Lisette Lapierre

Genetic Characterization of Antibiotic Resistance Genes Linked to Class 1 and Class 2 Integrons in Commensal Strains of Escherichia coli Isolated from Poultry and Swine

The aim of this research was to identify the presence of integrons among Escherichia coli strains isolated from poultry and swine and to characterize the topological association of these integrons with resistance genes and assess their potential ability to transfer these elements by conjugation. One hundred and seventy-two strains of E. coli were isolated. Their resistance to tetracycline, streptomycin, sulfamethoxazole-trimethoprim, ciprofloxacin, and enrofloxacin was studied by plate dilution. In resistant strains the presence of integrons and resistance genes was assessed by PCR. In the variable region, genes aadA1, dfrA1, and qnr were analyzed. Also, presence of tetA, tetB, and sul1 was assessed. Transference of these genes and integrons in vitro was evaluated by conjugation assays, using E. coli J53 Az(r) as recipient strain. Seventy-eight percent and 83% of the poultry and swine strains, respectively, were resistant to at least one of the studied antimicrobials. Of the isolated strains 91 presented integrons. Resistance genes detected within the integrons were aadA1, dfrA1, and sat1. Gene qnr was not detected. Genes tet and sul1 were identified in 105 and 53 strains, respectively. Seven strains transferred their resistance determinants by conjugation. The results verify the high percentage of antibiotic resistance in the E. coli strains isolated, and these represent a reservoir of resistance genes and integrons.

Molecular epidemiology and genetic diversity of Listeria monocytogenes isolates from a wide variety of ready-to-eat foods and their relationship to clinical strains from listeriosis outbreaks in Chile

Listeria monocytogenes is a pathogen transmitted through food that can cause severe infections in high-risk groups such as pregnant women, elderly, young children and immunocompromised individuals. It is a ubiquitous bacterium that can survive in harsh conditions, such as dry environments, at low temperatures, in brine conditions and at low pH values. It also has the capacity to form biofilms, which makes it particularly successful even in colonizing surfaces within food processing plants. This study analyzed the presence of L. monocytogenes in ready-to-eat food (RTE) such as sausage, cheese, fresh salads, and other types of raw food. 850 samples of refrigerated and packaged food collected in 2008 and 2009 were analyzed. It was found that 25% of these samples were contaminated with L. monocytogenes strains. Serotyping and virulence genes detection by polymerase chain reaction (PCR) identified that strains belonging to serotype 4b, and containing one or more genes encoded by pathogenicity island (LIPI-1), were significantly associated with specific food types. Furthermore, using pulse field gel electrophoresis (PFGE), it was possible to associate isolates from cheese with strains from clinical cases of listeriosis outbreaks that occurred during the same time period within the same geographic regions. In addition, a strong correlation was observed between isolates from frozen seafood and from clinical strains obtained from sporadic cases of listeriosis. In agreement with reports described in other countries, our results shown that Chilean strains of L. monocytogenes from food products include the most virulent serotypes, encoding for the main virulence genes of the LIPI-1, and were clonally related to clinical isolates from sporadic cases and outbreaks of listeriosis. In conclusion, we show that Chilean isolates of L. monocytogenes from RTE and raw food products can cause disease in humans, representing a public health risk that justifies permanent surveillance.

Withdrawal time of four pharmaceutical formulations of enrofloxacin in poultry according to different maximum residues limits.

To ensure delivery of safe animal products to consumers, the withdrawal time (WDT) of drugs must be respected. Property differences among pharmaceutical formulations, for the same drugs, can lead to differences in the WDTs estimation. The WDTs of four commercial formulations of enrofloxacin (ENRO) in broiler chickens, considering MRLs established by different countries, were studied. Two hundred-thirty-four broiler chicks were allotted among four groups; the formulations were orally administered daily with 10 mg/kg bw. After treatment, six chickens of each group and two controls were slaughtered daily until day 9 post-treatment. Samples of muscle and liver were collected, and analyzed using HPLC-MS-MS. The WDTs among formulations of ENRO showed differences of 24 and 48 h. Based on the European Community and Chile MRLs of 100 microg/kg (muscle) and 200 microg/kg (liver), the WDTs did not exceed 5 days. When Japan MRL was considered (10 microg/kg(,)), the WDTs increased up to 8 days. These results indicate that for WDTs determination, the differences among pharmaceutical formulations of a drug must be considered as well as the MRLs.

Depletion study of three formulations of flumequine in edible tissues and drug transfer into chicken feathers

To ensure the delivery of safe animal products to consumers, withdrawal times (WDT) of drugs must be respected. Drugs administered in therapies can also reach nonedible tissues (for humans) such as feathers; this transfer is of concern as feather meal is used in diets of food producing animals, being this a possible source of residue contamination of final products for human consumption. WDTs of three flumequine formulations (10%, 80% premix powder and 20% solution) as well as the transfer of this drug into feathers were determined. One hundred and twenty broiler chickens were allocated into four experimental groups (36 birds each). Three of them were treated with 24 mg/kg bw orally for five consecutive days of each flumequine formulation, whereas one group remained untreated (12 birds as control group). After the treatment ended, six chickens of each experimental group and two controls were slaughtered daily for 6 days. Samples of muscle, liver and feathers were collected and analyzed by liquid chromatography tandem mass spectrometry (LC MS/MS). The WDTs showed differences between formulations. Flumequine concentrations found in feathers remained high during WDT and after this period, thus suggesting that the WDTs estimated for the pharmaceutical formulation of flumequine do not guarantee the absence of this drug in chicken nonedible tissues such as feathers.

Study of enrofloxacin and flumequine residues depletion in eggs of laying hens after oral administration

Two groups of laying hens (each n=12) were administered 10 mg/kg enrofloxacin (ENRO) (group A) or 26.6 mg/kg flumequine (FLU) (group B) by gastric catheter daily for five consecutive days. A third group (n=6) was untreated controls. Eggs were collected from day one of treatment and up to 30 days after withdrawal of the drug. Egg white and yolk from each egg were separated, and ENRO, its metabolite ciprofloxacin (CIP) and FLU residues were analysed by a high-performance liquid chromatography method with fluorescence detection. The sum of ENRO and CIP was detectable in egg white on the first day of treatment in high-level concentrations (2007.7 μg/kg) and remained steady during administration. In egg yolk, residues were detectable at day one in lower concentrations (324.4 μg/kg), increasing to the end of treatment. After treatment, these residues decreased and were detectable up to day 8 in egg white, and day 10 in yolk. FLU residues during drug administration in white were detectable in high concentrations from day one to five (6788.4-6525.9 μg/kg), and in yolk, concentrations were lower during administration (629.6-853.9 μg/kg). After drug withdrawal, FLU residues remained longer in egg white (30 days) than in yolk (26 days). For both drugs, differences of concentrations between matrices were significant.

High prevalence of pathogenic Leptospira in alien American mink (Neovison vison) in Patagonia

BackgroundLeptospirosis is an important zoonosis with worldwide distribution caused by pathogenic bacteria of the genus Leptospira. The North American mink (Neovison vison) has an important role in the environmental contamination with Leptospira, as minks live in aquatic environments and are the predators of rodents.FindingsBlood and kidney samples were obtained from 57 minks in Southern Chile 39° S to 45° S. Pathogenic species of Leptospira were detected by PCR on 31/57 minks. To determine the species, we sequenced the 16S ribosomal RNA (rRNA) gene on nine of the positive samples. We predicted two pathogenic species: Leptospira interrogans (five samples) and Leptospira borgpetersenii (four samples).ConclusionsThis study showed that the American mink presents pathogenic species of Leptospira and confirm important environmental contamination of Patagonian rivers and lakes with pathogenic Leptospira.

Antimicrobial Resistance in Campylobacter spp.

The resistance of infectious agents to a broad range of antimicrobial drugs is a growing public health problem, which raises significant social concerns. This chapter describes the main antimicrobial resistance problems in developed and developing countries and the possible relationship between the resistances of Campylobacter strains found in poultry and humans. Determining the level of the problem is essential for control, formulating, and monitoring an effective response to antimicrobial resistance.

Determination of Chlortetracycline Residues, Antimicrobial Activity and Presence of Resistance Genes in Droppings of Experimentally Treated Broiler Chickens

Tetracyclines are important antimicrobial drugs for poultry farming that are actively excreted via feces and urine. Droppings are one of the main components in broiler bedding, which is commonly used as an organic fertilizer. Therefore, bedding becomes an unintended carrier of antimicrobial residues into the environment and may pose a highly significant threat to public health. For this depletion study, 60 broiler chickens were treated with 20% chlortetracycline (CTC) under therapeutic conditions. Concentrations of CTC and 4-epi-CTC were then determined in their droppings. Additionally, this work also aimed to detect the antimicrobial activity of these droppings and the phenotypic susceptibility to tetracycline in E. coli isolates, as well as the presence of tet(A), tet(B), and tet(G) resistance genes. CTC and 4-epi-CTC concentrations that were found ranged from 179.5 to 665.8 µg/kg. Based on these data, the depletion time for chicken droppings was calculated and set at 69 days. All samples presented antimicrobial activity, and a resistance to tetracyclines was found in bacterial strains that were isolated from these samples. Resistance genes tet(A) and tet(B) were also found in these samples.

The immune profile induced is crucial to determine the effects of immunocastration over gonadal function, fertility, and GnRH-I expression

Immunocastration or vaccination against the GnRH‐I hormone is a promising alternative to reproductive control in different animal species. Given the low immunogenicity of this hormone, the use of adjuvants becomes necessary.

Characterization of the epidemiology of bat-borne rabies in Chile between 2003 and 2013

Rabies is a zoonotic disease of great impact to public health. According to the World Health Organization, the country of Chile is currently declared free from human rabies transmitted by dogs. An epidemiological characterization and description was conducted using rabies data from 2003 to 2013 held by the National Program for Prevention and Control of Rabies from the Ministry of Health, consisting of bats samples reported as suspect and samples taken by active surveillance (bats brain tissue). Spatial autocorrelation analysis was performed using Local Indicators of Spatial Association (LISA) statistics, particularly Morans I index, for the detection of spatial clusters. Temporal descriptive analysis was also carried out. Nine hundred and twenty-seven positive cases were reported, presenting an average of 84 cases per year, mainly originated from passive surveillance (98.5%), whilst only 1.5% of cases were reported by active surveillance. Global positivity for the study period was 7.02% and 0.1% in passive and active surveillance respectively. Most of the cases were reported in the central zone of Chile (88.1%), followed by south zone (9.1%) and north zone (2.8%). At a regional level, Metropolitana (40.6%), Valparaíso (19.1%) and Maule (11.8%) regions reported the majority of the cases. Tadarida brasiliensis (92%) presented the majority of the cases reported, with viral variant 4 (82%) being most commonly diagnosed. Only two cases were detected in companion animals. The central zone presented a positive spatial autocorrelation (Morans I index=0.1537, 95% CI=0.1141-0.1933; p-value=0.02); north and south zones returned non-significant results (Morans I index=0.0517 and -0.0117, 95% CI=-0.0358-0.1392 and -0.0780-0.0546, and p-values=0.21 and 0.34 respectively). The number of rabies cases decreased between May and August (late fall and winter) and tended to increase during the hot season (December to March), confirmed with the evidence from Autocorrelation analysis and the Ljun-Box test (X2=234.85 and p-value<0.0001). Knowledge of animal rabies epidemiologic behaviour becomes relevant when designing prevention and control measures and surveillance programs. This is especially important considering the high impact to Public Health of this disease and that wildlife rabies in bats remains endemic in Chile.