Laura Mensa

Antimicrobial Susceptibility and Mechanisms of Resistance in Shigella and Salmonella Isolates from Children under Five Years of Age with Diarrhea in Rural Mozambique

ABSTRACT The antimicrobial susceptibility and mechanisms of resistance of 109 Shigella and 40 Salmonella isolates from children with diarrhea in southern Mozambique were assessed. The susceptibility to seven antimicrobial agents was tested by disk diffusion, and mechanisms of resistance were searched by PCR or colorimetric method. A high proportion of Shigella isolates were resistant to chloramphenicol (Chl) (52%), ampicillin (Amp) (56%), tetracycline (Tet) (66%), and trimethoprim-sulfamethoxazole (Sxt) (84%). Sixty-five percent of the isolates were multidrug resistant. Shigella flexneri isolates were more resistant than those of Shigella sonnei to Amp (66% versus 0.0%, P < 0.001) and Chl (61% versus 0.0%, P < 0.001), whereas S. sonnei isolates presented higher resistance to Tet than S. flexneri isolates (93% versus 64%, P = 0.02). Resistance among Salmonella isolates was as follows: Tet and Chl, 15% each; Sxt, 18%; and Amp, 25%. Only 3% of Salmonella isolates were resistant to nalidixic acid (Nal), and none to ciprofloxacin or ceftriaxone (Cro). Among Salmonella isolates, multiresistance was found in 23%. Among Shigella isolates, antibiotic resistance was related mainly to the presence of oxa-1-like β-lactamases for Amp, dfrA1 genes for Sxt, tetB genes for Tet, and Chl acetyltransferase (CAT) activity for Chl. Among Salmonella isolates, resistance was conferred by tem-like β-lactamases for Amp, floR genes and CAT activity for Chl, tetA genes for Tet, and dfrA1 genes for Sxt. Our data show that Shigella isolates are resistant mostly to the most available, inexpensive antibiotics by various molecular mechanisms but remain susceptible to ciprofloxacin, Cro, and Nal, which is the first line for empirical treatment of shigellosis in the country.

Hepatitis C virus receptors claudin-1 and occludin after liver transplantation and influence on early viral kinetics

Liver transplantation (LT) is a unique model to study hepatitis C virus (HCV) entry into hepatocytes. Recent in vitro studies suggest significant changes in the expression of the HCV receptors claudin‐1 and occludin after HCV infection. Our aims were: (1) to characterize claudin‐1 and occludin expression in grafts from LT recipients and (2) to explore their potential influence on early HCV kinetics and their changes after HCV infection. We included 42 HCV‐infected LT recipients and 19 uninfected controls. Claudin‐1 and occludin were detected in paraffin‐embedded liver biopsies obtained during reperfusion and 3 and 12 months after LT. HCV receptors were characterized by confocal immunofluorescence microscopy; quantification and colocalization studies were performed with dedicated software. Claudin‐1 and occludin expression were restricted to the apical pole of hepatocytes. There was a significant correlation between the amount of scavenger receptor B1 at the time of reperfusion and the HCV‐RNA decay during the first 24 hours following LT (r = 0.55, P = 0.007). Similarly, there was a significant correlation between the levels of claudin and occludin and the slope of HCV‐RNA increase during the first week after LT (r = 0.63, P = 0.005). Occludin and claudin‐1 levels increased significantly 12 months after LT (P = 0.03 and P = 0.007, respectively). The expression pattern of both proteins, however, remained unchanged, colocalizing strongly (60%‐94%) at the apical membrane of hepatocytes. Conclusions. HCV receptor levels at the time of LT seem to modulate early HCV kinetics. Hepatitis C recurrence after LT was associated with increased levels of claudin‐1 and occludin in the hepatocyte cell membrane, although it did not alter their localization within the tight junctions. (HEPATOLOGY 2011;.)

Hepatitis C virus superinfection of liver grafts: a detailed analysis of early exclusion of non-dominant virus strains

Liver transplantation (LT) of hepatitis C virus (HCV)-infected grafts into HCV-infected recipients leads to superinfection with two different virus strains. To characterize the virological outcomes of HCV superinfection immediately after LT, we performed phylogenetic analysis of a fragment of the NS5B gene in donor and recipient serum samples prospectively collected before and after LT, starting on day 1. In four of six cases, the donor strain finally prevailed, while in the remaining two cases, the native recipient strain overtook the donor quasispecies. Clonal sequence analysis showed that, in three cases, the expelled strain was undetectable 1 day after LT. Our study shows that superinfection with a different HCV strain can lead to the exclusion of one strain by the other as soon as the first day after LT. This would suggest that competition might not be limited to the replication level, but could also take place during virus entry.

Development of Escherichia coli rifaximin-resistant mutants: frequency of selection and stability.

OBJECTIVES To select rifaximin-resistant mutants of Escherichia coli and to establish the frequency of mutation, cross-resistance with other antimicrobial agents and the stability of the mutants obtained. METHODS Four E. coli isolates [two enteroaggregative E. coli (EAEC) and two enterotoxigenic E. coli (ETEC)] were used to obtain rifaximin-resistant mutants. The frequency of mutation in the presence of rifaximin, rifampicin and ciprofloxacin was established by growth on plates containing serial dilutions of antibiotic above the bacterial MIC. To determine the stability of rifaximin resistance, 28 selected resistant mutants were grown for 20 consecutive cultures on antibiotic-free plates. Every 10 days, the MICs of rifaximin, chloramphenicol, nalidixic acid and ciprofloxacin were established. RESULTS The frequency of mutation in the presence of rifaximin ranged between 5.7 x 10(-7) and 1.6 x 10(-6) in the case of the ETEC isolates, and between 2.0 x 10(-8) and 9.3 x 10(-8) in the case of the EAEC isolates; the frequency of mutation in the presence of rifampicin was in the order of 10(-8) and no mutant in the presence of ciprofloxacin was obtained. Twenty-six out of 28 selected mutants exhibited resistance levels around or higher than 256 mg/L. In all cases, the resistance was stable, and no reversion towards the original parental MIC was observed. In no case was the MIC of chloramphenicol, nalidixic acid or ciprofloxacin affected. CONCLUSIONS Rifaximin has a low level of resistance selection, although it may select stable highly resistant mutants in a single step. Periodical surveillance of the levels of rifaximin resistance is required to detect the possible appearance of rifaximin-resistant clinical isolates. Further studies to characterize in-depth the mechanisms of stable resistance to rifaximin are necessary.

Antimicrobial resistance in Shigella spp. causing traveller's diarrhoea (1995–2010): A retrospective analysis

BACKGROUND Shigellosis is a global human health problem causing an important morbidity among travellers returning from tropical areas. This study was aimed to describe the evolution of antimicrobial resistance profile in Shigella spp. isolated between the years 1995-2010 in patients with travellers diarrhoea (TD) returning from tropical areas. METHODS The levels of antimicrobial resistance were tested in a total of 191 Shigella spp. isolated during the period from 1995 to 2010. RESULTS A decrease of cases of diarrhoea caused by Shigella has been observed in recent years. A wide spectrum of antibiotic resistance was observed among Shigella spp. These isolates showed high levels of resistance to tetracycline (84%), co-trimoxazole (75.5%), and ampicillin (45.5%). The resistance was low to ciprofloxacin (2.1%), azithromycin (3.9%) and furazolidone (8.4%). According to the period, in the case of ampicillin, amoxicillin plus clavulanic acid, chloramphenicol, values of resistance were significantly decreasing from 1995-2000 to 2001-2010, (62.5% vs. 28.4%, 19.8% vs. 6.6%, 23.4 vs. 10.4%, respectively). Meanwhile in nalidixic acid and tetracycline the evolution of resistance has increased over time. CONCLUSIONS A decrease in the isolation number of Shigella spp. causing TD has been observed. Differential trends in the evolution of the levels of resistance to the tested antibacterial agents have been observed.

Interplay between Basic Residues of Hepatitis C Virus Glycoprotein E2 with Viral Receptors, Neutralizing Antibodies and Lipoproteins

Positively-charged amino acids are located at specific positions in the envelope glycoprotein E2 of the hepatitis C virus (HCV): two histidines (H) and four arginines (R) in two conserved WHY and one RGERCDLEDRDR motifs, respectively. Additionally, the E2 hypervariable region 1 (HVR1) is rich in basic amino acids. To investigate the role(s) of these residues in HCV entry, we subjected to comparative infection and sedimentation analysis cell culture-produced (HCVcc, genotype 2a) wild type virus, a panel of alanine single-site mutants and a HVR1-deletion variant. Initially, we analyzed the effects of these mutations on E2-heparan sulfate (HS) interactions. The positive milieu of the HVR1, formulated by its basic amino acids (key residues the conserved H386 and R408), and the two highly conserved basic residues H488 and R648 contributed to E2-HS interactions. Mutations in these residues did not alter the HCVcc-CD81 entry, but they modified the HCVcc-scavenger receptor class B type I (SR-BI) dependent entry and the neutralization by anti-E2 or patients IgG. Finally, separation by density gradients revealed that mutant viruses abolished partially or completely the infectivity of low density particles, which are believed to be associated with lipoproteins. This study shows that there exists a complex interplay between the basic amino acids located in HVR1 and other conserved E2 motifs with the HS, the SR-BI, and neutralizing antibodies and suggests that HCV-associated lipoproteins are implicated in these interactions.

Cell Culture Replication of a Genotype 1b Hepatitis C Virus Isolate Cloned from a Patient Who Underwent Liver Transplantation

The introduction of the genotype 2a isolate JFH1 was a major breakthrough in the field of hepatitis C virus (HCV), allowing researchers to study the complete life cycle of the virus in cell culture. However, fully competent culture systems encompassing the most therapeutically relevant HCV genotypes are still lacking, especially for the highly drug-resistant genotype 1b. For most isolated HCV clones, efficient replication in cultured hepatoma cells requires the introduction of replication-enhancing mutations. However, such mutations may interfere with viral assembly, as occurs in the case of the genotype 1b isolate Con1. In this study, we show that a clinical serum carrying a genotype 1b virus with an exceptionally high viral load was able to infect Huh7.5 cells. Similar to previous reports, inoculation of Huh7.5 cells by natural virus is very inefficient compared to infection by cell culture HCV. A consensus sequence of a new genotype 1b HCV isolate was cloned from the clinical serum (designated Barcelona HCV1), and then subjected to replication studies. This virus replicated poorly in a transient fashion in Huh7.5 cells after electroporation with in vitro transcribed RNA. Nonetheless, approximately 3 weeks post electroporation and thereafter, core protein-positive cells were detected by immunofluorescence. Surprisingly, small amounts of core protein were also measurable in the supernatant of electroporated cells, suggesting that HCV particles might be assembled and released. Our findings not only enhance the current method of cloning in vitro HCV replication-competent isolates, but also offer valuable insights for the realization of fully competent culture systems for HCV.

Hepatitis C virus infection inhibits P-body granule formation in human livers

BACKGROUND & AIMS Decoding the myriad of interactions that hepatitis C virus (HCV) establishes with infected cells is mandatory to obtain a complete understanding of HCV biology and its associated pathogenesis. We and others have previously found that HCV infection disrupts the formation of P-bodies in cell culture. These are cytoplasmic RNA granules with key roles in post-transcriptional regulation of gene expression. Therefore, P-body disruption might have consequences beyond viral propagation. However, whether P-body disruption occurs also in vivo is unknown. Aim of this study was to address this important issue. METHODS Formalin-fixed paraffin-embedded liver biopsies from four groups of patients (healthy donors, patients with non-virus related liver inflammation, HCV- and HBV-infected patients) were immunostained to detect DDX6 and Dcp1, two core P-body components. Changes in the localization of these proteins were assessed by confocal microscopy. RESULTS HCV specifically inhibited P-body formation in hepatocytes from human livers regardless of viral genotype, inflammation grade or whether the infection was recent or long established. Importantly, this alteration was reversed once HCV was eliminated by therapy. Furthermore, we observed in vivo an unexpected heterogeneity in P-body composition, which might reflect functional specializations. CONCLUSIONS This is the first comprehensive in vivo P-body analysis that links a pathogenic condition to P-body alterations. Because of their role in gene expression, the alteration of P-bodies should be further studied to understand fully complex HCV-associated pathologies.

HEV infection in two referral centers in Spain: epidemiology and clinical outcomes.

BACKGROUND AND OBJECTIVES Hepatitis E virus (HEV) is one of the major causes of icteric hepatitis worldwide. In industrialized countries it is considered an emerging disease, as a growing number of autochthonous cases have been reported in recent years. Occasional extrahepatic manifestations have been described in the setting of HEV infection. STUDY DESIGN To characterize the epidemiological pattern and clinical outcomes of new cases of HEV infection diagnosed in two referral centers during the period 2011-2013. RESULTS During the study period, four cases of self-limited acute hepatitis E after travel to endemic areas were recorded, as well as five cases of HEV infection after solid organ transplantation. Four patients failed to spontaneously clear the virus and received ribavirin monotherapy; all of them had HEV genotype-3. Ribavirin was effective in inhibiting HEV replication, although in one patient a virological relapse occurred after the end of therapy. Finally, we report a case of HEV-genotype-3 related agranulocytosis in an immunocompetent patient, resulting in a fatal outcome; this is the first case reported of its kind. CONCLUSION Diagnosis of HEV infection needs to be taken into consideration in patients with acute or chronic hepatitis in whom other etiologies have been excluded. Although hematological complications related to acute HEV infection are infrequent, these may affect any of the bone marrow series, even after viral clearance.

Fitness and molecular mechanisms of resistance to rifaximin in in vitro selected Escherichia coli mutants.

AIMS This study sought to analyze the molecular mechanisms contributing to the development of rifaximin (Rfx) resistance in vitro in Escherichia coli. Twenty-eight Rfx-resistant mutants as well as four clinical isolates of E. coli were analyzed. The results obtained show that mutations in the rpoB gene and overexpression of Phe-Arg-β-naphthylamide (PAβN)-inhibitible efflux pump were implicated in Rfx resistance. RESULTS Amino acid substitutions at position 516 of the β-subunit of RNA polymerases were the most frequently obtained (53.6% of the mutants). The efflux pump inhibitor decreased the minimal inhibitory concentration (MIC) of 71.43% (20/28) of the mutant strains. CONCLUSIONS Mutations studied in the rpoB gene and overexpression of PAβN-inhibitible efflux pumps contribute to Rfx resistance (together or not), whereas alterations in porin levels do not seem to have a relevant role in the acquisition of Rfx resistance.