Enhanced Antibacterial Activity of Repurposed Mitomycin C and Imipenem in Combination with the Lytic Phage vB_KpnM-VAC13 against Clinical Isolates of Klebsiella pneumoniae.

Klebsiella pneumoniae is an opportunistic Gram-negative pathogen that employs different strategies (resistance and persistence) to counteract antibiotic treatments. This study aimed to search for new means of combatting imipenem-resistant and persister strains of K. pneumoniae by repurposing the anticancer drug mitomycin C as an antimicrobial agent and by combining the drug and the conventional antibiotic imipenem with the lytic phage vB_KpnM-VAC13. Several clinical K. pneumoniae isolates were characterized, and an imipenem-resistant isolate (harboring OXA-245 ß-lactamase) and a persister isolate were selected for study. The mitomycin C and imipenem MICs for both isolates were determined by the broth microdilution method. Time-kill curve data were obtained by optical density at 600 nm (OD600) measurement and CFU enumeration in the presence of each drug alone and with the phage. The frequency of occurrence of mutants resistant to each drug and the combinations was also calculated, and the efficacy of the combination treatments was evaluated using an in vivo infection model (Galleria mellonella). The lytic phage vB_KpnM-VAC13 and mitomycin C had synergistic effects on imipenem-resistant and persister isolates, both in vitro and in vivo. The phage-imipenem combination successfully killed the persisters but not the imipenem-resistant isolate harboring OXA-245 ß-lactamase. Interestingly, the combinations decreased the emergence of in vitro resistant mutants of both isolates. Combinations of the lytic phage vB_KpnM-VAC13 with mitomycin C and imipenem were effective against the persister K. pneumoniae isolate. The lytic phage-mitomycin C combination was also effective against imipenem-resistant K. pneumoniae strains harboring OXA-245 ß-lactamase.

In vitro activity of a polyhexanide-betaine solution against high-risk clones of multidrug-resistant nosocomial pathogens.

OBJECTIVE: To determine the in vitro activity of a polyhexanide-betaine solution against collection strains and multidrug-resistant (MDR) nosocomial isolates, including high-risk clones. METHODS: We studied of 8 ATCC and 21 MDR clinical strains of Staphylococcus aureus, Enterococcus faecium, Enterococcus faecalis, Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa, including the multiresistant high-risk clones. The MICs and MBCs of a 0.1% polyhexanide-0.1% betaine solution were determined by microdilution. For each species, strains with the highest MICs were selected for further experiments. The dilution-neutralization test (PrEN 12054) was performed by incubating bacterial inocula of 106CFU/mL for 1min with undiluted 0.1% polyhexanide-betaine solution. The CFUs were counted after neutralization. Growth curves and time-kill curves at concentrations of 0.25, 1, 4, and 8×MIC, were performed. MICs of recovered strains were determined when regrowth was observed in time-kill studies after 24h of incubation. Strains with reduced susceptibility were selected by serial passage on plates with increasing concentrations of polyhexanide-betaine, and MICs were determined. RESULTS: Polyhexanide-betaine MIC range was 0.5-8mg/L. MBCs equalled or were 1 dilution higher than MICs. The dilution-neutralization method showed total inoculum clearance of all strains. In time-kill curves, no regrowth was observed at 4×MIC, except for S. aureus (8×MIC). Increased MICs were not observed in time-kill curves, or after serial passages after exposure to polyhexanide-betaine. CONCLUSIONS: Polyhexanide-betaine presented bactericidal activity against all MDR clinical isolates tested, including high-risk clones, at significantly lower concentrations and time of activity than those commercially used.

Epidemiological and clinical features associated with colonisation/infection by Acinetobacter baumannii with phenotypic heterogeneous resistance to carbapenems.

The objective of this study was to identify risk factors for the acquisition of Acinetobacter baumannii with phenotypic heterogeneous resistance (PHR) to carbapenems and to determine whether these factors are similar to those associated with A. baumannii not showing this phenotype. Microbiological and clinical data from 211 patients included in the GEIH-Ab 2000 project were used. Isolates of A. baumannii were studied for their susceptibility to imipenem (IPM) by microdilution and for PHR to IPM as determined by the presence of colonies growing within the inhibition zone of IPM disks. Isolates were divided into three groups: (i) IPM-PHR isolates, i.e. susceptible and non-susceptible A. baumannii displaying PHR to IPM; (ii) non-IPM-PHR isolates, i.e. susceptible A. baumannii showing an inhibition halo but no colonies growing within it; and (iii) IPM-FR isolates, i.e. fully resistant A. baumannii displaying no halo of inhibition. IPM-PHR isolates of A. baumannii were more commonly isolated from respiratory tract samples and less commonly from urine, and were more frequently causes of infection than were IPM-FR isolates. Independent risk factors identified in patients with IPM-PHR isolates were Intensive Care Unit admission, surgery, and previous use of piperacillin/tazobactam or carbapenems, whilst risk factors for IPM-FR and IPM-PHR were previous use of cephalosporins and isolation from a urine sample. In conclusion, risk factors associated with colonisation/infection by isolates of A. baumannii with PHR to carbapenems are similar to those previously described for isolates resistant to carbapenems.

Nosocomial outbreak of infection with pan-drug-resistant Acinetobacter baumannii in a tertiary care university hospital.

OBJECTIVE: To describe what is, to our knowledge, the first nosocomial outbreak of infection with pan-drug-resistant (including colistin-resistant) Acinetobacter baumannii, to determine the risk factors associated with these types of infections, and to determine their clinical impact. DESIGN: Nested case-control cohort study and a clinical-microbiological study. SETTING: A 1,521-bed tertiary care university hospital in Seville, Spain. PATIENTS: Case patients were inpatients who had a pan-drug-resistant A. baumannii isolate recovered from a clinical or surveillance sample obtained at least 48 hours after admission to an intensive care unit (ICU) during the time of the epidemic outbreak. Control patients were patients who were admitted to any of the "boxes" (ie, rooms that partition off a distinct area for a patient's bed and the equipment needed to care for the patient) of an ICU for at least 48 hours during the time of the epidemic outbreak. RESULTS: All the clinical isolates had similar antibiotic susceptibility patterns (ie, they were resistant to all the antibiotics tested, including colistin), and, on the basis of repetitive extragenic palindromic-polymerase chain reaction, it was determined that all of them were of the same clone. The previous use of quinolones and glycopeptides and an ICU stay were associated with the acquisition of infection or colonization with pan-drug-resistant A. baumannii. To control this outbreak, we implemented the following multicomponent intervention program: the performance of environmental decontamination of the ICUs involved, an environmental survey, a revision of cleaning protocols, active surveillance for colonization with pan-drug-resistant A. baumannii, educational programs for the staff, and the display of posters that illustrate contact isolation measures and antimicrobial use recommendations. CONCLUSIONS: We were not able to identify the common source for these cases of infection, but the adopted measures have proven to be effective at controlling the outbreak.