Emergence of Ceftazidime-Avibactam Resistance and Decreased Virulence in Carbapenem-Resistant ST11 Klebsiella pneumoniae During Antibiotics Treatment.

Introduction: Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a serious threat to human public health. Ceftazidime-avibactam (CZA) is currently one of the few effective antibiotics for carbapenem-resistant Enterobacteriaceae (CRE). Methods and Results: Here, we analyzed two longitudinal Klebsiella pneumoniae clinical isolates (FK8578, FK8695) that were isolated from an ICU patient during antimicrobial treatment. Broth microdilution method, whole-genome sequencing (WGS) and comparative genomic analysis were used to elucidate the dynamics and mechanisms of antibiotic resistance. String test, quantification of capsule, biofilm inhibition test and Galleria mellonella (G. mellonella) infection model were used to explore the changes in virulence of the two clinical isolates. During antibiotic treatment, CRKP FK8578 underwent a series of drug resistance and virulence changes, including CZA resistance, carbapenem susceptibility and virulence attenuation. The results of WGS showed that mutation of bla KPC-2 to bla KPC-33 was responsible for the change of drug resistance phenotype between FK8578 and FK8695. pLVPK-like virulence plasmid without siderophore synthesis operon was identified in the two strains. On the other hand, the loss of hypermucoviscosity phenotype in the FK8695 strain may be related to a single nucleotide deletion of the rmpA gene, which would further lead to a decrease in virulence. Virulence results showed that compared with FK8578, FK8695 was negative in the string test, with decreased capsular production, smaller amounts of biofilm formation and higher survival rate of G. mellonella. Conclusion: This is the first report of CZA resistance and decreased virulence in ST11 CRKP strains during antimicrobial treatment. It is urgent to monitor CZA resistance and timely adjust anti-infective treatment strategies.

Hypervirulent carbapenem-resistant Klebsiella pneumoniae causing highly fatal meningitis in southeastern China.

Klebsiella pneumoniae (K. pneumoniae) is one of the most common causes of bacterial meningitis worldwide. The purpose of this study was to investigate the clinical and microbiological characteristics of K. pneumoniae meningitis, as well as the association of antimicrobial resistance, virulence, and patient prognosis. The clinical data of patients with K. pneumoniae meningitis from 2014 to 2020 in a tertiary teaching hospital were retrospectively evaluated. Antimicrobial susceptibility profiles were performed by the agar dilution method and broth microdilution method. The isolates were detected for virulence-related genes, resistance genes, capsular serotypes, and molecular subtypes. A total of 36 individuals with K. pneumoniae meningitis were included in the study, accounting for 11.3% (36/318) of all cases of bacterial meningitis. Of the 36 available isolates, K1, K47, and K64 were tied for the most frequent serotype (7/36, 19.4%). MLST analysis classified the isolates into 14 distinct STs, with ST11 being the most common (14/36, 38.9%). Carbapenem resistance was found in 44.4% (16/36) of the isolates, while hypervirulent K. pneumoniae (HvKP) was found in 66.7% (24/36) of the isolates. The isolates of hypervirulent carbapenem-resistant K. pneumoniae (Hv-CRKP) were then confirmed to be 36.1% (13/36). Importantly, individuals with meningitis caused by Hv-CRKP had a statistically significant higher mortality than the other patients (92.3%, 12/13 vs. 56.5%, 13/23; P < 0.05). The high percentage and fatality of K. pneumoniae-caused meningitis, particularly in Hv-CRKP strains, should be of significant concern. More effective surveillance and treatment solutions will be required in future to avoid the spread of these life-threatening infections over the world.

A Selective Medium for Screening Ceftazidime/Avibactam Resistance in Carbapenem-Resistant Enterobacterales.

Ceftazidime/avibactam (CZA) is an alternative antibiotic used for the treatment of infections caused by carbapenem-resistant Enterobacterales (CRE). However, the CZA-resistant CRE strains have been detected worldwide. Therefore, it is critical to screen CZA-resistant CRE strains in colonized patients or a specific population so as to rapidly implement infection control measures to limit their transmission. In this study, we developed a Salmonella-Shigella (SS) CZA-selective medium and assessed its performance to screen for clinical CZA-resistant CRE isolates in both pure-strain specimens and stool samples. A total of 150 non-duplicated isolates, including 75 CZA-susceptible and 75 CZA-resistant CRE pathogens, were tested by using the broth microdilution method and the SS CZA medium, respectively. The bacterial suspensions were serially diluted in the SS CZA medium, which showed excellent screening performance in both pure CZA-resistant CRE strain and the stool samples with the lowest detection limit of 101-102 and 101-103 CFU/ml, respectively. Notably, none of the susceptible isolates showed growth even at the highest dilution concentration of 108 CFU/ml. Most importantly, the SS CZA medium demonstrated excellent performance in screening simulated clinical polymicrobial specimens. Moreover, its screening performance was unaffected by the different resistance determinants for tested isolates. Cumulatively, our data suggest that the SS CZA medium can be used as a promising selective medium to screen CZA-resistant CRE, irrespective of their resistance mechanisms.

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

BACKGROUND: OXA-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. METHODS: We 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. RESULTS: Between 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. CONCLUSION: In 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.

Combining Colistin with Furanone C-30 Rescues Colistin Resistance of Gram-Negative Bacteria in Vitro and in Vivo.

The spread of multidrug-resistant (MDR) Gram-negative bacteria (GNB) has led to serious public health problems worldwide. Colistin, as a "last resort" for the treatment of MDR bacterial infections, has been used significantly in recent years and has led to the continuous emergence of colistin-resistant strains. In this study, we aimed to investigate the synergistic effect on the antimicrobial and antibiofilm activities of a colistin/furanone C-30 combination against colistin-resistant GNB in vitro and in vivo. According to antimicrobial resistance profiles, most of the colistin-resistant strains we collected showed MDR phenotypes. The checkerboard method and time-kill curve showed that the combination with furanone C-30 increases the antibacterial activity of colistin significantly. In addition, the furanone C-30/colistin combination can not only inhibit the formation of bacterial biofilm but also has a better eradication effect on preformed mature biofilms. The result of scanning electron microscopy (SEM) demonstrated that the furanone C-30/colistin combination led to a significant reduction in the number of cells in biofilms. Furthermore, furanone C-30 at 50 µg/ml did not cause any additional toxicity to RAW264.7 cells according to a cytotoxicity assay. In in vivo infection experiments, the furanone C-30/colistin combination increased the survival rate of infected Galleria mellonella larvae as well as decreased the microbial load in a mouse thigh infection model. The synergistic effect of the furanone C-30/colistin combination against colistin-resistant GNB is encouraging, and this work may shed light on a new therapeutic approach to combat colistin-resistant pathogens. IMPORTANCE Colistin is among the few antibiotics effective against multidrug-resistant Gram-negative bacteria (GNB) clinical isolates. However, colistin-resistant GNB strains have emerged in recent years. Therefore, the combination of colistin and nonantibacterial drugs has attracted much attention. In this study, the furanone C-30/colistin combination showed good antibacterial and antibiofilm activity in vitro and in vivo. In addition, increased membrane permeability leads to the synergistic effect of the furanone C-30/colistin combination. Because of the low cytotoxicity of furanone C-30, this combination has good application prospects in clinical anti-infective therapy. This finding might shed light on the discovery of combination therapy for infections caused by colistin-resistant GNB pathogens.

The prevalence and functional characteristics of CrpP-like in Pseudomonas aeruginosa isolates from China.

Modifying enzyme-CrpP and its variants reduced the MICs of fluoroquinolones in Pseudomonas aeruginosa. This study investigated the dissemination and functional characteristics of CrpP-like in P. aeruginosa from China. The positive rate of crpP-like genes in 228 P. aeruginosa was 25.4% (58/228), and 6 new crpP-like genes were determined. Transformation experiments showed that CrpP-like had a low effect on CIP and LEV susceptibility. The genetic of crpP-positive was diverse. Furthermore, the mean expression level of crpP was no statistical difference between fluoroquinolone-susceptible and -resistant group (P > 0.05). CrpP-like may not play a significant role in fluoroquinolone resistance in P. aeruginosa.

Synergistic Activity and Biofilm Formation Effect of Colistin Combined with PFK-158 Against Colistin-Resistant Gram-Negative Bacteria.

PURPOSE: The emergence of colistin resistance among Gram-negative bacteria (GNB) poses a serious public health threat. Therefore, it is necessary to enhance the antibacterial activity of colistin through the combination with other drugs. In this study, we demonstrated the synergistic activity and the possible synergy mechanism of colistin with PFK-158 against colistin-resistant GNB, including non-fermenting bacteria and Enterobacteriaceae. PATIENTS AND METHODS: Thirty-one colistin-resistant GNB, including Pseudomonas aeruginosa (n = 9), Acinetobacter baumannii (n = 5), Escherichia coli (n = 8) and Klebsiella pneumoniae (n = 9), were collected as the experimental strains and the minimum inhibitory concentrations (MICs) of colistin, other routine antimicrobial agents and PFK-158 against all strains were determined by the broth microdilution method. The synergistic activity of colistin with PFK-158 was assessed by the checkerboard assay and time-kill assay. The biofilm formation assay and scanning electron microscopy were used to demonstrate the biofilm formation effect of colistin with PFK-158 against colistin-resistant GNB. RESULTS: The results of the checkerboard assay showed that when colistin was used in combination with PFK-158, synergistic activity was observed against the 31 colistin-resistant GNB. The time-kill assay presented a significant killing activity of colistin with PFK-158 against the 9 colistin-resistant GNB selected randomly, including Pseudomonas aeruginosa (n = 6), Acinetobacter baumannii (n = 1), Escherichia coli (n = 1), and Klebsiella pneumoniae (n = 1). The biofilm formation assay and scanning electron microscopjihy showed that colistin with PFK-158 can effectively suppress the formation of biofilm and reduce the cell arrangement density of biofilm against most experimental strains. CONCLUSION: The results of the performed experiments suggest that the combination of colistin and PFK-158 may be a potential new choice as a new antibiofilm group for the treatment of infections caused by the colistin-resistant GNB.

Evaluation of NitroSpeed-Carba NP test for rapid identification among different classes of carbapenemases in Enterobacterales and Pseudomonas aeruginosa.

OBJECTIVES: This study aimed to evaluate the performance of the NitroSpeed-Carba NP test for detecting carbapenemases in the clinical strains of Enterobacterales and Pseudomonas aeruginosa (P. aeruginosa), and analyze its advantages and limitations. METHODS: The antimicrobial susceptibility tests were performed according to the agar dilution method. Using the modified carbapenemase inactivation method (mCIM), polymerase chain reaction (PCR), and sequencing, the production of carbapenemase and the prevalence of genes were studied. The NitroSpeed-Carba NP test was performed to detect different types of carbapenemases in Enterobacterales and P. aeruginosa. The results of PCR and sequencing were used as the gold standard. RESULTS: Among 144 carbapenemase-producing and 54 carbapenemase-negative strains of Enterobacterales and P. aeruginosa, the NitroSpeed-Carba NP test correctly detected 143 of 144 carbapenemase producers and 51 of 54 non-carbapenemase producers. Moreover, the sensitivity and specificity of all tested isolates were 99.31% and 94.44%, respectively (99.28% and 92.86% for Enterobacterales; 100% and 100% for P. aeruginosa). The sensitivity was 100% for class A (56 of 56), 100% for class B (60 of 60), and 100% for class D (27 of 27). CONCLUSIONS: The results suggest that NitroSpeed-Carba NP test is a simple and valuable assay that could be used as a rapid and stable detection method to identify the carbapenemases in Enterobacterales and P. aeruginosa strains.

Resistance and Heteroresistance to Colistin in Escherichia coli Isolates from Wenzhou, China.

BACKGROUND: Colistin is being administered as last-line therapy for patients that have failed to respond to other available antibiotics that are active against Escherichia coli. The underlying mechanisms of colistin resistance and heteroresistance remain largely uncharacterized. The present study investigated the mechanisms of resistance and heteroresistance to colistin in Escherichia coli isolates from Wenzhou, China. MATERIALS AND METHODS: Colistin resistance was detected by the broth microdilution method (BMD). Colistin heteroresistance was determined by population analysis profiles (PAPs). The polymerase chain reaction (PCR) was conducted to detect mcr-1, mcr-2, mcr-3, pmrA, pmrB, phoP, phoQ and mgrB, and quantitative real-time PCR (qRT-PCR) was used to determine the expression levels of mcr-1, pmrC, pmrA and pmrB. Lipid A characterization was conducted by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). RESULTS: 0.69% (2/291) of Escherichia coli strains were resistant to colistin, whereas the heteroresistance rate reached 1.37% (4/291). mcr-1, the mobile colistin-resistance gene, was present in the two resistant isolates. The substitutions in PmrB were detected in the two heteroresistant isolates. The transcripts levels of the pmrCAB operon were upregulated in two of the heteroresistant isolates. carbonylcyanide m-chlorophenylhydrazone (CCCP) was able to reverse colistin resistance of all isolates tested and exhibited a significantly higher effect on colistin-heteroresistant isolates. MALDI-TOF MS indicated that the additional phosphoethanolamine (PEtn) moieties in lipid A profiles were present both in colistin-resistant and heteroresistant isolates. CONCLUSION: The present study was the first to investigate the differential mechanisms between colistin resistance and heteroresistance. The development of colistin heteroresistance should be addressed in future clinical surveillance.

Evaluation of resazurin-based assay for rapid detection of polymyxin-resistant gram-negative bacteria.

BACKGROUND: Colistin resistance is considered a serious problem due to a lack of alternative antibiotics. The Rapid ResaPolymyxin Acinetobacter/Pseudomonas NP test is a resazurin reduction-based technique that relies on the visual detection of bacterial growth in the presence of a defined concentration of colistin. The aim of this study was to evaluate the performance of the Rapid ResaPolymyxin Acinetobacter/Pseudomonas NP test in the detection of colistin susceptibility in common clinical Gram-negative bacteria. RESULTS: A total of 253 clinical isolates from a teaching hospital, including Acinetobacter baumanii (n = 58, 8 colistin-resistant), Pseudomonas aeruginosa (n = 61, 11 colistin-resistant), Klebsiella pneumoniae (n = 70, 20 colistin-resistant) and Escherichia coli (n = 64, 14 colistin-resistant) were tested in this study. The sensitivity and specificity of the Rapid ResaPolymyxin Acinetobacter/Pseudomonas NP test compared to Broth microdilution method was 100 and 99%, respectively. CONCLUSIONS: Our results suggest that Rapid ResaPolymyxin Acinetobacter/Pseudomonas NP test could be used as an accurate detection method for colistin resistance.