Antimicrobial activity of copper (II) complex with 1,2-bis [(1,3-diphenylpyrazol-4-yl)methyl] diaminoethane / Antimicrobial activity of copper (II) complex with 1,2-bis [(1,3-diphenylpyrazol-4-yl)methyl] diaminoethane

Background: Multi-resistant strains multiply daily, populate farms, hospitals and other ecological niches around the world, and cause serious infections in animals and humans, often leading to a fatal outcome. Researchers of all profiles are investigating intensively to fi nd new substances with antimicrobial activity. In the period between 1981 and 2002, 163 new chemical compounds were approved for use as drugs. Synthesized compounds have become much more interesting than the natural ones in the production of new antimicrobial agents. Some of these synthesized compounds are Copper (II) complex. The antimicrobial properties of copper were known in ancient Egypt (2000 BC), where it was used to sterilize water and wounds. Copper is still interesting for todays research. Materials, Methods & Results: Antimicrobial activity was tested using a microdilution method according to Clinical and Laboratory Standards Institute. The percentage of surviving bacteria was calculated in comparison to the number of bacteria placed in each well. Based on these results, using the Excel software package from Microsoft Office 2007, graphs were generated that showed the percentage of surviving bacteria depending on the corresponding effective concentrations of the tested substance. The function, which was used to approximate the experimental results, was determined using the Power Trendline supp

Background: Multi-resistant strains multiply daily, populate farms, hospitals and other ecological niches around the world, and cause serious infections in animals and humans, often leading to a fatal outcome. Researchers of all profiles are investigating intensively to fi nd new substances with antimicrobial activity. In the period between 1981 and 2002, 163 new chemical compounds were approved for use as drugs. Synthesized compounds have become much more interesting than the natural ones in the production of new antimicrobial agents. Some of these synthesized compounds are Copper (II) complex. The antimicrobial properties of copper were known in ancient Egypt (2000 BC), where it was used to sterilize water and wounds. Copper is still interesting for todays research. Materials, Methods & Results: Antimicrobial activity was tested using a microdilution method according to Clinical and Laboratory Standards Institute. The percentage of surviving bacteria was calculated in comparison to the number of bacteria placed in each well. Based on these results, using the Excel software package from Microsoft Office 2007, graphs were generated that showed the percentage of surviving bacteria depending on the corresponding effective concentrations of the tested substance. The function, which was used to approximate the experimental results, was determined using the Power Trendline supp

Antimicrobial activity of copper (II) complex with 1,2-bis [(1,3-diphenylpyrazol-4-yl)methyl] diaminoethane / Antimicrobial activity of copper (II) complex with 1,2-bis [(1,3-diphenylpyrazol-4-yl)methyl] diaminoethane

Background: Multi-resistant strains multiply daily, populate farms, hospitals and other ecological niches around the world, and cause serious infections in animals and humans, often leading to a fatal outcome. Researchers of all profiles are investigating intensively to fi nd new substances with antimicrobial activity. In the period between 1981 and 2002, 163 new chemical compounds were approved for use as drugs. Synthesized compounds have become much more interesting than the natural ones in the production of new antimicrobial agents. Some of these synthesized compounds are Copper (II) complex. The antimicrobial properties of copper were known in ancient Egypt (2000 BC), where it was used to sterilize water and wounds. Copper is still interesting for todays research. Materials, Methods & Results: Antimicrobial activity was tested using a microdilution method according to Clinical and Laboratory Standards Institute. The percentage of surviving bacteria was calculated in comparison to the number of bacteria placed in each well. Based on these results, using the Excel software package from Microsoft Office 2007, graphs were generated that showed the percentage of surviving bacteria depending on the corresponding effective concentrations of the tested substance. The function, which was used to approximate the experimental results, was determined using the Power Trendline supp

Background: Multi-resistant strains multiply daily, populate farms, hospitals and other ecological niches around the world, and cause serious infections in animals and humans, often leading to a fatal outcome. Researchers of all profiles are investigating intensively to fi nd new substances with antimicrobial activity. In the period between 1981 and 2002, 163 new chemical compounds were approved for use as drugs. Synthesized compounds have become much more interesting than the natural ones in the production of new antimicrobial agents. Some of these synthesized compounds are Copper (II) complex. The antimicrobial properties of copper were known in ancient Egypt (2000 BC), where it was used to sterilize water and wounds. Copper is still interesting for todays research. Materials, Methods & Results: Antimicrobial activity was tested using a microdilution method according to Clinical and Laboratory Standards Institute. The percentage of surviving bacteria was calculated in comparison to the number of bacteria placed in each well. Based on these results, using the Excel software package from Microsoft Office 2007, graphs were generated that showed the percentage of surviving bacteria depending on the corresponding effective concentrations of the tested substance. The function, which was used to approximate the experimental results, was determined using the Power Trendline supp

Antimicrobial activity of copper (ii) complex with 1, 2-bis [(1, 3-diphenylpyrazol-4-yl)methyl] diaminoethane

Background: Multi-resistant strains multiply daily, populate farms, hospitals and other ecological niches around the world, and cause serious infections in animals and humans, often leading to a fatal outcome. Researchers of all profiles are investigating intensively to find new substances with antimicrobial activity. In the period between 1981 and 2002, 163 new chemical compounds were approved for use as drugs. Synthesized compounds have become much more interesting than the natural ones in the production of new antimicrobial agents. Some of these synthesized compounds are Copper (II) complex. The antimicrobial properties of copper were known in ancient Egypt (2000 BC), where it was used to sterilize water and wounds. Copper is still interesting for today's research. Materials, Methods & Results: Antimicrobial activity was tested using a microdilution method according to Clinical and Laboratory Standards Institute. The percentage of surviving bacteria was calculated in comparison to the number of bacteria placed in each well. Based on these results, using the Excel software package from Microsoft Office 2007, graphs were generated that showed the percentage of surviving bacteria depending on the corresponding effective concentrations of the tested substance. The function, which was used to approximate the experimental results, was determined using the Power Trendline supplement from the Microsoft Excel program. Cytotoxicity (growth inhibition) was evaluated by tetrazolium colorimetric MTT assay, after exposure of cells to the tested compound for 48 h. Inhibition of growth was expressed as a percentage of cytotoxicity and calculated according to the following equation: (1-A test/A control) x 100. MBC99.9 and MIC99 of the test substance were lowest for Arcanobacterium haemolyticum being 0.2 mg/L and 0.0054 mg/L, respectively. The highest values were obtained for Arcanobacterium pyogenes and methicillin-resistant Staphylococcus aureus (MRSA) 488.002 mg/L and 20.2 mg/L. MIC80 for all four strains ranged from 0.00002 to 0.0023 mg/L. Measured values for MIC99 are 0.00545 mg/L for A. haemolyticum, 0.0443199 mg/L for R. equi, 0.0520712 mg/L for S. aureus and 2.36378 mg/L for A. pyogenes. Values for MIC99.9 ranged from 0.236134 to 488,002 mg/L. Most of the MIC values obtained in this study are significantly lower than those reported by other researchers. The values we obtained were lower as compared to MIC values for standard antibiotics, which were considered acceptable by the relevant institutions. This speaks in favor of a stronger antibacterial effect of our tested substances. In regards to cytotoxicity, the obtained MIC80 doses were lower than toxic, whereas MIC90 could be classified as low-toxic (less than 0.0625 µM), except of Arcanobacterium pyogenes only. According to the IC50 values, the compound Cu (L) Br2·MeOH was 6.4-fold and 4.8-fold more potent against HCT116 cells compared to normal lung fi broblasts and SW620 cells, respectively. Discussion: Copper (II) complex with an arylpyrazole ligand exhibits strong antibacterial properties, and it shows bacteriostatic effect at concentrations where there is no cytotoxic effect in normal human cells. The emergence of multi-resistant strains of pathogenic bacteria is a growing problem worldwide. Therefore, each new compound with potential antimicrobial activity, especially if it is not cytotoxic in effective dosage, deserves the attention of the scientific community. In this paper, we presented a newly synthesized substance with such properties.

Clinical importance and representation of toxigenic and non-toxigenic Clostridium difficile cultivated from stool samples of hospitalized patients

The aim of this study was to fortify the clinical importance and representation of toxigenic and non-toxigenic Clostridium difficile isolated from stool samples of hospitalized patients. This survey included 80 hospitalized patients with diarrhea and positive findings of Clostridium difficile in stool samples, and 100 hospitalized patients with formed stool as a control group. Bacteriological examination of a stool samples was conducted using standard microbiological methods. Stool sample were inoculated directly on nutrient media for bacterial cultivation (blood agar using 5% sheep blood, Endo agar, selective Salmonella Shigella agar, Selenite-F broth, CIN agar and Skirrow's medium), and to selective cycloserine-cefoxitin-fructose agar (CCFA) (Biomedics, Parg qe tehnicologico, Madrid, Spain) for isolation of Clostridium difficile. Clostridium difficile toxin was detected by ELISA-ridascreen Clostridium difficile Toxin A/B (R-Biopharm AG, Germany) and ColorPAC ToxinA test (Becton Dickinson, USA). Examination of stool specimens for the presence of parasites (causing diarrhea) was done using standard methods (conventional microscopy), commercial concentration test Paraprep S Gold kit (Dia Mondial, France) and RIDA®QUICK Cryptosporidium/Giardia Combi test (R-Biopharm AG, Germany). Examination of stool specimens for the presence of fungi (causing diarrhea) was performed by standard methods. All stool samples positive for Clostridium difficile were tested for Rota, Noro, Astro and Adeno viruses by ELISA - ridascreen (R-Biopharm AG, Germany). In this research we isolated 99 Clostridium difficile strains from 116 stool samples of 80 hospitalized patients with diarrhea. The 53 (66.25%) of patients with diarrhea were positive for toxins A and B, one (1.25%) were positive for only toxin B. Non-toxigenic Clostridium difficile isolated from samples of 26 (32.5%) patients. However, other pathogenic microorganisms of intestinal tract cultivated from samples of 16 patients. Examination of cultivated colonies revealed that most of cultivated species belonged to genera of Campylobacter spp., Salmonella spp., and Candida spp.. In control group, toxigenic Clostridium difficile cultivated from stool samples of two patients (2%) and non-toxigenic Clostridium difficile from samples of five patients (5%). This research confirmed clinical importance of toxigenic Clostridium difficile found in liquid stool samples of hospitalized patient, and the possibility of asymptomatic carriage in 2% of patients with formed stool.

Clinical importance and representation of toxigenic and non-toxigenic Clostridium difficile cultivated from stool samples of hospitalized patients

The aim of this study was to fortify the clinical importance and representation of toxigenic and non-toxigenic Clostridium difficile isolated from stool samples of hospitalized patients. This survey included 80 hospitalized patients with diarrhea and positive findings of Clostridium difficile in stool samples, and 100 hospitalized patients with formed stool as a control group. Bacteriological examination of a stool samples was conducted using standard microbiological methods. Stool sample were inoculated directly on nutrient media for bacterial cultivation (blood agar using 5% sheep blood, Endo agar, selective Salmonella Shigella agar, Selenite-F broth, CIN agar and Skirrow's medium), and to selective cycloserine-cefoxitin-fructose agar (CCFA) (Biomedics, Parg qe tehnicologico, Madrid, Spain) for isolation of Clostridium difficile. Clostridium difficile toxin was detected by ELISA-ridascreen Clostridium difficile Toxin A/B (R-Biopharm AG, Germany) and ColorPAC ToxinA test (Becton Dickinson, USA). Examination of stool specimens for the presence of parasites (causing diarrhea) was done using standard methods (conventional microscopy), commercial concentration test Paraprep S Gold kit (Dia Mondial, France) and RIDA®QUICK Cryptosporidium/Giardia Combi test (R-Biopharm AG, Germany). Examination of stool specimens for the presence of fungi (causing diarrhea) was performed by standard methods. All stool samples positive for Clostridium difficile were tested for Rota, Noro, Astro and Adeno viruses by ELISA - ridascreen (R-Biopharm AG, Germany). In this research we isolated 99 Clostridium difficile strains from 116 stool samples of 80 hospitalized patients with diarrhea. The 53 (66.25%) of patients with diarrhea were positive for toxins A and B, one (1.25%) were positive for only toxin B. Non-toxigenic Clostridium difficile isolated from samples of 26 (32.5%) patients. However, other pathogenic microorganisms of intestinal tract cultivated from samples of 16 patients. Examination of cultivated colonies revealed that most of cultivated species belonged to genera of Campylobacter spp., Salmonella spp., and Candida spp.. In control group, toxigenic Clostridium difficile cultivated from stool samples of two patients (2%) and non-toxigenic Clostridium difficile from samples of five patients (5%). This research confirmed clinical importance of toxigenic Clostridium difficile found in liquid stool samples of hospitalized patient, and the possibility of asymptomatic carriage in 2% of patients with formed stool.

A proposal of a diagnostic protocol for isolation of Corynebacterium ulcerans from cows milk / A proposal of a diagnostic protocol for isolation of Corynebacterium ulcerans from cows milk

Background: Literature about presence Corynebacterium ulcerans in milk samples from cows with mastitis is rare and in the literature there are only a few reports. In this study the isolation and identification of Corynebacterium ulcerans from mastitis in dairy cows were done. Also, optimization of diagnostic protocols to identify Corynebacterium ulcerans was performed.Materials, Methods & Results: The investigation was performed at the cattle farm that is characterized by closed housing system diary Holstein-Friesian cows during an outbreak of acute mastitis. Milk samples from 298 lactating cows were collected in sterile sampling tubes. Before the collection of quarter milk samples, the udder was thoroughly cleaned with soap and water and rubbed to dry. All collected milk samples were examined for mastitis using California mastitis test, which was carried out by the method first described by Schalm and Noorlander. Equal volumes (5 mL) of commercial CMT reagent and quarter milk were mixed and the changes in milk fluidity and viscosity were observed. Sample portions (0.1 mL each) were inoculated on 10% sheep blood agar, Endo agar and Sabouraud agar as well as on thioglycolate medium and nutrient broth. Primary plates were incubated for 3 days at 37o C in aerobic conditions. Cultural, morphological and conventional biochemical testing was done. The survey was complemented by d

Background: Literature about presence Corynebacterium ulcerans in milk samples from cows with mastitis is rare and in the literature there are only a few reports. In this study the isolation and identification of Corynebacterium ulcerans from mastitis in dairy cows were done. Also, optimization of diagnostic protocols to identify Corynebacterium ulcerans was performed.Materials, Methods & Results: The investigation was performed at the cattle farm that is characterized by closed housing system diary Holstein-Friesian cows during an outbreak of acute mastitis. Milk samples from 298 lactating cows were collected in sterile sampling tubes. Before the collection of quarter milk samples, the udder was thoroughly cleaned with soap and water and rubbed to dry. All collected milk samples were examined for mastitis using California mastitis test, which was carried out by the method first described by Schalm and Noorlander. Equal volumes (5 mL) of commercial CMT reagent and quarter milk were mixed and the changes in milk fluidity and viscosity were observed. Sample portions (0.1 mL each) were inoculated on 10% sheep blood agar, Endo agar and Sabouraud agar as well as on thioglycolate medium and nutrient broth. Primary plates were incubated for 3 days at 37o C in aerobic conditions. Cultural, morphological and conventional biochemical testing was done. The survey was complemented by d

A proposal of a diagnostic protocol for isolation of Corynebacterium ulcerans from cows milk / A proposal of a diagnostic protocol for isolation of Corynebacterium ulcerans from cows milk

Background: Literature about presence Corynebacterium ulcerans in milk samples from cows with mastitis is rare and in the literature there are only a few reports. In this study the isolation and identification of Corynebacterium ulcerans from mastitis in dairy cows were done. Also, optimization of diagnostic protocols to identify Corynebacterium ulcerans was performed.Materials, Methods & Results: The investigation was performed at the cattle farm that is characterized by closed housing system diary Holstein-Friesian cows during an outbreak of acute mastitis. Milk samples from 298 lactating cows were collected in sterile sampling tubes. Before the collection of quarter milk samples, the udder was thoroughly cleaned with soap and water and rubbed to dry. All collected milk samples were examined for mastitis using California mastitis test, which was carried out by the method first described by Schalm and Noorlander. Equal volumes (5 mL) of commercial CMT reagent and quarter milk were mixed and the changes in milk fluidity and viscosity were observed. Sample portions (0.1 mL each) were inoculated on 10% sheep blood agar, Endo agar and Sabouraud agar as well as on thioglycolate medium and nutrient broth. Primary plates were incubated for 3 days at 37o C in aerobic conditions. Cultural, morphological and conventional biochemical testing was done. The survey was complemented by d

Background: Literature about presence Corynebacterium ulcerans in milk samples from cows with mastitis is rare and in the literature there are only a few reports. In this study the isolation and identification of Corynebacterium ulcerans from mastitis in dairy cows were done. Also, optimization of diagnostic protocols to identify Corynebacterium ulcerans was performed.Materials, Methods & Results: The investigation was performed at the cattle farm that is characterized by closed housing system diary Holstein-Friesian cows during an outbreak of acute mastitis. Milk samples from 298 lactating cows were collected in sterile sampling tubes. Before the collection of quarter milk samples, the udder was thoroughly cleaned with soap and water and rubbed to dry. All collected milk samples were examined for mastitis using California mastitis test, which was carried out by the method first described by Schalm and Noorlander. Equal volumes (5 mL) of commercial CMT reagent and quarter milk were mixed and the changes in milk fluidity and viscosity were observed. Sample portions (0.1 mL each) were inoculated on 10% sheep blood agar, Endo agar and Sabouraud agar as well as on thioglycolate medium and nutrient broth. Primary plates were incubated for 3 days at 37o C in aerobic conditions. Cultural, morphological and conventional biochemical testing was done. The survey was complemented by d

A proposal of a diagnostic protocol for isolation of Corynebacterium ulcerans from cow's milk

Background: Literature about presence Corynebacterium ulcerans in milk samples from cows with mastitis is rare and in the literature there are only a few reports. In this study the isolation and identification of Corynebacterium ulcerans from mastitis in dairy cows were done. Also, optimization of diagnostic protocols to identify Corynebacterium ulcerans was performed. Materials, Methods & Results: The investigation was performed at the cattle farm that is characterized by closed housing system diary Holstein-Friesian cows during an outbreak of acute mastitis. Milk samples from 298 lactating cows were collected in sterile sampling tubes. Before the collection of quarter milk samples, the udder was thoroughly cleaned with soap and water and rubbed to dry. All collected milk samples were examined for mastitis using California mastitis test, which was carried out by the method first described by Schalm and Noorlander. Equal volumes (5 mL) of commercial CMT reagent and quarter milk were mixed and the changes in milk fluidity and viscosity were observed. Sample portions (0.1 mL each) were inoculated on 10% sheep blood agar, Endo agar and Sabouraud agar as well as on thioglycolate medium and nutrient broth. Primary plates were incubated for 3 days at 37ºC in aerobic conditions. Cultural, morphological and conventional biochemical testing was done. The survey was complemented by double CAMP and plasma coagulation tube test. All 14 isolates developed a synergistic haemolysis with Rhodococcus equi (ATCC 6939) and inverse CAMP phenomenon with Staphylococcus aureus and coagulated rabbit plasma. Final diagnosis was confirmed using API Coryne V 2.0 and software program by BioMerieux1, revealing an identity rate of 99.9%, accuracy rate T = 1, test count = 0. Discussion: The first fourteen isolates of Corynebacterium ulcerans have been identified in our country, on the basis of a diagnostic protocol that is proposed in this paper. In our experience double CAMP test, rabbit plasma coagulation, catalase, oxidase tests and selected biochemical parameters, are sufficient as a diagnostic minimum. In the diagnostics of bacterial agents in cow mastitis, the attention of a bacteriologist is mostly limited to most widespread agents of mastitis, the isolation of which is mandatory pursuant to national legislation (Staphylococcus aureus and Streptococcus agalactiae). A more important reason for "missing" Corynebacterium ulcerans in the diagnosis is its colonial morphology that could resemble organisms of the genus Staphylococcus. Complex and expensive diagnostic procedure that is not available to most laboratories is also responsible for the small number of reports of isolation C. ulcerans. Furthermore, in routine work C. ulcerans could be misidentified with Staphylococcus intermedius, because of cultural similarity, positive plasma coagulation tube test and absence of manitol fermentation of both species. This paper is a report on isolation and identification of Corynebacterium ulcerans from milk of cows with mastitis, as well as a suggestion of a diagnostic protocol available for routine work in most veterinary microbiology laboratory. Therefore we suggest as the diagnostic protocol double CAMP test to be used as a complementary method to rabbit plasma coagulation tube test.