Capsule Polysaccharide Conjugate Vaccine against Diarrheal Disease Caused by Campylobacter jejuni

The capsule polysaccharide (CPS) of Campylobacter jejuni is one of the few identified virulence determinants of this important human pathogen. Since CPS conjugate vaccines have been so effective against other mucosal pathogens, we evaluated this approach using CPSs from two strains of C. jejuni, 81-176 (HS23 and HS36 serotype complex) and CG8486 (HS4 serotype complex). The CPSs of 81-176 and CG8486 were independently linked to the carrier protein CRM₁₉₇ by reductive amination between an aldehyde(s), strategically created at the nonreducing end of each CPS, and accessible amines of CRM₁₉₇. In both cases, the CPS:CRM₁₉₇ ratio used was 2:1 by weight. Mass spectrometry and gel electrophoresis showed that on average, each glycoconjugate preparation contained, at least in part, two to five CPSs attached to one CRM₁₉₇. When administered subcutaneously to mice, these vaccines elicited robust immune responses and significantly reduced the disease following intranasal challenge with the homologous strains of C. jejuni. The CPS_81-176-CRM₁₉₇ vaccine also provided 100% protection against diarrhea in the New World monkey Aotus nancymaae following orogastric challenge with C. jejuni 81-176.Campylobacter jejuni, a member of the epsilon division of the Proteobacteria, is a major cause of bacterial diarrhea worldwide (16). In North America, the organism is among the leading bacterial causes of food-borne illness, with an estimated incidence of 25 to 50 per 100,000. In some developing countries, the incidence of C. jejuni diarrhea has been reported to be as high as 40,000/100,000 (12, 16, 42). Campylobacteriosis is also associated with a number of important sequelae, including reactive arthritis, Reiter''s syndrome, irritable bowel syndrome, and Guillain-Barré syndrome (GBS) (40). GBS is thought to occur as a result of molecular mimicry of the outer lipooligosaccharide (LOS) regions of some strains of C. jejuni with human gangliosides (23, 60, 61). Thus, antibodies directed against LOS during infection can result in an autoimmune disease that affects peripheral nerves.There are no licensed vaccines against C. jejuni, although several have undergone preclinical and clinical development (55). One of the main obstacles to vaccine development is the association of C. jejuni with GBS, a fact that precludes whole cell-based approaches and also poses safety concerns for human challenge studies. Although C. jejuni strain 81-176 has been fed to human volunteers (10; D. Tribble, personal communication), these studies predated the awareness of the association of C. jejuni with GBS and the knowledge that the LOS of this strain contained ganglioside mimics (19). A clinical isolate of C. jejuni lacking ganglioside mimicry has recently been characterized and is currently being used to develop a human challenge model (45). Another hurdle in vaccine development is that C. jejuni has proven particularly recalcitrant to studies on molecular pathogenesis. Despite extensive study and numerous genome sequences (15, 21, 43, 45, 46), there are few defined virulence factors that could be targeted as subunit vaccines.Early structural analyses by Aspinall and coworkers revealed the presence of polysaccharides (PSs) in several Campylobacter species that were not associated with an expected lipopolysaccharide (LPS) component but that were more akin to teichoic acid PSs and capsule PSs (CPSs) (1, 3, 5, 6). More recent genomic analysis provided further evidence that C. jejuni was indeed encapsulated (43), and subsequent structural studies confirmed that the C. jejuni PSs, previously designated as LPS O-chain PSs, were in fact CPSs. Thus, unlike most other gram-negative enteric pathogens, C. jejuni does not express an LPS (O-chain→core→lipid A), instead producing an LOS (core→lipid A) and a CPS (1). Additional genetic studies have indicated that C. jejuni CPS modulates the invasion of intestinal epithelial cells (7, 8) and serum resistance (8). Moreover, CPS is a component of the Penner or heat-stable (HS) serotyping scheme (8, 30). CPSs are unusual surface structures for enteric pathogens, but given the clinical success of CPS conjugate vaccines against other mucosal pathogens (14, 24, 32-35, 57), we hypothesized that a CPS conjugate vaccine would protect against diarrheal disease by C. jejuni. Here, we report on the synthesis of two prototype C. jejuni CPS conjugate vaccines based on the well-characterized strains 81-176 (HS23 and HS36) (10, 21) and CG8486 (HS4 complex) (46) (Fig. ​(Fig.1).1). The well-characterized diphtheria toxin mutant CRM₁₉₇ was used as the carrier protein (56). This 63-kDa protein is an approved carrier for licensed pneumococcal vaccines and has been shown to be safe and effective in numerous clinical trials (27, 48, 51). The C. jejuni CPS conjugate vaccines synthesized in this study were immunogenic in mice and reduced the disease following intranasal challenge (9) with the homologous strain of C. jejuni. We also show here that the 81-176 CPS conjugate vaccine is immunogenic and 100% protective against diarrheal disease in New World monkeys (26).Open in a separate windowFIG. 1.Chemical structures and conjugation schemes to CRM₁₉₇ of C. jejuni strain 81-176 (A) and C. jejuni strain 8486 CPSs (B).     

Characterization of a Campylobacter jejuni VirK Protein Homolog as a Novel Virulence Determinant

Campylobacter jejuni is a leading cause of food-borne illness in the United States. Despite significant recent advances, its mechanisms of pathogenesis are poorly understood. A unique feature of this pathogen is that, with some exceptions, it lacks homologs of known virulence factors from other pathogens. Through a genetic screen, we have identified a C. jejuni homolog of the VirK family of virulence factors, which is essential for antimicrobial peptide resistance and mouse virulence.Campylobacter jejuni is a leading cause of infectious diarrhea in industrialized and developing countries (2, 67). Although most often self-limiting, C. jejuni infections can also lead to severe disease and harmful sequelae, such as Guillain-Barré syndrome (4, 55). Despite the significant progress made during the past few years, the mechanisms of C. jejuni pathogenesis remain poorly understood. A number of potential virulence factors have been identified, and in some cases, their role in virulence and/or colonization has been demonstrated in animal models of infection. For example, motility has been shown to be crucial in order for C. jejuni to colonize or cause disease in several animal models of infection (1, 15, 30, 54). A variety of surface structures, such as adhesins (34, 40, 64) and polysaccharides (5, 6), and glycosylation systems (38, 74), which presumably modify some of these surface structures, have also been shown to be important for infection. Additional studies have revealed the importance of specific metabolic pathways in C. jejuni growth both in vitro and within animals (16, 25, 31, 60, 76). The ability of C. jejuni to invade and survive within nonphagocytic cells has also been proposed to be an important virulence determinant (21, 41, 57, 58, 68, 75, 80).The available genome sequences of several C. jejuni strains have provided significant insight into C. jejuni physiology and metabolism (22, 32, 62, 63, 65). Remarkably, however, analysis of these C. jejuni genome sequences has revealed very few homologs of common virulence factors from other pathogens. A notable exception is the toxin CDT (cytolethal distending toxin), which is also encoded by several other important bacterial pathogens (36, 44, 45). In this paper we describe the identification of a transposon insertion mutant in C. jejuni 81-176, which results in increased susceptibility to antimicrobial peptides and a significant defect in the ability of the organism to cause disease in an animal model of infection. The insertion mutant was mapped to the CJJ81176_1087 open reading frame (Cj1069 in the C. jejuni NCT 11168 reference strain), which encodes a protein with very significant amino acid sequence similarity to the VirK (DUF535) family of virulence factors (13, 20, 56).     

Identification of Campylobacter jejuni Genes Involved in Its Interaction with Epithelial Cells

Campylobacter jejuni is the leading cause of infectious gastroenteritis in industrialized nations. Its ability to enter and survive within nonphagocytic cells is thought to be very important for pathogenesis. However, little is known about the C. jejuni determinants that mediate these processes. Through an extensive transposon mutagenesis screen, we have identified several loci that are required for C. jejuni efficient entry and survival within epithelial cells. Among these loci, insertional mutations in aspA, aspB, and sodB resulted in drastic reduction in C. jejuni entry and/or survival within host cells and a severe defect in colonization in an animal model. The implications of these findings for the understanding of C. jejuni-host cell interactions are discussed.Campylobacter jejuni is one of the most important causes of food-borne illness in industrialized nations and diarrhea in children in developing countries (45, 67). Despite its importance as a pathogen, its virulence mechanisms are just beginning to be understood. The ability of C. jejuni to enter nonphagocytic cells is thought to be very important for its pathogenesis (77). Correlation between the cultured-cell invasiveness of Campylobacter strains and the severity of the disease outcome has been reported (10, 11, 33, 47), and studies have visualized C. jejuni inside intestinal epithelial cells during human infections (68). C. jejuni can enter and survive within a variety of cultured cell lines (37, 49, 71, 73). Studies have revealed unique aspects in the cell biology of C. jejuni entry. For example, it has been shown that C. jejuni entry does not require an intact actin cytoskeleton although it requires an intact microtubular network (49). Other studies have implicated Rho-family GTPases in C. jejuni entry (39). However, little is known about the bacterial determinants specifically involved in mediating entry and intracellular survival. Although several studies have identified C. jejuni mutants exhibiting various degrees of deficiency in their ability to enter cultured cells (1, 2, 5, 13, 17, 25, 27, 29-31, 52, 63), there is no evidence indicating that the identified gene products directly mediate the entry process. Nonmotile mutants exhibit a drastic decrease in their ability to invade cultured cells (17, 70, 75). However, it is unclear whether the flagellar structure is directly involved in triggering bacterial internalization or whether the severe entry defect of nonmotile mutants indicates that motility per se is required for entry. A mutation in the pflA gene, which results in paralyzed flagella, has been shown to be defective for entry (75), suggesting that motility and not the flagellar structure itself is required for entry. However, the actual role of PflA is unknown, and therefore it is still possible that the phenotype of the pflA mutation may be due to functions other than its putative role in motility. We have carried out a mutagenesis screen to identify C. jejuni genes that are required for entry and/or survival within host cells. Among the identified loci, insertional mutations in aspA, aspB, and sodB resulted in a drastic reduction in C. jejuni entry and/or survival within host cells and a severe colonization defect in an animal model. Although our studies did not provide evidence for a direct involvement of these loci in the cell entry process, these findings highlight the importance of C. jejuni basic metabolism in its ability to interact with host cells.     

Clonal Analysis of Colonizing Group B Streptococcus,Serotype IV,an Emerging Pathogen in the United States

Colonizing group B Streptococcus (GBS) capsular polysaccharide (CPS) type IV isolates were recovered from vaginal and rectal samples obtained from 97 (8.4%) nonpregnant women of 1,160 women enrolled in a U.S. multicenter GBS vaccine study from 2004 to 2008. Since this rate was much higher than the rate of prevalence of 0.4 to 0.6% that we found in previous studies, the isolates were analyzed by using surface protein profile identification, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) to characterize them and identify trends in DNA clonality and divergence. Of the 101 type IV isolates studied, 53 expressed α and group B protective surface (BPS) proteins, 27 expressed BPS only, 20 expressed α only, and 1 had no detectable surface proteins. The isolates spanned three PFGE macrorestriction profile groups, groups 37, 38, and 39, of which group 37 was predominant. The isolates in group 37 expressed the α and BPS proteins, while those in groups 38 and 39 expressed the α protein only, with two exceptions. MLST studies of selective isolates from the four protein profile groups showed that isolates expressing α,BPS or BPS only were of a new sequence type, sequence type 452, while those expressing α only or no proteins were mainly of a new sequence type, sequence type 459. Overall, our study revealed a limited diversity in surface proteins, MLST types, and DNA macrorestriction profiles for type IV GBS. There appeared to be an association between the MLST types and protein expression profiles. The increased prevalence of type IV GBS colonization suggested the possibility that this serotype may emerge as a GBS pathogen.Group B Streptococcus (GBS) (Streptococcus agalactiae) is a leading cause of neonatal infection in the United States, with maternal vaginal or rectal colonization often resulting in the transmission of GBS to the infant during the perinatal period (8, 23). GBS isolates are classified according to nine capsular polysaccharide (CPS) types: types Ia, Ib, and II to VIII and the recently proposed type IX (9, 15, 21, 23, 46, 52). Isolates that do not express any of the known CPS types are designated nontypeable (NT) (2, 6, 21, 40). In addition to CPS, GBS may express one or more surface-localized proteins, including the α and β components of the c protein (24); the alpha-like R proteins, specifically R1, R4(Rib), and R1,R4 (also known as Alp3) (14, 17, 19, 30, 40); and the group B protective surface (BPS) protein (12). Certain protein profiles are associated with each capsular polysaccharide CPS type (2), for example, the c(α only) protein with types Ia and II, c(α + β) with type Ib, and R4(Rib) with type III (2, 14). BPS, expressed by fewer than 3% of colonizing isolates, can be found alone or with another protein in type Ia, II, and V isolates (12, 14).In the United States, the predominant serotypes over the past 2 decades, constituting 70 to 75% of all GBS isolates, have been type Ia, type III, and the more recently emerged type V (14, 15, 20, 52). The remaining isolates consisted primarily of types Ib and II, with types IV, VI, VII, and VIII making up a small fraction of the isolates. We found type IV to represent between 0.4 and 0.6% of colonizing GBS isolates (14, 15), but only rare type IV isolates were found in invasive GBS disease during that same time period (14, 43, 52).In contrast to the previously low percentage of type IV isolates reported for the United States, recent studies in the United Arab Emirates, Turkey, and Zimbabwe showed large proportions of type IV isolates among their GBS isolates. In the United Arab Emirates, type IV was the predominant serotype among colonized pregnant women, representing 26.3% of the GBS isolates (1). In eastern Turkey, it was the second most common serotype, at 8.3%, among colonizing isolates (10), and in Zimbabwe, it was the fourth most common serotype, comprising 5.1% of GBS isolates from colonized pregnant women and 4.0% of all GBS isolates from various sites, including blood and cerebrospinal fluid (CSF), from hospitalized patients (36).Immunization studies of humans (3, 28) and protection studies with mice (37) have shown the potential of vaccines against the common GBS serotypes to prevent invasive neonatal GBS disease through the vaccination of pregnant women (3, 28). The GBS strains described here are from a phase II randomized, double-blinded clinical trial of a GBS serotype III-tetanus toxoid (CPS III-TT) vaccine to prevent the vaginal acquisition of GBS type III in nonpregnant women in three areas of the United States: Pittsburgh (PA), Georgia, and Texas (S. Hillier, unpublished data). Because we found type IV isolates for almost 10% of these patients, we examined the type IV isolates for surface proteins and clonality.Pulsed-field gel electrophoresis (PFGE) was used in this analysis because it is a widely used method that can further characterize GBS isolates within particular CPS type and/or protein profile groups (2, 4, 6, 48). Multilocus sequence typing (MLST) was performed in order to assess the general relatedness of strains within and across laboratories (25, 50). Together, the discriminatory power of PFGE and the objectivity of MLST gave insight into the GBS type IV population genetic structure and the identification of emerging clones (2, 5, 13, 18, 19).     

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.     

Diversity of Group B Streptococcus Serotypes Causing Urinary Tract Infection in Adults

Serotypes of group B streptococcus (GBS) that cause urinary tract infection (UTI) are poorly characterized. We conducted a prospective study of GBS UTI in adults to define the clinical and microbiological characteristics of these infections, including which serotypes cause disease. Patients who had GBS cultured from urine over a 1-year period were grouped according to symptoms, bacteriuria, and urinalysis. Demographic data were obtained by reviewing medical records. Isolates were serotyped by latex agglutination and multiplex PCR-reverse line blotting (mPCR/RLB). Antibiotic susceptibilities were determined by disc diffusion. GBS was cultured from 387/34,367 consecutive urine samples (1.1%): 62 patients had bacteriuria of >10⁷ CFU/liter and at least one UTI symptom; of these patients, 31 had urinary leukocyte esterase and pyuria (others not tested), 50 (81%) had symptoms consistent with cystitis, and 12 (19%) had symptoms of pyelonephritis. Compared with controls (who had GBS isolated without symptoms), a prior history of UTI was an independent risk factor for disease. Increased age was also significantly associated with acute infection. Serotyping results were consistent between latex agglutination and mPCR/RLB for 331/387 (85.5%) isolates; 22 (5.7%) and 7 (1.8%) isolates were nontypeable with antisera and by mPCR/RLB, respectively; and 45/56 (80.4%) isolates with discrepant results were typed by mPCR/RLB as belonging to serotype V. Serotypes V, Ia, and III caused the most UTIs; serotypes II, Ib, and IV were less common. Nontypeable GBS was not associated with UTI. Erythromycin (39.5%) and clindamycin (26.4%) resistance was common. We conclude that a more diverse spectrum of GBS serotypes causes UTI than previously recognized, with the exception of nontypeable GBS.Group B streptococcus (GBS) is a leading cause of infection in newborns, pregnant women, and older persons with chronic medical illness (3, 8). In addition to maternal cervicovaginal colonization and neonatal infection that results from the vertical transmission of bacteria from mothers to their infants, GBS can also cause urinary tract infection (UTI). The spectrum of GBS UTI includes asymptomatic bacteriuria (ABU), cystitis, pyelonephritis, urethritis, and urosepsis (6, 8, 10, 20, 23, 26). GBS ABU is particularly common among pregnant women, although those most at risk for cystitis due to GBS appear to be the elderly and immunocompromised individuals (8, 9, 25). Despite the uropathogenic nature of GBS, the clinical and microbiological features of GBS UTI, including risk factors for disease and whether there is a tendency for particular GBS serotypes to cause UTI, are poorly understood.Clinically, UTI due to GBS may be indistinguishable from UTI caused by other uropathogens (25). However, a recent study of multiple uropathogens and host characteristics highlighted unique frequencies of host characteristics in UTI groups defined by the causal organism (37). This suggests that the clinical and microbiological features of UTI may differ depending on the infecting uropathogen. GBS colonization of the urinary tract in women most likely occurs by an ascending route from the vagina, where GBS can persist asymptomatically. While the overall prevalence of GBS UTI in the adult population remains unclear, GBS bacteriuria during pregnancy occurs at rates of between 1 and 3.5% (4, 23, 41). Many of these episodes represent ABU (2, 18); however, GBS ABU is considered to be a surrogate for heavy maternal colonization (29, 42) and is currently recommended for intrapartum antibiotic chemoprophylaxis (23, 34). In addition, up to 7% of pregnancies may be complicated by GBS UTI, and GBS reportedly accounts for approximately 10% of all cases of pyelonephritis during pregnancy (25, 28). GBS UTI may also contribute to chorioamnionitis (1), premature onset of labor (24), and an increased risk of vertical transmission of GBS (29, 42).Several studies have also reported high rates of GBS UTI in nonpregnant adults (8, 9, 25, 39). In one study, GBS was cultured from 39% of all cases of symptomatic UTI among nursing home residents >70 years of age (40). Other studies reported that GBS UTI may account for up to one-third of all invasive infections due to GBS in adults (9, 12, 19, 26). Several independent surveys have reported the recovery of GBS from between 1 and 2% of all UTI cases (7, 26, 30). GBS UTI may also account for up to 7% of late-onset disease in neonates (43). Thus, while there is an increasing amount of data regarding the prevalence of GBS UTI in adults, little is known regarding the clinical and microbiological features associated with these infections or the GBS serotypes that cause UTI. In this study, we carried out a single-center analysis of adult patients at the University of Alabama at Birmingham Hospital between August 2007 and August 2008 who had GBS cultured from urine during routine assessments for UTI to define the clinical and microbiological characteristics of GBS UTI including which serotypes cause disease.     

Phosphoethanolamine Substitution of Lipid A and Resistance of Neisseria gonorrhoeae to Cationic Antimicrobial Peptides and Complement-Mediated Killing by Normal Human Serum

The capacity of Neisseria gonorrhoeae to cause disseminated gonococcal infection requires that such strains resist the bactericidal action of normal human serum. The bactericidal action of normal human serum against N. gonorrhoeae is mediated by the classical complement pathway through an antibody-dependent mechanism. The mechanism(s) by which certain strains of gonococci resist normal human serum is not fully understood, but alterations in lipooligosaccharide structure can affect such resistance. During an investigation of the biological significance of phosphoethanolamine extensions from lipooligosaccharide, we found that phosphoethanolamine substitutions from the heptose II group of the lipooligosaccharide β-chain did not impact levels of gonococcal (strain FA19) resistance to normal human serum or polymyxin B. However, loss of phosphoethanolamine substitution from the lipid A component of lipooligosaccharide, due to insertional inactivation of lptA, resulted in increased gonococcal susceptibility to polymyxin B, as reported previously for Neisseria meningitidis. In contrast to previous reports with N. meningitidis, loss of phosphoethanolamine attached to lipid A rendered strain FA19 susceptible to complement killing. Serum killing of the lptA mutant occurred through the classical complement pathway. Both serum and polymyxin B resistance as well as phosphoethanolamine decoration of lipid A were restored in the lptA-null mutant by complementation with wild-type lptA. Our results support a role for lipid A phosphoethanolamine substitutions in resistance of this strict human pathogen to innate host defenses.Neisseria gonorrhoeae causes over 60 million cases of the sexually transmitted disease gonorrhea each year worldwide (6). Although most infections are uncomplicated and usually restricted to the lower urogenital tract, more invasive forms of disease that result in significant medical complications can occur. For example, entrance of N. gonorrhoeae into the bloodstream followed by dissemination, termed disseminated gonococcal infection (DGI), can occur in 1 to 3% of cases when particular strains are endemic in the community (26, 33, 38).In contrast to gonococcal strains that cause pelvic inflammatory disease or salpingitis in women and to a lesser extent uncomplicated, urogenital tract infections in men or women, DGI strains can stably resist the bactericidal action of normal human serum (NHS) (33, 38). In some cases the bactericidal activity of NHS is mediated by natural immunoglobulin M (IgM) antibodies (14) that activate the classical complement pathway (CCP) (16, 40, 41). Bactericidal activity in NHS is an important host defense mechanism for prevention of invasive bloodstream disease due to the pathogenic Neisseria species (gonococci and meningococci) (9, 32, 34). For example, patients with defects in their terminal complement components often have recurrent bacteremias with these strict human pathogens (26). Although the multiplicity of mechanisms by which gonococci can resist killing by NHS remains to be fully defined, there is evidence that certain stably serum-resistant gonococci fail to bind bactericidal IgM (36). Further, a strong correlation exists between the ability of gonococci to bind the CCP regulatory protein C4b binding protein (C4BP), which dampens activation of the CCP, and stably serum-resistant phenotypes of N. gonorrhoeae (29).Variations in surface structures of the pathogenic neisseriae have been invoked as being important in the capacity of these strict human pathogens to resist innate host defenses that function during infection both at mucosal surfaces and in the bloodstream. For instance, the structure of the lipooligosaccharide (LOS) possessed by gonococci (53, 54) and meningococci (17, 27) can vary at high frequencies (1-3, 13, 37, 48). LOS can also be modified by sialylation (43) or by the addition of phosphoethanolamine (PEA) to the heptose (HepII) group in the β-chain of the core oligosaccharide (2, 24, 25, 52) as well as to the 1 and 4′ positions of lipid A (8). Changes in LOS structure can have a profound impact on bacterial interactions with host cells and/or defensive systems (18, 20, 35, 45, 46). As an example, the NHS resistance expressed by certain gonococci can be lost by high-frequency, spontaneous mutations within the lgtABCDE operon (39), which encodes the glycosyl transferases responsible for extending the LOS α-chain (13).We hypothesized that the PEA substitutions of gonococcal LOS could influence the susceptibility of this pathogen to mediators of innate host defense. In support of this hypothesis, previous work (28) has shown that PEA attached to position 6 of HepII of meningococcal LOS can form an amide linkage with complement component C4b and enhances susceptibility of N. meningitidis to the bactericidal action of NHS. Additionally, PEA attached to the lipid A of meningococcal LOS enhances bacterial resistance to cationic antimicrobial peptides (CAMPs), including the human cathelicidin LL-37 (49), but does not influence resistance to killing by NHS (8). Based on these examples with meningococci, we tested whether loss of PEA from the HepII group of the LOS β-chain or lipid A would alter gonococcal susceptibility to NHS or CAMPs. We report that loss of PEA substitution of lipid A significantly increases gonococcal susceptibility to both polymyxin B (PB) and NHS and propose that the presence of PEA on lipid A contributes to the ability of gonococci to resist mediators of innate host defense.     

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.     

Assessment of the Duration of Protection in Campylobacter jejuni Experimental Infection in Humans

A human Campylobacter jejuni infection model provided controlled exposure to assess vaccine efficacy and investigate protective immunity for this important diarrheal pathogen. A well-characterized outbreak strain, C. jejuni 81-176, was investigated using a volunteer experimental infection model to evaluate the dose range and duration of protection. Healthy Campylobacter-seronegative adults received C. jejuni strain 81-176 via oral inoculation of 10⁵, 10⁷, or 10⁹ CFU (5 adults/dose), which was followed by clinical and immunological monitoring. Based on dose range clinical outcomes, the 10⁹-CFU dose (n = 31) was used to assess homologous protection at 28 to 49 days (short-term veterans [STV]; n = 8) or 1 year (long-term veterans [LTV]; n = 7) after primary infection. An illness dose effect was observed for naïve subjects (with lower doses, 40 to 60% of the subjects were ill; with the 10⁹-CFU dose, 92% of the subjects were ill) along with complete protection for the STV group and attenuated illness for the LTV group (57%). Partial resistance to colonization was seen in STV (25% of the subjects were not infected; 3-log-lower maximum excretion level). Systemic and mucosal immune responses were robust in naïve subjects irrespective of the dose or the severity of illness. In contrast, in STV there was a lack of circulating antibody-secreting cells (ASC), reflecting the local mucosal effector responses. LTV exhibited comparable ASC responses to primary infection, and anamnestic fecal IgA responses likely contributed to self-resolving illness prior to antibiotic treatment. Campylobacter antigen-dependent production of gamma interferon by peripheral blood mononuclear cells was strongly associated with protection from illness, supporting the hypothesis that TH1 polarization has a primary role in acquired immunity to C. jejuni. This study revealed a C. jejuni dose-related increase in campylobacteriosis rates, evidence of complete short-term protection that waned with time, and immune response patterns associated with protection.Campylobacter species, the most common of which is Campylobacter jejuni, are zoonotic food- and waterborne bacterial enteropathogens (1, 2, 52). The gastrointestinal tracts of animals used for food, such as chickens, are the reservoirs for these common organisms (1). Worldwide, C. jejuni is among the most frequent causes of diarrhea, including traveler''s diarrhea, and the spectrum of illness ranges from mild watery diarrhea to febrile dysentery (6, 14, 16, 21). Evidence for acquired immunity against C. jejuni has been obtained from epidemiologic studies performed in developing countries that documented that there is a decline in the incidence of disease with increasing age that is accompanied by a shift in the illness-to-infection ratio for children between 2 and 5 years old, development of resistance to colonization, and a shorter excretion period during convalescence (12, 44, 45). Age-related increases in C. jejuni-specific serology coincide with acquisition of resistance to infection and illness (9, 12, 45). In addition, a lower incidence of Campylobacter-associated diarrhea was observed in infants whose mothers had colostral Campylobacter-specific secretory IgA antibodies in their breast milk (13).Evidence for acquired immunity against C. jejuni has also been obtained from studies performed in industrialized countries. Reduced C. jejuni-associated diarrhea rates correlated with increased levels of Campylobacter-specific IgA antibody in chronic consumers of raw milk on dairy farms compared to individuals exposed to raw milk for the first time, as well as a lower risk of diarrhea for travelers to regions where C. jejuni is hyperendemic (10, 11, 51). Black and colleagues performed the initial study of an experimental C. jejuni infection in humans (5a, 7). A human Campylobacter infection model provided controlled exposure coupled with predefined endpoints to assess the efficacy of a candidate vaccine and to investigate pathogenesis and immunity. C. jejuni 81-176, a milk-borne outbreak strain (26), was one of the two strains investigated. This study documented the pathogenicity of C. jejuni; however, low and variable rates of attack (40 to 60%) without an illness dose response (10⁶ to 10⁹ CFU delivered in skim milk) were observed (5a, 7). Infection with or without illness induced serologic and intestinal antibody responses. Higher prechallenge C. jejuni-specific (acid-extracted protein) serologic and jejunal fluid IgA levels in noninfected subjects than in infected subjects were observed, as were increased levels of jejunal fluid IgA during rechallenge in subjects who remained well after a second exposure.A rare (approximately 1 in 1,000 to 3,000 C. jejuni enteritis cases [30, 42]) but potentially life-threatening complication of Campylobacter infection is Guillain-Barré syndrome (GBS), a postinfectious polyneuropathy that is a leading cause of paralysis (32, 49). Research evidence supports the hypothesis that the C. jejuni-GBS association is due to molecular mimicry, where peripheral nerve gangliosides share epitopes with C. jejuni outer lipooligosaccharide (LOS) cores, leading to a misdirected and harmful immune response (17, 25, 53, 54). Prestudy characterization of the challenge strain revealed no evidence of ganglioside mimicry associated with GBS pathogenesis (12).The campylobacteriosis clinical outcomes observed in the study of Black et al. were not sufficiently frequent or predictable based on the dose to support evaluation of vaccine efficacy. In the current study, two modifications were included, inoculum delivery with bicarbonate buffer and C. jejuni-specific serologic screening, based on post hoc analysis of data from the previous study (3). The change in the method of inoculum delivery was based on evidence obtained with a human Shigella infection model, which showed that 11/12 (92%) naïve subjects developed clinical illness when 1.4 × 10³ CFU was delivered with bicarbonate buffer (2 g NaHCO₃ in 150 ml distilled water), compared to attack rates of 50 to 60% (upper limits) with challenge doses between 5 × 10³ and 1 × 10⁸ CFU in skim milk in previous studies (27).In this study we report a refined human C. jejuni 81-176 infection model which demonstrated that there was a dose-related increase in campylobacteriosis rates and provided evidence of complete short-term protection that waned with time and cell-mediated immune response patterns that were associated with protection. This work improves the model for future application and provides directions for additional refinements.(This study was presented in part at the 10th International Congress of Immunology, New Delhi, India, 1998, and at the 10th International Workshop on Campylobacter, Helicobacter and Related Organisms, Baltimore, MD, 1999.)     

Typing of Campylobacter jejuni Isolates from Dogs by Use of Multilocus Sequence Typing and Pulsed-Field Gel Electrophoresis

Campylobacter is a major cause of human gastroenteritis worldwide. Risk of Campylobacter infection in humans has been associated with many sources, including dogs. This study aimed to investigate whether C. jejuni carried by dogs could potentially be a zoonotic risk for humans and if there were common sources of C. jejuni infection for both humans and dogs. Multilocus sequence typing (MLST) together with macrorestriction analysis of genomic DNA using SmaI and pulsed-field gel electrophoresis (PFGE) were both used to analyze 33 C. jejuni isolates obtained from various dog populations, including those visiting veterinary practices and from different types of kennels. MLST data suggested that there was a large amount of genetic diversity between dog isolates and that the majority of sequence types found in isolates from these dogs were the same as those found in isolates from humans. The main exception was ST-2772, which was isolated from four samples and could not be assigned to a clonal complex. The most commonly identified clonal complex was ST-45 (11 isolates), followed by ST-21 (4 isolates), ST-508 (4 isolates), and ST-403 (3 isolates). The profiles obtained by macrorestriction PFGE were largely in concordance with the MLST results, with a similar amount of genetic diversity found. The diversity of sequence types found within dogs suggests they are exposed to various sources of C. jejuni infection. The similarity of these sequence types to C. jejuni isolates from humans suggests there may be common sources of infection for both dogs and humans. Although only a small number of household dogs may carry C. jejuni, infected dogs should still be considered a potential zoonotic risk to humans, particularly if the dogs originate from kennelled or hunt kennel dog populations, where the prevalence may be higher.Campylobacter species are among the most commonly reported bacterial cause of human gastroenteritis (2, 5, 10, 44). The majority of such infections are caused by C. jejuni and C. coli. There is some evidence of an elevated risk of Campylobacter infection in humans associated with dog or pet ownership (1, 17, 38).Multilocus sequence typing (MLST) is a tool used to investigate relationships between Campylobacter spp., particularly C. jejuni. It is useful for investigating possible reservoir hosts and host associations and also for studying the molecular epidemiology of the disease (11, 28). The C. jejuni clonal complexes ST-21 and ST-45 and others, such as ST-61, are often isolated from human cases of campylobacteriosis (12, 31). The complexes ST-21 and ST-45 are commonly found in chickens, cattle, water, and wildlife, and ST-61 has been particularly associated with cattle (7, 18, 22, 36).Some other C. jejuni sequence types isolated from humans have not yet been associated with cattle and poultry, and the sources of these need further investigation (31). In contrast, some sequence types are strongly associated with certain animal hosts (6) but are not usually isolated from humans (12, 13, 15, 31).Whether or not dogs are a possible source of C. jejuni infection for humans is not fully understood. Macrorestriction pulsed-field gel electrophoresis (PFGE) has been used in studies to compare C. jejuni isolates found in both dogs and humans (29), and in some cases the dog and the owner did share an identical strain (8). There is currently little MLST data available for Campylobacter spp. isolated from dogs (16; http://pubmlst.org/campylobacter [accessed 5 February 2009]), although ST-45 infection in humans has also been significantly associated with contact with pet cats and dogs (22).The aims of this study were to examine 33 C. jejuni isolates obtained from dogs from various populations using MLST in order to determine whether strains of C. jejuni carried by dogs appear to be different from those found in humans and other species and to determine possible sources of canine infection. PFGE with SmaI digestion was also performed with the isolates in order to investigate the epidemiology of the disease within the different dog populations.     

Real-Time PCR Assay Provides Reliable Assessment of Intrapartum Carriage of Group B Streptococcus

The objective of this study was to determine the reliability of the real-time PCR assay for determining the group B Streptococcus (GBS) status of women in labor. In this prospective study we compared the results of culture and PCR testing of vaginal and rectal samples collected by nursing staff when women were in labor. Patients'' charts were also reviewed to obtain relevant information about pregnancy risk factors. Our results demonstrated a sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 90.5%, 96.1%, 86.4%, and 97.4%, respectively, for rapid PCR. Of the 196 women evaluated, 29 (14.8%) presented with unknown GBS status, 11 (37.9%) of whom received unnecessary intrapartum antibiotics. The rapid real-time PCR test was robust and was able to reliably detect the presence of GBS in women in labor within 1 h of specimen submission to the laboratory. We recommend that the rapid PCR test be targeted to women who present in labor with unknown GBS status. In cases where the laboratory does not offer 24-h availability of testing, sample collection followed by PCR testing the next morning is still valuable and provides reliable results 24 to 48 h faster than culture and will aid appropriate decision-making regarding continuing or stopping antibiotics for neonates of women with unknown GBS status.Streptococcus agalactiae, the group B Streptococcus (GBS) species, colonizes the genitourinary and/or gastrointestinal tract of 10 to 40% of pregnant women (2, 8, 11, 14, 16, 18, 20, 22, 24). GBS causes early-onset sepsis (EOS) in newborns and can be responsible for severe morbidity and mortality in infected neonates (2, 8, 11, 14, 24). Early-onset sepsis in neonates due to GBS has been shown to be preventable if intrapartum antimicrobial prophylaxis is given to mothers who carry GBS (5, 9, 19). Providing that there has been at least 2 h of exposure to the prophylactic antibiotics during labor, the risk of GBS colonization and EOS in neonates born to mothers who carry GBS is significantly reduced (9). Current American and Canadian guidelines recommend that routine rectal/vaginal screening be performed on pregnant women at 35 to 37 weeks of gestation to determine their GBS status (20, 22). If the screening or urine cultures during pregnancy indicate that GBS is present, or if the women have previously given birth to an infant with invasive GBS disease, intrapartum prophylaxis should be given to the mother (20, 22). If the GBS status is unknown and the woman is in labor, risk factors are used to determine whether intrapartum antibiotics for GBS should be administered or not (20).Although the rate of GBS neonatal EOS decreased remarkably after the introduction of intrapartum prophylaxis recommendations, a sizable number of newborns still develop GBS disease, particularly preterm infants (15). This is significant since women who deliver preterm usually do not get the chance to be screened for GBS at 35 to 37 weeks of gestation, and therefore, their GBS status is mostly unknown at labor. Also of concern are reports indicating that as many as two-thirds of infants who developed GBS EOS were born to mothers who were negative for GBS upon prenatal screening (19). On the other hand, evidence shows that the increasing use of intrapartum prophylactic antibiotics has shifted the profile of causative agents of EOS toward Gram-negative organisms (15). Moreover, the administration of maternal intrapartum antibiotics has been linked to the emergence of resistant strains of bacteria in recent years (3, 23).Such data reveal the inadequate use of prophylactic antibiotics despite screening at 35 to 37 weeks of gestation. By following current GBS recommendations, a considerable portion of pregnant women are still treated inappropriately, either as a result of the failure to identify a subset of pregnant women who are colonized with GBS at delivery or by providing unnecessary prophylactic antibiotics to a portion of GBS-negative women. One study showed that the risk-based approach exposes 65 to 85% of women with risk factors who are GBS negative to antibiotics inappropriately (14).While culture has always been the standard method for GBS detection, this method also carries some limitations. Using culture as a diagnostic tool, it usually takes 24 to 72 h for results to become available. Furthermore, even when culture is performed as a screening tool during weeks 35 to 37 of pregnancy, difficulties such as the availability of results at the time of labor or change in the status of GBS colonization can be encountered. Also, women with no prenatal care and ones who deliver preterm do not have access to prenatal GBS screening (21).There is a need for a rapid test that would facilitate in-labor testing for GBS so that women who present with unknown GBS status can be given intrapartum antibiotics only if they carry GBS. A rapid PCR test has been approved by both the FDA and Health Canada for in-labor testing for GBS, but its position in everyday GBS practice remained unclear (i.e., to replace current culture screening or to provide the test for all or a subset of women in labor).The objectives of this study were to determine whether the real-time PCR assay was robust enough when samples were collected by the routine nursing staff (and not study personnel) and whether the turnaround time when offered to women in labor was fast enough to still provide value in diagnostic laboratories that were not functional 24 h a day.     

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.     

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.     

Trends in Methicillin-Resistant Staphylococcus aureus Anovaginal Colonization in Pregnant Women in 2005 versus 2009

In 2005, the prevalence of methicillin-resistant Staphylococcus aureus (MRSA) anovaginal colonization in pregnant women at our center (Columbia University Medical Center) was 0.5%, and MRSA-colonized women were less likely to carry group B streptococcus (GBS). In this study, our objectives were to identify changing trends in the prevalence of MRSA and methicillin-susceptible S. aureus (MSSA) anovaginal colonization in pregnant women, to assess the association between MRSA and GBS colonization, and to characterize the MRSA strains. From February to July 2009, Lim broths from GBS surveillance samples were cultured for S. aureus. MRSA strains were identified by resistance to cefoxitin and characterized by MicroScan, staphylococcal cassette chromosome mec (SCCmec) typing, pulsed-field gel electrophoresis (PFGE), spa typing, and Panton-Valentine leukocidin PCR. A total of 2,921 specimens from different patients were analyzed. The prevalences of MSSA, MRSA, and GBS colonization were 11.8%, 0.6% and 23.3%, respectively. GBS colonization was associated with S. aureus colonization (odds ratio [OR], 1.9; 95% confidence interval [95% CI], 1.5 to 2.4). The frequencies of GBS colonization were similar in MRSA-positive (34.2%) versus MRSA-negative patients (21.8%) (P = 0.4). All MRSA isolates from 2009 and 13/14 isolates from 2005 were SCCmec type IV or V, consistent with community-associated MRSA; 12/18 (2009) and 0/14 (2005) isolates were the USA300 clone. Levofloxacin resistance increased from 14.3% (2005) to 55.6% (2009) (P = 0.028). In conclusion, the prevalence of MRSA anovaginal colonization in pregnant women in New York City, NY, remained stable from 2005 to 2009, and USA300 emerged as the predominant clone with a significant increase in levofloxacin-resistant isolates.The rapid spread of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) across the United States has been associated primarily with the dissemination of a specific clone that has the pulsed-field gel electrophoresis (PFGE) pattern termed USA300 (20). CA-MRSA can cause infections in pregnant and postpartum women and outbreaks in newborn nurseries and neonatal intensive care units (NICUs) (5, 22, 26, 32, 35, 36, 38). CA-MRSA strains, including USA300, have replaced health care-associated (HA)-MRSA as the predominant strains isolated from infants in some NICUs (6, 35). CA-MRSA infections appear to be increasing in otherwise healthy neonates in the nursery (18, 38) who may acquire S. aureus from health care workers or from their mothers and other family members (19, 23, 25, 28).Vertical transmission from mothers to infants at delivery has also been proposed as a possible mechanism of acquisition of CA-MRSA (1, 2, 7, 28). S. aureus has been reported to colonize the vagina in 4 to 22% of pregnant women (2, 4, 9, 13, 14). In 2005, following an outbreak of USA300 in postpartum women at our medical center (Columbia University Medical Center), we determined that the prevalence of methicillin-susceptible S. aureus (MSSA) anovaginal colonization was 16.6% and the prevalence of MRSA colonization was 0.5% (9). Overall, 93% of MRSA strains were staphylococcal cassette chromosome mec (SCCmec) type IV or V, which is consistent with CA-MRSA (9). More recent studies conducted in other locales have suggested that the prevalence of anovaginal colonization with MRSA is increasing, with reported rates ranging from 3.5 to 10.4% (2, 13). The association of MRSA colonization with group B streptococcus (GBS) colonization is less clear; some reports have shown an increased rate of MRSA colonization in GBS-positive women (2), while others have shown a decreased rate (8, 32).Reports suggesting an increasing prevalence of MRSA among pregnant women, coupled with the recent emergence of USA300 in our NICU (6), led us to question whether the epidemiology of MRSA colonization was also changing in pregnant women in our population in New York City, NY. The objectives of this study were to determine the current prevalence of MRSA and MSSA colonization in pregnant women, to assess the association between MRSA and GBS colonization, and to use molecular methods to characterize MRSA strains from the current study and compare those with strains from our 2005 study.     

Recombinant PorA,the Major Outer Membrane Protein of Campylobacter jejuni,Provides Heterologous Protection in an Adult Mouse Intestinal Colonization Model

Immunity against Campylobacter jejuni, a major food-borne pathogen causing diarrhea, is largely serotype specific. The major outer membrane protein (MOMP) of C. jejuni, PorA, is a common antigen with the potential to provide broad protection. Adult BALB/c mice were orally immunized with a recombinant glutathione S-transferase (GST) fused to PorA prepared from Campylobacter jejuni C31 (O:6,7) (GST-PorA) combined with a modified heat-labile enterotoxin of Escherichia coli as an adjuvant and later orally challenged with C31 strain or three heterologous strains: 48 (O:19), 75 (O:3), and 111 (O:1,44). Protection from colonization with the challenge organism was studied by fecal screening daily for 9 days. Serum and intestinal lavage fluid antibodies against the vaccine and Sarkosyl-purified MOMP from C31 were measured by using an enzyme-linked immunosorbent assay. The vaccine produced robust antibody responses against both antigens in serum and secretion. Since strain C31 was a poor colonizer, homologous protection could not be studied. The protective efficacies of heterologous strains were 43% (for strain 48, P < 0.001), 29% (for strain 75, P < 0.005), and 42% (for strain 111, P < 0.001) for the 9-day period compared to control mice given phosphate-buffered saline. Thus, PorA provided appreciable protection against colonization with heterologous serotypes.Campylobacter jejuni is a food-borne pathogen and a leading cause of diarrhea worldwide (19, 33, 34). Campylobacteriosis is associated with a number of important sequelae, including Guillain-Barre syndrome, Reiter''s syndrome, reactive arthritis, and irritable bowel syndrome (10, 37). C. jejuni also contributes to significant mortality of children in developing countries (18). In view of significant morbidity, mortality, and economic burden associated with C. jejuni infection, the control and prevention of campylobacteriosis are a priority. One possible tool for the prevention of campylobacteriosis is vaccination. It has been observed that C. jejuni infection results in the acquisition of immunity. However, immunity in humans seems to be serotype (Penner serotype) specific (13, 36). There are numerous serotypes of C. jejuni according to the Penner serotyping scheme, which is based on the lipopolysaccharide capsule (25). Therefore, vaccines based on common antigens that are shared by all serotypes seem attractive for broad protection. One such antigen is the major outer membrane protein (MOMP) of C. jejuni, PorA, which is present in abundant quantity in the organism. There are three conformational forms of MOMP: folded monomer (35 kDa), denatured monomer (45 kDa), and the native trimer (120 to 140 kDa). Only the folded monomer and the native trimer have pore-forming activities (44). The MOMP is involved in ion transport across the bacterial cell wall and adhesion of the bacterium to the intestinal mucosa (30, 41). It consists of 18 β-strands connected by short periplasmic turns and nine external loops. The external loops are antigenically variable (44). This is evident in the extreme genetic diversity of the porA gene encoding the MOMP (17, 44). However, the β-strands representing the conserved regions have common antigenic epitopes for different strains (16, 44). The sera of patients recovering from C. jejuni infection (8, 12, 35), as well as those of animals immunized with C. jejuni (21), possess antibodies to MOMP. Therefore, PorA seems to be an appropriate antigen that could be investigated as a possible subunit vaccine.There are no easily handled, widely available, and inexpensive natural animal models of C. jejuni diarrhea. Laboratory mice are widely available, inexpensive, and easy to handle. Oral feeding of the adult mouse with C. jejuni does not result in diarrhea in the animal. However, the organism colonizes the intestine, with shedding of the organism for several days in the stool. The organism can be isolated from the blood as well (7, 9). Thus, the adult mouse can be used as a colonization model of C. jejuni infection.Since the PorA of C. jejuni has never been investigated as a vaccine candidate, we studied a recombinant PorA in the form of a fusion protein in the adult mouse colonization model for broad protection against colonization. We studied fecal excretion of the challenge organisms and serum and intestinal antibody responses to the vaccine.     

LuxS Promotes Biofilm Maturation and Persistence of Nontypeable Haemophilus influenzae In Vivo via Modulation of Lipooligosaccharides on the Bacterial Surface

Nontypeable Haemophilus influenzae (NTHI) is an extremely common airway commensal which can cause opportunistic infections that are usually localized to airway mucosal surfaces. During many of these infections, NTHI forms biofilm communities that promote persistence in vivo. For many bacterial species, density-dependent quorum-signaling networks can affect biofilm formation and/or maturation. Mutation of luxS, a determinant of the autoinducer 2 (AI-2) quorum signal pathway, increases NTHI virulence in the chinchilla model for otitis media infections. For example, bacterial counts in middle-ear fluids and the severity of the host inflammatory response were increased in luxS mutants compared with parental strains. As these phenotypes are consistent with those that we have observed for biofilm-defective NTHI mutants, we hypothesized that luxS may affect NTHI biofilms. A luxS mutant was generated using the well-characterized NTHI 86-028NP strain and tested to determine the effects of the mutation on biofilm phenotypes in vitro and bacterial persistence and disease severity during experimental otitis media. Quantitation of the biofilm structure by confocal microscopy and COMSTAT analysis revealed significantly reduced biomass for NTHI 86-028NP luxS biofilms, which was restored by a soluble mediator in NTHI 86-028NP supernatants. Analysis of lipooligosaccharide moieties using an enzyme-linked immunosorbent assay and immunoblotting showed decreased levels of biofilm-associated glycoforms in the NTHI 86-028NP luxS strain. Infection studies showed that NTHI 86-028NP luxS had a significant persistence defect in vivo during chronic otitis media infection. Based on these data, we concluded that a luxS-dependent soluble mediator modulates the composition of the NTHI lipooligosaccharides, resulting in effects on biofilm maturation and bacterial persistence in vivo.Haemophilus influenzae is a gram-negative bacterium that frequently inhabits the nasopharynx and upper airways of children and healthy adults (17). Nontypeable H. influenzae (NTHI) strains lacking capsular polysaccharides are the strains carried most frequently and can cause opportunistic infections of the airway mucosa that include bronchitis, sinusitis, and otitis media (OM) (16). OM is among the most common pediatric infections and affects the majority of all children by the age of 3 years (31). NTHI is one of the leading causes of OM (6, 37), including chronic and recurrent OM infections that are generally not resolved by antibiotic treatment or host immunity (39). The chronic or recurrent presentations of OM have long been thought of as involving bacterial persistence within a biofilm (7). Biofilms have been directly observed both in the chinchilla experimental model for OM (12, 25, 26, 29, 56) and in middle-ear tissues from patients with OM (19).Most factors associated with NTHI disease promote evasion of host clearance and persistence in vivo (16). These factors include specific subtypes of the lipooligosaccharides (LOS) on the bacterial surface (4, 5), many of which contain structures found on host cells and are thought to mask the organism from host immune defenses (34). Previous work by our group and other groups has elucidated the role of the LOS modifications in NTHI persistence during biofilm growth (25, 26, 29, 44). Specifically, LOS forms decorated with sialic acid and phosphorylcholine predominate in vivo (3, 46, 53, 54) and are known to promote biofilm formation and/or maturation and persistence in vivo (25, 26, 29, 44).Quorum sensing refers to density-dependent changes in the phenotype(s) of bacterial populations that occur due to accumulation of a soluble signaling mediator (27). For example, various homoserine lactone derivatives mediate quorum signaling for some gram-negative bacteria, whereas gram-positive species typically produce peptide signal molecules (51). Autoinducer 2 (AI-2) is a furanosyl borate diester molecule that was first identified as a quorum signal molecule in Vibrio harveyi (13). AI-2 is produced by many different bacterial species and thus has been referred to as an interspecies signaling system (51). The genetic determinant of AI-2 production is luxS, which is part of the methionine-homocysteine cycle (20). Homologs of luxS have been identified in numerous bacterial species, including H. influenzae (42), and have been implicated in biofilm formation, maturation, or dispersal in several different species (20). For NTHI, luxS mutants are known to have increased virulence in the chinchilla infection model for OM (8). In this study, an isogenic luxS mutant was generated using the well-characterized NTHI 86-028NP strain background, and the effect of AI-2 on NTHI biofilm formation, maturation, and persistence in vivo was assessed.     

Three Doses of an Experimental Detoxified L3-Derived Lipooligosaccharide Meningococcal Vaccine Offer Good Safety but Low Immunogenicity in Healthy Young Adults

This open, randomized phase I study evaluated the safety and reactogenicity of an experimental meningococcal serogroup B (MenB) vaccine obtained from outer membrane vesicle detoxified L3-derived lipooligosaccharide. Healthy young adults (n = 150) were randomized to receive either experimental vaccine (provided in five formulations, n = 25 in each group) or VA-Mengoc-BC (control, n = 25) administered on a 0- to 6-week/6-month schedule. Serum bactericidal assays performed against three MenB wild-type strains assessed the immune response, defined as a 4-fold increase from pre- to postvaccination. No serious adverse events related to vaccination were reported. Pain at the injection site, fatigue, and headache were the most commonly reported adverse events. Solicited adverse events graded level 3 (i.e., preventing daily activity) were pain (up to 17% of the test subjects versus 32% of the controls), fatigue (up to 12% of the test subjects versus 8% of the controls), and headache (up to 4% of any group). Swelling graded level 3 (greater than 50 mm) occurred in up to 4% of the test subjects versus 8% of the controls. The immune responses ranged from 5% to 36% across experimental vaccines for the L3 H44-76 strain (versus 27% for the control), from 0% to 11% for the L3 NZ98/124 strain (versus 23% for the control), and from 0% to 13% for the L2 760676 strain (versus 59% for the control). All geometric mean titers were below those measured with the control vaccine. The five experimental formulations were safe and well tolerated but tended to be less immunogenic than the control vaccine.Meningococcal diseases caused by Neisseria meningitidis are a significant health burden throughout the world, leading to death and permanent sequelae (15). Whereas polysaccharide or polysaccharide conjugate vaccines are effective against serogroups A, C, Y, and W135, N. meningitidis serogroup B (MenB) remains a major cause of death and morbidity throughout the world, infants less than 1 year of age being affected the most (5, 8). Serogroup B outbreaks were reported in Europe, Latin America, Australia, New Zealand, and the United States (3, 7, 22, 33). Immunization against MenB presents a challenge, as the capsular polysaccharide is poorly immunogenic in humans (4) and shares molecular mimicry with human antigens (11), which guided the search for outer membrane vesicle (OMV) vaccines (16).Three MenB OMV vaccines with PorA protein as the dominant antigen have been brought to the market (VA-Mengoc-BC [Finlay Institute], MeNZB [Chiron], and MenBvac [Norwegian Institute of Public Health]), but although they have shown protection against PorA-heterologous strains in older children and adults, protection of the youngest is mostly against PorA-homologous MenB strains and their accessibility is geographically limited (7, 9, 18, 21, 25, 26, 31, 34, 36, 37). To be immunogenic in the pediatric and adult populations, a more comprehensive MenB vaccine should include antigens inducing cross-reactive serum bactericidal antibodies (SBA) against a broad spectrum of circulating strains (16, 17, 20, 21, 35). That could best be achieved with non-PorA vaccines (20).Natural immunity against MenB is also induced by protein and lipooligosaccharide (LOS) antigens (28), but proteins and LOS may vary substantially across meningococcal strains. However, at least 70% of invasive MenB isolates express LOS of immunotype L3,7 (19, 27, 29, 30). Hence, GlaxoSmithKline (GSK) Biologicals has developed an experimental vaccine based on the LOS L3 immunotype that was shown to induce bactericidal antibodies in preclinical studies (39). Two detoxified LOS type 3 MenB experimental vaccines differing by the length of the LOS were developed. Such formulations have shown good safety and immunogenicity during preclinical and toxicological studies (39).The primary objective of this study was to evaluate the safety and reactogenicity of several formulations of the experimental vaccines given to healthy young adults. The secondary objective was to assess the immunogenicity of the different formulations.     

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.