Laura Carrilero

Large-Scale Screening of a Targeted Enterococcus faecalis Mutant Library Identifies Envelope Fitness Factors

Spread of antibiotic resistance among bacteria responsible for nosocomial and community-acquired infections urges for novel therapeutic or prophylactic targets and for innovative pathogen-specific antibacterial compounds. Major challenges are posed by opportunistic pathogens belonging to the low GC% Gram-positive bacteria. Among those, Enterococcus faecalis is a leading cause of hospital-acquired infections associated with life-threatening issues and increased hospital costs. To better understand the molecular properties of enterococci that may be required for virulence, and that may explain the emergence of these bacteria in nosocomial infections, we performed the first large-scale functional analysis of E. faecalis V583, the first vancomycin-resistant isolate from a human bloodstream infection. E. faecalis V583 is within the high-risk clonal complex 2 group, which comprises mostly isolates derived from hospital infections worldwide. We conducted broad-range screenings of candidate genes likely involved in host adaptation (e.g., colonization and/or virulence). For this purpose, a library was constructed of targeted insertion mutations in 177 genes encoding putative surface or stress-response factors. Individual mutants were subsequently tested for their i) resistance to oxidative stress, ii) antibiotic resistance, iii) resistance to opsonophagocytosis, iv) adherence to the human colon carcinoma Caco-2 epithelial cells and v) virulence in a surrogate insect model. Our results identified a number of factors that are involved in the interaction between enterococci and their host environments. Their predicted functions highlight the importance of cell envelope glycopolymers in E. faecalis host adaptation. This study provides a valuable genetic database for understanding the steps leading E. faecalis to opportunistic virulence.

Haemophilus influenzae Clinical Isolates with Plasmid pB1000 Bearing blaROB-1: Fitness Cost and Interspecies Dissemination

ABSTRACT Plasmid pB1000 is a mobilizable replicon bearing the blaROB-1 β-lactamase gene that we have recently described in Haemophilus parasuis and Pasteurella multocida animal isolates. Here we report the presence of pB1000 and a derivative plasmid, pB1000′, in four Haemophilus influenzae clinical isolates of human origin. Pulsed-field gel electrophoresis showed unrelated patterns in all strains, indicating that the existence of pB1000 in H. influenzae isolates is not the consequence of clonal dissemination. The replicon can be transferred both by transformation and by conjugation into H. influenzae, giving rise to recipients resistant to ampicillin and cefaclor (MICs, ≥64 μg/ml). Stability experiments showed that pB1000 is stable in H. influenzae without antimicrobial pressure for at least 60 generations. Competition experiments between isogenic H. influenzae strains with and without pB1000 revealed a competitive disadvantage of 9% per 10 generations for the transformant versus the recipient. The complete nucleotide sequences of nine pB1000 plasmids from human and animal isolates, as well as the epidemiological data, suggest that animal isolates belonging to the Pasteurellaceae act as an antimicrobial resistance reservoir for H. influenzae. Further, since P. multocida is the only member of this family that can colonize both humans and animals, we propose that P. multocida is the vehicle for the transport of pB1000 between animal- and human-adapted members of the Pasteurellaceae.

Klebsiella pneumoniae sequence type 11 from companion animals bearing ArmA methyltransferase, DHA-1 β-lactamase, and QnrB4.

ABSTRACT Seven Klebsiella pneumoniae isolates from dogs and cats in Spain were found to be highly resistant to aminoglycosides, and ArmA methyltransferase was responsible for this phenotype. All isolates were typed by multilocus sequence typing (MLST) as ST11, a human epidemic clone reported worldwide and associated with, among others, OXA-48 and NDM carbapenemases. In the seven strains, armA was borne by an IncR plasmid, pB1025, of 50 kb. The isolates were found to coproduce DHA-1 and SHV-11 β-lactamases, as well as the QnrB4 resistance determinant. This first report of the ArmA methyltransferase in pets illustrates their importance as a reservoir for human multidrug-resistant K. pneumoniae.

Fitness cost and interference of Arm/Rmt aminoglycoside resistance with the RsmF housekeeping methyltransferases.

ABSTRACT Arm/Rmt methyltransferases have emerged recently in pathogenic bacteria as enzymes that confer high-level resistance to 4,6-disubstituted aminoglycosides through methylation of the G1405 residue in the 16S rRNA (like ArmA and RmtA to -E). In prokaryotes, nucleotide methylations are the most common type of rRNA modification, and they are introduced posttranscriptionally by a variety of site-specific housekeeping enzymes to optimize ribosomal function. Here we show that while the aminoglycoside resistance methyltransferase RmtC methylates G1405, it impedes methylation of the housekeeping methyltransferase RsmF at position C1407, a nucleotide that, like G1405, forms part of the aminoglycoside binding pocket of the 16S rRNA. To understand the origin and consequences of this phenomenon, we constructed a series of in-frame knockout and knock-in mutants of Escherichia coli, corresponding to the genotypes rsmF+, ΔrsmF, rsmF+ rmtC+, and ΔrsmF rmtC+. When analyzed for the antimicrobial resistance pattern, the ΔrsmF bacteria had a decreased susceptibility to aminoglycosides, including 4,6- and 4,5-deoxystreptamine aminoglycosides, showing that the housekeeping methylation at C1407 is involved in intrinsic aminoglycoside susceptibility in E. coli. Competition experiments between the isogenic E. coli strains showed that, contrary to expectation, acquisition of rmtC does not entail a fitness cost for the bacterium. Finally, matrix-assisted laser desorption ionization (MALDI) mass spectrometry allowed us to determine that RmtC methylates the G1405 residue not only in presence but also in the absence of aminoglycoside antibiotics. Thus, the coupling between housekeeping and acquired methyltransferases subverts the methylation architecture of the 16S rRNA but elicits Arm/Rmt methyltransferases to be selected and retained, posing an important threat to the usefulness of aminoglycosides worldwide.

SatR Is a Repressor of Fluoroquinolone Efflux Pump SatAB

ABSTRACT Streptococcus suis is an emerging zoonotic agent responsible for high-mortality outbreaks among the human population in China. In this species, the ABC transporter SatAB mediates fluoroquinolone resistance when overexpressed. Here, we describe and characterize satR, an open reading frame (ORF) encoding a MarR superfamily regulator that acts as a repressor of satAB. satR is cotranscribed with satAB, and its interruption entails the overexpression of the pump, leading to a clinically relevant increase in resistance to fluoroquinolones.

H2S mediates interbacterial communication through the air reverting intrinsic antibiotic resistance

Hydrogen sulfide, a gas classically considered as a by-product of cellular metabolism, is today recognized as a crucial gasotransmitter in Eukaryotes. Moreover, most bacteria harbor the eukaryotic orthologous genes for H2S synthesis, and these genes have been linked to different metabolic pathways. Some bacteria, however, produce high amounts of H2S in their extracellular space, a characteristic classically used for identification purposes. This is the case of Salmonella Typhimurium, which produces H2S by its phsABC operon. Here we show that extracellular release of H2S by S. Typhimurium is solely dependent on its phsABC operon. Furthermore, we show that S. Typhimurium and other H2S-producing bacteria can interact with physically distant bacteria through H2S production. We demonstrate how H2S can revert intrinsic cephalosporin resistance of Enterococccus faecalis and Enterococcus faecium to complete susceptibility. This study constitutes a significant step in the study of bacterial interplay and niche competition. Furthermore, as H2S releasing drugs have already been designed, our results open the way to future therapeutic alternatives for the treatment of infections caused by enterococci, multiresistant pathogens for which no treatments are clinically available. Author Summary It has been known for decades that bacteria can communicate with each other through the diffusion of metabolites in the media. However, the capacity of a bacterium to interact with other physically distant cell is a recent discovery of the 21st century. In this work we show how some well-studied bacteria, as it is Salmonella spp., interacts with other bacteria thanks to the compound hydrogen sulfide (H2S) that they produce and release to the environment. In our study we have designed novel techniques that allow us to study the interaction between two bacteria, and we have seen that Salmonella is able to affect other species that is even 1 cm away, i.e., a distance corresponding to 10.0000 times its own size. What is more astonishing is that Enterococcus, when exposed to the H2S, is dramatically becomes susceptible to many antibiotics, to which it is supposed to be naturally resistant. Enterococcus spp. are responsible for life-threatening infections in hospitals worldwide. Thus, our observations reveal that bacteria can communicate through the air with H2S, and that this molecule can make bacteria that are highly resistant to antibiotics susceptible to antibiotics, making untreatable infections treatable with current antibiotics.