A cumulative experience examining the effect of natural and synthetic antimicrobial peptides vs. Chlamydia trachomatis.

We tested the activity of 48 structurally diverse antimicrobial peptides against Chlamydia trachomatis, serovar L2. The peptides' activity against C. trachomatis, serovar L2 was measured in 48-h McCoy cell shell vial assays. Peptides of 16-20 amino acids were more active than larger peptides, such as defensins. Beta-sheet protegrins, as well as alpha-helical peptides such as novispirin (G-10) were equally active. Enantiomers were as active as native structures. Moderate-sized circular mini-defensins were less effective against C. trachomatis. Moderate-sized cationic peptides may be useful in microbicide preparations designed to prevent chlamydial infection.

Activity of Novispirin G-10, a novel antimicrobial peptide against Chlamydia trachomatis and vaginosis-associated bacteria.

We tested the activity of Novispirin G-10, a novel antimicrobial alpha-helical octadecapeptide structurally related to cathelicidins and other innate immunity peptides, against Chlamydia trachomatis serovars L2, D, and E and three organisms associated with bacterial vaginosis (BV). The peptide's activity against C. trachomatis was measured in 48-h shell vial assays with McCoy cell targets. Exposure to 100 micro g/ml of Novispirin G-10 reduced the infectivity of serovars D and E by 99.4-100% and serovar L2 by 91.7-99.1%. At the same concentration of 100 micro g/ml, Novispirin G-10 rapidly killed >99% of Mobiluncus curtisii, Gardnerella vaginalis, and Prevotella bivia, in standard colony-forming unit (CFU) assays. Given its simple structure and relative lack of cytotoxic and hemolytic activity, Novispirin G-10 may be a useful component of microbicide preparations designed to prevent chlamydial infection and/or remediate the abnormal vaginal flora associated with BV.

Evaluation of the inactivation of infectious Herpes simplex virus by host-defense peptides.

A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide microplate assay was adapted to screen for the ability of 20 host-defense peptides to inactivate herpes simplex virus type 1 and type 2. The procedure required minimal amounts of material, was reproducible, and was confirmed with standard antiviral testing techniques. In screening tests, with the exception of melittin, a highly cytotoxic and hemolytic peptide found in bee venom, the alpha-helical peptides in our test panel (magainins, cecropins, clavanins, and LL-37) caused little viral inactivation. Several beta-sheet peptides (defensins, tachyplesin, and protegrins) inactivated one or both viruses, sometimes with remarkable selectivity. Two peptides were identified as having antiviral activity against both viruses, indolicidin (a tryptophan-rich peptide from bovine neutrophils) and brevinin-1 (a peptide found in frog skin). The antiviral activity of these two peptides was confirmed with standard antiviral assays. Interestingly, the antiviral activity of brevinin-1 was maintained after reduction and carboxamidomethylation, procedures that abolished its otherwise prominent hemolytic and cytotoxic effects.

Leptospiral outer membrane proteins OmpL1 and LipL41 exhibit synergistic immunoprotection.

New vaccine strategies are needed for prevention of leptospirosis, a widespread human and veterinary disease caused by invasive spirochetes belonging to the genus Leptospira. We have examined the immunoprotective capacity of the leptospiral porin OmpL1 and the leptospiral outer membrane lipoprotein LipL41 in the Golden Syrian hamster model of leptospirosis. Specialized expression plasmids were developed to facilitate expression of leptospiral proteins in Escherichia coli as the membrane-associated proteins OmpL1-M and LipL41-M. Although OmpL1-M expression is highly toxic in E. coli, this was accomplished by using plasmid pMMB66-OmpL1, which has undetectable background expression without induction. LipL41-M expression and processing were enhanced by altering its lipoprotein signal peptidase cleavage site to mimic that of the murein lipoprotein. Active immunization of hamsters with E. coli membrane fractions containing a combination of OmpL1-M and LipL41-M was found to provide significant protection against homologous challenge with Leptospira kirschneri serovar grippotyphosa. At 28 days after intraperitoneal inoculation, survival in animals vaccinated with both proteins was 71% (95% confidence interval [CI], 53 to 89%), compared with only 25% (95% CI, 8 to 42%) in the control group (P < 0.001). On the basis of serological, histological, and microbiological assays, no evidence of infection was found in the vaccinated survivors. The protective effects of immunization with OmpL1-M and LipL41-M were synergistic, since significant levels of protection were not observed in animals immunized with either OmpL1-M or LipL41-M alone. In contrast to immunization with the membrane-associated forms of leptospiral proteins, hamsters immunized with His(6)-OmpL1 and His(6)-LipL41 fusion proteins, either alone or in combination, were not protected. These data indicate that the manner in which OmpL1 and LipL41 associates with membranes is an important determinant of immunoprotection.

A comparison of the early inflammatory effects of an agr-/sar- versus a wild type strain of Staphylococcus aureus in a rat model of endophthalmitis.

PURPOSE: We examined the ability of a wild type and an isogenic mutant strain of Staphylococcus aureus, deficient in the production of hemolysins and lipase (agr (-)/sar (-)), to induce endophthalmitis and inflammatory cell infiltration into the eye at 6, 24 and 48 hours after injection in a rat model of endophthalmitis. METHODS: Rat eyes were injected with 25 microl of viable S. aureus or sterile saline. Eyes were graded for clinical signs of inflammation daily, removed and processed for standard histologic analysis 6, 24 and 48 hours after injections. Comparisons of clinical scores and mean inflammatory cell numbers were made between S. aureus and control injected eyes. RESULTS: Both experimental groups developed clinical signs of endophthalmitis and demonstrated infiltration of inflammatory cells at 24 and 48 hours. Clinical inflammation in the Mutant I group was less than the wild type group at these times and significantly less at 48 hours (p<0.05). No statistically significant difference in the number of inflammatory cells was detected between the wild type and Mutant I injected eyes at 24 hours. At 48 hours, inflammatory cells increased by 75.0% in the wild type group and decreased by 19.0% in the Mutant I group and a statistically significant difference was seen between these two groups (p<0.05). At all times, the majority of inflammatory cells were neutrophils. By 48 hours, an increase in monocytes-macrophages was noted. CONCLUSION: Both strains of S. aureus induced clinical signs of inflammation and inflammatory cell infiltration. Clinical inflammation and inflammatory cell numbers were less in rats injected with the Mutant I strain. These results suggest that hemolysins and lipase may be important in the early induction phase of the inflammatory response.

Bacterial species identification after DNA amplification with a universal primer pair.

The diagnosis of bacterial infections can be difficult and time consuming. Rapid and reliable molecular triage of potentially infected patients, particularly the young and the elderly, would prevent unnecessary hospitalizations, reduce associated medical costs, and improve the quality of care. Polymerase chain reaction (PCR) amplification utilizing a universal bacterial primer pair, followed by hybridization with species-specific probes, would allow rapid identification of the presence or absence of bacterial DNA, along with an identification of the bacterial species present. Molecular microbiological analyses will require access to bacterial strain standards that can be catalogued and distributed to clinical laboratories. We amplified template DNA in filter paper spots containing boiled bacteria from 14 clinical isolates using a universal primer pair for the 16S ribosomal RNA (rDNA) coding sequence. Species-specific probes were hybridized to the amplification products for bacterial species identification. We conclude that template DNA can be identified with species-specific probes after universal bacterial amplification with a single primer pair. We also demonstrate a rapid and efficient method for the long-term storage and cataloguing of bacterial DNA for use in quality control at clinical laboratories adopting molecular diagnostic methodologies. We speculate that PCR amplification combined with species-specific probe hybridization not only will represent an improvement over culture-based methods in terms of speed, sensitivity, and cost, but will also allow for the identification of unculturable bacteria and emerging or reemerging pathogenic organisms.

Expression and distribution of leptospiral outer membrane components during renal infection of hamsters.

The outer membrane of pathogenic Leptospira species grown in culture media contains lipopolysaccharide (LPS), a porin (OmpL1), and several lipoproteins, including LipL36 and LipL41. The purpose of this study was to characterize the expression and distribution of these outer membrane antigens during renal infection. Hamsters were challenged with host-derived Leptospira kirschneri to generate sera which contained antibodies to antigens expressed in vivo. Immunoblotting performed with sera from animals challenged with these host-derived organisms demonstrated reactivity with OmpL1, LipL41, and several other proteins but not with LipL36. Although LipL36 is a prominent outer membrane antigen of cultivated L. kirschneri, its expression also could not be detected in infected hamster kidney tissue by immunohistochemistry, indicating that expression of this protein is down-regulated in vivo. In contrast, LPS, OmpL1, and LipL41 were demonstrated on organisms colonizing the lumen of proximal convoluted renal tubules at both 10 and 28 days postinfection. Tubular epithelial cells around the luminal colonies had fine granular cytoplasmic LPS. When the cellular inflammatory response was present in the renal interstitium at 28 days postinfection, LPS and OmpL1 were also detectable within interstitial phagocytes. These data establish that outer membrane components expressed during infection have roles in the induction and persistence of leptospiral interstitial nephritis.

Quantitative cytomegalovirus DNA level in the blood and its relationship to cytomegalovirus retinitis in patients with acquired immune deficiency syndrome.

OBJECTIVE: A pilot study was performed to determine whether relationships exist between changes in a quantitative solution hybridization assay for cytomegalovirus (CMV) DNA in the blood and development of CMV retinitis, development of nonocular CMV disease, or reactivation of pre-existing CMV retinitis lesions. DESIGN: Observational case series. PARTICIPANTS: Two groups of human immunodeficiency virus-infected patients: 10 CMV antibody-positive patients with CD4+ T-lymphocyte counts of less than 50 ml and no CMV disease at baseline and 11 patients with CMV retinitis but no extraocular CMV disease at baseline. INTERVENTION: Quantitative changes in leukocyte-associated CMV DNA levels were observed over time. Anti-CMV therapies were based on clinical findings only. MAIN OUTCOME MEASURES: Development of CMV end-organ disease or change in activity of pre-existing CMV retinitis lesions was measured. RESULTS: Among patients with no CMV disease at baseline, four had CMV disease develop during follow-up (three cases of CMV retinitis, one case of presumed CMV esophagitis); all had CMV DNA levels greater than 5000 genomes/ml before the onset of CMV disease. The remaining six patients had levels less than 5000 genomes/ml throughout follow-up (P = 0.05). Among patients with CMV retinitis at baseline, all whose CMV DNA blood levels rose more than tenfold had extraocular CMV disease or reactivation of CMV retinitis develop. Raised CMV DNA blood levels were not seen in every patient with clinical reactivation of CMV retinitis. CONCLUSION: Elevated or rising CMV DNA blood levels appear to be associated with the development of CMV disease in individuals with low CD4+ T-lymphocyte counts. In patients with CMV retinitis, rising levels appear to be associated with the development of extraocular CMV disease or reactivation of CMV retinitis. Conversely, reactivation of CMV retinitis also may occur in the absence of changes in CMV DNA blood levels. Further studies are warranted to determine whether changes in CMV blood levels can be used as a guide for preemptive therapy to prevent reactivation of CMV retinitis lesions or to help choose between local and systemic therapy for management of reactivations.

Critical evaluation of 4-week incubation for fungal cultures: is the fourth week useful?

To determine the benefit of a 4-week incubation for mycology cultures, we evaluated all positive cultures during the fourth week of incubation in a 1-year period. Of 3,855 positive mycology cultures (yeast, 82%; molds, 18%), 62 (1.6%) were positive during the fourth week (yeast, 42%; molds, 58%). Only 15 of the 62 cultures (24%) were considered clinically relevant (2 isolates from invasive fungal infection and 13 isolates from cutaneous mycosis). With the exception of those from skin samples, isolates recovered during the fourth week are rarely important for patient care.

Something's rotten: a nosocomial outbreak of malodorous Pseudomonas aeruginosa.

From July 1994 through November 1996, a phenotypically unique strain of Pseudomonas aeruginosa producing a pungent, "rotten-potato" odor and a positive lysine decarboxylase reaction was isolated from 39 patients at UCLA Medical Center (Los Angeles). Most cases (95%) were in intensive care units and had clinical infections (72%). Most isolates (74%) were recovered from cultures of respiratory secretions. To determine risk factors for acquisition of the organism, 23 cases were compared with 23 randomly selected controls matched by service and isolate date. Multivariate analysis revealed that isolation of malodorous P. aeruginosa was associated with mechanical ventilation of > 24 hours' duration (odds ratio [OR] = 9.4; P = .001) and transfer from an outside hospital (OR = 5.7; P = .04). DNA from outbreak strains hybridized to P. aeruginosa-specific toxin A and phospholipase C gene probes and all outbreak isolates tested were found to be identical by use of pulsed-field gel electrophoresis. An unusual phenotypic characteristic of the strain led to the recognition of a nosocomial outbreak of P. aeruginosa infection associated with mechanical ventilation.

Microbial evaluation: 139 implants removed from symptomatic patients.

Possible adverse effects of microbial organisms have been implicated in symptomatic silicone implant patients. In the literature, numerous authors have investigated the possible role of infection with respect to implant problems. To date, various bacterial species have been reported, including Staphylococcus aureus, Staphylococcus epidermidis, peptostreptococci, and Clostridium perfringens. Infections in polyurethane-coated prostheses also have been shown to prolong morbidity. Antibiotic use has been relatively empirical in this regard. The purpose of this study was, first, to determine the frequency, type, and clinical relevance of microbial colonization on implant surfaces removed from symptomatic patients and, second, to determine possible effects of microbial colonization on implant integrity (gel bleed, rupture). A total of 139 implants from 72 symptomatic patients were entered into the prospective clinical study between February of 1993 and July of 1994 at the UCLA Medical Center. The implant shell types included smooth (79 percent), polyurethane (8 percent), textured (7 percent), and smooth and Dacron (6 percent). The implant locations were subglandular (71 percent), submuscular (28 percent), and subcutaneous (1 percent). Of the 139 implants removed, 69 percent were intact and 31 percent were ruptured. Forty-seven percent of 139 implants were culture-positive. Propionibacterium acnes was isolated most frequently (57.5 percent), followed by Staphylococcus epidermidis (41 percent), and then Escherichia coli (1.5 percent). No fungal infections were identified. Culture positivity was not significantly associated with systemic symptoms. Sixty-seven percent of the positive culture implants were intact; 33 percent were ruptured. The frequency (47 percent) and types (P. acnes and S. epidermidis) of microbial colonization are determined in symptomatic silicone implant patients.

Protegrins: structural requirements for inactivating elementary bodies of Chlamydia trachomatis.

We tested 20 protegrins against Chlamydia trachomatis serovar L2 (L2/434/Bu). Five of the protegrins had native structures; the others included nonamidated, enantiomeric, and truncated variants and peptides with <2 disulfide bonds. Antichlamydial activity resided principally in residues 5 to 15 of native protegrin PG-1, and optimal activity required both intramolecular disulfide bonds.

Susceptibility of Chlamydia trachomatis to protegrins and defensins.

We compared the susceptibilities of Chlamydia trachomatis elementary bodies (EBs) to human defensin HNP-2 and porcine protegrin PG-1, cysteine-rich beta-sheet antimicrobial peptides produced by mammalian leukocytes. Although both peptides protected McCoy cell monolayers from infection by chlamydial EBs, protegrins were especially potent. Protegrin-mediated inactivation of chlamydiae occurred rapidly, was relatively independent of the presence of serum, and was effective against serovars L2, D, and H. Protegrin-treated EBs showed striking morphological changes, with obvious damage to their limiting membranes and loss of their cytoplasmic contents and nucleoid. Their effectiveness against chlamydial EBs and other sexually transmitted pathogens combined with their relative lack of cytotoxicity suggests that protegrins and related molecules could serve as prototypes for topical agents to prevent sexually transmitted chlamydial infection.

Direct hybridization and amplification applications for the diagnosis of infectious diseases.

We are entering an exciting new era of molecular diagnostics in the clinical microbiology laboratory. A number of perspectives are presented in this review. First presented was a discussion of molecular diagnostics for detection of the bacterium, Chlamydia trachomatis. This is especially relevant since the tests available for this organism represent the forefront of commercial systems. Also, these tests exemplify the difficulties and advantages inherent to future molecular diagnostics for all types of disease processes. Next, a discussion of the techniques thus far employed in the field of clinical microbiology is presented. Obvious overlap exists with other areas of molecular pathology. However, the emphasis is on which techniques have proven most useful in identifying infectious agents. Finally, the features of a successful clinical microbiology diagnostics laboratory are presented, including test component requirements, laboratory personnel, quality assurance techniques, and physical laboratory setting. It is hoped that helpful advice and references are provided that will assist individual clinical laboratories as they enter the field of molecular diagnostics of infectious diseases.

Defining the unknown: molecular methods for finding new microbes.

The purpose of this article is to describe specific applications of molecular diagnostics that are currently identifying suspected or unidentified microbial pathogens. The techniques reviewed include (i) the use of specific primers and PCR to identify new microbes, (ii) PCR amplification of conserved 16S rRNA sequence with subsequent identification of specific internal sequence from the candidate bacterial pathogen, and (iii) an exciting new modification of PCR called RDA or "reverse PCR" that can identify unique infectious agents in diseased tissue. The field will continue to expand rapidly and, it is hoped, contribute to a better understanding of the microbial environment with which humans coexist. Also, molecular techniques will eventually be applied in the demonstration of pathogenesis by the various newly identified microbial pathogens.

Clinical significance of intracapsular fluid in patients' breast implants.

Clinical reports on the incidence and clinical significance of intracapsular fluid are lacking in the literature. It remains unknown whether the presence of intracapsular fluid has any relation to implant infection or colonization. The purpose of this study was to determine the frequency and type of intracapsular fluid, specifically, whether intracapsular fluid causes implant infection, implant rupture, or bacterial colonization. A total of 139 implants from 72 symptomatic patients were entered into the prospective clinical study. Our study demonstrated the presence of intracapsular fluid in 21 of 139 (15%) implants. Positive microbial cultures were identified in 39% of the implants in the positive intracapsular fluid group, compared to 43% in the negative fluid group. There was no statistically significant difference between these groups. Also, no adverse clinical relationship was demonstrated between local symptoms and presence of intracapsular fluid. There was, however, a positive trend toward the presence of fluid when implant shell types were nonsmooth (polyurethane and textured silicone implants). Further studies are indicated to elucidate the fluid production mechanism and possible secretory activity of prosthetic capsules interfacing the textured breast implant surface.

The glycyl-tRNA synthetase of Chlamydia trachomatis.

Aminoacyl-tRNA synthetases specifically charge tRNAs with their cognate amino acids. A prototype for the most complex aminoacyl-tRNA synthetases is the four-subunit glycyl-tRNA synthetase from Escherichia coli, encoded by two open reading frames. We examined the glycyl-tRNA synthetase gene from Chlamydia trachomatis, a genetically isolated bacterium, and identified only a single open reading frame for the chlamydial homolog (glyQS). This is the first report of a prokaryotic glycyl-tRNA synthetase encoded by a single gene.

Rabbit model of Lyme borreliosis: erythema migrans, infection-derived immunity, and identification of Borrelia burgdorferi proteins associated with virulence and protective immunity.

Erythema migrans (EM), persistent skin infection, and visceral dissemination can be induced reproducibly in the adult male New Zealand White rabbit by intradermal injection of as few as 10(3) Borrelia burgdorferi. EM was found to persist for 7 +/- 3 d. Skin culture positivity (infection) cleared within a mean of 6.7 +/- 1.4 wk after infection and similarly visceral infection was not demonstrated after 8 wk; infection-derived immunity to intradermal challenge was evident 5 mo after initial infection. The extent of the protection against EM and dermal infection induced by untreated infection was directly related to the extent of prior in vitro passage of the B31 strain. Initial infection with as few as 4 x 10(3) B31 passage 4 induced complete protection against EM and skin infection upon subsequent challenge with 4 x 10(7) B31, passage 4. Initial infection with B31 passage 27 led to partial protection against EM along with complete protection against skin infection. Initial infection with passage 47 led to partial protection against EM, but conferred no protection against skin infection. Using serum from rabbits fully immune to reinfection, we defined a set of B. burgdorferi proteins present in virulent B31, but absent in the avirulent American Type Culture Collection B31 strain, termed "va" for virulent strain associated. The va proteins of B31 passages 1, 27, and 47 differed strikingly, thus raising the possibility that these changes may relate in a causal way to the differences in induction of protective immunity observed.

Application of PCR to multiple specimen types for diagnosis of cytomegalovirus infection: comparison with cell culture and shell vial assay.

Human cytomegalovirus (CMV) is a herpesvirus that is responsible for significant morbidity and mortality in congenitally infected infants and immunocompromised patients. Antiviral therapies are available, thus making timely diagnosis of significant importance to at-risk patients. A PCR system was devised. The newly devised system, unlike previously described systems, can be applied to a wide variety of specimen types in a clinical microbiology laboratory setting. Specimens from all sites routinely accepted for CMV culture were shown to be acceptable for CMV PCR. Sensitivity and specificity were established in comparison with those of both monolayer culture and shell vial assay (SVA). The sensitivity and specificity of PCR for detection of CMV in specimens exclusive of urine and blood were 97.5 (77 of 79 specimens) and 87.2% (41 of 47 specimens), respectively. The sensitivity and specificity of PCR for urine and blood specimens were 100 (10 of 10) and 95.7% (45 of 47) and 66.7 (4 of 6) and 78.8% (41 of 52), respectively. Discrepancies of positive PCR results with negative culture or SVA results occurred for specimens flanked chronologically by other culture- or SVA-positive specimens and were likely culture failures, increasing the specificity (100%) of PCR. Discrepancies of negative PCR results with positive culture or SVA results occurred in specimens with few cells or infectious foci by SVA or culture and may represent sampling variability associated with low virus titers.

Analysis of genomic DNA from Chlamydia trachomatis for Dam and Dcm methylation.

Chlamydia trachomatis is a Gram-negative eubacterium with a dimorphic developmental cycle and obligate intracellular growth in the eucaryotic host. The Dam transmethylase of Escherichia coli methylates at the N6 position of adenine in the sequence 5'-GATC-3' and the Dcm transmethylase adds methyl groups to the C5 position of the internal cytosines in the sequences 5'-CCWGG-3'. In contrast to E. coli, C. trachomatis DNA appears to have unmethylated Dam sites and only low level Dcm methylation.