How to: screening for mcr-mediated resistance to colistin.

BACKGROUND: Colistin belongs to the last-resort antibiotics. The discovery of plasmid-bound colistin resistance mediated by the mcr-gene(s) is of great concern because, given its biological potential, there is a risk of its rapid spread. OBJECTIVES: To discuss the current literature on the methods for the screening for mcr-mediated resistance to colistin. SOURCES: Literature was drawn from a search of PubMed from 1 January 2016 to 26 April 2021. CONTENT: The selective culture-based or culture-independent approach can be used for the screening of mcr-mediated resistance to colistin in clinical samples. Rapid Polymyxin NP, Colistin Drop or Colistin Agar Spot tests are applicable for the selection of isolates with a suspected resistance to colistin that has to be confirmed by broth microdilution. The mcr-mediated resistance to colistin can be confirmed by the detection of the causal gene(s) or by phenotype using EDTA-colistin broth disc elution; production of the MCR-1 enzyme can be confirmed with lateral flow immunoassay, using matrix-assisted laser desorption/ionization time-of flight or liquid chromatography-based mass spectrometry. Whole-genome sequencing (WGS) is the ultimate typing method. When a WGS platform is not available at a healthcare facility, a WGS-outsourced service, in combination with freely available bioinformatics tools, allows for the characterization of the mcr-gene(s) carrying isolates. IMPLICATIONS: mcr-mediated colistin resistance should be monitored through active targeted screening. The broth microdilution method is required for colistin susceptibility testing but as only a selected number of clinical isolates are tested, colistin resistance, including mcr-mediated, may remain undetected. In mcr-1-positive Escherichia coli isolates, the MIC to colistin can range from 2 to 8 mg/L, so it is proposed that Enterobacterales with a colistin MIC of 2 mg/L should also be included in the mcr-mediated colistin resistance screening and those with a confirmed mcr-genotype and/or MCR-phenotype should be considered to be colistin-resistant.

The Intestinal Carriage of Plasmid-Mediated Colistin-Resistant Enterobacteriaceae in Tertiary Care Settings.

Background: In order to estimate the prevalence of plasmid borne colistin resistance and to characterize in detail the mcr-positive isolates, we carried out a sentinel testing survey on the intestinal carriage of plasmid-mediated colistin-resistant Enterobacteriaceae in hospitalized patients. Methods: Between June 2018 and September 2019, 1922 faecal samples from hospitalised patients were analysed by selective culture in presence of colistin (3.5 mg/L), and in parallel by direct detection of the mcr-1 to mcr-8 genes by qPCR. The mcr-positive isolates were characterised by whole-genome sequencing. Results: The prevalence of the mcr-1 gene was 0.21% (n = 4/1922); the mcr-2 to 8 genes were not detected. The mcr-1 gene was found to be localised in the IncX4 (n = 3) and IncHI2 (n = 1) plasmid type. One Escherichia coli isolate was susceptible to colistin due to the inactivation of the mcr-1 gene through the insertion of the IS2 element; however, the colistin resistance was inducible by culture in low concentrations of colistin. One human mcr-1 positive E. coli isolate was related genetically to the mcr-1 E. coli isolate derived from turkey meat of Czech origin. Conclusions:mcr-mediated colistin resistance currently poses little threat to patients hospitalised in Czech healthcare settings. The presence of the mcr-1 gene in the human population has a possible link to domestically produced, retail meat.

[Methods for detecting plasmid-borne colistin resistance mediated by the mcr genes].

BACKGROUND: Colistin is a last-resort antibiotic used for the treatment of infections caused by multidrug-resistant Gram-negative pathogens. The emergence of plasmid-borne colistin resistance mediated by the mcr genes poses a risk of its spread and its occurrence should be monitored. The aim of this study was to discuss possible detection methods and their reliability in screening for this type of resistance. MATERIAL AND METHODS: The reliability of the disk diffusion method, Rapid Polymyxin NP test and two types of gradient tests for the screening of mcr-mediated colistin resistance was evaluated on 16 human and two reference isolates of Escherichia coli (colistin-susceptible and colistin-resistant with chromosomally-mediated resistance or harboring the mcr-1 gene). Broth microdilution was the reference method used for the determination of colistin resistance. RESULTS: Targeted screening for colistin-resistant strains is best performed with a selective agar medium supplemented with colistin. In cultured isolates, suspected colistin resistance should always be confirmed by reliable methods. There was 100 percent agreement between both the gradient methods and the Rapid Polymyxin NP test and the reference broth microdilution method. When using the disk diffusion method with 10µg and 50µg disks, only 11 % and 33 % of results were correct, respectively. Therefore, disk diffu-sion is inappropriate for colistin resistance screening. CONCLUSION: Prospective prevalence studies of intestinal carriage of colistin-resistant Enterobacterales among Czech hospitalized patients and travelers do not yet indicate the spread of strains with mcr-mediated plasmid-borne colistin resistance. However, the high prevalence of strains carrying the mcr genes in raw meat products poses a risk of exposure for their consumers. The detection of mcr-harbouring colistin-resistant strains cannot be expected during routine microbiological testing, without using reliable but expensive methods. A suitable alternative method for active monitoring of colistin resistance is the use of selective agar media with colistin followed by verification of the resistance in the obtained isolates.

The colonisation of Czech travellers and expatriates living in the Czech Republic by colistin-resistant Enterobacteriaceae and whole genome characterisation of E. coli isolates harbouring the mcr-1 genes on a plasmid or chromosome: A cross-sectional study.

BACKGROUND: Travellers were recognized as a risk cohort that can be colonized by mcr-1-mediated colistin-resistant Enterobacteriaceae. We aimed to investigate the carriage of mcr-mediated colistin resistance in Enterobacteriaceae in Czech travellers or expatriates residing temporarily in the Czech Republic. METHODS: Between August 2018 and September 2019, the stool samples were cultured in enrichment broth. The enriched cultures were tested for the presence of the mcr-1-8 genes and inoculated onto selective agar with colistin. Colistin-resistant Enterobacteriaceae were tested for the presence of the mcr-1-8 genes; the mcr-positive isolates were characterised by whole genome sequencing. RESULTS: From the 177 stool samples, 15 colistin-resistant Enterobacteriaceae isolates were cultured (7.9%); two of the E. coli isolates carried the mcr-1 gene (1.1%). In the E. coli multilocus sequence type (ST) 156, the mcr-1 gene was located in an ISApl1-mcr-1-orf-ISApl1 (Tn6330) and incorporated into the chromosome; in the E. coli ST23 isolate, the mcr-1 gene was harboured by the plasmid IncX4. Both of the mcr-1 positive E. coli isolates were multidrug-resistant and one isolate was an extended-spectrum ß-lactamase producer (blaCTX-M-27). CONCLUSION: Patients with an international travel history should be monitored for the carriage of the mcr-1 gene in order to prevent its dissemination into healthcare settings.

The molecular epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) in the Czech Republic.

OBJECTIVES: To gain data on the current molecular epidemiology and resistance of MRSA in the Czech Republic. METHODS: Between September 2017 and January 2018, a total of 441 single-patient MRSA isolates were collected from 11 Czech hospitals and analysed by spa typing, SCCmec typing, antibiotic susceptibility testing, detection of the PVL toxin and the arcA gene. RESULTS: Of all MRSA isolates, 81.41% (n = 359) belonged to the CC5-MRSA clone represented by the spa types t003 (n = 136), t586 (n = 92), t014 (n = 81), t002 (n = 20) and other spa types (n = 30); a majority of the CC5 isolates (n = 348, 96.94%) carried SCCmec type II. The occurrence of CC5-MRSA was more likely in older inpatients and associated with a healthcare origin (P < 0.001). The CC5-MRSA isolates were resistant to more antimicrobial drugs compared with the other MRSAs (P < 0.001). Interestingly, t586 was detected in blood samples more often than the other spa types and, contrary to other spa types belonging to CC5-MRSA, t586 was not associated with patients of advanced age. Other frequently found lineages were CC8 (n = 17), CC398 (n = 11) and CC59 (n = 10). The presence of the PVL was detected in 8.62% (n = 38) of the MRSA isolates. CONCLUSIONS: The healthcare-associated CC5-MRSA-II lineage (t003, t586, t014) was found to be predominant in the Czech Republic. t586 is a newly emerging spa type in the Czech Republic, yet reported rarely in other countries. Our observations stress the need for MRSA surveillance in the Czech Republic in order to monitor changes in MRSA epidemiology.

[The phenomenon of bacterial persistence to antibiotics].

Bacterial persistence in clinical microbiology is a phenomenon where the bacterial subpopulation of any bacterial strain, without having been exposed to an antibiotic, is already persistent to it. In clinical bacterial strains, persistence is not tested at all and the role of this phenomenon in the treatment of bacterial infections has not yet been evaluated. Therefore, the aim of the article is to highlight the significance of this probably global phenomenon in the treatment of bacterial infections with antibiotics. Also described are the mechanisms of its origin and some manner that could potentially reduce the frequency of these antibiotic-resistant bacterial cells in the bacterial population.

Incapacity for work of employees of the selected metallurgical plant related to age.

Evaluation of the health state of employees in relation to the working environment is one of the main tasks of occupational medicine. The specified results represent a part of a methodically oriented project which was focused on evaluation of: age structures of employees in the metallurgical plant being monitored in comparison with the age structure in the whole region, evaluation of the health status of employees in the plan by means of absolute and relative indicators of morbidity and even their dependence on age. The analysis was carried out during three successive years in approximately 10 000 employees of a metallurgical plant. The selected six groups of diagnoses represented from 40 % to 50 % of all employees suffering from a disease. Particularly the following diagnoses according to the International Classification of Diseases were involved there: I10-I15 (hypertensive disease), I20-I25 (ischaemic heart disease), J00-J06 (acute infections of upper respiratory airways), K20-K31 (diseases of oesophagus, stomach and duodenum), M15-M19 (arthroses) and M50-M54 (dorsopathies). The age structure of employees related to men and women was considerably statistically significantly different from the age structure of the region. From the economic point of view on incapacity for work, we consider the number of days of absence from work for the reason of the disease as an indicator with the highest informative value. We noted two dominative maxima in men and women for this characteristic. A proportion of patients with acute infections of upper respiratory airways ranged from 40 % to 50 % out of total cases of diagnoses we have followed up, the highest number of patients was in men aged 20-24 with a subsequent significant decrease. We did not find any noticeable dependence on age for this diagnosis in women, only a slight increase in older age categories was evident. Dorsopathies are the second most frequent group; they were from 30 to 38 % out of all monitored diseases. The age dependence was evident especially in women aged 40+. Considering duration of incapacity for work and its repeatability, dorsopathies seem to be the most relevant health problem in employees in heavy industry, and especially in women. The participation of the remaining four selected groups of diseases is relatively low. Both in men and women, the hypertensive disease ranged from 1.9 %-3.7 %, ischaemic heart disease from 0.4 %-6.5 %, diseases of oesophagus, stomach and duodenum from 5.3 %-9.0 % and arthroses from 4.7 %-6.8 % in three years in question.