Alain Reynaud

Escherichia coli: Epidemiology and Analysis of Risk Factors for Infections Caused by Resistant Strains

This study analyzes the epidemiology of hospital and community-acquired infections caused by Escherichia coli. The antimicrobial resistance pattern was used to characterize the isolates, and a prospective observational study was performed to assess the relationship between antimicrobial use and bacterial resistance. The study was conducted during a 3-month period in a 1,200-bed tertiary care hospital in Nantes, France. An E. coli infection was diagnosed in 3.8% of the patients (507 of 13,384) admitted to the hospital between 1 January and 31 March 1996. Of the 507 isolates, 205 (40.4%) were resistant to at least one antimicrobial; 40% were resistant to amoxicillin, 30% to amoxicillin/clavulanate, 38% to ticarcillin, and 16% to trimethoprim-sulfamethoxazole, while resistance to other antimicrobials was low. Prior receipt of antimicrobial and/or immunosuppressive therapy was significantly associated with infection caused by a resistant organism.

Characterization and amino acid sequence analysis of a new oxyimino cephalosporin-hydrolyzing class A β-lactamase from Serratia fonticola CUV

Serratia fonticola CUV produces two isoenzymes (forms I and II) with beta-lactamase activity which were purified by a five-step procedure. The isoenzymes had identical kinetic parameters and isoelectric point (pI = 8.12). They were characterized by a specific activity towards benzylpenicillin of 1650 U/mg. The beta-lactamase hydrolyzed benzylpenicillin, amoxycillin, ureidopenicillins, first- and second-generation cephalosporins. Carboxypenicillins and isoxazolylpenicillins were hydrolyzed to a lesser extent. Towards cefotaxime and ceftriaxone (third-generation cephalosporins), the S. fonticola enzyme exhibited catalytic efficiencies much higher than those of MEN-1 and extended-spectrum TEM derivative beta-lactamases. The beta-lactamase from S. fonticola was markedly inhibited by beta-lactamase inhibitors such as clavulanic acid, sulbactam and tazobactam. The purified isoenzymes were digested by trypsin, endoproteinase Asp-N and chymotrypsin. Amino acid sequence determinations of the resulting peptides allowed the alignment of 267 amino acid residues (Swiss-Prot, accession number P 80545) for form I beta-lactamase. Form II is five residues shorter than form I at its N-terminus. From amino acid sequence comparisons, S. fonticola CUV beta-lactamase was found to share more than 69.3% identity with the chromosomally encoded beta-lactamases of Klebsiella oxytoca, Proteus vulgaris, Citrobacter diversus and the plasmid-mediated enzymes MEN-1 and Toho-1. Therefore, the oxyimino cephalosporin-hydrolyzing beta-lactamase of S. fonticola belongs to Amblers class A. Contribution of the serine at ABL 237 in the broad-spectrum activity of these beta-lactamases is discussed.

Chromosomally encoded cephalosporin-hydrolyzing β-lactamase of Proteus vulgaris RO104 belongs to Ambler's class A

Proteus vulgaris RO104 strain produces a chromosomally encoded beta-lactamase that confers resistance to various beta-lactam antibiotics including methoxyimino third-generation cephalosporins. The beta-lactamase hydrolyzes first- and second-generation cephalosporins efficiently and cefotaxime to a lesser extent. Catalytic activity is inhibited by low concentrations of clavulanic acid and sulbactam. By its broad-spectrum substrate profile, beta-lactamase of Proteus vulgaris RO104 belongs to the group 2e defined by Bush. The protein purified to homogeneity by a four-step procedure was characterized by a pI of 8.31 and a specific activity of 1200 U/mg. The beta-lactamase was digested by trypsin, endoproteinase Asp-N and chymotrypsin. Amino-acid sequence determinations of the resulting peptides allowed the alignment of the 271 amino-acid residues of the protein which did not contain any cysteine residue. From amino-acid sequence comparisons, Proteus vulgaris RO104 beta-lactamase was found to share about 68% identity with the chromosomally mediated beta-lactamases of Klebsiella oxytoca D488 and E23004. Therefore, the cephalosporin-hydrolyzing beta-lactamase of Proteus vulgaris RO104 belongs to Amblers class A.

Occurrence of ST23 Complex Phylogroup A Escherichia coli Isolates Producing Extended-Spectrum AmpC β-Lactamase in a French Hospital

ABSTRACT Extended-spectrum AmpC β-lactamase (ESAC) Escherichia coli producers were investigated over a 5-year period. Eleven isolates presenting a strong ampC promoter and different strategic AmpC mutations, including two newly described modifications (A292V and an L-A-A insertion at 295), were characterized. All the isolates belonged to phylogenetic group A and to the ST23 complex.

Emergence of high ampicillin-resistant Enterococcus faecium isolates in a kidney transplant ward: role of antibiotic pressure and cross transmission.

The epidemiology of patients associated with ampicillin-resistant Enterococcus faecium (ARE) was investigated by combining both clinical approach and molecular analysis in a kidney transplant patients ward. A case-control study was performed to identify risk factors for ARE by matching each patient with ARE with two control patients without any isolated E. faecium strain. ARE isolates were characterized by pulsed-field gel electrophoresis. From June 2004 to May 2006, 18 cases with clinical ARE samples were detected and compared with 35 control patients. By univariate analysis, recurrent urinary tract infections (UTIs) (odds ratio [OR], 4.9; 95% confidence interval [CI], 1.0-25.6), mean number of hospitalization days in the last year (p < 0.003), pyelonephritis or UTI (OR, 9.6; 95% CI, 2.2-46.1), oral third-generation cephalosporin use (OR, 12.42; 95% CI, 2.04-109.1), and fluoroquinolone use (OR, 4.4; 95% CI, 1.1-18.2) were significantly associated with ARE urinary tract colonization. By conditional logistic regression, hospitalization >21 days within 1 year (adjusted OR [aOR], 6.9; 95% CI, 1.0-46.5), recent medical history of pyelonephritis or UTI (aOR, 8.6; 95% CI, 1.5-49.1), and prior oral third-generation cephalosporin use (aOR, 13.1; 95% CI, 1.2-142.6) were identified as independent factors associated with ARE urinary tract colonization. Genotyping revealed a heterogeneous epidemiological situation with two major clones in patients hospitalized in successive rooms and 10 different single pulsotypes. Emergence of highly resistant enterococcal strains is a collateral damage from antibiotic prescription and represents a potential source of patient-to-patient transmission. Combining epidemiological approach and molecular analysis is a powerful tool to delineate mechanisms of emerging resistance. Improving our knowledge on ARE emergence in high antibiotic pressure hospital wards is a key factor to better control these colonizations/infections and to prevent the emergence of vancomycin-resistant E. faecium.

Chitinophaga terrae bacteremia in human.

To the Editor: The genus Chitinophaga, first described by Sangkhobol and Skerman in 1981, belongs to the phylum Bacteroidetes (formerly the Cytophaga-Flexibacter-Bacteroides group), which includes filamentous, chitinolytic, gliding bacteria that transform into spherical bodies upon aging (1). This genus contains 10 environmental species that demonstrate similarities in 16S rDNA sequence and in phenotypic and chemotaxonomic data (menaquinone, fatty acids, hydroxy fatty acid, and polyamine) (2–5). Chitinophaga terrae, originally isolated from soil in South Korea, was first described in 2007 (3,4). Here we report a case of bacteremia due to C. terrae in a severely immunosuppressed woman. On July 31, 2008, a 51-year-old woman was admitted to the emergency department at Nantes University Hospital in Nantes, France, because of a slowly growing left cheek mass associated with weight loss and change of general state. Physical examination showed several cutaneous infiltrated nodules, bilateral axillary adenopathies, and hepatosplenomegaly. Deteriorating renal function led to intermittent hemodialysis. Histopathology of skin and renal biopsies revealed a diffuse, high–grade, large B-cell lymphoma with cutaneous localization. Systemic CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) chemotherapy and methotrexate intrathecal chemotherapy were begun August 9. She developed bone marrow aplasia 3 days later, shortly followed by the onset of pyrexia. Corynebacterium pseudotuberculosis was isolated from 3 blood samples, 2 drawn using the central venous catheter (CVC) and 1 from peripheral blood. Consecutively, serotype O1 Pseudomonas aeruginosa strain was isolated from cultures of urine and fecal specimens. The empirical antimicrobial drug treatment started with piperacillin-tazobactam, ciprofloxacin, and teicoplanin on August 16 and was replaced by imipenem, ciprofloxacin, and teicoplanin on August 21. On August 26, the patient was admitted to the medical intensive care unit (MICU) after indications of toxic epidermal necrolysis. Blood analysis showed pancytopenia with thrombocytopenia (26 × 109/L), anemia (hemoglobin 8.7 g/dL), and profound leukopenia (0.01 × 109/L). Antimicrobial drug treatment was changed to an association of noncytotoxic drugs, i.e., aztreonam, amikacin, and teicoplanin. Despite this broad-spectrum antimicrobial therapy, 4 aerobic blood cultures (1 drawn August 29, 2 on September 2, and 1 on September 3), 2 drawn using the CVC and 2 from a peripheral site, yielded gram-negative bacilli (our laboratory reference no. NTS8639) after 2 days’ incubation. The CVC was removed September 3 and sent to the laboratory for culture. The catheter tip was immersed in 2 mL of brain heart infusion agar, and semiquantitative culture was performed on the blood agar plate using 100 µL of the solution. The culture remained negative. On September 2, treatment was changed to imipenem, trimethoprim-sulfamethoxazole, and teicoplanin. Additionally, diagnosis of invasive pulmonary aspergillosis led to changing caspofungin prophylaxis to voriconazole. Trimethoprim-sulfamethoxazole treatment was stopped September 9, and imipenem was stopped September 25, a week after bone marrow recovery. The patient was discharged from MICU on October 7. Yellow-pigmented colonies grew on bromocresol purple lactose agar plate after 48 hours of incubation at 37°C and appeared as thin gram-negative bacilli after gram-staining was performed. The nonfermenting, nonmotile, oxidase-positive bacterium could grow at various pH values (pH 6.0, 7.3, and 8.0) and at different temperatures (30, 37, and 40°C). The semi-automatic Api 20NE gallery (bioMerieux, Marcy l’Etoile, France) identified the strain as Sphingomonas paucimobilis, whereas the ID-GNB card of the VITEK 2 system (bioMerieux) identified the bacterium as Sphingobacterium thalpophilum. The 16S rDNA amplification and sequencing were performed with universal primers 27f and 1378r as previously described (6). The 1366-bp sequence matched that of C. terrae with 100% similarity, according to BIBI (Bioinformatic Bacteria Identification, http://umr5558-sud-str1.univ-lyon1.fr/lebibi/lebibi.cgi) or BLAST (www.ncbi.nlm.nih.gov) analysis. Phylogenetic analysis with either the neighbor-joining or maximum-parsimony algorithm embedded the NTS8639 strain to the genus Chitinophaga and the species C. terrae (Figure). The biochemical characteristics of the bacterium corresponded to those previously described for C. terrae by Kim and Jung (3). The strain reduced nitrate to nitrite, produced N-acetyl-β-glucosamidase, phosphatase, α- and β-galactosidases, α- and β-glucosidases, and assimilated L-arabinose, L-fucose, D-glucose, maltose, D-mannose, D-melibiose, L-rhamnose, sucrose and salicin with Api 20NE, Api ID32GN (bioMerieux), and ID-GNB biochemical galleries. Unlike S. paucimobilis, the strain was positive for nitrate reductase and L-rhamnose. Also, negative reaction for urease and L-fucose assimilation differentiated the strain from S. thalpophilum. At the species level, the bacterium grew well at 37°C, unlike Chitinophaga arvensicola, and showed positive oxidase reaction and nitrate reduction, unlike Chitinophaga ginsengisegetis (2,5). Figure Neighbor-joining (NJ) tree showing the phylogenetic placement of strain NTS8639 (in boldface) among members of the Chitinophaga terrae species. Twenty-one 16S rRNA gene sequences selected from the GenBank database were aligned with that of strain NTS8639 ... Disk diffusion tests showed that the bacterium was multiresistant to antimicrobial drugs, including most of the β-lactams, aminoglycosides, fluoroquinolones, colistin, fosfomycin, and tigecyclin. It remained susceptible to amoxicillin-clavulanate, ticarcillin-clavulanate, carbapenems, and trimethoprim-sulfamethoxazole. Interpretation of the susceptibility test was impossible before 48 hours incubation, i.e., 4 days after the first positive blood culture. We describe in this report a case of human bacteremia due to C. terrae. This environmental organism behaving as an opportunistic pathogen was able to produce infection in a severely immunosuppressed woman. The source of bacteremia was not clearly established. Catheter-related bacteremia was not confirmed by culture of the CVC tip sent to the laboratory on September 3. Virulence factors contributing to the pathogenicity of C. terrae have not yet been well defined. The infection could have been favored by the immunosuppressive therapy, the profound leukopenia, and the extensive cutaneous detachment subsequently associated with methotrexate overdose in this patient. Unlike most susceptible environmental organisms, this bacterium probably was assisted by its multiresistance to antimicrobial drugs in producing infection. The intrinsic or acquired resistance of C. terrae to antimicrobial drugs has not yet been fully elucidated. The lack of commercially available biochemical gallery databases makes correct identification of this environmental organism difficult. It also underlines the usefulness of 16S rDNA sequencing for identification of unusual gram-negative bacilli isolated from immunocompromised hosts (7).

Methicillin-susceptible Staphylococcus aureus CC398: First description in prosthetic joint infection and genetic background comparison with nasal carriage isolates ☆

Few reports described infections with CC398 methicillin-susceptible Staphylococcus aureus (MSSA). We compared the genetic background of CC398 MSSA strains from nasal carriage and knee arthroplasty infection. DNA microarray analysis shows acquisition of particular adhesin, iron capture system and immune defense evasion mechanisms. These characteristics could explain pathogenesis in this type of infection.

Genotypic and phenotypic characterization of Staphylococcus epidermidis causing chronic relapsing prosthetic joint infections

Abstract Twenty-one isolates of Staphylococcus epidermidis from 9 patients with persistent prosthetic joint infections were analysed by pulsed-field gel electrophoresis and antibiotic susceptibility assays. In 7 of these cases, the S. epidermidis isolate was different from that of the initial episode. In 1 further case, the superinfection was polyclonal. Recurrence, i.e., renewed isolation of a clone identical to that of an initial episode, occurred in 3 cases, 1 of which was in the absence of superinfection. A high degree of antibiotic resistance was demonstrated, including methicillin in 17 of 21 strains. In conclusion, a frequent occurrence of superinfection and a high degree of resistance make management of these infections complex.

Correlation between the VITEK2 system and cefoxitin disk diffusion for the daily detection of oxacillin resistance in a large number of clinical Staphylococcus aureus isolates

The aim of the present study was to compare the performance of the new VITEK2 AST-P551 card with the cefoxitin disk diffusion method for the daily detection of methicillin resistance with a high number of Staphylococcus aureus clinical isolates. Detection of the PBP2a protein or mecA gene was performed for each discordant case. Seventy (3.3%) isolates out of 2,107 clinical strains showed discordant results, two very major errors, four major errors and 64 minor errors. Fifty-nine (84%) discordant results were resolved, with a final overall agreement of 99.5%. Eleven (0.5%) strains remained discordant (minor error [mE]). Four of 370 MRSA strains were misclassified as susceptible in daily practice by the cefoxitin disk diffusion method. All of these strains were resistant to aminoglycosides and/or fluoroquinolones. The VITEK2 system is highly reliable for methicillin resistance detection at the routine level. Oxacillin-susceptible classified clinical strains with associated resistance patterns required attention.

Relapse of Serratia marcescens Sternal Osteitis 15 Years after the First Episode

ABSTRACT Sternal osteitis, a potential consequence of cardiac surgery, remains rare. The bacteria involved belong mostly to the genus Staphylococcus. Sternal infections caused by Serratia marcescens are exceptional. We report an unusual recurrence of sternal infection with S. marcescens, 15 years after the initial episode. The identities of the isolates were determined by genomic analysis.