Emergence of extended-spectrum ß-lactamase (ESBL) and/or carbapenemase producing Enterobacteriaceae (CPE) and their antimicrobial resistance.

OBJECTIVES: We focused on detecting the most frequent resistance mechanisms in selected multidrug-resistant (MDR) strains and determining their antimicrobial resistance. BACKGROUND: MDR pathogens pose urgent public health threat due to limited treatment options, rigorous control measures and significant mortality. METHODS: We confirmed extended-spectrum ß-lactamase (ESBL) and carbapenemase producing Enterobacteriaceae through guidelines, as well following ß-lactamases: AmpC by cloxacillin, class A carbapenemase with phenylboronic acid, class B metallo-ß-lactamase with ethylenediaminetetraacetic acid. Multilocus sequence typing was used to investigate 20 Escherichia coli strains. RESULTS: Overall 205 mostly ESBL Escherichia coli demonstrated resistance against amikacin (4.7 %), tigecycline (1.2 %), and no resistance to ceftazidime/avibactam, meropenem, nitrofurantoin and fosfomycin. Out of 41 Klebsiella species (spp.), 37 (90.2 %) showed carbapenemase activity, 13 (35.1 %) of class A and 24 (64.9 %) of class B. Resistance was following: meropenem 66.7 %, tigecyclin 10.2 % and colistin 0 %. From Enterobacter spp. 21 strains, 14 (66.7 %) were ESBL, 5 produced ESBL and/or AmpC and 2 were MDR. We ascertained 14 (70 %) E. coli sequence type - ST131. CONCLUSIONS: The study revealed various resistance mechanisms in concert with different agents and association of specific ST131 within E. coli. These characteristics considerably contribute to emergence of antimicrobial resistance (Tab. 4, Ref. 30).

Influence of vitamin D on the expression of mRNA of cytokines in the mucosa of inflammatory bowel disease patients.

The aim of this study was to analyze the influence of 25(OH)VD serum concentration on the expression of mRNA cytokines (IL-6, IL-8, IL-12, IL-17, IL-23, TNFα, CCR1, CCR2, CCR5, CCR9, CCL5, TLR2, TLR4, TLR5, CD207 ,CD206, FoxP3) in mucosa of IBD patients. The cohort consisted of 86 IBD patients (48 CD and 38 UC) followed at the IBD center of University Hospital Bratislava-Ruzinov. We performed colonoscopy in each patient and took biopsies from mucosa of sigma and terminal ileum. Serum concentration of 25(OH)VD was assessed at the time of colonoscopy. mRNA was extracted from mucosal biopsy samples for each cytokine. Then we analyzed the correlation between VD and the expression of mRNA of cytokines from biopsies samples.  In CD we observed a significant positive correlation of serum concentration 25(OH)VD and the expression mRNA level of IL-6. There was also trend towards significant positive correlation of the expression mRNA of TNFα, IL-10, IL-23, TLR 2 in inflamed mucosa of terminal ileum as well as the expression mRNA of CCR5 and CCR1 in non-inflamed mucosa from terminal ileum. We also found a trend towards positive correlation between 25(OH)VD and the expression mRNA of IL-23, TLR4, CD 207, CCR1, CCR5 and CD 206 in non-inflamed mucosa of sigma in UC.VD significantly correlated with the levels of expression of several inflammatory cytokines including TNFα in colonic mucosa of patients with IBD (Tab. 4, Fig. 3, Ref. 31).

The speciation and genotyping of Cronobacter isolates from hospitalised patients.

The World Health Organization (WHO) has recognised all Cronobacter species as human pathogens. Among premature neonates and immunocompromised infants, these infections can be life-threatening, with clinical presentations of septicaemia, meningitis and necrotising enterocolitis. The neurological sequelae can be permanent and the mortality rate as high as 40-80%. Despite the highlighted issues of neonatal infections, the majority of Cronobacter infections are in the elderly population suffering from serious underlying disease or malignancy and include wound and urinary tract infections, osteomyelitis, bacteraemia and septicaemia. However, no age profiling studies have speciated or genotyped the Cronobacter isolates. A clinical collection of 51 Cronobacter strains from two hospitals were speciated and genotyped using 7-loci multilocus sequence typing (MLST), rpoB gene sequence analysis, O-antigen typing and pulsed-field gel electrophoresis (PFGE). The isolates were predominated by C. sakazakii sequence type 4 (63%, 32/51) and C. malonaticus sequence type 7 (33%, 17/51). These had been isolated from throat and sputum samples of all age groups, as well as recal and faecal swabs. There was no apparent relatedness between the age of the patient and the Cronobacter species isolated. Despite the high clonality of Cronobacter, PFGE profiles differentiated strains across the sequence types into 15 pulsotypes. There was almost complete agreement between O-antigen typing and rpoB gene sequence analysis and MLST profiling. This study shows the value of applying MLST to bacterial population studies with strains from two patient cohorts, combined with PFGE for further discrimination of strains.

Identification and characterization of Cronobacter strains isolated from powdered infant foods.

UNLABELLED: Cronobacter spp. (formerly Enterobacter sakazakii) is responsible for rare but fatal cases of infection in neonates and immunocompromised infants. The aim of our study was to characterize Cronobacter strains isolated from powdered infant foods in Public Health Authority of the Slovak Republic in 2009-2010. Powdered infant food products have been analysed using currently available standard method ISO/TS 22964: 2006 for the detection of Cronobacter spp. complemented with qPCR confirmation of positive strains. Thirteen Cronobacter strains were isolated from more than 900 powdered infant formulae, milk-based and cereal-based powdered weaning food products. The strains were assigned to five biogroups and ten multilocus sequence typing (MLST) sequence types. In total, twelve strains were identified as Cronobacter sakazakii and one strain as Cronobacter dublinensis. Multiple strains originated from parallel isolation were obtained in three samples and the variability between strains from the same food was observed twice. The results are in agreement with the hypothesis that the Cronobacter contamination detected in infant powdered food is low and originating in various accidental sources. SIGNIFICANCE AND IMPACT OF THE STUDY: This study characterized Cronobacter strains isolated from powdered infant formulae and weaning foods by biotyping and multilocus sequence typing. The later method was shown to be more discriminative and suitable for both species identification and subtyping. Low level (0·9%) of Cronobacter positivity was observed in 916 samples. Multiple sequence types were observed among strains isolated from the same food product. This highlights that multiple isolates from each single sample should be analysed in epidemiological studies, since more than one genetic subtype may be present.

Occurrence of aminoglycoside-modifying-enzyme genes aac(6')-aph(2"), aph(3'), ant(4') and ant(6) in clinical isolates of Enterococcus faecalis resistant to high-level of gentamicin and amikacin.

The genes coding for 4 aminoglycoside-modifying enzymes AAC(6')-APH(2"), APH(3'), ANT(4') and ANT(6) were determined in 44 Slovak clinical isolates of Enterococcus faecalis with high-level resistance to gentamicin (HLGR, collection 1) and 48 E. faecalis isolates with resistance to amikacin (AR, collection 2). The occurrence of spotted genes was (collection 1 vs. collection 2): aac(6)-aph(2") 81.8 vs. 8.3 %, ant(4') 52.3 vs. 81.3 %, aph(3') 50 vs. 56.3 % and ant(6) 6.8 vs. 4.2 %, the most frequent combinations of genes in the HLGR collection were aac(6')-aph(2") + ant(4') and aac(6')-aph(2") + aph(3). In contrast, the aph(3') + ant(4') gene profile was predominant in AR isolates. None of the isolates contained all four AGME genes simultaneously.

Detection of transgene copy number by analysis of the T1 generation of tobacco plants with introduced P3 gene of potato virus A.

Real-time PCR, namely the deltadeltaCt method was used to determine the relative copy number of Potato virus A (PVA) P3 gene in the genome of the T1 generation of 18 transgenic tobacco lines. These results were compared with segregation ratios of kanamycin (Km)-resistant phenotype in T1 plants of each line and were found to be, in general, concordant. All the five lines with the Mendelian segregation ratio of 3:1 carried one gene copy. In 12 of 13 lines with uneven segregation more inserted gene copies were detected. Only for one line the real-time PCR and phenotype segregation differed. According to our results the real-time PCR of T1 generation may be used as supplementary method of estimation of the number of transgene copies in the case of nonavailability of the original T0 plants.

Detection of Listeria monocytogenes by polymerase chain reaction oriented to inlB gene.

Primers were designed for the detection of Listeria monocytogenes by the polymerase chain reaction oriented to specific sequences of the inlB gene encoding an internalin. At optimized reaction conditions, 100% sensitivity (on a panel of 33 strains of L. monocytogenes) and 100% specificity (on panels of 15 strains of other Listeria spp. and 41 other bacteria), were determined for the inlB-L/R primers. The detection limit of PCR with these primers was 10(4) cfu/ml and was not affected by up to 10(8) cfu/ml of L. innocua.

PCR-based methods for identification of Enterococcus species.

Two DNA-based techniques were used for species identification of enterococci. PvuII digestion of the genus-specific PCR product yielded four different restriction profiles among 20 enterococcal species; one of them was species-specific for E. faecium. In the second case, 32 reference strains belonging to 20 enterococcal species were divided to 12 groups by amplification of internal transcribed spacer of rRNA operon. Interspecies and some intraspecies profile variability was determined. Both methods gave similar results.

Antibiotic resistance and virulence factors among clinical and food enterococci isolated in Slovakia.

The resistance to antibiotics and the distribution of virulence factors in enterococci isolated from traditional Slovak sheep cheese bryndza was compared with strains from human infections. The occurrence of 4 enterococcal species was observed in 117 bryndza-cheese isolates. The majority of strains were identified as E. faecium (76 %) and E. faecalis (23 %). Several strains of E. durans and 1 strain of E. hirae were also present. More than 90 % of strains isolated from 109 clinical enterococci were E. faecalis, the rest belonged to E. faecium. The resistance to 6 antimicrobial substances (ampicillin, ciprofloxacin, higher concentration of gentamicin, nitrofurantoin, tetracycline and vancomycin) was tested in clinical and food enterococci. A higher level of resistance was found in clinical than in food strains and E. faecium had a higher resistance than E. faecalis; no resistance to vancomycin was detected. The occurrence of 3 virulence-associated genes, cylA (coding for hemolysin), gelE (coding for gelatinase) and esp (coding for surface protein) was monitored. Differences were found in the distribution of cylA gene between clinical and bryndza-cheese E. faecalis strains; in contrast to clinical strains (45 %), cylA gene was detected in 22 % of food isolates. The distribution of 2 other virulence factors, gelE and esp, was not significantly different in the two groups of E. faecalis strains. cylA and gelE genes were not detected in E. faecium but more than 70 % of clinical E. faecium were positive for esp, even thought none of the 79 E. faecium cheese isolates contained this gene.

Quantification of Escherichia coli by kinetic 5'-nuclease polymerase chain reaction (real-time PCR) oriented to sfmD gene.

AIMS: A kinetic 5'-nuclease polymerase chain reaction (real-time PCR) for the quantification of Escherichia coli was developed. METHODS AND RESULTS: Specific primers and a fluorogenic probe oriented to sfmD gene, encoding a putative outer membrane export usher protein, were designed. The PCR system was highly specific and sensitive for E. coli, as determined with 37 non-E. coli strains (exclusivity, 100%) and 24 E. coli strains (inclusivity, 100%). When used in real-time PCR, linear calibration lines were obtained in the range from 10(2) to 10(8) CFU ml(-1) for three E. coli strains. Salmonella Enteritidis (10(6) CFU ml(-1)) or Citrobacter freundii (10(6) CFU ml(1)) had no effect on quantification of E. coli by the method. CONCLUSIONS: The developed real-time PCR is suitable for rapid quantification of E. coli. SIGNIFICANCE AND IMPACT OF THE STUDY: In connection to an appropriate sample preparation technique, the method is suitable for food safety and technological hygiene applications.

Construction of Lactose-Utilizing Xanthomonas campestris with a Mini-Mu Derivative.

Xanthomonas campestris is not able to grow in lactose media. The lactose operon from Escherichia coli as part of a mini-Mu phage was integrated at random sites in the chromosome of this bacterium. Clones expressing (beta)-galactosidase were selected. The resulting strain X. campestris 204, is suitable for production of xanthan gum directly from lactose.