A TdT-driven amplification loop increases CRISPR-Cas12a DNA detection levels.

Recent findings on CRISPR-Cas enzymes with collateral DNAse/RNAse activity have led to new and innovative methods for pathogen detection. However, many CRISPR-Cas assays necessitate DNA pre-amplification to boost sensitivity, restricting their utility for point-of-care applications. Achieving higher sensitivity without DNA pre-amplification presents a significant challenge. In this study, we introduce a Terminal deoxynucleotidyl Transferase (TdT)-based amplification loop, creating a positive feedback mechanism within the CRISPR-Cas12a pathogen detection system. Upon recognizing pathogenic target DNA, Cas12a triggers trans-cleavage of a FRET reporter and a specific enhancer molecule oligonucleotide, indicated by the acronym POISER (Partial Or Incomplete Sites for crRNA recognition). POISER comprises half of a CRISPR-RNA recognition site, which is subsequently elongated by TdT enzymatic activity. This process, involving pathogen recognition-induced Cas12a cleavage and TdT elongation, results in a novel single-stranded DNA target. This target can subsequently be recognized by a POISER-specific crRNA, activating more Cas12a enzymes. Our study demonstrates that these POISER-cycles enhance the signal strength in fluorescent-based CRISPR-Cas12a assays. Although further refinement is desirable, POISER holds promise as a valuable tool for the detection of pathogens in point-of-care testing, surveillance, and environmental monitoring.

Expression of virulence and antimicrobial related proteins in Burkholderia mallei and Burkholderia pseudomallei.

BACKGROUND: Burkholderia mallei and Burkholderia pseudomallei are both potential biological threat agents. Melioidosis caused by B. pseudomallei is endemic in Southeast Asia and Northern Australia, while glanders caused by B. mallei infections are rare. Here we studied the proteomes of different B. mallei and B. pseudomallei isolates to determine species specific characteristics. METHODS: The expressed proteins of 5 B. mallei and 6 B. pseudomallei strains were characterized using liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). Subsequently, expression of potential resistance and virulence related characteristics were analyzed and compared. RESULTS: Proteome analysis can be used for the identification of B. mallei and B. pseudomallei. Both species were identified based on >60 discriminative peptides. Expression of proteins potentially involved in antimicrobial resistance, AmrAB-OprA, BpeAB-OprB, BpeEF-OprC, PenA as well as several other efflux pump related proteins and putative ß-lactamases was demonstrated. Despite, the fact that efflux pump BpeAB-OprB was expressed in all isolates, no clear correlation with an antimicrobial phenotype and the efflux-pump could be established. Also consistent with the phenotypes, no amino acid mutations in PenA known to result in ß-lactam resistance could be identified. In all studied isolates, the expression of virulence (related) factors Capsule-1 and T2SS was demonstrated. The expression of T6SS-1 was demonstrated in all 6 B. pseudomallei isolates and in 2 of the 5 B. mallei isolates. In all, except one B. pseudomallei isolate, poly-beta-1,6 N-acetyl-D-glucosamine export porin (Pga), important for biofilm formation, was detected, which were absent in the proteomes of B. mallei. Siderophores, iron binding proteins, malleobactin and malleilactone are possibly expressed in both species under standard laboratory growth conditions. Expression of multiple proteins from both the malleobactin and malleilactone polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) clusters was demonstrated in both species. All B. pseudomallei expressed at least seven of the nine proteins of the bactobolin synthase cluster (bactobolin, is a ribosome targeting antibiotic), while only in one B. mallei isolate expression of two proteins of this synthase cluster was identified. CONCLUSIONS: Analyzing the expressed proteomes revealed differences between B. mallei and B. pseudomallei but also between isolates from the same species. Proteome analysis can be used not only to identify B. mallei and B. pseudomallei but also to characterize the presence of important factors that putatively contribute to the pathogenesis of B. mallei and B. pseudomallei.

Combining CRISPR-Cas12a with terminal deoxynucleotidyl transferase dependent reporter elongation for pathogen detection using lateral flow test strips.

CRISPR-Cas (CC)-based detection technologies have some exceptional features, which hold the promise of developing into the next-generation diagnostic platforms. One of these features is the ability to trigger non-specific single-stranded DNA/RNA cleavage activity after specific target recognition and Cas enzyme activation. This cleavage activity can be visualized either by single-stranded DNA/RNA fluorescence resonance energy transfer quenching reporters or via lateral flow strips, which separate and detect the cleaved reporters. In a previous study, we reported coupling CC-cleavage activity with the enzyme terminal deoxynucleotidyl transferase (TdT) that elongates cleaved ssDNA reporter fragments with dTTP nucleotides. These elongated poly(thymine) tails then act as scaffolds for the formation of copper nanoparticles which generate a bright fluorescent signal upon UV excitation. In the current study, we visualize the poly(thymine) tails on lateral flow strips, using different combinations of biotinylated or fluorescein-labeled nucleotides, various reporters, and capture oligos. One particular approach, using a fluorescein reporter, reached a target sensitivity of <1 pM and was named Cas activity assay on a strip and was tested using Bacillus anthracis genomic DNA.

Bright fluorescent nucleic acid detection with CRISPR-Cas12a and poly(thymine) templated copper nanoparticles.

Fluorescence-based diagnostic tools are attractive and versatile tests with multiple advantages: ease of use, sensitivity and rapid results. The advent of CRISPR-Cas technology has created new avenues for the development of diagnostic testing tools. In this study, by effectively combining the specific functions of two enzymes, CRISPR-Cas12a and terminal deoxynucleotidyl transferase (TdT), we developed a DNA detection assay that generates copper nanoparticles (CuNPs) that are easily visible to the naked eye under UV-light; we named this detection assay Cas12a Activated Nuclease poly-T Reporter Illuminating Particles (CANTRIP). Upon specific target DNA recognition by Cas12a, single-stranded DNA (ssDNA) reporter oligos with blocked 3'-ends are cut into smaller ssDNA fragments, thereby generating neo 3'-hydroxyl moieties. TdT subsequently elongates these newly formed ssDNA fragments, incorporating only dTTP nucleotides, and these poly(thymine)-tails subsequently function as scaffolds for the formation of CuNPs. These CuNPs produce a bright fluorescent signal upon UV excitation, and thus, this bright orange signal indicates the presence of target DNA, which in this proof-of-concept study consisted of anthrax lethal factor plasmid DNA. CANTRIP, which combines two detection platforms consisting of CRISPR-Cas12a and fluorescent CuNPs into a single reaction, appears to be a robust, low-cost and simple diagnostic tool.

Untargeted accurate identification of highly pathogenic bacteria directly from blood culture flasks.

To improve the preparedness against exposure to highly pathogenic bacteria and to anticipate the wide variety of bacteria that can cause bloodstream infections (BSIs), a safe, unbiased and highly accurate identification method was developed. Our liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method can identify highly pathogenic bacteria, their near-neighbors and bacteria that are common causes of BSIs directly from positive blood culture flasks. The developed Peptide-Based Microbe Detection Engine (http://proteome2pathogen.com) relies on a two-step workflow: a genus-level search followed by a species-level search. This strategy enables the rapid identification of microorganisms based on the analyzed proteome. This method was successfully used to identify strains of Bacillus anthracis, Brucella abortus, Brucella melitensis, Brucella suis, Burkholderia pseudomallei, Burkholderia mallei, Francisella tularensis, Yersinia pestis and closely related species from simulated blood culture flasks. This newly developed LC-MS/MS method is a safe and rapid method for accurately identifying bacteria directly from positive blood culture flasks.

Identification of microorganisms grown in blood culture flasks using liquid chromatography-tandem mass spectrometry.

AIM: Bloodstream infections are a common cause of disease and a fast and accurate identification of the causative agent or agents of bloodstream infections would aid the start of adequate treatment. MATERIALS & METHODS: A liquid chromatography-tandem mass spectrometry (LC-MS/MS) shotgun proteomics method was developed for the identification of bacterial species directly from blood cultures that were simulated by inoculating blood culture bottles with single or multiple clinically relevant microorganisms. RESULTS: Using LC-MS/MS, the single species were correctly identified in 100% of the blood cultures, whereas for polymicrobial infections, 78% of both species were correctly identified in blood cultures. CONCLUSION: The LC-MS/MS method allows for the identification of the causative agent of positive blood cultures.

A Study of the Variation in the Salivary Peptide Profiles of Young Healthy Adults Acquired Using MALDI-TOF MS.

A cross-sectional observational study was conducted to evaluate the inter-individual variation in the MALDI-TOF MS peptide profiles of unstimulated whole saliva in a population of 268 systemically healthy adults aged 18-30 yr (150 males and 118 females) with no apparent caries lesions or periodontal disease. Using Spectral Clustering, four subgroups of individuals were identified within the study population. These subgroups were delimited by the pattern of variation in 9 peaks detected in the 2-15 kDa m/z range. An Unsupervised Feature Selection algorithm showed that P-C peptide, a 44 residue-long salivary acidic proline-rich protein, and three of its fragments (Fr. 1-25, Fr. 15-35 and Fr. 15-44) play a central role in delimiting the subgroups. Significant differences were found in the salivary biochemistry of the subgroups with regard to lysozyme and chitinase, two enzymes that are part of the salivary innate defense system (p < 0.001). These results suggest that MALDI-TOF MS salivary peptide profiles may relate information on the underlying state of the oral ecosystem and may provide a useful reference for salivary disease biomarker discovery studies.

Rapid and reliable discrimination between Shigella species and Escherichia coli using MALDI-TOF mass spectrometry.

E. coli-Shigella species are a cryptic group of bacteria in which the Shigella species are distributed within the phylogenetic tree of E. coli. The nomenclature is historically based and the discrimination of these genera developed as a result of the epidemiological need to identify the cause of shigellosis, a severe disease caused by Shigella species. For these reasons, this incorrect classification of shigellae persists to date, and the ability to rapidly characterize E. coli and Shigella species remains highly desirable. Until recently, existing matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) assays used to identify bacteria could not discriminate between E. coli and Shigella species. Here we present a rapid classification method for the E. coli-Shigella phylogroup based on MALDI-TOF MS which is supported by genetic analysis. E. coli and Shigella isolates were collected and genetically characterized by MLVA. A custom reference library for MALDI-TOF MS that represents the genetic diversity of E. coli and Shigella strains was developed. Characterization of E. coli and Shigella species is based on an approach with Biotyper software. Using this reference library it was possible to distinguish between Shigella species and E. coli. Of the 180 isolates tested, 94.4% were correctly classified as E. coli or shigellae. The results of four (2.2%) isolates could not be interpreted and six (3.3%) isolates were classified incorrectly. The custom library extends the existing MALDI-TOF MS method for species determination by enabling rapid and accurate discrimination between Shigella species and E. coli.

Simultaneous Identification of Multiple ß-Lactamases in Acinetobacter baumannii in Relation to Carbapenem and Ceftazidime Resistance, Using Liquid Chromatography-Tandem Mass Spectrometry.

Shotgun proteomics using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was applied to detect ß-lactamases in clinical Acinetobacter baumannii isolates. The correlation of the detection of ß-lactamase proteins (rather than PCR detection of the corresponding genes) with the resistance phenotypes demonstrated an added value for LC-MS/MS in antimicrobial susceptibility testing.

Rapid and generic identification of influenza A and other respiratory viruses with mass spectrometry.

The rapid identification of existing and emerging respiratory viruses is crucial in combating outbreaks and epidemics. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a rapid and reliable identification method in bacterial diagnostics, but has not been used in virological diagnostics. Mass spectrometry systems have been investigated for the identification of respiratory viruses. However, sample preparation methods were laborious and time-consuming. In this study, a reliable and rapid sample preparation method was developed allowing identification of cultured respiratory viruses. Tenfold serial dilutions of ten cultures influenza A strains, mixed samples of influenza A virus with human metapneumovirus or respiratory syncytial virus, and reconstituted clinical samples were treated with the developed sample preparation method. Subsequently, peptides were subjected to MALDI-TOF MS and liquid chromatography tandem mass spectrometry (LC-MS/MS). The influenza A strains were identified to the subtype level within 3h with MALDI-TOF MS and 6h with LC-MS/MS, excluding the culturing time. The sensitivity of LC-MS/MS was higher compared to MALDI-TOF MS. In addition, LC-MS/MS was able to discriminate between two viruses in mixed samples and was able to identify virus from reconstituted clinical samples. The development of an improved and rapid sample preparation method allowed generic and rapid identification of cultured respiratory viruses by mass spectrometry.

OmpU as a biomarker for rapid discrimination between toxigenic and epidemic Vibrio cholerae O1/O139 and non-epidemic Vibrio cholerae in a modified MALDI-TOF MS assay.

BACKGROUND: Cholera is an acute diarrheal disease caused by Vibrio cholerae. Outbreaks are caused by a genetically homogenous group of strains from serogroup O1 or O139 that are able to produce the cholera toxin. Rapid detection and identification of these epidemic strains is essential for an effective response to cholera outbreaks. RESULTS: The use of ferulic acid as a matrix in a new MALDI-TOF MS assay increased the measurable mass range of existing MALDI-TOF MS protocols for bacterial identification. The assay enabled rapid discrimination between epidemic V. cholerae O1/O139 strains and other less pathogenic V. cholerae strains. OmpU, an outer membrane protein whose amino acid sequence is highly conserved among epidemic strains of V. cholerae, appeared as a discriminatory marker in the novel MALDI-TOF MS assay. CONCLUSIONS: The extended mass range of MALDI-TOF MS measurements obtained by using ferulic acid improved the screening for biomarkers in complex protein mixtures. Differences in the mass of abundant homologous proteins due to variation in amino acid sequences can rapidly be examined in multiple samples. Here, a rapid MALDI-TOF MS assay was developed that could discriminate between epidemic O1/O139 strains and other less pathogenic V. cholerae strains based on differences in mass of the OmpU protein. It appeared that the amino acid sequence of OmpU from epidemic V. cholerae O1/O139 strains is unique and highly conserved.

Indigenous Infection with Francisella tularensis holarctica in The Netherlands.

We report here the first case of indigenous tularemia detected in The Netherlands, a nonendemic country, since 1953. Whole genome DNA sequence analysis assigned the isolate BD11-00177 to the genomic group B.FTNF002-00, which previously has been exclusively reported from Spain, France, Italy, Switzerland, and Germany. The patient had not been abroad for years, which implies that this is an indigenous infection. The current case might predict an upcoming distribution of Francisella tularensis holarctica genomic group B.FTNF002-00 in Europe.

Rapid identification of Bacillus anthracis spores in suspicious powder samples by using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).

Rapid and reliable identification of Bacillus anthracis spores in suspicious powders is important to mitigate the safety risks and economic burdens associated with such incidents. The aim of this study was to develop and validate a rapid and reliable laboratory-based matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis method for identifying B. anthracis spores in suspicious powder samples. A reference library containing 22 different Bacillus sp. strains or hoax materials was constructed and coupled with a novel classification algorithm and standardized processing protocol for various powder samples. The method's limit of B. anthracis detection was determined to be 2.5 × 10(6) spores, equivalent to a 55-µg sample size of the crudest B. anthracis-containing powder discovered during the 2001 Amerithrax incidents. The end-to-end analysis method was able to successfully discriminate among samples containing B. anthracis spores, closely related Bacillus sp. spores, and commonly encountered hoax materials. No false-positive or -negative classifications of B. anthracis spores were observed, even when the analysis method was challenged with a wide range of other bacterial agents. The robustness of the method was demonstrated by analyzing samples (i) at an external facility using a different MALDI-TOF MS instrument, (ii) using an untrained operator, and (iii) using mixtures of Bacillus sp. spores and hoax materials. Taken together, the observed performance of the analysis method developed demonstrates its potential applicability as a rapid, specific, sensitive, robust, and cost-effective laboratory-based analysis tool for resolving incidents involving suspicious powders in less than 30 min.

Draft genome sequence of Francisella tularensis subsp. holarctica BD11-00177.

Francisella tularensis is a facultative intracellular bacterium in the class Gammaproteobacteria. This strain is of interest because it is the etiologic agent of tularemia and a highly virulent category A biothreat agent. Here we describe the draft genome sequence and annotation of Francisella tularensis subsp. holarctica BD11-00177, isolated from the first case of indigenous tularemia detected in The Netherlands since 1953. Whole genome DNA sequence analysis assigned this isolate to the genomic group B.FTNF002-00, which previously has been exclusively reported from Spain, France, Italy, Switzerland and Germany. Automatic annotation of the 1,813,372 bp draft genome revealed 2,103 protein-coding and 46 RNA genes.

Reduced expression of PBP-2A by neonatal mecA-positive coagulase-negative staphylococci (CoNS) blood isolates: ß-lactams are useful first-line agents for the treatment of neonatal CoNS sepsis, restricting the use of vancomycin.

OBJECTIVES: Vancomycin use for neonatal coagulase-negative staphylococci (CoNS) sepsis is based on a high CoNS carriage rate of mecA, encoding penicillin-binding protein (PBP)-2a, with low affinity for, and associated with resistance to, ß-lactam antibiotics. The relationship between mecA gene carriage, phenotypic expression of the gene by PBP-2a production and in vitro resistance to the ß-lactam antibiotics oxacillin, cefazolin and amoxicillin/clavulanate was determined for 85 CoNS blood isolates randomly obtained from our collection of isolates from neonates with CoNS sepsis. METHODS: The relationship between mecA gene carriage, phenotypic expression of the gene by PBP-2a production and in vitro resistance to the ß-lactam antibiotics oxacillin, cefazolin and amoxicillin/clavulanate was determined for randomly obtained CoNS blood isolates from our collection of isolates from neonates with CoNS sepsis. The mecA gene was detected using multiplex PCR, and PBP-2a expression was determined using a latex agglutination (LA) test (Oxoid). ß-Lactam susceptibility was determined using the Phoenix automated system and, in addition, by Etest with interpretation of MIC values according to the reference MIC breakpoints adopted from the CLSI guidelines M100-S20, Infobase™. RESULTS: Among 85 CoNS blood isolates, 73 (86%) were mecA positive and 12 (14%) were mecA negative. None of the mecA-negative isolates expressed PBP-2a and all were ß-lactam susceptible. All mecA-positive CoNS isolates were oxacillin resistant, although most oxacillin MICs were not very high, ranging from 2 to 8 mg/L for the majority of isolates. Only 8/73 (11%) mecA-positive CoNS isolates had oxacillin MICs ≥32 mg/L (range 32 to >256 mg/L). mecA-positive CoNS blood isolates, although fully resistant to oxacillin, were almost universally susceptible to cefazolin and amoxicillin/clavulanate, which was associated with a low expression rate of PBP-2a. CONCLUSIONS: ß-Lactam antibiotics are useful for the treatment of neonatal CoNS sepsis, reserving vancomycin for selected cases.

Reliable identification at the species level of Brucella isolates with MALDI-TOF-MS.

BACKGROUND: The genus Brucella contains highly infectious species that are classified as biological threat agents. The timely detection and identification of the microorganism involved is essential for an effective response not only to biological warfare attacks but also to natural outbreaks. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is a rapid method for the analysis of biological samples. The advantages of this method, compared to conventional techniques, are rapidity, cost-effectiveness, accuracy and suitability for the high-throughput identification of bacteria. Discrepancies between taxonomy and genetic relatedness on the species and biovar level complicate the development of detection and identification assays. RESULTS: In this study, the accurate identification of Brucella species using MALDI-TOF-MS was achieved by constructing a Brucella reference library based on multilocus variable-number tandem repeat analysis (MLVA) data. By comparing MS-spectra from Brucella species against a custom-made MALDI-TOF-MS reference library, MALDI-TOF-MS could be used as a rapid identification method for Brucella species. In this way, 99.3% of the 152 isolates tested were identified at the species level, and B. suis biovar 1 and 2 were identified at the level of their biovar. This result demonstrates that for Brucella, even minimal genomic differences between these serovars translate to specific proteomic differences. CONCLUSIONS: MALDI-TOF-MS can be developed into a fast and reliable identification method for genetically highly related species when potential taxonomic and genetic inconsistencies are taken into consideration during the generation of the reference library.

Evolution in quantum leaps: multiple combinatorial transfers of HPI and other genetic modules in Enterobacteriaceae.

Horizontal gene transfer is a key step in the evolution of Enterobacteriaceae. By acquiring virulence determinants of foreign origin, commensals can evolve into pathogens. In Enterobacteriaceae, horizontal transfer of these virulence determinants is largely dependent on transfer by plasmids, phages, genomic islands (GIs) and genomic modules (GMs). The High Pathogenicity Island (HPI) is a GI encoding virulence genes that can be transferred between different Enterobacteriaceae. We investigated the HPI because it was present in an Enterobacter hormaechei outbreak strain (EHOS). Genome sequence analysis showed that the EHOS contained an integration site for mobile elements and harbored two GIs and three putative GMs, including a new variant of the HPI (HPI-ICEEh1). We demonstrate, for the first time, that combinatorial transfers of GIs and GMs between Enterobacter cloacae complex isolates must have occurred. Furthermore, the excision and circularization of several combinations of the GIs and GMs was demonstrated. Because of its flexibility, the multiple integration site of mobile DNA can be considered an integration hotspot (IHS) that increases the genomic plasticity of the bacterium. Multiple combinatorial transfers of diverse combinations of the HPI and other genomic elements among Enterobacteriaceae may accelerate the generation of new pathogenic strains.

Yersiniabactin reduces the respiratory oxidative stress response of innate immune cells.

Enterobacteriaceae that contain the High Pathogenicity Island (HPI), which encodes the siderophore yersiniabactin, display increased virulence. This increased virulence may be explained by the increased iron scavenging of the bacteria, which would both enhance bacterial growth and limit the availability of iron to cells of the innate immune system, which require iron to catalyze the Haber-Weiss reaction that produces hydroxyl radicals. In this study, we show that yersiniabactin increases bacterial growth when iron-saturated lactoferrin is the main iron source. This suggests that yersiniabactin provides bacteria with additional iron from saturated lactoferrin during infection. Furthermore, the production of ROS by polymorphonuclear leukocytes, monocytes, and a mouse macrophage cell line is blocked by yersiniabactin, as yersiniabactin reduces iron availability to the cells. Importantly, iron functions as a catalyst during the Haber-Weiss reaction, which generates hydroxyl radicals. While the physiologic role of the Haber-Weiss reaction in the production of hydroxyl radicals has been controversial, the siderophores yersiniabactin, aerobactin, and deferoxamine and the iron-chelator deferiprone also reduce ROS production in activated innate immune cells. This suggests that this reaction takes place under physiological conditions. Of the tested iron chelators, yersiniabactin was the most effective in reducing the ROS production in the tested innate immune cells. The likely decreased bacterial killing by innate immune cells resulting from the reduced production of hydroxyl radicals may explain why the HPI-containing Enterobacteriaceae are more virulent. This model centered on the reduced killing capacity of innate immune cells, which is indirectly caused by yersiniabactin, is in agreement with the observation that the highly pathogenic group of Yersinia is more lethal than the weakly pathogenic and the non-pathogenic group.

Identification of resistance and virulence factors in an epidemic Enterobacter hormaechei outbreak strain.

Bacterial strains differ in their ability to cause hospital outbreaks. Using comparative genomic hybridization, Enterobacter cloacae complex isolates were studied to identify genetic markers specific for Enterobacter cloacae complex outbreak strains. No outbreak-specific genes were found that were common in all investigated outbreak strains. Therefore, the aim of our study was to identify specific genetic markers for an Enterobacter hormaechei outbreak strain (EHOS) that caused a nationwide outbreak in The Netherlands. Most EHOS isolates carried a large conjugative plasmid (pQC) containing genes encoding heavy-metal resistance, mobile elements, pili-associated proteins and exported proteins as well as multiple-resistance genes. Furthermore, the chromosomally encoded high-pathogenicity island (HPI) was highly associated with the EHOS strain. In addition, other DNA fragments were identified that were associated with virulence: three DNA fragments known to be located on a virulence plasmid (pLVPK), as well as phage- and plasmid-related sequences. Also, four DNA fragments encoding putative pili with the most homology to pili of Salmonella enterica were associated with the EHOS. Finally, four DNA fragments encoding putative outer-membrane proteins were negatively associated with the EHOS. In conclusion, resistance and putative virulence genes were identified in the EHOS that may have contributed to increased epidemicity. The high number of genes detected in the EHOS that were related to transferable elements reflects the genomic plasticity of the E. cloacae complex and may explain the emergence of the EHOS in the hospital environment.

Genomic diversity within the Enterobacter cloacae complex.

BACKGROUND: Isolates of the Enterobacter cloacae complex have been increasingly isolated as nosocomial pathogens, but phenotypic identification of the E. cloacae complex is unreliable and irreproducible. Identification of species based on currently available genotyping tools is already superior to phenotypic identification, but the taxonomy of isolates belonging to this complex is cumbersome. METHODOLOGY/PRINCIPAL FINDINGS: This study shows that multilocus sequence analysis and comparative genomic hybridization based on a mixed genome array is a powerful method for studying species assignment within the E. cloacae complex. The E. cloacae complex is shown to be evolutionarily divided into two clades that are genetically distinct from each other. The younger first clade is genetically more homogenous, contains the Enterobacter hormaechei species and is the most frequently cultured Enterobacter species in hospitals. The second and older clade consists of several (sub)species that are genetically more heterogeneous. Genetic markers were identified that could discriminate between the two clades and cluster 1. CONCLUSIONS/SIGNIFICANCE: Based on genomic differences it is concluded that some previously defined (clonal and heterogenic) (sub)species of the E. cloacae complex have to be redefined because of disagreements with known or proposed nomenclature. However, further improved identification of the redefined species will be possible based on novel markers presented here.