Molecular characterization of metallo ß-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 ß-lactamases (MBL) production, extended spectrum ß-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.

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.

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).

Fluorescent amplified fragment length polymorphism (FAFLP) based molecular epidemiology of hospital infections in a tertiary care setting in Hyderabad, India.

Bacterial isolates from respiratory and urinary tract infections in an Indian hospital setting were genotyped using FAFLP analysis. The 77 different isolates analyzed belonged to five genera namely Escherichia, Staphylococcus, Pseudomonas, Enterobacter and Pantoea. Before carrying out FAFLP analysis all the isolates were subjected to16S-23S ribosomal RNA-based species identification. Cluster analysis of FAFLP profiles of 77 isolates generated five groups corresponding to five bacterial genera that are used in the study. Further analyses of the dendrograms revealed efficient species and strain differentiation. Cluster analysis identified genetically distant clones among the clinical isolates of Staphylococcus aureus, two distinct genetic lineages among the Escherichia coli strains and a single cluster of closely related Pseudomonas aeruginosa isolates. Ribosomal spacer region amplification identified different species accurately but intraspecies discrimination could not be accomplished completely. Comparison of FAFLP profiles of our isolates, with a pilot database of validated strains, was very useful in identification and worked better in conjunction with dendrogram analysis.

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.

Rescue of TNFalpha-inhibited neuronal cells by IGF-1 involves Akt and c-Jun N-terminal kinases.

Proinflammatory cytokines, especially tumor necrosis factor alpha (TNFalpha), is a pleiotropic mediator of a diverse array of physiologic and neurologic functions and is upregulated during various inflammatory and neurodegenerative diseases. A common survival response during such situations is the increased expression of the hormone insulin-like growth factor 1 (IGF-1). Although it was thought previously that the mechanisms of TNFalpha and IGF-1 action were unrelated, it has been shown that low doses of TNFalpha can inhibit the survival effects of IGF-1 in mouse cerebellar granule neurons. We used a neuronal cell line SH-SY5Y, which underwent apoptosis in response to TNFalpha and this process could be reversed substantially by IGF-1. Crosstalk between signaling pathways of these two factors was found at various points downstream of their signal transduction. To determine the mechanisms of IGF-1-mediated rescue, we looked at the MAP kinases, which are known to be involved in IGF-1 as well as TNFalpha signaling. The c-Jun N-terminal kinase pathway, which is known normally to promote cell death, was found to actually promote survival of TNFalpha-mediated cell death. Inhibiting the c-Jun survival pathway completely reversed the rescue mediated by IGF-1. In addition, the Akt pathway played an equally important role in this rescue.

Simplified panel of assimilation tests for identification of Acinetobacter species.

A total of 66 Acinetobacter isolates obtained from JIPMER hospital wards were subjected to phenotypic identification schemes involving 25-test and a simplified 13-test panel of carbon utilization or assimilation tests. Reference strains belonging to different DNA groups (n=24) were also tested. Identification was done using numerical approach based on a matrix constructed of phenotypic data published elsewhere and the strains were assigned to different DNA groups according to classification of Tjernberg & Ursing. Sixty-six strains tested represented 10 DNA groups in matrix of large test panel; at a probability level of 0.95. Much simplified scheme of 13 assimilation test panel failed to differentiate some isolates with in A. calcoaceticus-A. baumannii complex (Acb-complex) unlike extended panel. In all, from the large panel 95% of isolates were identified correctly among all the isolates and it did not identify 5% of isolates. From the small panel, a total of 89% of isolates were identified correctly and it could not identify 11% of isolates. Reduced number of assimilation tests to 13 from the large panel bought reduction in identification percentage rate by only 6%. It is impossible for many bacterial diagnostic labs worldwide to perform large panel of carbon utilization tests in routine practice. Simplified panel of assimilation tests suggested here seems to be the best alternative method for identification of Acinetobacter species.