Detection of Antibiotic Resistance and Biofilm- Producing Ability of Staphylococcus Species in Clinical Isolates

Introduction

Staphylococcus aureus is a pathogen that can infect human as well as animals. The infections can range from skin and soft tissue infection to serious life-threatening illnesses like endocarditis, septicemia, and toxin-mediated shock syndrome in human, and skin infections and mastitis caused by S. aureus are reported in animals.¹ Staphylococcus aureus has many virulence factors that help the organism survive and initiate infection inside the host. These factors act in different ways to colonize, invade, and spread themselves. This is the primary reason for S. aureus being able to attack immunologically competent hosts.² The ability of the organism to develop biofilm enhances the severity of infections caused by S. aureus as well as increases the antibiotic resistance.³ The steps of biofilm formation involve adhesion of the organism to the surface, which is aided by the expression of various microbial surface components that recognize adhesive matrix molecules. These molecules can bind to various extracellular matrix components, including elastin, fibronectin A and B, laminin, collagen, fibrinogen, and clumping factors. These proteins can connect to the cell wall or other surfaces by using similar signal sequences. They can cover medical equipment and start the creation of proteins like biofilm matrix proteins in a bacterial matrix.^4,5 Several Staphylococcus species now exhibit significant virulence traits due to the production of biofilm.⁵ The biofilm serves as a protective cover that provides a favorable environment to the bacteria and allows them to survive against adverse conditions.⁶ In addition to its ability to form biofilm on medical devices, which leads to the removal of devices, S. aureus can form biofilm inside the human body. This leads to infection associated with resistance to antimicrobial agents and failure of the treatment resulting in removal of the infected tissue to control the infection.⁷ The resistance caused by biofilm is due to the presence of bacteria inside the biofilm; they remain dormant during the antimicrobial therapy and later emerge as resistant strains. Another mechanism that leads to resistance to antibiotics is the inability of antimicrobials to penetrate through the layers of biofilm.⁸ Numerous coagulase negative staphylococci (CoNs) species, including S. epidermidis, have emerged as the primary carriers of nosocomial infections, particularly those that affect newborns, people with immunological disorders, and patients receiving care in the intensive care unit who utilize catheters or other prosthetic devices.⁹ Catheter-associated infections may be caused by CoNs that produce biofilm that affects immunocompromised patients, including premature infants, neutropenic cancer patients, elderly people with serious underlying diseases, hospitalized patients undergoing invasive procedures, and people with permanent plastic devices.¹⁰ Methicillin resistant S. aureus (MRSA) is largely found to be resistant to common available antibiotics. One of the common reasons for high mortality and morbidity in MRSA infections are biofilm producers.¹¹ In hospitals, the biofilm-producing pathogens are becoming a serious concern, and the existing antimicrobial agents are failing to treat these pathogens. The increase in multidrug-resistant organisms with biofilm is weakening the health care policy.¹² Therefore, the development of clinically applicable biofilm experimental modalities is urgently required since biofilm plays a significant role in the slow healing of wounds.¹³

In our study, 81 isolates belonging to the genus Staphylococcus were investigated for the presence of biofilm forming activity by three different methods such as the tube adherence method, Congo red agar method, and tissue culture plate method. Antibiotic resistance was compared between the biofilm producers and nonproducers. We found that the multidrug resistance was common among the biofilm producers and not all the strains showed presence of biofilm by all the methods used in this study.

Materials and Methods

Congo Red Agar Method

The detection of biofilm by the Congo red agar method was done by using the method given in a previous study. The test organisms are inoculated into a special media prepared using brain–heart infusion medium supplemented with sucrose (5%) and Congo red dye. After the inoculation, the plates were incubated at 37°C for 24 to 48 hours. The test was interpreted positive for biofilm if the colonies were black in color with dry crystalline consistency. Weakly positive showed pink colonies with occasional black color in the center of the colonies (►Fig. 1A).¹⁸

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Fig. 1

(A) Biofilm producers show black metallic colonies on Congo red agar. (B) Biofilm producers show material adhering to the bottom. (C) Tissue culture plate method.

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Result

In this study we included 81 isolates of Staphylococcus isolated from various clinical samples. The distribution of S. aureus and CoNs among the different types of samples is shown in ►Fig. 1. Of the 81 samples, S. aureus was isolated from 44 (54%) samples and CoNs from 37 (45.6%) samples. Staphylococcus aureus was mostly isolated from pus (n = 17) and CoNs were present mostly in the nasal swabs (n = 17).

Out of the 44 S. aureus found 27 were MSSA and 17 were MRSA and out of the total 37 CoNs, 18 were identified as S. epidermidis, 16 as S. haemolyticus and 3 as S. saprophyticus (►Fig. 2).

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Fig. 2

Distribution of staphylococci isolated from various clinical samples.

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Discussion

Staphylococcus aureus is present on the skin and mucous membrane of the nasal cavity of human beings. It can cause multiple infections like septicemia, pneumonia, urinary tract infection, and skin infections in human. Production of biofilm by them is an important virulence factor that allows the bacteria to adapt to the adverse conditions and difficult to treat. This is also responsible for antimicrobial resistance. This calls for the need of removal of biofilm-producing organisms from medical devices and for the detection of biofilm-producing ability of the organism.²¹

In our study, we included 81 isolates of Staphylococcus species. The isolates were tested for their ability to produce biofilm as a virulence factor by three different methods. With regard to these three techniques, the Congo red agar method detected 43 (53%) biofilm producers among the isolates and 38 nonproducers; the tube adherence method detected 40 (49%) positive biofilm detections and 41 nonproducers, and the tissue culture plate method detected 52 (64%) producers and 29 nonproducers. Looking at the positivity rate given by various methods, the microtiter plate method is highly efficient. In a study conducted by Amita Jain in 2009, the microtiter plate method is used as the gold standard test for biofilm to evaluate the sensitivity and specificity of Congo red agar method.²³ Tube adherence on the other hand has given the least rate of positivity; this could be due to weak biofilm producers which fail to adhere well to the tube. Most of the biofilm positive strains showed resistance to multiple antibiotics. CoNs have shown resistance against many antibiotics like clindamycin, levofloxacin, and trimethoprim-sulfamethoxazole. MSSA and MRSA also showed significant degrees of resistance to antibiotics like ciprofloxacin, erythromycin, and amoxicillin- clavulanic acid.

Conclusion

Clinically isolated strains of S. aureus recovered from infection in hospitalized patients show high levels of biofilm formation. Higher rates of antibiotic resistance were demonstrated by biofilm manufacturers compared with nonbiofilm manufacturers. The biofilm formation of S. aureus, MRSA, and CoNs can lead to high rates of antibiotic resistance, making treatment difficult. Biofilm screening must be done to prevent it. This study detects the phenotypic ability of Staphylococcus species to produce biofilm; a genotypic detection of associated genes can give better results.