Despite low incidence, POE remains a serious complication [1
]. All patients included in this study required pars plana vitrectomy with intraocular antibiotics and corticosteroid injection. One patient required therapeutic penetrating keratoplasty with intraocular lens explantation after nine days of treatment. While intraocular amikacin and vancomycin with dexamethasone were administered at the time of presentation in all patients, two patients required repeat injections of ceftazidime and dexamethasone. All patients were given ciprofloxacin eye drops topically along with mydriatics and prednisolone acetate eye drops. Six patients were also treated with oral ciprofloxacin and corticosteroid. The outcome was satisfactory in five of our patients and it was poor in remaining four patients.
Outbreaks of P. aeruginosa
POE most likely have an exogenous origin, as they are not normal commensal on skin and conjunctiva [1
]. Understanding the relative importance of the routes of colonization is crucial for the development of effective preventive remedies against P. aeruginosa
POE. The source of infection and routes of contamination are important issues in any hospital [2
]. In the present study, a reservoir source was suspected in each of these independent outbreaks. Following a thorough sampling of operating room environment and equipment, P. aeruginosa
were isolated from phacoprobe at the first hospital and from the internal tubing of phacoemulsification surgical equipment in the second hospital. All the isolates obtained from the clinical and environmental sources from both the outbreaks showed identical antibiotic susceptibility patterns, implying a single source for both the outbreaks, which was most unlikely given the distance in time of occurrence and separate physical location of the hospitals. On the other hand, FAFLP analysis clearly showed that there were two independent sources involved. Molecular typing methods are necessary for confirming the source of an outbreak [1
]. In the present study, FAFLP conclusively identified the isolate from surgery equipment to be the source for infection in both the outbreaks. Nosocomial contamination and acquisition was proven in this study. Clustering of the isolates was close enough to explain their clonal expansion in the respective hospital settings. Distinct identity of the two strain types involved in the outbreaks was obvious with 18% genetic difference between them. This was highly significant with additional distinctive phenotypic characteristics like green metallic sheen and mucoid colonies, notwithstanding the similarity in antibiotic susceptibility.
DNA typing methods have emerged as more practical and reliable option for the investigation of outbreaks. AFLP analysis has been reported to provide sufficient discriminatory power comparable to the PFGE for the investigation of P. aeruginosa
]. Our study provides additional proof for the above observations. Minor variations are more frequent in P. aeruginosa
isolates with abundant mutations and recombination that contemplates wide spectrum of microevolution in a shorter duration [5
]. These variations were more evident in our study wherein only 6 to 8 fragment difference was observed within the isolates of different amplitypes. Distribution of fragments among different isolates within each cluster (Amplitype A & B) did not vary significantly.
Comparative in silico
analysis with predictive annotation of sequenced PA01 strain showed lack of amplification of the 6 to 12 genomic regions in many isolates belonging to both the clusters (L1445, L1447, L1449, L1450, L1455, OT3, L1130, L1147 and OT1) and this might be certainly due to the mutation (indels or point mutation) in the DNA region bearing EcoRI
restriction sites. Genetic polymorphisms mapped to genes coding for probable porin gene (PA4137), probable ATP-binding component of ABC transporter (PA4909) and COG functional prediction of ORFs PA1091 and PA3350 as glycerophosphate transferase involved in teichoic acid biosynthesis and flagellar based body P-ring biosynthesis protein respectively, appears to be significant as all these four gene products are localized in cell envelope and outer membrane of the organism [12
]. In addition, COG prediction of ORF PA4203 as probable lysR transcriptional regulator, is known to be very similar to the periplasmic binding proteins. Modification of these genes in above-mentioned isolates might possibly indicate enhanced ability of these isolates to cause POE through alterations of their outer membrane porin and efflux proteins [13
Genetic polymorphisms mapped to ORFs PA4137, PA4909 and PA2228 seem to be very important. PA4137 is 52% similar to the oprE gene product of P. aeruginosa
that is homologous to OprD, which is known to be involved in permeability of imipenem and basic amino acids [14
]. PA4909 is 74% homologous to braG gene product of P
, which is cell membrane protein bearing ATP- binding domains [16
]. COG prediction of PA2228 as AmpC, beta-lactamase class C family of protein is usually associated with beta lactam resistance in P. aeruginosa
]. Lack of amplification of these three regions in the closely related clones of amplitype A and B appears to be due to mutation under different stress conditions prevailing in the host. Further studies are clearly needed to ascertain functional role of these polymorphisms in ocular infection.