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Due to drug addiction and the emergence of antibiotic resistance in pathogens, the disease load and medication intake have risen worldwide. The alternative treatment for drug-resistant infections is Nano formulation-based antimicrobial agents. The plant extract of Conocarpus Lancifolius fruits was used to synthesize silver nanoparticles in the current study, and it was further employed as an antimicrobial and anticancer agent. Nanoparticles have been characterized by UV–visible spectrometer revealed the notable peak of λmax = 410–442 nm, which confirms the reduction of silver ion to elemental silver nanoparticles, and the biological moieties in the synthesis were further confirmed by FTIR analysis. The stability and crystalline nature of materials were approved by XRD analysis and expected the size of the nanomaterials of 21 to 173 nm analyzed by a nanophox particle-size analyzer. In vitro, synthesized materials act as an antibacterial agent against Streptococcus pneumonia and Staphylococcus aureus. The inhibition zones of 18 and 24 mm have been estimated to be antibacterial activity against both bacteria. The potency of up to 100% of AgNPs for bacterial strains was incubated overnight at 60 μg/ml. Based on our results, biogenic AgNPs reveal significant activity against fungal pathogen Rhizopusus stolonifera and Aspergillus flavus that cause leading infectious diseases. Additionally, nanomaterials were biocompatible and demonstrated the potential anticancer activities against MDA MB-231 cells after 24-hour exposure.

Microbial levan has great potential as a functional biopolymer in different fields including foods, feeds, cosmetics, and the pharmaceutical and chemical industries. In this study, a good levan producer bacterial strain of Pseudomonas fluorescens strain ES, isolated from soil in Egypt in a previous study, was used. Levan production by this strain was optimized using Plackett-Burman experimental design (PBD) to screen the critical factors of several process variables and Centered Central Composite Design (CCD) was applied for further estimation of the relationship between the variables and the response as well as optimization of the levels. Plackett-Burman (P-B) design showed a p-value 0.0144 less than 0.05 indicated the significance of the model. Sucrose, potassium dihydrogen phosphate, yeast extract and pH value showed the most significant effect on levan concentration at the values of 89.17, 65.83, 24.17, and 15.83, respectively. The purified levan polymer was characterized using different Physico-chemical methods such as Fourier Transform Infrared Spectrometer (FTIR), Nuclear magnetic resonance (NMR), and High-Performance Liquid Chromatography (HPLC) to determine the main composition and functional groups in the obtained polymer. HPLC results indicated that the polymer purification increased the percentage of fructose residue from 75 up to 89. Furthermore, 1H and 13C NMR spectroscopy analysis showed great matching between the obtained signal for our polymer with that reported in other people’s work. The obtained levan polymer exhibited cytotoxic activity against Human epidermoid Skin carcinoma and Hepatocellular carcinoma with IC50 of 469 and 222.7 µg/ml, respectively. Antioxidant activity was determined using DPPH assay and the percentage of inhibition at 1000 µg/ml was found to be

Biologically synthesized metal-oxide nanoparticles (NPs) are in focus for their multifarious applications. In the current study, the green synthesis of copper oxide NPs (CuO NPs) is achieved using the fresh biomass of Cylindrospermum stagnale after its molecular characterization. Physical characterization of CuO NPs by several analytical techniques including UV–Vis analysis, FT-IR spectroscopy, SEM and TEM microscopy revealed spherical shaped crystalline CuO-NPs capped by functional groups of cyanobacterium biomolecules with slight aggregation. CuO NPs were found significantly antimicrobial against pathogens with MICs of 1.5, 2.4, 1.7, 2.5, and 0.6 mM against Candida albicans, Klebsiella pneumoniae, Enterobacter cloacae, Pseudomonas aeruginosa, Escherichia coli, respectively. Moreover, cyanobacterium-capped CuO NPs were cytotoxic against HepG2 cell line and effective against larvae of Aedes ageypti, Anopheles subpictus Grassi and Culex quinquefasciatus. The synthesized CuO NPs were effective due to their high surface area and fairly small size 12.21 nm. Due to adequate biological activities of cyanobacterium CuO NPs even at lower concentrations, we prospect these NPs for clinical applications.

The viral RNA of SARS-Coronavirus-2 is known to be contaminating municipal wastewater. We aimed to assess if COVID-19 disease is spreading through wastewater. We studied the amount of viral RNA in raw sewage and the efficiency of the sewage treatment to remove the virus. Sewage water was collected before and after the activated sludge process three times during summer 2020 from three different sewage treatment plants. The sewage treatment was efficient in removing SARS-CoV-2 viral RNA. Each sewage treatment plant gathered wastewater from one hospital, of which COVID-19 admissions were used to describe the level of disease occurrence in the area. The presence of SARS-CoV-2 viral RNA-specific target genes (N1, N2, and E) was confirmed using RT-qPCR analysis. However, hospital admission did not correlate significantly with viral RNA. Moreover, viral RNA loads were relatively low, suggesting that sewage might preserve viral RNA in a hot climate only for a short time.

Hana Sonbol,1 Afrah E Mohammed,1 Shereen M Korany2 1Department of Biology, College of Science, Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia; 2Botany and Microbiology Department, Faculty of Science, Helwan University, Cairo, 11795, EgyptCorrespondence: Afrah E Mohammed, Department of Biology, College of Science, Princess Nourah bint Abdulrahman University, P.O. Box 84428, Riyadh, 11671, Saudi Arabia, Tel +966 566675853, Email AFAMohammed@pnu.edu.saIntroduction and Objectives: Biogenic agents in nanoparticles fabrication are gaining great interest due to their lower possible negative environmental impacts. The present study aimed to isolate fungal strains from deserts in Saudi Arabia and assess their ability in silver nanoparticles (AgNPs) fabrication and evaluate their antibacterial effect.Methods: Soil fungi were identified using 18s rDNA, and their ability in NPs fabrication was assessed as extracellular synthesis, then UV-vis spectroscopy, dynamic light scattering (DLS), energy-dispersive X-ray spectroscopy, and transmission electron microscopy were used for AgNPs characterization. The antibacterial activity of fungal-based NPs was assessed against one Gram-positive methicillin-resistant S. aureus (MRSA) and three Gram-negative bacteria (E. coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae). Ultrastructural changes caused by fungal-based NPs on K. pneumoniae were investigated using TEM along with SDS-PAGE for protein profile patterns.Results: The three fungal isolates were identified as Phoma sp. (MN995524), Chaetomium globosum (MN995493), and Chaetomium sp. (MN995550), and their filtrate reduced Ag ions into spherical P-AgNPs, G-AgNPs, and C-AgNPs, respectively. DLS data showed an average size between 12.26 and 70.24 nm, where EDX spectrums represent Ag at 3.0 keV peak. G-AgNPs displayed strong antibacterial activities against Klebsiella pneumoniae, and the ultrastructural changes caused by NPs were noted. Additionally, SDS-PAGE analysis of treated K. pneumoniae revealed fewer bands compared to control, which could be related to protein degradation.Conclusion: Present findings have consequently developed an eco-friendly approach in NPs formation by environmentally isolated fungal strains to yield NPs as antibacterial agents.Keywords: nanostructure, soil fungi, Phoma sp., Chaetomium sp., antibacterial, structural changes

CONTEXT: Exploring clinical characteristics of coronavirus disease-19 (COVID-19) in children may help in prevention and treatment guidelines. AIMS: The aim of the to describe the spectrum of pediatric COVID-19 in Saudi Arabia. SETTINGS And DESIGN: A multicenter, retrospective, cross-sectional study involving pediatric COVID-19 patients across all Saudi regions. METHODS: All patients aged between 2 months and 18 years with a confirmed diagnosis of COVID-19 were included. The primary end point was the hospitalization. STATISTICAL ANALYSIS USED: Descriptive statistics were used to describe the baseline demographic data and clinical characteristics. Numerical data were explored using Kolmogorov–Smirnov test and Shapiro–Wilk test, while Chi-square or Fisher's exact test were used for categorical data. RESULTS: Among the 654 pediatric COVID-19 patients, 4.7% (n = 31) were hospitalized, with one patient only needing pediatric intensive care admission. Sex, breastfeeding, birth status, and the patients' living environment showed no significant association with hospitalization. Most children (80.3%, n = 525) were symptomatic, with two symptoms that were significantly associated with admission, namely, vomiting (P = 0.007) and nausea (P = 0.026). History of admission within the last year was identified in 10.4% (n = 68) children but had no association with worse outcome. The median duration of hospitalization for the entire group was 5.5 days, with longest hospital stay for age group 7–12 years (median 6 days). CONCLUSIONS: COVID-19 is usually a milder disease in children. Although having preexisting medical conditions was linked to a longer hospitalization, it was not associated with worse outcome. Continuous surveillance will allow additional characterization of the burden and outcomes of pediatric COVID-19-associated hospitalizations.

Several studies isolated fungal and bacterial species from extreme environments, such as Sabkha and hot deserts, as their natural habitat, some of which are of medicinal importance. Current research aimed investigating the microbial (fungi and bacteria) diversity and abundance in Sabkha and desert areas in Saudi Arabia. Soil samples from nine different geographical areas (Al-Aushazia lake, AlQasab, AlKasar, Tabuk, Al-Kharj, Al-Madina, Jubail, Taif and Abqaiq) were collected and cultured for microbial isolation. Isolated fungi and bacteria were identified by molecular techniques (PCR and sequencing). Based on 18S rDNA sequencing, 203 fungal species belonging to 33 genera were identified. The most common fungal genera were Fusarium, Alternaria, Chaetomium, Aspergillus Cochliobolus and Pencillium, while the most common species were Chaetomium globosum and Fusarium oxysporum. By 16S rDNA sequencing 22 bacterial species belonging to only two genera, Bacillus and Lactobacillus, were identified. The most commonly isolated bacterial species were Bacillus subtilis and Lactobacillus murinus. Some fungal species were confined to specific locations, such as Actinomyces elegans, Fusarium proliferatum, Gymnoascus reesii and Myzostoma spp. that were only isolated from Al-Aushazia soil. AlQasab soil had the highest microbial diversity among other areas with abundances of 23.5% and 4.4% of total fungi, and bacteria, respectively. Findings of this study show a higher degree of fungal diversity than that of bacteria in all studied areas. Further studies needed to investigate the connection between some isolated species and their habitat ecology, as well as to identify those of medicinal importance.

The unregulated discharge of nanoparticles (NPs) from various nanotechnology industries into the environment is expected to alter the composition and physiological functions of soil microbiota. Considering this knowledge gap, the impact of five NPs (Ag, ZnO, CuO, Al2O3, and TiO2) differing in size and morphology on growth behavior and physiological activity of Azotobacter chroococcum, Bacillus thuringiensis, Pseudomonas mosselii, and Sinorhizobium meliloti were investigated. Various biochemical and microscopic approaches were adopted. Interestingly, all bacterial strains were found sensitive to Ag-NPs and ZnO-NPs but showed tolerance toward CuO, Al2O3, and TiO2-NPs. The loss of cellular respiration due to NPs was coupled with a reduction in population size. ZnO-NPs at 387.5 μg mL-1 had a maximum inhibitory impact on A. chroococcum and reduced its population by 72%. Under Ag-NP stress, the reduction in IAA secretion by bacterial strains followed the order S. meliloti (74%) > P. mosselii (63%) > A. chroococcum (49%). The surface of bacterial cells had small- or large-sized aggregates of NPs. Also, numerous gaps, pits, fragmented, and disorganized cell envelopes were visible. Additionally, a treated cell surface appeared corrugated with depressions and alteration in cell length and a strong heterogeneity was noticed under atomic force microscopy (AFM). For instance, NPs induced cell roughness for P. mosselii followed the order 12.6 nm (control) > 58 nm (Ag-NPs) > 41 nm (ZnO-NPs). TEM analysis showed aberrant morphology, cracking, and disruption of the cell envelope with extracellular electron-dense materials. Increased permeability of the inner cell membrane caused cell death and lowered EPS production. Ag-NPs and ZnO-NPs also disrupted the surface adhering ability of bacteria, which varied with time and concentration of NPs. Conclusively, a plausible mechanism of NP toxicity to bacteria has been proposed to understand the mechanistic basis of ecological interaction between NPs and resourceful bacteria. These results also emphasize to develop strategies for the safe disposal of NPs.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly contagious with various possible routes of transmission, resulting in high mortality globally. Controversy exists regarding the vertical transmission of the SARS-CoV-2 infection to fetuses of COVID-19-infected women. The aim of this study was to investigate the possibility of the vertical transmission of SARS-CoV-2 from COVID-19-infected mothers to their neonates.We prospectively collected demographical and clinical characteristics of 31 COVID-19 positive pregnant women and their neonates. All mothers and neonates were tested for SARS-CoV-2 infection using the real-time polymerase chain reaction on nasopharyngeal swabs and breast milk samples. Antenatal and placental abnormalities were ultrasonically and histopathologically examined. In cord blood samples, the immunoglobins (Ig) M and IgG were estimated qualitatively.The women's mean age and gestational age were 31 years and 38 weeks, respectively, with 58% undergoing an elective cesarean section. Gestational diabetes was reported in 29% of cases, 64.5% of women were medically free and only 16.12% were symptomatic. A normal antenatal ultrasound was observed in 77.42% of cases. Nine cord blood samples were positive for IgG. Villous infarction (24%), villous agglutination, and chorangiosis (51%), accelerated villous maturation (21%) and reduced and hypercoiling were reported for 6.97% of the umbilical cords. Three newborns had possible vertical transmission of SARS-CoV-2 infection, of which, two were preterm and IUFD. The third neonate was born full-term, admitted to NICU and later discharged in good health.Our findings support the possibility of the direct vertical transmission of the SARS-CoV-2 infection to neonates from infected mothers. Further studies with a larger sample size are required to validate the current findings.

The antimicrobial activities of certain spices and herbs are well-documented. Herbs and spices are often exposed to bacterial contamination, spore-forming bacteria in particular. The spores introduced during the production process result in a contamination that causes food alteration during storage. The aim of this study was to isolate spore-forming bacteria from commercially available spices and herbs, such as ginger, cinnamon, black pepper, curcumin, saffron, and clove. The API 50 CHB/E medium was used to identify different genera of spore-forming bacteria isolated from samples. We found that these belonged to the genus Bacillus, such as Bacillus cereus, which was isolated from cinnamon, and Bacillus subtilis, and isolated from curcumin and black pepper. The third species belonged to the genus Brevibacillus, Brevibacillus laterosporus, which was isolated from ginger. The D-values (decimal reduction) of each bacterium were determined at 80, 85, 90, 95, and 100 °C. The Z-value was determined for each bacterium. The whole-cell protein profiles of the identified bacteria were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Since spore-forming bacterial contamination of herbs and spices can occur during their harvest, exsiccation, or through cross-contamination during packaging, we propose different methods for spore elimination.

Human campylobacteriosis can be transmitted from animals to human either directly or through contaminated food. So, food safety plays a critical role in preventing the prevalence of food-borne diseases. Probiotics microorganisms are considered one of the most promising new strategies in controlling gastrointestinal tract (GIT) infection. In this study, two probiotic strains Lactobacillus delbrueckii subsp. bulgaricus DSM 20080 and Bifidobacterium bifidum were investigated as anti-Campylobacter agent both in vitro and in vivo. Where they showed elevated antibacterial activity when used as mixed culture with a significant downregulation in the mice intestinal gene expression of IL-1β mRNA and TNF-α mRNA induced by Campylobacter jejuni. Besides, the histopathological investigation of mice intestinal tissue showed great morphological changes in the infected groups with C. jejuni compared with Probiotics treated group that showed healthy intestinal sections with well-defined and enhanced villi. Generally, our findings demonstrate that probiotics have a beneficial and significant effect in the reduction and treatment of destructive effect of C. jejuni on mice gut.

One strategy to manage resistant pathogens and develop potential anticancer drugs is the search for new, promising, and cost-effective medicinal benefits in the field of bioactive metabolites derived from mushrooms. In the current study, Egyptian cultivated

COVID-19 pandemic has severe impacts on human health and economy worldwide. Aerosols and droplets are the major routes of transmission of SARS-CoV-2 coronavirus causing COVID-19 disease. However, wastewater is a possible transmission pathway. Therefore, many studies have been published about the relation of wastewater and COVID-19 disease. Many studies have shown the presence of viral RNA in wastewater throughout the world recently. Therefore, research on wastewater treatments and disinfection methods are needed. Communities must make sure that the virus is not transmitted via treated wastewater. This review focuses on the Saudi Arabian wastewater treatment and disinfection techniques to assess the possibility of SARS-CoV-2 transmission through wastewaters. In view of the current pandemic situation, the wide analysis of wastewater treatments in Saudi Arabia is needed. The review gives guidelines to develop the wastewater treatment in Saudi Arabia.

Twenty fungal strains belonging to 17 species and isolated from male scalp hair were tested for their capacity to hydrolyze keratinous material from chicken feather. The identification of the three most efficient species was confirmed by sequencing of the internal transcribed spacer (ITS) region of rDNA. Activities of fungal keratinases produced by Aspergillus stelliformis (strain AUMC 10920), A. sydowii (AUMC 10935), and Fusarium brachygibbosum (AUMC 10937) were 113, 120, and 130 IU mg−1 enzymes, respectively. The most favorable conditions were at pH 8.0 and 50 °C. Keratinase activity was markedly inhibited by EDTA and metal ions Ca+2, Co+2, Ni+2, Cu+2, Fe+2, Mg+2, and Zn+2, with differences between the fungal species. To the best of our knowledge, this is the first study on the activity of keratinase produced by A. stelliformis, A. sydowii, and F. brachygibbosum. F. brachygibbosum keratinase was the most active, but the species is not recommended because of its known phytopathogenicty. Aspergillus sydowii has many known biotechnological solutions and here we add another application of the species, as producer of keratinases. We introduce A. stelliformis as new producer of active fungal keratinases for biotechnological solutions, such as in the management of keratinous waste in poultry industry.

COVID-19 disease was announced as a global pandemic in March 2020 by the World health organization (WHO). Saudi Arabia was among the first countries to enforce restriction measures such as closing schools, remote working, and a travel ban. We aim to evaluate the impact of the COVID-19 pandemic on people’s depression in Saudi Arabia. A cross-sectional online survey of 1109 participants was conducted during the curfew between 18th of May and 11th of June 2020. An online questionnaire included questions about the commitment to follow the precautionary measures, knowledge on COVID-19, and depression. Depression was assessed with the Impact of Event Scale-Revised method. Females, unmarried individuals, elderly persons, parents of young children, unemployed, and small families were more likely to be depressed. Education level did not explain the differences in depression. However, the more knowledge the participants had about COVID-19 the better they followed the restrictions. A regression analysis revealed that the commitment of a person to follow the restrictions increased his/her depression symptoms. Attention should be paid to different groups of people in future psychiatric planning.

Green synthesized nanoparticles (NPs) have emerged as a new and promising alternative to overcome the drug resistance problem. Peculiar nano-specific features of palladium NPs (Pd-NPs) offer invaluable possibilities for clinical treatment. Due to the development of multi-drug resistance (MDR) in pathogenic bacteria and the prevalence of cancers, use of algae-mediated Pd-NPs could be a prospective substitute. Therefore, Pd-NPs were synthesized by a one-step, cost-effective, and environmentally friendly green method using the extract from a brown alga, Padina boryana (PB-extract), and evaluated for their antibacterial, antibiofilm, and anticancer activities. Pd-NPs were physicochemically characterized for size, shape, morphology, surface area, charge, atomic composition, crystal structure, and capping of Pd-NPs by PB-extract biomolecules by various techniques. The data revealed crystalline Pd-NPs with an average diameter of 8.7 nm, crystal size/structure of 11.16 nm/face-centered cubic, lattice d-spacing of 0.226 nm, 28.31% as atomic percentage, surface area of 16.1 m2/g, hydrodynamic size of 48 nm, and zeta-potential of − 28.7 ± 1.6 mV. Fourier-transform infrared spectroscopy (FT-IR) analysis revealed the role of PB-extract in capping of Pd-NPs by various functional groups such as –OH, C=C, C–O, and C–N from phenols, aliphatic hydrocarbons, aromatic rings, and aliphatic amine. Out of 31, 23 compounds were found involved in biosynthesis by Gas chromatography–mass spectrometry (GC–MS) analysis. Isolated strains were identified as MDR Staphylococcus aureus, Escherichia fergusonii, Acinetobacter pittii, Pseudomonas aeruginosa, Aeromonas enteropelogenes, and Proteus mirabilis and Pd-NPs exhibited strong antibacterial/antibiofilm activities against them with minimum inhibitory concentration (MIC) in the range of 62.5–125 μg/mL. Moreover, cell viability assays showed concentration-dependent anti-proliferation of breast cancer MCF-7 cells. Pd-NPs also enhanced mRNA expression of apoptotic marker genes in the order: p53 (5.5-folds) > bax (3.5-folds) > caspase-3 (3-folds) > caspase-9 (2-folds) at 125 μg/mL. This study suggested the possible role of PB-extract capped Pd-NPs for successful clinical management of MDR pathogens and breast cancer cells.

Soil is considered an extensively explored ecological niche for microorganisms that produce useful biologically active natural products suitable for pharmaceutical applications. The current study aimed at investigating biological activities and metabolic profiles of three fungal strains identified from different desert sites in Saudi Arabia. Soil fungal isolates were collected from AlQasab, Tabuk, and Almuzahimiyah in Saudi Arabia and identified. Furthermore, their antibacterial activity was investigated against Staphylococcus aureus, Enterococcus faecalis, Klebsiella pneumonia, and Escherichia coli in blood, nutrient, and Sabouraud dextrose agars. Moreover, fungal extracts were evaluated on cell viability/proliferation against human breast carcinoma and colorectal adenocarcinoma cells. To identify the biomolecules of the fungal extracts, High-performance liquid chromatography HPLC–DAD coupled to analytical LC–QTOF-MS method was employed for fungal ethyl acetate crude extract. Identified fungal isolates, Chaetomium sp. Bipolaris sp. and Fusarium venenatum showed varied inhibitory activity against tested microbes in relation to crude extract, microbial strain tested, and growth media. F. venenatum showed higher anticancer activity compared to Chaetomium sp. and Bipolaris sp. extracts against four of the tested cancer cell lines. Screening by HPLC and LC/MS-QTOF identified nine compounds from Chaetomium sp. and three from Bipolaris sp. however, for F. venenatum extracts compounds were not fully identified. In light of the present findings, some biological activities of fungal extracts were approved in vitro, suggesting that such extracts could be a useful starting point to find compounds that possess promising agents for medical applications. Further investigations to identify exact biomolecules from F. venenatum extracts are needed.

The unregulated discharge of nanoparticles (NPs) from various nanotechnology industries into the environment is expected to alter the composition and physiological functions of soil microbiota. Considering this knowledge gap, the impact of five NPs (Ag, ZnO, CuO, Al2O3, and TiO2) differing in size and morphology on growth behavior and physiological activity of Azotobacter chroococcum, Bacillus thuringiensis, Pseudomonas mosselii, and Sinorhizobium meliloti were investigated. Various biochemical and microscopic approaches were adopted. Interestingly, all bacterial strains were found sensitive to Ag-NPs and ZnO-NPs but showed tolerance toward CuO, Al2O3, and TiO2-NPs. The loss of cellular respiration due to NPs was coupled with a reduction in population size. ZnO-NPs at 387.5 μg mL–1 had a maximum inhibitory impact on A. chroococcum and reduced its population by 72%. Under Ag-NP stress, the reduction in IAA secretion by bacterial strains followed the order S. meliloti (74%) > P. mosselii (63%) > A. chroococcum (49%). The surface of bacterial cells had small- or large-sized aggregates of NPs. Also, numerous gaps, pits, fragmented, and disorganized cell envelopes were visible. Additionally, a treated cell surface appeared corrugated with depressions and alteration in cell length and a strong heterogeneity was noticed under atomic force microscopy (AFM). For instance, NPs induced cell roughness for P. mosselii followed the order 12.6 nm (control) > 58 nm (Ag-NPs) > 41 nm (ZnO-NPs). TEM analysis showed aberrant morphology, cracking, and disruption of the cell envelope with extracellular electron-dense materials. Increased permeability of the inner cell membrane caused cell death and lowered EPS production. Ag-NPs and ZnO-NPs also disrupted the surface adhering ability of bacteria, which varied with time and concentration of NPs. Conclusively, a plausible mechanism of NP toxicity to bacteria has been proposed to understand the mechanistic basis of ecological interaction between NPs and resourceful bacteria. These results also emphasize to develop strategies for the safe disposal of NPs.

In 2013, Enterobacter helveticus, Enterobacter pulveris and Enterobacter turicensis, were reclassified as Cronobacter helveticus, Cronobacter pulveris and Cronobacter zurichensis, respectively. Previously these species had been used as negative controls for some Cronobacter detection assays. This study examined cultural, biochemical and molecular Cronobacter detection and identification assays, with emphasis on the new species. Additionally, 32 Cronobacter genomes were examined for the presence of PCR target genes using the BLAST function of the online Cronobacter PubMLST facility. The results of the cultural methods varied and no single medium was able to correctly detect all Cronobacter spp. Since the supporting databases have not been updated to include the Cronobacter genus, Enterobacter sakazakii was returned for four strains of the newly reclassified species with ID32E and none with API 20E. PCR probes targeting rpoB and ompA could not correctly identify the new Cronobacter spp., due to primer specificity or absent target genes. As neonates have been identified as a high-risk group for infection, international standards require the absence of all Cronobacter species in powdered infant formula. However, many conventional detection methods cannot correctly identify the newly recognized species. Conversely, DNA sequence-based methods can adapt to taxonomic revisions and will likely become more common.

This study applied the Cronobacter spp. multilocus sequence typing (MLST) scheme to three strain collections, then known as Enterobacter sakazakii, which had been isolated between 1988 and 2009 from 14 countries. The results revealed the predominance (85%) of Cronobacter sakazakii (72 strains) in all three collections. The remaining strains were Cronobacter turicensis (10%), Cronobacter malonaticus (4%), and Cronobacter muytjensii (1%). No strains of Cronobacter dublinensis, Cronobacter universalis or Cronobacter condimenti were identified. Twenty-one out of seventy-two C. sakazakii strains were in the clinically significant ST4 clonal complex, and were found in all three strain collections. These results confirm C. sakazakii ST4 is one of the predominant clonal complexes over the past 20 years in several parts of the world. Further understanding of the ecosystem and sources of the organism may be used for the development of improved intervention strategies in the diary industry.

Cronobacter (previously known as Enterobacter sakazakii ) is a diverse bacterial genus consisting of seven species: C. sakazakii , C. malonaticus , C. turicensis , C. universalis , C. muytjensii , C. dublinensis , and C. condimenti . In this study, we have used a multilocus sequence typing (MLST) approach employing the alleles of 7 genes ( atpD , fusA , glnS , gltB , gyrB , infB , and ppsA ; total length, 3,036 bp) to investigate the phylogenetic relationship of 325 Cronobacter species isolates. Strains were chosen on the basis of their species, geographic and temporal distribution, source, and clinical outcome. The earliest strain was isolated from milk powder in 1950, and the earliest clinical strain was isolated in 1953. The existence of seven species was supported by MLST. Intraspecific variation ranged from low diversity in C. sakazakii to extensive diversity within some species, such as C. muytjensii and C. dublinensis , including evidence of gene conversion between species. The predominant species from clinical sources was found to be C. sakazakii. C. sakazakii sequence type 4 (ST4) was the predominant sequence type of cerebral spinal fluid isolates from cases of meningitis.

Background\ud Species of Cronobacter are widespread in the environment and are occasional food-borne pathogens associated with serious neonatal diseases, including bacteraemia, meningitis, and necrotising enterocolitis. The genus is composed of seven species: C. sakazakii, C. malonaticus, C. turicensis, C. dublinensis, C. muytjensii, C. universalis, and C. condimenti. Clinical cases are associated with three species, C. malonaticus, C. turicensis and, in particular, with C. sakazakii multilocus sequence type 4. Thus, it is plausible that virulence determinants have evolved in certain lineages.\ud \ud Methodology/Principal Findings\ud We generated high quality sequence drafts for eleven Cronobacter genomes representing the seven Cronobacter species, including an ST4 strain of C. sakazakii. Comparative analysis of these genomes together with the two publicly available genomes revealed Cronobacter has over 6,000 genes in one or more strains and over 2,000 genes shared by all Cronobacter. Considerable variation in the presence of traits such as type six secretion systems, metal resistance (tellurite, copper and silver), and adhesins were found. C. sakazakii is unique in the Cronobacter genus in encoding genes enabling the utilization of exogenous sialic acid which may have clinical significance. The C. sakazakii ST4 strain 701 contained additional genes as compared to other C. sakazakii but none of them were known specific virulence-related genes.\ud \ud Conclusions/Significance\ud Genome comparison revealed that pair-wise DNA sequence identity varies between 89 and 97% in the seven Cronobacter species, and also suggested various degrees of divergence. Sets of universal core genes and accessory genes unique to each strain were identified. These gene sequences can be used for designing genus/species specific detection assays. Genes encoding adhesins, T6SS, and metal resistance genes as well as prophages are found in only subsets of genomes and have contributed considerably to the variation of genomic content. Differences in gene content likely contribute to differences in the clinical and environmental distribution of species and sequence types.