Hubert P. Endtz

Prospective Study of the Performance of Vibrational Spectroscopies for Rapid Identification of Bacterial and Fungal Pathogens Recovered from Blood Cultures

ABSTRACT Rapid identification of microbial pathogens reduces infection-related morbidity and mortality of hospitalized patients. Raman spectra and Fourier transform infrared (IR) spectra constitute highly specific spectroscopic fingerprints of microorganisms by which they can be identified. Little biomass is required, so that spectra of microcolonies can be obtained. A prospective clinical study was carried out in which the causative pathogens of bloodstream infections in hospitalized patients were identified. Reference libraries of Raman and IR spectra of bacterial and yeast pathogens highly prevalent in bloodstream infections were created. They were used to develop identification models based on linear discriminant analysis and artificial neural networks. These models were tested by carrying out vibrational spectroscopic identification in parallel with routine diagnostic phenotypic identification. Whereas routine identification has a typical turnaround time of 1 to 2 days, Raman and IR spectra of microcolonies were collected 6 to 8 h after microbial growth was detected by an automated blood culture system. One hundred fifteen samples were analyzed by Raman spectroscopy, of which 109 contained bacteria and 6 contained yeasts. One hundred twenty-one samples were analyzed by IR spectroscopy. Of these, 114 yielded bacteria and 7 were positive for yeasts. High identification accuracy was achieved in both the Raman (92.2%, 106 of 115) and IR (98.3%, 119 of 121) studies. Vibrational spectroscopic techniques enable simple, rapid, and accurate microbial identification. These advantages can be easily transferred to other applications in diagnostic microbiology, e.g., to accelerate identification of fastidious microorganisms.

Investigating Microbial (Micro)colony Heterogeneity by Vibrational Spectroscopy

ABSTRACT Fourier transform infrared and Raman microspectroscopy are currently being developed as new methods for the rapid identification of clinically relevant microorganisms. These methods involve measuring spectra from microcolonies which have been cultured for as little as 6 h, followed by the nonsubjective identification of microorganisms through the use of multivariate statistical analyses. To examine the biological heterogeneity of microorganism growth which is reflected in the spectra, measurements were acquired from various positions within (micro)colonies cultured for 6, 12, and 24 h. The studies reveal that there is little spectral variance in 6-h microcolonies. In contrast, the 12- and 24-h cultures exhibited a significant amount of heterogeneity. Hierarchical cluster analysis of the spectra from the various positions and depths reveals the presence of different layers in the colonies. Further analysis indicates that spectra acquired from the surface of the colonies exhibit higher levels of glycogen than do the deeper layers of the colony. Additionally, the spectra from the deeper layers present with higher RNA levels than the surface layers. Therefore, the 6-h colonies with their limited heterogeneity are more suitable for inclusion in a spectral database to be used for classification purposes. These results also demonstrate that vibrational spectroscopic techniques can be useful tools for studying the nature of colony development and biofilm formation.

The crucial role of Campylobacter jejuni genes in anti-ganglioside antibody induction in Guillain-Barre syndrome

Molecular mimicry of Campylobacter jejuni lipo-oligosaccharides (LOS) with gangliosides in nervous tissue is considered to induce cross-reactive antibodies that lead to Guillain-Barre syndrome (GBS), an acute polyneuropathy. To determine whether specific bacterial genes are crucial for the biosynthesis of ganglioside-like structures and the induction of anti-ganglioside antibodies, we characterized the C. jejuni LOS biosynthesis gene locus in GBS-associated and control strains. We demonstrated that specific types of the LOS biosynthesis gene locus are associated with GBS and with the expression of ganglioside-mimicking structures. Campylobacter knockout mutants of 2 potential GBS marker genes, both involved in LOS sialylation, expressed truncated LOS structures without sialic acid, showed reduced reactivity with GBS patient serum, and failed to induce an anti-ganglioside antibody response in mice. We demonstrate, for the first time, to our knowledge, that specific bacterial genes are crucial for the induction of anti-ganglioside antibodies.

Host Specificity of Vancomycin-Resistant Enterococcus faecium

Amplified-fragment length polymorphism (AFLP) analysis was used to investigate the genetic relationships among 255 vancomycin-resistant Enterococcus faecium (VREF) strains isolated from hospitalized patients, nonhospitalized persons, and various animal sources. Four major AFLP genogroups (A-D) were discriminated. The strains of each taxon shared >/=65% of the restriction fragments. Most isolates recovered from nonhospitalized persons (75%) were grouped together with all pig isolates in genogroup A. Most isolates from hospitalized patients (84%), a subset of veal calf isolates (25%), and all isolates from cats and dogs clustered in genogroup C. Most isolates from chickens (97%) and turkeys (86%) were grouped in genogroup B, whereas most veal calf isolates (70%) clustered in genogroup D. Therefore, VREF strains are predominantly host-specific, and strains isolated from hospitalized patients are genetically different from the prevailing VREF strains present in the fecal flora of nonhospitalized persons.

In Vitro Activities of Ertapenem (MK-0826) against Recent Clinical Bacteria Collected in Europe and Australia

ABSTRACT Ertapenem (MK-0826, L-749,345) is a 1-β-methyl carbapenem with a long serum half-life. Its in vitro activity was determined by broth microdilution against 3,478 bacteria from 12 centers in Europe and Australia, with imipenem, cefepime, ceftriaxone, and piperacillin-tazobactam used as comparators. Ertapenem was the most active agent tested against members of the familyEnterobacteriaceae, with MICs at which 90% of isolates are inhibited (MIC90s) of ≤1 μg/ml for all species. Ertapenem also was more active than imipenem against fastidious gram-negative bacteria and Moraxella spp.; on the other hand, ertapenem was slightly less active than imipenem against streptococci, methicillin-susceptible staphylococci, and anaerobes, but its MIC90s for these groups remained ≤0.5 μg/ml.Acinetobacter spp. and Pseudomonas aeruginosawere also much less susceptible to ertapenem than imipenem, and mostEnterococcus faecalis strains were resistant. Ertapenem resistance, based on a provisional NCCLS MIC breakpoint of ≥16 μg/ml, was seen in only 3 of 1,611 strains of the familyEnterobacteriaceae tested, all of them Enterobacter aerogenes. Resistance was also seen in 2 of 135 anaerobes, comprising 1 Bacteroides fragilis strain and 1Clostridium difficile strain. Ertapenem breakpoints for streptococci have not been established, but an unofficial susceptibility breakpoint of ≤2 μg/ml was adopted for clinical trials to generate corresponding clinical response data for isolates for which MICs were as high as 2 μg/ml. Of 234 Streptococcus pneumoniae strains tested, 2 required ertapenem MICs of 2 μg/ml and one required an MIC of 4 μg/ml, among 67 non-Streptococcus pyogenes, non-Streptococcus pneumoniae streptococci, single isolates required ertapenem MICs of 2 and 16 μg/ml. These streptococci also had diminished susceptibilities to other β-lactams, including imipenem as well as ertapenem. The Etest and disk diffusion gave susceptibility test results in good agreement with those of the broth microdilution method for ertapenem.

Rapid identification of Candida species by confocal Raman microspectroscopy

ABSTRACT Candida species are important nosocomial pathogens associated with high mortality rates. Rapid detection and identification of Candida species can guide a clinician at an early stage to prescribe antifungal drugs or to adjust empirical therapy when resistant species are isolated. Confocal Raman microspectroscopy is highly suitable for the rapid identification of Candida species, since Raman spectra can be directly obtained from microcolonies on a solid culture medium after only 6 h of culturing. In this study, we have used a set of 42 Candida strains comprising five species that are frequently encountered in clinical microbiology to test the feasibility of the technique for the rapid identification of Candida species. The procedure was started either from a culture on Sabouraud medium or from a positive vial of an automated blood culture system. Prior to Raman measurements, strains were subcultured on Sabouraud medium for 6 h to form microcolonies. Using multivariate statistical analyses, a high prediction accuracy (97 to 100%) was obtained with the Raman method. Identification with Raman microspectroscopy may therefore be significantly faster than identification with commercial identification systems that allow various species to be identified and that often require 24 to 48 h before a reliable identification is obtained. We conclude that confocal Raman microspectroscopy offers a rapid, accurate, and easy-to-use alternative for the identification of clinically relevant Candida species.

Structure of Campylobacter jejuni lipopolysaccharides determines antiganglioside specificity and clinical features of Guillain-Barré and Miller Fisher patients

ABSTRACT Ganglioside mimicry in the lipopolysaccharide (LPS) fraction of Campylobacter jejuni isolated from Guillain-Barré syndrome (GBS) and Miller Fisher syndrome (MFS) patients was compared with isolates from patients with an uncomplicated enteritis. The antibody response to C. jejuni LPS and gangliosides in neuropathy patients and controls was compared as well. LPS from GBS and MFS-associated isolates more frequently contained ganglioside-like epitopes compared to control isolates. Almost all neuropathy patients showed a strong antibody response against LPS and multiple gangliosides in contrast to enteritis patients. Isolates from GBS patients more frequently had a GM1-like epitope than isolates from MFS patients. GQ1b-like epitopes were present in all MFS-associated isolates and was associated with anti-GQ1b antibody reactivity and the presence of oculomotor symptoms. These results demonstrate that the expression of ganglioside mimics is a risk factor for the development of post-Campylobacter neuropathy. This study provides additional evidence for the hypothesis that the LPS fraction determines the antiganglioside specificity and clinical features in post-Campylobacter neuropathy patients.

Prospective Multicenter Evaluation of a New Immunoassay and Real-Time PCR for Rapid Diagnosis of Clostridium difficile-Associated Diarrhea in Hospitalized Patients

ABSTRACT In a prospective multicenter study, 367 fecal samples from 300 patients with diarrhea were tested for Clostridium difficile-associated diarrhea (CDAD) with a new immunochromatography assay for toxins A and B (ICTAB), a real-time PCR on the toxin B gene, and the cell cytotoxicity assay. Twenty-three (6.2%) of the 367 fecal samples were positive by the cell cytotoxicity assay. With the cell cytotoxicity assay as the “gold standard,” the sensitivity, specificity, positive predictive value, and negative predictive value for the ICTAB assay and real-time PCR were 91, 97, 70, and 99%, and 87, 96, 57 and 99%, respectively. In conclusion, both the ICTAB and the real-time PCR can be implemented as rapid screening methods for patients suspected of having CDAD.

Cholera Outbreaks Caused by an Altered Vibrio cholerae O1 El Tor Biotype Strain Producing Classical Cholera Toxin B in Vietnam in 2007 to 2008

ABSTRACT Vibrio cholerae O1 isolates collected during cholera outbreaks occurring from late 2007 to early 2008 in northern Vietnam were revealed to represent an altered strain containing the RS1 element followed by a CTX prophage harboring El Tor type rstR and classical ctxB on the large chromosome.

Axonal variant of Guillain-Barré syndrome associated with Campylobacter infection in Bangladesh

Background: Campylobacter jejuni enteritis is the predominant bacterial infection preceding Guillain-Barré syndrome (GBS), an acute postinfectious immune-mediated polyradiculoneuropathy. The purpose of this study was to define the clinical phenotype of GBS and the relation with preceding C jejuni infections in Bangladesh. Methods: We performed a prospective matched case-control hospital surveillance including 100 patients fulfilling the National Institute of Neurological Disorders and Stroke criteria for GBS from 2006 to 2007 in the Dhaka area of Bangladesh. Detailed clinical, electrophysiologic, serologic, and microbiologic data were obtained with a follow-up of 6 months. Results: GBS affected predominantly young adult males living in rural areas. Sixty-nine percent of the patients had clinical evidence of a preceding infection. The most frequent symptom was diarrhea (36%). The majority of patients had a pure motor variant of GBS (92%) with relatively infrequent cranial nerve involvement (30%). Twenty-five percent of patients required respiratory support. Electrophysiologic studies showed that 67% of patients had an axonal variant of GBS. Eleven patients (14%) died, and 23 (29%) remained severely disabled during the follow-up. Positive C jejuni serology was found in an unprecedented high frequency of 57% as compared with 8% in family controls and 3% in control patients with other neurologic diseases (p < 0.001). C jejuni infection was significantly associated with serum antibodies to the gangliosides GM1 and GD1a, axonal neuropathy, and greater disability. Conclusions: We report an unusually high frequency of the axonal variant of Guillain-Barré syndrome in Bangladesh, associated with preceding Campylobacter jejuni infection, severe residual disability, and high mortality.