[This corrects the article DOI: 10.3389/fcimb.2024.1407246.].

• Publication type
• Published Erratum MeSH

INTRODUCTION: In the battle against multidrug-resistant bacterial infections, ceftazidime- avibactam (CZA) stands as a pivotal defense, particularly against carbapenemresistant (CR) Gram-negative pathogens. However, the rise in resistance against this drug poses a significant threat to its effectiveness, highlighting the critical need for in-depth studies about its resistance mechanisms. METHODS: This research focuses on the genomic characterization of CR- and CZA-resistant Escherichia coli (n=26) and Klebsiella pneumoniae (n=34) strains, harboring the blaNDM and/or blaOXA-48-like genes, at a major Lebanese tertiary care medical center, using whole genome sequencing (WGS). RESULTS: Our findings revealed a notable prevalence of blaNDM in all K. pneumoniae strains isolates, with 27 of these also harboring blaOXA-48. On the other hand, E. coli strains predominantly carried the blaNDM-5 gene. Whole genome sequencing (WGS) identified a predominance of ST383 among K. pneumoniae strains, which possessed a multi-replicon IncFIB-IncHI1B plasmid harboring the blaNDM-5. Additionally, various Inc group plasmids in K. pneumoniae across multiple sequence types were found to carry the blaNDM. Similarly, diverse STs of E. coli were observed to carry blaNDM-5 on different plasmids. DISCUSSION: The study underscores NDM carbapenemases as a paramount resistance mechanism in Lebanon,jeopardizing critical last-resort treatments. It also illuminates the role of varied sequence types and mobile genetic elements in the spread of NDM resistance,stressing the urgent need for strategies to mitigate this threat, especially in nosocomial infections.

• MeSH
• Anti-Bacterial Agents * pharmacology   MeSH
• Azabicyclo Compounds * pharmacology   MeSH
• Bacterial Proteins genetics   metabolism   MeSH
• beta-Lactamases * genetics   metabolism   MeSH
• Ceftazidime * pharmacology   MeSH
• Tertiary Care Centers MeSH
• Carbapenem-Resistant Enterobacteriaceae genetics   drug effects   isolation & purification   MeSH
• Escherichia coli * genetics   drug effects   MeSH
• Drug Combinations * MeSH
• Genome, Bacterial MeSH
• Carbapenems * pharmacology   MeSH
• Klebsiella pneumoniae * genetics   drug effects   MeSH
• Humans MeSH
• Microbial Sensitivity Tests MeSH
• Drug Resistance, Multiple, Bacterial * genetics   MeSH
• Plasmids genetics   MeSH
• Gene Transfer, Horizontal MeSH
• Whole Genome Sequencing * MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Lebanon MeSH

A 2-year national genomic screening in the Czech Republic identified a notable prevalence of the New Delhi metallo-β-lactamase 5 (NDM-5)-producing Escherichia coli sequence type 38 (ST38) in the city of Brno. Forty-two ST38 E. coli isolates harbored the blaNDM-5 gene on the chromosome. Virulence factors confirmed the persistence of these isolates through biofilm formation. Single Nucleotide Polymorphisms (SNPs)-based phylogeny and CRISPR assay typing showed minimal genomic variations, implying a clonally driven outbreak. Results suggest that this high-risk clone may impose a nationwide problem.

• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• beta-Lactamases * genetics   MeSH
• Biofilms growth & development   MeSH
• Disease Outbreaks * MeSH
• Escherichia coli * genetics   drug effects   isolation & purification   enzymology   MeSH
• Virulence Factors genetics   MeSH
• Phylogeny MeSH
• Genome, Bacterial MeSH
• Genomics methods   MeSH
• Escherichia coli Infections * microbiology   epidemiology   MeSH
• Polymorphism, Single Nucleotide * MeSH
• Humans MeSH
• Microbial Sensitivity Tests MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Czech Republic MeSH

OBJECTIVE: Here we describe a novel IncFIA plasmid harbouring mcr-10 gene in a clinical Enterobacter ludwigii strain isolated at the University Hospital in Pilsen in the Czech Republic. METHODS: The strain was subjected to antibiotic susceptibility testing. Whole genome sequencing was performed using Illumina for short-read sequencing and Oxford Nanopore Technologies for long-read sequencing followed by hybrid assembly. The resulting genome was used to detect species using average nucleotide identity, resistance genes, plasmid replicon and MLST (using centre for genomic epidemiology databases; ResFinder, PlasmidFinder and MLST, respectively) and virulence genes using VFDB. RESULTS: Τhe strain showed susceptibility against tetracycline, cefuroxime and chloramphenicol, and it was susceptible to the second and third generation of cephalosporins, carbapenems and colistin. Genome analysis identified the strain as E. ludwigii sequence type ST20 and located the mcr-10 gene on an IncFIA (HI1)/IncFII (Yp) plasmid (pI9455333_MCR10; 129 863 bp). Upon blasting the nucleotide sequence of pI9455333_MCR10 against the NCBI database, no similar plasmid sequence was detected, implying a novel plasmid structure. Nevertheless, it showed a partial similarity with pRHBSTW-00123_3 and FDAARGOS 1432, which were detected in Enterobacter cloacae complex (ECC) strains in wastewater samples in 2017 in UK and in 2021 in the United States, respectively, and pEC81-mcr, which was detected in a clinical Escherichia coli strain in 2020 in China. Moreover, I9455333cz genome carried virulence genes coding for curli fibers, fimbrial adherence determinants, siderophore aerobactin, iron uptake proteins and regulators of sigma factor. CONCLUSION: In conclusion, we identified a novel IncF plasmid harbouring mcr-10 gene in a clinical Enterobacter ludwigii strain. To our knowledge, this is the first clinical report of mcr-10 in the Czech Republic.

• MeSH
• Anti-Bacterial Agents * pharmacology   MeSH
• Bacterial Proteins genetics   MeSH
• Tertiary Care Centers * MeSH
• Enterobacter * genetics   drug effects   isolation & purification   MeSH
• Enterobacteriaceae Infections * microbiology   MeSH
• Humans MeSH
• Microbial Sensitivity Tests * MeSH
• Drug Resistance, Multiple, Bacterial genetics   MeSH
• Multilocus Sequence Typing MeSH
• Plasmids * genetics   MeSH
• Whole Genome Sequencing MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Geographicals
• Czech Republic MeSH

• MeSH
• Bacterial Proteins * genetics   MeSH
• beta-Lactamases * genetics   analysis   MeSH
• Lactones metabolism   MeSH
• Humans MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization * methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Letter MeSH

Paenibacillus larvae and Melissococcus plutonius represent the most threatening bacterial diseases of honeybee (Apis mellifera)-American and European foulbrood, respectively. For efficient control of those diseases, rapid and accurate detection of the pathogens is crucial. Therefore, we developed a novel multiplex PCR method simultaneously detecting both pathogens. To design and optimize multiplex PCR reaction, four strains of P. larvae representing four ERIC genotypes I-IV (strain DSM 7030-ERIC I, DSM 25430-ERIC II, LMG 16252-ERIC III, DSM 3615-ERIC IV) were selected. Those strains were fully sequenced using long-read sequencing (Sequel I, Pacific Biosciences). For P. larvae, the multicopy insertion sequence IS256 identified in all genotypes of P. larvae was selected to provide high sensitivity. M. plutonius was detected by plasmid pMP1 sequence and the virulence verified by following detection of ETX/MTX2 toxin responsible for pore formation in the cell membrane. As an internal control, a gene encoding for major royal jelly protein 1 specific for honeybees was selected. The method was validated on 36 clinical specimens collected from the colonies suffering from American and European foulbrood in the Czech Republic. Based on the results, sensitivity of PCR was calculated to 93.75% and specificity to 100% for P. larvae diagnosed from hive debris and 100% sensitivity and specificity for honeybee workers and larval scales as well as for diseased brood infected by M. plutonius.

• MeSH
• Enterococcaceae * MeSH
• Larva microbiology   MeSH
• Multiplex Polymerase Chain Reaction methods   MeSH
• Paenibacillus larvae * genetics   MeSH
• Paenibacillus * genetics   MeSH
• Plasmids genetics   MeSH
• DNA Transposable Elements MeSH
• Bees genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH

Enterobacterales produkující karbapenemázu (CPE – Carbapenemase-Producing Enterobacterales) představují globální zdravotnický problém. V Evropě dochází zejména v jižních a východních státech ke zvyšování podílu enterobakterií rezistentních vůči karbapenemům. Od roku 2021 roste také v České republice výskyt kmenů s produkcí karbapenemázy, změnila se i struktura zastoupení jednotlivých enzymů, zejména ve prospěch metalobetalaktamázy typu NDM. Tato situace je zřejmě následkem epidemie covid-19 a zhoršenou epidemickou situací ve zdravotnických zařízeních.

Carbapenemase-producing Enterobacterales (CPE) are a global health problem. In Europe, the proportion of carbapenem resistant enterobacteria is increasing, particularly in the southern and eastern countries. Since 2021, there has also been a widespread increase in the occurrence of CPE in the Czech Republic, the structure of carbapenemases has also shifted towards higher proportion of the NDM type. This situation is apparently the result of the COVID-19 epidemic and the worsened epidemic situation in healthcare facilities.

Health-care associated infections (HCAIs) significantly influence current medicine and may limit the progress in many medical procedures. Treatment of such infections has been increasingly complicated by the spread of antimicrobial resistance (AMR). To reduce risks of inappropriate initial therapy, the availability of rapid diagnostic microbiological assays is of utmost importance. Especially, functional diagnostics (i.e., detection of enzyme activity) is important in era of horizontal gene transfer. Functional assays for detection of selected bacterial toxins (ToxB of C. difficile and YopT of Y. enterocolitica) using affinity MALDI will be developed and validated. Simultaneously, functional assay for detection of carbapenemase activity developed in the laboratory of co-investigator will be further optimized and validated. Further, a new assay for functional detection of lipid A modification detecting colistin resistance will be also developed. All of those assays and tests represent a challenge for symptom-based microbiology and functional detection of important antibiotic resistance. Introduction of such assays to routine diagnostics will help to detect potentially unknown pathogens emerged because of horizontal gene transfer.

Infekce spojené se zdravotní péčí (HCAI) významně ovlivňují současnou medicínu a limitují rozvoj některých invazivních zákroků. Léčba HCAI je obvykle komplikovaná rezistencí původců na dostupná antibiotika. Ke snížení nevhodné iniciální terapie, která často vede k selhání léčby, je nutné se zaměřit na vývoj nových rychlých diagnostických testů. Především se jedná o metody funkční diagnostiky, které reagují na současný stav horizontálního toku genetické informace. V rámci řešení projektu budou vyvinuty a validovány testy pro detekci některých bakteriálních toxinů (např. toxin B produkovaný Clostridium difficile, YopT produkovaný Yersinia enterocolitica) pomocí metody afinitní MALDI-TOF hmotnostní spektrometrie. Zároveň budou optimalizovány metody pro detekci karbapenemáz dříve vyvinuté v laboratoři spoluřešitele. Pro detekci kolistinové rezistence bude vyvinuta metoda umožňující detekci modifikace lipidu A. Všechny zmíněné metodiky reagují na urgentní potřebu rutinní mikrobiologické diagnostiky a umožní detekovat i potenciálně neznámé patogeny s ohledem na horizontální přenos genetické informace.

• Keywords
• antibiotická terapie, antibiotic therapy, Kolistin, colistin, antibiotic resistance, Hmotnostní spektrometrie, Mass Spectrometry, antibiotická rezistence, karbapenemázy, lipid A, carbapenemase, lipid A, Funkční diagnostika, Functional diagnostics,

• NML Publication type
• závěrečné zprávy o řešení grantu AZV MZ ČR

Antimicrobial resistance is well-known to be a global health and development threat. Due to the decrease of effective antimicrobials, re-evaluation in clinical practice of old antibiotics, as fosfomycin (FOS), have been necessary. FOS is a phosphonic acid derivate that regained interest in clinical practice for the treatment of complicated infection by multi-drug resistant (MDR) bacteria. Globally, FOS resistant Gram-negative pathogens are raising, affecting the public health, and compromising the use of the antibiotic. In particular, the increased prevalence of FOS resistance (FOSR) profiles among Enterobacterales family is concerning. Decrease in FOS effectiveness can be caused by i) alteration of FOS influx inside bacterial cell or ii) acquiring antimicrobial resistance genes. In this review, we investigate the main components implicated in FOS flow and report specific mutations that affect FOS influx inside bacterial cell and, thus, its effectiveness. FosA enzymes were identified in 1980 from Serratia marcescens but only in recent years the scientific community has started studying their spread. We summarize the global epidemiology of FosA/C2/L1-2 enzymes among Enterobacterales family. To date, 11 different variants of FosA have been reported globally. Among acquired mechanisms, FosA3 is the most spread variant in Enterobacterales, followed by FosA7 and FosA5. Based on recently published studies, we clarify and represent the molecular and genetic composition of fosA/C2 genes enviroment, analyzing the mechanisms by which such genes are slowly transmitting in emerging and high-risk clones, such as E. coli ST69 and ST131, and K. pneumoniae ST11. FOS is indicated as first line option against uncomplicated urinary tract infections and shows remarkable qualities in combination with other antibiotics. A rapid and accurate identification of FOSR type in Enterobacterales is difficult to achieve due to the lack of commercial phenotypic susceptibility tests and of rapid systems for MIC detection.

• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Drug Resistance, Bacterial genetics   MeSH
• Escherichia coli genetics   MeSH
• Fosfomycin * pharmacology   MeSH
• Klebsiella pneumoniae genetics   MeSH
• Escherichia coli Proteins * genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH

OBJECTIVES: Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is a widely used method for bacterial species identification. Incomplete databases and mass spectral quality (MSQ) still represent major challenges. Important proxies for MSQ are the number of detected marker masses, reproducibility, and measurement precision. We aimed to assess MSQs across diagnostic laboratories and the potential of simple workflow adaptations to improve it. METHODS: For baseline MSQ assessment, 47 diverse bacterial strains, which are challenging to identify by MALDI-TOF MS, were routinely measured in 36 laboratories from 12 countries, and well-defined MSQ features were used. After an intervention consisting of detailed reported feedback and instructions on how to acquire MALDI-TOF mass spectra, measurements were repeated and MSQs were compared. RESULTS: At baseline, we observed heterogeneous MSQ between the devices, considering the median number of marker masses detected (range = [2-25]), reproducibility between technical replicates (range = [55%-86%]), and measurement error (range = [147 parts per million (ppm)-588 ppm]). As a general trend, the spectral quality was improved after the intervention for devices, which yielded low MSQs in the baseline assessment as follows: for four out of five devices with a high measurement error, the measurement precision was improved (p-values <0.001, paired Wilcoxon test); for six out of ten devices, which detected a low number of marker masses, the number of detected marker masses increased (p-values <0.001, paired Wilcoxon test). DISCUSSION: We have identified simple workflow adaptations, which, to some extent, improve MSQ of poorly performing devices and should be considered by laboratories yielding a low MSQ. Improving MALDI-TOF MSQ in routine diagnostics is essential for increasing the resolution of bacterial identification by MALDI-TOF MS, which is dependent on the reproducible detection of marker masses. The heterogeneity identified in this external quality assessment (EQA) requires further study.

• MeSH
• Bacteria * MeSH
• Laboratories * MeSH
• Humans MeSH
• Workflow MeSH
• Reproducibility of Results MeSH
• Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH