Endolysin EN572-5 as an alternative to treat urinary tract infection caused by Streptococcus agalactiae.

Streptococcus agalactiae (Group B Streptococcus, GBS) is an opportunistic pathogen causing urinary tract infection (UTI). Endolysin EN572-5 was identified in prophage KMB-572-E of the human isolate Streptococcus agalactiae KMB-572. The entire EN572-5 gene was cloned into an expression vector and the corresponding recombinant protein EN572-5 was expressed in Escherichia coli in a soluble form, isolated by affinity chromatography, and characterized. The isolated protein was highly active after 30 min incubation in a temperature range of - 20 °C to 37 °C and in a pH range of 5.5-8.0. The endolysin EN572-5 lytic activity was tested on different Streptococcus spp. and Lactobacillus spp. The enzyme lysed clinical GBS (n = 31/31) and different streptococci (n = 6/8), and also exhibited moderate lytic activity against UPEC (n = 4/4), but no lysis of beneficial vaginal lactobacilli (n = 4) was observed. The ability of EN572-5 to eliminate GBS during UTI was investigated using an in vitro model of UPSA. After the administration of 3 µM EN572-5, a nearly 3-log decrease of urine bacterial burden was detected within 3 h. To date, no studies have been published on the use of endolysins against S. agalactiae during UTI. KEY POINTS: • A lytic protein, EN572-5, from a prophage of a human GBS isolate has been identified. • This protein is easily produced, simple to prepare, and stable after lyophilization. • The bacteriolytic activity of EN572-5 was demonstrated for the first time in human urine.

Carbapenem-Resistant Klebsiella pneumoniae in COVID-19 Era-Challenges and Solutions.

The COVID-19 era brought about new medical challenges, which, together with nosocomial bacterial infections, resulted in an enormous burden for the healthcare system. One of the most alarming nosocomial threats was carbapenem-resistant Klebsiella pneumoniae (CRKP). Monitoring CRKP incidence and antimicrobial resistance globally and locally is vitally important. In a retrospective study, the incidence of CRKP in the pre-COVID-19 period (2017-2019) and the COVID-19 pandemic (2020-2022) was investigated in the Central Military Hospital in Ruzomberok, Slovak Republic. The relative incidence of CRKP significantly increased during the COVID-19 period-by 4.8 times, from 0.18 to 0.76%. At the same time, 47% of CRKP-positive patients also had COVID-19. Twenty-six KPC and sixty-nine NDM-producing isolates were identified. CRKPs isolated in the year 2022 were submitted to whole genome sequencing, and their susceptibility was tested to cefiderocol, ceftazidime-avibactam, imipenem-relebactam and meropenem-vaborbactam, with excellent results. KPC-producing isolates were also highly susceptible to colistin (92%). The NDM isolates revealed lower susceptibility rates, including only 57% colistin susceptibility. ST-307 prevailed in KPC and ST-11 in NDM isolates. Despite the excellent activity of new antimicrobials, rational antibiotic policy must be thoroughly followed, supported by complementary treatments and strict anti-epidemic precautions.

High Emergence of Multidrug-Resistant Sequence Type 131 Subclade C2 among Extended-Spectrum ß-Lactamase (ESBL)-Producing Escherichia coli Isolated from the University Hospital Bratislava, Slovakia.

The expansion of sequence type 131 (ST131) extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli (E. coli) represents major worldwide challenges. E. coli strains originating from healthcare facilities (labeled No. 1 and No. 2) of the University Hospital Bratislava (UHB) were analyzed for ST131 emergence, including its (sub)lineages and clonal relatedness. Antimicrobial resistance was determined in most strains. Of a total of 354 E. coli strains, 263 (74.3%) belonged to ST131; of these, 177 (67.3%) were from No. 1. Generally, among 260 ST131 E. coli, clades A/B were confirmed in 20 (7.7%), while clade C was noted in 240 (92.3%) strains; within them, subclades were detected as follows: C0 (17; 7.1%), C1 (3; 1.2%), and C2 (220; 91.7%). Among fifteen randomly selected E. coli strains that were investigated for ST and clonal relatedness, seven STs were identified: eight (53.3%) ST131, two (13.3%) ST73, and one each (6.7%) of ST10, ST12, ST14, ST1193, and ST1196. From No. 1, two ST131 in the first internal clinic and one ST131 from No. 2 in the aftercare department were highly clonally related, suggesting possible epidemiological association. Antimicrobial resistance was as follows: ciprofloxacin 93.8%, ceftazidime 78.4%, meropenem 0%, fosfomycin 2.9% and nitrofurantoin 1.4%. Prevention of ESBL-producing E. coli dissemination, especially for ST131 clade C2, is inevitably necessary for reducing drug resistance and decreasing healthcare-associated infections.

Complete Genome Sequence of Cronobacter dublinensis Bacteriophage vB_Cdu_VP8.

Cronobacter dublinensis is a Gram-negative pathogen that is capable of causing infection in humans. In this announcement, we describe the characterization of bacteriophage vB_Cdu_VP8, which is able to lyse a Cronobacter dublinensis strain. Related to phages belonging to the genus Muldoonvirus, such as Muldoon and SP1, vB_Cdu_VP8 contains 264 predicted protein-coding genes and 3 tRNAs.

Complete Genome Sequence of New Cronobacter-Specific Bacteriophage Dev_CS701.

Cronobacter is a ubiquitous Gram-negative bacterium linked with serious infections. In this report, we describe the characterization of Cronobacter phage Dev_CS701, which was isolated from wastewater. Related to phages belonging to the Straboviridae family and Pseudotevenvirus genus, such as vB_CsaM_IeB, Dev_CS701 contains 257 predicted protein-coding genes and a tRNA gene.

Characterization and the host specificity of Pet-CM3-4, a new phage infecting Cronobacter and Enterobacter strains.

Bacteria belonging to Cronobacter and Enterobacter genera are opportunistic pathogens responsible for infections in immunocompromised patients including neonates. Phage therapy offers a safe method for pathogen elimination, however, phages must be well characterized before application. In the present study we isolated four closely related bacteriophages from the subfamily Tevenvirinae infecting Cronobacter and Enterobacter strains. Bacteriophage Pet-CM3-4 which was isolated on C. malonaticus strain possessed broader host specificity than other three phages with primary Enterobacter hosts. Based on genome sequences all these phages have been assigned to the genus Karamvirus. We also studied factors influencing the host specificity of Pet-CM3-4 phage and its host range mutant Pet-CM3-1 and observed that a lysine to glutamine substitution in the long tail fiber adhesin was the reason of the Pet-CM3-1 reduced host specificity. By characterization of phage-resistant mutants from transposon library of C. malonaticus KMB-72 strain we identified that LPS is the receptor of both phages. C. malonaticus O:3 antigen is the receptor of Pet-CM3-1 phage and the Pet-CM3-4 phage binds to structures of the LPS core region. Obtained results will contribute to our understanding of biology and evolution of Tevenvirinae phages.

Next-Generation Sequencing of Carbapenem-Resistant Klebsiella pneumoniae Strains Isolated from Patients Hospitalized in the University Hospital Facilities.

Carbapenem-resistant (CR) Klebsiella pneumoniae represents an urgent worldwide threat. We focused on CR K. pneumoniae in selected facilities of the University Hospital Bratislava (UHB) to investigate sequence types (STs), clonal relatedness, and antimicrobial resistance. Suspected carbapenem-nonsusceptible K. pneumoniae strains were obtained from hospitalized patients. Further examination included carbapenemase confirmation, cgMLST, and quantitative susceptibility testing. Of the total 41 CR K. pneumoniae strains, 26 (63.4%) were determined as ST11 in hospital No. 1; of these ST11, 22 (84.6%) were found in the first internal clinic. Six (14.6%) ST258 and three (7.3%) ST584 occurred in hospital No. 2; the most, i.e., four (66.7%), ST258 were detected in the geriatric clinic. In hospital No. 3, we found two (4.8%) ST584 and one (2.4%) ST258. Of the ST11 set, 24 (92.3%) produced NDM-1. Furthermore, seven (87.5) ST258 and five (100%) ST584 strains generated KPC-2. Antimicrobial resistance was as follows: ertapenem 97.6%, meropenem 63.4%, tigecycline 7.3%, eravacycline 7.3%, and colistin 2.5%. We revealed a presumably epidemiological association of ST11 with transmission, particularly in the first internal clinic of hospital No. 1, while ST258 and ST584 were related to interhospital dissemination between medical facilities No. 2 and No. 3. It is essential to prevent the circulation of these pathogens within and between healthcare facilities.

A novel phage-encoded endolysin EN534-C active against clinical strain Streptococcus agalactiae GBS.

Streptococcus agalactiae (Group B Streptococcus, GBS) is primarily known as a major neonatal pathogen. In adults, these bacteria often colonize the gastrointestinal and urogenital tracts. Treatment of infections using antibiotics is often complicated by recurrences caused by multi-resistant streptococci. Endolysin EN534 from prophage A2 of human isolate Streptococcus agalactiae KMB-534 has a modular structure consisting of two terminal catalytic domains, amidase_3 and CHAP, and one central binding domain, LysM. The EN534 gene was cloned into an expression vector, and the corresponding recombinant protein EN534-C was expressed in Escherichia coli in a soluble form and isolated by affinity chromatography. The lytic activity of this endolysin was tested on cell wall substrates from different GBS serotypes, B. subtilis, L. jensenii, and E. coli. The enzyme lysed streptococci, but not beneficial vaginal lactobacilli. The isolated protein is stable in a temperature range of 20-37 °C. Calcium ions enhanced the activity of the enzyme in the pH range from 5.0 to 8.0. The exolytic activity of EN534-C was observed by time-lapse fluorescence microscopy on a S. agalactiae CCM 6187 substrate. Recombinant endolysin EN534-C may have the potential to become an antimicrobial agent for the treatment of S. agalactiae infections.

Susceptibility of Staphylococcus aureus strains to commercial therapeutic phage preparations.

OBJECTIVES: The aim of this study was to determine the susceptibility of Staphylococcus aureus strains to commercial phage preparations. The strains were isolated from clinical patients as well as from nasal mucosa of healthy carriers. BACKGROUND: The elevating number of antibiotic-resistant S. aureus strains present a therapeutic challenge, especially in high-risk patients. One of the promising ways to solve this problem is phage therapy. METHODS: Susceptibility of 111 carrier strains of S. aureus (4 strains were methicillin-resistant; MRSA) and 81 clinical isolates from bloodstream or skin and soft tissue infections (28 were MRSA) to four commercial phage preparations was assessed in vitro by spot assay. The clonality of S. aureus strains was determined by spa typing. RESULTS: Spa typing revealed 97 distinct spa types. A proportion of 73-80 % of the tested S. aureus strains were revealed to have in vitro phage susceptibility, depending on the clonal affiliation of the strains and phage preparation tested. The susceptibility to phage preparations was significantly higher in MRSA strains (p < 0.001). CONCLUSIONS: In vitro results indicate a promising therapeutic potential of the tested commercial anti-staphylococcal phage preparations. They could be applied to a broad spectrum of bacterial clones, and have an excellent activity especially against MRSA strains (Tab. 2, Fig. 2, Ref. 43).

Characterization of Anti-Bacterial Effect of the Two New Phages against Uropathogenic Escherichia coli.

Urinary tract infections (UTIs) are among the events that most frequently need medical intervention. Uropathogenic Escherichia coli are frequently their causative agents and the infections are sometimes complicated by the presence of polyresistant nosocomial strains. Phage therapy is a tool that has good prospects for the treatment of these infections. In the present study, we isolated and characterized two bacteriophages with broad host specificity against a panel of local uropathogenic E. coli strains and combined them into a phage cocktail. According to genome sequencing, these phages were closely related and belonged to the Tequatrovirus genus. The newly isolated phages showed very good activity on a panel of local clinical E. coli strains from urinary tract infections. In the form of a two-phage cocktail, they were active on E. coli strains belonging to phylogroups B2 and D, with relatively lower activity in B1 and no response in phylogroup A. Our study is a preliminary step toward the establishment of a national phage bank containing local, well-characterized phages with therapeutic potential for patients in Slovakia.

Aspergillus niger Environmental Isolates and Their Specific Diversity Through Metabolite Profiling.

We present a biological profile of 16 Aspergillus niger environmental isolates from different types of soils and solid substrates across a pH range, from an ultra-acidic (<3.5) to a very strongly alkaline (>9.0) environment. The soils and solid substrates also differ in varying degrees of anthropic pollution, which in most cases is caused by several centuries of mining activity at old mining sites, sludge beds, ore deposits, stream sediments, and coal dust. The values of toxic elements (As, Sb, Zn, Cu, Pb) very often exceed the limit values. The isolates possess different macro- and micromorphological features. All the identifications of Aspergillus niger isolates were confirmed by molecular PCR analysis and their similarity was expressed by RAMP analysis. The biochemical profile of isolates based on FF-MicroPlate tests from the Biolog system showed identical biochemical reactions in 50 tests, while in 46 tests the utilisation reactions differed. The highest similarity of strains isolated from substrates with the same pH, as well as the most suitable biochemical tests for analysis of the phenotypic similarity of isolated strains, were confirmed when evaluating the biochemical profile using multicriterial analysis in the Canoco program. The isolates were screened for mycotoxin production by thin-layer chromatography (TLC), as well. Two of them were able to synthesise ochratoxin A, while none produced fumonisins under experimental conditions. Presence of toxic compounds in contaminated sites may affect environmental microscopic fungi and cause the genome alteration, which may result in changes of their physiology, including the production of different (secondary) metabolites, such as mycotoxins.

Impact of DNA extraction methods on 16S rRNA-based profiling of bacterial communities in cheese.

Numerous factors associated with sample preparation, DNA extraction, primer choice, sequencing platform and data analysis can affect the accuracy of 16S rRNA sequencing results. The DNA extraction method is considered critical for the success of sequencing as it can be the source of considerable variations in the analysis of the microbiome. In this study, the impact of various DNA extraction methods on the results of analysis of bacterial communities in cheese was evaluated. DNA was isolated from Mozzarella as a model cheese using optimized bead-based homogenization followed by different extraction procedures. Five commercial kits and two open-formula DNA extraction protocols were evaluated for amplicon sequencing of a 16S rRNA fragment of ~1460 bp. In addition, model cheese samples artificially contaminated by defined concentrations of Listeria monocytogenes and Escherichia coli, as representatives of Gram positive and Gram negative bacteria, were analysed. Six out of seven DNA extraction procedures were found to be able to provide amplifiable bacterial DNA suitable for 16S rRNA sequence analysis, but individual extraction procedures led to variable results. In particular, lysis supported with bead-beating led to a higher proportion of G+ bacteria in relative abundance profiles, probably because of the more efficient cell wall disruption. Artificially added bacterial species were reliably detected with a quantitative response. The results demonstrated a risk in comparing the data on bacterial communities in cheese when different DNA extraction protocols are used and highlighted the need to choose a standardized approach when comparison across multiple sequencing runs is required.

Characterization of Clinical and Carrier Streptococcus agalactiae and Prophage Contribution to the Strain Variability.

Streptococcus agalactiae (group B Streptococcus, GBS) represents a leading cause of invasive bacterial infections in newborns and is also responsible for diseases in older and immunocompromised adults. Prophages represent an important factor contributing to the genome plasticity and evolution of new strains. In the present study, prophage content was analyzed in human GBS isolates. Thirty-seven prophages were identified in genomes of 20 representative sequenced strains. On the basis of the sequence comparison, we divided the prophages into eight groups named A-H. This division also corresponded to the clustering of phage integrase, even though several different integration sites were observed in some relative prophages. Next, PCR method was used for detection of the prophages in 123 GBS strains from adult hospitalized patients and from pregnancy screening. At least one prophage was present in 105 isolates (85%). The highest prevalence was observed for prophage group A (71%) and satellite prophage group B (62%). Other groups were detected infrequently (1-6%). Prophage distribution did not differ between clinical and screening strains, but it was unevenly distributed in MLST (multi locus sequence typing) sequence types. High content of full-length and satellite prophages detected in present study implies that prophages could be beneficial for the host bacterium and could contribute to evolution of more adapted strains.

Seasonal changes of circulating 25-hydroxyvitamin D correlate with the lower gut microbiome composition in inflammatory bowel disease patients.

Higher probability of the development of Crohn's disease (CD) and ulcerative colitis (UC) as a possible consequence of the north-south gradient has been recently suggested. Living far north or south of the equator is manifested in fluctuation of vitamin D (vitD) levels depending on the season in both healthy and affected individuals. In the present study we investigate the possible link between the seasonal serum vitD level to the microbial composition of the lower gut of Inflammatory Bowel disease (IBD) patients using 16S rRNA sequencing. Decrease of serum vitD level in winter/spring season in a cohort of 35 UC patients and 39 CD patients was confirmed. Low gut microbiota composition of patients with IBD correlated with the serum level of 25(OH)D that directly coupled to seasonal variability of the sunshine in the central European countries. It is supposed to be related to increased abundance of Actinobacteria and Proteobacteria in UC and Actinobacteria, Fusobacteria, Firmicutes and Bacteroidetes in CD. In summer/autumn period, we observed a reduction in abundance of bacterial genera typical for inflammation like Eggerthella lenta, Fusobacterium spp., Bacteroides spp., Collinsella aerofaciens, Helicobacter spp., Rhodococcus spp., Faecalibacterium prausnitzii; and increased abundance of Pediococcus spp. and Clostridium spp. and of Escherichia/Shigella spp.

Characterization of Dev-CD-23823 and Dev-CT57, new Autographivirinae bacteriophages infecting Cronobacter spp.

Cronobacter spp. are opportunistic pathogenic bacteria responsible for severe infections in neonates. Powdered infant formula has been confirmed to be the source of infection in some cases. Bacteriophages offer a safe means for eliminating this pathogen. In the present study, we characterized two closely related Cronobacter-specific bacteriophages of the proposed genus "GAP227virus". The phages Dev-CD-23823 and Dev-CT57 possessed broad host specificity, as they infected 88% and 80% of the Cronobacter strains tested. Genome sequence comparisons of phages Dev-CD-23823 and Dev-CT57 showed different levels of similarity to the prototype GAP227 phage. The Dev-CT57 phage was highly similar, whereas the Dev-CD-23823 phage showed only 75% sequence identity. A phylogenic tree based on the RNA polymerase (RNAP) gene from selected representatives of the subfamily Autographivirinae confirmed the grouping of Dev-CD-23823, Dev-CT57 and GAP227 in one cluster together with phages PP2, Phi80-18 and PhiR8-01. A common conserved motif was also detected in the RNAP promoters of these phages. The functional activity of these RNAP promoters was confirmed experimentally using a promoter probe vector, and a phage-specific signal was observed; however, some cross-specificity of Dev-CD-23823 and Dev-CT57 promoters was also detected. These results will contribute to our understanding of the biology and evolution of Autographivirinae phages.

Genome analysis of lactic acid bacterial strains selected as potential starters for traditional Slovakian bryndza cheese.

Genomes of 21 strains of lactic acid bacteria isolated from Slovakian traditional cheeses were sequenced on an Illumina MiSeq platform. Subsequently, they were analysed regarding taxonomic classification, presence of genes encoding defence systems, antibiotic resistance and production of biogenic amines. Thirteen strains were found to carry genes encoding at least one bacteriocin, 18 carried genes encoding at least one restriction-modification system, all strains carried 1-6 prophages and 9 strains had CRISPR-Cas systems. CRISPR-Cas type II-A was the most common, containing 0-24 spacers. Only 10% spacers were found to be homological to known bacteriophage or plasmid sequences in databases. Two Enterococcus faecium strains and a Lactococcus lactis strain carried antibiotic resistance genes. Genes encoding for ornithine decarboxylase were detected in four strains and genes encoding for agmatine deiminase were detected in four strains. Lactobacillus paraplantarum 251 L appeared to be the most interesting strain, as it contained genes encoding for two bacteriocins, a restriction-modification system, two CRISPR-Cas systems, four prophages and no genes connected with antibiotic resistance or production of biogenic amines.

Benzalkonium chloride tolerance of Listeria monocytogenes strains isolated from a meat processing facility is related to presence of plasmid-borne bcrABC cassette.

Listeria monocytogenes is a serious foodborne pathogen capable of persisting in food processing environments. Tolerance to disinfectants used in industrial settings constitutes an important factor of Listeria survival. In the present study, the mechanism of tolerance to benzalkonium chloride (BAC) was investigated in 77 L. monocytogenes isolates from a meat facility. By PCR approach, the mdrL and lde chromosomal efflux pump genes were detected in all isolates. No isolate was positive for qacH and emrE genes. However, the bcrABC cassette was present in 17 isolates of serogroup IIa possessing the same AscI/ApaI pulsotype, the operon being localized on a plasmid. The significant relation of BAC tolerance with bcrABC presence was confirmed as all bcrABC positive isolates showed the highest minimal inhibitory concentration (MIC) values for BAC and increased sensitivity to BAC was observed after plasmid curing. No effect of the efflux pump inhibitor reserpine on BAC tolerance in bcrABC positive strains was observed in contrast to all bcrABC negative strains. Lower ethidium bromide efflux in bcrABC positive isolates compared to bcrABC negative and plasmid-cured L. monocytogenes isolates was observed. The expression of bcrABC genes was BAC-induced. The confirmed effect of bcrABC to increased BAC tolerance, coupled with its plasmid location, may be an important factor in potential dissemination of the biocide resistance among Listeria species. The understanding of molecular mechanisms of biocide tolerance should help to improve control measures to prevent further spread of L. monocytogenes in food production environments with frequent use of BAC.

Characterisation of Cronobacter strains isolated from hospitalised adult patients.

Bacteria belonging to the genus Cronobacter are opportunistic pathogens known for causing rare but serious infections in neonates, including meningitis, necrotising enterocolitis and sepsis. Cronobacter infections occur also in adult populations, however, they generally have milder manifestations and their prevalence is uncertain. In this study, the presence of Cronobacter strains from adult patients in the University Hospital in Bratislava was investigated and overall 18 confirmed isolates from 321 patients (5.3%) were recovered. No Cronobacter positive sample was detected in 215 sputum samples from outpatients. The highest occurrence of Cronobacter strains was observed from stroke patients and this may be associated with an abnormal swallowing ability. The isolated strains belonged to the species Cronobacter sakazakii and Cronobacter malonaticus. In silico genotyping (MLST, CRISPR-cas array profiling) of whole genome sequences assigned the strains to three different MLST clones. The majority (12/18) of the isolated strains were sequence type ST513 or single locus variants ST514 and ST515, thereby being members of C. sakazakii pathovar clonal complex CC4. However, according to core genome MLST analysis the ST513-ST515 strains created a unique cluster substantially different from other CC4 strains. The isolated strains were susceptible to 18 tested antibiotics. All strains possess a genomic island encoding for increased thermal tolerance. As Cronobacter strains are frequently present in dried foods of plant origin, spread of a specific clone within a hospital may be caused by food transmission and may be facilitated by its tolerance to environmental stresses such as desiccation and temperature.

Contribution of the thermotolerance genomic island to increased thermal tolerance in Cronobacter strains.

Cronobacter spp. are opportunistic pathogens associated with serious infections in neonates. Increased stress tolerance, including the thermotolerance of some Cronobacter strains, can promote their survival in production facilities and thus raise the possibility of contamination of dried infant formula which has been identified as a potential source of infection. Some Cronobacter strains contain a genomic island, which might be responsible for increased thermotolerance. By analysis of Cronobacter sequenced genomes this determinant was found to be present in only 49/73 Cronobacter sakazakii strains and in 9/14 Cronobacter malonaticus strains. The island was also found in 16/17 clinical isolates originating from two hospitals. Two configurations of the locus were detected; the first one with the size of 18 kbp containing the thrB-Q genes and a shorter version (6 kbp) harbouring only the thrBCD and thrOP genes. Strains containing the thermotolerance island survived significantly better at 58 °C comparing to a C. sakazakii isogenic mutant lacking the island and strains with the longer version of the island were 2-10 times more tolerant than those with the shortened sequence. The function of the genomic island was further confirmed by its cloning into a low-copy vector and transforming it into the isogenic mutant. Different levels of rpoS, encoding for stress-response sigma factor, expression were also associated with variability in strain thermotolerance.

Characterization of Cronobacter spp. isolated from food of plant origin and environmental samples collected from farms and from supermarkets in the Czech Republic.

The Cronobacter genus (previously known as Enterobacter sakazakii) comprises seven species (Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter muytjensii, Cronobacter turicensis, Cronobacter dublinensis, Cronobacter universalis and Cronobacter condimenti)which cause serious infections in neonates and immunocompromised people.Most of the documented outbreaks of these bacteria have been associated with consumption of contaminated powdered infant formula. The plant environment is considered to be the natural habitat of these bacteria. Therefore, a total number of 563 samples of vegetables, fruit, water and environmental swabs were collected from local farms and supermarkets in the Czech Republic and investigated for the presence of Cronobacter spp. The obtained 45 isolates (8.0%) were further characterized by phenotyping (antimicrobial resistance, capsule and pigment production) and genotyping (fusA sequencing,MLST, PCR-serotyping) methods. Most of the Cronobacter isolates (42.2%) were identified as C. sakazakii, followed by C. turicensis (31.1%), C. dublinensis (22.2%), C. malonaticus (2.2%) and C. universalis (2.2%). The 25 identified sequence types, out of which 17 were unique for only one strain, indicated a high diversity of strains. C. sakazakii sequence type 4 (ST 4), which has been associated with many cases of meningitis, was isolated only in one case. A strong association of C. turicensis and C. dublinensis with the plant environment can be deduced from our results.