Effects of N-acetyl-L-cysteine on the membrane vesicle release and growth of respiratory pathogens.

Bacterial infections contribute to the disease progression of chronic obstructive pulmonary disease by stimulating mucus production in the airways. This increased mucus production and other symptoms are often alleviated when patients are treated with mucolytics such as N-acetyl-L-cysteine (NAC). Moreover, NAC has been suggested to inhibit bacterial growth. Bacteria can release membrane vesicles (MVs) in response to stress, and recent studies report a role for these proinflammatory MVs in the pathogenesis of airways disease. Yet, until now it is not clear whether NAC also affects the release of these MVs. This study set out to determine whether NAC, at concentrations reached during high-dose nebulization, affects bacterial growth and MV release of the respiratory pathogens non-typeable Haemophilus influenzae (NTHi), Moraxella catarrhalis (Mrc), Streptococcus pneumoniae (Spn) and Pseudomonas aeruginosa (Psa). We observed that NAC exerted a strong bacteriostatic effect, but also induced the release of proinflammatory MVs by NTHi, Mrc and Psa, but not by Spn. Interestingly, NAC also markedly blunted the release of TNF-α by naive macrophages in response to MVs. This suggests that the application of NAC by nebulization at a high dosage may be beneficial for patients with airway conditions associated with bacterial infections.

Prevention of influenza virus induced bacterial superinfection by standardized Echinacea purpurea, via regulation of surface receptor expression in human bronchial epithelial cells.

Viral infections may predispose the airways to secondary bacterial infections that can lead to unfavorable progression of principally self-limiting illnesses. Such complicated respiratory infections include pneumonia, bronchitis, sinusitis, acute otitis media, and sepsis, which cause high morbidity and lethality. Some of the pathogenic consequences of viral infections, like the expression of bacterial adhesion receptors and the disturbance of physical barrier integrity due to inflammation, may create permissive conditions for co-infections. Influenza virus A (H3N2) is a major pathogen that causes secondary bacterial infections and inflammation that lead to pneumonia. The herbal medicine Echinacea purpurea, on the other hand, has been widely used to prevent and treat viral respiratory infections, and recent clinical data suggest that it may prevent secondary infection complications as well. We investigated the role of standardized E. purpurea (Echinaforce® extract or EF) on H3N2-induced adhesion of live nontypeable Haemophilus influenzae (NTHi) and Staphylococcus aureus, along with the expression of bacterial receptors, intracellular adhesion molecule-1 (ICAM-1), fibronectin, and platelet activating factor receptor (PAFr), by BEAS-2B cells. Inflammatory processes were investigated by determining the cellular expression of IL-6 and IL-8 and the involvement of Toll-like receptor (TLR-4) and NFκB p65. We found that influenza virus A infection increased the adhesion of H. influenzae and S. aureus to bronchial epithelial cells via upregulated expression of the ICAM-1 receptor and, to some extent, of fibronectin and PAFr. Echinaforce (EF) significantly reduced the expression of ICAM-1, fibronectin, and PAFr and consequently the adhesion of both bacterial strains. EF also effectively prevented the super-expression of inflammatory cytokines by suppressing the expression of NFκB and possibly TLR-4. These results indicate that E. purpurea has the potential to reduce the risk of respiratory complications by preventing virus-induced bacterial adhesion and through the inhibition of inflammation super-stimulation (cytokine storms).

Oldenlandia diffusa Extract Inhibits Biofilm Formation by Haemophilus influenzae Clinical Isolates.

Oldenlandia diffusa has been empirically used as a therapeutic adjunct for the treatment of respiratory infections. To establish the basic evidence of its clinical usefulness, antimicrobial and biofilm inhibitory activities of an O. diffusa extract were examined against clinical isolates of Haemophilus influenzae, a major causative pathogen of respiratory and sensory organ infections. No significant growth inhibitory activity was observed during incubation for more than 6 h after the extract addition into a culture of H. influenzae. On the other hand, biofilm formation by H. influenzae, evaluated by a crystal violet method, was significantly and dose-dependently inhibited by the O. diffusa extract. Furthermore, the mRNA level of the biofilm-associated gene luxS of H. influenzae significantly decreased soon after the extract addition, and the suppressive effect continued for at least 2 h. At 2 h after the addition of the O. diffusa extract, the autoinducer in the culture supernatant was also significantly reduced by the O. diffusa extract in a dose-dependent manner. These results revealed that O. diffusa extract shows inhibitory activity against luxS-dependent biofilm formation but has no antimicrobial activity against planktonic cells of H. influenzae. Thus, O. diffusa extract might be useful as an adjunctive therapy for the treatment of respiratory infections caused by H. influenzae.

Copper(II)-Bis(Thiosemicarbazonato) Complexes as Antibacterial Agents: Insights into Their Mode of Action and Potential as Therapeutics.

There is increasing interest in the use of lipophilic copper (Cu)-containing complexes to combat bacterial infections. In this work, we showed that Cu complexes with bis(thiosemicarbazone) ligands [Cu(btsc)] exert antibacterial activity against a range of medically significant pathogens. Previous work using Neisseria gonorrhoeae showed that Cu(btsc) complexes may act as inhibitors of respiratory dehydrogenases in the electron transport chain. We now show that these complexes are also toxic against pathogens that lack a respiratory chain. Respiration in Escherichia coli was slightly affected by Cu(btsc) complexes, but our results indicate that, in this model bacterium, the complexes act primarily as agents that deliver toxic Cu ions efficiently into the cytoplasm. Although the chemistry of Cu(btsc) complexes may dictate their mechanism of action, their efficacy depends heavily on bacterial physiology. This is linked to the ability of the target bacterium to tolerate Cu and, additionally, the susceptibility of the respiratory chain to direct inhibition by Cu(btsc) complexes. The physiology of N. gonorrhoeae, including multidrug-resistant strains, makes it highly susceptible to damage by Cu ions and Cu(btsc) complexes, highlighting the potential of Cu(btsc) complexes (and Cu-based therapeutics) as a promising treatment against this important bacterial pathogen.

Evaluation of the EUCAST disc diffusion susceptibility testing method for Haemophilus influenzae based on the resistance mechanism to ß-lactam antibiotics.

OBJECTIVES: EUCAST developed an antibiotic susceptibility testing method for Haemophilus influenzae. We assessed the EUCAST testing method and EUCAST clinical breakpoints and newly proposed epidemiological cut-off values against H. influenzae clinical isolates with known molecular mechanisms of resistance to ß-lactam antibiotics. METHODS: In total, 89 clinical isolates were used: 30 were ß-lactamase negative with PBP3 mutations (gBLNAR), 20 were ß-lactamase positive without PBP3 mutations (gBLPAR), 15 were ß-lactamase positive with PBP3 mutations (gBLPACR), and 24 were ß-lactamase negative without resistance mechanism (gBLNAS). Twelve different ß-lactam antibiotics and disc charges were tested. RESULTS: None of the discs tested fully separated between gBLNAS and gBLNAR populations. According to EUCAST clinical zone diameter breakpoints, overall the best values of sensitivity and specificity were obtained with cefuroxime 30 µg and amoxicillin/clavulanic acid 2/1 µg discs for detection of gBLNAR and gBLPACR populations, although a previous ß-lactamase test was needed. Other antibiotic discs could be suitable for epidemiological purposes, such us penicillin 10 U for separating gBLNAR isolates and cefoxitin 30 µg for detection of gBLPACR isolates. By Etest using the EUCAST method, the EUCAST MIC clinical breakpoints for ampicillin and amoxicillin/clavulanic acid showed high specificity, but low sensitivity, for the detection of genotypes with mutations in PBP3. CONCLUSIONS: The main genotypes of ß-lactam-resistant H. influenzae can be separated by using the EUCAST disc diffusion method, although it should be noted that overlapping between populations with and without PBP3 mutations is common.

Evaluation of pharmacokinetic/pharmacodynamic relationships of PD-0162819, a biotin carboxylase inhibitor representing a new class of antibacterial compounds, using in vitro infection models.

The present study investigated the pharmacokinetic/pharmacodynamic (PK/PD) relationships of a prototype biotin carboxylase (BC) inhibitor, PD-0162819, against Haemophilus influenzae 3113 in static concentration time-kill (SCTK) and one-compartment chemostat in vitro infection models. H. influenzae 3113 was exposed to PD-0162819 concentrations of 0.5 to 16× the MIC (MIC = 0.125 µg/ml) and area-under-the-curve (AUC)/MIC ratios of 1 to 1,100 in SCTK and chemostat experiments, respectively. Serial samples were collected over 24 h. For efficacy driver analysis, a sigmoid maximum-effect (E(max)) model was fitted to the relationship between bacterial density changes over 24 h and corresponding PK/PD indices. A semimechanistic PK/PD model describing the time course of bacterial growth and death was developed. The AUC/MIC ratio best explained efficacy (r(2) = 0.95) compared to the peak drug concentration (C(max))/MIC ratio (r(2) = 0.76) and time above the MIC (T>MIC) (r(2) = 0.88). Static effects and 99.9% killing were achieved at AUC/MIC values of 500 and 600, respectively. For time course analysis, the net bacterial growth rate constant, maximum bacterial density, and maximum kill rate constant were similar in SCTK and chemostat studies, but PD-0162819 was more potent in SCTK than in the chemostat (50% effective concentration [EC(50)] = 0.046 versus 0.34 µg/ml). In conclusion, basic PK/PD relationships for PD-0162819 were established using in vitro dynamic systems. Although the bacterial growth parameters and maximum drug effects were similar in SCTK and the chemostat system, PD-0162819 appeared to be more potent in SCTK, illustrating the importance of understanding the differences in preclinical models. Additional studies are needed to determine the in vivo relevance of these results.

Strong antibacterial effect of miswak against oral microorganisms associated with periodontitis and caries.

BACKGROUND: The chewing stick (miswak) is used for oral hygiene in many parts of the world. In addition to the mechanical removal of plaque, an antibacterial effect has been postulated; however, tests of miswak extract from Salvadora persica (Arak) disclosed only low to moderate antibacterial effects. This may be attributable to the extraction process. Our aim was to test in vitro the antibacterial effect of miswak pieces, without extraction, on bacteria implicated in the etiology of periodontitis and caries. METHODS: Miswak pieces were standardized by size and weight (0.07 and 0.14 g) and tested against Streptococcus mutans, Lactobacillus acidophilus, Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans), Porphyromonas gingivalis, and, as a reference, Haemophilus influenzae. The miswak pieces were tested in two ways: embedded in the agar plate or suspended above the agar plate. RESULTS: The inhibitory effect was most pronounced on P. gingivalis, A. actinomycetemcomitans, and H. influenzae, less on S. mutans, and least on L. acidophilus. Suspended miswak had comparable or stronger effects than miswak embedded in agar. The 0.14-g suspended miswak exhibited significantly greater inhibition on A. actinomycetemcomitans and H. influenzae than the 0.14-g miswak embedded in agar (P<0.01 and P<0.001, respectively). CONCLUSIONS: Miswak embedded in agar or suspended above the agar plate had strong antibacterial effects against all bacteria tested. The antibacterial effect of suspended miswak pieces suggests the presence of volatile active antibacterial compounds.

Investigating the antimicrobial activity of natural honey and its effects on the pathogenic bacterial infections of surgical wounds and conjunctiva.

Antimicrobial activities of 10-100% (wt/vol) concentrations of new honey, stored honey, heated honey, ultraviolet-exposed honey, and heated stored honey were tested against common human pathogens, including Escherichia coli, Entrobacter cloacae, Pseudomonas aeruginosa, Shigella dysenteriae, Klebsiella sp., Haemophilus influenzae, Proteus sp., Staphylococcus aureus, Streptococcus hemolyticus group B, and Candida albicans. Antimicrobial activity of honey was tested in acidic, neutral, or alkaline media. These were compared with similar concentrations of glucose in nutrient broth. Surgical wounds were made on the dorsum of mice and infected with S. aureus or Klebsiella sp. The wounds were treated with local application of honey four times a day or appropriate antibiotics and compared with control values. Bacterial conjunctivitis due to E. coli, Proteus sp., S. aureus, Klebsiella sp., and P. aeruginosa was induced in rats. Conjunctival application of honey four times a day or appropriate antibiotics was used for treatment and compared with control values. Growth of all the isolates was completely inhibited by 30-100% honey concentrations. The most sensitive microbes were E. coli, P. aeruginosa, and H. influenzae. Glucose showed less antimicrobial activity than honey, and many microbes showed positive culture even in 100% glucose. Heating to 80 degrees C for 1 hour decreased antimicrobial activity of both new and stored honey. Storage of honey for 5 years decreased its antimicrobial activity, while ultraviolet light exposure increased its activity against some of the microorganisms. Antimicrobial activity of honey was stronger in acidic media than in neutral or alkaline media. Single doses of honey used to prepare the 60% concentration in nutrient broth were bacteriocidal for P. aeruginosa and bacteriostatic for S. aureus and Klebsiella sp. during certain periods. Local application of raw honey on infected wounds reduced redness, swelling, time for complete resolution of lesion, and time for eradication of bacterial infection due to S. aureus or Klebsiella sp. Its potency was comparable to that of local antibiotics. Honey application into infective conjunctivitis reduced redness, swelling, pus discharge, and time for eradication of bacterial infections due to all the isolates tested.

Synthesis and SAR evaluation of oxadiazolopyrazines as selective Haemophilus influenzae antibacterial agents.

The parallel synthesis and antibacterial activity of 5-hydroxy[1,2,5] oxadiazolo[3,4-b]pyrazines is reported. The compounds were synthesized by condensing diaminofurazan with alpha-keto acids to give a variety of aryl-substituted analogues. Halogenated phenyl groups at C-6 give rise to the greatest Haemophilus influenzae antibacterial activity.

Surveillance of resistance in bacteria causing community-acquired respiratory tract infections.

Bacterial resistance to antibiotics in community-acquired respiratory tract infections is a serious problem and is increasing in prevalence world-wide at an alarming rate. Streptococcus pneumoniae, one of the main organisms implicated in respiratory tract infections, has developed multiple resistance mechanisms to combat the effects of most commonly used classes of antibiotics, particularly the beta-lactams (penicillin, aminopenicillins and cephalosporins) and macrolides. Furthermore, multidrug-resistant strains of S. pneumoniae have spread to all regions of the world, often via resistant genetic clones. A similar spread of resistance has been reported for other major respiratory tract pathogens, including Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pyogenes. To develop and support resistance control strategies it is imperative to obtain accurate data on the prevalence, geographic distribution and antibiotic susceptibility of respiratory tract pathogens and how this relates to antibiotic prescribing patterns. In recent years, significant progress has been made in developing longitudinal national and international surveillance programs to monitor antibiotic resistance, such that the prevalence of resistance and underlying trends over time are now well documented for most parts of Europe, and many parts of Asia and the Americas. However, resistance surveillance data from parts of the developing world (regions of Central America, Africa, Asia and Central/Eastern Europe) remain poor. The quantity and quality of surveillance data is very heterogeneous; thus there is a clear need to standardize or validate the data collection, analysis and interpretative criteria used across studies. If disseminated effectively these data can be used to guide empiric antibiotic therapy, and to support-and monitor the impact of-interventions on antibiotic resistance.

Simultaneous respiratory tract colonization by multiple strains of nontypeable haemophilus influenzae in chronic obstructive pulmonary disease: implications for antibiotic therapy.

Nontypeable Haemophilus influenzae often causes exacerbations of chronic obstructive pulmonary disease (COPD), and these exacerbations are frequently treated with oral antibiotics. The goals of this study were to determine the frequency of the simultaneous presence of multiple strains of H. influenzae in sputum and to measure the MICs of antibiotics for the isolates. In a prospective study, adults with COPD were seen monthly. Sputum cultures were obtained, and individual colonies were subjected to genomic DNA typing and MIC determinations. Multiple strains of H. influenzae were present simultaneously in the sputum of 26.3% of adults with COPD. In 64.5% of these, MICs of >/=1 antibiotic varied by >/=4-fold among the strains. Therefore, multiple strains of H. influenzae are frequently present simultaneously in the sputum of adults with COPD, and the antimicrobial susceptibility of different strains in the same sputum sometimes differs.

Long-term persistence of ciprofloxacin-resistant Haemophilus influenzae in patients with cystic fibrosis.

Ciprofloxacin has been a major advance in the treatment of chronic respiratory infections. Three patients with cystic fibrosis and colonized by 5 nontypeable Haemophilus influenzae strains exhibiting low- (MIC, 2 microg/mL) and high-level ciprofloxacin resistance (MICs, 16-32 microg/mL) are described. The patients had received several courses of ciprofloxacin. These MICs represent a decrease in ciprofloxacin susceptibility of 200-3200 times. Molecular epidemiologic methods demonstrated that 2 patients were chronically colonized by their own ciprofloxacin-resistant strains for > or = 15-17 months. Three strains showed simultaneous resistance to ampicillin and chloramphenicol by enzyme inactivation, and 2 had ampicillin resistance without beta-lactamase activity. These data suggest that the emergence and long-term persistence of ciprofloxacin-resistant H. influenzae in patients with cystic fibrosis can be a consequence of antibiotic treatment.

Lack of expression of the global regulator OxyR in Haemophilus influenzae has a profound effect on growth phenotype.

A pBR322-based library of chromosomal DNA from the nontypeable Haemophilus influenzae TN106 was screened for the expression of transferrin-binding activity in Escherichia coli. A recombinant clone expressing transferrin-binding activity contained a 3.7-kb fragment of nontypeable H. influenzae DNA. Nucleotide sequence analysis of this insert revealed the presence of two complete open reading frames encoding proteins of approximately 26 and 34 kDa. Mini-Tn10kan transposon mutagenesis at different sites within the open reading frame encoding the 34-kDa protein resulted in the abolition of transferrin-binding activity in the recombinant E. coli clone. The deduced amino acid sequence of the 34-kDa protein had 70% identity with the OxyR protein of E. coli; this latter macromolecule is a member of the LysR family of transcriptional activators. When a mutated H. influenzae oxyR gene was introduced into the chromosome of the wild-type H. influenzae strain by allelic exchange, the resulting oxyR mutant still exhibited wild-type levels of transferrin-binding activity but was unable to grow on media containing the heme precursor protoporphyrin IX (PPIX) in place of heme. This mutant also exhibited reduced growth around disks impregnated with heme sources. Supplementation of the PPIX-based growth media with catalase or sodium pyruvate resulted in normal growth of the H. influenzae oxyR mutant. Provision of the wild-type H. influenzae oxyR gene in trans also permitted the growth of this mutant on a PPIX-based medium. Exogenously supplied catalase restored the growth of this mutant with heme sources to nearly wild-type levels. These results indicate that expression of a wild-type OxyR protein by H. influenzae is essential to allow this organism to protect itself against oxidative stresses in vitro.

Influence of pili, fibrils, and capsule on in vitro adherence by Haemophilus influenzae type b.

Haemophilus influenzae type b is an encapsulated bacterium that initiates infection by colonizing the upper respiratory epithelium. In vitro studies indicate that H. influenzae type b is capable of expressing two morphologically distinct filamentous adhesive structures, referred to as pili and fibrils, respectively. In this study, we examined adherence to a variety of human epithelial-cell types and demonstrated that pili and fibrils have separate cellular binding specificities. In addition, we found that capsular material inhibits fibril recognition of the host-cell surface. This inhibitory effect was reduced when bacteria were grown to stationary phase, reflecting diminished encapsulation. However, when growth medium was supplemented with Mg2+, stationary-phase organisms were relatively heavily encapsulated and non-adherent. These observations suggest that encapsulation can be modulated in response to growth phase or environmental signals. It is possible that encapsulation is down-modulated early in the infectious process in order to avoid interfering with colonization. In contrast, encapsulation may be up-modulated between hosts and during bacteremia, where it appears to confer a selective advantage. We speculate that this model may also apply to other encapsulated pathogens.

[A growth supplement for "fastidious germs"]. / Suplemento de crecimiento para "gérmenes fastidiosos".

A growth supplement for "fastidious germs" was produced at the Laboratory of Acute Respiratory Infections of "Pedro Kourí" Institute of Tropical Medicine. This supplement was studied by computerized optic spectrophotometry, and chemical composition was determined. The efficacy of this supplement for the culture of Haemophilus influenzae type b, was evidenced using 100 strains, and it was proven that it can be used in concentrations raging from 1 to 10%.

Modification in penicillin-binding proteins during in vivo development of genetic competence of Haemophilus influenzae is associated with a rapid change in the physiological state of cells.

By using whole-cell labeling assay with 125I-penicillin V, we observed a reduction in the binding of the radiolabeled beta-lactam to four or five penicillin-binding proteins (PBPs) in Haemophilus influenzae cells cultivated under specific conditions. PBPs 3A, 3B, 4, and 6 were altered after the growth of bacteria in diffusion chambers implanted in the peritoneal cavity of rats. PBP 2 was also modified when cells were cultivated in human cerebrospinal fluids. Because this observation may have important consequences on the efficacy of beta-lactams during antibiotic therapy, we characterized the physiological state of bacteria cultivated in animals in the hope of explaining how such important changes in cell properties develop in vivo. Since the development of natural genetic competence occurs at the stationary phase of growth in H. influenzae, we used a DNA transformation assay to evaluate the physiological state of bacteria grown in diffusion chambers implanted in rats. Chromosomal DNA isolated from an antibiotic-resistant donor strain was mixed with bacteria in diffusion chambers. At different times during a 5-h incubation period, recipient bacteria were collected from the chambers, CFU were determined by plate counting, and antibiotic-resistant transformants were isolated on selective plates. Genetic competence rapidly developed in cells grown in rats, and the frequency of transformation by test DNA was elevated. Electron microscopy revealed an irregular cell shape and blebs at the surface of bacteria cultivated in animals and in cerebrospinal fluids. In an attempt to induce a similar physiological state in vitro, we supplemented broth cultures with cyclic AMP or synchronized cultures by a nutritional upshift. No changes in PBPs were observed with supplemental cyclic AMP or during a single cell cycle. Finally, a reduction in the affinity of PBPs for 125I-penicillin V identical to that observed in bacteria grown in rats was observed in cells isolated from the stationary phase of growth in vitro. These results clearly indicate that H. influenzae cells grown in animals undergo a rapid change to a physiological state similar to that found in late-stationary-phase cultures in vitro. This observation indicates that the rational design of future and improved antibiotic therapy of H. influenzae infections should consider cell properties of slow-growing or latent bacteria.

Broth microdilution testing of Haemophilus influenzae with haemophilus test medium versus lysed horse blood broth. Canadian Haemophilus Study Group.

Broth microdilution testing of 702 community-acquired isolates of Haemophilus influenzae from across Canada was performed with both Mueller-Hinton broth supplemented with 3% lysed horse blood broth (LHB) (BBL Microbiology Systems, Cockeysville, Md.) and haemophilus test medium (HTM). The prevalence of beta-lactamase production was found to be 26% with no regional variation. MICs determined with LHB tended to be higher than those with HTM, but interpretive errors due to these differences were observed only rarely with trimethoprim-sulfamethoxazole (n = 5), cefaclor (n = 8), and cefamandole (n = 3). The interobserver variability in MIC determinations was found to be greater when LHB was used than when HTM was used. There was no difference in intraobserver variability between the two medium formulations. beta-Lactamase-positive isolates developed false resistance to amoxicillin-clavulanate 2 weeks after microdilution panels of both types of medium were stored at -20 degrees C but not when panels were stored at -70 degrees C. In conclusion, this study supports the use of HTM rather than LHB for sensitivity testing of H. influenzae because of its lower rate of interobserver variability and its ability to support the growth of these organisms, which is comparable to that of LHB.

Experimental pneumonia due to Haemophilus influenzae: observations on pathogenesis and treatment.

A model of pneumonia due to Haemophilus influenzae type b was developed in mice and used for exploration of the pathophysiology of the infection and evaluation of the efficacy of five antimicrobial agents. Adult C57BL/6 mice were challenged with 3 X 10(9) cfu of H influenzae by intratracheal inoculation. Mice given placebo or no treatment experienced a uniformly bacteremic and fatal infection. Animals given ampicillin, cefamandole, chloramphenicol, erythromycin plus sulfisoxazole, or fludalanine plus pentizidone (MK 0641/MK 0642, an investigational combination drug) survived at a higher rate than did controls (P less than .001 at 72 hr for each antibiotic). However, survival rates for the various antibiotic-treated groups were similar. Viable organisms were eradicated from the lungs of antibiotic-treated mice more quickly than from the lungs of controls (P less than .001 at 24 hr for each drug). Studies of pulmonary clearance revealed significant differences among regimens; the order of efficacy (from most to least) was ampicillin, chloramphenicol, erythromycin/sulfisoxazole, cefamandole, and fludalanine / pentizidone . This model represents an appropriate system for evaluation of invasive pulmonary infection caused by H influenzae type b. Of the antibiotics assessed, ampicillin was most active in vivo.

A comparison of chloramphenicol and ampicillin as bactericidal agents for Haemophilus influenzae type B.

In tests of bactericidal action against H. influenzae type b strains isolated from patients with meningitis, chloramphenicol was found to be far more reliable than ampicillin in dealing with large inocula, and more rapidly effective against both large and relatively small inocula. These findings provide a laboratory explanation for the somewhat better record of chloramphenicol as an agent for treatment of haemophilus meningitis.

Molecular events accompanying the fixation of genetic information in Haemophilus heterospecific transformation.

Heterospecific transformation between Haemophilus influenzae and H. parainfluenzae was investigated by isopycnic analysis of deoxyribonucleic acid (DNA) extracts of (3)H-labeled transforming cells that had been exposed to (32)P-labeled, heavy transforming DNA. The density distribution of genetic markers from the resident DNA and from the donor DNA was determined by transformation assay of fractions from CsCl gradients, both species being used as recipients. About 50% of the (32)P atoms in H. parainfluenzae donor DNA taken up by H. influenzae cells were transferred to resident DNA, and only a small amount of the label was lost under conditions of little cell growth. There was less transfer in the reciprocal cross, and almost half of the donor label was lost. In both crosses, the transferred donor material transformed for the donor marker considerably more efficiently when assayed on the donor species than on the recipient species, indicating that at least some of the associated (32)P atoms are contained in relatively long stretches of donor DNA. When the transformed cultures were incubated under growth conditions, the donor marker associated with recipient DNA transformed the donor species with progressively decreasing efficiency. The data indicate that the low heterospecific transformation between H. influenzae and H. parainfluenzae may be due partly to events occurring before association of donor and resident DNA but results mostly from events that occur after the association of the two DNA preparations.