To investigate the epidemiology of nontypeable Haemophilus influenzae in the respiratory tract of cystic fibrosis (CF) patients, H. influenzae isolates from sputum specimens of 40 CF patients were analyzed longitudinally for 2 years. The isolates were characterized by analysis of the major outer membrane protein (MOMP) patterns. MOMP variant H. influenzae strains were discriminated from distinct strains by randomly amplified polymorphic DNA analysis of genomic DNA. Multiple H. influenzae strains and MOMP variant strains were isolated from single sputum specimens of 29 patients. In 22 patients, a distinct H. influenzae strain persisted over time (median persistence, 8 months; range 2-24). In general, the appearance of MOMP variant strains did not coincide with the occurrence of exacerbations.

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Summary. Non-capsulate strains of Haemophilus injluenzae were genotyped by analysis of variable DNA segments obtained by amplification of genomic DNA with the polymerase chain reaction (PCR fingerprinting). Discrete fragments of 100-2000 bp were obtained. The reproducibility of the procedure was assessed by comparing : (i) the fingerprints of 16 colonies of a single H. injluenzae strain; (ii) isolates obtained from individual sputum samples (a total of 57 H. injluenzae isolates from three cystic fibrosis patients); and (iii) 17 isolates collected during an outbreak of H. injluenzae infection in a local pulmonary rehabilitation centre. The discriminatory power of the method was demonstrated by showing that the PCR fingerprints of eight unrelated H. injluenzae strains from sputum samples of patients with chronic obstructive pulmonary disease (COPD) and 32 strains from cystic fibrosis patients were all different. These 40 isolates also differed with respect to their restriction fragment length polymorphisms (RFLP) and major outer-membrane protein (MOMP) composition. Twelve MOMP antigenic strain variants from sputum samples of five COPD patients had identical PCR fingerprints and RFLPs. It was concluded that PCR fingerprinting is a reliable and reproducible method for genotyping non-capsulate strains of H . injluenzae. The discriminatory power of PCR fingerprinting was similar to that of RFLP analysis, but the results of PCR fingerprinting were easier to interpret.

We investigated the relationship between susceptibility to β-lactam antibiotics and variation in the major outer membrane protein P2 (OmpP2; also called porin) of persistent nonencapsulatedHaemophilus influenzae isolated from cystic fibrosis patients. Nine OmpP2 variants were selected from two distinctH. influenzae strains from two patients extensively treated with β-lactam antibiotics. The variants differed in their susceptibilities to at least two β-lactam antibiotics. By detergent extraction and column chromatography, OmpP2 was purified from two variants that were derived from strain 70 and that differed notably in their susceptibilities to β-lactam antibiotics. The proteins were reconstituted into black lipid membranes for measurement of porin function. OmpP2 from the more resistant isolate (isolate 70b) had a smaller channel conductance than OmpP2 of the more susceptible isolate (isolate 70f). DNA sequencing of ompP2 of these isolates revealed single nonsynonymous base differences; there were changes in the amino acid sequence corresponding to surface-exposed loops 4, 5, 6, and 8. Changes in loops 4, 5, and 6 were previously shown to result in antigenic differences. Beside these mutations, variants of strain 70 showed additional mutations in loop 1 and nonexposed loop 3. Taken together, our results suggest that in variants of strain 70, nonsynonymous point mutations accumulated both in the sequences ofompP2 coding for antigen-variable loops and in other loops, notably, loops 1 and 3. The latter changes are suggested to affect the permeability of the porin channel.

The conformational stability of holo-lipoamide and apo-lipoamide dehydrogenase from Azotohucter vinelandii was studied by thermoinactivation, unfolding and limited proteolysis. The oxidized holoenzyme is thermostable, showing a melting temperature, t, = 80°C. The thermal stability of the holoenzyme drastically decreases upon reduction. Unlike the oxidized and lipoamide two-electron reduced enzyme species, the NADH four-electron reduced enzyme is highly sensitive to unfolding by urea. Loss of energy transfer from Trp199 to flavin reflects the unfolding of the oxidized holoenzyme by guanidine hydrochloride. Unfolding of the monomeric apoenzyme is a rapid fully reversible process, following a simple two-state mechanism. The oxidized and two-electron reduced holoenzyme are resistant to limited proteolysis by trypsin and endoproteinase Glu-C. Upon cleavage of the apoenzyine or four-electron reduced holoenzyme by both proteases, large peptide fragments (molecular mass > 40 kDa) are transiently produced. Sequence studies show that limited trypsinolysis of the NADH-reduced enzyme starts mainly at the C-terminus of Arg391. In the apoenzyme, limited proteolysis by endoproteinase Glu-C starts from the C-terminus at the carboxyl ends of Glu4.59 and/ or Glu435. From crystallographic data it is deduced that the susceptible amino acid peptide bonds are situated near the subunit interface. Thus, these bonds are inaccessible to the proteases in the dimeric enzyme and become accessible after monomerization. It is concluded that reduction of lipoamide dehydrogenase to the four-electron reduced state(s) is accompanied by conformational changes promoting subunit dissociation.Dihydrolipoamide dehydrogenase is a member of the class of flavoprotein disulfide oxidoreductases [l]. The dimeric enzyme catalyzes the NAD+-dependent oxidation of dihydrolipoyl groups, which in vivo are covalently attached to the lipoate acyltransferase component of different multienzyme complexes [2]. The active site of the enzyme is composed of both subunits, each containing FAD and a redox-active disulfide group [3]. Mechanistically and kinetically, lipoamide dehydrogenase from pig heart and Escherichiu coli have been most extensively characterized [4, 51. These enzymes differ strongly with rcspect to the stabilization of different twoelectron reduced enzyme species [6-91. Inhibition of the E . coli enzyme by the product NADH was shown to be mainly due to over-reduction of the enzyme to the inactive fourelectron reduced state [lo].Recent

We analyzed the antimicrobial susceptibilities of Haemophilus influenzae isolates from 157 sputum specimens prospectively collected from 39 cystic fibrosis (CF) patients during a 2-year study. These isolates were characterized by random amplified polymorphic DNA analysis and major outer membrane protein (MOMP) analysis to identify H. influenzae strains and MOMP variants and to assess their persistence in the respiratory tract. Among the 247H. influenzae isolates, 16 (6.5%) produced β-lactamase. The 231 β-lactamase-negative isolates represented 85H. influenzae strains, 61 MOMP variants derived from 27 of these strains, and 85 persistent isolates identical to strains or MOMP variants. All β-lactamase-negative isolates were tested for susceptibility to ampicillin, amoxicillin-clavulanic acid, cefuroxime, cefotaxime, cefaclor, imipenem, tetracycline, and trimethoprim-sulfamethoxazole by disk diffusion testing. Eleven (13%)H. influenzae strains, 18 (30%) MOMP variants, and 30 (35%) persistent isolates were resistant to one or more of the antibiotics tested. Antimicrobial susceptibility was decreased among MOMP variants and persistent isolates compared to nonpersistentH. influenzae strains, and changes in susceptibility occurred irrespective of MOMP variation. We conclude that the decreased antimicrobial susceptibility of H. influenzae during persistence contributes to the poor eradication of H. influenzae from the respiratory tracts of CF patients.

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