Detekcija gena virulencije otoka patogenosti cag i gena dupA izolata Helicobacter pylori nakon višestruke neuspjele eradikacijske terapije

BACKGROUND Certain virulence genes including cagA located in the pathogenicity island cagPAI (eng. citotoxin pathogenicity island) of Helicobacter pylori are associated with gastroduodenal diseases and thus the variation of cagPAI might influence various clinical syndroms. According to some studies dupA gene located in plastic region (eng. plasticity region) of H. pylori is associated with development of duodenal ulcer and has protective role against development of atrophy and gastric metaplasia. AIM The aim of this study was to determine the frquency of virulence genes within cagPAI island (Apcag, cagA1, cagA2, cagA3, cagM, cagT, cagE, LEC, tnpA i tnpB) and dupA gene in Helicobacter pylori isolates obtained from patients after multiple unsuccessful antimicrobial therapy, and to analyze the correlation between the presence of these genes and endoscopic diagnosis and pathohistological alterations of gastric mucosa. MATERIALS/METHODS Virulence genes of 103 H. pylori isolates were detected by PCR. According to endoscopid diagnosis the patients were classified into three groups: non-ulcer dyspepsia (NUD) (n=69), erosio/ulcus ventriculi (EUV) (n=22) and erosio/ulcus duodeni (EUD) (n=12). Pathohistological findins were interpreted according to Sydney classification sheme. RESULTS Single prevalence of cagPAI gene was 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 6.3% i LEC 48.5%. The frequency of dupA was 34. There was no correlation between 10 analysed genes of cagPAI island with endoscopic diagnosis (P>0.16). The presence of CagA, Apcag i cagM gene was associated with higher grade of pathohistological parameters of gastritisa (p<0.05). DupA gene was not found in patients with duodenal ulcer disease from Nortwest Croatia. DupA gene was significantly more frequent in patients with non-ulcer dyspepsia and gastric ulcer disease (P=0.016). There was no statistically significant difference in gastritis intensity score either in antrumu (P=0.434), or in corpus (P=0.084) in relation to the presence of dupA gene. More then 50% of the isolates were resistant to both macrolides and metronidazol. CONCLUSION Our study demonstrated high frequency of cagA, Apcag i cagM genes in patient’s isolates and their correlation with high grade of pathohistological alterations in gastric mucosa, representing a risk factor for development of ulcer, premalignant and malignant diseases. There was no correlation between the presence of dupA gene and duodenal ulcer. Moreover, no significant difference in the frequency of dupA gene according to the type of gastritis was found. Repeated attempt to eradicate H. pylori seems to be the best choice for the patient. Concerning the unsatisfactory efficiency of triple therapy based on clarithromycine due to increasing resistance, new therapeutic options based on the new treatment strategies should be implemented in order to increase the level of eradication (new antibiotics or combination of antibiotics)

Mobile phones – potential infectors in the hospital

Cilj rada je bio ustanoviti mikrobiološku čistoću mobilnih telefona zdravstvenih djelatnika Opće bolnice Zabok. U jednom danu kao slučajan uzorak izabrali smo 40 zdravstvenih djelatnika Opće bolnice Zabok koji su u tom trenutku imali mobilni telefon uz sebe. Bakterijska kontaminacija dokazana je u 50% ispitanih mobilnih telefona. Iz 16 (40%) obrisaka mobilnih telefona izolirani su koagulaza negativni stafilokoki (KNS), iz 2 (5%) obriska izoliran je Corynebacterium sp., a iz po jednog obriska izoliran je meticilin senzitivni Staphylococcus aureus (2,5%) i Acinetobacter baumannii (2,5%). Rezultati su pokazali da su mobilni telefoni bili kontaminirani različitim bakterijama i treba naglasiti i zaključiti da sama higijena ruku zdravstvenih djelatnika, a uz upotrebu mobilnih telefona, nije dovoljna, ako se povremeno ne dezinficira i vanjska površina mobilnog telefona.  Abstract The aim of the project was to ascertain the microbiological level of cleanliness on the mobile phones belonging to the healthcare staff of the Zabok General hospital. During the course of one day, 40 health care staff of Zabok General hospital who possessed a mobile phone on their person were randomly picked. The tests for bacterial contamination were positive on fifty percent of the tested health care staffs mobile phones. From 16 (40%) swabs we isolated coagulase­negative staphylococci(CNS), Corynebacterium sp. was isolated from 2 (5%) swabs. We also isolated a meticillin­sensitive Staphylococcus aureus from one swab (2,5%) and an Acinetobacter baumannii from another one (2,5%). These results showed that mobile phones are contaminated with various bacteria and it is important to stress that mere hand washing is not enough for the hospital staff who use mobile phones, if they do not disinfect the surfaces of their phones from time to time

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

EVOLUTION OF BETA-LACTAM ANTIBIOTIC RESISTANCE IN ENTEROBACTER SPP. IN CROATIA

Rezistencija na cefalosporine proširenog spektra u Enterobacter spp. nastaje zbog indukcije ili derepresije AmpC-β-laktamaze, produkcije β-laktamaza proširenog spektra (ESBL) ili karbapenemaza. Cilj istraživanja bio je utvrditi mehanizme rezistencije na β-laktamske antibiotike na kolekciji od 58 izolata prikupljenih metodom slučajnog odabira u tri klinička centra u Hrvatskoj i Kantonalnom zavodu za javno zdravstvo Zenica u Bosni i Hercegovini u razdoblju od 2008. do 2011. godine, kao i utvrditi evoluciju rezistencije na tu skupinu antibiotika tijekom perioda istraživanja. Pretpostavka je istraživanja da će izolati Enterobacter spp. rezistentni na ceftazidim pokazivati različite mehanizme rezistencije od inducibilne i dereprimirane AmpC-β-laktamaze do β-laktamaza proširenog spektra, a u kasnijim godinama i karbapenemaza. Očekivana je i razlika u fenotipu i mehanizmima rezistencije između različitih centara. Osjetljivost na antibiotike testirana je metodom dilucije u bujonu, a geni rezistencije testirani su PCR-om. Plazmidi su karakterizirani tipizacijom replikona PCR-om. Istraživanje je pokazalo dominaciju β-laktamaza proširenog spektra iz porodice CTX-M u kombinaciji s derepresijom AmpC-β-laktamaze kao dominantan mehanizam rezistencije na cefalosporine proširenog spektra. Plazmidi koji su kodirali ESBL pripadali su različitim inkompatibilnim grupama. U izolatima iz KBC-a Zagreb zapažena je u posljednjoj godini istraživanja (2010.) pojava prvih metalo-β-laktamaza iz VIM-serije, što pokazuje evoluciju rezistencije na cefalosporine proširenog spektra od dereprimiranih AmpC-β-laktamaza i ESBL-a na početku istraživanja do karbapenemaza na njegovu kraju.Enterobacter spp. develops resistance to expanded-spectrum cephalosporins by induction or derepression of chromosomal AmpC β-lactamase, or production of extended-spectrum β-lactamases (ESBLs) or carbapenemases. The aim of the study was to analyze the mechanisms of resistance to expanded-spectrum cephalosporins and the evolution of resistance mechanism during the study period (2008–2011) on a collection of 58 randomly collected Enterobacter spp. strains from three hospital centers in Croatia and Bosnia and Herzegovina during 2008-2010. The antibiotic susceptibility was determined by broth microdilution method according to CLSI. Resistance genes were determined by PCR. Plasmids were characterized by PCR-based replicon typing (PBRT). The hypothesis of the study was that there will be multiple mechanisms of ceftazidime resistance involved, from inducible and derepressed AmpC β-lactamases to extended-spectrum β-lactamases and carbapenemases at the end of the study. The isolates from different centers were expected to express different phenotypes and mechanisms of resistance. The study showed the predominance of derepressed AmpC β-lactamases combined with ESBLs belonging to CTX-M family as a mechanism of resistance to expanded-spectrum cephalosporins. The emergency of MBLs was reported in the last year of the study in University Hospital Center Zagreb. The plasmids encoding ESBLs belonged to different incompatibility groups. This points out to the evolution of β-lactam resistance in Enterobacter spp. from derepressed AmpC β-lactamases and ESBL to carbapenemases

Epidemic spread of OXA-48 beta-lactamase in Croatia

PURPOSE: A dramatic increase in OXA-48 β-lactamase was observed recently not only in large hospital centres, but also in smaller suburban hospital centres in geographic areas bordering Croatia. The aim of the study was to analyse the epidemiology, the mechanisms of antibiotic resistance and the routes of spread of OXA-48 carbapenemase in Croatia. ----- METHODS: Carbapenemase and other β-lactamase and fluoroquinolone resistance genes were detected by PCR and sequencing. Whole-genome sequencing (WGS) was performed on five representative isolates. The isolates were genotyped by PFGE. ----- RESULTS: Forty-eight isolates positive for OXA-48, collected from seven hospital centres in Croatia from May 2016 to May 2017, were analysed (40 Klebsiella pneumoniae, 5 Enterobacter cloacae, 2 Escherichia coli and one Citrobacter freundii). Thirty-three isolates were ESBL positive and harboured group 1 CTX-M 1 β-lactamases. In addition to the β-lactam resistance genes detected by PCR (blaSHV-1, blaOXA-48 and blaOXA-1), WGS of five representative isolates revealed the presence of genes encoding aminoglycoside resistance, aadA2 and aph3-Ia, fluoroquinolone resistance determinants aac(6)Ib-c, oqxA and oqxB, the sulfonamide resistance gene sul1, and fosA (fosfomycin resistance). IncL plasmid was found in all isolates. Two K. pneumoniae isolates belonged to ST16, two E. cloacae to ST66 and E. coli to ST354. K. pneumoniae isolates were allocated to five clusters by PFGE which occured in different hospitals, indicating epidemic spread. ----- CONCLUSIONS: The OXA-48-positive organisms found in this study showed wide variability in antibiotic susceptibility, β-lactamase content and PFGE banding patterns. This study revealed a switch from the predominance of VIM-1 in 2012-2013 to that of OXA-48 in the 2015 to 2017

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