Comparative genotyping of Campylobacter jejuni by amplified fragment length polymorphism,multilocus sequence typing,and short repeat sequencing: strain diversity,host range,and recombination

Three molecular typing methods were used to study the relationships among 184 Campylobacter strains isolated from humans, cattle, and chickens. All strains were genotyped by amplified fragment length polymorphism (AFLP) analysis, multilocus sequence typing (MLST), and sequence analysis of a genomic region with short tandem repeats designated clustered regularly interspaced short palindromic repeats (CRISPRs). MLST and AFLP analysis yielded more than 100 different profiles and patterns, respectively. These multiple-locus typing methods resulted in similar genetic clustering, indicating that both are useful in disclosing genetic relationships between Campylobacter jejuni isolates. Group separation analysis of the AFLP analysis and MLST data revealed an unexpected association between cattle and human strains, suggesting a common source of infection. Analysis of the polymorphic CRISPR region carrying short repeats allowed about two-thirds of the typeable strains to be distinguished, similar to AFLP analysis and MLST. The three methods proved to be equally powerful in identifying strains from outbreaks of human campylobacteriosis. Analysis of the MLST data showed that intra- and interspecies recombination occurs frequently and that the role of recombination in sequence variation is 50 times greater than that of mutation. Examination of strains cultured from cecum swabs revealed that individual chickens harbored multiple Campylobacter strain types and that some genotypes were found in more than one chicken. We conclude that typing of Campylobacter strains is useful for identification of outbreaks but is probably not useful for source tracing and global epidemiology because of carriage of strains of multiple types and an extremely high diversity of strains in animals.     

Let them fly or light them up: matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry and fluorescence in situ hybridization (FISH)

This review focuses on clinical bacteriology and by and large does not cover the detection of fungi, viruses or parasites. It discusses two completely different but complementary approaches that may either supplement or replace classic culture-based bacteriology. The latter view may appear provocative in the light of the actual market penetration of molecular genetic testing in clinical bacteriology. Despite its elegance, high specificity and sensitivity, molecular genetic diagnostics has not yet reached the majority of clinical laboratories. The reasons for this are manifold: Many microbiologists and medical technologists are more familiar with classical microbiological methods than with molecular biology techniques. Culture-based methods still represent the work horse of everyday routine. The number of available FDA-approved molecular genetic tests is limited and external quality control is still under development. Finally, it appears difficult to incorporate genetic testing in the routine laboratory setting due to the limited number of samples received or the lack of appropriate resources. However, financial and time constraints, particularly in hospitals as a consequence of budget cuts and reduced length of stay, lead to a demand for significantly shorter turnaround times that cannot be met by culture-dependent diagnosis. As a consequence, smaller laboratories that do not have the technical and personal equipment required for molecular genetic amplification techniques may adopt alternative methods such as fluorescence in situ hybridization (FISH) that combines easy-to-perform molecular hybridization with microscopy, a technique familiar to every microbiologist. FISH is hence one of the technologies presented here. For large hospital or reference laboratories with a high sample volume requiring massive parallel high-throughput testing we discuss matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) of nucleic acids, a technology that has evolved from the post-genome sequencing era, for high-throughput sequence variation analysis (1, 2).     

Near-haploid clones in a malignant fibrous histiocytoma

Near-haploid solid tumors are very rare. In a storiform-pleomorphic malignant fibrous histiocytoma (MFH) of bone, we found three cell populations: one with a near-haploid, a second with a near-diploid, and a third with a near-tetraploid chromosome number. The near-haploid cells had few structural rearrangements: i(12p) and t(13q21q) in one clone, and these two and an additional t(19;?)(p11;?) in another clone. One structurally normal copy of all chromosomes was also present, except that the only chromosome 13 was involved in the t(13q21q). There were also two near-diploid clones, one without the t(19;?) and one with a single copy of this derivative chromosome. This is the first MFH reported to have a near-haploid modal chromosome number, and also the first tumor with i(12p) among bone and soft tissue tumors.     

Oxygen metabolites induced by phorbol myristate acetate increase lateral diffusion of wheat germ agglutinin-labeled glycoconjugates in human polymorphonuclear leukocytes

To assess the general effects of protein kinase C (PKC) activation on cell membrane receptor mobility in human neutrophilic polymorphonuclear leukocytes (PMNLs), the lateral diffusion of fluoresceinated succinylated wheat germ agglutinin (S-WGA-FITC)-labeled membrane glycoconjugates was measured using fluorescence recovery after photobleaching (FRAP). Activation of PKC was achieved by incubating the PMNLs with different concentrations (5–100 nM) of phorbol myristate acetate (PMA). The membrane effects of dimethyl sulfoxide (DMSO), another possible membrane perturbant, were also studied. We found that PMA treatment ( 10 nM) increased the glycoconjugate diffusion coefficient (D) 2–2.5-fold. The mobile fraction (R) remained constant, around 30%. With DMSO, no effect on the diffusion was seen. The increase in lateral mobility due to cell stimulation with PMA was totally inhibited by catalase (200 units/ml) but only partly with superoxide dismutase (2000 units/ml). Exogenous hydrogen peroxide (0.01–5 mM) had no effect on glycoconjugate mobility in unstimulated cells. We therefore propose that activation of PKC mediates augmented mobility of glycoconjugate receptors in PMNL, a reaction that seems to be critically dependent on formation of reactive oxygen metabolites. The results indicate that endogenous formation of reactive metabolites upon receptor stimulation may have a general effect on receptor mobility.     

Identification of an N-linked glycan in the V1-loop of HIV-1 gp120 influencing neutralization by anti-V3 antibodies and soluble CD4

Summary Glycosylation is necessary for HIV-1 gp120 to attain a functional conformation, and individual N-linked glycans of gp120 are important, but not essential, for replication of HIV-1 in cell culture. We have constructed a mutant HIV-1 infectious clone lacking a signal for N-linked glycosylation in the V1-loop of HIV-1 gp120. Lack of an N-linked glycan was verified by a mobility enhancement of mutant gp120 in SDS-gel electrophoresis. The mutated virus showed no differences in either gp120 content per infectious unit or infectivity, indicating that the N-linked glycan was neither essential nor affecting viral infectivity in cell culture. We found that the mutated virus lacking an N-linked glycan in the V1-loop of gp120 was more resistant to neutralization by monoclonal antibodies to the V3-loop and neutralization by soluble recombinant CD4 (sCD4). Both viruses were equally well neutralized by ConA and a conformation dependent human antibody IAM-2G12. This suggests that the N-linked glycan in the V1-loop modulates the three-dimensional conformation of gp120, without changing the overall functional integrity of the molecule.     

Isolation of human immunodeficiency virus (HIV) from plasma during primary HIV infection

Human immunodeficiency virus (HIV) has been isolated from plasma in 6 of 7 patients showing clinical symptoms of a primary HIV infection. Parallel cultures from peripheral blood mononuclear cells (PBMC) yielded virus in 5 patients. In one case, virus could only be isolated from the cerebrospinal fluid but not from peripheral blood. Detectable viremia was transient and preceded the appearance of HIV specific antibodies. After cessation of acute symptoms, the frequency of HIV isolations was similar to that of asymptomatic carriers (23 and 26%, respectively). The role of the immune response in terminating detectable viremia remains to be established.     

Karyotypic evolution in Ph-positive chronic myeloid leukemia in relation to management and disease progression

In a prospective study of 32 patients with chronic myeloid leukemia the frequency of chromosome abnormalities in addition to the Philadelphia chromosome (Ph) increased when the disease progressed. Before metamorphosis, 10 patients (31%) had developed additional abnormalities. Such abnormalities were present in three of them at the time of diagnosis; in the other seven, they were detected late in the chronic phase. New clonal abnormalities heralded or accompanied a more malignant phase of the disorder, usually a blastic leukemia. During metamorphosis, 78% of the patients had additional abnormalities, which in 68% of these cases comprised at least one of +8, +22q- or i(17q). Clones with additional abnormalities disappeared in eight cases, either spontaneously or in association with cytostatic therapy during the chronic or blastic phase. Involvement of chromosome #8, usually in the form of a trisomy, was found in 7 of 12 patients treated with busulfan, but was not found in any of the 10 hydroxyurea-treated patients, of whom 8 were splenectomized early during the chronic phase. Cells from the spleen, obtained by fine needle aspiration or splenectomy were cytogenetically examined in 18 cases during the chronic phase, but abnormalities in addition to the Ph were noted in only one patient, who was examined in the late chronic phase. The same abnormalities were present in bone marrow cells of this patient.