No abstract

AimTo determine attrition and predictors of academic success among medical students at University of Split, Croatia.MethodsWe analysed academic records of 2054 students enrolled during 1979–2008 period.ResultsWe found that 26% (533/2054) of enrolled students did not graduate. The most common reasons for attrition were ‘personal’ (36.4%), transfer to another medical school (35.6%), and dismissal due to unsatisfactory academic record (21.2%). Grade point average (GPA) and study duration of attrition students were significantly associated with parental education. There were 1126 graduates, 395 men and 731 women. Their average graduation GPA was 3.67±0.53 and study duration 7.6±2.44 years. During 5-year curriculum only 6.4% (42/654) of students graduated in time, and 55% (240/472) of students graduated in time after curriculum was extended to 6 years. Variables predicting whether a student will graduate or not were high school grades, entrance exam score and year of enrollment. Significant predictors of graduation grades were high school grades and entrance exam score. Entrance exam score predicted length of studying.ConclusionPreadmission academic qualifications and year of enrollment predict academic success in medical school. More attention should be devoted to high attrition.

The lactose operon from Lactobacillus casei is regulated by very tight glucose repression and substrate induction mechanisms, which made it a tempting candidate system for the expression of foreign genes or metabolic engineering. An integrative vector was constructed, allowing stable gene insertion in the chromosomal lactose operon of L. casei. This vector was based on the nonreplicative plasmid pRV300 and contained two DNA fragments corresponding to the 3 end of lacG and the complete lacF gene. Four unique restriction sites were created, as well as a ribosome binding site that would allow the cloning and expression of new genes between these two fragments. Then, integration of the cloned genes into the lactose operon of L. casei could be achieved via homologous recombination in a process that involved two selection steps, which yielded highly stable food-grade mutants. This procedure has been successfully used for the expression of the E. coli gusA gene and the L. lactis ilvBN genes in L. casei. Following the same expression pattern as that for the lactose genes, ␤-glucuronidase activity and diacetyl production were repressed by glucose and induced by lactose. This integrative vector represents a useful tool for strain improvement in L. casei that could be applied to engineering fermentation processes or used for expression of genes for clinical and veterinary uses.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has lately been implemented as a solid technology for rapid microorganism identification in microbiology laboratories. This study compares two methods for bacterial separation from 85 positive blood culture before MALDI-TOF MS: (1) a conventional method that we used in our laboratory to prepare bacteria for susceptibility testing and (2) a new commercialized technique (Sepsityper). There were no significant differences in the identification of Gram-negative bacilli regardless of the bacterial separation method used. However, identification was greater for Gram-positive cocci when the Sepsityper method was used (84.15% vs. 100% in the identification to a genus level in staphylococci and 57.14% vs. 85.71% in the identification to a genus level of enterococci with the in-house and Sepsityper methods, respectively). Therefore, the Sepsityper method to prepare bacteria from a positive blood culture is more adequate for the further identification of Gram-positive cocci by MALDI-TOF MS.

No abstract

Francisella tularensis is a category A bioterror pathogen which in some cases can cause a severe and fatal human infection. Very few virulence factors are known in this species due to the difficulty in working with it as well as the lack of tools for genetic manipulation. This work describes the construction of a shuttle vector that can replicate in Escherichia coli and F. tularensis as well as two distinct promoter trap constructs based on the shuttle vector backbone. Replication in F. tularensis is based on the promiscuous origin of replication from the Staphylococcus aureus plasmid pC194. We demonstrate the novel plasmids can coexist with established F. tularensis vectors based on the pFNL10 plasmid, the current workhorse of F. tularensis genetics. Our promoter trap can identify promoters that are activated during intracellular growth and survival. These new vectors provide additional tools for the genetic manipulation of F. tularensis.

The lactose operon, lacTEGF, of Lactobacillus casei ssp. casei ATCC393 [pLZ15-] is encoding an antiterminator protein (LacT), the elements (LacE and LacF) of the lactose-specific phosphotransferase system (PTS) and a phospho-β-galactosidase (LacG). The lac operon is repressed by glucose and fructose and is induced by lactose, through the PTS/CcpA signal transduction system and an antiterminator mechanism, respectively. Furthermore, the antiterminator activity of LacT is also negatively modulated possibly by a PTS-mediated phosphorylation event. These strong regulatory mechanisms have been used in this work for the design of expression systems. Hence, Bacillus licheniformis α-amylase has been efficiently expressed from pIAβ5lacamy on lactose grown cells. Furthermore, a food-grade mutant, expressing Lactococcus lactis acetohydroxy acid synthase genes (ilvBN), was obtained with an integrative system, developed using lacG and lacF as homologous sequences for recombination. As a result, ilvBN genes were integrated in tandem between lacG and lacF in the chromosome and were co-ordinately expressed with the genes of the lactose operon. Lactobacillus / lactose expression system / replicative vector / integrative vector Résumé-Usage des éléments régulateurs de l'opéron lactose pour l'expression de gènes chez Lactobacillus casei. Les gènes du lactose Lactobacillus casei ssp. casei ATCC393 [pLZ15-] sont regroupés dans un même opéron, lacTEGF, codant pour un antiterminateur (LacT), pour les éléments spécifiques du transport du lactose par le système phosphotransferase dépendant du phosphoénolpyruvate (PTS) (LacE et LacF) et pour une phospho-β-galactosidase (LacG). L'opéron lac est soumis à une répression par le glucose et le fructose via le système de transduction de signal PTS/CcpA et à une induction par le lactose au moyen d'un mécanisme d'antiterminaison. De plus, l'activité de l'antiterminateur est aussi négativement modulée possiblement par un événement de phosphorylation étant impliqué le PTS. Dans ce travail, on a utilisé ces mécanismes régulateurs pour la construction de systèmes d'expression. Avec le vecteur pIAβ5lacamy, on a obtenu l'expression de l'α-amylase de Bacillus licheniformis à partir des éléments régulateurs de l'opéron lactose. On a aussi construit un vecteur intégratif utilisant les gènes lacG et lacF comme séquence homologue

In low-G+C Gram-positive bacteria, the regulatory protein CcpA has been shown to play a major part in the so-called carbon catabolite repression (CCR) process, as well as in the induction of basic metabolic genes, for which it is considered a global regulator. A strain of Lactobacillus casei that carried a complete deletion of ccpA has been constructed and used to test the effect of CCR on N-acetylglucosaminidase activity and growth performance of a collection of seven CcpA mutations obtained by site-directed mutagenesis. The replaced amino acids were located in the DNA-and cofactor (P-Ser-HPr)-binding domains. Mutations in the DNA-binding domain lacked CCR, as found in Bacillus megaterium. However, mutations in the cofactor-binding domain of L. casei CcpA had a different phenotype to that observed in the previous studies with B. megaterium. Two of them, S80L and T307I, displayed a significant hyper-repression, an effect never reported before for CcpA. Comparison of growth capabilities provided by the different mutants and their ability to sustain CCR demonstrated that CCR, at least on the enzymic activity tested, and the growth defect caused by the CcpA mutations are unrelated features. INTRODUCTIONIn the industrially relevant lactic acid bacterium Lactobacillus casei, the preferential utilization of carbon sources is controlled by the mechanism of carbon catabolite repression (CCR) (Monedero et al., 1997). As in other low-G+C Gram-positive bacteria, CCR takes place through the binding of the transcriptional repressor CcpA (Miwa et al., 1994;Hueck et al., 1995; Egeter & Brückner, 1996;Lokman et al., 1997;Leboeuf et al., 2000) to an operator sequence called cre (catabolite responsive element) (Fujita et al., 1995;Aung-Hilbrich et al., 2002). CcpA binding to cre sequences is markedly enhanced by its co-repressor, the Ser-46 phosphorylated HPr protein (P-Ser-HPr), a key component of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) Jones et al., 1997). This links CCR to the sugar transport process via the PTS (Brückner & Titgemeyer, 2002). Some small molecules, such as fructose 1,6-bisphosphate, glucose 6-phosphate or NADP, can also influence the interaction between CcpA and cre (Gosseringer et al., 1997;Kim et al., 1998). Therefore, the major role attributed to CcpA was related to CCR in the presence of rapidly metabolizable carbon sources.CcpA inactivation pleiotropically affects the expression of approximately 8 % of the genes in Bacillus subtilis (Moreno et al., 2001). Genes of very important metabolic pathways are regulated by CcpA, such as citB and citZ contributing to the Krebs cycle (Kim et al., 2002), gltAB for ammonium assimilation (Faires et al., 1999) or the ilv-leu operon for branched-chain amino-acid biosynthesis (Ludwig et al., 2002). Other studies revealed that CcpA in Gram-positive bacteria also regulates carbon catabolite activation (CCA) of acetoin secretion, acetate biosynthesis genes in B. subtilis (Turinsky et al., 2000) and glycolytic genes in B. subtilis, Lactococcus lact...