María Tomás

Antimicrobial Resistance and Virulence: a Successful or Deleterious Association in the Bacterial World?

SUMMARY Hosts and bacteria have coevolved over millions of years, during which pathogenic bacteria have modified their virulence mechanisms to adapt to host defense systems. Although the spread of pathogens has been hindered by the discovery and widespread use of antimicrobial agents, antimicrobial resistance has increased globally. The emergence of resistant bacteria has accelerated in recent years, mainly as a result of increased selective pressure. However, although antimicrobial resistance and bacterial virulence have developed on different timescales, they share some common characteristics. This review considers how bacterial virulence and fitness are affected by antibiotic resistance and also how the relationship between virulence and resistance is affected by different genetic mechanisms (e.g., coselection and compensatory mutations) and by the most prevalent global responses. The interplay between these factors and the associated biological costs depend on four main factors: the bacterial species involved, virulence and resistance mechanisms, the ecological niche, and the host. The development of new strategies involving new antimicrobials or nonantimicrobial compounds and of novel diagnostic methods that focus on high-risk clones and rapid tests to detect virulence markers may help to resolve the increasing problem of the association between virulence and resistance, which is becoming more beneficial for pathogenic bacteria.

Quorum quenching quandary: resistance to antivirulence compounds.

Quorum sensing (QS) is the regulation of gene expression in response to the concentration of small signal molecules, and its inactivation has been suggested to have great potential to attenuate microbial virulence. It is assumed that unlike antimicrobials, inhibition of QS should cause less Darwinian selection pressure for bacterial resistance. Using the opportunistic pathogen Pseudomonas aeruginosa, we demonstrate here that bacterial resistance arises rapidly to the best-characterized compound that inhibits QS (brominated furanone C-30) due to mutations that increase the efflux of C-30. Critically, the C-30-resistant mutant mexR was more pathogenic to Caenorhabditis elegans in the presence of C-30, and the same mutation arises in bacteria responsible for chronic cystic fibrosis infections. Therefore, bacteria may evolve resistance to many new pharmaceuticals thought impervious to resistance.

Identification and Broad Dissemination of the CTX-M-14 β-Lactamase in Different Escherichia coli Strains in the Northwest Area of Spain

ABSTRACT During the course of a molecular epidemiology study of mechanisms of antibiotic resistance in the area served by our hospital (516,000 inhabitants), we isolated the gene encoding CTX-M-14 β-lactamase. Thirty clinical strains (27 Escherichia coli and 3 Klebsiella pneumoniae isolates) with a phenotype of extended-spectrum β-lactamase were collected from January to October 2001 and studied for the presence of the CTX-M-14 β- lactamase gene. By isoelectric point determination, PCR, and nucleotide sequencing, we detected the presence of this gene in 17 E. coli strains belonging to 15 different genotypes (REP-PCR) causing infections in 17 different patients. Epidemiological studies based on medical records did not suggest any relationship between the patients infected with these E. coli strains and, interestingly, 7 of 30 patients harboring strains with extended-spectrum β-lactamases never had contact with the hospital environment before the clinical E. coli isolation. Conjugation experiments revealed that this gene was plasmid mediated in the 17 E. coli strains, and plasmid restriction fragment length polymorphisms showed 9 different patterns in the 17 E. coli strains. By PCR, the sequence of the tnpA transposase gene of the insert sequence ISEcp-1 was detected in all the plasmids harboring the CTX-M-14 gene. These results strongly suggest that plasmid dissemination between different E. coli strains in addition to a mobile element (transposon) around the β-lactamase gene may be involved in the spreading of the CTX-M-14 gene. This study reinforces the hypothesis that the epidemiology of the prevalence of the β-lactamase genes is changing and should alert the medical community to the increase in the emergence of the CTX-M β-lactamases worldwide.

Efflux Pumps, OprD Porin, AmpC β-Lactamase, and Multiresistance in Pseudomonas aeruginosa Isolates from Cystic Fibrosis Patients

ABSTRACT Expression of ampC, oprD, mexA, mexC, mexE, and mexX was studied in 25 Pseudomonas aeruginosa isolates from cystic fibrosis patients, including 14 isolates of the Liverpool epidemic strain. Overexpressed mexA or ampC and reduced oprD were associated with β-lactam resistance. A specific combination of mexR, nalC, and nalD mutations occurred in 11 Liverpool strain isolates, including 7 with upregulated mexA.

Cloning and Functional Analysis of the Gene Encoding the 33- to 36-Kilodalton Outer Membrane Protein Associated with Carbapenem Resistance in Acinetobacter baumannii

ABSTRACT We investigated a multiresistant strain of Acinetobacter baumannii isolated in our hospital. Analysis of the N-terminal peptide sequence of the outer membrane proteins (OMPs) purified from the strain allowed us to clone and sequence the nucleotides of the gene encoding the 33- to 36-kDa OMP associated with carbapenem resistance in A. baumannii

High-Level Resistance to Ceftazidime Conferred by a Novel Enzyme, CTX-M-32, Derived from CTX-M-1 through a Single Asp240-Gly Substitution

ABSTRACT A clinical strain of Escherichia coli isolated from pleural liquid with high levels of resistance to cefotaxime, ceftazidime, and aztreonam harbors a novel CTX-M gene (blaCTX-M-32) whose amino acid sequence differs from that of CTX-M-1 by a single Asp240-Gly substitution. Moreover, by site-directed mutagenesis we demonstrated that this replacement is a key event in ceftazidime hydrolysis

Contribution of Efflux Pumps, Porins, and β-Lactamases to Multidrug Resistance in Clinical Isolates of Acinetobacter baumannii

ABSTRACT We investigated the mechanisms of resistance to carbapenems, aminoglycosides, glycylcyclines, tetracyclines, and quinolones in 90 multiresistant clinical strains of Acinetobacter baumannii isolated from two genetically unrelated A. baumannii clones: clone PFGE-ROC-1 (53 strains producing the OXA-58 β-lactamase enzyme and 18 strains with the OXA-24 β-lactamase) and clone PFGE-HUI-1 (19 strains susceptible to carbapenems). We used real-time reverse transcriptase PCR to correlate antimicrobial resistance (MICs) with expression of genes encoding chromosomal β-lactamases (AmpC and OXA-51), porins (OmpA, CarO, Omp33, Dcap-like, OprB, Omp25, OprC, OprD, and OmpW), and proteins integral to six efflux systems (AdeABC, AdeIJK, AdeFGH, CraA, AbeM, and AmvA). Overexpression of the AdeABC system (level of expression relative to that by A. baumannii ATCC 17978, 30- to 45-fold) was significantly associated with resistance to tigecycline, minocycline, and gentamicin and other biological functions. However, hyperexpression of the AdeIJK efflux pump (level of expression relative to that by A. baumannii ATCC 17978, 8- to 10-fold) was significantly associated only with resistance to tigecycline and minocycline (to which the TetB efflux system also contributed). TetB and TetA(39) efflux pumps were detected in clinical strains and were associated with resistance to tetracyclines and doxycycline. The absence of the AdeABC system and the lack of expression of other mechanisms suggest that tigecycline-resistant strains of the PFGE-HUI-1 clone may be associated with a novel resistance-nodulation-cell efflux pump (decreased MICs in the presence of the inhibitor Phe-Arg β-naphthylamide dihydrochloride) and the TetA(39) system.

Whole Transcriptome Analysis of Acinetobacter baumannii Assessed by RNA-Sequencing Reveals Different mRNA Expression Profiles in Biofilm Compared to Planktonic Cells

Acinetobacter baumannii has emerged as a dangerous opportunistic pathogen, with many strains able to form biofilms and thus cause persistent infections. The aim of the present study was to use high-throughput sequencing techniques to establish complete transcriptome profiles of planktonic (free-living) and sessile (biofilm) forms of A . baumannii ATCC 17978 and thereby identify differences in their gene expression patterns. Collections of mRNA from planktonic (both exponential and stationary phase cultures) and sessile (biofilm) cells were sequenced. Six mRNA libraries were prepared following the mRNA-Seq protocols from Illumina. Reads were obtained in a HiScanSQ platform and mapped against the complete genome to describe the complete mRNA transcriptomes of planktonic and sessile cells. The results showed that the gene expression pattern of A . baumannii biofilm cells was distinct from that of planktonic cells, including 1621 genes over-expressed in biofilms relative to stationary phase cells and 55 genes expressed only in biofilms. These differences suggested important changes in amino acid and fatty acid metabolism, motility, active transport, DNA-methylation, iron acquisition, transcriptional regulation, and quorum sensing, among other processes. Disruption or deletion of five of these genes caused a significant decrease in biofilm formation ability in the corresponding mutant strains. Among the genes over-expressed in biofilm cells were those in an operon involved in quorum sensing. One of them, encoding an acyl carrier protein, was shown to be involved in biofilm formation as demonstrated by the significant decrease in biofilm formation by the corresponding knockout strain. The present work serves as a basis for future studies examining the complex network systems that regulate bacterial biofilm formation and maintenance.

Detection of Pseudomonas aeruginosa isolates producing VEB-type extended-spectrum β-lactamases in the United Kingdom

OBJECTIVES The aim of this study was to investigate the presence of VEB enzymes among Pseudomonas spp. referred to the UKs national reference laboratory and with phenotypic evidence of extended-spectrum beta-lactamase (ESBL) production. METHODS Antibiograms were analysed for Pseudomonas spp. referred from November 2003 to November 2007. Isolates with >/=4-fold ceftazidime/clavulanate synergy were screened for bla(VEB) alleles. Genes encoding metallo-beta-lactamases (bla(MBL)) were sought in isolates with positive imipenem/EDTA synergy tests. Selected PCR products were sequenced. PFGE of SpeI-digested genomic DNA was used to compare isolates. RESULTS Forty-nine (3.7%) of 1338 Pseudomonas spp. were considered potential ESBL producers; 40 were recovered for molecular testing. bla(VEB) alleles were detected in 32 Pseudomonas aeruginosa isolates, comprising diverse PFGE types, from 12 UK hospitals and 1 in India. One UK centre referred 15 isolates with VEB-1 enzyme; these were serotype O15, representing a single PFGE-defined strain that also produced VIM-10 metallo-carbapenemase. This strain was resistant to all beta-lactams, aminoglycosides and ciprofloxacin, remaining susceptible only to colistin (MICs </=1 mg/L). Two other P. aeruginosa isolates co-producing both VEB and VIM enzymes were received from two other UK hospitals; one isolate represented inter-hospital spread of the O15 strain and the second was distinct. CONCLUSIONS VEB enzymes have not been reported previously in the UK, but were produced by 80% of Pseudomonas spp. with phenotypic evidence of ESBL production. They co-existed with VIM carbapenemases in two strains, with one responsible for a major hospital outbreak. The incidence of ESBLs may be underestimated in Pseudomonas because ESBL phenotypes can be masked by other beta-lactam resistance mechanisms.

Role of quorum sensing in bacterial infections

Quorum sensing (QS) is cell communication that is widely used by bacterial pathogens to coordinate the expression of several collective traits, including the production of multiple virulence factors, biofilm formation, and swarming motility once a population threshold is reached. Several lines of evidence indicate that QS enhances virulence of bacterial pathogens in animal models as well as in human infections; however, its relative importance for bacterial pathogenesis is still incomplete. In this review, we discuss the present evidence from in vitro and in vivo experiments in animal models, as well as from clinical studies, that link QS systems with human infections. We focus on two major QS bacterial models, the opportunistic Gram negative bacteria Pseudomonas aeruginosa and the Gram positive Staphylococcus aureus, which are also two of the main agents responsible of nosocomial and wound infections. In addition, QS communication systems in other bacterial, eukaryotic pathogens, and even immune and cancer cells are also reviewed, and finally, the new approaches proposed to combat bacterial infections by the attenuation of their QS communication systems and virulence are also discussed.