Pathobiology and innate immune responses of gallinaceous poultry to clade 2.3.4.4A H5Nx highly pathogenic avian influenza virus infection.

In the 2014-2015 Eurasian lineage clade 2.3.4.4A H5 highly pathogenic avian influenza (HPAI) outbreak in the U.S., backyard flocks with minor gallinaceous poultry and large commercial poultry (chickens and turkeys) operations were affected. The pathogenesis of the first H5N8 and reassortant H5N2 clade 2.3.4.4A HPAI U.S. isolates was investigated in six gallinaceous species: chickens, Japanese quail, Bobwhite quail, Pearl guinea fowl, Chukar partridges, and Ring-necked pheasants. Both viruses caused 80-100% mortality in all species, except for H5N2 virus that caused 60% mortality in chickens. The surviving challenged birds remained uninfected based on lack of clinical disease and lack of seroconversion. Among the infected birds, chickens and Japanese quail in early clinical stages (asymptomatic and listless) lacked histopathologic findings. In contrast, birds of all species in later clinical stages (moribund and dead) had histopathologic lesions and systemic virus replication consistent with HPAI virus infection in gallinaceous poultry. These birds had widespread multifocal areas of necrosis, sometimes with heterophilic or lymphoplasmacytic inflammatory infiltrate, and viral antigen in parenchymal cells of most tissues. In general, lesions and antigen distribution were similar regardless of virus and species. However, endotheliotropism was the most striking difference among species, with only Pearl guinea fowl showing widespread replication of both viruses in endothelial cells of most tissues. The expression of IFN-γ and IL-10 in Japanese quail, and IL-6 in chickens, were up-regulated in later clinical stages compared to asymptomatic birds.

Bioaerosol exposure to personnel in a clinical environment absent patients.

Nosocomial infections pose a significant and escalating threat to both patients and healthcare workers (HCWs). By their nature, hospitals induce antibiotic resistance in virulent and commensal strains, leading to increasingly severe hospital-acquired infections. This study measured environmental exposure experienced by domestic staff cleaning vacated patient rooms of a community hospital to bacteria in ambient bioaerosols. While they cleaned the room, participants wore an N95 filtering facepiece respirator (FFR), from which coupons were cut and bacteria were extracted, cultured and enumerated. Extrapolation to the full area of the respirator yielded measured exposures of 0.2-1.4 × 10(4) colony-forming units/hour, of which ∼97% collected on the front layer of the N95, suggesting a possible role for minimal respiratory protection in nonpatient environments. Random resistance testing of 1.6% of the isolates showed that ∼70% of both Gram-positive and Gram-negative organisms exhibited resistance to oxacillin and ∼9% of the Gram-positives displayed resistance to vancomycin. These data provide an estimate for mask bioaerosol loading that can be used in risk modeling and to refine strategies for reuse of FFRs during critical shortages.

Fluoroquinolone therapy in a rabbit model of post-LASIK methicillin-resistant Staphylococcus aureus keratitis.

PURPOSE: To develop a rabbit model of post-laser in situ keratomileusis (LASIK) methicillin-resistant Staphylococcus aureus (MRSA) keratitis for studying fluoroquinolone prophylaxis and treatment. SETTING: Department of Microbiology, University of Mississippi Medical Center, Jackson, Mississippi, USA. METHODS: An MRSA keratitis isolate (5 microL, 500 colony forming units [CFU]) was inoculated underneath a corneal flap. Bacterial growth and pathology were determined by quantitative cultures (CFU) and slitlamp examination, respectively. The effectiveness of commercial moxifloxacin and gatifloxacin formulations was compared in 3 regimens: prophylaxis (4 drops before inoculation), early therapy (single drop hourly from 4 to 9 hours postinfection), and late therapy (single drop hourly from 10 to 15 hours postinfection). Zones of bacterial inhibition to known in vivo antibiotic concentrations were determined. RESULTS: Bacteria grew to a maximum of approximately 10(6) CFU/cornea within 10 hours postinfection. The slitlamp examination scores showed pathologic changes beginning 10 hours postinfection and progressed throughout the infection. For prophylaxis, eyes treated with moxifloxacin had significantly fewer CFU than gatifloxacin-treated eyes or untreated controls (both P < or = .0001). During early treatment, the antibiotics were equally effective in reducing CFU relative to untreated controls (P < or = .0001). In late treatment, gatifloxacin and moxifloxacin caused significant reductions in CFU relative to untreated controls (P < or = .0007 and P < or = .0001, respectively). Moxifloxacin produced zones of bacterial inhibition significantly larger than those produced by gatifloxacin. CONCLUSIONS: Methicillin-resistant S aureus inoculation beneath a rabbit corneal flap produced an infection that was useful for quantitative microbiological studies. A significant advantage in using moxifloxacin relative to gatifloxacin was observed in prophylaxis of keratitis (P = .0001).

The efficacy of recombinant turkey herpesvirus vaccines targeting the H5 of highly pathogenic avian influenza virus from the 2014-2015 North American outbreak.

The outbreak of highly pathogenic avian influenza virus in North American poultry during 2014 and 2015 demonstrated the devastating effects of the disease and highlighted the need for effective emergency vaccine prevention and control strategies targeted at currently circulating strains. This study evaluated the efficacy of experimental recombinant turkey herpesvirus vector vaccines with three different inserts targeting the hemagglutinin gene of an isolate from the recent North American influenza outbreak. White leghorn chickens were vaccinated at one day of age and challenged with A/Turkey/Minnesota/12582/2015 H5N2 at 4 weeks of age. Birds were analyzed for survival, viral shedding at two and four days after infection, and specific antibody prior to challenge and from surviving birds. The three experimental vaccines demonstrated 100%, 45% and 15% survival with the most effective vaccine significantly reducing oral and cloacal viral shedding compared to all other groups and generated specific antibody prior to challenge with highly pathogenic avian influenza virus. More studies are needed using diverse H5Nx highly pathogenic virus isolates to fully determine the breadth of coverage against possible exposure strains, as well as possible impact of maternally derived antibody on protection and vaccine efficacy.

Maternal antibody inhibition of recombinant Newcastle disease virus vectored vaccine in a primary or booster avian influenza vaccination program of broiler chickens.

Maternally-derived antibodies (MDA) provide early protection from disease, but may interfere with active immunity in young chicks. In highly pathogenic avian influenza virus (HPAIV)-enzootic countries, broiler chickens typically have MDA to Newcastle disease virus (NDV) and H5 HPAIV, and their impact on active immunity from recombinant vectored vaccines is unclear. We assessed the effectiveness of a spray-applied recombinant NDV vaccine with H5 AIV insert (rNDV-H5) and a recombinant turkey herpesvirus (HVT) vaccine with H5 AIV insert (rHVT-H5) in commercial broilers with MDA to NDV alone (MDA:AIV-NDV+) or to NDV plus AIV (MDA:AIV+NDV+) to provide protection against homologous HPAIV challenge. In Experiment 1, chicks were spray-vaccinated with rNDV-H5 at 3 weeks (3w) and challenged at 5 weeks (5w). All sham-vaccinated progeny lacked AIV antibodies and died following challenge. In rNDV-H5 vaccine groups, AIV and NDV MDA had completely declined to non-detectable levels by vaccination, enabling rNDV-H5 spray vaccine to elicit a protective AIV antibody response by 5w, with 70-78% survival and significant reduction of virus shedding compared to shams. In Experiment 2, progeny were vaccinated with rHVT-H5 and rNDV-H5 at 1 day (1d) or 3w and challenged at 5w. All sham-vaccinated progeny lacked AIV antibodies and died following challenge. In rHVT-H5(1d) vaccine groups, irrespective of rNDV-H5(3w) boost, AIV antibodies reached protective levels pre-challenge, as all progeny survived and virus shedding significantly decreased compared to shams. In contrast, rNDV-H5-vaccinated progeny had AIV and/or NDV MDA at the time of vaccination (1d and/or 3w) and failed to develop a protective immune response by 5w, resulting in 100% mortality after challenge. Our results demonstrate that MDA to AIV had minimal impact on the effectiveness of rHVT-H5, but MDA to AIV and/or NDV at the time of vaccination can prevent development of protective immunity from a primary or booster rNDV-H5 vaccine.

Staphylococcus Alpha-Toxin Action on the Rabbit Iris: Toxic Effects and Their Inhibition.

PURPOSE: Staphylococcus aureus infection of the anterior chamber can occur after cataract surgery, causing inflammation and extensive damage to the iris. Alpha-toxin, the most potent S. aureus corneal toxin, was tested as a possible mediator of damage to the iris, and alpha-toxin anti-serum and a chemical toxin inhibitor were tested as potential pathology-reducing agents. METHODS: The hemolytic activity of alpha-toxin and its inhibition by a chemical inhibitor or anti-serum were quantified in vitro. Purified alpha-toxin, heat-inactivated toxin, or alpha-toxin plus normal serum, alpha-toxin anti-serum, or the chemical inhibitor, methyl-ß-cyclodextrin-cholesterol (CD-cholesterol), was injected into the rabbit anterior chamber. Pathological changes were photographed, quantified by slit-lamp examination (SLE) scoring, and further documented by histopathological analysis. RESULTS: At five hours post-injection, eyes injected with alpha-toxin or heat-inactivated toxin had a mean SLE score of 7.3 ± 0.59 or 0.84 ± 0.19, respectively. Active toxin caused moderate to severe iris edema, severe erosion of the iris, and mild to moderate fibrin accumulation in the anterior chamber. Alpha-toxin plus anti-serum or CD-cholesterol, in contrast to alpha-toxin alone, caused less iris edema and epithelium sloughing as well as significantly lower SLE scores than eyes receiving alpha-toxin alone (p ≤ 0.019). CONCLUSION: Alpha-toxin caused extensive iris damage and inflammation, and either anti-alpha-toxin anti-serum or CD-cholesterol was able to significantly reduce toxin-mediated damage and inflammation.

Identification and potency of cyclodextrin-lipid inhibitors of Staphylococcus aureus α-toxin.

PURPOSE: Staphylococcus aureus, a leading cause of bacterial keratitis, secretes α-toxin, a cytotoxin active on the corneal epithelium. This study describes the production and testing of chemical inhibitors of α-toxin action. METHODS: Purified α-toxin was titered by its ability to lyse rabbit erythrocytes in buffered saline (PBS). To prepare potential toxin inhibitors, each of 18 lipids was incorporated into a complex with methyl-ß-cyclodextrin (MßCD) or hydroxypropyl-ß-cyclodextrin (HPßCD). Serial dilutions of each lipid-cyclodextrin (CD-lipid) complex were mixed with α-toxin prior to the addition of rabbit erythrocytes. Select CD-lipid complexes were mixed with 12 hemolytic units (HU) α-toxin and injected into the rabbit corneal stroma so the resulting corneal erosions could be measured at 4 and 8 hours post-injection (PI). Eyes injected with toxin alone, MßCD, or HPßCD alone served as controls. RESULTS: Neither form of CD alone inhibited α-toxin. Of the 36 complexes prepared, 6 lipid-CD complexes were found to inhibit >100 HU of α-toxin. Four lipid complexes able to inhibit >200 HU of α-toxin were tested in toxin-injected corneas; at 4 and 8 hours PI, the complexes of cholesterol or lanosterol with MßCD and squalene or desmosterol with HPßCD caused a significant reduction in the corneal erosion size as compared to eyes injected with α-toxin alone (P ≤ 0.05). CONCLUSIONS: Specific lipid inclusion complexes with either MßCD or HPßCD demonstrated a significant inhibition of α-toxin in both in vitro and in vivo assays. Changes in either the cyclodextrin or lipid of a complex affected the inhibitory activity.

Staphylococcus aureus infection of the rabbit cornea following topical administration.

PURPOSE: To determine the ability of diverse S. aureus strains to infect the rabbit cornea following topical inoculation, with special emphasis on a strain of unusual virulence. MATERIALS AND METHODS: S. aureus strains (5 × 10(5) colony forming units; CFU) were topically applied onto scarified rabbit corneas or 100 CFU were intrastromally injected into rabbit corneas. Eyes were scored by slit lamp examination (SLE) and corneas were cultured to determine the log CFU. Polymorphonuclear leukocytes (PMN) were quantified by myeloperoxidase assays and corneas underwent histopathological analysis. Hemolysin titers of S. aureus strains were determined and S. aureus interactions with rabbit tears or human corneal epithelial cells were investigated. RESULTS: All strains injected into the cornea produced high SLE scores and multi-log increases in CFU. Following topical inoculation, four strains produced low SLE scores with no bacterial replication. One strain (UMCR1) topically infected the cornea, causing high SLE scores, extensive PMN infiltration, and multi-log increases in CFU. Histopathologic analysis demonstrated a PMN influx into the UMCR1-infected cornea, destruction of the corneal epithelium, and severe edema. Strain UMCR1 did not demonstrate a high hemolysin titer or resistance to the bactericidal activity of rabbit tears, but did invade human corneal epithelial cells with relatively high efficiency. CONCLUSIONS: One S. aureus strain demonstrated the ability to topically infect the rabbit cornea. This strain was previously found to be unique in its ability to infect the anterior chamber and conjunctiva, suggesting that a key mechanism may be employed to overcome the host defenses of these three ocular sites.

Diverse virulence of Staphylococcus aureus strains for the conjunctiva.

PURPOSE: To determine the virulence of Staphylococcus aureus strains in the rabbit conjunctiva. METHODS: Three strains of methicillin-sensitive S. aureus (8325-4, Newman, and UMCR1) and two strains of methicillin-resistant S. aureus (70490 and MW2) were analyzed. Rabbit bulbar conjunctivas (n ≥ 6 per group) were injected with 10(5) colony forming units (CFU) in 10 µl. Eyes were photographed and analyzed for pathology at 20 hr postinfection (PI) using slit lamp examination (SLE) to measure five parameters on a scale from 0 (normal) to 4 (severe): injection, chemosis, iritis, corneal edema, and pinpoint conjunctival hemorrhages. The parameter grades were added to produce a SLE score. Bacteria were enumerated and histopathological analysis was done at 20 hr PI. Myeloperoxidase assays were performed on conjunctival swabs (n ≥ 3 per strain) at 0 and 20 hr PI. RESULTS: Conjunctivas injected with 8325-4 or Newman had SLE scores of 1.67 ± 0.12 and 0.81 ± 0.16, respectively. Strain 70490 produced an average SLE score of 2.94 ± 0.47, whereas MW2 produced a score of 5.04 ± 0.73. UMCR1 produced severe conjunctivitis having a SLE score of 13.25 ± 0.80. Only strain UMCR1 grew in the conjunctiva showing a 2.7 log increase in CFU; all other strains remained near the inoculated numbers or decreased as much as 1.85 logs. Myeloperoxidase activity was greatest in the tear film of UMCR1 infected eyes with over one million PMN present at 20 hr PI. CONCLUSIONS: Only one S. aureus strain, UMCR1, was able to cause a reproducible severe conjunctivitis. This conjunctival infection could be used to test new antimicrobials and to help understand the pathogenesis of conjunctivitis, especially in terms of overcoming the host defenses.

Sustained anti-staphylococcal effect of lysostaphin in the rabbit aqueous humor.

PURPOSE: Staphylococcus aureus is an important cause of ocular infections including endophthalmitis. The purpose of this study was to determine the ability of a relatively large molecule, such as lysostaphin, to remain in the rabbit aqueous humor for extended periods while retaining its bactericidal activity. METHODS: Lysostaphin, gatifloxacin, or Tris-buffered saline (TBS) was injected into the rabbit anterior chamber. Aqueous humor was sampled at 0.5, 1, 2, 3, and 4 days after injection, and then assayed for bactericidal activity against S. aureus. The anterior chamber of treated eyes was also challenged 2 days after treatment by an infection of S. aureus. The surviving bacteria were quantified to determine bactericidal effectiveness in the anterior chamber. The aqueous humor of lysostaphin injected eyes was assayed by western blot for the presence of the molecule 2 days post-injection. RESULTS: The bactericidal activity of lysostaphin was confirmed by lysis of S. aureus and sensitivity to zinc. Eyes injected with lysostaphin showed no adverse reactions. Aqueous humor of gatifloxacin injected eyes demonstrated no greater effectiveness than that of TBS injected eyes in vitro at any time point assayed, whereas lysostaphin injected eyes retained potent bactericidal activity for at least 3 days. In an in vivo challenge, the anterior chamber of lysostaphin injected eyes retained significant bactericidal activity for at least 2 days after treatment, whereas gatifloxacin injected eyes demonstrated no significant difference from those injected with TBS. Western blot analysis demonstrated the presence of lysostaphin in the aqueous humor 2 days post-injection. CONCLUSIONS: Lysostaphin demonstrated the ability to remain in the aqueous humor for days while maintaining its bactericidal activity, an indication that a high molecular weight antimicrobial can provide prolonged prophylactic protection of the anterior chamber.

Penetration and effectiveness of prophylactic fluoroquinolones in experimental methicillin-sensitive or methicillin-resistant Staphylococcus aureus anterior chamber infections.

PURPOSE: To determine the effectiveness of moxifloxacin and besifloxacin prophylactic therapy for experimental Staphylococcus aureus infections originating in the rabbit anterior chamber. SETTING: Microbiology Department, University of Mississippi Medical Center, Jackson, Mississippi, USA. DESIGN: Experimental study. METHODS: Minimum inhibitory concentrations (MICs) of moxifloxacin 0.5% and besifloxacin 0.6% for methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) strains were determined. Eyes were treated with moxifloxacin, a moxifloxacin alternative formulation 0.5%, or besifloxacin (45 µL) 30 minutes or 60 minutes before anterior chamber infection (10(6) colony-forming units [CFUs]). Aqueous humor was removed 30 minutes after infection for quantification of antibiotic and bacteria. RESULTS: The MIC for both organisms was 0.06 µg/mL for moxifloxacin and 0.03 µg/mL for besifloxacin. In MSSA infections, the untreated eyes contained 5.18 log CFU/mL, which was similar to besifloxacin-treated eyes with either treatment (P≥.1091). Eyes treated with moxifloxacin or moxifloxacin alternative formulation contained significantly fewer CFUs than untreated controls or besifloxacin-treated eyes with either treatment (P≤.0020). The aqueous humor in eyes treated with moxifloxacin or moxifloxacin alternative formulation contained significantly more drug than besifloxacin-treated eyes at both prophylactic time points (P≤.0012). In MRSA infections, the untreated eyes contained 4.91 log CFU/mL, which was similar to besifloxacin-treated eyes with either treatment (P≥.5830). Eyes treated with moxifloxacin or moxifloxacin alternative formulation contained significantly fewer CFUs than untreated controls or besifloxacin-treated eyes at both prophylactic time points (P≤.0008). CONCLUSIONS: Moxifloxacin had greater in vivo effectiveness against MSSA and MRSA than besifloxacin. The aqueous antibiotic concentrations suggest limited penetration by besifloxacin, accounting for its lack of effectiveness.

The effectiveness of an improved combination therapy for experimental Staphylococcus aureus keratitis.

INTRODUCTION: antibiotic and steroid combination therapies, such as tobramycin with dexamethasone, are often used in ophthalmology to treat or prevent infection and inflammation. The purpose of this study was to use a model of Staphylococcus aureus keratitis to quantify and compare the effectiveness of a standard tobramycin and dexamethasone combined therapy, with each drug individually, and with a new formulation of the two drugs in a xanthan gum vehicle. METHODS: rabbit corneas were intrastromally injected with a methicillin-sensitive S. aureus (MSSA) or a methicillin-resistant S. aureus (MRSA) strain. Rabbit eyes were treated every hour from 10 to 15 hours postinfection (PI) with 0.1% dexamethasone, 0.3% tobramycin, 0.3% tobramycin with 0.1% dexamethasone, or 0.3% tobramycin with 0.05% dexamethasone in a xanthan gum vehicle (ST). Slit lamp examinations (SLE) were performed on infected eyes and pathology scored at 15 hours PI. At 16 hours PI, colony forming units (CFUs) per cornea were quantified. RESULTS: the CFUs in eyes treated with dexamethasone alone were similar to untreated control eyes for MSSA or MRSA infections. All other treatment groups had significantly less CFUs per cornea than untreated eyes. The eyes treated with the ST formulation had significantly fewer CFUs per cornea than all other treatment groups when infected with MSSA or MRSA. The SLE scores of MSSA or MRSA infected eyes treated with tobramycin alone were similar to untreated control eyes. All other treatment groups had significantly lower SLE scores than untreated controls eyes, but were not significantly different from each other. CONCLUSION: the results of this study demonstrated that the tobramycin and dexamethasone combination therapy with a xanthan gum vehicle has an improved bactericidal effectiveness compared to the commercially available formulation, and maintains a similar anti-inflammatory effect while containing half the amount of steroid.

A highly virulent Staphylococcus aureus: rabbit anterior chamber infection, characterization, and genetic analysis.

PURPOSE: To describe and characterize a Staphylococcus aureus strain with unique virulence that overcomes host defenses of the rabbit anterior chamber and mimics clinical cases of postcataract surgery endophthalmitis. METHODS: Nine isolates of S. aureus were tested to determine their viability in the rabbit anterior chamber. Growth of UMCR1 in the anterior chamber was established and expressed as log colony-forming units per milliliter of aqueous humor. Pathologic changes produced by UMCR1 were documented by photographs, slit lamp examination, histopathologic analysis, and quantification of neutrophils. UMCR1 was characterized by antibiotic susceptibility, biochemical tests, ribotyping, genome restriction mapping, and multilocus sequence typing (MLST). RESULTS: UMCR1 was the only S. aureus strain that grew within the anterior chamber, reaching log 6.97 ± 0.18 CFU/mL by 16 hours after infection. Pathologic changes included conjunctival injection, chemosis, corneal edema, severe iritis, fibrin accumulation, and a 193-fold increase in neutrophils by 16 hours after infection. UMCR1 was only resistant to sulfamethoxazole and, like other S. aureus isolates, polymyxin B. UMCR1 also had biochemical reactions and a ribotype pattern typical of S. aureus. The genomic reconstruction analysis of UMCR1 was most similar to strains MW2 and MSSA476. MLST revealed a 1 in 3198 nucleotide difference between UMCR1 and strains MW2 and MSSA476. CONCLUSIONS: This study describes a unique S. aureus strain that overcomes host defenses and replicates in the anterior chamber. The survival and growth of this organism could be used for studies of S. aureus pathogenesis, host defenses, and effectiveness of antibiotics within the anterior chamber.

Chemical inhibition of alpha-toxin, a key corneal virulence factor of Staphylococcus aureus.

PURPOSE: alpha-Toxin mediates extreme corneal damage during Staphylococcus aureus keratitis. Chemical inhibition of this toxin was sought to provide relief from toxin-mediated pathology. METHODS: Inhibition of alpha-toxin by phosphate-buffered saline (PBS), 0.1% methyl-beta-cyclodextrin (CD), or CD plus cholesterol (0.1%, CD-cholesterol) was assayed by hemolysis of rabbit erythrocytes. Pathologic changes in rabbit corneas injected with 12 hemolytic units of alpha-toxin suspended in PBS, 1% CD, or 1% CD-cholesterol were compared over time. Rabbit corneas injected with 10(2) colony forming units (CFU) of S. aureus were treated from 7 to 13 hours postinfection (PI) with a total of 15 drops of CD-cholesterol, CD, or PBS. Slit lamp examination (SLE) and measurement of erosions were performed at 13 hours PI and bacteria were quantified at 14 hours PI. RESULTS: Toxin-mediated lysis of erythrocytes was inhibited up to 16,000-fold in the presence of CD-cholesterol compared with CD or PBS. Eyes injected with alpha-toxin mixed with CD-cholesterol had, at 7 hours postinjection, significantly smaller erosions than eyes injected with alpha-toxin in PBS or alpha-toxin mixed with CD (P = 0.0090 and P = 0.0035, respectively). Eyes infected with S. aureus and treated with CD-cholesterol had significantly lower SLE scores than eyes treated with CD or PBS (P or= 0.0648). CONCLUSIONS: CD-cholesterol is a potent inhibitor of alpha-toxin activity in vitro and an effective means to arrest corneal damage during S. aureus keratitis.