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Background: Currently, antimicrobial agents are widely used in both animals and agriculture, causing the crisis of multidrug-resistant (MDR) bacteria. In this study we surveyed for 4 important antimicrobial-resistant bacteria: extended-spectrum β-lactamase (ESBL)-producing Escherichia coli, Klebsiella pneumoniae and Salmonella spp., and methicillin-resistant Staphylococcus aureus (MRSA) from the environment around chicken and pig farms., Methods: Forty-four soils, 44 water and 15 bootstrap samples were collected from upstream and downstream of the riverside close to 22 animal farms in 2020 (9 farms) and 2023 (13 farms). The samples were cultured in appropriate media and isolated colonies were further identified. Antimicrobial susceptibility and ESBL production were tested by disk diffusion method except for colistin susceptibility determined by broth disk elution test. Common resistance genes were tested by polymerase chain reaction (PCR) methods., Results: A total of 123 E. coli (n = 59) and Klebsiella species (n = 64) were identified and 25 isolates of them were MDR; 21 and 4 isolates from the downstream and upstream areas respectively. In addition, 13 isolates were ESBL producers, of which 12 isolates were from the downstream area. The most common ESBL gene among both organisms was bla CTX-M , whereas colistin resistance gene, mcr-1, was found in 2 E. coli isolates. Salmonella spp. and MRSA were not isolated from any sample., Conclusions: Antimicrobial-resistant bacteria were found in the environment around the animal farms especially from the downstream area, suggesting the urgent need of judicious use of antimicrobial agents in animal farms., Competing Interests: Declaration of Competing Interest All authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Extended-spectrum β-lactamase-producing Enterobacterales (ESBL-PE), particularly Escherichia coli and Klebsiella pneumoniae, have been consistently associated with treatment failure, high mortality and morbidity. The emergence of carbapenem resistance among ESBL-PE strains exacerbates the antimicrobial resistance. However, data are very limited in developing countries as Burkina Faso. This study aimed to determine the prevalence of carbapenemase and AmpC-β-lactamase production among ESBL-producing E. coli (ESBL-Ec) and Klebsiella spp. (ESBL-K) isolated from patients' stool in Burkina Faso., Materials and Methods: From January 2020 to June 2022, we isolated 277 ESBL-PE from patients' stool in five hospitals in Burkina Faso. The strains were isolated on ESBL-selective chromogenic media and identified using API20E. The isolates were tested against 15 antimicrobial agents using the disc-diffusion method on Mueller-Hinton (MH) agar. ESBL production was confirmed by double disc synergy method. Carbapenemase and AmpC-β-lactamase production and phenotypic co-resistance were determined., Results: Among the 277 ESBL-PE strains isolated, 203 were E. coli, and 74 were Klebsiella spp. Of these bacteria, 2.9% were carbapenemase producers and 6.5% were AmpC-β-lactamase producers. The carbapenemase producers were detected at tertiary and secondary hospitals, mainly in hospitalized patients and females, whereas AmpC-β-lactamase producers were detected at all levels of healthcare, predominantly in non-hospitalized patients, male, and under 15 years of age. The co-resistance rates were as high as 82% for fluoroquinolones, 91% for aminoglycosides, and 94% for sulfonamides. Fosfomycin resistance was 2.5% for ESBL-Ec and 50% for ESBL-K., Conclusion: This study showed that ESBL-PEs co-produce carbapenemase and/or AmpC-β-lactamase. High co-resistances were reported for commonly used antibiotic agents. Therefore, screening for carbapenem-resistant Enterobacterales (CRE) carriage is necessary to limit its spread within hospitals., Competing Interests: Declarations. Ethical aspects: This study is part of the AMRIWA project, which received approval from the health research Ethics Committee of Burkina Faso (N°153-12-2018/CERS). Informed consents were obtained from patients or parents of minor patients for their participation in the study. The study was conducted in accordance with the declaration of Helsinki. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

β-Galactosidases are highly desirable in various biotechnological applications. However, research on those obtained from Klebsiella strains has been noticeably restricted. The present investigation centers on the isolation, purification, and characterization of a β-galactosidase enzyme derived from Klebsiella michiganensis (GALB5582Y). Additionally, the study aims to immobilize GALB5582Y onto functionalized graphene oxide (GO)-based polystyrene electrospun nanofibrous membranes (ENMs). The ultimate goal is to enhance the enzyme's transgalactosylation and catalytic efficiency, thereby expanding its range of potential applications. The GALB5582Y gene was sequenced, revealing a 3354 bp sequence that encodes 1024 amino acids. This discovery provides vital information about the gene's structural arrangement. The effectiveness of functionalized graphene oxide (GO)-based engineered nanomaterials (ENMs) in immobilising GALB5582Y was confirmed using SEM, FTIR, and XRD investigations. Significant stability was reported during assessments, with the enzyme activity remaining extended. Additionally, it was shown that the enzyme was efficiently distributed across the surface of the ENM. Although there have been breakthroughs in enzyme production and immobilisation techniques, there is still room for improvement in maximizing the effectiveness of GALB5582Y immobilisation and increasing the yield of galactooligosaccharides (GOS). This calls for additional investigation and refinement., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objectives: Klebsiella spp. are leading causes of nosocomial infections. Their ability to harbour antimicrobial resistance genes makes them an important public health threat. This study aimed to report the genomic background of carbapenemase-producing Klebsiella quasipneumoniae (HV55B) and Klebsiella michiganensis (HV55D) strains isolated from fresh vegetables destined for hospitalized inpatients., Methods: Microbiological and molecular methods were used to isolate and identify the strains, which were submitted to the antimicrobial susceptibility test and pH tolerance assays. Whole genome sequencing was performed on MiSeq and NextSeq platforms, and online available tools were applied to bioinformatic analysis of clinically relevant information., Results: Both isolates were considered multidrug-resistant and tolerated pH ≥ 4 for 24 h. HV55B belonged to sequence type (ST) ST668, and presented a broad resistome and plasmids from four incompatibility groups. HV55D belonged to ST40. Both strains HV55B and HV55D were genetically close to isolates responsible for human infections around the world, which stands for the plausibility of such bacteria to cause disease in patients of the studied institution., Conclusions: Our results confirm the presence of carbapenemase-producing Klebsiella spp. in fresh foodstuffs intended for hospitalized inpatients' consumption. The genomes characterized here also provide clinically and genomically relevant information to forthcoming epidemiological surveillance studies., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Klebsiella michiganensis is an emerging hospital-acquired bacterial pathogen, particularly strains harboring plasmid-mediated carbapenemase genes. Here, we recovered and characterized a multidrug-resistant strain, bla KPC-2 -producing Klebsiella michiganensis LS81, which was isolated from the abdominal drainage fluid of a clinical patient in China, and further characterized the co-harboring plasmid. K. michiganensis LS81 tested positive for the bla KPC-2 genes by PCR sequencing, with bla KPC-2 located on a plasmid as confirmed by S1 nuclease pulsed-field gel electrophoresis combined with Southern blotting. In the transconjugants, the bla KPC-2 genes were successfully transferred to the recipient strain E. coli EC600. Whole-genome sequencing and bioinformatics analysis confirmed that this strain belongs to sequence type 196 (ST196), with a complete genome comprising a 5,926,662bp circular chromosome and an 81,451bp IncM2 plasmid encoding bla KPC-2 (designated pLS81-KPC). The IncM2 plasmid carried multiple β-lactamase genes such as bla TEM-1B , bla CTX-M-3 , and bla KPC-2 inserted in truncated Tn 6296 with the distinctive core structure IS Kpn27 - bla KPC-2 -IS Kpn6 . A comparison with 46 K. michiganensis genomes available in the NCBI database revealed that the closest phylogenetic relative of K. michiganensis LS81 is a clinical isolate from a wound swab in the United Kingdom. Ultimately, the pan-genomic analysis unveiled a substantial accessory genome within the strain, alongside significant genomic plasticity within the K. michiganensis species, emphasizing the necessity for continuous surveillance of this pathogen in clinical environments., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Song, Long, Ye, Yang, Wu, He, Wang, Li, Li, Deng, Li and Yuan.)

Objective: To investigate the distribution of carbapenem-resistant Enterobacterales (CRE) in the community and to describe the genomic characteristics., Methods: CRE screened from fecal samples in healthy people at the health examination center of a tertiary hospital in China underwent Whole genome sequencing (WGS) to analyze genotypic characteristics of CRE. The flanking DNA sequence of blaNDM-5 and mcr1.1 genes were analyzed by Gcluster software., Results: A total of 7187 fecal samples were screened, and CRE carriage was detected in 0.4 % of the sampled population. In total, 30 Escherichia coli, one Citrobacter freundii and one Klebsiella aerogene were screened. The 30 carbapenem-resistant Escherichia coli (CREC) isolates displayed slight resistance to amikacin (13.3 %) and aztreonam (20.0 %). All the CRE isolates contained blaNDM, and blaNDM-5 (84.4 %) was the most common one. B1 (n = 11) and A (n = 7) were predominant phylogroups. Furthermore, 34 distinct plasmid replicons, 67 different VFs, 22 distinct STs, 17 different FimH types, 26 O:H serotypes as well as 74 MGEs including 61 insertion sequences and 13 transposons were identified. The flanking DNA sequence analysis of blaNDM-5 and mcr1.1 genes indicates the key role of horizontal transfer of blaNDM-5 in the CRE development evidenced by diverse STs and phylogenetic tree., Conclusion: E. coli was the most predominant CRE isolates in community setting, and blaNDM (blaNDM-5) was the main CHβL encoding genes. The high prevalence of ARGs was associated with high resistance to commonly used antimicrobials. Besides, the genetic diversity of these isolates suggested the key role of blaNDM horizontal transfer in the CRE development. Thus, active screening of blaNDM in communities is particularly important for the prevention and control of CRE., Competing Interests: Declaration of competing interest The authors declare that they have no competing interest., (Copyright © 2024. Published by Elsevier Ltd.)

Polyacrylamide (PAM) is a high molecular weight polymer with extensive applications. However, inefficient natural degradation of PAM results in its environmental accumulation. Here, using multi-omics analysis, we constructed the PAM biodegradation pathway in Klebsiella sp. PCX, an efficient PAM-degrading bacterium. Subsequently, two unclassified amidohydrolases (PCX00451 and PCX04581) were identified as key factors for rapid PAM biodegradation, both of which possessed much higher hydrolysis efficiency for PAM than for small molecule amide compounds. Besides, crystal structures of PCX00451 and PCX04581 were solved. Both two amidohydrolases were consisted with a twisted triosephosphateisomerase (TIM)-barrel and a smaller β-sandwich domain. And their binding pockets were in the conserved metal center of TIM-barrel domain. Moreover, Asp267 of PCX00451 and Asp282 of PCX04581 were examined as active sites for acid/base catalysis. Our research characterized the molecular mechanisms of two efficient amidohydrolases, providing theoretical basis and valuable tools for PAM bioremediation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The genomic comparison of two Klebsiella michiganensis clinical isolates recovered from the same patient, one resistant to piperacillin-tazobactam and intermediate to cefotaxime, the other resistant to ceftazidime but susceptible to piperacillin-tazobactam, revealed one mutation in the bla OXY-1-24 gene accounting for a L169M substitution in the Ω loop. Cloning experiment in Escherichia coli demonstrated the contribution of this mutation to the hydrolysis spectrum extension towards ceftazidime and cefepime, whereas the resistance to piperacillin-tazobactam was reduced. To the best of our knowledge, this study shows for the first time that ceftazidime resistance can occur in vivo from OXY-1 precursor by structural alteration., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Infections with Enterobacterales (E) are increasingly difficult to treat due to antimicrobial resistance. After ceftriaxone replaced chloramphenicol (CHL) as empiric therapy for suspected sepsis in Malawi in 2004, extended-spectrum beta-lactamase (ESBL)-E rapidly emerged. Concurrently, resistance to CHL in Escherichia coli and Klebsiella spp. decreased, raising the possibility of CHL re-introduction. However, many phenotypically susceptible isolates still carry CHL acetyltransferase (cat) genes. To understand the molecular mechanisms and stability of this re-emerging CHL susceptibility we use a combination of genomics, phenotypic susceptibility assays, experimental evolution, and functional assays for CAT activity. Here, we show that of 840 Malawian E. coli and Klebsiella spp. isolates, 31% have discordant CHL susceptibility genotype-phenotype, and we select a subset of 42 isolates for in-depth analysis. Stable degradation of cat genes by insertion sequences leads to re-emergence of CHL susceptibility. Our study suggests that CHL could be reintroduced as a reserve agent for critically ill patients with ESBL-E infections in Malawi and similar settings and highlights the ongoing challenges in inferring antimicrobial resistance from sequence data., (© 2024. The Author(s).)

Klebsiella species commonly reside in dairy cattle guts and are consistently exposed to beta-lactam antibiotics, including ceftiofur, which are frequently used on the U.S. dairy farms. This may impose selection pressure and result in the emergence of extended-spectrum beta-lactamase (ESBL)-producing strains. However, information on the status and antimicrobial resistance (AMR) profile of ESBL- Klebsiella spp. in the U.S. dairy farms is largely unknown. This study aimed to determine the prevalence and AMR profile of ESBL- Klebsiella spp. and the factors affecting their occurrence in dairy cattle farms. Rectal fecal samples ( n = 508) and manure, feed, and water samples ( n = 64) were collected from 14 dairy farms in Tennessee. Samples were directly plated on CHROMagar ESBL, and presumptive Klebsiella spp. were confirmed using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Antimicrobial susceptibility testing was performed on the isolates against panels of 14 antimicrobial agents from 10 classes using minimum inhibitory concentration. Of 572 samples, 57 (10%) were positive for ESBL- Klebsiella spp. The fecal prevalence of ESBL- Klebsiella spp. was 7.2% (95% CI: 6.5-8.0). The herd-level fecal prevalence of ESBL- Klebsiella spp. was 35.7% (95% CI: 12.7-64.8). The fecal prevalence of ESBL- Klebsiella spp. was significantly higher in calves than in cows and higher in cows with higher parity (≥3) as compared to cows with low parity ( P < 0.001). Most (96.5%, n = 57) ESBL- Klebsiella spp. were resistant to ceftriaxone. The highest level of acquired co-resistance to ceftriaxone in ESBL- Klebsiella spp. was to sulfisoxazole (66.7%; 38/57). About 19% of ESBL-K lebsiella spp. were multidrug resistant. The presence of ESBL-producing Klebsiella spp. in dairy cattle, feed, and water obtained from troughs could play a crucial epidemiological role in maintaining and spreading the bacteria on farms and serving as a point source of transmission., Importance: We collected 572 samples from dairy farms, including rectal feces, manure, feed, and water. We isolated and identified extended-spectrum beta-lactamase (ESBL)- Klebsiella spp. and conducted an antimicrobial susceptibility test and analyzed different variables that may be associated with ESBL- Klebsiella spp. in dairy farms. The results of our study shed light on how ESBL- Klebsiella spp. are maintained through fecal-oral routes in dairy farms and possibly exit from the farm into the environment. We determine the prevalence of ESBL- Klebsiella spp. and their antimicrobial susceptibility profiles, underscoring their potential as a vehicle for multiple resistance gene dissemination within dairy farm settings. We also collected data on variables affecting their occurrence and spread in dairy farms. These findings have significant implications in determining sources of community-acquired ESBL- Enterobacteriaceae infections and designing appropriate control measures to prevent their spread from food animal production systems to humans, animals, and environments., Competing Interests: The authors declare no conflict of interest.

Biocatalytic degradation of non-hydrolyzable plastics is a rapidly growing field of research, driven by the global accumulation of waste. Enzymes capable of cleaving the carbon-carbon bonds in synthetic polymers are highly sought-after as they may provide tools for environmentally friendly plastic recycling. Despite some reports of oxidative enzymes acting on non-hydrolyzable plastics, including polyethylene or poly(vinyl chloride), the notion that these materials are susceptible to efficient enzymatic degradation remains controversial, partly driven by a general lack of studies independently reproducing previous observations. Here, we attempt to replicate two recent studies reporting that deconstruction of polyethylene and poly(vinyl chloride) can be achieved using an insect hexamerin from Galleria mellonella (so-called "Ceres") or a bacterial catalase-peroxidase from Klebsiella sp., respectively. Reproducing previously described experiments, we do not observe any activity on plastics using multiple reaction conditions and multiple substrate types. Digging deeper into the discrepancies between the previous data and our observations, we show how and why the original experimental results may have been misinterpreted., (© 2024. The Author(s).)

Objective: The aim of this study is to characterise the molecular characteristics of NDM-producing Enterobacterales, which have been on the increase in recent years in Japan, where IMP-producing bacteria are dominant among carbapenemase-producing Enterobacterales., Methods: We collected 21 strains of NDM-producing Enterobacterales detected between 2015 and 2022 at five hospitals in Tokyo and performed illumina whole genome sequencing. For the seven selected strains, nanopore long-read sequencing was also performed to characterise the plasmids harbouring bla NDM ., Results: Fourteen strains were Escherichia coli and all carried bla NDM-5 . Among these strains, eight and three were sequence type (ST) 410 and ST167, respectively, and both groups of strains were spread clonally in different hospitals. Two strains of Klebsiella pneumoniae ST147 carrying bla NDM-1 were detected in a hospital, and these strains had also spread clonally. The remainder included Enterobacter hormaechei, Klebsiella quasipneumoniae, Citrobacter amalonaticus, and Klebsiella michiganensis. Plasmid analysis revealed that an identical IncX3 plasmid harbouring bla NDM-5 was shared among four strains of different bacterial species (E. coli, C. amalonaticus, K. michiganensis, and E. hormaechei) detected at the same hospital. In addition, a Klebsiella quasipneumoniae strain detected at a different hospital also carried an IncX3 plasmid with a similar genetic structure., Conclusions: Nosocomial spread of multiple multidrug-resistant global clones and transmission of IncX3 plasmids harbouring bla NDM-5 among multiple species were detected as the major pathways of spread of NDM-producing Enterobacterales in Tokyo. Early detection of carriers and measures to prevent nosocomial spread are important to prevent further spread of NDM-producing organisms., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objective: Despite the increasing reports of bla NDM in Enterobacterales in Brazil, comprehensive whole genome sequencing (WGS) data remain scarce. To address this knowledge gap, our study focuses on the characterization of the genome of an New Delhi Metallo-β-lactamase (NDM)-1-producing Klebsiella quasipneumoniae subsp. quasipneumoniae (KQPN) clinical strain isolated in Brazil., Methods: The antimicrobial susceptibility profile of the A-73.113 strain was performed by agar dilution or broth microdilution following the Brazilian Antimicrobial Susceptibility Testing Committee/European Committee on Antimicrobial Susceptibility Testing recommendations. WGS was performed using the Illumina® NextSeq platform and the generated reads were assembled using the SPAdes software. The sequences obtained were submitted to the bioinformatics pipelines to determine the sequence type, resistome, plasmidome, and virulome., Results: The A-73.113 strain was identified as KQPN and was susceptible to polymyxins (MICs, ≤0.25 µg/mL), tigecycline (MIC, 0.5 µg/mL), ciprofloxacin (MIC, 0.5 µg/mL), and levofloxacin (MIC, 1 µg/mL). WGS analysis revealed the presence of genes conferring resistance to β-lactams (bla NDM-1 , bla CTX-M-15 , bla OXA-9 , bla OKP-A-5 , bla TEM-1 ), aminoglycosides [aph(3')-VI, aadA1, aac(6')-Ib], and fluoroquinolones (oqxAB, qnrS1, aac(6')-Ib-cr]. Additionally, the presence of the plasmid replicons Col(pHAD28), IncFIA(HI1), IncFIB(K) (pCAV1099-114), IncFIB(pQil), and IncFII(K), as well as virulence-encoding genes fimABCDEFGHIK (type 1 fimbria), pilW (type IV pili), iutA (aerobactin), entABCDEFS/fepABCDG/fes (Ent siderophores), iroE (salmochelin), and allABCDRS (allantoin utilization) was verified. Furthermore, we found that the A-73.113 strain belongs to ST1040., Conclusions: Here we report the genomic characteristics of an NDM-1-producing KQPN ST1040 strain isolated from blood cultures in Brazil. These data will enhance our comprehension of how this species contributes to the acquisition and dissemination of bla NDM-1 in Brazilian nosocomial settings., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Klebsiella pneumoniae species complex (KpSC) is an important disseminator of carbapenemase-encoding genes, mainly bla KPC-2 and bla NDM-1 , from hospitals to the environment. Consequently, carbapenem-resistant strains can be spread through the agrifood system, raising concerns about food safety. This study therefore aimed to isolate carbapenem-resistant KpSC strains from the agricultural and environmental sectors and characterize them using phenotypic, molecular, and genomic analyses., Results: Klebsiella pneumoniae and Klebsiella quasipneumoniae strains isolated from soils used for lemon, guava, and fig cultivation, and from surface waters, displayed an extensive drug-resistance profile and carried bla KPC-2 , bla NDM-1 , or both. In addition to carbapenemase-encoding genes, KpSC strains harbor a broad resistome (antimicrobial resistance and metal tolerance) and present putative hypervirulence. Soil-derived K. pneumoniae strains were assigned as high-risk clones (ST11 and ST307) and harbored the bla KPC-2 gene associated with Tn4401b and Tn3-like elements on IncN-pST15 and IncX5 plasmids. In surface waters, the coexistence of bla KPC-2 and bla NDM-1 genes was identified in K. pneumoniae ST6326, a new carbapenem-resistant regional Brazilian clone. In this case, bla KPC-2 with Tn4401a isoform and bla NDM-1 associated with a Tn125-like transposon were located on different plasmids. Klebsiella quasipneumoniae ST526 also presented the bla NDM-1 gene associated with a Tn3000 transposon on an IncX3 plasmid., Conclusion: These findings provide a warning regarding the transmission of carbapenemase-positive KpSC across the agricultural and environmental sectors, raising critical food safety and environmental issues. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Phytate (inositol hexaphosphate) is the major storage form of phosphorus (P) in nature, and phytases catalyze the hydrolysis of P from phytate and the formation of inositol phosphate isomers. In this study, a bacterium that produces phytase was isolated in a phytase screening medium. The bacterium was identified as Klebsiella sp. using phenotypic and molecular techniques. The PhyK phytase gene was successfully amplified from the genome, inserted into the pET-21a (+) vector, and expressed as a recombinant protein in E. Coli BL21. The efficiency of a laboratory phytase (Lab-Ph, PhyK phytase) was determined and compared with a commercial phytase (Com-Ph, Quantum Blue 40P phytase, AB Vista) under an in vitro digestion assay. The native signal peptide effectively facilitated the translocation of the protein to the periplasmic space of E. Coli BL21, resulting in the proper folding of the protein and the manifestation of desirable enzyme activity. The Lab-Ph displayed the temperature and pH optima at 50 °C and 5 respectively. In addition, the Lab-Ph was inactivated at 80 °C. Under an in vitro digestion assay condition, Lab-Ph improved the P solubility coefficient in broiler diets. In comparison, the Com-Ph significantly increased the P solubility coefficient even when compared with the Lab-Ph. In summary, this study has shown that Lab-Ph possesses the necessary biochemical properties to be used in various industrial applications. However, Lab-Ph is extremely sensitive to heat treatment. The Lab-Ph and Com-Ph under an in vitro digestion assay improved the solubility coefficient of P in the broiler diet., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024. Published by Elsevier Inc.)

Engineering enzyme-substrate binding pockets is the most efficient approach for modifying catalytic activity, but is limited if the substrate binding sites are indistinct. Here, we developed a 3D convolutional neural network for predicting protein-ligand binding sites. The network was integrated by DenseNet, UNet, and self-attention for extracting features and recovering sample size. We attempted to enlarge the dataset by data augmentation, and the model achieved success rates of 48.4%, 35.5%, and 43.6% at a precision of ≥50% and 52%, 47.6%, and 58.1%. The distance of predicted and real center is ≤4 Å, which is based on SC6K, COACH420, and BU48 validation datasets. The substrate binding sites of Klebsiella variicola acid phosphatase (KvAP) and Bacillus anthracis proline 4-hydroxylase (BaP4H) were predicted using DUnet, showing high competitive performance of 53.8% and 56% of the predicted binding sites that critically affected the catalysis of KvAP and BaP4H. Virtual saturation mutagenesis was applied based on the predicted binding sites of KvAP, and the top-ranked 10 single mutations contributed to stronger enzyme-substrate binding varied while the predicted sites were different. The advantage of DUnet for predicting key residues responsible for enzyme activity further promoted the success rate of virtual mutagenesis. This study highlighted the significance of correctly predicting key binding sites for enzyme engineering., (© The Author(s) 2024. Published by Oxford University Press.)

Introduction: The study of Klebsiella quasipneumoniae, Klebsiella variicola, and AmpC production in extended-spectrum β-lactamase (ESBL)-producing Klebsiella in Japan is limited, and existing data are insufficient. This study aims to characterize Klebsiella species, determine AmpC production rates, and analyze antimicrobial resistance patterns in ESBL-producing Klebsiella isolates in Japan., Methods: A total of 139 clinical isolates of ESBL-producing Klebsiella were collected in Japan, along with their corresponding antimicrobial susceptibility profiles. The isolates were identified using a web-based tool. ESBL genes within the isolates were identified using multiplex PCR. Screening for AmpC-producing isolates was performed using cefoxitin disks, followed by multiplex PCR to detect the presence of AmpC genes. Antimicrobial resistance patterns were analyzed across the predominant ESBL genotypes., Results: The web-based tool identified 135 isolates (97.1%) as Klebsiella pneumoniae and 4 (2.9%) as K. quasipneumoniae subsp. similipneumoniae, with no instances of K. variicola detected. Among K. pneumoniae, the CTX-M-1 group emerged as the predominant genotype (83/135, 61.5%), followed by K. quasipneumoniae subsp. similipneumoniae (3/4, 75.0%). The CTX-M-9 group was the second most prevalent genotype in K. pneumoniae (45/135, 33.3%). The high resistance rates were observed for quinolones (ranging from 46.7% to 63.0%) and trimethoprim/sulfamethoxazole (78.5%). The CTX-M-1 group exhibited higher resistance to ciprofloxacin (66/83, 79.5%) compared to the CTX-M-9 group (18/45, 40.0%), a trend also observed for levofloxacin and trimethoprim/sulfamethoxazole. Among the 16 isolates that tested positive during AmpC screening, only one K. pneumoniae isolates (0.7%) were confirmed to carry the AmpC gene., Conclusion: Klebsiella pneumoniae with the CTX-M-1 group is the most common ESBL-producing Klebsiella in Japan and showed a low proportion of AmpC production. These isolates are resistant to quinolones and trimethoprim/sulfamethoxazole, highlighting the challenge of managing this pathogen. The findings underscore the importance of broader research and continuous monitoring to address the resistance patterns of ESBL-producing Klebsiella., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Watanabe et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Codonopsis pilosula is a perennial herbaceous liana with medicinal value. It is critical to promote Codonopsis pilosula growth through effective and sustainable methods, and the use of plant growth-promoting bacteria (PGPB) is a promising candidate. In this study, we isolated a PGPB, Klebsiella michiganensis LDS17, that produced a highly active 1-aminocyclopropane-1-carboxylate deaminase from the Codonopsis pilosula rhizosphere. The strain exhibited multiple plant growth-promoting properties. The antagonistic activity of strain LDS17 against eight phytopathogenic fungi was investigated, and the results showed that strain LDS17 had obvious antagonistic effects on Rhizoctonia solani , Colletotrichum camelliae , Cytospora chrysosperma , and Phomopsis macrospore with growth inhibition rates of 54.22%, 49.41%, 48.89%, and 41.11%, respectively. Inoculation of strain LDS17 not only significantly increased the growth of Codonopsis pilosula seedlings but also increased the invertase and urease activities, the number of culturable bacteria, actinomycetes, and fungi, as well as the functional diversity of microbial communities in the rhizosphere soil of the seedlings. Heavy metal (HM) resistance tests showed that LDS17 is resistant to copper, zinc, and nickel. Whole-genome analysis of strain LDS17 revealed the genes involved in IAA production, siderophore synthesis, nitrogen fixation, P solubilization, and HM resistance. We further identified a gene ( koyR ) encoding a plant-responsive LuxR solo in the LDS17 genome. Klebsiella michiganensis LDS17 may therefore be useful in microbial fertilizers for Codonopsis pilosula . The identification of genes related to plant growth and HM resistance provides an important foundation for future analyses of the molecular mechanisms underlying the plant growth promotion and HM resistance of LDS17., Importance: We comprehensively evaluated the plant growth-promoting characteristics and heavy metal (HM) resistance ability of the LDS17 strain, as well as the effects of strain LDS17 inoculation on the Codonopsis pilosula seedling growth and the soil qualities in the Codonopsis pilosula rhizosphere. We conducted whole-genome analysis and identified lots of genes and gene clusters contributing to plant-beneficial functions and HM resistance, which is critical for further elucidating the plant growth-promoting mechanism of strain LDS17 and expanding its application in the development of plant growth-promoting agents used in the environment under HM stress., Competing Interests: The authors declare no conflict of interest.

Background: The emergence and spread of β-lactamase-producing Klebsiella spp. has been associated with a substantial healthcare burden resulting in therapeutic failures. We sought to describe the proportion of phenotypic resistance to commonly used antibiotics, characterize β-lactamase genes among isolates with antimicrobial resistance (AMR), and assess the correlates of phenotypic AMR in Klebsiella spp. isolated from stool or rectal swab samples collected from children being discharged from hospital., Methods: We conducted a cross-sectional study involving 245 children aged 1-59 months who were being discharged from hospitals in western Kenya between June 2016 and November 2019. Whole stool or rectal swab samples were collected and Klebsiella spp. isolated by standard microbiological culture. β-lactamase genes were detected by PCR whilst phenotypic antimicrobial susceptibility was determined using the disc diffusion technique following standard microbiology protocols. Descriptive analyses were used to characterize phenotypic AMR and carriage of β-lactamase-producing genes. The modified Poisson regression models were used to assess correlates of phenotypic beta-lactam resistance., Results: The prevalence of β-lactamase carriage among Klebsiella spp. isolates at hospital discharge was 62.9% (154/245). Antibiotic use during hospitalization (adjusted prevalence ratio [aPR] = 4.51; 95%CI: 1.79-11.4, p < 0.001), longer duration of hospitalization (aPR = 1.42; 95%CI: 1.14-1.77, p < 0.002), and access to treated water (aPR = 1.38; 95%CI: 1.12-1.71, p < 0.003), were significant predictors of phenotypically determined β-lactamase. All the 154 β-lactamase-producing Klebsiella spp. isolates had at least one genetic marker of β-lactam/third-generation cephalosporin resistance. The most prevalent genes were bla CTX-M 142/154 (92.2%,) and bla SHV 142/154 (92.2%,) followed by bla TEM 88/154 (57.1%,) and bla OXA 48/154 (31.2%,) respectively., Conclusion: Carriage of β-lactamase producing Klebsiella spp. in stool is common among children discharged from hospital in western Kenya and is associated with longer duration of hospitalization, antibiotic use, and access to treated water. The findings emphasize the need for continued monitoring of antimicrobial susceptibility patterns to inform the development and implementation of appropriate treatment guidelines. In addition, we recommend measures beyond antimicrobial stewardship and infection control within hospitals, improved sanitation, and access to safe drinking water to mitigate the spread of β-lactamase-producing Klebsiella pathogens in these and similar settings., (© 2024. The Author(s).)

Introduction: Beta-lactamase producing bacterial infection has been on surge due to selection pressure and injudicious antibiotics usage. Organisms that co-produced more than one beta lactamase enzyme posed diagnostic challenges which may result in inadequate treatment. To date, there is no standardised guideline offering phenotypic detection of AmpC β-lactamase. The purpose of this study was to determine the prevalence of ESBLs, AmpC β-lactamase and co-producer organisms in a teaching hospital., Materials and Methods: Three hundred and four isolates of E. coli and Klebsiella sp. had been selected via convenient sampling. These isolates were identified using conventional laboratory methods and their antimicrobial susceptibilities were determined using disc diffusion method. Those isolates were then proceeded with ESBL confirmatory test, cloxacillin-containing Muller Hinton confirmatory test, modified double disk synergy test and AmpC disk test., Results: Out of 304 isolates, 159 isolates were E. coli and 145 were Klebsiella sp. The prevalence of organisms which co-produced AmpC β-lactamase and ESBL enzymes were 3.0%. Besides that, 39 cefoxitin resistant and three cefoxitin susceptible isolates (13.8%) were proven to produce AmpC β-lactamase through AmpC disk test. Through the CLSI confirmatory test, 252 (82.9%) isolates were identified as ESBLs producers and the prevalence increased slightly when cloxacillin-containing Muller Hinton were used. Only three ESBLs positive organisms were positive for modified double disk synergy test., Conclusion: Distinguishing between AmpC β-lactamase and ESBL-producing organisms has epidemiological significance as well as therapeutic importance. Moreover, AmpC β-lactamase and ESBLs co-producing organisms can lead to false negative ESBL confirmatory test. Therefore, knowing the local prevalence can guide the clinician in navigating the treatment.

We report here a hypermucoviscous, New Delhi metallo-β-lactamase 1 (NDM-1) and imipenemase 4 (IMP-4) carbapenemases-coproducing Klebsiella variicola isolate obtained from a pediatric patient. This strain was resistant to carbapenems and most other β-lactams. Although hypermucoviscous, this strain possessed attenuated virulence according to serum killing assay and Galleria mellonella infection model. Notably, two copies of bla NDM-1 were contained on two tandem IS CR1 elements and coexisted with bla IMP-4 in a novel hybrid multidrug resistance plasmid. This is the first description of the coexistence of bla NDM-1 and bla IMP-4 in a single plasmid of hypermucoviscous K. variicola. IMPORTANCE As an important member of the Klebsiella pneumoniae complex, Klebsiella variicola is poorly studied as an emerging human pathogen. We, for the first time, report a unique K. variicola isolated from a pediatric patient in China. This isolate exhibited hypermucoviscosity, a classic hypervirulence characteristic of K. pneumoniae, and contained multiple carbapenem-resistant genes, including bla IMP-1 and bla NDM-1 . Interestingly, these antimicrobial resistance genes were located on a novel hybrid plasmid, and our results suggested that this plasmid might have been introduced from K. pneumoniae and undergone a series of integration and recombination evolutionary events. Overall, our study provides more insight into K . variicola and highlights its superior capability to acquire and maintain foreign resistance genes.

Convergence of virulence and antibiotic-resistance has been reported in Klebsiella pneumoniae , but not in Klebsiella variicola . We, hereby, report the detection and genomic characterization of hypervirulent and hypermucoviscous K. pneumoniae and K. variicola recovered in Chile from health-care associated infections, which displayed resistance to broad-spectrum cephalosporins. One hundred forty-six K. pneumoniae complex isolates were screened by hypermucoviscosity by the "string test." Two hypermucoid isolates, one hypermucoviscous K. pneumoniae (hmKp) and one K. variicola (hmKv), were further investigated by whole-genome sequencing. In vivo virulence was analyzed by the Galleria mellonella killing assay. In silico analysis of hmKp UCO-494 and hmKv UCO-495 revealed the presence of multiple antibiotic-resistance genes, such as bla CTX-M-1 , bla DHA-1 and bla LEN-25 among others clinically relevant resistance determinants, including mutations in a two-component regulatory system related to colistin resistance. These genetic features confer a multidrug-resistant (MDR) phenotype in both strains. Moreover, virulome in silico analysis confirmed the presence of the aerobactin gene iutA , in addition to yersiniabactin and/or colicin V encoding genes, which are normally associated to high virulence in humans. Furthermore, both isolates were able to kill G. mellonella and displayed higher virulence in comparison with the control strain. In summary, the convergence of virulence and the MDR-phenotype in K. pneumoniae complex members is reported for the first time in Chile, denoting a clinical problem that deserves special attention and continuous surveillance in South America.

In a cohort of 257 infants with congenital heart disease admitted to the pediatric intensive care unit, 22 infants had positive cultures for extended-spectrum beta-lactamase or AmpC Gram-negative bacteria. These infants had longer exposure to broad-spectrum antibiotics, greater support with invasive devices and longer intensive care and hospital lengths of stay., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background and Objective: The global antibiotic resistance threat posed by ESBL and AmpC-producing Gram-Negative Bacteria (GNB) is a public health menace that rolls back the gains of 'One Health'. This study investigated the antibiogram and prevalence of AmpC and ESBL genes in Escherichia coli, Klebsiella spp. and Pseudomonas spp. from poultry and abattoir milieus in Enugu and Ebonyi States, Nigeria., Materials and Methods: Isolation, identification and characterization of GNB from samples (150 abattoirs and 300 poultry) were done using standard microbiological techniques. Antimicrobial Susceptibility Testing (AST), as well as phenotypic screening for ESBL and AmpC enzymes, was performed using the Kirby-Bauer disc diffusion technique. PCR technique was used to screen isolated GNB for AmpC and ESBL genes., Results: Exactly 42 E. coli and 8 Klebsiella spp. isolate from poultry samples and another 5 P. aeruginosa isolates from abattoir samples were phenotypically confirmed to be ESBL-producers. AmpC enzymes were phenotypically detected in 8 E. coli and 13 P. aeruginosa isolates from poultry samples. All ESBL and AmpC-positive bacteria exhibited high resistance frequencies to tested antibiotics, especially to the carbapenems and cephalosporins. ESBL genes (CTX-M, SHV-1, TEM) and AmpC genes (ACC-M, MOX-M, DHA-M) were harbored by the isolated GNB in this study. Overall, the DHA-M and CTX-M genes, mediating AmpC and ESBL production respectively were the most prevalent genes harbored by the tested GNB., Conclusion: This study reported that AmpC and ESBL genes are harbored by Gram-negative bacteria (E. coli, Klebsiella species and P. aeruginosa) that emanated from poultry and abattoir milieus.

Antimicrobial resistance is a global emergency which needs one health approach to address. The present study was conducted to detect the prevalence of beta-lactamase and biofilm-producing Klebsiella strains in rectal swabs (n = 624) collected from healthy dogs, cats, sheep and goats reared as companion or household animals in India. The dogs and cats were frequently exposed to third- or fourth-generation cephalosporins for therapy. The sheep and goats were occasionally exposed to antibiotics and had environmental exposure. Phenotypical ESBL (n = 93) and ACBL (n = 88)-producing Klebsiella were isolated significantly more (P < 0·05) from companion animals than household animals. Majority of the Klebsiella possessed bla CTX-M-15 . The sequences bla CTX-M-15.2 , bla CTX-M-197 and bla CTX-M-225 are reported first time from the companion animals. All ACBL-producing isolates possessed bla AmpC . The present study detected 65·8% of Klebsiella strains as biofilm producers possessing the studied biofilm associated genes. The isolates showed phenotypical resistance against chloramphenicol, tetracycline, doxycycline, co-trimoxazole, ampicillin, cefotaxime/clavulanic acid. The present study showed that companion and household animals (dogs, cats, sheep, goats) may act as a carrier of ESBL/biofilm-producing, multi-drug resistant, high-risk clonal lineage of Klebsiella., (© 2020 The Society for Applied Microbiology.)

Objective: The emergence of carbapenem-resistant Escherichia coli and Klebsiella species is a global threat. We aimed to compare two phenotypic methods and evaluate the genotypic method for the detection of beta-lactamases produced by E. coli and Klebsiella spp., Materials and Methods: One hundred and twenty-six E. coli and Klebsiella isolates were examined for phenotypic production of beta-lactamases by using disc diffusion, combined disc test (CDT) and modified carbapenem inactivation method (mCIM). All strains were also studied for the presence of various genes by polymerase chain reaction., Results: Out of 126 isolates, 96% of the isolates were extended-spectrum β-lactamase (ESBL) producers based on the presence of various ESBL genes. CDT method showed higher number of total (89%) carbapenemases in comparison to mCIM (81%). Among carbapenemases none of the isolates were Klebsiella pneumoniae carbapenemase producer by CDT, while 69% isolates were metallo-beta-lactamase (MBL) producers. Another method, mCIM/ethylene diamine tetraacetic acid mCIM showed 100% agreement for MBL detection. As regards, AmpC and class D carbapenemases; 0.04% and 16% positivity was detected, respectively, based on CDT method. Molecular analysis revealed 91% of the isolates harbouring carbapenemase genes. bla NDM was the most common gene detected followed bybla OXA-48 . Nine of the bla NDM -positive isolates also possessed bla OXA-48 gene., Conclusion: Our finding shows high percentages of ESBL and carbapenemases in E. coli and Klebsiella spp. Among phenotypic methods, CDT seems to be a better choice as prevalence of carbapenemases shows lots of variation in our country. For Class B enzymes, both CDT and mCIM/eCIM can be used in the routine laboratories., Competing Interests: None

Introduction. Klebsiella rods, belonging to the family Enterobacteriaceae , are generally opportunistic pathogens commonly associated with nosocomial infections, especially in intensive care units. Interestingly, strains of this genus also show multi-drug resistance. In recent years, multiple studies have indicated that the prevalence of carbapenem resistance has increased rapidly among Klebsiella representatives. Aim. The aim of this study was to assess the usefulness of selected phenotypic and genotypic methods for the detection of the most important carbapenemases in Klebsiella strains. Methodology. The study involved 51 Klebsiella strains. The ability to produce carbapenemases was determined by phenotypic methods (double disc synergy test, test with four discs and three inhibitors, CarbaNP test, culture on chromogenic medium, panels of automatic method - Phoenix, CIM test and modified Hodge test). The potential for carbapenemase synthesis was also evaluated using real-time PCR, detecting bla VIM/IMP , bla KPC , bla NDM and bla OXA-48 genes. Results. Using the phenotypic methods, positive results were obtained for all of the analysed strains. Using PCR, carbapenemase synthesis potential was confirmed on the molecular level; the bla VIM gene was detected in 23 strains, the bla NDM gene in 26 strains and the bla OXA-48 gene in two strains. Conclusion. There was complete agreement between the carbapenemases detected by the genetic method and the results obtained with phenotypic methods.

We evaluated the in vitro effectiveness of temocillin and several commonly used antimicrobials against Enterobacterales bacteria in isolates from Polish patients. We tested 400 isolates: 260 extended-spectrum β-lactamase (ESBL)- and/or ampC β-lactamase (AmpC)-producing isolates; 40 Klebsiella pneumoniae carbapenemase (KPC)-producing isolates; and 100 ESBL-, AmpC-, and KPC-negative isolates. The minimal inhibitory concentrations (MICs) of temocillin and 16 other antimicrobials were determined by reference microdilution. We also determined the activities of fosfomycin and ceftazidime/avibactam in KPC-producing isolates. The antibiotic sensitivities were interpreted according to EUCAST, BSAC, and CLSI criteria. Overall, 91% of the isolates were susceptible to temocillin using the urinary tract infection breakpoint (≤ 32 mg/L), and 61.8% were susceptible using the systemic infection breakpoint (≤ 8 mg/L). Meropenem and imipenem were the most active drugs (MIC 50 values of 0.06 and 0.5 mg/L, respectively). Colistin and ertapenem (both MIC 50  = 0.12 mg/L) were less active than meropenem or imipenem, but some strains were 77% susceptible to each of them. Among the KPC-producing isolates, 42.5% had MIC values of ≤ 32 mg/L (urinary tract infection breakpoint), but 100% were resistant to temocillin (systemic infection breakpoint). Ceftazidime/avibactam was active against 100% of the KPC-producing isolates, and fosfomycin was active against 40%. The empirical susceptibility rate observed among the urinary isolates suggests that temocillin may be considered as an alternative to carbapenems in the absence of KPC-producing bacteria. With regard to isolates from other sources, temocillin might be useful as a documented therapy agent or an empirical treatment in hospitals with a low prevalence of ESBL/AmpC-producing strains.

The New Delhi metallo-β-lactamase (NDM-1) mediates resistance to β-lactam antibiotics. NDM-1 was likely formed as the result of a gene fusion between sequences encoding the first six amino acids of cytoplasm-localised aminoglycosidase, AphA6, and a periplasmic metallo-β -lactamase. We show that NDM-1 has an atypical signal peptide and is inefficiently secreted. Two new bla NDM-1 alleles that have polymorphisms in the signal peptide; NDM-1(P9R), a proline to arginine substitution, and NDM-2, a proline to alanine substitution (P28A) were studied. Here, we show that both the P9R and P28A substitutions improve secretion compared to NDM-1 and display higher resistance to some β-lactam antibiotics. Mass spectrometry analysis of these purified NDM proteins showed that the P28A mutation in NDM-2 creates new signal peptide cleavage sites at positions 27 and 28. For NDM-1, we detected a signal peptide cleavage site between L21/M22 of the precursor protein. We find no evidence that NDM-1 is a lipoprotein, as has been reported elsewhere. In addition, expression of NDM-2 improves the fitness of E. coli, compared to NDM-1, in the absence of antibiotic selection. This study shows how optimization of the secretion efficiency of NDM-1 leads to increased resistance and increased fitness., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Although it has been proved that abiotic processes can transform tetracycline (TEC), little is known about how microbial processes may degrade TEC in aquatic environment. The objective of this study is to investigate the biodegradation pathway of TEC by strain Klebsiella sp. SQY5 and molecular mechanism of TEC resistance under the aerobic conditions. Effects of mycelium, intracellular, and extracellular enzyme on TEC degradation process were explored, suggesting that mycelium contributed the most of TEC degradation with a maximum efficiency of 58.64%. Biodegradation characteristic of TEC and its degradation products were studied. The results showed that nine possible biodegradation products were identified, and a potential biodegradation pathway was proposed including the removal of methyl, carbonyl, and amine groups. The functional genes of this bacterium were also determined by genomics, and analysis indicated that functional genes that could be relevant to hydrolysis, ring opening and oxidation played an important role in the process of TEC biodegradation. Results from this study can provide a theoretical basis for better estimating the fate, transportation, and degradation of antibiotics in aquatic environment., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objectives: To describe the population structure, molecular epidemiology and genetic context of blaKPC-2-bearing Klebsiella pneumoniae., Methods: Isolates (n = 157) were retrospective, phenotypically carbapenem-resistant blaKPC-positive K. pneumoniae, collected from public hospitals. WGS was performed on the Illumina platform. Phylogenomic analysis, screening of resistance and virulence genes, and comparison of the genetic environment of blaKPC were carried out., Results: Based on core-tree phylogeny, 67.5% of the isolates were K. pneumoniae and the remainder comprised Klebsiella quasipneumoniae. No Klebsiella variicola strains were observed. Only a single K. pneumoniae carbapenemase (KPC) variant type, blaKPC-2, was seen. MLSTs were diverse and did not comprise the 'traditional' KPC clonal group (CG) 258. blaKPC-2 was associated with a non-Tn4401 element (NTE) in >99% of genomes. Screening for four key virulence loci: yersiniabactin (ybt), aerobactin (iuc), salmochelin (iro) and colibactin (clb) as well as ICEKp (virulence-associated integrative conjugative element of K. pneumoniae), revealed the lack of virulence factors and ICEKp within K. quasipneumoniae. Amongst the K. pneumoniae, there were 32 ybt+ isolates (32/106, 30.2%) and, of these, 8 isolates were also clb+ (7.5%). K. pneumoniae serotypes K1 and K2, the majority of capsular serotype seen in patients with invasive liver abscess syndrome, were detected at 4.5% (7/157)., Conclusions: Results suggest that dissemination of blaKPC-2 is driven by NTEKPC in non-ST258 isolates. The detection of blaKPC-2K. pneumoniae serotypes K1/K2 carrying virulence factors, albeit in low numbers, reflects the worrisome convergence of carbapenem resistance and hypervirulence in K. pneumoniae., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objectives: The emergence and dissemination of carbapenem-resistant Enterobacteriaceae (CRE) is an important public health problem. This study aimed to understand the prevalence and mechanisms of carbapenem resistance in clinically important members of Enterobacteriaceae in rural South India., Methods: Routine clinical isolates of Escherichia coli and Klebsiella spp. were tested for Ertapenem (ETP) non-susceptibility by the disk diffusion method over a 3-year period (2012-2014). The ETP non-susceptible isolates were preserved, and tested for the MIC of carbapenems and the carriage of major carbapenemase-encoding genes. Representative genes were sequenced and selective isolates were tested for the production of carbapenemase by carbapenem inactivation method., Results: A total of 444 ETP non-susceptible isolates were identified in increasing incidence over the study period. Among them, MIC 50 and MIC 90 of carbapenems (excluding ETP) were 0.25-0.5μg/mL and 8-16μg/mL, respectively, and the prevalence of non-ETP carbapenem resistance was estimated as 3%. Among the 177 tested isolates, 65 (37%) had one or more carbapenemase-encoding genes with a predominance of New Delhi Metallo-β-lactamase (NDM; 32 of 65; 49.2%)., Conclusions: This study documented the MIC range for carbapenems, prevalence and mechanisms of carbapenem resistance among Enterobacteriaceae in rural South India. It substantiated NDM as a leading mechanism of carbapenem resistance and highlighted the importance of MIC testing in patient management., (Copyright © 2019 International Society for Antimicrobial Chemotherapy. Published by Elsevier Ltd. All rights reserved.)

The emergence and spread of aminoglycoside-resistant bacteria are a public health concern. The acquisition of the genes encoding 16S rRNA methylases, such as armA, rmtA, and rmtB, confers high-level resistance to aminoglycosides. However, the prevalence has not been well investigated in Japanese veterinary fields. To determine the prevalence of 16S rRNA methylases in animals, we detected 16S rRNA methylases genes in Gram-negative bacteria from animals. Here, we report the isolation of rmtB amd armA from two of the 446 Escherichia coli (0.5%) and one of the 103 Klebsiella spp. isolates (1.0%) from companion animals, respectively. However, none of the isolations were observed from 2445 E. coli isolates derived from livestock in Japan. The prevalence of 16S rRNA methylases in animals, especially in companion animals, should be carefully monitored in Japanese veterinary fields to avoid the spreading of the genes.

Here, the draft genome of simultaneous nitrification-denitrification strain (SND) Klebsiella sp. KSND revealed possible existence of genes involved in N-assimilation and -dissimilation pathways. The change levels of genes under defined N-sources were analyzed by Quantitative Real-Time PCR. It suggested that NH 4 + -assimilation via NADP-glutamate dehydrogenase pathway would occur preferentially. NirBD genes were tightly regulated in a lower level, so that nitrite was rapidly consumed for detoxication by denitrification. Three types of nitrate reductase homologues are surprisingly present in KSND, whereas the dominant nitrate reduction for assimilation and denitrification processes mediates by NapA-type nitrate reductase. Nitric oxide reductase homologues FlRd and FlRd-red provide an adequate capacity for NO detoxification. The recombinant hydroxylamine reductase showed high activity in hydroxylamine to generate ammonium, which might contribute to detoxification mechanism in nitrogen cycling. Overall, this study firstly provides valuable insights into the genes expression and enzyme action, which helps understanding the mechanism of SND processes., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Several emerging pathogens have arisen as a result of selection pressures exerted by modern health care. Klebsiella quasipneumoniae was recently defined as a new species, yet its prevalence, niche, and propensity to acquire antimicrobial resistance genes are not fully described. We have been tracking inter- and intraspecies transmission of the Klebsiella pneumoniae carbapenemase (KPC) gene, bla KPC , between bacteria isolated from a single institution. We applied a combination of Illumina and PacBio whole-genome sequencing to identify and compare K. quasipneumoniae from patients and the hospital environment over 10- and 5-year periods, respectively. There were 32 bla KPC -positive K. quasipneumoniae isolates, all of which were identified as K. pneumoniae in the clinical microbiology laboratory, from 8 patients and 11 sink drains, with evidence for seven separate bla KPC plasmid acquisitions. Analysis of a single subclade of K. quasipneumoniae subsp. quasipneumoniae ( n  = 23 isolates) from three patients and six rooms demonstrated seeding of a sink by a patient, subsequent persistence of the strain in the hospital environment, and then possible transmission to another patient. Longitudinal analysis of this strain demonstrated the acquisition of two unique bla KPC plasmids and then subsequent within-strain genetic rearrangement through transposition and homologous recombination. Our analysis highlights the apparent molecular propensity of K. quasipneumoniae to persist in the environment as well as acquire carbapenemase plasmids from other species and enabled an assessment of the genetic rearrangements which may facilitate horizontal transmission of carbapenemases., (Copyright © 2019 Mathers et al.)

Background: This study aimed to identify the predictors and build a prediction score for community-onset bloodstream infections (CO-BSIs) caused by extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella species., Methods: All CO-BSIs caused by E. coli and Klebsiella species from 2012 to 2015 were grouped into derivation (BSIs from 2012 to 2014) and validation (BSIs in 2015) cohorts. A prediction score was built using the coefficients of the multivariate logistic regression model from the derivation cohort., Results: The study included 886 CO-BSIs (594 and 292 in the derivation and validation cohorts, respectively). The independent predictors of CO-BSIs caused by ESBL-producing E. coli and Klebsiella species included: 1) identification of ESBL-producing microorganisms from any clinical culture within one year of admission, 2) beta-lactam or fluoroquinolone treatment within 30 days (with 2 or more courses within 90 days; with 1 course within 90 days), 3) hospitalization within one year, 4) the presence of an indwelling urinary catheter at the time of admission. The area under the curve (AUC) of the clinical prediction score was 0.72 (95% confidence interval [CI], 0.68-0.77). In the validation cohort, the AUC was 0.70 (95% CI, 0.63-0.77)., Conclusions: The results of this study suggest a simple and easy-to-use scoring system to predict CO-BSIs caused by ESBL-producing E. coli and Klebsiella species., Competing Interests: The authors have no potential conflicts of interest to disclose., (© 2019 The Korean Academy of Medical Sciences.)

The emergence and spread of multi-drug-resistant (MDR), extended-spectrum beta-lactamase (ESBL) producing carbapenem-resistant members of Enterobacteriaceae family has become a worldwide health problem. Carbapenem resistance caused by blaKPC, blaNDM gene regions are sporadic and blaOXA-48 gene region is endemic in our country. The aim of this study was to determine the presence of blaOXA-232, blaOXA-181, blaOXA-162, blaOXA-204, blaOXA-244, blaOXA-163, blaOXA-245 genes in OXA-48 like carbapenemase producing Klebsiella pneumoniae isolates. The isolates used in this study were provided from the Medical Microbiology Laboratory collection of Sakarya University Sakarya Training and Research Hospital. Identification and antibiotic susceptibility tests were determined by the VITEK 2® automated system (biomerieux, France) and the carbapenemase production of isolates was determined by the modified Hodge test. Minimal inhibitor concentration (MIC) values were determined with broth microdilution method. The isolates containing the blaOXA-48-like gene region were identified by real-time polymerase chain reaction (Rt-PCR) method using consensus primers. In "High Resolution Melting Analysis (HRMA)" method carried out by using "Type-it HRM PCR" (Qiagen, Hilden, Germany) kit, isolates which showed a deviation in melting temperatures (Tm) were selected with the suspicion of OXA-48 variant. The sequence analysis (ABI 3500, Applied Biosystems, USA) was carried out to determine which variants were present in these isolates. Compatibility of MIC values was determined between VITEK 2® and the microdilution method with the rate of 82% for imipenem, 77% for meropenem and 90% for ertapenem in carbapenemase-producing K.pneumoniae isolates. In 45 of 100 K.pneumoniae isolates, the blaOXA-48-like gene region was found to be positive by the Rt-PCR method. For the determination of OXA-48 variants, these 45 isolates were evaluated by HRMA method. The sequence analysis revealed that 41 (91.2%) isolates contained blaOXA-48/blaOXA-245 gene regions, while 2 (4.4%) isolates were found to contain blaOXA-181 gene regions and 2 (4.4%) isolates were found to contain blaOXA-244 gene regions. This is the first study to determine OXA-48 and OXA-244 positivity in blaOXA-48-like gene regions in Turkey. As a result of this study, the OXA-48-like gene region was found to be 45%, of which 4.4% had blaOXA-181 and 4.4% had blaOXA-244 gene regions. The detection of blaOXA-48-like gene regions will guide for the selection of antibiotics in critical patient groups.

The bacterial pathogen Klebsiella pneumoniae comprises several phylogenetic groups (Kp1 to Kp7), two of which (Kp5 and Kp7) have no taxonomic status. Here we show that group Kp5 is closely related to Klebsiella variicola (Kp3), with an average nucleotide identity (ANI) of 96.4%, and that group Kp7 has an ANI of 94.7% with Kp1 (K. pneumoniae sensu stricto). Biochemical characteristics and chromosomal beta-lactamase genes also distinguish groups Kp5 and Kp7 from other Klebsiella taxa. We propose the names Klebsiella africanensis for Kp7 (type strain, 200023 T  = CIP 111653 T ) and K. variicola subsp. tropicalensis for Kp5 (type strain, 1266 T  = CIP 111654 T )., (Copyright © 2019 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Carbapenemase-producing organisms have been an immense public health problem in recent years. Combined disc test (CDT) is a simple and widely used phenotypic method for carbapenemase detection, especially in developing countries. This study evaluates the performance of modified combined disc test (mCDT), a novel and 4 times cheaper method than CDT. In total, 572 (15.5%) Klebsiella spp. including 81 (14.2%) carbapenemase producers were isolated from 3993 clinical samples. Both mCDT and CDT showed similar sensitivity, specificity, positive predictive value, and negative predictive value for the differentiation of Class A, B, and D carbapenemase-producing Klebsiella spp., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Purpose: The aim of this study was to determine the prevalence and risk factors for community-acquired urinary tract infections (CA-UTIs) caused by extended-spectrum β-lactamase (ESBL) producing Escherichia coli and Klebsiella species., Materials and Methods: The patients diagnosed with CA-UTIs caused by E. coli or Klebsiella spp. were included in the study. All of the patients were compared to demographic characteristics, underlying diseases, urinary tract pathology, history of hospitalization, use of antibiotics according to ESBL positivity., Results: A total of 322 urine isolates were studied. Sixty-six patients (37.1%) of a total of 178 patients were ESBL positive E. coli and Klebsiella spp. Being over the age of sixty (odds ratio [OR], 1.90; p=0.03), history of renal stone (OR, 3.00; p=0.03), urinary tract anatomical of physiological disorder (OR, 2.17; p=0.01), urologic intervention (OR, 3.43; p<0.001), history of urinary tract surgery (OR, 3.10; p=0.01), history of urinary catheterization (OR, 3.43; p<0.001), and hospitalization for last 1 year (OR, 3.70; p=0.01) and antibiotic usage in the last 3 months (OR, 1.90; p=0.04) were found as significant risk factors for the producing of ESBL. However, gender and underlying disease were not related for ESBL production., Conclusions: In present study, high rate of ESBL positivity was detected in CA-UTIs. The increasing of infections caused by ESBL positive E. coli and Klebsiella spp. are bringing together a lot of the problem, such as antibiotic resistance and reducing treatment options for outpatients. Identification of underlying risk factors would be important for the development of preventive strategies., Competing Interests: CONFLICTS OF INTEREST: The authors have nothing to disclose.

Aminoglycosides are a class of antibiotics that play a key role in antimicrobial treatment of Multidrug resistant (MDR) Gram-negative bacilli, typically in combination with β-lactams. Ribosomal 16S RNA modification by methyltransferases (e.g. RmtG) is an aminoglycoside resistance mechanism that, along with the occurrence carbapenem-resistant Enterobacteriaceae (CRE), has become a clinical concern. In Brazil, rmtG genes were initially reported in Klebsiella pneumoniae, and monitoring isolates from other species carrying this gene is critical for epidemiological studies and to prevent dissemination. Here we report the presence of rmtG in Klebisella aerogenes D3 and characterize its genetic context in comparison to isolates from other species. Further, we performed a phylogenetic reconstruction of 900 16S rRNA methyltransferases (16S-RMTases) and methyltransferase-related proteins. We show that, in K. aerogenes D3, rmtG co-occurs with sul2, near a transposon with an IS91-like insertion sequence. Resistome analysis revealed the co-production of RmtG and CTX-M-59. Ongoing surveillance of 16S-RMTases is crucial to delay the dissemination of such multiresistant isolates. Our results also highlight the reduction in treatment options for CRE infections, as well as the need of expanding prevention measures of these pathogens worldwide.

Overcoming recalcitrance of lignin has motivated bioprospecting of high-yielding enzymes from environmental ligninolytic microorganisms associated with lignocellulose degrading-systems. Here, we performed isolation of 21 ligninolytic strains belonging to the genus Klebsiella spp., driven by the presence of lignin in the media. The fastest-growing strains (FP10-5.23, FP10-5.22 and P3TM1) reached the stationary phase in approximately 24 h, in the media containing lignin as the main carbon source. The strains showed biochemical evidence of ligninolytic potential in liquid- and solid media-converting dyes, which the molecular structures are similar to lignin fragments. In liquid medium, higher levels of dye decolorization was observed for P3TM.1 in the presence of methylene blue, reaching 98% decolorization in 48 h. The highest index values (1.25) were found for isolates P3TM.1 and FP10-5.23, in the presence of toluidine blue. The genomic analysis revealed the presence of more than 20 genes associated with known prokaryotic lignin-degrading systems. Identification of peroxidases (lignin peroxidase-LiP, dye-decolorizing peroxidase-DyP, manganese peroxidase-MnP) and auxiliary activities (AA2, AA3, AA6 and AA10 families) among the genetic repertoire suggest the ability to produce extracellular enzymes able to attack phenolic and non-phenolic lignin structures. Our results suggest that the Klebsiella spp. associated with fresh water and soil may play important role in the cycling of recalcitrant molecules in the Caatinga (desert-like Brazilian biome), and represent a potential source of lignin-degrading enzymes with biotechnological applications.

Objective: The objective of this study is to determine the prevalence of rectal carriage of plasmid- and chromosome-encoded AmpC β-lactamase-producing Escherichia coli and Klebsiella spp. in patients in a Dutch teaching hospital between 2013 and 2016., Methods: Between 2013 and 2016, hospital-wide yearly prevalence surveys were performed to determine the prevalence of AmpC β-lactamase-producing E. coli and Klebsiella spp. rectal carriage. Rectal swabs were taken and cultured using an enrichment broth and selective agar plates. All E. coli and Klebsiella spp. isolates were screened for production of AmpC β-lactamase using phenotypic confirmation tests and for the presence of plasmid-encoded AmpC (pAmpC) genes. E. coli isolates were screened for chromosome-encoded AmpC (cAmpC) promoter/attenuator alterations., Results: Fifty (2.4%) of 2,126 evaluable patients were identified as rectal carrier of AmpC β-lactamase-producing E. coli. No carriage of AmpC β-lactamase producing Klebsiella spp. was found. Nineteen (0.9%) patients harboured isolates with pAmpC genes and 30 (1,4%) patients harboured isolates with cAmpC promoter/attenuator alterations associated with AmpC β-lactamase overproduction. For one isolate, no pAmpC genes or cAmpC promotor/attenuator alterations could be identified. During the study period, a statistically significant decline in the prevalence of rectal carriage with E. coli with cAmpC promotor/attenuator alterations was found (p = 0.012). The prevalence of pAmpC remained stable over the years., Conclusions: The prevalence of rectal carriage of AmpC-producing E. coli and Klebsiella spp. in patients in Dutch hospitals is low and a declining trend was observed for E. coli with cAmpC promotor/attenuator alterations., Competing Interests: The authors have declared that no competing interests exist.

Background: The aim of this study was to evaluate the modified Carba NP test to differentiate KPC (Klebsiella pneumoniae carbapenemase)- and MBL (metallo-β-lactamase)-producing Klebsiella species., Methods: A total of 508 non-duplicate clinical isolates of Klebsiella spp. were processed by modified Carba NP and combined disc tests which were further confirmed by conventional polymerase chain reaction (PCR), a gold standard method for statistical analysis., Results: Modified Carba NP test demonstrated 91.7% sensitivity, 100% specificity, 100% positive predictive value (PPV) and 99.8% negative predictive value (NPV) for KPC and 96.7%, 100%, 100%, and 99.5% for MBL detection, respectively., Conclusion: The performance of modified Carba NP test was significantly better than combined disc test, fulfilling the requirement of simple and rapid test for clinical applications., (© 2018 Wiley Periodicals, Inc.)

Community-acquired carriage and infections due to extended-spectrum beta-lactamase producing Enterobacteriaceae (ESBL-E) are increasing worldwide, resulting in increased morbidity, mortality and healthcare costs. The origins of community-acquired ESBL-E carriage and infections remain unclear. Bean sprouts are a potential source of Enterobacteriaceae for the community, as illustrated by outbreaks of pathogenic Enterobacteriaceae in the past. The current study focuses on contamination of retail bean sprouts with ESBL-E in the Netherlands. Of 131 bean sprout samples purchased between 2013 and 2016, 25 (19%) were contaminated with ESBL-E. The detected isolates were almost exclusively Klebsiella spp. and co-resistance to other antibiotics was observed frequently. Over time there was substantial genetic diversity between isolates. On the other hand, isolates from samples closely matched in time were frequently clonally related, indicative of batch contamination. Remarkably, no Escherichia coli was found. In conclusion, bean sprouts frequently harbor ESBL-E, which is a potential source for consumers., Competing Interests: The authors have declared that no competing interests exist.

Background: The presence of extended-spectrum β-lactamase (ESBL) in Escherichia coli, Klebsiella species, and Proteus mirabilis (EKP) is of great microbiological and clinical importance. The study dealing with the direct impact of ESBL producers on the outcome of patients with community-onset bacteremia is lacking., Methods: Adults with community-onset EKP bacteremia were recruited retrospectively during a 6-year period. ESBL producers were determined according to ESBL phenotype. ESBL patients were compared on a 1:2 basis with non-ESBL patients by using propensity-score matching (PSM) calculated based on independent predictors of 28-day mortality., Results: Of the 1141 eligible adult patients, 65 (5.7%) caused by ESBL producers. Significant differences between the two groups were discovered in the proportions of patients with critical illness (a Pitt bacteremia score ≥ 4) at bacteremia onset, inappropriate empirical antibiotic therapy, bacteremia because of urosepsis and pneumonia, and several comorbidities. In a PSM analysis after controlling for six independent predictors-critical illness at bacteremia onset, underlying fatal comorbidities (McCabe classification), inappropriate empirical antibiotic therapy, comorbidities with liver cirrhosis, bacteremia because of urosepsis and pneumonia-a appropriate matching between two groups (ESBL group, 60 patients; non-ESBL group, 120) were observed in age, causative microorganism, bacteremia severity, major comorbidities, comorbidity severity, and major bacteremia source. Consequently, a strong relationship between ESBL producers and poor prognosis was highlighted., Conclusions: The adverse influence of ESBL producers on clinical outcomes was presented with respect to adults with community-onset EKP bacteremia. Establishing a predictive scoring algorithm for identifying patients at risk of ESBL-producer infections is crucial., (Copyright © 2017. Published by Elsevier B.V.)

This cross-sectional study was conducted in the Department of Microbiology of Dhaka Medical College, Dhaka, Bangladesh, from January 2015 to December 2015 to investigate the antibiotic resistance pattern and prevalence of genes encoding aminoglycoside-modifying enzymes in Escherichia coli and Klebsiella species isolated from a tertiary care hospital of Bangladesh. A total of 166 Escherichia coli, Klebsiella pneumoniae and Klebsiella oxytoca were isolated from urine, wound swab, pus, sputum and blood samples of patients of Dhaka Medical College Hospital. Antibiotic susceptibility test was performed by disk-diffusion technique. Genes encoding aminoglycoside-modifying enzymes [aac(3)-IIa, aac(6')-Ib, ant(2'')-Ia, aac(3)-Ia] among the isolated Escherichia coli and Klebsiella species were detected by polymerase chain reaction. The most prevalent aminoglycoside-modifying enzyme gene found was aac (3)-IIa (60.24%), followed by aac(6')-Ib (57.23%), ant(2'')-Ia (12.05%) and aac(3)-Ia (10.84%). Colistin and tigecycline were the most effective antibacterial agents against aminoglycoside-modifying enzymes followed by imipenem. The results of this study showed high proportion of aminoglycoside-modifying enzymes producing Escherichia coli and Klebsiella species in Bangladesh.

Rheumatoid arthritis (RA) is an autoimmune inflammatory disease. Early diagnosis can prevent joint erosion. However, available biomarkers do not always allow for clear distinction between RA and non-RA individuals. It has become known that bacteria/viruses are among the environmental triggers that initiate RA via multiple molecular mechanisms. Thus, to better understand the role of bacteria in RA, we synthetized 6 peptidomimetics of bacterial ureases' flap region. These peptides were then used to distinguish RA patients from healthy people sera by immunoblotting. Most patients' sera were bound to peptidomimetic characteristic for Enterobacter sp. and Klebsiella sp. flap urease. We also found similarities between peptidomimetic sequence and human proteins connected with RA. This pilot study suggests that bacteria may trigger RA via mechanism of molecular mimicry of urease to host proteins and ureases flap peptidomimetics may be potential candidate as a new additional diagnostic test., (Copyright © 2018 European Peptide Society and John Wiley & Sons, Ltd.)

Background: In recent years, the world has seen a surge in Enterobacteriaceae resistant to broad-spectrum beta-lactam antibiotics due to the production of extended-spectrum beta-lactamases (ESBLs) or plasmid-mediated AmpC (pAmpC) enzymes. Data on the epidemiology of cephalosporin-resistant Enterobacteriaceae in Sub-Saharan Africa are still limited., Methods: Two hundred seventy-five non-repetitive stool samples were collected from Mozambican university students of both sexes. Samples were cultured on MacConkey agar with and without ceftriaxone (1 mg/L) for selection of third-generation cephalosporin-resistant isolates, which were subjected to antimicrobial susceptibility testing by disc diffusion, characterization of resistance genes by PCR and ERIC-PCR analysis for strain clonality., Results: Among the 275 students, 55 (20%) carried a total of 56 E. coli (n = 35) and Klebsiella spp. (n = 21) isolates resistant to ceftriaxone and phenotypically positive for ESBL- and/or pAmpC-production. Forty-three percent of the isolates (24/56) contained only ESBL genes, 11% (6/56) only pAmpC genes, and 36% (20/56) both ESBL and pAmpC genes. The remaining six isolates were negative for the CTX-M/pAmpC genes included in the test panel. E. coli and Klebsiella spp. combined demonstrated 70% resistance to tetracycline and co-trimoxazole, 63% to ceftazidime and 34% to ciprofloxacin. In total, 89% of ESBL/pAmpC-positive isolates were defined as multi-resistant by being resistant to three or more antibiotic classes. ERIC-PCR fingerprinting demonstrated low similarity among isolates. None of the participants reported recent hospitalization and just 12.5% had taken antibiotics 3 months prior to the study., Conclusion: This study demonstrated 20% colonization with multi-resistant E. coli and Klebsiella spp. among Mozambican students with a diversity of ESBL and pAmpC genes. Colonization was not related to prior hospitalization or antimicrobial consumption.