Burkholderia cenocepacia ST-250 in cystic fibrosis patients in Switzerland: Genomic investigation of transmission routes.

This report describes the characterization of Burkholderia cenocepacia isolates belonging to sequence type (ST)-250, detected in eight patients with cystic fibrosis (CF) in Switzerland. We retrospectively analyzed 18 isolates of B. cenocepacia ST-250 isolated between 2003 and 2015 by whole-genome sequencing and evaluated clinical and epidemiological data. Single nucleotide polymorphism analysis of the B.°cenocepacia ST-250 lineage showed that the isolates from all patients cluster tightly, suggesting that this cluster has a recent common ancestor. Epidemiological investigations showed that six out of eight patients acquired B.°cenocepacia ST-250 in the years 2001-2006, where participation in CF summer camps was common. Two patients were siblings. Genomic relatedness of the B. cenocepacia ST-250 isolates supported transmission by close contact, however, a common source or nosocomial routes cannot be excluded. With respect to the fatal outcome in six patients, our study shows the importance of infection control measurements in CF patients with B.°cenocepacia.

Outbreak of Burkholderia cenocepacia in an intensive care unit of a tertiary care hospital identified by pulsed-field gel electrophoresis.

Nosocomial outbreaks of Burkholderia cepacia complex, transmitted through contaminated medical surfaces or equipment have been reported. Pulsed-field Gel Electrophoresis (PFGE) is recognized as the "gold standard" for molecular subtyping, yet studies on clonal relationships in India are limited. PFGE was used to study the clonal relationships of 22 isolates of Burkholderia cenocepacia from 12 patients admitted to a critical care unit during 2 months (November and December 2021). PFGE revealed three different profiles with 15 isolates belonging to a single cluster suggesting a common source within the hospital, emphasizing the need for preventive measures to control B. cenocepacia transmission.

Fourier Transform Infrared Spectroscopy for Typing Burkholderia cenocepacia ET12 Isolates.

The IR Biotyper and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) using ClinProTools software (MALDI-TOF MS-ClinProTools) are two novel typing methods that rely on the analysis of carbohydrate and peptide residues in intact bacterial cells. These two methods have shown promising results in the rapid and accurate typing of bacteria. In this study, we evaluated these novel typing methods in comparison with genotypic typing for cluster analysis of Burkholderia cenocepacia epidemic strain ET12, isolated from adult cystic fibrosis patients. Sixty-six isolates of B. cenocepacia were used in this study, 35 of which were identified as the ET12 strain and 31 as non-ET12 strains by repetitive-element PCR (rep-PCR). Twelve isolates were used for the creation of typing models using IR Biotyper and MALDI-TOF MS-ClinProTools, and 54 isolates were used for external validation of the typing models. The IR Biotyper linear discriminant analysis (LDA) model had a diagnostic sensitivity of 84.6% for typing the epidemic strain, ET12. At a cutoff of 70%, MALDI-TOF MS-ClinProTools had 87.5% diagnostic sensitivity in detecting the ET12 strain (P = 1.00). Both methods had a diagnostic specificity of ≥80% for detecting the ET12 strain. In conclusion, IR Biotyper and MALDI-TOF MS-ClinProTools offer rapid typing using proteomics and analysis of small cellular molecules with a low running cost. Our pilot study showed suboptimal accuracy of both methods for typing outbreak strains of B. cenocepacia. Extending the spectral region analyzed by the IR Biotyper can improve the accuracy and has the potential of improving the generalizability of this technique for typing other organisms. IMPORTANCE Respiratory infections due to Burkholderia cenocepacia, particularly the ET12 epidemic strain, are considered sentinel events for persons with cystic fibrosis, as they are often associated with person-to-person transmission and accelerated decline in lung function and early mortality. Current typing methods are generally only available at reference centers, with long turn-around-times, which can affect the identification of outbreaks and critical patient triage. This pilot study aims to add to the growing literature illustrating the potential utility of Fourier transform infrared spectroscopy (FTIR), a novel rapid method, for the successful typing of clinically significant bacteria. In this study, we evaluated its utility to discriminate between the ET12 clone and non-ET12 isolates of B. cenocepacia and compared it to proteomics cluster analysis using MALDI-TOF MS and ClinProTools software. Both methods had encouraging but suboptimal accuracy (≥85% sensitivity and ≥83% specificity), which will likely be improved by extending the spectral region analyzed by the IR Biotyper with updated software.

Genomic analysis of Burkholderia cenocepacia isolated from a liver abscess in a patient with cystic fibrosis.

Burkholderia cenocepacia complex is associated with high transmissibility, virulence, and poor prognosis in cystic fibrosis (CF) patients. However, extrapulmonary infections are rare. We investigated the genome of a B. cenocepacia IIIA isolated from a liver abscess in a Brazilian CF patient and compared it to strain J2315. The whole genome was sequenced, and contigs were annotated by Rapid Annotation using Subsystem Technology. The Pathosystems Resource Integration Center was used to map antimicrobial and virulence genes. The genomic island (GIs) analysis was performed using two prediction methods, and the presence of putative plasmids and insertion sequences (ISs) was investigated. The isolate was confirmed as B. cenocepacia IIIA to ST-28 (ET12 lineage). A total of 64 genes for antimicrobial resistance and 47 genes related to virulence were identified. Among the virulence factors, there was a predominance of factors related to the invasion mechanism, to the flagellar biosynthesis protein, and to the RNA polymerase sigma factor for flagellar operon (cdpA). Two IS families (IS3 and IS5) and only one plasmid were found. On average 56 GIs were predicted by at least one of the methods applied. Comparative analysis showed resistance mechanisms and virulence factors revealing invasive determinants used by B. cenocepacia IIIA (ET12) in the process of disease spread to other infection sites (extrapulmonary) of highly virulent strains in CF patients.

Biofilm formation by strains of Burkholderia cenocepacia lineages IIIA and IIIB and B. gladioli pv. alliicola associated with onion bacterial scale rot.

The Burkholderia genus has high ecological and nutritional versatility, having species capable of causing diseases in animals, humans, and plants. During chronic infections in humans, biofilm formation is a characteristic often associated with strains from different species of this genus. However, there is still no information on the formation of biofilms by plant pathogenic strains of B. cenocepacia (Bce) lineages IIIA and IIIB and B. gladioli pv. alliicola (Bga), which are associated with onion bacterial scale rot in the semi-arid region of northeast Brazil. In this study, we performed an in vitro characterization of biofilm formation ability in different culture media by the phytopathogenic strains of Bce and Bga and investigated its relationship with swarming motility. Our results indicated the existence of an intraspecific variation in biofilm formation capacity in vitro by these bacteria and the existence of a negative correlation between swarming motility and biofilm formation for strains of Bce lineage IIIB. In addition, histopathological analyses performed using optical microscopy and scanning electron microscopy revealed the formation of biofilm in vivo by Bce strains in onion tissues.

Isolation and characterization of Burkholderia cenocepacia CR318, a phosphate solubilizing bacterium promoting corn growth.

Plant-growth promoting rhizobacteria benefit crop health and growth through various mechanisms including phosphate and potassium solubilisation, and antimicrobial activity. Previously, we sequenced the genome of bacterial strain Burkholderia cenocepacia CR318, which was isolated from the roots of the starch corn (Zea mays L.) in London, Ontario, Canada. In this work, the species identity of this isolate is confirmed by recA phylogeny and in silico DNA-DNA hybridization (isDDH), and its plant-growth promoting characteristics are described. B. cenocepacia CR318 exhibited strong activity of inorganic phosphate and potassium solubilization. It significantly promoted the growth of corn plants and roots by solubilizing inorganic tricalcium phosphate under greenhouse conditions. Functional analysis of the complete B. cenocepacia CR318 genome revealed genes associated with phosphate metabolism such as pstSCAB encoding a high affinity inorganic phosphate-specific transporter, and the pqqABCDE gene cluster involved in the biosynthesis of pyrroloquinoline quinone (PQQ), which is a required cofactor for quinoprotein glucose dehydrogenase (Gdh). However, it appears that B. cenocepacia CR318 lacks the quinoprotein Gdh which can produce gluconic acid to solubilize inorganic phosphate. Overall, these findings provide an important step in understanding the molecular mechanisms underlying the plant growth promotion trait of B. cenocepacia CR318.

The Eno Gene of Burkholderia cenocepacia Strain 71-2 is Involved in Phosphate Solubilization.

Bacterial strain 71-2 with phosphate-solubilizing activity was isolated from tobacco rhizosphere and classified as Burkholderia cenocepacia based on sequence analyses of 16S rRNA and recA genes. To learn phosphate-solubilizing mechanisms of 71-2, mutants showing reduced solubilizing phosphate activity were obtained using the EZ-Tn5 transposon. Mutant 71-2-MT51 was reduced in the solubilizing phosphate content to 34.36% as compared with the wild-type strain 71-2. The disrupted gene in 71-2-MT51 was cloned and sequenced, and the putative protein from the gene shared 65.26% identity to protein sequences of enolase from Escherichia coli, which suggests the gene encodes an enzyme of enolase. Complementation analyzing showed that Eno was responsible for phosphate solubilizing for B. cenocepacia strain 71-2. To our knowledge, this is the first report of Eno involved in phosphate solubilizing in B. cenocepacia as well as in other bacteria.

TssA from Burkholderia cenocepacia: expression, purification, crystallization and crystallographic analysis.

TssA is a core component of the type VI secretion system, and phylogenetic analysis of TssA subunits from different species has suggested that these proteins fall into three distinct clades. Whilst representatives of two clades, TssA1 and TssA2, have been the subjects of investigation, no members of the third clade (TssA3) have been studied. Constructs of TssA from Burkholderia cenocepacia, a representative of clade 3, were expressed, purified and subjected to crystallization trials. Data were collected from crystals of constructs of the N-terminal and C-terminal domains. Analysis of the data from the crystals of these constructs and preliminary structure determination indicates that the C-terminal domain forms an assembly of 32 subunits in D16 symmetry, whereas the N-terminal domain is not involved in subunit assocation.

Clinical characteristics of bacteraemia caused by Burkholderia cepacia complex species and antimicrobial susceptibility of the isolates in a medical centre in Taiwan.

This study investigated the clinical characteristics and outcomes of bacteraemia due to Burkholderia cepacia complex (BCC) species among 54 patients without cystic fibrosis from January 2013 to February 2015. BCC isolates were identified to the species level by the Bruker Biotyper MALDI-TOF MS system and by sequencing analysis of the 16S rRNA and recA genes. Antimicrobial susceptibilities of the isolates were determined by the agar dilution method. Sequencing of the recA gene in the 54 blood isolates revealed 37 (68.5%) isolates of B. cenocepacia, 9 (16.7%) of B. cepacia, 4 (7.4%) of B. multivorans and one isolate each of B. arboris, B. pseudomultivorans, B. seminalis, and B. vietnamiensis. The overall performance of the Bruker Biotyper MALDI-TOF MS system for correctly identifying the 54 BCC isolates to the species level was 79.6%, which was better than that (16.7%) by 16S RNA sequencing analysis. Bacteraemic pneumonia (n = 23, 42.6%) and catheter-related bacteraemia (n = 21, 38.9%) were the most common types of infection. Higher rates of ceftazidime and meropenem resistance were found in B. cepacia isolates (33.3% and 22.2%, respectively) than in isolates of B. cenocepacia (21.6% and 10.8%, respectively) and other species (12.5% and 12.5%, respectively). Overall, the 30-day mortality rate was 38.9% (21/54). Bacteraemia caused by BCC species other than B. cenocepacia and B. cepacia (adjusted odds ratio [aOR] 20.005, P = 0.024) and high SOFA score (aOR 1.412, P = 0.003) were predictive of higher 30-day mortality. Different BCC species are associated with different outcomes of bacteraemia and exhibit different susceptibility patterns.

Outbreak of health care-associated Burkholderia cenocepacia bacteremia and infection attributed to contaminated sterile gel used for central line insertion under ultrasound guidance and other procedures.

BACKGROUND: We report an outbreak of Burkholderia cenocepacia bacteremia and infection in 11 patients predominately in intensive care units caused by contaminated ultrasound gel used in central line insertion and sterile procedures within 4 hospitals across Australia. METHODS: Burkholderia cenocepacia was first identified in the blood culture of a patient from the intensive care unit at the Gold Coast University Hospital on March 26, 2017, with 3 subsequent cases identified by April 7, 2017. The outbreak response team commenced investigative measures. RESULTS: The outbreak investigation identified the point source as contaminated gel packaged in sachets for use within the sterile ultrasound probe cover. In total, 11 patient isolates of B cenocepacia with the same multilocus sequence type were identified within 4 hospitals across Australia. This typing was the same as identified in the contaminated gel isolate with single nucleotide polymorphism-based typing, demonstrating that all linked isolates clustered together. CONCLUSION: Arresting the national point-source outbreak within multiple jurisdictions was critically reliant on a rapid, integrated, and coordinated response and the use of informal professional networks to first identify it. All institutions where the product is used should look back at Burkholderia sp blood culture isolates for speciation to ensure this outbreak is no larger than currently recognized given likely global distribution.

Whole-Genome Sequencing of Three Clonal Clinical Isolates of B. cenocepacia from a Patient with Cystic Fibrosis.

Burkholderia cepacia complex bacteria are amongst the most feared of pathogens in cystic fibrosis (CF). The BCC comprises at least 20 distinct species that can cause chronic and unpredictable lung infections in CF. Historically the species B. cenocepacia has been the most prevalent in CF infections and has been associated in some centers with high rates of mortality. Modeling chronic infection by B. cenocepacia in the laboratory is challenging and no models exist which effectively recapitulate CF disease caused by BCC bacteria. Therefore our understanding of factors that contribute towards the morbidity and mortality caused by this organism is limited. In this study we used whole-genome sequencing to examine the evolution of 3 clonal clinical isolates of B. cenocepacia from a patient with cystic fibrosis. The first isolate was from the beginning of infection, and the second two almost 10 years later during the final year of the patients' life. These isolates also demonstrated phenotypic heterogeneity, with the first isolate displaying the mucoid phenotype (conferred by the overproduction of exopolysaccharide), while one of the later two was nonmucoid. In addition we also sequenced a nonmucoid derivative of the initial mucoid isolate, acquired in the laboratory by antibiotic pressure. Examination of sequence data revealed that the two late stage isolates shared 20 variant nucleotides in common compared to the early isolate. However, despite their isolation within 10 months of one another, there was also considerable variation between the late stage isolates, including 42 single nucleotide variants and three deletions. Additionally, no sequence differences were identified between the initial mucoid isolate and its laboratory acquired nonmucoid derivative, however transcript analysis indicated at least partial down regulation of genes involved in exopolysaccharide production. Our study examines the progression of B. cenocepacia throughout chronic infection, including establishment of sub-populations likely evolved from the original isolate, suggestive of parallel evolution. Additionally, the lack of sequence differences between two of the isolates with differing mucoid phenotypes suggests that other factors, such as gene regulation, come into play in establishing the mucoid phenotype.

SNaPBcen: a novel and practical tool for genotyping Burkholderia cenocepacia.

Burkholderia cenocepacia is the most prevalent and feared member of the Burkholderia cepacia complex in lung infections of cystic fibrosis (CF). Genotyping and monitoring of long-term colonization are critical at clinical units; however, the differentiation of specific lineages performed by multilocus sequence typing (MLST) is still limited to a small number of isolates due to the high cost and time-consuming procedure. The aim of this study was to optimize a protocol (the SNaPBcen assay) for extensive bacterial population studies. The strategy used for the SNaPBcen assay is based on targeting single nucleotide polymorphisms (SNPs) located in MLST genes instead of sequencing full MLST sequences. Nonpolymorphic and redundant MLST positions were eliminated, and a set of 24 polymorphisms included in the SNaPBcen assay ensures a high-resolution genomic characterization. These polymorphisms were identified based on the comparative analysis of 137 B. cenocepacia MLST profiles available online (http://pubmlst.org/bcc/). The group of 81 clinical isolates of B. cenocepacia examined in this study using the SNaPBcen assay revealed 51 distinct profiles, and a final discriminatory power of 0.9997 compared with MLST was determined. The SNaPBcen assay was able to reveal isolates with microvariations and the presence of multiple clonal variants in patients chronically colonized with B. cenocepacia. Main phylogenetic subgroups IIIA, IIIB, and IIIC of B. cenocepacia could be separated by the Gl94R polymorphism included in the panel. The SNaPBcen assay proved to be a rapid and robust alternative to the standard MLST for B. cenocepacia, allowing the simultaneous analysis of multiple polymorphisms following amplification and mini-sequencing reactions.

An outbreak of Burkholderia cenocepacia associated with contaminated chlorhexidine solutions prepared in the hospital.

From October to December 2007, an outbreak of Burkholderia cenocepacia occurred in a secondary care hospital. The 19 B cenocepacia isolated from the patients, the chlorhexidine solutions of each different ward, and the purified water that diluted these solutions exhibited an identical pulsed-field gel electrophoresis pattern. Inadequate preparation of chlorhexidine solutions diluted with contaminated purified water may have resulted in an outbreak of B cenocepacia. Adequate preparation of chlorhexidine solutions should be emphasized.

Novel diagnostic PCR assay for Burkholderia cenocepacia epidemic strain ST32 and its utility in monitoring infection in cystic fibrosis patients.

BACKGROUND: A highly transmissible Burkholderia cenocepacia sequence type (ST) 32 strain caused a major outbreak at the Prague Cystic Fibrosis (CF) Centre in the late 1990s and early 2000s. Because a large number of CF patients were affected by ST32, a rapid and easy-to-use diagnostic tool for ST32 infection was urgently needed for the detection of new cases as well as for long-term surveillance. The present study sought to identify unique DNA sequences within the ST32 genome to develop an ST32 strain-specific PCR assay. METHODS: Genomic subtractive hybridisation between B. cenocepacia ST32 and the closely related genome-sequenced strain B. cenocepacia ST28 identified a 325 bp long region that was absent in all but one Burkholderia strain, as demonstrated by our newly designed PCR. RESULTS: Out of 57 strains, only B. cenocepacia ST33 cross-reacted with ST32, resulting in a PCR specificity of 98.2%. This specificity was further tested by various genotyping methods, which revealed the practical indistinguishibility of ST32 and ST33. The PCR sensitivity, checked on a panel of 50 ST32 clinical isolates, was 100%. A closer examination of the ST32-specific sequence revealed no significant homology apart from a fragment of the ISBmu3 transposase. CONCLUSIONS: This novel ST32-specific PCR assay allows the rapid and reliable detection of a globally distributed B. cenocepacia epidemic strain. Its routine use is especially well suited to infection surveillance programs for CF populations with a high rate of ST32 infection. This PCR method can also be used to detect ST33, a clonal variant of ST32.

Insertion sequence evolutionary patterns highlight convergent genetic inactivations and recent genomic island acquisitions among epidemic Burkholderia cenocepacia.

The Burkholderia cenocepacia B&B clone was found previously to be responsible for an epidemic outbreak within an intensive care unit in France. This clone belongs to the ST32 clonal complex, which is one of the most prevalent among French cystic fibrosis patients and is known to be related to the highly virulent ET12 clonal complex. Genomic repartition biases of insertion sequences (ISs) were investigated to improve our understanding of the evolutionary events leading to B. cenocepacia diversification and the emergence of such epidemic lineages. IS were used for tracking convergent genetic inactivations and recent DNA acquisitions. B. cenocepacia IS families and subgroups were compared in terms of genetic diversity and genomic architecture using fully sequenced genomes, PCR screening and DNA blot analysis. These analyses revealed several features shared by the B&B and ET12 epidemic clones. IS elements showed a frequent localization on genomic islands (GI) and indicated convergent evolution towards inactivation of certain loci. The IS407 subgroup of the IS3 family was identified as a good indicator of recently acquired GIs in clone ET12. Several IS407 elements showed strain-specific or clonal complex-specific localizations. IS407 DNA probing of a DNA library built from the B. cenocepacia B&B epidemic clone led to the identification of a recently acquired IS407-tagged GI likely to be conjugative and integrative. The B&B clone showed significant differences in its IS architecture from that of ST32 strains isolated from Czech cystic fibrosis patients.

Differences in strategies to combat osmotic stress in Burkholderia cenocepacia elucidated by NMR-based metabolic profiling.

AIMS: To investigate mechanisms of osmotic tolerance in Burkholderia cenocepacia, a member of the B. cepacia complex (Bcc) of closely related strains, which is of clinical as well as environmental importance. METHODS AND RESULTS: We employed NMR-based metabolic profiling (metabolomics) to elucidate the metabolic consequences of high osmotic stress for five isolates of B. cenocepacia. The strains differed significantly in their levels of osmotic stress tolerance, and we identified three different sets of metabolic responses with the strains least impacted by osmotic stress exhibiting higher levels of the osmo-protective metabolites glycine-betaine and/or trehalose. Strains either increased concentrations or had constitutively high levels of these metabolites. CONCLUSIONS: Even within the small set of B. cenocepacia isolates, there was a surprising degree of variability in the metabolic responses to osmotic stress. SIGNIFICANCE AND IMPACT OF THE STUDY: The metabolic responses, and hence osmotic stress tolerance, vary between different B. cenocepacia isolates. This study provides a first look into the potentially highly diverse physiology of closely related strains of one species of the Bcc and illustrates that physiological or clinically relevant phenotypes are unlikely to be inferable from genetic relatedness within this species group.

The second RNA chaperone, Hfq2, is also required for survival under stress and full virulence of Burkholderia cenocepacia J2315.

Burkholderia cenocepacia J2315 is a highly virulent and epidemic clinical isolate of the B. cepacia complex (Bcc), a group of bacteria that have emerged as important pathogens to cystic fibrosis patients. This bacterium, together with all Bcc strains and a few other prokaryotes, is unusual for encoding in its genome two distinct and functional Hfq-like proteins. In this work, we show results indicating that the 188-amino-acid Hfq2 protein is required for the full virulence and stress resistance of B. cenocepacia J2315, despite the presence on its genome of the functional 79-amino-acid Hfq protein encoded by the hfq gene. Similar to other Hfq proteins, Hfq2 is able to bind RNA. However, Hfq2 is unique in its ability to apparently form trimers in vitro. Maximal transcription of hfq was observed in B. cenocepacia J2315 cells in the early exponential phase of growth. In contrast, hfq2 transcription reached maximal levels in cells in the stationary phase, depending on the CepR quorum-sensing regulator. These results suggest that tight regulation of the expression of these two RNA chaperones is required to maximize the fitness and virulence of this bacterium. In addition, the ability of Hfq2 to bind DNA, not observed for Hfq, suggests that Hfq2 might play additional roles besides acting as an RNA chaperone.

Burkholderia cenocepacia in cystic fibrosis: epidemiology and molecular mechanisms of virulence.

Burkholderia cepacia complex (Bcc) bacteria have gained notoriety as pathogens in cystic fibrosis (CF) because they are difficult to identify and treat, and also have the ability to spread between CF individuals. Of the 17 formally named species within the complex, Burkholderia multivorans and Burkholderia cenocepacia dominate in CF. Multilocus sequence typing has proven to be a very useful tool for tracing the global epidemiology of Bcc bacteria and has shown that B. cenocepacia strains with high transmissibility, such as the ET-12 strain (ST-28) and the Czech strain (ST-32), have spread epidemically within CF populations in Canada and Europe. The majority of research on the molecular pathogenesis of Bcc bacteria has focused on the B. cenocepacia ET-12 epidemic lineage, with gene mutation, genome sequence analysis and, most recently, global gene expression studies shedding considerable light on the virulence and antimicrobial resistance of this pathogen. These studies demonstrate that the ability of B. cenocepacia to acquire foreign DNA (genomic islands, insertion sequences and other mobile elements), regulate gene expression via quorum sensing, compete for iron during infection, and mediate antimicrobial resistance and inflammation via its membrane and surface polysaccharides are key features that underpin the virulence of different strains. With the wealth of molecular knowledge acquired in the last decade on B. cenocepacia strains, we are now in a much better position to develop strategies for the treatment of pathogenic colonization with Bcc and to answer key questions on pathogenesis concerning, for example, the factors that trigger the rapid clinical decline in CF patients.

Taxonomic parsing of bacteriophages using core genes and in silico proteome-based CGUG and applications to small bacterial genomes.

A combined genomics and in situ proteomics approach can be used to determine and classify the relatedness of organisms. The common set of proteins shared within a group of genomes is encoded by the "core" set of genes, which is increasingly recognized as a metric for parsing viral and bacterial species. These can be described by the concept of a "pan-genome", which consists of this "core" set and a "dispensable" set, i.e., genes found in one or more but not all organisms in the grouping. "CoreGenesUniqueGenes" (CGUG) is a web-based tool that determines this core set of proteins in a set of genomes as well as parses the dispensable set of unique proteins in a pair of viral or small bacterial genomes. This proteome-based methodology is validated using bacteriophages, aiding the reevaluation of current classifications of bacteriophages. The utility of CGUG in the analysis of small bacterial genomes and the annotation of hypothetical proteins is also presented.