Use of Fourier-transform infrared spectroscopy for real-time outbreak investigation of OXA-48-producing Escherichia coli.

BACKGROUND: Efficient infection control during carbapenem-resistant Enterobacterales outbreaks demands rapid and simple techniques for outbreak investigations. WGS, the current gold standard for outbreak identification, is expensive, time-consuming and requires a high level of expertise. Fourier-transform infrared (FTIR) spectroscopy (IR Biotyper) is a rapid typing method based on infrared radiation applied to samples, which provides a highly specific absorption spectrum. OBJECTIVES: To investigate an outbreak of OXA-48-producing Escherichia coli in real-time using FTIR and subsequently compare the results with WGS. METHODS: Twenty-one isolates were collected during a nosocomial outbreak, and identification and antibiotic susceptibilities were confirmed by VITEK®2. FTIR was conducted for all isolates, and nine representative isolates were sequenced. RESULTS: FTIR was able to correctly determine the clonal relatedness of the isolates and to identify the outbreak cluster, as confirmed by WGS. By WGS, isolates in the main FTIR cluster belonged to the same MLST type and core-genome MLST type, and they harboured similar plasmids and resistance genes, whereas the singletons external to the FTIR cluster had different genetic content. CONCLUSIONS: FTIR can operate as a rapid, efficient and reliable first-line tool for outbreak investigations during a real-time ongoing E. coli outbreak, which can contribute to limiting the spread of pathogens.

Identification and control of two outbreaks of unrelated New Delhi metallo-ß-lactamase-producing carbapenem-resistant Escherichia coli traced to the same endoscope defect.

We report 2 outbreaks of genetically unrelated carbapenem-resistant New Delhi metallo-ß-lactamase-producing Escherichia coli caused by contaminated duodenoscopes. Using endoscopes with disposable end caps, adherence to the manufacturer's reprocessing instructions, routine audits, and manufacturer evaluation are critical in preventing such outbreaks.

Phenotypic and Genomic Characterization of Nine String-Positive Carbapenem-Resistant Acinetobacter baumannii Isolates from Israel.

A positive "string test" indicates the ability of bacterial colonies grown on agar plates to form viscous strings of >5 mm when stretched. This phenotype is strongly associated with hypervirulence in Klebsiella pneumoniae but has never been described in carbapenem-resistant Acinetobacter baumannii (CRAB), an emerging human pathogen of high clinical significance. In this work, we screened 1,000 CRAB isolates, among which we identified and characterized 9 string-positive CRAB (stCRAB) isolates. Phenotypic and genotypic analyses revealed that the isolates were not phylogenetically related and possessed different antibiotic resistance and virulence profiles. Transmission electron microscopy (TEM) showed the presence of capsule in string-positive isolates. String-positive isolates were more motile but did not form more biofilm than non-string-positive isolates. They were less virulent in a murine thigh fitness model and a Galleria mellonella survival assay. In conclusion, here, we describe string-positive A. baumannii isolates and their phenotypic and molecular characteristics. We found that unlike K. pneumoniae, stCRAB isolates were not associated with increased virulence. IMPORTANCE Acinetobacter baumannii has been considered a major health care threat in recent years. Despite many efforts, the pathogenesis and molecular mechanism of A. baumannii virulence remain poorly understood. Moreover, the plasticity of its genome frequently gives rise to new and more virulent isolates. Our current study is of significant importance as it concerns a previously undescribed A. baumannii phenotype. The string-positive phenotype is strongly associated with increased fitness and virulence in other Gram-negative bacteria such as K. pneumoniae. Although no clear correlation with virulence or fitness was found in our 9 stCRAB isolates, this could have been due to the limited statistical power of our research. We suggest that this phenotype should be taken into consideration as due to its genome plasticity, the next change can give rise to string-positive and hypervirulent strains, as is known for K. pneumoniae. Additional future research is needed regarding its possible consequences.

Large-scale WGS of carbapenem-resistant Acinetobacter baumannii isolates reveals patterns of dissemination of ST clades associated with antibiotic resistance.

OBJECTIVES: To describe the population genetics and antibiotic resistance gene distribution of carbapenem-resistant Acinetobacter baumannii (CRAB) isolates causing infections in three Mediterranean countries. METHODS: Isolates were collected during the 2013-17 AIDA clinical trial in six hospitals in Israel, Greece and Italy. WGS, bioinformatic characterization and antibiotic resistance profiling were performed. RESULTS: In the 247 CRAB isolates characterized in this study, ST distribution varied by country: 29/31 (93.5%) Greek isolates, 34/41 (82.9%) Italian isolates and 70/175 (40.0%) Israeli isolates belonged to ST2. The identified ST2 isolates included eight distinct clades: 2C, 2D and 2H were significantly more common in Italy, while 2F was unique to Greece. The uncommon ST3 was not present among Greek isolates and constituted only 5/41 (12%) Italian isolates. On the other hand, it was much more common among Israeli isolates: 78/175 (44.6%) belonged to ST3. The vast majority of isolates, 240/247 (97.2%), were found to harbour acquired carbapenemases, primarily blaOXA-23. The chromosomal oxaAb (blaOXA-51-like) and ampC genes characteristic of this organism were also ubiquitous. Most (96.4%) ST3 isolates carried a broad-host-range plasmid IncP1α. CONCLUSIONS: The geographical differences in CRAB populations support the theory that clonal spread of CRAB leads to endemicity in hospitals and regions. The close association between antibiotic resistance genes and clades, and between plasmids and STs, suggest that de novo creation of MDR A. baumannii is rare. The clustering of antibiotic resistance genes and plasmids that is unique to each clade/ST, and nearly uniform within clades/STs, suggests that horizontal transmission is rare but crucial to the clade's/ST's success.

YMAP: a pipeline for visualization of copy number variation and loss of heterozygosity in eukaryotic pathogens.

The design of effective antimicrobial therapies for serious eukaryotic pathogens requires a clear understanding of their highly variable genomes. To facilitate analysis of copy number variations, single nucleotide polymorphisms and loss of heterozygosity events in these pathogens, we developed a pipeline for analyzing diverse genome-scale datasets from microarray, deep sequencing, and restriction site associated DNA sequence experiments for clinical and laboratory strains of Candida albicans, the most prevalent human fungal pathogen. The YMAP pipeline (http://lovelace.cs.umn.edu/Ymap/) automatically illustrates genome-wide information in a single intuitive figure and is readily modified for the analysis of other pathogens with small genomes.

Phylogenetic- and genome-derived insight into the evolution of N-glycosylation in Archaea.

N-glycosylation, the covalent attachment of oligosaccharides to target protein Asn residues, is a post-translational modification that occurs in all three domains of life. In Archaea, the N-linked glycans that decorate experimentally characterized glycoproteins reveal a diversity in composition and content unequaled by their bacterial or eukaryal counterparts. At the same time, relatively little is known of archaeal N-glycosylation pathways outside of a handful of model strains. To gain insight into the distribution and evolutionary history of the archaeal version of this universal protein-processing event, 168 archaeal genome sequences were scanned for the presence of aglB, encoding the known archaeal oligosaccharyltransferase, an enzyme key to N-glycosylation. Such analysis predicts the presence of AglB in 166 species, with some species seemingly containing multiple versions of the protein. Phylogenetic analysis reveals that the events leading to aglB duplication occurred at various points during archaeal evolution. In many cases, aglB is found as part of a cluster of putative N-glycosylation genes. The presence, arrangement and nucleotide composition of genes in aglB-based clusters in five species of the halophilic archaeon Haloferax points to lateral gene transfer as contributing to the evolution of archaeal N-glycosylation.

By their genes ye shall know them: genomic signatures of predatory bacteria.

Predatory bacteria are taxonomically disparate, exhibit diverse predatory strategies and are widely distributed in varied environments. To date, their predatory phenotypes cannot be discerned in genome sequence data thereby limiting our understanding of bacterial predation, and of its impact in nature. Here, we define the 'predatome,' that is, sets of protein families that reflect the phenotypes of predatory bacteria. The proteomes of all sequenced 11 predatory bacteria, including two de novo sequenced genomes, and 19 non-predatory bacteria from across the phylogenetic and ecological landscapes were compared. Protein families discriminating between the two groups were identified and quantified, demonstrating that differences in the proteomes of predatory and non-predatory bacteria are large and significant. This analysis allows predictions to be made, as we show by confirming from genome data an over-looked bacterial predator. The predatome exhibits deficiencies in riboflavin and amino acids biosynthesis, suggesting that predators obtain them from their prey. In contrast, these genomes are highly enriched in adhesins, proteases and particular metabolic proteins, used for binding to, processing and consuming prey, respectively. Strikingly, predators and non-predators differ in isoprenoid biosynthesis: predators use the mevalonate pathway, whereas non-predators, like almost all bacteria, use the DOXP pathway. By defining predatory signatures in bacterial genomes, the predatory potential they encode can be uncovered, filling an essential gap for measuring bacterial predation in nature. Moreover, we suggest that full-genome proteomic comparisons are applicable to other ecological interactions between microbes, and provide a convenient and rational tool for the functional classification of bacteria.

Contribution of lateral gene transfer to the gene repertoire of a gut-adapted methanogen.

Methanobrevibacter smithii is the most abundant archaeon in the human colon. As most of its neighbors are bacterial species, it is expected that lateral gene acquisition from bacteria might have contributed to the evolution and adaptation of this archaeon. We performed a tree-based genome-wide survey of putative lateral gene transfer products in M. smithii, using a phylogenetic pipeline. Over 15% of the coding genes of M. smithii are inferred to be bacterial in origin, based on this analysis. Laterally acquired genes have had the largest contribution to surface functions, and encode glycosyl-transferases and adhesin-like proteins. In addition, several important ABC transporters, especially metal transporters are of bacterial origin. Thus, bacterial genes contributed to the host-adaptation by allowing a larger variety of surface structures and increasing the efficiency of metal ion uptake in the competitive gut niche.

CRISPR loci reveal networks of gene exchange in archaea.

BACKGROUND: CRISPR (Clustered, Regularly, Interspaced, Short, Palindromic Repeats) loci provide prokaryotes with an adaptive immunity against viruses and other mobile genetic elements. CRISPR arrays can be transcribed and processed into small crRNA molecules, which are then used by the cell to target the foreign nucleic acid. Since spacers are accumulated by active CRISPR/Cas systems, the sequences of these spacers provide a record of the past "infection history" of the organism. RESULTS: Here we analyzed all currently known spacers present in archaeal genomes and identified their source by DNA similarity. While nearly 50% of archaeal spacers matched mobile genetic elements, such as plasmids or viruses, several others matched chromosomal genes of other organisms, primarily other archaea. Thus, networks of gene exchange between archaeal species were revealed by the spacer analysis, including many cases of inter-genus and inter-species gene transfer events. Spacers that recognize viral sequences tend to be located further away from the leader sequence, implying that there exists a selective pressure for their retention. CONCLUSIONS: CRISPR spacers provide direct evidence for extensive gene exchange in archaea, especially within genera, and support the current dogma where the primary role of the CRISPR/Cas system is anti-viral and anti-plasmid defense. OPEN PEER REVIEW: This article was reviewed by: Profs. W. Ford Doolittle, John van der Oost, Christa Schleper (nominated by board member Prof. J Peter Gogarten).

The origins of eukaryotic-like proteins in Legionella pneumophila.

Legionella pneumophila, the causative agent of Legionnaires' disease, is known to be an intracellular pathogen of multiple species of protozoa and is assumed to have co-evolved with these organisms for millions of years. Genome sequencing of L. pneumophila strains has revealed an abundance of eukaryotic-like proteins (ELPs). Here, we study the evolution of these ELPs, in order to investigate their origin. Thirty-four new ELPs were identified, based on a higher similarity to eukaryotic proteins than to bacterial ones. Phylogenetic analyses demonstrated that both lateral gene transfer from eukaryotic hosts and bacterial genes that became eukaryotic-like by gradual adaptation to the intracellular milieu or gene fragment acquisition, contributed to the existing repertoire of ELPs, which comprise over 3% of the putative proteome of L. pneumophila strains. A PCR survey of 72 L. pneumophila strains showed that most ELPs were conserved in nearly all of these strains, indicating that they are likely to play important roles in this species. Genes of different evolutionary origin have distinct patterns of selection, as reflected by their ratio of a synonymous vs. synonymous mutations. One ELP is common to several strains of Legionella, but outside this genus has homologs only in Acanthamoeba polyphaga mimivirus, indicating that gene exchange involving eukaryotic viruses and intracellular bacterial pathogens may also contribute to the evolution of virulence in either or both of these groups of organisms. Information on selection patterns and eukaryotic-like status was combined as a novel approach to predict type IV secretion system effectors of Legionella, which represent promising targets for future study.