The oxazolidinones (linezolid and tedizolid) are last-resort antimicrobial agents used for the treatment of severe infections in humans caused by MDR Gram-positive bacteria. They bind to the peptidyl transferase centre of the bacterial ribosome inhibiting protein synthesis. Even if the majority of Gram-positive bacteria remain susceptible to oxazolidinones, resistant isolates have been reported worldwide. Apart from mutations, affecting mostly the 23S rDNA genes and selected ribosomal proteins, acquisition of resistance genes (cfr and cfr-like, optrA and poxtA), often associated with mobile genetic elements [such as non-conjugative and conjugative plasmids, transposons, integrative and conjugative elements (ICEs), prophages and translocatable units], plays a critical role in oxazolidinone resistance. In this review, we briefly summarize the current knowledge on oxazolidinone resistance mechanisms and provide an overview on the diversity of the mobile genetic elements carrying oxazolidinone resistance genes in Gram-positive and Gram-negative bacteria.

Oxazolidinones: mechanisms of resistance and mobile genetic elements involved.

Escherichia coli ST1193 is an emerging global multidrug (MDR) high-risk clone and an important cause of community-onset urinary and bloodstream infections. ABSTRACT Escherichia coli ST1193 is an emerging global multidrug (MDR) high-risk clone and an important cause of community-onset urinary and bloodstream infections. ST1193 is imitating E. coli ST131, the most successful MDR clone of all time. Both clones emerged in the early 1990s by acquiring quinolone resistance-determining region (QRDR) mutations, IncF plasmids, virulence factors, and type 1 pilus (fimH) recombination. They are the only MDR clones that are dominant among unselected E. coli populations. ST131 is the most frequent clone and ST1193 the second most frequent clone among fluoroquinolone/cephalosporin-resistant E. coli isolates. Both clones have played pivotal roles in the global spread of MDR E. coli. ST1193 originated from ST clonal complex 14 (STc14), is lactose nonfermenting, belongs to phylogenetic group B2, and contains the O type O75. Global ST1193 prevalence has been increasing since 2012, even replacing ST131 in certain regions. blaCTX-M genes are rapidly expanding among ST1193 isolates, a scenario that occurred with ST131 during the 2000s. A validated PCR will enable global surveys to determine the extent of ST1193 among One Health E. coli isolates. The rapid emergence of ST1193 is concerning and is adding to the public health burden of MDR E. coli clones. Basic mechanistic, evolutionary, surveillance, and clinical studies are urgently required to investigate the success of ST1193. Such information will aid with management and prevention strategies. The medical community can ill afford to ignore the spread of another global successful MDR high-risk E. coli clone, especially one that is following in the footsteps of E. coli ST131.

Escherichia coli ST1193: Following in the Footsteps of E. coli ST131

Lactic acid bacteria (LAB) were isolated and characterized from chhurpi, a traditionally produced cheese product of Sikkim Himalaya. Enterococcus durans was the most dominant LAB making up 34% of the identified LAB, followed by Lactobacillus delbrueckii (25%). Chhurpi was produced using milk curdling LAB strains followed by simulated in vitro gastrointestinal digestion of chhurpi. Evaluation of protein hydrolysis and antioxidant activity was performed. Chhurpi produced using Lactobacillus delbrueckii WS4 had a relatively higher yield (10.4%), protein hydrolysis, and antioxidant activity as compared to other starter strains (P < 0.05). Gastrointestinal digestion of chhurpi increased the antioxidant activity, with the highest activity observed for gastrointestinal digests of chhurpi produced by using Lb. delbrueckii WS4. The EC50 for DPPH radical scavenging activity of chhurpi produced by using Lb. delbrueckii WS4 was observed to decrease significantly (from 239.16 to 48.87 mg sample/ml) after digestion with pepsin and pancreatin (P < 0.05). Protein hydrolysis and antioxidant activity in whey were comparable to that of chhurpi, indicating the potential of using the chhurpi whey to develop nutraceuticals. This is the first report on evaluating the antioxidant activity of chhurpi produced using defined LAB strains and the effect of gastrointestinal digestion on chhurpi functionality. 

Characterization of native lactic acid bacteria from traditionally fermented chhurpi of Sikkim Himalayan region for the production of chhurpi cheese with enhanced antioxidant effect

Lactic acid bacteria (LAB) were isolated and characterized from chhurpi, a traditionally produced cheese product of Sikkim Himalaya. Enterococcus durans was the most dominant LAB making up 34% of the identified LAB, followed by Lactobacillus delbrueckii (25%). Chhurpi was produced using milk curdling LAB strains followed by simulated in vitro gastrointestinal digestion of chhurpi. Evaluation of protein hydrolysis and antioxidant activity was performed. Chhurpi produced using Lactobacillus delbrueckii WS4 had a relatively higher yield (10.4%), protein hydrolysis, and antioxidant activity as compared to other starter strains (P

Clostridioides difficile is the most common cause of antibiotic-associated gastrointestinal infections. Capillary electrophoresis (CE)-PCR ribotyping is currently the gold standard for C. difficile typing but lacks the discriminatory power to study transmission and outbreaks in detail. 

Comparison of Whole-Genome Sequence-Based Methods and PCR Ribotyping for Subtyping of Clostridioides difficile

Objectives Fosfomycin is an important antibiotic for the treatment of MDR Enterobacteriaceae infections. High susceptibility rates are, however, threatened by the spread of plasmids encoding fosfomycin-modifying enzymes. In this study, we sought to characterize the genetic context of fosA in plasmids from Escherichia coli and Klebsiella spp. isolates recovered from food, wastewater and surface water in Switzerland. Methods E. coli and Klebsiella spp. isolates collected between 2012 and 2019 in Switzerland were screened for fosfomycin resistance. Presence of fosA was verified by PCR and sodium phosphonoformate (PPF) disc potentiation testing, and transferability was tested using conjugation assays. Whole-genome sequences including complete fosA-containing plasmids were determined using long- and short-read sequencing. Results In 11 E. coli and two Klebsiella spp. isolates, high-level fosfomycin resistance was mediated by plasmids containing fosA3 (n = 12) or fosA8 (n = 1). Four isolates harboured a near-identical 45 kb IncN plasmid with fosA3, while replicon types varied in the remaining plasmids. The fosA genes were typically embedded in IS26-bounded transposition units and frequently located in the proximity of blaCTX-M transposition units. Conclusions Although fosfomycin resistance rates are currently low, the presence of fosA-encoding plasmids circulating in the Enterobacteriaceae population suggests that fosfomycin resistance may rapidly spread upon increased selection pressure. Transposition mobility of fosA and co-location on plasmids with other resistance genes may further promote its dissemination.

Characteristics of fosA-carrying plasmids in E. coli and Klebsiella spp. isolates originating from food and environmental samples

Clostridioides difficile causes nosocomial outbreaks which can lead to severe and even life-threatening colitis. Rapid molecular diagnostic tests allow the identification of toxin-producing, potentially hypervirulent strains, which is critical for patient management and infection control. PCR-ribotyping has been used for decades as the reference standard to investigate transmission in suspected outbreaks. However, the introduction of whole genome sequencing (WGS) for molecular epidemiology provides a realistic alternative to PCR-ribotyping. In this transition phase it is crucial to understand the strengths and weaknesses of the two technologies, and to assess their correlation. We aimed to investigate ribotype prediction from WGS data, and options for analysis at different levels of analytical granularity. Ribotypes cannot be directly determined from short read Illumina sequence data as the rRNA operons including the ribotype-defining ISR fragments collapse in genome assemblies, and comparison with traditional PCR-ribotyping results becomes impossible. Ribotype extraction from long read Oxford nanopore data also requires optimization. We have compared WGS-based typing with PCR-ribotyping in nearly 300 clinical and environmental isolates from Switzerland, and in addition from the Enterobase database (n=1778). Our results show that while multi-locus sequence type (MLST) often correlates with a specific ribotype, the agreement is not complete, and for some ribotypes the resolution is insufficient. Using core genome MLST (cgMLST) analysis, there is an improved resolution and ribotypes can often be predicted within clusters, using cutoffs of 30-50 allele differences. The exceptions are ribotypes within known ribotype complexes such as RT078/RT106, where the genome differences in cgMLST do not reflect the ribotype segregation. We show that different ribotype clusters display different degrees of diversity, which could be important for the definition of ribotype cluster specific cutoffs. WGS-based analysis offers the ultimate resolution to the SNP level, enabling exploration of patient-to-patient transmission. PCR-ribotyping does not sufficiently discriminate to prove nosocomial transmission with certainty. We discuss the associated challenges and opportunities in a switch to WGS from conventional ribotyping for C. difficile.

/pdf/transition-from-pcr-ribotyping-to-whole-genome-sequencing-ix82ayurhn.pdf

Transition From PCR-Ribotyping to Whole Genome Sequencing Based Typing of Clostridioides difficile

Objectives The global dissemination of vancomycin-resistant enterococci (VRE) has become a serious public-health concern. Although outbreaks are typically caused by nosocomial transmission, contaminated food and water may contribute to the spread of VRE. The aim of this study was to assess the presence of VRE in flowing surface water bodies in Switzerland and to characterise the isolates. Methods Surface water was sampled from rivers, streams and canals throughout Switzerland and was screened for the presence of VRE. Whole-genome sequencing was used to identify antimicrobial resistance genes and the phylogenetic similarity of the obtained isolates. Results VRE were detected in 6 (3.1%) of 191 water samples. The six VRE-containing samples were all collected near treated wastewater discharge sites. The six isolates were identified as Enterococcus faecium sequence type 133 (ST133) and harboured the vancomycin resistance-conferring vanA gene cluster on transposon Tn1546. They showed a close phylogenetic relationship to ST133 swine faecal isolates obtained during a previously reported screening in Switzerland. Conclusion Our results suggest that surface water contributes to the environmental dissemination of VRE. Repeated identification of ST133 clones in geographically distinct water sampling sites and swine faecal samples collected in slaughterhouses may indicate a local dominance of this VRE lineage in Switzerland.

Spread of vancomycin-resistant Enterococcus faecium ST133 in the aquatic environment in Switzerland.

Linezolid is an important last-resort antibiotic for the treatment of multi-drug resistant enterococci. The aim of this study was to further characterize the genetic context of optrA and poxtA in ten florfenicol-resistant enterococci isolated from flowing surface water. In most genomes, optrA and poxtA were embedded in transposition units integrated into plasmids or into the chromosomal radC. For the first time a chromosomally integrated optrA in an Enterococcus raffinosus isolate is described.

Genetic Context of optrA and poxtA in Florfenicol-Resistant Enterococci Isolated from Flowing Surface Water in Switzerland.

Bacillus cytotoxicus belongs to the Bacillus cereus group that also comprises the foodborne pathogen Bacillus cereus sensu stricto, Bacillus anthracis causing anthrax, as well as the biopesticide Bacillus thuringiensis. The first B. cytotoxicus was isolated in the context of a severe food poisoning outbreak leading to fatal cases of diarrheal disease. Subsequent characterization of the outbreak strain led to the conclusion that this Bacillus strain was highly cytotoxic and eventually resulted in the description of a novel species, whose name reflects the observed toxicity: B. cytotoxicus. However, only a few isolates of this species have been characterized with regard to their cytotoxic potential and the role of B. cytotoxicus as a causative agent of food poisoning remains largely unclear. Hence, the aim of this study was to gain further insights into the toxicity of B. cytotoxicus. To this end, 19 isolates were obtained from mashed potato powders and characterized by toxin gene profiling and Vero cell cytotoxicity assays. All isolates harbored the cytK1 (cytotoxin K1) gene and species-specific variants of the nhe (non-hemolytic enterotoxin) gene. The isolates exhibited low or no toxicity towards Vero cells. Thus, this study indicates that the cytotoxic potential of B. cytotoxicus may be potentially lower than initially assumed.

https://www.mdpi.com/2072-6651/13/4/234/pdf

Michael Biggel

Papers

Characteristics of fosA-carrying plasmids in E. coli and Klebsiella spp. isolates originating from food and environmental samples

Transition From PCR-Ribotyping to Whole Genome Sequencing Based Typing of Clostridioides difficile

Spread of vancomycin-resistant Enterococcus faecium ST133 in the aquatic environment in Switzerland.

Genetic Context of optrA and poxtA in Florfenicol-Resistant Enterococci Isolated from Flowing Surface Water in Switzerland.

Further Insights into the Toxicity of Bacillus cytotoxicus Based on Toxin Gene Profiling and Vero Cell Cytotoxicity Assays.