Identification of Mycobacterium pseudoshottsii in the Eastern Mediterranean.

Among the numerous pathogenic nontuberculous mycobacteria (NTM), which may cause disease in both poikilothermic and homoeothermic organisms, members of the unique clade Mycobacterium ulcerans/Mycobacterium marinum (MuMC) may cause disease in both fish and humans. Here, we describe the emergence of Mycobacterium pseudoshottsii, one of the four MuMC members, in Israel. For many years, M. marinum was the dominant NTM that was diagnosed in Israel as a fish pathogen. To the best of our knowledge, this is the first isolation and genomic characterization of M. pseudoshottsii infecting edible fish from two different fish species farmed in offshore sea cages in the eastern Mediterranean as well as in a recirculating aquaculture system in Israel. We compared the M. pseudoshottsii whole-genome sequences to all available genomic sequences of MuMC in free, publicly accessible databases. IMPORTANCE Mycobacterium pseudoshottsii was first detected in 1997 in the USA, infecting wild striped bass (Morone saxatilis). Since then, several reports from different countries worldwide have shown its capacity to become established in new regions as well as its pathogenicity to saltwater and euryhaline finfish of different genera. Our phylogenetic analysis revealed that the Mycobacterium ulcerans/Mycobacterium marinum clade (MuMC) is divided into two main branches: one that includes M. marinum and M. pseudoshottsii, and the second, which includes other M. marinum isolates as well as two isolates of M. shottsii. Our results reinforce the proposition that the geographical distribution of M. pseudoshottsii is much more extensive than is commonly believed. The emergence of M. pseudoshottsii in different parts of the world and its pathogenic traits that affect finfish of different genera may be a cause for concern among fish farmers, researchers, and environmental organizations.

Prevalence and Molecular Characterization of Extended-Spectrum ß-Lactamase Producing Enterobacterales in Healthy Community Dogs in Israel.

BACKGROUND: antimicrobial resistance is a global problem in human and veterinary medicine. We aimed to investigate the extended spectrum ß-lactamase-producing Enterobacterales (ESBL-PE) gut colonization in healthy community dogs in Israel. METHODS: Rectal swabs were sampled from 145 healthy dogs, enriched, plated on selective plates, sub-cultured to obtain pure cultures, and ESBL production was confirmed. Bacterial species and antibiotic susceptibility profiles were identified. WGS was performed on all of the ESBL-PE isolates and their resistomes were identified in silico. Owners' questionnaires were collected for risk factor analysis. RESULTS: ESBL-PE gut colonization rate was 6.2% (n = 9/145, 95% CI 2.9-11.5). Overall, ten isolates were detected (one dog had two isolates); the main species was Escherichia coli (eight isolates), belonging to diverse phylogenetic groups-B1, A and C. Two isolates were identified as Citrobacter braakii, and C. portucalensis. A phylogenetic analysis indicated that all of the isolates were genetically unrelated and sporadic. The isolates possessed diverse ESBL genes and antibiotic-resistance gene content, suggesting independent ESBL spread. In a multivariable risk factor analysis, coprophagia was identified as a risk factor for ESBL-PE gut colonization (p = 0.048, aOR = 4.408, 95% CI 1.014-19.169). CONCLUSIONS: healthy community dogs may be colonized with ESBL-PE MDR strains, some of which were previously reported in humans, that carry wide and diverse resistomes and may serve as a possible source for AMR.

The activity of the stress modulated Arabidopsis ubiquitin ligases PUB46 and PUB48 is partially redundant.

The Arabidopsis ubiquitin ligases PUB46, PUB47 and PUB48 are encoded by paralogus genes. Single gene pub46 and pub48 mutants display increased drought sensitivity compared to wild type (WT) suggesting that each has specific biological activity. The high sequence homology between PUB46 and PUB48 activity suggested that they may also share some aspects of their activity. Unfortunately, the close proximity of the PUB46 and PUB48 gene loci precludes obtaining a double mutant required to study if they are partially redundant by crossing the available single mutants. We thus applied microRNA technology to reduce the activity of all three gene products of the PUB46-48 subfamily by constructing an artificial microRNA (aMIR) targeted to this subfamily. Expressing aMIR46-48 in WT plants resulted in increased drought-sensitivity, a phenotype resembling that of each of the single pub46 and pub48 mutants, and enhanced sensitivity to methyl viologen, similar to that observed for the pub46 mutant. The WT plants expressing aMIR46-48 plants also revealed reduced inhibition by ABA at seed germination, a phenotype not evident in the single mutants. Expressing aMIR46-48 in pub46 and pub48 mutants further enhanced the drought sensitivity of each parental single mutant and of WT expressing aMIR46-48. These results suggest that the biological activities of PUB46 and PUB48 in abiotic stress response are partially redundant.

High-resolution multilocus sequence typing for Chlamydia trachomatis: improved results for clinical samples with low amounts of C. trachomatis DNA.

BACKGROUND: Several Multilocus Sequence Typing (MLST) schemes have been developed for Chlamydia trachomatis. Bom's MLST scheme for MLST is based on nested PCR amplification and sequencing of five hypervariable genes and ompA. In contrast to other Chlamydia MLST schemes, Bom's MLST scheme gives higher resolution and phylogenetic trees that are comparable to those from whole genome sequencing. However, poor results have been obtained with Bom's MLST scheme in clinical samples with low concentrations of Chlamydia DNA. RESULTS: In this work, we present an improved version of the scheme that is based on the same genes and MLST database as Bom's MLST scheme, but with newly designed primers for nested-1 and nested-2 steps under stringent conditions. Furthermore, we introduce a third primer set for the sequencing step, which considerably improves the performance of the assay. The improved primers were tested in-silico using a dataset of 141 Whole Genome Sequences (WGS) and in a comparative analysis of 32 clinical samples. Based on cycle threshold and melting curve analysis values obtained during Real-Time PCR of nested-1 & 2 steps, we developed a simple scoring scheme and flow chart that allow identification of reaction inhibitors as well as to predict with high accuracy amplification success. The improved MLST version was used to obtain a genovars distribution in patients attending an STI clinic in Tel Aviv. CONCLUSIONS: The newly developed MLST version showed great improvement of assay results for samples with very low concentrations of Chlamydia DNA. A similar concept could be applicable to other MLST schemes.

HiSpike Method for High-Throughput Cost Effective Sequencing of the SARS-CoV-2 Spike Gene.

The changing nature of the SARS-CoV-2 pandemic poses unprecedented challenges to the world's health systems. Emerging spike gene variants jeopardize global efforts to produce immunity and reduce morbidity and mortality. These challenges require effective real-time genomic surveillance solutions that the medical community can quickly adopt. The SARS-CoV-2 spike protein mediates host receptor recognition and entry into the cell and is susceptible to generation of variants with increased transmissibility and pathogenicity. The spike protein is the primary target of neutralizing antibodies in COVID-19 patients and the most common antigen for induction of effective vaccine immunity. Tight monitoring of spike protein gene variants is key to mitigating COVID-19 spread and generation of vaccine escape mutants. Currently, SARS-CoV-2 sequencing methods are labor intensive and expensive. When sequence demands are high sequencing resources are quickly exhausted. Consequently, most SARS-CoV-2 strains are sequenced in only a few developed countries and rarely in developing regions. This poses the risk that undetected, dangerous variants will emerge. In this work, we present HiSpike, a method for high-throughput cost effective targeted next generation sequencing of the spike gene. This simple three-step method can be completed in < 30 h, can sequence 10-fold more samples compared to conventional methods and at a fraction of their cost. HiSpike has been validated in Israel, and has identified multiple spike variants from real-time field samples including Alpha, Beta, Delta and the emerging Omicron variants. HiSpike provides affordable sequencing options to help laboratories conserve resources for widespread high-throughput, near real-time monitoring of spike gene variants.

Extended spectrum ß lactamase-producing Enterobacteriaceae shedding by race horses in Ontario, Canada.

BACKGROUND: We aimed to investigate the prevalence, molecular epidemiology and prevalence factors for Extended Spectrum ß-Lactamase-producing Enterobacteriaceae (ESBL-E) shedding by race horses. A cross-sectional study was performed involving fecal samples collected from 169 Thoroughbred horses that were housed at a large racing facility in Ontario, Canada. Samples were enriched, plated on selective plates, sub-cultured to obtain pure cultures and ESBL production was confirmed. Bacterial species were identified and antibiotic susceptibility profiles were assessed. E. coli sequence types (ST) and ESBL genes were determined using multilocus sequence type (MLST) and sequencing. Whole genome sequencing was performed to isolates harboring CTX-M-1 gene. Medical records were reviewed and associations were investigated. RESULTS: Adult horses (n = 169), originating from 16 different barns, were sampled. ESBL-E shedding rate was 12% (n = 21/169, 95% CI 8-18%); 22 ESBL-E isolates were molecularly studied (one horse had two isolates). The main species was E. coli (91%) and the major ESBL gene was CTX-M-1 (54.5%). Ten different E. coli STs were identified. Sixty-four percent of total isolates were defined as multi-drug resistant. ESBL-E shedding horses originated from 8/16 different barns; whereas 48% (10/21) of them originated from one specific barn. Overall, antibiotic treatment in the previous month was found as a prevalence factor for ESBL-E shedding (p = 0.016, prevalence OR = 27.72, 95% CI 1.845-416.555). CONCLUSIONS: Our findings demonstrate the potential diverse reservoir of ESBL-E in Thoroughbred race horses. Multi-drug resistant bacteria should be further investigated to improve antibiotic treatment regimens and equine welfare.

Draft Genome Sequences of Cronobacter muytjensii Cr150, Cronobacter turicensis Cr170, and Cronobacter sakazakii Cr611.

The sequencing and bioinformatics analyses of isolates Cr150, Cr170, and Cr611 from powdered infant formula indicate that the three strains represent new members in the Cronobacter muytjensii, Cronobacter turicensis, and Cronobacter sakazakii groups, respectively.

Draft Genome Sequence of Cronobacter sakazakii Cr268, Isolated from Infant Powder Formula.

Cronobacter sakazakii is an emerging pathogen that causes meningitis, bacteremia, sepsis, and necrotizing enterocolitis in premature infants. Strain Cr268 was isolated from imported powdered infant formula in 2009 during routine microbial examination according to ISO-22964 ("Microbiology of the food chain-horizontal method for the detection of Cronobacter spp."). Isolate Cr268 was confirmed to be C. sakazakii by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and standard biochemical analysis. Here, we announce its genome, which represents a new member in the C. sakazakii group.