Virulence and Antibiotic Resistance Profiles of Cronobacter sakazakii and Enterobacter spp. Involved in the Diarrheic Hemorrhagic Outbreak in Mexico

Cronobacter spp. are bacterial pathogens that cause neonatal meningitis, septicemia, and necrotizing enterocolitis in infants with a lethality rate of 40–80%. Powdered infant formulas (PIF) have been implicated as the main vehicles of transmission. This pathogen can also cause infection through contaminated expressed breast milk, and it has been recovered from neonatal feeding tubes of neonates not fed reconstituted PIF and milk kitchen areas. This study analyzed antibiotic resistance profiles and the tissue virulence tests of Cronobacter sakazakii and Enterobacter spp. recovered from PIF, infant fecal matter‘s, and milk kitchen environment involved in a diarrheic hemorrhagic outbreak in 2011 in Mexico. The strains isolated from the outbreak had similar antibiotic resistance profiles and pathogenicity irrespective of isolation site, however, C. sakazakii strains isolated from PIF showed significantly higher invasive profiles than Enterobacter spp. (p = 0.001) and 83% were resistant to more than one antibiotic. The findings of this study can be used to complement existing information to better control Cronobacter and Enterobacter spp. contamination in PIF production, prevent its transmission, and improve infant food safety.


INTRODUCTION
Cronobacter infections are associated with adults and infants (Bowen and Braden, 2006;Holy and Forsythe, 2014;Alsonosi et al., 2015;Forsythe, 2018). Nevertheless, infections of premature neonates are of particular concern are due to their high lethality rate of between 40 and 80% . The clinical manifestation of this pathogen in infants is mainly meningitis, septicemia, and necrotizing enterocolitis (Nazarowec-White and Farber, 1999;Van Acker et al., 2001;Baumbach et al., 2009;Hariri et al., 2013b) although diarrheal and urinary infections have also been observed (Friedemann, 2009). From 2003to 2009 cases of Cronobacter spp. infection were identified in 6 states of the United States, especially among children <5 years of age (Patrick et al., 2014), indeed, Cronobacter is the genus that is the most commonly involved in cases of illness associated mainly with the consumption of contaminated powdered infant formula (PIF) rehydrated (Food Agriculture Organization of the United Nations [FAO] and World Health Organization [WHO], 2004[WHO], , 2006 although, additional possible reservoirs from preparation utensils and the environment have been recognized (Friedemann, 2008;Siqueira-Santos et al., 2013;Holy and Forsythe, 2014) and contaminated expressed breast milk, where C. malonaticus strain was isolated from a breast abscess (Bowen et al., 2017). Additionally, C. sakazakii has been isolated from the enteral feeding tubes of neonates not fed reconstituted infant formula (Hurrell et al., 2009).
There are recommended biochemical methods to identify Cronobacter spp. (Api20E, ID32E, BIOLOG microarray, Vitek 2 System), but these can only be used for presumptive identification and they can have accuracy level as low as 43% (Cetinkaya et al., 2012;Joseph et al., 2013;Jackson and Forsythe, 2016). Several PCR primers have been proposed to identify members of the genus Cronobacter by amplifying specific sequences of variable and conserved regions of the 16S rRNA of the bacteria (Lehner et al., 2004;Hassan et al., 2007). Specific primers for the rpoB gene encoding the β region of the polymerase enzyme have been proposed for identifying Cronobacter species, but have not taken into account changes in the taxonomy of the species, giving false positive results with some Enterobacter species (Jackson et al., 2015;Jackson and Forsythe, 2016). Baldwin et al. (2009) developed a 7-loci multilocus sequence typing (MLST) scheme for Cronobacter speciation and genotyping. The MLST scheme has an open access database 1 that contains >2,400 strains and >350 whole genomes along with corresponding metadata and updates according to changes in taxonomy. This approach has led to the recognition of clonal complexes (CC) within the Cronobacter genus. Of special significance is the recognition of the Cronobacter sakazakii CC4 pathovar which is strongly associated with neonatal meningitis cases (Joseph and Forsythe, 2011;Sonbol et al., 2013;Hariri et al., 2013a;Forsythe et al., 2014;Forsythe, 2018). Jackson et al. (2015) provided the re-evaluation of a previous study done by Flores et al. (2011) of C. sakazakii outbreak caused by consuming contaminated reconstituted PIF in Mexico, which had used phenotyping and rpoB PCR probe method to identify the isolates, whereas Jackson et al. (2015) used DNA sequencing, and showed that the strains were E. hormaechei and Enterobacter spp. (undesignated species), demonstrating for the first time, the possible transmission of Enterobacter from PIF to infants (Jackson et al., 2015). This possible transmission suggests that these organisms may pose a risk to infants consuming rehydrated PIF (Jackson et al., 2015). In fact, this risk was estimated by Parra- Flores et al. (2016) in a risk based assessment under a probabilistic approach of reconstituted PIF contaminated with different inoculum size of Cronobacter, differing heat treatment to prepare the PIF and storage temperature. 1 www.PubMLST.org/cronobacter/ Important aspects to be considered in the severity and prognosis of Cronobacter infection are the presence of antibioticresistance (Caubilla- Kilonzo-Nthenge et al., 2012;Xu et al., 2015), and virulence factors (Townsend et al., 2008). Such virulence factors can include iron acquisition and the invasiveness and adhesion in cell lines such as HEp-2 and CaCo-2 (Pagotto et al., 2003;Mange et al., 2006;Grim et al., 2012;Almajed and Forsythe, 2016).
The aim of this work was to evaluate and compare the virulence and antibiotic resistance profiles of the Cronobacter sakazakii and Enterobacter spp. involved in the diarrheic hemorrhagic outbreak in Mexico in 2011.

Sequencing of fusA Gene
The methodology described by Baldwin et al. (2009) was followed using PCR CORE Kit QIAGEN (Cat No. 201225) solutions. Amplified products were sent to MACROGEN in Korea for sequencing. The sequenced products were analyzed with the Gentle software and later aligned with the ClustalW software. A phylogenetic tree was constructed using the maximum likelihood method with the MEGA7 software. Identification was performed with the free access online database https://pubmlst. org/cronobacter/ and BLASTn (NCBI).
Virulence Determination of Cronobacter and Enterobacter spp.

Adherence Assay
HEp-2 cells were cultured in Eagle's minimal medium (MEM) (In Vitro, Mexico) supplemented with 10% fetal bovine serum (FBS) (Gibco, United States) and without antibiotics. The cells were prepared in T75 cm 2 flasks (Sarstedt, Germany) and grown for 24 h at 37 • C and 5% CO 2 . Upon reaching FIGURE 1 | The phylogenetic tree of fusA sequencing of Cronobacter sakazakii, Enterobacter hormaechei, and Enterobacter spp. The tree with the highest log likelihood (-1450.2262) is shown. Initial tree using the Maximum Composite Likelihood (MCL). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 24 nucleotide sequences. Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated. There were a total of 401 positions in the final dataset and analyses were conducted in MEGA7.
Frontiers in Microbiology | www.frontiersin.org confluency, cells were disaggregated with 0.25% trypsin (In Vitro, Mexico) and sown into 24 well plates from a 1 ml suspension containing 2.5 × 10 6 HEp-2 cells per ml (Sarstedt, Germany). The monolayers with 70-80% confluency were washed three times with phosphate-buffered saline (PBS) and 900 µl of MEM were added. Isolates were previously cultured overnight in 1% tryptone, and 100 µl bacterial suspensions (10 8 cells per ml) were added to each well. Plates were incubated for 3 h at 37 • C and 5% CO 2 . For quantitative assays, bacteria were removed by adding 1 ml 0.1% Triton X-100 (Amresco, United States), and serial 10-fold dilutions were plated onto tryptone soy agar (TSA) to determine the colony-forming units (CFU) of bacteria bound to HEp-2 cells. Triplicate assays were performed. Enteroaggregative E. coli O42 (EAEC) was used as the positive control. Escherichia coli K-12 HB101 was the negative control (Cruz et al., 2011). This assay was repeated three times and the results were expressed as the means ± SD of the data.

Invasion Assays
The HEp-2 cell lines were prepared according to the procedure described in the adherence assay section. After 3-h incubation, the infected monolayers were washed three times with PBS and incubated with 1 ml MEM with lysozyme 300 µg/ml and gentamycin 100 µg ml −1 (In Vitro, Mexico). Samples were washed once with PBS. For quantitative assays, cells were washed three times with PBS, detached with 1 ml 0.1% Triton X-100 and plated on TSA. Invasion frequencies were calculated as the number of bacteria surviving incubation with gentamycin divided by the total number of bacteria present in the absence of this antibiotic (bacterial adherence). Enteroinvasive E. coli 1192 and E. coli HB101 (K-12) were used as positive and negative controls, respectively. This assay was repeated three times and the results were expressed as the means ± standard deviation of the data (Cruz et al., 2011).

RESULTS AND DISCUSSION
This study is an extension of a previous study by Flores et al. (2011) and Jackson et al. (2015), who suggested that, after the fusA sequence analysis, isolates from the outbreak in Mexico 2011 were a mixture of C. sakazakii, Enterobacter hormaechei, and Enterobacter spp. This was also corroborated by the phylogenetic analysis that clearly showed differences with other strains (Figure 1). Currently, the fusA sequencing method enables higher speciation accuracy because it follows the whole genome phylogeny and adjusts to taxonomic changes Xu et al., 2014;Alsonosi et al., 2015;Jackson et al., 2015). Several studies have confirmed that Cronobacter strains can be effectively eliminated by antibiotics, however, prolonged use of antibiotics, inappropriate dose, quantity and length of application are undesirable as it may result in the development of Cronobacter antibiotic resistance (Pérez et al., 2007;Langdon et al., 2016;Fei et al., 2017;Kardos, 2017). Therefore, it is interesting to determine some degree of association in the resistance profiles among strains from food products, environment, and fecal matter of colonized individuals exhibiting or not exhibiting symptoms or from a foodborne outbreak. This aspect is considered part of the objective of the present study because the strains were isolated from PIF, surfaces on which PIF was prepared (Supplementary Figure S1), and fecal matter of infants associated to an outbreak occurred in Mexico (Flores et al., 2011). This evaluation enables the design of treatment strategies for exposed individuals, especially those who are hypersensitive such as infants and the elderly. Although, there is considerable speculation about the source of PIF contamination. Some researchers suggest that the sources are either the environment of the production plants (Craven et al., 2010;Jacobs et al., 2011;Fei et al., 2015;Jing-Hua et al., 2015) or the ingredients (i.e., starch) used to prepare the PIF, which are the probable vehicles of transmission ( and World Health Organization [WHO] (2004[WHO] ( , 2006 risk assessments on the microbiological safety of infant formula also recognized that other members of Enterobacteriaceae were recoverable from PIF and could put infants at risk even though no confirmed cases had been confirmed at that time. In our study, the 24 strains were identified as C. sakazakii (5), Enterobacter hormaechei (3), and Enterobacter spp. (16). In general, 83% (20/24) of the isolated strains were resistant to 1-7 antibiotics. Eight percent (2/24) were resistant to 5 antibiotics and 37% (9/24) resistant to 3 antibiotics. Sixty-five percent (16/24) of the resistant strains were isolated from PIF ( Table 1).
Eighty percent of C. sakazakii (4/5) strains were resistant to cephalothin ( Table 2). It is important to assess the antibiotic resistance profile of Cronobacter spp., associated to those product (PIFs and infant products) consumed by high risk population whose are immunologically vulnerable. Molloy et al. (2009) reported that of 33 C. sakazakii strains isolated in the environment, 51% were resistant to cephalothin. Kleiman et al. (1981) also reported a moderate resistance to cephalothin in isolated strains in a case of meningoencephalitis.
For E. hormaechei, 100% (3/3) were resistant to cephalothin and ampicillin, 33% (1/3) to cefotaxime and ceftriaxone, and 66% (2/3) to nitrofurantoin. The Enterobacter spp. strains were resistant to cephalothin and ampicillin. The resistance values for ampicillin and cephalothin are higher than those previously reported (Kim et al., 2008;Molloy et al., 2009). Lai (2001) reported isolated strains were resistant to the first and second cephalosporin generation. The same situation was found in the present study with the Cronobacter sakazakii and Enterobacter strains isolated from PIF, milk kitchen surfaces, and fecal matter. Resistance was 26% (4/16) for cefotaxime, 13% (2/16) for ceftriaxone, and 26% (4/16) for cefepime ( Table 2). This suggests that ß-lactamase production should be further monitored as recommended by the Food Agriculture Organization of the United Nations [FAO] and World Health  Organization [WHO], 2008, especially since the resistant strains were isolated from PIF. Caubilla-  analyzed Cronobacter sakazakii strains from an outbreak with fatalities in a neonatal intensive care unit in France; they found one pulsotype that was associated with the three fatal cases. These were later shown to be the pathovar C. sakazakii CC4 (Joseph and Forsythe, 2011;Masood et al., 2015). In addition, two of these isolates had extended-spectrum ß-lactamase activity. A recent study evaluated the antimicrobial and desiccation resistance of Cronobacter sakazakii (Caubilla Barron and Forsythe, 2007), and Cronobacter malonaticus isolates from powdered infant formula and processing environments showed that the 70 Cronobacter strains, representing 19 sequence types, were susceptible to the most of the antibiotics except for amoxicillin-clavulanate, ampicillin, and cefazolin (Fei et al., 2017) which is in accordance with our results.
Our findings indicate that hospitalized infants were unpurposed and accidentally exposed to Cronobacter and Enterobacter spp. for 2 months. This fact could increase the susceptibility to suffer an infection by this pathogen, especially if this pathogen has a variety of virulence factors which aid in tissue adhesion, invasion and host cell injury. In addition, the results of this study indicate the hospitalized infants were unpurposed and accidentally exposed to Cronobacter and Enterobacter spp. which were able to adhere and invade human cells (HEp-2 cell line) in vitro. This was shown using twelve selected strains which had been isolated from PIF, work surfaces, and fecal matter (Figure 2). Due to funding limitations it was impossible to carry out more strains analysis.
Adherence is one of the events that enables bacteria to colonize and invade the host cells; it is a property associated with bacterial pathogenesis, especially of intracellular pathogens (Pizarro-Cerdá and Cossart, 2006;Cruz et al., 2011). In our study, adherence mean values in Cronobacter spp., Enterobacter hormaechei, and Enterobacter spp. HEp-2 cells were 22, 23, and 19 × 10 4 CFU/mL and with no significant differences among them (p > 0.05). Mean invasion values were 3.3, 5.2, and 2.5%, respectively; E. hormaechei (p = 0.001) was significantly more invasive than C. sakazakii. In general, 100% of the evaluated strains had adherence capacity and 75% were invasive in HEpcells; these values were similar to the results reported by Mange et al. (2006) and Townsend et al. (2008). Cruz et al. (2011) found five species of Cronobacter spp. (C. sakazakii, C. malonaticus, C. dublinensis, C. muytjensii, and C. genomospecies (current universalis), all of which had the capacity to adhere to HEp-2 cell lines. The C. sakazakii strains from a human source exhibited higher adherence values compared to strains of the same species isolated from other sources. Furthermore, when the invasion capacity of C. sakazakii was evaluated, it was found that 35% of the isolates were invasive and apparently more efficient than the other evaluated Cronobacter spp. species.
The C. sakazakii and E. hormaechei strains evaluated in our study were invasive; however, Enterobacter spp. only had 33% of invasive strains, which is of concern because the virulence trait is in isolated PIF strains. Reports of several outbreaks of sepsis in neonatal intensive care units in Brazil and the United States (Campos et al., 2007;Townsend et al., 2008) have shown that E. hormaechei is clinically significant, indeed an outbreak of E. hormaechei occurred among premature infants in the intensive care nursery (ICN) at the Hospital of the University of Pennsylvania between November 29, 1992and March 17, 1993(Wenger et al., 1997. Cronobacter species adhered to HEp-2, Caco-2 and brain microvascular endothelial cells, producing two distinctive adherence patterns, a diffuse and a localized adhesion (Mange et al., 2006;Cruz et al., 2011). Moreover, it has been suggested that the outer membrane proteins OmpA and OmpX from C. sakazakii are involved in basolateral invasion of human enterocyte-like Caco-2 and intestinal epithelial cells Singamsetty et al., 2008).
In conclusion, all isolated strains showed resistant to more than one antibiotic (cephalothin, ampicillin, cefotaxime, and ceftriaxone) independent of the source of isolation. In addition, C. sakazakii strains isolated from PIF were significantly more invasive than Enterobacter spp. Individually; E. hormaechei was more invasive than C. sakazakii and Enterobacter spp.
The knowledge generated in the present work can be used to complement existing information to better control Cronobacter and Enterobacter spp. contamination in PIF production, prevent its transmission, and improve infant food safety. This information should support regulatory and health authorities in their microbial surveillance measures and improve neonatal and infant health.

AUTHOR CONTRIBUTIONS
JP-F conceived the experiments. JP-F, JS-S, AC-C, and JA designed the experiments. JP-F, AC-C, and JS-S conducted the laboratory work. VJ and JA provided data analysis. JP-F, AC-C, JS-S, SF, VJ, and JA drafted the manuscript. EJ revised the manuscript and data analysis. All the authors reviewed and approved the final manuscript.

FUNDING
Funding was provided by the Research Directorate of the Universidad del Bío-Bío, Projects 161720 3/R, 091824/R, and GI 171220/EF and Consejo Nacional de Ciencia y Tecnología, México project 98625.

ACKNOWLEDGMENTS
We thank the Santander Research Mobility Grant for providing financial support. We also wish to thank Suzanne Théberge for technical assistance with the manuscript.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2018.02206/full#supplementary-material FIGURE S1 | Milk kitchen floor plan: (A) PIF bottles preparation area (sterile), and (B) Washing and disinfection area.