KPC Genes Research Articles

Rapid detection of enterobacteria in wastewater treated by microalgal consortia using loop-mediated isothermal amplification (LAMP).

In the present study, nine Enterobacteriaceae species present in wastewater were isolated and identified, and loop-mediated isothermal amplification (LAMP) was developed for the detection of Enterobacteriaceae by designing primers based on the mcr-1, KPC, OXA-23, and VIM genes, which are recognized markers of antimicrobial resistance (AMR) transmission during microalgal bioremediation treatment. The developed assays successfully detected four strains positive for mcr-1 gene-asociated resistance (Acinetobacter baylyi, Klebsiella pneumoniae, Morganella morganii, and Serratia liquefaciens), three strains for KPC gene-associated resistance (Acinetobacter sp., Escherichia coli 15499, and Morganella morganii), seven strains for OXA-23 gene-associated resistance (Acinetobacter baylyi, Enterobacter hormaechi, Enterobacter cloacae, Escherichia coli 15922, Escherichia coli 51446, Morganella morganii, and Serratia liquefaciens), and three strains for resistance to the VIM gene-associated resistance (Acinetobacter baylyi, Acinetobacter sp., and Enterobacter hormaechi) from a single colony. A reduction in microbiological load of 93.6% was achieved at 15 colony-forming units (CFU) mL-1, utilizing EMB agar and LAMP values of 0.142 ± 0.011 for the mcr-1 gene, 0.212 ± 0.02 for the KPC gene, 0.233 ± 0.006 for the OXA-23 gene, and 0.219 ± 0.035 for the VIM gene. Furthermore, bioremediation efficiency values of 71.6% and 75% for total nitrogen and phosphorus, respectively, were observed at 72 h of treatment in open pond microalgal remediation systems (MRS). This study demonstrated that the LAMP technique is faster and more sensitive than traditional detection methods, such as CFU, for Enterobacteriaceae. Consequently, this method may be considered for the detection of microbiological quality indicators within the water treatment industry.

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.

First Report of Carbapenem-Resistant Klebsiella michiganensis Co-Harboring bla KPC-2 and TmexCD2-ToprJ2 Isolated from Wastewater at a Tertiary Hospital in Beijing.

Klebsiella michiganensis is an emerging human pathogen that causes nosocomial infections. Its prevalence and spread in the environment should not be ignored. This study identified and characterized Klebsiella michiganensis co-harboring bla KPC-2 and TmexCD2-ToprJ2 in hospital wastewater samples. Twelve K. michiganensis strains were isolated from wastewater samples collected at a tertiary hospital in Beijing, China. The genomic characteristics of K. michiganensis strains were analyzed using whole-genome sequences, providing information on the comparison between the genome of K. michiganensis strains and the reference genome, antibiotic resistance genes (ARGs), virulence genes, secretion systems, and mobile genetic elements (plasmids, insertion sequences [ISs], and prophages). Genome analysis showed that the twelve multi-drug resistant (MDR) strains carried a variety of ARGs and virulence genes, as well as four macromolecular secretion systems (T1SS, T2SS, T5aSS, T5bSS, and T4aP). The genetic environments of both the TmexCD2-ToprJ2 gene cluster and bla KPC-2 gene contained ISs. The plasmids carrying TmexCD2-ToprJ2 gene cluster of nine strains in clade 1 and two strains in clade 2 were annotated as IncR plasmid and rep_cluster_1254 type, respectively. The plasmids carrying bla KPC-2 in 10 strains in clade 1 were identified as IncU, and the plasmids carrying bla KPC-2 in the k11 and k12 strains in clade 2 were IncU and IncX6. The phylogenetic tree and heatmap revealed that the secretion system of type VI (T6SSi) existed in 10 strains in clade 1, and Type IV (T4SS) only existed in the k11 strain in clade 2. In addition, K. michiganensis strains carried 13 plasmids, 14 ISs, and 138 prophages. In this study, the whole genome sequencing demonstrated the diversity of K. michiganensis genome despite 12 K. michiganensis strains from a hospital wastewater, which lays the foundation for further genetic research and drug resistance gene transmission.

Carbapenem-Resistant Infections in Neonates and Children in Latin America: A Literature Review.

Carbapenems are broad-spectrum beta-lactam antibiotics that are increasingly being used worldwide to treat multidrug-resistant infections, but since their introduction, carbapenem resistance has emerged. This phenomenon has been well documented in the adult population, but there is a paucity of evidence from the neonatal and pediatric populations. A literature search of carbapenem-resistant infections in Latin American neonates and children was conducted via PubMed/Medline and SCOPUS: 551 titles were screened, and 17 articles were included in the review. The most commonly reported predominant isolate was Klebsiella pneumoniae (11 of 17 studies). Genotypic data were available in 10 of 17 studies, and the KPC gene was the most commonly reported resistance gene. The mortality rate ranged from 13% to 52.6%. Carbapenem-resistant infections are prevalent in children and neonates in Latin America and are associated with high rates of mortality, highlighting the need for enhanced antimicrobial stewardship and surveillance within these populations.

Genetic diversity and antibiotic resistance patterns of Escherichia coli isolates causing septicemia: A phylogenetic typing and PFGE analysis

IntroductionThis study aims to analyze clinical isolates of E. coli causing septicemia across various phylogroups utilizing the PFGE method. Materials and methodsA total of 100 clinical isolates were collected. The presence of CTX-M, TEM, SHV, KPC, MBL and OXA-48 genes was detected by PCR. Additionally, phylotyping, serotyping, and virulence-typing assay were done by PCR and PFGE methods to investigate the genetic diversity of the isolates. ResultsThe O1 serotype and the HlyA gene were the most prevalent serotype and virulence gene, respectively. Notably, 34% of the isolates harbored SHV, TEM, and CTX-M-1 β-lactamase genes. All isolates showed resistance to amoxicillin and tetracycline, but no resistance to fosfomycin was seen. The most and least common phylotypes, according to PFGE analysis, belonged to phylogroups B2 and B1, respectively. ConclusionThe data offers valuable insights into the genetic diversity and antibiotic resistance patterns of E. coli isolates responsible for septicemia.

Molecular Detection of Beta-Lactamase Genes (KPC and CTX-M) of Pseudomonas aeruginosa in Diabetic Foot Ulcers Patients

Background: diabetic foot ulcers (DFUs ) are an inflammatory disease caused by a bacterial infection such as Pseudomonas aeruginosa (P. aeruginosa), provided with genetic virulence factors. This study aims to detect KPC and CTX-M genes of P. aeruginosa isolated from chronic DFU patients. Methods: One hundred twenty DFU patients were recruited from Al-Sader Medical City and Al-Hakim General Hospital in Najaf province from September to January 2021 to collect ear swaps. The Primary identification depends on Gram stain and biochemical tests. We also used the Vitek 2 system to make another identification and examine resistance to antibiotics. A polymerase chain reaction (PCR) was used to amplify the required genes. Furthermore, gel electrophoresis was performed to show the amplified genes. Results: The results have revealed that 100 samples (83.3%) give a positive outcome, while 20 samples (16.6%)are negative for culture bacteria. Only 17(17 %) of the 100 clinical specimens showed positive tests for P. aeruginosa. However, the gene, CTX-M gene is found in 58.8% of P. aeruginosa isolates, while the KPC gene is not found in the isolates. Conclusion: Our findings suggest that P. aeruginosa is one of the most common causes of DFU infection in Iraqi patients and The levels of MDR of P. aeruginosa isolates in Najaf hospitals were undoubtedly high. Most bacterial isolates show the presence of the CTX-M gene. The presence of these genes is one of the virulence factors and probably partly explains the resistance to antibiotics.

Establishment of a platform based on dual RPA combined with CRISPR/Cas12a for the detection of Klebsiella pneumoniae and its KPC resistance gene.

Carbapenem resistant Klebsiella pneumoniae (CRKP) can cause serious hospital- and community-acquired infections. Treatment for CRKP infection is limited, resulting in prolonged hospitalization and high consultation costs. The KPC genotype has the highest detection rate of CRKP, and its mortality rate is higher than the overall mortality rate of CRKP. However, traditional testing methods have disadvantages such as long time and reliance on complex and sophisticated instruments, which are not conducive to rapid screening for CRKP. Therefore, this study aimed to establish a detection platform for early screening of CRKP so that effective antimicrobial therapy could be administered promptly to prevent the widespread spread of CRKP. We integrated dual RPA with CRISPR/Cas12a to establish a dual platform for the detection of K. pneumoniae (Kp) rcsA-specific gene and KPC resistance gene. Four result reading methods were established, including fluorescence detection (FD), blue light irradiation detection (BLID), ultraviolet irradiation detection (UID), and lateral flow test strips (LFTS). For the rcsA gene, the LOD of FD was 1 × 10pg/μL, and the other three methods could detect 1 × 101pg/μL of bacterial DNA. As for the KPC gene, four resultant readout methods were able to detect 1 × 102pg/μL of bacterial DNA. In 59 clinical strains tested, the dual RPA-CRISPR/Cas12a detection of the rcsA had 100% sensitivity, specificity, and accuracy compared to the culture method. Compared with the drug sensitivity test, the sensitivity of dual RPA-CRISPR/Cas12a detection for the KPC was 85.71%, the specificity was 100%, and the accuracy was 94.92%. In summary, our dual RPA-CRISPR/Cas12a platform proved to be rapid, precise, and convenient for the efficient detection of Kp with KPC in the laboratory or at the point of care.

Molecular Characterization of Carbapenemase (Blakpc-2) Gene From Klebsiella Pneumoniae Involved in Urinary Tract Infection in Al-Najaf City

Background: The human gastrointestinal tract, eyes, respiratory system, and genitourinary tract are all home to K. pneumonia. KPCs, or Klebsiella pneumonia carbapenemases, are β-lactamase enzyme types that have the ability to hydrolyze β-lactam drugs. Because of many co-morbid diseases, immunological suppression, and critical sickness, this KPC-producing strain infection can be fatal. In this study K. pneumonia was isolated from urinary tract infected patients, Where seven clinical urine specimen obtained from patients has UTI were gathered from different diagnostic Al-Sajjad Hospital in Najaf province the KPC gene was amplified by PCR. In this present study, a particular primer derived from the antibiotic sensitivity (blaKPC-2) gene is used to screen the urine samples collected for Klebsiella pneumoniae stains utilizing culture techniques then sequence analysis of the blaKPC-2 gene was conducted in order to gain a fresh understanding of the genetic variations. It's interesting to note that, while comparing our strains' blaKPC-2 open reading frame to previously released sequences, we discovered 6 mutations. Therefore, of the seven samples analyzed, three exhibited a positive band. The purpose of the primer was to amplify the bacteria's blaKPC-2 antibiotic sensitivity gene. Conclusion: In this work, we verified a PCR technique for blaKPC-2 gene identification that is quick, sensitive, and specific. Out of the seven samples analyzed, three exhibited a positive band. The purpose of the primer was to amplify the bacteria's blaKPC-2 antibiotic sensitivity gene.

Resistance Genes and Mortality in Carbapenem-resistant Klebsiella pneumoniae Bacteremias: Effects of the COVID-19 Pandemic

Emerging carbapenem-resistant Klebsiella pneumoniae (K. pneumoniae) (CRKP) bacteremias are presenting significant public health risks due to limited treatment options and increased mortality. K. pneumoniae isolates exhibit carbapenem resistance rates that vary from 25% to 50% throughout the European continent, including our country. To assess the characteristics of CRKP bacteremia, a condition that has recently demonstrated an increasing prevalence in our center. We sought to ascertain the resistance rates of isolated strains to antibiotics other than carbapenems, identify the responsible carbapenemase genes, evaluate the efficacy of antibiotics, determine mortality rates, explore clonality among strains, and investigate the influence of the COVID-19 pandemic on all these factors. Retrospective observational study. This study included patients aged 18 and older who had experienced meropenem-resistant K. pneumoniae bacteremia. Meropenem resistance was confirmed by employing the Kirby-Bauer disk diffusion method. Meropenem minimum inhibitory concentration (MIC) levels were determined using the gradient test, while colistin MIC levels were ascertained using the disk elution technique. Carbapenemase genes were evaluated via colony polymerase chain reaction (PCR), and clonality analysis was performed using the arbitrarily primed PCR technique. The study comprised 230 patients, with a mean age of 63.1 ± 15.9 years, of whom 58.7% were male. Oxacillinase-48 (OXA-48) was detected in 74.8% of the patients, New Delhi metallo-beta-lactamase (NDM) in 12.6%, OXA-48 + NDM in 7.8%, and KPC in 4.8%. The 14-day and 30-day mortality rates were 57% and 69.6%, respectively. Multivariate analysis of the 30-day mortality revealed several crucial factors, including bacteremia development in the intensive care unit, the occurrence of bacteremia during the COVID-19 pandemic, polymicrobial bacteremia, the use of indwelling intravenous catheters, a platelet count of ≤ 140,000/μl, procalcitonin levels of ≥ 6 μg/l, and a Charlson comorbidity score ≥ 3. Notably, the OXA-48 and KPC genes were upregulated significantly during the COVID-19 pandemic, while the NDM gene groups were downregulated. Additionally, both 14-day and 30-day mortality rates increased significantly. In this study, the most prevalent carbapenemase gene was OXA-48; however, there has been a recent increase in KPC genes. No dominant epidemic strain was identified through clonality analysis. The clustering rate was 68% before the pandemic, increasing to 85.7% during the pandemic. The significance of infection control measures is underscored by the rise in both clustering and mortality rates during the COVID-19 pandemic.

Characterisation of carbapenemase-producing Gram-negative bacilli among clinical isolates in a tertiary care centre in Kerala, South India

Background: Infections caused by carbapenem-resistant Gram-negative bacteria are a cause for concern due to the limited choice of antibiotics available for their treatment. Aims, Settings and Design: This study screened multidrug-resistant (MDR) Gram-negative bacilli isolated from clinical samples over a period of one year, for carbapenem resistance and characterised them using phenotypic methods such as combined disc diffusion test (CDDT), modified Hodge test (MHT), E-test for metallo-beta-lactamase (MBL) and molecular method, PCR. Materials and Methods: Two hundred and ten MDR Gram-negative bacilli were screened for carbapenem resistance using Imipenem and Meropenem disc diffusion. These were further checked for carbapenemase production by CDDT, MHT and E-test for MBL. Those positive by E-test were subjected to PCR. Uniplex PCR for New Delhi metallo-beta-lactamase-1 was used for Escherichia coli and Klebsiella pneumoniae , and multiplex PCR for Imipenemase and Verona imipenemase was used for Pseudomonas aeruginosa and Acinetobacter baumannii isolates. Results: Twenty-three (11%) isolates were found to be carbapenem-resistant and included E. coli (six) K. pneumoniae (three), P. aeruginosa (five) and A. baumannii (nine). Seventeen (74%) isolates were positive by phenotypic methods and were subjected to PCR. Out of eight Enterobacteriaceae isolates subjected to PCR, all were positive for bla NDM gene. All were negative for bla KPC gene. All five A. baumannii isolates subjected to PCR were found to contain bla VIM gene. Two out of four P. aeruginosa isolates were positive for bla IMP , one was positive for bla VIM gene. One P. aeruginosa isolate was positive for both bla IMP and bla VIM gene. Conclusions: In view of the increasing resistance of Gram-negative bacilli to carbapenems, rational use of antibiotics needs to be emphasised.

Genomic and Phenotypic Analysis of bla KPC-2 Associated Carbapenem Resistance in Klebsiella aerogenes: Insights into Clonal Spread and Resistance Mechanisms Across Hospital Departments in Beijing.

This study conducted an phenotypic and whole-genome sequencing analysis with Klebsiella aerogenes to elucidate its clinical epidemiological characteristics, antimicrobial resistance (AMR) phenotype, biofilm formation ability and hemolytic activity testing, AMR genes and phylogenetic relationships, so as to provide a further understanding of the intra-hospital strain transmission. Samples were collected from a hospital in Beijing between 2020 and 2022. All strains underwent bacterial identification, antimicrobial susceptibility testing (AST) using the VITEK-2 compact system. Biofilm formation ability and hemolytic activity were tested. Second-generation sequencing was applied to all strains, with those carrying the bla KPC gene were selected for third-generation sequencing. Whole-genome analysis identified resistance genes, plasmid types, MLST typing, and phylogenetic relationships. Plasmids were assembled to detect plasmid structures and AMR gene location. Among the 42 K. aerogenes isolates, 21 were carbapenem-resistant K. aerogenes (CRKA). All strains exhibited strong biofilm formation and no hemolytic activity. Most were sourced from sputum (83.3%). CRKA demonstrated extensive resistance to antibiotics, particularly β-lactamase inhibitors and Cefotetan. This resistance pattern was closely associated with the presence of an IncFII(pHN7A8) plasmid, which carried multiple resistance genes, including bla KPC-2, bla CTX-M-65, bla TEM-1, rmtB and a large number of mobile elements. The majority of CRKA strains clustered within the same branch of the phylogenetic tree, exhibiting minimal single nucleotide polymorphism (0-13 SNPs) differences, and they shared the same sequence type (ST292), resistance genes, and plasmids, originating from different departments, suggesting clonal transmission among the hospital. Our research reveals that the clonal transmission of CRKA occurs across various departments within the hospital. The widespread resistance observed in CRKA, attributed to the presence of bla KPC and ESBLs genes, underscores the need for heightened vigilance to prevent the further dissemination of CRKA within the hospital and, potentially, throughout the wider community.

Whole Genome Sequencing to Identify Multiple Clusters of Carbapenemase-Producing Enterobacterales Cases – Colorado, 2022-2023

Background: The Colorado Department of Public Health and Environment (CDPHE) detected an increase in Klebsiella pneumoniae carbapenemase-producing carbapenem-resistant Enterobacterales (KPC-CRE) infections in October 2022. We investigated patient epidemiological links and isolate relatedness to characterize interfacility transmission of KPC-CRE in the Denver metro area and inform regional prevention strategies. Methods: We defined a case as polymerase chain reaction detection of KPC from clinical or screening specimens collected during January 2022 – January 2023. Cases were identified through statewide CRE surveillance and carbapenemase testing at the CDPHE laboratory and counted once within a 30-day period. Medical records were reviewed to identify healthcare facility admissions and patient facility overlap in the 12 months prior to sample collection. Whole genome sequencing (WGS) was performed for 34 patients with available KPC-CRE isolates using short- and long-read sequencing techniques. We performed multi-locus sequence typing, generated genome phylogenetic trees, and compared plasmid contig sequences to identify relatedness between KPC-CRE isolates. Clusters were defined as ≥2 genetically related isolates of the same organism or carbapenemase plasmid, from different patients. Results: We identified 48 cases (34 clinical and 14 screening) among 39 patients (figure). Patients had a mean age of 52 years (range 16-86) and median of three healthcare facility admissions (range 1-14). Twenty-eight patients (72%) were male. We identified 16 (41%) patients with epidemiological links to one acute care hospital (ACH), 11 (28.2%) patients to one long-term acute care hospital (LTACH), and four (10.2%) patients to each of two ventilator-capable skilled nursing facilities (vSNF). Five distinct clusters of KPC-CRE were identified by WGS among 23 patients (E. hormaechei, two distinct E. cloacae clusters, K. pneumoniae, and K. oxytoca) with linkages to ten healthcare facilities, including two vSNFs, two LTACHs, and six ACHs. Three distinct KPC genes were identified among the clusters: KPC-2, KPC-3, and KPC-4. Genomes assembled from long reads identified identical or similar KPC-gene-containing plasmids across different species or sequence types, suggesting horizontal gene transfer of KPC. Conclusions: Multiple KPC-CRE strains co-circulated and were associated with patient movement between acute and post-acute care settings. WGS allowed us to identify multi-facility clusters. Time and location of carbapenemase acquisition were challenging to determine for genetically related isolates when epidemiologic links could not be determined from medical records. This could be due to undetected cases. We notified healthcare facilities of their shared transmission risk and advocated for improved attention to infection control, carbapenemase screening, and communication upon patient transfer.

Molecular study of Klebsiella pneumoniae antibiotic resistance genes isolates from wound infections

K. pneumoniae, which produces Carbapenemases, is of public health importance because of its resistance to antimicrobials. K. Pneumoniae is identified phenotypically by its large pink mucus appearance on MacConkey agar because of lactose fermentation and by bacteriological, microscopic, and biochemical tests using the VITK 2 device. Antimicrobial susceptibility testing was performed by Kirby-Bauer disc diffusion. Klebsiella pneumoniae represented 5.3% (n = 16). It is highly resistant to antibiotics used to treat wound infections and shows the highest rate of resistance with cefepime 11(68.8%), followed by Ertapenem 9(56.3%), Doripenem and Imipenem 7(43.8%), and Meropenem 6(37.5%). Carbapenemases resistance was identified by phenotype through the Hodge test and also detection of Carbapenemases genes by polymerase chain reaction. The results showed the presence of the bla SHV 16 gene (100%) in K. pneumoniae isolates. The results showed that there were 4 isolates (25%) positive for the bla AMPC gene. The results also showed the presence of the bla KPC gene in two isolates (12.5%). The bla GES gene was not found in K. pneumoniae isolates. In general, the isolates were resistant to carbapenem and cefepime antibiotics. Five random samples were examined by publishing the results of the sequence analysis by BLAST in (NCBI) and the Mega 7 polymorphism detection program: it was found that there was no difference between the sequences, but rather the match was with the bacterium Klebsiella pneumoniae.

Inhibitory Potential of Bifidobacterium longum FB1-1 Cell-Free Supernatant against Carbapenem-Resistant Klebsiella pneumoniae Drug Resistance Spread.

The widespread dissemination of carbapenem-resistant Klebsiella pneumoniae (CRKP) and its drug resistance transfer poses a global public health threat. While previous studies outlined CRKP's drug resistance mechanism, there is limited research on strategies inhibiting CRKP drug resistance spread. This study investigates the potential of Bifidobacterium longum (B. longum) FB1-1, a probiotic, in curbing the spread of drug resistance among CRKP by evaluating its cell-free supernatant (CFS) for antibacterial activity. Evaluating the inhibitory effect of FB1-1 CFS on CRKP drug resistance spread involved analyzing its impact on drug resistance and virulence gene expression; drug resistance plasmid transfer FB1-1 CFS exhibited an MIC range of 125 μL/mL against CRKP. After eight hours of co-culture, CFS achieved a 96% and 100% sterilization rate at two and four times the MIC, respectively. At sub-inhibitory concentrations (1/2× MIC), FB1-1 CFS reduced the expression of the bla_KPC gene, which is pivotal for carbapenem resistance, by up to 62.13% across different CRKP strains. Additionally, it markedly suppressed the expression of the uge gene, a key virulence factor, by up to 91%, and the fim_H gene, essential for bacterial adhesion, by up to 53.4%. Our study primarily focuses on determining the inhibitory effect of FB1-1 CFS on CRKP strains harboring the bla_KPC gene, which is a critical resistance determinant in CRKP. Furthermore, FB1-1 CFS demonstrated the ability to inhibit the transfer of drug resistance plasmids among CRKP strains, thus limiting the horizontal spread of resistance genes. This study highlights FB1-1 CFS's inhibitory effect on CRKP drug resistance spread, particularly in strains carrying the bla_KPC gene, thus offering a novel idea and theoretical foundation for developing antibacterial drugs targeting CRKP resistance.

Molecular mechanisms responsible KPC-135-mediated resistance to ceftazidime-avibactam in ST11-K47 hypervirulent Klebsiella pneumoniae

ABSTRACT Ceftazidime-avibactam resistance attributable to the bla KPC-2 gene mutation is increasingly documented in clinical settings. In this study, we characterized the mechanisms leading to the development of ceftazidime-avibactam resistance in ST11-K47 hypervirulent Klebsiella pneumoniae that harboured the bla KPC-135 gene. This strain possessed fimbriae and biofilm, demonstrating pathogenicity. Compared with the wild-type KPC-2 carbapenemase, the novel KPC-135 enzyme exhibited a deletion of Glu168 and Leu169 and a 15-amino acid tandem repeat between Val262 and Ala276. The bla KPC-135 gene was located within the Tn6296 transposon truncated by IS26 and carried on an IncFII/IncR-type plasmid. Compared to the bla KPC-2-positive cloned strain, only the MIC of ceftazidime increased against bla KPC-135-positive K. pneumoniae and wasn’t inhibited by avibactam (MIC 32 μg/mL), while clavulanic acid and vaborbactam demonstrated some inhibition. Kinetic parameters revealed that KPC-135 exhibited a lower Km and kcat/Km with ceftazidime and carbapenems, and a higher (∼26-fold) 50% inhibitory concentration with avibactam compared to KPC-2. The KPC-135 enzyme exerted a detrimental effect on fitness relative to the wild-type strain. Furthermore, this strain possessed hypervirulent determinants, which included the IncHI1B/FIB plasmid with rmpA2 and expression of type 1 and 3 fimbriae. In conclusion, we reported a novel KPC variant, KPC-135, in a clinical ST11-K47 hypervirulent K. pneumoniae strain, which conferred ceftazidime-avibactam resistance, possibly through increased ceftazidime affinity and decreased avibactam susceptibility. This strain simultaneously harboured resistance and virulence genes, posing an elevated challenge in clinical treatment.

Performance comparison of BD Phoenix CPO detect panel with Cepheid Xpert Carba-R assay for the detection of carbapenemase-producing Klebsiella pneumoniae isolates

BackgroundWe aimed to compare the performance of carbapenemase classification in carbapenem-resistant Klebsiella pneumoniae (CRKP) obtained using the BD Phoenix CPO Detect panel (CPO panel) and Cepheid Xpert Carba-R assays. We analyzed 55 CRKP strains from clinical specimens collected between November 2020 and November 2022. The CPO panel was used to detect both antibiotic susceptibility and phenotypic carbapenemase classes, while Xpert Carba-R was employed to identify KPC, NDM, VIM, OXA-48, and IMP genes. Due to the limited availability of molecular kits, we arbitrarily selected 55 isolates, identified as carbapenemase-producing according to the CPO panel and with meropenem minimum inhibitory concentration values > 8 mg/L.ResultsAccording to the Xpert Carba-R assay, 16 of the 55 isolates (29.1%) were categorised as Ambler Class A (11 of which matched CPO panel Class A identification); three isolates (5.5%) were identified as Class B and 27 isolates (49.1%) as Class D (in both cases consistent with CPO panel B and D classifications). A further eight isolates (14.5%) exhibited multiple carbapenemase enzymes and were designated as dual-carbapenemase producers, while one isolate (1.8%) was identified as a non-carbapenemase-producer. The CPO panel demonstrated positive and negative percent agreements of 100% and 85.7% for Ambler Class A, 100% and 100% for Class B, and 96.4% and 100% for Class D carbapenemase detection, respectively.ConclusionWhile the CPO panel’s phenotypic performance was satisfactory in detecting Class B and D carbapenemases, additional confirmatory testing may be necessary for Class A carbapenemases as part of routine laboratory procedures.

Carbapenemases Produced by Multidrug-Resistant Strains of <i>Klebsiella Pneumoniae</i> Isolated from Intensive Care Patients

Relevance Klebsiella pneumoniae is one of the main pathogens of nosocomial infections. Hospital strains of this pathogen are characterized by a high frequency of resistance to many antibiotics, including carbapenems. The main mechanism for the formation of resistance to carbapenems is the production of carbapenemases by bacteria. To date, K. pneumoniae is considered one of the main “distributors” of clinically important antibiotic resistance genes.Aim of the study To study the frequency of occurrence of the most common carbapenemase genes in multiresistant K.pneumoniae strains isolated from patients of intensive care units in an emergency hospital.Material and methods 4708 samples of various types of clinical material from patients of 5 intensive care units of the N.V. Sklifosovsky Research Institute for Emergency Medicine were analyzed. Microbiological studies were carried out using standard generally accepted methods. For the purposes of this study, unique sequential K.pneumoniae strains resistant to imipenem and/or meropenem were selected. DNA isolation was carried out using the RIBO-prep kit (Russia). Carbapenemase genes were detected by real-time PCR using the kits of reagents “AmpliSens MDR-MBL-FL” and “AmpliSens MDR-KPC/OXA-48-FL” on a “Rotor Gene” device (Corbett Research, Australia).Results Etiologically significant microorganisms were detected in 64.7% of the studied samples. K. pneumoniae was isolated in a quarter of the samples. 194 unique carbapenem-resistant strains of K.pneumoniae were selected. Of these, 11.3% of the genes of the studied carbapenemases were not detected. In 38.1% of strains, 1 carbapenemase was detected, in 29.9% — two and in 20.6% — three or more. Among the strains with one carbapenemase gene, OXA-48 (19.1%) and CATTLE (13.4%) producers prevailed. Strains producing only NDM betalactamase were found in 5.7% of cases. Isolated allocation of VIM and IMP was not detected. In 34%, metallobectalamases were isolated in combination with serine carbapenemases. The production of serine carbapenemases alone was detected in 48.5% of the strains. Depending on the specialization of the intensive care unit, there are differences in the frequency of detection of serine and metallobetalactamases in strains of carbapenem-resistant Klebsiella.Conclusion K. pneumoniae is the causative agent of nosocomial infections in 25% of cases. In 11.3% of carbapenem-resistant strains, the production of KPC, OXA-48, NDM, VIM and IMP genes was not detected. When developing algorithms for antibacterial therapy, it is necessary to take into account that from 25.7% to 60.6% of K. pneumoniae strains in different intensive care units are the producers of metallobetalactamases.

High Prevalence of Carbapenem-Resistant Enterobacterales Producing OXA-48 among Carbapenem-Resistant Isolates in a Regional Hospital in Central Taiwan.

In response to the increasing number of carbapenem-resistant Enterobacterales (CRE), we investigated carbapenemase-producing Klebsiella pneumoniae and non-K. pneumoniae epidemiology and genetics. We collected 76 clinical Enterobacterales and 4 stool surveillance Escherichia coli isolates resistant to ertapenem or imipenem. Using polymerase chain reaction (PCR) and DNA sequencing, we assessed carbapenemases, extended-spectrum β-lactamases, and AmpC β-lactamases. Molecular typing via pulsed-field gel electrophoresis (PFGE) and conjugation experiments were conducted to examine resistance gene transfer. Among the 80 isolates, 96.2% harbored at least one carbapenemase gene, with blaOXA-48 in 87.5%. KPC-2 and IMP-8 carbapenemases were found in 15.0 and 22.5% of the isolates, respectively, with 27.5% having 2 or more carbapenemase genes. The PFGE analysis revealed the presence of diverse genotypes. PCR-based plasmid replicon typing identified IncA/C as the most prevalent type among K. pneumoniae isolates (26/29), and IncF and IncFIB among E. coli isolates (22/28). Conjugal transfer was successful for plasmids encoding OXA-48, CTX-M-3, CTX-M-14, CMY-2, and other β-lactamases, except the KPC-2 gene. In conclusion, our study highlights high carbapenemase prevalence in CRE, primarily OXA-48. Multiple carbapenemases within strains were common, and PFGE showed diverse patterns in these carbapenem-resistant isolates.

Evaluation of Carbapenemase-Resistant Enterobacteriaceae by Disk Diffusion Test and Polymerase Chain Reaction

Background: Carbapenemase-resistant Enterobacteriaceae is a major threat to public health. These microorganisms are resistant to all types of beta-lactam antibiotics. Methods: This cross-sectional study was conducted on 51 Enterobacteriaceae isolates from clinical samples in Sina Hospital and Tehran Heart Center in Iran from 2016 to 2018. Antibiotic susceptibility test was performed by disk diffusion method. Carbapenem-resistant isolates were identified by the Modified Hodge Test (MHT) and Polymerase Chain Reaction (PCR) for surveying the presence of VIM, NDM, IMMP, and OXA-48 genes. Results: Out of 51 clinical samples, 38 isolates were positive for both MHT and PCR tests, and 5 isolates were negative in both tests. The results of both tests are similar in 84.3% of the isolates. Conclusion: The MHT is an appropriate and easy method for approving carbapenemase production. Also, a laboratory can detect the carbapenemase production by identification of the KPC genes.

Genomic Characteristics of a Carbapenem-Resistant Klebsiella pneumoniae Co-Carrying bla NDM-5 and bla KPC-2 Capsular Type KL25 Recovered from a County Level Hospital in China.

Hypervirulent carbapenem-resistant K. pneumoniae (hv-CRKP) has been spreading rapidly worldwide. Here, we investigated the genomic characteristics of ST11 K. pneumoniae isolate SM117 with capsular serotype KL25, co-carrying bla NDM-5, two copies of bla KPC-2 and multiple plasmid-borne virulence genes from a county level hospital in China. Antimicrobial susceptibility of K. pneumoniae SM117 was evaluated. The Illumina NovaSeq 6000 and Oxford Nanopore MinION platforms were applied to sequence the genome and then de novo assembled. The genome sequence was annotated using the NCBI Prokaryotic Genome Annotation Pipeline and further subjected to identify the sequence type (ST), capsular type, antibiotic resistance genes, plasmid replicon types and virulence genes. The phylogenetic analysis was performed based on the core genome single nucleotide polymorphisms (cgSNPs) using CSI Phylogeny 1.4, and further visualized by Interactive Tree of Life (iTOL) V5 web server. The whole-genome sequence of K. pneumoniae SM117 is made up of eight contigs totaling 6,104,486 bp, contain a 5,612,620 bp single chromosome and seven plasmids. The isolate was assigned to ST11 with capsular serotype KL25, co-carrying including bla NDM-5, bla KPC-2 and multiple plasmid-borne virulence genes including rmpA2 and aerobactin genes iucABCD-iutA. The coexistence of bla KPC and bla NDM in K. pneumoniae strains exhibit a high degree of resistance to β-lactam antibiotics. The strain SM117 also carries multiple antibiotic resistance genes, making it resistant to all antibiotics except polymyxin. The closest relative of K. pneumoniae C793 was identified in 2023 from a hospital surface sample in Zhejiang, China, with just 52 SNPs difference. This study reported the genomic characteristics of a multidrug-resistant ST11 K. pneumoniae with capsular serotype KL25, co-carrying bla NDM-5, two copies of bla KPC-2 genes and multiple plasmid-borne virulence genes in China. These findings will provide important knowledge of the antibiotic resistance mechanisms, genomic epidemiological characteristics and transmission dynamics of multidrug-resistant K. pneumoniae.