Association Between Biofilm Formation and Extended-Spectrum Beta-Lactamase Production in Klebsiella pneumoniae Isolated from Fresh Fruits and Vegetables.

The presence of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae in fresh fruits and vegetables is a growing public health concern. The primary objective of this study was to investigate the relationship between biofilm formation and extended-spectrum ß-lactamase (ESBL) production in K. pneumoniae strains obtained from fresh fruits and vegetables. Out of 120 samples analysed, 94 samples (78%) were found to be positive for K. pneumoniae. Among the K. pneumoniae strains isolated, 74.5% were from vegetables, whereas the remaining (25.5%) were from fresh fruits. K. pneumoniae isolates were resistant to at least three different classes of antibiotics, with ceftazidime (90%) and cefotaxime (70%) showing the highest resistance rates. While the high occurrence of ESBL-producing and biofilm-forming K. pneumoniae strains were detected in vegetables (73.5% and 73.7%, respectively), considerable amounts of the same were also found in fresh fruits (26.5% and 26.3%, respectively). The results further showed a statistically significant (P < 0.001) association between biofilm formation and ESBL production in K. pneumoniae strains isolated from fresh fruits and vegetables. Furthermore, the majority (81%) of the ESBL-producing strains harbored the blaCTX-M gene, while a smaller proportion of strains carried the blaTEM gene (30%), blaSHV gene (11%) or blaOXA (8%). This study highlights the potential public health threat posed by K. pneumoniae in fresh fruits and vegetables and emphasizes the need for strict surveillance and control measures.

The detection of KPC-2, NDM-1, and VIM-2 carbapenemases in international clones isolated from fresh vegetables highlights an emerging food safety issue.

Resistance to carbapenems emerged in clinical settings and has rapidly spread to other sectors, such as food and the environment, representing a One Health problem. In this regard, vegetables contaminated by critical priority pathogens have raised global concerns. Here, we have performed a whole-genome sequence-based analysis of extensively drug-resistant Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa strains isolated from cabbage, spinach, and lettuce, respectively. Genomic analysis revealed the emergence of international and high-risk clones belonging to ST340, ST155, and ST233, harboring a broad resistome to clinically important antimicrobials. In this context, K. pneumoniae, E. coli, and P. aeruginosa strains carried blaKPC-2, blaNDM-1, and blaVIM-2, respectively. The blaKPC-2 gene with a non-Tn4401 element (NTEKPC-Ic) was located on an IncX3-IncU plasmid, while the blaVIM-2 gene was associated with a Tn402-like class 1 integron, In559, on the chromosome. Curiously, the blaNDM-1 gene coexisted with the blaPER-2 gene on an IncC plasmid and the regions harboring both genes contained sequences of Tn3-like element ISKox2-like family transposase. Comparative genomic analysis showed interspecies and clonal transmission of carbapenemase-encoding genes at the human-animal-environmental interface. These findings raise a food safety alert about hospital-associated carbapenemase producers, supporting that fresh vegetables can act as a vehicle for the spread of high-risk clones.

Analysis of a new phage, KZag1, infecting biofilm of Klebsiella pneumoniae: genome sequence and characterization.

BACKGROUND: This study investigates the effectiveness of the bacteriophage KZag1 against drug-resistant Klebsiella pneumoniae, aiming to assess its potential as a therapeutic agent. The novelty lies in the characterization of KZag1, a Myovirus with specific efficacy against multidrug-resistant K. pneumoniae strains. This highlights the significance of exploring alternative strategies, particularly phage therapy, in addressing biofilm-associated infections. METHODS: KZag1, characterized by a typical Myovirus structure with a 75 ± 5 nm diameter icosahedral head and a 15 ± 5 nm short tail, was evaluated in experimental trials against 15 strains of K. pneumoniae. The infection cycle duration was determined to be 50 min, resulting in an estimated burst size of approximately 83 plaque-forming units per colony-forming unit (PFU/CFU). Stability assessments were conducted within a pH range of 4 to 12 and temperatures ranging from 45°C to 60°C. Biofilm biomass reduction was observed, particularly at a multiplicity of infection (MOI) of 10. RESULTS: KZag1 demonstrated infection efficacy against 12 out of 15 tested K. pneumoniae strains. The phage exhibited stability across a broad pH range and at elevated temperatures. Notably, treatment with KZag1 significantly reduced K. pneumoniae biofilm biomass, emphasizing its potential in combating biofilm formation. Genomic analysis revealed a complete genome of 157,089 base pairs with a GC content of 46.38%, encompassing 203 open reading frames (ORFs) and a cysteine-specific tRNA sequence. Comparison with phage GP4 highlighted similarities, with KZag1 having a longer genome by approximately 4829 base pairs and a higher GC content by approximately 0.93%. Phylogenetic analysis classified KZag1 within the Myoviridae family. CONCLUSION: The efficacy of KZag1 against K. pneumoniae biofilm suggests its potential as a therapeutic candidate, especially for drug-resistant infections. Further clinical research is warranted to explore its synergy with other treatments, elucidate genomic traits, compare with Myoviridae phages, and understand its host interactions. These findings underscore the promising role of KZag1 in addressing drug-resistant bacterial infections.

The Rising Tide of Antibiotic Resistance: A Study on Extended-Spectrum Beta-Lactamase and Carbapenem-Resistant Escherichia coli and Klebsiella pneumoniae.

BACKGROUND: The global spread of extended-spectrum beta-lactamase (ESBL)-producing and carbapenem-resistant Enterobacterales (CRE) poses a significant concern. Acquisition of antimicrobial resistance genes leads to resistance against several antibiotics, limiting treatment options. We aimed to study ESBL-producing and CRE transmission in clinical settings. METHODS: From clinical samples, 227 ESBL-producing and CRE isolates were obtained. The isolates were cultured on bacterial media and confirmed by VITEK 2. Antibiograms were tested against several antibiotics using VITEK 2. The acquired resistance genes were identified by PCR. RESULTS: Of the 227 clinical isolates, 145 (63.8%) were Klebsiella pneumoniae and 82 (36.1%) were Escherichia coli; 76 (33.4%) isolates were detected in urine, 57 (25.1%) in pus swabs, and 53 (23.3%) in blood samples. A total of 58 (70.7%) ESBL-producing E. coli were resistant to beta-lactams, except for carbapenems, and 17.2% were amikacin-resistant; 29.2% of E. coli isolates were resistant to carbapenems. A total of 106 (73.1%) ESBL-producing K. pneumoniae were resistant to all beta-lactams, except for carbapenems, and 66.9% to ciprofloxacin; 38 (26.2%) K. pneumoniae were resistant to carbapenems. Colistin emerged as the most effective antibiotic against both bacterial types. Twelve (20.6%) E. coli isolates were positive for blaCTX-M, 11 (18.9%) for blaTEM, and 8 (33.3%) for blaNDM. Forty-six (52.3%) K. pneumoniae isolates had blaCTX-M, 27 (18.6%) blaTEM, and 26 (68.4%) blaNDM. CONCLUSION: This study found a high prevalence of drug-resistant ESBL-producing and CRE, highlighting the need for targeted antibiotic use to combat resistance.

Distribution and molecular characterization of carbapenemase-producing gram-negative bacteria in Henan, China.

This study aimed to investigate the epidemiological characteristics and trends over time of carbapenemase-producing (e.g., KPC, NDM, VIM, IMP, and OXA-48) Gram-negative bacteria (CPGNB). Non-duplicated multi-drug resistant Gram-negative bacteria (MDRGNB) were collected from the First Affiliated Hospital of Zhengzhou University from April 2019 to February 2023. Species identification of each isolate was performed using the Vitek2 system and confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry according to the manufacturer's instructions. PCR detected carbapenem resistance genes in the strains, strains carrying carbapenem resistance genes were categorized as CPGNB strains after validation by carbapenem inactivation assay. A total of 5705 non-repetitive MDRGNB isolates belonging to 78 different species were collected during the study period, of which 1918 CPGNB were validated, with the respiratory tract being the primary source of specimens. Epidemiologic statistics showed a significant predominance of ICU-sourced strains compared to other departments. Klebsiella pneumoniae, Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa were the significant CPGNB in Henan, and KPC and NDM were the predominant carbapenemases. Carbapenem-resistant infections in Henan Province showed an overall increasing trend, and the carriage of carbapenemase genes by CPGNB has become increasingly prevalent and complicated. The growing prevalence of CPGNB in the post-pandemic era poses a significant challenge to public safety.

Investigation of genotyping and phenotyping characteristics of carbapenem-resistant Klebsiella pneumoniae isolates.

Klebsiella pneumoniae (K. pneumoniae) is a major cause of healthcare-associated infections and plays a prominent role in the widespread antibiotic resistance crisis. Accurate identification of carbapenemases is essential to facilitate effective antibiotic treatment and reduce transmission of K. pneumoniae. This study aimed to detect carbapenemase production in carbapenem-resistant K. pneumoniae strains using phenotypic and genotypic methods. A total of 67 carbapenem-resistant K. pneumoniae strains obtained from various clinical samples were utilized for identification and antimicrobial susceptibility by the Vitek 2 Compact system (Biomerieux, France). Carbapenemase production was determined by using the Polymerase chain reaction, Blue-carba test (BCT) and Carbapenem inactivation method (CIM). Out of the isolates, 59 (88.1%) were positive bla OXA-48, 16 (23.9%) bla IMP, and five (7.5%) were positive bla NDM. No bla KPC genes were detected. The CIM identified 62 (92.5%), BCT identified 63 (94%) of PCR-positive isolates. The sensitivity and specificity of the BCT and the CIM were determined to be 96.7%, 40%, and 96.7%, 25% respectively. The bla OXA-48 gene was found to be the most prevalent in K. pneumoniae isolates. Early identification of carbapenem resistance plays a vital role in designing effective infection control strategies and mitigating the emergence and transmission of carbapenem resistance, thus reducing healthcare-associated infections.

Molecular Prevalence and Geographical Variations of Carbapenem-Resistant Klebsiella pneumoniae ST15 Isolates in a Tertiary Hospital in Ningbo, China.

BACKGROUND In China, the most prevalent type of CRKP is ST11, but the high-risk clone ST15 has grown in popularity in recent years, posing a serious public health risk. Therefore, we investigated the molecular prevalence characteristics of ST15 CRKP detected in a tertiary hospital in Ningbo to understand the current potential regional risk of ST15 CRKP outbreak. MATERIAL AND METHODS We collected and evaluated 18 non-duplicated CRKP strains of ST15 type for antibiotic resistance. Their integrons, virulence genes, and resistance genes were identified using polymerase chain reaction (PCR), and their homology was determined using MALDI-TOF MS. RESULTS The predominant serotype of 18 ST15 CRKP strains was K5. ST15 CRKP exhibited the lowest antimicrobial resistance to Cefoperazone/sulbactam (11.1%), followed by trimethoprim/sulfamethoxazole (22.2%). Resistance gene testing revealed that 14 out of 18 ST15 CRKP strains (77.8%) carried Klebsiella pneumoniae carbapenemase 2 (KPC-2), whereas all ST15 CRKP integrons were of the intI1 type. Furthermore, virulence gene testing revealed that all 18 ST15 CRKP strains carried ybtS, kfu, irp-1, and fyuA genes, followed by the irp-2 gene (17 strains) and entB (16 strains). The homology analysis report showed that 2 clusters had closer affinity, which was mainly concentrated in classes C and D. CONCLUSIONS The ST15 CRKP antibiotic resistance rates demonstrate clear geographical differences in Ningbo. Additionally, some strains carried highly virulent genes, indicating a possible evolution towards carbapenem-resistant highly virulent strains. To reduce the spread of ST15 CRKP, we must rationalize the clinical use of antibiotics and strengthen resistance monitoring to control nosocomial infections.

Genome mining of Lactiplantibacillus plantarum PA21: insights into its antimicrobial potential.

BACKGROUND: The dramatic increase of antimicrobial resistance in the healthcare realm has become inexorably linked to the abuse of antibiotics over the years. Therefore, this study seeks to identify potential postbiotic metabolites derived from lactic acid bacteria such as Lactiplantibacillus plantarum that could exhibit antimicrobial properties against multi-drug resistant pathogens. RESULTS: In the present work, the genome sequence of Lactiplantibacillus plantarum PA21 consisting of three contigs was assembled to a size of 3,218,706 bp. Phylogenomic analysis and average nucleotide identity (ANI) revealed L. plantarum PA21 is closely related to genomes isolated from diverse niches such as dairy products, food, and animals. Genome mining through the BAGEL4 and antiSMASH database revealed four bacteriocins in a single cluster and four regions of biosynthetic gene clusters responsible for the production of bioactive compounds. The potential probiotic genes indirectly responsible for postbiotic metabolites production were also identified. Additionally, in vitro studies showed that the L. plantarum PA21 cell-free supernatant exhibited antimicrobial activity against all nine methicillin-resistant Staphylococcus aureus (MRSA) and three out of 13 Klebsiella pneumoniae clinical isolates tested. CONCLUSION: Results in this study demonstrates that L. plantarum PA21 postbiotic metabolites is a prolific source of antimicrobials against multi-drug resistant pathogens with potential antimicrobial properties.

AcrAB-TolC efflux pump overexpression and tet(A) gene mutation increase tigecycline resistance in Klebsiella pneumoniae.

Tigecycline-non-susceptible Klebsiella pneumoniae (TNSKP) is increasing and has emerged as a global public health issue. However, the mechanism of tigecycline resistance remains unclear. The objective of this study was to investigate the potential role of efflux pump system in tigecycline resistance. 29 tigecycline-non-susceptible Klebsiella pneumoniae (TNSKP) strains were collected and their minimum inhibitory concentrations (MIC) were determined by the broth microdilution method. The ramR, acrR, rpsJ, tet(A), and tet(X) were amplified by polymerase chain reaction (PCR). The mRNA expression of different efflux pump genes and regulator genes were analyzed by real-time PCR. Additionally, KP14 was selected for genome sequencing. KP14 genes without acrB, oqxB, and TetA were modified using suicide plasmids and MIC of tigecycline of KP14 with target genes knocked out was investigated. It was found that MIC of tigecycline of 20 out of the 29 TNSKP strains decreased by over four folds once combined with phenyl-arginine-ß-naphthylamide dihydrochloride (PaßN). Most strains exhibited upregulation of AcrAB and oqxAB efflux pumps. The strains with acrB, oqxB, and tetA genes knocked out were constructed, wherein the MIC of tigecycline of KP14∆acrB and KP14∆tetA was observed to be 2 µg/mL (decreased by 16 folds), the MIC of tigecycline of KP14ΔacrBΔTetA was 0.25 µg/mL (decreased by 128 folds), but the MIC of tigecycline of KP14∆oqxB remained unchanged at 32 µg/mL. The majority of TNSKP strains demonstrated increased expression of AcrAB-TolC and oqxAB, while certain strains showed mutations in other genes associated with tigecycline resistance. In KP14, both overexpression of AcrAB-TolC and tet(A) gene mutation contributed to the mechanism of tigecycline resistance.

Prevalence of the CRISPR-cas system and its association with antibiotic resistance in clinical Klebsiella pneumoniae isolates.

BACKGROUND AND OBJECTIVE(S): CRISPR-Cas is a prokaryotic adaptive immune system that protects bacteria and archaea against mobile genetic elements (MGEs) such as bacteriophages plasmids, and transposons. In this study, we aimed to assess the prevalence of the CRISPR-Cas systems and their association with antibiotic resistance in one of the most challenging bacterial pathogens, Klebsiella pneumoniae. MATERIALS AND METHODS: A total of 105 K. pneumoniae isolates were collected from various clinical infections. Extended-spectrum ß-lactamases (ESBLs) phenotypically were detected and the presence of ESBL, aminoglycoside-modifying enzymes (AME), and CRISPR-Cas system subtype genes were identified using PCR. Moreover, the diversity of the isolates was determined by enterobacterial repetitive intergenic consensus (ERIC)-PCR. RESULTS: Phenotypically, 41.9% (44/105) of the isolates were found to be ESBL producers. A significant inverse correlation existed between the subtype I-E CRISPR-Cas system's presence and ESBL production in K. pneumoniae isolates. Additionally, the frequency of the ESBL genes blaCTX-M1 (3%), blaCTX-M9 (12.1%), blaSHV (51.5%), and blaTEM (33.3%), as well as some AME genes such as aac(3)-Iva (21.2%) and ant(2'')-Ia (3%) was significantly lower in the isolates with the subtype I-E CRISPR-Cas system in comparison to CRISPR-negative isolates. There was a significant inverse correlation between the presence of ESBL and some AME genes with subtype I-E CRISPR-Cas system. CONCLUSION: The presence of the subtype I-E CRISPR-Cas system was correlated with the antibiotic-resistant gene (ARGs). The isolates with subtype I-E CRISPR-Cas system had a lower frequency of ESBL genes and some AME genes than CRISPR-negative isolates.

Transcriptomic responses of extensively drug resistant Klebsiella pneumoniae to N-acetyl cysteine reveals suppression of major biogenesis pathways leading to bacterial killing and biofilm eradication.

AIMS: Carbapenemase-producing Klebsiella pneumoniae is categorized as a "critical global priority-one" pathogen by WHO and new and efficient treatment options are warranted. This study aims to assess the antibacterial and antibiofilm potential of N-acetyl cysteine (NAC), against clinical isolates of extensively drug resistant (XDR) K. pneumoniae and elucidate the mechanism of killing. METHODS AND RESULTS: XDR-K. pneumoniae were isolated from patients admitted to Madras Medical Mission Hospital, India. Antibiofilm activity of NAC was checked using in vitro continuous flow model and RNA sequencing was done using Illumina Novoseq. Data quality was checked using FastQC and MultiQC software. Our findings revealed that NAC at a concentration of 100 mg/ml was safe, and could inhibit the growth and completely eradicate mature biofilms of all XDR-K. pneumoniae isolates. Transcriptomic responses in XDR-K. pneumoniae to NAC showed significant downregulation of the genes associated with crucial biogenesis pathways, including electron transport chain and oxidoreductase activity besides a specific cluster of genes linked to ribosomal proteins. CONCLUSIONS: Our results indicate that NAC kills the XDR- K. pneumoniae clinical isolates by shutting the overall metabolism and, hence, successfully eradicate in vitro biofilms formed on catheters.

Whole genome sequencing analysis of seven unknown resistance mechanisms of carbapenem-resistant Klebsiella pneumoniae strains resistance to ceftazidime-avibactam.

AIMS: To investigate alternative resistance mechanisms among seven ceftazidime-avibactam (CZA)-resistant carbapenem-resistant Klebsiella pneumoniae (CRKP) strains lacking common antimicrobial resistance genes (ARGs) using whole genome sequencing. METHODS AND RESULTS: ARG and virulence factors (VFs) were screened using the ARG database CARD and the VF database, respectively, and identified using genomic annotation data with BLAST+. Six strains were ST11 sequence types (STs), and one was ST2123. ST11 strains harbored more ARGs than the ST2123 strains. All seven strains carried multiple ARGs with efflux-mediated antibiotic resistance, including oqxA, oqxB, tet (A), qacEdltal, CRP, H-NS, Kpn-E, F, G, H, acrA, LptD, acrB, acrD, cpxA, mdtB, and mdtC. These efflux-mediated ARGs were identified in most strains and even all strains. Whole genome sequencing revealed that the ST11 strain carried multiple potential prophages, genomic islands, and integrative and conjugative elements, while the ST2123 strain carried an independent potential prophages and a genomic island. CONCLUSIONS: Whole genome sequencing analysis revealed that these seven CZA-resistant CRKP strains lacking common ARGs exhibited efflux-mediated antibiotic resistance-associated ARGs. The main mechanism by which CRKP resists CZA is antibiotic inactivation. Except for tet (A), no ARGs and validation experiments related to efflux were found. This study's results provide a new possibility for the resistance mechanism of CRKP to CZA, and we will verify this conclusion through experiments in the future.

Genomic and immunocyte characterisation of bloodstream infection caused by Klebsiella pneumoniae.

OBJECTIVES: The aim of this study was to evaluate the characteristics of immunocyte associated with bloodstream infection (BSI) caused by Klebsiella pneumoniae (Kpn). METHODS: Patients with BSI-Kpn were included from 2015 to 2022 in our hospital. Immunocyte subpopulations of enrolled BSI-Kpn patients were tested on the same day of blood culture using multicolor flow cytometry analysis. Antibiotic susceptibility test was determined by agar dilution or broth dilution method. All included isolates were subjected to whole genome sequencing and comparative genomics analysis. Clinical and genetic data were integrated to investigate the risk factors associated with clinical outcome. RESULTS: There were 173 patients with non-duplicate BSI-Kpn, including 81 carbapenem-resistant Kpn (CRKP), 30 extended-spectrum ß-lactamases producing Kpn (ESBL-Kpn), 62 none CRKP or ESBL-Kpn (S-Kpn). Among 68 ST11-CRKP isolates, ST11-O2v1:KL64 was the most common serotypes cluster (77.9%, 53/68), followed by ST11-OL101: KL47 (13.2%, 9/68). Compared with CSKP group, subpopulations of immunocyte in patients with CRKP were significantly lower (P < 0.01). In patients with ST11-O2v1:KL64 BSI-Kpn, the level of cytotoxic T lymphocytes (CD3 + CD8 +) is the highest, while the B lymphocytes (CD3-CD19 +) was the least. In addition, the level of immunocyte in patients with Kpn co-harbored clpV-ybtQ-qacE were lower than that in patients with Kpn harbored one of clpV, ybtQ or qacE and without these three genes. Furthermore, co-existence of clpV-ybtQ-qacE was independently associated with a higher risk for 30-day mortality. CONCLUSIONS: The results demonstrate that patients with BSI-CRKP, especially for ST11-O2v1:KL64, exhibit lower leukomonocyte counts. In addition, BSI-Kpn co-harbored clpV-ybtQ-qacE is correlated to higher 30-day mortality.

Prevalence of ST1049-KL5 carbapenem-resistant Klebsiella pneumoniae with a blaKPC-2 and blaNDM-1 co-carrying hypertransmissible IncM1 plasmid.

Infection caused by KPC and NDM carbapenemases co-producing Klebsiella pneumoniae (KPC_NDM_CRKP) poses serious public health concerns. Here, we elucidate the prevalence of a hypertransmissible lncM1 plasmid, pKPC_NDM, co-carrying blaKPC-2 and blaNDM-1 genes in sequence type 1049 K_locus 5 (ST1049-KL5) KPC_NDM_CRKP isolates. Genetic and clonal relatedness analyses using pulsed-field gel electrophoresis, single nucleotide polymorphism analysis and core genome multilocus sequence typing suggested clonal dissemination of ST1049-KL5 KPC_NDM_CRKP strains in our hospital. Whole genome sequencing identified an identical 76,517 bp- blaKPC-2 and blaNDM-1 genes co-carrying IncM1 plasmid pKPC_NDM and a pLVPK-like hypervirulent plasmid in all ST1049-KL5 KPC_NDM_CRKP isolates. pKPC_NDM shared 100% identity with a previously sequenced plasmid CRKP35_unnamed4, demonstrating high transferability in conjugation assay, with conjugation frequencies reaching 10-4 and 10-5 in Escherichia coli and K. pneumoniae recipients, respectively. It also maintained favorable stability and flexible compatibility, with retention rates exceeding 80% after 10 days of continuous passage, and could be compatible with pre-existing blaKPC- or blaNDM-carrying plasmids in recipient strains. This study summarizes the characteristics of KPC_NDM_CRKP outbreaks and highlights the importance of ongoing surveillance and infection control strategies to address the challenges posed by ST1049 K. pneumoniae strains.

Occurrence of carbapenem-resistant hypervirulent Klebsiella pneumoniae in oysters in Egypt: a significant public health issue.

BACKGROUND: The global dissemination of critical-priority carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKp) via food sources represents a significant public health concern. Epidemiological data on CR-hvKp in oysters in Egypt is limited. This study aimed to investigate the potential role of oysters sold in Egypt as a source for carbapenem-resistant K. pneumoniae (CRKP), hypervirulent K. pneumoniae (hvKp), and CR-hvKp and assess associated zoonotic risks. METHODS: A sample of 330 fresh oysters was randomly purchased from various retail fish markets in Egypt and divided into 33 pools. Bacteriological examination and the identification of Klebsiella pneumoniae were performed. Carbapenem resistance in K. pneumoniae isolates was determined by phenotypic and molecular methods. Additionally, the presence of hypervirulent K. pneumoniae was identified based on virulence gene markers (peg-344, rmpA, rmpA2, iucA, and iroB), followed by a string test. The clustering of CR-hvKp strains was carried out using R with the pheatmap package. RESULTS: The overall prevalence of K. pneumoniae was 48.5% (16 out of 33), with 13 isolates displaying carbapenem resistance, one intermediate resistance, and two sensitive. Both carbapenem-resistant K. pneumoniae and carbapenem-intermediate-resistant K. pneumoniae strains exhibited carbapenemase production, predominantly linked to the blaVIM gene (68.8%). HvKp strains were identified at a rate of 62.5% (10/16); notably, peg-344 was the most prevalent gene. Significantly, 10 of the 13 CRKP isolates possessed hypervirulence genes, contributing to the emergence of CR-hvKp. Moreover, cluster analysis revealed the clustering of two CR-hvKp isolates from the same retail fish market. CONCLUSION: This study provides the first insight into the emergence of CR-hvKp among oysters in Egypt. It underscores the potential role of oysters as a source for disseminating CR-hvKp within aquatic ecosystems, presenting a possible threat to public health.

International and regional spread of carbapenem-resistant Klebsiella pneumoniae in Europe.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) are of particular concern due to the spread of antibiotic resistance genes associated with mobile genetic elements. In this study, we collected 687 carbapenem-resistant strains recovered among clinical samples from 41 hospitals in nine Southern European countries (2016-2018). We identified 11 major clonal lineages, with most isolates belonging to the high-risk clones ST258/512, ST101, ST11, and ST307. blaKPC-like was the most prevalent carbapenemase-encoding gene (46%), with blaOXA-48 present in 39% of isolates. Through the combination and comparison of this EURECA collection with the previous EuSCAPE collection (2013-2014), we investigated the spread of high-risk clones circulating in Europe exhibiting regional differences. We particularly found blaKPC-like ST258/512 in Greece, Italy, and Spain, blaOXA-48 ST101 in Serbia and Romania, blaNDM ST11 in Greece, and blaOXA-48-like ST14 in Türkiye. Genomic surveillance across Europe thus provides crucial insights for local risk mapping and informs necessary adaptions for implementation of control strategies.

Microbial metagenomic shifts in children with acute lymphoblastic leukaemia during induction therapy and predictive biomarkers for infection.

BACKGROUND: Emerging evidence has indicated a link between the gut microbiota and acute lymphoblastic leukaemia (ALL). However, the acute changes in gut microbiota during chemotherapy and the predictive value of baseline gut microbiota in infectious complication remain largely unknown. METHODS: Faecal samples (n = 126) from children with ALL (n = 49) undergoing induction chemotherapy were collected at three timepoints, i.e., initiation of chemotherapy (baseline, T0), 7 days (T1) and 33 days (T2) after initiation of chemotherapy. Gut microbiome profile was performed via metagenomic shotgun sequencing. The bioBakery3 pipeline (Kneaddata, Metaphlan 3 and HUMAnN) was performed to assign taxonomy and functional annotations. Gut microbiome at T0 were used to predict infection during chemotherapy. RESULTS: The microbial diversities and composition changed significantly during chemotherapy, with Escherichia coli, Klebsiella pneumoniae and Bifidobacterium longum being the most prominent species. The microbial metabolic pathways were also significantly altered during chemotherapy, including the pathway of pyruvate fermentation to acetate and lactate, and assimilatory sulfate reduction pathway. The receiver operating characteristic (ROC) models based on Bifidobacterium longum at T0 could predict infectious complications during the first month of chemotherapy with the area under the curve (AUC) of 0.720. CONCLUSIONS: Our study provides new insights into the acute changes in microbial and functional characteristics in children with ALL during chemotherapy. The baseline gut microbiota could be potential biomarkers for infections during chemotherapy. TRIAL REGISTRATION: The study was approved by the Ethics Committee of Zhujiang Hospital, Southern Medical University (2021-KY-171-01) and registered on http://www.chictr.org.cn (ChiCTR2200065406, Registration Date: November 4, 2022).

Type IV-A3 CRISPR-Cas systems drive inter-plasmid conflicts by acquiring spacers in trans.

Plasmid-encoded type IV-A CRISPR-Cas systems lack an acquisition module, feature a DinG helicase instead of a nuclease, and form ribonucleoprotein complexes of unknown biological functions. Type IV-A3 systems are carried by conjugative plasmids that often harbor antibiotic-resistance genes and their CRISPR array contents suggest a role in mediating inter-plasmid conflicts, but this function remains unexplored. Here, we demonstrate that a plasmid-encoded type IV-A3 system co-opts the type I-E adaptation machinery from its host, Klebsiella pneumoniae (K. pneumoniae), to update its CRISPR array. Furthermore, we reveal that robust interference of conjugative plasmids and phages is elicited through CRISPR RNA-dependent transcriptional repression. By silencing plasmid core functions, type IV-A3 impacts the horizontal transfer and stability of targeted plasmids, supporting its role in plasmid competition. Our findings shed light on the mechanisms and ecological function of type IV-A3 systems and demonstrate their practical efficacy for countering antibiotic resistance in clinically relevant strains.

sRNA expression profile of KPC-2-producing carbapenem-resistant Klebsiella pneumoniae: Functional role of sRNA51.

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) has significant challenges to human health and clinical treatment, with KPC-2-producing CRKP being the predominant epidemic strain. Therefore, there is an urgent need to identify new therapeutic targets and strategies. Non-coding small RNA (sRNA) is a post-transcriptional regulator of genes involved in important biological processes in bacteria and represents an emerging therapeutic strategy for antibiotic-resistant bacteria. In this study, we analyzed the transcription profile of KPC-2-producing CRKP using RNA-seq. Of the 4693 known genes detected, the expression of 307 genes was significantly different from that of carbapenem-sensitive Klebsiella pneumoniae (CSKP), including 133 up-regulated and 174 down-regulated genes. Both the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and Gene Ontology (GO) analysis showed that these differentially expressed genes (DEGs) were mainly related to metabolism. In addition, we identified the sRNA expression profile of KPC-2-producing CRKP for the first time and detected 115 sRNAs, including 112 newly discovered sRNAs. Compared to CSKP, 43 sRNAs were differentially expressed in KPC-2-producing CRKP, including 39 up-regulated and 4 down-regulated sRNAs. We chose sRNA51, the most significantly differentially expressed sRNA in KPC-2-producing CRKP, as our research subject. By constructing sRNA51-overexpressing KPC-2-producing CRKP strains, we found that sRNA51 overexpression down-regulated the expression of acrA and alleviated resistance to meropenem and ertapenem in KPC-2-producing CRKP, while overexpression of acrA in sRNA51-overexpressing strains restored the reduction of resistance. Therefore, we speculated that sRNA51 could affect the resistance of KPC-2-producing CRKP by inhibiting acrA expression and affecting the formation of efflux pumps. This provides a new approach for developing antibiotic adjuvants to restore the sensitivity of CRKP.