Molecular Identification of Bacterial Species from Musca domestica l. and Chrysomya megachepala l. in luwuk City, Central sulawesi, indonesia

House flies (Musca domestica l.) and green flies (Chrysomya megachepala l.) are two species of flies that are vectors of many types of bacteria in humans and animals throughout the world. the research was conducted to determine the sequence of the 16S rRNA gene and thus to conduct the phylogenic position, the bacteria isolated from Musca domestica and Chrysomya megachepala L used the 16S rRNA gene as a barcode. The bacteria identified were bacteria that have been tested to produce antibiotics. Pure cultures of each of the seven bacterial isolates were used for DNA analysis dan molecular identification. The results showed that the S1 isolate had a 90% similarity with the Bifidobacterium minimum [NR 044692.2]. Isolate S2, showing similarity of 86% with Alcaligenes faecalis [CP021883.1]. Isolate S3 has similarty 94% Brucella melitensis bv. [NR_076080.2]. Isolate S4 has a similarity of 99% with Sphingobacterium sp. [LK931720.1]. Isolate S5 has a similarity of 100% with Bacillus sp. [MH071158.1]. Isolate S6 has a similarity of 97% with Gamma proteobacterium [KJ127178.1]. While isolate S7 has a similarity of 100% Enterobacter asburiae [MH071322.1]. Species of bacteria identified from green flies and house flies that inhabit the city of Luwuk have never been reported to be associated with flies.

domestica and C. megacephala are two species of flies that live in human settlements. Both species of flies have evolved with various species of pathogenic bacteria in humans, so it is very potential to transmit new species of pathogenic bacteria in humans 1,12 . Furthermore, C. megacephala is able to transmit 11-12 times more bacterial colonies than Musca domestica 11,13 .
House flies potential mechanical or biological vectors for the dissemination of pathogenic and multidrug-resistant bacteria 8,15,16 .
In spite of these pathogenic microbes do not infect house flies. House flies have developed self-defense mechanisms against pathogenic microbes associated with the mouth organ, digestive tract and body surface 9,17 . Researchers from Macquarie University found antibiotics produced by microbes associated with flies, at each stage of fly development 18 . It should be suspected that house flies play a role in spreading antibiotic-resistant bacteria 3,19,20 . For this reason, research into the identification of bacteria from flies in various places in the world needs to be done continuously.
Isolation and identification of bacteria in house flies from various geographical locations is important to solve the types of pathogenic bacteria associated with flies. In this study, flies were obtained from the city of Luwuk. Luwuk is a coastal city in Central Sulawesi which, according to data from the Central Sulawesi Provincial Health Office in 2018, has a high digestive tract pathogenic infection. The location of the fly sample was obtained from several characteristics of the fly's habitat, namely the traditional market area, residential areas, and hospitals. This research was conducted to identify molecular species of bacterial isolates from flies from various habitats in the city of Luwuk. The bacteria identified are bacteria that in previous studies have been known to produce antibiotics against other pathogenic bacteria.

MATeRTIALS AND MeTHoDS Samples
Flies samples were taken from two locations in the city of Luwuk namely hospital waste dumps and traditional market waste dumps. Flies are caught with insect catching nets, directly preserved in sterile bottles sampels. Based on morphological identification, house flies and green flies were preserved separately in bottles.

Isolate bacteria from flies Isolation of bacteria from wings of flies
The flies were preserved with sterile bottles samples before being used as a source of bacterial inoculum, preserved again at sterile tube 60 minutes After 60 minutes, the fly is transferred to a sterile petri dish with a filter paper mat to remove residue on the surface of the fly's body. Next, the flies were put into a test tube containing 9 ml of physiological NaCl, incubated for 15 minutes. In a test tube containing flies, 1 ml of suspension is taken, then poured into a petri dish. Subsequently, the petri dish was added to the nutrient agar medium with a volume of 15 ml and then homogenized. The culture was incubated at 35 0 C for 2 x 24 hours. Each isolate that grew, was distinguished by colony shape, color, and edge of the colony in pure culture on two nutrient agar and incubated for 1 x 24 hours at 35°C. Colonies that grow in the subculture on sloping agar are then used for morphological analysis, gram staining, biochemical tests and DNA analysis.

Isolate bacteria from the fly's digestive tract
Flies were preserved with 95% ethanol before being used as a source of bacterial inoculums, preserved again on 95% ethanol in new bottles for 60 minutes. After 60 minutes, flies are placed on sterile petri dishes with sterile filter paper to absorb the remaining ethanol on the surface of the fly's body. Then, flies are noticed in the new sterile petri dish. Using a surgical instrument, cut longitudinally from the posterior direction (head) to the tail so that it is separated into two parts. Sterile cotton is applied from the head to the fly's body parts, then inoculated in agar nutrient media. The culture was incubated at 35°C for 1 x 24 hours. Each isolate that grew, was distinguished by colony shape, color, and edge of the colony in pure culture on two nutrient agar and incubated for 1 x 24 hours at 35°C. gram, biochemical testing and DNA analysis.

Molecular identification Preparation of Bacterial Isolate
Prior to DNA extraction and Purification, the purified bacterial isolate was previously inoculated into agar nutrient media and incubated for 1 x 24 hours at room temperature. Bacteria were isolated from the digestive tract of the green fly (Chrysomya megachepala) and the wing of the house fly (Musca domestica L.). Growing bacterial colonies are used as a source of bacterial cell suspension for DNA analysis. extraction and Purification of DNA Bacterial suspension from pure culture is prepared aseptically. The bacterial isolate DNA was extracted with Presto TM Mini gDNA Bacteria Kit Geneaid as described in the manufacturer's protocol, with modification by researchers. Suspension of bacterial isolates was taken as much (1 x 109 cells) / 200 µl, then put into a 1.5 ml sample tube. Subsequently added 200 µl of positive gram buffer (+), lysozyme (± 0.8 mg / 200 µl), then vortex until lysosim dissolves. Incubated at 37°C for 90 minutes. Next, add 20 µl proteinase K, then vortex. Incubate at 600C for 10 minutes, during incubation, the tube is rocked every 5 minutes. Next, add 200 µl GB of the buffer, mix with vortex for 10 seconds. Incubate at 70°C for 10 minutes, making sure the lisis is clear. Meanwhile, heat the elution on a thermostat with a temperature of 70°C. 200 µl of ethanol are added, which mixes quickly.
The GD column is inserted into the 2 ml collection tube, then transfer the mixture to the GD column. Centrifugation was carried out at 14000 -16000 g for 2 minutes. The collection tube is separated from the GD column, then enter the GD column in the new collection tube. 400 µl of W1 buffer was added to the GD column, then centrifuged at 16000 g for 30 seconds. Move the GD column to the new collection tube, then add 600 µl wash buffer to the GD column. Centrifuge at 16,000 g for 30 seconds, then move the GD column to the new collection tube. Centrifuge for 3 minutes at 16000 g to ensure dry/clean matrix column. Move the GD column to the new microcentrifuge tube. Add 100 µl of heated elution buffer and incubate for 3 minutes. Centrifuge at 16,000 g for 30 seconds. Transfer the total DNA to the DNA sample tube that has been previously labeled.

Analysis of DNA Concentration and Purity
The results of total DNA extraction were then analyzed for concentration and purity using a Lambda 35 Perkin Elmer UV / VIS Spectrometer. DNA purity can be seen with A260 / A280 ratio values between 1. 8 Table 2 and Table 3). Visualization of PCR products using the agarose electrophoresis method of 1.8%. Electrophoresis results were observed using a UV Chamber to obtain electrogram images.

Sequence processing and analysis
Sequencing uses ABI PRISM 3730xl  22 . The phylogeny tree construction was also carried out online at the NCBI website.

ReSuLTS
A total of seven bacterial isolates were successfully isolated and cultured pure. One isolate came from the digestive tract of a green fly and six isolates were successfully grown from the wing of a house fly (Table 4).
Five isolates had a white colony, one isolate had a grayish-white colony and one isolate had a yellow colony. As for the colony shape characteristics, two isolates had an irregular colony shape, three-round isolates, one isolate in the form of diffuse filaments and one isolate in the form of fine filaments. The characteristics of isolate cells were obtained by four gram-positive isolates, two gram-negative isolates and one unidentified isolate (   (Table 6).
Concentration and purity of total bacterial DNA greatly influence the success of 16S rRNA gene amplification by PCR method 24,25 . The optimal total DNA concentration based on the kit protocol used is 30 µg / µl up to 60 µg / µl, while for total DNA purity it is in the range of 1.7 -2.0 (A260 / A280). Reach a conclusion, the concentration of DNA of seven bacterial fly isolates was good. The total DNA purity obtained is quite good. Modified protocols at the immersion stage of proteinase-K and RNAase have been shown to increase concentration but not to total DNA purity.
However, the quality of the total bacterial DNA as a template will be known after the 16S rRNA gene amplification was performed using the PCR method.

PCR and visualization of amplicon16S rRNA by electrophoresis method
Visualization of 16S rRNA gene amplicons of seven bacterial isolates in the range of 700 bp to 1500 bp. Isolates S1 was visualized at 780 bp, Isolate S2 800 bp, Isolate S3 815 bp, Isolate S4 890 bp, Isolate S5 1500 bp, Isolate S6 1097 bp, and    Isolate S7 1500 bp. Based on the band formed, it indicates that the 16S rRNA gene amplification process was successful in all bacterial isolates (Fig. 2).

Sequencing
The sequencing of 16S RNA gene nucleotides from seven bacterial fly isolates was carried out by the sequencing method. The sequencing product in the form of seq file, from Singapore's FIRST BASE Laboratory, is read using the Geneous program. The consensus area of the 16S rRNA gene sequence was obtained using the Bioedit Program. >Consensus isolate s1 G

C G C C T G T T T A A C A A A A A C A T AT G G C T T T G C A A A AT C G A A A G AT G A G G TATA A G G C C TG A C A C C TG C C C G G TG C TG G A A G G T TA A G A G G G G AT G T C AT C C G C A A G G A G A A G AT TG A ATC G A A S C C C C A G TA A A CGGCKGCCGTAACTATAACGGTCCTAAKGYA GCAAATTCCTTGTCGGGAAGTTCCGACCTGC ACSAATGGTGTAACGATCTGGGCGCTGTCTCRC CRGAKCKCRGTSAAAKTGGGAKCGGTGAASAC G C C G AT TA C C C G A A C R G G A C R G A A A S A C C
TTCTTT AGAAAAAG GGGGG GCCAGGAA GT TCGAATAAAAGGGGCAAGGT T TAACGCA  CAGATTCATCGTGACGCCAATTTATTAGACTGAAG  GGTTTCCCCTTGTCGGCCCCGGCTCCAAGACC  TTCAGTCAACGCAAAGTGGCGAACCGTT GGCATGGG  GCC CCGGAAAGCCCCAAAGAACATGCTTGATGG  TTCATTGACCGAGCTG CGCCCCTTCAAAGTGAGGA  CCTGAGTGAGGAGATCCTCAGCCCAAGGGCAT  GATTGAGCAAGTGGCCGTGTTAATGGTAGCGCTGCC  AGCAGAAAGTTCAGCATCTGAAGGACGTCGGCTC  CAGCGTGAACTATCGCTTGATTGACGATCAGGGCCAG  TC CCGCGAGGTGTGAATTACATGCTGC CTGTGGA  GCTGGATGGCACCTGGTGTTTCTGGCTGGTA  TGCGTTTGTAGTTCGGCGAGCCGTTACGGTA  TGTGCGTTTCCCGGATGACGACAAGGGTTCCT  G TC G G  TATCCAGAGCCGTTTTGCAGACGTTTGCGCCAGCC  GGTTTTG ATGGGTT GATCAGCGGTGTTC CAGAGGC  TGAGCGA GAGAGTCT TCTGGG TCTGCAATTCCCAT  GATTCAGTTCACGCCGTCGGAATTGCGTGATG  CGATCAG GCCGCAACAAGCTTGGCCCGATCCAGAT  CCCCGTCAGGAAAATAGCGCTCACACTCGCGGGATT  GAGTCTGATGGACGATCATTCGCCAGCTCACGTGG  GTGCCGGCCTTCGGCAGAGGCGGCTCTGTACA  GCTGGGAGCAATATCAGTCCCGGGGGTA CAGGTTGA  GTGCTGGCCTGGCATGGACGATCACCATCAGT  CAGGTTCTCTACGTTGATGGGGCGCAGCTCGGT  CTTCTGTATCGTCGATGCGTGTTCGTGGGTGCGG  ACTTGGCCTCCCATGCAAGTCAGCTCCATTTGCC  ACATAGCGTAAAGTGTCGGAACCAGGGCTTTCCC  AGGGATAGCCTTGCATGAAAGGGATAGTTCAGCCG  TCGTAAA   >Consensus isolate s3  TTTTTAATCTATCGCCTGTTCGCCTGTTT  CCAGCAATA TAGGGCTCTGCGAAGTCGCAAGTCGAC  GTATAGGGTCTGACGCCTGCCCGGTGCTGGAAGGTT  AAGAGGAGAGGTGCAAGCCTTGAATTSWAKCCCCA  GTAAACGGCGGCCGTAACTATAACGGTCCTAASGTA  SCGAAATTCCTTGTCKKGYAAGTTYCGACCTGCMC  GAATGGCGTAACGACTTCMCCGCTGTYTCCAACGC  ASACTCAGTGAAATTGAATTYCCCGTGAAGATGCGG  GGTTCCTGCGGTTAGACGGAAAGACCCCGTGCACC  TTTACTATAGCTTTACMCTGGCATTTGTGGCGACATG  TGTAGGATAGGTGGTAGACTTTGAAGCGKAGGCG  CCAGCCTTCGTGGAGTCATCCTTGAAATACCACCCTT  ATCGTCATAGATGTCTAACTGCGACCCGTATATCCGG  GTCCAGGACCGTGTATGGTGGGTAGTTTGACTGGGG  CGGTCGCCTCCTAAAGAGTAACGGAGGCGCGCRATG  GTGGGCTCAGAACGGTCGGAAATCGTTCGTCGAGT  GCAATGGCATAAGCCCGCCTGACTGCWAGACAGA  CAAGTCGAGCAGAGACGAAAGTCGGTCATAGTGAT  CCRGTGGTCCCGTRTGGAAGGGCCATCGCTCAACGG  ATAAAAGGTACGCCGGGGATAACAGGCTGATGACC  CCCAAGAGTCCATATCGACGGAGTTGTTTGGCACCT  CGATGTTGGATTAAAAGATTAATT  >Consensus isolate s4  CCTGTTTTCGCCTGTTTTACAAAAACATA  TGGCTTTGCAAAATCGAAAGATGAGGTATAAGGCCT  GACACCTGCCCGGTGCTGGAAGGTTAAGAGGGAGA  TGTCATCGCAAGAGAAGCATTGAATCGAAGCCCCAG  TAAACGGCGGCCGTAA CTATAACGGTCCTAAGGTAGC  GAAATTCCTTGTCGGGTAAGTTCCGACCTGCACGAA  TGGTGTAACGATCTGGGCGCTGTYTCAGCCATGAGCT  CGGTGAAATTGTGGTATCGGTGAAGACGCCGAT  TACCCGCAACGGGACGGAAAGACCCCATGCACCTT  CACTATAGCTTAACATTGAAATTGGGTACAGGATG  TG TA G G ATA G G C G G G A G ATG T TG A A G TG G  CTTCGCCAGGAGTCATGGAATCAACCTTGAA  ATACCGCCCTTTCTGTATTCGGTTTCTAACTCG  GCCATGCCGAGGACATTGTTTGGTGGGTAGTTT  GACTGGGGTGGTCGCCTCCAAAAAGGTAACGGA  GGCTTTCAAAGGTAAGCTCAGTACGCTTGGTAA  CCGTACGTGGAGTGCAATGGCATAAGCTTGCTTGA  CTGTGAGACCAACAAGTCGACCAGGGTCGAAAGA  CGGACATAGTGATCCGGTGGTTCTGTATGGAAGGG  CCATCGCTCAAAGGATAAAAGGTACGCTGGGGATAA  CAGGCTGATCTCCCCCAAGAGCTCATATCGACGGG  GAGGTTTGGCACCTCTATGTTGGATTAAAATATAC  >Consensus Isolate S5 TAAGAGCTTGCTCTTATGAAGTTAGCG GCGGACGGGTGAGTAACACGTGGGTAACCTG CCCATAAGACTGGGATAACTCCGGGAAACCG TA G C  TAGTTGGTGAGGTAACGGCTCACCAAGGCAA  CGATGCGTAGCCGACCTGAGAGGGTGATCGG  CCACACTGGGACTGAGACACGGCCCAGACTCC  TACGGGAGGCAGCAGTA GGGAATCTTCCGCAATGG  ACGAAAGTCTGACGGAGCAACGCCGCGTGAGT  GATGAAGGCTTTCGGGTCGTAAAACTCTGTT  GTTAGGGAAGAACAAGTGCTAGTTGAATAAGC  TGGCACCTTGACGGTACCTAACCAGAAAGCCA  CGGCTAACTACGTGCCAGCAGCCGCGGTAAT  ACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGC  GTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATG  TGAAAGCCCACGGCTCAACCGTGGAGGGTCAT  TGGAAACTGGGAGACTTGAGTGCAGAAGAG  GAAAGTGGAATTCCATGTGTAGCGGTGAAATG  CGTAGAGA TATGGAGGAACA CCAGTGGCGAAGGC  GACTTTCTGGTCT GTAACTGACACTGAGGCGC  >Consensus Isolate S6  AGCTAGTATCCATCGTTTACGGCGTGGA  CTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTT  CGTTCATCAGCGTCAATAAATACGTAGTAACCTGCCTT  CGCAATTGGTATT CCATGTAATATCTAAGCATTTCACC  GCTACACTACATATTCTAGTTACTTCCATACTACTCA  A

G C TA G A C A G TAT C A AT G G C A G T G T C T T AGTTGAGCTAAGAAATTTCACCTCTGACTTAT C C TA A C C G C C TA C G A A C C C T T TA A A C C C A A TAATTCCGGATAACGCTCGCATCCTCCGTATTACCG CGGCTGCTGGCACGGAGTTAGCCGATCCTTATTC >Consensus isolate s7
A A G C G T TA ATC G G A AT TA C TG G G C  GTAAAGCG CACGCAGGCGGTC TGTCAAGTCGGATG  TGAAATCCCCGGGCTCAACCTGGGAACTGCATTCG  AAACTGGCAGGCTAGAGTCTTGTAGAGGGGGGT  AGAATTCCAGGTGTAGCGGTGAAATGCGTAGA  GATCTGGAGG AATACCGGTGGCGAAGGCGGCCC  CCTGGACAAAGACTGACGCTCAGGTGCGAAA  GCGTGGGGAGCAAACAGGAT TAGATACCCT  GGTAGTCCACGCCGTAAACGATGTCGACTTGG  A G G T TG TG C C C T TG A G G C G TG G C T TC C G G  AGCTAACGCGT TAAGTCGACCGCCTGGGGA  GTACGGCCGCAAGGTTAAAACTCAAATGAATTG  ACGGGGGCCCGCACAAGCGGTGGAGCATGTGGT  TTAATTCGATGCAACGCGAAGAACCTTACCTAC  TCTTGACATCCAGAGAACTTTCCAGAGATGGAT  TGGTGCCTTCGGGAACTCTGAGACAGGTGCTGCA  TGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGG  TTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTT  GCCAGCGGTTCGGCCGGGAACTCAAAGGAGAC   TG C C AGTG ATA A AC TG G AG G A AG GTG G G G  ATGACGTCAAGTCATCATGGCCCTTACGAGTAG  GGCTACACACGTGCTACAATGGCGCATACAAAG  AGAAGCGACCTCGCGAGAGCAAGCGGACCTCA  TAAAGTGCGTCGTAGTCCGGATTGGAGTCTG  CAACTCGACTCCATGAAGTCGGAATCGCTAGTAAT  CGTAGATCAGAATGCTACGGTGAATACGTTCCCGG  GCCTTGTACACACC

DISCuSSIoN
House flies have long been known as vectors of various types of diseases caused by bacteria. The bacteria that are transmitted by house flies to humans are very dependent on habitat. Identification of bacteria by conventional methods not only requires a long time but also the accuracy of the species identified is still low. Molecular identification using the 16S rRNA gene has been widely used in determining the position of bacterial species. Bacterial identification based on morphology and biochemical analysis has been apply before on Musca domestica and Chrysomya megachepala that lives in Luwuk City, Central Sulawesi, Indonesia. Molecular identification can determine the position of bacterial species that exist in flies and construct phylogeny trees of these bacteria. Knowing the species of bacteria in flies is very important to overcome the transmission of pathogenic bacteria conducts by flies.
The phylogeny tree construction aims to determine the evolutionary history of a species, diversity characteristics, predict novelty of genes, break pathogenic microbial strains and other purposes 20 . The phylogeny construction is based on consideration of the type or model of the phylogeny tree, the sequence that becomes the comparison and calculation of sequence data 21 . In this present study, the term reconstruction is used because the phylogeny tree was rebuilt based on the 16S rRNA gene sequence of bacteria from flies with BLAST sequences that were recorded in the NCBI gene bank. The phylogeny reconstruction was executed using two models. It aims to compare and ensure phylogeny trees were formed. The phylogeny tree built using the Neighbor-Joining model has topography similar to the minimum evolution model (Fig. 3 in Fig. 4).
Based on the 16S rRNA gene in constructing phylogenic trees, isolate S1 and S2 isolate have the closest evolutionary relationship with Brucella sp. Isolate S1 and Isolate S2 form the same node, thus having a very close evolutionary relationship. Isolate S3 has the closest evolutionary relationship with Bifidobacterium minimum even though it is in a different node. S4 isolate has the closest evolutionary relationship with Empedobacter brevis. Isolate S5 has the closest evolutionary relationship with Achromobacter xylosoxidans. Isolate S6 has the closest evolutionary relationship with isolate S5, then with Achromobacter xylosoxidans. Isolate S7 has the closest evolutionary relationship to Alcaligenes faecalis ( Fig. 2 and Fig. 3).
From the results of this study, it is known that the similarity of 16S rRNA gene sequences is different from the close evolutionary relationship of each bacterial isolate obtained from green flies and house flies in Luwuk. The alignment of 16S rRNA sequences using BLAST (https://www.ncbi. nlm.nih.gov/) is based on the similarity in the sequence of nucleotide sequences of each isolate with similar sequences that have been recorded in the NCBI gene bank. In contrast to phylogeny reconstruction, it was built based on the close evolutionary relationship of each isolate based on 16S RNA sequences 22,[24][25][26] . Species of bacteria identified from green flies and house flies that inhabit the city of Luwuk have never been reported to be associated with flies.
Research conducted by Park et al. 2019 states that the genus Staphylococcus and Weissella were predominantly found on the external portion of house flies however, in this study neither species of bacteria was found 4 . The genus Pseudomonas and E. coli were found the most on the surface of the body of the fly that lives in the hospital 8 . In this study, the bacteria isolated from flies that live in hospital waste bins were very different from those commonly found. Bacterial species found based on molecular identification are Bifidobacterium minimum (Isolate 1), Alcaligenes faecalis (Isolate 2), Brucella melitensis bv.

CoNCLuSIoN
Based on the alignment analysis of the BLAST method, the 16S RNA Isolate S1 gene sequence showed a 90% similarity with Bifidobacterium minimum [ Based on the evolutionary relationship on phylogeny trees formed using 16 S RNA sequences and BLAST sequences, isolates S1 and isolates S2 have the closest evolutionary relationship with Brucella sp. Isolate S3 has the closest evolutionary relationship with Bifidobacterium minimum. Isolate S4 has the closest evolutionary relationship with Empedobacter brevis. Isolate S5 has the closest evolutionary relationship with Achromobacter xylosoxidans. Isolate S6 has the closest evolutionary relationship with isolate S5, then Achromobacter xylosoxidans. Isolate S7 has the closest evolutionary relationship to Alcaligenes faecalis. Bacteria found in house flies and green flies from the city of Luwuk were first reported to be associated with flies.

ACKNoWLeDgMeNTS
We would like to acknowledge Prof. Dr. Ir. Max Tulung, MS for his assistance in the