Ted L. Hadfield

The pathology of diphtheria.

Diphtheria is an acute, communicable disease caused by Corynebacterium diphtheriae. The disease is generally characterized by local growth of the bacterium in the pharynx with pseudomembrane formation or, less commonly, in the stomach or lungs; systemic dissemination of toxin then invokes lesions in distant organs. Acute disease of the upper respiratory tract usually involves one or more of the following: tonsillar zones, larynx, soft palate, uvula, and nasal cavities. A recent epidemic in Russia emphasized the role of vaccination in reducing disease in children and adults.

Detection of Bacillus anthracis DNA by LightCycler PCR

ABSTRACT Anthrax is a zoonotic disease that is also well recognized as a potential agent of bioterrorism. Routine culture and biochemical testing methods are useful for the identification of Bacillus anthracis, but a definitive identification may take 24 to 48 h or longer and may require that specimens be referred to another laboratory. Virulent isolates of B. anthracis contain two plasmids (pX01 and pX02) with unique targets that allow the rapid and specific identification of B. anthracis by PCR. We developed a rapid-cycle real-time PCR detection assay for B. anthracis that utilizes the LightCycler instrument (LightCycler Bacillus anthracis kit; Roche Applied Science, Indianapolis, Ind.). PCR primers and probes were designed to identify gene sequences specific for both the protective antigen (plasmid pX01) and the encapsulation B protein (plasmid pX02). The assays (amplification and probe confirmation) can be completed in less than 1 h. The gene encoding the protective antigen (pagA) was detected in 29 of 29 virulent B. anthracis strains, and the gene encoding the capsular protein B (capB) was detected in 28 of 29 of the same strains. Three avirulent strains containing only pX01 or pX02, and therefore only pagA or pagB genes, could be detected and differentiated from virulent strains. The assays were specific for B. anthracis: the results were negative for 57 bacterial strains representing a broad range of organisms, including Bacillus species other than anthracis (n = 31) and other non-Bacillus species (n = 26). The analytical sensitivity demonstrated with target DNA cloned into control plasmids was 1 copy per μl of sample. The LightCycler Bacillus anthracis assay appears to be a suitable method for rapid identification of cultured isolates of B. anthracis. Additional clinical studies are required to determine the usefulness of this test for the rapid identification of B. anthracis directly from human specimens.

Bartonella vinsonii subsp. berkhoffii subsp. nov., isolated from dogs ; Bartonella vinsonii subsp. vinsonii ; and emended description of Bartonella vinsonii

Two bacterial strains, one isolated from the blood of a dog with valvular endocarditis and one isolated from the blood of a healthy dog, were similar to Bartonella species, as determined by a number of phenotypic criteria, including growth characteristics, biochemical reactions, and cell wall fatty acid composition. The results of 16S rRNA gene sequence similarity studies confirmed that these strains are closely related and belong in the genus Bartonella and that Bartonella vinsonii is their closest relative (the 16S rRNA of isolate 93-C01T [T = type strain] was 99.37% identical to the 16S rRNA of the type strain of B. vinsonii, the 16S rRNA of isolate G7464 was 99.61% identical to the 16S rRNA of the type strain, and the 16S rRNAs of the dog isolates were 99.77% identical to each other). The 16S rRNAs of both strains contained a 12-base insertion that was not present in the 16S rRNA of the type strain of any Bartonella species. DNA relatedness tests revealed that these strains were related at the species level to the type strain of B. vinsonii. They were, however, significantly more closely related to each other than to B. vinsonii. On the basis of their unique 16S rRNA sequence insertion, their preferentially high level of relatedness, and their similar origins (dogs), we believe that strains 93-C01(T) and G7464 should be placed in a separate subspecies of B. vinsonii, for which we propose the name B. vinsonii subsp. berkhoffii subsp. nov. The type strain of B. vinsonii subsp. berkhoffii is strain 93-C01 (= ATCC 51672). The description of B. vinsonii is emended to accommodate the new subspecies, and B. vinsonii subsp. vinsonii is described.

Interactions between Brucella melitensis and Human Phagocytes: Bacterial Surface O-Polysaccharide Inhibits Phagocytosis, Bacterial Killing, and Subsequent Host Cell Apoptosis

ABSTRACT Brucellae are gram-negative intracellular pathogens that survive and multiply within host phagocytic cells. Smooth organisms present O-polysaccharides (OPS) on their surface. The wboA gene, which codes for the enzyme glycosyl transferase, is essential for the assembly of O-chain in Brucella. Deletion of wboA in smooth, virulent B. melitensis 16M results in a rough mutant designated WRR51. Unlike B. abortus, both smooth and rough strains of B. melitensis are resistant to complement-mediated killing. To determine the role of surface OPS in the interactions of B. melitensis with monocytes/macrophages (M/M), 16M and WRR51 were transformed with the plasmid pBBR1MCS-6y encoding green fluorescent protein, and the transformants were used to infect human mononuclear phagocytes with and without fresh human serum as a source of complement. Human monocytes were cultured in the presence of macrophage colony-stimulating factor to allow their differentiation into macrophages during the course of infection. Intracellular bacteria were easily visualized using fluorescence microscopy. Infection in M/M, identified by surface staining and fate of infected phagocytes, was quantitated by flow cytometry. Rough bacteria were internalized, with no requirement for opsonization by serum, at a higher rate than smooth organisms. Smooth B. melitensis survived and multiplied for at least 6 days inside M/M, but rough organisms were eliminated by death of the infected cells. In human monocytes cultured for 1 day without serum in order to trigger the apoptotic pathway, infection by rough brucellae accelerated phagocyte death; smooth brucellae inhibited apoptosis. This study suggests that the presence of surface OPS on live B. melitensis benefits the bacterium by preventing the death of macrophages, Brucellas preferred target for intracellular replication.

Detection of Smallpox Virus DNA by LightCycler PCR

ABSTRACT A 300-bp plasmid fragment of the hemagglutinin gene was used as target DNA to develop a rapid real-time LightCycler (Roche Applied Science, Indianapolis, Ind.) PCR assay for laboratory detection of smallpox virus. PCR primers and probes were designed specifically for detection of smallpox virus DNA, but all viruses of the genus Orthopoxvirus tested could be detected by use of the hemagglutinin gene target sequence. Base pair mismatches in the 204-bp amplicon allowed discrimination of cowpox virus (melting temperature [Tm], 56.40°C), monkeypox virus (Tm, 56.24°C), and vaccinia virus (Tm, 56.72°C), including the Dryvax vaccine strain, from smallpox virus (Tm, 62.45°C) by melting curve analysis. The analytical sensitivity was 5 to 10 copies of target DNA per sample. The assay was specific for members of the genus Orthopoxvirus; the DNAs of herpes simplex virus and varicella-zoster virus were not detected by the smallpox virus LightCycler PCR.

Pathogenic bacteria: Their detection and differentiation by rapid lipid profiling with pyrolysis mass spectrometry

Abstract Pyrolysis mass spectrometry (Py–MS) can be used to profile methylated fatty acids from bacterial pathogens without a chromatographic step. An in situ thermal hydrolysis and methylation (THM) step incorporated into the Py–MS analysis of whole bacteria reduces the sample preparation time from 60 min to less than 1 min. Detection of four bacterial pathogens with a field-portable aerosol-sampling Py–ion trap MS using FAME profiles from whole bacteria is demonstrated with a total analysis time of less than 10 min/lipid profile.

A rapid approach for the detection of dipicolinic acid in bacterial spores using pyrolysis/mass spectrometry.

Curie-point pyrolysis/triple quadrupole mass spectrometry and micro-tube furnace pyrolysis/quadrupole ion trap mass spectrometry have been used to detect dipicolinic acid (DPA) in sporulated whole bacteria. DPA in whole cells of sporulated Bacillus anthracis reacted in situ during pyrolysis with tetramethylammonium hydroxide to form the dimethyl ester derivative of DPA, dimethyl-2,6-dipicolinate (mDPA). The mDPA was identified by its positive-ion electron ionization fragmentation pattern and confirmed with tandem mass spectrometry. In an oxidative pyrolysis/quadrupole ion trap instrument, the mDPA mass spectrum showed characteristic positive-ion electron ionization fragmentation along with a significant [M+1]+ ion due to self-chemical ionization. The characteristic collision-induced dissociation fragments of mDPA were used to establish the presence of sporulation in B. anthracis whole cells at a concentration of 2.2 x 10(7) CFU (colony-forming units)/mL using the triple quadrupole instrument. The total time for analysis, including sample preparation, was less than 10 minutes for both instruments.

Cat Scratch Disease Bacilli in the Conjunctiva of Patients with Parinaud's Oculoglandular Syndrome

Unilateral conjunctivitis with regional lymphadenitis has been designated Parinauds oculoglandular syndrome (POGS). The cause has been attributed to various infectious agents, especially leptothrix and the unidentified agent of cat scratch disease (CSD). In 1983-84 the cause of CSD in lymph node and skin was established as small pleomorphic gram-negative bacilli. We now report identical bacteria in the conjunctival lesions of 9 of 24 patients with POGS. This confirms the belief that the CSD bacillus is a major cause of POGS.

Detection of Francisella tularensis within Infected Mouse Tissues by Using a Hand-Held PCR Thermocycler

ABSTRACT The diagnosis of human cases of tularemia often relies upon the demonstration of an antibody response to Francisella tularensis or the direct culturing of the bacteria from the patient. Antibody response is not detectable until 2 weeks or more after infection, and culturing requires special media and suspicion of tularemia. In addition, handling live Francisella poses a risk to laboratory personnel due to the highly infectious nature of this pathogen. In an effort to develop a rapid diagnostic assay for tularemia, we investigated the use of TaqMan 5′ hydrolysis fluorogenic PCR to detect the organism in tissues of infected mice. Mice were infected to produce respiratory tularemia. The fopA and tul4 genes of F. tularensis were amplified from infected spleen, lung, liver, and kidney tissues sampled over a 5-day period. The samples were analyzed using the laboratory-based Applied Biosystems International 7900 and the Smiths Detection-Edgewood BioSeeq, a hand-held portable fluorescence thermocycler designed for use in the field. A comparison of culturing and PCR for detection of bacteria in infected tissues shows that culturing was more sensitive than PCR. However, the results for culture take 72 h, whereas PCR results were available within 4 h. PCR was able to detect infection in all the tissues tested. Lung tissue showed the earliest response at 2 days when tested with the ABI 7900 and in 3 days when tested with the BioSeeq. The results were in agreement between the ABI 7900 and the BioSeeq when presented with the same sample. Template preparation may account for the loss of sensitivity compared to culturing techniques. The hand-held BioSeeq thermocycler shows promise as an expedient means of forward diagnosis of infection in the field.

The use of biomarker compounds for the identification of bacteria by pyrolysis-mass spectrometry

Abstract Several recent advances in using biomarkers for bacteria characterization are discussed. A study using Gram-positive and Gram-negative bacteria has been conducted to compare both gas chromatographic and mass spectrometric analyses of the separately isolated and methylated fatty acids to an in situ reaction in which the saponification and methylation of the fatty acids is achieved using tetramethylammoninium hydroxide (TMAH). Principal components analysis of the three data sets showed the same three cluster patterns. In situ methylation using TMAH has also been conducted on free nucleotides, oligonucleotides, DNA, various amino acids, and oligopeptides. An increase in the volatility of important biomarkers was observed in all analyses which leads to an increase in the information content of the data. High resolution mass spectrometry has also been applied to the peaks corresponding to a series of biomarker compounds. The data show that most nominal masses are composed of several contributing species.