Regulation of AmpC-Driven ß-Lactam Resistance in Pseudomonas aeruginosa: Different Pathways, Different Signaling.

The hyperproduction of the chromosomal AmpC ß-lactamase is the main mechanism driving ß-lactam resistance in Pseudomonas aeruginosa, one of the leading opportunistic pathogens causing nosocomial acute and chronic infections in patients with underlying respiratory diseases. In the current scenario of the shortage of effective antipseudomonal drugs, understanding the molecular mechanisms mediating AmpC hyperproduction in order to develop new therapeutics against this fearsome pathogen is of great importance. It has been accepted for decades that certain cell wall-derived soluble fragments (muropeptides) modulate AmpC production by complexing with the transcriptional regulator AmpR and acquiring different conformations that activate/repress ampC expression. However, these peptidoglycan-derived signals have never been characterized in the highly prevalent P. aeruginosa stable AmpC hyperproducer mutants. Here, we demonstrate that the previously described fragments enabling the transient ampC hyperexpression during cefoxitin induction (1,6-anhydro-N-acetylmuramyl-pentapeptides) also underlie the dacB (penicillin binding protein 4 [PBP4]) mutation-driven stable hyperproduction but differ from the 1,6-anhydro-N-acetylmuramyl-tripeptides notably overaccumulated in the ampD knockout mutant. In addition, a simultaneous greater accumulation of both activators appears linked to higher levels of AmpC hyperproduction, although our results suggest a much stronger AmpC-activating potency for the 1,6-anhydro-N-acetylmuramyl-pentapeptide. Collectively, our results propose a model of AmpC control where the activator fragments, with qualitative and quantitative particularities depending on the pathways and levels of ß-lactamase production, dominate over the repressor (UDP-N-acetylmuramyl-pentapeptide). This study represents a major step in understanding the foundations of AmpC-dependent ß-lactam resistance in P. aeruginosa, potentially useful to open new therapeutic conceptions intended to interfere with the abovementioned cell wall-derived signaling.IMPORTANCE The extensive use of ß-lactam antibiotics and the bacterial adaptive capacity have led to the apparently unstoppable increase of antimicrobial resistance, one of the current major global health challenges. In the leading nosocomial pathogen Pseudomonas aeruginosa, the mutation-driven AmpC ß-lactamase hyperproduction stands out as the main resistance mechanism, but the molecular cues enabling this system have remained elusive until now. Here, we provide for the first time direct and quantitative information about the soluble cell wall-derived fragments accounting for the different levels and pathways of AmpC hyperproduction. Based on these results, we propose a hierarchical model of signals which ultimately govern ampC hyperexpression and resistance.

The Cpx envelope stress response modifies peptidoglycan cross-linking via the L,D-transpeptidase LdtD and the novel protein YgaU.

The Cpx envelope stress response mediates a complex adaptation to conditions that cause protein misfolding in the periplasm. A recent microarray study demonstrated that Cpx response activation led to changes in the expression of genes known, or predicted, to be involved in cell wall remodeling. We sought to characterize the changes that the cell wall undergoes during activation of the Cpx pathway in Escherichia coli. Luminescent reporters of gene expression confirmed that LdtD, a putative l,d-transpeptidase; YgaU, a protein of unknown function; and Slt, a lytic transglycosylase, are upregulated in response to Cpx-inducing conditions. Phosphorylated CpxR binds to the upstream regions of these genes, which contain putative CpxR binding sites, suggesting that regulation is direct. We show that the activation of the Cpx response causes an increase in the abundance of diaminopimelic acid (DAP)-DAP cross-links that involves LdtD and YgaU. Altogether, our data indicate that changes in peptidoglycan structure are part of the Cpx-mediated adaptation to envelope stress and indicate a role for the uncharacterized gene ygaU in regulating cross-linking.

Protección inducida por nanococleatos derivados de proteoliposomas de Leptospira interrogans serovar Canicola / Induced protection by nanocochleates derived from proteoliposomes from Leptospira interrogans serovar canicola

Desde los años 20 del pasado siglo, hasta el presente, en el mundo se han desarrollado y empleado vacunas de células enteras contra la leptospirosis que confieren una corta inmunidad; la mayoría no adyuvadas y dirigidas, fundamentalmente, contra los diferentes serogrupos de la especie Leptospira interrogans, contenidos en las preparaciones. Numerosos han sido los intentos realizados para lograr una formulación vacunal más pura, efectiva, de amplio espectro y duración de la protección que las bacterinas de células enteras inactivadas. Sin embargo, hasta el momento no se ha registrado ninguna vacuna con tales características. En el presente trabajo se obtuvieron antígenos de membrana externa a partir de una cepa cubana autóctona (Cepa 87, L. interrogans serovar Canicola), mediante una modificación de la tecnología para la producción de vesículas de membrana, patentada por investigadores del Instituto Finlay. Estos antígenos con estructura nanoproteoliposómica fueron formulados/adyuvados mediante diferentes estrategias, logrando cinco preparaciones con estructura coclear, que constituyen nanopartículas de aproximadamente 100 a 150 nm de largo y entre 15 a 30 nm de diámetro. Los inmunógenos se inocularon en el biomodelo Mesocrisetus aureatus, con dos dosis e intervalo de seis semanas. El reto fue realizado con 100,000 DL 50. Los resultados demuestran que las nuevas formulaciones vacunales confieren protección frente al reto homólogo y fueron capaces de eliminar el estado de portador, lo que unido a la robustez del método de preparación, el mayor nivel de pureza, en comparación con las bacterinas, y la no necesidad del hidróxido de aluminio, las convierten en una alternativa de interés para continuar su desarrollo(AU)

Whole cell Leptospirosis vaccines have been developed and used from the 20`s of last century up today , most of them are not adjuvated and do not confer a long-lasting immunity. These vaccines are mainly against different serogroups of Leptospira interrogans contained in the preparation. Though several attempts to obtain a vaccinal formulation more effective, purer, with wider spectrum and longer protection than the bacterines of inactivated whole cells have been made no vaccine with these characteristics have been registered yet. This paper deals with the obtainment of outer membrane antigens from a Cuban autochthonous strain (Strain 87, L. interrogans serovar Canicola), by a modification of the technology for the production of outer membrane vesicles, patented by researchers from Finlay institute These antigens with nanoproteoliposomic structure were formulated/adjuvated by different strategies, obtaining five preparations with cochlear structures which are nanoparticles of approximately 100 to 150 nm of lenght and from 15 to 30 nm of diameter. Two doses of the immunogens are inoculated in the biomodel Mesocrisetus aureatus, with an interval of six weeks. The challenge was carried out with 100.000 DL 50 . Results showed that the new vaccinal formulations confer protection against the homologous challenge and were able to eliminate the carrier status. This, together with the robutness of the preparation method, the higher level of purity, compared to the bacterines, and the no need of aluminium hydroxide make the formulations an alternative of interest for continuing their development(AU)

Antimicrobial resistance determinants among anaerobic bacteria isolated from footrot.

Antibiotic resistance has been evaluated among 36 Gram negative and anaerobic bacilli (10 Bacteroides, 11 Prevotella, 7 Porphyromonas and 8 Fusobacterium strains) isolated from clinical cases of caprine and ovine footrot (necrotic pododermatitis). The initial analysis on this bacterial consortium evaluates the relationships existing among antimicrobial resistance determinants, phenotype expression and mobilization potential. The Bacteroides strains were generally resistant to penicillins, first-generation cephalosporins, tetracycline and erythromycin, and expressed low level of ß-lactamase activity. The main determinants found among the Bacteroides strains were cepA and tetQ genes, conferring resistance to ß-lactams and tetracycline, respectively. A general susceptibility to ß-lactams was shown for most Prevotella, Porphyromonas and Fusobacterium strains, where none of the ß-lactamase genes described in Bacteroides was detected. Resistance to tetracycline and/or erythromycin was found among the three bacterial groups. Although tetQ genes were detected for several Prevotella and Porphyromonas strains, a unique ermF positive was revealed among Prevotella strains. The expression of resistance markers was not related with the polymorphism of their coding sequences. However, the finding of sequence signatures for conjugative transposons in the vicinities of tetQ and ermF suggests a mobilization potential that might have contributed to the spread of antimicrobial resistance genes.

Molecular analysis of the effector mechanisms of cefoxitin resistance among Bacteroides strains.

OBJECTIVES: The characterization of Bacteroides strains with regard to the cfxA gene, the MTn4555 mobilizable transposon, the role of penicillin-binding proteins (PBPs) and heterogeneous cefoxitin resistance. METHODS: Eighty-four randomly selected and 11 heterogeneously or highly cefoxitin-resistant Bacteroides isolates were included. Agar dilution and Etest methods were used for the determination of cefoxitin MICs. PCR experiments and nucleotide sequencing were used to detect the cfxA gene and the molecular features of MTn4555. Cefoxitin-binding experiments to determine its affinity (IC(50)) for PBPs and cefoxitinase assays were also applied. Southern blotting was used to determine the copy number of the cfxA genes. RESULTS: Sixteen strains from the random collection proved to be positive for cfxA, and the MIC distribution for the cfxA-negative and -positive strains did not display a clear separation. The majority of the cfxA-positive strains in this collection harboured a 1.2 kb common region at the 3' end of MTn4555. This region encoded an open reading frame that exhibited homology to abortive phage infection proteins (AbiD). The cfxA genes were transferable only at low frequencies in conjugation experiments. In PBP affinity studies, the PBP-A and PBP3 species were largely insensitive to cefoxitin, whereas the other PBP species were affected at very low concentrations. Seven of the heterogeneously resistant strains were positive for cfxA and most of them had mutations in the regulatory regions of cfxA. CONCLUSIONS: Major and minor roles for Bacteroides fragilis PBPs and the CfxA cefoxitinase, respectively, were inferred. The role of the newly recognized abiD may be to control the copy number of cfxA.