Differences in tetracycline resistance determinant carriage among Shigella flexneri and Shigella sonnei are not related to different plasmid Inc-type carriage.

OBJECTIVES: The aim of this study was to establish the prevalence of the most common molecular mechanisms involved in tetracycline resistance as well as their relationship with plasmid incompatibility (Inc) groups in a collection of Shigella spp. causing traveller's diarrhoea. METHODS: Tetracycline susceptibility was established in 187 Shigella spp. (74 Shigella flexneri and 113 Shigella sonnei), of which 153 isolates were recovered as a confirmed cause of traveller's diarrhoea. The prevalence of the tet(A), tet(B) and tet(G) genes was analysed by PCR. Eighteen plasmid Inc groups was determined in a subset of 59 isolates. RESULTS: Among 154 tetracycline-resistant isolates, 122 (79.2%) harboured at least tet(A) or tet(B). The tet(B) gene was the most frequently detected, being present in 70 isolates (45.5%), whilst tet(A) was detected in 57 isolates (37.0%). The tet(G) gene was present in only 11 (7.2%) isolates. Moreover, the tet(A) gene was more frequent in S. sonnei (P=0.0007), whilst the tet(B) gene was more frequent in S. flexneri (P<0.0001). Plasmids belonging to Inc group B (P<0.05) were significantly more frequent among S. flexneri, whilst those belonging to groups K, FIC and FIIA (P<0.05) were preferentially detected among S. sonnei. CONCLUSION: The prevalence of the tet(A) and tet(B) genes differed between S. sonnei and S. flexneri. Moreover, the prevalence of plasmid Inc groups in S. flexneri and S. sonnei differed. However, no relationship was found between the two phenomena.

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.

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.

IFNL4 polymorphism predicts response to hepatitis C treatment after liver transplantation.

BACKGROUND: Recent studies in chronic hepatitis C patients have shown that rs368234815 polymorphism nearby IL28B is a better predictor of response to antiviral treatment with pegylated interferon and ribavirin than IL28B polymorphisms (rs12979860 and rs8099917). Its effect could be related to interferon lambda 4 (IFNL4), a protein which seems to confer some paradoxical disadvantages in hepatitis C virus (HCV) immune response. OBJECTIVES: To assess the role of IFNL4 rs368234815 polymorphism on the response to antiviral treatment after liver transplantation (LT). STUDY DESIGN: IFNL4 and IL28B polymorphisms were genotyped in 86 HCV-infected LT recipients and in their donors; all patients had undergone antiviral treatment with pegylated interferon and ribavirin after LT. RESULTS: IFNL4 polymorphism strongly correlated with IL28B ones (p < 0.001). The favorable IFNL4 genotype (TT/TT) was significantly more frequent among donors than recipients (60% donors vs. 22% recipients, p <0.001). Recipient TT/TT genotype was associated with a higher sustained virological response rate after LT (p = 0.024). Nevertheless, the highest sustained virological response frequency was found when both donors and recipients had favorable genotypes (73% vs. 25%, p = 0.002), suggesting a role for donor genotype. CONCLUSIONS: Our study demonstrates that IFNL4 rs368234815 polymorphism is an important predictor of response to antiviral treatment in the LT setting. These findings warrant further studies on IFNL4 role in immune response against HCV.

Early periportal sinusoidal fibrosis is an accurate marker of accelerated HCV recurrence after liver transplantation.

BACKGROUND & AIMS: Significant liver fibrosis (F ⩾ 2) and portal hypertension (hepatic venous pressure gradient [HVPG] ⩾ 6 mmHg) 1 year after liver transplantation (LT) are predictors of severe hepatitis C recurrence. Periportal sinusoidal fibrosis (SF) is an early expression of the fibrogenic process in response to liver injury. We aimed to evaluate whether SF in early liver biopsies represents an early and accurate marker for identifying patients with severe HCV recurrence after LT. METHODS: A total of 101 HCV LT patients with early biopsy (<6 months), and HVPG measurement and/or liver biopsy one year after LT were included. Early biopsies were stained with Sirius Red and SF was graded semi-quantitatively. Results were compared between groups (significant SF vs. non-significant SF) and correlated with the development of severe HCV recurrence one year after LT. RESULTS: Patients with early significant SF had older donor age and higher necroinflammatory activity (NIA). The presence of early significant SF enabled identification of 78.9% and 90.6% of patients with F ⩾ 2 and HVPG ⩾ 6 mmHg, respectively, one year after LT. Donor age and NIA were independent predictors of significant fibrosis (F ⩾ 2) one year after LT, whereas donor age, ALT (3 months), NIA, and SF grade were independent predictors of portal hypertension (HVPG ⩾ 6). CONCLUSIONS: Significant SF in early biopsies is a good predictor of severe hepatitis C recurrence. This histological finding, when combined with simple variables, may be useful to select the best candidates for early antiviral therapy after LT.

Imaging of hepatitis C virus infection in liver grafts after liver transplantation.

BACKGROUND & AIMS: The detection of native hepatitis C virus (HCV) antigens in liver tissue may be relevant to diagnostic purposes and to better understand the pathogenesis of HCV infection. The aim of our study was to characterize HCV antigens in liver grafts. METHODS: We selected 32 liver transplant (LT) recipients with recurrent hepatitis C. HCV core and NS5A antigens were detected in formalin-fixed, paraffin-embedded (FFPE) liver biopsies obtained immediately after graft reperfusion (negative controls), during the acute phase of HCV infection (1-6 months) and during follow-up (7-74 months) after LT. Viral antigens were assessed by immunohistochemistry and confocal microscopy. RESULTS: All reperfusion biopsies were negative for both antigens. Core protein was detected in 75% and 33% of acute phase and follow-up biopsies, respectively. HCV antigens were not detected in any of the 10 samples from patients who cleared HCV after antiviral treatment. Immunostaining was hepatocellular, with a granular cytoplasmic pattern and a wide spectrum of intensity. We found a significant association between viral load and the presence of HCV core-positive hepatocytes (p=0.004). NS5A colocalized strongly with core (66%) and adipophilin (36%), supporting the localization of core and NS5A around lipid droplets. A detailed three-dimensional analysis showed that NS5A surrounded the core and adipophilin-positive areas. CONCLUSIONS: HCV antigens can be detected in FFPE liver biopsies by immunohistochemistry. The in vivo colocalization of core and NS5A proteins around the lipid droplets supports that the latter may play a role in virus particle production, similar to what reported in vitro.

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 R4°8), 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.

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-ß-naphthylamide (PAßN)-inhibitible efflux pump were implicated in Rfx resistance. RESULTS: Amino acid substitutions at position 516 of the ß-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 PAßN-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.

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 receptors claudin-1 and occludin after liver transplantation and influence on early viral kinetics.

UNLABELLED: 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.

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.

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

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 beta-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 beta-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

Antimicrobial susceptibility and mechanisms of resistance in Shigella and Salmonella isolates from children under five years of age with diarrhea in rural Mozambique.

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 beta-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 beta-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.

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.

In vitro antimicrobial activity of rifaximin against enteropathogens causing traveler's diarrhea.

The in vitro activity of rifaximin against 84 diarrheagenic Escherichia coli and 11 Shigella sonnei causing traveler's diarrhea was evaluated. The MIC of rifaximin ranged between <0.007 and 32 mg/L; other agents tested had an MIC of >256 mg/L in most cases. The results showed the potential use of rifaximin to treat these infections.