Anita Forslund

Distribution and Content of Class 1 Integrons in Different Vibrio cholerae O-Serotype Strains Isolated in Thailand

ABSTRACT In this study, 176 clinical and environmental Vibrio cholerae strains of different O serotypes isolated in Thailand from 1982 to 1995 were selected and studied for the presence of class 1 integrons, a new group of genetic elements which carry antibiotic resistance genes. Using PCR and DNA sequencing, we found that 44 isolates contained class 1 integrons harboring the aadB,aadA2, blaP1, dfrA1, anddfrA15 gene cassettes, which encode resistance to gentamicin, kanamycin, and tobramycin; streptomycin and spectinomycin; β-lactams; and trimethoprim, respectively. Each cassette array contained only a single antibiotic resistance gene. Although resistance genes in class 1 integrons were found in strains from the same epidemic, as well as in unrelated non-O1, non-O139 strains isolated from children with diarrhea, they were found to encode only some of the antibiotic resistance expressed by the strains. Serotype O139 strains did not contain class 1 integrons. However, the appearance and disappearance of the O139 serotype in the coastal city Samutsakorn in 1992 and 1993 were associated with the emergence of a distinct V. cholerae O1 strain which contained the aadA2resistance gene cassette. A 150-kb self-transmissible plasmid found in three O1 strains isolated in 1982 contained the aadB gene cassette. Surprisingly, several strains harbored two integrons containing different cassettes. Thus, class 1 integrons containing various resistance gene cassettes are distributed among differentV. cholerae O serotypes of mainly clinical origin in Thailand.

Analysis of Vibrio vulnificus from market oysters and septicemia cases for virulence markers.

ABSTRACT Representative encapsulated strains of Vibrio vulnificus from market oysters and oyster-associated primary septicemia cases (25 isolates each) were tested in a blinded fashion for potential virulence markers that may distinguish strains from these two sources. These isolates were analyzed for plasmid content, for the presence of a 460-bp amplicon by randomly amplified polymorphic DNA PCR, and for virulence in subcutaneously (s.c.) inoculated, iron-dextran-treated mice. Similar percentages of market oyster and clinical isolates possessed detectable plasmids (24 and 36%, respectively), produced the 460-bp amplicon (45 and 50%, respectively), and were judged to be virulent in the mouse s.c. inoculation-iron-dextran model (88% for each). Therefore, it appears that nearly all V. vulnificus strains in oysters are virulent and that genetic tests for plasmids and specific PCR size amplicons cannot distinguish between fully virulent and less virulent strains or between clinical and environmental isolates. The inability of these methods to distinguish food and clinical V. vulnificus isolates demonstrates the need for alternative subtyping approaches and virulence assays.

Persistence and leaching potential of microorganisms and mineral N in animal manure applied to intact soil columns.

ABSTRACT Pathogens may reach agricultural soils through application of animal manure and thereby pose a risk of contaminating crops as well as surface and groundwater. Treatment and handling of manure for improved nutrient and odor management may also influence the amount and fate of manure-borne pathogens in the soil. A study was conducted to investigate the leaching potentials of a phage (Salmonella enterica serovar Typhimurium bacteriophage 28B) and two bacteria, Escherichia coli and Enterococcus species, in a liquid fraction of raw pig slurry obtained by solid-liquid separation of this slurry and in this liquid fraction after ozonation, when applied to intact soil columns by subsurface injection. We also compared leaching potentials of surface-applied and subsurface-injected raw slurry. The columns were exposed to irrigation events (3.5-h period at 10 mm h−1) after 1, 2, 3, and 4 weeks of incubation with collection of leachate. By the end of incubation, the distribution and survival of microorganisms in the soil of each treatment and in nonirrigated columns with injected raw slurry or liquid fraction were determined. E. coli in the leachates was quantified by both plate counts and quantitative PCR (qPCR) to assess the proportions of culturable and nonculturable (viable and nonviable) cells. Solid-liquid separation of slurry increased the redistribution in soil of contaminants in the liquid fraction compared to raw slurry, and the percent recovery of E. coli and Enterococcus species was higher for the liquid fraction than for raw slurry after the four leaching events. The liquid fraction also resulted in more leaching of all contaminants except Enterococcus species than did raw slurry. Ozonation reduced E. coli leaching only. Injection enhanced the leaching potential of the microorganisms investigated compared to surface application, probably because of a better survival with subsurface injection and a shorter leaching path.

Leaching of Cryptosporidium parvum Oocysts, Escherichia coli, and a Salmonella enterica Serovar Typhimurium Bacteriophage through Intact Soil Cores following Surface Application and Injection of Slurry

ABSTRACT Increasing amounts of livestock manure are being applied to agricultural soil, but it is unknown to what extent this may be associated with contamination of aquatic recipients and groundwater if microorganisms are transported through the soil under natural weather conditions. The objective of this study was therefore to evaluate how injection and surface application of pig slurry on intact sandy clay loam soil cores influenced the leaching of Salmonella enterica serovar Typhimurium bacteriophage 28B, Escherichia coli, and Cryptosporidium parvum oocysts. All three microbial tracers were detected in the leachate on day 1, and the highest relative concentration was detected on the fourth day (0.1 pore volume). Although the concentration of the phage 28B declined over time, the phage was still found in leachate at day 148. C. parvum oocysts and chloride had an additional rise in the relative concentration at a 0.5 pore volume, corresponding to the exchange of the total pore volume. The leaching of E. coli was delayed compared with that of the added microbial tracers, indicating a stronger attachment to slurry particles, but E. coli could be detected up to 3 months. Significantly enhanced leaching of phage 28B and oocysts by the injection method was seen, whereas leaching of the indigenous E. coli was not affected by the application method. Preferential flow was the primary transport vehicle, and the diameter of the fractures in the intact soil cores facilitated transport of all sizes of microbial tracers under natural weather conditions.

Survival of Salmonella spp. and fecal indicator bacteria in Vietnamese biogas digesters receiving pig slurry.

Abstract Small-scale biogas digesters are widely promoted worldwide as a sustainable technology to manage livestock manure. In Vietnam, pig slurry is commonly applied to biogas digesters for production of gas for electricity and cooking with the effluent being used to fertilize field crops, vegetables and fish ponds. Slurry may contain a variety of zoonotic pathogens, e.g. Salmonella spp., which are able to cause disease in humans either through direct contact with slurry or by fecal contamination of water and foods. The objective of this study was to evaluate the survival of Salmonella spp. and the fecal indicator bacteria, enterococci, E. coli, and spores of Clostridium perfringens in biogas digesters operated by small-scale Vietnamese pig farmers. The serovar and antimicrobial susceptibility of the Salmonella spp. isolated were also established. The study was conducted in 12 farms (6 farms with and 6 farms without toilet connected) located in Hanam province, Vietnam. Sampling of pig slurry and biogas effluent was done during two seasons. Results showed that the concentration of enterococci, E. coli, and Clostridium perfringens spores was overall reduced by only 1–2log10-units in the biogas digesters when comparing raw slurry and biogas effluent. Salmonella spp. was found in both raw slurry and biogas effluent. A total of 19 Salmonella serovars were identified, with the main serovars being Salmonella Typhimurium (55/138), Salmonella enterica serovar 4,[5],12:i:- (19/138), Salmonella Weltevreden (9/138) and Salmonella Rissen (9/138). The Salmonella serovars showed similar antimicrobial resistance patterns to those previously reported from Vietnam. When promoting biogas, farmers should be made aware that effluent should only be used as fertilizer for crops not consumed raw and that indiscriminate discharge of effluent are likely to contaminate water recipients, e.g. drinking water sources, with pathogens. Relevant authorities should promote safe animal manure management practices to farmers and regulations be updated to ensure food safety and public health.

Escherichia coli Contamination of Lettuce Grown in Soils Amended with Animal Slurry

A pilot study was conducted to assess the transfer of Escherichia coli from animal slurry fertilizer to lettuce, with E. coli serving as an indicator of fecal contamination and as an indicator for potential bacterial enteric pathogens. Animal slurry was applied as fertilizer to three Danish agricultural fields prior to the planting of lettuce seedlings. At harvest, leaves (25 g) of 10 lettuce heads were pooled into one sample unit (n = 147). Soil samples (100 g) were collected from one field before slurry application and four times during the growth period (n = 75). E. coli was enumerated in slurry, soil, and lettuce on 3M Petrifilm Select E. coli Count Plates containing 16 mg/liter streptomycin, 16 mg/liter ampicillin, or no antimicrobial agent. Selected E. coli isolates (n = 83) originating from the slurry, soil, and lettuce were genotyped by pulsed-field gel electrophoresis (PFGE) to determine the similarity of isolates. The slurry applied to the fields contained 3.0 to 4.5 log CFU/g E. coli. E. coli was found in 36 to 54% of the lettuce samples, streptomycin-resistant E. coli was found in 10.0 to 18.0% of the lettuce samples, and ampicillin-resistant E. coli in 0 to 2.0% of the lettuce samples (the detection limit was 1 log CFU/g). The concentration of E. coli exceeded 2 log CFU/g in 19.0% of the lettuce samples. No E. coli was detected in the soil before the slurry was applied, but after, E. coli was present until the last sampling day (harvest), when 10 of 15 soil samples contained E. coli. A relatively higher frequency of E. coli in lettuce compared with the soil samples at harvest suggests environmental sources of fecal contamination, e.g., wildlife. The higher frequency was supported by the finding of 21 different PFGE types among the E. coli isolates, with only a few common PFGE types between slurry, soil, and lettuce. The frequent finding of fecal-contaminated lettuce indicates that human pathogens such as Salmonella and Campylobacter can be present and represent food safety hazards.

Redistribution and persistence of microorganisms and steroid hormones after soil-injection of swine slurry

The redistribution and fate of contaminants in pig slurry after direct injection were investigated at two field sites, Silstrup (sandy clay loam) and Estrup (sandy loam), in Denmark. Intact soil samples were collected for up to seven weeks after slurry injection and concentrations of Salmonella Typhimurium Bacteriophage 28B (phage 28B), Escherichia coli, steroid hormones and other slurry components (water, volatile solids, chloride and mineral N) determined in and around the injection slit. The two experiments at Silstrup and Estrup differed with respect to slurry solid content (6.3 vs. 0.8%), as well as soil clay content (27 vs. 15%) and differed considerably with respect to the initial redistribution of slurry-borne contaminants in soil. The transport of microorganisms from the slurry injection slit to the surrounding soil was much lower than that of mineral N and chloride due to attachment and entrapment. The redistribution of E. coli was more affected by site-specific conditions compared to phage 28B, possibly due to the larger cell size of E. coli. The overall recovery of phage 28B was 0.8-4%, and of E. coli 0.0-1.3% in different samples, by the end of the study. Nine different steroid hormones were detected in the slurry slit, and a slow redistribution to the surrounding soil was observed. Overall recovery of estrogens was 0.0 to 6.6% in different samples. The study showed that the combination of soil and slurry properties determined the initial spreading of contaminants, and hence the potential for subsequent leaching.

Characterization of Salmonella spp. from wastewater used for food production in Morogoro, Tanzania

Wastewater use for crop irrigation and aquaculture is commonly practiced by communities situated close to wastewater treatment ponds. The objective of this study was to characterize Salmonella spp. and their antimicrobial susceptibility patterns among isolates from wastewater and Tilapia fish. A total of 123 Salmonella spp. isolates were isolated from 52 water and 21 fish intestinal samples. Genotyping of Salmonella spp. isolates was done by Pulsed-field Gel Electrophoresis (PFGE). Antimicrobial susceptibility testing was done by the minimal inhibitory concentration (MIC) technique. A total of 123 Salmonella spp. isolates represented 13 different serovars and 22 PFGE groups. Salmonella serovars showed resistance to 8 out of 14 antimicrobials; sulfamethaxazole (94%), streptomycin (61%), tetracycline (22%), ciprofloxacin and nalidixic acid (17%), trimethoprim (11%); gentamycin and chloramphenicol (6%). Salmonella Kentucky, S. Chandans, S. Durban and S. Kiambu showed multiple antimicrobial resistance to 7, 6 and 3 antimicrobials, respectively. This study has demonstrated that wastewater at the study sites is contaminated with Salmonella spp. which are resistant to common antimicrobials used for treatment of diseases in humans. Wastewater may, therefore, contaminate pristine surface water bodies and foodstuffs including fish and irrigated crops as well as food handlers.