Tuberculosis in Ukrainian War Refugees and Migrants in the Czech Republic and Slovakia: A Molecular Epidemiological Study.

BACKGROUND: The war in Ukraine has led to significant migration to neighboring countries, raising public health concerns. Notable tuberculosis (TB) incidence rates in Ukraine emphasize the immediate requirement to prioritize approaches that interrupt the spread and prevent new infections. METHODS: We conducted a prospective genomic surveillance study to assess migration's impact on TB epidemiology in the Czech Republic and Slovakia. Mycobacterium tuberculosis isolates from Ukrainian war refugees and migrants, collected from September 2021 to December 2022 were analyzed alongside 1574 isolates obtained from Ukraine, the Czech Republic, and Slovakia. RESULTS: Our study revealed alarming results, with historically the highest number of Ukrainian tuberculosis patients detected in the host countries. The increasing number of cases of multidrug-resistant TB, significantly linked with Beijing lineage 2.2.1 (p < 0.0001), also presents substantial obstacles to control endeavors. The genomic analysis identified the three highly related genomic clusters, indicating the recent TB transmission among migrant populations. The largest clusters comprised war refugees diagnosed in the Czech Republic, TB patients from various regions of Ukraine, and incarcerated individuals diagnosed with pulmonary TB specialized facility in the Kharkiv region, Ukraine, pointing to a national transmission sequence that has persisted for over 14 years. CONCLUSIONS: The data showed that most infections were likely the result of reactivation of latent disease or exposure to TB before migration rather than recent transmission occurring within the host country. However, close monitoring, appropriate treatment, careful surveillance, and social support are crucial in mitigating future risks, though there is currently no evidence of local transmission in EU countries.

A smooth tubercle bacillus from Ethiopia phylogenetically close to the Mycobacterium tuberculosis complex.

The Mycobacterium tuberculosis complex (MTBC) includes several human- and animal-adapted pathogens. It is thought to have originated in East Africa from a recombinogenic Mycobacterium canettii-like ancestral pool. Here, we describe the discovery of a clinical tuberculosis strain isolated in Ethiopia that shares archetypal phenotypic and genomic features of M. canettii strains, but represents a phylogenetic branch much closer to the MTBC clade than to the M. canettii strains. Analysis of genomic traces of horizontal gene transfer in this isolate and previously identified M. canettii strains indicates a persistent albeit decreased recombinogenic lifestyle near the emergence of the MTBC. Our findings support that the MTBC emergence from its putative free-living M. canettii-like progenitor is evolutionarily very recent, and suggest the existence of a continuum of further extant derivatives from ancestral stages, close to the root of the MTBC, along the Great Rift Valley.

Tuberculosis: current challenges and beyond.

Despite being a preventable and curable disease, tuberculosis (TB) is still a major global health threat and the second leading cause of death due to an infectious agent worldwide. All the efforts invested to end TB have resulted overall in rather slow decreases in TB incidence and mortality rates, which have been further negatively affected by the ongoing coronavirus disease 2019 (COVID-19) pandemic. While the majority of targets of the End TB Strategy remain off track, and we have not yet overcome the disruptions caused by the COVID-19 pandemic, recent conflicts such as the ongoing war in Ukraine are threatening the decrease of the burden of TB even further. To get back on track and get closer to ending TB, we need urgent, global, well-structured and committed multi-sectoral actions that go beyond national and global TB programmes with the support of deep investments in research and facilitation of equitable and rapid implementation of innovation worldwide.

Whole-genome sequence analysis of clinically isolated carbapenem resistant Escherichia coli from Iran.

BACKGROUND: The emergence of carbapenem-resistant Enterobacterales (CRE) continues to threaten public health due to limited therapeutic options. In the current study the incidence of carbapenem resistance among the 104 clinical isolates of Escherichia coli and the genomic features of carbapenem resistant isolates were investigated. METHODS: The susceptibility to imipenem, tigecycline and colistin was tested by broth dilution method. Susceptibility to other classes of antimicrobials was examined by disk diffusion test. The presence of blaOXA-48, blaKPC, blaNDM, and blaVIM carbapenemase genes was examined by PCR. Molecular characteristics of carbapenem resistant isolates were further investigated by whole-genome sequencing (WGS) using Illumina and Nanopore platforms. RESULTS: Four isolates (3.8%) revealed imipenem MIC of ≥32 mg/L and positive results for modified carbapenem inactivation method and categorized as carbapenem resistant E. coli (CREC). Colistin, nitrofurantoin, fosfomycin, and tigecycline were the most active agents against all isolates (total susceptibility rate of 99, 99, 96 and 95.2% respectively) with the last three compounds being found as the most active antimicrobials for carbapenem resistant isolates (susceptibility rate of 100%). According to Multilocus Sequence Type (MLST) analysis the 4 CREC isolates belonged to ST167 (n = 2), ST361 (n = 1) and ST648 (n = 1). NDM was detected in all CREC isolates (NDM-1 (n = 1) and NMD-5 (n = 3)) among which one isolate co-harbored NDM-5 and OXA-181 carbapenemases. WGS further detected blaCTX-M-15, blaCMY-145, blaCMY-42 and blaTEM-1 (with different frequencies) among CREC isolates. Co-occurrence of NDM-type carbapenemase and 16S rRNA methyltransferase RmtB and RmtC was found in two isolates belonging to ST167 and ST648. A colistin-carbapenem resistant isolate which was mcr-negative, revealed various amino acid substitutions in PmrB, PmrD and PhoPQ proteins. CONCLUSION: About 1.9% of E. coli isolates studied here were resistant to imipenem, colistin and/or amikacin which raises the concern about the outbreaks of difficult-to-treat infection by these emerging superbugs in the future.

Genomic features of in vitro selected mutants of Escherichia coli with decreased susceptibility to tigecycline.

OBJECTIVES: The increase in multidrug-resistant bacteria has reached an alarming rate globally, making it necessary to understand the underlying mechanisms mediating resistance in order to discover new therapeutics. Tigecycline (TGC) is a last-resort antimicrobial agent for the treatment of serious infections caused by extensively drug-resistant Enterobacteriaceae. METHODS: The TGC-resistant Escherichia coli mutants were obtained by exposing three different TGC-susceptible isolates belonging to ST131 (n = 2) and ST405 (n = 1) to increasing concentrations of TGC. The genetic alterations associated with reduced susceptibility to TGC were identified using whole genome sequencing. The fitness cost of TGC resistance acquisition, as well as incidence of cross-resistance, was also investigated. RESULTS: The TGC minimum inhibitory concentrations (MICs) of in vitro selected mutants were elevated 8 to 32 times compared with ancestral strains. Inactivating mutations (frameshift and nonsense) or amino acid substitutions were identified in genes encoding proteins with diverse functions, including AcrAB efflux pump or its regulators (lon and marR), Lipopolysaccharides (LPS) inner core biosynthesis enzymes (waaQ and eptB), ribosomal S9 protein (rpsI), and RNA polymerase ß subunit. In most cases (but not all), acquisition of TGC resistance was associated with a fitness cost. While TGC resistance development was associated with cross-resistance to other members of the tetracycline family and chloramphenicol, hypersensitivity to nitrofurantoin was identified among heptose III-less LPS mutants. CONCLUSION: TGC resistance among the studied mutants was found to be multifactorial with extrusion by efflux transports being the most common mechanism. The LPS inner core biosynthesis pathway, as well as ribosomal S9 protein, could be additional targets for TGC resistance.

Ancient and recent differences in the intrinsic susceptibility of Mycobacterium tuberculosis complex to pretomanid.

OBJECTIVES: To develop a robust phenotypic antimicrobial susceptibility testing (AST) method with a correctly set breakpoint for pretomanid (Pa), the most recently approved anti-tuberculosis drug. METHODS: The Becton Dickinson Mycobacterial Growth Indicator Tube™ (MGIT) system was used at six laboratories to determine the MICs of a phylogenetically diverse collection of 356 Mycobacterium tuberculosis complex (MTBC) strains to establish the epidemiological cut-off value for pretomanid. MICs were correlated with WGS data to study the genetic basis of differences in the susceptibility to pretomanid. RESULTS: We observed ancient differences in the susceptibility to pretomanid among various members of MTBC. Most notably, lineage 1 of M. tuberculosis, which is estimated to account for 28% of tuberculosis cases globally, was less susceptible than lineages 2, 3, 4 and 7 of M. tuberculosis, resulting in a 99th percentile of 2 mg/L for lineage 1 compared with 0.5 mg/L for the remaining M. tuberculosis lineages. Moreover, we observed that higher MICs (≥8 mg/L), which probably confer resistance, had recently evolved independently in six different M. tuberculosis strains. Unlike the aforementioned ancient differences in susceptibility, these recent differences were likely caused by mutations in the known pretomanid resistance genes. CONCLUSIONS: In light of these findings, the provisional critical concentration of 1 mg/L for MGIT set by EMA must be re-evaluated. More broadly, these findings underline the importance of considering the global diversity of MTBC during clinical development of drugs and when defining breakpoints for AST.

Mutation in mgrB is the major colistin resistance mechanism in Klebsiella pneumoniae clinical isolates in Tehran, Iran.

Colistin is considered as one of a last resort antimicrobial agent against multidrug-resistant Gram-negative bacteria including Escherichia coli and Klebsiella pneumoniae. However, the recent emergence of colistin resistance (ColR) worldwide that severely restricts therapeutic options is a serious threat to global public health. In this study we have investigated the molecular determinants in ColR K. pneumoniae isolates collected from clinical specimens. A total of 98 E. coli and 195 K. pneumoniae clinical isolates were collected from two hospitals from August 2018 to December 2019 in Tehran, Iran. Colistin susceptibility and minimum inhibitory concentrations (MIC) were determined according to the Clinical and Laboratory Standards Institute by disk diffusion method, and microdilution method, respectively. For isolates with colistin MIC ≥4 µg mL-1, PCR was performed for the detection of mcr-1 to mcr-4 genes. Moreover, nucleotide sequences of mgrB, phoP, phoQ, pmrA, and pmrB genes were determined by sequencing. Finally, the transcriptional level of pmrK and pmrC genes was evaluated by quantitative reverse transcription PCR (RT-qPCR). None of the E. coli isolates were resistant to colistin while 21 out 195 K. pneumoniae isolates were identified as resistant, 19 of which carried mutation in the mgrB gene. Three different mutations were observed in the pmrB gene in 3 K. pneumoniae isolates. None of the ColR isolates showed alternations in pmrA, phoP, and phoQ genes. Furthermore, none of the plasmid-encoding genes were detected. Transcriptional level of the pmrK gene increased in all ColR isolates meanwhile, pmrC overexpression was detected in 16 out 21 (76.19%) isolates. Eventually, all ColR isolates were susceptible to tigecycline. Our results demonstrated that the alternation of mgrB gene is the main mechanism related to colistin resistance among ColR K. pneumoniae isolates in this study.

Assessment of the GenoType MTBDRsl VER 2.0 compared to the phenotypic drug susceptibility testing and whole genome sequencing for the rapid detection of resistance to fluoroquinolone and second-line injectable drugs among rifampicin-resistant Mycobacterium tuberculosis isolates.

Molecular techniques have considerable advantages for rapid detection, a reduction of infectiousness, prevention of further resistance development and surveillance of drug-resistant TB. MTBDRsl VER 2.0 was used to detect resistance to second-line anti-tuberculosis drugs on 35 rifampicin-resistant M. tuberculosis (RR-MTB) isolates compared to the minimum inhibitory concentrations (MICs) and whole genome sequencing (WGS). The MTBDRsl VER 2.0 (Hain Life Science, Nehren, Germany) and WGS (San Diego, CA, USA) were performed for tracing mutations in resistant-related genes involved in resistance to fluoroquinolone (FLQ) and second-line injectable drugs. The broth microdilution method using 7H9 Middlebrook media supplemented with OADC was used to determine the MICs. The MTBDRsl VER 2.0 correctly detected 5/6 (83.3%) of FLQ-resistant strains. The MUT1 A1401G (seven strains) and MUT2 G1484T (one strain) mutations in rrs gene were detected in eight AMK/KAN/CAP-resistant strains. Four low-level KAN-resistant strains with the G-10A/C-12T (three strains) and eis C-14T (one strain) mutations in eis gene was diagnosed using MTBDRsl VER 2.0. Five errors were found in detecting resistance to kanamycin and capreomycin compared to the phenotypic drug susceptibility testing and WGS. Failling wild-type bands without improved mutant bands did not indicate a reliable resistance. WGS could efficiently resolve the discrepancies of the results. MTBDRsl showed better performance in detecting XDR strains than pre-XDR.

Genomic analysis of cardiac surgery-associated Mycobacterium chimaera infections in Italy.

One hundred and twenty-two Mycobacterium chimaera strains isolated in Italy from cardiac surgery-related patients, cardiac surgery-unrelated patients and from heater-cooler units, were submitted to whole-genome sequencing and to subsequent SNP analysis. All but one strains isolated from cardiac surgery-related patients belonged to Subgroup 1.1 (19/23) or Subgroup 1.8 (3/23). Only 28 out of 79 strains isolated from heater-cooler units belonged to groupings other than 1.1 and 1.8. The strains isolated from cardiac surgery-unrelated patients were instead distributed across the phylogenetic tree. Our data, the first on isolates from Italy, are in agreement with a recent large genomic study suggesting a common source, represented by strains belonging to Subgroups 1.1 and 1.8, of cardiac surgery-related Mycobacterium chimaera infections. The strains belonging to groupings other than 1.1 and 1.8 isolated from heather-cooler units evidently resulted from contaminations at hospital level and had no share in the Mycobacterium chimaera outbreak. One Mycobacterium chimaera strain investigated in this study proved distant from every previously known Mycobacterium chimaera Groups (1, 2, 3 and 4) and we propose to assign to a novel group, named "Group 5".

Whole Genome Sequencing Results Associated with Minimum Inhibitory Concentrations of 14 Anti-Tuberculosis Drugs among Rifampicin-Resistant Isolates of Mycobacterium Tuberculosis from Iran.

Accurate and timely detection of drug resistance can minimize the risk of further resistance development and lead to effective treatment. The aim of this study was to determine the resistance to first/second-line anti-tuberculosis drugs in rifampicin/multidrug-resistant Mycobacterium tuberculosis (RR/MDR-MTB) isolates. Molecular epidemiology of strains was determined using whole genome sequencing (WGS)-based genotyping. A total of 35 RR/MDR-MTB isolates were subjected to drug susceptibility testing against first/second-line drugs using 7H9 Middlebrook in broth microdilution method. Illumina technology was used for paired-end WGS applying a Maxwell 16 Cell DNA Purification kit and the NextSeq platform. Data analysis and single nucleotide polymorphism calling were performed using MTBseq pipeline. The genome-based resistance to each drug among the resistant phenotypes was as follows: rifampicin (97.1%), isoniazid (96.6%), ethambutol (100%), levofloxacin (83.3%), moxifloxacin (83.3%), amikacin (100%), kanamycin (100%), capreomycin (100%), prothionamide (100%), D-cycloserine (11.1%), clofazimine (20%), bedaquiline (0.0%), and delamanid (44.4%). There was no linezolid-resistant phenotype, and a bedaquiline-resistant strain was wild type for related genes. The Beijing, Euro-American, and Delhi-CAS were the most populated lineage/sublineages. Drug resistance-associated mutations were mostly linked to minimum inhibitory concentration results. However, the role of well-known drug-resistant genes for D-cycloserine, clofazimine, bedaquiline, and delamanid was found to be more controversial.

Factors and Characteristics of Workplace Violence Against Nurses: A Study in Iran.

Violence against nurses is a serious problem that can affect negatively the quality of nursing care. The extent of violence against nurses in Iran and the factors leading to this violence have not been known. Thus, the aim of this study was to investigate all forms of violence against nurses in Shahrekord hospitals in 2014. In this 2014 study, 100 nurses working in Shahrekord's Hajar, Kashani, and Social Security branch hospitals were studied. Data were collected through standardized questionnaires on workplace violence, as well as demographic data, in health units on five types of violence, including physical and verbal violence, intimidation and bullying, ethnic violence, and violation of chastity. The analysis was done by SPSS (Version 19) software. All nurses indicated that during some period of their work, they had been subjected to at least one type of violence; the highest prevalence of mental violence was belonged to the subtype of intimidation and bullying (91%). The primary agents of violence against nurses were patients and their relatives. Factors such as gender, age, work experience, and nursing shifts played important roles in the distribution of violence. In ethnic violence, the only factor affecting nurses was race (p < .05). The highest rate of violence against nurses was due to mental or psychological violence. To minimize violence in hospitals, authorities should consider appropriate preventive strategies, good management, proper protective measures, and public education.

The Anti-Mycobacterial Activity Of Ag, ZnO, And Ag- ZnO Nanoparticles Against MDR- And XDR-Mycobacterium tuberculosis.

BACKGROUND: Nowadays, tuberculosis (TB) is one of the top ten leading causes of mortality worldwide. The emergence of multidrug-resistant (MDR) - and extensively drug-resistant (XDR) - Mycobacterium tuberculosis (M. tuberculosis) is identified as one of the most challenging threats to TB control. Thus, new and safe nano-drugs are urgently required for the elimination of TB. The aim of this study was to investigate the anti-bacterial effects of Ag, ZnO, and Ag-ZnO nanoparticles (NPs) on MDR- and XDR-M. tuberculosis. MATERIALS AND METHODS: In this study, Ag, ZnO, and Ag-ZnO NPs were synthesized by the chemical reduction and chemical deposition methods. NPs were characterized using ultraviolet-visible spectroscopy, dynamic light scattering, and transmission electron microscopy. Then, various dilutions of NPs were prepared and their minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined against M. tuberculosis strains using the broth microdilution and agar microdilution methods. Finally, MTT test and cell culture assay were performed. RESULTS: The effects of concentrations of 1-128 µg/mL Ag NPs, ZnO NPs, 2Ag: 8ZnO, 8Ag:2ZnO, 3Ag: 7ZnO, 7Ag:3ZnO, and 5Ag:5ZnO on M. tuberculosis strains were investigated. MIC results showed the inhibitory effect of 1 µg/mL of all NPs against XDR-M. tuberculosis. In addition, the concentrations of 4 µg/mL Ag, 8 µg/mL 5Ag:5ZnO, 8 µg/mL 7Ag:3ZnO, 32 µg/mL 3Ag:7ZnO, 16 µg/mL 8Ag:2ZnO, and 64 µg/mL 2Ag:8ZnO inhibited MDR-M. tuberculosis growth. However, MBC results indicated the inability of Ag, ZnO and Ag-ZnO NPs, either in combination or alone, to kill MDR- or XDR-M. tuberculosis. CONCLUSION: To the best of our knowledge, this is the first study to evaluate the effects of Ag and ZnO NPs against MDR and XDR strains of M. tuberculosis. According to the results, Ag and ZnO NPs showed bacteriostatic effects against drug-resistant strains of M. tuberculosis. Therefore, these NPs may be considered as promising anti-mycobacterial nano-drugs. However, further studies are required to affirm the bactericidal effects of these NPs against TB.

Acquisition of Cross-Resistance to Bedaquiline and Clofazimine following Treatment for Tuberculosis in Pakistan.

We report on the first six cases of acquired resistance to bedaquiline in Pakistan. Seventy sequential isolates from 30 drug-resistant-tuberculosis patients on bedaquiline-containing regimens were retrospectively tested for bedaquiline resistance by MIC testing and by the detection of mutations in relevant genes. We documented cases failing therapy that developed specific mutations in Rv0678 and had increased MICs associated with cross-resistance to clofazimine during treatment. This study underlines the relevance of surveillance programs following the introduction of new drugs.

Isoniazid Resistance in Mycobacterium tuberculosis Is a Heterogeneous Phenotype Composed of Overlapping MIC Distributions with Different Underlying Resistance Mechanisms.

MIC testing using the Bactec mycobacteria growth indicator tube system 960 of 70 phylogenetically diverse, isoniazid-resistant clinical strains of Mycobacterium tuberculosis revealed a complex pattern of overlapping MIC distributions. Whole-genome sequencing explained most of the levels of resistance observed. The MIC distribution of strains with only inhA promoter mutations was split by the current concentration endorsed by the Clinical and Laboratory Standards Institute to detect low-level resistance to isoniazid and is, consequently, likely not optimally set.