Escherichia coli XL10-Gold Bacteria Produce Bacteriophage
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Abstract:
Site-directed mutagenesis (SDM) is a powerful method to introduce specific mutations into DNA sequences ([1][1]). Package inserts in commercial kits for SDM sold by Agilent Technologies (QuikChange methods) recommend the use of XL10-Gold ultracompetent bacteria ([2][2]). We find, however, that XL10-Avian Pathogenic Escherichia coli (APEC) cause colibacillosis leading to significant economic losses in the poultry industry. This laboratory-based study aimed at establishing stocks of avian pathogenic Escherichia coli lytic bacteriophages, for future development of cocktail products for colibacillosis management. The study determined the antibiotic susceptibility; phylogenetic categories, occurrence of selected serotypes and virulence genes among Escherichia coli stock isolates from chicken colibacillosis cases; and evaluated bacteriophage activity against the bacteria. Escherichia coli characterization was done through phenotypic and multiplex PCR methods. Bacteriophage isolation and preliminary characterization was achieved using the spot assay and overlay plating techniques. Fifty-six (56) isolates were phenotypically confirmed as E . coli and all exhibited resistance to at least one antimicrobial agent; while multi-drug resistance (at least three drugs) was encountered in 50 (89.3%) isolates. The APEC isolates mainly belonged to phylogroups A and D, representing 44.6% and 39.3%, respectively; whereas serotypes O1, O2 and O78 were not detected. Of the 56 isolates, 69.6% harbored at least one virulence gene, while 50% had at least four virulence genes; hence confirmed as APEC. Virulence genes, ompT and iutA were the most frequent in 33 (58.9%) and 32 (57.1%) isolates respectively; while iroN least occurred in 23 (41.1%) isolates. Seven lytic bacteriophages were isolated and their host range, at 1×10 8 PFU/ml, varied from 1.8% to 17.9% of the 56 APEC isolates, while the combined lytic spectrum was 25%. Phage stability was negatively affected by increasing temperatures with both UPEC04 and UPEC10 phages being undetectable at 70°C; whereas activity was detected between pH 2 and 12. The high occurrence of APEC isolates resistant against the commonly used antibiotics supports the need for alternative strategies of bacterial infections control in poultry. The low host range exhibited by the phages necessitates search for more candidates before in-depth phage characterization and application.
Pathogenic Escherichia coli
Lytic cycle
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Escherichia coli bacteria are gram-negative bacteria in form of single or paired cells it is included Enterobacteriaceae family and intestinal normal flora. Laboratory tests conducted for the calculation of the number of germs can be done using cultivation techniques using a loop and micropipet. The aim of this research is to know the difference of number of bacteria bacteria on calibrated loop and micropipet to colony of Escherichia coli bacteria. This research is an analytical observation study with cross sectional design. The hypothesis was tested using independent t test with 95% confidence level. The results showed the average number colonies of Escherichia coli bacteria growing on PCA media using calibrated loop technique was 138,25 CFU/mL where as the average number of colonies of Escherichia coli bacteria grown on PCA media using micropipet technique was 104,56 CFU/mL, and significant value of p = 0,001. Furthemore, the result of data analysis showed that there were no any differences in the number of colonies of Escherichia coli bacteria in planting using calibrated loop technique and micropipet at incubation temperature of 36�C.
Escherichia
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Aims: To isolate bacteria from bovine gastrointestinal tract and investigate their inhibitory effect on Escherichia coli O157:H7 in vitro. Methods and Results: A total of 2400 bacterial colonies were isolated from cattle colonic mucous membrane. Thirteen strains demonstrated the ability to inhibit the growth of E. coli O157:H7. From these, seven were screened for the presence of virulence factors as: stx1, stx2, ehxA, eae, st1a and lt1 by polymerase chain reaction. The selected bacteriocin-producing bacteria showed susceptibility to most of the antibiotics used. Conclusions: The strains of E. coli isolated, which exhibit inhibitory activity on E. coli O157:H7 growth by the production of inhibitory substances, may be useful in the control of this pathogen in reservoirs. An important characteristic of these strains was the absence of any of the virulence factors assayed and the susceptibility to most of the antibiotics used for Gram-negative bacteria. Significance and Impact of the Study: These microorganisms might be used as probiotic bacteria to reduce the carriage of E. coli O157:H7 in cattle, thus limiting the contamination of carcasses at slaughter and subsequently the contamination of foods and the transfer of this pathogen to man.
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Myoviridae
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Pathogenic Escherichia coli
Incubation period
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Salmonella enterica
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Abstract Background: Extended spectrum beta-lactamase (ESBL)-producing enterobacteria pose a major hazard to public health. Due to the possibility of genetic transfer, ESBL genes might spread to pathogenic enterobacterial strains. Thus, information on possible genetic transfer between enterobacteria is of high interest. It was therefore the aim of this in vitro study to screen the capacity of a wide range of Enterobacteriaceae for time dependent differences in conjugation with five ESBL-producing Escherichia ( E.) coli strains. Results: Conjugation frequencies for five potential E. coli donor strains, producing the enzymes CTX-M-1, CTX-M-15, SHV-12, TEM-1, TEM-52 and CMY-2, and six potential recipient strains ( E. coli, Serratia marcescens subsp. marcescens , Enterobacter cloacae, Salmonella Typhimurium and Proteus mirabilis ) were obtained. Hence, different combinations of donor and recipient strains were co-incubated for between 0 and 22 hours and spread on selective agar. Conjugation frequencies were calculated as transconjugants per donor. Some of the donor and recipient strain combinations did not show plasmid transfer within 22 hours. Hence, the recipient Proteus mirabilis did not accept plasmids from any of the given donors and E. coli ESBL10716 was not able to transfer its plasmid to any recipient . Enterobacter cloacae only accepted the plasmids from the donors E. coli ESBL10708 and E. coli ESBL10716 while E. coli ESBL10708 did not transfer its plasmid to Serratia marcescens subsp. marcescens . E. coli IMT11716 on the other hand did not perform conjugation with the donor E. coli ESBL10689. The remaining mating pairs differed in conjugation frequency, ranging from log -5 to -8.5 transconjugants/donor. The earliest conjugation events were detected after 4 hours. However, some mating pairs turned positive only after 22 hours co-incubation. Conclusion: The results of this study suggest that conjugation is a frequent event in the spread of ESBL genes among commensal and pathogen bacteria. This should be considered when addressing antibiotic resistance issues.
Enterobacter cloacae
Citrobacter freundii
Serratia
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Abstract Background Extended spectrum beta-lactamase (ESBL)-producing enterobacteria pose a major hazard to public health. Due to the possibility of genetic transfer, ESBL genes might spread to pathogenic enterobacterial strains. Thus, information on possible genetic transfer between enterobacteria is of high interest. It was therefore the aim of this in vitro study to screen the capacity of a wide range of Enterobacteriaceae for differences in conjugation at different time points with five ESBL-producing Escherichia coli strains. Results Conjugation frequencies for five potential E. coli donor strains producing the enzymes CTX-M-1, CTX-M-15, SHV-12, TEM-1, TEM-52 and CMY-2, and six potential recipient strains commonly detected in the gastrointestinal tract of poultry ( E. coli, Serratia marcescens subsp. marcescens , Enterobacter cloacae, Salmonella ( S. ) enterica serovar Typhimurium and Proteus mirabilis ) were obtained. Different combinations of donor and recipient strains were co-incubated for between 0 and 22 h and spread on selective agar. Conjugation frequencies were calculated as transconjugants per donor. Some donor and recipient strain combinations did not perform plasmid transfer within 22 h. Hence, the recipient Proteus mirabilis did not accept plasmids from any of the given donors and the E. coli ESBL10716 donor was unable to transfer its plasmid to any recipient . Enterobacter cloacae only accepted the plasmids from the donors E. coli ESBL10708 and E. coli ESBL10716 while E. coli ESBL10708 did not transfer its plasmid to Serratia marcescens subsp. marcescens . E. coli IMT11716 on the other hand did not perform conjugation with the donor E. coli ESBL10689. The remaining mating pairs differed in conjugation frequency, ranging from 10 − 5 to 10 − 9 transconjugants/donor. The earliest conjugation events were detected after 4 h. However, some mating pairs turned positive only after 22 h of coincubation. Conclusion A suitable mating pair for future in vivo studies to combat transfer of antibiotic resistance to pathogenic bacteria in broiler chicken was determined. The results of this study also suggest that the kinetic of conjugation differs between mating pairs and is independent of species origin. This should be considered when performing conjugation experiments.
Enterobacter cloacae
Salmonella enterica
Citrobacter freundii
Serratia
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Challenging 1-day-old White Leghorn chicks perorally with 2.6 x 10(1) to 2.6 x 10(5) Escherichia coli O157:H7 bacteria per chick resulted in cecal colonization at all levels. Two of six chicks inoculated with only 2.6 x 10(1) E. coli O157:H7 bacteria carried 10(3) to 10(4) E. coli O157:H7 bacteria per g of cecal tissue when sacrificed 3 months postinoculation. E. coli O157:H7 colonization persisted at least 10 to 11 months when chicks were administered 10(8) E. coli O157:H7 bacteria. Eggs from five hens that were fecal shedders of E. coli O157:H7 until the termination of the study (10 to 11 months) were assayed for E. coli O157:H7. The organism was isolated from the shells of 14 of 101 (13.9%) eggs but not from the yolks and whites. Considering that chicks can be readily colonized by small populations of E. coli O157:H7 and continue to be long-term shedders, it is possible that chickens and hen eggs can serve as vehicles of this human pathogen.
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Consumption of water contaminated with pathogenic bacteria is a major cause of water-borne diseases. To address this challenge, we have developed a novel and sensitive sensing scheme for the rapid detection of bacteria (Escherichia coli B40) on a fiber-optic platform using bacteriophage (T4) as a bio-recognition element. The novelty of our sensing scheme is that instead of bacteriophages, bacteria (analyte) were first captured on the sensing surface and then the sensing surface was subjected to bacteriophages for specific detection of bacteria. The sensor was subjected to 100 to 107 cfu/mL of E. coli B40 spiked in a lake water matrix, and the least concentration of bacteria that could be easily detected was found to be 1000 cfu/mL. The control studies were performed with nonhost bacteria Pseudomonas aeruginosa. Bacteriophage T4, being specific to its host E. coli B40, did not interact with P. aeruginosa captured on the sensing probe, giving a negligible nonspecific response. Due to the specificity of bacteriophages to its host bacteria, it is possible to use this scheme to carry out the detection of specific bacteria in a mixed sample (containing a combination of bacteria) using bacteriophages specific to it. The sensor was able to detect E. coli B40 (target bacteria) even in the presence of a very high concentration (1000 times higher) of P. aeruginosa (nontarget bacteria).
Pathogenic bacteria
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Non-antibiotic measures are needed to reduce the rate of infections due to multidrug-resistant organisms (MDROs), including by eliminating the commensal reservoir that underlies such strains' dissemination and leads to recurrent infections. Here, we tested a cocktail of pre-selected bacteriophages and an engineered microcin C7-producing probiotic Escherichia coli Nissle-1917 strain for their ability to reduce gut colonization by an E. coli strain from sequence type 131 (ST131)-H30R, which is the major clonal group of MDROs among extraintestinal clinical E. coli isolates. Although the bacteriophage cocktail was highly effective against ST131-H30R strains both in vitro and in a murine model of subcutaneous sepsis, it was only weakly and transiently effective against gut colonization by the target ST131-H30R strain (0.5 log10 decrease on d + 1: p < 0.001; no significant effect on d + 4 and beyond). The probiotic strain, while also highly active against ST131-H30R in vitro, was ineffective against ST131-H30R gut colonization despite its abundant presence in feces. Nonetheless, despite failing as decolonizing agents when administered separately, when co-administered the bacteriophage cocktail and probiotic strain exhibited striking synergy against ST131-H30R gut colonization. This combinatory effect was most pronounced on d + 1 (3.3 log10 target strain decrease: p < 0.001), and persisted until d + 7 (0.5 log10 decrease; p < 0.02.). Although by d + 10 the ST131-H30R load was fully restored, these findings provide proof of concept for combined bacteriophage-plus-probiotic administration to reduce or, possibly, to prevent gut colonization with MDROs in high-risk individuals.
Strain (injury)
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