Therapeutic activity of ceftazidime and eleven other beta-lactam antibiotics against experimental Haemophilus influenzae, type b meningitis

Twelve beta-lactams (11 cephalosporins and aztreonam) were compared in an infant rat model of Haemophilus influenzae, type b meningitis. Ceftazidime was the most effective compound tested and showed greater activity than cefuroxime, latamoxef (moxalactam) and cefotaxime--all of which have been used successfully in clinical haemophilus meningitis in children.     

Tolerance of Haemophilus influenzae to beta-lactam antibiotics.

Two hundred clinical isolates of Haemophilus influenzae were tested for tolerance (MBC/MIC greater than or equal to 32) to ampicillin and cefotaxime by broth dilution tests. Of 200 strains, 9 were tolerant to ampicillin, and 10 were tolerant to cefotaxime. Tolerant organisms were identified in both systemic and nonsystemic infections and among different biotypes and serotypes of H. influenzae. These tolerant isolates were compared with nontolerant isolates by broth dilution and killing curves with log-phase and stationary-phase inocula. Both tolerant and nontolerant bacteria in log phase were killed more rapidly by antibiotics than bacteria in stationary-phase growth. When tested against 11 different beta-lactams, several patterns of tolerance were observed. Six of the ten strains were tolerant to aztreonam, four were tolerant to cefuroxime, three were tolerant to cefamandole, and two were tolerant to cefoxitin. Strain H130 was tolerant to all beta-lactam antibiotics studied. None of the 10 tolerant H. influenzae isolates were tolerant to chloramphenicol, rifampin, tobramycin, ciprofloxacin, enoxacin, and trimethoprim-sulfamethoxazole. Although the clinical significance of tolerance is not determined, this study suggests that the bactericidal activity (MBC) of beta-lactam antibiotics against H. influenzae should be determined in cases of severe infections in which clinical response is slow or unsatisfactory.     

Mechanism of resistance of an ampicillin-resistant, beta-lactamase-negative clinical isolate of Haemophilus influenzae type b to beta-lactam antibiotics

The mechanism of non-beta-lactamase-mediated beta-lactam resistance in a clinical isolate of Haemophilus influenzae type b was studied. This clinical isolate showed up to a 32-fold increase in MICs of a wide variety of beta-lactams, including moxalactam and cefotaxime, although no beta-lactamase activity was detected, even after attempted induction. Transformation of broad-spectrum beta-lactam resistance into ampicillin-susceptible H. influenzae RDnov was accomplished. Examination of the outer membrane protein profile of the resistant parent by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of Triton X-100-extracted membranes revealed an unusual major outer membrane protein band at a molecular weight of 45,000. This outer membrane protein profile did not transform with beta-lactam resistance. Permeability differences were noted between the resistant strain and the nonisogenic susceptible strain of H. influenzae, although these penetration differences were not transformed. Comparison of the penicillin-binding protein profile of a resistant transformant with that of a susceptible parent with both whole-membrane preparations and whole-cell labeling, revealed a major reduction in binding affinity to penicillin-binding proteins 3a and 3b (molecular weights, 68,000 and 65,000, respectively). Thus, alteration in penicillin-binding proteins 3a and 3b correlated with the beta-lactam resistance.     

Activities of newer beta-lactam antibiotics against ampicillin, chloramphenicol, or multiply-resistant Haemophilus influenzae

The susceptibilities of singly or multiply-resistant clinical isolates of Haemophilus influenzae were determined by agar dilution to aztreonam, imipenem, and six third-generation cephalosporins. These included selected isolates that were resistant to ampicillin only, chloramphenicol only, and four isolates that were resistant to ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole. All of the isolates were highly susceptible to these newer beta-lactam antibiotics. Isolates resistant to trimethoprim-sulfamethoxazole and/or chloramphenicol had susceptibilities similar to those of strains resistant only to ampicillin. Ceftriaxone, ceftizoxime, and cefotaxime were the most active of the study antibiotics (MIC90 = 0.004-0.016 micrograms/ml), and were also bactericidal at concentrations no more than twice the minimum inhibitory concentration (MIC). Minimum inhibitory concentrations of cefoperazone increased dramatically with only a 10-fold increase in inoculum size of beta-lactamase producing strains, while MICs of the other new agents were not significantly affected by elevation of the inoculum. These new antibiotics appear to be promising candidates for therapy of infections due to resistant H. influenzae.     

Effect of antibiotics on adherence of Haemophilus influenzae type b.

During antibiotic therapy for serious Haemophilus influenzae type b infections in children, respiratory mucosal colonization with this organism is suppressed but not eradicated. To define possible mechanisms contributing to this suppression, the ability of six antibiotics to influence the adherence of H. influenzae type b to human epithelial cells was investigated. In assays in which the organisms were grown in broth containing 0.5 X the MIC of rifampin, ampicillin, clindamycin, chloramphenicol, lincomycin, or trimethoprim-sulfamethoxazole, all drugs except rifampin significantly reduced bacterial adherence. In assays in which nonreplicating organisms were exposed to the antibiotics, all six drugs reduced the adherence of the bacteria. In assays in which the epithelial cells were exposed to the antibiotics, all drugs reduced bacterial adherence. In addition, the presence of ampicillin, chloramphenicol, lincomycin, or trimethoprim-sulfamethoxazole appeared to facilitate the release of organisms adherent to epithelial cells. Thus, antibiotics appear to inhibit adherence of H. influenzae type b to human epithelial cells and may interfere with bacterial or epithelial cell binding sites. These observations may explain the suppression of H. influenzae type b mucosal colonization that occurs during antibiotic treatment of patients with systemic H. influenzae type b infections.     

Susceptibility of Haemophilus influenzae to chloramphenicol and eight beta-lactam antibiotics

We examined the minimal inhibitory concentrations and minimal bactericidal concentrations of chloramphenicol, ampicillin, ticarcillin, cefamandole, cefazolin, cefoxitin, cefotaxime, ceforanide, and moxalactam for 100 isolates of Haemophilus influenzae, 25 of which produced beta-lactamase. Susceptibility was not influenced by the capsular characteristic of the organism. The mean minimal inhibitory concentrations of cefamandole, ticarcillin, and ampicillin for beta-lactamase-producing strains were 3-, 120-, and 400-fold higher than their respective mean minimal inhibitory concentrations for beta-lactamase-negative strains. No such difference was noted for the other antibiotics. We performed time-kill curve studies, using chloramphenicol, ampicillin, cefamandole, cefotaxime, and moxalactam with two concentrations of the antimicrobial agents (4 or 20 times the minimal inhibitory concentrations) and two inoculum sizes (10(4) or 10(6) colony-forming units per ml). The inoculum size had no appreciable effect on the rate of killing of beta-lactamase-negative strains. The rates at which beta-lactamase-producing strains were killed by chloramphenicol, cefotaxime, and moxalactam was not influenced by the inoculum size. Whereas cefamandole in high concentrations was able to kill at 10(6) colony-forming units/ml of inoculum, it had only a temporary inhibiting effect at low drug concentrations. Methicillin and the beta-lactamase inhibitor CP-45,899 were able to neutralize the inactivation of cefamandole by a large inoculum of beta-lactamase-producing H. influenzae.     

Effect of inoculum size on the susceptibility of Haemophilus influenzae b to beta-lactam antibiotics.

The current prevalence of ampicillin-resistant Haemophilus influenzae b meningitis requires accurate knowledge of susceptibility to alternative antibiotics. One variable affecting susceptibility is inoculum size. We studied the susceptibility of 200 clinical isolates of H. influenzae b to ampicillin, carbenicillin, and cefamandole at inocula of 10(5) and 10(7) CFU by two techniques. Fifty ampicillin-susceptible and fifty ampicillin-resistant strains were tested for susceptibility to ampicillin by broth dilution while 100 of each were tested by agar dilution. An inoculum effect was found, being greatest with the ampicillin-resistant strains. The range of minimal inhibitory concentrations for the resistant strains was 25 to 800 microgram of ampicillin per ml at an inoculum of 10(5) and 2,000 to less than 6,000 microgram of ampicillin at 10(7); 1.0 to 150 microgram of carbenicillin per ml at 10(5) and 6.2 to 2,000 microgram of carbenicillin per ml at 10(7); 0.4 to 2.0 microgram of cefamandole at 10(5) and 1.0 to 125 microgram/ml at 10(7). Because of this inoculum effect, we would not recommend the use of carbenicillin or cefamandole for therapy of ampicillin-resistant H. influenzae meningitis.     

Haemophilus influenzae type b vaccine

Haemophilus influenzae type b (Hib) vaccine has been included in the national Immunization Program in more than 120 countries around the world and its effectiveness and safety have been confirmed. In Japan, Hib vaccine was recently licensed in December 2008. Because it has included in the category of voluntary vaccine and the burden of out-of-pocket expense is a major concern for caregivers, payment coverage through the local government funded by the temporal national budget has been initiated since November 2010. It is important to make an effort to fill the "vaccine gap" between Japan and other developed countries and increase a vaccine coverage rate including Hib vaccine to decrease vaccine preventable diseases in Japanese children.     

Comparative activities of LY 164846 and other orally administered beta-lactam antibiotics against Haemophilus influenzae.

LY 164846, a new oral cephalosporin, demonstrated very good inhibitory and bactericidal activities against Haemophilus influenzae irrespective of the production of beta-lactamase by the test strains. However, its activity was not markedly superior to that of either Augmentin (amoxicillin plus clavulanate) or cefaclor.     

Beta-lactamase-positive strains of Haemophilus influenzae: susceptibility to and inactivation of beta-lactam antibiotics

Susceptibility and time-kill studies were done with low and high inocula of both beta-lactamase-positive and -negative strains of Haemophilus influenzae with cefamandole, ampicillin, cefoperazone, mezlocillin, moxalactam, and ceftriaxone. Bioassay was done to test for antibiotic inactivation by beta-lactamase-positive strains. All six antibiotics were highly active against the low inoculum (10(4) to 10(5) colony-forming units/ml) of beta-lactamase-negative strains; ceftriaxone, moxalactam, and cefoperazone were equally active against the same inoculum concentration of beta-lactamase-positive strains. In contrast, cefamandole, mezlocillin, and ampicillin were less active against the low inoculum of beta-lactamase-positive H influenzae. A marked inoculum effect occurred with the high inoculum (10(7) to 10(8) CFU/ml) with all six antibiotics, regardless of beta-lactamase production. In time-kill studies, marked differences in bacterial killing resulted after low and high inocula. Ampicillin, cefamandole, cefoperazone, and mezlocillin were rapidly inactivated by the high inoculum of beta-lactamase-positive H influenzae.     

Comparative activities of antibiotics against intracellular non-typeable Haemophilus influenzae

INTRODUCTION: Non-typeable Haemophilus influenzae (NTHi) is a major bacterial pathogen of community-acquired respiratory tract infection and is usually found extracellularly, although studies have revealed that NTHi may possess the ability to invade human epithelial cells where it is then protected against attack by the local immune system and partly against the effect of antibiotics. The aim of the present study was to assess the ability of ampicillin, azithromycin, telithromycin, ciprofloxacin and moxifloxacin, five antibiotics in common clinical use, to kill NTHi within bronchial epithelial cells. METHODS: Confluent human bronchial epithelial cells were infected with NTHi 77, a particularly invasive clinical strain. Extracellular bacterial cells were killed with gentamicin and the intracellular bacteria were incubated with antibiotics at concentrations of 1 mg/l or 10 mg/l for 4 h or 8 h. Viable intracellular bacteria were counted after lysis of the epithelial cells. RESULTS: With the exception of ampicillin, all the antibiotics caused significant reduction of intracellular bacteria at concentrations of 10 mg/l and exposure for 4 h or at 1 mg/l for 8 h. At 1 mg/l, moxifloxacin eliminated 94% of intracellular NTHi after 4 h and 98% after 8 h; ciprofloxacin, azithromycin and telithromycin only achieved killing indices below 75 after 4 h but 86-90% killing after 8 h. At 10 mg/l, moxifloxacin, ciprofloxacin, telithromycin and azithromycin were able to achieve 99.7%, 96.3%, 86.7% and 74.7% eradication of intracellular bacteria, respectively, after exposure for 4 h. CONCLUSION: These results demonstrate the rapid antibacterial efficacy of moxifloxacin against intracellular NTHi in vitro. Moxifloxacin, which combines high extracellular and intracellular activities, could be an important tool in the treatment of recurrent respiratory tract infections.     

Influence of beta-lactamase-producing strains of Branhamella catarrhalis and Haemophilus influenzae on certain beta-lactam antibiotics

Ampicillin, benzylpenicillin, cefaclor and cefuroxime were exposed to strains of Branhamella catarrhalis and Haemophilus influenzae with and without ability to produce beta-lactamase. The antibiotics were dissolved in phosphate buffer at pH 6, 7 and 8 and the mean enzyme activity was calculated from decrease in peak heights by the HPLC technique. Cefuroxime was the most stable drug regardless of pH. For the other antibiotics, changes in pH influenced the results. In infectious processes factors like pH and pCO2 show some variation. This fact may influence the interaction between beta-lactams and beta-lactamases.     

Effect of inoculum size on Haemophilus influenzae type b susceptibility to new and conventional antibiotics

Thirty-three Haemophilus influenzae type b isolates, including beta-lactamase acetyltransferase-positive strains, were tested by microtiter broth dilution for susceptibility to eight beta-lactam compounds and chloramphenicol. All antibiotics except ampicillin and chloramphenicol were highly bactericidal against all isolates at an inoculum of 10(5) CFU/ml. However, at an inoculum of 10(5) CFU/ml, the minimal bactericidal concentrations of all drugs except ceftriaxone were above levels usually achievable in cerebrospinal fluid. Results of time-kill studies confirmed this inoculum effect. In vivo studies are needed to test the clinical impact of these observations.     

Childhood meningitis and Haemophilus influenzae type b vaccines

Effective vaccines against Haemophilus influenzae type b (Hib) disease in children are available. If overseas experience is any indication, the incidence of invasive Hib disease in infants and children will fall substantially over the next few years. In the absence of signs of raised intracranial pressure, lumbar puncture is generally indicated in children with suspected meningitis prior to therapy. Infants and children with suspected meningitis should be given dexamethasone preferably before or within twenty minutes of the first dose of antibiotics in order to prevent the sequelae of deafness and neurological damage. The treatment of choice for neonatal meningitis is ampicillin and cefotaxime, but the efficacy of dexamethasone in preventing long-term sequelae in neonates is not yet established. The treatment of choice in older infants and children is cefotaxime or ceftriaxone. Antibiotic therapy should be continued for seven days for uncomplicated meningococcal meningitis and 10 days for Hib and pneumococcus in infants and children, and for 14 to 21 days for neonatal meningitis. Chemoprophylaxis of selected contacts with rifampicin is recommended.     

Susceptibility of Haemophilus influenzae type b to ampicillin-sulbactam.

Seventy-five strains of Haemophilus influenzae type b, including 45 beta-lactamase-positive strains, were tested by MIC and time kill studies for susceptibility to ampicillin-sulbactam at various ratios. beta-Lactamase-negative strains were inhibited by lower concentrations of ampicillin-sulbactam than beta-lactamase-positive organisms. beta-Lactamase-negative strains showed a decrease in CFU per milliliter by a factor of 10(4) after a 24-h incubation with ampicillin-sulbactam. beta-Lactamase-positive isolates showed an initial decrease by a factor of up to 10(2) CFU per milliliter, but by the end of incubation these isolates grew to approximately the same cell density as the antibiotic-free control regardless of ampicillin-sulbactam ratios. Caution should be exercised in the use of this combination in treatment of meningitis, in which a high bacterial density is commonly encountered.     

Genotyping of Haemophilus influenzae type b in pre-vaccination era

Identification of Haemophilus influenzae type b (Hib) in asymptomatic carriers is critical to control the spread of disease. This study was conducted between January 2008 and August 2011 as part of a birth cohort study in Sado Island, Japan, to elucidate the prevalence of Hib and its clones in a specific region. Nasopharyngeal cultures were obtained from 349 subjects at 4-, 7-, 10-, 18-, and 36-month health checkups and analyzed for H. influenzae. The Hib and nontypeable H. influenzae detection rates ranged from 0 to 1.5% (12 isolates) and from 7.9 to 32.9%, respectively. Twelve pediatric patients diagnosed with invasive or non-invasive Hib infections during the study period were also enrolled. The Hib isolates were analyzed for carriage of the beta-lactamase gene and ftsI mutations, and multilocus sequence type (MLST, ST type). Of the 24 Hib isolates, 18 (75%) were ST54, 5 (21%) were ST190, and 1 isolate (4%) was ST95. All of the ST190 isolates were genetically beta-lactamase-negative ampicillin-susceptible isolates, while all but one of the ST54 isolates were genetically beta-lactamase-positive amoxicillin/clavulanic acid-resistant isolates. The geographic distribution of Hib isolates in the study period was scattered. There were 2 day-care cases and 1 family case of Hib infection. The ST54 and ST190 strains circulated in Sado Island and were detected in both asymptomatic carriers and patients. We note that surveillance of healthy subjects to identify Hib carriers is important to understand the transmission of Hib.     

Role of cerebrospinal fluid pleocytosis and Haemophilus influenzae type b capsule on blood brain barrier permeability during experimental meningitis in the rat.

The influence of leukocytes and Haemophilus influenzae type b (Hib) capsule on blood brain barrier permeability (BBBP) to circulating 125I-albumin in normal and leukopenic rats was assessed after intracisternal inoculation of encapsulated (Rd-/b+/02) or unencapsulated (Rd-/b-/02) isogenic strains of Hib. Both normal and leukopenic animals had increased BBBP 18 h after inoculation, with normal rats demonstrating significantly increased BBBP after challenge with the encapsulated strain. Despite cerebrospinal fluid (CSF) pleocytosis in normal rats, CSF bacterial concentrations were not lower. Normal rats cleared unencapsulated Rd-/b-/02 more effectively than leukopenic rats, with BBBP correlating with CSF bacterial density and not leukocyte concentrations. Challenge with heat-killed Rd-/b+/02 resulted in increased BBBP in both normal and leukopenic rats, with greater BBBP at higher bacterial concentrations. The data suggest: (a) significant increases in BBBP occur in the near absence of CSF leukocytes; (b) CSF leukocytes can augment changes in BBBP; (c) type b capsule inhibits host clearance mechanisms within the CSF; and (d) BBBP appears to correlate with bacterial concentrations within the CSF.     

New protection against Haemophilus influenzae type b infections in infants and young children.

Invasive Haemophilus influenzae type b (Hib) infections carry high morbidity and mortality, primarily due to meningitis among infants less than 1 year of age. Two new vaccines, HbOC and PRP-OMP, have been developed and licensed to prevent invasive Hib infections in infants and young children. New recommendations advise clinicians to begin immunization for Hib at 2 months of age and to complete the designated series. This article details the new Hib immunization schedule for all pediatric clients between 2 months and 5 years. Additionally, public-health measures toward the prevention of serious pediatric morbidity and mortality are covered.     

A permeability barrier as a mechanism of chloramphenicol resistance in Haemophilus influenzae.

Chloramphenicol resistance in Haemophilus influenzae occurs most frequently via plasmid-mediated chloramphenicol acetyltransferase production. We studied four strains with high-level chloramphenicol resistance (MIC greater than 20 micrograms/ml) which did not have detectable chloramphenicol acetyltransferase activity. The chloramphenicol resistance determinant was transformed into a chloramphenicol-susceptible laboratory H. influenzae strain from each of the four wild-type strains, enabling isogenic comparisons. By thin-layer chromatography and a bioassay, there was no evidence of non-chloramphenicol acetyltransferase modification of chloramphenicol. In vitro protein synthesis in the presence of chloramphenicol was equivalently inhibited in the chloramphenicol-resistant transformants and in the susceptible recipient. Chloramphenicol uptake by these strains during logarithmic growth was compared by high-pressure liquid chromatographic quantitation; at chloramphenicol concentrations of 5, 10, and 20 micrograms/ml the four transformants showed a decreased rate of uptake of chloramphenicol compared with the isogenic chloramphenicol-susceptible recipient. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of outer membrane proteins revealed a markedly diminished 40-kilodalton protein in the resistant transformants. We propose that the mechanism of chloramphenicol resistance in these strains is a relative permeability barrier due to the loss of an outer membrane protein.