Genesis of BRO beta-lactamase-producing Moraxella catarrhalis: evidence for transformation-mediated horizontal transfer

The dramatic rise in BRO-producing M. catarrhalis strains observed in the last decades is without precedence. The aim of this study was to elucidate the events that led to the emergence of BRO-1 and BRO-2 β-lactamases. Previously, we showed bro1 and bro2 to be >99% identical. Data presented here suggested that bro2 was acquired by a fortuitous event and inserted between M. catarrhalis genes orf1 and orf3 . Subsequently, bro1 evolved from bro2 . Promoter-up mutations increased fitness of bro2 , explaining its present predominance. The highly conserved nature of bro compared with orf1 and orf3 suggested that acquisition has occurred relatively recently. The random distribution of bro among M. catarrhalis fingerprint types indicated that bro has spread by horizontal transfer. Sequence analysis revealed that 80–200 bp is generally cotransferred with bro , serving as regions of homology that target bro to the same chromosomal locus. A region of 160 bases upstream of bro1 lacked polymorphism, indicating it was derived from the original strain that acquired bro2 . We observed that bro was readily transferred by transformation between M. catarrhalis strains in vitro , suggesting a mechanism by which bro has disseminated. In conclusion, we have been able to reconstruct the steps that led to the emergence of BRO-producing M. catarrhalis .     

Etest for assessing the susceptibility of filamentous fungi

The purpose of this study was to evaluate the Etest as an in vitro antifungal susceptibility test method for different moulds originating from human samples and from the environment. A total of 50 isolates (1 Acremonium, 18 Aspergillus, 2 Cladosporium, 1 Epicoccum, 15 Penicillium, 2 Scopulariopsis and 11 Trichoderma strains) were tested by the Etest. Forty-six of the tested moulds (92%) were resistant to fluconazole with minimal inhibitory concentrations (MICs) > or = 256 microg ml(-1). There were strains resistant to ketoconazole among Aspergillus niger, A. ochraceus and Cladosporium spp. with MICs > 32 microg ml(-1). For fluconazole, no differences were observed using two different inocula, while for itraconazole, ketoconazole and amphotericin B, a 1 or less step 2-fold dilution difference in MIC was seen for the most of 10 selected strains. The MICs of fluconazole and amphotericin B obtained for Trichoderma strains by the Etest and the agar dilution method were also compared. MICs for fluconazole were in agreement, while MICs for amphotericin B were higher with 1 or 2 steps of 2-fold dilutions for most of Trichoderma strains in the case of the agar dilution method.     

Alterations in surface hydrophobicity ofAcinetobacter baumannii induced by meropenem

Six strains ofAcinetobacter baumannii out of eleven strains tested revealed a strong hydrophobic character. This was demonstrated by adherence of bacteria to xylene in the range of 90–94%. Changes in surface hydrophobicity of these strains were studied after treatment with meropenem at subinhibitory concentrations (sub-MICs) (1/4, 1/8, 1/16 or 1/32 of the MICs). All strains showed a reduced adherence to xylene after the action of meropenem at 1/4 or 1/16 of the MICs. Hydrophobicity of the treated bacteria was decreased to 1.3–70% (1/16 of the MICs) or to 12–86% (1/4 of the MICs), depending on the strain. A decrease in surface hydrophobicity of three strains was also observed after their exposure to meropenem at 1/8 of the MICs (to 18–71% of the control values). Meropenem at 1/32 of the MICs practically did not affect bacterial hydrophobic properties, with the exception of one strain.     

Moraxella catarrhalis in chronic and relapsing respiratory tract infections in children]

Examination of 700 children with chronic and relapsing respiratory tract infections showed that during the period from 1996 to 2003 Moraxella catarrhalis strains were isolated from the sputum of 5.5-9.7% of the patients. The frequency of the emergence was the third after Haemophilus influenzae and Streptococcus pneumoniae. In healthy children M. catarrhalis was isolated in 2.7% of the cases. The most frequent detection of M. catarrhalis was stated in children under 1 year (4.5%). The antibiotic susceptibility tests revealed that the majority of the M. catarrhalis isolates had beta-lactamase activity, were resistant to benzylpenicillin, ampicillin and lincomycin and highly susceptible to amoxycillin/clavulanate, macrolides, certain cephalosporins and levofloxacin. The isolates were most frequent in the patients of the rather severe contingent (congenital lung disease, alveolitis, chronic pneumonia, bronchial asthma). In such patients the bronchoobstructive syndrome was more frequent (46.6%). High frequency of the affection of the upper respiratory tracts in the examined children was stated (62.1%).     

Influence of subinhibitory concentrations of cefotaxime, imipenem and ciprofloxacin on adhesion of Escherichia coli strains to polystyrene

The present study investigated the ability of sub MICs of cefotaxime, imipenem and ciprofloxacin to interfere with adhesion of E. coli strains to polystyrene (selected polymer used in studies on microorganisms' adhesion). It was observed that cefotaxime and imipenem at 1/2 and 1/4 MICs decreased the adherence of E. coli strains to polystyrene significantly. 1/2, 1/4 and 1/8 MICs of ciprofloxacin generally decreased the adhesive properties of E. coli strains, but two E. coli strains showed a noticeable enhancement of adhesion after incubation at sub MICs of this antibiotic.     

Possible relationship of PFGE patterns of Moraxella catarrhalis between hospital- and community-acquired respiratory infections in a community hospital

We describe a prospective study of molecular analysis of Moraxella catarrhalis isolated from a community hospital. Our study was designed to investigate the possible relationship of pulsed-field gel electrophoresis (PFGE) patterns of M. catarrhalis between hospital- and community-acquired respiratory infections. A nosocomial outbreak of M. catarrhalis was observed between September 2000 and September 2001. During the study period, 40 strains of M. catarrhalis were isolated from a total of 32 patients with respiratory infections (26 strains from 18 inpatients, and 14 strains from 14 outpatients). We compared the PFGE patterns in 40 strains of M. catarrhalis isolated from the respiratory tract of the study patients. The genomic types of M. catarrhalis were classified into three PFGE patterns (A, B, and C). Interestingly, the nosocomial outbreak of M. catarrhalis included two patterns (A and B). Of the three patterns, two patterns (A and B) were found in both inpatients and outpatients. More interestingly, two subtypes of pattern B (B1 and B4) were simultaneously found in both inpatients and outpatients. Our results indicated that PFGE with SmaI chromosomal digestion is a suitable technique to establish the inter-strain genetic relatedness of M. catarrhalis, and suggested that the outbreak of M. catarrhalis occasionally included miscellaneous PFGE patterns. The results also showed that PFGE patterns of M. catarrhalis isolates were similar between hospital- and community-acquired respiratory infections. Analysis of the subtypes suggested that there might be some association between hospital- and community-acquired respiratory infections caused by M. catarrhalis.     

替加环素与多粘菌素B对泛耐药鲍曼不动杆菌体外研究

目的为治愈耐舒普深的泛耐药鲍曼不动杆菌（PDR-Ab）提供新药。方法替加环素采用二倍琼脂稀释法,多粘菌素B用E-test条法测分离目标菌株100株的最低抑菌浓度（M IC）,并用WHONET 5.4软件分析数据。结果多粘菌素B对耐舒普深的泛耐药鲍曼不动杆菌株的M IC值分布情况为：1.5 mg/L为2株,1.0 mg/L为9株,0.75 mg/L为9株,0.5 mg/L为38株,0.38 mg/L为34株,0.25 mg/L为8株,均为敏感;替加环素对耐舒普深的泛耐药鲍曼不动杆菌株的M IC值分布情况为：≥32 mg/L为0株、16 mg/L为2株、8 mg/L为3株、4 mg/L为4株、2 mg/L为6株、1 mg/L为9株、0.5 mg/L为30株、0.25 mg/L为33株、0.125 mg/L为9株、0.06 mg/L为2株、0.03 mg/L为2株,敏感率为95.0%,中敏率为3.0%,耐药率为2.0%。结论替加环素或多粘菌素B是目前对耐舒普深的泛耐药鲍曼不动杆菌最有效药物之一。     

The UspA1 protein and a second type of UspA2 protein mediate adherence of Moraxella catarrhalis to human epithelial cells in vitro

The UspA1 and UspA2 proteins of Moraxella catarrhalis are structurally related, are exposed on the bacterial cell surface, and migrate as very high-molecular-weight complexes in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Previous analysis of uspA1 and uspA2 mutants of M. catarrhalis strain 035E indicated that UspA1 was involved in adherence of this organism to Chang conjunctival epithelial cells in vitro and that expression of UspA2 was essential for resistance of this strain to killing by normal human serum (C. Aebi, E. R. Lafontaine, L. D. Cope, J. L. Latimer, S. R. Lumbley, G. H. McCracken, Jr., and E. J. Hansen, Infect. Immun. 66:3113-3119, 1998). In the present study, isogenic uspA1, uspA2, and uspA1 uspA2 mutations were constructed in three additional M. catarrhalis strains: 012E, TTA37, and 046E. The uspA1 mutant of strain 012E had a decreased ability to attach to Chang cells. However, inactivation of the uspA1 gene in both strain TTA37 and strain 046E did not cause a significant decrease in attachment ability. Inactivation of the uspA2 gene of strain TTA37 did result in a loss of attachment ability. Nucleotide sequence analysis revealed that the predicted protein encoded by the uspA2 genes of both strains TTA37 and 046E had a N-terminal half that resembled the N-terminal half of UspA1 proteins, whereas the C-terminal half of this protein was nearly identical to those of previously characterized UspA2 proteins. The gene encoding this "hybrid" protein was designated uspA2H. PCR-based analysis revealed that approximately 20% of M. catarrhalis strains apparently possess a uspA2H gene instead of a uspA2 gene. The M. catarrhalis uspA1, uspA2, and uspA2H genes were cloned and expressed in Haemophilus influenzae cells, which were used to prove that both the UspA1 and UspA2H proteins can function as adhesins in vitro.     

Fimbriation, hemagglutination and adherence properties of fresh clinical isolates of Branhamella catarrhalis.

This study investigated the fimbriation on 24 fresh clinical isolates of Branhamella catarrhalis by electron microscopy. All the strains were isolated from patients with respiratory infections. The Branhamella catarrhalis strains were classified into three groups according to the grade of fimbriation. Among these 24 strains the incidence of densely fimbriated, moderately fimbriated and sparsely fimbriated isolates were 12 (50%), 7 (29%) and 5 (21%), respectively. After five-times serial subculture on Brain Heart Infusion agar, the average number of fimbriae per bacteria was decreased from 174 to 114 in the densely fimbriated strain and from 48 to 10 in the moderately fimbriated strain. Moreover, 20% of the population became non-fimbriated in moderately fimbriated strain after the serial subculture. In strains with higher hemagglutination titer the number of fimbriae was significantly (P < 0.04) more than in strains with lower hemagglutination titer.     

Towards understanding the functional role of the glycosyltransferases involved in the biosynthesis of Moraxella catarrhalis lipooligosaccharide

The glycosyltransferase enzymes (Lgts) responsible for the biosynthesis of the lipooligosaccharide-derived oligosaccharide structures from Moraxella catarrhalis have been investigated. This upper respiratory tract pathogen is responsible for a spectrum of illnesses, including otitis media (middle ear infection) in children, and contributes to exacerbations of chronic obstructive pulmonary disease in elderly patients. To investigate the function of the glycosyltransferase enzymes involved in the biosynthesis of lipooligosaccharide of M. catarrhalis and to gain some insight into the mechanism of serotype specificity for this microorganism, mutant strains of M. catarrhalis were produced. Examination by NMR and MS of the oligosaccharide structures produced by double-mutant strains (2951lgt1/4Delta and 2951lgt5/4Delta) and a single-mutant strain (2951lgt2Delta) of the bacterium has allowed us to propose a model for the serotype-specific expression of lipooligosaccharide in M. catarrhalis. According to this model, the presence/absence of Lgt4 and the Lgt2 allele determines the lipooligosaccharide structure produced by a strain. Furthermore, it is concluded that Lgt4 functions as an N-acetylglucosylamine transferase responsible for the addition of an alpha-D-GlcNAc (1-->2) glycosidic linkage to the (1-->4) branch, and also that there is competition between the glycosyltransferases Lgt1 and Lgt4. That is, in the presence of an active Lgt4, GlcNAc is preferentially added to the (1-->4) chain of the growing oligosaccharide, instead of Glc. In serotype B strains, which lack Lgt4, Lgt1 adds a Glc at this position. This implies that active Lgt4 has a much higher affinity/specificity for the beta-(1-->4)-linked Glc on the (1-->4) branch than does Lgt1.     

Genotypic and phenotypic relatedness of 80 strains of Branhamella catarrhalis of worldwide origin

Abstract 80 clinical Branhamella catarrhalis strains of worldwide origin were examined for genotypic relatedness and phenotypic characteristics. Using a quantitative bacterial dot method for DNA-DNA hybridization the strains were found to form a homogeneous group with ΔT_m-values ranging from 0.0–2.3°C. In Minibact-N, an identification kit for oxidase positive, Gram-negative diplococci using eight phenotypic characteristics, all isolates were correctly identified and also demonstrated complete homogeneity except for β-lactamase production. Type strains representing the genera Branhamella, Moraxella and Neisseria were also examined for comparison. B. catarrhalis strain NCTC 4103-known to be atypical-had a ΔT_m-value of 5.7°C and produced γ-glutamylaminopeptidase, in contrast to all other B. catarrhalis strains. In GN MicroPlate^TM, a kit which tests utilizable carbon sources, B. catarrhalis strains were found to be able to utilize up to 16 to 95 carbon sources.     

Effect of aminoglycosides on surface hydrophobicity of Acinetobacter baumannii

Effects of amikacin, gentamicin, netilmicin and tobramycin at subinhibitory concentrations (sub-MICs) (11/4, 1/8, 1/16 or 1/32 of their MICs) on the cell surface hydrophobicity of two Acinetobacter baumannii strains (7194 and 16265) were evaluated. Hydrophobicity was determined by two different methods - by adherence of bacteria to hydrocarbon (xylene) and by aggregation of bacteria in ammonium sulphate solutions at various concentrations. The adherence of A. baumannii strains to xylene decreased, mainly, after treatment with netilmicin at 1/4, 1/8 or 1/16 of the MIC (to 6.4%, 17.0% or 24.5% of the control value) (strain 7194) and after treatment with amikacin and gentamicin at 1/4 of their MICs (to 58.4% or 54.4%) (strain 16265). A decrease in surface hydrophobicity of exposed strains under these conditions was shown in salting-out test, too. Tobramycin reduced hydrophobic properties of A. baumannii strains at all tested sub-MICs to only a small extent.     

嗜水气单胞菌对四环素类药物诱导耐药表型及机理研究

【目的】初步探讨利用四环素类药物体外诱导嗜水气单胞菌耐药后,嗜水气单胞菌对四环素类药物敏感性的变化及其耐药机制。【方法】筛选临床分离嗜水气单胞菌的四环素类敏感株,从含有1/4×MIC的强力霉素的TSA固体培养基开始,等比2倍提高诱导药物质量浓度对受试菌进行连续传代培养,以获得高耐诱导株;测定诱导菌对强力霉素和16种非诱导药物的最小抑菌浓度(MIC)及添加外排泵抑制剂N-甲基吡咯烷酮(NMP)后的MIC,分析其敏感性变化与外排作用的关系;提取诱导菌的DNA,PCR扩增其5个tet基因并测序。【结果】诱导后菌株对强力霉素的MIC显著升高,对非诱导四环素类药物也有不同程度提高,对氟喹诺酮类药物的MIC比诱导前增加几十至上千倍;对氨基糖苷类药物和利福平的MIC则有不同程度的降低;添加NMP后,所有诱导菌株对强力霉素的MIC值均有不同程度的下降;四环素类耐药基因的检测结果表明,在诱导后7号菌株中同时检测到tet A和tet E;在诱导前后的2号菌株中检测到tet C;在诱导前后的1、3、4、5、6、7号菌株中均检测到tet E。【结论】本研究表明tet E基因可能是介导气单胞菌分离株对四环素类药物耐药的优势基因,为阐明嗜水气单胞菌对四环素类药物耐药机制及耐药性与耐药基因之间的关系提供理论依据。     

A prospective study of intrafamilial transmission and antimicrobial susceptibility of Moraxella catarrhalis

Moraxella catarrhalis has been recognized as a particularly threatening respiratory tract pathogen in humans. A prospective study was performed to investigate which strains of M. catarrhalis can be transmitted within families; the study also addressed features of antimicrobial susceptibility. Seventy-five strains were isolated from six participants between July 2002 and February 2004, including 73 that were verified as beta-lactamase-producing strains. Antimicrobial susceptibility was tested for six types of antibiotics and no treatment issues were found. Pulsed-field gel electrophoresis (PFGE) was performed on all strains and 25 independent PFGE patterns were detected. The dominant pattern L (defined in the present study) was found in 21 (28%) of strains that were continuously recovered from children from the same family over an 8-month period. Strains with the patterns G, J, L, M, R, S, U, and W seemed to spread among the children, but there was no evidence of child-parent transmission. In the present study, the characteristics of M. catarrhalis within families have been documented, and PFGE profiles found to reveal alternating colonization and intrafamilial transmission.     

Electron microscopic observation of Branhamella catarrhalis

The hemagglutination (HA) test was done on 85 strains of Branhamella catarrhalis, isolated from sputum of patients with respiratory infections; 53% were HA positive strains. Three HA positive and three HA negative strains were selected and were observed under the electron microscope. The bacterial cell wall appeared to be lobulated and its total thickness was about 38 nm. The nuclear region consisted of whorls or fibrils and dense bodies. Five strains were fimbriated and one strain was nonfimbriated. The size of fimbriae was about 68 nm in length and 4.5 nm in width. The fimbriae of Branhamella catarrhalis were densely arranged and peritrichous in distribution. There was no change of fimbriation between broth and agar cultures.     

Complement resistance is a virulence factor of Branhamella (Moraxella) catarrhalis

Abstract The purpose of this study was to investigate complement resistance in Branhamella (Moraxella) catarrhalis isolated from healthy schoolchildren or sputum-producing adult patients. Two techniques were used: a serum bactericidal assay as the gold standard and an easier ‘culture and spot’ test. Children (age 4–13; n = 303) and patients ( n = 1047) showed high colonization/infection rates with B. catarrhalis (31% and 19%, respectively). Complement resistance or intermediate sensitivity occurred frequently in patient isolates (62% and 27%, respectively) and less often in children (33% and 8.5%, respectively; P ⪡ 0.0001). In young children (age 4–5 years), the proportion of complement-resistant strains was around 50%. Complement resistance in B. catarrhalis is associated with illness and may hence be considered a virulence factor.     

The C. elegans CBFbeta homologue BRO-1 interacts with the Runx factor, RNT-1, to promote stem cell proliferation and self-renewal

In this report, we investigate the C. elegans CBFbeta homologue, BRO-1. bro-1 mutants have a similar male-specific sensory ray loss phenotype to rnt-1 (the C. elegans homologue of the mammalian CBFbeta-interacting Runx factors), caused by failed cell divisions in the seam lineages. Our studies indicate that BRO-1 and RNT-1 form a cell proliferation-promoting complex, and that BRO-1 increases both the affinity and specificity of RNT-1-DNA interactions. Overexpression of bro-1, like rnt-1, leads to an expansion of seam cell number and co-overexpression of bro-1 and rnt-1 results in massive seam cell hyperplasia. Finally, we find that BRO-1 appears to act independently of RNT-1 in certain situations. These studies provide new insights into the function and regulation of this important cancer-associated DNA-binding complex in stem cells and support the view that Runx/CBFbeta factors have oncogenic potential.     

Comparison of E test and agar dilution methods for determining antibiotic susceptibilities of anaerobic bacteria and viridans streptococci isolated from blood

The antimicrobial susceptibilities of 311 strains of anaerobic bacteria and 140 strains of aerobic bacteria, isolated from blood after dental extraction, were determined by the E test and compared with the results obtained by the agar dilution method on PDM-ASM 2 agar. E test MICs agreed within +/-1 dilution step to the agar dilution MICs in 93%, 52%, 90%, 94% of the tested anaerobes for penicillin V, cefaclor, clindamycin and erythromycin, respectively. For aerobic bacteria the agreement was > or = 90% for the antibiotics tested. The in vitro activities of penicillin V, clindamycin and erythromycin were higher than the activity of cefaclor against the majority of bacteria tested.