the released chlorosalicylates. Neither of the organisms was able to grow with chlorodibenzofurans alone. Degradation of 2-chloro- and 3-chlorodibenzofuran proceeded to the end products 5-chloro- and 4-chlorosalicylate, respectively, when the initial dioxygenase of Sphingomonas sp RW 16 attacked the unchlorinated aromatic ring of the heterocyclic dibenzofuran molecule. 2-Hydroxypenta-2,4-dienoate, formed upon meta-cleavage of the intermediary chlorotrihydroxybiphenyls, served as a growth substrate for the sphingomonad. Presumably, most of the chlorosalicylates were excreted and degraded further by Pseudomonas sp RW10. Mineralization of both chlorosalicylates proceeded through a converging pathway, via 4-chlorocatechol, and protoanemonin. Chlorosalicylates were mineralized by the pseudomonad only when their concentration in the culture medium was below 1.5 mM. In the case of initial dioxygenation taking place on the chlorinated aromatic ring, salicylate and chlorinated hydroxypentadienoates should be formed. The metabolic fate of putative chlorohydroxypentadienoates is not clear; ie, they may be channeled into unproductive catabolism and, thus, represent the critical point in the breakdown of the carbon of these two chlorodibenzofurans by Sphingomonas sp RW16.

Lessons learned from Sphingomonas species that degrade abietane triterpenoids.

Abietane terpenoid-degrading organisms include Sphingomonas spp which inhabit natural environments and biological treatment systems. An isolate from the high Arctic indicates that these organisms occur far from trees which synthesize abietanes and suggests that some of these organisms can occupy a niche in hydrocarbon-degrading soil communities. Abietane-degrading Sphingomonas spp provide additional evidence that the phylogeny of this genus is independent of the catabolic capabilities of its members. Studies of Sphingomonas sp DhA-33 demonstrate that biological treatment systems for pulp mill effluents have the potential to mineralize abietane resin acids. On the other hand, these studies indicate that some chlorinated dehydroabietic acids are quite recalcitrant. Strain DhA-33 grows relatively well on some chlorinated dehydroabietic acids but transforms others to stable metabolites. Using strain DhA-33, a novel method was developed to measure the metabolic activity of an individual population within a complex microbial community. Oligonucleotide hybridization probes were used to assay the 16S rRNA:rDNA ratio of DhA-33 as it grew in an activated sludge community. However, this method proved not to be sufficiently sensitive to measure naturally occurring resin acid-degrading populations. We propose that the same approach can be modified to use more sensitive assays.