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Objectives: Protegrins are a family of natural peptides from the innate immune system of vertebrates, with broad-spectrum antimicrobial activity. However, the toxicity and haemolysis of protegrin-1 (PG-1) at low concentrations renders it useless for therapeutic application. We rationally designed PLP-3, a novel synthetic PG-1-like peptide, comprising key activity features of protegrins in a constrained bicyclic structure. Our main objective was to investigate PLP-3's activity against MDR strains of Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae and to analyse its haemolysis and cytotoxicity.Methods: Peptide synthesis was performed via solid phase and intramolecular ligation in solution, and the correct folding of the peptide was verified by circular dichroism. Antimicrobial activity was performed through broth microdilution. The test panel contained 45 bacterial strains belonging to A. baumannii, P. aeruginosa and K. pneumoniae (15 strains per species) comprising colistin-resistant and MDR strains. Cytotoxicity was assessed by XTT cell viability assays using HeLa and A549 cells and haemolysis of human erythrocytes.Results: PLP-3 was successfully synthesized, and its antiparallel β-sheet conformation was confirmed. Antimicrobial activity screening showed MIC90 values of 2 mg/L for A. baumannii, 16 mg/L for K. pneumoniae and 8 mg/L for P. aeruginosa. The haemolysis IC50 value was 48.53 mg/L. Cytotoxicity against human HeLa and A549 cells showed values of ca. 200 mg/L in both cell lines resulting in a 100-fold selectivity window for bacterial over human cells.Conclusions: PLP-3 has potent antimicrobial activity, especially against A. baumannii, while maintaining low haemolysis and toxicity against human cell lines at antimicrobial concentrations. These characteristics make PLP-3 a promising peptide with an interesting therapeutic window. [ABSTRACT FROM AUTHOR]

Background: Carbapenemase-producing Vibrio spp., which exhibit an XDR phenotype, have become increasingly prevalent and pose a severe threat to public health.Objectives: To investigate the genetic characteristics of NDM-1-producing Vibrio spp. isolates and the dissemination mechanisms of blaNDM-1 in Vibrio.Methods: A total of 1363 non-duplicate Vibrio spp. isolates collected from shrimp samples in China were subjected to antimicrobial susceptibility tests and screened for blaNDM-1. The blaNDM-1-positive isolates were further characterized by PFGE, MLST, conjugation and WGS using Illumina and Nanopore platforms. Plasmid stability and fitness cost were assessed using Escherichia coli J53, Klebsiella pneumoniae Kpt80 and Salmonella spp. SA2051 as recipient strains.Results: In total, 13 blaNDM-1-positive isolates were identified, all exhibiting MDR. WGS analysis revealed that the 13 blaNDM-1 genes were all associated with a derivative of Tn125. Plasmid analysis revealed that six blaNDM-1 genes were located in IncC plasmids and the other seven were carried by plasmids of two different novel types. Conjugation and plasmid stability assays showed that only the IncC plasmids could be transferred to all the recipient strains and could be stably maintained in the hosts.Conclusions: The emergence of the novel plasmids has contributed to the variable genetic contexts of blaNDM-1 in Vibrio spp. and IncC plasmids harbouring the blaNDM-1 gene could facilitate the spread of such genes between Vibrio spp. and other zoonotic pathogens, leading to a rapid dissemination of blaNDM-1 in bacterial pathogens worldwide. [ABSTRACT FROM AUTHOR]

This review describes the translational in vivo and non-clinical pharmacokinetics/pharmacodynamics (PK/PD) research that supported clinical trialling and subsequently licensing approval of ceftazidime/avibactam, a new β-lactam/β-lactamase inhibitor combination aimed at the treatment of infections by Enterobacterales and Pseudomonas aeruginosa. The review thematically follows on from the co-published article, Nichols et al. (J Antimicrob Chemother 2022; 77: 2321-40). Avibactam protected ceftazidime in animal models of infection with ceftazidime-resistant, β-lactamase-producing bacteria. For example, a single subcutaneous dose of ceftazidime at 1024 mg/kg yielded little effect on the growth of ceftazidime-resistant, blaKPC-2-carrying Klebsiella pneumoniae in the thighs of neutropenic mice (final counts of 4 × 108 to 8 × 108 cfu/thigh). In contrast, co-administration of avibactam in a 4:1 ratio (ceftazidime:avibactam) was bactericidal in the same model (final counts of 2 × 104 to 3 × 104 cfu/thigh). In a rat abdominal abscess model, therapy with ceftazidime or ceftazidime/avibactam (4:1 w/w) against blaKPC-2-positive K. pneumoniae resulted in 9.3 versus 3.3 log cfu/abscess, respectively, after 52 h. With respect to PK/PD, in Monte Carlo simulations, attainment of unbound drug exposure targets (ceftazidime fT>8 mg/L and avibactam fT>1 mg/L, each for 50% of the dosing interval) for the labelled dose of ceftazidime/avibactam (2 and 0.5 g, respectively, q8h by 2 h IV infusion), including dose adjustments for patients with impaired renal function, ranged between 94.8% and 99.6% of patients, depending on the infection modelled. [ABSTRACT FROM AUTHOR]

Objectives: To investigate the in vitro activity of fosfomycin, colistin and combinations thereof against planktonic and biofilm cultures of Gram-negative pathogens, mostly showing MDR phenotypes, at concentrations achievable via inhalation of aerosolized drugs.Methods: Activity against planktonic cultures was tested by the chequerboard assay with 130 strains, including 52 Pseudomonas aeruginosa, 47 Klebsiella pneumoniae, 19 Escherichia coli, 7 Stenotrophomonas maltophilia and 5 Acinetobacter baumannii. Activity against biofilm cultures was tested by biofilm chequerboard and quantitative antibiofilm assays with a subset of 20 strains. In addition, 10 of these strains were tested in mutant prevention concentration (MPC) assays.Results: Against planktonic cultures, synergism between fosfomycin and colistin was detected with a minority (10%) of strains (eight K. pneumoniae and five P. aeruginosa), while antagonism was never observed. Synergism between fosfomycin and colistin against biofilms was observed with the majority of tested strains (16/20 in biofilm chequerboard assays, and 18/20 in the quantitative antibiofilm assays), including representatives of each species and regardless of their resistance genotype or phenotype. Furthermore, combination of fosfomycin and colistin was found to significantly reduce the MPC of individual drugs.Conclusions: Fosfomycin and colistin in combination, at concentrations achievable via inhalation of nebulized drugs, showed notable synergy against MDR Gram-negative pathogens grown in biofilm, and were able to reduce the emergence of fosfomycin- and colistin-resistant subpopulations. [ABSTRACT FROM AUTHOR]

Objectives: To characterize a clinical Klebsiella pneumoniae isolate from China co-harbouring tet(X4), blaOXA-181 and the aerobactin operon on an IncFIBk-FII-X3-ColKP3 hybrid plasmid.Methods: A tigecycline-resistant strain was recovered from the intestinal sample of a patient. It was subjected to antimicrobial susceptibility testing, conjugation assay, virulence testing, WGS, bioinformatics analysis, plasmid stability testing and fitness cost testing.Results: The strain K. pneumoniae T877 was resistant to tigecycline, intermediate to piperacillin/tazobactam and ertapenem, and positive for tet(X), blaOXA-181 and the virulence-associated operon iutAiucABCD, which were located on the same plasmid, named pKPT877-hybrid. It was 99.96% identical to the IncFIBk-FII plasmid pSCH6109-Vir (accession number CP050860) from K. pneumoniae strain SCH6109 at 96% coverage with the absence of a 50 kb region on pKPT877-hybrid; this region was highly homologous to the 51 kb IncX3-ColKP3-type, blaOXA-181-carrying plasmid pOXA181-191773 (accession number CP080367). Plasmid pKPT877-hybrid was conjugatively transferable to the ST11 K. pneumoniae strains FJ8 and KP04. pKPT877-hybrid did not have a significant impact on the fitness cost and could be maintained stably in T877.Conclusions: We report for the first time (to the best of our knowledge) the co-transfer of last-line antibiotic resistance determinants [tet(X4) and blaOXA-181] and the aerobactin operon (iutAiucABCD) by a mobile IncFIBk-FII-X3-ColKP3 hybrid plasmid, which can be stably maintained in K. pneumoniae strains, even in the absence of antibiotic selective pressure. Once the plasmid transfers to a K. pneumoniae with porin deficiency, the strain might have high levels of resistance to carbapenems and tigecycline, which are the last line of defence against infections. Heightened and continuous efforts are needed to control its dissemination. [ABSTRACT FROM AUTHOR]

Background: Carbapenem-resistant Enterobacterales (CRE) are highly concerning MDR pathogens. Horizontal transfer of broad-host-range IncN plasmids may contribute to the dissemination of the Klebsiella pneumoniae carbapenemase (KPC), spreading carbapenem resistance among unrelated bacteria. However, the population structure and genetic diversity of IncN plasmids has not been fully elucidated.Objectives: We reconstructed blaKPC-harbouring IncN plasmid genomes to characterize shared gene content, structural variability, and putative horizontal transfer within and across patients and diverse bacterial clones.Methods: We performed short- and long-read sequencing and hybrid assembly on 45 CRE isolates with blaKPC-harbouring IncN plasmids. Eight serial isolates from two patients were included to assess intra-patient plasmid dynamics. Comparative genomic analysis was performed to assess structural and sequence similarity across plasmids. Within IncN sublineages defined by plasmid MLST and kmer-based clustering, phylogenetic analysis was used to identify closely related plasmids.Results: Comparative analysis of IncN plasmid genomes revealed substantial heterogeneity including large rearrangements in serial patient plasmids and differences in structure and content across plasmid clusters. Within plasmid sublineages, core genome content and resistance gene regions were largely conserved. Closely related plasmids (≤1 SNP) were found in highly diverse isolates, including ten pST6 plasmids found in eight bacterial clones from three different species.Conclusions: Genomic analysis of blaKPC-harbouring IncN plasmids revealed the presence of several distinct sublineages as well as substantial host diversity within plasmid clusters suggestive of frequent mobilization. This study reveals complex plasmid dynamics within a single plasmid family, highlighting the challenge of tracking plasmid-mediated transmission of blaKPC in clinical settings. [ABSTRACT FROM AUTHOR]

Objectives: Ertapenem has proven to be an effective antimicrobial; however, increasing enzyme-mediated resistance has been noted. Combination with zidebactam, a β-lactam enhancer, is restorative. Human-simulated regimens (HSRs) of ertapenem and zidebactam alone and in combination (WCK 6777; 2 g/2 g q24h) were assessed for efficacy against carbapenemase-producing Klebsiella pneumoniae (CP-KP) in the pneumonia model.Methods: Infected ICR mice were rendered neutropenic and exposed to various doses of ertapenem and zidebactam alone and in combination to develop the HSRs that were subsequently confirmed in additional pharmacokinetic studies. Twenty-one CP-KP (KPC or OXA-48-like producers) with WCK 6777 MICs of 1-8 mg/L were utilized. Mice were treated for 24 h with saline or HSRs of ertapenem, zidebactam and WCK 6777. Efficacy was defined as change in mean lung bacterial density relative to 0 h.Results: Confirmatory pharmacokinetic analysis showed agreement between predicted human exposures (%fT>MIC) and those achieved in vivo for all three HSRs. The 0 h bacterial density across all isolates was 6.69 ± 0.31 log10 cfu/lungs. At 24 h, densities increased by 2.57 ± 0.50, 2.2 ± 0.60 and 2.05 ± 0.71 log10 cfu/lungs in the 24 h control, ertapenem HSR and zidebactam HSR groups, respectively. Overall, 18/21 of the isolates exposed to the WCK 6777 HSR displayed a killing profile that exceeded the translational benchmark for efficacy of a 1 log10 cfu reduction. Among the remaining three isolates, two displayed ∼0.5 log10 kill and stasis was observed in the third.Conclusions: Human-simulated exposures of WCK 6777 demonstrated potent in vivo activity against CP-KP, including those with WCK 6777 MICs up to 8 mg/L. [ABSTRACT FROM AUTHOR]

Objectives: The novel carbapenem/β-lactamase inhibitor combination imipenem/cilastatin/relebactam has been developed for the treatment of infections due to carbapenemase-producing Enterobacteriaceae (CPE). Herein, we describe the in vivo evolution of imipenem/cilastatin/relebactam resistance in longitudinal intra-patient Klebsiella pneumoniae carbapenemase (KPC)-producing Klebsiella pneumoniae (KPC-Kp) strains isolated from a patient following ceftazidime/avibactam-based treatments.Methods: WGS analysis was performed on KPC-Kp strains isolated at different times and during antimicrobial treatments from the same patient. Genome assemblies were performed using a hybrid approach using Illumina iSeq 100 and Minion Oxford Nanopore platforms. Subpopulation analysis and allele frequency determination was performed by mapping Illumina reads to blaKPC.Results: During antimicrobial treatment, resistance to ceftazidime/avibactam was observed following 16 days of antimicrobial therapy. WGS results showed that all KPC-Kp exhibited a low SNP rate of divergence, belonged to ST512 and shared similar antimicrobial resistance and porin gene patterns. Genetic analysis demonstrated that the first ceftazidime/avibactam-resistant KPC-Kp strain harboured a blaKPC-53 gene in a Tn4401 transposon moved from IncFII(K) to a 43 kb IncX3 plasmid, while a imipenem/cilastatin/relebactam-resistant strain exhibited two copies of the Tn4401 transposon in IncFII(K) and IncX3 plasmids, resulting in an increased blaKPC copy number. Of note, frequency analysis demonstrated that imipenem/cilastatin/relebactam-resistant KPC-Kp consisted of mixed subpopulations harbouring blaKPC-40 and blaKPC-53 alleles.Conclusions: Our results show the in vivo evolution of genetic rearrangement conferring resistance to imipenem/relebactam in a patient with KPC-Kp infection and treated with different ceftazidime/avibactam-based treatments. The rapid development of mutations and the high adaptability of its genome highlight the potential threat of KPC-Kp. [ABSTRACT FROM AUTHOR]

Objectives: To evaluate the performance of two rapid antimicrobial susceptibility testing (RAST) methods to determine ceftazidime/avibactam susceptibility directly from blood cultures (BCs).Methods: A total of 246 Escherichia coli or Klebsiella pneumoniae isolates were tested for ceftazidime/avibactam susceptibility directly from BC bottles using EUCAST RAST and Etest® RAST. Results obtained after 4, 6 and 8 h of incubation were compared with those obtained by reference broth microdilution on pure overnight subcultures.Results: In total, the proportion of readable zones after 4 h of incubation was 96.7% and reached 100% after 6 and 8 h of incubation. EUCAST RAST yielded >98% of categorical agreement (CA) with all reading times. Major error (ME) and very major error (VME) rates were inferior to 3%, for each of the reading times. The proportion of results in the area of technical uncertainty (ATU) was almost similar (3.8%-4.1%) at the different reading times. DET-RAST yielded 97.5%, 98% and 99.6% of CA with readings at 4, 6 and 8 h, respectively. One (0.6%) ME was observed at each reading time, whereas five (5.9%) and four (4.5%) VMEs were observed analysing readings at 4 and 6 h, respectively. No VME was observed with readings at 8 h.Conclusions: EUCAST RAST was accurate to determine ceftazidime/avibactam susceptibility of carbapenemase-producing K. pneumoniae and E. coli directly from BC bottles. DET-RAST has the advantage of determining MIC values and avoiding ATU results but showed to be an accurate method only with reading at 8 h. [ABSTRACT FROM AUTHOR]

Background: Increased use of colistin in healthcare necessitates studies on the trend of colistin resistance and the underlying mechanisms.Objectives: To understand the susceptibility trend and molecular mechanisms of colistin resistance in neonatal isolates over a 12 year period.Methods: Colistin susceptibility, mRNA expression, whole genome sequence and mutational analysis was performed. Phylogenomic comparison with a global collection of colistin-resistant Klebsiella pneumoniae strains (n = 70) was done.Results: Of 319 Enterobacterales (K. pneumoniae and Escherichia coli) studied, colistin resistance was found in 9 K. pneumoniae (2.8%). The transmissible colistin resistance gene, mcr, was absent. Colistin-resistant K. pneumoniae belonged to diverse sequence types (ST14/37/101/147/716) and exhibited multiple mechanisms of colistin resistance including overexpression of the two-component systems (TCS) (phoP/Q, pmrA/B), and AcrAB-TolC pump and its regulators. Mutations in TCS, mgrB, pumps, repressors, and lipopolysaccharide-modifying genes were detected. Phylogenomic comparison revealed that this global collection of colistin-resistant K. pneumoniae was diverse, with the presence of epidemic and international clones. Mutations in mgrB and TCS noted in global strains were comparable to the study strains. Co-occurrence of carbapenem resistance (n = 61, 87%) was observed in global strains. Co-existence of dual carbapenemases (blaNDM-5 with blaOXA-48/181) in multiple lineages within different replicons was found in neonatal colistin-resistant study isolates only.Conclusions: Colistin resistance both in study and global strains is multifaceted and attributed to mutations in chromosomal genes leading to lipopolysaccharide modification or efflux of colistin through pumps. With no transmissible mcr, prevalence of colistin-resistant strains was low in this unit. Colistin-resistant strains with dual carbapenemases causing sepsis are alarming as they are practically untreatable. [ABSTRACT FROM AUTHOR]

Background: Consecutive Polish regions have become endemic for NDM-1-producing Klebsiella pneumoniae ST11, followed by K. pneumoniae ST147. Since 2017 a significant increase in NDM-positive Enterobacter hormaechei cases has been observed.Objectives: To investigate the origin and character of this increase in NDM-positive E. hormaechei.Methods: The analysis included 160 NDM-producing Enterobacter cloacae complex isolates, recovered in 2015-20 in 37 centres of 9/16 regions. These were typed by PFGE and MLST, and screened by PCR-mapping for NDM-1-encoding Tn125-like elements. Forty-four isolates were sequenced by MiSeq. Species identification was based on whole-genome average nucleotide identity; clonality and phylogeny were inferred by SNP approaches. The structural plasmid analysis was done for 12 isolates sequenced by MinION.Results: The isolates belonged to 11 STs, predominantly ST89 (65.6%), followed by ST146 (15.6%), ST198 (7.5%) and ST1303 (3.7%), representing different E. hormaechei subspecies. Most of the isolates contained the Tn125A variant of the K. pneumoniae ST11 lineage, and several had Tn125F of the ST147. Individual E. hormaechei genotypes represented various epidemiological situations, from sporadic cases to single-hospital, city and regional outbreaks, including one caused by ST89 organisms with 82 cases in 17 centres. Acquisitions of the Tn125A/Tn125F determinants by the E. hormaechei strains occurred around 10 times and were plasmid-mediated, with a significant plasmid rearrangement in case of Tn125F.Conclusions: The increase in E. hormaechei NDM-1 cases in Poland is a consequence of the uncontrolled spread of NDM-1-producing K. pneumoniae genotypes. Several E. hormaechei lineages have acquired NDM-encoding plasmids in different locales which started 'secondary' progressive outbreaks. [ABSTRACT FROM AUTHOR]

Objectives: Resistance levels of Gram-negative bacteria producing OXA-48 carbapenemase can vary greatly and some of them can even be categorized as susceptible to imipenem and meropenem according to EUCAST breakpoints. This study aimed to reveal resistance mechanisms leading to varying levels of resistance to carbapenems in Klebsiella pneumoniae with blaOXA-48 submitted to the German National Reference Centre for MDR Gram-negative bacteria.Methods: Meropenem-susceptible clinical blaOXA-48-bearing K. pneumoniae isolates were put under gradually increasing selective pressure of meropenem. Clinical isolates and spontaneous meropenem-resistant mutants were whole-genome sequenced with Illumina and Oxford Nanopore Technology. Identified mutations apart from porin mutations were genetically constructed in the original clinical isolates using CRISPR/Cas. Clinical isolates and mutants were analysed for MICs, growth rates and expression of porins on mRNA and protein levels.Results: Mutations associated with meropenem resistance were predominantly found in ompK36, but in some cases ompK36 was unaffected. In two mutants, ISs within the rpoE (sigma factor E; σE) operon were detected, directly in or upstream of rseA. These IS1R elements were then inserted into the same position of the susceptible clinical isolates using CRISPR/Cas. CRISPR-rseA-rseB-rseC mutants showed higher resistance levels to carbapenems and cephalosporins, reduced growth rates and reduced expression of major porins OmpK36 and OmpK35 in quantitative RT-PCR and SDS-PAGE.Conclusions: Enhanced synthesis of σE leads to increased resistance to cephalosporins and carbapenems in clinical K. pneumoniae isolates. This effect could be based upon remodelling of expression patterns of outer membrane proteins. The up-regulated σE stress response also leads to a significant reduction in growth rates. [ABSTRACT FROM AUTHOR]

Introduction: The use of oral fosfomycin for urinary tract infections (UTIs) caused by non-Escherichia coli uropathogens is uncertain, including Klebsiella pneumoniae, the second most common uropathogen.Methods: A multicompartment bladder infection in vitro model was used with standard media and synthetic human urine (SHU) to simulate urinary fosfomycin exposure after a single 3 g oral dose (fAUC0-72 16884 mg·h/L, t½ 5.5 h) against 15 K. pneumoniae isolates including ATCC 13883 (MIC 2 to >1024 mg/L) with a constant media inflow (20 mL/h) and 4-hourly voiding of each bladder. The impact of the media (CAMHB + G6P versus SHU) on fosfomycin MIC measurements, drug-free growth kinetics and regrowth after fosfomycin administration was assessed. A low and high starting inoculum (5.5 versus 7.5 log10 cfu/mL) was assessed in the bladder infection model.Results: Compared with CAMHB, isolates in SHU had a slower growth rate doubling time (37.7 versus 24.1 min) and reduced growth capacity (9.0 ± 0.3 versus 9.4 ± 0.3 log10 cfu/mL), which was further restricted with increased inflow rate (40 mL/h) and more frequent voids (2-hourly). Regrowth was commonly observed in both media with emergence of fosfomycin resistance promoted by a high starting inoculum in CAMHB (MIC rise to ≥1024 mg/L in 13/14 isolates). Resistance was rarely detected in SHU, even with a high starting inoculum (MIC rise to ≥1024 mg/L in 2/14 isolates).Conclusions: Simulated in an in vitro UTI model, the regrowth of K. pneumoniae urinary isolates was inadequately suppressed following oral fosfomycin therapy. Efficacy was further reduced by a high starting inoculum. [ABSTRACT FROM AUTHOR]

Background: WHO considers ESBL- and carbapenemase-producing Klebsiella pneumoniae a major global concern. In animals, ESBL- and carbapenemase-producing K. pneumoniae of human-related ST11, ST15 and ST307 have been reported, but not in the context of large WGS-based One Health investigations.Objectives: To perform comparative phylogenomics on a large collection of multidrug-resistant (MDR) K. pneumoniae recovered from diseased companion animals and humans.Methods: MDR K. pneumoniae (n = 105) recovered from companion animals in France during 2010-18 were phenotypically characterized. All isolates were whole-genome sequenced using the NovaSeq technology and phylogenomic analysis across animal and human K. pneumoniae was performed using appropriate pipelines.Results: bla CTX-M-15, blaDHA-1 and blaOXA-48 were strongly associated with IncFIIk, IncR and IncL plasmids, respectively. When compared with human K. pneumoniae genomes, four groups of closely related French human and animal isolates belonging to ST11, ST15 and ST307 were detected, suggesting the circulation of clones between the human and animal sectors at country level. A large cluster of 31 ST11-KL105 animal isolates from France and Switzerland suggested it corresponds to a sub-lineage that is particularly well-adapted to the animal host.Conclusions: This study demonstrates the spread of blaCTX-M-15-carrying ST15 and ST307, and blaDHA-1-carrying ST11 K. pneumoniae clones in animal populations. ST11 was the main vector of blaOXA-48/IncL, despite the absence of carbapenem use in French animals. Comparative phylogenomics suggests cross-transmission of K. pneumoniae sub-lineages more prone than others to colonize/infect the animal host. Our data also evidenced the emergence of convergent hypervirulent and MDR K. pneumoniae in animals. [ABSTRACT FROM AUTHOR]

Background: Transportation of carbapenem-resistant plasmids contributes to carbapenem resistance in Gram-negative bacteria. KPC enzymes are the most clinically important enzymes among carbapenem-resistant Klebsiella pneumoniae, whereas the rate of blaKPC in Escherichia coli is low. The CRISPR-Cas system and restriction-modification system (R-M system) in bacteria defend against invading genomes. Currently, the role of the immune systems in the low rate of KPC-producing E. coli remains unclear.Objectives: We investigated the relationship between immune systems and the low detection rate of blaKPC in E. coli.Methods: We searched for blaKPC among 1039 E. coli whole genomes available in GenBank using nucleotide BLAST. CRISPR-Cas systems and the R-M system were detected in all strains having the ST as blaKPC-positive strains. Nucleotide BLAST was used to search for protospacers on blaKPC plasmids. A conjugation assay was performed to determine whether the R-M system influences the acquisition of blaKPC plasmids by E. coli.Results: ST131 was the dominant ST of KPC-producing E. coli and IncN was the main plasmid type (12/32). CRISPR-Cas systems were frequently present in E. coli carrying blaKPC. Furthermore, CRISPR-Cas systems in E. coli didn't target plasmids with blaKPC. Type I R-M systems were rare in KPC-producing E. coli, but significantly over-represented in KPC-negative strains. E. coli DH5α with hsdR deletion accepted blaKPC-carrying plasmids, whereas those with hsdR complementation impeded blaKPC-carrying plasmid conjugation.Conclusions: Horizontal transmission of blaKPC occurs among E. coli. The type I R-M system is associated with the defence against blaKPC plasmid transport into E. coli. [ABSTRACT FROM AUTHOR]

Objectives: To use the nationwide Norwegian surveillance programme on resistant microbes in humans (NORM) to address longitudinal changes in the population structure of Klebsiella pneumoniae isolates from 2001-15, focusing on the emergence and dissemination of ESBL-producing K. pneumoniae in Norway.Methods: Among blood (n = 6124) and urinary tract (n = 5496) surveillance isolates from 2001-15, we used Illumina technology to whole genome sequence 201 ESBL-producing isolates from blood (n = 130) and urine (n = 71), and 667 non-ESBL isolates from blood. Complete genomes for four isolates were resolved with Oxford Nanopore sequencing.Results: In a highly diverse collection, Klebsiella variicola ssp. variicola caused 24.5% of Klebsiella pneumoniae species complex (KpSC) bacteraemias. ESBL production was limited to K. pneumoniae sensu stricto (98.5%). A diverse ESBL population of 57 clonal groups (CGs) were dominated by MDR CG307 (17%), CG15 (12%), CG70 (6%), CG258 (5%) and CG45 (5%) carrying blaCTX-M-15. Yersiniabactin was significantly more common in ESBL-positive (37.8%) compared with non-ESBL K. pneumoniae sensu stricto isolates (12.7%), indicating convergence of virulence and resistance determinants. Moreover, we found a significantly lower prevalence of yersiniabactin (3.0%, 37.8% and 17.3%), IncFIB (58.7%, 87.9% and 79.4%) and IncFII plasmid replicons (40.5%, 82.8% and 54.2%) in K. variicola ssp. variicola compared with ESBL- and non-ESBL K. pneumoniae sensu stricto isolates, respectively.Conclusions: The increase in Norwegian ESBL-producing KpSC during 2010-15 was driven by CG307 and CG15 carrying blaCTX-M-15. K. variicola ssp. variicola was a frequent cause of invasive KpSC infection, but rarely carried ESBLs. [ABSTRACT FROM AUTHOR]

Objectives: Tigecycline is a last-resort antibiotic used to treat lethal infections caused by carbapenem-resistant Enterobacterales; however, plasmid-borne tigecycline resistance tmexCD-toprJ gene clusters can confer tigecycline resistance. The aim of the study was to identify novel subtypes and the spread of tmexCD-toprJ.Methods: Five non-duplicate isolates of different species, carrying tmexCD-toprJ gene clusters or novel subtypes, were isolated from patients across China between November 2018 and June 2019. WGS was performed using Illumina and Nanopore platforms. A phylogenetic tree was constructed using a dataset of 77 sequences carrying the tmexCD-toprJ gene clusters, 72 of which were downloaded from NCBI with a blastn identity cut-off of 95%.Results: We detected six different transfer units and two novel subtypes (tmexC1D1.2-toprJ1 and tmexC2D2.2-toprJ2) of the tmexCD-toprJ gene clusters. Among the six transfer units, three were mediated by IS26, while the rest were presumably mediated by Tn5393, hypothetical integrases (xerD-hp clusters-umuC-integrases-tnfxB2-tmexC2D2-toprJ2-umuC) and hypothetical units (hp-hp-hp-tnfxB2-tmexC2D2.2-toprJ2-ΔTn5393-Tn6292). Moreover, two tmexCD-toprJ-like gene clusters co-located on the same plasmid with blaNDM in five isolates. Phylogenetic analysis revealed that tmexCD-toprJ gene clusters may have originated in Pseudomonas spp., being mainly distributed in Pseudomonas spp. and Klebsiella spp. (64/77). Most tmexCD-toprJ gene clusters in Enterobacterales were located on plasmids, indicating that the gene clusters have a high inter-species transfer risk after transfer to Enterobacterales.Conclusions: In summary, to the best of our knowledge, this is the first report of tmexCD-toprJ gene clusters being isolated from Enterobacter cloacae and Klebsiella oxytoca, revealing that these multiple transfer units should be further studied because of their clinical significance. [ABSTRACT FROM AUTHOR]

Objectives: To determine the transmission and molecular characteristics of blaNDM-producing Escherichia coli between companion animals and their healthcare providers at veterinary clinics in Guangzhou, China.Methods: A total of 359 samples from companion animals and their healthcare providers were collected at 14 veterinary clinics in Guangzhou, China. Genomic characteristics and clonal relationships for blaNDM-positive E. coli and complete plasmid sequences were characterized based on WGS data from combined Illumina and MinION platform reads.Results: Forty-five blaNDM-positive bacteria were recovered from companion animals (n = 43) and their healthcare providers (n = 2) at 10 veterinary clinics. Overall, E. coli (73.3%, 33/45) and Klebsiella pneumoniae (13.3%, 6/45) were the most prevalent species among the seven species of blaNDM-positive bacteria. Four blaNDM variants (blaNDM-1, blaNDM-4, blaNDM-5 and blaNDM-7) were identified in 45 blaNDM-positive bacteria and blaNDM-5 was the most prevalent (77.8%, 35/45). WGS indicated that the most prevalent STs were ST405 (8/33), ST453 (6/33), ST457 (6/33) and ST410 (5/33) among the 33 blaNDM-positive E. coli isolates. Phylogenomics and PFGE analysis revealed that clonal spread of blaNDM-positive ST453 E. coli isolates between companion animals and their healthcare providers was evident. In addition, two novel IncFIB plasmids carrying blaNDM-4 (pF765_FIB and pG908_FIB) were found in this study and indicated that IS26 may promote the horizontal transmission of blaNDM between different plasmid types.Conclusions: In this study we conducted a large-scale investigation on the prevalence of blaNDM-positive E. coli isolates from companion animals and their healthcare providers and revealed the clonal spread of blaNDM-positive E. coli isolates between these two groups. [ABSTRACT FROM AUTHOR]

Background: Aminoglycoside-containing regimens may be an effective treatment option for infections caused by carbapenem-resistant Klebsiella pneumoniae (CR-Kp), but aminoglycoside-resistance genes are common in these strains. The relationship between the aminoglycoside-resistance genes and aminoglycoside MICs remains poorly defined.Objectives: To identify genotypic signatures capable of predicting aminoglycoside MICs for CR-Kp.Methods: Clinical CR-Kp isolates (n = 158) underwent WGS to detect aminoglycoside-resistance genes. MICs of amikacin, gentamicin, plazomicin and tobramycin were determined by broth microdilution (BMD). Principal component analysis was used to initially separate isolates based on genotype. Multiple linear regression was then used to generate models that predict aminoglycoside MICs based on the aminoglycoside-resistance genes. Last, the performance of the predictive models was tested against a validation cohort of 29 CR-Kp isolates.Results: Among the original 158 CR-Kp isolates, 91.77% (145/158) had at least one clinically relevant aminoglycoside-resistance gene. As a group, 99.37%, 84.81%, 82.28% and 10.76% of the CR-Kp isolates were susceptible to plazomicin, amikacin, gentamicin and tobramycin, respectively. The first two principal components explained 72.23% of the total variance in aminoglycoside MICs and separated isolates into four groups with aac(6')-Ib, aac(6')-Ib', aac(6')-Ib+aac(6')-Ib' or no clinically relevant aminoglycoside-resistance genes. Regression models predicted aminoglycoside MICs with adjusted R2 values of 56%-99%. Within the validation cohort, the categorical agreement when comparing the observed BMD MICs with the predicated MICs was 96.55%, 89.66%, 86.21% and 82.76% for plazomicin, gentamicin, amikacin and tobramycin, respectively.Conclusions: Susceptibility to each aminoglycoside varies in CR-Kp. Detection of aminoglycoside-resistance genes may be useful to predict aminoglycoside MICs for CR-Kp. [ABSTRACT FROM AUTHOR]

Objectives: To compare the in vitro activity of plazomicin and two older aminoglycosides (gentamicin and amikacin) against 180 isolates of Escherichia coli and Klebsiella pneumoniae, including subsets of 60 non-ESBL-producing, 60 ESBL-producing and 60 carbapenem-resistant (46 carrying blaOXA-48, 11 carrying blaNDM and 3 carrying blaOXA-48 and blaNDM) strains.Methods: MICs of plazomicin, gentamicin and amikacin were determined by a gradient diffusion method. Gentamicin and amikacin MICs were interpreted according to CLSI criteria and EUCAST breakpoint tables. Plazomicin MICs were interpreted using FDA-defined breakpoints.Results: All non-ESBL-producing and ESBL-producing isolates were susceptible to plazomicin. The plazomicin susceptibility rate (71.7%) in carbapenem-resistant isolates was significantly higher than those observed for gentamicin (45%) and amikacin (56.7% and 51.7% according to CLSI and EUCAST breakpoints, respectively). Gentamicin, amikacin and plazomicin susceptibility rates (35.6% for gentamicin; 44.4% and 37.8% for amikacin according to CLSI and EUCAST breakpoints, respectively; 64.4% for plazomicin) in carbapenem-resistant K. pneumoniae were significantly lower than those observed for carbapenem-resistant E. coli isolates (73.3% for gentamicin; 93.3% for amikacin and plazomicin). Gentamicin, amikacin and plazomicin susceptibility rates for blaNDM-positive isolates were lower than those observed for blaOXA-48-positive isolates, but differences were not statistically significant. Among the isolates that were non-susceptible to both gentamicin and amikacin, the plazomicin susceptibility rate was less than 30%.Conclusions: Although plazomicin showed excellent in vitro activity against carbapenem-susceptible isolates, the plazomicin resistance rate increased to 35.6% among carbapenem-resistant K. pneumoniae and further increased to 45.5% among blaNDM-positive isolates. [ABSTRACT FROM AUTHOR]

Objectives: Ceftazidime/avibactam is not active against MBL-producing bacteria. Combining ceftazidime/avibactam or avibactam with aztreonam can counter the resistance of MBL-producing Enterobacterales. The aim of this study was to evaluate whether the addition of avibactam could reduce or close the mutant selection window (MSW) of aztreonam in Escherichia coli and Klebsiella pneumoniae harbouring MBLs; MSW is a pharmacodynamic (PD) parameter for the selection of emergent resistant mutants.Methods: In vitro susceptibility of 19 clinical isolates to ceftazidime/avibactam, aztreonam alone, and in co-administration (aztreonam/ceftazidime/avibactam and aztreonam/avibactam) was determined, as well as the mutant prevention concentration (MPC). The fraction of time within 24 h that the free drug concentration was within the MSW (fTMSW) and the fraction of time that the free drug concentration was above the MPC (fT>MPC) in both plasma and epithelial lining fluid (ELF) were determined from simulations of 10 000 profiles. The joint PTA was used to derive a joint cumulative fraction of response (CFR).Results: All isolates were resistant to ceftazidime/avibactam or aztreonam. Combining aztreonam and avibactam or ceftazidime/avibactam resulted in synergistic bactericidal activities against all isolates. Synergism was primarily due to the aztreonam/avibactam combination. For aztreonam/avibactam dosing regimens evaluated in clinical trials, fT>MPC values were >90% and >80%, whereas fTMSW measures were <10% and <20% in plasma and ELF, respectively. The CFR was 100% for aztreonam/avibactam against the collection of clinical isolates.Conclusions: Effective antimicrobial combination optimized the PD parameters measuring selection for emergent mutants by increasing fT>MPC and reducing fTMSW. [ABSTRACT FROM AUTHOR]

Background: The combination aztreonam/avibactam is currently under Phase 3 trials for the treatment of serious infections caused by Gram-negative bacteria including those with MBLs.Objectives: To investigate the resistance mechanisms in Enterobacterales exhibiting aztreonam/avibactam MICs of ≥4 mg/L.Methods: Among 8787 Enterobacterales, 17 (0.2%) isolates exhibited an aztreonam/avibactam MIC of ≥4 mg/L. Isolates were sequenced and screened for β-lactamases. Sequences of porins, penicillin-binding protein 3 (PBP3) and expression levels of AmpC and AcrA were evaluated.Results: Eleven (11/4154 isolates; 0.26%) Escherichia coli, three (3/1981; 0.15%) Klebsiella pneumoniae and three (3/628; 0.5%) Enterobacter cloacae were identified. All E. coli showed either an 'YRIK' or 'YRIN' insertion in PBP3. In general, these isolates carried blaCMY and/or blaCTX-M variants, except for one isolate from Korea that also produced NDM-5 and one isolate from Turkey that produced OXA-48. Two DHA-1-producing K. pneumoniae overexpressed acrA and had a premature stop codon in either OmpK35 or OmpK36, whereas a third K. pneumoniae carried blaPER-2 and had a premature stop codon in OmpK35. All three E. cloacae expressed AmpC at levels ≥570-fold, but sequence analysis did not reveal known amino acid alterations associated with decreased avibactam binding or increased hydrolysis of β-lactams. Minor amino acid polymorphisms within OmpC, OmpF and PBP3 were noted among the E. cloacae.Conclusions: A small number of isolates (0.2%) met the inclusion criteria. E. coli showed altered PBP3 as the most relevant resistance mechanism, whereas K. pneumoniae had multiple resistance mechanisms. Further investigations are needed to clarify resistance in E. cloacae. [ABSTRACT FROM AUTHOR]

Background: There is a growing interest in the rapid genotypic identification of antimicrobial resistance (AMR). In routine diagnostics, we detected multiple KPC-positive Escherichia coli (KPC-Ec) with discordant phenotypic meropenem susceptibility from a single patient's blood cultures, which prompted a more thorough investigation.Objectives: We investigated the potential clinical relevance of, and the mechanism behind, discordant phenotypic and genotypic meropenem susceptibility in KPC-Ec.Methods: WGS was used to perform a comparative analysis of the isolates' genetic characteristics and their blaKPC-2 locus. Expression of blaKPC-2 was determined by quantitative PCR and the potency of meropenem hydrolysis was determined using a semi-quantitative carbapenem inactivation method. An in vivo infection assay using Galleria mellonella was performed to assess the potential clinical relevance of KPC expression in E. coli.Results: Despite the presence of blaKPC-2, three of five isolates were susceptible to meropenem (MICVITEK2 ≤ 0.25 mg/L), while two isolates were resistant (MICVITEK2 ≥ 16 mg/L). The isolates with high MICs had significantly higher blaKPC-2 expression, which corresponds to phenotypic meropenem inactivation. The genetic environment of blaKPC-2, which may impact KPC production, was identical in all isolates. In vivo infection assay with G. mellonella suggested that meropenem was effective in reducing mortality following infection with low-expressing KPC-Ec.Conclusions: Our findings clearly highlight a limitation of genotypic AMR prediction for blaKPC. For the time being, genotypic AMR prediction requires additional analysis for accurate antibiotic therapy decision-making. [ABSTRACT FROM AUTHOR]

Objectives: ESBL-producing Escherichia coli and Klebsiella pneumoniae are pathogens of increasing importance in Canada and elsewhere in the world. The purpose of this study was to phenotypically and molecularly characterize ESBL-producing E. coli and K. pneumoniae clinical isolates obtained from patients attending Canadian hospitals over a 12 year period.Methods: Isolates were collected between January 2007 and December 2018 as part of an ongoing national surveillance study (CANWARD). ESBL production was confirmed using the CLSI (M100) phenotypic method. Susceptibility testing was carried out using custom broth microdilution panels, and all isolates underwent WGS.Results: In total, 671 E. coli and 141 K. pneumoniae were confirmed to be ESBL producers. The annual proportion of ESBL-producing isolates increased for both E. coli (from 3.3% in 2007 to 11.2% in 2018; P < 0.0001) and K. pneumoniae (from 1.3% in 2007 to 9.3% in 2018; P < 0.0001). The most frequent STs were ST131 for E. coli [62.4% (419/671) of isolates] and ST11 [7.8% (11/141)] and ST147 [7.8% (11/141)] for K. pneumoniae. Overall, 97.2% of ESBL-producing E. coli and K. pneumoniae isolates were MDR. blaCTX-M-15 predominated in both ESBL-producing E. coli (62.3% of isolates) and ESBL-producing K. pneumoniae (48.9% of isolates).Conclusions: The proportion of ESBL-producing E. coli, especially ST131, and K. pneumoniae, especially ST11 and ST147, in Canada increased significantly from 2007 to 2018. Continued prospective surveillance of these evolving MDR and at times XDR pathogens is imperative. [ABSTRACT FROM AUTHOR]

Objectives: Bacteria that acquire plasmids incur a biological cost. Despite this fact, clinical Enterobacteriaceae isolates commonly contain multiple co-existing plasmids harbouring carbapenemase genes.Methods: Six different plasmids carrying blaNDM-1, blaNDM-5, blaCTX-M-15, blaKPC-2, blaOXA-181 and blaOXA-232 genes were obtained from Klebsiella pneumoniae and Escherichia coli clinical isolates. Using the E. coli DH5α strain as recipient, 14 transconjugants with diverse plasmid combinations (single or double plasmids) were generated. For each of these, the effects of plasmid carriage on the bacterial host were investigated using in vitro and in vivo competition assays; additionally, the effects were investigated in the context of biofilm formation, serum resistance and survival inside macrophages. Transcriptomic changes in single- and double-plasmid recipients were also investigated.Results: Increased in vitro and in vivo competitiveness was observed when two plasmids carrying blaNDM-1 and blaOXA-232 were co-introduced into the host bacteria. However, DH5α::pNDM5 + pOXA232 and other double-plasmid recipients did not show such competitiveness. DH5α::pNDM5 + pOXA181 did not show any fitness cost compared with a plasmid-free host and single-plasmid transconjugants, while both the double-plasmid recipients with pCTXM15 or pKPC2 exhibited a fitness burden. The double-plasmid recipient DH5α::pNDM1 + pOXA232 also exhibited increased biofilm formation, serum resistance and survival inside macrophages. Transcriptomic analysis revealed that the genes of DH5α::pNDM1 + pOXA232 involved in metabolic pathways, transport and stress response were up-regulated, while those involved in translation were down-regulated.Conclusions: Our study suggests that bacterial strains can gain fitness through the acquisition of multiple plasmids harbouring antibiotic resistance genes, which may be mediated by transcriptomic changes in the chromosomal genes of the bacterial host. [ABSTRACT FROM AUTHOR]

Objectives: To establish the epidemiological cut-off values (ECOFFs) for cefoselis against Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Proteus mirabilis and Pseudomonas aeruginosa.Methods: We collected 2288 non-repetitive clinical isolates from five laboratories throughout four cities in China. The cefoselis MICs and inhibition zone diameters for all isolates were established using the broth microdilution method and the disc diffusion method following EUCAST guidelines. MIC ECOFFs were determined by visual estimation and ECOFFinder software. Zone diameter ECOFFs were set if a high correlation of MICs and inhibition zone diameters was found by Pearson correlation. Zone diameter ECOFFs were finally determined by the visual estimate method.Results: MICs of cefoselis were distributed from 0.008 to >256 mg/L for the four Enterobacterales species and from 0.25 to >256 mg/L for P. aeruginosa. MIC ECOFFs were 0.125 mg/L for E. coli, K. pneumoniae and P. mirabilis, 0.25 mg/L for E. cloacae and 32 mg/L for P. aeruginosa. A high correlation of MICs and zone diameters was observed for all Enterobacterales (|r| > 0.8, P < 0.001) and a relatively high correlation was found for P. aeruginosa (|r| = 0.71, P < 0.001). The zone diameter ECOFF was 24 mm for E. cloacae, E. coli and K. pneumoniae, 26 mm for P. mirabilis and 21 mm for P. aeruginosa.Conclusions: We determined MIC and zone diameter ECOFFs for cefoselis against four Enterobacterales species and P. aeruginosa. The establishment of ECOFFs for cefoselis provides clinicians with helpful guidance to differentiate WT and non-WT pathogens. [ABSTRACT FROM AUTHOR]

Objectives: This was a comparative assessment of WCK 5222 (cefepime/zidebactam 2/1 g as a 1 h infusion every 8 h) efficacy using human-simulated plasma and ELF exposures against serine-carbapenemase-producing Klebsiella pneumoniae in the neutropenic murine pneumonia model.Methods: Ten clinical isolates were utilized: eight were serine-carbapenemase-producing (KPC, n = 4; OXA-48-like, n = 4) Enterobacterales with WCK 5222 MICs (1:1) ranging from 1 to 4 mg/L; and two were previously studied MDR isolates serving as quality controls. Lungs of mice were inoculated with 50 μL of 107 cfu/mL. Treatment mice received human-simulated regimens of cefepime, zidebactam or WCK 5222 derived from plasma or epithelial lining fluid (ELF) profiles obtained from healthy subjects. Lung bacterial densities resulting from the humanized exposures in plasma and ELF were compared.Results: Initial lung bacterial densities ranged from 6.06 to 6.87 log10 cfu/lungs, with a mean bacterial burden increase to 9.06 ± 0.42 after 24 h. Human-simulated plasma and ELF exposures of cefepime and zidebactam monotherapy had no activity. Human-simulated WCK 5222 plasma exposures resulted in a >1 log10 cfu/lungs reduction in bacterial burden for all isolates. Humanized WCK 5222 ELF exposures achieved a >1 log10 cfu/lungs reduction for all isolates. While statistically significant differences in bacterial burden reduction were observed between the plasma and ELF exposures for WCK 5222 in 5/8 isolates, all treatments achieved the translational kill target of a >1 log10 cfu reduction.Conclusions: Clinically achievable WCK 5222 plasma and ELF exposures produced in vivo killing of carbapenem-resistant Enterobacterales in the neutropenic murine pneumonia model that is predictive of efficacy in humans. [ABSTRACT FROM AUTHOR]

Background: Recent studies indicated that the monosubstituted deoxystreptamine aminoglycoside apramycin is a potent antibiotic against a wide range of MDR Gram-negative pathogens.Objectives: To evaluate the in vitro activity of apramycin against carbapenem-resistant Klebsiella pneumoniae (CRKp) isolates from New York and New Jersey, and to explore mechanisms of apramycin resistance.Methods: Apramycin MICs were determined by broth microdilution for 155 CRKp bloodstream isolates collected from 2013 to 2018. MLST STs, wzi capsular types and apramycin resistance gene aac(3')-IV were examined by PCR and Sanger sequencing. Selected isolates were further characterized by conjugation experiments and WGS.Results: Apramycin MIC50/90 values were 8 and >128 mg/L for CRKp isolates, which are much higher than previously reported. Twenty-four isolates (15.5%) were apramycin resistant (MIC ≥64 mg/L) and they were all from the K. pneumoniae ST258 background. The 24 apramycin-resistant K. pneumoniae ST258 strains belonged to six different capsular types and 91.7% of them harboured the apramycin resistance gene aac(3')-IV. Sequencing analysis showed that different ST258 capsular type strains shared a common non-conjugative IncR plasmid, co-harbouring aac(3')-IV and blaKPC. A novel IncR and IncX3 cointegrate plasmid, p59494-RX116.1, was also identified in an ST258 strain, demonstrating how apramycin resistance can be spread from a non-conjugative plasmid through cointegration.Conclusions: We described a high apramycin resistance rate in clinical CRKp isolates in the New York/New Jersey region, mainly among the epidemic K. pneumoniae ST258 strains. The high resistance rate in an epidemic K. pneumoniae clone raises concern regarding the further optimization and development of apramycin and apramycin-like antibiotics. [ABSTRACT FROM AUTHOR]

Objectives: Carbapenem-resistant Klebsiella pneumoniae (CR-KP) infections represent severe threats to public health worldwide. The aim of this study was to assess potential synergistic interaction between tigecycline and aminoglycosides via in vitro and in vivo studies.Methods: Antibiotic resistance profiles and molecular characteristics of 168 CR-KP clinical isolates were investigated by susceptibility testing, PCR and MLST. Chequerboard tests and time-kill assays were performed for 20 CR-KP isolates to evaluate in vitro synergistic effects of tigecycline combined with aminoglycosides. A tissue-cage infection model of rats was established to evaluate in vivo synergistic effects. Different doses of tigecycline and aminoglycosides alone or in combination were administered for 7 days via tail vein injection. Antibiotic efficacy was evaluated in tissue-cage fluid and emergence of resistance was screened.Results: The chequerboard tests showed that this combination displayed synergistic or partial synergistic activity against CR-KP. The time-kill assays further demonstrated that strong synergistic effects of such a combination existed against isolates that were susceptible to both drugs but for resistant isolates no synergy was observed if clinical pharmacokinetics were taken into consideration. The in vivo study showed that the therapeutic effectiveness of combination therapies was better than that of monotherapy for susceptible isolates, suggesting in vivo synergistic effects. Furthermore, combinations of tigecycline with an aminoglycoside showed significant activity in reducing the occurrence of tigecycline-resistant mutants.Conclusions: Compared with single drugs, tigecycline combined with aminoglycosides could exert synergistic effects and reduce the emergence of tigecycline resistance. Such a combination might be an effective alternative when treating CR-KP infections in clinical practice. [ABSTRACT FROM AUTHOR]

Objectives: To define characteristics of Klebsiella pneumoniae complex (hereafter KP) isolates from healthy pigs, farm workers and their household members in Thailand.Methods: A total of 839 individual rectal swabs from pigs on 164 farms and 271 faecal samples of humans working on pig farms and persons living in the same household in Khon Kaen, Thailand were screened for gut colonization by KP. Genomic sequences were investigated for antibiotic resistance and virulence genes. Phylogenetic analyses were performed in addition to comparison with isolates from previous studies from Thailand.Results: KP was detected in approximately 50% of pig and human samples. In total, 253 KP isolates were obtained: 39% from pigs, 34% from farmers and 26% from individuals living on the same farm but without animal contact. MLST revealed high genetic diversity with 196 different STs distributed over four phylogroups (Kp1 to Kp4). Low prevalence of ESBL-KP (7.5%) and colistin-resistant KP (3.2%) was observed among pigs and humans. Remarkably, four convergent MDR and hypervirulent strains were observed: one from pigs (ST290) and three from humans [ST35, ST3415 (strain 90CP1), ST17 (strain 90CM2)]. Sharing of KP clones among pigs and humans was identified for some STs including ST4788, ST661, ST3541 and ST29.Conclusions: The study indicated a low prevalence of ESBL and mcr genes among KP isolated from pigs and healthy humans in Thailand and suggested the possibility of zoonotic transmission for a subset of circulating KP clones. [ABSTRACT FROM AUTHOR]

Objectives: Fosfomycin is an important antibiotic for the treatment of MDR Enterobacteriaceae infections. High susceptibility rates are, however, threatened by the spread of plasmids encoding fosfomycin-modifying enzymes. In this study, we sought to characterize the genetic context of fosA in plasmids from Escherichia coli and Klebsiella spp. isolates recovered from food, wastewater and surface water in Switzerland.Methods: E. coli and Klebsiella spp. isolates collected between 2012 and 2019 in Switzerland were screened for fosfomycin resistance. Presence of fosA was verified by PCR and sodium phosphonoformate (PPF) disc potentiation testing, and transferability was tested using conjugation assays. Whole-genome sequences including complete fosA-containing plasmids were determined using long- and short-read sequencing.Results: In 11 E. coli and two Klebsiella spp. isolates, high-level fosfomycin resistance was mediated by plasmids containing fosA3 (n = 12) or fosA8 (n = 1). Four isolates harboured a near-identical 45 kb IncN plasmid with fosA3, while replicon types varied in the remaining plasmids. The fosA genes were typically embedded in IS26-bounded transposition units and frequently located in the proximity of blaCTX-M transposition units.Conclusions: Although fosfomycin resistance rates are currently low, the presence of fosA-encoding plasmids circulating in the Enterobacteriaceae population suggests that fosfomycin resistance may rapidly spread upon increased selection pressure. Transposition mobility of fosA and co-location on plasmids with other resistance genes may further promote its dissemination. [ABSTRACT FROM AUTHOR]

Objectives: Long-term care facilities (LTCFs) may act as a reservoir of ESBL-producing Enterobacterales (ESBL-E) and carbapenemase-producing Enterobacterales (CPE) for hospitals and the general population. In this study, we estimated the prevalence and molecular epidemiology of rectal carriage with ESBL-E and CPE in residents of Dutch LTCFs between March 2018 and December 2018.Methods: LTCFs were geographically selected across the country. For each LTCF, a random sample of residents were tested for ESBL-E and CPE in 2018. To identify risk factors for high carriage prevalence and/or individual carriage, characteristics of LTCFs and of a subset of the tested residents were collected. WGS was conducted on isolates from LTCFs with an ESBL-E prevalence of >10% and all CPE isolates to identify institutional clonal transmission.Results: A total of 4420 residents of 159 LTCFs were included. The weighted mean ESBL-E prevalence was 8.3% (95% CI: 6.8-10.0) and no CPE were found. In 53 LTCFs (33%), where ESBL-E prevalence was >10%, MLST using WGS (wgMLST) was performed. This included 264 isolates, the majority being Escherichia coli (n = 224) followed by Klebsiella pneumoniae (n = 30). Genetic clusters were identified in more than half (30/53; 57%) of high ESBL-positive LTCFs. Among the E. coli isolates, blaCTX-M-15 (92/224; 41%) and blaCTX-M-27 (40/224; 18%) were the most prevalent ESBL-encoding genes. For K. pneumoniae isolates, the most common was blaCTX-M-15 (23/30; 80%).Conclusions: The estimated prevalence of ESBL-E rectal carriage in Dutch LTCFs is 8.3% and resistance is observed mainly in E. coli with predominance of blaCTX-M-15 and blaCTX-M-27. ESBL-E prevalence in LTCFs seems comparable to previously reported prevalence in hospitals and the general population. [ABSTRACT FROM AUTHOR]

Objectives: Infections with carbapenem-resistant Enterobacterales (CRE) are an emerging problem in pets and a major threat to public health. We determined the genetic relationships among carbapenemase-producing Klebsiella pneumoniae (CPKp) strains causing infections in hospitalized pets in a veterinary clinic and those found in the environment.Methods: WGS was performed with both the Illumina and Nanopore platforms. Searches of genetic features were performed using several databases and bioinformatics tools, and phylogeny was assessed by whole-genome MLST (wgMLST) using SeqSphere and SNP calling with Snippy.Results: WGS analysis of the CPKp strains identified all environmental and almost all animal strains as the high-risk clone ST11, with the exception of two strains that belonged to ST307. All CPKp belonged to novel complex types (CTs) and carried a conjugative 63 kb IncL plasmid encoding the carbapenemase gene blaOXA-48, yersiniabactin and other virulence factors. Although all CPKp ST11 strains carried additional similar IncR plasmids harbouring multiple antimicrobial resistance genes (ARGs), such as the plasmid-mediated blaDHA-1 AmpC gene, some structural variations were observed. The two ST307 strains carried identical 156 kb MDR IncFIB(K) plasmids with several ARGs, including the blaCTX-M-15 ESBL gene. Both wgMLST and cgSNP analysis confirmed that CPKp strains of the same ST were genetically highly related independent of the source of isolation.Conclusions: This study demonstrated that the clinical CPKp strains were highly related to those contaminating the clinical environment. These findings confirmed nosocomial spread and highlight veterinary hospitals as a source of CPKp, which may further spread to animals, the environment and humans. [ABSTRACT FROM AUTHOR]

Background: Ceftriaxone is the preferred treatment for bacteraemia caused by non-MDR (antibiotic-susceptible) Klebsiella pneumoniae. Excessive and widespread ceftriaxone use creates selection pressure for ESBLs. Cefazolin is an alternative, although there are theoretical concerns that SHV-1 β-lactamase in K. pneumoniae may inactivate cefazolin in an inoculum-dependent manner.Objectives: In this retrospective study, we investigated the outcomes in K. pneumoniae bacteraemia patients treated with IV cefazolin versus IV ceftriaxone as definitive therapy.Methods: A total of 917 patients infected with K. pneumoniae from 1 January to 31 December 2016 in three public acute care hospitals in Singapore were screened for study eligibility. Consecutive unique episodes of monomicrobial bacteraemia caused by cefazolin- and/or ceftriaxone-susceptible K. pneumoniae were analysed (n = 284).Results: There were 143 patients (50.4%) in the cefazolin group and 141 patients (49.6%) in the ceftriaxone group. Demographics, baseline illness severity and risk factors for healthcare-associated bacteraemia were comparable in the two treatment groups. The primary outcome of 28 day all-cause mortality was not significantly different between the cefazolin and ceftriaxone groups (10.5% versus 7.1%, P = 0.403). Both in the crude analysis and using a multivariable logistic regression model with inverse probability weighting based on propensity score, cefazolin treatment was not associated with increased risk of 28 day mortality (OR 1.51 with ceftriaxone as the reference group, 95% CI 0.67-3.53; adjusted OR 1.55, 95% CI 0.33-7.40).Conclusions: Cefazolin may be a ceftriaxone-sparing alternative treatment for antibiotic-susceptible K. pneumoniae bacteraemia. This observation may provide sufficient clinical equipoise for a randomized controlled trial. [ABSTRACT FROM AUTHOR]

Background: A patient repeatedly developed bacteraemia despite the continuous use of antibiotics. We obtained two Klebsiella pneumoniae isolates from the patient's blood on Days 72 and 105 after hospitalization. Each of the two isolates belonged to ST45, but while the first isolate was susceptible to most antibiotics, the second one was resistant to multiple drugs including carbapenems.Objectives: To identify the genetic differences between the two isolates and uncover alterations formed by the within-host bacterial evolution leading to the antimicrobial resistance.Methods: Whole-genome comparison of the two isolates was carried out to identify their genetic differences. We then profiled their outer membrane proteins related to membrane permeability to drugs. To characterize a ramR gene mutation found in the MDR isolate, its WT and mutant genes were cloned and expressed in the MDR isolate.Results: The two isolates showed only three genomic differences, located in mdoH, ramR and upstream of ompK36. In the MDR isolate, a single nucleotide substitution in the ompK36 upstream region attenuated OmpK36 expression. A single amino acid residue insertion in RamR in the MDR isolate impaired its function, leading to the down-regulation of OmpK35 and the subsequent up-regulation of the AcrAB-TolC transporter, which may contribute to the MDR.Conclusions: We identified very limited genomic changes in the second K. pneumoniae clone during within-host evolution, but two of the three identified mutations conferred the MDR phenotype on the clone by modulating drug permeability. [ABSTRACT FROM AUTHOR]

The near-identical plasmid sequences and epidemiological data suggest that the ST585 strains were the donors of the resistance plasmid to ST218 strains. In this study, we found that the hvKP clone ST218 developed carbapenem resistance in a single patient over time by acquiring the I bla i SB KPC sb plasmid. [Extracted from the article]

Objectives: Carbapenemase-producing Enterobacterales (CPE) are increasingly recognized in nosocomial infections, also affecting ICU patients. We aimed to characterize the carbapenemase-producing Serratia marcescens (CPSm) isolates recovered in our hospital in Madrid (Spain) between March 2016 and December 2018.Methods: Overall, 50 isolates from clinical and epidemiological surveillance samples were recovered from 24 patients admitted to the medical ICU and 10 non-ICU-related patients based on their phenotypic resistance. Carbapenemase characterization, antibiotic susceptibility, PFGE clonal relatedness, plasmid characterization, WGS (Illumina-NovaSeq 6000) and phylogenetic analysis were performed.Results: A single isolate was finally considered for each patient, except for Patient 8 that was colonized by two different isolates (n = 35). Isolates were characterized as VIM-1 (n = 29) or OXA-48 producers (n = 6). Up to seven genetic lineages were found by PFGE, with dominance of two clones. Plasmid characterization confirmed that almost all CPSm carried the same ∼60 kb IncL OXA-48- or VIM-1-encoding plasmid, which was related to the globally disseminated IncL-pOXA-48a. WGS allowed plasmid reconstruction with two variants: IncL-pVIM-1 (∼65 kb) and IncL-pOXA-48 (∼62 kb). blaOXA-48-Tn1999 (∼5 kb) was the unique antibiotic resistance gene in pOXA-48, whereas pVIM-1 plasmids (∼8 kb) harboured a class 1 integron containing 5'-blaVIM-1+aacA4+dfrB1+aadA1+catB2+qacEDelta1+sul1-3'.Conclusions: Our results confirm the dissemination of CPSm within our institution in both ICU and non-ICU environments, representing two prevalent CPSm clones, and the same IncL-pOXA-48 plasmid previously described in other Enterobacterales, but containing the blaVIM-1 gene. This also reinforces the relevance of species different from Klebsiella pneumoniae or Escherichia coli in the CPE landscape and circulating lineages and plasmids in local CPE epidemiology. [ABSTRACT FROM AUTHOR]

Background: Mecillinam (amdinocillin) is active against Gram-negative bacteria. Clinical data on the efficacy of IV mecillinam for severe urinary tract infections is sparse.Objectives: To assess the effectiveness of targeted IV mecillinam compared with other β-lactams for bacteraemia with Escherichia coli and Klebsiella spp. and a urinary tract focus.Patients and Methods: We performed a retrospective cohort study at five university hospitals in the Capital Region of Denmark from 1 January 2012 to 31 December 2017. We used Cox proportional hazard regression to compare the primary composite endpoint (all-cause mortality or bacteraemia recurrence within 30 days) between patients treated with mecillinam versus ampicillin, cefuroxime, piperacillin/tazobactam and meropenem.Results: We included 1129 patients in the primary analysis, of which 146 were given IV mecillinam as targeted treatment. We found no significant difference in the primary endpoint between patients treated with mecillinam versus ampicillin and cefuroxime, but found a higher risk for the primary endpoint in the piperacillin/tazobactam and meropenem groups, with adjusted HRs of 2.22 (95% CI 1.24-3.97, P < 0.01) and 2.48 (95% CI 1.04-5.93, P = 0.04), respectively, compared with mecillinam.Conclusions: The results of this study suggest that IV mecillinam may be a suitable targeted treatment for bacteraemia with a urinary tract focus. However, these results need confirmation by randomized controlled studies. [ABSTRACT FROM AUTHOR]

Objectives: Microbial resistance exhibits dependency patterns between different antibiotics, termed cross-resistance and collateral sensitivity. These patterns differ between experimental and clinical settings. It is unclear whether the differences result from biological reasons or from confounding, biasing results found in clinical settings. We set out to elucidate the underlying dependency patterns between resistance to different antibiotics from clinical data, while accounting for patient characteristics and previous antibiotic usage.Methods: Additive Bayesian network modelling was employed to simultaneously estimate relationships between variables in a dataset of bacterial cultures derived from hospitalized patients and tested for resistance to multiple antibiotics. Data contained resistance results, patient demographics and previous antibiotic usage, for five bacterial species: Escherichia coli (n = 1054), Klebsiella pneumoniae (n = 664), Pseudomonas aeruginosa (n = 571), CoNS (n = 495) and Proteus mirabilis (n = 415).Results: All links between resistance to the various antibiotics were positive. Multiple direct links between resistance of antibiotics from different classes were observed across bacterial species. For example, resistance to gentamicin in E. coli was directly linked with resistance to ciprofloxacin (OR = 8.39, 95% credible interval 5.58-13.30) and sulfamethoxazole/trimethoprim (OR = 2.95, 95% credible interval 1.97-4.51). In addition, resistance to various antibiotics was directly linked with previous antibiotic usage.Conclusions: Robust relationships among resistance to antibiotics belonging to different classes, as well as resistance being linked to having taken antibiotics of a different class, exist even when taking into account multiple covariate dependencies. These relationships could help inform choices of antibiotic treatment in clinical settings. [ABSTRACT FROM AUTHOR]

Objectives: To assess the impact of ESBL production on mortality and length of hospital stay (LOS) of community-onset infections due to Escherichia coli or Klebsiella pneumoniae.Methods: A population-based cohort study including all adult patients hospitalized with a first-time community-onset E. coli or K. pneumoniae bacteraemia or urinary tract infection in the North Denmark Region between 2007 and 2017. For each bacterial agent, we computed 1 year Kaplan-Meier survival curves and cumulative incidence functions of LOS, and by use of Cox proportional hazard regression we computed HRs as estimates of 30 day and 1 year mortality rate ratios (MRRs) and LOS among patients with and without ESBL-producing infections.Results: We included 24 518 cases (among 22350 unique patients), of whom 1018 (4.2%) were infected by an ESBL-producing bacterium. The 30 day cumulative mortality and adjusted MRR (aMRR) in patients with and without ESBL-producing isolates was as follows: E. coli bacteraemia (n = 3831), 15.8% versus 14.0%, aMRR = 1.01 (95% CI = 0.70-1.45); E. coli urinary tract infection (n = 17151), 9.5% versus 8.7%, aMRR = 0.97 (95% CI = 0.75-1.26); K. pneumoniae bacteraemia (n = 734), 0% versus 17.2%, aMRR = not applicable; and K. pneumoniae urinary tract infection (n = 2802), 13.8% versus 10.7%, aMRR = 1.13 (95% CI = 0.73-1.75). The 1 year aMRR remained roughly unchanged. ESBL-producing E. coli bacteraemia was associated with an increased LOS compared with non-ESBL production.Conclusions: ESBL production was not associated with an increased short- or long-term mortality in community-onset infections due to E. coli or K. pneumoniae, yet ESBL-producing E. coli bacteraemia was associated with an increased LOS. [ABSTRACT FROM AUTHOR]

Objectives: This study was designed to understand the prevalence of antibiotic-resistant bacteria in the Beijing subway environment and the potential transmission of carbapenem-resistant Enterobacteriaceae in a public transportation environment.Methods: Carbapenem-resistant isolates were selected on brain heart infusion agar supplemented with meropenem (0.5 mg/L) and antimicrobial susceptibility testing was conducted using the broth microdilution method. WGS analyses were conducted for 11 Klebsiella pneumoniae isolates to identify resistance genes. The genetic relationships among the isolates were evaluated by MLST and PFGE.Results: We identified 11 carbapenem-resistant K. pneumoniae isolates from the Beijing subway environment. WGS revealed three STs among the 11 isolates, with 9 isolates classified as ST726 and containing a blaNDM-5-carrying IncX3 plasmid. The genetic environment of blaNDM-5 was very similar to that observed in other blaNDM-5-containing clinical isolates.Conclusions: The presence of carbapenem-resistant Enterobacteriaceae in a public transportation environment is concerning and indicates that regular antimicrobial resistance surveillance is urgent and necessary. [ABSTRACT FROM AUTHOR]

Background: The increased incidence of polymyxin-resistant MDR Klebsiella pneumoniae has become a major global health concern.Objectives: To characterize the lipid A profiles and metabolome differences between paired polymyxin-susceptible and -resistant MDR K. pneumoniae clinical isolates.Methods: Three pairs of K. pneumoniae clinical isolates from the same patients were examined [ATH 7 (polymyxin B MIC 0.25 mg/L) versus ATH 8 (64 mg/L); ATH 15 (0.5 mg/L) versus ATH 16 (32 mg/L); and ATH 17 (0.5 mg/L) versus ATH 18 (64 mg/L)]. Lipid A and metabolomes were analysed using LC-MS and bioinformatic analysis was conducted.Results: The predominant species of lipid A in all three paired isolates were hexa-acylated and 4-amino-4-deoxy-l-arabinose-modified lipid A species were detected in the three polymyxin-resistant isolates. Significant metabolic differences were evident between the paired isolates. Compared with their corresponding polymyxin-susceptible isolates, the levels of metabolites in amino sugar metabolism (UDP-N-acetyl-α-d-glucosamine and UDP-N-α-acetyl-d-mannosaminuronate) and central carbon metabolism (e.g. pentose phosphate pathway and tricarboxylic acid cycle) were significantly reduced in all polymyxin-resistant isolates [fold change (FC) > 1.5, P < 0.05]. Similarly, nucleotides, amino acids and key metabolites in glycerophospholipid metabolism, namely sn-glycerol-3-phosphate and sn-glycero-3-phosphoethanolamine, were significantly reduced across all polymyxin-resistant isolates (FC > 1.5, P < 0.05) compared with polymyxin-susceptible isolates. However, higher glycerophospholipid levels were evident in polymyxin-resistant ATH 8 and ATH 16 (FC > 1.5, P < 0.05) compared with their corresponding susceptible isolates.Conclusions: To our knowledge, this study is the first to reveal significant metabolic perturbations associated with polymyxin resistance in K. pneumoniae. [ABSTRACT FROM AUTHOR]

Objectives: To compare bacterial killing and the emergence of resistance to piperacillin/tazobactam, administered by intermittent versus prolonged infusion (i.e. extended or continuous), for ceftriaxone-resistant Klebsiella pneumoniae clinical isolates in an in vitro dynamic hollow-fibre infection model (HFIM).Methods: K. pneumoniae 68 (Kp68; MIC = 8 mg/L, producing SHV-106 and DHA-1) and K. pneumoniae 69 (Kp69; MIC = 1 mg/L, producing CTX-M-14) were studied in the HFIM over 7 days (initial inoculum ~107 cfu/mL). Six piperacillin/tazobactam dosing regimens for Kp68 (4/0.5 g 8 hourly as 0.5 and 4 h infusions, 12/1.5 g/24 h continuous infusion, 4/0.5 g 6 hourly as 0.5 and 3 h infusions and 16/2 g/24 h continuous infusion) and three piperacillin/tazobactam dosing regimens for Kp69 (4/0.5 g 8 hourly as 0.5 and 4 h infusions and 12/1.5 g/24 h continuous infusion) were simulated (piperacillin clearance = 14 L/h, creatinine clearance = 100 mL/min). Total and resistant populations and MICs were quantified/determined.Results: For Kp68, all simulated dosing regimens exhibited approximately 4 log10 of bacterial killing at 8 h followed by regrowth to approximately 1011 cfu/mL within 24 h. The MICs for resistant subpopulations exceeded 256 mg/L at 72 h. Similarly, for Kp69, all simulated dosing regimens exhibited approximately 4 log10 of bacterial killing over 8 h; however, only the continuous infusion prevented bacterial regrowth.Conclusions: Compared with intermittent infusion, prolonged infusion did not increase initial bacterial killing and suppression of regrowth of plasmid-mediated AmpC- and ESBL-producing K. pneumoniae. However, continuous infusion may suppress regrowth of some ESBL-producing susceptible K. pneumoniae, although more data are warranted to confirm this observation. [ABSTRACT FROM AUTHOR]

Objectives: To investigate the nationwide prevalence of mcr-1-positive Klebsiella pneumoniae (MCRPKP) strains among healthy adults in China and identify their phenotypic and genomic characterizations.Methods: A total of 7401 rectal swab samples were collected from healthy individuals in 30 hospitals located in 30 provinces and municipalities of mainland China in 2016. Colistin-resistant bacteria were enriched in colistin-supplemented lysogeny broth. MCRPKP strains were isolated and characterized with MALDI-TOF MS, PCR analysis and antimicrobial susceptibility testing. The genomic characteristics of MCRPKP strains were determined by WGS and bioinformatics analysis.Results: Seven MCRPKP strains and one mcr-1-positive Klebsiella variicola strain were selectively isolated from six locales (three from Henan and one from each of Tianjin, Jiangxi, Yunnan, Gansu and Tibet). Antimicrobial susceptibility testing results indicated that all mcr-1-positive strains were susceptible to meropenem, aztreonam and ceftazidime/avibactam. WGS analysis suggested these strains belonged to seven distinct STs: ST15, ST1425, ST1462, ST273, ST307, ST391 and ST37-SLV. mcr-1 genes were carried by diverse plasmids, including IncHI2 (n = 3), IncX4 (n = 2), IncHI2/IncN (n = 1), IncFIB (n = 1) and one other plasmid type. Two ST15 strains harboured both mcr-1 and mcr-8 genes, which has not been reported before.Conclusions: Our data indicated a low prevalence of mcr-1-positive Klebsiella strains (0.11%, 8/7401) in healthy individuals in mainland China and most of these strains remained susceptible to clinically important antibiotics. The prevalence and coexistence of mcr-1 and mcr-8 in K. pneumoniae may further threaten public health through either the food chain or environmental routes. [ABSTRACT FROM AUTHOR]

Objectives: To determine trends, seasonality and the association between community antibiotic use and antimicrobial resistance (AMR) in Escherichia coli and Klebsiella pneumoniae in urinary tract infections.Methods: We analysed Dutch national databases from January 2008 to December 2016 regarding antibiotic use and AMR for nitrofurantoin, trimethoprim, fosfomycin and ciprofloxacin. Antibiotic use was expressed as DDD/1000 inhabitant-days (DID) and AMR was expressed as the percentage of resistance from total tested isolates. Temporal trends and seasonality were analysed with autoregressive integrated moving average (ARIMA) models. Each antibiotic use-resistance combination was cross-correlated with a linear regression of the ARIMA residuals.Results: The trends of DID increased for ciprofloxacin, fosfomycin and nitrofurantoin, but decreased for trimethoprim. Similar trends were found in E. coli and K. pneumoniae resistance to the same antibiotics, except for K. pneumoniae resistance to ciprofloxacin, which decreased. Resistance levels peaked in winter/spring, whereas antibiotic use peaked in summer/autumn. In univariate analysis, the strongest and most significant cross-correlations were approximately 0.20, and had a time delay of 3-6 months between changes in antibiotic use and changes in resistance. In multivariate analysis, significant effects of nitrofurantoin use and ciprofloxacin use on resistance to these antibiotics were found in E. coli and K. pneumoniae, respectively. There was a significant association of nitrofurantoin use with trimethoprim resistance in K. pneumoniae after adjusting for trimethoprim use.Conclusions: We found a relatively low use of antibiotics and resistance levels over a 9 year period. Although the correlations were weak, variations in antibiotic use for these four antibiotics were associated with subsequent variations in AMR in urinary pathogens. [ABSTRACT FROM AUTHOR]

Background: Omadacycline, an aminomethylcycline, was approved in 2018 for the treatment of acute bacterial skin and skin structure infections and community-acquired bacterial pneumonia. In a Phase Ib study, around 34% of the absorbed dose of omadacycline was shown to be excreted in urine-an important property for urinary tract infection (UTI) treatment. Therefore, omadacycline has been studied in two Phase II trials for the treatment of uncomplicated UTIs and acute pyelonephritis. The activity of omadacycline against UTI pathogens in human urine is important to understand in this context.Objectives: To study the in vitro activity of omadacycline against UTI pathogens in human urine supplemented with calcium and magnesium.Methods: Omadacycline activity was compared with that of levofloxacin against the urinary pathogens Escherichia coli, Klebsiella pneumoniae and Staphylococcus saprophyticus in standard medium, pooled normal human urine and neutral pH-adjusted pooled normal human urine spiked with calcium or magnesium at concentrations consistent with hypercalcaemia and hypermagnesaemia.Results: The activities of omadacycline and levofloxacin against these urinary pathogens were lower in urine relative to standard medium; addition of Mg2+ to broth and urine had a further negative impact on omadacycline activity, whereas the addition of Ca2+ had less of an impact. Levofloxacin activity was not substantially reduced in either broth or urine by the addition of divalent cations.Conclusions: The activity of omadacycline against UTI organisms was lower in urine relative to standard medium and was negatively impacted by magnesium. Omadacycline displayed slightly reduced activity when excess calcium was present, but, overall, the differences were ≤2-fold. These observations should be considered along with the pharmacokinetics of the agent for clinical context. [ABSTRACT FROM AUTHOR]

Background: KPC-producing Klebsiella pneumoniae of clonal group 258 are prominent in healthcare settings in many regions of the world. The blaKPC gene is mostly carried by a multireplicon IncFIIk-IncFI plasmid suspected to be highly compatible and stable in this genetic background. Here, we analysed the genetic diversity of an ST512 K. pneumoniae population in a single patient.Methods: Twelve K. pneumoniae isolates (n = 5 from urine samples and n = 7 from rectal swabs) were recovered from one patient over a 2 month period. Antimicrobial susceptibility testing, plasmid extraction and WGS were performed on all isolates. The first K. pneumoniae isolate, D1, was used as a reference for phylogenetic analysis.Results: Antimicrobial susceptibility testing, plasmid analysis and WGS revealed concomitant carriage of carbapenem-resistant and carbapenem-susceptible K. pneumoniae isolates of ST512, with the absence of the entire blaKPC-carrying plasmid in the susceptible population. Furthermore, 14 other genetic events occurred within the genome, including 3 chromosomal deletions (of 71 kb, 33 kb and 11 bp), 2 different insertions of ISKpn26 and 9 SNPs. Interestingly, most of the events occurred in the same chromosomal region that has been deleted independently several times, probably after homologous recombination involving 259 bp repeated sequences.Conclusions: Our study revealed (to the best of our knowledge) the first case of in vivo blaKPC-carrying plasmid curing and a wide within-patient genetic diversity of a single K. pneumoniae ST512 clone over a short period of carriage. This within-patient diversity must be taken into account when characterizing transmission chains using WGS during nosocomial outbreaks. [ABSTRACT FROM AUTHOR]