BACKGROUND: Cronobacter spp. are Gram-negative, facultative-anaerobic, non-spore forming, enteric coliform bacteria, which belongs to the Enterobacteriaceae family. Cronobacter spp. are opportunistic pathogens that have brought rare but life-threatening infections such as meningitis, necrotizing enterocolitis and bloodstream infections in neonates and infants. Information on the diversity, pathogenicity and virulence of Cronobacter species obtained from various sources is still relatively scarce and fragmentary. The aim of this study was to examine and analyse different pathogenicity and virulence factors among C. sakazakii and C. malonaticus strains isolated from clinical samples. METHODS: The thirty-six clinical Cronobacter strains have been used in this study. This bacterial collection consists of 25 strains of C. sakazakii and 11 strains of C. malonaticus, isolated from different clinical materials. Seven genes (ompA, inv, sip, aut, hly, fliC, cpa) were amplified by PCR. Moreover, the motility and the ability of these strains to adhere and invade human colorectal adenocarcinoma (HT-29) and mouse neuroblastoma (N1E-115) cell lines were investigated. RESULTS: Our results showed that all tested strains were able to adhere to both used cell lines, HT-29 and N1E-115 cells. The invasion assay showed that 66.7% (24/36) of isolates were able to invade N1-E115 cells while 83% (30/36) of isolates were able to invade HT-29 cells. On the average, 68% of the C. sakazakii strains exhibited seven virulence factors and only 18% in C. malonaticus. All strains amplified ompA and fliC genes. The other genes were detected as follow: sip 97% (35/36), hlyA 92% (33/36), aut 94% (34/36), cpa 67% (24/36), and inv 69% (25/36). CONCLUSIONS: C. sakazakii and C malonaticus strains demonstrate the diversity of the virulence factors present among these pathogens. It is necessary to permanently monitor the hospital environment to appropriately treat and resolve cases associated with disease. Furthermore, in-depth knowledge is needed about the source and transmission vehicles of pathogens in hospitals to adopt pertinent prevention measures.

• Klíčová slova
• Adherence, Clinical strains, Cronobacter malonaticus, Cronobacter sakazakii, Invasion, Virulence factors,
• MeSH
• bakteriální adheze MeSH
• bakteriologické techniky MeSH
• buněčné linie MeSH
• Cronobacter genetika   izolace a purifikace   patogenita   MeSH
• cytologické techniky MeSH
• endocytóza MeSH
• enterobakteriální infekce mikrobiologie   MeSH
• epitelové buňky mikrobiologie   MeSH
• faktory virulence genetika   MeSH
• lidé MeSH
• myši MeSH
• nemoci přenášené potravou mikrobiologie   MeSH
• polymerázová řetězová reakce MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• faktory virulence MeSH

AIMS: To study the occurrence of Cronobacter spp. in foods and to investigate the phenotypic properties of the strains isolated. METHODS AND RESULTS: A total of 53 strains of Cronobacter spp. isolated from 399 food samples were identified using conventional biochemical methods and MALDI-TOF mass spectrometry. Foods of plant origin were the most frequently contaminated samples. No Cronobacter spp. were found in infant milk formula, wheat-based infant food, pasteurized and raw cow milk, mincemeat, chicken, chickpea and potato dumpling powder. The individual species were identified as Cronobacter sakazakii (54·7%), Cronobacter malonaticus (28·4%), Cronobacter dublinensis (7·5%), Cronobacter muytjensii (7·5%) and Cronobacter turicensis (1·9%). Cronobacter sakazakii and C. malonaticus belong to biotype 1, 2, 2a, 3, 4 and 5, 5a, respectively. Cronobacter dublinensis strains were subdivided into biotypes 6 and 12. All strains were resistant to erythromycin and two of them were resistant to both erythromycin and tetracycline. CONCLUSIONS: Cronobacter spp. were isolated from various food samples pre-eminently of plant origin and dried food ingredients. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings will increase and detail our knowledge of the presence and diversity of Cronobacter spp. in foods.

• MeSH
• Cronobacter sakazakii izolace a purifikace   MeSH
• Cronobacter klasifikace   izolace a purifikace   MeSH
• DNA bakterií MeSH
• enterobakteriální infekce epidemiologie   mikrobiologie   MeSH
• lidé MeSH
• potravinářská mikrobiologie * MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika epidemiologie   MeSH
• Názvy látek
• DNA bakterií MeSH

Cronobacter spp. are bacterial pathogens that affect children and immunocompromised adults. In this study, we used multilocus sequence typing (MLST) to determine sequence types (STs) in 11 Cronobacter spp. strains isolated from retail foods, 29 strains from dust samples obtained from vacuum cleaners, and 4 clinical isolates. Using biochemical tests, species-specific polymerase chain reaction, and MLST analysis, 36 strains were identified as Cronobacter sakazakii, and 6 were identified as Cronobacter malonaticus. In addition, one strain that originated from retail food and one from a dust sample from a vacuum cleaner were identified on the basis of MLST analysis as Cronobacter dublinensis and Cronobacter turicensis, respectively. Cronobacter spp. strains isolated from the retail foods were assigned to eight different MLST sequence types, seven of which were newly identified. The strains isolated from the dust samples were assigned to 7 known STs and 14 unknown STs. Three clinical isolates and one household dust isolate were assigned to ST4, which is the predominant ST associated with neonatal meningitis. One clinical isolate was classified based on MLST analysis as Cronobacter malonaticus and belonged to an as-yet-unknown ST. Three strains isolated from the household dust samples were assigned to ST1, which is another clinically significant ST. It can be concluded that Cronobacter spp. strains of different origin are genetically quite variable. The recovery of C. sakazakii strains belonging to ST1 and ST4 from the dust samples suggests the possibility that contamination could occur during food preparation. All of the novel STs and alleles for C. sakazakii, C. malonaticus, C. dublinensis, and C. turicensis determined in this study were deposited in the Cronobacter MLST database available online ( http://pubmlst.org/cronobacter/).

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• Cronobacter sakazakii klasifikace   izolace a purifikace   metabolismus   MeSH
• Cronobacter klasifikace   izolace a purifikace   metabolismus   MeSH
• enterobakteriální infekce mikrobiologie   MeSH
• fylogeneze MeSH
• lidé MeSH
• mikrobiologie životního prostředí * MeSH
• molekulární typizace MeSH
• multilokusová sekvenční typizace MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• potravinářská mikrobiologie * MeSH
• prach * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH
• Názvy látek
• bakteriální proteiny MeSH
• prach * MeSH

BACKGROUND: Until recently, members of the Cronobacter genus (formerly known as Enterobacter sakazakii) were a relatively unknown cause of nosocomial infections. However, their association with infant infections, particularly through the consumption of contaminated reconstituted infant formula in neonatal intensive care units, has resulted in international efforts to improve neonatal health care. AIM: To investigate current understanding of this emergent group of bacterial pathogens and the steps taken to reduce neonatal infection. METHODS: A literature review was undertaken to determine current knowledge of the Cronobacter genus with respect to recent taxonomic revisions, sources and clinical relevance. FINDINGS: The majority of severe neonatal meningitis infections are associated with one of the 10 Cronobacter spp., the clonal complex known as C. sakazakii sequence type 4. International efforts by the Food and Agriculture Organization-World Health Organization (WHO) to reduce the risk of neonatal infection by this organism have resulted in improved microbiological safety of powdered infant formula (PIF), but revised guidelines for feeding practices have been problematic. In addition, the majority of infections occur in the adult population and the sources are unknown. CONCLUSION: International improvements in the microbiological safety of PIF and advice on feeding practices have focused on improving neonatal health care following the heightened awareness of Cronobacter infections in this particular age group. These measures are also likely to reduce neonatal exposure to other opportunistic bacterial pathogens, but a number of unresolved issues remain with respect to the practicalities of feeding premature neonates safely while following WHO advice.

• Klíčová slova
• Cronobacter, Healthcare infections,
• MeSH
• Cronobacter izolace a purifikace   MeSH
• enterobakteriální infekce epidemiologie   mikrobiologie   MeSH
• infekce spojené se zdravotní péčí epidemiologie   mikrobiologie   MeSH
• kojenec MeSH
• lidé MeSH
• meningitida bakteriální epidemiologie   mikrobiologie   MeSH
• náhražky mateřského mléka MeSH
• nemoci přenášené potravou epidemiologie   mikrobiologie   MeSH
• novorozenec MeSH
• objevující se infekční nemoci epidemiologie   mikrobiologie   MeSH
• zdravotnická zařízení MeSH
• Check Tag
• kojenec MeSH
• lidé MeSH
• novorozenec MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Members of the genus Cronobacter are responsible for severe infections in infants and immunosuppressed individuals. Although several virulence factors have been described, many proteins involved in the pathogenesis of such infections have not yet been mapped. This study is the first to fractionate Cronobacter sakazakii cells into outer membrane, inner membrane, periplasmic, and cytosolic fractions as the basis for improved proteome mapping. A novel method was designed to prepare the fractionated samples for protein identification. The identification was performed via one-dimensional electrophoresis-liquid chromatography electrospray ionization tandem mass spectrometry. To determine the subcellular localization of the identified proteins, we developed a novel Python-based script (Subcelloc) that combines three web-based tools, PSORTb 3.0.2, CELLO 2.5, and UniProtKB. Applying this approach enabled us to identify 1,243 C. sakazakii proteins, which constitutes 28% of all predicted proteins and 49% of all theoretically expressed outer membrane proteins. These results represent a significant improvement on previous attempts to map the C. sakazakii proteome and could provide a major step forward in the identification of Cronobacter virulence factors. IMPORTANCECronobacter spp. are opportunistic pathogens that can cause rare and, in many cases, life-threatening infections, such as meningitis, necrotizing enterocolitis, and sepsis. Such infections are mainly linked to the consumption of contaminated powdered infant formula, with Cronobacter sakazakii clonal complex 4 considered the most frequent agent of serious neonatal infection. However, the pathogenesis of diseases caused by these bacteria remains unclear; in particular, the proteins involved throughout the process have not yet been mapped. To help address this, we present an improved method for proteome mapping that emphasizes the isolation and identification of membrane proteins. Specific focus was placed on the identification of the outer membrane proteins, which, being exposed to the surface of the bacterium, directly participate in host-pathogen interaction.

• Klíčová slova
• Cronobacter, cell subfractionation, food pathogen, membrane proteomics,
• MeSH
• Cronobacter sakazakii * MeSH
• Cronobacter * MeSH
• faktory virulence metabolismus   MeSH
• kojenec MeSH
• lidé MeSH
• náhražky mateřského mléka mikrobiologie   MeSH
• novorozenec MeSH
• potravinářská mikrobiologie MeSH
• proteiny vnější bakteriální membrány metabolismus   MeSH
• proteom metabolismus   MeSH
• proteomika MeSH
• Check Tag
• kojenec MeSH
• lidé MeSH
• novorozenec MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• faktory virulence MeSH
• proteiny vnější bakteriální membrány MeSH
• proteom MeSH

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.

• Klíčová slova
• Cronobacter spp., Heat stress, Thermotolerance, Thermotolerance island, rpoS,
• MeSH
• bakteriální geny MeSH
• biologická adaptace genetika   MeSH
• Cronobacter klasifikace   genetika   metabolismus   MeSH
• enterobakteriální infekce mikrobiologie   MeSH
• genom bakteriální * MeSH
• genomové ostrovy * MeSH
• infekce spojené se zdravotní péčí MeSH
• klonování DNA MeSH
• lidé MeSH
• multilokusová sekvenční typizace MeSH
• plazmidy genetika   MeSH
• pořadí genů MeSH
• reakce na tepelný šok genetika   MeSH
• teplota * MeSH
• testy genetické komplementace MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Cronobacter malonaticus is a member of the genus Cronobacter which is considered an opportunistic pathogen. The significance of C. malonaticus has recently increased since it was documented to be involved in several serious neonatal infections. However, the virulence factors of C. malonaticus including their ability to adhere, invade and overcome host barriers have not been studied before. Unlike previous Cronobacter research, this study is mainly focused on C. malonaticus and is aimed to investigate its virulence characteristics that enable this species to cause adult and neonatal infections. Altogether, 20 strains were included in this study (19 clinical and one environmental strain). Our data showed that the clinical C. malonaticus has an ability to adhere and invade Caco-2, HBMEC, A549 and T24 cell lines. Moreover, the result showed that certain strains of C. malonaticus (including 1827 and 2018) were able to persist well in macrophages. However, ST7 strains 1827 and 2018 proved to be the most invasive strains among all used strains. The CDC strain 1569 (ST307) which was isolated from the blood of a fatal neonatal case showed also significant results in this study as it was able to invade all used human cells and survive and replicate within microphages. Finally, the findings of this study confirm the potential ability of C. malonaticus to cause serious infections in neonates or adults such as necrotising enterocolitis, meningitis, bacteraemia, pneumonia and urinary tract infection.

• Klíčová slova
• Adherence, Cronobacter malonaticus, Invasion, Macrophages, Pathogenicity,
• MeSH
• bakteriální adheze * MeSH
• biologické modely MeSH
• buněčné linie MeSH
• Cronobacter izolace a purifikace   patogenita   MeSH
• endocytóza * MeSH
• enterobakteriální infekce mikrobiologie   MeSH
• epitelové buňky mikrobiologie   MeSH
• lidé MeSH
• makrofágy mikrobiologie   MeSH
• virulence MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Cronobacter species are Gram-negative opportunistic pathogens that can cause serious infections in neonates. The lipopolysaccharides (LPSs) that form part of the outer membrane of such bacteria are possibly related to the virulence of particular bacterial strains. However, currently there is no clear overview of O-antigen diversity within the various Cronobacter strains and links with virulence. In this study, we tested a total of 82 strains, covering each of the Cronobacter species. The nucleotide variability of the O-antigen gene cluster was determined by restriction fragment length polymorphism (RFLP) analysis. As a result, the 82 strains were distributed into 11 previously published serotypes and 6 new serotypes, each defined by its characteristic restriction profile. These new serotypes were confirmed using genomic analysis of strains available in public databases: GenBank and PubMLST Cronobacter. Laboratory strains were then tested using the current serotype-specific PCR probes. The results show that the current PCR probes did not always correspond to genomic O-antigen gene cluster variation. In addition, we analyzed the LPS phenotype of the reference strains of all distinguishable serotypes. The identified serotypes were compared with data from the literature and the MLST database (www.pubmlst.org/cronobacter/). Based on the findings, we systematically classified a total of 24 serotypes for the Cronobacter genus. Moreover, we evaluated the clinical history of these strains and show that Cronobacter sakazakii O2, O1, and O4, C. turicensis O1, and C. malonaticus O2 serotypes are particularly predominant in clinical cases.

• MeSH
• Cronobacter chemie   klasifikace   genetika   izolace a purifikace   MeSH
• DNA bakterií genetika   MeSH
• DNA fingerprinting MeSH
• enterobakteriální infekce mikrobiologie   MeSH
• genetická variace * MeSH
• genotyp MeSH
• lidé MeSH
• multigenová rodina MeSH
• multilokusová sekvenční typizace MeSH
• O-antigeny analýza   genetika   MeSH
• oligonukleotidové sondy MeSH
• polymerázová řetězová reakce MeSH
• polymorfismus délky restrikčních fragmentů MeSH
• séroskupina MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA bakterií MeSH
• O-antigeny MeSH
• oligonukleotidové sondy MeSH

In the last decade, strains of the genera Franconibacter and Siccibacter have been misclassified as first Enterobacter and later Cronobacter Because Cronobacter is a serious foodborne pathogen that affects premature neonates and elderly individuals, such misidentification may not only falsify epidemiological statistics but also lead to tests of powdered infant formula or other foods giving false results. Currently, the main ways of identifying Franconibacter and Siccibacter strains are by biochemical testing or by sequencing of the fusA gene as part of Cronobacter multilocus sequence typing (MLST), but in relation to these strains the former is generally highly difficult and unreliable while the latter remains expensive. To address this, we developed a fast, simple, and most importantly, reliable method for Franconibacter and Siccibacter identification based on intact-cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Our method integrates the following steps: data preprocessing using mMass software; principal-component analysis (PCA) for the selection of mass spectrum fingerprints of Franconibacter and Siccibacter strains; optimization of the Biotyper database settings for the creation of main spectrum projections (MSPs). This methodology enabled us to create an in-house MALDI MS database that extends the current MALDI Biotyper database by including Franconibacter and Siccibacter strains. Finally, we verified our approach using seven previously unclassified strains, all of which were correctly identified, thereby validating our method.IMPORTANCE We show that the majority of methods currently used for the identification of Franconibacter and Siccibacter bacteria are not able to properly distinguish these strains from those of Cronobacter While sequencing of the fusA gene as part of Cronobacter MLST remains the most reliable such method, it is highly expensive and time-consuming. Here, we demonstrate a cost-effective and reliable alternative that correctly distinguishes between Franconibacter, Siccibacter, and Cronobacter bacteria and identifies Franconibacter and Siccibacter at the species level. Using intact-cell MALDI-TOF MS, we extend the current MALDI Biotyper database with 11 Franconibacter and Siccibacter MSPs. In addition, the use of our approach is likely to lead to a more reliable identification scheme for Franconibacter and Siccibacter strains and, consequently, a more trustworthy epidemiological picture of their involvement in disease.

• Klíčová slova
• Franconibacter, Siccibacter, intact-cell MALDI-TOF mass spectrometry, “pseudo-Cronobacter”,
• MeSH
• bakteriální proteiny genetika   MeSH
• Cronobacter chemie   klasifikace   genetika   izolace a purifikace   MeSH
• Enterobacteriaceae chemie   klasifikace   genetika   izolace a purifikace   MeSH
• enterobakteriální infekce mikrobiologie   MeSH
• fylogeneze MeSH
• lidé MeSH
• multilokusová sekvenční typizace metody   MeSH
• spektrometrie hmotnostní - ionizace laserem za účasti matrice metody   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• Názvy látek
• bakteriální proteiny MeSH

Cronobacter spp. have been recognized as causative agents of various severe infections in pre-term or full-term infants as well as elderly adults suffering from serious underlying disease or malignancy. A surveillance study was designed to identify antibiotic resistance among clinical Cronobacter spp. strains, which were isolated from patients of two hospitals between May 2007 and August 2013. Altogether, 52 Cronobacter spp. isolates were analyzed. Although MALDI-TOF mass spectrometry recognized all Cronobacter sakazakii and Cronobacter malonaticus strains, it could not identify Cronobacter muytjensii strain. Nevertheless, all strains were identified as Cronobacter spp. using multilocus sequence typing (MLST). Strains were tested against 17 types of antibiotics, using the standard microdilution method according to the 2018 European Committee on Antimicrobial Susceptibility Testing criteria. Three Cronobacter species were identified as C. sakazakii (n = 33), C. malonaticus (n = 18), and C. muytjensii (n = 1); all isolates were susceptible to all tested antibiotics. All strains were PCR-negative for bla TEM, bla SHV, and bla CTX-M β-lactamase genes, as well. Even though the results of this study showed that Cronobacter spp. isolates were pan-susceptible, continued antibiotic resistance surveillance is warranted. Cronobacter spp. have been recognized as causative agents of various severe infections in pre-term or full-term infants as well as elderly adults suffering from serious underlying disease or malignancy. A surveillance study was designed to identify antibiotic resistance among clinical Cronobacter spp. strains, which were isolated from patients of two hospitals between May 2007 and August 2013. Altogether, 52 Cronobacter spp. isolates were analyzed. Although MALDI-TOF mass spectrometry recognized all Cronobacter sakazakii and Cronobacter malonaticus strains, it could not identify Cronobacter muytjensii strain. Nevertheless, all strains were identified as Cronobacter spp. using multilocus sequence typing (MLST). Strains were tested against 17 types of antibiotics, using the standard microdilution method according to the 2018 European Committee on Antimicrobial Susceptibility Testing criteria. Three Cronobacter species were identified as C. sakazakii (n = 33), C. malonaticus (n = 18), and C. muytjensii (n = 1); all isolates were susceptible to all tested antibiotics. All strains were PCR-negative for bla TEM, bla SHV, and bla CTX-M β-lactamase genes, as well. Even though the results of this study showed that Cronobacter spp. isolates were pan-susceptible, continued antibiotic resistance surveillance is warranted.

• MeSH
• antibakteriální látky farmakologie   MeSH
• Cronobacter sakazakii klasifikace   účinky léků   genetika   MeSH
• Cronobacter klasifikace   účinky léků   MeSH
• genotyp MeSH
• gramnegativní bakteriální infekce mikrobiologie   MeSH
• lidé MeSH
• mikrobiální testy citlivosti MeSH
• mnohočetná bakteriální léková rezistence MeSH
• multilokusová sekvenční typizace MeSH
• polymerázová řetězová reakce MeSH
• techniky typizace bakterií MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Geografické názvy
• Polsko MeSH
• Názvy látek
• antibakteriální látky MeSH