Role of adhesion forces in mechanosensitive channel gating in Staphylococcus aureus adhering to surfaces.

Mechanosensitive channels in bacterial membranes open or close in response to environmental changes to allow transmembrane transport, including antibiotic uptake and solute efflux. In this paper, we hypothesize that gating of mechanosensitive channels is stimulated by forces through which bacteria adhere to surfaces. Hereto, channel gating is related with adhesion forces to different surfaces of a Staphylococcus aureus strain and its isogenic ΔmscL mutant, deficient in MscL (large) channel gating. Staphylococci becoming fluorescent due to uptake of calcein, increased with adhesion force and were higher in the parent strain (66% when adhering with an adhesion force above 4.0 nN) than in the ΔmscL mutant (40% above 1.2 nN). This suggests that MscL channels open at a higher critical adhesion force than at which physically different, MscS (small) channels open and contribute to transmembrane transport. Uptake of the antibiotic dihydrostreptomycin was monitored by staphylococcal killing. The parent strain exposed to dihydrostreptomycin yielded a CFU reduction of 2.3 log-units when adhering with an adhesion force above 3.5 nN, but CFU reduction remained low (1.0 log-unit) in the mutant, independent of adhesion force. This confirms that large channels open at a higher critical adhesion-force than small channels, as also concluded from calcein transmembrane transport. Collectively, these observations support our hypothesis that adhesion forces to surfaces play an important role, next to other established driving forces, in staphylococcal channel gating. This provides an interesting extension of our understanding of transmembrane antibiotic uptake and solute efflux in infectious staphylococcal biofilms in which bacteria experience adhesion forces from a wide variety of surfaces, like those of other bacteria, tissue cells, or implanted biomaterials.

Dihydrostreptomycin Directly Binds to, Modulates, and Passes through the MscL Channel Pore.

The primary mechanism of action of the antibiotic dihydrostreptomycin is binding to and modifying the function of the bacterial ribosome, thus leading to decreased and aberrant translation of proteins; however, the routes by which it enters the bacterial cell are largely unknown. The mechanosensitive channel of large conductance, MscL, is found in the vast majority of bacterial species, where it serves as an emergency release valve rescuing the cell from sudden decreases in external osmolarity. While it is known that MscL expression increases the potency of dihydrostreptomycin, it has remained unclear if this effect is due to a direct interaction. Here, we use a combination of genetic screening, MD simulations, and biochemical and mutational approaches to determine if dihydrostreptomycin directly interacts with MscL. Our data strongly suggest that dihydrostreptomycin binds to a specific site on MscL and modifies its conformation, thus allowing the passage of K+ and glutamate out of, and dihydrostreptomycin into, the cell.

Tmc1 Point Mutation Affects Ca2+ Sensitivity and Block by Dihydrostreptomycin of the Mechanoelectrical Transducer Current of Mouse Outer Hair Cells.

The transduction of sound into electrical signals depends on mechanically sensitive ion channels in the stereociliary bundle. The molecular composition of this mechanoelectrical transducer (MET) channel is not yet known. Transmembrane channel-like protein isoforms 1 (TMC1) and 2 (TMC2) have been proposed to form part of the MET channel, although their exact roles are still unclear. Using Beethoven (Tmc1(Bth/Bth)) mice, which have an M412K point mutation in TMC1 that adds a positive charge, we found that Ca(2+) permeability and conductance of the MET channel of outer hair cells (OHCs) were reduced. Tmc1(Bth/Bth) OHCs were also less sensitive to block by the permeant MET channel blocker dihydrostreptomycin, whether applied extracellularly or intracellularly. These findings suggest that the amino acid that is mutated in Bth is situated at or near the negatively charged binding site for dihydrostreptomycin within the permeation pore of the channel. We also found that the Ca(2+) dependence of the operating range of the MET channel was altered by the M412K mutation. Depolarization did not increase the resting open probability of the MET current of Tmc1(Bth/Bth) OHCs, whereas raising the intracellular concentration of the Ca(2+) chelator BAPTA caused smaller increases in resting open probability in Bth mutant OHCs than in wild-type control cells. We propose that these observations can be explained by the reduced Ca(2+) permeability of the mutated MET channel indirectly causing the Ca(2+) sensor for adaptation, at or near the intracellular face of the MET channel, to become more sensitive to Ca(2+) influx as a compensatory mechanism. SIGNIFICANCE STATEMENT: In the auditory system, the hair cells convert sound-induced mechanical movement of the hair bundles atop these cells into electrical signals through the opening of mechanically gated ion channels at the tips of the bundles. Although the nature of these mechanoelectrical transducer (MET) channels is still unclear, recent studies implicate transmembrane channel-like protein isoform 1 (TMC1) channels in the mammalian cochlea. Using a mutant mouse model (Beethoven) for progressive hearing loss in humans (DFNA36), which harbors a point mutation in the Tmc1 gene, we show that this mutation affects the MET channel pore, reducing its Ca(2+) permeability and its affinity for the permeant blocker dihydrostreptomycin. A number of phenomena that we ascribe to Ca(2+)-dependent adaptation appear stronger, in compensation for the reduced Ca(2+) entry.

The Role of Flies in Disseminating Plasmids with Antimicrobial-Resistance Genes Between Farms.

Dissemination of antimicrobial resistance is a major global public health concern. To clarify the role of flies in disseminating antimicrobial resistance between farms, we isolated and characterized tetracycline-resistant Escherichia coli strains isolated from flies and feces of livestock from four locations housing swine (abattoir, three farms) and three cattle farms. The percentages of isolates from flies resistant to tetracycline, dihydrostreptomycin, ampicillin, and chloramphenicol (80.8%, 61.5%, 53.8%, and 50.0%, respectively) and those from animal feces (80.5%, 78.0%, 41.5%, and 46.3%, respectively) in locations housing swine were significantly higher than those from cattle farms (p<0.05). The rates of resistance in E. coli derived from flies reflected those derived from livestock feces at the same locations, suggesting that antimicrobial resistance spreads between livestock and flies on the farms. The results of pulsed-field gel electrophoresis (PFGE) analysis showed that, with a few exceptions, all E. coli isolates differed. Two pairs of tetracycline-resistant strains harbored similar plasmids with the same tetracycline-resistance genes, although the origin (fly or feces), site of isolation, and PFGE patterns of these strains differed. Therefore, flies may disseminate the plasmids between farms. Our results suggest that flies may be involved not only in spreading clones of antimicrobial-resistant bacteria within a farm but also in the widespread dissemination of plasmids with antimicrobial resistance genes between farms.

Isolated Schwannoma of the Olfactory Groove: A Case Report

Introduction Schwannoma of the olfactory groove is an extremely rare tumor that can share a differential diagnosis with meningioma or neuroblastoma. Objectives The authors present a case of giant schwannoma involving the anterior cranial fossa and ethmoid sinuses. Case Report The patient presented with a 30-month history of left nasal obstruction, anosmia, and sporadic ipsilateral bleeding. Computed tomography of the paranasal sinuses revealed expansive lesion on the left nasal cavity extending to nasopharynx up to ethmoid and sphenoid sinuses bilaterally with intraorbital and parasellar extension to the skull base. Magnetic resonance imaging scan confirmed the expansive tumor without dural penetration. Biopsy revealed no evidence of malignancy and probable neural cell. Bifrontal craniotomy was performed combined with lateral rhinotomy (Weber-Ferguson approach), and the lesion was totally removed. The tumor measured 8.0 4.3 3.7 cm and microscopically appeared as a schwannoma composed of interwoven bundles of elongated cells (Antoni A regions)mixed with less cellular regions (Antoni B). Immunohistochemical study stained intensively for vimentin and S-100. Conclusion Schwannomas of the olfactory groove are extremely rare, and the findings of origin of this tumor is still uncertain but recent studies point most probably to the meningeal branches of trigeminal nerve or anterior ethmoidal nerves. .

Streptomycin potency is dependent on MscL channel expression.

The antibiotic streptomycin is widely used in the treatment of microbial infections. The primary mechanism of action is inhibition of translation by binding to the ribosome, but how it enters the bacterial cell is unclear. Early in the study of this antibiotic, a mysterious streptomycin-induced potassium efflux preceding any decrease in viability was observed; it was speculated that this changed the electrochemical gradient such that streptomycin better accessed the cytoplasm. Here we use a high-throughput screen to search for compounds targeting the mechanosensitive channel of large conductance (MscL) and find dihydrostreptomycin among the 'hits'. Furthermore, we find that MscL is not only necessary for the previously described streptomycin-induced potassium efflux, but also directly increases MscL activity in electrophysiological studies. The data suggest that gating MscL is a novel mode of action of dihydrostreptomycin, and that MscL's large pore may provide a mechanism for cell entry.

Transduction without tip links in cochlear hair cells is mediated by ion channels with permeation properties distinct from those of the mechano-electrical transducer channel.

Tip links between adjacent stereocilia are believed to gate mechano-electrical transducer (MET) channels and mediate the electrical responses of sensory hair cells. We found that mouse auditory hair cells that lack tip links due to genetic mutations or exposure to the Ca(2+) chelator BAPTA can, however, still respond to mechanical stimuli. These MET currents have unusual properties and are predominantly of the opposite polarity relative to those measured when tip links are present. There are other striking differences, for example, the channels are usually all closed when the hair cell is not stimulated and the currents in response to strong stimuli can be substantially larger than normal. These anomalous MET currents can also be elicited early in development, before the onset of mechano-electrical transduction with normal response polarity. Current-voltage curves of the anomalous MET currents are linear and do not show the rectification characteristic of normal MET currents. The permeant MET channel blocker dihydrostreptomycin is two orders of magnitude less effective in blocking the anomalous MET currents. The findings suggest the presence of a large population of MET channels with pore properties that are distinct from those of normal MET channels. These channels are not gated by hair-bundle links and can be activated under a variety of conditions in which normal tip-link-mediated transduction is not operational.

Distribution of antimicrobial-resistant lactic acid bacteria in natural cheese in Japan.

To determine and compare the extent of contamination caused by antimicrobial-resistant lactic acid bacteria (LAB) in imported and domestic natural cheeses on the Japanese market, LAB were isolated using deMan, Rogosa and Sharpe (MRS) agar and MRS agar supplemented with six antimicrobials. From 38 imported and 24 Japanese cheeses, 409 LAB isolates were obtained and their antimicrobial resistance was tested. The percentage of LAB resistant to dihydrostreptomycin, erythromycin, and/or oxytetracycline isolated from imported cheeses (42.1%) was significantly higher than that of LAB resistant to dihydrostreptomycin or oxytetracycline from cheeses produced in Japan (16.7%; P=0.04). Antimicrobial resistance genes were detected in Enterococcus faecalis (tetL, tetM, and ermB; tetL and ermB; tetM) E. faecium (tetM), Lactococcus lactis (tetS), Lactobacillus (Lb.), casei/paracasei (tetM or tetW), and Lb. rhamnosus (ermB) isolated from seven imported cheeses. Moreover, these E. faecalis isolates were able to transfer antimicrobial resistance gene(s). Although antimicrobial resistance genes were not detected in any LAB isolates from Japanese cheeses, Lb. casei/paracasei and Lb. coryniformis isolates from a Japanese farm-made cheese were resistant to oxytetracycline (minimal inhibitory concentration [MIC], 32 µg/mL). Leuconostoc isolates from three Japanese farm-made cheeses were also resistant to dihydrostreptomycin (MIC, 32 to >512 µg/mL). In conclusion, the present study demonstrated contamination with antimicrobial-resistant LAB in imported and Japanese farm-made cheeses on the Japanese market, but not in Japanese commercial cheeses.

The resting transducer current drives spontaneous activity in prehearing mammalian cochlear inner hair cells.

Spontaneous Ca(2+)-dependent electrical activity in the immature mammalian cochlea is thought to instruct the formation of the tonotopic map during the differentiation of sensory hair cells and the auditory pathway. This activity occurs in inner hair cells (IHCs) during the first postnatal week, and the pattern differs along the cochlea. During the second postnatal week, which is before the onset of hearing in most rodents, the resting membrane potential for IHCs is apparently more hyperpolarized (approximately -75 mV), and it remains unclear whether spontaneous action potentials continue to occur. We found that when mouse IHC hair bundles were exposed to the estimated in vivo endolymphatic Ca(2+) concentration (0.3 mm) present in the immature cochlea, the increased open probability of the mechanotransducer channels caused the cells to depolarize to around the action potential threshold (approximately -55 mV). We propose that, in vivo, spontaneous Ca(2+) action potentials are intrinsically generated by IHCs up to the onset of hearing and that they are likely to influence the final sensory-independent refinement of the developing cochlea.

Prevalence and antimicrobial susceptibility of Salmonella in Japanese broiler flocks.

This study determined the prevalence and antimicrobial susceptibility of Salmonella isolated from broiler flocks in Japan. Caecal dropping samples were collected from 288 broiler flocks between November 2007 and February 2010. Salmonella was prevalent in 248 (86·1%) broiler flocks. The top three serovars were S. Infantis, S. Manhattan and S. Schwarzengrund. S. Infantis was found in all regions tested in this study. However, S. Manhattan and S. Schwarzengrund were frequently found only in the western part of Japan. High antimicrobial resistance rates were observed against oxytetracycline (90·2%), dihydrostreptomycin (86·7%) and ampicillin (36·5%), and 258 (90·5%) of 285 isolates were resistant to two or more antimicrobial agents. Interestingly, 26·3% of isolates were resistant to ceftiofur, especially 38·1% of S. Infantis isolates, although its use in broilers has not been approved in Japan. This study showed that Salmonella is highly prevalent (86·1%) in Japanese broiler flocks, that 90·5% of Salmonella isolates were multidrug-resistant, and that S. Infantis frequently exhibited resistance to cephalosporin antimicrobial agents.

Collaborative study for the establishment of the 3rd international standard for dihydrostreptomycin.

An international collaborative study was organised to establish the World Health Organization (WHO) 3rd International Standard (IS) for dihydrostreptomycin. Eleven laboratories from different countries participated in the collaborative study. The potency of the candidate batch, a freeze-dried preparation, was estimated by microbiological assays with sensitive microorganisms. To ensure continuity between consecutive batches of the WHO IS, the 2nd IS for dihydrostreptomycin was used as standard. Based on the results of the study, the 3rd IS for dihydrostreptomycin was adopted at the meeting of the WHO Expert Committee on Biological Standardisation (ECBS) in 2011 with an assigned anti-microbiological activity of 19425 International Units (IU) per vial. The 3rd IS for dihydrostreptomycin is available from the EDQM.

Susceptibility of bacterial pathogens against lincomycin/spectinomycin (1/2), penicillin G/neomycin (1/1), and penicillin G/dihydrostreptomycin (1/1) as determined in the BfT-GermVet monitoring program 2004-2006.

A total of 368 bacterial pathogens, including 72 coagulase-positive and coagulase-variable staphylococci, 97 beta-haemolytic streptococci, 51 Escherichia coli, 75 Pasteurella multocida, 25 Mannheimia haemolytica, 25 Pseudomonas aeruginosa, and 23 Arcanobacterium pyogenes, were investigated for their susceptibility to the three combinations of antimicrobial agents lincomycin/spectinomycin (1/2), penicillin G/neomycin (1/1), and penicillin G/dihydrostreptomycin (1/1) in comparison to their susceptibility to the corresponding single substances. When comparing the minimum inhibitory concentrations (MICs) determined for any of the three combinations with those for the single substances, the lowest MIC of one of the two substances usually determined the MIC of the combination.This observation was made for all three combinations and all bacterial pathogens tested.Thus, it is assumed that the combination of lincomycin with spectinomycin as well as that of penicillin with either neomycin or dihydrostreptomycin resulted in an extended spectrum of target bacterial pathogens rather than in an increase in antimicrobial efficacy.

Clinical mastitis in Norwegian herds after a combined selective dry-cow therapy and teat-dipping trial.

The objective of this study was to see if introduction of a 2-yr combined selective dry-cow therapy and teat-dipping trial would reduce clinical mastitis (CM) events in 164 Norwegian dairy herds. Three different penicillin or penicillin/dihydrostreptomycin-based dry-cow treatments, and 3 different teat-dipping regimens (negative control, iodine teat dip, or an external teat sealant) were independently and randomly allocated to each herd. Complete lactations both before and during the trial were investigated. Altogether, 1,005 CM cases were recorded in the lactations before the trial and 924 cases were recorded during the trial. Bacteriological milk samples were available from 784 of the 924 CM cases during the trial. Among these, Staphylococcus aureus were isolated from 47.4%, Streptococcus dysgalactiae from 22.5%, Escherichia coli from 10.7%, and coagulase-negative staphylococci from 6.3%. In addition, 12.5% cases were bacteriological negative, and the remainder of the CM cases were caused by other microbes. The different models were analyzed using Cox regression analysis with PROC PHREG and a positive stable frailty model in the SAS macro. Separate models were made for cows housed in tie-stalls and free-stalls. Parity had a significant impact on the CM risk in both type of stalls. Older cows (parity > 3) had the highest hazard ratio of contracting CM in tie-stalls (1.68) and free-stalls (2.18) compared with parity 1. The CM risk decreased significantly (13%) in tie-stalls and by 18% in free-stalls. In tie-stalls, iodine-dipped cows had a significantly lower chance (21%) of getting CM compared with the negative control and the use of external teat sealant. The same trend was seen in free-stalls; however, the differences were not significant. Compared with CM before the trial, the reduction of CM was 15% during the trial.

Depolarization of cochlear outer hair cells evokes active hair bundle motion by two mechanisms.

There is current debate about the origin of mechanical amplification whereby outer hair cells generate force to augment the sensitivity and frequency selectivity of the mammalian cochlea. To distinguish contributions to force production from the mechanotransducer (MET) channels and somatic motility, we have measured hair bundle motion during depolarization of individual outer hair cells in isolated rat cochleas. Depolarization evoked rapid positive bundle deflections that were reduced by perfusion with the MET channel blocker dihydrostreptomycin, with no effect on the nonlinear capacitance that is a manifestation of prestin-driven somatic motility. However, the movements were also diminished by Na salicylate and depended on the intracellular anion, properties implying involvement of the prestin motor. Furthermore, depolarization of one outer hair cell caused motion of neighboring hair bundles, indicating overall motion of the reticular lamina. Depolarization of solitary outer hair cells caused cell-length changes whose voltage-activation range depended on the intracellular anion but were insensitive to dihydrostreptomycin. These results imply that both the MET channels and the somatic motor participate in hair bundle motion evoked by depolarization. It is conceivable that the two processes can interact, a signal from the MET channels being capable of modulating the activity of the prestin motor.

Comparison of Campylobacter isolated from humans and food-producing animals in Japan.

AIMS: To compare the antimicrobial resistance, serotypes and flagellin gene types of Campylobacter isolated from humans and food-producing animals and thereby facilitate elucidation of the origin of Campylobacter causing human infection in Japan. METHODS AND RESULTS: The MIC values of ampicillin, dihydrostreptomycin, gentamicin, erythromycin, oxytetracycline, nalidixic acid and enrofloxacin for Campylobacter isolated from humans (134 isolates), cattle (38 isolates), pigs (69 isolates), layers (84 isolates) and broilers (51 isolates) were compared. The MIC(90) values of ampicillin for Campylobacter jejuni isolates from poultry were higher than those from humans and cattle. Campylobacter coli that was resistant to dihydrostreptomycin and erythromycin was observed at a higher frequency in humans and pigs than in poultry. The restriction fragment profiles of flaA of human, bovine and broiler isolates were analysed by clustering, and the isolates were classified into five clusters. Cluster I contained only human and bovine isolates. Clusters III, IV and V contained human, bovine and broiler isolates. CONCLUSIONS: Campylobacter isolates from humans included isolates that exhibited characteristics identical to those of the bovine, porcine and poultry isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: In addition to poultry, cattle and pigs are believed to be sources of campylobacteriosis in Japan.

Induction of the MexXY efflux pump in Pseudomonas aeruginosa is dependent on drug-ribosome interaction.

MexXY is an inducible efflux system that contributes to the natural resistance of Pseudomonas aeruginosa to antibiotics. Experiments involving real-time PCR after reverse transcription in reference strain PAO1 showed concentration-dependent induction of gene mexY by various ribosome inhibitors (e.g., chloramphenicol, tetracycline, macrolides, and aminoglycosides) but not by antibiotics acting on other cellular targets (e.g., beta-lactams, fluoroquinolones). Confirming a functional link between the efflux system and the translational machinery, ribosome protection by plasmid-encoded proteins TetO and ErmBP increased the resistance of a DeltamexAB-oprM mutant of PAO1 to tetracycline and erythromycin, respectively, as well as the concentrations of both drugs required to induce mexY. Furthermore, spontaneous mutations resulting in specific resistance to dihydrostreptomycin or spectinomycin also raised the minimal drug concentration for mexXY induction in strain PAO1. While strongly upregulated in a PAO1 mutant defective in gene mexZ (which codes for a putative repressor of operon mexXY), gene mexY remained inducible by agents such as tetracycline, chloramphenicol, and spectinomycin, suggesting additional regulatory loci for mexXY. Altogether, these data demonstrate physiological interplays between MexXY and the ribosome and are suggestive of an alternative function for MexXY beyond antibiotic efflux.

Tonic mechanosensitivity of outer hair cells after loss of tip links.

Tip links - the extracellular connectors between the distal ends of adjacent stereocilia - are essential for the fast mechanical gating of hair-cell transducer channels. Transduction in the absence of tip links was investigated for outer hair cells of the adult guinea-pig cochlea by patch-clamp recordings of the whole-cell current during mechanical stimulation of the hair bundle. Loss of tip links induced by application of BAPTA led to permanently opened transducer channels, as evidenced by a constant inward current, loss of response to sinusoidal mechanical deflection of the hair bundle and block by the open-channel blocker dihydrostreptomycin (100 microM). Step deflection of the hair bundle (200-500 nm) in the inhibitory direction exponentially reduced this current to a constant value with time constant, tau(on), of the order of seconds. The current returned exponentially to the pre-stimulus level with time-constant, tau(off), also of the order of seconds. tau(on) was dependent on the inter-stimulus interval, Deltat, such that reducing this interval below about 40 s resulted in an exponentially faster response. tau(off) was independent of Deltat. Application of the calcium ionophore, ionomycin (10 microM), showed that tau(on) became independent of Deltat after saturating elevation of the intracellular Ca(2+) concentration. Flash-photolytic release of intracellular caged calcium (25-microM NP-EGTA/AM) showed that tau(on) is dependent on intracellular Ca(2+) concentration. These experiments imply an intracellular, calcium-dependent gating mechanism for hair-cell transducer channels.

Antimicrobial susceptibilities of Campylobacter isolated from food-producing animals on farms (1999-2001): results from the Japanese Veterinary Antimicrobial Resistance Monitoring Program.

Nationwide monitoring of antimicrobial resistance in Campylobacter isolated from food-producing animals on farms was performed in Japan. A total of 468 Campylobacter isolates were obtained during the period from June 1999 to March 2001. Campylobacter species showed high frequencies of resistance to oxytetracycline and dihydrostreptomycin. The frequencies of resistance in Campylobacter coli to aminoglycosides, macrolides, tetracycline and quinolones were higher than those in Campylobacter jejuni to the same drugs. All of the C. jejuni isolates were susceptible to macrolide antibiotics, whereas 48.4% of the C. coli isolates were resistant to macrolides. Resistance to fluoroquinolones was observed in C. jejuni isolates from broilers (12%) and layers (2.6%), indicating that the level of fluoroquinolone resistance in Japan could be ranked as low.

[Interaction of aminoglycosides and the other antibiotic on selected bacterial strains]. / Ocena oddzialywania aminoglikozydu z antybiotykiem innej grupy na wybrane szczepy bakteryjne.

The aim of this study was to analyse the activity interaction of aminoglycosides (gentamicin, kanamycin, streptomycin and dihydrostreptomycin) when combined with other antibiotics (lincomycin, benzylpenicillin, amoxicillin, cephalexin, spectinomycin and erythromycin), on selected clinical bacterial strains. The checkerboard method has been selected from the traditional assays for the measurement of antibiotic interaction. Checkerboard results for all strains demonstrated synergism for nine cases (9/112--8%). Additive effects were predominant--73/112--65.2%. In 12.5% neutral effects were shown, but in 11.6% of combinations FIC indexes were not possible to calculate, because of the resistance of clinical strains to the highest concentration of at least one antibiotic. The best results were achieved for combinations of dihydrostreptomycin with procaine penicillin because of higher number of cases synergy effect was observed. Antagonism of aminoglycosides and beta-lactams in case of gentamicin and amoxicillin for E. coli and E. cloacea strains were shown. Potential activity for combination of streptomycin and erythromycin was shown.

[Comparison of checkerboard and time-kill methods for the analysis of two antibiotics combined]. / Porównanie metod szachownicy i "time-kill" stosowanych do badania zestawienia dwóch antybiotyków.

Activity interaction analysis of two antibiotics by two methods: checkerboard and "time-kill" was compared during this study. Combinations of procaine penicillin, polymyxin B and bacitracin with neomycin and procaine penicillin with dihydrostreptomycin were examined. Checkerboard method is the most widely used technique for antimicrobials interactions analyses. The "time-kill" method, performed by the broth macrodilution technique, provides a dynamic picture of antimicrobial action and interaction over time (based on serial colony counts). Differences of "time-kill" method and the checkerboard technique, allow single visual examination (after 16 to 24 hours of incubation). Additive and inhibition effects were observed in combinations of neomycin with beta-lactam antibiotic (procaine penicillin) and peptide antibiotics (bacitracin and polymyxin B) on clinical strain S. Enteritidis IL 35 "Time-kill" method also confirmed observations mentioned above. In combinations of procaine penicillin with dihydrostreptomycin on strains E. coli IL 531 and E. coli IL 256 synergy effects on checkerboard technique were noticed. Such observation was not confirmed by the "time-kill" method. The methodologies and definitions of synergism are variable and not standardized. This situation should be improved, because comparison of the results obtained by different methods becomes a very difficult task.