Impact of strain typing methods on assessment of relationship between paired nares and wound isolates of methicillin-resistant Staphylococcus aureus.

The anterior nares are the site of choice for the Veterans Administration methicillin-resistant Staphylococcus aureus (MRSA) surveillance program; however, a correlation between nares colonization and concomitant wound infections has not been well established. The purpose of this study was 3-fold: to determine the relatedness of MRSA isolates from 40 paired wound and nares specimens by four different strain typing methods, to determine concordance of typing methods, and to establish a baseline of MRSA types at this medical center. Isolates were typed by repetitive PCR (rep-PCR) (DiversiLab System; DL) and SpectraCell Raman analysis (SCRA) (commercially available methods that can be performed within a clinical lab), pulsed-field gel electrophoresis (PFGE), and an antibiotic susceptibility profile (AB). Whole-genome optical mapping (WGM) (OpGen, Inc.) was performed on selected isolates. All methods agreed that 26 pairs were indistinguishable and four pairs were different. Discrepant results were as follows: 4 where only SCRA was discordant, 3 where only AB was discordant, 2 where both DL and AB were discordant, and 1 where both DL and SCRA were discordant. All WGM agreed with PFGE. After discrepancy resolution, 80% of the pairs were indistinguishable and 20% were different. A total of 56% of nares results were nonpredictive if negative nares and positive wound cultures are included. Methods agreed 85 to 93% of the time; however, congruence of isolates to a clade was lower. Baseline analysis of types showed that 15 pairs were unique to single patients (30 strains, 38%; 47% of the matching pairs). Twenty-five strains (30%) represented a single clade identical by PFGE, SCRA, and DL, decreasing specificity. Typing method and institutional type frequency are important in assessing MRSA strain relatedness.

Rapid typing of extended-spectrum ß-lactamase- and carbapenemase-producing Escherichia coli and Klebsiella pneumoniae isolates by use of SpectraCell RA.

Enterobacteriaceae are important pathogens of both nosocomial and community-acquired infections. In particular, strains with broad-spectrum beta-lactamases increasingly cause problems in health care settings. Rapid and reliable typing systems are key tools to identify transmission, so that targeted infection control measures can be taken. In this study, we evaluated the performance of Raman spectroscopic analysis (RA) for the typing of multiresistant Escherichia coli and Klebsiella pneumoniae isolates using the SpectraCell RA bacterial strain analyzer (River Diagnostics). Analysis of 96 unrelated isolates revealed that RA generated highly reproducible spectra and exhibited a discriminatory power that is comparable to pulsed-field gel electrophoresis. Furthermore, adequate results were obtained for three collections of clinical isolates. RA was able to discriminate outbreak-related isolates from isolates that were not involved in an outbreak or transmission. Furthermore, it was found that the RA approach recognized clones, irrespective of the extended-spectrum ß-lactamase type. It can be concluded that RA is a suitable typing technique for E. coli and K. pneumoniae isolates. Combining high reproducibility, speed, and ease-of-use, this technique may play an important role in monitoring the epidemiology of these important nosocomial species.

Optical fingerprinting in bacterial epidemiology: Raman spectroscopy as a real-time typing method.

Hospital-acquired infections (HAI) increase morbidity and mortality and constitute a high financial burden on health care systems. An effective weapon against HAI is early detection of potential outbreaks and sources of contamination. Such monitoring requires microbial typing with sufficient reproducibility and discriminatory power. Here, a microbial-typing method is presented, based on Raman spectroscopy. This technique provides strain-specific optical fingerprints in a few minutes instead of several hours to days, as is the case with genotyping methods. Although the method is generally applicable, we used 118 Staphylococcus aureus isolates to illustrate that the discriminatory power matches that of established genotyping techniques (numerical index of diversity [D]=0.989) and that concordance with the gold standard (pulsed-field gel electrophoresis) is high (95%). The Raman clustering of isolates was reproducible to the strain level for five independent cultures, despite the various culture times from 18 h to 24 h. Furthermore, this technique was able to classify stored (-80 degrees C) and recent isolates of a methicillin-resistant Staphylococcus aureus-colonized individual during surveillance studies and did so days earlier than established genotyping techniques did. Its high throughput and ease of use make it suitable for use in routine diagnostic laboratory settings. This will set the stage for continuous, automated, real-time epidemiological monitoring of bacterial infections in a hospital, which can then be followed by timely corrective action by infection prevention teams.

A novel approach to correct variations in Raman spectra due to photo-bleachable cellular components.

Bacterial typing by Raman spectroscopy is based on small spectral differences that exist between strains, due to differences in their overall molecular composition. These strain-specific spectral differences can be obscured by sources of non-specific signal variance. One such source is the signal contribution of microbial pigments that can vary strongly in intensity. Examples of such pigments are carotenoids in Staphylococcus aureus, and other pigments in Pseudomonas aeruginosa and Mycobacterium lentiflavum. The variance in the intensity of these pigments greatly overshadows strain-specific differences, and therefore lowers spectral reproducibility and causes misclassification of microbial strains. Here a method is presented to determine the spectral signature of pigments of which the relative signal contribution decreases under laser irradiation; so-called photo-bleachable pigments. These signatures are used to eliminate signal variance caused by these pigments by means of the extended multiplicative scatter correction algorithm and spectral interferent subtraction. Application of this method increases the reproducibility of the spectra of microorganisms that contain such pigments to the extent that reproducible identification of samples at strain level is achieved.

Discriminating basal cell carcinoma from perilesional skin using high wave-number Raman spectroscopy.

An expanding body of literature suggests Raman spectroscopy is a promising tool for skin cancer diagnosis and in-vivo tumor border demarcation. The development of an in-vivo diagnostic tool is, however, hampered by the fact that construction of fiber optic probes suitable for Raman spectroscopy in the so-called fingerprint region is complicated. In contrast, the use of the high wave-number region allows for fiber optic probes with a very simple design. We investigate whether high wave-number Raman spectroscopy (2800 to 3125 cm(-1)) is able to provide sufficient information for noninvasive discrimination between basal cell carcinoma (BCC) and noninvolved skin. Using a simple fiber optic probe, Raman spectra are obtained from 19 BCC biopsy specimens and 9 biopsy specimens of perilesional skin. A linear discriminant analysis (LDA)-based tissue classification model is developed, which discriminates between BCC and noninvolved skin with high accuracy. This is a crucial step in the development of clinical dermatological applications based on fiber optic Raman spectroscopy.

Current trends in the epidemiological typing of clinically relevant microbes in Europe.

A questionnaire-based survey was performed to accumulate data on methodologies used in microbiology laboratories involved in epidemiological typing. Genotyping by PFGE and MLST are currently clearly preferred over phenotyping. The overall wish is to increase the activities by over 20% and additional resources would be used to invest in real-time typing.

Rapid epidemiological analysis of Acinetobacter strains by Raman spectroscopy.

From earlier publications, we noticed that Raman spectra could potentially be used for subspecies identification of microorganisms. Here we evaluated the technique for its use as a typing tool of Acinetobacter species, using a collection of well-characterised strains from five hospital outbreaks. The strains were previously analysed using molecular techniques as cell envelope protein profiling and ribotyping. In this study, we have typed the strains by AFLP analysis and Raman spectroscopy. We compared the results using hierarchical cluster analysis, which showed highly similar groupings by both techniques. There seemed to be some misclassification between two sets of outbreak strains in the Raman analysis. We ascribe this to the clonal relationship between the strains of both outbreaks, described earlier. This results from a highly similar biochemical composition of the strains involved, and hence a highly similar Raman spectrum. We conclude that Raman spectroscopy could be an easy-to-use alternative in epidemiological studies of Acinetobacter strains and a promising starting point for the development of epidemiological studies in general.

Epidemiology of Staphylococcus aureus harboring the mecA or Panton-Valentine leukocidin genes in hospitals in Java and Bali, Indonesia.

Data of Staphylococcus aureus carriage in Indonesian hospitals are scarce. Therefore, the epidemiology of S. aureus among surgery patients in three academic hospitals in Indonesia was studied. In total, 366 of 1,502 (24.4%) patients carried S. aureus. The methicillin-resistant S. aureus (MRSA) carriage rate was 4.3%, whereas 1.5% of the patients carried Panton-Valentine leukocidin (PVL)-positive methicillin-sensitive S. aureus (MSSA). Semarang and Malang city (odds ratio [OR] 9.4 and OR 9.0), being male (OR 2.4), hospitalization for more than 5 days (OR 11.708), and antibiotic therapy during hospitalization (OR 2.6) were independent determinants for MRSA carriage, whereas prior hospitalization (OR 2.5) was the only one risk factor for PVL-positive MSSA carriage. Typing of MRSA strains by Raman spectroscopy showed three large clusters assigned type 21, 24, and 38, all corresponding to ST239-MRSA-SCCmec type III. In conclusion, MRSA and PVL-positive MSSA are present among patients in surgical wards in Indonesian academic hospitals.

Towards Raman-based epidemiological typing of Pseudomonas aeruginosa.

Raman spectra of bacteria can be used as highly specific fingerprints, enabling discrimination at strain level. Pseudomonas aeruginosa strains can be strongly pigmented, making it difficult to obtain high quality spectra of such isolates due to high fluorescent spectral backgrounds. Furthermore, the spectra that could be measured with acceptable quality often showed large spectral variations limiting the reproducibility required for strain level discrimination. P. aeruginosa produces a characteristic yellowish green fluorescent pigment, called pyoverdin. Applying a washing procedure to reduce the amount of fluorescent pigment, enabled the highly pigmented isolates to be measured with sufficient spectral quality. Isolation of the pigment/pyoverdin spectral features, together with spectral scaling methods improved reproducibility. It will be important to analyze the range of the spectral variations that can occur and ensure the correction of all of these factors to obtain the highest reproducibility required for strain level typing.

Raman spectroscopic typing reveals the presence of carotenoids in Mycoplasma pneumoniae.

Raman spectroscopy has previously been demonstrated to be a highly useful methodology for the identification and/or typing of micro-organisms. In this study, we set out to evaluate whether this technology could also be applied as a tool to discriminate between isolates of Mycoplasma pneumoniae, which is generally considered to be a genetically highly uniform species. In this evaluation, a total of 104 strains of M. pneumoniae were analysed, including two reference strains (strains M129 and FH), and 102 clinical isolates, which were isolated between 1973 and 2005 and originated from various countries. By Raman spectral analysis (Raman typing) of this strain collection, we were able to reproducibly distinguish six different clusters of strains. An unequivocal correlation between Raman typing and P1 genotyping, which is based on sequence differences in the P1 (or MPN141) gene of M. pneumoniae, was not observed. In the two major Raman clusters that we identified (clusters 3 and 6, which together harboured 81 % of the strains), the different P1 subtypes were similarly distributed, and approximately 76 % isolates were of subtype 1, approximately 20 % of subtype 2 and approximately 5 % of variant 2a. Nevertheless, a relatively high prevalence of P1 subtype 2 strains was found in clusters 2 and 5 (100 %), as well as in cluster 1 (75 %) and cluster 4 (71 %); these clusters, however, harboured a small number of strains. Only two of the strains (2 %) could not be typed correctly. Interestingly, analysis of the Raman spectra revealed the presence of carotenoids in M. pneumoniae. This finding is in line with the identification of M. pneumoniae genes that have similarity with genes involved in a biochemical pathway leading to carotenoid synthesis, i.e. the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. Therefore, we hypothesize that M. pneumoniae hosts an MEP-like pathway for carotenoid synthesis. We conclude that Raman spectroscopy is a convenient tool for discriminating between M. pneumoniae strains, and that it presents a promising supplement to the current methods for typing of this bacterium.

Monitoring poly(3-hydroxybutyrate) production in cupriavidus necator DSM 428 (H16) with raman spectroscopy.

This study explored the potential of Raman spectroscopy for the analysis of poly(3-hydroxybutyrate) (PHB) in bacteria. PHB can be formed in large amounts by certain bacteria as a storage material and is of high importance for industrial biodegradable plastic production. Raman spectra were collected from Cupriavidus necator DSM 428 (H16), from its non-PHB-producing mutant strain C. necator DSM 541, and from pure PHB, in order to determine at which Raman shifts a contribution of PHB in bacterial spectra can be expected. The Raman band intensity at ca. 1734 cm(-1) appeared to be suitable for the monitoring of PHB production and consumption. These intensities were linearly related to the PHB concentration (mg L(-1) culture) determined by parallel HPLC analysis. Therefore, Raman spectroscopy is considered as a fast and noninvasive technique for the determination and monitoring of the PHB content in bacteria.

Toxin profiles and resistances to macrolides and newer fluoroquinolones as epidemicity determinants of clinical isolates of Clostridium difficile from Warsaw, Poland.

Amplified fragment length polymorphism genotypes, antibiotic resistance profiles, and toxin profiles of Clostridium difficile strains from Warsaw were determined. The isolates segregate in six major genotypes, coinciding with toxin profiles. Most of the toxin A-negative toxin B-positive toxin CDT-negative strains possess ermB, and several strains were resistant to fluoroquinolones. Resistograms and toxin types of C. difficile strains are epidemicity determinants.

Discrimination between nontumor bladder tissue and tumor by Raman spectroscopy.

We have applied Raman spectroscopy to discriminate between nontumor and tumor bladder tissue and to determine the biochemical differences therein. Tissue samples from 15 patients were collected, and frozen sections were made for Raman spectroscopy and histology. Twenty-five pseudocolor Raman maps were created in which each color represents a cluster of spectra measured on tissue areas of similar biochemical composition. For each cluster, the cluster-averaged spectrum (CAS) was calculated and classified as tumor and nontumor in accordance to pathohistology. Unguided hierarchical clustering was applied to display heterogeneity between and within groups of nontumor and tumor CAS. A linear discriminant analysis model was developed to discriminate between CAS from tumor and nontumor. The model was tested by a leave-one-patient-out validation, 84 of the 90 CAS (93%) were correctly classified with 94% sensitivity and 92% specificity. Biochemical differences between tumor and nontumor CAS areas were analyzed by fitting spectra of pure compounds to the CAS. Nontumor CAS showed higher collagen content while tumor CAS were characterized by higher lipid, nucleic acid, protein, and glycogen content. Raman spectroscopy enabled effective discrimination between tumor and nontumor bladder tissue based on characterized biochemical differences, despite heterogeneity expressed in both tumor and nontumor CAS.