Carbapenem-resistant Klebsiella Pneumoniae Research Articles

Comparative genomic analysis of plasmids harboring bla OXA-48-like genes in Klebsiella pneumoniae.

The emergence and spread of carbapenem-resistant Klebsiella pneumoniae (CRKP) is a serious medical problem worldwide. Acquired OXA-48-like carbapenemases encoded by plasmids are important causes of carbapenem resistance in K. pneumoniae. To explore the links between plasmids and bla OXA-48-like genes in K. pneumoniae, we systematically analyzed the variants of bla OXA-48-like plasmid replicon types, phylogenetic patterns, geographic distribution, conjugative transfer regions, and the genetic environments surrounding bla OXA-48-like of 191 bla OXA-48-like-harboring plasmids, which were identified from 4451 plasmids of K. pneumoniae downloaded from GenBank. Our results showed that seven different variants of bla OXA-48-like genes were identified from the 191 bla OXA-48-like-harboring plasmids in K. pneumoniae, with bla OXA-48, bla OXA-232, and bla OXA-181 being highly prevalent. In K. pneumoniae, bla OXA-48 was mainly carried by the composite transposon Tn1999.2 located on IncL/M-type conjugative plasmids, which were mainly geographically distributed in Switzerland, Germany, and China. In K. pneumoniae, the blaOXA-232 gene was mainly carried by 6.1-kb ColKP3-type mobilizable plasmids, which were mainly isolated in India. In K. pneumoniae, bla OXA-181 was mainly carried by a group of 50-kb ColKP3-IncX3 hybrid conjugative plasmids and a group of small ColKP3-type mobilizable plasmids with lengths of 5.9-9.3 kb, the former was sporadically discovered in China, South Korea, India, and Czech Republic, while the latter was almost all isolated in India. In addition, five bla OXA-245-harboring 65.9-kb IncL plasmids of K. pneumoniae isolated in Spain were found to have the genetic context of bla OXA-245 more complicated than that of bla OXA-48-harboring IncL/M-type plasmids, with two copies of IS1R inserted both upstream and downstream of bla OXA-245-lysR. These findings enhance our understanding of the genetic diversity of bla OXA-48-like-harboring plasmids in K. pneumoniae.

Carbapenem-resistant Klebsiella pneumoniae: Risk Factors for Isolation Among Hospitalized Patients

Background: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is an important healthcare-associated pathogen. This study aimed to identify factors associated with CRKP isolation among hospitalized patients, describe molecular epidemiology, and mortality associated with CRKP isolation. Methods: We performed a case-control study at two university-affiliated teaching hospitals. We included 150 patients (30 cases and 120 controls). Each patient with CRKP, a case-patient, was matched with four controls by admission facility, admission date, age, and sex. Controls, patients without CRKP, were randomly selected from a computerized list of inpatients whose admission date was the same as that of the case, within 48 hours of the date of the initial positive culture. We calculated the risk of in-hospital death as the number of deaths divided by the number of cases and evaluated the risk of mortality associated with the site of positive culture. Molecular epidemiology investigation using comparison of restricted DNA patterns of CRKP by pulsed-field gel electrophoresis (PFGE) was conducted. Results: A greater proportion of cases than controls had undergone an invasive procedure, including use of a central vein catheter, or mechanical nutrition by tube feeding. Pre-admission treatment within two months with the following antibiotic classes was associated with CRKP isolation: carbapenems, fluoroquinolones, anti-pseudomonal penicillins, and cephalosporins. The molecular analysis indicated that over 90% of isolates shared similar PFGE patterns. CRKP isolation was associated with significantly higher in-hospital mortality in comparison to controls. Positive cultures from sites other than urine were associated with substantially higher mortality than was a positive urine culture (RR= 4.0). Conclusions: The use of multiple broad-spectrum antibiotics, multiple comorbid conditions and poor performance status are important risk factors for developing CRKP in a hospitalized population.

421. In vitro and in vivo synergistic antimicrobial activities of the combinations of meropenem, colistin, tigecycline, and ceftolozane/tazobactam against carbapenem-resistant Klebsiella pneumoniae

Abstract Background The aim of this study was to evaluate the in vitro and in vivo activities of various antimicrobial combinations against carbapenem-resistant Klebsiella pneumoniae (CRKP). Methods The in vitro activity of six two-drug combinations against CRKP isolates collected from the rectal swab samples of patients with intestinal CRKP colonization was evaluated using the checkerboard method and time-kill assay to identify potential synergistic and bactericidal two-drug combinations against CRKP isolates. The in vivo efficacy of the combinations of colistin, meropenem and ceftolozane/tazobactam (C/T) as therapeutic options, was assessed in a mouse model of intraperitoneal CRKP sepsis. Antimicrobial susceptibility and genotypes of carbapenem-resistance were also analyzed. Results A total of 5 clinical isolates of CRKP were collected from nonduplicate patients with CRKP infection. Of 5 CRKP isolates, 4 carried the blaKPC-2-type carbapenem-hydrolyzing enzymes except 1 blaNDM-1-type. Antimicrobial susceptibility rates were tigecycline 80%, colistin 40%, and C/T 0%, respectively. The checkerboard assay showed that in vitro synergistic activity against CRKP isolates was 90% for the meropenem-tigecycline combinations and 20% for colistin-C/T combinations. None of the various combinations of the six antibiotics showed a synergistic effect in the time-kill assay. However, the meropenem-colistin (100%) and colistin-C/T (100%) combinations showed bactericidal effects in the time-kill assay (using an antibiotic concentration of 1× MIC) (Table). The survival rates of the mice was 80% (4/5) at 48 h in the treated group using low dose combinations of colistin (10 mg/kg) and C/T (25 mg/kg), unlike both antibiotics, which were 60% in a monotherapy with the same dose (Figure). Comparison of checkerboard assay and time-kill assay Comparison of checkerboard assay and time-kill assay associated with the bactericidal activity of two-drug combinations (1 × MIC) against each carbapenem-resistant Klebsiella pneumoniae isolate In vivo survival tests for antimicrobial combinations Comparison of the survival rates of the mice in the treated group using antimicrobial combinations Conclusion In conclusion, the present in vivo study demonstrated that the low-dose combination of colistin and C/T may be a promising alternative to nephrotoxic high-dose colistin alone for treating CRKP infections. However, it was difficult to predict this possibility in in vitro tests, and even there is great discordance of antimicrobial synergistic activities between the checkerboard microdilution and time-kill assays. Disclosures All Authors: No reported disclosures.

Clinical Outcomes and Risk Factors for Death following Carbapenem-Resistant Klebsiella pneumoniae Infection in Solid Organ Transplant Recipients.

Infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP) are associated with significant morbidity and mortality. Among solid organ transplant recipients (SOTRs), clinical outcomes and risk factors for death following such infections remain not well documented. A single-center retrospective study was performed. All SOTRs with a CRKP infection at the First Affiliated Hospital of Zhengzhou University between 1 January 2018 and 31 December 2021 were included. Multivariable Cox regression was performed to determine risk factors for death following CRKP infection. We identified 94 SOTRs with CRKP infection, with a median age of 50 years old. CRKP infections resulted in 38.3% of overall 30-day mortality. On multivariable analysis, independent risk factors for death following CRKP infection included older age (hazard ratio [HR], 1.044; 95% confidence interval [CI], 1.007 to 1.083; P = 0.02), allograft failure (HR, 3.962; 95% CI, 1.628 to 9.644; P = 0.002), and septic shock (HR, 8.512; 95% CI, 3.294 to 21.998; P < 0.001). Receiving appropriate targeted therapy was associated with a reduced hazard of death (HR, 0.245; 95% CI, 0.111 to 0.543; P = 0.001). Our study characterized the clinical features and mortality in SOTRs with CRKP infection. The protective effects of appropriate targeted therapy highlight the importance of assessing how antibiotic choices affect the clinical outcomes among SOTRs. IMPORTANCE Carbapenem-resistant Klebsiella pneumoniae (CRKP) infections are increasingly identified in solid organ transplant recipients (SOTRs), but data on the clinical outcomes and risk factors for death following such infections remain limited. Here, we reported CRKP infection was associated with 38.3% of overall 30-day mortality in SOTRs. Independent risk factors for death after CRKP infection included older age, allograft failure, and septic shock. Appropriate targeted therapy was important for alleviating the impact of CRKP infections on these SOTRs.

Urban wastewater contributes to the emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) in an urban receiving river in eastern India.

The present study revealed the emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) and the associated driving factors in an urban river system surrounding Cuttack city, Odisha. The high contamination factor and contamination degree indicate poor water quality. The CRKP isolates showed 100% resistance against piperacillin, amoxicillin-clavulanic acid, piperacillin-tazobactam, ceftriaxone, ceftazidime, meropenem, and imipenem but less resistance to colistin (12.85%). Among the CRKP isolates, carbapenemase genes blaNDM, blaOXA-48-like, and blaKPC were detected in 94.28%, 35%, and 10% of isolates, respectively. The resistance genes (blaNDM, blaTEM, and blaCTX-M) were found to be significantly correlated with toxic metals (As, Cd, Co, Cu, Fe, Mn, Pb) (P<0.05). Detection of virulence factors (yersiniabactin and aerobactin) and capsular serotypes (K1, K2, and K54 types) explain the pathogenicity of CRKP isolates. Enterobacterial repetitive intergenic consensus-PCR based molecular typing separated the CRKP strains into 13 clusters, of which VI and XI clusters showed similar resistance and virulence determinants, indicating the dissemination of clones from wastewater to the river system. Our results provide first-hand information on assessing risks to public health posed by the CRKP isolates and toxic metals in the Kathajodi River. Molecular surveillance of nearby hospitals for the prevalence of CRKP will help trace their transmission route.

Diversity of Circulating NKT Cells in Defense against Carbapenem-Resistant Klebsiella Pneumoniae Infection.

Nosocomial infection caused by carbapenem-resistant Klebsiella pneumonia (CRKP) infection has become a global public health problem. Human NK and NKT cells in peripheral immune responses are recognized as occupying a critical role in anti-bacterial immunity. Through performed scRNA-seq on serial peripheral blood samples from 3 patients with CRKP undergoing colonization, infection, and recovery conditions, we were able to described the immune responses of NK and NKT cells during CRKP infection and identified a mechanism that could contribute to CRKP clearance. The central player of CRKP infection process appears to be the NKT subset and CD56hiNKT subset which maintained immune competence during CRKP colonization. With time, CRKP leads to the loss of NK and CD160hiNKT cells in peripheral blood, resulting in suppressed immune responses and increased susceptibility to opportunistic infection. In summary, our study identified a possible mechanism for the CRKP invasion and to decipher the clues behind the host immune response that influences CRKP infection pathogenesis.

Molecular Epidemiology of Carbapenem-Resistant Klebsiella pneumoniae in a Tertiary Hospital in Northern China.

In recent years, carbapenem-resistant Klebsiella pneumoniae (CRKP) has emerged rapidly in China with the abuse and overuse of antibiotics, and infections caused by CRKP pose a serious threat to global public health safety. The present study aimed to explore the epidemiological characteristics of CRKP isolates in Northern China and to elucidate their drug resistance mechanisms. 45 CRKP strains were consecutively collected at a teaching hospital from March 1st, 2018 to June 30th, 2018. Antimicrobial susceptibility was determined by the VITEK2 compact system and microbroth dilution method. Polymerase chain reaction (PCR) and sequencing were used to analyze multilocus sequence typing (MLST), drug resistance determinants, and plasmid types. The transfer of resistance genes was determined by conjugation. All statistical analysis was performed using SPSS 22.0 software. All 45 isolates showed multidrug resistance (MDR). MLST analysis showed ST11 (48.9%, 22/45) was the most frequent type. All of the 45 CRKP isolates contained carbapenemase genes, extended-spectrum β-lactamase (ESBL) genes, and plasmid-mediated quinolone resistance (PMQR) genes. For carbapenemase genes, KPC-2 (93.3%, 42/45) was the main genotype, and followed by GES (37.8%, 17/45) and NDM-1 (11.1%, 5/45). Plasmid typing analysis showed that IncFII and IncFIB were the most prevalent plasmids. The carbapenem resistance rate of K.pneumoniae was 11.4% and ICU was the main CRKP infection source. ST11 is the most frequent sequence type and KPC-2 is the predominant carbapenemase of CRKP strains in Northern China. KPC-2-ST11 are representative clonal lineages.

Molecular Epidemiology, Clinical Characteristics and Risk Factors for Bloodstream Infection of Multidrug-Resistant Klebsiella pneumoniae Infections in Pediatric Patients from Tianjin, China.

The data on pediatrics with Multidrug-Resistant (MDR) Klebsiella pneumoniae infections are scarce. This study aims to investigate the molecular epidemiology of MDR Klebsiella pneumoniae, detect the mechanism of drug resistance, and determine the clinical risk factors for carbapenem-resistant Klebsiella pneumonia (CRKP) bloodstream infections (BSIs) in a children's hospital. A total of 62 strains were collected from Tianjin Children's Hospital. Carba NP and polymerase chain reactions (PCR) were performed to detect MDR mechanisms. Multilocus sequence typing (MLST) was used for analyzing strain homology. Clinical data were collected and logistic regression was used for BSI risk factors. ST11 was the principal ST among the CRKP isolates clinically, accounting for 56.45% (35/62); there were also 57.14% (20/35) ST11 CRKP strains co-carrying bla NDM-5 and bla KPC-2, which were resistant to most of the tested antibiotics, being susceptible only to cotrimoxazole and tigecycline. The clinical data showed that 72.73% (40/55) of children with CRKP infection had serious underlying diseases; 20.00% (11/55) patients developed BSIs with the potential to cause multiple organ failure, shock and death. The logistic regression showed that the risk of BSIs caused by CRKP strain infections in children with hematological malignancies after chemotherapy was 7 times that of other children (95%Cl: 1.298-45.415, P=0.025). ST11 was the prevalent clone in our hospital. The emergence of ST11 CRKP co-carrying bla NDM-5 and bla KPC-2 should be a cause for alarm as they were resistant to most of the tested antibiotics. CRKP strain infections are mainly occurring in young immunocompromised patients and the chemotherapy for hematological malignancies is an independent risk factor for BSIs.

Evidence of Sharing of Carbapenem-Resistant Klebsiella pneumoniae Strains Between Intensive Care Unit Patients and the Environment.

Carbapenem-resistant Klebsiella pneumoniae (CR-KP) has emerged as an important public health threat. Intestinal colonization with CR-KP increases the risk of infection and death, especially in intensive care unit patients. To clarify the source of colonizing bacteria is very important to prevent the spread of CR-KP, so the purpose of this study was to explore the relationship between the ward environment and intestinal colonization of CR-KP. In this study, 353 environmental swabs from ICU (Intensive Care Unit) wards and 241 anal swab samples from ICU patients were collected and screened on MacConkey plates containing 2 μg/mL ertapenem, and the origin and genotype of CR-KP were analyzed by PCR and sequencing. The sequence type of the strains was also obtained by multi-locus sequence type (MLST) analysis, and plasmid conjugation test was used to clarify whether CR-KP can promote the transmission of drug resistance genes through plasmid integration and rearrangement. A total of 20 CR-KP environmental strains and 7 intestinal strains were obtained, most of which were bla OXA-48 resistant genotypes. Four different STs were identified by multi-locus sequence type (MLST) analysis, among which the large logarithm was ST485 type, and PFGE clustering showed that the similarity between them was >85%. In the plasmid transcoupling assay, we report that one of the Klebsiella pneumoniae drug-resistant plasmids was successfully transferred to E. coli, indicating that it may promote the spread of drug-resistant genes through plasmid integration and rearrangement. Our research suggests that the environment may be a potential source of CR-KP and that there is a need for us to adopt more effective disinfection measures.

Risk Factors and Molecular Mechanism of Polymyxin B Resistance in Carbapenem-Resistant Klebsiella pneumoniae Isolates from a Tertiary Hospital in Fujian, China.

The emergence of polymyxin B resistance among carbapenem-resistant Klebsiella pneumoniae (CRKP) causes clinical treatment to be more difficult. We aimed to investigate the risk factors and resistance mechanisms in the polymyxin resistant CRKP (PR-CRKP) strains. From January 2021 to January 2022, 239 CRKP strains were selected, all of which were analyzed using antimicrobial susceptibility testing and clinical data. Polymerase chain reaction (PCR) was performed for the detection of resistance genes. RT-qPCR was used to quantify transcriptional levels of polymyxin resistance genes. Risk factors for polymyxin B resistant isolates were identified by logistic regression analysis. The resistance rate of polymyxin B was 5.02%. In all CRKP strains, 41.84% came from the ICU. The percentage of carbapenemase producing strains was 93.72%. The main carbapenem resistance gene was blaKPC (90.79%). In the 12 strains of PR-CRKP screened, pmrB and pmrK were overexpressed in all samples which were linked with polymyxin B resistance. Multivariate analysis showed that coronary heart disease may be an independent risk factor predisposing patients to polymyxin B resistance. We determine the multifaceted mechanism and risk factors of polymyxin B resistance in CRKP. Polymyxin resistance is a complex and changing problem, and more research is required.

Tandem Repeat of bla NDM-1 and Clonal Dissemination of a fosA3 and bla KPC-2 Co-Carrying IncR-F33: A-: B- Plasmid in Klebsiella pneumoniae Isolates Collected in a Southwest Hospital in China, 2010-2013.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) has been widespread in coastal cities of eastern China since 2009. However, how CRKP spreads and evolves in southwest China is unclear. We investigated the genetic characteristics and dissemination mechanisms of carbapenemase genes in forty-one non-repetitive CRKP isolates collected from a southwest hospital, Kunming, Yunnan, during 2010-2013. Drug susceptibilities were analyzed by using VITEK 2 compact system. Genetic relationships were ascertained based on multilocus sequence typing (MLST) and Pulsed-field gel electrophoresis (PFGE) analysis. Genetic backgrounds of bla KPC-2 and bla NDM-1 were revealed by DNA walking and high-throughput sequencing. All isolates were highly resistant to common antibiotics except for tigecycline. In total, 34 bla KPC-2, 3 bla NDM-1, 1 bla IMP-4 and 3 bla IMP-26 genes were identified and KP67 plasmid 1 co-harbored bla NDM-1 and bla IMP-26. Five sequence types, namely ST11, ST290, ST340, ST395 and ST437, were recognized by MLST. Surprisingly, bla KPC-2 was only detected in ST11 strains. We described a clonal dissemination of fosA3-positive IncR-IncF33:A-:B- multireplicon plasmid carrying the gene cassettes IS26-ΔTn3-ISKpn27-bla KPC-2-ΔISKpn6-korC-klcA-ΔrepB-Tn1721 in all ST11 isolates. Three bla NDM-1 positive isolates belonged to three different ST types and their bla NDM-1 genetic backgrounds were also distinct. Interestingly, the flanking regions of bla NDM-1 in KP67 and KP72 were duplicated into one to five copies in a form of tandem repeat by the transposition of IS91 like element. The bla NDM-1 of KP82 was carried on a common IncX3 plasmid. This study described the early epidemiological characteristics of bla KPC-2/bla NDM-1-carrying CRKP, and reported a new tandem repeat pattern of bla NDM-1 cluster in Yunnan. These findings extend our knowledge on the carbapenemase gene evolutions.

Antimicrobial treatment of monomicrobial phenotypic carbapenem-resistant Klebsiella pneumoniae bacteremia: Two are better than one.

Infections caused by carbapenem-resistant Klebsiella pneumoniae (CRKP) are emerging worldwide. The optimal treatment for CRKP infections is challenging for clinicians because therapeutic agents are greatly limited. A retrospective study of CRKP monomicrobial bacteremia was conducted at a medical center between 2010 and 2016. The use of at least one or more drugs with invitro activity against the blood isolates was defined as appropriate combination therapy. The logistic regression model and propensity score analysis was used to assess clinical effects of therapeutic strategies. The 30-day crude mortality was the primary end point. Two hundred and three patients were eligible and the 30-day mortality rate was 37.9% (77 patients). As compared with monotherapy, empirical (11.6 vs. 57.3%, p<.001) or definitive (26.5% vs. 48.6%, p=.001) combination antibiotic therapy showed a lower 30-day mortality rate independently. The propensity score analysis showed that those receiving combination therapy had less clinical (p≤.001) or microbiological failure (p=.003) and a lower 30-day mortality rate (p<.001). Among various regimens of definitive therapy, the 30-day mortality rate was the lowest among patients with appropriate combination therapy 23.6%, (p<.001; by log rank test). The primary outcome was similar in those with definitive carbapenem-containing and carbapenem-sparing combination regimens (p=.81). The presence or absence of carbapenemase production did not affect the mortality rate (p=.26). Combination therapy, regardless of carbapenem-containing or carbapenem-sparing regimens, was associated with a favorable outcome.

Competitive Transmission of Carbapenem-Resistant Klebsiella pneumoniae in a Newly Opened Intensive Care Unit.

We conducted a 6-month prospective study in a newly opened ICU for high-resolution tracking of carbapenem-resistant Klebsiella pneumoniae (CRKP) through environmental surveillance, patient screening, and genome sequencing. Among all ICU patients (n = 348) screened, 3.5% carried CRKP on admission and 16.3% acquired CRKP thereafter. CRKP was not detected in the environment until 10 weeks and was then isolated from 98 of 2,989 environmental samples (3.3%). The first CRKP isolate from rectal swabs (n = 37) and the first clinical isolate (n = 8) of each patient as well as the 98 isolates from environmental were subjected to whole-genome sequencing. The 143 CRKP isolates from patients and environment samples were assigned to four sequence types, with ST11 dominating (95.8%) and further divided into 14 clones, suggesting introduction of multiple clones. Subsequent CRKP transmission was complex and dynamic with 10 clones found in multiple patients and seven also detected in the environment. Two particular ST11 clones caused extensive (≥5 rooms) and persistent (≥10 weeks) environmental contamination. Both clones were associated with patients who carried CRKP throughout their prolonged ICU stay. Such "super-contaminators" are a priority for isolation and environmental surveillance. IMPORTANCE Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a global challenge for human health. In health care settings, patients have frequent interactions with other patients and the environment, rendering challenges for untangling the introduction and transmission of CRKP. We conducted a prospective surveillance study in a newly opened ICU for high-resolution tracking of CRKP. Our study demonstrated the dynamic, complicated transmission of CRKP and has important findings that may help to curb its spread in health care settings. First, compliance with basic measures such as routine environment cleaning and postdischarge terminal cleaning is needed to minimize the environmental contamination-driven spread. Second, active screening could demonstrate the scale of the problem, and room transfer of patients with CRKP should be prohibited whenever possible. Third, the priority for single-room isolation should be given to patients with prolonged carriage of CRKP, especially in resource-limited settings. Good infection control practice lays a foundation for tackling multidrug-resistant organisms like CRKP.

Molecular typing and drug resistance analysis of carbapenem-resistant Klebsiella pneumoniae from paediatric patients in China.

There are few studies on paediatric carbapenem-resistant Klebsiella pneumoniae (CRKP) in China. The present study investigated the molecular epidemiological and drug resistance characteristics of CRKP from paediatric patients in China to provide a reference for the prevention and control of CRKP infection. In total, 20 nonrepetitive clinical CRKP isolates were collected between February 2019 and February 2020 in a tertiary hospital in China. Strain identification and drug susceptibility testing were carried out using the VITEK® 2 Compact Bacterial Identification and Monitoring System. Sequence typing, phylogenetic relationships, and antibiotic resistance-associated genes were analysed by whole genome sequencing (WGS). sequence typing (MLST) and Core genome multilocus sequence typing (cgMLST) analysis revealed the most frequently represented were ST2407-CT3536 (30%), ST76-CT5893 (25%), and ST309-CT7864 (25%). All 20 CRKP isolates were divided into three clusters. All isolates were highly resistant to a variety of β-lactams and were highly susceptible to quinolones, aminoglycosides, and sulphonamides. All isolates mainly carried the carbapenem resistance genes blaNDM-1 and blaKPC-2, among which 10 isolates carried both blaNDM-1 and blaKPC-2 simultaneously. Sequence typing, phylogenetic relationships, and antibiotic resistance genes can be determined using WGS technology. This can guide CRKP infection control and clinical treatment for paediatric patients.

In vitro and in vivo activity of meropenem+avibactam against MBL-producing carbapenem-resistant Klebsiella pneumoniae

Background Antibiotic resistance has become a public health problem to be solved worldwide and metallo-β-lactamase (MBL)-producing bacteria make this problem even more challenging. Methods : The interactions of meropenem (MEM) in combination with avibactam (AVI) in growth inhibition on MBL-producing carbapenem-resistant Klebsiella pneumoniae (CRKP) strains were tested. In vitro interactions of MEM+AVI were tested using the microdilution chequerboard assay and time-kill curves. In vivo interactions of MEM+AVI were tested using the Galleria mellonella model. Results All strains were multi-drug resistant strains and six of them were proved to produce MBLs. We show that the combination of MEM+AVI generates profound synergistic effects on growth inhibition of all strains, which was better than that of MEM+vaborbactam or imipenem+relebactam. Time-kill curves further confirmed the potent synergistic antibacterial effects of MEM+AVI against MBL-producing CRKP strains. Galleria mellonella studies were consistent with in vitro analysis. Combining MEM with AVI improved survival rates and mean survival days were obviously prolonged compared to the drug alone and the untreated controls. Conclusions To our knowledge, this study is the first report of MEM+AVI collaborating against MBL-producing CRKP strains. Our findings showed that the combination of MEM+AVI has the potential for antibiotic drug development to combat MBL-producing pathogens.

Synergistic Effects of Some Methoxyflavones Extracted from Rhizome of Kaempferia parviflora Combined with Gentamicin against Carbapenem-Resistant Strains of Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii.

The present study aimed to investigate the antibacterial activity of ethanolic Kaempferia parviflora extracts and the combined effects of the plant's specific compounds with gentamicin against clinical strains of carbapenem-resistant Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. The minimal inhibitory concentrations (MIC) of gentamicin and Kaempferia parviflora extracts against the tested bacterial strains were determined by using broth microdilution. The combined effects of Kaempferia parviflora extract and gentamicin were investigated by using a checkerboard assay and expressed as a fractional inhibitory concentration index (FICI). Crude ethanolic extract of Kaempferia parviflora showed the lowest median values of MIC towards the tested isolates (n = 10) of these tested bacteria at doses of 64 µg/mL, compared to those of other Kaempferia extracts. Among the isolated compounds, only three compounds, namely 3,5,7-trimethoxyflavone, 3,5,7,3'4'-pentamethoxyflavone, and 5,7,4'-trimethoxyflavone, were identified by NMR structural analysis. According to their FICIs, the synergistic effects of gentamicin combined with 3,5,7,3'4'-pentamethoxyflavone were approximately 90%, 90%, and 80% of tested carbapenem-resistant Klebsiella pneumoniae (CRKP), Pseudomonas aeruginosa (CRPA), and Acinetobacter baumannii (CRAB), respectively. The present study concluded that 3,5,7,3'4'-pentamethoxyflavone extracted from Kaempferia parviflora potentiated the antibacterial action of gentamicin to combat bacterial resistance against the tested bacteria.

Interruption of Capsular Polysaccharide Biosynthesis Gene wbaZ by Insertion Sequence IS903B Mediates Resistance to a Lytic Phage against ST11 K64 Carbapenem-Resistant Klebsiella pneumoniae.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a major challenge for clinical management worldwide with limited antimicrobial options. Phages are considered an alternative option. Here, we isolated and identified a phage able to lyse ST11-K64 CRKP, the major type in China. This phage has a narrow host range, only lysing ST11-K64 CRKP, and inhibits the growth of host strains for 3 h forming large clear plaques (3.0 to 6.0 mm in diameter) with a surrounding halo. This phage exhibited excellent stability in different temperatures and pH and did not contain any virulence, lysogenic, antimicrobial resistance genes nor tRNA, meeting the criteria for phage therapy. Genomic analysis revealed that it represents a novel species of the Przondovirus genus according to ICTV standards. However, phage-resistant bacterial mutants emerged after 4-h exposure. Compared to the parental strain, phage-resistant mutants showed nonmucoid appearance and exhibited significantly reduced virulence for Galleria mellonella larva. Three randomly selected phage-resistant mutants were genome sequenced. Interruption of capsular polysaccharide biosynthesis-associated gene wcaJ or wbaZ by IS903B alone or an IS903-formed composite transposon was identified. Interruption of wcaJ is a known phage resistant mechanism, while that of wbaZ is not. By complementing the intact wbaZ, the phage susceptibility was restored, confirming the role of wbaZ interruption in phage resistance. This highlights that alteration in the capsular polysaccharide biosynthesis gene cluster, which could be due to transposable elements, is a major mechanism for resistance to Przondovirus phages in CRKP. Noncapsule-targeting phages may be combined for improving phage therapy against CRKP. IMPORTANCE Phage therapy is an alternative approach against multidrug resistant microorganisms such as carbapenem-resistant Klebsiella pneumoniae (CRKP), which represents a major challenge for treatment due to very limited options of antimicrobial agents. For optimizing phage therapy, more new lytic phages are needed. Here, we isolated and characterized a phage of a novel species able to rapidly lyse a major type of CRKP without carrying any virulence, lysogenic, antimicrobial resistance genes. This phage is therefore suitable for clinical treatment. However, phage-resistant mutants of CRKP strains were observed after exposure. We found a new mechanism, i.e., interruption of a capsular polysaccharide biosynthesis gene wbaZ by an insertion sequence-formed composite transposon. Our study demonstrates the capsular polysaccharide biosynthesis gene cluster as a major source of resistance to certain lytic phages in CRKP. This requires more studies to counter phage resistance. Our studies also highlight the critical role of insertion sequences in phage resistance.

The Genetic Characteristics and Carbapenem Resistance Mechanism of ST307 Klebsiella pneumoniae Coharbouring blaCMY-6, blaOXA-48, and a Truncated blaNDM-1.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a common nosocomial pathogen causing severe infectious diseases, and ST307 CRKP is an emerging clone. In this study, we collected five ST307 CRKP isolates, evaluated their antimicrobial susceptibility using microbroth dilution, and their clonality and population structure by PFGE, cgMLST, and SNP-based phylogenetic analysis. Then, the genome characteristics, such as antimicrobial resistance genes and plasmid profiles, were studied by subsequent genomic analysis. The plasmid transfer ability was evaluated by conjugation, and the carbapenem resistance mechanism was elucidated by gene cloning. The results showed that all five ST307 CRKP isolates harboured blaCMY-6, blaOXA-48, and blaNDM-1; however, the end of the blaNDM-1 signal peptide was interrupted and truncated by an IS10 element, resulting in the deactivation of carbapenemase. The ST307 isolates were closely related, and belonged to the globally disseminated clade. blaOXA-48 and blaNDM-1 were located on the different mobilisable IncL/M- and IncA/C2-type plasmids, respectively, and either the pOXA-48 or pNDM-1 transconjugants were ertapenem resistant. Gene cloning showed that blaCMY-6 could elevate the MICs of carbapenems up to 64-fold and was located on the same plasmid as blaNDM-1. In summary, ST307 is a high-risk clone type, and its prevalence should be given additional attention.

In-vitro Activity of Ceftazidime-Avibactam Against Carbapenem-Resistant K. pneumoniae in a Tertiary-Care Center in Al-Ahsa, KSA

Abstract Introduction/Objective Carbapenem-resistant Enterobacteriaceae (CRE) represents an urgent global threat. Klebsiella pneumoniae is a common cause of various clinical infections showing increased rates of resistance. Carbapenem-resistant Klebsiella pneumoniae (CR-KP) is often multi-drug resistant and sometimes becomes pan-drug resistant causing serious infections with substantial morbidity, mortality and cost. Ceftazidime-avibactam (CZA) is considered to be a good alternative to standard CRE therapy. In our region, CRE is mostly due to OXA-type carbapenemases which are inhibited by CZA, however, metallo-beta-lactamases (MBLs) are being increasingly reported and against which CZA is inactive. The aim of this study is to determine the activity of CZA against CRE-KP isolated in our hospital. Methods/Case Report Single-center, prospective study on consecutive non-duplicate CRE-KP isolates recovered from various clinical specimens in 2020 and 2021. BD Phoenix® system was used for initial Identification and susceptibility testing. E-test was done to confirm susceptibility and determine the minimum inhibitory concentration (MIC) for CZA. The standards of the Clinical and Laboratory Standards Institute were followed. Results (if a Case Study enter NA) 95 CRE-KP isolates were collected. CRE-KP was mostly isolated from: Respiratory samples: 36% (n=34), bedsores: 28% (n=27), urine: 23% (n=22) and 11% (10 isolates) were retrieved from blood cultures. 65% of patients were admitted to the intensive care unit (ICU), 13% were in long-term care wards and 13% were admitted for management of infected bedsores. 88 isolates were susceptible to CZA (93%) with MIC50= 1.5 μg/mL and MIC90= 4.0 μg/mL. 26 isolates (27%) were sensitive only to CZA. Susceptibility to tigecycline was 54%, amikacin 18% and gentamicin 17%. Other antimicrocbials including ceftolozane-tazobactam were basically ineffective. Conclusion Ceftazidime-avibactam shows excellent in-vitro activity against CR-KP isolates tested in this study. Further clinical studies on treatment efficacy and emergence of resistance are needed. Moreover, with the recent increase in reported MBLs-mediated resistance in our region, infection control measures, resistance-surveillance programs, and molecular testing may prove essential in the fight against carbapenem-resistant Enterobacteriaceae.

Molecular epidemiology and mechanism of Klebsiella pneumoniae resistance to ertapenem but not to other carbapenems in China.

Resistance to only ertapenem is an unusual phenotype of carbapenem-resistant Klebsiella pneumoniae (CRKP). This study aimed to investigate the molecular epidemiology and underlying mechanism involved in ertapenem resistance of K. pneumoniae strains that are susceptible to meropenem and imipenem. Among the 697 K. pneumoniae strains isolated from 11 grade A hospitals in China, 245 were CRKP strains, of which 18 strains resistant only to ertapenem were isolated. The genotypes, phenotypes, drug resistance homology, and drug sensitivity were analyzed; moreover, the expressions of efflux pump components and outer membrane proteins were assessed. The whole genomes of these 18 strains were sequenced and analyzed for mutations leading to drug resistance. The results revealed that ertapenem resistance may be related to ramR mutation. The function of ramR was confirmed using gene complementation to the original strain to determine the mechanism underlying ertapenem resistance of K. pneumoniae strains. In total, 7.4% of the tested CRKP strains were resistant only to ertapenem. None of these strains contained carbapenemase genes. Of the 18 ertapenem-resistant strains, 17 expressed the efflux pump, and outer membrane protein expression was reduced or absent in 4 strains. Whole-genome sequencing revealed the presence of mutations that introduced premature ramR codons stop in 14 strains (77.78%). When a functional copy of ramR was restored in the 14 strains, the minimum inhibitory concentration of ertapenem decreased, inhibition of efflux pumps was not detected, and the expression of outer membrane protein OmpK35 was either increased or was restored. These findings reveal the existence of ertapenem-resistant K. pneumoniae exhibiting no clonal transmission between strains. Mutations in ramR were demonstrated to cause efflux pump inhibition and over-expression of outer membrane protein OmpK35 in some strains, which is implicated in ertapenem resistance only in K. pneumoniae.