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Article

The Potential Role of rpoS and ompR in the Acid Resistance and Desiccation Tolerance of Cronobacter malonaticus Strains

by
Abdlrhman M. Alsonosi
1,*,
Khaled M. Ibrahim
2,
Bassam A. Elgamoudi
3,*,
Mahmoud B. Agena
4 and
Stephen J. Forsythe
5
1
Microbiology Department, Faculty of Medicine, Sebah University, Sebha P.O. Box 1000, Libya
2
Microbiology Department, Libyan Biotechnology Research Center, Tripoli P.O. Box 30313, Libya
3
Institute for Biomedicine and Glycomics, Griffith University, Southport 4215, Australia
4
Libyan Medical Research Center, Zawia P.O. Box 20311, Libya
5
Foodmicrobe.com Ltd., Nottingham NG12 5GY, UK
*
Authors to whom correspondence should be addressed.
Microbiol. Res. 2025, 16(3), 53; https://doi.org/10.3390/microbiolres16030053
Submission received: 24 December 2024 / Revised: 25 January 2025 / Accepted: 22 February 2025 / Published: 25 February 2025
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Versions Notes

Abstract

:
In this study, the acid resistance and desiccation tolerance of 20 strains of Cronobacter malonaticus were explored, and their genetic variances with respect to their survival in stressful conditions were identified by genomic analysis. The strains showed significant variances in acid tolerance when exposed to simulated gastric acid (pH 3.5) for 2 h. Strain 685 demonstrated less viability, suggesting greater susceptibility. Desiccation in infant formula also yielded sub-lethally injured cells, with variable strain recovery, highlighting strain 685 as the strain with the lowest recovery. Strains were determined to contain single nucleotide polymorphisms (SNPs) in the ompR and rpoS genes, suggesting loss-of-function mutations and potentially elucidating the stress sensitivity mechanism of strain 685. This study underscores the importance of genetic factors in C. malonaticus resilience and the necessity for developed detection methods for assessing food safety risk, especially in relation to powdered infant formulas. Our results provide important information on the pathogenic potential of C. malonaticus and help guide future research priorities to mitigate risks associated with foodborne pathogens.

1. Introduction

The Cronobacter genus, consisting of seven distinct species, has garnered significant attention in the field of food safety and public health, particularly due to its association with severe infections in vulnerable populations, such as neonates and adults. Among these species, Cronobacter sakazakii and Cronobacter malonaticus have been implicated in the majority of clinical cases. Notably, outbreaks of neonatal infections linked to C. sakazakii have frequently been traced back to powdered infant formula (PIF), whereas C. malonaticus has been associated more commonly with adult infections [1,2]. It is crucial to understand the epidemiology and pathogenic capabilities of these two species in order to formulate effective strategies for reducing the risks associated with Cronobacter infections.
Multilocus sequence typing has been instrumental in elucidating the genetic relationships among different Cronobacter pathovars. Specific clonal complexes have been identified, such as C. sakazakii clonal complex CC4, which is strongly associated with neonatal meningitis, and C. malonaticus clonal complex CC7, which is predominantly linked to adult infections [3]. However, it is important to note that C. malonaticus is not exclusively a pathogen of adults; it has also been implicated in neonatal infections. This fact highlights the importance of this species, necessitating greater efforts to understand its pathogenic potential [4,5,6,7]. The presence of C. malonaticus in various dry food products, including powdered infant formula, powdered milk, and spices, highlights the need for vigilant monitoring and risk assessment in food safety practices [8,9]. The ability of Cronobacter species to persist in these environments is mostly attributed to several physiological traits that confer resistance against adverse conditions. For example, Cronobacter species can survive for extended periods exceeding one year in PIF, which raises concerns regarding their potential to cause infections upon the rehydration of these products [9].
A significant factor contributing to Cronobacter’s survival is its ability to produce high quantities of capsular polysaccharides. These polysaccharides play a critical role in protecting the bacteria from desiccation, a key stressor during food processing [10,11]. Furthermore, research indicates that Cronobacter can tolerate desiccation better than many other members of the Enterobacteriaceae family [12]. During food processing, bacterial cells can be injured by various stressors, such as drying and exposure to high temperatures. While some of these injured cells may survive under harsh conditions, they can suffer damage to essential cellular structures, including the cell wall, cytoplasmic membrane, and ribosomes, as well as DNA, RNA, and enzymes. As a result, sub-lethally injured bacterial cells are characterised by their inability to grow on both general and selective media [13,14].
Once dried foods like PIF are rehydrated, these injured cells can potentially repair themselves, posing a risk of infection in infants upon ingestion [10,13]. Importantly, sub-lethally injured bacteria may not grow on selective media due to the presence of inhibitory agents like bile salts, which can lead to a false sense of microbiological safety. Consequently, dried food products might appear safe while harbouring viable pathogens, endangering consumer health.
To assess the risks of persistent bacterial pathogens in PIF, Caubilla-Barron and Forsythe [10] conducted an investigation involving various Enterobacteriaceae, including Salmonella, Citrobacter, and Cronobacter (previously known as Enterobacter sakazakii). However, it is noteworthy that none of the Cronobacter strains utilised in their study were identified as C. malonaticus, highlighting a gap in understanding the full spectrum of risk associated with this genus. The survival of Cronobacter spp. in their host due to severe environmental conditions results from innate factors such as acid and desiccation tolerance. For example, the new C. malonaticus Py79 strain showed significant acid and desiccation susceptibility, closely related to the sigma factor σS (RpoS) and a transcriptional activator protein (OmpR) stress response regulatory factor. It is believed that RpoS cascade regulation can potentially extend the shelf life of acid- and desiccation-sensitive strains of C. malonaticus. RpoS is essential for the bacterial general stress response (GSR), in which cells are protected from environmental stresses such as extreme temperatures, osmotic shock, desiccation, and low pH. RpoS enables the transition from exponential growth to a stationary phase, allowing bacteria to adapt to environmental challenges and increasing their potential for pathogenicity [15,16].
Mutations or variations in rpoS can significantly impact a bacterium’s stress tolerance, overall fitness, and virulence [17]. When the bacterial cells are exposed to environmental stresses, the RpoS level increases. Then, RpoS recognises and binds to the promoters of hundreds of stationary-phase genes, which prompt the expression of many genes encoding proteins, including RNA polymerase and alternative sigma factors that might be involved in the environmental stress response [18,19]. Moreover, the proportion of RpoS in E. coli increased when the cells were exposed to lower pH values; RpoS-deficient mutants displayed sensitivity to the acid treatment. In addition, transcriptome analysis of different growth phases revealed major changes in the mRNA levels of stationary-phase genes under low-pH conditions in C. sakazakii, suggesting that these genes may be involved in the acid resistance of C. sakazakii. A previous study reported that RpoS enabled C. sakazakii to survive desiccation, but the role of RpoS in desiccation tolerance in members of the C. malonaticus group requires investigation [17,20,21]. It has been stated that the expression of the rpoS, hfq, and ompA genes is upregulated when C. sakazakii enters the viable but nonculturable (VBNC) state after a 2 h period of desiccation tolerance. C. sakazakii, in this VBNC state, can maintain its virulence while avoiding detection by standard colony-based assays, which typically depend on culturing the bacteria. The regulation of rpoS plays a critical role in controlling the bacterial mechanisms that enable the transition into the VBNC state. This capability to enter the VBNC state enables Cronobacter to survive under unfavourable environmental circumstances while retaining its pathogenic potential [20].
Moreover, the ompR gene encodes a transcriptional activator protein essential for responding to environmental stresses, particularly acid and osmotic challenges [16,21]. As part of a two-component regulatory system with the sensor kinase EnvZ, ompR modulates the expression of outer membrane proteins and other stress-responsive genes, enhancing the bacterium’s capacity to survive in hostile conditions. Up to the present, the role of OmpR in the C. malonaticus group has only been associated with the responses to bile and stresses, and it found that changing the expression of OmpR in C. sakazakii only resulted in different acid resistance. Foodborne pathogens’ ability to cope with the acidic environment of the human gastrointestinal tract is crucial for their survival and pathogenicity. Cronobacter not only resists this acidity, but studies have found that it can grow at low pH values, where growth occurs at pH as low as 4.2 [22,23]. Food products are particularly sensitive to these populations, and this makes it even harder to find a method for staying away from products that make you ill.
Genetic factors also play a significant role in the stress tolerance of Cronobacter species. Several genes associated with desiccation and osmotic stress response have been identified. Notably, the yihUTRSQVO gene cluster, integral to metabolism and carbohydrate transport, has been linked to desiccation resistance in C. sakazakii SP291 [24,25]. Additionally, osmoprotectant ABC transporter genes such as yehZYXW contribute to the organism’s survival in stressful environments. Yen et al. [25] further reported various genes responsible for regulating osmotolerance, including five osmotolerance regulation genes—yiaD, osmY, ompA, aqpZ, and glpF—and five genes associated with osmotic stress, such as osmB and osmO. Therefore, this study’s results provide new evidence for the role of OmpR in desiccation tolerance in both C. sakazakii and the C. malonaticus group. They also mean that OmpR plays a wider range of roles in the Cronobacter genus than previously reported.
This study aims to investigate the generation of sub-lethally injured cells during the desiccation of 20 strains of C. malonaticus and their ability to tolerate acidic conditions in powdered infant formula. In parallel, this research will examine the roles of the ompR and rpoS genes, enhancing our understanding of the survival mechanisms employed by this potentially harmful pathogen.

2. Materials and Methods

2.1. Bacterial Strains and Growth Conditions

This study used 20 C. malonaticus strains selected from the Cronobacter PubMLST database to cover the available sequence types (STs) of C. malonaticus (Table 1). All strains were obtained from the Nottingham Trent collection (Nottingham Trent University, Nottingham, UK). Bacterial cells were cultured aerobically at 37 °C on tryptone soya agar (TSA) (Thermo Fisher Scientific, Cambridge, UK) and violet red bile glucose agar (VRBGA) (Thermo Fisher Scientific, Cambridge, UK), using TSA as a non-selective medium and VRBGA as a selective medium. These are commonly employed in regulatory detection and phenotypic methods for identifying the Cronobacter genus.

2.2. Genetic Analysis

All the genomes of the 20 C. malonaticus strains were sequenced by Source BioScience (https://www.selectscience.net/company/source-bioscience, accessed on 1 February 2025). These sequences are currently accessible in the Cronobacter PubMLST database (http://www.pubmlst.org/cronobacter, accessed on 1 February 2025). Screening for the presence and absence of genes of interest, as well as genome comparison, was performed to reveal the presence of unique genes or regions that may contribute to physiological features. The Artemis Comparison Tool (ACT) [26] was used for comparative analysis. The nucleotide sequence of each gene of interest was retrieved in FASTA format. This sequence was then compared against the 20 C. malonaticus genomes using the Basic Local Alignment Search Tool (BLAST), which facilitates the identification of gene regions by aligning the DNA sequence with known gene databases. The genome search was conducted using the Cronobacter PubMLST database [27]. To identify single nucleotide polymorphisms (SNPs) in the 20 C. malonaticus strains, their genetic sequences were aligned using MEGA (Molecular Evolutionary Genetics Analysis) software, version 6. Following alignment, SNPs were detected by examining the nucleotide differences across the sequences to identify positions where variations occurred between strains. This approach enabled the identification of specific SNPs, which were then analysed for their potential biological significance and contribution to strain-specific characteristics. Phylogenetic trees were constructed using MEGA software to examine the evolutionary relationships among the 20 C. malonaticus strains. The trees were based on sequence variation, highlighting the impact of SNPs on the genetic divergence between the strains.

2.3. Acid Resistance

The effect of acid with a low pH on C. malonaticus was performed as described by Edelson-Mammel et al. [28]. A single colony from a fresh TSA plate was transferred into 5 mL of TSB and incubated overnight at 37 °C in a shaking incubator set to 150 rpm. A liquid infant formula (Cow & Gate Premium 1) was adjusted to a pH value of 3.5 units with 1 M hydrochloric acid to mimic stomach acid. Then, 4 mL of overnight culture were inoculated into 15 mL of the acidified infant formula, distributed into five sterile tubes, and incubated in a water bath at 37 °C. Tubes were then used to determine viable cells after 0, 15, 30, 60, 90, and 120 min. At a specific time, 200 μL from each tube was transferred, serial diluted in 1800 µL of normal saline, and plated on TSA plates using the Miles–Misra technique. The TSA plates were incubated at 37 °C overnight before the enumeration of bacterial cells.

2.4. Generation of Sub-Lethally Injured C. malonaticus Following Desiccation

One colony of each C. malonaticus strain (overnight culture) was inoculated into 5ml of liquid infant formula (Cow & Gate Premium 1) and incubated in a shaking incubator at 37 °C for 20 h. The overnight bacterial growth was diluted to 1:10 using the same liquid infant formula and adjusted to a cell density of approximately 1011 CFU/mL, and then 200 µL of the suspension was transferred into 6-well plates. The plates were left overnight in a class II cabinet at room temperature. After desiccation, the content of each well was resuspended in 2 mL of sterile distilled water (SDW). The viable counts were determined using the Miles and Misra method on VRBGA as a selective medium and TSA as a non-selective medium for the total viable count. After desiccation, the number of sub-lethally injured C. malonaticus cells was calculated as the difference in recovery on TSA and VRBGA. The number of undetected C. malonaticus cells, assumed to be sub-lethally injured bacterial cells in each medium, was calculated by subtracting the bacterial cell recovery of each strain on either TSA or VRBGA from the initial inoculum of each strain [13].

2.5. Statistical Analysis

Statistical analysis was performed using an unpaired t-test and one-way and two-way ANOVA (GraphPad Prism Software Version 5.0) to determine the reproducibility of the independent experiments. All experiments conducted in this study were repeated at least twice.

3. Results

3.1. Multiple Alignments of ompR and rpoS Genes and Their Homologues

The analysis of nucleotide differences across the sequences of 20 C. malonaticus strains revealed an SNP variation between strain 685 and the other strains (Figure 1A,B). This finding was further validated using MEGA software, where the manual curation of the ompR and rpoS gene sequence alignments identified a single SNP difference in each gene sequence (Figure 2 and Figure 3).

3.2. Acid Stress Response in C. malonaticus

Twenty strains of Cronobacter malonaticus were investigated for their tolerance to pH 3.5 (Figure 4). The initial viable count for all strains ranged between 5.69 log10 CFU/mL and 6.26 log10 CFU/mL. Over the 2 h period of the experiment, there was variation in the strains’ ability to withstand acidic conditions at pH 3.5. Strains ST7, ST11, ST84, and ST307 were able to survive this level of acidity throughout the 2 h exposure. In contrast, the three ST60 strains showed resistance to the acid during the first hour, but after 60 min, their viable count decreased by approximately 2-log at 120 min. Strain 685, corresponding to ST129, began to show a decline in viable cell count after 30 min and decreased to about 3.5 log10 CFU/mL by 60 min. By 90 min, this strain could not survive the acidic conditions.
The ompR gene sequences from the 20 C. malonaticus strains were aligned to identify sequence variations, revealing that strain 685 had a unique sequence. As shown in Figure 1A, strain 685 clusters separately, indicating a distinct nucleotide sequence. This strain was sensitive to the acid at pH 3.5 after 30 min, and SNP analysis revealed differences at a specific nucleotide position (102) (Figure 2). Additionally, the rpoS gene was present in all tested C. malonaticus strains. The rpoS gene sequences of C. malonaticus strains, along with C. sakazakii strain NTU 658 (known to be sensitive to acid stress), were aligned to detect any sequence variations. Notably, C. malonaticus strain 685, which showed sensitivity to acid stress in this study (Figure 4), clustered with C. sakazakii strain 658 (Figure 1B). Both strains shared SNPs at several positions in the rpoS gene, such as 264, 429, 432, 462, 552, and 615 (Figure 3A–E); however, the nucleotide analysis also showed differences between these two strains in some positions such as 861 (Figure 3F). This suggests the possibility of a loss-of-function mutation in the rpoS gene of C. malonaticus strain 685.

3.3. Sub-Lethal Injury and Desiccation

C. malonaticus cells generated following desiccation in infant formula were tested on TSA and VRBGA to assess the viability of the recovered cells (Figure 5). The inoculum for tested strains was approximately 8 log10 CFU/mL, and all the strains displayed a greater recovery on TSA (p < 0. 05) than in VRBGA (Figure 5). On TSA plates, the cell recovery ranged from 5.9 to 6.8 log10 CFU/mL, with strain 685 showing the lowest recovery. Cell recovery on VRBGA ranged from 2.5 to 6.3 log10 CFU/mL. Strains 1827 and 893 had the highest cell recovery rate, at about 6.3 log10 CFU/mL, while strain 685 showed the lowest recovery rate, at 2.5 log10 CFU/mL. The remaining strains recovered from 4 to 5.9 log10 CFU/mL on VRBGA.
Figure 6 shows the differences between the non-viable cells, those not detected on TSA, and those not detected on VRBGA. The number of dead cells not detected on TSA agar was ~1 log10 CFU/mL for most strains, except strain 685, which had a greater decrease in viability of ~2 log10 CFU/mL. However, on VRBGA, the number of non-detected cells (sub-lethally injured and dead cells) varied between the strains. The largest loss in viability was shown by strains 565 and 685 with 4 and 5.5 log10 CFU/mL decreases, respectively. The smallest decrease in viability loss was shown by strain 1827 (1.5 log10 CFU/mL decrease). The viability loss for the remaining 17 strains ranged between 2 and 3 log10 CFU/mL.
The differences in recovery on TSA and VRBGA after desiccation reflected the number of sub-lethally injured cells. As shown in Table 2, the highest number of sub-lethal injured cells (3.37 log10 CFU/mL) was shown by strain 685 (ST129), followed by strain 565 (ST7), which showed 2.85 log10 CFU/mL. In contrast, strain 1827 (ST7) showed the lowest number of sub-lethally injured cells, which was 0.36 log10 CFU/mL (Table 2). There was a clear variation among ST7 strains regarding the number of injured bacterial cells; however, less variation was observed among ST11 and ST60 (Table 2).

4. Discussion

This study evaluated the ability of C. malonaticus to withstand stressful environments, including acidic and desiccation conditions. Additionally, genomic analysis was performed to investigate the potential roles of related genes. Thus, the results provide crucial insights into the genetic and phenotypic variability in C. malonaticus strains, particularly regarding their responses to stress.
Generating sub-lethally injured cells through desiccation in infant formula revealed crucial differences in the recovery capabilities of various C. malonaticus strains. All strains tested in this study exhibited significantly higher recovery rates on TSA than on VRBGA (as shown in Figure 5). This difference suggests that the medium’s composition significantly affects post-desiccation viability assessments. TSA, a rich medium, appears to provide a more favourable environment for the recovery of sub-lethally injured cells, aligning with previous findings indicating that nutrient-rich media can better support the recovery of stressed bacteria [29,30].
The notable recovery of strains 1827 and 893, with counts reaching approximately 6.3 log10 CFU/mL, contrasted sharply with the significantly lower recovery of strain 685, which only achieved 2.5 log10 CFU/mL on VRBGA. This suggests that strain 685 may have sustained more severe damage during desiccation or possesses intrinsic weaknesses impairing recovery. The differences in viability loss and recovery between TSA and VRBGA also reflect the extent of sub-lethal injury experienced by the strains. As indicated in Figure 5 and Figure 6, strain 685 exhibited the lowest recovery rates. It showed a substantial decrease in viability, further supporting the idea that this strain is particularly vulnerable to desiccation stress. The significant loss of viability observed in strain 685 may be linked to its genetic makeup, as sub-lethal injury can impact subsequent stress responses and overall fitness.
Desiccation compromises bacterial cell membranes, decreasing viability [10,31]. The complex matrix of PIF, containing proteins, lactose, and fats, may confer some protection to the bacteria during desiccation, enhancing survival [32]. Additionally, the ability of sub-lethally injured cells to repair damage and restore virulence traits poses a significant risk for consumers, emphasising the need for sensitive detection methods that account for these injured cells. The observed variability among the C. malonaticus strains, particularly within sequence types ST7 and ST60, underscores the genetic diversity within these sequence types. While strains from ST7 showed a range of recovery rates, strain 1827 displayed notably low numbers of sub-lethally injured cells, suggesting that genetic factors play a substantial role in determining resilience to desiccation. In contrast, less variability among ST11 and ST60 strains indicates a more uniform response to desiccation stress within these groups. In addition to phenotypic assessments, the genetic analysis of the rpoS gene provided insights into the potential underlying mechanisms of stress tolerance in C. malonaticus. Therefore, variations in the rpoS gene may affect the strain’s ability to withstand environmental stresses, as previous studies have linked rpoS mutations to increased sensitivity to desiccation and other stresses [16,17]. Genetic analysis in this study confirmed the presence of single nucleotide polymorphisms (SNPs) in the rpoS gene of strain 685 at several positions (264, 429, 432, 462, 552, and 615), differing from other strains, suggesting a possible alteration in the gene’s function. This alteration is more likely to correlate with the observed phenotypic weakness, such as the decreased recovery rate of this particular strain. Thus, the identified SNPs in the rpoS gene of strain 685 raise intriguing questions regarding their functional implications.
On the other hand, when ingested, foodborne pathogens such as Cronobacter encounter stomach acid, which plays a significant role in their survival. The ability of C. malonaticus to thrive in acidic conditions is crucial for its survival during gastrointestinal transit, enabling it to invade intestinal cells and cause disease. To simulate this acidic environment, the infant formula media was buffered to a pH of 3.5, as the human stomach pH ranges from 1.5 to 6 depending on the types of food consumed [33]. Our survival analysis revealed a heterogeneous response to acid exposure within the C. malonaticus species. While most strains demonstrated the ability to withstand simulated gastric pH conditions, strain 685 exhibited relatively low tolerance to rectified gastric acid, likely due to specific mutations in the rpoS and ompR genes. Cronobacter is adept at surviving in various environmental conditions, including acidic environments, and studies have shown a positive correlation between RpoS activity and resistance to acid, alkaline, osmotic, and oxidative stresses [17].
The detection of SNP differences in the rpoS gene sequence of C. malonaticus strain 685 compared to other strains that could not tolerate acidic conditions in this study confirms the correlation of this gene with the ability of C. malonaticus to survive in an acidic environment. The ompR gene, known to play a critical role in acid stress response in related genera such as Salmonella and E. coli [34], may also contribute to this stress resistance. Mutations in the rpoS gene have been linked to reduced stress tolerance in several bacterial species. In E. coli, such mutations impair the ability to survive environmental stressors like acid, heat, and osmotic stress [35]. Similarly, in Salmonella enterica, rpoS mutations decrease survival under acidic conditions and reduce virulence [36]. Mutations in the ompR gene, which regulates outer membrane porin expression, are also linked to antibiotic resistance in E. coli [37], as well as impacting stress resistance in both E. coli and Salmonella enterica [34].
The analysis of the ompR gene sequence in acid-sensitive C. malonaticus strain 685 also revealed an SNP difference compared to other strains. This SNP was detected at position 102 of the nucleotide sequence. Therefore, this difference may play also a significant role in the ability of C. malonaticus to tolerate acidic conditions. The potential loss of function of rpoS and ompR in this study was confirmed phenotypically, as most strains maintained viability at pH 3.5 throughout the 2 h experiment, while strain 685 exhibited a significant decline in cell counts after just 30 min and ultimately succumbed to the acidic conditions. This phenomenon aligns with the rpoS and ompR phylogenetic trees in Figure 1, where the susceptible C. malonaticus strain 685 is shown to cluster alone in Figure 1A, while in Figure 1B, it clusters with the acid-susceptible C. sakazakii strain 658.
This study highlights genetic differences among C. malonaticus strains, particularly in their resilience to desiccation and acidity. Strain 685 showed poor recovery, likely due to mutations in rpoS and ompR. The possibility of a loss-of-function mutation in the rpoS and ompR genes of C. malonaticus strain 685 should be further investigated, as these mutations could explain its poor recovery in acidic and dry conditions. Future studies should focus on the functional activity of RpoS and OmpR to better understand their role in strain survival. These genetic variations affect strain resilience, with some strains showing stronger survival abilities. The findings emphasise the need for sensitive recovery assays, especially in food products like infant formula. Given the bacterium’s ability to survive in harsh gastrointestinal environments, improving detection and safety protocols is essential. Understanding these genetic variations will aid in developing better food safety interventions. This research is crucial for protecting vulnerable populations.

Author Contributions

A.M.A.: research design, methodology, formal analysis, validation, and writing—original draft preparation. K.M.I., B.A.E., M.B.A. and S.J.F.: supervision, validation, project administration, and writing—review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

This study used the Cronobacter PubMLST open-access database, as described by Jolley et al. (2018) [33]. Wellcome Open Res 3:124. All relevant data, including sequence types (STs), serotypes, and whole-genome sequences, are available via the Cronobacter PubMLST open-access database, https://pubmlst.org/bigsdb?db=pubmlst_cronobacter_isolates, accessed on 29 January 2018.

Acknowledgments

We thank the Libyan Ministry of Higher Education and Sebha University for their support.

Conflicts of Interest

Author Stephen J. Forsythe was employed by Foodmicrobe.com Ltd. The remaining authors declare that the research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

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Microbiolres 16 00053 g001
Figure 1. (A) Maximum likelihood tree for the ompR gene (720 bp), the proposed acid tolerance gene, for C. malonaticus strains. The NTU strain ID, gene name, and sequence type (ST) number are shown. The alignment was constructed using MEGA6. (B) Maximum likelihood tree for the rpoS gene sequence (993 bp), the proposed stress gene, for C. malonaticus strains. C. sakazakii 658 and SP291 were added for comparison purposes. The NTU strain ID, name of gene, and sequence type (ST) number are shown. Alignment was performed using MEGA6.
Figure 1. (A) Maximum likelihood tree for the ompR gene (720 bp), the proposed acid tolerance gene, for C. malonaticus strains. The NTU strain ID, gene name, and sequence type (ST) number are shown. The alignment was constructed using MEGA6. (B) Maximum likelihood tree for the rpoS gene sequence (993 bp), the proposed stress gene, for C. malonaticus strains. C. sakazakii 658 and SP291 were added for comparison purposes. The NTU strain ID, name of gene, and sequence type (ST) number are shown. Alignment was performed using MEGA6.
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Figure 2. Manual curation of the ompR gene sequence alignment using MEGA software. The figure above represents the cropped segments of screenshots combined as one image showing only the sections where variations in the ompR gene sequence were observed. The highlighted column contains a nucleotide at the end where the single nucleotide polymorphisms (SNPs) occur in C. malonaticus strain 685. The shaded letters indicate the single nucleotide polymorphism (SNP) changes in C. malonaticus strains.
Figure 2. Manual curation of the ompR gene sequence alignment using MEGA software. The figure above represents the cropped segments of screenshots combined as one image showing only the sections where variations in the ompR gene sequence were observed. The highlighted column contains a nucleotide at the end where the single nucleotide polymorphisms (SNPs) occur in C. malonaticus strain 685. The shaded letters indicate the single nucleotide polymorphism (SNP) changes in C. malonaticus strains.
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Microbiolres 16 00053 g003
Figure 3. Sequence alignment of the rpoS gene using MEGA software. The figure represents variations in the rpoS gene sequence. The highlighted column in (A–F) contains nucleotides in the first and second rows, where the single nucleotide polymorphism (SNP) changes are shared between C. sakazakii strain NTU658 and C. malonaticus strain 685. The letters enclosed in dashed-line boxes indicate the single nucleotide polymorphism (SNP) variations in C. malonaticus strains.
Figure 3. Sequence alignment of the rpoS gene using MEGA software. The figure represents variations in the rpoS gene sequence. The highlighted column in (A–F) contains nucleotides in the first and second rows, where the single nucleotide polymorphism (SNP) changes are shared between C. sakazakii strain NTU658 and C. malonaticus strain 685. The letters enclosed in dashed-line boxes indicate the single nucleotide polymorphism (SNP) variations in C. malonaticus strains.
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Figure 4. Survival of C. malonaticus after exposure to pH 3.5. Survival was measured for up to 2 h at 0, 15, 30, 60, 90, and 120 min. NTU numbers of C. malonaticus strains with their representative lines are shown on the right of this figure. Strains 15, 687, and 689 showed a decrease in viable cell count by about 2-log at 120 min. Strain 685 started to decrease after 30 min to about 3.5 log10 CFU/mL at 60 min and could not survive at 90 min.
Figure 4. Survival of C. malonaticus after exposure to pH 3.5. Survival was measured for up to 2 h at 0, 15, 30, 60, 90, and 120 min. NTU numbers of C. malonaticus strains with their representative lines are shown on the right of this figure. Strains 15, 687, and 689 showed a decrease in viable cell count by about 2-log at 120 min. Strain 685 started to decrease after 30 min to about 3.5 log10 CFU/mL at 60 min and could not survive at 90 min.
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Figure 5. Viable cell counts for C. malonaticus on TSA and VRBGA after desiccation, followed by reconstitution in infant formula.
Figure 5. Viable cell counts for C. malonaticus on TSA and VRBGA after desiccation, followed by reconstitution in infant formula.
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Figure 6. After desiccation, the count of undetected bacterial cells on TSA and VRBGA was determined following reconstitution in infant formula.
Figure 6. After desiccation, the count of undetected bacterial cells on TSA and VRBGA was determined following reconstitution in infant formula.
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Table 1. C. malonaticus strains used in this study.
Table 1. C. malonaticus strains used in this study.
Strain No.STSourceCountryYear of Isolation
5657Faecal isolateUSA1973
6817Breast abscess isolateUSA1977
6887SputumCzech Republic2004
9837Infant formulaBrazil2007
15587Faecal isolateCzech RepublicUnknown
18277Cannula (blood)Czech Republic2007
18307Throat swabCzech Republic2007
18337Faecal isolateCzech Republic2010
18357Throat swabCzech Republic2012
20187SputumCzech Republic2013
20207Faecal isolateCzech Republic2013
50711Faecal isolateCzech Republic1984
51211ClinicalCzech Republic1983
51411ClinicalCzech Republic1983
1560Faecal isolateCzech Republic2003
68760SputumCzech Republic2004
68960Faecal isolateCzech Republic2005
154584Faecal isolateCzech RepublicUnknown
685129Blood isolateUSA1977
1569307Blood isolateUSA2011
Table 2. Comparison of viable counts of C. malonaticus on TSA and VRBGA after desiccation, followed by reconstitution in infant formula.
Table 2. Comparison of viable counts of C. malonaticus on TSA and VRBGA after desiccation, followed by reconstitution in infant formula.
Isolate IDSTNon-Detected Cells on VRBGA in log CFU/mLNon-Detected Cells on TSA
in log CFU/mL		Sub-Lethally Injured Cells in log CFU/mL
56573.931.082.85
68173.301.272.02
68872.321.061.26
89371.821.210.62
155873.101.181.92
182771.481.120.36
183071.971.180.79
183372.031.300.73
183572.071.200.87
201871.941.200.74
202072.221.191.02
507112.841.191.65
512112.851.281.57
514112.691.321.37
1545842.201.320.88
15602.551.171.38
687603.061.211.86
689602.791.201.59
6851295.382.013.37
15693072.651.451.20
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Alsonosi, A.M.; Ibrahim, K.M.; Elgamoudi, B.A.; Agena, M.B.; Forsythe, S.J. The Potential Role of rpoS and ompR in the Acid Resistance and Desiccation Tolerance of Cronobacter malonaticus Strains. Microbiol. Res. 2025, 16, 53. https://doi.org/10.3390/microbiolres16030053

AMA Style

Alsonosi AM, Ibrahim KM, Elgamoudi BA, Agena MB, Forsythe SJ. The Potential Role of rpoS and ompR in the Acid Resistance and Desiccation Tolerance of Cronobacter malonaticus Strains. Microbiology Research. 2025; 16(3):53. https://doi.org/10.3390/microbiolres16030053

Chicago/Turabian Style

Alsonosi, Abdlrhman M., Khaled M. Ibrahim, Bassam A. Elgamoudi, Mahmoud B. Agena, and Stephen J. Forsythe. 2025. "The Potential Role of rpoS and ompR in the Acid Resistance and Desiccation Tolerance of Cronobacter malonaticus Strains" Microbiology Research 16, no. 3: 53. https://doi.org/10.3390/microbiolres16030053

APA Style

Alsonosi, A. M., Ibrahim, K. M., Elgamoudi, B. A., Agena, M. B., & Forsythe, S. J. (2025). The Potential Role of rpoS and ompR in the Acid Resistance and Desiccation Tolerance of Cronobacter malonaticus Strains. Microbiology Research, 16(3), 53. https://doi.org/10.3390/microbiolres16030053

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