Shazia Akbar Ansari* , Rakhshanda Baqai** , Muhammad Rizwan Memon*** , MubashirAziz**** , Muhammad Khalil Khan*****
The objective of this study was the detection and separation of S. aureus from blood cultures of patients undergoing oral surgical procedures.Antibiotic sensitivity pattern and biofilmformation of S. aureuswere also performed.
Total 250 patients undergoing oral surgical procedures were selected for bacteriological examination. 5ml of Blood sample was collected in blood culture bottles containing tryptone soya broth. Blood sample was incubated at 37°C for 7 days and after incubation subculturing was done on appropriate Media. The plates were then incubated at 37°C aerobically for 24 hours, after which isolated colonies were obtained. S.aureus was identified by Gram staining, colony morphology, pigment production, catalase, coagulase and often biochemical tests.Antibiotic susceptibilitywas performed by disc diffusion technique on isosensitivity agar. Strains of S.aureus were used for biofilm formation by simple tubemethod.With the help of spectrophotometer at 570 nm optical densitywasmeasured. S.aureus (ATTC2523)was analyzed for biofilmproduction.
Bacterial isolates in descending orderwere S.aureus 56%, E.coli 25%, Pseudomonas spp. 13%, S.typhi 4%and Shigella spp2%. S.aureuswas resistant to different antibiotics.Biofilmproduction of S.aureuswas detected in 16.17%of the S.aureus andmostly in associationwith antibiotic resistant bacteria.
S.aureus was the predominant group of bacteria isolated from blood cultures of dental patients. Increased antibiotic resistance of S.aureusmay be due to biofilmproduction resulting in persistent dental infections.
Staphylococcus aureus,Biofilm,Antibiotic resistance.
In hospitals and in our community, Staphylococcus aureus is amajor human pathogen and a primary cause of bloodstream infections. In the form of biofilm, S. aureus colonizes the teeth of patients. Biofilms are the most common mode of bacterial growth in nature and are also important in clinical and dental infections which are common in our population.
Most common dental diseases are gingivitis, periodontitis, dental caries, pulpitis, pulp necrosis, peripheral abscess, cellulites and pericoronitis. For the treatment of these different dental diseases, numerous dental procedures like tooth extraction, endodontic treatment, periodontal surgery, conservative dental procedures and scaling are performed, that causes significant bleeding and cause the spread of oral bacteria into the blood streamwhich result in bacteremia. S.aureus is a major human pathogen causing a wide range of infections. Over the last 25 years, the incidence of both community-acquired and hospital-acquired S. aureus infections has increased.
Dental diseases are caused by microorganismswhich are a part of normal flora that is present in the form of biofilm. Biofilm is defined as complex aggregation of microorganism surrounded by a protective and adhesive matrix of polymer substances that adheres to all surfaces either inert or living. Van Leeuwenhoek can be credited with the discovery ofmicrobial biofilmswho first observe microorganisms on tooth surfaces. It can form on a wide variety of abiotic hydrophobic and hydrophilic surfaces. These environments are oligotrophic rendering the microbes in a starved state . The biofilm regulate the developmental process that leads to a development of complex surface-attached bacterial community. This bacterial community has a number of distinct characteri sti cs including the product ion ofexopolysaccharides hydrodynamics of the bulk fluid. In this environment, biofilm formation can have profound negative and positive impact and as a consequence, it can increase the cost in term of both economics and human health Bacteria, in these environment, can grow as a biofilm.
Certain species (Spp) appear to have a predilection to form biofilms. Most of these Spp are members of the normal microflora of human beings and form biofilms at sites where they are found naturally. Streptococci form biofilms on the surfaces of teeth, are cariogenic and periodontal pathogenic bacteria Bacteria in a biofilm are very resistant to biocides.
Gene transfer between bacteria is known to be an important means by which antibiotic resistance and virulence factors are spread betweenmembers of the same and different Spp. Transfer of gene occurs in biofilms and only a very limited number are involved with human diseases. . In S aureus biofilm, S.aureus is an adaptable, pathogenic organism. It is an opportunistic pathogen and can infect humans resulting in a myriad of infections . The close contact between bacteria within a biofilm and the matrix may inhibit the penetration of antibiotics through the exopolysaccharidematrix
The phenotypic heterogeneity of bacteria in a biofilm suggests the presence of persister cells or antibioticresistant non-dividing cells which are able to reestablish the biofilm after the threat has passed The oral cavity may also harbor antibiotic-resistant organisms, which are incriminated in many extra-oral systemic infectious diseases.
Total 250 bacterial isolates were collected from blood cultures of dental patients, who underwent different oral surgical procedures. Clinical history and consent was obtained from every patient. Ethical committee of Fatima JinnahDental College and Hospital Karachi has given the approval of the study. Blood samples were collected for bacteriological examination immediately after the essential steps of the oral surgical procedures had been performed. 5ml Blood sample were collected in Tryptic Soya broth (diaphasic media) and incubated at 37°C for 7 days and then subcultured on blood agar and MacConkey’s agar.
The plates were incubated at 37°C aerobically, after 24 hours growth was observed. Colonies were identified by gram’s staining and biochemical test. Biochemical characteristics were observed by performing catalase, coagulase and other biochemical tests. With the help of disc diffusion technique on isosensitivity agar, antibiotic susceptibilitywas done Test was performed according to the clinical laboratory Standard Institute (CLSI) guidelines.
Antibiotic sensitivity test was done by using the discs of ampicillin , clindamycin, vancomycin , tetracycline , cephalexin and cefoxitin. Biofilm assay was done by simple tubemethod and analyzed by spectrophotometer. S aureus strain (ATTC 2523) was used to compare the biofilm production. 68 strains of S.aureus were selected for biofilm analysis. Sterilized plastic tubes were taken and under aseptic condition, isolated cultures were inoculated in 2ml TSB (tryptone soya broth) tubes. TSB tubes were then incubated at 37°C for 24 hours. After incubation, to each tube2ml ofTSBwith 2%glucosewere added. Tubes were reincubated at 37°C for 24 hours. The growth medium was discarded. Each tube was washed 3 times with Phosphate Buffer Saline (PBS) under aseptic condition to eliminate the unbound bacteria. To valuate the formation of biofilm, remaining attached bacteria were fixed with 2ml of 99% methanol for 15 minutes and biofilm were stained with 0.2 ml of 2% crystal violet. Excess stain was rinsed off by placing the tubes under running tap water. Tubes were air dried and the adherent cells were solubilized with 1.5ml of 33% glacial acetic acid.
The optical densities of each tube were determined at 570 nm by using spectrophotometer. The blank (negative control) was determined for each tube by measuring the optical density (OD) of a tube filled with PBS. For the positive control, pure cultures were used to measure the optical density. Results were recorded and OD of the isolated microorganisms was compared with the OD of pure cultures of S.aureus. If the OD of isolated and identified cultureswas grater than the OD of pure cultures then it indicated that isolated culture had the ability to formmorepowerful biofilmthan the pure culture
Total 114 S.aureus were isolated from blood cultures of different dental patients. 68 strains of S.aureus were used for biofilm production. Figure 1 indicates the bacterial isolates from blood cultures associated with oral surgical procedures. Bacteril isolated in descending order were S.aureus 56%, E.coli 25%, Pseudomonas Spp.13%, S.typhi4%and Shigella Spp.2%.
Figure 2 indicates the antibiotic sensitivity pattern of S.aureus. It was sensitive to ampicillin (98%), clindamycin (79%), vancomycin (77%), tetracycline (77%), cephalexin (71%), and cefoxime (47%).
S.aureus. ATCC culture of S.aureus (2523) was analyzed for biofilm production and it indicated weak biofilm production.
Dental infections are more common in our country due to poor oral hygiene that results inBiofilmproduction and high antibiotic resistance. It is therefore recommended to maintain the oral hygiene and control dental diseases with comprehensive mechanical and chemotherapeutic oral hygiene practices. Teaching patients to improve oral health by regular tooth brushing, interdental cleaning and use of antimicrobial mouthrinses and conduction of continuing dental education programs to prevent dental diseases and bacteremia during oral surgical procedures is recommended.
1. Lipton JA, Ship JA, Larach-Robinson D. Estimated prevalence and distribution of reported orofacial pain in the United States. JAm DentAssoc. 1993 ;124:115- 121.
2. Daly CG, Mitchell DH, Highfield JE, Grossberg DE, Stewart D. Bacteremia due to periodontal probi ng: a c linic al and microbi ol ogic al investigation. JPeriodontol. 2001;72:210-214.
3. McGowan JE., Shulman JA.: Blood stream invasion. 2nd ed.W.B. Saunders. 1998. 645-654 old
4. Steinberg JP, Clark CC, Hackman BO. Nosocomial and community-acquired Staphylococcus aureus
bacteremias from 1980 to 1 9 9 3 : i m p a c t o f intravascular devices and methicillin resistance. Clin InfectDis. 1996 ;23:255-259.
5. Bowden GH, Hardie JM, Slack GL. Microbial variations in approximal dental plaque. Caries
6. CoghlanA. Slime city.NewSci. 1996; 15: 32-36
7. Mazzola PG, Martins AM, Penna TC. Chemical resistance of the gram-negative bacteria to different
sanitizers in a water purification system. BMC Infect Dis. 2006 16;6:131.
8. O’Toole G, Kaplan HB, Kolter R. Biofilm formation as microbial development. Annu Rev Microbiol.
9. Jabra-Rizk MA, Falkler WA, Meiller TF. Fungal biofilms and drug resistance. Emerg Infect Dis.
10. Listgarten, BioLine, Cardiff M. Formation of dental plaque and other oral biofilms. In: Newman, H. N. & Wilson, M. (eds), Dental Plaque Revisited: oral biofilms in health and disease, 1999. 187-210.
11. Borenstein S.B. Microbiologically Influenced Corrosion Handbook, Industrial Press Inc., New
York. 2004. 12. Stefani S, Agodi A. Molecular epidemiology ofantibiotic resistance. I nt J Antimicrob Agents. 2000 ;13:143-153.
13. Marcus SL, Brumell JH, Pfeifer CG, Finlay BB. Salmonella pathogenicity islands: big virulence in
small packages.Microbes Infect. 2000 ;2:145-156.
14. Licht TR, Christensen BB, Krogfelt KA, Molin S. Plasmid transfer in the animal intestine and other
dynamic bacterial populations: the role of community structure and environment. Microbiology.
1999 ;145 :2615-2622.
15. Götz F,BannermanTand. SchleiferKH. The Gener a Staphylococcus andMacrococcus. Springer New
York,NY, 2006. 11-59.
16. Jefferson KK, Goldmann DA, Pier GB. Use of confocal microscopy to analyze the rate of vancomycin penetration through Staphylococcus aureus biofilms. Antimicrob Agents Chemother. 2005 ;49:2467-2473.
17. Lewis K. Persister cells, dormancy and infectious disease.Nat RevMicrobiol. 2007 ;5:48-56.
18. Suzuki J, Komatsuzawa H, Sugai M, Suzuki T, Kozai K, Miyake Y, Suginaka H, Nagasaka N. A
long-term survey of methicillin- res istant Staphylococcus aureus in the oral cavity of children.Microbiol Immunol. 1997;41:681-686.
19. Cirioni O, Giacometti A, Ghiselli R, Dell’Acqua G, Orlando F, Mocchegiani F, Silvestri C, Licci A,
Saba V, Scalise G, Balaban N. RNAIII-inhibiting peptide significantly reduces bacterial load and enhances the effect of antibiotics in the treatment o f c e n t r a l v e n o u s c a t h e t e r – a s s o c i a t e d Staphylococcus aureus infections. J Infect Dis. 2006 15;193:180-6.
20. Wenzel RP, Edmond MB. The impact of hospitalacquired bloodstream infections. Emerg Infect
21. American Academy of Pediatrics. Summaries of infectious diseases. Staphylococcal infections.
In: Pickering LK, ed. 2003 Red Book: Report of the Committee on Infectious Diseases, 26th ed.
Elk Grove Village, IL: American Academy of Pediatrics, 2003. 561-573.
22. Seifert H, Wisplinghoff H, Kaasch A, Achilles K, Langhorst A, Peyerl-Hoffmann G, Woehrmann A,
Fätkenheuer G, Salzberger B, Kern WV. [Epidemiology, cours e and prognosis of Staphylococcus aureus bacteremia–Preliminary r e s ul t s f r om t h e INSTINCT ( INva s i ve STaphylococcus aureus INfectionCohorT) cohort]. DtschMedWochenschr. 2008 ;133 :340-345.
23. Iwatsuki K, Yamasaki O, Morizane S, Oono T. Staphylococcal cutaneous infections: invasion, evasion and aggression. J Dermatol Sci. 2006 ;42:203-214.
24. Bagge N, Ciofu O, Skovgaard LT, Høiby N. Rapid development in vitro and in vivo of resistance to
ceftazidime in biofilm-growing Pseudomonas aeruginosa due to chromosomal beta-lactamase. APMIS. 2000 ;108:589-600.
25. Chandra J, Mukherjee PK, Leidich SD, Faddoul FF,HoyerLL,Douglas LJ,GhannoumMA.Antifungal
resistance of candidal biofilms formed on denture acrylic in vitro. JDent Res. 2001 ;80:903-908.
26. Xu KD, McFeters GA, Stewart PS. Biofilm resistance to antimicrobial agents. Microbiology. 2000 ;146
27. MonroeD. Looking for chinks in the armor of bacterial biofilms.PLoS Biol. 2007 ;5:e307.
28. Jefferson KK, Goldmann DA, Pier GB. Use of confocal microscopy to analyze the rate of vancomycin penetration through Staphylococcus aureus biofilms. Antimicrob Agents Chemother. 2005 ;49:2467-2473.
29. MonroeD. Looking for chinks in the armor of bacterial biofilms.PLoS Biol. 2007 ;5(:e307. 30. Rogers A H. Molecular Oral Microbiology. Caister Academic Press. (2008).
31. Sbordone L, Bortolaia C. Oral microbial biofilms a n d p l a q u e – r e l a t e d d i s e a s e s : mi c ro bi a l communities and their role in the shift from oral health to disease. ClinOral Investig. 2003 ;7:181-188.