[Extended-spectrum betalactamases producing Escherichia coli: a new health-care associated infection threat?]. / Escherichia coli producteurs de bêtalactamases à spectre étendu: de nouvelles menaces nosocomiales ?

OBJECTIVES: Study the health-care associated infection risk due to Extended-Spectrum Betalactamases Producing Escherichia coli (ESBL Esc) isolated from diagnostic samples. METHODS: Descriptive, longitudinal and prospective study of 104 diagnostic isolates of ESBL Esc, one per patient, identified in Amiens university hospital between February 1999 and December 2005. Patients (sex, age, contamination risk factor, antecedent hospitalization) and microbiological data were progressively collected, entered into EPI INFO 6.04dFr software (ENSP, France) database, and compared using the chi-square test and Wilcoxon rank sum test, as appropriate. A p value of less than 0.05 was considered significant. RESULTS: Diagnostic ESBL Esc isolates raised, per 1000 isolates of Esc, from 1.2 in 1999 to 6 in 2005. Global and acquired isolates number of ESBL Esc varied from 7 and 3 in 2002 to 25 and 19 in 2003 (P=0.22). ESBL Esc global and acquired incidence per 10(5) patient-days were, 0.8 and 0.6 in 1999 and 4.99 and 3.4 in 2005 (P<10(-6)), but rose from 0.6 acquired isolate in 2002 to 3.9 in 2003 (P=0.002). ESBL Esc, isolated from urines, stools, pulmonary, blood and surgical site samples of patients of>/=65 years aged (68.3%), were imipenem and latamoxef sensitive. Their acquisition risk factors found were hospitalization during the last 6 month period (40/104) and transfer from other institutions (20/104). CONCLUSION: ESBL Esc isolates, among ESBL-producing Enterobacteriaceae, constitute an escalating health-care associated risk in our institution. The research at admission time of ESBL-producing Enterobacteriaceae, mainly in acute geriatric wards, strict isolation precaution and hand hygiene observance, rational antibiotic usage, are the key actions to control their cross transmission. Nonetheless, other studies are needed to determine whether we are in front of an ESBL Esc new clone emergence.

Molecular diversity of Proteus mirabilis isolates producing extended-spectrum beta-lactamases in a French university hospital.

Between February 1997 and December 2002, 3340 hospitalised patients yielded samples positive for Proteus mirabilis, of whom 45 (1.3%) were colonised/infected by P. mirabilis producing extended-spectrum beta-lactamases (ESBLs). The gross incidence of patients colonised/infected by ESBL-producing P. mirabilis was 1.61/10(5) days of hospitalisation, with 20% of isolates being collected from patients in urology wards, most frequently (53.3%) from urine samples. Seventeen (37.7%) of the 43 isolates were obtained from samples collected within 48 h of hospitalisation, indicating that they were community-acquired. Isoelectric focusing assays and sequencing identified the TEM-24, TEM-92 and TEM-52 ESBLs. Pulsed-field gel electrophoresis revealed eight pulsotypes (I-VIII), with the two most common pulsotypes, IV and VI, comprising ten (23.3%) and 12 (26.6%) isolates, respectively. These pulsotypes were considered to represent epidemic strains and spread in various wards of the hospital.

Regional dissemination of Salmonella enterica serovar Enteritidis is season dependent.

OBJECTIVE: To carry out epidemiological typing of clinical isolates of Salmonella enterica serovar Enteritidis by pulsed-field gel electrophoresis (PFGE), random amplified polymorphic DNA (RAPD) and analysis of their antibiotic resistance. METHODS: Over a 12-month period, 44 Salmonella Enteritidis isolates, recovered from 40 patients admitted to the University Hospital Center of Amiens, France and from three outpatients, were characterized by the analysis of phenotypic and genotypic traits and clinical data from medical reports. RESULTS: Forty nontyphoidal salmonellosis episodes were diagnosed in hospitalized patients (34 episodes of gastroenteritis, two episodes of bacteremia not affecting other organs, one episodes of bacteremia plus urinary infection, one episodes of bacteremia plus gastroenteritis, one episodes of chronic colitis plus gastroenteritis and one episode of peritonitis), and three carriers were observed in outpatients. By means of PFGE, RAPD and antibiotic susceptibility patterns 44 isolates were subdivided into 16 clonally related groups. Two of them were predominantly implicated in the course of these infections, being responsible for two successive waves of infection, while the others were encountered sporadically.

[Evaluation of the efficacy of a multiresistant bacteria control programme in a teaching hospital, studying the evolution of methicillin-resistant Staphylococcus aureus incidence]. / Evaluation de l'efficacité d'une politique de lutte contre les bactéries multirésistantes dans un centre hospitalier universitaire par l'étude de l'évolution de l'incidence de Staphylococcus aureus résistant à la méticilline.

Methicillin-resistant Staphylococcus aureus (MRSA) constitute the most important multiresistant bacteria (MRB) recovered in French hospitals. Our objective was to measure these MRSA diffusion in our hospital to evaluate the MRB control programme which had been implemented in the beginning of 1999. This study was conducted in a teaching hospital containing 1800 beds, from February 1999 to January 2001. All MRSA isolated in clinical samples were included. Duplicates (same bacteria in the same patient) were excluded. The detection of methicillin-resistance was performed at 30 degrees C, by disk diffusion method. Incidence densities were determined with their 95% confidence interval (CI 95%). Their evolution by four-month period was evaluated with the chi-square test for trend. During the two-year period, 866 MRSA were isolated. The global incidence was 0,88 per 1000 patient-days (PD) (IC 95% = left open bracket 0,83-0,93 right open bracket ). For cases acquired in our hospital the incidence was 0,66 per 1000 PD, whereas it was 0,26 per 1000 PD for imported cases. Concerning the evolution of incidences, no significant trend was observed for global incidence. The incidence of acquired MRSA decreased during the first year, but increased thereafter. The incidence of imported MRSA increased with a significant trend (p < 10(-5)). The number of these imported MRSA isolated in our hospital was twice fold higher in 2000. This study emphasizes an important actual problem : the increase of patient colonization pressure at the time of admission in hospitals. This increase, which can be due in part to a community transmission, is responsible for a reduction of the efficacy of MRSA control programmes.

[Spread of Enterobacteriaceae producing broad-spectrum beta-lactamase and the development of their incidence over a 16-month period in a university hospital center]. / Diffusion des entérobactéries productrices de beta-lactamase à spectre élargi et évolution de leur incidence sur une période de 16 mois dans un centre hospitalier universitaire.

Enterobacteriaceae producing extended-spectrum beta-lactamases (ESBLE) constitute with methicillin-resistant Staphylococcus aureus the main multiresistant bacteria recovered in French hospitals. Our objectives were to evaluate these ESBLE diffusion in our teaching hospital and to follow their incidence during a 16-month period, whereas a control programme (barrier precautions) had been implemented in the beginning of 1999. This study was conducted in a teaching hospital containing 1800 beds, from February 1999 to May 2000. All ESBLE isolated in clinical or screening samples were included. Duplicates (same bacteria in the same patient) were excluded. The detection of the ESBL was performed with the double-disk diffusion test. Incidence densities were determined with their 95% confidence interval (CI95%). Their evolution by four-month period was evaluated with the chi-square test for trend. During the 16-month period, 229 ESBLE were isolated. The incidence was 0.35 per 1000 patient-days (PD) (CI95% = [0.30-0.40]) for the whole hospital. It was 0.47/1000 PD (CI95% = [0.38-0.56]) in medical wards, 0.29/1000 PD (CI95% = [0.20-0.38]) in surgical wards and 1.32/1000 PD (CI95% = [0.90-1.74]) in intensive care units. Enterobacter aerogenes strains represented more than 75% of all ESBLE, whereas Klebsiella pneumoniae stains represented only 8.6%. During the study, the incidence of ESBLE and the proportion of strains acquired in our hospital decreased significantly (p < 0.0001 and p < 0.001 respectively). Indeed, between the first eight-month period and the last one, the incidence of ESBLE acquired in our hospital decreased by 55%, whereas the incidence of imported strains increased slightly. This study shows that the diffusion of ESBLE concerns the entire hospital. The implementation of a control programme of the spread of multiresistant bacteria allowed us to reduce significantly the incidence of ESBLE. This incidence seemed to be stable for several months. The implementation of a policy which restricts antimicrobial use would allow us to complete the the efficacy of barrier precautions.

Detection of Chlamydia trachomatis in semen and urethral specimens from male members of infertile couples in Tunisia.

The sequelae to infection with Chlamydia trachomatis in women are an established cause of tubal infertility. However, little is known about chlamydial infection and male infertility. The main objective of this study was to evaluate the presence of asymptomatic C. trachomatis infections in urethral and semen specimens from the male members of infertile couples by means of four different methods: the direct fluorescence antibodies assay, cell culture, the Roche Cobas Amplicor polymerase chain reaction, and the presence of chlamydial local IgA antibodies by the recombinant antibody-enzyme-linked immunosorbent assay. One or more chlamydial infection markers were detected in 42 (45.7%) of the 92 examined urethral and semen specimens from the male partners of infertile couples. C. trachomatis was detected in 23.9% (22/92) of urethral specimens and in 35.9% (33/92) of semen specimens. Although there was a significant correlation between the detection of one or more chlamydial infection markers in urethral and semen specimens (p = 0.01), no significant correlation was found between the detection of C. trachomatis DNA in these samples. Furthermore, no significant association was found between the presence of chlamydial local IgA antibodies and the detection of C. trachomatis. The discrepancies in positive results found between some techniques for the detection of C. trachomatis in urethral and semen specimens might be explained by variations in the sensitivities and specificities of the tests carried out and the use of specimens from different anatomical locations. Our findings suggest that C. trachomatis seems to be widespread among the male partners of infertile couples in Tunisia. The detection of C. trachomatis in urethral or semen specimens can serve as a marker for the presence of this organism in the genital tract, which is not necessarily the cause of male infertility. The study of the correlation between the detection of chlamydial infection markers and the parameters of male fertility seems to be necessary in order to determine the direct link between chlamydial infection and male infertility and to choose the most efficient technique and most suitable specimen with which to diagnose C. trachomatis-associated male infertility.

Evaluation of the contribution of isolation precautions in prevention and control of multi-resistant bacteria in a teaching hospital.

From February 1999 to January 2000, a control programme to prevent the spread multi-resistant bacteria (MRB) was implemented in a French teaching hospital. This programme focused on methicillin-resistant Staphylococcus aureus (MRSA) and Enterobacteriaceae producing extended-spectrum beta-lactamases (ESBL), and was based on the application of barrier precautions (washing hands with antiseptic soaps, wearing disposable gloves and gowns, identifying MRB carriers). No changes in antibiotic policy occurred during the year. Our aim was to conduct an evaluation of this programme by measuring incidence rates. Concurrently, the effect of barrier precautions was estimated in an indirect way, by documenting the availability of barrier precautions in MRB carriers' rooms and by analysing the monthly correlation between the supply of such material and the theoretical cumulated length of MRB carriers' isolation in six randomized wards. All MRB isolated in hospitalized patients were recorded, and differentiated between acquisition in our hospital or from elsewhere. For the analysis of trends, the year was divided in three periods of four months. Over the year, the global MRB incidence was 1.26 per 1000 patient-days (PD) [95% confidence interval (95%CI)=1.16-1.36]. The MRSA incidence was 0.89 per 1000 PD (95%CI=0.81- 0.97) and the ESBL incidence was 0.38 per 1000 PD (95% CI=0.33-0.43). The MRB incidence decreased significantly in all types of specialties except for surgical wards. The incidence decreased by 17.9% for MRSA, 54.9% for ESBL and 34.8% for both MRB. Concurrently, the proportion of strains acquired in our hospital decreased for MRSA (P for trend > or = 0.05) and ESBL (P for trend > or = 0.01), whereas the incidence of imported strains increased slightly. The proportion of multiresistant strains in S. aureus (36.8%) and Enterobacter aerogenes (37.0%) remained similar throughout the year. Thus, the decrease of the incidence concerned both resistant and susceptible strains. The availability of antiseptic soaps increased significantly (P for trend > or = 0.01). The amount of antiseptic soap ordered and the theoretical lengths of isolation were correlated on a monthly basis (Spearman coefficient = 0.72; P > or = 0.02). These results shows the efficacy of such a programme of MRB containment in a large hospital, provided barrier nursing is instigated, together with the availability of such material as antiseptic soap, to allow implementation.

Dissecting the energetics of the apoflavodoxin-FMN complex.

Many flavoproteins are non-covalent complexes between FMN and an apoprotein. To understand better the stability of flavoproteins, we have studied the energetics of the complex between FMN and the apoflavodoxin from Anabaena PCC 7119 by a combination of site-directed mutagenesis, titration calorimetry, equilibrium binding constant determinations, and x-ray crystallography. Comparison of the strength of the wild type and mutant apoflavodoxin-FMN complexes and that of the complexes between wild type apoflavodoxin and shortened FMN analogues (riboflavin and lumiflavin) allows the dissection of the binding energy into contributions associated with the different parts of the FMN molecule. The estimated contribution of the phosphate is greatest, at 7 kcal mol(-1); that of the isoalloxazine is of around 5-6 kcal mol(-1) (mainly due to interaction with Trp-57 and Tyr-94 in the apoprotein) and the ribityl contributes least: around 1 kcal mol(-1). The stabilization of the complex is both enthalpic and entropic although the enthalpy contribution is dominant. Both the phosphate and the isoalloxazine significantly contribute to the enthalpy of binding. The ionic strength does not have a large effect on the stability of the FMN complex because, although it weakens the phosphate interactions, it strengthens the isoalloxazine-protein hydrophobic interactions. Phosphate up to 100 mM does not affect the strength of the riboflavin complex, which suggests the isoalloxazine and phosphate binding sites may be independent in terms of binding energy. Interestingly, we find crystallographic evidence of flexibility in one of the loops (57-62) involved in isoalloxazine binding.

Epidemiological study of an Acinetobacter baumannii outbreak by using a combination of antibiotyping and ribotyping.

From June to November 1994 (period 1) and from February to June 1995 (period 2), multiresistant Acinetobacter baumannii strains were isolated in intensive care units and surgical wards of the Amiens Teaching Hospital Center (Amiens, France). Eighteen isolates were obtained from 17 (1%) of 1,706 patients admitted during both of these periods, giving an incidence rate of nosocomial infection per 1,000 patient days of 0.6%. Of 17 infected patients, 9 had pneumonia, 3 had urinary tract infection, 2 had peritonitis, 1 had septicemia, 1 had a catheter infection, and 1 had pneumonia and urinary tract infection. According to typing results, four antibiotic resistance profiles were detected: a, b, c, and d; seven ribotypes were distinguished by both restriction enzymes EcoRI and SalI (A, B, C, D, E, F, and G). By combining antibiotyping and ribotyping, we obtained eight groups of strains (groups I to VIII). Group I contained five strains (strains 4, 5, 7, 8, and 9) which had antibiogram pattern a and ribopattern A and constituted the outbreak strains. The strains of group II (strains 3, 10, 11, 13, and 14) were closely related to outbreak strain A and appeared to be variants of ribotype A (A2 [strain 3]; A4 [strain 10]; A5 [strains 11, 13, and 14]). Groups III, IV, V, VI, VII, and VIII included strains which were epidemiologically unrelated to the strains of group I and were considered nonoutbreak strains.

Apoflavodoxin: structure, stability, and FMN binding.

Flavodoxins are one domain alpha/beta electron transfer proteins that participate in photosynthetic reactions. All flavodoxins carry a molecule of flavin mononucleotide (FMN), non-covalently bound, that confers redox properties to the protein. There are two structurally distinct flavodoxins, short ones and long flavodoxins; the latter contain an extra loop with unknown function. We have undertaken the study of the stability and folding of the apoflavodoxin from Anabaena (a long flavodoxin) and the analysis of the interaction between the apoflavodoxin and FMN. Our studies indicate that apoflavodoxin folds in a few seconds to a form that is competent in FMN binding. The stability of this apoflavodoxin is low and its urea denaturation can be described by a two-state mechanism. The role of the different parts of the apoflavodoxin in the stability and structure of the whole protein is being investigated using mutagenesis and specific cleavage to generate apoflavodoxin fragments. The X-ray structure of apoflavodoxin is very similar to that of its complex with FMN, the main difference being the conformation of the two aromatic residues that sandwich FMN in the complex. In apoflavodoxin these groups interact with each other so closing the FMN binding site. Despite this fact, apoflavodoxin binds FMN tightly and rapidly, and the resulting holoflavodoxin displays a high conformational stability. We have found that one role of the aromatic residues that interact with FMN is to help to retain bound the reduced form of the cofactor whose complex with apoflavodoxin is otherwise too weak.