Erysipelothrix amsterdamensis sp. nov., associated with mortalities among endangered seabirds.

Infectious diseases threaten endangered species, particularly in small isolated populations. Seabird populations on the remote Amsterdam Island in the Indian Ocean have been in decline for the past three decades, with avian cholera caused by Pasteurella multocida proposed as the primary driver. However, Erysipelothrix species have also been sporadically detected from albatrosses on Amsterdam Island and may be contributing to some of the observed mortality. In this study, we genomically characterized 16 Erysipelothrix species isolates obtained from three Indian yellow-nosed albatross (Thalassarche carteri) chick carcasses in 2019. Histological analyses suggest that they died of bacterial septicaemia. Two isolates were sequenced using both Illumina short-read and MinION long-read approaches, which - following hybrid assembly - resulted in closed circular genomes. Mapping of Illumina reads from the remaining isolates to one of these new reference genomes revealed that all 16 isolates were closely related, with a maximum of 13 nucleotide differences distinguishing any pair of isolates. The nucleotide diversity of isolates obtained from the same or different carcasses was similar, suggesting all three chicks were likely infected from a common source. These genomes were compared with a global collection of genomes from Erysipelothrix rhusiopathiae and other species from the same genus. The isolates from albatrosses were phylogenetically distinct, sharing a most recent common ancestor with E. rhusiopathiae. Based on phylogenomic analysis and standard thresholds for average nucleotide identity and digital DNA-DNA hybridization, these isolates represent a novel Erysipelothrix species, for which we propose the name Erysipelothrix amsterdamensis sp. nov. The type strain is A18Y020dT (=CIP 112216T=DSM 115297T). The implications of this bacterium for albatross conservation will require further study.

Fitness effects of blaCTX-M-15-harbouring F2:A1:B- plasmids on their native Escherichia coli ST131 H30Rx hosts.

OBJECTIVES: To investigate the fitness effects of large blaCTX-M-15-harbouring F2:A1:B- plasmids on their native Escherichia coli ST131 H30Rx hosts. METHODS: We selected five E. coli ST131 H30Rx isolates of diverse origin, each carrying an F2:A1:B- plasmid with the blaCTX-M-15 gene. The plasmid was eliminated from each isolate by displacement using an incompatible curing plasmid, pMDP5_cureEC958. WGS was performed to obtain complete chromosome and plasmid sequences of original isolates and to detect chromosomal mutations in 'cured' clones. High-throughput competition assays were conducted to determine the relative fitness of cured clones compared with the corresponding original isolates. RESULTS: We were able to successfully eliminate the F2:A1:B- plasmids from all five original isolates using pMDP5_cureEC958. The F2:A1:B- plasmids produced non-significant fitness effects in three isolates and moderate reductions in relative fitness (3%-4%) in the two remaining isolates. CONCLUSIONS: We conclude that F2:A1:B- plasmids pose low fitness costs in their E. coli ST131 H30Rx hosts. This plasmid-host fitness compatibility is likely to promote the maintenance of antibiotic resistance in this clinically important E. coli lineage.

Extended-spectrum beta-lactamase-producing Escherichia coli and antimicrobial resistance in municipal and hospital wastewaters in Czech Republic: Culture-based and metagenomic approaches.

Wastewaters serve as important hot spots for antimicrobial resistance and monitoring can be used to analyse the abundance and diversity of antimicrobial resistance genes at the level of large bacterial and human populations. In this study, whole genome sequencing of beta-lactamase-producing Escherichia coli and metagenomic analysis of whole-community DNA were used to characterize the occurrence of antimicrobial resistance in hospital, municipal and river waters in the city of Brno (Czech Republic). Cefotaxime-resistant E. coli were mainly extended-spectrum beta-lactamase (ESBL) producers (95.6%, n = 158), of which the majority carried blaCTX-M (98.7%; n = 151) and were detected in all water samples except the outflow from hospital wastewater treatment plant. A wide phylogenetic diversity was observed among the sequenced E. coli (n = 78) based on the detection of 40 sequence types and single nucleotide polymorphisms (average number 34,666 ± 15,710) between strains. The metagenomic analysis revealed a high occurrence of bacterial genera with potentially pathogenic members, including Pseudomonas, Escherichia, Klebsiella, Aeromonas, Enterobacter and Arcobacter (relative abundance >50%) in untreated hospital and municipal wastewaters and predominance of environmental bacteria in treated and river waters. Genes encoding resistance to aminoglycosides, beta-lactams, quinolones and macrolides were frequently detected, however blaCTX-M was not found in this dataset which may be affected by insufficient sequencing depth of the samples. The study pointed out municipal treated wastewater as a possible source of multi-drug resistant E. coli and antimicrobial resistance genes for surface waters. Moreover, the combination of two different approaches provided a more holistic view on antimicrobial resistance in water environments. The culture-based approach facilitated insight into the dynamics of ESBL-producing E. coli and the metagenomics shows abundance and diversity of bacteria and antimicrobial resistance genes vary across water sites.

Complete Nucleotide Sequences of mcr-4.3-Carrying Plasmids in Acinetobacter baumannii Sequence Type 345 of Human and Food Origin from the Czech Republic, the First Case in Europe.

Here, we describe two plasmids carrying mcr-4.3 in two Acinetobacter baumannii strains isolated from imported food and a clinical sample. The comparative analysis of these plasmids, with two other plasmids reported in the NCBI database, highlighted the common origin of the plasmidic structure carrying mcr-4.3 This is the first case of the mcr-4.3 gene in a A. baumannii strain isolated from a clinical case in Europe. We hypothesize that food import is initiating the spread in Czech Republic.

Fecal Carriage and Whole-Genome Sequencing-Assisted Characterization of CMY-2 Beta-Lactamase-Producing Escherichia coli in Calves at Czech Dairy Cow Farm.

The study aimed to monitor the fecal shedding of cefotaxime-resistant Escherichia coli (CREC) in a cohort of healthy calves on a dairy farm with documented antimicrobial usage and to characterize selected AmpC beta-lactamase-producing E. coli isolates. Fecal samples from 13 suckling calves (1-63 d of age; 113 samples in total) were repeatedly collected and cultivated on MacConkey agar with cefotaxime (2 mg/L). Resistant colonies were counted, and one colony obtained from the highest dilution of each fecal sample was identified by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry. Susceptibility to antimicrobials and production of AmpC and extended-spectrum beta-lactamase (ESBL) were tested. No ESBL-producing E. coli was found, but representative AmpC-positive E. coli isolates were subjected to further typing and whole-genome sequencing (WGS) for the analysis of clonal relationships, resistance genes, virulence factors, and plasmid replicons. High amounts of CREC were detected in the feces of all 13 calves during the study. The number of CREC colonies varied from 1.0 log10 to 8.0 log10 colony-forming unit per gram. Drops in CREC density or its discontinued shedding were recorded at the end of the study period. A total of 82 (94%, n = 87) CREC isolates were confirmed as AmpC producers and all but one showed resistance to multiple antimicrobials. Twenty-nine selected AmpC-positive E. coli isolates belonged to 12 and 13 unique rep-PCR fingerprints and pulsed-field gel electrophoresis types, respectively, highlighting the variation in E. coli genotypes in individual calves. WGS of 10 selected isolates showed diverse antimicrobial resistance and virulence gene content and the presence of a blaCMY-2 gene carried by an IncK2 plasmid. Clinically important multiresistant E. coli isolates belonging to emerging extraintestinal pathogenic E. coli ST69 and ST648 lineages were found. Our findings reinforce the urgency of efforts to prevent the spread of ESBL-/AmpC-producing bacteria in dairy cow farms.

Whole genome sequencing and function prediction of 133 gut anaerobes isolated from chicken caecum in pure cultures.

BACKGROUND: In order to start to understand the function of individual members of gut microbiota, we cultured, sequenced and analysed bacterial anaerobes from chicken caecum. RESULTS: Altogether 204 isolates from chicken caecum were obtained in pure cultures using Wilkins-Chalgren anaerobe agar and anaerobic growth conditions. Genomes of all the isolates were determined using the NextSeq platform and subjected to bioinformatic analysis. Among 204 sequenced isolates we identified 133 different strains belonging to seven different phyla - Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, Verrucomicrobia, Elusimicrobia and Synergistetes. Genome sizes ranged from 1.51 Mb in Elusimicrobium minutum to 6.70 Mb in Bacteroides ovatus. Clustering based on the presence of protein coding genes showed that isolates from phyla Proteobacteria, Verrucomicrobia, Elusimicrobia and Synergistetes did not cluster with the remaining isolates. Firmicutes split into families Lactobacillaceae, Enterococcaceae, Veillonellaceae and order Clostridiales from which the Clostridium perfringens isolates formed a distinct sub-cluster. All Bacteroidetes isolates formed a separate cluster showing similar genetic composition in all isolates but distinct from the rest of the gut anaerobes. The majority of Actinobacteria clustered closely together except for the representatives of genus Gordonibacter showing that the genome of this genus differs from the rest of Actinobacteria sequenced in this study. Representatives of Bacteroidetes commonly encoded proteins (collagenase, hemagglutinin, hemolysin, hyaluronidase, heparinases, chondroitinase, mucin-desulfating sulfatase or glutamate decarboxylase) that may enable them to interact with their host. Aerotolerance was recorded in Akkermansia and Cloacibacillus and was also common among representatives of Bacteroidetes. On the other hand, Elusimicrobium and the majority of Clostridiales were highly sensitive to air exposure despite their potential for spore formation. CONCLUSIONS: Major gut microbiota members utilise different strategies for gut colonisation. High oxygen sensitivity of Firmicutes may explain their commonly reported decrease after oxidative burst during gut inflammation.

Characterization of the Complete Nucleotide Sequences of IMP-4-Encoding Plasmids, Belonging to Diverse Inc Families, Recovered from Enterobacteriaceae Isolates of Wildlife Origin.

The complete nucleotide sequences of six IMP-4-encoding plasmids recovered from Enterobacteriaceae isolates of wildlife origin were characterized. Sequencing data showed that plasmids of different incompatibility groups (IncM, IncI1, IncF, and nontypeable [including an IncX5_2 and two pPrY2001-like]) carried the blaIMP-4-carrying integrons In809 or In1460. Most of the plasmids carried an mph(A) region, and chrA-like, aac(3)-IId, and blaTEM-1b genes. Finally, plasmid analysis revealed the involvement of two different IS26- and Tn1696-associated mechanisms in the mobilization of IMP-4-encoding integrons.

Characterization of KPC-Encoding Plasmids from Enterobacteriaceae Isolated in a Czech Hospital.

Ten Enterobacteriaceae isolates collected in a Czech hospital carried blaKPC-positive plasmids of different sizes (∼30, ∼45, and ∼80 kb). Sequencing revealed three types of plasmids (A to C) with the Tn4401a transposon. Type A plasmids comprised an IncR backbone and a KPC-2-encoding multidrug resistance (MDR) region. Type B plasmids were derivatives of type A plasmids carrying an IncN3-like segment, while type C plasmids were IncP6 plasmids sharing the same KPC-2-encoding MDR region with type A and B plasmids.

Complete Nucleotide Sequences of Two VIM-1-Encoding Plasmids from Klebsiella pneumoniae and Leclercia adecarboxylata Isolates of Czech Origin.

Two multidrug resistance (MDR) plasmids, carrying the VIM-1-encoding integron In110, were characterized. Plasmid pLec-476cz (311,758 bp), from a Leclercia adecarboxylata isolate, consisted of an IncHI1 backbone, a MDR region, and two accessory elements. Plasmid pKpn-431cz (142,876 bp), from a sequence type 323 (ST323) Klebsiella pneumoniae isolate, comprised IncFIIY-derived and pKPN3-like sequences and a mosaic region. A 40,400-bp sequence of pKpn-431cz was identical to the MDR region of pLec-476cz, indicating the en bloc acquisition of the VIM-1-encoding region from one plasmid by the other.

Characterization of the Complete Nucleotide Sequences of IncA/C2 Plasmids Carrying In809-Like Integrons from Enterobacteriaceae Isolates of Wildlife Origin.

A total of 18 Enterobacteriaceae (17 from gulls and 1 from a clinical sample) collected from Australia, carrying IncA/C plasmids with the IMP-encoding In809-like integrons, were studied. Seven plasmids, being representatives of different origins, plasmid sizes, replicon combinations, and resistance genes, were completely sequenced. Plasmid pEc158, identified in a clinical Escherichia coli ST752 isolate, showed extensive similarity to type 2 IncA/C2 plasmids. pEc158 carried none of the blaCMY-2-like region or ARI-B and ARI-A regions, while it contained a hybrid transposon structure. The six remaining plasmids, which were of wildlife origin, were highly similar to each other and probably were fusion derivatives of type 1 and type 2 A/C2 plasmids. The latter plasmids contained an ARI-B region and hybrid transposon structures. In all plasmids, hybrid transposon structures containing In809-like integrons were inserted 3,434 bp downstream of the rhs2 start codon. In all cases, the one outermost 38-bp inverted repeat (IR) of the transposon was associated with the Tn1696 tnp module, while the other outermost 38-bp IR of the transposon was associated with either a Tn6317-like module or a Tn21 mer module. However, the internal structure of the transposon and the resistance genes were different in each plasmid. These findings indicated that, for the specific periods of time and settings, different IncA/C2 plasmid types carrying In809-like elements circulated among isolates of wildlife and clinical origins. Additionally, they provided the basis for speculations regarding the reshuffling of IncA/C2 plasmids with In809-like integrons and confirmed the rapid evolution of IncA/C2 plasmid lineages.

Molecular Characterization of Carbapenemase-Producing Pseudomonas aeruginosa of Czech Origin and Evidence for Clonal Spread of Extensively Resistant Sequence Type 357 Expressing IMP-7 Metallo-ß-Lactamase.

The objective of this study was to perform molecular surveillance for assessing the spread of carbapenemase-producing Pseudomonas aeruginosa in Czech hospitals. One hundred thirty-six carbapenemase-producing isolates were recovered from 22 hospitals located throughout the country. Sequence type 357 (ST357) dominated (n = 120) among carbapenemase producers. One hundred seventeen isolates produced IMP-type (IMP-7 [n = 116] and IMP-1 [n = 1]) metallo-ß-lactamases (MßLs), 15 produced the VIM-2 MßL, and the remaining isolates expressed the GES-5 enzyme. The blaIMP-like genes were located in three main integron types, with In-p110-like being the most prevalent (n = 115). The two other IMP-encoding integrons (In1392 and In1393) have not been described previously. blaVIM-2-carrying integrons included In59-like, In56, and a novel element (In1391). blaGES-5 was carried by In717. Sequencing data showed that In-p110-like was associated with a Tn4380-like transposon inserted in genomic island LESGI-3 in the P. aeruginosa chromosome. The other integrons were also integrated into the P. aeruginosa chromosome. These findings indicated the clonal spread of ST357 P. aeruginosa, carrying the IMP-7-encoding integron In-p110, in Czech hospitals. Additionally, the sporadic emergence of P. aeruginosa producing different carbapenemase types, associated with divergent or novel integrons, punctuated the ongoing evolution of these bacteria.

Molecular Characterization of OXA-48-Like-Producing Enterobacteriaceae in the Czech Republic and Evidence for Horizontal Transfer of pOXA-48-Like Plasmids.

The aim of this study was to characterize the first cases and outbreaks of OXA-48-like-producing Enterobacteriaceae recovered from hospital settings in the Czech Republic. From 2013 to 2015, 22 Klebsiella pneumoniae isolates, 3 Escherichia coli isolates, and 1 Enterobacter cloacae isolate producing OXA-48-like carbapenemases were isolated from 20 patients. Four of the patients were colonized or infected by two or three different OXA-48-like producers. The K. pneumoniae isolates were classified into nine sequence types (STs), with ST101 being predominant (n = 8). The E. coli isolates were of different STs, while the E. cloacae isolate belonged to ST109. Twenty-four isolates carried blaOXA-48, while two isolates carried blaOXA-181 or blaOXA-232 Almost all isolates (n = 22) carried blaOXA-48-positive plasmids of a similar size (∼60 kb), except the two isolates producing OXA-181 or OXA-232. In an ST45 K. pneumoniae isolate and an ST38 E. coli isolate, S1 nuclease profiling plus hybridization indicated a chromosomal location of blaOXA-48 Sequencing showed that the majority of blaOXA-48-carrying plasmids exhibited high degrees of identity with the pOXA-48-like plasmid pE71T. Additionally, two novel pE71T derivatives, pOXA-48_30715 and pOXA-48_30891, were observed. The blaOXA-181-carrying plasmid was identical to the IncX3 plasmid pOXA181_EC14828, while the blaOXA-232-carrying plasmid was a ColE2-type plasmid, being a novel derivative of pOXA-232. Finally, sequencing data showed that the ST45 K. pneumoniae and ST38 E. coli isolates harbored the IS1R-based composite transposon Tn6237 containing blaOXA-48 integrated into their chromosomes. These findings underlined that the horizontal transfer of pOXA-48-like plasmids has played a major role in the dissemination of blaOXA-48 in the Czech Republic. In combination with the difficulties with their detection, OXA-48 producers constitute an important public threat.

Plasmid-mediated colistin resistance among human clinical Enterobacterales isolates: national surveillance in the Czech Republic.

The occurrence of colistin resistance has increased rapidly among Enterobacterales around the world. We performed a national survey of plasmid-mediated colistin resistance in human clinical isolates through a retrospective analysis of samples from 2009 to 2017 and a prospective sampling in 2018-2020. The aim of this study was to identify and characterize isolates with mcr genes from various regions of the Czech Republic using whole genome sequencing (WGS). Of all 1932 colistin-resistant isolates analyzed, 73 (3.8%) were positive for mcr genes. Most isolates carried mcr-1 (48/73) and were identified as Escherichia coli (n = 44) and Klebsiella pneumoniae (n = 4) of various sequence types (ST). Twenty-five isolates, including Enterobacter spp. (n = 24) and Citrobacter freundii (n = 1) carrying the mcr-9 gene were detected; three of them (Enterobacter kobei ST54) co-harbored the mcr-4 and mcr-9 genes. Multi-drug resistance phenotype was a common feature of mcr isolates and 14% (10/73) isolates also co-harbored clinically important beta-lactamases, including two isolates with carbapenemases KPC-2 and OXA-48. Phylogenetic analysis of E. coli ST744, the dominant genotype in this study, with the global collection showed Czech isolates belonged to two major clades, one containing isolates from Europe, while the second composed of isolates from diverse geographical areas. The mcr-1 gene was carried by IncX4 (34/73, 47%), IncHI2/ST4 (6/73, 8%) and IncI2 (8/73, 11%) plasmid groups. Small plasmids belonging to the ColE10 group were associated with mcr-4 in three isolates, while mcr-9 was carried by IncHI2/ST1 plasmids (4/73, 5%) or the chromosome (18/73, 25%). We showed an overall low level of occurrence of mcr genes in colistin-resistant bacteria from human clinical samples in the Czech Republic.

Development of submicromolar 17ß-HSD10 inhibitors and their in vitro and in vivo evaluation.

17ß-hydroxysteroid dehydrogenase type 10 (17ß-HSD10) is a multifunctional mitochondrial enzyme and putative drug target for the treatment of various pathologies including Alzheimer's disease or some types of hormone-dependent cancer. In this study, a series of new benzothiazolylurea-based inhibitors were developed based on the structure-activity relationship (SAR) study of previously published compounds and predictions of their physico-chemical properties. This led to the identification of several submicromolar inhibitors (IC50 ∼0.3 µM), the most potent compounds within the benzothiazolylurea class known to date. The positive interaction with 17ß-HSD10 was further confirmed by differential scanning fluorimetry and the best molecules were found to be cell penetrable. In addition, the best compounds weren't found to have additional effects for mitochondrial off-targets and cytotoxic or neurotoxic effects. The two most potent inhibitors 9 and 11 were selected for in vivo pharmacokinetic study after intravenous and peroral administration. Although the pharmacokinetic results were not fully conclusive, it seemed that compound 9 was bioavailable after peroral administration and could penetrate into the brain (brain-plasma ratio 0.56).

Interspecies Transmission of CMY-2-Producing Escherichia coli Sequence Type 963 Isolates between Humans and Gulls in Australia.

Escherichia coli sequence type 963 (ST963) is a neglected lineage closely related to ST38, a globally widespread extraintestinal pathogenic ST causing urinary tract infections (UTI) as well as sepsis in humans. Our current study aimed to improve the knowledge of this understudied ST by carrying out a comprehensive comparative analysis of whole-genome sequencing data consisting of 31 isolates from silver gulls in Australia along with another 80 genomes from public resources originating from geographically scattered regions. ST963 was notable for carriage of cephalosporinase gene blaCMY-2, which was identified in 99 isolates and was generally chromosomally encoded. ST963 isolates showed otherwise low carriage of antibiotic resistance genes, in contrast with the closely related E. coli ST38. We found considerable phylogenetic variability among international ST963 isolates (up to 11,273 single nucleotide polymorphisms [SNPs]), forming three separate clades. A major clade that often differed by 20 SNPs or less consisted of Australian isolates of both human and animal origin, providing evidence of zoonotic or zooanthropogenic transmission. There was a high prevalence of virulence F29:A-:B10 pUTI89-like plasmids within E. coli ST963 (n = 88), carried especially by less variable isolates exhibiting ≤1,154 SNPs. We characterized a novel 115,443-bp pUTI89-like plasmid, pCE2050_A, that carried a traS:IS5 insertion absent from pUTI89. Since IS5 was also present in a transposition unit bearing blaCMY-2 on chromosomes of ST963 strains, IS5 insertion into pUTI89 may enable mobilization of the blaCMY-2 gene from the chromosome/transposition unit to pUTI89 via homologous recombination. IMPORTANCE We have provided the first comprehensive genomic study of E. coli ST963 by analyzing various genomic and phenotypic data sets of isolates from Australian silver gulls and comparison with genomes from geographically dispersed regions of human and animal origin. Our study suggests the emergence of a specific blaCMY-2-carrying E. coli ST963 clone in Australia that is widely spread across the continent by humans and birds. Genomic analysis has revealed that ST963 is a globally dispersed lineage with a remarkable set of virulence genes and virulence plasmids described in uropathogenic E. coli. While ST963 separated into three clusters, a unique specific clade of Australian ST963 isolates harboring a chromosomal copy of AmpC ß-lactamase encoding the gene blaCMY-2 and originating from both humans and wild birds was identified. This phylogenetically close cluster comprised isolates of both animal and human origin, thus providing evidence of interspecies zoonotic transmission. The analysis of the genetic environment of the AmpC ß-lactamase-encoding gene highlighted ongoing evolutionary events that shape the carriage of this gene in ST963.

Population genomics of Bacillus anthracis from an anthrax hyperendemic area reveals transmission processes across spatial scales and unexpected within-host diversity.

Genomic sequencing has revolutionized our understanding of bacterial disease epidemiology, but remains underutilized for zoonotic pathogens in remote endemic settings. Anthrax, caused by the spore-forming bacterium Bacillus anthracis, remains a threat to human and animal health and rural livelihoods in low- and middle-income countries. While the global genomic diversity of B. anthracis has been well-characterized, there is limited information on how its populations are genetically structured at the scale at which transmission occurs, critical for understanding the pathogen's evolution and transmission dynamics. Using a uniquely rich dataset, we quantified genome-wide SNPs among 73 B. anthracis isolates derived from 33 livestock carcasses sampled over 1 year throughout the Ngorongoro Conservation Area, Tanzania, a region hyperendemic for anthrax. Genome-wide SNPs distinguished 22 unique B. anthracis genotypes (i.e. SNP profiles) within the study area. However, phylogeographical structure was lacking, as identical SNP profiles were found throughout the study area, likely the result of the long and variable periods of spore dormancy and long-distance livestock movements. Significantly, divergent genotypes were obtained from spatio-temporally linked cases and even individual carcasses. The high number of SNPs distinguishing isolates from the same host is unlikely to have arisen during infection, as supported by our simulation models. This points to an unexpectedly wide transmission bottleneck for B. anthracis, with an inoculum comprising multiple variants being the norm. Our work highlights that inferring transmission patterns of B. anthracis from genomic data will require analytical approaches that account for extended and variable environmental persistence, as well as co-infection.

PepMANDIS: A Peptide Selection Tool for Designing Function-Based Targeted Proteomic Assays in Complex Microbial Systems.

The majority of studies focusing on microbial functioning in various environments are based on DNA or RNA sequencing techniques that have inherent limitations and usually provide a distorted picture about the functional status of the studied system. Untargeted proteomics is better suited for that purpose, but it suffers from low efficiency when applied in complex consortia. In practice, the scanning capabilities of the currently employed LC-MS/MS systems provide limited coverage of key-acting proteins, hardly allowing a semiquantitative assessment of the most abundant ones from most prevalent species. When particular biological processes of high importance are under investigation, the analysis of specific proteins using targeted proteomics is a more appropriate strategy as it offers superior sensitivity and comes with the added benefits of increased throughput, dynamic range and selectivity. However, the development of targeted assays requires a priori knowledge regarding the optimal peptides to be screened for each protein of interest. In complex, multi-species systems, a specific biochemical process may be driven by a large number of homologous proteins having considerable differences in their amino acid sequence, complicating LC-MS/MS detection. To overcome the complexity of such systems, we have developed an automated pipeline that interrogates UniProt database or user-created protein datasets (e.g. from metagenomic studies) to gather homolog proteins with a defined functional role and extract respective peptide sequences, while it computes several protein/peptide properties and relevant statistics to deduce a small list of the most representative, process-specific and LC-MS/MS-amenable peptides for the microbial enzymatic activity of interest.

Whole genome sequencing of a clinical drug resistant Candida albicans isolate reveals known and novel mutations in genes involved in resistance acquisition mechanisms.

Candida albicans is an opportunistic pathogen accounting for the majority of cases of Candida infections. Currently, C. albicans are developing resistance towards different classes of antifungal drugs and this has become a global health burden that does not spare Lebanon. This study aims at determining point mutations in genes known to be involved in resistance acquisition and correlating resistance to virulence and ergosterol content in the azole resistant C. albicans isolate CA77 from Lebanon. This pilot study is the first of its kind to be implemented in Lebanon. We carried out whole genome sequencing of the azole resistant C. albicans isolate CA77 and examined 18 genes involved in antifungal resistance. To correlate genotype to phenotype, we evaluated the virulence potential of this isolate by injecting it into BALB/c mice and we quantified membrane ergosterol. Whole genome sequencing revealed that eight out of 18 genes involved in antifungal resistance were mutated in previously reported and novel residues. These genotypic changes were associated with an increase in ergosterol content but no discrepancy in virulence potential was observed between our isolate and the susceptible C. albicans control strain SC5314. This suggests that antifungal resistance and virulence potential in this antifungal resistant isolate are not correlated and that resistance is a result of an increase in membrane ergosterol content and the occurrence of point mutations in genes involved in the ergosterol biosynthesis pathway.

Frequency of mutations associated with resistance to first- and second-line drugs in multidrug-resistant Mycobacterium tuberculosis isolates.

INTRODUCTION: Tuberculosis is considered one of the most fatal diseases worldwide, with an estimation of 10.1 million cases. In this study, whole-genome sequencing was used to determine the genomic characterisation of 40 Mycobacterium tuberculosis isolates from patients with different nationalities hospitalised in the Czech Republic. MATERIALS AND METHODS: Susceptibility testing for first-line drugs was performed. DNA was sequenced using the Illumina MiSeq platform. Spoligotype single-nucleotide polymorphisms and mutations in antibiotic-resistant genes were detected, and phylogenetic analysis was performed. RESULTS: Samples showing phenotypic resistance to at least one drug were 12 to streptomycin, 11 to isoniazid, 7 to rifampicin, 6 to ethambutol and 5 to pyrazinamide. Phenotypic and genotypic profiles did not match in all cases, suggesting the presence of a novel mutation in some cases and a low expression of resistant genes in others. The presented phylogeny enables the correct assignation of M. tuberculosis lineages and sublineages. Our results suggest that the most dominant lineage in our samples was lineage 4 (33/40). CONCLUSION: To our knowledge, this is the first study using this approach to be done in the Czech Republic. Lineage 4 was the predominant lineage identified among our samples. Nevertheless, the dominance of Lineage 4 along with other lineages suggests that infections can originate from different sources.