Clonal background and routes of plasmid transmission underlie antimicrobial resistance features of bloodstream Klebsiella pneumoniae.

Bloodstream infections caused by the opportunistic pathogen Klebsiella pneumoniae are associated with adverse health complications and high mortality rates. Antimicrobial resistance (AMR) limits available treatment options, thus exacerbating its public health and clinical burden. Here, we aim to elucidate the population structure of K. pneumoniae in bloodstream infections from a single medical center and the drivers that facilitate the dissemination of AMR. Analysis of 136 short-read genome sequences complemented with 12 long-read sequences shows the population consisting of 94 sequence types (STs) and 99 clonal groups, including globally distributed multidrug resistant and hypervirulent clones. In vitro antimicrobial susceptibility testing and in silico identification of AMR determinants reveal high concordance (90.44-100%) for aminoglycosides, beta-lactams, carbapenems, cephalosporins, quinolones, and sulfonamides. IncF plasmids mediate the clonal (within the same lineage) and horizontal (between lineages) transmission of the extended-spectrum beta-lactamase gene blaCTX-M-15. Nearly identical plasmids are recovered from isolates over a span of two years indicating long-term persistence. The genetic determinants for hypervirulence are carried on plasmids exhibiting genomic rearrangement, loss, and/or truncation. Our findings highlight the importance of considering both the genetic background of host strains and the routes of plasmid transmission in understanding the spread of AMR in bloodstream infections.

Virulence gene distribution and molecular epidemiological characteristics of carbapenem-resistant Klebsiella pneumoniae in the ICU. / ICUç¢³é’éœ‰çƒ¯è€è¯è‚ºç‚Žå ‹é›·ä¼¯èŒçš„æ¯’åŠ›åŸºå› åˆ†å¸ƒåŠåˆ†å­æµè¡Œç— å­¦ç‰¹å¾.

OBJECTIVES: The drug-resistant genes carried by carbapenem-resistant Klebsiella pneumoniae (CRKP) limit clinical treatment options, and its virulence genes severely affect patient prognosis. This study aims to investigate the distribution of virulence genes, capsular serotypes, and molecular epidemiological characteristics of CRKP in ICU, to understand the characteristics of CRKP infections in ICU, and to provide a scientific basis for effective monitoring and control of CRKP infections in ICU. METHODS: A total of 40 non-duplicate strains of CRKP isolated from the ICU of Guangdong Provincial People's Hospital between January 2021 and December 2022 were collected and analyzed. Whole-genome sequencing was used to analyze the distribution of resistance genes, virulence genes, and capsular serotypes of the strains. The sequences of 7 housekeeping genes of CRKP genome were uploaded to the Klebsiella pneumoniae (KPN)multilocus sequence typing (MLST) database to determine the sequence types (STs) of the strains. RESULTS: The age of the 40 ICU CRKP-infected patients was (69.03±17.82) years old, with various underlying diseases, and there were 20 patients with improved clinical outcome and 20 patients with death. The isolated strains primarily originated from mid-stream urine and bronchoalveolar lavage fluid. Whole-genome sequencing results revealed that the strains predominantly carried blaKPC-1 (29 strains, 72.5%) and blaNDM-1 (6 strains, 15.0%), with 5 strains carrying both blaKPC-1 and blaNDM-1. Various virulence genes were detected, among which the carriage rates of genes such as entA, entB, entE, entS, fepA, fepC, fepG, yag/ecp, and ompA reached 100%, while the carriage rates of genes such as entD, fimB, iroB, iroD, fes,and pla were low. The CRKP strains isolated from ICU were predominantly ST11 (27 cases, 67.5%), with KL64 being the main capsular serotype (29 cases, 72.5%). A total of 23 ST11-KL64 CRKP strains were detected, accounting for 57.5%. CONCLUSIONS: The main type of ICU CRKP is ST11-KL64, carrying various virulence genes, primarily those related to iron absorption. Furthermore, blaKPC has shifted from blaKPC-2 to blaKPC-1. Therefore, close monitoring of the molecular epidemiological changes of CRKP is necessary, and strict control measures should be implemented to effectively curb the occurrence of CRKP infections.

Antibiotic resistance and epidemiological characteristics of polymyxin-resistant Klebsiella pneumoniae. / è€å¤šé»èŒç´ è‚ºç‚Žå ‹é›·ä¼¯èŒçš„è¯æ•ç‰¹å¾åŠæµè¡Œç— å­¦ç‰¹ç‚¹.

OBJECTIVES: The emergence of polymyxin-resistant Klebsiella pneumoniae (KPN) in clinical settings necessitates an analysis of its antibiotic resistance characteristics, epidemiological features, and risk factors for its development. This study aims to provide insights for the prevention and control of polymyxin-resistant KPN infections. METHODS: Thirty clinical isolates of polymyxin-resistant KPN were collected from the Third Xiangya Hospital of Central South University. Their antibiotic resistance profiles were analyzed. The presence of carbapenemase KPC, OXA-48, VIM, IMP, and NDM was detected using colloidal gold immunochromatography. Hypervirulent KPN was initially screened using the string test. Biofilm formation capacity was assessed using crystal violet staining. Combination drug susceptibility tests (polymyxin B with meropenem, tigecycline, cefoperazone/sulbactam) were conducted using the checkerboard method. Polymyxin-related resistance genes were detected by PCR. Multi-locus sequence typing (MLST) was performed for genotyping and phylogenetic tree construction. The study also involved collecting data from carbapenem-resistant (CR)-KPN polymyxin-resistant strains (23 strains, experimental group) and CR-KPN polymyxin-sensitive strains (57 strains, control group) to analyze potential risk factors for polymyxin-resistant KPN infection through univariate analysis and multivariate Logistic regression. The induction of resistance by continuous exposure to polymyxin B and colistin E was also tested. RESULTS: Among the 30 polymyxin-resistant KPN isolates, 28 were CR-KPN, all producing KPC enzyme. Four isolates were positive in the string test. Most isolates showed strong biofilm formation capabilities. Combination therapy showed additive or synergistic effects. All isolates carried the pmrA and phoP genes, while no mcr-1 or mcr-2 genes were detected. MLST results indicated that ST11 was the predominant type. The phylogenetic tree suggested that polymyxin-resistant KPN had not caused a hospital outbreak in the institution. The use of two or more different classes of antibiotics and the use of polymyxin were identified as independent risk factors for the development of polymyxin-resistant strains. Continuous use of polymyxin induced drug resistance. CONCLUSIONS: Polymyxin-resistant KPN is resistant to nearly all commonly used antibiotics, making polymyxin-based combination therapy a viable option. No plasmid-mediated polymyxin-resistant KPN has been isolated in the hospital. Polymyxin can induce resistance in KPN, highlighting the need for rational antibiotic use in clinical settings to delay the emergence of resistance.

Retrospective analysis of molecular characteristics, risk factors, and outcomes in carbapenem-resistant Klebsiella pneumoniae bloodstream infections.

BACKGROUND: Klebsiella pneumoniae (KP) is the second most prevalent Gram-negative bacterium causing bloodstream infections (BSIs). In recent years, the management of BSIs caused by KP has become increasingly complex due to the emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP). Although numerous studies have explored the risk factors for the development of CRKP-BSIs, the mortality of patients with KP-BSIs, and the molecular epidemiological characteristics of CRKP, the variability in data across different populations, countries, and hospitals has led to inconsistent conclusions. In this single-center retrospective observational study, we utilized logistic regression analyses to identify independent risk factors for CRKP-BSIs and factors associated with mortality in KP-BSI patients. Furthermore, a risk factor-based prediction model was developed. CRKP isolates underwent whole-genome sequencing (WGS), followed by an evaluation of microbiological characteristics, including antimicrobial resistance and virulence genes, as well as epidemiological characteristics and phylogenetic analysis. RESULTS: Our study included a total of 134 patients with KP-BSIs, comprising 50 individuals infected with CRKP and 84 with carbapenem-susceptible Klebsiella pneumoniae (CSKP). The independent risk factors for CRKP-BSIs were identified as gastric catheterization (OR = 9.143; CI = 1.357-61.618; P = 0.023), prior ICU hospitalization (OR = 4.642; CI = 1.312-16.422; P = 0.017), and detection of CRKP in non-blood sites (OR = 8.112; CI = 2.130-30.894; P = 0.002). Multivariate analysis revealed that microbiologic eradication after 6 days (OR = 3.569; CI = 1.119-11.387; P = 0.032), high Pitt bacteremia score (OR = 1.609; CI = 1.226-2.111; P = 0.001), and inappropriate empirical treatment after BSIs (OR = 6.756; CI = 1.922-23.753; P = 0.003) were independent risk factors for the 28-day mortality in KP-BSIs. The prediction model confirmed that microbiologic eradication after 6.5 days and a Pitt bacteremia score of 4.5 or higher were significant predictors of the 28-day mortality. Bioinformatics analysis identified ST11 as the predominant CRKP sequence type, with blaKPC-2 as the most prevalent gene variant. CRKP stains carried multiple plasmid-mediated resistance genes along with some virulence genes. Phylogenetic analysis indicated the presence of nosocomial transmission of ST11 CRKP within the ICU. CONCLUSIONS: The analysis of risk factors for developing CRKP-BSIs and the association between KP-BSIs and 28-day mortality, along with the development of a risk factor-based prediction model and the characterization of CRKP strains, enhances clinicians' understanding of the pathogens responsible for BSIs. This understanding may help in the timely administration of antibiotic therapy for patients with suspected KP-BSIs, potentially improving outcomes.

Emergence of silent NDM-1 carbapenemase gene in carbapenem-susceptible Klebsiella pneumoniae: Clinical implications and epidemiological insights.

The global dissemination of carbapenemase genes, particularly blaNDM-1, poses a significant threat to public health. While research has mainly focused on strains with phenotypic resistance, the impact of silent resistance genes has been largely overlooked. This study documents the first instance of silent blaNDM-1 in a cluster of clonally related carbapenem-susceptible K. pneumoniae strains from a single patient. Despite initial effectiveness of carbapenem therapy, the patient experienced four recurrent lung infections over five months, indicating persistent K. pneumoniae infection. Genomic sequencing revealed all strains harbored blaNDM-1 on the epidemic IncX3 plasmid. A deletion within the upstream promoter region (PISAba125) of blaNDM-1 hindered its expression, resulting in phenotypic susceptibility to carbapenems. However, in vitro bactericidal assays and a mouse infection model showed that K. pneumoniae strains with silent blaNDM-1 exhibited significant tolerance to carbapenem-mediated killing. These findings demonstrate that silent blaNDM-1 can mediate both phenotypic susceptibility and antibiotic tolerance. In silico analysis of 1986 blaNDM sequences showed that 1956 (98.5%) retained the original promoter PISAba125. Given that previous genomic sequencing typically targets carbapenem-resistant strains, accurately assessing the prevalence of silent blaNDM remains challenging. This study highlights the hidden threat of silent resistance genes to clinical antimicrobial therapy and calls for enhanced clinical awareness and laboratory detection.

Molecular epidemiology of string test-positive Klebsiella pneumoniae isolates in Huzhou, China, 2020-2023.

Objective: This study used whole-genome sequencing (WGS) to explore the genetic diversity, virulence factors, and antimicrobial resistance determinants of string test-positive Klebsiella pneumoniae (KP) over a 4-year surveillance period in Huzhou, China. Methods: In total, 632 clinical isolates were collected via hospital surveillance from 2020 to 2023; 100 were positive in the string test and these 100 strains were subjected to antimicrobial susceptibility testing using an agar dilution method followed by WGS. Results: The resistance rates to cefotaxime (77.0%), trimethoprim-sulfamethoxazole (67.0%), and nalidixic acid (64.0%) were high. Multilocus sequence typing revealed high genetic diversity; there were 33 sequence types (STs) and 15 capsular serotypes. The most common ST was ST23 (16.0%) and the most common capsular serotype was K1 (22.5%). Virulome analysis revealed among-strain differences in virulence factors that affected bacterial adherence, efflux pump action, iron uptake, nutritional factors, metabolic regulation, the secretion system, and toxin production. The Kleborate strain-specific virulence scores of all 100 string test-positive KPs were derived: 28 strains scored 5, 28 scored 4, 21 scored 3, 12 scored 1, and 11 scored 0. All 77 strains with scores of 3 to 5 contained the iucA gene. The phylogeny based on whole-genome single nucleotide polymorphisms (wgSNPs) indicated high clonality; the string test-positive KP strains were grouped into six clades. Closely related isolates in each genetic cluster usually shared STs. Conclusion: The present study highlights the significance of the KP iucA gene in terms of hypervirulence and the diverse genotypes of string test-positive KP strains isolated in Huzhou hospitals.

Antimicrobial resistance profiles and genome characteristics of Klebsiella isolated from the faeces of neonates in the neonatal intensive care unit.

Introduction. Klebsiella spp. are important bacteria that colonize the human intestine, especially in preterm infants; they can induce local and systemic disease under specific circumstances, including inflammatory bowel disease, necrotizing enterocolitis and colorectal cancer.Hypothesis. Klebsiella spp. colonized in the intestine of the neonates in the neonatal intensive care unit (NICU) may be associated with disease and antibiotic resistance, which will be hazardous to the children.Aim. Our aim was to know about the prevalence, antimicrobial resistance and genome characteristics of Klebsiella spp. in neonate carriers.Methodology. Genome sequencing and analysis, and antimicrobial susceptibility testing were mainly performed in this study.Results. The isolation rates of Klebsiella spp. strains were 3.7% (16/436) in 2014 and 4.3% (18/420) in 2021. Cases with intestinal-colonized Klebsiella spp. were mainly infants with low birth weights or those with pneumonia or hyperbilirubinemia. According to the core-pan genomic analysis, 34 stains showed gene polymorphism and a sequence type (ST) of an emerging high-risk clone (ST11). Eight strains (23.5%) were found to be resistant to 2 or more antibiotics, and 46 genes/gene families along with nine plasmids were identified that conferred resistance to antibiotics. In particular, the two strains were multidrug-resistant. Strain A1256 that is related to Klebsiella quasipneumoniae subsp. similipneumoniae was uncommon, carrying two plasmids similar to IncFII and IncX3 that included five antibiotic resistance genes.Conclusion. The prevention and control of neonatal Klebsiella spp. colonization in the NICU should be strengthened by paying increased attention to preventing antimicrobial resistance in neonates.

Genomic characterization of Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) strains circulating in three university hospitals in Northern Italy over three years.

OBJECTIVES: Genomic surveillance of Klebsiella pneumoniae carbapenemase-producing Klebsiella pneumoniae (KPC-Kp) is crucial for virulence, drug-resistance monitoring, and outbreak containment. METHODS: Genomic analysis on 87 KPC-Kp strains isolated from 3 Northern Italy hospitals in 2019-2021 was performed by whole genome sequencing (WGS), to characterize resistome, virulome, and mobilome, and to assess potential associations with phenotype resistance and clinical presentation. Maximum Likelihood and Minimum Spanning Trees were used to determine strain correlations and identify potential transmission clusters. RESULTS: Overall, 15 different STs were found; the predominant ones included ST307 (35, 40.2%), ST512/1519 (15, 17.2%), ST20 (12, 13.8%), and ST101 (7, 8.1%). 33 (37.9%) KPC-Kp strains were noticed to be in five transmission clusters (median number of isolates in each cluster: 5 [3-10]), four of them characterized by intra-hospital transmission. All 87 strains harbored Tn4401a transposon, carrying blaKPC-3 (48, 55.2%), blaKPC-2 (38, 43.7%), and in one case (1.2%) blaKPC-33, the latter gene conferred resistance to ceftazidime/avibactam (CZA). Thirty strains (34.5%) harbored porin mutations; of them, 7 (8.1%) carried multiple Tn4401a copies. These strains were characterized by significantly higher CZA minimum inhibitory concentration compared with strains with no porin mutations or single Tn4401a copy, respectively, even if they did not overcome the resistance breakpoint of 8 ug/mL. Median 2 (IQR:1-2) virulence factors per strain were detected. The lowest number was observed in ST20 compared to the other STs (p<0.001). While ST307 was associated with infection events, a trend associated with colonization events could be observed for ST20. CONCLUSIONS: Integration of genomic, resistance score, and clinical data allowed us to define a relative diversification of KPC-Kp in Northern Italy between 2019 and 2021, characterized by few large transmission chains and rare inter-hospital transmission. Our results also provided initial evidence of correlation between KPC-Kp genomic signatures and higher MIC levels to some antimicrobial agents or colonization/infection status, once again underlining WGS's importance in bacterial surveillance.

Investigation of gyrA and parC mutations and the prevalence of plasmid-mediated quinolone resistance genes in Klebsiella pneumoniae clinical isolates.

BACKGROUND: The emergence of fluoroquinolone resistance in clinical isolates of Klebsiella pneumoniae is a growing concern. To investigate the mechanisms behind this resistance, we studied a total of 215 K. pneumoniae isolates from hospitals in Bushehr province, Iran, collected between 2017 and 2019. Antimicrobial susceptibility test for fluoroquinolones was determined. The presence of plasmid mediated quinolone resistance (PMQR) and mutations in quinolone resistance-determining region (QRDR) of gyrA and parC genes in ciprofloxacin-resistant K. pneumoniae isolates were identified by PCR and sequencing. RESULTS: Out of 215 K. pneumoniae isolates, 40 were resistant to ciprofloxacin as determined by E-test method. PCR analysis revealed that among these ciprofloxacin-resistant isolates, 13 (32.5%), 7 (17.5%), 40 (100%), and 25 (62.5%) isolates harbored qnrB, qnrS, oqxA and aac(6')-Ib-cr genes, respectively. Mutation analysis of gyrA and parC genes showed that 35 (87.5%) and 34 (85%) of the ciprofloxacin-resistant isolates had mutations in these genes, respectively. The most frequent mutations were observed in codon 83 of gyrA and codon 80 of parC gene. Single gyrA substitution, Ser83→ Ile and Asp87→Gly, and double substitutions, Ser83→Phe plus Asp87→Ala, Ser83→Tyr plus Asp87→Ala, Ser83→Ile plus Asp87→Tyr, Ser83→Phe plus Asp87→Asn and Ser83→Ile plus Asp87→Gly were detected. In addition, Ser80→Ile and Glu84→Lys single substitution were found in parC gene. CONCLUSIONS: Our results indicated that 90% of isolates have at least one mutation in QRDR of gyrA orparC genes, thus the frequency of mutations was very significant and alarming in our region.

[Analysis of the molecular epidemiological characteristics of carbapenem-resistant Klebsiella pneumoniae in a hospital in Hunan Province].

To examine the molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae (CRKP) and investigate the horizontal transmission of blaKPC and blaNDM genes for the prevention and treatment of CRKP. A total of 49 clinically isolated CRKP strains were retrospectively analyzed from January to December 2022 at The First Hospital of Hunan University of Chinese Medicine. Phenotypic screening was performed using modified carbapenem inactivation assay (mCIM) and EDTA-carbapenem inactivation assay (eCIM). Polymerase chain reaction (PCR) was utilized to identify carbapenem resistance genes, ß-lactamase resistance genes, and virulence genes, while multi-locus sequence analysis (MLST) was employed to assess the homology of CRKP strains. Conjugation experiments were conducted to infer the horizontal transmission mechanism of blaKPC and blaNDM genes. The results showed that the study included 49 CRKP strains, with 44 carrying blaKPC and 8 carrying blaNDM, Three strains were identified as blaKPC+blaNDM-CRKP. In this study, 28 out of 49 CRKP strains (57.2%) were found to carry virulence genes. Additionally, one CRKP strain tested positive in the string test and was found to carry both Aerobactin and rmpA virulence genes. MLST results revealed a total of 5 ST types, with ST11 being predominant (41/49, 83.7%). Successful conjugation was observed in all 3 blaKPC-CRKP strains, while only 1 out of 3 blaNDM-CRKP strains showed successful conjugation. The transconjugant exhibited significantly reduced susceptibility to imipenem and cephalosporin antibiotics. In conclusion, the resistance mechanism of CRKP in this study is primarily attributed to the production of KPC enzymes, along with the presence of multiple ß-lactamase resistance genes. Additionally, there is a local prevalence of hv-CRKP and blaKPC+blaNDM-CRKP. blaKPC and blaNDM can be horizontally transmitted through plasmids, with varying efficiency among different strains.

Gut community structure as a risk factor for infection in Klebsiella pneumoniae-colonized patients.

The primary risk factor for infection with members of the Klebsiella pneumoniae species complex is prior gut colonization, and infection is often caused by the colonizing strain. Despite the importance of the gut as a reservoir for infectious K. pneumoniae, little is known about the association between the gut microbiome and infection. To explore this relationship, we undertook a case-control study comparing the gut community structure of K. pneumoniae-colonized intensive care and hematology/oncology patients. Cases were K. pneumoniae-colonized patients infected by their colonizing strain (N = 83). Controls were K. pneumoniae-colonized patients who remained asymptomatic (N = 149). First, we characterized the gut community structure of K. pneumoniae-colonized patients agnostic to case status. Next, we determined that gut community data is useful for classifying cases and controls using machine learning models and that the gut community structure differed between cases and controls. K. pneumoniae relative abundance, a known risk factor for infection, had the greatest feature importance, but other gut microbes were also informative. Finally, we show that integration of gut community structure with bacterial genotype data enhanced the ability of machine learning models to discriminate cases and controls. Interestingly, inclusion of patient clinical variables failed to improve the ability of machine learning models to discriminate cases and controls. This study demonstrates that including gut community data with K. pneumoniae-derived biomarkers improves our ability to classify infection in K. pneumoniae-colonized patients.IMPORTANCEColonization is generally the first step in pathogenesis for bacteria with pathogenic potential. This step provides a unique window for intervention since a given potential pathogen has yet to cause damage to its host. Moreover, intervention during the colonization stage may help alleviate the burden of therapy failure as antimicrobial resistance rises. Yet, to understand the therapeutic potential of interventions that target colonization, we must first understand the biology of colonization and if biomarkers at the colonization stage can be used to stratify infection risk. The bacterial genus Klebsiella includes many species with varying degrees of pathogenic potential. Members of the K. pneumoniae species complex have the highest pathogenic potential. Patients colonized in their gut by these bacteria are at higher risk of subsequent infection with their colonizing strain. However, we do not understand if other members of the gut microbiota can be used as a biomarker to predict infection risk. In this study, we show that the gut microbiota differs between colonized patients who develop an infection versus those who do not. Additionally, we show that integrating gut microbiota data with bacterial factors improves the ability to classify infections. Surprisingly, patient clinical factors were not useful for classifying infections alone or when added to microbiota-based models. This indicates that the bacterial genotype and the microbial community in which it exists may determine the progression to infection. As we continue to explore colonization as an intervention point to prevent infections in individuals colonized by potential pathogens, we must develop effective means for predicting and stratifying infection risk.

Correlation between Antibiotics-Resistance, Virulence Genes and Genotypes among Klebsiella pneumoniae Clinical Strains Isolated in Guangzhou, China.

Klebsiella pneumoniae is an important opportunistic pathogen causing community-acquired and hospital-acquired infections. This aim of this study was to analysis the antibiotic-resistance phenotypes, carbapenemase genes, virulence genes, and genotypes the 62 K. pneumoniae clinical isolates, and to explore the correlations between these isolates. The antimicrobial susceptibility profiles were determined using the BD Phoenix-100 system. Carbapenemase and virulence genes were detected using multiplex PCR. Out of the 62 K. pneumoniae clinical isolates, 79.0% were exhibited resistance to antibiotics, with 69.4% displaying multi-drug resistance. The rate of antibiotic-resistance was highest for penicillin (71.0%), followed by cephalosporins (66.1%), and lowest for carbapenems (29.0%). The detection rates of carbapenemase genes were as follows: KPC (56.5%), VIM (35.5%), and NDM (1.61%). Additionally, seven virulence genes were detected with the highest prevalence rates, of which entB and mrkD were at the top of the carrier rates with 95.2% each. The study classified 62 isolates into 13 clusters and 46 genotypes using ERIC-PCR. Cluster A6 exhibited the highest genetic diversity, comprising 20 strains and 13 genotypes. The statistical analysis revealed a strong correlation between MDR and resistance to penicillin and cephalosporin. Furthermore, genes related to siderophores were closely associated with mrkD. Genotypes identified by ERIC-PCR showed a negative correlation with allS. The study revealed a negative correlation between antibiotic resistance and genes kfu, ybtS, iutA, rmpA, and allS. Conversely, a positive correlation was observed between antibiotic resistance and genes entB and mrkD. The correlations identified in this study provide insights into the occurrence of hospital-acquired infections. The findings of this study may guide the prevention and control of K. pneumoniae outbreaks by utilizing appropriate medication.

Pathogenomics analysis of high-risk clone ST147 multidrug-resistant Klebsiella pneumoniae isolated from a patient in Egypt.

BACKGROUND: The emergence of multi-drug-resistant Klebsiella pneumoniae (MDR-KP) represents a serious clinical health concern. Antibiotic resistance and virulence interactions play a significant role in the pathogenesis of K. pneumoniae infections. Therefore, tracking the clinical resistome and virulome through monitoring antibiotic resistance genes (ARG) and virulence factors in the bacterial genome using computational analysis tools is critical for predicting the next epidemic. METHODS: In the current study, one hundred extended spectrum ß-lactamase (ESBL)-producing clinical isolates were collected from Mansoura University Hospital, Egypt, in a six-month period from January to June 2022. One isolate was selected due to the high resistance phenotype, and the genetic features of MDR-KP recovered from hospitalized patient were investigated. Otherwise, the susceptibility to 25 antimicrobials was determined using the DL Antimicrobial Susceptibility Testing (AST) system. Whole genome sequencing (WGS) using Illumina NovaSeq 6000 was employed to provide genomic insights into K. pneumoniae WSF99 clinical isolate. RESULTS: The isolate K. pneumoniae WSF99 was phenotypically resistant to the antibiotics under investigation via antibiotic susceptibility testing. WGS analysis revealed that WSF99 total genome length was 5.7 Mb with an estimated 5,718 protein-coding genes and a G + C content of 56.98 mol%. Additionally, the allelic profile of the WSF99 isolate was allocated to the high-risk clone ST147. Furthermore, diverse antibiotic resistance genes were determined in the genome that explain the high-level resistance phenotypes. Several ß-lactamase genes, including blaCTX-M-15, blaTEM-1, blaTEM-12, blaSHV-11, blaSHV-67, and blaOXA-9, were detected in the WSF99 isolate. Moreover, a single carbapenemase gene, blaNDM-5, was predicted in the genome, positioned within a mobile cassette. In addition, other resistance genes were predicted in the genome including, aac(6')-Ib, aph(3')-VI, sul1, sul2, fosA, aadA, arr-2, qnrS1, tetA and tetC. Four plasmid replicons CoIRNAI, IncFIB(K), IncFIB(pQil), and IncR were predicted in the genome. The draft genome analysis revealed the occurrence of genetic mobile elements positioned around the ARGs, suggesting the ease of dissemination via horizontal gene transfer. CONCLUSIONS: This study reports a comprehensive pathogenomic analysis of MDR-KP isolated from a hospitalized patient. These findings could be relevant for future studies investigating the diversity of antimicrobial resistance and virulence in Egypt.

Limited transmission of carbapenem-resistant Klebsiella pneumoniae between animals and humans: a study in Qingdao.

ABSTRACTDespite no carbapenem use in food animals, carbapenem-resistant Klebsiella pneumoniae (CRKP) perseveres within food animals, rising significant concerns regarding public health risks originating from these non-clinical reservoirs. To investigate the potential link between CRKP in food animals and its infections in humans, we conducted a cross-sectional study encompassing human clinical, meat products, and farm animals, in Qingdao city, Shandong province, China. We observed a relatively higher presence of CRKP among hospital inpatients (7.3%) compared to that in the meat products (2.7%) and farm animals (pig, 4.6%; chicken, 0.63%). Multilocus sequence typing and core-genome phylogenetic analyses confirm there is no evidence of farm animals and meat products in the clinical acquisition of K. pneumoniae isolates and carbapenem-resistant genes. However, potential transmission of K. pneumoniae of ST659 and IncX3 plasmid harbouring blaNDM-5 gene from pigs to pork and farm workers was observed. Our findings suggest a limited role of farm animals and meat products in the human clinical acquisition of K. pneumoniae, and the transmission of K. pneumoniae is more common within settings, than between them.

Geographical distribution, disease association and diversity of Klebsiella pneumoniae K/L and O antigens in India: roadmap for vaccine development.

Klebsiella pneumoniae poses a significant healthcare challenge due to its multidrug resistance and diverse serotype landscape. This study aimed to explore the serotype diversity of 1072 K. pneumoniae and its association with geographical distribution, disease severity and antimicrobial/virulence patterns in India. Whole-genome sequencing was performed on the Illumina platform, and genomic analysis was carried out using the Kleborate tool. The analysis revealed a total of 78 different KL types, among which KL64 (n=274/1072, 26 %), KL51 (n=249/1072, 24 %), and KL2 (n=88/1072, 8 %) were the most prevalent. In contrast, only 13 distinct O types were identified, with O1/O2v1 (n=471/1072, 44 %), O1/O2v2 (n=353/1072, 33 %), and OL101 (n=66/1072, 6 %) being the predominant serotypes. The study identified 114 different sequence types (STs) with varying serotypes, with ST231 being the most predominant. O serotypes were strongly linked with STs, with O1/O2v1 predominantly associated with ST231. Simpson's diversity index and Fisher's exact test revealed higher serotype diversity in the north and east regions, along with intriguing associations between specific serotypes and resistance profiles. No significant association between KL or O types and disease severity was observed. Furthermore, we found the specific association of virulence factors yersiniabactin and aerobactin (P<0.05) with KL types but no association with O antigen types (P>0.05). Conventionally described hypervirulent clones (i.e. KL1 and KL2) in India lacked typical virulent markers (i.e. aerobactin), contrasting with other regional serotypes (KL51). The cumulative distribution of KL and O serotypes suggests that future vaccines may have to include either ~20 KL or four O types to cover >85 % of the carbapenemase-producing Indian K. pneumoniae population. The results highlight the necessity for comprehensive strategies to manage the diverse landscape of K. pneumoniae strains across different regions in India. Understanding regional serotype dynamics is pivotal for targeted surveillance, interventions, and tailored vaccine strategies to tackle the diverse landscape of K. pneumoniae infections across India. This article contains data hosted by Microreact.

Genomic analysis of carbapenem- and colistin-resistant Klebsiella pneumoniae complex harbouring mcr-8 and mcr-9 from individuals in Thailand.

The surge in mobile colistin-resistant genes (mcr) has become an increasing public health concern, especially in carbapenem-resistant Enterobacterales (CRE). Prospective surveillance was conducted to explore the genomic characteristics of clinical CRE isolates harbouring mcr in 2015-2020. In this study, we aimed to examine the genomic characteristics and phonotypes of mcr-8 and mcr-9 harbouring carbapenem-resistant K. pneumoniae complex (CRKpnC). Polymerase chain reaction test and genome analysis identified CRKpnC strain AMR20201034 as K. pneumoniae (CRKP) ST147 and strain AMR20200784 as K. quasipneumoniae (CRKQ) ST476, harbouring mcr-8 and mcr-9, respectively. CRKQ exhibited substitutions in chromosomal-mediated colistin resistance genes (pmrB, pmrC, ramA, and lpxM), while CRKP showed two substitutions in crrB, pmrB, pmrC, lpxM and lapB. Both species showed resistance to colistin, with minimal inhibitory concentrations of 8 µg/ml for mcr-8-carrying CRKP isolate and 32 µg/ml for mcr-9-carrying CRKQ isolate. In addition, CRKP harbouring mcr-8 carried blaNDM, while CRKQ harbouring mcr-9 carried blaIMP, conferring carbapenem resistance. Analysis of plasmid replicon types carrying mcr-8 and mcr-9 showed FIA-FII (96,575 bp) and FIB-HI1B (287,118 bp), respectively. In contrast with the plasmid carrying the carbapenemase genes, the CRKQ carried blaIMP-14 on an IncC plasmid, while the CRKP harboured blaNDM-1 on an FIB plasmid. This finding provides a comprehensive insight into another mcr-carrying CRE from patients in Thailand. The other antimicrobial-resistant genes in the CRKP were blaCTX-M-15, blaSHV-11, blaOXA-1, aac(6')-Ib-cr, aph(3')-VI, ARR-3, qnrS1, oqxA, oqxB, sul1, catB3, fosA, and qacE, while those detected in CRKQ were blaOKP-B-15, qnrA1, oqxA, oqxB, sul1, fosA, and qacE. This observation highlights the importance of strengthening official active surveillance efforts to detect, control, and prevent mcr-harbouring CRE and the need for rational drug use in all sectors.

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.

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.

Molecular characterization and epidemiological investigation of colistin resistance in carbapenem-resistant Klebsiella pneumoniae in a tertiary care hospital in Tehran, Iran.

BACKGROUND: Carbapenemase-producing Klebsiella pneumoniae (CRKP) presents a significant challenge to antimicrobial therapy, especially when compounded by resistance to colistin. The objective of this study was to explore molecular epidemiological insights into strains of clinical K. pneumoniae that produce carbapenemases and exhibit resistance to colistin. Eighty clinical isolates of CRKP were obtained from Milad Hospital in Tehran, Iran. Antimicrobial susceptibility and colistin broth disk elution were determined. PCR assays were conducted to examine the prevalence of resistance-associated genes, including blaKPC, blaIMP, blaVIM, blaOXA-48, blaNDM and mcr-1 to -10. Molecular typing (PFGE) was used to assess their spread. RESULTS: Colistin resistance was observed in 27 isolates (33.7%) using the Broth Disk Elution method. Among positive isolates for carbapenemase genes, the most frequent gene was blaOXA-48, identified in 36 strains (45%). The mcr-1 gene was detected in 3.7% of the obtained isolates, with none of the other of the other mcr genes detected in the studied isolates. CONCLUSION: To stop the spread of resistant K. pneumoniae and prevent the evolution of mcr genes, it is imperative to enhance surveillance, adhere rigorously to infection prevention protocols, and implement antibiotic stewardship practices.

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.