Mobile genetic elements related to the diffusion of plasmid-mediated AmpC β-lactamases or carbapenemases from Enterobacteriaceae: findings from a multicenter study in Spain

We examined the genetic context of 74 acquired ampC genes and 17 carbapenemase genes from 85 out of 640 Enterobacteriaceae isolates collected in 2009. Using S1-PFGE and Southern hybridization, 37 out of 74 blaAmpC genes were located on large plasmids of different sizes belonging to six Inc groups. We used sequencing and PCR mapping to investigate the regions flanking the acquired ampC genes. The blaCMY-2like genes were associated with ISEcp1, the surrounding blaDHA genes were similar to Klebsiella pneumoniae plasmid pTN60013 associated with IS26 and the psp and sap operons, and blaACC-1 genes were associated with IS26 elements inserted into ISEcp1. All the carbapenemase genes (blaVIM-1, two blaIMP-22 and blaIMP-28) were located in class 1 integrons. Therefore, although plasmids are the main cause of the rapid dissemination of ampC genes among Enterobacteriaceae, we need to be aware that other mobile genetic elements, such as insertion sequences, transposons or integrons, can be involved in the mobilization of these genes of chromosomal origin. Additionally, three new integrons are described in this study (In846 to In848)

Biological Markers of Pseudomonas aeruginosa Epidemic High-Risk Clones

A limited number of Pseudomonas aeruginosa genotypes (mainly ST-111, ST-175, and ST-235), known as high-risk clones, are responsible for epidemics of nosocomial infections by multidrug-resistant (MDR) or extensively drug-resistant (XDR) strains worldwide. We explored the potential biological parameters that may explain the success of these clones. A total of 20 isolates from each of 4 resistance groups (XDR, MDR, ModR [resistant to 1 or 2 classes], and MultiS [susceptible to all antipseudomonals]), recovered from a multicenter study of P. aeruginosa bloodstream infections performed in 10 Spanish hospitals, were analyzed. A further set of 20 XDR isolates belonging to epidemic high-risk clones (ST-175 [n = 6], ST-111 [n = 7], and ST-235 [n = 7]) recovered from different geographical locations was also studied. When unknown, genotypes were documented through multilocus sequence typing. The biological parameters evaluated included twitching, swimming, and swarming motility, biofilm formation, production of pyoverdine and pyocyanin, spontaneous mutant frequencies, and the in vitro competition index (CI) obtained with a flow cytometry assay. All 20 (100%) XDR, 8 (40%) MDR, and 1 (5%) ModR bloodstream isolate from the multicenter study belonged to high-risk clones. No significant differences were observed between clonally diverse ModR and MultiS isolates for any of the parameters. In contrast, MDR/XDR high-risk clones showed significantly increased biofilm formation and mutant frequencies but significantly reduced motility (twitching, swimming, and swarming), production of pyoverdine and pyocyanin, and fitness. The defined biological markers of high-risk clones, which resemble those resulting from adaptation to chronic infections, could be useful for the design of specific treatment and infection control strategies

Mecanismos de regulación de AmpC en Pseudomonas aeruginosa: evaluación de nuevas dianas para evitar la resistencia a β-lactámicos

[spa] En Pseudomonas aeruginosa, la hiperproducción de AmpC es el mecanismo más frecuente de resistencia a penicilinas y cefalosporinas, y está conducida por mutaciones en ampD y/o dacB. En el primer capítulo de esta Tesis Doctoral, se demuestra que el efecto descrito en trabajos anteriores sobre la inactivación de ampD y/o dacB, es un principio general en Pseudomonas aeruginosa. En el segundo capítulo, se evalúan dos componentes de la vía de regulación de ampC: NagZ y AmpG, como dianas para prevenir la resistencia a β-lactámicos, demostrando que ambas inactivaciones restablecen completamente la sensibilidad y la expresión basal de la β-lactamasa ampC en mutantes ampD o dacB. Finalmente, el último capítulo de la tesis evalúa la interconexión del sistema regulador en dos componentes CreBC y la vía de regulación de ampC, y el efecto del sistema creBC sobre el fitnes; sugiriendo que la hiperexpresión de ampC, provocada por β-lactámico inductor, produce la inhibición del Sistema creBC y dicha inactivación reduce la eficacia biológica in vivo e in vitro