Skin microflora and bacterial infections of the skin.

The skin is a milieu for controlled bacterial growth. Skin supports the growth of commensal bacteria, which protect the host from pathogenic bacteria. Environmental and local factors, host immunity, and organism adherence and virulence are intricately related to cutaneous infection. Resident gram-positive bacteria include Staphylococcus, Micrococcus, and Corynebacterium sp. Staphylococcus aureus and Streptococcus pyogenes are notoriously pathogenic in the skin. In order for bacteria to be pathogenic, they must be able to adhere to, grow on, and invade the host. Bacteria possess numerous virulence genes that allow for growth in these privileged niches. Epidermal infections caused by S. aureus and S. pyogenes include impetigo and ecthyma. Dermal infections consist of erysipelas, cellulitis, and necrotizing fasciitis. The pilosebaceous unit is involved in folliculitis, furunculosis, and carbunculosis. Moreover, S. aureus and S. pyogenes produce toxins that may elicit a superantigen response, causing massive release of cytokines. Staphylococcal scalded skin syndrome, toxic shock syndrome, and scarlet fever are all superantigen-mediated. Gram-negative organisms such as Pseudomonas aeruginosa, Pasteurella multocida, Capnocytophaga canimorsus, Bartonella sp., Klebsiella rhinoscleromatis, and Vibrio vulnificus are not typical resident skin microflora but may cause cutaneous infection.

Cutaneous aspergillosis and acquired immunodeficiency syndrome.

BACKGROUND: Primary cutaneous aspergillosis is an uncommon finding in patients with acquired immunodeficiency syndrome (AIDS); only 13 cases have been reported in the literature. OBSERVATIONS: We describe 11 patients with primary cutaneous aspergillosis and AIDS. There does not seem to be an age, sex, race, or human immunodeficiency virus risk factor predisposition. This is a late manifestation of AIDS; patients typically have low CD4 counts (<0.050 x 10(9)/L [<50/microL]) and other AIDS-defining illnesses. The most frequent presentation is in patients with cytomegalovirus disease and neutropenia caused by ganciclovir therapy. Lesions developed at the site of occlusive dressings for an indwelling intravenous catheter site in 10 patients. Neutrophil counts may be normal at the time of diagnosis. A minor presentation is in the patient without neutropenia as a result of traumatic inoculation. Histological findings and/or culture results are required for diagnosis. Patients develop cutaneous lesions despite prophylactic therapy with fluconazole. Lesions can be treated with excision and lifelong therapy with itraconazole. CONCLUSION: Because of the potential morbidity and mortality of cutaneous aspergillosis, a high level of suspicion and prompt institution of therapy is required.

Cutaneous Cryptococcus infection and AIDS. Report of 12 cases and review of the literature.

BACKGROUND: Cryptococcal infections occur in 6% to 13% of patients with acquired immunodeficiency syndrome (AIDS), most commonly infecting the central nervous system. Cutaneous lesions have been described morphologically as umbilicated papules, nodules, and violaceous plaques and can mimic molluscum contagiosum and Kaposi's sarcoma. Cutaneous lesions can present months prior to other signs of systemic infection. OBSERVATIONS: Cases of infection with cutaneous Cryptococcus and AIDS were reviewed and compared with cases reported in the literature. Among patients with Cryptococcus infection and AIDS seen at our institutions, 5.9% had skin lesions. All patients with cutaneous lesions had systemic involvement. Women were less commonly infected than men. There was no apparent predisposition associated with age, race, or human immunodeficiency virus infection risk factors. The median CD4 helper T-cell count was 0.024 X 10(9)/L (24/microL), and 44% (16/36) of the patients had previous opportunistic infections. Lesions were most commonly seen on the head and neck (78% [36/46]) and often mimicked molluscum contagiosum (54% [25/46]). The median serum and cerebrospinal fluid cryptococcal antigen titers were 1:32,768 and 1:512, respectively. Patients in our group did well with therapy (one death at 6 weeks, compared with 38% [13/34] mortality in the literature). There was no correlation between onset of lesions, number of lesions, CD4 helper T-cell count, or histopathologic characteristics. CONCLUSIONS: Disseminated Cryptococcus infection in AIDS presents with cutaneous lesions in up to 6% of cases. Clinicians need to be aware of the varied morphologic characteristics, since cutaneous lesions may present well in advance of other signs of systemic infection.

Disseminated acanthamebiasis in patients with AIDS. A report of five cases and a review of the literature.

BACKGROUND: Acanthamoeba and Leptomyxida are free-living amebae that cause granulomatous amebic encephalitis, a rare, slowly progressive, fatal neurologic process seen in immunosuppressed hosts. In addition, these organisms produce disseminated cutaneous lesions and involve other organs, particularly in patients with the acquired immunodeficiency syndrome (AIDS). RESULTS: We report five cases of disseminated acanthamebiasis in patients with AIDS, each with cutaneous manifestations but lacking central nervous system involvement. The medial CD4+ T-cell count was 0.024 x 10(9)/L. Skin lesions included pustules, subcutaneous and deep dermal nodules, and ulcers, most often seen on the extremities and face. Histopathologically, both pustular and vasculitic changes were observed; in all cases, the microscopic identification of organisms was difficult because of the macrophagelike appearance of the microbes in routine sections. CONCLUSIONS: Skin lesions are the most common reported presentation of infections caused by Acanthamoeba and Leptomyxida organisms in patients with AIDS, a minority of whom have central nervous system manifestations. A high index of suspicion is necessary for both the dermatologist and the dermatopathologist. Prognosis is guarded, but early treatment using a combination of intravenous pentamidine and oral fluconazole, sulfadiazine, and flucytosine may be beneficial.

Transcription and decay of the lac messenger: role of an intergenic terminator.

Prior work has indicated that the polycistronic lacZYA mRNA of Escherichia coli is cleaved during decay at approximately intergenic sites (L. W. Lim and D. Kennell, J. Mol. Biol. 135: 369-390, 1979). In this work, we characterized the products by using probes specific for the different cistrons. This analysis indicated that six lac mRNA species are present in the following order of decreasing abundance: lacZ, -A, -ZYA, -ZY, -YA, and -Y. Very little lacYA and lacY mRNAs were present, whereas in cells induced to steady state, there was 10 times more lacZ than lacZYA mRNA. The lacZ mRNA appeared as a discrete species extending to a site in the lacZ-Y intergenic space (ca. residue 3150). This site is just distal to a potential rho-independent termination sequence. We examined the function of this sequence to determine whether it contributes to the distribution of the mRNAs. Although the termination sequence was shown to function in vitro, when it was recloned into an expression vector, no termination was seen in vivo. Moreover, direct examination of the kinetics of lac messenger synthesis revealed that after initiation, most transcription continued to the end of the operon. We conclude that during normal growth, the operon is transcribed in its entirety and that the individual lac mRNAs are formed by cleavage. These results confirm earlier work implying that the lac operon is transcribed in its entirety but are in conflict with several recent reports suggesting that internal termination occurs. Our findings indicate that the natural polarity of the operon (lacZ is expressed sixfold more strongly than lacA) is based on posttranslational effects and not on polarity of transcription.

Mapping the lacZ ribosome binding site by RNA footprinting.

The ribosome binding site of the Escherichia coli lacZ mRNA has been characterized by using an RNA footprinting technique. Purified E. coli 70S ribosomes and fMet-tRNA were incubated with mRNA, and the complex was treated with RNA-reactive reagents or RNases as probes. The protected sites on the mRNA were then mapped by extending a radioactive primer with reverse transcriptase. Dimethyl sulfate, diethyl pyrocarbonate, and 1,10-phenanthroline-copper ion oxidative complex were used as reagent probes; they detected interaction sites within the ribosome binding site. A region of approximately 35 nucleotides was protected by the ribosome, specifically across the Shine-Dalgarno region, around the fMet initiation codon, and at a region 7-12 nucleotides distal to the fMet codon. In addition, an enhanced reaction occurred between the fMet codon and the distal site. These results imply an internally selective interaction between the ribosome and the mRNA sequence. The enhanced reactivity of a site distal to the initiation site--flanked by the AUG codon and a site previously identified as conserved in a study of initiation sequences--may indicate a region where the mRNA is specifically exposed.

Scission of RNA by the chemical nuclease of 1,10-phenanthroline-copper ion: preference for single-stranded loops.

The scission of RNA by the chemical nuclease activity of 1,10-phenanthroline-copper (OP-Cu) has been studied using a lac mRNA fragment and tRNAphe as substrates. Since the chemical mechanism of scission involves oxidative attack on the ribose, scission is observed at all nucleotides including dihydrouridine and Y-bases. Specificity for single-stranded loop regions is apparent from the similarity of the reactivity of OP-Cu to the single-strand specific reagents dimethyl sulfate and diethyl pyrocarbonate using the fragment of lac mRNA as a substrate. Similar preference is observed in the reaction with tRNA although scission in the helical acceptor stem is also observed.

Inhibition of human immunodeficiency virus by using an oligonucleoside methylphosphonate targeted to the tat-3 gene.

Antiviral effects were characterized for two oligodeoxyribonucleoside methylphosphonates synthesized in an antisense (3'-TCTTAACC-5') or a sense (5'-AGAATTGG-3') orientation, based on the RNA sequence of the first splice acceptor site of the tat-3 gene of human immunodeficiency virus (HIV) (5'...AGAAUUGG...3'). The development of syncytial cells and supernatant reverse transcriptase was inhibited by a single exposure to the antisense HIV, and HIV RNA synthesis was inhibited by both antisense and sense methylphosphonates but not by a control herpes simplex virus antisense sequence.

Direct detection of HIV-1 RNA from AIDS and ARC patient samples.

Human immunodeficiency virus (HIV), formerly termed human T-lymphotropic virus (HTLVIII/LAV), is the etiological agent of acquired immune deficiency syndrome (AIDS). Direct detection of HIV-1 nucleic acid sequences in patient tissue or blood samples is possible in only a minor fraction of cases due to the low percentage of infected cells (Shaw et al., 1984). We report a modification of the polymerase chain reaction method (PCR) (Saiki et al., 1985), in which we amplify sequences from HIV-1 RNA templates, for the identification of HIV-1 in peripheral blood and tissue samples obtained from AIDS and AIDS-related complex (ARC) patients. This method of HIV-1 detection is at least six orders of magnitude more sensitive than standard nucleic acid detection methods and has direct clinical applications. In vitro tissue culturing of the virus is not required for HIV-1 detection. Using this technique, the sequence in the orfB region of HIV-1 has been amplified and detected from less than 1 microgram of total RNA prepared from a few milliliters of peripheral blood samples. This technique enables the rapid and unambiguous clinical detection of potential HIV-infected individuals and can be used to assay the efficacy of anti-HIV-1 drugs. To enhance the efficiency of this technique, we have appended the prokaryotic T7 RNA polymerase promoter sequence to one of the priming oligonucleotides. After several cycles of PCR with the promoter-containing oligo, a small aliquot of the reaction can be utilized to direct specific and efficient T7 RNA polymerase-mediated transcription of the amplified sequences, thus enhancing the sensitivity and simplifying the labor of the experiment.

Expression of a tRNA gene in the context of the lacZ mRNA.

Fusions of the gene for tyrosine suppressor tRNA, tyrT(Sup3), and the lacZ gene of Escherichia coli were constructed such that the tRNA gene could be expressed from either its own promoter or that of the lac operon. These chimeras, carried on phage M13 vectors, were tested for the expression of the tRNA in E. coli. The tRNA gene was expressed on the order of 10-fold more weakly from the lac promoter than from its own promoter. To examine whether pausing or premature termination of transcription played a role in determining the relative strength, the fusions were tested in a variety of genetic backgrounds and under different physiological conditions that uncouple transcription and translation. The expression of the tRNA was not enhanced in backgrounds in which polarity was weakened or under the other conditions tested, although a dependence on nusB function was observed when the tRNA was transcribed from the lac promoter. These results indicate that pausing or premature termination of transcription did not play a role in the weak expression of the gene fusions. The results further suggest that the transcription of the tyrT gene does not normally require relief from polarity as imposed by any of the known transcriptional termination systems, in contrast to the antitermination system thought to be involved in the expression of the rRNAs.