Campylobacter armoricus sp. nov., a novel member of the Campylobacter lari group isolated from surface water and stools from humans with enteric infection.

During a study on the prevalence and diversity of members of the genus Campylobacter in a shellfish-harvesting area and its catchment in Brittany, France, six urease-positive isolates of members of the genus Campylobacter were recovered from surface water samples, as well as three isolates from stools of humans displaying enteric infection in the same period. These strains were initially identified as members of the Campylobacter lari group by MALDI-TOF mass spectrometry and placed into a distinct group in the genus Campylobacter, following atpA gene sequence analysis based on whole-genome sequencing data. This taxonomic position was confirmed by phylogenetic analysis of the 16S rRNA, rpoB and hsp60 (groEL) loci, and an analysis of the core genome that provided an improved phylogenetic resolution. The average nucleotide identity between the representative strain CA656T (CCUG 73571T=CIP 111675T) and the type strain of the most closely related species Campylobacter ornithocola WBE38T was 88.5 %. The strains were found to be microaerobic and anaerobic, motile, non-spore-forming, Gram-stain-negative, spiral-shaped bacteria that exhibit catalase, oxidase and urease activities but not nitrate reduction. This study demonstrates clearly that the nine isolates represent a novel species within the C. lari group, for which the name Campylobacter armoricus is proposed. Here, we present phenotypic and morphological features of the nine strains and the description of their genome sequences. The proposed type strain CA656T has a 1.589 Mbp chromosome with a DNA G+C content of 28.5 mol% and encodes 1588 predicted coding sequences, 38 tRNAs, and 3 rRNA operons.

Investigating the host specificity of Campylobacter jejuni and Campylobacter coli by sequencing gyrase subunit A.

BACKGROUND: Surveillance and field investigations of Campylobacter infections require molecular tools with genetic markers appropriate for tracing purposes, i.e. based on the principle that some Campylobacter lineages acquire a host signature under adaptive selection pressure. We developed a sequence-based method targeting the quinolone resistance determining region within the subunit A of DNA gyrase (gyrA). Host specificity was evaluated by characterizing two collections of Campylobacter jejuni (N = 430) and Campylobacter coli (N = 302) originating from surface waters, domestic mammals and poultry. RESULTS: Based on nucleotide identity, a total of 80 gyrA alleles were observed. Thirty nine alleles assigned to C. coli encoding two peptides fell into three clades: two associated with surface waters and one associated with domestic mammals and poultry. The variability in GC content generated by synonymous mutations suggested that surface waters isolates originated from two distinct ecological niches. A total of 42 alleles were recorded from C. jejuni strains and encoded 8 peptides including one lying in a distinct lineage associated with wildlife. Seven of the 23 alleles encoding peptide #1 displayed the synonymous mutation G408A not identified in poultry isolates. By contrast, the substitution Ser22Gly observed in 4 different peptide groups was significantly associated with domestic birds (P = 0.001). The change in amino acid sequences Thr86Ile conferring resistance to quinolones was significantly associated with poultry (P < 0.001) in both C. jejuni and C. coli with 38.7% and 67.9% of quinolone-resistant strains, respectively. CONCLUSIONS: The gyrA typing method presented here is an informative tool as sequences appear to be predictive of particular ecological niches. Combined with multi-locus sequence typing, it could increase the resolution of source attribution, and combined with porA/flaA typing it could be suitable for detecting temporal clusters of human cases. All gyrA alleles identified were deposited in the freely accessible online database http://pubmlst.org/campylobacter.

Two-day detection of infectious enteric and non-enteric adenoviruses by improved ICC-qPCR.

In order to provide a more suitable response to public health concerns, we improved the detection of infectious human adenoviruses in water by optimising the commonly used integrated cell culture-PCR method. Risk evaluation studies seek for rapid detection of infectious adenoviruses, including the enteric types 40 and 41 that are considered as the second most common agents of gastroenteritis in children next to rotaviruses. The here-employed 293A cell line used for infectious status assessment showed its ability to multiply adenoviruses including type 41. Two modifications were moreover applied to the workflow for viral detection. The first occurred at the nucleic acid extraction step performed directly on all infected cells, while the second was the application of real-time quantitative PCR as detection tool. All adaptations led to a 3-day reduction of the response delay and an improved sensitivity especially for the enteric adenoviral types. The infectious status of laboratory strain types 2 and 41 was demonstrated by a more than 2-log10 increase in genome quantity. These conclusions were confirmed and reinforced by the analysis of water samples applying the improved assay. Naturally occurring infectious adenoviruses were detected in wastewater and river water, within 2 days. Types belonging to the species human adenoviruses C and type 31 were observed, but the most frequently identified type was 41 (71 % of identified sequences, n = 34). This highlights the usefulness of our method for a wide range of types, and especially for the most prevalent and public health-relevant enteric adenoviruses.

Metagenomic Strain-Typing Combined with Isolate Sequencing Provides Increased Resolution of the Genetic Diversity of Campylobacter jejuni Carriage in Wild Birds.

As the world's leading cause of human gastro-enteritis, the food- and waterborne pathogen Campylobacter needs to be intensively monitored through a One Health approach. Particularly, wild birds have been hypothesized to contribute to the spread of human clinical recurring C. jejuni genotypes across several countries. A major concern in studying epidemiological dynamics is resolving the large genomic diversity of strains circulating in the environment and various reservoirs, challenging to achieve with isolation techniques. Here, we applied a passive-filtration method to obtain isolates and in parallel recovered genotypes from metagenomic sequencing data from associated filter sweeps. For genotyping mixed strains, a reference-based computational workflow to predict allelic profiles of nine extended-MLST loci was utilized. We validated the pipeline by sequencing artificial mixtures of C. jejuni strains and observed the highest prediction accuracy when including obtained isolates as references. By analyzing metagenomic samples, we were able to detect over 20% additional genetic diversity and observed an over 50% increase in the potential to connect genotypes across wild-bird samples. With an optimized filtration method and a computational approach for genotyping strain mixtures, we provide the foundation for future studies assessing C. jejuni diversity in environmental and clinical settings at improved throughput and resolution.

Environmental dynamics of Campylobacter jejuni genotypes circulating in Luxembourg: what is the role of wild birds?

Campylobacter jejuni is the leading cause of bacterial gastroenteritis worldwide, but, unlike other foodborne pathogens, is not commonly reported as causing outbreaks. The population structure of the species is characterized by a high degree of genetic diversity, but the presence of stable clonally derived genotypes persisting in space and time, and potentially leading to diffuse outbreaks, has recently been identified. The spread of these recurring genotypes could be enhanced by wild birds, suspected to act as vectors for a wide range of microorganisms that can be transmissible to other animals or humans. This study assessed the genetic diversity of C. jejuni carriage in wild birds and surface waters to explore a potential link between these environments and the persistence over years of recurring lineages infecting humans in Luxembourg. These lineages corresponded to over 40 % of clinical isolates over a 4 year period from 2018 to 2021. While mainly exotic genotypes were recovered from environmental samples, 4 % of C. jejuni from wild birds corresponded to human recurring genotypes. Among them, a human clinical endemic lineage, occurring for over a decade in Luxembourg, was detected in one bird species, suggesting a possible contribution to the persistence of this clone and its multi-host feature. Whereas 27 % of wild birds were carriers of C. jejuni, confirming their role as spreader or reservoir, only three out of 59 genotypes overlapped with recurring human strains. While direct transmission of C. jejuni infection through wild birds remains questionable, they may play a key role in the environmental spreading of stable clones to livestock, and this issue merits further investigation.

Combination of MALDI-TOF Mass Spectrometry and Machine Learning for Rapid Antimicrobial Resistance Screening: The Case of Campylobacter spp.

While MALDI-TOF mass spectrometry (MS) is widely considered as the reference method for the rapid and inexpensive identification of microorganisms in routine laboratories, less attention has been addressed to its ability for detection of antimicrobial resistance (AMR). Recently, some studies assessed its potential application together with machine learning for the detection of AMR in clinical pathogens. The scope of this study was to investigate MALDI-TOF MS protein mass spectra combined with a prediction approach as an AMR screening tool for relevant foodborne pathogens, such as Campylobacter coli and Campylobacter jejuni. A One-Health panel of 224 C. jejuni and 116 C. coli strains was phenotypically tested for seven antimicrobial resistances, i.e., ciprofloxacin, erythromycin, tetracycline, gentamycin, kanamycin, streptomycin, and ampicillin, independently, and were submitted, after an on- and off-plate protein extraction, to MALDI Biotyper analysis, which yielded one average spectra per isolate and type of extraction. Overall, high performance was observed for classifiers detecting susceptible as well as ciprofloxacin- and tetracycline-resistant isolates. A maximum sensitivity and a precision of 92.3 and 81.2%, respectively, were reached. No significant prediction performance differences were observed between on- and off-plate types of protein extractions. Finally, three putative AMR biomarkers for fluoroquinolones, tetracyclines, and aminoglycosides were identified during the current study. Combination of MALDI-TOF MS and machine learning could be an efficient and inexpensive tool to swiftly screen certain AMR in foodborne pathogens, which may enable a rapid initiation of a precise, targeted antibiotic treatment.

Investigation of MALDI-TOF Mass Spectrometry for Assessing the Molecular Diversity of Campylobacter jejuni and Comparison with MLST and cgMLST: A Luxembourg One-Health Study.

There is a need for active molecular surveillance of human and veterinary Campylobacter infections. However, sequencing of all isolates is associated with high costs and a considerable workload. Thus, there is a need for a straightforward complementary tool to prioritize isolates to sequence. In this study, we proposed to investigate the ability of MALDI-TOF MS to pre-screen C. jejuni genetic diversity in comparison to MLST and cgMLST. A panel of 126 isolates, with 10 clonal complexes (CC), 21 sequence types (ST) and 42 different complex types (CT) determined by the SeqSphere+ cgMLST, were analysed by a MALDI Biotyper, resulting into one average spectra per isolate. Concordance and discriminating ability were evaluated based on protein profiles and different cut-offs. A random forest algorithm was trained to predict STs. With a 94% similarity cut-off, an AWC of 1.000, 0.933 and 0.851 was obtained for MLSTCC, MLSTST and cgMLST profile, respectively. The random forest classifier showed a sensitivity and specificity up to 97.5% to predict four different STs. Protein profiles allowed to predict C. jejuni CCs, STs and CTs at 100%, 93% and 85%, respectively. Machine learning and MALDI-TOF MS could be a fast and inexpensive complementary tool to give an early signal of recurrent C. jejuni on a routine basis.

Coupling of denaturing high-performance liquid chromatography and terminal restriction fragment length polymorphism with precise fragment sizing for microbial community profiling and characterization.

Terminal restriction fragment length polymorphism (T-RFLP) is used to monitor the structural diversity of complex microbial communities in terms of richness, relative abundance, and distribution of the major subpopulations and individual members. However, discrepancies of several nucleotides between expected and experimentally observed lengths of terminal restriction fragments (T-RFs), together with the difficulty of obtaining DNA sequence information from T-RFLP profiling, often prevent accurate phylogenetic characterization of the microbial community of interest. In this study, T-RFLP analysis of DNA from an artificial assembly of five bacterial strains was carried out with a combination of two size markers with different fluorescent tags. Precise sizing of T-RFs in the 50- to 500-nucleotide range was achieved by using the same dye for both samples and size markers. Phylogenetic assignment of the component microbial strains was facilitated by coupling T-RFLP to denaturing high-performance liquid chromatography (D-HPLC) of 16S RNA gene fragments followed by direct sequencing. The proposed coupling of D-HPLC and T-RFLP provides unambiguous characterization of microbial communities containing less than 15 microbial strains.

Investigating Major Recurring Campylobacter jejuni Lineages in Luxembourg Using Four Core or Whole Genome Sequencing Typing Schemes.

Campylobacter jejuni is the leading cause of bacterial gastroenteritis, which has motivated the monitoring of genetic profiles circulating in Luxembourg since 13 years. From our integrated surveillance using a genotyping strategy based on an extended MLST scheme including gyrA and porA markers, an unexpected endemic pattern was discovered in the temporal distribution of genotypes. We aimed to test the hypothesis of stable lineages occurrence by implementing whole genome sequencing (WGS) associated with comprehensive and internationally validated schemes. This pilot study assessed four WGS-based typing schemes to classify a panel of 108 strains previously identified as recurrent or sporadic profiles using this in-house typing system. The strain collection included four common lineages in human infection (N = 67) initially identified from recurrent combination of ST-gyrA-porA alleles also detected in non-human samples: veterinary (N = 19), food (N = 20), and environmental (N = 2) sources. An additional set of 19 strains belonging to sporadic profiles completed the tested panel. All the strains were processed by WGS by using Illumina technologies and by applying stringent criteria for filtering sequencing data; we ensure robustness in our genomic comparison. Four typing schemes were applied to classify the strains: (i) the cgMLST SeqSphere+ scheme of 637 loci, (ii) the cgMLST Oxford scheme of 1,343 loci, (iii) the cgMLST INNUENDO scheme of 678 loci, and (iv) the wgMLST INNUENDO scheme of 2,795 loci. A high concordance between the typing schemes was determined by comparing the calculated adjusted Wallace coefficients. After quality control and analyses with these four typing schemes, 60 strains were confirmed as members of the four recurrent lineages regardless of the method used (N = 32, 12, 7, and 9, respectively). Our results indicate that, regardless of the typing scheme used, epidemic or endemic signals were detected as reflected by lineage B (ST2254-gyrA9-porA1) in 2014 or lineage A (ST19-gyrA8-porA7), respectively. These findings support the clonal expansion of stable genomes in Campylobacter population exhibiting a multi-host profile and accounting for the majority of clinical strains isolated over a decade. Such recurring genotypes suggest persistence in reservoirs, sources or environment, emphasizing the need to investigate their survival strategy in greater depth.

MALDI-TOF Mass Spectrometry and Specific Biomarkers: Potential New Key for Swift Identification of Antimicrobial Resistance in Foodborne Pathogens.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) is today the reference method for direct identification of microorganisms in diagnostic laboratories, as it is notably time- and cost-efficient. In the context of increasing cases of enteric diseases with emerging multi-drug resistance patterns, there is an urgent need to adopt an efficient workflow to characterize antimicrobial resistance (AMR). Current approaches, such as antibiograms, are time-consuming and directly impact the "patient-physician" workflow. Through this mini-review, we summarize how the detection of specific patterns by MALDI-TOF MS, as well as bioinformatics, become more and more essential in research, and how these approaches will help diagnostics in the future. Along the same lines, the idea to export more precise biomarker identification steps by MALDI-TOF(/TOF) MS data towards AMR identification pipelines is discussed. The study also critically points out that there is currently still a lack of research data and knowledge on different foodborne pathogens as well as several antibiotics families such as macrolides and quinolones, and many questions are still remaining. Finally, the innovative combination of whole-genome sequencing and MALDI-TOF MS could be soon the future for diagnosis of antimicrobial resistance in foodborne pathogens.

Human Campylobacteriosis in Luxembourg, 2010-2013: A Case-Control Study Combined with Multilocus Sequence Typing for Source Attribution and Risk Factor Analysis.

Campylobacteriosis has increased markedly in Luxembourg during recent years. We sought to determine which Campylobacter genotypes infect humans, where they may originate from, and how they may infect humans. Multilocus sequence typing was performed on 1153 Campylobacter jejuni and 136 C. coli human strains to be attributed to three putative animal reservoirs (poultry, ruminants, pigs) and to environmental water using the asymmetric island model. A nationwide case-control study (2010-2013) for domestic campylobacteriosis was also conducted, including 367 C. jejuni and 48 C. coli cases, and 624 controls. Risk factors were investigated by Campylobacter species, and for strains attributed to different sources using a combined case-control and source attribution analysis. 282 sequence types (STs) were identified: ST-21, ST-48, ST-572, ST-50 and ST-257 were prevailing. Most cases were attributed to poultry (61.2%) and ruminants (33.3%). Consuming chicken outside the home was the dominant risk factor for both Campylobacter species. Newly identified risk factors included contact with garden soil for either species, and consuming beef specifically for C. coli. Poultry-associated campylobacteriosis was linked to poultry consumption in wintertime, and ruminant-associated campylobacteriosis to tap-water provider type. Besides confirming chicken as campylobacteriosis primary source, additional evidence was found for other reservoirs and transmission routes.

Quantifying potential sources of surface water contamination with Campylobacter jejuni and Campylobacter coli.

Campylobacter is the most common causative agent of human bacterial gastroenteritis and is frequently found in surface water, where it indicates recent contamination with animal faeces, sewage effluent, and agricultural run-off. The contribution of different animal reservoirs to surface water contamination with Campylobacter is largely unknown. In the Netherlands, the massive poultry culling to control the 2003 avian influenza epidemic coincided with a 44-50% reduction in human campylobacteriosis cases in the culling areas, suggesting substantial environment-mediated spread of poultry-borne Campylobacter. We inferred the origin of surface water Campylobacter jejuni and Campylobacter coli strains in Luxembourg and the Netherlands, as defined by multilocus sequence typing, by comparison to strains from poultry, pigs, ruminants, and wild birds, using the asymmetric island model for source attribution. Most Luxembourgish water strains were attributed to wild birds (61.0%), followed by poultry (18.8%), ruminants (15.9%), and pigs (4.3%); whereas the Dutch water strains were mainly attributed to poultry (51.7%), wild birds (37.3%), ruminants (9.8%), and pigs (1.2%). Attributions varied over seasons and surface water types, and geographical variation in the relative contribution of poultry correlated with the magnitude of poultry production at either the national or provincial level, suggesting that environmental dissemination of Campylobacter from poultry farms and slaughterhouses can be substantial in poultry-rich regions.

A Designed Experiments Approach to Optimizing MALDI-TOF MS Spectrum Processing Parameters Enhances Detection of Antibiotic Resistance in Campylobacter jejuni.

MALDI-TOF MS has been utilized as a reliable and rapid tool for microbial fingerprinting at the genus and species levels. Recently, there has been keen interest in using MALDI-TOF MS beyond the genus and species levels to rapidly identify antibiotic resistant strains of bacteria. The purpose of this study was to enhance strain level resolution for Campylobacter jejuni through the optimization of spectrum processing parameters using a series of designed experiments. A collection of 172 strains of C. jejuni were collected from Luxembourg, New Zealand, North America, and South Africa, consisting of four groups of antibiotic resistant isolates. The groups included: (1) 65 strains resistant to cefoperazone (2) 26 resistant to cefoperazone and beta-lactams (3) 5 strains resistant to cefoperazone, beta-lactams, and tetracycline, and (4) 76 strains resistant to cefoperazone, teicoplanin, amphotericin, B and cephalothin. Initially, a model set of 16 strains (three biological replicates and three technical replicates per isolate, yielding a total of 144 spectra) of C. jejuni was subjected to each designed experiment to enhance detection of antibiotic resistance. The most optimal parameters were applied to the larger collection of 172 isolates (two biological replicates and three technical replicates per isolate, yielding a total of 1,031 spectra). We observed an increase in antibiotic resistance detection whenever either a curve based similarity coefficient (Pearson or ranked Pearson) was applied rather than a peak based (Dice) and/or the optimized preprocessing parameters were applied. Increases in antimicrobial resistance detection were scored using the jackknife maximum similarity technique following cluster analysis. From the first four groups of antibiotic resistant isolates, the optimized preprocessing parameters increased detection respective to the aforementioned groups by: (1) 5% (2) 9% (3) 10%, and (4) 2%. An additional second categorization was created from the collection consisting of 31 strains resistant to beta-lactams and 141 strains sensitive to beta-lactams. Applying optimal preprocessing parameters, beta-lactam resistance detection was increased by 34%. These results suggest that spectrum processing parameters, which are rarely optimized or adjusted, affect the performance of MALDI-TOF MS-based detection of antibiotic resistance and can be fine-tuned to enhance screening performance.

Fine-Scale Spatial Heterogeneity in the Distribution of Waterborne Protozoa in a Drinking Water Reservoir.

BACKGROUND: The occurrence of faecal pathogens in drinking water resources constitutes a threat to the supply of safe drinking water, even in industrialized nations. To efficiently assess and monitor the risk posed by these pathogens, sampling deserves careful design, based on preliminary knowledge on their distribution dynamics in water. For the protozoan pathogens Cryptosporidium and Giardia, only little is known about their spatial distribution within drinking water supplies, especially at fine scale. METHODS: Two-dimensional distribution maps were generated by sampling cross-sections at meter resolution in two different zones of a drinking water reservoir. Samples were analysed for protozoan pathogens as well as for E. coli, turbidity and physico-chemical parameters. RESULTS: Parasites displayed heterogeneous distribution patterns, as reflected by significant (oo)cyst density gradients along reservoir depth. Spatial correlations between parasites and E. coli were observed near the reservoir inlet but were absent in the downstream lacustrine zone. Measurements of surface and subsurface flow velocities suggest a role of local hydrodynamics on these spatial patterns. CONCLUSION: This fine-scale spatial study emphasizes the importance of sampling design (site, depth and position on the reservoir) for the acquisition of representative parasite data and for optimization of microbial risk assessment and monitoring. Such spatial information should prove useful to the modelling of pathogen transport dynamics in drinking water supplies.

Tetrachloromethane-Degrading Bacterial Enrichment Cultures and Isolates from a Contaminated Aquifer.

The prokaryotic community of a groundwater aquifer exposed to high concentrations of tetrachloromethane (CCl4) for more than three decades was followed by terminal restriction fragment length polymorphism (T-RFLP) during pump-and-treat remediation at the contamination source. Bacterial enrichments and isolates were obtained under selective anoxic conditions, and degraded 10 mg·L(-1) CCl4, with less than 10% transient formation of chloroform. Dichloromethane and chloromethane were not detected. Several tetrachloromethane-degrading strains were isolated from these enrichments, including bacteria from the Klebsiella and Clostridium genera closely related to previously described CCl4 degrading bacteria, and strain TM1, assigned to the genus Pelosinus, for which this property was not yet described. Pelosinus sp. TM1, an oxygen-tolerant, Gram-positive bacterium with strictly anaerobic metabolism, excreted a thermostable metabolite into the culture medium that allowed extracellular CCl4 transformation. As estimated by T-RFLP, phylotypes of CCl4-degrading enrichment cultures represented less than 7%, and archaeal and Pelosinus strains less than 0.5% of the total prokaryotic groundwater community.

Spatial and temporal distribution of Cryptosporidium and Giardia in a drinking water resource: implications for monitoring and risk assessment.

Because of their significant public health impact, waterborne Cryptosporidium and Giardia have been monitored in surface water in order to assess microbial quality of water bodies used for drinking water production and/or for recreational purposes. In this context, sampling strategy is of key importance and should be representative enough to appropriately assess the related microbial risk. This, however, requires sound knowledge on the behaviour of both pathogens in water. In the present study, the spatial and temporal distribution of Cryptosporidium and Giardia was explored in the rural Upper-Sûre watershed used for drinking water production in Luxembourg. By subdividing it into three compartments including (i) sub-catchments, (ii) the Sûre River fed by the sub-catchments and (iii) the Upper-Sûre reservoir fed by the Sûre River, parasite distribution was assessed using sampling designs adapted to the hydro-dynamic characteristics of the respective compartments. Results highlighted the high spatial and temporal variability in parasite distribution at watershed scale, as well as the prevalence of Giardia over Cryptosporidium. Besides land use features and catchment characteristics, hydro-climatology appeared to be a major driver of parasite behaviour in the watershed. It introduced a seasonal trend in their occurrence, highest densities being detected during the wet season. Peaks of contamination triggered out by rainfall-induced runoff were further observed in the three compartments. In the Sûre River, Cryptosporidium and Giardia fluxes peaked at 10(9) and 10(10) (oo)cysts.d(-1), respectively, and were discharged into the drinking water reservoir, where they underwent a 2 to 3 log10 removal rate. Despite this, parasite fluxes entering the drinking water treatment plant were still high (10(6) to 10(7) (oo)cysts.d(-1)) and stressed on the need for improved watershed management upstream the water treatment barrier. The catchment-wide analysis described here constitutes a valuable tool for assessment of catchment microbial dynamics, especially within the framework of Water Safety Plans.

Microbial degradation of tetrachloromethane: mechanisms and perspectives for bioremediation.

Toxic man-made compounds released into the environment represent potential nutrients for bacteria, and microorganisms growing with such compounds as carbon and energy sources can be used to clean up polluted sites. However, in some instances, microorganisms contribute to contaminant degradation without any apparent benefit for themselves. Such cometabolism plays an important part in bioremediation, but is often difficult to control. Microbial degradation of tetrachloromethane (carbon tetrachloride, CCl(4)), a toxic ozone-depleting organic solvent mainly of anthropogenic origin, is only known to occur by cometabolic reduction under anoxic conditions. Yet no microbial system capable of using CCl(4) as the sole carbon source has been described. Microbial growth based on CCl(4) as a terminal electron acceptor has not been reported, although corresponding degradation pathways would yield sufficient energy. Known modes for the biodegradation of CCl(4) involve several microbial metabolites, mainly metal-bound coenzymes and siderophores, which are produced by facultative or strictly anaerobic bacteria and methanogenic Archaea. Recent reports have demonstrated that CCl(4) dechlorination rates are enhanced by redox-active organic compounds such as humic acids and quinones, which act as shuttles between electron-providing microorganisms and CCl(4) as a strong electron acceptor. The key factors underlying dechlorination of CCl(4), the practical aspects and specific requirements for microorganism-associated degradation of CCl(4) at contaminated sites and perspectives for future developments are discussed.

Methylobacterium genome sequences: a reference blueprint to investigate microbial metabolism of C1 compounds from natural and industrial sources.

BACKGROUND: Methylotrophy describes the ability of organisms to grow on reduced organic compounds without carbon-carbon bonds. The genomes of two pink-pigmented facultative methylotrophic bacteria of the Alpha-proteobacterial genus Methylobacterium, the reference species Methylobacterium extorquens strain AM1 and the dichloromethane-degrading strain DM4, were compared. METHODOLOGY/PRINCIPAL FINDINGS: The 6.88 Mb genome of strain AM1 comprises a 5.51 Mb chromosome, a 1.26 Mb megaplasmid and three plasmids, while the 6.12 Mb genome of strain DM4 features a 5.94 Mb chromosome and two plasmids. The chromosomes are highly syntenic and share a large majority of genes, while plasmids are mostly strain-specific, with the exception of a 130 kb region of the strain AM1 megaplasmid which is syntenic to a chromosomal region of strain DM4. Both genomes contain large sets of insertion elements, many of them strain-specific, suggesting an important potential for genomic plasticity. Most of the genomic determinants associated with methylotrophy are nearly identical, with two exceptions that illustrate the metabolic and genomic versatility of Methylobacterium. A 126 kb dichloromethane utilization (dcm) gene cluster is essential for the ability of strain DM4 to use DCM as the sole carbon and energy source for growth and is unique to strain DM4. The methylamine utilization (mau) gene cluster is only found in strain AM1, indicating that strain DM4 employs an alternative system for growth with methylamine. The dcm and mau clusters represent two of the chromosomal genomic islands (AM1: 28; DM4: 17) that were defined. The mau cluster is flanked by mobile elements, but the dcm cluster disrupts a gene annotated as chelatase and for which we propose the name "island integration determinant" (iid). CONCLUSION/SIGNIFICANCE: These two genome sequences provide a platform for intra- and interspecies genomic comparisons in the genus Methylobacterium, and for investigations of the adaptive mechanisms which allow bacterial lineages to acquire methylotrophic lifestyles.