Phylogenetic relationship of Gram Negative Bacteria of Enterobacteriaceae Family in the Positive Widal Blood Cultures based on 16S rRNA Gene Sequences

The purpose of this study was to analyze the phylogenetic relationship of Gram negative bacteria (3 strains of Salmonella typhi, 1 strain of Escherichia coli, 1 strain of Serratia marcescens, and 3 strains of Enterobacter cloacae) of Enterobacteriaceae family in positive Widal blood cultures based on 16S rRNA gene sequences. The results respectively showed that each two 16S rRNA gene clones of Serratia marcescens KD 08.4 had a close relationship with 16S rRNA gene of Serrratia marcescens ATCC 13880 (similarity: 99.53-99.8%), Eschericia coli BA 30.1 with Eschericia coli ATCC 11775T (similarity: 99.38-99.67%), Salmonella typhi BA 07.4, Salmonella typhi KD 30.4, and Salmonella typhi SA 02.2 with Salmonella typhi ATCC 19430T (similarity: 99.4-100%) as well as the isolates of Enterobacter cloacae SA 02.1, Enterobacter cloacae BA 45.4.1, one 16S rRNA gene clone of Enterobacter cloacae TG 03.5 with Enterobacter cloacae ATCC 23373 (similarity: 99.0-99.87%).


Introduction
The typhoid fever incidence rate in Indonesia had reached 358-810/100.000 population/year with the mortality rate of 1-5% of patients (Anonymous, 2007). In Semarang, typhoid fever had been in the third rank of 10 major diseases after Dengue Fever and Diarrhea and gastroenteritis (Anonymous, 2008). Typhoid fever was a serious systemic infectious disease that was possibly accompanied by a variety of diseases such as dengue fever and malaria (Gasem et al., 2002).
The clinical features of typhoid fever were unspecific that the gold standard diagnosis could not only depend on the clinical symptoms but it should also be supported by the laboratory diagnosis (Khoharo et al., 2010;Ley et al., 2010;Fadeel et al., 2011). The gold standard diagnosis of typhoid fever was by the finding of Salmonella typhi (S. typhi) in blood or bone marrow cultures (Khoharo et al., 2010;Ley et al., 2010). However, the facilities for culturing of bacteria were not always available, it was expensive, time consuming (seven days) and the result was frequently negative since the patients had consumed antibiotics. Widal test was a widely used laboratory test in Indonesia supporting the typhoid fever diagnosis as it was cheap, easy, fast, and simple. The sensitivity, specifi city, and predictive values of Widal test were various, due to the presence of anti-O and anti-H antibodies in patients infected by Salmonella sp., species of Enterobacteriaceae family member other than Salmonella sp. and malaria (Novianti, 2006;Beig et al., 2010). Darmawati et al., 2012, stated that there was bacterial species diversity of Enterobacteriaceae family members of such as S. typhi, Serratia marcescens, Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae in positive Widal blood cultures from Semarang. However, the phylogenetic relationship between species was unknown. Thus, the purpose of this study was to determine the phylogenetic relationship of Gram-negative bacillus bacteria of Enterobacteriaceae family members based on 16S rRNA gene sequences.

Bacterial Strains
There was a total of 8 isolates (3 isolates of Salmonella typhi, 1 isolate of Escherichia coli, 1 isolates of Serratia marcescens, and 3 isolates of Enterobacter cloacae) isolated from positive Widal blood samples of in and outpatients from Semarang (Tugurejo hospital, City Hospital of Semarang, Sultan Agung Islamic Hospital, Community Health Center of Bangetayu, and Community Health Center of Kedungmundu). The bacterial Identifi cation used API 20E and API 50CHB/E media (Darmawati, et al., 2012).

DNA bacterial Extraction, PCR amplifi cation, cloning, and DNA plasmid extraction with insertion and sequencing
DNA was extracted from eight bacterial strains used DNeasy Blood & Tissue Kits (Qiagen, K69504). The 16S rRNA gene amplification used Applied Biosystems GeneAmp PCR System 2400, 0.25μl Takara Ex Taq, 5μl 10X Ex Taq buffer, 4 μl dNTP Mixture (2.5 mM each), 2μl DNA template, 0.5μl primer 8F (1.0μM fi nal conc.), and 0.5μl primer 4192R (1.0μM final conc.), 37.75μl sterile deionized water, for a total volume of 50μl. The thermal cyling was as follows: denaturation at 95 o C for 30 sec, annealing at 55 o C for 30 sec, extension at 72 o C for 1,5 min, and fi nal extention 72 o C for 10 min for the total of 30 cycles. PCR products (1500bp) were visualilized through electrophoresis at 1% agarose gel with ethidium bromide added directly to the gel.
The amplifi ed DNA bands were purifi ed from agarose using glass powder method (Volgstein and Gillespie, 1979), ligated to T-Vector pMD20 (Takara Biotechnology), and transformated to E.coli DH5α. The plasmid DNA containing inserts was isolated, respectively amplified using primer M13 reverse, U515F, and M13-40 (Table 1). The amplified DNA were sequenced using primer M13 reverse, U515F, and M13-40. The DNA Sequencing was conducted with sequencer device of ABI Prism TM 310 Genetic Analizer. The sequenced data were in the form of electrophenogram files and base DNA arrangement.

Analysis and alignment of 16S rRNA gene sequences
The 16S rRNA sequence were analyzed and compared to the Gene Bank nucleotide database using Basic Local Alignment Search Tool (BLAST). The 16S rRNA Sequences of 8 bacterial strains were aligned using CLUSTAL X program.

Phylogenetic tree Construction
Phylogenetic tree was prepared using PHYLIP program, matrix similarity, and nucleotide difference of 16S rRNA between clones and strains analyzed with PHYDIT program.

Results And Discussion
T h e r e s u l t s o f 1 6 S r R N A g e n e amplifi cation of 8 isolates of Enterobacteriaceae family members were shown in Figure 1 while the results of phylogenetic relationship analysis based on 16S rRNA gene sequences were shown in Figure 2. There was a total of 15 sequences with each isolate consisted of two 16S rRNA gene sequences derived from two clones of 16S rRNA gene sequences except TG 03.5 isolate and 5 sequences derived from 16S rRNA sequences of Gram-negative bacillus bacteria of Enterobacteriaceae family (Gene Bank, NCBI) consisting of S. typhi strain types of ATCC 19430 T (accession no. Z47544), E. coli 11775 T (X80725.1), Ent. cloacae ATCC 23373 (HQ651841.1), Citrobacter freundii ATCC 8090 (AJ233408.1), Ser. marcescens ATCC 13880 (AB594756.1). Two strains as out group used Vibrio cholerae ATCC 14547 (NR_044050.1) from Vibrionaceae family (negative catalase and positive oxidase) and Pseudomonas aeruginosa ATCC 23993 (FJ652615.1) from Pseudomonadaceae family (positive catalase and oxidase).
After the 16S rRNA gene sequences was aligned with Clustal-X program, the phylogenetic trees were arranged using PHYLIP program The phylogenetic relationship analysis of 15 16S rRNA gene clones of 8 isolates ( Figure  2) was divided into fi ve clades. The fi rst clade consisted of 6 16S rRNA gene clones derived from 3 isolates (SA 02.2, KD 30.4, and BA 07.4) and 2 reference strains: S. typhi ATCC 19430 T and Ent. cloacae ATCC 23373. The similarity value of those six clones with S. typhi ATCC 19430 T was 99.4 to 100% with the difference of 0-9 nucleotide, shown in Table 2.   The number of 16S rRNA gene copies in bacteria was various (1-15) in each genome. Each copy had the size approximately 1500 bp. Marchandin et al. (2003) reported that the 16S rRNA gene sequences in each copy of every organism were identical. The nucleotide difference of 16S rRNA gene copies was called micro-heterogeneity. The 16S rRNA gene sequences in 2 different 16S rRNA gene clones from the same isolates showed the presence of nucleotide difference with the similarity value of 99-100%. The results of this study was similar with the fi ndings of Marchandin et al. (2003), that 4 16S rRNA gene copies in one bacterial strain of Veillonilla sp. ADV 360.1 showed two identical gene copies (similarity 100 %) and two various gene copies (similarity 98.5 to 99.8%).
The fourth clade consisted of one reference strain of Citrobacter freundii ATCC 8090. The fi fth clade consisted of 2 16S rRNA gene clones derived from isolate KD 08.4 (similarity 99.53-99.8 %) and one reference strains of Ser. marcescens ATCC 13880 with nucleotide difference number of 3-7 nucleotides, were shown in Table 5.
Based on the nucleotide similarity value, it could be concluded that isolate KD 08.4 was identifi ed as the isolate of Ser. marcescens KD 08.4 included as Ser. marcescens members.
The constructed phylogenetic trees based on Neighbor-Joining algorithm ( Saitou and Nei, 1987 ) Chang, et al. (1997). Besides using numerical systematic based on phenotypic characters and chemical systematic, the bacterial identifi cation could also conducted by using the molecular character systematic based on the nucleic acids, such as based on 16S rRNA genes. 16S rRNA genes were conserved genes which were present in all bacteria, that could be used to classify bacteria based on the kinship relationship. The genomic analysis was better than the protein analysis since it did not rely on certain genomic expression which encoded proteins and might result in phenotypic variation (Priest and Austin, 1995;Vandamme et al., 1996;Giammanco et al., 1999).
The results of phylogenic analysis based on 16S rRNA gene sequences showed that the isolates presented in positive Widal blood were Ser. marcescens KD 08.  Darmawati et al. (2013), showed that the classifi cation based on biochemical characters was congruent with the results of classifi cation based on total protein. This was also congruent with the classifi cation based on 16S rRNA gene sequences. It might happen as the biochemical characteristics was the refl ection of enzyme activities. The enzyme was functionally active protein as a result of translated expressed gene in a genome. Thus, the classification combining between the classifi cation based on phenotypic, chemical, and molecular might result in accurate classifi cation.