Vol. 7(29), pp. 3874-3884, 19 July, 2013 DOI: 10.5897/AJMR2013.5466 ISSN 1996-0808 ©2013 Academic Journals http://www.academicjournals.org/AJMR
African Journal of Microbiology Research
Full Length Research Paper
Identification of three related genera, Salmonella, Citrobacter and Proteus using API 20E, 16S-23S rDNA intergenic transcribed spacer fingerprinting and 16S rDNA sequencing Yousra Turki1*, Ines Mehri1, Amel Khessairi1, Kaouther Agrebi2, Abdennaceur Hassen1 and Hadda Ouzari2 1
Laboratoire Traitement et Recyclage des Eaux, Centre de Recherche et des Technologies des Eaux (C.E.R.T.E), Borj Cédria, 8020 Soliman, Tunisie. 2 Laboratoire Microorganisme et Biomolécules Actives, Département de Biologie, Faculté des Sciences de Tunis, Campus Universitaire, 2092, Tunis, Tunisie. Accepted 5 July, 2013
A total of 176 presumptive Salmonella strains isolated from different sources (food, environment, veterinary, and human) were analyzed by API 20E, serotyping, ITSF/ITSR-PCR and MINf/MINr-PCR. On the other hand, the 16S-23S rRNA intergenic transcribed spacer region was used to aid in their identification. Test results were confirmed by 16S rRNA sequencing. Of 176 suspected S. enterica isolates, API20E identified 105 (59.6%) as S. enterica, 42 (24%) as Citrobacter and 29 (16.4%) as Proteus. A total of 11 serotypes were found among 102 out of 105 Salmonella isolates. The remaining isolates were classified as untypeable by serotyping. When ITSF/ITSR-PCR and MINf/MINr-PCR were used, 101 out of 105 Salmonella isolates tested generated PCR products. Amplification of the spacer region between 16S-23S rRNA gene allowed discrimination of genera and facilitated species identification. Sequencing of 16S rRNA gene has identified 4 different species in proteus genus (P. mirabilis, P. penneri, P. vulgaris, and P. hausseri), 3 different species in Citrobacter genus (Citrobacter freundii, Citrobacter braakii, and Citrobacter youngae), and Salmonella enterica sub sp. enterica for all Salmonella isolates. The four suspect Salmonella isolates were definitively classified in Citrobacter genus. In conclusion, this study has demonstrated that API20E, serotyping associated to Salmonella specific PCR are accurate methods for S. enterica detection. In addition, PCR amplification of the 16S23S spacer region was suitable tool for identification of bacteria at genera and species level. Key words: Salmonella, API 20E, serotyping, MINf/MINr-PCR, ITSF/ITSR-PCR, 16S-23S intergenic spacer regions (ITS)-PCR, 16S rRNA. INTRODUCTION Salmonella spp. are ubiquitous enteric bacteria. These gram-negative rods are the etiologic agents of food-borne salmonellosis and also the agent that causes typhoid and
paratyphoid fevers (Baudart et al., 2000). Isolation and identification of Salmonella continues to be an important issue in clinical and applied microbiology. The conven-
*Corresponding author. E-mail:
[email protected]. Tel: +216 21576990.
Turki et al.
tional methods for the detection of Salmonella require multiple steps which may take up to 5 to 7 days (Chiu et al., 2005). The final identification and characterization of Salmonella enterica is based on biochemical reactions followed by serotyping (Popoff and Le Minor, 1997; Turki et al., 2012). However, considerable variation can be seen in the biotyping pattern. Thus, a minor proportion of presumptive S. enterica isolates identified by biochemical testing cannot be identified by subsequent serotyping procedures (Hoorfar et al., 1999). Indeed, the API 20E diagnostic, which detects 20 biochemical reactions, is a traditional method for the identification of S. enterica and other Enterobacteriaceae (Koneman et al., 1997). Previous studies of API 20E have reported both good (Swanson and Collins, 1980; O’Hara et al., 1992; Peele et al., 1997) and inaccurate (Aldridge et al., 1978; Aldridge and Hodges, 1981; Robinson et al., 1995; Jones et al., 2000) sample classifications. So, rapid and more sensitive methods for the detection of Salmonella species are required. Genetic identification systems may improve Salmonella identification (Hoorfar et al., 2000). PCR is an extremely sensitive test, able to amplify picogram quantities of DNA (Amavisit et al., 2001). In recent years, PCR methods have been successfully used to detect a number of pathogens including Salmonella (Hoorfar et al., 1999; Amavisit et al., 2001; Trokov and Augustin, 2003; Sanath Kumar et al., 2003; Chiu et al., 2005; Hassan et al., 2008). The rRNA genes are present in all organisms. Bacterial rRNA operons are made up of 16S rRNA, 23S rRNA and 5S rRNA genes. The copy number of rRNA gene clusters varies among bacterial species (Chiu et al., 2005). A molecular method for the detection of Salmonella enterica strains based on 16S rRNA sequence analysis was developed (Lin and Tsen, 1995, 1996), modified and improved by Trkov and Avgustin (2003). Likewise, Salmonella spp. 16S-23S based PCR primers were designed (Chiu et al., 2005). The 16S rDNA and 16S-23S rDNA Internal Transcribed Spacer (ITS) region have been the most popular target for bacterial taxonomy. The ITS regions exhibit a large degree of sequence and length variation. This diversity is due in part to variations in the number and type of tRNA sequences found within the spacer. Sequence polymorphisms can be used to recognize and to discriminate between different genus and species (Jensen et al., 1993; Christensen et al., 2000; Wang and Jayarao, 2001; Ouzari et al., 2008; Wang et al., 2008; Liguori et al., 2011). On the other hand, the 16S rRNA gene sequences have been widely used to assess to phylogenetic closeness among bacteria. In many case, the16S rRNA gene sequencing has been used to study the phylogenetic relationships between species, to examine the taxonomy of different subspecies and to identify the species and the subspecies (Toth et al., 2001; Lin et al., 2004). The aim of this study was to compare API 20E, serotyping, Salmonella specific-ITS based PCR, Salmonella
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specific-16S based PCR, and 16S-23S ribosomal DNA intergenic transcribed spacer polymorphisms in the identification of S. enterica and related genera such as Proteus and Citrobacter. Test results were validated by comparison with 16S rRNA gene sequencing. MATERIALS AND METHODS Bacterial strains A total of 176 enterobacterial strains described in the present study were collected in Tunisia from different sources (food, environment, veterinary and human) during 2006 to 2008. Samples were cultured for Salmonella by using the procedure described by Baudart et al. (2000) and Stevens et al. (2006). Presumptive Salmonella colonies were subcultured to tryptic soy agar plates (Difco, Detroit, MI). For each isolate, an API 20E strip (Biomerieux, France) was inoculated and incubated according to the manufacturer’s instruction. The likelihood of S. enterica was calculated using the manufacturer’s coding system, based on reactions to reagents in the 20 compartments. All isolates classified as S. enterica by API 20E with likelyhood values from 77.8 to 99.9% were serotyped for confirmation. Salmonella serotyping was achieved at the National Center for Enteropathogenic Bacteria at Pasteur institute, Tunis, Tunisia, on the basis of somatic O, phase 1 flagellar, and phase 2 flagellar antigens agglutination using commercial antisera (Bio-Rad, France) according to the White-Kauffman-Le Minor scheme Popoff (2001).
DNA extraction DNA was extracted according to the method of Chen and Kuo (1993) with some modification. Briefly, 3 ml of a fresh overnight culture grown in Luria-Bertani broth at 37°C for 16 h, and centrifuged at 12000 rpm for 3 min using (MPW-350R). The pellet was suspended in 200 µl of lysis buffer (40 mM, Tris-acetate, pH 7.8; 20 mM sodium acetate; 1 mM EDTA; 1% SDS) vortexed and incubated for 30 min at 37°C. 66 µl of 5 M NaCl was then added and the suspension was centrifuged at 12000 rpm for 10 min. The supernatant was mixed with 200 µl of chloroform and centrifuged 10 min at 12000 rpm. DNA from the upper aqueous phase was precipitate with 200 µl isopropanol, washed with 70% ethanol, dried for a short time and finally resuspended in 50 µl TER buffer (Tris/EDTA buffer with RNase) for PCR (Turki et al., 2012).
PCR amplification All Amplifications were carried out with a 1x Taq polymerase buffer, in a total reaction of 25 µl containing 1 mM of MgCl2, 0.1 mM dNTP, 0.17 µM of each primer, 1 U Taq DNA polymerase (Promega) and 1 µl of bacterial genomic DNA.
Salmonella-specific PCR Two Salmonella-specific PCR targeting the internal transcribed spacer (ITS) sequences located between 16S and 23S rDNA and the 16S rRNA were used in this study. Primers designed to amplify the 16S-23S ribosomal DNA intergenic spacers were ITSF (5’-TGC GGC TGG ATC ACC TCC TT-3’) and ITSR (5’- TAT AGC CCC ATC GTG TAG TCA GAA C-3’) described previously by Chiu et al. (2005). The DNA amplification generated PCR products with mole-
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cular weight bands equal to 312 bp. The Salmonella specific 16S rRNA PCR primers MINf (5’-ACG GTA ACA GGA AGM AG-3’), M = A/C and MINr (5’- TAT TAA CCA CAA CAC CT-3’) previously designed by Trkov and Avgustin (2003) were used and generated an approximatively 402 bp long PCR products. Amplification was performed using a thermocycler (UNO II-BioMetra) according to the following procedure. Initial denaturation at 94°C for 4 min followed by 40 cycles of PCR consisting of denaturation at 94°C for 30 s, annealing at 52°C for 30 s, and extension at 72°C for 45 s; in the last cycle, the extension time was 7 min at 72°C. The PCR product (3 µl) was analyzed on a 2% (w/v) agarose gel stained with ethidium bromide (1 mg/ml) at 90 V/cm for 30 min, and visualized under ultraviolet transillumination.
Amplification of the 16S-23S rDNA spacer region and the16SrDNA - Amplification of the 16S-23S spacer region of the rRNA is performed using the primers complementary to the conserved regions of the 16S-23S bacterial rRNA genes, as previously reported by Wheeler et al. (1996). The primers used were S-D-Bact1494-a-S-20 (5’-GTCGTAACAAGGTAGCCGTA-3’) and S-D-Bact0035-a-A-15 (5’-CAAGGCATCCACCGT-3’). - Amplification of the DNA 16S region was performed using primers S-D-Bact-0008-a-S-20 (5’-AGAGTTTGATCCTGGCTCAG-3’) and S-D-Bact-1495-a-A-20 (5’-CTACGGCTACCTTGTTACGA-3’) to amplify a 1486-bp fragment (Olsen et al., 1986). - Amplification was performed using a thermocycler (UNO IIBioMetra) according to the following procedure. Initial denaturation at 94°C for 4 min followed by 40 cycles of PCR consisting of denaturation at 94°C for 30 s, annealing at 45°C for 30 s, and extension at 72°C for 45 s; in the last cycle, the extension time was 7 min at 72°C. The PCR product (3 µl) was analyzed on a 2% (w/v) agarose gel stained with ethidium bromide (1 mg/ml) at 90 V/cm for 30 min, and visualized under ultraviolet transillumination.
Partial sequencing of the 16S-rDNA PCR products obtained from representative ITS groups (39 bacterial isolates) were purified with a Promega PCR purification Kit, and sequenced using an Applied Biosystems sequencer. Sequences of the PCR products obtained with the 16S F were aligned and corrected manually with Chromas Pro (version 1.34). The BLAST database of the National Center for Biotechnology Information (NCBI) was used to compare resolved sequences of the 39 isolates with 16S rDNA sequences data deposited in GenBank. Alignment of sequences was performed using the Clustal method (ClustalX 1.81). The NCBI Accession Numbers for the 16S rRNA gene sequences of 39 representative isolates determined in this study are listed in Table 2. Pseudomonas isolate (Ps12) was used as an out group.
Data analysis The number and the size of DNA amplified fragments generated were evaluated by visual inspection using the molecular-weight DNA marker. To determine significant differences in the patterns, the reproducibility of results was assessed by repetition of at least two independent assays. The data analysis was performed by using Gel Pro 32.
RESULTS AND DISCUSSION API 20E and Salmonella serotyping Complete and accurate identification of Salmonella and other members of the family Enterobacteriaceae is a subject of much concern and to be an important issue in clinical and applied microbiology (Robinson et al., 1995; Hoorfar et al., 1999; Nucera et al., 2006). Biochimical reactions are the basis of identification for Salmonella species, although, some variations and discrepancies can be seen in the biotyping patterns (Aldridge et al., 1978; Aldridge and Hodges, 1981; Nucera et al., 2006). In this study, 176 presumptive Salmonella isolates were classified by using API 20E which identified, 105 (59.6%) as S. enterica with likelihood values from 77.8 to 99.9%, 42 (24%) as Citrobacter and 29 (16.4%) as Proteus. The majority of isolates were recognized as non-lactose fermenters (Lac-) and hydrogen-sulfide producers (H2S+) colonies. However, most of Lac- / H2S+ colonies turn out not to be S. enterica, but related species such as Proteus and Citrobacter (Hoorfar et al., 1999; Nucera et al., 2006). Among 105 Salmonella strains classified by API20E, 11 serotypes (S. anatum, S. typhimurium, S. enteritidis, S. kentucky, S. amsterdam, S. montevideo, S. derby, S. mbandaka, S. zanzibar, S. gallinarum and S. newport) were found among 102 Salmonella isolates with dominance of serotype Kentucky. However, three isolates (143, 274, and 317) were not identified by the serotyping procedure, and were classified as untypeable strains. Additionally, one S. kentucky isolate (319) identified by API20E and serotyping was negative by using Salmonella specific PCR. These results were in agreement with several previous studies (Hoorfar et al., 1999; Nucera et al., 2006) which demonstrated that serotyping approach can present a risk of false positives. Salmonella specific PCR In order to increase the sensitivity and specificity of detection of Salmonella, several specific DNA probes were designed (Lin et Tsen, 1995, 1996; Olsen et al., 1995; Hoorfar et al., 1999; Kumar et al., 2003; Lin et al., 2004; Chiu et al., 2005). In our study, two pair of PCR primers (MINf/MINr) based on the 16S ribosomal RNA sequences and (ITSF/ITSR) based on ITS region of 16S23S rRNA sequences were used to detect Salmonella strains. PCR primers MINf/MINr were developed by a modification of the previously described PCR primer 16SFI (Lin and Tsen, 1996) and by constructing a new PCR primer. As primers ITSF/ITSR and MINf/MINr were used for Salmonella detection, the majority of Salmonella respectively. However, one isolate (319) classified as S. kentucky by serotyping was negative by PCR. On the other hand, isolates (143, 274 and 317) classified as
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Figure 1. (A) Lane 1-6: PCR products amplified from ITS region with ITSF/ITSR primers for Salmonella strains. (B) Lane 1-6: PCR products amplified from 16S rRNA gene region using MINf/MINr primers for Salmonella strains. M: molecular weight marker, 100 bp ladder.
a 1
b 2
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M
5
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7
8
9
10
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8
M 1000 pb
500 pb
Figure 2. ITS-PCR amplification patterns of strains belonging to Salmonella genus. (a) Lane 1-4: S. anatum strains, 5-10: S. enteritidis strains. (b) Lane 1-8: S. kentucky strains. Lane M: molecular weight marker (100 bp ladder).
Salmonella by API20E, and non-Salmonella strains (representing Citrobacter and Proteus genera) did not generate any bands (Figure 1). Analysis of API20E, serotyping and specific-PCR amplification showed excellent agreement between the results of Salmonella detection by PCR and serotyping, also between the results of Citrobacter and proteus PCR and API 20E. These results affirm previous studies finding MINf/MINr and ITSF/ITSR PCR to be an accurate tool in the detection of S. enterica isolates (Trkov and Avgustin, 2003; Chiu et al., 2005; Ben Abdallah et al., 2007). However, MINf/MINr and ITSF/ITSR PCR is able to identify only S. enterica and not other Enterobacteriaceae as does API 20E, thus limiting its identification to one specific pathogen. ITS –PCR fingerprinting Since enterobacterial genera represent a significant number of pathogenic microorganisms whose identification
and characterization is particularly important, polymorphisms of the spacer region between the 16S and 23S rRNA genetic loci were studied. PCR amplification of the ITS was run for all Salmonella, Citrobacter and Proteus strains. Salmonella strains Figure 2 shows the patterns of 16S-23S spacer amplication products for Salmonella serotypes. The comparison of the different profiles showed reproducible patterns consisting of 2 to 6 bands ranging from 425 to 850 bp. 11 different ITS patterns were produced, encompassing of 4 (S1), 20 (S2), 9 (S3), 36 (S4), 20 (S5), 4 (S6), 3 (S7), 2 (S8), 1 (S9), 1 (S10) and 1 (S11) isolates. These patterns were characterized by the same pair of fragments at 425 and 620 bp, which were common to all the stains. In addition, six variable bands were observed at 450, 470, 510, 575, 660 and 850 bp. The diversity of the profiles depends on the presence or the absence of these bands (Table 1).
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For example, two bands were observed at 510 and 850 bp in three strains of S. anatum and one strain of S. amsterdam (profile 1). Only one band was observed for one strain of S. anatum, six strains of S. enteritidis, seven strains of S. zanzibar, three strains of S. gallinarum, one strain of S. typhimurium and two strain of S. kentucky (profile 2). For the profile 3, two variable bands were observed at 450 and 470 bp for two strains of S. enteritidis, one strain of S. zanzibar and six strains of S. kentucky. Also, it was noted that different serotypes examined could be represented by the same profile and one serotype examined could by represented by different profiles. Proteus strains Figure 3A represents the patterns of 16S-23S spacer amplification products for 29 isolates belonging to Proteus genus. ITS-PCR showed reproducible patterns consisting of 2 to 5 bands ranging from 320 to 750 bp. Seven different ITS profiles were produced, encompassing of 9 (Pr1), 4 (Pr2), 3 (Pr3), 1 (Pr4), 7 (Pr5), 1 (Pr6) and 4 (Pr7) isolates. These patterns were characterized by common pair of bands at 475 and/or 590 bp. In addition, three variable bands were observed at 320, 550 and 750 bp (Table 1). Citrobacter strains Figure 4 represents the patterns of 16S-23S spacer amplification products for 42 stains belonging to Citrobacter genus. ITS-PCR showed reproducible patterns consisting of 4 to 6 fragments ranging from 320 to 1400 bp. Four different ITS patterns (C1 to C4) were obtained. These patterns were characterized by three common bands at 490, 620 and 690 bp. In addition, six variable bands were observed at 320, 580, 820, 1000, 1200 and 1400 bp (Table 1). The four suspect Salmonella isolates (143, 274, 317 and 319) were belonged to Citrobacter patterns: C1 (strain 274), C2 (strains 317 and 319) and C4 (strain 143). Both Salmonella specific PCR assays and ITS fingerprinting produced identical classification of these suspect isolates. These results were validated by comparison with 16S rRNA gene. The reliability of the results presented earlier was enhanced by complete agreement with the 16-23S PCR results as well as the 16S rRNA sequencing. In previous studies, PCR ribotyping based on the amplification of the intergenic spacer region sequences between the 16S and the 23S rRNA genes provide higher level of bacterial differentiation (Christensen et al., 2000; Wenner et al., 2002; Liguori et al., 2011). In the present study, amplification of the 16S-23S spacer regions of Salmonella, Proteus, and Citrobacter isolates showed high degree of polymorphism with 11, 7 and 4 different ITS patterns, respectively.
The different isolates generated a product profile containing elements which are characteristic of the genus. Salmonella strains were characterized by a common pair of fragments at 425 and 620 bp; Proteus and Citrobacter isolates demonstrate common bands at (475 and 590 bp) and at (490, 620 and 690 bp), respectively. This type of bands allowed the characterization of the genera. In addition, representative species selected from the genera Citrobacter and Proteus also demonstrated interspecies diversity. In example, C. youngae and C. braakii are characterized by bands at (820, 1400 bp) and at 1000 bp, respectively and can be used in the recognition of species. These results are in agreement with the findings of Jensen et al. (1993) and Clementino et al. (2001). According to these authors, the PCR amplification of the 16S-23S spacer region showed significant tool for the identification of different genera as well as many bacterial species. Basing on the result presented earlier, Salmonella isolates (143, 274, 317) which were negative by MINf/MINr and ITSF/ITSR PCR, classified by API20E as Salmonella at the likelihood levels about 89.4% and untypeable by serotyping were classified in Citrobacter genus. Moreover, the strain (319) identified as S. kentucky by serotyping belonged also to Citrobacter genus. These results were validated by comparison with 16S rRNA sequencing which classified C143, C317 and C319 isolates as Citrobacter freundii with 98 to 100% of sequence similarity and C274 as Citrobacter braakii with 99% of sequence similarity. 16S rDNA sequence analysis and phylogenetic tree A phylogenetic tree constructed by the neighbour-joining method and comparing the16S rRNA sequences is shown in Figure 5. On the basis of BLAST analysis of 16S rRNA gene similarity, several species belonging to Proteus and Citrobacter genera were recognized. In genus Proteus, four species were identified as P. mirabilis, P. Vulgaris, P. penneri and P. Hauseri. However, in genus Citrobacter, three species were identified as C. Freundii, C. youngae and C. braakii. A total of 5 isolates (P205, P105, P235, P238b and P209) belong to P. mirabilis, one isolate (P239) belongs to P. penneri, one isolate (P219b) belongs to P. vulgaris, one isolate (P207a) belongs to P. hauseri, six isolates (C111, C143, C154, C317, C319, C220) belong to C. freundii, one isolate (C23) belongs to C. youngae and one isolate (C274) belongs to C. braakii. The 16S rRNA sequencing classified the Salmonella suspect isolates (underlined strains) as C. freundii with 98 to 100% of sequence similarity and as C. braakii with 99% of sequence similarity. The isolate Pseudomonas mendocina (Ps12) was used in the phylogenetic tree as an out group. In genus Salmonella, all the isolates belonged to S. enterica specie (Table 2). Phylogenetic analy-ses of 40 representative isolates based on neighbour-joining method with 1000 bootstrap sampling
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Table 1. ITS patterns and sizes of spacer amplification products by genera and species.
Genera
Proteus
Citrobacter
ITS pattern 1 2 3 5 4 6 7
Size (bp) 475, 550, 590, 750 320, 475, 550, 590, 750 475, 550 320, 475 590, 750 475, 590 320, 475, 550, 590
Species/serotypes Proteus mirabilis
1
320, 490, 620, 690, 1000, 1200
Citrobacter brakii
2 3 4
320, 490, 580, 620, 690, 1200 490, 620, 690, 820, 1400 320, 490, 620, 690
Citrobacter freundii Citrobacter youngae Citrobacter freundii
1
425, 510, 620, 850
Proteus penneri Proteus vulgaris Proteus hauseri
S. anatum S. amsterdam
2
425, 620, 850
S. anatum S. enteritidis S. zanzibar S. gallinarum S. typhimurium S. kentucky
3
425, 450, 470, 620
S. enteritidis S. zanzibar S. kentucky
4
425, 450, 620, 660
S. kentucky S. amsterdam S. mbandaka S. typhimurium
5
425, 450, 620
S. kentucky S. amsterdam S. typhimurium
6
425, 620, 660
S. zanzibar S. montevideo S. typhimurium
7
425, 575, 620, 660, 850
S. derby S. newport
8 9 10 11
450, 620 450, 620, 660 425, 450, 575, 620, 660, 850 425, 450, 620, 850
S. S. S. S.
Salmonella
mbandaka kentucky montevideo mbandaka
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Table 2. GenBank accession numbers of 16S rRNA gene sequences of Salmonella, Proteus and Citrobacter representative isolates. C1-C4: Citrobacter patterns, Pr1-Pr7: Proteus patterns, S1-S9: Salmonella patterns.
Bacterial strains Ps12 C23 C111 C143 C154 C220 C274 C317 C319 Pr 105 Pr 205 Pr 207a Pr 209 Pr 219b Pr 235 Pr 238b Pr 239' S164 S169 S176 S179 S256 S257 S260 S279 S293 S294 S297 S302 S307 S308 S313 S324 S330 S331 S332 S334 S338 S352 S353
rDNA accession number JN118493 JN118494 JN118497 JN118498 JN118499 JN118500 JN118501 JN118502 JN118503 JN118504 JN118505 JN118506 JN118507 JN118508 JN118509 JN118510 JN118511 JN118512 JN118513 JN118514 JN118515 JN118516 JN118517 JN118518 JN118522 JN118525 JN118526 JN118527 JN118529 JN118530 JN118531 JN118532 JN118535 JN118536 JN118537 JN118538 JN118539 JN118540 JN118542 JN118543
Closed species Pseudomonas mendocina Citrobacter freundii Citrobacter freundii Citrobacter freundii Citrobacter freundii Citrobacter freundii Citrobacter freundii Citrobacter freundii Citrobacter freundii Proteus mirabilis Proteus mirabilis Proteus hauseri Proteus mirabilis Proteus vulgaris Proteus mirabilis Proteus mirabilis Proteus penneri Salmonella enterica subsp. enterica Salmonella enterica subsp. enterica Salmonella enterica subsp. enterica Salmonella enterica subsp. enterica Salmonella enterica subsp. enterica Salmonella enterica subsp. enterica Salmonella enterica subsp. enterica Salmonella enterica subsp. enterica Salmonella enterica subsp. enterica Salmonella enterica subsp. enterica Salmonella enterica subsp. enterica Salmonella enterica subsp. enterica Salmonella enterica subsp. enterica Salmonella enterica subsp. enterica Salmonella enterica subsp. enterica Salmonella enterica subsp. enterica Salmonella enterica subsp. enterica Salmonella enterica subsp. enterica Salmonella enterica subsp. enterica Salmonella enterica subsp. enterica Salmonella enterica subsp. enterica Salmonella enterica subsp. enterica Salmonella enterica subsp. enterica
resulted into four major clusters (Figure 5). Of 40 isolates, cluster I formed with 23 isolates representing the Salmonella genus; cluster II formed with 8 strain representing the Citrobacter genus; cluster III formed with 8 strains representing the Proteus genus, and cluster IV formed with one strain representing the Pseudomonas genus. The
Accession number FJ828887 GQ 983053 FJ405288 AB548830 EU545403 NR 028687 AF025368 JN118499 HQ677190 FJ581028 FJ581028 DQ885262 JN092590 AB545932 EF626945 GU569875 FJ971869 DQ344532 CP002614 CP002614 DQ344532 CP002614 DQ344532 DQ344532 HM007577 DQ344532 HM007577 DQ344532 AP011957 DQ344532 CP002614 DQ344532 AP011957 HM007577 DQ344532 FQ312003 DQ344532 DQ344531 CP002487 HQ268500
Similarity (%)
Size (bp)
Pattern
100 99 99 98 100 99 99 97 100 99 98 99 99 99 99 98 99 99 99 100 99 99 99 98 98 99 97 100 98 100 98 99 99 98 99 98 99 97 98 97
525 624 604 540 596 597 510 556 489 618 667 610 632 661 605 504 647 424 434 407 644 715 409 341 581 707 510 397 684 397 543 692 520 663 393 503 444 546 651 562
C3 C2 C4 C2 C2 C1 C2 C2 Pr2 Pr1 Pr7 Pr1 Pr6 Pr3 Pr5 Pr4 S5 S5 S4 S4 S4 S5 S2 S2 S4 S2 S3 S2 S5 S2 S1 S6 S9 S2 S6 S4 S7 S8 S6
main clusters obtained reflect the species and are generally concordant with the 16S-23S PCR results. Salmonella was genetically closer to Citrobacter such as Proteus. However, Pseudomonas strains used as out group appear clearly distant from the rest. The four strains C143, C317, C319 and C274 are definitively classified in
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M
1
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4
5
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7
1500 bp 1000 bp
500 bp
Figure 3. Lane 1-7: ITS-PCR amplification patterns of strains belonging to Proteus genus. Lane M: molecular weight marker (100 bp ladder).
M
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4
M 1500 bp
1000 bp
500 bp
Figure 4. Lane 1-4: ITS-PCR amplification patterns of strains belonging to Citrobacter genus. Lane M: molecular weight marker (100 bp ladder).
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Figure 5. Neighbor-joining phylogenetic tree of 16S rRNA sequences of 39 isolates and their closest phylogenetic relatives. Sequences of the compared strains were obtained from databases, and the accession numbers are in parenthesis. The tree topology was constructed using Clustal X (1.81).
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Citrobacter genus. Conclusion The results of this study have demonstrated that API20E, serotyping associated to Salmonella specific PCR using primers MINf/MINR and/or ITSF/ITSR are accurate methods for S. enterica detection. In addition, PCR amplification of the 16S-23S spacer region was suitable tool for identification of bacteria at genera and species level. It was found to be simple and rapid method compared to conventional method. The 16S rRNA gene sequencing allowed assignation of strains to species and subspecies, and aimed at more precise phylogeny. REFERENCES Aldridge KE, Gardne BB, Clark RSJ, Matsen JM (1978). Comparison of Micro-ID, API 20E, and conventional media systems in identification of Enterobacteriaceae. J. Clin. Microbiol. 7:507–513. Aldridge KE, Hodges RL (1981). Correlation studies of Entero-Set 20, API 20E, and conventional media systems for Enterobacteriaceae identification. J. Clin. Microbiol. 13:120–125. Amavisit P, Browning GF, Lightfoot D, Church S, Anderson GA, Anderson GA, Whithear KG, Markham PF (2001). Rapid PCR detection of Salmonella in horse faecal samples. Vet. Microbiol. 79:63-74. Baudart J, Lemarchand K, Brisabois A, Lebaron P (2000). Diversity of Salmonella strains isolated from the aquatic environment as determined by serotyping and amplification of the ribosomal DNA spacer regions. Appl. Environ. Microbiol. 66:1544-1552. Ben Abdallah F, Chaib K, Snoussi M, Bakhrouk A, Gaddour K (2007). Phenotypic variations and molecular identification of Salmonella enterica serovar Typhimurium cells under starvation in seawater. Curr. Microbiol. 55:485-491. Chen WP, Kuo TT (1993). A simple and rapid method for preparation of negative bacterial genomic DNA. Nucleic Acids Res. 21:2260. Chiu TC, Chen TR, Hwang WZ, Tsen HY (2005). Sequencing of internal transcribed spacer region of 16S-23S rRNA gene and designing of PCR primers for the detection of Salmonella spp. In food. Int. J. food Microbiol. 97: 259-265. Christensen H, Moller PL, Vogensen, FK, Olsen JE (2000). Squence variation of the 16S to 23S rRNA a spacer region in Salmonella enterica. Res. Microbiol. 151:37-42. Clementino MMC, DeFilippis I, Nascimento CR, Branquinho R, Rocha CL, Martins OB (2001). PCR analyes of tRNA intergenic spacer, 16s23S internal transcribed spacer, and randomly amplified polymorphic DNA reveal inter- and intraspecific relationships of Enterobacter cloacae strains. J. Clin. Microbiol. 39:3865-3870. Hoorfar J, Ahrens P, Radstrom P (2000). Automated 5-nuclease assay for identification of Salmonella enterica. J. Clin. Microbiol. 38:3429– 3435. Hoorfar J, Baggesen DL, Porting PH (1999). A PCR-based strategy for simple and rapid identification of rough presumptive Salmonella isolates. J. Microbiol. Meth. 35:77-84. Jensen MA, Webster JA, Straus N (1993). Rapid identification of bacteria on the basis of polymerase chain reaction- amplified ribosomal DNA spacer polymorphisms. Appl. Environ. Microbiol. 59:945-952. Jones YE, McLaren IM, Wray C (2000). Laboratory aspects of Salmonella, In C. Wray and A. Wray (ed.), Salmonella in domestic animals, CABI Publishing, Wallingford, United Kingdom, pp 393–405. Koneman EW, Allen SD, Janda WM, Schreckenberger PC, Winn WC (1997). The Enterobacteriaceae, In A. Allen, H. Collins, S. Deitch, H. Ewan, K. Rule, and K. Kelley-Luedtke (ed.), Color atlas and textbook of diagnostic microbiology, Lippincott-Raven Publishers, Philadelphia,
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