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KPC is a clinically significant serine carbapenemase in most countries, and its rapid spread threatens global public health. blaKPC transmission is commonly mediated by Tn4401 transposons. The blaKPC gene has also been found in non-Tn4401 elements (NTEKPC). To fill the gap in the understanding of the stability and dissemination of NTEKPC-carrying plasmids, we selected and characterized carbapenem-resistant bacteria isolated between 2009 and 2016 from a hospital for a retrospective study of their plasmids conjugation capacity, impact on fitness, and replication in different species. Different clones were selected using PFGE, and their genomes were sequenced using Illumina and Oxford Nanopore methods. Minimum inhibitory concentrations (MICs) were determined by broth microdilution. Plasmid copy numbers (PCNs) were determined using qPCR. Doubling time was used to analyze fitness change. Most isolates (67%, 33/49) carried blaKPC, of which 85% presented blaKPC in a NTEKPC. The 25 isolates selected presented the blaKPC gene in NTEKPC-IId in IncQ1-type plasmids, showing multispecies dissemination. IncQ1 plasmids were mobilizable and PCN seemed to be directly linked to the species, presenting a high-copy number, mainly in K. pneumoniae. No relationship was observed between IncQ1 PCN and carbapenems MIC values. IncQ1 and a conjugative plasmid from K. pneumoniae BHKPC10 were transferred to E. coli J53 without fitness changes, and MIC values were maintained for carbapenems despite the low transconjugant PCN. In addition to IncQ1 with NTEKPC, Enterobacter cloacae BHKPC28 contained the mcr-9 gene in an IncHI2/IncHI2A conjugative plasmid, which may help the mobility of IncQ1 and the dissemination of two resistance determinants to last-resort antibiotics. Understanding the interaction between plasmids and high-risk lineages can help develop new therapies to prevent the dissemination of resistance traits. [ABSTRACT FROM AUTHOR]

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]

Abstract Background Klebsiella pneumoniae infections have become a major cause of hospital acquired infection worldwide with the increased rate of acquisition of resistance to antibiotics. Carbapenem resistance mainly among Gram negative is an ongoing problem which causes serious outbreaks dramatically limiting treatment options. This prospective cross-sectional study was designed to detect bla KPC gene from carbapenem resistant K. pneumoniae. Materials and Methods A totally of 1118 different clinical specimens were screened and confirmed for KPC producing K. pneumoniae phenotypically using Meropenem (10 μg) disc. The bla KPC gene was amplified from the isolates of K. pneumoniae to detect the presence of this gene. Result Of the total samples processed, 18.6% (n = 36) were K. pneumoniae and among 36 K. pneumoniae, 61.1% (n = 22/36) were meropenem resistant. This study demonstrated the higher level of MDR 91.7% (n = 33) and KPC production 47.2% (n = 17) among K. pneumoniae isolates. The bla KPC gene was detected in 8.3% (n = 3) of meropenem resistant isolates. Conclusion Since the study demonstrates the higher level of MDR and KPC producing K. pneumoniae isolates that has challenged the use of antimicrobial agents, continuous microbiology, and molecular surveillance to assist early detection and minimize the further dissemination of bla KPC should be initiated. We anticipate that the findings of this study will be useful in understanding the prevalence of KPC-producing K. pneumoniae in Nepal.

Abstract Background Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) co-producing bla KPC and bla NDM poses a serious threat to public health. This study aimed to investigate the mechanisms underlying the resistance and virulence of CR-hvKP isolates collected from a Chinese hospital, with a focus on bla KPC and bla NDM dual-positive hvKP strains. Methods Five CR-hvKP strains were isolated from a teaching hospital in China. Antimicrobial susceptibility and plasmid stability testing, plasmid conjugation, pulsed-field gel electrophoresis, and whole-genome sequencing (WGS) were performed to examine the mechanisms of resistance and virulence. The virulence of CR-hvKP was evaluated through serum-killing assay and Galleria mellonella lethality experiments. Phylogenetic analysis based on 16 highly homologous carbapenem-resistant K. pneumoniae (CRKP) producing KPC-2 isolates from the same hospital was conducted to elucidate the potential evolutionary pathway of CRKP co-producing NDM and KPC. Results WGS revealed that five isolates individually carried three unique plasmids: an IncFIB/IncHI1B-type virulence plasmid, IncFII/IncR-type plasmid harboring KPC-2 and IncC-type plasmid harboring NDM-1. The conjugation test results indicated that the transference of KPC-2 harboring IncFII/IncR-type plasmid was unsuccessful on their own, but could be transferred by forming a hybrid plasmid with the IncC plasmid harboring NDM. Further genetic analysis confirmed that the pJNKPN26-KPC plasmid was entirely integrated into the IncC-type plasmid via the copy-in route, which was mediated by TnAs1 and IS26. Conclusion KPC-NDM-CR-hvKP likely evolved from a KPC-2-CRKP ancestor and later acquired a highly transferable bla NDM-1 plasmid. ST11-KL64 CRKP exhibited enhanced plasticity. The identification of KPC-2-NDM-1-CR-hvKP highlights the urgent need for effective preventive strategies against aggravated accumulation of resistance genes.

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]

Abstract Background The infection of carbapenem-resistant organisms was a huge threat to human health due to their global spread. Dealing with a carbapenem-resistant Serratia marcescens (CRSM) infection poses a significant challenge in clinical settings. This study aims to provide insights into strategies for controlling CRSM infection by exploring the transformation mechanism of carbapenem-resistance. Methods We used whole genome sequencing (WGS) to investigate the mechanism of carbapenem resistance in 14 S. marcescens isolates in vivo. The expression level of related genes and the minimum inhibitory concentration of meropenem (MICMEM) were also evaluated to confirm the mechanism of carbapenem resistance. Results Seven groups of S. marcescens, each consisting of two strains, were collected from a hospital and displayed a shift in MICMEM from low to high levels. Homology analysis revealed that the isolates in five groups were significantly different from the remaining two. WGS and experimental evidence indicated that four groups of strains developed carbapenem resistance by acquiring the bla KPC (obtaining group), while two groups (persisting group) increased the expression level of the bla KPC. In contrast, isolates in the last group (missing group) did not carry the bla KPC. All strains possessed multiple β-lactamase genes, including bla CTX−M−14, bla SRT−1, and bla SRT−2. However, only in the missing group, the carbapenem-resistant strain lost an outer membrane protein-encoding gene, leading to increased bla CTX−M−14 expression compared to the carbapenem-susceptible strain. Conclusion The study findings suggest that S. marcescens strains developed diverse carbapenem resistance in vivo through the evolution of drug resistance, rather than through clone replacement. We hypothesize that carbapenem resistance in S. marcescens was due to certain clonal types with a distinct mechanism.

Wastewater treatment plants (WWTPs) are recognized as important niches of antibiotic-resistant bacteria that can be easily spread to the environment. In this study, we collected wastewater samples from the WWTP of A Coruña (NW Spain) from April 2020 to February 2022 to evaluate the presence of Gram-negative bacteria harboring carbapenemase genes. Bacteria isolated from wastewater were classified and their antimicrobial profiles were determined. In total, 252 Gram-negative bacteria carrying various carbapenemase genes were described. Whole-genome sequencing was conducted on 55 selected carbapenemase producing isolates using Oxford Nanopore technology. This study revealed the presence of a significant population of bacteria carrying carbapenemase genes in WWTP, which constitutes a public health problem due to their risk of dissemination to the environment. This emphasizes the usefulness of WWTP monitoring for combating antibiotic resistance. Data revealed the presence of different types of sequences harboring carbapenemase genes, such as blaKPC-2, blaGES-5, blaGES-6, blaIMP-11, blaIMP-28, blaOXA-24, blaOXA-48, blaOXA-58, blaOXA-217, and blaVIM-2. Importantly, the presence of the blaKPC-2 gene in wastewater, several months before any clinical case was detected in University Hospital of A Coruña, suggests that wastewater-based epidemiology can be used as an early warning system for the surveillance of antibiotic-resistant bacteria. [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 Carbapenemase production is a global public health threat. Antimicrobial resistance (AMR) data analysis is critical to public health policy. Here we analyzed carbapenemase detection trends using the AMR Brazilian Surveillance Network. Methods Carbapenemase detection data from Brazilian hospitals included in the public laboratory information system dataset were evaluated. The detection rate (DR) was defined as carbapenemase detected by gene tested per isolate per year. The temporal trends were estimated using the Prais–Winsten regression model. The impact of COVID-19 on carbapenemase genes in Brazil was determined for the period 2015–2022. Detection pre- (October 2017 to March 2020) and post-pandemic onset (April 2020 to September 2022) was compared using the χ 2 test. Analyses were performed with Stata 17.0 (StataCorp, College Station, TX). Results 83 282 bla KPC and 86 038 bla NDM were tested for all microorganisms. Enterobacterales DR for bla KPC and bla NDM was 68.6% (41 301/60 205) and 14.4% (8377/58 172), respectively. P. aeruginosa DR for bla NDM was 2.5% (313/12 528). An annual percent increase for bla NDM of 41.1% was observed, and a decrease for bla KPC of −4.0% in Enterobacterales, and an annual increase for bla NDM of 71.6% and for bla KPC of 22.2% in P. aeruginosa. From 2020 to 2022, overall increases of 65.2% for Enterobacterales, 77.7% for ABC, and 61.3% for P. aeruginosa were observed in the total isolates. Conclusions This study shows the strengths of the AMR Brazilian Surveillance Network with robust data related to carbapenemases in Brazil and the impact of COVID-19 with a change in carbapenemase profiles with bla NDM rising over the years. [ABSTRACT FROM AUTHOR]

The most common resistance mechanism to carbapenems is the production of carbapenemases. In 2021, the Pan American Health Organization warned of the emergence and increase in new carbapenemase combinations in Enterobacterales in Latin America. In this study, we characterized four Klebsiella pneumoniae isolates harboring blaKPC and blaNDM from an outbreak during the COVID-19 pandemic in a Brazilian hospital. We assessed their plasmids' transference ability, fitness effects, and relative copy number in different hosts. The K. pneumoniae BHKPC93 and BHKPC104 strains were selected for whole genome sequencing (WGS) based on their pulsed-field gel electrophoresis profile. The WGS revealed that both isolates belong to ST11, and 20 resistance genes were identified in each isolate, including blaKPC-2 and blaNDM-1. The blaKPC gene was present on a ~56 Kbp IncN plasmid and the blaNDM-1 gene on a ~102 Kbp IncC plasmid, along with five other resistance genes. Although the blaNDM plasmid contained genes for conjugational transfer, only the blaKPC plasmid conjugated to E. coli J53, without apparent fitness effects. The minimum inhibitory concentrations (MICs) of meropenem/imipenem against BHKPC93 and BHKPC104 were 128/64 and 256/128 mg/L, respectively. Although the meropenem and imipenem MICs against E. coli J53 transconjugants carrying the blaKPC gene were 2 mg/L, this was a substantial increment in the MIC relative to the original J53 strain. The blaKPC plasmid copy number was higher in K. pneumoniae BHKPC93 and BHKPC104 than in E. coli and higher than that of the blaNDM plasmids. In conclusion, two ST11 K. pneumoniae isolates that were part of a hospital outbreak co-harbored blaKPC-2 and blaNDM-1. The blaKPC-harboring IncN plasmid has been circulating in this hospital since at least 2015, and its high copy number might have contributed to the conjugative transfer of this particular plasmid to an E. coli host. The observation that the blaKPC-containing plasmid had a lower copy number in this E. coli strain may explain why this plasmid did not confer phenotypic resistance against meropenem and imipenem. [ABSTRACT FROM AUTHOR]

Antimicrobial resistance (AMR) has become one of the greatest challenges worldwide, hampering the treatment of a plethora of infections. Indeed, the AMR crisis poses a threat to the achievement of the United Nations' Sustainable Development Goals and, due to its multisectoral character, a holistic approach is needed to tackle this issue. Thus, the investigation of environments beyond the clinic is of utmost importance. Here, we investigated thirteen strains of antimicrobial-resistant Aeromonas isolated from an urban estuary in Brazil. Most strains carried at least one antimicrobial resistance gene and 11 carried at least one heavy metal resistance gene. Noteworthy, four (30.7%) strains carried the blaKPC gene, coding for a carbapenemase. In particular, the whole-genome sequence of Aeromonas hydrophila strain 34SFC-3 was determined, revealing not only the presence of antimicrobial and heavy metal resistance genes but also a versatile virulome repertoire. Mobile genetic elements, including insertion sequences, transposons, integrative conjugative elements, and an IncQ1 plasmid were also detected. Considering the ubiquity of Aeromonas species, their genetic promiscuity, pathogenicity, and intrinsic features to endure environmental stress, our findings reinforce the concept that A. hydrophila truly is a "Jack of all trades" that should not be overlooked under the One Health perspective. [ABSTRACT FROM AUTHOR]

Las carbapenemasas se encuentran ampliamente distribuidas en nuestro país, tanto en bacilos gramnegativos fermentadores como no fermentadores. Durante 2021, se ha reportado incremento de cepas con estas enzimas. Con el objetivo de evaluar la doble producción de carbapenemasas en Enterobacterales y comunicar su circulación, fue puesta a punto una PCR convencional múltiple. Estudio retrospectivo en 128 aislamientos provenientes de 20 centros colaboradores de la Red Nacional de Vigilancia de la RAM (Capital, Central e interior del país), remitidos al LCSP entre febrero y setiembre de 2021, para confirmación y genotipificación de carbapenemasas. Se realizaron pruebas fenotípicas y colorimétricas con sustratos específicos, y pruebas genotípicas (PCR convencional múltiple) para la detección simultánea de varios genes de resistencia (bla NDM, bla KPC, bla OXA-48-like, bla IMP y bla VIM). De los 128 aislamientos estudiados, 107 correspondieron a Klebsiella pneumoniae, 14 a Enterobacter cloacae complex, entre otros; aislados en mayor frecuencia de muestras de orina (30%), respiratorias (30%), sangre y catéter (24%). Los genes de resistencia a los carbapenemes detectados fueron: bla NDM (77,3%), bla KPC (17,2%); siendo confirmada la doble producción de carbapenemasas en 7 aislamientos (5,5%) provenientes de 4 centros diferentes de la capital de país y uno de Central; 6 de ellas (K. pneumoniae) con bla NDM+bla KPC y 1 (E. cloacae complex) con bla NDM+bla OXA-48-like; confirmando circulación de Enterobacterales dobles productores de carbapenemasas en el país (KPC+NDM y OXA+NDM); hallazgos que obligan a proveer de capacidades de detección, de manera a que se puedan tomar medidas oportunas y eficaces de contención y control.

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]

This study investigated the use of real-time PCR melting curves for the diagnosis of blaKPC and blaNDM genes among the most frequently detected carbapenemase-producing Enterobacteriaceae in Korea. As a means of addressing the shortcomings of phenotype tests and conventional PCR. The modified Hodge test confirmed positivity in 25 of 35 strains, and carbapenemase inhibition testing confirmed positivity in 14 strains by meropenem+PBA or meropenem+EDTA. PCR analysis showed amplification products in 25 strains of Klebsiella pneumoniae carbapenemases (KPC), 10 of K. pneumoniae, 5 of E. coli, 5 of A. baumannii, 4 of P. aeruginosa, and 1 of P. putida. New Delhi metallo β-lactamase (NDM) identified amplification products in 8 strains, that is, 2 K. pneumoniae, 3 E. coli, 1 P. aeruginosa, 1 E. cloacae, and 1 P. retgeri strains. Real-time PCR melting curve analysis confirmed amplification in 25 strains of KPC and 8 strains of NDM, and these results were 100% consistent with PCR results. In conclusion, our findings suggest early diagnosis of carbapenem resistant Enterobacteriaceae by real-time PCR offers a potential means of antibacterial management that can prevent and control nosocomial infection spread.

Klebsiella pneumoniae is an important pathogen in nosocomial and community-acquired UTIs. It is showing a high resistance towards many drugs. This study aimed to estimate the percentage of K. pneumonia in Hospital acquired and Community acquired UTI, drug resistance, ESBL, and detect the resistance genes (bla KPC, bla CTXM, bla SHV) and presence of mutation in (bla KPC). A total of (200) urine samples were collected; K. pneumonia was cultured and isolated on MacConkey agar, antibiotic sensitivity testing and ESBL detection were performed by Vitek 2 system, then DNA was extracted, PCR and sequencing for KPC gene were done. There is 7% (14 isolates) of K. pneumonia (50% HAUTI and 50% CAUTI) within 21.43% positive ESBL were obtained. Percentage of positive bla SHV and bla CTXM genes were 100%, while positive bla KPC gene was 42.8%. KPC gene sequencing show high rate of mutation in comparison between positive isolates reach to 50 different nucleotides. This work has significantly increased the knowledge on the Klebsiella pneumonia causing HAUTI and CAUTI patients. [ABSTRACT FROM AUTHOR]

Carbapenem-resistant Enterobacterales (CRE) are pathogens that have been found in several countries, with a significant public health concern. Characterizing the mode of resistance and determining the prevailing clones are vital to the epidemiology of CRE in our community. This study was conducted to characterize the molecular mode of resistance and to determine the clonality of the CRE fecal isolates among community food handlers (FHs) vs. infected control patients (ICPs) in Kuwait. Fecal CRE isolates obtained from FHs and ICPs from September 2016 to September 2018 were analyzed for their resistance genes. Gene characterization was carried out by polymerase chain reaction (PCR) assays and sequencing. Clonality of isolates was established by multilocus sequence typing (MLST). Of the 681 and 95 isolates of the family Enterobacterales isolated from FHs and ICPs, 425 (62.4%) and 16 (16.8%) were Escherichia coli , and 18 (2.6%) and 69 (72.6%) were Klebsiella pneumoniae , respectively. A total of 36 isolates were CRE with a prevalence of 5.3% among FH isolates and 87 (91.6%) among the ICPs. Of these, carbapenemase genes were detected in 22 (61.1%) and 65 (74.7%) isolates, respectively (p < 0.05). The detected specific genes among FHs and ICPs were positive for bla KPC 19 (86.4%) and 35 (40.2%), and bla OXA 10 (45.5%) and 59 (67.8%), in addition to bla NDM 2 (9.1%) and 32 (36.8%), respectively. MLST assays of the E. coli and K. pneumoniae isolates revealed considerable genetic diversity and polyclonality as well as demonstrated multiple known ST types and eight novel sequence types. The study revealed a relatively high number of CRE harboring predominantly bla KPC-mediated CRE among the community FH isolates vs. predominant bla OXA genes among the ICPs. Those heterogeneous CRE isolates raise concerns and mandate more efforts toward molecular surveillance. A multinational study is recommended to monitor the spread of genes mediating CRE in the community of Arabian Peninsula countries. [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]

Klebsiella pneumoniae carbapenemase genes (bla KPC) play an important role in carbapenem-resistant Enterobacteriaceae in China. A rapid detection method for bla KPC genes and investigations into the molecular characteristics of bla KPC positive Klebsiella pneumoniae were necessary. In this study, an easy and rapid recombinase aided amplification assay (RAA) for bla KPC was established. This protocol could be completed at 39°C in 15–20 min. The sensitivity of this assay was determined as 48 copies per reaction, and the specificity was 100%. The bla KPC RAA method could be used for clinical diagnosis and epidemiological investigation. Among 801 fecal samples from inpatients, 34 bla KPC positive isolates were identified from each sample, of which 23 isolates were K. pneumoniae. ST11 with bla KPC-2 was the most prevalent type. All these strains were multidrug resistant and carried various virulence genes. Fecal carriage of bla KPC positive carbapenem-resistant K.pneumoniae poses significant challenges for public health control. [ABSTRACT FROM AUTHOR]

Several physicochemical and season factors have been related to the abundance of antibiotic resistance genes (ARGs) in wastewater treatment plants (WWTPs), considered hotspots of bacterial resistance. However, few studies on the subject have been carried out in tropical countries endemic for resistance mechanisms such as bla KPC. In this study, the occurrence of ARGs, particularly bla KPC, was determined throughout a WWTP, and the factors related to their abundance were explored. In 2017, wastewater samples were taken from a WWTP in Colombia every 15 days for 6 months, and a total of 44 samples were analyzed by quantitative real-time PCR. sul 1, sul 2, bla KPC, and erm B were found to be the most prevalent ARGs. A low average reduction of the absolute abundance ARGs in effluent with respect to influent was observed, as well as a greater absolute abundance of ARGs in the WWTP effluent in the rainy season. Factors such as temperature, pH, oxygen, total organic carbon (TOC), chemical oxygen demand (COD), and precipitation were significantly correlated with the absolute abundance of several of the ARGs evaluated. A generalized linear mixed-effects model analysis showed that dissolved oxygen and precipitation in the sampling day were important factors related to the absolute concentration of bla KPC over time. In conclusion, the abundance of ARGs in the WWTP could be influenced by endemic conditions and physicochemical and climatological parameters. Therefore, it is necessary to continuously monitor clinical relevant genes in WWTPs from different global regions, even more so in low-income countries where sewage treatment is limited. [ABSTRACT FROM AUTHOR]

Objective: To investigate the overall distributions of key virulence genes in Klebsiella pneumoniae , especially the hypervirulent bla KPC-positive K. pneumoniae (Hv- bla KPC(+)-KP). Methods: A total of 521 complete genomes of K. pneumoniae from GenBank were collected and analyzed. Multilocus sequence typing, molecular serotyping, antibiotic-resistance, virulence genes and plasmid replicon typing were investigated. Results: Positive rates of virulence genes highly varied, ranging from 2.9 (c-rmpA/A2) to 99.6% (entB). Totally 207 strains presented positive fimH , mrkD , entB and wzi and 190 showed positive fimH , mrkD , entB , irp2 and wzi , which were the two primary modes. A total of 94, 165 and 29 strains were denoted as hypervirulent K. pneumoniae (HvKP), bla KPC(+)-KP and Hv- bla KPC(+)-KP. ST11 accounted for 17 among the 29 Hv- bla KPC(+)-KP strains; Genes iucA , p-rmpA2 and p-rmpA were positive in 28, 26 and 18 Hv- bla KPC(+)-KP strains respectively. Among the 29 Hv- bla KPC(+)-KP strains exhibiting four super clusters from GenBank, IncHI1B plasmids carrying virulence genes and IncFII ones with bla KPC were responsible for both 23 strains respectively. Conclusions: Positive rates of virulence genes vary remarkably in K. pneumoniae. Genes iucA , p-rmpA2 and p-rmpA were primary ones inducing Hv- bla KPC(+)-KP. IncHI1B plasmids carrying virulence genes and IncFII ones with bla KPC constitute the primary combination responsible for Hv- bla KPC(+)-KP. The making of Hv- bla KPC(+)-KP is mostly via bla KPC(+)-KP acquiring another plasmid harboring virulence genes. [ABSTRACT FROM AUTHOR]

The predominance of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae was caused by the spread of ST258 clone. In Latin America, KPC was reported in 2006, with the isolation of genetically unrelated K. pneumoniae in Colombia. Since then, the expansion of blaKPC in either K. pneumoniae ST258 or other Enterobacteriaceae (ETB) species was increasingly reported. In this study, we characterized 89 KPC-producing Escherichia coli, Klebsiella oxytoca, Serratia marcescens, and Citrobacter freundii that were received between 2010 and 2014. The results revealed that all isolates harbored blaKPC-2. Moreover, the dissemination of KPC by non-K. pneumoniae was mainly caused by the dispersion of ETB mostly genetically unrelated. E. coli is a community pathogen that may serve as the vehicle for the spread of KPC into community settings. Recently, KPC was detected in E. coli ST131, an international epidemic and multidrug-resistant clone. We found that 5/29 KPC-producing E. coli belonged to ST131 and four were blaCTXM-15 producers. The detection of blaKPC in ST131 should be closely monitored to prevent further dissemination. The blaKPC is generally located within Tn4401 transposon capable of mobilization through transposition found in plasmids in ST258. Less is known about the diversity of blaKPC genetic elements that disseminate horizontally among other species of ETB. We found that 16/29 E. coli and 2/18 S. marcescens harbored blaKPC-2 in Tn4401a. In 71 isolates, blaKPC-2 was located amidst diverse Tn3-derived genetic elements bearing non-Tn4401 structure. Further studies on the plasmids that encode blaKPC-2 in these clinical isolates may provide additional insight into its transmission mechanisms. [ABSTRACT FROM AUTHOR]

The wide spread of Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae is great threat to public health in China. Plasmids are among the major factors mediating bla KPC gene dissemination. A total of 156 carbapenem-resistant Enterobacteriaceae (CRE) isolates were identified in a tertiary hospital in China. Six KPC-producing isolates, namely, E. coli (n = 2), E. asburiae (n = 1), C. freundii (n = 1), C. portucalensis (n = 1), and C. koseri (n = 1), tested positive for the pCKPC18-1-like untypeable plasmid, which was described recently in C. freundii. All 6 plasmids could be easily transferred into E. coli by chemical transformation or conjugation and were confirmed by sequencing to harbor bla KPC−2. Multilocus PCRs and EcoRI-RFLP revealed that the 6 untypeable plasmids belonged to 2 isoforms. High-throughput sequencing of representative plasmids (pCP40 and pEC86) led to the identification of 2 plasmids that shared the common backbone genes repA, DnaJ, StpA , and yafB , which were characteristic of the untypeable plasmid, and had similar bla KPC−2 genetic contexts of the Tn 3 -Tn 4401 chimera. Nucleotide comparison revealed high sequence identity of the 2 plasmids with previously reported bla KPC−2-carrying untypeable plasmids. In particular, the pCP40 plasmid from C. portucalensis and the pHS062105-3 plasmid from K. pneumoniae differed by only 20 single-nucleotide polymorphisms (SNPs). To the best of our knowledge, this is the first report of a bla KPC-harboring untypeable plasmid spread into E. coli, E. asburiae , and C. koseri strains in China. [ABSTRACT FROM AUTHOR]

Forty-five KPC-producing Enterobacteriaceae strains were isolated from multiple departments in a Chinese public hospital from 2014 to 2015. Genome sequencing of four representative strains, namely Proteus mirabilis GN2, Serratia marcescens GN26, Morganella morganii GN28, and Klebsiella aerogenes E20, indicated the presence of blaKPC-2-carrying IncX6 plasmids pGN2-KPC, pGN26-KPC, pGN28-KPC, and pE20-KPC in the four strains, respectively. These plasmids were genetically closely related to one another and to the only previously sequenced IncX6 plasmid, pKPC3_SZ. Each of the plasmids carried a single accessory module containing the blaKPC-2/3-carrying ΔTn6296 derivatives. The ΔTn6292 element from pGN26-KPC also contained qnrS, which was absent from all other plasmids. Overall, pKPC3_SZ-like blaKPC-carrying IncX6 plasmids were detected by PCR in 44.4% of the KPC-producing isolates, which included K. aerogenes, P. mirabilis, S. marcescens, M. morganii, Escherichia coli, and Klebsiella pneumoniae, and were obtained from six different departments of the hospital. Data presented herein provided insights into the genomic diversity and evolution of IncX6 plasmids, as well as the dissemination and epidemiology of blaKPC-carrying IncX6 plasmids among Enterobacteriaceae in a hospital setting. [ABSTRACT FROM AUTHOR]

Over a 5-month period between the end of June and the beginning of November in 2015, a KPC-producing Enterobacteriaceae outbreak occurred in a general hospital in Busan, South Korea, being associated with a total of 50 clinical isolates from 47 patients. Multilocus sequence typing and pulsed-field gel electrophoresis were carried out for strain typing and whole-genome sequencing was performed to characterize the plasmids. A clonal spread of K. pneumoniae sequence type 307 (ST307) carrying a self-transferable IncX3-type plasmid harboring bla KPC-2 was responsible for the outbreak. Sporadic emergence of K. pneumoniae ST697 carrying an IncFII-type plasmid and a ST11 isolate harboring a small plasmid devoid of any known origin of replication were observed to be associated with bla KPC-3 , but no further dissemination of these strains was identified. The results indicated a healthcare-associated infection associated with a bla KPC -harboring plasmid dissemination and a clonal spread of KPC-producing Enterobacteriaceae . [ABSTRACT FROM AUTHOR]

Objectives Production of carbapenemase, especially Klebsiella pneumoniae carbapenemases (KPC), is one of the antibiotic resistance mechanisms of Enterobacteriaceae such as Klebsiella oxytoca . This study aimed to investigate and identify KPC-producing K. oxytoca isolates using molecular and phenotypic methods. Methods A total of 75 isolates of K. oxytoca were isolated from various clinical samples, and were verified as K. oxytoca after performing standard microbiological tests and using a polymerase chain reaction (PCR) method. An antibiotic susceptibility test was performed using a disc diffusion method according to the Clinical and Laboratory Standards Institute guidelines. CHROMagar KPC chromogenic culture media was used to examine and confirm the production of the carbapenemase enzyme in K. oxytoca isolates; in addition, PCR was used to evaluate the presence of bla KPC gene in K. oxytoca strains. Results Of a total of 75 K. oxytoca isolates, one multidrug resistant strain was isolated from the urine of a hospitalized woman. This strain was examined to assess its ability to produce carbapenemase enzyme; it produced a colony with a blue metallic color on the CHROMagar KPC chromogenic culture media. In addition, the bla KPC gene was confirmed by PCR. After sequencing, it was confirmed and deposited in GenBank. Conclusion To date, many cases of KPC-producing Enterobacteriaceae, in particular K. pneumoniae , have been reported in different countries; there are also some reports on the identification of KPC-producing K. oxytoca . Therefore, to prevent the outbreak of nosocomial infections, the early detection, control, and prevention of the spread of these strains are of great importance. [ABSTRACT FROM AUTHOR]

Carbapenem resistance among Klebsiella pneumoniae, is an emerging problem worldwide. One of the main mechanisms of resistance to carbapenems is potential of Klebsiella pneumoniae to produce carbapenemase enzyme. The aim of this study was to determine the frequency of bla VIM, bla IMP and bla KPC genes among Carbapenem-Resistant Klebsiella pneumoniae (CRKP) isolated from Kurdistan and Isfahan hospitals. This study was carried out in Iran on 183 samples from Besat and Alzahra hospitals in 2017. Antibiotic susceptibility tests were performed by Kirby-Bauer disc diffusion. The Modified Hodge Test (MHT) was used to investigate the carbapenemase presence. The β -lactamases genes were detected by PCR method. The highest and the lowest rate of resistance were observed against cefotaxime (98.2%) and gentamicin (43.6%) respectively. Among 183 isolates, 134 (73.2 %) of them were positive in MHT. The prevalence of bla VIM, bla IMP and bla KPC genes was 4 (2.18%), 1 (0.5%) and 0% respectively. The prevalence of CRKP (Carbapenem-Resistant Klebsiella pneumonia) strains detected in this study is a major concern and the need of infection control process of care measures. Regarding to our findings, these gene has low prevalence in the our country, probably other mechanisms of resistance to Carbapenems , are involved.

bla KPC genes encoding resistance to carbapenems are increasingly widely reported and are now endemic in parts of several countries, but only one Klebsiella pneumoniae isolate carrying bla KPC-2 had previously been reported in Australia, in 2010. Here we characterised this isolate, six additional K. pneumoniae and one Escherichia coli carrying bla KPC and another K. pneumoniae lacking bla KPC , all isolated in Australia in 2012. Seven K. pneumoniae belonged to clonal complex (CC) 292, associated with bla KPC in several countries. Five with bla KPC-2 plus the isolate lacking a bla KPC gene were sequence type 258 (ST258) and the seventh was the closely related ST512 with bla KPC-3 . The eighth K. pneumoniae isolate, novel ST1048, and the E. coli (ST131) also carried bla KPC-2 . bla KPC genes were associated with the most common Tn 4401 a variant, which gives the highest levels of expression, in all isolates. The ST258 isolates appeared to share a similar set of plasmids, with IncFII K , IncX3 and ColE-type plasmids identified in most isolates. All K. pneumoniae isolates had a characteristic insertion in the ompK35 gene resulting in a frameshift and early termination, but only the ST512 isolate had a GlyAsp insertion in loop 3 of OmpK36 that may contribute to increased resistance. The clinical epidemiology of bla KPC emergence in Australia thus appears to reflect the global dominance of K. pneumoniae CC292 (and perhaps E. coli ST131). Some, but not all, patients carrying these isolates had previously been hospitalised outside Australia, suggesting multiple discrete importation events of closely related strains, as well as undetected nosocomial spread. [ABSTRACT FROM AUTHOR]

The worldwide dissemination of Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae ST258 demands a rapid PCR-based typing method to detect unique genes of the ST258 clone. This study evaluates a PCR developed by Adler et al. (2014) for the detection of ST258 in K. pneumoniae clinical isolates centered on the identification of the pilv-I and prp genes. We tested 143 clinical isolates from Argentina ( n = 109), Chile ( n = 1), Colombia ( n = 1), Costa Rica ( n = 2), Ecuador ( n = 5), El Salvador ( n = 2), Nicaragua ( n = 5), Panamá ( n = 2), Paraguay ( n = 2), Perú ( n = 3) and Trinidad and Tobago ( n = 11) recovered from 2006 to 2015. bla KPC , pilv-l and prp genes were detected by PCR and sequenced by standard procedures. ST258 and non-ST258 were defined by PFGE and/or MLST. Isolates were grouped according to PFGE patterns: 58 were compatible with ST258 (group 1) and 85 with non-ST258 (group 2). MLST study was done on an arbitrary selection of isolates. The pilv-l gene was present only in ST258 isolates, regardless of the presence of the bla KPC gene. Results for the prp gene were variable. Its presence did not define ST258. The pilv-I PCR had a sensitivity and specificity of 100%, respectively, for the detection of ST258 in the isolates under investigation. Given our findings, the pilv-I PCR could replace more time and resource consuming methods, allowing for more rapid detection of the circulating high risk K. pneumoniae clone ST258 in Latin American (LA) countries. [ABSTRACT FROM AUTHOR]