The species-specific mode of action of the antimicrobial peptide subtilosin against Listeria monocytogenes Scott A

Aims: To elucidate the molecular mechanism of action of the antimicrobial peptide subtilosin against the foodborne pathogen Listeria monocytogenes Scott A. Methods and Results: Subtilosin was purified from a culture of Bacillus amyloliquefaciens. The minimal inhibitory concentration of subtilosin against L. monocytogenes Scott A was determined by broth microdilution method. The effect of subtilosin on the transmembrane electrical potential (ΔΨ) and pH gradient (ΔpH), and its ability to induce efflux of intracellular ATP, was investigated. Subtilosin fully inhibited L. monocytogenes growth at a concentration of 19 μg ml^?1. Subtilosin caused a partial depletion of the ΔΨ and had a similar minor effect on the ΔpH. There was no significant efflux of intracellular ATP. Conclusion: Subtilosin likely acts upon L. monocytogenes Scott A by perturbing the lipid bilayer of the cellular membrane and causing intracellular damage, leading to eventual cell death. Subtilosin’s mode of action against L. monocytogenes Scott A differs from the one previously described for another human pathogen, Gardnerella vaginalis. Significance and Impact of the Study: This is the first report on the specific mode of action of subtilosin against L. monocytogenes and the first report of a bacteriocin with a species‐specific mode of action.     

Anti-bacterial activity of Lactobacillus plantarum strain SK1 against Listeria monocytogenes is due to lactic acid production

AIMS: The aim of this research was to investigate the potential of Lactobacillus plantarum strain SK1 for use as a biological control agent against Listeria monocytogenes and determine its mechanism of anti-listerial activity. METHODS AND RESULTS: Co-growth of Lact. plantarum SK1 and L. monocytogenes UMCC98 in MRS broth showed that anti-listerial activity of Lact. plantarum SK1 occurred during late log/early stationary phase of growth. This coincided with a reduction in broth pH to 4.26. Evidence obtained from the analysis of cell-free culture filtrates of strain SK1 grown in MRS broth using thin-layer chromatography and growth of L. monocytogenes in pH-adjusted culture filtrates suggested that the anti-listerial activity was due to lactic acid production alone. Trials of Lact. plantarum SK1 on radishes stored at 5 degrees C showed that it had statistically significant (P < 0.05) anti-listerial activity. CONCLUSIONS: The anti-listerial activity of Lact. plantarum SK1 was due to lactic acid production alone. A small-scale trial on radishes stored at 5 degrees C showed it to have significant anti-listerial activity in planta. SIGNIFICANCE AND IMPACT OF THE STUDY: This organism has potential as a biological control agent for L. monocytogenes.     

Cellular injuries upon exposure of Escherichia coli and Lactobacillus rhamnosus to high-intensity ultrasound

AIMS: To examine cellular injuries occurring in cells of Escherichia coli (Gram-negative bacteria) and Lactobacillus rhamnosus (Gram-positive bacteria) in response to a high-intensity ultrasound treatment using classical plate count technique and flow cytometry. METHOD AND RESULTS: According to plate count results, E. coli (D-value 8.3 min) was far more sensitive than L. rhamnosus (D-value 18.1 min) in their response to the ultrasound intensity applied (20 kHz, 17.6 W). The dye precursor carboxyfluorescein diacetate (cFDA) could freely diffuse across the cytoplasmic membrane of intact cells of Gram-positive bacteria L. rhamnosus, resulting in its intracellular enzymatic conversion and emission of green fluorescence. In contrast, the presence of an outer membrane on E. coli, which represents the class of Gram-negative bacteria, apparently disabled the penetration of viability marker cFDA. Ultrasound application on E. coli yielded in an increasing population with disintegrated outer membrane, which allowed penetration of cFDA and its intracellular enzymatic conversion as well as accumulation. In both organisms evaluated only a small population was labelled by propidium iodide upon exposure to ultrasound for up to 20 min. Within the experimental conditions investigated ultrasound did not considerably affect the cytoplasmic membrane, although according to plate count results viability loss occurred. CONCLUSIONS: The results compiled suggest, that ultrasound induced cell death, which may not be related to membrane damage. SIGNIFICANCE AND IMPACT OF THE STUDY: Limitation on the use of bacteriocins, which are aimed on destabilization of cytoplasmic membrane but inhibited by the outer membrane, could be overcome by ultrasound-assisted physical disruption of the outer membrane.     

Membrane permeability and antimicrobial kinetics of cecropin P1 against Escherichia coli

The interaction of cecropin P1 (CP1) with Escherichiacoli was investigated to gain insight into the time‐dependent antimicrobial action. Biophysical characterizations of CP1 with whole bacterial cells were performed using both fluorescent and colorimetric assays to investigate the role of membrane permeability and lipopolysaccharide (LPS) binding in lytic behavior. The kinetics of CP1 growth inhibition assays indicated a minimal inhibitory concentration (MIC) of 3 µM . Bactericidal kinetics at the MIC indicated rapid killing of E.coli (<30 min). Membrane permeability studies illustrated permeation as a time‐dependent event. Maximum permeability at the MIC occurred within 30 min, which correlates to the bactericidal action. Further investigation showed that the immediate permeabilizing action of CP1 is concentration‐dependent, which correlates to the concentration‐dependent nature of the inhibition assays. At the MIC and above, the immediate permeability was significant enough that the cells could not recover and exhibit growth. Below the MIC, immediate permeability was evident, but the level was insufficient to inhibit growth. Dansyl polymyxin B displacement studies showed LPS binding is essentially the same at all concentrations investigated. However, it does appear that only the immediate interaction is important, because binding continued to increase over time beyond cell viability. Our studies correlated CP1 bactericidal kinetics to membrane permeability suggesting CP1 concentration‐dependent killing is driven by the extent of the immediate permeabilizing action of the peptide. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Functional expression,purification, and antimicrobial activity of a novel antimicrobial peptide MLH in Escherichia coli

In this study, a novel heterozygous antimicrobial peptide MLH was synthesized, expressed, purified, and characterized. The peptide Md-cec-LL-37_Hp (MLH) was selected through bioinformatic analysis using musca domestica antimicrobial peptide (Cec-Med), human antimicrobial peptide LL-37, and helicobacter pylori antimicrobial peptide (Hp) as parent peptides. The target gene was synthesized by overlap extension PCR (SOE-PCR) and connected to the expression vector pET-32a (+), and the recombinant plasmid pET-32a-MLH was transformed to Escherichia coli for constructing pET-32a-MLH/BL21 (DE3). Isopropyl β-D-thiogalactoside (IPTG) was used to induce protein expression, and SDS-PAGE and western blot were adopted to test the target protein. And fermentation condition was optimized to get the mass expression of the fusion protein. The Ni²⁺ affinity chromatographic column was used to purify. Active heterozygous peptide was obtained after renaturation. Finally, the activity of the heterozygous antimicrobial peptide was identified. The fusion peptide showed significant antimicrobial effect on both E. coli and Staphylococcus aureus.     

Activity of natural antimicrobial compounds against Escherichia coli and Salmonella enterica serovar Typhimurium

AIMS: The objective of this study was to evaluate the inhibitory activity of several natural organic compounds alone or in combination with nisin against Escherichia coli and Salmonella Typhimurium. METHODS AND RESULTS: The minimum inhibitory concentration (MIC) of five natural organic compounds were determined, and the effect of their combinations with nisin was evaluated by the checkerboard assay using the Bioscreen C. As expected, nisin by itself showed no inhibition against either of the Gram-negative bacteria. Thymol was found to be the most effective with the lowest MIC values of 1.0 and 1.2 mmol 1-1 against Salm. Typhimurium and E. coli, respectively. After thymol, the antimicrobial order of the natural organic compounds was carvacrol > eugenol > cinnamic acid > diacetyl. However, the combination of nisin with the natural organic compounds did not result in the enhancement of their antimicrobial activities. On the contrary, combination of nisin with diacetyl against Salm. Typhimurium resulted in an antagonism of diacetyl activity. CONCLUSIONS: While the individual natural organic compounds showed inhibitory activity against the two Gram-negatives, their combinations with nisin showed no improvement of antimicrobial activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the potential of the natural organic compounds to control E. coli and Salm. Typhimurium.     

The microbial ecology of high-risk, chilled food factories; evidence for persistent Listeria spp. and Escherichia coli strains

AIMS: The intention of this study was to provide evidence of any Listeria spp. or Escherichia coli strain persistence, and to identify whether strains of these organisms adapt to specific environmental or product niches in food factories. METHODS AND RESULTS: A 3-year assessment of the microbial ecology of four, ready-to-eat food-processing factories was undertaken in which approx. 196 000 and 75 000 product and environmental samples were examined for Escherichia coli and Listeria spp. respectively. A total of 152 E. coli isolates (44 environmental and 108 product in 62 ribogroups) and 260 Listeria spp. isolates (174 environmental and 86 product in 30 ribogroups) were identified and ribotyped. The overall prevalence of E. coli (0.08%), all Listeria spp. (0.35%) and L. monocytogenes (0.23%) was very low. Some 10 E. coli ribogroups and 14 Listeria spp. ribogroups showed evidence for persistence, defined as the isolation of the same strain, from the same site, over a prolonged time period. The majority of E. coli strains were product niche oriented whilst the majority of Listeria spp. strains were environmental niche oriented. CONCLUSION: Current UK high-risk food factory designs, personnel hygiene and cleaning and disinfection regimes are sufficient to control Listeria spp. and E. coli to very low levels. SIGNIFICANCE AND IMPACT OF THE STUDY: Persistent strains of these organisms, however, can remain within factory high-risk production areas over considerable time periods, warranting an examination of the strain persistence mechanisms and alternative hygiene controls.     

Occurrence and molecular characteristics of antimicrobial resistance of Escherichia coli from broilers,pigs and meat products in Thailand and Cambodia provinces

Nine hundred and forty‐one samples were collected in Sa Keao, Thailand (n = 554) and Banteay Meanchey, Cambodia (n = 387) from July 2014 to January 2015. A total of 667 Escherichia coli isolates (381 isolates from Sa Keao and 286 isolates from Banteay Meanchey) were obtained and examined for antimicrobial susceptibility, class 1 integrons, ESBL genes and horizontal transfer of resistance determinants. Prevalence of E. coli in pig and broiler carcass samples from slaughterhouses and fresh markets was 36–85% in Sa Keao and 11–69% in Banteay Meanchey. The majority of these isolates were multidrug resistant (75.3%). Class 1 integrons were common in both Thai (47%) and Cambodian (62%) isolates, of which four resistance gene cassette arrays including aadA1, dfrA1‐aadA1, dfrA12‐aadA2 and aadA2‐linF were identified. Class 1 integrons in two broiler isolates from Sa Keao (dfrA12‐aadA2) and one broiler isolate from Banteay Meanchey (dfrA1‐aadA1) were horizontally transferable. Sixteen isolates were confirmed to be ESBL‐producing strains with ESBL gene bla_CTX‐M‐15, broad spectrum β‐lactamase gene bla_TEM‐1 and the AmpC gene bla_CMY‐2 being detected. The bla_TEM‐1 gene was most prevalent and located on a conjugative plasmid.     

Inactivation of Escherichia coli and Listeria monocytogenes on iceberg lettuce by dip wash treatments with organic acids

AIMS: To study and compare the efficacy of organic acids and chlorine dipping in inactivation of Escherichia coli and Listeria monocytogenes on fresh-cut iceberg lettuce. METHODS AND RESULTS: Fresh-cut iceberg lettuce leaves were inoculated with E. coli or L. monocytogenes. After inoculation, samples were stored at 4 degrees C for 24 h and dipped in organic acid or chlorine solutions for 2 and 5 min. E. coli and L. monocytogenes were enumerated on selective media. Treatment of fresh-cut iceberg lettuce with chlorine solution caused 1.0 and 2.0 log(10) CFU g(-1) reductions in the number of L. monocytogenes and E. coli, respectively. Maximum reduction for E. coli (about 2.0 log(10) CFU g(-1)) was obtained for samples dipped in lactic or citric acids while maximum reduction for L. monocytogenes (about 1.5 log(10) CFU g(-1)) was attained for samples dipped in lactic acid. CONCLUSIONS: Dipping of iceberg lettuce in 0.5% citric acid or 0.5% lactic acid solution for 2 min could be as effective as chlorine for reducing microbial populations on fresh-cut iceberg lettuce. SIGNIFICANCE AND IMPACT OF THE STUDY: Dipping in solutions containing organic acids is shown to be effective to reduce E. coli and L. monocytogenes on fresh-cut iceberg lettuce.     

Antimicrobial action of N‐(n‐dodecyl)diethanolamine on Escherichia coli: effects on enzymes and growing cultures

Aims: This study investigates the effects of N‐(n‐dodecyl)diethanolamine (DDA) on enzymes and growing cells of Escherichia coli NCIMB 8277. Methods and Results: Enzyme activities in the presence of DDA were determined by measuring substrate‐dependent oxygen consumption by whole cells, or of NADH formation or oxidation by cell extracts. Lysis of growing cells was followed by measuring changes in turbidity and cell count. DDA promptly arrested oxygen uptake on pyruvate and acetate, due to cofactor loss rather than to enzyme denaturation, since cell‐free glyceraldehyde‐3‐phosphate and NADH dehydrogenases remained active. Formate and succinate oxidation by membrane‐bound enzyme systems independent of cofactors was likewise unaffected. DDA lysed growing cells at rates related to drug concentration, pH, and the previous growth rate. Conclusions: Loss of cellular enzyme activity following addition of DDA is due to cofactor leakage and not to enzyme denaturation. Whereas nongrowing cells remain intact in the presence of DDA, actively‐growing organisms undergo lysis, consistent with autolysin action. Significance and Impact of the Study: Cell lysis, not normally observed with membrane‐active antimicrobials, also occurs with cetrimide, and may be dependent on the alkyl chain length in these compounds. The action on growing cells parallels that of penicillin and daptomycin, which bears a decanoyl residue that penetrates the cell membrane, causing leakage and membrane depolarization.     

Characterization of antimicrobial resistance among Escherichia coli isolates from chickens in China between 2001 and 2006

Escherichia coli is a common commensal bacterium and is regarded as a good indicator organism for antimicrobial resistance for a wide range of bacteria in the community and on farms. Antimicrobial resistance of E. coli isolated from chickens from 49 farms in China between 2001 and 2006 was studied. A total of 536 E. coli isolates were collected, and minimal inhibitory concentrations (MICs) of eight antimicrobials were determined by the broth microdilution method. Isolates exhibited high levels of resistance to ampicillin (80.2%), doxycycline (75.0%) and enrofloxacin (67.5%). Relatively lower resistance rates to cephalothin (32.8%), cefazolin (17.0%) and amikacin (6.5%) were observed. Strains were comparatively susceptible to colistin (MIC(50)=1 mug mL(-1)). A marked increase in isolates with elevated MICs for florfenicol was observed over the study period. Therefore, five resistance genes leading to the dissemination of phenicol resistance in the isolates (n=113) with florfenicol MICs>/=32 mug mL(-1) were analyzed. The gene floR was the most prevalent resistance gene and was detected in 92% of the 113 isolates, followed by the cmlA (53%), catA1 (23%) and catA2 (10%) genes. catA3 was not detected in these isolates. Eight isolates with florfenicol MICs=32 mug mL(-1) and one with MIC=64 mug mL(-1) were negative for the floR gene.     

The EnvZ protein of Salmonella typhimurium LT-2 and Escherichia coli K-12 is located in the cytoplasmic membrane

Abstract The osmoregulated expression of the porin proteins OmpC and OmpF in S. typhimurium and E. coli is dependent on the regulatory proteins OmpR and EnvZ. The function of the EnvZ protein is not clear. In order to establish the cellular location of EnvZ two different methods of buoyant sucrose density centrifugation was employed. The presence of EnvZ in the different fractions was visualised by immunoblotting. It was conclusively shown that the EnvZ protein is located in the cytoplasmic membrane fraction. The result is in agreement with the available sequence data which shows that the EnvZ polypeptide contains two long hydrophobic stretches.     

A high prevalence of antimicrobial resistant Escherichia coli isolated from pigs and a low prevalence of antimicrobial resistant E. coli from cattle and sheep in Great Britain at slaughter

The incidence of antimicrobial resistance and expressed and unexpressed resistance genes among commensal Escherichia coli isolated from healthy farm animals at slaughter in Great Britain was investigated. The prevalence of antimicrobial resistance among the isolates varied according to the animal species; of 836 isolates from cattle tested only 5.7% were resistant to one or more antimicrobials, while only 3.0% of 836 isolates from sheep were resistant to one or more agents. However, 92.1% of 2480 isolates from pigs were resistant to at least one antimicrobial. Among isolates from pigs, resistance to some antimicrobials such as tetracycline (78.7%), sulphonamide (66.9%) and streptomycin (37.5%) was found to be common, but relatively rare to other agents such as amikacin (0.1%), ceftazidime (0.1%) and coamoxiclav (0.2%). The isolates had a diverse range of resistance gene profiles, with tet(B), sul2 and strAB identified most frequently. Seven out of 615 isolates investigated carried unexpressed resistance genes. One trimethoprim-susceptible isolate carried a complete dfrA17 gene but lacked a promoter for it. However, in the remaining six streptomycin-susceptible isolates, one of which carried strAB while the others carried aadA, no mutations or deletions in gene or promoter sequences were identified to account for susceptibility. The data indicate that antimicrobial resistance in E. coli of animal origin is due to a broad range of acquired genes.     

The development of Escherichia coli and Listeria monocytogenes variants resistant to high‐pressure carbon dioxide inactivation

Aims: The objective of this study was to investigate whether bacterial cells could develop resistance (as a part of their adaptation strategy) to high‐pressure CO₂ (HPCD) inactivation. Methods and Results: Alternating cycles of exposure to pressurized CO₂ (10·5 MPa, 35°C, 400 min^?1, 70% working volume ratio during 10 min) and re‐growth of the surviving subpopulation were used to investigate possible increases in the resistance of Escherichia coli and Listeria monocytogenes to HPCD. The results show an increased resistance of both pathogens tested after seven cycles of inactivation. Increase in the resistance after 15 cycles resulted in a difference of 2·4 log CFU ml^?1 in log N₀/N_i when parental (N₀) and treated cultures (N_i) of E. coli and L. monocytogenes were compared. Conclusions: Current findings indicate the ability of micro‐organisms to adapt to HPCD preservation technology. Significance and Impact of the Study: The occurrence of HPCD‐resistant micro‐organisms could pose a new hazard to the safety and stability of HPCD‐processed foods.     

应用实时荧光定量PCR定量检测溃疡性结肠炎肠道大肠埃希菌、乳酸杆菌及双歧杆菌属的变化

目的应用实时荧光定量PCR（RT—PCR）法对溃疡性结肠炎（ulcerative colitis,UC）患者粪便中大肠埃希菌、乳酸杆菌及双歧杆菌属的数量进行定量检测分析。方法根据细菌的16SrDNA基因序列设计大肠埃希菌、乳酸杆菌及双歧杆菌属的种属特异性引物。收集溃疡性结肠炎患者及正常对照者新鲜粪便标本各35例,从待测粪便标本中提取细菌基因组DNA,进行实时荧光定量PCR反应,定量分析不同细菌的数量。结果正常对照组与病例组粪便中细菌数量分别为大肠埃希菌（4．62±1．10;5．27±1．02）、乳酸杆菌属（4．99±0．75;4．65±0．95）、双歧杆菌属（5．07±0．95;4．93±0．99）,病例组大肠埃希菌数量明显增多（t=2．540,P=0．013）,而乳酸杆菌及双歧杆菌属数量与正常组比较差异无统计学意义（t1=0．488,P,=0．530;t2=-0．533,P：=0．596）。结论溃疡性结肠炎患者粪便中大肠埃希菌的数量较正常对照明显增多,而乳酸杆菌及双歧杆菌属的数量无明显变化,提示大肠埃希菌与溃疡性结肠炎的发病或复发有关系,而乳酸及双歧杆菌属与此病的关系有待进一步研究。     

Stx genotypes and antimicrobial resistance profiles of Shiga toxin-producing Escherichia coli strains isolated from human infections, cattle and foods in Brazil

A total of 107 Shiga toxin-producing Escherichia coli strains (STEC) isolated from different origins in S?o Paulo, Brazil, and belonging to different serotypes were characterized regarding stx subtypes and susceptibility to antimicrobial agents. Most of the human STEC strains harbored stx1 (85.7%), while stx2, associated or not to stx1, was identified preferentially in the animal and food strains. None of the STEC strains carried stx1c. Some genotypes occurred exclusively among strains of bovine origin as stx2c, stx1+2+2c (16.5% each), and stx2d (0.9%), whereas stx2+2c2vha) was only identified among the O157:H7 human strains. Moreover, the stx(2c2vhb) subtype was found more frequently among bovine than human strains (39% vs. 4.8%). The highest frequencies of susceptibility to antimicrobial agents were observed among bovine (87%) and food (100%) STEC strains, while 47.6% of the human isolates were resistant to at least one drug. Multiresistance occurred among O111 STEC strains from human and bovine origin. The antimicrobials to which resistance was most frequently observed were tetracycline (90%) and streptomycin (75%) among human strains, and also sulphazotrin (88%) in animal strains. A few serotypes were commonly identified among STEC strains isolated from diverse sources in Brazil, but in general the strains presented distinct stx subtypes and/or antimicrobial resistance profiles.     

植物乳杆菌素L-1对单核细胞增生李斯特氏菌作用机理的研究

以凝胶层析纯化的植物乳杆菌素作用单核细胞增生李斯特氏菌,结果表明该细菌素可以导致能量化的敏感细胞胞内K 、无机磷离子、乳酸脱氢酶、紫外吸收物质和ATP发生不同程度的泄漏,相应地破坏了膜Δψ和部分ΔpH,引起PMF的耗散,结果导致细胞的死亡。综合所测指标,可以推测植物乳杆菌素L-1对单增李斯特氏菌的作用目标主要是细胞膜,通过形成非选择性孔洞使得选择性离子和小分子生命物质外泄,从而打破原有平衡,最终引起细胞的衰亡。