Phenotypic characterization of gram-positive cocci strains isolated from infections localized in the oro-maxillo-facial sphere

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1 Phenotypic characterization of gram-positive cocci strains isolated from infections localized in the oro-maxillo-facial sphere R.E. VLAD (1), M. TILINCA (2), E. MIRCIA (3), R.A. VLAD (4), Ș. STĂCESCU (5), G. HANCU (5,*), C. TĂNASE (6), M. MONEA (7), G. BĂNCESCU (1) 1 Department of Microbiology, Faculty of Dental Medicine, University of Medicine and Pharmacy Carol Davila, București, 2 Cell and Molecular Biology, Faculty of Medicine, University of Medicine and Pharmacy Tîrgu Mureș; 3 Department of Drugs Industry and Pharmaceutical Management, Faculty of Pharmacy, University of Medicine and Pharmacy Tîrgu Mureș, 4 Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Medicine and Pharmacy Tîrgu Mureș, 5 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Medicine and Pharmacy Tîrgu Mureș, 6 Department of Pharmaceutical Botany, Faculty of Pharmacy, University of Medicine and Pharmacy Tîrgu Mureș, 7 Department of Odontology and Oral Pathology, Faculty of Dental Medicine, University of Medicine and Pharmacy Tîrgu Mureș, *Corresponding author Gabriel Hancu, University of Medicine and Pharmacy Tîrgu Mureș, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Gh Marinescu 38, , Tîrgu Mureș, România, Tel: , gabriel.hancu@umftgm.ro Key words: oro-maxillo facial infections, Gram-positive cocci, phenotypic characterization, biochemical profiling. Summary Oro-maxillo-facial infections are can be a serious health problem not only in their frequency but also through the severe complications they may develop. Taking in consideration the implications of Grampositive cocci strains in regional and systemic pathology, this paper aims to present the phenotypic characters of these bacteria, while underlining the role they play in human pathology. The study was performed on 30 patients diagnosed during a year with infections located in the oro-maxillo-facial area. A total of 73 strains were isolated, of which 43 were Gram-positive strains. Biochemical profile of the strains was evaluated using the Rapid ID 32 STREP system. Introduction Oro-maxillo-facial infections are not rare and could be considered as a public health problem due to their great potential of spreading to important and vital anatomical structures, such as the respiratory system and mediastinum, increasing the risk of septicemia and death for the affected patients (Veronez et al., 2014). In terms of the incidence of oro-maxillo-facial infections, abscesses are by far on the first place. The abscesses are plurimicrobial, being produced usually by anaerobic bacteria and sometimes anaerobic bacteria. Among the anaerobic strains, a number of Gram-positive cohorts, especially oral streptococci, are the most important (Wang et al., 2005). The pathogenicity potential can be manifested by the streptococci when the host's antiinfective resistance is diminished or when accidentally the bacterium reaches normally sterile areas through various traumas, dental or surgical interventions. Here the body's defense mechanisms are limited, but there are favorable nutritional conditions for the bacterium (Sato et al., 2009). 23

2 The paper aims the phenotypic characterization of some strains of Grampositive cocci strains isolated from 30 cases of infections with localization in the oromaxillo-facial sphere. Materials and methods Harvesting and transport of pathological products The harvest was performed in a total of 30 patients diagnosed during a year with infections located in the oro-maxillo-facial area, at the Oro-Maxillo-Facial Surgery Clinic of the Faculty of Dental Medicine of the University of Medicine and Pharmacy "Carol Davila" - Bucharest. This was done by puncture and aspiration after local antiseptization. A 1-2 ml pus sample was harvested. The patient population consisted of 20 women and 10 men aged between 5 and 87 years. Samples were from the following types of infections: vestibular abscess (10 cases), submandibular abscess (4 cases), genian abscess (3 cases), peribazilar abscess (2 cases), lingual abscess (1 case), sublingual abscess (1 case), subtemporary abscess (1 case) and abscess maseterin (1 case). There were also cases of submandibular adenitis (3 cases), of acute supraventricular parotiditis (3 cases) and of sub-submandibular suppuration (1 case). Sample transport was performed in the syringe that was harvested, within less than one hour of sampling, in order to continue the microbiological investigation. Microscopic examination of the pathological product Colored Gram smears were made from the 30 samples of pus. These were examined under the optical microscope with the X100 immersion objective. The following working techniques have been followed: on a surface of about 1.5 cm² in the middle of a microscope blade a drop of pus was pushed in thin layer, avoiding reaching the edges of the blade; the microscope blade was left on the laboratory bench at room temperature until the smear was completely dried; the dry smear was fixed by passing the opposite face of the microscope blade 2-3 times for a few seconds through the tip of the Bunsen bulb flame; the smear was covered with a gentian violet solution, which was removed after one minute; the smear was covered with a solution of Lugol, which removed after 2 minutes; the smear was coated with an alcohol-acetone solution for 10 seconds, rotating and tilting the microscope blade to quickly remove dye traces; after 10 seconds, the smear was washed with water; the smear was then coated with a solution of fucin, diluted 1/10 extemporaneously with water, which was maintained for 30 seconds - 1 minute; after the second dye was removed, the smear was washed with water and the microscope blade was placed in a vertical position in a stand for drying. Seeding the pathological product and the isolation of microbial strains From each sample, a drop of pus was seeded using a bacterilogical loop on each of the three categories of used culture media: Columbia Gelose with 5% ram blood (GS), blood agar with Nalidixic acid and Sulfamethasine (NAS), and Schaedler agar with Menadione And 5% ram blood (SCH). Inoculum dispersion was performed in 4 sectors, with burning and cooling of the loop before starting the next sector for nonselective media (GS and SCH). The GS plates were incubated in a 5% CO2 enriched atmosphere using BBL CampyPakPlus (Becton Dickinson Microbiology Systems, USA) at 37 C for hours, and the NAS and SCH plates in anaerobic atmosphere, with the GasPakPlus system (Becton Dickinson Microbiology Systems, USA) at 37 C for 2 days for NAS and 7 days for SCH. Bacterial growth was studied with an stereomicroscope at 24 and 48 hours for GS and NAS, and for SCH at 7 days. From NAS and GS, 1-3 colonies of each type were replicated on 2 GS plates in the sector, one plaque being incubated aerobically and the other anaerobically at 37 C for hours. From SCH, 1-3 colonies of each type were replicated on 2 GS plates in the sector, one plate was incubated in 5% CO2 enriched atmosphere and the second anaerobic at 37 C for 48 hours. Identification of bacterial strains Isolated strains were categorized as: strictly aerobic strains, optionally anaerobic strains 24

3 and strictly anaerobic strains, depending on the possibility of growing or not in the presence of O2. Following the microscopic examination of Gram-stained smears, the isolates were divided into morphothinctorial categories, respectively in strains of: Grampositive coils in irregular piles, Gram-positive batch strains in strains, Gram-positive bacilli, Cocobacilli and bacilli Gram-negative bacilli. Identification continued at gender and species level only for Gram-positive strains. Results and discussions At the microscopic examination of Gram smears made from pus samples, a number of 28 smears were identified. The absence of bacterial flora was detected on two smears. The cultures were obtained from the 28 samples of pus on at least one of the used media. A total of 73 strains were isolated, of which 43 were Gram-positive strains (fig. 1). Of these, 24 were optional anaerobic strains of Gram-Positive cocci in chains, noncatalyso-negative, non-producing pyrrolidonyl arylamidase, sensitive to vancomycin with variable response to the bile-esculin test and the 6.5% NaCl tolerance test, consequently were included in the Streptococcus genus. Fig. 1. Distribution of the 43 Gram-positive batch strains. Identification at species level indicated the membership of Gram-positive cohorts in 5 bacterial genes. Streptococcus anginosus (3 strains), Streptococcus constellatus (2 strains), Staphylococcus aureus (3 strains), Streptococcus aureus (3 strains), Gemella morbillorum (1 strain) and Enterococcus faecalis (1 strain). Identification was performed using the Rapid ID 32 STREP system, because this method is standardized, based on a large number of biochemical tests, is easy and fast, giving the result in 4 hours. From the strains in the S. anginosus group, 9 developed on the agar-blood type S-type colonies and one M-type colony, all producing caramel-smelling cultures. One strain of S. constellatus and 4 strains of S. anginosus were non-hemolytic. A S. constellaus isolate and two S. anginosus isolates produced were beta-hemolytic on agar-blood, and another 4 S. anginosus isolates were alpha-hemolytic. All strains of S.oralis produced dimorphic-like colonies on blood-agar medium, most of the colonies were alpha-hemolytic and only a few nonhemolytic. The same dimorphic aspect has always been achieved in the repopulation of both categories of colonies on new culture media. The S. aureus isolates produced yellow-gold colored S colonies on the bloodyellowing-blood environment, and on Chapman medium decomposed mannitol. They produced complete beta hemolysis and were coagulase-positive. Strains of S. epidermidis produced white blood cell colonies on white blood cell agar: they were non-hemolytic and coagulase-negative. The strains of Gemella morbillorum and Enterococcus faecalis were alpha-hemolytic. Biochemical profile of the strains was evaluated using the Rapid ID 32 STREP system. All 10 strains belonging to the S. anginosus group produced: arginine dehydrolase, beta-glucosidase, alaninephenylalanine-proline arylamidase, alkaline phosphatase and acetoin by glucose fermentation (Voges-Proskauer positive test). The ability to synthesize beta-galactosidase was highlighted in 7 strains, none of which did hydrolyze sodium hypochlorite and did not develop: beta-glucuronidase, betagalactosidase, pyroglutamic arylamidase and 25

4 urease. Also all isolates fermented: sucrose and maltose, but not: sorbitol, D-arabitol, cyclodextrin, glycogen and melezitose. The 2 strains of S. constellatus produced: acetoin by glucose fermentation (Voges- Proskauer positive test), arginine dehydrolase, alkaline phosphatase and alaninephenylalaline-dehydrogenase. Also all isolates fermented: sucrose and maltose, but not: sorbitol, D-arabitol, cyclodextrin, glycogen and melezitose. Of the 12 strains belonging to the S. oralis group, 11 of them synthesized: betagalactosidase and alanine-phenylalanineproline arylamidase, and 6 of them alkaline phosphatase. No strain hydrolysed sodium hypochlorite and did not produce: arginine dehydrogenase, beta-glucuronidase, acetoin (Voges-Proskauer negativ). All 10 strains of staphylococci (7 strains of S. epidermidis and 3 strains of S. aureus) developed: urate, acetoin (Voges-Proskauer positive test), alkaline phosphatase and fermented glucose, fructose, maltose, lactose and sucrose. They also produced nitrate reduction. S. aureus isolates fermented trehalose and mannitol, but no staphylococcus strain fermented raffinose, arabinose and celibiose and did not produce pyrrolidonyl arylamidase. Gemella morbillorum produced alaninephenylalanine-proline arylamidase, pyroglutamic arylamidase and fermented sucrose and maltosis. It did not synthesize alkaline phosphatase, arginine dehydrogenase, beta-glucosidase, beta-galactosidase, betaglucuronidase, beta-galactosidase and did not produce acetoin (Voges-Proskauer negative test). Enterococcus faecalis synthesized: arginine dehydrogenase, beta-glucosidase and beta-galactosidase, alkaline phosphatase, alanine-phenylalanine-proline arylamidase and acetoin by glucose fermentation (Voces- Proskauer positive test). It did not produce beta-glucuronidase and urease and did not hydrolyse sodium hypochlorite. A number of 5 isolates of S. anginosus could be assigned to one of the tested Lancefield groups, and one isolated S. constellation and 6 S. anginosus isolates were declared ungrouped. At the same time, the strain of Gemella morbillorum has been declared ungrouped. We identified 4 strains of group F (3 non-hemolytic S. anginosus and one S.constellatus beta-hemolytic), a S. anginosus (nonhemolytic) strain (group C) and a E. faecalis (alpha-hemolytic) strain (group D). Conclusion This phenotypic characterization of isolated strains is particularly important for both proper species identification and for capturing certain particularities in order to establish the correct microbiological diagnosis and for the testing of antibiotic susceptibility (Flynn et al., 2006). Gram-positive cocci constituted more than half of the total number of microbial strains and were isolated in most cases together with different strains of bacilli or cocobacilli, especially with strictly anaerobic Gramnegative bacilli. Exclusive associations of Gram-positive batch strains belonging to different species were found in more than one-tenth of the investigated piogenic diseases, whereas isolation of a single etiological agent was found in only one case of vestibular abscess involving a strain of S. anginosus. Species identification indicated Gram-positive cohort strains belonging to 5 bacterial genes, with the net predominance of oral streptococci (especially S. anginosus and S. oralis), followed by P. micra, which is the only strictly anaerobic species isolates in this study. The representation of Gemella and Enterococcus species by a single isolate excluded the obtaining of particular conclusions regarding their phenotypic characterization. Unlike Staphylococcus and P. micra isolates, for which the phenotypic characteristics investigated proved to be very similar at the level of the respective species, in the group of S. anginosus the heterogeneity of the biochemical profile as well as the aspect of the colonies and antigenic characters can be observed. Frequent involvement of oral streptococci and peptostreptococci in the etiopathogenicity of oro-maxillo-facial infections highlights the 26

5 importance of accurately identifying species of Gram-positive cohort isolates of clinical significance. References Flynn, TR; Shanti, RM; Levi, MH; Adamo, AK; Kraut, RA; Trieger N.: Severe odontogenic infections, Part 1: Prospective report. Journal of Oral Maxillofacial Surgurery. 64: , Sato, FRL; Hajala, FAC; Freire Filho, FWV; Moreira, RWF; Moraes, M.: Eight-year retrospective study of odontogenic origin infections in a postgraduation program in oral and maxillofacial surgery. Journal of Oral Maxillofacial Surgurery. 67: , Veronez, B; de Matos, FP; Monnazzi, MS, Sverzut, AT; Sverzut, CE; Trivellato AE.: Maxillofacial infection. A retrospective evaluation of eight years. Brazilian Journal of Oral Sciences, 13(2), , Wang, J; Ahani, A; Pogrel, MA.: A five-year retrospective study of odontogenic maxillofacial infections in a large urban public hospital. International Journal of Oral Maxillofacial Surgerery, 34, ,