Occurrence, genetic characterization, and antibiotic susceptibility of Cronobacter spp. isolated from low water activity functional foods in Brazil.

Cronobacter spp. are bacterial pathogens isolated from a wide variety of foods. This study aims at evaluating the occurrence of Cronobacter spp. in low water activity functional food samples, detect the presence of virulence genes, and determine the antibiotic susceptibility of strains. From 105 samples, 38 (36.2%) were contaminated with Cronobacter spp. The species identified by polymerase chain reaction (PCR) and sequencing analyses (rpoB and fusA genes, respectively) were C. sakazakii (60.3%), C. dublinensis (25.4%), C. turincensis (9.5%), and C. malonaticus (4.8%). Nineteen fusA alleles were identified, including four new alleles. The virulence genes were identified by PCR and all isolates were positive for ompX and sodA genes, 60.3% to cpa gene, and 58.7% to hly gene. Using the disk diffusion method, antibiotic susceptibility to twelve antibiotics was assessed twice, separated by a 19-month period. In the first test, the isolates showed diverse antibiotic susceptibility profiles, with nineteen isolates (30.2%) being multi-drug resistant (resistant to three or more antibiotic classes), in the second, the isolates were susceptible to all antibiotics. Cronobacter spp. in functional foods demonstrates the need for continued investigation of this pathogen in foods, and further research is needed to clarify the loss of resistance of Cronobacter strains.

Incidence, evaluation of detection and identification methods, and antimicrobial resistance of Aeromonas spp. in ready-to-eat foods.

Aeromonas spp. are opportunistic and ubiquitous bacteria considered emerging pathogens that can cause infections in animals, especially fish, as well as humans. In humans, these bacteria are associated with gastroenteritis but can also be related to extraintestinal diseases. Its main infection route is through water, but it has been increasingly associated with foods. Their association with ready-to-eat foods may be a concern, especially because these products are for immediate consumption. This study aimed to investigate the prevalence of Aeromonas spp. in ready-to-eat foods (temakis, cheeses and minimally processed fruits) and to characterize the virulence profile and antimicrobial resistance of the isolates. The species A. hydrophila, A. caviae and A. veronii were identified using polymerase chain reaction (PCR), which was later compared with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). The performance of two isolation selective agars (starch-ampicillin agar-SAA and Aeromonas agar-AA) was also evaluated. Aeromonas spp. was isolated in 66.67 % (20/30) of temaki, 3.23 % (1/31) of fruits and none (0/30) of cheeses, observing high microorganism counts from <102 to 2.6 × 105 CFU/g. A. caviae (26.39 %) was the most prevalent species, followed by A. hydrophila (20.83 %) and A. veronii (8.34 %), and 44.44 % were classified as Aeromonas sp. The performance analysis between PCR and MALDI-TOF/MS for Aeromonas identification was not statistically significant, and the Kappa index showed moderate agreement (p < 0.01 and Kappa = 0.718). The SAA selective medium performed better than AA. We identified seventeen virulence profiles, and 59.72 % of the isolates had some of the genes studied. The aerA gene (47.2 %) was the most abundant, followed by act (41.7 %), hlyA and alt (38.9 %), and ast (18.1 %). A. hydrophila was the species most associated with these genes. The antimicrobial susceptibility test showed that 90 % of the isolates were resistant to amoxicillin-clavulanic acid, 17 % to tetracycline, 10 % to imipenem and 3 % to aztreonam. The results showed that temakis are carriers of potentially pathogenic Aeromonas spp. and therefore should be avoided by children, elderly individuals, pregnant women, and immunocompromised people. We also found strains resistant to antimicrobials, meaning that these microorganisms need constant monitoring.

Cronobacter spp. in foods of plant origin: occurrence, contamination routes, and pathogenic potential.

Cronobacter is an emerging bacterial pathogen associated with infections such as necrotizing enterocolitis, sepsis, and meningitis in neonates and infants, related to the consumption of powdered infant formula. In addition, this bacterium can also cause infections in adults by the ingestion of other foods. Thus, this review article aims to report the occurrence and prevalence of Cronobacter spp. in foods of plant origin, as well as the possible sources and routes of contamination in these products, and the presence of pathogenic strains in these foods. Cronobacter was present in a wide variety of cereal-based foods, vegetables, herbs, spices, ready-to-eat foods, and foods from other categories. This pathogen was also found in cultivation environments, such as soils, compost, animal feces, rice and vegetable crops, as well as food processing industries, and domestic environments, thus demonstrating possible contamination routes. Furthermore, sequence types (ST) involved in clinical cases and isolates resistant to antibiotics were found in Cronobacter strains isolated from food of plant origin. The identification of Cronobacter spp. in plant-based foods is of great importance to better elucidate the vehicles and routes of contamination in the primary production chain and processing facility, until the final consumption of the food, to prevent infections.

Virulence and DNA sequence analysis of Cronobacter spp. isolated from infant cereals.

Cronobacter spp. is an opportunistic pathogen that causes severe infections, affecting newborns and infants, and is also an emerging cause of hospital-acquired infection in elderly populations. These infections are mainly associated with the consumption of infant formulas, even though these bacteria have been isolated from other foods as well. Cronobacter spp. invades epithelial cells and escapes the immune response mechanisms, multiplying inside macrophages. However, the pathogenesis and virulence factors of these bacteria have not been fully elucidated and need to be further studied. Therefore, this study aimed to evaluate the ability of Cronobacter spp. strains isolated from infant cereals to invade and survive within macrophages, investigate the virulence phenotype using the Galleria mellonella model, and identify possible genes involved in bacterial pathogenesis through pan-genome analysis. All the isolates were able to invade macrophages and the survival of bacteria decreased over a 72 h period, with bacterial cell counts reaching up to 106 CFU/ml. Cronobacter sakazakii isolate 112 exhibited a similar mortality rate (40-70%) to the ATCC BAA 894 strain (Cronobacter sakazakii) in G. mellonella assay. In addition, some unique virulence genes (isolate 7, ada_2, tcmA_1, acrB_3; isolate 78, ampC_2, rihC_1 and isolate 112, fimH, ylpA, gtrA) were identified within isolates with the invasive profile in the in vivo and in vitro assays. Furthermore, isolates from different species were grouped into seven distinct clusters in the pan-genome analysis. The most virulent isolates (7, 78, and 112) were grouped in distinct subclusters in the cladogram. This work revealed potential Cronobacter spp. pathogenic strains recovered from infant cereals.

Isolation, comparison of identification methods and antibiotic resistance of Cronobacter spp. in infant foods.

Cronobacter spp. are opportunistic pathogens that cause serious infections, especially in infants, elderly, and immunocompromised people. Dehydrated infant foods are the main vehicle associated with infections caused by these bacteria. Thus, this study aims to investigate the occurrence of Cronobacter spp. in 152 commercial samples of dehydrated infant formulas (77 samples) and dehydrated infant cereals (75 samples), as well as characterize the isolates. Polymerase Chain Reaction (PCR) and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF/MS) methods for isolate identification were used, and their results compared. Furthermore, the susceptibility to 11 antibiotics was tested, and DNA sequencing of one isolate with multi-drug resistance was analyzed. No contamination in the infant formula samples was found, whereas 17.33% (13/75) of the infant cereal samples presented contamination with Cronobacter sakazakii. The identification results by PCR and MALDI-TOF/MS were divergent for some isolates. The antimicrobial resistance results showed a high incidence of resistance to cefazolin (94.4%) besides resistance to amoxicillin (9.45%), cefpodoxime (5.55%), streptomycin (1.35%), and trimethoprim/sulfamethoxazole (1.35%). Whole genome sequencing of one multi-drug resistant isolate showed six genes associated with antimicrobial resistance and an 82% possibility of being a human pathogen based on the presence of virulence factors. The presence of Cronobacter spp. in infant foods represents a risk for the infant's health. Moreover, the presence of a pathogenic multi-drug resistant isolate in infant's food reinforces the necessity of improving food safety policies to protect young children.

The photodynamic effect of methylene blue and toluidine blue on Candida albicans is dependent on medium conditions.

Due to the increased number of immunocompromised patients, the infections associated with the pathogen of the genus Candida and other fungi have increased dramatically. Photodynamic antimicrobial chemotherapy (PACT) has been presented as a potential antimicrobial therapy, in a process that combines light and a photosensitizing drug, which promotes a phototoxic response by the treated cells. In this work, we studied the effects of the different medium conditions during PACT, using either methylene blue (MB) or toluidine blue (TB) on Candida albicans. The inhibition of the growth produced by PACT was decreased for different pH values (6.0, 7.0, and 8.0) in a buffered medium. The phototoxic effects were observed only in the presence of saline (not buffered medium). PACT was modulated by calcium in a different manner using either MB or TB. Also when using MB both verapamil or sodium azide were able to decrease the phototoxic effects on the C. albicans. These results show that PACT is presented as a new and promising antifungal therapy, however, new studies are necessary to understand the mechanism by which this event occurs.