Characteristics and coordinated mechanisms of carbapenem heteroresistance in KPC-producing Enterobacteriaceae

Enterobacteriaceae strains producing the Klebsiella pneumoniae carbapenemase (KPC) have disseminated worldwide, causing an urgent threat to public health. KPC-producing strains often exhibit low-level carbapenem resistance, which may be missed by automated clinical detection systems. In these studies, eight Klebsiella pneumoniae strains, one Enterobacter cloacae strain and one Escherichia coli strain with heterogeneous resistance to imipenem were used to elucidate the factors leading from imipenem susceptibility (1-2ug/ml) to high-level resistance (>32ug/ml) as determined by clinical laboratory testing standards. These strains had highly similar heteroresistance phenotypes, yet were genetically diverse in their plasmid content other than carriage of the blaKPC-containing Tn4401. We showed that the heteroresistant strains had effective KPC-mediated inactivation of low levels of imipenem, combined with reduced or absent expression of ompK35, but as the imipenem concentration increased to 8- and 16-fold higher than the MIC, a majority (>99%) of the population was killed. Time-kill analysis with an inoculum as low as 3x106 cfu/ml showed that full recovery of the population occurred by 20 hours of incubation in the same drug concentrations. We showed that it was not the density of the cultures per se, but cells with a distinct physiology, present at a frequency of 2x10-7 to 3x10-6 in starting cultures, and selected by lethal concentrations of imipenem, that coordinate population recovery subsequent to the killing of the majority of the initial cells. Samples selected 2 hours after exposure to imipenem were as susceptible as the unexposed parental strain and produced the major outer membrane porin OmpK36. However, between 4 to 8 hours after exposure, OmpK36 became absent and the imipenem MIC increased at least 32-fold. Individual colonies isolated from cultures after 20 hours of exposure revealed both susceptible and resistant subpopulations. We hypothesize that these heterogeneous populations arose from the small population of cells that initially survived imipenem-mediated killing. There were two types of OmpK36 production among the K. pneumoniae heteroresistant strains. The majority (6 of 8) of the strains permanently abolished OmpK36 upon lethal imipenem exposure due to mobile insertion element interruptions in the coding region of ompK36. High-level imipenem resistance was maintained and OmpK36 remained absent even without continued carbapenem exposure. Two strains reverted to the heteroresistance phenotype and resumed production of OmpK36 once imipenem exposure was removed. Through transposon mutagenesis of a reverter-type heteroresistant K. pneumoniae strain, we showed that acquisition of specific nutrients was essential to abolish OmpK36 production and for population recovery. Addition of the KPC enzyme inhibitor phenylboronic acid (PBA) at any point during imipenem exposure prior to loss of OmpK36 inhibited subsequent OmpK36 loss and prevented population recovery, showing the essentiality of blaKPC in the expression of heteroresistance. These studies demonstrated the coordination between bacterial physiology, blaKPC and ompK36 expression that led to the rapid induction of high-level imipenem resistance from a population of bacteria that initially exhibited a carbapenem-susceptibility phenotype. The finding of specific nutritional requirements for full expression of heteroresistance highlights potential therapeutic avenues of exploration for these strains that pose an urgent and increasing threat to public health

Genomic Analysis of Factors Associated with Low Prevalence of Antibiotic Resistance in Extraintestinal Pathogenic Escherichia coli Sequence Type 95 Strains

Extraintestinal pathogenic Escherichia coli (ExPEC) strains belonging to multilocus sequence type 95 (ST95) are globally distributed and a common cause of infections in humans and domestic fowl. ST95 isolates generally show a lower prevalence of acquired antimicrobial resistance than other pandemic ExPEC lineages. We took a genomic approach to identify factors that may underlie reduced resistance. We fully assembled genomes for four ST95 isolates representing the four major fimH-based lineages within ST95 and also analyzed draft-level genomes from another 82 ST95 isolates, largely from the western United States. The fully assembled genomes of antibiotic-resistant isolates carried resistance genes exclusively on large (\u3e90-kb) IncFIB/IncFII plasmids. These replicons were common in the draft genomes as well, particularly in antibiotic-resistant isolates, but we also observed multiple instances of a smaller (8.3-kb) ampicillin resistance plasmid that had been previously identified in Salmonella enterica. Among ST95 isolates, pansusceptibility to antibiotics was significantly associated with the fimH6 lineage and the presence of homologs of the previously identified 114-kb IncFIB/IncFII plasmid pUTI89, both of which were also associated with reduced carriage of other plasmids. Potential mechanistic explanations for lineage- and plasmid-specific effects on the prevalence of antibiotic resistance within the ST95 group are discussed

Zidovudine enhances activity of carbapenems against NDM-1-producing Enterobacteriaceae.

OBJECTIVES: To investigate the efficacy of zidovudine in combination with carbapenems against NDM-1-producing Enterobacteriaceae. METHODS: MICs were determined using the broth microdilution method. The combinatory effects of zidovudine and carbapenems were examined using the chequerboard method and time-kill analysis. RESULTS: We found that the NDM-1-producing strains were resistant to all carbapenems tested. FIC index from chequerboard assay demonstrated that zidovudine synergized with carbapenems against all the NDM-1 strains. Time-kill analysis demonstrated significant synergistic activity when a low level of zidovudine was combined with meropenem. CONCLUSIONS: Zidovudine in combination with carbapenems produced synergistic activity against NDM-1 Enterobacteriaceae strains in vitro

Stress-Adaptive Responses Associated with High-Level Carbapenem Resistance in KPC-Producing Klebsiella pneumoniae

Carbapenem-resistant Enterobacteriaceae (CRE) organisms have emerged to become a major global public health threat among antimicrobial resistant bacterial human pathogens. Little is known about how CREs emerge. One characteristic phenotype of CREs is heteroresistance, which is clinically associated with treatment failure in patients given a carbapenem. Through in vitro whole-transcriptome analysis we tracked gene expression over time in two different strains (BR7, BR21) of heteroresistant KPC-producing Klebsiella pneumoniae, first exposed to a bactericidal concentration of imipenem followed by growth in drug-free medium. In both strains, the immediate response was dominated by a shift in expression of genes involved in glycolysis toward those involved in catabolic pathways. This response was followed by global dampening of transcriptional changes involving protein translation, folding and transport, and decreased expression of genes encoding critical junctures of lipopolysaccharide biosynthesis. The emerged high-level carbapenem-resistant BR21 subpopulation had a prophage (IS1) disrupting ompK36 associated with irreversible OmpK36 porin loss. On the other hand, OmpK36 loss in BR7 was reversible. The acquisition of high-level carbapenem resistance by the two heteroresistant strains was associated with distinct and shared stepwise transcriptional programs. Carbapenem heteroresistance may emerge from the most adaptive subpopulation among a population of cells undergoing a complex set of stress-adaptive responses

Genetic features of antimicrobial drug-susceptible extraintestinal pathogenic<i>Escherichia coli</i>pandemic sequence type 95

AbstractExtraintestinal pathogenicEscherichia coli(ExPEC) belonging to multilocus sequence type 95 (ST95) is one of the most geographically widespread ExPEC lineages causing bloodstream (BSI) and urinary tract infections (UTI). In contrast to other widespread ExPEC sequence types, a large proportion ST95 strains remains susceptible to all antimicrobial agents used to treat BSI or UTI. We aimed to identify genomic features of ST95 associated with its relatively high drug-susceptible frequency. We analyzed whole genomes of 1749 ST95 isolates, 80 from patients with BSI or UTI in Northern California, and 1669 isolates from the Enterobase database. We first compared whole-genome sequences (WGS) of 887 drug-susceptible strains and 862 strains resistant to one or more drugs (defined genotypically as strains harboring drug-resistance genes annotated in the ResFinder database) to identify genetic features associated with strains devoid of drug-resistance genes. We then conducted a pan-genome-wide association study on human clinical isolates of ST95, which included 553 UTI and BSI ST95 isolates. We found 44 accessory genes to be significantly associated with ST95 strains lacking drug resistance genes. Fifteen of these were not found in any of the WGSs of ST131 ExPEC strains, which are frequently multidrug-resistant. These genes were annotated to encode transporter or transfer systems and DNA repair polymerases. A large proportion of ST95 strains may have evolved to adapt to antibiotic-imposed stresses without acquiring drug-resistance genes.ImportanceDespite the increasing prevalence of antibiotic-resistantEscherichia colistrains that cause urinary tract and bloodstream infections, a major pandemic lineage of extraintestinal pathogenicE. coli(ExPEC) ST95 has a comparatively low frequency of drug resistance. We compared genomes of 1749 ST95 isolates to identify genetic features that may explain why most strains of ST95 resist becoming drug-resistant. Identification of such genomic features could contribute to the development of novel strategies to prevent the spread of antibiotic-resistant genes and devise new measures to control antibiotic-resistant infections.</jats:sec

Genomic Analysis of Factors Associated with Low Prevalence of Antibiotic Resistance in Extraintestinal Pathogenic Escherichia coli Sequence Type 95 Strains.

Extraintestinal pathogenic Escherichia coli (ExPEC) strains belonging to multilocus sequence type 95 (ST95) are globally distributed and a common cause of infections in humans and domestic fowl. ST95 isolates generally show a lower prevalence of acquired antimicrobial resistance than other pandemic ExPEC lineages. We took a genomic approach to identify factors that may underlie reduced resistance. We fully assembled genomes for four ST95 isolates representing the four major fimH-based lineages within ST95 and also analyzed draft-level genomes from another 82 ST95 isolates, largely from the western United States. The fully assembled genomes of antibiotic-resistant isolates carried resistance genes exclusively on large (&gt;90-kb) IncFIB/IncFII plasmids. These replicons were common in the draft genomes as well, particularly in antibiotic-resistant isolates, but we also observed multiple instances of a smaller (8.3-kb) ampicillin resistance plasmid that had been previously identified in Salmonella enterica. Among ST95 isolates, pansusceptibility to antibiotics was significantly associated with the fimH6 lineage and the presence of homologs of the previously identified 114-kb IncFIB/IncFII plasmid pUTI89, both of which were also associated with reduced carriage of other plasmids. Potential mechanistic explanations for lineage- and plasmid-specific effects on the prevalence of antibiotic resistance within the ST95 group are discussed. IMPORTANCE Antibiotic resistance in bacterial pathogens is a major public health concern. This work was motivated by the observation that only a small proportion of ST95 isolates, a major pandemic lineage of extraintestinal pathogenic E.&nbsp;coli, have acquired antibiotic resistance, in contrast to many other pandemic lineages. Understanding bacterial genetic factors that may prevent acquisition of resistance could contribute to the development of new biological, medical, or public health strategies to reduce antibiotic-resistant infections