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Background: To explore the plasmid characteristics and transfer mechanisms of an extensive drug resistant (XDR) clinical isolate, Citrobacter portucalensis L2724hy, co-producing blaSFO-1, blaNDM-1, and blaKPC-2. Methods: Species confirmation of L2724hy was achieved through 16S rRNA sequencing and Average Nucleotide Identity (ANI) analysis. Antimicrobial susceptibility testing (AST) employed the agar dilution and micro broth dilution methods. Identification of resistance genes was carried out by PCR and whole-genome sequencing (WGS). Essential resistance gene locations were verified by S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and southern hybridization experiments. Subsequent WGS data analysis delved into drug resistance genes and plasmids. Results: The confirmation of the strain L2724hy as an extensive drug-resistant Citrobacter portucalensis, resistant to almost all antibiotics tested except polymyxin B and tigecycline, was achieved through 16S rRNA sequencing, ANI analysis and AST results. WGS and subsequent analysis revealed L2724hy carrying blaSFO-1, blaNDM-1, and blaKPC-2 on plasmids of various sizes. The uncommon ESBL gene blaSFO-1 coexists with the fosA3 gene on an IncFII plasmid, featuring the genetic environment IS 26-fosA3-IS 26-ampR-blaSFO-1-IS 26. The blaNDM-1 was found on an IncX3 plasmid, coexisting with blaSHV-12, displaying the sequence IS 5-IS 3000-IS 3000-Tn 2-blaNDM-1-ble-trpF-dsbD-cutA-gros-groL, lacking ISAa125. The blaKPC-2 is located on an unclassified plasmid, exhibiting the sequence Tn 2-tnpR-ISKpn27-blaKPC-2-ISKpn6-korC. Conjugation assays confirmed the transferability of both blaNDM-1 and blaKPC-2. Conclusion: We discovered the coexistence of blaSFO-1, blaNDM-1, and blaKPC-2 in C. portucalensis for the first time, delving into plasmid characteristics and transfer mechanisms. Our finding highlights the importance of vigilant monitoring of drug-resistance genes and insertion elements in uncommon strains. [ABSTRACT FROM AUTHOR]

Background: Hospital wastewater (HWW) promotes the spread of carbapenem resistance genes (CRGs). Aeromonas carry a large number of CRGs in HWW, they may play a role as a suitable reservoir for CRGs, while resistomes in HWW are still poorly characterized regarding carbapenem resistant Aeromonas. Thus, the aim of the study was to evaluate the molecular epidemiological characteristics of carbapenem resistant Aeromonas in HWW. Methods: A total of 33 carbapenem resistant Aeromonas were isolated from HWW. Antimicrobial susceptibility testing and polymerase chain reaction (PCR) were used to assess the antimicrobial resistance profiles. Molecular typing was performed using enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) and multilocus sequence typing (MLST). The horizontal transmission mode of blaKPC was explored through conjugation and transformation experiments. The stability of blaKPC-IncP-6 plasmids was assessed through plasmid stability and in vitro competition test. The PCR mapping method was used to investigate the structural diversity of blaKPC. Results: The detection rates of blaKPC and cphA in Aeromonas were 97.0% and 39.4% respectively. Aeromonas caviae were grouped into 13 clusters by ERIC-PCR and 12 STs by MLST. Aeromonas veronii were grouped into 11 clusters by ERIC-PCR and 4 STs by MLST. 56.3% blaKPC were located on mobilizable IncP-6 plasmids. blaKPC-IncP-6 plasmid showed high stability and low cost fitness. Conclusion: Carbapenem resistant Aeromonas from HWW mainly carried blaKPC, which exhibited great structural diversity. Aeromonas might serve as reservoirs for blaKPC and blaKPC might spread mainly through transformation in HWW. [ABSTRACT FROM AUTHOR]

Background: Carbapenem-resistant P. aeruginosa (CRPA) is a common hospital-acquired bacterium. It exhibits high resistance to many antibiotics, including ceftazidime/avibactam and cefteolozane/tazobactam. The presence of carbapenem-resistant genes and co-existence Klebsiella pneumoniae carbapenemase (KPC) and metallo-β-lactamases (MBLs) further inactivated all β-lactams. Understanding the resistance genes of CRPA can help in uncovering the resistance mechanism and guiding anti-infective treatment. Herein, we reported a case of perianal infection with hypervirulent ST463 Pseudomonas aeruginosa. Case Presentation: The case is a 32-year-old acute myeloid leukemia (AML) patient with fever and septic shock during hematopoietic stem cell transplantation (HSCT), and the pathogen was finally identified as a highly virulent sequence type 463 (ST463) P. aeruginosa harboring carbapenem-resistant genes blaAFM-1 and blaKPC-2, which was detected in the bloodstream and originated from a perianal infection. The strain was resistant to ceftazidime/avibactam but successfully treated with polymyxin B, surgical debridement, and granulocyte engraftment after HSCT. The AML was cured during the 19-month follow-up. Conclusion: This case emphasizes the importance of metagenomic next-generation sequencing (mNGS) and whole-genome sequencing (WGS) in identifying microbes with rare resistant genes, and managing CRPA, especially in immunocompromised patients. Polymyxin B may be the least resistant option. [ABSTRACT FROM AUTHOR]

Introduction: Carbapenem-Resistant Enterobacteriaceae (CRE) has posed a significant threat to humans.The aim of this study was to investigate the molecular characteristics of blaKPC-producing Escherichia coli in a university-affiliated tertiary hospital.Methods: Polymerase chain reaction (PCR) and BLAST+ software were used to detect the prevalence of blaKPC in E. coli and Klebsiella pneumoniae. Whole-genome sequencing was performed for the blaKPC-harboring clinical E. coli isolates. Antimicrobial resistance genes, MLSTs, KPC-carrying plasmid typing and genetic environment of blaKPC were analyzed. A maximum likelihood core single nucleotide polymorphism (SNP)-based phylogeny tree was constructed to determine the evolutionary relationships within this ST131 collection. Conjugation experiments were performed to determine the mobilization of blaKPC. The minimal inhibitory concentrations of the common antimicrobial agents were determined using the broth microdilution method.Results: The prevalence of blaKPC in 424 clinical E. coli isolates and 1636 E. coli strains from GenBank database were 2.2% (45/2060) whereas the detection rate of blaKPC in K. pneumoniae from the GenBank database was 29.8% (415/1394). The blaKPC-harboring conjugants exhibited resistance to multiple β-lactams, except for cefepime-zidebactam and ceftazidime-avibactam. All blaKPC-carring E. coli isolates were susceptible to tigecycline and polymyxin B. ST131 was the dominant sequence type of blaKPC-carring E. coli, accounting for 40.0% (18/45). Most of the blaKPC-producing ST131 E. coli (89.5%,17/19) belonged to clade C ST131 lineage. Genetic environment analysis revealed that 57.8% (26/45) of blaKPC gene was linked to Tn 4401-associated structure ISKpn6-blaKPC-ISKpn7. IncN was the most common plasmid type in KPC-producing E. coli whereas IncFII was the dominant plasmid type in KPC-producing K. pneumoniae.Conclusion: The detection rate of blaKPC was lower in E. coli compared with K.pneumoniae. The dominant sequence and plasmid types of blaKPC-harboring isolates differed between E. coli and K. pneumoniae. Further studies about the role of the defense system in acquisition of KPC-plasmids in E. coli will be performed to provide new insights into the low prevalence of blaKPC. [ABSTRACT FROM AUTHOR]

Purpose: This research aims to profile ten novel strains of carbapenem-resistant Enterobacteriaceae (CRE) co-carrying blaKPC and blaNDM.Methods: Clinical CRE strains, along with corresponding medical records, were gathered. To ascertain the susceptibility of the strains to antibiotics, antimicrobial susceptibility tests were conducted. To validate the transferability and cost of fitness of plasmids, conjugation experiments and growth curves were employed. For determining the similarity between different strains, ERIC-PCR was utilised. Meanwhile, whole genome sequencing (WGS) was performed to characterise the features of plasmids and their evolutionary characteristics.Results: During the course of this research, ten clinical CRE strains co-carrying blaKPC and blaNDM were gathered. It was discovered that five out of these ten strains exhibited resistance to tigecycline. A closer examination of the mechanisms underlying tigecycline resistance revealed that tmexCD 1-toprJ 1, blaKPC-2, and blaNDM-1 existed concurrently within a single Citrobacter freundii strain (CF10). This strain, with a minimum inhibitory concentration (MIC) of 32 mg/L to tigecycline, was obtained from a sepsis patient. Furthermore, an investigation of genome evolution implied that CF10 belonged to a novel ST type 696, which lacked analogous strains. Aligning plasmids exposed that similar plasmids all had less than 70% coverage when compared to pCF10-tmexCD1, pCF10-KPC, and pCF10-NDM. It was also found that tmexCD 1-toprJ 1, blaKPC-2, and blaNDM-1 were transferred by Tn 5393, IS 5, and Tn 6296, respectively.Conclusion: This research presents the first report of coexistence of tmexCD 1-toprJ 1, blaKPC-2, and blaNDM-1 in a carbapenem and tigecycline-resistant C. freundii strain, CF10.Importance: Tigecycline is considered a "last resort" antibiotic for treating CRE infections. The ongoing evolution of resistance mechanisms to both carbapenem and tigecycline presents an alarming situation. Moreover, the repeated reporting of both these resistance mechanisms within a single strain poses a significant risk to public health. The research revealed that the genes tmexCD 1-toprJ 1, blaKPC-2, and blaNDM-1, which cause carbapenem and tigecycline-resistance in the same strain, were located on mobile elements, suggesting a potential for horizontal transmission to other Gram-negative bacteria. The emergence of such a multi-resistant strain within hospitals should raise significant concern due to the scarcity of effective antimicrobial treatments for these "superbugs". [ABSTRACT FROM AUTHOR]

Background: A growing body of evidence suggests that ceftazidime/avibactam (CZA) is a potential therapeutic option for carbapenem-resistant Klebsiella pneumoniae (CRKP) infections; however, resistant strains are increasingly emerged worldwide. Herein, we deemed to investigate the susceptibility profile of CRKP isolates from cancer patients to CZA and to identify the underlying resistance mechanisms. Methods: Clinical samples were obtained from adult patients admitted to the Oncology Center of Mansoura University, Mansoura, Egypt. The antibiotic susceptibility pattern of K. pneumoniae isolates to different antibiotics was tested by the modified Kirby Bauer's disc diffusion method. Minimum inhibitory concentrations of CZA were assessed using broth microdilution method. Screening for carbapenemase-producing strains was achieved by the modified Hodge test. Multiplex polymerase chain reactions (PCRs) were conducted for uncovering of carbapenemase-encoding genes (blaKPC, blaVIM, blaIMP, blaNDM-1, and blaOXA-48), and outer membrane porin genes (ompK35 and ompK36). Results: A total of 12 CZA-resistant isolates were identified out of 47 CRKP isolates (25.5%). The MIC50 and MIC90 of CZA against CRKP were 1 and 64 μg/mL, respectively. Risk factors for CZA resistance included chronic kidney disease, mechanical ventilation, longer length of hospital stay, and ICU admission. The multivariate logistic regression demonstrated that longer length of hospital stay (P=0.03) was the only independent predictor for acquisition of CZA-resistant isolates. The leading mechanism for CZA resistance was sustained by blaKPC (50%), meanwhile 16.7% and 8.3% of the CZA-resistant isolates harbored blaOXA-48 and blaOXA-48/blaNDM-1, respectively. The MBL-encoding genes blaNDM-1 and blaIMP were detected in 16.7% and 8.3% of the isolates, respectively. Absence of both ompK35 and ompK36 was observed in 58.3% of the CZA-resistant isolates. Conclusion: CZA has displayed superior in vitro activity against CRKP isolates in comparison to other antibiotics; however, thorough molecular characterization of resistant strains is highly recommended in future studies to detect and monitor the emergence of further tackling strains. [ABSTRACT FROM AUTHOR]

aim of this study was to use whole-genome sequencing to characterize Klebsiella pneumoniae SKp2F and Klebsiella variicola SKv2E, both carrying blaKPC, co-isolated from the same sputum specimen. Methods: Antimicrobial susceptibility testing was performed using microbroth dilution. Biofilm formation was determined by crystal violet staining and virulence was measured by a serum killing assay. Whole-genome sequencing of SKp2F and SKv2E was performed using an Illumina sequencer and the genetic characteristics were analyzed by computer. Results: SKp2F and SKv2E were sensitive only to tigecycline and polymyxin among the tested antibiotics. The biofilm-forming ability of SKv2E is stronger than that of SKp2F. The grades of serum resistance of SKp2F and SKv2E are 4 and 3. MLST analysis of the 6,115,610 bp and 5,403,687 bp of SKv2E and SKp2F showed associations with ST1615 and ST631, respectively. SKv2E carried 13 resistance genes (blaKPC-2, blaTEM-1A, blaLEN17, aadA16, arr-3, qnrB4, oqxA/B, dfrA27, sul1, tetD, fosA, qacEΔ 1) and SKp2F carried 23 (blaKPC-2, blaCTX-M-3, blaTEM-1B, blaCTX-M-65, blaSHV-27, aac(6ʹ)-IIa, rmtB, arr-3, aph(3ʹ)-Ia, aadA16, qnrS1, aac(6ʹ)-Ib-cr, qnrB91, oqxA/B, mph(A), tet(A), fosA, dfrA27, and two copies of qacEΔ 1-sul1). Most of them were carried by various mobile genetic elements, such as IncFIB(K)/IncFII(K)/IncFII(Yp), IncFII(K) plasmid, Tn6338, and In469. Both SKv2E and SKp2F carried a large number of virulence factors, including type 1 and 3 fimbriae, capsule, aerobactin (iutA), ent siderophore (entABCDEFS, fepABCDGfes), and salmochelin (iroE/iroEN). SKv2E also carried type IV pili (pilW), fimbrial adherence (steB, stfD), and capsule biosynthesis gene (glf). Conclusion: blaKPC-2-carrying K. variicola and K. pneumoniae, which carried multiple resistance genes, virulence factors, and highly similar mobile genetic elements, were identified from the same specimen, indicating that clinical samples may carry multiple bacteria. We should avoid misidentification, and bear in mind that resistance genes carrying mobile genetic elements can be transmitted or integrated between bacteria in the same host. [ABSTRACT FROM AUTHOR]