First report of Acinetobacter pittii acute community-acquired pneumonia in an immunocompetent patient in France following a heat wave.

BACKGROUND: In recent years, Acinetobacter baumannii-calcoaceticus complex (ABC) infections have attracted attention, mainly because of the impact of carbapenem-resistant isolates in hospital-acquired infections. However, acute community-acquired ABC infections are not uncommon in warm and humid countries, where they are responsible for community-acquired infections with specific clinical features. To date, such infection has not been reported in France. CASE PRESENTATION: We report the case of a 55-year-old non-immunocompromised patient living in France with no known risk factors for community-acquired ABC infections who presented pneumonia with bloodstream infection due to wild-type A. pittii. The outcome was favorable after 7 days of antibiotic treatment with cefepime. We confirmed bacterial identification with whole-genome sequencing, and we examined the A. pitii core-genome phylogeny for genomic clusters. CONCLUSIONS: This situation is uncommon in Europe and occurred after a heat wave in France with temperatures above 38 °C. Herein, we discuss the possibility that this pneumonia may be emerging in the current context of global warming.

The Population Genomics of Increased Virulence and Antibiotic Resistance in Human Commensal Escherichia coli over 30 Years in France.

Escherichia coli is a commensal species of the lower intestine but is also a major pathogen causing intestinal and extraintestinal infections that is increasingly prevalent and resistant to antibiotics. Most studies on genomic evolution of E. coli used isolates from infections. Here, instead, we whole-genome sequenced a collection of 403 commensal E. coli isolates from fecal samples of healthy adult volunteers in France (1980 to 2010). These isolates were distributed mainly in phylogroups A and B2 (30% each) and belonged to 152 sequence types (STs), the five most frequent being ST10 (phylogroup A; 16.3%), ST73 and ST95 (phylogroup B2; 6.3 and 5.0%, respectively), ST69 (phylogroup D; 4.2%), and ST59 (phylogroup F; 3.9%), and 224 O:H serotypes. ST and serotype diversity increased over time. The O1, O2, O6, and O25 groups used in bioconjugate O-antigen vaccine against extraintestinal infections were found in 23% of the strains of our collection. The increase in frequency of virulence-associated genes and antibiotic resistance was driven by two evolutionary mechanisms. Evolution of virulence gene frequency was driven by both clonal expansion of STs with more virulence genes ("ST-driven") and increases in gene frequency within STs independent of changes in ST frequencies ("gene-driven"). In contrast, the evolution of resistance was dominated by increases in frequency within STs ("gene-driven"). This study provides a unique picture of the phylogenomic evolution of E. coli in its human commensal habitat over 30 years and will have implications for the development of preventive strategies. IMPORTANCE Escherichia coli is an opportunistic pathogen with the greatest burden of antibiotic resistance, one of the main causes of bacterial infections and an increasing concern in an aging population. Deciphering the evolutionary dynamics of virulence and antibiotic resistance in commensal E. coli is important to understand adaptation and anticipate future changes. The gut of vertebrates is the primary habitat of E. coli and probably where selection for virulence and resistance takes place. Unfortunately, most whole-genome-sequenced strains are isolated from pathogenic conditions. Here, we whole-genome sequenced 403 E. coli commensals isolated from healthy French subjects over a 30-year period. Virulence genes increased in frequency by both clonal expansion of clones carrying them and increases in frequency within clones, whereas resistance genes increased by within-clone increased frequency. Prospective studies of E. coli commensals should be performed worldwide to have a broader picture of evolution and adaptation of this species.

Evidence of Sexual Transmission of Extended-Spectrum ß-Lactamase-Producing Enterobacterales: A Cross-sectional and Prospective Study.

BACKGROUND: Extended-spectrum ß-lactamase-producing Enterobacterales (ESBL-E) represent a major threat to public health. Little is known on their potential for sexual transmission. METHODS: We recruited individuals at a sexually transmitted infection and human immunodeficiency virus (HIV) outpatient clinic in Paris, France, in whom we evaluated the prevalence of ESBL-E intestinal carriage and, among those testing positive, the proportion with clearance 6 months thereafter. We compared carriage prevalence between groups using logistic regression adjusted for age, geographic origin, travel outside Europe, and antibiotic use in the past 6 months. RESULTS: A total of 2157 individuals participated, of whom 226 (10.5%) were ESBL-E carriers. The proportions of ESBL-E carriers varied across sexual groups and were as follows: HIV-negative men who have sex with men (MSM) and who were on preexposure prophylaxis (PrEP), 16.3% (41 of 251); HIV-negative MSM not on PrEP, 9.7% (47 of 487); HIV-positive MSM, 12.2% (61 of 500); HIV-negative men who have sex exclusively with women, 10.0% (44 of 439); and HIV-negative women who have sex with men, 6.9% (n = 33 of 480). After adjustment, ESBL-E prevalence was significantly higher in HIV-negative MSM on PrEP (P < .001) and HIV-positive MSM (P = .01) than in women who have sex with men. A higher number of sexual partners in the past 6 months was associated with ESBL-E carriage after adjustment (P = .004). Escherichia coli sequence type 14 and blaSHV-12-producing ESBL-E were observed only in MSM. Of 102 individuals with ESBL-E returning for testing, 26 (25%) had carriage at 6 months. CONCLUSION: ESBL-E carriage is more frequent in MSM undergoing PrEP or living with HIV and with increasing number of sexual partners. More research is warranted to understand the consequences of ESBL-E carriage in these populations and how transmission can be reduced.

Reduced Chlorhexidine Susceptibility Is Associated with Tetracycline Resistance tet Genes in Clinical Isolates of Escherichia coli.

Chlorhexidine is a widely used antiseptic in hospital and community health care. Decreased susceptibility to this compound has been recently described in Klebsiella pneumoniae and Pseudomonas aeruginosa, together with cross-resistance to colistin. Surprisingly, few data are available for Escherichia coli, the main species responsible for community and health care-associated infections. In order to decipher chlorhexidine resistance mechanisms in E. coli, we studied both in vitro derived and clinical isolates through whole-genome sequence analysis. Comparison of strains grown in vitro under chlorhexidine pressure identified mutations in the gene mlaA coding for a phospholipid transport system. Phenotypic analyses of single-gene mutants from the Keio collection confirmed the role of this mutation in the decreased susceptibility to chlorhexidine. However, mutations in mlaA were not found in isolates from large clinical collections. In contrast, genome wide association studies (GWAS) showed that, in clinical strains, chlorhexidine reduced susceptibility was associated with the presence of tetA genes of class B coding for efflux pumps and located in a Tn10 transposon. Construction of recombinant strains in E. coli K-12 confirmed the role of tetA determinant in acquired resistance to both chlorhexidine and tetracycline. Our results reveal that two different evolutionary paths lead to chlorhexidine decreased susceptibility: one restricted to in vitro evolution conditions and involving a retrograde phospholipid transport system; the other observed in clinical isolates associated with efflux pump TetA. None of these mechanisms provide cross-resistance to colistin. This work demonstrates the GWAS power to identify new resistance mechanisms in bacterial species.

Phylogroup stability contrasts with high within sequence type complex dynamics of Escherichia coli bloodstream infection isolates over a 12-year period.

BACKGROUND: Escherichia coli is the leading cause of bloodstream infections, associated with a significant mortality. Recent genomic analyses revealed that few clonal lineages are involved in bloodstream infections and captured the emergence of some of them. However, data on within sequence type (ST) population genetic structure evolution are rare. METHODS: We compared whole genome sequences of 912 E. coli isolates responsible for bloodstream infections from two multicenter clinical trials that were conducted in the Paris area, France, 12 years apart, in teaching hospitals belonging to the same institution ("Assistance Publique-Hôpitaux de Paris"). We analyzed the strains at different levels of granularity, i.e., the phylogroup, the ST complex (STc), and the within STc clone taking into consideration the evolutionary history, the resistance, and virulence gene content as well as the antigenic diversity of the strains. RESULTS: We found a mix of stability and changes overtime, depending on the level of comparison. Overall, we observed an increase in antibiotic resistance associated to a restricted number of genetic determinants and in strain plasmidic content, whereas phylogroup distribution and virulence gene content remained constant. Focusing on STcs highlighted the pauci-clonality of the populations, with only 11 STcs responsible for more than 73% of the cases, dominated by five STcs (STc73, STc131, STc95, STc69, STc10). However, some STcs underwent dramatic variations, such as the global pandemic STc131, which replaced the previously predominant STc95. Moreover, within STc131, 95 and 69 genomic diversity analysis revealed a highly dynamic pattern, with reshuffling of the population linked to clonal replacement sometimes coupled with independent acquisitions of virulence factors such as the pap gene cluster bearing a papGII allele located on various pathogenicity islands. Additionally, STc10 exhibited huge antigenic diversity evidenced by numerous O:H serotype/fimH allele combinations, whichever the year of isolation. CONCLUSIONS: Altogether, these data suggest that the bloodstream niche is occupied by a wide but specific phylogenetic diversity and that highly specialized extra-intestinal clones undergo frequent turnover at the within ST level. Additional worldwide epidemiological studies overtime are needed in different geographical and ecological contexts to assess how generalizable these data are.

Genomic characterization of 16S rRNA methyltransferase-producing Escherichia coli isolates from the Parisian area, France.

BACKGROUND: The resistance to all aminoglycosides (AGs) conferred by 16S rRNA methyltransferase enzymes (16S-RMTases) is a major public health concern. OBJECTIVES: To characterize the resistance genotype, its genetic environment and plasmid support, and the phylogenetic relatedness of 16S-RMTase-producing Escherichia coli from France. METHODS: We screened 137 E. coli isolates resistant to all clinically relevant AGs from nine Parisian hospitals for 16S-RMTases. WGS was performed on clinical isolates with high-level AG resistance (MIC ≥256 mg/L) and their transformants. RESULTS: Thirty of the 137 AG-resistant E. coli produced 16S-RMTases: 11 ArmA, 18 RmtB and 1 RmtC. The 16S-RMTase producers were also resistant to third-generation cephalosporins (90% due to a blaCTX-M gene), co-trimoxazole, fluoroquinolones and carbapenems (blaNDM and blaVIM genes) in 97%, 83%, 70% and 10% of cases, respectively. Phylogenomic diversity was high in ArmA producers, with 10 different STs, but a similar genetic environment, with the Tn1548 transposon carried by a plasmid closely related to pCTX-M-3 in 6/11 isolates. Conversely, RmtB producers belonged to 12 STs, the most frequent being ST405 and ST complex (STc) 10 (four and four isolates, respectively). The rmtB gene was carried by IncF plasmids in 10 isolates and was found in different genetic environments. The rmtC gene was carried by the pNDM-US plasmid. CONCLUSIONS: ArmA and RmtB are the predominant 16S-RMTases in France, but their spread follows two different patterns: (i) dissemination of a conserved genetic support carrying armA in E. coli with high levels of genomic diversity; and (ii) various genetic environments surrounding rmtB in clonally related E. coli.

Comparative Whole-Genome Phylogeny of Animal, Environmental, and Human Strains Confirms the Genogroup Organization and Diversity of the Stenotrophomonas maltophilia Complex.

The Stenotrophomonas maltophilia complex (Smc) comprises opportunistic environmental Gram-negative bacilli responsible for a variety of infections in both humans and animals. Beyond its large genetic diversity, its genetic organization in genogroups was recently confirmed through the whole-genome sequencing of human and environmental strains. As they are poorly represented in these analyses, we sequenced the whole genomes of 93 animal strains to determine their genetic background and characteristics. Combining these data with 81 newly sequenced human strains and the genomes available from RefSeq, we performed a genomic analysis that included 375 nonduplicated genomes with various origins (animal, 104; human, 226; environment, 30; unknown, 15). Phylogenetic analysis and clustering based on genome-wide average nucleotide identity confirmed and specified the genetic organization of Smc in at least 20 genogroups. Two new genogroups were identified, and two previously described groups were further divided into two subgroups each. Comparing the strains isolated from different host types and their genogroup affiliation, we observed a clear disequilibrium in certain groups. Surprisingly, some antimicrobial resistance genes, integrons, and/or clusters of attC sites lacking integron-integrase (CALIN) sequences targeting antimicrobial compounds extensively used in animals were mainly identified in animal strains. We also identified genes commonly found in animal strains coding for efflux systems. The result of a large whole-genome analysis performed by us supports the hypothesis of the putative contribution of animals as a reservoir of Stenotrophomonas maltophilia complex strains and/or resistance genes for strains in humans.IMPORTANCE Given its naturally large antimicrobial resistance profile, the Stenotrophomonas maltophilia complex (Smc) is a set of emerging pathogens of immunosuppressed and cystic fibrosis patients. As it is group of environmental microorganisms, this adaptation to humans is an opportunity to understand the genetic and metabolic selective mechanisms involved in this process. The previously reported genomic organization was incomplete, as data from animal strains were underrepresented. We added the missing piece of the puzzle with whole-genome sequencing of 93 strains of animal origin. Beyond describing the phylogenetic organization, we confirmed the genetic diversity of the Smc, which could not be estimated through routine phenotype- or matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF)-based laboratory tests. Animals strains seem to play a key role in the diversity of Smc and could act as a reservoir for mobile resistance genes. Some genogroups seem to be associated with particular hosts; the genetic support of this association and the role of the determinants/corresponding genes need to be explored.

Outbreak of an Uncommon Rifampin-resistant blaNDM-1Citrobacter amalonaticus Strain in a Digestive Rehabilitation Center: The Putative Role of Rifaximin.

We describe a sudden 2-week outbreak due to a blaNDM-1Citrobacter amalonaticus strain in a 22-bed digestive rehabilitation center. Three of the 5 colonized patients received long-term rifaximin treatment to prevent hepatic encephalopathy. The strains were genotypically identical, phenotypically resistant to rifampin, and harbored arr-3, a rifampin adenosine diphosphate-ribosyl transferase.

Characterization of the dynamics of human cytomegalovirus resistance to antiviral drugs by ultra-deep sequencing.

Prophylactic or preemptive treatment strategies are required to prevent human cytomegalovirus (CMV) infections in transplant recipients. However, treatment failure occurs when CMV resistant-associated variants (RAVs) are selected. Although the diversity of CMV is lower than that of RNA viruses, CMV appears to show some genetic instability, with possible minor emerging resistance that may be undetectable by Sanger sequencing. We aimed to examine CMV-resistance mutations over time by ultra-deep sequencing (UDS) and Sanger sequencing in a kidney transplant recipient experiencing CMV infection. This patient showed a transient response to three different antiviral drugs (valganciclovir, foscarnet, and maribavir) and four episodes of CMV resistance over two years. The full-length UL97 (2.3kpb) and partial UL54 (2.4kpb) CMV genes were studied by UDS and Sanger sequencing and linkage mutations calculated to determine RAVs. We detected four major and five minor resistance mutations. Minor resistant variants (2-20%) were detected by UDS, whereas major resistance substitutions (>20%) were identified by both UDS and Sanger method. We detected cross-resistance to three drugs, despite high CMV loads, suggesting that the fitness of the viral mutants was not impaired. In conclusion, CMV showed complex dynamic of resistance under antiviral drug pressure, as described for highly variable viruses. The emergence of successive RAVs constitutes a clinically challenging complication and contributes to the difficulty of therapeutic management of patients.

Added value of ultra-deep sequencing (UDS) approach for detection of genotypic antiviral resistance of herpes simplex virus (HSV).

Classically, Sanger sequencing is considered the gold standard for detection of HSV drug resistance mutations (DRMs). As a complementary method, ultra-deep sequencing (UDS) has an improved ability to detect minor variants and mixed populations. The aim of this work was to apply UDS performed on MiSeq® Illumina platform to the detection of HSV DRMs and to the evaluation of the subpopulation diversity in clinical samples in comparison with Sanger sequencing. A total of 59 HSV-positive clinical samples (31 HSV-1 and 28 HSV-2) recovered from 50 patients mainly immunocompromised (70%) were retrospectively analyzed. Remarkably, UDS analysis revealed significant differences of relative abundance according to the type of DRMs within TK and Pol: natural polymorphisms and amino acid changes associated with resistance to antivirals were identified as high-abundant mutations (>96%), whereas TK frameshifts conferring resistance to ACV were systematically detected at lower abundance (≈80%). This work also revealed that UDS can detect low-frequency DRMs and provides extensive information on viral population composition.

Performance assessment of a fully automated deep sequencing platform for HCV resistance testing.

BACKGROUND: International liver society guidelines recommended to perform HCV resistance testing at baseline of first-line therapy with certain combination regimens or prior to retreatment in patients previously exposed to a direct-acting antiviral (DAA) containing regimen. Currently, no standardized assays have been developed as purchasable kits for HCV resistance testing. The aim of this study was to evaluate the performance of the Sentosa SQ HCV Genotyping Assay, a novel deep sequencing-based assay, to identify resistance-associated substitutions (RASs) in the NS3 protease, NS5A protein domain I and NS5B polymerase regions for patients infected with HCV genotypes-1a and 1b. METHODS: Serum samples collected from patients with chronic hepatitis C infection who failed to achieve a sustained virological response after receiving a DAA-containing treatment regimen were extracted and sequenced by two methods including population sequencing of the NS3, NS5A and NS5B coding region reference method and the deep sequencing-based Sentosa SQ HCV Genotyping Assay. RESULTS: A high concordance rate with Sanger sequencing, the reference method, was found for the NS3, NS5A and NS5 coding regions, regardless of the genotype-1 subtypes. The deep sequencing-based assay was more sensitive than population sequencing to detect minority variants, representing less than 10% of the viral populations, but also some variants representing up to 30% of the viral quasispecies, as expected. CONCLUSIONS: The Sentosa SQ HCV Genotyping Assay can be confidently used in clinical practice in the indications of HCV resistance testing for these subtypes. Technical improvements are now required to allow for pangenotypic coverage.

Colistin resistance in Parisian inpatient faecal Escherichia coli as the result of two distinct evolutionary pathways.

BACKGROUND: Beyond plasmid-encoded resistance (mcr genes) prevalence in strain collections, large epidemiological studies to estimate the human burden of colistin-resistant Escherichia coli gut carriage are lacking. OBJECTIVES: To evaluate the prevalence of colistin-resistant E. coli carriage in inpatients and decipher the molecular support of resistance and the genetic background of the strains. METHODS: During a 3 month period in 2017, we prospectively screened patients in six Parisian hospitals for rectal carriage of colistin-resistant E. coli using a selective medium, a biochemical confirmatory test and MIC determination. WGS of the resistant strains and their corresponding plasmids was performed. RESULTS: Among the 1217 screened patients, 153 colistin-resistant E. coli strains were isolated from 152 patients (12.5%). The mcr-1 gene was identified in only seven isolates (4.6%) on different plasmid scaffolds. The genetic background of these MCR-1 producers argued for an animal origin. Conversely, the remaining 146 colistin-resistant E. coli exhibited a phylogenetic distribution corresponding to human gut commensal/clinical population structure (B2 and D phylogroup predominance); 72.6% of those isolates harboured convergent mutations in the PmrA and PmrB proteins, constituting a two-component system shown to be associated with colistin resistance. CONCLUSIONS: We showed that the occurrence at a high rate of colistin resistance in human faecal E. coli is the result of two distinct evolutionary pathways, i.e. the occurrence of chromosomal mutations in an endogenous E. coli population and the rare acquisition of exogenous mcr-1-bearing strains probably of animal origin. The involved selective pressures need to be identified in order to develop preventative strategies.

Genotypic and phenotypic characteristics of Escherichia coli involved in transfusion-transmitted bacterial infections: implications for preventive strategies.

BACKGROUND: Transfusion-transmitted bacterial infections (TTBIs) are the main residual infectious complications of transfusions. Escherichia coli and platelet (PLT) concentrates may be epidemiologically associated, leading to severe, if not lethal, TTBIs. We investigated the genotypic and phenotypic reasons for this clinically deleterious combination. STUDY DESIGN AND METHODS: We investigated a French national E. coli strain collection related to six independent episodes of TTBIs. Their phenotypic characterizations included antibiotic susceptibility testing, growth testing under different culture conditions, serum survival assays, and virulence in a sepsis mouse model. Their genotypic characterizations included polymerase chain reaction phylotyping, whole genome sequencing, and a subsequent in silico analysis. RESULTS: We highlighted a selection process of highly extraintestinal virulent strains, mainly belonging to the B2 phylogroup, adapted to the hostile environment (high citrate concentration and a bactericidal serum effect) of apheresis-collected platelet concentrates (PCs). Compared to controls, the E. coli TTBI strains grew faster in the PCs due to a superior ability to capture iron. The in vitro growth performances were highly compatible with blood-derived product real-life conditions, including storage conditions and delays. The consistent serum resistance of TTBI strains promotes their survival in both the donor's and the receiver's blood and in the PCs. CONCLUSION: This study pointed out that E. coli strains responsible for TTBI exhibit very specific traits. They belong to the extraintestinal pathogenic phylogroups and have a high intrinsic virulence. They can be resistant to complement, capture iron, and grow in the apheresis-collected PCs. These findings therefore support the reinforcement of the postdonation information.

Drug resistance among patients who acquired HIV infection in a preexposure prophylaxis trial.

BACKGROUND: The IPERGAY ANRS trial showed that on-demand preexposure prophylaxis (PrEP) with tenofovir (TDF) and emtricitabine (FTC) was highly effective in preventing HIV infection among highly exposed MSM. Here, we analyzed drug resistance-associated mutations (RAMs) among all participants who acquired HIV infection during this trial. METHODS: Resistance was analyzed on frozen plasma at the time of HIV diagnosis among participants enrolled in the double-blind and open-label phases of the ANRS IPERGAY trial. Reverse transcriptase sequencing was performed, using population-based and ultradeep sequencing (454 GS Flex). Adherence was measured by pill counting and by plasma tenofovir and FTC assay. RESULTS: During the trial, 31 participants were diagnosed with HIV-1 infection (subtype B, 64.5%), using antigen/antibody immune assay in 29 cases and plasma HIV RNA assay in two. The median plasma HIV-1 RNA level was 5.52 log10 copies/ml. Drug resistance was tested in 12 participants before starting PrEP, in six assigned to TDF/FTC group and in 13 assigned to placebo group. Primary resistance to nucleoside reverse transcriptase inhibitors (zidovudine) and/or nonnucleoside reverse transcriptase inhibitors was detected in six participants (19%; 95% confidence interval 7-42). No major or minor TDF-resistant or FTC-resistant variants were detected. CONCLUSION: No TDF or FTC resistance-associated mutations were found among participants who acquired HIV in the ANRS IPERGAY trial.

Ultra-deep sequencing improves the detection of drug resistance in cellular DNA from HIV-infected patients on ART with suppressed viraemia.

Background: Standard genotypic tests performed on HIV DNA from patients on suppressive ART, with previous resistance-associated mutations (RAMs) detected in their plasma, underestimate resistance. We thus compared ultra-deep sequencing (UDS) with bulk sequencing of DNA to detect RAMs previously identified in plasma. Methods: We sequenced the DNA of 169 highly treatment experienced patients with suppressed viraemia (ANRS 138-EASIER trial). Protease (PR) and reverse transcriptase (RT) genes from HIV DNA were sequenced by bulk sequencing and UDS, comparing 1% and 20% as thresholds of detection for UDS. Results: Patients were highly treatment experienced (13.6 years). UDS of DNA was successful for the RT and PR genes in 133 (79%) and 137 (81%) patients, respectively. The detection of RAMs was similar by bulk sequencing and UDS with a 20% cut-off. However, the detection of RAMs by UDS with a 1% cut-off was significantly higher than that of bulk sequencing for RT codons D67N (65.4% versus 52.3%), M184V (66.2% versus 52.3%), L210W (48.9% versus 36.4%) and T215Y (57.9% versus 42.1%) and PR codons M46I (46% versus 26%), I54L (12.4% versus 3.9%), V82A (44.5% versus 29.9%) and L90M (57.7% versus 42.5%). Conclusions: Genotypic resistance testing of cellular HIV DNA of well-controlled patients should use UDS technology with a sensitivity threshold of 1% to improve the detection of the resistant reservoir.

Are animals a source of Stenotrophomonas maltophilia in human infections? Contributions of a nationwide molecular study.

Stenotrophomonas maltophilia (Sm) is an archetypal environmental opportunistic bacterium responsible for health care-associated infections. The role of animals in human Sm infections is unknown. This study aims to reveal the genetic and phylogenetic relationships between pathogenic strains of Sm, both animal and human, and identify a putative role for animals as a reservoir in human infection. We phenotypically and genotypically characterized 61 Sm strains responsible for animal infections (mainly respiratory tract infections in horses) from a French nationwide veterinary laboratory network. We tested antimicrobial susceptibility and performed MLST and genogrouping using the concatenation of the seven housekeeping genes from the original MLST scheme. Excluding the eight untypeable strains owing to the lack of gene amplification, only 10 out of the 53 strains yielded a known ST (ST5, ST39, ST162, ST8, ST27, ST126, ST131). The genogroup distribution highlighted not only genogroups (genogroups 5 and 9) comprised exclusively of animal strains but also genogroups shared by human and animal strains. Interestingly, these shared genogroups were primarily groups 2 and 6, which have previously been identified as the two most frequent genogroups among human-pathogenic Sm strains, especially among respiratory pathogens. The antimicrobial susceptibility testing underlined the presence of acquired resistance: 18.8 and 7.5% of the tested isolates were resistant to the sulfonamide-trimethoprim combination and ciprofloxacin, respectively. Animal strains of Sm shared phylogenetic traits with some of the most successful human strains. The exact relationships between the human and animal strains, and the genetic support of these common traits, need to be determined.

A novel standardized deep sequencing-based assay for hepatitis C virus genotype determination.

Hepatitis C virus (HCV) genotype and subtype (1a/1b) identification is needed to tailor anti-HCV therapy. Currently available methods accurately identify the genotype and differentiate subtypes 1a from 1b. However, these assays have not been designed to identify other HCV subtypes, nor to recognize mixed genotype/subtype infections, emphasizing the need for a high-resolution system based on phylogenetic analysis of reads obtained by deep sequencing of a relevant genome region. The aim of this study was to evaluate the performance of the Sentosa SQ HCV Genotyping Assay, a novel deep sequencing-based assay targeting the HCV nonstructural 5B (NS5B) region, in clinical samples from patients with an indication for anti-HCV therapy. A high concordance rate with Sanger sequencing of the NS5B region, the reference method, was found for genotype 1 to 6 determination, 1a/1b subtype identification, and genotype 4, 5 and 6 subtyping. Discrepancies were seen essentially for HCV genotype 2 subtyping. Overall, the performance of the deep sequencing-based assay in generating the genotypes/subtype information needed to tailor anti-HCV treatment was adequate in this study. Further improvements, such as a longer NS5B fragment analyzed and enriching the database of reference prototype strains used for subtype assignment would make it a method of choice for HCV genotyping and subtyping for future clinical practice and research.

Utility of ultra-deep sequencing for detection of varicella-zoster virus antiviral resistance mutations.

We report the first application of ultra-deep sequencing (UDS) to varicella-zoster virus (VZV) genotypic antiviral testing in a case of acyclovir-resistant VZV infection initially detected by Sanger sequencing within a deeply immunocompromised heart transplant recipient. As added-value compared to Sanger analysis, UDS revealed complex dynamics of viral population under antiviral pressure. Varicella-zoster virus (VZV) is a ubiquitous human herpesvirus affecting populations worldwide. VZV is commonly acquired in youth whose primary infection usually manifests as benign varicella (chickenpox). After the initial infection, the virus establishes lifelong latency in sensory ganglia leading to a risk of subsequent reactivation. Reactivation usually results in the development of localized herpes zoster (HZ) lesions, a painful skin rash commonly known as shingles (Cohen, 2013). The incidence and severity of HZ increase with impaired specific cell-mediated immunity mainly as a result of increasing age, malignancy, immunodeficiency, organ transplantation, or immunosuppressive drug therapy (Cohen, 2013; Koo et al., 2014; Pavlopoulou et al., 2015). In particular, HZ remains a significant cause of morbidity among solid organ transplant (SOT) recipients, especially in patients undergoing heart transplantation (HT) compared with liver, kidney, or lung transplant recipients (Carby et al., 2007; Koo et al., 2014; Pavlopoulou et al., 2015). These particular individuals are at increased risk of primary infection, reactivation followed by dissemination with visceral involvement and associated with bacterial superinfection, and chronic recurrences (Cohen, 2013). VZV infections may also engender debilitating neuralgia among highly immunocompromised patients (Sampathkumar et al., 2009). HT is also associated with the risk of reactivation of other latent viruses belonging to the Herpesviridae family as herpes simplex virus (HSV). Currently licensed drugs to prevent or to cure HSV- or VZV-associated diseases target the viral DNA polymerase (Pol). Acyclovir (ACV) and its prodrug valacyclovir (VACV) are considered as the first-line therapy, whereas foscarnet (FOS) or cidofovir (CDV) constitute alternative options. After primophosphorylation by the viral thymidine kinase (TK), ACV targets the viral DNA polymerase and inhibits the viral genome replication by a chain termination mechanism. According to this mechanism of action, viral mutations conferring resistance to ACV have been mapped both in TK and Pol encoding genes. Viral mutations conferring resistance to FOS and CDV are only detected in Pol gene. VZV ACV-resistance is mostly mediated by TK alterations, consisting in either translational frameshifts, sometimes associated with premature stop codon, or amino acid substitutions. In the remaining cases, amino acid substitutions are detected within Pol (De et al., 2015; Piret and Boivin, 2014). Classically, Sanger sequencing has been recognized as the gold standard for the detection of drug resistance mutations (DRMs) within VZV TK and Pol genes (Perrier et al., 2016; Piret and Boivin, 2014). However, this approach cannot detect minor variants present at a frequency below 20%. Ultra-deep sequencing (UDS) has an enhanced sensitivity compared to Sanger method and allows quantitative evaluation of the viral mutants (Chin et al., 2013). We report here a case of VZV resistant infection in an HT recipient. Our retrospective study aimed at showing the utility of UDS for DRM detection as a complement of Sanger method.