Reinhard Zbinden

Etiologic Diagnosis of Infective Endocarditis by Broad-Range Polymerase Chain Reaction: A 3-Year Experience

We analyzed surgically resected endocardial specimens from 49 patients by broad-range PCR. PCR results were compared with (1) results of previous blood cultures, (2) results of culture and Gram staining of resected specimens, and (3) clinical data (Duke criteria). Molecular analyses of resected specimens and previous blood cultures showed good overall agreement. However, in 18% of patients with sterile blood cultures, bacterial DNA was found in the resected materials. When data from patients with definite or rejected cases of infective endocarditis (IE) were included, the sensitivity, specificity, and positive and negative predictive values of broad-range PCR were 82.6%, 100%, 100%, and 76.5%, respectively, overall, and 94.1%, 100%, 100%, and 90%, for cases of native valve endocarditis. The sensitivity, specificity, and positive and negative predictive values of culture of resected specimens from patients with native valve endocarditis were 17.6%, 88.9%, 75%, and 36.4%. We recommend broad-range PCR of surgically resected endocardial material in cases of possible IE, in cases of suspected IE in which blood cultures are sterile, and in cases in which organisms grow in blood cultures but only Duke minor criteria are met. We propose to add molecular techniques to the pathologic criteria of the Duke classification scheme.

Ribosomal DNA Sequencing for Identification of Aerobic Gram-Positive Rods in the Clinical Laboratory (an 18-Month Evaluation)

ABSTRACT We have evaluated over a period of 18 months the use of 16S ribosomal DNA (rDNA) sequence analysis as a means of identifying aerobic gram-positive rods in the clinical laboratory. Two collections of strains were studied: (i) 37 clinical strains of gram-positive rods well identified by phenotypic tests, and (ii) 136 clinical isolates difficult to identify by standard microbiological investigations, i.e., identification at the species level was impossible. Results of molecular analyses were compared with those of conventional phenotypic identification procedures. Good overall agreement between phenotypic and molecular identification procedures was found for the collection of 37 clinical strains well identified by conventional means. For the 136 clinical strains which were difficult to identify by standard microbiological investigations, phenotypic characterization identified 71 of 136 (52.2%) isolates at the genus level; 65 of 136 (47.8%) isolates could not be discriminated at any taxonomic level. In comparison, 16S rDNA sequencing identified 89 of 136 (65.4%) isolates at the species level, 43 of 136 (31.6%) isolates at the genus level, and 4 of 136 (2.9%) isolates at the family level. We conclude that (i) rDNA sequencing is an effective means for the identification of aerobic gram-positive rods which are difficult to identify by conventional techniques, and (ii) molecular identification procedures are not required for isolates well identified by phenotypic investigations.

Changes in bacterial isolates from burn wounds and their antibiograms: a 20-year study (1986-2005).

BACKGROUND Our aim is to elucidate shifts in the bacterial spectrum colonising burn wounds and corresponding antibiotic susceptibilities during a 20-year study period. METHODS Microbiological results from burn patients collected between 1986 and 2005 were analysed retrospectively. RESULTS Staphylococcus aureus was isolated most frequently (20.8%), followed by Escherichia coli (13.9%), Pseudomonas aeruginosa (11.8%), coagulase-negative staphylococci (CNS) (10.9%), Enterococcus sp. (9.7%), Enterobacter cloacae (5.6%), Klebsiella pneumoniae (5%), Acinetobacter sp. (3.2%), Proteus mirabilis (2%) and Stenotrophomonas maltophilia (1.4%). Susceptibility of S. aureus to broad-spectrum substances such as ciprofloxacin or penicillinase-stable penicillins has waned, others such as cotrimoxazole or netilmicin remained effective. Not a single resistance against vancomycin was recorded. Increases in methicillin-resistant S. aureus (MRSA) were pronounced (3% in 1986-1997 (the first of the three study periods) to 16% in 1998-2001 and 13% in 2002-2005). Results for methicillin-resistant CNS (MRCNS) show an even greater increase. P. aeruginosa has shown increasing susceptibility against netilmicin (1986-1989: 84%, 2002-2005: 95%). Susceptibility of P. aeruginosa to ceftazidime has decreased markedly. S. maltophilia has shown clinically relevant susceptibility mainly against ciprofloxacin. Acinetobacter sp. have shown little susceptibility to most antibiotics. Imipenem or meropenem have been very reliable reserve antibiotics throughout the study period for the fermenting Enterobacteriaceae (E. coli, K. pneumoniae, E. cloacae and P. mirabilis), with susceptibilities of or near 100%. CONCLUSION In-depth knowledge of the bacteria causing infectious complications and of their antibiotic susceptibilities is a prerequisite for treating burn patients. Our study shows shifts in the microbial spectrum and their antibiogram, which mandate frequent reassessments.

Turicibacter sanguinis gen. nov., sp. nov., a novel anaerobic, Gram-positive bacterium

An unknown, strictly anaerobic, Gram-positive, rod-shaped bacterium (strain MOL361T) was isolated from a blood culture of a febrile patient with acute appendicitis and characterized using phenotypic and molecular methods. Fatty acid analysis and biochemical examination indicated that the isolate most closely resembles members of the Gram-positive bacteria with low DNA G+C content. 16S rDNA sequencing revealed a relatively high overall similarity (97%) to an uncultured bacterium, but these two strains both exhibit low (<87%) 16S rDNA similarity to other bacteria. Phylogenetic analysis with different treeing methods showed that this strain forms a novel line of descent within the Gram-positive bacteria with low G+C content. Strain MOL361T is described as the type strain of a novel species within a new genus, Turicibacter sanguinis gen. nov., sp. nov.

Identification of Gram-Positive Cocci by Use of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry: Comparison of Different Preparation Methods and Implementation of a Practical Algorithm for Routine Diagnostics

ABSTRACT This study compared three sample preparation methods (direct transfer, the direct transfer-formic acid method with on-target formic acid treatment, and ethanol-formic acid extraction) for the identification of Gram-positive cocci with matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS). A total of 156 Gram-positive cocci representing the clinically most important genera, Aerococcus, Enterococcus, Staphylococcus, and Streptococcus, as well as more rare genera, such as Gemella and Granulicatella, were analyzed using a Bruker MALDI Biotyper. The rate of correct genus-level identifications was approximately 99% for all three sample preparation methods. The species identification rate was significantly higher for the direct transfer-formic acid method and ethanol-formic acid extraction (both 77.6%) than for direct transfer (64.1%). Using direct transfer-formic acid compared to direct transfer, the total time to result was increased by 22.6%, 16.4%, and 8.5% analyzing 12, 48, and 96 samples per run, respectively. In a subsequent prospective study, 1,619 clinical isolates of Gram-positive cocci were analyzed under routine conditions by MALDI-TOF MS, using the direct transfer-formic acid preparation, and by conventional biochemical methods. For 95.6% of the isolates, a congruence between conventional and MALDI-TOF MS identification was observed. Two major limitations were found using MALDI-TOF MS: the differentiation of members of the Streptococcus mitis group and the identification of Streptococcus dysgalactiae. The Bruker MALDI Biotyper system using the direct transfer-formic acid sample preparation method was shown to be a highly reliable tool for the identification of Gram-positive cocci. We here suggest a practical algorithm for the clinical laboratory combining MALDI-TOF MS with phenotypic and molecular methods.

Three Consecutive Outbreaks of Serratia marcescens in a Neonatal Intensive Care Unit

We investigated an outbreak of Serratia marcescens in the neonatal intensive care unit (NICU) of the University Hospital of Zurich. S. marcescens infection was detected in 4 children transferred from the NICU to the University Childrens Hospital (Zurich). All isolates showed identical banding patterns by pulsed-field gel electrophoresis (PFGE). In a prevalence survey, 11 of 20 neonates were found to be colonized. S. marcescens was isolated from bottles of liquid theophylline. Despite replacement of these bottles, S. marcescens colonization was detected in additional patients. Prospective collection of stool and gastric aspirate specimens revealed that colonization occurred in some babies within 24 hours after delivery. These isolates showed a different genotype. Cultures of milk from used milk bottles yielded S. marcescens. These isolates showed a third genotype. The method of reprocessing bottles was changed to thermal disinfection. In follow-up prevalence studies, 0 of 29 neonates were found to be colonized by S. marcescens. In summary, 3 consecutive outbreaks caused by 3 genetically unrelated clones of S. marcescens could be documented. Contaminated milk could be identified as the source of at least the third outbreak.

Epidemic Spread of a Single Clone of Methicillin-Resistant Staphylococcus aureus among Injection Drug Users in Zurich, Switzerland

We describe an outbreak of methicillin-resistant Staphylococcus aureus (MRSA) among injection drug users (IDUs). From August 1994 through December 1999, we registered 31 IDUs with MRSA infections (12 with soft-tissue infection, 7 with pneumonia [fatal in 1], 7 with endocarditis [fatal in 1], 2 with osteomyelitis, 2 with septic arthritis, and 1 with ulcerative tonsillitis), with a marked increase in the number of IDUs registered during 1998 and 1999. Of 31 patients, 15 (48%) were infected with human immunodeficiency virus. A point-prevalence study among IDUs who frequented outpatient facilities in Zurich revealed an MRSA carriage rate of 10.3% (range, 0%-28.6%) in various facilities. In all but 1 case, pulsed-field gel electrophoresis banding patterns of isolates obtained from these patients were indistinguishable from isolates of the initial 31 IDUs registered. Risk factors for MRSA carriage were disability and prior hospitalization in a hospice. In summary, MRSA became endemic in IDUs in Zurich as a result of the spread of a single clone. This clone caused major morbidity and was responsible for a lethal outcome in 2 cases.

Significance of Staphylococcus lugdunensis bacteremia: report of 28 cases and review of the literature

Background:Staphylococcus lugdunensis endocarditis has been associated with an aggressive course. The aim of this study was to determine factors associated with the development of endocarditis in patients with S. lugdunensis bacteremia.Methods:A retrospective analysis of all patients with S. lugdunensis bacteremia in three tertiary care centers in Switzerland was performed. Data regarding medical history, symptoms, and susceptibility of S. lugdunensis isolates were collected. Our results were reviewed in the context of the current literature.Results:A total of 28 patients with S. lugdunensis bacteremia were identified. Of the 13 patients with endocarditis, all were community acquired. Cardiac surgery was performed in 85% of these patients; mortality was 23%, reflecting the aggressive course of this disease. In contrast, in the 15 patients without endocarditis, no complications associated with S. lugdunensis bacteremia were observed. In 73%, a probable source was identified in the form of a venous catheter or other foreign device. Only three of these episodes were community acquired. No difference was observed in susceptibility of the S. lugdunensis isolates to penicillin, which was 77% in endocarditis isolates, and 87% in isolates of bacteremia without endocarditis, respectively.Conclusion:S. lugdunensis bacteremia is associated with endocarditis in up to 50% of patients. Every patient with community-acquired S. lugdunensis bacteremia should be carefully examined for signs of endocarditis. Once S. lugdunensis endocarditis is diagnosed, close monitoring is essential and surgical treatment should be considered early. In the nosocomial setting, endocarditis is far less frequent, and S. lugdunensis bacteremia is usually associated with a catheter or other foreign materials.

Detection of AmpC Beta-Lactamase in Escherichia coli: Comparison of Three Phenotypic Confirmation Assays and Genetic Analysis

ABSTRACT Two mechanisms account for AmpC activity in Escherichia coli, namely, mutations in the ampC promoter and attenuator regions resulting in ampC overexpression and acquisition of plasmid-carried ampC genes. In this study, we analyzed 51 clinical E. coli isolates with reduced susceptibility to amoxicillin-clavulanic acid, piperacillin-tazobactam, or extended-spectrum cephalosporins for the presence of AmpC production. Three phenotypic AmpC confirmation assays (cefoxitin-cloxacillin disk diffusion test, cefoxitin-EDTA disk diffusion test, and AmpC Etest) were compared for the detection of AmpC activity. All 51 isolates were characterized genetically by mutational analysis of the chromosomal ampC promoter/attenuator region and by PCR detection of plasmid-carried ampC genes. Altogether, 21/51 (41%) E. coli isolates were considered true AmpC producers. AmpC activity due to chromosomal ampC promoter/attenuator mutations was found in 12/21 strains, and plasmid-carried ampC genes were detected in 8/21 isolates. One strain contained both ampC promoter mutations and a plasmid-carried ampC gene. All three phenotypic tests were able to detect the majority (>90%) of AmpC-positive strains correctly. Cefoxitin resistance was found to be a discriminative parameter, detecting 20/21 AmpC-producing strains. Susceptibility to extended-spectrum cephalosporins, e.g., ceftriaxone, ceftazidime, and cefotaxime, was found in 9 of the 21 AmpC-positive strains. Considering the elevated zone diameter breakpoints of the 2010 CLSI guidelines, 2/21 AmpC-positive strains were categorized as susceptible to extended-spectrum cephalosporins.

Evaluation of the Bruker MALDI Biotyper for Identification of Gram-Positive Rods: Development of a Diagnostic Algorithm for the Clinical Laboratory

ABSTRACT Reported matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) identification rates of Gram-positive rods (GPR) are low compared to identification rates of Gram-positive cocci. In this study, three sample preparation methods were compared for MALDI-TOF MS identification of 190 well-characterized GPR strains: direct transfer, direct transfer-formic acid preparation, and ethanol-formic acid extraction. Using the interpretation criteria recommended by the manufacturer, identification rates were significantly higher for direct transfer-formic acid preparation and ethanol-formic acid extraction than for direct transfer. Reducing the species cutoff from 2.0 to 1.7 significantly increased species identification rates. In a subsequent prospective study, 215 clinical GPR isolates were analyzed by MALDI-TOF MS, and the results were compared to those for identification using conventional methods, with discrepancies being resolved by 16S rRNA and rpoB gene analysis. Using the direct transfer-formic acid preparation and a species cutoff of 1.7, congruencies on the genus and species levels of 87.4% and 79.1%, respectively, were achieved. In addition, the rate of nonidentified isolates dropped from 12.1% to 5.6% when using an extended database, i.e., the Bruker database amended by reference spectra of the 190 GPR of the retrospective study. Our data demonstrate three ways to improve GPR identification by the Bruker MALDI Biotyper, (i) optimize sample preparation using formic acid, (ii) reduce cutoff scores for species identification, and (iii) expand the database. Based on our results, we suggest an identification algorithm for the clinical laboratory combining MALDI-TOF MS with nucleic acid sequencing.