Examining the influence of environmental factors on Acanthamoeba castellanii and Pseudomonas aeruginosa in co-culture.

Exploration of interspecies interactions between microorganisms can have taxonomic, ecological, evolutionary, or medical applications. To better explore interactions between microorganisms it is important to establish the ideal conditions that ensure survival of all species involved. In this study, we sought to identify the ideal biotic and abiotic factors that would result in high co-culture viability of two interkingdom species, Pseudomonas aeruginosa and Acanthamoeba castellanii, two soil dwelling microbes. There have been limited studies showing long-term interactions between these two organisms as co-culture can result in high mortality for one or both organisms suggesting a predator-predator interaction may exist between them. In this study, we identified biotic and abiotic conditions that resulted in a high viability for both organisms in long-term co-culture, including optimizing temperature, nutrient concentration, choice of bacterial strains, and the initial ratio of interacting partners. These two species represent ideal partners for studying microbial interactions because amoebae act similarly to mammalian immune cells in many respects, and this can allow researchers to study host-pathogen interactions in vitro. Therefore, long-term interaction studies between these microbes might reveal the evolutionary steps that occur in bacteria when subjected to intense predation, like what occurs when pathogens enter the human body. The culture conditions characterized here resulted in high viability for both organisms for at least 14-days in co-culture suggesting that long-term experimental studies between these species can be achieved using these culture conditions.

Porphyromonas gingivalis Tyrosine Kinase Is a Fitness Determinant in Polymicrobial Infections.

Many pathogenic microbial ecosystems are polymicrobial, and community function can be shaped by interbacterial interactions. Little is known, however, regarding the genetic determinants required for fitness in heterotypic community environments. In periodontal diseases, Porphyromonas gingivalis is a primary pathogen, but only within polymicrobial communities. Here, we used a transposon sequencing (Tn-Seq) library of P. gingivalis to screen for genes that influence fitness of the organism in a coinfection murine abscess model with the oral partner species Streptococcus gordonii and Fusobacterium nucleatum. Genes impacting fitness with either organism were involved in diverse processes, including metabolism and energy production, along with cell wall and membrane biogenesis. Despite the overall similarity of function, the majority of identified genes were specific to the partner species, indicating that synergistic mechanisms of P. gingivalis vary to a large extent according to community composition. Only two genes were identified as essential for P. gingivalis fitness in abscess development with both S. gordonii and F. nucleatum: ptk1, encoding a tyrosine kinase, and inlJ, encoding an internalin family surface protein. Ptk1, but not InlJ, is required for community development with S. gordonii, and we found that the action of this kinase is similarly required for P. gingivalis to accumulate in a community with F. nucleatum. A limited number of P. gingivalis genes are therefore required for species-independent synergy, and the Ptk1 tyrosine kinase network may integrate and coordinate input from multiple organisms.

Bactericidal effects of high-energy visible light on common otitis media pathogens.

AIMS: This study assessed the use of high-energy, visible light on the survival rates of three bacteria commonly found in middle ear infections (i.e. otitis media; Streptococcus pneumoniae, Moraxella catarrhalis and Haemophilus influenzae). METHOD AND RESULTS: Bacteria were cultured and then subjected to a single, 4-h treatment of 405 nm wavelength light at two different intensities. All three bacteria species were susceptible to the light at clinically significant rates (>99.9% reduction). Bacteria were susceptible to the high-energy visible (HEV) light in a dose-dependent manner (lower survival rates with increased intensity and duration of exposure). CONCLUSIONS: The results suggest that HEV light may provide a non-surgical, non-pharmaceutical approach to the therapeutic treatment of otitis media. SIGNIFICANCE AN IMPACT OF THE STUDY: Given the growing concerns surrounding antibiotic resistance, this study demonstrates a rapid, alternative method for effective inactivation of bacterial pathogens partly responsible for instances of otitis media.

Porphyromonas gingivalis genes conferring fitness in a tobacco-rich environment.

Smokers are more likely than non-smokers to harbour Porphyromonas gingivalis, they are more susceptible to destructive periodontal disease and smokers may, ultimately, benefit from tobacco-specific preventive and treatment strategies. A Mariner transposon insertion library for P. gingivalis ATCC 33277 was exploited to define 256 genes as essential for P. gingivalis survival in a tobacco-rich environment. Genes whose products play roles in protein transport and catabolism, nicotinamide processing, protection against oxidative stress, drug resistance, and transcriptional regulation have all been identified as essential for CSE survival. Many of these tobacco-essential genes are also requisite for epithelial colonization and abscess formation, suggestive of a core stress-related P. gingivalis genome. Single-gene deletions in several of the TnSeq-implicated genes led to significantly reduced P. gingivalis fitness upon competition with the parent strain, under conditions of cigarette smoke extract-induced stress (1,000 ng/ml nicotine equivalents). This study identifies, for the first time, a subset of P. gingivalis genes required for surviving the plethora of insults present in cigarette smoke. Such conditionally essential genes may delineate bacterial persistence strategies and represent novel therapeutic foci for the prevention of P. gingivalis infection and related diseases in smokers and in general.

Detection of Volatile Compounds Emitted by Bacteria in Wounds Using Gas Sensors.

In this paper we analyze an experiment for the use of low-cost gas sensors intended to detect bacteria in wounds using a non-intrusive technique. Seven different genera/species of microbes tend to be present in most wound infections. Detection of these bacteria usually requires sample and laboratory testing which is costly, inconvenient and time-consuming. The validation processes for these sensors with nineteen types of microbes (1 Candida, 2 Enterococcus, 6 Staphylococcus, 1 Aeromonas, 1 Micrococcus, 2 E. coli and 6 Pseudomonas) are presented here, in which four sensors were evaluated: TGS-826 used for ammonia and amines, MQ-3 used for alcohol detection, MQ-135 for CO2 and MQ-138 for acetone detection. Validation was undertaken by studying the behavior of the sensors at different distances and gas concentrations. Preliminary results with liquid cultures of 108 CFU/mL and solid cultures of 108 CFU/cm2 of the 6 Pseudomonas aeruginosa strains revealed that the four gas sensors showed a response at a height of 5 mm. The ammonia detection response of the TGS-826 to Pseudomonas showed the highest responses for the experimental samples over the background signals, with a difference between the values ​​of up to 60 units in the solid samples and the most consistent and constant values. This could suggest that this sensor is a good detector of Pseudomonas aeruginosa, and the recording made of its values ​​could be indicative of the detection of this species. All the species revealed similar CO2 emission and a high response rate with acetone for Micrococcus, Aeromonas and Staphylococcus.

Pathophysiology of Escherichia coli pneumonia: Respective contribution of pathogenicity islands to virulence.

Ventilator-associated pneumonia (VAP) remains the most frequent life-threatening nosocomial infection. Enterobacteriaceae including Escherichia coli are increasingly involved. If a cumulative effect of pathogenicity islands (PAIs) has been shown for E. coli virulence in urinary tract or systemic infections, very little is known regarding pathophysiology of E. coli pneumonia. This study aimed to determine the role of each of the 7 PAIs present in pathogenic E. coli strain 536 in pneumonia pathophysiology. We used mutant strains to screen pathophysiological role of PAI in a rat pneumonia model. We also test individual gene mutants within PAI identified to be involved in pneumonia pathogenesis. Finally, we determined the prevalence of these genes of interest in E. coli isolates from feces and airways of ventilated patients. Only PAIs I and III were significantly associated with rat pneumonia pathogenicity. Only the antigen-43 (Ag43) gene in PAI III was significantly associated with bacterial pathogenicity. The prevalence of tested genes in fecal and airway isolates of ventilated patients did not differ between isolates. In contrast, genes encoding Ag43, the F17-fimbriae subunits, HmuR and SepA were more prevalent in VAP isolates with statistical significance for hmuR when compared to airway colonizing isolates. The E. coli PAIs involved in lung pathogenicity differed from those involved in urinary tract and bloodstream infections. Overall, extraintestinal E. coli virulence seems to rely on a combination of numerous virulence genes that have a cumulative effect depending on the infection site.

A putative lateral flagella of the cystic fibrosis pathogen Burkholderia dolosa regulates swimming motility and host cytokine production.

Burkholderia dolosa caused an outbreak in the cystic fibrosis clinic at Boston Children's Hospital and was associated with high mortality in these patients. This species is part of a larger complex of opportunistic pathogens known as the Burkholderia cepacia complex (Bcc). Compared to other species in the Bcc, B. dolosa is highly transmissible; thus understanding its virulence mechanisms is important for preventing future outbreaks. The genome of one of the outbreak strains, AU0158, revealed a homolog of the lafA gene encoding a putative lateral flagellin, which, in other non-Bcc species, is used for movement on solid surfaces, attachment to host cells, or movement inside host cells. Here, we analyzed the conservation of the lafA gene and protein sequences, which are distinct from those of the polar flagella, and found lafA homologs to be present in numerous ß-proteobacteria but notably absent from most other Bcc species. A lafA deletion mutant in B. dolosa showed a greater swimming motility than wild-type due to an increase in the number of polar flagella, but did not appear to contribute to biofilm formation, host cell invasion, or murine lung colonization or persistence over time. However, the lafA gene was important for cytokine production in human peripheral blood mononuclear cells, suggesting it may have a role in recognition by the human immune response.

Genes Contributing to Porphyromonas gingivalis Fitness in Abscess and Epithelial Cell Colonization Environments.

Porphyromonas gingivalis is an important cause of serious periodontal diseases, and is emerging as a pathogen in several systemic conditions including some forms of cancer. Initial colonization by P. gingivalis involves interaction with gingival epithelial cells, and the organism can also access host tissues and spread haematogenously. To better understand the mechanisms underlying these properties, we utilized a highly saturated transposon insertion library of P. gingivalis, and assessed the fitness of mutants during epithelial cell colonization and survival in a murine abscess model by high-throughput sequencing (Tn-Seq). Transposon insertions in many genes previously suspected as contributing to virulence showed significant fitness defects in both screening assays. In addition, a number of genes not previously associated with P. gingivalis virulence were identified as important for fitness. We further examined fitness defects of four such genes by generating defined mutations. Genes encoding a carbamoyl phosphate synthetase, a replication-associated recombination protein, a nitrosative stress responsive HcpR transcription regulator, and RNase Z, a zinc phosphodiesterase, showed a fitness phenotype in epithelial cell colonization and in a competitive abscess infection. This study verifies the importance of several well-characterized putative virulence factors of P. gingivalis and identifies novel fitness determinants of the organism.

Immune Recognition of the Epidemic Cystic Fibrosis Pathogen Burkholderia dolosa.

Burkholderia dolosa caused an outbreak in the cystic fibrosis (CF) clinic at Boston Children's Hospital from 1998 to 2005 and led to the infection of over 40 patients, many of whom died due to complications from infection by this organism. To assess whether B. dolosa significantly contributes to disease or is recognized by the host immune response, mice were infected with a sequenced outbreak B. dolosa strain, AU0158, and responses were compared to those to the well-studied CF pathogen Pseudomonas aeruginosa In parallel, mice were also infected with a polar flagellin mutant of B. dolosa to examine the role of flagella in B. dolosa lung colonization. The results showed a higher persistence in the host by B. dolosa strains, and yet, neutrophil recruitment and cytokine production were lower than those with P. aeruginosa The ability of host immune cells to recognize B. dolosa was then assessed, B. dolosa induced a robust cytokine response in cultured cells, and this effect was dependent on the flagella only when bacteria were dead. Together, these results suggest that B. dolosa can be recognized by host cells in vitro but may avoid or suppress the host immune response in vivo through unknown mechanisms. B. dolosa was then compared to other Burkholderia species and found to induce similar levels of cytokine production despite being internalized by macrophages more than Burkholderia cenocepacia strains. These data suggest that B. dolosa AU0158 may act differently with host cells and is recognized differently by immune systems than are other Burkholderia strains or species.

An Oxygen-Sensing Two-Component System in the Burkholderia cepacia Complex Regulates Biofilm, Intracellular Invasion, and Pathogenicity.

Burkholderia dolosa is a member of the Burkholderia cepacia complex (BCC), which is a group of bacteria that cause chronic lung infection in patients with cystic fibrosis (CF) and can be associated with outbreaks carrying high morbidity and mortality. While investigating the genomic diversity of B. dolosa strains collected from an outbreak among CF patients, we previously identified fixL as a gene showing signs of strong positive selection. This gene has homology to fixL of the rhizobial FixL/FixJ two-component system. The goals of this study were to determine the functions of FixLJ and their role in virulence in B. dolosa. We generated a fixLJ deletion mutant and complemented controls in B. dolosa strain AU0158. Using a fixK-lacZ reporter we found that FixLJ was activated in low oxygen in multiple BCC species. In a murine pneumonia model, the B. dolosa fixLJ deletion mutant was cleared faster from the lungs and spleen than wild-type B. dolosa strain AU0158 at 7 days post infection. Interestingly, the fixLJ deletion mutant made more biofilm, albeit with altered structure, but was less motile than strain AU0158. Using RNA-seq with in vitro grown bacteria, we found ~11% of the genome was differentially expressed in the fixLJ deletion mutant relative to strain AU0158. Multiple flagella-associated genes were down-regulated in the fixLJ deletion mutant, so we also evaluated virulence of a fliC deletion mutant, which lacks a flagellum. We saw no difference in the ability of the fliC deletion mutant to persist in the murine model relative to strain AU0158, suggesting factors other than flagella caused the phenotype of decreased persistence. We found the fixLJ deletion mutant to be less invasive in human lung epithelial and macrophage-like cells. In conclusion, B. dolosa fixLJ is a global regulator that controls biofilm formation, motility, intracellular invasion/persistence, and virulence.

Comprehensive identification of virulence factors required for respiratory melioidosis using Tn-seq mutagenesis.

Respiratory melioidosis is a disease presentation of the biodefense pathogen, Burkholderia pseudomallei, which is frequently associated with a lethal septicemic spread of the bacteria. We have recently developed an improved respiratory melioidosis model to study the pathogenesis of Burkholderia pseudomallei in the lung (intubation-mediated intratracheal [IMIT] inoculation), which more closely models descriptions of human melioidosis, including prominent septicemic spread from the lung and reduced involvement of the upper respiratory tract. We previously demonstrated that the Type 3 Secretion System cluster 3 (T3SS3) is a critical virulence determinant for B. pseudomallei when delivered directly into the lung. We decided to comprehensively identify all virulence determinants required for respiratory melioidosis using the Tn-seq phenotypic screen, as well as to investigate which virulence determinants are required for dissemination to the liver and spleen. While previous studies have used Tn-seq to identify essential genes for in vitro cultured B. pseudomallei, this represents the first study to use Tn-seq to identify genes required for in vivo fitness. Consistent with our previous findings, we identified T3SS3 as the largest genetic cluster required for fitness in the lung. Furthermore, we identified capsular polysaccharide and Type 6 Secretion System cluster 5 (T6SS5) as the two additional major genetic clusters facilitating respiratory melioidosis. Importantly, Tn-seq did not identify additional, novel large genetic systems supporting respiratory melioidosis, although these studies identified additional small gene clusters that may also play crucial roles in lung fitness. Interestingly, other previously identified virulence determinants do not appear to be required for lung fitness, such as lipopolysaccharide. The role of T3SS3, capsule, and T6SS5 in lung fitness was validated by competition studies, but only T3SS3 was found to be important for respiratory melioidosis when delivered as a single strain challenge, suggesting that competition studies may provide a higher resolution analysis of fitness factors in the lung. The use of Tn-seq phenotypic screening also provided key insights into the selective pressure encountered in the liver.

Development and evaluation of murine lung-specific disease models for Pseudomonas aeruginosa applicable to therapeutic testing.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen capable of causing a wide range of disease manifestations, including severe bacterial pneumonia. Recently, clinics have reported a rise in nosocomial infections with multidrug resistant (MDR) species, including MDR strains of P. aeruginosa. In order to quickly evaluate the efficacy of new therapeutics for MDR infections, highly reproducible and validated animal models need to be developed for pre-clinical testing. Here, we describe the characterization of two murine models to study MDR P. aeruginosa respiratory disease. We evaluated and compared these models using a non-invasive intratracheal instillation method and established the 50% lethal dose, course of infection, biometric parameters of disease and degree of pneumonia development for each model. Further, we tested meropenem as a proof-of-concept therapeutic and report efficacy data that suggests that the leukopenic model could serve a robust pre-clinical model to test novel therapeutics.

Genome Sequence of Porphyromonas gingivalis Strain HG66 (DSM 28984).

Porphyromonas gingivalis is considered a major etiologic agent in adult periodontitis. Gingipains are among its most important virulence factors, but their release is unique in strain HG66. We present the genome sequence of HG66 with a single contig of 2,441,680 bp and a G+C content of 48.1%.

Enhanced in vivo fitness of carbapenem-resistant oprD mutants of Pseudomonas aeruginosa revealed through high-throughput sequencing.

An important question regarding the biologic implications of antibiotic-resistant microbes is how resistance impacts the organism's overall fitness and virulence. Currently it is generally thought that antibiotic resistance carries a fitness cost and reduces virulence. For the human pathogen Pseudomonas aeruginosa, treatment with carbapenem antibiotics is a mainstay of therapy that can lead to the emergence of resistance, often through the loss of the carbapenem entry channel OprD. Transposon insertion-site sequencing was used to analyze the fitness of 300,000 mutants of P. aeruginosa strain PA14 in a mouse model for gut colonization and systemic dissemination after induction of neutropenia. Transposon insertions in the oprD gene led not only to carbapenem resistance but also to a dramatic increase in mucosal colonization and dissemination to the spleen. These findings were confirmed in vivo with different oprD mutants of PA14 as well as with related pairs of carbapenem-susceptible and -resistant clinical isolates. Compared with OprD(+) strains, those lacking OprD were more resistant to killing by acidic pH or normal human serum and had increased cytotoxicity against murine macrophages. RNA-sequencing analysis revealed that an oprD mutant showed dramatic changes in the transcription of genes that may contribute to the various phenotypic changes observed. The association between carbapenem resistance and enhanced survival of P. aeruginosa in infected murine hosts suggests that either drug resistance or host colonization can cause the emergence of more pathogenic, drug-resistant P. aeruginosa clones in a single genetic event.

A comprehensive analysis of in vitro and in vivo genetic fitness of Pseudomonas aeruginosa using high-throughput sequencing of transposon libraries.

High-throughput sequencing of transposon (Tn) libraries created within entire genomes identifies and quantifies the contribution of individual genes and operons to the fitness of organisms in different environments. We used insertion-sequencing (INSeq) to analyze the contribution to fitness of all non-essential genes in the chromosome of Pseudomonas aeruginosa strain PA14 based on a library of ∼300,000 individual Tn insertions. In vitro growth in LB provided a baseline for comparison with the survival of the Tn insertion strains following 6 days of colonization of the murine gastrointestinal tract as well as a comparison with Tn-inserts subsequently able to systemically disseminate to the spleen following induction of neutropenia. Sequencing was performed following DNA extraction from the recovered bacteria, digestion with the MmeI restriction enzyme that hydrolyzes DNA 16 bp away from the end of the Tn insert, and fractionation into oligonucleotides of 1,200-1,500 bp that were prepared for high-throughput sequencing. Changes in frequency of Tn inserts into the P. aeruginosa genome were used to quantify in vivo fitness resulting from loss of a gene. 636 genes had <10 sequencing reads in LB, thus defined as unable to grow in this medium. During in vivo infection there were major losses of strains with Tn inserts in almost all known virulence factors, as well as respiration, energy utilization, ion pumps, nutritional genes and prophages. Many new candidates for virulence factors were also identified. There were consistent changes in the recovery of Tn inserts in genes within most operons and Tn insertions into some genes enhanced in vivo fitness. Strikingly, 90% of the non-essential genes were required for in vivo survival following systemic dissemination during neutropenia. These experiments resulted in the identification of the P. aeruginosa strain PA14 genes necessary for optimal survival in the mucosal and systemic environments of a mammalian host.

Identification of T6SS-dependent effector and immunity proteins by Tn-seq in Vibrio cholerae.

Type VI protein secretion system (T6SS) is important for bacterial competition through contact-dependent killing of competitors. T6SS delivers effectors to neighboring cells and corresponding antagonistic proteins confer immunity against effectors that are delivered by sister cells. Although T6SS has been found in more than 100 gram-negative bacteria including many important human pathogens, few T6SS-dependent effector and immunity proteins have been experimentally determined. Here we report a high-throughput approach using transposon mutagenesis and deep sequencing (Tn-seq) to identify T6SS immunity proteins in Vibrio cholerae. Saturating transposon mutagenesis was performed in wild type and a T6SS null mutant. Genes encoding immunity proteins were predicted to be essential in the wild type but dispensable in the T6SS mutant. By comparing the relative abundance of each transposon mutant in the mutant library using deep sequencing, we identified three immunity proteins that render protection against killing by T6SS predatory cells. We also identified their three cognate T6SS-secreted effectors and show these are important for not only antibacterial and antieukaryotic activities but also assembly of T6SS apparatus. The lipase and muramidase T6SS effectors identified in this study underscore the diversity of T6SS-secreted substrates and the distinctly different mechanisms that target these for secretion by the dynamic T6SS organelle.

The single-nucleotide resolution transcriptome of Pseudomonas aeruginosa grown in body temperature.

One of the hallmarks of opportunistic pathogens is their ability to adjust and respond to a wide range of environmental and host-associated conditions. The human pathogen Pseudomonas aeruginosa has an ability to thrive in a variety of hosts and cause a range of acute and chronic infections in individuals with impaired host defenses or cystic fibrosis. Here we report an in-depth transcriptional profiling of this organism when grown at host-related temperatures. Using RNA-seq of samples from P. aeruginosa grown at 28°C and 37°C we detected genes preferentially expressed at the body temperature of mammalian hosts, suggesting that they play a role during infection. These temperature-induced genes included the type III secretion system (T3SS) genes and effectors, as well as the genes responsible for phenazines biosynthesis. Using genome-wide transcription start site (TSS) mapping by RNA-seq we were able to accurately define the promoters and cis-acting RNA elements of many genes, and uncovered new genes and previously unrecognized non-coding RNAs directly controlled by the LasR quorum sensing regulator. Overall we identified 165 small RNAs and over 380 cis-antisense RNAs, some of which predicted to perform regulatory functions, and found that non-coding RNAs are preferentially localized in pathogenicity islands and horizontally transferred regions. Our work identifies regulatory features of P. aeruginosa genes whose products play a role in environmental adaption during infection and provides a reference transcriptional landscape for this pathogen.

Genomic diversity of Escherichia isolates from diverse habitats.

Our understanding of the Escherichia genus is heavily biased toward pathogenic or commensal isolates from human or animal hosts. Recent studies have recovered Escherichia isolates that persist, and even grow, outside these hosts. Although the environmental isolates are typically phylogenetically distinct, they are highly related to and phenotypically indistinguishable from their human counterparts, including for the coliform test. To gain insights into the genomic diversity of Escherichia isolates from diverse habitats, including freshwater, soil, animal, and human sources, we carried out comparative DNA-DNA hybridizations using a multi-genome E. coli DNA microarray. The microarray was validated based on hybridizations with selected strains whose genome sequences were available and used to assess the frequency of microarray false positive and negative signals. Our results showed that human fecal isolates share two sets of genes (n>90) that are rarely found among environmental isolates, including genes presumably important for evading host immune mechanisms (e.g., a multi-drug transporter for acids and antimicrobials) and adhering to epithelial cells (e.g., hemolysin E and fimbrial-like adhesin protein). These results imply that environmental isolates are characterized by decreased ability to colonize host cells relative to human isolates. Our study also provides gene markers that can distinguish human isolates from those of warm-blooded animal and environmental origins, and thus can be used to more reliably assess fecal contamination in natural ecosystems.

A vast collection of microbial genes that are toxic to bacteria.

In the process of clone-based genome sequencing, initial assemblies frequently contain cloning gaps that can be resolved using cloning-independent methods, but the reason for their occurrence is largely unknown. By analyzing 9,328,693 sequencing clones from 393 microbial genomes, we systematically mapped more than 15,000 genes residing in cloning gaps and experimentally showed that their expression products are toxic to the Escherichia coli host. A subset of these toxic sequences was further evaluated through a series of functional assays exploring the mechanisms of their toxicity. Among these genes, our assays revealed novel toxins and restriction enzymes, and new classes of small, non-coding toxic RNAs that reproducibly inhibit E. coli growth. Further analyses also revealed abundant, short, toxic DNA fragments that were predicted to suppress E. coli growth by interacting with the replication initiator DnaA. Our results show that cloning gaps, once considered the result of technical problems, actually serve as a rich source for the discovery of biotechnologically valuable functions, and suggest new modes of antimicrobial interventions.

Evaluating the performance of oligonucleotide microarrays for bacterial strains with increasing genetic divergence from the reference strain.

DNA oligonucleotide microarrays (oligoarrays) are being developed continuously; however, several issues regarding the applicability of these arrays for whole-genome DNA-DNA strain comparisons (genomotyping) have not been investigated. For example, the extent of false negatives (i.e., no hybridization signal is observed when the amino acid sequence is conserved but the nucleotide sequence has diverged to a level that does not allow hybridization) remains speculative. To provide quantitative answers to such questions, we performed competitive DNA-DNA oligoarray (60-mer) hybridizations with several fully sequenced (tester) strains and a reference strain (whose genome was used to design the oligoarray probes) of the genus Burkholderia and compared the experimental results obtained to the results predicted based on bioinformatic modeling of the probe-target pair using the available sequences. Our comparisons revealed that the fraction of the total probes that provided experimental results consistent with the predicted results decreased substantially with increasing divergence of the tester strain from the reference strain. The fractions were 90.8%, 84.3%, and 77.4% for tester strains showing 96% 89%, and 80% genome-aggregate average nucleotide identity (ANI) to the reference strain, respectively. New approaches to determine gene presence or absence based on the hybridization signal, which outperformed previous approaches (e.g., 92.9% accuracy versus 86.0% accuracy) and to normalize across different array experiments are also described. Collectively, our results suggest that the performance of oligoarrays is acceptable for tester strains showing >90% ANI to the reference strain and provide useful guidelines for using oligoarray applications in environmental gene detection and gene expression studies with strains other than the reference strain.