Prevalence and Distribution of the hmw and hia Genes and the HMW and Hia Adhesins among Genetically Diverse Strains of Nontypeable Haemophilus influenzae

Nontypeable Haemophilus influenzae is a common cause of human disease and initiates infection by colonizing the upper respiratory tract. In previous work we identified high-molecular-weight adhesins referred to as HMW1 and HMW2, expressed by nontypeable strain 12, and determined that most strains of nontypeable H. influenzae express one or two antigenically related proteins. More recently, we determined that some strains lack HMW1- and HMW2-like proteins and instead express an adhesin called Hia. In the present study, we determined the prevalence and distribution of the hmw and hia genes in a collection of 59 nontypeable strains previously characterized in terms of genetic relatedness. Based on Southern analysis, 47 strains contained sequences homologous to the hmw1 and hmw2 genes and nine strains contained homologs to hia. No strain harbored both hmw and hia, and three strains harbored neither. Although the hmw and hia genes failed to define distinct genetic divisions, the hmw-deficient strains formed small clusters or lineages within the larger population structure. Additional analysis established that the IS1016 insertion element was uniformly absent from strains containing hmw sequences but was present in two-thirds of the hmw-deficient strains. As IS1016 is associated with the capsule locus (cap) in most encapsulated strains of H. influenzae, we speculate that hmw-deficient nontypeable strains evolved more recently from an encapsulated ancestor.

Haemophilus influenzae is a gram-negative bacterium that commonly inhabits the human upper respiratory tract. Isolates of H. influenzae are subdivided into encapsulated and nonencapsulated forms (11). Encapsulated strains express one of six structurally and antigenically distinct capsular polysaccharides, designated serotypes a to f (11). Nonencapsulated strains are defined on the basis of their failure to agglutinate with typing antisera against the known H. influenzae capsular structures and are referred to as nontypeable (11).Analysis by multilocus enzyme electrophoresis indicates that encapsulated strains are clonal and can be segregated into genetically related clusters, which are grouped into two major phylogenetic divisions (9, 10). Division I includes clusters of serotypes a, b, c, d, and e strains, and division II includes clusters of serotypes a, b, and f strains. All encapsulated strains of H. influenzae have genes necessary for encapsulation (cap genes). These genes are organized into functionally distinct regions, with serotype-specific DNA flanked by DNA common to all serotypes (7). In division I strains, the cap gene cluster lies between direct repeats of an insertion element referred to as IS1016 (5). Division II strains also contain one or more copies of IS1016, but in these strains IS1016 is not associated with cap genes (5).The population structure of nontypeable H. influenzae is less well defined. Musser et al. examined 65 epidemiologically distinct isolates by multilocus enzyme electrophoresis and found considerable heterogeneity, with each isolate corresponding to a unique electromorph type (8). Furthermore, comparison with 177 type b isolates revealed no overlap, suggesting that nontypeable strains are not simply phenotypic variants of common type b strains (8). More recently we studied a series of 123 pharyngeal isolates of nontypeable H. influenzae collected from healthy 3-year-old Finnish children (19). Among these isolates, one was a capsule-deficient type b strain. Interestingly, of the remaining 122 isolates, 31% hybridized with a probe containing the type b cap locus. These findings suggested that a subgroup of nontypeable strains might be more recent descendents of an encapsulated ancestor.In previous work directed at understanding the mechanism by which strains of nontypeable H. influenzae colonize the respiratory tract, we identified two high-molecular-weight adhesins referred to as HMW1 and HMW2 (2, 18). Both of these proteins are expressed by nontypeable H. influenzae 12 and show significant amino acid sequence similarity to one another (2). Preliminary studies suggested that most nontypeable strains express one or two proteins that are antigenically related to HMW1 and HMW2. Further analysis revealed that strains lacking HMW1- and HMW2-like proteins remain capable of efficient attachment to cultured epithelial cells (3). Consistent with this observation, we recently identified another nontypeable H. influenzae high-molecular-weight adhesin referred to as Hia (3). Based on Southern hybridization studies, encapsulated strains of H. influenzae lack hmw1- and hmw2-like genes but uniformly possess an allelic variant of hia (16). In serotype b strains, the counterpart to hia is associated with expression of short, thin surface appendages referred to as fibrils and is called hsf (13, 16).In the present study, we sought to determine the prevalence of the hmw and hia genes in a collection of nontypeable H. influenzae strains. In addition, we addressed the hypothesis that strains can be separated into genetically distinct divisions according to the adhesin they possess. Finally, we examined the possibility that strains lacking hmw genes are more likely to contain cap-specific sequences or IS1016 elements and might be more recent derivatives of an encapsulated line.In performing this study, we exploited the collection of nontypeable H. influenzae strains previously characterized by Musser et al. and defined in terms of genetic relatedness (8). Four biotype 4 strains originally isolated from the blood of newborn infants or women with obstetrical infections were excluded because DNA hybridization studies indicate that they represent a separate Haemophilus species (12), a fifth strain was excluded because it is now known to express a serotype e capsule (14), and a sixth strain was missing from the collection. The remaining 59 strains included in our analysis were recovered between 1937 and 1985 during episodes of otitis media, bacteremia, or sinusitis in children throughout the United States and Canada (8). Control strains included nontypeable H. influenzae 12 and 11 and H. influenzae type b strain Eagan. Strain 12 is the strain from which the hmw1 and hmw2 genes were originally cloned (2), and strain 11 is the strain from which hia was originally cloned (3). Strain Eagan contains an allelic variant of hia (referred to as hsf) and an intact type b cap locus (13, 15).In initial experiments, we examined the 59 strains by Southern analysis, probing with a 3.2-kb SpeI-EcoRI fragment that corresponds to the promoter and 5′ coding sequence of the H. influenzae 12 HMW1 structural gene (hmw1A). Hybridization conditions were as previously described (17). As summarized in Fig. ​Fig.1,1, 47 strains demonstrated specific high-molecular-weight bands of hybridization. In some cases, only a single band was discernible, while in others two bands were clearly present. Of note, in strain 12 the chromosomal fragments harboring hmw1A and hmw2A comigrated, giving the impression of a single band of hybridization. Figure ​Figure2A2A depicts a representative sampling of these strains. Nontypeable H. influenzae 11, known to lack hmw genes, served as a negative control. Open in a separate windowFIG. 1Genetic relationships between the 59 strains examined in this study. The dendrogram was generated previously by Musser et al. (8) by the average-linkage method of clustering from a matrix of coefficients of genetic distance, based on 15 metabolic enzymes. Circles indicate strains that hybridized with hmw but not hia, squares indicate strains that hybridized with hia but not hmw, and triangles indicate strains that failed to hybridize with either hia or hmw. Blackened symbols indicate strains that hybridized with pUO38 and IS1016. The numbers and letters to the right of the symbols are strain designations. Numbers in parentheses refer to level of adherence.Open in a separate windowFIG. 2Southern blotting performed with probes for hmw sequences and hia. Chromosomal DNA was digested with BglII, separated by agarose electrophoresis, and subjected to Southern analysis. (A) Southern analysis with a probe corresponding to the promoter and 5′ coding sequence of hmw1A. Strains by lane: 1, 12; 2, 11; 3, 3219B; 4, 3686; 5, 3242A; 6, 3219C; 7, 3655; 8, 1667; 9, 3246A; 10, 1484A; 11, 1674. Less chromosomal DNA was inadvertently loaded in lanes 7 and 9. (B) Southern analysis with a probe that represents an intragenic fragment of hia. Strains by lane: 1, 12; 2, 11; 3, 1862; 4, 3179B; 5, 3640; 6, 3A; 7, 1860; 8, 3230B; 9, 3248A; 10, 3230A; 11, 1396B. Comparable quantities of DNA were loaded in each lane.Next we probed chromosomal DNA from the 59 strains with a 1.6-kb SspI-StyI fragment that corresponds to an intragenic region of the H. influenzae 11 hia locus. Nontypeable H. influenzae 12, lacking an hia homolog, served as a negative control, while strain 11 was the positive control. As summarized in Fig. ​Fig.11 and illustrated in Fig. ​Fig.2B,2B, nine strains demonstrated specific bands of hybridization. Interestingly, the 47 strains with hmw sequences and the nine strains with an hia homolog were mutually exclusive but failed to segregate into genetically distinct divisions (Fig. ​(Fig.1).1). Nevertheless, the strains containing an hia homolog formed small clusters within the larger population structure.To gain additional insight into the evolutionary relationship between typeable and nontypeable strains of H. influenzae, we performed Southern analysis with pUO38, a pBR322 derivative that contains a complete set of the cap genes, including one copy of IS1016, from a phylogenetic division I H. influenzae type b strain (6). We refer to this set of genes, which includes sequences common to all encapsulated H. influenzae strains, as the cap locus. Of the 47 strains with hmw genes, none demonstrated hybridization with pUO38 (Fig. ​(Fig.1).1). In contrast, 8 of the remaining 12 strains, including 6 of the 9 strains with an hia gene, showed a single band of hybridization (Fig. ​(Fig.3).3). Further analysis revealed that in all strains, hybridization with IS1016 accounted entirely for the hybridization seen with pUO38 (not shown). H. influenzae type b strain Eagan served as a control for these blots and gave the predicted pattern of hybridizing bands, including bands of 2.1, 2.7, 4.4, 9.0, 10.7, and 16 kb with pUO38 and bands of 9.0, 10.7, and 16 kb with IS1016 (5, 19). Hybridization studies performed with radiolabeled pBR322 were negative for all strains (not shown). Open in a separate windowFIG. 3Southern blotting performed with radiolabeled pUO38. Chromosomal DNA was digested with EcoRI, separated by agarose electrophoresis, and subjected to Southern analysis. Strains by lane: 1, Eagan; 2, 1862; 3, 3179B; 4, 3640; 5, 3A; 6, 1860; 7, 3230B; 8, 3248A; 9, 3230A; 10, 1396B; 11, 3232A; 12, 3639.To determine whether the presence of hmw and hia sequences correlated with specific protein expression, whole-cell lysates of all 59 strains were examined by Western immunoblot analysis with a rabbit polyclonal antiserum reactive with HMW1 and HMW2 and a rabbit polyclonal antiserum raised against Hia. Forty-five of the 47 strains with hmw genes expressed one or two proteins that reacted with serum 25G (2), with the reactive proteins ranging in size between ∼105 and ∼160 kDa. Figure ​Figure4A4A shows a representative set of these strains. Among the 12 strains that failed to hybridize with hmw1A, none expressed proteins reactive with serum 25G. Control strains for these immunoblots included strain 12 (expressing HMW1 and HMW2) and strain 11 (lacking HMW proteins). Open in a separate windowFIG. 4Western immunoblotting performed with serum 25G against HMW1 and HMW2 or serum 36B against Hia. Whole-cell lysates were prepared, and proteins were separated on a 7.5% polyacrylamide gel. (A) Western analysis with serum 25G. Strains by lane: 1, 12; 2, 11; 3, 3219B; 4, 3686; 5, 3242A; 6, 3219C; 7, 3655; 8, 1667; 9, 3246A; 10, 1484A; 11, 1674; 12, 3894. (B) Western analysis with serum 36B. Strains by lane: 1, 12; 2, 11; 3, 11hia::kan; 4, 1862; 5, 3179B; 6, 3640; 7, 3A; 8, 1860; 9, 3230B; 10, 3248A; 11, 3230A; 12, 1396B.Analysis of the 59 strains with serum 36B (3), directed against Hia, revealed reactivity with all 9 strains containing an hia gene but with none of the remaining 50 isolates. Interestingly, in six of the nine reactive strains, multiple high-molecular-weight bands were detected, suggesting formation of multimers or aggregates (not shown). Formic acid treatment (4) resulted in elimination of the high-molecular-weight bands and generation of a predominant band ranging in size between ∼80 and ∼120 kDa (Fig. ​(Fig.4B).4B). Control strains for these blots again included strain 12 and strain 11, along with a strain 11 mutant deficient in expression of Hia.To examine whether expression of HMW or Hia proteins correlated with a capacity for adherence to human epithelial cells, we performed adherence assays with Chang epithelial cells. HMW1, HMW2, and Hia originally were identified as adhesins based on the ability to promote attachment to Chang cells. In the present study, adherence was measured by staining samples with Giemsa and then examining with light microscopy and counting the number of bacteria associated with 25 cells. Adherence was considered 3+ if the average number of bacteria per cell was greater than 25, 2+ if the average number was 10 to 25, and 1+ if the average number was 1 to 10. Strains were considered nonadherent if the average number of bacteria per cell was less than one. As summarized in Fig. ​Fig.1,1, 45 of the 47 strains with hmw genes, including all 45 containing proteins reactive with serum 25G, demonstrated appreciable in vitro adherence. In 43 of these strains, the level of adherence was moderate to high (2+ to 3+). All nine of the strains with an hia homolog demonstrated high-level adherence (3+). Interestingly, the three strains that were negative by Southern blotting with both hmw1A and hia were nonadherent.To summarize, Southern analysis of the 59 strains included in this study revealed three subsets, including 80% with an hmw1A or hmw2A homolog, 15% lacking hmw1A and hmw2A genes but harboring an hia homolog, and 5% lacking both hmw and hia sequences. Strains containing an hmw gene were uniformly devoid of IS1016, an insertion element associated with the cap locus in division I encapsulated H. influenzae. In contrast, nearly 70% of the hmw-deficient strains possessed one copy of this element.Based on our earlier observations that a subset of nontypeable strains contain the hia gene, which is allelic to the hsf locus found in all encapsulated H. influenzae strains (3, 16), and that 31% of pharyngeal isolates of nontypeable H. influenzae from healthy 3-year-old Finnish children demonstrated hybridization with the cap locus (19), we hypothesized that strains possessing an hia homolog would contain cap sequences as well, reflecting more recent evolution from an encapsulated ancestor. Indeed, in the present study we found that six of nine strains that hybridized with hia also hybridized with pUO38. Further analysis revealed that in all six of these strains, the hybridization with pUO38 was due entirely to the presence of an IS1016 element. It is possible that these strains experienced a deletion of capsule genes due to recombination between duplicated copies of IS1016, leaving one intact copy of IS1016. Such precedent exists in the form of the longtime laboratory strain Rd, which originally contained capsule genes flanked by IS1016 elements and expressed a serotype d capsule but now contains a single copy of IS1016 with no capsule genes and is nonencapsulated (5).Examination of the genetic relatedness between the 47 strains with hmw sequences and the 12 strains without such sequences revealed the absence of separable divisions. Nevertheless, the hmw-deficient strains were found to form clusters within the larger population structure. For example, strains 3179B and 3640, both of which hybridized with hia and IS1016, are more closely related to each other than they are to other strains in the collection. Similarly, strains 1860 (containing hia [hia+] but lacking IS1016 [IS1016−]), 3232A (hia−, IS1016+), and 3230B (hia+, IS1016+) form a second cluster of closely related strains, and strains 1136B (hia−, IS1016−), 3248A (hia+, IS1016−), and 3230A (hia+, IS1016+) form a third such cluster. Consistent with the evidence that strains 3179B and 3640 are closely related and the possibility that they have evolved from the same encapsulated ancestor, in both strains an ∼10-kb EcoRI fragment hybridizes with IS1016.Division I and division II encapsulated H. influenzae strains are separated from one another by a genetic distance of 0.66 (9, 10). As shown in Fig. ​Fig.1,1, the first 58 nontypeable strains in the collection we studied are separated by genetic distances that range between 0.08 and 0.64. Furthermore, the analysis by Musser et al. revealed that strains in positions 1 to 3 (strains 3219B, 3686, and 3242A [Fig. 1]) lie between type b strains belonging to clonal groups A1a and A2a, and strains in positions 4 to 22 (including strains 1862, 3179B, 3640, and 3A) lie between type b strains in clonal groups A2a and B1b (8, 9). Thus, 58 of the 59 nontypeable strains we examined, including 11 of the 12 hmw-deficient strains, are likely to be more closely related to division I encapsulated strains than they are to division II strains. One conclusion consistent with our observations and the dendrogram that exists for encapsulated strains is that strains 1862, 3179B, and 3640 all evolved from a type b ancestor. Similarly, superimposition of the dendrogram for encapsulated H. influenzae suggests that strain 3A might have evolved from a cluster B type b, type d, or type a strain; strains 1860, 3232A, and 3230B might have evolved from a cluster C type b or a cluster D type c strain; and strain 3639 might have evolved from a cluster E type c strain. The origins of strains 1136B, 3248A, 3230A, and 1396B are less clear, except that strain 1396B is more closely related to division II encapsulated strains, suggesting possible evolution from a type a, type b, or type f strain.To examine the correlation between the presence of hmw sequences and expression of HMW protein, we performed Western immunoblot analysis with an antiserum raised against HMW1 and reactive with both HMW1 and HMW2. All but two of the 47 strains with an hmw1A or hmw2A homolog reacted with the antiserum. Of the two nonreactive strains, neither demonstrated appreciable adherence to Chang epithelial cells, thus confirming the absence of significant protein expression. One possibility suggested from a recent study by Barenkamp (1) is that protein expression in these strains was turned off during the course of infection, perhaps related to antibody directed against the HMW proteins, which are known to be immunogenic (2). Whether these strains have intact hmw loci is presently under investigation.To conclude, our results indicate that 95% of nontypeable H. influenzae strains contain either hmw or hia sequences. Furthermore, it appears that hmw-deficient strains might have evolved more recently from an encapsulated progenitor. One possible model is that the primordial H. influenzae strain was nonencapsulated and spawned two separate but overlapping lineages. The first such lineage acquired hmw genes and remained nonencapsulated, while the second acquired hia and cap genes and became encapsulated. Subsequent mutations within the cap locus resulted in evolution of a relatively restricted set of nonencapsulated strains. Studies of additional strain collections may contribute to a better understanding of the prevalence of the hmw and hia genes and provide further insights into the evolutionary relationships between nontypeable and encapsulated forms.     

Characterization of genetic and phenotypic diversity of invasive nontypeable Haemophilus influenzae

The ability of unencapsulated (nontypeable) Haemophilus influenzae (NTHi) to cause systemic disease in healthy children has been recognized only in the past decade. To determine the extent of similarity among invasive nontypeable isolates, we compared strain R2866 with 16 additional NTHi isolates from blood and spinal fluid, 17 nasopharyngeal or throat isolates from healthy children, and 19 isolates from middle ear aspirates. The strains were evaluated for the presence of several genetic loci that affect bacterial surface structures and for biochemical reactions that are known to differ among H. influenzae strains. Eight strains, including four blood isolates, shared several properties with R2866: they were biotype V (indole and ornithine decarboxylase positive, urease negative), contained sequence from the adhesin gene hia, and lacked a genetic island flanked by the infA and ksgA genes. Multilocus sequence typing showed that most biotype V isolates belonged to the same phylogenetic cluster as strain R2866. When present, the infA-ksgA island contains lipopolysaccharide biosynthetic genes, either lic2B and lic2C or homologs of the losA and losB genes described for Haemophilus ducreyi. The island was found in most nasopharyngeal and otitis isolates but was absent from 40% of invasive isolates. Overall, the 33 hmw-negative isolates were much more likely than hmw-containing isolates to have tryptophanase, ornithine decarboxylase, or lysine decarboxylase activity or to contain the hif genes. We conclude (i) that invasive isolates are genetically and phenotypically diverse and (ii) that certain genetic loci of NTHi are frequently found in association among NTHi strains.     

Haemophilus influenzae in children with cystic fibrosis: antimicrobial susceptibility, molecular epidemiology, distribution of adhesins and biofilm formation

Haemophilus influenzae commonly infects the respiratory tract of patients with cystic fibrosis (CF), early in childhood. In this investigation, 79 H. influenzae isolates were recovered from the respiratory secretions of 64 CF patients (median age: 5 years) included in a 5-year follow-up study. Fifteen of the 64 patients contributed two or more H. influenzae isolates overtime. Serotyping, antibiotic susceptibility testing, genotyping, detection of both hmwA and hia adhesin genes and hypermutable strains was carried out. Biofilm formation ability was investigated. Most strains (72/79, 91.2%) were nonencapsulated or nontypeable (NTHi). Resistance to ampicillin (13.9%) and imipenem (17.7%) was the most detected. Few isolates (2.5%) exhibited the hypermutable phenotype. The NTHi strains showed 55 different genotypes, but 19 clusters of closely related strains were identified. Nine clusters included strains that cross-colonised several patients over a long-time period (mean: 3.7 years). Most patients with sequential isolates harboured strains genetically unrelated, but persistent colonisation with the same clone was observed in 37.5% of patients. Over 45% of NTHi strains contained hmwA-related sequences, 26.3%, hia, 8.3% both hmwA and hia, while 19.4% lacked both. A significant association was found between occurrence of an adhesive gene (irrespective of which) and both persistence (P < 0.0001) and long-term cross-colonisation (P < 0.0001). Mean biofilm level formed by the persistent strains was found significantly increased compared to non-persistent ones (P < 0.0001). Hia-positive strains produced significantly more biofilm than hmwA-carrying strains (P < 0.01). Although a high turnover of NTHi strains in FC patients was observed, distinct clones with increased capacity of persistence or cross-colonisation occurred.     

Antimicrobial Susceptibility Patterns of Aeromonas jandaei, A. schubertii, A. trota, and A. veronii Biotype veronii

Fifty-six isolates of four Aeromonas species, which have been documented as causative agents of human infections or isolated from human clinical specimens, were subjected to antimicrobial susceptibility testing using a MicroScan WalkAway conventional (overnight incubation) gram-negative panel. The four species tested and the number of isolates of each were as follows: Aeromonas jandaei, 17; A. schubertii, 12; A. trota, 15; and A. veronii biotype veronii, 12. All isolates of A. trota were susceptible to all antimicrobial agents tested, except cefazolin (20% of isolates were resistant) and cefoxitin (13% of isolates were resistant). All isolates of A. schubertii and A. veronii biotype veronii, as well as 88% of A. jandaei isolates, were resistant to ampicillin. Resistance to ampicillin-sulbactam ranged from 25% of A. schubertii strains to 100% of A. veronii biotype veronii strains. Cefazolin resistance ranged from 17% of A. veronii biotype veronii isolates to 59% of A. jandaei isolates. Imipenem resistance was detected in 65% of A. jandaei strains and 67% of A. veronii biotype veronii strains. A. jandaei displayed resistance to piperacillin and ticarcillin in 53 and 71% of the isolates, respectively. A. veronii biotype veronii strains were 100% susceptible to piperacillin and 100% resistant to ticarcillin. These antibiogram data may be useful in establishing the identification of these four species when members of the genus Aeromonas are isolated from human clinical sources.     

Characterization of IS1245 for Strain Typing of Mycobacterium avium

IS1245 is an insertion element widely prevalent among isolates of Mycobacterium avium. We used PvuII Southern blots to analyze IS1245 polymorphisms among 159 M. avium isolates (141 clinical isolates from 40 human immunodeficiency virus-infected patients plus 18 epidemiologically related environmental isolates) that represented 40 distinct M. avium strains, as resolved by previous studies by pulsed-field gel electrophoresis (PFGE). All 40 strains carried DNA homologous to IS1245 and thus were typeable. Twenty-five (63%) strains had ≥10 copies of the element, 6 (15%) had 4 to 9 copies, and 9 (23%) had only 1 to 3 copies. Among the last group of nine strains (each of which was distinct by PFGE analysis), IS1245 typing resolved only four patterns and thus provided poor discriminatory power. To evaluate the in vivo stability of IS1245, we analyzed 32 strains for which sets of 2 to 19 epidemiologically related isolates were available. For 19 (59%) of these sets, all isolates representing the same strain had indistinguishable IS1245 patterns. Within eight (25%) sets, one or more isolates had IS1245 patterns that differed by one or two fragments from the modal pattern for the isolates of that strain. Five (16%) sets included isolates whose patterns differed by three or more fragments; on the basis of IS1245 typing those isolates would have been designated distinct strains. IS1245 was stable during in vitro passage, suggesting that the variations observed represented natural translocations of the element. IS1245 provides a useful tool for molecular strain typing of M. avium but may have limitations for analyzing strains with low copy numbers or for resolving extended epidemiologic relationships.     

Nasopharyngeal Haemophilus influenzae carriage in Japanese children attending day-care centers

We conducted a prospective bacteriological survey to investigate antibiotic resistance-related genetic characteristics and the turnover of nasopharyngeal Haemophilus influenzae carriage in healthy children in day-care centers (DCCs). A total of 363 nasopharyngeal mucus samples were collected from children aged 0 to 6 years attending two DCCs in the summer of 2004 (n = 184) and the following winter (n = 179). We obtained 172 H. influenzae isolates and analyzed them by antimicrobial susceptibility testing, PCR for bla_TEM-1 and the penicillin-binding protein (PBP) gene, and pulsed-field gel electrophoresis (PFGE). The overall carriage rate was 47.4% (172/363), and 37.2% of the isolates (64/172) were ampicillin (AMP) resistant. All the resistant isolates had a PBP mutation(s), while only three isolates had TEM-1. The carriage rate was significantly higher in the winter than in the summer (56.4% and 38.6%, respectively), owing to the increase in the numbers of AMP-susceptible H. influenzae isolates in the winter. Children aged ≤3 years showed a higher rate of carriage of H. influenzae isolates with an AMP resistance gene(s) than those aged ≥4 years (21.9% and 12.6%, respectively). Forty-two strains with different PFGE patterns were obtained from among the 172 isolates. Only five strains were observed in both seasons. None of the strains isolated in the summer was isolated from the same carrier in the winter. Twenty-seven strains (64.3%) were isolated from two or more children, and 25 of these were each isolated from children belonging to the same DCC. These results indicate the spread of H. influenzae, particularly those with a PBP mutation(s), and the highly vigorous genetic turnover and substantial horizontal transmission of this pathogen in healthy children attending DCCs in Japan.     

Identification of Haemophilus influenzae Clones Associated with Invasive Disease a Decade after Introduction of H. influenzae Serotype b Vaccination in Italy

The introduction of Haemophilus influenzae serotype b (Hib) conjugate vaccines has changed the epidemiology of invasive H. influenzae disease, with a shift in the predominant serotype from Hib to nonencapsulated H. influenzae (ncHi). The objective of this study was to identify the genotypes/clones associated with invasive H. influenzae disease in Italy. Eighty-seven H. influenzae strains isolated in the years 2009 to 2011 within the National Surveillance of Invasive Bacterial Disease program were analyzed. Strains were characterized by serotyping, antimicrobial susceptibility testing, and multilocus sequence typing (MLST). Genetic polymorphisms in the bla_TEM gene promoter region as well as the occurrence of both adhesin genes (hmwA and hia) and the IgA1 protease-encoding gene (igaB) were also investigated. Of 87 strains, 67 were ncHi and 20 were encapsulated. Eleven strains were β-lactamase positive, harboring the bla_TEM gene. Most bla_TEM genes (10/11) were associated with a Pdel promoter region exhibiting a 135-bp deletion; the remaining strain possessed the Pa/Pb overlapping promoter. MLST analysis showed that encapsulated isolates were clonal, with each serotype sharing a few related sequence types (STs). Forty-six different STs were identified among the 67 ncHi strains. Despite this heterogeneity, a group of closely related STs (ST103, ST139, and ST145) encompassed almost 25% of all ncHi strains and 45.5% of the β-lactamase producers carrying the Pdel promoter. These major ST clones were found to be associated with the hmwA gene but not with the igaB gene. To conclude, although the heterogeneity of the ncHi population was confirmed, diffusion of major successful ST clones was documented.     

Potential of a Novel Protein, OMP26, from Nontypeable Haemophilus influenzae To Enhance Pulmonary Clearance in a Rat Model

A major outer membrane protein band of approximately 25 to 27 kDa is commonly observed in strains of Haemophilus influenzae. This study has investigated the potential of a 26-kDa protein (OMP26) from nontypeable H. influenzae (NTHI) as a vaccine candidate. OMP26 was used to immunize rats via intestinal Peyer’s patches, followed by an intratracheal boost. Immunization was found to significantly enhance bacterial clearance following pulmonary challenge with both the homologous NTHI strain and a different NTHI strain. Significant levels of anti-OMP26 were found in the serum and bronchoalveolar lavage from immunized rats, and isotypes of immunoglobulin G (IgG) were also measured in serum. Analysis of IgG isotypes present in serum following OMP26-immunization suggest that predominantly a T-helper 1-type response was induced. The OMP26 protein was amino-terminally sequenced and found to have no homology with the P5 of H. influenzae type b P5 or the fimbrin protein of NTHI, both can migrate upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis at similar molecular masses but OMP26 has 100% homology with a segment of the H. influenzae Rd genome. The results of this study suggest that OMP26 may be a suitable vaccine candidate against NTHI infection and warrants continued investigation and characterization.     

Pneumococcal Haemophilus influenzae protein D conjugate vaccine induces antibodies that inhibit glycerophosphodiester phosphodiesterase activity of protein D

Haemophilus influenzae outer membrane protein D (PD) is a glycerophosphodiester phosphodiesterase (GlpQ) activity-possessing virulence factor and a promising vaccine antigen, providing 35.3% efficacy against acute otitis media caused by nontypeable H. influenzae (NTHI) when it was used as a carrier protein in a novel pneumococcal PD conjugate (Pnc-PD) vaccine. To study if PD-induced protection against NTHI could be due to antibodies that inhibit or neutralize its enzymatic activity, a GlpQ enzyme inhibition assay was developed, and serum samples collected from Finnish infants before and after Pnc-PD vaccination were analyzed for enzyme inhibition and anti-PD immunoglobulin G (IgG) antibody concentration. Before vaccination at age 2 months, the majority (84%) of infants (n = 69) had no detectable anti-PD IgG antibodies, and all were enzyme inhibition assay negative (inhibition index, <20). At age 13 to 16 months, all infants receiving three or four doses of Pnc-PD had detectable anti-PD IgG antibodies and 36% (8/22 infants) of the infants receiving three doses and 26% (6/23 infants) of the infants receiving four doses of Pnc-PD were inhibition assay positive (inhibition index, ≥20). No significant rise in anti-PD IgG antibodies or enzyme inhibition among control vaccinees (n = 24) receiving three doses of hepatitis B vaccine was detected. A modest correlation (r_s, ~0.66) between anti-PD IgG concentration and enzyme inhibition was detected; however, their kinetics were clearly different. These data suggest that measurement of antibody responses that inhibit PD's enzymatic activity could be a useful tool for assessing Pnc-PD vaccine-induced protective immunity against NTHI.     

In vitro susceptibility of invasive isolates of Candida spp. to anidulafungin, caspofungin, and micafungin: six years of global surveillance

The echinocandins are being used increasingly as therapy for invasive candidiasis. Prospective sentinel surveillance for the emergence of in vitro resistance to the echinocandins among invasive Candida sp. isolates is indicated. We determined the in vitro activities of anidulafungin, caspofungin, and micafungin against 5,346 invasive (bloodstream or sterile-site) isolates of Candida spp. collected from over 90 medical centers worldwide from 1 January 2001 to 31 December 2006. We performed susceptibility testing according to the CLSI M27-A2 method and used RPMI 1640 broth, 24-h incubation, and a prominent inhibition endpoint for determination of the MICs. Of 5,346 invasive Candida sp. isolates, species distribution was 54% C. albicans, 14% C. parapsilosis, 14% C. glabrata, 12% C. tropicalis, 3% C. krusei, 1% C. guilliermondii, and 2% other Candida spp. Overall, all three echinocandins were very active against Candida: anidulafungin (MIC₅₀, 0.06 μg/ml; MIC₉₀, 2 μg/ml), caspofungin (MIC₅₀, 0.03 μg/ml; MIC₉₀, 0.25 μg/ml), micafungin (MIC₅₀, 0.015 μg/ml; MIC₉₀, 1 μg/ml). More than 99% of isolates were inhibited by ≤2 μg/ml of all three agents. Results by species (expressed as the percentages of isolates inhibited by ≤2 μg/ml of anidulafungin, caspofungin, and micafungin, respectively) were as follows: for C. albicans, 99.6%, 100%, and 100%; for C. parapsilosis, 92.5%, 99.9%, and 100%; for C. glabrata, 99.9%, 99.9%, and 100%; for C. tropicalis, 100%, 99.8%, and 100%; for C. krusei, 100%, 100%, and 100%; and for C. guilliermondii, 90.2%, 95.1%, and 100%. There was no significant change in the activities of the three echinocandins over the 6-year study period and no difference in activity by geographic region. All three echinocandins have excellent in vitro activities against invasive strains of Candida isolated from centers worldwide. Our prospective sentinel surveillance reveals no evidence of emerging echinocandin resistance among invasive clinical isolates of Candida spp.     

Clinical and epidemiological aspects of invasive Streptococcus pyogenes infections in Denmark during 2003 and 2004

Active surveillance of invasive group A streptococcal (GAS) infections was conducted in Denmark during 2003 and 2004 as a part of the Strep-EURO initiative. The main objective was to improve understanding of the epidemiology of invasive GAS disease in Denmark. During the 2 years, 278 cases were reported, corresponding to a mean annual incidence of 2.6 cases per 100,000 inhabitants. The vast majority of isolates, 253 (91%), were from blood, with the remaining 25 (9%) being from cerebrospinal fluid, joints, or other normally sterile sites. The mean case fatality rate (CFR) was 20%, with the rate being higher in patients more than 70 years of age (36.5%). For streptococcal toxic shock syndrome (STSS) and necrotizing fasciitis the CFRs were 53% and 25%, respectively. Out of 16 T types recorded, three predominated: T28 (23%), T1 (22%), and the cluster T3/13/B3264 (14%). Among 29 different emm types, emm28 and emm1 accounted for 51% of strains, followed by emm3 (11%), emm89 (7%), and emm12 (5.5%). Low resistance rates were detected for macrolide-lincosamide-streptogramin B (MLS_B) antibiotics (3%) and tetracycline (8%); two isolates exhibited coresistance to tetracycline and macrolides. Of nine pyrogenic exotoxin (superantigen) genes examined, speA and speC were identified in 58% and 40% of the strains, respectively; either of the genes was present in all strains causing STSS. Most strains harbored speG (99%). ssa was present in 14% of the isolates only. In Denmark, as in comparable countries, GAS invasive disease shows a sustained, high endemicity, with involvement of both established and emerging streptococcal emm and T types.     

Virulence factors in urinary Escherichia coli strains: phylogenetic background and quinolone and fluoroquinolone resistance

Quinolone- and fluoroquinolone-resistant Escherichia coli strains harbor fewer virulence factors than susceptible strains. The reasons underlying this correlation are incompletely understood. We investigated the phylogenetic background, the presence of the papC, hlyA, and cnf1 (pathogenicity island II_J96-associated), fimA, iss, and iutA genes, and the presence of type 1 fimbriae, P fimbriae, and hemolysin in 243 urinary E. coli isolates resistant only to quinolones (8%), resistant to both quinolones and fluoroquinolones (51%), or susceptible to both drugs (41%). Group B2 accounted for 56% of the isolates, showing a significantly higher prevalence among fluoroquinolone-susceptible strains than among resistant strains (65% versus 50% [P = 0.03]). hly and cnf1 were significantly more associated with susceptibility (P < 0.001) and with group B2 (P < 0.001 for group B2 versus groups A and D). However, within group B2, fluoroquinolone-resistant strains showed lower prevalences of papC, hlyA, and cnf1 than their susceptible counterparts (P < 0.001). In contrast, the incidence of iutA appeared higher for refractory isolates, including group B2, than for susceptible isolates (P < 0.001). Only in group B2 did fluoroquinolone-resistant strains reveal a lesser ability to agglutinate Saccharomyces cerevisiae (7%) than quinolone-resistant (87%) and susceptible (80%) isolates, despite uniform possession of fimA genes. No similar contrast emerged for expression of hemolysin and P fimbriae. Mutations conferring quinolone and fluoroquinolone resistance may thus require a particular genetic background, not strictly correlated with phylogenetic groups. More interestingly, the mutational event itself can affect the expression of type 1 fimbriae, at least in the prevalent and complex B2 strains.     

Nasopharyngeal Colonization with Nontypeable Haemophilus influenzae in Chinchillas

Colonization of the nasopharynx by a middle ear pathogen is the first step in the development of otitis media in humans. The establishment of an animal model of nasopharyngeal colonization would therefore be of great utility in assessing the potential protective ability of candidate vaccine antigens (especially adhesins) against otitis media. A chinchilla nasopharyngeal colonization model for nontypeable Haemophilus influenzae (NTHI) was developed with antibiotic-resistant strains. This model does not require coinfection with a virus. There was no significant difference in the efficiency of NTHI colonization between adult (1- to 2-year-old) and young (2- to 3-month-old) animals. However, the incidence of middle ear infection following nasopharyngeal colonization was significantly higher in young animals (83 to 89%) than in adult chinchillas (10 to 30%). Chinchillas that had recovered either from a previous middle ear infection caused by NTHI or from an infection by intranasal inoculation with NTHI were completely protected against nasopharyngeal colonization with a homologous strain and were found to be the best positive controls in protection studies. Systemic immunization of chinchillas with inactivated whole-cell preparations significantly protected animals not only against homologous NTHI colonization but also partially against heterologous NTHI infection. In all protected animals, significant serum anti-P6 and anti-HMW antibody responses were observed. The outer membrane P6 and high-molecular-weight (HMW) proteins appear to be promising candidate vaccine antigens to prevent nasopharyngeal colonization and middle ear infection caused by NTHI.     

Copy Number of Pilus Gene Clusters in Haemophilus influenzae and Variation in the hifE Pilin Gene

Brazilian purpuric fever (BPF)-associated Haemophilus influenzae biogroup aegyptius strain F3031 contains two identical copies of a five gene cluster (hifA to hifE) encoding pili similar to well-characterized Hif fimbriae of H. influenzae type b. HifE, the putative pilus tip adhesin of F3031, shares only 40% amino acid sequence similarity with the same molecule from type b strains, whereas the other four proteins have 75 to 95% identity. To determine whether pilus cluster duplication and the hifE_F3031 allele were special features of BPF-associated bacteria, we analyzed a collection of H. influenzae strains by PCR with hifA- and hifE-specific oligonucleotides, by Southern hybridization with a hifC gene probe, and by nucleotide sequencing. The presence of two pilus clusters was limited to some H. influenzae biogroup aegyptius strains. The hifE_F3031 allele was limited to H. influenzae biogroup aegyptius. Two strains contained one copy of hifE_F3031 and one copy of a variant hifE allele. We determined the nucleotide sequences of four hifE genes from H. influenzae biogroup aegyptius and H. influenzae capsule serotypes a and c. The predicted proteins produced by these genes demonstrated only 35 to 70% identity to the three published HifE proteins from nontypeable H. influenzae, serotype b, and BPF strains. The C-terminal third of the molecules implicated in chaperone binding was the most highly conserved region. Three conserved domains in the otherwise highly variable N-terminal putative receptor-binding region of HifE were similar to conserved portions in the N terminus of Neisseria pilus adhesin PilC. We concluded that two pilus clusters and hifE_F3031 were not specific for BPF-causing H. influenzae, and we also identified portions of HifE possibly involved in binding mammalian cell receptors.     

Typing of Human Mycobacterium avium Isolates in Italy by IS1245-Based Restriction Fragment Length Polymorphism Analysis

All but 2 of 63 Mycobacterium avium isolates from distinct geographic areas of Italy exhibited markedly polymorphic, multibanded IS1245 restriction fragment length polymorphism (RFLP) patterns; 2 isolates showed the low-number banding pattern typical of bird isolates. By computer analysis, 41 distinct IS1245 patterns and 10 clusters of essentially identical strains were detected; 40% of the 63 isolates showed genetic relatedness, suggesting the existence of a predominant AIDS-associated IS1245 RFLP pattern.     

Evaluation of Four DNA Typing Techniques in Epidemiological Investigations of Bovine Tuberculosis

DNA fingerprinting techniques were used to type 273 isolates of Mycobacterium bovis from Australia, Canada, the Republic of Ireland, and Iran. The results of restriction fragment length polymorphism (RFLP) analysis with DNA probes from IS6110, the direct repeat (DR), and the polymorphic GC-rich sequence (PGRS) were compared with those of a new PCR-based method called spacer oligonucleotide typing (spoligotyping) developed for the rapid typing of Mycobacterium tuberculosis (J. Kamerbeek et al., J. Clin. Microbiol. 35:907–914, 1997). Eighty-five percent of the isolates harbored a single copy of IS6110, and 81.5% of these carried IS6110 on the characteristic 1.9-kb restriction fragment. RFLP analysis with IS6110 identified 23 different types, RFLP analysis with the DR probe identified 35 types, RFLP analysis with the PGRS probe identified 77 types, and the spoligotyping method identified 35 types. By combining all results, 99 different strains could be identified. Isolate clusters were frequently associated within herds or were found between herds when epidemiological evidence confirmed animal movements. RFLP analysis with IS6110 was sufficiently sensitive for the typing of isolates with more than three copies of IS6110, but RFLP analysis with the PGRS probe was the most sensitive typing technique for strains with only a single copy of IS6110. Spoligotyping may have advantages for the rapid typing of M. bovis, but it needs to be made more sensitive.     

Diversity of Nontypeable Haemophilus influenzae Strains Colonizing Australian Aboriginal and Non-Aboriginal Children

Nontypeable Haemophilus influenzae (NTHI) strains are responsible for respiratory-related infections which cause a significant burden of disease in Australian children. We previously identified a disparity in NTHI culture-defined carriage rates between Aboriginal and non-Aboriginal children (42% versus 11%). The aim of this study was to use molecular techniques to accurately determine the true NTHI carriage rates (excluding other culture-identical Haemophilus spp.) and assess whether the NTHI strain diversity correlates with the disparity in NTHI carriage rates. NTHI isolates were cultured from 595 nasopharyngeal aspirates collected longitudinally from asymptomatic Aboriginal (n = 81) and non-Aboriginal (n = 76) children aged 0 to 2 years living in the Kalgoorlie-Boulder region, Western Australia. NTHI-specific 16S rRNA gene PCR and PCR ribotyping were conducted on these isolates. Confirmation of NTHI by 16S rRNA gene PCR corrected the NTHI carriage rates from 42% to 36% in Aboriginal children and from 11% to 9% in non-Aboriginal children. A total of 75 different NTHI ribotypes were identified, with 51% unique to Aboriginal children and 13% unique to non-Aboriginal children (P < 0.0001). The strain richness (proportion of different NTHI ribotypes) was similar for Aboriginal (19%, 65/346) and non-Aboriginal children (19%, 37/192) (P = 0.909). Persistent carriage of the same ribotype was rare in the two groups, but colonization with multiple NTHI strains was more common in Aboriginal children than in non-Aboriginal children. True NTHI carriage was less than that estimated by culture. The Aboriginal children were more likely to carry unique and multiple NTHI strains, which may contribute to the chronicity of NTHI colonization and subsequent disease.     

Experimental Infection of Mongolian Gerbils with Wild-Type and Mutant Helicobacter pylori Strains

Experimental Helicobacter pylori infection was studied in Mongolian gerbils with fresh human isolates that carry or do not carry cagA (cagA-positive or cagA-negative, respectively), multiply passaged laboratory strains, wild-type strain G1.1, or isogenic ureA, cagA, or vacA mutants of G1.1. Animals were sacrificed 1 to 32 weeks after challenge, the stomach was removed from each animal for quantitative culture, urease test, and histologic testing, and blood was collected for antibody determinations. No colonization occurred after ≥20 in vitro passages of wild-type strain G1.1 or with the ureA mutant of G1.1. In contrast, infection occurred in animals challenged with wild-type G1.1 (99 of 101 animals) or the cagA (25 of 25) or vacA (25 of 29) mutant of G1.1. Infection with G1.1 persisted for at least 8 months. All 15 animals challenged with any of three fresh human cagA-positive isolates became infected, in contrast to only 6 (23%) of 26 animals challenged with one of four fresh human cagA-negative isolates (P < 0.001). Similar to infection in humans, H. pylori colonization of gerbils induced gastric inflammation and a systemic antibody response to H. pylori antigens. These data confirm the utility of gerbils as an animal model of H. pylori infection and indicate the importance of bacterial strain characteristics for successful infection.     

Predominance of Mycobacterium tuberculosis EAI and Beijing Lineages in Yangon, Myanmar

Isolates of the Mycobacterium tuberculosis Beijing lineage are associated with high rates of transmission, hypervirulence and drug resistance. The Beijing lineage has been shown to dominate the tuberculosis (TB) epidemic in East Asia; however, the diversity and frequency of M. tuberculosis genotypes from Myanmar are unknown. We present the first comprehensive study describing the M. tuberculosis isolates circulating in Yangon, Myanmar. Thus, 310 isolates from pulmonary TB patients from Yangon, Myanmar, were genotyped by spoligotyping and IS6110-based restriction fragment length polymorphism analysis (IS6110 RFLP). The most frequent lineages observed were the East African-Indian (EAI; 48.4%; n = 150) and Beijing (31.9%; n = 99) lineages. Isolates belonging to the most frequent shared types (STs), ST1 (n = 98; Beijing), ST292 (n = 28; EAI), and ST89 (n = 11; EAI), had ≥75% similarity in their IS6110 patterns. Five of 11 Beijing isolates comprising five clusters with identical IS6110 RFLP patterns could be discriminated by mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) analysis. Of the 150 EAI isolates, 40 isolates (26.7%) had only one IS6110 copy, and 17 of these isolates could be discriminated by MIRU-VNTR analysis. The findings from this study suggest that although there is a predominance of the ancient EAI lineage in Yangon, the TB epidemic in Yangon is driven by clonal expansion of the ST1 genotype. The Beijing lineage isolates (21.4%) were more likely (P = 0.009) than EAI lineage isolates to be multidrug resistant (MDR) (1.3%; odds ratio, 3.2, adjusted for the patients' history of exposure to anti-TB drugs), suggesting that the spread of MDR Beijing isolates is a major problem in Yangon.