Safety assessment of Enterococcus lactis strains complemented with comparative genomics analysis reveals probiotic and safety characteristics of the entire species.

BACKGROUND: The gut microbiota is considered a rich source for potential novel probiotics. Enterococcus genus is a normal component of a healthy gut microbiota, suggesting its vital role. Nosocomial infections caused mainly by E. facalis and E. faecium have been attributed to the plasticity of the Enterococcus genomes. In this study, we assessed the probiotic and safety characteristics of two E. lactis strains isolated from the human gut microbiota using in-vitro and in silico approaches. Additionally, the safety of the E. lactis species was evaluated using comparative genomics analysis. RESULTS: The two E. lactis strains 10NA and 50NA showed resistance to bile salts and acid tolerance with antibacterial activity against Escherichia coli, Salmonella typhi, and Clostridioides difficile. For safety assays, the two strains did not display any type of hemolysis on blood agar, and the survival of Caco-2 cells was not significantly different (P-value > 0.05) compared to the control using cell free supernatants at 100% (v/v), 50% (v/v), 10% (v/v), and 5% (v/v) concentrations. Regarding antibiotic susceptibility, both strains were sensitive to vancomycin, tetracycline, and chloramphenicol. Comprehensive whole-genome analysis revealed no concerning associations between virulence or antibiotic resistance genes and any of the identified mobile genetic elements. Comparative genome analysis with closely related E. faecium species genomes revealed the distinctive genomic safety of the E. lactis species. CONCLUSIONS: Our two E. lactis strains showed promising probiotic properties in-vitro. Their genomes were devoid of any transferable antibiotic resistance genes. In silico comparative analysis confirmed the safety of the E. lactis species. These results suggest that E. lactis species could be a potential source for safer Enterococcus probiotic supplements.

Prevalence and In Vivo Assessment of Virulence in Shiga Toxin-Producing Escherichia coli Clinical Isolates from Greater Cairo Area.

Background: Shiga toxin-producing Escherichia coli (STEC) has been identified as an important etiologic agent of human disease in Egypt. Aims: To investigate the occurrence and describe the characterization as well as prevalence of STEC in Greater Cairo hospitals as well as molecular characterization of virulence and resistance genes. Methods: Four hundred seventy E. coli clinical isolates were collected from eight hospitals and analyzed by genotypic and phenotypic methods for STEC, followed by histopathological examination and scoring of different organs lesions. Results: The highest proportion of isolates was from urine (151 isolates), whereas the lowest was from splenic drain (3 isolates). In tandem, when serogrouping was performed, 15 serogroups were obtained where the most prevalent was O157 and the least prevalent was O151. All isolates were positive when screened for identity gene gad A, while only typable strains were screened for seven virulence genes stx1 (gene encoding Shiga toxin 1), stx2 (gene encoding Shiga toxin 2), tsh (gene encoding thermostable hemagglutinin), eaeA (gene encoding intimin), invE (gene encoding invasion protein), aggR (gene encoding aggregative adherence transcriptional regulator), and astA (aspartate transaminase) where the prevalence was 48%, 30%, 50%, 57%, 7.5%, 12%, and 58%, respectively. Of 254 typable isolates, 152 were STEC carrying stx1 or stx2 genes or both. Conclusions: Relying on in vivo comparison between different E. coli pathotypes via histopathological examination of different organs, E. coli pathotypes could be divided into mild virulent, moderate virulent, and high virulent strains. Statistical analysis revealed significant correlation between different serogroups and presence of virulence genes.

Acinetobacter baumannii, Klebsiella pneumoniae and Elizabethkingia miricola isolated from wastewater have biodegradable activity against fluoroquinolone.

Ciprofloxacin (CIP) and levofloxacin (LEV), widely used fluoroquinolone antibiotics, are often found in sewage from the sewage treatment plants and marine environment. In this study, CIP and LEV biodegrading bacterial consortia were obtained from industrial wastewater. Microorganisms in these consortia were identified as Acinetobacter baumannii (A. baumannii), Klebsiella pneumoniae (K. pneumoniae) and Elizabethkingia miricola (E. miricola). The impacts of the critical operating parameters on the elimination of CIP and LEV by bacterial consortia have been investigated and optimized to achieve the maximum levels of CIP and LEV biodegradation. Using liquid chromatography with tandem mass spectrometry (LC-MS-MS), possible degradation pathways for CIP and LEV were suggested by analyzing the intermediate degradation products. The role of the enzymes fluoroquinolone-acetylating aminoglycoside (6'-N-acetyltransferase) and cytochrome P450 (CYP450) in the breakdown of fluoroquinolones (FQs) was investigated as well. According to our findings, various biodegradation mechanisms have been suggested, including cleavage of piperazine ring, substitution of F atom, hydroxylation, decarboxylation, and acetylation, as the main biotransformation reactions. This study discovers the ability of non-reported bacterial strains to biodegrade both CIP and LEV as a sole carbon source, providing new insights into the biodegradation of CIP and LEV.

The inhibitory effect of dextranases from Bacillus velezensis and Pseudomonas stutzeri on Streptococcus mutans biofilm.

Background and Objectives: Dental caries is a breakdown of the teeth enamel due to harmful bacteria, lack of oral hygiene, and sugar consumption. The acid-producing bacterium Streptococcus mutans is the leading cause of dental caries. Dextranase is an enzyme that can degrade dextran to low molecular weight fractions, which have many therapeutic and industrial applications. The purpose of the present study was to isolate a novel dextranase-producing bacteria from a source (molasses). The cell-free extracts containing dextranases were tested as antibiofilm agents. Materials and Methods: Dextranase-producing bacteria were identified using phenotypic and genotypic methods such as 16S rRNA gene sequencing and enzymatic characterization. Results: The highest six dextranase-producing bacterial isolates were Bacillus species. The best conditions for dextranase productivity were obtained after 72 hours of culture time at pH 7. The addition of glucose to the medium enhanced the production of the enzymes. The cell-free extract of the six most active isolates showed remarkable activity against biofilm formation by Streptococcus mutans ATCC 25175. The highest inhibition activities reached 60% and 80% for Bacillus velezensis and Pseudomonas stutzeri, respectively. Conclusion: Therefore, our study added to the current dextranase-producing bacteria with potential as a source of dextranases.

A Study on Multidrug-Resistant Escherichia coli Clinical Isolates from Different Hospitals in Greater Cairo.

The biological fitness cost of antibiotic resistance is a key parameter in determining the rate of appearance and spread of antibiotic-resistant bacteria in Egypt. Our study aimed to investigate the prevalence of antibiotic resistance among Escherichia coli clinical isolates from Greater Cairo area hospitals. A total of 537 clinical isolates were recovered from samples of urine, diarrheal specimen, pus, wound culture, gastric wound, blood, drain culture, sputum, high vaginal swab, abscess, amniotic fluid, ventilator, burn swab, splenic drain culture, and unknown site of infection during different seasons. All isolates were subjected to phenotypic and genotypic susceptibility testing for colistin, nitrofurantoin, fosfomycin, and trimethoprim, quinolones, and ß-lactam resistance. Our results revealed that 42.7% of the isolates harbored at least one resistance encoding gene, 10% harboring 2, 0.6% harboring 3, and 0.85% harboring 4 resistance-encoding genes. PCR reported the prevalence of resistance genes as follows: bla-SHV 13.4%, mcr-1 0.6%, qnr-A 23.8%, fos-A 1.06%, nfs-A 3.6%, and dfr-A 25.5%. We reported that three isolates carried the mcr-1 gene encoding colistin resistance from three different hospitals. Upon performing sequencing and phylogenetic analysis on the three positive mcr-1 isolates (MT890587, MT890588, and MT890589), the three isolates showed 100% identity with themselves, with some strains from Egypt and Japan, and 99.9% identity with an isolate from China.

Expression levels of pro-inflammatory interleukin-8 and certain antimicrobial peptides in concurrent with bacterial conjunctivitis.

AIM: To detect the quantitative expression levels of the pro-inflammatory interleukin-8 (IL8), antimicrobial peptides human beta defense-2 (HBD2), and human beta defense-3 (HBD3) genes in bacterial conjunctivitis. METHODS: The human conjunctival epithelial cells were obtained using the impression cytology technique from healthy controls and patients. The genes expression levels were determined utilizing a reverse transcription quantitative polymerase chain reaction (RT-qPCR). The contribution of causative agent type, the number of isolates and severity of clinical features, in the increase of genes expression was also determined. RESULTS: The RT-qPCR showed that IL8, HBD2, and HBD3 expression increased in bacterial conjunctivitis as compared to healthy control (P<0.001). In gram-negative bacterial conjunctivitis, HBD2 was highly up-regulated (P<0.001) compared to other types of bacterial conjunctivitis. In mixed bacterial conjunctivitis, a direct correlation between HBD2 up-regulation and HBD3 up-regulation was observed (P<0.05). The severity of clinical features was related to the up-regulation of IL8 and HBD2 (P<0.05). CONCLUSION: IL8, HBD2, and HBD3 are immune-effectors in infectious conjunctivitis. HBD2 is active during different bacterial conjunctivitis but is more released with gram-negative bacteria compared to gram-positive bacteria. HBD3 is an obvious defender in different bacterial conjunctivitis.

SARS-CoV-2: The Path of Prevention and Control.

The 2019 coronavirus pandemic (COVID-19) continues to expand worldwide. Although the number of cases and the death rate among children and adolescents are reported to be low compared to adults, limited data have been reported. We urgently need to find the treatment and vaccine to stop the epidemic. Vaccine development is in progress, but any approved and effective vaccine for COVID-19 will take at least 12 to 18 months. The World Health Organization (WHO), the Center for Disease Control and Prevention (CDC), and the Food and Agriculture Organization (FAO) have issued instructions and strategies for controlling COVID-19 outbreak to the general public, physicians, travelers and injured patients to follow so that the transmission to a healthy population can be prevented. In this review, we summarize demographic data, clinical characteristics, complications and outcomes and finally prevention and control strategies for this serious pandemic.

Correction to: Hepatitis D virus seroprevalence in Egyptian HBsAg­positive children: a single­center study.

Unfortunately, the affiliation of author "Rania Abdelmonem Khattab" was published incorrectly in the original publication. The correct version of affiliation is updated here.

Hepatitis D virus seroprevalence in Egyptian HBsAg-positive children: a single-center study.

In this study, we investigated the seroprevalence of anti-hepatitis D virus (HDV) antibodies in hepatitis B surface antigen (HBsAg)-positive children after 25 years of obligatory vaccination of infants against hepatitis B virus. This cross-sectional study included 120 treatment-naïve HBsAg-positive children, with a male-to-female ratio of 1.8:1 and a mean age of 7.8 ± 3.8 years (range, 1-17 years). Mothers were positive for HBsAg in 96.6% of the cases. HBeAg-positive chronic infection was observed in 60% of the cases, HBeAg-positive chronic hepatitis in 12.5%, and HBeAg-negative chronic infection in 26.7%. Anti-HDV antibodies were not detected in any of the cases. Thus, there is a lack of anti-HDV antibodies in HBsAg-positive children, despite the current burden in adults.

Bacteria producing antimicrobials against Clostridium difficile isolated from human stool.

Clostridium difficile infection (CDI) is a common cause of morbidity and mortality in hospitalized patients worldwide. The major problem facing current treatment is multiple recurrences, prompting the need for alternative therapies. In this study we isolated bacterial species, from Egyptian individuals' stool, with antimicrobial activity against clinical isolates of C. difficile and tried to examine the nature of the produced antimicrobials. In vitro antibacterial activity against C. difficile was initially screened in 123 fecal samples cultures using an agar overlay method. The isolates with antimicrobial activity against C. difficile in addition to Clostridium isolates were identified using partial 16S rDNA gene sequencing analysis. The isolates acting against C. difficile belonged to Lactobacillus, Enterococcus and Clostridium genera. The concentrated cell-free supernatants (CFSs) from these bacterial isolates were examined for antimicrobial activity against C. difficile growth by broth dilution method. 10 x concentrated CFSs of five isolates showed inhibition for C. difficile growth which was significantly different (p < 0.001) from control. Lactobacillus agilis T99A and Clostridium butyricum T58A isolates were selected for further evaluation of the produced antimicrobials. The antimicrobial activity of 10x CFSs of the two isolates was stable after enzymatic treatment with proteinase K or heating treatments up to 90 °C or neutralizing pH. The spectrum of activity of the two isolates was evaluated using different gram-positive and gram-negative bacterial species and did not show antimicrobial activity against these species. Our results showed two unconventional bacterial isolates: L. agilis T99A and C. butyricum T58A producing extracellular thermo stable antimicrobial agents against C. difficile clinical isolates.

Importance of Studying the Levels of Hepcidin and Vitamin D in Egyptian Children with Chronic Hepatitis C.

BACKGROUND AND OBJECTIVE: Hepcidin is the key regulator of iron metabolism and is a significant biomarker for systemic inflammatory states. Vitamin D is a powerful immunomodulator and plays a significant role in the inflammatory responses and fibrosis occurring due to hepatitis C virus (HCV) infection. This study assessed the level of vitamin D and serum hepcidin and its expression in peripheral blood of children with chronic hepatitis C (CHC) and correlated them with other serum markers to reflect iron metabolism and liver disease severity. METHODS: A total of 100 children were included in this study: 50 with HCV infection and 50 healthy controls. Biochemical parameters together with vitamin D, hepcidin, and its expression were all measured. RESULTS: The level of hepcidin and its expression together with vitamin D and hepcidin-to-ferritin (H/F) ratios were significantly reduced in patients, but the iron and ferritin levels were higher (P<0.001). Serum hepcidin level showed significant positive correlation with hepcidin expression, HCV titer, iron, ferritin, and H/F ratio (r = 0.43, 0.31, 0.34, 0.28, and 0.91, respectively) but significant negative correlation with vitamin D (r = -0.37). Both hepcidin and ferritin were higher in patients with Child Pugh scores B and C than those with score A (P<0.001). CONCLUSION: Measuring serum hepcidin and its expression together with vitamin D levels in patients may have a prognostic value and is promising in the follow-up of the severity of liver disease.

Mining of Egypt's Red Sea invertebrates for potential bioactive producers.

OBJECTIVE: The objective of this work was to isolate bacteria from Red Sea invertebrates, determine their antimicrobial activity, and screen for the biosynthetic gene clusters [polyketides (PKs) and nonribosomal peptides (NRPs)] which could be involved in the production of bioactive secondary metabolites. RESULT: Eleven different samples of marine invertebrates' were collected from Egypt's Red Sea (El-Tor-Sharm El-Sheikh and Hurghada) by scuba diving, and a total 80 isolates of the associated microorganisms were obtained from the cultivation on six different cultural medium. Seven isolates of them showed an antimicrobial activity against five pathogenic reference strains, while the most active antimicrobial agent was isolate number HFF-8 which was 99% identical to Bacillus amyloliquefaciens. HFF-8's extract showed positive results against Gram negative bacteria, Gram positive bacteria and yeast. Moreover, the isolates gave positive bands when screened for the presence of PK synthase (PKS) I and II and NRP synthetase (NRPS) I and II biosynthetic genes, those biosynthetic fragments when cloned and sequenced were primitively predicted as biosynthetic fragments for kirromycin and leinamycin production by NaPDoS program with 56 and 55%, respectively. CONCLUSION: The Red Sea can provide a sustainable solution to combat bacterial resistance. The contribution of this work is that B. amyloliquefaciens was isolated from Heteroxenia fuscescens, Red Sea, Egypt. Moreover, the bacterial extract showed a broad spectrum with a potent antimicrobial activity.

Erratum to: Assessment of Vitamin D status in a group of Egyptian children with non alcoholic fatty liver disease (multicenter study).

[This corrects the article DOI: 10.1186/s12986-016-0112-z.].

Evaluation of patients with dry eye disease for conjunctival Chlamydia trachomatis and Ureaplasma urealyticum.

AIM: To determine the possibility of the development of dry eye disease (DED) as a result of persistent infection with Chlamydia trachomatis and Ureaplasma urealyticum in the conjunctiva of patients. METHODS: This study was conducted on 58 patients of age range 20-50y, diagnosed with DED confirmed by Schirmer I test and tear breakup time. The non-dry eye control group included 27 subjects of the same age. Ocular specimens were collected as conjunctival scrapings and swabs divided into three groups: the first used for bacterial culture, the second and third taken to detect Chlamydia trachomatis and Ureaplasma urealyticum by direct fluorescent antibody (DFA) assay and polymerase chain reaction (PCR) method. RESULTS: Chlamydia trachomatis was detected in 65.5% and 76% of DED patients by DFA and PCR methods respectively. Ureaplasma urealyticum was found in 44.8% of DED infected patients using the PCR method. Both organisms were identified in only 37.9% of DED patients found to be infected. Control subjects had a 22% detection rate of Chlamydia trachomatis by DFA assay versus a 7% detection rate by PCR; while Ureaplasma urealyticum was detected in 3.7% of the controls by PCR method. The conjunctival culture revealed that gram positive microorganisms represented 75% of isolates with coagulase negative Staphylococci the most common (50%) followed by Staphylococcus aureus (20%), whereas gram negative microorganisms occurred in 25% of cases, isolating Moraxella spp. as the most frequent organism. CONCLUSION: Our results tend to point out that Chlamydia trachomatis and Ureaplasma urealyticum were detected in a moderate percentage of patients with DED, and could be a fair possibility for its development. PCR is more reliable in detecting Chlamydia trachomatis than DFA technique. The presence of isolated conjunctival bacterial microflora can be of some potential value.

Study of the prevalence and association of ocular chlamydial conjunctivitis in women with genital infection by Chlamydia trachomatis, Mycoplasma genitalium and Candida albicans attending outpatient clinic.

AIM: To determine the association between chlamydial conjunctivitis and genital infection by Chlamydia trachomatis, Mycoplasma genitalium and Candida albicans, in addition to the possible relationship between cultured bacterial pathogens and oculogenital chlamydial infection. METHODS: This study was performed on 100 (50 symptomatic and 50 asymptomatic) women attending the Gynecological and Obstetric outpatient clinic of Alzahra hospital, Alazhar University. Simultaneously a conjunctival swab was taken from these patients. Polymerase chain reaction (PCR) was done on DNA extracted from both vaginal and conjunctival swab samples. Culture for both vaginal and conjunctival swabs was also done. RESULTS: Candida albicans was the predominant organism isolated by culture in 20% and 40% of conjunctival and vaginal swabs respectively. By the PCR method, ocular Chlamydia trachomatis was present in 60% of symptomatic women, while genital Chlamydia trachomatis infection was present in 30% of symptomatic women. The results of this method also indicated that 25/50 (50%) vaginal swabs were positive with PCR for Candida albicans versus 15/50 (30%) were PCR positive in conjunctival swab. Mycoplasma genitalium was present in only 10% of vaginal swabs. Concomitant oculogenital PCR positive results for Chlamydia trachomatis and Candida albicans were 30% and 28% respectively. CONCLUSION: Ocular Chlamydia trachomatis was associated with genital Chlamydia trachomatis in a high percentage of women followed by Candida albicans. Cultured bacterial organisms do not play a role in enhancement of Chlamydia trachomatis infection.

Immune response to Vi polysaccharide, heat-killed whole cells, and outer membrane protein of Salmonella typhi.

INTRODUCTION: Salmonella typhiVi capsular polysaccharide (ViCPS) is a licensed vaccine against typhoid fever in many countries; in Egypt, the killed whole-cell vaccine is still used. In this study, mice were used as an animal model to evaluate the immune response to ViCPS and other S. typhi antigens such as heat-killed whole cells and outer membrane protein (OMP). METHODOLOGY: The three antigens were laboratory prepared, injected into mice groups, and the humoral response was evaluated using the indirect whole-cell enzyme-linked immunosorbent assay (ELISA). The sensitivity of this assay was investigated using in situ or pre-heated whole cells as coating antigens. In addition, the effect of the immunization route for ViCPS was examined. RESULTS: Immunizing doses of heat-killed whole cells as well as ViCPS, 2 and 4 µg given subcutaneously (SC) and 4 µg given intraperitoneally (IP), showed significant immune response compared to controls. However, the responses to these doses were not significantly different from each other. The OMP showed a higher significant response. The sensitivity of indirect whole-cell ELISA was enhanced significantly by in situ heat treatment of the coating antigen rather than the pre-heated coating antigen. CONCLUSIONS: The three antigens showed significant immune response. The immune response to OMP was higher. Though heat-killed whole cells and ViCPS are almost similar in immunizing level, ViCPS is recommended. The SC route was more immunizing than the IP one. Furthermore, the sensitivity of the indirect whole-cell ELISA technique could be enhanced by in situ heat inactivation of the coating cells.