Juan-Carlos Galán

Th1 and Th17 hypercytokinemia as early host response signature in severe pandemic influenza.

IntroductionHuman host immune response following infection with the new variant of A/H1N1 pandemic influenza virus (nvH1N1) is poorly understood. We utilize here systemic cytokine and antibody levels in evaluating differences in early immune response in both mild and severe patients infected with nvH1N1.MethodsWe profiled 29 cytokines and chemokines and evaluated the haemagglutination inhibition activity as quantitative and qualitative measurements of host immune responses in serum obtained during the first five days after symptoms onset, in two cohorts of nvH1N1 infected patients. Severe patients required hospitalization (n = 20), due to respiratory insufficiency (10 of them were admitted to the intensive care unit), while mild patients had exclusively flu-like symptoms (n = 15). A group of healthy donors was included as control (n = 15). Differences in levels of mediators between groups were assessed by using the non parametric U-Mann Whitney test. Association between variables was determined by calculating the Spearman correlation coefficient. Viral load was performed in serum by using real-time PCR targeting the neuraminidase gene.ResultsIncreased levels of innate-immunity mediators (IP-10, MCP-1, MIP-1β), and the absence of anti-nvH1N1 antibodies, characterized the early response to nvH1N1 infection in both hospitalized and mild patients. High systemic levels of type-II interferon (IFN-γ) and also of a group of mediators involved in the development of T-helper 17 (IL-8, IL-9, IL-17, IL-6) and T-helper 1 (TNF-α, IL-15, IL-12p70) responses were exclusively found in hospitalized patients. IL-15, IL-12p70, IL-6 constituted a hallmark of critical illness in our study. A significant inverse association was found between IL-6, IL-8 and PaO2 in critical patients.ConclusionsWhile infection with the nvH1N1 induces a typical innate response in both mild and severe patients, severe disease with respiratory involvement is characterized by early secretion of Th17 and Th1 cytokines usually associated with cell mediated immunity but also commonly linked to the pathogenesis of autoimmune/inflammatory diseases. The exact role of Th1 and Th17 mediators in the evolution of nvH1N1 mild and severe disease merits further investigation as to the detrimental or beneficial role these cytokines play in severe illness.

Integron Content of Extended-Spectrum-β-Lactamase-Producing Escherichia coli Strains over 12 Years in a Single Hospital in Madrid, Spain

ABSTRACT The contribution of integrons to the dissemination of extended-spectrum β-lactamases (ESBL) was analyzed on all ESBL-producing Escherichia coli isolates from 1988 to 2000 at Ramón y Cajal Hospital. We studied 133 E. coli pulsed-field gel electrophoresis types: (i) 52 ESBL-producing clinical strains (C-ESBL) (16 TEM, 9 SHV, 21 CTX-M-9, 1 CTX-M-14, and 5 CTX-M-10); (ii) 43 non-ESBL blood clinical strains (C-nESBL); and (iii) 38 non-ESBL fecal isolates from healthy volunteers (V-nESBL). Class 1 integrons were more common among C-ESBL (67%) than among C-nESBL (40%) or V-nESBL (26%) (P < 0.001) due to the high number of strains with blaCTX-M-9, which is linked to an In6-like class 1 integron. Without this bias, class 1 integron occurrence would be similar in C-ESBL and C-nESBL groups (47% versus 40%). Occurrence of class 2 integrons was similar among clinical and community isolates (13 to 18%). No isolates contained class 3 integrons. The relatively low rate of class 1 integrons within transferable elements carrying blaTEM (23%) or blaSHV (33%) and the absence of class 2 integrons in all ESBL transconjugants mirror the assembly of translocative pieces containing blaTEM or blaSHV on local available transferable elements lacking integrons. The low diversity of class 1 integrons (seven types recovered in all groups) might indicate a wide dissemination of specific genetic elements in which they are located. In our environment, the spread of genetic elements encoding ESBL has no major impact on the dispersion of integrons, nor do integrons have a major impact on the spread of ESBL, except when blaESBL genes are within an integron platform such as blaCTX-M-9.

Dissemination and Persistence of blaCTX-M-9 Are Linked to Class 1 Integrons Containing CR1 Associated with Defective Transposon Derivatives from Tn402 Located in Early Antibiotic Resistance Plasmids of IncHI2, IncP1-α, and IncFI Groups

ABSTRACT This study analyzes the diversity of In60, a class 1 integron bearing CR1 and containing blaCTX-M-9, and its association with Tn402, Tn21, and classical conjugative plasmids among 45 CTX-M-9-producing clinical strains (41 Escherichia coli strains, 2 Klebsiella pneumoniae strains, 1 Salmonella enterica strain, and 1 Enterobacter cloacae strain). Forty-five patients in a Spanish tertiary care hospital were studied (1996 to 2003). The diversity of In60 and association of In60 with Tn402 or mercury resistance transposons were investigated by overlapping PCR assays and/or hybridization. Plasmid characterization included comparison of restriction fragment length polymorphism patterns and determination of incompatibility group by PCR-based replicon typing, sequencing, and hybridization. CTX-M-9 plasmids belonged to IncHI2 (n = 26), IncP-1α (n = 10), IncFI (n = 4), and IncI (n = 1) groups. Genetic platforms containing blaCTX-M-9 were classified in six types in relation to the In60 backbone and in eight subtypes in relation to Tn402 derivatives. They were associated with Tn21 sequences when located in IncP-1α or IncHI2 plasmids. Our study identified blaCTX-M-9 in a high diversity of CR1-bearing class 1 integrons linked to different Tn402 derivatives, often to Tn21, highlighting the role of recombination events in the evolution of antibiotic resistance plasmids. The presence of blaCTX-M-9 on broad-host-range IncP-1α plasmids might contribute to its dissemination to hosts that were not members of the family Enterobacteriaceae.

Evolutionary Trajectories of Beta-Lactamase CTX-M-1 Cluster Enzymes: Predicting Antibiotic Resistance

Extended-spectrum beta-lactamases (ESBL) constitute a key antibiotic-resistance mechanism affecting Gram-negative bacteria, and also an excellent model for studying evolution in real time. A shift in the epidemiology of ESBLs is being observed, which is characterized by the explosive diversification and increase in frequency of the CTX-M-type β-lactamases in different settings. This provides a unique opportunity for studying a protein evolutionary radiation by the sequential acquisition of specific mutations enhancing protein efficiency and fitness concomitantly. The existence of driver antibiotic molecules favoring protein divergence has been investigated by combining evolutionary analyses and experimental site-specific mutagenesis. Phylogenetic reconstruction with all the CTX-M variants described so far provided a hypothetical evolutionary scenario showing at least three diversification events. CTX-M-3 was likely the enzyme at the origin of the diversification in the CTX-M-1 cluster, which was coincident with positive selection acting on several amino acid positions. Sixty-three CTX-M-3 derivatives containing all combinations of mutations under positively selected positions were constructed, and their phenotypic efficiency was evaluated. The CTX-M-3 diversification process can only be explained in a complex selective landscape with at least two antibiotics (cefotaxime and ceftazidime), indicating the need to invoke mixtures of selective drivers in order to understand the final evolutionary outcome. Under this hypothesis, we found congruent results between the in silico and in vitro analyses of evolutionary trajectories. Three pathways driving the diversification of CTX-M-3 towards the most complex and efficient variants were identified. Whereas the P167S pathway has limited possibilities of further diversification, the D240G route shows a robust diversification network. In the third route, drift may have played a role in the early stages of CTX-M-3 evolution. Antimicrobial agents should not be considered only as selectors for efficient mechanisms of resistance but also as diversifying agents of the evolutionary trajectories. Different trajectories were identified using a combination of phylogenetic reconstructions and directed mutagenesis analyses, indicating that such an approach might be useful to fulfill the desirable goal of predicting evolutionary trajectories in antimicrobial resistance.

In Vitro Activity of Telithromycin against Spanish Streptococcus pneumoniae Isolates with Characterized Macrolide Resistance Mechanisms

ABSTRACT The susceptibilities to telithromycin of 203 Streptococcus pneumoniae isolates prospectively collected during 1999 and 2000 from 14 different geographical areas in Spain were tested and compared with those to erythromycin A, clindamycin, quinupristin-dalfopristin, penicillin G, cefotaxime, and levofloxacin. Telithromycin was active against 98.9% of isolates (MICs, ≤0.5 μg/ml), with MICs at which 90% of isolates are inhibited being 0.06 μg/ml, irrespective of the resistance genotype. The corresponding values for erythromycin were 61.0% (MICs, ≤0.25 μg/ml) and >64 μg/ml. The erm(B) gene (macrolide-lincosamide-streptogramin B resistance phenotype) was detected in 36.4% (n = 74) of the isolates, which corresponded to 93.6% of erythromycin-intermediate and -resistant isolates, whereas the mef(A) gene (M phenotype [resistance to erythromycin and susceptibility to clindamycin and spiramycin without blunting]) was present in only 2.4% (n = 5) of the isolates. One of the latter isolates also carried erm(B). Interestingly, in one isolate for which the erythromycin MIC was 2 μg/ml, none of these resistance genes could be detected. Erythromycin MICs forS. pneumoniae erm(B)-positive isolates were higher (range, 0.5 to >64 μg/ml) than those for erm(B)- andmef(A)-negative isolates (range, 0.008 to 2 μg/ml). The corresponding values for telithromycin were lower for both groups, with ranges of 0.004 to 1 and 0.002 to 0.06 μg/ml, respectively. The erythromycin MIC was high for a large number oferm(B)-positive isolates, but the telithromycin MIC was low for these isolates. These results indicate the potential usefulness of telithromycin for the treatment of infections caused by erythromycin-susceptible and -resistant S. pneumoniaeisolates when macrolides are indicated.

Haemophilus influenzae blaROB-1 Mutations in Hypermutagenic ΔampC Escherichia coli Conferring Resistance to Cefotaxime and β-Lactamase Inhibitors and Increased Susceptibility to Cefaclor

ABSTRACT The clinical use of cefaclor has been shown to enrich Haemophilus influenzae populations harboring cefaclor-hydrolyzing ROB-1 β-lactamase. Such a selective process may lead to the increased use of extended-spectrum cephalosporins or β-lactams plus β-lactamase inhibitors and, eventually, resistance to these agents, which has not previously been observed in H. influenzae. In order to establish which blaROB-1 mutations, if any, could confer resistance to extended-spectrum cephalosporins and/or to β-lactamase inhibitors, a plasmid harboring blaROB-1 was transformed into hypermutagenic strain Escherichia coli GB20 (ΔampC mutS::Tn10), and this construct was used in place of H. influenzae blaROB-1. Strain GB20 with the cloned gene was submitted to serial passages in tubes containing broth with increasing concentrations of selected β-lactams (cefotaxime or amoxicillin-clavulanate). Different mutations in the blaROB-1 gene were obtained during the passages in the presence of the different concentrations of the selective agents. Mutants resistant to extended-spectrum cephalosporins harbored either the Leu169→Ser169 or the Arg164→Trp164 substitution or the double amino acid change Arg164→Trp164 and Ala237→Thr237. ROB-1 mutants that were resistant to β-lactams plus β-lactamase inhibitors and that harbored the Arg244→Cys244 or the Ser130→Gly130 replacement were also obtained. The cefaclor-hydrolyzing efficiencies of the ROB-1 variants were strongly decreased in all mutants, suggesting that if blaROB-1 mutants were selected by cefaclor, this drug would prevent the further evolution of this β-lactamase toward molecular forms able to resist extended-spectrum cephalosporins or β-lactamase inhibitors.

Macrolide Resistance in Peptostreptococcus spp. Mediated by ermTR: Possible Source of Macrolide-Lincosamide-Streptogramin B Resistance in Streptococcus pyogenes

ABSTRACT Eighty percent (21 of 26) of macrolide-resistantPeptostreptococcus strains studied harbored theermTR gene. This methyltransferase gene is also the most frequently found gene among macrolide-lincosamide-streptogramin B-resistant Streptococcus pyogenes strains. Transfer of theermTR gene from Peptostreptococcus magnus to macrolide-susceptible S. pyogenes strains indicates that this resistance determinant may circulate among gram-positive aerobic and anaerobic species of the oropharyngeal bacterial flora.

In Vitro Selection of Variants Resistant to β-Lactams plus β-Lactamase Inhibitors in CTX-M β-Lactamases: Predicting the In Vivo Scenario?

ABSTRACT CTX-M β-lactamases are the most prevalent group of enzymes within the extended-spectrum β-lactamases (ESBL). The therapeutic options for CTX-M-carrying isolates are scarce, forcing the reexamination of the therapeutic possibilities of β-lactams plus β-lactamase inhibitors (BBLIs). Inhibitor-resistant CTX-M β-lactamases (IR-CTX-M) have not hitherto been described in natural isolates. In this study, 168 cultures of the hypermutagenic Escherichia coli GB20 strain carrying plasmid pBGS18 with different blaCTX-M genes were submitted to parallel experimental evolution assays in the presence of increasing concentrations of a combination of amoxicillin and clavulanate. Fourteen CTX-M β-lactamases belonging to the three most representative clusters (CTX-M-1, -2, and -9) and the two main phenotypes (cefotaxime resistance and cefotaxime-ceftazidime resistance) were studied. Three types of IR-CTX-M mutants were detected, having mutations S130G, K234R, and S237G, which are associated with different resistance patterns. The most frequently recovered mutation was S130G, which conferred the highest resistance levels to BBLIs (reaching 12 μg/ml for amoxicillin-clavulanate and 96 μg/ml for piperacillin-tazobactam when acquired by CTX-M-1 cluster enzymes). The S130G change also provided a clear antagonistic pleiotropy effect, strongly decreasing the enzymes activity against all cephalosporins tested. A double mutation, S130G L169S, partially restored the resistance against cephalosporins. A complex pattern observed in CTX-M-58, carrying P167S and S130G or K234R changes, conferred ESBL and IR phenotypes simultaneously. The K234R and S237G changes had a smaller effect in providing inhibitor resistance. In summary, IR-CTX-M enzymes might evolve under exposure to BBLIs, and the probability is higher for enzymes belonging to the CTX-M-1 cluster. However, this process could be delayed by antagonistic pleiotropy.

Molecular Surveillance of HIV-1 in Madrid, Spain: a Phylogeographic Analysis

ABSTRACT The molecular epidemiology of HIV-1 is constantly changing, mainly as a result of human migratory flows and the high adaptive ability of the virus. In recent years, Spain has become one of Europes main destinations for immigrants and one of the western European countries with the highest rates of HIV-positive patients. Using a phylogeographic approach, we have analyzed the relationship between HIV-1 variants detected in immigrant and native populations of the urban area of Madrid. Our project was based on two coincidental facts. First, resistance tests were extended to naïve and newly diagnosed patients, and second, the Spanish government legislated the provision of legal status to many immigrants. This allowed us to obtain a large data set (n = 2,792) from 11 Madrid hospitals of viral pol sequences from the two populations, and with this unique material, we explored the impact of immigration in the epidemiological trends of HIV-1 variants circulating in the largest Spanish city. The prevalence of infections by non-B HIV-1 variants in the studied cohort was 9%, rising to 25% among native Spanish patients. Multiple transmission events involving different lineages and subsubtypes were observed in all the subtypes and recombinant forms studied. Our results also revealed strong social clustering among the most recent immigrant groups, such as Russians and Romanians, but not in those groups who have lived in Madrid for many years. Additionally, we document for the first time the presence of CRF47_BF and CRF38_BF in Europe, and a new BG recombinant form found in Spaniards and Africans is tentatively proposed. These results suggest that the HIV-1 epidemic will evolve toward a more complex epidemiological landscape.

Fosfomycin and Rifampin Disk Diffusion Tests for Detection of Escherichia coli Mutator Strains

ABSTRACT A simple method, using commercial disks to detect Escherichia coli mutator strains, is proposed. The breakpoint for detecting strains with a mutation frequency ≥5 × 10−7 was established at ≥70 and ≥20 colonies in the inhibition zone of fosfomycin and rifampin disks, respectively, after seeding 100 μl of an overnight culture. Strains with <30 and <10 colonies in fosfomycin and rifampin inhibition zones are presumptively non-mutators.