Correlation Between Antimicrobial Resistance And Virulence Genes In Klebsiella Pneumoniae Isolates From Egypt.

Objectives: To genotypically assess the relationship between certain resistance and virulence determinants. Method: The cross-sectional study was conducted at Kafrelsheikh University, Egypt, from March 2019 to May 2021, and comprised pathologicalsamples, like blood,sputum, urine, vaginalswabs and wound swabs, that had been taken from patients who had never received treatment. The sample were collected from Kafrelsheikh and Mansoura University hospitals, and Klebsiella pneumoniae isolates were obtained. Resistance and virulence determinants were tested phenotypically. Uniplex polymerase chain reaction was used to evaluate the presence of several resistance accompanied genes and virulence genes in the isolates. Disc diffusion method was used to assess the isolates' susceptibility in accordance with the Clinical and Laboratory Standards Institute criteria for identifying diverse resistance patterns. RESULTS: There were 23 isolatesfrom 16 patients. Of the tested isolates, 22(95.65%)showed drug resistance; 19(82.6%) had multidrug resistance, and 3(13.04%) had extensive drug resistance. There was no case of pan drug resistance. CTX-M-15, NDM, CTX-M-1, VIM-1 and qnr B genes were detected in 14(60.86%), 13(56.5%), 6(26.08%), 6(26.08%) and 6(26.08%) isolates, respectively. Moreover, 6(26.08%) isolates exhibited extended-spectrum ß-lactamase producers, and 12(52.17%) ofsuch isolates contained both CTX-M-1 and CTX-M-15 genes, 6 and 33.3% contained CTX-M-1, CTX M-15 and fox genes. Type 3 fimbriae adhesin mrkD and mucoviscosity regulatory gene uge were found in the tested isolates. However, gene of iron uptake system kfu wasfound in 8(34.78%) isolates, and increased serum survival protein is and mucoviscosity accompanied gene magA were detected in 3(13.04%) isolates. A direct correlation was found among 5 from 8 Klebsiella pneumoniae virulence genes and antimicrobial resistance genes. CONCLUSIONS: There was a direct correlation between the existence of virulence factors and resistance to antimicrobials.

Coexistence Of Blaoxa-48, Blavim, And Blashv Genes In Klebsiella Pneumoniae And Escherichia Coli Isolated From Urinary Tract Infections: Microbiological And Epidemiological Analysis.

Objectives: To investigate antimicrobial resistance mechanisms of isolated bacterialstrains, and their correlation with virulence profile. Method: The cross-sectional study was conducted in January 2020 at outpatient health centres in Kafrelsheikh Governorate of Egypt, and comprised urine samples from patients regardless of age and gender. Midstream samples were collected into sterile swaps which were kept in ice-cooled boxes until transported to the laboratory within 5h. Antimicrobial resistance profile of the isolated Enterobacteriaceae was done using Kirby-Bauer disk diffusion method and was confirmed withVitek compact 2. The phenotypic of carbapenemases and extended-spectrum beta lactamase was determined, and polymerase chain reaction was used, as appropriate. Data was analysed using SPSS 20. RESULTS: Of the 199 patients, 101(50.7%) were females and 98(49.3%) were males. The majority 73(36.6%) were aged 30-50 years. Urinary tract infection was found in 68(34.2%) patients. In 28(41.2%) of these patients, there were 32 isolates of Enterobacterales; 21(65.62%) Klebsiella pneumoniae, 7(21.87%) Escherichia coli and 4(12.5%) Enterobacter cloacae. Of the 28(41.2%) patients, 24(85.7%) were infected with a single strain; 17(70.8%) Klebsiella pneumoniae, 4(16.7%) Escherichia coli and 3(12.5%) Enterobacter cloacae. In 3(10.7%) cases, there was co-infection with Escherichia coli and Klebsiella pneumoniae, and 1(3.6%)sample had mixed infection with Klebsiella pneumoniae and Enterobacter cloacae. The other 40(58.8%) patients had other causative agents. Housewives, agricultural workers and those aged >50 years had a higher risk of urinary tract infections(p<0.05) Among Klebsiella pneumonia isolates, 6(28.5%) possessed carbapenemase-related genes and 4(19.1%) extended-spectrum beta lactamase-related genes. The carbapenemase related genes were bla-Verona integron-encoded metallo beta lactamase 6(100%) bla-New Delhi metallo beta lactamase-1 4(66.6%) and bla-oxacillinase-48 2(33.3%). The 4(19.1%) cases of extended-spectrum beta lactamase related genes had bla-temoneira gene 3(75%) and bla-sulfhydryl variable gene 4(100%). In Escherichia coli isolates, bla-oxacillinase-48 and bla-Cefotaximase genes were observed in 2(28.5%) cases.Virulence genes uridine diphosphate glucose 4-epimerase, fimbrial adhesion and mannose-resistance adhesin of Klebsiella spp genes in Klebsiella pneumoniae isolates were positive in in 16(76.2%), 14(66.7%) and 10(47.6%) cases, respectively. All 21(100%) isolates of Klebsiella pneumoniae were negative for mucoviscosity-associated gene A. CONCLUSIONS: There was evidence of the coexistence of bla- oxacillinase-48, bla-Verona integron-encoded metallo beta lactamase and bla-sulfhydryl variable genes in Klebsiella pneumoniae and Escherichia coli isolates from mixed urinary tract infection samples.

Immune Response And Pathophysiological Features Of Klebsiella Pneumoniae In Mice.

Objectives: To assess the bacterial colonisation of mice organs and faeces infected with 3 strains of Klebsiella pneumoniae, to measure levels of tumour necrosisfactor-alpha, tumour necrosisfactor-beta and interleukin-6 in mice serum, and to evaluate immune response of mice infected with Klebsiella pneumoniae. Method: The animalstudy was conducted at Kafreslsheikh University, Egypt, in 2021, and comprised mice 5-7 weeks old who were infected with 3 strains of Klebsiella pneumoniae; K80uge+ (uri, kfu+, mrkD+; K68 gyrA+(gyrase A), mrkD+; and K84 uge+, kfu+, mrkD+". They were monitored for 14 days. The bacterial colonisation of mice livers, lungs, spleens and faeces were determined using culture on MacConkey agar. The percentage of neutrophils detected as cluster of differentiation 11b+ and cluster of differentiation 45+ in the mice serum was determined by flow cytometry. Levels of tumour necrosis factor-alpha and tumour necrosis factor-beta were measured using enzyme-linked immunosorbent assay. RESULTS: There were 4 sets of female mice [1 control and 3 infected groups for which 3 K. pneumoniae strains (K80 uge+, kfu+, mrkD+; K68 gyrA+, mrkD+; and K84 "uge+, kfu+, mrkD+)] weighing 13-24gm was used. Colonisation of mice organs and faeces was high after 24 hours then declined rapidly after 3 days, 10 days and 14 days in case of infection with capsulated and non-capsulated strains of bacteria. Livers, lungs and spleens showed maximum inflammation after 24 hours, then declined rapidly. Both cytokine production and organ inflammation increased after one day of infection. There was a significant correlation between the produced cytokines and histopathological changesin liver, lung and spleen. The neutrophils increase in case of infection with K84 and K80 was more than non-capsulated K68. CONCLUSIONS: Neutrophils were found to play an important role in the clearance and treatment of Klebsiella pneumoniae.

Overview on Cryptosporidium bovis and Its Effect on Calves in Some Governorates in Egypt.

The present study was conducted to elucidate the prevalence of Cryptosporidium bovis in suckling and weaned cattle calves (Bubalus bubalis) from different governorates in northern, middle, and southern Egypt, such as Behera, Menofia, Qaliubiya, Assiut, and Sohag; result revealed that from the overall examined fecal samples (n = 825), the overall prevalence was 7.27%, the highest significant infection rate was in young suckling calves less than one month (8.2%), and seasonally, winter season has the highest significant level (11.24%), but sex and locality were of no significant effect on the prevalence of infection in this study. Gene sequencing and phylogenetic analysis of the 18SSU-rRNA gene of the local bovine isolate were performed, and it was found that C. bovis genotype was highly similar to human isolate, which provoke the zoonotic transmission of bovine isolate to humans and identified as a potential source for human cryptosporidiosis infection in Egypt.

The Prevalence and Molecular Biology of Staphylococcus aureus Isolated from Healthy and Diseased Equine Eyes in Egypt.

This work aimed to characterize S. aureus isolates from the eyes of healthy and clinically affected equines in the Kafrelsheikh Governorate, Egypt. A total of 110 animals were examined for the presence of S. aureus, which was isolated from 33 animals with ophthalmic lesions and 77 healthy animals. We also investigated the antimicrobial resistance profile, oxacillin resistance mechanism, and the major virulence factors implicated in many studies of the ocular pathology of pathogenic S. aureus. The association between S. aureus eye infections and potential risk factors was also investigated. The frequency of S. aureus isolates from clinically affected equine eyes was significantly higher than in clinically healthy equids. A significant association was found between the frequency of S. aureus isolation from clinically affected equine eyes and risk factors including age and season but not with sex or breed factors. Antimicrobial resistance to common antibiotics used to treat equine eyes was also tested. Overall, the isolates showed the highest sensitivity to sulfamethoxazole (100%) and the highest resistance to cephalosporin (90.67%) and oxacillin (90.48%). PCR was used to demonstrate that mecA was present in 100% of oxacillin- and ß-lactam-resistant S. aureus strains. The virulence factor genes Spa (x region), nuc, and hlg were identified in 62.5%, 100%, and 56%, of isolates, respectively, from clinically affected equines eyes. The severity of the eye lesions increased in the presence of γ-toxin-positive S. aureus. The phylogenetic tree of the Spa (x region) gene indicated a relationship with human reference strains isolated from Egypt as well as isolates from equines in Iran and Japan. This study provides insight into the prevalence, potential risk factors, clinical pictures, zoonotic potential, antimicrobial resistance, and ß-lactam resistance mechanism of S. aureus strains that cause eye infection in equines from Egypt.

Epidemiological, Morphological, and Morphometric Study on Haemonchus spp. Recovered From Goats in Egypt.

Goats can be infected by multiple groups of external and internal parasites. Haemonchus spp. are among abomasal parasites that can result in higher mortality and several considerable economic losses in goats. Early detection of parasites and better understanding of the major risk factors associated with infection are among the main strategies for controlling the infection. Considering this, information on hemonchosis in goats from Egypt, and the contribution of goats in the maintenance of the epidemiological foci of the disease is limited. This study investigated the prevalence of Haemonchus species among 240 abomasum samples collected during postmortem examination of goat carcasses from Assiut Governorate, Egypt. Moreover, the association of the major risk factors to describe the epidemiological pattern of the disease was explored. This study demonstrated that 16.66% of abomasa samples harbored Haemonchus species. Additionally, age, sex, and sampling season were the most significant risk factors associated with infection. Following the variable factors under study, goats aged 1 year or older were at higher risk, with an infection rate of 22.14% (31 of 140), than those younger than 1 year (9%) [p = 0.008; odds ratio (OR) = 2.87; 95% confidence interval (CI), 1.30-6.35]. The infection rate was 25% (19 of 76) in males and 12.8% (21 of 164) in females [p = 0.024; odds ratio (OR) = 2.26; 95% confidence interval (CI), 1.13-4.53]. Moreover, the exposure to infection was higher in summer (22.22%) than in winter (8.33%) (p = 0.007; odds ratio (OR) = 0.318; 95% confidence interval (CI), 0.139-0.725). More importantly, three species of the parasite-Haemonchus contortus, Haemonchus placei, and Haemonchus longistipes-were identified for the first time, and the confirmation of the identification and morphological characterization of the worms was performed using light microscopy and SEM. Collectively, this study reveals interesting epidemiological, morphological, and morphometric findings associated with the occurrence of hemonchosis among goats in Egypt. This study suggests further research for exploring the major circulating species of the parasite in Egypt, which is mandatory for controlling the disease.

Epidemiological, Morphometric, and Molecular Investigation of Cystic Echinococcosis in Camel and Cattle From Upper Egypt: Current Status and Zoonotic Implications.

Cystic echinococcosis has been considered one of the major parasitic zoonoses which is associated with severe economic losses. The present study was undertaken to investigate the occurrence, organ distribution, cyst fertility, and viability of cystic echinococcosis in slaughtered camels and cattle from various abattoirs in Assiut Governorate, Egypt. The work also involved morphological, morphometric, and molecular identification of the parasite. The occurrence of hydatid cysts was investigated in total number of 100 lungs of camels and 574 liver and lungs of cattle admitted to three slaughterhouses at Assiut Governorate, Egypt. Moreover, several individual variable factors, including organ involvement, age, sex, and hydatid cyst characteristics, were studied to identify their possible association with the occurrence of the disease. Genomic DNA was extracted from the hydatid cysts, followed by molecular identification of the parasite through amplification of ribosomal DNA internal transcribed spacer (ITS) regions. Hydatid cysts were found in 6 camels (6%) out of 100 inspected camels, while 5 hydatid cysts (0.87%) were detected in a total number of 574 cattle examined. The parasite was detected exclusively in lungs of camels, while lungs were the main organ infected by the parasite in cattle and one hydatid cyst was found in the liver (0.17%). In camel, 66.7, 16.65, and 16.65%of detected cysts were fertile, sterile, and calcified, respectively, while in cattle, these percentages were 60, 20, and 20%, respectively. None of the studied variable factors were significantly associated with the occurrence of the disease in camels, with the exception that all cysts were found in the lung. Conversely, we found a significant association (P < 0.05) between the age and sex of the slaughtered cattle and the occurrence of hydatid cysts. In this respect, the rate of infection was higher in female cattle and those cattle more than 5 years (P < 0.05). The morphological, morphometric, and molecular studies confirmed the presence of the parasite. Taken together, our results concluded that camels and cattle play a potential role in maintaining the transmission cycle of this zoonotic parasite.

Mitotic Arrest-Deficient 2 Like 2 (MAD2L2) Interacts with Escherichia coli Effector Protein EspF.

Enteropathogenic (EPEC) and Enterohemorrhagic (EHEC) Escherichia coli are considered emerging zoonotic pathogens of worldwide distribution. The pathogenicity of the bacteria is conferred by multiple virulence determinants, including the locus of enterocyte effacement (LEE) pathogenicity island, which encodes a type III secretion system (T3SS) and effector proteins, including the multifunctional secreted effector protein (EspF). EspF sequences differ between EPEC and EHEC serotypes in terms of the number and residues of SH3-binding polyproline-rich repeats and N-terminal localization sequence. The aim of this study was to discover additional cellular interactions of EspF that may play important roles in E. coli colonization using the Yeast two-hybrid screening system (Y2H). Y2H screening identified the anaphase-promoting complex inhibitor Mitotic Arrest-Deficient 2 Like 2 (MAD2L2) as a host protein that interacts with EspF. Using LUMIER assays, MAD2L2 was shown to interact with EspF variants from EHEC O157:H7 and O26:H11 as well as EPEC O127:H6. MAD2L2 is targeted by the non-homologous Shigella effector protein invasion plasmid antigen B (IpaB) to halt the cell cycle and limit epithelial cell turnover. Therefore, we postulate that interactions between EspF and MAD2L2 serve a similar function in promoting EPEC and EHEC colonization, since cellular turnover is a key method for bacteria removal from the epithelium. Future work should investigate the biological importance of this interaction that could promote the colonization of EPEC and EHEC E. coli in the host.

Prevalence, antimicrobial resistance, and genotyping of Shiga toxin-producing Escherichia coli in foods of cattle origin, diarrheic cattle, and diarrheic humans in Egypt.

Shiga toxin-producing Escherichia coli (STEC) is a pathotype of E. coli that causes enteric and systemic diseases ranging from diarrhoea to severe hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). The emergence of multidrug-resistant (MDR) STEC from cattle sources has increased public health risk and limited treatment options. The prevalence of STEC was investigated in 200 raw food samples (milk and beef samples) and 200 diarrheic samples (cattle and human samples) in a matched region. The presence of stx genes (stx1 and stx2), carbapenemase-encoding genes (blaVIM, blaNDM-1, and blaIMP), and extended-spectrum ß-lactamase (ESBL)-encoding genes (blaTEM group, blaCTX-M1 group, and blaOXA-1 group) was screened by polymerase chain reaction (PCR). Antibiogram and Enterobacterial repetitive intergenic consensus (ERIC)-PCR were also conducted. STEC isolates were identified in 6.5% (13/200) of food samples [6% (6/100) of milk and 7% (7/100) of beef samples] and in 11% (22/200) of diarrheic cases [12% (12/100) of cattle and 10% (10/100) of human samples]. We found that O26 (4.5%, 18/400) and O111 (1.5%, 6/400) were the most prevalent STEC serovars and were found more commonly in diarrheic samples. STEC strains with both stx genes, stx2 only, and stx1 only genotypes were present in 62.9% (22/35), 20% (7/35), and 17.1% (6/35) of isolates, respectively. Carbapenemase-producing STEC (CP STEC) isolates were found in 1.8% (7/400) of samples [0.5% (1/200) of foods and 3% (6/200) of diarrheic cases]. The blaVIM gene was detected in all CP STEC isolates, and one human isolate carried the blaNDM-1 gene. ESBL-producing STEC strains were detected in 4.3% (17/400) of samples [1.5% (3/200) of food samples and 7% (14/200) of diarrheic cases]. The blaTEM, blaCTX-M1, and blaOXA-1 genes were detected in 42.9% (15/35), 28.6% (10/35), and 2.9% (1/35) of STEC isolates, respectively. Approximately half (51.4%, 18/35) of STEC isolates were MDR STEC; all CP STEC and ESBL-producing STEC were also MDR STEC. The highest antimicrobial resistance rates were found against nalidixic acid (51.4%) and ampicillin (48.6%), whereas the lowest rates were reported against gentamicin (5.7%) and ciprofloxacin (11.4%). MDR STEC strains were 5.3 times more likely to be found in diarrheic cases than in foods (P = 0.009, 95% CI 1.5-18.7). ERIC-PCR was used for genotyping STEC isolates into 27 different ERIC-types (ETs) with a discrimination index of 0.979. Five ETs showed clusters of 2-4 identical isolates that shared the same virulence and antibiotic resistance genetic profile. Human isolates matched food isolates in two of these ET clusters (the O26 CP STEC cluster and the O111 STEC cluster), highlighting the potential cross-species zoonotic transmission of these pathogens and/or their genes in the study region. This is the first detection of CP STEC in milk and diarrheic cattle in Egypt.

Enhanced Antibacterial Activity of Silver Nanoparticles Combined with Hydrogen Peroxide Against Multidrug-Resistant Pathogens Isolated from Dairy Farms and Beef Slaughterhouses in Egypt.

PURPOSE: The last few decades have witnessed a rapid and global increase in multidrug-resistant bacteria (MDR) emergence. METHODS: The aim of the current study is to isolate the most common MDR bacteria from dairy farms and beef slaughterhouses followed by evaluation of their antimicrobial resistance pattern and assessment of the antibacterial activity of AgNPs-H2O2 as an alternative to conventional antibiotics. In this regard, 200 samples were collected from two dairy farms and one beef slaughterhouse located in Dakhliya Governorate, Egypt. RESULTS: Interestingly, out of 120 collected samples from dairy farms, the prevalence of the isolated strains was 26.7, 23.3, 21.7, 16.7, and 11.7% for S. typhimurium, E. coli O157:H7, L. monocytogenes, K. pneumoniae and P. aeruginosa, respectively. Meanwhile, the overall prevalence was 30, 25, 22.5, 17.5, and 5% for E. coli O157:H7, L. monocytogenes, S. typhimurium, P. aeruginosa, and K. pneumoniae, respectively, for the 80 samples collected from a beef slaughterhouse. The antimicrobial susceptibility pattern elucidated that all isolated strains exhibited resistance to at least four of the tested antimicrobials, with multiple-antibiotic resistance index values (MAR) ranging between 0.44 and 0.88. Furthermore, the commercial AgNPs-H2O2 product was characterized by transmission electron microscopy (TEM) and zeta potential that showed spherical particles with a surface charge of -0.192 mV. The antimicrobial activity of synergized nano-silver (AgNP) with H2O2 product toward MDR strains was assessed via measuring minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and time-kill curve. CONCLUSION: The present data report high prevalence rates of MDR pathogens in dairy farms and abattoirs. More importantly, AgNPs-H2O2 exerted broad-spectrum bactericidal activity toward MDR bacterial strains, suggesting their promising usage as safe, ecofriendly, cost-effective antibacterial agents. To our knowledge, this study is a pioneer in investigating the potential alternative antimicrobial role of silver nanoparticles for control of multiple drug-resistant pathogens in Egypt.

Molecular Characterization and Developing a Point-of-Need Molecular Test for Diagnosis of Bovine Papillomavirus (BPV) Type 1 in Cattle from Egypt.

Bovine papillomatosis is a viral disease of cattle causing cutaneous warts. A diagnosis of this viral infection is very mandatory for combating the resulting economic losses. Given the limited data available about bovine papillomavirus (BPV) in Egypt, the present study involved the molecular diagnosis of bovine papillomavirus type-1 (BPV-1), -2, -4, -5, and -10 in cattle presenting cutaneous warts on the head and neck from New Valley Province, Egypt. The phylogenetic analysis of the detected types of BPV was also performed, followed by developing a point-of-need molecular assay for the rapid identification of identified BPV types. In this regard, a total of 308 cattle from private farms in Egypt were clinically examined, of which 13 animals presented cutaneous warts due to suspected BPV infection. The symptomatic animals were treated surgically, and biopsies from skin lesions were collected for BPV-1, -2, -4, -5, and -10 molecular identification using polymerase chain reaction (PCR). The presence of BPV-1 DNA was confirmed in 11 collected samples (84.6%), while BPV-2, -4, -5, and -10 were not detected. Sequencing of the PCR products suggested the Egyptian virus is closely related to BPV found in India. An isothermal nucleic acid amplification test (NAAT) with labeled primers specific for the BPV-1 L1 gene sequence, and based on recombinase polymerase amplification (RPA), in combination with a lateral flow strip assay for the detection of RPA products, was developed and tested. The point-of-need molecular assay demonstrated a diagnostic utility comparable to PCR-based testing. Taken together, the present study provides interesting molecular data related to the occurrence of BPV-1 in Egypt and reveals the genetic relatedness of the Egyptian BPV-1 with BPV-1 found in buffalo in India. In addition, a simple, low-cost combined test was also validated for diagnosis of the infection. The present study suggests the necessity of future investigations about the circulating strains of the virus among the cattle in Egypt to assess their genetic relatedness and better understand the epidemiological pattern of the disease.

Epidemiological and Molecular Investigation of Ocular Fungal Infection in Equine from Egypt.

Diagnosis and treatment of ocular fungal infection in equine seems very challenging for owners and clinicians. The present study aimed to identify and characterize fungal species isolated from the eyes of clinically healthy and diseased equines (N = 100) from Dakahlia Governorate, Egypt. The work also involved morphological and molecular characterization of the major fungal species. In addition, correlations between the occurrence of isolated fungi and some of the potential risk factors were also investigated. Interestingly, the prevalence rate of ocular mycosis in all examined equines in the study was 28% and there were major clinical signs associated with ocular fungal infection. Moreover, the identified fungal species included Aspergillus flavus, A. fumigatus, A. niger, Penicillium spp., Mucor spp., and Alternari spp. with a corresponding prevalence rate of 63.9%, 27.8%, 15.3%, 18.1%, 13.9%, and 4.2%, respectively, in healthy equine eyes, while their prevalence in diseased equine eyes was 57.1%, 32.1%, 21.4%, 7.1%, 3.6%, and 0%. Furthermore, a statistical significant association (p < 0.05) was found between the frequency of isolation of A. fumigatus and Penicillium and several risk factors (breed, sex, and ground type), while the remaining risk factors and occurrence of fungi were not statistically correlated. A subset of the Aspergillus species samples positive by polymerase chain reaction (PCR) were sequenced and their phylogenetic analysis identified three species of Aspergillus. Taken together, our study provides novel data related to the occurrence of ocular mycosis in equine in Egypt. Given the zoonotic potential of some identified fungi, our data may be helpful for implementation of novel diagnostic and therapeutic strategies for combating this sight-threatening infection in equine.

The interaction of Escherichia coli O157 :H7 and Salmonella Typhimurium flagella with host cell membranes and cytoskeletal components.

Bacterial flagella have many established roles beyond swimming motility. Despite clear evidence of flagella-dependent adherence, the specificity of the ligands and mechanisms of binding are still debated. In this study, the molecular basis of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium flagella binding to epithelial cell cultures was investigated. Flagella interactions with host cell surfaces were intimate and crossed cellular boundaries as demarcated by actin and membrane labelling. Scanning electron microscopy revealed flagella disappearing into cellular surfaces and transmission electron microscopy of S. Typhiumurium indicated host membrane deformation and disruption in proximity to flagella. Motor mutants of E. coli O157:H7 and S. Typhimurium caused reduced haemolysis compared to wild-type, indicating that membrane disruption was in part due to flagella rotation. Flagella from E. coli O157 (H7), EPEC O127 (H6) and S. Typhimurium (P1 and P2 flagella) were shown to bind to purified intracellular components of the actin cytoskeleton and directly increase in vitro actin polymerization rates. We propose that flagella interactions with host cell membranes and cytoskeletal components may help prime intimate attachment and invasion for E. coli O157:H7 and S. Typhimurium, respectively.

Evaluation of Bifidobacteria and Lactobacillus Probiotics as Alternative Therapy for Salmonella typhimurium Infection in Broiler Chickens.

Chicken Salmonella enterica serovars are enteric bacteria associated with massive public health risks and economic losses. There is a widespread antimicrobial resistance among S. enterica serotypes, and innovative solutions to antibiotic resistance are needed. We aimed to use probiotics to reduce antibiotic resistance and identify the major probiotic players that modify the early interactions between S. enterica and host cells. One-day-old cobb broiler chicks were challenged with S. typhimurium after oral inoculation with different probiotic strains for 3 days. The adherence of different probiotic strains to Caco-2 intestinal epithelial cells was studied in vitro. Lactobacillus (Lacticaseibacillus) casei ATTC334 and Bifidobacterium breve JCM1192 strains attached to Caco-2 cells stronger than B. infantis BL2416. L. casei ATTC334 and B. breve JCM1192 reduced S. typhimurium recovery from the cecal tonsils by competitive exclusion mechanism. Although B. infantis BL2416 bound poorly to Caco-2 epithelial cells, it reduced S. typhimurium recovery and increased IFN-γ and TNF-α production. L. casei ATTC334, B. breve JCM1192 and B. infantis BL2416 improved body weight gain and the food conversion rate in S. typhimurium-infected broilers. B. longum Ncc2785 neither attached to epithelial cells nor induced IFN-γ and TNF-α release and consequently did not prevent S. typhimurium colonization in broiler chickens. In conclusion, probiotics prevented the intestinal colonization of S. typhimurium in infected chickens by competitive exclusion or cytokine production mechanisms.

Inflammasome Activation in Bovine Peripheral Blood-Derived Macrophages Is Associated with Actin Rearrangement.

Inflammation is critical for infection control and acts as an arsenal defense mechanism against invading microbes through activation of the host immune system. It works via its inflammasome components to sense the dangerous invading microorganism and send messages to the immune system to destroy them. To date, the function of bovine macrophage inflammasome and its relationship with actin has not been identified. This study aimed to investigate the activation of bovine inflammasome by phase one flagellin from Salmonella typhimurium and its interaction with actin. Bovine monocyte-derived macrophages were prepared and challenged with S. typhimurium SL1344 phase one flagellin. The results demonstrated the relationship between the flagellin-based activation of inflammasome and actin rearrangement. The flagellin-based activation of inflammasome promoted the activation and co-localization of F-actin and the inflammasome complex. Actin was remodeled to different degrees according to the stage of inflammasome activation. The actin redistribution varied from polymerization to filopodia, while at the stage of pyroptotic cell death, actin was broken down and interacted with activated inflammasome complexes. In conclusion, flagellin-dependent inflammasome activation and actin localization to the inflammasome at the stage of pyroptotic cell death may be of importance for appropriate immune responses, pending further studies to explore the exact cross-linking between the inflammasome complex and actin.

Cefotaxime pharmacokinetics in Arabian camel (Camelus dromedarius) calves after single intravenous injection.

Cefotaxime is a third-generation broad-spectrum cephalosporin acting on a wide range of Gram-positive and Gram-negative bacteria. In this work, the pharmacokinetics of cefotaxime were determined in dromedary camel calves by single intravenous injection of 10 mg/kg b.w. Cefotaxime levels were estimated by ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS/MS). Cefotaxime pharmacokinetics in camel calves obeyed three-compartment kinetics model. There was a central compartment and two peripheral, one shallow and one deep compartment. The shallow compartment equilibrates very rapidly with distribution half-life (t1/2α) of 0.6 min, while the deep compartment has large distribution half-life (t1/2ß) of 42 min indicating slower uptake of cefotaxime. The elimination rate constant (γ = 0.04 h-1) and elimination half-life (t1/2 γ) = 15.46 h indicating slow elimination. In comparison with other animals, cefotaxime pharmacokinetics in camel calves showed potential wide distribution in multi-compartment, lower elimination constant, lower clearance and higher volume of distribution at steady state. This indicates substantial differences in cefotaxime pharmacokinetics in camel calves with a very characteristic ultra-rapid distribution into three-compartment and slow elimination.

An RNA-dependent mechanism for transient expression of bacterial translocation filaments.

The prokaryotic RNA chaperone Hfq mediates sRNA-mRNA interactions and plays a significant role in post-transcriptional regulation of the type III secretion (T3S) system produced by a range of Escherichia coli pathotypes. UV-crosslinking was used to map Hfq-binding under conditions that promote T3S and multiple interactions were identified within polycistronic transcripts produced from the locus of enterocyte effacement (LEE) that encodes the T3S system. The majority of Hfq binding was within the LEE5 and LEE4 operons, the latter encoding the translocon apparatus (SepL-EspADB) that is positively regulated by the RNA binding protein, CsrA. Using the identified Hfq-binding sites and a series of sRNA deletions, the sRNA Spot42 was shown to directly repress translation of LEE4 at the sepL 5' UTR. In silico and in vivo analyses of the sepL mRNA secondary structure combined with expression studies of truncates indicated that the unbound sepL mRNA is translationally inactive. Based on expression studies with site-directed mutants, an OFF-ON-OFF toggle model is proposed that results in transient translation of SepL and EspA filament assembly. Under this model, the nascent mRNA is translationally off, before being activated by CsrA, and then repressed by Hfq and Spot42.

Host species adaptation of TLR5 signalling and flagellin recognition.

Toll-like receptor 5 (TLR5) recognition of flagellin instigates inflammatory signalling. Significant sequence variation in TLR5 exists between animal species but its impact on activity is less well understood. Building on our previous research that bovine TLR5 (bTLR5) is functional, we compared human and bovine TLR5 activity and signalling in cognate cell lines. bTLR5 induced higher levels of CXCL8 when expressed in bovine cells and reciprocal results were found for human TLR5 (hTLR5) in human cells, indicative of host cell specificity in this response. Analysis of Toll/interleukin-1 receptor (TIR) sequences indicated that these differential responses involve cognate MyD88 recognition. siRNA knockdowns and inhibitor experiments demonstrated that there are some host differences in signalling. Although, PI3K activation is required for bTLR5 signalling, mutating bTLR5 F798 to hTLR5 Y798 within a putative PI3K motif resulted in a significantly reduced response. All ruminants have F798 in contrast to most other species, suggesting that TLR5 signalling has evolved differently in ruminants. Evolutionary divergence between bovine and human TLR5 was also apparent in relation to responses measured to diverse bacterial flagellins. Our results underscore the importance of species specific studies and how differences may alter efficacy of TLR-based vaccine adjuvants.

Capsular polysaccharide inhibits adhesion of Bifidobacterium longum 105-A to enterocyte-like Caco-2 cells and phagocytosis by macrophages.

BACKGROUND: Bifidobacterium longum 105-A produces markedly high amounts of capsular polysaccharides (CPS) and exopolysaccharides (EPS) that should play distinct roles in bacterial-host interactions. To identify the biological function of B. longum 105-A CPS/EPS, we carried out an informatics survey of the genome and identified the EPS-encoding genetic locus of B. longum 105-A that is responsible for the production of CPS/EPS. The role of CPS/EPS in the adaptation to gut tract environment and bacteria-gut cell interactions was investigated using the ΔcpsD mutant. RESULTS: A putative B. longum 105-A CPS/EPS gene cluster was shown to consist of 24 putative genes encoding a priming glycosyltransferase (cpsD), 7 glycosyltransferases, 4 CPS/EPS synthesis machinery proteins, and 3 dTDP-L-rhamnose synthesis enzymes. These enzymes should form a complex system that is involved in the biogenesis of CPS and/or EPS. To confirm this, we constructed a knockout mutant (ΔcpsD) by a double cross-over homologous recombination. Compared to wild-type, the ∆cpsD mutant showed a similar growth rate. However, it showed quicker sedimentation and formation of cell clusters in liquid culture. EPS was secreted by the ∆cpsD mutant, but had altered monosaccharide composition and molecular weight. Comparison of the morphology of B. longum 105-A wild-type and ∆cpsD by negative staining in light and electron microscopy revealed that the formation of fimbriae is drastically enhanced in the ∆cpsD mutant while the B. longum 105-A wild-type was coated by a thick capsule. The fimbriae expression in the ∆cpsD was closely associated with the disappearance of the CPS layer. The wild-type showed low pH tolerance, adaptation, and bile salt tolerance, but the ∆cpsD mutant had lost this survivability in gastric and duodenal environments. The ∆cpsD mutant was extensively able to bind to the human colon carcinoma Caco-2 cell line and was phagocytosed by murine macrophage RAW 264.7, whereas the wild-type did not bind to epithelial cells and totally resisted internalization by macrophages. CONCLUSIONS: Our results suggest that CPS/EPS production and fimbriae formation are negatively correlated and play key roles in the survival, attachment, and colonization of B. longum 105-A in the gut.

Epidemiological, molecular characterization and antibiotic resistance of Salmonella enterica serovars isolated from chicken farms in Egypt.

BACKGROUND: Salmonella is one of major causes of foodborne outbreaks globally. This study was conducted to estimate the prevalence, typing and antibiotic susceptibilities of Salmonella enterica serovars isolated from 41 broiler chicken farms located in Kafr El-Sheikh Province in Northern Egypt during 2014-2015. The clinical signs and mortalities were observed. RESULTS: In total 615 clinical samples were collected from broiler flocks from different organs (liver, intestinal content and gall bladder). Salmonella infection was identified in 17 (41%) broiler chicken flocks and 67 Salmonella isolates were collected. Recovered isolates were serotyped as 58 (86.6%) S. enterica serovar Typhimurium, 6 (9%) S. enterica serovar Enteritidis and 3 (4.5%) were non-typable. The significant high mortality rate was observed only in 1-week-old chicks. sopE gene was detected in 92.5% of the isolates which indicating their ability to infect humans. All S. enterica serovar Enteritidis isolates were susceptible to all tested antimicrobials. The phenotypically resistant S. enterica serovar Typhimurium isolates against ampicillin, tetracycline, sulphamethoxazole and chloramphenicol were harbouring BlaTEM, (tetA and tetC), (sul1 and sul3) and (cat1 and floR), respectively. The sensitivity rate of S. enterica serovar Typhimurium to gentamycin, trimethoprim/sulphamethoxazole and streptomycin were 100, 94.8, 89.7%, respectively. The silent streptomycin antimicrobial cassettes were detected in all Salmonella serovars. A class one integron (dfrA12, orfF and aadA2) was identified in three of S. enterica serovar Typhimurium strains. CONCLUSIONS: To the best of our knowledge, this study considered first report discussing the prevalence, genotyping, antibiotic susceptibility and public health significance of S. enterica serovars in broilers farms of different ages in Delta Egypt. Further studies are mandatory to verify the location of some resistance genes that are within or associated with the class one integron.