Pseudomonas chlororaphis is a bacterium used as a soil inoculant in agriculture and horticulture.... more Pseudomonas chlororaphis is a bacterium used as a soil inoculant in agriculture and horticulture. It can act as a biocontrol agent against certain phytopathogenic fungi via production of phenazine type antibiotics. Phytopathogenic fungi are responsible for several plant diseases in different medicinal plants and cause very important economic losses in Serbian plantation. Among them, Alternaria sp., Phoma sp. and Drechslera tetramera are known as major plant pathogens, whereby at least 20% of agricultural spoilage is caused by Alternaria species. In this study, we examined the antifungal activity of indigenous Pseudomonas chlororaphis isolates against the phytopathogenic fungi Alternaria sp., Phoma sp. and Drechslera tetramera, which had infected anise (Pimpinella anisum L., fam. Apiaceae), using in vitro inhibition tests. The obtained results showed that studied antibioticproducing Ps. chlororaphis isolates inhibited the tested fungi as follows: D. tetramera in the range from 3.85-6...
The genetic context of the bla NDM-1 gene in the genome of Pseudomonas aeruginosa MMA83 was inves... more The genetic context of the bla NDM-1 gene in the genome of Pseudomonas aeruginosa MMA83 was investigated. Sequencing of the cosmid selected for the bla NDM-1 gene revealed the presence of two bla NDM-1 copies in the genome of P. aeruginosa MMA83 in a unique genetic environment. Additionally, mating assays, DNA-DNA hybridization, and an S1 nuclease assay strongly suggest that the bla NDM-1 gene in P. aeruginosa MMA83 is chromosome borne.
The aim of this study was to detect genes for enterotoxins (hbla, entFM and bceT) and for emetic ... more The aim of this study was to detect genes for enterotoxins (hbla, entFM and bceT) and for emetic toxin (cer), to determine antibiotic resistance, and to estimate intraspecies diversity in B. cereus isolates by RAPD analysis. B. cereus was identified in 12 out of 117 indigenous Bacillus spp. using the classical microbiological methods and PCR. All isolates were resistant to penicillin and ampicillin, two to tetracyclin and four to trimethoprim-sulphamethoxazole. Also, all isolates produced inducible penicillinases and β-lactamase. Toxin genes were detected with PCR. EntFM and cer genes were present in all isolates, hbla in all, but two, and bceT in none. RAPD analysis was performed with four different primers, two of them designed for this study. The intraspecies diversity revealed 10 different patterns at the 90% similarity level. Two separate clusters were formed regardless of a soil type or utilization. The detection of genes encoding toxins in all B. cereus isolates indicated these bacteria as potentially pathogenic and seriously for human health. Regardless of a soil type or utilization, the RAPD analysis showed high intraspecies heterogeneity in B. cereus isolates. To the best of our knowledge, this is the first study to analyse the presence of entero-and emetic toxin genes and genetic heterogeneity in B. cereus isolates from different soil types and different soil utilization in Serbia.
To select isolates suitable for use in biological control of pathogenic fungi and as plant growth... more To select isolates suitable for use in biological control of pathogenic fungi and as plant growth promoting (PGP) agents, Pseudomonas spp., indigenous to the rhizosphere of wild alfalfa and clover plants growing on the old magmatic hill Vagan (Serbia), were isolated. Isolates were selected on the basis of intrinsic antibiotic resistance (IAR) and PGP traits: phosphate solubilization, production of siderophores, hydrogen cyanide (HCN), acyl homoserine lactones (AHLs), indole acetic acid (IAA) and enzymatic activities. The results of the genotyping by repetitive sequence-based polymerase chain reaction, using ERIC and (GTG) 5 primers, and phenotyping by IAR clustered the isolates in five groups, represented by two isolates from alfalfa (Q1 and Q16) and three from white clover (B25, B28 and B29). Isolates Q16 and B25 showed production of HCN, IAA and AHLs. Good phosphate solubilization and production of siderophores were observed on Q1 and Q16. The Q16 isolate exhibited high enzymatic activity. The most promising PGP isolates, Q16 and B25, showed the best antifungal activity against Trichoderma viride, and good antifungal effect against Aspergillus fumigatus and Aspergillus niger, while Penicillium verrucosum was the most resistant fungus. Pseudomonas sp. B25 exhibited higher antifungal potential than Q16. The selected isolates could be further investigated as biological control agents and tested in field conditions to confirm their PGP efficacy.
International Journal of Antimicrobial Agents, 2007
S162 17th ECCMID / 25th ICC, Posters production was detected by double-disc synergy tests and ESB... more S162 17th ECCMID / 25th ICC, Posters production was detected by double-disc synergy tests and ESBL Etests. MICs were determined and interpreted according to CLSI criteria. Isolates were screened for blaCTX-M genes using multiplex PCR and were compared by PFGE of XbaI-digested genomic DNA. Data were analysed using BioNumerics software. Results: A total of 177 ESBL-producing K. pneumoniae isolates were collected. Of these, 60 (33.9%), from 8 hospitals, were positive for blaCTX-M, all with group-1 enzymes. Clonal relationships among these 60 isolates were studied in comparison with 27 CTX-M-negative strains from the same hospitals. The isolates represented multiple strains, but several clusters of related isolates (>80% similarity) were observed, some of them including isolates from more than one centre. The largest cluster consisted of 26 clonally-related isolates with group-1 CTX-M enzymes, 24 of them from one hospital. This outbreak needs further investigation. Two isolates of this outbreak strain were identified in further hospitals, suggesting inter-site transmission. Most isolates had substantial resistance to both cefotaxime (>128 mg/L) and ceftazidime (>16 mg/L), possibly indicating CTX-M-15. Conclusion: K. pneumoniae with CTX-M enzymes are an emerging problem in Slovenian hospitals, but currently represent a minority of ESBL producers of this species. This contrasts with the dominance of CTX-M enzymes among ESBL-producing E. coli in most European countries. The epidemiology of K. pneumoniae with CTX-M enzymes was complex, with the spread of several strains within and between hospitals. Since K. pneumoniae is an important hospital pathogen this worrying development merits closer monitoring.
PER-1 extended-spectrum b-lactamase-producing Pseudomonas aeruginosa clinical isolates from Budap... more PER-1 extended-spectrum b-lactamase-producing Pseudomonas aeruginosa clinical isolates from Budapest, Hungary, and Belgrade, Serbia, were characterized by molecular methods. Two PER-1-positive isolates were recovered from sporadic cases in Budapest and a small cluster of PER-1-positive infections involving four patients were identified at a Belgrade hospital. A class 1 integron harbouring a bla OXA-2 b-lactamase gene and four other gene cassettes was detected in both the Budapest and the Belgrade isolates. The two P. aeruginosa isolates from Budapest also carried another class 1 integron containing bla OXA-74 , aac(6 0)-Ib-cr and cmlA7 genes in its variable region. The aac(6 0)-Ib-cr fluoroquinolone-acetylating aminoglycoside acetyltransferase gene is described here for the first time in P. aeruginosa. Multilocus sequence typing (MLST) revealed that the PER-1 positive P. aeruginosa isolates identified in this study display ST235, a sequence type that belongs to clonal complex CC11. Two bla PER-1-positive P. aeruginosa reference isolates from France and Belgium could also be assigned to complex CC11 by MLST. Our results underscore the role of complex CC11 in the dissemination of bla PER-1 among P. aeruginosa clinical isolates.
To evaluate the performance of the MB/BacT system (Organon Teknika) in comparison to Lowenstein-J... more To evaluate the performance of the MB/BacT system (Organon Teknika) in comparison to Lowenstein-Jensen (LJ) solid medium for recovery of mycobacteria from clinical specimens. Two thousand three hundred and ten specimens (1626 respiratory, 593 urine, 60 body fluids, five tissue and 26 others) were inoculated in MB/BacT (0.5 mL) and on two LJ slants (0.25 mL each). N-acetyl-l-cysteine-NaOH (final concentration 2%) was used for decontamination. Two hundred and fifty-one (10.8%) mycobacterial isolates [190 Mycobacterium tuberculosis complex (MTBC) and 61 non-tuberculous mycobacteria (NTM)] were detected. Of these 251 isolates, 234 (93.2%; 181 MTBC and 53 NTM) were detected in MB/BacT and 169 (67.3%; 154 MTBC and 15 NTM) on LJ. The mean (median) times to detection of MTBC by MB/BacT and LJ were 13.8 (13) and 22.1 (20) days, respectively, while overall contamination rates were 7.7% and 8.1%, respectively. Sensitivity and time to detection were significantly better with MB/BacT than with solid LJ medium.
Acta Microbiologica et Immunologica Hungarica, 2008
From the Central-East European region the first VIM metallo-beta-lactamase (MBL) producing Pseudo... more From the Central-East European region the first VIM metallo-beta-lactamase (MBL) producing Pseudomonas aeruginosa strains were published from Croatia, Poland and Hungary. The aim of this study was to assess the contribution of MBL-production to carbapenem-resistance among P. aeruginosa clinical isolates in the Military Medical Academy (MMA) in Belgrade, Serbia between August 2004 and September 2007. Only one P. aeruginosa isolate with strain number 722 proved MBL-positive that harboured a novel class 1 integron with a bla(VIM-2)-like cassette in the first position, followed by orfD, a putative gene with unknown function. Our data indicate that MBL-producing strains occur at a prevalence of less than 1% among imipenem-nonsusceptible P. aeruginosa clinical isolates in this Belgrade hospital. The newly identified VIM MBL-producing P. aeruginosa strain 722 could be assigned to serotype O11, and it was panresistant to all antimicrobials tested. The isolate displayed sequence type ST235 by multilocus sequence typing which is the founder sequence type of the previously identified international clonal complex CC11 that already contains bla(VIM)-positive isolates from Italy, Greece, Sweden, Hungary and Poland. In conclusion, this is the first report of VIM MBL-producing P. aeruginosa from Serbia and also of the occurrence of such isolates belonging to the international clonal complex CC11 in this country.
Pseudomonas chlororaphis is a bacterium used as a soil inoculant in agriculture and horticulture.... more Pseudomonas chlororaphis is a bacterium used as a soil inoculant in agriculture and horticulture. It can act as a biocontrol agent against certain phytopathogenic fungi via production of phenazine type antibiotics. Phytopathogenic fungi are responsible for several plant diseases in different medicinal plants and cause very important economic losses in Serbian plantation. Among them, Alternaria sp., Phoma sp. and Drechslera tetramera are known as major plant pathogens, whereby at least 20% of agricultural spoilage is caused by Alternaria species. In this study, we examined the antifungal activity of indigenous Pseudomonas chlororaphis isolates against the phytopathogenic fungi Alternaria sp., Phoma sp. and Drechslera tetramera, which had infected anise (Pimpinella anisum L., fam. Apiaceae), using in vitro inhibition tests. The obtained results showed that studied antibioticproducing Ps. chlororaphis isolates inhibited the tested fungi as follows: D. tetramera in the range from 3.85-6...
The genetic context of the bla NDM-1 gene in the genome of Pseudomonas aeruginosa MMA83 was inves... more The genetic context of the bla NDM-1 gene in the genome of Pseudomonas aeruginosa MMA83 was investigated. Sequencing of the cosmid selected for the bla NDM-1 gene revealed the presence of two bla NDM-1 copies in the genome of P. aeruginosa MMA83 in a unique genetic environment. Additionally, mating assays, DNA-DNA hybridization, and an S1 nuclease assay strongly suggest that the bla NDM-1 gene in P. aeruginosa MMA83 is chromosome borne.
The aim of this study was to detect genes for enterotoxins (hbla, entFM and bceT) and for emetic ... more The aim of this study was to detect genes for enterotoxins (hbla, entFM and bceT) and for emetic toxin (cer), to determine antibiotic resistance, and to estimate intraspecies diversity in B. cereus isolates by RAPD analysis. B. cereus was identified in 12 out of 117 indigenous Bacillus spp. using the classical microbiological methods and PCR. All isolates were resistant to penicillin and ampicillin, two to tetracyclin and four to trimethoprim-sulphamethoxazole. Also, all isolates produced inducible penicillinases and β-lactamase. Toxin genes were detected with PCR. EntFM and cer genes were present in all isolates, hbla in all, but two, and bceT in none. RAPD analysis was performed with four different primers, two of them designed for this study. The intraspecies diversity revealed 10 different patterns at the 90% similarity level. Two separate clusters were formed regardless of a soil type or utilization. The detection of genes encoding toxins in all B. cereus isolates indicated these bacteria as potentially pathogenic and seriously for human health. Regardless of a soil type or utilization, the RAPD analysis showed high intraspecies heterogeneity in B. cereus isolates. To the best of our knowledge, this is the first study to analyse the presence of entero-and emetic toxin genes and genetic heterogeneity in B. cereus isolates from different soil types and different soil utilization in Serbia.
To select isolates suitable for use in biological control of pathogenic fungi and as plant growth... more To select isolates suitable for use in biological control of pathogenic fungi and as plant growth promoting (PGP) agents, Pseudomonas spp., indigenous to the rhizosphere of wild alfalfa and clover plants growing on the old magmatic hill Vagan (Serbia), were isolated. Isolates were selected on the basis of intrinsic antibiotic resistance (IAR) and PGP traits: phosphate solubilization, production of siderophores, hydrogen cyanide (HCN), acyl homoserine lactones (AHLs), indole acetic acid (IAA) and enzymatic activities. The results of the genotyping by repetitive sequence-based polymerase chain reaction, using ERIC and (GTG) 5 primers, and phenotyping by IAR clustered the isolates in five groups, represented by two isolates from alfalfa (Q1 and Q16) and three from white clover (B25, B28 and B29). Isolates Q16 and B25 showed production of HCN, IAA and AHLs. Good phosphate solubilization and production of siderophores were observed on Q1 and Q16. The Q16 isolate exhibited high enzymatic activity. The most promising PGP isolates, Q16 and B25, showed the best antifungal activity against Trichoderma viride, and good antifungal effect against Aspergillus fumigatus and Aspergillus niger, while Penicillium verrucosum was the most resistant fungus. Pseudomonas sp. B25 exhibited higher antifungal potential than Q16. The selected isolates could be further investigated as biological control agents and tested in field conditions to confirm their PGP efficacy.
International Journal of Antimicrobial Agents, 2007
S162 17th ECCMID / 25th ICC, Posters production was detected by double-disc synergy tests and ESB... more S162 17th ECCMID / 25th ICC, Posters production was detected by double-disc synergy tests and ESBL Etests. MICs were determined and interpreted according to CLSI criteria. Isolates were screened for blaCTX-M genes using multiplex PCR and were compared by PFGE of XbaI-digested genomic DNA. Data were analysed using BioNumerics software. Results: A total of 177 ESBL-producing K. pneumoniae isolates were collected. Of these, 60 (33.9%), from 8 hospitals, were positive for blaCTX-M, all with group-1 enzymes. Clonal relationships among these 60 isolates were studied in comparison with 27 CTX-M-negative strains from the same hospitals. The isolates represented multiple strains, but several clusters of related isolates (>80% similarity) were observed, some of them including isolates from more than one centre. The largest cluster consisted of 26 clonally-related isolates with group-1 CTX-M enzymes, 24 of them from one hospital. This outbreak needs further investigation. Two isolates of this outbreak strain were identified in further hospitals, suggesting inter-site transmission. Most isolates had substantial resistance to both cefotaxime (>128 mg/L) and ceftazidime (>16 mg/L), possibly indicating CTX-M-15. Conclusion: K. pneumoniae with CTX-M enzymes are an emerging problem in Slovenian hospitals, but currently represent a minority of ESBL producers of this species. This contrasts with the dominance of CTX-M enzymes among ESBL-producing E. coli in most European countries. The epidemiology of K. pneumoniae with CTX-M enzymes was complex, with the spread of several strains within and between hospitals. Since K. pneumoniae is an important hospital pathogen this worrying development merits closer monitoring.
PER-1 extended-spectrum b-lactamase-producing Pseudomonas aeruginosa clinical isolates from Budap... more PER-1 extended-spectrum b-lactamase-producing Pseudomonas aeruginosa clinical isolates from Budapest, Hungary, and Belgrade, Serbia, were characterized by molecular methods. Two PER-1-positive isolates were recovered from sporadic cases in Budapest and a small cluster of PER-1-positive infections involving four patients were identified at a Belgrade hospital. A class 1 integron harbouring a bla OXA-2 b-lactamase gene and four other gene cassettes was detected in both the Budapest and the Belgrade isolates. The two P. aeruginosa isolates from Budapest also carried another class 1 integron containing bla OXA-74 , aac(6 0)-Ib-cr and cmlA7 genes in its variable region. The aac(6 0)-Ib-cr fluoroquinolone-acetylating aminoglycoside acetyltransferase gene is described here for the first time in P. aeruginosa. Multilocus sequence typing (MLST) revealed that the PER-1 positive P. aeruginosa isolates identified in this study display ST235, a sequence type that belongs to clonal complex CC11. Two bla PER-1-positive P. aeruginosa reference isolates from France and Belgium could also be assigned to complex CC11 by MLST. Our results underscore the role of complex CC11 in the dissemination of bla PER-1 among P. aeruginosa clinical isolates.
To evaluate the performance of the MB/BacT system (Organon Teknika) in comparison to Lowenstein-J... more To evaluate the performance of the MB/BacT system (Organon Teknika) in comparison to Lowenstein-Jensen (LJ) solid medium for recovery of mycobacteria from clinical specimens. Two thousand three hundred and ten specimens (1626 respiratory, 593 urine, 60 body fluids, five tissue and 26 others) were inoculated in MB/BacT (0.5 mL) and on two LJ slants (0.25 mL each). N-acetyl-l-cysteine-NaOH (final concentration 2%) was used for decontamination. Two hundred and fifty-one (10.8%) mycobacterial isolates [190 Mycobacterium tuberculosis complex (MTBC) and 61 non-tuberculous mycobacteria (NTM)] were detected. Of these 251 isolates, 234 (93.2%; 181 MTBC and 53 NTM) were detected in MB/BacT and 169 (67.3%; 154 MTBC and 15 NTM) on LJ. The mean (median) times to detection of MTBC by MB/BacT and LJ were 13.8 (13) and 22.1 (20) days, respectively, while overall contamination rates were 7.7% and 8.1%, respectively. Sensitivity and time to detection were significantly better with MB/BacT than with solid LJ medium.
Acta Microbiologica et Immunologica Hungarica, 2008
From the Central-East European region the first VIM metallo-beta-lactamase (MBL) producing Pseudo... more From the Central-East European region the first VIM metallo-beta-lactamase (MBL) producing Pseudomonas aeruginosa strains were published from Croatia, Poland and Hungary. The aim of this study was to assess the contribution of MBL-production to carbapenem-resistance among P. aeruginosa clinical isolates in the Military Medical Academy (MMA) in Belgrade, Serbia between August 2004 and September 2007. Only one P. aeruginosa isolate with strain number 722 proved MBL-positive that harboured a novel class 1 integron with a bla(VIM-2)-like cassette in the first position, followed by orfD, a putative gene with unknown function. Our data indicate that MBL-producing strains occur at a prevalence of less than 1% among imipenem-nonsusceptible P. aeruginosa clinical isolates in this Belgrade hospital. The newly identified VIM MBL-producing P. aeruginosa strain 722 could be assigned to serotype O11, and it was panresistant to all antimicrobials tested. The isolate displayed sequence type ST235 by multilocus sequence typing which is the founder sequence type of the previously identified international clonal complex CC11 that already contains bla(VIM)-positive isolates from Italy, Greece, Sweden, Hungary and Poland. In conclusion, this is the first report of VIM MBL-producing P. aeruginosa from Serbia and also of the occurrence of such isolates belonging to the international clonal complex CC11 in this country.
Uploads
Papers by Z. Lepsanovic