Inactivation and injury of Escherichia coli O157:H7 and Staphylococcus aureus by pulsed electric fields

Two pathogenic microorganisms Escherichia coli O157:H7 and Staphylococcus aureus, suspended in peptone solution (0.1% w/v) were treated with 12, 14, 16 and 20 kV/cm electric field strengths with different pulse numbers up to 60 pulses. Pulsed electric field (PEF) treatment at 20 kV/cm with 60 pulses provided nearly 2 log reduction in viable cell counts of E. coli O157:H7 and S. aureus. S. aureus cells were slightly more resistant than E.coli O157:H7 cells. The results related to the effect of initial cell concentration of E. coli O157:H7 on the PEF inactivation showed that more inactivation was obtained by decreasing initial cell concentration. Any possible injury by PEF was also investigated after applying 20 kV/cm electric field to the microorganisms. As a result, it was determined that there was 35.92 to 43.36% injury in E. coli O157:H7 cells, and 17.26 to 30.86% injury in S. aureus cells depending on pulse number. The inactivation results were also described by a kinetic model.     

Cephalexin-Induced Morphological Alterations in the Surface Structures of Staphylococcus aureus and Escherichia coli Demonstrated by Scanning Electron Microscopy

The scanning electron microscope was used to investigate the alterations in surface morphology of Staphylococcus aureus 209P and Escherichia coli NIH induced by the action of cephalexin known to interfere with cell-wall synthesis. Exposure to cephalexin produced a series of changes on the surface morphology in proportion to the concentrations of cephalexin added. Untreated S. aureus cells had smooth contours. Exposure to 1 μg/ml of cephalexin during the logarithmic phase of growth in S. aureus did not produce any detectable changes. Upon exposure of S. aureus to 5 μ/ml or 10 μg/ml, some cells were larger than normal and showed abnormal cell division-like structures in part. When S. aureus was exposed to 50 μg/ml, cell division was completely inhibited, and no formation of grape-like clusters was observed. Untreated E. coli cells appeared to have smooth and regular contours. E. coli propagated almost normally upon exposure of the organisms to 1 μg/ml of cephalexin. Filamentous structures were observed with the exposure of E. coli to 12.5 μg/ml or 25 μg/ml, but spheroplast-like structures were not observed. Exposure to 100 μg/ml of cephalexin resulted in the formation of marked filamentous cells and spheroplast-like structures having multiple small saccular outpouchings. Scanning electron microscope demonstrated more completely the morphological abnormalities induced by cephalexin.     

Effects of 50 Hz rotating magnetic field on the viability of Escherichia coli and Staphylococcus aureus

This study presents results of research on the influence of rotating magnetic field (RMF) of the induction of 30?mT and the frequency of 50?Hz on the growth dynamics and cell metabolic activity of E. coli and S. aureus, depending on the exposure time. The studies showed that the RMF caused an increase in the growth and cell metabolic activity of all the analyzed bacterial strains, especially in the time interval t?=?30 to 150?min. However, it was also found that the optical density and cell metabolic activity after exposition to RMF were significantly higher in S. aureus cultures. In turn, the study of growth dynamics, revealed a rapid and a significant decrease in these values from t?=?90?min) in the case of E. coli samples. The obtained results prove that RMF (B?=?30?mT, f?=?50?Hz) has a stimulatory effect on the growth and metabolic activity of E. coli and S. aureus. Furthermore, taking into account the time of exposure, stronger influence of RMF on the viability was observed in S. aureus cultures, which may indicate that this effect depends on the shape of the exposed cells.     

Antibacterial and membrane‐damaging activities of β‐bungarotoxin B chain

This study investigates whether the B chain of β‐bungarotoxin exerted antibacterial activity against Escherichia coli (Gram‐negative bacteria) and Staphylococcus aureus (Gram‐positive bacteria) via its membrane‐damaging activity. The B chain exhibited a growth inhibition effect on E. coli but did not show a bactericidal effect on S. aureus. The B‐chain bactericidal action on E. coli positively correlated with an increase in membrane permeability in the bacterial cells. Lipopolysaccharide (LPS) layer destabilization and lipoteichoic acid (LTA) biosynthesis inhibition in the cell wall increased the B‐chain bactericidal effect on E. coli and S. aureus. The B chain induced leakage and fusion in E. coli and S. aureus membrane‐mimicking liposomes. Compared with LPS, LTA notably suppressed the membrane‐damaging activity and fusogenicity of the B chain. The B chain showed similar binding affinity with LPS and LTA, whereas LPS and LTA binding differently induced B‐chain conformational change as evidenced by the circular dichroism spectra. Taken together, our data indicate that the antibacterial action of the B chain is related to its ability to induce membrane permeability and suggest that the LPS‐induced and LTA‐induced B‐chain conformational change differently affects the bactericidal action of the B chain. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Cloning vector system forNocardia spp.

An electro-transformation system has been developed forNocardia asteroides andNocardia corallina by using aNocardia-Escherichia coli shuttle vector. The shuttle vector, named pCY104, was constructed by joining a 2.5-kb crypticN. asteroides plasmid pCY101 with theE. coli plasmid pIJ4625. The resistance genes for kanamycin, chloramphenicol, and thiostrepton on plasmid pCY104 were expressed inN. asteroides andN. corallina. The transformation method was optimized forN. asteroides, and transformation efficiency of 8×10⁴ transformants per g plasmid DNA was achieved routinely.     

Primed Immune Responses to Gram-negative Peptidoglycans Confer Infection Resistance in Silkworms

A heightened immune response, in which immune responses are primed by repeated exposure to a pathogen, is an important characteristic of vertebrate adaptive immunity. In the present study, we examined whether invertebrate animals also exhibit a primed immune response. The LD₅₀ of Gram-negative enterohemorrhagic Escherichia coli O157:H7 Sakai in silkworms was increased 100-fold by pre-injection of heat-killed Sakai cells. Silkworms pre-injected with heat-killed cells of a Gram-positive bacterium, Staphylococcus aureus, did not have resistance to Sakai. Silkworms preinjected with enterohemorrhagic E. coli peptidoglycans, cell surface components of bacteria, were resistant to Sakai infection. Silkworms preinjected with S. aureus peptidoglycans, however, were not resistant to Sakai. Silkworms preinjected with heat-killed Sakai cells showed persistent resistance to Sakai infection even after pupation. Repeated injection of heat-killed Sakai cells into the silkworms induced earlier and greater production of antimicrobial peptides than a single injection of heat-killed Sakai cells. These findings suggest that silkworm recognition of Gram-negative peptidoglycans leads to a primed immune reaction and increased resistance to a second round of bacterial infection.     

Influence of Heterologously Expressed azurin from Pseudomonas aeruginosa on the Adhesion and Invasion of Pathogenic Bacteria to the Caco-2 Cell Line

This study proposed to investigate the effect of azurin on the major stages of pathogenesis (adhesion and invasion) of intestinal bacterial pathogens (Salmonella spp. and Escherichia coli) and epithelial pathogens (Staphylococcus aureus and Pseudomonas aeruginosa) on the human colorectal adenocarcinoma (Caco-2) cell line. Azurin protein was produced by cloning the azurin gene into pET21a and heterologous expression in E. coli BL21. The protein was purified using affinity chromatography and confirmed by Western blotting. The purified protein was evaluated by three experiments of adhesion and invasion assays, including exclusion, competition, and replacement. Azurin was observed to significantly inhibit the attachment and invasion of S. aureus, Salmonella spp., and E. coli, while no such inhibitory effects were observed on P. aeruginosa. In fact, the protein increased the adhesion of P. aeruginosa to the cell. In conclusion, our study proposes that azurin is a potential prophylactic or preventive helper candidate to inhibit the attachment and invasion of pathogenic bacteria to host cells and reduce the progression of the infection process. Our study also reveals the involvement of azurin in bacteria-host cell interactions, providing novel and important insights toward the elucidation of its biological function in this field. Thus, this study provides new opportunities to use azurin as an adjunct therapy against critical stages of infection by a wide range of pathogenic bacteria.

     

MODIFICATION OF LYSOZYME WITH OLEOYL CHLORIDE FOR BROADENING THE ANTIMICROBIAL SPECIFICITY

Lysozyme from egg white was modified by covalent attachment of an oleyl group to the free amino groups of lysozyme. The aim of the chemical modification was to develop an effective antimicrobial lysozyme derivative against both gram-negative and gram-positive bacteria. Lysozyme with various degrees of modification was obtained by changing oleoyl chloride/lysozyme mass ratio. Lysozyme derivatives evidently exhibited an antimicrobial effect against Escherichia coli (ATCC 29998). The modification slightly changed the antimicrobial effect of lysozyme derivative against Staphylococcus aureus (ATCC 121002). Since there was a positive correlation between the modification degree and the antimicrobial effect against E. coli, it was concluded that the change in antimicrobial behavior was due to an increase in hydrophobicity of the enzyme molecule enabling it to penetrate through the bacterial membrane of E. coli. It was also shown that oleoyl chloride with an MIC value of 10 mg/mL was effective against both E. coli and S. aureus.     

Molecular epidemiology of clinical and carrier strains of methicillin resistant Staphylococcus aureus (MRSA) in the hospital settings of north India

Background

The study was conducted between 2000 and 2003 on 750 human subjects, yielding 850 strains of staphylococci from clinical specimens (575), nasal cultures of hospitalized patients (100) and eye & nasal sources of hospital workers (50 & 125 respectively) in order to determine their epidemiology, acquisition and dissemination of resistance genes.

Methods

Organisms from clinical samples were isolated, cultured and identified as per the standard routine procedures. Susceptibility was measured by the agar diffusion method, as recommended by the Nat ional Committee for Clinical Laboratory Standards (NCCLS). The modified method of Birnboin and Takahashi was used for isolation of plasmids from staphylococci. Pulsed-field gel electrophoresis (PFGE) typing of clinical and carrier Methicillin resistant Staphylococcus aureus (MRSA) strains isolated during our study was performed as described previously.

Results

It was shown that 35.1% of Staphylococcus aureus and 22.5% of coagulase-negative staphylococcal isolates were resistant to methicillin. Highest percentage of MRSA (35.5%) was found in pus specimens (n = 151). The multiple drug resistance of all MRSA (n = 180) and Methicillin resistant Coagulase-negative Staphylococcus aureus (MRCNS) (n = 76) isolates was detected. In case of both methicillin-resistant as well as methicillin-sensitive Saphylococcal isolates zero resistance was found to vancomycin where as highest resistance was found to penicillin G followed by ampicillin. It was shown that the major reservoir of methicillin resistant staphylococci in hospitals are colonized/infected inpatients and colonized hospital workers, with carriers at risk for developing endogenous infection or transmitting infection to health care workers and patients. The results were confirmed by molecular typing using PFGE by SmaI-digestion. It was shown that the resistant markers G and T got transferred from clinical S. aureus (JS-105) to carrier S. aureus (JN-49) and the ciprofloxacin (Cf) and erythromycin (E) resistance seemed to be chromosomal mediated. In one of the experiments, plasmid pJMR1O from Staphylococcus aureus coding for ampicillin (A), gentamicin (G) and amikacin (Ak) resistance was transformed into Escherichia coli. The minimal inhibitory concentrations (MICs) for A and G were lower in E. coli than in S. aureus. However, the MIC for Ak was higher in E. coli transformants than in S. aureus.

Conclusion

There is a progressive increase in MRSA prevalence and multi-drug resistance in staphylococci. Vancomycin is still the drug of choice for MRSA infections. The major reservoir of methicillin resistant staphylococci in hospitals is colonized/infected inpatients and colonized hospital workers. Resistance transfer from staphylococci to E. coli as well as from clinical to carrier staphylococci due to antibiotic stress seemed to be an alarming threat to antimicrobial chemotherapy.     

Gram positivity induced in Escherichia coli by chloroform-methanol treatment

Summary Extraction of lipid by chloroform-methanol from Escherichia coli induces Gram positivity in the cells of this bacterium. It has been observed that approximately 1.5 times more lipid is extracted from Escherichia coli than from Staphylococcus aureus by this treatment. An increase of 1.5–1.8 fold in the retention of dye has been evident in the case of S. aureus where as this was 70–90 fold in the case of E. coli, which is 1/4th of the normal retention by S. aureus. The more lipid present in the cell wall or cell membrane, the less retention of dye seems to be exhibited by the bacteria. This also substantiates our model for Gram reaction presented in a previous communication.     

Enteric bacteria isolated from acute diarrheal patients in the Republic of Korea between the year 2004 and 2006

In an epidemiological survey of human enterobacterial infections in the Republic of Korea during three years from 2004 to 2006, we isolated 1,784 (6.2%, isolation rate of enteropathogens from stool samples) in 2004, 2,547 (9.5%) in 2005 and 3,506 bacteria (12.3%) from people who visited clinics. Among the isolated bacteria, pathogenic Escherichia coli, especially, EAEC was the most frequently identified pathogen in both urban and rural regions followed by Staphylococcus aureus, Salmonella species, Bacillus cereus, Vibrio parahaemolyticus, Campylobacter jejuni, Clostridium perfringens, and Shigella species. Distinct seasonality was found in V. parahaemolyticus species, while this pathogen showed no age-specific patterns. However, other bacteria, i.e., pathogenic E. coli, S. aureus, Salmonella spp., and B. cereus showed similar seasonality throughout the year, showing a slight increase in the infection rate during the summer months and high prevalence among children under 10 years of age and elder-age people. The antibiotic susceptibility patterns of pathogenic E. coli, Salmonella spp., and S. aureus showed high resistance to penicillins. However, both pathogenic E. coli and Salmonella spp. were susceptible to several cephems, imipenem, and amikacin. Moreover, S. aureus strains resistant to vancomycin were not found. In conclusion, these surveillances can play an important role for the control and prevention to the diseases originated by enteritis bacteria.     

Sensitivity of Mixed Populations of Staphylococcus aureus and Escherichia coli to Mercurials

Staphylococcus aureus was found to have a higher resistance to merbromin and mercuric chloride in the presence of Escherichia coli. The protective effect of the gram-negative organism on S. aureus was due to the production of extracellular glutathione and hydrogen sulfide and to an unequal distribution of the inhibitor between the two species. S. aureus did not significantly influence the resistance of E. coli to mercurials.     

Differences in Toll-like receptor expression and cytokine production after stimulation with heat-killed gram-positive and gram-negative bacteria

Innate immune surveillance in the blood is executed mostly by circulating monocytes, which recognize conserved bacterial molecules such as peptidoglycan and lipopolysaccharide. Toll-like receptors (TLR) play a central role in microbe-associated molecular pattern detection. The aim of this study was to compare the differences in TLR expression and cytokine production after stimulation of peripheral blood cells with heat-killed gram-negative and gram-positive human pathogens: Neisseria meningitidis, Escherichia coli, Staphylococcus aureus, and Streptococcus pneumoniae. We found that TLR2 expression is up-regulated on monocytes after stimulation with S. aureus, S. pneumoniae, E. coli, and N. meningitidis. Moreover, TLR2 up-regulation was positively associated with increasing concentrations of gram-positive bacteria, whereas higher concentrations of gram-negative bacteria, especially E. coli, caused a milder TLR2 expression increase when compared to low doses. Cytokines were produced in similar dose-dependent profiles regardless of the stimulatory pathogen; however, gram-negative pathogens induced higher cytokine levels when compared to gram-positive bacteria at the same density. These results indicate that gram-positive and gram-negative bacteria differ in their dose-dependent patterns of induction of TLR2 and TLR4, but not cytokine expression.     

血培养病原菌构成及耐药性分析

了解2008年至2012年哈尔滨医科大学附属第一医院血培养常见病原菌构成及耐药性。对血培养分离出的病原菌进行鉴定,用MIC法、K-B法测定药物敏感性,用WHONET 5.6统计软件进行细菌菌谱及耐药性分析。共分离出病原菌4 245株;其中凝固酶阴性葡萄球菌最多,947株占22.3%;其次为大肠埃希菌822株,肺炎克雷伯菌520株,鲍曼不动杆菌195株,金黄色葡萄球菌142株;耐甲氧西林凝固酶阴性葡萄球菌(MRCNS)和耐甲氧西林金黄色葡萄球菌(MRSA)检出率分别为81.1%、38.8%,未发现耐万古霉素、利奈唑胺及替考拉宁的凝固酶阴性葡萄球菌和金黄色葡萄球菌;大肠埃希菌及肺炎克雷伯菌产ESBLs检出率分别为55.2%、53.8%,对头孢哌酮/舒巴坦、哌拉西林/他唑巴坦、头孢替坦、头孢西丁、阿米卡星的耐药率较低,对碳青霉烯类抗菌药物存在耐药现象;鲍曼不动杆菌对抗菌药物的耐药率普遍较高。及时、准确地对血培养分离出的病原菌进行监测,以便指导临床合理用药,控制耐药株的产生。     

牦牛源产细菌素屎肠球菌的分离鉴定和益生特性

【背景】抗生素的滥用导致牦牛肠道常见病原菌耐药性增加,益生菌作为对抗耐药性细菌的新型武器,应用前景广阔。【目的】获取益生特性优良的牦牛源益生菌。【方法】将20份牦牛粪便样本在含0.5%CaCO3的MRS培养基上分离纯化,以大肠杆菌和金黄色葡萄球菌为指示菌,用牛津杯法筛选有抑菌活性的菌株;排除酸和过氧化氢后,经耐酸耐热试验和蛋白酶敏感试验筛选产细菌素菌株,用形态学和16S rRNA基因序列分析鉴定;通过对大肠杆菌、沙门氏菌等腹泻病原菌体外抑菌试验、耐模拟胃肠液、测定自聚集能力和疏水性及抗生素敏感试验分析益生特性。【结果】从20份牦牛粪便样本中共分离出11株产生溶钙圈的菌株,其中6株对大肠杆菌和金黄色葡萄球菌抑菌效果显著,经复筛得到2株产细菌素的乳酸菌SC6和SC9,经鉴定均为屎肠球菌(Enterococcus faecium)。其中SC9对腹泻病原菌抑菌效果明显,有良好的耐受性和肠道黏附能力,对5种常用抗生素均敏感。【结论】屎肠球菌SC9有一定的抗逆性和潜在的益生能力,具备作为益生菌的潜力。     

Bacteria induce expression of apoptosis in human spermatozoa

An increased number of sperm undergoing apoptosis has been observed during inflammatory processes in the male genital tract, which might be associated with elevated reactive oxygen species (ROS) levels. However, another factor to stimulate apoptosis could be the direct contact with bacteria or its products, even in the absence of ROS. The aim of this study was to investigate whether bacteria can directly initiate apoptosis in human spermatozoa. Human spermatozoa selected by density gradient centrifugation were incubated with polymorphonuclear granulocytes (PMN) isolated from blood and/or E. faecalis, E. coli or S. aureus. As ROS inductor in PMN, phorbol-12-myristate-13-acetate was used. After incubating the cells for 60 min at 37C, ROS were determined by chemiluminescence and phosphatidyl serine (PS) externalization was analyzed by flow cytometry with Annexin V-FITC and propidium iodide (PI). The increase in the percentage of spermatozoa Annexin V-FITC-positive/ PI-negative (early event of late apoptosis) was significant after the incubation with PMN plus PMA, PMN plus E. coli and E. coli alone. The percentage of spermatozoa Annexin V-FITC-positive/ PI-positive (apoptosis/necrosis) increased significantly in sperm incubated with E. coli and S. aureus(20.3% ± 3 and 13.6% ± 3.2 compared to sperm alone, 6% ± 0.5). Sperm incubated with PMN-PMA activated showed only a relative increase in apoptosis/necrosis (8.4% ± 1). Our results show that bacteria directly increase the PS externalisation in ejaculated human sperm. This way of inducing apoptosis does not require external ROS and may result from anyone of the molecular mechanisms that account for changes in motility, vitality and DNA integrity, that are characteristics of spermatozoa in male genital tract infection.     

High pressure homogenization inactivation of Escherichia coli and Staphylococcus aureus in phosphate buffered saline,milk and apple juice

High pressure homogenization (HPH) offers new opportunities for food pasteurization/sterilization. Escherichia coli and Staphylococcus aureus suspended in phosphate buffered saline (PBS) buffer, milk and apple juice at initial concentration of ~10⁶ log₁₀ CFU per ml were subjected to HPH treatments up to 200 MPa with inlet temperatures at 4–40°C. After HPH at 200 MPa with the inlet temperature at 40°C, the count of E. coli suspended in PBS, milk and apple juice reduced by 3·42, 3·67 and 3·19 log₁₀ CFU per ml respectively while the count of S. aureus decreased by 2·21, 1·02 and 2·33 log₁₀ CFU per ml respectively suggesting that S. aureus was more resistant. The inactivation data were well fitted by the polynomial equation. Milk could provide a protective effect for S. aureus against HPH. After HPH at 200 MPa with the inlet temperature at 20°C, the cell structure of E. coli was destroyed, while no obvious damages were found for S. aureus.     

Effect of ultra‐strong static magnetic field on bacteria: Application of Fourier‐transform infrared spectroscopy combined with cluster analysis and deconvolution

A new method based on Fourier‐transform infrared (FTIR) spectroscopy combined with cluster analysis and deconvolution was established to investigate the biological effect of an ultra‐strong static magnetic field (SMF) of 10.0 T on Escherichia coli and Staphylococcus aureus. FTIR spectroscopy was applied to characterize the spectroscopic fingerprints of these bacterial cells with or without the treatment of the SMF. After the calculation, the results of cluster analysis indicated that the SMF had significant effects on E. coli compared with S. aureus, and the effects were reflected by the changes of spectral region of 1500–1200 cm^?1. The deconvolution results of this major indication region showed that the composition and conformation of nucleic acid, protein, and fatty acid of E. coli were altered under the magnetic conditions. Bioelectromagnetics 30:500–507, 2009. © 2009 Wiley‐Liss, Inc.