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.

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.

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.

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.

Clinical and Microbiological Characteristics of Bacteremia Caused by Carbapenemase-producing Enterobacterales in Minami Ibaraki Area, Japan.

Although recent propagation of carbapenemase-producing Enterobacterales (CPE) has become a problem worldwide, the picture of CPE infection in Japan has not fully been elucidated. In this study, we examined clinical and microbiological characteristics of invasive CPE infection occurring at 8 hospitals in Minami Ibaraki Area between July 2001 to June 2017. Of 7294 Enterobacterales strains isolated from independent cases of bacteremia and/or meningitis, 10 (0.14%) were CPE (8 Enterobacter cloacae-complex, 1 Escherichia coli, and 1 Edwardsiella tarda）, all of which had the blaIMP-1 gene and susceptible to gentamicin and trimethoprim/sulfamethoxazole. These strains were isolated from 7 adult and 2 infant bacteremia (1 infant patient developed CPE bacteremia twice) after 2007. The most common portal of entry was intravenous catheters. All of the adult patients were recovered, while the infant patients eventually died. Genomic analyses showed that the 8 E. cloacae-complex strains were classified into 5 groups, each of which was exclusively detected in specific facilities at intervals of up to 3 years, suggesting persistent colonization in the facilities. This study showed that invasive CPE infection in the area was rare, caused by IMP-1-type CPE having susceptibility to various antibiotics, and nonfatal among adult patients.

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

BACKGROUND: We 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. RESULTS: According 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. CONCLUSION: While 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.

Genetic, virulence, and antimicrobial resistance characteristics associated with distinct morphotypes in ST11 carbapenem-resistant Klebsiella pneumoniae.

ST11 is the most common lineage among carbapenem-resistant Klebsiella pneumoniae (CRKP) infections in Asia. Diverse morphotypes resulting from genetic mutations are associated with significant differences in microbial characteristics among K. pneumoniae isolates. Here, we investigated the genetic determinants and critical characteristics associated with distinct morphotypes of ST11 CRKP. An ST11-KL47 CRKP isolate carrying a pLVPK-like virulence plasmid was isolated from a patient with a bloodstream infection; the isolate had the "mcsw" morphotype. Two distinct morphotypes ("ntrd" and "msdw") were derived from this strain during in vitro passage. Whole genome sequencing was used to identify mutations that cause the distinct morphotypes of ST11 CRKP. Transmission electron microscopy, antimicrobial susceptibility tests, growth assays, biofilm formation, virulence assays, membrane permeability assays, and RNA-seq analysis were used to investigate the specific characteristics associated with different morphotypes of ST11 CRKP. Compared with the parental mcsw morphotype, the ntrd morphotype resulted from mutation of genes involved in capsular polysaccharide biosynthesis (wza, wzc, and wbaP), a result validated by gene knockout experiments. This morphotype showed capsule deficiency and lower virulence potential, but higher biofilm production. By contrast, the msdw morphotype displayed competition deficiency and increased susceptibility to chlorhexidine and polymyxin B. Further analyses indicated that these characteristics were caused by interruption of the sigma factor gene rpoN by insertion mutations and deletion of the rpoN gene, which attenuated membrane integrity presumably by downregulating the phage shock protein operon. These data expand current understanding of genetic, virulence, and antimicrobial resistance characteristics associated with distinct morphotypes in ST11 CRKP.

Molecular epidemiology and antimicrobial resistance profiles of Klebsiella pneumoniae isolates from hospitalized patients in different regions of China.

Introduction: The increasing incidence of Klebsiella pneumoniae and carbapenem-resistant Klebsiella pneumoniae (CRKP) has posed great challenges for the clinical anti-infective treatment. Here, we describe the molecular epidemiology and antimicrobial resistance profiles of K. pneumoniae and CRKP isolates from hospitalized patients in different regions of China. Methods: A total of 219 K. pneumoniae isolates from 26 hospitals in 19 provinces of China were collected during 2019-2020. Antimicrobial susceptibility tests, multilocus sequence typing were performed, antimicrobial resistance genes were detected by polymerase chain reaction (PCR). Antimicrobial resistance profiles were compared between different groups. Results: The resistance rates of K. pneumoniae isolates to imipenem, meropenem, and ertapenem were 20.1%, 20.1%, and 22.4%, respectively. A total of 45 CRKP isolates were identified. There was a significant difference in antimicrobial resistance between 45 CRKP and 174 carbapenem-sensitive Klebsiella pneumoniae (CSKP) strains, and the CRKP isolates were characterized by the multiple-drug resistance phenotype.There were regional differences among antimicrobial resistance rates of K. pneumoniae to cefazolin, chloramphenicol, and sulfamethoxazole,which were lower in the northwest than those in north and south of China.The mostcommon sequence type (ST) was ST11 (66.7% of the strains). In addition, we detected 13 other STs. There were differences between ST11 and non-ST11 isolates in the resistance rate to amikacin, gentamicin, latamoxef, ciprofloxacin, levofloxacin, aztreonam, nitrofurantoin, fosfomycin, and ceftazidime/avibactam. In terms of molecular resistance mechanisms, the majority of the CRKP strains (71.1%, 32/45) harbored blaKPC-2, followed by blaNDM (22.2%, 10/45). Strains harboring blaKPC or blaNDM genes showed different sensitivities to some antibiotics. Conclusion: Our analysis emphasizes the importance of surveilling carbapenem-resistant determinants and analyzing their molecular characteristics for better management of antimicrobial agents in clinical use.

Characterizing carbapenemase-producing Escherichia coli isolates from Spain: high genetic heterogeneity and wide geographical spread.

Introduction: Carbapenemase-Producing Escherichia coli (CP-Eco) isolates, though less prevalent than other CP-Enterobacterales, have the capacity to rapidly disseminate antibiotic resistance genes (ARGs) and cause serious difficult-to-treat infections. The aim of this study is phenotypically and genotypically characterizing CP-Eco isolates collected from Spain to better understand their resistance mechanisms and population structure. Methods: Ninety representative isolates received from 2015 to 2020 from 25 provinces and 59 hospitals Spanish hospitals were included. Antibiotic susceptibility was determined according to EUCAST guidelines and whole-genome sequencing was performed. Antibiotic resistance and virulence-associated genes, phylogeny and population structure, and carbapenemase genes-carrying plasmids were analyzed. Results and discussion: The 90 CP-Eco isolates were highly polyclonal, where the most prevalent was ST131, detected in 14 (15.6%) of the isolates. The carbapenemase genes detected were bla OXA-48 (45.6%), bla VIM-1 (23.3%), bla NDM-1 (7.8%), bla KPC-3 (6.7%), and bla NDM-5 (6.7%). Forty (44.4%) were resistant to 6 or more antibiotic groups and the most active antibiotics were colistin (98.9%), plazomicin (92.2%) and cefiderocol (92.2%). Four of the seven cefiderocol-resistant isolates belonged to ST167 and six harbored bla NDM. Five of the plazomicin-resistant isolates harbored rmt. IncL plasmids were the most frequent (45.7%) and eight of these harbored bla VIM-1. bla OXA-48 was found in IncF plasmids in eight isolates. Metallo-ß-lactamases were more frequent in isolates with resistance to six or more antibiotic groups, with their genes often present on the same plasmid/integron. ST131 isolates were associated with sat and pap virulence genes. This study highlights the genetic versatility of CP-Eco and its potential to disseminate ARGs and cause community and nosocomial infections.

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.

Porin deficiency or plasmid copy number increase mediated carbapenem-resistant Escherichia coli resistance evolution.

This study investigated resistance evolution mechanisms of conjugated plasmids and bacterial hosts under different concentrations of antibiotic pressure. Ancestral strain ECNX52 was constructed by introducing the blaNDM-5-carrying IncX3 plasmid into E. coli C600, and was subjected to laboratory evolution under different concentrations of meropenem pressure. Minimal inhibitory concentrations and conjugation frequency were determined. Fitness of these strains was assessed. Whole genome sequencing and transcriptional changes were performed. Ancestral host or plasmids were recombined with evolved hosts or plasmids to verify plasmid or host factors in resistance evolution. Role of the repA mutation on plasmid copy number was determined. Two out of the four clones (EM2N1 and EM2N3) exhibited four-fold increase in MIC when exposed to a continuous pressure of 2 µg/mL MEM (1/32 MIC), by down regulating expression of outer membrane protein ompF. Besides, all four clones displayed four-fold increase in MIC and higher conjugation frequency when subjected to a continuous pressure of 4 µg/mL MEM (1/16 MIC), attributing to increasing plasmid copy number generated by repA D140Y (GAT→TAT) mutation. Bacterial hosts and conjugative plasmids can undergo resistance evolution under certain concentrations of antimicrobial pressure by reducing the expression of outer membrane proteins or increasing plasmid copy numbers.

Ceftazidime-Avibactam resistance in clinical isolates of carbapenem-resistant Klebsiella pneumoniae: A phenotypic and genotypic analysis.

OBJECTIVE: To find the prevalence of Ceftazidime-Avibactam (CAZ-AVI) resistant Klebsiella pneumoniae in clinical isolates and to determine the genes responsible for Ceftazidime-Avibactam resistance using PCR. METHODS: A total of 89 carbapenem resistant Klebsiella pneumoniae from various clinical samples were included in the study. CAZ-AVI resistance was tested using E-test. CAZ-AVI resistant strains were subjected to conventional PCR for detection of carbapenamase genes blaNDM- 1, blaOXA-48, blaVIM, blaIMP, blaKPC. RESULTS: Of the 89 isolates screened for CAZ-AVI resistance, 45(50.5%) isolates were found to be resistant. 42 isolates were subjected to PCR for detection of ß lactamase genes.34 isolates were positive for blaNDM-1 and all 42 isolates were positive for blaOXA-48. Co-expression of NDM-1 and OXA-48 was seen in 34 isolates. Sensitivity of mCIM test to identify a carbapenamse compared to PCR was 61.9%. Sensitivity of eCIM test to identify NDM-1 was 80%. CONCLUSION: CAZ-AVI was effective in vitro in 49.4% of the isolates. Indicating that CAZ-AVI is a promising addition to antibiotics against CRE as well as a carbapenem sparing drug in ESBL producing organisms. ß-Lactamase-related mutations are the main mechanism leading to CAZ-AVI resistance.

Rapid, culture-free detection of carbapenem-resistant Klebsiella pneumoniae in a case of bloodstream infection using genomics.

Timely diagnosis and treatment of sepsis is a major challenge faced by critical care specialists around the world. The traditional blood culture methods have a significant turnaround time which delays targeted therapy leading to poor prognosis. In the current study, we highlight the clinical utility of a genomics solution for diagnosis and management of bloodstream infections by combining the real-time DNA sequencing of Oxford Nanopore Technology with an automated genomic data analysis software. We identify a carbapenem-resistant Klebsiella pneumoniae directly from a blood sample in <24 hours and thereby prove the effectiveness of the test in early diagnosis of sepsis.

Enhancing resistance, but not virulence attributed to the high mortality caused by carbapenem-resistant Klebsiella pneumoniae.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has emerged as a global threat due to its high mortality in clinical patients. However, the specific mechanisms underlying this increased mortality remain unclear. The objective of this study is to investigate how the development of a resistance phenotype contributes to the significantly higher mortality associated with this pathogen. To achieve this, a collection of isogeneic strains was generated. The clinical carbapenem-susceptible K. pneumoniae (CSKP) strain HKU3 served as the control isolate, while HKU3-KPC was created through conjugation with a blaKPC-2-bearing plasmid and served as clinical CRKP strain. Using a sepsis model, it was demonstrated that both HKU3 and HKU3-KPC exhibited similar levels of virulence. Flow cytometry, RNA-seq, and ELISA analysis were employed to assess immune cell response, M1 macrophage polarization, and cytokine storm induction, revealing that both strains elicited comparable types and levels of these immune responses. Subsequently, meropenem was utilized to treat K. pneumoniae infection, and it was found that meropenem effectively reduced bacterial load, inhibited M1 macrophage polarization, and suppressed serum cytokine production during HKU3 (CSKP) infection. However, these effects were not observed in the case of HKU3-KPC (CRKP) infection. These findings provide evidence that the high mortality associated with CRKP is attributed to its enhanced survival within the host during antibiotic treatment, resulting in a cytokine storm and subsequent host death. The development of an effective therapy for CRKP infections could significantly reduce the mortality caused by this pathogen.

Molecular Epidemiology of Carbapenem-Resistant Klebsiella aerogenes in Japan.

Information regarding Klebsiella aerogenes haboring carbapenemase in Japan is limited. A comprehensive nationwide survey was conducted from September 2014 to December 2022, and 67 non-duplicate strains of carbapenem-resistant K. aerogenes were isolated from 57 healthcare facilities in Japan. Through genetic testing and whole-genome sequencing, six strains were found to possess carbapenemases, including imipenemase (IMP)-1, IMP-6, New Delhi metallo-ß-lactamase (NDM)-1, and NDM-5. The strain harboring blaNDM-5 was the novel strain ST709, which belongs to the clonal complex of the predominant ST4 in China. The novel integron containing blaIMP-1 featured the oxacillinase-101 gene, which is a previously unreported structure, with an IncN4 plasmid type. However, integrons found in the strains possessing blaIMP-6, which were the most commonly identified, matched those reported domestically in Klebsiella pneumoniae, suggesting the prevalence of identical integrons. Transposons containing blaNDM are similar or identical to the transposon structure of K. aerogenes harboring blaNDM-5 previously reported in Japan, suggesting that the same type of transposon could have been transmitted to K. aerogenes in Japan. This investigation analyzed mobile genetic elements, such as integrons and transposons, to understand the spread of carbapenemases, highlighting the growing challenge of carbapenem-resistant Enterobacterales in Japan and underscoring the critical need for ongoing surveillance to control these pathogens.

A CRISPR/Cas12a-Based System for Sensitive Detection of Antimicrobial-Resistant Genes in Carbapenem-Resistant Enterobacterales.

Antimicrobial-resistant (AMR) bacteria pose a significant global health threat, and bacteria that produce New Delhi metallo-ß-lactamase (NDM) are particularly concerning due to their resistance to most ß-lactam antibiotics, including carbapenems. The emergence and spread of NDM-producing genes in food-producing animals highlight the need for a fast and accurate method for detecting AMR bacteria. We therefore propose a PCR-coupled CRISPR/Cas12a-based fluorescence assay that can detect NDM-producing genes (blaNDM) in bacteria. Thanks to its designed gRNA, this CRISPR/Cas12a system was able to simultaneously cleave PCR amplicons and ssDNA-FQ reporters, generating fluorescence signals. Our method was found to be highly specific when tested against other foodborne pathogens that do not carry blaNDM and also demonstrated an excellent capability to distinguish single-nucleotide polymorphism. In the case of blaNDM-1 carrying E. coli, the assay performed exceptionally well, with a detection limit of 2.7 × 100 CFU/mL: 100 times better than conventional PCR with gel electrophoresis. Moreover, the developed assay detected AMR bacteria in food samples and exhibited enhanced performance compared to previously published real-time PCR assays. Thus, this novel PCR-coupled CRISPR/Cas12a-based fluorescence assay has considerable potential to improve current approaches to AMR gene detection and thereby contribute to mitigating the global threat of AMR.

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Objective: To investigate the clinical characteristics and molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolated from patients with bloodstream infections in a large tertiary-care general hospital in Southwest China. Methods: A total of 131 strains of non-repeating CRKP were collected from the blood cultures of patients who had bloodstream infections in 2015-2019. The strains were identified by VITEK-2, a fully automated microbial analyzer, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The minimum inhibitory concentration (MIC) was determined by microbroth dilution method. The common carbapenemase resistant genes and virulence factors were identified by PCR. Homology analysis was performed by multilocus sequencing typing. Whole genome sequencing was performed to analyze the genomic characteristics of CRKP without carbapenemase. Results: The 131 strains of CRKP showed resistance to common antibiotics, except for polymyxin B (1.6% resistance rate) and tigacycline (8.0% resistance rate). A total of 105 (80.2%) CRKP strains carried the Klebsiella pneumoniae carbapenemase (KPC) resistance gene, 15 (11.4%) strains carried the New Delhi Metallo-ß-lactamase (NDM) gene, and 4 (3.1%) isolates carried both KPC and NDM genes. Sequence typing (ST) 11 (74.0%) was the dominant sequence type. High detection rates for mrkD (96.2%), fimH (98.5%), entB (100%), and other virulence genes were reported. One hypervirulent CRKP strain was detected. The seven strains of CRKP that did not produce carbapenemase were shown to carry ESBL or AmpC genes and had anomalies in membrane porins OMPK35 and OMPK36, according to whole genome sequencing. Conclusion: In a large-scale tertiary-care general hospital, CRKP mainly carries the KPC gene, has a high drug resistance rate to a variety of antibiotics, and possesses multiple virulence genes. Attention should be paid to CRKP strains with high virulence.

National genomic epidemiology investigation revealed the spread of carbapenem-resistant Escherichia coli in healthy populations and the impact on public health.

BACKGROUND: Carbapenem-resistant Escherichia coli (CREC) has been considered as WHO priority pathogens, causing a great public health concern globally. While CREC from patients has been thoroughly investigated, the prevalence and underlying risks of CREC in healthy populations have been overlooked. Systematic research on the prevalence of CREC in healthy individuals was conducted here. We aimed to characterize CREC collected from healthy populations in China between 2020 and 2022 and to compare the genomes of CREC isolates isolated from healthy individuals and clinical patients. METHODS: We present a nationwide investigation of CREC isolates among healthy populations in China, employing robust molecular and genomic analyses. Antimicrobial susceptibility testing, whole-genome sequencing, and bioinformatics were utilized to analyze a cohort of CREC isolates (n = 113) obtained from fecal samples of 5 064 healthy individuals. Representative plasmids were extracted for third-generation nanopore sequencing. We previously collected 113 non-duplicate CREC isolates (59 in 2018, 54 in 2020) collected from ICU patients in 15 provinces and municipalities in China, and these clinical isolates were used to compare with the isolates in this study. Furthermore, we employ comparative genomics approaches to elucidate molecular variations and potential correlations between clinical and non-clinical CREC isolates. RESULTS: A total of 147 CREC isolates were identified from 5 064 samples collected across 11 provinces in China. These isolates were classified into 64 known sequence types (STs), but no dominant STs were observed. In total, seven carbapenemase genes were detected with blaNDM-5 (n = 116) being the most prevalent one. Genetic environments and plasmid backbones of blaNDM were conserved in CREC isolated from healthy individuals. Furthermore, we compared clinical and healthy human-originated CRECs, revealing noteworthy distinctions in 23 resistance genes, including blaNDM-1, blaNDM-5, and blaKPC (χ2 test, p < 0.05). Clinical isolates contained more virulence factors associated with iron uptake, adhesion, and invasion than those obtained from healthy individuals. Notably, CREC isolates generally found healthy people are detected in hospitalized patients. CONCLUSIONS: Our findings underscore the significance of healthy populations-derived CRECs as a crucial reservoir of antibiotic resistance genes (ARGs). This highlights the need for ongoing monitoring of CREC isolates in healthy populations to accurately assess the potential risks posed by clinical CREC isolates.

A challenging case of carbapenem resistant Klebsiella pneumoniae-related pyogenic liver abscess with capsular polysaccharide hyperproduction: a case report.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) infections are a major public health problem, necessitating the administration of polymyxin E (colistin) as a last-line antibiotic. Meanwhile, the mortality rate associated with colistin-resistant K. pneumoniae infections is seriously increasing. On the other hand, importance of administration of carbapenems in promoting colistin resistance in K. pneumoniae is unknown. CASE PRESENTATION: We report a case of K. pneumoniae-related pyogenic liver abscess in which susceptible K. pneumoniae transformed into carbapenem- and colistin-resistant K. pneumoniae during treatment with imipenem. The case of pyogenic liver abscess was a 50-year-old man with diabetes and liver transplant who was admitted to Abu Ali Sina Hospital in Shiraz. The K. pneumoniae isolate responsible for community-acquired pyogenic liver abscess was isolated and identified. The K. pneumoniae isolate was sensitive to all tested antibiotics except ampicillin in the antimicrobial susceptibility test and was identified as a non-K1/K2 classical K. pneumoniae (cKp) strain. Multilocus sequence typing (MLST) identified the isolate as sequence type 54 (ST54). Based on the patient's request, he was discharged to continue treatment at another center. After two months, he was readmitted due to fever and progressive constitutional symptoms. During treatment with imipenem, the strain acquired blaOXA-48 and showed resistance to carbapenems and was identified as a multidrug resistant (MDR) strain. The minimum inhibitory concentration (MIC) test for colistin was performed by broth microdilution method and the strain was sensitive to colistin (MIC < 2 µg/mL). Meanwhile, on blood agar, the colonies had a sticky consistency and adhered to the culture medium (sticky mucoviscous colonies). Quantitative real-time PCR and biofilm formation assay revealed that the CRKP strain increased capsule wzi gene expression and produced slime in response to imipenem. Finally, K. pneumoniae-related pyogenic liver abscess with resistance to a wide range of antibiotics, including the last-line antibiotics colistin and tigecycline, led to sepsis and death. CONCLUSIONS: Based on this information, can we have a theoretical hypothesis that imipenem is a promoter of resistance to carbapenems and colistin in K. pneumoniae? This needs more attention.