Identificationand analysis of the potential diagnosis target genes in Finegoldia magna genome sequence as a rapid detection of thepathogen in clinical specimens.
Abstract: Bioinformaticsdatabases and analytical search tools were used, in order to, design specificpolymerase chain reaction (PCR) primers for the amplification of oxypeptidase, transposase and autoinducer 2, genes obtained fromthe following Finegoldia magna clinicalstrains: AC 166, AC 167, DS 001.Amplicon products were produced by the primers designed to amplify autoinducer 2 gene in all tested straintypes. Furthermore, on sequencing, 358 bp, 359 bp and 138 bp amplicons producedby the autoinducer 2 primers on alltested strains, showed homology with the corresponding database referencestrains.
Thus, based on the results of this experiment, development of simple,rapid and highly specific PCR diagnostic clinical method can be made possible. Key words: Finegoldiamagna, autoinducer 2 gene/primers, 23SrRNA sequence, polymerase chain reaction (PCR), diabetic foot ulcers, gram-positive anaerobic cocci (GPAC) Introduction: Finegoldia magna (F. magna) or formerly Peptostreptococcusmagnus is an opportunistic microorganism which is frequently isolated fromdiabetes-related foot ulcers and abscesses where under favourable conditions itbecomes pathogenic.
In the wound, bacteria are present in the form ofmulti-layered polymicrobial communities surrounded by self-producedextracellular debris, i. e. bio-forms where aerobes, anaerobes, and funginormally co-exist.
“Approximately 70% ofF. magna strains recovered from human clinical materials co-exist with otherbacterial species such as group D streptococci, Staphylococcus, Bacteroides,and Fusobacterium” (Goto et al. 2008).
Presence of bio-forms makes healingdifficult, as the structure shields the encased cells from antimicrobial agentsand the host immune system, allowing bacteria to persist (Smith et al. 2016).Moreover, open wounds provide a perfect niche for bacterial growth.
Developmentof foot ulcerations also depends on a combination of the diabetes-associatedintrinsic factors such as micro-vascular disease causing poor extremity perfusion,peripheral neuropathy and impaired host immune response (Alexiadou et al.2012). Diabetic foot wounds is a commondebilitating complication of diabetes mellitus, ultimately affecting up to 50%of patients with both type 1 and 2 diabetes over life a time(Didac 2016). Proven by the frequent recovery ofF. magna from clinical specimensobtained from diabetic foot ulcers, it has the highest pathogenicity as well asthe highest antibiotic resistance among all gram-positiveanaerobic cocci (GPAC) (Misra et al.
2012; Frank et al. 2010; Wren 1996). Thestudy of Murphy and Frick 2013 demonstrated thatcompared to other GPAC like P. micra and P. harei, also tested, F. magna showedthe highest minimum inhibitory concentration (MIC50) required to inhibit thegrowth of 50% of the organisms and MIC90 values for penicillin G,amoxicillin-clavulanic acid, clindamycin, and tigecycline and it also had thehighest MIC90 values for levofloxacin and moxifloxacin.
Furthermore, diabetes-relatedfoot infections are the leading cause of lower extremity amputations (Amin2016). “The incidence of major amputationis 0.5-5.
0 per 1000 people with diabetes” (Jeffcoate and Harding 2003). “Diabetic foot infections remain one of the majorcomplications leading to a leg loss every 3 seconds due to amputations causingmental trauma and distress”. (Dharod 2010). The increasing longevity andconstantly growing population of diabetic patients have resulted in a greaternumber of diabetic foot infections that continue to be the major cause ofhospital admissions, mortality in patients with diabetes mellitus and financialstrain on the healthcare system (Moulik et al. 2003).
“The number of people with diabetes mellitus (DM) is estimated toexceed 640 million by the year 2040” (Adeghate et al. 2017). Aspathogenesis of foot ulceration is very complex, poorly understood and itsclinical presentation is variable, therefore, this experiment was focused onthe molecular analysis of F. magnagenome sequence that would allow identification of more specific antimicrobialtherapy that in turn would considerably improve quality of life, reducemorbidity among diabetic patients and engender considerable financial costs(Singh al. 2005). The objectives of this experimentwere the following: (i) using bioinformatics software Primer 3, designgene-specific primers for the amplification of specific fragments from F.
magnagenome sequence; (ii) establish a discriminating PCR amplification method forreliable and rapid detection of selected genes in F. magna genome whichpotentially can be useful for detection of F. magna in clinical practice.
Methods: Theexperiment was carried out in two independent directions, namely (i) Bioinformaticsand (ii) Microbiology, according to standard protocols (Russell and Sambrook2001). Step (i): relevant literature based webdatabases (PubMed based at the NCBI, The GenBank EMBL) were searched and F. magna complete genome sequence wasidentified, accession number: AP 008971.1 and three suitable genes encodedwithin F. magna genome: oxypeptidase-NC_010376, transposase-AP008971,autoinducer 2 production protein-AP008971 were also identified then theirsequences were selected. Gene sequences of the identified genes, displayed inthe section of supplementary data. NCBI (www.ncbi.
nih.nlm.gov) analytical tool(homology search sequence alignment tool) such as BLAST was accessed, in order totest the following hypothesis: selected genes are single copies, selected genesdo not belong to other microorganisms or humans (homology search) and they donot have any mutations. Bioinformatics software Primer 3 was used to designspecific gene primers (sequences were presented in Table 1). Step (ii): Clinical samples wereobtained from the laboratory of Dr.
M. Wren at University College London (UCL).F. magna DNA was extracted and purifiedby the standard phenol-chloroform method. Standard DNA extraction protocol wasused.
The AC166, AC167 and DS 001 strains of F. magna were used as a source of genomic DNA. Step (iii): Polymerase chain reaction(PCR) was carried out using oxypeptidase,transposase, and autoinducer 2gene oligonucleotide primers designed with Primer 3 web tool. Standard PCRMastermix was used. PCR controls for each set of samples included sterile water(negative control) and “universal” primers of known DNA sequence as positivecontrol which was of the following sequence: forward primer: 5′ GCG ATT TCY GAAYGG GGR AAC CC 3′ and reverse primer: 3′ TTC GCC TTT CCC TCA CGG TAC 5′, where R=A+G,Y=C+T. In general, PCR was performed with 30 cycles (at 92 C for 30 seconds, at60 C for 60 seconds and at 72 C for 90 seconds). In order to make all primersbind to the correct gene sequence, PCR run was repeated three times: with theannealing temperature (Tm) 60 C, then it was lowered to 55 C, and 45 C.PCR-amplified fragments were stained in ethidium-bromide and separated in 1%agarose gel (made according to the instructions) by electrophoresis for 1 hourat 100 V.
Expected size of amplicon products is 200 bp. Step (iv): For validation of PCRamplification (designed primers hybridised to the correct sequences),experimental strains of F. magna (AC 166,AC 167 and DS 001) were sequenced. PCR product was purified using QIAquickPCR purification kit. The instructions for the purification procedure wereobtained from Quiagen-QIAquick PCR (www.qiagen.com).
Identified base sequencesof the strains were presented in the section of supplementary data. Identified sequences (rRNA 23-S sequences) were compared using BLAST at NCBI databaseincluding GenBank and EMBL databases and also aligned with sequences of thestrains derived from the EMBI-EBI (www.ebi.ac.uk) database using CLUSTAL Wmultiple sequence alignment tool, in order to verify BLAST results.Phylogenetic analysis including phylogenetic tree estimation was performed. Results: Gelelectrophoresis (Fig.1): Theprimers which successfully amplified products were primers designed for the autoinducer 2 gene of strains AC 166, AC 167 and DS 001 and also for the oxypeptidasegene but only of the strain AC 166.
PCR products of the AC 166 and AC167 strains using autoinducer 2 gene primers were 260 bp and 290 bp in size, PCRproduct of DC 001 strain using autoinducer 2 gene primers were 247 bpand 290 bp. PCR product of AC 166strain using oxypeptidase geneprimers was 247 bp. Primers designed for transposasegene did not produce any amplicon product (bands). BLASTanalysis of F.
magna strains: rRNA 23-S sequences of F. magna experimental strains (AC 166, AC 167, DS 001) were analysed byBLAST against all available sequences. BLAST search showed that percentidentity ranged from 80% to 100% (mean 90%) to the NCBI database strains. BLASTalignment results were presented in the supplementary data section. Phylogeneticanalysis: Multiplesequence alignment i. e. CLUSTAL W pairwise alignment (Fig.
3) of theexperimental F. magna strains (AC 166, AC 167, DS 001) andEMBI-EBI database available strains showed that strains which were obtained inthe experiment have stronger phylogenetic relationship maximum 96.9% homologywith each other then with the corresponding strain(s) rRNAFMagtypestrain in the EMBI-EBI database maximum 54.3% homology.(Table 2). Homology between F.magnastrains is also reflected by the phylogenetic tree. (Fig.
2) Discussion: Determiningthe precise etiology of infection in diabetic patients aids in managementdecisions is of prognostic and epidemiological consequence, and may haveprofound public health and disease control impact. (Doern et al. 2000).Accurate identification of bacterial species is an essential task of theroutine microbiology laboratory, however conventional methods for identificationof F. magna rely upon microbiologicalculture and biochemical tests.
That can be lengthily and often produceambiguous results due to F. magna isa highly fastidious microorganism making its pathogenic potential oftendifficult to assess (Riggio et al. 2003; Lin et al.
2010). In addition, Murdocket al., (1998) reported that gas-liquid chromatography was introduced to be P. magna detection method but itsresults closely correlated with biochemical tests. Nowadays, there is a lack ofrapid, reliable and inexpensive diagnostic approach for P. magna detection. Thus, the lack of an accurate techniquesresults in F. magna is overlooked inculture leading to that diabetes-related soft tissue infections diagnosed lateor not at all.
Therefore, a clinical diagnosis should utilise gene sequencingas a reference method for bacterial identification (Schlaberg et al.2012). Currently, little sequence data isavailable for F. magna so PCR usinguniversal or specific primers followed by identification of the amplifiedproduct, mainly by sequencing, has enabled the rapid identification of F. magna (Jauhaneer et al. 2004;Fenollar and Didier 2004). In this study, primer design based onhomology amplified correctly autoinducer2 gene in all type strains tested, as demonstrated by the appearance of aPCR product of the expected size. To present quality and accuracy of PCR andprimer design, 23S rRNA sequence datawere determined for three strains of F.
magna (AC 166, AC 167, DS 001).Primers designed to detect autoinducer 2gene showed 100% specificity when validated against the sequenced experimentalstrains. The purpose of performing sequence similarity searches with the 23S rRNA sequence of all types ofstrains used of was to evaluate the accuracy of the results produced by theprograms such as BLAST and CLUSTAL W. Thus, according to the data generated inpresent experiment, 23S rRNAsequence-based analysis proved to be an accurate method in unambiguousdefinitive identification of clinically important isolates of F. magna. Also, it provides informationon the taxonomic relatedness and genomic identification of F. magna strains. “Theavailability of genotypic data now makes it possible to develop moleculartechniques for the detection and identification of GPAC” (Wildeboer-Velooet al.
2007). However, it is not without of some limitations. First of all, asdetermined by BLAST and CLUSTAL W strains obtained during present study havehigher sequence similarities with each other (maximum 96.9% homology) than withcorresponding EMBI database strains (maximum 54.3% homology). This can indicatethat newly sequenced strains may possibly have some changes (mutations)occurred in their DNA with time probably due to misbalance between pathogenicand commensal microflora caused by the wide antibiotic use, prosthesis use,increase in a number of diabetic, elderly and immunocompromised patients,therefore, making strains evolutionary distant. Another reason for lowersimilarity between the experimental and database strains is that the majorityof the established GPAC speciesincluding F. magna were uploaded tothe databases in the early 1990s.
As the methods used then may not were able toprovide the quality of sequences easily obtained probably due to variableuncorrected errors. Thirdly, databases may be incomplete with higher quality of23S rRNA sequences. The negativeresults of the present study i. e.
other two sets of primers failed to amplifygenes oxypeptidase and transposase selected for the experimentshould be interpreted as follows: either bacterial DNA was not present orextraction was insufficient or the product was degraded. The last assumption ismore likely as storage of the samples did not meet the temperature regimenrequirements due to an accidental power cut. The autoinducer 2 gene primers gave a good yield of amplicons as onePCR run was before the accident. In conclusion, using bioinformaticsand homology, specific PCR method to identify F. magna autoinducer 2 gene which is only a conserved sequence inthe F.
magna genome, can be developedand used as the diagnostic mean for rapid detection of F. magna directly in clinical specimens. In the face of increasingmicrobial antibiotic resistance, it can be helpful for antibiotic-resistantstrain identification and further prediction of antibacterial therapy. Reproducibility: Theexperiment was conducted once.
The results were verified.Acknowledgements: I would like to thank Dr.Pamela Greenwell and a laboratory assistant Karima Brimah for the great helpprovided for me during my work at the experiment. I also wish the bestachievements to the laboratory in all the future research.