Raman spectroscopy-based identification of nosocomial outbreaks of the clonal bacterium Escherichia coli.

DNA-based techniques are frequently used to confirm the relatedness of putative outbreak isolates. These techniques often lack the discriminatory power when analyzing closely related microbes such as E. coli. Here the value of Raman spectroscopy as a typing tool for E. coli in a clinical setting was retrospectively evaluated.

Raman spectroscopic analysis of the clonal and horizontal spread of CTX-M-15-producing Klebsiella pneumoniae in a neonatal intensive care unit.

Nosocomial outbreaks of extended-spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae are an increasing concern in neonatal intensive care units (NICUs). We describe an outbreak of ESBL-producing K. pneumoniae that lasted 5 months and affected 23 neonates in our NICU. Proton pump inhibitor and extended-spectrum cephalosporin exposure were significantly associated with the risk of ESBL-producing K. pneumoniae colonisation and/or infection. Thirty isolates recovered from clinical, screening and environmental samples in the NICU were studied by means of Raman spectroscopy, pulsed-field gel electrophoresis and repetitive extragenic palindromic polymerase chain reaction (rep-PCR). The Raman clustering was in good agreement with the results of the other two molecular methods. Fourteen isolates belonged to the Raman clone 1 and 16 to the Raman clone 3. Molecular analysis showed that all the strains expressed SHV-1 chromosomal resistance, plasmid-encoded TEM-1 and CTX-M-15 ß-lactamases. Incompatibility groups of plasmid content identified by PCR-based replicon typing indicated that resistance dissemination was due to the clonal spread of K. pneumoniae and horizontal CTX-M-15 gene transfer between the two clones.

The use of Raman spectroscopy in the epidemiology of methicillin-resistant Staphylococcus aureus of human- and animal-related clonal lineages.

In order to perform a cost-effective search and destroy policy for methicillin-resistant Staphylococcus aureus (MRSA), a quick and reliable typing method is essential. In an area with a high level of animal-related MRSA ST398, pulsed field gel electrophoresis (PFGE) typing and spa-typing are not sufficient to discriminate between co-incidental findings and true transmission of MRSA. This study is the first to retrospectively show the performance of Raman spectroscopy in 16 well-documented outbreaks. We analysed 525 isolates, 286 MRSA ST398 and 239 from other PFGE clusters with Raman spectroscopy. When epidemiologically linked isolates from the outbreaks were analysed with PFGE as the reference standard, Raman spectroscopy correctly identified 97% of cases that were indistinguishable from the index case. With Raman cluster analysis, the most dominant distinction was between MRSA ST398 and other MRSA of human clonal lineages. Within MRSA ST398, 22 different Raman clusters were identified. Raman typing correctly identified an ST398 (spa type t567) outbreak in a hospital setting. No direct correlation was observed between Raman clusters and spa types. We conclude that Raman spectroscopy is a quick and reliable method of MRSA typing, which can be used in outbreak settings and it is comparable to PFGE, with the added advantage that PFGE non-typeable isolates can also be readily typed using the same sample preparation protocol.

Proof of principle for successful characterization of methicillin-resistant coagulase-negative staphylococci isolated from skin by use of Raman spectroscopy and pulsed-field gel electrophoresis.

Coagulase-negative staphylococci (CNS) are among the most frequently isolated bacterial species in clinical microbiology, and most CNS-related infections are hospital acquired. Distinguishing between these frequently multiple-antibiotic-resistant isolates is important for both treatment and transmission control. In this study we used isolates of methicillin-resistant coagulase-negative staphylococci (MR-CNS) that were selected from a large surveillance study of the direct spread of MR-CNS. This strain collection was used to evaluate (i) Raman spectroscopy as a typing tool for MR-CNS isolates and (ii) diversity between colonies with identical and different morphologies. Reproducibility was high, with 215 of 216 (99.5%) of the replicate samples for 72 isolates ending up in the same cluster. The concordance with pulsed-field gel electrophoresis (PFGE)-based clusters was 94.4%. We also confirm that the skin of patients can be colonized with multiple MR-CNS types at the same time. Morphological differences between colonies from a single patient sample correlated with differences in Raman and PFGE types. Some morphologically indistinguishable colonies revealed different Raman and PFGE types. This indicates that multiple MR-CNS colonies should be examined to obtain a complete insight into the prevalence of different types and to be able to perform an accurate transmission analysis. Here we show that Raman spectroscopy is a reproducible typing system for MR-CNS isolates. It is a tool for screening variability within a collection of isolates. Because of the high throughput, it enables the analysis of multiple colonies per patient, which will enhance the quality of clinical and epidemiological studies.

Rapid identification of mycobacteria by Raman spectroscopy.

A number of rapid identification methods have been developed to improve the accuracy for diagnosis of tuberculosis and to speed up the presumptive identification of Mycobacterium species. Most of these methods have been validated for a limited group of microorganisms only. Here, Raman spectroscopy was compared to 16S rRNA sequencing for the identification of Mycobacterium tuberculosis complex strains and the most frequently found strains of nontuberculous mycobacteria (NTM). A total of 63 strains, belonging to eight distinct species, were analyzed. The sensitivity of Raman spectroscopy for the identification of Mycobacterium species was 95.2%. All M. tuberculosis strains were correctly identified (7 of 7; 100%), as were 54 of 57 NTM strains (94%). The differentiation between M. tuberculosis and NTM was invariably correct for all strains. Moreover, the reproducibility of Raman spectroscopy was evaluated for killed mycobacteria (by heat and formalin) versus viable mycobacteria. The spectra of the heat-inactivated bacteria showed minimal differences compared to the spectra of viable mycobacteria. Therefore, the identification of mycobacteria appears possible without biosafety level 3 precautions. Raman spectroscopy provides a novel answer to the need for rapid species identification of cultured mycobacteria in a clinical diagnostic setting.

Rapid identification of Candida spp. in peritonitis patients by Raman spectroscopy.

This prospective study evaluated Raman spectroscopy for the identification of clinically relevant Candida spp. in peritonitis patients. A Raman database was developed by measuring spectra from 93 reference strains belonging to ten different Candida spp. Clinical samples were obtained from the surgical department and intensive care unit of a tertiary university hospital. In total, 88 peritoneal specimens from 45 patients with primary, secondary or tertiary peritonitis were included. Specimens were cultured initially on a selective Sabouraud medium that contained gentamicin to suppress bacterial growth. For conventional identification, a chromogenic medium was used for presumptive identification, followed by use of the Vitek 2 system for definitive identification (requiring a total time of 48-96 h). Raman measurements were taken on overnight cultures from Sabouraud-gentamicin medium. Thirty-one samples were positive for Candida by culture. Using multivariate statistical analyses, a prediction accuracy of 90% was obtained for Raman spectroscopy, which appears to offer an accurate and rapid (12-24 h) alternative for the identification of Candida spp. in peritonitis patients. The reduced turn-around time is of great clinical importance for the treatment of critically ill patients with invasive candidiasis in intensive care units.

Effect of culture conditions on the achievable taxonomic resolution of Raman spectroscopy disclosed by three Bacillus species.

Confocal micro-Raman spectroscopy requires a minimum of sample handling and no reagents and allows fast identification of microorganisms. Since it reflects the overall molecular composition of the cells, it provides much more information than classical, microbial analyses. However, since the molecular makeup of a cell depends on culture conditions, it can be argued that this will affect the reproducibility and discrimination ability of Raman spectroscopy. We used Bacillus cereus, Bacillus pumilus, and Bacillus licheniformis, which are known to be clearly distinct from each other and each displaying important phenotypic heterogeneity, in a wide variety of culture conditions to analyze this. It is illustrated that the influence of culture conditions on the identification accuracy and taxonomic resolution of Raman spectroscopy is important though the effect on the final identification is limited within the set of stains studied. Furthermore, some conditions even allow for better discrimination than others. From a practical point of view, it is especially important that differences in culturing time (and culturing temperature) can be accommodated.

Pro- and non-coagulant forms of non-cell-bound tissue factor in vivo.

BACKGROUND: Concentrations of non-cell-bound (NCB; soluble) tissue factor (TF) are elevated in blood collecting in the pericardial cavity of patients during cardiopulmonary bypass (CPB). Previously, we reported microparticles supporting thrombin generation in such blood samples. In this study we investigated the extent of microparticle association of the NCB form of TF in pericardial and systemic blood, and whether this microparticle-associated form is active in thrombin generation compared with non-microparticle-bound, (fluid-phase) TF. METHODS: Systemic and pericardial blood samples were collected before and during CPB from six patients undergoing cardiac surgery. Microparticles were isolated by differential centrifugation and their thrombin-generating capacity measured in a chromogenic assay. Microparticle-associated and fluid-phase forms of NCB TF were measured by ELISA. Microparticle-associated TF was visualized by flow cytometry. RESULTS: In pericardial samples, 45-77% of NCB TF was microparticle-associated, and triggered factor VII (FVII)-mediated thrombin generation in vitro. Microparticles from systemic samples triggered thrombin generation independently of FVII, except at the end of bypass (P = 0.003). The fluid-phase form of TF did not initiate thrombin generation. Both forms of NCB TF were, at least in part, antigenically cryptic. CONCLUSIONS: We demonstrate the occurrence of two forms of NCB TF. One form, which is microparticle-associated, supports thrombin generation via FVII. The other form, which is fluid-phase, does not stimulate thrombin formation. We hypothesize that the microparticle-associated form of NCB TF may be actively involved in postoperative thromboembolic processes when pericardial blood is returned into the patients.

Prospective study of the performance of vibrational spectroscopies for rapid identification of bacterial and fungal pathogens recovered from blood cultures.

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.

Human cell-derived microparticles promote thrombus formation in vivo in a tissue factor-dependent manner.

BACKGROUND: Circulating microparticles of various cell types are present in healthy individuals and, in varying numbers and antigenic composition, in various disease states. To what extent these microparticles contribute to coagulation in vivo is unknown. OBJECTIVES: To examine the in vivo thrombogenicity of human microparticles. METHODS: Microparticles were isolated from pericardial blood of cardiac surgery patients and venous blood of healthy individuals. Their numbers, cellular source, and tissue factor (TF) exposure were determined using flow cytometry. Their in vitro procoagulant properties were studied in a fibrin generation test, and their in vivo thrombogenicity in a rat model. RESULTS: The total number of microparticles did not differ between pericardial samples and samples from healthy individuals (P = 0.786). In both groups, microparticles from platelets, erythrocytes, and granulocytes exposed TF. Microparticle-exposed TF antigen levels were higher in pericardial compared with healthy individual samples (P = 0.036). Pericardial microparticles were strongly procoagulant in vitro and highly thrombogenic in a venous stasis thrombosis model in rats, whereas microparticles from healthy individuals were not [thrombus weights 24.8 (12.2-41.3) mg vs. 0 (0-24.3) mg median and range; P < 0.001]. Preincubation of pericardial microparticles with an inhibitory antibody against human TF abolished their thrombogenicity [0 (0-4.4) mg; P < 0.01], while a control antibody had no effect [19.6 (12.6-53.7) mg; P > 0.05]. The thrombogenicity of the microparticles correlated strongly with their TF exposure (r = 0.9524, P = 0.001). CONCLUSIONS: Human cell-derived microparticles promote thrombus formation in vivo in a TF-dependent manner. They might be the direct cause of an increased thromboembolic tendency in various patient groups.

Identification of medically relevant microorganisms by vibrational spectroscopy.

In the recent years, vibrational spectroscopies (infrared and Raman spectroscopy) have been developed for all sorts of analyses in microbiology. Important features of these methods are the relative ease with which measurements can be performed. Furthermore, in order to obtain infrared or Raman spectra, there is only a limited amount of sample handling involved without the need for expensive chemicals, labels or dyes. Here, we review the potential application of vibrational spectroscopies for the use in medical microbiology. After describing some of the basics of the techniques, considerations on reproducibility and standardisation are presented. Finally, the use of infrared and Raman spectroscopy for the (rapid) identification of medically relevant microorganisms is discussed. It can be concluded that vibrational spectroscopies show high potential as novel methods in medical microbiology.

Rapid identification of Candida species by confocal Raman microspectroscopy.

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.

Investigating microbial (micro)colony heterogeneity by vibrational spectroscopy.

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.

Classification and identification of enterococci: a comparative phenotypic, genotypic, and vibrational spectroscopic study.

Rapid and accurate identification of enterococci at the species level is an essential task in clinical microbiology since these organisms have emerged as one of the leading causes of nosocomial infections worldwide. Vibrational spectroscopic techniques (infrared [IR] and Raman) could provide potential alternatives to conventional typing methods, because they are fast, easy to perform, and economical. We present a comparative study using phenotypic, genotypic, and vibrational spectroscopic techniques for typing a collection of 18 Enterococcus strains comprising six different species. Classification of the bacteria by Fourier transform (FT)-IR spectroscopy in combination with hierarchical cluster analysis revealed discrepancies for certain strains when compared with results obtained from automated phenotypic systems, such as API and MicroScan. Further diagnostic evaluation using genotypic methods-i.e., PCR of the species-specific ligase and glycopeptide resistance genes, which is limited to the identification of only four Enterococcus species and 16S RNA sequencing, the "gold standard" for identification of enterococci-confirmed the results obtained by the FT-IR classification. These results were later reproduced by three different laboratories, using confocal Raman microspectroscopy, FT-IR attenuated total reflectance spectroscopy, and FT-IR microspectroscopy, demonstrating the discriminative capacity and the reproducibility of the technique. It is concluded that vibrational spectroscopic techniques have great potential as routine methods in clinical microbiology.

Aprotinin administration in the pericardial cavity does not prevent platelet activation.

OBJECTIVES: Aprotinin is frequently administered systemically to patients undergoing cardiopulmonary bypass to inhibit activation of platelets and plasma protein systems and thus reduce postoperative blood loss. Two reports on local aprotinin administration, that is, into the pericardial cavity, also indicated improvement in postoperative blood loss, but the underlying mechanism was not investigated. We previously reported the disappearance of glycoprotein Ib from the platelet surface and the appearance of platelet-derived microparticles in the pericardial cavity of patients undergoing cardiopulmonary bypass as signs of platelet activation. Here, we investigated whether such local aprotinin administration reduced platelet activation. METHODS: In a double-blind study, 6 patients received aprotinin (500,000 KIU) into the pericardial cavity during the operation and 7 patients received a placebo. Platelet surface glycoprotein Ib expression, concentration of microparticles, and concentration of complexes of platelets with leukocytes, erythrocytes, or each other, were measured by flow cytometry. RESULTS: We confirmed the reduced glycoprotein Ib expression and the increased concentration of microparticles in the pericardial cavity, as previously reported, and found no increased concentration of platelet complexes. However, no differences between aprotinin and placebo treatments were observed in these platelet activation parameters in the pericardial cavity or the systemic circulation. CONCLUSION: We conclude that administration of aprotinin into the pericardial cavity during cardiopulmonary bypass and at concentrations similar to the systemic application does not reduce platelet activation in that compartment or the systemic circulation.

Raman spectroscopic method for identification of clinically relevant microorganisms growing on solid culture medium.

Routine clinical microbiological identification of pathogenic microorganisms is largely based on nutritional and biochemical tests. In the case of severely ill patients, the unavoidable time delay associated with such identification procedures can be fatal. We present a novel identification method based on confocal Raman microspectroscopy. With this approach it is possible to obtain Raman spectra directly from microbial microcolonies on the solid culture medium, which have developed after only 6 h of culturing for the most commonly encountered organisms. Due to the limited thickness of microcolonies, some of the underlying culture medium is sampled together with the bacteria. Spectra measured at different depths in a microcolony contain different amounts of the medium signal. A mathematical routine, involving vector algebra, is described for the nonsubjective correction of spectra for variable signal contributions of the medium. To illustrate the possibilities of our approach for the identification of microorganisms, Raman spectra were collected from 6-h microcolonies of five bacterial strains on solid culture medium. The classification results show that confocal Raman microspectroscopy has great potential as a powerful new tool in clinical diagnostic microbiology.

Disappearance of glycoprotein Ib from the platelet surface in pericardial blood during cardiopulmonary bypass.

OBJECTIVES: Several investigators have reported decreased expression of glycoprotein Ib on the platelet surface during coronary artery bypass grafting, but others could not confirm this finding. Because platelet glycoprotein Ib functions as an adhesion receptor for von Willebrand factor and other adhesive proteins, this decreased expression may explain excessive postoperative blood loss. In this study the expressions of glycoprotein Ib and other platelet activation markers were studied in the systemic and pericardial blood of seven patients undergoing coronary artery bypass grafting. Pericardial blood was recently shown to have high activation levels of fibrinolytic and coagulation pathways; we hypothesized that this local blood activation might be paralleled by extensive platelet activation and associated disappearance of glycoprotein Ib. METHODS: Expression of platelet surface antigens was determined by whole-blood double-label flow cytometry. RESULTS: Glycoprotein Ib expression in systemic blood decreased 10% (p = 0.03) from preoperative levels at the start of cardiopulmonary bypass and 30% (p = 0.04) before release of the aortic crossclamp. Expression in pericardial blood at these times decreased by 50% and 51%, respectively (p = 0.003, p = 0.009). No changes were observed in the expression of the platelet activation antigens CD62P (P-selectin, indicating platelet alpha-granular release) and CD63 (indicating lysosomal release) or in binding of monoclonal antibody PAC-1 (detecting the fibrinogen-binding receptor conformation of the glycoprotein IIb-IIIa complex). CONCLUSION: Glycoprotein Ib disappeared from the platelet surface during bypass grafting, most notably in pericardial blood. No increased expression of CD62P, CD63, or PAC-1 was found, indicating the absence of general platelet activation.

Cell-derived microparticles generated in patients during cardiopulmonary bypass are highly procoagulant.

BACKGROUND: Microparticles from platelets and other cells have been extensively studied and characterized in vitro. Although the level of platelet-derived microparticles is elevated in a variety of diseases, including cardiac surgery, virtually nothing is known about their functions in vivo. The aim of the present study was to investigate the procoagulant properties of microparticles generated in vivo. METHODS AND RESULTS: In 6 patients at the end of cardiopulmonary bypass, 14.8 x 10(9)/L (median; range, 9.7 to 27.4 x 10(9)/L) platelet-derived microparticles were present in pericardial blood, whereas blood obtained from the systemic circulation contained 1.6 x 10(9)/L (median; range, 0.4 to 8.9 x 10(9)/L) of such microparticles, as determined by flow cytometry. Microparticles stained positively for phosphatidylserine as determined with labeled annexin V. In contrast to systemic blood, pericardial blood contained not only microparticles of platelet origin but also microparticles that originated from erythrocytes, monocytes, or granulocytes, and other hitherto unknown cellular sources. Plasma prepared from pericardial blood and to a lesser extent plasma from systemic blood obtained at the same time, stimulated formation of thrombin in vitro. This activity of pericardial plasma was lost after removal of its microparticles by high-speed centrifugation, whereas the corresponding microparticle pellet was strongly procoagulant. The generation of thrombin in vitro involved a tissue factor/factor VII-dependent and factor XII-independent pathway. CONCLUSIONS: This study is the first to demonstrate that microparticles generated in vivo can stimulate coagulation.

Discrimination of Acinetobacter genomic species by AFLP fingerprinting.

AFLP is a novel genomic fingerprinting method based on the selective PCR amplification of restriction fragments. The usability of this method for the differentiation of genomic species in the genus Acinetobacter was investigated. A total of 151 classified strains (representing 18 genomic species, including type, reference, and field strains) and 8 unclassified strains were analyzed. By using a single set of restriction enzymes (HindIII and TaqI) and one particular set of selective PCR primers, all strains could be allocated to the correct genomic species and all groups were properly separated, with minimal intraspecific similarity levels ranging from 29 to 74%. Strains belonging to genomic species 8 (Acinetobacter lwoffii sensu stricto) and 9 grouped together in one cluster. The closely related DNA groups 1 (Acinetobacter calcoaceticus), 2 (Acinetobacter baumannii), 3 and 13TU (sensu Tjernberg & Ursing 1989) were clearly distinguishable, with intraspecific linkage levels above 50%. Strains of the independently described genomic species 13BJ (sensu Bouvet & Jeanjean 1989) and 14TU linked together at a relatively low level (33%). Although a previous DNA-DNA hybridization study seemed to justify the unification of these genomic species, AFLP analysis actually divides the 13BJ-14TU group into three well-separated subgroups. Finally, four unclassified strains obtained from diverse sources and origins grouped convincingly together, with a similarity linkage level of approximately 50%. These strains showed no similarities in their AFLP patterns with any of the other 155 strains studied and may represent a thus-far-undescribed Acinetobacter species. Based on these results, AFLP should be regarded as an important auxiliary method for the delineation of genomic species. Furthermore, because AFLP provides a detailed insight into the infraspecific structure of Acinetobacter taxa, the method also represents a highly effective means for the confirmation of strain identity in the epidemiology of acinetobacters.

Comparison of arbitrarily primed polymerase chain reaction and cell envelope protein electrophoresis for analysis of Acinetobacter baumannii and A. junii outbreaks.

Two successive Acinetobacter outbreaks in a neonatal intensive care unit were studied with arbitrarily primed polymerase chain reaction (AP-PCR), cell envelope protein electrophoresis (protein fingerprinting) and antibiotic susceptibility testing. AP-PCR fingerprinting and protein fingerprinting yielded identical clustering of the isolates studied. Susceptibility test results were useful for rapid recognition of the outbreaks, but clustering of several isolates was different from the clustering obtained with AP-PCR fingerprinting and protein fingerprinting. Typing results indicated that the two outbreaks, which occurred at a three-month interval, were each caused by a single strain, and that both strains differed from the strains prevailing in the hospital. The strain of one outbreak was identified as A. junii, a species commonly not involved in outbreaks. A. baumannii isolates collected from different departments of this hospital during a period of four years clustered into only five different types. Moreover, strains from different departments of a second hospital belonged to the type prevailing in the first hospital, although there were no apparent connections between the two institutions. This may indicate that only a limited number of strains of the A. calcoaceticus-baumannii complex are involved in nosocomial outbreaks.