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The pH of a microbiological culture is important for both cell growth and chitinase accumulation, but the optimal pH is not normally the same for both. The objective of this study was to investigate the effect of pH on chitinase production by Chitinolyticbacter meiyuanensis strain SYBC-H1 (ATCC BAA-2140) in a mineral medium. The results of batch culture at different pH values showed that the optimum pH for cell growth and chitinase production varied with time, although KOH produced the best results for cell growth and chitinase production, NaOH was chosen because of cost considerations. We designed a three-stage pH control strategy using NaOH as the neutralizing agent. Maximum cell growth (1.07 g dry cell weight/l) and maximum chitinase activity (13.6 U/ml) were observed after culture at 26°C for 72 h in a mineral medium. These values were greater by 129% and 162%, respectively, and the length of time to attain maximum chitinase activity was decreased by 12 h, compared with results from an earlier study (Hao et al., 2011b).

Two CHI genes from Chitinolyticbacter meiyuanensis SYBC-H1 encoding chitinases were identified and their protein 3D structures were predicted. According to the amino acid sequence alignment, CHI1 gene encoding 166 aa had a structural domain similar to the GH18 type II chitinase, and CHI2 gene encoding 383 aa had the same catalytic domain as the glycoside hydrolase family 19 chitinase. In this study, CHI2 chitinase were expressed in Escherichia coli BL21 cells, and this protein was purified by ammonium sulfate precipitation, DEAE-cellulose, and Sephadex G-100 chromatography. Optimal activity of CHI2 chitinase occurred at a temperature of 40 °C and a pH of 6.5. The presence of metal ions Fe(3+), Fe(2+), and Zn(2+) inhibited CHI2 chitinase activity, while Na⁺ and K⁺ promoted its activity. Furthermore, the presence of EGTA, EDTA, and β-mercaptoethanol significantly increased the stability of CHI2 chitinase. The CHI2 chitinase was active with p-NP-GlcNAc, with the Km and Vm values of 23.0 µmol/L and 9.1 mM/min at a temperature of 37 °C, respectively. Additionally, the CHI2 chitinase was characterized as an N-acetyl glucosaminidase based on the hydrolysate from chitin. Overall, our results demonstrated CHI2 chitinase with remarkable biochemical properties is suitable for bioconversion of chitin waste.

The widespread application of ω-transaminases as biocatalysts for chiral amine synthesis has been hampered by fundamental challenges, including unfavorable equilibrium positions and product inhibition. Herein, an efficient process that allows reactions to proceed in high conversion in the absence of by-product removal using only one equivalent of a diamine donor (ortho-xylylenediamine) is reported. This operationally simple method is compatible with the most widely used (R)- and (S)-selective ω-TAs and is particularly suitable for the conversion of substrates with unfavorable equilibrium positions (e.g., 1-indanone). Significantly, spontaneous polymerization of the isoindole by-product generates colored derivatives, providing a high-throughput screening platform to identify desired ω-TA activity., (© 2014 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.)

The eukaryotic heme oxygenases (HOs) (E.C. 1.14.99.3) convert heme to biliverdin, iron, and carbon monoxide (CO) in three successive oxygenation steps. Pathogenic bacteria require iron for survival and infection. Extracellular heme uptake from the host plays a critical role in iron acquisition and virulence. In the past decade, several HOs required for the release of iron from extracellular heme have been identified in pathogenic bacteria, including Corynebacterium diphtheriae, Neisseriae meningitides, and Pseudomonas aeruginosa. The bacterial enzymes were shown to be structurally and mechanistically similar to those of the canonical eukaryotic HO enzymes. However, the recent discovery of the structurally and mechanistically distinct noncanonical heme oxygenases of Staphylococcus aureus and Mycobacterium tuberculosis has expanded the reaction manifold of heme degradation. The distinct ferredoxin-like structural fold and extreme heme ruffling are proposed to give rise to the alternate heme degradation products in the S. aureus and M. tuberculosis enzymes. In addition, several "heme-degrading factors" with no structural homology to either class of HOs have recently been reported. The identification of these "heme-degrading proteins" has largely been determined on the basis of in vitro heme degradation assays. Many of these proteins were reported to produce biliverdin, although no extensive characterization of the products was performed. Prior to the characterization of the canonical HO enzymes, the nonenzymatic degradation of heme and heme proteins in the presence of a reductant such as ascorbate or hydrazine, a reaction termed "coupled oxidation", served as a model for biological heme degradation. However, it was recognized that there were important mechanistic differences between the so-called coupled oxidation of heme proteins and enzymatic heme oxygenation. In the coupled oxidation reaction, the final product, verdoheme, can readily be converted to biliverdin under hydrolytic conditions. The differences between heme oxygenation by the canonical and noncanonical HOs and coupled oxidation will be discussed in the context of the stabilization of the reactive Fe(III)-OOH intermediate and regioselective heme hydroxylation. Thus, in the determination of heme oxygenase activity in vitro, it is important to ensure that the reaction proceeds through successive oxygenation steps. We further suggest that when bacterial heme degradation is being characterized, a systems biology approach combining genetics, mechanistic enzymology, and metabolite profiling should be undertaken.

Biocatalytic approaches to the synthesis of optically pure chiral amines, starting from simple achiral building blocks, are highly desirable because such motifs are present in a wide variety of important natural products and pharmaceutical compounds. Herein, a novel one-pot ω-transaminase (TA)/monoamine oxidase (MAO-N) cascade process for the synthesis of chiral 2,5-disubstituted pyrrolidines is reported. The reactions proceeded with excellent enantio- and diastereoselectivity (>94 % ee; >98 % de) and can be performed on a preparative scale. This methodology exploits the complementary regio- and stereoselectivity displayed by both enzymes, which ensures that the stereogenic center established by the transaminase is not affected by the monoamine oxidase, and highlights the potential of this multienzyme cascade for the efficient synthesis of chiral building blocks., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Chitiniphilus shinanonensis type strain SAY3(T) is a strongly chitinolytic bacterium, originally isolated from the moat water in Ueda, Japan. To elucidate the chitinolytic activity of this strain, 15 genes (chiA-chiO) coding for putative chitin-degrading enzymes were isolated from a genomic library. Sequence analysis revealed the genes comprised 12 family 18 chitinases, a family 19 chitinase, a family 20 β-N-acetylglucosaminidase, and a polypeptide with a chitin-binding domain but devoid of a catalytic domain. Two operons were detected among the sequences: chiCDEFG and chiLM. The gene coding for the polypeptide (chiN) showed sequence similarity to family 19 chitinases and was successfully expressed in Escherichia coli. ChiN demonstrated a multi-domain structure, composed of the N-terminal, two chitin-binding domains connected by a Pro- and Thr-rich linker, and a family 19 catalytic domain located at the C-terminus. The recombinant protein rChiN catalyzed an endo-type cleavage of N-acetyl-d-glucosamine oligomers, and also degraded insoluble chitin and soluble chitosan (degree of deacetylation of 80%). rChiN exhibited an inhibitory effect on hyphal growth of the fungus Trichoderma reesei. The chitin-binding domains of ChiN likely play an important role in the degradation of insoluble chitin, and are responsible for a growth inhibitory effect on fungi., (Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Chitiniphilus shinanonensis strain SAY3(T) is a chitinolytic bacterium isolated from moat water of Ueda Castle in Nagano Prefecture, Japan. Fifteen genes encoding putative chitinolytic enzymes (chiA-chiO) have been isolated from this bacterium. Five of these constitute a single operon (chiCDEFG). The open reading frames of chiC, chiD, chiE, and chiG show sequence similarity to family 18 chitinases, while chiF encodes a polypeptide with two chitin-binding domains but no catalytic domain. Each of the five genes was successfully expressed in Escherichia coli, and the resulting recombinant proteins were characterized. Four of the recombinant proteins (ChiC, ChiD, ChiE, and ChiG) exhibited endo-type chitinase activity toward chitinous substrates, while ChiF showed no chitinolytic activity. In contrast to most endo-type chitinases, which mainly produce a dimer of N-acetyl-D-glucosamine (GlcNAc) as final product, ChiG completely split the GlcNAc dimer into GlcNAc monomers, indicating that it is a novel chitinase.

Alkaline exonuclease and single-strand DNA (ssDNA) annealing proteins (SSAPs) are key components of DNA recombination and repair systems within many prokaryotes, bacteriophages and virus-like genetic elements. The recently sequenced β-proteobacterium Laribacter hongkongensis (strain HLHK9) encodes putative homologs of alkaline exonuclease (LHK-Exo) and SSAP (LHK-Bet) proteins on its 3.17 Mb genome. Here, we report the biophysical, biochemical and structural characterization of recombinant LHK-Exo protein. LHK-Exo digests linear double-stranded DNA molecules from their 5'-termini in a highly processive manner. Exonuclease activities are optimum at pH 8.2 and essentially require Mg(2+) or Mn(2+) ions. 5'-phosphorylated DNA substrates are preferred over dephosphorylated ones. The crystal structure of LHK-Exo was resolved to 1.9 Å, revealing a 'doughnut-shaped' toroidal trimeric arrangement with a central tapered channel, analogous to that of λ-exonuclease (Exo) from bacteriophage-λ. Active sites containing two bound Mg(2+) ions on each of the three monomers were located in clefts exposed to this central channel. Crystal structures of LHK-Exo in complex with dAMP and ssDNA were determined to elucidate the structural basis for substrate recognition and binding. Through structure-guided mutational analysis, we discuss the roles played by various active site residues. A conserved two metal ion catalytic mechanism is proposed for this class of alkaline exonucleases., (© The Author(s) 2011. Published by Oxford University Press.)

Aim: To identify and characterize six chitinolytic bacterial strains isolated from ornamental fish., Methods and Results: Six different isolates of Deefgea chitinilytica were detected in healthy as well as diseased ornamental fish in Germany over a period of 2 years. Bacterial strains were identified using 16S rRNA partial gene sequencing and further characterized using different biochemical microtest systems and additional standard biochemical tests., Conclusion: We show that commercially available biochemical microtest systems are useful for identification of D. chitinilytica, supplemented by 16S rRNA partial gene sequencing. Furthermore, this study provides new information about the occurrence of D. chitinilytica, as this is the first isolation of D. chitinilytica from animals and first described isolation in Europe., Significance and Impact of the Study: Deefgea chitinilytica may be isolated regularly in fish diagnostic laboratories. Therefore, accurate identification of this bacterial species is important. Involvement of D. chitinilytica in opportunistic infections of aquatic organisms cannot be excluded and has to be further investigated., (© 2011 The Authors. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.)

Endotoxin [Lipopolysaccharide (LPS)/Lipooligosaccharide (LOS)] is an important virulence determinant in gram negative bacteria. While the genetic basis of endotoxin production and its role in disease in the pathogenic Neisseria has been extensively studied, little research has focused on the genetic basis of LOS biosynthesis in commensal Neisseria. We determined the genomic sequences of a variety of commensal Neisseria strains, and compared these sequences, along with other genomic sequences available from various sequencing centers from commensal and pathogenic strains, to identify genes involved in LOS biosynthesis. This allowed us to make structural predictions as to differences in LOS seen between commensal and pathogenic strains. We determined that all neisserial strains possess a conserved set of genes needed to make a common 3-Deoxy-D-manno-octulosonic acid -heptose core structure. However, significant genomic differences in glycosyl transferase genes support the published literature indicating compositional differences in the terminal oligosaccharides. This was most pronounced in commensal strains that were distally related to the gonococcus and meningococcus. These strains possessed a homolog of heptosyltransferase III, suggesting that they differ from the pathogenic strains by the presence a third heptose. Furthermore, most commensal strains possess homologs of genes needed to synthesize lipopolysaccharide (LPS). N. cinerea, a commensal species that is highly related to the gonococcus has lost the ability to make sialyltransferase. Overall genomic comparisons of various neisserial strains indicate that significant recombination/genetic acquisition/loss has occurred within the genus, and this muddles proper speciation.

A bacterial strain capable of degrading chitin, strain SAY3T, was isolated from moat water of Ueda Castle in Nagano Prefecture, Japan. The strain was gram-negative, curved rod-shaped, facultatively anaerobic, and motile with a single polar flagellum. It grew well with chitin as a sole carbon source. The cellular fatty acids profiles showed the presence of C16:1 omega7c and C16:0 as the major components. The G+C content of DNA was 67.6 mol% and Q-8 was the major respiratory quinone. A 16S rRNA gene sequence-based phylogenetic analysis showed the strain belonged to the family Neisseriaceae but was distantly related (94% identity) to any previously known species. Since the strain was clearly distinct from closely related genera in phenotypic and chemotaxonomic characteristics, it should be classified under a new genus and a new species. We propose the name Chitiniphilus shinanonensis gen. nov., sp. nov. The type strain is SAY3T (=NBRC 104970T=NICMB 14509T).

Available data indicate that the most common beta-lactamase produced by Branhamella catarrhalis is plasmid mediated. The same enzyme occurs in Moraxella nonliquefaciens, a commensal in the upper respiratory tract. The ability to produce the enzyme, which is known as BRO-1, can be transferred by conjugation from M. nonliquefaciens to B. catarrhalis. Since the first beta-lactamase-producing strains of B. catarrhalis appeared in 1977, the frequency of beta-lactamase production has increased rapidly; figures as high as 76% have been reported. The plasmid-mediated beta-lactamase TEM-1 occurs in several species of the genus Haemophilus. While the frequency of beta-lactamase production in H. influenzae is reported to be 10-15%, the incidence is significantly higher in non-pathogenic Haemophilus species. Both phenoxymethyl-penicillin and ampicillin promote the occurrence of beta-lactamase-producing strains, but the selective pressure exerted by ampicillin seems to be more pronounced. It may be possible to reduce the ecological effects of the penicillins by avoiding overdiagnosis of the most common bacterial infections of the respiratory tract, and by shortening the courses of antibiotic treatment.

The incidence of Branhamella catarrhalis in respiratory infections at City Hospital, Edinburgh from January 1981 to April 1984 is described. Beginning in January 1982 there was an increased incidence associated with a high proportion of beta-lactamase-producing strains. The number of these strains increased: from January 1981 to April 1983, 61% of strains produced beta-lactamase, and 83% produced beta-lactamase from January to April 1984. 53% of patients were infected in hospital. Environmental studies showed that 7% of staff and 8% of patients were carriers; there was also circumstantial evidence of ward and patient-to-patient infection. The antimicrobial susceptibility of 54 clinical strains was tested: all strains were resistant to trimethoprim but were susceptible to clavulanic acid plus amoxycillin, chloramphenicol, erythromycin, co-trimoxazole, cefotaxime and cefuroxime. beta-Lactamase-negative strains were uniformly susceptible to penicillin and ampicillin.

A rapid colorimetric method for the identification of pathogenic Neisseria (Identicult-Neisseria; Scott Laboratories, Inc.) based on beta-galactosidase, gamma-glutamylaminopeptidase, and gamma-prolylaminopeptidase is described. All 82 clinical isolates of Neisseria gonorrhoeae, 9 clinical isolates of N. meningitidis, and 5 clinical isolates of N. lactamica were correctly determined to the species level, as were 4 isolates of Branhamella catarrhalis. Reactions were prompt and easily interpreted. The system should be extremely useful in clinical laboratories.

Extracts were prepared from Micrococcus cryophilus, several strains of Branhamella catarrhalis and Neisseria spp. Esterases, NADP-dependent isocitrate dehydrogenase and malate dehydrogenase activities were assayed after electrophoresis of extracts of polyacrylamide gels. Except for Neisseria perflava and N. sicca which resolved activity bands for the acetate-esterase only, the remaining bacteria exhibited species-specific esterase patterns also for the propionate and butyrate substrates. The multiple esterase patterns from B. catarrhalis ATCC25238 were qualitatively and quantitatively different from those of B. catarrhalis ATCC23246. This finding and other evidence supports a taxonomic shift of the latter to a species level of that genus. The atypical neisserias N. caviae and N. ovis appeared to exhibit an intrageneric specificity in their esterase patterns with those from B. catarrhalis but not to the other Neisseria spp. tested. The malate dehydrogenase patterns from the atypical neisserias and B. catarrhalis ATCC23246 were qualitatively similar; however, the patterns of isocitrate dehydrogenase activity were variable for these species. Micrococcus cryophilus was distinct in its esterase and dehydrogenase bands, strongly suggesting its taxon unrelatedness to the genus Branhammella or the atypical neisserias. Of the enzymes assayed, esterase proved to be the most reliable for taxonomic identifications.

Two inexpensive screening tests were evaluated singly and in tandem for distinguishing Neisseria gonorrhoeae from other oxidase-positive microorganisms growing on selective gonococcal media. In tests of 728 cultures, including 460 N. gonorrhoeae, 4 Neisseria lactamica, 257 Neisseria meningitidis, and 7 Branhamella catarrhalis, both Superoxol (30% H2O2; J. T. Baker Chemical Co., Phillipsburg, N.J.) and amylase inhibition tests were 100% sensitive (positive) for 20-h cultures of N. gonorrhoeae. Singly, the Superoxol test was 92.7% specific for N. gonorrhoeae, compared with a specificity of 82.3% for the amylase inhibition test. By using tandem screening tests to distinguish gonococci, we achieved an overall specificity of 98.6%. Group A meningococci were the primary source of error in the Superoxol test, with 97% (37 of 38) strains producing gonococcal like reactions for catalase. From 5 to 20% of N. meningitidis serogroups X, Y, Z, and Z' and nontypable strains, as well as about 50% of B. catarrhalis and N. lactamica strains, were also strong catalase producers.

Branhamella catarrhalis has been implicated previously as a cause of bronchopulmonary infections. Sputum Gram's stain and culture results suggesting significant infection with beta-lactamase-producing strains of B. catarrhalis were correlated with a retrospective chart review of eight pediatric and ten adult patients. Preexisting pulmonary disease was observed in 12 patients; 5 had a history of aspiration; and 13 were intubated. Clinically, ten patients had pneumonia, five had bronchitis, and three manifested no disease. Only three sputum specimens grew a pure culture of B. catarrhalis, and six specimens yielded B. catarrhalis in the presence of normal upper respiratory flora. Analysis of broth microdilution susceptibility test results showed that 90% of the strains were inhibited at the following minimum inhibitory concentrations (MICs90): ampicillin, 8 micrograms/mL; cefotaxime, 0.5 microgram/mL; cefoxitin, 0.5 microgram/mL; cephalexin, 4 micrograms/mL; cephalothin, 8 micrograms/mL; chloramphenicol, 1 microgram/mL; clindamycin, 4 micrograms/mL; erythromycin, 0.25 microgram/mL; methicillin, 16 micrograms/mL; mezlocillin, 16 micrograms/mL; moxalactam less than or equal to 0.6 microgram/mL; penicillin, 16 micrograms/mL; piperacillin, 8 micrograms/mL; tetracycline, less than or equal to 0.3 microgram/mL; and trimethoprim/sulfamethoxazole, 1.6/30 micrograms/mL. Therapy may have been adequate in only eight (44%) of the cases. However, all but four of the patients, who died of unrelated causes, exhibited resolution of disease. The data indicate that Gram's stain and culture results of sputum specimens suggesting B. catarrhalis bronchopulmonary infection should be interpreted with caution by clinicians.

All 11 clinically significant isolates of Branhamella catarrhalis examined in this study were found to produce beta-lactamase. The enzyme was apparently not plasmid associated since extrachromosomal deoxyribonucleic acid was not detected in any of the strains. The beta-lactamase activity of all strains was significantly depressed by the beta-lactamase inhibitors clavulanic acid and CP 45899. Based on comparisons of relative susceptibility to various beta-lactam antibiotics, it was inferred that the beta-lactamase of B. catarrhalis was significantly more active against penicillin congeners than against cephalosporin congeners. Most strains were not inhibited by readily achievable serum concentrations of the pencillinase-sensitive penicillins, penicillin G, ampicillin, and amoxicillin. Methicillin was equally ineffective. With rare exceptions, most strains of B. catarrhalis were inhibited by achievable serum concentrations of seven cephalosporins (cephalothin, cephapirin, cephaloridine, cephalexin, cephamandole, cefaclor, and cefuroxin) and one cephamycin (cefoxitin). All strains were uniformly resistant to clindamycin but were inhibited by achievable serum concentrations of erythromycin, tetracycline, chloramphenicol, and trimethoprim-sulfamethoxazole. Comparison of geometric mean minimum inhibitory concentrations of all antimicrobial agents tested suggested that B. catarrhalis was most susceptible to cefoxitin, erythromycin, and tetracycline.

We tested 53 clinical isolates of Branhamella catarrhalis recovered from patients with respiratory symptoms to determine the susceptibility of the isolates to 25 antimicrobial agents, including the newer beta-lactam antibiotics. Of the 53 strains, 46 (86.7%) were beta-lactamase producers. All the strains were susceptible to the majority of the new penicillins and cephalosporins. The combinations of amoxacillin-clavulanic acid and ticarcillin-clavulanic acid were also very active against the beta-lactamase-producing strains.

A beta-lactamase positive Branhamella catarrhalis strain, B.c. 002 PcR, is described. The strain has the ability to transfer beta-lactamase production. The strain was isolated from nasopharynx in an adult patient with long-standing laryngitis. It showed atypical colony morphology.

A case of Branhamella catarrhalis bacteremia without known infectious focus in a granulocytopenic patient is reported. There were no unusual clinical features of bacteremia, such as purpura, in this case which distinguished it from bacteremia due to other bacteria. The isolated strain was beta-lactamase producing. The patient recovered uneventfully on antibiotics; at the same time, however, the bone marrow was also normalized.

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.

Over six months Branhamella catarrhalis was isolated in pure culture from the sputum of 81 patients with symptoms of acute respiratory tract infection. Of 38 patients who were infected in the community, over half required admission to hospital. The remaining 43 patients acquired the infection in hospital. Forty one of the 81 isolates produced beta-lactamase, 24 of these being hospital acquired infections. As a result 40% of patients who were treated with ampicillin did not respond. Most patients had chronic lung diseases or lung cancer or were taking corticosteroids. Three patients died and one required assisted ventilation; strains producing beta-lactamase were isolated in each case. Acute bronchitis developed in one previously healthy young non-smoker. It is concluded that B catarrhalis is an important pathogen of the lower respiratory tract which should be reported, and strains producing beta-lactamase should be identified. Otherwise, treatment with inappropriate antibiotics may result in increased morbidity or mortality.

Since 1978 we have taken an interest in lower respiratory tract infections associated with Branhamella catarrhalis in Christchurch, New Zealand. In a preliminary trial, 20 patients with bronchopulmonary infection caused by beta-lactamase-producing B. catarrhalis were treated with a combination tablet of amoxycillin 500 mg and clavulanic acid 125 mg ('Augmentin') 3 times daily for 5 days. Sputum cultures were negative for B. catarrhalis within 3 days in all patients. Two of 7 patients whose sputum cultures were positive for this organism at a review 2 to 4 weeks later were successfully treated with a further course of amoxycillin/clavulanic acid.

Since 1980, we have observed an epidemic of otitis media caused by Branhamella catarrhalis. This event was characterized by studying the nasopharyngeal colonization of infants and children with B. catarrhalis and the clinical presentation and therapeutic outcome of acute otitis media caused by this organism. Pharyngeal colonization with B. catarrhalis was commoner in winter than summer. B. catarrhalis was present in middle-ear fluid (MEF) of 17% of children with otitis media, and was commoner in fall and winter (20%) than in spring and summer (11%, P less than .05). Seventy-five percent of isolates produced beta-lactamase (Ravasio type). In five of 20 patients, treatment with beta-lactamase-susceptible agents failed to sterilize B. catarrhalis-infected MEF. All of these five patients were infected with beta-lactamase-producing strains. The increasing prominence of antibiotic-resistant B. catarrhalis in acute otitis media may lead to a reevaluation of initial antibiotic therapy for acute otitis media, particularly in winter or in areas where colonization with such strains is prevalent.

Branhamella catarrhalis is being increasingly identified as an important pulmonary pathogen, particularly in elderly patients with chronic obstructive lung disease. This gram-negative diplococcus can readily be isolated and detected in respiratory secretions. Laboratory identification requires care and beta-lactamase testing. Recognition of lower respiratory tract disease caused by B. catarrhalis is rewarding, since safe and effective therapy exists to treat these patients.

Beta-lactamase-producing isolates of Branhamella catarrhalis were first detected in France in 1977. The frequency of beta-lactamase producers has increased, especially since 1980. An agar iodometric test, a fast chromogenic test and an acidimetric test were used to assess the beta-lactamase-producing capabilities of 188 isolates of B. catarrhalis obtained mainly from sputum and the pharynx. Data from the first 2 procedures indicated positive beta-lactamase activity for all 49 strains of B. catarrhalis identified, but there were some discrepancies in the acidimetric test results. Evidence from a diffusion technique showed significant increases in the inhibition diameters surrounding filter discs impregnated with amoxycillin in the presence of clavulanic acid, or with ampicillin in the presence of sulbactam, compared with discs of the penicillins used alone. Two types of enzyme activity emerged from examination of isoelectric focusing patterns. Type I, having pI values of 5.35, 5.55 and 5.85, accounted for 87.2% of the enzyme-producing isolates. Type II, with pIs of 5.5, 5.9 and 6.25, occurred in 12.8% of isolates and appeared to be less widely distributed. The beta-lactamase inhibitors clavulanic acid and sulbactam in combination with benzylpenicillin produced potentiated effects, as demonstrated by significant reductions in MIC (33- and 44-fold decreases, respectively). Higher concentrations of each inhibitor similarly affected the MICs of amoxycillin. A weak synergy occurred with cefoxitin, a beta-lactamase-resistant beta-lactam antibiotic, and the 2 beta-lactamase inhibitors. Because B. catarrhalis has been shown to be a beta-lactamase-producing pathogenic organism, the addition of enzyme inhibitors, such as clavulanic acid and sulbactam, to standard therapy may be beneficial.

Branhamella catarrhalis strains resistant to commonly used penicillins, and presently isolated, produce a beta-lactamase. Most of these enzymes are chromosomally mediated, but a plasmid-mediated beta-lactamase has been described (enzyme BRO-1). With reference to isoelectric points, 7 different enzymes have been identified: 6 chromosomally mediated and 1 plasmid-mediated. Nevertheless, they have many common properties, such as being biosynthesised constitutively but with a low level of production. They have a penicillinase-type profile, and are strongly inhibited by clavulanic acid.

It was revealed earlier by the authors that N. meningitidis of the principal serological groups A, B, and C possessed neuraminidase activity. In the present work it was shown that this sign was genus-specific, and that this property was also possessed by the representatives of nonpathogenic neisseria habituating in the nasopharynx, However, the enzymatic activity in nonpathogenic neisseria was less pronounced than in meningococci, and required more prolonged contact of the enzymatic-substrate mixture for its detection. There was revealed a direct relationship between the neuraminidase activity and the growth phase of the microorganisms, whose maximum coincided with the exponential phase of bacterial growth, this apparently being associated with the extracellular character of its origin.

The plasmid-mediated Branhamella catarrhalis beta-lactamase BRO-1, also found in Moraxella nonliquefaciens, was characterized as regards substrate profile, isoelectric point and relative substrate affinity index (RSAI) to various substrates and compared in these aspects with the TEM-1 enzyme of Haemophilus influenzae. As measured by a biological assay and with high performance liquid chromatography (HPLC), BRO-1 was found to hydrolyse carbenicillin, mecillinam, methicillin and cefaclor with a higher rate than TEM-1. The only substrates having a relative rate of hydrolysis higher for TEM-1 than for BRO-1 were ampicillin and cephaloridine. The rates of hydrolysis registered with these two methods were comparable for all but 2 of 13 tested substrates. Isoelectric focusing yielded a main band at pH 5.6 and several satellite bands consistent with those reported by other authors for Branhamella enzymes having a substrate profile similar to that of BRO-1. A tenfold or higher difference in RSAI between BRO-1 and TEM-1 was recorded for five of the 15 compounds tested. BRO-1 seems to be the most common beta-lactamase in Bran. catarrhalis, irrespective of geographic origin. Its substrate profile, isoelectric pattern and RSAI differ from those of other known plasmid-mediated beta-lactamases described, thus justifying a specific designation.

The discovery that penicillinase production in Neisseria gonorrhoeae was plasmid mediated and the spread of the beta-lactamase encoding plasmids in gonococcal isolates since 1976, raise the possibility that a nonpathogenic indigenous bacterium could serve as a reservoir for these plasmids. We initiated studies to define the ability of commensal Neisseria species and Branhamella catarrhalis strains, as well as strains of the pathogen Neisseria meningitidis, to serve as recipients in conjugation with Neisseria gonorrhoeae. We found that with N. gonorrhoeae as the donor, 3 of 5 Neisseria cinerea, 2 of 5 Neisseria flava, 0 of 1 Neisseria flavescens, 1 of 3 Neisseria subflava, 0 of 6 B. catarrhalis, 0 of 7 Neisseria lactamica, 1 of 5 Neisseria mucosa, 1 of 7 Neisseria perflava/sicca, and 0 of 13 N. meningitidis strains gave detectable conjugation frequencies (greater than 10(-8). N. cinerea was the only species found to maintain the gonococcal conjugal plasmid (pLE2451). A N. cinerea transconjugant containing pLE2451 was observed to transfer both the beta-lactamase plasmid and pLE2451 to N. gonorrhoeae at high frequency.

The frequency of isolation of Branhamella catarrhalis from sputum in a laboratory in the southern part of the Netherlands is reviewed; at present 23% of positive cultures contain these organisms. Seasonal variations include a considerable reduction in frequency of isolation in the summer months. The possible role of heavy industry and air pollution is briefly discussed. Evidence for the direct pathogenicity of B. catarrhalis is presented, and although this may be considered as only circumstantial it is still convincing, particularly in acute otitis media and in acute purulent exacerbations of chronic respiratory disease. The possibility of indirect pathogenicity through beta-lactamase production is discussed as is the evidence obtained by the use of several new antimicrobial agents which were subsequently shown to have been inappropriate.

The beta-lactamases of Branhamella catarrhalis Ravasio and strain 1908 were readily inhibited by low concentrations of sulbactam, beta-halopenicillanic acids, MM 13902 and clavulanic acid. More detailed studies on the interaction of the Ravasio beta-lactamase with clavulanic acid demonstrated that the enzyme had high affinity for the inhibitor (Ki = 0.07 mumol/L) and was rapidly inhibited (t1/2 = 21 sec, kinhib. = 0.033/sec). Two types of enzyme-inhibitor complex were formed, a transiently stable species (t1/2 = 5.3 minutes at pH 7.3 and 37 degrees C) and a more stable species (t1/2 approximately equal to 2 hours at pH 7.3 and 37 degrees C). Irreversible inactivation of the enzyme was not achieved. Permeability studies on whole cells showed that beta-lactam antibiotics and beta-lactamase inhibitors readily penetrated the outer membrane of B. catarrhalis.

The lectin slide agglutination test for Neisseria gonorrhoeae has been modified and improved. Results show that wheat germ agglutinin and soybean lectin agglutinate 100% (193 of 193 tested) of clinical isolates of N. gonorrhoeae. Lectin-reactive meningococci can be readily identified by the hydrolysis of gamma-glutamyl-beta-naphthylamide. Branhamella catarrhalis, Neisseria lactamica, Neisseria sicca, Neisseria subflava, Neisseria perflava, and meningococcal serogroups A, B, C, X, Y, and Z do not interfere with the positive identification of N. gonorrhoeae. The frequently encountered problem of autoagglutination of members of the family Neisseriaceae may be circumvented by a short treatment of cellular suspensions with DNase. Based on agglutination assays, the enzyme treatment did not result in a loss of wheat germ agglutinin receptors from the bacteria. The lectin agglutination test, coupled with the gamma-glutamyl aminopeptidase assay, is proposed as a rapid and accurate means of identifying clinical isolates of gonococci.

A total of 5217 middle ear fluid (MEF) samples obtained from 1203 children with otitis media, aged 3 months to 6 years, were studied for the presence of Branhamella catarrhalis (Br) between Oct. 1977 and Sept. 1981. Br grew in 10.2% of 3497 MEFs of acute otitis media (AOM), with almost the same frequency in the very first and subsequent attacks. During the first 4 years of life the percentage did not vary much; among older children it seemed to decrease. The overall prevalence of Br in AOM did not change during the study period. Br alone grew in 72.4% of acute MEFs with Br; with other bacteria the respective figure was 82.9% (p less than 0.001). Acute attacks with bilateral Br were found in 22.8% of attacks with Br. In 1720 non-acute MEFs obtained at postacute control visits, Br was isolated in only 7.0%. The proportion of beta-lactamase-producing strains among the 2419 otitis-Br strains tested in two laboratories of the two study regions showed an increase from 27.1% and 21.1% in 1980 to 57.6% and 38.6% in 1983, respectively (p less than 0.001).

The clinical significance of Branhamella catarrhalis in respiratory infections is evaluated. 175 strains were isolated, mainly from the sputum, in 71 patients with respiratory infections. B. catarrhalis was most frequently isolated in mixed infections with Haemophilus influenzae (38.3%), H. influenzae plus Streptococcus pneumoniae (10.3%) or S. pneumoniae (9.7%). The rate of isolation of B. catarrhalis alone was as low as 5.1% and from mixed infections with Pseudomonas aeruginosa, Escherichia coli, and Enterobacter and/or Klebsiella species it was 36.6%. More than 10(7) cfu/ml of B. catarrhalis were isolated from 71.4% of cases. In 29 cases the organism was determined to be causative according to our criteria, most often in secondary infections in patients with complicated pneumoconiosis, chronic bronchitis and bronchiectasis. 29 of 47 strains (61.7%) produced beta-lactamase of the penicillinase type. Against these strains, penicillin antibiotics and first and second generation cephalosporin antibiotics (except cefroxadine) showed weak activity compared with their activity against non-beta-lactamase-producing strains. The third generation cephalosporins showed a uniform spectrum of activity against both groups of organisms.

A beta-lactamase-producing Branhamella catarrhalis was isolated in pure culture from the right middle ear aspiration of an otitis media in a 3-month-old girl. The patient responded well to cefamandole treatment.

Dihydrofolate reductases of five species of the family Neisseriaceae were compared by means of inhibition profiles, using several structurally different inhibitors, including trimethoprim (TMP) and pyrimethamine. All enzymes were seen to be highly susceptible to the folate analog aminopterin, but exhibited moderate susceptibility to all other inhibitors tested. Approximately 200-fold higher concentrations of TMP were needed to inhibit neisserial reductases as compared to the E. coli enzyme. Besides poor penetration this is assumed to be the main basis for the low susceptibility of neisseriae to TMP. In addition to TMP all other inhibitors were also moderately active or inactive in vitro. The enzymatic differences, as seen from inhibition profiles, were statistically significant but small among all species of the genus Neisseria. Branhamella catarrhalis on the other hand was seen to be far less related to the other neisseriae, as seen by the inhibition profile of its reductase, its dihydrofolate reductase conttent, as well as by its in vitro properties.

The in vitro activity of amoxicillin in the presence of clavulanic acid against clinical isolates of Haemophilus influenzae and Branhamella catarrhalis was assessed in comparison with ampicillin, amoxicillin, cefaclor and erythromycin. The isolates were selected so as to yield equal numbers of beta-lactamase producing and non-beta-lactamase producing strains of the two species. MICs obtained by agar dilution indicated that amoxicillin in the presence of clavulanic acid was the most active of the drugs tested. Clavulanic acid potentiated the activity of amoxicillin against beta-lactamase-producing strains of both Haemophilus influenzae and Branhamella catarrhalis. Further studies on a few strains of each species revealed that the beta-lactamase of Haemophilus influenzae (TEM-1) rapidly inactivated ampicillin and slowly inactivated cefaclor but not cefuroxime. The Branhamella catarrhalis enzyme rapidly inactivated cefaclor, ampicillin and to some extent cefuroxime. Clavulanic acid afforded protection against the beta-lactamase action of both species when beta-lactam antibiotics were added to bacterial cultures.

The incidence of Branhamella catarrhalis in the respiratory tract of adults, especially in the United States, is not known. During the 30-month period from January 1983 to June 1985, 4180 sputum and endotracheal samples from patients in a hospital for chest diseases were evaluated. All samples were acceptable for Gram-stain analysis and/or culture based on published cellular criteria. Using primarily Gram-stain directed cultures, 220 isolates of B. catarrhalis were identified in 180 patients, being present in 5.3% of all sputum cultures and 11.5% of those positive for a pathogen. B. catarrhalis was the fourth most common pathogen identified. It was found in pure culture (124) and mixed culture (96), the latter usually in association with Haemophilus influenzae or Streptococcus pneumoniae. Of the 220 B. catarrhalis isolates, 158 (71.8%) were positive for beta-lactamase. The number and incidence of B. catarrhalis varied, with the organism being most prevalent during the winter months. Despite its frequent presence in sputum, B. catarrhalis was not recovered from pleural fluid or blood during the same period. This study demonstrates the frequent presence of B. catarrhalis in the sputum of adults with chronic lung disease, although the role of this organism as a pathogen was not determined.

A strain of Branhamella catarrhalis isolated from sputum was shown to be resistant to penicillin and ampicillin. This strain produced a beta-lactamase which has been judged to be a novel enzyme, on the basis of substrat profil, isoelectric point and location of genetic determinant.

23 strains of Branhamella catarrhalis, mainly isolated from patients with acute exacerbations of chronic bronchitis, were studied. The strains were selected on the basis of a positive beta-lactamase reaction using the cefinase disc or the nitrocefin method. No bands were visible by isoelectric focusing of the crude enzyme extracts of strains showing a weak positive reaction with the cefinase method. In the 15 remaining strains, at least 6 different isoelectric focusing patterns could be detected. Four strains showed a pattern similar to that of the Ravasio strain, the most common type in a previous study. The most prevalent type in this study, present in 5 strains, demonstrated an isoelectric focusing pattern quite different from those described so far: a main band at pI 5.28 and 2 minor bands at pI 5.71 and 6.37. Two beta-lactamase-positive, but ampicillin-sensitive strains (MIC = 0.064 mg/L) showed different isoelectric focusing patterns, pI = 4.93 and 5.43, respectively, but similar substrate profiles.

Fifteen out of 89 clinical strains of Branhamella catarrhalis isolated from patients at the University Hospital of Zaragoza were resistant to aminoglycosides and other antimicrobials. In two strains, B. catarrhalis 220 and B. catarrhalis 115, the resistance to aminoglycosides was associated with synthesis of aminoglycoside-modifying enzymes, namely 3"-O-phosphotransferase [APH(3")] and 3'-O-phosphotransferase [APH(3')]. B. catarrhalis 115 was resistant to ampicillin, streptomycin, kanamycin, neomycin, butirosin, lividomycin, ribostamycin, paromomycin and trimethoprim-sulfamethoxazole and harboured a 32 megadalton (Md) plasmid. The resistance determinants of the latter were transferred to Neisseria subflava by conjugation and to Escherichia coli by transformation. The transconjugant strain presented an antibiotic resistance pattern similar to the donor strain and carried the same plasmid. The transformant strain acquired the 32 Md plasmid but presented, besides the resistance pattern already mentioned, resistance to tetracycline, gentamicin and tobramycin. Resistance to gentamicin and tobramycin was mediated by the synthesis of a 3-N-acetyltransferase. This resistance and the related enzyme were expressed neither in the donor B. catarrhalis strain nor in the transconjugant N. subflava strain.

Branhamella catarrhalis obtained from the sputum of 146 patients with lower respiratory tract disease and from middle ear fluids of 26 children with otitis media were evaluated for beta-lactamase activity and the enzymes were characterised by isoelectric focusing (IEF). 71% (103 of 146) of the sputum isolates and 77% (20 of 26) of the ear isolates produced beta-lactamase. By IEF, the beta-lactamases of 113 of 123 (92%) strains revealed patterns identical with the Ravasio type strain, having unique enzyme bands at pIs of 6.4 and 6.65. The remaining 10 isolates (8%) produced patterns similar to the 1908 type strain with a unique band of activity having a pI of 6.55. In addition, the 1908 types revealed a band of minor enzyme activity with a pI of 7.55 that was absent from the Ravasio types. All strains tested shared major enzyme bands with pIs of 5.1, 5.3, 5.55 and 6.1. These results indicate that the most common beta-lactamase(s) produced by clinical isolates of B. catarrhalis in the United States are similar to those produced by the Belgian Ravasio type strain.

The plasmid-mediated beta-lactamase in Branhamella catarrhalis (BRO-1), also occurring in Moraxella nonliquefaciens, differs from other known plasmid-mediated beta-lactamases in Gram-negative bacteria regarding substrate profile and isoelectric point. B. catarrhalis strains previously reported to produce beta-lactamases deviating from BRO-1 were tested, and the beta-lactamases did not differ significantly from BRO-1 in substrate profile, isoelectric point or relative substrate affinity index (RSAI). Further investigations of strains of various geographic origin should be undertaken. RSAI seems to be a useful tool for screening of beta-lactamases in B. catarrhalis since values for a large number of strains can easily be determined. The previously reported conjugational transfer of BRO-1 production within species B. catarrhalis and from M. nonliquefaciens to B. catarrhalis was confirmed. Four bands of extrachromosomal DNA were regularly detected by agarose gel electrophoresis in beta-lactamase-producing as well as in beta-lactamase-negative strains of B. catarrhalis and M. non-liquefaciens, provided that the excessive nuclease activity in the preparations was inhibited.

The enzymatic profiles of Neisseria gonorrhoeae, N. meningitidis, and related species were determined, using a total of 48 chromogenic substrates. Enzyme classes assayed for included glycosidases, aminopeptidases, phosphoamidases, proteases, lipases, esterases, and aryl sulfatase. A final test selection of 10 substrates, based upon their differential and reproducible characteristics, allowed the separation of N. gonorrhoeae and N. meningitidis from each other and from all species tested within 4 h after primary isolation on modified Thayer-Martin medium. The need for subculturing suspect colonies from modified Thayer-Martin medium to chocolate medium with a subsequent loss of 18 to 24 h of identification is eliminated.