Suraj B. Baloda

Bacterial antibiotic resistance levels in Danish farmland as a result of treatment with pig manure slurry

Resistance to tetracycline, macrolides and streptomycin was measured for a period of 8 months in soil bacteria obtained from farmland treated with pig manure slurry. This was done by spread plating bacteria on selective media (Luria Bertani (LB) medium supplemented with antibiotics). To account for seasonal variations in numbers of soil bacteria, ratios of resistant bacteria divided by total count on nonselective plates were calculated. Soil samples were collected from four different farms and from a control soil on a fifth farm. The control soil was not amended with animal manure. The occurrence of tetracycline-resistant bacteria was elevated after spread of pig manure slurry but declined throughout the sampling period to a level corresponding to the control soil. Higher load of pig manure slurry yielded higher occurrence of tetracycline resistance after spreading; however, the tetracycline resistance declined to normal occurrence defined by the tetracycline resistance occurrence in the control soil. Concentrations of tetracycline in soil and in pig manure slurry were measured using HPLC. No tetracycline exceeding the detection limit was found in soil samples. Manure slurry concentrations of tetracycline for three of the farms were 42, 81 and 698 microg/l, respectively. For streptomycin and macrolides, only minor variations in resistance levels were detected. Results obtained in this study thus indicate that tetracycline resistance levels in soil are temporarily influenced by the addition of pig manure slurry. The results indicate also that increased amount of pig manure slurry amendment may result in increased levels of tetracycline resistance in the soil.

Prevalence and diversity of Aeromonas and Vibrio spp. in coastal waters of Southern Italy.

A survey was undertaken to examine sea water and sediment for the presence of Vibrio and Aeromonas spp. along approximately 900 km of coast in Southern Italy during early and late summer. A quantitative analysis was also done to evaluate the water fecal contamination at the stations examined. The results indicate that all the investigated areas were submitted to a wide spatial fluctuation of fecal contamination and that Vibrio and Aeromonas spp. were present in both high and low fecal-contaminated stations. Sixty two percent of the investigated samples were positive for Aeromonas spp., while 42% of samples were positive for Vibrio spp. It was interesting to note that 38% of the positive stations for both Aeromonas and Vibrio spp. showed a fecal coliform contamination of water at < 10(2) cells 100 ml(-1). Thus, these findings support the hypothesis that the bacterial indicators (such as fecal coliforms) do not always satisfactorily reflect the hygienic quality of water. The presence of Vibrionaceae on copepods was also investigated. Copepods were sampled at a station located inside the harbour of the city of Naples and were found contaminated by V. cholerae non-O1, V. alginolyticus, V. fluvialis and A. caviae. Furthermore, the antibiotic resistance patterns of isolated bacteria showed the presence of a number of resistant strains among the isolates. In order to discriminate the isolates on the basis of their biochemical profiles and/or antibiotic resistance patterns, cluster analysis was carried out which showed that no unique assay could fully discern these isolates. However, the best discrimination resulted from complete pattern profile based on both biochemical profiles and antibiotic resistance patterns.

Antimicrobial resistance among Pseudomonas spp. and the Bacillus cereus group isolated from Danish agricultural soil

From four Danish pig farms, bacteria of Pseudomonas spp. and the Bacillus cereus group were isolated from soil and susceptibility towards selected antimicrobials was tested. From each farm, soil samples representing soil just before and after spread of animal waste and undisturbed agricultural soil, when possible, were collected. Soil from a well-characterized Danish farm soil (Højbakkegaard) was collected for comparison. The Pseudomonas spp. and B. cereus were chosen as representative for Gram-negative and Gram-positive indigenous soil bacteria to test the effect of spread of animal waste on selection of resistance among soil bacteria. No variations in resistance levels were observed between farms; but when the four differently treated soils were compared, resistance was seen for carbadox, chloramphenicol, nalidixan (nalidixic acid), nitrofurantoin, streptomycin and tetracycline for Pseudomonas spp., and for bacitracin, erythromycin, penicillin and streptomycin for the B. cereus group. Variations in resistance levels were observed when soil before and after spread of animal waste was compared, indicating an effect from spread of animal waste.

Isolation, and Virulence Profiles, of Aeromonas hydrophila Implicated in an outbreak of Food Poisoning in Sweden

A case of food poisoning outbreak involving Aeromonas hydrophila is reported in this study. A group of 27 people consumed a typical Swedish food “landgång” which is a type of smörgåsbord containing shrimps with mayonnaise, liver paté, ham, sausage, and legume salad which was purchased from a food store. Twenty‐two of the 27 persons became ill within 20‐34 hr of consumption of the food and reported the symptoms ranging from severe acute diarrhea, abdominal pain, headache, fever and vomiting. One person also fainted. The symptoms lasted for a couple of days. Of the remaining 5 healthy persons who consumed the left‐over food the next day, 2 became ill with similar symptoms. The bacteriological examination of left‐over food samples resulted in the isolation of A. hydrophila from shrimps with mayonnaise, smoked sausage, liver paté and boiled ham. The total number of A. hydrophila in these foods were log 106 to log >107 organisms per gram of food sample. A. hydrophila was however, not isolated from legume/mayonnaise salad samples. All the food samples tested showed low numbers of other expected food contaminating organisms such as coliforms at 37 C and 44 C, fecal streptococci, Staphylococcus aureus, fungi and yeast etc., while Bacillus cereus, Clostridium perfringens and Salmonella spp. were not detected in the food samples. Investigations of the virulence profiles of the A. hydrophila isolates showed their capacity to produce β‐hemolysin, cytotoxins, cytotonic toxins, enterotoxins, and adhesion to and invasion of human intestinal (Henle 407) cells in culture.

Ecological relationship between Aeromonas and Vibrio spp. and planktonic copepods in the coastal marine environment in Southern Italy

The colonisation of planktonic copepod integument by bacteria belonging to the family of Vibrionaceae is a well described phenomenon. In this study, besides reporting on the occurrence of Vibrionaceae and other enteropathogens, we further report on the bacterial attachment to the Estuarine copepod Acartia margalefi in a faecal polluted coastal lagoon near Naples, Southern Italy. In addition, we also performed a laboratory experiment to study the ability of 7 bacterial strains (Vibrio cholerae non-Ol, V. mimicus, V. parahaemolyticus, V. alginolyticus, Aeromonas hydrophila, Escherichia coli and Pseudomonas sp.) to colonise the copepod integument. For this laboratory study, 4 different species of copepods, namely Temora stylifera, A. clausi, Centropages typicus and Paracalanus parvus sampled from the Gulf of Naples (Southern Italy) were used. Scanning electron microscope (SEM) studies on the copepods sampled from the lagoon indicated that the bacterial attachment on the integument of Acartia margalefi was mainly on the ventro-lateral body region of the host and in the joints of the thoracic segments, as well as on the swimming and feeding appendages. This infestation, made by rod-like bacteria, was absent in winter but reached peak values of 70% frequency in June. The laboratory studies showed that while V. cholerae non-O1 and A. hydrophila attached on live and dead copepods, respectively, the V. parahaemolyticus, V. alginolyticus, V. mimicus, E. coli and Pseudomonas sp. failed to colonise either live or dead copepods. Thus, this study provides further valuable information about the ecological relationship between different microorganisms (pathogens) and copepods in the coastal marine environment in Southern Italy.

Cytotonic enterotoxins and cytotoxic factors produced by Salmonella enteritidis and Salmonella typhimurium

Strains of Salmonella enteritidis and Salmonella typhimurium isolated from human diarrheal cases produced heat-labile enterotoxin(s) and cytotoxic factor(s) which elongated, lysed or deformed Chinese hamster ovary cells in tissue culture. The toxin(s) caused fluid accumulation in ligated rabbit gut loops and produced increased skin permeability. Salmonella toxin produced by these strains does not cross-react immunologically with high titer Vibrio cholerae toxin antisera or heat-labile Escherichia coli enterotoxin antisera used in this study and does not bind to galactose--Sepharose gel. The activity of the toxin was not inhibited by GM1-ganglioside.

Prevalence and characterization of Aeromonas spp. isolated from foods in Uppsala, Sweden

Different food samples collected from randomly selected retail consumer shops in Uppsala (Central Sweden) were investigated for the presence of Aeromonas species. Forty-two percent (10/24) of the food samples contained organisms belonging to two Aeromonas spp. namely, Aeromonas hydrophila and Aeromonas sobria. The type of foods from which Aeromonas were isolated, were poultry, fish, beef and pork. None of the Aeromonas spp. were isolated from vegetables or raw milk samples. The Aeromonas isolates possessed the capacity to produce different potential virulence determinants such as haemolysin, cytotoxin, cytotonic toxin, enterotoxin and protease. Since A. hydrophila and A. sobria are considered to be potential human pathogens, it is concluded that in case foodborne infections are suspected or encountered, the role of Aeromonas should not be ruled out.

Isolation and virulence profiles of Aeromonas spp. from different municipal drinking water supplies in Sweden

Drinking water from three different municipal distribution systems in Sweden was investigated for the occurrence of Aeromonas spp. and identification of virulence factors of these organisms. Twenty-three of the 27 samples (85%) from the three distribution systems were positive for presumptive Aeromonas with a maximum of 860 cfu/100 ml drinking water. Of the 61 randomly selected presumptive Aeromonas isolates, 41 were identified as A. hydrophila and 20 as A. sobria . These Aeromonas isolates produced a variety of potential virulence factors such as haemolysin, cytotonic- and cytotoxic-toxin, enterotoxin and protease. These findings on the presence of Aeromonas in municipal drinking water supplies in Sweden show that the consumer can be exposed to high number of aeromonads. However, more epidemiological information needs to be gathered in this regard to elucidate their public health significance.

Detection of aerolysin gene in Aeromonas strains isolated from drinking water, fish and foods by the polymerase chain reaction

A polymerase chain reaction (PCR) technique was used to assay the presence of the aerolysin gene in a total of 89 Aeromonas hydrophila and A. sobria strains isolated from drinking water, fish and foods. These strains were also characterized for the production of virulence factors such as haemolysin, protease and cytotoxin. The primers used in the PCR targeted a 209-bp fragment of the aer gene coding for the beta-haemolysin and detected template DNA only in haemolytic A. hydrophila strains. The cell-free culture supernatants of these aerolysin-positive A. hydrophila strains were also cytotoxic to the HeLa and McCoy cells. The haemolytic A. sobria and non-haemolytic A. hydrophila were consistently negative in the PCR assay. Primer specificity was determined in the PCR by using a control haemolytic Escherichia coli, Streptococcus pyogenes and a restriction endonuclease assay. The PCR clearly identified the aerolysin-producing strains of A. hydrophila and may have application as a rapid species-specific virulence test.

Cell-Surface Properties of Enterotoxigenic and Cytotoxic Salmonella enteritidis and Salmonella typhimurium: Studies on Hemagglutination, Cell-Surface Hydrophobicity, Attachment to Human Intestinal Cells and Fibronectin-Binding

Thirteen Salmonella enteritidis and S. typhimurium strains with smooth or rough colony morphology were investigated for their surface properties based on hemagglutination (HA), hydrophobicity, and fibronectin‐binding profiles. The strains showed 5 different patterns of HA which was mannose‐sensitive. The rough strains possessed comparatively greater number of fimbriae than the corresponding smooth strains and also attached to human intestinal cells in greater numbers. The Salmonella strains used in this study interacted with fibronectin and its 29‐kDa N‐terminal fragment to varied extents. These properties may be helpful in broadening the prospective interaction capabilities of Salmonella organisms with the host surfaces.