Carbapenem-resistant Klebsiella Pneumoniae Isolates Research Articles

Using Whole Genome Sequencing to Investigate a Mock-Outbreak of Carbapenem-Resistant Klebsiella pneumoniae in Real-Time.

Healthcare associated infections due to carbapenem-resistant Klebsiella pneumoniae (CRKP) are a major concern in Portuguese hospitals. Whole genome sequencing (WGS) can improve infection control, but this practice is not routinely used by hospital clinical laboratories in Portugal. We simulated the investigation of a CRKP outbreak based on WGS, with the aim of determining, in the minimum possible time, genetic relatedness between CRKP clinical and environmental isolates. Ten CRKP clinical isolates routinely obtained in the hospital laboratory were used. Forty environmental samples - from sinks and sink drains of ward rooms - were collected. Environmental samples were plated on selective media and presumptive CRKP colonies were isolated. Total DNA was extracted from all putative CRKP isolates and sequenced. Clonal relatedness was determined by multi-locus sequence typing and core genome single nucleotide polymorphism analysis; the presence of carbapenemase genes was evaluated. Clinical isolates were characterized in 48 hours: eight strains were confirmed as CRKP, of which six were of ST13 and carried blaKPC-3. Environmental samples results were obtained in six days: eight CRKP were isolated from which five were of ST13 and carried blaKPC-3. Clinical and environmental ST13 isolates were highly related: ten (of 11) isolates differed from each other in < 0.001% of 2 172 367 core nucleotides. WGS can be used as a high-resolution effective tool to investigate healthcare associated infections and track routes of dissemination in real-time. In Portugal, routine use of WGS to improve infection control could thrive through collaborative initiatives between hospitals and research institutes.

In Vitro Activity of Plazomicin Against Carbapenem Resistant Klebsiella pneumoniae Strains.

In our study, we evaluated the in vitro activities of plazomicin, amikacin, gentamicin, and tobramycin among fifty carbapenem resistant Klebsiella pneumoniae (CR-Kp) isolates. Aminoglycoside resistance genes in selected CR-Kp strains were also examined. Minimum inhibitory concentration (MIC) of meropenem, plazomicin, tobramycin, gentamicin, and amikacin were determined by gradient test (G-test) method. In all strains carbapenemase activity was assessed by polymerase chain reaction (PCR). Aminoglycoside modifying enzyme (AME) genes in 14 CR-Kp strains that are resistant to at least one of tobramycin, gentamicin, and amikacin among fifty CR-Kp isolates, and 16S ribosomal methylase genes in 6 CR-Kp strains with plazomicin MIC ≥ 128 mg/L were investigated by PCR method. The most frequently detected carbapenemase enzyme in the strains in our study was OXA-48 (88%). Aminoglycoside susceptibilities of all isolates were determined; plazomicin 84%, amikacin 66%, gentamicin 50%, tobramycin 18%. The most common AME gene positivities were found, 93% (n = 13) ant(3')-I, 78% (n = 11) aac(6')-Ib, 57% (n = 8) aac(3')-IV, 42% (n = 6) aac(3')-IIa, and 29% (n = 4) aph(3')-VI. Most of the isolates examined for the presence of AME carry at least two or more AME genes. The most common 16S ribosomal methylase gene was rmtH. In our study, MIC values of ≥ 256 µg/mL were found in 6 (12%) of 50 isolates against amikacin, tobramycin, and gentamicin, including plazomicin. At least two 16S ribosomal methylase gene positivity has been shown in these 6 strains. In our study, increased in vitro efficacy of plazomicin was shown in CR-Kp isolates comparing to other aminoglycosides. Plazomicin is an effective treatment option against CR-Kp isolates and needs to be sup-ported by clinical studies.

Two Phenotypes of Klebsiella pneumoniae ST147 Outbreak from Neonatal Sepsis with a Slight Increase in Virulence.

PurposeSevere infection has been the leading causes of neonatal death, especially the emergency of multidrug-resistant bacteria such as carbapenem-resistant Enterobacteriaceae. This study aimed to investigate the outbreak of carbapenem-resistant Klebsiella pneumoniae (CR-KP) in neonatal wards and to explore the possible pathogenesis of Klebsiella pneumoniae.Materials and MethodsCR-KP were collected from neonatal ward of Chongqing Health Center for Women and Children between 2017 and 2019. Broth microdilution method was used to evaluate the antimicrobial activities in vitro, at the same time, the virulence of the strain was evaluated by in vitro and in vivo experiments. At last, prokaryotic chain specific transcriptome sequencing was conducted to explore the possible pathogenesis of CR-KP.ResultsIn this study, a total of 14 carbapenem-resistant-Klebsiella pneumoniae (CR-KP) strains were isolated from Chongqing Health Center for Women and Children, among which all CR-KP isolates were identified as NDM-1-producers. Molecular epidemiological studies revealed ST147 being the most common sequence type (ST). Moreover, we first found two phenotypes of K. pneumoniae with different virulence from the same specimen. Type I, which was a white and sticky colony had a slight increase in virulence with higher biofilm formation, serum resistance and virulence than Type II with gray colony. Compared with the Type II, 10 pathways were obviously changed in Type I especially amino acid metabolism, such as arginine and proline metabolism.ConclusionOur findings revealed a new potential threat of NDM-1-positive CR-KP with higher virulence in neonatal ICU ward. We found two phenotypes of K. pneumoniae with different virulent, which may be due to the difference expression of arginine and proline metabolism.

Using Whole Genome Sequencing to Trace, Control and Characterize a Hospital Infection of IMP-4-Producing Klebsiella pneumoniae ST2253 in a Neonatal Unit in a Tertiary Hospital, China.

Background: The purpose of this study is to use whole genome sequencing (WGS) combined with epidemiological data to track a hospital infection of the carbapenem-resistant Klebsiella pneumoniae (CRKP), which affected 3 neonatal patients in the neonatal intensive care unit (NICU).Methods: The minimum inhibitory concentrations for the antimicrobial agents were determined according to the guidelines of the Clinical and Laboratory Standards Institute. Beta-lactamases were investigated using the polymerase chain reaction and DNA sequencing. The transferability of the plasmid was investigated by a conjugation experiment. The clonal relationships were evaluated using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). WGS and single nucleotide polymorphisms (SNPs) analysis were performed on the CRKP isolates to investigate how the infection might progress.Results: Nine CRKP isolates were obtained from the NICU, seven from three patients, one from a duster cloth and one from the hand of a nurse, they all harbored blaIMP-4. Other resistance genes including blaKPC-2, blaIMP-4, blaSHV-1, blaTEM-1, blaCTX-M-15, and blaDHA-1 were also detected. PFGE analysis showed that IMP-4-producing K. pneumoniae were clonally related, and MLST assigned them to a new sequence type 2253. The SNP variations throughout the genome divided the 9 strains into three clades. Clade 1 comprised 7 strains (K1- K2 and K4-K8), whereas clade 2 and 3 consisted of only one strain each: K3 and K9, respectively.The sputum isolate K3 from patient 3 was the most distinct one differing from the other eight isolates by 239-275 SNPs.Conclusions: This is a report of using WGS to track a hospital infecion of IMP-4-producing K. pneumoniae ST2253 among neonates. Nosocomial surveillance systems are needed to limit the spread of the infection caused by these pathogens resulting from the environmental exposure in NICUs.

Clonal spread of carbapenem-resistant Klebsiella pneumoniae among patients at admission and discharge at a Vietnamese neonatal intensive care unit

BackgroundThe increasing prevalence of carbapenem-resistant Enterobacteriaceae (CRE) is a growing problem globally, particularly in low- to middle-income countries (LMICs). Previous studies have shown high rates of CRE colonisation among patients at hospitals in LMICs, with increased risk of hospital-acquired infections.MethodsWe isolated carbapenem-resistant Klebsiella pneumoniae (CRKP) from faecal samples collected in 2017 from patients at admission and discharge at a Vietnamese neonatal intensive care unit (NICU). 126 CRKP were whole-genome sequenced. The phylogenetic relationship between the isolates and between clinical CRKP isolates collected in 2012–2018 at the same hospital were investigated.ResultsNDM-type carbapenemase-(61%) and KPC-2-encoding genes (41%) were the most common carbapenem resistance genes observed among the admission and discharge isolates. Most isolates (56%) belonged to three distinct clonal clusters of ST15, carrying blaKPC-2, blaNDM-1 and blaNDM-4, respectively. Each cluster also comprised clinical isolates from blood collected at the study hospital. The most dominant ST15 clone was shown to be related to isolates collected from the same hospital as far back as in 2012.ConclusionsHighly resistant CRKP were found colonising admission and discharge patients at a Vietnamese NICU, emphasising the importance of continued monitoring. Whole-genome sequencing revealed a population of CRKP consisting mostly of ST15 isolates in three clonally related clusters, each related to blood isolates collected from the same hospital. Furthermore, clinical isolates collected from previous years (dating back to 2012) were shown to likely be clonally descended from ST15 isolates in the largest cluster, suggesting a successful hospital strain which can colonise inpatients.

Molecular characterisation of carbapenem- and tigecycline-resistant Klebsiella pneumoniae strains isolated from blood and bile samples

IntroductionThe number of carbapenem-resistant Klebsiella pneumoniae (CRKP) strains are increasing, further raising healthcare concerns worldwide. In this study, we isolated three CRKP strains from bile and blood samples of an elderly patient (90s) with acute cholangitis and characterised the features and antimicrobial resistance mechanism of CRKP isolates. MethodsThree CRKP isolates were characterised by Pulsed-field gel electrophoresis (PFGE), whole genome sequencing using the NovaSeq 6000, and antimicrobial susceptibility testing. Transcriptional levels of resistance-associated genes were measured by real-time RT-qPCR. ResultsPFGE analysis revealed highly similar patterns for these isolates. Furthermore, they showed resistance to not only carbapenem but also tigecycline. Genomic analysis of the blood isolate identified the exogenous resistance genes blaCTX-M14, tet(A), tet(D), opxAB, and qnrS1 but not any carbapenemase-encoding genes. In addition, nonsense mutations were found in both the outer membrane protein K36 (ompK36) and transcriptional regulator ramR, suggesting that this isolate developed multidrug resistance by acquiring both exogenous resistance genes and nonsense mutations. The extended-spectrum β-lactamase-producing carbapenem-susceptible K. pneumoniae isolate exhibited the same susceptibility pattern, except to β-lactams, as prior CRKP isolates. ConclusionsAntimicrobial susceptibility to carbapenem and tigecycline should be continuously monitored, because it might change from susceptible to resistant during another antimicrobial treatment, even if an isolate initially shows susceptibility, and the patient has not been exposed to these agents.

Aminoglycoside-resistance gene signatures are predictive of aminoglycoside MICs for carbapenem-resistant Klebsiella pneumoniae.

Aminoglycoside-containing regimens may be an effective treatment option for infections caused by carbapenem-resistant Klebsiella pneumoniae (CR-Kp), but aminoglycoside-resistance genes are common in these strains. The relationship between the aminoglycoside-resistance genes and aminoglycoside MICs remains poorly defined. To identify genotypic signatures capable of predicting aminoglycoside MICs for CR-Kp. Clinical CR-Kp isolates (n = 158) underwent WGS to detect aminoglycoside-resistance genes. MICs of amikacin, gentamicin, plazomicin and tobramycin were determined by broth microdilution (BMD). Principal component analysis was used to initially separate isolates based on genotype. Multiple linear regression was then used to generate models that predict aminoglycoside MICs based on the aminoglycoside-resistance genes. Last, the performance of the predictive models was tested against a validation cohort of 29 CR-Kp isolates. Among the original 158 CR-Kp isolates, 91.77% (145/158) had at least one clinically relevant aminoglycoside-resistance gene. As a group, 99.37%, 84.81%, 82.28% and 10.76% of the CR-Kp isolates were susceptible to plazomicin, amikacin, gentamicin and tobramycin, respectively. The first two principal components explained 72.23% of the total variance in aminoglycoside MICs and separated isolates into four groups with aac(6')-Ib, aac(6')-Ib', aac(6')-Ib+aac(6')-Ib' or no clinically relevant aminoglycoside-resistance genes. Regression models predicted aminoglycoside MICs with adjusted R2 values of 56%-99%. Within the validation cohort, the categorical agreement when comparing the observed BMD MICs with the predicated MICs was 96.55%, 89.66%, 86.21% and 82.76% for plazomicin, gentamicin, amikacin and tobramycin, respectively. Susceptibility to each aminoglycoside varies in CR-Kp. Detection of aminoglycoside-resistance genes may be useful to predict aminoglycoside MICs for CR-Kp.

Transmission Dynamics of Carbapenem-Resistant Klebsiella pneumoniae Sequence Type 11 Strains Carrying Capsular Loci KL64 and rmpA/rmpA2 Genes.

The presence and dissemination of carbapenem-resistant Klebsiella pneumoniae (CRKP) often cause life-threatening infections worldwide, but the therapeutic option is limited. In this study, whole-genome sequencing (WGS) was applied to assess the epidemiological characteristics and transmission dynamics of CRKP isolates recovered from two fetal outbreaks of nosocomial infections. Between April 2016 and March 2018, a total of 70 isolates of K. pneumoniae were collected from sterile samples in a tertiary hospital in Hangzhou, China. The minimal inhibitory concentrations (MICs) of 21 antimicrobial agents were determined using the broth microdilution methods. Pulsed-field gel electrophoresis (PFGE) was performed on 47 CRKP isolates, and 16 clonally related isolates were further characterized by Illumina sequencing. In addition, the complete genome sequences of three representative isolates (KP12, KP36, and KP37) were determined by Oxford Nanopore sequencing. The K. pneumoniae isolates were recovered from patients diagnosed with pulmonary infection, cancer, or encephalopathy. For all CRKP isolates, PFGE separated three clusters among all strains. The most predominant PFGE cluster contained 16 isolates collected from patients who shared close hospital units and represented a potential outbreak. All 16 isolates showed an extremely high resistance level (≥87.5%) to 18 antimicrobials tested but remain susceptible to colistin (CST). Multiple antimicrobial resistance and virulence determinants, such as the carbapenem resistance gene bla KPC-2, and genes encoding the virulence factor aerobactin and the regulator of the mucoid phenotype (rmpA and rmpA2), were observed in the 16 CRKP isolates. These isolates belonged to sequence type 11 (ST11) and capsular serotype KL64. A core genome single nucleotide polymorphism (cgSNP)-based phylogenetic analysis indicated that the 16 CRKP isolates could be partitioned into two separate clades (≤15 SNPs), suggesting the two independent transmission scenarios co-occurred. Moreover, a high prevalence of IncFIB/IncHI1B type virulence plasmid with the iroBCDN locus deleted, and an IncFII/IncR type bla KPC-2-bearing plasmid was co-harbored in ST11-KL64 CRKP isolates. In conclusion, our data indicated that the nosocomial dissemination of ST11-KL64 CRKP clone is a potential threat to anti-infective therapy. The development of novel strategies for surveillance, diagnosis, and treatment of this high-risk CRKP clone is urgently needed.

Outbreak of Multidrug-Resistant OXA-232-Producing ST15 Klebsiella pneumoniae in a Teaching Hospital in Wenzhou, China.

BackgroundOXA-232-producing carbapenem-resistant Klebsiella pneumoniae (CRKP) has the potential to become the “third epidemic” of carbapenem-resistant Klebsiella strain after KPC-2 and NDM in China. We investigated the first outbreak of CRKP in the First Affiliated Hospital of Wenzhou Medical University.MethodsWe collected 610 clinical isolates of CRKP from the First Affiliated Hospital of Wenzhou Medical University between January 2019 and September 2020 and screened them by Polymerase Chain Reaction (PCR). The multilocus sequence typing and pulsed-field gel electrophoresis were used to determine the genetic relatedness of the strains. The antimicrobial susceptibility test was performed to determine the drug resistance of the clinical isolates. The molecular mechanism underlying carbapenem resistance was elucidated by performing PCR and conjugation experiments. The virulence potential of the strains was determined by the string test, detection of virulence-associated genes and capsular serotypes, and Galleria mellonella larval infection model.ResultsBetween September 2019 and May 2020, 26 OXA-232-producing CRKP were obtained from 12 patients in our hospital. Ten patients were hospitalized in the intensive care units (ICU) and the overall mortality of the inpatients involved in the outbreak was 50% (6/12). Epidemiological investigations reported that all the OXA-232-producing CRKP strains belonged to the sequence type ST15 and can be clonally transmitted among the inpatients in the ICU. All the strains had low virulence and were resistant to commonly used clinical antibiotics except for ceftazidime/avibactam, colistin, and tigecycline. The OXA-232-producing CRKP was sensitive to triclosan and chlorhexidine, and its eradication from our hospital can be achieved by the use of disinfectants in the ICU.ConclusionIn our study, OXA-232-producing CRKP isolates appeared to be clonally transmitted and the sequence type ST15 was responsible for the outbreak. Therefore, effective measurements for the infection control of CRKP are urgently needed to prevent its epidemic in the nearby region in the future.

Factors behind the prevalence of carbapenem-resistant Klebsiella pneumoniae in pediatric wards.

The emergence of carbapenem-resistant Enterobacteriaceae made the treatment difficult, which has become a significant issue of public health. A sharp increase of carbapenem-resistance rate in Klebsiella pneumoniae was observed in a maternity and child health care hospital in Zunyi, China, in 2014.In 2015 to 2016, carbapenem-resistant Klebsiella pneumoniae (CRKp) isolated from all the clinical samples were analyzed to identify the carbapenem-resistance genes. They were then fingerprinted in order to determine their genetic relationship. Clinical data such as usage of imipenem in 2012 to 2016 and the nosocomial infection surveillance data were analyzed.Thirty-five isolates of CRKp out of 4328 various pathogens were obtained, and blaNDM-1 was identified to be the most common resistant gene present in the CRKp isolates. The fingerprint analysis identified 15 major clusters of CRKp isolates. The bacteria with close proximity relationship tended to be from the same wards. However, a few CRKp isolates from different wards were found to be genetically highly related. The clinical data showed a significantly higher usage of carbapenems in 2012 to 2013 before the CRKp rate sharply increased in 2014. The nosocomial infection surveillance showed an unexpectedly high rate of failures to meet the requirement of the hospital environment hygiene and hand hygiene in the neonatal ward.The increasing isolation rate of CRKp was associated with poorly regulated usage of carbapenems, impropriate medical practices, and the poor hospital environmental hygiene and hand hygiene.

The Emergence of Novel Sequence Type Strains Reveals an Evolutionary Process of Intraspecies Clone Shifting in ICU-Spreading Carbapenem-Resistant Klebsiella pneumoniae.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is an urgent public health problem worldwide, and its rapid evolution in the clinical environment has been a major concern. A total of 99 CRKP isolates spreading in the intensive care unit (ICU) setting were included and subjected to whole-genome sequencing, and their sequence types (STs), serotype loci, and virulence determinants were screened based on genome data. The phylogenetic structure was reconstructed based on the core genome multilocus sequence typing method. Regions of recombination were assessed. Biofilm formation, serum resistance assays, and a Galleria mellonella infection model were used to evaluate strain virulence. A novel ST, designated ST4496, emerged in the ICU and spread for 6 months before its disappearance. ST4496 was closely related to ST11, with only a single-allele variant, and ST11 is the most dominant clinical clone in China. Recombination events occurred at capsule biosynthesis loci and divided the strains of ST11 and its derivative ST4496 into three clusters, including ST11-KL47, ST11-KL64, and ST4496-KL47. The phylogenetic structure indicated that ST11-KL47 was probably the origin of ST11-related strain evolution and presented more diversity in terms of both sequence similarity and phenotypes. ST4496-KL47 cluster strains presented less virulence than ST11-KL64, which was probably one of the factors preventing the former from spreading widely. In conclusion, ST4496-KL47 was probably derived from ST11-KL47 via intraspecies shifting but was less competitive than ST11-KL64, which also evolved from ST11-KL47 and developed increased virulence via capsule biosynthesis locus recombination. ST11-KL64 has the potential to be the predominant CRKP clone in China.

Optical DNA Mapping of Plasmids Reveals Clonal Spread of Carbapenem-Resistant Klebsiella pneumoniae in a Large Thai Hospital.

Carbapenem-resistant Klebsiella pneumoniae (CR-KP) in patients admitted to hospitals pose a great challenge to treatment. The genes causing resistance to carbapenems are mostly found in plasmids, mobile genetic elements that can spread easily to other bacterial strains, thus exacerbating the problem. Here, we studied 27 CR-KP isolates collected from different types of samples from 16 patients admitted to the medical ward at Siriraj Hospital in Bangkok, Thailand, using next generation sequencing (NGS) and optical DNA mapping (ODM). The majority of the isolates belonged to sequence type (ST) 16 and are described in detail herein. Using ODM, we identified the plasmid carrying the blaNDM-1 gene in the ST16 isolates and the plasmids were very similar, highlighting the possibility of using ODM of plasmids as a surrogate marker of nosocomial spread of bacteria. We also demonstrated that ODM could identify that the blaCTX-M-15 and blaOXA-232 genes in the ST16 isolates were encoded on separate plasmids from the blaNDM-1 gene and from each other. The other three isolates belonged to ST147 and each of them had distinct plasmids encoding blaNDM-1.

Antibiotic resistance and virulence characteristics analysis of a carbapenem-resistant hypervirulent Klebsiella pneumoniae

Objective: To characterize the antibiotic resistance and virulence in a carbapenem-resistant Klebsiella pneumoniae (CRKP). Methods: A CRKP (designated K. pneumoniae C35) was isolated from a stool sample. The minimal inhibitory concentrations of antimicrobial agents were determined using the broth microdilution method. Whole-genome sequencing and genome analysis were performed to identify the antibiotic resistance and virulence genes. The genetic relationship among K. pneumoniae C35 and other CRKP isolates from our hospital was analyzed by single nucleotide polymorphism (SNP) typing of core genomes. Conjugation experiments were carried out by filter mating to evaluate the transferability and efficiency of resistance genes. The virulence phenotype was determined by Galleria mellonella infection model. Results: K. pneumoniae C35 exhibited resistance to the majority of tested antibiotics, especially carbapenems, sulbactam, and polymyxins. SNP typing showed that K. pneumoniae C35 shared a high degree of sequence homology with several CRKP isolates from different wards. This ST11 CRKP carried 13 resistance genes, including blaKPC-2, blaCTX-M-199, mcr-1, and tet(A) variant. blaKPC-2 gene was located on an IncFⅡ plasmid with>69 800 bp in size, blaCTX-M-199 and mcr-1 genes were located on an IncI2 plasmid (>64 800 bp), and tet(A) variant was located on an unknown Inc-type plasmid (83 628bp). All these three plasmids were conjugative. K. pneumoniae C35 was found to harbor rmpA, rmpA2, and iucABCD aerobactin-related genes, and was considered to be classic carbapenem-resistant hypervirulent K. pneumoniae (CR-hvKP). The virulence potential of this strain was confirmed in a Galleria mellonella infection model. The survival rate of the larvae injected with strain C35 at 48 h after infection was significantly lower than that of negative control strain (16.7% vs 80.0%). Conclusion: Multiple conjugative plasmids are identified in a faecal CR-hvKP. The IncI2 plasmid co-carrying both blaCTX-M-199 and mcr-1 genes is firstly identified in CR-hvKP. The emergence of such strain should be alerted and active surveillance is warranted.

Evaluation of the Organotellurium Compound AS101 for Treating Colistin- and Carbapenem-Resistant Klebsiella pneumoniae.

Colistin- and carbapenem-resistant Enterobacteriaceae cases are increasing at alarming rates worldwide. Drug repurposing is receiving greater attention as an alternative approach in light of economic and technical barriers in antibiotics research. The immunomodulation agent ammonium trichloro(dioxoethylene-O,O’-)tellurate (AS101) was repurposed as an antimicrobial agent against colistin- and carbapenem-resistant Klebsiella pneumoniae (CRKP). 134 CRKP isolates were collected between 2012 and 2015 in Taiwan. The in vitro antibacterial activities of AS101 was observed through broth microdilution, time-kill assay, and electron microscopy. Pharmaceutical manipulation and RNA microarray were applied to investigate these antimicrobial mechanisms. Caenorhabditis elegans, a nematode animal model, and the Institute for Cancer Research (ICR) mouse model was employed for the evaluation of in vivo efficacy. The in vitro antibacterial results were found for AS101 against colistin- and CRKP isolates, with minimum inhibitory concentration (MIC) values ranging from <0.5 to 32 μg/mL. ROS-mediated antibacterial activity eliminated 99.9% of bacteria within 2–4 h. AS101 also extended the median survival time in a C. elegans animal model infected with a colistin-resistant CRKP isolate and rescued lethally infected animals in a separate mouse model of mono-bacterial sepsis by eliminating bacterial organ loads. These findings support the use of AS101 as an antimicrobial agent for addressing the colistin and carbapenem resistance crisis.

Carbapenem-Resistant Klebsiella pneumoniae in Southwest China: Molecular Characteristics and Risk Factors Caused by KPC and NDM Producers.

BackgroundCarbapenem-resistant Klebsiella pneumoniae (CRKP) infection has attracted worldwide concern and became a serious challenge for clinical treatment. The aims of this study were to evaluate the molecular characteristics and risk factors for CRKP infection.MethodsAll the CRKP strains were screened for antimicrobial resistance genes, virulence genes, and integron by polymerase chain reaction (PCR). Plasmid typing was performed by plasmid conjugation assay and PCR-based replicon typing (PBRT). The genetic environments of blaKPC-2 and blaNDM-1 were analyzed by using overlapping PCR and molecular typing was performed by multi-locus sequence typing (MLST). Risk factors for CRKP infection were analyzed by logistic regression model.ResultsAll the 66 CRKP isolates were multidrug-resistant, but all of them were susceptible to tigecycline and polymyxin B. Among the CRKP isolates, 42 blaKPC-2-positive strains were identified carrying IncFII plasmids. Meanwhile, 24 blaNDM-positive strains were found on lncX3 plasmids, including 20 blaNDM-1 isolates and 4 blaNDM-5 isolates. Most of CRKP isolates contained several virulence genes and the class I integron (intl1). The genetic environments of blaKPC-2 and blaNDM-1 revealed that the conserved regions (tnpA-tnpR-ISkpn8-blaKPC-2) and (blaNDM-1-bleMBL-trpF-tat) were associated with the dissemination of KPC-2 and NDM-1. ST11 was the most common type in this work. Hematological disease, tracheal cannula, and use of β-lactams and β-lactamase inhibitor combination were identified as independent risk factors for CRKP infection.ConclusionThis study established the resistance pattern, molecular characteristics, clonal relatedness, and risk factors of CRKP infection. The findings of the novel strain that co-harboring blaNDM-5 and blaIMP-4, and the novel ST4495 indicated that the brand-new types have spread in Southwest China, emphasizing the prevent and control the further dissemination of CRKP isolates are highly needed.

Treatment of Donor-derived Carbapenem-resistant Klebsiella pneumoniae Infection after Renal Transplantation with Tigecycline and Extended-infusion Meropenem.

Donor-derived carbapenem-resistant Klebsiella pneumoniae (CRKP) infection has recently emerged as a critical early complication after renal transplantation. Although CRKP is usually sensitive to tigecycline, monotherapy with this drug is often less than effective. We investigated the efficacy of a combined regimen of tigecycline with high-dose, extended-infusion meropenem in the treatment of donor-derived CRKP infection after kidney transplantation. From Jan. 2016 to Dec. 2017, a total of 12 CRKP isolates were detected from cultures of the organ preservation solution used for soaking the donor kidneys at our institute. Probable or possible donor-derived infection (DDI) was identified in 8 transplant recipients. Clinical data were retrospectively analyzed. Klebsiella pneumoniae carbapenemase-2 (KPC-2)-producing CRKP was reported to be positive in organ preservation solution cultures at 3.5±0.9 days after transplantation, leading to surgical site (n=3), urinary tract (n=4), and/or bloodstream (n=2) infections in 8 recipients. The drug susceptibility tests showed that CRKP was sensitive to tigecycline, but resistant to meropenem. In 7 patients who received tigecycline combined with high-dose extended-infusion meropenem, DDIs were successfully cured. The length of hospital stay was 31 (18-129) days, and the serum creatinine at discharge was 105.8±16.7 µmol/L. The one remaining patient who received tigecycline combined with intravenous-drip meropenem died of septic shock. A median follow-up of 43 months (33-55) showed no recurrence of new CRKP infection in the 7 surviving recipients. It was suggested that a prompt and appropriate combination therapy using tigecycline with high-dose extended-infusion meropenem is effective in treating donor-derived KPC-2-producing CRKP infection after renal transplantation.

Resistance Phenotype and Molecular Epidemiology of Carbapenem-Resistant Klebsiella pneumoniae Isolates in Shanghai.

Background: The emergence and wide global spread of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates are of great concern, and the aim of this study was to investigate drug resistance, molecular epidemiology, and genetic relationship of CRKP isolates from patients in Shanghai, China. Methods: A retrospective study was conducted from April 2018 to July 2019, and a total of 133 CRKP isolates were collected. Antimicrobial susceptibility was determined by VITEK-2 automated microbiology analyzer platform (bioMérieux, France) and the broth microdilution method. Polymerase chain reaction assays were used to investigate the presence of drug resistance genes. A modified carbapenem inactivation method was performed to detect carbapenemases. Multilocus sequence typing and pulsed-field gel electrophoresis (PFGE) were conducted for genetic relatedness of 50 CRKP isolates selected. Results: Among 670 isolates of K. pneumoniae, 133 (19.9%) strains were identified as CRKP, of which, 76.7% (102/133) strains were isolated from intensive care units (ICUs). All the 133 CRKP isolates were found to be carbapenemase-producers and harbor blaKPC-2 gene. No other carbapenemase genes of blaNDM, blaOXA-48, blaVIM, and blaIMP were detected. Furthermore, β-lactamase genes of blaSHV, blaCTX, and blaTEM were the most common resistance-associated genes among these KPC-2 producing isolates. All the 133 CRKP strains displayed >95% of resistance to cephalosporins and carbapenems, except for gentamicin, trimethoprim-sulfamethoxazole, amikacin, tigecycline and colistin, and ceftazidime-avibactam. The most common sequence type was ST11, accounting for 90.0% of the 50 CRKP selected, followed by ST15 (10.0%). PFGE analysis clustered the 50 KPC-2-producing isolates into seven (A-G) distinct clonal clusters at 85% cutoff. Of which, A and G were the two major clusters, accounting for the majority of the strains collected in emergency ICU and neurosurgical ICU. And all the strains of clusters D and E were collected in cardiothoracic surgery ICU, except for one strain collected in one outpatient. Conclusion: The KPC-2-producing K. pneumoniae belonged to ST11 was widely disseminated in ICUs, and active and effective surveillance of infection control strategies was initiated to limit the spread of CRKP strains.

The Potential Use of Ceftazidime-Avibactam Against Carbapenem Resistant Klebsiella pneumoniae Clinical Isolates Harboring Different Carbapenemase Types in a Thai University Hospital

PurposeMBL and OXA-48 genes in carbapenem-resistant Enterobacterales (CRE) have emerged as a major public health problem worldwide, including Thailand. Due to the lack of susceptibility data and dosing regimens of ceftazidime-avibactam (CZA) against CRE in Thailand, especially in colistin-resistant era, we aimed to demonstrate in vitro susceptibility data of CZA and optimal dose based on Monte Carlo simulation of CZA to expand the treatment options.Patients and MethodsWe collected 49 carbapenem-resistant Klebsiella pneumoniae (CRKP) clinical isolates from unique patients at Phramongkutklao Hospital (June–October 2020). CZA disk diffusion and E-test testing were performed to obtain minimum inhibitory concentration (MIC). Each drug regimen was simulated using the Monte Carlo technique to calculate the probability of target attainment (PTA) and the cumulative fraction of response (CFR).ResultsThe most common genotypes of CRKP were blaOXA-48 (53.1%) and blaOXA-48 +blaNDM (42.8%). CZA showed 47.7% and 90.5% susceptible rate against all genotypes of carbapenemases and OXA-48 type CRKP isolates. The MIC50 and MIC90 of CZA against CRKP were 2 and >256 µg/mL. The categorical agreement (CA) between disk diffusion and E-test testing of CZA against CRKP was 95.4%. The CZA dosing regimens of 2.5 g infused 2–3 h every 8 h achieved ≥90% of the target of free ceftazidime plasma concentration over MIC (%fTime >MIC) ≥50% and 100% against isolates MICs of ≤8 and ≤8 µg/mL, respectively. The avibactam regimens also provided 100%fTime at 0.5 µg/mL. Based on CFR ≥90%, no CZA regimens were effective against all of the studied CRKP isolates except CRKP carrying OXA-48.ConclusionCZA exhibited a fairly susceptible rate among the OXA-48-positive isolates in Thailand. The current suggested dose of CZA with prolonged infusion appears appropriate to achieve the pharmacokinetic/pharmacodynamic targets of ceftazidime and avibactam against CRKP carrying blaOXA-48.

The Synergistic Activity and Optimizing Doses of Tigecycline in Combination with Aminoglycosides against Clinical Carbapenem-Resistant Klebsiella pneumoniae Isolates.

Carbapenem-resistant Enterobacteriaceae (CRE), especially carbapenem-resistant Klebsiella pneumoniae (CRKP), are among the largest pathogenic threats to humans. The available antibiotic treatment options for combating CRKP are limited. Colistin-resistant Enterobacteriaceae (CoRE) have also been reported worldwide, including in Thailand. Therefore, this study aimed (1) to determine minimum inhibitory concentrations (MICs) and synergistic activities of antibiotics of CRKP, and (2) to determine the probability target of attainment (PTA) and cumulative fraction of response (CFR) using pharmacokinetic/pharmacodynamic (PK/PD) data. Clinical CRKP isolates were obtained from Phramongkutklao Hospital (June to November 2020). Broth microdilution and checkerboard techniques were used to determine the mono- and synergistic activities of antibiotics. Carbapenemase and mcr-1 genes were also identified by polymerase chain reaction (PCR). The optimal antibiotic regimens were evaluated using Monte Carlo simulations. Forty-nine CRKP isolates were collected, 40 of which were CoRKP strains. The MIC50 and MIC90 of tigecycline, amikacin, and gentamicin were 1 and 2 µg/mL, 4 and 16 µg/mL, and 0.25 and 4 µg/mL, respectively. None of any isolates expressed the mcr-1 gene, whereas blaOXA-48 (53.1%) and blaOXA-48 plus blaNDM (42.9%) were detected. Synergistic activity was observed in 8.2% of isolates for tigecycline combined with amikacin or gentamicin. Additive activity was observed in 75.5% of isolates for tigecycline-amikacin and 69.4% for tigecycline-gentamicin, and no antagonism was observed. High-dose antibiotic regimens achieved the PTA target. The general recommended dose of combination regimens began with 200 mg tigecycline and 25 mg/kg amikacin, or 7 mg/kg gentamicin, followed by 100 mg tigecycline every 12 h and 15 mg/kg amikacin or 5 mg/kg gentamicin every 24 h. In conclusion, tigecycline plus aminoglycosides might be a potential regimen against CRKP and CoRKP. The appropriate combination regimen based on MIC-based dose adjustment can improve optimal antibiotic dosing. Further research via clinical studies will be necessary to confirm these results.

Distribution of fluoroquinolone resistance determinants in Carbapenem-resistant Klebsiella pneumoniae clinical isolates associated with bloodstream infections in China

BackgroundThe rate of fluoroquinolone (FQ) resistance among carbapenem-resistant Klebsiella pneumoniae (CRKP) is high. The present study aimed to investigate the distribution of fluoroquinolone resistance determinants in clinical CRKP isolates associated with bloodstream infections (BSIs).ResultsA total of 149 BSI-associated clinical CRKP isolates collected from 11 Chinese teaching hospitals from 2015 to 2018 were investigated for the prevalence of fluoroquinolone resistance determinants, including plasmid-mediated quinolone resistance (PMQR) genes and spontaneous mutations in the quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes. Among these 149 clinical CRKP isolates, 117 (78.5%) exhibited resistance to ciprofloxacin. The GyrA substitutions (Ser83 → IIe/Phe) and (Asp87 → Gly/Ala) were found among 112 (75.2%) of 149 isolates, while the substitution (Ser80 → IIe) of ParC was found in 111 (74.5%) of the 149 isolates. In total, 70.5% (105/149) of the CRKP isolates had at least two mutations within gyrA as well as a third mutation in parC. No mutations in the QRDRs were found in 31 ciprofloxacin susceptible CRKP isolates. Eighty-nine (56.9%) of 149 were found to carry PMQR genes including qnrS1 (43.0%), aac(6′)-Ib-cr (16.1%), qnrB4 (6.0%), qnrB2 (2.7%), and qnrB1 (1.3%). Nine isolates contained two or more PMQR genes, with one carrying four [aac(6′)-Ib-cr, qnr-S1, qnrB2, and qnrB4]. The co-existence rate of PMQR determinants and mutations in the QRDRs of gyrA and parC reached 68.5% (61/89). Seventy-four (83.1%, 74/89) PMQR-positive isolates harbored extended-spectrum beta-lactamase (ESBL)-encoding genes. Multilocus sequence typing (MLST) analysis demonstrated that the ST11 was the most prevalent STs in our study.ConclusionsMutations in the QRDRs of gyrA and parC were the key factors leading to the high prevalence of fluoroquinolone resistance among BSI-associated CRKP. The co-existence of PMQR genes and mutations in the QRDRs can increase the resistance level of CRKP to fluoroquinolones in clinical settings. ST11 CRKP isolates with identical QRDR substitution patterns were found throughout hospitals in China.