Coexistence of blaNDM-1, blaOXA-51, blaOXA-23, and armA in conjunction with novel mutations detected in RND efflux pump regulators in tigecycline resistant clinical isolates of Acinetobacter baumannii.

This study has investigated a total of 51 Acinetobacter baumannii isolates for the prevalence of resistant determinants in tigecycline susceptible and non-susceptible clinical isolates of A. baumannii. Antimicrobial susceptibility testing revealed 74% of isolates were tigecycline resistant. Mutations in RND-efflux pump regulatory genes and the expression of efflux pump genes were measured in tigecycline resistant isolates. There was a strong co-relation between the blaNDM-1 and armA wherein majority of the isolates that are positive for blaNDM-1 have also harbored armA. Compared with TSAB (tigecycline susceptible A. baumannii), TNAB (tigecycline non-susceptible A. baumannii) isolates show increased distribution of blaNDM-1 (P = 0.048), blaIMP-1 (P< 0.0001) and blaOXA-51 (P = 0.0029) carbapenemase genes. The variants of RND-efflux pump regulatory genes due to amino-acid mutations in adeS (F12S, K84E, W61R, N268H and Q299R) and adeL (G21R and Q262R) were identified in tigecycline resistant isolates as well as ISAba1 mediated disruption of adeN were observed causing overexpression of adeIJK efflux pump. Additionally, mutations in adeRS were also associated with increased expression of adeABC efflux pump. Besides, TNAB isolates showed significantly (P< 0.0001) higher ability of biofilm formation as compared to TSAB isolates. The tigecycline resistance due to mutations in contemporary A. baumannii isolates having a higher ability to form biofilm may pose therapeutic difficulties.

Comprehensive Analysis of Secondary Metabolites in Usnea longissima (Lichenized Ascomycetes, Parmeliaceae) Using UPLC-ESI-QTOF-MS/MS and Pro-Apoptotic Activity of Barbatic Acid.

Considering the importance of ultra-performance liquid chromatography-electrospray ionization-quadrupole time of flight-tandem mass spectrometry (UPLC-ESI-QTOF-MS/MS) hyphenated techniques for analysis of secondary metabolites from crude extracts, the present study was aimed at identification of secondary metabolites in acetone extract of the lichen Usnea longissima. From our study, 19 compounds were tentatively identified through comparison of exact molecular masses from their MS/MS spectra, mass fragmentation studies and comparison with literature data. In addition, potent cytotoxic activity of U. longissima extract prompted us to isolate four compounds, 18R-hydroxy-dihydroalloprotolichesterinic acid (19), neuropogolic acid (20), barbatic acid (21), and usnic acid (22) from this extract which were adequately identified through mass spectrometry and NMR spectroscopy. All four compounds displayed cytotoxic activity. Barbatic acid (21) manifested doxorubicin equivalent activity against A549 lung cancer cell line with IC50 of 1.78 µM and strong G0/G1 accumulation of cells. Poly ADP-ribose polymerase (PARP) cleavage confirmed that it induced cytotoxic activity via apoptosis. Finally, our work has discerned the depside, barbatic acid (21) from crude extract as a candidate anti-cancer molecule, which induces cell death by stepping up apoptosis.

New cycloartane type-triterpenoids from the areal parts of Caragana sukiensis and their biological activities.

A comprehensive re-investigation of aerial parts of Caragana sukiensis resulted in the isolation of twelve compounds (1-12) including three new cycloartane type triterpenoids (3-5) respectively. Chemical structures of the isolated compounds were established by analysis of their IR, HRMSESI, 1D and 2D NMR spectroscopic data. In addition, these compounds were evaluated for their cytotoxic activity against cancer lines (HeLa, A549, MCF-7, DU-145) and Human embryonic kidney cell line (HEK-293). The results indicated that compound 8 showed potent cytotoxic activity against A549 with IC50 value of 1.54 µM which is comparable to standard drug, doxorubicin. Further, flow cytometric analysis showed that compound 8 arrested the cell cycle in the Go/G1 phase leading to apoptotic cell death. In addition, Hoechst 33258 staining, Annexin V-FITC assay and measurement of mitochondrial membrane potential also suggested that 8 induced cell death by apoptosis. Further, all the isolates were also screened for their antifeedant and insecticidal activity against tobacco caterpillar (Spodoptera litura), using no-choice leaf disk method. Among screened compounds 1, 3, 4, and 6 showed potent antifeedancy with ED50 values of 0.59, 1.19, 0.67, and 1.68 µg/cm2. Overall, this study identified a novel class of cycloartane tritepenoids as potent cyotoxic agents as well as antifeedants.

Familiar trespassers in histopathology: An obstacle in diagnosis? A single-blind study.

BACKGROUND: Histopathologists encounter strange structures in tissue sections that appear unrelated to tissues, and these artifacts may be misinterpreted and misdiagnosed as pathological lesions. These substances may either be present within the tissues or can get implanted into tissue during biopsy procedure or laboratory handling or processing. AIMS: The aim of this study is to observe the microscopic appearance of different abnormal structures like commonly implanted food particles or easily incorporated substances during tissue processing with their probable histological misdiagnosis. MATERIALS AND METHODS: Certain food particles, suture materials, wood pieces, insects, and filter paper were intentionally introduced in the tissue specimens of uterus and lung. Following routine processing and hematoxylin and eosin staining, the slides were subjected to single-blind study and viewed under light and polarizing microscope. RESULTS: The vivid appearances of these structures lead to histological misdiagnosis. CONCLUSION: Knowledge and familiarity of these commonly encountered extraneous substances will help to prevent misinterpretation.

Emergence of carbapenem non-susceptible multidrug resistant Acinetobacter baumannii strains of clonal complexes 103(B) and 92(B) harboring OXA-type carbapenemases and metallo-ß-lactamases in Southern India.

The molecular epidemiology and carbapenem resistance mechanisms of clinical isolates of Acinetobacter baumannii obtained from a south Indian tertiary care hospital were investigated by repetitive extragenic palindromic sequence PCR (REP-PCR) and multi-locus sequence typing (MLST). Analysis of resistant determinants was achieved by PCR screening for the presence of genes encoding OXA-carbapenemases, metallo-ß-lactamases (MBLs) and efflux pumps. REP-PCR generated around eight clusters of high heterogeneity; of these, two major clusters (I and V) appeared to be clonal in origin. Analysis of representative isolates from different clusters by MLST revealed that most of the isolates belonged to sequence type 103 of CC103(B) . Second most prevalent ST belonged to clonal complex (CC) 92(B) which is also referred to as international clone II. Most of the isolates were multi-drug resistant, being susceptible only to polymyxin-B and newer quinolones. Class D ß-lactamases such as blaOXA-51-like (100%), blaOXA-23-like (56.8%) and blaOXA-24-like (14.8%) were found to be predominant, followed by a class B ß-lactamase, namely blaIMP-1 (40.7%); none of the isolates had blaOXA-58 like, blaNDM-1 or blaSIM-1 . Genes of efflux-pump adeABC were predominant, most of isolates being biofilm producers that were PCR-positive for autoinducer synthase gene (>94%). Carbapenem non-susceptible isolates were highly diverse and present throughout the hospital irrespective of type of ward or intensive care unit. Although previous reports have documented diverse resistant mechanisms in A. baumannii, production of MBL and OXA-type of carbapenamases were found to be the predominant mechanism(s) of carbapenem resistance identified in strains isolated from Southern India.

Multiple drug resistant carbapenemases producing Acinetobacter baumannii isolates harbours multiple R-plasmids.

BACKGROUND & OBJECTIVES: The nosocomial human pathogen Acinetobacter baumannii has high propensity to develop resistance to antimicrobials and to become multidrug resistant (MDR), consequently complicating the treatment. This study was carried out to investigate the presence of resistant plasmids (R-plasmids) among the clinical isolates of A. baumannii. In addition, the study was performed to check the presence of common ß-lactamases encoding genes on these plasmids. METHODS: A total of 55 clinical isolates of A. baumannii were included in the study and all were subjected to plasmid DNA isolation, followed by PCR to check the presence of resistance gene determinants such as blaOXA-23, blaOXA-51, blaOXA-58 and blaIMP-1 on these plasmids that encode for oxacillinase (OXA) and metallo-ß-lactamase (MBL) type of carbapenemases. Plasmid curing experiments were carried out on selected isolates using ethidium bromide and acridine orange as curing agents and the antibiotic resistance profiles were evaluated before and after curing. RESULTS: All the isolates were identified as A. baumannii by 16SrDNA amplification and sequencing. Plasmid DNA isolated from these isolates showed the occurrence of multiple plasmids with size ranging from 500bp to ≥25 kb. The percentage of blaOXA-51 and blaOXA-23 on plasmids were found to be 78 and 42 per cent, respectively and 20 isolates (36%) carried blaIMP-1 gene on plasmids. Significant difference was observed in the antibiograms of plasmid cured isolates when compared to their parental ones. The clinical isolates became susceptible to more than two antibiotic classes after curing of plasmids indicating plasmid borne resistance. INTERPRETATION & CONCLUSIONS: Our study determined the plasmid mediated resistance mechanisms and occurrence of different resistance genes on various plasmids isolated from MDR A. baumannii. The present findings showed the evidence for antibiotic resistance mediated through multiple plasmids in A. baumannii clinical isolates. This indicates towards a need for preventive measures to avert the dissemination of plasmid resistance determinants in clinical environments.

Changing Trend in the Antibiotic Resistance Pattern of Pseudomonas Aeruginosa Isolated from Wound Swabs of Out-Patients and in-Patients of a Tertiary Care Hospital.

CONTEXT: Pseudomonas aeruginosa is the most common gram negative bacteria associated with nosocomial infections. Active surveillance of trends in antibiotic resistance of P. aeruginosa is necessary for the selection of appropriate antimicrobial agent for empirical therapy. AIM: To assess the rates of antibiotic resistance and multidrug resistance among P. aeruginosa isolates and to observe the trend in its resistance pattern over a period of 5 years. MATERIALS AND METHODS: Pseudomonas aeruginosa isolated from wound swabs during January to June 2007 and January to June 2012 were included in the study. Isolates were identified by conventional tests and antibiotic susceptibility was determined by disc diffusion method according to CLSI guidelines. RESULTS: A total of 307 Pseudomonas aeruginosa isolates were included in the study. Among these isolates, 165 were isolated during Jan-June 2007 and 142 were isolated during Jan-June 2012. Among in-patients, there was a significant reduction in resistance rates of the isolates to ciprofloxacin (49% to 33%), ceftazidime (50% to 33%), meropenem (35% to 19%) and imipenem (28% to 14%) in 2012. Similarly, the rate of MDR Pseudomonas aeruginosa among the in-patients decreased from 37.9% in 2007 to 23.7% in 2012 (p value 0.0241). There was no significant difference in the resistance rates of the isolates from out-patients during the two study periods. CONCLUSION: There was a significant decreasing trend in the resistance rates of the isolates to ciprofloxacin, ceftazidime, meropenem and imipenem. Reduction in the use of ciprofloxacin could be probable reason for the decreased resistance among P. aeruginosa isolates, which needs to be further investigated.

Profile of ocular trauma in industries-related hospital.

INTRODUCTION: Ocular trauma is a worldwide cause of visual morbidity, a significant proportion of which occurs in the industrial workplace and includes a spectrum of simple ocular surface foreign bodies, abrasions to devastating perforating injuries causing blindness. Being preventable is of social and medical concern. AIM: A prospective case series study, to know the profile of ocular trauma at a hospital caters exclusively to factory employees and their families, to co-relate their demographic and clinical profile and to identify the risk factors. MATERIALS AND METHODS: Patients with ocular trauma who presented at ESIC Model hospital, Rajajinagar, Bangalore, from June 2010 to May 2011 were taken a detailed demographic data, nature and cause of injury, time interval between the time of injury and presentation along with any treatment received. Ocular evaluation including visual acuity, anterior and posterior segment findings, intra-ocular pressure and gonio-scopy in closed globe injuries, X-rays for intraocular foreign body, B-scan and CT scan were done. Data analyzed as per the ocular trauma classification group. The rehabilitation undertaken medically or surgically was analyzed. At follow-up, the final best corrected visual acuity was noted. RESULTS: A total of 306 cases of ocular trauma were reported; predominantly in 20-40 year age group (72.2%) and in men (75%). The work place related cases were 50.7%and of these, fall of foreign bodies led the list. Visual prognosis was poorer in road traffic accidents rather than work place injuries owing to higher occurrence of open globe injuries and optic neuropathy. Finally, 11% of injured cases ended up with poor vision. CONCLUSION: Targeting groups most at risk, providing effective eye protection, and developing workplace safety cultures may together reduce occupational eye injuries.

Significance of histopathology in leprosy patients with 1-5 skin lesions with relevance to therapy.

BACKGROUND: Patients with 1-5 skin lesions are clinically categorized as paucibacillary for treatment purposes. For betterment and adequate treatment of patients, this grouping needs further study. AIM: To study a group of leprosy patients with 1-5 skin lesions, compare clinical details with histopathological findings and bacteriological status of the skin to evaluate the relevance of this grouping. MATERIALS AND METHODS: Two-year study involving 31 patients of leprosy with 1-5 skin lesions was included in this study. A number of skin lesions were recorded. Skin biopsies were taken in all patients. The biopsies were evaluated for the type of pathology and acid fast bacilli (AFB) status. RESULTS: Of 31 patients, 19 (61.2%) had single skin lesion, 7 (22.5%) had two lesions, 4 (12.9%) had three lesions, and only one (3.22%) had four lesions, there were no patients with five lesions. Of the 31 patients, 30 (96.7%) were clinically diagnosed as borderline tuberculoid and one patient (3.22%) has tuberculoid leprosy. Skin smears were negative for AFB in all patients. The histological diagnoses were: TT 1 (3.22%), BT 24 (77.41%), and IL 6 (19.2%). AFB were found in 2 (6.45%) out of 31 skin biopsies. Clinicopathological correlation was 76.6% in the BT group. CONCLUSION: Tissue biopsy findings in 1-5 skin lesions which were not considered relevant for treatment purposes until now should be given a status in the categorization and assessment of severity of the disease. The significance of finding of AFB and histopathology of multibacillary (MB) type of leprosy in tissue biopsies, in patients grouped as PB should be resolved so that patients could be given the drug therapy and duration of therapy they warrant.

Clinicopathological evaluation of nonmelanoma skin cancer.

BACKGROUND: Basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), in combination, are referred to as nonmelanoma skin cancers (NMSCs). NMSC is not as extensively studied in the Asian population as it is in the Caucasian population. AIM: This study sought to evaluate the clinical and histopathologic aspects of NMSC from cases of cutaneous malignancies. MATERIALS AND METHODS: The present study is a descriptive analysis of NMSC specimens seen at Department of Pathology, SSIMS and RC, Davangere. Histologically diagnosed NMSC, i.e. BCC and SCC specimens from January 2005 to December 2009 were analyzed according to site distribution, risk factors and histological variants. RESULTS: Of the various specimens received during the 5year study period, 60 were histologically categorized as skin malignancies, of which 31(51.6%) cases were of NMSC. SCC was the most common NMSC constituting 26 (83.9%) cases and 5 NMSC cases (16.1%) were of BCC. The most common incidence was among the age group 60-80 years (80%) for BCC and 40-60 years (50%) for SCC. Head and neck was the most common site of presentation with predilection for face. Forty-six percent of SCC was histologically categorized as well differentiated, 42.3% as moderately differentiated and 11.5% as poorly differentiated. Most common histological variant of BCC was solid (nodular) type. CONCLUSION: NMSC often associated with greater morbidity, necessitating increased efforts to assess risk factors in individuals, to encourage periodic self-examination and professional evaluation of skin and to optimize strategies for earlier diagnosis and treatment.

Correlations between genotyping and antibiograms of clinical isolates of Pseudomonas aeruginosa from three different south Indian hospitals.

PURPOSE: To compare the molecular relationships and antibiograms of nosocomial isolates of Pseudomonas aeruginosa obtained from three different genres of hospitals located in Southern India, two located at Hyderabad (one private hospital and an ophthalmic hospital) and one in Puducherry (tertiary care teaching hospital). Each of these hospitals, which follow different infection control strategies and various problems associated with it, were investigated. MATERIALS AND METHODS: Antibiograms generated by disk diffusion susceptibility testing for clinically relevant antibiotics and genotyping through fluorescent amplified fragment length polymorphism analysis (fAFLP) were the tools used in the study. RESULTS: Molecular genotyping revealed a heterogeneous group of unrelated molecular clusters of P. aeruginosa strains having higher resistance that are apparently being endemic throughout the tertiary care teaching hospital. In eye care hospital, only a few distinct strains of P. aeruginosa predominating the study period were shown to be responsible for outbreaks. The third private hospital witnessed a group of resistant and persistent strains that might have clonally originated from a diverse collection of strains. CONCLUSIONS: The divergent kind of strains in our study suggests that there may be a direct link between the infection control practices followed in each hospital and kind of strains isolated in that particular setup. The study also emphasizes the need for maintaining infection control practices in hospitals with superior standards, failure of which might result in thriving of persistent P. aeruginosa clones in the hospitals.

Phenotypic and genotypic assays for detecting the prevalence of metallo-beta-lactamases in clinical isolates of Acinetobacter baumannii from a South Indian tertiary care hospital.

Nosocomial infections caused by Acinetobacter baumannii often prove difficult to treat owing to their multiple drug resistance. Carbapenems play a pivotal role in the management of severe Acinetobacter infections. However, reports of carbapenem resistance have been increasing alarmingly due to production of a variety of carbapenemases including metallo-beta-lactamases (MBLs). This study investigated by both phenotypic and genotypic assays the prevalence of MBLs in a total of 55 A. baumannii strains isolated from a South Indian tertiary care hospital. Random amplified polymorphic DNA (RAPD) genotyping and antimicrobial susceptibility testing for nine clinically relevant antibiotics was done for characterization of isolates. Phenotypic expression of MBLs was examined by a simple double disc synergy (DDS) test, and the presence of the most frequent MBL coding genes, bla(IMP1) and bla(VIM2), was checked by PCR. RAPD analysis generated six clusters of isolates and there was very little correlation between RAPD clusters and resistant profiles. Most of the isolates showed complete or high resistance to imipenem (100 %), meropenem (89 %), amikacin (80 %), cefotaxime (89 %) and ciprofloxacin (72 %). In addition, 44 % of isolates showed a high MIC level (> or =16 microg ml(-1)) for meropenem. Thirty-nine isolates (70.9 %) were positive for MBL production by the DDS test while bla(IMP1) gene amplification was seen only in 23 isolates (42 %). Interestingly, none of the isolates showed amplification of bla(VIM2). Further investigations on DDS-positive/PCR-negative isolates by spectrophotometric assay showed MBL activity in most of the isolates, suggesting involvement of other genes. The high incidence of isolates possessing MBL activity in the present study represents an emerging threat of complete resistance to carbapenems among Acinetobacter spp. in India.

Correlation between biofilm production and multiple drug resistance in imipenem resistant clinical isolates of Acinetobacter baumannii.

PURPOSE: To study the qualitative and quantitative methods for the investigation of biofilm formation and to examine the correlation between biofilm and antibiotic resistance among the clinical isolates of Acinetobacter baumannii . We also verified the association between biofilm and presence of extended spectrum beta-lactamases, particularly, bla PER-1 . METHODS: A total of 55 isolates were subjected to susceptibility testing by disc diffusion method for 13 clinically relevant antibiotics. Screening for biofilm production was done by both qualitative and quantitative methods through tube and microtitre plate assay respectively. The presence of bla PER-1 was checked by PCR. RESULTS: A. baumannii isolates showed very high resistance (>75%) to imipenem, cephotaxime, amikacin and ciprofloxacin. Only cefoperazone, netillin and norfloxacin were found to be effective agents. Results of microtitre and tube methods were concordant with 34 isolates (62%) showing biofilm formation. Resistance to four antibiotics such as amikacin (82% vs. 17.6%, P < 0.001), cephotaxime (88% vs. 11%, P P < 0.001), ciprofloxacin (70% vs. 29%, P =0.005) and aztreonam (38% vs. 11%, P =0.039) was comparatively higher among biofilm producers than non-biofilm producers. Microtitre assay additionally detected 14 weakly adherent isolates. Only 11 isolates had bla PER-1 gene and among these two were strong biofilm producers, while remaining were weakly adherent isolates. CONCLUSION: Microtitre plate method was found to be a more sensitive method for biofilm detection. This study demonstrates a high propensity among the clinical isolates of A. baumannii to form biofilm and a significant association of biofilms with multiple drug resistance. Presence of bla PER-1 appears to be more critical for cell adherence than for biofilm formation.

Correlation of endometrial ultrasonography and endometrial histopathology in patients with postmenopausal bleeding.

A retrospective study of 49 cases with persistent postmenopausal vaginal bleeding undergoing hysterectomy in the absence of postoperatively evident diagnosis of genital malignancy was carried out. Normal endometrium was the source of bleeding in 24 cases (48.9%) while 11 cases (22.4%) had evidence of endometritis. Endometrial hyperplasia and polyps were present in 12 cases (24.4%). Two cases were found to have malignancy in the surgical specimen, which were not evident on pre-operative endometrial biopsy. However, there was high degree of suspicion of malignancy in these 2 cases based on the endometrial histopathology and ultrasonographic endometrial thickness. By careful correlation of clinical findings, endometrial histopathology and ultrasound measurement of endometrial thickness, most cases with postmenopausal bleeding can be managed conservatively.

Malignant chondroid syringoma.

An adult female presented with a painful recurrent swelling on the posterior aspect of the scalp of six months' duration, which was clinically diagnosed as fibroma. An excision biopsy showed anaplastic epithelial cells arranged in irregular cords and trabeculae, mitosis, malignant chondroid areas, desmoplasia and perineural invasion, suggesting malignancy. Wide excision of the tumor was the only treatment given since there was no metastasis.

Germ cell tumors of the ovary: is there a role for aggressive cytoreductive surgery for nondysgerminomatous tumors?

Thirty-three patients with germ cell tumor of the ovary were seen at Kidwai Memorial Institute of Oncology (KMIO), Bangalore, between 1996 and 1999. Twelve patients had endodermal sinus tumor (EST), 11 dysgerminoma, seven mixed germ cell tumor, and three immature teratoma. Thirteen patients had bulky residual disease of >10 cm after the primary surgery. All but one patient received a combination of bleomycin, etoposide, and cisplatin (BEP) either as neoadjuvant (NACT, 3 cases) or as adjuvant therapy (28 cases). In the present study, all 11 patients with dysgerminoma achieved sustained complete remission (CR), irrespective of the size of residual disease at the time of chemotherapy. Four out of six cases (66.6%) with bulky nondysgerminomatous tumor achieved CR, which was sustained in three cases and one recurred. Fifteen of the remaining 16 (93.7%) nonbulky, nondysgerminomatous tumors achieved CR, which was sustained in 14 cases and recurred in one. This study indicates that there may be a role for aggressive cytoreductive surgery, either primary/interval or at the time of second-look laparotomy, in selected patients with nondysgerminomatous germ cell tumor of the ovary.

Bronchioloalveolar carcinoma mimicking miliary tuberculosis.

A case of bronchioloalveolar carcinoma, with widespread dissemination to both lungs and miliary mottling on chest X-ray is reported in a 40 year old male.

Chediak-Higashi syndrome.

A case of Chediak-Higashi syndrome is reported in a four-year-old boy who presented with recurrent chest infection, partial albinism, hyperpigmentation of the extremities and presence of giant granules in leucocytes and melanocytes in the skin. Parental consanguinity was present. Though uncommon, hyperpigmentation of sun exposed areas may be the initial symptom in Chediak-Higashi syndrome.