Occurrence, antimicrobial resistance and genomic features of Klebsiella pneumoniae from broiler chicken in Faisalabad, Pakistan.

Introduction: The dissemination of antimicrobial resistance (AMR) in critical priority pathogens is a significant threat. Non-clinical reservoirs of AMR, such as agriculture and food production facilities, may contribute to the transmission of clinically relevant pathogens such as multidrug-resistant (MDR) Klebsiella pneumoniae. There is currently very limited knowledge regarding the population structure and genomic diversity of K. pneumoniae in poultry production in Pakistan. Methods: We explored healthy broilers in a commercial farm from Faisalabad, Pakistan, and identified six K. pneumoniae strains from 100 broiler birds. We characterized the strains, determining clonality, virulence and antimicrobial resistance genes using next generation sequencing. Results: The evaluation of antimicrobial susceptibility revealed that all the strains were MDR. Genomic analysis showed that 3/6 strains belonged to ST152, harbouring acquired resistance aminoglycosides [aadA2, aph(4')-Ia], ß-lactams (blaSHV-187 , blaLAP2 ), fosfomycin (fosA6), tetracycline (tetA), trimethoprim (dfrA12), quinolone (qnrS1), sulphonamides (sul2) and phenicol (floR). All the strains harboured the efflux pump genes oqxA, oqxB, emrR, kpnG, kpnH, kpnF, baeR, mtdB and mtdC. All six strains encoded identical virulence profiles possessing six genes, i.e., ureA, iutA, entB, allS, fimH and mrkD. Phylogenomic analysis of the dominant sequence type (ST152) present in our dataset with publicly available genomes showed that the isolates clustered to strains mainly from human sources and could pose a potential threat to food safety and public health. Discussion: The combination of these findings with antimicrobial use data would allow a better understanding of the selective pressures that may be driving the spread of AMR. This is the first report of MDR K. pneumoniae isolated from broiler hens in Pakistan, and the finding suggests that routine surveillance of WHO critical priority pathogens in such settings would be beneficial to the development of effective control strategies to reduce AMR.

Prevalence and antimicrobial susceptibility pattern of Klebsiella pneumoniae isolated from various clinical specimens at the University of Gondar Comprehensive Specialized Hospital, Northwest Ethiopia.

BACKGROUND: K. pneumoniae become multidrug-resistant (MDR) and commonly poses a serious health threat to patients due to limited therapeutic options. As a result, determining the prevalence and antimicrobial susceptibility patterns of K. pneumoniae isolates from clinical specimens is substantial to patient diagnosis and treatment. METHODS AND MATERIALS: A retrospective cross-sectional study was conducted from July 2021 to July 2022 at the University of Gondar Comprehensive Specialized Hospital, Northwest Ethiopia. Sociodemographic and laboratory data were collected from registered books using a data collection sheet. All types of samples were collected and processed using standard procedures. Identification of K. pneumoniae was done using Gram stain, colony characterization on culture media, anda series of biochemical tests. Antimicrobial susceptibility testing was done by the Kirby Bauer disc diffusion technique. The data were entered using Epi-info version 7 and exported to SPSS version 20 for analysis. RESULTS: Among 2600 clinical specimens, 735 (28.3%) were positive for bacteria, and K. pneumoniae isolates accounted for 147 (20%). Most of them were isolated from neonates and mainly obtained from blood specimens (81.6%). These isolates were 100% resistant to Nalidixic acid, Cefotaxime, and Cefazolin. About 84% and 83.3% of the isolates were also resistant to Ceftriaxone and Tetracycline, respectively. However, they are sensitive to Nitrofurantoin (86.6%), Imipenem (85.7%), Meropenem (79%), and Amikacin (78.3%). The overall proportion of MDR K. pneumoniae isolates accounted for 57.1%. CONCLUSION: The magnitude of MDR K. pneumoniae was very alarming. Therefore, strengthening antimicrobial stewardship programs and antimicrobial surveillance practices is strongly recommended in the study area.

Genomic diversity and nutritional analysis of multi-drug resistant extended spectrum ß-lactamase Producing-Klebsiella pneumoniae genes isolated from mastitic cattle milk in district peshawar, Pakistan.

The increasing incidence of resistance extended spectrum-beta lactamase (ESBL) producing Klebsiella pneumonia become worldwide issue. The current study aimed to determine the genomic diversity of ESBL-producing K. pneumoniae in milk samples collected from cows with mastitis as well as their antibiotic sensitivity profiles and genetic identification in Peshawar, Pakistan. The california mastitis test (CMT) was initially used to verify the presence for mastitis in 700 collected milk samples. The molecular identification of the 16SrRNA gene confirmed 120/700 (17.14 %) propagation of K. pneumonia. Out of these isolates MDR ESBL-producing isolates were 60/120 (50 %). The lactose were found (M = 3.96 ± 0.28, SD = 2.19), followed by fats (M = 3.12 ± 0.11, SD = 0.90), protein (M = 5.97 ± 0.24, SD = 1.84), sodium (M = 55.74 ± 2.07, SD = 15.81), potassium (M = 138.5 ± 1.53, SD = 11.71), chloride (M = 0.74 ± 0.03, SD = 0.24), calcium (M = 10.27 ± 0.31, SD = 2.42), and chlorine (M = 2.80 ± 0.22, SD = 1.70), respectively. Amikacin (80 %), ceftazidime (71 %), and tetracycline (71 %) were shown to be the most effective antimicrobials against all of the isolates. The occurrence of the blaSHV gene was observed at 56.00 % whereas the blaTEM gene and blaCTX-M gene were 36.00 %, and 30.00 %. The distribution of blaCTX-M subgroup genes was followed by blaCTX-M-1 (38.00 %), blaCTX-M-9 (22.20 %), and blaCTX-M-15 (61.10 %). Co-occurrence of blaCTX-M+ blaSHV was (15.00 %), blaCTX-M+ blaTEM were (6.60 %), and blaSHV + blaTEM were (10.00 %), respectively. The inappropriate, prolonged and common use of antibiotics may apply selective pressure for propagation and the occurrence of resistant isolates.

Current trend of biapenem susceptibility and disc diffusion breakpoints in Enterobacterales and Pseudomonas aeruginosa.

INTRODUCTION: Biapenem has been recently approved by the Drug Controller General of India for the treatment of complicated urinary tract infections (cUTI). However, there are no assessment studies that evaluate the in-vitro activity of biapenem against contemporary ESBL-producing Indian Enterobacterales isolates. To determine the activity of biapenem against contemporary ESBLs and/or OXA-1/ampC producing Enterobacterales and Pseudomonas aeruginosa isolates. METHODOLOGY: Isolates were tested for susceptibility to biapenem and its comparators using the broth microdilution method. Presence of ESBLs (SHV, TEM, CTX-M) genes, OXA-1, and ampC genes (ACC, ACT, DHA, CIT/CMY, FOX) using multiplex PCR. RESULTS: Against ESBL with OXA-1 and/or ampC-producing E. coli, ESBL-K. pneumoniae, and cephalosporin-resistant P. aeruginosa, biapenem showed in-vitro activity similar to that of meropenem. Overall, a biapenem disc concentration of 10 µg provided no error rates for testing E. coli, K. pneumoniae, and P. aeruginosa isolates. CONCLUSION: It is more accurate to test biapenem at a 10 µg disc concentration and apply more stringent disc diffusion breakpoints for interpretation.

Natural products from food sources can alter the spread of antimicrobial resistance plasmids in Enterobacterales.

Antimicrobial resistance (AMR) poses a significant threat to global public health. Notably, resistance to carbapenem and extended-spectrum ß-lactam antibiotics in Gram-negative bacteria is a major impediment to treating infections. Genes responsible for antibiotic resistance are frequently carried on plasmids, which can transfer between bacteria. Therefore, exploring strategies to prevent this transfer and the prevalence of AMR plasmids is timely and pertinent. Here, we show that certain natural product extracts and associated pure compounds can reduce the conjugation of AMR plasmids into new bacterial hosts. Using our established high-throughput fluorescence-based flow cytometry assay, we found that the natural products were more active in reducing transmission of the IncK extended-spectrum ß-lactamase-encoding plasmid pCT in Escherichia coli EC958c, compared to Klebsiella pneumoniae Ecl8 carrying the IncFII carbapenemase-encoding plasmid pKpQIL. The exception was the natural product rottlerin, also active in K. pneumoniae. In classical conjugation assays, rottlerin also reduced the conjugation frequency of the IncFII bla NDM-1 carrying plasmid pCPE16_3 from a clinical K. pneumoniae isolate. Our data indicate that the natural products tested here, in their current molecular structure, reduced conjugation by a small amount, which is unlikely to achieve a large-scale reduction in AMR in bacterial populations. However, certain natural products like rottlerin could provide a foundation for further research into compounds with effective anti-plasmid activity.

Hypervirulent and carbapenem-resistant Klebsiella pneumoniae: A global public health threat.

The evolution of hypervirulent and carbapenem-resistant Klebsiella pneumoniae can be categorized into three main patterns: the evolution of KL1/KL2-hvKp strains into CR-hvKp, the evolution of carbapenem-resistant K. pneumoniae (CRKp) strains into hv-CRKp, and the acquisition of hybrid plasmids carrying carbapenem resistance and virulence genes by classical K. pneumoniae (cKp). These strains are characterized by multi-drug resistance, high virulence, and high infectivity. Currently, there are no effective methods for treating and surveillance this pathogen. In addition, the continuous horizontal transfer and clonal spread of these bacteria under the pressure of hospital antibiotics have led to the emergence of more drug-resistant strains. This review discusses the evolution and distribution characteristics of hypervirulent and carbapenem-resistant K. pneumoniae, the mechanisms of carbapenem resistance and hypervirulence, risk factors for susceptibility, infection syndromes, treatment regimens, real-time surveillance and preventive control measures. It also outlines the resistance mechanisms of antimicrobial drugs used to treat this pathogen, providing insights for developing new drugs, combination therapies, and a "One Health" approach. Narrowing the scope of surveillance but intensifying implementation efforts is a viable solution. Monitoring of strains can be focused primarily on hospitals and urban wastewater treatment plants.

Exploration of Antimicrobial Peptides in the Treatment of Gentamicin-Resistant Klebsiella pneumoniae Infection.

Introduction: The emergence of multidrug-resistant Klebsiella pneumoniae (K. pneumoniae) and the decline of effective antibiotics lead to the urgent need for new antibacterial agents. The aim of this study is to investigate the therapeutic effect of antimicrobial peptides against gentamicin-resistant (RT) K. pneumoniae and to screen effective antimicrobial peptides. Methods: In this study, the RT strains were induced by gradient gentamicin, and the RT strains were selected by detecting the expression levels of efflux pump genes, porin genes, and biofilm formation genes of the strains combined with their effects on the cells. Then the effects of four antimicrobial peptides on the efflux pump activity, biofilm formation level and cell condition after infection were detected to explore the effects of antimicrobial peptides on RT strains. Finally, the RT strain was used to induce a mouse model of pneumonia, and the four antimicrobial peptides were used to treat pneumonia mice for in vivo experiments. The pathological changes in lung tissues in each group were detected to explore the antimicrobial peptide with the most significant effect on the RT strain in vivo. Results: The results showed that the minimal inhibitory concentrations of the RT strains (strain C and strain I) were significantly higher than those of the wild-type strain, and the expression of efflux pump, porin and biofilm formation genes was significantly increased. The antimicrobial peptides could effectively inhibit the biofilm formation and efflux pump protein function of the RT strains. In addition, the antimicrobial peptides showed promising antibacterial effects both in vitro and in vivo. Discussion: Our study provided a theoretical basis for the treatment of gentamicin resistant K. pneumoniae infection with antimicrobial peptides, and found that KLA was significantly superior to LL37, Magainin I, KLA and Dermaseptin (10 µg/mL in cells, 50 µg in mice).

Solithromycin in Combination with Other Antimicrobial Agents Against the Carbapenem Resistant Klebsiella pneumoniae (CRKP).

Klebsiella pneumoniae is considered as the most common pathogen of hospital-acquired pneumonia. K. pneumoniae has emerged as the superbug which had shown multidrug resistance (MDR) as well as extensively drug resistance. Carbapenem resistant K. pneumoniae (CRKP) has become a menace for the treatment with monotherapy of the patients mainly admitted in intensive care units. Hence, in the present study we collected total 187 sputum isolates of K. pneumoniae and performed the antimicrobial susceptibility testing by using the automated Vitek-2 system and broth micro-dilution method (67 CRKP). The combination study of solithromycin with meropenem, colistin, cefotaxime, piperacillin and tazobactam, nitrofurantoin, tetracycline, levofloxacin, curcumin and nalidixic acid was performed by using checkerboard assay. We observed the high rate of resistance towards ampicillin, cefotaxime, ceftriaxone, cefuroxime and aztreonam. The colistin and tigecycline were the most sensitive drugs. The CRKP were 36%, maximum were from the patients of ICUs. The best synergistic effect of solithromycin was with meropenem and cefotaxime (100%), colistin and tetracycline (80%). So, these combinations can be a choice of treatment for the infections caused by MDR CRKP and other Gram-negative bacteria where the monotherapy could not work.

Screening the Rumen of Balochi Camel (Camelus dromedarius) and Cashmere Goat (Capra hircus) to Isolate Enzyme-Producing Bacteria as Potential Additives for Animal Feed.

Rumen microbiology has made a significant contribution to the discovery of biodegradation processes, which convert nutrients into energy for ruminants. Therefore, understanding the enzymatic potential in the rumen of different animal species is essential for developing efficient microbial feed additives. The aim of this study was to isolate enzyme-producing bacteria (EPBs) from the rumen of the Balochi camel (Camelus dromedarius) and Cashmere goat (Capra hircus) as potential additives for animal feed. The EPBs were screened based on the hydrolysis of carboxyl methyl cellulose, tannin, starch, and bovine serum albumin. The isolates were then subjected to enzyme activity assays and molecular characterization. Additionally, they were evaluated for their antagonistic effects, antibiotic susceptibility, and growth in acidic, bile, and saline media. Thirteen enzyme-producing strains were identified in the rumen of the camels and goats, belonging to the genera Klebsiella, Escherichia, Raoultella, Enterobacter and Pectobacterium. The highest and lowest tannase activities were recorded for Escherichia coli GHMGHE41 (10.46 Um/l-1) and Raoultella planticola GHMGHE15 (1.83 Um/l-1), respectively. Enterobacter cloacae GHMGHE18 (2.03 U/ml) was the most effective cellulolytic isolate, compared to Klebsiella strains (1.05 Um/l-1). The highest protease producer was Klebsiella pneumoniae GHMGHE13 (3.00 U/ml-1), while Escherichia coli GHMGHE17 (1.13 U/ml-1) had the lowest activity. Klebsiella pneumoniae GHMGHE13 (1.55 U/ml-1) and Enterobacter cloacae GHMGHE19 (1.26 U/ml-1) were the highest and lowest producers of amylase, respectively. The strains exhibited mixed responses to antibiotics and remained stable under stressful conditions. These findings indicate that ruminal EPBs have the potential to be used in animal feed, pending further in vivo studies.

Emergence of a clinical Klebsiella pneumoniae harboring an acrAB-tolC in chromosome and carrying the two repetitive tandem core structures for bla KPC-2 and bla CTX-M-65 in a plasmid.

Objective: The emergence of clinical Klebsiella pneumoniae strains harboring acrAB-tolC genes in the chromosome, along with the presence of two repetitive tandem core structures for bla KPC-2 and bla CTX-M-65 genes on a plasmid, has presented a significant clinical challenge. Methods: In order to study the detailed genetic features of K. pneumoniae strain SC35, both the bacterial chromosome and plasmids were sequenced using Illumina and nanopore platforms. Furthermore, bioinformatics methods were employed to analyze the mobile genetic elements associated with antibiotic resistance genes. Results: K. pneumoniae strain SC35 was found to possess a class A beta-lactamase and demonstrated resistance to all tested antibiotics. This resistance was attributed to the presence of efflux pump genes, specifically acrAB-tolC, on the SC35 chromosome. Additionally, the SC35 plasmid p1 carried the two repetitive tandem core structures for bla KPC-2 and bla CTX-M-65, as well as bla TEM-1 with rmtB, which shared overlapping structures with mobile genetic elements as In413, Tn3, and TnAs3. Through plasmid transfer assays, it was determined that the SC35 plasmid p1 could be successfully transferred with an average conjugation frequency of 6.85 × 10-4. Conclusion: The structure of the SC35 plasmid p1 appears to have evolved in correlation with other plasmids such as pKPC2_130119, pDD01754-2, and F4_plasmid pA. The infectious strain SC35 exhibits no susceptibility to tested antibioticst, thus effective measures should be taken to prevent the spread and epidemic of this strain.

Occurrence of multi-carbapenemase-producing Enterobacterales in a tertiary hospital in Madrid (Spain): A new epidemiologic scenario.

INTRODUCTION: Multi-carbapenemase-producing Enterobacterales (M-CPE) are increasingly described. We characterized the M-CPE isolates prospectively recovered in our hospital (Madrid, Spain) over two years (2021-2022). METHODS: We collected 796 carbapenem resistant Enterobacterales (CRE) from clinical and surveillance samples. Carbapenemase production was confirmed with phenotypic (immunochromatographic, disk diffusion) and molecular (PCR, WGS) techniques. Antimicrobial susceptibility was evaluated by a standard broth microdilution method. Clinical and demographic data were collected. RESULTS: Overall, 23 M-CPE (10 Klebsiella pneumoniae, 6 Citrobacter freundii complex, 3 Escherichia coli, 2 Klebsiella oxytoca, and 2 Enterobacter hormaechei) isolates were recovered from 17 patients (3% with CPE, 0.26-0.28 cases per 1000 admissions). OXA-48 + KPC-3 (7/23) and KPC-3 + VIM-1 (5/23) were the most frequent carbapenemase combinations. All patients had prior antibiotics exposure, including carbapenems (8/17). High resistance rates to ceftazidime/avibactam (14/23), imipenem/relebactam (16/23) and meropenem/vaborbactam (7/23) were found. Ceftazidime/avibactam + aztreonam combination was synergistic in all metallo-ß-lactamase producers. Clonal and non-clonal related isolates were found, particularly in K. pneumoniae (5 ST29, 3 ST147, 3 ST307) and C. freundii (3 ST8, 2 ST125, 1 ST563). NDM-1 + OXA-48 was introduced with the ST147-K. pneumoniae high-risk clone linked to the transfer of a Ukrainian patient. We identified four possible nosocomial clonal transmission events between patients of the same clone with the same combination of carbapenemases (KPC-3 + VIM-1-ST29-K. pneumoniae, NDM-1 + OXA-48-ST147-K. pneumoniae and KPC-2 + VIM-1-ST145-K. oxytoca). Carbapenemase-encoding genes were located on different plasmids, except for VIM-1 + KPC-2-ST145-K. oxytoca. Cross-species transmission and a possible acquisition overtime was found, particularly between K. pneumoniae and E. coli producing OXA-48 + KPC-3. CONCLUSION: M-CPE is an emerging threat in our hospital. Co-production of different carbapenemases, including metallo-ß-lactamases, limits therapeutic options and depicts the need to reinforce infection control measures.

Carbapenem-resistant Klebsiella pneumoniae strains isolated from clinical specimens in Siirt, Türkiye; molecular characterization and antimicrobial resistance genes detection.

This study aimed to molecularly identify carbapenem-resistant Klebsiella pneumoniae (CRKP) strains isolated from clinical samples and to determine antibiotic resistance genes. Only carbapenem-resistant strains were included in our study. Of the 35 CRKP strains, 18 (51.4%) were extensive drug, 11 (31.4%) were multi-drug, and 6 (17.1%) were pan-drug resistances. PCR amplification revealed that 25% of the strains carried the OXA-51, 20% the OXA-48, and %5 the OXA23 genes. Multilocus sequence typing (MLST) analysis based on seven house-keeping genes revealed sequence type 39. The capsule and O-antigen types were determined as KL103 and O2a, respectively. WGS analysis revealed the existence of ß-lactamase, aminoglycoside, sulfonamide, Phenicol, and Fosfomycin-resistant genes. While the K. pneumoniae OmpK37 gene was detected in all 3 strains, the OmpK36 gene was detected only in the CRSU20 strain. This study is important as it is the first study to perform molecular analysis of CRKP strains from Siirt, Türkiye.

Genomic characterization of Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) strains circulating in three university hospitals in Northern Italy over three years.

OBJECTIVES: Genomic surveillance of Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) is crucial for virulence, drug-resistance monitoring, and outbreak containment. METHODS: Genomic analysis on 87 KPC-Kp strains isolated from 3 Northern Italy hospitals in 2019-2021 was performed by whole genome sequencing (WGS), to characterize resistome, virulome, and mobilome, and to assess potential associations with phenotype resistance and clinical presentation. Maximum Likelihood and Minimum Spanning Trees were used to determine strain correlations and identify potential transmission clusters. RESULTS: Overall, 15 different STs were found; the predominant ones included ST307 (35, 40.2%), ST512/1519 (15, 17.2%), ST20 (12, 13.8%), and ST101 (7, 8.1%). 33 (37.9%) KPC-Kp strains were noticed to be in five transmission clusters (median number of isolates in each cluster: 5 [3-10]), four of them characterized by intra-hospital transmission. All 87 strains harbored Tn4401a transposon, carrying blaKPC-3 (48, 55.2%), blaKPC-2 (38, 43.7%), and in one case (1.2%) blaKPC-33, the latter gene conferred resistance to ceftazidime/avibactam (CZA). Thirty strains (34.5%) harbored porin mutations; of them, 7 (8.1%) carried multiple Tn4401a copies. These strains were characterized by significantly higher CZA minimum inhibitory concentration compared with strains with no porin mutations or single Tn4401a copy, respectively, even if they did not overcome the resistance breakpoint of 8 ug/mL. Median 2 (IQR:1-2) virulence factors per strain were detected. The lowest number was observed in ST20 compared to the other STs (p<0.001). While ST307 was associated with infection events, a trend associated with colonization events could be observed for ST20. CONCLUSIONS: Integration of genomic, resistance score, and clinical data allowed us to define a relative diversification of KPC-Kp in Northern Italy between 2019 and 2021, characterized by few large transmission chains and rare inter-hospital transmission. Our results also provided initial evidence of correlation between KPC-Kp genomic signatures and higher MIC levels to some antimicrobial agents or colonization/infection status, once again underlining WGS's importance in bacterial surveillance.

A New Casjensviridae Bacteriophage Isolated from Hospital Sewage for Inactivation of Biofilms of Carbapenem Resistant Klebsiella pneumoniae Clinical Isolates.

Klebsiella pneumoniae, a member of the ESKAPE pathogen group, is a prominent cause of hospital-acquired infections. The WHO has recognized carbapenem-resistant K. pneumoniae as a critical-one priority pathogen. These resilient superbugs have the ability to form biofilms and present a significant global threat. In the present study, we isolated and characterized a bacteriophage SAKp02, from hospital sewage, infectious to carbapenem-resistant K. pneumoniae patient isolates. SAKp02 could infect 43 of 72 clinical isolates, indicating a broad host spectrum. Whole genome analysis classified SAKp02 within the family Casjensviridae, with a 59,343 bp genome encoding 82 ORFs. Comparative genomic analysis revealed significant differences between SAKp02 and its closest viruses, indicating a distinct genetic makeup positioning it as a novel phage strain within the lineage. The SAKp02 genome comprises bacteriolytic enzymes, including holin, endolysin, and phage depolymerase, crucial for bacterial lysis and biofilm disruption. It reduced biofilm biomass by over threefold compared to the control and eradicated 99% of viable cells within a 4 h treatment period. Scanning electron microscopy corroborated the ability of the phage to dismantle biofilm matrices and lyse bacterial cells. Safe and effective treatments are warranted, and hence, the fully characterized lytic phages with therapeutic potential against drug-resistant clinical isolates of bacteria are needed. Our study is the first to report the antibacterial and antibiofilm activity of Casjensviridae phages, and our discovery of a novel K. pneumoniae phage broadens the arsenal against the bacteria.

Co-existence of two plasmids harboring transferable resistance-nodulation-division pump gene cluster, tmexCD1-toprJ1, and colistin resistance gene mcr-8 in Klebsiella pneumoniae.

BACKGROUND: The emergence of plasmid-mediated mobile colistin resistance (mcr) gene poses a great challenge to the clinical application of polymyxins. To date, mcr-1 to mcr-10 have been found in animals, humans, and the environment. Among them, mcr-8 was first identified in Klebsiella pneumoniae (K. pneumoniae) of swine origin, and then mcr-8.1 to mcr-8.5 were successively identified. Notably, K. pneumoniae is the major host of the mcr-8 gene in both animals and humans. This study aims to explore the characteristics of K. pneumoniae strains carrying the mcr-8 gene and tmexCD1-toprJ1 gene cluster and investigate the correlation between these two antibiotic resistance genes. METHODS: The isolates from the poultry farms and the surrounding villages were identified by mass spectrometer, and the strains positive for mcr-1 to mcr-10 were screened by polymerase chain reaction (PCR). The size of the plasmid and the antimicrobial resistance genes carried were confirmed by S1-nuclease pulsed-field gel electrophoresis (S1-PFGE) and Southern hybridization, and the transferability of the plasmid was verified by conjugation experiments. Antimicrobial susceptibility testing (AST) and whole genome sequencing (WGS) were used to characterize the strains. RESULTS: Two K. pneumoniae isolates (KP26 and KP29) displaying polymyxin resistance were identified as mcr-8 gene carriers. Besides that, tigecycline-resistant gene cluster tmexCD1-toprJ1 was also found on the other plasmid which conferred strain resistance to tigecycline. Through epidemiological analysis, we found that the mcr-8 gene has dispersed globally, circulating in the human, animals, and the environment. Furthermore, our analysis suggests that the coexistence of mcr-8 and tmexCD1-toprJ1 on a single plasmid might evolved through plasmid recombination. CONCLUSIONS: Although the mcr-8 and tmexCD1-toprJ1 gene clusters in the two strains of K. pneumoniae in this study were on two different plasmids, they still pose a potential threat to public health, requiring close monitoring and further study.

Emergence of blaNDM-5 and blaOXA-232 Positive Colistin- and Carbapenem-Resistant Klebsiella pneumoniae in a Bulgarian Hospital.

The rapid spread of carbapenemase-producing strains has led to increased levels of resistance among Gram-negative bacteria, especially enterobacteria. The current study aimed to collect and genetically characterize the colistin- and carbapenem-resistant isolates, obtained in one of the biggest hospitals (Military Medical Academy) in Sofia, Bulgaria. Clonal relatedness was detected by RAPD and MLST. Carbapenemases, ESBLs, and mgrB were investigated by PCR amplification and sequencing, replicon typing, and 16S rRNA methyltransferases with PCRs. Fourteen colistin- and carbapenem-resistant K. pneumoniae isolates were detected over five months. Six carbapenem-resistant and colistin-susceptible isolates were also included. The current work revealed a complete change in the spectrum of carbapenemases in Bulgaria. blaNDM-5 was the only NDM variant, and it was always combined with blaOXA-232. The coexistence of blaOXA-232 and blaNDM-5 was observed in 10/14 (72%) of colistin- and carbapenem-resistant K. pneumoniae isolates and three colistin-susceptible isolates. All blaNDM-5- and blaOXA-232-positive isolates belonged to the ST6260 (ST101-like) MLST type. They showed great mgrB variability and had a higher mortality rate. In addition, we observed blaOXA-232 ST14 isolates and KPC-2-producing ST101, ST16, and ST258 isolates. The colistin- and carbapenem-resistant isolates were susceptible only to cefiderocol for blaNDM-5- and blaOXA-232-positive isolates and to cefiderocol and ceftazidime/avibactam for blaOXA-232- or blaKPC-2-positive isolates. All blaOXA-232-positive isolates carried rmtB methylase and the colE replicon type. The extremely limited choice of appropriate treatment for patients infected with such isolates and their faster distribution highlight the need for urgent measures to control this situation.

Colistin resistance in ESBL- and Carbapenemase-producing Escherichia coli and Klebsiella pneumoniae clinical isolates in Cambodia.

OBJECTIVES: Despite the critical importance of colistin as a last-resort antibiotic, limited studies have investigated colistin resistance in human infections in Cambodia. This study aimed to investigate the colistin resistance and its molecular determinants among Extended-spectrum beta-lactamase (ESBL)- and carbapenemase-producing (CP) Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E. coli) isolated in Cambodia between 2016 and 2020. METHODS: E. coli (n = 223) and K. pneumoniae (n = 39) were tested for colistin minimum inhibitory concentration (MIC) by broth microdilution. Resistant isolates were subjected to polymerase chain reaction (PCR) for detection of mobile colistin resistance genes (mcr) and chromosomal mutations in the two-component system (TCS). RESULTS: Eighteen isolates (10 K. pneumoniae and 8 E. coli) revealed colistin resistance with a rate of 5.9% in E. coli and 34.8% in K. pneumoniae among ESBL isolates, and 1% in E. coli and 12.5% in K. pneumoniae among CP isolates. The resistance was associated with mcr variants (13/18 isolates, mcr-1, mcr-3, and mcr-8.2) and TCS mutations within E. coli and K. pneumoniae, with the first detection of mcr-8.2 in Cambodia, the discovery of new mutations potentially associated to colistin resistance in the TCS of E. coli (PhoP I47V, PhoQ N352K, PmrB G19R, and PmrD G85R) and the co-occurrence of mcr genes and colistin resistance conferring TCS mutations in 11 of 18 isolates. CONCLUSIONS: The findings highlight the presence of colistin resistance in ESBL- and CP- Enterobacteriaceae involved in human infections in Cambodia as well as chromosomal mutations in TCS and the emergence of mcr-8.2 in E. coli and K. pneumoniae. It underscores the need for continuous surveillance, antimicrobial stewardship, and control measures to mitigate the spread of colistin resistance.

Geographical distribution, disease association and diversity of Klebsiella pneumoniae K/L and O antigens in India: roadmap for vaccine development.

Klebsiella pneumoniae poses a significant healthcare challenge due to its multidrug resistance and diverse serotype landscape. This study aimed to explore the serotype diversity of 1072 K. pneumoniae and its association with geographical distribution, disease severity and antimicrobial/virulence patterns in India. Whole-genome sequencing was performed on the Illumina platform, and genomic analysis was carried out using the Kleborate tool. The analysis revealed a total of 78 different KL types, among which KL64 (n=274/1072, 26 %), KL51 (n=249/1072, 24 %), and KL2 (n=88/1072, 8 %) were the most prevalent. In contrast, only 13 distinct O types were identified, with O1/O2v1 (n=471/1072, 44 %), O1/O2v2 (n=353/1072, 33 %), and OL101 (n=66/1072, 6 %) being the predominant serotypes. The study identified 114 different sequence types (STs) with varying serotypes, with ST231 being the most predominant. O serotypes were strongly linked with STs, with O1/O2v1 predominantly associated with ST231. Simpson's diversity index and Fisher's exact test revealed higher serotype diversity in the north and east regions, along with intriguing associations between specific serotypes and resistance profiles. No significant association between KL or O types and disease severity was observed. Furthermore, we found the specific association of virulence factors yersiniabactin and aerobactin (P<0.05) with KL types but no association with O antigen types (P>0.05). Conventionally described hypervirulent clones (i.e. KL1 and KL2) in India lacked typical virulent markers (i.e. aerobactin), contrasting with other regional serotypes (KL51). The cumulative distribution of KL and O serotypes suggests that future vaccines may have to include either ~20 KL or four O types to cover >85 % of the carbapenemase-producing Indian K. pneumoniae population. The results highlight the necessity for comprehensive strategies to manage the diverse landscape of K. pneumoniae strains across different regions in India. Understanding regional serotype dynamics is pivotal for targeted surveillance, interventions, and tailored vaccine strategies to tackle the diverse landscape of K. pneumoniae infections across India. This article contains data hosted by Microreact.

Antibiotic resistance rates in hypervirulent Klebsiella pneumoniae strains: A systematic review and meta-analysis.

OBJECTIVES: In response to the growing global concerns regarding antibiotic resistance, we conducted a meta-analysis to assess the prevalence of antibiotic resistance in hypervirulent Klebsiella pneumoniae (hvKp) strains. METHODS: We conducted a meta-analysis of antibiotic resistance in the hvKp strains. Eligible studies published in English until April 10, 2023, were identified through a systematic search of various databases. After removing duplicates, two authors independently assessed and analysed the relevant publications, and a third author resolved any discrepancies. Data extraction included publication details and key information on antibiotic resistance. Data synthesis employed a random-effects model to account for heterogeneity, and various statistical analyses were conducted using R and the metafor package. RESULTS: This meta-analysis of 77 studies from 17 countries revealed the prevalence of antibiotic resistance in hvKp strains. A high resistance rates have been observed against various classes of antibiotics. Ampicillin-sulbactam faced 45.3% resistance, respectively, rendering them largely ineffective. The first-generation cephalosporin cefazolin exhibited a resistance rate of 38.1%, whereas second-generation cefuroxime displayed 26.7% resistance. Third-generation cephalosporins, cefotaxime (65.8%) and ceftazidime (57.1%), and fourth-generation cephalosporins, cefepime (51.3%), showed substantial resistance. The last resort carbapenems, imipenem (45.7%), meropenem (51.0%) and ertapenem (40.6%), were not spared. CONCLUSION: This study emphasizes the growing issue of antibiotic resistance in hvKp strains, with notable resistance to both older and newer antibiotics, increasing resistance over time, regional disparities and methodological variations. Effective responses should involve international cooperation, standardized testing and tailored regional interventions.

Temperatures above 37°C increase virulence of a convergent Klebsiella pneumoniae sequence type 307 strain.

Background: Convergence of Klebsiella pneumoniae (KP) pathotypes has been increasingly reported in recent years. These pathogens combine features of both multidrug-resistant and hypervirulent KP. However, clinically used indicators for hypervirulent KP identification, such as hypermucoviscosity, appear to be differentially expressed in convergent KP, potential outbreak clones are difficult to identify. We aimed to fill such knowledge gaps by investigating the temperature dependence of hypermucoviscosity and virulence in a convergent KP strain isolated during a clonal outbreak and belonging to the high-risk sequence type (ST)307. Methods: Hypermucoviscosity, biofilm formation, and mortality rates in Galleria mellonella larvae were examined at different temperatures (room temperature, 28°C, 37°C, 40°C and 42°C) and with various phenotypic experiments including electron microscopy. The underlying mechanisms of the phenotypic changes were explored via qPCR analysis to evaluate plasmid copy numbers, and transcriptomics. Results: Our results show a temperature-dependent switch above 37°C towards a hypermucoviscous phenotype, consistent with increased biofilm formation and in vivo mortality, possibly reflecting a bacterial response to fever-like conditions. Furthermore, we observed an increase in plasmid copy number for a hybrid plasmid harboring carbapenemase and rmpA genes. However, transcriptomic analysis revealed no changes in rmpA expression at higher temperatures, suggesting alternative regulatory pathways. Conclusion: This study not only elucidates the impact of elevated temperatures on hypermucoviscosity and virulence in convergent KP but also sheds light on previously unrecognized aspects of its adaptive behavior, underscoring its resilience to changing environments.