Antimicrobial resistance prediction by clinical metagenomics in pediatric severe pneumonia patients.

BACKGROUND: Antimicrobial resistance (AMR) is a major threat to children's health, particularly in respiratory infections. Accurate identification of pathogens and AMR is crucial for targeted antibiotic treatment. Metagenomic next-generation sequencing (mNGS) shows promise in directly detecting microorganisms and resistance genes in clinical samples. However, the accuracy of AMR prediction through mNGS testing needs further investigation for practical clinical decision-making. METHODS: We aimed to evaluate the performance of mNGS in predicting AMR for severe pneumonia in pediatric patients. We conducted a retrospective analysis at a tertiary hospital from May 2022 to May 2023. Simultaneous mNGS and culture were performed on bronchoalveolar lavage fluid samples obtained from pediatric patients with severe pneumonia. By comparing the results of mNGS detection of microorganisms and antibiotic resistance genes with those of culture, sensitivity, specificity, positive predictive value, and negative predictive value were calculated. RESULTS: mNGS detected bacterial in 71.7% cases (86/120), significantly higher than culture (58/120, 48.3%). Compared to culture, mNGS demonstrated a sensitivity of 96.6% and a specificity of 51.6% in detecting pathogenic microorganisms. Phenotypic susceptibility testing (PST) of 19 antibiotics revealed significant variations in antibiotics resistance rates among different bacteria. Sensitivity prediction of mNGS for carbapenem resistance was higher than penicillins and cephalosporin (67.74% vs. 28.57%, 46.15%), while specificity showed no significant difference (85.71%, 75.00%, 75.00%). mNGS also showed a high sensitivity of 94.74% in predicting carbapenem resistance in Acinetobacter baumannii. CONCLUSIONS: mNGS exhibits variable predictive performance among different pathogens and antibiotics, indicating its potential as a supplementary tool to conventional PST. However, mNGS currently cannot replace conventional PST.

Genome-Wide Investigation Reveals Potential Therapeutic Targets in Shigella spp.

Shigella stands as a major contributor to bacterial dysentery worldwide scale, particularly in developing countries with inadequate sanitation and hygiene. The emergence of multidrug-resistant strains exacerbates the challenge of treating Shigella infections, particularly in regions where access to healthcare and alternative antibiotics is limited. Therefore, investigations on how bacteria evade antibiotics and eventually develop resistance could open new avenues for research to develop novel therapeutics. The aim of this study was to analyze whole genome sequence (WGS) of human pathogenic Shigella spp. to elucidate the antibiotic resistance genes (ARGs) and their mechanism of resistance, gene-drug interactions, protein-protein interactions, and functional pathways to screen potential therapeutic candidate(s). We comprehensively analyzed 45 WGS of Shigella, including S. flexneri (n = 17), S. dysenteriae (n = 14), S. boydii (n = 11), and S. sonnei (n = 13), through different bioinformatics tools. Evolutionary phylogenetic analysis showed three distinct clades among the circulating strains of Shigella worldwide, with less genomic diversity. In this study, 2,146 ARGs were predicted in 45 genomes (average 47.69 ARGs/genome), of which only 91 ARGs were found to be shared across the genomes. Majority of these ARGs conferred their resistance through antibiotic efflux pump (51.0%) followed by antibiotic target alteration (23%) and antibiotic target replacement (18%). We identified 13 hub proteins, of which four proteins (e.g., tolC, acrR, mdtA, and gyrA) were detected as potential hub proteins to be associated with antibiotic efflux pump and target alteration mechanisms. These hub proteins were significantly (p < 0.05) enriched in biological process, molecular function, and cellular components. Therefore, the finding of this study suggests that human pathogenic Shigella strains harbored a wide range of ARGs that confer resistance through antibiotic efflux pumps and antibiotic target modification mechanisms, which must be taken into account to devise and formulate treatment strategy against this pathogen. Moreover, the identified hub proteins could be exploited to design and develop novel therapeutics against MDR pathogens like Shigella.

Distribution of bacteria and antimicrobial resistance in retail Nile tilapia (Oreochromis spp.) as potential sources of foodborne illness.

This study aimed to investigate AMR profiles of Aeromonas hydrophila, Salmonella spp., and Vibrio cholerae isolated from Nile tilapia (Oreochromis spp.) (n = 276) purchased from fresh markets and supermarkets in Bangkok, Thailand. A sample of tilapia was divided into three parts: fish intestine (n = 276), fish meat (n = 276), and liver and kidney (n = 276). The occurrence of A. hydrophila, Salmonella, and V. cholerae was 3.1%, 7.4%, and 8.5%, respectively. A high prevalence of these pathogenic bacteria was observed in fresh market tilapia compared to those from supermarkets (p < 0.05). The predominant Salmonella serovars were Paratyphi B (6.4%), followed by Escanaba (5.7%), and Saintpaul (5.7%). All isolates tested positive for the virulence genes of A. hydrophila (aero and hly), Salmonella (invA), and V. cholerae (hlyA). A. hydrophila (65.4%), Salmonella (31.2%), and V. cholerae (2.9%) showed multidrug resistant isolates. All A. hydrophila isolates (n = 26) exhibited resistant to ampicillin (100.0%) and florfenicol (100.0%), and often carried sul1 (53.8%) and tetA (50.0%). Salmonella isolates were primarily resistant to ampicillin (36.9%), with a high incidence of blaTEM (26.2%) and qnrS (25.5%). For V. cholerae isolates, resistance was observed against ampicillin (48.6%), and they commonly carried qnrS (24.3%) and tetA (22.9%). To identify mutations in the quinolone resistance determining regions (QRDRs), a single C248A point mutation of C248A (Ser-83-Tyr) in the gyrA region was identified in six out of seven isolates of Salmonella isolates. This study highlighted the presence of antimicrobial-resistant pathogenic bacteria in Nile tilapia at a selling point. It is important to rigorously implement strategies for AMR control and prevention.

Influence of PhoPQ and PmrAB two component system alternations on colistin resistance from non-mcr colistin resistant clinical E. Coli strains.

BACKGROUND: The current understanding of acquired chromosomal colistin resistance mechanisms in Enterobacterales primarily involves the disruption of the upstream PmrAB and PhoPQ two-component system (TCS) control caused by mutations in the regulatory genes. Interestingly, previous studies have yielded conflicting results regarding the interaction of regulatory genes related to colistin resistance in Escherichia coli, specifically those surrounding PhoPQ and PmrAB TCS. RESULTS: In our study, we focused on two clinical non-mcr colistin-resistant strains of E. coli, TSAREC02 and TSAREC03, to gain a better understanding of their resistance mechanisms. Upon analysis, we discovered that TSAREC02 had a deletion (Δ27-45) in MgrB, as well as substitutions (G206R, Y222H) in PmrB. On the other hand, TSAREC03 exhibited a long deletion (Δ84-224) in PhoP, along with substitutions (M1I, L14P, P178S, T235N) in PmrB. We employed recombinant DNA techniques to explore the interaction between the PhoPQ and PmrAB two-component systems (TCSs) and examine the impact of the mutated phoPQ and pmrB genes on the minimum inhibitory concentrations (MICs) of colistin. We observed significant changes in the expression of the pmrD gene, which encodes a connector protein regulated by the PhoPQ TCS, in the TSAREC02 wild-type (WT)-mgrB replacement mutant and the TSAREC03 WT-phoP replacement mutant, compared to their respective parental strains. However, the expressions of pmrB/pmrA, which reflect PmrAB TCS activity, and the colistin MICs remained unchanged. In contrast, the colistin MICs and pmrB/pmrA expression levels were significantly reduced in the pmrB deletion mutants from both TSAREC02 and TSAREC03, compared to their parental strains. Moreover, we were able to restore colistin resistance and the expressions of pmrB/pmrA by transforming a plasmid containing the parental mutated pmrB back into the TSAREC02 and TSAREC03 mutants, respectively. CONCLUSION: While additional data from clinical E. coli isolates are necessary to validate whether our findings could be broadly applied to the E. coli population, our study illuminates distinct regulatory pathway interactions involving colistin resistance in E. coli compared to other species of Enterobacterales. The added information provided by our study contribute to a deeper understanding of the complex pathway interactions within Enterobacterales.

Comprehensive identification of pathogenic microbes and antimicrobial resistance genes in food products using nanopore sequencing-based metagenomics.

Foodborne pathogens, particularly antimicrobial-resistant (AMR) bacteria, remain a significant threat to global health. Given the limitations of conventional culture-based approaches, which are limited in scope and time-consuming, metagenomic sequencing of food products emerges as a promising solution. This method provides a fast and comprehensive way to detect the presence of pathogenic microbes and antimicrobial resistance genes (ARGs). Notably, nanopore long-read sequencing provides more accurate bacterial taxonomic classification in comparison to short-read sequencing. Here, we revealed the impact of food types and attributes (origin, retail place, and food processing methods) on microbial communities and the AMR profile using nanopore metagenomic sequencing. We analyzed a total of 260 food products, including raw meat, sashimi, and ready-to-eat (RTE) vegetables. Clostridium botulinum, Acinetobacter baumannii, and Vibrio parahaemolyticus were identified as the top three foodborne pathogens in raw meat and sashimi. Importantly, even with low pathogen abundance, higher percentages of samples containing carbapenem and cephalosporin resistance genes were identified in chicken and RTE vegetables, respectively. In parallel, our results demonstrated that fresh, peeled, and minced foods exhibited higher levels of pathogenic bacteria. In conclusion, this comprehensive study offers invaluable data that can contribute to food safety assessments and serve as a basis for quality indicators.

Phenotypic and genotypic characterization of extended spectrum beta-lactamase producing E. coli harboring carbapenem and colistin-resistant genes from poultry farms in Egypt.

Background: eEscherichia coli (E. coli) bacteria that produce extended spectrum beta-lactamase (ESBL) is associated with a high prevalence of human illnesses worldwide. The emergence of resistance to carbapenem and colistin compounds poses further challenges to the treatment options for these illnesses. This study aimed to evaluate the phenotypic and genotypic pattern of resistance to carbapenem and colistin in ESBL-producing E. coli. Escherichia coli isolates collected from the respiratory tract of chickens in El-Sharkia government, Egypt. Methods: A total of 250 lung samples were collected from 50 poultry farms. These samples were then subjected to isolation, identification, and serotyping of E. coli. The presence of antimicrobial resistance was identified by disc diffusion testing. The occurrence of ESBL phenotypes was also assessed using the double disc synergy method. PCR/sequencing techniques were employed to examine the presence of ESBL (ß-lactamase (bla)-TEM, blaSHV, and blaCTX-M), colistin (mcr-1), and carbapenem (blaNDM, blaVIM, and blaKPC) resistance genes. Results: The findings revealed that 140 out of 250 (56%) were identified as E. coli. All E. coli isolates had a high level of multi-antimicrobial resistance (MAR) with an index value greater than 0.2, and 65.7% of them were confirmed to produce ESBL. Out of the 92 ESBL phenotypes, 55 (59.7%), 32 (34.7%), 18 (19.6%), and 37 (40.2%) isolates harbor b laTEM-3, b laSHV-4, b laCTX-M-1, a nd blaCTX-M-14 genes, respectively. The blaNDM-1 gene was identified in all 40 phenotypes that exhibited resistance to carbapenem, accounting for 28.5% of all strains of E. coli and 43.4% of ESBL isolates. The VIM and KPC genes were not detected in any of the samples. Furthermore, there was a significant prevalence of the mobilized colistin resistance (mcr)-1 gene, with 64 (69.5%) of the ESBL isolates exhibiting this gene. Conclusion: The prevalence of ESBL-producing E. coli, particularly those resistant to carbapenem and colistin, poses a significant public health risk in society.

Clinical manifestations, antimicrobial resistance and genomic feature analysis of multidrug-resistant Elizabethkingia strains.

BACKGROUND: Elizabethkingia is emerging as an opportunistic pathogen in humans. The aim of this study was to investigate the clinical epidemiology, antimicrobial susceptibility, virulence factors, and genome features of Elizabethkingia spp. METHODS: Clinical data from 71 patients who were diagnosed with Elizabethkingia-induced pneumonia and bacteremia between August 2019 and September 2021 were analyzed. Whole-genome sequencing was performed on seven isolates, and the results were compared with a dataset of 83 available Elizabethkingia genomes. Genomic features, Kyoto Encyclopedia of Genes and Genomes (KEGG) results and clusters of orthologous groups (COGs) were analyzed. RESULTS: The mean age of the patients was 56.9 ± 20.7 years, and the in-hospital mortality rate was 29.6% (21/71). Elizabethkingia strains were obtained mainly from intensive care units (36.6%, 26/71) and emergency departments (32.4%, 23/71). The majority of the strains were isolated from respiratory tract specimens (85.9%, 61/71). All patients had a history of broad-spectrum antimicrobial exposure. Hospitalization for invasive mechanical ventilation or catheter insertion was found to be a risk factor for infection. The isolates displayed a high rate of resistance to cephalosporins and carbapenems, but all were susceptible to minocycline and colistin. Genomic analysis identified five ß-lactamase genes (blaGOB, blaBlaB, blaCME, blaOXA, and blaTEM) responsible for ß-lactam resistance and virulence genes involved in stress adaptation (ureB/G, katA/B, and clpP), adherence (groEL, tufA, and htpB) and immune modulation (gmd, tviB, cps4J, wbtIL, cap8E/D/G, and rfbC). Functional analysis of the COGs revealed that "metabolism" constituted the largest category within the core genome, while "information storage and processing" was predominant in both the accessory and unique genomes. The unique genes in our 7 strains were mostly enriched in KEGG pathways related to microRNAs in cancer, drug resistance (ß-lactam and vancomycin), ABC transporters, biological metabolism and biosynthesis, and nucleotide excision repair mechanisms. CONCLUSION: The Elizabethkingia genus exhibits multidrug resistance and carries carbapenemase genes. This study presents a comparative genomic analysis of Elizabethkingia, providing knowledge that facilitates a better understanding of this microorganism.

Detection and genomic characterization of Klebsiella pneumoniae and Escherichia coli harboring tet(X4) in black kites (Milvus migrans) in Pakistan.

The emergence of plasmid-mediated tigecycline resistance gene tet(X4) among clinically relevant bacteria has promoted significant concerns, as tigecycline is considered a last-resort drug against serious infections caused by multidrug-resistant bacteria. We herein focused on the isolation and molecular characterization of tet(X4)-positive Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E. coli) in wild bird populations with anthropogenic interaction in Faisalabad, Pakistan. A total of 150 birds including black kites (Milvus migrans) and house crows (Corvus splendens) were screened for the presence of tigecycline resistance K. pneumoniae and E. coli. We found two K. pneumoniae and one E. coli isolate carrying tet(X4) originating from black kites. A combination of short- and long-read sequencing strategies showed that tet(X4) was located on a broad host range IncFII plasmid family in K. pneumoniae isolates whereas on an IncFII-IncFIB hybrid plasmid in E. coli. We also found an integrative and conjugative element ICEKp2 in K. pneumoniae isolate KP8336. We demonstrate the first description of tet(X4) gene in the WHO critical-priority pathogen K. pneumoniae among wild birds. The convergence of tet(X4) and virulence associated ICEKp2 in a wild bird with known anthropogenic contact should be further investigated to evaluate the potential epidemiological implications. The potential risk of global transmission of tet(X4)-positive K. pneumoniae and E. coli warrant comprehensive evaluation and emphasizes the need for effective mitigation strategies to reduce anthropogenic-driven dissemination of AMR in the environment.

[Virulence genes and antimicrobial resistance of Bacillus cereus in prepackaged pastries sold in Taizhou from 2020 to 2022].

OBJECTIVE: To investigate the virulence genes and antimicrobial resistance of Bacillus cereus from the pre-packaged pastries in Taizhou city. METHODS: 500 pre-packaged patries were collected in taizhou city market. 97 Bacillus cereus strains were detected from them by GB 4789.14-2014 method and identified with 4 houseking genes, then 13 virulence genes were detected by polymerase chain reaction(PCR)method and the antimicrobial resistance of Bacillus cereus to 19 antibiotics was detected by paper diffusion method. RESULTS: The result showed that the contamination rate of Bacillus cereus was 19.4% in 500 pre-packaged pastries. The detection rate of four housekeeping genes groEL, gyr B, rpoB and Vrr were 100%, 94.8%, 97.9% and 96.9%, respectively, and 89.7% at the same time. The virulence gene test result showed that the detection rate of nheABC, entFM, bceT, cytK and hblABCD were 91.8%, 88.7%, 61.9%, 51.6% and 25.8%, emetic virulence genes had the lowest detection rate, ces and EMl were 4.1%, cer was 5.2%. 97 Bacillus cereus strains show different degrees of drug resistance to 14 antimicrobials, the resistance rates to penicillin, ampicillin, cefotaxime and cotrimoxazole were higher than 95%, but they were completely sensitive to streptomycin, vancomycin and chloramphenicol. CONCLUSION: There is a risk of contamination by diarrhea-type Bacillus cereus and vomiting-type Bacillus cereus in prepackaged pastries in Taizhou. The isolated and identified Bacillus cereus has multiple-drug resistance.

Staphylococcus haemolyticus: An updated review on nosocomial infections, antimicrobial resistance, virulence, genetic traits, and strategies for combating this emerging opportunistic pathogen.

Staphylococcus haemolyticus, a key species of the Staphylococcus genus, holds significant importance in healthcare-associated infections, due to its notable resistance to antimicrobials, like methicillin, and proficient biofilms-forming capabilities. This coagulase-negative bacterium poses a substantial challenge in the battle against nosocomial infections. Recent research has shed light on Staph. haemolyticus genomic plasticity, unveiling genetic elements responsible for antibiotic resistance and their widespread dissemination within the genus. This review presents an updated and comprehensive overview of the clinical significance and prevalence of Staph. haemolyticus, underscores its zoonotic potential and relevance in the one health framework, explores crucial virulence factors, and examines genetics features contributing to its success in causing emergent and challenging infections. Additionally, we scrutinize ongoing studies aimed at controlling spread and alternative approaches for combating it.

High carriage of plasmid-mediated quinolone resistance (PMQR) genes by ESBL-producing and fluoroquinolone-resistant Escherichia coli recovered from animal waste dumps.

BACKGROUND: There has been a rise in the consumption of fluoroquinolones in human and veterinary medicine recently. This has contributed to the rising incidence of quinolone resistance in bacteria. This study aimed at the determination of the antibiotic resistance profile of ESBL-producing and fluoroquinolone-resistant E. coli (FQEC) isolated from animal waste obtained from the waste dumps of an agricultural farm and their carriage of genes encoding PMQR. METHODS AND RESULTS: Isolation of ESBL-producing E. coli from animal waste samples was done on CHROMagar ESBL, while presumptive isolates were purified, and identified via the detection of uidA gene. Susceptibility to a panel of ten antibiotics was done using the disc diffusion method, and detection of PMQR genes (qnrA, qnrB, qnrS, aac(6')-lb-cr, qepA and oqxAB) was done using monoplex and duplex PCR. Twenty-five ESBL-producing and FQEC were obtained from the cattle (6), piggery (7) and poultry (12) waste dumps of the farm. There was 100% resistance to cefpodoxime, cefotaxime, enrofloxacin, trimethoprim-sulfamethoxazole and penicillin by the isolates. The resistance to the other antibiotics was streptomycin (48%), ceftazidime (24%), while no isolate resisted amoxicillin-clavulanate and imipenem. The frequencies of PMQR genes detected were; qnrA (96%), oqxAB (96%), qnrB (92%), while  qnrS was detected in 88% (22) of the isolates. Aminoglycoside acetyltransferase (aac(6')-lb-cr) and quinolone efflux pump (qepA) were each detected in 20 (80%) of the isolates. CONCLUSIONS: This study showed that animal wastes disposed indiscriminately into dumps could be a budding 'hotspot' for multidrug resistant, ESBL-producing and fluoroquinolone-resistant E. coli carrying multiple genes encoding resistance to fluoroquinolone antibiotics.

Understanding associations between antimicrobial agents usage and antimicrobial resistance genes prevalence at the community level using wastewater-based epidemiology: A Spanish pilot study.

Understanding the development and spread of antimicrobial resistance (AMR) is important for combating this global threat for public health. Wastewater-based epidemiology (WBE) is a complementary approach to current surveillance programs that minimizes some of the existing limitations. The aim of the present study is to explore WBE for monitoring antibiotics and antibiotic resistance genes (ARGs) in wastewater samples collected during 2021/2022 from the city of Castellon (Spain). Eighteen commonly prescribed antibiotics have been selected and measured by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), with triple quadrupole mass analysers. Moreover, qPCR for specific ARGs has been performed to obtain information of these genes in co-presence with antibiotics. All selected ARGs, along with a total of 11 antibiotics, were identified. The highest population-normalized daily loads were observed for the macrolide azithromycin, followed by the quinolones ciprofloxacin and levofloxacin. Subsequently, daily consumption estimates based on wastewater data were compared with prescription data of antibiotics. Statistical analyses were conducted to explore if there is correlation between antibiotics and ARGs. While no correlations were found between antibiotics and their corresponding ARGs, certain correlations (p < 0.05) were identified among non-corresponding ARGs. In addition, a strong positive correlation was found between the sum of all antibiotics and the intl1 gene. Moreover, population-normalized ARG loads significantly correlate with the 16S rRNA-normalized ARG loads, serving as an indicator for population size. Results provide a baseline for future work and a proof-of-concept emphasising the need for future work and long-term surveillance, and highlight the need of similar programs at a regional and global levels worldwide.

The European livestock resistome.

Metagenomic sequencing has proven to be a powerful tool in the monitoring of antimicrobial resistance (AMR). Here, we provide a comparative analysis of the resistome from pigs, poultry, veal calves, turkey, and rainbow trout, for a total of 538 herds across nine European countries. We calculated the effects of per-farm management practices and antimicrobial usage (AMU) on the resistome in pigs, broilers, and veal calves. We also provide an in-depth study of the associations between bacterial diversity, resistome diversity, and AMR abundances as well as co-occurrence analysis of bacterial taxa and antimicrobial resistance genes (ARGs) and the universality of the latter. The resistomes of veal calves and pigs clustered together, as did those of avian origin, while the rainbow trout resistome was different. Moreover, we identified clear core resistomes for each specific food-producing animal species. We identified positive associations between bacterial alpha diversity and both resistome alpha diversity and abundance. Network analyses revealed very few taxa-ARG associations in pigs but a large number for the avian species. Using updated reference databases and optimized bioinformatics, previously reported significant associations between AMU, biosecurity, and AMR in pig and poultry farms were validated. AMU is an important driver for AMR; however, our integrated analyses suggest that factors contributing to increased bacterial diversity might also be associated with higher AMR load. We also found that dispersal limitations of ARGs are shaping livestock resistomes, and future efforts to fight AMR should continue to emphasize biosecurity measures.IMPORTANCEUnderstanding the occurrence, diversity, and drivers for antimicrobial resistance (AMR) is important to focus future control efforts. So far, almost all attempts to limit AMR in livestock have addressed antimicrobial consumption. We here performed an integrated analysis of the resistomes of five important farmed animal populations across Europe finding that the resistome and AMR levels are also shaped by factors related to bacterial diversity, as well as dispersal limitations. Thus, future studies and interventions aimed at reducing AMR should not only address antimicrobial usage but also consider other epidemiological and ecological factors.

Outbreak of NDM-5-producing Klebsiella pneumoniae ST307: an emerging high-risk antimicrobial resistance clone in Shanghai, China.

The high-risk clone Klebsiella pneumoniae ST307, associated with various carbapenem resistance genes, exhibits a global distribution and prevalence. However, in China, it has remained sporadic and has rarely been detected. In this study, we reported an outbreak caused by nine ST307 CRKP isolates harboring blaNDM-5 in Shanghai, China, in 2022. We employed antimicrobial susceptibility testing, conjugation assay, whole-genome sequencing (WGS) and comparative genomics, phylogenetic analysis, and fitness and virulence comparison to further characterize the isolates causing the outbreak. Besides blaNDM-5, these nine isolates co-carried blaCTX-M-15 and blaDHA-1, exhibiting nearly identical resistance profiles with high-level resistance to carbapenems and ceftazidime/avibactam, while showing susceptibility to colistin and tigecycline. blaNDM-5 was located on an IncX3 plasmid of 45,403 bp with a high frequency of conjugative ability. Phylogenetic and single-nucleotide polymorphism (SNP) analysis indicated the nature of clonal transmission with a maximum of five SNPs between these nine isolates, and they were closely related to strains obtained from the United States. ST307 isolates in our study showed a relatively lower virulence but higher growth rates and certain adaptability compared with ST11 isolates. Clinical investigation revealed that shared nursing staff in a mixed emergency intensive care unit ward and doctors' movement between wards might be responsible for the outbreak. The nonexistence before and sudden emergence of ST307 suggested that the currently circulating ST307 clone was a newly introduced superbug in our hospital. In conclusion, we revealed that blaNDM-5-producing ST307 CRKP isolates, a globally significant high-risk clone, are spreading in China, posing a substantial threat to public health.IMPORTANCEThe high-risk clone ST307, associated with various carbapenemases, including KPC, NDM, and OXA, has a global distribution. However, it is rarely reported in China, let alone causing outbreaks. Here, we found an outbreak caused by the clonal transmission of nine ST307 CRKP isolates. Clinical investigation revealed that shared nurses in a mixed emergency intensive care unit ward and doctors' movement between wards might be responsible for the outbreak. In our study, the nine NDM-5-producing ST307 isolates exhibited high-level resistance to carbapenems and ceftazidime-avibactam, high conjugative ability to Escherichia coli J53, and certain adaptability to environment, phylogenetically closet to the United States. All these features make ST307 clone the next successful clone comparable to ST11 clone in China. Therefore, it is imperative for us to vigilantly monitor the prevalence of carbapenem-resistant Klebsiella pneumoniae and promptly implement measures to control the spread of K. pneumoniae ST307 in China.

An Intra-Hospital Spread of Colistin-Resistant K. pneumoniae Isolates-Epidemiological, Clinical, and Genetic Analysis.

Background and Objective: Klebsiella pneumoniae appears to be a significant problem due to its ability to accumulate antibiotic-resistance genes. After 2013, alarming colistin resistance rates among carbapenem-resistant K. pneumoniae have been reported in the Balkans. The study aims to perform an epidemiological, clinical, and genetic analysis of a local outbreak of COLr CR-Kp. Material and Methods: All carbapenem-resistant and colistin-resistant K. pneumoniae isolates observed among patients in the ICU unit of Military Medical Academy, Sofia, from 1 January to 31 October 2023, were included. The results were analyzed according to the EUCAST criteria. All isolates were screened for blaVIM, blaIMP, blaKPC, blaNDM, and blaOXA-48. Genetic similarity was determined using the Dice coefficient as a similarity measure and the unweighted pair group method with arithmetic mean (UPGMA). mgrB genes and plasmid-mediated colistin resistance determinants (mcr-1, mcr-2, mcr-3, mcr-4, and mcr-5) were investigated. Results: There was a total of 379 multidrug-resistant K. pneumoniae isolates, 88% of which were carbapenem-resistant. Of these, there were nine (2.7%) colistin-resistant isolates in six patients. A time and space cluster for five patients was found. Epidemiology typing showed that two isolates belonged to clone A (pts. 1, 5) and the rest to clone B (pts. 2-4) with 69% similarity. Clone A isolates were coproducers of blaNDM-like and blaOXA-48-like and had mgrB-mediated colistin resistance (40%). Clone B isolates had only blaOXA-48-like and intact mgrB genes. All isolates were negative for mcr-1, -2, -3, -4, and -5 genes. Conclusions: The study describes a within-hospital spread of two clones of COLr CR-Kp with a 60% mortality rate. Clone A isolates were coproducers of NDM-like and OXA-48-like enzymes and had mgrB-mediated colistin resistance. Clone B isolates had only OXA-48-like enzymes and intact mgrB genes. No plasmid-mediated resistance was found. The extremely high mortality rate and limited treatment options warrant strict measures to prevent outbreaks.

Microevolution, reinfection and highly complex genomic diversity in patients with sequential isolates of Mycobacterium abscessus.

Mycobacterium abscessus is an opportunistic, extensively drug-resistant non-tuberculous mycobacterium. Few genomic studies consider its diversity in persistent infections. Our aim was to characterize microevolution/reinfection events in persistent infections. Fifty-three sequential isolates from 14 patients were sequenced to determine SNV-based distances, assign resistance mutations and characterize plasmids. Genomic analysis revealed 12 persistent cases (0-13 differential SNVs), one reinfection (15,956 SNVs) and one very complex case (23 sequential isolates over 192 months), in which a first period of persistence (58 months) involving the same genotype 1 was followed by identification of a genotype 2 (76 SNVs) in 6 additional alternating isolates; additionally, ten transient genotypes (88-243 SNVs) were found. A macrolide resistance mutation was identified from the second isolate. Despite high diversity, the genotypes shared a common phylogenetic ancestor and some coexisted in the same specimens. Genomic analysis is required to access the true intra-patient complexity behind persistent infections involving M. abscessus.

Prevalence and antimicrobial resistance of Enterobacteriaceae in the north of Kazakhstan.

Background: An increasing number of drugs are used each year in the treatment of small pets (cats and dogs), including medicines (cephalosporins and fluoroquinolones) used in human therapy. Aim: The purpose of this study was to isolate and explore the antibiotic resistance of opportunistic Enterobacteriaceae (Escherichia coli, Klebsiella, Proteus, Ci trobacter, Enterobacter) from cats and dogs, and to isolate resistance genes in the microorganisms. Methods: In 2021, 808 samples of biological material from small domestic animals were collected in veterinary clinics in Kostanay. From these, 210 microorganisms were isolated and identified. Results: A large majority of the strains sampled belonged to E. coli-149 (70.9%), Enterobacter-11 (5.2%), Klebsiella-28 (13.3%), Proteus-12 (5.7%) and 10 Citrobacter isolates (4.8%). In all isolates identified, antibiotic resistance/sensitivity was determined by disc-diffusion method to ampicillin, cefoxitin, gentamicin, levomycetin, tetracycline, ciprofloxacin, norfloxacin, ofloxacin, cefoperazone, cefpodoxime, streptomycin, kanamycin, doxycycline, gemifloxacin, nalidixic acid, furazolidone, furadonine, amoxicillin, and enrofloxacin. Conclusion: The study has demonstrated that the greatest number of Enterobacteriaceae were sensitive to the action of meropenem, which belongs to the group of beta-lactam antibiotics; resistance was demonstrated against tetracycline, doxycycline, ampicillin, amoxicillin, ofloxacin, and cefpodoxime. The most common genes encoding antimicrobial resistance were as follows: BlaTEM and OXA in 41 and 28 isolates, respectively, encoding resistance to beta-lactams; StrA and StrB in 45 and 48 isolates encoding aminoglycosides; and tetA and tetB in 43 and 28 isolates encoding tetracyclines. Obtained data demonstrate that uncontrolled and frequent use of beta-lactam and tetracycline antibacterials, in cats and dogs, results in the spread of genotypic resistance among micro-organisms of the family Enterobacteriaceae.

Compensatory evolution in NusG improves fitness of drug-resistant M. tuberculosis.

Drug-resistant bacteria are emerging as a global threat, despite frequently being less fit than their drug-susceptible ancestors1-8. Here we sought to define the mechanisms that drive or buffer the fitness cost of rifampicin resistance (RifR) in the bacterial pathogen Mycobacterium tuberculosis (Mtb). Rifampicin inhibits RNA polymerase (RNAP) and is a cornerstone of modern short-course tuberculosis therapy9,10. However, RifR Mtb accounts for one-quarter of all deaths due to drug-resistant bacteria11,12. We took a comparative functional genomics approach to define processes that are differentially vulnerable to CRISPR interference (CRISPRi) inhibition in RifR Mtb. Among other hits, we found that the universally conserved transcription factor NusG is crucial for the fitness of RifR Mtb. In contrast to its role in Escherichia coli, Mtb NusG has an essential RNAP pro-pausing function mediated by distinct contacts with RNAP and the DNA13. We find this pro-pausing NusG-RNAP interface to be under positive selection in clinical RifR Mtb isolates. Mutations in the NusG-RNAP interface reduce pro-pausing activity and increase fitness of RifR Mtb. Collectively, these results define excessive RNAP pausing as a molecular mechanism that drives the fitness cost of RifR in Mtb, identify a new mechanism of compensation to overcome this cost, suggest rational approaches to exacerbate the fitness cost, and, more broadly, could inform new therapeutic approaches to develop drug combinations to slow the evolution of RifR in Mtb.

Antimicrobial resistance, ß-lactamase genotypes, and plasmid replicon types of Shiga toxin-producing Escherichia coli isolated from different animal hosts.

AIMS: The primary objective of this study was to analyze antimicrobial resistance (AMR), with a particular focus on ß-lactamase genotypes and plasmid replicon types of Shiga toxin-producing Escherichia coli (STEC) strains originating from various animal hosts. METHODS AND RESULTS: A total of 84 STEC strains were isolated from cattle (n = 32), sheep/goats (n = 26), pigeons (n = 20), and wild animals (n = 6) between 2010 and 2018 in various regions of Iran. The Kirby-Bauer susceptibility test and multiple polymerase chain reaction (PCR) panels were employed to elucidate the correlation between AMR and plasmid replicon types in STEC isolates. The predominant replicon types were IncFIC and IncFIB in cattle (46.8%), IncFIC in sheep/goats (46.1%), IncA/C in pigeons (90%), and IncP in wild animals (50%). STEC of serogroups O113, O26, and O111 harbored the IncFIB (100%), IncI1 (80%), and IncFIC + IncA/C (100%) plasmids, respectively. A remarkable AMR association was found between ciprofloxacin (100%), neomycin (68.7%), and tetracycline (61.7%) resistance with IncFIC; amoxicillin + clavulanic acid (88.8%) and tetracycline (61.7%) with IncA/C; ciprofloxacin (100%) with IncFIB; fosfomycin (85.7%) and sulfamethoxazole + trimethoprim (80%) with IncI1. IncI1 appeared in 83.3%, 50%, and 100% of the isolates harboring blaCTX-M, blaTEM, and blaOXA ß-lactamase genes, respectively. CONCLUSIONS: The emergence of O26/IncI1/blaCTX-M STEC in cattle farms poses a potential risk to public health.