Recent actuality about Bacillus cereus and human milk bank: a new sensitive method for microbiological analysis of pasteurized milk.

Three cases of Bacillus cereus infection or colonization occurred in the same region in France, and milk from the milk bank was suspected as a possible common source of contamination. All Batches delivered to the three cases complied with the requirements of the bacteriological reference method recommended by good practices guidelines. Still, a retrospective analysis with a more sensitive method showed one batch to contain B. cereus, however straincomparison revealed no epidemiological link betweenisolates from patients and those from the milk. Consequently, in accordance with the precautionary principle, we developed a new sensitive method for the screening of pasteurized milk for pathogenic bacteria. From January 1 to August 31, 2017, 2526 samples of pasteurized milk were prospectively included in the study. We showed that a 20 mL sample of pasteurized milk incubated for 18 h at 37 °C under aerobic conditions was favoring the detection of B. Cereus. The nonconformity rate was 6.3% for the reference method and 12.6% for the improved method (p < 0.0001). Nonconformity was due to the presence of B. cereus in 88.5% of cases for the improved method and 53% of cases for the reference method (p < 0.0001). Thus our new method is improves the microbiological safety of the product distributed and only moderately increases the rate of bacteriological nonconformity .

[Applications of MALDI-TOF technology in clinical microbiology]. / Applications de la technologie MALDI-TOF en microbiologie clinique.

Until now, the identification of micro-organisms has been based on the cultural and biochemical characteristics of bacterial and fungal species. Recently, Mass Spectrometry type Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF MS) was developed in clinical microbiology laboratories. This new technology allows identification of micro-organisms directly from colonies of bacteria and fungi within few minutes. In addition, it can be used to identify germs directly from positive blood culture bottles or directly from urine samples. Other ways are being explored to expand the use of MALDI-TOF in clinical microbiology laboratories. Indeed, some studies propose to detect bacterial antibiotic resistance while others compare strains within species for faster strain typing. The main objective of this review is to update data from the recent literature for different applications of MALDI-TOF technique in microbiological diagnostic routine.

Evaluation of the Andromas matrix-assisted laser desorption ionization-time of flight mass spectrometry system for identification of aerobically growing Gram-positive bacilli.

Matrix-associated laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a rapid and simple microbial identification method. Previous reports using the Biotyper system suggested that this technique requires a preliminary extraction step to identify Gram-positive rods (GPRs), a technical issue that may limit the routine use of this technique to identify pathogenic GPRs in the clinical setting. We tested the accuracy of the MALDI-TOF MS Andromas strategy to identify a set of 659 GPR isolates representing 16 bacterial genera and 72 species by the direct colony method. This bacterial collection included 40 C. diphtheriae, 13 C. pseudotuberculosis, 19 C. ulcerans, and 270 other Corynebacterium isolates, 32 L. monocytogenes and 24 other Listeria isolates, 46 Nocardia, 75 Actinomyces, 18 Actinobaculum, 11 Propionibacterium acnes, 18 Propionibacterium avidum, 30 Lactobacillus, 21 Bacillus, 2 Rhodococcus equi, 2 Erysipelothrix rhusiopathiae, and 38 other GPR isolates, all identified by reference techniques. Totals of 98.5% and 1.2% of non-Listeria GPR isolates were identified to the species or genus level, respectively. Except for L. grayi isolates that were identified to the species level, all other Listeria isolates were identified to the genus level because of highly similar spectra. These data demonstrate that rapid identification of pathogenic GPRs can be obtained without an extraction step by MALDI-TOF mass spectrometry.

Activation of ErbB2 receptor tyrosine kinase supports invasion of endothelial cells by Neisseria meningitidis.

ErbB2 is a receptor tyrosine kinase belonging to the family of epidermal growth factor (EGF) receptors which is generally involved in cell differentiation, proliferation, and tumor growth, and activated by heterodimerization with the other members of the family. We show here that type IV pilus-mediated adhesion of Neisseria meningitidis onto endothelial cells induces tyrosyl phosphorylation and massive recruitment of ErbB2 underneath the bacterial colonies. However, neither the phosphorylation status nor the cellular localization of the EGF receptors, ErbB3 or ErbB4, were affected in infected cells. ErbB2 phosphorylation induced by N. meningitidis provides docking sites for the kinase src and leads to its subsequent activation. Specific inhibition of either ErbB2 and/or src activity reduces bacterial internalization into endothelial cells without affecting bacteria-induced actin cytoskeleton reorganization or ErbB2 recruitment. Moreover, inhibition of both actin polymerization and the ErbB2/src pathway totally prevents bacterial entry. Altogether, our results provide new insight into ErbB2 function by bringing evidence of a bacteria-induced ErbB2 clustering leading to src kinase phosphorylation and activation. This pathway, in cooperation with the bacteria-induced reorganization of the actin cytoskeleton, is required for the efficient internalization of N. meningitidis into endothelial cells, an essential process enabling this pathogen to cross host cell barriers.

Is it time to reconsider initial antibiotic treatment strategies for severe urinary tract infections in Europe?

Until recently, most reported cases of bacteraemia caused by multidrug-resistant strains of Enterobacteriacae producing an extended-spectrum beta-lactamase (ESBL) in Europe have been nosocomial in origin. However, increasing numbers of reports of community-acquired bacteraemia and urinary tract infection caused by ESBL-producing microorganisms suggest that the geographical origin of patients should be taken into account as a risk-factor for possible ESBL production. Early identification of patients at high-risk of infection with ESBL-producing microorganisms, based on their geographical origin and travel history, should help to optimise initial antibiotic treatment strategies for severe urinary tract infections in Europe.

Host and bacterial determinants of initial severity and outcome of Escherichia coli sepsis.

A 1-year prospective cohort study of all episodes of Escherichia coli bacteraemia in two French university hospitals was conducted to assess simultaneously the influence of host and bacterial determinants on the initial severity and outcome of E. coli sepsis. Clinical data (community-acquired/nosocomial infection, immune status, underlying disease, primary source of infection, severity sepsis scoring and outcome), phylogenetic groups (A, B1, D and B2), nine virulence factors (VFs) (papC, papGII, papGIII, sfa/foc, hlyC, cnf1, iucC, fyuA and iroN) and the antibiotic susceptibility of isolates were investigated. All VFs except iucC were significantly more prevalent (p <0.05) among the B2 group isolates. The non-B2 isolates were more frequently resistant to antibiotics than were B2 isolates (p <0.05). There were significantly more B2 isolates from immunocompetent than from immunocompromised patients (p <0.05). No bacterial or host determinants influenced the initial severity of sepsis. Multivariate analysis revealed that the presence of papGIII, septic shock at baseline and a non-urinary tract origin of sepsis were associated independently with a fatal outcome (p 0.04, <0.0001 and 0.04, respectively). A factorial analysis of correspondence allowed two populations of isolates to be distinguished: those belonging to the B2 group were associated more frequently with susceptibility to antibiotics, community-acquired infection, a urinary tract origin and immunocompetent hosts; those belonging to the A, B1 or D groups were associated more frequently with resistance to antibiotics, a nosocomial origin, a non-urinary tract source and immunocompromised hosts. Although no influence of host or bacterial determinants on the initial severity of sepsis was detected, bacterial and host determinants both influenced the outcome of E. coli sepsis significantly.

Untargeted next-generation sequencing-based first-line diagnosis of infection in immunocompromised adults: a multicentre, blinded, prospective study.

OBJECTIVE: Infections are the major cause of morbidity and mortality in immunocompromised patients. Improving microbiological diagnosis in these patients is of paramount clinical importance. METHODS: We performed this multicentre, blinded, prospective, proof-of-concept study, to compare untargeted next-generation sequencing with conventional microbiological methods for first-line diagnosis of infection in 101 immunocompromised adults. Patients were followed for 30 days and their blood samples, and in some cases nasopharyngeal swabs and/or biological fluids, were analysed. At the end of the study, expert clinicians evaluated the results of both methods. The primary outcome measure was the detection rate of clinically relevant viruses and bacteria at inclusion. RESULTS: Clinically relevant viruses and bacteria identified by untargeted next-generation sequencing and conventional methods were concordant for 72 of 101 patients in samples taken at inclusion (κ test=0.2, 95% CI 0.03-0.48). However, clinically relevant viruses and bacteria were detected in a significantly higher proportion of patients with untargeted next-generation sequencing than conventional methods at inclusion (36/101 (36%) vs. 11/101 (11%), respectively, p <0.001), and even when the latter were continued over 30 days (19/101 (19%), p 0.003). Untargeted next-generation sequencing had a high negative predictive value compared with conventional methods (64/65, 95% CI 0.95-1). CONCLUSIONS: Untargeted next-generation sequencing has a high negative predictive value and detects more clinically relevant viruses and bacteria than conventional microbiological methods. Untargeted next-generation sequencing is therefore a promising method for microbiological diagnosis in immunocompromised adults.

Extracellular bacterial pathogens and small GTPases of the Rho family: an unexpected combination.

Even in the case of extracellular bacterial pathogens, it is becoming increasingly clear that successful colonization does not limit itself to passive attachment on the surface of human cells; a dialogue takes place between bacteria and infected cells. These pathogens modulate cellular functions to their advantage, leading to survival and proliferation at the cell surface. Furthermore, there is increasing evidence that a variety of extracellular pathogens activate small GTPases of the Rho family during adhesion, placing these regulators at the center of the interaction between these bacteria and their infected host.

Interaction mechanisms of encapsulated meningococci with eucaryotic cells: what does this tell us about the crossing of the blood-brain barrier by Neisseria meningitidis?

An important feature of Neisseria meningitidis is its ability to invade the meninges. This requires that bacteria cross the blood-brain barrier (BBB), which is one of the tightest barriers of the body. N. meningitidis has, therefore, evolved very sophisticated means by which it circumvents the physical properties of this cellular barrier. Recent advances have allowed the identification of several steps that might occur in the interaction of N. meningitidis with the BBB and the transit of the bacteria to the meninges.

Meningococcal pathogenesis: at the boundary between the pre- and post-genomic eras.

Meningococcal disease remains an important public health burden worldwide and, indeed, cause of death, particularly in poorer countries. The rapidly progressive nature of infections means that antibiotic therapy often comes too late. Vaccines are of limited efficacy in infants, one of the most vulnerable age groups, and do not exist for bacteria of serogroup B. Hence, much remains to be achieved in terms of vaccine design and the understanding of the pathogenesis of meningococcal disease. The causative bacterium, Neisseria meningitidis, is usually a commensal of the nasopharynx. Factors that lead to the invasion of the bloodstream, often followed by the crossing of the blood-brain barrier and meningitis, may be partly host- and partly bacterium-dependent, but are ill-understood. It is hoped that, taken together with the fundamental knowledge gained from biochemical and genetic studies, the huge amount of new information made available with the recent publication of the genome sequences will help to unlock more of the secrets of the lifestyle and pathogenic potential of this still poorly understood pathogen.

Type-4 pili and meningococcal adhesiveness.

The ability to interact with non-phagocytic cells is a crucial virulence attribute of the meningococcus. Pili play a major role in this process and are the only means yet discovered by which capsulated bacteria may adhere to cells. Pilus-mediated adhesion is a two-step process which requires (i) the expression of the adhesin PilC1 and (ii) the expression of an appropriate pilin variant. Some pilin variants have the ability to modify the degree of adhesiveness through the formation of bundles of pili which increases bacteria-bacteria interactions.

Do pathogenic neisseriae need several ways to modify the host cell cytoskeleton?

Neisseria meningitidis and Neisseria gonorrhoeae are human pathogens which have to interact with mucosa and/or cellular barriers for their life cycle. Even though they both give rise to dramatically different diseases, most of the mechanisms mediating cellular interactions are common to N. meningitidis and N. gonorrhoeae. This suggests that bacterial cell interactions may be essential not only for pathogenesis but also for other aspects of the bacterial life cycle that are common to both N. meningitidis and N. gonorrhoeae. Opacity proteins and pili are two major components identified as transducing signals to host cells, thus leading to cytoskeleton modifications. This manuscript will review the recent developments concerning the mechanisms mediating cellular interactions of pathogenic Neisseria and will tentatively put them into the perspective of pathogenesis and bacterial life cycle.

Severe encephalitis resulting from coinfections with HIV and JC virus.

We observed 3 cases of progressive multifocal leukoencephalopathy (PML) among frozen CNS samples obtained at autopsy from 102 adult AIDS patients. In 2 patients, PML was associated with severe HIV encephalitis. In those 2 cases, the areas of extensive JC-induced demyelination were massively infiltrated by HIV infected macrophages/microglial cells with evidence for localized increase of HIV encephalitis in PML lesions. Using immunohistochemistry and in situ hybridization, we demonstrated that each virus infects, in a latent or productive fashion, different CNS cell populations. Therefore, the extension of HIV encephalitis could not be related to an intracellular transactivation of 1 virus by the other. However, the results are consistent with dissemination of viral infection by the recruitment of HIV-infected macrophages to damaged areas of the brain. This phenomenon might be generalized to other pathogens that are frequently associated with HIV CNS infection. Early detection and treatment of opportunistic CNS lesions could be important to prevent extension of HIV encephalitis.

Nucleotide sequence of rmpB, a Klebsiella pneumoniae gene that positively controls, colanic biosynthesis in Escherichia coli.

Production of colanic acid in Escherichia coli is regulated by two negative regulators, Lon and RcsC, and by two positive regulators, RcsA and RcsB. Two genes of Klebsiella pneumoniae, rmpA and rmpB, have been shown to positively control colanic acid synthesis in E. coli. While colanic acid production is activated by RmpA only in a lon strain of E. coli, a plasmid carrying both rmpA and rmpB can stimulate colanic acid synthesis in a Lon+ strain. In this work, we present the determination of the nucleotide sequence of rmpB and, on the basis of comparison of the predicted RmpA and RmpB sequences with those of RcsA, B and C and two-component regulatory proteins, we propose that RmpA acts as a transcriptional activator of the structural genes involved in colanic acid biosynthesis.

Cloning and characterization of the Neisseria meningitidis rfaC gene encoding alpha-1,5 heptosyltransferase I.

We cloned and characterized the Neisseria meningitidis rfaC gene which encodes an enzyme, alpha-1,5 heptosyltransferase I, involved in the synthesis of the deep-core of the lipooligosaccharide. The N. meningitidis rfaC mutant, obtained by an allelic exchange, produced lipooligosaccharide which migrated faster in sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the lipooligosaccharide isolated from the wild-type N. meningitidis. The N. meningitidis rfaC mutant was not affected by growth in a rich microbiological medium and did not show any defect in adhesion to epithelial cell lines. Conversely, the rfaC mutant was attenuated in the infant rat model of meningococcemia, thus confirming the importance of intact lipooligosaccharide in the virulence of N. meningitidis.

Invasion of the Central Nervous System by Neisseria meningitidis.

Neisseria meningitidis is an extracellular pathogen responsible for septicemia and meningitis. The occurrence of meningitis requires that bacteria cross the blood-brain barrier (BBB) and induce an inflammatory response within the sub arachnoid space. The mechanisms that lead to the development of cerebrospinal fluid (CSF) pleocytosis once bacteria have reached the CSF have been studied using several animal models. These mechanisms are similar among extracellular pathogens responsible for meningitis (i.e., Haemophilus influenzae type b, Streptococcuspneumoniae, and N. meningitidis). The in situ production of cytokines is the primary event leading to transmigration of leucocytes through the BBB (1-4).

[Meningitis due to Bacillus cereus in an infant with Reye syndrome]. / Méningite à Bacillus cereus chez un nourrisson au décours d'un syndrome de Reye.

OBSERVATION: We report the case of a 2.5-month-old infant with Bacillus cereus meningitis who was initially admitted for Reye syndrome. Gram positive bacteria was isolated in CSF and shown to be located inside the polymorphonuclears. This pathogen was further identified by sequencing of the 16S RNA. Early administration of imipenem in association with amikacin resulted in a rapid recovery. No obvious immune defect or invasive procedure could be assessed. CONCLUSION: Although Bacillus cereus is mainly associated with contamination, repeated isolations of this bacteria may be due to true infection.

[Rapid epidemiological characterization of Neisseria meningitidis using polymerization chain reaction from biological samplings]. / Caractérisation épidémiologique rapide de Neisseria meningitidis par réaction de polymérisation en chaîne à partir des prélèvements biologiques.

OBJECTIVES: Due to the spread of the meningococcal infections, a good epidemiological surveillance is needed. Prophylactic measures should be undertaken because of the high transmissibility of these bacteria. One problem which hinders the epidemiological characterization is that the responsible strain should be isolated. The aim of this work is to develop a rapid and non culture typing method of Neisseria meningitidis. METHODS: Six cerebrospinal fluids were obtained from 5 different patients with meningococcal meningitis. A specific locus, pil A, for N. meningitidis was amplified by polymerization chain reaction (PCR). The polymorphism of this locus was then analyzed by digesting the PCR products with one of three different restriction enzymes. RESULTS: The polymorphism of this locus allowed us to establish the clonal relationships between the meningococcal strains involved in the infection. Three CSF corresponded to epidemiological strains. CONCLUSION: This typing method allows a rapid and less expensive epidemiological characterization of meningococcal infections. Moreover, it is a non culture typing method.

Evaluation of two matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems for the identification of Candida species.

Candida spp. are responsible for severe infections in immunocompromised patients and those undergoing invasive procedures. The accurate identification of Candida species is important because emerging species can be associated with various antifungal susceptibility spectra. Conventional methods have been developed to identify the most common pathogens, but have often failed to identify uncommon species. Several studies have reported the efficiency of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for the identification of clinically relevant Candida species. In this study, we evaluated two commercially available MALDI-TOF systems, Andromas™ and Bruker Biotyper™, for Candida identification in routine diagnosis. For this purpose, we investigated 1383 Candida isolates prospectively collected in eight hospital laboratories during routine practice. MALDI-TOF MS results were compared with those obtained using conventional phenotypic methods. Analysis of rDNA gene sequences with internal transcribed regions or D1-D2 regions is considered the reference standard for identification. Both MALDI-TOF MS systems could accurately identify 98.3% of the isolates at the species level (1359/1383 for Andromas™; 1360/1383 for Bruker Biotyper™) vs. 96.5% for conventional techniques. Furthermore, whereas conventional methods failed to identify rare or emerging species, these were correctly identified by MALDI-TOF MS. Both MALDI-TOF MS systems are accurate and cost-effective alternatives to conventional methods for mycological identification of clinically relevant Candida species and should improve the diagnosis of fungal infections as well as patient management.