Identification the following Finegoldia magna clinical strains:

Identification
and analysis of the potential diagnosis target genes in Finegoldia magna genome sequence as a rapid detection of the
pathogen in clinical specimens.

 

Abstract:

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Bioinformatics
databases and analytical search tools were used, in order to, design specific
polymerase chain reaction (PCR) primers for the amplification of oxypeptidase, transposase and autoinducer 2, genes obtained from
the following Finegoldia magna clinical
strains: AC 166, AC 167, DS 001.
Amplicon products were produced by the primers designed to amplify autoinducer 2 gene in all tested strain
types. Furthermore, on sequencing, 358 bp, 359 bp and 138 bp amplicons produced
by the autoinducer 2 primers on all
tested strains, showed homology with the corresponding database reference
strains. 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:  Finegoldia
magna, autoinducer 2 gene/primers, 23S
rRNA sequence, polymerase chain reaction (PCR), diabetic foot ulcers, gram-positive anaerobic cocci (GPAC)

 

Introduction:

 

Finegoldia magna (F. magna) or formerly Peptostreptococcus
magnus is an opportunistic microorganism which is frequently isolated from
diabetes-related foot ulcers and abscesses where under favourable conditions it
becomes pathogenic. In the wound, bacteria are present in the form of
multi-layered polymicrobial communities surrounded by self-produced
extracellular debris, i. e. bio-forms where aerobes, anaerobes, and fungi
normally co-exist. “Approximately 70% of
F. magna strains recovered from human clinical materials co-exist with other
bacterial species such as group D streptococci, Staphylococcus, Bacteroides,
and Fusobacterium” (Goto et al. 2008). Presence of bio-forms makes healing
difficult, as the structure shields the encased cells from antimicrobial agents
and the host immune system, allowing bacteria to persist (Smith et al. 2016).
Moreover, open wounds provide a perfect niche for bacterial growth. Development
of foot ulcerations also depends on a combination of the diabetes-associated
intrinsic 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 common
debilitating 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 of
F. magna from clinical specimens
obtained from diabetic foot ulcers, it has the highest pathogenicity as well as
the highest antibiotic resistance among all gram-positive
anaerobic cocci (GPAC) (Misra et al. 2012; Frank et al. 2010; Wren 1996). The
study of Murphy and Frick 2013 demonstrated that
compared to other GPAC like P. micra and P. harei, also tested, F. magna showed
the highest minimum inhibitory concentration (MIC50) required to inhibit the
growth of 50% of the organisms and MIC90 values for penicillin G,
amoxicillin-clavulanic acid, clindamycin, and tigecycline and it also had the
highest MIC90 values for levofloxacin and moxifloxacin.  

         Furthermore, diabetes-related
foot infections are the leading cause of lower extremity amputations (Amin
2016). “The incidence of major amputation
is 0.5-5.0 per 1000 people with diabetes” (Jeffcoate and Harding 2003). “Diabetic foot infections remain one of the major
complications leading to a leg loss every 3 seconds due to amputations causing
mental trauma and distress”. (Dharod 2010).

          The increasing longevity and
constantly growing population of diabetic patients have resulted in a greater
number of diabetic foot infections that continue to be the major cause of
hospital admissions, mortality in patients with diabetes mellitus and financial
strain on the healthcare system (Moulik et al. 2003). “The number of people with diabetes mellitus (DM) is estimated to
exceed 640 million by the year 2040” (Adeghate et al. 2017).      

As
pathogenesis of foot ulceration is very complex, poorly understood and its
clinical presentation is variable, therefore, this experiment was focused on
the molecular analysis of F. magna
genome sequence that would allow identification of more specific antimicrobial
therapy that in turn would considerably improve quality of life, reduce
morbidity among diabetic patients and engender considerable financial costs
(Singh al. 2005).

          The objectives of this experiment
were the following: (i) using bioinformatics software Primer 3, design
gene-specific primers for the amplification of specific fragments from F. magna
genome sequence; (ii) establish a discriminating PCR amplification method for
reliable and rapid detection of selected genes in F. magna genome which
potentially can be useful for detection of F. magna in clinical practice.

 

Methods:

 

The
experiment was carried out in two independent directions, namely (i) Bioinformatics
and (ii) Microbiology, according to standard protocols (Russell and Sambrook
2001).

          Step (i): relevant literature based web
databases (PubMed based at the NCBI, The GenBank EMBL) were searched and F. magna complete genome sequence was
identified, accession number: AP 008971.1 and three suitable genes encoded
within F. magna genome: oxypeptidase-NC_010376, transposase-AP008971,
autoinducer 2 production protein-AP008971 were also identified then their
sequences were selected. Gene sequences of the identified genes, displayed in
the section of supplementary data. NCBI (www.ncbi.nih.nlm.gov) analytical tool
(homology search sequence alignment tool) such as BLAST was accessed, in order to
test the following hypothesis: selected genes are single copies, selected genes
do not belong to other microorganisms or humans (homology search) and they do
not have any mutations. Bioinformatics software Primer 3 was used to design
specific gene primers (sequences were presented in Table 1).

          Step (ii): Clinical samples were
obtained from the laboratory of Dr. M. Wren at University College London (UCL).
F. magna DNA was extracted and purified
by the standard phenol-chloroform method. Standard DNA extraction protocol was
used. 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 2
gene oligonucleotide primers designed with Primer 3 web tool. Standard PCR
Mastermix was used. PCR controls for each set of samples included sterile water
(negative control) and “universal” primers of known DNA sequence as positive
control which was of the following sequence: forward primer: 5′ GCG ATT TCY GAA
YGG 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, at
60 C for 60 seconds and at 72 C for 90 seconds). In order to make all primers
bind to the correct gene sequence, PCR run was repeated three times: with the
annealing 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 hour
at 100 V. Expected size of amplicon products is 200 bp.

          Step (iv): For validation of PCR
amplification (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 QIAquick
PCR purification kit. The instructions for the purification procedure were
obtained from Quiagen-QIAquick PCR (www.qiagen.com). Identified base sequences
of the strains were presented in the section of supplementary data.  Identified sequences (rRNA 23-S sequences) were compared using BLAST at NCBI database
including GenBank and EMBL databases and also aligned with sequences of the
strains derived from the EMBI-EBI (www.ebi.ac.uk) database using CLUSTAL W
multiple sequence alignment tool, in order to verify BLAST results.
Phylogenetic analysis including phylogenetic tree estimation was performed.

 

Results: 

 

Gel
electrophoresis (Fig.1):

The
primers 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 oxypeptidase
gene 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, PCR
product of DC 001 strain using autoinducer 2 gene primers were 247 bp
and 290 bp. PCR product of AC 166
strain using oxypeptidase gene
primers was 247 bp. Primers designed for transposase
gene did not produce any amplicon product (bands). 

 

BLAST
analysis of F. magna strains:

rRNA 23-S sequences of F. magna experimental strains (AC 166, AC 167, DS 001) were analysed by
BLAST against all available sequences. BLAST search showed that percent
identity ranged from 80% to 100% (mean 90%) to the NCBI database strains. BLAST
alignment results were presented in the supplementary data section.

 

Phylogenetic
analysis:

Multiple
sequence alignment i. e. CLUSTAL W pairwise alignment (Fig.3) of the
experimental F. magna strains (AC 166, AC 167, DS 001) and
EMBI-EBI                                                       
database available strains showed that strains which were obtained in
the experiment have stronger phylogenetic relationship maximum 96.9% homology
with each other then with the corresponding strain(s) rRNAFMagtypestrain in the EMBI-EBI database maximum 54.3% homology.
(Table 2). Homology between F.magna
strains is also reflected by the phylogenetic tree. (Fig.2)

 

 

Discussion:

 

Determining
the precise etiology of infection in diabetic patients aids in management
decisions is of prognostic and epidemiological consequence, and may have
profound public health and disease control impact. (Doern et al. 2000).
Accurate identification of bacterial species is an essential task of the
routine microbiology laboratory, however conventional methods for identification
of F. magna rely upon microbiological
culture and biochemical tests. That can be lengthily and often produce
ambiguous results due to F. magna is
a highly fastidious microorganism making its pathogenic potential often
difficult to assess (Riggio et al. 2003; Lin et al. 2010). In addition, Murdock
et al., (1998) reported that gas-liquid chromatography was introduced to be P. magna detection method but its
results closely correlated with biochemical tests. Nowadays, there is a lack of
rapid, reliable and inexpensive diagnostic approach for P. magna detection. Thus, the lack of an accurate techniques
results in F. magna is overlooked in
culture leading to that diabetes-related soft tissue infections diagnosed late
or not at all. Therefore, a clinical diagnosis should utilise gene sequencing
as a reference method for bacterial identification (Schlaberg et al.
2012). 

          Currently, little sequence data is
available for F. magna so PCR using
universal or specific primers followed by identification of the amplified
product, 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 on
homology amplified correctly autoinducer
2 gene in all type strains tested, as demonstrated by the appearance of a
PCR product of the expected size. To present quality and accuracy of PCR and
primer design, 23S rRNA sequence data
were determined for three strains of F.
magna (AC 166, AC 167, DS 001).
Primers designed to detect autoinducer 2
gene showed 100% specificity when validated against the sequenced experimental
strains. The purpose of performing sequence similarity searches with the 23S rRNA sequence of all types of
strains used of was to evaluate the accuracy of the results produced by the
programs such as BLAST and CLUSTAL W. Thus, according to the data generated in
present experiment, 23S rRNA
sequence-based analysis proved to be an accurate method in unambiguous
definitive identification of clinically important isolates of F. magna. Also, it provides information
on the taxonomic relatedness and genomic identification of F. magna strains. “The
availability of genotypic data now makes it possible to develop molecular
techniques for the detection and identification of GPAC” (Wildeboer-Veloo
et al. 2007). However, it is not without of some limitations. First of all, as
determined by BLAST and CLUSTAL W strains obtained during present study have
higher sequence similarities with each other (maximum 96.9% homology) than with
corresponding EMBI database strains (maximum 54.3% homology). This can indicate
that newly sequenced strains may possibly have some changes (mutations)
occurred in their DNA with time probably due to misbalance between pathogenic
and 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 lower
similarity between the experimental and database strains is that the majority
of the established GPAC species
including F. magna were uploaded to
the databases in the early 1990s. As the methods used then may not were able to
provide the quality of sequences easily obtained probably due to variable
uncorrected errors. Thirdly, databases may be incomplete with higher quality of
23S rRNA sequences. The negative
results of the present study i. e. other two sets of primers failed to amplify
genes oxypeptidase and transposase selected for the experiment
should be interpreted as follows: either bacterial DNA was not present or
extraction was insufficient or the product was degraded. The last assumption is
more likely as storage of the samples did not meet the temperature regimen
requirements due to an accidental power cut. The autoinducer 2 gene primers gave a good yield of amplicons as one
PCR run was before the accident.

           In conclusion, using bioinformatics
and homology, specific PCR method to identify F. magna autoinducer 2 gene which is only a conserved sequence in
the F. magna genome, can be developed
and used as the diagnostic mean for rapid detection of F. magna directly in clinical specimens. In the face of increasing
microbial antibiotic resistance, it can be helpful for antibiotic-resistant
strain identification and further prediction of antibacterial therapy.

 

 

Reproducibility: The
experiment 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 help
provided for me during my work at the experiment. I also wish the best
achievements to the laboratory in all the future research.