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 blaTEM, blaSHV, and blaCTX-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 blaTEM, blaSHV, and blaCTX-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
Bacteria from the genus Cronobacter are opportunistic foodborne pathogens that can cause severe infections. More rapid, cost-effective and reliable methods are still required for the species identification of Cronobacter spp. In this study, we present a novel PCR-RFLP-based method that uses a newly designed pair of primers for the PCR-amplification of a partial rpoB gene sequence (1635 bp). The amplified products of DNA from 80 Cronobacter strains were separately digested with three restriction endonucleases (Csp6I, HinP1I, MboI). Using the obtained restriction patterns, a PCR-RFLP identification system was created to enable differentiation between all seven currently-known Cronobacter species. The functionality of our method was successfully verified on real food samples. Moreover, the relationships between the Cronobacter species were determined via a phylogenetic tree created from the RFLP patterns.
- MeSH
- bakteriální geny * MeSH
- Cronobacter klasifikace genetika MeSH
- DNA bakterií MeSH
- DNA primery MeSH
- DNA řízené RNA-polymerasy genetika MeSH
- fylogeneze MeSH
- kontaminace potravin analýza MeSH
- polymerázová řetězová reakce ekonomika metody MeSH
- polymorfismus délky restrikčních fragmentů * MeSH
- potravinářská mikrobiologie * MeSH
- restrikční endonukleasy typu II metabolismus MeSH
- sekvenční analýza DNA MeSH
- Publikační typ
- časopisecké články 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.
- 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
- práce podpořená grantem MeSH
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.
- MeSH
- antibakteriální látky farmakologie MeSH
- Cronobacter klasifikace genetika izolace a purifikace MeSH
- genotyp MeSH
- kojenec MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- multilokusová sekvenční typizace MeSH
- náhražky mateřského mléka mikrobiologie MeSH
- novorozenec MeSH
- ovoce mikrobiologie MeSH
- polymerázová řetězová reakce MeSH
- potravinářská mikrobiologie * MeSH
- techniky typizace bakterií MeSH
- zelenina mikrobiologie MeSH
- zemědělství MeSH
- Check Tag
- kojenec MeSH
- lidé MeSH
- novorozenec MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika 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.
- 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 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
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
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 MeSH
