Emerging ciprofloxacin-resistant Pseudomonas aeruginosa

PURPOSE: To report a clinical series of ciprofloxacin-resistant ocular isolates of Pseudomonas aeruginosa from a tertiary care ophthalmic center. METHODS: Review of in vitro sensitivities of all ocular isolates of P. aeruginosa be tween July 1991 and September 1998. In vitro resistance was defined as a minimum inhibitory concentration of 4 or more microg per ml. RESULTS: Nine of 423 ocular isolates of P. aeruginosa showed in vitro resistance to ciprofloxacin. From 1991 to 1994, 0.44% (1/227) of ocular isolates were resistant to ciprofloxacin, whereas from 1995 to 1998, 4.1% (8/ 196) of ocular isolates showed in vitro resistance (P = .014). CONCLUSIONS: Ciprofloxacin-resistant P. aeruginosa has been identified in recent clinical ocular specimens. Ciprofloxacin resistance among ocular isolates of P. aeruginosa is a local and worldwide concern.     

Overview of mechanisms of antibiotic resistance in Pseudomonas aeruginosa: an ocular perspective

Treatment of Pseudomonas aeruginosa eye infections often becomes a challenge due to the ability of this bacterium to be resistant to antibiotics via intrinsic and acquired mechanisms. Transfer of resistance due to interchangeable genetic elements is an important mechanism for the rapid transfer of antibiotic resistance in this pathogen. As a result, drug‐resistant strains are becoming increasingly prevalent worldwide. This review systematically analyses data from recent publications to describe the global prevalence and antibiotic sensitivity of ocular P. aeruginosa. Thirty‐seven studies were selected for review from PubMed‐based searches using the criteria ‘microbial keratitis OR eye infection AND Pseudomonas aeruginosa AND antibiotic resistance’ and limiting to papers from 2011 onward, to demonstrate the antibiotic resistance from isolates from around the world. Subsequently, we reviewed the ways in which P. aeruginosa can become resistant to antibiotics. Both the rate of isolation of bacteria in general (79 per cent of cases), and prevalence of P. aeruginosa (68 per cent of all isolates) were highest in contact lens‐related microbial keratitis. The average resistance rate to common ocular antibiotics such as ciprofloxacin (9 per cent), gentamicin (22 per cent) and ceftazidime (13 per cent) remained relatively low. However, there were large variations in resistance rates reported in studies from different countries, for example resistance to ciprofloxacin reached up to 33 per cent. We next reviewed the types of mobile genetic elements (MGEs) such as plasmids, integrons and transposons that are frequently associated with drug resistance in P. aeruginosa. MGEs are important for the transmission of resistance to beta‐lactams and aminoglycosides and recently have been shown to be potential factors for the transmission of fluoroquinolone resistance. Studies on the molecular mechanisms of resistance transfer in ocular P. aeruginosa have begun to be reported and will provide valuable information on the emergence of new antibiotic resistance and potential to treat resistant strains.     

Pseudomonas aeruginosa exotoxin A and keratitis in mice

PURPOSE: To determine the importance of Pseudomonas aeruginosa exotoxin A (ETA) as a virulence factor in corneal disease. METHODS: Isogenic mutants deficient in ETA were constructed in P. aeruginosa strains PAO1 and ATCC 19660 by allelic exchange and then evaluated for virulence in a mouse model of bacterial keratitis. The effect of ETA on adherence to scarified corneal epithelium was assessed in an in vitro organ culture model. RESULTS: Mutants of either P. aeruginosa PAO1 or 19660, deficient in ETA, adhered to wounded corneal tissue and initiated ocular disease similar to that in wild-type strains. However, in contrast to wild-type strains, ETA mutants were quickly cleared from the eye, inflammation diminished, and the cornea healed. CONCLUSIONS: Although ETA has no effect on the ability of P. aeruginosa to adhere to corneal wounds or to initiate Pseudomonas keratitis, it is crucial for the organism to persist in the eye and ultimately cause disease.     

Molecular analysis of Pseudomonas aeruginosa protease IV expressed in Pseudomonas putida

PURPOSE: In this study, the protease IV gene of Pseudomonas aeruginosa was expressed in the nonocular pathogenic host, Pseudomonas putida, to elucidate the molecular properties and virulence contribution of the enzyme. Recent determination of the protease IV gene sequence suggests that the protein of 463 amino acids contains a signal sequence, a propeptide domain, and a mature protease. The only form of this protein that has been detected previously is the extracellular mature protease. METHODS: The protease IV gene was cloned and expressed in a protease IV-negative Pseudomonas species, P. putida. The cloned protease IV gene product was analyzed to identify biochemical, enzymatic, and immunologic properties and its contribution to corneal virulence. RESULTS: P. putida expressing the cloned protease IV gene had significantly greater extracellular enzyme activity than P. aeruginosa. These P. putida cell extracts produced a protein with the same molecular mass as mature protease IV and two other polypeptides representing larger precursors, all of which were recognized by protease IV-specific antibodies. P. putida producing protease IV, relative to P. putida with the vector alone, caused a threefold increase in ocular inflammation and tissue damage when intrastromally injected into rabbit corneas. CONCLUSIONS: The present study demonstrates for the first time that protease IV is synthesized as a large precursor that is processed intracellularly through an intermediate form and secreted into the extracellular milieu as a mature protease. The results also confirm a significant correlation between production of protease IV and corneal virulence.     

Contamination of contact-lens-related sources with Pseudomonas aeruginosa

The studies conducted sought to estimate contamination of contact lens (CL) storage cases and commercial solutions with Pseudomonas aeruginosa as well as to investigate everyday sources, which the CL wearer uses or comes into contact with. The studies revealed that 13. 1% of storage lens cases and 5.1% of commercial solutions in use were positive for the bacterium. Ten hermetically sealed CL solutions were tested for possible contamination, but these all proved to be sterile. In addition, environmental samples, of both a solid and liquid nature, yielded relatively high contamination rates with this opportunistic bacterium. Results showed that common sources, which the CL wearer is in contact with, possess P. aeruginosa and this may indeed predispose to corneal infections.     

Type III secretion system-associated toxins, proteases, serotypes, and antibiotic resistance of Pseudomonas aeruginosa isolates associated with keratitis

The association between possession of toxin gene-related type III secretory system, protease profiles, O serotypes, and antibiotic resistance patterns was characterized genetically and phenotypically in 46 keratitis isolates of Pseudomonas aeruginosa. There was no significant difference in exoU or exoS prevalence among the keratitis strains. Distinct protease profiles were seen in isolates harboring either exoU or exoS genes. One hundred percent (13/13) of serotype E (O:11) strains contained type III secretion system-associated cytotoxin gene exoU. Multidrug resistance was identified in 4% of Australian and 29% of Indian isolates. None of the Australian isolates was resistant to ciprofloxacin. In general, the rate of multidrug resistance in the exoU positive cytotoxic and serotype E (O:11) strains was significantly higher than in exoS positive invasive strains (p < 0.01). The results suggest that multidrug resistance may be more commonly associated with the corneal isolates of P. aeruginosa having type III secretion system-associated cytotoxin gene exoU and belonging to serotype E (O:11) group.     

Pseudomonas aeruginosa Protease IV: A Corneal Virulence Factor of Low Immunogenicity

Purpose: To study antibody production to Pseudomonas aeruginosa protease IV (PIV) for immunoassay development and to assess the possible role of antibody in arresting corneal damage. Methods: Rabbits were immunized with PIV, urea-soluble recombinant PIV (rPIV), or precipitated rPIV. Antibody was analyzed by ELISA and Western blotting. Antibody-mediated inhibition of PIV activity was tested by colorimetric assay and during keratitis by slit-lamp examination of infected eyes. Results: Antibody was not produced after PIV immunization but was induced by rPIV. Rabbits immunized first with soluble and then precipitated rPIV produced high titers (log₁₀) to rPIV (4.28 ± 0.09) and significantly higher titers to PIV (3.90 ± 0.06) compared to the other immunized groups. Antibody to rPIV reacted with PIV, but neither neutralized enzyme activity in vitro nor protected infected rabbits in vivo. Conclusions: The present study demonstrates that PIV is a virulence factor which can escape a protective immune response.     

Ciprofloxacin susceptibility of Pseudomonas aeruginosa isolates from keratitis

AIM: To examine the ciprofloxacin susceptibility of 106 Pseudomonas aeruginosa eye isolates from the United Kingdom, Denmark, India, the United States, and Australia, and to determine the molecular mechanisms of resistance. METHODS: Ciprofloxacin susceptibility was tested by an agar dilution method; genomic DNA corresponding to the quinolone target genes gyrA and parC, and the regulatory genes mexR and nfxB controlling drug efflux systems, was amplified by PCR and sequenced; multilocus enzyme electrophoresis was performed to examine the genetic relation among resistant strains. RESULTS: Three out of 90 keratitis isolates (3.3%), one from the United Kingdom and two from India, exhibited MIC values of 16 mg/l or 32 mg/l. The UK isolate had a mutation in gyrA (Thr83Ile), whereas the two Indian isolates showed mutations in both gyrA (Thr83Ile) and parC (Ser87Leu). The remaining isolates from keratitis, endophthalmitis, contact lens associated red eye (CLARE), and contact lens storage cases showed MIC values below 1 mg/l. Several allelic forms of gyrA and a single variation in the mexR gene product were detected in 10 ciprofloxacin susceptible strains. CONCLUSIONS: The vast majority of eye isolates of P aeruginosa from European countries are fully susceptible to ciprofloxacin and the concentration of ciprofloxacin eye drops used for local treatment (3000 mg/l) exceeds MIC values for strains recorded as resistant. Mutations in more than one target gene were associated with higher MIC values.     

Adhesion of Pseudomonas aeruginosa ocular isolates to mucin

The purpose of this study was to compare the adhesion of Pseudomonas aeruginosa ocular isolates to mucin. An adhesion assay was developed using biotin‐labelled P. aeruginosa strains (two corneal ulcer, two acute red eye, one asymptomatic and one standard strains) incubated with porcine gastric mucin immobilized on a nitrocellulose membrane. The adhesion was semiquantified using densitometry. The results showed that all P. aeruginosa strains tested were able to adhere to mucin to various extents with three strains (one corneal ulcer, one acute red eye, one asymptomatic) binding significantly greater than the negative control (P < 0.1). Results suggest that ocular strains of P. aeruginosa strains differ in their adhesion to mucin but this did not correlate with the pathogenic origin of the strain. It is concluded that the adhesion of P. aeruginosa strains to mucin alone may not be a principal determinant of pathogenesis but may be a contributing factor along with other bacterial virulence traits.     

Survival of Pseudomonas aeruginosa in M-K preserved corneas

AIM: To present seven eyes of suspected donor to host transmitted Pseudomonas sp corneal graft infection after corneal and scleral graft leading to corneal melting within 24 hours, in a span of 10 months. METHODS: Case series. Seven eyes, operated for either penetrating or lamellar keratoplasty or scleral patch graft for different indications and which developed massive corneal/corneoscleral infection within 24 hours, were studied prospectively. RESULTS: Pseudomonas aeruginosa, resistant to almost all antibiotics except polymyxin B in all and vancomycin in two, was identified as the causative organism from all the specimens obtained from the infected graft. CONCLUSION: Post-keratoplasty infection is a disaster. The source of early infection is invariably iatrogenic. Use of empirical antibiotics in the media is not always sufficient to prevent such infection. Thus, measures must be taken in the form of strict maintenance of asepsis and revision of antibiotics added to the storage medium. Further, early recognition and energetic therapy for such infection could reduce the ophthalmic morbidity.     

Resistance of Pseudomonas aeruginosa to modern eye-banking technique

Rabbit eyes were inoculated with different concentrations of Pseudomonas aeruginosa. Copious irrigation and topical Neosporin solution decreased surface contamination by 90% as compared to controls. Ps. aeruginosa added to M-K medium and stored at 4 degree C remained viable for 6 days and multiplied rapidly when the medium was incubated at 35 degree C. Adding penicillin and streptomycin (125 units/ml each) suppressed, but did not prevent this growth. Gentamicin (100 micrograms/ml) suppressed growth in all vials by 72 hours at 4 degree C. These studies show that gentamicin is clearly superior to penicillin and streptomycin in preventing Ps. aeruginosa contamination of donor corneas. It is suggested that donor eyes and M-K media be routinely cultured upon arrival at the eye-bank and the time of surgery in order to avoid or immediately treat gentamicin-resistant donor cornea contamination.