Effectiveness of fluoroquinolones against Mycobacterium abscessus in vivo.

PURPOSE: To determine the effectiveness of fluoroquinolones against Mycobacterium abscessus in vivo. METHODS: M. abscessus growth was determined quantitatively in rabbit corneas after intrastromal bacterial injection (10(4) CFU/cornea; n >or= 4 corneas per group). Eyes were treated topically with 0.3% ciprofloxacin, 0.5% levofloxacin, or 0.5% moxifloxacin by three protocols: (1) 1 drop of antibiotic applied hourly for 10 hr on day 3 postinfection (PI); (2) 1 drop applied every 2 hr for 10 hr on days 2 and 3 PI; or (3) 1 drop applied every 2 hr for 10 hr on days 1, 2, and 3 PI. Corneas were cultured 1 hr after the last topical drop. Results are expressed as the log CFU. RESULTS: Bacteria in control group reached maximal numbers in vivo by day 3 PI (approximately 6 logs CFU/cornea). Treatment of infected eyes on day 3 with moxifloxacin or levofloxacin resulted in approximately 2.0 log decrease in CFU/cornea relative to the untreated control. Treatment on days 2 and 3 with moxifloxacin or levofloxacin resulted in approximately 3.0 and 2.5 log CFU/cornea decrease, respectively. Ciprofloxacin had no effect on bacterial load. Treatment on days 1, 2, and 3 with moxifloxacin resulted in a 5.5 log CFU decrease, whereas treatment with levofloxacin or ciprofloxacin resulted in a approximately 4.0 log CFU decrease. CONCLUSIONS: Moxifloxacin, and to a lesser extent levofloxacin and ciprofloxacin, demonstrated significant effectiveness for reducing the number of M. abscessus in vivo, suggesting the potential usage of these agents in prevention of M. abscessus keratitis.

Effectiveness of ciprofloxacin and ofloxacin in a prophylaxis model of Staphylococcus keratitis.

PURPOSE: To determine the effectiveness of prophylactic fluoroquinolone treatment against staphylococci in a rabbit keratitis model. METHODS: Prophylactic ciprofloxacin or ofloxacin was applied as one topical drop 15 minutes before infection or as one drop at three time points (19, 17, and 15 minutes) before infection. In a second experiment, rabbits were treated with two, three, or four drops of ciprofloxacin 1 hour before infection. Approximately 250 colony-forming units (CFUs) of Staphylococcus aureus were injected intrastromally, and CFUs were determined 5 hours after infection. RESULTS: The CFUs per cornea in all treatment groups were significantly less than the 5.6 +/- 0.11 log CFUs per cornea in the untreated group ( p < or = 0.0001). Rabbit eyes treated 15 minutes before infection with Ciloxan or Ocuflox had 0.96 +/- 0.48 log CFUs per cornea (three of six sterile corneas) or 1.26 +/- 0.31 log CFUs per cornea (one of six sterile corneas), respectively ( p = 0.5226). Eyes treated with Ciloxan 19, 17, and 15 minutes before infection had 0.0 +/- 0.0 log CFUs per cornea, and all eyes were sterile, whereas eyes treated with Ocuflox had 0.98 +/- 0.48 log CFUs per cornea and two of six eyes sterile ( p = 0.0435). Eyes treated 1 hour before infection with two, three, or four drops of Ciloxan had 2.61 +/- 0.69 log CFUs, 1.23 +/- 0.32 log CFUs, or 0.85 +/- 0.28 log CFUs per cornea, respectively, which was significantly less than untreated eyes ( p < or = 0.0001). CONCLUSIONS: Multiple topical drops of a fluoroquinolone administered prophylactically were effective for subsequent staphylococcal ocular infection.

Effect of famciclovir on herpes simplex virus type 1 corneal disease and establishment of latency in rabbits.

Famciclovir (FCV) is efficacious in the treatment of acute herpes zoster and recurrent genital infections but has not been used to treat ocular herpes simplex virus (HSV) infections. We evaluated the efficacy of orally administered FCV in treating HSV-1 epithelial keratitis and determined its effects on the establishment of latency and subsequent reactivation. Rabbits were inoculated with HSV-1 strain 17 syn+ and treated twice daily with increasing concentrations of FCV (60 to 500 mg/kg of body weight). This resulted in a significant, dose-dependent improvement in keratitis scores, as well as prolonged survival. Regardless of the dose of drug used, all groups exhibited the high rates of spontaneous and induced reactivation characteristic of 17syn+. The efficacy of 250 mg of FCV per kg was also compared to topical treatment with 1% trifluorothymidine (TFT). Although TFT treatment was more effective at reducing eye disease, FCV-treated rabbits had a better survival rate. Real-time quantitative PCR analysis of rabbit trigeminal ganglia (TG) demonstrated that FCV significantly reduced the HSV-1 copy number compared to that after treatment with TFT or the placebo but not in a dose-dependent manner. In summary, oral FCV treatment significantly reduces the severity of corneal lesions, reduces the number of HSV-1 genomes in the TG, improves survival, and therefore may be beneficial in reducing the morbidity of HSV keratitis in the clinic.

A cAMP response element within the latency-associated transcript promoter of HSV-1 facilitates induced ocular reactivation in a mouse hyperthermia model.

Herpes simplex virus type 1 (HSV-1) recombinant strain 17CRE contains a site-directed mutation in the 7-bp CRE consensus sequence located 38 nucleotides upstream of the transcription start site. Scarified mouse corneas received inoculations of 17syn+ (parent), 17CRE, and rescue 17CREr. Slit lamp examination of herpetic lesions and tear film swabs containing infectious virus showed that 17CRE had the same acute phenotype as 17syn+ and 17CREr. At 4 weeks, when the corneas had healed and latency was established, mice received hyperthermic shock. Eye swabs taken 24 h after hyperthermia showed that 17CRE reactivated significantly less than 17syn+ and 17CREr, while no significant differences were found in HSV-1 DNA genome copy numbers and latent virus in the trigeminal ganglia. These results are evidence that this CRE site in the LAT promoter facilitates ocular HSV-1 reactivation in mice.

Stress-associated immunomodulation and herpes simplex virus infections.

Stress has been shown to modulate an individual's immune system through the release of certain signal molecules such as catecholamines, cytokines and glucocorticoids. These signal molecules can significantly alter the host immune system and leave it susceptible to a primary or recurrent viral infection. Focusing on herpes simplex virus types-1 and -2 as examples, the authors explain how stress-associated immunomodulation can influence the recurrence of herpes simplex viral infections. Specific signal molecules such as epinephrine, interleukin-6, cyclic adenosine monophosphate, glucocorticoids and prostaglandins are upregulated during episodes of acute and chronic stress and have been implicated as effectors of herpes simplex viral reactivation and recurrent disease. The authors suggest that the release of immunomodulating signal molecules due to stress can compromise the host's cellular immune response and trigger herpes simplex viral reactivation.

Pseudomonas aeruginosa protease IV enzyme assays and comparison to other Pseudomonas proteases.

Pseudomonas aeruginosa secretes multiple proteases that have been implicated as virulence factors and the detection of each specific enzyme can be difficult to determine. Unlike the three Pseudomonas enzymes that have been well characterized (elastase A, elastase B, and alkaline protease), the activity of protease IV in multiple assays has yet to be described. This study defines new assays for Pseudomonas proteases and compares protease IV activity to the activities of elastase A, elastase B, and alkaline protease. Six in vitro assays were studied: zymography, elastin congo red assay, staphylolytic assay, colorimetric peptide assay, solid-phase colorimetric peptide assay, and poly-l-lysine degradation. Casein zymography distinguished protease IV from elastase B and alkaline protease, and gelatin zymography differentiated all four proteases. The elastin congo red assay detected mainly elastase B while the staphylolytic assay was specific for elastase A. Protease IV activity was assayed specifically by the colorimetric assay and two new assays, the solid-phase colorimetric assay and degradation of poly-L-lysine in the presence of EDTA. Alkaline protease could be specifically assayed by poly-L-lysine degradation in the presence of N-alpha-p-tosyl-L-lysine chloromethyl ketone. The results identified three specific assays for protease IV, a new assay specific for alkaline protease, and showed that protease IV has a distinct enzymatic specificity relative to the three other Pseudomonas proteases.

Identification of a novel 0.7-kb polyadenylated transcript in the LAT promoter region of HSV-1 that is strain specific and may contribute to virulence.

Herpes Simplex virus expresses latency-associated transcripts (LATs) the function of which remains obscure despite increasing knowledge of their structure and expression. Upstream of the LAT coding region is a region of the genome that is poorly characterized although it lies in an area that is responsible for modulation of reactivation efficiency in two different animal models. Transcript mapping with strains 17, McKrae, KOS, and F has revealed strain differences in this region of the viral genome. Strain 17 and McKrae expressed a novel polyadenylated 0.7-kb transcript that is absent from KOS and F. This transcript is expressed in the LAT direction and has the kinetics of a true late gene during the lytic cycle of infection. A deletion mutant, 17DeltaBsa, which does not express the 0.7-kb RNA, is less virulent than the parental strain 17. A rescuant with F sequence (17DeltaBsa/RF) shows virulence similar to F, whereas a rescuant with strain 17 sequence (17DeltaBsa/R17) is similar to strain 17. Virulence is altered by deletion or substitution in the region encoding the 0.7-kb transcript (BsaI-BsaI); however, reactivation in the mouse explant cocultivation assay or the adrenergically induced rabbit reactivation model remained unchanged. The importance of this region for virulence is discussed.

HSV-1 migration in latently infected and naive rabbits after penetrating keratoplasty.

PURPOSE: To investigate the migration of herpes simplex virus type 1 (HSV-1) between latently infected and naive corneal tissues and trigeminal ganglion (TG) in rabbits after penetrating keratoplasty (PKP) and transcorneal epinephrine iontophoresis. METHODS: Two mutants, genetically constructed from HSV-1 strain 17syn+, were used to inoculate rabbit corneas: 17deltaPst, a latency associated transcript (LAT) negative, low-reactivating virus and 17Pr, a high-reactivating, LAT-positive rescuant of 17deltaPst. Latently infected rabbits were given corneal allografts from naive rabbits, and naive rabbits received grafts from latently infected rabbits. Ninety days after PKP, groups of the transplanted rabbits were induced to reactivate by transcorneal epinephrine iontophoresis, but others were not induced. Viral shedding was monitored by tear film cultures. Rabbits were killed 5 days after iontophoresis. Transplanted grafts, recipient corneal rims, and corresponding TG were obtained. Nucleic acids were extracted and amplified for detection of HSV-1 DNA and viral gene transcription. RESULTS: In naive rabbits receiving grafts transplanted from rabbits latently infected with 17Pr (LAT+), 3 of 6 corneal rims contained HSV DNA after induction. In contrast, none of the 5 corneal rims from naive rabbits receiving grafts from rabbits latent with 17deltaPst (LAT-) contained viral DNA. Viral DNA and gene transcripts were detected in 2 of 6 TG from naive rabbits that received grafts from 17Pr (LAT+) latently infected rabbits. In recipient corneal rims and TG of latently infected rabbits receiving grafts from naive rabbits, viral DNA concentration was significantly greater with induced reactivation, compared with the results in noninduced rabbits. The amount of viral DNA in naive grafts transplanted into 17Pr (LAT+) latently infected rabbits was significantly higher with induction than without induction (P = 0.018). More viral DNA and viral gene transcripts were found in tissues from rabbits latently infected with 17Pr (LAT+) than in rabbits latently infected with 17deltaPst (LAT-). CONCLUSIONS: Corneas from latently infected rabbits contain HSV-1 DNA that can replicate after induced reactivation. Viral migration can occur in both anterograde and retrograde directions between the transplanted graft and the recipient corneal rim and TG. The LAT negative HSV-1 construct 17deltaPst has a significantly reduced ability to replicate and migrate.

Identical 371-base-pair deletion mutations in the LAT genes of herpes simplex virus type 1 McKrae and 17syn+ result in different in vivo reactivation phenotypes.

The herpes simplex virus type 1 (HSV-1) LAT gene is the only viral gene abundantly transcribed during latency. LAT null mutants created with strains McKrae and 17syn+ are impaired for both in vivo spontaneous and in vivo-induced reactivation. Thus, LAT is essential for efficient in vivo-induced and spontaneous reactivation. Different investigators have studied two LAT mutants containing a StyI-StyI region deletion corresponding to LAT nucleotides 76 to 447. One mutant, dLAT371 (parent strain, McKrae), had parental high frequencies of spontaneous reactivation. In vivo-induced reactivation was not examined. The other mutant, 17DeltaSty (parent strain, 17syn+), had parental frequencies of in vitro reactivation following cocultivation of explanted ganglia but reduced frequencies of in vivo-induced reactivation. Spontaneous reactivation frequency was not reported for 17DeltaSty. These combined results suggested the possibility that in vivo spontaneous reactivation and in vivo-induced reactivation may map to different regions within the LAT domain. We now report that dLAT371 has in vivo-induced reactivation frequencies of the parent and that 17DeltaSty has reduced frequencies of in vivo spontaneous reactivation. Thus, dLAT371 demonstrated the parental phenotype for both in vivo spontaneous and -induced reactivation while the apparently identical 17DeltaSty was impaired for both in vivo spontaneous and -induced reactivation. These results suggest that one or more differences between the genetic backgrounds of McKrae and 17syn+ result in different in vivo reactivation phenotypes of otherwise identical deletion mutations and that McKrae may have compensating sequences sufficient to overcome the loss of the StyI-StyI region of the LAT transcript.

Protein-protein interactions in the assembly of Shigella flexneri invasion plasmid antigens IpaB and IpaC into protein complexes.

Shigella flexneri is a facultative intracellular bacterial pathogen that invades human colonic epithelial cells by a process called pathogen-induced phagocytosis. Pathogen entry requires three virulence plasmid-encoded proteins called invasion plasmid antigens (Ipa) B, C and D which are secreted upon bacterial contact with a host cell. Following their secretion, IpaB and IpaC are found within a complex of proteins that may also contain IpaA and IpaD. Previous work has shown that exogenously added recombinant IpaC is sufficient for promoting the uptake of S. flexneri in gentamicin-protection assays. It is shown here that purified recombinant Ipa proteins can also be used to investigate the formation of Ipa protein complexes in vitro. The protein-protein contacts involved in the formation of Ipa complexes appear to include previously undescribed IpaC-IpaC interactions in addition to a strong association between IpaB and IpaC. IpaD does not appear to interact with either IpaB or IpaC in vitro although it is possible that recombinant IpaD forms homodimers that are stabilized by disulfide bridges involving this protein's single cysteine residue. This investigation represents the first characterization of the biochemistry of Ipa complex assembly.

Cloning, expression, and affinity purification of recombinant Shigella flexneri invasion plasmid antigens IpaB and IpaC.

Shigella flexneri and related enteropathogenic bacteria are important agents of bacillary dysentery, a potentially life-threatening illness for children in underdeveloped regions of the world. Onset of shigellosis stems from S. flexneri invasion of colonic epithelial cells, leading to localized cell death and inflammation. Invasion plasmid antigens (Ipa) B, C, and D are three secreted proteins encoded by the large virulence plasmid of S. flexneri that have been implicated as essential effectors of this cell invasion process. These proteins are expressed as part of the ipa operon and are among the major targets of the host immune response to shigellosis. Biochemical characterization of the Ipa invasins has been complicated by the fact they have not been purified in the quantities needed for detailed in vitro analysis. Here we describe the first cloning, expression, and extensive purification of IpaB and IpaC fusion proteins from Escherichia coli for use in dissecting of the protein biochemistry of S. flexneri pathogenesis. A variety of approaches were used to prepare significant quantities of these proteins in their soluble forms, including the use of different host cell lines, modification of bacterial growth conditions, and the use of alternative plasmid expression vectors. Now that these Ipa proteins are available in a highly pure form, it will be possible to initiate studies on their important biological and immunological properties as well as their recruitment into high-molecular-weight protein complexes. Together with IpaD (purified as part of a previous study), these purified proteins will be useful for: (a) exploring properties of the host immune response to S. flexneri invasion, (b) elucidating the specific biochemical properties that lead to pathogen internalization, (c) analyzing the importance of specific Ipa protein complexes in host cell invasions, and (d) monitoring, or perhaps even augmenting, the efficacy of live oral vaccines in human trials.

Soluble invasion plasmid antigen C (IpaC) from Shigella flexneri elicits epithelial cell responses related to pathogen invasion.

Shigella flexneri invades colonic epithelial cells by pathogen-induced phagocytosis. The three proposed effectors of S. flexneri internalization are invasion plasmid antigens B (IpaB), IpaC, and IpaD, which are encoded on the pathogen's 230-kb virulence plasmid and translocated to the extracellular milieu via the Mxi-Spa translocon. To date, there are no definitive functional data for any purified Ipa protein. Here, we describe the first characterization of highly purified recombinant IpaC, which elicits numerous epithelial cell responses related to events that take place during pathogen invasion. 125I-labeled IpaC binds cultured Henle 407 intestinal cells with an apparent dissociation constant in the low micromolar range. Moreover, incubation of epithelial cells with IpaC results in general changes in cellular phosphoprotein content, demonstrating this protein's ability to influence cellular protein kinase activities. These results contrast dramatically with those seen for recombinant IpaD, which does not bind to or induce detectable changes in the normal activities of cultured epithelial cells. In addition to influencing host cell activities, preincubation of epithelial cells with purified IpaC enhances uptake of S. flexneri by host cells. A similar result is seen when the cells are preincubated with a highly concentrated water extract of virulent S. flexneri 2a (strain 2457T). Interestingly, soluble IpaC also appears to promote uptake of the noninvasive S. flexneri 2a strain BS103. Purified IpaD failed to enhance the uptake of virulent S. flexneri and did not facilitate uptake of BS103. Taken together, the data suggest that IpaC is a potential effector of the host cell biological activities and may be responsible for entry of S. flexneri into target cells.

Structural analysis of invasion plasmid antigen D (IpaD) from Shigella flexneri.

Invaison plasmid antigen D (IpaD) and water-extracted outer-membrane proteins (OMPs) from Shigella flexneri were used to investigate some of the structural relationships of this pathogen's invasions. Extracellular presentation of the three invasion plasmid antigens (Ipa) B, C, and D is required for the S. flexneri invasive phenotype; however, little is known of the structural properties of these essential virulence components. Biochemical data suggest IpaB, C, and D present in S. flexneri OMPs from soluble protein complexes and IpaD may be able to form large homopolymeric complexes.