High-resolution genome profiling differentiated Staphylococcus epidermidis isolated from patients with ocular infections and normal individuals

Purpose: To investigate the potential phenotypic and genetic differences among the Staphylococcus epidermidis isolates obtained from control subjects (lower conjunctival sac; n = 14) with those from patients with keratitis (corneal scrapings; n = 18) or endophthalmitis (vitreous; n = 24). Methods: Biofilm-forming capacity was detected by PCR for the icaAB gene and phenotyping by microtiter plate assay and congo red agar plate. Genotyping was performed by using fluorescence-amplified fragment length polymorphism (FAFLP) and in silico analysis of the FAFLP profiles. Results: Biofilm phenotyping (congo red agar/microtiter plate) differentiated disease-causing strains from control subjects. PCR assays (mecA, icaAB) were not useful in differentiating disease-causing strains from that of control subjects. The biofilm-forming capability appeared more critical in the pathogenesis of keratitis than in that of endophthalmitis. Cluster analysis of FAFLP data generated 11 clusters comprising 4 major clusters (I, II, III, and V) and 7 minor ones. FAFLP analysis clearly showed clustering of most of the commensal isolates in cluster I, separate from keratitis and endophthalmitis isolates. In silico analysis mapped signature bands to genes such as ebh, tagD, ptsI, and sepA, which might have a significant role in transforming less virulent populations of S. epidermidis to more virulent ones. Conclusions: The population dynamics of S. epidermidis revealed that there are significant genetic variations that can be detected through FAFLP between ocular disease causing isolates and the commensal population

Escherichia coli in hospitalised patients

Background Extended spectrum betalactamase (ESBL)-producing organisms are a major cause of hospital-acquired infections. ESBL-producing Escherichia coli (E. coli) have been recovered from the hospital environment. These drug-resistant organisms have also been found to be present in humans as commensals. The present investigation intended to isolate ESBL-producing E. coli from the gut of already infected patients; to date, only a few studies have shown evidence of the gut microflora as a major source of infection. Aims This study aimed to detect the presence of ESBL genes in E.coli that are isolated from the gut of patients who have already been infected with the same organism. Methods A total of 70 nonrepetitive faecal samples were collected from in-patients of our hospital. These in-patients were clinically diagnosed and were culture-positive for ESBL-producing E. coli either from blood, urine, or pus. Standard microbiological methods were used to detect ESBL from clinical and gut isolates. Genes coding for major betalactamase enzymes such as blaCTX-M, blaTEM, and blaSHV were investigated by polymerase chain reaction (PCR). Results ESBL-producing E. coli was isolated from 15 (21 per cent) faecal samples of the 70 samples that were cultured. PCR revealed that out of these 15 isolates, the blaCTX-M gene was found in 13 (86.6 per cent) isolates, the blaTEM was present in 11 (73.3 per cent) isolates, and blaSHV only in eight (53.3 per cent) isolates. All 15 clinical and gut isolates had similar phenotypic characters and eight of the 15 patients had similar pattern of genes (blaTEM, blaCTX-M, and blaSHV) in their clinical and gut isolates. Conclusion Strains with multiple betalactamase genes that colonise the gut of hospitalised patients are a potential threat and it may be a potential source of infection

High-resolution genotyping of Pseudomonas aeruginosa strains linked to acute post cataract surgery endophthalmitis outbreaks in India

BACKGROUND: Investigation of two independent outbreaks of post cataract surgery endophthalmitis identified the reservoir of epidemic strains of P. aeruginosa. METHODS: Patient isolates cultured from vitreous fluid of all the nine cases and from the peripheral devices of phacoemulsification machine were subjected to high-resolution Fluorescent Amplified Fragment Length Polymorphism (FAFLP) analysis. RESULTS: FAFLP based genotyping of the isolates confirmed nosocomial transmission. Although biochemical characterization and antibiotic susceptibility profiles grouped all the isolates together, FAFLP based genotyping revealed that, all the outbreak isolates were derived from 2 different strains, with independent origins. One group of isolates was traced to phacoprobe and the second one to the internal tubing system of the phacoemulsification machine used in cataract surgery. In silico analysis indicated possible evolution in both the clusters of P. aeruginosa isolates due to genetic polymorphisms. The polymorphisms were mapped to gene products (cell envelope, outer membrane proteins) possibly having significant role in pathogenesis. CONCLUSION: The present study is probably the first one to apply FAFLP typing successfully to investigate outbreaks of postoperative endophthalmitis (POE) in an ophthalmic setting, which was able to identify the source, and helped to make rational decisions on sterilization procedures that halted more cases of infection in these hospitals

Fluorescent amplified fragment length polymorphism (FAFLP) genotyping demonstrates the role of biofilm-producing methicillin-resistant periocular Staphylococcus epidermidis strains in postoperative endophthalmitis

BACKGROUND: An observational case series was used to study the virulence characteristics and genotypes of paired Staphylococcus epidermidis isolates cultured from intraocular samples and from periocular environment of patients with postcataract surgery endophthalmitis. METHODS: Eight S. epidermidis isolates were obtained from three patients (2 from patients #1 and 2 and 4 from patient #3) whose vitreous and/or anterior chamber (AC) specimens and preoperative lid/conjunctiva samples were culture positive. Cultures were identified by API-Staph phenotypic identification system and genotypically characterized by Fluorescent Amplified Fragment Length Polymorphism (FAFLP) and checked for their antimicrobial susceptibility. The isolates were tested for biofilm-production and methicillin-resistance (MR) by PCR amplification of icaAB and mecA gene respectively. RESULTS: Four out of eight S. epidermidis strains showed multiple drug resistance (MDR). All the eight strains were PCR positive for mecA gene whereas seven out of eight strains were positive for icaAB genes. In all three patients FAFLP typing established vitreous isolates of S. epidermidis strains to be indistinguishable from the strains isolated from the patient's conjunctival swabs. However, from patient number three there was one isolate (1030b from lid swab), which appeared to be nonpathogenic and ancestral having minor but significant differences from other three strains from the same patient. This strain also lacked icaAB gene. In silico analysis indicated possible evolution of other strains from this strain in the patient. CONCLUSION: Methicillin-resistant biofilm positive S. epidermidis strains colonizing the conjunctiva and eyelid were responsible for postoperative endophthalmitis (POE)

Ag/ZnO/Au 3D hybrid structured reusable SERS substrate as highly sensitive platform for DNA detection

Ultra-sensitive hybrid Silver/Zinc oxide/Gold (Ag/ZnO/Au) structure based three dimensional (3D) surface enhanced Raman scattering (SERS) substrates have been prepared by three step fabrication process using thermal evaporation, hydrothermal growth, and sputtering techniques. The size and inter-particle (IP) gap of decorated Au nanoparticles (NPs) on ZnO nanorods (NRs) in the sub-nanometer range have been achieved through varying the sputtering time of Au. The superhydrophobic nature, the formation of the Schottky barrier at ZnO/Au interface and the broad optical absorption spectrum facilitated towards higher SERS activity of Ag/ZnO/Au hybrid structures. The higher SERS activity of 3D SERS substrate as compared with two dimensional (2D) SERS substrate has been studied. The good SERS signal reproducibility of 3D hybrid structures have been explored through Raman mapping. Higher SERS enhancement factor (EF) of 1 x 10(10) has been achieved with a limit of detection (LOD) up to 10(-16) M and 10 ng/mu L for Rhodamine-6 G (Rh6G) and lambda DNA (lambda-DNA), respectively. The degradation of Rh6G and lambda-DNA molecules have been studied through photocatalytic degradation process to explore the reusability of the SERS substrates up to 10 and 4 times, respectively, with maintaining good SERS signal reproducibility. This metal/semiconductor/metal hybrid structure based SERS substrate with reusable capability indicates potential application towards biosensor for the detection of biologically important molecules at very low concentration level

Annals of Clinical Microbiology and Antimicrobials High-resolution genotyping of Pseudomonas aeruginosa strains linked to acute post cataract surgery endophthalmitis outbreaks in India

Abstract Background: Investigation of two independent outbreaks of post cataract surgery endophthalmitis identified the reservoir of epidemic strains of P. aeruginosa

High-resolution genotyping of <it>Pseudomonas aeruginosa </it>strains linked to acute post cataract surgery endophthalmitis outbreaks in India

Abstract Background Investigation of two independent outbreaks of post cataract surgery endophthalmitis identified the reservoir of epidemic strains of P. aeruginosa. Methods Patient isolates cultured from vitreous fluid of all the nine cases and from the peripheral devices of phacoemulsification machine were subjected to high-resolution Fluorescent Amplified Fragment Length Polymorphism (FAFLP) analysis. Results FAFLP based genotyping of the isolates confirmed nosocomial transmission. Although biochemical characterization and antibiotic susceptibility profiles grouped all the isolates together, FAFLP based genotyping revealed that, all the outbreak isolates were derived from 2 different strains, with independent origins. One group of isolates was traced to phacoprobe and the second one to the internal tubing system of the phacoemulsification machine used in cataract surgery. In silico analysis indicated possible evolution in both the clusters of P. aeruginosa isolates due to genetic polymorphisms. The polymorphisms were mapped to gene products (cell envelope, outer membrane proteins) possibly having significant role in pathogenesis. Conclusion The present study is probably the first one to apply FAFLP typing successfully to investigate outbreaks of postoperative endophthalmitis (POE) in an ophthalmic setting, which was able to identify the source, and helped to make rational decisions on sterilization procedures that halted more cases of infection in these hospitals.</p

Molecular characterization of metallo β-lactamase producing multidrug resistant Pseudomonas aeruginosa from various clinical samples

Introduction: Pseudomonas aeruginosa is a potent opportunistic nosocomial human pathogen among Gram-negative bacteria causing various life-threatening infections in patients from Intensive Care Units. This bacterium has become resistant to almost all commonly available antibiotics with limited treatment options. Multi drug resistant P. aeruginosa (MDRPA) is a major cause of concern among hospital acquired infections. It uses distinctive resistant mechanisms virtually to all the available antibiotics such as Metallo β-lactamases (MBL) production, extended spectrum β-lactamase production (ESBL), up regulation of efflux systems related genes and decreased outer membrane permeability. This study was carried out to find one the predominant resistance mechanisms among MDRPA and the prevalence of corresponding resistance genes. Materials and Methods: MDRPA isolates collected from various clinical samples for a period of 1-year (November 2009-Octo ber 2010) were included to detect the predominant mechanism of resistance using phenotypic and molecular methods. Molecular characterization of all these isolates was done by polymerase chain reaction (PCR) for the presence of blaVIM-2, blaIMP-1, blaOXA-23, and blaNDM-1 genes with specific primers. Results: Among 75 MDRPA isolates 84% (63) were MBL producers. Molecular characterization studied by PCR showed the presence of blaVIM-2 gene in 13% of MBL producers. Conclusion: The prevalence of MBLs has been increasing worldwide, particularly among P. aeruginosa, leading to severe limitations in the therapeutic options for the management. Thus, proper resistance screening measures and appropriate antibiotic policy can be strictly adopted by all the healthcare facility providers to overcome these superbugs