Pierre-Alexandre Drèze

Diversity of bacterial type II toxin–antitoxin systems: a comprehensive search and functional analysis of novel families

Type II toxin–antitoxin (TA) systems are generally composed of two genes organized in an operon, encoding a labile antitoxin and a stable toxin. They were first discovered on plasmids where they contribute to plasmid stability by a phenomenon denoted as ‘addiction’, and subsequently in bacterial chromosomes. To discover novel families of antitoxins and toxins, we developed a bioinformatics approach based on the ‘guilt by association’ principle. Extensive experimental validation in Escherichia coli of predicted antitoxins and toxins increased significantly the number of validated systems and defined novel toxin and antitoxin families. Our data suggest that toxin families as well as antitoxin families originate from distinct ancestors that were assembled multiple times during evolution. Toxin and antitoxin families found on plasmids tend to be promiscuous and widespread, indicating that TA systems move through horizontal gene transfer. We propose that due to their addictive properties, TA systems are likely to be maintained in chromosomes even though they do not necessarily confer an advantage to their bacterial hosts. Therefore, addiction might play a major role in the evolutionary success of TA systems both on mobile genetic elements and in bacterial chromosomes.

Sequential multiplex PCR assay for determining capsular serotypes of colonizing S. pneumoniae

BackgroundAsymptomatic nasopharyngeal carriage represents an important biological marker for monitoring pneumococcal serotype distribution and evaluating vaccine effects. Serotype determination by conventional method (Quellung reaction) is technically and financially challenging. On the contrary, PCR-based serotyping represents a simple, economic and promising alternative method.MethodWe designed a novel multiplex PCR assay for specific detection of the 30 classical colonizing S. pneumoniae serogroups/types. This multiplex assay is composed of 7 consecutive PCR reactions and was validated on a large and recent collection of Streptococcus pneumoniae isolated during a prospective study conducted in Belgium at the time of PCV7 adoption.ResultsThe multiplex PCR assay allowed the typing of more than 94% of the isolates of a collection of pneumococci isolated from Belgian preschool attendees (n = 332). Seventy-five percent of the isolates were typed after 3 subsequent PCR reactions. Results were in agreement with the Quellung identification.ConclusionOur novel multiplex assay is an accurate and reliable method which can be used in place of the conventional method for S. pneumoniae carriage studies.

The development of several organs and appendages is impaired in mice lacking Sp6.

SP6 belongs to the SP/KLF family of transcription factors, characterized by a DNA‐binding domain composed of three zinc fingers of the C2H2 type. The Sp6 gene generates two different transcripts, termed Sp6 and epiprofin, which differ in the first exon and encode the same SP6 protein. These transcripts are mainly expressed in the skin, the teeth, and the limb buds of embryos and also in the adult lungs. To gain insight into the biological function of the SP6 protein, we knocked out the gene by eliminating the full coding region. The resulting Sp6 null mice are nude, lack functional teeth, and present limb and lung malformations. We also showed that the identified abnormalities are associated with apoptotic misregulations. In conclusion, this work indicates that Sp6 plays a critical role in the development of several epithelium‐containing organs or appendages, possibly by regulating apoptosis. Developmental Dynamics 237:883–892, 2008.

Characterization of a novel temperate phage originating from a cereulide-producing Bacillus cereus strain.

A novel temperate bacteriophage was isolated from a Bacillus cereus cereulide-producing strain and named vB_BceS-IEBH. vB_BceS-IEBH belongs to the Siphoviridae family. The complete genome sequence (53 kb) was determined and annotated. Eighty-seven ORFs were detected and for 28, a putative function was assigned using the ACLAME database. vB_BceS-IEBH replicates as a plasmid in the prophage state. Accordingly, a 9-kb plasmid-like region composed of 13 ORFs was identified. A fragment of around 2000 bp comprising an ORF encoding a putative plasmid replication protein was shown to be self-replicating in Bacillus thuringiensis. Mass spectrometry analysis of the purified vB_BceS-IEBH particle identified 8 structural proteins and enabled assignment of a supplementary ORF as being part of the morphogenesis module. Genome analysis further illustrates the diversity of mobile genetic elements and their plasticity within the B. cereus group.

Characterization of Novel Phages Isolated in Coagulase-Negative Staphylococci Reveals Evolutionary Relationships with Staphylococcus aureus Phages

Despite increasing interest in coagulase-negative staphylococci (CoNS), little information is available about their bacteriophages. We isolated and sequenced three novel temperate Siphoviridae phages (StB12, StB27, and StB20) from the CoNS Staphylococcus hominis and S. capitis species. The genome sizes are around 40 kb, and open reading frames (ORFs) are arranged in functional modules encoding lysogeny, DNA metabolism, morphology, and cell lysis. Bioinformatics analysis allowed us to assign a potential function to half of the predicted proteins. Structural elements were further identified by proteomic analysis of phage particles, and DNA-packaging mechanisms were determined. Interestingly, the three phages show identical integration sites within their host genomes. In addition to this experimental characterization, we propose a novel classification based on the analysis of 85 phage and prophage genomes, including 15 originating from CoNS. Our analysis established 9 distinct clusters and revealed close relationships between S. aureus and CoNS phages. Genes involved in DNA metabolism and lysis and potentially in phage-host interaction appear to be widespread, while structural genes tend to be cluster specific. Our findings support the notion of a possible reciprocal exchange of genes between phages originating from S. aureus and CoNS, which may be of crucial importance for pathogenesis in staphylococci.

Nosocomial transmission of necrotising fasciitis

Department of Bacteriology (F Sablier MPh, D Allemeersch MPh), Department of Pediatrics (T Slaouti MD), and Department of Plastic Surgery (P E El Fouly MD), Cliniques de l’Europe, Brussels, Belgium; Laboratory of Bacterial Genetics and Physiology, IBMM, Faculte des Sciences, Universite Libre de Bruxelles, Gosselies, Belgium (P-A Dreze BSc, L Van Melderen PhD, P R Smeesters MD); and Infectious Diseases Department, Hopital Universitaire des Enfants Reine Fabiola, Universite Libre de Bruxelles, Belgium (P R Smeesters MD)

Group A Streptococcus virulence and host factors in two toddlers with rheumatic fever following toxic shock syndrome

BACKGROUND Rheumatic fever (RF) classically occurs after group A Streptococcus (GAS) pharyngitis in children aged over 5 years in developing countries. The present report describes the bacterial and host determinants in non-related toddlers who developed RF diagnostic criteria after toxic shock syndrome (TSS). METHODS AND RESULTS A 13-month-old boy and a 14-month-old girl presented GAS TSS. After several weeks, multiple subcutaneous nodules as well as migratory polyarthritis or monoarthritis developed in both children, fulfilling Jones criteria of RF. The relevance of the Jones criteria for very young children is, however, debatable and their use might lead to the unnecessary prescribing of secondary prophylaxis. A molecular analysis of both bacterial and host factors was carried out in an attempt to decipher the combination that could have led to such uncommon, but very similar presentations. The two GAS isolates belonged to the usual, although distinct, invasive emm-types 1 and 3. Both isolates carried a wide set of prophage-encoded virulence factors, with only the speG and speA superantigen-encoding genes in common. Both patients shared the HLA DQB1*0301 allele, which has been associated with susceptibility to GAS necrotizing fasciitis. CONCLUSIONS Our study exemplifies the particularity of RF in young children and the complex role of superantigens and streptodornases in GAS-related pathologies.

Contrasting epistatic interactions between rat quantitative trait loci controlling mammary cancer development

We previously defined quantitative trait loci (QTLs) that control susceptibility to 7,12-dimethylbenz(α)anthracene-induced mammary carcinoma in SPRD-Cu3 (susceptible) and WKY (resistant) rats. Two of these QTLs, assigned to chromosomes (Chr) 10 and 18, control tumor growth rate and invasiveness. In this study we characterized a congenic strain in which a large segment of WKY Chr 10 was introduced in the SPRD-Cu3 genetic background and demonstrated that this chromosome segment controls this tumor trait. The WKY allele at this QTL (Mcsta1) reduces the growth rate of the fastest growing tumors by 26%. We also previously showed that two SPRD-Cu3-WKY congenic strains containing a WKY chromosome segment derived either from Chr 5 or from Chr 18 exhibit a reduction in tumor multiplicity (QTLs Msctm1 and Mcstm2, respectively) (with no reduction in tumor growth rate in the Chr 18 congenic). In this study we generated a double congenic strain, which contains the two WKY differential segments from Chr 5 and 18, to determine how these two segments interact with one another. Interestingly, two types of epistatic interactions were found: no additive effect was seen with respect to tumor multiplicity, while a reduction in tumor growth rate was observed. It thus appears that WKY alleles located on Chr 5 and Chr 8 interact epistatically in a contrasting manner to modulate tumor multiplicity (in a nonadditive manner) and growth rate (in a synergic manner). Tumor growth rate is thus influenced by two QTLs, on Chr 10 (Mcsta1) and on Chr 18 (Mcsta2), the action of the latter being dependent on the presence of the Chr5 QTL (Mcstm1). The expression level of positional and functional candidate genes was also analyzed. On Chr 5, Pla2g2a is subject to a syntenic control while expression of the Tp53 (Chr 10) and Pmai1/Noxa (Chr 18) genes appears to be controlled by several mammary cancer resistance QTLs.

Carriage-associated Streptocccus pneumoniae serotype 1 in Brussels, Belgium.

Streptococcus pneumoniae serotype 1 presents a high invasiveness index and is seldom isolated from its niche, the nasopharynx. We report an unusual serotype 1 carriage in a healthy pediatric population at the time of the heptavalent pneumococcal vaccine adoption in Belgium. Our sampling period coincides with an epidemic wave of serotype 1 invasive pneumococcal infections. Invasive and colonizing isolates were characterized by both antibiotic resistance profile and multilocus sequence typing and were shown to share the same backbone (ST306 and ST350).

Group A Streptococcus Colonies from a Single Throat Swab Can Have Heterogeneous Antimicrobial Susceptibility Patterns.

This study describes for the first time heterogeneity of antibiotic resistance profiles among group A Streptococcus isolates originating from a single throat swab in patients with acute pharyngitis. For each throat swab, 10 group A Streptococcus colonies were randomly selected from the primary plate and subcultured to a secondary plate. These isolates were characterized by various phenotypic and genotypic methods. Our results demonstrated that differing antibiotic resistance profiles were present in 19% of pediatric patients with acute pharyngitis before antimicrobial treatment. This heterogeneity likely resulted from horizontal gene transfer among streptococcal isolates sharing the same genetic background. As only a minority of colonies displayed antibiotic resistance among these heterogeneous samples, a classical diagnostic antibiogram would have classified them in most instances as “susceptible,” although therapeutic failure could be caused by the proliferation of resistant strains after initiation of antibiotic treatment.