Evidence for gene silencing in Haemophilus influenzae.

Evidence for gene silencing of Haemophilus influenzae involved a beta-subunit of RNA polymerase. The gene presumed silenced was rifampin resistance. The evidence that it was silencing, rather than dominance of a rifampin-sensitive marker, was that it took place when the rifampin resistance marker was on both a plasmid and the chromosome, without the presence of a rifampin-sensitive marker, as judged by lack of transformation of a rifampin-resistant cell to rifampin sensitivity by the plasmid. In addition, three compounds that are known to decrease gene silencing in eukaryotes (trichostatin A, sodium butyrate and 5-azacytidine) also decreased the presumed silencing in H. influenzae. Silencing of rifampin-resistant Escherichia coli did not take place with the plasmid from H. influenzae.

Factors influencing capacitance-based monitoring of microbial growth.

Microbiological impedance devices are routinely used by food and manufacturing industries, and public health agencies to measure microbiological growth. Factors contributing to increases and decreases in capacitance at the culture medium-electrode interface are poorly understood. To objectively evaluate the effects of temperature, cell density and medium conductivity on capacitance, admittance values from an impedance device were standardized; capacitance was converted to susceptance to allow unit comparisons with conductance. Although increases in temperature increased susceptance, a linear relationship could not be established between the change of susceptance with temperature and conductance of the medium. Cell density by itself had no measureable effect on susceptance or conductance, indicating that cells did not impede the movement of ions in the medium or around the electrode. In a low conductivity medium, increases in conductance by the addition of ions resulted in a concomitant increase of susceptance values. However, in a high conductivity medium, increases in conductance resulted in little or no increase of susceptance values because ions saturated the electrode surface. Susceptance increased when Escherichia coli, Pseudomonas aeruginosa, Alcaligenes faecalis and Staphylococcus aureus were grown in high conductivity media because protons produced by metabolically active bacteria balance more charge on the electrode than other ions. Increases in susceptance due to bacterial growth and metabolism in low conductivity media were attributed to both increases in protons and ionic metabolites. These results indicate that capacitance may provide a better measure of microbial growth and metabolism than conductance.

Human immunodeficiency virus testing for patient-based and population-based diagnosis.

Laboratory testing for human immunodeficiency virus (HIV) has been introduced for individual patient-based diagnosis as well as high-risk and low-risk population-based screening. The choice of test, confirmatory algorithm, and interpretative criteria used depend on the clinical setting. In the context of general population-based testing, factors affecting test performance will have to be considered carefully in the development of testing policy.

An analysis of the complete nucleotide sequence of the Haemophilus ducreyi broad-host-range plasmid pLS88.

We present an analysis of the complete nucleotide sequence of pLS88, a naturally occurring, 4.8-kb broad-host-range plasmid isolated from Haemophilus ducreyi and encoding resistance to sulfonamides, streptomycin, and kanamycin. Sequence analysis of the genes encoding sulfonamide and streptomycin resistance revealed homology to the RSF1010 sulII and strA genes. The sulII-strA intergenic region of pLS88 has a 38-bp deletion identical to that of the RSF1010-like plasmid pHD8.1, isolated from Actinobacillus pleuropneumoniae. The kanamycin resistance gene shows strong homology to Tn903, but lacks the inverted repeats of the transposon. No other genes have been identified. The region downstream of the kanamycin resistance gene shows homology to the RSF1010 oriV region; however, this region is not essential to plasmid replication. The ori of pLS88 is contained within a 1060-bp region and does not appear to contain structures typical of plasmid origins. This region is flanked by DNA showing strong homology to regions both upstream and downstream of the Haemophilus influenzae ROB-1 beta-lactamase gene. Because of the small size of the origin, pLS88 appears to resemble the structure of narrow-host-range plasmids, but replicates, via an as yet unidentified mechanism, as a broad-host-range plasmid.

Clarification of the structure of the ampicillin-resistance plasmid RSF0885 from Haemophilus influenzae HR-885 serotype b.

The nonconjugative ampicillin-resistance plasmid RSF0885 has been reported to be as small as 2.9 MDa and as large as 4.1 MDa with at least two restriction enzyme maps reported. In addition, the source of the original plasmid has been reported to be Haemophilus influenzae and Haemophilus parainfluenzae. Characterization of the source strains and sequencing data of the plasmids revealed that H. influenzae serotype b was the original source strain and that IS1-K in the larger plasmid was presumably acquired when the smaller plasmid was maintained in Escherichia coli in S. Falkow's laboratory during the late 1970s.

Sequence analysis of two deletion mutants in the hisC gene of Salmonella typhimurium.

Limited sequencing of the terminal region of the hisC gene in two deletion mutants involving the hisC gene of Salmonella typhimurium was carried out after polymerase chain reaction amplification of the appropriate region, using oligonucleotide primers selected from the published sequence of the histidine operon from this organism. his2648 was shown to have a 34 base pair deletion in the terminal region of the hisC gene between the P2 promoter and the Shine-Delgarno sequence of the hisB gene. hisHB22 was shown to have a 1.4 kilobase deletion extending from the TGA termination codon of the hisC gene to the middle of the hisH gene. The sequence data were consistent with previous genetic and phenotypic characterization of these strains.

Major outbreak of pertussis in northern Alberta, Canada: analysis of discrepant direct fluorescent-antibody and culture results by using polymerase chain reaction methodology.

A major outbreak of 5,683 cases of pertussis occurred in northern Alberta, Canada, from December 1989 to January 1991. The outbreak highlighted a number of problems with current methods of pertussis diagnosis. In particular, an exceptionally high proportion of direct fluorescent-antibody (DFA)-positive, culture-negative specimens (88.4%) was identified. We took this opportunity to use polymerase chain reaction (PCR) methodology to examine whether the low culture rates were due to specimens containing dead organisms or whether the DFA results represented high numbers of false-positive results. A set of primer sequences within a Bordetella pertussis-specific repetitive element was used to amplify proteinase K extracts of B. pertussis DNA recovered from 279 submitted slides inoculated at the point of collection with nasopharyngeal material obtained from pernasal swabs. The PCR data corroborated the culture results: 84.6% of DFA-positive, culture-negative specimens were similarly PCR negative. At least three different bacterial species that were significantly cross-reactive with the commercial DFA reagent were identified in clinical specimens and in pure culture, providing one possible explanation for the false-positive DFA results. These results and other limitations of current diagnostic techniques underline the urgent need for a new DFA reagent with improved specificity and a standardized means of measuring the patient antibody response for the diagnosis of pertussis.

The sexually transmitted diseases laboratory in a framework of population-based diagnostics.

Laboratory support for the diagnosis and treatment of sexually transmitted diseases has traditionally been within a patient-based diagnostic paradigm. Tests and interpretative criteria developed within this paradigm may not be appropriate for laboratories supporting population-based STD control programs. As STD control strategies expand to population-based levels, the present patient-based laboratory models will have to be modified to meet these increased demands.

Use of electroporation to construct isogenic mutants of Haemophilus ducreyi.

Little is known about the genetics of Haemophilus ducreyi, the etiologic agent of chancroid. To develop a method for constructing isogenic mutants of this organism that could be utilized in pathogenesis-related studies, electroporation techniques were evaluated as a means of introducing DNA into this organism. Electroporation of the plasmid shuttle vector pLS88 into H. ducreyi yielded approximately 10(6) antibiotic-resistant transformants per microgram of plasmid DNA. Studies of the feasibility of moving mutated genes into H. ducreyi were initiated by using NotI linker insertion and mini-Tn10kan mutagenesis techniques to introduce insertion mutations into cloned H. ducreyi genes encoding cell envelope antigens. In the former case, a gene encoding chloramphenicol acetyltransferase was then inserted into the NotI linker site created in the cloned H. ducreyi gene. The recombinant Escherichia coli strains containing these mutated plasmids no longer expressed the homologous H. ducreyi cell envelope antigens, as evidenced by their lack of reactivity with monoclonal antibody probes for these H. ducreyi proteins. Subsequent electroporation of both circular and linearized forms of plasmids carrying these mutated H. ducreyi genes into the homologous wild-type strain of H. ducreyi yielded antibiotic-resistant transformants which also lacked reactivity with the cell envelope antigen-specific monoclonal antibodies. Southern blot analysis confirmed that homologous recombination had occurred in these monoclonal antibody-unreactive transformants, resulting in the replacement of the wild-type allele with the mutated allele. Allelic exchange was most efficient when linear DNA molecules were used for electroporation. These results indicate that electroporation methods can be utilized to construct isogenic mutants of H. ducreyi.

Identification of a ROB-1 beta-lactamase in Haemophilus ducreyi.

A collection of 100 clinical isolates of Haemophilus ducreyi from Thailand were all found to harbor a 5.4-kb plasmid, designated pTH126, which was shown to contain the bla ROB-1 gene. Restriction enzyme analysis and DNA-DNA hybridization studies confirmed that pTH126 was similar to the ROB-1 beta-lactamase plasmid pVM105 from Actinobacillus pleuropneumoniae. In approximately one-half of the isolates, pTH126 was found together with pHD131, which mediates TEM-1 beta-lactamase production.

Heterotrophic plate counts of surface water samples by using impedance methods.

Membrane filtration, spread plating, and pour plating are conventional methods used to determine the heterotrophic plate counts of water samples. Impedance methods were investigated as an alternative to conventional methods, since sample dilution is not required and the bacterial count can be estimated within 24 h. Comparisons of impedance signals obtained with different water samples revealed that capacitance produced faster detection times than conductance. Moreover, the correlation between heterotrophic plate count and detection time was highest (r = 0.966) when capacitance was used. Linear and quadratic regressions of heterotrophic plate count and impedance detection time were affected by incubation temperatures. Regressions between heterotrophic plate counts based on conventional methods and detection times of water samples incubated at less than or equal to 25 degrees C had R2 values of 0.878 to 0.933. However, regressions using detection times of water samples incubated at greater than or equal to 30 degrees C had lower R2 values, even though water samples produced faster detection times. Comparisons between broth-based versions of R2A medium and plate count agar revealed that the latter correlated highly with heterotrophic plate count, provided that water samples were incubated at 25 degrees C and impedance measurements were conducted with the capacitance signal (r = 0.966). When the linear regression of this relationship was tested with 100 water samples, the correlation between predicted and actual log10 CFU milliliter-1 was 0.869. These results indicate that impedance methods provide a suitable alternative to conventional methods.

Klebsiella pneumoniae gastroenteritis masked by Clostridium perfringens.

An unusual food-borne outbreak of gastroenteritis associated with contaminated turkey occurred at a catered company meal. The average incubation period was 10 h, and the predominant symptoms were watery diarrhea and cramps. Vomiting did not occur. Initial epidemiological features and cultures from turkey and feces of infected patients suggested that the causative agent was Clostridium perfringens, but Klebsiella pneumoniae of capsular type K15 was also isolated in large numbers from both the turkey and feces of the same patients. Plasmid analysis and enterotoxin results supported the role of K. pneumoniae as the causative agent in this outbreak. Organisms other than commonly identified pathogens should not be ignored if present in high concentrations in both food and feces of infected persons.

Biology of Haemophilus ducreyi.

The etiological agent of the sexually transmitted genital ulcer disease chancroid was first described in 1889 by Auguste Ducrey following repeated autoinoculation of purulent ulcer material from a series of patients. The organism was isolated on artificial media a decade later but has remained difficult to isolate consistently, resulting in controversy over its characteristics and role as the causative agent of chancroid. Because of its fastidious growth requirements, including unknown components in blood, the organism was included in the original description of the genus Haemophilus. Requirement for exogenous hemin and limited phenotypic characteristics, including structural and antigenic properties, suggested that Haemophilus ducreyi was a valid member of the genus Haemophilus. Recent studies of respiratory quinones, deoxyribonucleic acid hybridization, and competition for homologous transformation of the type species, H. influenzae, suggest that H. ducreyi is unrelated to any of the present species of the family Pasteurellaceae, which includes members of the genera Haemophilus, Actinobacillus, and Pasteurella. This review summarizes the early studies with H. ducreyi and our current knowledge of the microbiology of this important human pathogen.

Characterization of a multiple antibiotic resistance plasmid from Haemophilus ducreyi.

Plasmid pLS88 from a clinical isolate of Haemophilus ducreyi encoded resistance determinants for sulfonamides and streptomycin related to those of RSF1010 and for kanamycin related to Tn903 but lacked the inverted repeats of the transposon. Its host range included Haemophilus influenzae, Actinobacillus pleuropneumoniae, and Escherichia coli; and it was compatible with pDM2 and RSF1010.

Comparison of whole-cell protein electrophoretic profiles of Haemophilus influenzae: implementation of a microcomputer mainframe linked system and description of a new similarity coefficient.

A microcomputer mainframe linked system is described which allows video camera data capture and storage of one-dimensional whole-cell protein electrophoresis gel images, processing of normalized traces to produce a similarity matrix, and analysis of the matrix using the commercial cluster analysis program CLUSTAN. A new similarity coefficient is introduced which takes into account both band position and intensity. Forty-five strains of Haemophilus influenzae, including the eight biotypes and six serotypes, were analyzed using this system. Results demonstrated groupings which are consistent with known genetic relationships.

Transfer of plasmid-mediated ampicillin resistance from Haemophilus to Neisseria gonorrhoeae requires an intervening organism.

Haemophilus species have been implicated as the source of plasmid-mediated ampicillin resistance in Neisseria gonorrhoeae. Previous attempts to transfer conjugally the resistance plasmids from Haemophilus species to N. gonorrhoeae have met with limited success. Using both biparental and triparental mating systems, it was found that transfer will occur if the commensal Neisseria species, Neisseria cinerea, is used as a transfer intermediate. This organism stably maintains resistance plasmids of Haemophilus and facilitates transfer of these plasmids to N. gonorrhoeae, in a triparental mating system, at a transfer frequency of 10(-8). Both Haemophilus ducreyi and N. gonorrhoeae carry mobilizing plasmids capable of mediating conjugal transfer of the same resistance plasmids. However, restriction endonuclease mapping and DNA hybridization studies indicate that the mobilizing plasmids are distinctly different molecules. Limited homology is present within the transfer region of these plasmids.

Susceptibility of Haemophilus aegyptius to trooleandomycin: lack of taxonomic value.

Two hundred and nine strains of Haemophilus influenzae and Haemophilus aegyptius were screened for trooleandomycin susceptibility. Four strains were shown to be sensitive to the drug. Of these four, two were Haemophilus aegyptius (ATCC 11116, NCTC 8134), and the other two were Haemophilus influenzae biotype I (1-605) and IV (80-212. One strain of Haemophilus aegyptius (NCTC 8135) was resistant to trooleandomycin. Restriction enzyme assays and DNA homology were carried out to establish relationships between the strains. It is concluded that trooleandomycin susceptibility has no taxonomic value to differentiate between Haemophilus aegyptius and biotype III Haemophilus influenzae.

Characterization of a Haemophilus ducreyi mobilizing plasmid.

The OriV site of Haemophilus ducreyi mobilizing plasmid pHD147, determined by replication in Escherichia coli polA, is located close to the OriT site. The OriT site, located by recombination-proficient and -deficient cells, and the OriV site map in a region of pHD147 homologous to the beta-lactamase-specifying plasmids of H. ducreyi and Neisseria gonorrhoeae.