Colistin is a last-resort antibiotic for the treatment of infections caused by multidrug and carbapenem-resistant Gram-negative bacteria. Colistin resistance has been emerging and multiple outbreaks ...have been reported in Europe and elsewhere. It has been most frequently reported in carbapenem-resistant K. pneumoniae. In this study, 24 multidrug and colistin-resistant clinical isolates (14 K. pneumoniae, one E. aerogenes, one E. cloacae, and eight A. baumannii) were collected from four hospitals in Croatia from 2013 to 2018, in order to analyse the molecular epidemiology and mechanisms of antibiotic resistance. β-lactamase and carbapenemase genes were detected by PCR. Genotyping was done on selected isolates by rep-PCR. Whole genome sequencing (WGS) was performed to discover possible molecular mechanisms for the observed colistin resistance. All isolates, except two K. pneumoniae isolates, were extensively drug resistant. Ten out of 16 (63%) K. pneumoniae isolates possessed blaOXA-48, which is the most common carbapenem resistance gene in Croatia and in other parts of Europe. All A. baumannii isolates possessed the OXA-23-like carbapenem hydrolysing oxacillinase and five turned out to be pandrug-resistant. Colistin resistance was most likely chromosomally mediated. After sequence analysis, none of the isolates were found to possess any of the mcr gene variants. Several previously reported mutations were found in PmrB, PhoP, PhoQ, and MgrB, which are associated with colistin resistance. In the global phylogenetic analysis, DNA mutations causing mutations in the MgrB protein were present mostly in lineages comprising colistin resistant isolates, and the second most prevalent mutation (K3X) was also encountered in our isolates. In addition, based on genotyping by rep-PCR, the spread of colistin resistance is most likely to be clonal. Most importantly, the presence of colistin resistance together with carbapenemase genes in extensively drug resistant isolates poses real threats in the use of carbapenems and colistin to fight infections.
•Genetic analysis of colistin resistant isolates in Croatia.•In total, 66.7% (8/12) Enterobacteriaceae and 71.4% (5/7) A. baumannii isolates were XDR.•No evidence for plasmid-mediated colistin resistance mechanisms.•Global phylogenetic analysis of the mgrB and lpxC genes.
Resistance to carbapenems in
has become a matter of the highest concern in the last decade. Recently,
harboring multiple carbapenemases were detected in three hospital centers in Croatia and in the ...outpatient setting, posing a serious therapeutic challenge for clinicians. In this study, we analyzed eight
and two
complex isolates with multiple carbapenemases, with regard to antibiotic susceptibility, β-lactamase production and plasmid content. The isolates demonstrated uniform resistance to amoxicillin/clavulanate, piperacillin/tazobactam, cefuroxime, ceftazidime, cefotaxime, ceftriaxone and ertapenem. Among novel β-lactam/inhibitor combinations, ceftazidime/avibactam exhibited moderate activity, with 50% of isolates susceptible. All isolates demonstrated resistance to imipenem/cilastatin/relebactam, and all but one to ceftolozane/tazobactam. Four isolates exhibited a multidrug-resistant phenotype (MDR), whereas six were allocated to an extensively drug-resistant phenotype (XDR). OKNV detected three combinations of carbapenemases: OXA-48+NDM (five isolates), OXA-48+VIM (three isolates) and OXA-48+KPC (two isolates). Inter-array testing identified a wide variety of resistance genes for β-lactam antibiotics:
,
,
,
,
,
, aminoglycosides:
6,
,
,
and
, fluoroquinolones:
A,
B and
S, sulphonamides:
1 and
2 and trimethoprim:
.
genes were reported for the first time in Croatia. This study demonstrated the ability of
and
to acquire various resistance determinants under the selection pressure of antibiotics widely used during the COVID-19 pandemic. The novel inter-array method showed good correlation with OKNV and PCR, although some discrepancies were found.
Carbapenemases involved in acquired carbapenem resistance in Enterobacteriaceae belong to Ambler class A serin β-lactamases, class B metallo-β-lactamases (MBL) or class D OXA-48-like β-lactamases. ...The aim of the present study was to analyse the molecular epidemiology and the mechanisms and routes of spread of class B and class D carbapenemases in Croatia.
In total 68 isolates were analyzed. Antibiotic susceptibility was determined by broth microdilution method. PCR was used to detect antibiotic-resistance genes. Genotyping was performed by rep-PCR and MLST.
Sixty-five isolates were found to harbour VIM-1 carbapenemase, seven of which were positive also for NDM-1, while two strains harboured only NDM-1. OXA-48 was detected in three isolates, two of which coproduced VIM-1. Thirty-six strains possessed additional CTX-M-15 β-lactamase whereas 64 were positive for TEM-1. CMY was found in 18 Citrobacter freundii isolates and DHA-1 in one Enterobacter cloacae isolate. Four different plasmid-incompatibility groups were found: A/C, L/M, N and FIIAs. Unlike C. freundii and E. cloacae, Klebsiella pneumoniae showed high diversity of rep-PCR patterns. E. cloacae and C. freundii predominantly belonged to one large clone which was allocated to ST105 and ST24, respectively.
Three different types of carbapenemases were identified showing the complexity of CRE in Croatia.
•In total 68 carbapenem-resistant isolates were analyzed: 34 Enterobacter cloacae, 18 Citrobacter freundii, 15 Klebsiella pneumoniae and 1 Klebsiella oxytoca•Sixty-five isolates were found to harbour VIM-1 carbapenemase, seven of which were positive also for NDM-1, while two strains harboured only NDM-1.•Three different types of carbapenemases were identified showing the complexity of carbapenemase producing Enterobacteriaceae in Croatia.•Four different plasmid-incompatibility groups were found: A/C, L/M, N and FIIAs.•K. pneumoniae isolates showed high diversity of rep-PCR patterns in contrast to E. cloacae and C. freundii.
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.
•Metronidazole resistance could be selected in nim-negative Bacteroides fragilis group strains.•In vitro induction of metronidazole resistance in B. fragilis group strains may be due to physiological ...changes.•Global downregulation of the cellular pathway may explain the lower lactate dehydrogenase (LDH) activity of the induced strains.•Genetic mutations in a B. fragilis strain that demonstrated an emergent increase in LDH activity could be responsible for the activity increase.
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 8mg/L to 96mg/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 49.1U/mg and 222.0U/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.
Purpose
Aim of this study was to investigate the prevalence of methicillin-resistant
Staphylococcus aureus
(MRSA), extended-spectrum (ESBL) and plasmid-mediated AmpC beta-lactamase producing ...Gram-negative bacteria in children.
Methods
Antibiotic susceptibility of MRSA and beta-lactamase producing Gram-negative bacteria was determined by disc diffusion and broth microdilution methods according to CLSI guidelines. Methicillin resistance was confirmed by the presence of
mecA
gene by PCR. The genetic characterization of
S. aures
was performed using
spa
-typing and the algorithm based upon repeat pattern (BURP). Double-disk synergy test was used to screen for ESBL production. PCR was used to detect
bla
ESBL alleles. Genetic relatedness of the strains was tested by pulsed-field gel electrophoresis (PFGE).
Results
Among 23 MRSA, 12 (52.2 %) were obtained from newborns. MLST CC152 (
spa
-CC 355–595) (Balkan clone) was the most prevalent, 20 (87 %) cases. Among 24 beta-lactamase producing Gram-negative bacteria, 10 (41.7 %) were obtained from each newborns and one-year-old children; 14 (58.3 %) were from urine. Among 11
Klebsiella
strains isolated from urine eight (73 %) produced CTX-M-15, and one CTX-M-3 beta-lactamase. Twenty (83 %) of CTX-M producers were coproduced by other types of beta-lactamases. Fifteen (65.2 %) MRSA isolates were clonally related. Five clones among 13
K.
pneumoniae
isolates were detected by PFGE suggesting clonal spread of β-lactamase producing Gram-negative bacteria.
Conclusion
Pediatric infections caused by clonal spread of MRSA and beta-lactamase-producing Gram-negative bacteria are of major concern. Proper infection control measures should be implemented in order to avoid the transmission and major outbreaks.
We report the first outbreak caused by Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae sequence type 258 (ST 258) clone that occurred in Croatia from May to December 2012.
23 ...carbapenem-resistant K. pneumoniae isolates were recovered from clinical and screening specimens of 12 patients hospitalized in a regional hospital. The first isolates from the 12 patients were typed using pulsed-field gel electrophoresis and multilocus sequence typing.
PCR detection showed that all isolates harbored the bla
gene, together with bla
and bla
.
The rapid detection of KPC-producing K. pneumoniae and vigorous implementation of infection control measures were necessary to successfully control the outbreak.
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 endoscopic 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. Keywords: Helicobacter pylori, genotyping, cag pathogenicity island, cagA
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