Survival of pathogenic bacteria in various freshwater sediments

Four human-associated bacteria, Pseudomonas aeruginosa, Salmonella newport, Escherichia coli, and Klebsiella pneumoniae, were tested for survival in five freshwater sediments. Bacterial survival in continuous-flow chambers was monitored over 14-day periods on sediments ranging from organically rich high-clay fractions to organically poor sandy fractions. Bacterial die-off ranged from 1 to 5 orders of magnitude in sediments. E. coli survived as long as or longer than S. newport. P. aeruginosa and K. pneumoniae tended to survive longer than E. coli. Survival of E. coli and S. newport was greater in sediments containing at least 25% clay. Good reproducibility allowed the development of linear models to describe die-off rates.     

Survival of pathogenic bacteria in various freshwater sediments.

Four human-associated bacteria, Pseudomonas aeruginosa, Salmonella newport, Escherichia coli, and Klebsiella pneumoniae, were tested for survival in five freshwater sediments. Bacterial survival in continuous-flow chambers was monitored over 14-day periods on sediments ranging from organically rich high-clay fractions to organically poor sandy fractions. Bacterial die-off ranged from 1 to 5 orders of magnitude in sediments. E. coli survived as long as or longer than S. newport. P. aeruginosa and K. pneumoniae tended to survive longer than E. coli. Survival of E. coli and S. newport was greater in sediments containing at least 25% clay. Good reproducibility allowed the development of linear models to describe die-off rates.     

Specific detection of Salmonella spp. by multiplex polymerase chain reaction.

Three sets of oligonucleotide primers were used in the polymerase chain reaction (PCR) assay to detect Salmonella species. phoP primers specific to the phoP/phoQ loci of coliform pathogenic bacteria such as Salmonella, Shigella, Escherichia coli, and Citrobacter species served as presumptive indicators of enteric bacteria. In addition to the phoP primers, the Hin and the H-1i primers, which targeted a 236-bp region of hin/H2 and a 173-bp region of the H-1i flagellin gene, respectively, were used. Both Hin and H-1i primers are specific to motile Salmonella species and are not present in Shigella, E. coli, or Citrobacter species. Thus, by multiplex PCR amplification, Salmonella species including Salmonella typhi, Salmonella typhimurium, Salmonella paratyphi A, and Salmonella enteritidis can be specifically detected. Optimal reaction conditions have been described to demonstrate this specific, sensitive detection of Salmonella species. By using agarose gel electrophoresis for detection of the PCR-amplified products, the sensitivity of detection was 10(2) CFU after 25 cycles of PCR and 1 (10(0)) CFU after a 50-cycle double PCR. The efficacy of these primers was demonstrated on environmental isolates which had previously been confirmed as Salmonella species by the use of conventional cultural techniques. In addition, positive amplifications resulted from Salmonella species in environmental samples including soil and water.     

Selection of Salmonella typhimurium as an indicator for pathogen regrowth potential in composted biosolids

In order to select a suitable indicator for monitoring the pathogen regrowth potential of composted biosolids, the growth kinetics of selected bacteria were investigated. Growth parameters of six serovars of Salmonella and three strains of Escherichia coli in sterilized compost were compared. Seeded Salmonella and E. coli grew rapidly, reaching population densities of more than 10(8) g-1 after 30 h of incubation. The specific growth rates of Salmonella serovars and E. coli strains were similar and varied from 0.49 to 0.55 h-1. The specific growth rate of the Salm. Typhimurium isolates was significantly higher than the other bacterial strains. It was concluded that an antibiotic-resistant strain of Salm. Typhimurium can be used as an indicator for a pathogen regrowth potential test.     

Paw oedema test for detection of Salmonella enterotoxin : modification and standardization

Paw oedema test (POT) was standardized with modifications for the detection of Salmonella enterotoxin. Instead of measuring the weight of the inoculated paws after amputating the limbs at 48 hr post-inoculation, percent relative thickness of the order of 121 +/- 3.8% at 24-48 hr was found to be a better index. This test yielded parallel results to rabbit ligated ileal loop (RLIL) technique. The test was positive with enterotoxic crude cell lysates (CL) and cell free-culture-supernatants (CFCS) of S. newport and S. typhimurium, partially purified and purified enterotoxin of S. newport and purified cholera toxin. The test was found to be specific in that non-enterotoxic CFCS did not cause significant increase in the thickness. Minimum detection level of purified S. newport enterotoxin was estimated to be as low as 20 micrograms. Thus, the modified POT was considered to be an effective and economical bioassay model for the detection of Salmonella enterotoxin.     

Chemical modification of microcin J25 with diethylpyrocarbonate and carbodiimide: evidence for essential histidyl and carboxyl residues

In this paper we compared the antibacterial activity of native microcin J25, a peptide antibiotic, with the activities of two analogues obtained by chemical modifications. In the first analogue, the negative charge of glutamic carboxyl group was specifically blocked with an L-glycine methyl ester and in the second the histidine imidazole ring was carbethoxylated. Both analogues decreased notably its antibiotic activity against Escherichia coli and Salmonella newport, strains sensible to the native microcin J25. The biological activity of the carbethoxylated analogue was completely recovered after treatment with hydroxylamine. The extreme importance of both polar residues could be interpreted as specific structural features indispensable for the peptide transportation into the cell, extrusion outside the cell or alternatively to inhibit the RNA-polymerase.     

致病性大肠埃希菌和沙门菌毒力岛基因的双重PCR检测方法的建立

目的实现对致病性大肠埃希菌（E．coli）、沙门菌（Salmonella）的同时检测,建立快速灵敏的双重PCR检测方法。方法以致病性大肠埃希菌和沙门菌毒力岛基因为研究对象,根据GenBank发表的大肠埃希菌和沙门菌毒力岛基因序列,分别设计合成了大肠埃希菌毒力岛irpl、irl）2和fyuA,沙门菌毒力岛mgtC、sseL和sopB等6对引物,以禽致病性大肠埃希菌（CVCC1565）菌株和沙门菌（ATCC9150）菌株的核酸混合物为模板,经引物特异性试验,引物组合,成功建立了快速鉴别检测致病性大肠埃希菌和沙门菌的双重PCR方法。结果特异性试验结果显示,引物irpl、irp2和fyuA仅能扩增出大肠埃希菌（CVCC1565）的特异性片段,大小分别是799、414和948bp;引物mgtC、sseL和sopB仅能扩增出沙门菌（ATCC9150）的特异性片段,大小分别是500、269和1000bp。敏感性试验结果表明大肠埃希菌和沙门菌的最低检测限分别为2．2×101CFU／mL和2．0×101CFU／mL。结论本研究建立的双重PCR方法具有特异性强、敏感性高、快速简便等特点,可用于致病性大肠埃希菌和沙门菌的联合检测与鉴别诊断。     

Antibiotic resistance in coagulase-negative staphylococci isolated from Cope''s gray treefrogs (Hyla chrysoscelis)

Microcin J25 (MccJ25) is a cyclic peptide of 21 unmodified amino acid residues produced by a fecal strain of Escherichia coli. It has previously been shown that the antibiotic activity of this peptide is mainly directed to Enterobacteriaceae, including several pathogenic E. coli, Salmonella and Shigella strains. In this paper we show that MccJ25 acts on the cytoplasmic membrane of Salmonella newport cells producing alteration of membrane permeability, and the subsequent gradient dissipation, that initiate the inhibition of process, such as oxygen consumption. These results, taken together with our in vitro observations [Rintoul et al. (2000) Biochim. Biophys. Acta 1509, 65-72], strongly suggest that the disruption of the cytoplasmic membrane gradient is closely related to the bactericidal activity of MccJ25 in S. newport.     

Isolation of salmonellae and other potential pathogens from the freshwater aquarium snail Ampullaria.

The freshwater aquarium snail (Ampullaria spp.) was demonstrated to carry as many as 10(8) viable mesophilic bacteria per g of meat plus shell. Some 16 genera of bacteria were identified, with gram negatives predominating. Enrichment culture techniques enabled the isolation of salmonellae from 24 to 42 lots of 200 g each. The salmonellae comprised eight different serotypes, including Salmonella newport, Salmonella saint-paul, and Salmonella infantis. This association of salmonellae with snails may contribute to cases of human salmonellosis, since other aquarium species have already been shown to contribute to many such cases. The snails were also found to commonly harbor Pseudomonas aeruginosa and, occasionally, Edwardsiella tarda.     

MULTIPLEX DETECTION OF ESCHERICHIA COLI AND SALMONELLA ENTERITIDIS BY USING QUANTUM DOT-LABELED ANTIBODIES

In this study, we demonstrated the simultaneous detection of Escherichia coli and Salmonella enteritidis, by coupling immunomagnetic separation (IMS) with quantum dots (QDs) labeling. QDs having different emission wavelengths were conjugated with anti- E. coli and anti- Salmonella antibodies. QD–antibody conjugates were used to label immunomagnetically separated bacteria and the fluorescence intensities were measured for enumerations of both species. The concentrations of primary antibodies used in IMS, the ratio of QDs to antibodies during the conjugation and the concentration of QD–antibody conjugates used in labeling were optimized to enhance the sensitivity of the assay. After labeling bacteria with QDs, the quenching observed between bead–bacteria complex and QDs was eliminated by separating QDs from the complex using sodium dodecyl sulfate solution. The fluorescence intensities due to the capturing of different concentrations of bacteria were measured and the working ranges were found to be 5 × 10² to 5 × 10⁵ cfu/mL for E. coli and 4  ×  10² to 4  ×  10⁵ cfu/mL for S. enteritidis .

PRACTICAL APPLICATIONS

In this study, antibody-conjugated multicolor quantum dots (QDs) were used for simultaneous detection of Escherichia coli and Salmonella enteritidis . The results of this study indicate that QD labels can be used in multiplex, rapid and selective detection of bacteria with detection limits comparable with those of many novel methods in cases where the assay conditions are optimized. Furthermore, the assay can be modified for the simultaneous detection of more than two species through using QD labels having different emission wavelengths.     

Mode of Salmonella and Escherichia coli O157:H7 inactivation by a stabilized oxychloro-based sanitizer

AIM: To determine the mechanisms by which a stabilized oxychloro (SOC)-based sanitizer, applied to decontaminate seeds destined for sprout production, inactivates Escherichia coli O157:H7 ph1 and Salmonella serotype Meleagridis. MATERIALS AND RESULTS: The action of SOC on the metabolism, membrane and DNA integrity of Salmonella and E. coli O157:H7 was studied. In both pathogens, there was an oxidative burst and depletion of intracellular glutathione (GSH) upon initial exposure to 200 ppm SOC. Metabolic activity, measured via bioluminescence, decreased over a 4-h period in E. coli O157:H7 ph1 cells exposed to SOC. Membrane integrity, assessed through viability staining, decreased progressively over 23 h when exposed to SOC. The appearance of auxotrophic mutants suggested that DNA damage had also occurred. Enzymes rich in disulfide bonds (alkaline phosphatase and protease) were sensitive to the chlorite-based sanitizer. Through challenging other microbial types, it was found that Gram positive had higher tolerance to SOC than Gram negatives with the exception of Salmonella. MS2 bacteriophage was highly sensitive; however, Bacillus endospores were not inactivated by SOC. CONCLUSIONS: SOC inactivates E. coli O157:H7 and Salmonella through GSH oxidation and disruption of disulfide bonds. Ultimately, membrane damage resulting from prolonged exposure to SOC leads to the loss of cell viability. SIGNIFICANCE AND IMPACT OF THE STUDY: The results provide a basis for understanding why extended treatment times are required to inactivate bacteria using SOC.     

Enzyme immunoassay in which a myeloma protein is used for detection of salmonellae

An enzyme immunoassay (EIA) in which an immunoglobulin A monoclonal antibody from a myeloma (MOPC 467) is used was developed to detect the presence of Salmonella organisms. This myeloma protein binds to a flagellar determinant of the organisms but is not directed toward the H antigens. Of 100 strains tested, 94% were detectable with this antibody. The EIA, used with MOPC 467, is quick, sensitive, and specific, showing virtually no cross-reactivity to other enteric organisms. Initial screening of antibody reactivity was performed by Ouchterlony gel diffusion with the supernatants of heat-treated Salmonella cultures. After this, an EIA was performed on the heat extracts with the myeloma protein, which had been directly coupled to alkaline phosphatase. A positive reaction was indicated by the production of a yellow color after the addition of a substrate (p-nitrophenylphosphate), and this was quantitated by determining the absorbance at 405 nm. The EIA proved to be slightly more sensitive than the Ouchterlony analysis. The sensitivity of the EIA is such that as few as 10(6) Salmonella organisms per ml were detected. This concentration was easily obtained after a 24-h preenrichment incubation of the sample. Mixtures of Salmonella strains with a 10 x concentration of Escherichia coli did not prevent detection of the Salmonella strains. This EIA can be successfully used to detect contamination of foods, as it was used to detect the intentional contamination of infant formula in these studies. Indications are that the EIA is sensitive enough to detect Salmonella strains in M broth subcultures taken directly from a preenrichment culture. Testing of samples could thus be completed 36 h after culture initiation, rather than after 96 h, the time currently needed.     

Enzyme immunoassay in which a myeloma protein is used for detection of salmonellae.

An enzyme immunoassay (EIA) in which an immunoglobulin A monoclonal antibody from a myeloma (MOPC 467) is used was developed to detect the presence of Salmonella organisms. This myeloma protein binds to a flagellar determinant of the organisms but is not directed toward the H antigens. Of 100 strains tested, 94% were detectable with this antibody. The EIA, used with MOPC 467, is quick, sensitive, and specific, showing virtually no cross-reactivity to other enteric organisms. Initial screening of antibody reactivity was performed by Ouchterlony gel diffusion with the supernatants of heat-treated Salmonella cultures. After this, an EIA was performed on the heat extracts with the myeloma protein, which had been directly coupled to alkaline phosphatase. A positive reaction was indicated by the production of a yellow color after the addition of a substrate (p-nitrophenylphosphate), and this was quantitated by determining the absorbance at 405 nm. The EIA proved to be slightly more sensitive than the Ouchterlony analysis. The sensitivity of the EIA is such that as few as 10(6) Salmonella organisms per ml were detected. This concentration was easily obtained after a 24-h preenrichment incubation of the sample. Mixtures of Salmonella strains with a 10 x concentration of Escherichia coli did not prevent detection of the Salmonella strains. This EIA can be successfully used to detect contamination of foods, as it was used to detect the intentional contamination of infant formula in these studies. Indications are that the EIA is sensitive enough to detect Salmonella strains in M broth subcultures taken directly from a preenrichment culture. Testing of samples could thus be completed 36 h after culture initiation, rather than after 96 h, the time currently needed.     

Rapid detection of Escherichia coli and enterococci in recreational water using an immunomagnetic separation/adenosine triphosphate technique

Aims:  The aim of this study was to examine a rapid method for detecting Escherichia coli and enterococci in recreational water.
Methods and Results:  Water samples were assayed for E. coli and enterococci by traditional and immunomagnetic separation/adenosine triphosphate (IMS/ATP) methods. Three sample treatments were evaluated for the IMS/ATP method: double filtration, single filtration, and direct analysis. Pearson's correlation analysis showed strong, significant, linear relations between IMS/ATP and traditional methods for all sample treatments; strongest linear correlations were with the direct analysis ( r  = 0·62 and 0·77 for E. coli and enterococci, respectively). Additionally, simple linear regression was used to estimate bacteria concentrations as a function of IMS/ATP results. The correct classification of water-quality criteria was 67% for E. coli and 80% for enterococci.
Conclusions:  The IMS/ATP method is a viable alternative to traditional methods for faecal-indicator bacteria.
Significance and Impact of the Study:  The IMS/ATP method addresses critical public health needs for the rapid detection of faecal-indicator contamination and has potential for satisfying US legislative mandates requiring methods to detect bathing water contamination in 2 h or less. Moreover, IMS/ATP equipment is considerably less costly and more portable than that for molecular methods, making the method suitable for field applications.     

基因芯片技术检测3种食源性致病微生物方法的建立

建立一种运用多重PCR和基因芯片技术检测和鉴定志贺氏菌、沙门氏菌、大肠杆菌O157的方法, 为3种食源性致病菌的快速检测和鉴定提供了准确、快速、灵敏的方法。分别选取编码志贺氏菌侵袭性质粒抗原H基因(ipaH)、沙门氏菌肠毒素(stn)基因和致泻性大肠杆菌O157志贺样毒素(slt)基因设计引物和探针, 进行三重PCR扩增, 产物与含特异性探针的芯片杂交。对7种细菌共26株菌进行芯片检测, 仅3种菌得到阳性扩增结果, 证明此方法具有很高的特异性。3种致病菌基因组DNA和细菌纯培养物的检测灵敏度约为8 pg。对模拟食品样品进行直接检测, 结果与常规细菌学培养结果一致, 检测限为50 CFU/mL。结果表明：所建立的基因芯片检测方法特异性好, 灵敏度高, 为食源性致病菌的检测提供了理想手段, 有良好的应用前景。     

水蛭常见病原菌的分离与鉴定

从表现软体、水肿、出血和硬结等特征的患病宽体金线蛭(Whitmania pigra)中分离到13株细菌分离物。通过培养特征、形态学观察及生化试验,确定了所分离的细菌中有6株为大肠杆菌(占46．15％),4株为变形杆菌(占30．77％),3株为沙门氏菌(占23．08％)。动物回归试验表明,接种后10d内大肠杆菌、沙门氏菌和变形杆菌对水蛭的致死率分别为100％(4／4)、100％(4／4)和25％(1／4);药敏试验结果表明,这三种细菌普通对复合磺胺、氯霉素、丁胺卡那霉素、头孢哌酮、丙氟哌酸、氟哌酸敏感。研究结果表明,本病是由多个病因导致的一种临床综合症。因此,在进行药物治疗时,应选用多种药物联合以达到最佳的治疗效果。     

Development of Radiation Resistance in Salmonella Cultures

Cultures of Salmonella subjected to repeated cycles of gamma-irradiation and subculture developed radiation resistance sooner at a low cycling dose ( approximately 1% survival) compared to a high cycling dose ( approximately 0.001% survival). Radioresistant cells in a population of radiosensitive cells of Salmonella newport or S. typhimurium could be selected by the double-irradiation plate method. The frequency of radioresistant cells in a population of S. newport was found to be about 1 per 8.9 million. Radio-resistant cells obtained by cyclic irradiation and subculture were larger (plumper) than the parent strain and showed a marked pleomorphism.     

Are growth rates of Escherichia coli in batch cultures limited by respiration?

Batch cultures of Escherichia coli were grown in minimal media supplemented with various carbon sources which supported growth at specific growth rates from 0.2 to 1.3/h. The respiration rates of the cultures were measured continuously. With few exceptions, the specific rate of oxygen consumption was about 20 mmol of O2/h per g (dry weight), suggesting that the respiratory capacity was limited at this value. The adenosine triphosphate (ATP) required for the production of cell material from the different carbon sources was calculated on the basis of known ATP requirements in the biochemical pathways and routes of macromolecular synthesis. The calculated ATP requirements, together with the measured growth rates and growth yields on the different carbon sources, were used to calculate the rate of ATP synthesis by oxidative phosphorylation. This rate was closely related to the respiration rate. We suggest that aerobic growth of E. coli in batch cultures is limited by the rate of respiration and the concomitant rate of ATP generation through oxidative phosphorylation.     

Involvement of apolipophorin III in antibacterial defense of Galleria mellonella larvae

Apolipophorin III (apoLp-III) is an abundant hemolymph protein involved in lipid transport and immune response in insects. We investigated involvement of apoLp-III in the antibacterial response in Galleria mellonella larvae. Immune challenge with Gram-negative (Escherichia coli, Klebsiella pneumoniae) and Gram-positive (Micrococcus luteus) bacteria led to an increase in the level of apoLp-III in G. mellonella hemolymph, 0.5-2h and 8h after treatment, respectively. ApoLp-III purified from larval hemolymph as well as that present in hemolymph extracts adsorbed on the surface of different bacteria. The adsorption capacity of apoLp-III on bacterial cells prompted us to investigate the effect of this phenomenon on bacterial growth. Our results demonstrate antibacterial activity of apoLp-III against selected Gram-positive and Gram-negative bacteria in vitro. Among bacteria tested, Salmonella typhimurium and K. pneumoniae were the most sensitive to apoLp-III. LIVE/DEAD staining of bacteria incubated with purified apoLp-III revealed their growth inhibition; however, neither morphological changes in the cell shape nor formation of cell aggregates was noticed. The results suggest that apoLp-III is a multifunctional protein in G. mellonella hemolymph.     

Operon Coordination in Different Bacterial Hosts

Coordination of gene expression in the lac operon was compared in Escherichia coli and Salmonella typhimurium as an approach to detecting possible differences in protein synthesis or membrane structure between organisms. Either a wild-type F' lac pro(AB) episome or the same episome with a polar mutation in one of the lac genes was introduced into pro(-) derivatives of the two strains of bacteria. Activity assays showed that the beta-galactosidase levels were only slightly lower in the S. typhimurium cells than in E. coli cells, whereas the transacetylase levels were significantly higher in S. typhimurium for all of the lac markers tested. Galactoside transport activities were always comparable in the two strains of bacteria; this latter result indicates that the cell envelopes of E. coli and S. typhimurium do not differ sufficiently to affect the membrane-associated lac transport system. It was found, however, that the specific transport activity is very sensitive to culture age in both bacteria, and decreases rapidly in cultures past the mid-exponential phase of growth.