Fabien Rallu

Acid‐ and multistress‐resistant mutants of Lactococcus lactis : identification of intracellular stress signals

Lactococcus lactis growth is accompanied by lactic acid production, which results in acidification of the medium and arrest of cell multiplication. Despite growth limitation at low pH, there is evidence that lactococci do have inducible responses to an acid pH. In order to characterize the genes involved in acid tolerance responses, we selected acid‐resistant insertional mutants of the L. lactis strain MG1363. Twenty‐one independent characterized mutants were affected in 18 different loci, some of which are implicated in transport systems or base metabolism. None of these genes was identified previously as involved in lactococcal acid tolerance. The various phenotypes obtained by acid stress selection allowed us to define four classes of mutants, two of which comprise multistress‐resistant strains. Our results reveal that L. lactis has several means of protecting itself against low pH, at least one of which results in multiple stress resistance. In particular, intracellular phosphate and guanine nucleotide pools, notably (p)ppGpp, are likely to act as signals that determine the level of lactococcal stress response induction. Our results provide a link between the physiological state of the cell and the level of stress tolerance and establish a role for the stringent response in acid stress response regulation.

Sensitivities of Antigen Detection and PCR Assays Greatly Increased Compared to That of the Standard Culture Method for Screening for Group B Streptococcus Carriage in Pregnant Women

ABSTRACT Group B streptococcus (GBS) is a major cause of serious infections in neonates. The 2002 revised guidelines of the Centers for Disease Control and Prevention (CDC) for the prevention of perinatal GBS disease recommend that all pregnant women be screened for GBS carriage at between 35 and 37 weeks of gestation and that intrapartum antibiotic prophylaxis be given to carriers. We studied the performances of four different GBS detection assays in the context of antenatal screening. Between May and August 2004, the 605 vaginorectal swab specimens received at our bacteriology laboratory for GBS antenatal detection were tested by the four assays. The standard culture method was done according to the CDC recommendations. The three experimental assays performed with the growth from the selective enrichment (LIM) broth (Todd-Hewitt broth with 15 μg/ml nalidixic acid and 10 μg/ml colistin) after overnight incubation were a GBS antigen detection assay (PathoDx) and two PCR assays (for cfb and scpB). The most accurate assay was the scpB PCR (sensitivity, 99.6%; specificity, 100%), followed by the cfb PCR (sensitivity, 75.3%; specificity, 100%), GBS antigen detection (sensitivity, 57.3%; specificity, 99.5%), and standard culture (sensitivity, 42.3%; specificity, 100%). The GBS antigen detection assay was found to be more sensitive than the standard culture method, and moreover, the assay has a low cost and is easy to perform in all obstetrical centers which have access to the most basic of diagnostic microbiology services. We believe that antigen detection on incubated LIM broth should replace the standard culture method for screening for GBS carriage at 35 to 37 weeks of gestation. The impact of the greater sensitivities of PCR assays on the diminution of neonatal GBS infections remains to be demonstrated.

The problem of hypernegative supercoiling and R-loop formation in transcription.

DNA supercoiling and topoisomerases have long been known to affect transcription initiation. In many studies, topA mutants were used to perturb chromosomal supercoiling. Although such studies clearly revealed that supercoiling could significantly affect gene expression, they did not tell much about the essential function(s) of DNA topoisomerase I, encoded by topA. Indeed, the topA mutants used in these studies were growing relatively well, although this gene is normally essential for growth. These mutants were either carrying a topA allele with enough residual activity to permit growth, or if deleted for the topA gene, they were carrying a compensatory mutation allowing them to grow. We have recently used a set of isogenic strains carrying a conditional gyrB mutation that allowed us to study the real effects of losing topoisomerase I activity on cell physiology. The results of our work show that an essential function of topoisomerase I is related to transcription, more precisely to inhibit R-loop formation. This is in agreement with a series of biochemical studies that revealed a role for topoisomerase I in inhibiting R-loop formation during transcription in the presence of DNA gyrase. In addition, our studies may have revealed an important role for DNA supercoiling in modulating gene expression, not only at the level of transcription initiation but also during elongation. In this paper, we will first discuss global and local supercoiling, then we will address the topic of R-loop formation and finally, we will review the subject of hypersupercoiling and R-loop formation in gene expression. Whenever possible, we will try to make correlations with growth phenotypes, since such correlations reveal the essential function of DNA topoisomerase I.

Effects of RNA polymerase modifications on transcription‐induced negative supercoiling and associated R‐loop formation

Transcription in the absence of topoisomerase I, but in the presence of DNA gyrase, can result in the formation of hypernegatively supercoiled DNA and associated R‐loops. In this paper, we have used several strategies to study the effects of elongation/termination properties of RNA polymerase on such transcription‐induced supercoiling. Effects on R‐loop formation were exacerbated when cells were exposed to translation inhibitors, a condition that stimulated the accumulation of R‐loop‐dependent hypernegative supercoiling. Translation inhibitors were not acting by decreasing (p)ppGpp levels as the absence of (p)ppGpp in spoT relA mutant strains had little effect on hypernegative supercoiling. However, an rpoB mutation leading to the accumulation of truncated RNAs considerably reduced R‐loop‐dependent hypernegative supercoiling. Transcription of an rrnB fragment preceded by a mutated and inactive boxA sequence to abolish the rrnB antitermination system also considerably reduced R‐loop‐dependent supercoiling. Taken together, our results indicate that RNA polymerase elongation/termination properties can have a major impact on R‐loop‐dependent supercoiling. We discuss different possibilities by which RNA polymerase directly or indirectly participates in R‐loop formation in Escherichia coli. Finally, our results also indicate that what determines the steady‐state level of hypernegatively supercoiled DNA in topA null mutants is likely to be complex and involves a multitude of factors, including the status of RNA polymerase, transcription–translation coupling, the cellular level of RNase HI, the status of DNA gyrase and the rate of relaxation of supercoiled DNA.

Bacteriology of Amniotic Fluid in Women With Suspected Cervical Insufficiency

OBJECTIVE To determine the prevalence of mid-trimester microbial invasion of the amniotic cavity (MIAC) in women with suspected cervical insufficiency. METHODS A prospective observational cohort study was performed in women with suspected cervical insufficiency and visible fetal membranes who were undergoing amniocentesis to rule out MIAC between 16 and 26 weeks of gestation. Women with preterm premature rupture of membranes, regular uterine contractions, or who had a cervical cerclage were excluded. Gram staining of amniotic fluid, glucose and lactate dehydrogenase (LDH) levels in amniotic fluid, and aerobic and anaerobic amniotic fluid cultures were performed, along with polymerase chain reaction (PCR) for the detection of Ureaplasma and Mycoplasma species. RESULTS Fifteen women with a mean gestational age of 22.6 +/- 2.3 weeks were included in the study. The diagnosis of MIAC was confirmed in 47% (7/15), of whom 20% (3/15) were infected with more than one bacterial strain and 33% (5/15) with Ureaplasma species. According to receiver-operator curve analyses, amniotic fluid levels of glucose were associated with MIAC (P = 0.02), but not amniotic fluid LDH (P = 0.25). CONCLUSION MIAC is present in approximately one half of women with suspected cervical insufficiency and visible fetal membranes at speculum examination.

Comparison of Three Different Methods for Detection of Shiga Toxin-Producing Escherichia coli in a Tertiary Pediatric Care Center

ABSTRACT Shiga toxin-producing Escherichia coli (STEC) is a well-known cause of sporadic and epidemic food-borne gastroenteritis. A low infectious dose, approximately 10 microorganisms, is sufficient to cause disease that may lead to hemolytic-uremic syndrome. The objective of this study was to compare the performances of an in-house real-time PCR, a commercial enzyme immunoassay (EIA) (Premier EHEC; Meridian Bioscience), and culture on sorbitol MacConkey agar for the detection of STEC in a tertiary care pediatric hospital. Of 632 stool samples tested, 21 were positive for STEC. All were detected by PCR, 6 were detected by EIA, and only 5 O157 STEC isolates were identified by culture. Among the 15 specimens falsely negative by EIA, there were 9 Stx1, 2 Stx2, and 4 Stx1 and Stx2 STEC isolates. The latter group included 2 O157 STEC isolates that would have been missed if only EIA had been performed. To our knowledge, this is the first prospective study performed in a pediatric hospital which demonstrates the superiority of PCR over EIA for the detection of STEC. We conclude that PCR is specific and more sensitive than EIA. PCR should be considered for routine use in clinical settings where molecular detection facilities are available. Its lower limit of detection, equivalent to the infectious dose, is an obvious advantage for patient care and public health surveillance.

RNase HI overproduction is required for efficient full-length RNA synthesis in the absence of topoisomerase I in Escherichia coli

It has long been known that Escherichia coli cells deprived of topoisomerase I (topA null mutants) do not grow. Because mutations reducing DNA gyrase activity and, as a consequence, negative supercoiling, occur to compensate for the loss of topA function, it has been assumed that excessive negative supercoiling is somehow involved in the growth inhibition of topA null mutants. However, how excess negative supercoiling inhibits growth is still unknown. We have previously shown that the overproduction of RNase HI, an enzyme that degrades the RNA portion of an R‐loop, can partially compensate for the growth defects because of the absence of topoisomerase I. In this article, we have studied the effects of gyrase reactivation on the physiology of actively growing topA null cells. We found that growth immediately and almost completely ceases upon gyrase reactivation, unless RNase HI is overproduced. Northern blot analysis shows that the cells have a significantly reduced ability to accumulate full‐length mRNAs when RNase HI is not overproduced. Interestingly, similar phenotypes, although less severe, are also seen when bacterial cells lacking RNase HI activity are grown and treated in the same way. All together, our results suggest that excess negative supercoiling promotes the formation of R‐loops, which, in turn, inhibit RNA synthesis.

The origin of Fusobacterium nucleatum involved in intra-amniotic infection and preterm birth

Objective. To evaluate the potential oral origin of Fusobacterium nucleatum found in amniotic fluid of women at high risk of preterm birth. Methods. A transversal study nested into a cohort study of women with preterm labor and/or preterm premature rupture of membranes was undergone. Women with the presence of F. nucleatum in the amniotic fluid and their respective partners were invited to be examined for their periodontal health after delivery, and samples of saliva and subgingival plaque were collected. For each couple, specific PCR detection of Fusobacterium species was performed on each oral sample, and the DNA sequences were compared with the one obtained from amniotic fluid. Results. Three women, all in preterm labor with intact membranes, were included. Intra-amniotic sludge was observed in all of them. A strain of F. nucleatum with 100% sequence identity with the strain detected in the amniotic fluid was found in the oral samples of one of them and of two partners. Conclusion. This study suggests that intra-amniotic F. nucleatum could originate from the patients or the partners oral microflora.

CLINICAL AND MICROBIOLOGIC CHARACTERISTICS OF GROUP A STREPTOCOCCAL NECROTIZING FASCIITIS IN CHILDREN

An increase in the incidence of Group A streptococcal necrotizing fasciitis has recently been observed in Montréal, Canada. Clinical features of children hospitalized for invasive Group A streptococcal infections and various virulence factor genes of the bacteria were concomitantly analyzed. It was determined that varicella and presence of speC gene in group A streptococcal strains were associated with necrotizing fasciitis.

Invasive pneumococcal disease after implementation of a reduced three-dose pneumococcal conjugate vaccine program: a pediatric tertiary care center experience

Following the implementation of a government-sponsored reduced three-dose (2 + 1) heptavalent conjugate pneumococcal vaccine (PCV7) program, we report a 61.4% decrease in the number of cases of invasive pneumococcal diseases (IPD) treated at our institution. Four years after the implementation of the three-dose reduced vaccine program, only 7.4% of IPD were caused by PCV7 serotypes, and there was an increase in the proportion of IPD caused by nonPCV7 serotypes; serotype 19A represented 40.7% of the strains isolated during the last year of the study. These results, similar to those previously observed with a regular four-dose (3 + 1) PCV7 schedule, are reassuring as to the effectiveness of a reduced three-dose (2 + 1) PCV7 program. Increasing numbers of IPD caused by nonPCV7 serotypes warrant the use of a new conjugate pneumococcal vaccine that contains serotype 19A.