Colistin Resistance Mechanisms in Human Salmonella enterica Strains Isolated by the National Surveillance Enter-Net Italia (2016–2018)

Background: A collection of human-epidemiologically unrelated S. enterica strains collected over a 3-year period (2016 to 2018) in Italy by the national surveillance Enter-Net Italia was analysed. Methods: Antimicrobial susceptibility tests, including the determination of minimal inhibitory concentrations (MICs) for colistin, were performed. Colistin resistant strains were analysed by PCR to detect mobile colistin resistance (mcr) genes. In mcr-negative S. enterica serovar Enteritidis strains, chromosomal mutations potentially involved in colistin resistance were identified by a genomic approach. Results: The prevalence of colistin-resistant S. enterica strains was 7.7%, the majority (87.5%) were S. Enteritidis. mcr genes were identified only in one strain, a S. Typhimurium monophasic variant, positive for both mcr-1.1 and mcr-5.1 genes in an IncHI2 ST4 plasmid. Several chromosomal mutations were identified in the colistin-resistant mcr-negative S. Enteritidis strains in proteins involved in lipopolysaccharide and outer membrane synthesis and modification (RfbN, LolB, ZraR) and in a component of a multidrug efflux pump (MdsC). These mutated proteins were defined as possible candidates for colistin resistance in mcr-negative S. Enteritidis of our collection. Conclusions: The colistin national surveillance in Salmonella spp. in humans, implemented with genomic-based surveillance, permitted to monitor colistin resistance, determining the prevalence of mcr determinants and the study of new candidate mechanisms for colistin resistance.

Penicillinase plasmid Australia type in Neisseria gonorrhoeae isolated in Poland

Purpose Neisseria gonorrhoeae is an etiological agent of gonorrhea which remains a major public health problem the mechanisms that determine resistance to drugs of the beta-lactam class, which are recommended for the treatment of gonorrhea, are currently the most important problem in its treatment. Chromosomal mutations are responsible for resistance to ceftriaxone and cefepime. The possibility of mutations in the gene encoding beta-lactamase (blaTEM) in the penicillinase plasmid may also turn out to be a serious threat. Methods The occurrence of resistance encoded on penicillinase plasmid has been investigated. For this purpose, the susceptibility of bacteria was determined and the gene for resistance to beta-lactams as well as the plasmids themselves was typed. Results Of the 333 strains tested, 21 (6.3%) had the beta-lactamase gene and produced penicillinase. Two of the beta-lactamase: TEM-1 and TEM-135 occurred among the tested strains of N. gonorrhoeae. Most of the known penicillinase plasmid types of N. gonorrhoeae were demonstrated: the Asian, the African, the Toronto/Rio plasmids and Australian variants. Conclusions In the first 3 years, TEM-1 beta-lactamases dominated in N. gonorrhoeae, which were replaced by TEM-135 in the following years of the study. Not all molecular methods are capable of varying the types of penicillinase plasmids. A particularly noteworthy observation is the fact that the Australia-type of penicillinase plasmid (3270 bp) was identified for the first time in Europe, and the second time in the world.

Selection of AmpC β-lactamase variants and metallo-β-lactamases leading to ceftolozane/tazobactam and ceftazidime/avibactam-resistance during treatment of MDR/XDR Pseudomonas aeruginosa infections

Infections caused by ceftolozane/tazobactam and ceftazidime/avibactam-resistant P. aeruginosa infections are an emerging concern. We aimed to analyze the underlying ceftolozane/tazobactam and ceftazidime/avibactam resistance mechanisms in all MDR/XDR P. aeruginosa isolates recovered during one year (2020) from patients with a documented P. aeruginosa infection. Fifteen isolates showing ceftolozane/tazobactam and ceftazidime/avibactam resistance were evaluated. Clinical conditions, previous positive cultures and β-lactams received in the previous month were reviewed for each patient. MICs were determined by broth microdilution. MLSTs and resistance mechanisms were determined using short- and long-read WGS. The impact of PDCs on β-lactam resistance was demonstrated by cloning into an ampC -deficient PAO1 derivative (PAOΔC) and construction of 3D models. Genetic support of acquired β-lactamases was determined in silico from high-quality hybrid assemblies. In most cases, the isolates were recovered after treatment with ceftolozane/tazobactam or ceftazidime/avibactam. Seven isolates from different STs owed their β-lactam resistance to chromosomal mutations and all displayed specific substitutions in PDC: Phe121Leu and Gly222Ser, Pro154Leu, Ala201Thr, Gly214Arg, ΔGly203-Glu219 and Glu219Lys. In the other eight isolates, the ST175 clone was overrepresented (6 isolates) and associated with IMP-28 and IMP-13, whereas two ST1284 isolates produced VIM-2. The cloned PDCs conferred enhanced cephalosporin resistance. 3D PDC models revealed rearrangements affecting residues involved in cephalosporin hydrolysis. Carbapenemases were chromosomal (VIM-2) or plasmid-borne (IMP-28, IMP-13), and associated with class-1 integrons located in Tn402-like transposition modules. Our findings highlight that cephalosporin/ß-lactamase inhibitors are potential selectors of MDR/XDR P. aeruginosa strains producing PDC variants or metallo-ß-lactamases. Judicious use of these agents is encouraged.

Prevalence and Molecular Characterization of Fluoroquinolone-Resistant Escherichia coli in Healthy Children

Faecal E. coli can act as reservoirs for resistance genes. Here, we analyzed prevalence of drug resistance in faecal E. coli isolated from healthy children at a single kindergarten in Beijing, China, then used whole genome sequencing to characterize fluoroquinolone-non-susceptible strains. Our results revealed high resistance to ampicillin (54.0%), trimethoprim/sulphurmethoxazole (47.5%) and tetracycline (58.9%) among 576 faecal E. coli isolates, 49.2% of which exhibited multidrug resistance. A total of 113 E. coli isolates were not susceptible to ciprofloxacin, with four sequence types, namely ST1193 (25.7%), ST773 (13.3%), ST648 (8.8%) and ST131 (7.1%) found to be the most prevalent (54.9%). With regards to resistance to quinolones, we detected chromosomal mutations in gyrA, parC, and parE in 111 (98.2%), 105 (92.9%), and 67 (61.1%) isolates, respectively. blaCTX-M (37.2%) was the major ESBL gene, whereas blaCTX-M-14 (12.4%) and blaCTX-M-27 (11.5%) were the most frequent subtypes. A total of 90 (79.6%) ExPEC and 65 (57.5%) UPEC isolates were classified. Overall, these findings revealed clonal spread of certain prevalent STs, namely ST1193, ST773, ST648 and ST131 E. coli isolates in healthy children within a single kindergarten in Beijing, China, affirming the seriousness of the multidrug resistance problem and potential pathogenicity of E. coli isolates in healthy children. Therefore, there is an urgent need for increased surveillance to enhance control of this problem.

Use of growth hormone in region 19p13.3 microduplication syndrome in girl with central early puberty: a clinical case report

Chromosomal mutations involving 19p13.3 have been described as pathogenic. clinical and phenotypic features can include, in most cases, psychomotor development delay, microcephaly, typical facial appearance, hand and foot anomalies, umbilical hernia, hypotonia, and low percentage of lean mass. The main types of mutation found on this chromosome are deletion or duplication. Short stature is often the cause of medical demand and the use of exogenous GH for patients with this syndrome is not beneficial. This article reports the case of a 5-year-old girl who sought medical help due to short stature and was diagnosed with this syndrome. Furthermore, this case study may contribute to the dissemination in the medical community about the association of this genetic mutation with the child's clinical condition, warning about this syndrome, and the possibility of the occurrence of early puberty. This study was analyzed and approved by the Research Ethics Committee (CEP) according to a substantiated opinion number 4.765.113.

Genotypic Diversity of Ciprofloxacin Nonsusceptibility and Its Relationship with Minimum Inhibitory Concentrations in Nontyphoidal Salmonella Clinical Isolates in Taiwan

This study analyzed the genetic diversity of ciprofloxacin (CIP) nonsusceptibility and the relationship between two major mechanisms and minimum inhibitory concentrations (MICs) of CIP in nontyphoidal Salmonella (NTS). Chromosomal mutations in quinolone resistance-determining regions (QRDRs) and plasmid-mediated quinolone resistance (PMQR) genes were searched from ResFinder, ARG-ANNOT, and PubMed for designing the sequencing regions in gyrA, gyrB, parC, and parE, and the 13 polymerase chain reactions for PMQR genes. We found that QRDR mutations were detected in gyrA (82.1%), parC (59.0%), and parE (20.5%) but not in gyrB among the 39 isolates. Five of the 13 PMQR genes were identified, including oqxA (28.2%), oqxB (28.2%), qnrS (18.0%), aac(6′)-Ib-cr (10.3%), and qnrB (5.1%), which correlated with the MICs of CIP within 0.25–2 μg/mL, and it was found that oxqAB contributed more than qnr genes to increase the MICs. All the isolates contained either QRDR mutations (53.8%), PMQR genes (15.4%), or both (30.8%). QRDR mutations (84.6%) were more commonly detected than PMQR genes (46.2%). QRDR mutation numbers were significantly associated with MICs (p < 0.001). Double mutations in gyrA and parC determined high CIP resistance (MICs ≥ 4 μg/mL). PMQR genes contributed to intermediate to low CIP resistance (MICs 0.25–2 μg/mL), thus providing insights into mechanisms underlying CIP resistance.

Chromosomal mutations in high-yield Holstein cows

The results of the research of somatic chromosomal instability: aneuploidy and polyploidy in the blood cells of high-yield Holstein cows with the productivity of more than 9000 kg have been presented in the article. The research was conducted in JSC Vaganovo, Promyshlennovsky District, Kemerovo Region on the population of Holstein cows. Sample preparation was carried out accord- ing to P. Moorhead et al. Samples were stained using the Romanowsky-Giemsa method. The authors studied 6068 metaphase plates in a group of 30 healthy animals. Aneuploidy was counted according to the practice of N.P. Bochkov et al. The authors also analysed the ecological situation in the area of keeping and breeding cattle. Studies of soil, fodder, organs and tissues in farm animals of different species confirm that heavy metal and chemical pollutants are not contaminated in Western Siberia, and their levels are within sanitary norms. Therefore, the ecological situation in the study area should be considered safe. The study shows that the frequency of polyploidy was 0.59% in the studied sample. Tetraploidy, triploidy, and hexaploidy rates were 0.33; 0.18 and 0.06, respectively, the frequency of true hypoploidy was 0.73, and the number of diploid cells was 84.7%. The data obtained can be tentatively applied as average values and can also be used to evaluate the interior of the animals.

The bystander effect formation in the meristem of Allium cepa L. plants with different genotype

Aim. Obtaining and analysis of cytogenetic parameters in meristem cells of Allium cepa L. seedling sprouting from unirradiated seeds, while germinating them with seeds, that were exposed to γ-radiation in different doses. Methods. Cytogenetic analysis included analysis of mitotic activity, anaphase method of chromosome mutations, nucleolar analysis. The F-test was used to compare the control and experimental samples. Results. The formation of the bystander effect is shown in the case of joint germination of irradiated and non-irradiated seeds. The severity of bystander effect increases with increasing irradiation dose. Conclusions. Irradiation of seeds with a dose of gamma radiation of 40 Gy causes the formation of radiation hormesis by the criterion of mitotic activity in the root meristem cells of seedlings, and the irradiation with a dose of 10 Gy does not affect the level of cell proliferation. The bystander effect formation occurs during the joint germination of intact and irradiated seeds as an increase in the level of mitotic activity, and an increase in the number of chromosomal mutations. Keywords: bystander effect, γ-radiation, meristem, mitotic activity, chromosomal abnormalities, micronuclei.

Structural Variations in the Genome of Potato Varieties of the Ural Selection

Potato (Solanum tuberosum L.) is the third most common plant crop in the world. Many studies, such as those using marker-assisted selection (MAS), are devoted to the genomic evaluation of potato. However, the nucleotide composition of some markers has not been described yet, and some regions of the genome remain unknown. Thus, the development of new marker systems for further genetic selection is required. Whole-genome sequencing and the search for structural variants (SVs) should further develop molecular genetic studies of the potato crop. In this paper, we will show the first results of our studies of the three backcrossed varieties Alaska, Argo, and Shah, which were selected for sequencing. Alaska is a patented variety with confirmed resistance to pathogens, while Argo and Shah are new perspective varieties. We sequenced genomes of these varieties using a nanopore platform. As a result, we identified more than 24,000 authentic structural variants with lengths varying from 4 bp to 100 Mbp. Deletion was found to be the most common type of structural variant in the genome and the genes. The majority of SVs are located in non-coding regions, including introns. However, a quarter of the genes of the sequenced varieties have some chromosomal mutations. Some genes responsible for resistance to abiotic stress and pathogens were duplicated, while genes of nucleic acid polymerization and few metabolic proteins were deleted.

Bacterial resistance to CRISPR-Cas antimicrobials

In the age of antibiotic resistance and precise microbiome engineering, CRISPR-Cas antimicrobials promise to have a substantial impact on the way we treat diseases in the future. However, the efficacy of these antimicrobials and their mechanisms of resistance remain to be elucidated. We systematically investigated how a target E. coli strain can escape killing by episomally-encoded CRISPR-Cas9 antimicrobials. Using Cas9 from Streptococcus pyogenes (SpCas9) we studied the killing efficiency and resistance mutation rate towards CRISPR-Cas9 antimicrobials and elucidated the underlying genetic alterations. We find that killing efficiency is not correlated with the number of cutting sites or the type of target. While the number of targets did not significantly affect efficiency of killing, it did reduce the emergence of chromosomal mutations conferring resistance. The most frequent target of resistance mutations was the plasmid-encoded SpCas9 that was inactivated by bacterial genome rearrangements involving translocation of mobile genetic elements such as insertion elements. This resistance mechanism can be overcome by re-introduction of an intact copy of SpCas9. The work presented here provides a guide to design strategies that reduce resistance and improve the activity of CRISPR-Cas antimicrobials.