Pathogenicity of live bacteria and extracellular products of motile Aeromonas isolated from eels

The pathogenic activities in vitro and in vivo of live bacteria and extracellular products (ECP) of 24 motile Aeromonas strains were investigated. Most Aer. hydrophila and Aer. jandaei isolates were pathogenic for eels (LD₅₀ 10^5·4-10^7·6 cfu fish^-1) but no Aer. sobria , Aer. caviae and Aer. allosaccharophila caused mortality in eels at doses of > 10^8·4 cfu fish^-1. Of these Aeromonas strains, Aer. hydrophila and Aer. jandaei in particular produced elastases and haemolysins against fish erythrocytes. ECP from Aer. hydrophila and Aer. jandaei caused degenerative changes in fish cell lines and were strongly toxic for eels (LD⁵⁰ 1·0–3·2 μg (g fish)^-1) reproducing the symptoms associated with natural disease. ECP from non-pathogenic species were inactive on fish cell lines as well as being poorly lethal for eels (LD⁵⁰ > 9·2 μg (g fish)^-1). All these biological activities of Aeromonas ECP were lost after heat treatment. These findings indicate differences between pathogenic and non-pathogenic Aeromonas species with respect to the expression of virulence factors, and show that elastases, haemolysins and exotoxins play a leading role in the pathogenicity of motile Aeromonas for eels.     

Susceptibility to chlorine of Aeromonas hydrophila strains

The susceptibility of five Aeromonas hydrophila strains and one Escherichia coli strain to chlorine was studied under carefully controlled laboratory conditions. Of the Aer. hydrophila strains, two were from untreated water, two from tap water (immediately downstream of a water treatment plant) and one from the DSM collection. The study included disinfectant concentration (0·1, 0·2 and 0·5 mg l⁻¹), pH (6, 7 and 8) and temperature (4, 21 and 32 °C) as controlled variables. The results indicated that the untreated water strains, the DSM strain and the E. coli strain were inactivated within 1 min of chlorine treatment. The strains from chlorinated water (TW11 and TW27) showed a different susceptibility to chlorine disinfection, the rate of inactivation being greater at pH 6 than at pH 8 for both strains. Under the standard conditions of temperature 21 °C, pH 7 and chlorine concentration 0·2 mg l⁻¹, an increase or decrease of approximately 1 log unit in the number of bacteria did not affect the kill rate of the strains TW11 and TW27.     

Serological and conductimetric assays for the detection of Pseudomonas syringae pathovar pisi in pea seeds

Test protocols for detecting Pseudomonas syringae pv. pisi , the causal agent of bacterial blight, in pea seeds are generally based on dilution-plating assays. These assays are usually very specific and reliable, but are time-consuming and laborious. Tests suited for large scale screening of seed lots are therefore needed. Conductimetric assays, immunofluorescence microscopy (IF) and an enzyme-linked immunosorbent assay (ELISA) for detecting Ps. syr. pv. pisi in pea seed extracts were compared with dilution-plating by two extraction methods, viz. 6 h soaking of seeds and 2 h soaking of flour of ground pea seeds in water. In general, the detection of Ps. syr. pv. pisi with conductimetric, IF and dilution-plating assays in the suspension water of the ground and 2 h-soaked pea samples was less sensitive than detection in suspension water of the 6 h-soaked pea seeds. The detection threshold of these assays varied per seed lot between 0 and 4.08 log cfu ml^-1 for the 6 h soaking procedure. The detection threshold of ELISA varied for both extraction methods generally between 4.08 and 6.08 log cfu ml^-1. Detection times recorded in conductimetric assays correlated well (— 0.89 < r < —0.98) with the log colony-forming units of Ps. syr. pv. pisi added to seed extracts at 27 as well as 17°. However, confirmation of results by isolation on semi-selective media after conductimetry was more successful at 17° than at 27°, because of the relatively lower activity of saprophytic Pseudomonas spp. at this temperature.     

Virulence markers in Aeromonas hydrophila and Aeromonas veronii biovar sobria isolates from freshwater fish and from a diarrhoea case

AIMS: To evaluate the public health significance of representative strains of two Aeromonas spp., mainly from freshwater fish, on the basis of production of virulence-associated factors and presence of the haemolytic genes aerA and hlyA. METHODS AND RESULTS: Eleven strains of Aer. hydrophila, three strains of Aer. veronii biovar sobria (all from freshwater fish) and one strain of Aer. hydrophila from human diarrhoea were tested for potential virulence traits and for the presence of the haemolytic genes aerA and hlyA. Ten Aer. hydrophila isolates were aerA(+)hlyA(+) and two aerA(+)hlyA(-). Aeromonas veronii biovar sobria isolates were aerA(-)hlyA(-). Strains from the three genotypes showed enterotoxic activity in the suckling mouse assay. At 28 degrees C, four Aer. hydrophila fish strains could be considered as potentially virulent (possessing at least two of these characteristics: haemolytic, cytotoxic and enterotoxic). One Aer. veronii biovar sobria strain and the clinical isolate were cytotoxic on Vero cells. When grown at 4 degrees C, these six isolates fulfilled virulence criterion, but at 37 degrees C, only one fish strain, an Aer. hydrophila, did. CONCLUSIONS: The potential health risk derived from the presence of Aer. hydrophila and Aer. veronii biovar sobria in ice-stored freshwater fish should not be underestimated. SIGNIFICANCE AND IMPACT OF THE STUDY: Expression of virulence factors is affected by temperature incubation and not always related to the presence of haemolytic genes.     

Minimum inhibitory concentrations of intestinal Escherichia coli from broiler chickens after oral administration of apramycin

This study describes the influence of apramycin on minimum inhibitory concentrations (MIC) of intestinal Escherichia coli in young broiler chickens, after oral administration of the antibiotic at a dosage equivalent to a prophylactic course of treatment for 10 d. The bacteria were isolated from cloacal swabs and caecal contents. MICs were determined by agar dilution procedures. MIC of apramycin for the investigated strains ranged from 1 μg ml^-1 to 16 μg ml^-1. Strains obtained from undosed birds mainly had MIC values of 1 μg ml^-1. MIC values of 8 μg ml^-1 or more were recorded only among isolates obtained from chickens which had received apramycin. Administration of apramycin resulted in a slight but statistically significant increase in the average MIC. Statistically higher average MICs were recorded among isolates from cloacal swabs 10 d after withdrawal until the end of the experiment. For strains from caecal contents, this was demonstrated only on one sampling occasion, 15 d after withdrawal.     

Survival of Aeromonas hydrophila, Aeromonas caviae and Aeromonas sobria in soil

The survival of mesophilic Aeromonas spp. in soil in the presence or absence of indigenous microflora was evaluated in a laboratory study. Two cytotoxic ( Aer. hydrophila and Aer. caviae ) and one invasive ( Aer. sobria ) clinical isolate strains were selected for this study. After contamination of sterile or unsterilized soil with the three strains of Aeromonas , the number of living cells was determined over at least 5 months. For all strains the survival curves were characterized by an initial re-growth followed by a slow inactivation of bacteria, with significant differences due to the presence of indigenous microflora. The times necessary to achieve a 95% reduction of the initial population were > 140, 113 and 62 d in sterilized soil respectively for Aer. caviae, Aer. hydrophila and Aer. sobria , while the corresponding times in unsterilized soil were 42, 38 and 11 d. All strains preserved the virulence factors for the entire period of the study. These results suggest that the soil may be an important reservoir for Aeromonas spp. and, thus, may play an important role in the epidemiology of Aeromonas -associated human infections.     

Effect of Iysozyme concentration, heating at 90°C, and then incubation at chilled temperatures on growth from spores of non-proteolytic Clostridium botulinum

The heat treatment necessary to inactivate spores of non-proteolytic Clostridium botulinum in refrigerated, processed foods may be influenced by the occurrence of lysozyme in these foods. Spores of six strains of non-proteolytic Cl. botulinum were inoculated into tubes of an anaerobic meat medium, to give 10⁶ spores per tube. Hen egg white lysozyme (0–50 μg ml^-1) was added, and the tubes were given a heat treatment equivalent to 19·8 min at 90°C, cooled, and incubated at 8°, 12°, 16° and 25°C for up to 93 d. In the absence of added lysozyme, neither growth nor toxin formation were observed. A 6–D inactivation was therefore achieved. In tubes to which lysozyme (5–50 μg ml^-1) had been added prior to heating, growth and toxin formation were observed. With lysozyme added at 50 μg ml^-1, growth was first observed after 68 d at 8°C, 31 d at 12°C, 24 d at 16°C, and 9 d at 25°C. Thus, in these circumstances, a heat treatment equivalent to 19·8 min at 90°C was not sufficient, on its own, to give a 6–D inactivation. A combination of the heat treatment, maintenance at less than 12°C, and a shelf-life not more than 4 weeks reduced the risk of growth of non-proteolytic Cl. botulinum by a factor of 10⁶.     

Colonization of Vibrio pelagius and Aeromonas caviae in early developing turbot (Scophthalmus maximus L.) larvae

Polyclonal antisera made in rabbits against whole washed cells of Vibrio pelagius and Aeromonas caviae were used for detection of these bacterial species in the rearing water and gastrointestinal tract of healthy turbot ( Scophthalmus maximus ) larvae exposed to V. pelagius and/or Aer. caviae . The results demonstrated that this method is suitable for detection of V. pelagius and Aer. caviae in water samples and larvae at population levels higher than 10³ ml⁻¹ and 10³ larva⁻¹. Populations of aerobic heterotrophic bacteria present in the gastrointestinal tract of turbot larvae, estimated using the dilution plate technique, increased from approximately 4 × 10² bacteria larva⁻¹ on day 3 post-hatching to approximately 10⁵ bacteria fish⁻¹ 16 days post-hatching. Sixteen days after hatching, Vibrio spp. accounted for approximately 3 × 10⁴ cfu larva⁻¹ exposed to V. pelagius on days 2, 5 and 8 post-hatching. However, only 10³ of the Vibrio spp. belonged to V. pelagius . When larvae were exposed to Aer. caviae on day 2 post-hatching, the gut microbiota of 5-day old larvae was mainly colonized by Aeromonas spp. (10⁴ larva⁻¹), of which 9 × 10³ belonged to Aer. caviae . Later in the experiment, at the time when high mortality occurred, 9 × 10⁵ Aer. caviae were detected. Introduction of V. pelagius to the rearing water seemed to improve larval survival compared with fish exposed to Aer. caviae and with the control group. It was therefore concluded that it is beneficial with regard to larval survival to introduce bacteria ( V. pelagius ) to the rearing water.     

Interaction between the effects of bacteria and dry storage on the opening and water relations of cut rose flowers

W.G. VAN DOORN AND K. D'HONT. 1994. Flowering stems of four rose cultivars (Sonia, Madelon, Jacaranda and Frisco) were placed in aqueous suspensions of bacteria at 10⁴ and 10⁸ colony-forming units (cfu) ml^-1 for 24 h at 5C, then stored dry or held in water for 24 h at 8C and subsequently placed in vase-water at 20C. The effects of these treatments on vase-water uptake were similar to the effects on flower opening. In Sonia and Madelon roses flower opening was negatively affected both by 10⁸ cfu ml^-1 of bacteria and by dry storage. No effect was found at 10⁴ cfu ml^-1, but this concentration had a detrimental effect on flower opening when combined with dry storage. Although flower development in Jacaranda roses was not affected by the bacteria treatments it was inhibited by dry storage. This inhibition was progressively greater when the stems had previously been pulse-treated with a larger number of bacteria. Flower opening in Frisco roses was not affected by even the highest concentration of bacteria, nor by the period of dry storage. It is concluded that placing flowers in water containing bacteria (up to 10⁸ cfu ml^-1) may not always have a negative effect on flower development in cut rose flowers but, together with the effects of dry storage, the presence of even a low number of exogenous bacteria (10⁴ cfu ml^-1) inhibits the development in several cultivars. Such bacterial counts are nearly always found in samples of water used for standing roses during distribution to the consumers.     

Effect of chill and freezing temperatures on survival of Vibrio parahaemolyticus inoculated in homogenates of oyster meat

Survival of Vibrio parahaemolyticus was determined in oyster meat homogenates at various temperatures. (4°C, 0°C, -18°C and -24°C) and bacterial levels (10², 10⁴, 10⁵ and 10⁷ ml^-1). In all cases, the numbers of V. parahaemolyticus were a logarithmic function of log time. This study indicates that high numbers of V. parahaemolyticus can be inactivated at low temperatures. The time of total inactivation depends on the initial number of micro-organisms and incubation temperature. It is possible to use this information to determine the storage time necessary to reduce V. parahaemolyticus hazards in fish.     

Hypocholesterolemic effect of Eubacterium coprostanoligenes ATCC 51222 in rabbits

Recently, a unique bacterium, Eubacterium coprostanoligenes ATCC 51222, that reduces cholesterol to coprostanol was isolated. Because coprostanol is absorbed poorly, it was hypothesized that oral administration of Eu. coprostanoligenes might decrease cholesterol concentration in blood because the micro-organisms will decrease the absorption of endogenous and dietary cholesterol by conversion to coprostanol. To test the hypothesis, three adult New Zealand White rabbits received 4 ml of Eu. coprostanoligenes suspension ( ca 2 times 10⁷ cells ml^-1) daily per os for 10 d; three other adult New Zealand White rabbits received the same dosage of boiled bacterial suspension. Plasma cholesterol concentration of experimental rabbits (183.3 ± 11.0 mg dl^-1, mean ± S.E.) was significantly lower ( P < 0.001) than that of controls (248.8 ± 12.3 mg dl^-1, mean ± S.E.). The coprostanol-to-cholesterol ratios in contents of digestive tracts of experimental rabbits were greater than those of controls. The data indicate that oral administration of Eu. coprostanoligenes caused a significant hypocholesterolemic effect in rabbits and that this effect can be explained by the conversion of cholesterol to coprostanol in the intestine.     

Antibacterial effect of protamine assayed by impedimetry

Impedimetric measurements were used to assay the antibacterial effect of protamine. A good linear correlation between the impedance detection time and the initial cell counts was obtained ( r = 0.99, n = 2). As basic peptides may cause clumping of cells, this correlation curve was used when estimating the cell number after protamine treatment, rather than colony counts.
Protamine from salmon killed growing Gram-positive bacteria and significantly inhibited growth of Gram-negative bacteria in Tryptone Soy Broth (TSB) at 25°C. In general Gram-positive bacteria were more sensitive to protamine than Gram-negative bacteria; the minimum inhibitory concentrations (MIC) determined for Gram-positive strains varied from 20 to 1000 μ ml^-1 and for Gram-negative strains from 500 μ ml^-1 to more than 4000 μ ml^-1.
The effect of protamine on non-growing Listeria monocytogenes Scott A suspended in buffer was not lethal as was the effect on growing cells; however, protamine (50–500 μg ml^-1) killed the Gram-negative fish spoilage bacteria Shewanella putrefaciens when the live cells were suspended in buffer.     

Antibacterial activity of anacardic acid and totarol, alone and in combination with methicillin, against methicillinresistant Staphylococcus aureus

The inhibitory and bactericidal activities of anacardic acid and totarol, alone and in combination with methicillin, were investigated against methicillin-resistant Staphylococcus aureus (MRSA). The growth of two MRSA strains was inhibited by 6·25 μg ml^-1 of anacardic acid and 0·78 μg ml^-1 of totarol. The time-kill curve study showed that these two compounds were bactericidal against MRSA. Anacardic acid killed MRSA cells more rapidly than totarol, and no viable cells were detected after being exposed to 6·25 μg ml^-1 of anacardic acid for 6 h. Anacardic acid showed bactericidal activity against MRSA at any stage of growth, and also even when cell division was inhibited by chloramphenicol. In the combination studies, the minimal inhibitory concentration (MIC) of methicillin was lowered from 800 to 1·56 μg ml^-1 for MRSA ATCC 33591, and from 800 to 6·25 μg ml^-1 for MRSA ATCC 33592, by combining with 1/2 X MIC of anacardic acid. The time-kill curves demonstrated synergistic bactericidal activities for these combinations.     

The enzymatic reaction for the production of panose and isomaltose by glucosyltransferase from Aureobasidium

Glucosyltransferase from Aureobasidium produced 212 mg ml^-1 of glucosyl-oligosaccharides (panose: Glcα1→6Glcα1→4Glc 189 mg ml^-1 and isomaltose: Glcα1→6Glc 23 mg ml^-1) from maltose: Glcα1→4Glc at a high concentration (500 mg ml^-1) and the efficiency of production was 42-4%. The enzymatic reaction from maltose to panose is reversible but that from panose to isomaltose is not.     

The effect of essential oils of basil on the growth of Aeromonas hydrophila and Pseudomonas fluorescens

Basil essential oils, including basil sweet linalool (BSL) and basil methyl chavicol (BMC), were screened for antimicrobial activity against a range of Gram-positive and Gram-negative bacteria, yeasts and moulds using an agar well diffusion method. Both essential oils showed antimicrobial activity against most of the micro-organisms examined except Clostridium sporogenes , Flavimonas oryzihabitans , and three species of Pseudomonas . The minimum inhibitory concentration (MIC) of BMC against Aeromonas hydrophila and Pseudomonas fluorescens in TSYE broth (as determined using an indirect impedance method) was 0·125 and 2% (v/v), respectively; the former was not greatly affected by the increase of challenge inoculum from 10³ to 10⁶ cfu ml⁻¹. Results with resting cells demonstrated that BMC was bactericidal to both Aer. hydrophila and Ps. fluorescens . The growth of Aer. hydrophila in filter-sterilized lettuce extract was completely inhibited by 0·1% (v/v) BMC whereas that of Ps. fluorescens was not significantly affected by 1% (v/v) BMC. In addition, the effectiveness of washing fresh lettuce with 0·1 or 1% (v/v) BMC on survival of natural microbial flora was comparable with that effected by 125 ppm chlorine.     

黄沙鳖白底板病病原菌的分离鉴定及6种毒力基因检测

用常规方法从患典型白底板病黄沙鳖的心脏、肝脏等处进行细菌的接种分离, 通过人工感染确定分离菌株的致病性, API 20NE、16S rRNA基因序列分析进行病原菌鉴定和确定其系统发育地位, K-B纸片扩散法进行药敏试验, PCR检测病原菌的6种毒力基因。试验结果, 共分离到13株病原菌, 其中嗜水气单胞菌9株, 温和气单胞菌4株。在9株嗜水气单胞菌中, 有5株与Aeromonas hydrophila ATCC 7966菌株亲源关系最近, 4株与Aeromonas hydrophila北京株QDC01的亲源关系最近; 而4株温和气单胞菌与Aeromonas sobria ATCC 43979的亲源关系最近。药敏试验结果, 仅头孢哌酮对13株病原菌都高度敏感, 来源于不同养殖区域的病原菌药敏结果相差较大。6种毒力基因的阳性率, Aer、Act和ahp均为100%, hly和Alt为92.31%, ahal为76.92%; 毒力基因型共有4种, 嗜水气单胞菌主要为hly+Aer+Alt+Act+ahal+ahp+基因型, 而温和气单胞菌主要为 hly+Aer+Alt－Act+ahal+ahp+基因型, 同时携带hly基因的菌株其致病力更强。       

The effect of age on the thermal resistance of arthroconidia from Chrysosporium inops

J. L. KINDERLERER. 1996. Food-borne members of the genus Chrysosporium have been isolated relatively infrequently. The heat resistance of arthroconidia of the xerophilic fungus, Chrysosporium inops Carmichael, was determined in 0.1% peptone at 66C. The survival curve was sigmoid in shape. The initial lag period was due to the chains of arthroconidia. Thermal inactivation occurred when one viable conidium was left per chain. The presence of chains of arthroconidia was confirmed with the cryo scanning electron microscope. The decimal reduction times were obtained from the regression line of the linear death phase for the heat-sensitive spores. The decimal reduction time (D₆₆) increased with increasing spore age. It was 1.67 min for 3-week-old spores, 1.95 min for 4-week-old spores and 5.49 min for 6-week-old spores. The older spores could recover from thermal death if they were given sufficient time. There was a significant increase in D₆₆ value for 6-week-old spores from 3.97 min to 5.49 when the counts were obtained after 14 d incubation (compared to counts after incubation for 10 d). This effect was not seen for the 3- and 4-week-old spores. There was a small population of heat-resistant spores. The initial population of arthroconidia was greater than log 7 cfu ml^-1. After heating for 1 h at 66C approximately log 2.2 cfu ml^-1 survived. These survivors represented approximately 0.001% of the original population.     

Detection of low levels of specific Salmonella species by fluorescent antibodies and flow cytometry

The use of fluorescently-labelled monoclonal antibodies, with detection by multi-parameter flow cytometry, was investigated for the rapid detection of salmonellas in pure cultures. Accurate detection of specific Salmonella serotypes was demonstrated down to levels of below 10⁴ cells ml^-1 (within 30 min) and 1 cell ml^-1 (after 6 h non-selective pre-enrichment). This level of sensitivity was attainedeven in the presence of high levels of other bacterial species that would otherwise have interfered with the results. With combinations of different antibodies, each with a unique fluorescent label, simultaneous analysis for two species was possible.     

Electrotransfection of Propionibacterium freudenreichii TL 110

The first highly efficient protocol is described for the electrotransfection of Propionibacterium freudenreichii with DNA phage. The transfection efficiency is 7 times 10⁵ transfectants per μg of DNA under optimal conditions. Optimized parameters included the field strength (12.5 kV, 200 Ohms, 25 μF), phage DNA concentration (1 μg ml^-1) and cell density (1.5 times 10¹⁰ cells ml^-1). Growth in the presence of glycine and harvesting of cells during the early exponential growth phase increased the transfection efficiency. This electrotransfection protocol is of importance for the genetic improvement of dairy propionibacteria.     

Detection of metallo-β-lactamases-encoding genes in environmental isolates of Aeromonas hydrophila and Aeromonas jandaei

Aims:  To determine the prevalence and expression of metallo-β-lactamases (MBL)-encoding genes in Aeromonas species recovered from natural water reservoirs in southeastern Brazil.
Methods and Results:  Eighty - seven Aeromonas isolates belonging to Aeromonas hydrophila ( n  =   41) and Aer. jandaei ( n  =   46) species were tested for MBL production by the combined disk test using imipenem and meropenem disks as substrates and EDTA or thioglycolic acid as inhibitors. The presence of MBL genes was investigated by PCR and sequencing using new consensus primer pairs designed in this study. The cphA gene was found in 97·6% and 100% of Aer. hydrophila and Aer. jandaei isolates, respectively, whereas the acquired MBL genes bla _IMP , bla _VIM and bla _SPM-1 were not detected. On the other hand, production of MBL activity was detectable in 87·8% and 10·9% of the cphA -positive Aer. hydrophila and Aer. jandaei isolates respectively.
Conclusions:  Our results indicate that cphA seems to be intrinsic in the environmental isolates of Aer. hydrophila and Aer. jandaei in southeastern Brazil, although, based on the combined disk test, not all of them are apparently able to express the enzymatic activity.
Significance and Impact of the Study:  These data confirm the presence of MBL-producing Aeromonas species in natural water reservoirs. Risk of waterborne diseases owing to domestic and industrial uses of freshwater should be re-examined from the increase of bacterial resistance point of view.