Influence of Thiazolidine-2,4-Dione Derivatives with Azolidine or Thiosemicarbazone Moieties on Haemophilus spp. Planktonic or Biofilm-Forming Cells.

Biofilm, naturally formed by microorganisms as integrated surface-bound communities, is one of the reasons for the development of antimicrobial resistance. Haemophilus spp. are common and representative opportunistic Gram-negative rods forming from the upper respiratory tract microbiota. The aim of this paper was to evaluate the influence of thiazolidine-2,4-dionebased azolidine and chlorophenylthiosemicarbazone hybrids against both planktonic and biofilm-forming Haemophilus spp. cells. The in vitro activity against planktonic and biofilm-forming cells of the tested compounds were evaluated by using the broth microdilution method. These activities were detected against reference and clinical strains of Haemophilus spp. on the basis of MICs (minimal inhibitory concentrations) and MBICs (minimal biofilm inhibitory concentrations). In addition, anti-adhesive properties of these compounds were examined. The target compounds showed potential activity against planktonic cells with MIC = 62.5⁻500 mg/L and biofilm-forming cells with MBIC = 62.5⁻1000 mg/L. The observed anti-adhesive properties of the tested compounds were reversible during long-term incubation in a lower concentration of compounds.

Haemophilus-Dominant Nasopharyngeal Microbiota Is Associated With Delayed Clearance of Respiratory Syncytial Virus in Infants Hospitalized for Bronchiolitis.

The relation of nasopharyngeal microbiota to the clearance of respiratory syncytial virus (RSV) in infants hospitalized for bronchiolitis is not known. In a multicenter cohort, we found that 106 of 557 infants (19%) hospitalized with RSV bronchiolitis had the same RSV subtype 3 weeks later (ie, delayed clearance of RSV). Using 16S ribosomal RNA gene sequencing and a clustering approach, infants with a Haemophilus-dominant microbiota profile at hospitalization were more likely than those with a mixed profile to have delayed clearance, after adjustment for 11 factors, including viral load. Nasopharyngeal microbiota composition is associated with delayed RSV clearance.

An isolate of Haemophilus haemolyticus produces a bacteriocin-like substance that inhibits the growth of nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi) frequently colonises the upper respiratory tract and is an important cause of respiratory infections. Resistance to antibiotics is an emerging trend in NTHi and alternative prevention or treatment strategies are required. Haemophilus haemolyticus is a common commensal occupying the same niche as NTHi and, if able to produce substances that inhibit NTHi growth, may have a role as a probiotic. In this study, ammonium sulphate extracts from broth culture of 100 H. haemolyticus isolates were tested for the presence of substances inhibitory to NTHi using a well diffusion assay. One isolate produced a substance that consistently inhibited the growth of NTHi. The substance was inactivated by protease enzymes and had a molecular size of ca. 30 kDa as determined by size exclusion chromatography. When the substance was tested against bacteria from eight Gram-negative and three Gram-positive genera, only Haemophilus spp. were inhibited. Quantitative PCR testing showed the substance to be different to 'haemocin', the previously described bacteriocin of H. influenzae type b. These molecular characteristics, together with narrow-spectrum activity, suggest the substance may be a novel bacteriocin, and there is potential for this H. haemolyticus isolate to function as a probiotic for reduction of colonisation and subsequent infection with NTHi.

Culture and PCR detection of Haemophilus influenzae and Haemophilus haemolyticus in Australian Indigenous children with bronchiectasis.

A PCR for protein D (hpd#3) was used to differentiate nontypeable Haemophilus influenzae (NTHI) from Haemophilus haemolyticus. While 90% of nasopharyngeal specimens and 100% of lower-airway specimens from 84 Indigenous Australian children with bronchiectasis had phenotypic NTHI isolates confirmed as H. influenzae, only 39% of oropharyngeal specimens with phenotypic NTHI had H. influenzae. The nasopharynx is therefore the preferred site for NTHI colonization studies, and NTHI is confirmed as an important lower-airway pathogen.

Chloramphenicol is a substrate for a novel nitroreductase pathway in Haemophilus influenzae.

The p-nitroaromatic antibiotic chloramphenicol has been used extensively to treat life-threatening infections due to Haemophilus influenzae and Neisseria meningitidis; its mechanism of action is the inhibition of protein synthesis. We found that during incubation with H. influenzae cells and lysates, chloramphenicol is converted to a 4-aminophenyl allylic alcohol that lacks antibacterial activity. The allylic alcohol moiety undergoes facile re-addition of water to restore the 1,3-diol, as well as further dehydration driven by the aromatic amine to form the iminoquinone. Several Neisseria species and most chloramphenicol-susceptible Haemophilus species, but not Escherichia coli or other gram-negative or gram-positive bacteria we examined, were also found to metabolize chloramphenicol. The products of chloramphenicol metabolism by species other than H. influenzae have not yet been characterized. The strains reducing the antibiotic were chloramphenicol susceptible, indicating that the pathway does not appear to mediate chloramphenicol resistance. The role of this novel nitroreductase pathway in the physiology of H. influenzae and Neisseria species is unknown. Further understanding of the H. influenzae chloramphenicol reduction pathway will contribute to our knowledge of the diversity of prokaryotic nitroreductase mechanisms.

Bactericidal monoclonal antibody against Moraxella catarrhalis lipooligosaccharide cross-reacts with Haemophilus Spp.

Monoclonal antibodies (MAbs) against lipooligosaccharide (LOS) determinants after immunization of BALB/c mice with heat inactivated Moraxella catarrhalis serotype A were generated. MAb 219A9 was specific for a common epitope of A, B, and C M. catarrhalis serotypes in ELISA and immunofluorescent test (IFT). In both tests it also cross-reacted with whole bacteria and LPS antigens isolated from non-typeable H. influenzae and H. parainfluenzae strains. IgM antibody clone 219A9 possessed a strong bactericidal effect against the three serotypes in the presence of complement. Our results demonstrate that antibodies directed to a single LOS epitope common for A, B, and C serotype could be highly protective. This suggests that the common determinants are very promising in the development of LOS-based vaccine against M. catarrhalis. The cross-reactions of MAb 219A9 with Haemophilus spp. also show that immunization could result in immune response to epitopes conserved in other important respiratory pathogens.

Activity of LBM415 compared to those of 11 other agents against Haemophilus species.

When tested against 254 Haemophilus influenzae strains, LBM415, a peptide deformylase inhibitor, gave MIC50 and MIC90 values of 2.0 microg/ml and 8.0 microg/ml, respectively. The MICs were independent of beta-lactam or quinolone susceptibility and the presence or absence of macrolide efflux or ribosomal protein mutations. The MICs of LBM415 against 23 H. parainfluenzae strains were similar to those against H. influenzae. In contrast, erythromycin, azithromycin, and clarithromycin gave unimodal MIC distributions, and apart from beta-lactamase-negative, ampicillin-resistant strains, all strains were susceptible to the beta-lactams tested. Apart from selected quinolone-resistant strains, all strains were susceptible to ciprofloxacin, levofloxacin, gatifloxacin, moxifloxacin, and gemifloxacin. Resistance to trimethoprim-sulfamethoxazole was common. The potencies of all drugs against 23 H. parainfluenzae strains were similar to those against H. influenzae. Time-kill studies with 10 Haemophilus strains showed LBM415 to be bactericidal at 2 x the MIC against 8 of 10 strains after 24 h. For comparison, the macrolides and beta-lactams were bactericidal against 8 to 10 strains each at 2 x the MIC after 24 h. Quinolones were bactericidal against all 10 strains tested at 2 x the MIC after 24 h. Against six H. influenzae strains, postantibiotic effects for LBM415 lasted between 0.8 and 2.2 h. In multistep resistance selection studies, LBM415 produced resistant clones in 7 of the 10 strains tested, with MICs ranging from 4 to 64 microg/ml. No mutations in deformylase (def) and formyltransferase (fmt) genes were detected in any of the LBM415-resistant mutants.

Direct detection of bacterial pathogens in brain abscesses by polymerase chain reaction amplification and sequencing of partial 16S ribosomal deoxyribonucleic acid fragments.

OBJECTIVE: To evaluate the feasibility of detecting bacterial pathogens directly from the clinical brain abscess specimens by polymerase chain reaction (PCR) amplification and sequencing of bacterial 16S ribosomal deoxyribonucleic acid (rDNA). METHODS: A total of 14 specimens were tested by both culture and PCR amplification, targeting the full-length or a partial region of 16S rDNA. 16S rDNA is known to be conserved in bacteria. Sequencing of partial-length and full-length 16S rDNA was performed. The sequence data were compared with known sequences of 16S rDNA in the National Center for Biotechnology Information GenBank by using the Basic Local Alignment Search Tool (BLAST) algorithm. The species with the best match of similarity were regarded as the pathogenic species in the samples. We also developed a Streptococcus-specific multiplex PCR analysis for identifying members of the Streptococcus species, the most common pathogen of brain abscesses. RESULTS: The 10 culture-positive specimens were all PCR-positive for partial 16S rDNA, but only seven were positive for full-length 16S rDNA amplification. Bacterial DNA was not detected in the remaining four specimens with a negative culture. Species identification by phenotypes from culture was in agreement with that by sequencing results of partial-length (or full-length) 16S rDNA. The Streptococcus-specific PCR analysis could detect Streptococcus species correctly in one step. CONCLUSION: Bacterial 16S rDNA sequences provide reliable clues to the identification of unknown pathogens. PCR analysis of 16S rDNA and sequencing may identify pathogens to the species level directly from brain abscesses. This approach is rapid and is useful especially in the identification of slow-growing and fastidious organisms.

[Selective culture media for Haemophilus bacteria]. / Selektivnaia pitatel'naia sreda dlia bakterii roda Haemophilus.

A selectivity factor was specified to the previously developed nutrient medium meant for the cultivation of Haemophilus bacteria--CAE, which is based on the acid hydrolysate of casein, enzyme hydrolysate of animal blood (aminopeptide) and an extract of nutrient yeast. The above nutrient medium containing additionally growth factors V and X, glucose and bacitracin was shown to fit well for the primary sowing of Haemophilus bacteria and it can be used in diagnostic examinations.

Eustachian tube gland tissue changes are related to bacterial species in acute otitis media.

BACKGROUND AND OBJECTIVE: Prior investigations have shown that the number of mucus producing goblet cells in the middle ear and Eustachian tube (ET) mucosa is highly increased during and up to at least six months after experimental acute otitis media (AOM) caused by Streptococcus pneumoniae (SP). Further, the volume of the mucus producing paratubal gland components is increased up to 3 months after the acute infection. These changes may in conjunction with a deteriorated ET function predispose a subsequent development of secretory otitis media. The present investigation compares changes in goblet cell density and gland structures of the ET during and after AOM caused by various bacteria typically encountered in this disease, with emphasis on potential differences due to bacterial species. METHODS: Rat models of AOM caused by SP, non-typeable or type b Haemophilus influenzae (NTHI/HIB) or Moraxella catarrhalis (MC) were studied longitudinally up to 6 months after bacterial challenge. The ET was dissected and decalcified, paraffin embedded and serially sectioned, followed by PAS/alcian blue staining. The goblet cell density and the paratubal gland composition and volume were determined morphometrically in every 20th section, using a light microscope. RESULTS: Regardless of bacterial species, the ET goblet cell density was increased from day 8 and peaked day 16, followed by some degree of normalisation, although not reaching normal numbers within the 6 month period, except for MC. The highest increase was seen in AOM caused by the non-typeable Haemophilus strain, followed by HIB, SP and MC. Except with MC, pathological intra-epithelial glands formed and goblet cells were found in mucosal areas normally devoid of these. In all species but MC, the volume of the paratubal glands progressed to peak 16 days post-inoculation, followed by a gradual normalisation. The volume was still increased 3 months after the acute infection, but completely normalised after 6 months. The increase was primarily due to hypertrophy of the mucous gland components and highest in AOM caused by the Haemophilus species, followed by SP. CONCLUSION: The Eustachian tube goblet cell density is increased during and up to at least six months after AOM regardless of bacterial species, except when employing MC, by which the density was increased for a few weeks only. Except in AOM caused by MC, the volume of the ET glands increases during and up to at least 3 months after infection, primarily due to hypertrophy of the mucous gland components. The non-typeable Haemophilus strain induced the highest increase of both goblet cell density and mucous gland volume. The increased secretory capacity of the ET following AOM may by excessive mucus secretion contribute to the deteriorated ET function found after AOM and thus predispose, sustain or aggravate middle ear disease.

Evaluation of survival of Actinobacillus pleuropneumoniae and Haemophilus parasuis in four liquid media and two swab specimen transport systems.

OBJECTIVE: To determine duration and rates of recovery of Actinobacillus pleuropneumoniae and Haemophilus parasuis from 4 liquid media and 2 swab specimen transport systems and compare findings with those of Escherichia coli. SAMPLE POPULATION: One strain each of A pleuropneumoniae (biovar 1, serotype 1), H parasuis (serovar 5), and E coli (serotype O149:K91:H19). PROCEDURE: Strains were incubated in brain heart infusion broth supplemented with horse serum and other nutrients or in horse serum alone, with and without nicotinamide-adenine dinucleotide in both instances, for 150 days at 4 degrees C or room temperature (21 degrees C). Similarly, strains were tested in Stuart and Amies transport systems after storage at room temperature for 8 days. RESULTS: Colony counts greater than those of the initial inoculum were observed after incubation in horse serum for A pleuropneumoniae but not for H parasuis. Overall, incubation at 4 degrees C in the 4 liquid media resulted in longer recovery duration and higher rates than at room temperature. Culture of H parasuis resulted in lower recovery rates and shorter durations of recovery than culture of A pleuropneumoniae, except for culture in horse serum. Haemophilus parasuis survived longer than A pleuropneumoniae in the transport systems, and all organisms survived longer in the Amies system. CONCLUSIONS AND CLINICAL RELEVANCE: Survival of A pleuropneumoniae and H parasuis indicated that horse serum prolongs survivability, which may result in exposure of more animals during a prolonged period. The Amies system might be a good choice for collection of clinical samples from animals, especially for recovery of H parasuis.

Levels of nicotinamide adenine dinucleotide in extracellular body fluids of pigs may be growth-limiting for Actinobacillus pleuropneumoniae and Haemophilus parasuis.

During infection, nutrient deprivation can alter bacterial phenotype. This, in turn, may have implications for pathogenesis and prophylaxis. Actinobacillus pleuropneumoniae (biotype 1) and Haemophilus parasuis, respiratory tract pathogens of swine, are both V-factor-dependent. The concentrations of V factor in the extracellular fluids of pigs are unknown and may limit the growth of these bacteria in vivo. The aim of this study was to determine the concentrations of nicotinamide adenine dinucleotide (NAD) in select porcine body fluids and to compare the availability of NAD in vivo with the affinities of the organisms for this compound. Levels in plasma, tissue fluids (peritoneal, pleural, synovial, and cerebrospinal), and laryngeal, tracheal, and lung washings were determined with an enzymatic cycling assay. We concluded that, although the NAD supply in the respiratory tract is probably not growth-limiting, it may become limiting if the organisms are disseminated.

Evolution of the paralogous hap and iga genes in Haemophilus influenzae: evidence for a conserved hap pseudogene associated with microcolony formation in the recently diverged Haemophilus aegyptius and H. influenzae biogroup aegyptius.

Certain non-capsulate strains belonging to the Haemophilus influenzae/Haemophilus aegyptius complex show unusually high pathogenicity, but the evolutionary origin of these virulent phenotypes, termed H. influenzae biogroup aegyptius, is as yet unknown. The aim of the present study was to elucidate the mechanisms of evolution of two paralogous genes, hap and iga, which encode the adhesion and penetration Hap protein and the IgA1 protease respectively. Partial sequencing of hap and iga genes in a comprehensive collection of strains belonging to the H. influenzae/H. aegyptius complex revealed considerable genetic polymorphism and pronounced mosaic-like patterns in both genes, but no evidence of intrastrain recombination between the two genes. A conserved hap pseudogene was present in all strains of H. aegyptius and H. influenzae biogroup aegyptius, each of which constituted distinct subpopulations as revealed by phylogenetic analysis. There was no evidence for a second, functional copy of the hap gene in these strains. The perturbed expression of the Hap serine protease appears to be associated with the formation of elongated bacterial cells growing in chains and a distinct colonization pattern on conjunctival cells, previously termed microcolony formation. The fact that individual hap pseudogenes differed from the ancestral sequence by zero to two positions within a 1.5 kb stretch suggests that the silencing event happened approximately 2000-11,000 years ago. Divergence of H. aegyptius and H. influenzae biogroup aegyptius occurred subsequent to this genetic event. The loss of Hap protein expression may be one of the genetic events that facilitated exploitation of the conjunctivae as a new niche.

Haemophilus segnis polymicrobial and monomicrobial bacteraemia identified by 16S ribosomal RNA gene sequencing.

This paper reports a case of Haemophilus segnis polymicrobial bacteraemia and a case of H. segnis monomicrobial bacteraemia identified by 16S ribosomal RNA gene sequencing. In the first case, a gram-negative aerobic coccobacillus was isolated with Streptococcus intermedius and S. sanguis from the blood culture of a 32-year-old intravenous drug addict with left thoracic empyema. In the second case, a gram-negative aerobic coccobacillus was isolated from the blood culture of an 82-year-old woman with Clostridium difficile colitis and septicaemic shock. Both gram-negative coccobacilli grew on chocolate agar as colonies of 1 mm in diameter after incubation for 24 h at 37 degress C in air with CO2 5%, but only to pinpoint sizes on blood agar under the same incubation conditions. Both strains were factor V-dependent, but not factor X-dependent. For the first isolate, the Vitek system (NHI) showed that it was 56% likely to be Actinobacillus actinomycetemcomitans and 40% Neisseria subflava; whereas the API system (NH) showed that it was 58% likely to be H. aphrophilus/paraphrophilus and 42% H. parainfluenzae. For the second isolate, the Vitek system (NHI) showed that it was 95% likely to be H. influenzae VIII; whereas the API system (NH) showed that it was 58% likely to be H. aphrophilus/paraphrophilus and 42% H. parainfluenzae. 16S rRNA gene sequencing showed that there were four base differences between isolate 1 and H. segnis and two base differences between isolate 2 and H. segnis, indicating that both isolates most closely resembled a strain of H. segnis. Only two cases of H. segnis bacteraemia were found in the English scientific literature, one in a case of infective endocarditis and the other in a case of pancreatic abscess. Including the present two cases, the overall mortality of H. segnis bacteraemia was 50%.

rrn operons in Haemophilus parainfluenzae and mosaicism of conserved and species-specific sequences in the 16S-23S rDNA long spacer.

The mosaic organisation of short-sequence boxes was analysed in the cloned and sequenced long ribosomal spacer (547 bp) of Haemophilus parainfluenzae GR. Comparison and alignment of both the long and the short spacer were performed in H. parainfluenzae and H. influenzae Rd. The long spacer contained two tRNA genes (tRNA(Ala) and tRNA(Ile)) which are highly homologous to the corresponding genes found in the spacers of other species, such as Haemophilus spp., Actinobacillus spp., and Plesiomonas shigelloides. At the 3' end of tRNA(Ala) a putative ribosomal spacer loop was found, showing a strong secondary structure. Pulsed field gel electrophoresis (PFGE) analysis after restriction of the genome of H. parainfluenzae GR with I-Ceu I and subsequent polymerase chain reaction (PCR) analysis of PFGE-separated DNA fragments demonstrated that the H. parainfluenzae genome contained six operons and that the long spacer was present in three copies of them. Two short DNA segments were identified as being species-specific, allowing us to design PCR primers which were useful in the molecular identification of H. parainfluenzae isolates.

The nose as bacterial reservoir: important differences between the vestibule and cavity.

UNLABELLED: The difference between the spectra of potential bacterial pathogens (PBPs) in the nasal vestibule and cavity has not been taken into account in clinical studies. PURPOSE: Since one can anticipate different flora in different kinds of mucosae, the authors compared bacterial species in the vestibule with those of the cavity. SUBJECTS AND METHOD: A total of 534 healthy male clerical workers in a downtown Lucerne office building were examined with fractionated swabs. RESULTS: PBPs, notably Staphylococcus aureus, were found in 412 subjects and surprisingly, differences in flora of the two sites were noted in 130 of them: PBPs were observed in the vestibule and not in the cavity in 85 of the subjects, and in 45 of them, the reverse was true. CONCLUSION: The practical implications of these findings are considerable regarding infection control in patients at increased infection risk.

Increased susceptibility to gingival colonization by specific HACEK microbes in children with congenital heart disease.

It is well established that infective endocarditis (IE) involving the HACEK (Hemophilus, Actinobacillus, Cardiobacter, Eikenella, Kingella) group of microbes occurs in patients with congenital heart defects (CHD) and in those with prosthetic grafts. Dental caries and gingival disease have been presumed to be the focus of microbial shedding. The purpose of this study was to determine if children with CHD had a more severe gingival inflammatory condition and harbored the HACEK group of microbes to a greater extent than normal children. Two groups of 12 age and sex matched children were selected for this study. The experimental group consisted of twelve children with CHD, 1-1/2 to 8 years of age. The control group consisted of 12 healthy children 2 to 8 years of age. Each child had a gingival index score recorded as described by Massler. Subgingival cultures were obtained. Gingival samples were cultured for HACEK microbes and total Streptococcus (spp) using standard techniques. Fisher's exact test was performed with significance defined at P < 0.05. Children with CHD had more severe gingival inflammatory index than the control group (P < 0.05). 8/12 CHD patient had Actinobacillus actinomycetemcomitans (A.a.) as compared with 2/12 controls (P < 0.05). Furthermore, all cyanotic CHD patients (4/4) had A.a. whereas, only 2/12 controls did (P < 0.05). 4/12 CHD patients harbored Eikenella corrodens (E.c.) compared to 1/12 controls (N.S.). There was no significant difference in colonization with E.c. or A.a. between cyanotic and acyanotic patients. No significant difference in total Streptococcus (spp) was found between the two groups. This study suggests that children with CHD have a more severe gingival inflammatory index and are colonized with specific HACEK microbes more so than normal children.

Initial lung lesions in two calves experimentally infected with Haemophilus somnus.

The initial lung lesions in two calves intrabronchially inoculated with Haemophilus somnus are described. The animals were euthanized within 7 h after challenge. The in situ location of H. somnus and accompanying lesions were examined by light microscopy, immunohistochemistry and transmission electron microscopy (TEM). Inoculation with H. somnus resulted in the development of acute pulmonary lesions within 3.5 h. H. somnus antigen was demonstrated only within the luminal spaces of the airways and in one area of bronchio-associated lymphoid tissue (BALT). As observed by TEM, the bacteria were phagocytized by both neutrophils and alveolar macrophages. Antigen was never demonstrated in the pulmonary intravascular macrophages.

Cloning and sequencing of a 16S/23S ribosomal spacer from Haemophilus parainfluenzae reveals an invariant, mosaic-like organisation of sequence blocks.

A 16S/23S ribosomal spacer from a Haemophilus parainfluenzae rrn locus was cloned and sequenced. Analysis of PCR-amplified genomic fragments showed that this region is strongly conserved among unrelated isolates; computer analysis of database homologies showed that the spacer consists of sequence blocks, arranged in a mosaic-like structure, with strong homologies with analogous blocks present in the spacer regions of Haemophilus influenzae, Haemophilus ducreyi and Actinobacillus spp. It also contains a tRNA(Glu) gene, which is highly homologous to tRNA(Glu) genes found in spacers of other species. These data strongly support the hypothesis that recombination events are involved in the organisation of the sequence of the spacer, as a result of horizontal gene transfer.