Anders Dalsgaard

Distribution and in-vitro transfer of tetracycline resistance determinants in clinical and aquatic Acinetobacter strains.

Following characterisation by phenotypic tests and amplified ribosomal DNA restriction analysis (ARDRA), 50 tetracycline-resistant (MIC > or = 16 mg/L) Acinetobacter strains from clinical (n = 35) and aquatic (n = 15) samples were analysed by PCR for tetracycline resistance (Tet) determinants of classes A-E. All the clinical strains were A. baumannii; most (33 of 35) had Tet A (n = 16) or B (n = 17) determinants, and only two did not yield amplicons with primers for any of the five tetracycline resistance determinants. The aquatic strains belonged to genomic species other than A. baumannii, and most (12 of 15) did not contain determinants Tet A-E. Strains negative for Tet A-E were also negative for Tet G and M; further analysis of two aquatic strains with specific primers for Tet O and Tet Y and degenerate primers for Tet M-S-O-P(B)-Q also showed negative results. Transfer of tetracycline resistance was tested for 20 strains with three aquatic Acinetobacter strains and Escherichia coli K-12 as recipients. Transfer of resistance was demonstrated between aquatic strains from distinct ecological niches, but not from clinical to aquatic strains, nor from any Acinetobacter strain to E. coli K-12. Most transconjugants acquired multiple relatively small plasmids (<36 kb). Transfer did not occur when DNA from the donor strains was added to the recipient cultures and was not affected by deoxyribonuclease I, suggesting a conjugative mechanism. It is concluded that Tet A and B are widespread among tetracycline-resistant A. baumannii strains of clinical origin, but unknown genetic determinants are responsible for most tetracycline resistance among aquatic Acinetobacter spp. These differences, together with the inability of clinical strains to transfer tetracycline resistance in vitro to aquatic strains, contra-indicate any important flow of tetracycline resistance genes between clinical and aquatic acinetobacter populations.

Impact of Integrated Fish Farming on Antimicrobial Resistance in a Pond Environment

ABSTRACT Integrated fish farming combines livestock production with fish farming. Animal manure is shed directly into a fish pond as fertilizer and supports the growth of photosynthetic organisms. The livestock, mainly chickens and pigs, is often fed feed containing growth promoters. In this study we investigated the impact of integrated fish farming on the levels of antimicrobial-resistant bacteria in a pond environment. One integrated broiler chicken-fish farm was studied for 2 months immediately after the start of a new fish production cycle. A significant increase over time in the resistance to six different antimicrobials was found for the indicator organism Acinetobacter spp. isolated from composite water-sediment samples. The initial resistance levels prior to the new production cycle were 1 to 5%. After 2 months the levels of resistance to oxytetracycline and sulfamethoxazole reached 100%, and the levels of resistance to ciprofloxacin were more than 80%. The long-term effects of resistance on integrated farming were studied on seven additional farms. The resistance levels were particularly high among Enterococcus spp. and were also high among Acinetobacter spp. isolated from water-sediment samples compared to the resistance levels at four control farms. In conclusion, integrated fish farming seems to favor antimicrobial-resistant bacteria in the pond environment. This could be attributed to the selective pressure of antimicrobials in the pond environment and/or to the introduction of antimicrobial-resistant bacteria from animal manure. Potential risks to human health were not addressed in this study and remain to be elucidated.

The effects of tertiary wastewater treatment on the prevalence of antimicrobial resistant bacteria

The effects of tertiary wastewater treatment on the prevalence of antimicrobial resistant bacteria were investigated in two large-scale municipal treatment plants during a period of six months. Total and relative numbers of resistant bacteria were determined in raw sewage, treated sewage and anaerobically digested sludge by bacteriological counts on media selective for coliforms (MacConkey agar) and Acinetobacter spp. (Baumann agar). In addition, the level of antimicrobial susceptibility was determined by the disc-diffusion method in 442 Acinetobacter isolates identified by colony hybridisation with a genus-specific DNA probe. Independent of the different antibiotics and media used, the total numbers of resistant bacteria in treated sewage were 10-1000 times lower than in raw sewage. Based on linear regression analysis of data on bacteriological counts, the prevalences of antimicrobial-resistant presumptive coliforms and Acinetobacter spp. in treated sewage and digested sludge were not significantly higher compared with raw sewage. On the contrary at one plant, statistically significant decreases were observed in the prevalence of ampicillin-resistant presumptive Acinetobacter spp. (p = 0.0188) following sewage treatment, and in the prevalence of either ampicillin-resistant presumptive Acinetobacter spp. (p = 0.0013) or ampicillin- and gentamicin-resistant presumptive coliforms (p = 0.0273 and p = 0.0186) following sludge treatment. The results obtained by bacteriological counts were confirmed by antimicrobial susceptibility testing of Acinetobacter isolates. Based on logistic regression analysis, isolates from treated sewage and digested sludge were generally not significantly more resistant compared with isolates from raw sewage. Based on these evidences, it was concluded that tertiary wastewater treatment did not result in a selection of antimicrobial resistant bacteria.

Fishborne Zoonotic Intestinal Trematodes, Vietnam

These parasites are an unrecognized food safety risk in a population with a tradition of eating raw fish.

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.

Prevalence of fishborne zoonotic parasites in important cultured fish species in the Mekong Delta, Vietnam

A seasonal investigation on the occurrence of fishborne zoonotic trematodes (FZT) in economically important mono-cultured hybrid catfish and giant gouramy was conducted in the Mekong Delta of Vietnam. Fish from carp poly-culture and intensive small-scale integrated vegetable–aquaculture–animal husbandry farming (VAC) systems were also examined. No FZT metacercariae were found in any mono-cultured hybrid catfish. FZT metacercariae were common, however, in fish from the other three systems: All metacercariae belonged to the Heterophyidae family of trematodes, Haplorchis pumilio, H. taichui, Centrocestus formosanus and Stellantchasmus falcatus. The FZT prevalence was 1.7% in mono-cultured giant gouramy, 6.6% in carp from the poly-culture and 3.0% in fish raised in the VAC system. H. pumilio was the most common FZT species constituting more than 58.0% of all metacercariae recovered. The prevalence of infection was significantly higher in the flooding season compared to the non-flooding season for both giant gouramy and fish reared in the VAC system. FZT intensity was greatest in fish from carp poly-culture, particularly in the flooding season. The results indicate that certain fish production systems are at risk for FZT, and control approaches will benefit from understanding these risk factors.

Antimicrobial resistance of intestinal Aeromonas spp. and Enterococcus spp. in fish cultured in integrated broiler-fish farms in Thailand

Abstract Integrated fish farming in Southeast Asia combines intensive husbandry with extensive aquaculture. The feed of the livestock, most importantly chicken and pigs, is supplemented with various antimicrobials for growth promoting and prophylactic and therapeutic treatment. Animal manure is excreted into the ponds and fertilizes the water and this supports the growth of photosynthetic organisms. Antimicrobials and antimicrobial residues enter the pond and may establish a selective pressure in favour of antimicrobial-resistant bacteria. Intestinal samples were taken from fish collected from eight integrated broiler-fish farms and nine control farms culturing fish only. Levels of resistance to chloramphenicol (CHL), ciprofloxacin (CIP), erythromycin (ERY), oxytetracycline (OTC), streptomycin (STR) and sulfamethoxazole (SMZ) were determined in two groups of indicator organisms, i.e. Aeromonas spp. and Enterococcus spp. Resistance was determined by the disk diffusion method and for Aeromonas spp. also by plate counts on agar supplemented with antimicrobials. Differences in levels of antimicrobial resistance were analysed by logistic regression with each individual farm included as random effect. For Enterococcus spp., significant higher levels of resistance to chloramphenicol (8%), erythromycin (91%, odds ratio (OR) 35), oxytetracycline (75%, OR 14), and streptomycin (72%, OR 31) were found among fish intestinal samples from integrated farms compared to Enterococcus spp. derived from control farms (0%, 23%, 16%, and 31%, respectively). Resistance among the Aeromonas spp. was low ( Aeromonas spp. isolated in integrated farms was 37% but not significant ( P =0.053) compared to control farms (13%). Resistance determination based on plate counting did not reveal any significant differences between Aeromonas spp. in fish intestinal samples from integrated and control farms. The results indicated that the practice of integrated fish farming may lead to higher levels of antimicrobial-resistant bacteria in the intestine of the fish. However, they also showed that the use of two groups of different indicator organisms gave different outcomes, thus stressing the importance of using different indicators when assessing antimicrobial resistance in the aquatic environment.

A high proportion of Vibrio cholerae strains isolated from children with diarrhoea in Bangkok, Thailand are multiple antibiotic resistant and belong to heterogenous non-O1, non-O139 O-serotypes

Results of a surveillance on cholera conducted with patients seen at the Children Hospital in Bangkok, Thailand from August 1993 to July 1995 are presented. Annually, isolation rates for Vibrio cholerae varied between 1.7 and 4.4% of patients with diarrhoea. V. cholerae O1 serotype Ogawa accounted for between 31 and 47% of patients cultured positive for V. cholerae, whereas the O139 serotype dominated in early 1994 after which it disappeared. Non-O1, non-0139 strains were isolated at similar rates as serotype O1 in 1993 and 1994, but accounted for 69% of V. cholerae culture positive specimens in 1995. However, the annual proportion of the isolation of non-O1, non-O139 strains showed little variation and remained low between 1.0 and 1.3%. Serotyping of 69 epidemiological unrelated non-O1, non-O139 strains produced 37 different O-serotypes. BglI ribotyping of serotypes containing more than two strains demonstrated a high degree of heterogeneity within and between serotypes, except seven serotype O37 strains which showed an identical ribotype suggesting clonality. None of the 69 strains hybridized with a cholera toxin probe and only two strains hybridized with a heat-stable enterotoxin probe. Susceptibility testing to 12 antibiotics showed that 40 of 69 (58%) non-O1, non-O139 strains were resistant to colistin, streptomycin and sulphisoxazole and 28 of 69 (41%) were multiple antibiotic resistant (MAR; > or = 4 antibiotics). Although 26 of 69 (38%) strains contained one or more plasmids, the plasmids were of low molecular weights and did not seem to encode antibiotic resistance. The results of the present study showed that a high proportion of heterogenous MAR V. cholerae non-O1, non-O139 strains were isolated from children at the hospital. With reference to the emergence of V. cholerae O139 in 1992, we suggest that non-O1, non-O139 strains should be monitored carefully to detect new serotypes with a possible epidemic potential, but also to determine the development and mechanism of antibiotic resistance.

Faecal and protozoan parasite contamination of water spinach (Ipomoea aquatica) cultivated in urban wastewater in Phnom Penh, Cambodia

Objective  To identify the level of contamination with thermotolerant coliforms (ThC), intestinal helminth eggs and protozoan parasites in water spinach (Ipomoea aquatica) cultivated in a wastewater‐fed lake in Phnom Penh, Cambodia.

High prevalence of Ancylostoma ceylanicum hookworm infections in humans, Cambodia, 2012

Preventative chemotherapy without community hygiene and animal health programs may be leading to emergence of this zoonosis.