[Identification of bacteria from blood cultures by fluorescence in situ hybridization]. / Prukaz bakteriálních agens z hemokultur metodou fluorescencní in situ hybridizace.

The diagnosis of bacterial agents of sepsis from blood cultures is crucial for the subsequent treatment of this condition. The aim of this study was to compare Gram stain, culture, and biochemical identification (conventional methods) and fluorescence in-situ hybridisation (FISH) that detects microorganisms from positive blood cultures using specific probes. Another aim was to evaluate the potential of this method for use in clinical practice. Altogether 71 samples of positive blood cultures were tested by FISH. Blood cultures were also processed in the conventional way using the BACTEC analyser. The bacteria recovered were inoculated on solid media and then identified biochemically. The results obtained by the conventional methods and HemoFISH were not 100% concordant. The sensitivity of HemoFISH was 90.1%. Very good results were achieved for staphylococci and enterobacteria. FISH identification failed in three cases because the hybridization probes were not able to bind to bacterial rRNA. The FISH bacterial identification is faster than the conventional methods, but should be confirmed by the latter.

Plasma discharge and time-dependence of its effect to bacteria.

Several types of plasma discharge have been proven to have a capacity for sterilization. Our goal is to introduce new nonthermal plasma pencil. We used it to sterilize different microbial populations with differing ages. We used a plasma discharge of the following characteristics: radio frequency barrier discharger at atmospheric pressure with a working frequency of 13.56 MHz, and the working gas used was argon. We performed 110 tests with the following microbial populations: Pseudomonas aeruginosa, Staphylococcus aureus, Proteus species, and Klebsiella pneumoniae. All populations were inoculated on the previous day and also on the day of our experiment. We made our evaluations the following day and also after 5 days, with all our microbial populations. Eradication of microbial populations is dependent on the plasma discharge exposure time in all cases. With regard to freshly inoculated microbes, we were able to sterilize agar with intensive exposure lasting for 10 s of colonies Pseudomonas, Proteus, and Klebsiella. The most resistant microbe seems to be S. aureus, which survives 5 s of coherent exposure in half of the cases. Using the lightest plasma discharge exposure, we achieved a maximum of 10(4)-10(5) CFU/mL (colony-forming unit - CFU). Regarding older microbial populations inoculated the day before the experiment, we can only decrease population growth to 10(5) CFU/mL approximately, but never completely sterilize. The plasma discharge with our characteristics could be used for the sterilization of the aforementioned superficially growing microbes, but does not sufficiently affect deeper layers and thus seems to be a limitation for eradication of the already erupted colonies.

[Antibiotic resistance and biofilm formation in Staphylococcus saprophyticus strains isolated from urine]. / Staphylococcus saprophyticus--jeho rezistence k vybraným antibiotikum a tvorba biofilmu u kmenu izolovaných z moce.

Eighty-seven Staphylococcus saprophyticus strains isolated from urine of 87 patients with cystitis were examined in 2005-2009. All strains were tested for resistance to vancomycin, nitrofurantoin, doxycycline, oxacillin, amoxicillin/clavulanate, cefoxitin and sulfamethoxazole/trimethoprim and for biofilm formation by a modified Christensen method. None of the tested strains of S. saprophyticus showed resistance to vancomycin, while 2 strains (2.3 %) were resistant to nitrofurantoin, 9 (10.3%) to doxycycline, 20 (23.0 %) to oxacillin, 6 (6.9%) to amoxicillin/clavulanate, 6 (6.9%) to cefoxitin and 1 (1.1%) to sulfamethoxazole/trimethoprim. S. saprophyticus was detected as the causative agent of cystitis in 0.4 % of 20,375 culture positive urine samples analyzed in our laboratory between 2005 and 2009. Most 67 (77.0%) S. saprophyticus strains were recovered from women, particularly from young women. Biofilm formation was detected in 16 (18.4 %) out of 87 S. saprophyticus strains.

[Antibiotic resistance and biofilm formation in Pseudomonas aeruginosa strains isolated from patients with urinary tract infections]. / Pseudomonas aeruginosa, její rezistence k vybraným antibiotikum a tvorba biofilmu u kmenu izolovaných od pacientu s infekcí mocových cest.

Altogether 118 Pseudomonas aeruginosa strains isolated from urine of patients with urinary tract infection were tested by the disk diffusion method for susceptibility to ciprofloxacin, ofloxacin, gentamicin, amikacin, colistin, meropenem, imipenem, piperacillin/tazobactam and ceftazidime. All strains were also screened for biofilm formation using a modified Christensen method. Eighty-eight, i.e., 74.6%, of the tested strains were resistant to ofloxacin, 86 (72.9%) to ciprofloxacin and 70 (59.3%) to gentamicin. Forty strains (33.9%) were resistant to imipenem, 42 strains (35.6%) to meropenem, 14 strains (11.9%) to amikacin, 2 strains (1.7%) to colistin, 35 strains (29.7%) to piperacillin/tazobactam and 41 strains (34.7%) to ceftazidime. Co-resistance to ofloxacin, ciprofloxacin and gentamicin was detected in 67 strains (56.8%) while 12 strains (10.2%) were resistant to most tested antibiotics, with the exception of amikacin and colistin. Biofilm formation was found in 41 strains (34.7%), more precisely in 23 of 46 inpatient strains and 18 of 72 outpatient strains. Eight (66.6%) of 12 polyresistant strains were biofilm producers.

[Detection of AmpC beta-lactamases in Gram-negative bacteria isolated from urine]. / Detekce beta-laktamáz typu AmpC u gramnegativních bakterií izolovaných z moce.

AmpC beta-lactamases, enzymes produced by a range of Gram-negative bacteria and able to hydrolyse penicillins, monobactams, cephalosporins and cephamycins, are not inhibited by beta-lactamase inhibitors. They are spread via plasmids and pose a huge epidemiological and diagnostic problem. The production of AmpC B-lactamases was tested in various Gram-negative bacteria. Of strains isolated from urine of patients diagnosed with urinary tract infection, we selected those that were resistant to third generation cephalosporins and did not produce extended-spectrum beta-lactamases (ESBL). The production of inducible AmpC was assayed by modified DDST (double disk synergy test) and that of constitutive AmpC was tested on agar containing oxacillin as an AmpC inhibitor. All constitutive AmpC producers were also tested for susceptibility to amikacin, piperacillin/tazobactam, cefoperazon/sulbactam, imipenem, meropenem and colistin by the disk diffusion method on MH-agar. Altoghether 119 strains (68 strains of Klebsiella pneumoniae, 20 strains of Escherichia coli and 12 strains of Enterobacter aerogenes) were tested. We detected 38 constitutive AmpC producers, including primarily K. pneumoniae (17 strains), E. coli (10 strains) and E. aerogenes (4 strains). Thirty-two strains were inducible AmpC producers (21 K pneumoniae strains, 6 Morganella morganii strains and 3 E. aerogenes strains). Two K. pneumoniae strains were ESBL/AmpC coproducers. The constitutive AmpC producers showed relatively good susceptibility to cefepim (84.2%) and carbapenems.

Antibiotic synergy against biofilm-forming Pseudomonas aeruginosa.

Eight antibiotics (aztreonam, ceftazidim, cefoperazon, cefepim, netilmicin, amikacin, ofloxacin and ciprofloxacin) exhibited antimicrobial activity individually and/or in combinations against 20 wild-type biofilm-forming strains of Pseudomonas aeruginosa. The strains were less susceptible in biofilm; in 10 strains antibiotic synergy was observed for the combination of aztreonam and ciprofloxacin. Synergy was also demonstrated in the case of beta-lactams and aminoglycosides, beta-lactams and fluoroquinolones, aminoglycosides and fluoroquinolones, and for monobactams and beta-lactams although the strains were resistant to the individual antibiotics. Synergism or partial synergism was found with one or more antibiotic combinations against 32.4% of isolates.

Determination of minimal regrowth concentration (MRC) in clinical isolates of various biofilm-forming bacteria.

Based on the ability to attach to polymeric surfaces, the formation of biofilms was determined in 5 wild-type strains (Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumanii, Escherichia coli, Staphylococcus warneri). Using modified Christensen method, minimum regrowth concentration (MRC) of piperacillin, piperacillin-tazobactam, cefoperazon, ceftazidim, cefepim, meronem, ciprofloxacin, netilmicin and amikacin for Gram-negative and of ampicillin-sulbactam, chloramphenicol, tetracycline, clindamycin, vancomycin and teicoplanin for Gram-positive bacteria was estimated in trypticase-soy broth medium after a 1-d growth on polystyrene microtiter plates. Adherent bacterial populations exhibited reduced antimicrobial susceptibility, which was not shown in submerged cultures. Our results indicate that MRC can predict therapeutic outcome of antibiotic treatment better than the minimum inhibitory concentration tests commonly used.

[Biofilms and their significance in medical microbiology]. / Biofilmy a jejich význam v lékarské mikrobiologii.

Microorganisms are able to adhere to various surfaces and to form there a three-dimensional structure known as biofilm. In biofilms, microbial cells show characteristics and behaviours different from those of plankton cells. Intercellular signalizations of the quorum-sensing type regulate interaction between members of the biofilm. Bacteria embedded in the biofilm can escape and form well known planktonic forms, that are obviously only a part of the bacterial life cycle. Bacteria adhere also to medically important surfaces such as catheters, either urinary or intravenous ones, artificial heart valves, orthopedic implants and so on and contribute to device-related infections like cystitis, catheter-related sepsis, endocarditis etc. Once a biofilm has been established on a surface, the bacteria harboured inside are less exposed to the host's immune response and less susceptible to antibiotics. As an important cause of nosocomial infections the biofilm must remain in the centre of the microbiologist's attention.