Klebsiella pneumoniae ST147 harboring blaNDM-1, multidrug resistance and hypervirulence plasmids.

The spread of hypervirulent (hv) and carbapenem-/multidrug-resistant Klebsiella pneumoniae is an emerging problem in healthcare settings. The New Delhi metallo-ß-lactamase-1 (blaNDM-1) is found in Enterobacteriaceae including K. pneumoniae. The blaNDM-1 is capable of hydrolyzing ß-lactam antibiotics which are used for treatment of severe infections caused by multidrug-resistant Gram-negative bacteria. This is associated with the unacceptably high mortality rate in immunocompromised burn injury patients. This study reports on the characterization of blaNDM-1 gene and virulence factors in hv carbapenem-/multidrug-resistant K. pneumoniae ST147 in the burns unit of a tertiary teaching hospital during routine surveillance. Two K. pneumoniae strains were obtained from wounds of burn-infected patients from May 2020 to July 2021. The hypervirulence genes and genetic context of the blaNDM-1 gene and mobile genetic elements potentially involved in the transposition of the gene were analyzed. We identified a conserved genetic background and an IS26 and open reading frame flanking the blaNDM-1 gene that could suggest its involvement in the mobilization of the gene. The plasmid harbored additional antibiotic resistance predicted regions that were responsible for resistance to almost all the routinely used antibiotics. To ensure the identification of potential outbreak strains during routine surveillance, investigations on resistance genes and their environment in relation to evolution are necessary for molecular epidemiology.IMPORTANCEData obtained from this study will aid in the prompt identification of disease outbreaks including evolving resistance and virulence of the outbreak bacteria. This will help establish and implement antimicrobial stewardship programs and infection prevention protocols in fragile health systems in countries with limited resources. Integration of molecular surveillance and translation of whole-genome sequencing in routine diagnosis will provide valuable data for control of infection. This study reports for the first time a high-risk clone K. pneumoniae ST147 with hypervirulence and multidrug-resistance features in Ghana.

Understanding blaNDM-1 gene regulation in CRKP infections: toward novel antimicrobial strategies for hospital-acquired pneumonia.

BACKGROUND: The escalating challenge of Carbapenem-resistant Klebsiella pneumoniae (CRKP) in hospital-acquired pneumonia (HAP) is closely linked to the blaNDM-1 gene. This study explores the regulatory mechanisms of blaNDM-1 expression and aims to enhance antibacterial tactics to counteract the spread and infection of resistant bacteria. METHODS: KP and CRKP strains were isolated from HAP patients' blood samples. Transcriptomic sequencing (RNA-seq) identified significant upregulation of blaNDM-1 gene expression in CRKP strains. Bioinformatics analysis revealed blaNDM-1 gene involvement in beta-lactam resistance pathways. CRISPR-Cas9 was used to delete the blaNDM-1 gene, restoring sensitivity. In vitro and in vivo experiments demonstrated enhanced efficacy with Imipenem and Thanatin or Subatan combination therapy. RESULTS: KP and CRKP strains were isolated with significant upregulation of blaNDM-1 in CRKP strains identified by RNA-seq. The Beta-lactam resistance pathway was implicated in bioinformatics analysis. Knockout of blaNDM-1 reinstated sensitivity in CRKP strains. Further, co-treatment with Imipenem, Thanatin, or Subactam markedly improved antimicrobial effectiveness. CONCLUSION: Silencing blaNDM-1 in CRKP strains from HAP patients weakens their Carbapenem resistance and optimizes antibacterial strategies. These results provide new theoretical insights and practical methods for treating resistant bacterial infections.

Intestinal Colonization Due to Carbapenem-Resistant Enterobacteriaceae Among Hematological Malignancy Patients in India: Prevalence and Molecular Charecterisation.

Faecal carriage of Carbapenem-resistant Enterobacteriaceae (CRE) is being observed as an important risk factor for bacteremia among patients with hematological malignancies. A prospective surveillance study was conducted among these patients to determine the gut colonization of CRE. Rectal/perianal swabs were collected to isolate CRE. Carbapenem resistance was detected by disk diffusion, modified-Hodge, Carba-NP test, and PCR for bla NDM-1, bla KPC, bla OXA-48, bla VIM, bla IMP genes. A total of 209 CRE isolates were identified from 151 patients. E. coli was the most common (83.2%) CRE identified, followed by Klebsiella spp. (9.6%). The majority of CRE were observed resistant to ertapenem (86%). bla NDM-1 was the most common gene (57.3%), followed by bla OXA-48 (37.8%). 26.8% isolates found to carry both bla NDM-1 and bla OXA-48 genes. CRE is increasingly observed to cause bacteremia among hematological malignancy patients due to increased colonization. Screening for gut CRE colonization is necessary to guide empirical therapy and apply infection control measures among these patients.

Detection of NDM-1 producing Klebsiella pneumoniae ST15 and ST147 in Iran during 2019-2020.

Carbapenems are employed to treat infections caused by Gram-negative bacteria including Klebsiella pneumoniae. This research is aimed to perform phenotypic detection of ß-lactamases and molecular characterization of NDM-1 positive K. pneumoniae isolates. Another objective is to investigate NDM-1 producing K. pneumoniae among children in Iran. From 2019 to 2020, altogether 60 K. pneumoniae isolates were acquired from various patients in certain Iranian hospitals. Antimicrobial susceptibility testing was performed by disk diffusion and broth microdilution methods. In addition, mCIM and eCIM were used to confirm the production of carbapenemases and metallo-beta-lactamases (MBLs), respectively. Detection of resistance genes namely, blaNDM-1, blaIMP, blaVIM, blaKPC, blaOXA-48-like, blaCTX-M, blaSHV, blaTEM, and mcr-1 was performed by PCR and confirmed by DNA sequencing. Multilocus sequence typing (MLST) was employed to determine the molecular typing of the strains. According to the findings, the highest rate of carbapenem resistance was detected against doripenem 83.3% (50). Moreover, 31.7% (19) were resistant to colistin. Further to the above, altogether 80% (48) were carbapenemase-producing isolates and among them 46.7% (28) of the isolates were MBL and 33.3% (20) isolates were serine ß-lactamase producer. According to the PCR results, 14 isolates produced blaNDM-1. Remarkably, four blaNDM-1 positive isolates were detected in children. In addition, these isolates were clonally related as determined by MLST (ST147, ST15). Altogether ten blaNDM-1 positive isolates were ST147 and four blaNDM-1 positive isolates were ST15. Based on the results, the emergence of NDM-producing K. pneumoniae among children is worrying and hence, it is necessary to develop a comprehensive program to control antibiotic resistance in the country.

Molecular characterization and antimicrobial susceptibility profile of New Delhi metallo-beta-lactamase-1-producing Escherichia coli among hospitalized patients.

BACKGROUND: Carbapenemase-producing microorganisms are becoming a major concern among hospital-acquired infections. There is also increased multidrug resistance seen among these isolates. AIMS: We have conducted this study to determine the prevalence of New Delhi metallo-beta-lactamase-1 (NDM-1) gene-producing Escherichia coli among hospitalized patients in a tertiary care hospital in Northern India. SETTINGS AND DESIGN: The study was conducted in the Department of Microbiology with the tertiary care hospital settings. It was a prospective cross-sectional observational study conducted during January 2014-August 2014. MATERIALS AND METHODS: A total of 500 nonduplicate E. coli samples were processed. The isolates with reduced susceptibility to ertapenem, i.e., zone diameter between 19 and 21 mm, were considered carbapenemase producers. These isolates were subjected to modified Hodge test for phenotypic confirmation. Polymerase chain reaction was performed on all the screened isolates for molecular detection of NDM-1 gene. STATISTICAL ANALYSIS USED: Chi-square test was used to analyze the data and P < 0.05 was considered statistically significant. RESULTS: Out of 500 E. coli isolates, 61 (12.2%) were screened for carbapenemase production. 47 (9.4%) isolates were positive by modified Hodge test and 36 (7.2%) isolates showed the presence of blaNDM-1 gene (P < 0.05). CONCLUSION: There is an increased prevalence of NDM-1 gene-producing E. coli isolates. These carbapenemase-producing isolates are more resistant to other group of antibiotics (aminoglycosides, fluoroquinolones along with ß-lactam group). Early detection of blaNDM-1 gene can help in choosing the effective treatment options for hospitalized patients in time, thereby reducing the risk of mortality.

Emergence of NDM-1 among carbapenem-resistant Klebsiella pneumoniae in Iraqi hospitals.

Carbapenems are the last drugs of choice apart from colistin against serious infections caused by Gram-negative bacteria. However, there are increasing number of reports indicating prevailing emergence of metallo-ß-lactamase (MBL)-producing clinical isolates worldwide and among them New Delhi MBL (NDM) is the most prevalent one. This study reports NDM-1 for the first time among Klebsiella pneumoniae from hospitalized patients in Baghdad, Iraq. Fifty-five clinical isolates of K. pneumoniae resistant to carbapenem were investigated from burned wounds, sputum, and blood samples. The susceptibility to different antibiotics was tested by VITEK-2 system. All strains were multidrug-resistant and they showed nine different antimicrobial-resistant patterns (A-I) and the most effective antibiotic on these strains was levofloxacin (85.45%). The phenotypic detection of carbapenemases by MASTDISCS D70C revealed 29 (52.73%) strains were MBL-producing, out of 55 were carbapenem-resistant K. pneumoniae strains. The blaNDM-1 and other MBL genes were detected by conventional PCR and the result showed 37 (67.27%) strains positive for blaNDM-1 gene and only 5 (9.1%) strains harbored blaIMP gene, while all strains were negative for blaVIM, blaSIM, blaGIM, and blaSPM genes. Our results showed the coexistence of both blaNDM-1 and blaIMP genes in three strains of K. pneumoniae, while indicated widespread NDM-1 in Baghdad, Iraq. Hence, it is necessary to follow proper infection control practices and physicians should be aware of the patients with such risk factors.

Dissemination of blaOXA-23, blaOXA-24, blaOXA-58, and blaNDM-1 Genes of Acinetobacter baumannii Isolates from Four Tertiary Hospitals in Thailand.

Acinetobacter baumannii is a major threat to public health due to the emergence and dissemination of antibiotic-resistant strains. The purpose of this study was to determine the molecular epidemiology of antibiotic-resistant A. baumannii isolates collected from four tertiary hospitals in Thailand during the period November 2013-February 2015. We screened 339 A. baumannii, nonrepetitive clinical isolates to determine drug susceptibility. Among all isolates, we found that 7.9% was nondrug-resistant A. baumannii (NR-AB). Carbapenem-resistant A. baumannii (CR-AB) strains accounted for 84.9% of the total isolates, with extensively drug-resistant A. baumannii (XDR-AB) accounting for 7.9% of the total isolates. We further investigated class D carbapenemase genes using multiplex-PCR amplification and class B metallo-ß-lactamase genes, including blaIMP, blaVIM, and blaNDM-1 genes, using PCR and sequencing methods. We found that 300 (88.5%) isolates carried acquired class D carbapenemase genes, including blaOXA-23 (82.6%), blaOXA-24 (0.3%), and blaOXA-58 (6.5%). The genes blaIMP and blaVIM were not detected in any isolates. The blaNDM-1 was detected in 31 isolates from two hospitals (9.1%). All of the blaNDM-1-positive A. baumannii (NDM-AB) had ISAba125 sequences upstream of the blaNDM-1 gene. A coexistence of three resistance genes-blaOXA-23-blaOXA-58-blaNDM-1-was found in one isolate. A repetitive element palindromic-PCR (REP-PCR) revealed that all A. baumannii isolates were genetically diverse and could be divided into 33 genotypes. Only three genotypes were found to be predominant in all hospitals. Data from our study indicate the widespread emergence of multiple resistance determinants in A. baumannii isolates in Thailand, suggesting the need for more stringent infection control measures.

Study on plasmid-mediated blaNDM-1 gene in carbapenem-resistant Citrobacter freundii / 中国感染与化疗杂志

Objective To investigate the transmission of blaNDM-1 gene in carbapenem-resistant Citrobacter freundii. Methods A total of 18 strains of NDM-1-producing C. freundii were collected from the First Affiliated Hospital of Kunming Medical University during the period from June 2012 to October 2014. The isolates were identified and subjected to antimicrobial susceptibility testing with VITEK 2 System. Conjugation experiments, pulsed-field gel electrophoresis (PFGE) and Southern blot hybridization were performed to determine the transferability of plasmids. Results The antibiotic susceptibility results showed that all the NDM-1-producing C. freundii isolates were resistant to penicillins, cephalosporins and carbapenems. All isolates exhibited different level resistance to other antibiotics. Conjugation experiments revealed that the plasmids harboring blaNDM-1 in 13 strains were transformed into E. coli 600, and exhibited carbapenem resistance. PFGE and Southern blot hybridization found that blaNDM-1 was located on a 33.3 kb plasmid in 16 isolates and on 33.3-54.7 kb plasmid in 2 isolates. Conclusions Our findings suggest that plasmids contribute to the horizontal dissemination of blaNDM-1 gene in carbapenemresistant C. freundii.

Clonal diversity and detection of carbapenem resistance encoding genes among multidrug-resistant Acinetobacter baumannii isolates recovered from patients and environment in two intensive care units in a Moroccan hospital.

BACKGROUND: Carbapenem-resistant Acinetobacter baumannii has recently been defined by the World Health Organization as a critical pathogen. The aim of this study was to compare clonal diversity and carbapenemase-encoding genes of A. baumannii isolates collected from colonized or infected patients and hospital environment in two intensive care units (ICUs) in Morocco. METHODS: The patient and environmental sampling was carried out in the medical and surgical ICUs of Mohammed V Military teaching hospital from March to August 2015. All A. baumannii isolates recovered from clinical and environmental samples, were identified using routine microbiological techniques and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry. Antimicrobial susceptibility testing was performed using disc diffusion method. The carbapenemase-encoding genes were screened for by PCR. Clonal relatedness was analyzed by digestion of the DNA with low frequency restriction enzymes and pulsed field gel electrophoresis (PFGE) and the multi locus sequence typing (MLST) was performed on two selected isolates from two major pulsotypes. RESULTS: A total of 83 multidrug-resistant A. baumannii isolates were collected: 47 clinical isolates and 36 environmental isolates. All isolates were positive for the blaOXA51-like and blaOXA23-like genes. The coexistence of blaNDM-1/blaOXA-23-like and blaOXA 24-like/blaOXA-23-like were detected in 27 (32.5%) and 2 (2.4%) of A. baumannii isolates, respectively. The environmental samples and the fecally-colonized patients were significantly identified (p < 0.05) as the most common sites of isolation of NDM-1-harboring isolates. PFGE grouped all isolates into 9 distinct clusters with two major groups (0007 and 0008) containing up to 59% of the isolates. The pulsotype 0008 corresponds to sequence type (ST) 195 while pulsotype 0007 corresponds to ST 1089.The genetic similarity between the clinical and environmental isolates was observed in 80/83 = 96.4% of all isolates, belonging to 7 pulsotypes. CONCLUSION: This study shows that the clonal spread of environmental A. baumannii isolates is related to that of clinical isolates recovered from colonized or infected patients, being both associated with a high prevalence of the blaOXA23-like and blaNDM-1 genes. These findings emphasize the need for prioritizing the bio-cleaning of the hospital environment to control and prevent the dissemination of A. baumannii clonal lineages.

Construction of blaNDM-1gene deletion mutant of Enterobacter cloacae and analysis of its biological characteristics / 中华微生物学和免疫学杂志

Objective To construct a blaNDM-1gene deletion mutant of Enterobacter cloacae and to analyze its biological characteristics. Methods The blaNDM-1gene deletion mutant was constructed by using Red homologous recombination technology and verified by PCR and RT-qPCR. Antimicrobial susceptibility profiles,growth curves and in vitro competition abilities of the original strain and the blaNDM-1gene deletion mutant were analyzed. Results PCR,DNA sequencing and RT-qPCR showed that the blaNDM-1gene dele-tion mutant was successfully constructed. Antimicrobial susceptibility test showed that the original strain was resistant to imipenem,meropenem and ertapenem, while the blaNDM-1gene deletion mutant was sensitive to all. The original strain and the blaNDM-1gene deletion mutant had similar growth curves in Luria-Bertani liq-uid medium. In vitro competition experiment revealed that the competitive index of them was 0.69. Conclu-sion Red homologous recombination technology can be used to knockout the blaNDM-1gene of Enterobacter cloacae,which is associated with antimicrobial resistance and competitiveness.

Separation and confirmation of nine Enterobacteriaceae strains that carry the blaNDM-1 gene.

The aim of the present study was to confirm the existence of carbapenem-resistant Enterobacteriaceae carrying the blaNDM-1 gene in clinics in Hainan province, China. Collected clinical bacterial isolates that were Enterobacteriaceae strains suspected of producing carbapenemase were used as experimental strains. Drug resistance to imipenem, meropenem and other antibacterial agents was tested. Imipenem/imipenem inhibitor (IP/IPI) E-testing was conducted to identify the bacterial strains that produced metallo-ß-lactamases. The blaNDM-1 drug resistance gene was amplified by polymerase chain reaction (PCR), and agarose gel electrophoresis (AGE) and sequencing were conducted to identify the products. The species of the strains carrying the blaNDM-1 gene were determined using a biochemical identification system. Through the IP/IPI E-test, 21 of the 30 collected Enterobacteriaceae strains were found to be positive, indicating that 70% of the strains produced metallo-ß-lactamases. Following blaNDM-1 gene PCR amplification, AGE and sequencing tests confirmed that nine of the strains carried the blaNDM-1 drug resistance gene. The biochemical identification system indicated that four of the strains were Klebsiella pneumoniae, two were Escherichia coli, two were Enterobacter cloacae and one was Enterobacter aerogenes. Drug susceptibility testing in vitro demonstrated that the strains were 100% resistant to a broad spectrum antibiotic plus lactamase inhibitor, cephalosporins and carbapenems. However, they had high sensitivity rates to polymyxin B and tigecycline of 100 and 88.9%, respectively. The sensitivity rate to amikacin was also high at 77.8%, whereas sensitivity to ciprofloxacin and gentamicin was moderate at rates of 44.4 and 33.3% respectively. This clinical study of Enterobacteriaceae strains that carry the blaNDM-1 gene in Hainan shows a bacterial tolerance that is different from that in previous studies, which requires further in-depth study.