Genetic diversity and antimicrobial susceptibility profile of multidrug-resistant strains of Acinetobacter calcoaceticus-A. baumannii complex in a teaching hospital

Abstract

Healthcare Associated Infections (HAIs) due to Acinetobacter calcoaceticus-A. baumannii Complex (ACB) are a major public health problem, impacting on morbidity, mortality, length of stay and costs associated with patient care. These pathogens accumulate diverse mechanisms of resistance to antimicrobials, which contributes to their maintenance in the hospital environment. It has been reported in national and worldwide literature a significant increase in rates of resistance to antimicrobial agents in Acinetobacter, including carbapenems, one of the last antimicrobial classes with satisfactory activity against this agent. Such resistance is mainly mediated by oxacilinase enzymes, which currently present more than 150 variants. The drugs tigecycline, polymyxin B, colistin, minocycline, ampicillin-sulbactam, sulbactam, and tetracycline are alternatives to the treatment of ACB infections, because their activities are still preserved, which raises the monitoring of this susceptibility profile. The definition of the origin of circulating strains is a requirement for action to control and contain the spread of bacteria resistant to antibiotics. For that, molecular techniques will be an important tool for monitoring and for epidemiological research.In 2008, it was first verified the presence of a peculiar multi-drug resistance A. baumannii that presented resistance to carbapenems and the remaining drugs (except gentamicin, tobramycin and polimixin B). The index case was identified as a transferred patient from a hospital in a city near to the hospital we are investigating. The new resistance phenotype spread over the hospital and it is detected in A. baumannii even nowadays.Thus, the aim of this project is to evaluate the genetic diversity and antimicrobial susceptibility profile of multidrug-resistant strains of the ACB Complex circulating in a teaching hospital in Brazil from 2007 to 2014. We will study all the strains of the ACB Complex available isolated from bloodstream infection, from different patients, attended at a university hospital from 2007 to 2014, totalling 134 strains. The genes of the major oxacilinases (OXA-23, OXA-24, OXA-51, OXA-58, OXA-143) will be searched by a multiplex PCR reaction. In negative samples for OXA/ISAba1, we will search for other regulatory sequences of ISAba type. blaOXA-51 subtypes will be determined. To appoint the genetic diversity among the isolates, the technique of pulsed field gel electrophoresis (PFGE) will be used (Seifert et al, 2005). Also, the ancestry and global distribution of pulsotypes will be determined by multilocus analysis sequence typing (MLST) (Pasteur Institute and Bartual scheme, Oxford University). Sequences will be submitted to comparative analysis with those available in the database provided by these two institutions, by using the software BioNumerics 7.5 (Applied Maths). Also, we will perform the 3-locus typing (3-LST) of A. baumannii pulsotypes (Turton et al, 2007) to elucidate the major circulating clonal groups by variants of ompA gene (encodes the outer membrane porin A), EUSC (involved in biofilm formation) and blaOXA-51-like (A. baumannii intrinsic carbapenemase encoder). The susceptibility profile to the drugs tigecycline, polymyxin B, colistin, minocycline, ampicillin-sulbactam, sulbactam and tetracycline will be evaluated by agar dilution and/or broth microdilution techniques.To characterize the genetic context of oxacilinases detected in this study, approaches such as cloning of resistance genes, or inverse PCR, will be used to identify the genes present both upstream and downstream of the resistance genes, and determine whether the resistance genes are present on transposons. Moreover, if we verify that several genes are allocated in plasmids, plasmid replicase genes will be identified to determine the types of plasmid present in the isolates (To be executed in collaboration with Dr. Benjamin Evans, Anglia Ruskin University). (AU)