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Article

The Genotyping Diversity and Hemolytic Activity of Cronobacter spp. Isolated from Plant-Based Food Products in Poland

by
Monika Garbowska
1,*,
Anna Berthold-Pluta
1,
Lidia Stasiak-Różańska
1,
Antoni Pluta
1,
Stephen Forsythe
2 and
Ilona Stefańska
3
1
Department of Technology and Food Assessment, Division of Milk Technology, Institute of Food Sciences, Warsaw University of Life Sciences—SGGW, 02-787 Warsaw, Poland
2
FoodMicrobe.com, Keyworth, Nottinghamshire NG12 5GY, UK
3
Department of Preclinical Sciences, Institute of Veterinary Medicine, Warsaw University of Life Sciences—SGGW, 02-787 Warsaw, Poland
*
Author to whom correspondence should be addressed.
Foods 2023, 12(20), 3873; https://doi.org/10.3390/foods12203873
Submission received: 21 September 2023 / Revised: 15 October 2023 / Accepted: 19 October 2023 / Published: 23 October 2023
(This article belongs to the Special Issue Risk Identification, Rapid Detection, Prevention and Control of Foodborne Pathogenic Microorganisms)
Review Reports Versions Notes

Abstract

:
The present study aimed to determine the genotyping diversity and hemolytic properties of 24 strains of Cronobacter spp. (15 Cronobacter sakazakii, 6 Cronobacter malonaticus, 2 Cronobacter turicensis, and 1 Cronobacter condimenti) isolated from commercial ready-to-eat leaf vegetables, sprouts, nuts, and dried fruits. The multilocus sequence typing (MLST) method was used to determine the sequence types (ST) and clonal complexes (CC) of these strains. The study demonstrated the high genotypic diversity of the Cronobacter genus bacteria isolated from plant-based foods. Five novel sequence types (804, 805, 806, 807, and 808) and the presence of novel alleles in the ppsA, gltB, gyrB, and infB loci were detected. In total, 16 of the 24 strains were assigned to the sequence types ST99, ST258, ST17, ST648, ST21, ST494, and ST98. One C. sakazakii strain (s12) isolated from alfalfa sprouts was assigned to the clonal complex CC4, which encompasses strains often associated with severe infections leading to meningitis in infants. In addition, 87.5% and 16.7% of the Cronobacter spp. strains showed β-hemolysis of equine and sheep red blood cells, respectively. The presence of the pathogenic species C. sakazakii, C. malonaticus, and C. turicensis in ready-to-eat plant-derived food products shows they are potential sources of infection, especially to those with compromised immunity, which substantiates their further multi-faceted characterization. The significance of this study may prove useful not only in epidemiological investigations, but also in assessing the risk of infections caused by the presence of Cronobacter.

1. Introduction

Cronobacter bacteria are motile, facultatively anaerobic, Gram-negative rods of the family Enterobacteriaceae. The history of this genus is relatively short, as it was only in 2007 that organisms previously classified on the basis of their phenotypic features as Enterobacter sakazakii (and even earlier as yellow-pigment-producing Enterobacter cloacae) were assigned to the new genus Cronobacter, then composed of four species. However, as knowledge of the diversity of the genus improved, it was expanded to the currently agreed composition of seven species. Of the seven Cronobacter species, C. sakazakii, C. malonaticus, and C. turicensis have been recognized as important human pathogens resulting in opportunistic infections [1,2]. These bacteria can cause severe, invasive infections in preterm infants, low-birth-weight infants, neonates hospitalized in intensive care, and immunocompromised infants, as well as immunocompromised adults and the elderly [2,3]. Infections caused by Cronobacter spp. in infants are associated with a high mortality rate, ranging from 40 to 80% [4,5], with frequent neurological complications observed in 20% of convalescents [6,7]. Cronobacter bacteria cause bacteremia, necrotizing enterocolitis, meningitis, and sepsis in neonates and infants, and wound and urinary tract infections in adults [7,8]. In 2002, the International Commission for Microbiological Specification for Foods (ICMSF) placed Cronobacter (then known as Enterobacter sakazakii) in group I.B., which covers pathogenic bacteria that are a “severe hazard for restricted populations, life threatening or substantial chronic sequelae or long duration” [9]. The number of Cronobacter infections reported in the literature is relatively low and is most likely underestimated, as it is generally not a mandated notifiable disease [7,10].
The prevalence of Cronobacter spp. in various food products has been confirmed in investigations conducted across many countries [11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26], with C. sakazakii being the most frequently isolated species. The presence of pathogenic C. sakazakii, C. malonaticus, and C. turicensis in ready-to-eat food products (RTE) makes them putative sources of infections [16,19,20,21,22,23,27]. Cronobacter spp. have been isolated from a range of plant-origin foods, including ready-to-eat vegetables, cereals, and nuts [19,23,28]. Productive sources of Cronobacter strains are fresh or dried herbs and spices [29]. Although the bacterium has been isolated from various plant-based food products, no foodborne infections have been reported. The majority of Cronobacter infections occur in the adult population, but are less severe. Cases of Cronobacter infection in all age groups are probably under-reported for a number of reasons, such as misidentification as Enterobacter cloacae [29]. To date, very little is known about the genotyping diversity and hemolytic activity possessed by such plant-origin Cronobacter. The characterization of strains from plant-based foods with genomic features similar to clinically relevant strains of different Cronobacter spp. sequence types (STs) suggests that these foods may serve as potential vehicles for the transmission of opportunistic pathogens. Therefore, it is necessary to understand the genotyping diversity of Cronobacter spp. associated with plant-based foods to improve food safety.
As an intestinal pathogen, the main route of Cronobacter entry into the human body is through the consumption of contaminated food. These pathogens show a high tolerance to stressful environments, being one of the most heat-tolerant members of the Enterobacteriaceae family [5], extremely resistant to low water activity of the environment [30,31], able to tolerate acidic conditions as low as pH 4.2 [7] and produce biofilms that increase their survival under food production conditions [32]. After entering the host, the infection strategy consists of the following stages: (i) the colonization of the mucosa (intestinal, respiratory, or the urinary tract epithelia); (ii) the circumvention, subversion, and exploitation of host defenses; (iii) systemic spread and multiplication (within the blood or phagocytes); and (iv) host damage (through the expression of toxins and/or damage due to the proinflammatory modulation of the human immune system) [8].
Various hemolysins and hemolysis-related genes have been reported in Cronobacter genomes by Joseph et al. [4]. The hemolysin gene (hly) was present in all the genomes, with the only exceptions being C. sakazakii strain 701 and C. malonaticus strain 507. Most of the strains had two copies of the hemolysin gene and the hemolysin activator protein precursor gene [4]. Cruz et al. [33] identified the hemolysin gene (hly) as a hemolysin III homolog (COG1272), and Jang et al. [34], studying 390 strains, showed that all seven species tested possessed the hemolysin III COG1272 gene homolog. Additionally, three other hemolysin genes were identified, including genes encoding the cystathionine β-synthase (CBS) domain containing hemolysin, putative hemolysin, and 21 kDa hemolysin [34]. Umeda et al. [35] reported that all analyzed Cronobacter strains exhibited β-hemolytic activity against guinea pig, horse, and rabbit erythrocytes and that 92.9% of the strains were capable of the α-hemolysis of sheep erythrocytes. The further characterization of individual Cronobacter species, including strains occurring in food, is, however, needed to establish their hemolytic capacity. Results related to the hemolytic activity of Cronobacter spp. could extend the knowledge in this field, enabling the assessment of whether other closely related species commonly misidentified as Cronobacter (like Franconibacter helveticus, Franconibacter pulveris, Siccibacter colletis, and Siccibacter turicensis) differ in their hemolytic phenotypes from the Cronobacter genus species [36].
Multi-locus sequence typing (MLST) is a genetic method recommended for the characterization, differentiation, and typing of many microorganisms. Conventional MLST genotypes bacterial strains according to loci (commonly ~500 nucleotides) for seven housekeeping genes, and it enables the recognition of the sequence types (ST) and clonal complexes (CC) of the tested Cronobacter strains and thus the assessment of their genetic diversity and clinical significance [4,37].
The genetic basis of the virulence of Cronobacter spp. strains has not been fully elucidated, but some sequence types have been found to be associated with specific types of infection [37]. Infant meningitis due to Cronobacter spp. is more frequently caused by strains of C. sakazakii belonging to the CC4 clonal complex (especially ST4), whereas C. sakazakii ST12 is strongly associated with cases of necrotizing enterocolitis in infants. Infections in children and adults are mainly caused by the clonal complexes of C. sakazakii CC4 and C. malonaticus CC7, respectively [2,37].
Since the severity of infections due to Cronobacter spp. may be related to the genotype of the strain that caused the infection, it is important to determine the STs of isolates from both clinical and food sources. Therefore, the present study aimed to identify the sequence types of Cronobacter spp. strains isolated from commercial ready-to-eat leaf vegetables, sprouts, nuts, and dried fruits. The genotypic analysis of the strains could prove useful not only in epidemiological investigations, but also in their risk assessments.

2. Materials and Methods

2.1. Materials

The study was conducted with 24 strains of Cronobacter spp., including 15 strains of C. sakazakii (9n, 10n, 11m, s12, s14, s21, s22, s41, s42, s44, s45, s47, s48, lv25, and lv27), 6 C. malonaticus (5n, 6n, 7n, 8n, 12m, and lv31), 2 C. turicensis (1n and lv54), and 1 C. condimenti (s37). These isolates were from the bacterial collection of the Division of Milk Technology, Warsaw University of Life Sciences, Poland. The isolation of these strains from plant-derived food products, including ready-to-eat leaf vegetables, sprouts, nuts, and dried fruits, was described in our previous studies (Table 1) [19,23].
The Cronobacter spp. strains were stored frozen on Tryptone Soy Broth (TSB) (Oxoid Argenta, Poznań, Poland) with a 10% glycerol addition at a temperature of −40 °C. They were recovered from the frozen state by transferring 0.1 mL of a defrosted culture onto TSB of a given strain to 10 mL of sterile TSB, with incubation at a temperature of 35 °C for 24 h. Afterwards, each strain was inoculated onto Tryptone Soy Agar (TSA) medium (Oxoid Argenta, Poznań, Poland).

2.2. MLST Analysis

The MLST typing followed the methodology of Baldwin et al. [38]. The seven loci analyzed were glutaminyl tRNA synthetase gene (glnS), glutamate synthase large subunit gene (gltB), ATP synthase beta chain (atpD), DNA gyrase beta subunit (gyrB), phosphoenolpyruvate synthase A (ppsA), the gene encoding the translation initiation factor IF-2 (infB), and the gene encoding the translocase protein of the elongation factor EF-G (fusA). The gene fragments were amplified using primers and PCR conditions according to the protocol available in the Cronobacter MLST database https://pubmlst.org/organisms/cronobacter-spp/primers (accessed on 25 May 2020). The genomic DNA was isolated using the GenElute Bacterial Genomic DNA Kit (Sigma Aldrich, Poznań, Poland), according to the manufacturer’s instructions. The PCR and sequencing primers were synthesized at Eurofins Genomics (Ebersberg, Germany). The PCR was performed using the Phusion High-Fidelity PCR Master Mix with the HF buffer kit (ThermoFisher Scientific, Poland), in a total volume of 50 µL containing 20–40 ng of template DNA and 10 pmol of each primer. The polymerase used in the study was characterized by a 50-fold lower error insertion frequency compared to the standard Taq polymerase. The amplification products were purified using the GenElute PCR Clean-Up Kit (Sigma-Aldrich) or EXOSAP (ThermoFisher Scientific). The purified amplicons, obtained using standard and alternative primers (https://pubmlst.org/organisms/cronobacter-spp/primers and https://pubmlst.org/static/organisms/cronobacter-spp/Cronobacter_alternative_primers.pdf) (accessed on 20 June 2020), were sequenced in both directions (Eurofins Genomics). Afterwards, the obtained nucleotide sequences were compared with the sequences deposited in the Cronobacter MLST database (https://pubmlst.org/cronobacter/) (accessed on 17 October 2020). Alleles were determined for the seven loci, which identified their sequence type and clonal complex. Novel alleles and sequence types were assigned by the MLST database curator, Prof. Stephen Forsythe.

2.3. Hemolytic activity of Cronobacter spp. Strains

Hemolytic capability was determined on Columbia agar plates with a 5% addition of horse blood (COH) or sheep blood (COS) (Biomerieux, Warsaw Poland). Various hemolytic types were identified using the following reference strains: Streptococcus pneumoniae ATCC 6305, Bacillus cereus ATCC 14579, and Listeria innocua ATCC 33090 (Oxoid Argenta, Poznań Poland).
Single colonies of Cronobacter bacteria grown on TSA medium were transferred using a sterile, disposable loop to a blood agar plates and incubated at 37 °C. The hemolysis zones on the plates were measured after 96 h of incubation. The appearance of a green zone around the colony was recorded as α-hemolysis, a transparent zone around the colony was recorded as β-hemolysis, and no changes on the plate was defined as γ-hemolysis [39].

3. Results and Discussion

3.1. MLST Analysis

The MLST analysis results confirmed the earlier identification of all the tested strains obtained using RFLP-PCR: 15 strains belonged to the C. sakazakii species, 6 strains to the C. malonaticus species, 2 strains to the C. turicensis species, and one strain to the C. condimenti species. Interestingly, this profiling was consistent with the results of earlier intraspecies strain differentiation conducted with the RAPD-PCR method, indicating that the strains sharing the same RAPD pattern concurred with the same sequence type [19,23].
The present study showed that the 24 strains of Cronobacter spp. isolated from plant-derived foods (ready-to-eat leaf vegetables, sprouts, nuts, and dried fruits) belonged to 14 different sequence types (ST) (Table 2).
The main STs identified were C. sakazakii ST99 (40%, 6/15), which was isolated from various sprouts, and C. malonaticus ST258 (66.7%, 4/6), which was isolated from various nuts. Interestingly, out of the 24 strains, six novel sequence types were defined: ST806 and ST808 in the C. turicensis 1n and lv54 strains; ST805 and ST807 in the C. malonaticus 12m and lv31 strains; and ST804 in the C. sakazakii s41 and s42 strains. In the case of the C. malonaticus 12m strain, the analyses showed the presence of a novel allele in the ppsA locus and the closest match to the 355 allele with four differences (A231G, G291A, T387G, and T438C). The C. turicensis lv54 strain showed novel alleles in as many as four loci, i.e., gltB, gyrB, infB, and ppsA. The sequencing chromatograms indicates as the closest match pps159 (C48T, C57T, C405G, and T459C), gltB64 (A252G, T351C, and T480C), infB80 (C21G), and gyrB31 (C96T and T138C). The remaining detected sequence types included: ST4, ST17, ST648, ST21, ST494 (C. sakazakii), and ST98 (C. condimenti). Among the 15 C. sakazakii isolates, 4 (s12, 9n, 10m, and 11m,) had STs: ST4, ST17, and ST494, assigned to isolates from clinical sources in the PubMLST Cronobacter database (accessed: 10 May 2023).
It is notable that C. sakazakii strains belonging to the sequence type ST4 and the clonal complex CC4 are often associated with severe infections leading to meningitis in neonates, children, and adults. These strains have been isolated from milk powder, food products, ice creams, and powdered infant formulas [2,37]. In the present study, the only ST4 strain was C. sakazakii s12 (6.6%, 1/15), which was isolated from alfalfa sprouts. Wang et al. [40] analyzed 84 Cronobacter spp. isolates obtained from foods imported to Beijing in 2006–2015 and identified mainly the pathogenic sequence types ST1 and ST4 in the case of 31.67% (19/60) and 21.67% (13/60) of C. sakazakii strains, respectively, as well as the ST7 type in 70.59% (12/17) of C. malonaticus strains. The sequence type ST1 belongs to the clonal complex 1 of C. sakazakii (CC1), which is the second major ST in the PubMLST database after ST4 (CC4). ST4 (C. sakazakii) and ST7 (C. malonaticus) were the predominant STs identified by Li et al. [41] for the Cronobacter spp. isolated from wet rice and flour products in China. Similarly, Fei et al. [42] determined the sequence types ST4 (19/56, 33.9%), ST1 (12/56, 21.4%), and ST64 (11/56, 16.1%) in C. sakazakii as dominant for Cronobacter spp. isolated from powdered infant formula collected from Chinese retail markets. The results of this study may provide a theoretical basis for investigating the transmission routes and genotyping diversity of Cronobacter spp. and developing more effective methods for preventing this organism.

3.2. Hemolytic Activity of Cronobacter spp.

The hemolytic activity of the Cronobacter strains was determined (Table 3). In total, 87.5% and 16.7% of the Cronobacter spp. strains were capable of β-hemolysis on the culture medium with the addition of horse and sheep red blood cells, respectively.
A greater capability for β-hemolysis was determined on the horse blood agar, as indicated by a zone width ranging from 1.0 to 3.5 mm, than on the sheep blood agar—a zone width ranging from 1.0 to 1.2 mm. Regardless of ST and CC, all the C. sakazakii strains produced β-hemolysis on the horse blood agar and 86.7% of the strains caused α-hemolysis on the sheep blood agar. Both C. sakazakii strains (s41 and s42) belonging to ST 804 were capable of causing β–hemolysis on both blood agars. In turn, both analyzed C. turicensis (1n and lv54) strains assigned to different STs (806 and 808) were α-hemolytic on the horse blood agar and weakly β-hemolytic on the sheep blood agar. All the analyzed strains of C. malonaticus were capable of α-hemolysis on the sheep blood agar, while one strain assigned to ST 807 was α-hemolytic on both blood agar media. In the case of C. condimenti s37, β-hemolysis was shown on the horse blood agar, whereas it caused α-hemolysis on the sheep blood agar. All the Cronobacter strains assigned to a given ST or CC showed the same hemolytic activity profile on both blood agar media. The obtained results of the hemolytic activity of the Cronobacter strains indicated that the type of hemolysis was not species-specific, but was a strain-dependent feature.
The presence of various hemolysins and hemolysin-related genes in Cronobacter spp. has been described by many authors [33,34,43]. Fakruddin et al. [44] showed hemolytic activity on human blood agar with two out of six C. sakazakii strains isolated from food samples, whereas Rajani et al. [45] demonstrated β-hemolysis on bovine blood agar after a 4-day incubation at a temperature of 37 °C in the case of all 11 C. sakazakii isolates tested. Furthermore, Cui et al. [46] found that 27 of 31 (87%) Cronobacter isolates were not capable of hemolysis, except for C. sakazakii SC26, C. malonaticus SD16, C. malonaticus SD26, and C. muytjensii SD83, as indicated by the cleared zones produced around them. Finally, Umeda et al. [35] reported that 57 (100%) Cronobacter strains exhibited β-hemolytic activity against guinea pig, horse, and rabbit erythrocytes, and that 92,9% of the strains were capable of the α-hemolysis of sheep erythrocytes. It is known that a gene encoding a hemolysin is present in Cronobacter spp. [33], however, whether this hemolysin is active and associated with cytotoxicity has not yet been clarified. This requires conducting more in-depth genetic studies to assign the functionality of these various hemolysin genes to the appropriate phenotype.

4. Conclusions

The present study demonstrated the considerable genotypic diversity of Cronobacter strains isolated from plant-based ready-to-eat foods. Five novel sequence types (804, 805, 806, 807, and 808) were detected and novel alleles were found in four loci: ppsA, gltB, gyrB, and infB. Therefore, future studies should aim to detect more new STs, which, in the study of the genotypic diversity of Cronobacter spp., are beneficial for monitoring some sources and will enable the development of more effective methods to control these bacteria. The sequence type of one C. sakazakii strain (s12) was found to be ST4, which is strongly associated with meningitis in newborns. It was also found that 87.5% of the Cronobacter spp. strains were capable of β-hemolysis on the culture medium with horse red blood cells and 16.7% on the medium with sheep red blood cells. The presence of the pathogenic species C. sakazakii, C. malonaticus, and C. turicensis in ready-to-eat plant-derived food products shows they are potential sources of infection, especially to those with compromised immunity, which substantiates their further multi-faceted characterization [47]. The fact that active hemolysins appear in isolates from plant-based food seems to be disturbing, as this feature is usually associated with pathogens originating from clinical sources.

Author Contributions

Conceptualization, M.G.; methodology, M.G. and I.S.; investigation, M.G. and I.S.; writing—original draft preparation, M.G.; writing—review and editing, M.G., A.B.-P., I.S., S.F., A.P., L.S.-R.; visualization, M.G.; supervision, M.G.; project administration, M.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by National Science Centre, research project No. DEC-2019/03/X/NZ1/01583 and the APC was funded by Warsaw University of Life Sciences.

Data Availability Statement

Data are contained within the article.

Conflicts of Interest

The authors declare no conflict of interest. Author Stephen Forsythe was employed by the company FoodMicrobe.com Ltd., Adams Hill, Keyworth, Nottinghamshire NG12 5GY, UK. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that this study received funding from National Science Centre (Poland). The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.

References

  1. FAO/WHO. Enterobacter sakazakii (Cronobacter spp.) in Powdered Follow-up Formulae. In Microbiological Risk Assessment Series No. 15. Food and Agriculture Organization of the United Nations; World Health Organization: Rome, Italy, 2008. [Google Scholar]
  2. Forsythe, S.J. Updates on the Cronobacter genus. Annu. Rev. Food Sci. Technol. 2018, 9, 23–44. [Google Scholar] [CrossRef] [PubMed]
  3. Carvalho, G.G.; Calarga, A.P.; Zorgi, N.E.; Astudillo-Trujillo, C.A.; Pardini Gontijo, M.T.; Brocchi, M.; Giorgio, S.; Kabuki, D.Y. Virulence and DNA sequence analysis of Cronobacter spp. isolated from infant cereals. Int. J. Food Microbiol. 2022, 376, 109745. [Google Scholar] [CrossRef]
  4. Joseph, S.; Forsythe, S.J. Insights into the emergent bacterial pathogen Cronobacter spp., generated by multilocus sequence typing and analysis. Front. Microbiol. 2012, 3, 397. [Google Scholar] [CrossRef] [PubMed]
  5. Yang, H.Y.; Kim, S.K.; Choi, S.Y.; You, D.H.; Lee, S.C.; Bang, W.S.; Yuk, H.G. Effect of acid, desiccation and heat stresses on the viability of Cronobacter sakazakii during rehydration of powdered infant formula and in simulated gastric fluid. Food Control 2015, 50, 336–341. [Google Scholar] [CrossRef]
  6. Shi, C.; Sun, Y.; Liu, Z.; Guo, D.; Sun, H.; Sun, Z.; Chen, S.; Zhnag, W.; Wen, Q.; Peng, X.; et al. Inhibition of Cronobacter sakazakii virulence factors by citral. Sci. Rep. 2017, 7, 43243. [Google Scholar] [CrossRef]
  7. Phair, K.; Pereira, S.G.; Kealey, C.; Fanning, S.; Brady, D.B. Insights into the mechanisms of Cronobacter sakazakii virulence. Microb. Pathog. 2022, 169, 105643. [Google Scholar] [CrossRef]
  8. Jang, H.; Gopinath, G.R.; Eshwar, A.; Srikumar, S.; Scott, N.; Gangiredla, J.; Isha, R.; Isha, P.R.; Finkelstein, S.B.; Negrete, F.; et al. The secretion of toxins and other exoproteins of Cronobacter: Role in virulence, adaption, and persistence. Microorganisms 2020, 8, 229. [Google Scholar] [CrossRef]
  9. International Commission on Microbiological Specifications for Foods (ICMSF). Microorganisms in Food 7. In Microbiological Testing in Food Safety Management; Kluwer Academic/Plenum Publishers: New York, NY, USA, 2002; pp. 168–169. [Google Scholar]
  10. Henry, M.; Fouladkhah, A. Outbreak history, biofilm formation, and preventive measures for control of Cronobacter sakazakii in infant formula and infant care settings. Microorganisms 2019, 7, 77. [Google Scholar] [CrossRef]
  11. Iversen, C.; Forsythe, S.J. Isolation of Enterobacter sakazakii and other Enterobacteriaceae from powdered infant formula milk and related products. Food Microbiol. 2004, 21, 771–776. [Google Scholar] [CrossRef]
  12. Kandhai, M.C.; Heuvelink, A.E.; Reij, M.W.; Beumer, R.R.; Dijk, R.; van Tilburg, J.J.H.C.; van Schothorst, M.; Gorris, L.G.M. A study into the occurrence of Cronobacter spp. in The Netherlands between 2001 and 2005. Food Control 2010, 21, 1127–1136. [Google Scholar] [CrossRef]
  13. Kim, J.-B.; Park, Y.-B.; Kang, S.-H.; Lee, M.-J.; Kim, K.-C.; Jeong, H.-R.; Kim, D.-H.; Yoon, M.-H.; Lee, J.-B.; Oh, D.-H. Prevalence, genetic diversity, and antibiotic susceptibility of Cronobacter spp. (Enterobacter sakazakii) isolated from Sunshik, its ingredients and soils. Food Sci. Biotechnol. 2011, 20, 941–948. [Google Scholar] [CrossRef]
  14. Lee, Y.-D.; Park, J.-H.; Chang, H. Detection, antibiotic susceptibility and biofilm formation of Cronobacter spp. from various foods in Korea. Food Control 2012, 24, 225–230. [Google Scholar] [CrossRef]
  15. Ogihara, H.; Kiribe, N.; Fukuda, N.; Furukawa, S.; Morinaga, Y.; Igimi, S. Cronobacter spp. in commercially available dried food in Japan. Biocontrol. Sci. 2014, 19, 209–213. [Google Scholar] [CrossRef]
  16. Garbowska, M.; Berthold-Pluta, A.; Stasiak-Różańska, L. Microbiological quality of selected spices and herbs including the presence of Cronobacter spp. Food Microbiol. 2015, 49, 1–5. [Google Scholar] [CrossRef]
  17. Mohammed, M.A.; Sallam, K.I.; Tamura, T. Prevalence, identification and molecular characterization of Cronobacter sakazakii isolated from retail meat products. Food Control 2015, 53, 206–211. [Google Scholar] [CrossRef]
  18. Vojkovska, H.; Karpiskova, R.; Orieskova, M.; Drahovska, H. Characterization of Cronobacter spp. isolated from food of plant origin and environmental samples collected from farms and from supermarkets in the Czech Republic. Int. J. Food Microbiol. 2016, 217, 130–136. [Google Scholar] [CrossRef]
  19. Berthold-Pluta, A.; Garbowska, M.; Stefańska, I.; Pluta, A. Microbiological quality of selected ready-to-eat leaf vegetables, sprouts and non-pasteurized fresh fruit-vegetable juices including the presence of Cronobacter spp. Food Microbiol. 2017, 65, 221–230. [Google Scholar] [CrossRef]
  20. Li, C.; Zeng, H.; Zhang, J.; Luo, D.; Chen, M.; Lei, T.; Yang, X.; Wu, H.; Cai, S.; Ye, Y.; et al. Cronobacter spp. isolated from aquatic products in China: Incidence, antibiotic resistance, molecular characteristic and CRISPR diversity. Int. J. Food Microbiol. 2020, 335, 108857. [Google Scholar] [CrossRef]
  21. Carvalho, G.G.; Calarga, A.P.; Teodoro, J.R.; Queiroz, M.M.; Astudillo-Trujillo, A.C.; Levy, C.E.; Brocchi, M.; Kabuki, D.Y. Isolation, comparison of identification methods and antibiotic resistance of Cronobacter spp. in infant foods. Food Res. Int. 2020, 137, 109643. [Google Scholar] [CrossRef]
  22. Zeng, H.; Li, C.; Ling, N.; Zhang, J.; Chen, M.; Lei, T.; Wu, S.; Yang, X.; Luo, D.; Ding, Y.; et al. Prevalence, genetic analysis and CRISPR typing of Cronobacter spp. isolated from meat and meat products in China. Int. J. Food Microbiol. 2020, 321, 108549. [Google Scholar] [CrossRef]
  23. Berthold-Pluta, A.; Garbowska, M.; Stefańska, I.; Stasiak-Różańska, L.; Aleksandrzak-Piekarczyk, T.; Pluta, A. Microbiological quality of nuts, dried and candied fruits, including the prevalence of Cronobacter spp. Pathogens 2021, 10, 900. [Google Scholar] [CrossRef] [PubMed]
  24. Parra-Flores, J.; Maury-Sintjago, E.; Rodriguez-Fernández, A.; Acuña, S.; Cerda, F.; Aguirre, J.; Holy, O. Microbiological Quality of Powdered Infant Formula in Latin America. J. Food Prot. 2020, 83, 534–541. [Google Scholar] [CrossRef] [PubMed]
  25. Pakbin, B.; Brück, W.M.; Allahyari, S.; Rossen, J.W.A.; Mahmoudi, R. Antibiotic resistance and molecular characterization of Cronobacter sakazakii strains isolated from powdered infant formula milk. Foods 2022, 11, 1093. [Google Scholar] [CrossRef] [PubMed]
  26. Fei, P.; Xing, M.; Feng, Y.; Liu, S.; Chang, Y.; Wang, Y.; Yu, Y.; Shi, E.; Zhang, Y.; Bian, X.; et al. Cronobacter sakazakii in goat milk-based infant formula from shaanxi province, China. Foodborne Pathog. Dis. 2022, 19, 304–310. [Google Scholar] [CrossRef]
  27. Xu, X.; Li, C.; Wu, Q.; Zhang, J.; Huang, J.; Yang, G. Prevalence, molecular characterization, and antibiotic susceptibility of Cronobacter spp. in Chinese ready-to-eat foods. Int. J. Food Microbiol. 2015, 204, 17–23. [Google Scholar] [CrossRef]
  28. Lou, X.; Liu, T.; Zhang, W.; Yu, H.; Wang, H.; Song, S.; Chen, Q.; Fang, Z. The occurrence and distribution characteristics of Cronobacter in diverse cereal kernels, flour, and flour-based products. Food Microbiol. 2019, 84, 103269. [Google Scholar] [CrossRef]
  29. Holý, O.; Forsythe, S. Cronobacter spp. as emerging causes of healthcare-associated infection. J. Hosp. Infect. 2014, 86, 169–177. [Google Scholar] [CrossRef]
  30. Burgess, C.M.; Gianotti, A.; Gruzdev, N.; Holah, J.; Knøchel, S.; Lehner, A.; Margas, E.; Schmitz Esser, S.; Saldinger, S.S.; Tresse, O. The response of foodborne pathogens to osmotic and desiccation stresses in the food chain. Int. J. Food Microbiol. 2016, 221, 37–53. [Google Scholar] [CrossRef]
  31. Srikumar, S.; Cao, Y.; Yan, Q.; Van Hoorde, K.; Nguyen, S.; Cooney, S.; Gopinath, G.R.; Tall, B.D.; Sivasankaran, S.K.; Lehner, A.; et al. RNA sequencing-based transcriptional overview of xerotolerance in Cronobacter sakazakii SP291. Appl. Environ. Microbiol. 2019, 85, e01993-18. [Google Scholar] [CrossRef]
  32. Ling, N.; Forsythe, S.; Wu, Q.; Ding, Y.; Zhang, J.; Zeng, H. Insights into Cronobacter sakazakii biofilm formation and control strategies in the food industry. Engineering 2020, 6, 393–405. [Google Scholar] [CrossRef]
  33. Cruz, A.; Xicohtencatl-Cortes, J.; Gonzalez-Pedrajo, B.; Bobadilla, M.; Eslava, C.; Rosas, I. Virulence traits in Cronobacter species isolated from different sources. Can. J. Microbiol. 2011, 57, 735–744. [Google Scholar] [CrossRef]
  34. Jang, H.; Chase, H.R.; Gangiredla, J.; Grim, C.J.; Patel, I.R.; Kothary, M.H.; Jackson, S.A.; Mammel, M.K.; Carter, L.; Negrete, F.; et al. Analysis of the molecular diversity among Cronobacter species isolated from filth flies using targeted PCR, pan genomic DNA microarray, and whole genome sequencing analyses. Front. Microbiol. 2020, 11, 561204. [Google Scholar] [CrossRef] [PubMed]
  35. Umeda, N.S.; De Filippis, I.; Forsythe, S.J.; Brandão, M.L.L. Phenotypic characterization of Cronobacter spp. strains isolated from foods and clinical specimens in Brazil. Food Res. Int. 2017, 102, 61–67. [Google Scholar] [CrossRef] [PubMed]
  36. Svobodová, B.; Vlach, J.; Junková, P.; Karamonová, L.; Blažková, M.; Fukal, L. Novel method for reliable identification of Siccibacter and Franconibacter strains: From “Pseudo-Cronobacter” to new Enterobacteriaceae genera. Appl. Environ. Microbiol. 2017, 83, e00234-17. [Google Scholar] [CrossRef] [PubMed]
  37. Forsythe, S.J.; Dickins, B.; Jolley, K.A. Cronobacter, the emergent bacterial pathogen Enterobacter sakazakii comes of age; MLST and whole genome sequence analysis. BMC Genom. 2014, 15, 1121–1134. [Google Scholar] [CrossRef]
  38. Baldwin, A.; Loughlin, M.; Caubilla-Barron, J.; Kucerova, E.; Manning, G.; Dowson, C.; Forsythe, S. Multilocus sequence typing of Cronobacter sakazakii and Cronobacter malonaticus reveals stable clonal structures with clinical significance which do not correlate with biotypes. BMC Microbiol. 2009, 9, 223. [Google Scholar] [CrossRef]
  39. Buxton, R. Blood Agar Plates and Hemolysis Protocols. Am. Soc. Microbiol. 2005, 15, 1–9. Available online: https://asm.org/getattachment/7ec0de2b-bb16-4f6e-ba07-2aea25a43e76/protocol-2885.pdf (accessed on 10 April 2020).
  40. Wang, Q.; Forsythe, S.J.; Zhao, X.-J.; Wanga, Z.-W.; Li, D.; Ma, D.; Cao, J.-Y.; Zeng, J. Species identification and molecular characterization of Cronobacter spp. isolated from food imported over nine years into Beijing, China. Food Microbiol. 2019, 82, 11–19. [Google Scholar] [CrossRef]
  41. Li, Q.; Li, C.; Ye, Q.; Gu, Q.; Wu, S.; Zhang, Y.; Wei, X.; Xue, L.; Chen, M.; Zeng, H.; et al. Occurrence, molecular characterization and antibiotic resistance of Cronobacter spp. isolated from wet rice and flour products in Guangdong, China. Curr. Res. Food Sci. 2023, 7, 100554. [Google Scholar] [CrossRef]
  42. Fei, P.; Jiang, Y.; Jiang, Y.; Yuan, X.; Yang, T.; Chen, J.; Wang, Z.; Kang, H.; Forsythe, S.J. Prevalence, Molecular Characterization, and Antibiotic Susceptibility of Cronobacter sakazakii Isolates from Powdered Infant Formula Collected from Chinese Retail Markets. Front. Microbiol. 2017, 8, 2026. [Google Scholar] [CrossRef]
  43. Joseph, S.; Desai, P.; Ji, Y.; Cummings, C.A.; Shih, R.; Degoricija, L.; Rico, A.; Brzoska, P.; Hamby, S.E.; Masood, N.; et al. Comparative analysis of genome sequences covering the seven Cronobacter species. PLoS ONE 2012, 7, e49455. [Google Scholar] [CrossRef] [PubMed]
  44. Fakruddin, M.; Rahaman, M.; Ahmed, M.M.; Hoque, M.M. Stress tolerant virulent strains of Cronobacter sakazakii from food. Biol. Res. 2014, 47, 63. [Google Scholar] [CrossRef] [PubMed]
  45. Rajani, C.S.R.; Chaudhary, A.; Swarna, A.; Puniya, A.K. Identification and virulence of Enterobacter sakazakii. J. Food Ind. Microbiol. 2016, 2, 108. [Google Scholar] [CrossRef]
  46. Cui, J.; Hu, J.; Du, X.; Yan, C.; Xue, G.; Li, S.; Cui, Z.; Huang, H.; Yuan, J. Genomic analysis of putative virulence factors affecting cytotoxicity of Cronobacter. Front. Microbiol. 2020, 10, 3104. [Google Scholar] [CrossRef] [PubMed]
  47. Yong, W.; Guo, B.; Shi, X.; Cheng, T.; Chen, M.; Jiang, X.; Ye, Y.; Wang, J.; Xie, G.; Ding, J. An investigation of an acute gastroenteritis outbreak: Cronobacter sakazakii, a potential cause of food-borne illness. Front. Microbiol. 2018, 9, 2549. [Google Scholar] [CrossRef] [PubMed]
Table 1. Strains of Cronobacter spp. used in the study and their origin.
Table 1. Strains of Cronobacter spp. used in the study and their origin.
IsolateSpeciesOrigin
s12C. sakazakiiAlfalfa sprouts
9nC. sakazakiiBrazilian nuts
10nC. sakazakiiBrazilian nuts
14C. sakazakiiAlfalfa sprouts
s44C. sakazakiiMix of sprouts
s45C. sakazakiiMix of sprouts
s21C. sakazakiiLeek sprouts
s47C. sakazakiiMix of sprouts
s48C. sakazakiiMix of sprouts
s22C. sakazakiiLeek sprouts
11mC. sakazakiiMixes of dried fruits, seeds, and nuts
lv25C. sakazakiiRucola
lv27C. sakazakiiEndive escarola
s41C. sakazakiiSunflower sprouts
s42C. sakazakiiSunflower sprouts
5nC. malonaticusHazelnuts
6nC. malonaticusCashew nuts
7nC. malonaticusPini nuts
8nC. malonaticusMacadamia nuts
12mC. malonaticusMixes of dried fruits, seeds, and nuts
lv31C. malonaticusLambs lettuce
1nC. turicensisAlmonds
lv54C. turicensisMix of leaf vegetables
s37C. condimentiSmall radish sprouts
Table 2. Comparison of 7-loci MLST for 24 strains from 4 Cronobacter species.
Table 2. Comparison of 7-loci MLST for 24 strains from 4 Cronobacter species.
PubMLST IDIsolate SpeciesatpDfusAglnSgltBgyrBinfBppsASTCC
4062s12C. sakazakii513355444
40639nC. sakazakii3121651620141717
406410nC. sakazakii3121651620141717
4065s14C. sakazakii31113181117132121
4066s44C. sakazakii3852542165739999
4067s45C. sakazakii3852542165739999
4068s21C. sakazakii3852542165739999
4069s47C. sakazakii3852542165739999
4070s48C. sakazakii3852542165739999
4071s22C. sakazakii3852542165739999
407211mC. sakazakii118242201556261494-
4073lv25C. sakazakii175112027521234333648-
4074lv27C. sakazakii175112027521234333648-
3574s41C. sakazakii31120942701368804-
3575s42C. sakazakii31120942701368804-
40755nC. malonaticus891310781035160258-
40766nC. malonaticus891310781035160258-
40777nC. malonaticus891310781035160258-
40788nC. malonaticus891310781035160258-
313912mC. malonaticus6476471016381805-
3576lv31C. malonaticus38109459374807-
35731nC. turicensis461474221237193318806-
3140lv54C. turicensis4654314279265382808-
1896s37C. condimenti24869628634214798-
New assigned allele number and STs are underlined and in bold.
Table 3. Hemolysis of horse blood and sheep blood agars by Cronobacter spp. strains.
Table 3. Hemolysis of horse blood and sheep blood agars by Cronobacter spp. strains.
IsolateSpeciesHemolysis (Zone in mm)
Horse Blood AgarSheep Blood Agar
s12C. sakazakiiβ (2.9)α
9nC. sakazakiiβ (2.4)α
10nC. sakazakiiβ (2.8)α
s14C. sakazakiiβ (2.7)α
s44C. sakazakiiβ (3.3)α
s45C. sakazakiiβ (2.8)α
s21C. sakazakiiβ (2.4)α
s47C. sakazakiiβ (2.5)α
s48C. sakazakiiβ (2.8)α
s22C. sakazakiiβ (3.2)α
11mC. sakazakiiβ (1.0)α
lv25C. sakazakiiβ (2.5)α
lv27C. sakazakiiβ (1.1)α
s41C. sakazakiiβ (3.5)β (1.2)
s42C. sakazakiiβ (3.1)β (1.0)
5nC. malonaticusβ (1.9)α
6nC. malonaticusβ (1.0)α
7nC. malonaticusβ (2.6)α
8nC. malonaticusβ (2.4)α
12mC. malonaticusβ (1.0)α
lv31C. malonaticusαα
1nC. turicensisαβ (1.0)
lv54C. turicensisαβ (1.0)
s37C. condimentiβ (1.0)α
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Garbowska, M.; Berthold-Pluta, A.; Stasiak-Różańska, L.; Pluta, A.; Forsythe, S.; Stefańska, I. The Genotyping Diversity and Hemolytic Activity of Cronobacter spp. Isolated from Plant-Based Food Products in Poland. Foods 2023, 12, 3873. https://doi.org/10.3390/foods12203873

AMA Style

Garbowska M, Berthold-Pluta A, Stasiak-Różańska L, Pluta A, Forsythe S, Stefańska I. The Genotyping Diversity and Hemolytic Activity of Cronobacter spp. Isolated from Plant-Based Food Products in Poland. Foods. 2023; 12(20):3873. https://doi.org/10.3390/foods12203873

Chicago/Turabian Style

Garbowska, Monika, Anna Berthold-Pluta, Lidia Stasiak-Różańska, Antoni Pluta, Stephen Forsythe, and Ilona Stefańska. 2023. "The Genotyping Diversity and Hemolytic Activity of Cronobacter spp. Isolated from Plant-Based Food Products in Poland" Foods 12, no. 20: 3873. https://doi.org/10.3390/foods12203873

APA Style

Garbowska, M., Berthold-Pluta, A., Stasiak-Różańska, L., Pluta, A., Forsythe, S., & Stefańska, I. (2023). The Genotyping Diversity and Hemolytic Activity of Cronobacter spp. Isolated from Plant-Based Food Products in Poland. Foods, 12(20), 3873. https://doi.org/10.3390/foods12203873

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