Low Rates of Pseudomonas aeruginosa Misidentification in Isolates from Cystic Fibrosis Patients

Pseudomonas aeruginosa is an important cause of pulmonary infection in cystic fibrosis (CF). Its correct identification ensures effective patient management and infection control strategies. However, little is known about how often CF sputum isolates are falsely identified as P. aeruginosa. We used P. aeruginosa-specific duplex real-time PCR assays to determine if 2,267 P. aeruginosa sputum isolates from 561 CF patients were correctly identified by 17 Australian clinical microbiology laboratories. Misidentified isolates underwent further phenotypic tests, amplified rRNA gene restriction analysis, and partial 16S rRNA gene sequence analysis. Participating laboratories were surveyed on how they identified P. aeruginosa from CF sputum. Overall, 2,214 (97.7%) isolates from 531 (94.7%) CF patients were correctly identified as P. aeruginosa. Further testing with the API 20NE kit correctly identified only 34 (59%) of the misidentified isolates. Twelve (40%) patients had previously grown the misidentified species in their sputum. Achromobacter xylosoxidans (n = 21), Stenotrophomonas maltophilia (n = 15), and Inquilinus limosus (n = 4) were the species most commonly misidentified as P. aeruginosa. Overall, there were very low rates of P. aeruginosa misidentification among isolates from a broad cross section of Australian CF patients. Additional improvements are possible by undertaking a culture history review, noting colonial morphology, and performing stringent oxidase, DNase, and colistin susceptibility testing for all presumptive P. aeruginosa isolates. Isolates exhibiting atypical phenotypic features should be evaluated further by additional phenotypic or genotypic identification techniques.The accurate identification of Pseudomonas aeruginosa is a critical component of cystic fibrosis (CF) patient management. Once established within CF lungs, P. aeruginosa is rarely eradicated, leading to increased treatment requirements and an accelerated decline in pulmonary function, quality of life, and life expectancy (10, 13, 27). Emerging evidence indicates that aggressive antipseudomonal therapy at the time of initial acquisition may eliminate P. aeruginosa, preventing the development of chronic infection for months or even years (37). Similarly, separating patients with P. aeruginosa from other CF patients may reduce the spread of multiple-antibiotic-resistant strains capable of person-to-person transmission (16). Such strategies are contingent upon the early and correct identification of these organisms (30).While there is much emphasis on misidentifying P. aeruginosa as another species (39), less attention is paid to falsely identifying other species as P. aeruginosa. Nevertheless, accurate identification of P. aeruginosa is important, as this may avoid prolonged and sometimes unnecessary antibiotic treatments, which could select for other antibiotic-resistant pathogens (6). Similarly, in CF clinics where cohort isolation is practiced as an infection control measure, false identification could mean exposure of the CF patient to potentially transmissible bacteria (2, 17, 28, 33).While most clinical strains of P. aeruginosa are easily identified, respiratory isolates from patients with CF can present a taxonomic challenge (15, 24). Phenotypic identification of P. aeruginosa from patients with CF is often complicated by slow growth, auxotrophic metabolic activity, loss of pigment production, multiple antibiotic resistance, atypical colonial morphology, and development of mucoid exopolysaccharide (14, 25). Commercial identification platforms are also considered unreliable (18, 21, 39). Moreover, CF respiratory secretions may contain other nonfermenting gram-negative bacilli, such as Achromobacter, Stenotrophomonas, and Burkholderia species, which can further impede the identification of P. aeruginosa (29, 32, 35, 39).Although several molecular strategies have been developed recently (1, 35, 39), most clinical microbiology laboratories still identify P. aeruginosa by traditional phenotypic techniques. However, there are few published data describing the frequency at which bacterial species in CF sputum are falsely identified as P. aeruginosa by phenotypic methods. In this study, we used P. aeruginosa-specific duplex real-time (PAduplex) PCR assays, phenotypic analysis, amplified rRNA gene restriction analysis (ARDRA), and partial 16S rRNA gene sequence analysis to assess the rate and extent of misidentification of P. aeruginosa isolates in CF sputum by Australian clinical microbiology laboratories.     

Virulence and Cellular Interactions of Burkholderia multivorans in Chronic Granulomatous Disease

Chronic granulomatous disease (CGD) patients are susceptible to life-threatening infections by the Burkholderia cepacia complex. We used leukocytes from CGD and healthy donors and compared cell association, invasion, and cytokine induction by Burkholderia multivorans strains. A CGD isolate, CGD1, showed higher cell association than that of an environmental isolate, Env1, which correlated with cell entry. All B. multivorans strains associated significantly more with cells from CGD patients than with those from healthy donors. Similar findings were observed with another CGD pathogen, Serratia marcescens, but not with Escherichia coli. In a mouse model of CGD, strain CGD1 was virulent while Env1 was avirulent. B. multivorans organisms were found in the spleens of CGD1-infected mice at levels that were 1,000 times higher than those found in Env1-infected mice, which was coincident with higher levels of the proinflammatory cytokine interleukin-1β. Taken together, these results may shed light on the unique susceptibility of CGD patients to specific pathogens.Chronic granulomatous disease (CGD) is a rare primary immunodeficiency resulting from genetic defects in the phagocyte NAPDH oxidase. It is characterized by life-threatening infections caused by specific bacteria and fungi, leading to pneumonias, tissue abscesses, and exuberant granuloma formation (38). The Burkholderia cepacia complex (Bcc) includes at least 10 distinct species and is a leading cause of bacterial infections in CGD (44). Patients with cystic fibrosis (CF) also develop Bcc infections with various outcomes, ranging from no change in clinical course to a more rapid deterioration of lung function to the dreadful cepacia syndrome, which is characterized by necrotizing pneumonia and sepsis (25, 45). Interestingly, Bcc rarely causes infection in healthy individuals, but it can infect patients undergoing bronchoscopies and other procedures (4).Within the Bcc, Burkholderia cenocepacia and Burkholderia multivorans are commonly isolated from CF and non-CF patients (4, 32); the rate of B. multivorans infection now exceeds that of B. cenocepacia at several CF centers (15). In contrast to the high transmissibility of some CF B. cenocepacia strains (i.e., the epidemic lineage ET12) (24, 25), CF B. multivorans infections likely reflect independent acquisitions from unrelated sources (24). Curiously, unlike B. cenocepacia, B. multivorans has been recovered from environmental samples only rarely (1, 24), and it is the most frequently found species among CGD patients (16, 17).The mechanisms by which the Bcc causes disease specifically in CF are not known. Bcc isolates can survive within macrophages (28, 33) and respiratory epithelial cells (5, 21) and can invade epithelial cells in vivo (8, 10) and persist in the lung (9, 10). Cell infection assays using monocytes, macrophages, and epithelial cells (10, 11, 29, 46) show great variability among individual Bcc strains, with no clear correlation between those isolated from CF patients and those isolated from the environment (22). For the most part, these studies have been carried out using tissue culture models (28, 29, 43) and, in some cases, CF human or CF mouse cell systems (34, 35).Much less is known about the interaction between the Bcc and CGD despite the availability of animal models for the disease (20, 31). B. cenocepacia induced the necrosis of human CGD neutrophils but not normal controls (6). Similarly to healthy people, normal mice are resistant to the Bcc and usually show only transient infections upon inoculation (8, 37). On the other hand, CGD mice are highly susceptible to Bcc infection and show clinical signs that are similar to those of the human disease (20, 31, 37).To address why B. multivorans is a pathogen in CGD, we initiated studies with strains isolated from CGD patients and CGD cells. Strains of B. multivorans differed in cell association and cell entry. We found a preferential association of bacteria with CGD instead of normal leukocytes as shown by microscopy and culture techniques. This preferential association is shared by another CGD pathogen, Serratia marcescens, but not by Escherichia coli. Finally, we demonstrate dramatic differences in virulence in B. multivorans strains in a mouse model of CGD.     

Burkholderia cenocepacia Creates an Intramacrophage Replication Niche in Zebrafish Embryos,Followed by Bacterial Dissemination and Establishment of Systemic Infection

Bacteria belonging to the “Burkholderia cepacia complex” (Bcc) often cause fatal pulmonary infections in cystic fibrosis patients, yet little is know about the underlying molecular mechanisms. These Gram-negative bacteria can adopt an intracellular lifestyle, although their ability to replicate intracellularly has been difficult to demonstrate. Here we show that Bcc bacteria survive and multiply in macrophages of zebrafish embryos. Local dissemination by nonlytic release from infected cells was followed by bacteremia and extracellular replication. Burkholderia cenocepacia isolates belonging to the epidemic electrophoretic type 12 (ET12) lineage were highly virulent for the embryos; intravenous injection of <10 bacteria of strain K56-2 killed embryos within 3 days. However, small but significant differences between the clonal ET12 isolates K56-2, J2315, and BC7 were evident. In addition, the innate immune response in young embryos was sufficiently developed to control infection with other less virulent Bcc strains, such as Burkholderia vietnamiensis FC441 and Burkholderia stabilis LMG14294. A K56-2 cepR quorum-sensing regulator mutant was highly attenuated, and its ability to replicate and spread to neighboring cells was greatly reduced. Our data indicate that the zebrafish embryo is an excellent vertebrate model to dissect the molecular basis of intracellular replication and the early innate immune responses in this intricate host-pathogen interaction.In the 1980s, bacteria belonging to the “Burkholderia cepacia complex” (Bcc) emerged as opportunistic pathogens in immunocompromised patients, particularly patients with cystic fibrosis (CF) and chronic granulomatous disease (CGD) (42, 60). Formerly classified into genomovars, Bcc bacteria are now grouped in 17 species (25), all of which have been found in CF patients. Although Pseudomonas aeruginosa is the most common CF pathogen, Bcc infections are associated with poorer clinical prognosis, with Burkholderia cenocepacia and Burkholderia multivorans being the most prevalent species. After an unpredictable period of colonization, the bacteria can cause acute, fatal necrotizing pneumonia and septicemia, known as “cepacia syndrome,” in a some colonized patients (20, 60). The intrinsic multiple resistance of Bcc to antibiotics makes it very difficult to treat. Several epidemic outbreaks have resulted from the rapid spread of certain strains of B. cenocepacia that were highly transmissible between CF patients through both hospital and social contacts (19, 69), and the best-described outbreaks were caused by members of the electrophoretic type 12 (ET12) lineage identified in Canadian and United Kingdom CF populations in the late 1980s (20, 29).Over the past decade much progress has been made in understanding the epidemiology of clinical infections (42), and information concerning the pathogenesis and genetics of virulence has begun to provide clues about how Bcc causes disease. Although several virulence factors, including hemolysin, proteases, siderophores, lipopolysaccharide, flagella, cable pili, a type III secretion system, catalases, superoxide dismutase, and quorum sensing, have been identified (for a review, see reference 45), the molecular mechanisms of this disease are still largely unknown. Important questions are how the bacteria evade host immune responses, how they persist, what triggers the often fatal septicemia, what the bacterial intracellular survival strategy is, and how the bacteria disseminate.B. cenocepacia has been detected inside airway and alveolar epithelial cells, macrophages, and neutrophils in the lungs of CF patients (62, 72) and has been documented to be present intracellularly in murine models (8, 63, 72). Using cell culture infection models, it was shown that B. cenocepacia can invade and survive in both professional and nonprofessional phagocytes, as well as in amoebae (4, 46, 47, 61), by actively interfering with phagosome maturation (35) and delaying phagosome acidification (34). Recently, it was shown that the delay in acidification and phagolysosomal fusion is more compromised in cystic fibrosis transmembrane conductance regulator (CFTR)-negative macrophages (36) and that assembly of the NADPH-oxidase complex at the phagosomal membrane is compromised in B. cenocepacia-infected macrophages, which is also further delayed by inhibition of the CFTR (30). The ability of B. cenocepacia to invade and survive intracellularly as well as its ability to resist toxicity by reactive oxygen species in highly inflamed lungs (37) has been proposed to contribute significantly to its pathogenesis. However, the key question of whether B. cenocepacia can replicate intracellularly has been difficult to answer conclusively due to the intrinsic antibiotic resistance of this organism, which complicates the use of classical gentamicin protection assays (35). Colocalization with cellular markers in infected bronchial epithelial cells suggested that B. cenocepacia replicates inside endoplasmic reticulum-derived vacuoles (65). However, studies of the cell biology of Bcc infection are lagging far behind research on intracellular pathogens such as Legionella pneumophila (28) and Brucella (7), and new research tools are indispensable for increasing our knowledge of the cellular biology of infection by this group of important pathogens.To address the complexity of chronic respiratory infections, a number of mammalian infection models, such as the rat agar bead and mouse cftr^−/− models, have been developed (9, 63, 70, 74). Alternative nonvertebrate animal models, such as Caenorhabditis elegans, have been developed to study Bcc virulence (6, 32), and recently larvae of the wax moth were shown to be valuable for examining Bcc virulence (67). Nematode models have been instrumental in identifying bacterial virulence factors and showing fundamental conservation of virulence mechanisms for infection of evolutionarily divergent hosts with, for instance, P. aeruginosa (41). The invertebrate immune system, however, shows many differences from the immune system of humans, and other models are needed to address specific questions related to the innate immune response to a specific pathogen in great detail. The ability to mount an effective adaptive immune response, involvement of the complement system, and development of complex hematopoietic cell lineages are restricted to vertebrates.Originally established as a powerful model for developmental biology and human genetics, the zebrafish (Danio rerio) has emerged as a remarkably good nonmammalian vertebrate model to study development of the immune system and infectious diseases (48, 76-78, 80). A growing number of both Gram-positive and Gram-negative bacteria, including both natural pathogens of fish and true human pathogens, have been found to infect zebrafish (3, 10, 56, 76, 83). The zebrafish has many useful features as a model system, and the number of available cellular, molecular, and genetic tools, such as forward and reverse genetic screens and antisense techniques using morpholinos, is rapidly increasing. Importantly, proteins with significant homology to major factors in inflammation in humans, such as Toll-like receptors (TLR), the complement system, proinflammatory cytokines, acute-phase response proteins, and counterparts of the mammalian viral and bacterial interferon-dependent defense functions, are present in fish (2, 40, 49, 68, 75, 82). Whereas an adaptive immune system develops at later stages during development, an innate immune system resembling that of mammals is already developing in young embryos (13, 23, 33, 38).In this study, we exploited the transparency of the zebrafish embryo to visualize Bcc infections in real time. We show here that several Bcc species are highly virulent by establishing an intracellular replication niche in macrophages, followed by dissemination and bacteremia. In addition, a cepR quorum-sensing mutant was attenuated, and we observed differences in virulence between strains obtained from a panel of Bcc clinical isolates.     

EuroCareCF Quality Assessment of Diagnostic Microbiology of Cystic Fibrosis Isolates

The identification of microbial species from respiratory specimens and their susceptibility to antimicrobial agents are among the most important diagnostic measures of care for patients with cystic fibrosis (CF). Under the umbrella of EuroCareCF, two quality assurance trials of CF microbiology were performed in 2007 and 2008. Nine formulations with CF bacterial isolates were dispatched. A total of 31/37 laboratories from 18/21 European countries participated in the 2007 and 2008 trials. The common CF pathogens Pseudomonas aeruginosa and Staphylococcus aureus were correctly identified by almost all participants in both trials, even if the strains presented uncommon phenotypes. Burkholderia cenocepacia IIIB and Burkholderia vietnamensis CF isolates, however, were correctly assigned to the species level by only 26% and 27% of the laboratories, respectively. Emerging pathogens such as Achromobacter xylosoxidans, Inquilinus limosus, and Pandoraea pnomenusa were also not detected or were misclassified by many laboratories. One participant correctly identified all CF isolates in both trials. The percentages of correct classifications (susceptible, intermediate, resistant) by antimicrobial susceptibility testing ranged from 55 to 100% (median, 96%) per isolate and drug. The shortcomings in the diagnostics of rare and emerging pathogens point to the need for continuing education in CF microbiology and suggest the establishment of CF microbiology reference laboratories.The monogenic disorder cystic fibrosis (CF) predisposes individuals to chronic airway infections with opportunistic bacterial pathogens (16, 18, 19, 23). The bacteria most frequently isolated from the sputum of patients with CF by standard aerobic microbiological methods are Staphylococcus aureus, noncapsulated Haemophilus influenzae, and Pseudomonas aeruginosa (6). Individuals with CF are, moreover, susceptible to chronic respiratory tract infection with gram-negative bacterial species, which are intrinsically resistant to a broad range of antimicrobial agents and which are usually poor airway colonizers and not pathogenic for healthy persons (7). These rare and/or emerging pathogens in CF include Stenotrophomonas maltophilia (1); Achromobacter (Alcaligenes) xylosoxidans (15); Inquilinus limosus (20); and several species within the genera Burkholderia (12, 13), Ralstonia (12), and Pandoraea (11). Recent 16S rRNA gene profiling of CF respiratory secretions uncovered a further layer of complexity of CF microbiology (17). The bacterial community within the CF lung was found to be polymicrobial in nature and to include a range of anaerobic species primarily within the genera Prevotella, Veillonella, Propionibacterium, and Actinomyces (17, 21, 25).A further characteristic feature of CF isolates that impedes the straightforward identification of taxa is their broad spectrum of numerous and often atypical phenotypes (4, 9, 14, 22). For example, a P. aeruginosa clone may diversify in CF lungs into different morphotypes (14), such as small-colony variants, alginate-overproducing mucoid variants, nonpigmented variants, or colonies with visible autolysis or autoaggregative behavior, all of which carry other adaptive mutations, metabolic features, and antimicrobial susceptibility patterns.Present day CF microbiology services play a central role in the management of CF. Sensitive issues are the detection of transmissible pathogens, the emergence of multidrug-resistant variants, and the control of the efficacy of hygienic measures. To master these tasks, the clinical microbiology laboratory should have profound knowledge of the recent progress in the molecular taxonomy of CF pathogens, particularly among the betaproteobacteria, and the broad spectrum of uncommon phenotypes of isolates, particularly those from elderly CF patients (4). These demands are not trivial, and hence, the authors organized two quality assurance trials to address the issue of whether current knowledge in CF microbiology is translated into the microbiology services provided by the CF clinic. The trials asked for the species identification and antimicrobial susceptibilities of isolates from CF airways. Laboratories from 26 European countries which provide CF microbiology services for the largest CF centers in their home country were invited to participate. The trials identified shortcomings in the detection of rare and/or emerging pathogens which point to the need for continuing education in CF microbiology to promptly translate state-of-the-art knowledge into the daily practice of the clinical microbiology laboratory.     

Identification of Specific and Universal Virulence Factors in Burkholderia cenocepacia Strains by Using Multiple Infection Hosts

Over the past few decades, strains of the Burkholderia cepacia complex have emerged as important pathogens for patients suffering from cystic fibrosis. Identification of virulence factors and assessment of the pathogenic potential of Burkholderia strains have increased the need for appropriate infection models. In previous studies, different infection hosts, including mammals, nematodes, insects, and plants, have been used. At present, however, the extent to which the virulence factors required to infect different hosts overlap is not known. The aim of this study was to analyze the roles of various virulence factors of two closely related Burkholderia cenocepacia strains, H111 and the epidemic strain K56-2, in a multihost pathogenesis system using four different model organisms, namely, Caenorhabditis elegans, Galleria mellonella, the alfalfa plant, and mice or rats. We demonstrate that most of the identified virulence factors are specific for one of the infection models, and only three factors were found to be essential for full pathogenicity in several hosts: mutants defective in (i) quorum sensing, (ii) siderophore production, and (iii) lipopolysaccharide biosynthesis were attenuated in at least three of the infection models and thus may represent promising targets for the development of novel anti-infectives.The Burkholderia cepacia complex (BCC) comprises a group of the following 17 formally named bacterial species: Burkholderia cepacia, Burkholderia multivorans, Burkholderia cenocepacia, Burkholderia stabilis, Burkholderia vietnamiensis, Burkholderia dolosa, Burkholderia ambifaria, Burkholderia anthina, Burkholderia pyrrocinia, Burkholderia ubonensis, Burkholderia latens, Burkholderia diffusa, Burkholderia arboris, Burkholderia seminalis, Burkholderia metallica, Burkholderia lata, and Burkholderia contaminans (46, 69, 70, 71). Strains of the BCC are ubiquitously distributed in nature and have been isolated from soil, water, the rhizosphere of plants, industrial settings, hospital environments, and infected humans. Some BCC strains have enormous biotechnological potential and have been used for bioremediation of recalcitrant xenobiotics, plant growth promotion, and biocontrol purposes. At the same time, however, BCC strains have emerged as problematic opportunistic pathogens in patients with cystic fibrosis (CF) and in immunocompromised individuals (12, 19, 44, 46). The clinical outcomes of BCC infections range from asymptomatic carriage to a fulminant and fatal pneumonia, the so-called cepacia syndrome (30). Apart from acquisition from the environment, patient-to-patient transmission and indirect nosocomial acquisition from contaminated surfaces have caused several outbreaks within and between regional CF centers (55). Although all BCC species have been isolated from both environmental and clinical sources, B. cenocepacia and B. multivorans are most commonly found in clinical samples (12, 44).Members of the BCC not only are opportunistic pathogens of humans but also can cause infections in a diverse range of species, including animals, nematodes, and plants (59). This allowed the development of various infection models, using the mouse or rat, the nematode Caenorhabditis elegans, onions, or the alfalfa plant as an infection host. More recently, larvae of the wax moth Galleria mellonella have been used as infection hosts of BCC strains (58). These models have been employed to investigate the virulence of different BCC species as well as of mutants to understand the importance of specific genes in disease. These infection models have also been applied to studies of host response, gene therapy, antimicrobial delivery, and immunization for prevention of BCC lung disease (6, 32, 51).Previous work has identified several virulence factors that may play a role in infections caused by BCC strains. Some isolates have been demonstrated to be capable of surviving within eukaryotic cells, such as respiratory epithelial cells, macrophages, and amoebae (7, 50, 56). Other virulence factors that have been identified by the use of different infection models include the quorum-sensing system (40), biofilm formation (14), iron-chelating siderophores (16), proteases (15), type III and IV secretion systems (20, 24, 67), melanin production (77), catalase (38), lipopolysaccharide (LPS) (41), cable pili and flagella (57, 68), surface exopolysaccharides (11), a lysR regulator (4), capsule (29), and intrinsic antimicrobial resistance (10). Recently, a phenylacetic acid catabolic pathway was shown to be required for full pathogenicity of B. cenocepacia in the C. elegans infection model (37).At present, knowledge on the importance of these factors in different infection hosts is scarce. This study was initiated to identify both host-specific and conserved mechanisms of pathogenicity in C. elegans, G. mellonella, alfalfa, and murine infection models. We demonstrate that some virulence factors are important for pathogenicity in more than one infection model, while other factors were found to be host specific. N-Acyl homoserine lactone (AHL)-dependent quorum sensing (QS) was identified as a highly conserved regulatory mechanism for expression of pathogenic traits. Siderophore production and intact LPS were important for virulence in all animal models. However, we also identified several virulence factors that were required for pathogenesis in only one of the models.     

Molecular Identification and Susceptibility of Trichosporon Species Isolated from Clinical Specimens in Qatar: Isolation of Trichosporon dohaense Taj-Aldeen,Meis & Boekhout sp. nov.

Trichosporon species have been reported as emerging pathogens and usually occur in severely immunocompromised patients. In the present work, 27 clinical isolates of Trichosporon species were recovered from 27 patients. The patients were not immunocompromised, except for one with acute myeloid leukemia. Sequence analysis revealed the isolation of Trichosporon dohaense Taj-Aldeen, Meis & Boekhout sp. nov., with CBS 10761^T as the holotype strain, belonging to the Ovoides clade. In the D1-D2 large-subunit rRNA gene analysis, T. dohaense is a sister species to T. coremiiforme, and in the internal transcribed spacer analysis, the species is basal to the other species of this clade. Molecular identification of the strains yielded 17 T. asahii, 3 T. inkin, 2 T. japonicum, 2 T. faecale, and 3 T. dohaense isolates. The former four species exhibited low MICs for five antifungal azoles but showed high MICs for amphotericin B. T. dohaense demonstrated the lowest amphotericin B MIC (1 mg/liter). For the majority of T. asahii isolates, amphotericin B MICs were high (MIC at which 90% of isolates were inhibited [MIC₉₀], ≥16 mg/liter), and except for fluconazole (MIC₉₀, 8 mg/liter), the azole MICs were low: MIC₉₀s were 0.5 mg/liter for itraconazole, 0.25 mg/liter for voriconazole, 0.25 mg/liter for posaconazole, and 0.125 mg/liter for isavuconazole. The echinocandins, caspofungin and anidulafungin, demonstrated no activity against Trichosporon species.Trichosporon species are yeast-like fungi, widely distributed in nature and commonly isolated from soil and other environmental sources, which have been involved in a variety of opportunistic infections and have been recognized as emerging fungal pathogens in immunocompromised hosts (19, 79, 80). Disseminated Trichosporon infections are potentially life-threatening and are often fatal in neutropenic patients (7, 22). Although uncommon, pathogenic species of this genus have been reported increasingly, mostly in patients with malignant diseases (3, 6, 9, 10, 11, 20, 32, 44, 47, 48, 63, 77), neonates (18, 56, 84), a bone marrow transplant recipient (22), a solid organ transplant recipient (50), and patients with human immunodeficiency virus (34, 35, 46). Trichosporon has also been reported to cause fungemia (5, 9, 25, 29, 30, 33, 53, 62). Members of the genus Trichosporon have occasionally been implicated as nail pathogens (16, 28, 74) and in subcutaneous infections (66). Trichosporon is considered an opportunistic agent, and therefore, recovery of Trichosporon species capable of growing at 37°C, especially from immunocompromised patients, should be regarded as potentially significant. Several reports have addressed the difficulty of identifying Trichosporon to the species level by physiological and biochemical characteristics (2, 64); therefore, molecular methods based on the sequencing of the internal transcribed spacer (ITS) have been developed (15, 69, 71, 72).In the present paper, we report the isolation of Trichosporon species from clinical specimens over a 4-year period in Qatar, the poor performance of biochemical identification methods, the significance of molecular identification, and the antifungal susceptibility data for the isolates. While investigating the molecular identification of Trichosporon species, we found three strains that do not match any of the published strains in the literature. We describe this organism as Trichosporon dohaense Taj-Aldeen, Meis & Boekhout, sp. nov., the name proposed for this species.     

A Decade of Burkholderia cenocepacia Virulence Determinant Research

The Burkholderia cepacia complex (Bcc) is a group of genetically related environmental bacteria that can cause chronic opportunistic infections in patients with cystic fibrosis (CF) and other underlying diseases. These infections are difficult to treat due to the inherent resistance of the bacteria to antibiotics. Bacteria can spread between CF patients through social contact and sometimes cause cepacia syndrome, a fatal pneumonia accompanied by septicemia. Burkholderia cenocepacia has been the focus of attention because initially it was the most common Bcc species isolated from patients with CF in North America and Europe. Today, B. cenocepacia, along with Burkholderia multivorans, is the most prevalent Bcc species in patients with CF. Given the progress that has been made in our understanding of B. cenocepacia over the past decade, we thought that it was an appropriate time to review our knowledge of the pathogenesis of B. cenocepacia, paying particular attention to the characterization of virulence determinants and the new tools that have been developed to study them. A common theme emerging from these studies is that B. cenocepacia establishes chronic infections in immunocompromised patients, which depend more on determinants mediating host niche adaptation than those involved directly in host cells and tissue damage.Burkholderia cenocepacia is a motile, rod-shaped, metabolically diverse Gram-negative betaproteobacterium (168, 169) that is widespread in the environment, particularly within the rhizosphere (8), and is also an opportunistic pathogen causing chronic lung infections in patients with cystic fibrosis (CF) as well as other immunocompromised patients (169). Using recA sequencing and multilocus sequence typing, B. cenocepacia isolates can be subdivided into four distinct lineages, IIIA, IIIB, IIIC, and IIID (168). To date the majority of clinical isolates belong to the IIIA, IIIB, and IIID lineages (4, 96, 110, 168). Isolates from the IIIB and IIIC lineages may be readily cultivated from the natural environment (8, 96, 127, 168). However, even in the absence of culturable IIIA and IIID lineage bacteria from soil, members of these lineages can be detected in soil using non-culture-based methods (127), suggesting that they may also be present in soil but in low abundance. B. cenocepacia is one of at least 17 phenotypically similar species known as the Burkholderia cepacia complex (Bcc) (168-171). Although almost all the Bcc species have been isolated from CF patients, B. cenocepacia was initially the species most commonly isolated from patients with CF (97, 153) and associated with epidemic spread between CF patients (96). For these reasons, B. cenocepacia was the main focus of research groups studying the molecular biology, pathogenesis, and antibiotic resistance of Bcc bacteria. However, in recent years, Burkholderia multivorans has overtaken B. cenocepacia as the most common Bcc isolate in American and United Kingdom CF patients (102, 133). Some B. multivorans strains are widely distributed and have been associated with outbreaks (9). This article reviews our current understanding of the virulence determinants of B. cenocepacia as well as the tools developed to study them. Since Bcc bacteria are resistant to many clinically useful antibiotics (1, 22, 57, 118, 163), the study of virulence determinants is important for identifying bacterial processes that could be targeted by novel antibiotics or alternative anti-infective therapies.(A portion of this work appears in S.A.L.''s Ph.D. thesis.)     

Achromobacter xylosoxidans Genomic Characterization and Correlation of Randomly Amplified Polymorphic DNA Profiles of Cystic Fibrosis Patients

Achromobacter xylosoxidans is an emerging pathogen increasingly being isolated from respiratory samples of cystic fibrosis (CF) patients. Its role and clinical significance in lung pathogenesis have not yet been clarified. The aim of the present study was to genetically characterize A. xylosoxidans strains isolated from CF patients by use of randomly amplified polymorphic DNA (RAPD) profiles and to look for a possible correlation between RAPD profiles and the patients'' clinical features, such as their spirometry values, the presence of concomitant chronic bacterial flora at the time of isolation, and the persistent or intermittent presence of A. xylosoxidans strains. A set of 106 strains of A. xylosoxidans were typed by RAPD analysis, and their profiles were analyzed by agglomerative hierarchical classification (AHC) and associated with the patient characteristics mentioned above by factorial discriminant analysis (FDA). The overall results obtained in this study showed that (i) there is a marked genetic relationship between strains isolated from the same patients at different times, (ii) characteristic RAPD profiles are associated with different predicted classes for forced expiratory volume in 1 s (FEV1%), (iii) some characteristic RAPD profiles are associated with different concomitant chronic flora (CCF) profiles, and (iv) there is a significant division of RAPD profiles into “persistent strains” and “intermittent strains” of A. xylosoxidans. These findings seem to imply that the lung habitats found in CF patients are capable of shaping and selecting the colonizing bacterial flora, as seems to be the case for the A. xylosoxidans strains studied.Cystic fibrosis (CF) is the most common lethal genetic disease, causing a chronic infection of the respiratory tract, which in turn leads to progressive respiratory deficiency (6, 15). Pseudomonas aeruginosa is the most frequently found Gram-negative pathogen in the sputa of patients with CF, while Staphylococcus aureus is the most frequently found Gram-positive one. Recently, new pathogens have also emerged, such as Burkholderia cepacia complex, Stenotrophomonas maltophilia, and Achromobacter xylosoxidans (3, 9, 18, 19, 20, 24). Although the clinical significance of A. xylosoxidans is not yet clear, it is increasingly being isolated from the sputum cultures of CF patients. Tan et al. (22) found that 2.3% of CF patients had at least 3 positive cultures for A. xylosoxidans during a 6-month period. The U.S. Cystic Fibrosis Foundation''s National Patient Registry reported an increase of 4.5%, from 1995 to 2002 (1, 2), in the frequency of isolation of this microorganism from CF patients. Recently, A. xylosoxidans has been considered a nosocomial pathogen, particularly in immunocompromised patients, causing a variety of infections, including bacteremia, meningitis, pneumonia, and peritonitis (8, 23, 25). Achromobacter spp. are aerobic, nonfermentative, Gram-negative bacilli (5, 7, 21) that are frequently misidentified by routine laboratory tests, thus seriously compromising control measures related to epidemiology studies. These microorganisms are often highly resistant to various antibiotics, including β-lactams, quinolones, aminoglycosides, and carbapenems, all commonly used for the management of lung infection in CF patients.Considering the importance of bacterial lung infections in CF patients, our goals were (i) to assess the genetic relationships among isolated A. xylosoxidans strains by randomly amplified polymorphic DNA (RAPD) analysis and (ii) to use multivariate analysis techniques to look for possible correlations between A. xylosoxidans RAPD profiles and patients'' clinical features (predicted classes for forced expiratory volume in 1 s [FEV1%], presence of concomitant chronic flora [CCF] during the isolation step, and persistent or intermittent presence of A. xylosoxidans). The study was conceived in order to give a picture of adaptive changes of A. xylosoxidans during lung infection in patients with CF and to improve our knowledge about this emerging pathogenic species, highlighting its potential role in CF disease.     

Pseudomonas aeruginosa Alginate Promotes Burkholderia cenocepacia Persistence in Cystic Fibrosis Transmembrane Conductance Regulator Knockout Mice

Pseudomonas aeruginosa, a major respiratory pathogen in cystic fibrosis (CF) patients, facilitates infection by other opportunistic pathogens. Burkholderia cenocepacia, which normally infects adolescent patients, encounters alginate elaborated by mucoid P. aeruginosa. To determine whether P. aeruginosa alginate facilitates B. cenocepacia infection in mice, cystic fibrosis transmembrane conductance regulator knockout mice were infected with B. cenocepacia strain BC7 suspended in either phosphate-buffered saline (BC7/PBS) or P. aeruginosa alginate (BC7/alginate), and the pulmonary bacterial load and inflammation were monitored. Mice infected with BC7/PBS cleared all of the bacteria within 3 days, and inflammation was resolved by day 5. In contrast, mice infected with BC7/alginate showed persistence of bacteria and increased cytokine levels for up to 7 days. Histological examination of the lungs indicated that there was moderate to severe inflammation and pneumonic consolidation in isolated areas at 5 and 7 days postinfection in the BC7/alginate group. Further, alginate decreased phagocytosis of B. cenocepacia by professional phagocytes both in vivo and in vitro. P. aeruginosa alginate also reduced the proinflammatory responses of CF airway epithelial cells and alveolar macrophages to B. cenocepacia infection. The observed effects are specific to P. aeruginosa alginate, because enzymatically degraded alginate or other polyuronic acids did not facilitate bacterial persistence. These observations suggest that P. aeruginosa alginate may facilitate B. cenocepacia infection by interfering with host innate defense mechanisms.Respiratory failure due to lung infection is the major cause of mortality in cystic fibrosis (CF) patients. CF airways are colonized by more than one opportunistic bacterial pathogen, and Pseudomonas aeruginosa is a major pathogen. The other opportunistic bacterial pathogens that are frequently isolated from CF airways include Haemophilus influenzae, Staphylococcus aureus, the Burkholderia cepacia complex (BCC), Stenotrophomonas maltophilia, and methicillin-resistant S. aureus (7). Most individuals with CF experience a characteristic age-related pattern of pulmonary colonization and intermittent exacerbations involving H. influenzae and S. aureus, followed by P. aeruginosa (4, 5). Similarly, accumulating evidence suggests that P. aeruginosa can promote colonization by less commonly observed bacteria, such as S. maltophilia, Achromobacter xylosoxidans, and Mycobacterium abscessus (43). P. aeruginosa has also been implicated in promoting BCC pathogenesis by increasing the adherence of BCC to respiratory epithelial cells and upregulating the expression of BCC virulence factors (17, 31, 32).Chronic P. aeruginosa infections are often associated with a mucoid phenotype due to the production of large quantities of the acidic exopolysaccharide alginate (5). Alginate is an important extracellular virulence factor and has been shown to impair host innate defenses related to phagocytes (1, 13, 15, 18, 26, 30). In CF airways, P. aeruginosa is found in the airway lumen, and hence one may expect large amounts of alginate in airways along with host products. Sputum samples from CF patients have been shown to contain 50 to 200 μg/ml alginate (23, 30). In fact, it is likely that there are much higher concentrations of alginate in CF airways, as sputum samples are mixed with host secretions and hence the concentration of alginate may be underestimated. Since BCC infection generally occurs in patients who have been chronically colonized with mucoid P. aeruginosa, we hypothesized that alginate in the airways may prevent detection of BCC by phagocytes and facilitate colonization of CF lungs by BCC. To test this hypothesis, we infected gut-corrected CF mice with Burkholderia cenocepacia strain BC7 suspended in either phosphate-buffered saline (PBS) (BC7/PBS), P. aeruginosa alginate (BC7/alginate), or enzymatically degraded alginate (BC7/ED-alginate) and examined the persistence of bacteria and the associated lung inflammation. We also examined the effects of alginate on phagocytosis of B. cenocepacia by macrophages and neutrophils and the proinflammatory responses of airway epithelial cells to B. cenocepacia infection.     

Rapid identification of mycobacterial whole cells in solid and liquid culture media by matrix-assisted laser desorption ionization-time of flight mass spectrometry

Mycobacterial identification is based on several methods: conventional biochemical tests that require several weeks for accurate identification, and molecular tools that are now routinely used. However, these techniques are expensive and time-consuming. In this study, an alternative method was developed using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). This approach allows a characteristic mass spectral fingerprint to be obtained from whole inactivated mycobacterial cells. We engineered a strategy based on specific profiles in order to identify the most clinically relevant species of mycobacteria. To validate the mycobacterial database, a total of 311 strains belonging to 31 distinct species and 4 species complexes grown in Löwenstein-Jensen (LJ) and liquid (mycobacterium growth indicator tube [MGIT]) media were analyzed. No extraction step was required. Correct identifications were obtained for 97% of strains from LJ and 77% from MGIT media. No misidentification was noted. Our results, based on a very simple protocol, suggest that this system may represent a serious alternative for clinical laboratories to identify mycobacterial species.The genus Mycobacterium encompasses over 100 species. The most important mycobacterial diseases are predominantly caused by the Mycobacterium tuberculosis complex. The incidence of other mycobacterial diseases due to nontuberculous mycobacteria (NTM) appears to be increasing in recent years due to the increasing number of immunocompromised individuals (1). Because the treatment of these infections differs depending on the isolated species, the correct and rapid identification of causative organisms is essential.Conventional methods for the identification of mycobacteria were classically based on biochemical tests. They required several weeks for adequate growth, and sometimes accurate identification was not possible. Difficulties, such as the lack of adequate reproducibility, the variability of phenotypes, and the fact that phenotype information is limited to common species, may lead to ambiguous or erroneous results (29). New strategies have been developed in the last decades using molecular biology tools (6, 10, 16, 24). The techniques based on DNA hybridization are sensitive, fast, and simple, but the available commercial assays (AccuProbe; Gen-Probe, San Diego, CA) are able to identify only four species and two complexes of mycobacteria (10). Techniques requiring amplification followed by a hybridization step on a solid support are more complete than probes, but commercially available kits are limited to 5 (GenoType MTBC; Hain Lifescience GmbH, Nehren, Germany), 16 (Inno-LiPa Mycobacteria v2; Innogenetics, Gent, Belgium), or 30 (GenoType Mycobacterium; Hain Lifescience GmbH, Germany) species (19, 22, 30). Systems based on sequencing or enzymatic restriction targeting the hsp65, 16S rRNA, sod, and rpoB genes allow good identification of all mycobacteria at the species level but remain limited to specialized laboratories (14, 17, 23, 31, 36, 37). In addition, they are expensive and time-consuming and require qualified operators (20). Recently, alternatives based on the analysis of mycolic acid by high-performance liquid chromatography (HPLC) (2) or electrospray ionization-tandem mass spectrometry analysis (28) have been proposed. However, these methods are still labor-intensive.Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) allows rapid identification of the most frequently isolated bacteria grown on solid medium by the identification of species-specific profiles obtained from isolated colonies (3-5). This technique is now routinely used in a few laboratories (26, 34). Some authors have used MALDI-TOF MS for rapid identification of Mycobacterium species (12, 18, 21). However, the techniques used require several steps, such as 16S rRNA gene-based techniques (18), cell extractions (12), or a statistical analysis (12, 21). In addition, the number of tested strains in these studies is less than 40, encompassing a maximum of 13 species. It was first reported by Hettick et al. that the analysis of mycobacterial whole cells by MALDI-TOF MS could be used for identification. However, the limited number of strains prevented the engineering of a useful database (11).Recently, we engineered a strategy to identify bacteria using MALDI-TOF MS, based on the choice of a limited number of species-specific profiles (3, 5). The aim of the present work is to extend this strategy to the identification of mycobacterial strains without cell extraction. This method will allow us to have a rapid, accurate, and inexpensive identification tool in routine laboratories. The first step was to build a complete database for mycobacterial species isolated in human pathology. This database was then validated by using clinical strains cultivated in solid and liquid media.     

Identification of Legionella Species by Use of an Oligonucleotide Array

The genus Legionella contains a diverse group of motile, asaccharolytic, nutritionally fastidious gram-negative rods. Legionella pneumophila is the most important human pathogen, followed by L. micdadei, L. longbeachae, L. dumoffii, and other rare species. Accurate identification of Legionella spp. other than L. pneumophila is difficult because of biochemical inertness and phenotypic identity of different species. The feasibility of using an oligonucleotide array for identification of 18 species of Legionella was evaluated in this study. The method consisted of PCR amplification of the macrophage infectivity potentiator mip gene, followed by hybridization of the digoxigenin-labeled PCR products to a panel of 30 oligonucleotide probes (16- to 24-mers) immobilized on a nylon membrane. A collection of 144 target strains (strains we aimed to identify) and 50 nontarget strains (44 species) were analyzed by the array. Both test sensitivity (144/144 strains) and specificity (50/50 strains) of the array were 100%. The whole procedure for identification of Legionella species by the array can be finished within a working day, starting from isolated colonies. It was concluded that species identification of clinically relevant Legionella spp. by the array method is very reliable and can be used as an accurate alternative to conventional or other molecular methods for identification of Legionella spp.The genus Legionella currently contains 50 validly named species (http://www.dsmz.de/bactnom/bactname.htm), and among them, 20 have been found to be human pathogens (6, 10). Legionnaires'' disease (LD) is caused mainly by inhalation of aerosols generated from water sources contaminated with Legionella spp. (6, 40). While most species of Legionella are normal environmental flora, many are implicated in opportunistic infections in immunocompromised patients (14). Pulmonary infections caused by Legionella may be subclinical or severe (27), and the fatality rate can approach 50% in immunocompromised patients (49).Legionella pneumophila accounts for about 85 to 90% of cases of LD (6, 26, 49). Other Legionella spp. implicated in human infections include L. micdadei, L. longbeachae, L. dumoffii, and some less encountered species, such as L. anisa, L. bozemanae, L. feeleii, and L. wadsworthii (49). L. pneumophila is normally identified by immunofluorescent-antibody assay. A specific FDA-cleared fluorescein isothiocyanate-labeled monoclonal antibody (Bio-Rad, Hercules, CA) for all serogroups of L. pneumophila and fluorescein isothiocyanate-labeled polyclonal antisera specific for L. pneumophila serogroup 1 (m-TECH, Atlanta, GA) are commercially available (6). Accurate identification of Legionella spp. other than L. pneumophila and L. pneumophila serogroup 1 can be quite difficult due to serological cross-reactivities between serogroups and species, biochemical inertness, and phenotypic identity of different species (6). Legionella isolates which fail to react with L. pneumophila antibodies are recommended to be identified by public health or reference laboratories (6). Antigen detection in urine specimens is also commonly used in hospitals for diagnosing infection caused by L. pneumophila (46).Molecular approaches have been developed to provide more rapid and accurate identification of Legionella spp. These methods include PCR (20, 25, 34), gene probe hybridization (24, 41), restriction fragment length polymorphism analysis (21, 38), and sequence analysis of the rRNA gene (47) and the macrophage infectivity potentiator gene mip (35, 41). Since diagnostic delay may result in increased mortality for patients with LD (15), real-time PCR assay has been a focus of many studies in recent years (5, 13, 14, 17, 19, 34, 36, 41, 48). However, with real-time PCR assay, only L. pneumophila and a very limited number of Legionella spp. can be detected or identified.Recently, DNA array technology has been applied to identify a wide variety of bacteria that are difficult to be differentiated by phenotypic traits or whose identification may take a long time (12, 31, 43). This study aimed to develop an oligonucleotide array based on mip gene sequences to identify 18 species of Legionella that have been found to cause human infections in the literature (10).     

Identification of Clinically Important Anaerobic Bacteria by an Oligonucleotide Array

Anaerobic bacteria can cause a wide variety of infections, and some of these infections can be serious. Conventional identification methods based on biochemical tests are often lengthy and can produce inconclusive results. An oligonucleotide array based on the 16S-23S rRNA intergenic spacer (ITS) sequences was developed to identify 28 species of anaerobic bacteria and Veillonella. The method consisted of PCR amplification of the ITS regions with universal primers, followed by hybridization of the digoxigenin-labeled PCR products to a panel of 35 oligonucleotide probes (17- to 30-mers) immobilized on a nylon membrane. The performance of the array was determined by testing 310 target strains (strains which we aimed to identify), including 122 reference strains and 188 clinical isolates. In addition, 98 nontarget strains were used for specificity testing. The sensitivity and the specificity of the array for the identification of pure cultures were 99.7 and 97.1%, respectively. The array was further assessed for its ability to detect anaerobic bacteria in 49 clinical specimens. Two species (Finegoldia magna and Bacteroides vulgatus) were detected in two specimens by the array, and the results were in accordance with those obtained by culture. The whole procedure of array hybridization took about 8 h, starting with the isolated colonies. The array can be used as an accurate alternative to conventional methods for the identification of clinically important anaerobes.Anaerobic bacteria are important human pathogens, and infections caused by these bacteria can be serious and life-threatening (6). A recent report from the Mayo Clinic (Rochester, MN) revealed an overall increase in the incidence of anaerobic bacteremias of 74% from 2001 to 2004 compared to that from 1993 to 1996 (20), although the same trend was not found in community hospitals or in an European countries (2, 11). The commonly isolated anaerobic bacteria are the members of the Bacteroides fragilis group and Peptostreptococcus, Clostridium, and Fusobacterium species (3, 6, 20).Most clinical laboratories use differential biochemical tests for the identification of anaerobic microorganisms (35). However, Simmon et al. (31) found that 24% of the isolates of anaerobic bacteria recovered from blood cultures were misidentified and that 10% isolates were not identified to the species level by phenotypic characteristics. A rapid commercial kit, the Rapid ID 32A kit (bioMérieux, Marcy l''Etoile, France), was evaluated for its ability to identify strains in the Bacteroides fragilis group. The results showed that only 78.4% of the strains were correctly identified to the species level without supplemental tests (15). The success of the Rapid ID 32A system for species identification varied with different taxa (10), and a low identification rate (50%) was observed for fusobacteria (16). Veillonella isolates are relatively easily identified to the genus level, but the differentiation of Veillonella isolates at the species level remains difficult and inconclusive due to the lack of discriminatory tests (14). In recent years, increasing antimicrobial resistance for some anaerobic bacteria (1, 13, 33) were noted, especially for species in the B. fragilis group (40). The rapid identification of anaerobic bacteria and the administration of appropriate antimicrobials play crucial roles in preventing mortality and morbidity in patients (6).Molecular methods have emerged as accurate alternatives for the identification of anaerobic bacteria (21, 22, 34, 36). Approximately 9% isolates of bacteremic anaerobes could not be identified to the species level by 16S rRNA gene sequencing, although all isolates were correctly assigned to the genus level (31). Other molecular identification methods targeting the rRNA operon include PCR (32), real-time PCR (26), PCR-restriction fragment length polymorphism analysis (39), and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (37).The intergenic spacer (ITS) region separating the 16S and 23S rRNA genes has been suggested to be a good candidate for use for the identification of aerobic and anaerobic bacteria (8, 19, 42). Moreover, the DNA array technology has been applied to the identification of a variety of microorganisms (12, 17, 41). The aim of the study described here was to develop an oligonucleotide array based on the ITS sequences to identify 28 clinically important species of anaerobes and Veillonella.     

Development and Application of Multiprobe Real-Time PCR Method Targeting the hsp65 Gene for Differentiation of Mycobacterium Species from Isolates and Sputum Specimens

We developed a multiprobe real-time PCR assay targeting hsp65 (HMPRT-PCR) to detect and identify mycobacterial isolates and isolates directly from sputum specimens. Primers and probes for HMPRT-PCR were designed on the basis of the hsp65 gene sequence, enabling the recognition of seven pathogenic mycobacteria, including Mycobacterium tuberculosis, M. avium, M. intracellulare, M. kansasii, M. abscessus, M. massiliense, and M. fortuitum. This technique was applied to 24 reference and 133 clinical isolates and differentiated between all strains with 100% sensitivity and specificity. Furthermore, this method was applied to sputum specimens from 117 consecutive smear-positive patients with smear results of from a trace to 3+. These results were then compared to those obtained using the rpoB PCR-restriction analysis method with samples from cultures of the same sputum specimens. The HMPRT-PCR method correctly identified the mycobacteria in 89 samples (76.0%, 89/117), and moreover, the sensitivity level was increased to 94.3% (50/53) for sputa with an acid-fast bacillus score equal to or greater than 2+. Our data suggest that this novel HMPRT-PCR method could be a promising approach for detecting pathogenic mycobacterial species from sputum samples and culture isolates routinely in a clinical setting.Of the known species in the genus Mycobacterium, Mycobacterium tuberculosis is the most common and most important pathogen, causing 2 million deaths and over 8 million cases of tuberculosis worldwide annually (2, 3, 4, 7). In addition to M. tuberculosis, infections with nontuberculosis mycobacteria (NTM) can also cause clinical problems. Because of the different pathogenic potentials and susceptibilities of different mycobacterial species, the treatments of mycobacterial infections are different (13, 30, 33, 34). Thus, it is very important to differentiate between mycobacteria at the species level during early-stage diagnostics.Instead of a culture-based identification scheme, which may take 4 to 6 weeks or longer to identify slowly growing mycobacteria, PCR-based protocols (sequencing or PCR-restriction analysis [PRA]) targeting chronometer molecules, such as 16S rRNA (5, 6, 28), hsp65 (17, 19, 25), and rpoB (1, 16, 21), have been widely used to identify mycobacteria. However, in spite of the successful application of these conventional PCR-based methods to culture isolates, there are some drawbacks in their direct application to clinical specimens. This is especially true for sputum samples, which also contain numbers of commensal bacteria from the respiratory tract, producing confusing results by the simultaneous amplification of both commensals and mycobacterial strains. We have recently developed several methods for mycobacterial species identification based on amplification of hsp65 gene sequences directly from sputum samples (15, 27). Limitations due to the intrinsic features of conventional PCR prevented feasible identification of mycobacterial species from sputum samples using this method.The use of the real-time PCR assay in the diagnosis of many infectious diseases has been increasing, as it represents an appealing alternative to conventional PCR. It is an improvement over conventional methods because of its increased sensitivity and specificity, low contamination risk, and ease of performance and speed (8). In particular, fluorescence resonance energy transfer (FRET)-based real-time PCR permits not only the simultaneous identification of multiple target species but also the direct identification of target species from primary specimens such as sputum specimens through melting curve analysis of the amplification product (8). These characteristics of FRET-based real-time PCR provide a useful advantage for the identification of mycobacteria from sputum samples. Recently, several real-time PCR-based methods for mycobacterial detection and identification have been developed and evaluated (9, 22, 23, 26, 29). However, direct application of the real-time PCR-based method to primary specimens was generally limited to M. tuberculosis alone (11, 26). So far, a method which can simultaneously identify several pathogenic NTM as well as M. tuberculosis from primary sputum samples in a single reaction has not been developed.In the present study, we sought to develop a multiprobe real-time PCR targeting the hsp65 gene (HMPRT-PCR) based on melting curve analysis (HybProbes). This enabled the simultaneous identification of several pathogenic mycobacteria, including M. tuberculosis, in a single PCR performed on cultures and sputum samples. The usefulness of these methods was evaluated by blindly applying them to cultured and sputum samples.     

Variation in Susceptibility of Bloodstream Isolates of Candida glabrata to Fluconazole According to Patient Age and Geographic Location in the United States in 2001 to 2007

We examined the susceptibilities to fluconazole of 642 bloodstream infection (BSI) isolates of Candida glabrata and grouped the isolates by patient age and geographic location within the United States. Susceptibility of C. glabrata to fluconazole was lowest in the northeast region (46%) and was highest in the west (76%). The frequencies of isolation and of fluconazole resistance among C. glabrata BSI isolates were higher in the present study (years 2001 to 2007) than in a previous study conducted from 1992 to 2001. Whereas the frequency of C. glabrata increased with patient age, the rate of fluconazole resistance declined. The oldest age group (≥80 years) had the highest proportion of BSI isolates that were C. glabrata (32%) and the lowest rate of fluconazole resistance (5%).Candidemia is without question the most important of the invasive mycoses (6, 33, 35, 61, 65, 68, 78, 86, 88). Treatment of candidemia over the past 20 years has been enhanced considerably by the introduction of fluconazole in 1990 (7, 10, 15, 28, 29, 31, 40, 56-58, 61, 86, 90). Because of its widespread usage, concern about the development of fluconazole resistance among Candida spp. abounds (2, 6, 14, 32, 47, 53, 55, 56, 59, 60, 62, 80, 86). Despite these concerns, fluconazole resistance is relatively uncommon among most species of Candida causing bloodstream infections (BSI) (5, 6, 22, 24, 33, 42, 54, 56, 65, 68, 71, 86). The exception to this statement is Candida glabrata, of which more than 10% of BSI isolates may be highly resistant (MIC ≥ 64 μg/ml) to fluconazole (6, 9, 15, 23, 30, 32, 36, 63-65, 71, 87, 91). Suboptimal fluconazole dosing practices (low dose [<400 mg/day] and poor indications) may lead to an increased frequency of isolation of C. glabrata as an etiological agent of candidemia in hospitalized patients (6, 17, 29, 32, 35, 41, 47, 55, 60, 68, 85) and to increased fluconazole (and other azole) resistance secondary to induction of CDR efflux pumps (2, 11, 13, 16, 43, 47, 50, 55, 69, 77, 83, 84) and may adversely affect the survival of treated patients (7, 10, 29, 40, 59, 90). Among the various Candida species, C. glabrata alone has increased as a cause of BSI in U.S. intensive care units since 1993 (89). Within the United States, the proportion of fungemias due to C. glabrata has been shown to vary from 11% to 37% across the different regions (west, midwest, northeast, and south) of the country (63, 65) and from <10% to >30% within single institutions over the course of several years (9, 48). It has been shown that the prevalence of C. glabrata as a cause of BSI is potentially related to many disparate factors in addition to fluconazole exposure, including geographic characteristics (3, 6, 63-65, 71, 88), patient age (5, 6, 25, 35, 41, 42, 48, 63, 82, 92), and other characteristics of the patient population studied (1, 32, 35, 51). Because C. glabrata is relatively resistant to fluconazole, the frequency with which it causes BSI has important implications for therapy (21, 29, 32, 40, 41, 45, 56, 57, 59, 80, 81, 86, 90).Previously, we examined the susceptibilities to fluconazole of 559 BSI isolates of C. glabrata and grouped the isolates by patient age and geographic location within the United States over the time period from 1992 to 2001 (63). In the present study we build upon this experience and report the fluconazole susceptibilities of 642 BSI isolates of C. glabrata collected from sentinel surveillance sites throughout the United States for the time period from 2001 through 2007 and stratify the results by geographic region and patient age. The activities of voriconazole and the echinocandins against this contemporary collection of C. glabrata isolates are also reported.     

Proteomic Identification of OprL as a Seromarker for Initial Diagnosis of Pseudomonas aeruginosa Infection of Patients with Cystic Fibrosis

Identification of new immunogenic antigens that diagnose initial Pseudomonas aeruginosa infections in patients with cystic fibrosis (CF) alone or as an adjunct to microbiology is needed. In the present study, a proteomic analysis was performed to obtain a global assessment of the host immune response during the initial P. aeruginosa infection of patients with CF. Matrix-assisted laser desorption ionization-time of flight mass spectrometry was used to identify outer membrane protein L (OprL), a non-type III secretion system (TTSS) protein, as an early immunogenic protein during the initial P. aeruginosa infection of patients with CF. Longitudinal Western blot analysis of sera from 12 of 14 patients with CF detected antibodies to OprL during the initial P. aeruginosa infection. In addition, also detected were antibodies to ExoS, ExoU, or ExoS and ExoU, the latter indicating sequential P. aeruginosa infections during initial infections. Detection of serum reactivity to OprL, along with proteins of the TTSS, and in conjunction with microbiology may diagnose initial P. aeruginosa infections in patients with CF.Cystic fibrosis (CF) is an autosomal recessive multisystem disease which is caused by mutations in the CF transmembrane conductance receptor. Patients with CF have chronic respiratory infections which are the primary cause of morbidity and premature mortality (16). Patients with CF are infected with bacterial pathogens on an age-dependent timeline (16). Typically, Staphylococcus aureus and nonencapsulated Haemophilus influenzae are the first isolates from infants with CF (34, 35). However, Pseudomonas aeruginosa infections in children with CF are associated with progressive lung disease (30, 33). Microbiology is used for the diagnosis of P. aeruginosa, but successful P. aeruginosa isolation can be complicated in nonexpectorating populations of infants and young children with CF (4). The diagnosis and eradication of the initial P. aeruginosa infection with antibiotics to prevent chronic infection and mucoid transformation are important, since this diagnosis influences the quality of life and long-term patient survival (1, 2, 7, 11, 29, 30). Non-culture-based tests, like serology, should assist microbiology in the early diagnosis of P. aeruginosa infection.P. aeruginosa serology continues to be challenging without defined commercially available antigens licensed in the United States that reflect the molecular pathogenesis of P. aeruginosa upon adaptation to the host environment (13). Høiby (24) and Döring and Høiby (10) have detected an antibody response against a pool of antigens from common P. aeruginosa serotypes. Elevating antibody titers against this pool of antigens correlated with worsening P. aeruginosa infections and a poor clinical prognosis. The clinical progression of CF lung disease may be a reflection of the molecular pathogenesis of P. aeruginosa. Recent studies have correlated serological reactivity and known P. aeruginosa virulence factors. West et al. (42) showed that during the initial P. aeruginosa infection of children with CF, detection of serum antibodies to exotoxin A (ETA) and a P. aeruginosa lysate occurred earlier than detection of serum antibodies to elastase or alkaline phosphatase; subsequently, Corech et al. (6) detected antibodies to components of the type III secretion system (TTSS) at a time similar to that of P. aeruginosa Sup and earlier than ETA, showing the potential of measuring the antibody response to components of the TTSS as an indication of initial infection with P. aeruginosa in children with CF. This also indicated a role for TTSS in the initial P. aeruginosa pathogenesis of the CF lung.In the present study, a proteomic analysis was performed to obtain a global assessment of the host immune response during the initial P. aeruginosa infection of patients with CF. The goal was to identify a cellular component of P. aeruginosa that elicits an early immune response to P. aeruginosa infection to provide a stable immunogenic indication of P. aeruginosa infection relative to P. aeruginosa virulence factors that may fluctuate in expression during the course of P. aeruginosa infection, especially following transition from the acute to the chronic infection phase (41). Outer membrane protein L (OprL), a non-TTSS protein, was identified as an early immunogenic protein in the initial P. aeruginosa infection of patients with CF.     

High-Throughput Identification of Bacteria and Yeast by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry in Conventional Medical Microbiology Laboratories

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is suitable for high-throughput and rapid diagnostics at low costs and can be considered an alternative for conventional biochemical and molecular identification systems in a conventional microbiological laboratory. First, we evaluated MALDI-TOF MS using 327 clinical isolates previously cultured from patient materials and identified by conventional techniques (Vitek-II, API, and biochemical tests). Discrepancies were analyzed by molecular analysis of the 16S genes. Of 327 isolates, 95.1% were identified correctly to genus level, and 85.6% were identified to species level by MALDI-TOF MS. Second, we performed a prospective validation study, including 980 clinical isolates of bacteria and yeasts. Overall performance of MALDI-TOF MS was significantly better than conventional biochemical systems for correct species identification (92.2% and 83.1%, respectively) and produced fewer incorrect genus identifications (0.1% and 1.6%, respectively). Correct species identification by MALDI-TOF MS was observed in 97.7% of Enterobacteriaceae, 92% of nonfermentative Gram-negative bacteria, 94.3% of staphylococci, 84.8% of streptococci, 84% of a miscellaneous group (mainly Haemophilus, Actinobacillus, Cardiobacterium, Eikenella, and Kingella [HACEK]), and 85.2% of yeasts. MALDI-TOF MS had significantly better performance than conventional methods for species identification of staphylococci and genus identification of bacteria belonging to HACEK group. Misidentifications by MALDI-TOF MS were clearly associated with an absence of sufficient spectra from suitable reference strains in the MALDI-TOF MS database. We conclude that MALDI-TOF MS can be implemented easily for routine identification of bacteria (except for pneumococci and viridans streptococci) and yeasts in a medical microbiological laboratory.Identification of bacteria and yeasts is generally based on conventional phenotypic methods, encompassing culture and growth patterns on specific media, Gram staining, and morphological and biochemical characteristics. Although results of Gram staining can be achieved within minutes, complete identification usually takes 1 or more days. In addition, tests may be difficult to interpret or inconclusive and require specialized staff. Recent molecular methods for microbial identification, such as real-time PCR, sequence analysis, or microarray analysis, have found some application in bacteriology. However, these methods do not provide the complete solution in routine bacterial identifications. To optimize care of patients with infectious diseases, there still is an urgent need for rapid and simple techniques for microbial identification.Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been used to analyze many different biological molecules. The application of microbial identification based on species-specific spectra of peptides and protein masses by mass spectrometry was first reported about 30 years ago (1). By further improvement of the technique, a rapid, accurate, easy-to-use, and inexpensive method has become available for identification of microorganisms (4, 14, 27). MALDI-TOF MS can be used for accurate and rapid identification of various microorganisms, such as Gram-positive bacteria (2, 3, 9, 10, 22, 26), Enterobacteriaceae (5), nonfermenting bacteria (6, 19-21), mycobacteria (12, 16, 24), anaerobes (10, 23), and yeasts (18, 25). Most studies have reported on MALDI-TOF MS identification of a single strain or family of microorganisms in a research setting. Only one study applied MALDI-TOF MS for identification of bacteria—but not yeasts—in conventional microbiology settings but did not evaluate the results for individual bacteria at the species level (27). In the present study, identification of bacteria by MALDI-TOF MS was extensively evaluated for both bacterial and yeast species identification in an academic medical microbiologic laboratory.     

Identification of Potential Diagnostic Markers among Burkholderia cenocepacia and B. multivorans Supernatants

Patients with cystic fibrosis (CF) are susceptible to chronic respiratory infections with a number of bacterial pathogens. Among them, the Burkholderia cepacia complex (Bcc) bacteria, consisting of nine related species, have emerged as problematic CF pathogens due to their antibiotic resistance, incidence of nosocomial infection, and person-to-person transmission. Bcc organisms present the clinical microbiologist with a diagnostic dilemma due to the lack of phenotypic biochemical or growth-related characterization tests that reliably distinguish among these organisms. The complex taxonomy of the Bcc species colonizing the CF respiratory tract makes accurate identification problematic. Despite the clinical implications of Bcc identification, a clinical laboratory differentiation of species within the Bcc is lacking. Additionally, no commercial assays are available to further identify the Bcc species. In the current study, secretory proteins present in the cultured supernatants of Burkholderia cenocepacia and Burkholderia multivorans were analyzed by two-dimensional gel electrophoresis (2-DE), followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). To assess differential expression, protein spots of B. cenocepacia and B. multivorans that were unique or displayed different intensities were chosen for MALDI-TOF MS analysis. In total, 341 protein spots were detected, of which 23 were unique to each species, demonstrating that potential diagnostic candidates between these two members of the Bcc exist.The Burkholderia cepacia complex (Bcc) consists of nine genetically distinct but phenotypically similar Gram-negative bacilli distinguished by their role as important agents of pulmonary disease in cystic fibrosis (CF) patients (12, 16). Associated with increased morbidity and mortality, Bcc bacteria make eradication very difficult due to their ability to establish infection and maintain chronic colonization in the CF lung (6, 9, 18, 22). Furthermore, these organisms are capable of person-to-person spread, presenting the threat of nosocomial acquired infection. Highly variable and unpredictable, clinical outcomes of Bcc infection range from asymptomatic carriage to bacteremic disease and death (12). This variation is speculated to be the result of genomovar heterogeneity and differential expression of a virulence factor(s) during Bcc infection. Although all species of the Bcc have been recovered from CF patients, Burkholderia cenocepacia and Burkholderia multivorans are the most prevalent, accounting for 85% of pulmonary infections (16).Establishing accurate methods of Bcc identification is paramount for the implementation of infection control measures and appropriate treatment of infected and/or exposed CF patients. Furthermore, species identification within the Bcc is vital not only for surgical and clinical management of CF patients but also for the elucidation of the group''s epidemiology. Bcc organisms present clinical microbiologists with a diagnostic dilemma due to the lack of phenotypic biochemical or growth-related characterization tests that can reliably distinguish among these organisms. While commercially available multitest kits have been utilized in clinical laboratories for Bcc identification, previous studies have shown these kits to be unreliable (8, 15, 19, 25). Misidentification rates of the Bcc among United States CF treatment centers (11% were false positive, and 36% were false negative) suggest the need for improved diagnostics (15). Currently, accurate identification of the Bcc and species within the complex require molecular typing techniques limited to special reference laboratories. These expensive and technically challenging assays further delay the appropriate turnaround time necessary for proper patient management. These inherent limitations have resulted in the need for alternative methods of Bcc species identification.In both CF patients and murine models of pulmonary infection, different species (B. cenocepacia and B. multivorans) within the Bcc exhibit unique patterns of virulence (2, 3). Proteomic analyses of the Bcc species B. cenocepacia, Burkholderia vietnamiensis, B. multivorans, and Burkholderia ambifaria have revealed differential expression of secretory proteins (2). These differences may account for the various degrees of pathogenicity exhibited by Bcc species. Thus, species-specific secretory proteins would be ideal biomarkers for diagnostic assays and targets for vaccines and therapeutic agents. Elucidation of these unique proteins can be achieved through characterization and comparative analysis of Bcc species secretomes. In the current study, secretory proteins present in the cultured supernatants of B. cenocepacia and B. multivorans were analyzed by two-dimensional gel electrophoresis (2-DE), followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). To assess differential expression, protein spots of B. cenocepacia and B. multivorans that were unique or displayed different intensities were chosen for MALDI-TOF MS analysis.     

Clinical Relevance of the Recently Described Species Aeromonas aquariorum

Twenty-two human extraintestinal isolates (11 from blood) and three isolates recovered from patients with diarrhea were genetically characterized as Aeromonas aquariorum, a novel species known only from ornamental fish. The isolates proved to bear a considerable number of virulence genes, and all were resistant to amoxicillin (amoxicilline), cephalothin (cefalotin), and cefoxitin. Biochemical differentiation from the most relevant clinical species is provided.Members of the genus Aeromonas are responsible for producing intestinal and extraintestinal infections worldwide (1, 10). The most common clinical presentation of Aeromonas is diarrhea followed by localized soft-tissue infections and bacteremia. Bacteremia mainly occurs in patients with underlying diseases, i.e., hepatobiliary disorders, cancer, and diabetes. In patients with hepatobiliary disorders, especially in those with cirrhosis, fatal necrotizing fasciitis can be produced despite surgical intervention and antibiotic therapy (1, 10).The pathogenicity of Aeromonas has been associated with numerous virulence factors, including the aerolysin/hemolysin group of genes, the cytotonic enterotoxins Ast and Alt (4, 9, 19), the cytotoxin encoded by the act gene (9), and a type III secretion system (TTSS) (8, 20, 26). The TTSS is a virulence mechanism that delivers toxins (AexT among others) directly into the host cell and induces apoptosis (8, 27).Currently, the genus contains 22 species (A. Alperi, A. J. Martínez-Murcia, W. C. Ko, A. Monera, M. J. Saavedra and M. J. Figueras, submitted for publication). Although A. hydrophila is the most referenced species in the clinical literature, recent studies using molecular identification methods have demonstrated that A. veronii and A. caviae are more prevalent (3, 10, 18, 24).In a set of extraintestinal isolates received in our laboratory from Taiwan for genetic identification, we have identified 22 as belonging to A. aquariorum, a recently described species from aquarium water and the skin of ornamental fish (17). In addition, three clinical strains isolated from patients with diarrhea in Spain were also identified as belonging to this species. Since this is a poorly known species, we have considered it of interest to study the molecular characterization, antibiotic susceptibility, and virulence potential of these strains in order to provide complementary data to differentiate A. aquariorum from other, commonly encountered clinical species.     

An Engineered Human Antibody Fab Fragment Specific for Pseudomonas aeruginosa PcrV Antigen Has Potent Antibacterial Activity

Pseudomonas aeruginosa is an opportunistic pathogen that can cause acute lung injury and mortality through the delivery of exotoxins by the type III secretion system (TTSS). PcrV is an important structural protein of the TTSS. An engineered human antibody Fab fragment that binds to the P. aeruginosa PcrV protein with high affinity has been identified and has potent in vitro neutralization activity against the TTSS. The instillation of a single dose of Fab into the lungs of mice provided protection against lethal pulmonary challenge of P. aeruginosa and led to a substantial reduction of viable bacterial counts in the lungs. These results demonstrate that blocking of the TTSS by a Fab lacking antibody Fc-mediated effector functions can be sufficient for the effective clearance of pulmonary P. aeruginosa infection.Pseudomonas aeruginosa is an opportunistic pathogen that causes both acute and chronic infections in compromised individuals. It is a frequent causative agent of bacteremia in burn victims (32) and immunocompromised patients (18). It is also the most common cause of nosocomial gram-negative pneumonia (7, 25), especially in mechanically ventilated patients (25), and is the most prevalent pathogen in the lungs of individuals with cystic fibrosis (CF) (10, 17, 20). In CF, P. aeruginosa infection follows a well-established pattern of recurrent pulmonary infection in early childhood leading to the establishment of chronic infection in older CF patients, where it is a major contributing factor in the progressive decline in lung function and disease exacerbations leading to respiratory failure (10, 17).Morbidity and mortality associated with P. aeruginosa infections remain high despite the availability of antibiotics to which the bacterium is sensitive, and antibiotic resistance is an increasingly common problem in nosocomial infections (6).The type III secretion system (TTSS) is an important virulence determinant of P. aeruginosa in animal models of infection (15) and is required for the systemic spread of P. aeruginosa in a mouse pulmonary challenge model (31). The expression of a functional TTSS also correlates with poor prognosis in clinical infections (26, 27). This needle-like structure comprises a complex secretion and translocation machinery to inject a set of up to four different exotoxins (ExoS, ExoT, ExoU, and ExoY) directly into the cytoplasm of eukaryotic cells (9, 33, 34). Various strains of P. aeruginosa secrete different exotoxins. In addition, the TTSS can mediate direct cytotoxicity toward macrophages and neutrophils in the absence of exotoxins, a process called “oncosis,” requiring bacterial swarming in response to macrophage factors and leading to a direct perforation of the cell membranes (3, 4). In all of these functions of the TTSS, the needle tip protein, PcrV, is an essential component of the translocation apparatus.Antisera raised against PcrV in rabbits have been shown to block the translocation of Pseudomonas exotoxins into mammalian cells (12, 28) and to protect against lethality in a mouse model of acute pulmonary Pseudomonas infection (28, 29). Polyclonal anti-PcrV antibodies have also been shown to reduce lung damage and protect against bacteremia and septic shock in rat and rabbit pulmonary infection models (29) and to protect burned mice from infection (22). A mouse monoclonal anti-PcrV antibody, monoclonal antibody (MAb) 166, with potent neutralizing activity in mouse and rat models of Pseudomonas infection has also been described (11). This antibody inhibits the function of the TTSS in cell-based assays (11, 12). The MAb acts to prevent sepsis and mortality in an acute pulmonary infection model in mice when delivered either systemically or by intratracheal administration (11) and reduces lung damage due to Pseudomonas in a rat model (8). The antibody has activity when dosed either prophylactically or therapeutically in these models both as whole immunoglobulin G (IgG) and as a Fab fragment, indicating that the inhibition of TTSS function is sufficient to inhibit lung damage in pulmonary Pseudomonas infections.Here, we identify an engineered human antibody Fab fragment specific for the P. aeruginosa PcrV protein which competes with MAb 166 for binding to the same epitope on PcrV. The human Fab shows potent TTSS-neutralizing activity, equivalent to the activity of MAb 166, in cellular cytotoxicity assays. This Fab shows potent in vivo activity in protecting mice from potentially lethal doses of P. aeruginosa. In addition, the Fab mediates a substantial clearance of bacteria from the lungs of infected mice, suggesting that antagonism of the TTSS is sufficient not only to prevent damage to the pulmonary epithelium but also to restore normal immunological clearance mechanisms for the effective resolution of Pseudomonas infection in the complete absence of antibody effector functions.