Annals of Microbiology, 58 (3) 387-394 (2008)
Molecular typing of staphylococcal communities isolated during municipal solid waste composting process Olfa BOUZAIANE1*, Mohamed Salah ABBASSI2, Maher GTARI1, Ons BELHAJ1, Naceur JEDIDI1, Assia Ben Hassen2, Abdennaceur HASSEN1 1Institut
National de Recherche Scientifique et Technique, Laboratoire de Traitement et recyclage, B.P. 95 - 2050, Hammam Lif, Tunisie; des Laboratoires, Centre National de Greffe de Moelle Osseuse, Tunis, Tunisie
2Services
Received 15 February 2008 /Accepted 16 June 2008
Abstract - This study investigates the molecular typing occurrence of staphylococcal communities during composting process of municipal solid waste. One hundred staphylococcal strains were isolated during the composting process and analysed either by phenotypic and genotypic methods. Amplified ribosomal DNA restriction analysis (ARDRA), internal transcripted spacer-polymerase chain reaction (ITS-PCR) and pulsed-field gel electrophoresis (PFGE) were used as molecular tools. During the digestion phase of composting process three staphylococcal species were recovered: Staphylococcus xylosus (48%), Staphylococcus lentus (40%), and Staphylococcus hominis (12%). In the maturation phase in addition to the latter species four species were also recovered at low frequencies: Staphylococcus lugdunensis, Staphylococcus haemolyticus, Staphylococcus warneri and Staphylococcus sciuri. Therefore, the composition of staphylococcal communities was temperature-, and phase dependant. The molecular methods showed that the ARDRA was not able to differentiate between strains of the same species. However, the ITS-PCR and the PFGE methods allowed interspecies and intraspecies discrimination, respectively. Key words: Staphylococcus, compost of municipal solid waste, ARDRA, ITS-PCR, PFGE.
INTRODUCTION Composting transforms organic matter into a stable product consisting of humus-like substances. The end product is available for agricultural use. The composting process is used to treat yard waste, manure, sewage sludge and also municipal solid waste. Pathogens microorganisms found in municipal solid waste can be viruses, bacteria and protozoa or helminths (Strauch, 1991). As they are heat-sensitive, the temperature increment during the composting process should eliminate them, leading microbial pathogen-free end product, what is defined as disinfection or sanitisation. Several parameters such as humidity and oxygen have an influence on the heat increase. For this reason, sanitisation efficiency depends on the composting method used (Pereira-Neto et al., 1987). Therefore, there are many microbes that escape and resist to the heat treatment in composting process (Hassen et al., 2001). Among these microbes we notice spores or bacteria that are either spore forming (Clostridium, Bacillus spp.) or very resistant to desiccation like Staphylococcus. Staphylococci are among the most wide spread bacteria and are ubiquitous in the environment. In addition, they are well known nosocomial pathogens associated with multiple antimicrobial resistance mechanisms. For many years Staphylococcus aureus was the only species recognised as an important human pathogen, however, recently coagulase-negative staphylococci (CNS) have been emerged in * Corresponding author. Phone: 21671788436; Fax: 21671410740; E-mail:
[email protected]
clinical sets as a causative agents of several invasive infections (Kloos and Bannerman, 1999). Staphylococcus saprophyticus is believed to cause opportunistic infections in the female urinary tract (UTI) (Marrie et al., 1982). Staphylococcus haemolyticus and Staphylococcus epidermidis the second most frequently encountered species have been reported to cause myocarditis, sepsis, peritonitis, and UTI (John et al., 1978). Staphylococcus hominis, Staphylococcus warneri, Staphylococcus capitis, Staphylococcus simulans, Staphylococcus cohnii, Staphylococcus xylosus, Staphylococcus saccharophyticus have been associated, less frequently, with various infections (Martin et al., 1989; Westblom et al., 1990; Kamath et al., 1992). Coagulase-negative staphylococci are the most common bacteria isolated from the compost of municipal solid waste (Hassen et al., 2001). The emergence of CNS as human pathogens and reservoirs of antimicrobial resistance determinants requires their rapid and reliable identification in order to have an early prediction of the potential pathogenicity or antibiotic susceptibility of each isolate (Kloos and Bannerman, 1999; Lina et al., 2000). In recent years, several manual and automated methods for identification of staphylococci have been described, but they are time consumer (at least 30 h) as they require microbial growth. Furthermore, commercially available panels, which are based on functional differences in metabolic pathways, do not allow a reliable distinction between different CNS. A number of PCR-based methods for the species-specific have been developed combining speed, reliability, and low cost. Amplified ribosomal DNA restriction analysis (ARDRA) and ribosome spacer PCR or internal tran-
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scribed spacer-PCR (ITS-PCR) was tested by several authors for the identification of staphylococci from different origins (Mendoza et al., 1998; Bes et al., 2000). The aims of this present study were: (i) to identify the staphylococcal communities during composting process, and (ii) to characterise them by ARDRA, ITS-PCR, and PFGE methods in order to assess suitable method(s) of molecular typing.
MATERIALS AND METHODS Composting process. The study was carried out in the experimental composting plant of Beja City, north east of Tunisia; during the period between June and October 2003. In each year 1000 tons of solid waste, were treated in this plant. Municipal solid wastes collected from various sites of the Beja City and forwarded through two series of windrows with a pyramidal shape (3.0 m x 2.5 m x 1.5 m, length x width x height). At first, the digestion phase takes place in windrow, then wastes were refined by mechanical sorting of the inert matter (mostly plastics and glasses). Finally, 65 days of composting, sawdust and green wastes were added to enhance the microbial activity of digestion phase and to enhance biodegradation under optimal conditions. Temperature and humidity were controlled daily, and windrow was turned and watered (humidity regulatory adjusted to 50%) as soon as the inner temperature of the pile reached or exceed 65 °C. These operations of turning and watering were performed almost twice per month on average. Sampling of organic wastes during the composting cycle. Ten samples (approximately 5 kg) were collected every 15 days from 5 to 139 days of composting from ten randomly selected locations in the windrow by digging a small pit to 10 m depth with a shovel. In each sampling, samples were mixed thoroughly and three portions of 1 kg each were separated. The first portion was stored at 0-20 to constitute a collection of samples, the second was for pH determination, and the third was for microbiological analyses. Temperature and pH determination during the composting process. Temperature inside the windrow was measured, every day during the composting period, with a special sensing device stuck introduced to 60 cm depth in randomly selected points. For pH, 400 g of municipal solid waste (MSW) or compost were placed in an Erlenmeyer flask containing 2 l of distilled water and stirred for 3-5 min. The mixture was allowed to settle for 5 min and the pH was measured using a pH meter. For dry weight, 400 g of fresh MSW was dried at 105 °C until the weight remained stable. Microbiological assays. Preparation of compost suspension. A 50 g aliquot of fresh waste or compost, from the 1 kg sample, was taken and homogenised with 950 ml sterile distilled water for 2 h under mechanical shaking (Edmund Buhler shaker type Kl-2, Germany) as reported by Sikora et al. (1983) and Hachicha et al. (1993). Shaking was to remove the maximum of micro-organisms from their organomineral substrates. The suspensions were used for staphylococcal counts. Staphylococci counts and identification. In our study we investigated only the staphylococcal community during the composting process. Staphylococci were counted by the plate pouring technique on Baird-Parker agar as reported previously (Parente
O. Bouzaiane et al.
et al., 2001). Briefly, from the prepared suspension a 0.5 ml volume was poured and spread out on Baird-Parker agar and incubated at 37 °C during 48 h. Since other genus from the family Micrococcaceae, such as Kocuria, can grow on Baird-Parker agar, only colonies with typical staphylococcal morphology were considered: convex brilliant, black colonies encircled by a clear halo. Each colony was purified on Mueller-Hinton agar, incubated at 37 °C for 24 h, followed by Gram stain, catalase activity and coagulase test in coagulase mannitol broth supplemented with freeze-dried rabbit plasma (Diagnostic Pasteur, Paris). Results were expressed as the number of colony-forming units (UFC) per gram of compost or waste. Identification of selected isolates to the species level was determined by studying carbohydrates fermentation and enzymatic activities using biochemical tests in API Staph (bioMérieux, France). Biochemical profiles (API patterns) of the isolated were compared and strains were classified according to their profiles. Genotypic investigations. The study was carried out with a total of 100 isolates of Staphylococcus. They were collected from the different steps of composting of municipal solid waste and from compost. Staphylococcus aureus ATCC 25923 and S. epidermidis CCM 2124 were used as controls. ARDRA method. Preparation of crude cell extracts was carried out by the phenol-chlorophorm method as previously reported (Sambrook et al., 1989). Synthetic oligonucleotide universal primers 16S-1492 R (5’-tacgg (ct) taccttgttacgactt-3’) and 16S-27 F (5’agagtttgatc (ac) tg gctcag-3’) were used to amplify the 16 S rDNA. PCR products were purified to eliminate factors that might inhibit enzymatic digestion. Five endonucleases were used in this study: AluI, HaeIII, TaqI, RsaI, and MspI (Promega, France). A 8-µl sample of purified PCR product, 2-µl of buffer, 0.25-µl of BSA (100 µg/ml of BSA), and 1 U of each enzyme were combined in one reaction tube (20 µl as final solution adjusted with sterile distiller water) and incubated at least 4 h as recommended by the supplier (Promega). Digested PCR products were electrophoresed through 6% polyacrylamide gel. Then, gels were stained with ethidium bromide and photographed. ITS-PCR method. PCR analysis of 16S-23S intergenic spacer region length (ITS-PCR) was performed according to the method of Mendoza et al. (1998). Amplification was modified as follow: after an initial step of denaturation during 5 min at 94 °C, 40 amplification cycles consisting of: denaturation at 94 °C for 30 s, annealing at 45 °C for 30 s, and elongation at 72 °C for 45 s; the last cycle was followed by a 7 min at 72 °C for final elongation. The PCR amplification patterns were electrophoresed on standard 2.5% agarose gels in 0.5x Tris-borate-EDTA buffer and stained for 30 min in 0.5 mg/l solution of ethidium bromide. Separation in 6% polyacrylamide gels was performed in 1x Tris-borate-EDTA buffer for 10 to 14 h at 100 V. After electrophoresis, gels were stained with ethidium bromide and photographed. The various profiles obtained by ARDRA as well as those obtained for the ITS-PCR were treated by the software “gel Pro”. Profiles were analysed by the MVSP software (Kovach Computing Service, http://www.kovcomp.com). PFGE method. The protocol for the preparation of chromosomal DNA was modified from that described by Tammy et al. (1995). Cells were grown overnight in 5 ml of Brain Heart Infusion broth at 37 °C. Cells from 1 ml of each culture were collected by centrifugation and washed in 2 ml of TE buffer (100 mM Tris-HCl, 100 mM EDTA; pH 8) at 8000 x g for 5 min at 4 °C. Cells were re-
Ann. Microbiol., 58 (3), 387-394 (2008)
Humidity (HR %) Organic matter (OM %)
60
Number of staphylococcus/g of compost
6
Temperature
70
Temperature
Digestion
Maturation
80
5 4
50 40
3
30
HR, MO (%)
Digestion
389
2
20 1
10 0
5
9
20
34
48
62
76
90
111
125
139
0
Maturation
3,5 10 7 3,0 10 7 2,5 10 7 2,0 107 1,5 107 1,0 107 5 106 0
9
5
20
34
Time (day)
48
62
76
90
111
125
139
Time (day)
FIG. 1 - Progress of temperature, humidity and organic matter during composting process.
FIG. 2 - Evolution of staphylococci composting process.
species
suspended in 200 µl of EC buffer (6 mM Tris-HCl, 1 M NaCl, 100 mM EDTA, 0.5% Brij 58 (Sigma), 0.2% sodium desoxycholate (Difco Laboratories), 0.5% sodium N-lauroyl sarcosine (USB); pH 7.5) containing 100 µg/ml of lysostaphin and 20 µg/ml of RNase (Sigma). This suspension was mixed with an equal volume of 2% low-melting-point agarose (Gibco, France) at 55 °C. The mixture was transferred into sample plug molds (Bio-Rad Laboratories, Richmond, Calif) and refrigerated for 20 min. Sample plugs were incubated overnight at 37 °C in 1 ml of lysis buffer EC, followed by a further overnight incubation at 55 °C in proteolysis buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.5) containing 200 µg/ml of proteinase K. The plugs were washed three times (30 min each) in 10 ml TE buffer (10 mM Tris-HCl, 1 mM EDTA; pH 8), with gentile shaking at room temperature and stored in TE buffer at 4 °C until further analysis is done. One-quarter of a sample plug was placed in a microcentrifuge tube containing 175 µl of sterilised deionised water, 20 µl of buffer J, 100 µg/ml of BSA, and 30 U of SmaI (Promega, France), and the mixture was incubated overnight at 25 °C. The plug was then incubated in 1 ml of TE buffer at 4 °C for 1 h. Sample plugs were loaded in a 1% agarose gel in 0.5X TBE buffer. Electrophoresis was performed with the CHEF-DRIII apparatus (Bio-Rad) using the following conditions: pulse times ranged from 5 s to 40 s during 22 h at 6.0 V/ cm at 14 °C. Lambda DNA PFGE Marker (Amersham Pharmacia
Biotech, Piscataway, NJ) was used as size standards in the first lane on each gel. Gels were stained with a solution of ethidium bromide (0.5 µg/ml) and digitalized with UVItec apparatus equipment. DNA banding patterns were compared visually using the criteria of Tenover et al. (1995).
Results Physico-chemical parameters of composting process In this study the temperature progress vary according the two phases of composting process, digestion and maturation (Fig. 1). The phase of digestion start with a mesophilic phase in which the temperature reached 42 °C. During this mesophilic step, the humidity rate was up to 45%. After 20 days of composting, the temperature reached 65 °C and the thermophilic step started. In this step the humidity decreased significantly. Then, the temperature decreased gradually to reach 40 °C. At the 62 days, and after the addition of sawdust and green wastes in order to enhance the microbial activity, the maturation phase took place. In this phase, like in the digestion phase, the temperature increased gradually to reach 50 °C, stabilised for a short period then decreased. In this phase there was also mesophilic and thermophilic steps.
Table 1 - Staphylococci composition during composting cycle Stage of composting
during
Sampling
Time (day)
Digestion phase
E1 E2 E3 E4 E5 E6 E7
5 9 20 34 48 62 76
Identification using API Staph S. S. S. S. S. S. S.
lentus, lentus, lentus, lentus, lentus, lentus, lentus,
Maturation phase
E8
90
E9
111
E10
125
E11
139
S. S. S. S. S. S. S. S.
lentus, S. xylosus, S. hominis, lugdunensis lentus, S. xylosus, S. hominis, sciuri, S. haemolyticus lentus, S. xylosus, S. hominis sciuri, S. haemolyticus, S. warneri lentus, S. xylosus, S. hominis, sciuri, S. haemolyticus, S. warneri
S. S. S. S. S. S. S.
xylosus, xylosus, xylosus, xylosus, xylosus, xylosus, xylosus,
S. S. S. S. S. S. S.
hominis hominis hominis hominis hominis hominis hominis
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Enumeration and isolation of staphylococci The number and distribution of staphylococci isolates during the different steps of composting are shown in Fig. 2. During the mesophilic step of the digestion phase the number of staphylococci increased to reach 3 x 107 CFU/g dry matter, however, during the thermophilic phase it decreased significantly. This decrease continued until the sixth week of the process. During the maturation phase and after application of sawdust, the number of staphylococci re-increased to reach 2 x 107 CFU/g of dry matter during the thermophilic step, and re-decreased at the end of the process near 0.5 x 105 CFU/g of dry matter.
FIG. 3 - Simplified dendrogram showing the relationship between the 100 staphylococci and 2 references strains obtained by analysis of phenotypical tests. The dendrogram is based on unweighted pair group method with arithmetic average (UPGMA) using the simple matching coefficient. The cut off level for clustering the strains is 78%. Each tie represents one biochemical profile. A: S. lentus; B: S. xylosus; C: S. haemolyticus; D: S. xylosus; E: S. xylosus, S. sciuri; F: S. xylosus; G: S. warneri; H: S. hominis, S. lugdunensis; I: S. xylosus. In parenthesis number of strains grouped in each clusters.
Species occurrence Among the staphylococcal strains, one hundred isolates were randomly chosen for further investigations. These isolates were identified to the species level by the API Staph. Species and numbers of strains in each step of composting cycle identification are listed in Table 1. It is noteworthy that the distribution of recovered species is phase-varying. Indeed, during the digestion phase three species were recovered: S. lentus, S. xylosus, and S. hominis. Whereas, in the maturation phase and in addition to the later three species, S. lugdunensis, S. haemolyticus, S. sciuri and S. warneri were also recovered (Table 1). Phenotypic profiles On the basis of biochemical tests in API Staph, it was possible to distinguish 28 biochemical profiles among 100
A
B
C
D
E
FIG. 4 - Polyacrylamide gel of 16S gene digestion products by AluI (A), TaqI (B), HaeIII (C), MspI (D) and RsaI (E) enzymes. M: molecular weight markers of 50 pb.
Ann. Microbiol., 58 (3), 387-394 (2008)
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102 x bp 20 10
5
1
S. xylosus (45) S. lentu s (32) S. hominis (9) S. sciuri (4) S. warneri (4) S. ha emolyticus (3)
S. lugdunensis (3)
FIG. 5 - ARDRA dendrogram showing the relationship between the Staphylococcus strains obtained from the digestion of 16S rDNA amplified products gene by HaeIII, TaqI, RsaI, AluI and MspI.
FIG. 6 - Schematic representation of PCR-amplified 16S23S rDNA spacer region from 100 Staphylococcus strains representing 7 species. The number of strains tested is given in parentheses.
strains. The biochemical tests that can differentiate strains were: D(+)-mannose, maltose, lactose, trehalose, xylitol, D(+)-melibiose, nitrate reduction, phosphatase, acetoin production, raffinose, D(+)-xylose, saccharose, methylα-D-glucopyranoside, N-acetylgycosamine, arginine hydrolysis, urease. Simplified dendrogram obtained by cluster analysis (Fig. 3) each tie represent one biochemical profile. Nine clusters were observed with 78% of similarity (A, B, C, D, E, F, G, H and I). Staphylococcus xylosus, have 10 biochemical profiles; S. lentus, have 11 biochemical profiles, S. haemolyticus have 3 biochemical profiles, S. hominis, S. lugdunensis, S. sciuri and S. warneri have each 1 biochemical profile.
patterns discriminating between strains. DNA digestion with AluI, classified S. xylosus and S. lentus strains each into 3 patterns. However, S. hominis, S. lugdunensis, S. haemolyticus, S. sciuri and S. warneri strains were not polymorphic since a specific unique pattern was observed for strains of each species. The dendrogamme of similarity obtained by the combination of the different profiles obtained by the five independent digestions showed 8 clusters (up to 80% of similarity). Interestingly, strains of the same species have been shown to belong to different clusters, similarly some clusters contained strains of different species (Fig. 5). During the digestion phase, the 14 S. xylosus strains were classified in 1 genomic haplotype A (Table 2). In maturation phase, the 31 S. xylosus strains showed 2 genomic haplotypes: 20 strains with haplotype A and 11 strains exhibited the haplotype B (Table 2). The ten S. lentus strains, found during the digestion phase presented 2 haplotypes, A1 (7 strains) and B1 (5 strains). In addition to latter haplotypes, two new types were found in 5 strains among the 20 strains isolated during maturation phase, named C1 and D1 (Table 2). The nine S. hominis strains isolated during all the composting process showed only one genomic haplotype A2 (Table 2).
ARDRA method DNAs were extracted and amplified with universal 16S rDNA primers producing a single band of similar size (approximately 1500 bp) for each strain. The five endonucleases used gave polymorphic profiles (Fig. 4). However, we observed that DNA digestion by MspI enzyme yielded a unique pattern in all staphylococcal strains (Fig. 4d). In addition, this was also observed with little exception after DNA digestion with HaeIII, TaqI, RsaI, and MspI were strains from different species have been shown as indistinguishable (Fig. 4b, 4c, and 4d). In contrast, DNA digestion with AluI (Fig. 4a) was able not only to efficiently digest the amplified products, but also gave restriction
ITS-PCR method PCR amplification of 16S-23S intergenic spacer region yielded 7 different patterns (Fig. 6). Indeed strains within
TABLE 2 - Staphylococci identification Phase
Species
ARDRA
PFGE
Digestion
S. S. S. S.
A (14) A1 (7); B1 (5) A2 (3) A (20); B (11)
A (3); B (4); C (4); D (3); E (2) A1 (2); B1 (2); C1 (3); D1 (2); E1 (3) A2 (1); B2 (1); C2 (1) A (7); B (6); C (5); D (5); E (4); F (4) A1 (4); B1 (3); C1 (3); D1 (2); E1 (2); F1 (2); G1 (2); H1 (2) A2 (2); B2 (2); C2 (1); D2 (1) A3 (3) A4 (4) A5 (3) A6 (4)
Maturation
xylosus (14) lentus (12) hominis (3) xylosus (31)
S. lentus (20)
A1 (9); B1 (6); C1 (3); D1 (2)
S. S. S. S. S.
A2 A3 A4 A5 A6
hominis (6) lugdunensis (3) warneri (4) haemolyticus (3) sciuri (4)
( ): strains number; A, B, C, etc.: haplotypes.
(6) (3) (4) (3) (4)
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O. Bouzaiane et al.
A
B
M 1 2 3 4 5 6 7 8
M 1 2 3 4 5 6
C M 1
2
3
4
FIG. 7 - PFGE patterns of S. lentus (A), S. xylosus (B) and S. hominis (C). M: molecular size standard (lambda oligomers). each species presented a specific PCR pattern made of 3-8 fragments whose sizes ranged from 300 to 1500 bp. PCR products ranged in sizes from 400 to 1500 bp for S. warneri, 350 to 1000 bp for S. xylosus. Staphylococcus lugduensis showed special pattern with three close DNA fragments with sizes ranging from approximately 630 to 800 bp. Amplification of the 16S-23S spacer region of Staphylococcus species does not produce any common fragment that can be assigned as genus specific. PFGE method In the digestion phase S. xylosus strains showed five PFGE patterns, and S. lentus strains were also classified into five genotypes. The three strains of S. hominis were unrelated and exhibited each a unique PFGE pattern (Fig. 7, Table 2). However, in the maturation phase the of S. xylosus strains fell into six PFGE 6 patterns, and strains of S. lentus and S. hominis felled into eight and four PFGE patterns, respectively. It is worth to note that the related strains of S. xylosus, S. lentus, and S. hominis observed during the digestion phase were also observed during the maturation phase. A unique PFGE pattern was observed among strains belonging to the remaining species, S. lugdunensis, S. haemolyticus, S. sciuri and S. warneri.
Discussion Composting is a self-heating, aerobic, solid phase, biodegradative process of organic waste materials. The composting process at the microbial level involves several interrelated factors, namely temperature, ventilation (O2
imputed), moisture content and available nutrients. Based on temperature, the process of aerobic composting can be divided into three major steps, a mesophilic-heating step, a thermophilic step and a cooling step (Mustin, 1987). During the mesophilic step, the temperature and the water content increased as a consequence of biodegradation of organic compounds. The temperature increment is the consequence of the organic matter oxidation (Hassen et al., 2001). The mesophilic step is followed by the thermophilic step. The latter step occurred between days 20 and days 34 of the composting process. As mentioned by Hachicha et al. (1992) and Marrug et al. (1993), a temperature above 60 °C seriously affect the decomposition rate of the organic waste as a result of a reduction in microbiological activity. The temperature started to decrease after day 111, this decrease led to the depletion of organic matters and the carbon/azotes (C/N) ratio tended to stabilise. By the end of the composting process, the average temperature inside the windrow showed a decrement and reached approximately 30 °C at the end of the process (Ben Ayed et al., 2007). Staphylococci are ubiquitous bacteria and S. aureus is one of the main causes of collective toxic infections of food. In addition, it also generates cutaneous infections that represent a risk for compost handlers and agriculturists during farm compost spreading. Therefore the occurrence of this species at the end product of composting represents a real threat of public health. In our study, we found that the total number of staphylococci decreased from 3 x 107 CFU/g of dry matter at the mesophilic phase of digestion step to 0.5 x 105 CFU/g dry matter at the end of the maturation step. This means that the composting treatment can not eliminate these bacteria and that Staphylococcus
Ann. Microbiol., 58 (3), 387-394 (2008)
species support hostile conditions such as high temperature and reduction of nutriments. During the digestion, the colonies that have taken at random and identified by API Staph, belong to three species: S. xylosus, S. lentus, and S. hominis. However, at the maturation phase and after the addition of sawdust and green wastes there was appearance of other species: S. lugdunensis, S. haemolyticus, S. sciuri and S. warneri. It seems that the three species recovered in the digestion step are the most staphylococcal species in solid waste, and the other four species recovered during the maturation phase were added with the sawdust. Alternatively, the application of sawdust change the condition of the composting cycle and permit the growth of new staphylococci species that were present in the first phase but non culturable. Interestingly, S. epidermidis and S. aureus have not been detected in any phase. These results were in concordance with those reported by Hassen et al., 2001. The detected species are rarely implicated in human infections, in contrast to S. epidermidis and S. aureus. Dendrogram constricted on the basis of biochemical profiles exhibited by staphylococcal isolates showed that strains belonging to S. xylosus, S. lentus and S. hominis demonstrated the highest variability. Interestingly, these species were found along the composting process. Therefore, it seems that these species have an intrinsic ability of adaptation in hostile conditions. Modulation of metabolic pathways according to environmental conditions could have enhanced the survival ability of strains belonging to these species. ITS-PCR based strategy for studying the length polymorphisms of the 16S-23S rDNA spacer regions has been proved useful for both epidemiological and taxonomic investigations (Gürtler et al., 1993; Jensen et al., 1993). In our study, we found that the ITS-PCR profiles were specific for each of the Staphylococcus species examined. These results were in agreement with the biochemical and phenotypical methods previously used to identify these strains as reported by Freney et al. (1988). The ITS-PCR method did not allow detection of intraspecies polymorphism among species. Therefore, this technique could be used for the confirmation of the biochemical identification. The ARDRA analysis by five restriction enzymes (HaeIII, TaqI, RsaI, AluI and MspI) allowed the identification of 8 clusters. AluI was shown to be the best enzyme that yields distinct fragments; this enzyme permitted the detection of the highest diversity among strains. Many authors have reported a high diversity of staphylococcal strains using ARDRA methods either in environmental or clinical sets (Hoppe-Seylar et al., 2004). It is worthy to note that this technique is not able to differentiate between strains of different species. Indeed, same strains of different species had indistinguishable pattern, and contrarily, strains belonging to the same species had different patterns. The small number of identified ARDRA-generated clusters could be attributed in part, to the conserved property of the sequences encoding 16S rRNA in the genome of staphylococci. Nevertheless, we can not exclude the pressure selection during the composting process permitting the persistence of some clones. DNA digestion by SmaI enzyme, gave DNA patterns of 10 to 15 bands with sizes ranging from 10 to 700 kb. PFGE method showed that strains isolated during the phase of digestion persist during the phase of maturation. These results support the low diversity in 16S rDNA and the high genomic variability in non vital genomic DNA sequences (cut sites of SmaI enzyme) revealed by PFGE (Hoppe-Seyler et al., 2004). It is very interesting to note that in our case the PFGE method is not used only, as in clinical sets, for molecular typing but also to asses the genetic variability of strains under the composting process. In this view
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and similar to other bacteria submitted to hostile conditions (Ben Kahla-Nakbi et al., 2008) the genome seemed to be invariable under stressing conditions. The adaptation response to these hard conditions might touch other molecular mechanisms such as the sigma factor (Van Schaik and Abee, 2005). In conclusion, during composting process of municipal wastes we found seven species of Staphylococcus. The human pathogens S. aureus and S. epidermidis species were not found. The ITS-PCR method was able to yield a specific pattern for each species and confirm biochemical identification. ARDRA did not give discrimination between strains belonging to different species; therefore it could not be used as molecular method to assess staphylococcal communities in the compost. The PFGE has allowed the detection of persistent clones during the composting process. Acknowledgements The present study is a part of the 1999-2002 research programs “Municipal Solid Waste Treatment and Compost Agricultural Re-use” which is supported by the Tunisian state secretariat of Scientific Research and Technology.
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