Specific interaction between Group B Streptococcus CC17 hypervirulent clone and phagocytes.

Streptococcus agalactiae also named Group B Streptococcus (GBS) is the most significant pathogen causing invasive infections, such as bacteremia and meningitis, in neonates. Worldwide epidemiological studies have shown that a particular clonal complex (CC) of capsular serotype III, the CC17, is strongly associated with meningitis in neonates and is therefore, designated as the hypervirulent clone. Macrophages are a permissive niche for intracellular bacteria of all GBS clones. In this study, we deciphered the specific interaction of GBS CC17 strains with macrophages. Our study revealed that CC17 strains are phagocytosed at a higher rate than GBS non-CC17 strains by human monocytes and macrophages both in cellular models and in primary cells. CC17-enhanced phagocytosis is due to an initial enhanced-attachment step to macrophages mediated by the CC17-specific surface protein HvgA and the PI-2b pilus (Spb1). We showed that two different inhibitors of scavenger receptors (fucoidan and poly(I)) specifically inhibited CC17 adhesion and phagocytosis while not affecting those of non-CC17 strains. Once phagocytosed, both CC17 and non-CC17 strains remained in a LAMP-1 positive vacuole that ultimately fuses with lysosomes where they can survive at similar rates. Finally, both strains displayed a basal egress which occurs independently from actin and microtubule networks. Our findings provide new insights into the interplay between the hypervirulent GBS CC17 and major players of the host's innate immune response. This enhanced adhesion, leading to increased phagocytosis, could reflect a peculiar capacity of the CC17 lineage to subvert the host immune defenses, establish a niche for persistence or disseminate.

Deciphering the role of the conjugate's phycoerythrin label in complement-mediated interference occurring in HLA single antigen Luminex bead assays.

Complement-mediated interference is a well described phenomenon in single antigen bead (SAB) Luminex assay that leads to falsely low or negative results for anti-HLA antibody (Ab). In a context of high amount of Ab, the enrichment of the Ab around the bead can lead to complement cascade activation and deposition, thereafter impairing Ab detection. EDTA is now routinely used to circumvent this interference. In this report, we attempted to decipher the role of the phycoerythrin (PE) label conjugated to the secondary Ab in this interference. Indeed, PE is a huge molecule (240 kDa) that could participate to limiting access of the conjugate to its Ab target on the bead. To this purpose, 22 sera displaying complement interference without pre-treatment with EDTA were compared on SAB assay with three detection strategies: the recommended PE-conjugated secondary Ab (IgGPE), an Alexa Fluor 532-conjugated Ab (IgGAF) bearing a tiny 724 Da fluorochrome, and a biotinylated Ab followed by PE-conjugated streptavidin (IgGBiot). Complement interference occurred with the three detection methods, but its depth, defined by the percentage of MFI loss with neat serum, was the highest for IgGPE. Our study highlighted the partial role of the PE fluorochrome in complement interference in SAB assays.

Type II Fatty Acid Synthesis Pathway and Cyclopropane Ring Formation Are Dispensable during Enterococcus faecalis Systemic Infection.

Enterococcus faecalis, a multiple antibiotic-resistant Gram-positive bacterium, has emerged as a serious nosocomial pathogen. Here, we used a genetic approach to characterize the strategies used by E. faecalis to fulfill its requirements for endogenous fatty acid (FA) synthesis in vitro and in vivo. The type II fatty acid synthesis (FASII) pathway is encoded by two operons and two monocistronic genes. Expression of all of these genes is repressed by exogenous FAs, which are incorporated into the E. faecalis membrane and modify its composition. Deletion of nine genes of the 12-gene operon abolished growth in an FA-free medium. Addition of serum, which is lipid rich, restored growth. Interestingly, the E. faecalis membrane contains cyclic fatty acids that modify membrane properties but that are unavailable in host serum. The cfa gene that encodes the cyclopropanation process is located in a locus independent of the FASII genes. Its deletion did not alter growth under the conditions tested, but yielded bacteria devoid of cyclic FAs. No differences were observed between mice infected with wild-type (WT) or with FASII or cyclopropanation mutant strains, in terms of bacterial loads in blood, liver, spleen, or kidneys. We conclude that in E. faecalis, neither FASII nor cyclopropanation enzymes are suitable antibiotic targets. IMPORTANCE Membrane lipid homeostasis is crucial for bacterial physiology, adaptation, and virulence. Fatty acids are constituents of the phospholipids that are essential membrane components. Most bacteria incorporate exogenous fatty acids into their membranes. Enterococcus faecalis has emerged as a serious nosocomial pathogen that is responsible for urinary tract infections, bacteremia, and endocarditis and is intrinsically resistant to numerous antibiotics. E. faecalis synthesizes saturated and unsaturated fatty acids, as well as cyclic fatty acids that are not found in the human host. Here, we characterized mutant strains deficient in fatty acid synthesis and modification using genetic, biochemical, and in vivo approaches. We conclude that neither the fatty acid synthesis pathway nor the cyclopropanation enzyme are suitable targets for E. faecalis antibiotic development.

Invasive group B Streptococcus infections in non-pregnant adults: a retrospective study, France, 2007-2019.

OBJECTIVES: Group B Streptococcus (GBS) (Streptococcus agalactiae) is a pathogen of growing importance in adults. The objective of this study was to describe the features of invasive infections by GBS in non-pregnant adults. METHODS: GBS infections were reported to the national reference centre for streptococci. Clinical information was abstracted from questionnaires. Capsular typing, identification of the hypervirulent CC-17 clone, and antibiotic susceptibility testing were performed for all GBS isolates. Multi-locus sequence typing and assignment to clonal complexes (CCs) was performed on a representative sample of 324 isolates. RESULTS: In total, 1960 GBS invasive infections were analysed from 2007 to 2019. The median age at onset was 71 years old (range 18-103). The main manifestation was bacteraemia without focus (54.5%). Meningitis was more frequent in patients under 40 (26/180, 14.4% versus 78/1780, 4.4%, p < 0.0001). Capsular types Ia, Ib, II, III and V accounted for 91.0% of the cases (1786/1960). CC-1, -10, -17, -19 and -23 accounted for 96.3% (312/324) of the cases. Capsular type III and CC-17 were overrepresented in meningitis (38/104, 36.5%, p < 0.001 and 22/104, 21.2%, p 0.01, respectively). All isolates were susceptible to ß-lactam antibiotics. Resistance to erythromycin (32.7%) and clindamycin (26.3%) remained stable, whereas decreased susceptibility to fluoroquinolones increased, reaching 2.7% in 2019 (p for trend 0.002). CONCLUSIONS: This work highlights the susceptibility of the elderly to GBS infections and differences in the clinical manifestations according to the patients' age and GBS type. In agreement with worldwide reports on emerging multidrug-resistant GBS, it reinforces the need for a continued surveillance of GBS epidemiology.

Multidrug-Resistant Hypervirulent Group B Streptococcus in Neonatal Invasive Infections, France, 2007-2019.

We analyzed group B Streptococcus (GBS) neonatal invasive infections reported during 2007-2019 in France. The hypervirulent clonal complex (CC) 17 GBS was responsible for 66% (827/1,262) of cases. The role of CC17 GBS increased over time (p for trend = 0.0001), together with the emergence of a multidrug-resistant CC17 GBS sublineage.

Perinatal hormones favor CC17 group B Streptococcus intestinal translocation through M cells and hypervirulence in neonates.

Group B Streptococcus (GBS) is the leading cause of invasive bacterial neonatal infections. Late-onset diseases (LOD) occur between 7 and 89 days of life and are largely due to the CC17 GBS hypervirulent clone. We studied the impact of estradiol (E2) and progesterone (P4), which impregnate the fetus during pregnancy, on GBS neonatal infection in cellular and mouse models of hormonal exposure corresponding to concentrations found at birth (E2-P4 C0) and over 7 days old (E2-P4 C7). Using representative GBS isolates, we show that E2-P4 C7 concentrations specifically favor CC17 GBS meningitis following mice oral infection. CC17 GBS crosses the intestinal barrier through M cells. This process mediated by the CC17-specific surface protein Srr2 is enhanced by E2-P4 C7 concentrations which promote M cell differentiation and CC17 GBS invasiveness. Our findings provide an explanation for CC17 GBS responsibility in LOD in link with neonatal gastrointestinal tract maturation and hormonal imprint.

Biofilm production by Haemophilus influenzae and Streptococcus pneumoniae isolated from the nasopharynx of children with acute otitis media.

BACKGROUND: Biofilm production by Haemophilus influenzae and Streptococcus pneumoniae has been implicated in the pathogenesis of otitis media, mainly in chronic and recurrent cases. We studied the "in vitro" biofilm production by these 2 species isolated alone or together from the nasopharynx of children with acute otitis media. METHODS: The studied strains were from 3 pneumococcal conjugate vaccine (PCV) periods: pre-PCV7, post-PCV7/pre-PCV13 and post-PCV13. A modified microtiter plate assay with crystal violet stain was used to study the biofilm production of 182 H. influenzae and 191 S. pneumoniae strains. RESULTS: Overall, 117/181 (64.6%) H. influenzae and 128/191 (66.8%) S. pneumoniae strains produced biofilm. The proportion of biofilm-producing H. influenzae strains was greater with than without the isolation of S. pneumoniae in the same sample (75.5% vs 52.3%, p = 0.001). Conversely, the proportion of biofilm-producing S. pneumoniae strains was not affected by the presence or not of H. influenzae (66.3% vs 67.4%). S. pneumoniae serotypes 6B, 15B/C, 19A, 35F and 35B were the better biofilm producers (80%). Serotypes 11A, 14, 15A, 19F and 19A were more associated with H. influenzae biofilm-producing strains. Overall, 89/94 (94.6%) of cases with combined isolation showed biofilm production by S. pneumoniae or H. influenzae. CONCLUSION: This study emphasizes the high proportion of biofilm production by H. influenzae and S. pneumoniae strains isolated from the nasopharynx of children with acute otitis media, which reinforces the results of studies suggesting the importance of biofilm in the pathogenesis of acute otitis media.

Molecular diagnosis of Helicobacter pylori infection in gastric biopsies: Evaluation of the Amplidiag® H. pylori + ClariR assay.

BACKGROUND: Adapted treatments for Helicobacter pylori infection, guided by determining antimicrobial resistance, are associated with high eradication rates. We evaluated the performance of the Amplidiag® H. pylori + ClariR PCR assay (Amplidiag® ) for detecting H. pylori and its clarithromycin resistance from gastric biopsies taken during endoscopy in comparison to culture and our "in-house" PCR. MATERIALS AND METHODS: A total of 127 gastric biopsies were analyzed (98 adults; 29 children). Culture, PCR Amplidiag® , and in-house PCR were performed in parallel. The in-house PCR combined amplification and sequencing of a 267-bp fragment of the H. pylori 23S rRNA gene. Discrepancies were controlled by amplification of glmM gene. RESULTS: For detection of H. pylori, Amplidiag® and the in-house PCR were concordant in 118 of 127 of cases: 66 negative and 52 positive. Discrepancies were observed in nine cases, all with low bacterial load: Amplidiag® did not detect seven biopsies positive on in-house PCR but detected two positive biopsies that were negative on in-house PCR. Among the 19 of 52 (36%) H. pylori cases resistant to clarithromycin, only four biopsies with mixed populations exhibited discordant results between the two PCR methods. The A2142T mutation was not detected by Amplidiag® . With the in-house PCR and amplified glmM gene as the reference method, the sensitivity and specificity of Amplidiag® was 88.5% (95% confidence interval 83-94.1) and 100%. CONCLUSION: This study demonstrated the high sensitivity of the PCR-based Amplidiag® H. pylori test, especially with low H. pylori load, and the probability of its clarithromycin resistance analysis. For clinical use, a well-designed trial with a large scale of samples may still be needed.

Molecular characterization of Helicobacter pylori resistance to rifamycins.

INTRODUCTION: Antibiotic resistance is a major contributing factor in treatment failure of Helicobacter pylori eradication. Rifabutin (RB) is a rescue treatment and rifampicin (RP) is used to screen RB resistance in vitro. The aim of this study was to evaluate the rate of rifamycins resistance and to determine the mutations in the rpoB gene conferring resistance to discuss the current break point. METHODS: Antimicrobial susceptibility to RP was first determined by E-test for 1015 H. pylori isolates. RP and RB MICs were then determined by agar dilution method for strains with MIC of RP > 1 mg/L, and the rpoB gene was sequenced. RESULTS: Overall, 54 of 1015 strains exhibited a RP MIC > 1 mg/L by agar dilution method. Among these 54 strains, 10 had MICs of RP > 4 mg/L and RB ≥ 1 mg/L. They all carried at least one mutation in the rpoB gene at codons 530, 538, 540, 525 in the RP resistance-determining region (RRDR). Implication of the mutation L547F was confirmed by site-directed mutagenesis experiment. In contrast, among the 44 H. pylori isolates with a MIC of RP comprised between 2 and 4 mg/L, only 4 of 44 (9%) strains exhibited a mutation in rpoB, but outside RRDR (codons 470, 499, 636, or 657). For 31 of 44 tested strains, the RB MICs were ≤0.064 mg/L. CONCLUSION: These results suggest that H. pylori isolates should be classified as resistant to RP for MICs > 4 mg/L. We considered that the optimal cut off for RB was ≥0.125 mg/L. We report a new mutation responsible for rifamycins, resistance, L547F.

Changing Epidemiology of Group B Streptococcus Susceptibility to Fluoroquinolones and Aminoglycosides in France.

Group B Streptococcus (GBS) is the leading cause of neonatal invasive infections and an emerging pathogen in the elderly. Our objectives were to describe the evolution of GBS resistance to antibiotics in France and to investigate the emergence of fluoroquinolone (FQ)-resistant isolates. A total of 8,757 unrelated GBS isolates were collected and tested for antibiotic susceptibility from 2007 to 2014 according to EUCAST recommendations. All isolates were susceptible to penicillin G, amoxicillin, and vancomycin. Resistance to macrolides decreased from 47.0% to 30.0%, whereas high-level resistance to aminoglycosides, especially amikacin, increased from 6.4% to 8.8% and 24 isolates (0.3%) were highly resistant to gentamicin. FQ resistance gradually increased from 0.2% in 2007 (n = 1) to 1.5% in 2014 (n = 18, P < 0.01). Capsular polysaccharide (CPS) genotyping, multilocus sequence typing, and sequencing of the quinolone resistance-determining region (QRDR) showed that GBS isolates of sequence type 19 (ST-19) CPS type V were largely overrepresented in FQ-resistant isolates (n = 30, 45.5%). All 30 strains displayed the same QRDR mutations and were often associated with cross-resistance to macrolides (93.3%) and gentamicin (30%). In conclusion, we report the rise of FQ- and aminoglycoside-resistant GBS in France over an 8-year study period, an evolution likely linked to the clonal expansion of ST-19 CPS V-resistant isolates. This study emphasizes the need for a continuous surveillance of GBS epidemiology and antibiotic susceptibility.

Environmental fatty acids enable emergence of infectious Staphylococcus aureus resistant to FASII-targeted antimicrobials.

The bacterial pathway for fatty acid biosynthesis, FASII, is a target for development of new anti-staphylococcal drugs. This strategy is based on previous reports indicating that self-synthesized fatty acids appear to be indispensable for Staphylococcus aureus growth and virulence, although other bacteria can use exogenous fatty acids to compensate FASII inhibition. Here we report that staphylococci can become resistant to the FASII-targeted inhibitor triclosan via high frequency mutations in fabD, one of the FASII genes. The fabD mutants can be conditional for FASII and not require exogenous fatty acids for normal growth, and can use diverse fatty acid combinations (including host fatty acids) when FASII is blocked. These mutants show cross-resistance to inhibitors of other FASII enzymes and are infectious in mice. Clinical isolates bearing fabD polymorphisms also bypass FASII inhibition. We propose that fatty acid-rich environments within the host, in the presence of FASII inhibitors, might favour the emergence of staphylococcal strains displaying resistance to multiple FASII inhibitors.

Molecular characterization of Streptococcus agalactiae isolates harboring small erm(T)-carrying plasmids.

Among 1,827 group B Streptococcus (GBS) strains collected between 2006 and 2013 by the French National Reference Center for Streptococci, 490 (26.8%) strains were erythromycin resistant. The erm(T) resistance gene was found in six strains belonging to capsular polysaccharides Ia, III, and V and was carried by the same mobilizable plasmid, which could be efficiently transferred by mobilization to GBS and Enterococcus faecalis recipients, thus promoting a broad dissemination of erm(T).

Erm(X)-mediated resistance to macrolides, lincosamides and streptogramins in Actinobaculum schaalii.

OBJECTIVES: Actinobaculum schaalii is a Gram-positive bacillus increasingly reported as a causative agent of urinary tract infections as well as invasive infections, mainly in the elderly and patients with underlying urological conditions. Since little is known about the molecular basis of antimicrobial resistance in A. schaalii, the aim of this study was to investigate resistance to macrolides, lincosamides and streptogramins (MLS) in this emerging pathogen. METHODS: A total of 32 A. schaalii clinical isolates from France and Switzerland were studied. MICs of erythromycin, spiramycin, lincomycin, clindamycin and quinupristin/dalfopristin were determined by the agar dilution method. Resistance genes erm(A), erm(B), erm(C), erm(F), erm(G), erm(X), msr(A) and mef(A) were screened by PCR. The genetic environment was determined by random cloning and PCR mapping. RESULTS: Out of 32 isolates tested, 21 were highly resistant to erythromycin, spiramycin, lincomycin and clindamycin (MICs >256 mg/L), whereas 11 exhibited low MICs (MICs < 0.12 mg/L). On the other hand, quinupristin/dalfopristin remained active against all the isolates. An inducible MLSB resistance phenotype was noted in all cases. The erm(X) gene was detected among all resistant strains, whereas none was detected in susceptible strains. Analysis of genetic support and environment revealed that erm(X) was probably part of the chromosome of A. schaalii. CONCLUSIONS: This study is the first molecular characterization of MLS resistance in A. schaalii. In all cases, it was due to the presence of erm(X), a methylase gene previously identified in other clinically relevant Gram-positive bacilli.

Emergence of NDM-1-producing Klebsiella pneumoniae in Morocco.

OBJECTIVES: To analyse the mechanisms responsible for multidrug resistance in three carbapenem-resistant Klebsiella pneumoniae isolates recovered from two hospitalized patients and an outpatient from the same hospital in Rabat, Morocco. METHODS: PCR and sequencing were used to search for ß-lactamase genes. Clonal relationships between the isolates were analysed by multilocus sequence typing and PFGE. RESULTS: A history of prior hospitalization in the same ward was determined for two of the three patients. The three isolates of K. pneumoniae belonged to the same sequence type (ST), ST15, and were clonally related. All three isolates carried the bla(NDM-1) gene and co-expressed the extended-spectrum ß-lactamases CTX-M-15 and SHV-5, as well as the narrow-spectrum ß-lactamases SHV-1, OXA-1, OXA-9 and TEM-1. The bla(NDM-1) gene was located on an ~250 kb non-typeable plasmid co-harbouring the bla(OXA-1) and bla(CTX-M-15) genes. No link with the Indian subcontinent could be established for the three patients. CONCLUSIONS: This work further emphasizes the spread of NDM-1 producers worldwide.

Rapid and accurate identification of species belonging to the Candida parapsilosis complex by real-time PCR and melting curve analysis.

Candida parapsilosis is the second most frequent Candida species isolated from blood cultures. Since 2005, C. parapsilosis has been divided into three distinct species based on genetic traits: Candida parapsilosis, Candida metapsilosis and Candida orthopsilosis. The aim of this study was to develop a rapid real-time PCR assay able to distinguish these closely related species via a melting curve analysis. This identification method was optimized by using reference strains and well-characterized clinical isolates of Candida species. A single set of consensus primers was designed to amplify a 184 bp portion of the SADH gene in order to identify species based on the unique melt profile resulting from DNA sequence variations from each species of the complex. PCR products were detected with SYBR Green fluorescent dye and identification was established by melting curve analysis. For validation of the technique, a total of 116 clinical isolates, phenotypically identified as C. parapsilosis, were tested by real-time PCR and results were further compared with PCR-RFLP patterns of the SADH gene, used as the reference method. The melting curve analysis of amplified DNA could differentiate between C. parapsilosis (83.5 °C), C. metapsilosis (82.9 °C) and C. orthopsilosis (82.1 °C), with a sensitivity and specificity comparable to those of the reference method. One hundred and fourteen C. parapsilosis and two C. orthopsilosis isolates were identified among the clinical isolates. This method provides a simple, rapid and reliable identification of species belonging to the C. parapsilosis complex. This novel approach could be helpful for clinical and epidemiological investigations.