Antibiotic resistance, molecular characterizations, and clinical manifestations of Campylobacteriosis at a military medical center in Hawaii from 2012-2016: a retrospective analysis.

Hawaii has one of the highest incidences of Campylobacteriosis in the United States, but there remains little published data on circulating strains or antimicrobial resistance. We characterized 110 clinical Campylobacter isolates (106 C. jejuni, 4 C. coli) processed at Tripler Army Medical Center in Honolulu, HI from 2012-2016. Twenty-five percent of C. jejuni isolates exhibited fluoroquinolone (FQ) resistance, compared with 16% for tetracycline (TET), and 0% for macrolides. Two of the four C. coli isolates were resistant to FQ, TET, and macrolides. C. jejuni isolates further underwent multilocus sequence typing, pulsed-field gel electrophoresis, and molecular capsular typing. Nineteen capsule types were observed, with two capsule types (HS2 and HS9) being associated with FQ resistance (p < 0.001 and p = 0.006, respectively). HS2 FQ-resistant isolates associated with clonal complex 21, possibly indicating clonal spread in FQ resistance. Macrolides should be considered for treatment of suspect cases due to lack of observed resistance.

Hepatitis C Virus Frameshift/Alternate Reading Frame Protein Suppresses Interferon Responses Mediated by Pattern Recognition Receptor Retinoic-Acid-Inducible Gene-I.

Hepatitis C virus (HCV) actively evades host interferon (IFN) responses but the mechanisms of how it does so are not completely understood. In this study, we present evidence for an HCV factor that contributes to the suppression of retinoic-acid-inducible gene-I (RIG-I)-mediated IFN induction. Expression of frameshift/alternate reading frame protein (F/ARFP) from HCV -2/+1 frame in Huh7 hepatoma cells suppressed type I IFN responses stimulated by HCV RNA pathogen-associated molecular pattern (PAMP) and poly(IC). The suppression occurred independently of other HCV factors; and activation of interferon stimulated genes, TNFα, IFN-λ1, and IFN-λ2/3 was likewise suppressed by HCV F/ARFP. Point mutations in the full-length HCV sequence (JFH1 genotype 2a strain) were made to introduce premature termination codons in the -2/+1 reading frame coding for F/ARFP while preserving the original reading frame, which enhanced IFNα and IFNß induction by HCV. The potentiation of IFN response by the F/ARFP mutations was diminished in Huh7.5 cells, which already have a defective RIG-I, and by decreasing RIG-I expression in Huh7 cells. Furthermore, adding F/ARFP back via trans-complementation suppressed IFN induction in the F/ARFP mutant. The F/ARFP mutants, on the other hand, were not resistant to exogenous IFNα. Finally, HCV-infected human liver samples showed significant F/ARFP antibody reactivity, compared to HCV-uninfected control livers. Therefore, HCV F/ARFP likely cooperates with other viral factors to suppress type I and III IFN induction occurring through the RIG-I signaling pathway. This study identifies a novel mechanism of pattern recognition receptor modulation by HCV and suggests a biological function of the HCV alternate reading frame in the modulation of host innate immunity.

Real time application of whole genome sequencing for outbreak investigation - What is an achievable turnaround time?

Whole genome sequencing (WGS) is increasingly employed in clinical settings, though few assessments of turnaround times (TAT) have been performed in real-time. In this study, WGS was used to investigate an unfolding outbreak of vancomycin resistant Enterococcus faecium (VRE) among 3 patients in the ICU of a tertiary care hospital. Including overnight culturing, a TAT of just 48.5 h for a comprehensive report was achievable using an Illumina Miseq benchtop sequencer. WGS revealed that isolates from patient 2 and 3 differed from that of patient 1 by a single nucleotide polymorphism (SNP), indicating nosocomial transmission. However, the unparalleled resolution provided by WGS suggested that nosocomial transmission involved two separate events from patient 1 to patient 2 and 3, and not a linear transmission suspected by the time line. Rapid TAT's are achievable using WGS in the clinical setting and can provide an unprecedented level of resolution for outbreak investigations.

Ethanol and reactive species increase basal sequence heterogeneity of hepatitis C virus and produce variants with reduced susceptibility to antivirals.

Hepatitis C virus (HCV) exhibits a high level of genetic variability, and variants with reduced susceptibility to antivirals can occur even before treatment begins. In addition, alcohol decreases efficacy of antiviral therapy and increases sequence heterogeneity of HCV RNA but how ethanol affects HCV sequence is unknown. Ethanol metabolism and HCV infection increase the level of reactive species that can alter cell metabolism, modify signaling, and potentially act as mutagen to the viral RNA. Therefore, we investigated whether ethanol and reactive species affected the basal sequence variability of HCV RNA in hepatocytes. Human hepatoma cells supporting a continuous replication of genotype 1b HCV RNA (Con1, AJ242652) were exposed to ethanol, acetaldehyde, hydrogen peroxide, or L-buthionine-S,R-sulfoximine (BSO) that decreases intracellular glutathione as seen in patients. Then, NS5A region was sequenced and compared with genotype 1b HCV sequences in the database. Ethanol and BSO elevated nucleotide and amino acid substitution rates of HCV RNA by 4-18 folds within 48 hrs which were accompanied by oxidative RNA damage. Iron chelator and glutathione ester decreased both RNA damage and mutation rates. Furthermore, infectious HCV and HCV core gene were sufficient to induce oxidative RNA damage even in the absence of ethanol or BSO. Interestingly, the dn/ds ratio and percentage of sites undergoing positive selection increased with ethanol and BSO, resulting in an increased detection of NS5A variants with reduced susceptibility to interferon alpha, cyclosporine, and ribavirin and others implicated in immune tolerance and modulation of viral replication. Therefore, alcohol is likely to synergize with virus-induced oxidative/nitrosative stress to modulate the basal mutation rate of HCV. Positive selection induced by alcohol and reactive species may contribute to antiviral resistance.

Hepatocyte NAD(P)H oxidases as an endogenous source of reactive oxygen species during hepatitis C virus infection.

UNLABELLED: Oxidative stress has been identified as a key mechanism of hepatitis C virus (HCV)-induced pathogenesis. Studies have suggested that HCV increases the generation of hydroxyl radical and peroxynitrite close to the cell nucleus, inflicting DNA damage, but the source of reactive oxygen species (ROS) remains incompletely characterized. We hypothesized that HCV increases the generation of superoxide and hydrogen peroxide close to the hepatocyte nucleus and that this source of ROS is reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase 4 (Nox4). Huh7 human hepatoma cells and telomerase-reconstituted primary human hepatocytes, transfected or infected with virus-producing HCV strains of genotypes 2a and 1b, were examined for messenger RNA (mRNA), protein, and subcellular localization of Nox proteins along with the human liver. We found that genotype 2a HCV induced persistent elevations of Nox1 and Nox4 mRNA and proteins in Huh7 cells. HCV genotype 1b likewise elevated the levels of Nox1 and Nox4 in telomerase-reconstituted primary human hepatocytes. Furthermore, Nox1 and Nox4 proteins were increased in HCV-infected human liver versus uninfected liver samples. Unlike Nox1, Nox4 was prominent in the nuclear compartment of these cells as well as the human liver, particularly in the presence of HCV. HCV-induced ROS and nuclear nitrotyrosine could be decreased with small interfering RNAs to Nox1 and Nox4. Finally, HCV increased the level of transforming growth factor beta 1 (TGFbeta1). TGFbeta1 could elevate Nox4 expression in the presence of infectious HCV, and HCV increased Nox4 at least in part through TGFbeta1. CONCLUSION: HCV induced a persistent elevation of Nox1 and Nox4 and increased nuclear localization of Nox4 in hepatocytes in vitro and in the human liver. Hepatocyte Nox proteins are likely to act as a persistent, endogenous source of ROS during HCV-induced pathogenesis.

Ethanol enhances hepatitis C virus replication through lipid metabolism and elevated NADH/NAD+.

UNLABELLED: Ethanol has been suggested to elevate HCV titer in patients and to increase HCV RNA in replicon cells, suggesting that HCV replication is increased in the presence and absence of the complete viral replication cycle, but the mechanisms remain unclear. In this study, we use Huh7 human hepatoma cells that naturally express comparable levels of CYP2E1 as human liver to demonstrate that ethanol, at subtoxic and physiologically relevant concentrations, enhances complete HCV replication. The viral RNA genome replication is affected for both genotypes 2a and 1b. Acetaldehyde, a major product of ethanol metabolism, likewise enhances HCV replication at physiological concentrations. The potentiation of HCV replication by ethanol is suppressed by inhibiting CYP2E1 or aldehyde dehydrogenase and requires an elevated NADH/NAD(+) ratio. In addition, acetate, isopropyl alcohol, and concentrations of acetone that occur in diabetics enhance HCV replication with corresponding increases in the NADH/NAD(+). Furthermore, inhibiting the host mevalonate pathway with lovastatin or fluvastatin and fatty acid synthesis with 5-(tetradecyloxy)-2-furoic acid or cerulenin significantly attenuates the enhancement of HCV replication by ethanol, acetaldehyde, acetone, as well as acetate, whereas inhibiting beta-oxidation with beta-mercaptopropionic acid increases HCV replication. Ethanol, acetaldehyde, acetone, and acetate increase the total intracellular cholesterol content, which is attenuated with lovastatin. In contrast, both endogenous and exogenous ROS suppress the replication of HCV genotype 2a, as previously shown with genotype 1b. CONCLUSION: Therefore, lipid metabolism and alteration of cellular NADH/NAD(+) ratio are likely to play a critical role in the potentiation of HCV replication by ethanol rather than oxidative stress.

Redox regulation of hepatitis C in nonalcoholic and alcoholic liver.

Hepatitis C virus (HCV) is an RNA virus of the Flaviviridae family that is estimated to have infected 170 million people worldwide. HCV can cause serious liver disease in humans, such as cirrhosis, steatosis, and hepatocellular carcinoma. HCV induces a state of oxidative/nitrosative stress in patients through multiple mechanisms, and this redox perturbation has been recognized as a key player in HCV-induced pathogenesis. Studies have shown that alcohol synergizes with HCV in the pathogenesis of liver disease, and part of these effects may be mediated by reactive species that are generated during hepatic metabolism of alcohol. Furthermore, reactive species and alcohol may influence HCV replication and the outcome of interferon therapy. Alcohol consumption has also been associated with increased sequence heterogeneity of the HCV RNA sequences, suggesting multiple modes of interaction between alcohol and HCV. This review summarizes the current understanding of oxidative and nitrosative stress during HCV infection and possible combined effects of HCV, alcohol, and reactive species in the pathogenesis of liver disease.

Redox modulation of the hepatitis C virus replication complex is calcium dependent.

Reactive species and perturbation of the redox balance have been implicated in the pathogenesis of many viral diseases, including hepatitis C. Previously, we made a surprising discovery that concentrations of H(2)O(2) that are nontoxic to host cells disrupted the hepatitis C virus (HCV) replication complex (RC) in Huh7 human hepatoma cells in a manner that suggested signaling. Here, we show that H(2)O(2) and interferon-gamma have comparable effects on the HCV subgenomic and genomic RNA replication in Huh7 cells. H(2)O(2) induced a gradual rise in the intracellular calcium concentration ([Ca(2+)](i)). Both rapid and sustained suppression of HCV RNA replication by H(2)O(2) depended on this calcium elevation. The peroxide-induced [Ca(2+)](i) elevation was independent of extracellular calcium and derived, at least in part, from the endoplasmic reticulum. Likewise, the suppression of the HCV RC by H(2)O(2) was independent of extracellular calcium but required an intracellular calcium source. Other agents that elevated [Ca(2+)](i) could also suppress the HCV RC, suggesting that calcium elevation might be sufficient to suppress HCV RNA replication. In conclusion, oxidants may modulate the HCV RC through calcium. Effects on the infectivity and the morphogenesis of HCV remain to be determined. These findings suggest possible regulatory roles for redox and calcium signaling during viral infections.