The Minimum Inhibitory Concentration

Published Date: 02 Nov 2017

Atulya M1, Jesil Mathew. A1*, Koshy John2

1Dept. of Pharmaceutical Biotechnology, Manipal College of Pharmaceutical Sciences, Manipal, Udupi, Karnataka, 576104.

2 Dept. of Veterinary Microbiology, College of Veterinary and Animal Sciences, Pookot, Wayanad, Kerala

*Corresponding author: Dr. Jesil Mathew. A, Email: [email protected],

Tel: Fax:

ABSTRACT

Bacterial biofilm formation provides the bacteria inherent resistance to antibiotic chemotherapy and helps in the persistence of infection. The dental plaque is a dental biofilm which gets calcified and becomes dental tartar. The biofilm formation results in the development of periodontal diseases. Biofilm in the oral cavity of dog can affect the general health of the dog and the bacterial toxins can damage the visceral organs engaged in filtration of toxins. It can also be detrimental to human health when transferred via animal bites. Organisms were isolated from canine dental tartar and were identified as Pseudomonas fragi (43%), Citrobacter koseri (14%), Streptococcus mutans (33%) and yeast (10%). Biofilms were produced in microtiter wells and quantified using crystal violet staining. The minimum inhibitory concentration and minimum biofilm eradication concentration were compared to determine the change in antimicrobial susceptibility pattern of dental tartar isolates when they go from the planktonic to the biofilm mode of growth. The bacteria in biofilm phase of growth exhibited significantly higher antibiotic resistance compared to the planktonic state of the same organisms.

Key words: Dental tartar, Biofilm, Crystal violet assay, Minimum biofilm eradication concentration

INTRODUCTION

Dental plaque is a dental biofilm which is the primary etiology of periodontitis. This is a diverse microbial community adhered to the host surface, consisting of genetically distinct types of bacteria that live in close juxtaposition (Williams 2008). These oral microbial plaque communities are composed of gram positive and gram negative bacteria embedded in polymeric substances of bacterial and salivary origin. The biofilm forming ability is recognized as the factor responsible for the persistence of microbes in the host, which helps in evading the host immune response and allows the bacteria to survive at adverse situations (Clutterbuck, Woods et al. 2007). Biofilm formation in turn alters the microenvironment of its own inhabitants which then leads to additional alterations in gene expression and further maturation of the biofilm (Jefferson 2004). In course of time the dental plaque on the tooth surface get deposited with carbonates and phosphates of calcium and magnesium, and forms the hardened yellow/ brown layer called dental calculus/ dental tartar (Marsh 1995). The calculus serves as a trap for the further plaque development and this also results in periodontitis. Dental tartar is encountered in approximately 85% of adult dogs (Auvil 2002).

The plaque micro biota to a greater extend depends on the endogenous nutrients supplied by the host rather than by the exogenous factors in the diet (Lappin-Scott and Costerton 2003). The component species in the biofilm reaches a microbial homeostasis through numerous microbial interactions which includes both synergism and antagonism (Marsh 2003).

MATERIALS AND METHODS

A study was conducted to investigate the biofilm production and antibiotic susceptibility of microorganisms responsible for dental tartar and associated lesions. The media and other chemicals used were purchased from M/S Himedia laboratories unless otherwise mentioned. Dental swabs were collected from fourteen canines with dental tartar, belonging to different breeds which were otherwise quite normal. Sub gingival pooled samples were collected from four sites in each case. Sterile Brain Heart Infusion Agar plates were inoculated and incubated at 370C aerobically for 24hrs. Identification of bacteria was done as per the standard protocols prescribed in Bergy’s Manual (Holt, Krieg et al. 1994). The details of isolates are given in table no.1.

Table No.1 Details of clinical isolates

Serial No.

Age (years)

Breed

Clinical signs/ Symptoms reported

Micro organism

1

1.5

Non descript

Halitosis

P. fragi & S.mutans

2

9

Dachshund

Gingivitis,halitosis

P.fragi

3

9

Spitz

Halitosis,discoloration of tooth

P.fragi

4

3.5

Labrador

Halitosis,discoloration of cheek tooth

P.fragi & S.mutans

5

8

Dachshund

Halitosis

C.koseri & S.mutans

6

6

Cocker spaniel

Halitosis,gingivitis

P.fragi & S.mutans

7

6

Non descript

Halitosis, gingivitis

P.fragi & S.mutans

8

8

Doberman

Halitosis

C.koseri

9

10

Dachshund

Halitosis, gingivitis

P.fragi

10

10

Spitz

Halitosis,discoloration of cheek tooth, gingivitis

P.fragi & S.mutans

11

10

Spitz

Halitosis, discoloration of cheek tooth

C.koseri & S.mutans

12

7.5

Cocker spaniel

Halitosis, gingivitis

Malassezia pachydermatis

13

14

Dachshund

Halitosis

P.fragi

14

6

Labrador

Halitosis, gingivitis

Malassezia pachydermatis

The slime producing ability of all the bacterial isolates were determined phenotypically using congo red agar (CRA) (Arciola, Campoccia et al. 2002). Reading was taken based on the reference scale for slime production (Freeman, Falkiner et al. 1989). Quantification of biofilm formation was done by modified microtiterplate assay (Stepanovic, Vukovic et al. 2000) where the strains were grown in tryptic soy broth(TSB). This is a biofilm biomass assay. Six replicates of the test were performed and an equal number of controls were kept. The mean OD reading of the control well was deducted from all the test readings. Optical density was measured at 590nm using an ELISA plate reader as the biofilms of the tested strains showed maximum absorbance at this wavelength. The OD values less than 0.12, between 0.12 to 0.24 and greater than 0.24 were considered to be non/weak, moderate and high biofilm producers respectively (Stepanovic, Vukovic et al. 2000). The biofilm production of the bacteria which were found together was also determined and tested the significance by statistical analysis (one way ANOVA) using Graph pad prism 4.

Antibiogram of the isolates was done by disc diffusion method (Bauer, Kirby et al. 1966). The antibiotics and their concentration per disc are given in table no.2. Interpretation was done in accordance to performance standards for antimicrobial disk susceptibility tests of NCCLS (Lalitha and Vellore 2005). Minimum inhibitory concentration (MIC) for planktonic bacteria, for four broad spectrum antibiotics viz. gentamicin, ceftriaxone, ciprofloxacin and cefotaxim were determined according to the standard CLSI guidelines (Clinical and Laboratory Standards 2006). The MIC values were compared with the suggested ranges for MIC determination for planktonic bacteria.

Table No.2 Antibiotics and their concentration per disc

Antibiotic

Concentration

per disc

Ceftriaxone (Ci)

30 µg

Cephotaxim (Ce)

30µg

Chloramphenicol (C)

30µg

Ciprofloxacin (Cf)

5µg

Enrofloxacin (Ex)

10 µg

Erythromycin (E)

15 µg

Gentamicin (G)

30 µg

Biofilm susceptibility testing

Minimum biofilm inhibitory concentration (MBEC) was carried out in 96 well microtiter plate (Ceri, Olson et al. 1999). The testing was carried out using cation-adjusted Mueller-Hinton broth (CAMHB). Biofilms were produced by inoculating 100 µl of one in hundred dilutions of overnight cultures followed by 18-24hr incubation at 37°C. The planktonic bacteria were removed and rinsed the wells with phosphate buffered saline. Serial dilutions of antibiotic solutions were prepared in a seperate microtiter plate and was transferred to the corresponding wells. The antibiotic concentration in the first well was 5mg/ml. Incubated for 18-24hrs and discarded the culture. The wells were rinsed twice with phosphate buffered saline and added 100 µl of fresh sterile TSB to the wells. The wells were sonicated for 5minutes in a bath sonicator and incubated for 24hrs. The absorbance was measured at 650nm using an ELISA plate reader (ELX 800 Biotek). The minimal concentration of antibiotic required to eradicate the biofilm was taken as MBEC.

RESULTS

A total of twenty one dental tartar isolates were obtained and were identified as P. fragi (43%), C. koseri (14%), S. mutans (33%) and yeast (10%). P. fragi and C. koseri were found individually as well as in association with S. mutans. Further studies were carried out only with respect to the bacterial isolates.

In Congo red agar all the P. fragi isolates, two C. koseri isolates and four S. mutans isolates gave black colonies with dry crystalline consistency. All the other isolates produced bordeaux colonies with smooth consistency. The crystal violet assay showed that all the P. fragi isolates were with high biofilm producing ability (0.65 ± 0.057), two C. koseri isolates with moderate (0.205 ± 0.071) and one with weak biofilm production. About 57% of the S. mutans isolates were high biofilm production (0.495 ± 0.122) and the rest were weak biofilm producers (0.09 ± 0.059). Values are expressed as (mean ± S.D.). The quantitative evaluation biofilm production is given in fig.1. The microtiter plate assay gave confining results with respect to the chromatic subjective evaluation. A synergestic effect was found when the organisms in combination were allowed to produce biofilm. The biofilm production of S. mutans in combination with P. fragi and C. koseri was found to be (1.22 ± 0.17) and (1.09 ± 0.08) respectively. The biofilm production of these two combinations were found to be significantly high compared to the individual values (P <0.01).

Fig.1 Biofilm production by crystal violet assay using microtiter plate

Pf1

Pf2

Pf3

Pf4

Pf5

Pf6

Pf7

Pf8

Pf9

Ck1

Ck2

Ck3

Sm1

Sm2

Sm3

Sm4

Sm5

Sm6

Sm7

0.00

0.25

0.50

0.75

1.00

Isolates

Absorbance at 590nm

Pf1-Pf9: P.fragi isolates

Ck1-Ck3: C.koseri isolates

Sm1-Sm7: S.mutans isolates

Upon antibiogram using diffusion method isolates showed high sensitivity towards Chloramphenicol (90%), Ciprofloxacin (90%), Enrofloxacin (90%), Ceftriaxone (80%) and low sensitivity towards Gentamicin (66%), Cephotaxim (66%) and Sulphadiazine (50%). The minimum biofilm eradication concentration of the isolates was found to be much higher compared to the MIC range of the planktonic bacteria. The results are given in table no.3.

Table No.3 The Minimum inhibitory concentration of biofilm bacteria

Isolate No.

Anibiotic

Gentamicin

Ceftriaxone

Ciprofloxacin

Cefotaxim

MIC range*

MIC

MBEC

MIC range*

MIC

MBEC

MIC range*

MIC

MBEC

MIC range*

MIC

MBEC

Pf1

0.06-128

<2

16

0.5-128

2

32

0.015-128

0.1

64

0.5-128

2

64

Pf2

4

512

0.1

32

2

16

1

16

Pf3

0.1

128

2

8

1

16

0.5

8

Pf4

1

32

4

16

0.1

32

0.5

8

Pf5

0.5

4

0.3

8

0.3

4

1

2

Pf6

0.1

16

4

8

0.3

16

0.3

32

Pf7

2

>512

2

128

0.5

8

0.1

32

Pf8

0.1

64

2

64

0.1

4

0.1

64

Pf9

0.1

8

0.3

8

1

2

1

4

Ck1

0.03-128

0.3

4

0.001-128

<0.1

4

0.004-128

1

2

0.004-128

0.1

4

Ck2

0.3

8

0.5

4

0.5

8

<0.01

2

Ck3

0.3

32

0.1

8

2

4

0.5

4

Sm1

1-128

4

8

0.008-0.12

0.3

4

0.12-4

0.3

32

0.12-4

1

8

Sm2

0.1

128

2

64

0.5

64

4

8

Sm3

2

8

<0.1

32

0.5

16

2

4

Sm4

1

16

0.3

16

0.3

8

0.3

8

Sm5

4

128

0.1

64

2

64

4

64

Sm6

4

32

2

8

2

4

2

64

Sm7

0.1

64

4

4

1

<2

0.3

8

Pf1-Pf9: P.fragi isolates, Ck1-Ck3: C.koseri isolates, Sm1-Sm7: S.mutans isolates

MIC: Minimum inhibitory concentration

MBEC: Minimum biofilm eradication concentration

All the values are given in µg/ml

*The suggested MIC range for MIC determination of planktonic bacteria(Andrews 2001).

For P.fragi, C.koseri, S.mutans isolates the MIC range for Pseudomonas spp., Enterobacteriaceae and Hemolytic streptococci were considered respectively.

DISCUSSION

Biofilm in the oral cavity of dogs can be a risk to the host itself; this may affect other organs especially those organs involved in screening of bacterial toxins. The plaque formation also leads to periodontal diseases (DuPont 1998). Pseudomonas species is a major species involved in canine periodontal diseases (Riggio, Lennon et al. 2011) and even though they are non-motile, they were found to be slime producers (Sashara and Zottola 1993). C.koseri is an opportunistic pathogen and was reported to be associated with many other conditions such as myocarditis in boxers (Cassidy, Callanan et al. 2002). S. mutans is a major etiological agent in the development of human and animal dental caries (Hamada and Slade 1980; Kolenbrander 2000; Yoshida, Ansai et al. 2005) and this forms biofilm (Li, Tang et al. 2002) for survival and persistence on the natural ecosystem, the dental plaque (Li, Lau et al. 2001). The increased proportions of obligately anaerobic bacteria, including Gram-negative proteolytic species is in agreement with the earlier studies (Marsh 2003). The yeast isolate, Malassezia pachydermatis, causing bilateral dermatitis in dogs can also be present in healthy dogs (Bond, Saijonmaa Koulumies et al. 1995).

The high biofilm producing ability of P. fragi and S. mutans when in combination may be one of the reasons for being the major etiology of dental infections. The biofilm bacteria were found to be much more resistant to the antibiotics than the planktonic bacteria and in some cases they can be upto 1000 fold resistant to antibiotics than their planktonic counterparts (O'Toole, Kolter et al. 2001). Since the organisms behaved very differently when they are in the biofilm community, the antibiotic sensitivity they have expressed in the disc diffusion method won’t be reliable for the treatment strategy. This oral biofilm of dog may also infect humans when transferred via bites (Williams, Lewis et al. 2011). C. koseri was reported to cause dog bite wound infections (Abrahamian and Goldstein 2011). By this there is an increased risk of transfer of antibiotic resistance inherited by the biofilm bacteria in dog, to other host specific pathogens in man.