blaKPC Genes Research Articles

Dissemination of IncQ1 Plasmids Harboring NTEKPC-IId in a Brazilian Hospital

KPC is a clinically significant serine carbapenemase in most countries, and its rapid spread threatens global public health. blaKPC transmission is commonly mediated by Tn4401 transposons. The blaKPC gene has also been found in non-Tn4401 elements (NTEKPC). To fill the gap in the understanding of the stability and dissemination of NTEKPC-carrying plasmids, we selected and characterized carbapenem-resistant bacteria isolated between 2009 and 2016 from a hospital for a retrospective study of their plasmids conjugation capacity, impact on fitness, and replication in different species. Different clones were selected using PFGE, and their genomes were sequenced using Illumina and Oxford Nanopore methods. Minimum inhibitory concentrations (MICs) were determined by broth microdilution. Plasmid copy numbers (PCNs) were determined using qPCR. Doubling time was used to analyze fitness change. Most isolates (67%, 33/49) carried blaKPC, of which 85% presented blaKPC in a NTEKPC. The 25 isolates selected presented the blaKPC gene in NTEKPC-IId in IncQ1-type plasmids, showing multispecies dissemination. IncQ1 plasmids were mobilizable and PCN seemed to be directly linked to the species, presenting a high-copy number, mainly in K. pneumoniae. No relationship was observed between IncQ1 PCN and carbapenems MIC values. IncQ1 and a conjugative plasmid from K. pneumoniae BHKPC10 were transferred to E. coli J53 without fitness changes, and MIC values were maintained for carbapenems despite the low transconjugant PCN. In addition to IncQ1 with NTEKPC, Enterobacter cloacae BHKPC28 contained the mcr-9 gene in an IncHI2/IncHI2A conjugative plasmid, which may help the mobility of IncQ1 and the dissemination of two resistance determinants to last-resort antibiotics. Understanding the interaction between plasmids and high-risk lineages can help develop new therapies to prevent the dissemination of resistance traits.

Molecular Epidemiological Characteristics of bla IMP-4-Carrying Klebsiella pneumoniae ST-11 in Hospitalized Patients.

To investigate the molecular epidemiology and risk factors of carbapenem-resistant Klebsiella pneumoniae (CRKP) infection. Patient's clinical data and CRKP strains were collected from November 2017 to December 2018 at a tertiary hospital in Wuhan, China. The antimicrobial susceptibilities, carbapenem-resistant genes, multi-locus sequence typing (MLST), homologous analysis, and risk factors for CRKP were determined. A total of 203 CRKP strains were isolated, and 98.5% (200/203) of patients were nosocomially infected. The mortality rate was 17.7% (36/203). All 203 strains were confirmed as carbapenemases -producing strains. The most predominant carbapenemase gene was bla IMP-4 (81.3%, 165/203), followed by bla KPC-2 (25.1%, 51/203) and bla NDM-1 (23.2%, 47/205). Of the 203 strains, 28 (13.8%) had both bla KPC-2 and bla IMP-4 genes, 23 (11.3%) had both bla IMP-4 and bla NDM-1 genes, 20 (9.9%) had bla KPC-2, bla IMP-4 and bla NDM-1 three genes. MLST analysis showed that there were 48 ST typologies (including 7 new STs), of which ST-11 was the most prevalent (59.6%, 121/203). Phylogenetic analysis showed that 203 CRKP isolates came from 7 clusters and exhibited a strong correlation with the isolation source. eBURST analyses indicated that CRKP isolates have undergone different evolutionary processes. Patients with ST-11 CRKP underwent more mechanical ventilation (50% vs 32.9%, P=0.020) and gastric catheterization (15.7% vs 6.1%, P=0.042) within 3 months before sample collection, and also had higher drug-resistance rate than non-ST-11 CRKP. Comparing with CSKP (carbapenem-sensitive Klebsiella pneumoniae), gastrointestinal disease (odds ratio [OR]=6.168, P=0.003), nosocomial infection (OR=5.573, P=0.012), antibiotic exposure (OR=4.131, P=0.004), urinary catheterization (OR=3.960, P=0.031) and venous/arterial catheterization (OR=2.738, P=0.026) within the preceding 3 months were independent risk factors for CRKP infection. The IMP-4 was the most predominant carbapenemase and bla IMP-4 bearing Klebsiella pneumoniae ST-11 was spreading in the hospital. Nosocomial infections, antibiotic exposure, and urinary and venous/arterial catheterization within 3 months were the risk factors for developing CRKP infection.

Antimicrobial resistance and carbapenemase dissemination in Pseudomonas aeruginosa isolates from Libyan hospitals: a call for surveillance and intervention

ABSTRACT Pseudomonas aeruginosa is a multidrug-resistant bacterium capable of forming biofilms. This study aimed to assess resistance of clinical isolates from Libyan hospitals to antipseudomonal antibiotics, the prevalence of selected extended-spectrum β-lactamases and carbapenemase genes among these isolates, and the microorganisms’ capacity for alginate and biofilm production. Forty-five isolates were collected from four hospitals in Benghazi and Derna, Libya. Antimicrobial susceptibility was determined using agar disc diffusion. The presence of resistance genes (blaCTXM, blaTEM, blaSHV-1, blaGES-1, blaKPC, and blaNDM ) was screened using PCR. Biofilm formation was quantified via the crystal violet assay, while alginate production was measured spectrophotometrically. Resistance to antipseudomonal antibiotics ranged from 48.9% to 75.6%. The most prevalent resistance gene was blaNDM (26.7%), followed by blaGES-1 (17.8%). Moreover, all isolates demonstrated varying degrees of biofilm-forming ability and alginate production. No statistically significant correlation was found between biofilm formation and alginate production. The dissemination of resistant genes in P. aeruginosa, particularly carbapenemases, is of great concern. This issue is compounded by the bacteria’s biofilm-forming capability. Urgent intervention and continuous surveillance are imperative to prevent further deterioration and the catastrophic spread of resistance among these formidable bacteria.

Emergence and clonal dissemination of KPC-2- and NDM-1-coharboring Citrobacter freundii in China with an IncR plasmid.

The rise of carbapenem-resistant Enterobacteriaceae coharboring KPC-2 and NDM-1 poses a significant public health threat. KPC-2-NDM-1-Citrobacter freundii is rarely reported in clinical settings. In this study, we report the largest cohort of eight KPC-2-NDM-1-C. freundii isolated from children with urinary tract infections. Comprehensive characterization, including antimicrobial susceptibility testing, plasmid conjugation, whole-genome sequencing, and comparative genomics, was conducted for these clinical strains. Antimicrobial susceptibility testing indicated that all strains were resistant to meropenem [minimal inhibitory concentration (MICs) ≥ 8 µg/mL] and imipenem (MICs > 8 µg/mL). Genotyping and comparative genomics analyses identified the eight KPC-2-NDM-1- C. freundii belonging to ST523, exhibiting a close relationship characterized by 45 single nucleotide polymorphisms. Whole-genome sequencing and plasmid analysis confirmed the presence of blaKPC-2 and blaNDM-1 on an IncR plasmid named pC275-2 with 46,050 bp. The blaNDM-1 was integrated within the blaNDM-1 related region, which includes ΔISAba125, blaNDM-1, ble-MBL, trpF, dsbD, and ISCR1, while the blaKPC-2 gene was located on a novel non-Tn4401 genetic element. The blaKPC-2 genetic architectures containing blaKPC-2 differed from classical structures, underscoring the ongoing evolution of these genetic elements.IMPORTANCEOur study described the largest cohort to date of eight ST523 KPC-2-NDM-1-C. freundii isolated from children with urinary tract infections. The cooccurrence of KPC-2, a serine β-lactamases, and NDM-1, a metallo-β-lactamase on an IncR plasmid pC275-2 from a clinical carbapenem-resistant C. freundii. The conserved insertion structure mediated the blaNDM-1, and the propagation of blaKPC-2 gene with a new genetic background using IncR plasmid in clinical settings promotes the emergence of superbugs necessitating vigilant monitoring. Our research detected that ST523 KPC-2- NDM-1-C. freundii were disseminated in a children's hospital. The potential spread of an IncR plasmid within the hospital raises concerns about the pandemic potential of this clone that produces two carbapenemases: KPC-2 and NDM-1. Further investigations will be necessary to control and prevent the spread of KPC-2-NDM-1-C. freundiis.

Detection of KPC-3 producing Escherichia coli ST410 in Volos, Greece.

Escherichia coli A382 was isolated in July 2024 from a positive blood culture obtained from the central venous catheter of a male patient undergoing chemotherapy at the Hospital of Volos, Thessaly, Greece. Whole-genome sequencing analysis revealed that the isolate A382 is E. coli belonging to the ST410 high-risk clone, which co-harbors the blaKPC-3 and blaSHV-182 genes on an IncX3 plasmid. It also harbors blaTEM-1 and has five replicons, as follows: IncX3, IncQ1, CoIRNAI, IncF1A, and IncFIB. Complete genome analysis revealed that E. coli A382 isolate carries a range of virulence factors (iutA, iucC, fimH, fdeC, yehA, yehD, yehC, yehB, cgs, ahha, ccI, hlyE, papC, irp2, fyuA, lpfA, and nlpl) and many other non-beta-lactam resistance determinants, including dfrA14 and sul2, but it is susceptible to aminoglycosides, nitrofurantoin, tigecycline, colistin and ceftazidime-avibactam. In conclusion in this study, we describe the phenotypic and genome characteristics of an extensively drug-resistant E. coli ST410.

Decadal Evolution of KPC-related plasmids in Pseudomonas aeruginosa high-risk clone ST463 in Zhejiang, China

Klebsiella pneumoniae carbapenemase-producing Pseudomonas aeruginosa (KPC-PA) isolates have quickly expanded in China, especially the high-risk clone ST463. We aimed to explore the evolution of KPC-related plasmids driving ST463 clone success. Whole-genome sequencing of 1258 clinical P. aeruginosa strains (2011–2020) identified 106 ST463-PA isolates, with a KPC prevalence of 90.6%. Early on (2011-2012), ST463-PA obtained the KPC-encoding type II (pT2-KPC) or type I plasmid (pT1-KPC) to overcome carbapenem stress. Between 2012 and 2017, pT1-KPC plasmid dominated due to its lower fitness costs and IS26-driven blaKPC amplification ability. By 2017-2020, large fragment deletions in pT1-KPC formed pT1del-KPC plasmid. It conferred even lower fitness costs, enhanced blaKPC-2 gene stability, and greater copy-number flexibility, while maintaining horizontal transmission ability. Consequently, pT1del-KPC plasmid finally succeeded, making ST463 the dominant ST in China. Our findings highlight evolutionary pressures driving ST463 dominance and emphasize the need for targeted strategies to control its spread and antibiotic resistance development.

Characterization of a Novel KPC-2 Variant, KPC-228, Conferring Resistance to Ceftazidime-Avibactam in an ST11-KL64 Hypervirulent Klebsiella pneumoniae.

With the widespread clinical use of ceftazidime-avibactam (CZA), reports of resistance have increased continuously, posing immense threats to public health worldwide. In this study, we explored the underlying mechanisms leading to the development of CZA resistance in an ST11-KL64 hypervirulent Klebsiella pneumoniae CRE146 that harbored the blaKPC-228 gene. Twelve carbapenem-resistant Klebsiella pneumoniae (CRKP) strains were isolated from the same patient, including K. pneumoniae CRE146. Whole genome sequencing (WGS), phylogenetic analysis, blaKPC gene cloning and pACYC-KPC construction assays were conducted to further explore the molecular mechanisms of CZA resistance. Quantitative siderophore production assay, string test, capsule quantification and Galleria mellonella in vivo infection model were applied to verify the level of pathogenicity of K. pneumoniae CRE146. This strain carried key virulence factors, iutA-iucABCD operon and rmpA gene. Compared to the wild-type KPC-2 carbapenemase, the novel KPC-228 enzyme exhibited a deletion of four amino acids in the Ω-loop (del_167-170_ELNS). In addition, the emergence of CZA resistance appeared to be associated with drug exposure, and we observed the in vivo evolution of wild-type KPC-2 to KPC-228 and then the reversion to its original wild-type KPC-2. The blaKPC-228 gene was located within the double IS26 flanking the ISKpn6-blaKPC-228-ISKpn27 core structure and carried on an IncFII/IncR-type plasmid. Notably, CRE146 exhibited high-level resistance to CZA (64/4 mg/L) but increased susceptibility to meropenem (1 mg/L) and imipenem (0.5 mg/L) respectively. PACYC-KPC plasmids were constructed and expressed in K. pneumoniae ATCC13883. Compared to K. pneumoniae ATCC13883 harboring blaKPC-2, K. pneumoniae ATCC13883 harboring blaKPC-228 exhibited a high-level resistance to CZA (32/4 mg/L) and increased susceptibility to meropenem (1 mg/L) and imipenem (0.5 mg/L). Interestingly, K. pneumoniae ATCC13883 harboring blaKPC-228 showed a significant decrease in their resistance to all β-lactamases tested except CZA and ceftazidime. In conclusion, we reported a novel KPC variant, KPC-228, in a clinical ST11-KL64 hypervirulent K. pneumoniae strain, which conferred CZA resistance, possibly through enhancing ceftazidime affinity and reducing avibactam binding. The blaKPC-228 can mutate back to blaKPC-2 under carbapenem pressure, which was very detrimental to clinical treatment. This strain carried both resistance and virulence genes, posing a major challenge in clinical management.

Emergent Escherichia coli of the highly virulent B2-ST1193 clone producing KPC-2 carbapenemase in ready-to-eat vegetables.

Critical priority carbapenem-resistant pathogens constitute a worldwide public health problem. Escherichia coli (E. coli) ST1193 is an emerging high-risk clone that demonstrates prolonged gut persistence, and association with community-onset urinary and bloodstream infections. The purpose of this study is to report microbiological and genomic data on the emergence of KPC-2-producing E. coli ST1193 in ready-to-eat (RTE) vegetables. RTE vegetables were purchased from markets in southeastern Brazil. Epiphytic and endophytic Gram-negative bacteria displaying resistance to broad-spectrum beta-lactams were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Whole-genome sequence was conducted using the Illumina NextSeq platform. Antimicrobial susceptibility, conjugation, and acid tolerance assays were performed. Virulence behaviour was evaluated using the Galleria mellonella (G. mellonella) infection model. Epiphytic KPC-2-producing E. coli belonging to pandemic ST1193 was identified in RTE arugula. Genomic analysis predicted clinically relevant genes conferring resistance to β-lactams, fluoroquinolones, hazardous heavy metals, pesticides, disinfectants, and chlorine sanitizer. The blaKPC-2 gene was carried by a conjugative IncF plasmid. Acid tolerance of E. coli KPC-2/ST1193 during exposure to pH 2.0 was confirmed, being associated with gadWX and ibaG pH tolerance genes, supporting survival to stomach acid prior to reaching the small intestine, and potential for a dietary mode of host colonization. Virulent behaviour was supported by wide virulome of the highly virulent phylogroup B2, whereas single nucleotide polymorphisms of core genes (cgSNP)-based phylogenomics revealed clonal relationship with healthcare-associated lineages circulating in the United States, China, Mexico, France, and Brazil. We report the occurrence of KPC-2-producing E. coli of the highly virulent B2-ST1193 clone in RTE vegetables, highlighting a possible route of dissemination of the World Health Organization (WHO) priority pathogens to humans. © 2024 The Author(s). Published by Elsevier Ltd on behalf of International Society for Antimicrobial Chemotherapy.

Assessing the efficacy of postbiotics derived from Lactobacillus plantarum on antibiotic resistance genes in nosocomial pathogens such as Enterococcus faecalis and Pseudomonas aeruginosa.

This study investigated the antibacterial and anti-biofilm properties of postbiotics derived from Lactobacillus plantarum and their effect on the expression of antibiotic resistance genes (ermB and blaKPC) in Enterococcus faecalis and Pseudomonas aeruginosa, respectively. Cell-free supernatants (CFSs) were analyzed through gas chromatography-mass spectrometry (GC-MS), which showed that butyric acid (14.31%) was the major compound, other metabolites present in CFSs included lactic acid (5.94%), hdroxyacetone (5,21%), benzoic acid (3.12%), Pyrrolo[1,2-a] pyrazine-1,4-dione (1.91%), 2,3-Butanediol (1.04%), and 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one (0.73.%). To investigate the effect of postbiotics on bacterial growth and biofilm formation, minimal inhibitory concentration (MIC) and microtiter plate assays were used. MIC results showed that resistant En. faecalis and P. aeruginosa can grow at concentrations of 2.5and 5mg/ml, respectively, after exposure to postbiotics. Furthermore, the microtiter plate results showed that postbiotics significantly reduced biofilm formation: 51%, 45%, and 39% in En. faecalis and 46%, 38%, and 27% in P. aeruginosa at different concentrations. Real-time polymerase chain reaction also confirmed the reduction of resistance genes (ermB; P=0.007 and blaKPC; P=0.02) expression. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay showed that the cell survival rate was 80%. These findings suggest that postbiotics from L. plantarum may be a promising approach for combating bacterial growth, biofilm formation, and antibiotic resistance.

Effect of Meropenem on Conjugative Plasmid Transfer in Klebsiella pneumoniae.

Plasmid-mediated resistance is a major mechanism that contributes to the gradual decline in the effectiveness of antibiotics from different classes, including carbapenems. Antibiotics can significantly contribute to the efficiency of plasmid transfer between bacterial strains. To investigate the potential effect of an antibiotic on the efficacy of conjugative plasmid transfer, we conducted mating experiments with Klebsiella pneumoniae strains. Donor strains of K. pneumoniae that carry plasmids with blaKPC or blaOXA-48 carbapenemase genes and recipient plasmid-free K. pneumoniae strains were used in matings. Matings were conducted on the agar with or without meropenem at 1/8×, 1/4×, or 1/2×MIC against the respective recipients. In the second part of our study, we investigated the pharmacodynamic properties of meropenem against transconjugant strains of K. pneumoniae, which were obtained in the first part of this study. As a result, at a concentration equivalent to 1/8×MIC, meropenem primarily inhibited conjugation among K. pneumoniae strains, while at a concentration equal to 1/2×MIC, it facilitated conjugation. Transconjugants derived from K. pneumoniae with intermediate MICs failed to respond to simulated treatment with meropenem using prolonged infusion and a high-dose regimen. This finding suggests that such transconjugants may potentially pose a risk if involved in an infectious process.

Salmonella enterica Serovar Infantis KPC-2 Producer: First Isolate Reported in Ecuador.

Antimicrobial resistance is currently considered a public health threat. Carbapenems are antimicrobials for hospital use, and Enterobacterales resistant to these β-lactams have spread alarmingly in recent years, especially those that cause health care-associated infections. The blaKPC gene is considered one of the most important genetic determinants disseminated by plasmids, promoting horizontal gene transfer. This study describes, for the first time in Ecuador, and worldwide, the presence of a blaKPC-2 gene in an isolate of Salmonella enterica serovar Infantis from a clinical sample. Through whole-genome sequencing, we characterized the genetic determinants of antimicrobial resistance in this Salmonella ST-32 strain. Our results showed the presence of several resistance genes, including blaCTX-M-65, and a conjugative plasmid Kpn-WC17-007-03 that may be responsible for the horizontal transference of these resistance mechanisms.

Molecular characterization of ST15 carbapenem-resistant Klebsiella pneumoniae isolated in a single patient

The carbapenem-resistant K. pneumoniae (CRKP) poses a serious threat to antibiotic applicability and public health. During treatment, K. pneumoniae (KP) frequently exhibits shifts in drug-resistant phenotypes, complicating clinical treatment as it transitions from sensitivity to resistance. In this study, we analyzed the clinical and molecular characteristics of drug resistance changes in KP strains isolated from a single patient, and the potential mechanisms underlying these resistance changes. Antimicrobial susceptibility test and string test were conducted to evaluate the resistant and virulent characterization of the strains. Pulsed field gel electrophoresis (PFGE) was used to investigate the homology relationship between the strains. The whole genome sequencing and phylogenetic analysis of 9 representative isolates was also performed. The transfer ability of the drug-resistant plasmid was studied by plasmid conjugation experiment. The transconjugants were verified by PCR amplification of specific genes, antimicrobial susceptibility test and PFGE. Our results revealed that 9 KP strains, isolated from the same patient, exhibited 'resistance-sensitivity-resistance-sensitivity' alternately to carbapenems. The differences in DNA fingerprint bands among the nine KP isolates were ≤ 3, which can be classified as the same PFGE type. Phylogenetic analysis showed that these 9 strains constituted a distinct branch within the phylogenetic tree. All nine KP strains belonged to the ST15-KL19 clone. Six of the strains were classified as CRKP, all of which carried eleven drug resistance genes: oqxB, oqxA, fosA6, aac(3)-lld, blaSHV-28, blaKPC-2, blaOXA-1, mph(A), tet(A), catB3 and aac(6')-lb-cr, mediating drug resistance to quinolones, fosfomycin, aminoglycosides, β-lactam, carbapenems, macrolides and chloramphenicol, belonging to multi-drug resistant bacteria. The carbapenem-resistant plasmid p2-KP3762-1 was found to transfer within species, from CRKP to hypervirulent K. pneumoniae (hvKP) RJF293HA, carbapenem-sensitive K. pneumoniae (CSKP) KP3657 and E. coli C600 at a frequency of (1.19±1.58) ×10-6, (1.09±1.38) ×10-7 and (10.9±9.53) ×10-6 respectively, resulting in the dissemination of carbapenem resistance genes. In this study, KP strains isolated from a single patient exhibited an alternating phenotype of resistant-sensitive-resistant-sensitive to carbapenems. The 9 KP isolates share a high degree of genetic similarity. The plasmid p2-KP3762-1, harboring the carbapenem resistance gene blaKPC-2, may undergo inter-strain and inter-clone transfer via conjugation in the patient during treatment. Furthermore, our findings suggest that the pathogens in this patient are likely to have a common ancestral origin.

Detection and characterization of carbapenemase-producing Enterobacteriaceae in cancer patients: first Sri Lankan report of blaVIM in Enterobacteriaceae.

Carbapenem-resistant Enterobacteriaceae (CRE) are an important cause of infections in cancer patients. The proportion of carbapenem resistance and the types of carbapenemase-encoding genes in Enterobacteriaceae isolated from cancer patients were determined in this study. Bacteria isolated from adult, in-ward cancer patients with lower respiratory tract infections (LRTI), skin and soft tissue infections (SSTI), or urinary tract infections (UTI) were included in the study. Enterobacteriaceae were identified up to the species level by API® 20E test kits. Carbapenem resistance was defined as non-susceptibility to either imipenem or meropenem in the disc diffusion test. Major carbapenemase-encoding genes (blaKPC, blaNDM, blaOXA-48, blaIMP, and blaVIM) were detected by the GeneXpert® Carba-R real-time PCR instrument. Enterobacteriaceae comprised 57% (94/165) of the bacterial isolates. Carbapenem resistance among Enterobacteriaceae was 46.8% (44/94). Klebsiella pneumoniae (65.9%, 29/44) was the predominant CRE isolate followed by Escherichia coli (25%, 11/44). The majority of CRE isolates (72.7%, 32/44) had a meropenem MIC of ≥ 32 µg/mL. Carbapenemase-encoding genes were identified in 43 of the 44 CRE isolates. blaNDM was the most prevalent carbapenemase-encoding gene and was detected in 67.4% (29/43) of Enterobacteriaceae isolates. No isolate was positive for blaIMP. Sixteen (37.2%) isolates co-harbored more than one carbapenemase-encoding gene. Two Enterobacteriaceae isolates were found to harbor blaVIM. Nearly all CRE isolated in this study were carbapenemase producers. This study documented the emergence of blaVIM harboring Enterobacteriaceae for the first time in Sri Lanka.

Ceftazidime-avibactam tolerance and persistence among difficult-to-treat KPC-producing Klebsiella pneumoniae clinical isolates from bloodstream infections.

Tolerance and persistence occur "silently" in bacteria categorized as susceptible by antimicrobial susceptibility testing in clinical microbiology laboratories. They are different from resistance phenomena, not well-studied, and often remain unnoticeable. We aimed to investigate and characterize ceftazidime-avibactam (CZA) tolerance/persistence in 80 Klebsiella pneumoniae isolates from bloodstream infections. We used the Tolerance Disk Test (TDtest) to detect CZA tolerance/persistence and investigate the avibactam (AVI) influence on them, and time-kill assays with minimal duration for killing (MDK) determination to characterize/differentiate CZA tolerance from persistence, for selected isolates. Whole genome sequencing was performed for 49/80 selected isolates to investigate genes related to beta-lactam tolerance/persistence and resistance as well as phylogeny studies. Tolerance/persistence to CZA was detected in 48/80 (60%) isolates, all extensively drug-resistant (XDR) or multidrug-resistant, carbapenem-resistant K. pneumoniae (CRKp), KPC producers, and previously categorized as susceptible (not resistant) to CZA. No heteroresistance was detected. CZA tolerance/persistence occurred due to ceftazidime tolerance/persistence and was not related to AVI in the CZA combination. 5/11 isolates were characterized as CZA-tolerant and 5/11 as CZA-persistent. The single (1/11) XDR and CRKp non-KPC producer was truly susceptible. All the CZA-tolerant/persistent isolates (ST11, ST258, ST340, ST437, ST16, ST17, and ST307) harbored the carbapenemase-encoding gene blaKPC-2. Mutation in only two genes (rpoS and degQ) related to beta-lactam tolerance/persistence was found in only 7/49 CZA-tolerant/persistent isolates, suggesting the presence of yet unknown beta-lactam tolerance/persistence genes. Among the K. pneumoniae bloodstream isolates studied, 60%, previously categorized as susceptible to CZA, were, actually, tolerant/persistent to this antibiotic, all these KPC producers.

NDM-1 and KPC-3 co-producing Klebsiella pneumoniae ST512 in bronchial secretion from a patient in an intensive care unit of a Greek Tertiary Care Hospital.

This study investigated a strain of Klebsiella pneumoniae, identified as GRTHES, which exhibited extensive antibiotic resistance. The strain was resistant to all beta-lactams, including combinations withnewer agents such as meropenem/vaborbactam and imipenem/relebactam, as well as to aminoglycosides, fluoroquinolones, fosfomycin, trimethoprim-sulfamethoxazole and colistin. It remained susceptible to tigecycline. Whole-genome sequencing was performed by Ion Torrent platform on the K.pneumoniae strain. Genomic analysis revealed a genome length of 5,808,650 bp and a GC content of 56.9%. Advanced sequencing techniques and bioinformatic tools were used to assess resistance genesand plasmid replicons, highlighting the emergence of multidrug resistance and virulence traits. Thestrain carried blaNDM-1 and blaKPC-3 genes and was designated to KL107 O2afg type. Colistin resistance-associated mgrB/pmrB gene mutations were present, and the strain also harbored yersiniabactin-encoding ybt gene. Our findings provide insights into the genomic context of blaNDM-1 and blaKPC-3 carbapenemase-producing K. pneumoniae and emphasize the importance of continuous surveillance and novel therapeutic strategies to combat multidrug-resistant bacterial infections. It is the first time that an NDM-1 and KPC-3 co-producing strain of K. pneumoniae ST512 is identified in Greece. This study highlights the essential role of genomic surveillance as a proactive strategy to control the spread of carbapenemase-producing K. pneumoniae isolates, particularly when key antimicrobial resistance genes, such as blaNDM-1 and blaKPC-3, are plasmid-mediated. Detailed characterization of these isolates could reveal plasmid similarities that facilitate adaptation and transmission within and between hospitals. Although data on patient movements are limited, it is plausible that carbapenem-resistant isolate was selected to co-produce KPC and NDM through plasmid acquisition.

Analysis of the virulence of a lethal, carbapenem-resistant hypervirulent KPC-33-producing Klebsiella pneumoniae: Emergence of ST11-KL64 hv-CRKP in ICU

ObjectiveHypervirulent and carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) poses a serious threat to public health. Here, we analyse a case of systemic infection caused by a hv-CRKP, which ultimately led to the patient's death from sepsis. And a total of 30 CRKPs were analyzed to elucidate the molecular epidemiological features of CRKPs in the hospital, and to provide a basis for clinical anti-infective therapy. MethodsIn this case, a total of 7 K. pneumoniae strains were isolated from the blood, sputum, urine, and feces of the patient. The Vitek-2 compact system was used to identify the strains and perform antimicrobial susceptibility testing. Biofilm formation, siderophore production assays and Galleria mellonella infection model were used to verify the virulence phenotypes of the strains in the case. Whole-genome sequencing was conducted on the four hv-CRKP isolated from different samples in the case and 26 other CRKP collected in our hospital from September to November in 2022, using the Illumina Hiseq 6000 high-throughput sequencing platform to analyse the resistance and virulence genes. ResultsIn the case, after 7 days of treatment with ceftazidime-avibactam (CZA), the resistance profile of the strains changed. The strain that was initially sensitive to CZA developed to resistant, resistant to imipenem (IPM) developed to sensitive, and resistant to meropenem (MEM) developed to intermediate. Whole-genome sequencing revealed that the four strains in the case were all ST11-KL64 K. pneumoniae, and the change in resistance phenotype was due to the mutation from blaKPC-2 to blaKPC-33. KPN7 had a total of six plasmids, with siderophore-related genes iucABCD and iutA, and mucoid phenotype-related gene rmpA2 located on plasmid p4-KPN7; resistance genes blaKPC-33, blaTEM-1B, and blaCTX-M-65 located on plasmid p5-KPN7; and virulence genes fim, irp, iutA, and ybt located on the chromosome. Biofilm formation and siderophore production assays confirmed that the seven K. pneumoniae strains isolated in this case had strong biofilm formation and siderophore production capabilities. Galleria mellonella Infection Model showed that KPN4 and KPN7 was phenotypically highly virulent and KPN7 performed lower virulence compared to KPN4. Apart from the 4 hv-CRKP strains, other 26 CRKP strains all carried blaKPC-2, and 69.2% (18/26) were ST-11 and 30.8%(8/26) were ST-15. And 83.3% (15/18) were ST11-KL64 strains, followed by ST11-KL25 strains 11.1%(2/18) and ST11-KL47 strain 5.6%(1/18). All the eight ST-15 strains were KL-19. ConclusionThe ST11-KL64 hv-CRKP clone spread widely in ICU carried numerous resistance and virulence genes, and under antibiotic pressure, they easily underwent mutations resulting in changes in resistance phenotypes, especially in mutations of blaKPC-2 gene in acquiring resistance to CZA. Therefore, clinical attention should be paid to such strains, and the use of antibiotics should be adjusted promptly based on the susceptibility of the strains to antimicrobial agents.

Genomic characterization of a bla KPC-2-producing IncM2 plasmid harboring transposon ΔTn6296 in Klebsiella michiganensis.

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 Tn6296 with the distinctive core structure ISKpn27-bla KPC-2-ISKpn6. 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.

Antibiotic-resistant genes derived from commercial organic fertilizers are transported to balconies of residential buildings by express delivery.

The rise in antibiotic-resistant genes (ARGs) has recently become a pressing issue, with livestock manure identified as a significant source of these genes. Yet, the distribution of fertilizers derived from livestock manure sold online, potentially containing high levels of ARGs and antibiotic-resistant bacteria (ARB), is often not considered. Our study involved a random survey of commercial organic fertilizers available on online marketplaces, focusing on 13 common ARGs and 2 integrons (intI1, intI2). We found significant ARGs linked to sulfonamides, macrolides, and tetracycline in the 20 fertilizer samples we tested. The gene copy numbers for ermC, sul2, and tetL were exceptionally high, reaching up to 1011 copies per gram of fertilizer in specific samples. Additionally, 18 out of 20 samples contained the critical β-lactam resistance genes blaTEM and blaKPC, with gene copy numbers up to 1010 copies/g. Integrons, intI1, and intI2 were present in all samples, with abundances ranging from 103 to 1010copies/g. We categorized the 20 samples into three types for further analysis: poultry manure, livestock manure, and earthworm manure. Our findings indicated a high presence of ARGs in poultry manure compared to a lower occurrence in earthworm manure. The study also showed a strong correlation between integrons and specific ARGs. This research underscores the potential risk of commercial organic fertilizers as a pathway for spreading ARGs from the animal breeding environment to human settings through express transportation.

Overexpression of β-lactamase genes (blaKPC, blaSHV) and novel CirA deficiencies contribute to decreased cefiderocol susceptibility in carbapenem-resistant Klebsiella pneumoniae before its approval in China.

Cefiderocol (FDC) is an effective antibiotic that is used to treat severe infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP). The mechanisms underlying FDC resistance and molecular epidemiology in China remain unclear. We collected 477 non-duplicate CRKP clinical isolates in central China and characterized their susceptibility to FDC, virulence genes, and sequence typing. The overall FDC susceptibility rate of CRKP was 99.2% in central China, which was higher than that in North America and Europe (96.1%), with MIC50/90 values of 1/2 mg/L. The decrease in FDC susceptibility in central China was concentrated in the ST11 CRKP-carrying virulence plasmids. Whole-genome sequencing (WGS) and quantitative reverse transcription PCR (qRT-PCR) experiments showed that serine β-lactamases, especially highly expressed KPC and SHV, substantially decreased FDC susceptibility in four FDC non-susceptible isolates (two resistant and two intermediate isolates). Notably, different CirA deficiencies, p.E450GfsTer16 and p.E133Ter, were found in both of the resistant isolates. In contrast, global WGS data indicate that the resistance mechanisms in North America and Europe were primarily associated with NDM and KPC variants, predominantly found in ST307 and ST147. Overall, FDC exhibits excellent activity against CRKP in central China, with resistance mechanisms primarily related to high KPC and SHV expression, along with deficiencies in CirA, frequently observed in ST11. This is remarkably different from the situation in North America and Europe and will directly impact the choice of clinical interventions. Additionally, the surveillance of FDC resistance in China is imperative.

Public health concern of antimicrobial resistance and virulence determinants in E. coli isolates from oysters in Egypt

The emergence of critical-priority E. coli, carrying a wide array of resistance and virulence factors through food sources, poses a significant challenge to public health. This study aimed to investigate the potential role of oysters sold in Egypt as a source for E. coli, identify their resistance and virulence-associated gene profiles, and assess associated zoonotic risks. A total of 33 pooled fresh oyster samples were obtained from various retail fish markets in Egypt and examined bacteriologically for the presence of E. coli. Antimicrobial resistance was performed by the disk-diffusion method, and the multiple antibiotic resistance index (MAR) was calculated. All isolates were screened for extended-spectrum beta-lactamase (ESBL) (blaTEM, blaSHV, blaCTX−M, and blaOXA−1), plasmid-mediated AmpC blaCMY−2, and carbapenemases (blaKPC, blaNDM, blaVIM, and blaOXA−48) genes by Polymerase chain reaction. Moreover, the presence of virulence-encoding genes was investigated. The virulent MDR strains were clustered using R with the pheatmap package. The prevalence of E. coli was 72.7% (24 out of 33), with 66.7% of the isolates classified as multi-drug resistant, and 75% exhibited MAR values exceeding the 0.2 threshold. Different antimicrobial sensitivity phenotypes and genotype profiles were identified in E. coli isolates. The most prevalent gene detected among all isolates was blaTEM (22/24, 91.7%). Notably, all non-ESBL producers were positive for blaCMY2. Carbapenem-resistant and carbapenem-intermediate strains were carbapenemase producers, with the predominance of the blaKPC gene (11/24, 45.8%). Remarkably, twelve out of sixteen virulence genes were identified, with papC (21/24, 87.5%) and sfa (16/24, 66.7%) genes being the most prevalent. Most isolates carry virulence genes primarily associated with extra-intestinal pathogenic E. coli (ExPEC) (87.5%) and enteropathogenic (EPEC) (70.8%) pathotypes. Four E. coli isolates exhibit cluster patterns. This study provides the first insight into the emergence of virulent MDR E. coli among oysters in Egypt. It underscores the potential role of oysters as a source for disseminating these strains within aquatic ecosystems, presenting a possible threat to public health.