Efficacy of solar disinfection of Escherichia coli, Shigella flexneri, Salmonella Typhimurium and Vibrio cholerae

AIMS: To determine the efficacy of solar disinfection (SODIS) for enteric pathogens and to test applicability of the reciprocity law. METHODS AND RESULTS: Resistance to sunlight at 37 degrees C based on F99 values was in the following order: Salmonella Typhimurium>Escherichia coli>Shigella flexneri>Vibrio cholerae. While F90 values of Salm. Typhimurium and E. coli were similar, F99 values differed by 60% due to different inactivation curve shapes. Efficacy seemed not to be dependent on fluence rate for E. coli stationary cells. Sensitivity to mild heat was observed above a temperature of 45 degrees C for E. coli, Salm. Typhimurium and Sh. flexneri, while V. cholerae was already susceptible above 40 degrees C. CONCLUSIONS: Salmonella Typhimurium was the most resistant and V. cholerae the least resistant enteric strain. The reciprocity law is applicable for stationary E. coli cells irradiated with sunlight or artificial sunlight. SIGNIFICANCE AND IMPACT OF THE STUDY: Escherichia coli might not be the appropriate indicator bacterium to test the efficacy of SODIS on enteric bacteria and the physiological response to SODIS might be different among enteric bacteria. The applicability of the reciprocity law indicates that fluence rate plays a secondary role in SODIS efficacy. Stating inactivation efficacy with T90 or F90 values without showing original data is inadequate for SODIS studies.     

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.     

Adaptive responses of Salmonella enterica serovar Typhimurium DT104 and other S. Typhimurium strains and Escherichia coli O157 to low pH environments

AIMS: Cattle are a known main reservoir for acid-resistant Escherichia coli O157 and Salmonella enterica serovar Typhimurium DT104. We studied the response of S. Typhimurium DT104 to extreme low pH environments and compared their response to that of acid-resistant E. coli O157 and other S. Typhimurium phage types. METHODS AND RESULTS: Bacteria were grown in nutrient-rich medium and subsequently acid challenged at pH 2.5. We found that stationary phase cultures of various S. Typhimurium strains were able to survive a challenge for 2 h at pH 2.5. As in E. coli, the ability of S. Typhimurium to survive at pH 2.5 was shown to be dependent on the presence of amino acids, specifically arginine. The amount of proton pumping H+/ATPase, both in E. coli O157 and S. Typhimurium strains, was lower when grown at pH values <6 than after growth at pH 7.5. Cyclo fatty acid content of membranes of bacteria grown at pH values <6 was higher than that of membranes of bacteria grown at pH 7.5. CONCLUSIONS: Various S. Typhimurium strains, both DT104 and non-DT104, are able to survive for a prolonged period of time at pH 2.5. Their response to such low pH environment is seemingly similar to that of E. coli O157. SIGNIFICANCE AND IMPACT OF THE STUDY: Food-borne pathogens like S. Typhimurium DT104 and E. coli O157 form a serious threat to public health since such strains are able to survive under extreme low pH conditions as present in the human stomach. The emergence these acid-resistant strains suggests the presence of a selection barrier. The intestinal tract of ruminants fed a carbohydrate-rich diet might be such a barrier.     

Influence of temperature fluctuations on Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium in cow manure

The effects of four average temperatures (7, 16, 23 and 33 degrees C) and daily oscillations with three amplitudes (0, +/-4, +/-7 degrees C) on the survival of the enteropathogens Escherichia coli O157:H7 and Salmonella serovar Typhimurium were investigated in small microcosms. Manure was inoculated with a green fluorescent protein transformed strain of either pathogen at 10(7) cells g(-1) dry weight. Samples were collected immediately after inoculation, and 1 and 2 weeks after inoculation for E. coli O157:H7, and immediately and after 2 and 3 weeks for Salmonella serovar Typhimurium. Population densities were determined by dilution plating and direct counting. In addition, total bacterial CFUs were determined. Growth and survival data were fitted to a modified logistic model. Analysis of the estimated parameter values showed that E. coli O157:H7 survived for shorter periods of time and was more sensitive to competition by the native microbial community than Salmonella serovar Typhimurium. Survival of both pathogens significantly declined with increasing mean temperatures and with increasing amplitude in daily temperature oscillations. The results indicated that responses of enteropathogens to fluctuating temperatures cannot be deduced from temperature relationships determined under constant temperatures.     

Survival and gene expression of enterotoxigenic Escherichia coli during long‐term incubation in sea water and freshwater

Aims: In this study, the main objective was to verify the hypothesis of induction of ‘viable but non‐culturable’ (VBNC) forms of enterotoxigenic Escherichia coli (ETEC) during incubation in water. Methods and Results: Six clinically isolated ETEC strains were studied. Viable counts showed culturable ETEC bacteria for up to 3 months in freshwater but only two out of six strains were culturable in seawater at this time point. Although the bacterial cells remained intact, no production or secretion of heat‐labile (LT) or heat‐stable (ST) enterotoxins was observed using GM1‐ELISA methods. However, genes encoding ETEC toxins (STh and LT), colonization factors (CS7 and CS17), gapA and 16S RNA were expressed during 3 months in both sea water and freshwater microcosms as determined by real‐time RT‐PCR on cDNA derived from the bacteria. Conclusions: Clinically isolated ETEC strains can survive for long periods in both sea water and freshwater. The bacterial cells remain intact, and the gene expression of virulence genes and genes involved in metabolic pathways are detected after 3 months. Significance and Impact of the Study: These results indicate that ETEC bacteria can enter a VBNC state during stressful conditions and suggest that ETEC has the potential to be infectious after long‐term incubation in water.     

肠毒素大肠杆菌定居因子CS3和肠毒素融合抗原基因在减毒鼠伤寒沙门氏菌中的共表达

不耐热肠毒素（LT）和耐热肠毒素（ST）是产肠毒素大肠杆菌的主要致病因素,CS3为该菌的优势定居因子,是定居因子CFA/Ⅱ菌毛抗原的共有抗原组分。采用基因操作技术将编码CS3和融合肠毒素蛋白基因转化到减毒鼠伤寒沙门氏菌疫苗侯选株X4072中进行表达。用重组菌株口服免疫小鼠后,免疫动物能产生抗CS3、LT和ST的血清抗体。特别有意义的是,所产生的抗ST抗体能中和天然ST的生物活性。这一结果为研究载体疫苗防治肠毒素大肠杆菌腹泻疾病奠定了基础。     

选择性富集沙门氏菌、副溶血弧菌和霍乱弧菌的共增菌培养基SVV

本研究通过单因素试验和响应面分析试验建立了能够选择性富集沙门氏菌、副溶血弧菌和霍乱弧菌的共增菌培养基SVV,采用平板计数法及三重荧光PCR技术验证了SVV的增菌效果。结果表明:SVV能同时富集以不同浓度比例混合的3种目标菌,37oC振荡培养18h后,菌体浓度达到105~108CFU/mL;SVV强烈抑制大部分的非目标菌;用荧光PCR方法检测经过37oC振荡培养18h的10份人工接种样品和608份实际样品,结果表明目标菌在SVV中增殖18h后菌量达到检测限以上,SVV联合荧光PCR检测方法的检出率为4.06%,比传统检测方法(3.78%)高,无假阴性和假阳性。SVV可望应用于水产品中沙门氏菌、副溶血弧菌和霍乱弧菌检测前的增菌处理,可简化检测过程,有效克服漏检,提高检出率。     

Artificial small RNA-mediated growth inhibition in Escherichia coli and Salmonella enterica serovar Typhimurium

We developed a synthetic RNA approach to identify growth inhibition sequences by cloning random 24-nucleotide (nt) sequences into an arabinose-inducible expression vector. This vector expressed a small RNA (sRNA) of ∼140 nt containing a 24 nt random sequence insert. After transforming Escherichia coli with the vector, 10 out of 954 transformants showed strong growth defect phenotypes and two clones caused cell lysis. We then examined growth inhibition phenotypes in the Salmonella Typhimurium LT2 strain using the twelve sRNAs that exerted an inhibitory effect on E. coli growth. Three of these clones showed strong growth inhibition phenotypes in S. Typhimurium LT2. The most effective sRNA contained the same insert (N1) in both bacteria. The 24 nt random sequence insert of N1 was abundant in guanine residues (ten out of 24 nt), and other random sequences causing growth defects were also highly enriched for guanine (G) nucleotides. We, therefore, generated clones that express sRNAs containing a stretch of 16 to 24 continuous guanine sequences (poly-G₁₆, -G₁₈, -G₂₀, -G₂₂, and -G₂₄). All of these clones induced growth inhibition in both liquid and agar plate media and the poly-G₂₀ clone showed the strongest effect in E. coli. These results demonstrate that our sRNA expression system can be used to identify nucleotide sequences that are potential candidates for oligonucleotide antimicrobial drugs.     

The survival of Salmonella enterica serovar Typhimurium DT104 and total viable counts on beef surfaces at different relative humidities and temperatures

Aims:  The aim of this study was to investigate changes in Salmonella and total viable count (TVC) survival on beef carcass surfaces stored for 72 h under different combinations of relative humidity (i.e. RH 75% or 96%) and temperature (5°C or 10°C).
Methods and Results:  The influence of low water activity ( a _w) and temperature on the survival and growth of Salmonella enterica serovar Typhimurium DT104 and the aerobic mesophilic flora on meat pieces from different sites on beef carcasses was investigated, under controlled conditions (75% or 96% RH; 5 or 10°C) in an environmental cabinet. Salmonella counts declined during storage at low a _w (75% RH) conditions at 5°C or 10°C. Salmonella counts increased during storage at high a _w (96% RH) at 10°C only. At 5°C, TVCs increased during storage at high a _w, but not at low a _w. TVCs increased on all samples from carcasses stored at high or low a _w at 10°C, except those samples taken from areas of surface fat.
Conclusions:  This suggests that substrate composition dictates growth rates under low a _w conditions. The results are discussed in terms of the possible protective effects of substrate osmolyte accumulation in bacterial survival and/or growth.
Significance and Impact of the Study:  The data obtained in this study provides useful insights on the influence of a _w and temperature on pathogen survival on meat surfaces at chill temperature.     

Seasonal effects on the survival characteristics of Salmonella Typhimurium and Salmonella Derby in pig slurry during storage

AIMS: Studies were carried out to investigate the survival of Salmonella Typhimurium and Salmonella Derby in pig slurry during summer and winter seasons. METHODS AND RESULTS: Pig slurry samples collected from a commercial fattening house were inoculated with a broth culture of Salmonella Typhimurium and Salmonella Derby, each at a level of log(10) 5.0 CFU ml(-1) and log(10) 2.0 CFU ml(-1). At the higher inoculum level, S. Typhimurium and S. Derby survived for 34 and 23 days, respectively in the summer, and 58 and 46 days, respectively in the winter. Survival at the lower inoculum level for S. Typhimurium and S. Derby was 19 and 16 days, respectively, in the summer and 24 days for both in the winter. CONCLUSIONS: The survival of S. Typhimurium and S. Derby observed in this study indicates that a 2-month holding period of pig slurry, prior to land spreading, may be adequate if separate storage facilities are provided. SIGNIFICANCE AND IMPACT OF THE STUDY: Despite difficulties correlating laboratory studies with on-farm conditions, pig slurry may not represent a major source of transmission of Salmonella spp. in the farm environment in Ireland.     

Survival of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium in manure and manure‐amended soil under tropical climatic conditions in Sub‐Saharan Africa

Aims: To establish the fate of Escherichia coli O157:H7 and Salmonella Typhimurium in manure and manure‐amended agricultural soils under tropical conditions in Sub‐Saharan Africa. Methods and Results: Survival of nonvirulent E. coli O157:H7 and Salm. Typhimurium at 4 and 7 log CFU g^?1 in manure and manure‐amended soil maintained at ≥80% r.h. or exposed to exclusive field or screen house conditions was determined in the Central Agro‐Ecological Zone of Uganda. Maintaining the matrices at high moisture level promoted the persistence of high‐density inocula and enhanced the decline of low‐density inocula in the screen house, but moisture condition did not affect survival in the field. The large majority of the survival kinetics displayed complex patterns corresponding to the Double Weibull model. The two enteric bacteria survived longer in manure‐amended soil than in manure. The 7 log CFU g^?1E. coli O157:H7 and Salm. Typhimurium survived for 49–84 and 63–98 days, while at 4 log CFU g^?1, persistence was 21–28 and 35–42 days, respectively. Conclusions: Under tropical conditions, E. coli O157:H7 and Salm. Typhimurium persisted for 4 and 6 weeks at low inoculum density and for 12 and 14 weeks at high inoculum density, respectively. Significance and Impact of the Study: Persistence in the tropics was (i) mostly shorter than previously observed in temperate regions thus suggesting that biophysical conditions in the tropics might be more detrimental to enteric bacteria than in temperate environments; (ii) inconsistent with published data isothermally determined previously hence indicating the irrelevance of single point isothermal data to estimate survival under dynamic temperature conditions.     

Cell-cell interactions influence resistance and survival of Salmonella serotype Typhimurium to environmental stress

AIMS: The aim of this work was to study the effects of prolonged nutrient stress on survival, cell interactions and resistance to inimical processes in Salmonella serotype Typhimurium. METHODS AND RESULTS: Salmonella Typhimurium cells were subjected to prolonged incubation in the stationary phase of growth and the properties of starved cells (old) were investigated with reference to those of exponentially-growing cells (young). Competition experiments between old and young cells revealed cell-cell interactions that influenced stationary phase survival and response of the bacterium to heat stress. During prolonged incubation of cells, cycles of resistance and sensitivity to heat stress were identified. Competition experiments between old and young cells revealed that the resistance of young cells to heat increased to levels more like those of stationary phase cells than growing cells. The presence of old cells influenced the phenotype of young cells, possibly by means of cell-cell interactions. There was no evidence for the involvement of any extracellularly-produced factors in this phenomenon, but a requirement that the old competitor cells be viable could be demonstrated. CONCLUSIONS: It is proposed that the complex interactions within stationary phase cultures of Salm. Typhimurium may be due to cycles of mutation in concert with an as yet undefined interaction between old cells and growing ones. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides evidence for active and diverse responses to nutrient stress within populations of Salm. Typhimurium that promote survival and that may be important for the success of this bacterium as a pathogen.     

Kinetic model‐based prediction of the persistence of Salmonella enterica serovar Typhimurium under tropical agricultural field conditions

Aim: Present a kinetic model‐based approach for using isothermal data to predict the survival of manure‐borne enteric bacteria under dynamic conditions in an agricultural environment. Methods and Results: A model to predict the survival of Salmonella enterica serovar Typhimurium under dynamic temperature conditions in soil in the field was developed. The working hypothesis was that the inactivation phenomena associated with the survival kinetics of an organism in an agricultural matrix under dynamic temperature conditions is for a large part due to the cumulative effect of inactivation at various temperatures within the continuum registered in the matrix in the field. The modelling approach followed included (i) the recording of the temperature profile that the organism experiences in the field matrix, (ii) modelling the survival kinetics under isothermal conditions at a range of temperatures that were registered in the matrix in the field; and (iii) using the isothermal‐based kinetic models to develop models for predicting survival under dynamic conditions. The time needed for 7 log CFU g^?1Salmonella Typhimurium in manure and manure‐amended soil to reach the detection limit of the enumeration method (2 log CFU g^?1) under tropical conditions in the Central Agro‐Ecological Zone of Uganda was predicted to be 61–68 days and corresponded with observed CFU of about 2·2–3·0 log CFU g^?1, respectively. The Bias and Accuracy factor of the prediction was 0·71–0·84 and 1·2–1·4, respectively. Conclusions: Survival of Salm. Typhimurium under dynamic field conditions could be for 71–84% determined by the developed modelling approach, hence substantiating the working hypothesis. Significance and Impact of the Study: Survival kinetic models obtained under isothermal conditions can be used to develop models for predicting the persistence of manure‐borne enteric bacteria under dynamic field conditions in an agricultural environment.     

Long-term survival and plasmid maintenance of Escherichia coli in marine microcosms

Abstract The survival pattern and plasmid maintenance of Escherichia coli was examined in an artificial seawater microcosm. It was found that the three strains of E. coli (EK3C, H10407 and 34309) included in the study were able to maintain a portion of cells in the culturable phase for at least 3 years in artificial seawater. Along with retaining culturability, that portion of the cell population also maintained their indigenous plasmids over the 3-year period. It is concluded that cells of E. coli maintaining culturability in seawater are selectively adapted to the salinity of seawater, remaining in a culturable state. The results of the study are significant in that it has been assumed by many public health authorities that E. coli cannot survive, without nutrient addition, in seawater for long periods of time, i.e., years of exposure to seawater.