Carbapenem-resistant K. Pneumoniae Isolates Research Articles

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

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

Clonal transmission of blaIMP-4-carrying ST196 Klebsiella pneumoniae isolates mediated by the IncN plasmid in China

ObjectivesTo investigate the clinical and molecular epidemiological characteristics of blaIMP-4-carrying Klebsiella pneumoniae in a tertiary hospital in China. MethodsTen carbapenem-resistant K. pneumoniae (CRKP) isolates carrying the blaIMP-4 gene were collected. Molecular characteristics were analysed using whole-genome sequencing. Plasmid conjugation experiments were used to analyse conjugation of the plasmids. We compared and analysed K. pneumoniae-carrying blaIMP-4 genomic datasets obtained from the National Center for Biotechnology Information (NCBI) with the strains in this study. ResultsAll 10 CRKP isolates carrying blaIMP-4 were collected from 10 adult patients in the respiratory intensive care unit. These strains were only sensitive to polymyxins and tigecycline due to them simultaneously carrying multiple resistance genes, namely blaOKP-A-5, fosA, oqxA, and oqxB. Notably, R29 harboured two carbapenemase genes (blaNDM-1 and blaIMP-4). These strains had similar drug-resistant phenotypes and genes, all belonging to sequence type (ST)196. Additionally, the patients had experienced spatiotemporal intersection during hospitalization, suggesting that these strains underwent clonal transmission, but they belonged to different clonal clusters from the blaIMP-4-positive K. pneumoniae currently published in the NCBI. Among the 10 strains, blaIMP-4 was located on the IncN plasmid, and six strains had successfully transferred the plasmid to the recipient strain EC600 through plasmid conjugation. ConclusionsThe blaIMP-4-positive ST196 CRKP isolate showed clonal distribution in the respiratory intensive care unit, which was mediated by the IncN plasmid. Consequently, there should be increased monitoring of carbapenem-resistant strains in clinical settings to prevent and control its transmission.

Phenotypic, molecular, and in silico characterization of coumarin as carbapenemase inhibitor to fight carbapenem-resistant Klebsiella pneumoniae

BackgroundCarbapenems represent the first line treatment of serious infections caused by drug-resistant Klebsiella pneumoniae. Carbapenem-resistant K. pneumoniae (CRKP) is one of the urgent threats to human health worldwide. The current study aims to evaluate the carbapenemase inhibitory potential of coumarin and to test its ability to restore meropenem activity against CRKP. Disk diffusion method was used to test the antimicrobial susceptibility of K. pneumoniae clinical isolates to various antibiotics. Carbapenemase genes (NDM-1, VIM-2, and OXA-9) were detected using PCR. The effect of sub-MIC of coumarin on CRKP isolates was performed using combined disk assay, enzyme inhibition assay, and checkerboard assay. In addition, qRT-PCR was used to estimate the coumarin effect on expression of carbapenemase genes. Molecular docking was used to confirm the interaction between coumarin and binding sites within three carbapenemases.ResultsK. pneumoniae clinical isolates were found to be multi-drug resistant and showed high resistance to meropenem. All bacterial isolates harbor at least one carbapenemase-encoding gene. Coumarin significantly inhibited carbapenemases in the crude periplasmic extract of CRKP. The checkerboard assay indicated that coumarin-meropenem combination was synergistic exhibiting a fractional inhibitory concentration index ≤ 0.5. In addition, qRT-PCR results revealed that coumarin significantly decreased carbapenemase-genes expression. Molecular docking revealed that the binding energies of coumarin to NDM1, VIM-2, OXA-48 and OXA-9 showed a free binding energy of -7.8757, -7.1532, -6.2064 and − 7.4331 Kcal/mol, respectively.ConclusionCoumarin rendered CRKP sensitive to meropenem as evidenced by its inhibitory action on hydrolytic activity and expression of carbapenemases. The current findings suggest that coumarin could be a possible solution to overcome carbapenems resistance in CRKP.

Genomic insights and antimicrobial resistance profiles of CRKP and non-CRKP isolates in a Beijing geriatric medical center: emphasizing the blaKPC-2 carrying high-risk clones and their spread.

The escalating resistance of Klebsiella pneumoniae, a prevalent pathogen in healthcare settings, especially its carbapenem-resistant K. pneumoniae (CRKP), to a wide array of antibiotics, notably β-lactams, constitutes a formidable challenge for healthcare and global public health management. This research compared the resistance phenotypes and genomic profiles of CRKP and Non-CRKP isolates in a Beijing hospital, focusing on high-risk blaKPC-2 gene-bearing CRKP clones and the structure of mobile genetic elements facilitating their spread across hospital departments. Forty K. pneumoniae isolates were collected from various departments of the hospital and subjected to antimicrobial susceptibility testing and whole-genome sequencing to analyze their resistance phenotypes and genomic features. The study revealed that among the 31 CRKP isolates, ST11 is the most common sequence type, with K47 and OL101 being the dominant capsule types, primarily observed in the respiratory department. In terms of antimicrobial susceptibility: 87.5% of the isolates exhibited multidrug resistance (MDR), with a high resistance rate of 30% against tigecycline. All CRKP isolates demonstrated resistance to multiple drug classes (≥5 CLSI classes). Non-CRKP isolates also showed high resistance rates to minocycline and doxycycline (77.8%). the ST11-KL47-OL101 type emerged as the predominant clone among the CRKP isolates carrying the blaKPC-2 gene. This dominance appears to be mediated by the pKpnR03_2 plasmid, which harbors not only blaKPC-2 and rmtb but also gene clusters pertinent to iron transport and arsenic resistance. These isolates, clustering in the C3 clade of the phylogenetic tree, exhibited minor genetic variations and close evolutionary relationships, suggesting a plasmid-driven spread across various hospital departments. In summary, our study highlights the extensive spread of antibiotic-resistant K. pneumoniae across various departments in our hospital, with a particular emphasis on the dominant clonal proliferation of the ST11-KL47-OL101 CRKP strain. This finding underscores the significant role of plasmid-mediated gene transfer in the evolution and dissemination of resistant strains within hospital environments. The study emphasizes the necessity for ongoing surveillance of antibiotic resistance and genomic analysis in hospital settings to effectively monitor and manage these challenges.

Metagenomics next-generation sequencing (mNGS) reveals emerging infection induced by Klebsiella pneumoniaeniae

ObjectivesThe increasing emergence of hypervirulent Klebsiella pneumoniae (hv-Kp) and carbapenem-resistant K. pneumoniae (CR-Kp) is a serious and substantial public health problem. The use of the last resort antimicrobials, tigecycline and polymyxin to combat infections is complicated by the expanding repertoire of newly-identified CR-hvKp. The transmission and co-occurrence of the corresponding antimicrobial resistance and virulence determinants are largely unknown. The aim of this study was to investigate the dissemination and dynamics of CR-Kp and its antibiotic resistance in a hospitalised patient. MethodsMetagenomic next-generation sequencing (mNGS) was conducted for different specimens collected from an elderly male hospitalised patient. CR-Kp strains were examined using antibiotic susceptibility and string testing. Antimicrobial and virulence genes were annotated using whole-genome sequencing (WGS). ResultsA clinical case of a patient infected with a variety of CR-Kp isolates was reported. The co-occurrence of KPC-2 and NDM-1 in the patient was revealed. The CR-Kp isolates, such as BALF2, and Sputum T1 and T3, were classified into ST11 and ST147, respectively. The genetic signature (iuc operon) of hypervirulence was identified in strain T1, although string testing indicated its intermediate virulence. ConclusionsIn this study, multiple infections of CR-Kp isolates were revealed by mNGS, and their dissemination was attributed to plasmid variations, mgrB inactivation and integrative conjugative elements (ICEs). Furthermore, the finding indicated one likely convergence to form CR-hvKp, different from acquisition of carbapenem-resistance determinants in hvKp. A combination of mNGS and WGS is beneficial for clinical diagnosis and anti-infection therapy, and facilitates a better understanding of genetic variants conferring antimicrobial and virulence properties.

Molecular Screening of Carbapenem-Resistant K. pneumoniae (CRKP) Clinical Isolates for Concomitant Occurrence of Beta-Lactam Genes (CTX-M, TEM, and SHV) in the Kingdom of Bahrain.

The emergence of extended-spectrum β-lactamase-producing Klebsiella pneumoniae, including CRKP infections, has resulted in significant morbidity and mortality worldwide. We aimed to explore the presence of bla genes (CTX-M, TEM, and SHV) in CRKP isolates. A total of 24 CRKP isolates were randomly selected from the Salmaniya Medical Complex Microbiology Laboratory. These isolates, which were positive for carbapenemases, were further explored for CTX-M, TEM, and SHV genes using PCR. All the CTX-M PCR amplicons were sent for sequencing. To determine genetic relatedness, molecular typing by ERIC-PCR was performed. The bla gene testing demonstrated that a significant proportion of these isolates harbored SHV, CTX-M, and TEM genes (100%, 91.6%, and 45.8%), respectively. Bioinformatic analyses confirmed CTX-M-15 in these isolates. ERIC-PCR analysis showed three clusters demonstrating genetic relatedness. The study findings reveal the concomitant carriage of the SHV and CTX-M-15 and a comparatively lower carriage of TEM genes in CRKP isolates. Our findings highlight the significance of routinely reporting the presence of antibiotic resistance genes along with regular antibiotic sensitivity reports, as this will aid clinicians in prescribing appropriate antibiotics.

Mortality in KPC-producing Klebsiella pneumoniae bloodstream infections: a changing landscape.

To assess the impact of carbapenem resistance on mortality in Klebsiella pneumoniae bloodstream infection (BSI) in the era of novel β-lactam/β-lactamase inhibitor combinations. Retrospective study of patients with K. pneumoniae BSI between January and August 2020 in 16 centres (CARBANEW study within the MULTI-SITA project). Overall, 426 patients were included: 107/426 (25%) had carbapenem-resistant K. pneumoniae (CR-Kp) BSI and 319/426 (75%) had carbapenem-susceptible K. pneumoniae (CS-Kp) BSI. Crude cumulative 30 day mortality was 33.8% and 20.7% in patients with, respectively, CR-Kp BSI and CS-Kp BSI (P = 0.027). Carbapenemase production or carbapenemase-encoding genes were detected in 84/98 tested CR-Kp isolates (85.7%), mainly KPC (78/84; 92.9%). Ceftazidime/avibactam was the most frequently used appropriate therapy for CR-Kp BSI (80/107; 74.7%). In multivariable analyses, variables showing an unfavourable association with mortality after correction for multiple testing were age-adjusted Charlson comorbidity index (HR 1.20; 95% CI 1.10-1.31, P < 0.001) and Pitt score (HR 1.33; 95% CI 1.15-1.55, P < 0.001), but not carbapenem resistance (HR 1.28, 95% CI 0.74-2.22, P = 0.410). In a propensity score-matched analysis, there was no difference in mortality between patients appropriately treated with ceftazidime/avibactam for CR-Kp BSI and patients appropriately treated with other agents (mainly meropenem monotherapy or piperacillin/tazobactam monotherapy) for CS-Kp BSI (HR 1.07; 95% CI 0.50-2.29, P = 0.866). Our results suggest that the increased mortality in CR-Kp BSI compared with CS-Kp BSI is not (or no longer) dependent on the type of therapy in areas where ceftazidime/avibactam-susceptible KPC-producing isolates are the most prevalent type of CR-Kp.

Isolation of Hv-CRKP with co-production of three carbapenemases (blaKPC, blaOXA-181 or OXA-232, and blaNDM-1) and a virulence plasmid: a study from a Chinese tertiary hospital.

The worldwide dissemination of K. pneumoniae isolates is a significant public health concern, as these organisms possess a unique capacity to acquire genetic elements encoding both resistance and hypervirulence. This study aims to investigate the epidemiological, resistance, and virulence characteristics of K. pneumoniae isolates that carry both virulence plasmids and blaOXA-48-like genes in a tertiary hospital in China. A total of 217 clinical isolates of carbapenem-resistant K. pneumoniae (CRKP) were collected between April 2020 and March 2022. The antimicrobial susceptibility test was conducted to evaluate the drug resistance profile. All isolates were screened for the presence of genes encoding carbapenemases (blaKPC, blaNDM, blaIMP, blaVIM, and blaOXA-48-like), ESBLs genes (blaCTX-M, blaSHV, blaTEM), and virulence plasmid pLVPK-borne genes (rmpA, rmpA2, iucA, iroB, and peg344) using polymerase chain reaction (PCR) amplification. Clonal lineages were assigned using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). The plasmid incompatibility groups were identified using PCR-based replicon typing (PBRT). The transferability of carbapenemase-encoding plasmids and pLVPK-like virulence plasmids was assessed via conjugation. The plasmid location of rmpA2 was determined using S1-Pulsed Field Gel Electrophoresis (S1-PFGE) and southern blotting hybridization. The virulence potential of the isolates was assessed using the string test, capsular serotyping, serum killing assay and a Galleria mellonella larval infection model. Of the 217 CRKP clinical isolates collected, 23% were identified as carrying blaOXA-48-like genes. All blaOXA-48-like isolates exhibited resistance to commonly used clinical antimicrobial agents, except for ceftazidime/avibactam, colistin, tigecycline, trimethoprim-sulfamethOXAzole, polymyxin B, and nitrofurantoin. The main common OXA-48-like carbapenemase enzymes were found to be blaOXA-181 and blaOXA-232. MLST and PFGE fingerprinting analysis revealed clonal transmission and plasmid transmission. OXA-48-like producing CRKP isolates mainly clustered in K64 ST11 and K47 ST15. Results of the string Test, serum killing assay (in vitro) and Galleria mellonella infection model (in vivo) indicated hypervirulence. PBRT showed that the blaOXA-181 and blaOXA-232 producing hypervirulent carbapenem-resistant Klebsiella pneumoniae (Hv-CRKP) were mainly carried on ColE-type, IncF, and IncX3. Eight clinical isolates of hv-CRKP were identified as carrying three carbapenem-resistant genes (blaKPC, blaOXA-181 or OXA-232, and blaNDM-1). Moreover, Southern blotting hybridization revealed that all eight isolates had a pLVPK-like virulent plasmid (138.9-216.9 kb) with an uneven number and size of plasmid. In our investigation, we have observed the emergence of hv-CRKP carrying blaOXA-48-like genes, which identified two genetic relationships: clonal transmission and plasmid transmission. PBRT analysis showed that these genes were mainly carried on ColE-type, IncF, and IncX3 plasmids. These isolates have been shown to be hypervirulent in vitro and in vivo. Additionally, eight clinical isolates of hv-CRKP were identified as carrying three carbapenem-resistant genes (blaKPC, blaOXA-181 or OXA-232, and blaNDM-1) and carrying a pLVPK-like virulent plasmid. Hence, our findings highlight the need for further investigation and active surveillance of hypervirulent OXA-48-like producing Hv-CRKP isolates to control their transmission.

High Frequency of blaOXA-48like Producing Klebsiella pneumoniae Isolated from Nosocomial Infection in Azerbaijan, Iran.

Klebsiella pneumoniae is one of the significant agents of hospital-acquired infections. In recent years, carbapenem-resistant K. pneumoniae (CRKP) isolates have been found in numerous epidemics of nosocomial infections. This study aimed to determine carbapenem resistance mechanisms and molecular epidemiological of CRKP infections in Azerbaijan, Iran. A total of 50 non-duplicated CRKP from January 2020 to December 2020 were isolated form Sina and Imam Reza Hospitals in Tabriz, Iran. Antimicrobial susceptibility testing was performed by the disk-diffusion method. The carbapenem resistance mechanisms were determined by the phenotypic and PCR procedures. CRKP isolates were typed by the Random Amplified Polymorphic DNA PCR (RAPD-PCR) technique. Amikacin was the most effective antibiotics against CRKP isolates. AmpC overproduction was observed in five CRKP isolates. Efflux pump activity was found in one isolate by the phenotypic method. Carba NP test could find carbapenemases genes in 96% of isolates. The most common carbapenemases gene in CRKP isolates were blaOXA-48-like (76%) followed by blaNDM (50%), blaIMP (22%), blaVIM (10%), and blaKPC (10%). The outer membrane protein genes (OmpK36 and OmpK35) were identified in 76% and 82% of CRKP isolates, respectively. RAPD-PCR analysis yielded 37 distinct RAPD-types. Most blaOXA-48-like positive CRKP isolates were obtained from patients hospitalized in intensive care unit (ICU) wards with urinary tract infections. The blaOXA-48-like is the main carbapenemase among CRKP isolates in this area. Most blaOXA-48-like producer CRKP strains were collected from the ICU ward and urine samples. To control infections due to CRKP, a strict control program in hospital settings is required.

Prevalence of intestinal colonization and nosocomial infection with carbapenem-resistant Enterobacteriales in children: a retrospective study.

We investigated the epidemiological surveillance of the intestinal colonization and nosocomial infection of carbapenem-resistant Enterobacteriales (CRE) isolates from inpatients, which can provide the basis for developing effective prevention. A total of 96 CRE strains were collected from 1,487 fecal samples of hospitalized children between January 2016 and June 2017, which were defined as the "CRE colonization" group. In total, 70 CRE clinical isolates were also randomly selected for the comparison analysis and defined as the "CRE infection" group. The antimicrobial susceptibility of all strains was determined by the microdilution broth method. Polymerase chain reaction (PCR) was used to analyze carbapenemase genes, plasmid typing, and integrons. Multilocus sequence typing was further used to determine clonal relatedness. In the "CRE colonization" group, Klebsiella pneumoniae was mostly detected with a rate of 42.7% (41/96), followed by Escherichia coli (34.4%, 33/96) and Enterobacter cloacae (15.6%, 15/96). The ST11 KPC-2 producer, ST8 NDM-5 producer, and ST45 NDM-1 producer were commonly present in carbapenem-resistant K. pneumoniae (CRKPN), carbapenem-resistant E. coli (CRECO), and carbapenem-resistant E. cloacae (CRECL) isolates, respectively. In the "CRE infection" group, 70% (49/70) of strains were K. pneumoniae, with 21.4% E. cloacae (15/70) and 5.7% E. coli (4/70). The ST15 OXA-232 producer and ST48 NDM-5 producer were frequently observed in CRKPN isolates, while the majority of NDM-1-producing CRECL isolates were assigned as ST45. Phylogenetic analysis showed that partial CRE isolates from intestinal colonization and nosocomial infection were closely related, especially for ST11 KPC-2-producing CRKPN and ST45 NDM-1-producing CRECL. Furthermore, plasmid typing demonstrated that IncF and IncFIB were the most prevalent plasmids in KPC-2 producers, while IncX3/IncX2 and ColE were widely spread in NDM producer and OXA-232 producer, respectively. Then, class 1 integron intergrase intI1 was positive in 74.0% (71/96) of the "CRE colonization" group and 52.9% (37/70) of the "CRE infection" group. This study revealed that CRE strains from intestinal colonization and nosocomial infection showed a partial correlation in the prevalence of CRE, especially for ST11 KPC-2-producing CRKPN and ST45 NDM-1-producing CRECL. Therefore, before admission, long-term active screening of rectal colonization of CRE isolates should be emphasized.

Carbapenem-resistant hypermucoviscous Klebsiella pneumoniae clinical isolates from a tertiary hospital in China: Antimicrobial susceptibility, resistance phenotype, epidemiological characteristics, microbial virulence, and risk factors.

Hypervirulent and multidrug-resistant Klebsiella pneumoniae poses a significant threat to public health. We aimed to determine the common carbapenemase genotypes and the carriage patterns, main antibiotic resistance mechanisms, and in vitro susceptibility of clinical isolates of carbapenem-resistant K. pneumoniae (CRKP) to ceftazidime/avibactam (CZA) for the reasonable selection of antimicrobial agents and determine whether hypermucoviscous (HMV) phenotype and virulence-associated genes are key factors for CRKP colonization and persistence. Antibiotics susceptibility of clinical CRKP isolates and carbapenemase types were detected. CRKP isolates were identified as hypermucoviscous K. pneumoniae (HMKP) using the string test, and detection of virulence gene was performed using capsular serotyping. The bla KPC-2, bla NDM, bla IMP, and/or bla OXA-48-like were detected in 96.4% (402/417) of the isolates, and the bla KPC-2 (64.7%, 260/402) was significantly higher (P<0.05) than those of bla NDM (25.1%), bla OXA-48-like (10.4%), and bla IMP (4.2%). Carriage of a single carbapenemase gene was observed in 96.3% of the isolates, making it the dominant antibiotic resistance genotype carriage pattern (P < 0.05). Approximately 3.7% of the isolates carried two or more carbapenemase genotypes, with bla KPC-2 + bla NDM and bla NDM + bla IMP being the dominant multiple antibiotic resistance genotype. In addition, 43 CRKP isolates were identified as HMKP, with a prevalence of 10.3% and 2.7% among CRKP and all K. pneumoniae isolates, respectively. Most clinical CRKP isolates were isolated from elderly patients, and carbapenemase production was the main mechanism of drug resistance. Tigecycline and polymyxin B exhibited exceptional antimicrobial activity against CRKP isolates in vitro. Furthermore, bla KPC-2, bla NDM, and bla OXA-48-like were the main carbapenemase genes carried by the CRKP isolates. CZA demonstrated excellent antimicrobial activity against isolates carrying the single bla KPC-2 or bla OXA-48-like genotype. Capsular serotype K2 was the main capsular serotype of the carbapenem-resistant HMKP isolates. Survival rates of Galleria mellonella injected with K. pneumoniae 1-7 were 20.0, 16.7, 6.7, 23.3, 16.7, 3.3, and 13.3, respectively. Therefore, worldwide surveillance of these novel CRKP isolates and carbapenem-resistant HMKP isolates as well as the implementation of stricter control measures are needed to prevent further dissemination in hospital settings.

Carbapenem-Resistant Klebsiella pneumoniae: Diversity, Virulence, and Antimicrobial Resistance.

BackgroundKlebsiella pneumoniae (K. pneumoniae) is one of the most important pathogens in nosocomial infections. It has resistance to most antibiotics, even carbapenem, resulting in restricted therapeutic options.PurposeWe tried to assess the antimicrobial resistance and virulence fitness of carbapenem-resistant K. pneumoniae (CRKP) in addition to their phenotypic and genotypic diversity.Materials and MethodsThe conventional methods, automated Vitek-32 system, and antimicrobial susceptibility pattern were used to detect CRKP isolates. Virulence and resistance genes profiles were created by using PCR technique. The correlation analysis was done by using R-program.ResultsThe antimicrobial resistance profile for all our K. pneumoniae isolates was shocking as the MDR and CRKP were the most prominent phenotypes. Unfortunately, high degrees of heterogeneity among our CRKP isolates were recorded, as 97.5% of them were differentiated into different clusters. We found a negative correlation between the existence of virulence and antimicrobial resistance genes. In contrast to sputum and urine CRPK isolates, the blood isolates showed high antimicrobial resistance and low virulence fitness. Finally, K. pneumoniae creates several outbreaks and crises in Egypt owing to the highly heterogeneity and the wide spread of multidrug-resistant (MDR) and multi-virulent CRKP phenotypes.ConclusionOur results are significant and alarming to health organizations throughout the world for the severity and heterogeneity of K. pneumoniae infections. Therefore, the traditional method for treatment of CRKP infections must be renewed. Additionally, the treatment protocols must be well correlated with the site of infections, phenotypes, and genotypes of CRKP strains.

Genomic analysis of the emerging carbapenem-resistant Klebsiella pneumoniae sequence type 11 harbouring Klebsiella pneumoniae carbapenemase (KPC) in Oman

Background and objectiveThe presence of carbapenemase enzymes among Enterobacterales is the main mechanism to reduce susceptibility to a wide range of antibiotics. Carbapenemase enzymes such as the Klebsiella pneumoniae carbapenemase (KPC) hydrolyse beta-lactam antibiotics group, which includes carbapenem, leads to fewer treatment options. We aim to describe the first report of carbapenem-resistant K. pneumoniae (CRKP) sequence type (ST) 11 harbouring KPC in Oman. Material and methodsFive confirmed CRKP isolates were isolated from clinical samples during the period of January 2019 till December 2019. Strains were genotyped by pulse field gel electrophoresis (PFGE) for genetic relatedness. Whole genome sequencing (WGS) was performed to observe relationships with global strains using multilocus sequence typing (MLST). Antimicrobial genes, capsular loci-K-types, plasmids types and virulence genes were also identified using whole genome sequence data. ResultsAll five CRKP were determined to have blaKPC-2 with or without blaOX-A48 and blaNDM-2. The molecular genotyping by PFGE showed 100% similarity among the five isolates. The MLST allelic profile analysis clonally clustered our strains with SL-258, CG-11 and ST11 mainly reported from South Asia. Further molecular characterization of the capsular K-locus and O-locus genes, revealed the strains to belong to KL-47 type and OL101 type respectively. The core genome typing suggests that our strains were clonally related to Chinese strains with less than five chromosomal nucleotides differences. ConclusionEpidemiological and molecular analyses confirmed that these KPC-producing K. pneumoniae strains are from a single clone that caused multiple nosocomial infections in one health institution. This finding highlights the importance to sustain the surveillance and infection prevention efforts and to step up active screening to prevent the spread of nosocomial infection.

Prevalence and antimicrobial susceptibility profiles of ESBL-producing Klebsiella Pneumoniae from broiler chicken farms in Shandong Province, China

Klebsiella pneumoniae (K. pneumoniae) is a conditionally pathogenic bacterium present in the intestinal or the respiratory tract of animals, and it is a common factor in acquired infections and a major threat to public health. Increased production of extended-spectrum β-lactamases (ESBLs) has become a serious issue in the treatment of K. pneumoniae infections. In this study, we examined the serotypes and antibiotic resistance profiles of K. pneumoniae isolated from broiler chickens on farms in Shandong Province, China. The K. pneumoniae isolation rate was 4.67% (33/707), and the serotype Capsular K54 (42.42%, 14/33) was the most prevalent serotype in broilers in Shandong. The antimicrobial susceptibility assay revealed that the 33 isolates were resistant to 28 antimicrobial drugs to varying degrees; among these, the highest resistance rate was observed for tetracyclines (90.91%), and the lowest rate of resistance was observed for moxifloxacin and fosfomycin (0%). The multidrug resistance (MDR) rate was 87.88% (29/33). The carrying rate of β-lactam-resistance genes was as high as 100%, with blaSHV having the highest rate (93.94%). It is worth noting that one carbapenem-resistant K. pneumoniae (CRKP) isolate carrying blaNDM-1 and one colistin-resistant K. pneumoniae (COLR-KP) isolate carrying mcr-3 were found in broiler chickens. This study indicates that ESBL-producing CRKP isolates and COLR-KP isolates have emerged on poultry farms in Shandong and could be a potential threat to food safety and public health.

Outcomes and Risk Factors of Bloodstream Infections Caused by Carbapenem-Resistant and Non-Carbapenem-Resistant Klebsiella pneumoniae in China.

PurposeTo compare antimicrobial resistance, virulence, clinical characteristics, and risk factors between carbapenem-resistant K. pneumoniae (CRKP) and carbapenem-susceptible K. pneumoniae (CSKP) isolates from patients with bloodstream infections (BSIs) in China.Patients and MethodsThe clinical data of 103 patients with K. pneumoniae BSI from 10 hospitals were retrospectively analyzed. The minimum inhibitory concentrations of 15 antibiotics against the bacteria were determined. A Galleria mellonella infection model was used to evaluate virulence of the isolates. Kaplan–Meier curves were calculated to evaluate the 28-day and in-hospital survival rates of the isolates. The risk factors for CRKP and CSKP infection and respective mortality rate were evaluated by univariate analysis, and independent risk factors were evaluated using the multivariate logistic regression model.ResultsOur results indicated that CRKP isolates were more resistant to most tested antibiotics than CSKP isolates. The G. mellonella infection model was used to demonstrate that CRKP isolates were more virulent than CSKP isolates. We found that in-hospital deaths occurred in 39.3% (22/56) of patients with CRKP BSIs and were significantly higher than those in patients with CSKP infections (19.1%, 9/47). Patients infected with CRKP isolates had poorer outcomes than those infected with the CSKP strains. For in-hospital mortality of CRKP BSIs, the independent risk factors included carbapenem-resistant Enterobacterales bacteremia and length of hospitalization after the onset of BSI.ConclusionOur findings confirm that CRKP isolates are more drug-resistant than CSKP isolates and are associated with poorer outcomes. To prevent CRKP infection, strict infection control strategies and active surveillance should be implemented in hospitals.

Changes in molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae in the intensive care units of a Greek hospital, 2018-2021.

The spread of multi-drug resistant (MDR) Gram-negative bacteria, including Klebsiella pneumoniae, constitutes a global threat. The most frequent mechanism of acquired carbapenem resistance is the production of carbapenemases, especially KPC, NDM, VIM, IMP and OXA-48. We analyzed the epidemiological trend of carbapenem resistance genes of carbapenem-resistant K. pneumoniae (CRKP) strains isolated from critically ill patients in a Greek tertiary hospital. The study included 150 CRKP isolates collected from 116 (77.4%) patients hospitalized in the adult ICU and 17 (11.3%) each in the pediatric and the two neonatal ICUs between March 2018 and March 2021. Identification and antimicrobial susceptibility testing were performed using VITEK-2. A multiplex lateral flow immunoassay was used for the detection of carbapenemases, while the detection of bla VIM, bla KPC, bla NDM, bla IMP and bla OXA-48-like genes was achieved by multiplex PCR. The bla NDM was mainly detected in adults (54/116, 46.9%), while in children the most often detected gene was bla KPC (24/34, 70.6%). The predominant carbapenem resistance gene during 2018-2019 was bla KPC alone or in combination with bla VIM, reaching 44.4% in 2019, while during 2020-2021 the detection of bla NDM prevailed significantly, reaching 45.5 and 60.7% for 2020 and 2021, respectively. A shift in the molecular epidemiology of CRKP was seen during 2018-2021, which is probably associated with the recent excessive empiric use of newer antimicrobials. Surveillance studies and proper and strict implementation of infection control measures are highly needed to decrease the spread of MDR bacteria, including CRKP.

Molecular characterization of carbapenemase producing Klebsiella pneumoniae strains by multiplex PCR and PFGE methods: The first K.pneumoniae isolates co-producing OXA-48/KPC and KPC/NDM in Turkey

IntroductionCarbapenems are frequently used in the treatment of multidrug-resistant infections caused by Klebsiella pneumoniae. The aim of the study is to definition and incidence of transferable carbapenemase genes of carbapenem resistant K. pneumoniae (CRKP) and to determine clonal relatedness of these strains in tertiary care hospital in Turkey. MethodsIdentification of all 100 K. pneumoniae isolates and low sensitivity to any of the carbapenem group antibiotics were determined by Vitek-2 (BioMérieux, France). The frequency of carbapenemase genes (blaOXA-48, blaNDM, blaKPC, blaVIM,blaIMP) and extended spectrum beta-lactamase (ESBL) genes (blaCTX-M, blaSHV, blaTEM) which frequently detected in Turkey, have been investigated by multiplex polymerase chain reaction (PCR). Clonal relatedness was determined using Pulsed-field gel electrophoresis(PFGE). ResultsNinety five isolates carried at least one of the carbapenemase genes (81.05% blaOXA-48, 38.9% blaNDM, 9.47% blaKPC,1.05% blaVIM). One isolate was carried the blaOXA-48+KPC and the two isolates were carried the blaKPC+NDM. PFGE demonstrated the presence of 24 pulse types and 63.09% of the isolates were in four main pulse types. ConclusionsThis study demonstrated the incidence of blaNDM is beginning to reach endemic levels, in addition to blaOXA-48 found endemic in Turkey. To our knowledge, this is the first report of the co-production of these two genes (blaKPC + NDM and blaOXA-48 + KPC) in CRKP isolates.

Co-existence of NDM-1 and OXA-48 genes in Carbapenem Resistant Klebsiella pneumoniae clinical isolates in Kafrelsheikh, Egypt.

The noteworthy spread of carbapenem-resistant K. pneumoniae (CR-KP) isolates represents a significant safety threat. Determination of the carbapenemase genes incidence among CR-KP clinical isolates in Kafrelsheikh, Egypt. A total of 230 K. pneumoniae isolates were recovered from four hospitals in Kafrelsheikh, Egypt. Susceptibility testing was conducted using Kirby-Bauer method and automated-Vitek2 system. CR-KP isolates were tested using modified Hodge test (MHT) and combined disk synergy test. PCR and DNA sequencing were conducted for CR-KP isolates to recognize the included carbapenemase-genes. Out of 230 K. pneumoniae isolates, 50 isolates presented resistance to carbapenem (meropenem). All 50 CR-KP isolates were multidrug-resistant (MDR). Genes like blaNDM-1 and blaOXA-48 were the only detected genes among CR-KP with an incidence of 70.0% and 52.0%, respectively. Up to 74.0% of the tested isolates carried at least one of the two recorded genes, among them 48.0% co-harbored both blaNDM-1 and blaOXA-48 genes. The accession-numbers of sequenced blaNDM-1 and blaOXA-48 genes were MG594615 and MG594616, respectively. This study reported a high incidence of MDR profile with the emergence of blaNDM-1 and blaOXA-48 genes co-existence in CR-KP isolates in Kafrelsheikh, Egypt. Hence, more restrictions should be applied against the spread of such serious pathogens.

The Detection of Hypermucoviscous Carbapenem-Resistant Klebsiella pneumoniae from a Tertiary Teaching Hospital in Malaysia and Assessment of Hypermucoviscous as Marker of Hypervirulence.

Background: Hypermucoviscous carbapenem-resistant Klebsiella pneumoniae (hmCRKp) is emerging globally and approaching the worst-case scenario in health care system. Aims: The main objective in this study was to determine the hypermucoviscous characteristics among the carbapenem-resistant K. pneumoniae (CRKp) isolated from a teaching hospital in Malaysia. The association of hypermucoviscous phenotype with the virulence traits and clinical presentations were also investigated. Methods: A retrospective study was conducted in University Malaya Medical Centre (UMMC). The presence of hypermucoviscous K. pneumoniae was identified among a collection of CRKp clinical isolates (first isolate per patient) from 2014 to 2015 using string test. Correlation between clinical and microbial characteristics of the hmCRKp was investigated. Results: A total of nine (7.5%) hmCRKp were detected among 120 CRKp isolates. Majority of the isolates were hospital acquired or health care-associated infections. None of the patients had typical pyogenic liver abscess. All of the hmCRKp isolates harbored carbapenemase genes and were multidrug resistant. K1/K serotype, peg-344, allS, and magA were not identified among hmCRKp isolates, whereas aerobactin siderophore receptor gene (iutA), iroB, rmpA, and rmpA2 were detected. Only three hmCRKp isolates were resistant to serum bactericidal. Conclusions: All the isolates presented inconclusive evidence for the interpretation of hypervirulence. Therefore, more study should be performed in the future to have a better understanding of the virulence mechanisms in correlation with the clinical and microbial determinants.

The Plasmid-Borne tet(A) Gene Is an Important Factor Causing Tigecycline Resistance in ST11 Carbapenem-Resistant Klebsiella pneumoniae Under Selective Pressure

The emergence and prevalence of tigecycline-resistant Klebsiella pneumoniae have seriously compromised the effectiveness of antimicrobial agents in the treatment of infections. To explore the role of the plasmid-borne tet(A) gene in tigecycline resistance in carbapenem-resistant K. pneumoniae (CRKP), a total of 63 CRKP isolates were collected from a tertiary hospital in Hangzhou, China. The minimum inhibitory concentration (MIC) of tigecycline, mutation rate of tet(A) gene, genetic surroundings of tet(A)-carrying transmissible plasmid and the contribution of tet(A) mutation to tigecycline resistance were analyzed using antimicrobial susceptibility test, whole-genome sequencing, tigecycline resistance evolution experiment, and plasmid conjugation experiment. Our results showed that 52.4% (33 isolates) of the test isolates carried the tet(A) gene; among them, 75.8% (25 isolates) exhibited a tigecycline non-susceptible phenotype (MIC = 4 mg/L). Three clonal groups (cluster I, cluster II, and cluster III) were identified in these tet(A)-bearing isolates. All 17 isolates belonged to serotype KL21 (cluster I), which differed by only 13 SNPs, suggesting a clonal spread of tet(A)-positive ST11 K. pneumoniae with serotype KL21 occurred in the sampling hospital. The induction of tigecycline resistance experiments showed that 71.4% of strains evolved tet(A) mutations and developed a high-level tigecycline resistance. Eight amino acid substitutions were identified in these mutants. The most common amino acid substitution was A370V, followed by S251A and G300E. Twelve isolates carrying tet(A) mutants succeeded in the filter mating experiment with a conjugation efficiency of 10–3–10–8. Tigecycline MICs in E. coli EC600 transconjugants with a mutated tet(A) were 2 to 8-fold higher than those in E. coli EC600 transconjugants with a wild-type tet(A). One ColRNAI/IncFII type and two IncFII type tet(A)-bearing conjugative plasmids were identified in this study, including a class 1 integron containing multiple antibiotic resistance genes, i.e., tet(A), qnrS1, blaLAP–2, catA2, sul2, and dfrA14. Our study revealed the wide-spread situation of plasmid-borne tet(A) gene in clinical CRKP, and mutation of tet(A) is a potential driven force that lead to tigecycline resistance.