Evaluation of polymyxin B in combination with 13 other antibiotics against carbapenemase-producing Klebsiella pneumoniae in time-lapse microscopy and time-kill experiments.

OBJECTIVES: This study aimed to explore the interactions of polymyxin B in combination with 13 other antibiotics against carbapenemase-producing Klebsiella pneumoniae. METHODS: Five clinical isolates of multidrug-resistant K. pneumoniae producing KPC-2, KPC-3, NDM-1, OXA-48 and VIM-1 carbapenemases were used. Polymyxin B was tested alone and in combination with amikacin, aztreonam, cefepime, chloramphenicol, ciprofloxacin, fosfomycin, linezolid, meropenem, minocycline, rifampicin, temocillin, thiamphenicol and trimethoprim. Inhibition of growth during antibiotic exposure was evaluated in 24-hr automated time-lapse microscopy experiments. Combinations that showed positive interactions were subsequently evaluated in static time-kill experiments. RESULTS: All strains carried multiple (≥9) resistance genes as determined by whole-genome sequencing. In the initial screening the combination of polymyxin B and minocycline was most active with enhanced activity compared with the single antibiotics detected against all strains. Positive interactions were also observed with polymyxin B in combination with rifampicin and fosfomycin against four of five strains and less frequently with other antibiotics. Time-kill experiments demonstrated an additive or synergistic activity (1-2 log10 or ≥2 log10 CFU/mL reduction, respectively, compared with the most potent single antibiotic) with 21 of 23 tested combinations. However, because of regrowth, only 13 combinations were synergistic at 24 hr. Combinations with minocycline or rifampicin were most active, each showing synergy and bacteriostatic or bactericidal effects resulting in 1.93-3.97 and 2.55-5.91 log10 CFU/mL reductions, respectively, after 24 hr against four strains. DISCUSSION: Polymyxin B in combination with minocycline, rifampicin or fosfomycin could be of potential clinical interest. Time-lapse microscopy showed some discrepancy in results compared with the time-kill data but was useful for screening purposes.

Automated time-lapse microscopy a novel method for screening of antibiotic combination effects against multidrug-resistant Gram-negative bacteria.

OBJECTIVES: Antibiotic combinations are often used for carbapenemase-producing Enterobacteriaceae (CPE) but more data are needed on the optimal selection of drugs. This study aimed to evaluate the feasibility of a novel automated method based on time-lapse microscopy (the oCelloScope, Philips BioCell A/S, Allerød, Denmark) to determine in vitro combination effects against CPE and to discuss advantages and limitations of the oCelloScope in relation to standard methods. METHODS: Four Klebsiella pneumoniae and two Escherichia coli were exposed to colistin, meropenem, rifampin and tigecycline, alone and in combination. In the oCelloScope experiments, a background corrected absorption (BCA) value of ≤8 at 24 h was used as a primary cut-off indicating inhibition of bacterial growth. A new approach was used to determine synergy, indifference and antagonism based on the number of objects (bacteria) in the images. Static time-kill experiments were performed for comparison. RESULTS: The time-kill experiments showed synergy with 12 of 36 regimens, most frequently with colistin plus rifampin. BCA values ≤8 consistently correlated with 24-h bacterial concentrations ≤6 log10 CFU/mL. The classification of combination effects agreed with the time-kill results for 33 of 36 regimens. In three cases, the interactions could not be classified with the microscopy method because of low object counts. CONCLUSIONS: Automated time-lapse microscopy can accurately determine the effects of antibiotic combinations. The novel method is highly efficient compared with time-kill experiments, more informative than checkerboards and can be useful to accelerate the screening for combinations active against multidrug-resistant Gram-negative bacteria.

Evaluation of antibacterial activities of colistin, rifampicin and meropenem combinations against NDM-1-producing Klebsiella pneumoniae in 24 h in vitro time-kill experiments.

OBJECTIVES: To investigate the activity of colistin alone or in double and triple combination with rifampicin and meropenem against NDM-1-producing Klebsiella pneumoniae. METHODS: Eight isolates of NDM-1-producing K. pneumoniae were exposed to clinically relevant antibiotic concentrations in 24 h time-kill experiments. Three colistin concentrations were used for two of the strains. Resistance development was assessed with population analysis and sequencing of the mgrB and pmrB genes. RESULTS: Initial killing was achieved with colistin alone, but with considerable regrowth at 24 h. Combinations including colistin and rifampicin were bacteriostatic or bactericidal against all strains. Colistin plus meropenem was bactericidal against one strain, but, overall, meropenem showed little additive effects. Higher concentrations of colistin did not enhance antibacterial activity. Resistant populations and deletion or mutations in the mgrB and pmrB genes were frequently detected in endpoint samples after exposure to colistin alone. CONCLUSIONS: Based on the results of this and previous studies, the combination of colistin and rifampicin seems promising and should be further explored in vivo and considered for clinical evaluation. Meropenem seems less useful in the treatment of infections caused by high-level carbapenem-resistant NDM-1-producing K. pneumoniae. Higher colistin concentrations did not result in significantly better activity, suggesting that combination therapy might be superior to monotherapy also when colistin is prescribed using high-dose regimens in accordance with current recommendations.

Evaluation of double- and triple-antibiotic combinations for VIM- and NDM-producing Klebsiella pneumoniae by in vitro time-kill experiments.

Combination therapy is recommended for infections with carbapenemase-producing Klebsiella pneumoniae. However, limited data exist on which antibiotic combinations are the most effective. The aim of this study was to find effective antibiotic combinations against metallo-beta-lactamase-producing K. pneumoniae (MBL-KP). Two VIM- and two NDM-producing K. pneumoniae strains, all susceptible to colistin, were exposed to antibiotics at clinically relevant static concentrations during 24-h time-kill experiments. Double- and triple-antibiotic combinations of aztreonam, ciprofloxacin, colistin, daptomycin, fosfomycin, meropenem, rifampin, telavancin, tigecycline, and vancomycin were used. Synergy was defined as a ≥2 log10 decrease in CFU/ml between the combination and its most active drug after 24 h, and bactericidal effect was defined as a ≥3 log10 decrease in CFU/ml after 24 h compared with the starting inoculum. Synergistic or bactericidal activity was demonstrated for aztreonam, fosfomycin, meropenem, and rifampin in double-antibiotic combinations with colistin and also for aztreonam, fosfomycin, and rifampin in triple-antibiotic combinations with meropenem and colistin. Overall, the combination of rifampin-meropenem-colistin was the most effective regimen, demonstrating synergistic and bactericidal effects against all four strains. Meropenem-colistin, meropenem-fosfomycin, and tigecycline-colistin combinations were not bactericidal against the strains used. The findings of this and other studies indicate that there is great potential of antibiotic combinations against carbapenemase-producing K. pneumoniae. However, our results deviate to some extent from those of previous studies, which might be because most studies to date have included KPC-producing rather than MBL-producing strains. More studies addressing MBL-KP are needed.

The dorsal cell, one class of primary sensory neuron in the lamprey spinal cord. I. Touch, pressure but no nociception--a physiological study.

The dorsal cells in the lamprey spinal cord are primary sensory neurons. The cells were classified by Martin and Wickelgren in 1971 into 3 different groups, touch, pressure and nociceptive, according to their responses to mechanical stimulation of the skin. While confirming the presence of touch and pressure cells in the present study, we found no evidence for the existence of nociceptive spinal dorsal cells. Further we show that touch and pressure cells have different response latencies to a 40-ms hyperpolarizing current pulse. Measured from the end of the pulse to the initiation of the action potential, touch cells have a response latency shorter than 11 ms, whereas the pressure cells have a response latency longer than 11 ms.

The dorsal cell, one class of primary sensory neuron in the lamprey spinal cord. II. A light- and electron microscopical study.

Dorsal cells, are primary sensory neurons located in the lamprey spinal cord. They are of two types conveying touch (T) and pressure (P) and they have an ascending and/or a descending axon which joins the dorsal column. Dorsal cells have no initial axon segment. In the electron microscope synaptic boutons with spherical synaptic vesicles were found in contact with the cell membrane of T-cells while no boutons have been observed on P-cells. No other morphological differences between T- and P-cells could be detected ultrastructurally or in the light microscope. No output synapses have been observed from the dorsal cell bodies or the small processes extending from the cell body.

An ultrastructural study of the synaptology of gamma-motoneurones during the postnatal development in the cat.

The postnatal development of cat triceps surae gamma-motoneurones, retrogradely labelled with horseradish peroxidase (HRP), was studied light and electron microscopically. The mean diameter of the cell bodies of the gamma-motoneurones increased by about 25% from birth to the adult stage, which was much less than the increase in cell body diameter of alpha-motoneurones (about 45%). Throughout development the only bouton types apposing the gamma-motoneurones were the F- and S-types, with flattened and spherical synaptic vesicles, respectively. Thus, the C-, M- and T-types of boutons seen on a alpha-motoneurones. The number of boutons on the gamma-motoneurone cell bodies seemed to decrease postnatally. This decrease was only moderate for S-type boutons but substantial for F-type boutons. In contrast, the number of boutons on the proximal dendrites appeared to increase and this was most evident for S-type boutons. The mentioned postnatal changes in synaptology were more differentiated with regard to bouton type and part of the neurones under study than what could be inferred from earlier studies on the postnatal development of alpha-motoneurones. These changes also occurred later than in alpha-motoneurones. The relative dominance of F-type boutons with probable inhibitory actions on the immature gamma-motoneurone may explain the previously demonstrated poor encoding of muscle length by muscle spindles during the first postnatal weeks in the kitten.

Ultrastructural observations on beaded alpha-motoneuron dendrites.

Beaded dendrites of alpha-motoneurons intracellularly labelled with horseradish peroxidase (HRP) were studied ultrastructurally in eight adult cats. For comparison, adjacent unlabelled beaded dendrites of unknown origin were also included in the study. Electron microscopy revealed no signs of degeneration or poor fixation according to common criteria. With the exception of the HRP-reaction product no difference in structure was observed between labelled and unlabelled beaded dendrites. Both the beads and their interconnecting segments were postsynaptic to boutons of normal appearance containing spherical (S-type boutons) or flattened vesicles (F-type boutons). The values for synaptic covering and synaptic packing density of the beaded dendritic regions, which usually were located in the periphery of the dendritic trees, were clearly lower than values obtained previously for cell bodies and proximal dendrites of alpha-motoneurons.

Electron microscopic observations on the synaptology of cat sciatic gamma-motoneurons after intracellular staining with horseradish peroxidase.

Four cat sciatic motoneurons with axon conduction velocities below 30 m/s, and thus considered to be of the gamma-type, were intracellularly labelled with horseradish peroxidase (HRP) and subsequently studied in the electron microscope. The labelled neurons were apposed by synaptic terminals with spherical (S-type) and flattened vesicles (F-type) but not by large terminals with spherical vesicles (M- and C-types) seen on alpha-motoneurons. Quantitative analysis of a complete series of ultrathin sections through one of the neurons showed that the synaptic covering on the cell body (24.2%) was considerably larger than what has been reported for triceps surae gamma-motoneurons, but within the range of values for gamma-motoneurons in the thoracic region of the spinal cord.

Light microscopic observations on cat Renshaw cells after intracellular staining with horseradish peroxidase. I. The axonal systems.

Five intracellularly HRP-stained Renshaw cells were subjected to light microscopic analysis of the trajectories, branching patterns, and projections of the axonal systems. The cell bodies were located ventrally in lamina VII. In three neurons the axon originated from the cell body and in the remaining two cells from a dendrite. After a 600-870-microns distance the axons entered the ventral funiculus, where all of them continued rostrally. Two axons also gave off a caudal branch in the funiculus. The diameters of the main axons varied between 2.1 and 10.0 microns. The main axons gave off one to four first-order collaterals before entering the ventral funiculus and up to three collaterals could be seen to originate from the same node of Ranvier. In the ventral funiculus up to five first-order collaterals could be traced from the same main axon. The axon collateral trees were often very extensive and daughter branches up to the 22nd order were observed. The distance between two successive branching points varied between 4 and 410 microns. A large number of boutonlike swellings were found along (59%) or at the ends of the collateral branches. At the most, 1,278 swellings originated from a single axon collateral tree. Most of the swellings were located in lamina IX, but they also appeared ventrally and dorsolaterally in lamina VII.

Light microscopic observations on cat Renshaw cells after intracellular staining with horseradish peroxidase. II. The cell bodies and dendrites.

The cell bodies and dendritic trees of five lumbosacral Renshaw cells of adult cats were studied in the light microscope (LM) after intracellular injection with horseradish peroxidase (HRP). The cell bodies were all located in the ventral part of lamina VII. The dendrites extended up to 0.7 mm from the cell body into the neighbouring parts of laminae VIII and IX as well as into more dorsal parts of lamina VII. The dendritic branching was sparse and about half the dendrites were unbranched. The mean diameter of the cell body was positively correlated to both the combined and mean diameters of the first-order dendrites. Between four and eight dendrites originated from the cell bodies. The number of dendritic end-branches, the combined dendritic length, the mean dendritic length from the cell body to the termination of the end branches, the distance from the cell body to the termination of the most remote end-branch, the dendritic surface area, and the dendritic volume all correlated positively with the diameter of the parent first-order dendrite. The dendritic tapering was somewhat more pronounced in the Renshaw cells than previously observed in alpha- and gamma-motoneurons. The present data are discussed in relation to previous morphological observations on Renshaw cells and alpha- and gamma-motoneurons.

An ultrastructural study of cat lumbosacral gamma-motoneurons after retrograde labelling with horseradish peroxidase.

Twelve retrogradely horseradish peroxidase (HRP)-labelled triceps surae motoneurons of gamma size (mean cell body diameter less than 38 micron) were studied ultrastructurally. The contours of the cell bodies, as observed in the transverse midnucleolus plane, were elongated to rounded. The axons identified all originated from the cell body. The mean diameter of the stem dendrites was 4.5 micron. A substantial part of the cell membrane was covered by glial extensions. The boutons and synaptic contacts apposing the gamma-motoneurons could be classified into two categories on the basis of the type of synaptic vesicles: S-type boutons with spherical synaptic vesicles and F-type boutons with flattened vesicles. In each neuron, the values for mean length and mean area of apposition, percentage synaptic covering, and packing density of S-type, F-type, and S+F-type boutons were estimated on the cell body and in two dendritic compartments. In comparison with alpha-motoneurons and Renshaw cells, the cell bodies of the gamma-motoneurons were covered by smaller and strikingly fewer boutons of both the S- and F-types. The values for percentage synaptic covering and packing density of boutons on the proximal dendrites were also lower for gamma-motoneurons than for both alpha-motoneurons and Renshaw cells, although the differences were less pronounced than on the cell body. No boutons of the C-, M-, and T-types described for alpha-motoneurons were found on the gamma-motoneurons.

The edge cell, a possible intraspinal mechanoreceptor.

In the lateral edge of the "white matter" in the lamprey spinal cord, there is a group of nerve cells referred to as edge cells. The results of a combined physiological, light microscopical, and electron microscopical study suggest that these cells serve as intraspinal mechanoreceptors. Edge cells are depolarized on stretch of the lateral margin of the spinal cord, and they have nestlike ramifications in this region oriented in a rostrocaudal plane. These cells exhibit a close structural similarity with the crayfish stretch receptor.

An ultrastructural study of serially sectioned Renshaw cells. III. Quantitative distribution of synaptic boutons.

The quantitative distribution of synaptic boutons on 17 presumed Renshaw cells has been studied ultrastructurally. All 17 neurons were postsynaptic to axon collateral boutons of intracellularly HRP-stained triceps surae alpha-motoneurons and were located in lamina VII, ventromedially to the main motor nuclei. In each of the presumed Renshaw cells, the values for mean length and mean area of apposition, percentage synaptic covering, and packing density of S-type, F-type, and S + F-type boutons were estimated on the cell body and in two dendritic compartments. The F/S percentage synaptic covering ratio was also calculated. The previously demonstrated differences within the present group of neurons, with respect to the site of axonal origin, were not accompanied by any corresponding differences in the quantitative distribution of synaptic boutons. However, it is suggested that the presumed Renshaw cells may possibly fall into two categories with respect to the F/S percentage synaptic covering ratio. The results are discussed in relation to previous studies on the neuronal architecture and synaptic types on the same presumed Renshaw cells, as well as in relation to earlier observations on the quantitative distribution of boutons on central neurons, particularly spinal alpha-motoneurons.

An ultrastructural study of serially sectioned Renshaw cells. II. Synaptic types.

Boutons and synaptic contacts on 17 presumed Renshaw cells were studied ultrastructurally. All 17 neurons were postsynaptic to axon collateral boutons of intracellularly HRP-stained triceps and surae alpha-motoneurons and located in lamina VII, ventromedially to the main motor nuclei. The boutons and synaptic contacts could be classified into two main categories on the basis of synaptic vesicles: S-type boutons with spherical synaptic vesicles and F-type boutons with flattened vesicles, the alpha-motoraxon collateral boutons falling into the S-category. In addition, some S-type boutons containing neurofilaments and some being apposed by small presynaptic boutons were observed. The results are discussed to earlier observations on the synaptology of central neurons, particularly spinal alpha-motoneurons.

An ultrastructural study of serially sectioned Renshaw cells. I. Architecture of the cell body, axon hillock, initial axon segment and proximal dendrites.

Seventeen neurons which were postsynaptic to axon collateral boutons of intracellularly HRP-stained triceps surae alpha-motoneurons were studied ultrastructurally. All 17 neurons were situated in lamina VII, ventro-medially to the main motor nuclei. This and other facts support the assumption that the observed neurons are morphological correlates to the physiologically defined Renshaw cells. The contours of the cell bodies, as observed in the midnucleolus plane, were elongated. The axons originated either from the cell bodies or from dendrites. The number of dendrites of each neuron varied between 3 and 7. The appearance of the presumed Renshaw cells was also compared with that of a larger sample of neurons from the ventral part of lamina VII which was studied light microscopically in semithin sections. It was suggested that the Renshaw cells belong to the larger and more elongated neurons in the area.

An ultrastructural study of the synaptic contacts of alpha 1-motoneuron axon collaterals. II. Contacts in lamina VII.

Horseradish peroxidase (HRP) was injected intracellularly in triceps surae alpha-motoneurons. The axons and axon collaterals of these neurons were traced light and electron microscopically. Synaptic boutons of collaterals in the ventral part of Rexed's lamina VII were studied ultrastructurally. The boutons exhibited spherical synaptic vesicles and made synaptic contacts with cell bodies and proximal dendrites of neurons assumed to be Renshaw cells and with dendrites of unknown origin. The observations are discussed in relation to earlier qualitative and quantitative studies on the other known synaptic contacts of the alpha-motor axons, both in the central and peripheral nervous system.

An ultrastructural study of the synaptic contacts of alpha-motoneurone axon collaterals. I. Contacts in lamina IX and with identified alpha-motoneurone dendrites in lamina VII.

Horseradish peroxidase (HRP) was injected intracellularly in triceps surae alpha-motoneurones. The axons and axon collaterals of these neurones were traced light and electron microscopically. Synaptic boutons of collaterals, in Rexed's lamina IX or in synaptic contact with HRP-stained motoneurone dendrites in lamina VII, were studied ultrastructurally. The boutons exhibited spherical synaptic vesicles and made synaptic contacts of two different types with HRP-stained alpha-motoneurone dendrites in lamina IX and VII, dendrites and cell bodies of large neurones in lamina IX, dendrites of unknown origin in lamina IX and with one cell body of a medium size neurone in lamina IX. The observations are discussed in relation to earlier qualitative and quantitative studies on the synaptology of cat spinal alpha-motoneurones.