PATIENTS AND METHODS: Materials and methods: In the present study, the lytic bacteriophage, k3w7, isolated by the host Klebsiella pneumoniae kP2 was characterised using transmission electron microscope (TEM), plaque assay, and restriction digestive enzyme to investigate mor¬phology, host spectrum, bacteriophage life cycle and stability accordingly.
RESULTS: Results and conclusions: As shown by TEM, k3w7 was observed to have the characteristic of icosahedral heads 100 nm and contractile sheaths 120 nm suggesting it belongs to the family of myoviridae.The Investigation has done on the phage growth cycle showed a short latent period of 20 min and a burst size of approximately 220 plaque forming units per infected cell. Stability test showed the phage was stable over a wide range of pH and temperatures. According to restriction analysis, k3w7 had 50 -kb double-stranded DNA genome as well as the heterogeneous nature of genetic material. These findings suggest that K3W7 has a potential use in therapy against infections caused by K. pneumonia produces carbapenemase.
MATERIALS AND METHODS: A prospective epidemiologic and microbiologic study was conducted of MRKP isolated from the blood and wound of a boy with necrotizing fasciitis after a 7-day course of ceftazidime and amikacin. In the following 2 weeks, phenotypically similar MRKP were isolated from the blood cultures of four other patients and rectal swabs of another three patients and two liquid soap samples located in the same ward.
RESULTS: Antimicrobial profiles demonstrated that all the isolates were resistant to ceftazidime, sensitive to imipenem and ciprofloxacin, and confirmed to be extended-spectrum beta-lactamase producers. Plasmids of varying molecular weights were present in all isolates. In eight of these isolates, which included four from blood, there were common large molecular weight plasmids ranging from 80 kb to 100 kb. Pulsed-field gel electrophoresis analysis using XbaI demonstrated six different DNA profiles, A to F. Profile A was shared by two blood culture isolates and were related by 91%. Profile B was found in one rectal swab isolate and one isolate from liquid soap and were related by 94%. Profile C was shared by one blood isolate and one liquid soap isolate and showed 100% relatedness. Profiles D, E, and F each were demonstrated by one blood isolate and two rectal swab isolates, respectively. These showed only 65% relatedness.
CONCLUSIONS: The MRKP strains in this outbreak were not clonal in origin. The decline of the outbreak after 4 weeks was attributed to the reemphasis of standard infection control procedures and the implementation of a program that addressed sites of environmental contamination.
METHODS: Sixteen healthy adult participants donned one antimicrobial surgical glove and one non-antimicrobial surgical glove randomly allocated to their dominant and non-dominant hand following a crossover design. During a 2-h wear time, participants performed standardized finger and hand movements. Thereafter, the interior surface of excised fingers of the removed gloves was challenged with 8.00 log10 cfu/mL S. aureus (ATCC 6538) or K. pneumoniae (ATCC 4352), respectively. The main outcome measure was the viable mean log10 cfu counts of the two glove groups after 5 min contact with the interior glove's surface.
RESULTS: When comparing an antimicrobial glove against an untreated reference glove after 2-h simulated use wear-time, a mean reduction factor of 6.24 log10 (S. aureus) and 6.22 log10 (K. pneumoniae) was achieved after 5 min contact.
CONCLUSION: These results demonstrate that wearing antibacterial gloves on hands does not negatively impact their antibacterial activity after 2-h of wear. This may have a potential benefit for patient safety in case of glove puncture during surgical procedures.
METHODS: Regulators were overproduced using a pBAD expression vector. Antibiotic susceptibility was measured using disc testing. Envelope permeability was estimated using a fluorescent dye accumulation assay. Porin and efflux pump production was quantified using proteomics and validated using real-time quantitative RT-PCR.
RESULTS: Envelope permeability and antibiotic disc inhibition zone diameters both reduced during overproduction of RamA and to a lesser extent RarA or SoxS, but did not change following overproduction of MarA. These effects were associated with overproduction of the efflux pumps AcrAB (for RamA and SoxS) and OqxAB (for RamA and RarA) and the outer membrane protein TolC (for all regulators). Effects on porin production were strain specific.
CONCLUSIONS: RamA is the most potent regulator of antibiotic permeability in K. pneumoniae, followed by RarA then SoxS, with MarA having very little effect. This observed relative potency correlates well with the frequency at which these regulators are reportedly overproduced in clinical isolates.
Objectives: To define, in an otherwise isogenic background, the relative effects of OqxR and RamR loss-of-function mutations on envelope protein production, envelope permeability and antimicrobial susceptibility. We also investigated the clinical relevance of an OqxR loss-of-function mutation, particularly in the context of β-lactam susceptibility.
Methods: Envelope permeability was estimated using a fluorescent dye accumulation assay. Antimicrobial susceptibility was measured using disc testing. Total envelope protein production was quantified using LC-MS/MS proteomics and quantitative RT-PCR was used to measure transcript levels.
Results: Loss of RamR or OqxR reduced envelope permeability in K. pneumoniae by 45%-55% relative to the WT. RamR loss activated AcrAB efflux pump production ∼5-fold and this reduced β-lactam susceptibility, conferring ertapenem non-susceptibility even in the absence of a carbapenemase. In contrast, OqxR loss specifically activated OqxAB efflux pump production >10 000-fold. This reduced fluoroquinolone susceptibility but had little impact on β-lactam susceptibility even in the presence of a β-lactamase.
Conclusions: Whilst OqxR loss and RamR loss are both seen in K. pneumoniae clinical isolates, only RamR loss significantly stimulates AcrAB efflux pump production. This means that only RamR mutants have significantly reduced β-lactamase-mediated β-lactam susceptibility and therefore represent a greater clinical threat.
Methods: Envelope permeability was estimated using a fluorescent dye accumulation assay. β-Lactam susceptibility was measured using disc testing. Total envelope protein production was quantified using LC-MS/MS proteomics and transcript levels were quantified using real-time RT-PCR.
Results: RamA overproduction enhanced β-lactamase-mediated β-lactam resistance, in some cases dramatically, without altering β-lactamase production. It increased production of efflux pumps and decreased OmpK35 porin production, though micF overexpression showed that OmpK35 reduction has little impact on envelope permeability. A survey of K. pneumoniae bloodstream isolates revealed ramA hyperexpression in 3 of 4 carbapenemase producers, 1 of 21 CTX-M producers and 2 of 19 strains not carrying CTX-M or carbapenemases.
Conclusions: Whilst RamA is not a key mediator of antibiotic resistance in K. pneumoniae on its own, it is potentially important for enhancing the spectrum of acquired β-lactamase-mediated β-lactam resistance. LC-MS/MS proteomics analysis has revealed that this enhancement is achieved predominantly through activation of efflux pump production.