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The rapid spread of carbapenemase-producing strains has led to increased levels of resistance among Gram-negative bacteria, especially enterobacteria. The current study aimed to collect and genetically characterize the colistin- and carbapenem-resistant isolates, obtained in one of the biggest hospitals (Military Medical Academy) in Sofia, Bulgaria. Clonal relatedness was detected by RAPD and MLST. Carbapenemases, ESBLs, and mgrB were investigated by PCR amplification and sequencing, replicon typing, and 16S rRNA methyltransferases with PCRs. Fourteen colistin- and carbapenem-resistant K. pneumoniae isolates were detected over five months. Six carbapenem-resistant and colistin-susceptible isolates were also included. The current work revealed a complete change in the spectrum of carbapenemases in Bulgaria. blaNDM-5 was the only NDM variant, and it was always combined with blaOXA-232. The coexistence of blaOXA-232 and blaNDM-5 was observed in 10/14 (72%) of colistin- and carbapenem-resistant K. pneumoniae isolates and three colistin-susceptible isolates. All blaNDM-5- and blaOXA-232-positive isolates belonged to the ST6260 (ST101-like) MLST type. They showed great mgrB variability and had a higher mortality rate. In addition, we observed blaOXA-232 ST14 isolates and KPC-2-producing ST101, ST16, and ST258 isolates. The colistin- and carbapenem-resistant isolates were susceptible only to cefiderocol for blaNDM-5- and blaOXA-232-positive isolates and to cefiderocol and ceftazidime/avibactam for blaOXA-232- or blaKPC-2-positive isolates. All blaOXA-232-positive isolates carried rmtB methylase and the colE replicon type. The extremely limited choice of appropriate treatment for patients infected with such isolates and their faster distribution highlight the need for urgent measures to control this situation. [ABSTRACT FROM AUTHOR]

The global emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) has become a critical public healthcare concern due to treatment challenges and high mortality. In recent years, there has been an increase in cases of CRKP co-producing New Delhi metallo-β-lactamases (NDM) and oxacillinase 48 (OXA-48)-like carbapenemases in the US. The aim of this study was to correlate the clinical and genomic characteristics of CRKP co-producing NDM and OXA-48-like carbapenemases isolated from patients in Southern California since 2016. Whole-genome sequencing was performed on clinical isolates obtained from various sources, including blood, abdominal fluid, wounds, and urine. Genetic diversity was observed in these CRKP, including ST-14, ST-16, ST-167, ST-437, ST-2096, and ST-2497 lineages. Phylogenetic analysis revealed two closely related clusters (ST-14 and ST-2497), with single nucleotide polymorphism (SNP) differences ranging from 0 to 36, suggesting a possible local spread of these CRKP. Significant antimicrobial resistance (AMR) genes were identified in these CRKP, including blaNDM-1, blaNDM-5, blaOXA-232, blaOXA-181, blaCTX-M-15, armA, tet(A), and tet(D). Moreover, pColKP3-type and Inc-type plasmids known to harbor AMR genes were also detected in these isolates. Most of the patients infected with this rare type of CRKP died, although their severe comorbidities also played important roles in their demise. Our study highlighted the extremely limited treatment options and poor clinical outcomes associated with these dual-carbapenemase-producing CRKP. Real-time genomic surveillance of these unusual and deadly CRKP can provide critical information for infection prevention and treatment guidance.

ABSTRACTOXA-232 is one of the most common OXA-48-like carbapenemase derivatives and is widely disseminated in nosocomial settings across countries. The blaOXA-232 gene is located on a 6-kb non-conjugative ColKP3-type plasmid, while the dissemination of blaOXA-232 into different Enterobacterales species and the polyclonal dissemination of OXA-232-producing K. pneumoniae revealed the horizontal transfer of blaOXA-232. However, it’s still unclear how this non-conjugative ColKP3 plasmid could facilitate the mobilization of blaOXA-232. Here, we observed the in vivo intraspecies transfer of blaOXA-232 during a nosocomial outbreak of OXA-232-producing K. pneumoniae. We demonstrated the presence of ColKP3 OXA-232 plasmid in the outer membrane vesicles (OMVs) derived from clinical isolates, and OMVs could facilitate the horizontal transfer of blaOXA-232 among Enterobacterales. In contrast, for the most prevalent carbapenemase genes, including blaKPC-2 and blaNDM-1, though the presence of carbapenemase genes and plasmid backbones in the vesicular lumen was observed, OMVs couldn’t promote effective transformation, probably due to the low copy number of plasmids in clinical isolates and the low number of plasmids loaded into vesicles. Conjugation assay revealed that the epidemic IncX3 NDM-1 and IncFII(pHN7A8)/IncR KPC-2 plasmids were conjugative and could be horizontally transferred via independent conjugation or with the help of a co-existent conjugative plasmid. For the large-size and low-copy number conjugative plasmids carrying carbapenemase genes, OMVs-mediated gene exchange may only serve as an alternative pathway for horizontal transfer. In conclusion, diverse mobilization strategies were employed by plasmids harbouring carbapenemase genes, and plasmids display a proper choice of mobility pathway due to their individual properties.

The rapid spread of carbapenemase-producing strains has led to increased levels of resistance among Gram-negative bacteria, especially enterobacteria. The current study aimed to collect and genetically characterize the colistin- and carbapenem-resistant isolates, obtained in one of the biggest hospitals (Military Medical Academy) in Sofia, Bulgaria. Clonal relatedness was detected by RAPD and MLST. Carbapenemases, ESBLs, and mgrB were investigated by PCR amplification and sequencing, replicon typing, and 16S rRNA methyltransferases with PCRs. Fourteen colistin- and carbapenem-resistant K. pneumoniae isolates were detected over five months. Six carbapenem-resistant and colistin-susceptible isolates were also included. The current work revealed a complete change in the spectrum of carbapenemases in Bulgaria. blaNDM-5 was the only NDM variant, and it was always combined with blaOXA-232. The coexistence of blaOXA-232 and blaNDM-5 was observed in 10/14 (72%) of colistin- and carbapenem-resistant K. pneumoniae isolates and three colistin-susceptible isolates. All blaNDM-5- and blaOXA-232-positive isolates belonged to the ST6260 (ST101-like) MLST type. They showed great mgrB variability and had a higher mortality rate. In addition, we observed blaOXA-232 ST14 isolates and KPC-2-producing ST101, ST16, and ST258 isolates. The colistin- and carbapenem-resistant isolates were susceptible only to cefiderocol for blaNDM-5- and blaOXA-232-positive isolates and to cefiderocol and ceftazidime/avibactam for blaOXA-232- or blaKPC-2-positive isolates. All blaOXA-232-positive isolates carried rmtB methylase and the colE replicon type. The extremely limited choice of appropriate treatment for patients infected with such isolates and their faster distribution highlight the need for urgent measures to control this situation.

Abstract Background OXA-232-producing Klebsiella pneumoniae was first identified in China in 2016, and its clonal transmission was reported in 2019. However, there are no prevalence and genotypic surveillance data available for OXA-232 in China. Therefore, we investigated the trends and characteristics of OXA-232 type carbapenemase in Zhejiang Province, China from 2018 to 2021. Methods A total of 3278 samples from 1666 patients in the intensive care units were collected from hospitals in Zhejiang Province from 2018 to 2021. Carbapenem-resistant isolates were initially selected by China Blue agar plates supplemented with 0.3 μg/ml meropenem, and further analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry identification, immune colloidal gold technique, conjugation experiment, antimicrobial susceptibility testing and whole genome sequencing. Results A total of 79 OXA-producing strains were recovered, with the prevalence increased from 1.8% [95% confidence interval (CI): 0.7–3.7%] in 2018 to 6.0% (95% CI: 4.4–7.9%) in 2021. Seventy-eight strains produced OXA-232 and one produced OXA-181. The bla OXA-232 gene in all strains was located in a 6141-bp ColKP3-type non-conjugative plasmid and the bla OXA-181 gene was located in a 51,391-bp ColKP3/IncX3-type non-conjugative plasmid. The bla OXA-232-producing K. pneumoniae was dominated (75/76) by isolates of sequence type 15 (ST15) that differed by less than 80 SNPs. All OXA-producing strains (100%, 95% CI: 95.4–100.0%) were multidrug-resistant. Conclusions From 2018 to 2021, OXA-232 is the most prevalent OXA-48-like derivative in Zhejiang Province, and ST15 K. pneumoniae isolates belonging to the same clone are the major carriers. The transmission of ColKP3-type plasmid to E. coli highlighted that understanding the transmission mechanism is of great importance to delay or arrest the propagation of OXA-232 to other species.

We report here a fatal case of carbapenem-resistant Klebsiella pneumoniae (CRKP) infections in a renal transplant patient without a travel history in the prior year, from whom 2 genetically different CRKP (sequence type 14 [ST14] and ST2497) strains carrying the same plasmids and antimicrobial resistance genes, including blaNDM-1, blaOXA-232, blaCTX-M-15, armA, and tet(D), were isolated from blood and the abdominal cavity. The isolates were susceptible to colistin, tigecycline, eravacycline, and cefiderocol, which was used to treat the CRKP in combination with ceftazidime-avibactam and polymyxin B and resulted in bacterial clearance. Despite the aggressive treatment, the patient died of ischemic colitis and multiorgan failure.

During 2013–2021, increased prevalence of oxacillinase 232–producing Enterobacterales was observed in France, mostly driven by its emergence in Klebsiella pneumoniae. Whole-genome sequencing identified that oxacillinase 232–producing K. pneumoniae belonged to 14 sequence types (STs), among which 2 polyclonal high-risk clones, ST-231 and ST-2096, were overrepresented.

Background: OXA-232-producing Klebsiella pneumoniae was first identified in China in 2016, and its clonal transmission was reported in 2019. However, there are no prevalence and genotypic surveillance data available for OXA-232 in China. Therefore, we investigated the trends and characteristics of OXA-232 type carbapenemase in Zhejiang Province, China from 2018 to 2021. Methods: A total of 3278 samples from 1666 patients in the intensive care units were collected from hospitals in Zhejiang Province from 2018 to 2021. Carbapenem-resistant isolates were initially selected by China Blue agar plates supplemented with 0.3 μg/ml meropenem, and further analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry identification, immune colloidal gold technique, conjugation experiment, antimicrobial susceptibility testing and whole genome sequencing. Results: A total of 79 OXA-producing strains were recovered, with the prevalence increased from 1.8% [95% confidence interval (CI): 0.7–3.7%] in 2018 to 6.0% (95% CI: 4.4–7.9%) in 2021. Seventy-eight strains produced OXA-232 and one produced OXA-181. The blaOXA-232 gene in all strains was located in a 6141-bp ColKP3-type non-conjugative plasmid and the blaOXA-181 gene was located in a 51,391-bp ColKP3/IncX3-type non-conjugative plasmid. The blaOXA-232-producing K. pneumoniae was dominated (75/76) by isolates of sequence type 15 (ST15) that differed by less than 80 SNPs. All OXA-producing strains (100%, 95% CI: 95.4–100.0%) were multidrug-resistant. Conclusions: From 2018 to 2021, OXA-232 is the most prevalent OXA-48-like derivative in Zhejiang Province, and ST15 K. pneumoniae isolates belonging to the same clone are the major carriers. The transmission of ColKP3-type plasmid to E. coli highlighted that understanding the transmission mechanism is of great importance to delay or arrest the propagation of OXA-232 to other species. [ABSTRACT FROM AUTHOR]

During 2013-2021, increased prevalence of oxacillinase 232-producing Enterobacterales was observed in France, mostly driven by its emergence in Klebsiella pneumoniae. Whole-genome sequencing identified that oxacillinase 232-producing K. pneumoniae belonged to 14 sequence types (STs), among which 2 polyclonal high-risk clones, ST-231 and ST-2096, were overrepresented. [ABSTRACT FROM AUTHOR]

Objectives: To characterize one OXA-232-producing wzi93-KL112-O1 carbapenem-resistant Klebsiella pneumoniae (CRKP) co-harboring chromosomal blaCTX-M-15 and one rmpA2-associated virulence plasmid. Methods: Minimum inhibitory concentrations (MICs) were measured via broth microdilution method. Conjugation, chemical transformation, string test and Galleria mellonella infection model experiments were also conducted. Wholegenome sequencing (WGS) was performed on the Illumina and Nanopore platforms. Antimicrobial resistance determinants were identified using ABRicate program with ResFinder database. Insertion sequences (ISs) were identified using ISfinder. Bacterial virulence factors were identified using virulence factor database (VFDB). Wzi, capsular polysaccharide (KL) and lipoolygosaccharide (OCL) were analyzed using Kleborate with Kaptive. Phylogenetic analysis of 109 ST15 K. pneumoniae strains was performed using core genome multilocus sequence typing (cgMLST) on the Ridom SeqSphere+ server. MLST, replicons type, SNP strategies and another cgMLST analysis for 45 OXA-232-producing K. pneumoniae strains were further conducted using BacWGSTdb server. Results: K. pneumoniae KPTCM strain belongs to ST15 with wzi93, KL112 and O1. It possessed a multidrug-resistant (MDR) profile and was resistant to carbapenems (meropenem and ertapenem), ciprofloxacin and amikacin. Virulence assays demonstrated KPTCM strain possesses a low virulence phenotype. WGS revealed it contained one circular chromosome and nine plasmids. The carbapenemase-encoding gene blaOXA-232 was located in a 6141-bp ColKP3-type non-conjugative plasmid and flanked by DISEcp1 and DlysR-DereA. Interestingly, blaCTX-M-15 was located in the chromosome mediated by ISEcp1-based transposon Tn2012. Importantly, it harbored a rmpA2-associated pLVPK-like virulence plasmid with iutA-iucABCD gene cluster and one IS26-mediated MDR fusion plasmid according to 8-bp (AGCTGCAC or GGCCTTTG) target site duplications (TSD). Based on the cgMLST and SNP analysis, data showed OXA-232-producing ST15 K. pneumoniae isolates were mainly isolated from China and have evolved in recent years. Conclusions: Early detection of CRKP strains carrying chromosomal blaCTX-M-15, OXA-232 carbapenemase and pLVPK-like virulence plasmid is recommended to avoid the extensive spread of this high-risk clone. [ABSTRACT FROM AUTHOR]

Huaiyu Jia,1 Ying Zhang,2 Jianzhong Ye,1 Wenya Xu,1 Ye Xu,1 Weiliang Zeng,2 Wenli Liao,1 Tao Chen,1 Jianming Cao,2 Qing Wu,1 Tieli Zhou1 1Department of Clinical Laboratory, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang Province, People’s Republic of China; 2Department of Medical Laboratory Science, School of Laboratory Medicine and Life Science, Wenzhou Medical University, Wenzhou, Zhejiang Province, People’s Republic of ChinaCorrespondence: Tieli Zhou; Qing WuDepartment of Clinical Laboratory, the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, People’s Republic of ChinaTel/Fax +86-0577-8668-9885Email wyztli@163.com; wuqing830@163.comBackground: 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.Keywords: Klebsiella pneumoniae, carbapenem-resistance, virulence, OXA-232, outbreak

ABSTRACT: Objectives: The aims of this study were to infer the phylogenetic relationship of OXA-232-producing Klebsiella pneumoniae (OXA232Kp) strains collected from a Chinese hospital and to determine the composition and genetic background of antimicrobial resistance genes (ARGs) among these strains. Methods: Three non-duplicate OXA232Kp strains were collected from a Chinese hospital. Whole-genome sequencing was used to determine their genome sequences and then a genomic comparison of ARG-carrying genetic elements from the three strains with related sequences was performed. Phylogenetic analysis was conducted by constructing a maximum-likelihood phylogenetic tree. Results: Compared with other Chinese sequence type 15 (ST15)-OXA232Kp strains, the three ST15-OXA232Kp strains in this study could be divided into a single subgroup in phylogenetic relationship. The composition and genetic background of ARGs were identical in the three strains. Three ARG-carrying genetic elements or multidrug resistance (MDR) regions were determined, including a truncated Tn2013-like IS-based transposition unit, a unit transposition Tn6867b and a 40.9-kb MDR region. Conclusion: This study reported clonal dissemination of ST15-OXA232Kp strains carrying multiple ARGs in a Chinese hospital. A comprehensive evolutionary and genomics analysis provided a deeper understanding of OXA232Kp. Further surveillance and study should be advocated to prevent the dissemination of OXA232Kp strains in China.

The global emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) has become a critical public healthcare concern due to treatment challenges and high mortality. In recent years, there has been an increase in cases of CRKP co-producing New Delhi metallo-β-lactamases (NDM) and oxacillinase 48 (OXA-48)-like carbapenemases in the US. The aim of this study was to correlate the clinical and genomic characteristics of CRKP co-producing NDM and OXA-48-like carbapenemases isolated from patients in Southern California since 2016. Whole-genome sequencing was performed on clinical isolates obtained from various sources, including blood, abdominal fluid, wounds, and urine. Genetic diversity was observed in these CRKP, including ST-14, ST-16, ST-167, ST-437, ST-2096, and ST-2497 lineages. Phylogenetic analysis revealed two closely related clusters (ST-14 and ST-2497), with single nucleotide polymorphism (SNP) differences ranging from 0 to 36, suggesting a possible local spread of these CRKP. Significant antimicrobial resistance (AMR) genes were identified in these CRKP, including blaNDM-1, blaNDM-5, blaOXA-232, blaOXA-181, blaCTX-M-15, armA, tet(A), and tet(D). Moreover, pColKP3-type and Inc-type plasmids known to harbor AMR genes were also detected in these isolates. Most of the patients infected with this rare type of CRKP died, although their severe comorbidities also played important roles in their demise. Our study highlighted the extremely limited treatment options and poor clinical outcomes associated with these dual-carbapenemase-producing CRKP. Real-time genomic surveillance of these unusual and deadly CRKP can provide critical information for infection prevention and treatment guidance.

This study reported the identification of a novel ceftazidime-avibactam-resistant KPC-2 variant, KPC-123, in a Citrobacter koseri isolated from a patient in a Chinese hospital following ceftazidime-avibactam treatment of infection caused by OXA-232-producing Klebsiella pneumoniae. This novel KPC-123 consisting of 302 amino acids differs from KPC-2 by two insertions after positions 179 (ins179_TY) and 270 (ins270_DDKHSEA), respectively. Conjugation and cloning experiments confirmed that KPC-123 was able to confer high-level resistance to ceftazidime and ceftazidime/avibactam (MICs of 128 mg/L and 64/4 mg/L, respectively) and elevated MIC values of cefotaxime, cefepime, and aztreonam (4 mg/L, 2 mg/L, and 4 mg/L, respectively) but retained susceptibility to carbapenems. Whole-genome sequencing and genomic analysis revealed that bla KPC−123 within the "IS Kpn27 - bla KPC-IS Kpn6 " structure was located on a 93,814-bp conjugative plasmid that was almost identical to a bla KPC−2-carrying plasmid harbored in a K. pneumoniae isolate from the same sampling site of the patient, suggesting the transfer and in vivo evolution of this bla KPC-carrying plasmid. Hence, active surveillance of ceftazidime/avibactam resistance and the underlying mechanisms, which may facilitate the prevention and control of the dissemination of resistance, is needed. [ABSTRACT FROM AUTHOR]

BackgroundWhole-genome sequencing (WGS) is an emerging and powerful technique by which to perform epidemiological studies in outbreak situations.MethodsWGS was used to identify and evaluate an outbreak of OXA-232-expressing carbapenem-resistant Klebsiella pneumoniae (CRKP) transmitted to 16 patients over the course of 40 weeks via endoscopic retrograde cholangiopancreatography procedures at a single institution. WGS was performed on 32 OXA-232 CRKP isolates (1-7 per patient) and single-nucleotide variants (SNVs) were analyzed, with reference to the index patient's isolate.ResultsInterhost genetic diversity of isolates was between 0 and 15 SNVs during the outbreak; molecular clock calculations estimated 12.31 substitutions per genome per year (95% credibility interval, 7.81-17.05). Both intra- and interpatient diversification at the plasmid and transposon level was observed, significantly impacting the antibiogram of outbreak isolates. The majority of isolates evaluated (n = 27) harbored a blaCTX-M-15 gene, but some (n = 5) lacked the transposon carrying this gene, which resulted in susceptibility to aztreonam and third- and fourth-generation cephalosporins. Similarly, an isolate from a colonized patient lacked the transposon carrying rmtF and aac(6')lb genes, resulting in susceptibility to aminoglycosides.ConclusionsThis study broadens the understanding of how bacteria diversify at the genomic level over the course of a defined outbreak and provides reference for future outbreak investigations.

ABSTRACT Here, a program was designed to surveil the colonization and associated infection of OXA-232-producing carbapenem-resistant Klebsiella pneumoniae (CRKP) (OXA-232-CRKP) in an intensive care unit (ICU) and to describe the epidemiological characteristics during surveillance. Samples were sourced from patient and environment colonization sites in the ICU from August to December 2019. During the surveillance, 106 OXA-232-CRKP strains were isolated from 8,656 samples of colonization sites, with an average positive rate of 1.22%. The rate from patient colonization sites was 3.59% (60/1,672 samples), over 5 times higher than that of the environment (0.66% [46/6,984 samples]). Rectal swabs and ventilator-related sites had the highest positive rates among patient and environment colonization sites, respectively. Six of the 15 patients who had OXA-232-CRKP at colonization sites suffered from OXA-232-CRKP-related infections. Patients could obtain OXA-232-CRKP from the environment, while long-term patient colonization was mostly accompanied by environmental colonization with subsequent infection. Antimicrobial susceptibility testing presented similar resistance profiles, in which all isolates were resistant to ertapenem but showed different levels of resistance to meropenem and imipenem. Whole-genome sequencing and single-nucleotide polymorphism (SNP) analysis suggested that all OXA-232-CRKP isolates belonged to the sequence type 15 (ST15) clone and were divided into two clades with 0 to 45 SNPs, sharing similar resistance genes, virulence genes, and plasmid types, indicating that the wide dissemination of OXA-232-CRKP between the environment and patients was due to clonal spread. The strains all contained β-lactam resistance genes, including blaOXA-232, blaCTX-M-15, and blaSHV-106, and 75.21% additionally carried blaTEM-1. In brief, wide ST15 clonal spread and long-term colonization of OXA-232-CRKP between patients and the environment were observed, with microevolution and subsequent infection. IMPORTANCE OXA-232 is a variant of OXA-48 carbapenemase, which has been increasingly reported in nosocomial outbreaks in ICUs. However, the OXA-232-CRKP transmission relationship between the environment and patients in ICUs was still not clear. Our study demonstrated the long-term colonization of OXA-232-CRKP in the ICU environment, declared that the colonization was a potential risk to ICU patients, and revealed the possible threat that this OXA-232-CRKP clone would bring to public health. The wide dissemination of OXA-232-CRKP between the environment and patients was due to ST15 clonal spread, which presented a multidrug-resistant profile and carried disinfectant resistance genes and virulence clusters, posing a challenge to infection control. The study provided a basis for environmental disinfection, including revealing common environmental colonization sites of OXA-232-CRKP and suggesting appropriate usage of disinfectants to prevent the development of disinfectant resistance.

This study reported the identification of a novel ceftazidime-avibactam-resistant KPC-2 variant, KPC-123, in a Citrobacter koseri isolated from a patient in a Chinese hospital following ceftazidime-avibactam treatment of infection caused by OXA-232-producing Klebsiella pneumoniae. This novel KPC-123 consisting of 302 amino acids differs from KPC-2 by two insertions after positions 179 (ins179_TY) and 270 (ins270_DDKHSEA), respectively. Conjugation and cloning experiments confirmed that KPC-123 was able to confer high-level resistance to ceftazidime and ceftazidime/avibactam (MICs of 128 mg/L and 64/4 mg/L, respectively) and elevated MIC values of cefotaxime, cefepime, and aztreonam (4 mg/L, 2 mg/L, and 4 mg/L, respectively) but retained susceptibility to carbapenems. Whole-genome sequencing and genomic analysis revealed that blaKPC−123 within the “ISKpn27-blaKPC-ISKpn6” structure was located on a 93,814-bp conjugative plasmid that was almost identical to a blaKPC−2-carrying plasmid harbored in a K. pneumoniae isolate from the same sampling site of the patient, suggesting the transfer and in vivo evolution of this blaKPC-carrying plasmid. Hence, active surveillance of ceftazidime/avibactam resistance and the underlying mechanisms, which may facilitate the prevention and control of the dissemination of resistance, is needed.

A prospective, multicentre observational cohort study of carbapenem-resistant Klebsiella spp. (CRK) bloodstream infections was conducted in Turkey from June 2018 to June 2019. One hundred eighty-seven patients were recruited. Single OXA-48-like carbapenemases predominated (75%), followed by OXA-48-like/NDM coproducers (16%). OXA-232 constituted 31% of all OXA-48-like carbapenemases and was mainly carried on ST2096. Thirty-day mortality was 44% overall and 51% for ST2096. In the multivariate cox regression analysis, SOFA score and immunosuppression were significant predictors of 30-day mortality and ST2096 had a non-significant effect. All OXA-48-like producers remained susceptible to ceftazidime-avibactam. [ABSTRACT FROM AUTHOR]

Abstract Background Bacterial isolates with multiple plasmids harbouring different carbapenemase genes have emerged and been identified repeatedly, despite a general notion that plasmids confer fitness cost in bacterial host. In this study, we investigated the effects of plasmids with carbapenemase genes on the fitness and virulence of bacteria. Methods Different plasmids harbouring the carbapenemase genes, bla NDM-1 and bla OXA-232, were isolated from a carbapenem-resistant K. pneumoniae strain. Each plasmid was conjugated into the Escherichia coli strain DH5α, and a transconjugant with both plasmids was also obtained by transformation. Their in vitro competitive ability, biofilm formation, serum resistance, survival ability within macrophage and fruit fly, and fly killing ability were evaluated. Results The transconjugants with a single plasmid showed identical phenotypes to the plasmid-free strain, except that they decreased fly survival after infection. However, significantly increased fitness, virulence and biofilm production were observed consistently for the transconjugant with both plasmids, harbouring bla NDM-1 and bla OXA-232. Conclusions Our data indicate that bacteria carrying multiple plasmids encoding different carbapenemases may have increased fitness and virulence, emphasizing the need for diverse strategies to combat antimicrobial resistance.

Background & objectives OXA-232 is a five amino acid substitution variant of OXA-48 and is reported in carbapenem-resistant Klebsiella pneumoniae (CRKP), which is associated with nosocomial infections among immunocompromised patients in the intensive care unit. This study aimed to characterise blaOXA-232 in CRKP of clinical origin and investigate its transcriptional response against sub-inhibitory levels of carbapenems. Methods CRKP was isolated from blood (pathogens) and stool cultures (colonisers) of neonates and was characterized for blaOXA-232. Co-existing resistance determinants were investigated in blaOXA-232 positive isolates, followed by horizontal gene transferability assay and PCR-based replicon typing (PBRT). Cloning of blaOXA-232 was performed, and expression of blaOXA-232 in the isolates and their clones under sub-inhibitory concentrations of carbapenems was checked via RT-PCR. Mobile genetic elements associated with blaOXA-232 were investigated, followed by DNA fingerprinting through enterobacterial repetitive intergenic consensus (ERIC) PCR. Results blaOXA-232 with co-carriage of extended-spectrum beta-lactamases (ESBLs), sulphonamides and quinolones were identified in seven CRPK isolates recovered from blood samples of neonates. Transformation and cloning of blaOXA-232 was successful. The sub-inhibitory concentration of carbapenems induces elevated expression of this resistant determinant. ISEcp1 was associated with blaOXA-232 in the upstream region within two haplotypes of CRKP isolates of clinical origin. Interpretation & conclusions Selective carbapenem pressure resulted in higher expression of this gene, which could account for treatment failure. With frequent reports of occurrence among clinical isolates, monitoring and further investigation of this novel variant are necessary to understand its transmission dynamics and to thwart its further dissemination.

Background: The incidence of hypervirulent (hv) carbapenem-resistant (CR) Klebsiella pneumoniae (Kp) is increasing globally among various clones and is also responsible for nosocomial infections. The CR-hvKp is formed by the uptake of a virulence plasmid by endemic high-risk clones or by the uptake of plasmids carrying antimicrobial resistance genes by the virulent clones. Here, we describe CR-hvKp from India belonging to high-risk clones that have acquired a virulence plasmid and are phenotypically unidentified due to lack of hypermucoviscosity. Methods: Twenty-seven CRKp isolates were identified to possess rmpA2 by whole-genome sequencing; and resistance and virulence determinants were characterized. By in silico protein modeling (and validation), protein backbone stability analysis, and coarse dynamics study, the fitness of RmpA, RmpA2, and aerobactin-associated proteins-IucA and IutA, were determined to establish a reliable marker for clinical identification of CR-hvKp. Results: The CR-hvKp belonged to multidrug-resistant (MDR) high-risk clones such as CG11, CG43, ST15, and ST231 and carried OXA-232 as the predominant carbapenemase followed by NDM. The virulence plasmid belonged to IncHI1B replicon type and carried frameshifted and truncated rmpA and rmpA2. This resulted in a lack of hypermucoviscous phenotype. However, functional aerobactin was expressed in all high-risk clones. In silico analysis portrayed that IucA and IutA were more stable than classical RmpA. Furthermore, IucA and IutA had lower conformational fluctuations in the functional domains than the non-functional RmpA2, which increases the fitness cost of the latter for its maintenance and expression among CR-hvKp. Hence, RmpA and RmpA2 are likely to be lost among CR-hvKp owing to the increased fitness cost while coding for essential antimicrobial resistance and virulence factors. Conclusion: Increasing incidence of convergence of AMR and virulence is observed among K. pneumoniae globally, which warrants the need for reliable markers for identifying CR-hvKp. The presence of non-functional RmpA2 among high-risk clones highlights the significance of molecular identification of CR-hvKp. The negative string test due to non-functional RmpA2 among CR-hvKp isolates challenges phenotypic screening and faster identification of this pathotype. This can potentially be counteracted by projecting aerobactin as a stable, constitutively expressed, and functional marker for rapidly evolving CR-hvKp. [ABSTRACT FROM AUTHOR]

BackgroundThe incidence of hypervirulent (hv) carbapenem-resistant (CR) Klebsiella pneumoniae (Kp) is increasing globally among various clones and is also responsible for nosocomial infections. The CR-hvKp is formed by the uptake of a virulence plasmid by endemic high-risk clones or by the uptake of plasmids carrying antimicrobial resistance genes by the virulent clones. Here, we describe CR-hvKp from India belonging to high-risk clones that have acquired a virulence plasmid and are phenotypically unidentified due to lack of hypermucoviscosity.MethodsTwenty-seven CRKp isolates were identified to possess rmpA2 by whole-genome sequencing; and resistance and virulence determinants were characterized. By in silico protein modeling (and validation), protein backbone stability analysis, and coarse dynamics study, the fitness of RmpA, RmpA2, and aerobactin-associated proteins-IucA and IutA, were determined to establish a reliable marker for clinical identification of CR-hvKp.ResultsThe CR-hvKp belonged to multidrug-resistant (MDR) high-risk clones such as CG11, CG43, ST15, and ST231 and carried OXA-232 as the predominant carbapenemase followed by NDM. The virulence plasmid belonged to IncHI1B replicon type and carried frameshifted and truncated rmpA and rmpA2. This resulted in a lack of hypermucoviscous phenotype. However, functional aerobactin was expressed in all high-risk clones. In silico analysis portrayed that IucA and IutA were more stable than classical RmpA. Furthermore, IucA and IutA had lower conformational fluctuations in the functional domains than the non-functional RmpA2, which increases the fitness cost of the latter for its maintenance and expression among CR-hvKp. Hence, RmpA and RmpA2 are likely to be lost among CR-hvKp owing to the increased fitness cost while coding for essential antimicrobial resistance and virulence factors.ConclusionIncreasing incidence of convergence of AMR and virulence is observed among K. pneumoniae globally, which warrants the need for reliable markers for identifying CR-hvKp. The presence of non-functional RmpA2 among high-risk clones highlights the significance of molecular identification of CR-hvKp. The negative string test due to non-functional RmpA2 among CR-hvKp isolates challenges phenotypic screening and faster identification of this pathotype. This can potentially be counteracted by projecting aerobactin as a stable, constitutively expressed, and functional marker for rapidly evolving CR-hvKp.

Antibiotic resistance is a global health issue, with Klebsiella pneumoniae (KP) posing a particular threat due to its ability to acquire resistance to multiple drug classes rapidly. OXA-232 is a carbapenemase that confers resistance to carbapenems, a class of antibiotics often used as a last resort for treating severe bacterial infections. The study reports the earliest known identification of six OXA-232-producing KP strains that were isolated in Zhejiang, China, in 2008 and 2009 within a hospital, two years prior to the first reported identification of OXA-232 in France. The four KP strains carry the OXA-232 gene and exhibit hypervirulent loci, suggesting a broader temporal and geographical spread and integration of this resistance and virulence than previously recognized with implications for public health. Global analysis of all OXA-232-bearing KP strains revealed that OXA-232-encoding plasmids are conservative, while the strains were very diverse suggesting the plasmid mediated transmission of this carbapenemase genes. Importantly, a large proportion of the OXA-232-bearing KP strains also carried virulence plasmids, in particular the recent emergence of ST15 type of KP that carried both OXA-232-encoding plasmids and hypervirulent (hv) plasmids in China since 2019, highlighting the importance of the emergence of this type of KP strains in clinical setting. The early detection and investigations of OXA-232 in these strains warrants the retrospective studies to uncover the true timeline of antibiotic resistance spread, which could provide valuable insights for shaping future strategies to tackle the global health crisis. [ABSTRACT FROM AUTHOR]

Abstract Background The spread and outbreak of Enterobacteriaceae producing OXA-48-like carbapenemases have become more and more prevalent in China. Results A total of 62 non-duplicated OXA-232-producing K. pneumoniae (OXA232Kp) were isolated between 2015 and 2017. An outbreak of OXA232Kp was observed in burn ICU. The 62 OXA232Kp isolates were all belongs to ST15 and categorized into two PFGE types (A and B). Type A was dominated of the isolates, which contained 61 clinical isolates and divided into 10 subtypes (A1-A10). In addition, most of OXA232Kp strains exhibited low-level carbapenems resistance. All strains carried a 6141 bp ColKP3 plasmid harboring the bla OXA-232 gene which is highly homologous to other bla OXA-232-bearing plasmids involved in other studies in eastern China. Conclusions In this study, clone transmission of OXA232Kp ST15was observed. Highly significant homology among the bla OXA-232-bearing plasmids indicated the important role of the 6.1 kb ColE-like plasmid on the prevalence of bla OXA-232 gene in China.

For addressing the issue of antimicrobial drug resistance in developing countries, it is important to investigate the characteristics of carbapenemase-producing organisms. We aimed to genetically characterize a carbapenemase-producing Klebsiella pneumoniae (CPKP) isolated in the intensive care unit of a tertiary hospital in Bangladesh. The number of CPKP isolates were 43/145 (30%), of which pandrug-resistant (PDR) strains were 14%. These carbapenemases were New Delhi metallo-beta-lactamase (NDM)-1 (53%), NDM-5 (14%), oxacillinase (OXA)-181 (12%), OXA-232 (10%), NDM-5 + OXA-181 (5%), and NDM-5 + OXA-232 (2%). Many CPKP isolates harbored a variety of resistance genes, and the prevalence of 16S rRNA methyltransferase was particularly high (91%). The 43 CPKP isolates were classified into 14 different sequence types (STs), and the common STs were ST34 (26%), ST147 (16%), ST11 (9%), ST14 (9%), ST25 (7%), and ST231 (7%). In this study, PDR strains were of three types, ST147, ST231, and ST14, and their PDR rates were 57, 33, and 25%, respectively. The spread of the antimicrobial drug resistance of CPKP in Bangladesh was identified. In particular, the emergence of PDR is problem, and there may be its spread as a superbug of antimicrobial treatment.

For addressing the issue of antimicrobial drug resistance in developing countries, it is important to investigate the characteristics of carbapenemase-producing organisms. We aimed to genetically characterize a carbapenemase-producing Klebsiella pneumoniae (CPKP) isolated in the intensive care unit of a tertiary hospital in Bangladesh. The number of CPKP isolates were 43/145 (30%), of which pandrug-resistant (PDR) strains were 14%. These carbapenemases were New Delhi metallo-beta-lactamase (NDM)-1 (53%), NDM-5 (14%), oxacillinase (OXA)-181 (12%), OXA-232 (10%), NDM-5 + OXA-181 (5%), and NDM-5 + OXA-232 (2%). Many CPKP isolates harbored a variety of resistance genes, and the prevalence of 16S rRNA methyltransferase was particularly high (91%). The 43 CPKP isolates were classified into 14 different sequence types (STs), and the common STs were ST34 (26%), ST147 (16%), ST11 (9%), ST14 (9%), ST25 (7%), and ST231 (7%). In this study, PDR strains were of three types, ST147, ST231, and ST14, and their PDR rates were 57, 33, and 25%, respectively. The spread of the antimicrobial drug resistance of CPKP in Bangladesh was identified. In particular, the emergence of PDR is problem, and there may be its spread as a superbug of antimicrobial treatment. [ABSTRACT FROM AUTHOR]

BackgroundIn recent years, the emergence of multidrug resistant hypervirulent K. pneumoniae (MDR hvKp) isolates poses severe therapeutic challenge to global public health. The present study used the complete genome sequence of two MDR hvKp isolates belonging to ST23 to characterize the phylogenetic background and plasmid diversity.MethodsTwo hvKp isolates from patients with bacteremia were sequenced using Ion Torrent PGM and Oxford Nanopore MinION platforms and assembled by hybrid genome assembly approach. Comparative genomics approaches were used to investigate the population structure, evolution, virulence, and antimicrobial resistance of MDR hvKp strains.ResultsThe study isolates exhibited typical features of hvKp phenotypes associated with ST23. The convergence of multidrug resistance and hypervirulence were attributed by the presence of multiple plasmids including a 216 kb virulence plasmid and MDR plasmids belonging to IncA/C2, IncFIB, IncX3, and ColKP3 groups. The insertion of catA1 gene into virulence plasmid was observed along with genetic factors such as aerobactin, salmochelin, and rmpA2 that confer hvKp’s hypervirulent phenotype. The core genome single nucleotide polymorphism (SNP) phylogenetic analyses of the isolates showed the evolution of ST23 hvKp was predominantly driven by ICEKp acquisitions.ConclusionTo the best of our knowledge, this is the first report of MDR hvKp isolates of ST23 with insertion of catA1 gene into the virulence plasmid which presents the possibility of hotspot integration sites on the plasmids to aid acquisition of AMR genes. ST23 is no longer confined to susceptible strains of hvKp. Our findings emphasize the need for more studies on recombinant events, plasmid transmission dynamics and evolutionary process involving hvKp.

Dongxing Tian, Fen Pan, Chun Wang, Yan Sun, Hong Zhang Department of Clinical Laboratory, Shanghai Children’s Hospital, Shanghai Jiaotong University, Shanghai, China Background: Carbapenem-resistant Klebsiella pneumoniae (CRKP) has caused wide global disseminations and serious clinical outcomes in pediatric patients, and the purpose of this study was to analyze drug resistance, molecular epidemiology, and clinical characteristics of CRKP from children in Shanghai, China.Methods: A retrospective study was conducted from January 2016 to December 2017, and a total of 170 CRKP isolates were collected. Antimicrobial susceptibility was determined by the broth microdilution method. MAST D73C and polymerase chain reaction were used for the analysis of carbapenemase types. Multilocus sequence typing of K. pneumoniae was performed for genetic relationship. Clinical data were also reviewed.Results: Of the 170 CRKP isolates, blaOXA-232 was mainly detected with a proportion of 42.35%, followed by blaNDM-1 (20.59%), blaKPC-2 (17.65%), blaNDM-5 (16.47%), and blaIMP-4 (1.18%). The predominant gene was blaOXA-232 in 2016 (54.46%; 55/101) and blaNDM-1 in 2017 (31.88%; 22/69). All these 170 CRKP isolates showed high resistance to cephalosporins and carbapenems (>95%), except for tigecycline and colistin. Sixteen distinct sequence types were observed with ST15 being mostly identified (41.76%). Most CRKP harboring OXA-232 type carbapenemase belonged to ST15, while NDM-1 type belonged to ST37 and KPC-2 type belonged to ST11. Furthermore, other β-lactamase genes including blaTEM, blaCTX-M, and DHA-1 were also found in this study. Clinical data reviewed that more than half of the patients produced clinical infections (112/170), mainly lower respiratory tract (58/112) and bloodstream (21/112) infections. A majority of these children had received therapy of antibiotics before CRKP isolation, especially for carbapenems (76/170) and β-lactam/β-lactamase inhibitor combinations (91/170).Conclusions: Our data revealed the increasing incidence of OXA-232-producing K. pneumoniae from pediatric patients in Shanghai, and infection control measures should be conducted to limit the spread of CRKP strains. Keywords: Klebsiella pneumoniae, carbapenemases, drug resistance, OXA-232, NDM-5, children

Background: In recent years, the emergence of multidrug resistant hypervirulent K. pneumoniae (MDR hvKp) isolates poses severe therapeutic challenge to global public health. The present study used the complete genome sequence of two MDR hvKp isolates belonging to ST23 to characterize the phylogenetic background and plasmid diversity. Methods: Two hvKp isolates from patients with bacteremia were sequenced using Ion Torrent PGM and Oxford Nanopore MinION platforms and assembled by hybrid genome assembly approach. Comparative genomics approaches were used to investigate the population structure, evolution, virulence, and antimicrobial resistance of MDR hvKp strains. Results: The study isolates exhibited typical features of hvKp phenotypes associated with ST23. The convergence of multidrug resistance and hypervirulence were attributed by the presence of multiple plasmids including a 216 kb virulence plasmid and MDR plasmids belonging to IncA/C2, IncFIB, IncX3, and ColKP3 groups. The insertion of catA1 gene into virulence plasmid was observed along with genetic factors such as aerobactin, salmochelin, and rmpA2 that confer hvKp's hypervirulent phenotype. The core genome single nucleotide polymorphism (SNP) phylogenetic analyses of the isolates showed the evolution of ST23 hvKp was predominantly driven by ICE Kp acquisitions. Conclusion: To the best of our knowledge, this is the first report of MDR hvKp isolates of ST23 with insertion of catA1 gene into the virulence plasmid which presents the possibility of hotspot integration sites on the plasmids to aid acquisition of AMR genes. ST23 is no longer confined to susceptible strains of hvKp. Our findings emphasize the need for more studies on recombinant events, plasmid transmission dynamics and evolutionary process involving hvKp. [ABSTRACT FROM AUTHOR]

Background: Carbapenem non-susceptible Enterobacterales (CNSE) can be broadly divided into those that produce carbapenemases (carbapenemase-producing Enterobacterales (CPE)), and those that harbour other mechanisms of resistance (non-carbapenemase-producing CNSE (NCP-CNSE)).Aim: To determine the predictors of CNSE nosocomial incidence rates according to their mechanism of resistance.Methods: A time-series analysis was conducted (July 2013 to December 2018) to evaluate the relationship in time between hospital antibiotic use and the percentage of adherence to hand hygiene with the CNSE rates.Findings: In all, 20,641 non-duplicated Enterobacterales isolates were identified; 2.2% were CNSE. Of these, 48.1% and 51.9% were CPE and NCP-CNSE, respectively. Of the CPE, 78.3% possessed a blaOXA-232 gene. A transfer function model was identified for CNSE, CPE, and OXA-232 CPE that explained 20.8%, 19.3%, and 24.2% of their variation, respectively. According to the CNSE and CPE models, an increase in piperacillin-tazobactam (TZP) use of 1 defined daily dose (DDD) per 100 hospital patient-days (HPD) would lead to an increase of 0.69 and 0.49 CNSE and CPE cases per 10,000 HPD, respectively. The OXA-232 CPE model estimates that an increase of 1 DDD per 100 HPD of TZP use would lead to an increase of 0.43 OXA-232 CPE cases per 10,000 HPD. A transfer function model was not identified for NCP-CNSE, nor was there an association between the adherence to handhygiene and the CNSE rates.Conclusion: The use of TZP is related in time with the CPE nosocomial rates, mostly explained by its effect on OXA-232 CPE. [ABSTRACT FROM AUTHOR]

A total of 345 unique (one isolate per patient) clinical carbapenem-resistant Klebsiella pneumoniae isolates were recovered in our hospital over the past 3 years (2016–2018), with 325, 14, and 6 isolates carried blaKPC-2, blaNDM-1, and blaOXA-232, respectively. The six OXA-232-Kp isolates were recovered in neurosurgery intensive care unit in 2018. All OXA-232-Kp belonged to ST15, differed by one or two pulsed-field gel electrophoresis (PFGE) bands, and belonged to the same clone. However, three isolates (RJ18-04-6) displayed elevated carbapenem resistance, with imipenem minimum inhibitory concentration (MIC) of 16–32 μg/mL. In contrast, other three isolates (RJ18-01-3) had imipenem MIC of 0.5–1 μg/mL. S1-PFGE revealed two different plasmid profiles and all strains had at least four plasmids. Strain RJ18-01 and RJ18-06 were selected for whole-genome sequencing, and a ColE2-type 6,141 bp blaOXA-232-carrying plasmid was identified. No blaOXA-232 gene was located on chromosome or other plasmids. Furthermore, no β-lactamase resistance gene other than blaCTX-M-15 was identified. Polymerase chain reaction-based sequencing of blaOXA-232-carrying plasmid was performed, and all OXA-232-Kp shared identical blaOXA-232-carrying plasmid sequences. The expression and copy number of blaOXA-232 in RJ18-04-6 were significantly higher than those of other isolates. The hydrolysis activity of nitrocefin and carbapenems were about six-fold higher in RJ18-04-6. Since only one copy of blaOXA-232 gene was spotted on the plasmid, the elevated carbapenem resistance could be attributed to the increased copy number of blaOXA-232-carrying plasmids. OmpK35 porin deficiency was observed in one isolate with decreased carbapenem susceptibility and two isolates with elevated carbapenem resistance, suggesting that OmpK35 deficiency did not significantly alter carbapenem MICs in OXA-232-Kp. [ABSTRACT FROM AUTHOR]

Background: Bacterial isolates with multiple plasmids harbouring different carbapenemase genes have emerged and been identified repeatedly, despite a general notion that plasmids confer fitness cost in bacterial host. In this study, we investigated the effects of plasmids with carbapenemase genes on the fitness and virulence of bacteria. Methods: Different plasmids harbouring the carbapenemase genes, blaNDM-1 and blaOXA-232, were isolated from a carbapenem-resistant K. pneumoniae strain. Each plasmid was conjugated into the Escherichia coli strain DH5α, and a transconjugant with both plasmids was also obtained by transformation. Their in vitro competitive ability, biofilm formation, serum resistance, survival ability within macrophage and fruit fly, and fly killing ability were evaluated. Results: The transconjugants with a single plasmid showed identical phenotypes to the plasmid-free strain, except that they decreased fly survival after infection. However, significantly increased fitness, virulence and biofilm production were observed consistently for the transconjugant with both plasmids, harbouring blaNDM-1 and blaOXA-232. Conclusions: Our data indicate that bacteria carrying multiple plasmids encoding different carbapenemases may have increased fitness and virulence, emphasizing the need for diverse strategies to combat antimicrobial resistance. [ABSTRACT FROM AUTHOR]

OXA-232 is one of the most common OXA-48-like carbapenemase derivatives and is widely disseminated in nosocomial settings across countries. The bla OXA-232 gene is located on a 6-kb non-conjugative ColKP3-type plasmid, while the dissemination of bla species and the polyclonal dissemination of OXA-232-producing OXA-232 into different Enterobacterales species and the polyclonal dissemination of OXA-232-producing K. pneumoniae revealed the horizontal transfer of bla OXA-232 during a nosocomial outbreak of OXA-232-producing bla . We demonstrated the presence of ColKP3 OXA-232 plasmid in the outer membrane vesicles (OMVs) derived from clinical isolates, and OMVs could facilitate the horizontal transfer of OXA-232 . Here, we observed the in vivo intraspecies transfer of bla OXA-232 during a nosocomial outbreak of OXA-232-producing K. pneumoniae . We demonstrated the presence of ColKP3 OXA-232 plasmid in the outer membrane vesicles (OMVs) derived from clinical isolates, and OMVs could facilitate the horizontal transfer of bla OXA-232 among Enterobacterales . In contrast, for the most prevalent carbapenemase genes, including bla KPC-2 and bla NDM-1 , though the presence of carbapenemase genes and plasmid backbones in the vesicular lumen was observed, OMVs couldn't promote effective transformation, probably due to the low copy number of plasmids in clinical isolates and the low number of plasmids loaded into vesicles. Conjugation assay revealed that the epidemic IncX3 NDM-1 and IncFII(pHN7A8)/IncR KPC-2 plasmids were conjugative and could be horizontally transferred via independent conjugation or with the help of a co-existent conjugative plasmid. For the large-size and low-copy number conjugative plasmids carrying carbapenemase genes, OMVs-mediated gene exchange may only serve as an alternative pathway for horizontal transfer. In conclusion, diverse mobilization strategies were employed by plasmids harboring carbapenemase genes, and plasmids display a proper choice of mobility pathway due to their individual properties.

Background: The spread and outbreak of Enterobacteriaceae producing OXA-48-like carbapenemases have become more and more prevalent in China. Results: A total of 62 non-duplicated OXA-232-producing K. pneumoniae (OXA232Kp) were isolated between 2015 and 2017. An outbreak of OXA232Kp was observed in burn ICU. The 62 OXA232Kp isolates were all belongs to ST15 and categorized into two PFGE types (A and B). Type A was dominated of the isolates, which contained 61 clinical isolates and divided into 10 subtypes (A1-A10). In addition, most of OXA232Kp strains exhibited low-level carbapenems resistance. All strains carried a 6141 bp ColKP3 plasmid harboring the blaOXA-232 gene which is highly homologous to other blaOXA-232-bearing plasmids involved in other studies in eastern China. Conclusions: In this study, clone transmission of OXA232Kp ST15was observed. Highly significant homology among the blaOXA-232-bearing plasmids indicated the important role of the 6.1 kb ColE-like plasmid on the prevalence of blaOXA-232 gene in China. [ABSTRACT FROM AUTHOR]

Background: Carbapenem-resistant Klebsiella pneumoniae (CRKP) has caused wide global disseminations and serious clinical outcomes in pediatric patients, and the purpose of this study was to analyze drug resistance, molecular epidemiology, and clinical characteristics of CRKP from children in Shanghai, China.Methods: A retrospective study was conducted from January 2016 to December 2017, and a total of 170 CRKP isolates were collected. Antimicrobial susceptibility was determined by the broth microdilution method. MAST D73C and polymerase chain reaction were used for the analysis of carbapenemase types. Multilocus sequence typing of K. pneumoniae was performed for genetic relationship. Clinical data were also reviewed.Results: Of the 170 CRKP isolates, blaOXA-232 was mainly detected with a proportion of 42.35%, followed by blaNDM-1 (20.59%), blaKPC-2 (17.65%), blaNDM-5 (16.47%), and blaIMP-4 (1.18%). The predominant gene was blaOXA-232 in 2016 (54.46%; 55/101) and blaNDM-1 in 2017 (31.88%; 22/69). All these 170 CRKP isolates showed high resistance to cephalosporins and carbapenems (>95%), except for tigecycline and colistin. Sixteen distinct sequence types were observed with ST15 being mostly identified (41.76%). Most CRKP harboring OXA-232 type carbapenemase belonged to ST15, while NDM-1 type belonged to ST37 and KPC-2 type belonged to ST11. Furthermore, other β-lactamase genes including blaTEM, blaCTX-M, and DHA-1 were also found in this study. Clinical data reviewed that more than half of the patients produced clinical infections (112/170), mainly lower respiratory tract (58/112) and bloodstream (21/112) infections. A majority of these children had received therapy of antibiotics before CRKP isolation, especially for carbapenems (76/170) and β-lactam/β-lactamase inhibitor combinations (91/170).Conclusions: Our data revealed the increasing incidence of OXA-232-producing K. pneumoniae from pediatric patients in Shanghai, and infection control measures should be conducted to limit the spread of CRKP strains. [ABSTRACT FROM AUTHOR]

Aim: The purpose of the study was to determine the epidemiology of carbapenemase genes among carbapenem-resistant Enterobacteriaceae and evaluate the Carba NP and modified carbapenem inactivation method (mCIM) tests in their detection. Materials and Methods: A total of 287 nonduplicated Enterobacteriaceae isolates, which were at least resistant to one of the carbapenems, were identified and detected for carbapenemase genes by multiplex PCR covering blaKPC, blaNDM, blaVIM, blaIMP, and blaOXA-48-like. All positive genes were then sequenced. These isolates were phenotypically tested for the production of carbapenemases by mCIM and Carba NP tests to evaluate the efficacy of these methods. Results: Seven species of carbapenem-resistant isolates mainly Klebsiella pneumoniae, Escherichia coli, and Enterobacter cloacae were detected. Of these isolates, three families of carbapenemase genes, including blaNDM (blaNDM-1, -4, -5, -9), blaOXA (blaOXA-48, -181, -232), and blaIMP-14, were found. Of these, 223 (77.70%) carried at least one of the carbapenemase genes. The blaNDM was detected in 160/223 (71.75%) isolates, of which 153/160 (95.63%) were the blaNDM-1. Three types of the blaOXA-48-like group, blaOXA-48, blaOXA-181, and blaOXA-232, were found, 91/104 (87.5%) harbored the blaOXA-232. In addition, 25.11% (56/223) of the carbapenemase-producing isolates harbored a combination of blaNDM and blaOXA-48-like. Phenotypic detection methods, mCIM and Carba NP, showed 100% sensitivity and specificity to blaNDM, blaIMP-14, and blaOXA-48, while the mCIM was positive in all blaOXA-181 and blaOXA-232 isolates, only 12.5% (1/8) and 28.95% (11/38), respectively, were detected by the Carba NP test. Conclusions: This study revealed a unique prevalence of carbapenemase genes in Bangkok, Thailand, as well as demonstrated the efficacy and limitation of phenotypic detection methods of carbapenemase in the area where blaNDM-1 and blaOXA-232 were predominant. [ABSTRACT FROM AUTHOR]

Background. Whole-genome sequencing (WGS) is an emerging and powerful technique by which to perform epidemiological studies in outbreak situations. Methods. WGS was used to identify and evaluate an outbreak of OXA-232-expressing carbapenem-resistant Klebsiella pneumoniae (CRKP) transmitted to 16 patients over the course of 40 weeks via endoscopic retrograde cholangiopancreatography procedures at a single institution. WGS was performed on 32 OXA-232 CRKP isolates (1-7 per patient) and single-nucleotide variants (SNVs) were analyzed, with reference to the index patient's isolate. Results. Interhost genetic diversity of isolates was between 0 and 15 SNVs during the outbreak; molecular clock calculations estimated 12.31 substitutions per genome per year (95% credibility interval, 7.81-17.05). Both intra- and interpatient diversification at the plasmid and transposon level was observed, significantly impacting the antibiogram of outbreak isolates. The majority of isolates evaluated (n = 27) harbored a blaCTX-M-15 gene, but some (n = 5) lacked the transposon carrying this gene, which resulted in susceptibility to aztreonam and third- and fourth-generation cephalosporins. Similarly, an isolate from a colonized patient lacked the transposon carrying rmtF and aac(6')lb genes, resulting in susceptibility to aminoglycosides. Conclusions. This study broadens the understanding of how bacteria diversify at the genomic level over the course of a defined outbreak and provides reference for future outbreak investigations. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae, a notorious pathogen for opportunistic health care-associated infections, represents increasing multidrug resistance, particularly to carbapenems. OXA-232 carbapenemase, as a variant of OXA-48, has been increasingly reported worldwide. ST231, an epidemic, multidrug resistant (MDR) K. pneumoniae clone in south and southeast Asia, has been found in other regions, including Europe. In the study, five OXA-232 carbapenemase-producing Klebsiella pneumoniae isolates, four of which belong to sequence type 231 (ST231) and one of which belongs to ST15, were isolated from two hospitals in China. All isolates displayed a MDR phenotype, being susceptible to only polymyxin B and colistin, and the bla OXA-232 gene was located on a ColKP3-type nonconjugative plasmid of 6.1 kb. A phylogenetic analysis of the global ST231 K. pneumoniae isolates ( n  = 231) suggested that the four ST231 isolates from this study gathered with strains from south Asia (especially India), indicating that the emerging Chinese ST231 clone was more closely related to south Asia isolates and might have spread from south Asia, where ST231 was a successful epidemic clone. Virulence assays suggested that the four ST231 strains were not highly virulent, as they displayed significantly lower virulence potential, compared with a ST23 K1 hypervirulent isolate in a G. mellonella infection and in mouse intraperitoneal infection models, although three ST231 strains harbored a plasmid-borne aerobactin-encoding iuc gene cluster. This is the first report of ST231 K. pneumoniae clinical strains bearing bla OXA-232 carbapenemase, being a vital resistance mechanism against carbapenems, has recently been increasingly reported. In China, the identified OXA-232-producing K. pneumoniae isolates almost belonged to ST15 and were not hypervirulent, despite harboring a virulence plasmid. Here, we report the first occurrence in China of a MDR OXA-232-producing K. pneumoniae ST231 clone that is an epidemic ST type in south and southeast Asia. A phylogenetic analysis indicated that this emerging Chinese ST231 clone was more closely related to Indian isolates. The occurrence of this clone may have been driven through the transnational importation of Indian ST231 K. pneumoniae clones. Moreover, this study is the first to assess the virulence potential of ST231 clones that have never been estimated in previous studies. While the high burden of MDR K. pneumoniae is concerning, genomic surveillance can shed light on the transmission chains of novel MDR clones, and active surveillance should be enforced to restrict the spread of MDR isolates.OXA-232 in China, and it highlights the emergence of the ST231 clone causing bloodstream infections in a health care setting as well as calls attention to the transmission of this emerging clone in China. IMPORTANCE OXA-232 carbapenemase, being a vital resistance mechanism against carbapenems, has recently been increasingly reported. In China, the identified OXA-232-producing K. pneumoniae isolates almost belonged to ST15 and were not hypervirulent, despite harboring a virulence plasmid. Here, we report the first occurrence in China of a MDR OXA-232-producing K. pneumoniae ST231 clone that is an epidemic ST type in south and southeast Asia. A phylogenetic analysis indicated that this emerging Chinese ST231 clone was more closely related to Indian isolates. The occurrence of this clone may have been driven through the transnational importation of Indian ST231 K. pneumoniae clones. Moreover, this study is the first to assess the virulence potential of ST231 clones that have never been estimated in previous studies. While the high burden of MDR K. pneumoniae is concerning, genomic surveillance can shed light on the transmission chains of novel MDR clones, and active surveillance should be enforced to restrict the spread of MDR isolates.

Background: Klebsiella pneumoniae subsp. pneumoniae KP617 is a pathogenic strain that coproduces OXA-232 and NDM-1 carbapenemases. We sequenced the genome of KP617, which was isolated from the wound of a Korean burn patient, and performed a comparative genomic analysis with three additional strains: PittNDM01, NUHL24835 and ATCC BAA-2146. Results: The complete genome of KP617 was obtained via multi-platform whole-genome sequencing. Phylogenetic analysis along with whole genome and multi-locus sequence typing of genes of the Klebsiella pneumoniae species showed that KP617 belongs to the WGLW2 group, which includes PittNDM01 and NUHL24835. Comparison of annotated genes showed that KP617 shares 98.3% of its genes with PittNDM01. Nineteen antibiotic resistance genes were identiied in the KP617 genome: blaOXA-1 and blaSHV-28 in the chromosome, blaNDM-1 in plasmid 1, and blaOXA-232 in plasmid 2 conferred resistance to beta-lactams; however, colistin- and tetracycline-resistance genes were not found. We identiied 117 virulence factors in the KP617 genome, and discovered that the genes encoding these factors were also harbored by the reference strains; eight genes were lipopolysaccharide-related and four were capsular polysaccharide-related. A comparative analysis of phage-associated regions indicated that two phage regions are speciic to the KP617 genome and that prophages did not act as a vehicle for transfer of antimicrobial resistance genes in this strain. Conclusions: Whole-genome sequencing and bioinformatics analysis revealed similarity in the genome sequences and content, and diferences in phage-related genes, plasmids and antimicrobial resistance genes between KP617 and the references. In order to elucidate the precise role of these factors in the pathogenicity of KP617, further studies are required. [ABSTRACT FROM AUTHOR]

Ceftazidime-avibactam exhibits good in vitro activity against carbapenem resistant Klebsiella carrying OXA-48-like enzymes. We tested two hundred unique carbapenem resistant Klebsiella blood stream isolates (71% with single OXA-48-like carbapenemases, including OXA-48, n = 62; OXA-232, n = 57; OXA-244, n = 17; OXA-181, n = 5) that were collected as part of a multicentre study against ceftazidime-avibactam using Etest (bioMérieux, Marcyl'Étoile, France), 10/4 μg disc (Thermo Fisher) and Sensititre Gram Negative EURGNCOL Plates (Lyophilized panels, Sensititre, Thermo Fisher) with the aim of comparing the performances of the Etest and disc to that of Sensititre. Ceftazidime-avibactam MIC 50/90 was 2/>16 mg/L for the entire collection and was 2/4 mg/L for single OXA-48-like producers. Categorical and essential agreements between the Etest and Sensititre were 100% and 97%, respectively. Categorical agreement between the disc and Sensititre was 100%. Etest and 10/4 μg discs are suitable alternatives to Sensititre for ceftazidime-avibactam sensitivity testing for OXA-48-like producers. [ABSTRACT FROM AUTHOR]

Introduction. Aminoglycosides are used for the treatment of carbapenemase-producing Klebsiella pneumoniae (CPK) infections. 16S rRNA methyltransferases (RMTs) confer resistance to all aminoglycosides and are often cocarried with NDM. Hypothesis/Gap Statement. There is a dart of studies looking at the aminoglycoside resistance mechanisms for invasive CPK isolates, particularly in OXA-48 endemic settings. Aim. We aimed to determine the prevalence of RMTs and their association with beta lactamases and MLSTs amongst aminoglycoside-resistant CPK bloodstream isolates in an OXA-48 endemic setting. Methodology. CPK isolates ( n =181), collected as part of a multicentre cohort study, were tested for amikacin, gentamicin and tobramycin susceptibility using custom-made sensititre plates (GN2XF, Thermo Fisher Scientific). All isolates were previously subjected to whole-genome sequencing. Carbapenemases, RMTs, MLSTs and plasmid incompatibility groups were detected on the assembled genomes. Results. Of the 181 isolates, 109(60 %) were resistant to all three aminoglycosides, and 96 of 109(88 %) aminoglycoside-resistant isolates carried an RMT (85 ArmA, 10 RmtC, 4 RmtF1; three isolates cocarried ArmA and RmtC). Main clonal types associated with ArmA were ST2096 (49/85, 58 %) and ST14 (24/85, 28 %), harbouring mainly OXA-232 and OXA-48 +NDM, respectively. RmtC was cocarried with NDM (5/10) on ST395, and NDM +OXA-48 or NDM +KPC (4/10) on ST14, ST15 and ST16. All RMT producers also carried CTX-M-15, and the majority cocarried SHV-106, TEM-150 and multiple other antibiotic resistance genes. The majority of the isolates harboured a combination of IncFIB, IncH and IncL/M type plasmids. Non-NDM producing isolates remained susceptible to ceftazidime-avibactam. Conclusion. Aminoglycoside resistance amongst CPK bloodstream isolates is extremely common and mainly driven by clonal spread of ArmA carried on ST2096 and ST14, associated with OXA-232 and OXA48 +NDM carriage, respectively.

Eleven carbapenem-resistant Klebsiella pneumoniae isolates were recovered from the military hospital of Tunis, Tunisia. Three of these isolates carried the carbapenemase bla OXA-232 gene recently identified in France. These isolates were clonally related and co-expressed the extended-spectrum ß-lactamase, CTX-M-15. This work identified the occurrence of OXA-232 producers in North-African countries. [ABSTRACT FROM AUTHOR]