Emergence of ceftazidime-avibactam resistance in blaKPC-33-harboring ST11 Klebsiella pneumoniae in pediatric patient.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses immense threats to the health of infected patients worldwide, especially in children. This study reports the infection caused by CRKP in a pediatric intensive care unit (PICU) child and its drug-resistant mutation during the treatment. Twelve Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae strains were isolated from the child. Broth microdilution method, plasmid transformation assay, and whole genome sequencing (WGS) were performed to investigate the antimicrobial susceptibility, resistance mechanisms, and genetic structural features of CRKPs. The results showed that twelve strains were highly resistant to most available antimicrobial agents. Among them, K. pneumoniae FD11 and K. pneumoniae FD12 were resistant to ceftazidime-avibactam (CZA, MIC>64mg/L) and restored the carbapenem susceptibility (IMP, MIC=0.25mg/L; MEM, MIC=2mg/L). The patient improved after treatment with CZA in combination with aztreonam. Plasmid transformation assay demonstrated that the blaKPC-33-positive transformant increased MICs of CZA by at least 33-fold and 8-fold compared with the recipient E.coli DH5α and blaKPC-2-positive transformants. WGS analysis revealed that all strains belonged to the ST11-KL64 type and showed highly homologous (3 to 26 single nucleotide polymorphisms (SNPs)). A single base mutation (G532T) of blaKPC-2 resulted in a tyrosine to aspartic acid substitution at Ambler amino acid position 179 (D179Y), which conferred CZA resistance in K. pneumoniae. This is the first report of a drug-resistant mutation evolving into blaKPC-33 during the treatment of blaKPC-2-positive CRKP in pediatric infected patients. It advises clinicians that routine sequential antimicrobial susceptibility testing and KPC genotyping are critical during CZA therapy in children infected with CRKP.

Coexistence of a novel chromosomal integrative and mobilizable element Tn7548 with two blaKPC-2-carrying plasmids in a multidrug-resistant Aeromonas hydrophila strain K522 from China.

OBJECTIVES: Herein, we detected one multidrug-resistant Aeromonas hydrophila strain K522 co-carrying two blaKPC genes together with a novel chromosomal integrative and mobilizable element (IME) Tn7548 from China. To reveal the genetic characteristics of the novel reservoir of blaKPC-2 and IME in Aeromonas, a detailed genomic characterization of K522 was performed, and a phylogenetic analysis of Tn7412-related IMEs was carried out. METHODS: Carbapenemases were detected by using the immunocolloidal gold technique and antimicrobial susceptibility was tested by using VITEK 2. The whole genome sequences of K522 were analyzed using phylogenetics, detailed dissection and comparison. RESULTS: Strain K522 carried a Tn7412-related chromosomal IME Tn7548 and three resistance plasmids pK522-A-KPC, pK522-B-KPC, and pK522-MOX. A phylogenetic tree of 82 Tn7412-related IMEs was constructed and five families of IMEs were divided. These IMEs shared four key backbone genes int, repC, and hipAB, and carried various profiles of antimicrobial resistance genes (ARGs). pK522-A-KPC and pK522-B-KPC carried blaKPC-2 and belonged to IncG and unclassified type plasmid, respectively. The blaKPC-2 regions of these two plasmids were the truncated version derived from Tn6296, resulting in the carbapenem resistance of K522. CONCLUSIONS: We first reported A. hydrophila harboring a novel Tn7412-related IME Tn7548 together with two blaKPC-2 carrying plasmids and an MDR plasmid. Three of these four MGEs discovered in A. hydrophila K522 were novel. The emergence of novel MGEs carrying ARGs indicated the rapid evolution of the resistance gene vectors in A. hydrophila under selection pressure and would contribute to the further dissemination of various ARGs in Aeromonas.

Global phylogeography and genomic characterization of blaKPC and blaNDM-positive clinical Klebsiella aerogenes isolates from China, 2016-2022.

Carbapenem-resistant Klebsiella aerogenes (CRKA), being one of the members of carbapenem-resistant Enterobacteriaceae (CRE), has caused great public health concern, but with fewer studies compared to other CRE members. Furthermore, studies on phylogenetic analysis based on whole genome Single-Nucleotide Polymorphism (SNP) of CRKA were limited. Here, 20 CRKA isolates (11 blaKPC-2-bearing and 9 blaNDM-1/5-harboring) were characterized by antimicrobial susceptibility testing, conjugation assay, whole genome sequencing (WGS) and bioinformatics analysis. Additionally, the phylogeographic relationships of K. aerogenes were further investigated from public databases. All isolates were multidrug-resistant (MDR) bacteria, and they demonstrated susceptibility to colistin. Most blaKPC-2 or blaNDM-1/5-carrying plasmids were found to be conjugative. Phylogenetic analysis revealed the clonal dissemination of K. aerogenes primarily occurred within clinical settings. Notably, some strains in this study showed the potential for clonal transmission, sharing few SNPs between K. aerogenes and KPC- and/or NDM-positive K. aerogenes isolated from various countries. The STs of K. aerogenes strains had significant diversity. WGS analysis showed that the IncFIIK plasmid was the most prevalent carrier of blaKPC-2, and, blaNDM-1/5 were detected on the IncX3 plasmids. The Tn6296 and Tn3000 transposons were most common vehicles for facilitating the transmission of blaKPC-2 and blaNDM-1/5, respectively. This study highlights the importance of continuous screening and surveillance by WGS for analysis of drug-resistant strains in hospital settings, and provide clinical information that supports epidemiological and public health research on human pathogens.

Genomic characterization of a WHO critical priority isolate Enterobacter kobei ST2070 harboring OXA-10, KPC-2, and CTX-M-12 recovered from a water irrigation channel in Ecuador.

The discharge of untreated or partially treated wastewater can have detrimental impacts on the quality of water bodies, posing a significant threat to public health and the environment. In Ecuador, previous research indicates a high prevalence of antimicrobial resistant (AMR) bacteria in surface waters affected by human activities, including irrigation channels. In this study, we analyzed sediment samples collected from an irrigation channel utilized for agricultural purposes in northern Ecuador, using microbiological techniques and whole-genome sequencing (WGS). Our investigation revealed the first documented occurrence of E. kobei in Ecuador and the initial report of environmental E. kobei ST2070. Furthermore, we identified the coexistence of OXA-10-type class D ß-lactamase and KPC-2-type class A ß-lactamase in the E. kobei isolate (UTA41), representing the first report of such a phenomenon in this species. Additionally, we detected various antibiotic resistance genes in the E. kobei UTA41 isolate, including blaCTX-M-12, fosA, aac(6')-lb, sul2, msr(E), and mph(A), as well as virulence genes such as bacterial efflux pump and siderophore biosynthesis genes. We also identified two intact prophage regions (Entero_186 and Klebsi_phiKO2) in the isolate. Our study presents the first evidence of E. kobei isolate containing two carbapenemase-encoding genes in environmental samples from Latin America. This finding indicates the potential spread of critical-priority bacteria in water samples originating from anthropogenic sources, such as urban wastewater discharges and livestock facilities.

Levofloxacin-induced MexS mutation triggers imipenem-relebactam resistance in a KPC-producing Pseudomonas aeruginosa.

OBJECTIVES: Imipenem-relebactam (IMR), a novel ß-lactam/ß-lactamase inhibitor combination, is recommended for infections caused by difficult-to-treat Pseudomonas aeruginosa. This study aimed to investigate the evolution trajectory of IMR resistance under the selection of levofloxacin in P. aeruginosa. METHODS: Antimicrobial susceptibility testing, complete genome sequencing and gene manipulation experiments were performed. Quantitative reverse transcription PCR for specific genes and porin levels were detected. Evolution trajectory was simulated in vitro by induction assay. RESULTS: P. aeruginosa HS347 and HS355 were isolated from abdominal drainage of two neighbouring patients (S and Z) undergoing surgery of colon carcinoma in Shanghai, China, with the latter patient having received levofloxacin. They were closely related ST16 strains, and both carried blaKPC-2 plasmids highly similar to those of P. aeruginosa endemic clones from Zhejiang province, where patient Z had received enteroscopy before this admission. Acquisition of resistance was observed for both IMR and fluoroquinolones in HS355, likely prompted by treatment with levofloxacin. The T274I substitution in MexS (putative oxidoreductase), upregulated efflux pump operon mexEF-oprN and decreased production of porin OprD leading to cross-resistance to fluoroquinolones and IMR, which was also verified by in vitro mutant selection under levofloxacin selection. CONCLUSIONS: The emergence of a rare blaKPC-2-plasmid-bearing ST16 clone implies the horizonal spread and inter-regional dissemination of a high-risk plasmid-clone combination, representing a public health challenge. Levofloxacin exposure can select for mexS inactivating mutation, which in turn leads to IMR resistance phenotype, implicating the role of an unrelated, widely used antimicrobial agent in insidiously triggering the development of cross resistance to a latest ß-lactam/ß-lactamase inhibitor combination.

Comparison of the Antibiotic Resistance of Escherichia coli Populations from Water and Biofilm in River Environments.

Antibiotic-resistant, facultative pathogenic bacteria are commonly found in surface water; however, the factors influencing the spread and stabilization of antibiotic resistance in this habitat, particularly the role of biofilms, are not fully understood. The extent to which bacterial populations in biofilms or sediments exacerbate the problem for specific antibiotic classes or more broadly remains unanswered. In this study, we investigated the differences between the bacterial populations found in the surface water and sediment/biofilm of the Mur River and the Drava River in Austria. Samples of Escherichia coli were collected from both the water and sediment at two locations per river: upstream and downstream of urban areas that included a sewage treatment plant. The isolates were subjected to antimicrobial susceptibility testing against 21 antibiotics belonging to seven distinct classes. Additionally, isolates exhibiting either extended-spectrum beta-lactamase (ESBL) or carbapenemase phenotypes were further analyzed for specific antimicrobial resistance genes. E. coli isolates collected from all locations exhibited resistance to at least one of the tested antibiotics; on average, isolates from the Mur and Drava rivers showed 25.85% and 23.66% resistance, respectively. The most prevalent resistance observed was to ampicillin, amoxicillin-clavulanic acid, tetracycline, and nalidixic acid. Surprisingly, there was a similar proportion of resistant bacteria observed in both open water and sediment samples. The difference in resistance levels between the samples collected upstream and downstream of the cities was minimal. Out of all 831 isolates examined, 13 were identified as carrying ESBL genes, with 1 of these isolates also containing the gene for the KPC-2 carbapenemase. There were no significant differences between the biofilm (sediment) and open water samples in the occurrence of antibiotic resistance. For the E. coli populations in the examined rivers, the different factors in water and the sediment do not appear to influence the stability of resistance. No significant differences in antimicrobial resistance were observed between the bacterial populations collected from the biofilm (sediment) and open-water samples in either river. The different factors in water and the sediment do not appear to influence the stability of resistance. The minimal differences observed upstream and downstream of the cities could indicate that the river population already exhibits generalized resistance.

Analysis of distinct blaKPC-encoding plasmids in an Enterobacter kobei strain recovered from recreational coastal water.

In this study we present an in-depth characterization of two blaKPC-2 encoding plasmids found in the Enterobacter kobei FL23 strain recovered from recreational coastal water. The plasmids belong to distinct incompatibility groups and carry a diverse collection of resistance genes. Furthermore, the genetic context of the blaKPC-2 gene was different in each of them. While pEkFL23-IncX3 presents a new Tn4401k, a new isoform, similar to Tn4401b but with a truncated tnpA and a deleted tnpR; pEkFL23-IncU/P6 carries a new isoform of a non-Tn4401 element (NTEKPC), named NTEKPC-IIh. Its difference from NTEKPC-IId is the truncated Tn3 resolvase upstream blaKPC-2. Capacity of conjugation, maintenance rates and fitness cost of both replicons were also assessed. Both were transferred after mating assays, whereas only pEkFL23-IncX3 was transferred under the adverse conditions of Marine broth at 25 °C as a mating platform. A remarkable stability of both plasmids was observed in the parental and transconjugant strains. Finally, both replicons did not impose a significant fitness cost to their transformant hosts, with pEkFL23-IncU/P6 conferring a statistically significant (p < 0.05) advantage in head-to-head competitions. Our findings show that E. kobei FL23 is a disquieting case of a carbapenem-resistant bacteria identified in a community setting, being a possible silent disseminator of two seemingly stable and metabolic weightless multidrug resistance plasmids.

ST11 KPC-2-Producing Klebsiella pneumoniae Isolated from Patient with Acute Myelocytic Leukemia.

Background: The emergence of the ST11-CRKP (ST11-CRKP) strain is expected to become a serious public health problem in China. As one of the most serious complications in patients with acute myeloid lymphoma, infections can cause systemic infection and life-threatening sepsis, seriously affecting the morbidity, mortality, and quality of life of patients. Thus, ST11-CRKP infections in patients with acute myeloid lymphoma are worthy of our attention. Aim: To investigate the occurrence and genetic characteristics of the ST11-CRKP from a patient with acute myeloid lymphoma. Methods: Species identification was determined by MALDI-TOF MS. Antimicrobial susceptibility testing (AST) was conducted by VITEK 2 system with AST-N335 panel. Whole-genome sequencing was performed on the Illumina NovaSeq 6000 platform. Phylogenetic analyses were performed using Snippy based on the core-genome SNPs. Findings: S1 nuclease pulsed-field gel electrophoresis (S1-PFGE), Southern blot and Whole-genome analysis indicated blaKPC-2 genes were located on plasmids with a conserved genetic environment. Moreover, the eight ST11-CRKP strains carry a variety of antimicrobial resistance genes (ARGs) and virulence factors. The ability of biofilm formation of eight strains was verified by a crystal violet assay. Core genome single-nucleotide polymorphism (cgSNP) analysis suggesting a possible bacterial translocation event. Conclusion: We performed a comprehensive analysis of ST11-CRKP strains from a patient with acute myelocytic leukemia. Our study emphasized the need for continuous surveillance of ST11-CRKP in the clinic especially in the immunocompromised population.

Genome sequencing unveils blaKPC-2-harboring plasmids as drivers of enhanced resistance and virulence in nosocomial Klebsiella pneumoniae.

The threat posed by Klebsiella pneumoniae in healthcare settings has worsened due to the evolutionary advantages conferred by blaKPC-2-harboring plasmids (pKPC-2). However, the specific evolutionary pathway of nosocomial K. pneumoniae carrying pKPC-2 and its transmission between patients and healthcare environments are not yet well understood. Between 1 August and 31 December 2019, 237 ST11 KPC-2-producing-carbapenem-resistant K. pneumoniae (CRKP) (KPC-2-CRKP) were collected from patient or ward environments in an intensive care unit and subjected to Illumina sequencing, of which 32 strains were additionally selected for Nanopore sequencing to obtain complete plasmid sequences. Bioinformatics analysis, conjugation experiments, antimicrobial susceptibility tests, and virulence assays were performed to identify the evolutionary characteristics of pKPC-2. The pKPC-2 plasmids were divided into three subgroups with distinct evolutionary events, including Tn3-mediated plasmid homologous recombination, IS26-mediated horizontal gene transfer, and dynamic duplications of antibiotic resistance genes (ARGs). Surprisingly, the incidence rates of multicopy blaKPC-2, blaSHV-12, and blaCTX-M-65 were quite high (ranging from 27.43% to 67.01%), and strains negative for extended-spectrum ß-lactamase tended to develop multicopy blaKPC-2. Notably, the presence of multicopy blaSHV-12 reduced sensitivity to ceftazidime/avibactam (CZA), and the relative expression level of blaSHV-12 in the CZA-resistant group was 6.12 times higher than that in the sensitive group. Furthermore, a novel hybrid pKPC-2 was identified, presenting enhanced virulence levels and decreased susceptibility to CZA. This study emphasizes the notable prevalence of multicopy ARGs and provides a comprehensive insight into the intricate and diverse evolutionary pathways of resistant plasmids that disseminate among patients and healthcare environments.IMPORTANCEThis study is based on a CRKP screening program between patients and ward environments in an intensive care unit, describing the pKPC-2 (blaKPC-2-harboring plasmids) population structure and evolutionary characteristics in clinical settings. Long-read sequencing was performed in genetically closely related strains, enabling the high-resolution analysis of evolution pathway between or within pKPC-2 subgroups. We revealed the extremely high rates of multicopy antibiotic resistance genes (ARGs) in clinical settings and its effect on resistance profile toward novel ß-lactam/ß-lactamase inhibitor combinations, which belongs to the last line treatment choices toward CRKP infection. A novel hybrid pKPC-2 carrying CRKP with enhanced resistance and virulence level was captured during its clonal spread between patients and ward environment. These evidences highlight the threat of pKPC-2 to CRKP treatment and control. Thus, surveillance and timely disinfection in clinical settings should be practiced to prevent transmission of CRKP carrying threatful pKPC-2. And rational use of antibiotics should be called for to prevent inducing of pKPC-2 evolution, especially the multicopy ARGs.

Emergence of KPC-2 and NDM-5-coproducing hypervirulent carbapenem-resistant Klebsiella pneumoniae with high-risk sequence types ST11 and ST15.

The emergence of Klebsiella pneumoniae carbapenemase-2 (KPC-2) and New Delhi metallo-ß-lactamase (NDM)-coproducing hypervirulent carbapenem-resistant Klebsiella pneumoniae (KPC-2-NDM-hv-CRKP) poses a certain threat to public health. Currently, only a few sporadic reports of such double-positive hv-CRKPs were available. In this study, we isolated two KPC-2-NDM-5-hv-CRKPs from elderly patients with serious underlying diseases and poor prognoses. We found both FK3122 and FK3127 were typical multidrug-resistant (MDR) isolates, exhibiting high-level resistance to both carbapenems and novel ß-lactamase inhibitors ceftazidime/avibactam. Notably, FK3122 is even resistant to cefiderocol due to multiple blaNDM-5 elements. Besides the MDR phenotype, A549 human lung epithelial cells and Galleria mellonella infection model all indicated that FK3122 and FK3127 were highly pathogenic. According to the whole-genome sequencing analysis, we observed over 10 resistant elements, and the uncommon co-existence of blaKPC-2, blaNDM-5, and virulence plasmids in both two isolates. Both virulence plasmids identified in FK3122 and FK3127 shared a high identity with classical virulence plasmid pK2044, harboring specific hypervirulent factors: rmpA and iuc operon. We also found that the resistance and virulence plasmids in FK3127 could not only be transferred to Escherichia coli EC600 independently but also together as a co-transfer, which was additionally confirmed by the S1-pulsed-field gel electrophoresis plasmid profile. Moreover, polymorphic mobile genetic elements were found surrounding resistance genes, which may stimulate the mobilization of resistance genes and result in the duplication of these elements. Considering the combination of high pathogenicity, limited therapy options, and easy transmission of KPC-2-NDM-5-hv-CRKP, our study emphasizes the need for underscores the imperative for ongoing surveillance of these pathogens.IMPORTANCEHypervirulent Klebsiella pneumoniae drug resistance has increased gradually with the emergence of carbapenem-resistant hypervirulent K. pneumoniae (hv-CRKP). However, little information is available on the virulence characteristics of the New Delhi metallo-ß-lactamase (NDM) and Klebsiella pneumoniae carbapenemase-2 (KPC-2) co-producing K. pneumoniae strains. In this study, we obtained two KPC-2-NDM-hv-CRKPs from elderly patients, each with distinct capsule types and sequence types: ST11-KL64 and ST15-KL24; these ST-type lineages are recognized as classical multidrug-resistant (MDR) K. pneumoniae. We found these KPC-2-NDM-hv-CRKPs were not only typical MDR isolates, including resistance to ceftazidime/avibactam and cefiderocol, but also displayed exceptionally high levels of pathogenicity. In addition, these high-risk factors can also be transferred to other isolates. Consequently, our study underscores the need for ongoing surveillance of these isolates due to their heightened pathogenicity, limited therapeutic options, and potential for easy transmission.

Clinical and molecular characteristics of Klebsiella pneumoniae infection in a tertiary general hospital of Wuhan, China.

OBJECTIVES: The aim of this study was to investigate the clinical and molecular characteristics of Klebsiella pneumoniae infection from a tertiary general hospital in Wuhan, China. METHODS: From December 2019 to August 2022, 311 non-duplicate isolates of K. pneumoniae were collected from a tertiary hospital in Wuhan. These comprised 140 carbapenem-resistant K. pneumoniae (CRKP) isolates and 171 carbapenem-susceptible K. pneumoniae (CSKP) isolates. The clinical characteristics of patients with K. pneumoniae infection were retrospectively collected. Polymerase chain reaction (PCR) assays were used to identify the main carbapenem resistance genes, virulence genes and multi-locus sequence typing (MLST) profiles of the isolates, and the Galleria mellonella infection model was used to determine their virulence phenotypes. RESULTS: Independent risk factors for CRKP infection were hypertension, neurological disorders, being admitted to the intensive care unit (ICU) and prior use of antibiotics. Patient with CRKP infection had higher mortality than those with CSKP infection (23.6% vs 14.0%, P < 0.05). One hundred and two sequence types (STs) were identified among the K. pneumoniae isolates, and the most prevalent ST type was ST11 (112/311, 36.0%). All of the ST11 isolates were CRKP. Among the 112 ST11 isolates, 105 (93.8%) harboured the carbapenem resistance gene blaKPC-2 (ST11-KPC-2), and of these isolates, 78 (74.3%, 78/105) contained all of the four virulence genes, namely rmpA, rmpA2, iroN and iucA, suggesting that these genes were widespread among the isolates responsible for K. pneumoniae infections. CONCLUSION: In this study, ST11-KPC-2 was responsible for most of the K. pneumoniae infection cases. Carbapenem resistance rather than the co-occurrence of the virulence genes rmpA, rmpA2, iroN and iucA was associated with K. pneumoniae infection-related mortality during hospitalisation. Furthermore, a high proportion of ST11-KPC-2 isolates carried all of the four virulence genes.

Predominant transmission of KPC-2 carbapenemase in Germany by a unique IncN plasmid variant harboring a novel non-transposable element (NTE KPC -Y).

IMPORTANCE: Current infection control protocols assume that the spread of KPC-2 carbapenemase-producing Enterobacterales (KPC2-CPE) by detected carriers to other in-house patients is through clonal transmission and can be restricted by implementing containment measures. We examined the presence of the bla KPC-2 gene in different genera and species of Enterobacterales isolated from humans at different hospitals and surface waters between 2013 and 2019 in Germany. We found that a single IncN[pMLST15] plasmid carrying the bla KPC-2 gene on a novel non-Tn4401-element (NTEKPC-Y), flanked by an adjacent region encoding 12 other antibiotic resistance genes, was uniquely present in multiple species of KPC2-CPE isolates. These findings demonstrate the selective impact of specific IncN plasmids as major drivers of carbapenemase dissemination and suggest "plasmid-based endemicity" for KPC2-CPE. Studies on the dynamics of plasmid-based KPC2-CPE transmission and its presence in persistent reservoirs need to be urgently considered to implement effective surveillance and prevention measures in healthcare institutions.

Emergency of the plasmid co-carrying blaKPC-2 and blaNDM-1 genes in carbapenem-resistant hypervirulent Klebsiella pneumoniae.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a prevalent issue in China, with its spread primarily attributed to the presence of the plasmid-borne carbapenemase genes, blaKPC and blaNDM. However, instances of plasmids containing both blaKPC-2 and blaNDM-1have never been reported. METHODS: In this study, the genomic and microbiological characteristics of hybrid plasmids containing both blaKPC-2 and blaNDM-1 were identified in Chinese clinical CRKP isolates by Illumina combined with ONT nanopore sequencing technology. RESULTS: The newly identified plasmid was formed via IS26-mediated recombination and has been shown to be transferable to Escherichia coli. It substantially elevates the minimum inhibitory concentration (MIC) of meropenem by 4000-fold in E. coli, surpassing the MIC values observed in E. coli strains that carry either blaKPC-2 and blaNDM-1 alone, as previously demonstrated in our study. Notably, the co-occurrence of the KPC-NDM fusion plasmid and a pLVPK-like virulence plasmid was observed in these organisms. In vivo experiments revealed that the isolates harbouring the pLVPK-like virulence plasmid exhibited a significantly higher lethality rate in Galleria mellonella. CONCLUSIONS: The increased antibiotic resistance brought by this novel fusion plasmid and its accompanying virulence factors pose a serious potential threat to human health and deserve our vigilance.

Prevalence of carbapenemases among Gram-negative bacteria in Tunisia: first report of KPC-2 producing Acinetobacter baumannii.

INTRODUCTION: The rapid evolution of the antibacterial resistance problem worldwide, including the Mediterranean countries, constitutes a real threat to public health. This study aims to characterize carbapenemase encoding genes among Gram-negative bacteria collected from some Tunisian hospitals. METHODOLOGY: Twenty-two clinical carbapenem-resistant Gram-negative bacteria were recovered, and identified by the matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) method. Antibiotic resistance was tested by disk diffusion method on Muller-Hinton Agar. The minimum inhibitory concentration (MIC) for imipenem was revealed by the E-test method. Carbapenemase encoding genes were screened by polymerase chain reaction (PCR). Genetic relatedness was performed by the pulsed field gel electrophoresis (PFGE) method. RESULTS: Our isolates, identified as K. pneumoniae (n = 7), P. mirabilis (n = 1), A. baumannii (n = 13), and P. aeruginosa (n = 1), presented high MIC values for imipenem. Enterobacerales were resistant to carbapenems due to OXA-48 production. Only, four K. pneumoniae harbored the blaNDM-1 gene. VIM-2 production was detected in P. aeruginosa. However, OXA-23 production was observed in A. baumannii isolates, one of which co-produced the KPC-2 enzyme that was identified for the first time in Tunisia in this species. A high genetic diversity was demonstrated by pulsed-field gel electrophoresis in K. pneumoniae and A. baumannii after XbaI and ApaI digestion respectively. CONCLUSIONS: Our findings highlight the spread of various unrelated clones of carbapenemase-producers in some Tunisian hospitals as well as the spread of several carbapenemase types.

Prevalence of intestinal colonization and nosocomial infection with carbapenem-resistant Enterobacteriales in children: a retrospective study.

Objective: We investigated the epidemiological surveillance of the intestinal colonization and nosocomial infection of carbapenem-resistant Enterobacteriales (CRE) isolates from inpatients, which can provide the basis for developing effective prevention. Methods: A total of 96 CRE strains were collected from 1,487 fecal samples of hospitalized children between January 2016 and June 2017, which were defined as the "CRE colonization" group. In total, 70 CRE clinical isolates were also randomly selected for the comparison analysis and defined as the "CRE infection" group. The antimicrobial susceptibility of all strains was determined by the microdilution broth method. Polymerase chain reaction (PCR) was used to analyze carbapenemase genes, plasmid typing, and integrons. Multilocus sequence typing was further used to determine clonal relatedness. Results: In the "CRE colonization" group, Klebsiella pneumoniae was mostly detected with a rate of 42.7% (41/96), followed by Escherichia coli (34.4%, 33/96) and Enterobacter cloacae (15.6%, 15/96). The ST11 KPC-2 producer, ST8 NDM-5 producer, and ST45 NDM-1 producer were commonly present in carbapenem-resistant K. pneumoniae (CRKPN), carbapenem-resistant E. coli (CRECO), and carbapenem-resistant E. cloacae (CRECL) isolates, respectively. In the "CRE infection" group, 70% (49/70) of strains were K. pneumoniae, with 21.4% E. cloacae (15/70) and 5.7% E. coli (4/70). The ST15 OXA-232 producer and ST48 NDM-5 producer were frequently observed in CRKPN isolates, while the majority of NDM-1-producing CRECL isolates were assigned as ST45. Phylogenetic analysis showed that partial CRE isolates from intestinal colonization and nosocomial infection were closely related, especially for ST11 KPC-2-producing CRKPN and ST45 NDM-1-producing CRECL. Furthermore, plasmid typing demonstrated that IncF and IncFIB were the most prevalent plasmids in KPC-2 producers, while IncX3/IncX2 and ColE were widely spread in NDM producer and OXA-232 producer, respectively. Then, class 1 integron intergrase intI1 was positive in 74.0% (71/96) of the "CRE colonization" group and 52.9% (37/70) of the "CRE infection" group. Conclusion: This study revealed that CRE strains from intestinal colonization and nosocomial infection showed a partial correlation in the prevalence of CRE, especially for ST11 KPC-2-producing CRKPN and ST45 NDM-1-producing CRECL. Therefore, before admission, long-term active screening of rectal colonization of CRE isolates should be emphasized.

In vitro activity of cefiderocol, a siderophore cephalosporin, against carbapenem-resistant hypervirulent Klebsiella pneumoniae in China.

Cefiderocol is a siderophore cephalosporin that binds ferric iron and utilizes iron transporters to cross the cell membrane. Hypervirulent Klebsiella pneumoniae (hvKp) is known to produce more siderophores; in this case, the uptake of cefiderocol may be decreased. Therefore, the objective of this study was to evaluate the in vitro activity of cefiderocol against hvKp isolates. A total of 320 carbapenem-resistant K. pneumoniae (CRKp) isolates were collected in China between 2014 and 2022, including 171 carbapenem-resistant hvKp (CR-hvKp) and 149 carbapenem-resistant classical K. pneumoniae (CR-cKp). Quantitative detection of siderophores showed that the average siderophore production of CR-hvKp (234.6 mg/L) was significantly higher than that of CR-cKp (68.9 mg/L, P < 0.001). The overall cefiderocol resistance rate of CR-hvKp and CR-cKp was 5.8% (10/171) and 2.7% (4/149), respectively. The non-susceptible rates of both cefiderocol and siderophore production of CR-hvKp isolates were higher than those of CR-cKp in either NDM-1- or KPC-2-producing groups. The MIC90 and MIC50 for CR-hvKp and CR-cKp were 8 mg/L and 2 mg/L and 4 mg/L and 1 mg/L, respectively. The cumulative cefiderocol MIC distribution for CR-hvKp was significantly lower than that of CR-cKp isolates (P = 0.003). KL64 and KL47 consisted of 53.9% (83/154) and 75.7% (53/70) of the ST11 CR-hvKp and CR-cKp, respectively, and the former had significantly higher siderophore production. In summary, cefiderocol might be less effective against CR-hvKp compared with CR-cKp isolates, highlighting the need for caution regarding the prevalence of cefiderocol-resistant K. pneumoniae strains, particularly in CR-hvKp isolates.

Genomics analysis of KPC-2 and NDM-5-producing Enterobacteriaceae in migratory birds from Qinghai Lake, China.

The study examined the epidemiological characteristics of carbapenem-resistant Enterobacteriaceae (CRE) isolated from migratory birds and surroundings in Qinghai Lake, China. We identified 69 (15.7%) CRE isolates from a total of 439 samples including 29 (6.6%) blaNDM-5 Escherichia coli and 40 (9.1%) blaKPC-2 Klebsiella pneumoniae. WGS analysis indicated that ST746, ST48, ST1011, and ST167 were the primary sequence types (ST) for blaNDM-5 E. coli, while all blaKPC-2 K. pneumoniae were ST11 and harbored numerous antibiotic resistance gene types including blaCTX-M, qnrS, and rmtB. A phylogenetic tree based on core genomes revealed that blaNDM-5 E. coli was highly heterogeneous while the blaKPC-2 K. pneumoniae was highly genetically similar within the group and to human Chinese isolates. IncX3, IncHI2, and IncFIB-HI2 plasmid replicon types were associated with blaNDM-5 spread, while IncFII-R and IncFII plasmids mediated blaKPC-2 spread. We also identified IncFII-R hybrid plasmids most likely formed by IS26-mediated integration of IncFII into IncR plasmid backbones. This also facilitated the persistence of IncFII-R plasmids and antibiotic resistance genes including blaKPC-2. In addition, all of the blaKPC-2 K. pneumoniae isolates harbored a pLVKP-like virulence plasmid carrying a combination of two or more hypervirulence markers that included peg-344, iroB, iucA, rmpA, and rmpA2. This is the first description of ST11 K. pneumoniae that co-carried blaKPC-2- and pLVKP-like virulence plasmids from migratory birds. The blaKPC-2 K. pneumoniae carried by migratory birds displayed high genetic relatedness to human isolates highlighting a high risk of transmission of these K. pneumoniae. KEY POINTS: • Multidrug resistance plasmids (blaKPC-2, bla436NDM-5, bla CTX-M, qnrS, and rmtB). • Co-occurrence of plasmid-mediated resistance and virulence genes. • High similarity between migratory bird genomes and humans.

Shift in the dominant sequence type of carbapenem-resistant Klebsiella pneumonia infection from ST278-NDM-1 to ST11-KPC-2 in neonatal patients in a children's hospital in Shanghai, China, 2017-2021.

OBJECTIVES: To investigate the clinical characteristics and molecular epidemiology of CRKP infection in neonatal patients in a children's hospital in China from 2017 to 2021. METHODS: Species identification and antibiotic susceptibilities were tested with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and VITEK 2 systems. The clinical data were collected from medical records. Carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates were investigated by antimicrobial susceptibility testing, carbapenemase genes and multilocus sequence typing. RESULTS: Six kinds of resistant genes and 23 STs were detected. BlaNDM-1 (n=83, 55.3%) was the predominant carbapenemase gene, followed by blaKPC-2 (n=45, 30.0%), blaNDM-5 (n=7, 4.7%), blaIMP-38 (n=6, 4.0%). BlaNDM-1 was predominant in 2017 and 2018, whereas blaKPC-2 increased in 2019 and became the predominant gene from 2020 to 2021. ST11 accounted for most infections (n=35, 23.3%), followed by ST278 (n=23, 15.3%), ST17 (n=17, 11. 3%) and ST2735 (n=16, 10.7%). ST278 and ST17 were predominant in 2017 and 2018, whereas ST11 increased in 2019 and became the predominant sequence type from 2020 to 2021. Compared with blaNDM-1, the CRKP strains producing blaKPC-2 were characterized by high resistance to gentamicin, amikacin and levofloxacin and the change trend of drug resistance rate before and after COVID-19 was consistent with that of blaNDM-1 and blaKPC-2. CONCLUSIONS: The main sequence type of CRKP infection changed dynamically from ST278-NDM-1 to ST11-KPC-2 during the years 2017-2021 in the newborns. Antibiotic exposure and the prevalence of COVID-19 since 2020 may have led to changes in hospital population and lead to the changes.

bla KPC-2 overexpression and bla GES-5 carriage as major imipenem/relebactam resistance mechanisms in Pseudomonas aeruginosa high-risk clones ST463 and ST235, respectively, in China.

Pseudomonas aeruginosa high-risk clones pose severe threats to public health. Here, we characterize the imipenem/relebactam (IR) resistance mechanisms in P. aeruginosa high-risk clones sequence type 235 (ST235) and ST463 in China. Minimum inhibitory concentrations (MICs) were determined, and Illumina short-read sequencing was performed for 1,168 clinical carbapenem-resistant P. aeruginosa (CRPA) isolates. The gene copy number and expression level were analyzed by Illumina sequencing depth and reverse transcription-quantitative PCR, respectively. Resistance conferred by bla GES-5 was evaluated by cloning experiments. ST463 and ST235 accounted for 9.8% (115/1,168) and 4.5% (53/1,168) of total isolates, respectively, and showed high frequencies of extensively drug-resistant and difficult-to-treat resistant phenotypes. The overall IR-resistant rate in CRPA was 21.0% (245/1,168). However, the IR resistance rate was 81.7% (94/115) in ST463-PA and 52.8% (28/53) in ST235-PA. Of the ST463 isolates, 92.2% (106/115) were Klebsiella pneumoniae carbapenemase-producing P. aeruginosa (KPC-PA), and all 94 IR-resistant ST463-PA produced KPC-2. Compared to IR-susceptible ST463 KPC-2-PA, IR-resistant ST463 KPC-2-PA exhibited significantly higher bla KPC-2 copy numbers and expression levels. In ST463 KPC-2-PA, 16 mg/L relebactam resulted in additional fourfold reductions in imipenem MIC50/90 values compared to 4 mg/L relebactam. In ST235, 1.9% (1/53) carried bla IMP carbapenemase and 54.7% (29/53) carried bla GES carbapenemase. Other than the IMP producer, all 27 IR-resistant ST235-PA produced GES-5. Cloning experiments revealed that imipenem resistance in bla GES-5-carrying PAO1 transformants was generally unaffected by relebactam. In conclusion, IR-resistant CRPA isolates in China were mainly distributed in P. aeruginosa high-risk clones ST463 and ST235. The major underlying IR resistance mechanisms were bla KPC-2 overexpression and bla GES-5 carriage.

Carbapenem-resistant Citrobacter freundii harboring blaKPC-2 and blaNDM-1: a study on their transferability and potential dissemination via generating a transferrable hybrid plasmid mediated by IS6100.

Introduction: The increase in clinical Enterobacteriaceae with dual carbapenemase has become a serious healthcare concern. It is essential to characterize the transferability and potential dissemination of blaKPC-2- and blaNDM-1-coharboring carbapenem-resistant Citrobacter freundii (CRCF). Methods: Four blaKPC-2- and blaNDM-1-coharboring CRCF strains were collected from our surveillance of the prevalence of carbapenem-resistant Enterobacteriaceae. The isolates were assessed using species identification, antimicrobial susceptibility testing, conjugation assays, whole-genome sequencing, plasmid stability, and fitness costs. Clonality, genome, plasmidome, and phylogeny were analyzed to reveal potential dissemination. Results: Three ST523 blaKPC-2- and blaNDM-1-coharboring CRCF strains, collected from the same hospital within 1 month, exhibited high homology (both identity and coverage >99%), implying clonal dissemination and a small-scale outbreak. Moreover, the blaKPC-2 and blaNDM-1 genes were coharbored on an IncR plasmid, probably generated by a blaKPC-2-harboring plasmid acquiring blaNDM-1, in these three strains. Importantly, the IncR plasmid may form a transferable hybrid plasmid, mediated by IS6100 via transposition, with another IncFII plasmid included in the same C. freundii strain. Furthermore, the blaKPC-2 and blaNDM-1 of the fourth CRCF strain are located on two different non-transferable plasmids lacking complete transfer elements. Additionally, throughout the course of the 10-day continuous passage, the genetic surroundings of blaNDM-1 in four CRCF strains were gradually excised from their plasmids after the 8th day, whereas they maintained 100% retention for blaKPC-2. Genome and plasmidome analyses revealed that blaKPC-2- or blaNDM-1-harboring C. freundii were divergent, and these plasmids have high homology to plasmids of other Enterobacteriaceae. Conclusion: Clonal dissemination of ST523 blaKPC-2- and blaNDM-1-coharboring CRCF strains was detected, and we first reported blaKPC-2 and blaNDM-1 concomitantly located on one plasmid, which could be transferred with mediation by IS6100 via transposition. Continued surveillance should urgently be implemented.