Antibodies Specific for the Hia Adhesion Proteins of Nontypeable Haemophilus influenzae Mediate Opsonophagocytic Activity

The Hia autotransporter proteins are highly immunogenic surface adhesins expressed by nontypeable Haemophilus influenzae (NTHI). The objective of our study was to assess the opsonophagocytic activity of anti-Hia antibodies against homologous and heterologous NTHI. A segment of the hia gene that encodes a surface-exposed portion of the H. influenzae strain 11 Hia protein was cloned into a pGEMEX-2 expression vector. Escherichia coli JM101 was transformed with the resulting pGEMEX-Hia BstEII del recombinant plasmid, and recombinant fusion protein was recovered. An immune serum against recombinant GEMEX-Hia (rGEMEX-Hia)-mediated killing of the homologous NTHI strain 11 at a 1:160 titer and five heterologous Hia-expressing strains at titers of ≥1:40. Immune serum did not mediate killing of two Hia-knockout strains whose hia genes were inactivated but did mediate killing of one knockout strain at a high titer after the strain was transformed with a plasmid containing the hia gene. Immune serum did not mediate killing of HMW1/HMW2-expressing NTHI strains, which do not express the Hia adhesin. However, when two representative HMW1/HMW2-expressing strains were transformed with the plasmid containing the hia gene, they expressed abundant Hia and were susceptible to killing by the immune serum. Immune serum did not mediate killing of HMW1/HMW2-expressing strains transformed with the plasmid without the hia gene. Our results demonstrate that the Hia proteins of NTHI are targets of opsonophagocytic antibodies and that shared epitopes recognized by such antibodies are present on the Hia proteins of unrelated NTHI strains. These data argue for the continued investigation of the Hia proteins as vaccine candidates for the prevention of NTHI disease.Otitis media remains a significant health problem for children in this country and elsewhere in the world (10, 11). Most children in the United States have had at least one episode of otitis by their third birthdays, and one-third have had three or more episodes (34). In addition to the short-term morbidity and costs of this illness, the potential for delay or disruption of normal speech and language development in children with persistent middle ear effusions is a subject of considerable concern (33, 41). Experts in the field have strongly recommended that efforts be made to develop safe and effective vaccines for the prevention of otitis media in young children (20). Although the total prevention of disease will be a difficult goal to achieve, the prevention of even a portion of cases would be beneficial, given the magnitude and costs of the problem.Bacteria, usually in pure culture, can be isolated from middle ear exudates in approximately two-thirds of the cases of acute otitis media (16, 35). Streptococcus pneumoniae is the most common bacterial pathogen recovered in all age groups, with isolation rates commonly ranging from 35% to 40% (16, 35). Nontypeable Haemophilus influenzae (NTHI) is the second-most-common bacterium recovered and accounts for 20% to 30% of the cases of acute otitis media and a larger percentage of the cases of chronic and recurrent disease (26). Interestingly, since the introduction of the pneumococcal conjugate vaccine as part of the regular childhood vaccine schedule, NTHI has become an even more common cause of acute and recurrent middle ear disease, often surpassing S. pneumoniae in the frequency of recovery from middle ear specimens (12, 26). Many different antigens have been suggested as possible NTHI vaccine candidates (1, 3, 18, 29, 30, 42). Outer membrane proteins appear to be the principal targets of bactericidal and protective antibodies (22), and as a group, they have been the major focus of vaccine development efforts. Table ​Table11 summarizes the relevant characteristics of some of the leading vaccine candidates currently under active investigation.

TABLE 1.

Potential vaccine antigens of NTHI

In Vitro Activity of Seven Systemically Active Antifungal Agents against a Large Global Collection of Rare Candida Species as Determined by CLSI Broth Microdilution Methods

Five Candida species (C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, and C. krusei) account for over 95% of invasive candidiasis cases. Some less common Candida species have emerged as causes of nosocomial candidiasis, but there is little information about their in vitro susceptibilities to antifungals. We determined the in vitro activities of fluconazole, voriconazole, posaconazole, amphotericin B, anidulafungin, caspofungin, and micafungin against invasive, unique patient isolates of Candida collected from 100 centers worldwide between January 2001 and December 2007. Antifungal susceptibility testing was performed by the CLSI M27-A3 method. CLSI breakpoints for susceptibility were used for fluconazole, voriconazole, anidulafungin, caspofungin, and micafungin, while a provisional susceptibility breakpoint of ≤1 μg/ml was used for amphotericin and posaconazole. Of 14,007 Candida isolates tested, 658 (4.7%) were among the less common species. Against all 658 isolates combined, the activity of each agent, expressed as the MIC₅₀/MIC₉₀ ratio (and the percentage of susceptible isolates) was as follows: fluconazole, 1/4 (94.8%); voriconazole, 0.03/0.12 (98.6%); posaconazole, 0.12/0.5 (95.9%); amphotericin, 0.5/2 (88.3%); anidulafungin, 0.5/2 (97.4%); caspofungin, 0.12/0.5 (98.0%); and micafungin, 0.25/1 (99.2%). Among the isolates not susceptible to one or more of the echinocandins, most (68%) were C. guilliermondii. All isolates of the less common species within the C. parapsilosis complex (C. orthopsilosis and C. metapsilosis) were susceptible to voriconazole, posaconazole, anidulafungin, caspofungin, and micafungin. Over 95% of clinical isolates of the rare Candida species were susceptible to the available antifungals. However, activity did vary by drug-species combination, with some species (e.g., C. rugosa and C. guilliermondii) demonstrating reduced susceptibilities to commonly used agents such as fluconazole and echinocandins.More than 95% of Candida bloodstream infections (BSI) are caused by five species: C. albicans, C. glabrata, C. parapsilosis, C. tropicalis, and C. krusei (83). The in vitro activities of available antifungal agents against these five species have been documented extensively (21, 25, 64, 83, 91), whereas very little is known regarding the susceptibility profiles of the less frequently isolated Candida species (8, 40, 75-77, 94).Among the Candida strains reported to cause BSI, more than 17 different species have been identified (31, 32, 84). Aside from the five most common species noted above, the remaining species include C. lusitaniae, C. guilliermondii, C. kefyr, C. pelliculosa, C. famata, C. lipolytica, and C. rugosa (Table ​(Table1)1) (31, 77, 84). Many of these species have been observed to occur in nosocomial clusters and/or to exhibit innate or acquired resistance to one or more established antifungal agents (6, 9, 23, 27, 36, 48, 49, 79, 80, 87, 88, 106). In addition, the use of molecular identification methods has resulted in the identification of new species within larger species complexes (e.g., C. dubliniensis within the C. albicans complex and C. orthopsilosis and C. metapsilosis within the C. parapsilosis complex) (43-46, 100). In vitro susceptibility data specific to those newly described species groups are also lacking.

TABLE 1.

Distribution of 14,007 isolates of Candida spp. from blood and other normally sterile sites, 2001 to 2007

Genetic Characterization of Genogroup I Norovirus in Outbreaks of Gastroenteritis

In this study, we demonstrate that differences within the P2 domain of norovirus genogroup I (GI) strains can be used to segregate outbreaks which are unrelated, whereas complete conservation within this region allows tracking of strains that are part of a single outbreak and likely to have a common source.Noroviruses (NoVs) are members of the Caliciviridae family (7) and the leading cause of outbreaks of acute gastroenteritis worldwide (14). NoV outbreaks are frequently associated with semiclosed or closed institutions such as hospitals and homes for the elderly (11, 22), but outbreaks also occur in other settings, including eating establishments, cruise ships, concert halls (2, 10, 20), and schools (16). Transmission of NoVs is usually person to person (15), although food and water (1, 3, 5, 9, 17, 18, 21) and environmental or airborne contamination (6, 19) have all been implicated in transmission.Human NoVs are genetically diverse, and three distinct genogroups (GI, GII, and GIV) and many genotypes/genetic clusters exist (8, 13, 26). Diversity among NoVs is generated through the accumulation of point mutations associated with the error-prone nature of RNA replication and genetic recombination involving the exchange of sequences between related RNA viruses.The NoV capsid is divided into the S domain, which constitutes the 5′ end (amino acids [aa] 1 to 225), and the P (protruding) domain (aa 226 to 530) (23). The P domain can be further subdivided into two subdomains, P1 and P2. The P2 domain is the hypervariable region of the capsid and corresponds to the most exposed area likely to be involved in immune recognition and attachment. Due to the high diversity within this region, it is not possible to design a single cross-reactive primer pair capable of amplifying all genotypes within a genogroup; therefore, genotype-specific primers are required in order to amplify this region, as previously seen for NoV genogroup II (25).Fecal samples were collected from genogroup I outbreaks of gastroenteritis as part of the ongoing National Surveillance Programme of the molecular epidemiology of norovirus genotypes.Outbreaks were defined as including two or more cases of gastroenteritis linked in place and time. A new outbreak was arbitrarily defined as occurring at least 7 days after the last case in a previous outbreak or as occurring in a different patient care unit such as a ward or hospital.Outbreak 714124 occurred in a nursing home in Blackburn, Lancashire, United Kingdom, in August 2007; outbreak 414003 occurred in a restaurant in Chorley, Lancashire, in January 2004 (4); outbreak 512057 occurred in a bar/restaurant in Liverpool, United Kingdom, in November 2005; outbreak 612057 occurred in a nursing home and outbreak 612058 occurred in a hotel in Liverpool in November 2006; outbreak E/2005/UK occurred in a cruise ship in December 2007; outbreak Q1/2007/US occurred in a cruise ship in the United States in February 2007; and outbreak Newquay/2008/UK occurred in Newquay, Cornwall, United Kingdom, in August 2008.Fecal specimens were prepared as previously described, and nucleic acid extraction, Norovirus detection through amplification of a small region spanning the open reading frame 1/2 (Orf1/2) junction, and genotyping through sequence analysis of the S domain were all performed as previously described (4).Oligonucleotide primers for the amplification of a region encompassing the P2 domain of NoV GI genotypes 1 to 7 were designed from alignments of complete Orf2 nucleotide sequence data. See Table ​Table11 for primer sequences and positions and amplification conditions. Separate monoplex reactions were carried out for each of the genotypes, and amplicons were separated by agarose gel electrophoresis and sequenced directly after purification using the same P domain genotype-specific primers.

TABLE 1.

Norovirus genogroup I genotype-specific primers for amplification of a region encompassing the P2 domain^a

Evaluation of Chimeric Japanese Encephalitis and Dengue Viruses for Use in Diagnostic Plaque Reduction Neutralization Tests

The plaque reduction neutralization test (PRNT) is a specific serological test used to identify and confirm arbovirus infection in diagnostic laboratories and monitor immunological protection in vaccine recipients. Wild-type (wt) viruses used in the PRNT may be difficult to grow and plaque titrate, such as the dengue viruses (DENV), and/or may require biosafety level 3 (BSL3) containment, such as West Nile virus (WNV), St. Louis encephalitis virus (SLEV), and Japanese encephalitis virus (JEV). These requirements preclude their use in diagnostic laboratories with only BSL2 capacity. In addition, wt JEV falls under the jurisdiction of the select-agent program and can be used only in approved laboratories. The chimeric vaccine viruses ChimeriVax-WNV and -SLEV have previously been shown to elicit antibody reactivity comparable to that of parental wt WNV and SLEV. ChimeriVax viruses provide advantages for PRNT, as follows: they grow more rapidly than most wt flaviviruses, produce large plaques, require BSL2 conditions, and are not under select-agent restrictions. We evaluated the ChimeriVax-DENV serotype 1 (DENV1), -DENV2, -DENV3, -DENV4, and -JEV for use in PRNT on sera from DENV- and JEV-infected patients and from JEV vaccine recipients. Serostatus agreement was 100% between the ChimeriVax-DENV serotypes and wt prototype DENV and 97% overall with ChimeriVax-JEV compared to prototype Nakayama JEV, 92% in a subgroup of JEV vaccine recipients, and 100% in serum from encephalitis patients naturally infected with JEV. ChimeriVax-DENV and -JEV plaque phenotype and BSL2 requirements, combined with sensitive and specific reactivity, make them good substitutes for wt DENV and JEV in PRNT in public health diagnostic laboratories.Flaviviruses are medically important pathogens that are significant causes of disease throughout the temperate and tropical regions of the world. In Asia, over 3 billion people live in areas where they are at risk of being infected with Japanese encephalitis virus (JEV), and JEV infections have become the leading cause of pediatric encephalitis in Asia, with as many as 50,000 cases and 15,000 deaths per year (49, 50). The four serotypes of dengue virus (DENV) have emerged in recent years to reach pandistribution throughout the tropics and subtropics of the Americas, Asia, and Africa, resulting in over 100 million dengue fever cases and hundreds of thousands of cases of the more severe dengue hemorrhagic fever/dengue shock syndrome (14-17, 37).Laboratory diagnosis of flavivirus infection is primarily serological, using detection of virus-specific immunoglobulin M (IgM) in an IgM antibody-capture enzyme-linked immunosorbent assay (MAC ELISA), ideally from paired acute and convalescent specimens but in practice from a single acute-phase serum specimen or cerebrospinal fluid specimen (6, 35, 36, 54). This method is sensitive and relatively specific. However, there is considerable cross-reactivity of antibodies elicited in the immune response to conserved regions of the flavivirus envelope (E) protein, which may cause false-positive results in the MAC ELISA and confound diagnosis in areas where multiple flaviviruses cocirculate (45). Specificity can be improved somewhat through differential diagnosis by cross-testing specimens against multiple flaviviruses simultaneously in a standardized MAC ELISA format, but it may be difficult to distinguish among flaviviruses by MAC ELISA alone, even those from different antigenic complexes such as JEV and DENV (7, 24, 35, 42).The plaque reduction neutralization test (PRNT) is a specific serological assay that is used at the CDC/Division of Vector-Borne Infectious Diseases (DVBID) diagnostic laboratory to confirm infection and differentiate among flaviviruses in primary flavivirus infections (4, 11, 46-48). Neutralization assays are also used to monitor protective immunity in vaccinees (26, 38). In the PRNT procedure, the serological specimen (generally serum) is mixed with live virus, and if virus-specific neutralizing antibodies are present in the serum, they bind to the virus to form a complex. The mixture is then inoculated onto a monolayer of cells. Virus bound up in an antibody-virus complex is inhibited from infecting the cells; i.e., it is neutralized. Consequently, laboratories conducting these assays must have tissue culture capability, considerable technical expertise in growing and plaque titrating the flaviviruses, which have a wide range of growth rates, and appropriate biosafety level laboratory conditions in which to grow the virus. JEV, West Nile virus (WNV), and St. Louis encephalitis virus (SLEV) require biosafety level 3 (BSL3) containment, which precludes their use in many public health diagnostic laboratories with only BSL2 capacity. In addition, JEV falls under the jurisdiction of the select-agent program and can be used only in select-agent-registered laboratories (8).Acambis, Inc. (now a part of Sanofi Pasteur), has developed chimeric vaccine viruses for JEV, WNV, SLEV, and the four serotypes of DENV, based on the attenuated yellow fever (YF) vaccine virus 17D (YF-VAX), with the genes encoding the premembrane (prM) and E proteins of the YF 17D virus replaced with those of heterologous flaviviruses, i.e., JEV, WNV, SLEV, and DENV (3, 9, 19, 20, 39). Previously, the ChimeriVax-WNV and -SLEV were shown to be functionally comparable to prototype WNV and SLEV in the PRNT (44). The ChimeriVax viruses have many advantages over the prototype wild-type (wt) viruses in the PRNT; most importantly for this application, they can be used under BSL2 containment. They are plaque purified and produce large-sized, relatively uniform plaques which are phenotypically similar to those of the YF 17D virus but with specific reactivity to the heterologous prM-E protein insert. They grow at relatively the same rate, which has allowed the procedure to be standardized at the CDC/DVBID so that when using multiple flaviviruses in differential diagnosis, the second overlay can be applied to all the PRNT plates on the same day (Table ​(Table1).1). This generally shortens the test duration compared to that of the wt flaviviruses.

TABLE 1.

PRNT₉₀ double agarose overlay procedure, showing the number of days following application of first overlay until application of second overlay

Internet-Accessible DNA Sequence Database for Identifying Fusaria from Human and Animal Infections

Because less than one-third of clinically relevant fusaria can be accurately identified to species level using phenotypic data (i.e., morphological species recognition), we constructed a three-locus DNA sequence database to facilitate molecular identification of the 69 Fusarium species associated with human or animal mycoses encountered in clinical microbiology laboratories. The database comprises partial sequences from three nuclear genes: translation elongation factor 1α (EF-1α), the largest subunit of RNA polymerase (RPB1), and the second largest subunit of RNA polymerase (RPB2). These three gene fragments can be amplified by PCR and sequenced using primers that are conserved across the phylogenetic breadth of Fusarium. Phylogenetic analyses of the combined data set reveal that, with the exception of two monotypic lineages, all clinically relevant fusaria are nested in one of eight variously sized and strongly supported species complexes. The monophyletic lineages have been named informally to facilitate communication of an isolate''s clade membership and genetic diversity. To identify isolates to the species included within the database, partial DNA sequence data from one or more of the three genes can be used as a BLAST query against the database which is Web accessible at FUSARIUM-ID (http://isolate.fusariumdb.org) and the Centraalbureau voor Schimmelcultures (CBS-KNAW) Fungal Biodiversity Center (http://www.cbs.knaw.nl/fusarium). Alternatively, isolates can be identified via phylogenetic analysis by adding sequences of unknowns to the DNA sequence alignment, which can be downloaded from the two aforementioned websites. The utility of this database should increase significantly as members of the clinical microbiology community deposit in internationally accessible culture collections (e.g., CBS-KNAW or the Fusarium Research Center) cultures of novel mycosis-associated fusaria, along with associated, corrected sequence chromatograms and data, so that the sequence results can be verified and isolates are made available for future study.In addition to being the single most important genus of toxigenic phytopathogens (40), Fusarium (Hypocreales, Ascomycota) has emerged over the past 3 decades as one of the most important genera of filamentous fungi responsible for deeply invasive, opportunistic infections in humans (83). Clinically, fusarioses in immunocompetent patients typically present as superficial infections, such as onychomycosis and trauma-associated keratitis, or locally invasive infections, such as sinusitis, catheter-associated peritonitis, pneumonia, or diabetic cellulitis (77). The 2005-2006 keratitis outbreaks within the United States and Asia, however, were unusual in that they were linked to the use of a novel soft contact lens cleaning solution, which was subsequently removed from the market (11). In contrast, immunocompromised or immunosuppressed patients who are persistently and profoundly neutropenic may acquire life-threatening angioinvasive, hematogenously disseminated fusarial infections associated with high morbidity and mortality rates (15). The high mortality of immunosuppressed patients is due in part to the broad resistance of most fusaria to the spectrum of antifungals currently available (1, 4-7, 56); liposomal amphotericin B shows the greatest efficacy among the drugs currently in use (3, 17, 66).A series of molecular phylogenetic studies has led to the important conceptual advance that morphological species recognition within Fusarium (22, 38, 47) greatly underestimates its species diversity (49, 50, 53, 54-57, 59, 70, 85). This finding is not too surprising, given that phenotypic methods for identifying fusaria rely on relatively few morphological and cultural characters (75). Based on an extensive literature review, Nucci and Anaissie (48) recently recorded 12 morphospecies associated with fusarial infections within the immunocompromised patient population. However, phylogenetic species recognition based on genealogical concordance of multilocus DNA sequence data (herein referred to as GCPSR) (79) has identified at least 69 clinically important Fusarium species (Table ​(Table1)1) (49, 54, 56, 57, 70, 85). Phylogenetic species in these studies were recognized if they received ≥70% maximum parsimony (MP) bootstrap support (78) from the majority of the individual gene partitions and the combined data set and if their monophyly was not contradicted by analyses of any of the individual single-gene partitions.

TABLE 1.

Fusaria subjected to DNA MLST

Performance of MicroScan WalkAway and Vitek 2 for Detection of Oxacillin Resistance in a Set of Methicillin-Resistant Staphylococcus aureus Isolates with Diverse Genetic Backgrounds

Of 104 genotypically diverse methicillin-resistant Staphylococcus aureus (MRSA) isolates tested with the MicroScan WalkAway (Pos MIC 24 panel) and Vitek 2 (AST-P549 card) systems, 7 and 6 isolates, respectively, showed an oxacillin MIC of ≤2mg/liter. Most of these MRSA isolates were community acquired. However, if the cefoxitin screen of AST-P549 was also considered, MRSA detection failed for only one isolate.The prevalence of methicillin-resistant Staphylococcus aureus (MRSA) has increased over the last years. Reliable detection of MRSA is important since a false report of a patient''s isolate as methicillin susceptible would result in inadequate therapy with probably fatal consequences (2). Whereas MRSA infections formerly occurred almost exclusively in hospitalized patients, community-acquired MRSA (cMRSA) isolates have been reported recently in patients without any previous contact with the health care system (7).Many laboratories rely on automatic susceptibility testing methods that use oxacillin MIC testing, oxacillin breakpoint detection in the presence of salt, or cefoxitin MIC testing as markers for the presence of methicillin resistance. Many studies have investigated the detection of MRSA by the Vitek 2 system (3, 4, 8, 11, 12, 13, 15, 17); however, data for the performance of the MicroScan WalkAway system in MRSA detection are scarce (17).Most studies evaluating the performance of Vitek 2 used consecutive clinical strains (3, 8, 11, 12, 15), but this approach may be biased by the overrepresentation of locally predominant clones and may not predict performance in other geographical areas. We therefore used a collection of MRSA strains with distinct pulsed-field gel electrophoresis (PFGE) patterns to study MRSA detection using the MicroScan WalkAway and Vitek 2 systems.From 1998 to 2006, noncopy MRSA isolates (n = 1,516), initially identified by oxacillin screening agar or Vitek, from four hospitals in the Bochum area were collected and typed by PFGE as described previously (5). Of these, 120 isolates with different PFGE patterns were chosen. The patterns were interpreted according to the criteria of Tenover et al. (18), and isolates grouped into PFGE types and subtypes.For susceptibility tests, isolates from frozen storage were subcultured twice on Columbia blood agar at 37°C in 5% CO₂ before being tested with the Vitek 2 system using the AST-P549 card and the MicroScan WalkAway system using the Pos MIC 24 panel.Whenever results for oxacillin in the Vitek 2 or MicroScan WalkAway system or for the cefoxitin screen in the Vitek 2 system were not indicative of MRSA, a mecA PCR was performed from colonies growing on purity control plates of both automatic systems and a S. aureus-specific PCR for SA442 (16) was used as an internal positive control. In addition, the Panton-Valentine leukocidin (PVL)-coding genes lukS-PVL and lukF-PVL were detected by PCR (9). SCCmec typing (10) and spa typing (6) were performed as described previously.Loss of mecA during storage of isolates could be demonstrated in 16 of 120 isolates by PCR (14), a proportion that is similar to that described before (19). Of the remaining 104 true MRSA isolates, 95 were detected as MRSA with both automatic systems.An oxacillin MIC of ≤2 mg/liter was measured for six isolates with the Vitek 2 test and for seven isolates with the WalkAway test (Table ​(Table1);1); thus, those isolates would not have been detected as MRSA based on oxacillin MICs alone. Microdilution performed according to CLSI methods (1) showed resistant oxacillin MICs for all but one of these isolates, whereas by Etest on Mueller-Hinton agar with 2% NaCl, oxacillin MICs of ≥4 mg/liter were found for only two isolates. Microcolonies were the only indication for MRSA in most of the remaining strains, demonstrating the challenge of detecting MRSA in those isolates. The cefoxitin screen incorporated in the Vitek 2 AST-P549 card was positive for five of six isolates not detected by oxacillin MIC. Thus, cefoxitin testing together with oxacillin MIC testing clearly leads to better MRSA detection. Cefoxitin MICs of ≥16 mg/liter and ≥4 mg/liter were also found by microdilution and Etest.

TABLE 1.

All test results for MRSA isolates with negative cefoxitin screen in Vitek 2 or oxacillin MIC of ≤2 mg/liter in Vitek 2 or MicroScan WalkAway assay^a

Structural Determinants of Autoproteolysis of the Haemophilus influenzae Hap Autotransporter

Haemophilus influenzae is a gram-negative bacterium that initiates infection by colonizing the upper respiratory tract. The H. influenzae Hap autotransporter protein mediates adherence, invasion, and microcolony formation in assays with respiratory epithelial cells and presumably facilitates colonization. The serine protease activity of Hap is associated with autoproteolytic cleavage and extracellular release of the Hap_S passenger domain, leaving the Hap_β C-terminal domain embedded in the outer membrane. Cleavage occurs most efficiently at the LN1036-37 peptide bond and to a lesser extent at three other sites. In this study, we utilized site-directed mutagenesis, homology modeling, and assays with a peptide library to characterize the structural determinants of Hap proteolytic activity and cleavage specificity. In addition, we used homology modeling to predict the S1, S2, and S4 subsite residues of the Hap substrate groove. Our results indicate that the P1 and P2 positions at the Hap cleavage sites are critical for cleavage, with leucine preferred over larger hydrophobic residues or other amino acids in these positions. The substrate groove is formed by L263 and N274 at the S1 subsite, R264 at the S2 subsite, and E265 at the S4 subsite. This information may facilitate design of approaches to block Hap activity and interfere with H. influenzae colonization.Haemophilus influenzae is a gram-negative coccobacillus that typically colonizes the nasopharynxes of children and adults. In addition, this organism is an important cause of localized respiratory tract and invasive disease. Nonencapsulated strains cause otitis media, sinusitis, conjunctivitis, and exacerbations of respiratory symptoms in individuals with underlying lung disease, bronchiectasis, and cystic fibrosis (21, 29). Encapsulated strains are an important cause of bacteremic diseases, including sepsis and meningitis (29). Colonization of the upper respiratory tract represents an early step in the pathogenesis of all Haemophilus disease and requires adherence to respiratory epithelium (19). Adherence is facilitated by a number of adhesins, including Hap, Hia, Hsf, HMW1/HMW2, P5, pili, and lipooligosaccharide (2, 18, 21, 26, 27).The Hap adhesin is ubiquitous among isolates of H. influenzae and is a member of the autotransporter family of virulence factors that have been recognized among many gram-negative bacteria (10). Autotransporters are synthesized as precursor proteins with three functional regions, namely, an N-terminal signal sequence, an internal passenger domain, and a C-terminal β-barrel domain (11). The signal sequence targets the precursor protein to the inner membrane and is then cleaved. The C-terminal β-barrel domain inserts into the outer membrane and facilitates presentation of the passenger domain on the bacterial cell surface. Depending upon the protein, the passenger domain remains covalently attached to the β-barrel domain, is cleaved but remains loosely attached to the β-barrel domain, or is cleaved and released entirely from the cell surface (10-12). Although diverse autotransporters share a similar structural organization and a common secretion mechanism, they vary widely in function, possibly reflecting adaptations to particular bacterial pathogenic niches. Autotransporters may function as adhesins mediating tissue tropism, as proteases involved in tissue degradation, as toxins causing host tissue damage, or as mediators of serum resistance (11).Hap is synthesized as a 155-kDa preprotein encompassing a 110-kDa passenger domain, Hap_S, and a 45-kDa β-barrel domain, Hap_β. The Hap_S passenger domain harbors adhesive activity that has been shown to promote interactions with human respiratory cells, as well as with extracellular matrix proteins such as fibronectin, laminin, and collagen IV (7). Hap_S is also responsible for bacterial aggregation via Hap-Hap interactions, contributing to microcolony formation (5). Adherence to epithelial cells and bacterial aggregation are mediated by the C-terminal 311 amino acids of Hap_S, whereas interaction with extracellular matrix proteins is mediated by the C-terminal 511 amino acids of Hap_S (7).Beyond possessing adhesive activities, the Hap_S passenger domain functions as a protease that directs the autoproteolytic cleavage of Hap_S from Hap_β, resulting in release of Hap_S from the bacterial cell surface (6). Hap autoproteolysis has been determined to occur at least partly through intermolecular cleavage on the surface of the bacterium and involves a catalytic triad consisting of residues His98, Asp140, and Ser243. Ser243 is part of the GDSGS motif that is characteristic of many serine proteases (6, 13). In wild-type Hap, cleavage occurs most abundantly at the L1036-N1037 peptide bond, which is referred to as the primary cleavage site (13). Site-directed mutagenesis of this site and N-terminal sequencing of the resulting cleaved Hap fragments has identified three additional cleavage sites, including L1046-T1047, F1077-A1078, and F1067-S1068, termed the secondary, tertiary, and quaternary cleavage sites, respectively (see Table ​Table2)2) (6). Alignment of the amino acid sequences at these cleavage sites has revealed a consensus target sequence motif that consists of (Q/R)(A/S)X(L/F) at the P4 through P1 positions (see Table ​Table2)2) (6).

TABLE 2.

Alignment of sequences at the Hap cleavage sites

Genotypic Characterization of Enterocytozoon bieneusi in Specimens from Pigs and Humans in a Pig Farm Community in Central Thailand

We determined that 15.7% of pigs and 1.4% of humans in a pig farm community in central Thailand harbored Enterocytozoon bieneusi. Genotyping of E. bieneusi from pigs showed genotypes O, E, and H. However, only genotype A was found in human subjects. This indicates nonzoonotic transmission of E. bieneusi in this community.Enterocytozoon bieneusi is an opportunistic organism causing diarrhea in human immunodeficiency virus (HIV)-positive patients, in whom it has a prevalence of 2 to 50% (5). The infection not only has been reported to occur in immunocompromised hosts but also has been found in healthy individuals (14, 20). This organism can infect a broad range of animals (4, 12, 16, 18, 22, 23). Genotypes of E. bieneusi in humans and animals are differentiated using the polymorphisms of the internal transcribed spacer (ITS) sequence of the rRNA gene (4, 11). To date, at least 70 ITS genotypes have been reported to infect humans and animals (2, 6). The zoonotic nature of E. bieneusi was confirmed because ITS genotypes found in domestic and wild animals had been reported to occur in immunosuppressed hosts (22). In Thailand, we reported genotypes E, O, and PigEBITS 7, which have previously been identified in pigs (3, 4) and in Thai HIV-infected patients (9). This study aimed to identify the ITS genotypes of E. bieneusi in pigs and humans who worked in or lived near pig farms to investigate the transmission of E. bieneusi among these host species.A cross-sectional study of E. bieneusi infection was conducted in a community in Nakorn Pathom Province, Central Thailand, January 2005. This community is composed primarily of four pig farms, a residential area, and a school. The residential area, but not the school, was near the pig farms. Fecal specimens were collected from school children and those who were living in this community, including pig farmers. Fecal specimens were also collected from pigs of four farms and examined for E. bieneusi. The study was approved by the Ethics Committee of the Royal Thai Army, Medical Department. Informed consent was obtained from each adult individual and from parents of school children before enrollment in the study.Fecal specimens from pigs and humans were examined for microsporidian spores using gram-chromotrope staining under light microscopy (13). DNA was prepared from water-ethyl acetate-concentrated stool specimens using FTA filter paper (Whatman Bioscience, United Kingdom) as previously described (21). Amplification of the ITS region of the small-subunit rRNA gene was performed using primers under conditions described by Katzwinkel-Wladarsch et al. (8). For specimens with PCR-negative results, PCR amplification was repeated at least twice. DNA sequencing was conducted by Macrogen Inc., Seoul, Republic of Korea. Nucleotide sequences were determined using the Sequencher program (Gene Codes Corporation, Inc.), and multiple alignment was performed using Clustal X 1.83 for Windows (24). The genotype of each specimen was confirmed by determining the homology of the sequenced PCR product with the published sequence.A total of 268 pig fecal samples were collected. Pigs aged between 21 days and 22 months were examined for E. bieneusi infection. Microsporidial spores were found in 0.7% of pig fecal samples using gram-chromotrope staining, while a greater prevalence of E. bieneusi infection, 15.7%, was detected by PCR. The prevalences of E. bieneusi infection among the four farms and different age groups are presented in Table ​Table1.1. A significantly higher prevalence of E. bieneusi was found in pigs aged 2 to 3.9 months than in pigs of other age groups (chi-square test, P < 0.001). Multivariate analysis confirmed that pigs aged 2 to 3.9 months had a 5.3-times-greater risk of infection than pigs in other age groups (95% confidence interval, 2.6 to 10.6; P < 0.001). Of these 42 E. bieneusi-positive samples, 21 (50%) were successfully characterized by sequencing analysis, and the organism was identified as being of genotypes E (12 samples [57.1%]), O (8 samples [38.1%]), and H (1 sample [4.8%]).

TABLE 1.

Prevalence of E. bieneusi positivity in pig specimens as determined by PCR

Outbreak of Febrile Respiratory Illness Associated with Adenovirus 11a Infection in a Singapore Military Training Camp

Outbreak cases of acute respiratory disease (ARD) associated with subspecies B2 human adenovirus 11a (HAdV-11a) infection were detected during 2005 in a military basic training camp in Singapore. The Singapore HAdV-11a strain is highly similar to other Asian strains of HAdV-11, including strain QS-DLL, which is responsible for the recently described 2006 outbreak of ARD in China.Due to unique risk factors that include crowding and increased physical and psychological stress, military recruits in training are highly susceptible to outbreaks of acute respiratory disease (ARD), which are most often caused by viruses (26, 29). Human adenovirus (HAdV) infections have been recognized for decades as being important causes of ARD among military trainees in North America, Europe, and Asia (5, 8, 10, 15, 25, 27, 30, 32). The HAdV serotypes most frequently associated with ARD in both military and civilian communities include subspecies B1 HAdV-3, HAdV-7, and HAdV-21 and species E HAdV-4. The association of subspecies B2 HAdV infection with ARD has historically been rarely reported and restricted mostly to closed community outbreaks, some of which were documented among military trainees (1, 2, 8, 24, 36).From 11 January 2005 to 14 October 2005, a total of 226 male participants aged 18 to 24 years were enrolled in a study designed to detect and characterize viral agents responsible for ARD in the Singapore military recruit population. Analysis of laboratory data collected during this period identified influenza virus as the primary causative viral agent of respiratory illness among Singapore recruits in training (51 influenza virus-positive isolates out of 226 tested cases [22.5%]). HAdV-associated ARD cases were detected sporadically between January and October 2005. Thirty symptomatic trainees (13.3%) tested positive for HAdV during this period by a PCR assay described previously by Echavarria and colleagues (6). The temporal distribution of confirmed HAdV-associated cases of ARD is shown in Fig. ​Fig.1.1. Cases of adenovirus-associated ARD were detected between February and June 2005 and in October 2005.Open in a separate windowFIG. 1.Temporal distribution of HAdV-associated cases of ARD diagnosed among Singapore military recruits during 2005.One case of coinfection with influenza A virus was detected among these patients. Recruits testing positive for HAdV were between 19 and 21 years old. The examination of clinical characteristics of the HAdV-positive cases of ARD showed that 23.3% of the recruits reported shortness of breath, 50% reported nasal congestion, 80% reported a headache, 80% reported body ache, and 13.3% reported signs of nausea or vomiting. The identified influenza A virus coinfection did not increase the severity of the respiratory symptoms. One patient presented with additional symptoms of conjunctivitis, but no eye swab samples were collected. Adenovirus isolation was accomplished for 27 of the 30 positive clinical specimens. All virus isolates were initially typed as species B HAdVs by PCR as described previously by Metzgar and colleagues (22). Isolates were further characterized by restriction enzyme analysis and sequencing of the hexon and fiber genes as described previously by Kajon and Erdman (13). Digestion with BamHI identified 26 of 27 isolates as belonging to subspecies B2 HAdVs and one isolate as being HAdV-3. Digestion of viral DNAs with BclI, BglII, BstEII, DraI, HindIII, PstI, SmaI, and XbaI determined that the 26 subspecies B2 isolates were identical and identified them as corresponding to genome type 11a (Fig. ​(Fig.2A)2A) (17). The HAdV-3 isolate was identified as belonging to genome type 3a2 (Fig. ​(Fig.2B)2B) (16).Open in a separate windowFIG. 2.(A) Restriction enzyme analysis of a representative HAdV-11a isolate (SNG1222). (B) Restriction enzyme analysis of HAdV-3a2 isolate SNG1206. M, 1-kbp and 100-bp molecular markers.By using the primer sets described in Table ​Table1,1, identical hexon and fiber sequences were obtained for three randomly selected HAdV-11a isolates (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ607010","term_id":"241992696","term_text":"FJ607010"}}FJ607010 and {"type":"entrez-nucleotide","attrs":{"text":"FJ603103","term_id":"284808702","term_text":"FJ603103"}}FJ603103 for isolate SNG1218, accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ607011","term_id":"241992698","term_text":"FJ607011"}}FJ607011 and {"type":"entrez-nucleotide","attrs":{"text":"FJ603104","term_id":"284808704","term_text":"FJ603104"}}FJ603104 for isolate SNG1222, and accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ607012","term_id":"241992700","term_text":"FJ607012"}}FJ607012 and {"type":"entrez-nucleotide","attrs":{"text":"FJ603105","term_id":"284808706","term_text":"FJ603105"}}FJ603105 for isolate SNG1223). Alignment of the sequences of the hexon gene corresponding to hypervariable regions 1 to 7 using the Basic Local Alignment Search Tool (BLAST) optimized for highly similar sequences (megablast) against the NCBI GenBank database (http://www.ncbi.nlm.nih.gov) showed the three examined viruses to correspond to serotype 11 (19, 28). By using ClustalW implemented in Lasergene (DNASTAR, Inc., Madison, WI), the sequences for both the hexon and the fiber genes of the Singapore HAdV-11a strain were found to be highly similar to those reported for Asian and Middle Eastern HAdV-11 strains circulating over the last 2 decades in association with respiratory disease (Table ​(Table2).2). In addition, the complete genomic sequence obtained at the Walter Reed Army Institute of Research for isolate SNG1222 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ597732","term_id":"257122613","term_text":"FJ597732"}}FJ597732) was found to be 99.9% identical to that reported previously for strain QS-DLL, isolated in China in 2006 during an outbreak of ARD (33) (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"FJ643676","term_id":"256028986","term_text":"FJ643676"}}FJ643676).The identified differences between these two genomes are listed in Table ​Table2.2. The results of this study suggest that, in contrast to other geographic locations where HAdV-3, -4, -7, and -21 rank among the most prevalent serotypes, HAdV-11 may be a relatively more important causative agent of ARD in military training facilities in Singapore. The circulation of HAdV-11 in South East Asia has been documented since the early 1960s in association with conjunctivitis, pharyngoconjuctival fever, ARD, and hemorrhagic cystitis among immunocompromised individuals (7, 9, 14, 17, 24, 31, 34-36). The work of Wadell and colleagues demonstrated the existence of two main clusters of relative homology for HAdV-11 genome types: the cluster of prototype-like genomic variants with a tropism for the renal epithelium, and the a-like cluster, with a distinct tropism for the respiratory tract. In addition to their unique restriction site maps, the p-like and a-like genomes differ in the sequences of the fiber gene involving the receptor binding domains (17, 20, 21). As noted by others previously (33), the fiber of the 11a-like genomes is more closely related to the fiber of the prototype strain of HAdV-14, de Wit (99.5% identity), than to the fiber of the prototype strain of HAdV-11, Slobitski (94.4% identity), suggesting that this genomic variant of HAdV-11 is an intertypic recombinant 11-14.

TABLE 1.

Primers used for amplification and sequencing of hexon and fiber genes of Singapore HAdV-11 isolates^a

In Vitro Activity of Anidulafungin and Other Agents against Esophageal Candidiasis-Associated Isolates from a Phase 3 Clinical Trial

The efficacy of anidulafungin, an echinocandin antifungal agent with potent anti-Candida activity, in treating esophageal candidiasis was tested in a double-blind study versus oral fluconazole. Isolates were identified and tested for susceptibility. Candida albicans represented >90% of baseline isolates. The MIC₉₀ of anidulafungin for all strains was 0.06 mg/liter.Anidulafungin is an echinocandin antifungal agent with broad-spectrum activity against Candida species (2, 12, 13, 17, 19, 20), including fluconazole-resistant strains (10, 16); concentration-dependent fungicidal activity; and a long postantifungal effect in vitro and in animal infection models (1, 6, 7, 10, 16, 18). It is available in the United States for intravenous treatment of esophageal candidiasis, a debilitating opportunistic infection among persons with HIV infection (9) for which cross-resistance among azoles may limit treatment options (4, 14). In patients treated with the anidulafungin dosage regimen for esophageal candidiasis (100-mg loading dose followed by 50 mg daily, half of the dosage used for invasive candidiasis), the steady-state mean maximum and minimum plasma concentrations were 4.2 and 1.6 μg/ml, respectively (Eraxis US package insert). Thus, anidulafungin may be a useful alternative to both amphotericin B and the azole antifungal agents in treating severe oral and esophageal candidiasis in persons with HIV infection and AIDS. We determined the in vitro activity of anidulafungin against clinical isolates of Candida spp. from esophageal candidiasis patients, most of them HIV infected, enrolled in a large (601 patients) phase 3 randomized, comparative, double-blind, double-dummy clinical study. The comparator was oral fluconazole, 200 mg administered on day 1 followed by 100 mg daily for 14 to 21 days.Candida isolates obtained from endoscopic biopsy specimens or brushings (11) were sent to a reference laboratory for identification, using standard methods (8), and susceptibility testing. Standard antifungal powders included anidulafungin (Vicuron, Inc., King of Prussia, PA), fluconazole (Pfizer, New York, NY), voriconazole (Pfizer), caspofungin (Merck, Whitehouse Station, PA), flucytosine (Sigma, St. Louis, MO), amphotericin B (Sigma), and itraconazole (Janssen, Beerse, Belgium). Preparation of stock solutions and broth microdilution susceptibility testing were as detailed in CLSI document M27-A2 (5, 15) for all agents except amphotericin B (tested in antibiotic medium 3). Incubation at 35°C was for 24 h (echinocandins) and 48 h (azoles, amphotericin B, and flucytosine). MICs, determined using a reading mirror, were defined as a prominent decrease in turbidity (ca. 50%), except for amphotericin B (complete growth inhibition).Overall, 96% of patients in both treatment arms were infected with C. albicans at baseline, with or without additional Candida species. A majority of the non-C. albicans species isolated at baseline were present in mixed infection with C. albicans. The predominance of C. albicans is characteristic of esophageal candidiasis (3). A total of 441 unique baseline isolates were received by the reference laboratory, including 411 of Candida albicans, 23 of Candida glabrata, 3 of Candida tropicalis, 2 of Candida krusei, and one isolate each of Candida pelliculosa and Candida lusitaniae.Anidulafungin had potent activity against these isolates (Table ​(Table1).1). Its MIC₉₀ was 0.06 μg/ml, and 99% of strains were inhibited by 0.12 μg/ml. The MIC distribution for caspofungin was similar. Micafungin was not available for testing at the time at which the study was conducted. For all of the azoles, susceptibility was greater than 90%. The MIC_50/90 of fluconazole for the 23 C. glabrata isolates was 8/16 μg/ml, respectively. Fluconazole-resistant strains included 3 of C. albicans and 1 of C. glabrata (MIC, ≥64 μg/ml) as well as the 2 of C. krusei (considered resistant irrespective of MIC). The MIC range of anidulafungin for these 6 isolates was 0.015 to 0.06 μg/ml. As noted previously, there is no cross-resistance between azoles and echinocandins (10, 13, 20).

TABLE 1.

In vitro susceptibilities of 441 esophageal isolates of Candida spp. to anidulafungin and six other systemically active antifungal agents

Novel Use of Tryptose Sulfite Cycloserine Egg Yolk Agar for Isolation of Clostridium perfringens during an Outbreak of Necrotizing Enterocolitis in a Neonatal Unit

Clostridium perfringens has been associated with necrotizing enterocolitis (NEC), which is a serious disease of neonates. Our study describes the novel use of selective tryptose sulfite cycloserine with egg yolk agar (TSC-EYA) during a nursery outbreak. This medium provides a rapid, sensitive, and accurate presumptive identification of C. perfringens.Necrotizing enterocolitis (NEC) is the most common acquired disease affecting the gastrointestinal system of neonates, with low-birth-weight babies at highest risk (20, 22). Clinical features of NEC range from mild intestinal signs such as abdominal distension (stage 1), to radiological signs of pneumatosis (stage 2), to advanced disease (stage 3) involving severe abdominal distension, hypotension, and peritonitis (1, 20). The underlying pathophysiology of NEC is poorly understood but is likely to be secondary to multiple injuries to the neonate gut through hypoxia-ischemia, hyperosmolar feeds, and infection (20, 24).No single infectious agent has been consistently identified as a cause of NEC, but Enterobacteriaceae (Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae), viruses (rotavirus, coronavirus, echovirus, norovirus), and clostridial species have all been implicated (4, 23, 25, 30). The pathology of NEC resembles gas gangrene of the intestine caused by Clostridium perfringens, which produces a range of extracellular toxins (10, 11, 19, 27), and colonization with C. perfringens has been shown to be associated with both sporadic and nursery outbreaks of NEC (2-4). However, it is unclear whether C. perfringens is the causative agent of NEC or a marker of intestinal changes associated with the disease (2, 3, 16).Culture for C. perfringens is not usually undertaken for neonatal feces, as C. perfringens is considered a part of the normal fecal flora, with up to 35% of preterm neonates colonized within the first 2 weeks of life (2, 30). In addition, isolation using conventional media such as horse blood agar (HBA), if required, is difficult without the use of selective supplements (2, 17). However, C. perfringens is also a major cause of human food poisoning, and when implicated in food-borne outbreaks, the causative bacterium can be recovered and enumerated by the use of highly selective media such as tryptose sulfite cycloserine agar, which provides a rapid presumptive morphological identification of C. perfringens (6, 8, 9, 31).The neonatal unit at Monash Medical Centre has the capacity for 50 neonates and includes 18 level III (ventilated) neonatal intensive care unit (NICU) beds. The unit has a stable background NEC rate of about 6/1,000 admissions per year. From 1 January to 30 June 2008, 15 neonates were diagnosed with NEC (modified Bell stage 2 and above), increasing the yearly rate to 32/1,000 admissions and raising concerns of an outbreak. Cases were defined as neonates who met the NEC stage 2 or 3 criteria, and controls were defined as current neonates who were in the unit without NEC at the peak June outbreak period (1).Fecal samples were collected from 11 neonates with NEC and 45 without NEC (current controls) from 6 to 20 June 2008. Microbiological investigations were undertaken for possible bacterial and viral pathogens (28).Fecal samples (n = 56) were also cultured for C. perfringens using HBA (Oxoid CM 0331) and incubated for 48 h at 35°C in anaerobic jars. Colonies were examined for anaerobic hemolytic Gram-positive rods. Presumptive identification of C. perfringens was determined by using the reverse CAMP test (RC) (7, 12, 13). All isolates exhibiting hemolysis were confirmed using the RapID ANA II panel (Remel, Kansas) and by 16S rRNA gene sequencing.Fecal samples were also directly cultured on tryptose sulfite cycloserine with egg yolk agar (TSC-EYA), which consisted of perfringens agar base (CM0587; Oxoid) with 5% egg yolk emulsion and d-cycloserine, for 24 h at 35°C (Media Preparation Unit, University of Melbourne) (5, 9, 14, 15). Black, lecithinase-positive or -negative colonies were presumptively identified as C. perfringens and confirmed as described above. To aid in the recovery of clostridia from a background mixture of bacteria, fecal samples were also heat shocked at 60°C for 20 min and then cultured on TSC-EYA (17). Since some C. perfringens isolates are known to be heat sensitive, a third method was used whereby samples were pretreated with ethanol for 1 h before being cultured on TSC-EYA (17, 18, 21).All phenotypically black colonies that exhibited lecithinase activity and were RC positive underwent 16S rRNA gene sequence analysis and multiplex PCR toxinotyping. The preparation of genomic DNA from clostridial isolates and multiplex PCR including primer pair sequences used for genotyping were done as previously described (26, 29).Direct inoculation onto TSC-EYA, which identified four more culture-positive neonates than did standard culture on HBA, was the most sensitive method examined (Table ​(Table1).1). Both heat shock and ethanol shock were less effective approaches for the isolation of C. perfringens than direct plating on TSC-EYA. No positive results were found by other methods using neonate samples that were negative on TSC-EYA. Direct plating on TSC-EYA was also rapid, saving 24 to 48 h compared to standard culture on HBA. In total, C. perfringens was isolated using TSC-EYA from 10 of 56 (18%) of the study subjects: 3 of 11 (27%) NEC cases and 7 of 45 (16%) controls (odds ratio [OR], 1.69; P = 0.46).

TABLE 1.

Clostridium perfringens isolated from neonatal fecal samples

Human α-Defensins Inhibit Hemolysis Mediated by Cholesterol-Dependent Cytolysins

Many pathogenic gram-positive bacteria release exotoxins that belong to the family of cholesterol-dependent cytolysins. Here, we report that human α-defensins HNP-1 to HNP-3 acted in a concentration-dependent manner to protect human red blood cells from the lytic effects of three of these exotoxins: anthrolysin O (ALO), listeriolysin O, and pneumolysin. HD-5 was very effective against listeriolysin O but less effective against the other toxins. Human α-defensins HNP-4 and HD-6 and human β-defensin-1, -2, and -3 lacked protective ability. HNP-1 required intact disulfide bonds to prevent toxin-mediated hemolysis. A fully linearized analog, in which all six cysteines were replaced by aminobutyric acid (Abu) residues, showed greatly reduced binding and protection. A partially unfolded HNP-1 analog, in which only cysteines 9 and 29 were replaced by Abu residues, showed intact ALO binding but was 10-fold less potent in preventing hemolysis. Surface plasmon resonance assays revealed that HNP-1 to HNP-3 bound all three toxins at multiple sites and also that solution-phase HNP molecules could bind immobilized HNP molecules. Defensin concentrations that inhibited hemolysis by ALO and listeriolysin did not prevent these toxins from binding either to red blood cells or to cholesterol. Others have shown that HNP-1 to HNP-3 inhibit lethal toxin of Bacillus anthracis, toxin B of Clostridium difficile, diphtheria toxin, and exotoxin A of Pseudomonas aeruginosa; however, this is the first time these defensins have been shown to inhibit pore-forming toxins. An “ABCDE mechanism” that can account for the ability of HNP-1 to HNP-3 to inhibit so many different exotoxins is proposed.Polymorphonuclear neutrophils (PMNs) contain three α-defensin peptides, called HNP-1, -2, and -3 (18, 59). They have almost identical sequences (XCYCRIPACIAGERRYGTCIYQGRLWAFCC), where “X” is alanine in HNP-1, aspartic acid in HNP-3, and absent in HNP-2. Collectively, HNP-1 to HNP-3 comprise 30 to 50% of total protein in a human PMN''s primary (“azurophil”) granules and 5 to 7% of the cell''s total protein (60). The concentration of HNP-1 to HNP-3 in azurophil granules approximates 50 mg/ml, ensuring that a PMN''s phagocytic vacuoles also contain high HNP concentrations (17). Human PMNs have small amounts of one additional α-defensin, HNP-4 (69), whose sequence differs substantially from HNP-1 to HNP-3 (Fig. ​(Fig.1;1; Table ​Table1).1). The other human α-defensins, HD-5 and HD-6, are primarily expressed in small intestinal Paneth cells and are believed to protect the intestinal tract against food- and waterborne pathogens, to modulate the intestinal flora, and to be key factors in the pathogenesis of inflammatory bowel disease in genetically susceptible humans (3, 67). Although the studies described below focused on human α-defensins, some studies also examined human β-defensin peptides hBD-1, hBD-2, and hBD-3. hBD-1 is expressed constitutively by epithelial cells and keratinocytes throughout the body (76). hBD-2 and hBD-3, which are typically inducible, are also widely expressed. hBD-3 differs from the other α- and β-defensins studied here in two important respects, as follows: its exceptionally high cationicity (net charge, +11) and the relative salt insensitivity of its antimicrobial activity (25, 49).Open in a separate windowFIG. 1.The sequences of six human α-defensins are shown, with their conserved residues boxed.

TABLE 1.

Properties of cytolysins used in this study^a

Molecular Epidemiology of Streptococcus pneumoniae Serogroup 6 Isolates from Fijian Children,Including Newly Identified Serotypes 6C and 6D

Multilocus sequence typing (MLST) was applied to all unique serotype 6C and 6D isolates and a random selection of serotype 6B and 6A isolates from nasopharyngeal swabs from Fijian children enrolled in a recent vaccine trial. The results suggest that Fijian serotype 6D has arisen independently from both serotypes 6A/C and 6B.Infection with Streptococcus pneumoniae is a leading cause of death in children worldwide (15, 19, 25). S. pneumoniae comprises 48 capsular serogroups containing more than 90 serotypes. Serogroup 6 classically consisted of serotypes 6A and 6B. The 7-valent conjugate vaccine (Prevnar; PCV7) includes the 6B antigen and confers some cross-protection against serotype 6A but not 6C (20, 22). Serotype 6C is important in carriage and invasive disease, and its prevalence has increased following widespread use of PCV7 (4, 5, 9, 10, 14, 16, 24).The existence of serotype 6D has been postulated (8) and was created experimentally (2), but naturally occurring isolates have not been identified (2, 8, 17) until recently, when we identified 14 naturally occurring serotype 6D isolates from nasopharyngeal swabs from Fijian children (11). Recently, two serotype 6D isolates have been found in Korean children (1).Initially, serotype 6C was postulated to have arisen from a single, possibly recent, evolutionary event in which serotype 6A wciN was replaced by serotype C wciN (21). Subsequent analyses, predominantly by multilocus sequence typing (MLST), have shown that 6C is genetically diverse, believed to be a consequence of multiple separate conversion events or a single event occurring sufficiently early in pneumococcal evolution (3, 4, 8, 9, 17).Serotype 6C is usually associated with clonal complexes (CCs) containing predominantly serotype 6A, and less commonly, 6B or non-serogroup-6 serotypes (3, 4, 9, 17). To date, MLST has been conducted on two serotype 6D isolates from Korea (ST282) (1), two from China (ST982 and ST4190), and one from Australia (ST4241) (http://spneumoniae.mlst.net). However, the molecular epidemiology of serotype 6D isolates is otherwise uncharacterized.In this study, we conducted MLST of serogroup 6 isolates to determine the genetic diversity and likely evolutionary origin of serotype 6C and 6D isolates.S. pneumoniae was isolated and identified as described elsewhere (18) from children aged 6 to 18 months participating in the Fiji Pneumococcal Project (FiPP) who had received 0, 1, 2, or 3 doses of PCV7 and 0 or 1 dose of 23-valent pneumococcal polysaccharide vaccine (PPV23) (23). Isolates were serotyped by a multiplex PCR-based reverse line blot (mPCR/RLB) assay and/or quellung reaction (before factor serum 6d was available), plus serogroup 6 serotype-specific PCR as previously described (11, 12).MLST was applied to all unique serotype 6C and 6D strains from the FiPP study (n = 52 and 24, respectively), of which 24 and 14 isolates, respectively, were included in our previous report (without ST results) (11). For comparison, we performed MLST on a subset of randomly selected serotype 6A (n = 16) and 6B (n = 17) isolates from the FiPP study.Fresh 18- to 24-h subcultures of S. pneumoniae isolates were suspended in nuclease-free water (Ambion), and genomic DNA was extracted using the DNeasy blood and tissue kit (Qiagen) per the manufacturer''s instructions. MLST was performed using primer pairs described by the Centers for Disease Control and Prevention (http://www.cdc.gov/ncidod/biotech/strep/alt-MLST-primers.htm) (aroE, recP, spi, xpt and ddl) or Enright and Spratt (6) (gdh and gki), except as described below.PCRs were conducted with 25-μl volumes containing approximately 5 ng of genomic DNA, 1 U of AmpliTaq DNA polymerase (Applied Biosystems), 1 × PCR buffer II (50 mM KCl, 10 mM Tris-HCl [pH 8.3]), 3.0 mM MgCl₂, 250 μM each deoxynucleoside triphosphate (dNTP), 0.5 μM forward primer, and 0.5 μM reverse primer (Sigma-Aldrich). PCR cycling conditions were a 5-min hold at 94°C, followed by 35 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 45 s, and a final extension at 72°C for 5 min. Some isolates which produced no or small amounts of PCR product from spi (seven 6C and three 6D isolates) and/or recP (three 6C and one 6A isolate) were successfully amplified with primers described by Enright and Spratt (6) at an annealing temperature of 52°C in 4.5 mM MgCl₂.Amplicons were sequenced in both directions using capillary separation on the ABI 3730xl DNA analyzer with ABI BigDye Terminator labeling (version 3.1) (Australian Genome Research Facility) using the same primers as for amplification. Contiguous sequences were formed and edited using Sequencher 4.9 (Gene Codes Corporation).Allelic profiles and sequence types (STs) were obtained and compared to those of other isolates in the MLST database (http://spneumoniae.mlst.net). Relationships between STs were explored using eBURST version 3 software (Imperial College, London), which is available at the MLST website. For this study, a group was defined as two or more isolates which shared alleles at six of seven loci. Using this stringent definition, a group also defined a clonal complex (CC). Bootstrap analyses of the CC founder are also presented where appropriate.Generally, Fijian serogroup 6 isolates were highly clonal, with the predominant clone in each serotype representing >44% of the isolates. This is not surprising, given that all strains were isolated from children of similar ages over a relatively short period of time in a small, geographically isolated area.Serotype 6A isolates included STs 490 (7/16), 4778, 4779, 499, and 460 (Table ​(Table1).1). ST490 is predicted to be the CC founder (bootstrap value, 96%) and contains predominantly serotype 6A isolates in the MLST database. ST4778 and ST4779 are newly identified in this study and were not assigned to a CC. The only other ST499 isolate in the database (serotype 6A isolate from Finland) was also not assigned to a CC. ST460 is predicted to be a CC founder (bootstrap value, 88%). Most serotype 6A isolates (10/16) belonged to STs which contain only serotype 6A in the database (i.e., ST490, ST499, and ST460).

TABLE 1.

Distribution of STs and eBURST analysis for 109 serotype 6 isolates from Fijian children