[Molecular epidemiological characterization of hypervirulent carbapenem-resistant Klebsiella pneumoniae in a hospital in Henan Province from 2020 to 2022].

Objective: The study investigated the clinical distribution, antimicrobial resistance and epidemiologic characteristics of hypervirulent Carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) in a hospital in Henan Province to provide a scientific basis for antibiotic use and nosocomial infection prevention and control. Methods: A retrospective analysis of the clinical data from the cases was carried out in this study. Clinical data of patients infected with the CRKP strain isolated from the clinical microbiology laboratory of Henan Provincial Hospital of Traditional Chinese Medicine from January 2020 to December 2022 were retrospectively analyzed. A string test, virulence gene screening, serum killing, and a G. mellonella infection model were used to screen hv-CRKP isolates. The clinical characteristics of hv-CRKP and the drug resistance rate of hv-CRKP to twenty-five antibiotics were analyzed using WHONET 5.6. Carbapenemase phenotypic characterization of the hv-CRKP was performed by colloidal gold immunochromatographic assay, and Carbapenemase genotyping, multi-locus sequence typing (MLST) and capsular serotyping of hv-CRKP isolates were performed by PCR and Sanger sequencing. Results: A total of non-duplicate 264 CRKP clinical isolates were detected in the hospital from 2020 to 2022, and 23 hv-CRKP isolates were detected, so the corresponding detection rate of hv-CRKP was 8.71% (23/264). The hv-CRKP isolates in this study were mainly from the intensive care unit (10/23) and neurosurgery department (8/23), and the main sources of hv-CRKP isolates were sputum (10/23) and bronchoalveolar lavage fluid (6/23). The hv-CRKP isolates in this study were highly resistant to ß-lactam antibiotics, fluoroquinolones and aminoglycosides, and were only susceptible to colistin, tigecycline and ceftazidime/avibactam. The detection rate of the blaKPC-2 among 23 hv-CRKP isolates was 91.30% (21/23) and none of the class B and class D carbapenemases were detected. Results of MLST and capsular serotypes showed that ST11 type hv-CRKP was the dominant strain in the hospital, accounting for 56.52% (13/23), and K64 (9/13) and KL47 (4/13) were the major capsular serotypes. Conclusion: The hv-CRKP isolates from the hospital are mainly from lower respiratory tract specimens from patients admitted to the intensive care department and the drug resistance is relatively severe. The predominant strains with certain polymorphisms are mainly composed of the KPC-2-producing ST11-K64 and ST11-KL47 hv-CRKP isolates in the hospital.

Clinical, biological and genome-wide comparison of carbapenem-resistant Klebsiella pneumoniae with susceptibility transformation to polymyxin B during therapy.

OBJECTIVES: The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) is a major clinical concern, and polymyxin B (PMB) is a 'last resort' antibiotic for its treatment. Understanding the effects of drug susceptibility transformation in CRKP-infected patients undergoing PMB treatment would be beneficial to optimize PMB treatment strategies. METHODS: We retrospectively collected data from patients infected with CRKP and treated with PMB from January 2018 to December 2020. CRKPs were collected before and after PMB therapy, and patients were classified into the 'transformation' group (TG) and 'non-transformation' group (NTG) by the shift of susceptibility to PMB. We compared clinical characteristics between these groups, and further analysed the phenotypic and genome variation of CRKP after PMB susceptibility transformation. RESULTS: A total of 160 patients (37 in the TG and 123 in the NTG) were included in this study. The duration of PMB treatment before PMB-resistant K. pneumoniae (PRKP) appearance in TG was even longer than the whole duration of PMB treatment in NTG (8 [8] vs. 7 [6] days; p 0.0496). Compared with isogenic PMB-susceptible K. pneumoniae (PSKP), most PRKP strains had missense mutations in mgrB (12 isolates), yciC (10 isolates) and pmrB (7 isolates). The competition index of 82.4% (28/34) of PRKP/PSKP pairs was <67.6% (23/34), and 73.5% (25/34) of PRKP strains showed a higher 7-day lethality in Galleria mellonella and a greater ability to resist complement-dependent killing than their corresponding PSKP, respectively. CONCLUSION: Low dose with longer PMB treatment durations may be associated with the emergence of polymyxin resistance. The evolution of PRKP is predominantly mediated by an accumulation of mutations, including those in mgrB, yciC, and pmrB. Lastly, PRKP exhibited reduced growth and increased virulence compared with parental PSKP.

A Systems-Based Analysis of Mono- and Combination Therapy for Carbapenem-Resistant Klebsiella pneumoniae Bloodstream Infections.

Antimicrobial resistance is a global threat. As "proof-of-concept," we employed a system-based approach to identify patient, bacterial, and drug variables contributing to mortality in patients with carbapenem-resistant Klebsiella pneumoniae (CRKp) bloodstream infections exposed to colistin (COL) and ceftazidime-avibactam (CAZ/AVI) as mono- or combination therapies. Patients (n = 49) and CRKp isolates (n = 22) were part of the Consortium on Resistance Against Carbapenems in Klebsiella and other Enterobacteriaceae (CRACKLE-1), a multicenter, observational, prospective study of patients with carbapenem-resistant Enterobacterales (CRE) conducted between 2011 and 2016. Pharmacodynamic activity of mono- and combination drug concentrations was evaluated over 24 h using in vitro static time-kill assays. Bacterial growth and killing dynamics were estimated with a mechanism-based model. Random Forest was used to rank variables important for predicting 30-day mortality. Isolates exposed to COL+CAZ/AVI had enhanced early bacterial killing compared to CAZ/AVI alone and fewer incidences of regrowth compared to COL and CAZ/AVI. The mean coefficient of determination (R2) for the observed versus predicted bacterial counts was 0.86 (range: 0.75 - 0.95). Bacterial subpopulation susceptibilities and drug mechanistic synergy were essential to describe bacterial killing and growth dynamics. The combination of clinical (hypotension), bacterial (IncR plasmid, aadA2, and sul3) and drug (KC50) variables were most predictive of 30-day mortality. This proof-of-concept study combined clinical, bacterial, and drug variables in a unified model to evaluate clinical outcomes.

High prevalence of mgrB-mediated colistin resistance among carbapenem-resistant Klebsiella pneumoniae is associated with biofilm formation, and can be overcome by colistin-EDTA combination therapy.

The global prevalence of colistin-resistant Klebsiella pneumoniae (ColRkp) facilitated by chromosomal and plasmid-mediated Ara4N or PEtN-remodeled LPS alterations has steadily increased with increased colistin usage for treating carbapenem-resistant K. pneumoniae (CRkp). Our study demonstrated the rising trend of ColRkp showing extensively and pandrug-resistant characteristics among CRkp, with a prevalence of 28.5%, which was mediated by chromosomal mgrB, pmrB, or phoQ mutations (91.5%), and plasmid-mediated mcr-1.1, mcr-8.1, mcr-8.2 alone or in conjunction with R256G PmrB (8.5%). Several genetic alterations in mgrB (85.1%) with increased expressions of Ara4N-related phoPQ and pmrK were critical for establishing colistin resistance in our isolates. In this study, we discovered the significant associations between extensively drug-resistant bacteria (XDR) and pandrug-resistant bacteria (PDR) ColRkp in terms of moderate, weak or no biofilm-producing abilities, and altered expressions of virulence factors. These ColRkp would therefore be very challenging to treat, emphasizing for innovative therapy to combat these infections. Regardless of the underlying colistin-resistant mechanisms, colistin-EDTA combination therapy in this study produced potent synergistic effects in both in vitro and in vivo murine bacteremia, with no ColRkp regrowth and improved animal survival, implying the significance of colistin-EDTA combination therapy as systemic therapy for unlocking colistin resistance in ColRkp-associated bacteremia.

Epidemiology and clinical characteristics of infection/colonization due to carbapenemase-producing Enterobacterales in neonatal patients.

BACKGROUND: The aim of this study was to elucidate the epidemiological features of carbapenemase-producing Enterobacterales (CPE) in the pediatric and neonatal patients, to describe clinical characteristics of neonatal patients with CPE infections, and to assess risk factors for neonatal rectal colonization with CPE. RESULTS: A total of 439 carbapenem-resistant Enterobacterales (CRE) isolates recovered from 367 infant patients were characterised, including 397 isolates of Klebsiella pneumoniae (KP) and 42 isolates of Escherichia coli (EC). Carbapenemase gene blaNDM-1 was the most commonly detected, accounting for 86.56% (n = 380), followed by blaKPC-2 (9.11%, 40) and blaIMP-4 (4.33%, 19). MLST analysis showed 17 different STs detected within CPKP isolates, with ST20, ST2068, ST36 and ST17 being the most frequently isolated types. Eleven STs were identified within CPEC isolates, with ST325 being the dominant types. Eight isolates of NDM-1 producing KP, belonging to ST23, were identified as having hypervirulent traits. The main infections caused by CPE were pneumonia (n = 90) and sepsis (n = 16). All infected patients received monotherapy, with meropenem and ciprofloxacin being the most commonly used antibiotics. All pneumonia patients were cured or improved after treatment. Of the 16 patients with sepsis, 9 were cured or improved, 3 died, and 4 abandoned treatment without any clinical improvement. The rectal prevalences of CPE in the 0-3 days old (DO), the 4-28 DO, and the 29 DO-1 year old groups were decreased from 15.31%, 27.37% and 14.29% in the first stool screening period to 11.78%, 19.59% and 4.07% in the second stool screening period, respectively. Multivariate analysis showed that cesarean section, acidosis, respiration failure, gastric lavage and enema were independent risk factors for rectal colonization in the 0-3 DO group, whereas cesarean section, cephalosporins, gastric lavage and residence in rural area were independently associated with rectal colonization in the 4-28 DO group. The implementation of a series of evidence-based control measures eventually contained the CPE transmission. CONCLUSIONS: Continued vigilance, epidemiological studies, and multimodal infection prevention strategies are urgently needed due to frequent importations.

Emergence of ceftazidime-avibactam resistance due to a novel blaKPC-2 mutation during treatment of carbapenem-resistant Klebsiella pneumoniae infections.

OBJECTIVE: Klebsiella pneumoniae carbapenemase (KPC)-producing K.pneumoniae has represented a serious health problem in worldwide. The resistance to ceftazidime-avibactam (CAZ-AVI) began to emerge since its approval in 2015. We aim to explore the resistance mechanism of CAZ-AVI. METHODS: Phenotypic test and whole-genome sequencing (WGS) analysis were performed in KP-HX0917 and KP-HX1016 Klebsiella pneumoniae isolates, collected from the same patient following treatment with CAZ-AVI. RESULTS: We report a case of emergence of CAZ-AVI resistance in ST 11 KPC-2-producing K. pneumoniae (KP-HX1016) during 14 days of exposure with CZA-AVI. Molecular analysis highlighted the A533C mutation in the blaKPC-2 gene, resulting a D179A substitution in protein sequence, which restored the hydrolysis ability of imipenem and meropenem, but not for ertapenem, and the result of phenotypic test was negative. However, KP-HX0917 produced serine-carbapenemase by phenotypic detection and lost its capacity of hydrolyzing carbapenems. CONCLUSION: The emergence of CAZ-AVI resistance should arouse our attention, the susceptibility testing should be followed by a combination of phenotypic and molecular methods, to make sure that no potential carbapenemase-producing bacteria are missed.

Acquisition of a Stable and Transferable blaNDM-5-Positive Plasmid With Low Fitness Cost Leading to Ceftazidime/Avibactam Resistance in KPC-2-Producing Klebsiella pneumoniae During Treatment.

The emergence and prevalence of carbapenem-resistant Enterobacteriaceae (CRE) have drawn worldwide attention. Ceftazidime/avibactam (CAZ/AVI) gives us a valuable alternative strategy to treat CRE infections. Unfortunately, CAZ/AVI resistance could occur during CAZ/AVI treatment. The CAZ/AVI-resistant Carbapenem-resistant Klebsiella pneumoniae (CR-KP) (KP137060) and earlier CAZ/AVI-susceptible isolate (KP135194) from the same hospitalized patient were collected at Fujian Medical University Union Hospital between October and November 2019. In this study, CAZ/AVI MICs of CAZ/AVI-susceptible and -resistant isolates (KP135194 and KP137060) were 4 mg/L and 128 mg/L, respectively; and the two isolates had the same antibiotic resistance pattern to other carbapenems. Two strains were then submitted for whole-genome sequencing and bioinformatic analysis. ompK36 was not detected in two isolates. No mutation was observed in blaKPC-2, ompK35 and ompK37 in this study and there was no significant difference of the expression in blaKPC-2, ompK35 and ompK37 between the two isolates (p>0.05). Two isolates were sequence type 11 and harbored blaKPC-2, blaSHV-182 and blaTEM-1B. Compared with KP135194, KP137060 harbored an additional blaNDM-5 positive plasmid. blaNDM-5 gene could be successfully transferred into E. coli J53 at a conjugation frequency of 1.14×10-4. Plasmid stability testing showed that blaKPC-2- and blaNDM-5-harboring plasmids were still stably maintained in the hosts. Growth assay and growth competition experiments showed there was no significant difference in fitness cost between two CR-KP isolates. Our study described the acquisition of a blaNDM-5-harboring plasmid leading to resistance to ceftazidime/avibactam in KPC-2-producing Klebsiella pneumoniae during treatment. This phenomenon deserves further exploration.

Carbapenem-resistant Enterobacter cloacae complex in a tertiary Hospital in Northeast China, 2010-2019.

BACKGROUND: Carbapenem-resistant Enterobacter cloacae complex (CREC) is a new emerging threat to global public health. The objective of the study was to investigate the clinical characteristics and molecular epidemiology of CREC infections in the medical center of northeast China. METHODS: Twenty-nine patients were infected/colonized with CREC during a ten-year period (2010-2019) by WHONET analysis. Antibiotic susceptibilities were tested with VITEK 2 and micro broth dilution method (for polymyxin B and tigecycline). Carbapenemase encoding genes, ß-lactamase genes, and seven housekeeping genes for MLST were amplified and sequenced for 18 cryopreserved CREC isolates. Maximum likelihood phylogenetic tree was built with the concentrated sequences to show the relatedness between the 18 isolates. RESULTS: There was a rapid increase in CREC detection rate during the ten-year period, reaching 8.11% in 2018 and 6.48% in 2019. The resistance rate of CREC isolates to imipenem and meropenem were 100.0 and 77.8%, however, they showed high sensitivity to tigecycline, polymyxin B and amikacin. The 30-day crude mortality of CREC infection was 17.4%, indicating that it may be a low-virulence bacterium. Furthermore, molecular epidemiology revealed that ST93 was the predominant sequence type followed by ST171 and ST145, with NDM-1 and NDM-5 as the main carbapenemase-encoding genes. Moreover, E. hormaechei subsp. steigerwaltii and E. hormaechei subsp. oharae were the main species, which showed different resistance patterns. CONCLUSION: Rising detection rate of CREC was observed in a tertiary hospital, which showed heterogeneity in drug resistance patterns, resistance genes, and MLST types. Effective infection prevention and control measures should be taken to reduce the spread of CREC.

[Clinical application of mCIM and eCIM combined detection of carbapenemase-producing enterobacteriaceae].

Objective: To explore the clinical application value of modified carbapenem inactivation test (mCIM) combined with EDTA-modified carbapenem inactivation test (eCIM) for detecting the carbapenemase of CRE isolated from infected patients in clinical diagnosis and infection control of CRE infection. Methods: Drug resistance of seventy eight non-repetitive enterobacteriaceae resistant to carbapenem antibiotics, which were isolated from clinically infected patients from January 2017 to December 2017 in the Tianjin Medical University General Hospital, was retrospectively analyzed. Meanwhile, Vitek2 Compact automatic bacterial identification instrument was used to identify the species and detect its minimum inhibitory concentration (MIC) for ertapenem and imipenem. Carbapenemase genes blaKPC, blaNDM, blaIMP and blaVIM were detected by PCR test, the genotype was determined by gene sequencing as the gold standard, and mCIM Combined eCIM test was used for carbapenemase detection of collected bacteria. Using PCR results as the standard, the sensitivity, specificity, positive predictive value, negative predictive value and Cohen' Kappa value of mCIM and eCIM were calculated, and the consistency of the combined detection results of mCIM and eCIM and PCR results was checked. Results: Seventy eight carbapenem-resistant Enterobacteriaceae were detected by PCR, 74 carbapenemase gene positive and 4 negative, including 60 blaKPC-2 positive, 2 blaNDM-1 positive and blaNDM-5 positive Of the 7 strains, 3 strains were positive for blaNDM-9, 1 strain was positive for blaIMP-4, and 1 strain was both positive for blaKPC-2 and blaNDM-1. The sensitivity of mCIM to detect carbapenemase is 100%, the specificity is 100%, and the Kappa value is 1.000; the combined detection of mCIM and eCIM for serine carbapenemase has a sensitivity of 100%, the specificity is 100%, and the Kappa value is 1.000; The sensitivity of combined detection of metallo-ß-lactamase is 92.9%, the specificity is 100%, and the Kappa value is 0.955. Conclusions: The combined test of mCIM and eCIM which is used to detect carbapenemase in CRE costs low, doesn't require special reagents and equipment, has strong practicability, simple operation, and easy interpretation of results. According to the different genotypes of CRE bacteria, it provides important clinical diagnostic evidence for clinical CRE diagnosis, precise antimicrobial treatment and infection control.

Detection of Carbapenemase and CTX-M Encoding Genes Directly from Bronchoalveolar Lavage Using the CRE and ESBL ELITe MGB Assays: Toward Early and Optimal Antibiotic Therapy Management of Critically Ill Patients with Pneumonia.

The detection of carbapenemase extended-spectrum ß-lactamase (ESBL)-producing Enterobacterales (EB) has become a major issue among critically ill patients, especially due to their impact on appropriate antimicrobial therapy. This study aimed at evaluating the potential contribution of molecular assays to early optimization of empirical antibiotic therapy among critically ill patients with carbapenemase- and/or CTX-M-producing EB pneumonia. The CRE and ESBL ELITe MGB® assays were evaluated directly on 197 bronchoalveolar lavage (BAL) samples obtained from 120 patients. Molecular results were then compared to routine culture-based diagnostic results, and a retrospective analysis of the therapeutic antimicrobial management was performed. Among the 197 clinical specimens, blaKPC-like and blaCTX-M-like were detected in 20 (10.2%) and 12 (6.1%) specimens belonging to 15 and 11 patients, respectively. Positive predictive value (PPV) and negative predictive value (NPV) of the CRE ELITe MGB Kit were 85% [95% confidence interval [CI]: 64.9-94.6] and 100%, respectively. PPV and NPV of the ESBL ELITe MGB Kit were 75% [95% CI: 49.4-90.2] and 100%, respectively. Retrospective analysis of the therapeutic antimicrobial management at the time of BAL collection showed that in ∼50% of patients with carbapenemase- and CTX-M-producing EB pneumonia empirical antibiotic therapy could have been optimized at least 48-72 hr earlier if positive molecular data had been used. The CRE and ESBL ELITe MGB assays might be an interesting tool for expediting optimization of empirical antibiotic therapy in critically ill patients with pneumonia, depending on local epidemiology of antibiotic resistance, patient risk stratification for EB infection, and availability of an antimicrobial stewardship team.

Successful control of the first carbapenem-resistant Klebsiella pneumoniae outbreak in a Chinese hospital 2017-2019.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is considered as a serious global threat. CRKPs occurred only sporadically in the Second Hospital of Shanxi Medical University. Our study aimed to investigate and control the first outbreak of CRKP in our hospital occurred between October 2017 and August 2019. METHODS: The antimicrobial stewardship (AMS) workers have been implemented control measures properly. Clinical and epidemiological data were retrospectively collected from medical records. Carbapenemase genes were detected by modified carbapenem inactivation method (mCIM) test and the EDTA-modified carbapenem inactivation method (eCIM) test. Resistance genes were identified by polymerase chain reaction (PCR) and sequencing. Genetic relatedness was studied by multilocus sequence typing (MLST). RESULTS: During the outbreak, 31 patients were infected with CRKP isolates. 20 (64.5%) patients were infected with KPC-2 and/or NDM-1 producing K. pneumoniae. Mostly MLST-sequence types belonged to ST11 (21/31). The outbreak was two major K. pneumoniae clusters present in epidemiologically linked patients. CONCLUSIONS: Setting up AMS workers is potentially a highly efficient strategy for the successful control of the outbreak. A multimodal and multidisciplinary infection control strategy proved to be crucial. The emergence of CRKP in our hospital emphasizes the importance of continuous monitoring of these isolates, which helps to limit the spread of CRKPs and improve the level of management.

The new perspective of old antibiotic: In vitro antibacterial activity of TMP-SMZ against Klebsiella pneumoniae.

BACKGROUND/PURPOSE: Trimethoprim-sulfamethoxazole (TMP-SMZ) is broadly administered to treat multiple infections, and the paucity of effective treatment alternatives for infections caused by Klebsiella pneumoniae has led to a renewed interest in TMP-SMZ. The aim of this study is to evaluate the antibacterial efficacy of TMP-SMZ against K. pneumoniae. METHODS: The resistance genes of K. pneumoniae clinical isolates were investigated by PCR, followed by conjugation experiments and multilocus sequence typing. RESULTS: The resistance rate of K. pneumoniae to TMP-SMZ decreased over the collection period from 26.7% (88/330) to 16.9% (56/332). The high carrying rates (173/175, 98.9%) of resistance determinants (sul genes or dfr genes) were the main mechanisms of TMP-SMZ resistance isolates, with sul1 (142/175, 81.1%) and dfrA1 (119/175, 68.0%). Only class 1 integron was detected, the prevalence of which in TMP-SMZ resistant K. pneumoniae was 63.4% (111/175). CONCLUSION: These results provided insights into the antimicrobial efficacy of TMP-SMZ against K. pneumoniae, also illustrating the wide distribution of SMZ and TMP resistance genes among resistant K. pneumoniae. Simultaneously, the present study highlights the significance of reasonable administration and effective continued monitoring.

In vivo study assessed meropenem and amikacin combination therapy against carbapenem-resistant and carbapenemase-producing Enterobacteriaceae strains.

BACKGROUND: Carbapenem-resistant Enterobacteriaceae (CRE), especially for carbapenemase-producing Enterobacteriaceae (CPE), is an emerging cause that pose a significant threat to public health. However, efficient therapy has not been established. We assessed the antimicrobial efficacy of meropenem (MEPM) and amikacin (AMK) combination therapy. MATERIAL AND METHODS: Total eight isolates of Escherichia coli or Klebsiella pneumoniae, including CRE and/or CPE have carbapenemase genes were used. The relationship between phenotype and in vivo efficacy was assessed in neutropenic murine thigh infection model. Efficacy was determined using the change in bacterial density and survival rate. RESULTS: The combination therapy showed enhanced antimicrobial activities against CRE+/CPE+ and CRE+/CPE-K. pneumoniae isolates than MEPM monotherapy (0.63 ± 0.04 vs. 2.56 ± 0.24 ⊿log10 cfu/mL, p < 0.05; -1.05 ± 0.15 vs. -0.48 ± 0.30 ⊿log10 cfu/mL, p < 0.05). Likewise, the combination therapy showed enhanced antimicrobial activities against CRE+/CPE+ and CRE+/CPE-E. coli isolates than MEPM monotherapy (0.90 ± 0.68 vs. 1.86 ± 0.23 ⊿log10 cfu/mL, p < 0.05; -1.81 ± 0.06 vs. -0.88 ± 0.23 ⊿log10 cfu/mL, p < 0.05). Also, combination therapy group showed similar to higher survival rates in CRE + E. coli infection mice, compared to MEPM monotherapy group. CONCLUSION: Our results are the first supportive data to threat CRE infections with combination therapy of MEPM and AMK with in vivo model. The current results verify the promising utility of the combination therapy with MEPM and AMK against CRE isolates with a wide range of MEPM MICs.

Efficacy of colistin alone and in various combinations for the treatment of experimental osteomyelitis due to carbapenemase-producing Klebsiella pneumoniae.

OBJECTIVES: In a new experimental model of carbapenemase-producing Klebsiella pneumoniae osteomyelitis we evaluated the efficacy of colistin alone and in various combinations and examined the emergence of colistin-resistant strains and cross-resistance to host defence peptides (HDPs). METHODS: KPC-99YC is a clinical strain with intermediate susceptibility to meropenem (MIC = 4 mg/L) and full susceptibility to gentamicin, colistin and tigecycline (MICs = 1 mg/L) and fosfomycin (MIC = 32 mg/L). Time-kill curves were performed at 4× MIC. Osteomyelitis was induced in rabbits by tibial injection of 2 × 108 cfu. Treatment started 14 days later for 7 days in seven groups: (i) control; (ii) colistin; (iii) colistin + gentamicin; (iv) colistin + tigecycline; (v) colistin + meropenem; (vi) colistin + meropenem + gentamicin; and (vii) colistin + fosfomycin. RESULTS: In vitro, colistin was rapidly bactericidal, but regrowth occurred after 9 h. Combinations of colistin with meropenem or fosfomycin were synergistic, whereas combination with tigecycline was antagonistic. In vivo, colistin alone was not effective. Combinations of colistin with meropenem or fosfomycin were bactericidal (P < 0.001) and the addition of gentamicin enhanced the efficacy of colistin + meropenem (P = 0.025). Tigecycline reduced the efficacy of colistin (P = 0.007). Colistin-resistant strains emerged in all groups except colistin + fosfomycin and two strains showed cross-resistance to HDP LL-37. CONCLUSIONS: In this model, combinations of colistin plus meropenem, with or without gentamicin, or colistin plus fosfomycin were the only effective therapies. The combination of colistin and tigecycline should be administered with caution, as it may be antagonistic in vitro and in vivo.

Rapid Increase in Carbapenemase-Producing Enterobacteriaceae in Retail Meat Driven by the Spread of the blaNDM-5-Carrying IncX3 Plasmid in China from 2016 to 2018.

The presence and molecular characteristics of carbapenemase-producing Enterobacteriaceae (CPE) among meat products in China were investigated. A total of 110 carbapenem-resistant Enterobacteriaceae (CRE) isolates, including 94 Escherichia coli and 10 Klebsiella pneumoniae isolates, were identified from 105 of 794 (13.2%) samples. The positive rates markedly increased from 2016 (9.4%) to 2018 (22.2%). Only blaNDM genes were detected; 79.1% of blaNDM genes were carried by IncX3 plasmids. Routine monitoring of carbapenemase-producing Enterobacteriaceae in the animal food supply is highly recommended.

Synergetic Effects of Combined Treatment of Colistin With Meropenem or Amikacin on Carbapenem-Resistant Klebsiella pneumoniae in vitro.

The purpose of this study was to investigate the synergistic and bactericidal effects of combinations of colistin with meropenem or amikacin in vitro and provide laboratory data needed for development of therapeutic strategies for the treatment of carbapenem-resistant Klebsiella pneumoniae (CRKP) infection. We found that minimum inhibitory concentration (MIC) of colistin, meropenem and amikacin were 2~32, 4~256, and 1~16384 µg/ml, respectively. The minimum bactericidal concentration of the antibiotics was either 1× or 2×MIC. Treatments of 6 CRKP isolates at 1 µg/ml colistin completely killed 2 of them and suppressed 4 others growth. 4 CRKP isolates at 16 µg/ml meropenem or amikacin completely killed and suppressed 2 others growth. 2 CRKP isolates showed synergic effects in all colistin combination and 3 CRKP isolates showed synergic effects in part of colistin combination. Our data suggest that colistin in combination with either meropenem or amikacin could be a valid therapeutic option against colistin-resistant CRKP isolates. Moreover, the combination of colistin-amikacin is less expensive to treat CRKP infections in Eastern Heilongjiang Province.

In vivo evolution of resistant subpopulations of KPC-producing Klebsiella pneumoniae during ceftazidime/avibactam treatment.

Objectives: KPC-producing Klebsiella pneumoniae (KPC-Kp) represent a serious problem worldwide. Herein, we describe the evolution of ceftazidime/avibactam resistance by sequencing longitudinal clinical isolates from a patient with KPC-Kp bloodstream infection undergoing ceftazidime/avibactam treatment. Methods: WGS was performed on one ceftazidime/avibactam-susceptible KPC-Kp (BOT-CA-S) and two phenotypically different ceftazidime/avibactam-resistant KPC-Kp with low (BOT-CA-R) and high (BOT-EMO) carbapenem MICs. The population diversity was assessed by the frequency of allele mutations and population analysis profiles (PAPs). Results: Phylogenetic analysis demonstrated clonal relatedness of the KPC-Kp isolates, all belonging to the clone ST1519. The D179Y mutation in blaKPC-3 was detected in both of the ceftazidime/avibactam-resistant KPC-Kp, whereas it was absent in the ceftazidime/avibactam-susceptible isolate. The mutation emerged independently in the two ceftazidime/avibactam-resistant isolates and was associated with a significant reduction in carbapenem MICs in BOT-CA-R, but not in BOT-EMO. WGS analysis revealed that the frequency of the D179Y mutation was 96.32% and 51.05% in BOT-CA-R and BOT-EMO, respectively. PAP results demonstrated that carbapenem resistance in BOT-EMO was due to the coexistence of mixed subpopulations harbouring WT and mutated blaKPC-3. A bacterial subpopulation with high ceftazidime/avibactam resistance for BOT-EMO KPC-Kp showed low carbapenem MICs, whereas a subpopulation with high meropenem resistance had a low MIC of ceftazidime/avibactam. Conclusions: Our analysis indicates that mixed subpopulations of ceftazidime/avibactam-resistant KPC-Kp emerge after ceftazidime/avibactam treatment. The evolution of different subpopulations that are highly resistant to ceftazidime/avibactam likely contributes to treatment failure, thereby highlighting the need for combination treatment strategies to limit selection of ceftazidime/avibactam-resistant KPC-Kp subpopulations.

Prevalence and drug resistance characteristics of carbapenem-resistant Enterobacteriaceae in Hangzhou, China.

With the abuse of antimicrobial agents in developing countries, increasing number of carbapenem-resistant Enterobacteriaceae (CRE) attracted considerable public concern. A retrospective study was conducted based on 242 CRE strains from a tertiary hospital in Hangzhou, China to investigate prevalence and drug resistance characteristics of CRE in southeast China. Bacterial species were identified. Antimicrobial susceptibility was examined by broth microdilution method or epsilometer test. Resistant ß-lactamase genes were identified by polymerase chain reaction and sequencing. Genotypes were investigated by phylogenetic analysis. Klebsiella pneumoniae and Escherichia coli were the most prevalent types of species, with occurrence in 71.9% and 21.9% of the strains, respectively. All strains exhibited high resistance (> 70%) against ß-lactam antibiotics, ciprofloxacin, trimethoprim-sulfamethoxazole, and nitrofurantoin but exhibited low resistance against tigecycline (0.8%) and minocycline (8.3%). A total of 123 strains harbored more than two kinds of ß-lactamase genes. blaKPC-2, blaSHV-11, blaTEM-1, and blaCTX-M-14 were the predominant genotypes, with detection rates of 60.3%, 61.6%, 43.4%, and 16.5%, respectively, and were highly identical with reference sequences in different countries, indicating potential horizontal dissemination. IMP-4 was the most frequent class B metallo-lactamases in this study. In conclusion, continuous surveillance and effective prevention should be emphasized to reduce spread of CRE.