Surveillance of antibiotic resistance of Streptococcus pneumoniae in the Czech Republic, respiratory study results, 2010-2017.

AIM: The purpose of the surveillance performed from October to December in 2010-2017 was to monitor the trends in the susceptibility to beta-lactam and macrolide antibiotics in Streptococcus pneumoniae isolates from respiratory tract infections in the Czech Republic. MATERIAL AND METHODS: Between 42 and 55 laboratories participated in the study every year. Consecutive non-duplicate pneumococcal isolates from relevant microbiological specimens from patients with community-acquired bacterial respiratory tract infection were sequentially included in the study. Laboratories recorded qualitative results of penicillin and erythromycin susceptibility testing; susceptibility to antibiotics was determined by the disk diffusion method. Penicillin non-susceptible and/or erythromycin resistant isolates were referred to the National Reference Laboratory for Antibiotics, where the minimum inhibitory concentration of each antibiotic was tested using the broth microdilution method, and their serotyping was performed in the National Reference Laboratory for Streptococcal Infections. Twenty-six isolates from 2017 were analysed by the multilocus sequence typing method. RESULTS: In total, 7 491 pneumococcal strains were examined, of which 53.7% (4 023) were from the upper respiratory tract and 47.7% (3 573) from children under 15 years of age. Non-susceptibility to penicillin decreased from 2.6% in 2010 to 1.2% in 2017, while resistance to erythromycin increased from 7.4% to 9.7% over the same period. Penicillin non-susceptible isolates were mostly of serotypes 19A, 19F, and 15A. Macrolide resistant but penicillin susceptible isolates were predominantly represented by serotypes 19A and 3. The presence of the Taiwan19F-14 clone was confirmed in penicillin non-susceptible isolates by MLST, and the most frequently identified sequence type (ST) in macrolide resistant isolates was ST416 classified into the Netherlands15B-37 clone. CONCLUSIONS: The respiratory study of antibiotic resistance in S. pneumoniae confirmed the decreasing trend of resistance to penicillin but revealed a growing resistance to macrolide antibiotics in the Czech Republic. The results of our study confirm that antibiotic resistance in the vaccination era is associated primarily with the non-vaccine serotypes, and the clonal expansion of macrolide resistant serotype 19A was apparently supported by the growing prescription of macrolide antibiotics.

Prevalence study on carbapenemase-producing Escherichia coli and Klebsiella pneumoniae isolates in Czech hospitals--results from Czech Part of European Survey on Carbapenemase--Producing Enterobacteriaceae (EuSCAPE).

OBJECTIVE: One of the most important threats of current medicine is the spread of multiresistant Gram-negative bacteria. We report here data from a six-month prevalence study on carbapenemase-producing K. pneumoniae and E. coli performed in Czech hospitals participating on European Survey on Carbapenemase-Producing Enterobacteriaceae (EuSCAPE). METHODS: Ten hospitals covering all regions of the Czech Republic were selected. During the study period (1st November 2013 to 30th April 2014), first ten carbapenem non-susceptible isolates of K. pneumoniae or E. coli isolated from non-surveillance specimens (i.e., blood, lower respiratory tract secretions, urine, puncture fluids, and wound secretions) of single successive patients were collected. Successive carbapenem-susceptible isolates of the same species were also preserved as controls. Susceptibility to 15 antibiotics was determined using EUCAST recommendations. Carbapenemase activity was detected by MALDI-TOF MS meropenem hydrolysis assay. Positive isolates were subjected for molecular typing (multi-locus sequence typing, identification of carbapenemase gene). RESULTS: During the study period, thirty non-susceptible isolates (K. pneumoniae n=28, E. coli n=2) were identified in 5 hospitals. Only two of them were confirmed to be carbapenemase producers. A NDM-1-producing K. pneumoniae ST11 was recovered from a patient, transferred from Ukraine, being injured during a Maidan revolution. The second isolate, an OXA-48-producing K. pneumoniae, belonging to ST101, was recovered from a patient admitted to a hospital for an ischemic stroke. CONCLUSIONS: This study again confirmed that the Czech Republic still belongs to the countries with low prevalence of carbapenemase-producing Enterobacteriaceae (CPE). Cases of CPE are usually restricted to an import from high-prevalence countries or countries with unknown epidemiological situation.

Minimum inhibitory concentrations of erythromycin and other antibiotics for Czech strains of Bordetella pertussis.

AIM OF THE STUDY: To test the susceptibility to first-line and alternative antibiotics of 70 Bordetella pertussis (B. pertussis) strains recovered from patients with whooping cough through national pertussis surveillance in the Czech Republic (CR) in 1967-2010. MATERIAL AND METHODS: The minimum inhibitory concentrations (MICs) of erythromycin, clarithromycin, azithromycin, ciprofloxacin, and co-trimoxazole were tested by the reference agar dilution method on Bordet-Gengou agar with 15 % defibrinated sheep blood. RESULTS: Each of the 70 study strains was inhibited by two concentrations of erythromycin and azithromycin (0.06 and 0.12 mg/l) and by three concentrations of clarithromycin (0.03, 0.06, and 0.12 mg/l), with the highest concentration of the MIC range being 0.12 mg/l for all these similar antibiotics. Tested in a 2-fold geometric dilution series, the concentration of erythromycin required to inhibit 90 % of the study strains (MIC90) was one dilution step lower (0.06 mg/l) than those of clarithromycin and azithromycin (0.12 mg/l). All study strains were inhibited by a single concentration of ciprofloxacin (0.06 mg/l) and two concentrations of co-trimoxazole (0.12 and 0.25 mg/l). CONCLUSION: The panel of 70 Czech strains of B. pertussis appears to be homogeneous in terms of the MICs of the antibiotics tested, with two to three low concentrations being effective against all strains. To be inhibited, no strain required a higher concentration of erythromycin, clarithromycin, azithromycin, ciprofloxacin, or co-trimoxazole.

[Resistance monitoring working group: resistance to erythromycin, ciprofloxacin, and tetracycline in human isolates of Campylobacter spp. in the Czech Republic tested by the EUCAST standard method]. / Rezistence k erytromycinu, ciprofloxacinu a k tetracyklinu u humánních izolátu Campylobacter spp. v Ceské republice, vysetrená standardní metodou EUCAST.

STUDY AIM: To determine the frequency of Campylobacter spp. isolated from humans in the Czech Republic and to test their susceptibility to antimicrobials commonly used to treat campylobacteriosis by the standard EUCAST method. MATERIAL AND METHODS: Consecutive Campylobacter isolates recovered from clinical specimens in 49 microbiological laboratories within one month in 2013 were identified using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Susceptibility to erythromycin, ciprofloxacin, and tetracycline was tested by the microdilution method and the results were interpreted based on the EUCAST clinical breakpoints to differentiate between susceptible and resistant strains. RESULTS: Of the study set of 769 Campylobacter spp. strains, 90.1 % were assigned to C. jejuni, 9.8 % to C. coli, and a single strain to C. fetus (0.1 %). Except one blood isolate of C. jejuni, all other isolates were recovered from the stool. Ciprofloxacin resistance (MIC > 0.5 mg/l) was detected in 61.9 % strains of C. jejuni and in 72.0 % strains of C. coli, tetracycline resistance (MIC > 2 mg/l) was detected in 32.0 % of strains of both species, and erythromycin resistance was found in 0.3 % of strains of C. jejuni (MIC > 8 mg/l) and in 2.7 % of strains of C. coli (MIC > 4 mg/l). A C. coli strain was multidrug resistant (i.e. resistant to all three antimicrobials tested). CONCLUSIONS: Despite the fact that most Campylobacter infections in humans cure on their own, the resistance of the causative strains to the antimicrobials of choice and alternative agents needs to be studied because of its relevance to the treatment of severe cases that require antibiotics. Resistance to macrolides was found rather infrequently in this study in both C. jejuni (0.1 %) and C. coli (2.7 %) strains. Nevertheless, alarming is ciprofloxacin resistance confirmed in 61.9 % of C. jejuni strains and 72.0 % C. coli strains. As the species C. coli is more often resistant to antimicrobials than C. jejuni and ciprofloxacin along with other fluoroquinolones is commonly used to treat severe food-borne and generalized infections, it is crucial to identify the Campylobacter strains to the species level and to test their susceptibility to relevant antibiotics by a valid and reproducible method to be able to provide an effective therapy.

Carbapenemase-producing Klebsiella pneumoniae in the Czech Republic in 2011.

Carbapenemase-producing Enterobacteriaceae and Pseudomonas spp. are increasingly reported in many countries all over the world. Due to the resistance of those bacteria to almost all antibiotics (e.g.beta-lactams, aminoglycosides, fluoroquinolones),treatment options are seriously limited. In the Czech Republic, the incidence of carbapenemase-producing Enterobacteriaceae seems to be low, restricted to only three cases detected between 2009 and 2010.Here, we describe molecular typing of 15 carbapenemase-producing Klebsiella pneumoniae isolates identified in the Czech Republic during 2011. Five VIM-1-producing isolates belonging to sequence type (ST)11 and one VIM-4-producing isolate of ST1029 have been detected. blaVIM-1 and blaVIM-4 as a part of class 1 integrons were chromosomally located or carried by a plasmid belonging to A/C replicon type (blaVIM-4). KPC-3-producing isolates of ST512, recovered from six patients, caused an outbreak. Three more isolates producing KPC-2 enzyme belonged to ST258. Both blaKPCgenes were part of the Tn4401a transposon carried on plasmids of the pKpQIL type. The isolates were resistant to all antibiotics tested except colistin and/or gentamicin.Four of these 15 strains were recovered from patients repatriated to the Czech Republic from Greece and Italy. This is the first report of outbreaks caused by carbapenemase-producing Enterobacteriaceae in the Czech Republic.

Carriage of methicillin-resistant Staphylococcus aureus in veterinary personnel.

A survey of 280 attendees at a veterinary meeting in the Czech Republic in 2008 revealed a carriage rate of 0.7% for methicillin-resistant Staphylococcus aureus (MRSA). The two strains isolated were of distinct genetic lineages, carried type IV SCCmec determinants and were negative for Panton-Valentine leukocidin genes. The MRSA positivity rates for veterinarians in the Czech Republic is considerably lower than reported elsewhere.

Species identification of Campylobacter jejuni ssp. jejuni and C. coli by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and PCR.

The Campylobacter species strains (n = 42; isolated from clinical samples and deposited in Czech National Collection of Type Cultures, Prague) originally phenotypically (and biochemically) identified as Campylobacter jejuni were re-classified using molecular biological and mass spectrometric methods. Whole-cell MALDI-TOF MS (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) separated the isolates into two genetically related strains--C. jejuni (n = 26) and C. coli (n = 16) and, moreover, distinguished the intimate details in the group of tested strains. It also made it possible to create the MALDI-TOF MS dendrogram; similarly, the spectral characteristics were used for the 3D cluster analysis. Polymerase chain reaction (PCR) confirmed the results obtained by mass spectrometry. Both methods (PCR and MALDI-TOF MS) gave the same results which supports their suitability in the rapid and accurate Campylobacter-species determination.

Emergence of a novel macrolide-resistant Streptococcus pyogenes emm53 strain.

An unusual emm53, T-28/T-non-typeable, iMLS(B) phenotype clone represented a substantial proportion (28.6%) of invasive erythromycin-resistant group A streptococcus (GAS) isolates in the Czech Republic during 2003. Clonal analysis of emm53 isolates between 2001 and 2004 revealed four pulsed-field gel electrophoresis (PFGE) patterns and two emm subtypes. Isolates produced identical PFGE patterns regardless of their invasiveness and/or tetracycline resistance. Multilocus sequence typing classified all isolates as ST340. An ST5 (emm83) isolate, a potential ancestor of ST340, was isolated in the Czech Republic from an impetigo patient in 1988. The Czech emm53/ST340 isolates shared only three of seven alleles with the original Lancefield emm53/ST11 isolate.

Dissemination of a capsular and antibiotype variant of the England-9 pneumococcal clone in the Czech Republic.

Forty-one clinical isolates of serotype 19F Streptococcus pneumoniae resistant to chloramphenicol and/or tetracycline, isolated in the Czech Republic between 1996 and 2005, were analysed by pulsed-field gel electrophoresis and multilocus sequence typing. All but two isolates belonged to a single cluster represented by sequence type 423, a double-locus variant of clone England(14)-9. Interestingly, these two isolates differed from the dominant clone in capsular type as well as antibiotic susceptibility profile.

[Detection of MRSA in a group of 752 strains of S. aureus using a cefoxitin disk]. / Odlisení MRSA v souboru 752 kmenu S. aureus pomocí disku s cefoxitinem.

A set of 752 S. aureus strains including 665 fresh clinical isolates, 82 collection strains from the NRL for staphylococci and three control strains for external quality assessment were tested for susceptibility to oxacillin by three routine phenotypic methods with oxacillin (agar screening method, dilution micromethod and disk diffusion method) and a new method with a 30 micrograms cefoxitin disk. Gene mecA coding for oxacillin resistance was detected by PCR, PBP2a gene product was detected by latex agglutination. All of 218 oxacillin resistant strains--MRSA (methicillin resistant S. aureus)--gave inhibition zones of 6-19 mm around the cefoxitin disk, i.e. zones within the range set up for oxacillin resistant strains, eight out of these strains showing false oxacillin susceptibility in one or more phenotypic tests. It can be stated that the presence of an inhibition zone of < 20 mm around the 30 micrograms cefoxitin disk allows for reliable differentiation between MRSA and oxacillin susceptible S. aureus.

[Resistance to macrolides in the species Streptococcus pyogenes in the Czech Republic in 1996-2003]. / Rezistence k makroliduim u druhu Streptococcus pyogenes v Ceské republice v období let 1996-2003.

The study of the prevalence of erythromycin resistance in 22 169 S. pyogenes strains in the Czech Republic in 1996-2003 on the background of rough data on the nationwide consumption of macrolide antibiotics confirmed that the exponential growth of resistance observed in 1998-2001 copied with a delay the rise in macrolide antibiotic consumption recorded in 1992-1995. The highest frequency of erythromycin resistance was found in 2001 (16.5%) with a subsequent decrease to 14.5% in 2002 and to 9.1% in 2003. The drop in resistance followed the stagnation in macrolide consumption and its decrease by 17% in 2002. Upward and downward trends in macrolide resistance in different regions and age groups copied the nationwide trends with some quantitative differences that could not be analyzed in view of the lack of detailed data on antibiotic consumption. A 99.5% concordance was found between the results of the phenotypic method and those of detection of genes coding for constitutive, inducible and efflux resistance to macrolide-lincosamide-streptograminB (MLSB) antibiotics. In 2001 when the highest erythromycin resistance was recorded in the Czech Republic, most of the tested strains (91.2%) showed resistance to all MLSB antibiotics, with macrolide efflux (susceptibility to lincosamides and 16-membered macrolides was conserved) being implicated in resistance of 8.8% of the strains only. In 2003, the number of erythromycin resistant strains decreased and the resistance mechanism was ascribed to macrolide efflux in 26.8% of them. Almost all of the strains with constitutive or induced MLSB resistance are also resistant to either tetracycline or bacitracin or both. In the light of S. pyogenes resistance to bacitracin, the bacitracin disk is not usable in preliminary identification any more.

Update on the major clonal types of methicillin-resistant Staphylococcus aureus in the Czech Republic.

The purpose of our study was the molecular characterization of methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in 21 hospitals in the Czech Republic in the period 2000-2002 and comparison with previous results from 1996-1997. Strains were analyzed by pulsed-field gel electrophoresis (PFGE) of SmaI digests and ribotyping of HindIII digests hybridized with a 16S-23S DNA probe. The prevalence of the most clinically important macrolide (ermA, ermB, ermC, and msrA) and aminoglycoside (aph3', ant4', and aac6'-aph2") resistance genes was evaluated as well. Selected isolates representative of each clonal type were analyzed by multilocus sequence typing and by a multiplex PCR method capable of identifying the structural type of the staphylococcal cassette chromosome mec (SCCmec) carried by the bacteria. Our results document the displacement of the Brazilian clone (ST239, SCCmec type IIIA, PFGE type B, ribotype H1) by a new clone that we named "Czech clone" (ST239, SCCmec type IIIA, PFGE type F, ribotype H6) and the maintenance of the Iberian clone (ST247, SCCmec type IA, PFGE type A, ribotype H2) exclusively in one hospital in the Czech Republic. In addition, we found a correlation between the distribution of aminoglycoside resistance genes and MRSA clonal types.