The virtue of training: extending phage host spectra against vancomycin-resistant Enterococcus faecium strains using the Appelmans method.

Phage therapy has (re)emerged as a serious possibility for combating multidrug-resistant bacterial infections, including those caused by vancomycin-resistant Enterococcus faecium strains. These opportunistic pathogens belong to a specific clonal complex 17, against which relatively few phages have been screened. We isolated a collection of 21 virulent phages growing on these vancomycin-resistant isolates. Each of these phages harbored a typical narrow plaquing host range, lysing at most 5 strains and covering together 10 strains of our panel of 14 clinical isolates. To enlarge the host spectrum of our phages, the Appelmans protocol was used. We mixed four out of our most complementary phages in a cocktail that we iteratively grew on eight naive strains from our panel, of which six were initially refractory to at least three of the combined phages. Fifteen successive passages permitted to significantly improve the lytic activity of the cocktail, from which phages with extended host ranges within the E. faecium species could be isolated. A single evolved phage able to kill up to 10 of the 14 initial E. faecium strains was obtained, and it barely infected nearby species. All evolved phages had acquired point mutations or a recombination event in the tail fiber genetic region, suggesting these genes might have driven phage evolution by contributing to their extended host spectra.

Web-based prediction of antimicrobial resistance in enterococcal clinical isolates by whole-genome sequencing.

Besides phenotypic antimicrobial susceptibility testing (AST), whole genome sequencing (WGS) is a promising alternative approach for detection of resistance phenotypes. The aim of this study was to investigate the concordance between WGS-based resistance prediction and phenotypic AST results for enterococcal clinical isolates using a user-friendly online tools and databases. A total of 172 clinical isolates (34 E. faecalis, 138 E. faecium) received at the French National Reference Center for enterococci from 2017 to 2020 were included. AST was performed by disc diffusion or MIC determination for 14 antibiotics according to CA-SFM/EUCAST guidelines. The genome of all strains was sequenced using the Illumina technology (MiSeq) with bioinformatic analysis from raw reads using online tools ResFinder 4.1 and LRE-finder 1.0. For both E. faecalis and E. faecium, performances of WGS-based genotype to predict resistant phenotypes were excellent (concordance > 90%), particularly for antibiotics commonly used for treatment of enterococcal infections such as ampicillin, gentamicin, vancomycin, teicoplanin, and linezolid. Note that 100% very major errors were found for quinupristin-dalfopristin, tigecycline, and rifampicin for which resistance mutations are not included in databases. Also, it was not possible to predict phenotype from genotype for daptomycin for the same reason. WGS combined with online tools could be easily used by non-expert clinical microbiologists as a rapid and reliable tool for prediction of phenotypic resistance to first-line antibiotics among enterococci. Nonetheless, some improvements should be made such as the implementation of resistance mutations in the database for some antibiotics.

Genetic features of the poxtA linezolid resistance gene in human enterococci from France.

OBJECTIVES: To describe the prevalence of poxtA among clinical linezolid-resistant enterococci (LRE) collected in France from 2016 to 2020 and to extensively characterize its genetic supports and environments. METHODS: All LRE clinical isolates received at the National Reference Centre for Enterococci from French hospitals between 2016 and 2020 were included. LRE isolates were screened for linezolid resistance genes (cfr-like, optrA and poxtA) by real-time PCR and phenotypically characterized. A collection of 11 representative poxtA-positive isolates (10 Enterococcus faecium and 1 Enterococcus faecalis) underwent WGS by hybrid assembly combining short-read (Illumina MiSeq) and long-read (MinION) approaches. Transferability of poxtA was attempted by filter-mating experiments. RESULTS: Out of 466 LRE received at the National Reference Centre for Enterococci over the period, 47 (10.1%) were poxtA-positive, including 42 E. faecium. The 11 isolates characterized by WGS were confirmed to be epidemiologically unrelated by core genome analysis and eight different STs were assigned to E. faecium isolates. The poxtA gene was found to be plasmid carried and flanked by IS1216E transposase genes in all isolates and frequently linked with fexB, tet(M) and tet(L). A total of seven distinct poxtA-harbouring plasmids were obtained after hybrid assembly and plasmid transfer of poxtA was successful in three cases. For the two poxtA/optrA-positive isolates, those genes were carried by different plasmids. CONCLUSIONS: The poxtA gene has been circulating among clinical enterococci in France since at least 2016, mostly in E. faecium and independently from optrA. The poxtA-carrying plasmids often co-carried resistance genes to phenicols and tetracyclines, and could have been co-selected through their veterinary use.

Molecular and functional analysis of the novel cfr(D) linezolid resistance gene identified in Enterococcus faecium.

OBJECTIVES: To characterize the novel cfr(D) gene identified in an Enterococcus faecium clinical isolate (15-307.1) collected from France. METHODS: The genome of 15-307.1 was entirely sequenced using a hybrid approach combining short-read (MiSeq, Illumina) and long-read (GridION, Oxford Nanopore Technologies) technologies in order to analyse in detail the genetic support and environment of cfr(D). Transfer of linezolid resistance from 15-307.1 to E. faecium BM4107 was attempted by filter-mating experiments. The recombinant plasmid pAT29Ωcfr(D), containing cfr(D) and its own promoter, was transferred to E. faecium HM1070, Enterococcus faecalis JH2-2 and Escherichia coli AG100A. RESULTS: As previously reported, 15-307.1 belonged to ST17 and was phenotypically resistant to linezolid (MIC, 16 mg/L), vancomycin and teicoplanin. A hybrid sequencing approach confirmed the presence of several resistance genes including vanA, optrA and cfr(D). Located on a 103 kb plasmid, cfr(D) encoded a 357 amino acid protein, which shared 64%, 64%, 48% and 51% amino acid identity with Cfr, Cfr(B), Cfr(C) and Cfr(E), respectively. Both optrA and cfr(D) were successfully co-transferred to E. faecium BM4107. When expressed in E. faecium HM1070 and E. faecalis JH2-2, pAT29Ωcfr(D) did not confer any resistance, whereas it was responsible for an expected PhLOPSA resistance phenotype in E. coli AG100A. Analysis of the genetic environment of cfr(D) showed multiple IS1216 elements, putatively involved in its mobilization. CONCLUSIONS: Cfr(D) is a novel member of the family of 23S rRNA methyltransferases. While only conferring a PhLOPSA resistance phenotype when expressed in E. coli, enterococci could constitute an unknown reservoir of cfr(D).

Performance of commercial methods for linezolid susceptibility testing of Enterococcus faecium and Enterococcus faecalis.

BACKGROUND: Linezolid-resistant enterococci (LRE) causing infections that are challenging to treat are rising, highlighting the need for reliable screening of LRE clinical isolates. OBJECTIVES: To evaluate the ability of the broth microdilution (BMD) method for LRE detection and to assess the performance of seven commercially available techniques for linezolid susceptibility testing. METHODS: A collection of 100 clinical isolates (80 Enterococcus faecium and 20 Enterococcus faecalis), including 20 optrA-positive isolates, 17 poxtA-positive isolates and 1 optrA/poxtA-positive E. faecium isolate, were studied. MICs were determined after 18 h [Day 1 (D1)] and 42 h [Day 2 (D2)] of incubation and interpreted following EUCAST and CLSI guidelines, which currently provide different interpretative breakpoints. Performance of commercial techniques was compared with BMD results. RESULTS: MIC50/D1 and MIC50/D2 were both 8 mg/L, while MIC90/D1 and MIC90/D2 were 16 and 32 mg/L, respectively. MICD1 values for poxtA-positive isolates were lower than those for optrA-positive isolates. Proportions of susceptible isolates at D1 and D2 were 48% and 41%, respectively, according to EUCAST breakpoints and 35% and 13%, respectively, according to CLSI criteria (the proportions of isolates categorized as intermediate following CLSI recommendations were 13% and 28% at D1 and D2, respectively). Percentage susceptibility assessed by the commercially available techniques was always higher. The four commercial methods allowing MIC determination provided an overall essential agreement of ≥90% at D1. Categorical agreement and error rates were generally improved at D2. CONCLUSIONS: Non-automated methods (Sensititre and UMIC) and, to a lesser extent, gradient strip Etest appear to show an acceptable correlation with the BMD reference method for the detection of isolates with low MICs of linezolid after prolonged incubation.

Emergence of optrA-mediated linezolid resistance in enterococci from France, 2006-16.

OBJECTIVES: To describe the epidemiological trend of linezolid-resistant enterococci (LRE) collected in France from 2006 to 2016 and to extensively characterize LRE isolates. METHODS: The National Reference Center for Enterococci (NRC-Enc) received enterococcal isolates suspected to be VRE and/or LRE from all French hospitals between 2006 and 2016. LRE isolates were phenotypically characterized and their genomes were entirely sequenced by Miseq (Illumina). Transfer of linezolid resistance was attempted by filter mating experiments. RESULTS: Out of 3974 clinical isolates of enterococci received at the NRC-Enc over the period, 9 (0.2%) were LRE (MICs 8 to >32 mg/L), including 6 Enterococcus faecium and 3 Enterococcus faecalis. This overall prevalence significantly increased over the study period, reaching 0.8% in 2016. The five LRE isolated before 2016 were vanA-positive E. faecium whereas strains isolated in 2016 (one E. faecium and three E. faecalis) were susceptible to vancomycin. None of these isolates was part of an outbreak, while E. faecium strains were assigned to four different STs [17 (1), 80 (3), 412 (1) and 650 (1)] and all three E. faecalis belonged to ST480. Except for the strain isolated in 2010, all LRE were positive for optrA, which was located on plasmids (5/8) or in the chromosome (3/8). Plasmid transfer of optrA was successful in three cases. CONCLUSIONS: There has been a significant increase in the prevalence of LRE in France over time; this is due to the spread of optrA among E. faecium and E. faecalis human clinical isolates (VRE or not).

The diagnosis of pertussis: which method to choose?

Despite the introduction of routine vaccination against pertussis for more than a half century, leading to a drastic decline in the number of reported cases, pertussis continues to be an important respiratory disease afflicting unvaccinated infants and previously vaccinated children as well as adults in whom immunity has waned. The diagnosis of pertussis is challenging and accurate laboratory identification of Bordetella infections remains problematic. Common laboratory diagnostic methods used for pertussis diagnosis include culture, direct-fluorescent-antibody testing (DFA), serology and polymerase chain reaction (PCR). Culture of Bordetella pertussis is highly specific but fastidious and has limited sensitivity. DFA provides a much more rapid result, but has the disadvantage of poor sensitivity and specificity. Serology is not useful in infants. In older persons, it is hampered by the limitations of paired sera and it provides mainly a retrospective diagnosis. Such limitations of conventional diagnosis testing have led to the development of PCR assays. Notwithstanding its lack of standardization, PCR has been found to be more sensitive and more specific than other methods. In this report, we aimed to review current knowledge about the available diagnostic methods and tests that accurately diagnose pertussis.

Investigation of a vanA linezolid- and vancomycin-resistant Enterococcus faecium outbreak in the Southwest Indian Ocean (Reunion Island).

INTRODUCTION: Dual resistance to linezolid and glycopeptides is a milestone reached by certain extensively drug-resistant (XDR) enterococci. This paper describes the molecular and epidemiological investigations of a linezolid-resistant and vancomycin-resistant Enterococcus faecium (E. faecium) (LVREf) outbreak in the French overseas territory of Reunion Island (Indian Ocean). METHODS: All vancomycin-resistant Enterococcus (VRE) isolates detected on Reunion Island between 2015 and 2019 were included in the study. The VRE isolates were phenotypically characterised and genetically explored by whole-genome sequencing (WGS). RESULTS: Sixteen vancomycin-resistant E. faecium (VREf) isolates were retrieved between 2015 and 2019. Seven isolates obtained in 2019 were involved in the outbreak. These seven LVREf isolates from the 2019 outbreak at the University Hospital of Reunion Island (UHRI) were suspected to be related to a linezolid-susceptible VREf strain imported from India. An epidemiological link was highlighted for six of the seven outbreak cases. All the LVREf outbreak isolates were obtained from rectal swabs (colonisation) and resistant to vancomycin (MIC > 128 mg/L) and linezolid (MIC 8-32 mg/L); one isolate was also resistant to daptomycin (MIC 8 mg/L). The seven outbreak isolates were positive for the vanA and optrA genes and belonged to ST761. CONCLUSIONS: These results argue for the strict application of control and prevention measures for VRE clones at high risk of spread, particularly in areas such as Reunion Island where the risk of importation from the Indian subcontinent is high. The regional spread of optrA linezolid-resistance genes in VRE isolates is a matter of concern, due to possibility of treatment failure.

In vitro activity of eravacycline and mechanisms of resistance in enterococci.

Eravacycline (ERC), the first fluorocycline, is a new tetracycline with superior activity to tigecycline (TGC) against many bacterial species. This work aimed to determine the in vitro activity of ERC compared with other tetracyclines against enterococcal clinical isolates and to analyse corresponding resistance mechanisms. A collection of 60 enterococcal strains was studied: 54 epidemiologically unrelated clinical isolates (46 Enterococcus faecium and 8 Enterococcus faecalis) including 42 vancomycin-resistant enterococci (VRE) (33 vanA and 9 vanB), 3 in vitro TGC-resistant mutants (E. faecium AusTig, HMtig1 and HMtig2) and 3 reference wild-type strains (E. faecium Aus0004 and HM1070, E. faecalis ATCC 29212). In vitro susceptibility was determined using Etest strips (for ERC) or by broth microdilution (for TGC, doxycycline, minocycline and tetracycline). Resistance genes [tet(M), tet(L), tet(O) and tet(S)] were screened by PCR for TGC- and/or ERC-resistant strains as well as sequencing of the rpsJ gene (encoding ribosomal protein S10). MIC50/90 values were 0.016/0.08, ≤0.03/0.5, 4/32, 8/16 and 32/>32 mg/L for ERC, TGC, doxycycline, minocycline and tetracycline, respectively. According to EUCAST guidelines, nine strains were categorised as resistant to TGC (MIC, 0.5-8 mg/L), including four E. faecium vanA(+) strains also resistant to ERC (MIC, 0.19-1.5 mg/L). These four strains all possessed at least one mutation in rpsJ and two tet determinants: tet(M) + tet(L) (n = 2); and tet(M) + tet(S) (n = 2). Although ERC has excellent in vitro activity against enterococci (including VRE), emergence of resistance is possible due to combined mechanisms (rpsJ mutations + tet genes).

Prevalence of Bordetella pertussis and Bordetella parapertussis infections in Tunisian hospitalized infants: results of a 4-year prospective study.

The prevalence of pertussis in Tunisia remains undetermined essentially because of the unavailability of a basic laboratory diagnostic service. Specific diagnostic tools were applied for the first time in a Tunisian prospective study in order to get a first estimation of the prevalence of Bordetella pertussis/parapertussis infections and to evaluate their use to determine the epidemiologic characteristics of these infections in Tunisian infants. Between 2007 and 2011, a total of 626 samples from 599 infants aged <1 year with and without pertussoid cough were investigated for the presence of B. pertussis/parapertussis using culture and real-time polymerase chain reaction (PCR). The real-time PCR (RT-PCR) targets include IS481 commonly found in B. pertussis, B. bronchiseptica, and B. holmesii; IS1001 specific of B. parapertussis, in combination with the pertussis toxin promoter region gene (ptx) of B. pertussis; and the recA gene specific of B. holmesii. When possible, patients' household contacts provided nasopharyngeal aspirates (NPAs) for RT-PCR detection of B. pertussis/parapertussis or single-serum samples for anti-PT IgG quantification. All except 1 NPAs were negative by conventional culture, whereas PCR gave positive signals for 126 specimens (21%): B. pertussis, B. parapertussis, and Bordetella spp. were detected in 82%, 6%, and 4% of the samples, respectively. The simultaneous presence of B. pertussis and B. parapertussis was noted in 8% of the cases. Pertussis was reported throughout the year with a peak during the summer of the year 2009. The prevalence of Bordetella infection was 20% between 2007 and 2011. Most of these cases corresponded to patients younger than 6 months who received <3 doses of pertussis vaccine. Among the household contacts enrolled in the study, mothers seemed to be the likely source of infection. This study showed that pertussis is still prevalent in Tunisia and that the disease remains a public health problem affecting not only infants but also adults. Given this situation, sensitive and specific laboratory tests are needed to improve the accuracy of pertussis diagnosis.

The new health legacy: when pertussis becomes a heritage transmitted from mothers to infants.

Despite high vaccination coverage rates, there has been a gradual increase in reported pertussis cases. Although whooping cough affects all ages, young infants continue to suffer the greatest pertussis disease burden. Adolescents and adults are the primary source of infection for young babies. In this paper, we report two cases involving the likely transmission of pertussis from mothers to infants in Tunisia.