versão On-line ISSN 1678-4162
Arq. Bras. Med. Vet. Zootec. v.59 n.4 Belo Horizonte ago. 2007
Eliminação de Escherichia coli Shigatoxigênica não-O157 em compostagem de esterco bovino
V.P. GonçalvesI; J.M. MarinII, *
IAluna de pós-graduação - FCAV-UNESP Jaboticabal, SP
IIFaculdade de Odontologia de Ribeirão Preto - USP Av. Café, s/n 14040-904 Ribeirão Preto, SP
To determine the fate of Shiga toxigenic Escherichia coli (STEC) non-O 157 in composted manure from naturally colonized cattle, fresh manure was obtained from three cows carrying non-O157 STEC strains possessing the stx2 gene. Two composting systems were used: a 0.6m deep cave opened in the soil and an one meter high solid manure heap in a pyramidal architecture. Every day, for the 10 first days, and every five days for a month, one manure sample from three different points in both systems was collected and cultured to determine the presence of E. coli and the presence of the stx 2 gene in the cells. The temperature was verified at each sampling. STEC non-O157 E. coli cells survived for 8, 25 and 30 days at 42, 40 and 38ºC, respectively, in the deep cave and 4, 4 and 7 days at 65, 58 and 52ºC, respectively, in the heap, during the composting manure. Temperature and indigenous microorganisms appear to contribute to pathogen disappearance in the composting system. It is concluded that both composting systems were efficient to eliminate STEC cells. Land application of composted manure should minimize environmental risk associated with the dissemination of the pathogen.
Keywords: bacterial reduction, manure, STEC, environment, composting system
Determinou-se o tempo necessário para a eliminação de Escherichia coli Shigatoxigênica (STEC) não-O157 em esterco bovino composto, obtido de fezes frescas de três vacas portadoras de cepas STEC não-O157 que apresentavam o gene stx 2. Foram utilizados dois sistemas de compostagem, o primeiro foi um buraco de 0,6m escavado no solo e o segundo um monte apresentando uma arquitetura piramidal com um metro de altura. Todos os dias, durante os primeiros 10 dias e a cada cinco dias durante um mês, uma amostra de três pontos diferentes dos dois sistemas de compostagem foram coletadas e semeadas para determinar a presença de E. coli e a presença do gene stx 2 nas células, sendo que em cada coleta a temperatura do sistema de compostagem foi determinada. Células de STEC não-O157 sobreviveram por 8, 25 e 30 dias nas temperaturas de 42, 40 e 38ºC, respectivamente, no sistema enterrado no solo, enquanto que no sistema de monte as células foram detectadas por 4, 4 e 7 dias em temperaturas de 65, 58 e 52ºC, respectivamente. A temperatura e os microrganismos presentes na microbiota do sistema de compostagem parecem ser os responsáveis pela eliminação do patógeno. Pode-se concluir que os dois sistemas de compostagem utilizados mostraram-se eficientes na eliminação de células de STEC. A aplicação de esterco após compostagem deve diminuir o risco de contaminação ambiental e a disseminação do patógeno.
Palavras-chave: redução bacteriana, esterco, STEC, ambiente, sistema de compostagem
Spreading of bovine manure is a common soil fertilization procedure, often used in vegetable production in Brazil (Pereira Neto, 1996). A potential risk arising from the disposal of animal waste of fecal origin is the spreading of enteric pathogens (Peel, 1997).
Animals from which food is derived are recognized as reservoirs of many significant food-borne pathogens, including Shiga-toxigenic Escherichia coli O157: H7 and other non-O157 strains (Kudva et al., 1998; Elder et al., 2000). Shiga toxin-producing E. coli (STEC) organisms of different serotypes have been increasingly isolated from diseased humans and from domestic animals (Karmali, 1989; Beutin et al., 1993). Many of these isolates were typical STEC, belonging to serotypes O26, O111 and O157, but most belonged to serotype O157:H7, which can cause severe diseases including hemorrhagic colitis and hemolityc-uremic syndrome in humans (Paton and Paton, 1998). The Shiga toxin exists in two antigenically different forms: stx1 and stx2. The genes encoding stx are located in lysogenic bacteriophages. Unless appropriately processed, manure is a potential biohazard capable of transmitting infective agents to both humans and animals (Jones, 1980; Tauxe, 1997).
Laboratory studies have shown that E. coli can survive for up to several months in experimentally inoculated animal feces (Kudva et al., 1998; Bolton et al., 1999; Jiang et al., 2002). The principal manner of disinfection during composting is based on time-temperature relationships that destroy pathogens, although antagonistic microorganisms may also play a role in the process (Himathongkham and Riemann, 1999; Larney et al., 2003). The temperature evolution pattern in the composting system can be divided into four different phases: mesophilic, thermophilic, cooling and maturation. After mixing of the compost material, the temperature raises rapidly reaching a maximum within nine days, after which it gradually declines to the ambient level over about 30 days (Ishii et al., 2000).
Cross-contamination of manure produce or improperly composted manure used for soil improvement may be a source of pathogen contamination during preharvesting. Competition with soil microorganisms and adverse environmental conditions can influence pathogen survival, but little information regarding the degree to which Shiga toxigenic E. coli non-O157 cells can survive in manure-amended soils is available. In the present study, the objective was to determine the fate of STEC non-O157 cells in cattle manure in two different composting systems.
MATERIALS AND METHODS
During June 2005, anal swabs were collected from the rectum of bovines in a herd of 50 healthy adult beef cows in the Northwestern region of São Paulo State, Brazil. The swabs were transferred to a bottle containing Stuart Transport Medium1 and were aerobically cultured on MacConkey agar1, as soon as possible. E. coli isolates were identified based on the colony characteristics, Gram staining and biochemical profile (Farmer, 1999). At least five colonies per plate were selected for further analysis. The isolates were serotyped for serotype O157 using the Latex Agglutination test kit2. Negative strains were considered non-O157. E. coli EDL 933 strain was used as a positive control for O157 serotype.
A loopfull of cells from each of the five colonies above selected was collected, mixed and overnight grown in Luria Bertani broth3, at 37ºC. Bacteria were pelleted from 1.5ml of broth, suspended in 200µl sterile distilled water and boiled for 10min. Following centrifugation of the lysate, a 150µl sample of the supernatant was stored at -20ºC as a template DNA stock (Keskimaki et al., 2001). Specific primers were used to amplify sequences of stx1 and stx2 genes. Primer sequences, predicted sizes of the amplified products, and the specific conditions were carried out according to those described by Orden et al. (1998). Briefly, amplification of bacterial DNA was performed with 50µl volumes containing 10µl of the prepared sample supernatant; 150ng each of the oligonucleotide primers; 0.2mM (each) d ATP, d GTP, d CTP and d TTP; 5 µl PCR 10x reaction buffer4 and 2.5U of Taq DNA Polymerase4. The PCR was performed at 94ºC for 2min for one cycle, followed by 30 cycles of 94ºC for 1min, 55ºC for 1min and 72ºC for 1min. The amplified product was visualized by standard submarine gel electrophoresis of 10µl of the final reaction mixture on a 1.5% agarose gel. Amplified DNA fragments of specific sizes were located by UV fluorescence after staining with ethidium bromide. Molecular size markers (Hae III digest of X 174 DNA4) were included in each gel.
Fresh manure was obtained from three cows that had been confirmed to have non-O157 STEC strains possessing the stx2 gene. In the herd examined this was the only stx gene detected in the eliminated fecal bacterial cells. The collected manure was hand-mixed in a bucket, covered with a sterile plastic and added to a composting system. A 0.6m deep cave was opened in the soil and filled with three layers of grass (10cm), sawdust (1-2cm) and manure free of STEC cells (5cm) with 40% humidity respectively. This arrangement was repeated three times. On the top layer, a 10cm layer of grass was placed. To avoid cross-contamination the selected manure was introduced in three plastic tubes (PVC ¾) which were placed into the cave at 20cm (CT), 40cm (CM), 60cm (CB) in the layers described above (Fig. 1b). One side of the tube was closed by lid and the other was kept open with an adapted rubber to permit the introduction of the swabs inside the system. A thermometer was adapted at the swab to register the temperature at the collecting time. The cave was completely covered with a wide black plastic sheet. Every day, for the 10 first days, and every five days for a month, three manure samples were collected from the top, the middle and the bottom layers of the composting hole and cultured to determine the presence of E. coli. After the first month, samples were collected once every month for four months. The other system was a manure heap with pyramidal architecture with the same layers scheduled as in cave, but with a 20cm grass layer and a 10cm manure layer. The same schedule of sampling collecting was done using the top (HT), the middle (HM) and the bottom (HB) layers (Fig. 1a). To verify the climatic effects, a small manure pile was deposited directly on a sheet of wood over a grass pasture and left exposed to the environment. This was designated the pile, and samples were collected in the same way as those of the composting hole. Samples of 5g manure were taken to the laboratory in ice-cold nutrient broth5. After an overnight period of aerated incubation at 37ºC, a loop of the broth was inoculated onto MacConkey agar and incubated overnight at 37ºC. Ten colonies per plate were selected for E. coli confirmation as described above. After that, a loopfull of each colony was inoculated and grown overnight into LB broth. The template DNA stock was obtained as described above. The presence of bacteria containing the stx 2 gene was assayed by PCR as mentioned.
A survey of E. coli survival and the presence of stx 2 gene in the composting cave and composting heap systems and also in the pile is shown in Table 1. Bacteria showing the stx 2 gene could be detected in the top layer of the composting heap until the fourth day and in the cave until the thirstiest day; in the middle layer, until the fourth day and twenty-fifth days in heap and cave, respectively; and in the bottom layer, until the seventh and eighth days in the heap and cave, respectively. The temperature in the center of the composting hole reached 40ºC, 72 hours after mixing of the manure, and reached a maximum of 45ºC within 10 days; it then gradually declined reaching that of the ambient. The variation of the temperature between the top and the bottom layers was 40ºC±2ºC, following the same rate of decline (Fig. 2b). While the temperature in the heap top reached a maximum of 65ºC between the sixth and ninth days, in the middle layer the maximum was 58ºC on the ninth day and in the bottom, it was 52ºC between the sixth and ninth days (Fig. 2a). However, in the pile the temperature was smaller and reached only a maximum of 33ºC during the experimental period (Fig. 2c) and the bacteria carrying the stx 2 gene could be detected for 60 days.
There is a growing public concern about the link between livestock production and soil contamination by pathogenic bacteria. This is especially true for the application of raw manure, which is potentially capable of spreading pathogens to a wider environment (Peel, 1997; Kudva et al., 1998). The percentage of STEC serotypes in cattle faeces ranged from 6% in a US study (Cray et al., 1996) to 71% in a French study (Pradel et al., 2000). Absence of routine cultural detection methods for non-O157:H7 means that many countries do not have data on the prevalence of non-O157:H7 and it is certain that there is considerable underreporting on these pathogens (Duffy, 2003).
E. coli O157:H7 in faeces outdoors on grass under ambient conditions in Ireland was capable of long-term survival (99 days) in faeces and in the underlying soil. The pathogen could be recovered directly from faeces on the grass for 50 days (Bolton et al., 1999). Using inoculated samples, a maximum survival time of 70 days in manure was observed by Wang et al. (1996). Mukherjee et al. (2006) reported an acquisition of E. coli O157:H7 by a child from garden soil recently fertilized with cattle manure and verified that the bacteria survived for more than two months in the grass. These data agree with the 60 days reported in the present work
Although the elimination of pathogens by composting has been well documented (Deportes et al., 1998; Tiquia et al., 2002; Larney et al., 2003), composting times and temperatures required to achieve elimination or reduction of the number of E. coli vary widely. Turner (2002) demonstrated inactivation of E. coli in farmyard manure, pig feces, and cereal straw already after 2h at 55ºC. In contrast, Lau and Ingham (2001) reported that E. coli could be cultured from bovine manure kept for 19 weeks at 21ºC. In the present study, E. coli could be cultured at every condition tested after up to 120 days, however the non-O157 STEC- stx2-gene strains were not found after 30 days, indicating that competition among the bacteria associated with the temperature seem to be very important aspects. Moreover, apparently the STEC strains are more sensitive to high temperatures than ordinary E. coli.
There have been few studies on the persistence of E. coli non-O157: H7 serotype in animal feces. However, one study which investigated the survival of E. coli O26, O111 and O157 in bovine feces (Fukushima et al., 1999) reported that storage at 5, 15, and 25ºC permitted the pathogens to survive for up to eight weeks at 25ºC. This agrees with the results obtained from the pile in this study. Recently, Fremaux et al. (2007) reported the elimination of non-0157 STEC strains submitted to composting in manure heaps after nine and 16 days at 65 and 56ºC, respectively, also similar with the results obtained in the present study.
Kuhnert et al. (2005) in Switzerland reported that 45.0 % of bovine STEC-positive faeces contained the stx 2 gene. This agrees with reports on Australian dairy cattle (Cobbold and Desmarchelier, 2001) and also with is reported in the present study. The stx 2 gene is assumed to be more important in developing disease than the stx 1 gene. Significantly, the stx2 gene is also associated with the majority of STEC strains causing human disease (Boerlin et al., 1999).
It is well-known that STEC is widespread in animal feces; therefore, application of untreated manure to vegetable cultures may pose a risk for the transmission of STEC to food. Large quantities of livestock waste are spread over agricultural soil in Brazil. Thus, small reductions in the prevalence of zoonotic agents would significantly lower the risk of pathogen dissemination following the spreading of manure. To conclude, it was shown that STEC strains non-O157 carrying the stx 2 gene can survive in a composting system for four to 30 days, depending on the temperature reached during composting. This system should be a good alternative to minimize the risk of pathogens spreading over agricultural soil.
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Recebido em 5 de outubro de 2006
Aceito em 25 de junho de 2007
* Corresponding author (autor para correspondência)
1 BBL/Becton Dickinson Sparks, MD - USA
2 Oxoid - Basingstoke - UK
3 Difco Oxford - UK
4 Amersham Biosciences - Buckinghamshire - UK
5 Sigma Chemical Co - St Louis, Mo - USA