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Evaluation of API NH, a new 2-hour system for identification of Neisseria and Haemophilus species and Moraxella catarrhalis in a routine clinical laboratory.

API NH is a new 2-h system (bioMérieux, La Balme-les-Grottes, France) for the identification of most Neisseria and Haemophilus spp. of clinical significance and of Moraxella catarrhalis and for the detection of penicillinase production. Furthermore, this system allows the biotyping of Haemophilus influenzae and Haemophilus parainfluenzae. Three hundred eighteen strains belonging to these species, previously identified by conventional methods, were tested. Among the 305 strains belonging to species included in the data base, 225 (73.8%) were identified without additional tests, 79 (25.9%) were correctly identified after extra tests, and 1 strain (0.3%) was misidentified. For 131 (90.3%) of the 145 H. influenzae and H. parainfluenzae strains, results of biotyping were in agreement with results of standard methods. API NH is an accurate and reliable method for the routine identification of these bacteria in a clinical laboratory, for biotyping of Haemophilus spp., and for the detection of penicillinase-producing strains. The system is ready to use and time saving; inoculation of the system and reading of results are easy.

Bacterial Typing Techniques↗

Disk diffusion versus broth microdilution susceptibility testing of Haemophilus species and Moraxella catarrhalis using seven oral antimicrobial agents: application of updated susceptibility guidelines of the National Committee for Clinical Laboratory Standards.

Susceptibility testing of Haemophilus species and Moraxella catarrhalis is medium and inoculum dependent. Seven oral agents, ampicillin, amoxicillin-clavulanic acid, cefaclor, loracarbef, cefuroxime-axetil, cefixime, and erythromycin, were tested against 400 beta-lactamase-positive and -negative clinically significant respiratory strains of Haemophilus species and 100 strains of M. catarrhalis. Sources of the strains included teaching and regional hospitals and a private laboratory. All strains were tested by broth microdilution and disk diffusion in haemophilus test medium for Haemophilus species and Mueller-Hinton broth and agar for M. catarrhalis. Appropriate National Committee for Clinical Laboratory Standards (NCCLS) standards were followed. For Haemophilus species, by disk diffusion and broth microdilution, respectively, 27 and 27% of strains were resistant to ampicillin, 37 and 5% were resistant to erythromycin, 3 and 0.5% were resistant to cefaclor, 2 and 0.5% were resistant to loracarbef, and 0% were resistant to cefuroxime-axetil, cefixime, and amoxicillin-clavulanic acid. beta-Lactamase-negative ampicillin-resistant strains were not observed. Of M. catarrhalis strains, 56% were resistant to ampicillin by disk diffusion and 95% were resistant by broth microdilution. This species was susceptible to all other agents tested by either method. The disagreements between disk diffusion results and MICs for cefaclor, ampicillin, cefuroxime, and loracarbef that occurred with use of the 1990 NCCLS tables were resolved when the 1992 NCCLS tables were used.

Administration, Oral↗

Genetic diversity among strains of Moraxella catarrhalis: analysis using multiple DNA probes and a single-locus PCR-restriction fragment length polymorphism method.

Moraxella (Branhamella) catarrhalis, a causative agent of otitis media, sinusitis, and exacerbation of bronchitis, has acquired widespread ability to produce beta-lactamase and can be nosocomially transmitted. The typing methods used in epidemiological analyses of M. catarrhalis are not optimal for genetic analyses. Two methods, a multiple-locus Southern blot (SB) method and a single-locus PCR-restriction fragment length polymorphism (RFLP) method, were developed and used to assess genetic diversity and potential clinical and geographic relationships in M. catarrhalis. Nine randomly cloned M. catarrhalis DNA fragments were used as probes of SBs containing DNA from 54 geographically and clinically diverse strains. For comparison, a PCR-RFLP method was developed as a quick, inexpensive, and discriminating alternative. A highly variable 3.7-kb genomic region (M46) was cloned and sequenced, and 3.5 kb of the cloned DNA was targeted for PCR amplification. DNAs from the 54 strains were subjected to PCR-RFLP. SB analysis distinguished all strains that had no apparent epidemiological linkage (40 of 54), and PCR-RFLP distinguished fewer strains (21 of 54). Epidemiologically linked strains appeared genetically identical by both methods. PCR-RFLP was compared to pulsed-field gel electrophoresis (PFGE) for 8 of the 54 strains and 23 additional strains. PCR-RFLP distinguished fewer strains than PFGE typing (16 of 31 versus 20 of 31 strains), but PCR-RFLP was more useful for inferring interstrain relatedness. Separate cluster analyses of multilocus SB and single locus PCR-RFLP data showed high genetic diversity within and across geographic locations and clinical presentations. The resultant dendrograms were not entirely concordant, but both methods often gave similar strain clusters at the terminal branches. High genetic diversity, nonconcordance of cluster analyses from different genetic loci, and shared genotypes among epidemiologically linked strains support a hypothesis of high recombination relative to spread of clones. Single-locus PCR-RFLP may be suitable for short-term epidemiological studies, but the SB data demonstrate that greater strain discrimination may be obtained by sampling variation at multiple genomic sites.

Adult↗

Comparison of randomly amplified polymorphic DNA analysis and pulsed-field gel electrophoresis for typing of Moraxella catarrhalis strains.

Randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) for the analysis of 13 Moraxella catarrhalis isolates, 11 successive strains isolated from sputa of five children and 2 isolates obtained the same day from twins, were compared. RAPD and PFGE both yielded nine types from the 13 isolates, showing a chronic colonization with one strain in three patients and a successive colonization with different strains in two patients. The promising results obtained with RAPD should be confirmed with a larger number of strains, but RAPD seems as suitable as PFGE for the typing of M. catarrhalis.

Adolescent↗

Activity of gatifloxacin against Haemophilus influenzae and Moraxella catarrhalis, including susceptibility test development, E-test comparisons, and quality control guidelines for H. influenzae.

In vitro antimicrobial activity and susceptibility testing interpretation criteria and quality control were studied for gatifloxacin, a new 8-methoxy fluoroquinolone, tested against Haemophilus influenzae. Moraxella catarrhalis (600 strains) and H. influenzae (1,400 strains) from the SENTRY Antimicrobial Surveillance Program in North America (Canada and the United States) were also tested against gatifloxacin and 12 other antimicrobial agents. Gatifloxacin (MIC at which 90% of the isolates are inhibited [MIC90], </=0.03 microg/ml; 100.0% of strains inhibited at </=2 microg/ml) was the most active agent tested against H. influenzae and was similar to four comparison fluoroquinolones (MICs, </=0.03 to 2 microg/ml) against M. catarrhalis. A subset of 300 recent clinical isolates of H. influenzae were tested by using media (Haemophilus Test Medium agar and broth) and procedures recommended by the National Committee for Clinical Laboratory Standards (NCCLS) and with the E-test (AB BIODISK, Solna, Sweden). Gatifloxacin (MIC50, 0.008 microg/ml) was slightly more active than levofloxacin, and E-test results were generally elevated by 0.5 log2 dilution step compared to reference MICs. The gatifloxacin 5-microg disk test produced zone diameters that were routinely above 30 mm for H. influenzae strains, corresponding to gatifloxacin MICs of 0.008 or 0. 016 microg/ml. The gatifloxacin susceptibility breakpoint proposed for nonfastidious species (</=2 microg/ml; >/=18 mm) was also suggested for H. influenzae testing. No interpretive errors were observed. Quality control guidelines for H. influenzae ATCC 49247 were determined by using the NCCLS M23-T3 (1998) study design. The results from the nine-laboratory protocol suggested the following control ranges: for broth microdilution tests, 0.004 to 0.03 microg/ml; for disk diffusion testing, 33 to 41 mm. Gatifloxacin appears to be a potent anti-Haemophilus fluoroquinolone compound with in vitro testing interpretive criteria that will produce accurate results (disk diffusion, broth microdilution, and E-test).

Anti-Bacterial Agents↗

Isolation of Moraxella canis from an ulcerated metastatic lymph node.

Moraxella canis was isolated in large numbers from an ulcerated supraclavicular lymph node of a terminal patient, who died a few days later. Although the patient presented with septic symptoms and with a heavy growth of gram-negative diplococci in the lymph node, blood cultures remained negative. M. canis is an upper-airway commensal from dogs and cats and is considered nonpathogenic for humans, although this is the third reported human isolate of this species.

DNA, Ribosomal↗

Assessment of the nasopharyngeal bacterial flora of rhesus macaques: moraxella, Neisseria, haemophilus, and other genera.

The nasopharyngeal bacterial flora of healthy rhesus macaques was surveyed for the presence of Neisseria and Haemophilus species, as well as Moraxella catarrhalis. M. catarrhalis was found both in healthy rhesus macaques and in possibly immunocompromised rhesus macaques. Several Haemophilus spp. that are part of the normal nasopharyngeal bacterial flora of humans were found in many animals; these Haemophilus species included H. parahaemolyticus, H. segnis, and H. parainfluenzae. While Haemophilus influenzae was not identified, it is possible that the identification of H. influenzae types may have been thwarted by the growth of less fastidious species. A number of animals harbored Neisseria spp. such as N. sicca. However, Neisseria meningitidis was not found. In summary, it appears as though the rhesus macaque may be used as a model for M. catarrhalis infections. Moreover, in view of the susceptibility of macaques to organisms of the Haemophilus and Neisseria genera, it is possible that these animals may also accurately model nontypeable H. influenzae and N. meningitidis infections.

Animals↗

Production of BRO beta-lactamases and resistance to complement in European Moraxella catarrhalis isolates.

Of the 419 Moraxella catarrhalis isolates collected during the 1997-1999 European SENTRY surveillance study, 385 (92%) were beta-lactamase positive. Twenty-two (5.7%) produced BRO-2 beta-lactamase. Twenty-one new mutations were found in the putative promoter region of the bro genes. Nineteen percent of all isolates tested were complement sensitive. Resistance to beta-lactams is not linked to the phylogenetic lineages associated with susceptibility to complement.

Complement System Proteins↗

Posttrabeculectomy endophthalmitis caused by Moraxella nonliquefaciens.

Moraxella nonliquefaciens, a commensal organism of the upper respiratory tract, is generally considered to have low pathogenic potential. We report here two cases of severe endophthalmitis occurring 9 years and 2 months after glaucoma filtration surgery, respectively. Apart from sulfonamide, very low MICs were recorded for several antibiotics tested. Identification was based on phenotypic characteristics in combination with sequencing of the 16S rRNA gene.

Aged↗

Bacteremia due to Moraxella atlantae in a cancer patient.

A gram-negative alkaline phosphatase- and pyrrolidone peptidase-positive rod-shaped bacterium (CCUG 45702) was isolated from two aerobic blood cultures from a female cancer patient. No identification could be reached using phenotypic techniques. Amplification of the tRNA intergenic spacers revealed fragments with lengths of 116, 133, and 270 bp, but no such pattern was present in our reference library. Sequencing of the 16S rRNA gene revealed its identity as Moraxella atlantae, a species isolated only rarely and published only once as causing infection. In retrospect, the phenotypic characteristics fit the identification as M. atlantae (formerly known as CDC group M-3). Comparative 16S rRNA sequence analysis indicates that M. atlantae, M. lincolnii, and M. osloensis might constitute three separate genera within the MORAXELLACEAE: After treatment with amoxicillin-clavulanic acid for 2 days, fever subsided and the patient was dismissed.

Adenocarcinoma↗

Survival of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis frozen in skim milk- tryptone-glucose-glycerol medium.

In STGG (skim milk, tryptone, glucose, glycerol) medium at -80 degrees C, Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis isolates survived for at least 3 years, and the same species have survived in nasopharyngeal swabs for at least 1.5 years. At -20 degrees C, S. pneumoniae and M. catarrhalis survived for 1.5 years, but H. influenzae survived for only 2 months.

Animals↗

Characteristics of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis isolated from the nasopharynges of asymptomatic children and molecular analysis of S. pneumoniae and H. influenzae strain replacement in the nasopharynx.

Nasopharyngeal carriage of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis in 226 children in different settings (in a crèche [day care center], in an orphanage, and at home) during two seasons (winter and spring) was studied. The rates of carriage of S. pneumoniae and H. influenzae were markedly higher in the crèche and in the orphanage than in the home setting (e.g., 56.5, 63.3, and 25.9%, respectively, for S. pneumoniae in winter). Approximately 80% of the S. pneumoniae isolates identified in the crèche and in the orphanage belonged to the serotypes represented in the seven-valent pneumococcal vaccine, and 4.4% of the children were colonized by H. influenzae type b. Almost all H. influenzae isolates were fully susceptible to the antimicrobial agents tested, and only five (3.6%) produced beta-lactamase; in contrast, 100% of the M. catarrhalis isolates were beta-lactamase positive. Among S. pneumoniae isolates, 36.2% were nonsusceptible to penicillin (PNSP) and 11.8% were fully resistant to penicillin (PRP). All PNSP isolates were obtained from children at the crèche and at the orphanage but not among children brought up at home, and all PRP isolates showed a multiresistant phenotype. Colonization by PRP isolates correlated well with prior treatment with beta-lactams. For the majority of children colonized at both sampling times, strain replacement of S. pneumoniae and H. influenzae was observed; long-term colonization by a single strain was rare.

Anti-Bacterial Agents↗

Multiplex PCR assay that identifies the major lipooligosaccharide serotype expressed by Moraxella catarrhalis clinical isolates.

A heterologous cluster of glycosyltransferase genes was identified in the three Moraxella catarrhalis LOS serotype strains. Multiple PCR primers designed to this region amplified products that differentiate between the serotypes more rapidly and efficiently than previously described serological analyses. This assay will be valuable for clinical and research-based studies.

Adult↗

Moraxella infectious arthritis: first report in an adult.

The first occurrence of septic arthritis due to moraxella in an adult is reported. The clinical presentation mimicked disseminated gonococcaemia with associated gonococcal arthritis except for an atypical rash. Diagnosis was made by culture.

Adult↗

The skin as the source of Acinetobacter and Moraxella species occurring in blood cultures.

A study was made of the flora of the skin in hospital inpatients and healthy people to demonstrate the presence of non-fermenting Gram-negative rods of the Acinetobacter and Moraxella group. These organisms were found to be present on the skin of 34.3% of inpatients and occurred even more commonly in those patients with kidney disease. It was also present on the skin of 20% of a group of healthy members of staff. This rather high rate of skin carriage is thought to account for the not infrequent occurrence of this organism in blood cultures.

Acinetobacter↗

Outbreak of Moraxella catarrhalis in a respiratory unit.

The clinical and epidemiological features of an outbreak of nosocomial Moraxella catarrhalis infection in a respiratory unit are described. Six isolates from five patients were shown to be indistinguishable by immunoblotting and restriction endonuclease analysis and different from 11 other, unrelated clinical strains.

Cross Infection↗