Pseudomonas aeruginosa is the most relevant pathogen producing chronic lung infections in patients with chronic underlying diseases such as cystic fibrosis (CF), bronchiectasis, and chronic obstructive pulmonary disease (COPD). Hypermutable (or mutator) P. aeruginosa strains, characterized by increased (up to 1,000-fold) spontaneous mutation rates due to alterations of the DNA mismatch repair (MMR) system have been found at high frequencies in the lungs of CF patients, but their role in other chronic processes is still unknown. Sixty-two P. aeruginosa isolates from 30 patients with underlying non-CF chronic respiratory diseases (22 with bronchiectasis and 8 with COPD) and documented chronic infection were studied. Antibiotic susceptibility profiles and mutation frequencies were determined, and complementation assays using the cloned wild-type mutS gene and molecular epidemiology studies (pulsed-field electrophoresis, [PFGE]) were performed with these strains. Thirty-three (53%) of the isolates were hypermutable, and 17 (57%) of the 30 patients were colonized by hypermutable strains. Strains from 11 of the 17 patients were found to be defective in the MMR mutS gene by complementation assays. Interpatient transmission of strains was ruled out by PFGE. Multipleantimicrobial resistance was documented in 42% of the hypermutable strains in contrast to 0% resistance in the nonhypermutable strains (P < 0.0001). Hypermutable P. aeruginosa strains are extremely prevalent in chronic infections in contrast to what has been described in acute processes, suggesting a role of hypermutation in bacterial adaptation for long-term persistence. Furthermore, hypermutation is found to be a key factor for the development of multiple-antimicrobial resistance, and therefore these findings are expected to have important consequences for the treatment of chronic infections.

) it was shown that Pseudomonas aeruginosa undergoes intense genetic adaptation during chronic respiratory infection (CRI) in cystic fibrosis (CF) patients. We used the same collection of isolates to explore the role of hypermutation in this process, since one of the hallmarks of CRI is the high prevalence of DNA mismatch repair (MMR) system-deficient mutator strains. The presence of mutations in 34 genes (many of them positively linked to adaptation in CF patients) in the study collection of 90 P. aeruginosa isolates obtained longitudinally from 29 CF patients was not homogeneous; on the contrary, mutations were significantly concentrated in the mutator lineages, which represented 17% of the isolates (87% MMR deficient). While sequential nonmutator lineages acquired a median of only 0.25 mutation per year of infection, mutator lineages accumulated more than 3 mutations per year. On the whole-genome scale, data for the first fully sequenced late CF isolate, which was also shown to be an MMR-deficient mutator, also support these findings. Moreover, for the first time the predicted amplification of mutator populations due to hitchhiking with adaptive mutations in the course of natural human infections is clearly documented. Interestingly, increased accumulation of mutations in mutator lineages was not a consequence of overrepresentation of mutations in genes involved in antimicrobial resistance, the only adaptive trait linked so far to hypermutation in CF patients, demonstrating that hypermutation also plays a major role in P. aeruginosa genome evolution and adaptation during CRI.Chronic respiratory infection (CRI) with Pseudomonas aeruginosa is the main driver of morbidity and mortality in cystic fibrosis (CF) patients (4, 15). The establishment of P. aeruginosa CRI is mediated by a complex adaptive process that includes physiological changes produced by the activation of specific regulatory pathways, including the induction of the biofilm mode of growth or the differential expression of virulence genes (39), and genetic changes leading to selection of an important number of adaptive mutations required for long-term persistence (19,27,34).Although CF patients with CRI are generally infected by a single P. aeruginosa strain that in most cases persists throughout the patient's life (32), one of the hallmarks of such infections is the emergence and fixation over time of multiple phenotypic variants of the underlying clonal populations (24), a process known as adaptive radiation (30). Many of the selected phenotypes have been clearly linked to adaptation to the lung environment that favors lifelong persistence of CRI (27). Indeed, once the adaptation stage is reached and the CRI is fully established, eradication is generally no longer possible. At this point, with resignation, the therapeutic goals change from attempting to cure the infection to slowing the decline of lung function and improving the patient's quality of life.The intense genetic adaptation process that takes place during the establishment of CRI has r...

Development of resistance to the antipseudomonal penicillins and cephalosporins mediated by hyperproduction of the chromosomal cephalosporinase AmpC is a major threat to the successful treatment of Pseudomonas aeruginosa infections. Although ampD inactivation has been previously found to lead to a partially derepressed phenotype characterized by increased AmpC production but retaining further inducibility, the regulation of ampC in P. aeruginosa is far from well understood. We demonstrate that ampC expression is coordinately repressed by three AmpD homologues, including the previously described protein AmpD plus two additional proteins, designated AmpDh2 and AmpDh3. The three AmpD homologues are responsible for a stepwise ampC upregulation mechanism ultimately leading to constitutive hyperexpression of the chromosomal cephalosporinase and high-level antipseudomonal ␤-lactam resistance, as shown by analysis of the three single ampD mutants, the three double ampD mutants, and the triple ampD mutant. This is achieved by a three-step escalating mechanism rendering four relevant expression states: basal-level inducible expression (wild type), moderate-level hyperinducible expression with increased antipseudomonal ␤-lactam resistance (ampD mutant), high-level hyperinducible expression with high-level ␤-lactam resistance (ampD ampDh3 double mutant), and very high-level (more than 1,000-fold compared to the wild type) derepressed expression (triple mutant). Although one-step inducible-derepressed expression models are frequent in natural resistance mechanisms, this is the first characterized example in which expression of a resistance gene can be sequentially amplified through multiple steps of derepression.

Vaccines based on outer membrane vesicles (OMV) were developed more than 20 years ago against Neisseria meningitidis serogroup B. These nano-sized structures exhibit remarkable potential for immunomodulation of immune responses and delivery of meningococcal antigens or unrelated antigens incorporated into the vesicle structure. This paper reviews different applications in OMV Research and Development (R&D) and provides examples of OMV developed and evaluated at the Finlay Institute in Cuba. A Good Manufacturing Practice (GMP) process was developed at the Finlay Institute to produce OMV from N. meningitidis serogroup B (dOMVB) using detergent extraction. Subsequently, OMV from N. meningitidis, serogroup A (dOMVA), serogroup W (dOMVW), and serogroup X (dOMVX) were obtained using this process. More recently, the extraction process has also been applied effectively for obtaining OMV on a research scale from Vibrio cholerae (dOMVC), Bordetella pertussis (dOMVBP), Mycobacterium smegmatis (dOMVSM), and BCG (dOMVBCG). The immunogenicity of the OMV has been evaluated for specific antibody induction, and together with functional bactericidal and challenge assays in mice has shown their protective potential. dOMVB has been evaluated with non-neisserial antigens, including with a herpes virus type 2 glycoprotein, ovalbumin, and allergens. In conclusion, OMV are proving to be more versatile than first conceived and remain an important technology for development of vaccine candidates.

The molecular mechanisms of ␤-lactam resistance mediated by AmpC hyperproduction in natural strains of Pseudomonas aeruginosa were investigated in a collection of 10 isogenic, ceftazidime-susceptible and -resistant pairs of isolates, each sequentially recovered from a different intensive care unit patient treated with ␤-lactams. All 10 ceftazidime-resistant mutants hyperproduced AmpC (␤-lactamase activities were 12-to 657-fold higher than those of the parent strains), but none of them harbored mutations in ampR or the ampC-ampR intergenic region. On the other hand, six of them harbored inactivating mutations in ampD: four contained frameshift mutations, one had a C3T mutation, creating a premature stop codon, and finally, one had a large deletion, including the complete ampDE region. Complementation studies revealed that only three of the six ampD mutants could be fully transcomplemented with either ampD-or ampDE-harboring plasmids, whereas one of them could be transcomplemented only with ampDE and two of them (including the mutant with the deletion of the ampDE region and one with an ampD frameshift mutation leading to an ampDE-fused open reading frame) could not be fully transcomplemented with any of the plasmids. Finally, one of the four mutants with no mutations in ampD could be transcomplemented, but only with ampDE. Although the inactivation of AmpD is found to be the most frequent mechanism of AmpC hyperproduction in clinical strains, our findings suggest that for certain types of mutations, AmpE plays an indirect role in resistance and that there are other unknown genes involved in AmpC hyperproduction, with at least one of them apparently located close to the ampDE operon.

From January 1979 to December 1990 we studied the susceptibility of 1,492 pneumococcal strains isolated from adult patients in Bellvitge Hospital, Barcelona, Spain, to nine antimicrobial agents. Among clinically significant pneumococci, the incidence of penicillin-resistant strains increased from 4.3% in 1979 to 40% in 1990, and that of erythromycin-resistant strains also rose from 0% in 1979 to 9.4% in 1990. On the other hand, the incidence of strains resistant to tetracycline decreased from 76.1% to 37.6%, as did that of chloramphenicol-resistant strains, from 56.5% to 29.4%. The incidence of co-trimoxazole-resistant strains was about 40% throughout the study. Even more alarming was the finding that about 70% of penicillin-resistant strains showed multiple resistance to non-beta-lactam antibiotics. All pneumococci were susceptible to vancomycin, and all but six were susceptible to rifampin. We observed that isolates from cerebrospinal fluid and the respiratory tract were significantly more resistant to penicillin than were isolates from blood. The majority of strains (95%) belonged to serogroups or serotypes included in the 23-valent pneumococcal vaccine and 77.6% of penicillin-resistant strains belonged to groups 23, 6, 9, and 19.

All (236) Pseudomonas aeruginosa isolates resistant to imipenem and/or meropenem collected during a multicenter (127-hospital) study in Spain were analyzed. Carbapenem-resistant isolates were found to be more frequently resistant to all ␤-lactams and non-␤-lactam antibiotics than carbapenem-susceptible isolates (P < 0.001), and up to 46% of the carbapenem-resistant isolates met the criteria used to define multidrug resistance (MDR). Pulsed-field gel electrophoresis revealed remarkable clonal diversity (165 different clones were identified), and with few exceptions, the levels of intra-and interhospital dissemination of clones were found to be low. Carbapenem resistance was driven mainly by the mutational inactivation of OprD, accompanied or not by the hyperexpression of AmpC or MexAB-OprM. Class B carbapenemases (metallo-␤-lactamases [MBLs]) were detected in a single isolate, although interestingly, this isolate belonged to one of the few epidemic clones documented. The MBL-encoding gene (bla VIM-2 ), along with the aminoglycoside resistance determinants, was transferred to strain PAO1 by electroporation, demonstrating its plasmid location. The class 1 integron harboring bla VIM-2 was characterized as well, and two interesting features were revealed: intI1 was found to be disrupted by a 1.1-kb insertion sequence, and a previously undescribed aminoglycoside acetyltransferaseencoding gene [designated aac(6)-32] preceded bla VIM-2 . AAC(6)-32 showed 80% identity to AAC(6)-Ib and the recently described AAC(6)-31, and when aac(6)-32 was cloned into Escherichia coli, it conferred resistance to tobramycin and reduced susceptibility to gentamicin and amikacin. Despite the currently low prevalence of epidemic clones with MDR, active surveillance is needed to detect and prevent the dissemination of these clones, particularly those producing integron-and plasmid-encoded MBLs, given their additional capacity for the intra-and interspecies spread of MDR.

Our purpose was to study prospectively the causes, routes of infection, and frequency of catheter-related sepsis in patients on total parenteral nutrition. From January 1981 to January 1984, cultures of 135 subclavian catheters from 135 adult patients were done by quantitative and semiquantitative methods. Twenty patients (14.8%) had catheter-related sepsis. Fourteen episodes (70%) stemmed from an colonized hub. Skin infection (Staphylococcus aureus, 2 cases), total parenteral nutrition mixture contamination (Enterobacter cloacae, 2 cases), and hematogenous seeding of the catheter tip (Yersinia enterocolitica, 1 case, and Streptococcus faecalis, 1 case) accounted for the remaining six septic episodes. The catheter hub is, in our experience, the most common site of origin of organisms causing catheter tip infection and bacteremia.