A Chimeric Penicillin Binding Protein 2X Significantly Decreases in Vitro Beta-Lactam Susceptibility and Increases in Vivo Fitness of Streptococcus pyogenes.

All tested strains of Streptococcus pyogenes (group A streptococcus, GAS) remain susceptible to penicillin. However, GAS strains with amino acid substitutions in penicillin-binding proteins that confer decreased susceptibility to beta-lactam antibiotics have been identified recently. This discovery raises concerns about emergence of beta-lactam antibiotic resistance in GAS. Whole genome sequencing recently identified GAS strains with a chimeric penicillin-binding protein 2X (PBP2X) containing a recombinant segment from Streptococcus dysgalactiae subspecies equisimilis (SDSE). To directly test the hypothesis that the chimeric SDSE-like PBP2X alters beta-lactam susceptibility in vitro and fitness in vivo, an isogenic mutant strain was generated and virulence assessed in a mouse model of necrotizing myositis. Compared with naturally occurring and isogenic strains with a wild-type GAS-like PBP2X, strains with the chimeric SDSE-like PBP2X had reduced susceptibility in vitro to nine beta-lactam antibiotics. In a mouse model of necrotizing myositis, the strains had identical fitness in the absence of benzylpenicillin treatment. However, mice treated intermittently with a subtherapeutic dose of benzylpenicillin had significantly more colony-forming units recovered from limbs infected with strains with the chimeric SDSE-like PBP2X. These results show that mutations such as the PBP2X chimera may result in significantly decreased beta-lactam susceptibility and increased fitness and virulence. Expanded diagnostic laboratory surveillance, genome sequencing, and molecular pathogenesis study of potentially emergent beta-lactam antibiotic resistance among GAS are needed.

Determination of amoxicillin and cotrimoxazole concentrations in sputum of patients with cystic fibrosis.

In the management of cystic fibrosis, treatments against Staphylococcus aureus and Haemophilus influenzae such as amoxicillin or cotrimoxazole have to be prescribed and the antibiotherapy's efficacy may be linked to the concentration that reaches the infected site. As cystic fibrosis patients present disturbed pharmacokinetics parameters, drug monitoring would be relevant to assess the lung distribution of antibiotics and to optimize dosing regimens. In this context, the aim of the study was to develop and validate HPLC-based methods for the determination of both antibiotics in bronchial sputum from cystic fibrosis patients, in order to assess the distribution of the drugs into the lungs. Plasma proteins were precipitated by acetonitrile and amoxicillin concentrations in sputum were determined by HPLC coupled with tandem-mass spectrometry. Following liquid extraction with ethyl acetate, cotrimoxazole was quantified by HPLC using ultraviolet detection. Both methods were rapid, specific, accurate and reproducible. The method was applied to patient samples. In three treated patients, concentrations of amoxicillin in sputum were similar and below the lower limit of quantification (0.1 µg/g) and in six patients, sputum concentrations up to 11.1 and 6.4 µg/g were measured for sulfamethoxazole and trimethoprim, respectively.

Opportunistic Pulmonary Bordetella hinzii Infection after Avian Exposure.

We report 2 cases of pulmonary Bordetella hinzii infection in immunodeficient patients. One of these rare cases demonstrated the potential transmission of the bacteria from an avian reservoir through occupational exposure and its persistence in humans. We establish bacteriologic management of these infections and suggest therapeutic options if needed.

First case of disseminated infection with Nocardia cerradoensis in a human.

Here we report in a human, a renal transplant patient, the first disseminated infection with Nocardia cerradoensis, isolated after a brain biopsy. Species identification was based on 16S rRNA, gyrB, and hsp65 gene analyses. Antibiotic treatment was successful by combining carbapenems and aminoglycosides and then switching to oral trimethoprim-sulfamethoxazole.

Protective effect of IgM against colonization of the respiratory tract by nontypeable Haemophilus influenzae in patients with hypogammaglobulinemia.

BACKGROUND: Primary immunoglobulin deficiencies lead to recurrent bacterial infections of the respiratory tract and bronchiectasis, even with adequate immunoglobulin replacement therapy. It is not known whether patients able to secrete IgM (eg, those with hyper-IgM [HIgM] syndrome) are as susceptible to these infections as patients who lack IgM production (eg, those with panhypogammaglobulinemia [PHG]). OBJECTIVE: This study is aimed at identifying specific microbiological and clinical (infections) characteristics that distinguish immunoglobulin-substituted patients with PHG from patients with HIgM syndrome. METHODS: A cohort of patients with HIgM syndrome (n = 25) and a cohort of patients with PHG (n = 86) were monitored prospectively for 2 years while receiving similar polyvalent immunoglobulin replacement therapies. Regular bacterial analyses of nasal swabs and sputum were performed, and clinical events were recorded. In parallel, serum and saliva IgM antibody concentrations were measured. RESULTS: When compared with patients with PHG, patients with HIgM syndrome were found to have a significantly lower risk of nontypeable Haemophilus influenzae carriage in particular (relative risk, 0.39; 95% CI, 0.21-0.63). Moreover, patients with HIgM syndrome (including those unable to generate somatic hypermutations of immunoglobulin genes) displayed anti-nontypeable H influenzae IgM antibodies in their serum and saliva. Also, patients with HIgM syndrome had a lower incidence of acute respiratory tract infections. CONCLUSIONS: IgM antibodies appear to be microbiologically and clinically protective and might thus attenuate the infectious consequences of a lack of production of other immunoglobulin isotypes in patients with HIgM syndrome. Polyvalent IgG replacement therapy might not fully compensate for IgM deficiency. It might thus be worth adapting long-term antimicrobial prophylactic regimens according to the underlying B-cell immunodeficiency phenotype.

Analysis of the Genomics and Mouse Virulence of an Emergent Clone of Streptococcus dysgalactiae Subspecies equisimilis.

Streptococcus dysgalactiae subsp. equisimilis is a bacterial pathogen that is increasingly recognized as a cause of severe human infections. Much less is known about the genomics and infection pathogenesis of S. dysgalactiae subsp. equisimilis strains compared to the closely related bacterium Streptococcus pyogenes. To address these knowledge deficits, we sequenced to closure the genomes of seven S. dysgalactiae subsp. equisimilis human isolates, including six that were emm type stG62647. Recently, for unknown reasons, strains of this emm type have emerged and caused an increasing number of severe human infections in several countries. The genomes of these seven strains vary between 2.15 and 2.21 Mbp. The core chromosomes of these six S. dysgalactiae subsp. equisimilis stG62647 strains are closely related, differing on average by only 495 single-nucleotide polymorphisms, consistent with a recent descent from a common progenitor. The largest source of genetic diversity among these seven isolates is differences in putative mobile genetic elements, both chromosomal and extrachromosomal. Consistent with the epidemiological observations of increased frequency and severity of infections, both stG62647 strains studied were significantly more virulent than a strain of emm type stC74a in a mouse model of necrotizing myositis, as assessed by bacterial CFU burden, lesion size, and survival curves. Taken together, our genomic and pathogenesis data show the strains of emm type stG62647 we studied are closely genetically related and have enhanced virulence in a mouse model of severe invasive disease. Our findings underscore the need for expanded study of the genomics and molecular pathogenesis of S. dysgalactiae subsp. equisimilis strains causing human infections. IMPORTANCE Our studies addressed a critical knowledge gap in understanding the genomics and virulence of the bacterial pathogen Streptococcus dysgalactiae subsp. equisimilis. S. dysgalactiae subsp. equisimilis strains are responsible for a recent increase in severe human infections in some countries. We determined that certain S. dysgalactiae subsp. equisimilis strains are genetically descended from a common ancestor and that these strains can cause severe infections in a mouse model of necrotizing myositis. Our findings highlight the need for expanded studies on the genomics and pathogenic mechanisms of this understudied subspecies of the Streptococcus family.

Integrative Reverse Genetic Analysis Identifies Polymorphisms Contributing to Decreased Antimicrobial Agent Susceptibility in Streptococcus pyogenes.

Identification of genetic polymorphisms causing increased antibiotic resistance in bacterial pathogens traditionally has proceeded from observed phenotype to defined mutant genotype. The availability of large collections of microbial genome sequences that lack antibiotic susceptibility metadata provides an important resource and opportunity to obtain new information about increased antimicrobial resistance by a reverse genotype-to-phenotype bioinformatic and experimental workflow. We analyzed 26,465 genome sequences of Streptococcus pyogenes, a human pathogen causing 700 million infections annually. The population genomic data identified amino acid changes in penicillin-binding proteins 1A, 1B, 2A, and 2X with signatures of evolution under positive selection as potential candidates for causing decreased susceptibility to ß-lactam antibiotics. Construction and analysis of isogenic mutant strains containing individual amino acid replacements in penicillin-binding protein 2X (PBP2X) confirmed that the identified residues produced decreased susceptibility to penicillin. We also discovered the first chimeric PBP2X in S. pyogenes and show that strains containing it have significantly decreased ß-lactam susceptibility. The novel integrative reverse genotype-to-phenotype strategy presented is broadly applicable to other pathogens and likely will lead to new knowledge about antimicrobial agent resistance, a massive public health problem worldwide. IMPORTANCE The recent demonstration that naturally occurring amino acid substitutions in Streptococcus pyogenes PBP2X are sufficient to cause severalfold reduced susceptibility to multiple ß-lactam antibiotics in vitro raises the concern that these therapeutic agents may become compromised. Substitutions in PBP2X are common first-step mutations that, with the incremental accumulation of additional adaptive mutations within the PBPs, can result in high-level resistance. Because ß-lactam susceptibility testing is not routinely performed, the nature and extent of such substitutions within the PBPs of S. pyogenes are poorly characterized. To address this knowledge deficit, polymorphisms in the PBPs were identified among the most comprehensive cohort of S. pyogenes genome sequences investigated to date. The mutational processes and selective forces acting on the PBPs were assessed to identify specific substitutions likely to influence ß-lactam susceptibility and to evaluate factors posited to be impediments to resistance emergence.

Diagnosis of Listeria monocytogenes meningoencephalitis by real-time PCR for the hly gene.

Listeria monocytogenes is a bacterial pathogen that can invade the central nervous system (CNS), causing meningoencephalitis and brain abscesses. The diagnosis of CNS listeriosis, based on the isolation of the bacteria in the cerebrospinal fluid (CSF), can be difficult because of previous antibiotic treatment and a low number of bacteria in the CSF. To improve the sensitivity of microbiological diagnosis, we have developed a real-time PCR assay for detecting and quantifying L. monocytogenes DNA in the CSF. The designed primers specifically amplify the L. monocytogenes hly gene, which encodes listeriolysin O, a pore-forming cytolysin. The PCR assay for the hly gene (PCR-hly) provides reproducible quantitative results over a wide dynamic range of concentrations and was highly sensitive while detecting a single gene copy/ml. By assaying a large panel of bacterial species, including species secreting pore-forming cytolysin, we determined the specificity of the PCR-hly, which exclusively detects the L. monocytogenes DNA. We then analyzed 214 CSF samples from patients suspected of having CNS listeriosis. PCR-hly was positive in all cases in which L. monocytogenes was isolated by culture. Positive PCR-hly of the CSF was also obtained for five additional, clinically confirmed cases of CNS listeriosis for which bacterial cultures were negative presumably due to previous treatment with antibiotics. As a complement to classical bacteriological CSF culture, our designed real-time PCR-hly assay proved to be valuable by enhancing the rapidity and the accuracy of the diagnosis of CNS infection by L. monocytogenes. In addition, the quantitative results provided may, in some instances, be useful for the follow-up of patients under treatment.

Listeria monocytogenes: a rare complication of ventriculoperitoneal shunt in children.

We report a case of ventriculoperitoneal (VP) shunt infection in a 3-year-old boy caused by the food-borne pathogen Listeria monocytogenes, subsequent to acute peritonitis. This unusual presentation of central nervous system (CNS) listeriosis underlines the ability of the bacteria to form and survive within biofilms on indwelling medical devices. Bacterial persistence may lead to treatment failure and spreading. We highlight the helpfulness of specific quantitative real-time PCR for the hly gene (PCR-hly) for the diagnosis and follow-up of such infections in detecting bacterial persistence within medical devices despite effective antibiotic treatment. Only the surgical replacement of the VP shunt will resolve the infection.

The complete genome sequence of Corynebacterium pseudotuberculosis FRC41 isolated from a 12-year-old girl with necrotizing lymphadenitis reveals insights into gene-regulatory networks contributing to virulence.

BACKGROUND: Corynebacterium pseudotuberculosis is generally regarded as an important animal pathogen that rarely infects humans. Clinical strains are occasionally recovered from human cases of lymphadenitis, such as C. pseudotuberculosis FRC41 that was isolated from the inguinal lymph node of a 12-year-old girl with necrotizing lymphadenitis. To detect potential virulence factors and corresponding gene-regulatory networks in this human isolate, the genome sequence of C. pseudotuberculosis FCR41 was determined by pyrosequencing and functionally annotated. RESULTS: Sequencing and assembly of the C. pseudotuberculosis FRC41 genome yielded a circular chromosome with a size of 2,337,913 bp and a mean G+C content of 52.2%. Specific gene sets associated with iron and zinc homeostasis were detected among the 2,110 predicted protein-coding regions and integrated into a gene-regulatory network that is linked with both the central metabolism and the oxidative stress response of FRC41. Two gene clusters encode proteins involved in the sortase-mediated polymerization of adhesive pili that can probably mediate the adherence to host tissue to facilitate additional ligand-receptor interactions and the delivery of virulence factors. The prominent virulence factors phospholipase D (Pld) and corynebacterial protease CP40 are encoded in the genome of this human isolate. The genome annotation revealed additional serine proteases, neuraminidase H, nitric oxide reductase, an invasion-associated protein, and acyl-CoA carboxylase subunits involved in mycolic acid biosynthesis as potential virulence factors. The cAMP-sensing transcription regulator GlxR plays a key role in controlling the expression of several genes contributing to virulence. CONCLUSION: The functional data deduced from the genome sequencing and the extended knowledge of virulence factors indicate that the human isolate C. pseudotuberculosis FRC41 is equipped with a distinct gene set promoting its survival under unfavorable environmental conditions encountered in the mammalian host.

Low penetrance, broad resistance, and favorable outcome of interleukin 12 receptor beta1 deficiency: medical and immunological implications.

The clinical phenotype of interleukin 12 receptor beta1 chain (IL-12Rbeta1) deficiency and the function of human IL-12 in host defense remain largely unknown, due to the small number of patients reported. We now report 41 patients with complete IL-12Rbeta1 deficiency from 17 countries. The only opportunistic infections observed, in 34 patients, were of childhood onset and caused by weakly virulent Salmonella or Mycobacteria (Bacille Calmette-Guérin -BCG- and environmental Mycobacteria). Three patients had clinical tuberculosis, one of whom also had salmonellosis. Unlike salmonellosis, mycobacterial infections did not recur. BCG inoculation and BCG disease were both effective against subsequent environmental mycobacteriosis, but not against salmonellosis. Excluding the probands, seven of the 12 affected siblings have remained free of case-definition opportunistic infection. Finally, only five deaths occurred in childhood, and the remaining 36 patients are alive and well. Thus, a diagnosis of IL-12Rbeta1 deficiency should be considered in children with opportunistic mycobacteriosis or salmonellosis; healthy siblings of probands and selected cases of tuberculosis should also be investigated. The overall prognosis is good due to broad resistance to infection and the low penetrance and favorable outcome of infections. Unexpectedly, human IL-12 is redundant in protective immunity against most microorganisms other than Mycobacteria and Salmonella. Moreover, IL-12 is redundant for primary immunity to Mycobacteria and Salmonella in many individuals and for secondary immunity to Mycobacteria but not to Salmonella in most.

Complete Genome Sequences of Two Strains of Streptococcus pyogenes Belonging to an Emergent Clade of the Genotype emm89 in Brittany, France.

The frequency of infections due to Streptococcus pyogenes M/emm89 strains is increasing, presumably due to the emergence of a genetically distinct clone. We sequenced two emm89 strains isolated in Brittany, France, in 2009 and 2010 from invasive and noninvasive infections, respectively. Both strains belong to a newly emerged emm89 clade 3 clone.

Avrilella dinanensis gen. nov., sp. nov., a novel bacterium of the family Flavobacteriaceae isolated from human blood.

Polyphasic taxonomic analysis was performed on a novel bacterium, designated UR159T, isolated in 2016 from human blood of a septic patient hospitalized in France. Preliminary 16S rRNA gene sequence-based phylogenetic analysis indicated that strain UR159T belonged to the family Flavobacteriaceae, forming a distinct phyletic line distantly related (<94% sequence similarity) to known species of the family. Further phenotypic, chemotaxonomic and genomic analyses were performed. Cells were non-motile, oxidase-negative, catalase-positive Gram-negative rods. It was strictly aerobic yielding yellow-pigmented colonies, and was metabolically rather inert. Major fatty acids were iso-branched fatty acids, predominantly iso-C15:0 (55.5%) and iso-C17:1ω9c (8.8%). Whole genome sequencing revealed a 2.3-Mbp genome encoding a total of 2262 putative genes with a genomic DNA G+C content at 37.6mol%. The average nucleotide identity (ANI) and in silico DNA-DNA hybridization (isDDH) values between strain UR159T and the most closely related members of the Flavobacteriaceae family were <75% and <39%, respectively, much below the established cut-offs for ANI (<95-96%) and isDDH (<70%) for species and genus delineation. Average Amino Acid Identity (AAI) percentages were also estimated and were lower than 65% (cut-off proposed for genus delineation for uncultivated prokaryotes) in all cases, except for F. marinum that was just at the limit (65.1%). Based on these findings, we propose it as a new genus and species, Avrilella dinanensis gen. nov., sp. nov. (type strain UR159T=CIP 111616T=DSM 105483T).

A prospective survey of Streptococcus pyogenes infections in French Brittany from 2009 to 2017: Comprehensive dynamic of new emergent emm genotypes.

Streptococcus pyogenes or group A Streptococcus (GAS) causes diseases ranging from uncomplicated pharyngitis to life-threatening infections. It has complex epidemiology driven by the diversity, the temporal and geographical fluctuations of the circulating strains. Despite the global burden of GAS diseases, there is currently no available vaccination strategy against GAS infections. This study, based on a longitudinal population survey, aimed to understand the dynamic of GAS emm types and to give leads to better recognition of underlying mechanisms for the emergence of successful clones. From 2009 to 2017, we conducted a systematic culture-based diagnosis of GAS infections in a French Brittany population with a prospective recovery of clinical data. The epidemiological analysis was performed using emm typing combined with the structural and functional cluster-typing system for all the recovered strains. Risk factors for the invasiveness, identified by univariate analysis, were computed in a multiple logistic regression analysis, and the only independent risk factor remaining in the model was the age (OR for the entire range [CI95%] = 6.35 [3.63, 11.10]; p<0.0001). Among the 61 different emm types identified, the most prevalent were emm28 (16%), emm89 (15%), emm1 (14%), and emm4 (8%), which accounted for more than 50% of circulating strains. During the study period, five genotypes identified as emm44, 66, 75, 83, 87 emerged successively and belonged to clusters D4, E2, E3, and E6 that were different from those gathering "Prevalent" emm types (clusters A-C3 to 5, E1 and E4). We previously reported significant genetic modifications for emm44, 66, 83 and 75 types resulting possibly from a short adaptive evolution. Herein we additionally observed that the emergence of a new genotype could occur in a susceptible population having specific risk factors or probably lacking a naturally-acquired cluster-specific immune cross-protection. Among emergent emm types, emm75 and emm87 tend to become prevalent with a stable annual incidence and the risk of a clonal expansion have to be considered.

Actinomyces in chronic granulomatous disease: an emerging and unanticipated pathogen.

BACKGROUND: Chronic granulomatous disease (CGD) is a rare inherited disease of the phagocyte NADPH oxidase system that causes defective production of toxic oxygen metabolites, impaired bacterial and fungal killing, and recurrent life-threatening infections, mostly by catalase-producing organisms. We report for the first time, to our knowledge, chronic infections with Actinomyces species in 10 patients with CGD. Actinomycosis is a chronic granulomatous condition that commonly manifests as cervicofacial, pulmonary, or abdominal disease, caused by slowly progressive infection with oral and gastrointestinal commensal Actinomyces species. Treatment of actinomycosis is usually simple in immunocompetent individuals, requiring long-term, high-dose intravenous penicillin, but is more complicated in those with CGD because of delayed diagnosis and an increased risk of chronic invasive or debilitating disease. METHODS: Actinomyces was identified by culture, staining, 16S ribosomal DNA polymerase chain reaction, and/or a complement fixation test in 10 patients with CGD. RESULTS: All 10 patients presented with a history of fever and elevated inflammatory signs without evident focus. Diagnosis was delayed and clinical course severe and protracted despite high-dose intravenous antibiotic therapy and/or surgery. These results suggest an unrecognized and unanticipated susceptibility to weakly pathogenic Actinomyces species in patients with CGD because these are catalase-negative organisms previously thought to be nonpathogenic in CGD. CONCLUSIONS: Actinomycosis should be vigorously sought and promptly treated in patients with CGD presenting with uncommon and prolonged clinical signs of infection. Actinomycosis is a catalase-negative infection important to consider in CGD.

Listeriolysin O: a key protein of Listeria monocytogenes with multiple functions.

Cholesterol-dependent cytolysins (CDCs) are produced by a large number of pathogenic Gram-positive bacteria. Most of these single-chain proteins are secreted in the extracellular medium. Among the species producing CDCs, only two species belonging to the genus Listeria (Listeria monocytogenes and Listeria ivanovii) are able to multiply intracellularly and release their toxins in the phagosomal compartment of the infected host cell. This review provides an updated overview on the importance of listeriolysin O (LLO) in the pathogenicity of L. monocytogenes, focusing mainly on two aspects: (1) the structure-function relationship of LLO and (2) its role in intra- and extracellular signalling. We first examine the specific sequence determinants, or protein domains, that make this cytolysin so well adapted to the intracellular lifestyle of L. monocytogenes. The roles that LLO has in cellular signalling events in the context of relations to pathogenesis are also discussed.

Full Sequencing and Genomic Analysis of Three emm75 Group A Streptococcus Strains Recovered in the Course of an Epidemiological Shift in French Brittany.

While the incidence and invasiveness of type emm75 group A Streptococcus (GAS) infections increased in French Brittany during 2013, we sequenced and analyzed the genomes of three independent strains isolated in 2009, 2012, and 2014, respectively. In this short-term evolution, genomic analysis evidenced mainly the integration of new phages encoding virulence factors.

Corynebacterium pseudotuberculosis necrotizing lymphadenitis in a twelve-year-old patient.

We report the case of a 12-year-old girl with Corynebacterium pseudotuberculosis necrotizing lymphadenitis. Awareness of this disease is critical for the diagnosis given that corynebacteria are often considered as skin contaminants. The isolate was highly susceptible to antibiotics but only ciprofloxacin, rifampin and gentamicin exerted bactericidal activity.

Genome Sequence of the Uncommon Streptococcus pyogenes M/emm66 Strain STAB13021, Isolated from Clonal Clustered Cases in French Brittany.

Here, we announce the complete annotated genome sequence of the invasive Streptococcus pyogenes strain M/emm66, isolated in 2013 from a subcutaneous abscess in new clustered cases in French Brittany.