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.     

Cohort Study of Molecular Identification and Typing of Mycobacterium abscessus,Mycobacterium massiliense,and Mycobacterium bolletii

Mycobacterium abscessus is the most common cause of rapidly growing mycobacterial chronic lung disease. Recently, two new M. abscessus-related species, M. massiliense and M. bolletii, have been described. Health care-associated outbreaks have recently been investigated by the use of molecular identification and typing tools; however, very little is known about the natural epidemiology and pathogenicity of M. massiliense or M. bolletii outside of outbreak situations. The differentiation of these two species from M. abscessus is difficult and relies on the sequencing of one or more housekeeping genes. We performed extensive molecular identification and typing of 42 clinical isolates of M. abscessus, M. massiliense, and M. bolletii from patients monitored at the NIH between 1999 and 2007. The corresponding clinical data were also examined. Partial sequencing of rpoB, hsp65, and secA led to the unambiguous identification of 26 M. abscessus isolates, 7 M. massiliense isolates, and 2 M. bolletii isolates. The identification results for seven other isolates were ambiguous and warranted further sequencing and an integrated phylogenetic analysis. Strain relatedness was assessed by repetitive-sequence-based PCR (rep-PCR) and pulsed-field gel electrophoresis (PFGE), which showed the characteristic clonal groups for each species. Five isolates with ambiguous species identities as M. abscessus-M. massiliense by rpoB, hsp65, and secA sequencing clustered as a distinct group by rep-PCR and PFGE together with the M. massiliense type strain. Overall, the clinical manifestations of disease caused by each species were similar. In summary, a multilocus sequencing approach (not just rpoB partial sequencing) is required for division of M. abscessus and closely related species. Molecular typing complements sequence-based identification and provides information on prevalent clones with possible relevant clinical aspects.Rapidly growing mycobacteria (RGM) are ubiquitous organisms increasingly emerging as important human pathogens. Mycobacterium abscessus is commonly associated with wound infections and abscess formation and is the most frequent RGM causing chronic lung disease, often in immunocompromised patients (15, 22, 24). M. abscessus is also notable for its resistance to treatment and the poor clinical outcome of infection with the organism (22, 24). Within the past decade, two new species of mycobacteria closely related to M. abscessus, M. massiliense and M. bolletii, have been described (1, 3). Information on the pathogenic role of M. massiliense and M. bolletii is still scant. Recent reports have described the isolation of M. massiliense from two patients in the United States (29) and one patient in Italy (35) and, lately, the identification of M. massiliense and M. bolletii among South Korean isolates (18). Both M. massiliense and M. bolletii have also been linked to health care-associated outbreaks (8, 19, 37).The species-level identification of RGM can provide the first indication of antibiotic susceptibility and can suggest the appropriate type of patient management. For example, M. abscessus is more resistant to many antibiotics both in vivo and in vitro than M. fortuitum and M. mucogenicum, but it is usually susceptible to amikacin and clarithromycin (6, 15, 24). M. massiliense was originally reported to be distinguishable from M. abscessus and related species by its susceptibility to doxycycline (3); however, resistant isolates have since been described (19, 37), suggesting that antibiotic susceptibility results may not reliably differentiate among these closely related species.Although 16S rRNA gene sequencing has been used for the identification of nontuberculous mycobacteria (NTM), including RGM, it has limited value in distinguishing among some closely related species (9, 14). Therefore, the use of several other gene targets for the identification of mycobacteria has been proposed (2, 5, 11, 23, 25, 31, 32, 39, 41). Discrimination among M. abscessus, M. massiliense, and M. bolletii (which have identical 16S rRNA gene sequences) has proven to be difficult, with sequencing of different gene targets often providing conflicting results. Among these gene targets, partial sequencing of rpoB has increasingly been used (1, 19, 29, 37).Genotypic analysis of NTM has proven useful not only in the investigation of outbreaks and pseudo-outbreaks (38) but also in characterizing the molecular epidemiology of strains, and in assessing clonal distribution and expansion (4, 7, 13, 17). In particular, molecular typing has recently been used for the characterization of health care-related outbreaks of M. massiliense and M. bolletii (19, 37).We sought to perform a thorough molecular investigation, including strain identification and typing, for a series of 42 clinical isolates (CIs) of M. abscessus, M. massiliense, and M. bolleti from patients monitored in our institution between 1999 and 2007. A retrospective patient chart review assessed demographics, underlying conditions, and clinical history.The 42 CIs and 3 type strains were subjected to multilocus sequence analysis, including sequencing of rpoB, hsp65, secA, and the internally transcribed spacer (ITS) region. The relatedness among the isolates was assessed by use of an automated repetitive-sequence-based PCR (rep-PCR) and pulsed-field gel electrophoresis (PFGE). This is the most extensive molecular characterization of non-outbreak-related isolates from patients with M. abscessus, M. massiliense, and M. bolletii infections.     

Hag Mediates Adherence of Moraxella catarrhalis to Ciliated Human Airway Cells

Moraxella catarrhalis is a human pathogen causing otitis media in infants and respiratory infections in adults, particularly patients with chronic obstructive pulmonary disease. The surface protein Hag (also designated MID) has previously been shown to be a key adherence factor for several epithelial cell lines relevant to pathogenesis by M. catarrhalis, including NCIH292 lung cells, middle ear cells, and A549 type II pneumocytes. In this study, we demonstrate that Hag mediates adherence to air-liquid interface cultures of normal human bronchial epithelium (NHBE) exhibiting mucociliary activity. Immunofluorescent staining and laser scanning confocal microscopy experiments demonstrated that the M. catarrhalis wild-type isolates O35E, O12E, TTA37, V1171, and McGHS1 bind principally to ciliated NHBE cells and that their corresponding hag mutant strains no longer associate with cilia. The hag gene product of M. catarrhalis isolate O35E was expressed in the heterologous genetic background of a nonadherent Haemophilus influenzae strain, and quantitative assays revealed that the adherence of these recombinant bacteria to NHBE cultures was increased 27-fold. These experiments conclusively demonstrate that the hag gene product is responsible for the previously unidentified tropism of M. catarrhalis for ciliated NHBE cells.Moraxella catarrhalis is a gram-negative pathogen of the middle ear and lower respiratory tract (29, 40, 51, 52, 69, 78). The organism is responsible for ∼15% of bacterial otitis media cases in children and up to 10% of infectious exacerbations in patients with chronic obstructive pulmonary disease (COPD). The cost of treating these ailments places a large financial burden on the health care system, adding up to well over $10 billion per annum in the United States alone (29, 40, 52, 95, 97). In recent years, M. catarrhalis has also been increasingly associated with infections such as bronchitis, conjunctivitis, sinusitis, bacteremia, pneumonia, meningitis, pericarditis, and endocarditis (3, 12, 13, 17-19, 24, 25, 27, 51, 67, 70, 72, 92, 99, 102-104). Therefore, the organism is emerging as an important health problem.M. catarrhalis infections are a matter of concern due to high carriage rates in children, the lack of a preventative vaccine, and the rapid emergence of antibiotic resistance in clinical isolates. Virtually all M. catarrhalis strains are resistant to β-lactams (34, 47, 48, 50, 53, 65, 81, 84). The genes specifying this resistance appear to be gram positive in origin (14, 15), suggesting that the organism could acquire genes conferring resistance to other antibiotics via horizontal transfer. Carriage rates as high as 81.6% have been reported for children (39, 104). In one study, Faden and colleagues analyzed the nasopharynx of 120 children over a 2-year period and showed that 77.5% of these patients became colonized by M. catarrhalis (35). These investigators also observed a direct relationship between the development of otitis media and the frequency of colonization. This high carriage rate, coupled with the emergence of antibiotic resistance, suggests that M. catarrhalis infections may become more prevalent and difficult to treat. This emphasizes the need to study pathogenesis by this bacterium in order to identify vaccine candidates and new targets for therapeutic approaches.One key aspect of pathogenesis by most infectious agents is adherence to mucosal surfaces, because it leads to colonization of the host (11, 16, 83, 93). Crucial to this process are surface proteins termed adhesins, which mediate the binding of microorganisms to human cells and are potential targets for vaccine development. M. catarrhalis has been shown to express several adhesins, namely UspA1 (20, 21, 59, 60, 77, 98), UspA2H (59, 75), Hag (also designated MID) (22, 23, 37, 42, 66), OMPCD (4, 41), McaP (61, 100), and a type 4 pilus (63, 64), as well as the filamentous hemagglutinin-like proteins MhaB1, MhaB2, MchA1, and MchA2 (7, 79). Each of these adhesins was characterized by demonstrating a decrease in the adherence of mutant strains to a variety of human-derived epithelial cell lines, including A549 type II pneumocytes and Chang conjunctival, NCIH292 lung mucoepidermoid, HEp2 laryngeal, and 16HBE14o-polarized bronchial cells. Although all of these cell types are relevant to the diseases caused by M. catarrhalis, they lack important aspects of the pathogen-targeted mucosa, such as the features of cilia and mucociliary activity. The ciliated cells of the respiratory tract and other mucosal membranes keep secretions moving out of the body so as to assist in preventing colonization by invading microbial pathogens (10, 26, 71, 91). Given this critical role in host defense, it is interesting to note that a few bacterial pathogens target ciliated cells for adherence, including Actinobacillus pleuropneumoniae (32), Pseudomonas aeruginosa (38, 108), Mycoplasma pneumoniae (58), Mycoplasma hyopneumoniae (44, 45), and Bordetella species (5, 62, 85, 101).In the present study, M. catarrhalis is shown to specifically bind to ciliated cells of a normal human bronchial epithelium (NHBE) culture exhibiting mucociliary activity. This tropism was found to be conserved among isolates, and analysis of mutants revealed a direct role for the adhesin Hag in binding to ciliated airway cells.     

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.     

Lack of Insertional-Deletional Polymorphism in a Collection of Mycobacterium ulcerans Isolates from Ghanaian Buruli Ulcer Patients

Mycobacterium ulcerans causes the devastating infectious skin disease Buruli ulcer and has a monomorphic population structure. The resolution of conventional genetic fingerprinting methods is therefore not sufficient for microepidemiological studies aiming to characterize transmission pathways. In a previous comparative genomic hybridization analysis with a microarray covering part of the M. ulcerans genome, we have found extensive insertional-deletional sequence polymorphisms among M. ulcerans isolates of diverse geographic origins that allowed us to distinguish between strains coming from different continents. Since large numbers of insertion sequences are spread over the genome of African M. ulcerans strains, we reasoned that these may drive large sequence polymorphisms in otherwise clonal local mycobacterial populations. In this study, we used a printed DNA microarray covering the whole genome of the Ghanaian M. ulcerans reference strain Agy99 for comparative genomic hybridization. The assay identified multiple regions of difference when DNA of a Japanese M. ulcerans strain was analyzed. In contrast, not a single insertional-deletional genomic variation was found within a panel of disease isolates coming from an area of Ghana where Buruli ulcer is endemic. These results indicate that, despite the expectations deduced from other mycobacterial pathogens, only analyses of single nucleotide polymorphisms will have the potential to differentiate local populations of M. ulcerans.Buruli ulcer (BU) is a severe skin disease characterized by large ulcerative lesions, often leading to deformity and the crippling of extremities. While this neglected tropical disease occurs in more than 30 countries worldwide, it currently has its major focus in West Africa, where the prevalence is greater than that of leprosy, and incidence rates still may be underestimated (61). The social impact of BU endemicity in rural settings of Africa, where children are most affected, is dramatic. Patients often have only limited access to health care and tend to report to health care facilities at advanced stages of disease (20, 49). BU is often concentrated in areas close to stagnant or slow-moving waters. The mode of transmission is not fully understood (13, 36, 44-46, 59, 60), partly because conventional molecular fingerprinting methods have not sufficiently high resolution for microepidemiological analyses (1-4, 10, 11, 23, 24, 27, 33, 51, 53, 55-57).Mycobacterium tuberculosis and related pathogenic mycobacteria exhibit major large sequence polymorphism (LSP)-based interstrain genomic variations (5, 9, 12, 34, 58). From these genomic insertional-deletional (InDel) markers, evolutionary scenarios were drawn for M. tuberculosis, the M. tuberculosis complex, Mycobacterium bovis, the M. bovis BCG vaccine strains, and mycolactone-producing mycobacteria related to Mycobacterium ulcerans (6-8, 25, 27, 38, 50). In recent years, proposed phylogenies were confirmed and refined by whole-genome sequence analysis identifying single nucleotide polymorphisms (SNPs) (16, 17, 21, 22). Combinations of all identified genomic polymorphisms are currently being used for studying mycobacterial evolution and transmission pathways, helping in the prevention of epidemics (34, 37).Comparative genomic hybridization (CGH) analysis of M. ulcerans clinical isolates of diverse geographic origins also revealed extensive LSPs (47). The identified transposable element-associated InDel recombination events are indicative of progressive genome shrinking in M. ulcerans, which has emerged from the environmental mycobacterium Mycobacterium marinum by acquisition of a large virulence plasmid (52). An in-depth comparison of the recently published M. ulcerans genome of strain Agy99 to the progenitor species strain M. marinum M revealed LSPs, named M. ulcerans regions of difference (MURDs) (54). Analysis of large InDel polymorphisms allowed us to distinguish between two distinct lineages: (i) the “classical” lineage representing the most pathogenic genotypes, consisting of those that come from Africa, Australia, and Southeast Asia; and (ii) an “ancestral” lineage comprising strains from China and Japan, South America, and Mexico (29). A total of 209 copies of the insertion sequence (IS) IS2404 and 83 copies of IS2606 have been found interspersed in the genome of the M. ulcerans reference strain Agy99 from Ghana (54). Due to achievements using LSPs in other mycobacterial pathogens, we reasoned that this high abundance of transposable elements may destabilize the general architecture of the genome of African M. ulcerans strains, resulting in a rapid accumulation of LSPs in otherwise monomorphic populations of the pathogen. To assess this experimentally, we performed CGH analyses using a comprehensive whole-genome microarray. Specifically, we tried to identify LSPs within a panel of M. ulcerans strains isolated from the BU lesions of 37 patients from several districts in Ghana between the years 2002 and 2004.     

Biochip System for Rapid and Accurate Identification of Mycobacterial Species from Isolates and Sputum

The accurate detection of mycobacterial species from isolates and clinical samples is important for pathogenic diagnosis and treatment and for disease control. There is an urgent need for the development of a rapid, simple, and accurate detection method. We established a biochip assay system, including a biochip, sample preparation apparatus, hybridization instrument, chip washing machine, and laser confocal scanner equipped with interpretation software for automatic diagnosis. The biochip simultaneously identified 17 common mycobacterial species by targeting the differences in the 16S rRNA. The system was assessed with 64 reference strains and 296 Mycobacterium tuberculosis and 243 nontuberculous mycobacterial isolates, as well as 138 other bacteria and 195 sputum samples, and then compared to DNA sequencing. The entire biochip assay took 6 h. The concordance rate between the biochip assay and the DNA sequencing results was 100%. In conclusion, the biochip system provides a simple, rapid, reliable, and highly accurate clinical assay for determination of mycobacterial species in a 6-h procedure, from either culture isolates or sputum samples, allowing earlier pathogen-adapted antimicrobial therapy in patients.Mycobacterium tuberculosis is an important pathogen; it is responsible for 1.3 to 1.7 million mortalities worldwide in 2007 (26). However, the incidence of opportunistic infections by nontuberculous mycobacteria (NTM) has gradually increased, causing a number of different NTM diseases (4, 5, 14). This apparent increase is thought to be the consequence of several factors; the increased use of diagnostic methods that can identify these agents has contributed to this number, while the AIDS epidemic is a principal cause (18). A report on a nationwide random survey in China on the epidemiology of tuberculosis in 2000 revealed that 11.1% of the 441 bacterial strains isolated from patient sputum were NTM (15). The low level of identification of NTM is partially due to the use of conventional methods, which rely on growth characteristics, colony morphology, pigment production, and biochemical tests. Because of the slow growth of mycobacteria, the methods are time-consuming, taking 4 to 8 weeks to complete. Extensive experience in the interpretation of the results of biochemical tests is also required, and the identity of specific species from cultures is often difficult to determine, resulting in possible misidentification. As a result of these numerous limitations, conventional methods are not widely used by the majority of clinical laboratories in China. However, the correct identification of NTM is clinically important because most NTM are naturally resistant to first-line antituberculosis drugs, and different species of NTM are sensitive to different antibiotics drugs. Thus, there is an urgent need for the development of a rapid, simple, and accurate method for mycobacterium species identification (21).Recently, different nucleic acid based molecular assays such as DNA sequencing (1), microarray assay (6, 16, 25), PCR-restriction fragment length polymorphism (RFLP) assays (23), and commercial kits such as Accuprobe (Gen-Probe, San Diego, CA) (2), GenoType (Hain, Germany), (19, 20), and INNO-LiPA (Innogenetics, Belgium) (24), have emerged as rapid tools for species identification. The molecular identification methods are based on the polymorphisms in the 16S rRNA (1) or 16S-23S rRNA spacer regions (24) or 23S rRNA (20) or hsp65 genes (12). The 16S rRNA gene sequences of most mycobacterial species are well known and can be found in online databases. Although the molecular methods mentioned above have greatly improved the diagnosis of NTM, there is still a need for a quick, semiautomated or fully automated, total solution-based system to meet the high-throughput demands of busy clinics, particularly for large centers with numerous patients.We report here a rapid diagnosis solution that includes a biochip, apparatus for sample preparation, chip hybridization, washing and data acquisition, and dedicated software for automated diagnosis. The biochip is designed to detect differences in 16S rRNA sequences for mycobacteria species identification. The entire process is semiautomatic and high throughput. The automated determination of species could also eliminate some elements of operator error.     

Mannheimia haemolytica and Its Leukotoxin Cause Neutrophil Extracellular Trap Formation by Bovine Neutrophils

Mannheimia haemolytica is an important member of the bovine respiratory disease complex, which is characterized by abundant neutrophil infiltration into the alveoli and fibrin deposition. Recently several authors have reported that human neutrophils release neutrophil extracellular traps (NETs), which are protein-studded DNA matrices capable of trapping and killing pathogens. Here, we demonstrate that the leukotoxin (LKT) of M. haemolytica causes NET formation by bovine neutrophils in a CD18-dependent manner. Using an unacylated, noncytotoxic pro-LKT produced by an ΔlktC mutant of M. haemolytica, we show that binding of unacylated pro-LKT stimulates NET formation despite a lack of cytotoxicity. Inhibition of LKT binding to the CD18 chain of lymphocyte function-associated antigen 1 (LFA-1) on bovine neutrophils reduced NET formation in response to LKT or M. haemolytica cells. Further investigation revealed that NETs formed in response to M. haemolytica are capable of trapping and killing a portion of the bacterial cells. NET formation was confirmed by confocal microscopy and by scanning and transmission electron microscopy. Prior exposure of bovine neutrophils to LKT enhanced subsequent trapping and killing of M. haemolytica cells in bovine NETs. Understanding NET formation in response to M. haemolytica and its LKT provides a new perspective on how neutrophils contribute to the pathogenesis of bovine respiratory disease.Mannheimia haemolytica is a member of the bovine respiratory disease complex (BRD), causing a severe fibrinous pleuropneumonia sometimes referred to as shipping fever. The pneumonia is characterized by intense neutrophil infiltration in alveoli, intra-alveolar hemorrhage, fibrin deposition, and consolidation of the lungs (42, 56). The importance of neutrophils in the production of inflammatory mediators, recruitment of other leukocytes, and lung damage (17, 56, 67, 74) was demonstrated in calves that were depleted of neutrophils before challenge with M. haemolytica (10, 56). Neutrophil-depleted calves displayed less lung pathology than did control calves infected with M. haemolytica (10, 56). From these data, it is clear that neutrophils are a key player in the pathology of bovine pleuropneumonia; however, the mechanisms by which they contribute to host defense and tissue destruction are not clearly defined.The most important virulence factor for M. haemolytica is its leukotoxin (LKT), a 104-kDa exotoxin produced during logarithmic-phase growth (18, 32). LKT is a member of the repeats-in-toxin (RTX) toxin family of exoproteins produced by a wide variety of Gram-negative bacteria, including Escherichia coli, Actinobacillus pleuoropneumoniae, and Aggregatibacter actinomycetemcomitans (70). RTX toxins are characterized by a C-terminal glycine-rich nonapeptide repeat region (-G-G-X-G-X-D-X-U-X, where U is a hydrophobic residue) that binds calcium (Ca²⁺). The latter is required for membrane binding and cytotoxicity (30, 70). RTX toxins can insert into the plasma membrane of target cells, causing lysis and necrotic cell death (30, 70). The N-terminal domain contains amphipathic and hydrophobic domains believed to be required for pore stabilization and formation, respectively (70). More recently, it was shown that LKT also causes apoptosis via a caspase 9-dependent pathway and that LKT is internalized and transported via the cytoskeleton to mitochondria (4-6).The leukotoxin operon contains the genes lktC, lktA, lktB, and lktD (36, 37, 58). lktA encodes the inactive pro-LKT protein that is not cytotoxic until acylated (62) by the transacylase encoded by lktC. lktB and lktD encode proteins responsible for leader sequence-independent secretion of LKT from the bacterial cell (36, 37, 58). The acylated LKT then binds the CD18 chain of the β₂-integrin lymphocyte function-associated antigen 1 (LFA-1) (3, 21-26, 33, 40, 41, 44, 55, 63) on ruminant leukocytes. LKT binding to amino acids 5 to 17 of the signal sequence of CD18 is required for cell death and restricts cytotoxicity to ruminant leukocytes, because the signal sequence for CD18 is not present on mature leukocytes from other mammalian species (55). Other investigators have shown that both the pro- form and mature LKT are capable of binding CD18, although the pro-LKT does not cause cytotoxicity (62). No biological role has been assigned to the pro- form of LKT.Recently, several authors have shown that human neutrophils are able to undergo a form of cell death, called NETosis, that is distinct from apoptosis and necrosis (12, 13, 31, 51, 69). NETosis is defined as the release of nuclear DNA from an activated neutrophil into the extracellular environment, with little concomitant release of lactate dehydrogenase (LDH) (12). The extracellular DNA and associated proteins (e.g., histones) released by activated neutrophils have been termed neutrophil extracellular traps (NETs) (12). There are four steps leading to NET formation. These are neutrophil activation, nuclear envelope degradation, mixing of nuclear DNA with cytosolic proteins, and extrusion of the DNA-protein mixture from the cell (31). Treatment of human neutrophils with interleukin-8 (IL-8), phorbol 12-myristate 13-acetate (PMA), or lipopolysaccharide (LPS) causes NET formation (12, 31, 69). NET formation also occurs in response to prokaryotic and eukaryotic pathogens (12, 35, 64). To date, no bacterial exotoxin has been shown to cause NET formation.NETs are composed of extracellular DNA that is studded with antimicrobial proteins. The latter include nuclear histones and primary, secondary, and tertiary granular components such as neutrophil elastase, myeloperoxidase, lactoferrin, and gelatinase (51, 69). When neutrophils become activated and commit to NET formation, they also are capable of trapping and killing pathogens. To date, NETs have been shown to kill a variety of Gram-negative and Gram-positive bacteria, fungi, and protozoans (2, 7-9, 12, 13, 15, 19, 20, 27, 28, 31, 34, 35, 43, 50-53, 59, 64, 67, 70). Here, we examine if M. haemolytica and its LKT cause NET formation by bovine neutrophils and whether NETs are capable of trapping and killing M. haemolytica cells in vitro.     

Acute Immune Response to Mycobacterium massiliense in C57BL/6 and BALB/c Mice

Mycobacterium massiliense is an environmental opportunistic pathogen that has been associated with soft tissue infection after minor surgery. We studied the acute immune response of C57BL/6 and BALB/c mice infected intravenously with 10⁶ CFU of an M. massiliense strain isolated from a nosocomial infection in Brazil. The results presented here show that M. massiliense is virulent and pathogenic to both C57BL/6 and BALB/c mice, inducing a granulomatous inflammatory reaction that involves the activation of macrophages, dendritic cells, and natural killer cells induced by gamma interferon and interleukin-17 (IL-17) in C57BL/6 mice and by IL-12 in BALB/c mice.Mycobacteria that do not belong to the complex Mycobacterium tuberculosis are known as nontuberculous mycobacteria (NTM) or atypical mycobacteria. NTM are ubiquitous microorganisms found worldwide in soil and water (3, 23, 38). These environmental mycobacteria are considered emerging and environmental opportunistic pathogens (6, 23).Mycobacterium massiliense is an environmental nonphotochromogenic, rapidly growing Mycobacterium strain that has been associated with soft-tissue infection after minor surgery or intramuscular injection (3, 5, 17, 22, 26, 46) and with pulmonary infection due to diseases, such as cystic fibrosis (29, 41). This species differs only slightly from Mycobacterium abscessus, sharing a 99.6% sequence identity of their 16S rRNA genes; genetic differences can be observed by comparative sequence analysis of the rpoB and hsp65 genes (1, 25, 42). Infections with these agents tend to respond poorly to macrolide-based chemotherapy (3), even though the organisms are susceptible to clarithromycin (15, 44, 47).M. massiliense infection mainly affects immunocompetent individuals and occasionally is associated with disseminated disease (8). An outbreak of M. massiliense occurred in Goiania, Brazil, where 30 individuals were infected after undergoing knee joint and laparoscopic surgery (5). Despite the fact that the infected individuals were from different hospitals, a unique M. massiliense strain was identified and characterized by pulsed-field gel electrophoresis.Disease pathogenesis involves host-pathogen interactions that directly affect parasite clearance. Typically, when environmental bacteria are passively introduced into the host, rapid bacterial clearance occurs due to an efficient innate immune response (30). Nonetheless, accidental infections with M. massiliense have been described as having a chronic evolution and, in some cases, the disease is disseminated irrespective of the host''s immune status. Such findings raise the possibility that this species is more virulent and/or pathogenic than other environmental mycobacteria, such as M. chelonae and M. abscessus.Recently, a murine model of M. abscessus infection was described, and isogenic mice were shown to be good models to address the immune response of the host (34, 39). In the present study, we analyzed the immune response of C57BL/6 and BALB/c mice infected with a clinical isolate of M. massiliense obtained from the recent outbreak in Goiania, Brazil. We show here that M. massiliense is virulent and pathogenic to both C57BL/6 and BALB/c mice, inducing a granulomatous inflammatory reaction that involves the activation of macrophages, dendritic cells (DCs), and natural killer (NK) cells induced mainly by gamma interferon (IFN-γ) and interleukin-17 (IL-17) in C57BL/6 mice and by IL-12 in BALB/c mice.     

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.     

Acanthamoeba culbertsoni Elicits Soluble Factors That Exert Anti-Microglial Cell Activity

Acanthamoeba culbertsoni is an opportunistic pathogen that causes granulomatous amoebic encephalitis (GAE), a chronic and often fatal disease of the central nervous system (CNS). A hallmark of GAE is the formation of granulomas around the amoebae. These cellular aggregates consist of microglia, macrophages, lymphocytes, and neutrophils, which produce a myriad of proinflammatory soluble factors. In the present study, it is demonstrated that A. culbertsoni secretes serine peptidases that degrade chemokines and cytokines produced by a mouse microglial cell line (BV-2 cells). Furthermore, soluble factors present in cocultures of A. culbertsoni and BV-2 cells, as well as in cocultures of A. culbertsoni and primary neonatal rat cerebral cortex microglia, induced apoptosis of these macrophage-like cells. Collectively, the results indicate that A. culbertsoni can apply a multiplicity of cell contact-independent modes to target macrophage-like cells that exert antiamoeba activities in the CNS.Acanthamoeba culbertsoni belongs to a group of free-living amoebae, such as Balamuthia mandrillaris, Naegleria fowleri, and Sappinia pedata, that can cause disease in humans (46, 56). Acanthamoeba spp. are found worldwide and have been isolated from a variety of environmental sources, including air, soil, dust, tap water, freshwater, seawater, swimming pools, air conditioning units, and contaminated contact lenses (30). Trophozoites feed on bacteria and algae and represent the infective form (47, 56). However, under unfavorable environmental conditions, such as extreme changes in temperature or pH, trophozoites transform into a double-walled, round cyst (22, 45).Acanthamoeba spp. cause an infection of the eye known as amoebic keratitis (AK), an infection of the skin referred to as cutaneous acanthamoebiasis, and a chronic and slowly progressing disease of the central nervous system (CNS) known as granulomatous amoebic encephalitis (GAE) (22, 23, 30, 56). GAE is most prevalent in humans who are immunocompromised (30, 33, 40) and has been reported to occur among individuals infected with the human immunodeficiency virus (HIV) (28). It has been proposed that Acanthamoeba trophozoites access the CNS by passage through the olfactory neuroepithelium (32) or by hematogenous spread from a primary nonneuronal site of infection (23, 24, 33, 53).In immune-competent individuals, GAE is characterized by the formation of granulomas. These cellular aggregates consist of microglia, macrophages, polymorphonuclear cells, T lymphocytes, and B lymphocytes (24, 30). The concerted action of these immune cells results in sequestration of amoebae and is instrumental in slowing the progression of GAE. This outcome is consistent with the observation that granulomas are rarely observed in immunocompromised individuals (34) and in mice with experimentally induced immune suppression following treatment with the cannabinoid delta-9-tetrahydrocannabinol (Δ⁹-THC) (8).Microglia are a resident population of macrophages in the CNS. These cells, along with CNS-invading peripheral macrophages, appear to play a critical early effector role in the control of Acanthamoeba spread during GAE (4, 5, 29, 31). In vitro, microglia have been shown to produce an array of chemokines and cytokines in response to Acanthamoeba (31, 51). However, these factors appear not to have a deleterious effect on these amoebae (29).Acanthamoeba spp. produce serine peptidases, cysteine peptidases, and metallopeptidases (1, 2, 9, 10, 14, 16, 18, 19, 21, 25, 26, 37, 38, 41, 42, 52). In the present study, it is demonstrated that serine peptidases secreted by A. culbertsoni degrade chemokines and cytokines that are produced by immortalized mouse BV-2 microglia-like cells. In addition, soluble factors present in cocultures of A. culbertsoni and BV-2 cells induced apoptosis of the BV-2 cells. Collectively, these results suggest a mode through which A. culbertsoni can evade immune responsiveness in the CNS.     

Kinetics of a Tuberculosis-Specific Gamma Interferon Release Assay in Military Personnel with a Positive Tuberculin Skin Test

Treatment of latent Mycobacterium tuberculosis infection on the basis of the tuberculin skin test (TST) result is inaccurate due to the false-positive TST results that occur after Mycobacterium bovis BCG vaccination or exposure to nontuberculous mycobacteria (NTM). Gamma interferon release assays (IGRAs) are based on M. tuberculosis-specific antigens. In a previous study among BCG-naïve military employees, a positive TST result after deployment was mostly associated with a negative IGRA result, suggesting exposure to NTM. Data regarding the kinetics of IGRAs are limited and controversial. The present study aimed to reassess the rate of false-positive TST results and to evaluate the kinetics of the Quantiferon TB Gold In-Tube assay (QFT-Git) in military personnel with a positive TST result. QFT-Git was performed at the time of inclusion in the study and was repeated after 2, 6, 12, and 18 or 24 months. Of 192 participants, 17 were recruits and 175 were screened after deployment (n = 169) or because of travel or health care work. Baseline positive QFT-Git results were observed in 7/17 (41.2%) and 12/174 (6.9%) participants, respectively. During follow-up, a negative QFT-Git result remained negative in 163/165 (98.8%) participants. Of 18 subjects with an initial positive QFT-Git result, reversion to a negative result occurred in 1/6 (16%) recruits, whereas it occurred in 8/12 (66%) subjects after deployment or with other risk factors (P = 0.046). The quantitative result was significantly lower in subjects with reversion than in those with consistent positive results (P = 0.017). This study confirmed a low rate of positive QFT-Git results among military personnel with a positive TST result after deployment, supporting the hypothesis of exposure to NTM. Reversion of the majority of initially low-positive QFT-Git results indicates that QFT-Git may be useful for the diagnosis of later reinfections.Each year, about 3,000 Dutch army personnel are deployed to regions where tuberculosis (TB) is highly endemic. Screening of military personnel for latent Mycobacterium tuberculosis infection (LTBI) has thus far been based on the tuberculin skin test (TST). The Netherlands is a country with a low prevalence of TB, with a yearly incidence of 5.9 cases/100,000 population in 2007, only one-third of which occurred among native Dutch persons (Tuberculosis in The Netherlands 2007 [www.kncvtbc.nl]). Personnel are screened by the TST upon initial recruitment into the army, after deployment, or in the presence of other risk factors for TB exposure. Military personnel with TST conversion are prescribed isoniazid for 6 months to prevent TB disease. The risk of progression from untreated LTBI to active TB is generally believed to be about 10%, with half of the cases occurring within 2 years after infection. However, the risks observed in different studies comparing subjects treated with isoniazid or placebo varied widely, depending on the setting and the characteristics of the study population (38). A major disadvantage of the current policy is that a substantial proportion of TST conversions in this setting are thought to be caused by exposure to nontuberculous mycobacteria (NTM), skewing the risk-benefit ratio of preventive treatment (8). In addition, increasing proportions of the Dutch population and Dutch military recruits originate from countries where M. bovis BCG vaccination is routinely used. In BCG-vaccinated Dutch military personnel or those with a previous positive TST result, TST is not performed, as a rule, and chest radiography is used as an alternative, but radiography lacks sensitivity for the detection of LTBI. Finally, a positive TST result often remains positive, thus precluding the detection of reinfection.In order to overcome the disadvantages of the TST, gamma interferon (IFN-γ) release assays (IGRAs) that use M. tuberculosis-specific antigens and that are not affected by BCG and most NTM were developed (2-4, 32, 34). In contact investigations, the results of IGRAs had a better correlation with measures of exposure (5, 19, 21, 43). Since 2005, IGRAs have increasingly been used for the detection of LTBIs either as a replacement of or as adjunct to the TST (18, 26, 28). Of the two presently commercially available IGRAs, the Quantiferon TB Gold In-Tube assay (QFT-Git) is a robust whole-blood-based test suitable for use for large-scale testing (5, 7, 22). In a previous study, QFT-Git was positive for only a minority of military personnel with a positive TST result after deployment (13). Those results were considered to be related to NTM exposure, in accordance with the high proportion of false-positive TST responses assessed by dual skin testing of army recruits by the use of tuberculin and atypical sensitin (8). The destinations of deployment at the time of the earlier study were mainly Iraq and Bosnia (13), but the destination has changed to Afghanistan in the past few years. As the incidence of TB is higher in Afghanistan than in most of the countries where the military personnel were deployed, in order to justify a change in treatment policy, the previously observed low rate of positive IGRA results in association with a positive TST result needed to be studied in the current setting. In the previous study (13), QFT-Git was performed only once, and the subjects were not assessed for eventual later conversion or reversion. Previous studies of the kinetics of IGRAs gave variable and partly conflicting results (9, 12, 14, 15, 17, 20, 29, 30, 35-37, 40), although the main trend was for high-positive results to usually remain positive, and reversion can occur when the results are low or moderately positive (12, 15, 20, 29, 30, 33, 35-37, 42). In a setting of LTBIs, the relevance of follow-up testing by IGRAs may lie in the possibility of detecting later reinfection if reversion to a negative result has been documented.The aims of this study were to study the kinetics of QFT-Git during at least 6 months of follow-up in order to evaluate the possibility of detection of later reinfection and to confirm the previously observed very low rate of positive QFT-Git results in military personnel with a positive TST result after deployment.     

Rapid Identification of Mycobacterium tuberculosis and Nontuberculous Mycobacteria by Multiplex,Real-Time PCR

The rapid identification of mycobacteria from culture is of primary importance for the administration of empirical antibiotic therapy and for the implementation of public health measures, yet there are few commercially available assays that can easily and accurately identify the mycobacteria in culture in a timely manner. Here we report on the development of a multiplex, real-time PCR assay that can identify 93% of the pathogenic mycobacteria in our laboratory in two parallel reactions. The mycobacteria identified by this assay include the Mycobacterium tuberculosis complex (MTC), the M. avium complex (MAC), the M. chelonae-M. abscessus group (MCAG), the M. fortuitum group (MFG), and M. mucogenicum. The primer targets included the 16S rRNA gene and the internal transcribed spacer. The assay was initially validated with a repository of reference strains and was subsequently tested with 314 clinical cultures identified by the AccuProbe assay or high-performance liquid chromatography. Of the 314 cultures tested, multiplex, real-time PCR produced congruent results for 99.8% of the 1,559 targets evaluated. The sensitivity and the specificity were each 99% or greater for MTC (n = 96), MAC (n = 97), MCAG (n = 68), and M. mucogenicum (n = 9) and 95% and 100%, respectively, for MFG (n = 19). We conclude that this multiplex, real-time PCR assay is a useful diagnostic tool for the rapid and accurate identification of MTC and clinically relevant nontuberculous mycobacteria.Mycobacterium tuberculosis, the causative agent of tuberculosis, is among the leading infectious causes of death in developing nations (23). In resource-rich countries, where tuberculosis is not endemic, nontuberculous mycobacteria (NTM) are responsible for the majority of mycobacterial infections in both immunocompromised and immunocompetent individuals (14). The clinical gravity of mycobacterial infections necessitates rapid isolation by culture and timely identification by the appropriate diagnostic assays. Rapid identification not only serves to focus empirical antibiotic therapy and thus avoid unnecessary drug exposure but also may aid with the appropriate respiratory isolation and prevention of secondary cases (15).Few molecular methods commercially available in the United States easily and rapidly identify mycobacteria in culture. Although the AccuProbe assay by Gen-Probe (San Diego, CA) is sufficient for the identification of isolates of the Mycobacterium tuberculosis complex (MTC) and the M. avium complex (MAC) (37), it lacks probe sets specific for rapidly growing mycobacteria, such as the M. chelonae-M. abscessus group (MCAG) and the M. fortuitum group (MFG), which make up approximately 30% of the pathogenic mycobacteria in the Clinical Microbiology Laboratory of Stanford Hospital (Stanford, CA). MCAG consists of M. chelonae, M. abscessus, and M. immunogenum; MFG consists of M. fortuitum, M. senegalense, M. farcinogenes, M. porcinum, M. septicum, M. peregrinum, and M. alvei (11). For these isolates, other methods, such as biochemical reactions (6, 33), high-performance liquid chromatography (HPLC) (13), and DNA sequencing (17), are required for identification. To address the need for a simple and rapid molecular assay with a broader identification scope, we developed a multiplex, real-time PCR assay that can identify 93% of the pathogenic mycobacterial isolates (both slowly growing and rapidly growing) recovered in the Clinical Microbiology Laboratory of Stanford Hospital. This assay simplifies the identification of MTC and MAC and also accommodates the identification of rapidly growing mycobacteria. Although conventional PCR-based and singleplex, real-time PCR assays for the identification of MTC and NTM have previously been reported (3, 7, 18, 19, 25, 29, 30, 32, 35), multiplex, real-time PCR assays with simple interpretative criteria have not been developed. A multiplex, real-time PCR assay for the identification of 18 mycobacterial species, including MTC and rapidly growing mycobacteria, was recently described (21); however, the complex nature of this assay may preclude its use in the routine laboratory. The only simple application of PCR for the identification of rapidly growing mycobacteria consists of PCR-restriction analysis (8, 36). Multiplex, real-time PCR has several crucial advantages over conventional PCR. Besides the ease of use and rapid availability of results, it also eliminates the need for postamplification handling and thus the potential contamination of the laboratory.Here we describe the development and validation of a multiplex, real-time PCR assay for the simple identification of MTC and clinically relevant NTM.     

Rapid Mycobacterial Liquid Culture-Screening Method for Mycobacterium avium Complex Based on Secreted Antigen-Capture Enzyme-Linked Immunosorbent Assay

Sensors in automated liquid culture systems for mycobacteria, such as MGIT, BacT/Alert 3D, and Trek ESP II, flag growth of any type of bacteria; a positive signal does not mean that the target mycobacteria are present. All signal-positive cultures thus require additional and often laborious testing. An immunoassay was developed to screen liquid mycobacterial cultures for evidence of Mycobacterium avium complex (MAC). The method, called the MAC-enzyme-linked immunosorbent assay (ELISA), relies on detection of MAC-specific secreted antigens in liquid culture. Secreted MAC antigens were captured by the MAC-ELISA with polyclonal anti- Mycobacterium avium subsp. paratuberculosis chicken immunoglobulin Y (IgY), detected using rabbit anti-MAC IgG, and then revealed using horseradish peroxidase-conjugated goat anti-rabbit IgG. When the MAC-ELISA was evaluated using pure cultures of known mycobacterial (n = 75) and nonmycobacterial (n = 17) organisms, no false-positive or false-negative MAC-ELISA results were found. By receiver operator characteristic (ROC) analysis of 1,275 previously identified clinical isolates, at the assay optimal cutoff the diagnostic sensitivity and specificity of the MAC-ELISA were 92.6% (95% confidence interval [95% CI], 90.3 to 94.5) and 99.9% (95% CI, 99.2 to 100), respectively, with an area under the ROC curve of 0.992. Prospective evaluation of the MAC-ELISA with an additional 652 clinical samples inoculated into MGIT ParaTB medium and signaling positive per the manufacturer''s instructions found that the MAC-ELISA was effective in determining those cultures that actually contained MAC species and warranting the resources required to identify the organism by PCR. Of these 652 MGIT-positive cultures, the MAC-ELISA correctly identified 96.8% (of 219 MAC-ELISA-positive cultures) as truly containing MAC mycobacteria, based on PCR or high-performance liquid chromatography (HPLC) as reference tests. Only 6 of 433 MGIT signal-positive cultures (1.4%) were MAC-ELISA false negative, and only 7 of 219 MGIT signal-negative cultures (3.2%) were false positive. The MAC-ELISA is a low-cost, rapid, sensitive, and specific test for MAC in liquid cultures. It could be used in conjunction with or independent of automated culture reading instrumentation. For maximal accuracy and subspecies-specific identification, use of a confirmatory multiplex MAC PCR is recommended.Members of the Mycobacterium avium complex (MAC) are a family of intracellular bacterial pathogens causing significant disease in both animals and humans. The complex contains four subspecies of M. avium: M. avium subsp. avium, M. avium subsp. paratuberculosis, M. avium subsp. hominissuis, and M. avium subsp. silvaticum (24, 35). Mycobacterium intracellulare is also a member of the complex (20, 35).The clinical importance of MAC infection has increased in recent decades because of the greater population of immunocompromised individuals with longer life expectancies, immunosuppressive chemotherapy, and the spread of human immunodeficiency virus infection (8, 20, 25, 27). With AIDS patients, the incidence of disseminated mycobacterial infection caused by MAC strains can reach up to 50% (19). Although these mycobacterial infections are not often characterized to subspecies, it appears that M. avium subsp. hominissuis is most often involved with AIDS patients (3, 4, 18, 24, 35). In addition, M. avium subsp. hominissuis causes infection in a subset of patients without an obvious immune defect (13) or underlying pulmonary disease and in children with lymphadenitis or cystic fibrosis (31). In virtually all cases, these organisms are believed to be of environmental origin: surface water, tap water, soil, dust, or food (22, 24, 29, 38). M. avium subsp. avium, ubiquitous in the environment and more virulent than M. avium subsp. hominissuis, is distinguished by the insertion element IS901 (24). While capable of infecting multiple animal species, M. avium subsp. avium is commonly isolated from birds as one of the causes of avian tuberculosis (26, 32). M. avium subsp. silvaticum, also called the “wood pigeon bacillus,” is uncommonly isolated but reported to cause enteritis in ruminants as well as disseminated infection in other hosts (33).M. avium subsp. paratuberculosis infection causes paratuberculosis (Johne''s disease) characterized by chronic granulomatous enteritis in animals, most often ruminants (9, 21). This organism grows very slowly in vitro (slower than most “slow-growing” mycobacteria), is dependent on mycobactin for growth in vitro, and is alone in containing IS900 in its genome (15, 16, 23). M. avium subsp. paratuberculosis has a broad host range and is implicated by some in the pathogenesis of Crohn''s disease in humans (1, 12). The inability of M. avium subsp. paratuberculosis to produce the siderophore mycobactin renders it incapable of replication in the environment, with the possible exception of inside free-living amoeba, and so it is considered an obligate parasite of animals and possibly humans (6). Paratuberculosis has emerged as a common and costly disease for the dairy industry (16). Surveys indicate that at least 68% of U.S. dairy herds are M. avium subsp. paratuberculosis infected (36).Microbiological culture remains a mainstay for diagnosis of mycobacterial infections, since it has greater sensitivity than PCR-based methods and yields the living isolates necessary for antibiotic susceptibility testing and molecular epidemiology. Because culture on conventional solid bacteriological media is laborious and slow, liquid culture-based mycobacterial detection systems, such the Bactec, MGIT, Trek ESP, and BacT/Alert 3D systems, have become commonplace in clinical laboratories, offering the advantages of automation and shorter detection times from clinical samples (5, 7, 17, 37). However, a positive signal during culture with any of these systems is simply a nonspecific indication of any sort of microbial growth (37). Thus, specimen processing and decontamination protocols to selectively kill nonmycobacterial microflora in the clinical or environmental samples are key components for an effective assay (7, 34). Although a number of different protocols have been described (7, 11, 28, 34), a standard protocol specifically designed for optimal recovery of MAC has not yet been established.Numerous PCRs are performed in our laboratory in response to these signal-positive cultures; in the last year, approximately 45% did not contain the pathogen of interest, MAC (unpublished data). This sample management approach is inefficient and labor-intensive.To better focus PCR resources on those cultures most likely to contain MAC, a novel enzyme-linked immunosorbent assay (ELISA) was designed to detect secreted MAC antigens in culture medium fluid. This assay, called the MAC-ELISA, was then evaluated for analytical and diagnostic specificity and sensitivity, first using pure cultures and then cultures derived from clinical samples.     

Amino Acid Changes in Elongation Factor Tu of Mycoplasma pneumoniae and Mycoplasma genitalium Influence Fibronectin Binding

Mycoplasma pneumoniae and Mycoplasma genitalium are closely related organisms that cause distinct clinical manifestations and possess different tissue predilections despite their high degree of genome homology. We reported earlier that surface-localized M. pneumoniae elongation factor Tu (EF-Tu_Mp) mediates binding to the extracellular matrix component fibronectin (Fn) through the carboxyl region of EF-Tu. In this study, we demonstrate that surface-associated M. genitalium EF-Tu (EF-Tu_Mg), in spite of sharing 96% identity with EF-Tu_Mp, does not bind Fn. We utilized this finding to identify the essential amino acids of EF-Tu_Mp that mediate Fn interactions by generating modified recombinant EF-Tu proteins with amino acid changes corresponding to those of EF-Tu_Mg. Amino acid changes in serine 343, proline 345, and threonine 357 were sufficient to significantly reduce the Fn binding of EF-Tu_Mp. Synthetic peptides corresponding to this region of EF-Tu_Mp (EF-Tu_Mp 340-358) blocked both recombinant EF-Tu_Mp and radiolabeled M. pneumoniae cell binding to Fn. In contrast, EF-Tu_Mg 340-358 peptides exhibited minimal blocking activity, reinforcing the specificity of EF-Tu-Fn interactions as mediators of microbial colonization and tissue tropism.Many pathogens express surface proteins that facilitate colonization and cellular invasion (12, 39, 44, 49, 55). The human mycoplasmas, Mycoplasma pneumoniae and Mycoplasma genitalium, have genome sizes of 816,394 bp (20) and 580,070 bp (12), respectively, with the latter considered the smallest self-replicating biological cell (14, 38). These bacterial pathogens possess terminal tip-like structures comprised of specific membrane adhesins and adherence-related accessory proteins that mediate surface parasitism of target cells (5) and are essential for virulence (4). While adherence of virulent M. pneumoniae is mediated primarily by tip organelle-associated adhesins (10, 24), the absence of these proteins in hemadsorption-negative mutants (HA⁻ class II mutants) (17) still permits detectable adherence (18), suggesting the involvement of alternative mechanisms by which mycoplasmas bind to host cells.Recently, we showed that M. pneumoniae surface-associated elongation factor Tu (EF-Tu_Mp; MPN665) and the pyruvate dehydrogenase E1 beta subunit (MPN392) interact with fibronectin (Fn) (11). In addition, we demonstrated that HA⁻ class II mutants also bind Fn through EF-Tu (11). Fn is an abundantly available pathogen target (22) that exists in soluble form in blood fluids and plasma and in fibrillar form in the extracellular matrix (56). M. pneumoniae could readily access the extracellular matrix through virulence-related determinants following epithelial cell damage (29) and could directly bind to subepithelial tissue targets through EF-Tu interactions with Fn. Furthermore, these distinct pathogenic pathways may also contribute to the ability of M. pneumoniae to invade and to establish intracellular and perinuclear residence (9, 57).Detailed analyses of EF-Tu_Mp-Fn interactions revealed the critical role of the carboxyl region of EF-Tu (amino acids 192 to 219 and 314 to 394) in Fn recognition (3). Other mycoplasmas with tip organelles, such as Mycoplasma penetrans and Mycoplasma gallisepticum, have been reported to bind Fn through a 65-kDa protein (13) and the PlpA and Hlp3 proteins (34).Following our initial findings of EF-Tu_Mp-Fn interactions, surface-associated EF-Tu proteins from other microorganisms, including Lactobacillus johnsonii, Listeria monocytogenes, and Pseudomonas aeruginosa, were reported to bind mucin (16), fibrinogen (43), plasminogen, and factor H (32). Since EF-Tu is one of the most highly conserved proteins in mycoplasmas, it has been used to create an EF-Tu sequence-based mycoplasma phylogeny tree. This allows the classification of the human pathogens, M. genitalium and M. pneumoniae, along with M. gallisepticum, a poultry pathogen, in the same group (28). M. pneumoniae is an established pathogen of the respiratory tract (54) but has also been isolated from the urogenital tract (15). M. genitalium, an emerging sexually transmitted disease pathogen (27, 51), has also been associated with respiratory (6) and joint (50) pathologies. It has been suggested that the tissue-specific tropisms and pathogenic mechanisms of these two mycoplasmas are determined by genetic distinctions between them (19). Most of the open reading frames proposed for M. genitalium are present in M. pneumoniae. Overall, M. pneumoniae and M. genitalium share 67.4% average identity at the amino acid level, while conserved housekeeping proteins exhibit 70 to 97% identity (19). Among the latter proteins, EF-Tu displays a high sequence identity (96%).In this study, we compared EF-Tu-Fn binding between M. pneumoniae and M. genitalium and discovered biological and biochemical differences that facilitated the identification of key amino acids responsible for these interactions. Such distinctions provide evidence of unique colonization capabilities of these bacteria.     

Use of Moraxella catarrhalis Lipooligosaccharide Mutants To Identify Specific Oligosaccharide Epitopes Recognized by Human Serum Antibodies

Moraxella catarrhalis is a causative agent of otitis media in children and lower respiratory tract infections in adults suffering from chronic obstructive pulmonary disease (COPD). This strict human pathogen continues to be a significant cause of disease in this broad spectrum of patients because there is no available vaccine. Although numerous putative vaccine antigens have been described, little is known about the human immune response to M. catarrhalis infection in vivo. Human serum antibodies are directed at a number of surface proteins, and lipooligosaccharides (LOS) and detoxified LOS may be an effective immunogen in mice. In this study, we used a specific LOS-based enzyme-linked immunosorbent assay (ELISA), containing the three major M. catarrhalis serotypes together with a complete series of truncated LOS mutants, to detect the development of new antibodies to specific regions of the oligosaccharide molecule. We compared serum samples from COPD patients who had recently cleared an M. catarrhalis infection to serum samples collected prior to their infection. Variability in the antibody response to LOS was observed, as some patients developed serotype-specific antibodies, others developed antibodies to the LOS of each serotype, others developed broadly cross-reactive antibodies, and some did not develop new antibodies. These newly developed human antibodies are directed at both side chains and core structures in the LOS molecule. This LOS-based ELISA can be used to dissect the human antibody response to both internal and external carbohydrate epitopes, thus providing a better understanding of the humoral immune response to M. catarrhalis LOS epitopes developed during natural infection.Chronic obstructive pulmonary disease (COPD) is currently the fourth leading cause of death in the United States and Europe. The morbidity and mortality associated with exacerbations of COPD together with the associated health care-related costs of $32 billion reported in the United States in 2002 demonstrate that there is a need for a better understanding of the etiology and pathogenesis of these events (6, 36, 43). Bacteria have been isolated in large numbers from the lower respiratory tract during exacerbations, and up to 50% of COPD exacerbations are due to a bacterial agent, primarily nontypeable Haemophilus influenzae, Moraxella catarrhalis, and Streptococcus pneumoniae (31). M. catarrhalis accounts for up to 10% of these infections in adults, and this strictly human pathogen is currently among the three most prominent causes of otitis media in children (13, 15, 28). Some of the primary reasons why M. catarrhalis continues to cause disease can be attributed to the fact that greater than 90% of the clinical isolates express beta-lactamase, there is a high frequency of recurrent disease observed for children that have recovered from infection, and there is a lack of a vaccine (13, 27, 42, 47). Thus, the identification of potential drug targets and vaccine antigens is clearly a priority.One of the major problems hindering the identification of putative vaccine antigens involves the fact that M. catarrhalis is a strictly human pathogen, and the human immune response to this bacterium remains poorly understood. Previous studies investigated the production of new antibodies against different bacterial pathogens in patients suffering from COPD and lower respiratory tract infections. These patients exhibited increased antibody responses to bacterial outer membrane proteins (OMP) and surface-exposed lipooligosaccharides (LOS) after clearing the bacterial strain following an exacerbation (4, 29, 38, 51). Human serum immunoglobulin G (IgG), sputum IgA, or salivary IgA antibodies against M. catarrhalis surface proteins such as UspA1, UspA2, Hag, TbpB, CopB, OMP CD, OMP E, and OMP G1b have been developed (1, 3, 25, 28, 29). In addition, new antibodies to LOS have also been detected in some COPD patients (3, 28, 29).The LOS structure of M. catarrhalis has been well studied. There are three major serotypes, serotypes A, B, and C, previously defined by polyclonal antisera and structural analyses (8-10, 23, 46). The LOS glycosyltransferase (lgt) genes that encode the enzymes required to transfer carbohydrate residues to the M. catarrhalis LOS molecule were previously identified and characterized (11, 33, 41, 49). In addition, the lgt present in a given strain of M. catarrhalis can be used to identify the specific LOS serotype of that isolate using our previously described multiplex PCR method (12). Serotypes A and B are the predominant glycoforms expressed by most clinical isolates analyzed to date (12, 46). In recently reported animal studies, other researchers suggested that detoxified M. catarrhalis LOS has potential as a vaccine antigen in a mouse pulmonary clearance model (16, 19, 52, 53). While these data are both valuable and interesting, it is sometimes difficult to link observations of animals to those of humans (34).Currently, we have constructed a panel of defined LOS mutants that are defective in the expression of each specific glycosyltransferase gene identified in all three major M. catarrhalis LOS serotypes. These truncations are a comprehensive set of mutations with various oligosaccharide (OS) chain lengths representing most, if not all, possible LOS epitopes (11, 33, 41, 49). Purified LOS samples from these mutants were used in enzyme-linked immunosorbent assays (ELISAs) to assess the development of new human antibodies to all LOS epitopes developed following an M. catarrhalis infection. ELISAs were previously employed to determine levels of antibody to Neisseria meningitidis lipopolysaccharide (LPS), including inner core mutations, in patients following disease (35). Thus, this LOS-based ELISA system with the full set of mutations has the potential to determine the regions of the LOS molecule that elicit new antibodies in both children and adults following infection, providing a unique opportunity to analyze the human immune response to these major surface glycolipids in the native host.     

Tracking the Emerging Human Pathogen Pseudallescheria boydii by Using Highly Specific Monoclonal Antibodies

Pseudallescheria boydii has long been known to cause white grain mycetoma in immunocompetent humans, but it has recently emerged as an opportunistic pathogen of humans, causing potentially fatal invasive infections in immunocompromised individuals and evacuees of natural disasters, such as tsunamis and hurricanes. The diagnosis of P. boydii is problematic since it exhibits morphological characteristics similar to those of other hyaline fungi that cause infectious diseases, such as Aspergillus fumigatus and Scedosporium prolificans. This paper describes the development of immunoglobulin M (IgM) and IgG1 κ-light chain monoclonal antibodies (MAbs) specific to P. boydii and certain closely related fungi. The MAbs bind to an immunodominant carbohydrate epitope on an extracellular 120-kDa antigen present in the spore and hyphal cell walls of P. boydii and Scedosporium apiospermum. The MAbs do not react with S. prolificans, Scedosporium dehoogii, or a large number of clinically relevant fungi, including A. fumigatus, Candida albicans, Cryptococcus neoformans, Fusarium solani, and Rhizopus oryzae. The MAbs were used in immunofluorescence and double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) to accurately differentiate P. boydii from other infectious fungi and to track the pathogen in environmental samples. Specificity of the DAS-ELISA was confirmed by sequencing of the internally transcribed spacer 1 (ITS1)-5.8S-ITS2 rRNA-encoding regions of environmental isolates.Pseudallescheria boydii is an infectious fungal pathogen of humans (7, 16, 40, 58, 59). It is the etiologic agent of white grain mycetoma in immunocompetent humans (7) and has emerged over recent years as the cause of fatal disseminated infections in individuals with neutropenia, AIDS, diabetes, renal failure, bone marrow or solid organ transplants, systemic lupus erythematous, and Crohn''s disease; in those undergoing corticosteroid treatment; and in leukemia and lymphoma patients (1, 2, 3, 18, 27, 31, 32, 34, 36, 37, 38, 47, 49, 52). The fungus is the most prevalent species after Aspergillus fumigatus in the lungs of cystic fibrosis patients (8), where it causes allergic bronchopulmonary disease (5) and chronic lung lesions simulating aspergillosis (24). Near-drowning incidents and recent natural disasters, such as the Indonesian tsunami in 2004, have shown P. boydii and the related species Scedosporium apiospermum and Scedosporium aurantiacum to be the causes of fatal central nervous system infections and pneumonia in immunocompetent victims who have aspirated polluted water (4, 11, 12, 21, 22, 25, 30, 33, 57). Its significance as a potential pathogen of disaster evacuees has led to its recent inclusion in the Centers for Disease Control and Prevention list of infectious etiologies in persons with altered mental statuses, central nervous system syndromes, or respiratory illness.P. boydii is thought to be an underdiagnosed fungus (60), and misidentification is one of the reasons that the mortality rate due to invasive pseudallescheriasis is high. Detection of invasive P. boydii infections, based on cytopathology and histopathology, is problematic since it can occur in tissue and bronchoalveolar and bronchial washing specimens with other hyaline septated fungi, such as Aspergillus and Fusarium spp. (7, 23, 53, 60), which exhibit similar morphological characteristics upon microscopic examination (2, 23, 24, 28, 37, 44, 53, 60). Early diagnosis of infection by P. boydii and differentiation from other agents of hyalohyphomycosis is imperative, since it is refractory to antifungal compounds, such as amphotericin B, that are commonly administered for the control of fungal infections (10, 39, 58).The immunological diagnosis of Pseudallescheria infections has focused on the detection of antigens by counterimmunoelectrophoresis, and by immunohistological techniques using polyclonal fluorescent antibodies, but cross-reactions with antigens from other fungi, such as Aspergillus species, occurs (7, 19, 23). Pinto and coworkers (41, 42) isolated a peptidorhamnomannan from hyphae of P. boydii and proposed the antigen as a diagnostic marker for the pathogen. Cross-reactivity with Sporothrix schenckii and with Aspergillus have, however, been noted (23, 41). Furthermore, it is uncertain whether a similar antigen is present in the related pathogenic species S. prolificans, an important consideration in patient groups susceptible to mixed Scedosporium infections (6, 18).Hybridoma technology allows the production of highly specific MAbs that are able to differentiate between closely related species of fungi (54, 55, 56). The purpose of this paper is to report the development of MAbs specific to P. boydii and certain closely related species and their use to accurately discriminate among P. boydii, A. fumigatus, and other human pathogenic fungi by using immunofluorescence and double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs).Currently, the natural environmental habitat of P. boydii is unknown, but nutrient-rich, brackish waters, such as estuaries, have been suggested (9, 17). In combination with a semiselective isolation procedure, I show how the DAS-ELISA can be used to rapidly and accurately track the pathogen in naturally infested estuarine muds, and in doing so illustrate the potential of the DAS-ELISA as a diagnostic platform for detection of P. boydii and related species within the Pseudallescheria complex.     

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.     

Ail Binding to Fibronectin Facilitates Yersinia pestis Binding to Host Cells and Yop Delivery

Yersinia pestis, the causative agent of plague, evades host immune responses and rapidly causes disease. The Y. pestis adhesin Ail mediates host cell binding and is critical for Yop delivery. To identify the Ail receptor(s), Ail was purified following overexpression in Escherichia coli. Ail bound specifically to fibronectin, an extracellular matrix protein with the potential to act as a bridge between Ail and host cells. Ail expressed by E. coli also mediated binding to purified fibronectin, and Ail-mediated E. coli adhesion to host cells was dependent on fibronectin. Ail expressed by Y. pestis bound purified fibronectin, as did the Y. pestis adhesin plasminogen activator (Pla). However, a KIM5 Δail mutant had decreased binding to host cells, while a KIM5 Δpla mutant had no significant defect in adhesion. Furthermore, treatment with antifibronectin antibodies decreased Ail-mediated adhesion by KIM5 and the KIM5 Δpla mutant, indicating that the Ail-fibronectin interaction was important for cell binding. Finally, antifibronectin antibodies inhibited the KIM5-mediated cytotoxicity of host cells in an Ail-dependent fashion. These data indicate that Ail is a key adhesin that mediates binding to host cells through interaction with fibronectin on the surface of host cells, and this interaction is important for Yop delivery by Y. pestis.The three species of Yersinia pathogenic for humans, Yersinia enterocolitica, Y. pseudotuberculosis, and Y. pestis, cause distinct diseases. Y. pseudotuberculosis and Y. enterocolitica typically cause acute gastroenteritis and mesenteric lymphadenitis. On the other hand, Y. pestis, the causative agent of the plague, is one of the most deadly human infectious diseases (8). Y. pestis is a close relative of Y. pseudotuberculosis, diverging only 1,500 to 20,000 years ago (1). To accommodate flea-borne transmission, Y. pestis has acquired two unique plasmids not harbored by enteropathogenic Yersinia species. All three pathogenic Yersinia species inject cytotoxic Yersinia outer proteins (Yops) into host cells via the Ysc type III secretion system (TTSS) to establish an infection (11). Host cell contact is essential for engagement of the TTSS and secretion of Yops (9, 54). Within the host cell, Yops effect actin rearrangements, inhibit phagocytosis, and block proinflammatory signals (4, 40, 42). Both Y. enterocolitica and Y. pseudotuberculosis express the well-studied adhesin molecules invasin (Inv) and YadA, capable of mediating Yop delivery (9, 54). However, Y. pestis does not express either adhesin due to an IS1541 element insertion within inv (58) and a frameshift mutation in yadA (44, 55). Y. pestis has a number of other adhesins capable of mediating host cell interaction. Both the pH 6 antigen (Psa [29, 63]) and plasminogen activator (Pla [28]) of Y. pestis have been shown to be adhesins. Psa is a tightly regulated pilus expressed at a pH of <6.7 and 37°C (52, 67) and is known to bind to β-linked galactosylated glycosphingolipids (46), low-density lipoprotein (31), and human IgG (69). Pla, expressed at 26°C but further induced at 37°C (49), is known to bind to several extracellular matrix components (23, 28, 30). The putative autotransporter, YapC, is also capable of mediating cell adhesion when it is expressed in Escherichia coli (15), as is the pilus encoded by the chaperone/usher system locus y0561-0563 (16), but neither yapC nor y0561-0563 results in significantly decreased adhesion when they are deleted from Y. pestis (15, 16).Recently, an additional adhesin of Y. pestis, Ail (adherence and invasion locus), was determined to facilitate cell binding (14, 25). Ail (encoded by y1324) is a 21.5-kDa outer membrane protein of the OmpX family that is predicted to have eight transmembrane domains and four extracellular loops extending above the surface of the bacterium (17, 65). Ail homologues include OmpX of Escherichia coli (32) and Enterobacter cloacae (61), PagC in Salmonella (53), and Opa proteins from Neisseria (10). Ail from Y. enterocolitica has been studied previously and shown to have three activities: cell adhesion, cell invasion (36), and the ability to confer serum resistance (5, 51) by binding to complement regulatory proteins (24). The residues for all three activities have been mapped to particular amino acids in the surface-exposed loops (35). Y. pseudotuberculosis Ail also confers adhesion and invasion functions (T. M. Tsang and E. S. Krukonis, unpublished data) and serum resistance (68), although the two amino acid changes between Y. pseudotuberculosis Ail and Y. pestis Ail result in decreased adhesion and invasion mediated by the former (Tsang and Krukonis, unpublished). More recently, Y. pestis Ail was also shown to mediate cell adhesion (14, 25), autoaggregation (25), and serum resistance (3, 24, 25) and to facilitate Yop delivery to host cells (14). Furthermore, Y. pestis Ail is required for virulence, as a Y. pestis Δail mutant has a >3,000-fold increase in the 50% lethal dose (14). A Y. pestis Δail mutant shows reduced binding to both epithelial and phagocytic human-derived cell lines, and in a mouse model of infection, a Y. pestis KIM5 Δail mutant colonizes host tissue to much lower levels than the parental KIM5 strain (14). Over the course of 7 days, the Δail mutant is cleared from the host (14). Together, these data demonstrate that Ail is an important adhesin that contributes to colonization and virulence.Cell adhesion is important for the establishment of a successful infection. Adhesion is also significant in Y. pestis pathogenesis because host cell contact is required for the production and translocation of the Yop effector proteins (48, 54). Bacteria can bind directly to host cell receptors (21) or use molecules like extracellular matrix (ECM) components to mediate attachment to host cells (12, 22, 30, 45, 57, 64). Common components of the cellular matrix that facilitate bacterial binding include fibronectin (22, 28, 64), collagen (23, 45), and laminin (28, 30, 45). Interactions between bacteria and ECM can lead to bridge-like attachments to host cells.Fibronectin is a large glycoprotein that is a key structural component in many tissues. This ∼220-kDa protein is commonly found as a dimer that is linked by two disulfide bonds located near the C terminus. Fibronectin is a complex molecule made up of three types of modular repeating units (43, 47). Fibronectin can bind to many substrates, including collagen (13), integrin receptors on host cells (50, 56), and heparin (60). Additionally, fibronectin contains a binding site for several bacterial pathogens at the N-terminal end of the molecule (39, 59).A number of fibronectin binding proteins on bacterial pathogens have been identified and studied, including SigB from Staphylococcus aureus (34), protein F from Streptococcus pyogenes (41), and YadA from Y. pseudotuberculosis (12, 19) and Y. enterocolitica (64). Binding of Y. pseudotuberculosis YadA to fibronectin allows Y. pseudotuberculosis to utilize β₁ integrins on the surface of host cells for invasion (12). Given the key role of Y. pestis Ail in cell adhesion, Yop delivery, and virulence, we sought to determine the component on host cells to which Ail binds.Although Ail has been studied extensively in other Yersinia species, the substrate on host cells with which Ail interacts is not known. In this study, we used a purified Y. pestis Ail to identify the extracellular matrix component, fibronectin, as a protein bound by Ail. Furthermore, Ail-mediated binding to host cells through fibronectin is important for the delivery of Yop effector proteins.