Haemophilus influenzae penicillin-binding proteins 1a and 3 possess distinct and opposite temperature-modulated penicillin-binding activities
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Abstract:
Upon investigation of penicillin-binding proteins (PBPs) in Haemophilus influenzae strains, five H. influenzae and seven other Haemophilus strains were tested for whole-cell penicillin binding at either 37 or 42 degrees C. Binding of [35S]penicillin G to H. influenzae PBPs 3a and 3b was drastically reduced at 42 degrees C, while PBP 1a showed a temperature-modulated increase in penicillin binding. Further investigation revealed that growth at 42 degrees C causes altered electrophoretic mobility of PBP 3a on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and that cell labeling performed at 42 degrees C showed the differential penicillin binding to target proteins. All Haemophilus spp. tested showed a similar temperature modulation of penicillin binding. Growth measurement and cell viability studies performed at 42 degrees C permitted correlation of PBP 3 temperature sensitivity to H. influenzae resistance to moxalactam at 42 degrees C, and the probable correlation of PBP 1a increased penicillin binding to the more rapid antibacterial activity of penicillin G against H. influenzae at 42 degrees C. Microscopic examination of Haemophilus cells grown at 42 degrees C revealed filamentous cell formation, supporting a role of PBP 3 in the septation process. Results of this study demonstrate that wild-type H. influenzae strains (and possibly all other Haemophilus spp.) possess PBPs 1a and 3, which have distinct and opposite temperature-modulated penicillin-binding activities.Keywords:
Penicillin binding proteins
Haemophilus spp. are small, Gram-negative bacilli that can be found as part of the normal microbiota of the upper respiratory, gastrointestinal, and genital tracts. Within this genus, most infections are caused by Haemophilus influenzae. Haemophilus influenzae may or may not be encapsulated. Capsules are the basis for serogrouping Haemophilus influenzae into six groups, designated groups a through f. Nonencapsulated strains are referred to as nontypeable Haemophilus influenzae (NTHi). Capsules are a major virulence factor of this organism, since they are antiphagocytic, although NTHi strains can cause human disease. Molecular techniques have been employed to identify Haemophilus influenzae directly in clinical specimens, in particular cerebrospinal fluid, as well as Haemophilus ducreyi in suspected cases of chancroid. Identification of Haemophilus from positive blood cultures is also available as part of commercial multiplex assays.
Haemophilus ducreyi
Chancroid
Haemophilus parainfluenzae
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Indole spot tests using isolated, nonhemolytic colonies of Haemophilus species were positive for 90 of 151 (60%) respiratory isolates of Haemophilus influenzae, whereas 67 to 72 (93%) isolates of H. influenzae from cerebrospinal fluid and blood specimens were indole positive. Only 4 of 117 (3%) Haemophilus parainfluenzae isolates were positive for indole spot tests. Thus, indole-positive, nonhemolytic Haemophilus isolates in respiratory cultures can be presumptively identified as H. influenzae.
Haemophilus parainfluenzae
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Two hundred and nine strains of Haemophilus influenzae and Haemophilus aegyptius were screened for trooleandomycin susceptibility. Four strains were shown to be sensitive to the drug. Of these four, two were Haemophilus aegyptius (ATCC 11116, NCTC 8134), and the other two were Haemophilus influenzae biotype I (1-605) and IV (80-212. One strain of Haemophilus aegyptius (NCTC 8135) was resistant to trooleandomycin. Restriction enzyme assays and DNA homology were carried out to establish relationships between the strains. It is concluded that trooleandomycin susceptibility has no taxonomic value to differentiate between Haemophilus aegyptius and biotype III Haemophilus influenzae.
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Permeabilized cells of Haemophilus influenzae incorporate wall precursors into murein material in an ampicillin-sensitive reaction. In resistant transformants that contain the low antibiotic affinity penicillin-binding proteins (PBPs) 4 and 5, the sensitivity of this incorporation reaction to ampicillin is proportionally lower, suggesting a catalytic role for these proteins in wall synthesis. We conclude that, analogous to the reaction in Escherichia coli, PBPs 4 and 5 of H. influenzae have transpeptidase activity.
Penicillin binding proteins
Amp resistance
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There has not previously been an objective comparison of medium formulations for the primary isolation of Haemophilus species. This study was undertaken to evaluate the components required for the optimal growth of large, easily identifiable colonies of these bacteria. We compared six medium bases and seven supplements for their ability to support the growth of 86 strains of Haemophilus influenzae and 17 strains of other species of Haemophilus. By using a growth index that combines colony size and the dilution factor, a formulation of GC agar base with 1% yeast autolysate and 5% sheep blood (chocolated) promoted the growth of large, easily recognizable colonies of H. influenzae and other Haemophilus species. This medium was designated GCYSB. The addition of hematin to supplements that supplied NAD (or factor V) to the medium was inhibitory to the growth of all of the Haemophilus species tested. In a clinical comparison of GCYSB with routinely used chocolate agar medium in two laboratories for the primary isolation of Haemophilus species, overall GCYSB promoted better growth of 124 strains of H. influenzae and H. parainfluenzae. GCYSB is easy to prepare and inexpensive compared with the ease of preparation and expense of other Haemophilus isolation media.
Chocolate agar
Haemophilus parainfluenzae
Isolation
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A study was performed to obtain a better understanding of the diversity and ecology of members of the family Pasteurellaceae in the porcine respiratory tract. A collection of 132 V factor-dependent strains of Pasteurellaceae selected from porcine field isolates mainly from the respiratory tract were subjected to detailed characterization. In addition to the three hitherto recognized species Actinobacillus pleuropneumoniae, Haemophilus parasuis, and Haemophilus taxon "minor group," three distinct taxa were observed. Some of these taxa, which are provisionally designated taxa D, E, and F, would by traditional criteria be mistaken for H. parasuis but differed by several biochemical characteristics. To study the ecology of the V factor-dependent species, swabs from the nasal and oral cavities of 29 pigs were cultivated on selective and nonselective media. By studying approximately 30 isolates from each sample, the distribution and relative proportion of the individual taxa were determined. A. pleuropneumoniae was detected in samples from the tonsil areas of only two acutely ill animals. H. parasuis was isolated from the nasal cavities of four out of nine healthy pigs but from the oral cavities of only two animals. In contrast, taxon "minor group" and taxa D, E, and F were present in the oral cavities of the majority of pigs but were not detected in samples from their nasal cavities. The results indicate that all the observed V factor-dependent species of Pasteurellaceae except A. pleuropneumoniae, are members of the resident microflora of various mucosal surfaces of the porcine upper respiratory tract.
Actinobacillus
Respiratory tract
Actinobacillus pleuropneumoniae
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Haemophilus parainfluenzae
Isolation
Chocolate agar
Respiratory tract
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The penicillin-binding protein (PBP) profile of Haemophilus ducreyi was determined by a whole-cell-labeling assay. Only two major PBPs, of molecular weights 90,000 (PBP 1) and 38,500 (PBP 2), were detected in six of eight strains studied. Competition binding experiments and the attendant morphological effects suggested that PBP 1 was either a functional amalgamation or a lack of resolution of two proteins equivalent to PBPs 1 and 3 of Escherichia coli.
Haemophilus ducreyi
Penicillin binding proteins
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Objective Investigate isolation rate and analyze resistance of Haemophilus mfluenzae and parainfluenzae during 2000～2001 in our hospital. Methods Samples were inoculated in the Chocolatc Haemophilus Agar, the strains were identified and β-lactamase and resistance test were carried out. Results In 974 samples from respiratory tract we found 8 strains of Haemophilus influcnzae, with β-lactamase(+) strains being 0, and 13 strains of Haemophilus parainfluenzae β-lactamases 7.69 in 2000. In 845 samples we found 25 strains of Haemophilus influenzae, and with β lxctamase(+) strans 24 and Haemophilus parain fluenzae 120 strains with β-lactamase 17.5 and were found 1 strain β-lactamase negative in 2001. Conclusion Haemophilus is a very important infectious agent in respiratory tract, and the culture medium quality is a key factor to isolation rate of Haemophilus. In 2001 Haemophilus infuenzae β-lactamases(+) stain was 24, but it was unusual to find multi-resistance. Haemophilus parainfluenzae resistance is more complex than that of Haemophilus influenzae, it occurs by other mechanisms and is more difficult in terms of treatment. The correct use of antibiotics is the best way to decrease drug resistance.
Haemophilus parainfluenzae
Chocolate agar
Isolation
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Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of lipopolysaccharide (LPS) was performed to assess the usefulness of this technique for the epidemiologic analysis of Haemophilus influenzae type b isolates. LPS samples were prepared from isolates which had been passaged either in vitro or in infant rats. Preparations from paired isolates from a number of epidemiologically related clinical situations also were examined. The gel patterns of LPS prepared on different occasions from an individual isolate were stable. However, the LPS gel patterns changed in 5 of 14 (36%) of the passaged isolates, and differences in gel patterns also were observed among epidemiologically related isolates. The variability in LPS electrophoretic patterns of individual isolates indicated that this technique is not useful for the epidemiologic analysis of H. influenzae type b disease.
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