Molecular genetic basis of antimicrobial agent resistance in Mycobacterium tuberculosis: 1998 update.

The molecular signature of mediastinal large B-cell lymphoma differs from that of other diffuse large B-cell lymphomas and shares features with classical Hodgkin lymphoma

The primary theme emerging from molecular genetic work conducted with Mycobacterium tuberculosis and several other mycobacterial species is that resistance is commonly associated with simple nucleotide alterations in target chromosomal genes rather than with acquisition of new genetic elements encoding antibiotic-altering enzymes. Mutations in an 81-bp region of the gene (rpoB) encoding the beta subunit of RNA polymerase account for rifampin resistance in 96% of M. tuberculosis and many Mycobacterium leprae isolates. Streptomycin resistance in about one-half of M. tuberculosis isolates is associated with missense mutations in the rpsL gene coding for ribosomal protein S12 or nucleotide substitutions in the 16S rRNA gene (rrs). Mutations in the katG gene resulting in catalase-peroxidase amino acid alterations nad nucleotide substitutions in the presumed regulatory region of the inhA locus are repeatedly associated with isoniazid-resistant M. tuberculosis isolates. A majority of fluoroquinolone-resistant M. tuberculosis isolates have amino acid substitutions in a region of the DNA gyrase A subunit homologous to a conserved fluoroquinolone resistance-determining region. Multidrug-resistant isolates of M. tuberculosis arise as a consequence of sequential accumulation of mutations conferring resistance to single therapeutic agents. Molecular strategies show considerable promise for rapid detection of mutations associated with antimicrobial resistance. These approaches are now amenable to utilization in an appropriately equipped clinical microbiology laboratory.

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Antimicrobial agent resistance in mycobacteria: molecular genetic insights.

The determination of antimicrobial susceptibility of a clinical isolate, especially with increasing resistance, is often crucial for the optimal antimicrobial therapy of infected patients. Nucleic acid-based assays for the detection of resistance may offer advantages over phenotypic assays. Examples are the detection of the methicillin resistance-encoding mecA gene in staphylococci, rifampin resistance in Mycobacterium tuberculosis, and the spread of resistance determinants across the globe. However, molecular assays for the detection of resistance have a number of limitations. New resistance mechanisms may be missed, and in some cases the number of different genes makes generating an assay too costly to compete with phenotypic assays. In addition, proper quality control for molecular assays poses a problem for many laboratories, and this results in questionable results at best. The development of new molecular techniques, e.g., PCR using molecular beacons and DNA chips, expands the possibilities for monitoring resistance. Although molecular techniques for the detection of antimicrobial resistance clearly are winning a place in routine diagnostics, phenotypic assays are still the method of choice for most resistance determinations. In this review, we describe the applications of molecular techniques for the detection of antimicrobial resistance and the current state of the art.

https://courses.washington.edu/pabio568/files/fluit.pdf

Molecular Detection of Antimicrobial Resistance

Nonpigmented and late-pigmenting rapidly growing mycobacteria (RGM) are increasingly isolated in clinical microbiology laboratories. Their accurate identification remains problematic because classification is labor intensive work and because new taxa are not often incorporated into classification databases. Also, 16S rRNA gene sequence analysis underestimates RGM diversity and does not distinguish between all taxa. We determined the complete nucleotide sequence of the rpoB gene, which encodes the bacterial β subunit of the RNA polymerase, for 20 RGM type strains. After using in-house software which analyzes and graphically represents variability stretches of 60 bp along the nucleotide sequence, our analysis focused on a 723-bp variable region exhibiting 83.9 to 97% interspecies similarity and 0 to 1.7% intraspecific divergence. Primer pair Myco-F-Myco-R was designed as a tool for both PCR amplification and sequencing of this region for molecular identification of RGM. This tool was used for identification of 63 RGM clinical isolates previously identified at the species level on the basis of phenotypic characteristics and by 16S rRNA gene sequence analysis. Of 63 clinical isolates, 59 (94%) exhibited 3% partial rpoB gene sequence divergence from the corresponding type strain; they belonged to three taxa related to M. mucogenicum, Mycobacterium smegmatis, and Mycobacterium porcinum. For M. abscessus and M. mucogenicum, this partial sequence yielded a high genetic heterogeneity within the clinical isolates. We conclude that molecular identification by analysis of the 723-bp rpoB sequence is a rapid and accurate tool for identification of RGM.

rpoB-Based Identification of Nonpigmented and Late-Pigmenting Rapidly Growing Mycobacteria

We report on the identification of mutations associated with streptomycin resistance in Mycobacterium tuberculosis. Two isolates (3656 and 3976) showed a wild-type ribosomal protein, S12, but exhibited a single point mutation at 16S rRNA position 491 (C-->T) or 512 (C-->T), respectively. Sequence analysis of a third isolate (2438) revealed a single base change at 16S rRNA position 904 (A-->G). This position is equivalent to invariant position 913 of the Escherichia coli 16S rRNA gene, an A-->G transition of which has been shown previously to impair streptomycin binding and streptomycin-induced misreading in vivo. Surprisingly, strain 2438 harbors an additional mutation in the ribosomal protein S12 (Lys-88-->Gln).

Genetic alterations in streptomycin-resistant Mycobacterium tuberculosis: mapping of mutations conferring resistance.

We investigated the contribution of the polysialic acid capsule and of terminal lipooligosaccharide (LOS) sialylation to the pathogenicity of Neisseria meningitidis in vivo using a set of defined isogenic mutants of the N. meningitidis strain B 1940 deficient in either capsule synthesis or LOS sialylation. Furthermore a spontaneous capsule-deficient variant was investigated, which was capable of switching on the capsule synthesis at a frequency of 3 x 10(-3) in vitro. Infection of infant rats with the wild-type strain revealed a high potential to cause bacteremia. This potential was attenuated in the capsule-phase variable mutant (LOS sialylation+). However, using a mutant irreversibly deficient in capsule synthesis, but expressing a sialylated LOS, bacteremia could only be achieved using 10(6) times higher numbers of bacteria when compared to the wild-type. The unencapsulated bacteria were located extracellularly upon examination of blood smears, suggesting that defense mechanisms, i.e. phagocytosis, directed against unencapsulated meningococci were exhausted using very high infecting doses. Interestingly, when infant rats were infected with encapsulated meningococci which were unable to sialylate the LOS, bacteremia could never be achieved, even with an infective dose as high as 10(8) colony forming units (CFU). Despite the presence of capsular polysaccharide this mutant was phagocytosed by peritoneal phagocytes, as was the unencapsulated, LOS-sialylated mutant, suggesting that the inability to cause bacteremia was due to a higher susceptibility to the action of the complement system, which is virtually unsaturable. We conclude that in the infant rat model of meningococcal infection both forms of sialic acid on the bacterial cell surface are indispensable for systemic survival.

Sialic acids of both the capsule and the sialylated lipooligosaccharide of Neisseria meningitis serogroup B are prerequisites for virulence of meningococci in the infant rat

IFP 35 Is an interferon-induced leucine zipper protein that undergoes interferon-regulated cellular redistribution

The acid cytokeratin K17 is inducible by interferon-gamma (IFN-gamma), a characteristic unique for cytokeratins analysed so far. In this report, we analysed the molecular basis of K17 expression by IFN-gamma in epithelial cells. The 5'-flanking region of the K17 gene (positions -1762 to -13), cloned in front of a chloramphenicol acetyl transferase (CAT) reporter gene construct, conferred responsiveness to IFN-gamma but not IFN-alpha in transient transfection assays. Sequence analysis revealed three putative gamma-interferon activation sites (GAS). Band-shift assays and transient transfections with CAT reporter gene constructs were used to characterize and to dissect the functional importance of each of the putative GAS elements. In the band shift assay, GAS3 (positions -1528 to -1515) was found to bind GAF/STAT91 and to compete with tryptophanyl-tRNA synthetase (IFP53/WRS)-GAS for binding to GAF; in contrast, GAS1 (positions -183 to -171) and GAS2 (positions -290 to -277) were neither able to bind to nor to compete for GAF/STAT91. However, deletion constructs and mutational analysis of CAT reporter gene constructs harbouring the 5'-flanking region (positions -1762 to -111) in front of the heterologous promoter revealed that the distal GAS3 site was dispensible, but that alteration of the GAS1 element rendered the promoter uninducible by IFN-gamma. Surprisingly, transfection of a CAT-reporter gene construct harbouring a promoter segment (positions -111 to +13) devoid of the GAS elements revealed enhanced CAT-gene expression upon IFN-gamma treatment. The interaction of GAS1 with the interferon-responsive promoter region in the physiological context remains to be clarified.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1995.tb20370.x

Transcriptional activation of psoriasis-associated cytokeratin K17 by interferon-gamma. Analysis of gamma-interferon activation sites.

Mycoplasma hominis has been recognized as an extragenital pathogen in immunocompromised hosts, including kidney and heart transplant recipients; patients with systemic autoimmune diseases, lymphomas, or leukemia; and patients who have undergone cardiac surgery or who have experienced trauma [1, 2]. Liver transplantation has rarely been reported to increase host susceptibility to intraabdominal M hominis infection [1, 3, 4]. We describe two liver transplant recipients with extragenital M. hominis infection, and we emphasize the need for physicians to be aware of the pathogenicity of M. hominis for this group of patients. The isolation of M. hominis from the pleural space and the abdominal surgical wound in the first case and from an intrahepatic abscess in the second one indicates that M hominis infection in sites other than the peritoneal cavity should be considered. We furthermore propose the use of sequence determination of the tuf gene [5-7] for rapid species diagnosis to accelerate clinical decisions. A 48-year-old male patient underwent orthotopic liver transplantation for treatment of hepatocellular carcinoma and liver cirrhosis associated with hepatitis B virus. The patient developed fever, pleural effusions, and signs of abdominal surgical wound infection in the fifth week after transplantation. The results of standard bacteriologic examination of the pleural fluid and of wound swabs were unremarkable; after 3-4 days, translucent pinpoint colonies appeared on Columbia agar and on chocolate agar. Subculture on supplemented mycoplasma agar (Oxoid, Basingstoke, UK) revealed colonies with the \"fried-egg\" appearance that is typical for mycoplasmas after 2 days. Before specific antibiotic therapy could be initiated, the patient developed fulminant and fatal Klebsiella oxytoca pneumonia that did not respond to initial treatment with piperacillin and tobramycin. The mycoplasma isolates were nonglycolytic and hydrolyzed arginine. The isolates were confirmed to be M hominis by amplification of the gene encoding the elongation factor Tu (tuf) and by direct sequence determination of the PCR product as described previously [5-7]. Although the clinical relevance of the pleural and wound infection remained unclear in this patient's case, our experience prompted us to culture relevant extragenital specimens obtained from liver transplant recipients in mycoplasma agar (this agar was used in addition to standard diagnostic culture media). The value of using this agar was confirmed since 2 months later M hominis was isolated from swab specimens from the peritoneal cavity of a female 29-year-old drug addict who had heptitis B virus—induced acute liver failure that had been treated by liver transplantation. Peritonitis and fever had developed 4 weeks after transplantation due to partial necrosis of the bile duct, which was treated surgically by a hepatojejunostomy.

Ulrich Vogel

Papers

Genetic alterations in streptomycin-resistant Mycobacterium tuberculosis: mapping of mutations conferring resistance.

Sialic acids of both the capsule and the sialylated lipooligosaccharide of Neisseria meningitis serogroup B are prerequisites for virulence of meningococci in the infant rat

IFP 35 Is an interferon-induced leucine zipper protein that undergoes interferon-regulated cellular redistribution

Transcriptional activation of psoriasis-associated cytokeratin K17 by interferon-gamma. Analysis of gamma-interferon activation sites.

Extragenital Mycoplasma hominis Infection in Two Liver Transplant Recipients