Molekularna analiza gena nim i inducibilne rezistencije na metronidazol u kliničkih izolata grupe Bacteroides fragilis [Molecular analysis of nim genes and inducible metronidazole resistance in Bacteroides fragilis clinical isolates]

The study is essentially based on detection of nim genes in B. fragilis group strains, in vitro induction of resistance to metronidazole in strains which are phenotypically sensitive to metronidazole and measurement of LDH activity in strains with induced resistance to metronidazole. All strains were nim-gene negative, as well as sensitive to metronidazole. In-vitro induction of metronidazole resistance is selected in nim-negative strains, after repeated exposure to subinhibitory concentrations of metronidazole incorporated into growth medium. The MIC values for metronidazole of the induced strains ranged from 8 to 96 mg/L. Only one B. fragilis strain with induced resistance to metronidazole demonstrated an emergent increase in LDH activity. We believe that genetic mutations were responsible for the increased activity. A significant decrease in LDH activity of the most other strains was contrary to previous findings in which, underlying higher metronidazole MICs, an increase in LDH activity compensated for the decreased activity of PFOR complex. These findings could be explained if the induction caused only physiologic and not genetic changes. These results provide the first insights into the mechanisms of metronidazole resistance during the exposure of nim-negative strains to antibiotic, that have been observed, but not investigated in detail, by other authors

Detection of virulence gene belonging to cag pathogenicity island in Helicobacter pylori isolates after multiple unsuccessful eradication therapy in Northwest Croatia

Background: Some of the genes belonging to cag pathogenicity island (cagPAI) in Helicobacter pylori were found to be associated with an increased severity of gastric mucosal inflammation that might lead to the development of gastroduodenal disease. Aim: The aim of our study was to define a group of patients based on the frequency of virulence genes of cagPAI island and comparison with pathohistological alterations of gastric mucosa who need to be subjected to further eradication therapy after previous unsuccessful eradication therapy and in spite of benign endoscopic findings. Material and methods: In total 103 H. pylori isolates were analysed. Genes encoding virulence factors were detected by PCR with primers for 10 loci in cagPAI: Apcag (cagA promotor region), cagA1, cagA2, cagA3, cagM, cagT, cagE, LEC, tnpA and tnpB. The patients who provided isolates were classified into three clinical categories: non-ulcer dyspepsia (n=69), erosio/ulcus ventriculi (n=22) and erosio/ulcus duodeni (n=12). Results: 16 strains (15.5%) were negative for all tested genes. 87 (84.5%) of the isolates had parcially deleated cagPAI. None of the isolates possessed all 10 genes. The frequency of single cagPAI genes were as follows: Apcag 63.1%, cagA1 71.8%, cagA2 69.9%, cagA3 5.8%, cagM 71.8%, cagE 75,7%, cagT 68%, tnpA 9.7%, tnpB 7.8% i LEC 48.5%. No statistically significant difference was observed between the presence of any cagPAI genes and endosopic diagnosis (p>0.16). The presence of CagA2, Apcag and cagM showed statistically significant correlation with higher level of patohistological parameters of gastritis (p<0.05). Conclusions: H. pylori isolates with positive cagA, Apcag and cagM genes are correlated to higher degree of patohistological lesions of gastric mucosa; without statistically significant correlation with endoscopic diagnosis

Usporedba dviju različitih metoda za testiranje osjetljivosti na tigeciklin u Acinetobacter baumannii

Tigecycline susceptibility testing (TST) presents a tremendous challenge for clinical microbiologists. Previous studies have shown that the Epsilometer test (E-test) and Vitek 2 automated system significantly overestimate the minimum inhibitory concentrations for tigecycline resistance compared to the broth microdilution method (BMM). This leads to very major errors or false susceptibility (i.e. the isolate is called susceptible when it is actually resistant). The aim of this study was to compare E-test against BMM for TST in carbapenem-resistant and carbapenem-susceptible Acinetobacter (A.) baumannii and to analyze changes in tigecycline susceptibility between two time periods (2009-2012 and 2013-2014), with BMM as the gold standard. Using the EUCAST criteria, the rate of resistance to tigecycline for the OXA-23 MBL-positive, OXA-23 MBL-negative and carbapenemase-negative strains for BMM was 54.5% (6/11), 29.4% (5/17) and 2.7% (1/37), respectively; the OXA-24/40 and OXA-58 producing organisms did not exhibit any resistance. With E-test, all OXA-23 MBL-positive organisms (11/11), 23.5% (4/17) of OXA-23 MBL-negative, and 4.1% of OXA-24/40 (3/74) strains displayed tigecycline resistance; there were no resistant strains among the OXA-58 and carbapenemase-negative isolates. Resistance emerged in the bacterial isolates from 2013 to 2014. Although tigecycline does not display cross-resistance, the highest rates of resistant A. baumannii isolates were observed among those producing VIM MBL, regardless of the testing method. These findings suggest that the commercial E-test does not provide reliable results for TST of A. baumannii. Further confirmation with the dilution method should be recommended, particularly in cases of serious infections.Testiranje osjetljivosti na tigeciklin (TST) je velik izazov za kliničke mikrobiologe. Prethodna istraživanja su pokazala da E-test i Vitek 2 daju veće vrijednosti minimalne inhibitorne koncentracije tigeciklina u odnosu na dilucijsku metodu, što uzrokuje vrlo veliku grešku (engl. very major error, što znači da je rezistentan izolat proglašen osjetljivim). Cilj istraživanja bio je usporediti dvije metode za testiranje osjetljivosti na tigeciklin (E-test i bujonska dilucijska metoda) u karbapenem osjetljvim i karbapenem rezistentnim izolatima Acinetobacter (A.) baumannii s različitim tipovima karbapenem-hidrolizirajućih oksacilinazama i analizirati promjenu u stopama osjetljivosti na tigeciklin u dva razdoblja istraživanja (2009.-2012. i 2013.-2014.). Dilucija u bujonu je bila referentna metoda. Testiranje osjetljivosti na tigeciklin je provedeno E-testom i bujonskom mikrodilucijskom metodom. Prema kriterijima EUCAST-a stopa rezistencije bila je 54,5% (6/11) za OXA-23 MBL-pozitivne sojeve, 29,4% (5/17) za OXA-23 MBL-negativne sojeve i 2,7% (1/37) za karbapenemaza-negativne sojeve uz bujonsku mikrodilucijsku metodu. OXA-24/40 i OXA-58 producirajući sojevi nisu iskazivali rezistenciju. E-testom su svi OXA-23 MBL pozitivni organizmi (11/11), 23,5% (4/17) OXA-23 MBL negativnih i 4,1% OXA-24/40 (3/74) pokazivali rezistenciju na tigeciklin. Svi OXA-58 pozitivni i karbapenemaza-negativni sojevi su bili osjetljivi na tigeciklin u E-testu. Rezistencija na tigeciklin se pojavila u razdoblju od 2013. do 2014. godine. Iako tigeciklin ne pokazuje križnu rezistenciju s drugim antibioticima najviše stope rezistencije su zapažene među VIM-pozitivnim izolatima bez obzira na metodu testiranja. Prema rezultatima našega istraživanja komercijalni E-test ne daje pouzdane rezultate TST u A. baumannii. Potrebna je potvrda dilucijskom metodom, osobito kod teških infekcija

Molecular analysis of nim genes and inducible metronidazole resistance in Bacteroides fragilis clinical isolates

Ovaj rad se temelji na probiru nim gena rezistencije unutar Bacteroides fragilis grupe, in vitro indukciji rezistencije na metronidazol u sojevima koji su fenotipski osjetljivi na metronidazol i mjerenju aktivnosti laktat dehidrogenaze (LDH) u sojevima s induciranom rezistencijom na metronidazol. Nim geni rezistencije i njihov produkt 5-nitroimidazol reduktaza najviše su istraživani mehanizam rezistencije unutar B. fragilis grupe. Djelovanjem 5-nitroimidazol reduktaze metronidazol se pretvara u aminoderivat, koji je netoksičan za DNK bakterijske stanice. Osim što su svi ispitivani sojevi osjetljivi na metronidazol, niti u jednom nije detektiran nim (A-E) gen rezistencije. Iz tog razloga nije moguća tipizacija istih RFLP metodom, niti potvrda hipoteze o nimA genu, kao najčešćem tipu nim gena. In vitro indukcija rezistencije na metronidazol postignuta je u sojevima bez nim gena, nakon višekratnog izlaganja sojeva subinhibitornim koncentracijama metronidazola u hranjivoj podlozi. Vrijednost minimalne inhibitorne koncentracije (MIK) metronidazola u induciranim sojevima bila je u rasponu 8 - 96 mg/L. Mogućnost indukcije rezistencije u sojevima bez nim gena, uz najvišu vrijednosti MIK-a metronidazola 96 mg/L, ne potvrđuje ni hipotezu o indukciji rezistencije u sojevima s nim genom i vrijednosti MIK-a metronidazola ≥256 mg/L. U jednom jedinom soju s induciranom rezistencijom na metronidazol izmjeren je značajan porast vrijednosti LDH. U većine ostalih induciranih sojeva izmjereni pad vrijednosti LDH je u suprotnosti s dosadašnjim nalazima gdje se uz povišeni MIK metronidazola bilježi porast aktivnosti LDH kao kompenzacija smanjene aktivnosti PFOR kompleksa. Pretpostavlja se da je u dotičnom soju sa značajno povišenom vrijednosti LDH riječ o genetskoj mutaciji proteina koji su uključeni u proces redukcije metronidazola, a da u svim ostalim sojevima indukcija rezistencije uzrokuje samo promjene u regulaciji fizioloških procesa, vezanim za redukciju metronidazola. Rezultati ovog rada donose tek početne spoznaje u mehanizme rezistencije u procesu indukcije rezistencije na metronidazol u B. fragilis grupi bez nim gena i zahtijevaju daljnje napore u proučavanju istih nakon in vitro izlaganja sojeva antibiotiku.The study is essentially based on detection of nim genes in B. fragilis group strains, in vitro induction of resistance to metronidazole in strains which are phenotypically sensitive to metronidazole and measurement of LDH activity in strains with induced resistance to metronidazole. All strains were nim-gene negative, as well as sensitive to metronidazole. In-vitro induction of metronidazole resistance is selected in nim-negative strains, after repeated exposure to subinhibitory concentrations of metronidazole incorporated into growth medium. The MIC values for metronidazole of the induced strains ranged from 8 to 96 mg/L. Only one B. fragilis strain with induced resistance to metronidazole demonstrated an emergent increase in LDH activity. We believe that genetic mutations were responsible for the increased activity. A significant decrease in LDH activity of the most other strains was contrary to previous findings in which, underlying higher metronidazole MICs, an increase in LDH activity compensated for the decreased activity of PFOR complex. These findings could be explained if the induction caused only physiologic and not genetic changes. These results provide the first insights into the mechanisms of metronidazole resistance during the exposure of nim-negative strains to antibiotic, that have been observed, but not investigated in detail, by other authors

Molecular analysis of nim genes and inducible metronidazole resistance in Bacteroides fragilis clinical isolates

Ovaj rad se temelji na probiru nim gena rezistencije unutar Bacteroides fragilis grupe, in vitro indukciji rezistencije na metronidazol u sojevima koji su fenotipski osjetljivi na metronidazol i mjerenju aktivnosti laktat dehidrogenaze (LDH) u sojevima s induciranom rezistencijom na metronidazol. Nim geni rezistencije i njihov produkt 5-nitroimidazol reduktaza najviše su istraživani mehanizam rezistencije unutar B. fragilis grupe. Djelovanjem 5-nitroimidazol reduktaze metronidazol se pretvara u aminoderivat, koji je netoksičan za DNK bakterijske stanice. Osim što su svi ispitivani sojevi osjetljivi na metronidazol, niti u jednom nije detektiran nim (A-E) gen rezistencije. Iz tog razloga nije moguća tipizacija istih RFLP metodom, niti potvrda hipoteze o nimA genu, kao najčešćem tipu nim gena. In vitro indukcija rezistencije na metronidazol postignuta je u sojevima bez nim gena, nakon višekratnog izlaganja sojeva subinhibitornim koncentracijama metronidazola u hranjivoj podlozi. Vrijednost minimalne inhibitorne koncentracije (MIK) metronidazola u induciranim sojevima bila je u rasponu 8 - 96 mg/L. Mogućnost indukcije rezistencije u sojevima bez nim gena, uz najvišu vrijednosti MIK-a metronidazola 96 mg/L, ne potvrđuje ni hipotezu o indukciji rezistencije u sojevima s nim genom i vrijednosti MIK-a metronidazola ≥256 mg/L. U jednom jedinom soju s induciranom rezistencijom na metronidazol izmjeren je značajan porast vrijednosti LDH. U većine ostalih induciranih sojeva izmjereni pad vrijednosti LDH je u suprotnosti s dosadašnjim nalazima gdje se uz povišeni MIK metronidazola bilježi porast aktivnosti LDH kao kompenzacija smanjene aktivnosti PFOR kompleksa. Pretpostavlja se da je u dotičnom soju sa značajno povišenom vrijednosti LDH riječ o genetskoj mutaciji proteina koji su uključeni u proces redukcije metronidazola, a da u svim ostalim sojevima indukcija rezistencije uzrokuje samo promjene u regulaciji fizioloških procesa, vezanim za redukciju metronidazola. Rezultati ovog rada donose tek početne spoznaje u mehanizme rezistencije u procesu indukcije rezistencije na metronidazol u B. fragilis grupi bez nim gena i zahtijevaju daljnje napore u proučavanju istih nakon in vitro izlaganja sojeva antibiotiku.The study is essentially based on detection of nim genes in B. fragilis group strains, in vitro induction of resistance to metronidazole in strains which are phenotypically sensitive to metronidazole and measurement of LDH activity in strains with induced resistance to metronidazole. All strains were nim-gene negative, as well as sensitive to metronidazole. In-vitro induction of metronidazole resistance is selected in nim-negative strains, after repeated exposure to subinhibitory concentrations of metronidazole incorporated into growth medium. The MIC values for metronidazole of the induced strains ranged from 8 to 96 mg/L. Only one B. fragilis strain with induced resistance to metronidazole demonstrated an emergent increase in LDH activity. We believe that genetic mutations were responsible for the increased activity. A significant decrease in LDH activity of the most other strains was contrary to previous findings in which, underlying higher metronidazole MICs, an increase in LDH activity compensated for the decreased activity of PFOR complex. These findings could be explained if the induction caused only physiologic and not genetic changes. These results provide the first insights into the mechanisms of metronidazole resistance during the exposure of nim-negative strains to antibiotic, that have been observed, but not investigated in detail, by other authors

Molecular analysis of nim genes and inducible metronidazole resistance in Bacteroides fragilis clinical isolates

Ovaj rad se temelji na probiru nim gena rezistencije unutar Bacteroides fragilis grupe, in vitro indukciji rezistencije na metronidazol u sojevima koji su fenotipski osjetljivi na metronidazol i mjerenju aktivnosti laktat dehidrogenaze (LDH) u sojevima s induciranom rezistencijom na metronidazol. Nim geni rezistencije i njihov produkt 5-nitroimidazol reduktaza najviše su istraživani mehanizam rezistencije unutar B. fragilis grupe. Djelovanjem 5-nitroimidazol reduktaze metronidazol se pretvara u aminoderivat, koji je netoksičan za DNK bakterijske stanice. Osim što su svi ispitivani sojevi osjetljivi na metronidazol, niti u jednom nije detektiran nim (A-E) gen rezistencije. Iz tog razloga nije moguća tipizacija istih RFLP metodom, niti potvrda hipoteze o nimA genu, kao najčešćem tipu nim gena. In vitro indukcija rezistencije na metronidazol postignuta je u sojevima bez nim gena, nakon višekratnog izlaganja sojeva subinhibitornim koncentracijama metronidazola u hranjivoj podlozi. Vrijednost minimalne inhibitorne koncentracije (MIK) metronidazola u induciranim sojevima bila je u rasponu 8 - 96 mg/L. Mogućnost indukcije rezistencije u sojevima bez nim gena, uz najvišu vrijednosti MIK-a metronidazola 96 mg/L, ne potvrđuje ni hipotezu o indukciji rezistencije u sojevima s nim genom i vrijednosti MIK-a metronidazola ≥256 mg/L. U jednom jedinom soju s induciranom rezistencijom na metronidazol izmjeren je značajan porast vrijednosti LDH. U većine ostalih induciranih sojeva izmjereni pad vrijednosti LDH je u suprotnosti s dosadašnjim nalazima gdje se uz povišeni MIK metronidazola bilježi porast aktivnosti LDH kao kompenzacija smanjene aktivnosti PFOR kompleksa. Pretpostavlja se da je u dotičnom soju sa značajno povišenom vrijednosti LDH riječ o genetskoj mutaciji proteina koji su uključeni u proces redukcije metronidazola, a da u svim ostalim sojevima indukcija rezistencije uzrokuje samo promjene u regulaciji fizioloških procesa, vezanim za redukciju metronidazola. Rezultati ovog rada donose tek početne spoznaje u mehanizme rezistencije u procesu indukcije rezistencije na metronidazol u B. fragilis grupi bez nim gena i zahtijevaju daljnje napore u proučavanju istih nakon in vitro izlaganja sojeva antibiotiku.The study is essentially based on detection of nim genes in B. fragilis group strains, in vitro induction of resistance to metronidazole in strains which are phenotypically sensitive to metronidazole and measurement of LDH activity in strains with induced resistance to metronidazole. All strains were nim-gene negative, as well as sensitive to metronidazole. In-vitro induction of metronidazole resistance is selected in nim-negative strains, after repeated exposure to subinhibitory concentrations of metronidazole incorporated into growth medium. The MIC values for metronidazole of the induced strains ranged from 8 to 96 mg/L. Only one B. fragilis strain with induced resistance to metronidazole demonstrated an emergent increase in LDH activity. We believe that genetic mutations were responsible for the increased activity. A significant decrease in LDH activity of the most other strains was contrary to previous findings in which, underlying higher metronidazole MICs, an increase in LDH activity compensated for the decreased activity of PFOR complex. These findings could be explained if the induction caused only physiologic and not genetic changes. These results provide the first insights into the mechanisms of metronidazole resistance during the exposure of nim-negative strains to antibiotic, that have been observed, but not investigated in detail, by other authors

Lactate dehydrogenase activity in Bacteroides fragilis group strains with induced resistance to metronidazole

Abstract The aims of this study were to induce in vitro metronidazole resistance in nim-negative Bacteroides fragilis group strains and to determine the lactate dehydrogenase (LDH) activity of the induced strains. A collection of B. fragilis group strains were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF/MS). Minimum inhibitory concentrations (MICs) for metronidazole were determined by the agar dilution technique. The presence of nim genes was screened by PCR. A sample of 52 nim-negative metronidazole-susceptible strains were selected at random and were exposed to metronidazole in the resistance induction experiment. LDH activity was measured by spectrophotometry. Of the 52 selected strains, 12 (23.1%) acquired resistance to metronidazole. MICs ranged from 8 mg/L to 96 mg/L. Eight of the twelve induced strains displayed decreased LDH activity, whilst only one expressed a significant increase in LDH activity with LDH values of 49.1 U/mg and 222.0 U/mg, respectively. In conclusion, in vitro induction of metronidazole resistance could be selected in nim-negative B. fragilis group strains. A statistically significant decrease in LDH activity was in contrast to previous findings in which, underlying higher metronidazole MICs, an increase in LDH activity compensated for the decreased activity of pyruvate-ferredoxin oxidoreductase (PFOR). These findings could be explained if the induction caused only physiological and not genetic changes. We believe that genetic mutations in the B. fragilis strain that demonstrated an emergent increase in LDH activity were responsible for the increased activity

Hidden Carbapenem Resistance in OXA-48 and Extended-Spectrum β-Lactamase-Positive

The purpose of this study was to report the identification OXA-48 carbapenemase in seven extended-spectrum β-lactamase (ESBL)-positive Escherichia coli clinical isolates, fully susceptible to all carbapenems by disk diffusion and E-test methods, but with borderline minimal inhibitory concentration (MIC) values of ertapenem. This report points to the necessity for determination of carbapenem MICs in ESBL-positive E. coli isolates and additional phenotypic testing for carbapenemases in all isolates with borderline ertapenem MIC defined by EUCAST. The isolates showed a high level of resistance to expanded-spectrum cephalosporins because of the production of an additional ESBL belonging to CTX-M family. All isolates and their respective tranconjugants were found to possess L plasmid. Pulsed-field gel electrophoresis analysis revealed two clusters containing highly related isolates. The global spread of multidrug-resistant E. coli should be monitored closely because of the ability of isolates to rapidly obtain additional antibiotic resistance traits such as plasmid-mediated OXA-48 genes