Benoît Jaulhac

Matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry identifies 90% of bacteria directly from blood culture vials

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is now widely used for marker/multi-biomarker detection in medical diagnosis. We tested a new protocol for bacterial identification from blood culture broths in hospital routine by using collection tubes with separator gels on 503 included samples examined over 3 months, where 1.5 mL was injected by a syringe into BD Vacutainer tubes from BACTEC-positive bottles, before processing for bacterial protein extraction. Samples were loaded in duplicate onto the MALDI MS target, allowing a series of 12 samples to be processed in duplicate within 80 min by using Biflex III and BioTyper 2.0 software (Bruker). Including polymicrobial samples, 193 of 213 of Gram-negative bacteria (91.08%) and 284 of 319 of Gram-positive bacteria (89.02%) were correctly identified at the species level. Enterobacteriaceae constituted 35.15% of all species found, Staphylococaceae 37.96%, Streptococaceae and Enterococaceae 20.85%, Pseudomonadaceae 1.69%, and anaerobes 2.44%. In most of the polymicrobial samples, one of the species present was identified (80.9%). Seven isolates remained misidentified as Streptococcus pneumoniae, all belonging to Streptococcus mitis. Staphylococcus aureus was identified better when grown on anaero-aerobic medium, and MALDI BioTyper identification scores as low as 1.4 were pertinent, provided that four successive proposals of the same species were given. This new protocol correlates with conventional microbiology procedures by up to 90%, and by >95% for only monomicrobial samples, and provides a decreased turn-around time for identification of bacteria isolated from blood cultures, making this technology suitable also for blood cultures, with less delay and cost decreases in bacterial diagnostics, and favouring better care of patients.

Comparison of Mycosis IC/F and Plus Aerobic/F Media for Diagnosis of Fungemia by the Bactec 9240 System

ABSTRACT Fungemia is associated with a high mortality rate. We compared the performance of the Mycosis IC/F selective fungal medium and the Plus Aerobic/F standard bacteriological medium for the diagnosis of fungemia on the Bactec 9240 automatic system. We retrospectively analyzed 550 blood culture pairs composed of one Mycosis IC/F vial and one Plus Aerobic/F vial, drawn in 187 patients with fungemia. The positivity rate by vial was significantly higher on Mycosis IC/F medium than on Plus Aerobic/F medium (88.0% versus 74.9%, P < 0.0001). The positivity rate for fungus detection on Plus Aerobic/F medium fell to 26.9% when bacteria were present in the same vial. The positivity rate by patient was also significantly higher on Mycosis IC/F medium than on Plus Aerobic/F medium (92.5% versus 75.9%, P < 0.0001). A marked superiority of Mycosis IC/F medium was demonstrated for diagnosis of Candida glabrata fungemia (31 of 31, 100%, versus 18 of 31, 58.1%, P < 0.0001). The mean detection time was significantly shorter on Mycosis IC/F medium than on Plus Aerobic/F medium (28.9 ± 22.2 h versus 36.5 ± 24.6 h, P < 0.0001). The mean time saving was 8.8 h for Candida albicans and 43.7 h for C. glabrata. Mycosis IC/F medium enabled more sensitive and earlier diagnosis, particularly for the two strains most frequently responsible for fungemia, C. albicans and C. glabrata, and also in the event of the concomitant presence of both yeasts and bacteria. In patients with risk factors, it would thus appear to be sensible to draw a Mycosis IC/F vial in addition to the standard bacteriological vials.

Autoantigen, innate immunity, and T cells cooperate to break B cell tolerance during bacterial infection

Autoantibody production during infections is considered to result from nonspecific activation of low-affinity autoreactive B cells. Whether this can lead to autoimmune disease remains uncertain. We show that chronic infection by Borrelia burgdorferi of Tg animals expressing human rheumatoid factor (RF) B cells (of low or intermediate affinities) in the absence or in the constitutive presence of the autoantigen (represented here by chimeric IgG with human constant region) breaks their state of immunological ignorance, leading to the production of RFs. Surprisingly, this production was more pronounced in intermediate-affinity RF Tg mice co-expressing the autoantigen. This overproduction was mediated by immune complexes and involved synergistic signaling between the B cell receptor and Toll-like receptors and T cell help. These findings indicate that chronic infection can activate autoreactive B cells with significant affinity and creates conditions that can drive them to differentiate into memory cells. Such cells may have some physiological yet undetermined role, but in autoimmune-prone individuals, this scenario may initiate autoimmunity.

Disease expression of Lyme borreliosis in northeastern France.

Abstract Since very little is known about the clinical expression of Lyme borreliosis in Western Europe, a 3-year prospective study was conducted that included all patients seen for suspected Lyme borreliosis at the Strasbourg University Hospital in northeastern France. The diagnosis of Lyme borreliosis was made on the basis of the presence of erythema migrans or on the basis of another suggestive clinical manifestation and laboratory confirmation. A total of 132 patients, 70 women and 62 men, mean age 54 years, had Lyme borreliosis according to these criteria. Within this study group, 77% of the patients were regularly exposed to tick bites and 64% could remember one. Erythema migrans, the most frequent clinical manifestation, occurred in 60% of the patients and was the only sign of Lyme borreliosis in 40%. Lymphocytoma and acrodermatitis chronica atrophicans were rare (1 and 3 patients, respectively). Nervous system involvement (mainly radiculoneuropathy), the second most common clinical manifestation, was found in 40% of the patients and was the only sign of Lyme borreliosis in 22%. Musculoskeletal involvement was present in 26% of the patients and was an isolated finding in 14%. During the study period, no patient was diagnosed with Lyme carditis. There was serological evidence of Lyme borreliosis in 75% of the cases and direct evidence of borrelial infection in 10 (7.5%). The results show that the clinical expression of Lyme borreliosis in northeastern France is similar to that in other European countries but different from that in North America.

Tick Saliva Represses Innate Immunity and Cutaneous Inflammation in a Murine Model of Lyme Disease

Lyme borreliosis is an arthropod-borne disease transmitted by the Ixodes tick. This spirochetal infection is first characterized by a local cutaneous inflammation, the erythema migrans. The skin constitutes a key interface in the development of the disease. During Borrelia inoculation, tick saliva affects the innate and adaptive immunity of the vertebrate host skin. Some key mediators of innate immunity such as antimicrobial peptides (cathelicidin and defensin families) have been identified as important initiators of skin inflammation. We analyzed the role of tick saliva on integumental innate immunity using different protocols of Borrelia infection, via syringe or direct tick transmission. When syringe inoculation was used, Borrelia triggered skin inflammation with induction of CRAMP, the mouse cathelicidin, and tumor necrosis factor-alpha. However, when Borrelia was transmitted directly via the tick, we observed a significant repression of inflammatory genes, suggesting a critical role of tick saliva in skin innate immunity. For all the protocols tested, a peak of intense Borrelia multiplication occurred in the skin between days 5 and 15, before bacterial dissemination to target organs. We conclude that Borrelia pathogens specifically use the tick saliva to facilitate their transmission to the host and that the skin constitutes an essential interface in the development of Lyme disease.

Analysis of bacterial DNA in synovial tissue of Tunisian patients with reactive and undifferentiated arthritis by broad-range PCR, cloning and sequencing.

IntroductionBacteria and/or their antigens have been implicated in the pathogenesis of reactive arthritis (ReA). Several studies have reported the presence of bacterial antigens and nucleic acids of bacteria other than those specified by diagnostic criteria for ReA in joint specimens from patients with ReA and various arthritides. The present study was conducted to detect any bacterial DNA and identify bacterial species that are present in the synovial tissue of Tunisian patients with reactive arthritis and undifferentiated arthritis (UA) using PCR, cloning and sequencing.MethodsWe examined synovial tissue samples from 28 patients: six patients with ReA and nine with UA, and a control group consisting of seven patients with rheumatoid arthritis and six with osteoarthritis (OA). Using broad-range bacterial PCR producing a 1,400-base-pair fragment from the 16S rRNA gene, at least 24 clones were sequenced for each synovial tissue sample. To identify the corresponding bacteria, DNA sequences were compared with sequences from the EMBL (European Molecular Biology Laboratory) database.ResultsBacterial DNA was detected in 75% of the 28 synovial tissue samples. DNA from 68 various bacterial species were found in ReA and UA samples, whereas DNA from 12 bacteria were detected in control group samples. Most of the bacterial DNAs detected were from skin or intestinal bacteria. DNA from bacteria known to trigger ReA, such as Shigella flexneri and Shigella sonnei, were detected in ReA and UA samples of synovial tissue and not in control samples. DNA from various bacterial species detected in this study have not previously been found in synovial samples.ConclusionThis study is the first to use broad-range PCR targeting the full 16S rRNA gene for detection of bacterial DNA in synovial tissue. We detected DNA from a wide spectrum of bacterial species, including those known to be involved in ReA and others not previously associated with ReA or related arthritis. The pathogenic significance of some of these intrasynovial bacterial DNAs remains unclear.

Antialarmin Effect of Tick Saliva during the Transmission of Lyme Disease

ABSTRACT Tick saliva has potent immunomodulatory properties. In arthropod-borne diseases, this effect is largely used by microorganisms to increase their pathogenicity and to evade host immune responses. We show that in Lyme borreliosis, tick salivary gland extract and a tick saliva protein, Salp15, inhibit in vitro keratinocyte inflammation induced by Borrelia burgdorferi sensu stricto or by the major outer surface lipoprotein of Borrelia, OspC. Chemokines (interleukin-8 [IL-8] and monocyte chemoattractant protein 1 [MCP-1]) and several antimicrobial peptides (defensins, cathelicidin, psoriasin, and RNase 7) were downregulated. Interestingly, antimicrobial peptides (AMPs) transiently inhibited bacterial motility but did not kill the organisms when tested in vitro. We conclude that tick saliva affects the chemotactic properties of chemokines and AMPs on immune cells and has an antialarmin effect on human primary keratinocytes. Alarmins are mediators that mobilize and activate antigen-presenting cells. Inhibition of cutaneous innate immunity and of the migration of immune cells to the site of the tick bite ensures a favorable environment for Borrelia. The bacterium can then multiply locally and, subsequently, disseminate to the target organs, including joints, heart, and the central nervous system.

Bacterial epidemiology and antimicrobial resistance in ascitic fluid: a 2-year retrospective study.

The bacterial epidemiology of bacterascites and spontaneous bacterial peritonitis is evolving. Four hundred and eleven strains isolated from ascites in cirrhotic patients from 5 French hospitals were isolated in 2006 and 2007. Of these, 114 were definitely associated with spontaneous bacterial peritonitis. The proportion of Gram-positive and Gram-negative agents was quite similar, even after excluding coagulase-negative staphylococci, or when considering only definite spontaneous bacterial peritonitis or community-acquired strains. Staphylococci and Escherichia coli were the most frequent pathogens, but enterococci were also involved in nearly 15% of the cases. Among the E. coli, 28% were intermediate or resistant to amoxicillin+clavulanate, 5.3% expressed cephalosporinases or extended β-lactamases and 17.3% were intermediate or resistant to fluoroquinolones. Resistance to methicillin was observed in 27% of Staphylococcus aureus. Cefotaxime and amoxicillin–clavulanate remained the most effective ‘single’ agents, however on less than 70% of isolates. Some combinations (such as cefotaxime+amoxicillin) extended coverage to a further 15% of strains. Since inadequate empiric antibiotic therapy is associated with increased mortality, these combinations may be of great interest as first-line treatment, even though they may also lead to the development of antimicrobial resistance. Repeated epidemiological surveys and new clinical trials are thus needed.

Broad-range PCR, cloning and sequencing of the full 16S rRNA gene for detection of bacterial DNA in synovial fluid samples of Tunisian patients with reactive and undifferentiated arthritis

IntroductionBroad-range rDNA PCR provides an alternative, cultivation-independent approach for identifying bacterial DNA in reactive and other form of arthritis. The aim of this study was to use broad-range rDNA PCR targeting the 16S rRNA gene in patients with reactive and other forms of arthritis and to screen for the presence of DNA from any given bacterial species in synovial fluid (SF) samples.MethodsWe examined the SF samples from a total of 27 patients consisting of patients with reactive arthritis (ReA) (n = 5), undifferentiated arthritis (UA) (n = 9), rheumatoid arthritis (n = 7), and osteoarthritis (n = 6) of which the latter two were used as controls. Using broad-range bacterial PCR amplifying a 1400 bp fragment from the 16S rRNA gene, we identified and sequenced at least 24 clones from each SF sample. To identify the corresponding bacteria, DNA sequences were compared to the EMBL (European Molecular Biology Laboratory) database.ResultsBacterial DNA was identified in 20 of the 27 SF samples (74, 10%). Analysis of a large number of sequences revealed the presence of DNA from more than one single bacterial species in the SF of all patients studied. The nearly complete sequences of the 1400 bp were obtained for most of the detected species. DNA of bacterial species including Shigella species, Escherichia species, and other coli-form bacteria as well as opportunistic pathogens such as Stenotrophomonas maltophilia and Achromobacter xylosoxidans were shared in all arthritis patients. Among pathogens described to trigger ReA, DNA from Shigella sonnei was found in ReA and UA patients. We also detected DNA from rarely occurring human pathogens such as Aranicola species and Pantoea ananatis. We also found DNA from bacteria so far not described in human infections such as Bacillus niacini, Paenibacillus humicus, Diaphorobacter species and uncultured bacterium genera incertae sedis OP10.ConclusionsBroad-range PCR followed by cloning and sequencing the entire 16S rDNA, allowed the identification of the bacterial DNA environment in the SF samples of arthritic patients. We found a wide spectrum of bacteria including those known to be involved in ReA and others not previously associated with arthritis.

Emergence of human granulocytic anaplasmosis in France.

In France, only one case of tick-borne human granulocytic anaplasmosis has been described in the literature (in 2003). Here, we report 5 new human cases of Anaplasma phagocytophilum infection from north-eastern France diagnosed in our laboratory in south-eastern France by serology and molecular biology. This increase is directly related to more physician interest in this disease and the implementation of a new PCR tool.