K Prashanth

Phenotypic and genotypic assays for detecting the prevalence of metallo-β-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.

The expanding spectrum of human infections caused by Kocuria species: a case report and literature review

Although not previously known to cause human infections, Kocuria species have now emerged as human pathogens, mostly in compromised hosts with severe underlying disease. Recently, there has been an increasing incidence of different types of Kocuria infections reported, most likely due to the adoption of better identification methods. Here, we report a case of peritonitis caused by Kocuria rosea in a diabetic nephropathy patient who was on continuous ambulatory peritoneal dialysis. Sepsis and peritonitis caused by K. rosea in our case yielded two identical Kocuria isolates from the peritoneal dialysate fluid within a period of three days. The infection was subsequently resolved by antibiotic treatment and catheter removal. In addition to reporting this case, we herein review the literature concerning the emergence of Kocuria as a significant human pathogen. The majority of cases were device-related, acquired in the hospital or endogenous, and different Kocuria species appear to share a common etiology of peritonitis. The overall disease burden associated with Kocuria appears to be high, and the treatment guidelines for diseases associated with Kocuria have not yet been clearly defined.Although not previously known to cause human infections, Kocuria species have now emerged as human pathogens, mostly in compromised hosts with severe underlying disease. Recently, there has been an increasing incidence of different types of Kocuria infections reported, most likely due to the adoption of better identification methods. Here, we report a case of peritonitis caused by Kocuria rosea in a diabetic nephropathy patient who was on continuous ambulatory peritoneal dialysis. Sepsis and peritonitis caused by K. rosea in our case yielded two identical Kocuria isolates from the peritoneal dialysate fluid within a period of three days. The infection was subsequently resolved by antibiotic treatment and catheter removal. In addition to reporting this case, we herein review the literature concerning the emergence of Kocuria as a significant human pathogen. The majority of cases were device-related, acquired in the hospital or endogenous, and different Kocuria species appear to share a common etiology of peritonitis. The overall disease burden associated with Kocuria appears to be high, and the treatment guidelines for diseases associated with Kocuria have not yet been clearly defined.

The quorum sensing molecule N-acyl homoserine lactone produced by Acinetobacter baumannii displays antibacterial and anticancer properties

Abstract Secretory N-acyl homoserine lactones (AHLs) mediate quorum sensing (QS) in bacteria. AHLs are shown to be inhibitory for an unrelated group of bacteria and might mimic host signalling elements, thereby subverting the regulatory events in host cells. This study investigated the AHL produced by Acinetobacter baumannii and analysed its effect on other bacterial species and mammalian cells. Chemically characterized AHL had an m/z value of 325 with a molecular formula C18H31NO4 and showed its inhibitory potential against Staphylococcus aureus. Molecular docking studies identified D-alanine-D-alanine synthetase A, a cell wall synthesizing enzyme of S. aureus having a strong binding affinity towards AHL. Electron microscopy showed the disruption and sloughing off of the S. aureus cell wall when treated with AHL. In vitro experiments revealed that this bacteriostatic AHL showed time-dependent activity and induced apoptosis in cancer cell lines. This compound could be a potential structural backbone for constructing new AHL analogues against S. aureus. The findings emphasize the need to re-evaluate all previously characterized AHLs for any additional new biological functions other than QS.

Emergence of carbapenem non‐susceptible multidrug resistant Acinetobacter baumannii strains of clonal complexes 103B and 92B harboring OXA‐type carbapenemases and metallo‐β‐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‐β‐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 CC103B. Second most prevalent ST belonged to clonal complex (CC) 92B 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 β‐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 β‐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.

Disruption of tetR type regulator adeN by mobile genetic element confers elevated virulence in Acinetobacter baumannii

ABSTRACT Acinetobacter baumannii is an important human pathogen and considered as a major threat due to its extreme drug resistance. In this study, the genome of a hyper-virulent MDR strain PKAB07 of A. baumannii isolated from an Indian patient was sequenced and analyzed to understand its mechanisms of virulence, resistance and evolution. Comparative genome analysis of PKAB07 revealed virulence and resistance related genes scattered throughout the genome, instead of being organized as an island, indicating the highly mosaic nature of the genome. Many intermittent horizontal gene transfer events, insertion sequence (IS) element insertions identified were augmenting resistance machinery and elevating the SNP densities in A. baumannii eventually aiding in their swift evolution. ISAba1, the most widely distributed insertion sequence in A. baumannii was found in multiple sites in PKAB07. Out of many ISAba1 insertions, we identified novel insertions in 9 different genes wherein insertional inactivation of adeN (tetR type regulator) was significant. To assess the significance of this disruption in A. baumannii, adeN mutant and complement strains were constructed in A. baumannii ATCC 17978 strain and studied. Biofilm levels were abrogated in the adeN knockout when compared with the wild type and complemented strain of adeN knockout. Virulence of the adeN knockout mutant strain was observed to be high, which was validated by in vitro experiments and Galleria mellonella infection model. The overexpression of adeJ, a major component of AdeIJK efflux pump observed in adeN knockout strain could be the possible reason for the elevated virulence in adeN mutant and PKB07 strain. Knocking out of adeN in ATCC strain led to increased resistance and virulence at par with the PKAB07. Disruption of tetR type regulator adeN by ISAba1 consequently has led to elevated virulence in this pathogen.

Draft Genome Sequence of a Multidrug-Resistant Acinetobacter baumannii PKAB07 Clinical Strain from India Belonging to Sequence Type 195

ABSTRACT Acinetobacter baumannii has emerged as one of the most common nosocomial pathogens and is considered to be a significant threat to public health worldwide. Here, we present the draft genome sequence of a multidrug-resistant clinical strain of A. baumannii PKAB07 isolated from a wound infection in India during 2011 to 2012.

Simultaneous gut colonisation and infection by ESBL-producing 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 non-repetitive 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 bla CTX-M , bla TEM, and bla SHV 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 bla CTX-M gene was found in 13 (86.6 per cent) isolates, the bla TEM was present in 11 (73.3 per cent) isolates, and bla SHV 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 (bla TEM, bla CTX-M, and bla SHV) 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.

Antibiotic Resistance, Biofilms and Quorum Sensing in Acinetobacter Species

Acinetobacter is a Gram-negative coccobacillus that is strictly aerobic, nonmotile, catalase positive and oxidase negative. It is ubiquitous in nature, being found in soil and water. Members of the genus Acinetobacter have now clearly emerged as opportunistic nosocomial pathogens (Forster et al., 1998). Bacteremia, pneumonia, meningitis, urinary tract and surgical wound infections are the most common infections caused by this organism (Cisneros et al., 2002; Dijkshoorn et al., 2007). The taxonomy of the genus Acinetobacter has undergone extensive revision during the last two decades, and at least 31 named and unnamed species have now been described (Dijkshoorn et al., 2007). Of these, Acinetobacter baumannii and the closely related unnamed genomic species 3 and 13 sensu Tjernberg and Ursing (13TU) species were the most clinically relevant. In recent years, multidrug-resistant (MDR) A. baumannii are increasingly held responsible for nosocomial infections and MDR A. baumannii clones are spreading into new geographic areas with increasing number of strains acquiring many resistance genes (Navon venezia et al., 2005). Unfortunately, newer extended-spectrum ┚-lactamases and different carbapenemases are emerging fast, leading to pan-resistant strains of A. baumannii.

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.

Simple and economical method for identification and speciation of Staphylococcus epidermidis and other coagulase negative Staphylococci and its validation by molecular methods

Coagulase-negative staphylococci (CoNS) have been increasingly recognized as a clinically important group of species that can cause several opportunistic nosocomial infections. There are at least 47 known species of Staphylococci and to differentiate all these species >40 biochemical tests need to be performed. The present study was able to refine the CoNS identification process by using only five tests to identify S. epidermidis from the rest and used six other tests to identify eleven other clinically significant CoNS species. A total of 242 CoNS isolates were collected from tertiary care hospitals and included in the study. The five-biochemical test scheme devised based on mathematical probability derived from a computer algorithm included fermentation of mannitol, maltose, mannose, trehalose and novobiocin susceptibility to differentiate S. epidermidis from other CoNS species. The remaining CoNS isolates other than S. epidermidis were further characterized with the help of six additional tests, which identified another eleven species. Species-specific PCR and 16SrDNA sequencing were used to confirm and validate the identification scheme. Species-specific PCR and 16SrDNA sequencing showed 100% agreement with non-divergent phenotypic test results, indicating that the five selected assays are highly specific for identifying S. epidermidis. In conclusion, this study used only 11 tests to identify most of the clinically significant CoNS that can reduce cost and time. This scheme is easy to perform in any laboratory with basic resources, the results of this study were validated using more accurate molecular methods such as PCR and 16S rDNA typing to confirm the utility of the proposed scheme.