Moraxella catarrhalis: A Clinical Microbiology Review PDF

CLINICAL MICROBIOLOGY REVIEWS, Jan. 2002, p. 125–144 Vol. 15, No. 1 0893-8512/02/$04.00⫹0 DOI: 10.1128/CMR.15.1.125–144.2002 Copyright © 2002, American Society for Microbiology. All Rights Reserved. Moraxella catarrhalis: from Emerging to Established Pathogen Cees M. Verduin,1* Cees Hol,2 André Fleer,3 Hans van Dijk,3 and Alex van Belkum1 Department of Medical Microbiology & Infectious Diseases, Erasmus University Medical Center Rotterdam EMCR, 3015 GD Rotterdam,1 Department of Medical Microbiology, Eemland Hospital, 3800 BM Amersfoort,2 and Eijkman- Winkler Institute for Microbiology, Infectious Diseases and Inflammation, Utrecht University Medical Center, University Hospital G04.614, 3508 GA Utrecht,3 The Netherlands INTRODUCTION.......................................................................................................................................................125 TAXONOMY...............................................................................................................................................................126 ISOLATION AND IDENTIFICATION....................................................................................................................127 EPIDEMIOLOGY.......................................................................................................................................................128 Conventional and Molecular Typing Systems....................................................................................................128 Carriage....................................................................................................................................................................129 DISEASES IN CHILDHOOD...................................................................................................................................129 Sinusitis....................................................................................................................................................................129 Otitis Media.............................................................................................................................................................130 Lower Respiratory Tract Infections.....................................................................................................................130 Other Infections......................................................................................................................................................131 INFECTIONS IN ADULTS.......................................................................................................................................131 Laryngitis.................................................................................................................................................................131 Bronchitis and Pneumonia....................................................................................................................................131 Nosocomial Infections............................................................................................................................................132 ANTIMICROBIAL SUSCEPTIBILITY....................................................................................................................132 ␤-Lactamase Production........................................................................................................................................132 CELL WALL STRUCTURES....................................................................................................................................133 Lipooligosaccharides..............................................................................................................................................133 Peptidoglycan...........................................................................................................................................................133 Outer Membrane Proteins.....................................................................................................................................134 Pericellular Structures...........................................................................................................................................134 Capsule.....................................................................................................................................................................135 VIRULENCE...............................................................................................................................................................135 Adherence.................................................................................................................................................................135 Animal Models........................................................................................................................................................135 Complement Resistance.........................................................................................................................................136 IMMUNITY.................................................................................................................................................................137 Antibody Responses to Whole Bacteria...............................................................................................................137 Lipooligosaccharide Immunogenicity...................................................................................................................138 Immunogenicity of Outer Membrane Proteins...................................................................................................138 Local Antibody Response.......................................................................................................................................138 Vaccines....................................................................................................................................................................138 CONCLUDING REMARKS......................................................................................................................................139 ACKNOWLEDGMENTS...........................................................................................................................................139 REFERENCES............................................................................................................................................................139 INTRODUCTION ple (48, 108, 132, 168). Moreover, M. catarrhalis is an impor- Moraxella (Branhamella) catarrhalis, formerly called Neisse- tant cause of lower respiratory tract infections, particularly in ria catarrhalis or Micrococcus catarrhalis, is a gram-negative, adults with chronic obstructive pulmonary disease (COPD) aerobic diplococcus frequently found as a commensal of the (48, 108, 168). In immunocompromized hosts, the bacterium upper respiratory tract (124, 126; G. Ninane, J. Joly, P. Piot, can cause a variety of severe infections including pneumonia, and M. Kraytman, Letter, Lancet ii:149, 1997). Over the last 20 endocarditis, septicemia, and meningitis (48, 63, 72). In addi- to 30 years, the bacterium has emerged as a genuine pathogen tion, hospital outbreaks of respiratory disease due to M. ca- and is now considered an important cause of upper respiratory tarrhalis have been described (188, 200), now establishing the tract infections in otherwise healthy children and elderly peo- bacterium as a nosocomial pathogen. Because M. catarrhalis has long been considered a harmless commensal (48, 124, 126), relatively little is known about its pathogenic characteristics * Corresponding author. Mailing address: Department of Medical and virulence factors, although developments in this field of Microbiology & Infectious Diseases, Erasmus University Medical Cen- ter Rotterdam EMCR, Dr. Molewaterplein 40, 3015 GD Rotterdam, research have accelerated over the past 5 years. The Netherlands. Phone: 31-10-4633510. Fax: 31-10-4633875. E-mail: The emergence of M. catarrhalis as a pathogen in the last [email protected]. decade, together with the increasing prevalence of ␤-lacta- 125 126 VERDUIN ET AL. CLIN. MICROBIOL. REV. FIG. 1. The bacterial species M. catarrhalis and its close relatives. (A) Position of the ␥ proteobacteria in the prokaryotic kingdom. M. catarrhalis is within this class of organisms. (B) More detailed positioning of M. catarrhalis in the order Pseudomonales. Panel A reprinted from reference 106 with permission of the publisher; the data in panel B are derived from Bergey’s Manual of Determinative Bacteriology, 8th ed. (R. E. Buchanan and N. E. Gibbons, ed.), The Williams & Wilkins Co. Baltimore, Md. (web-accessible version). mase-producing strains, has renewed interest in this bacterial Micrococcus catarrhalis actually contained two distinct species, species. In this review, we will summarize important features of Neisseria cinerea and N. catarrhalis (16). These species could be this organism, focusing on microbial epidemiology, virulence, separated based on the results of nitrate and nitrite reduction immunity, and clinical and molecular-pathogenic aspects of and tributyrin conversion testing. Because of the wide phylo- infections caused by this organism. genetic separation between N. catarrhalis and the so-called “true” Neisseria species, observed by a variety of a methods, the TAXONOMY bacterium was moved to the new genus Branhamella in honour In the past, M. catarrhalis was considered a nonpathogenic of Sara E. Branham (49). In 1984, B. catarrhalis was reassigned member of the resident flora of the nasopharynx. It was one of to the genus Moraxella as Moraxella (Branhamella) catarrhalis the species belonging to the so-called nongonococcal, nonme- (34). This genus now contains both coccoid and rod-shaped ningococcal neisseriae, considered to be members of the nor- bacteria, which are genetically related. The position of M. mal flora. The name of the species has caused considerable catarrhalis in the prokaryotic kingdom is shown in Fig. 1. DNA confusion. The bacterium was first described in 1896 (98) and sequencing has substantiated the validity of the current taxo- was called Micrococcus catarrhalis. Later it was renamed Neis- nomic classification (84, 191). Many scientists preferred the seria catarrhalis. In 1963, Berger showed that the original genus name Branhamella catarrhalis, and in several recent publica- VOL. 15, 2002 MORAXELLA CATARRHALIS 127 FIG. 1—Continued. tions this name is primarily used. As a means of resolving this genus. Consequently, M. catarrhalis is the currently preferred semantic problem, Catlin (47) has proposed the formation of a name for this bacterial species. new family, Branhamaceae, to accommodate the genera Moraxella and Branhamella. However, comparison of 16S ISOLATION AND IDENTIFICATION rDNA sequences of Moraxella spp. and these from bacterial species in related genera has demonstrated the close relation Isolation of M. catarrhalis from clinical specimes, e.g., spu- of M. catarrhalis to M. lacunata subsp. lacunata and to a “false” tum, can be complicated by the presence of nonpathogenic Neisseria species, N. ovis. In addition, M. catarrhalis appears to neisseriae. Selective agar media have been used to isolate M. be more closely related to Acinetobacter spp. than to Neisseria catarrhalis with some success. For example, acetazolamide, spp. (84). On the basis of these results, the latter authors which reduces the growth of Neisseria species when used under conclude that there is no rationale for a separate Branhamella aerobic conditions, and the antimicrobial components vanco- 128 VERDUIN ET AL. CLIN. MICROBIOL. REV. mycin, trimethoprim, and amphotericin B may be included in ear effusions can be performed in a single working day (113). an agar medium to inhibit the growth of the normal flora (71, Furthermore, the sensitivity of the PCR tests corresponds to 227). six or seven genome equivalents, making PCR an unrivaled Over the years, the following criteria have been used to diagnostic assay (195). However, it has to be emphasized that unambiguously distinguish M. catarrhalis from other bacterial the technical demands of PCR are still beyond the capacities of species: Gram stain; colony morphology; lack of pigmentation many routine microbiology laboratories, although improve- of the colony on blood agar; oxidase production; DNase pro- ments in robotics and other forms of laboratory automation duction; failure to produce acid from glucose, maltose, su- are fast bridging the current gap between theory and practice. crose, lactose, and fructose; growth at 22°C on nutrient agar; failure to grow on modified Thayer-Martin medium; and, fi- EPIDEMIOLOGY nally, reduction of nitrate and nitrite (76, 214). However, it has For several reasons, epidemiological studies of M. catarrhalis been shown that growth at 22°C and failure to grow on mod- are difficult. Practical typing systems have become available ified Thayer-Martin medium are not reliable parameters for only recently, and the lack of reliable serological tests is at least the correct identification of M. catarrhalis (76). Also, Jönsson partly to blame for this problem. Moreover, the clinical interest et al. (128) showed that colony morphology, Gram stain, and in M. catarrhalis is only relatively recent, and many laboratories oxidase production were an insufficient group of characteristics did not report M. catarrhalis as a pathogen, especially when a to permit correct and final identification of M. catarrhalis in well-recognized pathogen (e.g., S. pneumoniae or H. influen- cultures derived from sputum samples. It should be noted, zae) was present as well. In addition, as mentioned above, the however, that the Gram stain still plays a crucial role both in isolation of M. catarrhalis from sputa is complicated by the the isolation of the bacterium from clinical material (e.g., spu- presence of nonpathogenic neisseriae. Thus, the use of selec- tum) and in its subsequent identification. In typical Gram tive agar media could be advantageous (71, 227). stains, M. catarrhalis presents itself as a gram-negative diplo- coccus with flattened abutting sides. The bacterium has a ten- dency to resist destaining. Colonies on blood agar are nonhe- Conventional and Molecular Typing Systems molytic, round, opaque, convex, and greyish white. The colony Several phenotyping strategies have been described for ep- remains intact when pushed across the surface of the agar. The idemiological typing of M. catarrhalis, although none of these bacteria are oxidase positive, but additional tests are needed has been accepted internationally. Serological typing of lipo- for routine identification. Positive reactions for DNase produc- polysaccharide (LPS) (230), isoelectric focusing of ␤-lactamase tion, reduction of nitrate and nitrite, and tributyrin hydrolysis proteins (179), and electrophoretic profiling of outer mem- are valuable differentiating characteristics (48, 214, 217). Ac- brane proteins (13) have been described but have never been cording to Catlin (48), the identity of M. catarrhalis is best used in large-scale studies. Besides these and some other confirmed by positive reactions in at least three of these dif- regularly employed phenotyping procedures (66, 190), other ferentiating tests, since none of them is 100% sensitive or methods based on nucleic acid polymorphism have become specific by itself. available more recently. Comparison of restriction endonu- Modern DNA technology has opened new avenues for the clease analysis with phenotyping (57) has indicated that detection of M. catarrhalis in clinical materials (e.g., middle ear restriction endonuclease analysis of genomic DNA can be used effusion) without the need for bacterial culture. In particular, successfully for delineation of disease outbreaks (134, 188). PCR tests for M. catarrhalis have been both designed and used Also, macrorestriction enzymes and pulsed-field gel electro- for clinical purposes, with direct detection of M. catarrhalis phoresis have been used to shed light on matters of epidemi- DNA by PCR being concordant with culture and endotoxin ological concern (237, 254). An example of the use of PFGE to detection. However, DNA assays yield significantly more pos- document patient-to-patient transmission is shown in Fig. 2. itive results than does culture when, for instance, middle ear The use of strain-specific DNA probes has also been docu- effusions are analyzed, which suggests superior sensitivity of mented (14, 243). Recent studies identified amplified fragment the DNA amplification assays (70). The clinical relevance of length polymorphism analysis and automated ribotyping as PCR has been validated extensively in the chinchilla model for useful typing procedures (32, 235). Moreover, when these two otitis media. This animal model was instrumental in demon- strategies were used, complement-resistant strains of M. ca- strating the quick and effective effusion-mediated clearance of tarrhalis were found to form a distinct clonal lineage within the DNA and dead M. catarrhalis bacteria from the middle ear species (Fig. 3) (32, 235). These observations are in agreement cleft, implying that in this case a positive PCR result was with earlier studies identifying M. catarrhalis as a genetically indicative of the presence of viable bacteria (193). Moreover, heterogeneous species from which successful clones occasion- PCR has also been reliably used for the detection of mixed ally proliferate (85). Expansion of such competitive types has infections in the same experimental-infection model (11), also been documented during distinct periods and in particular thereby substantiating the applicability of multiplex PCR ap- geographic regions (157). Frequent horizontal gene transfer proaches for the detection of mixed bacterial infections, e.g., seems possible (31, 150), and in relation to the observations with M. catarrhalis, Haemophilus influenzae, and Streptococcus made for complement resistance, it may be postulated that pneumoniae in a single amplification assay. This approach has other phenotypic traits could be acquired through cross-spe- even been successful for culture-negative effusion (194). Re- cies gene acquisition. For instance, Bootsma et al. (31) cently, clinical evaluation of a multiplex PCR specific for the demonstrated that the barrier between M. catarrhalis and above three pathogens plus Alloiococcus otitidis demonstrated gram-positive microorganisms may be occasionally crossed by that reliable DNA amplification-based diagnostics of middle antimicrobial resistance genes. VOL. 15, 2002 MORAXELLA CATARRHALIS 129 ratory infection (40, 196, 230, 231). Klingman et al. (139) investigated the colonization of the respiratory tract of patients with bronchiectasis. A subset of these patients was repeatedly colonized with different M. catarrhalis strains. The patients were colonized with the same strain for an average of 2.3 months as determined by restriction fragment length polymor- phism patterns, and colonization with a new strain did not correlate with changes in clinical status. Although not studied in detail, there are indications that adults with chronic lung disease are colonized at a higher rate than are healthy adults (169). DISEASES IN CHILDHOOD M. catarrhalis is now considered an important pathogen in FIG. 2. Pulsed-field gel electrophoresis of SpeI-digested M. ca- respiratory tract infections, both in children and in adults with tarrhalis DNA. Twenty nosocomial isolates were analyzed, which re- underlying COPD. Occasionally, the bacterium causes sys- sulted in the identification of several clusters of indiscriminate strains. temic disease, e.g., meningitis and sepsis (2, 48, 59, 75). Bac- As indicated at the top, isolates 1 and 2 and isolates 17 and 18 are identical, which fits well with the fact that the strains were isolated teremia due to M. catarrhalis should be considered especially in from the same patients on separate occasions. Strains 5, 10, 11, and 13 febrile children with an underlying immune dysfunction and an were isolated from different patients hospitalized during overlapping upper respiratory tract infection (2). In addition, M. catarrhalis time intervals in the same pediatric department. It was concluded that may be the single cause of sinusitis, otitis media, tracheitis, patient-to-patient transmission occurred in this setting. bronchitis, pneumonia, and, less commonly, ocular infections in children. In children, nasopharyngeal colonization often Carriage precedes the development of M. catarrhalis-mediated disease (89). Below we summarize the clinical features of childhood The M. catarrhalis carriage rate in children is high (up to disease. 75%) (89, 230, 231). In contrast, the carriage rate of M. ca- tarrhalis in healthy adults is very low (about 1 to 3%) (69; T. Ejlartsen, Letter, Eur. J. Clin. Microbiol. Infect. Dis., 10:89, Sinusitis 1991). This inverse relationship between age and colonization has been known since 1907 (9) and is still present today (81; C. Sinus development is a process that may take up to 20 years, Hol, C. M. Verduin, E. van Dijke, J. Verhoef, and H. van Dijk, although the ethmoid and maxillary sinuses are already present Letter, Lancet 341:1281, 1993). At present, there is no good at birth; the development of sphenoid and frontal sinuses starts explanation for the difference in rates of colonization between in the first few years of life (55). Sinusitis is a very common children and adults; one explanation may be the age-depen- infection in early childhood, accounting for about 5 to 10% of dent development of secretory immunoglobulin A (IgA). Re- upper respiratory tract infections (239, 240; E. R. Wald, Edi- markably, IgG antibody levels do not correlate with the state of torial, Pediatr. Ann. 27:787–788, 1998). It is often underdiag- colonization or with lower respiratory tract infection with M. nosed in children because the symptoms are nonspecific. In catarrhalis in children (82). Interestingly, nasopharyngeal car- addition, physical examination and radiology are of little value riage rates are significantly higher in winter and autumn than in young children, and an etiologic diagnosis requires culturing in spring and summer (230). an aspirate of sinus secretions (28). In acute sinusitis (where Monthly or bimonthly sampling of the nasopharynges of symptoms are present for 10 to 30 days) and subacute disease children (n ⫽ 120) by Faden et al. (89) revealed the presence (30 to 120 days), S. pneumoniae, H. influenzae, and M. catarrha- of M. catarrhalis in 77.5% of subjects at least once during the lis are the most frequently isolated bacterial pathogens (27, 28, first 2 years of life. Furthermore, these authors showed a clear 46, 239, 240; Wald, Editorial). S. pneumoniae is found in 30 to relationship between the frequency of colonization and the 40% of patients, while H. influenzae and M. catarrhalis each development of otitis media. A small Japanese study revealed account for approximately 20% of cases. Interestingly, in chil- that colonization in children attending a day care center is dren with asthma, the same distribution of bacterial pathogens highly dynamic (254). Although clusters of genotypes could be is found (238), although Goldenhersch et al. (103) isolated M. discerned and seemed to persist for periods of 2 to 6 weeks, catarrhalis as the predominant pathogen in subacute or chronic frequent changes in the nature of individual colonizing strains sinusitis (symptoms present for more than 30 days) in children of M. catarrhalis were observed. Of note, rates of isolation of with respiratory allergy. It has been suggested that there is a M. catarrhalis are much higher in fall and winter than in spring possible underestimation of isolation rates for M. catarrhalis, and summer. This seasonal difference in isolation is less pro- since the bacterium stops growing in environments with re- nounced with S. pneumoniae or H. influenzae (230) but is quite duced oxygen concentrations, a condition frequently present common in viral infections. during sinusitis and otitis media (39, 204). This would indicate Other authors have described a relationship between the an even greater role for M. catarrhalis in the etiology of these frequency of colonization and the occurrence of upper respi- infectious diseases. 130 VERDUIN ET AL. CLIN. MICROBIOL. REV. FIG. 3. Dendrogram constructed on the basis of RiboPrint pattern types obtained for 13 complement-sensitive and 2 complement-resistant strains of M. catarrhalis (235). Selected were those RiboPrint patterns that are representative of the diverse genogroups that could be identified. The resistant strains appear to be a more homogeneous group (only 2 closely related types encountered among 47 strains) than are the complement-sensitive strains. The tree was constructed in the BioNumerics program developed by Applied Maths (Kortrijk, Belgium). On the basis of Pearson coefficients and unweighted pair group method using arithmetic averages (UPGMA), patterns were normalized using molecular size markers coanalyzed during pattern creation. The 40 to 100 scale above the dendrogram indicates the percent identity between fingerprints compared. The fully automated RiboPrinter has been developed and marketed by Qualicon, a Dupont subsidiary (Warwick, United Kingdom). Otitis Media middle ear during infection (8). In a study using PCR, M. catarrhalis DNA was detected in 46.4% of pediatric chronic Acute otitis media (AOM) is a very frequent infection in middle ear effusion specimens (n ⫽ 97), compared to 54.6% children: before the age of 1 year, around 50% of children have for H. influenzae DNA and 29.9% for S. pneumoniae DNA experienced at least one period of AOM. This proportion rises (194). A large percentage (48%) of specimens was PCR posi- to 70% at the age of 3 years (136, 222; M. L. Kabongo, Letter, tive and culture negative, whereas all culture-positive speci- Am. Fam. Physician 40:34, 39, 1989). Undoubtedly, it is the mens were also PCR positive. It is very unlikely that the PCR- most serious and frequent infection caused by M. catarrhalis in positive yet culture-negative specimens reflect the persistence children, and as such M. catarrhalis causes tremendous mor- of DNA from old infections (10, 193, 194). The severity of bidity and requires the widespread use of antibiotics (20, 58, symptoms and numbers of bacteria in middle ear fluid appear 88, 89, 97, 136, 137, 230). While not frequently encountered as to be lower for M. catarrhalis than for S. pneumoniae or H. a pathogen, M. catarrhalis has been recognized as a specific influenzae (87). pathogen in AOM for nearly 70 years (109). Since 1980, a marked increase has been reported in the isolation of M. ca- Lower Respiratory Tract Infections tarrhalis from middle-ear exudates (26, 141, 155, 213). This increase in M. catarrhalis isolation to approximately 15 to 20% Although lower respiratory tract infections in children are a (187) has been accompanied by the appearance of ␤-lacta- common cause of morbidity and even mortality among chil- mase-producing strains, which now account for approximately dren worldwide, obtaining a microbiological diagnosis is noto- 90 to 95% of isolates. However, the exact magnitude of this riously difficult. Most studies use combinations of serological apparent increase in isolation rates may not have been ade- and conventional microbiological (e.g., culture- or PCR-based) quately measured yet (155), since tympanocentesis and culture methods. Many of these methods have only been used within a of middle ear fluid are not performed routinely. Patel et al. research setting and are not always reliable or readily available (187) cultured the middle ear fluids of 99 children with AOM to clinicians. As a consequence, data concerning the role of M. and isolated S. pneumoniae, nontypeable H. influenzae, and M. catarrhalis in lower respiratory tract infections are not conclu- catarrhalis from 39, 30, and 25% of subjects, respectively. sive. Lower respiratory tract infections due to M. catarrhalis Again, the isolation rates for M. catarrhalis might be an under- appear to be relatively rare during childhood, with most infec- estimation, given the relatively anaerobic environment of the tions occurring in children below the age of 1 year (35). Korppi VOL. 15, 2002 MORAXELLA CATARRHALIS 131 et al. (140) have investigated the seroconversion to M. ca- Laryngitis tarrhalis in patients who were hospitalized with middle (laryn- M. catarrhalis is the most common bacterial species isolated gitis, tracheitis, bronchitis) and lower respiratory tract infec- from adult patients with laryngitis. Schalén et al. (209, 210) tions. They found seroconversion in only 4 (5%) of 76 children found that of 40 adults with this disease, 22 were infected by M. who had M. catarrhalis-positive nasopharyngeal aspirate cul- catarrhalis (55%), compared to 0 of 40 healthy adults. Even so, tures compared to 4 (1%) of 373 children who had negative the exact role of M. catarrhalis, either as an innocent bystander cultures. According to their results, M. catarrhalis is not a likely or as a causal microorganism in the pathogenesis of adult cause of these infections in children. However, in contrast to laryngitis, is not fully understood. these findings, several other studies have indeed implicated M. catarrhalis in lower respiratory tract infections in children. Bronchitis and Pneumonia First, M. catarrhalis has been isolated in pure culture from secretions obtained by tracheal aspiration in neonates, infants, M. catarrhalis is not a common cause of lower respiratory and children with pneumonia (15, 21, 107, 155). Underlying tract infections in healthy adults. However, the bacterium bronchopulmonary dysplasia has been suggested as a predis- causes pulmonary infections in three separate clinical settings posing factor in these cases (15, 60). Second, in a prospective (169): (i) in COPD patients, (ii) pneumonia in the elderly, and study combining microbiological and clinical criteria, M. ca- (iii) as a nosocomial respiratory tract pathogen. tarrhalis was identified as a significant respiratory pathogen in M. catarrhalis is a common cause of exacerbations in COPD children (35). Third, both local and systemic antibody re- (35, 43, 64, 83, 108, 160, 165, 178, 182, 192, 208). In COPD and sponses to M. catarrhalis infection have been documented in otitis media, only S. pneumoniae and nontypeable H. influenzae several studies (25, 51, 90, 91, 101, 102). Pneumonia in children are isolated more often than M. catarrhalis, yet the frequency can be complicated by bacteremia with M. catarrhalis (59, 123, of isolation of M. catarrhalis from sputa has risen during the 224). For example, Ioannidis et al. (123) have presented data past 10 to 15 years (35, 64, 160). This rise cannot be ascribed on 58 cases of M. cattarhalis bacteremia, including cases in 28 only to an increased awareness in the laboratory (64). One children younger than 12 years. Most patients (ca. 70%) had an study has shown M. catarrhalis to be the single most isolated underlying disease (malignancy and/or neutropenia, underly- pathogen in COPD (218). Sarubbi et al. (208) reviewed all ing respiratory tract disorder), and an associated respiratory respiratory tract cultures (n ⫽ 16,627) performed over a period tract infection was identified in half of the patients. In children of 42 months and identified M. catarrhalis in 2.7% (n ⫽ 457) of with bacteremia, skin lesions such as purpuric and petechial these cultures. In this study, M. catarrhalis was found to be the rash were frequent. Of 58 patients, 12 died (21%), including 4 second most commonly isolated respiratory tract pathogen af- of 5 patients with endocarditis and 4 of 7 patients who did not ter nontypeable H. influenzae but ranking before S. pneu- moniae. In addition, these and many other authors (62, 64, 69, receive therapy. In conclusion, although the current literature 192, 208, 251, 252) demonstrated striking seasonality, with does not provide a definite answer, the available data suggest winter and spring being the periods with the greatest incidence that M. catarrhalis can be involved in lower respiratory tract of M. catarrhalis isolation. This pattern is not found with S. infections in children. pneumoniae or H. influenzae (64, 69). Preceding viral respira- tory tract infection caused by respiratory syncytial virus, for Other Infections example, could be a factor in the seasonal variations which have been observed with M. catarrhalis infections, although this M. catarrhalis has been implicated as a cause of bacterial hypothesis remains untested (69, 229). tracheitis in childhood (23, 36, 86, 155; V. K. Wong and W. H. The typical clinical picture of an M. catarrhalis respiratory Mason, Letter, Pediatr. Infect. Dis. J. 6:945–946, 1987), for infection is that of tracheobronchitis, presenting with cough which preceding viral infection has been considered a signifi- and production of purulent sputum. Pneumonia caused by M. cant predisposing factor (Wong and Mason, Letter). In addi- catarrhalis tends to be a relatively mild disease. It differs from tion, a role for this microorganism has been suggested in con- bronchitis by the presence of mostly lower-lobe infiltrates on junctivitis and keratitis (152, 155), although reports on ocular chest X rays (108, 182, 218, 252). High fever, pleuritic pain, and infections have been rare (1, 152, 247; R. L. Bergren, W. S. toxic states are uncommon, as are empyema and bacteremia Tasman, R. T. Wallace, and L. J. Katz, Letter, Arch. Ophthal- (108, 182, 192, 218, 252). Collazos et al. (59) reviewed 15 cases mol. 111:1169–1170, 1993). Finally, one case of fatal meningitis of bacteremic pneumonia due to M. catarrhalis that had been due to M. catarrhalis has been reported (63). reported in the literature. These cases (nine in adults and six in children) were similar in both characteristics and clinical symp- toms to those described for patients with bronchitis or pneu- INFECTIONS IN ADULTS monia without bacteremia. The mortality rate for these bacte- remic cases was 13.3%. An even larger review by Ioannidis et M. catarrhalis has been associated with a variety of clinical al. in 1995 (123) described the clinical spectrum of M. catarrha- syndromes in adults; the most frequent are discussed in more lis bacteremia in 58 patients. Predisposing factors were present detail below. It has to be emphasized, however, that M. ca- in more than 70% of the patients and included neutropenia, tarrhalis can also manifest itself as a pathogen in the nosoco- malignancy, and respiratory impairment, either alone or in mial setting. A rare but very serious and frequently lethal combination. In this study, maculopapular rash appeared to be infection with M. catarrhalis appears to be endocarditis (123, a relatively rare symptom and was most frequently seen in 180, 219). patients with neutropenia. Mortality was high (29%) among 132 VERDUIN ET AL. CLIN. MICROBIOL. REV. patients with underlying respiratory disease, and the infection spiratory wards. They also showed that considerable contami- was more severe when the patient was coinfected with other nation of the environment with M. catarrhalis may occur, respiratory tract pathogens. The overall mortality related to implying a possible aerosol-mediated mode of dissemination. respiratory infection appears to be relatively low (around 10% Thus, important questions remain to be answered with regard [12, 108, 252]). Even so, M. catarrhalis pneumonia often occurs to the nosocomial spread of M. catarrhalis, including the iden- in patients with end-stage pulmonary or malignant disease, and tification of the reservoir of infection and the mode(s) of trans- the short-term mortality in some patient categories is as high as mission. Person-to-person transmission (122, 161, 188, 200) 45% (252). Most patients are elderly (older than 65 years), and and spread from environmental sources (44, 122) have been 90 to 95% of patients have underlying cardiopulmonary dis- implicated in nosocomial transmission on the basis of circum- ease (12, 108, 252), with COPD being present in the majority stantial evidence; of possible significance is the observation of cases. Many patients appear to be malnourished (252). A that the bacterium is able to survive in expectorated sputum for large percentage (⬎70%) are smokers or exsmokers (69). Men at least 3 weeks (44). Nursery schools are sites where frequent appear to be at greater risk than women, although this obser- exchanges of strains may occur (254). Preliminary data do vation could be confounded by, for instance, smoking habits reveal that this may indeed be important in the epidemiology (12, 59, 69, 108, 182). of M. catarrhalis carriage (unpublished observations). Research into the colonization and infection of bronchiec- tasis patients with M. catarrhalis over time has indicated that a ANTIMICROBIAL SUSCEPTIBILITY subset of patients (around 20%) appeared to be chronically Apart from its almost universal ␤-lactamase-mediated resis- colonized with M. catarrhalis, sometimes consecutively with tance to penicillins and its inherent resistance to trimethoprim, four different strains. A causal relation between isolation of the M. catarrhalis remains universally sensitive to most antibiotics bacterium and exacerbations could not be proven, although its used in the treatment of respiratory infections (18, 119, 159). A presence in a large proportion of patients suggests a causal role recent large international study, the Alexander Project 1996– (139). 1997, revealed that 100% of isolates were susceptible to amoxi- An additional organism can also be isolated from about 40 cillin-clavulanic acid, cefixime, chloramphenicol, ciprofloxacin, to 50% of sputum cultures; in most cases, S. pneumoniae or H. and ofloxacin (92). For some antibiotics (cefaclor, ceftriaxone, influenzae are isolated (12, 108, 182, 192). For several reasons, and doxycyclin) a small increase (⬍0.5%) in the incidence of it is important to define the role of M. catarrhalis in these resistant strains was noted over the years. The clinical rele- mixed infections, particularly with respect to the adequate vance of this increase is still unknown. Of note, strains that management of patients and specific antibiotic therapy. In a produce ␤-lactamase are expected to be resistant to penicillin, mixed infection with S. pneumoniae, for example, should treat- ampicillin, amoxicillin, and piperacillin (18, 33, 99, 130). ment for M. catarrhalis be considered at all, or can antibiotic treatment be targeted at the pneumococcus alone? ␤-Lactamase Production Nosocomial Infections Before 1970, no M. catarrhalis isolate was observed to pro- duce ␤-lactamase (48, 245); the first ␤-lactamase-positive That nosocomial infections could be caused by M. catarrhalis strain was isolated in 1976 (245). By 1980, however, 75% of M. has been suggested by several investigators (15, 19, 35, 60, 67, catarrhalis isolates from the United States produced ␤-lacta- 107, 108, 188, 200). In the past it has been difficult to confirm mase (244). By 1990, about 80% of respiratory M. catarrhalis the spread of the organism among hospitalized patients, be- isolates from the United States (130) and over 90% of isolates cause of the lack of a reliable typing system. Furthermore, from England and Scotland were positive for ␤-lactamase (99). because of the mildness of the disease, nosocomial spread can Recent studies from Australia, Europe and the United States be overlooked or simply disregarded. Patterson et al. (188) all noted ␤-lactamase production in over 90% of isolates (74, used restriction endonuclease analysis to confirm an outbreak 95, 154, 223, 231, 242, 251). Walker et al. (242) investigated in a hospital unit. Strains from five patients and two staff trends in antibiotic resistance of M. catarrhalis isolates (n ⫽ members yielded identical genotype patterns when this tech- 375) in a single hospital over a 10-year period (1984 to 1994). nique was used. During the investigation of another putative During this period, the number of isolates showing ␤-lacta- outbreak, immunoblotting with normal human serum was com- mase production increased from 30 to 96%. Moreover, a trend bined with restriction endonuclease analysis to type M. ca- toward reduced susceptibility to four ␤-lactam antibiotics, pen- tarrhalis strains. Six M. catarrhalis isolates from a cluster of icillin G, ceftriaxone, amoxicillin-clavulanic acid, and imi- infections involving five patients in a respiratory unit were penem, but not cefamandole, was observed (although this was shown to be identical to each other and different from other, not clinically relevant). For of penicillin and ceftriaxone, this unrelated strains from the same institution (200). Both meth- trend was due to an increased frequency of ␤-lactamase-posi- ods provided good discrimination between strains, but they tive isolates. However, the increase in the MIC of amoxicillin- were not always in complete agreement (166, 200). Thus, the clavulanic acid and imipenem was not due to the increased use of more than one typing technique was recommended. frequency of ␤-lactamase-positive strains but occurred mainly Another useful option would be restriction endonuclease anal- within the group of ␤-lactamase-positive strains. These obser- ysis with several enzymes rather than just one. Clear vehicles of vations indicate either (i) a selection for more efficient ␤-lac- bacterial dissemination have not yet been identified in the tamases, (ii) a more efficient production of a ␤-lactamase, or clinical setting. However, Ikram et al. (122) found the nosoco- (iii) selection for additional resistance determinants. Given the mial spread of M. catarrhalis to be common, especially in re- high percentage of strains that produce ␤-lactamase and de- VOL. 15, 2002 MORAXELLA CATARRHALIS 133 spite the fact that successful amoxicillin treatment of patients infected with ␤-lactamase-positive M. catarrhalis has been re- ported, clinicians should assume that all isolates of M. catarrha- lis are resistant to amoxicillin, ampicillin, piperacillin, and pen- icillin (73, 147). In M. catarrhalis two types of ␤-lactamases can be found that are phenotypically identical: the BRO-1 and BRO-2 types. Both are membrane associated, and they differ by only a single amino acid. The enzymes are encoded by chromosomal genes, and these genes can be relatively easily transferred from cell to cell by conjugation (159, 245). Fortunately, both enzymes are readily inactivated by ␤-lactamase inhibitors, and all isolates FIG. 4. Schematic structure of the LOS moieties that cover the are still susceptible to amoxicillin in combination with clavu- surface of the M. catarrhalis cells. Three main serotypes, A, B, and C, lanic acid (119, 159). BRO-1 is associated with higher MICs can be discerned, which differ in the nature of the R group. Abbrevi- ations: D-Galp, D-galactose phosphate; Kdo, 2-keto-3-deoxyoctonate; than is BRO-2; the difference is attributed to the production of GlcpNac, N-acetyllactosamine. more enzyme as a consequence of the higher transcriptional activity of the BRO-1 gene. BRO-1 is the most common en- zyme and is present in ca. 90% of ␤-lactamase-positive strains ences imposed by the presence of serotype-specific LOS struc- (159, 245). Recent studies have shown that the ␤-lactamase of tures (Fig. 4) (79, 118). There is a common polysaccharide M. catarrhalis is lipidated, suggesting a gram-positive origin. M. inner core in serotypes A, B, and C, which can, at least in part, catarrhalis is the first gram-negative bacterial species posses- explain the existing cross-reactivity between the serotypes. The sing such a lipidated BRO-type ␤-lactamase (31). This adds to antigenic specificities of the three serotypes are caused by the complexity of the dissemination of antibiotic resistance differences in terminal sugars of one of the branches (118). traits among M. catarrhalis strains in the sense that there seems Moreover, a structural overlap was documented with the LPS to be a possibility for the acquisition of genes even from the moieties from species of the Neisseria and Haemophilus groups. gram-positive gene pool. The G⫹C content of the BRO genes The LOS of serotype B and C contain oligosaccharide chains provides additional proof of their non-Moraxella origin and of variable length. This could be due to phase-variable expres- suggests a recent acquisition event. The lack of a genetic bar- sion of the biosynthetic genes, as suggested by the presence of rier between gram-negative and -positive bacterial species is a tandem repeats (189). Another explanation offered is that vari- reason for clinical concern, and additional research on the ations in the activity of enzymes involved in cell wall assembly mechanism of DNA uptake by M. catarrhalis is certainly war- (influenced by environmental factors or growth rate, for exam- ranted. ple) result in a different oligosaccharide (118). LOS is also ␤-Lactamase from M. catarrhalis not only protects the bac- present in culture supernatants of M. catarrhalis as a part of teria producing the enzyme but also is thought to inactivate subcellular elements called blebs. These small vesicles may penicillin therapy of concomitant infections by serious airway facilitate the distribution of LOS in the host environment. pathogens such as S. pneumoniae and/or nontypeable H. influ- Whether these structures serve some physiological function is enzae (37, 38, 42, 115). This phenomenon is referred to as the currently unknown. LOS serotype A, once adequately detoxi- indirect pathogenicity of M. catarrhalis. Indeed, in such circum- fied, can be used as a vaccine when conjugated to a protein stances, treatment failures have been reported (187, 230), carrier (120). Increases in the levels of anti-LOS IgG are ob- demonstrating the importance of reporting not only pure but served on immunization. The increased levels of antibodies also mixed cultures positive for M. catarrhalis (246). enhanced clearance of bacteria from the lungs of mice after an aerosol-mediated M. catarrhalis infection. Detoxified M. ca- CELL WALL STRUCTURES tarrhalis LOS conjugated to the high-molecular-weight (HMW) surface protein of nontypeable H. influenzae provides a poten- Lipooligosaccharides tially very interesting bivalent vaccine (see also below). Lipooligosaccharide (LOS) is an important virulence factor Peptidoglycan of gram-negative bacteria. M. catarrhalis LOS appears to be semirough, meaning that it contains only one repeating O Regarding the peptidoglycan, the studies by Keller et al. antigen at best (96, 118). In addition, it appears to be more (135) indicate that M. catarrhalis organisms have a multilay- antigenically conserved among strains than does the LOS of ered peptidoglycan architecture. This peptidoglycan layer was other gram-negative bacteria (171). This suggests that it will shown to be responsible for the extraordinary capacity of the probably not serve as a useful basis for a typing system (71). organism to trigger various functional capacities of macro- Even so, Vaneechoutte et al. were able to distinguish three phages. Secretion of tumor necrosis factor and nitrite metab- LOS types, A, B, and C, by enzyme-linked immunosorbent olism plus the cells’ tumoricidal activity were clearly enhanced. assay; these types accounted for more that 95% of all strains. This triggering capacity could be, at least partially, an expla- Type A represents the great majority of strains (61%), with nation for the low virulence of M. catarrhalis. It seems as if types B and C containing more limited numbers of strains (29 peptidoglycan is involved in some sort of suicidal activity, and and 5%, respectively); 5% of strains remain unidentified (228). further studies into the basic mechanisms of this phenomenon The various types can be discriminated by biophysical differ- are certainly needed. 134 VERDUIN ET AL. CLIN. MICROBIOL. REV. Outer Membrane Proteins from the saliva and tracheobronchial mucin (22). The CD protein is a potential vaccine candidate (253), and antibodies In contrast to other nonenteric gram-negative bacterial spe- raised in mice enhanced clearance in a pulmonary challenge cies, the outer membrane protein (OMP) profiles of different model (176). M. catarrhalis strains show a high degree of similarity. Using M. catarrhalis expresses both transferrin and lactoferrin re- sucrose gradient purification of M. catarrhalis outer mem- ceptors on its surface, named transferrin-binding proteins A branes and sodium dodecyl sulfate-polyacrylamide gel electro- and B (TbpA and TbpB) and lactoferrin-binding proteins A phoresis, Murphy and coworkers identified eight major pro- and B (LbpA and LbpB) (30), respectively. These proteins are teins, designated OMPs A through H, ranging from 21 to 98 partially homologous at the genetic level. In addition, homol- kDa (13, 170, 177). OMPs C and D appeared to be two dif- ogous proteins of the lactoferrin-binding proteins as well as the ferent stable forms of the same protein, the CD protein. The transferrin-binding proteins are found in Neisseria spp., Hae- strong degree of similarity of OMP profiles explains why sero- mophilus spp., and other gram-negative bacteria. These pro- typing systems on the basis of OMP profiles are of little epi- teins provide the cell with the capacity to acquire iron by demiological use. On the other hand, these well-conserved sequestering it from host carrier proteins (5, 45, 78). The surface proteins could be interesting vaccine candidates. In receptors themselves appear to be significant virulence factors, recent years the genes for several of these outer membrane since mutation analysis of the transferrin receptor has demon- proteins have been mapped and characterized in more detail. strated an impaired growth capacity for the mutated strain. In addition to OMPs A to H, Murphy and Klingman de- The receptors are also immunogenic and may be interesting scribed a novel OMP, designated HMW-OMP or ubiquitous vaccine candidates (54, 255). Several genes are associated with surface protein UspA (138). UspA has recently been shown to this iron acquisition machinery, with some functioning as as- be encoded by two different genes, which share the coding sociate receptors and others functioning as facilitating factors potential for a homologous, internal protein domain of more (148). Molecular knockout of the gene for transferrin-binding than 90% amino acid sequence homology (4). Additional protein TbpB revealed that in the presence of a TbpB-specific UspA-like genes have been discovered (144). Mutation in the monoclonal antibody and human complement, the mutant re- UspA1-encoding gene resulted in an attenuated phenotype: sisted killing, in contrast to the wild type, which was rapidly adherence capacities of the deletion mutant were significantly killed (149). However, the epitope recognized by the mono- decreased (3). Furthermore, it was demonstrated that UspA2 clonal antibody was surface expressed in only one of three is essential for complement resistance (3, 144). Protein purifi- clinical isolates. cation studies provided proof that the UspA1 protein binds The OMP E antigen appears to be of low immunogenicity, specifically to HEp-2 cells and has an affinity for fibronectin but it does possess universally surface-expressed epitopes in (163). The UspA2 protein, on the other hand, preferentially different M. catarrhalis strains (24). It is a relatively highly binds to vitronectin. Both purified proteins are immunogenic conserved protein, for which no definite function has yet been in mice, and immunized animals clear bacteria from their lungs defined, although it may have a function in the uptake of more rapidly than do nonimmunized mice (163). Genetic stud- nutrients (i.e., fatty acids) by the bacterium (173). In addition, ies have shown that intraspecies variability in the genes can be this recent study found an increased sensitivity to complement- attributed mainly to variation in regions of repetitive DNA in mediated killing in a knockout mutant of OMP E. the genes (61). In addition, electron microscopy of M. catar- Important goals for present and future investigations are to rhalis strains has revealed that the UspA1 and UspA2 pro- determine the antigenic variability of OMPs, to find antigens teins present as “lollipop-shaped” structures protruding from that generate protective antibodies, and to determine the pre- the bacterial surface (114). Interestingly, the structure of the cise function of these proteins in the pathogenesis of diseases Yersinia adhesin (YadA) protein of Yersinia enterocolitica, a caused by the bacterium. protein involved in adhesion and defence against complement- mediated killing, has a very similar overall structural organi- Pericellular Structures zation and function (114, 207). Moreover, many related genes have been identified in the genomes of a wide variety of bac- The attachment of bacteria to mucosal epithelial cells is terial species, suggesting that the proteins serve essential and often mediated by pili or fimbriae. Some studies have provided universally required functions. Although the UspA1 and UspA2 evidence for the expression of pili by M. catarrhalis (156), antigens currently are the best-studied M. catarrhalis proteins, whereas others have been unable to demonstrate their pres- their vaccine potential still is matter of ongoing investigations ence (7, 110). Consequently, some strains may be pilus positive (see also below). whereas others have been proven to lack pili (7, 202). Pili are The heat-modifiable CD OMP could be cloned and ex- composed of polymerized protein subunits called pilins. Marrs pressed in Escherichia coli. The gene appeared to be strictly and Weir (156) found several characteristics that point to the conserved among M. catarrhalis strains (175). Homology was presence of type 4 (MePhe) pili in M. catarrhalis. In addition, found with the porin F protein (OprF) of Pseudomonas spp. electron microscopic data revealed that besides pili similar to (R. de Mot and J. Vanderleyden, Letter, Mol. Microbiol. 13: those of type 4, an additional non-type 4 class of pili exists. 379–380, 1994) and with an OmpA-like protein from Acineto- Elucidation of the prevalence and role of these pili in the bacter spp. (184), indicating transspecies conservation that is pathogenesis and host response to M. catarrhalis requires fur- generally associated with functional importance. The CD pro- ther study (171), although preliminary studies have already tein was found to be involved in binding purified human mucin revealed that fimbriated bacteria bind more efficiently to lower from the nasopharynx and middle ear but not in binding mucin bronchial epithelial cells than nonfimbriated bacteria do (201). VOL. 15, 2002 MORAXELLA CATARRHALIS 135 Capsule adherence study with HEp-2 cell cultures demonstrated that strains derived from infections adhere more efficiently than do The presence of a polysaccharide capsule has been previ- mere colonizers (94). In addition, data from this latter study on ously suggested (7). Capsules are considered to be an impor- experimental periodate treatment suggested that bacterial ad- tant virulence factor in both gram-positive and gram-negative herence in this artificial system appears to be mediated by bacteria. Unlike the situation in many other bacterial patho- microbial carbohydrate moieties. Of interest, adherence of M. gens, the capsule is not detectable when colonies of M. ca- catarrhalis appeared to be stimulated by neutrophil defensins, tarrhalis are examined on agar plates. More research is neces- peptides with broad-spectrum antimicrobial activity, released sary to definitely demonstrate the presence of a capsule and to from activated neutrophils during inflammation, suggesting define its role, if any, in virulence. that defensin-mediated adherence contributes to persistence of infection, for instance in COPD patients (104). VIRULENCE A recent study by Ahmed et al. (6) investigated the influence of charge on adherence. Although bacteria and epithelial cells In general, the pathogenicity and virulence of a microorgan- are both negatively charged, interaction between the negatively ism are determined by its ability to avoid host defense mech- charged surface of M. catarrhalis cells and positively charged anisms. Smith (215) recognizes five cardinal requirements for a domains called microplicea on pharyngeal epithelial cells was bacterium to be virulent: (i) binding, colonization, and infec- found. More research, especially into the role of proteins like tion of mucous surfaces; (ii) entry into host tissues; (iii) mul- fibronectin, vitronectin, and plasminogen in adhesion, is tiplication in the in vivo environment; (iv) interference with needed. The contradictory nature of some of the current ob- host defense mechanisms; and (v) production of damage to the servations only strengthens this suggestion. host. Relatively little is known about the precise virulence traits of M. catarrhalis. Below, information will be provided on bacterial adherence and models of infection, whereas comple- Animal Models ment resistance will be presented as an example for some of The low virulence of M. catarrhalis in laboratory animals has these general virulence features. It has to be emphasized, how- hampered protection experiments and pathogenicity studies in ever, that a complete insight into the full virulence gene rep- rats and mice. Although several studies have been conducted ertoire is still lacking. For example, it has been demonstrated with different animal species, reports describing a reliable in- on the basis of DNA hybridisation studies that M. catarrhalis fection model are scarce. A reproducible, but rather artificial, harbors homologues of phase-variable H. influenzae virulence model was presented by Lee et al. (J. C. Lee, J. C. Hamel, D. genes (189). The precise nature of these genes has yet to be Staperd, and C. W. Ford, Abstr. 93rd Gen. Meet. Am. Soc. elucidated, which implies that on the basis of relatively Microbiol., p. 50, abstr. B-137, 1993), who were able to isolate straightforward cloning experiments, several new virulence live M. catarrhalis from a specific mouse strain, C3H/HeN. genes could well be identified in the near future. Bacteria, suspended in brain heart infusion broth supple- mented with 8% brewer’s yeast and 0.2% Tween 80, were Adherence inoculated via an intraperitoneal route. Infection resulted in It is noteworthy that only a small number of studies on the high mortality and facilitated antibiotic efficacy studies. In an- precise interaction between M. catarrhalis receptors and hu- other murine model (236), designed to study phagocytic re- man antigens have been undertaken. An elegant study was sponses and clearance mechanisms after endotracheal chal- presented by Reddy et al. (198). Using a purified middle ear lenge with M. catarrhalis, a high influx of polymorphonuclear mucin glycoprotein, they showed that only the CD protein of leukocytes into the lungs was noted. Bacteria were cleared M. catarrhalis was capable of establishing a specific interaction from the lungs within 24 to 48 h, and the animals remained with the sialo version of the human protein. A follow-up study healthy. A deficiency in complement component C5 resulted in from the same laboratory revealed immense heterogeneity in a minor delay in clearance. A similar and most frequently used the interaction between upper respiratory tract pathogens and animal model is a mouse model for the study of pulmonary human mucins (22). In this study, the CD protein of M. ca- clearance of M. catarrhalis (226). This model consisted of tarrhalis was shown to specifically attach to the mucin mole- transoral inoculation of bacteria into the lungs under anesthe- cules from the nasopharynx and middle ear but not to mucin sia and operative exposure of the trachea. Enumeration of from the saliva and tracheobronchial mucin. Interactions such viable bacteria in the lungs involved aseptic removal and ho- as these represent the first steps in the process of bacterial mogenization of the lungs, followed by serial dilution and plat- colonization and infection. The general mechanism of cellular ing on agar media. This model permits an evaluation of the adherence of M. catarrhalis to host cell surfaces has been stud- interaction of bacteria with lower respiratory tract epithelium ied by Rikitomi et al. (202). The presence or absence of fim- and the precise assessment of pathologic changes in the lungs. briae did not influence the capacity of the bacterium to adhere As an example, using this model, MacIver et al. (151) obtained or to cause hemagglutination. Indeed, the mechanisms of bind- evidence that immunization with M. catarrhalis-derived outer ing appeared different for adherence and hemagglutination. membrane vesicles gives rise to a systemic IgG antibody re- Another study found no differences between the source of the sponse which is accompanied by enhanced clearance of M. isolate (blood or lungs) and hemagglutination (129). Further- catarrhalis from the lungs, Kyd et al. (143) used a model of more, these investigators showed that attachment was not de- mucosal immunization involving direct inoculation of killed termined primarily by lectin-carbohydrate interactions. In con- bacteria into the Peyer’s patch followed by an intratracheal trast to the findings of these investigators, an in vitro booster with dead M. catarrhalis. Enhanced clearance of bac- 136 VERDUIN ET AL. CLIN. MICROBIOL. REV. teria from the lungs was observed, correlating with higher the biosynthesis of the Gal␣1-4Gal␤1-4Glc LOS epitope, re- levels of specific IgA and IgG in serum and bronchoalveolar sults in enhanced susceptibility to serum-mediated killing lavage fluid. A clear disadvantage of the above models is their (256). Apparently, deviant LOS structures render strains more complex and invasive nature, requiring operation techniques susceptible to complement attack. The structural details facil- and general anesthesia. In addition, the clearance of M. ca- itating these interactions are still unknown. Given that com- tarrhalis from the lungs of mice is relatively rapid (within 6 to plement resistance is considered an important virulence factor 24 h), most probably as a result of the low virulence of M. of neisseriae (68, 127, 153, 199), the similarity between mem- catarrhalis for laboratory animals. Moreover, since M. catarrha- bers of the neisseriae and M. catarrhalis makes complement lis inhabits the upper respiratory tract, inhalation models are resistance and the underlying mechanism an important subject preferred over intraperitoneal, endotracheal, or transoral in- for further study. oculation models. An initial report describing a putatively ef- The clinical relevance of complement resistance was shown fective and reproducible inhalation model in mice was recently for a group of strains isolated from the sputa of elderly persons published. Moreover, passive and active immunization studies (167). Complement resistance can be considered a virulence in this animal model documented improved pulmonary clear- factor of M. catarrhalis: the majority of strains (89%) isolated ance of M. catarrhalis bacteria (120, 121). from lower respiratory tract infections are resistant to comple- Useful infection models in rats have been described only in ment-mediated killing, whereas strains from the upper respi- the past 2 years. A purulent otitis media could be induced in ratory tract of children are mostly sensitive (58%) (117; Hol Sprague-Dawley rats, for instance (248). This infection pro- et al., Letter). Several other authors have tested M. catarrhalis gressed in a relatively mild fashion, lasting for about 1 week. strains for complement resistance (39, 51, 129, 216; R. E. Winn On immunization, a protection rate of 50% or more was in- and S. L. Morse, Abstr. 84th Annu. Meet. Am. Soc. Microbiol., duced. Using the same model, a clear increase in the density of p. 28, 1984). Complement-resistant strains inhibit the terminal goblet cells in the middle ear up to 60 days after inoculation of pathway of complement, i.e., formation of the membrane at- bacteria was found, suggesting a highly increased mucosal se- tack complex of complement (233). The binding of human vi- cretory capacity (50). In another rat model, inhalation of heat- tronectin, an inhibitor of the terminal pathway of complement, killed M. catarrhalis cells clearly affected the laryngeal mucosa appears to play a crucial role in complement resistance of M. (125), resulting in a clinical syndrome reminiscent of laryngo- catarrhalis (234). In Fig. 5 the binding of human vitronectin to tracheitis in children. The studies mentioned above suggest complement-resistant and complement-sensitive strains of M. that rats may provide an infection model that is more interest- catarrhalis is shown. HMW-OMP, also known as ubiquitous ing than was previously thought. surface protein A (composed of two separate proteins, UspA1 Inoculation of M. catarrhalis into the middle ear of chinchil- and UspA2), appears to play a major role (C. M. Verduin, H. J. las and gerbils gave rise to effusion, but no live bacteria could Bootsma, C. Hol, A. Fleer, M. Jansze, K. L. Klingman, T. F. be recovered from the middle ear after 24 h (77). Later studies, Murphy, and H. Van Dijk, Abstr. 95th Gen. Meet. Am. Soc. however, revealed the feasibility of studying otitis media in the Microbiol., p. 189, abstr. B-137, 1995). Indeed, it was shown chinchilla model. Although chinchillas are not generally avail- that vitronectin binds to UspA2 (163). Furthermore, a UspA2 able, PCR would not have reached it current state of applica- mutant strain of M. catarrhalis was sensitive to complement- bility without the studies in these animals (10, 11, 193). mediated killing, whereas the parent strain and an isogenic In conclusion, despite several drawbacks, mouse models of mutant with a mutation in UspA1 were resistant (3). pulmonary clearance appear useful in studies of the effects of Another study (112) suggested that OMP CopB/OMP B2 is vaccination with several M. catarrhalis antigens on clearance of involved in the resistance of M. catarrhalis to killing by normal the bacteria. The rat models appear promising. Suitable animal human serum. An isogenic mutant not expressing CopB was models to study the pathogenicity of infection by M. catarrhalis killed by normal human serum, whereas the wild-type parent in any detail are not yet available. This is primarily because strain survived. In addition, the researchers showed that the rodents, the best accessible laboratory animals, tend to resist CopB- mutant strain was less able to survive in the lungs of infection with this microorganism. mice (112). Recently, it has been shown that inactivation of the CopB-encoding gene inhibits iron acquisition from lactoferrin Complement Resistance and transferrin (5), although this may be due to an indirect effect (29). In addition, CopB had significant homology to Complement resistance is considered an important virulence TonB-dependent OMPs, among which is the N. gonorrhoeae factor of many gram-negative bacteria, which may explain why outer membrane protein FrpB. These proteins bind to and gram-negative bacteria isolated from the blood are largely transport several ligands from the environment into the intra- complement resistant; these strains are also especially success- cellular compartment of the bacterium. These functions are ful in establishing animal models of infection (41, 203). In controlled by the TonB proteins, which are thought to be general, rough strains of gram-negative bacteria, producing involved in energy transduction. Yet another OMP, OMP E, LPS devoid of O-specific side chains, are highly susceptible to has also been shown to be involved in complement resistance. C5b–9-mediated killing whereas smooth strains, which synthe- An M. catarrhalis OMP E knockout mutant showed a clear size complete LPS, are often complement resistant (221). Since increase in serum sensitivity (173). the LPS of M. catarrhalis is of the rough type (96), it presum- We conclude that complement resistance in M. catarrhalis ably does not play a major role in complement resistance. probably is a highly multifactorial process, from the perspec- However, Zaleski et al. recently showed that inactivation of tives of both the host and the pathogen. Within the near future, galE, a gene encoding a UDP-glucose-4-epimerase involved in additional bacterial genes involved in the defense against the VOL. 15, 2002 MORAXELLA CATARRHALIS 137 FIG. 5. Electron micrographs of the binding of human vitronectin to complement-resistant (left) and complement-sensitive (right) strains of M. catarrhalis. Immunogold labeling using antibodies specific for human vitronectin revealed that this protein is effectively bound to the resistant cells whereas the sensitive strain fails to bind a significant quantity of the human matrix protein. Note that the vitronectin protein seems to be attached toward the boundaries of the cellular matrix, which may correlate with the protruded orientation of the vitronectin-binding ubiquitous surface proteins (UspA). complement system may be discovered, requiring a major re- tarrhalis, and nontypeable H. influenzae appeared to be re- search effort to integrate the individual contributions of all the moved from the lungs of mice through various mechanisms different molecules into an overall mechanistic scheme. (186). It was found that, compared to other species, M. ca- tarrhalis was cleared relatively slowly from the lungs, and a IMMUNITY more pronounced, 400-fold increase in numbers of polymor- phonuclear leukocytes in the lungs was observed (186). In M. catarrhalis-related immunology is a rather confusing area addition, it was shown that there was stimulation of adherence of the literature. M. catarrhalis infections are restricted to mu- of M. catarrhalis by neutrophil defensins, peptides with broad- cosal surfaces and are not systemic. Therefore, the correlation spectrum antimicrobial activitity that are released from acti- between systemic antibody responses and protection against vated neutrophils during inflammation, suggesting that defen- this type of infections is not as straightforward as with systemic sin-mediated adherence contributes to persistence of infection, infections caused by other species of gram-negative bacteria. In for instance in COPD patients (104). addition, technical differences in the assays used in different Below we will summarize the literature covering the anti- studies may account for the lack of consistent results (90, 102). body responses of humans to whole bacteria and several dif- In general, surface structures of the bacterium are the main ferent antigens of M. catarrhalis. target for an antibody response, and the recognition of major targets for a protective antibody response is of clear impor- Antibody Responses to Whole Bacteria tance for the development of an efficacious vaccine. Many aspects of immunity to respiratory tract infections Several authors have investigated antibody responses to M. caused by M. catarrhalis are still unknown; they may include catarrhalis in different patient cohorts. Chapman et al. (51) local factors as mucociliary clearance, aerodynamics, alveolar showed that 18 (90%) of 20 adult patients with lower respira- macrophage activity, complement-mediated killing, and surfac- tory tract infections due to M. catarrhalis, as defined by strict tant activity. These factors play important roles in host defense clinical criteria, had bactericidal antibodies in their convales- against oropharyngeal pathogens (225). The development of cent-phase sera whereas only (37%) of 19 had bactericidal an inflammatory response or specific antibody response may, antibody present in their acute-phase sera. Black and Wilson however, augment these host defense mechanisms (225). As an (25) obtained essentially the same results in a larger-scale example, in COPD patients, local host defense against respi- study dealing with IgG antibodies in acute- and convalescent- ratory pathogens is relatively poor, and although M. catarrhalis phase and control sera from adults with bronchopulmonary is not a normal inhabitant of the upper respiratory tract in disease. Likewise, an enzyme-linked immunosorbent assay adults (69; Ejlerten, Letter), infections caused by M. catarrhalis study showed that 10 of 19 children with AOM to M. catarrhalis are frequent in these patients. This points to an important role had an increase in serum IgG antibody titers to the bacterium for these local defense mechanisms in nonspecific clearance of (146). Comparable results were found in the study of Faden et this bacterium. al. (90): 8 of 14 young children (younger than 2 years) with Bacterial clearance and phagocytic cell responses have been otitis media (57%) showed a rise in the levels of serum anti- shown to differ among bacterial species. Streptococci, M. ca- body to their own M. catarrhalis isolate (90). In a recent study 138 VERDUIN ET AL. CLIN. MICROBIOL. REV. of infants with otitis media, a specific IgG response (mainly suggests that there is no immune selective pressure. Still, the IgG1 and IgG3) was detected in 10 of 12 children aged 8 purified antigen could be a promising vaccine candidate (174, months or older compared to 1 of 6 younger children. In 253). Helminen et al. (111) also presented evidence that UspA addition, immunoblotting revealed four immunodominant may be a target for protective antibodies in humans. Chen et OMPs, UspA, CopB, TbpB, and a protein of 60 kDa, probably al. (53) immunized mice with purified UspA and subsequently OMP CD (158). challenged these mice intratracheally with M. catarrhalis. Six Antibodies to M. catarrhalis are very low or absent in chil- hours after challenge, approximately 50% fewer bacteria were dren younger than 1 year, and the development of an antibody isolated from the lungs of the immunized mice than from the response in children, especially of the IgG3 subclass, correlates lungs of the nonimmunized control mice. In addition, antibod- with a decrease in colonization. In addition, antibodies to ies induced complement-dependent bacterial killing of heter- OMPs of M. catarrhalis, mainly of the IgG3 subclass, appear ologous M. catarrhalis strains (53). The finding that IgG3 is a around the age of 4 years (56, 102). Furthermore, failure to major contributing factor in the immune response to M. ca- produce significant levels of IgG3 antibodies against M. ca- tarrhalis was confirmed by a study by Chen et al. of the immune tarrhalis predisposes to infection with the bacterium (100). In response of healthy adults and children to UspA1 and UspA2 addition, all adults appeared to have antibodies to M. catarrha- (52). In a small cohort of children suffering from otitis media, lis (56, 102). Data gathered during studies focusing on single- antibodies specific for UspA1 and UspA2 could be identified protein responses indicate that normal children and adults (206). The amounts of these specific antibodies varied strongly develop a systemic, M. catarrhalis-specific IgG response that with age (205). IgG antibody titers to UspA were low during may be protective. Again, this may in part explain the differ- the first 2 years of life and reached a maximum only during ences between children and adults when the colonization rate adulthood, whereas no specific IgA to UspA could be detected is considered (9, 52, 56, 89, 102; Ejlersten, Letter). in nasopharyngeal secretions of young children. Considering the age-dependent differences in antibody prevalence, the Lipooligosaccharide Immunogenicity question on whether to vaccinate against M. catarrhalis remains relevant. With regard to LOS, it was reported that antibody responses to these surface structures constitute a major part of the hu- Local Antibody Response moral immune response during infection with M. catarrhalis. This antibody response is not serotype specific but is directed Only a few investigators have studied the development of to common epitopes of the LOS of different M. catarrhalis antibody responses in the middle ear fluid of children with serotypes (185, 197). Hence, M. catarrhalis LOS may be of otitis media (89, 90, 133, 220). IgG and IgA appeared to be interest for evaluation as a possible vaccine candidate. The produced locally in the majority of patients, but antibodies usefulness of the LOS surface structures for the development derived from serum were also detected in middle ear fluids of of a vaccine requires more knowledge about the role of these patients with otitis media. Faden et al. (90) showed that middle structures in the pathogenesis of disease and the accompanying ear fluid IgG, IgM, or IgA antibody was produced in 100, 29, immune response, although preliminary results are already and 71% of the children, respectively. Of interest, many chil- promising (105, 120). dren with local antibodies in their middle ear fluid did not develop a systemic antibody response. Local antibodies may Immunogenicity of Outer Membrane Proteins play an important role in the recovery from and prevention of AOM (102). Much research nowadays is focused on the identification and In a study focusing on local IgA antibodies to UspA in the characterization of OMPs of M. catarrhalis as suitable vaccine nasopharyngeal secretions of children colonized by M. ca- candidates. OMP B1, CopB/OMP B2, LbpB, OMP CD, OMP tarrhalis, no response was detected (205). E, OMP G, TbpB, and UspA have all been mentioned as Since M. catarrhalis is a primarily mucosal pathogen, more potential vaccine candidates (53, 111, 158, 168, 172, 173, 175, detailed studies of local immune responses are urgently 212, 253, 255). In contrast, no antibody response to TbpA or needed, and the role of IgA antibodies in resistance to M. LbpA could be detected in convalescent-phase sera from pa- catarrhalis infection clearly needs more attention. tients with pulmonary infections, limiting their role as vaccine candidates (255). Hansen and coworkers showed that CopB/ Vaccines OMP B2 is a target for antibodies that increase pulmonary clearance in the mouse (111). Another study has demonstrated The development of vaccines for the prevention of M. ca- that CopB is essentially well conserved and that most strains tarrhalis-mediated disease is currently a hot topic. The most react with CopB-specific monoclonal antibodies (212). How- promising vaccine candidates have recently been reviewed by ever, certain regions in the protein show interstrain variability; McMichael (162). Most of the molecules that have raised peo- therefore, if this protein is to be developed into a candidate ple’s hopes have been described in the previous section of this vaccine, only its conserved regions should be targeted. Sethi et review and need not be reiterated here. However, the combi- al. (211) found a predominant antibody response to a minor nation of data that is available in today’s literature suggests 84-kDa OMP, designated OMP B1, OMP CD does not appear that the development of an M. catarrhalis vaccine is well under to be an immunodominant antigen, as indicated by the fact that way: animal models of infection have been developed and there is an absence of a new antibody response to this protein described, and several vaccine candidate molecules have been after exacerbation of M. catarrhalis infection in COPD pa- studied with respect to prevalence and genetic conservation tients. Furthermore, the high degree of sequence conservation among different isolates. Although new candidate molecules VOL. 15, 2002 MORAXELLA CATARRHALIS 139 are regularly brought forward (93), relatively little or nothing is 4. Aebi, Ć., I. Maciver, J. L. Latimer, L. D. Cope, M. K. Stevens, S. E. Thomas, G. H. McCracken, Jr., and E. J. Hansen. 1997. A protective epitope of known about the optimal routes for vaccine delivery or Moraxella catarrhalis is encoded by two different genes. Infect Immun. whether there is a need for adjuvants. A recent study, using a 65:4367–4377. rat model, suggests that the mucosal route of delivery for M. 5. Aebi, C., B. Stone, M. Beucher, L. D. Cope, I. Maciver, S. E. Thomas, G. H. McCracken, Jr., P. F. Sparling, and E. J. Hansen. 1996. Expression of the catarrhalis is more effective than systemic immunization (142). CopB outer membrane protein by Moraxella catarrhalis is regulated by iron However, no decisive data are available. It will probably still and affects iron acquisition from transferrin and lactoferrin. Infect. Immun. take more than a decade before the first vaccines for genuine 64:2024–2030. 6. Ahmed, K., T. Nakagawa, Y. Nakano, G. Martinez, A. Ichinose, C. H. clinical use will become available. Zheng, M. Akaki, M. Aikawa, and T. Nagatake. 2000. Attachment of Morax- ella catarrhalis occurs to the positively charged domains of pharyngeal epithelial cells. Microb. Pathog. 28:203–209. CONCLUDING REMARKS 7. Ahmed, K., N. Rikitomi, A. Ichinose, and K. Matsumoto. 1991. Possible presence of a capsule in Branhamella catarrhalis. Microbiol. Immunol. 35: It has become evident over the past decades that M. ca- 361–366. tarrhalis has significant pathogenic potential. Classical antibi- 8. Anonymous. 1994. Otitis media bacteriology and immunology. Pediatr. In- fect. Dis. J. 13(Suppl.):20–22. otic treatment alleviates the clinical burden, but

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