Transmission of Burkholderia cepacia complex: evidence for new epidemic clones infecting cystic fibrosis patients in Italy.

To analyze national prevalence, genomovar distribution, and epidemiology of the Burkholderia cepacia complex in Italy, 225 putative B. cepacia complex isolates were obtained from 225 cystic fibrosis (CF) patients attending 18 CF centers. The genomovar status of these isolates was determined by a polyphasic approach, which included whole-cell protein electrophoresis and recA restriction fragment length polymorphism (RFLP) analysis. Two approaches were used to genotype B. cepacia complex isolates: BOX-PCR fingerprinting and pulsed-field gel electrophoresis (PFGE) of genomic macrorestriction fragments. A total of 208 (92%) of 225 isolates belonged to the B. cepacia complex, with Burkholderia cenocepacia as the most prevalent species (61.1%). Clones delineated by PFGE were predominantly linked to a single center; in contrast, BOX-PCR clones were composed of isolates collected either from the same center or from different CF centers and comprised multiple PFGE clusters. Three BOX-PCR clones appeared of special interest. One clone was composed of 17 B. cenocepacia isolates belonging to recA RFLP type H. These isolates were collected from six centers and represented three PFGE clusters. The presence of insertion sequence IS 1363 in all isolates and the comparison with PHDC reference isolates identified this clone as PHDC, an epidemic clone prominent in North American CF patients. The second clone included 22 isolates from eight centers and belonged to recA RFLP type AT. The genomovar status of strains with the latter RFLP type is not known. Most of these isolates belonged to four different PFGE clusters. Finally, a third clone comprised nine B. pyrrocinia isolates belonging to recA RFLP type Se 13. They represented three PFGE clusters and were collected in three CF centers.

Identification, characterization, and variable expression of a naturally occurring inhibitor protein of IS1106 transposase in clinical isolates of Neisseria meningitidis.

Transposition plays a role in the epidemiology and pathogenesis of Neisseria meningitidis. Insertion sequences are involved in reversible capsulation and insertional inactivation of virulence genes encoding outer membrane proteins. In this study, we have investigated and identified one way in which transposon IS1106 controls its own activity. We have characterized a naturally occurring protein (Tip) that inhibits the transposase. The inhibitor protein is a truncated version of the IS1106 transposase lacking the NH(2)-terminal DNA binding sequence, and it regulates transposition by competing with the transposase for binding to the outside ends of IS1106, as shown by gel shift and in vitro transposition assays. IS1106Tip mRNA is variably expressed among serogroup B meningococcal clinical isolates, and it is absent in most collection strains belonging to hypervirulent lineages.

Whole-genome organization and functional properties of miniature DNA insertion sequences conserved in pathogenic Neisseriae.

The chromosome of pathogenic Neisseriae is peppered by members of an abundant family of small DNA sequences known as Correia elements. These DNA repeats, that we call nemis (for neisseria miniature insertion sequences) can be sorted into two major size classes. Both unit-length (154-158 bp) and internally rearranged (104-108 bp) elements feature long terminal inverted repeats (TIRs), and can potentially fold into robust stem-loop structures. Nemis are (or have been) mobile DNA sequences which generate a specific 2-bp target site duplication upon insertion, and strictly recall RUP, a repeated DNA element found in Streptococcus pneumoniae. The subfamilies of 26L/26R, 26L/27R, 27L/27R and 27L/26R elements, found by wide-genome computer surveys in both the Neisseria meningitidis and the Neisseria gonorrhoeae genomes, originate from the combination of TIRs which vary in length (26-27 bp) as in sequence content (L and R types). In both species, the predominant subfamily is made by the 26L/26R elements. The number of nemis is comparable in the N. meningitidis Z2491 (A serogroup) and the MC58 (B serogroup) strains, but is sharply reduced in the N. gonorrhoeae strain F1090. Consequently, several genes which are conserved in the two pathogens are flanked by nemis DNA in the meningococcus genome only. More than 2/3 of nemis are interspersed with single-copy DNA, and are found at close distance from cellular genes. Both primer extension and RNase protection data lend support to the notion that nemis are cotranscribed with cellular genes and subsequently processed, at either one or both TIRs, by a specific endoribonuclease, which plausibly corresponds to RNase III.

Evolution and function of the neisserial dam-replacing gene.

Phase variation through slippage-like mechanisms involving homopolymeric tracts depends in part on the absence of Dam-methylase in several pathogenic isolates of Neisseria meningitidis. In Dam-defective strains drg (dam-replacing gene), flanked by pseudo-transposable small repeated elements (SREs), replaced dam. We demonstrate that drg encodes a restriction endonuclease (NmeBII) that cleaves 5'-GmeATC-3'. drg is also present in 50% of Neisseria lactamica strains, but in most of them it is inactive because of the absence of an SRE-providing promoter. This is associated with the presence of GATmeC, suggesting an alternative restriction-modification system (RM) specific for 5'-GATC-3', similar to Sau3AI-RM of Staphylococcus aureus 3A, Lactococcus lactis KR2 and Listeria monocytogenes.

A new vector for insertion of any DNA fragment into the chromosome of transformable Neisseriae.

A useful method for inserting any DNA fragment into the chromosome of Neisseriae has been developed. The method relies on recombination-proficient vector plasmid pNLE1, a pUC19 derivative containing (1) genes conferring resistance to ampicillin and erythromycin, as selectable markers; (2) a chromosomal region necessary for its integration into the Neisseria chromosome; (3) a specific uptake sequence which is required for natural transformation; (4) a promoter capable of functioning in Neisseria; and (5) several unique restriction sites useful for cloning. pNLE1 integrates into the leuS region of the neisserial chromosome at high frequencies by transformation-mediated recombination. The usefulness of this vector has been demonstrated by cloning the tetracycline-resistance gene (tet) and subsequently inserting the tet gene into the meningococcal chromosome.

Intracistronic transcription termination in polysialyltransferase gene (siaD ) affects phase variation in Neisseria meningitidis.

Expression of serogroup B meningococcal capsular polysaccharide is subject to frequent phase variation. A reversible +1/-1 frameshift mutation within a poly(dC) repeat altering the reading frame of the polysialyltransferase gene (siaD ), thereby causing premature arrest of translation, is responsible for loss of capsule expression. After analysis of transcription of the siaD gene from an encapsulated strain and from two unencapsulated derivatives, we have found that the siaD mRNA in the unencapsulated strains is reduced in size as a result of premature transcription termination at a cryptic Rho-dependent site within the proximal region of the siaD cistron. Termination is sensitive to bicyclomycin, a natural inhibitor of Rho activity. Bicyclomycin decreased the rates of capsule re-expression (off-on) without affecting the rates of loss of capsule expression (on-off). This finding suggested the existence of a novel mechanism linking transcription elongation termination and mutation frequency. A genetic system was therefore developed to measure phase variation of siaD-ermC' gene fusions in wild type and Rho-defective Escherichia coli strains. These studies demonstrated that in the Rho-defective E. coli strain readthrough transcription of the mutated siaD gene caused a fourfold lower off-on phase variation rate than in the congenic Rho+ strain. Analysis of phase variation of siaD-ermC' gene fusions in a DNA mismatch-defective E. coli strain suggests that the effect of transcription on mutation rates required a functional mismatch repair system.

Hypermutation in pathogenic bacteria: frequent phase variation in meningococci is a phenotypic trait of a specialized mutator biotype.

Expression of serogroup B meningococcal capsular polysaccharide undergoes frequent phase variation involving reversible frameshift mutations within a homopolymeric repeat in the siaD gene. A high rate of phase variation is the consequence of a biochemical defect in methyl-directed mismatch repair. The mutator phenotype is associated to the absence of DNA adenine methyltransferase (Dam) activity in all pathogenic isolates and in 50% of commensal strains. Analysis of the meningococcal dam gene region revealed that in all Dam- strains a gene encoding a putative restriction endonuclease (drg) that cleaves only the methylated DNA sequence 5'-GmeATC-3' replaced the dam gene. Insertional inactivation of the dam and/or drg genes indicated that high rates of phase variation and hypermutator phenotype are caused by absence of a functional dam gene.

Performance characteristics of an enzyme-linked immunosorbent assay for determining salivary immunoglobulin G response to Helicobacter pylori.

We evaluated the salivary immunoglobulin G (IgG) immune response to Helicobacter pylori in 70 subjects by enzyme-linked immunosorbent assay (ELISA). Subjects with a positive H. pylori culture showed significantly higher titers of antibodies than subjects with no detectable H. pylori: the overall sensitivity and specificity of the test were 84 and 90%, respectively. The detection of salivary anti-H. pylori IgG antibodies may be considered as an alternative to serum IgG detection for ease of sample collection or when blood samples are not available in screening of patients with dyspepsia.

Antibacterial activity in Pleurochaete squarrosa extract (Bryophyta).

An acetone extract of the moss Pleurochaete squarrosa was tested against eleven bacterial strains, some of which are pathogenous for man. The extract was active on some Gram-negative strains. Antibacterial activity of the extract, expressed as MICs, was compared with three reference antibiotics. Acute toxicity assay was performed in Balb C mice.

Effects of bicyclomycin on RNA- and ATP-binding activities of transcription termination factor Rho.

Bicyclomycin is a commercially important antibiotic that has been shown to be effective against many gram-negative bacteria. Genetic and biochemical evidence indicates that the antibiotic interferes with RNA metabolism in Escherichia coli by inhibiting the activity of transcription termination factor Rho. However, the precise mechanism of inhibition is not completely known. In this study we have used in vitro transcription assays to analyze the effects of bicyclomycin on the termination step of transcription. The Rho-dependent transcription termination region located within the hisG cistron of Salmonella typhimurium has been used as an experimental system. The possible interference of the antibiotic with the various functions of factor Rho, such as RNA binding at the primary site, ATP binding, and hexamer formation, has been investigated by RNA gel mobility shift, photochemical cross-linking, and gel filtration experiments. The results of these studies demonstrate that bicyclomycin does not interfere with the binding of Rho to the loading site on nascent RNA. Binding of the factor to ATP is not impeded, on the contrary, the antibiotic appears to decrease the apparent equilibrium dissociation constant for ATP in photochemical cross-linking experiments. The available evidence suggests that this decrease might be due to an interference with the correct positioning of ATP within the nucleotide-binding pocket leading b an inherent block of ATP hydrolysis. Possibly, as a consequence of this interference, the antibiotic also prevents ATP-dependent stabilization of Rho hexamers.

Induction of antibacterial activity by alpha-d-oligogalacturonides in Nephrolepis sp. (pteridophyta).

This paper presents data on the induction by alpha-d-oligogalacturonides (OG) of antibiotic activity in vitro by the fern Nephrolepis sp. The extracts from the fern grown aseptically, partly in a medium containing a mixture of OG and partly in a medium lacking OG, as control, were tested against several bacterial strains. The results show that the OG mixture promotes the production of antibiotic compounds. Comparing the present results with those on the antimicrobial properties of the same fern grown in a greenhouse, we discuss the hypothesis that the production of antibiotic substances can be elicited by different factors, such as products of synthesis or degradation of the biotic component of the soil or by OG (in axenic culture) that can mimic the effect of natural elicitors.

Detection of IgA against the mycobacterial antigen A60 in serum and saliva in patients with active pulmonary tuberculosis: preliminary results.

Humoral immune response against the mycobacterial antigen A60 was evaluated in 38 subjects: 13 healthy volunteers (Group I), 10 patients with a defined acute or chronic non tuberculous lung disease (Group II), 15 patients suffering from pulmonary tuberculosis (Group III). Saliva IgA in samples diluted in various concentrations (1:10, 1:30, 1:50) and serum IgG and IgA levels were measured by ELISA. Positive values of IgG were found in sera of 0/13 subjects from Group I, 1/10 from Group II, 12/15 from Group III; searching for IgA in serum was positive in 1/13 subjects from Group I, 2/10 from Group II, 11/15 from Group III, 1:30 dilution of saliva led to positive results in 0/13 subjects from Group I, 0/10 from Group II and 10/15 from Group III. The measurement of anti-A60 IgA levels in both saliva and serum might be a useful complement to serology based on detection of anti-A60 IgG in blood samples.

Characterization of the rho genes of Neisseria gonorrhoeae and Salmonella typhimurium.

We have cloned and sequenced the genomic regions encompassing the rho genes of Neisseria gonorrhoeae and Salmonella typhimurium. Rho factor of S. typhimurium has only three amino acid differences with respect to the Escherichia coli homolog. Northern (RNA) blots and primer extension experiments were used to characterize the N. gonorrhoeae rho transcript and to identify the transcription initiation and termination elements of this cistron. The function of the Rho factor of N. gonorrhoeae was investigated by complementation assays of rho mutants of E. coli and S. typhimurium and by in vivo transcription assays in polar mutants of S. typhimurium.

Cloning and characterization of a Neisseria gene homologous to hisJ and argT of Escherichia coli and Salmonella typhimurium.

We have isolated from a genomic library of the pathogenic Neisseriae gonorrhoeae T2 strain, a gene encoding a putative protein of 268 amino acids which exhibited significant similarity to the hisJ and argT gene products of Salmonella typhimurium and Escherichia coli, periplasmic proteins deputed to amino acid transport within the cell. The gene is transcribed as a monocistronic mRNA species of about 960 nucleotides flanked by regulatory elements for initiation and termination of transcription that are efficiently recognized in an E. coli host.

Evaluation of pefloxacin activity against recent clinical isolates.

The in-vitro antibacterial activity of pefloxacin, a new quinolone carboxylic acid, was tested against 1140 bacterial strains, recently clinically isolated, by measuring the minimum inhibitory concentrations. Comparisons were made with other quinolones (enoxacin, norfloxacin, flumekin, oxolinic acid, pipemidic acid) and other drugs (piperacillin, cefotaxime, ceftazidime, gentamicin, tobramycin, amikacin) widely used for the treatment of bacterial infections. Pefloxacin was very active against the tested species and was the most active drug against all the bacterial strains, with a geometric mean of MICs, a MIC 50 and MIC 90 of 0.27, 0.12 and 4 microg/ml respectively.

Evaluation of ciprofloxacin's activity against recent clinical isolates.

The in vitro antibacterial activity of ciprofloxacin, a new quinoline carboxylic acid, was tested against 1671 recently clinically isolated bacterial strains, by measuring the minimum inhibitory concentrations (MIC). Comparisons were made with other quinolones: nalidixic acid, norfloxacin, and other drugs: piperacillin, cefoxitin, cefotetan, ceftazidime, tobramycin, rifampin, tetracycline, chloramphenicol. Ciprofloxacin was very active against the tested species and was the most active drug against all the bacterial strains, with a geometric mean, a MIC50 and MIC90 of 0.27, 0.12 and 2 micrograms/ml, respectively.

Human antibody response to the 70-Kd common neisserial antigen in patients and carriers of meningococci or non-pathogenic Neisseria.

A surface antigen of 70 Kd, common to most Neisseria species, was previously described. This antigen is expressed and immunogenic in vivo in humans since 70-Kd-specific antibodies were detected in 22/26 patients with meningococcal meningitis and 12/17 healthy meningococcal carriers. Sera from convalescent patients and from carriers blocked the binding between mouse 70-Kd-specific serum and a strain of N. gonorrhoeae. The correspondence between inhibition-ELISA, using whole gonococci as solid phase, and detection of anti-70-Kd antibodies by Western blotting confirmed the specificity of the mouse serum. Non-pathogenic Neisseria species although possessing the 70-Kd structure elicited less frequently an antibody response in children. The immunogenicity of the 70-Kd antigen in humans supports its potential use as a vaccine component.

The 70-Kd neisserial common antigen is a surface-exposed, antigenically stable peptidic structure.

A previously described 70-Kd antigen present in all Neisseria gonorrhoeae strains tested and in most Neisseria species was characterized as a mercaptoethanol-and heat-stable protein. Using mouse polyclonal antisera specifically directed against this antigen, it was shown that it is a surface-exposed structure. The 70-Kd antigen was recovered from all gonococcal strains isolated from different anatomic sites in male and female partners, thus demonstrating its antigenic stability after in vivo transmission in humans.

Comparative evaluation of the new Titertek Enterobac Rapid Automated System (TTE-RAS) for identification of members of the family Enterobacteriaceae.

The Titertek Enterobac Rapid Automated System (TTE-RAS; Flow Laboratories, SpA, Milan, Italy), a new semiautomated system for the identification of members of the family Enterobacteriaceae, was compared with the API 20E system (API System P.A., Montalieu Vercieu, France) by using 284 clinically isolated strains that were previously identified by conventional methods. Six strains from the American Type Culture Collection (Rockville, Md.) were included to evaluate the reproducibility of identification by both systems. Correct identifications at the species level were 93.7% with TTE-RAS and 96.1% with API 20E. Although some of the features of the TTE-RAS data base were not satisfactory, we consider this new miniaturized system to be a very valuable tool for the rapid identification of the most frequently isolated opportunistic bacteria.