Strains Of Haemophilus Influenzae Research Articles

Trimethoprim resistance in Haemophilus influenzae is due to altered dihydrofolate reductase(s)

We characterized a highly purified preparation of the chromosomally encoded dihydrofolate reductase (DHFR) from a trimethoprim-susceptible (Tmp8; strain MAP) and two trimethoprim-resistant (TmpR) strains (MAP/47 and MAP/42) of Haemophilus influenzae. The enzymes were purified between 650- and 3000-fold by gel-filtration and dye-ligand chromatography. The apparent molecular mass of the three proteins was 18400 Da by PAGE under denaturing and nondenaturing conditions. Total enzyme activity was greater in all fractions from the TmpR strains compared with the Tmp8 isolate. The three enzymes had a similar Km for dihydrofolate (7, 9 and 5 microM) and NADPH (2, 5 and 6 microM). However, the Tmp IC50 (the concentration necessary for 50% inhibition of DHFR activity) for the Tmp8 strain MAP was 0.001 microM, whereas DHFR from the TmpR strains MAP/47 and MAP/42 had values of 0.1 microM and 0.3 microM respectively. The methotrexate IC50 of the MAP/42 DHFR was 0.06 microM in comparison with the enzyme from MAP (0.008 microM) and MAP/47 (0.007 microM). Isoelectric focusing indicated that the DHFR from MAP/42 had a different isoelectric point (pI 7.6) compared with the enzymes from MAP and MAP/47 (pI 7.3). Peptide mapping after digestion with trypsin revealed one major peptide fragment (7.9 kDa) in the DHFR of MAP and MAP/47 and three major tryptic fragments (7.9, 9.6 and 12.5 kDa) in DHFR from MAP/42. We conclude that trimethoprim resistance in H. influenzae results from overproduction of structurally altered DHFR(s).

Activity of a new carbapenem antibiotic, meropenem, against Haemophilus influenzae strains with beta-lactamase- and non-enzyme-mediated resistance to ampicillin

Meropenem was 8- to 32-fold more active (MIC for 90% of strains = 0.125 micrograms/ml) than imipenem against a collection of Haemophilus influenzae strains characterized as either susceptible to ampicillin or resistant to that agent by virtue of beta-lactamase or nonenzymatic mechanisms. MBCs and kinetic kill curve tests showed that meropenem (as well as imipenem, several cephalosporins, and amoxicillin-clavulanate) was bactericidal against all strains at or within four times the respective MICs. Thus, meropenem demonstrated greater inhibitory activity than imipenem and activity comparable to that of cefotaxime against these selected strains.

Double-blind comparison of cefixime and cefaclor in the treatment of acute otitis media in children

In a double-blind study cefixime, an oral cephalosporin of the third generation, was compared to cefaclor in the treatment of acute otitis media in 397 children aged 6 months to 12 years. Clinical evaluation was carried out at the beginning, at day 10-12 and day 28-35 after the start of the treatment. Specimens for bacterial culture and sensitivity testings were taken from the nasopharynx at the initial visit. Patients were randomized either to cefixime in a dose of 8 mg/kg/day or cefaclor in a dose 40 mg/kg/day in the proportion of 2 cefixime patients to 1 cefaclor patient. Two daily doses were administered for 7 days. At day 10-12, 93.5% in the cefixime group and 90.5% in the cefaclor group (p = 0.08) were clinically cured or improved. At day 28-35 the rate of cured or improved patients had decreased, mostly due to reinfections, to 90.1% in the cefixime group and to 86.6% in the cefaclor group (p = 0.12), respectively. 375 patients (69.9%) had positive bacterial culture in the nasopharynx of at least one strain of Haemophilus influenzae, Streptococcus pneumoniae, Branhamella (Moraxella) catarrhalis or combinations of these 3.73.6% of the B. catarrhalis strains were beta-lactamase producing and 11.4% of the H. influenzae strains, respectively. All isolated bacteria were sensitive to cefixime. Adverse events were reported in 17.9% in the cefixime and 10.6% in the cefaclor group. Most reactions were of moderate or mild nature and mostly affected skin or the gastrointestinal region. No serious adverse experiences occurred. In view of the good clinical results obtained cefixime seems to be at least as effective as cefaclor in the treatment of acute otitis media in children.

Killing kinetics of cefuroxime axetil against Haemophilus influenzae in an in-vitro model simulating serum concentration profiles after oral administration

We have used a dynamic in-vitro model to determine the killing kinetics of two formulations of cefuroxime axetil against 18 clinical strains of Haemophilus influenzae. The concentration of cefuroxime axetil was adjusted dynamically to simulate the mean serum profile in healthy volunteers following the administration of a single 250 mg dose in oral suspension or tablet form. Bacterial numbers were reduced by 3 log10 cfu/mL during the eight-hour experimental period with both dosage form simulations. Our results suggest that cefuroxime axetil, 250 mg in oral suspension or tablet form, may protect against the dissemination of H. influenzae during episodes of bacteraemia.

In-vitro activity of clarithromycin combined with its 14-hydroxy metabolite A-62671 against Haemophilus influenzae

Clarithromycin and its 14-hydroxy metabolite (A-62671), were tested against 20 strains of Haemophilus influenzae. Minimum inhibitory and bactericidal concentrations of the two compounds alone and in combination were determined in a microbroth system which allowed continuous turbidimetric measurement for the construction of growth curves. MICs of clarithromycin were 2-8 and 1-4 mg/l for A-62671 for the majority of strains. MBCs were identical to or one dilution higher than the respective MICs. Combinations of the two compounds were inhibitory or bactericidal at concentrations that were lower than the MICs or MBCs. On the basis of fractional inhibitory (FIC) or fractional bactericidal (FBC) concentration indices, the interactions between clarithromycin and its metabolite were defined as additive.

Effect of fusafungine on adherence of Haemophilus influenzae type b to human epithelial cells in vitro

Haemophilus influenzae, a normal host of the nasopharynx of humans, may become a pathogen. The first step of infection is adherence to epithelial cells of the nasopharynx through glycopeptidic adhesins, or pili. Adherence to human epithelial cells in continuous lines, HeLa and Hep2, of 8 piliated strains of Haemophilus influenzae isolated from human infections of the respiratory tract was studied in vitro in ihe presence of fusafungine, a local bacteriostatic antibiotic. When the bacteria were grown in the presence of 0.5 × the MIC, fusafungine afforded 45–75% of adherence inhibition, but this inhibitory effect did not parallel the MICs. In contrast, no significant effect could be observed either when epithelial cells were exposed to 0.5 × the MIC before use in the adherence assay, or when this assay was performed in the presence of 0.5 × the MIC of fusafungine. The partial adherence inhibition observed suggests that fusafungine interacts with the bacterial binding sites but that other mechanisms may contribute to the inhibitory process. This effect of fusafungine should prevent but not eradicate colonization of the nasopharyngeal mucosa by Haemophilus influenzae and may account for the therapeutic efficacy reported in infections of the respiratory tract due to Haemophilus influenzae.

Reproducibility of disc susceptibility tests with Haemophilus influenzae

Triplicate susceptibility tests were performed in each of three laboratories with 45 strains of Haemophilus influenzae and a single lot of Haemophilus test medium (HTM) agar plates. Using clarithromycin, erythromycin and chloramphenicol discs. zone sizes varied over ranges averaging only 4.3-5.8 mm. Ampicillin zone sizes were more variable, especially when beta-lactamase-producing strains were being tested, but the latter strains were consistently categorized as being resistant. A 94% interpretive consistency was observed with the two macrolides.

Protein D, an immunoglobulin D-binding protein of Haemophilus influenzae: cloning, nucleotide sequence, and expression in Escherichia coli

The gene for protein D, a membrane-associated protein with specific affinity for human immunoglobulin D, was cloned from a nontypeable strain of Haemophilus influenzae. The gene was expressed in Escherichia coli from an endogenous promoter, and the gene product has an apparent molecular weight equal to that of H. influenzae protein D (42,000). The complete nucleotide sequence of the gene for protein D was determined, and the deduced amino acid sequence of 364 residues includes a putative signal sequence of 18 amino acids containing a consensus sequence, Leu-Ala-Gly-Cys, for bacterial lipoproteins. The sequence of protein D shows no similarity to those of other immunoglobulin-binding proteins. Protein D is the first example of immunoglobulin receptors from gram-negative bacteria that has been cloned and sequenced.

Bacterial Colonization of tbe Upper Respiratory Tract and Its Association witb Acute Lower Respiratory Tract Infections in Higbland Cbildren of Papua New Guinea

Acute lower respiratory tract infection (ALRI) is the major cause of death among children in Papua New Guinea. This longitudinal study reports the bacteriologic findings for children observed in their hamlets. A total of 1,449 nasal swab specimens from 158 children less than 5 years of age who were studied intensively for 18 months were examined. Non-serotypable strains of Haemophilus influenzae were isolated from 91% of specimens, and serotypable strains were isolated from 35% (8% H. influenzae type b) of specimens. All children had acquired Streptococcus pneumoniae by the age of 3 months. The most frequently occurring serotypes of S. pneumoniae were 6, 19, and 23. Children more frequently carried invasive pneumococci during an episode of ALRI than when they were healthy. Also, children more frequently carried serotypable strains of H. influenzae during the 2 weeks preceding an episode of ALRI than when they were healthy. Between-children analyses showed that children who were susceptible to attacks of ALRI and those who were not susceptible had similar rates of carriage of bacteria.

Cross-infection by non-encapsulated Haemophilus influenzae

From October, 1988, to January, 1989, 18 patients admitted to an acute medical chest ward were infected with an ampicillin-resistant β-lactamase-producing strain of Haemophilus influenzae. All 18 isolates were non-encapsulated strains of biotype III and showed identical cell envelope protein profiles, as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The organism was not isolated from repeated environmental samples but there was strong circumstantial evidence that a spirometer was a common iatrogenic source of the cross-infection.

In vitro activity of 9 antimicrobial agents against 177 strains of Haemophilus influenzae isolated from hospitalized patients

In vitro activity of 4 commonly used and 5 new antibiotics was examined against 177 strains of Haemophilus influenzae. All strains were collected from various sites in patients with clinical infections. The study confirms that several newer antibiotics are useful alternatives to older drugs, as measured by in vitro activity. Ciprofloxacin and ofloxacin were the most active agents, (MIC90 0.012 micrograms/ml and 0.05 micrograms/ml respectively), followed by aztreonam (MIC90 0.1 micrograms/ml) and cefuroxime (MIC90 0.8 micrograms/ml). A new macrolide, azithromycin (CP 62,993), was more active than erythromycin, MIC90 1.6 micrograms/ml vs 6.4 micrograms/ml. Beta-lactamase production was detected in 4.5% (8/177) of the strains. In vitro activity was the same against strains collected in 1985 and 1988. No increase in beta-lactamase production was recorded.

Urogenital, maternal and neonatal isolates of Haemophilus influenzae: identification of unusually virulent serologically non-typable clone families and evidence for a new Haemophilus species

A collection of 117 strains of Haemophilus influenzae, including 112 non-typable isolates recovered predominantly in the USA and France from genital, obstetric and neonatal sources, was characterized by the electrophoretic mobilities of 10 metabolic enzymes. Eighty-six distinctive multilocus chromosomal genotypes (electrophoretic types, ETs) were distinguished on the basis of allele profiles at the enzyme loci. Isolates of five allied biotype IV ETs were highly divergent from all other strains and hybridization of chromosomal DNA revealed that they undoubtedly represent a previously unrecognized species of Haemophilus. Isolates representing these ETs were recovered predominantly from obstetric infections and serious neonatal diseases and apparently possess specific tropism for the genital tract. Strains of these five ETs were present in samples from both the USA and France, but only in the USA did they cause bacteraemia and meningitis, an occurrence which probably reflects differences in patient management between the two countries. Although strains assigned to H. influenzae (sensu stricto) were strongly polymorphic in multilocus enzyme genotype, 69% of isolates recovered from patients with meningitis and/or septicaemia were assigned to only two clone families, a result suggesting that some serologically nontypable strains of H. influenzae originating from the genital tract are unusually virulent.

Ampicillin killing curve patterns for ampicillin-susceptible nontypeable Haemophilus influenzae strains by the agar dilution plate count method

Ampicillin killing curve patterns for 20 strains of ampicillin-susceptible nontypeable Haemophilus influenzae were determined by the agar dilution plate count method. The paradoxical effect was detected in the 24-h killing curve patterns for each strain. For the biphasic effect, minimum survivor percentages (maximum killing) occurred over a narrow range of ampicillin concentrations immediately above the MIC, with survivor percentages then rising rapidly to peak at approximately 1-log10-unit increment higher. The 24-h minimum survivor percentages for the 20 strains ranged from approximately 0.01% (rapid killing) to greater than 10% (slow killing). In comparison with the previous results for typeable strains, the present findings suggest that nontypeable stains are, on average, killed much more slowly. Based on the initial 24-h killing curve patterns for the 20 strains, 4 strains were selected as putative representatives of the range of bactericidal responses encountered. These strains were then studied to examine the reproducibility of the 24-h patterns and to determine sequential killing curves. These patterns were found to be reproducible and served to characterize the relative killing responses of the strains. In the sequential studies of three of the four strains, tiny colonies having the gross and microscopic characteristics of L-forms were found to be present on the agar dilution plate count plates prior to the application of penicillinase at 48 and 72 h. Such colonies reverted to vegetative forms within 24 to 48 h after application of penicillinase to the panels. Of particular interest was the observation that the paradoxical effect was manifested both by the L-form colonies and by the reverted vegetative colonies. The late development of L-forms was observed for both rapidly and slowly killed strains.

Haemocin, the bacteriocin produced by Haemophilus influenzae: species distribution and role in colonization

Four hundred thirty-eight strains of Haemophilus influenzae were examined for production of and sensitivity to haemocin, a bacteriocin produced by some members of this species. Whereas 199 of 212 (94%) type b isolates produced haemocin, 131 of 134 (98%) nontypeable and 91 of 92 (99%) encapsulated non-type b isolates were sensitive to haemocin. Among strains previously genetically characterized by multilocus enzyme electrophoresis, haemocin production was detected in type b isolates belonging to 25 of 29 (86%) clonally distinct electrophoretic types. None of 60 clonally distinct nontypeable strains produced this substance, and all were sensitive to it in vitro. The genes encoding haemocin production were transformed independently of the genes necessary for capsule expression from a prototypic type b strain to a nontypeable strain. After intranasal inoculation of infant rats with an equal mixture of a non-haemocin-producing strain and its haemocin-producing transformant, organisms capable of haemocin production predominated in both nasopharyngeal and blood cultures. These data demonstrate that haemocin production is strongly associated with type b encapsulated members of this species and suggest a mechanism by which haemocin might play a role in host nasopharyngeal colonization by this pathogen.

Disk diffusion susceptibility testing ofHaemophilus influenzae using Haemophilus test medium

A three-center collaborative study was performed with the aim of determining the reproducibility of disk diffusion and broth microdilution susceptibility tests of Haemophilus influenzae with 19 different antimicrobial agents using a newly described medium, Haemophilus Test Medium. The results of both methods were found to be highly reproducible. Using Haemophilus Test Medium, inhibitory zone diameters and MICs were then determined for 150 test strains of Haemophilus influenzae, selected with an emphasis on known mechanisms of resistance. Regression analysis of zone diameter-MIC comparisons was used to define zone diameter interpretive criteria and MIC correlates for Haemophilus Test Medium disk diffusion tests performed with Haemophilus spp. for the following antimicrobial agents: ampicillin, amoxicillin/clavulanate, ampicillin/sulbactam, cefaclor, cefuroxime, cefamandole, ceftriaxone, cefotaxime, ceftizoxime, ceftazidime, cefixime, trimethoprim/sulfamethoxazole, rifampin, chloramphenicol, tetracycline, imipenem, aztreonam and ciprofloxacin.

Haemophilus infection in cystic fibrosis.

Twenty seven patients with cystic fibrosis under the age of 12 years and 27 matched patients with asthma were followed up in a prospective study for one year. The isolation rate of non-capsulated strains of Haemophilus influenzae from cough swabs and sputum specimens taken at routine clinic visits every two months was significantly greater in cystic fibrosis than in asthma. Haemophilus para-influenzae was equally common in both groups. During exacerbations the isolation rate of H influenzae in cystic fibrosis was significantly greater than at other times, whereas in asthma there was no significant difference. The distribution of biotypes of H influenzae and H parainfluenzae was similar in the two groups. In cystic fibrosis, biotype I was associated with exacerbations. Biotype V was more common than in previous studies, but was not associated with exacerbations.

In-vitro activity of cefpodoxime against pathogens responsible for community-acquired respiratory tract infections

The in-vitro activity of cefpodoxime, the active compound of the ester prodrug, cefpodoxime proxetil, was compared with that of other antibiotics. The susceptibility of bacterial isolates from patients with respiratory tract infections was determined by an agar dilution method. The MIC90s of cefpodozime for ampicillin-sensitive and beta-lactamase-producing strains of Haemophilus influenzae were 0.12 and 0.25 mg/l, respectively; the MIC90s for ampicillin-resistant non-beta-lactamase-producing strains was 1 mg/l. Time-kill curves of cefpodoxime against ampicillin-sensitive and ampicillin-resistant beta-lactamase producing strains showed a time-dependent bactericidal activity. The MIC90s for ampicillin-sensitive and ampicillin-resistant Branhamella catarrhalis were 0.50 and 1 mg/l, respectively. The MIC90s for penicillin-sensitive pneumococci, beta-haemolytic streptococci and Streptococcus agalactiae were 0.06, 0.06 and 0.12 mg/l, respectively. The inhibitory activity against penicillin-resistant pneumococci was limited: the MIC90 was 4 mg/l.

Oral Cephalosporins in Perspective

Oral cephalosporins, after 25 years of use, continue to present the clinician with a therapeutic challenge. The older agents have been extensively prescribed for ambulatory adult and pediatric patients with a wide variety of infections caused by gram-positive and some gram-negative organisms. The newer agents, cefaclor, cefuroxime axetil, and cefixime, have increased in vitro activity against beta-lactamase-secreting strains of Haemophilus influenzae and Branhamella catarrhalis which has made them more popular for the treatment of otitis media and respiratory tract infections in children. The new agents are also more active against most gram-negative organisms. However, clinical trials have failed to show a clear-cut superiority over older, proven therapy when used to treat infections of the respiratory tract, middle ear, skin and soft tissue, urinary tract, and bone and joints when caused by sensitive organisms. Published reports of clinical trials continue to support the recommendation that oral cephalosporins, especially the newer and more expensive agents, be reserved for second- or third-line therapy when amoxicillin, penicillin V, or trimethoprim/sulfamethoxazole have either failed or produced patient intolerance. Erythromycin/sulfisoxazole and amoxicillin/clavulanate potassium are equally efficacious and also less expensive than cefaclor, cefuroxime axetil, and cefixime and could be considered second-line therapy prior to the use of the newer cephalosporins for infections in the ambulatory patient.

一次医療における呼吸器感染症の研究 (II) 個人開業医における呼吸器由来病原細菌の分布と45薬剤に対する感受性

The bacteriology of the isolates from the sputum or the throat swab of patients with respiratory infections visiting a doctor in private practice in Sendai city during the period from March in 1988 to February in 1989 was documented, and their sensitivity to 45 antimicrobial agents was determined. Of the 568 patients, 514 cases had acute pharyngitis, 8 cases each had acute tonsillitis and acute bronchitis, 7 cases were acute pneumonia, 6 cases had herpangina, 18 cases had hand-foot-mouth disease with the signs of respiratory infections, 5 cases had varicella with the signs of respiratory infections and 2 cases were mumps with the signs of respiratory infections. Three hundred strains of potential (greater than or equal to 10(7) CFU/ml) pathogens were recovered from 293 of the 568 cases, which consisted of 124 strains of Haemophilus influenzae, 58 strains of Streptococcus pneumoniae, 45 strains of Staphylococcus aureus, 26 strains of Branhamella catarrhalis, 25 strains of Streptococcus pyogenes, 9 strains of Klebsiella pneumoniae and 13 strains of other species, not including non-fermentile gram-negative bacteria such as Pseudomonas aeruginosa and Acinetobacter calcoaceticus. Staphylococcus aureus and other strains were documented simultaneously in 6 out of 7 cases in which multi-organisms were recovered. Many strains of Staphylococcus aureus were isolated from young patients throughout the year. On the other hand many strains of Branhamella catarrhalis were isolated from elderly patients in winter. The sensitivity of 45 antimicrobial agents of 231 of 300 strains was determined by sensitivity disks (EIKEN, Japan). No strain of the Haemophilus influenzae in this study was resistant to ampicillin. None of the Streptococcus pneumoniae and Streptococcus pyogenes was resistant to ampicillin or cefazolin. None of the Staphylococcus aureus was resistant to cloxacillin, cefazolin, gentamicin or ofloxacin. We conclude from the above results that antibiotic-resistant strains are found presumably only in a very few cases in primary care clinic.

Spread of non-typable multiply resistantHaemophilus influenzae in a South African hospital

In July 1987 non-typable Haemophilus influenzae strains resistant to both ampicillin and chloramphenicol were isolated from the endotracheal aspirate of two children with pneumonia at Baragwanath Hospital, Johannesburg, South Africa. A study was therefore undertaken to determine the carriage rates of Haemophilus influenzae strains in the nasopharynx of children and staff in the index ward and in three control wards. Using a disc diffusion and an agar dilution method the susceptibility was determined of 100 isolates to ampicillin, chloramphenicol, erythromycin, rifampicin, amoxicillin/clavulanic acid, gentamicin, cefaclor, cefotaxime, tetracycline, sulphamethoxazole, trimethoprim and trimethoprim/sulphamethoxazole (1:19). The overall carriage rate of Haemophilus influenzae on admission was 76%. In the index ward, children carrying multiply resistant strains differed from the other children in that there was a longer mean duration of hospitalization, a lower proportion of males, and a higher proportion who had previously received antibiotics. All ampicillin resistant strains were shown to produce beta-lactamase. Only four isolates belonged to serotype b, of which three were ampicillin resistant and chloramphenicol sensitive while one was resistant to both drugs. Nasopharyngeal spread of resistant non-typable strains of Haemophilus influenzae was demonstrated to affect the management of paediatric patients in the hospital.