Chemosystematic of Enterobacteriaceae Familia Obtained from Blood Cultures Based on Total Protein Pro fi les

The purpose of this study was to determine the chemosystematic of 14 strains of bacteria in blood cultures from Semarang using 1 reference strain S. typhi NCTC 786, based on the total protein profi les with the similarity relationship analysis based on Simple Matching Coeffi cient (SSM) analysis and algorithm method of unweighted pair group with averages (UPGMA) presented in a dendrogram. The results showed that the chemosystematic based on the total protein profi les using SDS-PAGE method can classify the member of bacterial strains of each species. The Clusters respectively consist of 4 strains of S. typhi (similarity: 89.7%), 2 strains of Ser. marcescens (similarity: 89.7%), two strains of E. coli, and one strain of Salmonella ssp, S. typhi NCTC 786 (similarity: 100%). Those three incorporated clusters had the similarity value of 75.3%. Those four strains of Ent. cloacae composed in one cluster (similarity: 100%) are incorporated in a cluster consisting of one strain of Kleb. pneumoniae (similarity: 92.9%). Both clusters were incorporated in a cluster consisting of S. typhi NCTC 786 (similarity: 67.9%).


Introduction
I n S e m a r a n g , t y p h o i d f e v e r is determined as the third of 10 major diseases after Dengue Fever, diarrhea, and gastroenteritis (Anonymous, 2008).Typhoid fever is a systemic infectious disease caused by Salmonella typhi bacteria (S. typhi), with no specifi c clinical symptoms, thus the gold standard diagnosis of this disease should be conducted in laboratory tests (Koharo et al., 2010;Ley et al., 2010;Fadeel et al., 2011).
Widal test is considered as the simplest, easiest, cheapest and fastest laboratory test.However, its sensitivity, specificity and prediction value are varies because anti-O and anti-H can be found in patients infected by the member of Enterobacteriaceae familia species beside S. typhi (Novianti, 2006;Beig et al., 2010).The fi nding of S. typhi in blood cultures or in bone marrow is the gold standard of typhoid fever (Khoharo et al., 2010;Ley et al., 2010).Blood culture success rate varies from 40% to 89% compared to S. typhi isolation success rate.The success of obtaining S. typhi isolates from positive Widal blood cultures was 10.74% (Amarantini et al.2009).These showed that there are other bacteria types beside S. typhi such as Salmonella ssp., Escherichia coli (E.coli), Enterobacter cloacae (Ent.cloacae), Serratia marcescens (Ser.marcescens), and Klebsiella pneumoniae (Kleb.pneumoniae) (Darmawati et al., 2012).
Identification of species and strains diversity of bacteria can be conducted with polyphasic systematic approach, by combining the systematic of phenetics numeric, and molecular (Sembiring, 2004;Vandamme et al., 1996).This approach uses phenotypic and genotypic characteristics.The biochemical and chemical characteristics of total protein profi le bacterial cell with sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) method can be used as chemical classifi cation by numerical methods (Berber, 2004;Berber and Yenidunya, 2005;Amarantini, 2010;Yanti, 2011).The Diversity of bacterial species including Enterobacteriaceae familia and strain diversity of the member of each species can be classifi ed based on the physiological characteristic differences, such as the ability of fermenting various carbohydrates, and its resistance to many antibiotics (Darmawati et al., 2011;Darmawati et al., 2012).This shows the existence of genetic variations in both inter-species and inter-individual member of each species.
Thus, the objective of was to classify bacteria of Enterobacteriaceae familia members based on its phenetics numeric in positive Widal blood cultures from Semarang based on its total protein profiles using UPGMA algorithm (unweighted pair group method with averages), so it enable to describe the similarities and differences of total protein profi les as genetic expression owned by each individual member of each species of bacteria, including Enterobacteriaceae that further can describe its morphology.

The soluble total protein isolation and protein separation
The soluble total protein was obtained by growing a colony of bacteria in 100 ml of BHI liquid, incubated at 37˚ C for 18 h.Bacteria cultures were then centrifuged at the temperature of 4˚C, at the speed of 3000 rpm for 20 min.Presipitans were washed by suspending them using 0.1 M PBS (Phosphate buffered saline) at pH 7.4.These washings were conducted 3 times.the presipitans then were re-suspended in 1.5 ml PBS at pH 7.4.The bacterial suspensions were sonicated 6 times in 30 sec with the rest period of 30 sec at 4˚C. repeating duty cycle Amplitude used was 0.7.The results of bacterial sonicated suspension were centrifuged at the temperature of 4 ˚C, at the speed of 12 000 rpm for 20 min.Supernatant obtained was the soluble total protein cells which protein concentration was readily measured using the reagent of Protein Assay CBB solution (5x) (Nacalai Tesque Inc.: code 29440-44, Kyoto Japan).The soluble protein absorption was then measured at a wavelength of 595 nm and separated using SDS-PAGE methods with ATTO corporation ATTA Tokyo Japan (AE-6530M/AE-6530P), stained with 0.25% Coomassie Brilliant Blue R250.

Data analysis
Data coding, unit character coding was done by giving a score.Positive unit character (+) was given score 1 while the negative unit character (-) was given score 0. The edited

Results and Discussion
The separated soluble total protein bacterial cells by SDS-PAGE (Figure 1A and 2A), was visualized with diagrammatic representation (Figure 1B  T h e D e n d r o g r a m s h o w i n g t h e similarity relationship between 9 strains of Enterobacteriaceae familia members and 1 reference strain S. typhi NCTC 786 (Figure 3) was composed into two clusters.The fi rst cluster consists of four strains of S. typhi composed of 3 incorporated sub-clusters with the similarity value of 89.7%.The fi rst sub-cluster consists of two strains (KD 30.3 and BA 07.4) with the similarity value of 93.1%, the second and third sub-clusters each respectively consists of 1 strain; KD 30.3 and 02.2 SA.The second cluster was composed of two incorporated sub-clusters with the similarity value of 81.0%.The first subcluster consists of two S.marcescens strains (KD 08.4 and KD 08.5) with the similarity value of 89.7%, and the second sub-cluster was composed of four strains of bacteria consisting of two E. coli strains (BA 30.1 and BA 30.2), 1 Salmonella ssp.strain BA 30.5 and one reference strain of S. typhi NCTC 786 with incorporated similarity value of 100%.The incorporated similarity value of the fi rst and second clusters is 75.3%.
The similarity relationship between 5 strains of Enterobacteriaceae familia members and 1 reference strain of S. typhi NCTC 786 is shown in the dendrogram (Figure 4) composed of two incorporated clusters with the similarity value of 67.9%.The fi rst cluster is composed of two sub-clusters; the fi rst sub-cluster consists of 4 strains of Ent.cloacae (BA 45.4.1, TG 03.5, KT 16, SA 02.1) with the similarity value of 100%, while the second sub-cluster consists of one strain of Kleb.pneumoniae KD 58.4.
Dendrogram (Figures 3 and 4) may indicate the diversity of bacteria species of Enterobacteriaceae familia members in clusters or sub-clusters levels.The strain members of S. typhi, Ser.marcescens, E. coli and Salmonella ssp.have higher similarity (75.3%) upon the reference strain of S. typhi NCTC 786 than the strain members of Ent.cloacae and Kleb.pneumoniae upon the same reference strains (67.9%).Both dendrogram composed based on the total protein profi le has a similar topology with the dendrogram based on phenotypic characters characterized using API 20E and API 50CHB/E media (Darmawati et al., 2012).The results of this study are also in accordance with several other studies that the total protein profi le analysis using SDS-PAGE method is identical to identification and classification results based on the biochemical characteristics (Amarantini, 2010;Yanti, 2011). Vandamme et al. (1996) states that the total protein profiles obtained with SDS-PAGE method can be used to compare and classify a number of strains.In addition, some studies have also shown that the total protein profi les have a high similarity with the results of DNA-DNA hybridization.
The results of classifi cation based on biochemical characteristics seem congruent with the classification results based on the total protein profiles.These because biochemical characteristics are the enzymatic activity refl ections that enzyme is composed of protein while protein is genome refl ection, thus the classifi cation based on biochemical characteristics and total protein profi les will result an accurate classifi cation.