Molecular characterization and resistance profile of nosocomial Acinetobacter baumannii intensive care unit of tertiary care hospital in Bangladesh.

This study was designed to investigate the resistance profile along with the genetic background of resistance to beta-lactam antibiotics among the nosocomial A. baumannii in Bangladesh. A. baumannii was confirmed by detecting blaoXA-51-like. Antibiotic susceptibility was determined by disk diffusion method. Agar dilution method was used to determine MIC of ceftazidime and imipenem. All A. baumannii were phenotypically screened for ampC, ESBL and MBL production. Genetic markers of antibiotic resistance. such as blaampC, blaOXA-51, 23, 40, 58 and 143, blaKPc, blaMp, blavi and blaNDM-j, genetic environment around blaADc and ISAbal upstream of blaoXA, were evaluated by PCR. Twenty-four (96%) A. baumannii were considered as MDR. 96% A. baumanii were resistant to amoxiclav, ceftazidime, ciprofloxacin and cefoxitin, 92% to cefotaxime and piperacillin-tazobactam, 88% to cefepime, amikacin and imipenem, 52% to sulbactam- cefoperazone and 40% were resistant to aztreonam. Everything were sensitive to colistin. The distribution of several beta-lactamase genes such as blaoxa-51 (100%), blaADC-like (92%), blaNDM-i (92%), EBC group (84%), blaoxa-23 (76%), blavm (72%), blacpc (44%), DHA group (24%), blaoxa-58 (16%), ACC group (8%) and CIT group (4%) were observed among the 25 A. baumannii. This is the first reported plasmid mediated ampC beta-lactamases in A. baumannii. blaoxa-51 was positive in 100%, blandm-i in 95.45%, blaoxa-23 in 77.27%, blavim in 72.73%, blakpc in 50% and blaOXA-58 in 18.18% of imipenem resistant isolates. MDR profile of nosocomial A. baumannii would highlight the importance of standard guideline of antimicrobials use and infection control policy in the hospitals of Bangladesh.

Insertion sequence disruption of adeR and ciprofloxacin resistance caused by efflux pumps and gyrA and parC mutations in Acinetobacter baumannii.

Acinetobacter baumannii is a pathogenic bacterium responsible for a wide range of infections in immunocompromised patients. This study examined the role of insertional inactivation of the adeR gene and its effect on adeABC gene expression along with characterisation of the gyrA and parC mutations involved in ciprofloxacin resistance in three A. baumannii clinical isolates and their derivatives. Primers designed for the detection of adeSRABC detected the presence of ISAba16, which disrupted the adeR gene in strain Ab12M, and ISAba1, which disrupted the same gene in strains Ab18 and Ab209. A second copy of ISAba1 was detected upstream of the adeA gene in Ab209 leading to AdeABC pump expression. AdeIJK pump expression was seen in all of the isolates but was not as significant as AdeABC expression. Minimum inhibitory concentrations of ciprofloxacin were ≥256 mg/L for all of the isolates and a decrease of ≥8-fold was seen following addition of the efflux pump inhibitor 1-(1-naphthylmethyl)-piperazine. Fluorometric analysis also demonstrated active efflux, with upregulation of adeIJK and some genes of the adeABC operon in some strains. Sequencing of the quinolone resistance-determining region of the gyrA and parC genes revealed a Ser83→Leu change in the gyrA gene and a novel change of Ser80→Trp in the parC gene of Ab12, Ab12M and Ab209; in Ab18 there was a Ser80→Leu change in parC. This study shows the multifactorial contribution of different mechanisms in A. baumannii leading to ciprofloxacin resistance.

ISAba825 controls the expression of the chromosomal bla(OXA-51-like) and the plasmid borne bla(OXA-58) gene in clinical isolates of Acinetobacter baumannii isolated from the USA.

Four non-repetitive, clonally related (ST114), carbapenem-resistant Acinetobacter baumannii strains isolated in the USA were examined to understand the mechanisms of carbapenem resistance including screening for the presence of an insertion sequence upstream of the bla(OXA-51-like) gene, which could be involved in the control and expression of the antibiotic-resistance gene. We observed that the main mechanisms of carbapenem resistance were the result of the over-expression of the bla(OXA-58-like) and the bla(OXA-65) gene, both of which had the presence of ISAba825 upstream of the genes. The importance of this element was shown by isolating plasmid-cured isogenic strains that had lost the plasmid with the ISAba825-bla(OXA-58-like) genes but during that same process also lost the chromosomal ISAba825 element present upstream of the bla(OXA-65) gene. A 16-fold decrease in minimum inhibitory concentration of imipenem and an eight-fold decrease in the minimum inhibitory concentration of meropenem were seen in the isogenic strains that lost the plasmid. The study presents the first report of ISAba825 simultaneously governing the bla(OXA-65) gene and the bla(OXA-58-like) gene expression and also highlights the importance of this element in carbapenem-sensitive isogenic strains, which were once carbapenem resistant.

Role of ISAba1 and ISAba125 in governing the expression of blaADC in clinically relevant Acinetobacter baumannii strains resistant to cephalosporins.

Acinetobacter baumannii is a multi-resistant opportunistic nosocomial pathogen responsible for several outbreaks worldwide. It can cause several infections at various sites of the body. One of the main infections caused by this bacterium is ventilator-associated pneumonia in patients in intensive care units. Treating these infections is becoming difficult because of the high resistance to antimicrobial agents. This study compared the expression of the chromosomally encoded bla(ADC) gene in isolates having ISAba1, ISAba125 and no insertion upstream of the bla(ADC) gene in A. baumannii clinical isolates. It showed that the expression of bla(ADC) was six times greater when ISAba125 was present upstream of the gene in comparison with the constitutively expressed bla(ADC) gene with no insertion present upstream. The study indicated that ISAba125 has better promoters than ISAba1 and this is responsible for the overexpression of the bla(ADC) gene as they share considerable homology to the well-established Escherichia coli promoters. The -10 box of ISAba125 formed a fusion promoter with the -35 box of the bla(ADC) gene causing the bla(ADC) gene to be significantly overexpressed. The ability to upregulate the expression of bla(ADC) with the assistance of different insertion elements such as ISAba1 and ISAba125 has become an important factor in A. baumannii resistance to cephalosporins.

Effect of frameshift mutagen acriflavine on control of resistance genes in Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative pathogenic bacterium that often exhibits a multidrug-resistant phenotype causing infections at various sites of the body and increasingly leading to septicaemic shock. This study evaluated the role of acriflavine, a frameshift mutagen, on the movement of insertion sequence ISAba1 in clinical isolates of A. baumannii, with the focus on changes in expression levels of the bla(ADC) and bla(OXA-51-like) genes. Resistance profiles were assessed with consideration of ISAba1 acting as a promoter upstream of the bla(ADC) or bla(OXA-51-like) gene. ISAba1 movement was observed in the acriflavine mutants Ab153M and Ab1225M. Ab153M exhibited an increase in the MIC values of carbapenems and ceftazidime, with ISAba1 gained upstream of the bla(ADC) and bla(OXA-51-like) genes, correlating with an increase in gene expression. Reduced expression of the 17, 23 and 25 kDa outer-membrane proteins (OMPs) was also observed in Ab153M. There was a significant decrease in MIC values of carbapenems with the loss of ISAba1 upstream of the bla(ADC) and bla(OXA-51-like) genes in strain Ab1225M, and a significant decrease in bla(OXA-51-like) gene expression and, to a lesser extent, in bla(ADC) expression. Ab1225M and a serially subcultured Ab1225 strain (Ab1225s) exhibited overexpression of the 17, 23, 25 and 27 kDa OMPs. There was a decrease in MIC values of the carbapenems and piperacillin/tazobactam but not of ceftazidime in Ab1225s, which had ISAba1 upstream of the bla(ADC) and bla(OXA-51-like) genes. A significant decrease in bla(OXA-51-like) expression was observed in Ab1225s, whereas the expression of bla(ADC) was similar to that in the Ab1225 parental strain. The attenuation in this strain may be due to overexpression of OMPs and it is clear that, even if ISAba1 is present upstream of an antibiotic resistance gene, it may not necessarily contribute towards the overexpression of antibiotic resistance genes (bla(OXA-51-like) in Ab1225s). Movement of the IS element within the A. baumannii chromosome may be an important regulatory mechanism employed by the bacterium under particular stress conditions, and the ability to upregulate the expression of antibiotic resistance genes is likely to be an important factor in the pathogenicity of this bacterium.

Quantitative chromatin pattern description in Feulgen-stained nuclei as a diagnostic tool to characterize the oligodendroglial and astroglial components in mixed oligo-astrocytomas.

The oligoastrocytoma, as a mixed glioma, represents a nosologic dilemma with respect to precisely defining the oligodendroglial and astroglial phenotypes that constitute the neoplastic cell lineages of these tumors. In this study, cell image analysis with Feulgen-stained nuclei was used to distinguish between oligodendroglial and astrocytic phenotypes in oligodendrogliomas and astrocytomas and then applied to mixed oligoastrocytomas. Quantitative features with respect to chromatin pattern (30 variables) and DNA ploidy (8 variables) were evaluated on Feulgen-stained nuclei in a series of 71 gliomas using computer-assisted microscopy. These included 32 oligodendrogliomas (OLG group: 24 grade II and 8 grade III tumors according to the WHO classification), 32 astrocytomas (AST group: 13 grade II and 19 grade III tumors), and 7 oligoastrocytomas (OLGAST group). Initially, image analysis with multivariate statistical analyses (Discriminant Analysis) could identify each glial tumor group. Highly significant statistical differences were obtained distinguishing the morphonuclear features of oligodendrogliomas from those of astrocytomas, regardless of their histological grade. When compared with the 7 mixed oligoastrocytomas under study, 5 exhibited DNA ploidy and chromatin pattern characteristics similar to grade II oligodendrogliomas, I to grade III oligodendrogliomas, and I to grade II astrocytomas. Using multifactorial statistical analyses (Discriminant Analysis combined with Principal Component Analysis). It was possible to quantify the proportion of "typical" glial cell phenotypes that compose grade II and III oligodendrogliomas and grade II and III astrocytomas in each mixed glioma. Cytometric image analysis may be an important adjunct to routine histopathology for the reproducible identification of neoplasms containing a mixture of oligodendroglial and astrocytic phenotypes.