J Arthropod-Borne Dis, June 2015, 9(1): 35–48 S Akbari et al.: Aerobic Bacterial Community … 35 Original Article Aerobic Bacterial Community of American Cockroach Periplaneta americana,a Step toward Finding Suitable Paratransgenesis Candidates Sanaz Akbari 1, *Mohammad Ali Oshaghi 2, Saedeh Sadat Hashemi-Aghdam 3, Sara Hajikhani 4, Ghazaleh Oshaghi 5, Mohammad Hasan Shirazi 4 1Department of Microbiology, Islamic Azad University, Damghan Branch, Damghan, Iran 2Department of Medical Entomology and Vector Control, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 3Department of Animal Science, Islamic Azad University, Damghan Branch, Damghan, Iran 4Department of Pathology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran 5Department of Nutrition, National Nutrition and Food Technology Research Institute (NNFTRI), Faculty of Nutrition Sciences and Food Technology, Shahid Beheshti University of Medical Sciences, Tehran, Iran (Received 29 Oct 2013; accepted 16 Apr 2014) Abstract Background: Cockroaches mechanically spread pathogenic agents, however, little is known about their gut microbiota. Identification of midgut microbial community helps targeting novel biological control strategies such as paratransgenesis. Here the bacterial microbiota of Periplaneta americana midgut, were identified and evaluated for finding proper paratransgenesis candidate. Methods: Midgut of specimens were dissected and cultivated in different media. The bacterial isolates were then identified using the phenotypic and 16S-rRNA sequencing methods. Results: The analytical profile index (API) kit showed presence of 11 bacterial species including: Escherichia coli, Shigella flexineri, Citrobacter freundii, E. vulneris, Enterobacter cloacae, Yersinia pseudotuberculosis, Y. intermedia, Leclericia adecarboxylata, Klebsiella oxytoca, K. planticola, and Rahnella aquatilis in the cockroach midguts. The first three species are potentially symbiotic whereas others are transient. The conventional plating method revealed presence of only four isolates of Salmonella, E. coli, and Proteus which in three cases mismatched with API and 16S-rRNA genotyping. The API correctly identified the four isolates as Shigella flexneri, Citrobacter freundii, and E. coli (n= 2). 16S-rRNA sequence analysis confirmed the API results; however the C. freundii se- quence was identical with C. murliniae indicating lack of genetic variation in the gene between these two closely related species. Conclusion: A low number of potentially symbiotic bacteria were found in the American cockroach midguts. Among them Enterobacter cloacae is a potential candidate for paratransgenesis approach whereas other bacteria are pathogens and are not useful for the approach. Data analysis showed that identification levels increase from the con- ventional to API and to genotyping respectively. Keywords: Periplaneta americana, Midgut bacteria, Enterobacter cloacae, 16s rRNA, Analytical profile index (API) kit Introduction Cockroaches are one of the most im- portant insects in medicine. They inhabit in the dirty environment such as home sewage and could contaminate human foods with pathogenic agents. They can spread germs mechanically as they move freely from areas that may harbor pathogenic organisms: for example, from sewers to food or food prepa- ration surfaces. A number of cockroaches have become pests and live in or around homes where they are omnivorous scaven- gers. The two most significant pest cock- roaches worldwide are the German cock- roach Blattella germanica and the American *Corresponding author: Dr Mohammad Ali Oshaghi, E-mail: moshaghi@sina.tums.ac.ir http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 35–48 S Akbari et al.: Aerobic Bacterial Community … 36 cockroach Periplaneta americana. German cockroach is more common inside homes particularly in kitchens while the later is found around the home close to water pipes and drainage systems (Bell et al. 2007, Hashemi-Aghdam and Oshaghi 2014). The presence of the American cockroach, in hu- man dwellings causes damage and distress worldwide. Cockroach body has bad smell se- cretions, which are one of the sources of al- lergens responsible for asthma (Arruda et al. 2001, Litonjua et al. 2001, Arlian 2002). A numerous pathogens counting 32 spe- cies of bacteria (including Salmonella and Shigella species), 15 species of fungi and moulds, 7 helminths (intestinal parasites), 2 protozoans, and 1 virus which are all harm- ful to humans being found in or on cock- roaches or in the faeces (Mille and Peters 2004, Pai et al. 2005, Zarchi and Vatani 2009, Allotey et al. 2009). Intestinal micro- bial communities of some omnivorous cock- roaches such as P. americana, and B. orientalis (Blattidae), B. germanica (Ectobiidae), and Eublaberus posticus (Blaberidae) have been examined using cultivation-based studies (Burgess et al. 1973, Cruden and Markovetz 1984, 1987, Tachbele et al. 2006, Vahabi et al. 2007, Akbari et al 2014). However, a com- prehensive analysis of the midgut microbiota of the American cockroach is so far lacking. The only cockroach whose gut microbiota has been characterized with cultivation-inde- pendent molecular methods is Turkistan cockroach Shelfordella lateralis (Schauer et al. 2012). Traditionally, control of cockroaches re- lies on application of different classes of in- secticides which is often associated with en- vironmental toxicity, adverse effects on hu- man health and the emergence of insect re- sistance (Limoee et al. 2006, Enayati and Motevalli-Haghi 2007). A new control strat- egy named paratransgenic, symbiotic or com- mensally microbes of host insects are trans- formed to express gene products that reduce fitness of insect or interfere with pathogen transmission (Hurwitz et al. 2011, Chavshin et al. 2012, Wang and Jacobs-Lorena 2013). These genetically modified microbes are re- introduced back to the insect where expres- sion of the engineered molecules decreases the host's fitness or ability to transmit the pathogen. Recently, Jiang et al. (2007) suc- cessfully utilized this strategy to develop an engineered densovirus (PfDNV) to control the smoky-brown cockroach (Periplaneta fuliginosa) nymphs. Identification of a causative pathogen is essential for the choice of treatment as well as evaluation of the presence of virulence factors and antibiotic resistance determinants for most infectious diseases. Methods for the identification and discrimination of bacterial isolates can be separated into traditional and molecular groups (Nazarowec-White and Farber 1999). The traditional microbiologi- cal techniques (phenotyping) are based on secondary characteristics of bacteria includ- ing staining, cultures, biochemical reactions, antibiograms, serotyping, and bacteriophage typing. Phenotypic methodologies still play a significant role in identifying, verifying, and providing antibiotic susceptibility test- ing for many microbial pathogens. However, the application of molecular techniques to microbiology has led to the development of new and rapid methods for the detection, identification and characterization of many microorganisms including bacteria. These applications are stepwise replacing or com- plementing phenotypic assays in microbiol- ogy laboratories (Weile and Knabbe 2009). The most common molecular typing meth- ods used in microbiology include chromo- somal DNA restriction analysis, plasmid typ- ing, ribotyping, pulsed-field gel electropho- resis (PFGE) and PCR-based methods such as randomly amplified polymorphic DNA (RAPD) typing, restriction fragment length polymorphisms (PCR-RFLP), and 16S-rRNA sequencing (Eisenstein 1990, Grant and Kroll http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 35–48 S Akbari et al.: Aerobic Bacterial Community … 37 1993, Maslow et al. 1993, Farber 1996, Krishna and Cunnion 2012, Chavshin et al. 2012). Other molecular techniques such as whole or partial genome sequencing, real-time PCR, microarrays are also being used (Weile and Knabbe 2009, Krishna and Cunnion 2012). The rDNA gene sequences are highly conserved within living organisms of the same species, but that they differ between organisms of other species. 16S-rRNA is com- monly used for taxonomic studies in bacteria because it is present in almost all bacteria, its function over time has not changed, and is large enough for informatics purposes (Barney et al. 2001, Harmsen and Karch 2004, Janda and Abbott 2007, Woo et al. 2008). In this study we analyzed the bacterial flora of American cockroach midgut and evaluat- ed their possible usefulness as proper candi- date for paratransgenesis approach. To iden- tify the potential symbiont bacteria two rou- tinely used commercial phenotypic methods namely the traditional methods (including staining, cultures, and biochemical reactions) and analytical profile index (API) biochem- ical fingerprinting kit compared to genotyp- ing (16S-rRNA sequencing) as a reference method. Materials and Methods Isolation and purification of cockroaches’ midgut bacteria Adult American cockroach specimens were collected alive from underground of house- holds and confectionary premises in center of Tehran, Iran. Specimens were trapped using small box or food-baited traps and transferred to the laboratory of School of Public Health, Tehran University of Medical Sciences (SPH-TUMS), Iran. The specimens were kept in freezer about 5 min until they became immobilized. Before dissection, the specimens were surface sterilized for 2 min in 70% ethanol. Bacterial isolation was con- ducted in a sterile environment on a sterile Petri-dish. The legs were then removed, and the alimentary canal was exposed by making a ventral incision extending from the termi- nal sternum to the prothorax. The intact midgut from each specimen was dissected and transferred to sterile tubes separately. To separate transient bacteria from potential sym- biotic bacteria, the midgut specimens were divided randomly into three following groups before homogenization: 1) intact midgut with its contents 2) removing midgut contents without washing, and 3) removing midgut contents with three times washing by PBS and distilled water. Phenotypic Identification Traditional method The midguts were homogenized with glass pestles in 200 μl PBS buffer. Homoge- nized midguts from P. americana adults were poured in a 1.7 ml micro tube contain- ing BHI broth and incubated at 37 °C for 24 h. The media was serially diluted in sterile PBS, and 0.1 to 0.5 ml of each dilution was spread on plates of four different media in- cluding Brain Heart Infusion broth (BHI broth), BHI agar, MC Conkey agar and Blood agar (Merck, Germany) media. Spread plates were placed in incubator until colo- nies developed (24 h). The colonies with different phenotype were selected for further characterization and were streaked for puri- fication onto fresh plates of the same me- dium from which they had been picked. Colonies grown on the media were purified by several sub-cultures and then were stored at 4 ºC for further analysis. A test tube con- taining BHI agar open near the dissection area constituted our sterility control during the dissection process. Besides, we used a laboratory strain of Escherichia coli as ref- erence bacterium in the study. Characteristics of the bacteria were eval- uated by routine microbiological methods. Cell morphology such as size, pigment, smoothness of colony’s surface, having reg- http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 35–48 S Akbari et al.: Aerobic Bacterial Community … 38 ular or irregular edges and hemolysis was observed by photonic microscopy after Gram staining. Identification of bacteria to genus level was performed by using Biochemical tests (motility, utilization of citrate, indole formation, lysine degradation, SH2 produc- tion and fermentation carbohydrate) accord- ing to methods described by Sneath (1984). Results of biochemical tests were compared with the test results with dichotomous keys. API biochemical fingerprinting kit This phenotypic identification was carried out using the API test kit of DS-DIF- ENTRO-24 (Microgene) which is commer- cially available in Iran as described by Maleki-Ravasan et al. (2013). Prior to test- ing, isolates were fresh cultured overnight at 37 °C on blood agar. Suspension was pre- pared from the colonies with the turbidity equivalent to 2 McFarland and injected to the wells, incubated for 24 h at 37 °C and reagents was added. Positive and negative results using created color changes were de- termined based on API kit protocol. Tests were carried out according to manufacturers’ instructions and results were interpreted us- ing the appropriate laboratory computer software or reference indices recommended by the manufacturer. Bacteria can be identi- fied to species level and sometimes to sub- species level. Genotypic identification Genomic DNA from fresh colonies incu- bated overnight in liquid cultures in nutrient broth was extracted using boiling method. For genotyping, a 1500bp of 16s rRNA gene was amplified with specific primers accord- ing to Weisburg et al. (1991) protocol. Am- plification was carried out on the isolates with the following PCR cycling conditions: an initial denaturation at 94 °C for 10 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 56.5 °C for 40 s, and extension at 72 °C for 40 s, and final extension at 72 °C for 10 min (Karimian et al. 2011). DNA sequencing The PCR products of the isolates were sequenced by Seqlab, Germany using the amplification primers. Consensus sequences obtained from forward and reverse sequenc- es and their homologies with the available sequence data in GenBank were tested by using the basic local alignment search tool (BLASTn) alignment program and the NCBI nucleotide database NCBI (www.ncbi.nlm. nih.gov/BLAST). Results Phenotypic identification Totally 45 adult American cockroaches were collected from the premises during the May-August 2011. The mid-guts of the spec- imens were homogenized individually and cultured in different media as described in M and M section. Totally 20 purified bacterial colonies in different media cultures were isolated from the midgut of the adult Amer- ican cockroaches. These colonies were identified as Esche- richia coli (n= 11), Salmonella (n= 7), and Proteus (n= 2) using the traditional methods. The classical method also identified the E. coli reference strain correctly. Details of bi- ochemical characters used for classical iden- tification of the isolates are shown in Table 1. Escherichia coli and Salmonella were the most prevalent isolates. When the isolates were tested using API kit, the number of ge- nus and species greatly raised and the specimens were categorized in eight genera of Escherichia, Citrobacter, Shigella, Yer- sinia, Klebsiella, Rahnella, Enterobacter, and Leclercia (Table 2). The API biochemical analysis revealed presence of 11 bacterial species in the midgut of the American cock- roaches, however, the bacterial community http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 35–48 S Akbari et al.: Aerobic Bacterial Community … 39 was varied in the number and composition when the midgut tested with or without its contents and or before and after washing with PBS. The number of species was seven, five, and three when the midgut tested with its contents, without its contents and no washing, and without its contents plus wash- ing with PBS respectively (Table 2). Esche- richia coli, Shigella flexneri, and Citrobacter freundii were present before and after wash- ing the midgut contents. The isolates that were present in the midgut after washing seem to be potentially symbiotic since the transient bacteria normally would wash out and the remaining presumably are symbiotic. Genotypic identification and its congruence with phenotypic As previously indicated the classical bac- teriological method identified the midgut bacteria as Salmonella, Proteus and E. coli. The classical method also identified the E. coli reference strain correctly (Table 1). We selected a single colony of Proteus, Salmo- nella (positive to citrate), Salmonella (nega- tive to citrate), and E. coli for further API biochemical fingerprinting and genotyping. The API kit using 24 biochemical tests (Table 3) identified the four isolates as S. flexneri, E. coli (n= 2), C. freundii, and the E. coli reference strain. This result was not in accordance with the traditional method in three out of four cases. The two Salmonella isolates were identified as S. flexneri and C. freundii, and the Proteus isolate identified as E. coli (Table 4). Then the four isolates plus the reference strain of E. coli were analyzed by genotyping using 16S rRNA PCR-direct sequencing. DNA extractions from the iso- lates contained enough bacterial DNA for PCR amplification. All samples except neg- ative control produced visible PCR products of about 1500 bp whereas the negative con- trols were blank. The four isolates that were biochemically (API kit) identified as S. flexneri, C. freundii and E. coli (n= 2) re- spectively, were identified accordingly as S. flexneri, C. murliniae/ C. freundii, and E. coli (n= 2) based on partial sequences of 16srRNA (Table 4). Their sequences with length of 766, 766, 764, and 768 bp were deposited in GenBank with accession numbers of KC 017349, KC017346, KC017348, and KC017 347 respectively. The correspondences of the phenotypic and genotypic methods as well as details of the homologous species and their homology percentage of the spe- cies identified in this study with the ones available in GenBank are shown in Table 4. Table 1. Details of biochemical characters used for classical identification of bacteria in the American cockroach midgut Species Biochemical characters SIM KIA and gas production Lysine H2S Citrate Indole Motility Salmonella + - R/Y + - + + E. coli + + Y/Y + + - - Proteus + - R/Y + - - - Salmonella + - R/Y + + + - E. coli Reference strain + + Y/Y + + - - http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 35–48 S Akbari et al.: Aerobic Bacterial Community … 40 Table 2. AIP Phenotypic identification of bacterial community of American cockroach midgut before and after washing and removing contents Intact Removing contents without Washing Removing contents with three times PBS Washing Bacterial species Escherichia coli Escherichia coli Escherichia coli Shigella flexineri Eischercia vulneris Shigella flexineri Citrobacter freundii Klebsiella planticola Citrobacter freundii Yersinia pseudotuberculosis Enterobacter cloacae Yersinia intermedia Rahnella aquatilis Klebsiella oxytoca Leclericia adecarboxylata Table 3. Details of reaction tests used in API kit for species identification of four bacteria isolated from midgut of the American cockroaches Results of reactionTest reactionNo. ++--Indole1 +++-VP2 --+-Citrate3 ----SH24 ++++Urease5 ---+Phenylalanine6 ++--Laysine7 ++--Argentine8 ----Ornitine9 ++--Malonate10 ----Glocuse11 ----Adonitole12 ----Arabinose13 --++Dolicitole14 ----Inositole15 ++++Lactose16 --++Maltose17 ++++Mannitol18 ++++Ramnose19 ----Sucrose20 ++++Surbitole21 ++++Trehalose22 ++--Eskoline23 ++--ONPG24 E.coliE.coliCitrobacter freundiiShigella flexneriIdentified species Table 4. Details of congruence between phenotypic and genotypic methods in diagnosis of bacteria isolated from the American cockroach midgut. AN: Accession number Classic API 16S rRNA sequencing (A.N in GenBank) Homolog species (A.N in GenBank) Homology Rate % Salmonella Shigella flexneri S. flexneri (KC017349) S. flexneri (HQ701686) S. flexneri (CP001383) S. flexneri (CP001386) 100 100 100 Proteus Escherichia coli E. coli (KC017348) E. coli (CP003034) E. coli (JN578644) S. sonnei (NR074894) 99 99 99 E. coli E. coli E. coli (KC017347) E. coli (JQ609683) E. coli (CP001925) E. coli (CP002967) E. coli (CP002970) E. coli (CP002291) 100 100 100 100 100 Salmonella Citrobacter freundii C. murliniae (KC017346) C. murliniae (JN092600) C. freundii (JX860618) 100 100 http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 35–48 S Akbari et al.: Aerobic Bacterial Community … 41 Discussion The results of this study using API kit revealed that the intact midgut of P. ameri- cana harbors fairly a diverse community of gram negative aerobic bacteria of Entero- bacteriaceae. However, most of these bac- teria were transient and acquired from the environment and or the food sources the cockroaches live and feed on as previously described by Kane and Breznak (1991). There- fore the non-transient bacteria comprise small part of the midgut community. The low number of non-transient (possibly sym- biotic) microbial community of the Amer- ican cockroaches found in this study concord to earlier perceptions of the cockroach midgut microbiota, which were based on cultivation-based studies that yielded mostly isolates of the genera Enterobacter, Klebsiella, and Citrobacter (Cruden and Markovetz 1987). It is worth mentioning that since the bac- terial community obtained by culture media is limited by the selectivity of the media employed, the species (number and diver- sity) estimated in this study are not definitely estimates of the total real community. Initial using of nonselective medium (BHI broth) to promote growth of bacteria generally fa- vored the growth of gram negative Entero- bacteriaceae in the media. However, it is the case for almost all of the studies analyzing the insect gut bacterial communities (Hillesland et al. 2008, Mukhopadhyay et al. 2012, Chavshin et al. 2012). Even the stud- ies that have implemented molecular tools used these tools only in the identification and analysis of isolated pure colonies from plate culture, not in the initial isolation of bacteria from the guts (Gouveia et al. 2008, Hillesland et al. 2008). The molecular tools used in both studies were implemented in the identification of bacterial colonies obtained by culturing, thereby limiting the findings to the small proportion of cultivable microbes. Taking into account the limitations of cul- ture dependent techniques makes these find- ings incomplete. The microbial community of different compartments of intestinal tract of cock- roaches including crop, midgut, rectum and colon of P. americana and Shelfordella lateralis (Turkistan cockroach) previously have been compared using culture media or molecular tools (Bignell 1977, Bracke et al. 1979, Schauer et al. 2012). These studies showed that each compartment harbor di- verse community of bacteria, where the anterior colon of the cockroaches contained the highest abundance of microorganisms. The diversity in different compartments of cockroach gut is related to the microbial ac- tivities, such as the accumulation of hy- drogen and the other microbial products, and the physiochemical characteristics of each part of the gut, such as pH and redox po- tential (Schauer et al. 2012 and references herein). Previous studies of cockroaches have reported a decrease in redox potential along the gut, with oxidizing conditions in crop and midgut and reducing conditions in the hindgut. The low redox potential in the hindgut lumen is consistent with the accu- mulation of hydrogen and the presence of a large and diverse community of Clostridiales (Bignell 1977, Vinokurov et al. 2007, Schauer et al. 2012). Besides, it is shown that in P. americana, the foregut is a site of consider- able lactate production owing to the abun- dance of lactic acid bacteria (Kane and Breznak 1991). In this study we focused only on the midgut of the American cock- roach because the midgut has an endodermic origin which does not destroy in molting (ecdysis) and hence its microbial community remains intact or is less prone to diminish during multiple molting of cockroach life span. This fact is important for selection of a http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 35–48 S Akbari et al.: Aerobic Bacterial Community … 42 proper candidate bacterium for paratransgenesis approach. In the present study Enterobacter cloacae was found among the transient bacteria after removing midgut content but did not re- mained after washing with PBS. However, this bacterium could be a potential candidate for paratransgesis approach because it is found as the normal gut flora of many hu- mans and is not usually a primary pathogen (Keller et al. 1998). Enterobacter cloacae have already been used for paratransgenesis and successfully could deliver, express, and spread foreign genes in termite colonies (Husseneder and Grace 2005) and sand fly Phlebotomus papatasi (Maleki-Ravasan et al. 2014). This species was genetically trans- formed with Defensin (small cysteine-rich cationic proteins found in both vertebrates and invertebrates) to reduce Leishmania par- asites in vitro conditions (Maleki-Ravasan et al. 2014). Enterobacter cloacae also has been transformed with an ice nucleation gene to reduce the mulberry pyralid moth, Glyphodes pyloalis (Watanabe et al. 2000). These docu- ments show that the species is amenable for transformation with foreign genes. In this study we also found that E. coli is possibly a symbiotic bacterium in the midgut of Amer- ican cockroaches. This bacterium was ge- netically manipulated and tested for paratransgenic approach (Riehle et al. 2007, Chavshin et al. 2013). However E. coli, and other two potential symbiotic bacteria S. flexneri and C. flexineri are not appropriate candidate for paratransgenesis approach due to their pathogenic effect that can cause diarrhea or meningitis in humans (Badger et al. 1999, Niyogi 2005). Further investigation need to test the utility of E. cloacae or to find another appropriate symbiont for ge- netic manipulation and delivering effector molecules to control and diminish cockroach pest populations. The results of this study showed the importance of choosing the correct identi- fication method for exact speciation of bac- terial species. Correct identification impacts directly on treatment outcomes and on the epidemiological analysis of emerging bac- terial infections in arthropod borne diseases. The present study revealed that 75% of clas- sical test systems including staining, cul- tures, and biochemical reactions, yielded wrong speciation results when compared to API kit and genotyping (Table 3). For ex- ample, classical method identified two iso- lates as Salmonella, however these two iso- lates were subsequently identified by API and genotyping as S. flexneri and C. murliniae which could mislead the diagnostician and subsequent treatment methods. On the other hand, species classification based on pheno- typic features is often time-consuming and is not always easy to carry out (Springer et al. 1995, Nagy et al. 2006, Erme et al. 2009). The classical methods cannot detect the het- erogeneity in species, are not reproducible, and challenge with limited database for phe- notypic characteristics for common species. Common strains are easily identified with charts or keys but when we are facing to rare or intermediate strains, identification is dif- ficult with these tools (Barkeley et al. 1984). Inability of classical method in recognition of some bacterial species is result of pheno- typic variation, phenotypic homogeneity with- out enough differential characteristics and tendency of traditional method toward estab- lished taxa. Also some species are complex (phenospecies) and have more than one DNA group then classic method cannot sep- arate them phenotypically (Janda and Abbot 2007). Besides, some bacteria particularly anaerobic ones are extremely slow growing or not cultivatable at all. One of the argu- ments for using classical tests is that they are less costly than API and genotyping, how- ever, the potential penalty of misidentifi- cation must be considered. On general there was a great congruence among the results of API and 16S rRNA http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 35–48 S Akbari et al.: Aerobic Bacterial Community … 43 genotyping: both methods identified the four selected isolates as S. flexneri, E. coli (n= 2), and C. freundii. However, the 16S rRNA sequencing revealed possible mismatching of C. freundii with C. murliniae because both species had identical sequences in the region sequenced. Studies of Geraghty et al. (2013) on the efficacy of routinely used phe- notypic methods compared to genotypic approaches for the identification of staph- ylococcal species showed that the API Staph 32 kit correctly identified all S. aureus isolates (11/11), but 83% (10/12) of the SIG species, and 66% (19/29) of the coagulase negative species. They concluded that alt- hough the API Staph 32 test performed with the highest degree of accuracy for the coagulase positive Staphylococci, however they are inadequate for the correct identifi- cation of both coagulase negative and coagulase positive staphylococcal species. The API kits are produced for the human diagnostics market and are interpreted against databases with reference strains of human origin. Therefore it is suggested that the reproducibility and reliability of these kits are uncertain when applied to none human origins. The API test is based on the evaluation of expression of genetically en- coded characteristics and erroneous identi- fication may be due to variable expression of biochemical traits within species (Blaiotta et al. 2010). Other disadvantages of API kits included long incubation period (18 h), lack of the required turbidity in suspension pro- duced, bubbles in kit’s well, incorrect in- cubation period which leads to error while reading the results of the color changes. Also it is necessary that the databases identifi- cation kits updated by their manufactures to include new genus and species (Laclaire and Facklam 2000). However, the biochemical API kit is less expensive than the molecular method and is more suits for most small clinical laboratories where the implemen- tation of advanced molecular techniques is not feasible owing to the high cost of instrumentation and reagents. Nowadays, DNA based methods, partic- ularly PCR techniques, for the detection and characterization of microorganisms have revolutionized diagnostic microbiology and are now part of routine specimen processing. These methods have now progressed beyond identification to detect antimicrobial re- sistance genes and provide public health information such as strain characterization by genotyping (Mellmann et al. 2003, Woo et al. 2003, 2008, Cloud et al. 2004, Speers 2006, Sibley et al. 2012). Literature show that 16S rDNA sequencing is essential and key tool for bacterial identification, partic- ularly important in the case of bacteria with unusual phenotypic profiles, rare bacteria, slow growing bacteria, uncultivable bacteria and culture-negative infections (Clarridge 2004, Woo et al. 2008). There are many con- served primers to amplify a region of a gene, such as the 16S rRNA bacterial gene, and the amplified product is usually sequenced and compared to about 4 million 16s RNA sequences from different bacteria in Internet databases such as GenBank (www.ncbi. nlm. nih.gov/Genbank), EMBL Data Library (www. ebi.ac.uk/embl), and the DNA Data Bank of Japan (www.ddbj.nig.ac.jp) with daily data exchange between them, and more special- ized high quality databases such as RIDOM (www.ridom-rdna.de/) for bacterial rDNA sequences used for mycobacterial speciation (Speers 2006). In spite of above mentioned advantages, experience showed that 16s rRNA gene sequences is able to identify most cases (>90%) at genus level but in species level decrease to 65 to 83% and 1 to 14 % of iso- lates remains unidentified (Mignard and Flandrois 2006). Some other limitations are 1) identifying of new taxa is impossible, 2) limited number of sequences or partial se- quences existence in the nucleotide database, 3) similarity of 16s rRNA gene sequence in some species, 4) error in nomenclature species http://jad.tums.ac.ir Published Online: July 16, 2014 J Arthropod-Borne Dis, June 2015, 9(1): 35–48 S Akbari et al.: Aerobic Bacterial Community … 44 and 5) use of sequences with gap to comparison (Hall et al. 2003, Bosshard et al. 2006). Conclusion This study suggests that midgut of Amer- ican cockroaches harbor few symbiotic bac- terial community at least in case of aerobic gram negative species. Except for E. cloa- cae, the other symbiotic isolates are not proper candidate for paratransgenesis and further studies need to find other proper candidates. 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