Carbapenem-resistant Klebsiella Pneumoniae Isolates Research Articles

Genomic Evolution of ST11 Carbapenem-Resistant Klebsiella pneumoniae from 2011 to 2020 Based on Data from the Pathosystems Resource Integration Center.

(1) Objective: ST11 carbapenem-resistant Klebsiella pneumoniae (CRKP) is widespread throughout the world, and the mechanisms for the transmission and evolution of major serotypes, ST11-KL47 and ST11-KL64, were analyzed to investigate the global distribution and evolutionary characteristics of ST11 CRKP; (2) Methods: The Pathosystems Resource Integration Center (PATRIC) database was downloaded and all K. pneumoniae from 2011 to 2020 were screened to obtain ST11 CRKP genome assemblies with basic information. The relationship of serotype evolution between KL47 and KL64 was then investigated using statistical and bioinformatic analysis; (3) Results: In total, 386 ST11 CRKP isolates were included for analysis. Blood (31.09%, 120/386), respiratory tract (23.06%, 89/386), and feces (20.21%, 78/386) were the major sources of samples. China was the leading country where ST11 CRKP was isolated. KL47 and KL64 were found to be the most prevalent serotypes. ST11-KL64 CRKP [median 78(P25~P75: 72~79.25)] had remarkably more virulence genes than the KL47 [median 63(P25~P75: 63~69)], and the distinction was statistically significant (p < 0.001). A differential comparison of virulence genes between KL47 and KL64 revealed 35 differential virulence genes, including rmpA/rmpA2, iucABCD, iutA, etc. The comparison of the recombination of serotype-determining regions between the two serotypes revealed that KL64 CRKP carried more nucleotide sequences in the CD1-VR2-CD2 region than KL47 CRKP. More nucleotide sequences added approximately 303 base pairs (bp) with higher GC content (58.14%), which might facilitate the evolution of the serotype toward KL64; (4) Conclusions: KL47 and KL64 have become the predominant serotypes of ST11 CRKP. KL64 CRKP carries more virulence genes than KL47 and has increased by approximately 303 bp through recombinant mutations, thus facilitating the evolution of KL47 to KL64. Stricter infection prevention and control measures should be developed to deal with the epidemic transmission of ST11-KL64 CRKP.

Reduced porin expression with EnvZ-OmpR, PhoPQ, BaeSR two-component system down-regulation in carbapenem resistance of Klebsiella Pneumoniae based on proteomic analysis.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has proven to be an urgent threat to human health. Proteomics (TMT/LC-MS/MS) and bioinformatics approaches were employed to explore the potential mechanisms underlying carbapenem resistance. Proteomic profiling of CRKP and susceptible KP (sKP) isolates revealed the involvement of outer membrane, beta-lactam resistance pathway, and two-component systems (TCSs) in carbapenem resistance. 27 CRKP strains and 27 susceptible Klebsiella pneumoniae strains were isolated from inpatients at the Second Xiangya Hospital, China to verify the mechanisms. Modified carbapenem inactivation method (mCIM) and PCR of common carbapenem resistance genes confirmed that 77.8% (21/27) of CRKP isolates were carbapenemase-producing. Porin decrease in CRKP isolates was found by SDS-PAGE and mRNA levels of major porins (OmpK35 and OmpK36). RT-qPCR detection of two-component systems (envZ, ompR, phoP, phoQ, baeS and baeR) revealed down-regulation of EnvZ-OmpR, PhoPQ, BaeSR TCSs. Expression of the TCSs, except ompR, were closely correlated with OMPs with the R-value >0.7. Together, this study reaffirmed the significance of the β-lactam resistance pathway in CRKP based on proteomic analysis. OmpK35/36 porin reduction and the controversial downregulation of EnvZ-OmpR, PhoPQ, and BaeSR TCSs were confirmed in carbapenem resistance of Klebsiella pneumoniae.

Arm race among closely-related carbapenem-resistant Klebsiella pneumoniae clones

Multiple carbapenem-resistant Klebsiella pneumoniae (CRKP) clones typically co-exist in hospital wards, but often certain clones will dominate. The factors driving this dominance are largely unclear. This study began from a genomic epidemiology analysis and followed by multiple approaches to identify the potential mechanisms driving the successful spread of a dominant clone. 638 patients in a 50-bed ICU were screened. 171 (26.8%) and 21 had CRKP from swabs and clinical specimens, respectively. Many (39.8% of those with ≥7-day ICU stay) acquired CRKP. After removing 18 unable to recover, 174 CRKP isolates were genome sequenced and belonged to six sequence types, with ST11 being the most prevalent (n = 154, 88.5%) and most (n = 169, 97.1%) carrying blaKPC-2. The 154 ST11 isolates belonged to 7 clones, with one (clone 1, KL64 capsular type) being dominant (n = 130, 84.4%). Clone 1 and the second-most common clone (clone 2, KL64, n = 15, 9.7%) emerged simultaneously, which was also detected by genome-based dating. Clone 1 exhibited decreased biofilm formation, shorter environment survival, and attenuated virulence. In murine gut, clone 1 outcompeted clone 2. Transcriptomic analysis showed significant upregulation of the ethanolamine operon in clone 1 when competing with clone 2. Clone 1 exhibited increased utilization of ethanolamine as a nitrogen source. This highlights that reduced virulence and enhanced ability to utilize ethanolamine may promote the success of nosocomial multidrug-resistant clones.

Genomic insights into blaNDM-carrying carbapenem-resistant Klebsiella pneumoniae clinical isolates from a university hospital in Thailand

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates is a serious threat to global health. Here, we elucidate the genetic features of blaNDM-carrying CRKP clinical isolates from a university hospital in Thailand. The entire genomes of 19 CRKP isolates were extracted and then sequenced using the MGISEQ200 platform. Using various bioinformatics tools, we analyzed the antimicrobial resistance (AMR), virulence factors, gene transfer, bacterial defense mechanisms, and genomic diversity of the CRKP isolates. The sequence type (ST) 16 was found in most of the isolates, along with carriages of the blaNDM-1, blaOXA-232, and blaCTX-M-15 genes. The IncFIB(pQil), Col440II, and ColKP3 plasmids were identified with high frequency. The CRKP isolates harbored genes encoding for virulence factors such as adherence, biofilm formation, immune evasion, and iron uptake. The CRISPR-Cas region in the CRKP9 isolate consisted of 28 distinct spacer sequences. The genomes of the CRKP isolates presented restriction-modification (R-M) sites (M.Kpn34618Dcm and M.Kpn928I) and integrated bacteriophage genomes (Klebsiella phage ST16-OXA48phi5.4 and Enterobacteria phage mEp390). Bottromycin and sactipeptides were also identified. The isolates could be separated into three clades according to STs and pairwise single nucleotide polymorphism (SNP) distance. Pairwise average nucleotide identity (ANI) values revealed intra-species. These findings support the importance of whole-genome sequencing (WGS) to the rapid and accurate genomic analysis of clinical isolates of CRKP.

Insertion sequence mediating mrgB disruption is the major mechanism of polymyxin resistance in carbapenem-resistant Klebsiella pneumoniae isolates from China

ObjectivesInfections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP) pose a huge health challenge worldwide. The aim of this study was to evaluate the incidence of polymyxin resistance in clinical CRKP isolates in China and to characterize the molecular mechanisms underlying these polymyxin-resistant CRKP (PR-CRKP) isolates. MethodsA total of 493 CRKP clinical isolates from patients were collected from six tertiary-care hospitals in China during 2017–2018. Minimum inhibitory concentrations of polymyxin B and colistin were determined using the broth microdilution method. PR-CRKP isolates were identified and subjected to whole-genome sequencing. Quantitative real-time PCR and structural modelling analysis were also performed. ResultsWe observed a 2.2% (11/493) polymyxin resistance rate in this multicentre cohort. Polymyxin B MICs ranged from 4 to 64 μg/mL and colistin MICs ranged from 8 to 128 μg/mL in 11 PR-CRKP isolates. Key genetic variations identified in PR-CRKP isolates involved eight disruptions (seven insertional inactivation by an insertion sequence [IS] element, one frameshift deletion) in mgrB, and three missense mutations in pmrA, pmrB, and phoP. ISKpn26 was the predominant IS (4/7), and three of these occurred in nucleotide position 74 in the mgrB gene. In addition, we reported a novel mutation S62R in pmrB that may confer polymyxin resistance in K. pneumoniae. ConclusionsOur findings highlight the multifaceted molecular mechanisms of polymyxin resistance in CRKP.

Rapid Detection of Carbapenem-Resistant Klebsiella pneumoniae Using Machine Learning and MALDI-TOF MS Platform.

BackgroundRapid detection of carbapenem-resistant Klebsiella pneumoniae (CRKP) is essential for specific antimicrobial therapy. Machine learning techniques combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) can be used as a rapid, reliable, sensitive, and low-cost species identification method.MethodsClinically collected K. pneumoniae were subjected to MALDI-TOF MS analysis. A random forest (RF) algorithm and non-linear support vector machine (SVM) were used to construct the RF, SVM, and dimension reduction (SVM-K) models, and their performance was assessed for accuracy, sensitivity, specificity, and area under the subject worker curve (AUC).ResultsThe RF, SVM and SVM-K models showed good classification performance with 0.88, 0.88, and 0.91 accuracy, 0.82, 0.85, and 0.89 sensitivity, 0.93, 0.92, and 0.94 specificity with an AUC of 0.9013, 0.9298, and 0.9356, respectively. For the SVM-K model, the optimal dimension reduction was 105 to 153, and the average accuracy was >0.9. The top 10 peak features of significance according to the RF algorithm with 6515 Da appeared in 56.8% of CRKP isolates and 5.3% of CSKP isolates, which indicated the best classification performance.ConclusionThe three RF, SVM, and SVM-K models showed excellent classification performance differentiating the CRKP from CSKP; the SVM-K model was the best. Data analysis with machine learning combined with MALDI-TOF MS can be employed as a rapid and inexpensive alternative to existing detection methods.

Epidemiology and Genomic Characteristics of Bloodstream Infection Caused by Carbapenem-Resistant Klebsiella pneumoniae With Decreased Susceptibility to Aztreonam/Avibactam in China.

Aztreonam/avibactam (AZA), as one of the novel β-lactamases and β-lactamase inhibitor combinations, is considered to be a promising option for bloodstream infection (BSI) of carbapenem-resistant Klebsiella pneumoniae (CR-Kp). However, decreased susceptibility of AZA activity in Enterobacterales has been reported. The aim of this study was to identify the mechanisms of BSI CR-Kp with decreased susceptibility of AZA (minimal inhibitory concentration above 16/4 mg/L) (AZAH-Kp). Nine BSI AZAH-Kp isolates were screened from 317 CR-Kp isolates in Blood Bacterial Resistant Investigation Collaborative System (BRICS) program. Whole genome sequencing, bioinformatics analysis, and the relative expression of blaKPC , ompK35, and ompK37 were explored for CR-Kp with decreased susceptibility to AZA. The results revealed that elevated inhibitory concentration of AZA has emerged in CR-Kp before previous clinical exposure. In addition, decreased AZA susceptibility was associated with higher KPC expression and changes in OmpK35-37.

Inhibitory effects of copper, brass, and stainless steel surfaces on multidrug-resistant microorganisms

Background: The aim of this study was to analyze the inhibitory effects of copper, brass (78% copper, 22% tin), and stainless steel surfaces on multidrug-resistant Acinetobacter baumannii (MRAB), extended-spectrum beta-lactamase (ESBL) Escherichia coli, and carbapenem-resistant Klebsiella pneumoniae (CRKP). Methods: MRAB, ESBL E. coli, and CRKP were isolated at Uijeongbu St. Mary’s Hospital in 2020. A. baumannii ATCC BAA-747, E. coli ATCC 25922, and K. pneumoniae ATCC 700603 were used as reference strains. The initial bacterial cell count of each inoculum was adjusted to 8 log CFU/mL using phosphate buffered saline, Copper, brass, and stainless steel plates were inoculated with 9 mL of MRAB, ESBL E. coli, and CRKP inoculum solutions. The bacterial cell count was measured from the beginning to the 20th day in an incubator maintained at 35°C. Results: MRAB, ESBL E. coli, and CRKP isolates were not detected on the copper and brass plates after 4, 5.5, and 6.5 hours, respectively. MRAB, ESBL E. coli, and CRKP isolates were not detected on the stainless steel plate after 15, 20, and 20 days, respectively. The bactericidal effects of copper and brass were much stronger than those of stainless steel. Conclusion: The use of copper and copper alloys should be considered to prevent crossinfection in hospitals.

Risk Factors for the Development of Colistin Resistance during Colistin Treatment of Carbapenem-Resistant Klebsiella pneumoniae Infections.

ABSTRACTColistin is one of the last-resort options for carbapenem-resistant Klebsiella pneumoniae (CRKP) infections if novel antibiotics are unavailable, where the development of colistin resistance during treatment represents a major challenge for clinicians. We aimed to investigate the risk factors associated with the development of colistin resistance in patients with CRKP infections following colistin treatment. We conducted a retrospective case-control study of patients with CRKP strains available before and after colistin treatment at a medical center in Taiwan, between October 2016 and November 2020. Cases (n = 35) included patients with an initial colistin-susceptible CRKP (ColS-CRKP) strain and a subsequent colistin-resistant CRKP (ColR-CRKP) strain. Controls (n = 18) included patients with ColS-CRKP as both the initial and subsequent strains. The 30-day mortality rate after the subsequent CRKP isolation was not different between cases and controls (12/35 [34%] versus 5/18 [28%] [P = 0.631]). blaKPC (n = 38) and blaOXA-48 (n = 11) accounted for the major mechanisms of carbapenem resistance. Alterations in mgrB were found in 18/35 (51%) ColR-CRKP strains, and mcr-1 was not detected in any of the strains. More patients received combination therapy in the control group than in the case group (17/18 versus 21/35 [P = 0.008]). The logistic regression model indicated that combination therapy with tigecycline was protective against the acquisition of colistin resistance (odds ratio, 0.17; 95% confidence interval, 0.05 to 0.62 [P = 0.008]). We observed that the inclusion of tigecycline in colistin treatment mitigated the risk of acquiring colistin resistance. These results offer insight into using the combination of tigecycline and colistin for the treatment of CRKP infections in antimicrobial stewardship.IMPORTANCE Treatment of carbapenem-resistant Klebsiella pneumoniae (CRKP) infections is challenging due to the limited options of antibiotics. Colistin is one of the last-resort antibiotics if novel antimicrobial agents are not available. It is crucial to identify modifiable clinical factors associated with the emergence of resistance during colistin treatment. Here, we found that the addition of tigecycline to colistin treatment prevented the acquisition of colistin resistance. Colistin-tigecycline combination therapy is therefore considered a hopeful option in antimicrobial stewardship to treat CRKP infections.

Molecular Epidemiology, Microbial Virulence, and Resistance of Carbapenem-Resistant Enterobacterales Isolates in a Teaching Hospital in Guangzhou, China.

Infection caused by carbapenem-resistant Enterobacterales (CRE) is a global public health problem. We performed whole-genome sequencing to investigate the molecular epidemiological characteristics of local CRE infections and understand the prevalence of hypervirulent carbapenem-resistant Klebsiella pneumoniae (CRKP). Analysis of multiLocus sequence typing (MLST), antibiotic resistance genes, plasmid replicons, virulence genes, and the genetic environment was also performed. Klebsiella pneumoniae (89, 60.95%) was the most common CRE species, primarily prevalent in the intensive care unit (36, 40.45%). Most CRE strains showed a high resistance rate to multiple antibiotics, especially cephalosporins and carbapenems. However, most of these isolates were susceptible to tigecycline (81.7%). Notably, the predominant sequence type (ST) of CRKP isolates was ST11 (80.90%, 72/89), with 93.05% as Klebsiella pneumoniae carbapenemase (KPC)-ST11. In Escherichia coli isolates, ST410 (21.43%, 6/28) was the predominant type, with approximately half carrying blaNDM-5, and importantly, the ST167 carbapenem-resistant Escherichia coli (CRECO) harbors both New Delhi metallo-β-lactamase (NDM)-5 and KPC-2. In Enterobacter cloacae isolates, three cases of ST88 were carrying the blaNDM-1 gene, and the ST594 carbapenem-resistant Enterobacter cloacae (CRECC) carrying NDM-1 and KPC-2 has also been identified. In addition, we found three novel STs, ST5386-ST5388. The IncFII (pHN7A8) (98.41%, 62/63) was the most common plasmid replicon type in KPC-2-producing CRKP strains, and the predominant plasmid ST of IncF was [f33:A-:B-] (n = 73). Two CRKP isolates were found to carry 4 virulence genes (iutA, iroB, rmpA, and rmpA2). As concluded, among CRKP strains, ST11 was the predominant ST with blaKPC-2, and a large proportion of CRKP strains co-harbor blaKPC-2, blaSHV, blaCTX-M, blaTEB-1B, and fosA. The predominant carbapenemase genes carried by CRECO and CRECC were blaNDM-1 and blaCTX-M, respectively.

The Rapid Emergence of Ceftazidime-Avibactam Resistance Mediated by KPC Variants in Carbapenem-Resistant Klebsiella pneumoniae in Zhejiang Province, China.

Ceftazidime-avibactam (CAV) is a new treatment option against carbapenem-resistant Klebsiella pneumoniae (CRKP) infections. However, the rapid emergence of CAV resistance mediated by KPC variants has posed a severe threat to healthcare after its clinical application. The characteristics of CAV resistance in CRKP strains needs to be determined in China. A total of 477 CRKP isolates were collected from 46 hospitals in Zhejiang Province from 2018 to 2021. The results demonstrated that CAV had a potent activity against 94.5% of all CRKP (451/477, 95% CI: 93.0–96.1%) and 86.0% of CRKP strains carrying blaKPC genes (410/477, 95% CI: 83.5–88.4%). A total of 26 CAV-resistant strains were found. Among these strains, sixteen harbored metallo-β lactamases, and two carried KPC-2 carbapenemase and mutated ompK35 and ompK36. Eight CRKP strains encoded KPC-33 or KPC-93, belonging to ST11, among which seven strains were detected in patients hospitalized in 2021 after exposure to CAV and one strain was associated with intra-hospital spread. CAV is a potent agent in vitro against CRKP strains. The rapid development of CAV resistance mediated by various KPC variants after a short period of CAV treatment has increased and brought difficulties in treating infections caused by CRKP strains, especially those belonging to ST11. The surveillance of bacterial resistance against CAV is highly recommended due to the steep development of CAV resistance and rapid evolution of KPC enzymes.

Disinfection Strategies for Carbapenem-Resistant Klebsiella pneumoniae in a Healthcare Facility.

Disinfectant resistance is evolving into a serious problem due to the long-term and extensive use of disinfectants, which brings great challenges to hospital infection control. As a notorious multidrug-resistant bacterium, carbapenem-resistant Klebsiella pneumoniae (CRKP) is one of the most common and difficult pathogens of nosocomial infection. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) tests of seven kinds of disinfectants (0.1% benzalkonium bromide, 4% aqueous chlorhexidine, 75% alcohol, entoiodine II, 2% glutaraldehyde, 2000 mg/L chlorine-containing disinfectants, and 3% hydrogen peroxide) were detected by the broth dilution method. Three efflux pump genes (oqxA, oqxB, and qacE∆1-sul1) were detected by PCR. The mean MIC value of aqueous chlorhexidine from the intensive care unit (ICU) (0.0034%) was significantly higher than that from non-ICUs (0.0019%) (p < 0.05). The positive rates of three efflux pump genes oqxA, oqxB and qacE∆1-sul1 were 60.9% (39/64), 17.2% (11/64) and 71.9% (46/64) in the detected CRKP isolates, respectively. This study discovered that CRKP strains demonstrated extensive resistance to clinical disinfectants and suggest that it is necessary to perform corresponding increases in the concentration of aqueous chlorhexidine and chlorine-containing disinfectants on the basis of current standards in the healthcare industry.

Three Novel Sequence Types Carbapenem-Resistant Klebsiella pneumoniae Strains ST5365, ST5587, ST5647 Isolated from Two Tertiary Teaching General Hospitals in Shanxi Province, in North China: Molecular Characteristics, Resistance and Virulence Factors.

BackgroundCarbapenem-resistant Klebsiella pneumoniae (CRKP) represents a significant threat to public health and has already drawn worldwide attention. Hence, we aim to comprehensively analyze the case condition, as well as molecular epidemiology, resistance and virulence of three CRKP isolates with new sequence types (STs).MethodsThree CRKP were collected from November 2019 to April 2021. The three patients’ clinical characteristics were analyzed through His system. In order to screen phenotype of metallo-carbapenemase, the modified Carbapenem Inactivation Method (mCIM) and EDTA-modified Carbapenem Inactivation Method (eCIM) were conducted. Three isolates were subjected to antimicrobial susceptibility testing (AST) using the agar dilution method or minimal broth dilution method. The string test, the sedimentation assay, biofilm formation and the serum resistance assay were performed as phenotypic experiments to assist in evaluating virulence. The presence of resistance and virulence genes were detected by Whole-Genome Sequencing (WGS). Serotypes and new STs were compared and determined by multi-locus sequence typing (MLST).ResultsOverall, all Klebsiella pneumoniae isolates were multi-resistant, but sensitive to tigecycline and colistin. Among them, all formed biofilms, strain 1 and strain 2 were classified as moderate-producers, while strain 3 as weak-producer. The results of the serum resistance assay indicated that only strain 2 was resistant. From WGS analysis, it showed that all isolates co-harbored multiple resistance genes, such as carbapenemase genes, sulfonamides, fluoroquinolones, aminoglycosides, and tetracyclines. Meanwhile, several virulence genes were also contained, including siderophores, fimbriae, capsule and lipopolysaccharides-associated genes. The serotypes of strain 1 and strain 2 manifested K35 and KL47, respectively.ConclusionThree novel ST5365, ST5587, ST5647 were first discovered in North China. Our study suggested that we should pay more attention to their resistance. And the results will help treat CRKP infections caused by these novel STs.

Evaluation of In Vitro Efficacy of Ceftazidime-Avibactam, Meropenem, and Colistin Single and Binary Combinations Against Carbapenem Resistant Klebsiella pneumoniae Strains Isolated from Various Clinical Specimens

It has been reported that many antibiotics used today, including the carbapenem group, fail to treat Klebsiella pneumoniae infections effectively. Despite many studies in recent years, the definitive treatment for carbapenem-resistant Klebsiella pneumoniae (CRKP) infections is still uncertain. In this study, it was aimed to investigate in vitro activities of colistin (COL) and meropenem (MEM), which are frequently used in the treatment of CRKP infections, and ceftazidime-avibactam (CZA), which is recently used in our country, alone or in combination against different CRKP isolates having different carbapenem resistance mechanisms andto analyze whether the presence of colistin resistance, which is an important problem in CRKP strains, influences the drug interaction results. This study was carried out in 42 K.pneumoniae isolates, which were isolated from various clinical samples as an infectious agent in Süleyman Demirel University Faculty of Medicine, Department of Medical Microbiology, Bacteriology Laboratory and whose carbapenem resistance was confirmed by carbapenemase inactivation test. The carbapenemase genes of the isolates were determined by the polymerase chain reaction (PCR) method. Antimicrobial susceptibilities of CRKP strains to CZA, MEM, and COL were determined by the broth microdilution method and in vitro synergy activities of dual combinations of these drugs were evaluated by checkerboard and time-kill methods. Statistical evaluation of categorical data was performed using Fisher's exact test, and p-value of less than 0.05 was considered statistically significant in terms of difference between the groups. Of the 42 CRKP isolates 34 (81%) were only OXA-48 positive, 5 (11.9%) were OXA-48+NDM and 3 (7.1%) were OXA-48+KPC positive. In the checkerboard test, synergy was detected against 97.6% of the isolates both with CZA+MEM and CZA+COL combinations, whereas this rate was 50% with MEM+COL. In the time-kill test, synergy was detected with CZA+MEM and CZA+COL combinations in the OXA-48 positive isolate and OXA-48+KPC positive isolate, while synergy was detected with CZA+COL and MEM+COL combinations in the OXA-48+NDM positive isolate. There was no significant relationship between whether the isolates were resistant to colistin or not and the checkerboard test results of antibiotic combinations (pCZA+MEM= 0.33, pCZA+COL= 0.11, pMEM+COL= 0.61). Results of our study revealed that the most common carbapenemase type in CRKP isolates was OXA-48 in our hospital, and the combinations of CZA with MEM and COL had high potential for synergism against these isolates.

An animal model of limitation of gut colonization by carbapenemase-producing Klebsiella pneumoniae using rifaximin

Current knowledge suggests that infection by carbapenem-resistant enterobacteria is preceded by gut colonization. It is hypothesized that colonization is eradicated by non-absorbable antibiotics like rifaximin. We investigated the effect of rifaximin against carbapenem-resistant Klebsiella pneumoniae (CRKP) in vitro and in a mouse model. We studied the in vitro efficacy of rifaximin against 257 CRKP clinical isolates, 188 KPC producers and 69 OXA-48 producers, by minimum inhibitory concentration and time-kill assays. We then developed a model of gut colonization by feeding 30 C57Bl6 mice with 108 cfu of one KPC-KP isolate for 7 days; mice were pre-treated orally with saline, omeprazole or ampicillin. Then, another 60 mice with established KPC-2 gut colonization received orally for 7 consecutive days rifaximin 180 mg/kg dissolved in ethanol and 4% bile or vehicle. On days 0, 3 and 7 stool samples were collected; mice were sacrificed for determination of tissue outgrowth. At a concentration of 1000 μg/ml rifaximin inhibited 84.8% of CRKP isolates. Α 3 × log10 decrease of the starting inoculum was achieved by 100, 250 and 500 μg/ml of rifaximin after 24 h against 25, 55 and 55% of isolates. Pre-treatment with ampicillin was necessary for gut colonization by KPC-KP. Treatment with rifaximin succeeded in reducing KPC-KP load in stool and in the intestine. Rifaximin inhibits at clinically meaningful gut concentrations the majority of CRKP isolates and is efficient against gut colonization by KPC-KP.

Epidemiology and genotypic characteristics of carbapenem resistant Enterobacterales in Henan, China: a multicentre study

To identify the carbapenemase and colistin resistance genes and epidemiological status of carbapenem-resistant Enterobacterales (CRE) among 7 secondary and 11 tertiary hospitals in Henan province, China. CRE isolates and clinical data of infected patients were collected from 7 secondary and 11 tertiary hospitals in Henan from July to September 2019 and analysed retrospectively. Polymerase chain reaction (PCR) and gene sequencing were performed to detect carbapenemase and colistin resistance genes; multilocus sequence typing was also performed. In total, 238 nonduplicate CRE isolates were collected mainly from the respiratory tract (54.20%) and blood (18.91%) of CRE-infected patients, half of them aged >65 years (45.80%). Carbapenem-resistant Klebsiella pneumoniae (CRKP) was the most common CRE (184 isolates, 77.31%) with constituent ratios of 84.38% and 72.54% in secondary and tertiary hospitals, respectively. In 184 CRKP isolates, blaKPC-2 (89.13%) was the dominant carbapenemase gene, and ST11 (71.74%) was the most prevalent sequence type (ST), with constituent ratios of 83.95% and 62.14% in secondary and tertiary hospitals, respectively. In 29 carbapenem-resistant Escherichia coli (CREC) isolates, blaNDM-5 (58.62%) and ST2 (31.03%) were prevalent. Four CRKP isolates and one CREC isolate were colistin-resistant and carried the plasmid-mediated mcr-1 gene. Our results showed a wide spread of CRKP-ST11 with KPC-2 carbapenemase in the analysed 18 hospitals. The CRKP constituent ratio, CRKP-STs, and CREC carbapenemase genes between secondary and tertiary hospitals showed significant differences. The emergence of a colistin-resistant CRKP with plasmid-mediated resistance gene mcr-1 is of serious concern due to the limited treatment options.

Surveillance of carbapenem-resistant Klebsiella pneumoniae in a paediatric hospital in China revealed the dynamics of carbapenemase and the prevalence of ST2735 K. pneumoniae.

Background. Carbapenem-resistant Klebsiella pneumoniae (CRKP) is increasingly isolated in paediatric wards, posing a severe threat to these vulnerable populations. This study investigated the clinical features, determinants of carbapenem resistance and clonal relatedness among CRKP in our hospital.Hypothesis. The prevalence of carbapenem-resistant K. pneumoniae in paediatric patients differs from the strains isolated from adult patients in carbapenemase and predominant clones.Aim. To investigate the pattern of carbapenemase and the clonal relationships between carbapenem-resistant Klebsiella pneumoniae in a paediatric hospital in Jiangxi Province.Methodology. Forty-five CRKP isolates were consecutively collected from October 2016 to October 2020. Medical records were reviewed to analyse clinical features. Detection of carbapenemase genes was used to determine CRKP resistance mechanisms and clonal relatedness among CRKP was identified through multi-locus sequence typing (MLST).Results. Forty-three (95.6 %) patients developed CRKP infection, and two (4.4 %) were colonized by CRKP in the urinary tract. The overall mortality rate was 13.3 %. In total, 42 (93.3 %) strains were positive for carbapenemase genes, and bla NDM (62.2 %) was the predominant gene. The MLST identified 24 different sequence types (STs) of CRKP, in which ST11 (n=8, 17.8 %) and ST2735 (n=8, 17.8 %) were the most common STs.Conclusion. The pattern of CRKP in paediatric patients reflects evolving changes. The ST2735 K. pneumoniae may present as a dangerous CRKP clone circulating in paediatric patients.

Detection of Colistin Resistance in Uropathogenic Carbapenem Resistant Klebsiella pneumoniae

Background: Colistin resistance among carbapenem-resistant Klebsiella pneumoniae (CRKp) strains at an accelerated rate poses a serious global threat which limits therapeutic options. Objective: In this study, 203 laboratory-archived uropathogenic K. pneumoniae isolates were screened for carbapenem and colistin resistance. Methods: Carbapenam resistance screening was performed using combined disc test. Colistin resistance was determined for CRKp isolates, according to clinical and laboratory standards institute- European Committee on Antimicrobial Susceptibility Testing guidelines. Polymerase Chain Reaction for carbapenamase genes (blaNDM-1, blaKPC-2 and blaOXA-1) and plasmid-mediated colistin resistance genes (mcr-1-2) were performed for the resistant isolates. Results: About 30% of the CRKp isolates were resistant to colistin; 57.1% and 35.7% of the isolates carried blaNDM-1 and blaOXA-1 gene, respectively. None of the isolates showed positive for blaKPC-2 and mcr (1-2) genes. Conclusion: Colistin resistance in the absence of a plasmid borne transfer mechanism was observed among our CRKp strains. Escalating colistin resistance is a crucial obstacle in treatment of K. pneumoniae infections and repurposing their usage is important to combat and control the resistance.

Molecular characterisation of carbapenem-resistant Klebsiella pneumoniae clinical isolates: preliminary experience from a tertiary care teaching hospital in the Himalayas.

There is a lack of whole-genome sequencing (WGS) data on multidrug-resistant (MDR) bacteria from the Uttarakhand region of India. The aim of this study was to generate WGS data of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates recovered from patients in Uttarakhand's tertiary care centre. A cross-sectional study included 29 MDR K. pneumoniae test isolates obtained from various clinical samples submitted to the bacteriology laboratory for culture and sensitivity testing from July 2018 to August 2019. After preliminary identification and antibiotic susceptibility testing, these isolates were subjected to WGS. A total of 27 of 29 isolates were CRKP. ST14 was the most common sequence type (n=8 [29.6%]). Carbapenem resistance was mainly encoded by OXA-48-like genes (21/27 [77.8%]). All isolates had a varied arsenal of resistance genes to different antibiotic classes. KL2 (9/27 [33.3%]) and KL51 (8/27 [29.6%]) were dominant K loci types. O1 and O2 together accounted for 88.9% (n=27) of CRKP isolates. Genes encoding yersiniabactin (ybt) and aerobactin (iuc) were identified in 88.9% (24/27) and 29.6% (8/27) of isolates. The predominant plasmid replicons present were ColKP3 (55.5%), IncFII(K) (51.8%) and IncFIB(pQil) (44.4%). This study emphasises the need for continued genomic surveillance of MDR bacteria that could be instrumental in developing treatment guidelines based on integrating phenotypic and molecular methods.

Molecular Epidemiology and Risk Factors of Carbapenem-Resistant Klebsiella Pneumoniae Bloodstream Infections in Wuhan, China

The clinical characteristics and microbiological data of patients with K. pneumoniae bloodstream infections (BSI) from January 2018 to December 2020 were retrospectively analyzed to study the molecular epidemiology of Carbapenem-resistant Klebsiella pneumoniae (CRKP). We also aimed to identify the risk factors for the development of CRKP BSI. This retrospective study was conducted at Renmin Hospital of Wuhan University from January 2018 to December 2020. The date of non-duplicate K. pneumoniae isolates isolated from blood samples was identified using the microbiology laboratory database. The data from patients diagnosed with K. pneumoniae BSI were collected and analyzed. From 2018 to 2020, there were 510 non-duplicated K. pneumoniae blood isolates, mainly distributed in the intensive care unit (ICU) (28.4%), that were identified in our research. These cases included 77 strains of CRKP and 433 strains of carbapenem-susceptible K. pneumoniae (CSKP). The resistance rate of K. pneumoniae to meropenem and imipenem increased from 11.2% in 2018 to 27.1% in 2020. Moreover, Compared with CSKP, all CRKP isolates showed multi-resistance to tested antibiotics. The phylogenetic analysis showed that the CRKP isolates could be grouped into four major clades, and multilocus sequence typing revealed that the isolates had considerable clonality. Overall, 8 sequence types (STs) of CRKP were detected, of which ST11 comprised the majority and clustered into clade 3. The most prevalent carbapenemase gene was blaKPC (87%) among the CRKP isolates, followed by blaNDM (9.1%) and blaIMP (1.3%). A total of 74 (16.6%) patients with CRKP BSI and 373 (83.4%) patients with CSKP BSI were categorized as the case and control groups. The mortality in the CRKP group was 44.6%, and 11.5% in CSKP group (P<0.001). A multivariate analysis that a long hospital stay before BSI (OR=1.42, 95% CI 1.02-4.31, P=0.011), ICU hospitalization history (OR=3.30, 95% CI 1.35-8.05, P=0.002), and prior use of carbapenems (OR=3.33, 95% CI 1.29-7.27, P=0.001) and antifungals (OR=2.81, 95% CI 1.24-6.04, P<0.001) were independent risk factors for CRKP BSI. ST11 is the predominant type of CRKP mediating inter-hospital transmission, and blaKPC is the main carbapenemase gene harboured by CRKP blood isolates. ICU stay, prolonged hospitalization before BSI, and prior use of carbapenems and antifungals were independent risk factors for acquiring CRKP BSI. Our study may provide insights into early infection control practices.