Iranian Journal of Microbiology
Volume:5 Issue: 3, Sep 2013

Helicobacter hepaticus was discovered in 1992 as a cause of liver cancer in the A/JCr mouse model. In susceptible mice, infection by H. hepaticus causes chronic gastrointestinal inflammation leading to neoplasia. It can also cause morphological changes in breast-glands leading to neoplasm and adenocarcinoma in mouse models. Studies performed on humans have revealed that H. hepaticus may also be a human pathogen since infection by H. hepaticus can be associated with cholecystitis, cholelithiasis and gallbladder cancer. H. hepaticus is a close relative of H. pylori, but it lacks the major virulence factors of H. pylori including vacoulating cytotoxin A (VacA) and cytotoxin associated gene (cagA). Moreover, SabA, AlpA, and BabA, three important adhesin proteins of H. pylori, are absent in its genome. In contrast, the genome of H. hepaticus contains genes encoding some orthologus virulence factors of Campylobacter jejuni such as cytolethal distending toxin (CDT), and PebI adhesin factor. Other genes including 16S rRNA, 18 KDa immunogenic protein, and urease structural subunits are related to H. pylori. Its genome contains a small island consisting of 71 Kbp named HHGI1, which probably encodes a secretion system type IV (T4SS), and some other virulence factors. As far as the immunogenic antigens are concerned, the lipopolysaccharide (LPS) and flagellin of H. hepaticus are weak stimulants of the immune system, while pro-inflammatory responses are mainly induced by its lipoproteins and most likely by the peptidoglycan. Concerning the multidrug efflux pumps, a homologue of H. pylori TolC, HefA, has been observed in H. hepaticus which contributes to resistance to amoxicillin and bile acids.

Acinetobacter baumannii is an aerobic non-motile Gram-negative bacterial pathogen that is resistant to most antibiotics. Carbapenems are the most common antibiotics for the treatment of infections caused by this pathogen. Mechanisms of antibiotic-resistance in A. baumannii are mainly mediated by efflux pumps-lactamases. The aim of this study was to determine antibiotic susceptibility, the possibility of existence of OXAs genes and fingerprinting by Pulsed-Field Gel Electrophoresis (PFGE) among clinical isolates of Acinetobacter collected from Kermanshah hospitals. One hundred and four isolates were collected from patients attending Imam Reza, Taleghani and Imam Khomeini hospitals of Kermanshah (Iran). Isolates were identified by biochemical tests and API 20NE kit. The susceptibility to different antibiotics was assessed with Kirby-Bauer disk diffusion method. PCR was performed for detection of blaOXA-23, blaOXA-24, blaOXA-51 and blaOXA-58 beta-lactamase genes. Clonal relatedness was estimated by PFGE (with the restriction enzyme Apa I) and DNA patterns were analyzed by Gel compare II 6.5 software. All isolates showed high-level of resistance to imipenem, meropenem as well as to other antimicrobial agents, while no resistance to polymyxin B, colistin, tigecylcine and minocycline was observed. The blaOXA-23like and blaOXA-24 like were found among 77.9% and 19.2% of the isolates, respectively. All isolates were positive for blaOXA-51, but none produced any amplicon for blaOXA-58. PFGE genotype analysis suggested the existence of eight clones among the 104 strains [A (n = 35), B (n = 29), C (n = 19), D (n = 10), E (n = 4), F (n = 3), G (n = 3), H (n = 1)]. Clone A was the dominant clone in hospital settings particularly infection wards so that the isolates in this group, compared to the other clones, showed higher levels of resistance to antibiotics. The blaOXA-51-like and blaOXA-23like were the predominant mechanisms of resistance to imipenem in A. baumannii. A high prevalence of clone A, B and C in different parts of the healthcare system showed that hospitalized patients should be safeguarded to prevent the spread of these clones. Early recognition of the presence of carbapenem-resistant A. baumannii clones is useful for preventing their spread within the hospital environment.

High-level gentamicin resistance (HLGR: MIC ≥ 500 μg/ml) in Enterococci is mediated by aminoglycoside modifying enzymes which is mainly encoded by aac(6′)-Ie-aph(2′′)-Ia gene. The aim of this study was to evaluate the frequency of aac(6′)-Ie-aph(2′′)-Ia gene in clinical isolates of Enterococcus facium and Enterococcus faecalis collected from hospitals in northwest of Iran. In the present study a total of 111 enterococcus isolates were collected from 4 hospitals during a two year period (July 2009-August 2011). Bacterial identification and species determination were carried out by standard biochemical tests. Antimicrobial susceptibility was evaluated by Kirby Bauer disc diffusion method. MICs were determined by agar dilution method. The frequency of aac(6′)Ie-aph(2″)Ia gene in the isolates was determined by PCR. The carriage of resistance gene on Tn5281 transposon was identified by long PCR and dot-blot hybridization methods. Antibiotic susceptibility tests revealed that the highest resistance was against streptomycin (74.77%) and erythromycin (67.58%) whereas the highest susceptibility was observed to vancomycin (81.1%). 36 isolates (32.43%) were identified as HLGR, 34(94.44%) of them had resistant gene in their genome. Long PCR studies revealed that 88 % of HLGR clinical isolates harboured Tn5281. The aac(6′)Ie-aph(2″)Ia resistance gene was present on Tn5281 transposon in all 32 isolates according to dot blot hybridization test. The results of this study indicated that aac(6′)Ie-aph(2″)Ia resistance gene is highly prevalent in gentamicin resistant isolates. Carriage of aac(6′)Ie-aph(2″)Ia resistance gene on Tn5281 transposable element suggests possible contribution of this transposone on dissemination of resistance gene among enterococcus isolates.

Due to contagiousness of pertussis, a rapid and sensitive method for diagnosis is required to initiate the treatment and interrupt its transmission. To detect B. pertussis strains, we used two real-time PCR targeting IS481 and BP283 sequences and compared factors influencing culture and real-time PCR results. Totally, 779 specimens were collected from patients among which 11 (1.4%) were culture positive. Using IS481 and BP283 primers, 122 (15.6%) and 100 (12.8%) were diagnosed as infected specimens respectively. There were significant relationships between the real-time PCR method for diagnosis of B. pertussis and age, sex and vaccination of patients before sampling. The real-time PCR is superior and much more sensitive than culture for diagnosis of B. pertussis. However, the sensitivity was improved when both IS481 and BP283 were used. Correct sampling and transportation of specimen also improved the detection rate in our research.

Brucellosis is a zoonotic disease and it’s still endemic in Iran. There are some reports regarding brucellosis infection in family members sharing same risk factors and remain unrecognized. However, few studies on the importance of family screening are available. We aimed to screen household members of index cases with acute brucellosis for detecting additional unrecognized cases in central province of Iran. 163 family members of 50 index cases were enrolled in the study. Standard Tube Agglutination Test (STA) and 2-mercaptoethanol (2ME) agglutination were checked in all samples. A case with STA titer ≥ 1:80, 2-mercaptoethanol (2ME) agglutination ≥ 40 and compatible signs and symptoms was considered positive for brucellosis. 15 (9.2%) of family members were seropositive for Brucella agglutinin and among them, 8 (53.3%) were asymptomatic and 7 (46.7%) were symptomatic. STA titer ranged from 1:80 to 1:640 in seropositive members. 4 of the 15 seropositive cases who identified by screening came from one index case with 6 family members. All symptomatic seropositive cases treated for Brucella infection and recovered without any complications in 6 months follow up. On the basis of our data, family members of brucellosis patients are at risk of disease acquisition, and screening of household members provides an effective way for early diagnosis and prompt treatment. However cost benefit of screening should be evaluated to reach definite decision for the implementation of the screening as a nationwide program.

Streptococcus pneumoniae is the most common cause of invasive infections among both young children and elderly people. Common serotypes causing invasive diseases and the emergence of carriers of Streptococcus pneumoniae in Iran is not yet known. Past-vaccine surveillance studies of serotype prevalence patterns in Iran are necessary to monitor the epidemiology of Streptococcus pneumoniae. Because of variation of pneumococcal serotypes in different geographical regions, in this study we evaluated common serotypes causing pneumococcal infections and healthy carrier children in Tehran by Multiplex PCR. A total of 150 nasopharyngeal swabs were collected from healthy children in Tehran between December 2011 and August a2012, and 100 clinical samples were collected. Identification was performed by biochemical and molecular tests. Serotyping was done by multiplex PCR. We designed primers based on the sequences available for the routine capsular types and combined them into six multiplex PCR. From 150 nasopharyngeal swabs, 40 isolates of Streptococcus pneumoniae were identified after identification tests. Thirty six clinical isolates were also detected among clinical samples. Four serotypes (19A, 6, 3, 23F) of S. pneumoniae accounted for 55.7% of both sets of strains isolated from nasal carriage and clinical samples. Serotype 19A was the most common serotype among both groups. The multiplex PCR approach was successfully adapted to identify serotypes from more than 91% of the isolates tested. Among S. pneumoniae isolates in Tehran, the most prevalent serotypes were similar among carriage and invasive isolates. Continued monitoring of common serotypes of Streptococcus pneumoniae is essential for future vaccine formulation in Iran.

Methicillin Resistant Staphylococcus aureus (MRSA) strains are divided into Community Associated (CA-) and Hospital Associated (HA-) MRSA. These strains vary in antimicrobial resistance and pathogenicity. S. aureus is one of the most common microorganisms in ocular infections. This study was aimed to determine antimicrobial resistance patterns and genetic characteristics of MRSA strains isolated from ocular infections in Iran. Out of 171 S. aureus strains isolated from various clinical samples during September-December 2011 at Mashhad Emam Reza Hospital, 3 were cultured from eye discharge samples. Antimicrobial resistance tests were performed with MIC and disk diffusion methods and also genetic evaluation was done with Staphylococcal Cassette Chromosome mec (SCCmec), Accessory Gene Regulator (agr) and Staphylococcal Protein A (spa) typing, Multi Locus Sequence Typing (MLST) and determination of toxin gene profile. All strains were MRSA and showed resistance to tetracycline, gentamicin and clindamycin too. Vancomycin, minocyclin and trimethoprim/sulfamethoxazole were effective on all ocular isolates. All isolates belonged to SCCmec IV type. MRSA1 belonged to ST239, CC8, Spa type t7688 and agrIII and had tst1 and hla toxin genes. MRSA2 belonged to ST239, CC8, Spa type t037 and agrI and had the hla toxin gene. Finally, MRSA3 belonged to ST291, CC398, Spa type t304, and agrI and had pvl and hla toxin genes. phenotypic and genotypic evaluation of the isolated MRSA strains revealed that these strains belong to endemic Asian and livestock related clones that could reach from other body sites or environment to the eye of patients and developed ocular infection.

Infectious diarrhoeal diseases are great problem throughout the world and are responsible for considerable morbidity and mortality. Shiga toxin-producing Escherichia coli (STEC) is a major cause of gastroenteritis that may be complicated by hemorrhagic colitis (HC) or the hemolytic uremic syndrome (HUS), which is the main cause of acute renal failure in children. Food-borne outbreaks associated with Shiga toxin-producing Escherichia coli have been well documented worldwide. The aim of this study was to investigate the prevalence of Shiga toxin-producing Escherichia coli (STEC) strains in raw milk samples. Raw milk samples collected from various cow farms in Kermanshah, Iran during June - September 2009 were investigated for STEC using PCR targeting stx1 and stx2 and then eaeA. Of 206 samples, 36 (17.47%) were contaminated with STEC. STEC isolates harbored 56.41% and 43.59% stx2 and stx1 gene respectively. In antibiotic resistance test, all strains were sensitive to ceftazidime, cefepime, gentamicin, imipenem and ciprofloxacin. 23.08% of isolates were resistat to tetracycline, and 38.5% of them showed intermediate sensitvity to cephalothin. The high presence of STEC in raw milk confirms the important role of raw milk as putative vehicle of infection to human. Moreover, this study suggests that the development of antibiotic resistant STEC must be a major concern in Iran and more studies are needed to identify the prevalence of STEC in other food samples.

Dental caries is still remained as a major health problem. This problem has created a new interest to search for new antimicrobial agents from various sources including medicinal plants. Since limited data is available so far regarding the antibacterial effect of Coriandrum sativum seed and Dentol Drop against Streptococcus mutans, this study aims to assess this activity. This experimental study was conducted in Shiraz University of Medical Sciences. In vitro comparison of antimicrobial activity of aqueous decoction of Coriandrum sativum seed and Dentol drop with chlorhexidine against Streptococcus mutans was evaluated using disk diffusion and broth microdilution assays. Positive and negative controls were considered. The data was statistically analyzed by applying Kruskal-Wallis and Tukey post-hoc test to compare the groups using SPSS software (version 17). Dentol drop showed a remarkable antibacterial activity, in comparison with chlorhexidine, against S. mutans in the disk diffusion (p value = 0.005), and broth microdilution assays (p value = 0.0001). Based on the results of this study, Coriandrum sativum seed did not posses any antibacterial property. Coriandrum sativum seed showed no anti-Streptococcus mutans activity. Dentol drop exhibited a remarkable antibacterial activity against S. mutans when tested in vitro. Dentol drop can be further studied as a preventive measure for dental caries.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an important enteric pathogen in human causing bloody or nonbloody diarrhea, which may be complicated by hemolytic uremic syndrome (HUS). Cattle are an important reservoir of EHEC. This research aims at vaccination with a divalent chimer protein composed of EspA120 and Intimin 282 and its preventive effect of EHEC O157 colonization in mice rectal epithelium. A divalent recombinant EspA-Intimin (EI) protein containing EspA120 and Intimin280 attached with a linker was amplified from a trivalent construct and cloned in pET-28a (+) vector. The immunization was conducted in mice after expression and purification of the recombinant EI (rEI). Mice subcutaneously immunized with rEI, elicited significant rEI specific serum IgG antibodies and showed significantly decreased E.coli O157:H7 shedding compared to the control group. The chimeric recombinant protein induced strong humoral response as well as protection against oral challenges with live E.coli O157:H7.

The outer membrane protein W (ompW) of Vibrio cholerae is involved in stimulating the im­mune response via induction of protective immunity. It also plays an important role in bacterial pathogenesis by increasing the adaptability of pathogenic strains. In this study we aimed to clone V. cholerae ompW gene in the strain X-33 of Pichia pastoris. A gene encoding ompW was cloned into the Ppicza vector downstream of alcohol oxidase promoter. Then recombinant vector was transformed into the genome of the strain X-33 of P. pastoris. After growth of zeocin-resistant transformants, clones were selected and subsequently confirmed for cloning by PCR enzymatic digestion and sequencing. PCR, enzymatic digestion and sequencing showed that the ompW gene was correctly cloned into P. pastoris ge­nome. Results of our study showed that the methylotrophic yeast P. pastoris can be considered as an appropriate host instead of mammalian and prokaryotic systems for cloning of ompW. As far as data show, this is the first time that ompW of V. cholera is cloned into the methylotrophic P. pastoris.

Early determination of antibacterial susceptibility increases the success of therapy, decreases unnecessary use of antibacterial agents and side-effects, and lowers the overall healthcare cost. A rapid colorimetric method for antibacterial susceptibility testing named Qui-Sensitest (Inovative Biotechnology Organization, Istanbul, Turkey) was evaluated in this study. Qui-Sensitest proved to be a reliable rapid test for determining antibacterial susceptibility having an overall agreement of 97.6% with Kirby Bauer disk diffusion test results for enteric bacteria with 0.4% of major discrepancies and 2.0% of minor discrepancy. Since the test makes the results available in 3.5-6 hours, it can provide the means for choosing the right treatment regimen the same day the infectious agent is isolated.Keywords:

Pantoea agglomerans is a Gram-negative rod in the Enterobacteriaceae family. It is reported as both commensal and opportunistic pathogen of animals and humans. This organism is potential candidates as powdered infant milk formula-borne opportunistic pathogen. The aim of our study was to perform isolation, identification and antimicrobial susceptibility pattern of Pantoea (Enterobacter) agglomerans strains isolated from consumed powdered infant formula milk (PIF) in NICU ward. A of total 125 powdered infant formula milk (PIF) samples were purchased from hospital drug stores between June 2011 to March 2012. P. agglomerans was isolated according to FDA method. For final confirmation, biochemical tests embedded in the API-20E system were used. The drug susceptibility test was performed using the disc diffusion method according to CLSI guidelines. Out of the 125 samples investigated, 8 (6.4%) samples were positive for P. agglomerans and these were uniformly susceptible to tigecycline, chloramphenicol, cefepime, levofloxacin, minocycline and colistin. Fifty percent of isolates were resistant to cefotaxime, moxifloxacin, cotrimoxazole and ticarcillin. Controlling the primary populations of P. agglomerans during the PIF production process and preventing post processing contamination, by using suitable microbiological guidelines, is accessible. Sanitary practices for the preparation of infant formula in both the home and hospitals should be carefully controlled.

Seborrheic dermatitis (SD) is a frequent disorder of the skin that is distinguished by the development of erythematous patches and yellow-gray scales. It is a multifactor disease that requires predisposing factors for its progress. Presence of these factors leads to reproduction of opportunistic yeast Malassezia spp.The aim of the present study was to isolate and identify distribution of Malassezia species on the scalp of SD patients in Ahvaz using modified Dixons agar. A total of 110 patients diagnosed with SD were sampled. The sampling was carried out by brushing the hair and collecting the dandruff in paper pockets. For identification of Malassezia species, the scalp scales were cultured in Dixons agar. A combination of different characteristics including yeast cell morphology, ability to grow on Sabouraud dextrose agar, catalase test and ability to utilize individual Tweens (20, 40, 60 & 80) were used for identification of species. Twenty-seven of 110 (24.5%) SD patients had positive cultures for Malassezia species of which 17 (63%) were male and 10 (37%) were female. The most commonly identified Malassezia species was M. globosa (40.7%) followed by M. pachydermatis (22.2%), M. furfur (11.1%) and M. restricta(7.4%) and Malassezia species (18.5%). Malassezia globosa was considered to be the most important orgaism involved in cases with Seborrheic dermatitisin this study.

Encephalitis can cause a severe public health problem. The main aim of this research was to evaluate the medical laboratory results of patients with Herpes Simplex Virus (HSV) encephalitis. Diagnosis of encephalitis for these patients was firstly based on a clinical profile for Herpes Simplex Encephalitis (HSE), plus either a detected HSV1&2-DNA by PCR in CSF or brain neuro-imaging results. Molecular testing on CSF showed that 15 patients (15%) had HSV infection, 5 patients (5%) had Varicella Zoster Virus (VZV) and one case was positive for Human Immunodeficiency Virus (HIV)-RNA in CSF. The cause of encephalitis in 79 out of 100 patients (79%) was unknown. The comparison of CSF analysis in HSV positives and negatives showed a significant increase of glucose and protein levels in HSV positives than negatives. The mortality rate was 46.6% (7/15) in patients with HSV encephalitis compared to 11.4% (10/85) in non-HSV encephalitis (P = 0.003). In the current study, 15% of cases were diagnosed as having HSV.

Leucine dehydrogenase (LeuDH; EC 1.4.1.9) belongs to the amino acid dehydrogenase family and isused as a biocatalyst in medical and pharmaceutical industries (1). This study reported deals with the isolation and characterization of LeuDH from a thermophilic bacterium isolated from Jask Port in the Province of Hormozgan. Aliquots of soil and water samples were cultured in LEU specific medium and thermophilc bacteria that exhibited LeuDH activity were isolated and characterized biochemically. The LeuDH was purified and characterized in regard to the effects of pH and temperature on the activity, as well as its molecular weight determination. A thermophilic bacterium, Citrobacter freundii strain JK-9 was identified and found to exhibit LeuDH activity. The enzyme characterization revealed that LeuDH exhibits higher activity at temperature range of 60 to 75°C (optimum of 60°C) and an optimum pH of activity at pH 10.5. The Km value of LeuDH is 1.2 mM, while its molecular weight is about 320 kDa, and consisted of eight subunits identical in molecular mass (40 kDa). Briefly, a thermostableLeuDH enzyme from a strain of C. freundii was isolated and characterized. Our data indicate that the C. freundii enzyme has potential for use in biotechnological applications.

The essential amino acid L-tryptophan can be produced by a condensation reaction between indole and L-serine, catalyzed by B. subtilis with tryptophan synthase activity. Application of the tryptophan is widespread in the biotechnology domain and is sometimes added to feed products as a food fortifier. The optimum concentration of the Iranian cane molasses was determined by measuring the amount of biomass after growth in 1 to 30 g/mL of molasses. The maximum amount of biomass was obtained in 10 g/mL molasses. Chromatographic methods, TLC and HPLC, were used to assay the amount of tryptophan produced in the presence of precursors of tryptophan production (indole and serine) and/or molasses. Our results indicate the importance of the Iranian cane molasses not only as carbon source, but also as a source of precursors for tryptophan production. This report evaluates the potential of cane molasses as an economical source for tryptophan production by B. subtilis, hence eliminating the requirement for additional serine and indole as precursors.

Astaxanthin, an orange-red carotenoid pigment, acts as a protective agent against oxidative damage to cells in vivo. The astaxanthin synthetase gene (crtS) size consists of 3995 bp. This gene has been suggested to catalyse β-carotene to astaxanthin in Phaffia rhodozyma. The aim of this research was to find any possible changes in this gene in two mutant strains, Gam1 and Gam2 (with high astaxanthin pigment production), previously created by gamma irradiation. The astaxanthin synthetase gene sequence of Phaffia rhodozyma in the NCBI Gene bank was used to design primer. In Gam1, this gene was amplified using primers Asta F1, Asta R2, Asta F3, Asta R4. In Gam2, primers asta F1, asta R4 were used to amplify the gene. The amplified fragments were 8 sequenced using primers Asta F1, Asta R1, Asta F2, Asta R2, Asta F3, Asta R3 and Asta F4, Asta R4. Astaxanthin synthetase gene from two mutant strains, Gam1 and Gam2 were amplified using PCR. The amplified products were sequenced and aligned using the ClustalW software. The comparison of this gene showed 98% and 99% similarities between the reference sequence and Gam1 and Gam2 mutant strains, respectively, whereas the comparison of this gene in Gam1 and Gam2 mutant strains showed 97% similarity. However, the deduced proteins showed 78% and 83% between the reference protein obtained from the wild type and Gam1 and Gam2, respectively. This similarity was 75% between the mutant strains.

Uttarakhand region is less explored, but possess a great biodiversity. This diversity can be explored for isolation and characterization of new actinomycetes strains for seeking antimicrobial molecules. It can therefore be predicted that novel bioactive metabolite producing actinomycetes can be discovered to combat multidrug resistant bacterial pathogens. Variations in the viable count of actinomycetes were accessed in different altitudes. Actinomycetes were isolated, indentified and screened for their antibacterial activity. The highest viable counts of actinomycetes were recorded in valleys followed by mid hills and high hills. A total of 512 actinomycetes were isolated which were found to belong the 14 different genera of actinomycetes. Mainly the genus Streptomyces was dominant in all the soil samples. Out of 512 isolates recovered, 23.44% exhibited antibacterial activity against one or more tested bacterial pathogens. Of these 56.67% showed activity against Gram-positive bacteria, 26.67% against Gram-negative bacteria while 16.67% showed broad spectrum activity. Isolate DV1S and GR9a-5 showed highest antibacterial properties against several multi-drug resistant bacterial pathogens and were identified using polyphasic approach. DV1S and GR9a-5 were found to be most closely related with S. massasporeus NBRC 12796T and Nocardia nova JCM 6044T respectively. The results of this study strongly support the idea that the viable count of actinomycetes varied greatly with altitude. The actinomycetes species isolated from valleys, mid hills and high hills possess significant capacity to produce compounds which are active against several drug resistant bacterial pathogens.

In the last few decades, losses of our cultural heritage due to biodeteriorationare beinghighly recognized. From museum objects to rock monuments, the microbial biodeterioration agents are found to be the most destructive. Possibilities for proper preservative measure(s) are always more when it is only a monument, statue, museum article, or pre-historic art in any small subterranean cave. Nevertheless, preservation/protection of the footprints occupying a big area, lying scattered in a very negligible manner requires safeguard against several deterioration factors; right from various physical, chemical and biological agents which are indeed interrelated to each other. In the present study, some microbial communities possibly responsible for deteriorating the rocks of Kabra-pahad, where the most famous pre-historic rock paints of India prevail have been identified. The diversity of fungi and bacteria present in the stone crust of the infected areas has been studied by employing standard laboratory methods.The cultivated cultures confirmed total fifteen fungal species, among which Aspergillus group were the most dominant. Among bacteria, total 80 numbers of colonies were observed that dominated by two major groups; Micrococcus.spp and Staphylococcus spp. The pre-historic footprint in the form of rock paints in Kabra-pahad of district Raigarh, Chhattisgarh, India is lying in a very deteriorated manner. In the present study, we have tried to identify few major deteriorating factors that are responsible for such degradation of our existing pre-historic footprints.