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Molecular typing of staphylococcal communities isolated during municipal solid waste composting process

Molecular typing of staphylococcal communities isolated during municipal solid waste composting... Annals of Microbiology, 58 (3) 387-394 (2008) Molecular typing of staphylococcal communities isolated during municipal solid waste composting process 1 2 1 1 1 2 Olfa BOUZAIANE *, Mohamed Salah ABBASSI , Maher GTARI , Ons BELHAJ , Naceur JEDIDI , Assia BEN HASSEN , Abdennaceur HASSEN Institut National de Recherche Scientifique et Technique, Laboratoire de Traitement et recyclage, B.P. 95 - 2050, Hammam Lif, Tunisie; Services des Laboratoires, Centre National de Greffe de Moelle Osseuse, Tunis, Tunisie Received 15 February 2008 /Accepted 16 June 2008 Abstract - This study investigates the molecular typing occurrence of staphylococcal communities during composting process of munic- ipal 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 proc- ess 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 compo- sition 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 clinical sets as a causative agents of several invasive infections (Kloos and Bannerman, 1999). Staphylococcus saprophyticus is believed to cause opportun- Composting transforms organic matter into a stable product consisting of humus-like substances. The end product is avail- istic infections in the female urinary tract (UTI) (Marrie et al., able for agricultural use. The composting process is used to treat 1982). Staphylococcus haemolyticus and Staphylococcus epider- yard waste, manure, sewage sludge and also municipal solid midis the second most frequently encountered species have been waste. Pathogens microorganisms found in municipal solid waste reported to cause myocarditis, sepsis, peritonitis, and UTI (John can be viruses, bacteria and protozoa or helminths (Strauch, et al., 1978). Staphylococcus hominis, Staphylococcus warneri, 1991). As they are heat-sensitive, the temperature increment Staphylococcus capitis, Staphylococcus simulans, Staphylococcus during the composting process should eliminate them, leading cohnii, Staphylococcus xylosus, Staphylococcus saccharophyticus microbial pathogen-free end product, what is defined as disinfec- have been associated, less frequently, with various infections tion or sanitisation. Several parameters such as humidity and (Martin et al., 1989; Westblom et al., 1990; Kamath et al., oxygen have an influence on the heat increase. For this reason, 1992). Coagulase-negative staphylococci are the most common bac- sanitisation efficiency depends on the composting method used (Pereira-Neto et al., 1987). Therefore, there are many microbes teria isolated from the compost of municipal solid waste (Hassen et al., 2001). The emergence of CNS as human pathogens and that escape and resist to the heat treatment in composting process (Hassen et al., 2001). Among these microbes we notice reservoirs of antimicrobial resistance determinants requires their spores or bacteria that are either spore forming (Clostridium, rapid and reliable identification in order to have an early predic- Bacillus spp.) or very resistant to desiccation like Staphylococcus. tion of the potential pathogenicity or antibiotic susceptibility of Staphylococci are among the most wide spread bacteria and are each isolate (Kloos and Bannerman, 1999; Lina et al., 2000). ubiquitous in the environment. In addition, they are well known In recent years, several manual and automated methods for nosocomial pathogens associated with multiple antimicrobial identification of staphylococci have been described, but they are resistance mechanisms. time consumer (at least 30 h) as they require microbial growth. For many years Staphylococcus aureus was the only species Furthermore, commercially available panels, which are based on recognised as an important human pathogen, however, recently functional differences in metabolic pathways, do not allow a reli- coagulase-negative staphylococci (CNS) have been emerged in able 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 restric- * Corresponding author. Phone: 21671788436; Fax: 21671410740; E-mail: olfa_bz2004@yahoo.fr tion analysis (ARDRA) and ribosome spacer PCR or internal tran- 388 O. Bouzaiane et al. scribed spacer-PCR (ITS-PCR) was tested by several authors for et al., 2001). Briefly, from the prepared suspension a 0.5 ml the identification of staphylococci from different origins (Mendoza volume was poured and spread out on Baird-Parker agar and et al., 1998; Bes et al., 2000). incubated at 37 °C during 48 h. Since other genus from the fam- The aims of this present study were: (i) to identify the sta- ily Micrococcaceae, such as Kocuria, can grow on Baird-Parker phylococcal communities during composting process, and (ii) to agar, only colonies with typical staphylococcal morphology were characterise them by ARDRA, ITS-PCR, and PFGE methods in considered: convex brilliant, black colonies encircled by a clear order to assess suitable method(s) of molecular typing. 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 MATERIALS AND METHODS freeze-dried rabbit plasma (Diagnostic Pasteur, Paris). Results were expressed as the number of colony-forming units (UFC) per Composting process. The study was carried out in the experi- gram of compost or waste. mental composting plant of Beja City, north east of Tunisia; Identification of selected isolates to the species level was during the period between June and October 2003. In each year determined by studying carbohydrates fermentation and enzy- 1000 tons of solid waste, were treated in this plant. Municipal matic activities using biochemical tests in API Staph (bioMérieux, solid wastes collected from various sites of the Beja City and France). Biochemical profiles (API patterns) of the isolated were forwarded through two series of windrows with a pyramidal compared and strains were classified according to their profiles. 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 Genotypic investigations. The study was carried out with a refined by mechanical sorting of the inert matter (mostly plastics total of 100 isolates of Staphylococcus. They were collected from and glasses). Finally, 65 days of composting, sawdust and green the different steps of composting of municipal solid waste and wastes were added to enhance the microbial activity of digestion from compost. Staphylococcus aureus ATCC 25923 and S. epi- phase and to enhance biodegradation under optimal conditions. dermidis CCM 2124 were used as controls. Temperature and humidity were controlled daily, and windrow was turned and watered (humidity regulatory adjusted to 50%) ARDRA method. Preparation of crude cell extracts was carried as soon as the inner temperature of the pile reached or exceed out by the phenol-chlorophorm method as previously reported 65 °C. These operations of turning and watering were performed (Sambrook et al., 1989). Synthetic oligonucleotide universal almost twice per month on average. primers 16S-1492 R (5’-TACGG (CT) TACCTTGTTACGACTT-3’) and 16S-27 F (5’AGAGTTTGATC (AC) TG GCTCAG-3’) were used to Sampling of organic wastes during the composting cycle. amplify the 16 S rDNA. PCR products were purified to eliminate Ten samples (approximately 5 kg) were collected every 15 days factors that might inhibit enzymatic digestion. Five endonucle- from 5 to 139 days of composting from ten randomly selected ases were used in this study: AluI, HaeIII, TaqI, RsaI, and MspI locations in the windrow by digging a small pit to 10 m depth with (Promega, France). A 8-μl sample of purified PCR product, 2-μl a shovel. In each sampling, samples were mixed thoroughly and of buffer, 0.25-μl of BSA (100 μg/ml of BSA), and 1 U of each three portions of 1 kg each were separated. The first portion was enzyme were combined in one reaction tube (20 μl as final solu- stored at 0-20 to constitute a collection of samples, the second tion adjusted with sterile distiller water) and incubated at least 4 h was for pH determination, and the third was for microbiological as recommended by the supplier (Promega). Digested PCR prod- analyses. ucts were electrophoresed through 6% polyacrylamide gel. Then, gels were stained with ethidium bromide and photographed. Temperature and pH determination during the compost- ing process. Temperature inside the windrow was measured, ITS-PCR method. PCR analysis of 16S-23S intergenic spacer every day during the composting period, with a special sensing region length (ITS-PCR) was performed according to the method device stuck introduced to 60 cm depth in randomly selected of Mendoza et al. (1998). Amplification was modified as follow: points. For pH, 400 g of municipal solid waste (MSW) or compost after an initial step of denaturation during 5 min at 94 °C, 40 were placed in an Erlenmeyer flask containing 2 l of distilled amplification cycles consisting of: denaturation at 94 °C for 30 s, water and stirred for 3-5 min. The mixture was allowed to settle annealing at 45 °C for 30 s, and elongation at 72 °C for 45 s; the for 5 min and the pH was measured using a pH meter. For dry last cycle was followed by a 7 min at 72 °C for final elongation. weight, 400 g of fresh MSW was dried at 105 °C until the weight The PCR amplification patterns were electrophoresed on standard remained stable. 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 Microbiological assays. in 6% polyacrylamide gels was performed in 1X Tris-borate-EDTA Preparation of compost suspension. A 50 g aliquot of fresh waste buffer for 10 to 14 h at 100 V. After electrophoresis, gels were or compost, from the 1 kg sample, was taken and homogenised stained with ethidium bromide and photographed. with 950 ml sterile distilled water for 2 h under mechanical shak- The various profiles obtained by ARDRA as well as those ing (Edmund Buhler shaker type Kl-2, Germany) as reported by obtained for the ITS-PCR were treated by the software “gel Sikora et al. (1983) and Hachicha et al. (1993). Shaking was to Pro”. Profiles were analysed by the MVSP software (Kovach remove the maximum of micro-organisms from their organo- Computing Service, http://www.kovcomp.com). mineral substrates. The suspensions were used for staphylococ- cal counts. PFGE method. The protocol for the preparation of chromosomal DNA was modified from that described by Tammy et al. (1995). Staphylococci counts and identification. In our study we inves- Cells were grown overnight in 5 ml of Brain Heart Infusion broth tigated only the staphylococcal community during the compost- at 37 °C. Cells from 1 ml of each culture were collected by cen- ing process. Staphylococci were counted by the plate pouring trifugation and washed in 2 ml of TE buffer (100 mM Tris-HCl, technique on Baird-Parker agar as reported previously (Parente 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) 389 Digestion Maturation Digestion Maturation 80 6 3,5 Temperature 70 Humidity (HR %) 3,0 Organic matter (OM %) 2,5 2,0 40 3 1,5 1,0 0 0 5 9 76 20 34 48 62 90 111 125 139 9 20 34 48 62 76 90 111 125 139 Time (day) Time (day) FIG. 1 - Progress of temperature, humidity and organic FIG. 2 - Evolution of staphylococci species during matter during composting process. composting process. suspended in 200 μl of EC buffer (6 mM Tris-HCl, 1 M NaCl, 100 Biotech, Piscataway, NJ) was used as size standards in the first mM EDTA, 0.5% Brij 58 (Sigma), 0.2% sodium desoxycholate lane on each gel. Gels were stained with a solution of ethidium (Difco Laboratories), 0.5% sodium N-lauroyl sarcosine (USB); pH bromide (0.5 μg/ml) and digitalized with UVItec apparatus equip- 7.5) containing 100 μg/ml of lysostaphin and 20 μg/ml of RNase ment. DNA banding patterns were compared visually using the (Sigma). This suspension was mixed with an equal volume of 2% criteria of Tenover et al. (1995). 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 RESULTS incubated overnight at 37 °C in 1 ml of lysis buffer EC, followed by a further overnight incubation at 55 °C in proteolysis buffer Physico-chemical parameters of composting process (10 mM Tris-HCl, 1 mM EDTA, pH 8.5) containing 200 μg/ml of In this study the temperature progress vary according the two proteinase K. The plugs were washed three times (30 min each) phases of composting process, digestion and maturation (Fig. 1). in 10 ml TE buffer (10 mM Tris-HCl, 1 mM EDTA; pH 8), with The phase of digestion start with a mesophilic phase in which gentile shaking at room temperature and stored in TE buffer at the temperature reached 42 °C. During this mesophilic step, 4 °C until further analysis is done. One-quarter of a sample plug the humidity rate was up to 45%. After 20 days of compost- was placed in a microcentrifuge tube containing 175 μl of steri- ing, the temperature reached 65 °C and the thermophilic step lised deionised water, 20 μl of buffer J, 100 μg/ml of BSA, and started. In this step the humidity decreased significantly. Then, 30 U of SmaI (Promega, France), and the mixture was incubated the temperature decreased gradually to reach 40 °C. At the 62 overnight at 25 °C. The plug was then incubated in 1 ml of TE days, and after the addition of sawdust and green wastes in order buffer at 4 °C for 1 h. Sample plugs were loaded in a 1% agar- to enhance the microbial activity, the maturation phase took ose gel in 0.5X TBE buffer. Electrophoresis was performed with place. In this phase, like in the digestion phase, the temperature the CHEF-DRIII apparatus (Bio-Rad) using the following condi- increased gradually to reach 50 °C, stabilised for a short period tions: pulse times ranged from 5 s to 40 s during 22 h at 6.0 V/ then decreased. In this phase there was also mesophilic and cm at 14 °C. Lambda DNA PFGE Marker (Amersham Pharmacia thermophilic steps. TABLE 1 - Staphylococci composition during composting cycle Stage of composting Sampling Time (day) Identification using API Staph Digestion phase E1 5 S. lentus, S. xylosus, S. hominis E2 9 S. lentus, S. xylosus, S. hominis E3 20 S. lentus, S. xylosus, S. hominis E4 34 S. lentus, S. xylosus, S. hominis E5 48 S. lentus, S. xylosus, S. hominis E6 62 S. lentus, S. xylosus, S. hominis E7 76 S. lentus, S. xylosus, S. hominis Maturation phase E8 90 S. lentus, S. xylosus, S. hominis, S. lugdunensis E9 111 S. lentus, S. xylosus, S. hominis, S. sciuri, S. haemolyticus E10 125 S. lentus, S. xylosus, S. hominis S. sciuri, S. haemolyticus, S. warneri E11 139 S. lentus, S. xylosus, S. hominis, S. sciuri, S. haemolyticus, S. warneri Temperature HR, MO (%) Number of staphylococcus/g of compost 390 O. Bouzaiane et al. 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 staphyloco- cci increased to reach 3 x 10 CFU/g dry matter, however, during the thermophilic phase it decreased significantly. This decrease continued until the sixth week of the process. During the matu- ration phase and after application of sawdust, the number of staphylococci re-increased to reach 2 x 10 CFU/g of dry matter during the thermophilic step, and re-decreased at the end of the process near 0.5 x 10 CFU/g of dry matter. 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 identifica- FIG. 3 - Simplified dendrogram showing the relationship tion are listed in Table 1. It is noteworthy that the distribution of between the 100 staphylococci and 2 references recovered species is phase-varying. Indeed, during the digestion strains obtained by analysis of phenotypical tests. The dendrogram is based on unweighted pair group phase three species were recovered: S. lentus, S. xylosus, and method with arithmetic average (UPGMA) using the S. hominis. Whereas, in the maturation phase and in addition to simple matching coefficient. The cut off level for the later three species, S. lugdunensis, S. haemolyticus, S. sciuri clustering the strains is 78%. Each tie represents and S. warneri were also recovered (Table 1). one biochemical profile. A: S. lentus; B: S. xylosus; C: S. haemolyticus; D: S. xylosus; E: S. xylosus, S. Phenotypic profiles sciuri; F: S. xylosus; G: S. warneri; H: S. hominis, S. lugdunensis; I: S. xylosus. In parenthesis num- On the basis of biochemical tests in API Staph, it was ber of strains grouped in each clusters. possible to distinguish 28 biochemical profiles among 100 A B C D 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) 391 10 x bp 20 10 5 1 S. xylosus (4 5) S. le ntu s (32) S. ho mi n is (9) S. sciuri (4) S. warneri (4) S. ha e mo lyticus (3) S. lu gdunensi s (3) FIG. 5 - ARDRA dendrogram showing the relationship be- FIG. 6 - Schematic representation of PCR-amplified 16S- tween the Staphylococcus strains obtained from the 23S rDNA spacer region from 100 Staphylococ- digestion of 16S rDNA amplified products gene by cus strains representing 7 species. The number of HaeIII, TaqI, RsaI, AluI and MspI. strains tested is given in parentheses. strains. The biochemical tests that can differentiate strains patterns discriminating between strains. DNA digestion were: D(+)-mannose, maltose, lactose, trehalose, xylitol, with AluI, classified S. xylosus and S. lentus strains each D(+)-melibiose, nitrate reduction, phosphatase, acetoin into 3 patterns. However, S. hominis, S. lugdunensis, S. production, raffinose, D(+)-xylose, saccharose, methyl- haemolyticus, S. sciuri and S. warneri strains were not A -D-glucopyranoside, N-acetylgycosamine, arginine polymorphic since a specific unique pattern was observed hydrolysis, urease. Simplified dendrogram obtained by for strains of each species. cluster analysis (Fig. 3) each tie represent one biochemical The dendrogamme of similarity obtained by the profile. Nine clusters were observed with 78% of similarity combination of the different profiles obtained by the five (A, B, C, D, E, F, G, H and I). Staphylococcus xylosus, have independent digestions showed 8 clusters (up to 80% of 10 biochemical profiles; S. lentus, have 11 biochemical similarity). Interestingly, strains of the same species have profiles, S. haemolyticus have 3 biochemical profiles, S. been shown to belong to different clusters, similarly some hominis, S. lugdunensis, S. sciuri and S. warneri have each clusters contained strains of different species (Fig. 5). 1 biochemical profile. During the digestion phase, the 14 S. xylosus strains were classified in 1 genomic haplotype A (Table 2). In maturation ARDRA method phase, the 31 S. xylosus strains showed 2 genomic haplotypes: DNAs were extracted and amplified with universal 20 strains with haplotype A and 11 strains exhibited the hap- 16S rDNA primers producing a single band of similar lotype B (Table 2). The ten S. lentus strains, found during the size (approximately 1500 bp) for each strain. The five digestion phase presented 2 haplotypes, A1 (7 strains) and B1 endonucleases used gave polymorphic profiles (Fig. 4). (5 strains). In addition to latter haplotypes, two new types were However, we observed that DNA digestion by MspI enzyme found in 5 strains among the 20 strains isolated during matura- yielded a unique pattern in all staphylococcal strains tion phase, named C1 and D1 (Table 2). The nine S. hominis (Fig. 4d). In addition, this was also observed with little strains isolated during all the composting process showed only exception after DNA digestion with HaeIII, TaqI, RsaI, and one genomic haplotype A2 (Table 2). MspI were strains from different species have been shown as indistinguishable (Fig. 4b, 4c, and 4d). In contrast, DNA ITS-PCR method digestion with AluI (Fig. 4a) was able not only to efficiently PCR amplification of 16S-23S intergenic spacer region digest the amplified products, but also gave restriction yielded 7 different patterns (Fig. 6). Indeed strains within TABLE 2 - Staphylococci identification Phase Species ARDRA PFGE Digestion S. xylosus (14) A (14) A (3); B (4); C (4); D (3); E (2) S. lentus (12) A1 (7); B1 (5) A1 (2); B1 (2); C1 (3); D1 (2); E1 (3) S. hominis (3) A2 (3) A2 (1); B2 (1); C2 (1) Maturation S. xylosus (31) A (20); B (11) A (7); B (6); C (5); D (5); E (4); F (4) A1 (4); B1 (3); C1 (3); D1 (2); E1 (2); F1 (2); S. lentus (20) A1 (9); B1 (6); C1 (3); D1 (2) G1 (2); H1 (2) S. hominis (6) A2 (6) A2 (2); B2 (2); C2 (1); D2 (1) S. lugdunensis (3) A3 (3) A3 (3) S. warneri (4) A4 (4) A4 (4) S. haemolyticus (3) A5 (3) A5 (3) S. sciuri (4) A6 (4) A6 (4) ( ): strains number; A, B, C, etc.: haplotypes. 392 O. Bouzaiane et al. AB C M 1 2 3 4 5 6 7 8 M 1 2 3 4 5 6 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 imputed), moisture content and available nutrients. Based 3-8 fragments whose sizes ranged from 300 to 1500 bp. on temperature, the process of aerobic composting can be PCR products ranged in sizes from 400 to 1500 bp for S. divided into three major steps, a mesophilic-heating step, warneri, 350 to 1000 bp for S. xylosus. Staphylococcus a thermophilic step and a cooling step (Mustin, 1987). lugduensis showed special pattern with three close DNA During the mesophilic step, the temperature and the water fragments with sizes ranging from approximately 630 to content increased as a consequence of biodegradation of 800 bp. Amplification of the 16S-23S spacer region of organic compounds. The temperature increment is the Staphylococcus species does not produce any common consequence of the organic matter oxidation (Hassen et al., fragment that can be assigned as genus specific. 2001). The mesophilic step is followed by the thermophilic step. The latter step occurred between days 20 and days PFGE method 34 of the composting process. As mentioned by Hachicha In the digestion phase S. xylosus strains showed five PFGE et al. (1992) and Marrug et al. (1993), a temperature patterns, and S. lentus strains were also classified into five above 60 °C seriously affect the decomposition rate of the genotypes. The three strains of S. hominis were unrelated organic waste as a result of a reduction in microbiological and exhibited each a unique PFGE pattern (Fig. 7, Table activity. The temperature started to decrease after day 2). However, in the maturation phase the of S. xylosus 111, this decrease led to the depletion of organic matters strains fell into six PFGE 6 patterns, and strains of S. lentus and the carbon/azotes (C/N) ratio tended to stabilise. and S. hominis felled into eight and four PFGE patterns, By the end of the composting process, the average respectively. It is worth to note that the related strains of temperature inside the windrow showed a decrement and S. xylosus, S. lentus, and S. hominis observed during the reached approximately 30 °C at the end of the process digestion phase were also observed during the maturation (Ben Ayed et al., 2007). phase. A unique PFGE pattern was observed among strains Staphylococci are ubiquitous bacteria and S. aureus is one belonging to the remaining species, S. lugdunensis, S. of the main causes of collective toxic infections of food. In addi- haemolyticus, S. sciuri and S. warneri. tion, 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 DISCUSSION product of composting represents a real threat of public health. In our study, we found that the total number of staphylococci Composting is a self-heating, aerobic, solid phase, decreased from 3 x 10 CFU/g of dry matter at the mesophilic biodegradative process of organic waste materials. The phase of digestion step to 0.5 x 10 CFU/g dry matter at the end composting process at the microbial level involves several of the maturation step. This means that the composting treat- interrelated factors, namely temperature, ventilation (O ment can not eliminate these bacteria and that Staphylococcus 2 Ann. Microbiol., 58 (3), 387-394 (2008) 393 species support hostile conditions such as high temperature and and similar to other bacteria submitted to hostile conditions (Ben reduction of nutriments. Kahla-Nakbi et al., 2008) the genome seemed to be invariable During the digestion, the colonies that have taken at random under stressing conditions. The adaptation response to these and identified by API Staph, belong to three species: S. xylosus, hard conditions might touch other molecular mechanisms such as S. lentus, and S. hominis. However, at the maturation phase the sigma factor (Van Schaik and Abee, 2005). and after the addition of sawdust and green wastes there was In conclusion, during composting process of municipal appearance of other species: S. lugdunensis, S. haemolyticus, S. wastes we found seven species of Staphylococcus. The human sciuri and S. warneri. It seems that the three species recovered pathogens S. aureus and S. epidermidis species were not found. in the digestion step are the most staphylococcal species in solid The ITS-PCR method was able to yield a specific pattern for waste, and the other four species recovered during the matura- each species and confirm biochemical identification. ARDRA did tion phase were added with the sawdust. Alternatively, the appli- not give discrimination between strains belonging to different cation of sawdust change the condition of the composting cycle species; therefore it could not be used as molecular method to and permit the growth of new staphylococci species that were assess staphylococcal communities in the compost. The PFGE has present in the first phase but non culturable. Interestingly, S. allowed the detection of persistent clones during the composting epidermidis and S. aureus have not been detected in any phase. process. These results were in concordance with those reported by Hassen et al., 2001. The detected species are rarely implicated in human Acknowledgements infections, in contrast to S. epidermidis and S. aureus. The present study is a part of the 1999-2002 research programs Dendrogram constricted on the basis of biochemical profiles “Municipal Solid Waste Treatment and Compost Agricultural exhibited by staphylococcal isolates showed that strains belong- Re-use” which is supported by the Tunisian state secretariat of ing to S. xylosus, S. lentus and S. hominis demonstrated the Scientific Research and Technology. highest variability. Interestingly, these species were found along the composting process. Therefore, it seems that these spe- cies have an intrinsic ability of adaptation in hostile conditions. REFERENCES Modulation of metabolic pathways according to environmental conditions could have enhanced the survival ability of strains Ben Ayed L., Hassen A., Jedidi N., SaidiI N., Bouzaiane O., Murano belonging to these species. F. (2007). Microbial C and N dynamics during compost- ITS-PCR based strategy for studying the length polymor- ing process of urban solid waste. Waste Manage. Res., 25: phisms of the 16S-23S rDNA spacer regions has been proved 24-29. useful for both epidemiological and taxonomic investigations (Gürtler et al., 1993; Jensen et al., 1993). In our study, we Ben Kahla-Nakbi A., Besbes A., Bakhrouf A. (2008). Survival found that the ITS-PCR profiles were specific for each of the of Vibrio fluvialis in seawater under starvation conditions. Staphylococcus species examined. These results were in agree- Microbiol. Res., 163: 323-328. ment with the biochemical and phenotypical methods previously Bes M., Guérin-Faublée V., Meugnier H., Etienne J., Freney J. used to identify these strains as reported by Freney et al. (1988). (2000). Improvement of the identification of staphylococci The ITS-PCR method did not allow detection of intraspecies isolated from bovine mammary infections using molecular polymorphism among species. Therefore, this technique could be methods. Vet. Microbiol., 71: 287-294. used for the confirmation of the biochemical identification. Coppola S., Mauriello G., Aponte M., Moschetti G., Villani F. The ARDRA analysis by five restriction enzymes (HaeIII, (2000). Microbial succession during ripening of Naples-type TaqI, RsaI, AluI and MspI) allowed the identification of 8 clusters. salami, a southern Italian fermented product. Meat Sci., 56: AluI was shown to be the best enzyme that yields distinct frag- 321-329. ments; this enzyme permitted the detection of the highest diver- sity among strains. Many authors have reported a high diversity Freney J., Brun Y., Bes M., Meugnier H., Grimont F., Grimont PAD., of staphylococcal strains using ARDRA methods either in environ- Nervi C., Fleurette J. (1988). Staphylococcus lugdunensis mental or clinical sets (Hoppe-Seylar et al., 2004). It is worthy sp. nov. and Staphylococcus schleiferi sp. nov. two species to note that this technique is not able to differentiate between from human clinical specimens. Int. J. Syst. Bacteriol., 38: strains of different species. Indeed, same strains of different spe- 168-172. cies had indistinguishable pattern, and contrarily, strains belong- Gürtler V. (1993). Typing of Clostridium difficile strains by ing to the same species had different patterns. The small number PCR-amplification of variable length 16S-23S rDNA spacer of identified ARDRA-generated clusters could be attributed in regions. J. Gen. Microbiol., 139: 3089-3097. part, to the conserved property of the sequences encoding 16S Hachicha R., Hassen A., Jedidi N., Kallali H. (1992). Optimal rRNA in the genome of staphylococci. Nevertheless, we can not conditions for municipal solid waste. Biocycle, 6: 76-77. exclude the pressure selection during the composting process permitting the persistence of some clones. Hachicha R., Jedidi N., Hassen A. (1993). Aspects hygiéniques DNA digestion by SmaI enzyme, gave DNA patterns of 10 de la fermentation aérobies des déchets urbains dans le to 15 bands with sizes ranging from 10 to 700 kb. PFGE method contexte tunisien. Arch. Inst. Pasteur Tunis, 70: 13-20. showed that strains isolated during the phase of digestion persist Hassen A., Belguith K., Jedidi N., Cherif A., Cherif M., Boudabbous during the phase of maturation. These results support the low A. (2001). Microbial characterization during composting of diversity in 16S rDNA and the high genomic variability in non vital municipal solid waste. Bioresource Technol., 80: 185-192. genomic DNA sequences (cut sites of SmaI enzyme) revealed Hoppe-Seyler T., Jaeger B., Bockelmann W., Noordman W., Geis by PFGE (Hoppe-Seyler et al., 2004). It is very interesting to A., Heller AKJ. (2004). Molecular identification and differen- 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 tiation of Staphylococcus species and strains of cheese origin, variability of strains under the composting process. In this view Syst. Appl. Microbiol., 27: 211-218. 394 O. Bouzaiane et al. Jensen MA., Webster JA., Straus N. (1993). Rapid identification Pereira-Neto J.T., Stentiford E.I., Mara D.D. (1987). Low cost of bacteria on the basis of polymerase chain reaction-ampli- controlled composting of refuse and sewage sludge. In: fied ribosomal DNA spacer polymorphisms. Appl. Environ. De Bertoldi M., Ferranti M.P., L’Hermite P., Zucconi F., Eds, Microbiol., 59: 945-952. Compost: Production, Quality and Use, Elsevier Applied Science Publishers Ltd, London and New York. John JF., Gramling PK., O’Dell NM. (1992). Species identification of coagulase-negative staphylococci from urinary tract infcec- Rossi F., Tofalo R., Torriani S., Suzzi G. (2001). Identification tions. J. Clin. Microbiol., 8: 435-437. by 16S-23S rDNA intergenic region amplification, Kamath U., Singer C., Isenberg HD. (1992). Clinical significance genotypic and phenotypic clustering of Staphylococcus of Staphylococcus warneri bacteremia. J. Clin. Microbiol., 30: xylosus strains from dry sausages. J. Appl. Microbiol., 261-264. 90: 365-371. Kloos W.E., Bannerman T.L. (1999). Staphylococcus and Sambrook J., Fritsch EF., Maniatis T. (1989). Molecular Cloning: nd Micrococcus. In: Murray P.R., Baron E.J., Pfaller M.A., Tenover A Laboratory Manual, 2 edn., Cold Spring Harbor Press, th F.C., Yolken R.H., Eds, Manual of Clinical Microbiology, 7 New York. edn, American Society for Microbiology, Washington, DC, pp. Sikora L.J., Ramirez M., Troeschel T. (1983). Laboratory com- 264-282. posure for simulation studies. J. Environ. Qual., 12: 219- Lina G., Etienne J., Vandenesch F. (2000). Biology and pathogenic- ity of staphylococci other than Staphylococcus aureus and S. Straub T.M., Pepper I.L., Gerba C.P. (1993). Hazards from patho- epidermidis. In: Fischetti V.A., Novick R.P., Ferretti J.J., Portnoy genic microorganisms in land-disposed sewage sludge. Rev. D.A., Rood J.I., Eds, Gram-Positive Pathogens, American Environ. Contam. T., 132: 55-91. Society for Microbiology, Washington, DC, pp. 450-462. Strauch D. (1991). Survival of pathogenic micro-organism and Mendoza M., Meugnier H., Bes M., Etienne J., Freney J. (1998). parasites in excreta, manure and sewage sludge. Rev. Sci. Identification of Staphylococcus species by 16S-23S rDNA Tech. Off. Int. Epiz., 10: 813-846. intergenic spacer PCR analysis. Int. J. Syst. Bacteriol., 48: 1049-1055. Tammy L.B., Gary A.H., Fred C.T., Michael M.J. (1995). Pulsed- Field Gel Electrophoresis as a replacement for bacteriophage Marrie TJ., Kwan C., Noble MA., West A., Duffield L. (1982). typing of Staphylococcus aureus. J. Clin. Microbiol., 33: Staphylococcus saprophyticus as a cause of urinary tract 551-555. infections. J. Clin. Microbiol., 16: 427-431. Tenover F.C., Arbeit R.D., Goering R.V., Mickelsen P.A., Murray Martin M.A., Pfaller M.A., Wenzel R.P. (1989). Coagulase-negative B.E., Persing D.H., Swaminathan B. (199 5). Interpreting staphylococcal bacteriemia. Ann. Intern. Med., 110: 9-16. chromosomal DNA restriction patterns produced by pulsed- Marrug C., Grebus M., Hassen R.C., Keener H.M., Hoitink H.A.J. field gel electrophoresis: criteria for bacterial strain typing. J. (1993). A kinetic model of yard waste composting process. Clin. Microbiol., 33: 2233-2239. Compost. Sci. Util., 1: 38-51. Van Schaik W., Abee T. (2005). The role of D in the stress Mustin M. (1987). Le Compost: Gestion de la Matière Organique. response of Gram-positive bacteria-targets for food preser- Editions François Dubusc, Paris. vation and safety. Curr. Opin. Biotech., 16: 218-224. Parente E., Martuscelli M., Gardini F., Grieco S., Crudele M.A., Westblom T.U., Gorse G.J., Milligan T.W., Schindzielorz A.H. Suzzi G. (2001). Evolution of microbial populations and biogenic amine production in dry sausages produced in (1990). Anaerobic endocarditis caused by Staphylococcus Southern Italy. J. Appl. Microbiol., 90: 882-891. saccharolyticus. J. Clin. Microbiol., 28: 2818-2819. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Molecular typing of staphylococcal communities isolated during municipal solid waste composting process

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Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
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Annals of Microbiology, 58 (3) 387-394 (2008) Molecular typing of staphylococcal communities isolated during municipal solid waste composting process 1 2 1 1 1 2 Olfa BOUZAIANE *, Mohamed Salah ABBASSI , Maher GTARI , Ons BELHAJ , Naceur JEDIDI , Assia BEN HASSEN , Abdennaceur HASSEN Institut National de Recherche Scientifique et Technique, Laboratoire de Traitement et recyclage, B.P. 95 - 2050, Hammam Lif, Tunisie; Services des Laboratoires, Centre National de Greffe de Moelle Osseuse, Tunis, Tunisie Received 15 February 2008 /Accepted 16 June 2008 Abstract - This study investigates the molecular typing occurrence of staphylococcal communities during composting process of munic- ipal 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 proc- ess 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 compo- sition 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 clinical sets as a causative agents of several invasive infections (Kloos and Bannerman, 1999). Staphylococcus saprophyticus is believed to cause opportun- Composting transforms organic matter into a stable product consisting of humus-like substances. The end product is avail- istic infections in the female urinary tract (UTI) (Marrie et al., able for agricultural use. The composting process is used to treat 1982). Staphylococcus haemolyticus and Staphylococcus epider- yard waste, manure, sewage sludge and also municipal solid midis the second most frequently encountered species have been waste. Pathogens microorganisms found in municipal solid waste reported to cause myocarditis, sepsis, peritonitis, and UTI (John can be viruses, bacteria and protozoa or helminths (Strauch, et al., 1978). Staphylococcus hominis, Staphylococcus warneri, 1991). As they are heat-sensitive, the temperature increment Staphylococcus capitis, Staphylococcus simulans, Staphylococcus during the composting process should eliminate them, leading cohnii, Staphylococcus xylosus, Staphylococcus saccharophyticus microbial pathogen-free end product, what is defined as disinfec- have been associated, less frequently, with various infections tion or sanitisation. Several parameters such as humidity and (Martin et al., 1989; Westblom et al., 1990; Kamath et al., oxygen have an influence on the heat increase. For this reason, 1992). Coagulase-negative staphylococci are the most common bac- sanitisation efficiency depends on the composting method used (Pereira-Neto et al., 1987). Therefore, there are many microbes teria isolated from the compost of municipal solid waste (Hassen et al., 2001). The emergence of CNS as human pathogens and that escape and resist to the heat treatment in composting process (Hassen et al., 2001). Among these microbes we notice reservoirs of antimicrobial resistance determinants requires their spores or bacteria that are either spore forming (Clostridium, rapid and reliable identification in order to have an early predic- Bacillus spp.) or very resistant to desiccation like Staphylococcus. tion of the potential pathogenicity or antibiotic susceptibility of Staphylococci are among the most wide spread bacteria and are each isolate (Kloos and Bannerman, 1999; Lina et al., 2000). ubiquitous in the environment. In addition, they are well known In recent years, several manual and automated methods for nosocomial pathogens associated with multiple antimicrobial identification of staphylococci have been described, but they are resistance mechanisms. time consumer (at least 30 h) as they require microbial growth. For many years Staphylococcus aureus was the only species Furthermore, commercially available panels, which are based on recognised as an important human pathogen, however, recently functional differences in metabolic pathways, do not allow a reli- coagulase-negative staphylococci (CNS) have been emerged in able 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 restric- * Corresponding author. Phone: 21671788436; Fax: 21671410740; E-mail: olfa_bz2004@yahoo.fr tion analysis (ARDRA) and ribosome spacer PCR or internal tran- 388 O. Bouzaiane et al. scribed spacer-PCR (ITS-PCR) was tested by several authors for et al., 2001). Briefly, from the prepared suspension a 0.5 ml the identification of staphylococci from different origins (Mendoza volume was poured and spread out on Baird-Parker agar and et al., 1998; Bes et al., 2000). incubated at 37 °C during 48 h. Since other genus from the fam- The aims of this present study were: (i) to identify the sta- ily Micrococcaceae, such as Kocuria, can grow on Baird-Parker phylococcal communities during composting process, and (ii) to agar, only colonies with typical staphylococcal morphology were characterise them by ARDRA, ITS-PCR, and PFGE methods in considered: convex brilliant, black colonies encircled by a clear order to assess suitable method(s) of molecular typing. 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 MATERIALS AND METHODS freeze-dried rabbit plasma (Diagnostic Pasteur, Paris). Results were expressed as the number of colony-forming units (UFC) per Composting process. The study was carried out in the experi- gram of compost or waste. mental composting plant of Beja City, north east of Tunisia; Identification of selected isolates to the species level was during the period between June and October 2003. In each year determined by studying carbohydrates fermentation and enzy- 1000 tons of solid waste, were treated in this plant. Municipal matic activities using biochemical tests in API Staph (bioMérieux, solid wastes collected from various sites of the Beja City and France). Biochemical profiles (API patterns) of the isolated were forwarded through two series of windrows with a pyramidal compared and strains were classified according to their profiles. 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 Genotypic investigations. The study was carried out with a refined by mechanical sorting of the inert matter (mostly plastics total of 100 isolates of Staphylococcus. They were collected from and glasses). Finally, 65 days of composting, sawdust and green the different steps of composting of municipal solid waste and wastes were added to enhance the microbial activity of digestion from compost. Staphylococcus aureus ATCC 25923 and S. epi- phase and to enhance biodegradation under optimal conditions. dermidis CCM 2124 were used as controls. Temperature and humidity were controlled daily, and windrow was turned and watered (humidity regulatory adjusted to 50%) ARDRA method. Preparation of crude cell extracts was carried as soon as the inner temperature of the pile reached or exceed out by the phenol-chlorophorm method as previously reported 65 °C. These operations of turning and watering were performed (Sambrook et al., 1989). Synthetic oligonucleotide universal almost twice per month on average. primers 16S-1492 R (5’-TACGG (CT) TACCTTGTTACGACTT-3’) and 16S-27 F (5’AGAGTTTGATC (AC) TG GCTCAG-3’) were used to Sampling of organic wastes during the composting cycle. amplify the 16 S rDNA. PCR products were purified to eliminate Ten samples (approximately 5 kg) were collected every 15 days factors that might inhibit enzymatic digestion. Five endonucle- from 5 to 139 days of composting from ten randomly selected ases were used in this study: AluI, HaeIII, TaqI, RsaI, and MspI locations in the windrow by digging a small pit to 10 m depth with (Promega, France). A 8-μl sample of purified PCR product, 2-μl a shovel. In each sampling, samples were mixed thoroughly and of buffer, 0.25-μl of BSA (100 μg/ml of BSA), and 1 U of each three portions of 1 kg each were separated. The first portion was enzyme were combined in one reaction tube (20 μl as final solu- stored at 0-20 to constitute a collection of samples, the second tion adjusted with sterile distiller water) and incubated at least 4 h was for pH determination, and the third was for microbiological as recommended by the supplier (Promega). Digested PCR prod- analyses. ucts were electrophoresed through 6% polyacrylamide gel. Then, gels were stained with ethidium bromide and photographed. Temperature and pH determination during the compost- ing process. Temperature inside the windrow was measured, ITS-PCR method. PCR analysis of 16S-23S intergenic spacer every day during the composting period, with a special sensing region length (ITS-PCR) was performed according to the method device stuck introduced to 60 cm depth in randomly selected of Mendoza et al. (1998). Amplification was modified as follow: points. For pH, 400 g of municipal solid waste (MSW) or compost after an initial step of denaturation during 5 min at 94 °C, 40 were placed in an Erlenmeyer flask containing 2 l of distilled amplification cycles consisting of: denaturation at 94 °C for 30 s, water and stirred for 3-5 min. The mixture was allowed to settle annealing at 45 °C for 30 s, and elongation at 72 °C for 45 s; the for 5 min and the pH was measured using a pH meter. For dry last cycle was followed by a 7 min at 72 °C for final elongation. weight, 400 g of fresh MSW was dried at 105 °C until the weight The PCR amplification patterns were electrophoresed on standard remained stable. 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 Microbiological assays. in 6% polyacrylamide gels was performed in 1X Tris-borate-EDTA Preparation of compost suspension. A 50 g aliquot of fresh waste buffer for 10 to 14 h at 100 V. After electrophoresis, gels were or compost, from the 1 kg sample, was taken and homogenised stained with ethidium bromide and photographed. with 950 ml sterile distilled water for 2 h under mechanical shak- The various profiles obtained by ARDRA as well as those ing (Edmund Buhler shaker type Kl-2, Germany) as reported by obtained for the ITS-PCR were treated by the software “gel Sikora et al. (1983) and Hachicha et al. (1993). Shaking was to Pro”. Profiles were analysed by the MVSP software (Kovach remove the maximum of micro-organisms from their organo- Computing Service, http://www.kovcomp.com). mineral substrates. The suspensions were used for staphylococ- cal counts. PFGE method. The protocol for the preparation of chromosomal DNA was modified from that described by Tammy et al. (1995). Staphylococci counts and identification. In our study we inves- Cells were grown overnight in 5 ml of Brain Heart Infusion broth tigated only the staphylococcal community during the compost- at 37 °C. Cells from 1 ml of each culture were collected by cen- ing process. Staphylococci were counted by the plate pouring trifugation and washed in 2 ml of TE buffer (100 mM Tris-HCl, technique on Baird-Parker agar as reported previously (Parente 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) 389 Digestion Maturation Digestion Maturation 80 6 3,5 Temperature 70 Humidity (HR %) 3,0 Organic matter (OM %) 2,5 2,0 40 3 1,5 1,0 0 0 5 9 76 20 34 48 62 90 111 125 139 9 20 34 48 62 76 90 111 125 139 Time (day) Time (day) FIG. 1 - Progress of temperature, humidity and organic FIG. 2 - Evolution of staphylococci species during matter during composting process. composting process. suspended in 200 μl of EC buffer (6 mM Tris-HCl, 1 M NaCl, 100 Biotech, Piscataway, NJ) was used as size standards in the first mM EDTA, 0.5% Brij 58 (Sigma), 0.2% sodium desoxycholate lane on each gel. Gels were stained with a solution of ethidium (Difco Laboratories), 0.5% sodium N-lauroyl sarcosine (USB); pH bromide (0.5 μg/ml) and digitalized with UVItec apparatus equip- 7.5) containing 100 μg/ml of lysostaphin and 20 μg/ml of RNase ment. DNA banding patterns were compared visually using the (Sigma). This suspension was mixed with an equal volume of 2% criteria of Tenover et al. (1995). 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 RESULTS incubated overnight at 37 °C in 1 ml of lysis buffer EC, followed by a further overnight incubation at 55 °C in proteolysis buffer Physico-chemical parameters of composting process (10 mM Tris-HCl, 1 mM EDTA, pH 8.5) containing 200 μg/ml of In this study the temperature progress vary according the two proteinase K. The plugs were washed three times (30 min each) phases of composting process, digestion and maturation (Fig. 1). in 10 ml TE buffer (10 mM Tris-HCl, 1 mM EDTA; pH 8), with The phase of digestion start with a mesophilic phase in which gentile shaking at room temperature and stored in TE buffer at the temperature reached 42 °C. During this mesophilic step, 4 °C until further analysis is done. One-quarter of a sample plug the humidity rate was up to 45%. After 20 days of compost- was placed in a microcentrifuge tube containing 175 μl of steri- ing, the temperature reached 65 °C and the thermophilic step lised deionised water, 20 μl of buffer J, 100 μg/ml of BSA, and started. In this step the humidity decreased significantly. Then, 30 U of SmaI (Promega, France), and the mixture was incubated the temperature decreased gradually to reach 40 °C. At the 62 overnight at 25 °C. The plug was then incubated in 1 ml of TE days, and after the addition of sawdust and green wastes in order buffer at 4 °C for 1 h. Sample plugs were loaded in a 1% agar- to enhance the microbial activity, the maturation phase took ose gel in 0.5X TBE buffer. Electrophoresis was performed with place. In this phase, like in the digestion phase, the temperature the CHEF-DRIII apparatus (Bio-Rad) using the following condi- increased gradually to reach 50 °C, stabilised for a short period tions: pulse times ranged from 5 s to 40 s during 22 h at 6.0 V/ then decreased. In this phase there was also mesophilic and cm at 14 °C. Lambda DNA PFGE Marker (Amersham Pharmacia thermophilic steps. TABLE 1 - Staphylococci composition during composting cycle Stage of composting Sampling Time (day) Identification using API Staph Digestion phase E1 5 S. lentus, S. xylosus, S. hominis E2 9 S. lentus, S. xylosus, S. hominis E3 20 S. lentus, S. xylosus, S. hominis E4 34 S. lentus, S. xylosus, S. hominis E5 48 S. lentus, S. xylosus, S. hominis E6 62 S. lentus, S. xylosus, S. hominis E7 76 S. lentus, S. xylosus, S. hominis Maturation phase E8 90 S. lentus, S. xylosus, S. hominis, S. lugdunensis E9 111 S. lentus, S. xylosus, S. hominis, S. sciuri, S. haemolyticus E10 125 S. lentus, S. xylosus, S. hominis S. sciuri, S. haemolyticus, S. warneri E11 139 S. lentus, S. xylosus, S. hominis, S. sciuri, S. haemolyticus, S. warneri Temperature HR, MO (%) Number of staphylococcus/g of compost 390 O. Bouzaiane et al. 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 staphyloco- cci increased to reach 3 x 10 CFU/g dry matter, however, during the thermophilic phase it decreased significantly. This decrease continued until the sixth week of the process. During the matu- ration phase and after application of sawdust, the number of staphylococci re-increased to reach 2 x 10 CFU/g of dry matter during the thermophilic step, and re-decreased at the end of the process near 0.5 x 10 CFU/g of dry matter. 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 identifica- FIG. 3 - Simplified dendrogram showing the relationship tion are listed in Table 1. It is noteworthy that the distribution of between the 100 staphylococci and 2 references recovered species is phase-varying. Indeed, during the digestion strains obtained by analysis of phenotypical tests. The dendrogram is based on unweighted pair group phase three species were recovered: S. lentus, S. xylosus, and method with arithmetic average (UPGMA) using the S. hominis. Whereas, in the maturation phase and in addition to simple matching coefficient. The cut off level for the later three species, S. lugdunensis, S. haemolyticus, S. sciuri clustering the strains is 78%. Each tie represents and S. warneri were also recovered (Table 1). one biochemical profile. A: S. lentus; B: S. xylosus; C: S. haemolyticus; D: S. xylosus; E: S. xylosus, S. Phenotypic profiles sciuri; F: S. xylosus; G: S. warneri; H: S. hominis, S. lugdunensis; I: S. xylosus. In parenthesis num- On the basis of biochemical tests in API Staph, it was ber of strains grouped in each clusters. possible to distinguish 28 biochemical profiles among 100 A B C D 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) 391 10 x bp 20 10 5 1 S. xylosus (4 5) S. le ntu s (32) S. ho mi n is (9) S. sciuri (4) S. warneri (4) S. ha e mo lyticus (3) S. lu gdunensi s (3) FIG. 5 - ARDRA dendrogram showing the relationship be- FIG. 6 - Schematic representation of PCR-amplified 16S- tween the Staphylococcus strains obtained from the 23S rDNA spacer region from 100 Staphylococ- digestion of 16S rDNA amplified products gene by cus strains representing 7 species. The number of HaeIII, TaqI, RsaI, AluI and MspI. strains tested is given in parentheses. strains. The biochemical tests that can differentiate strains patterns discriminating between strains. DNA digestion were: D(+)-mannose, maltose, lactose, trehalose, xylitol, with AluI, classified S. xylosus and S. lentus strains each D(+)-melibiose, nitrate reduction, phosphatase, acetoin into 3 patterns. However, S. hominis, S. lugdunensis, S. production, raffinose, D(+)-xylose, saccharose, methyl- haemolyticus, S. sciuri and S. warneri strains were not A -D-glucopyranoside, N-acetylgycosamine, arginine polymorphic since a specific unique pattern was observed hydrolysis, urease. Simplified dendrogram obtained by for strains of each species. cluster analysis (Fig. 3) each tie represent one biochemical The dendrogamme of similarity obtained by the profile. Nine clusters were observed with 78% of similarity combination of the different profiles obtained by the five (A, B, C, D, E, F, G, H and I). Staphylococcus xylosus, have independent digestions showed 8 clusters (up to 80% of 10 biochemical profiles; S. lentus, have 11 biochemical similarity). Interestingly, strains of the same species have profiles, S. haemolyticus have 3 biochemical profiles, S. been shown to belong to different clusters, similarly some hominis, S. lugdunensis, S. sciuri and S. warneri have each clusters contained strains of different species (Fig. 5). 1 biochemical profile. During the digestion phase, the 14 S. xylosus strains were classified in 1 genomic haplotype A (Table 2). In maturation ARDRA method phase, the 31 S. xylosus strains showed 2 genomic haplotypes: DNAs were extracted and amplified with universal 20 strains with haplotype A and 11 strains exhibited the hap- 16S rDNA primers producing a single band of similar lotype B (Table 2). The ten S. lentus strains, found during the size (approximately 1500 bp) for each strain. The five digestion phase presented 2 haplotypes, A1 (7 strains) and B1 endonucleases used gave polymorphic profiles (Fig. 4). (5 strains). In addition to latter haplotypes, two new types were However, we observed that DNA digestion by MspI enzyme found in 5 strains among the 20 strains isolated during matura- yielded a unique pattern in all staphylococcal strains tion phase, named C1 and D1 (Table 2). The nine S. hominis (Fig. 4d). In addition, this was also observed with little strains isolated during all the composting process showed only exception after DNA digestion with HaeIII, TaqI, RsaI, and one genomic haplotype A2 (Table 2). MspI were strains from different species have been shown as indistinguishable (Fig. 4b, 4c, and 4d). In contrast, DNA ITS-PCR method digestion with AluI (Fig. 4a) was able not only to efficiently PCR amplification of 16S-23S intergenic spacer region digest the amplified products, but also gave restriction yielded 7 different patterns (Fig. 6). Indeed strains within TABLE 2 - Staphylococci identification Phase Species ARDRA PFGE Digestion S. xylosus (14) A (14) A (3); B (4); C (4); D (3); E (2) S. lentus (12) A1 (7); B1 (5) A1 (2); B1 (2); C1 (3); D1 (2); E1 (3) S. hominis (3) A2 (3) A2 (1); B2 (1); C2 (1) Maturation S. xylosus (31) A (20); B (11) A (7); B (6); C (5); D (5); E (4); F (4) A1 (4); B1 (3); C1 (3); D1 (2); E1 (2); F1 (2); S. lentus (20) A1 (9); B1 (6); C1 (3); D1 (2) G1 (2); H1 (2) S. hominis (6) A2 (6) A2 (2); B2 (2); C2 (1); D2 (1) S. lugdunensis (3) A3 (3) A3 (3) S. warneri (4) A4 (4) A4 (4) S. haemolyticus (3) A5 (3) A5 (3) S. sciuri (4) A6 (4) A6 (4) ( ): strains number; A, B, C, etc.: haplotypes. 392 O. Bouzaiane et al. AB C M 1 2 3 4 5 6 7 8 M 1 2 3 4 5 6 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 imputed), moisture content and available nutrients. Based 3-8 fragments whose sizes ranged from 300 to 1500 bp. on temperature, the process of aerobic composting can be PCR products ranged in sizes from 400 to 1500 bp for S. divided into three major steps, a mesophilic-heating step, warneri, 350 to 1000 bp for S. xylosus. Staphylococcus a thermophilic step and a cooling step (Mustin, 1987). lugduensis showed special pattern with three close DNA During the mesophilic step, the temperature and the water fragments with sizes ranging from approximately 630 to content increased as a consequence of biodegradation of 800 bp. Amplification of the 16S-23S spacer region of organic compounds. The temperature increment is the Staphylococcus species does not produce any common consequence of the organic matter oxidation (Hassen et al., fragment that can be assigned as genus specific. 2001). The mesophilic step is followed by the thermophilic step. The latter step occurred between days 20 and days PFGE method 34 of the composting process. As mentioned by Hachicha In the digestion phase S. xylosus strains showed five PFGE et al. (1992) and Marrug et al. (1993), a temperature patterns, and S. lentus strains were also classified into five above 60 °C seriously affect the decomposition rate of the genotypes. The three strains of S. hominis were unrelated organic waste as a result of a reduction in microbiological and exhibited each a unique PFGE pattern (Fig. 7, Table activity. The temperature started to decrease after day 2). However, in the maturation phase the of S. xylosus 111, this decrease led to the depletion of organic matters strains fell into six PFGE 6 patterns, and strains of S. lentus and the carbon/azotes (C/N) ratio tended to stabilise. and S. hominis felled into eight and four PFGE patterns, By the end of the composting process, the average respectively. It is worth to note that the related strains of temperature inside the windrow showed a decrement and S. xylosus, S. lentus, and S. hominis observed during the reached approximately 30 °C at the end of the process digestion phase were also observed during the maturation (Ben Ayed et al., 2007). phase. A unique PFGE pattern was observed among strains Staphylococci are ubiquitous bacteria and S. aureus is one belonging to the remaining species, S. lugdunensis, S. of the main causes of collective toxic infections of food. In addi- haemolyticus, S. sciuri and S. warneri. tion, 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 DISCUSSION product of composting represents a real threat of public health. In our study, we found that the total number of staphylococci Composting is a self-heating, aerobic, solid phase, decreased from 3 x 10 CFU/g of dry matter at the mesophilic biodegradative process of organic waste materials. The phase of digestion step to 0.5 x 10 CFU/g dry matter at the end composting process at the microbial level involves several of the maturation step. This means that the composting treat- interrelated factors, namely temperature, ventilation (O ment can not eliminate these bacteria and that Staphylococcus 2 Ann. Microbiol., 58 (3), 387-394 (2008) 393 species support hostile conditions such as high temperature and and similar to other bacteria submitted to hostile conditions (Ben reduction of nutriments. Kahla-Nakbi et al., 2008) the genome seemed to be invariable During the digestion, the colonies that have taken at random under stressing conditions. The adaptation response to these and identified by API Staph, belong to three species: S. xylosus, hard conditions might touch other molecular mechanisms such as S. lentus, and S. hominis. However, at the maturation phase the sigma factor (Van Schaik and Abee, 2005). and after the addition of sawdust and green wastes there was In conclusion, during composting process of municipal appearance of other species: S. lugdunensis, S. haemolyticus, S. wastes we found seven species of Staphylococcus. The human sciuri and S. warneri. It seems that the three species recovered pathogens S. aureus and S. epidermidis species were not found. in the digestion step are the most staphylococcal species in solid The ITS-PCR method was able to yield a specific pattern for waste, and the other four species recovered during the matura- each species and confirm biochemical identification. ARDRA did tion phase were added with the sawdust. Alternatively, the appli- not give discrimination between strains belonging to different cation of sawdust change the condition of the composting cycle species; therefore it could not be used as molecular method to and permit the growth of new staphylococci species that were assess staphylococcal communities in the compost. The PFGE has present in the first phase but non culturable. Interestingly, S. allowed the detection of persistent clones during the composting epidermidis and S. aureus have not been detected in any phase. process. These results were in concordance with those reported by Hassen et al., 2001. The detected species are rarely implicated in human Acknowledgements infections, in contrast to S. epidermidis and S. aureus. The present study is a part of the 1999-2002 research programs Dendrogram constricted on the basis of biochemical profiles “Municipal Solid Waste Treatment and Compost Agricultural exhibited by staphylococcal isolates showed that strains belong- Re-use” which is supported by the Tunisian state secretariat of ing to S. xylosus, S. lentus and S. hominis demonstrated the Scientific Research and Technology. highest variability. Interestingly, these species were found along the composting process. Therefore, it seems that these spe- cies have an intrinsic ability of adaptation in hostile conditions. REFERENCES Modulation of metabolic pathways according to environmental conditions could have enhanced the survival ability of strains Ben Ayed L., Hassen A., Jedidi N., SaidiI N., Bouzaiane O., Murano belonging to these species. F. (2007). Microbial C and N dynamics during compost- ITS-PCR based strategy for studying the length polymor- ing process of urban solid waste. Waste Manage. Res., 25: phisms of the 16S-23S rDNA spacer regions has been proved 24-29. useful for both epidemiological and taxonomic investigations (Gürtler et al., 1993; Jensen et al., 1993). In our study, we Ben Kahla-Nakbi A., Besbes A., Bakhrouf A. (2008). Survival found that the ITS-PCR profiles were specific for each of the of Vibrio fluvialis in seawater under starvation conditions. Staphylococcus species examined. These results were in agree- Microbiol. Res., 163: 323-328. ment with the biochemical and phenotypical methods previously Bes M., Guérin-Faublée V., Meugnier H., Etienne J., Freney J. used to identify these strains as reported by Freney et al. (1988). (2000). Improvement of the identification of staphylococci The ITS-PCR method did not allow detection of intraspecies isolated from bovine mammary infections using molecular polymorphism among species. Therefore, this technique could be methods. Vet. Microbiol., 71: 287-294. used for the confirmation of the biochemical identification. Coppola S., Mauriello G., Aponte M., Moschetti G., Villani F. The ARDRA analysis by five restriction enzymes (HaeIII, (2000). Microbial succession during ripening of Naples-type TaqI, RsaI, AluI and MspI) allowed the identification of 8 clusters. salami, a southern Italian fermented product. Meat Sci., 56: AluI was shown to be the best enzyme that yields distinct frag- 321-329. ments; this enzyme permitted the detection of the highest diver- sity among strains. Many authors have reported a high diversity Freney J., Brun Y., Bes M., Meugnier H., Grimont F., Grimont PAD., of staphylococcal strains using ARDRA methods either in environ- Nervi C., Fleurette J. (1988). Staphylococcus lugdunensis mental or clinical sets (Hoppe-Seylar et al., 2004). It is worthy sp. nov. and Staphylococcus schleiferi sp. nov. two species to note that this technique is not able to differentiate between from human clinical specimens. Int. J. Syst. Bacteriol., 38: strains of different species. Indeed, same strains of different spe- 168-172. cies had indistinguishable pattern, and contrarily, strains belong- Gürtler V. (1993). Typing of Clostridium difficile strains by ing to the same species had different patterns. The small number PCR-amplification of variable length 16S-23S rDNA spacer of identified ARDRA-generated clusters could be attributed in regions. J. Gen. Microbiol., 139: 3089-3097. part, to the conserved property of the sequences encoding 16S Hachicha R., Hassen A., Jedidi N., Kallali H. (1992). Optimal rRNA in the genome of staphylococci. Nevertheless, we can not conditions for municipal solid waste. Biocycle, 6: 76-77. exclude the pressure selection during the composting process permitting the persistence of some clones. Hachicha R., Jedidi N., Hassen A. (1993). Aspects hygiéniques DNA digestion by SmaI enzyme, gave DNA patterns of 10 de la fermentation aérobies des déchets urbains dans le to 15 bands with sizes ranging from 10 to 700 kb. PFGE method contexte tunisien. Arch. Inst. Pasteur Tunis, 70: 13-20. showed that strains isolated during the phase of digestion persist Hassen A., Belguith K., Jedidi N., Cherif A., Cherif M., Boudabbous during the phase of maturation. These results support the low A. (2001). Microbial characterization during composting of diversity in 16S rDNA and the high genomic variability in non vital municipal solid waste. Bioresource Technol., 80: 185-192. genomic DNA sequences (cut sites of SmaI enzyme) revealed Hoppe-Seyler T., Jaeger B., Bockelmann W., Noordman W., Geis by PFGE (Hoppe-Seyler et al., 2004). It is very interesting to A., Heller AKJ. (2004). Molecular identification and differen- 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 tiation of Staphylococcus species and strains of cheese origin, variability of strains under the composting process. In this view Syst. Appl. Microbiol., 27: 211-218. 394 O. Bouzaiane et al. Jensen MA., Webster JA., Straus N. (1993). Rapid identification Pereira-Neto J.T., Stentiford E.I., Mara D.D. (1987). Low cost of bacteria on the basis of polymerase chain reaction-ampli- controlled composting of refuse and sewage sludge. In: fied ribosomal DNA spacer polymorphisms. Appl. Environ. De Bertoldi M., Ferranti M.P., L’Hermite P., Zucconi F., Eds, Microbiol., 59: 945-952. Compost: Production, Quality and Use, Elsevier Applied Science Publishers Ltd, London and New York. John JF., Gramling PK., O’Dell NM. (1992). Species identification of coagulase-negative staphylococci from urinary tract infcec- Rossi F., Tofalo R., Torriani S., Suzzi G. (2001). Identification tions. J. Clin. Microbiol., 8: 435-437. by 16S-23S rDNA intergenic region amplification, Kamath U., Singer C., Isenberg HD. (1992). Clinical significance genotypic and phenotypic clustering of Staphylococcus of Staphylococcus warneri bacteremia. J. Clin. Microbiol., 30: xylosus strains from dry sausages. J. Appl. Microbiol., 261-264. 90: 365-371. Kloos W.E., Bannerman T.L. (1999). Staphylococcus and Sambrook J., Fritsch EF., Maniatis T. (1989). Molecular Cloning: nd Micrococcus. In: Murray P.R., Baron E.J., Pfaller M.A., Tenover A Laboratory Manual, 2 edn., Cold Spring Harbor Press, th F.C., Yolken R.H., Eds, Manual of Clinical Microbiology, 7 New York. edn, American Society for Microbiology, Washington, DC, pp. Sikora L.J., Ramirez M., Troeschel T. (1983). Laboratory com- 264-282. posure for simulation studies. J. Environ. Qual., 12: 219- Lina G., Etienne J., Vandenesch F. (2000). Biology and pathogenic- ity of staphylococci other than Staphylococcus aureus and S. Straub T.M., Pepper I.L., Gerba C.P. (1993). Hazards from patho- epidermidis. In: Fischetti V.A., Novick R.P., Ferretti J.J., Portnoy genic microorganisms in land-disposed sewage sludge. Rev. D.A., Rood J.I., Eds, Gram-Positive Pathogens, American Environ. Contam. T., 132: 55-91. Society for Microbiology, Washington, DC, pp. 450-462. Strauch D. (1991). Survival of pathogenic micro-organism and Mendoza M., Meugnier H., Bes M., Etienne J., Freney J. (1998). parasites in excreta, manure and sewage sludge. Rev. Sci. Identification of Staphylococcus species by 16S-23S rDNA Tech. Off. Int. Epiz., 10: 813-846. intergenic spacer PCR analysis. Int. J. Syst. Bacteriol., 48: 1049-1055. Tammy L.B., Gary A.H., Fred C.T., Michael M.J. (1995). Pulsed- Field Gel Electrophoresis as a replacement for bacteriophage Marrie TJ., Kwan C., Noble MA., West A., Duffield L. (1982). typing of Staphylococcus aureus. J. Clin. Microbiol., 33: Staphylococcus saprophyticus as a cause of urinary tract 551-555. infections. J. Clin. Microbiol., 16: 427-431. Tenover F.C., Arbeit R.D., Goering R.V., Mickelsen P.A., Murray Martin M.A., Pfaller M.A., Wenzel R.P. (1989). Coagulase-negative B.E., Persing D.H., Swaminathan B. (199 5). Interpreting staphylococcal bacteriemia. Ann. Intern. Med., 110: 9-16. chromosomal DNA restriction patterns produced by pulsed- Marrug C., Grebus M., Hassen R.C., Keener H.M., Hoitink H.A.J. field gel electrophoresis: criteria for bacterial strain typing. J. (1993). A kinetic model of yard waste composting process. Clin. Microbiol., 33: 2233-2239. Compost. Sci. Util., 1: 38-51. Van Schaik W., Abee T. (2005). The role of D in the stress Mustin M. (1987). Le Compost: Gestion de la Matière Organique. response of Gram-positive bacteria-targets for food preser- Editions François Dubusc, Paris. vation and safety. Curr. Opin. Biotech., 16: 218-224. Parente E., Martuscelli M., Gardini F., Grieco S., Crudele M.A., Westblom T.U., Gorse G.J., Milligan T.W., Schindzielorz A.H. Suzzi G. (2001). Evolution of microbial populations and biogenic amine production in dry sausages produced in (1990). Anaerobic endocarditis caused by Staphylococcus Southern Italy. J. Appl. Microbiol., 90: 882-891. saccharolyticus. J. Clin. Microbiol., 28: 2818-2819.

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Annals of Microbiology – Springer Journals

Published: Nov 24, 2009

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