Epidemiological and clinical characterization of community, healthcare-associated and nosocomial colonization and infection due to carbapenemase-producing Klebsiella pneumoniae and Escherichia coli in Spain.

BACKGROUND: Community-acquired (CA) and healthcare-associated (HCA) infections caused by carbapenemase-producing Enterobacterales (CPE) are not well characterized. The objective was to provide detailed information about the clinical and molecular epidemiological features of nosocomial, HCA and CA infections caused by carbapenemase-producing Klebsiella pneumoniae (CP-Kp) and Escherichia coli (CP-Ec). METHODS: A prospective cohort study was performed in 59 Spanish hospitals from February to March 2019, including the first 10 consecutive patients from whom CP-Kp or CP-Ec were isolated. Patients were stratified according to acquisition type. A multivariate analysis was performed to identify the impact of acquisition type in 30-day mortality. RESULTS: Overall, 386 patients were included (363 [94%] with CP-Kp and 23 [6%] CP-Ec); in 296 patients (76.3%), the CPE was causing an infection. Acquisition was CA in 31 (8.0%) patients, HCA in 183 (47.4%) and nosocomial in 172 (48.3%). Among patients with a HCA acquisition, 100 (54.6%) had been previously admitted to hospital and 71 (38.8%) were nursing home residents. Urinary tract infections accounted for 19/23 (82.6%), 89/130 (68.5%) and 42/143 (29.4%) of CA, HCA and nosocomial infections, respectively. Overall, 68 infections (23%) were bacteremia (8.7%, 17.7% and 30.1% of CA, HCA and nosocomial, respectively). Mortality in infections was 28% (13%, 14.6% and 42.7% of CA, HCA and nosocomial, respectively). Nosocomial bloodstream infections were associated with increased odds for mortality (adjusted OR, 4.00; 95%CI 1.21-13.19). CONCLUSIONS: HCA and CA infections caused by CPE are frequent and clinically significant. This information may be useful for a better understanding of the epidemiology of CPE.

Carbapenemase-producing Enterobacterales strains causing infections in companion animals-Portugal.

An increase in Klebsiella pneumoniae carbapenem-resistant human nosocomial strains is occurring in Europe, namely with the blaOXA-48-like and blaKPC-like genes. We determined the prevalence of carbapenemase-producing Enterobacterales clinical strains in companion animals in Portugal and characterized their mobile genetic elements. Susceptibility data of a consecutive collection of 977 Enterobacterales clinical strains from a Portuguese private veterinary diagnostic laboratory were evaluated (January-December 2020). Additional phenotypical and genotypical assays were performed in a subset of 261 strains with a resistant phenotype. Whole-genome sequencing was performed for carbapenemase-producing strains. The frequency of carbapenemase-producing Enterobacterales clinical strains in companion animals in Portugal was 0.51% (n = 5/977). Thus, five strains were characterized: (i) one OXA-181-producing K. pneumoniae ST273, (ii) two KPC-3-producing K. pneumoniae ST147; (iii) one KPC-3-producing K. pneumoniae ST392; and (iv) one OXA-48-producing E. coli ST127. The blaKPC-3 gene was located on transposon Tn4401d on IncFIA type plasmid for the K. pneumoniae ST147 strains and on a IncN-type plasmid for the K. pneumoniae ST392 strain, while blaOXA-181 gene was located on an IncX3 plasmid. All de novo assembled plasmids and plasmid-encoded transposons harboring carbapenemase genes were homologous to those previously described in the human healthcare. No plasmid replicons were detected on the OXA-48-producing E. coli ST127. The dissemination of carbapenem resistance is occurring horizontally via plasmid spreading from the human high burden carbapenem resistance setting to the companion animal sector. Furthermore, companion animals may act as reservoirs of carbapenem resistance. Implementation of carbapenemase detection methods in routine clinical veterinary microbiology is urgently needed. IMPORTANCE: This is the first study on the prevalence of carbapenemase-producing Enterobacterales (CPE) clinical strains from companion animals in Portugal. Despite the generally low prevalence of CPE in companion animals, it is imperative for veterinary diagnostic laboratories to employ diagnostic methods for carbapenemase detection. The resemblance found in the mobile genetic elements transporting carbapenemase genes between veterinary medicine and human medicine implies a potential circulation within a One Health framework.

Genotypic Evolution of Klebsiella pneumoniae Sequence Type 512 during Ceftazidime/Avibactam, Meropenem/Vaborbactam, and Cefiderocol Treatment, Italy.

In February 2022, a critically ill patient colonized with a carbapenem-resistant K. pneumoniae producing KPC-3 and VIM-1 carbapenemases was hospitalized for SARS-CoV-2 in the intensive care unit of Policlinico Umberto I hospital in Rome, Italy. During 95 days of hospitalization, ceftazidime/avibactam, meropenem/vaborbactam, and cefiderocol were administered consecutively to treat 3 respiratory tract infections sustained by different bacterial agents. Those therapies altered the resistome of K. pneumoniae sequence type 512 colonizing or infecting the patient during the hospitalization period. In vivo evolution of the K. pneumoniae sequence type 512 resistome occurred through plasmid loss, outer membrane porin alteration, and a nonsense mutation in the cirA siderophore gene, resulting in high levels of cefiderocol resistance. Cross-selection can occur between K. pneumoniae and treatments prescribed for other infective agents. K. pneumoniae can stably colonize a patient, and antimicrobial-selective pressure can promote progressive K. pneumoniae resistome evolution, indicating a substantial public health threat.

Continuous infusion of meropenem-vaborbactam for a KPC-3-producing Klebsiella pneumoniae bloodstream infection in a critically ill patient with augmented renal clearance.

PURPOSE: To demonstrate the feasibility of continuous infusion of meropenem-vaborbactam to optimize the treatment of carbapenem-resistant Enterobacterales. METHODS: Report of a case of a Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae bloodstream infection comfirmed by whole genome sequencing and therapeutic drug monitoring (TDM) of meropenem. RESULTS: A patient with augmented renal clearance (ARC) went into septic shock caused by an ST11 KPC-3-producing K. pneumoniae bloodstream infection that was successfully treated with a continuous infusion of meropenem-vaborbactam at a dosage of 1 g/1 g q4h as a 4-h infusion. TDM confirmed sustained concentrations of meropenem ranging from 8 to 16 mg/L throughout the dosing interval. CONCLUSION: Continuous infusion of meropenem-vaborbactam was feasible. It could be appropriate for optimizing the management of critically ill patients with ARC, as it resulted in antibiotic concentrations above the minimum inhibitory concentration for susceptible carbapenem-resistant Enterobacterales (up to 8 mg/L) throughout the dosing interval.

Emergence and clonal dissemination of KPC-3-producing Pseudomonas aeruginosa in China with an IncP-2 megaplasmid.

BACKGROUND: Despite the global prevalence of Klebsiella pneumoniae Carbapenemase (KPC)-type class A ß-lactamases, occurrences of KPC-3-producing isolates in China remain infrequent. This study aims to explore the emergence, antibiotic resistance profiles, and plasmid characteristics of blaKPC-3-carrying Pseudomonas aeruginosa. METHODS: Species identification was performed by MALDI-TOF-MS, and antimicrobial resistance genes (ARGs) were identified by polymerase chain reaction (PCR). The characteristics of the target strain were detected by whole-genome sequencing (WGS) and antimicrobial susceptibility testing (AST). Plasmids were analyzed by S1-nuclease pulsed-field gel electrophoresis(S1-PFGE), Southern blotting and transconjugation experiment. RESULTS: Five P. aeruginosa strains carrying blaKPC-3 were isolated from two Chinese patients without a history of travelling to endemic areas. All strains belonged to the novel sequence type ST1076. The blaKPC-3 was carried on a 395-kb IncP-2 megaplasmid with a conserved structure (IS6100-ISKpn27-blaKPC-3-ISKpn6-korC-klcA), and this genetic sequence was identical to many plasmid-encoded KPC of Pseudomonas species. By further analyzing the genetic context, it was supposed that the original of blaKPC-3 in our work was a series of mutation of blaKPC-2. CONCLUSIONS: The emergence of a multidrug resistance IncP-2 megaplasmid and clonal transmission of blaKPC-3-producing P. aeruginosa in China underlined the crucial need for continuous monitoring of blaKPC-3 for prevention and control of its further dissemination in China.

Detection of carbapenemase- and ESBL-producing Klebsiella pneumoniae from bovine bulk milk and comparison with clinical human isolates in Italy.

Klebsiella pneumoniae is the most common Klebsiella species infecting animals and is one of the causing agents of mastitis in cows. The rise of antimicrobial resistance in K. pneumoniae, particularly in strains producing extended-spectrum ß-lactamases (ESBLs) and/or carbapenemases, is of concern worldwide. Recently (Regulation UE No 2022/1255), carbapenems and cephalosporins in combination with ß-lactamase inhibitors have been reserved only to human treatments in the European Union. The aim of this study was to investigate the role of cattle as carrier of human pathogenic carbapenem-resistant (CR) and ESBL-producing K. pneumoniae. On this purpose, a study involving 150 dairy farms in Parma province (Northern Italy) and 14 non replicate K. pneumoniae isolates from patients admitted at Parma University-Hospital was planned. Four multidrug resistant (MDR) K. pneumoniae strains were detected from 258 milk filters collected between 2019 and 2021. One carbapenemase KPC-3-positive K. pneumoniae ST307 (0.4 %; 95 % CI - 0.07 - 2.2) was detected in milk filters. The isolate also harboured OXA-9, CTX-M-15 and SHV-106 determinants, together with genes conferring resistance to aminoglycosides (aac(3')-IIa, aph (3″)-Ib, aph (6)-Id), fluoroquinolones (oqxA, oqxB, qnrB1), phosphonic acids (fosA6), sulphonamides (sul2), tetracyclines (tet(A)6) and trimethoprim (dfrA14). One KPC-3-producing K. pneumoniae ST307 was identified also among the human isolates, thus suggesting a possible circulation of pathogens out of the clinical settings. The remaining three bovine isolates were MDR ESBL-producing K. pneumoniae characterized by different genomic profiles: CTX-M-15, TEM-1B and SHV-187 genes (ST513); CTX-M-15 and SHV-145 (ST307); SHV-187 and DHA-1 (ST307). Occurrence of ESBL-producing K. pneumoniae in milk filters was 1.2 % (95 % CI 0.4-3.4). All the isolates showed resistance to aminoglycosides, 3rd-generation cephalosporins, and fluoroquinolones. Among the human isolates, two multidrug resistant ESBL-producing K. pneumoniae ST307 were found, thus confirming the circulation of this high-risk lineage between humans and cattle. Our findings suggest that food-producing animals can carry human pathogenic microorganisms harboring resistance genes against carbapenems and 3rd-generation cephalosporins, even if not treated with such antimicrobials. Moreover, on the MDR K. pneumoniae farms, the antimicrobial use was much higher than the Italian median value, thus highlighting the importance of a more prudent use of antibiotics in animal productions.

The Emerging Nosocomial Pathogen Klebsiella michiganensis: Genetic Analysis of a KPC-3 Producing Strain Isolated from Venus Clam.

The recovery and characterization of a multidrug-resistant, KPC-3-producing Klebsiella michiganensis that was obtained from Venus clam samples is reported in this study. A whole-genome sequencing (WGS) analysis using Illumina and Nanopore technologies of the K. michiganensis 23999A2 isolate revealed that the strain belonged to the new sequence type 382 (ST382) and carried seven plasmid replicon sequences, including four IncF type plasmids (FII, FIIY, FIIk, and FIB), one IncHI1 plasmid, and two Col plasmids. The FIB and FIIk plasmids showed high homology to each other and to multireplicon pKpQIL-like plasmids that are found in epidemic KPC-K. pneumoniae clones worldwide. The strain carried multiple ß-lactamase genes on the IncF plasmids: blaOXA-9 and blaTEM-1A on FIB, blaKPC-3 inserted in a Tn4401a on FIIK, and blaSHV-12 on FIIY. The IncHI1-ST11 harbored no resistance gene. The curing of the strain caused the loss of all of the bla genes and a rearrangement of the IncF plasmids. Conjugal transfer of the blaOXA-9, blaTEM-1A and blaKPC-3 genes occurred at a frequency of 5 × 10-7, using K. quasipneumoniae as a recipient, and all of the bla genes were transferred through a pKpQIL that originated from the recombination of the FIB and FIIk plasmids of the donor. A comparison with 31 K. michiganensis genomes that are available in the NCBI database showed that the closest phylogenetic relatives of K. michiganensis 23999A2 are an environmental isolate from soil in South Korea and a clinical isolate from human sputum in Japan. Finally, a pan-genome analysis showed a large accessory genome of the strain as well as the great genomic plasticity of the K. michiganensis species. IMPORTANCE Klebsiella michiganensis is an emerging nosocomial pathogen, and, so far, few studies describe isolates of clinical origin in the environment. This study contributes to the understanding of how the dissemination of carbapenem-resistance outside the hospital setting may be related to the circulation of pKpQIL-like plasmids that are derived from epidemic Klebsiella pneumoniae strains. The recovery of a carbapenem-resistant isolate in clams is of great concern, as bivalves could represent vehicles of transmission of pathogens and resistance genes to humans via the food chain. The study demonstrates the plasticity of K. michiganensis genome, which is probably useful to multiple environment adaptation and to the evolution of the species.

Emergence and clonal expansión of Klebsiella pneumoniae ST307, simultaneously producing KPC-3 and NDM-1

Abstract MDR Klebsiella pneumoniae ST307 is a high-risk clone, whose genetic features contribute to its adaptation to hospital environments and the human host. This study describesthe emergence and clonal dissemination of K. pneumoniae ST307, recovered during November2018 to February 2019 in a hospital in Buenos Aires city, which concurrently harbored KPC-3and NDM-1. These isolates were resistant to all -lactams and to the ceftazidime/avibactamcombination. Molecular studies showed that blaKPC-3was located in Tn4401a platform, whileblaNDM-1was surrounded upstream by ISKpn14 followed by a partial sequence of ISAba125 anddownstream by bleMBL-trpF, located in a 145.5 kb conjugative plasmid belonging to the Inc A/Cgroup. The dissemination of K. pneumoniae ST307 isolates co-producing KPC-3 and NDM-1 couldlead to a worrisome scenario due to the remarkable features of this clone and its resistanceprofile.

Resumen Klebsiella pneumoniae ST307 es un clon de alto riesgo, cuyas características genéticas contribuyen a su adaptación al entorno hospitalario y al huésped humano. Este estudio describe la emergencia y diseminación clonal de aislamientos de K. pneumoniae ST307 productores de KPC-3 y NDM-1, recuperados en un hospital de Buenos Aires. Estos aislamientos fueron resistentes a todos los p-lactámicos y a la combinación ceftacidima/avibactam. Los estudios moleculares evidenciaron que el contexto genético de blaKPC-3 se correspondió con el Tn4401a, mientras que blaNDM-1 estuvo flanqueado corriente arriba por ISKpn14 y una secuencia parcial de ISAba125 y corriente abajo por bleMBL - trpF, localizado a su vez en un plásmido conjugativo de 145.5 kb perteneciente al grupo Inc A/C. La emergencia de aislamientos de K. pneumoniae ST307 coproductores de KPC-3 y NDM-1 pone de manifiesto una situación altamente preocupante debido a las características de este clon y a su perfil de multirresistencia.

Klebsiella pneumoniae Carbapenemase Variants Resistant to Ceftazidime-Avibactam: an Evolutionary Overview.

First variants of the Klebsiella pneumoniae carbapenemase (KPC), KPC-2 and KPC-3, have encountered a worldwide success, particularly in K. pneumoniae isolates. These beta-lactamases conferred resistance to most beta-lactams including carbapenems but remained susceptible to new beta-lactam/beta-lactamase inhibitors, such as ceftazidime-avibactam. After the marketing of ceftazidime-avibactam, numerous variants of KPC resistant to this association have been described among isolates recovered from clinical samples or derived from experimental studies. In KPC variants resistant to ceftazidime-avibactam, point mutations, insertions and/or deletions have been described in various hot spots. Deciphering the impact of these mutations is crucial, not only from a therapeutic point of view, but also to follow the evolution in time and space of KPC variants resistant to ceftazidime-avibactam. In this review, we describe the mutational landscape of the KPC beta-lactamase toward ceftazidime-avibactam resistance based on a multidisciplinary approach including epidemiology, microbiology, enzymology, and thermodynamics. We show that resistance is associated with three hot spots, with a high representation of insertions and deletions compared with other class A beta-lactamases. Moreover, extension of resistance to ceftazidime-avibactam is associated with a trade-off in the resistance to other beta-lactams and a decrease in enzyme stability. Nevertheless, the high natural stability of KPC could underlay the propensity of this enzyme to acquire in vivo mutations conferring resistance to ceftazidime-avibactam (CAZavi), particularly via insertions and deletions.

Emergence and clonal expansion of Klebsiella pneumoniae ST307, simultaneously producing KPC-3 and NDM-1.

MDR Klebsiella pneumoniae ST307 is a high-risk clone, whose genetic features contribute to its adaptation to hospital environments and the human host. This study describes the emergence and clonal dissemination of K. pneumoniae ST307, recovered during November 2018 to February 2019 in a hospital in Buenos Aires city, which concurrently harbored KPC-3 and NDM-1. These isolates were resistant to all ß-lactams and to the ceftazidime/avibactam combination. Molecular studies showed that blaKPC-3 was located in Tn4401a platform, while blaNDM-1 was surrounded upstream by ISKpn14 followed by a partial sequence of ISAba125 and downstream by bleMBL-trpF, located in a 145.5kb conjugative plasmid belonging to the Inc A/C group. The dissemination of K. pneumoniae ST307 isolates co-producing KPC-3 and NDM-1 could lead to a worrisome scenario due to the remarkable features of this clone and its resistance profile.

Outbreak of IncX8 Plasmid-Mediated KPC-3-Producing Enterobacterales Infection, China.

Carbapenem-resistant Enterobacterales (CRE) infection is highly endemic in China; Klebsiella pneumoniae carbapenemase (KPC) 2-producing CRE is the most common, whereas KPC-3-producing CRE is rare. We report an outbreak of KPC-3-producing Enterobacterales infection in China. During August 2020-June 2021, 25 blaKPC-3-positive Enterobacteriale isolates were detected from 24 patients in China. Whole-genome sequencing analysis revealed that the blaKPC-3 genes were harbored by IncX8 plasmids. The outbreak involved clonal expansion of KPC-3-producing Serratia marcescens and transmission of blaKPC-3 plasmids across different species. The blaKPC-3 plasmids demonstrated high conjugation frequencies (10-3 to 10-4). A Galleria mellonella infection model showed that 2 sequence type 65 K2 K. pneumoniae strains containing blaKPC-3 plasmids were highly virulent. A ceftazidime/avibactam in vitro selection assay indicated that the KPC-3-producing strains can readily develop resistance. The spread of blaKPC-3-harboring IncX8 plasmids and these KPC-3 strains should be closely monitored in China and globally.

Successful Treatment of Bloodstream Infection due to a KPC-Producing Klebsiella Pneumoniae Resistant to Imipenem/Relebactam in a Hematological Patient.

Novel carbapenem-ß-lactamase inhibitor combination, imipenem/relebactam (IMI-REL), has been recently approved for treatment of infections with limited or no alternative treatment options. In this study, we described the emergence of the IMI-REL-resistance in a KPC-producing Klebsiella pneumoniae (KPC-Kp) strain collected from a hematological patient with no evidence of prior colonization. Interestingly, IMI-REL-resistance was associated with meropenem/vaborbactam (MER-VAB) cross-resistance but was not associated with cross-resistance to ceftazidime/avibactam (CAZ-AVI). Although treatment with CAZ-AVI and gentamicin completely eradicated the infection due KPC-Kp cross-resistance to IMI-REL and MER-VAB, the patient became colonized subsequently by KPC-Kp strains susceptible to IMI-REL and MER-VAB. Whole-genome sequencing performed by hybrid approach using Illumina and Oxford Nanopore platforms demonstrated that all KPC-Kp strains isolated from hematological patient belonged to the ST512 and were clonally related. Analysis of antimicrobial and porins genes demonstrated that cross-resistance to IMI-REL and MER-VAB was associated with increased blaKPC-3 copy number and truncated OmpK35 and OmpK36 with GD134-135 insertion. Phylogenetic analysis demonstrated that KPC-Kp cross-resistance to IMI-REL and MER-VAB was clonally related to a KPC-Kp resistant to IMI-REL as previously described, demonstrating the spread of this multidrug resistant clone in the hematological unit. In conclusion, the results presented in this study reported the emergence of cross-resistance to MER-VAB and IMI-REL in a KPC-Kp strain isolated from a hematological patient and highlight the potential development and diffusion of new multidrug resistance traits.

First Description of Ceftazidime/Avibactam Resistance in a ST13 KPC-70-Producing Klebsiella pneumoniae Strain from Portugal.

The combination of ceftazidime/avibactam (CZA) is a novel ß-lactam/ß-lactamase inhibitor with activity against Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales. Emerging cases caused by CZA-resistant strains that produce variants of KPC genes have already been reported worldwide. However, to the best of our knowledge, no CZA-resistant strains were reported in Portugal. In September 2019, a K. pneumoniae CZA-resistant strain was collected from ascitic fluid at a surgery ward of a tertiary University Hospital Center in Lisboa, Portugal. The strain was resistant to ceftazidime/avibactam, as well as to ceftazidime, cefoxitin, gentamicin, amoxicillin/clavulanic acid, and ertapenem, being susceptible to imipenem and tigecycline. A hypermucoviscosity phenotype was confirmed by string test. Whole-genome sequencing (WGS) analysis revealed the presence of an ST13 KPC70-producing K. pneumoniae, a KPC-3 variant, differing in two amino-acid substitutions (D179Y and T263A). The D179Y mutation in the KPC Ω-loop region is the most common amino-acid substitution in KPC-2 and KPC-3, further leading to CZA resistance. The second mutation causes a KPC-70 variant in which threonine replaces alanine (T263A). The CZA-resistant strain showed the capsular locus KL3 and antigen locus O1v2. Other important virulence factors were identified: fimbrial adhesins type 1 and type 3, as well as the cluster of iron uptake systems aerobactin, enterobactin, salmochelin, and yersiniabactin included in integrative conjugative element 10 (ICEKp10) with the genotoxin colibactin cluster. Herein, we report the molecular characterization of the first hypervirulent CZA-resistant ST13 KPC-70-producing K. pneumoniae strain in Portugal. The emergence of CZA-resistant strains might pose a serious threat to public health and suggests an urgent need for enhanced clinical awareness and epidemiologic surveillance.

Whole-Genome Sequencing Enables Molecular Characterization of Non-Clonal Group 258 High-Risk Clones (ST13, ST17, ST147 and ST307) among Carbapenem-Resistant Klebsiella pneumoniae from a Tertiary University Hospital Centre in Portugal.

The carbapenem-resistant Enterobacterales (CRE) strains have been identified by the World Health Organization as critical priority pathogens in research and development of diagnostics, treatments, and vaccines. However, recent molecular information about carbapenem-resistant K. pneumoniae (CRK) epidemiology in Portugal is still scarce. Thus, this study aimed to provide the molecular epidemiology, resistome, and virulome of CRK clinical strains recovered from a tertiary care hospital centre (2019-2021) using polymerase chain reaction (PCR) and the advanced molecular technique whole-genome sequencing (WGS). PCR amplification of carbapenemase genes was performed in 437 carbapenem-resistant K. pneumoniae strains. The most frequent carbapenemases were: KPC-3 (42%), followed by OXA-181 (20%), GES-5 (0.2%), and NDM-1 (0.2%). Additionally, 10 strains (2%) coproduced KPC-3 and OXA-181, and 1 strain coproduced KPC-3 and OXA-48 (0.2%). The genomic population structure of 68 strains characterized by WGS demonstrated the ongoing dissemination of four main high-risk clones: ST13, ST17, ST147, and ST307, while no clones belonging to the European predominant clonal groups (CG15 and CG258) were found. Moreover, we describe one K. pneumoniae ST39-KL62 that coproduced the NDM-1 carbapenemase and the extended-spectrum beta-lactamase CTX-M-15, and one K. pneumoniae ST29-KL54 GES-5 and BEL-1 coproducer. Furthermore, a high prevalence of iron siderophores were present in all CRK strains, with several strains presenting both colibactin and the hypermucoviscosity phenotype. Thus, the data presented here highlight an uncommon molecular epidemiology pattern in Portugal when compared with most European countries, further supporting the emergence and dissemination of nonclonal group 258 hypervirulent multidrug high-risk clones and the need to promote in-depth hospital molecular surveillance studies.

Impact of Ceftazidime-Avibactam Treatment in the Emergence of Novel KPC Variants in the ST307-Klebsiella pneumoniae High-Risk Clone and Consequences for Their Routine Detection.

The emergence of Klebsiella pneumoniae isolates carrying novel blaKPC variants conferring ceftazidime-avibactam (CAZ/AVI) resistance is being increasingly reported. We evaluated the accuracy of phenotypic methods commonly used in routine clinical laboratories in the detection of novel K. pneumoniae carbapenemase (KPC) enzymes. Additionally, we characterized by whole-genome sequencing (WGS) the KPC-ST307-K. pneumoniae isolates recovered in our hospital before and after CAZ/AVI therapy. Rectal colonization or infection by carbapenem-resistant KPC-3 K. pneumoniae isolates (imipenem MIC, 16 mg/L; meropenem MIC, 8 to >16 mg/L) and CAZ/AVI-susceptible isolates (CAZ/AVI MIC, 1 to 2 mg/L) were first detected in three intensive care unit (ICU) patients admitted between March 2020 and July 2020. KPC K. pneumoniae isolates with increased CAZ/AVI MICs (8 to 32 mg/L) and carbapenem susceptibility (imipenem and meropenem MIC, <1 mg/L) were recovered within 6 to 24 days after CAZ/AVI treatment. WGS confirmed that all KPC K. pneumoniae isolates belonged to the sequence type 307 (ST307) high-risk clone and carried identical antimicrobial resistance genes and virulence factors. The presence of the novel blaKPC-46, blaKPC-66, and blaKPC-92 genes was confirmed in the K. pneumoniae isolates with increased CAZ/AVI MICs and restored carbapenem activity. KPC production was confirmed by immunochromatography, the eazyplex Superbug CRE system, and the Xpert Carba-R assay in all KPC K. pneumoniae isolates, but not in any isolate using chromogenic agar plates for carbapenemase producers (ChromID-CARBA), the KPC/MBL/OXA-48 Confirm kit, and the ß-CARBA test. Nevertheless, all grew in chromogenic agar plates for extended-spectrum ß-lactamase (ESBL) producers (ChromID-ESBL). We report the failure of the most common phenotypic methods used for the detection of novel KPC carbapenemases but not of rapid molecular or immunochromatography assays, thus highlighting their relevance in microbiology laboratories.

Meropenem-Vaborbactam as Salvage Therapy for Ceftazidime-Avibactam-, Cefiderocol-Resistant ST-512 Klebsiella pneumoniae-Producing KPC-31, a D179Y Variant of KPC-3.

A 68-year-old man had recurrent bacteremia by Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae resistant to ceftazidime-avibactam and cefiderocol. The sequencing of a target region showed that it harbored a KPC-3 variant enzyme (D179Y; KPC-31), which confers resistance to ceftazidime-avibactam and restores meropenem susceptibility. The patient was successfully treated with meropenem-vaborbactam.

An Update on Wastewater Multi-Resistant Bacteria: Identification of Clinical Pathogens Such as Escherichia coli O25b:H4-B2-ST131-Producing CTX-M-15 ESBL and KPC-3 Carbapenemase-Producing Klebsiella oxytoca.

Wastewater treatment plants (WWTPs) are significant reservoirs of bacterial resistance. This work aims to identify the determinants of resistance produced by Gram-negative bacteria in the influent and effluent of two WWTPs in Portugal. A total of 96 wastewater samples were obtained between 2016 and 2019. The numbers of total aerobic and fecal contamination bacteria were evaluated, and genomic features were searched by polymerase chain reaction (PCR) and Next-Generation Sequencing (NGS). Enterobacteriaceae corresponded to 78.6% (n = 161) of the 205 isolates identified by 16sRNA. The most frequent isolates were Escherichia spp. (57.1%, n = 117), followed by Aeromonas spp. (16.1%, n = 33) and Klebsiella spp. (12.7%, n = 26). The remaining 29 isolates (14.1%) were distributed across 10 different genera. Among the 183 resistant genes detected, 54 isolates produced extended spectrum ß-lactamases (ESBL), of which blaCTX-M-15 was predominant (37 isolates; 68.5%). A KPC-3 carbapenemase-producing K. oxytoca was identified (n = 1), with blaKPC-3 included in a transposon Tn4401 isoform b. A higher number of virulence genes (VG) (19 genes) was found in the E. coli 5301 (O25b-ST131-B2) isolate compared with a commensal E. coli 5281 (O25b-ST410-A) (six genes). Both shared five VG [Enterobactin; Aerobactin, CFA/1 (clade α); Type1 (clade γ1); Type IV]. In conclusion, this work highlights the role of relevant clinical bacteria in WWTPs, such as KPC-3-producing K. oxytoca, and, for the first time, a CTX-M-15-producing Ochromobactrum intermedium, a human opportunistic pathogen, and a SED-1-producing Citrobacter farmeri, an uncommon CTX-M-type extended-spectrum beta-lactamase.

Cross-resistance to cefiderocol and ceftazidime-avibactam in KPC ß-lactamase mutants and the inoculum effect.

OBJECTIVES: Ceftazidime-avibactam (CZA) and cefiderocol are recently commercialized molecules active against highly drug-resistant bacteria, including carbapenem-resistant members of the Enterobacteriaceae. Mutants resistant to CZA have been described, notably in Klebsiella pneumoniae carbapenemase (KPC) producers. Considering the structural similarities between ceftazidime and cefiderocol, we hypothesized that resistance to CZA in KPC-producing members of the Enterobacterales may lead to cross-resistance to cefiderocol. METHODS: CZA-resistant mutants from three clinical isolates of the Enterobacterales carrying either blaKPC-2 or blaKPC-3 were selected in vitro. Mutants with increased MIC to CZA compared to the ancestral allele were cloned in a pBR322 plasmid and expressed in Escherichia coli TOP10. We evaluated the impact of these mutations on cefiderocol MICs and minimal bactericidal concentrations (MBCs), and we assessed the impact of bacterial inoculum size on cefiderocol MICs. RESULTS: We used 37 KPC mutants with increased CZA MICs. Of these, six have been described previously in clinical isolates. Compared to the wild-type alleles, increases in the cefiderocol MICs of 4- to 32-fold were observed for 75.6% of tested mutants (28/37), MICs reaching up to 4 mg/L in E. coli TOP10 for KPC-31 (D179Y-H274Y mutations). MBCs and MICs of cefiderocol were similar, confirming the bactericidal activity of this drug. Finally, when using higher inocula (107 CFU/mL), a large increase in cefiderocol MIC was observed, and all isolates were categorized as resistant. CONCLUSION: We observed that most of the CZA-resistant KPC variants have a possible impact on cefiderocol by increasing the cefiderocol MICs. In addition, cefiderocol is greatly impacted by the inoculum effect, suggesting that precautions should be taken when treating infections with a suspected high inoculum.

Ceftazidime-Avibactam Resistance Mutations V240G, D179Y, and D179Y/T243M in KPC-3 ß-Lactamase Do Not Alter Cefpodoxime-ETX1317 Susceptibility.

Mutations in KPC-2 and KPC-3 ß-lactamase can confer resistance to the ß-lactam/ß-lactamase inhibitor antibacterial intravenous drug combination ceftazidime-avibactam, introduced in 2015. Avibactam was the first of the diazabicyclooctane class of non-ß-lactam ß-lactamase inhibitors to be approved for clinical use. The orally bioavailable prodrug ETX0282 of the diazabicyclooctane ß-lactamase inhibitor ETX1317 is in clinical development in combination with the oral ß-lactam prodrug cefpodoxime proxetil for use against complicated urinary tract infections. We investigated the effects of 3 ceftazidime-avibactam resistance mutations in KPC-3 (V240G, D179Y, and D179Y/T243M) on the ability of ETX1317 to overcome KPC-3-induced cefpodoxime resistance. Isogenic Escherichia coli strains, each expressing the wild-type or a mutant KPC-3 at similar levels, retained susceptibility to cefpodoxime-ETX1317 (1:2) with essentially identical minimal inhibitory concentrations of 0.125-0.25 µg/mL cefpodoxime. The KPC-3 mutations had little or no effect on the kinact/Ki values for inhibition by each of 3 diazabicyclooctanes: avibactam, durlobactam (ETX2514), and ETX1317. The KM values for hydrolysis of cefpodoxime were similar for all 4 variants, but the kcat values of the D179Y and D179Y/T243M variants were much lower than those of the wild-type and V240G mutant enzymes. All 4 KPC-3 variants formed stable, reversibly covalent complexes with ETX1317, but dissociation of ETX1317 was much slower from the D179Y and D179Y/T243M mutants than from the wild-type and V240G mutant enzymes. Thus, the KPC-3 variants examined here that cause resistance to ceftazidime-avibactam do not cause resistance to cefpodoxime-ETX1317.

Genomic Epidemiology of Carbapenemase Producing Klebsiella pneumoniae Strains at a Northern Portuguese Hospital Enables the Detection of a Misidentified Klebsiella variicola KPC-3 Producing Strain.

The evolutionary epidemiology, resistome, virulome and mobilome of thirty-one multidrug resistant Klebsiella pneumoniae clinical isolates from the northern Vila Real region of Portugal were characterized using whole-genome sequencing and bioinformatic analysis. The genomic population structure was dominated by two main sequence types (STs): ST147 (n = 17; 54.8%) and ST15 (n = 6; 19.4%) comprising four distinct genomic clusters. Two main carbapenemase coding genes were detected (blaKPC-3 and blaOXA-48) along with additional extended-spectrum ß-lactamase coding loci (blaCTX-M-15, blaSHV-12, blaSHV-27, and blaSHV-187). Moreover, whole genome sequencing enabled the identification of one Klebsiella variicola KPC-3 producer isolate previously misidentified as K. pneumoniae, which in addition to the blaKPC-3 carbapenemase gene, bore the chromosomal broad spectrum ß-lactamase blaLEN-2 coding gene, oqxAB and fosA resistance loci. The blaKPC-3 genes were located in a Tn4401b transposon (K. variicolan = 1; K. pneumoniaen = 2) and Tn4401d isoform (K. pneumoniaen = 28). Overall, our work describes the first report of a blaKPC-3 producing K. variicola, as well as the detection of this species during infection control measures in surveillance cultures from infected patients. It also highlights the importance of additional control measures to overcome the clonal dissemination of carbapenemase producing clones.