Large quantities of liquid effluents are generated during slaughtering and meat processing services related to cleaning equipment and associated facilities and the high levels of organic material in these effluents can severely pollute aquatic environments. Liquid residue processing regimes utilize biological treatment technologies preceded by physical-chemical treatments. Some of these pretreatments use traditional chemical surfactant formulations (based on sodium alkyl benzene sulphonate) and common sanitizers (such as sodium hypochlorite), while others utilize biotechnological products (such as enzymes) (10). The continuous use of these sanitizing substances, however, can contribute to the incremental selection of resistant microorganisms. The continuous use of sanitizing substances can contribute to the selection of incremental resistant microorganisms similarly to antimicrobial resistance, which can help reduce membrane permeability and enzymatic inactivation of structures (8).
Pseudomonas spp. are gram-negative aerobic bacilli widely distributed in the natural environment and particularly abundant in soils and water; they are opportunistic and ubiquitous pathogens, probably due to their limited nutritional requirements and tolerance of adverse physical and chemical conditions including stream temperatures and sanitizers. Pseudomonas aeruginosa shows a wide spectrum of resistance to different classes of antimicrobial agents, including third and fourth-generation cephalosporins (cefepime) and carbapenems (imipenem and meropenem) (6, 7, 9).
One of the principal factors linked to the emergence of microbial resistance is the abusive and indiscriminate use of antimicrobial agents (3). The emergence of Pseudomonas spp. strains with variable and growing levels of antimicrobial resistance have generated considerable concern, and various studies have sought to characterize this resistance and establish risk parameters. This phenomenon is complex and has multiple causes, some of which have already been determined whereas others still need to be elucidated. The presence of Pseudomonas spp. in aquatic environments facilitates their dissemination, and their association with propitious conditions to antimicrobial multi-resistance may cause serious public health problems.
Antibiotics have been used to promote swine growth as well as for more traditional therapeutic treatments (12), and one of the consequences of this wide use of antimicrobial agents in animals is the development of resistance in pathogenic microorganisms and their posterior transmission to humans through food (14). The identification and monitoring of pathogenic microorganisms in effluents is laborious and expensive. Brazilian legislation, specifically Resolution No. 357 of the National Environment Council (CONAMA), does not require monitoring pathological bacteria in liquid effluents before their liberation into receptor rivers (5), although a study undertaken in recreational waters (6) recommended using P. aeruginosa as an indicator of water quality.
The present work was designed to determine the presence of Pseudomonas spp. during the different phases of processing slaughterhouse swine wastes in an Effluent Treatment Station (ETS) as well as their antimicrobial resistance profiles.
The slaughterhouse studied was located in the municipality of Dourados, Mato Grosso do Sul State (MS), Brazil. Approximately 1,536 animals are daily slaughtered there. The slaughtering process and subsequent meat processing procedures require approximately 2,050 l of water per animal, with a total average daily volume of approximately 3,145 m³. The average effluent volume into the final receiving water body is 35.8 l/s and the retention period in the ETS is approximately 20 days.
The ETS receives effluents that have received primary and secondary treatments, passing them through an Australian system of stabilization ponds comparing five sequential ponds: anaerobic (A1 and A2), facultative (F1 and F2), and polishing (P) ponds.
The study involved the collection of 80 water samples near the transfer ducts between each of the five ETS ponds during 16 days, covering the four seasons between November/2008 and December/2009. The samples were collected 10 cm below the water surface in sterile flasks, which were subsequently transported to the laboratory under refrigeration.
This research was undertaken following the norms established by the Standard Methods for the Examination of Water and Wastewater (1) with some adaptations. Asparagine broth was used for Pseudomonas spp. in the presumptive phase, and Pseudomonas was isolated on Cetrimide agar (Merck) using a spreading technique. The Pseudomonas spp. isolates were tested for antimicrobial susceptibility using the disk-diffusion method (2) and interpreted according to the Clinical and Laboratory Standards Institute protocols (4), except in the case of streptomycin (for which a cutoff was established based on the aminoglycoside group).
Pseudomonas spp. susceptibility to antimicrobials was tested using Laborclin® discs impregnated with: amikacin (30 μg), aztreonam (30 μg), cefepime (30 μg), ceftazidime (30 μg), ciprofloxacin (5 μg), streptomycin (10 μg), gentamicin (10 μg), imipenem (10 μg), meropenem (10 μg), piperacillin/tazobactam (100/10 μg), and tobramycin (10 μg).
The low levels of Pseudomonas spp. isolates encountered and the non-homogeneous distribution of the data did not permit the use of analysis of variance or other refined statistical tests, therefore, the results were examined using simple measures and percentage analyses.
The 80 water samples collected in the five ponds yielded 34 Pseudomonas spp. isolates, distributed throughout the ETS (Figure 1). The increasing presence of Pseudomonas spp. in industrial and hospital effluents, wells, rivers, and garbage dumps (6, 7, 11, 13) during the last 10 years makes them reservoirs and distribution points for potentially pathogenic isolates.
The 100 % reduction in Pseudomonas spp. isolates observed at the end of the treatment system demonstrated the efficiency of microorganism removal by ETS, and the low risk of their dissemination into the receptor river system (rivers themselves show a significant natural capacity for purifying effluents and reducing the numbers of the pathogenic microorganisms they carry). A survey undertaken from the head waters of a river at a point downstream of the inflow of effluents derived from a hospital showed the self-purification capacity of the system, as large quantities of Pseudomonas spp. isolates were identified near the effluent site, lower numbers further downstream of this area, but none in the headwaters (7).
The highest percentage of isolates identified in the present work (both in terms of total numbers and of microbes that demonstrated full or intermediate antimicrobial resistance profiles) were found in pond A1, decreasing successively in ponds A2, F1, and F2. This reduction was associated with the purification processes occurring during the passage of the effluents through the different treatment ponds (Figure 1).
The susceptibility profiles to the 11 drugs tested indicated that 16/34 (47.05 %) isolates showed susceptibility to all the antimicrobials tested. These results are compatible with data from natural aquatic environments (13) not containing any Pseudomonas spp. isolates resistant to nine different antimicrobials exhibiting less selection pressure for antimicrobial resistance in natural environments as compared to contaminated sites.
Individual evaluations of the antimicrobial agent isolates were susceptible to amikacin, piperacillin/ tazobactam, aztreonam, and imipenem (Table 1). These results draw attention to streptomycin (the first aminoglycoside isolated from Streptomyces griseus, 1944), the antibiotic exhibiting the greatest intermediate resistance profile and the second greatest resistance profile (only lower than cefepime) (Table 1). The observed absence of resistance to imipenem and to meropenem (2.94 %) corroborates the results of other workers in aquatic environments (6, 11).
Only one isolate showed combined resistance to three antibiotics (cefepime, ceftazidime and streptomycin). The resistance levels of Pseudomonas spp. isolates were also found to be low in a river in Belgium, in the surface waters of the Passo Fundo River in Rio Grande do Sul State, Brazil and in mineral water in Italy (7, 9, 11).
Fluoroquinolones are the drugs of choice in treating infections caused by Pseudomonas aeruginosa, although some β-lactams as ceftazidime, cefepime, aztreonam, imipenem, and piperacillin/tazobactam are also widely used. We observed resistance to ciprofloxacin (5.88 %), ceftazidime (2.94 %) and cefepime (20.58 %), which indicates the need for continued surveillance to avoid the propagation of resistant isolates.
The treatment system herein investigated significantly reduced Pseudomonas spp. populations in slaughterhouse effluents. None of the Pseudomonas spp. isolates investigated showed multiresistance to the antimicrobials tested. Furthermore, the risks of dissemination and subsequent contamination of receptor water bodies by microorganisms that could have multiresistance to antimicrobial agents appear to be very low. Nonetheless, constant monitoring will be necessary to prevent and control future contamination.
The authors would like to thank the Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado de Mato Grosso do Sul (FUNDECT) for their financial support.
This article was originally published in Revista Argentina de Microbiología (2013) 45: 57-60.