1. Antimicrobial resistance in gramnegative bacteria of medical interest..
Antimicrobial resistance is a current major health problem. A large proportion of the research activities in our group is related to the study of genetic and biochemical aspects of the mechanisms of resistance to antimicrobial agents (with emphasis on beta-lactams, quinolones and aminoglycosides) in gram-negative bacteria of medical importance. We consider both Enterobacteria (Escherichia coli, Klebsiella pneumoniae, Enterobacter spp., etc.) and non-fermenters (Pseudomonas aeruginosa, Actinetobacter baumannii, Stenotromophomonas maltophilia, Burkholderia cepacia complex, among others). Within this problem, we are particularly focused on multiresistance and low-level resistance. The clinical importance of multiresistant bacteria is capital, as there are quite few new agents under development aimed to fight against these pathogens. Our group is contributing to the “Resistance Program” of the Spanish network for research on infection diseases (Red Española de Investigación en RESEARCH AREAS Infection and Immunity Area Patología Infecciosa, REIPI, http://reipi.org/) supported by the Institute of Health Carlos III (ISCIII). This network has developed and is developing different multicenter studies on clinical and microbiological aspects of infections caused by resistant bacteria of clinical relevance, a major problem in many Spanish hospitals. Our collaboration on the study of the molecular bases of resistance has allowed obtaining new information on infections caused by Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii. Our group is also participating in another REIPI research program related to infections in transplanted patients. Our objectives include characterization of genes and mobile elements (plasmids, transposons, integrons, gene cassettes, etc.) involved in antimicrobial resistance. Our studies have contributed to the discovery of new beta-lactamases (i.e., the new oxacillinase OXA-207 in Acinetobacter pittii -a microorganism related to A. baumannii, which is also important as a nosocomial pathogen). Beta-lactams continue to be very frequently prescribed antibacterials. Among them, carbapenems (very broad-spectrum beta-lactams) are often considered the preferred therapeutic option against infections caused by different resistant gram-negative pathogens. Unfortunately, acquired resistance to carbapenems has been well documented, and is increasingly recognized among different gram-negative species. This is due very frequently to the production of enzymes (carbapenemases) hydrolyzing these agents. We are applying phenotypic and genotypic methods for study and characterization of carbapenem-resistance caused by metallo-beta-lactamases (MBL; including IMP-, VIM-, SPM- and NDM-types), or serine beta-lactamases of Ambler classes A and D. We are also interested in the characterization of these enzymes in environmental organisms, such as Pseudomonas putida and other taxonomically-related species such as P. monteilii, which represent reservoirs of multiresistance genes (including genes coding for MBL). We have reported nosocomial infections caused by strains of the later organisms producing the VIM-2 MBL in severely ill/immunocompromised patients. Fluoroquinolones are broad-spectrum antimicrobial agents. They are clinically useful for treating infections caused by (among others) gram-positive and gram-negative organisms. Because they are synthetic compounds, it was thought that transferable resistance was extremely unlike, as there are (presumably) not environmental producer organisms, which could represent a source of resistance genes. However, multiple variants of qnr (quinolone resistance) genes have been identified worldwide in a great variety of bacteria (particularly in gram-negative organisms). We were involved in the discovery of qnr genes, and the group is still active in the analysis of these genes and others causing plasmid-mediated quinolone resistance. The study of aminoglycoside-resistance in gram-negative bacteria (both Enterobacteria and non-fermenters) is another objective of our group. We are particularly interested in (1) aminoglycoside-modifying enzymes [N-acetyltransferases (AAC), O-phosphotransferases (APH) and O-nucleotidyltransferases (ANT)] which interferes with the ability of the corresponding substrate to interact with the ribosome, impairing its activity, and (2) ARN 16S methyl-transferases, which cause methylation at certain positions of the ribosome and cause high-level resistance to many clinically used aminoglycosides. Genes coding for these enzymes are frequently included in plasmid, which favors their dissemination among different bacterial hosts. An additional objective for us is the evaluation of the role of active efflux pumps in the intrinsic resistance of gram-negative bacteria to clinically relevant antimicrobial agents (including beta-lactams, quinolones, aminoglycosides and other families). Similarly, the group has great experience on the study of the importance of porins of enterobacteria (mainly K. pneumoniae, E. coli, Enterobacter spp.) in low-level resistance and in the evaluation of the regulation of the gene coding for the OprD porin of P. aeruginosa, involved in carbapenem resistance. Finally, in the context of the previously detailed information on mechanisms of antimicrobial resistance, we are also considering:
- Analysis of the relationship between antimicrobial resistance and virulence.
- Study of the molecular epidemiology of resistant bacteria, in particular on the problem of multiresistance in nosocomial infections.
- Implementation of massive sequencing technology for a better understanding of both resistance and virulence.
2. Mechanisms of pathogenicity in gram-negative bacteria of medical interest. . We are interested in deciphering the molecular me - chanisms implicated in the infections caused by Pseudomonas aeruginosa and Burkholderia cepacia. These bacteria are able to release huge quantities of hydrolytic enzymes inside host cells by means of complex secretion systems (TSS) partially inserted into the bacterial membrane. One of these systems is the type VI secretion system (T6SS), first identified in 2006. The T6SS is a one step mechanism that is used widely throughout gram-ne - gative bacterial species in injecting effector proteins and virulence factors from across the cytoplasm of a bacterial cell into a target cell (both bacterial and eukaryotic cells). Protein secretion is a key issue in bacterial pathoge - nesis and many of the proteins secreted by the cu - rrently known T6SS are also implicated in bacterial adherence and invasion, as well as growth and intra - cellular survival inside macrophages. We focus on new T6SS effector proteins of P. aerugi - nosa and several species of the B. cepacia complex. On the other hand, it is well known that the T6SS of P. aeruginosa is also implicated in biofilm formation and antimicrobial resistance. A biofilm can be defined as a structured community of bacterial cells enclosed in a self-produced polymeric matrix and adherent to an inert or living surface. Growth in biofilms enhan - ces the survival of bacterial populations in hospital environments and during host infections (i.e., in the presence of antibiotics), increasing the probability of causing nosocomial infections. Our group is also implicated in the discovery and characterization of quorum sensing regulators that may play a role in biofilm formation and dispersion. We use genomic and transcriptomic tools to understand the molecu - lar determinants that contribute to the virulence and antimicrobial resistance in these pathogens.
3. Host-Pathogen Interactions.
Acinetobacter spp. have become major pathogens in hospital-associated infections, especially in critical care settings such as intensive care units (ICUs). They can survive in the hospital environment for long periods and have a remarkable propensity to develop resistan - ce to multiple classes of antibiotics. This antibiotic re - sistance trend is a serious concern given the prospect of a further reduction in therapeutic options for infec - tions by these multi-drug-resistant bacteria. While the epidemiology and antibiotic resistance of the species A. baumannii have been extensively studied, the molecu - lar and genetic basis of A. baumannii, A. nosocomialis and A. pittii virulence remains poorly understood, and there is still lack of knowledge in host cell response to these bacteria. Answering the need for studies of the mechanisms involved in the antimicrobials and host-pathogen (H-P) relationship, we seek to develop a multi-disciplinary research on host-pathogen inte - ractions in these clinically relevant Acinetobacter spe - cies by using cell sorting, advanced microscopy, qPCR arrays, and, more recently, next generation sequencing. Our main objectives are:
1) To develop new tools for the study of host-pa - thogen interactions in Acinetobacter.
2) To study the impact of different antibiotics at subMICs on Acinetobacter species, biofilms, and on relevant host-pathogen interactions in vitro.
3) To unravel the dynamics and roles of vesi - cle-like droplets produced by Acinetobacter species on biofilms and during host-pathogen interactions.
4) To perform a detailed analysis of the immune responses of human immune and non-immune cells against Acinetobacter strains with diffe - rent phenotypes.
4. Antimicrobial Resistance mechanisms in gran-positive bacteria.
We currently face multiresistant infectious disease or - ganisms that are difficult and, sometimes, impossible to treat successfully. Multiresistance or multiple drug resistance (MDR) is often associated with mobile ge - netic elements implicated in horizontal gene transfer. This occurs both in gram-negative and gram-positive bacteria. For gram-positive bacteria, we focus our activity on ba - sic plasmid biology as well as in factors that allow the appearance and spread of multidrug resistant bacteria. Our model is Enterococcus, mainly E. faecalis and E. faecium. The most common nosocomial infections pro - duced by these organisms are urinary tract infections, endocarditis and bacteriemia. We study horizontal gene transfer (conjugation) of diverse mobile genetic elements such as the sex pheromone plasmids (present in 95% of clinical isolates of E. faecalis associated with hospital outbreaks with a transference rate near 100%). Also, we are studying the mechanisms of gene expres - sion regulation (activation or repression) in these sex pheromone plasmids, as the mechanisms that coordi - nately regulate basic plasmid processes (replication, partition, conjugation). We also perform the identification and characterization of plasmids present in multirresistant E. faecium clini - cal isolates carrying vancomycin resistant genes (and also resistance to aminoglycosides and macrolides).
5. Infections caused by coryneform bacteria.
We focus on antimicrobial resistance in several spe - cies of corynebacteria. These bacteria are widely dis - tributed in nature, and are found in the soil, water and on the skin of humans and animals. Several species cause disease in humans. The most important is C. diphteriae, but C. amycolatum, C. urealyticum, C. jeikeium and C. striatum are also oc - casionally cultured from clinical samples. In the last years, we have studied several aspects related to coryneform bacteria: antimicrobial resistance me - chanisms, their spread and their interactions with the host.
6. Diagnostic Methodology and Epidemiology
Our team is also implicated in the development and application of new diagnostic methods in clinical mi - crobiology, using genomic and proteomic techniques and nanotechnology. Molecular infectious disease testing has become an outstanding arm of our Service of Microbiology, in - cluding sequencing or direct detection of genes re - lated to microbial identification and resistance me - chanisms. For over 100 years, infectious diseases agents have been identified after they were growth in culture and their phenotypic traits were considered. In the mole - cular era, there is an opportunity to detect pathogens more rapidly and accurately based on their genetic signatures. Molecular methods, essentially those based upon the polymerase chain reaction (PCR), have become an indispensable tool in the diagnosis of infectious diseases. Over the past decade, there has been an explosion in the use of molecular tests to diagnose and manage infectious diseases. As a result, the Clinical Microbiology laboratory of the HUMV offers a growing and consolidated number of nucleic acid amplification tests (NAATs) for detec - tion and identification of bacterial, viral and fungal pathogens. A good example is the expertise we have acquired in gene amplification and sequencing of the 16S rDNA gene and of genes related with antibiotic resistance. We understand that, given the complexity of the mi - crobial world and the increasing sophistication of NAATs, the expertise is required not only for assay development and performance, but also for consul - tation. This rational improves patient care, reduces antibiotic usage, enhances test utilization, and in - creases laboratory and hospital efficiency. Recently, we have also incorporated an automated mass spectrometry microbial identification system that uses Matrix Assisted Laser Desorption Ioniza - tion Time-of-Flight (MALDI-TOF) technology and a comprehensive database of clinically relevant spe - cies for results in minutes. On the other side, it is sometimes important to analyze multiple isolates within a given species to determine whether they represent a single strain or multiple strains. The process of differentiating strains based on their phenotypic and genotypic di - fferences is known as ‘typing’. Genotyping methods involve the study of the microbial DNA. The develo - 189 Activity report 2015 188 Valdecilla Biomedical Research Institute Santander, Spain pment of molecular genotyping methods has revolu - tionized the possibility for classification of microor - ganisms at the sub-species level, which is crucial for deciding the molecular relatedness of isolates for epidemiological studies. In this aim, new PCR-based typing methods in the last years have supposed an important advance in determining the molecular epi - demiology of microorganisms. Major advantages of these methods are flexibility, technical simplicity and high discriminatory power. Although most of these PCR-based typing methods are less time-consuming and easy to perform and of interpretation, other approaches, however, depending on the species stu - died, are required to the study of the clonal relations - hip among microbial isolates. These include the pul - sed-field gel electrophoresis (PFGE, the current gold standard method for typing most bacterium and fun - gi), multilocus sequence typing (MLST), and variable number tandem repeat typing (VNRT). These typing methods are also performed in diffe - rent specific areas of our Clinical laboratory, and are useful in hospital infection control, epidemiological studies, and understanding the pathogenesis of in - fection.
7- in vitro activity of new antimicrobials.
Different organizations, including the WHO, have alerted that antimicrobial resistance is one of the main health problems in the world. One of the hi - gh-priority strategies to confront this menace is the development of new antimicrobial agents. Our group participates in multicenter studies pursuing the eva - luation of new antimicrobials, challenging a wide co - llection of bacterial clinical isolates. Also, we participate in the evaluation of reference automated methods that incorporate new antimi - crobial agents. In the last years, we have studied the activity of daptomycin, linezolid, chelocardin and cef - taroline, collaborating to establish MIC breakpoints and the interpretation of in vitro susceptibility tests to define clinical categories. Our group has also collaborated in the study of the variables that influence the in vitro activity of cef - taroline, a new broad-spectrum cephalosporin with activity against methicillin resistant Staphylococcus aureus (MRSA). We have also contributed to the in vitro study of a new antimicrobial, chelocardin, whose synthesis has been improved by genetic engineering and with promising in vitro activity against several species of multidrug resistant bacteria. These studies have been developed in collaboration with members of the University of Cantabria and the University of Lu - bljana (Slovenia).