Laura Hidalgo

Association of the novel aminoglycoside resistance determinant RmtF with NDM carbapenemase in Enterobacteriaceae isolated in India and the UK

OBJECTIVES 16S rRNA methyltransferases are an emerging mechanism conferring high-level resistance to clinically relevant aminoglycosides and have been associated with important mechanisms such as NDM-1. We sought genes encoding these enzymes in isolates highly resistant (MIC >200 mg/L) to gentamicin and amikacin from an Indian hospital and we additionally screened for the novel RmtF enzyme in 132 UK isolates containing NDM. METHODS All highly aminoglycoside-resistant isolates were screened for armA and rmtA-E by PCR, with cloning experiments performed for isolates negative for these genes. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was used to determine the methylation target of the novel RmtF methyltransferase. RmtF-bearing strains were characterized further, including susceptibility testing, PFGE, electroporation, PCR-based replicon typing and multilocus sequence typing of rmtF-bearing plasmids. RESULTS High-level aminoglycoside resistance was detected in 140/1000 (14%) consecutive isolates of Enterobacteriaceae from India. ArmA, RmtB and RmtC were identified among 46%, 20% and 27% of these isolates, respectively. The novel rmtF gene was detected in 34 aminoglycoside-resistant isolates (overall prevalence 3.4%), most (59%) of which also possessed a bla(NDM) gene; rmtF was detected in 6 NDM producers from the UK. It was found on different plasmid backbones. Four and two isolates showed resistance to tigecycline and colistin, respectively. CONCLUSIONS RmtF was often found in association with NDM in members of the Enterobacteriaceae and on diverse plasmids. It is of clinical concern that the RmtF- and NDM-positive strains identified here show additional resistance to tigecycline and colistin, current drugs of last resort for the treatment of serious bacterial infections.

Multiresistance in Pasteurella multocida Is Mediated by Coexistence of Small Plasmids

ABSTRACT In most gram-negative bacteria, acquired multiresistance is conferred by large plasmids compiling numerous antimicrobial resistance genes. Here, we show an evolutionary alternative strategy used by Pasteurella multocida to become resistant to multiple clinically relevant antibiotics. Thirteen β-lactam-resistant clinical isolates, concomitantly resistant to tetracyclines and/or streptomycin as well as to sulfonamides, were studied. Pulsed-field gel electrophoresis analysis revealed different profiles among the isolates, showing that clonal dissemination was not the sole event responsible for the spread of multiresistance. Each P. multocida strain carried two or three small plasmids between 4 and 6 kb in size. A direct association between resistance profile and plasmid content was found. Complete nucleotide sequencing of all plasmids revealed seven different replicons, six of them belonging to the ColE1 superfamily. All plasmids carried one, or a maximum of two, antimicrobial resistance determinants. Plasmids pB1000 and pB1002 bore blaROB-1, pB1001 carried tet(B), pB1003 and pB1005 carried sul2 and strA, pB1006 harbored tet(O), and p9956 bore the tet(H) gene. All plasmids except pB1002 and pB1006 were successfully transformed into Escherichia coli. pB1000, also involved in β-lactam resistance in Haemophilus parasuis (A. San Millan et al., Antimicrob. Agents Chemother. 51:2260-2264, 2007), was mobilized in E. coli using the conjugation machinery of an IncP plasmid. Stability experiments proved that pB1000 was stable in P. multocida but highly unstable in E. coli. In conclusion, blaROB-1 is responsible for β-lactam resistance in P. multocida in Spain. Coexistence and the spread of small plasmids are used by P. multocida to become multiresistant.

Survival and function of human thymic dendritic cells are dependent on autocrine Hedgehog signaling

The Hedgehog (Hh) family of signaling molecules functions in the development of numerous tissues during embryogenesis and has also been involved in adult self‐renewing tissues. Recent results have demonstrated that the different components of the Hh signaling pathway are expressed in the human thymus. In this study, we investigate whether thymic dendritic cells (DCs) are cell targets for Hh signaling. Both components of the Hh receptor, Patched and Smoothened, as well as other Hh‐binding proteins with modulating functions, are expressed by human thymic DCs. The expression of Gli1, Gli2, and Gli3 transcription factors suggests that the Hh signaling pathway is active in thymic DCs, and approximately one‐half of thymic DCs produces Sonic Hh (Shh). The culture of thymic DCs with Shh protects them from apoptosis [similarly to CD40 ligand (CD40L)], and these antiapoptotic effects are related to an up‐regulation of Bcl‐2 and Bcl‐XL protein expression. The addition of the Hh pathway inhibitor, cyclopamine, decreases DC viability and impairs their allostimulatory function in vitro. In addition, the blockade of the Hh signaling pathway by cyclopamine treatment abrogates the up‐regulation of HLA‐DR, CD86, CD80, and CD83 expression induced by CD40L on thymic DCs. Finally, we also show that after activation with CD40L thymic DCs down‐regulate the expression of Hh receptor components as well as Shh production. Taken together, these results suggest that the survival and function of thymic DCs are regulated by an autocrine Hh signaling.

CXCL12/CXCR4 signaling promotes human thymic dendritic cell survival regulating the Bcl-2/Bax ratio

CXCL12, a member of the chemokine CXC subfamily, and its physiologic receptor CXCR4 are essential for the development of various organs during embryonic development and are also involved in the control of cell survival, proliferation and migration in adult tissues. In the human thymus, CXCL12 is produced by epithelial cells located in the subcapsular and medullary regions and CXCR4 is expressed in different thymocyte subpopulations. Several results have demonstrated that CXCL12/CXCR4 signaling participates in different intrathymic processes including the control of human precursor cell survival and proliferation, and the exit of mature thymocytes to the periphery. In this study, we show that CXCL12 is also produced by human thymic dendritic cells (DCs), most of which express CXCR4 receptor. The addition of exogenous CXCL12 significantly inhibited the serum depletion-induced apoptosis in thymic DCs, and the treatment with neutralizing antibodies against CXCL12 or CXCR4 decreased their survival. The survival-promoting effect of CXCL12 was mediated by the up-regulation of Bcl-2 protein expression and the concomitant down-regulation of Bax protein expression. The higher viability of thymic DCs also enhanced their allostimulatory capacity. Taken together, the results suggest a new function of CXCL12 in the human thymus controlling the survival and functionality of thymic DCs.

16S rRNA Methyltransferase RmtC in Salmonella enterica Serovar Virchow

We screened Salmonella and Escherichia coli isolates, collected 2004–2008 in the United Kingdom, for 16S rRNA methyltransferases. rmtC was identified in S. enterica serovar Virchow isolates from clinical samples and food. All isolates were clonally related and bore the rmtC gene on the bacterial chromosome. Surveillance for and research on these resistance determinants are essential.

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.

The canonical BMP signaling pathway is involved in human monocyte-derived dendritic cell maturation

Bone morphogenetic proteins (BMPs), members of the transforming growth factor‐β superfamily, are multifunctional polypeptides regulating a broad spectrum of functions in embryonic and adult tissues. Recent reports have demonstrated that BMPs regulate the survival, proliferation and differentiation of several cell types in the immune system. In this study, we investigate the effects of BMP signaling activation on the capacity of human dendritic cells (DCs) to stimulate immune responses. Human DCs express type I and type II BMP receptors (BMPRIA, BMPRIB, type IA activin receptor, BMPRII) and BMP signal transduction molecules (Smad1, 5, and 8, as well as Smad4). On BMP stimulation, Id1–3 (inhibitor of differentiation 1–3/DNA binding) mRNA expression is upregulated and this effect can be blocked with the inhibitor dorsomorphin, showing that the canonical BMP signal transduction pathway is functionally active in DCs. BMP signaling activation promotes the phenotypic maturation of human DCs by increasing the expression of co‐stimulatory molecules and also CD83, programmed cell death ligand 1 (PD‐L1) and PD‐L2, and stimulates cytokine secretion, mainly interleukin‐8 and tumor necrosis factor‐α. Accordingly, BMP‐treated DCs exhibit an enhanced T‐cell stimulatory capacity. BMP signaling also enhances the survival of human DCs increasing the Bcl‐2/Bax ratio. Finally, the expression of Runx transcription factors is increased in mature DCs, and the mRNA levels of Runx1–3 are upregulated in response to BMP stimulation, indicating that Runx transcription factor family may mediate the effects of BMP signaling in human DC maturation.

Fluoroquinolone Efflux in Streptococcus suis Is Mediated by SatAB and Not by SmrA

ABSTRACT Streptococcus suis is an emerging zoonotic pathogen. With the lack of an effective vaccine, antibiotics remain the main tool to fight infections caused by this pathogen. We have previously observed a reserpine-sensitive fluoroquinolone (FQ) efflux phenotype in this species. Here, SatAB and SmrA, two pumps belonging to the ATP binding cassette (ABC) and the major facilitator superfamily (MFS), respectively, have been analyzed in the fluoroquinolone-resistant clinical isolate BB1013. Genes encoding these pumps were overexpressed either constitutively or in the presence of ciprofloxacin in this strain. These genes could not be cloned in plasmids in Escherichia coli despite strong expression repression. Finally, site-directed insertion of smrA and satAB in the amy locus of the Bacillus subtilis chromosome using ligated PCR amplicons allowed for the functional expression and study of both pumps. Results showed that SatAB is a narrow-spectrum fluoroquinolone exporter (norfloxacin and ciprofloxacin), susceptible to reserpine, whereas SmrA was not involved in fluoroquinolone resistance. Chromosomal integration in Bacillus is a novel method for studying efflux pumps from Gram-positive bacteria, which enabled us to demonstrate the possible role of SatAB, and not SmrA, in fluoroquinolone efflux in S. suis.