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.

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.

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.

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.

Chromosomal integration and plasmid fusion occurring in ST20 carbapenem-resistant Klebsiella pneumoniae isolates coharboring blaNDM-1 and blaIMP-4 induce resistance transmission and fitness variation.

To investigate the epidemiology of ST20 carbapenem-resistant Klebsiella pneumoniae (CRKP) in China, and further explore the genomic characteristics of blaIMP-4 and blaNDM-1 coharboring isolates and plasmid contributions to resistance and fitness. Seven ST20 CRKP isolates were collected nationwide, and antimicrobial susceptibility testing was performed. Antimicrobial resistance genes, virulence genes, and plasmid replicons were identified via whole-genome sequencing, and clonality assessed via core-genome multilocus sequence typing. Furthermore, we found four dual-metallo-ß-lactamases (MBL)-harbouring isolates, the gene location was detected by Southern blotting, and plasmid location analysis showed that blaIMP-4 was located on a separate plasmid, a self-conjugative fusion plasmid, or the bacterial chromosome. These isolates were subjected to long-read sequencing, the presence of blaIMP-4 in different locations was identified by genomic comparison, and transposon units were detected via inverse PCR. We subsequently found that blaIMP-4 on the fusion plasmid and bacterial chromosome was formed via intact plasmid recombination by the IS26 and ltrA, respectively, and the circular transposon unit was related to cointegration, however, blaIMP-4 in different locations did not affect the gene stability. The blaNDM-1-harbouring plasmid contributed to the increased resistance to ß-lactams and shortened survival lag time which was revealed in plasmid cured isolates. In summary, the K. pneumoniae ST20 clone is a high-risk resistant clone. With the use of ceftazidime/avibactam, MBL-positive isolates, especially dual-MBL-harbouring isolates, should be given additional attention.

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.

Intestinal carbapenem-resistant Klebsiella pneumoniae undergoes complex transcriptional reprogramming following immune activation.

Carbapenem-resistant Klebsiella pneumoniae (CR-Kp) is a significant threat to public health worldwide. The primary reservoir for CR-Kp is the intestinal tract. There, the bacterium is usually present at low density but can bloom following antibiotic treatment, mostly in hospital settings. The impact of disturbances in the intestinal environment on the fitness, survival, expansion, and drug susceptibility of this pathogen is not well-understood, yet it may be relevant to devise strategies to tackle CR-Kp colonization and infection. Here, we adopted an in vivo model to examine the transcriptional adaptation of a CR-Kp clinical isolate to immune activation in the intestine. We report that as early as 6 hours following host treatment with anti-CD3 antibody, CR-Kp underwent rapid transcriptional changes including downregulation of genes involved in sugar utilization and amino acid biosynthesis and upregulation of genes involved in amino acid uptake and catabolism, antibiotic resistance, and stress response. In agreement with these findings, treatment increased the concentration of oxidative species and amino acids in the mouse intestine. Genes encoding for proteins containing the domain of unknown function (DUF) 1471 were strongly upregulated, however their deletion did not impair CR-Kp fitness in vivo upon immune activation. Transcription factor enrichment analysis identified the global regulator cAMP-Receptor Protein, CRP, as a potential orchestrator of the observed transcriptional signature. In keeping with the recognized role of CRP in regulating utilization of alternative carbon sources, crp deletion in CR-Kp resulted in strongly impaired gut colonization, although this effect was not amplified by immune activation. Thus, following intestinal colonization, which occurs in a CRP-dependent manner, CR-Kp can rapidly respond to immune cues by implementing a well-defined and complex transcriptional program whose direct relevance toward bacterial fitness warrants further investigation. Additional analyses utilizing this model may identify key factors to tackle CR-Kp colonization of the intestine.

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.

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.

[Carbapenemase Genes, Virulence Genes, and Molecular Epidemiology of Carbapenem-Resistant Klebsiella pneumoniae Derived From Bloodstream Infections]. / è¡€æµæ„ŸæŸ“ç¢³é’éœ‰çƒ¯è€è¯è‚ºç‚Žå ‹é›·ä¼¯èŒçš„è€è¯åŸºå› å’Œæ¯’åŠ›åŸºå› åŠåˆ†å­æµè¡Œç— å­¦ç ”ç©¶.

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.

Expansion and transmission dynamics of high risk carbapenem-resistant Klebsiella pneumoniae subclones in China: An epidemiological, spatial, genomic analysis.

AIMS: Carbapenem-resistant Klebsiella pneumonia (CRKP) is a global threat that varies by region. The global distribution, evolution, and clinical implications of the ST11 CRKP clone remain obscure. METHODS: We conducted a multicenter molecular epidemiological survey using isolates obtained from 28 provinces and municipalities across China between 2011 and 2021. We integrated sequences from public databases and performed genetic epidemiology analysis of ST11 CRKP. RESULTS: Among ST11 CRKP, KL64 serotypes exhibited considerable expansion, increasing from 1.54% to 46.08% between 2011 and 2021. Combining our data with public databases, the phylogenetic and phylogeography analyses indicated that ST11 CRKP appeared in the Americas in 1996 and spread worldwide, with key clones progressing from China's southeastern coast to the inland by 2010. Global phylogenetic analysis showed that ST11 KL64 CRKP has evolved to a virulent, resistant clade with notable regional spread. Single-nucleotide polymorphism (SNP) analysis identified BMPPS (bmr3, mltC, pyrB, ppsC, and sdaC) as a key marker for this clade. The BMPPS SNP clade is associated with high mortality and has strong anti-phagocytic and competitive traits in vitro. CONCLUSIONS: The high-risk ST11 KL64 CRKP subclone showed strong expansion potential and survival advantages, probably owing to genetic factors.

Genomic insights into the evolution and mechanisms of carbapenem-resistant hypervirulent Klebsiella pneumoniae co-harboring blaKPC and blaNDM: implications for public health threat mitigation.

BACKGROUND: Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) co-producing blaKPC and blaNDM poses a serious threat to public health. This study aimed to investigate the mechanisms underlying the resistance and virulence of CR-hvKP isolates collected from a Chinese hospital, with a focus on blaKPC and blaNDM dual-positive hvKP strains. METHODS: Five CR-hvKP strains were isolated from a teaching hospital in China. Antimicrobial susceptibility and plasmid stability testing, plasmid conjugation, pulsed-field gel electrophoresis, and whole-genome sequencing (WGS) were performed to examine the mechanisms of resistance and virulence. The virulence of CR-hvKP was evaluated through serum-killing assay and Galleria mellonella lethality experiments. Phylogenetic analysis based on 16 highly homologous carbapenem-resistant K. pneumoniae (CRKP) producing KPC-2 isolates from the same hospital was conducted to elucidate the potential evolutionary pathway of CRKP co-producing NDM and KPC. RESULTS: WGS revealed that five isolates individually carried three unique plasmids: an IncFIB/IncHI1B-type virulence plasmid, IncFII/IncR-type plasmid harboring KPC-2 and IncC-type plasmid harboring NDM-1. The conjugation test results indicated that the transference of KPC-2 harboring IncFII/IncR-type plasmid was unsuccessful on their own, but could be transferred by forming a hybrid plasmid with the IncC plasmid harboring NDM. Further genetic analysis confirmed that the pJNKPN26-KPC plasmid was entirely integrated into the IncC-type plasmid via the copy-in route, which was mediated by TnAs1 and IS26. CONCLUSION: KPC-NDM-CR-hvKP likely evolved from a KPC-2-CRKP ancestor and later acquired a highly transferable blaNDM-1 plasmid. ST11-KL64 CRKP exhibited enhanced plasticity. The identification of KPC-2-NDM-1-CR-hvKP highlights the urgent need for effective preventive strategies against aggravated accumulation of resistance genes.

Incidence of carbapenem-resistant Escherichia coli ST2437 of clinical origin harbouring blaOXA-144 gene: a report from India.

AIMS: Carbapenem-resistant Escherichia coli has been categorized as a pathogen of critical priority by the World Health Organization as it is highly infectious with high mortality and morbidity rates and widespread transmission potential. Carbapenem resistance is primarily mediated by carbapenemase-encoding genes and, additionally, through intrinsic factors. In India, over the years, carbapenemase-encoding genes have been reported from diverse clinically significant pathogens. The present study identifies E. coli of clinical origin that harbours blaOXA-144. METHODS AND RESULTS: The study isolate was obtained from a tertiary referral hospital in northeast India. Carbapenemase production was investigated through culture on chromogenic agar and Rapidec Carba NP test as per manufacturer's instructions. Susceptibility of the isolate was performed by the Kirby-Bauer disc diffusion method and agar dilution method following CLSI guidelines. PCR targeting carbapenemase-encoding genes was performed, followed by transformation and conjugation experiments. Whole-genome sequencing of the isolate was done through the Illumina sequencing platform and the data were analysed using the Centre for Genomic Epidemiology database. BJD_EC180 is 6 919 180 bp in length and consists of six rRNA operons, 111 tRNA, and 6849 predicted protein-coding sequences. BJD_EC180 belonged to ST2437 and harboured the carbapenemase-encoding gene blaOXA-144 with ISAba1 upstream, along with multiple antibiotic resistance genes conferring clinical resistance towards beta-lactams, aminoglycosides, amphenicols, sulphonamides, tetracyclines, trimethoprim, and rifampin. CONCLUSIONS: Carbapenem-resistant E. coli harbouring blaOXA-144 associated with insertion sequence pose a serious health threat as their mobilization into carbapenem non-susceptible strains that will contribute to the resistance burden and therefore, needs urgent monitoring.

The role of silver nanoparticles alone and combined with imipenem on carbapenem-resistant Klebsiella pneumoniae.

AIMS: Carbapenem-resistant Klebsiella pneumoniae (CRKP) infections poses a significant threat to human health, necessitating urgent development of new antimicrobial agents. Silver nanoparticles (AgNPs), which are among the most widely used engineered nanomaterials, have been extensively studied. However, the impact of AgNPs on CRKP and the potential for drug resistance development remain inadequately explored. METHODS AND RESULTS: In this study, broth dilution method was used to determine the minimum inhibitory concentration (MIC) was determined using the broth dilution method. Results indicated MIC values of 93.1 ± 193.3 µg ml-1 for AgNPs, 2.3 ± 5.1 µg ml-1 for AgNO3, and 25.1 ± 48.3 µg ml-1 for imipenem (IMI). The combined inhibitory effect of AgNPs and IMI on CRKP was assessed using the checkerboard method. Moreover, after 6-20 generations of continuous culture, the MIC value of AgNPs increased 2-fold. Compared to IMI, resistance of Kl. pneumoniae to AgNPs developed more slowly, with a higher fold increase in MIC observed after 20 generations. Whole-genome sequencing revealed four nonsynonymous single nucleotide polymorphism mutations in CRKP after 20 generations of AgNP treatment. CONCLUSION: We have demonstrated that AgNPs significantly inhibit CRKP isolates and enhance the antibacterial activity of imipenem against Kl. pneumoniae. Although the development of AgNP resistance is gradual, continued efforts are necessary for monitoring and studying the mechanisms of AgNP resistance.

Genomic characterization of extended-spectrum beta-lactamase-producing and carbapenem-resistant Escherichia coli from urban wastewater in Australia.

This study investigates extended-spectrum beta-lactamase-producing and carbapenem-resistant Escherichia coli isolates from Sydney's wastewater. These isolates exhibit resistance to critical antibiotics and harbor novel resistance mechanisms. The findings highlight the importance of wastewater-based surveillance in monitoring resistance beyond the clinical setting.

Global phylogeography and genomic characterization of blaKPC and blaNDM-positive clinical Klebsiella aerogenes isolates from China, 2016-2022.

Carbapenem-resistant Klebsiella aerogenes (CRKA), being one of the members of carbapenem-resistant Enterobacteriaceae (CRE), has caused great public health concern, but with fewer studies compared to other CRE members. Furthermore, studies on phylogenetic analysis based on whole genome Single-Nucleotide Polymorphism (SNP) of CRKA were limited. Here, 20 CRKA isolates (11 blaKPC-2-bearing and 9 blaNDM-1/5-harboring) were characterized by antimicrobial susceptibility testing, conjugation assay, whole genome sequencing (WGS) and bioinformatics analysis. Additionally, the phylogeographic relationships of K. aerogenes were further investigated from public databases. All isolates were multidrug-resistant (MDR) bacteria, and they demonstrated susceptibility to colistin. Most blaKPC-2 or blaNDM-1/5-carrying plasmids were found to be conjugative. Phylogenetic analysis revealed the clonal dissemination of K. aerogenes primarily occurred within clinical settings. Notably, some strains in this study showed the potential for clonal transmission, sharing few SNPs between K. aerogenes and KPC- and/or NDM-positive K. aerogenes isolated from various countries. The STs of K. aerogenes strains had significant diversity. WGS analysis showed that the IncFIIK plasmid was the most prevalent carrier of blaKPC-2, and, blaNDM-1/5 were detected on the IncX3 plasmids. The Tn6296 and Tn3000 transposons were most common vehicles for facilitating the transmission of blaKPC-2 and blaNDM-1/5, respectively. This study highlights the importance of continuous screening and surveillance by WGS for analysis of drug-resistant strains in hospital settings, and provide clinical information that supports epidemiological and public health research on human pathogens.

Synergistic effects of ceftazidime/avibactam combined with meropenem in a murine model of infection with KPC-producing Klebsiella pneumoniae.

OBJECTIVES: The emergence and expansion of carbapenem-resistant Klebsiella pneumoniae infections is a concern due to the lack of 'first-line' antibiotic treatment options. The ceftazidime/avibactam is an important clinical treatment for carbapenem-resistant K. pneumoniae infections but there is an increasing number of cases of treatment failure and drug resistance. Therefore, a potential solution is combination therapies that result in synergistic activity against K. pneumoniae carbapenemase: producing K. pneumoniae (KPC-Kp) isolates and preventing the emergence of KPC mutants resistant to ceftazidime/avibactam are needed in lieu of novel antibiotics. METHODS: To evaluate their synergistic activity, antibiotic combinations were tested against 26 KPC-Kp strains. Antibiotic resistance profiles, molecular characteristics and virulence genes were investigated by susceptibility testing and whole-genome sequencing. Antibiotic synergy was evaluated by in vitro chequerboard experiments, time-killing curves and dose-response assays. The mouse thigh model was used to confirm antibiotic combination activities in vivo. Additionally, antibiotic combinations were evaluated for their ability to prevent the emergence of ceftazidime/avibactam resistant mutations of blaKPC. RESULTS: The combination of ceftazidime/avibactam plus meropenem showed remarkable synergistic activity against 26 strains and restored susceptibility to both the partnering antibiotics. The significant therapeutic effect of ceftazidime/avibactam combined with meropenem was also confirmed in the mouse model and bacterial loads in the thigh muscle of the combination groups were significantly reduced. Furthermore, ceftazidime/avibactam plus meropenem showed significant activity in preventing the occurrence of resistance mutations. CONCLUSIONS: Our results indicated that the combination of ceftazidime/avibactam plus meropenem offers viable therapeutic alternatives in treating serious infections due to KPC-Kp.

Whole genome sequence-based molecular characterization of blood isolates of carbapenem-resistant Enterobacter cloacae complex from ICU patients in Kolkata, India, during 2017-2022: emergence of phylogenetically heterogeneous Enterobacter hormaechei subsp. xiangfangensis.

Blood-borne infections caused by the carbapenem-resistant Enterobacter cloacae complex (CR-ECC) are major public threats with respect to the challenges encountered during treatment. This study describes the whole genome sequencing-based molecular characteristics of blood isolates (n = 70) of CR-ECC from patients admitted to the intensive care unit of tertiary care hospitals in Kolkata, India, during 2017-2022 with respect to species identification, antimicrobial resistance (AMR) profiling, mechanism of drug resistance, and molecular subtypes. Vitek2 MALDI and species-specific PCR identified Enterobacter hormaechei subsp. xiangfangensis (47.14%) as the emerging CR-ECC subspecies in Kolkata. The predominating carbapenemase and extended-spectrum ß-lactamase genes found were blaNDM-1 (51.42%) and blaCTX-M-15 (27%), respectively. Besides, blaNDM-4, blaNDM-5, blaNDM-7, blaCMH-3, blaSFO-1, blaOXA-181, blaOXA-232, blaKPC-3, and blaDHA-7 genes were also detected, which were not previously reported from India. A multitude of Class 1 integrons (including In180, In4874, In4887, and In4888, which were novel) and plasmid replicon types (IncFIB, IncFII, IncX3, IncHI1-HI2, IncC, and IncR) involved in AMR dissemination were identified. Reverse transcription-PCR and western blot revealed that carbapenem resistance in non-carbapenemase-producing CR-ECC isolates was contributed by elevated levels of ampC, overexpression of acrAB, and loss of ompF. A total of 30 distinct sequence types (STs) were ascertained by multi-locus sequence typing; of which, ST2011, ST2018, ST2055, ST2721, and ST2722 were novel STs. Pulsed-field gel electrophoresis analysis showed heterogeneity (69 pulsotypes with a similarity coefficient of 48.40%) among the circulating isolates, suggesting multiple reservoirs of infections in humans. Phylogenetically and genetically diverse CR-ECC with multiple AMR mechanisms mandates close monitoring of nosocomial infections caused by these isolates to forestall the transmission and dissemination of AMR.IMPORTANCEThe emergence and extensive dissemination of the carbapenem-resistant Enterobacter cloacae complex (CR-ECC) have positioned it as a critical nosocomial global pathogen. The dearth of a comprehensive molecular study pertaining to CR-ECC necessitated this study, which is the first of its kind from India. Characterization of blood isolates of CR-ECC over the last 6 years revealed Enterobacter hormaechei subsp. xiangfangensis as the most prevalent subsp., exhibiting resistance to almost all antibiotics currently in use and harboring diverse transmissible carbapenemase genes. Besides the predominating blaNDM-1 and blaCTX-M-15, we document diverse carbapenemase and AmpC genes, such as blaNDM-4, blaNDM-7, blaOXA-181, blaOXA-232, blaKPC-3, blaCMH-3, blaSFO-1, and blaDHA-7, in CR-ECC, which were not previously reported from India. Furthermore, novel integrons and sequence types were identified. Our findings emphasize the need for strengthened vigilance for molecular epidemiological surveillance of CR-ECC due to the presence of epidemic clones with a phylogenetically diverse and wide array of antimicrobial resistance genes in vulnerable populations.

Klebsiella pneumoniae ST147 harboring blaNDM-1, multidrug resistance and hypervirulence plasmids.

The spread of hypervirulent (hv) and carbapenem-/multidrug-resistant Klebsiella pneumoniae is an emerging problem in healthcare settings. The New Delhi metallo-ß-lactamase-1 (blaNDM-1) is found in Enterobacteriaceae including K. pneumoniae. The blaNDM-1 is capable of hydrolyzing ß-lactam antibiotics which are used for treatment of severe infections caused by multidrug-resistant Gram-negative bacteria. This is associated with the unacceptably high mortality rate in immunocompromised burn injury patients. This study reports on the characterization of blaNDM-1 gene and virulence factors in hv carbapenem-/multidrug-resistant K. pneumoniae ST147 in the burns unit of a tertiary teaching hospital during routine surveillance. Two K. pneumoniae strains were obtained from wounds of burn-infected patients from May 2020 to July 2021. The hypervirulence genes and genetic context of the blaNDM-1 gene and mobile genetic elements potentially involved in the transposition of the gene were analyzed. We identified a conserved genetic background and an IS26 and open reading frame flanking the blaNDM-1 gene that could suggest its involvement in the mobilization of the gene. The plasmid harbored additional antibiotic resistance predicted regions that were responsible for resistance to almost all the routinely used antibiotics. To ensure the identification of potential outbreak strains during routine surveillance, investigations on resistance genes and their environment in relation to evolution are necessary for molecular epidemiology.IMPORTANCEData obtained from this study will aid in the prompt identification of disease outbreaks including evolving resistance and virulence of the outbreak bacteria. This will help establish and implement antimicrobial stewardship programs and infection prevention protocols in fragile health systems in countries with limited resources. Integration of molecular surveillance and translation of whole-genome sequencing in routine diagnosis will provide valuable data for control of infection. This study reports for the first time a high-risk clone K. pneumoniae ST147 with hypervirulence and multidrug-resistance features in Ghana.