RESEARCH ARTICLE

Comparative antibiotic susceptibility profiling of S. haemolyticus recovered from frequency hand-touched surfaces of hospital settings and urban built environments of central India

Anushri Keshri, Varaprasad Kolla*

Amity Institute of Biotechnology, Amity University Chhattisgarh, Raipur, Chhattisgarh, 493225, India.

Authors Email: Anug7797@gmail.com; vkolla@rpr.amity.edu

*Corresponding Author Email- vkolla@rpr.amity.edu

Introduction

The study of S. haemolyticus is crucial due to its increasing role in human infections and its remarkable ability to acquire resistance to multiple antibiotics. Although traditionally S. haemolyticus is considered less virulent than Staphylococcus aureus, it is associated with severe conditions, including bacteremia, endocarditis, and infections in implanted medical devices (Eltwisy HO et al.). Its pathogenicity is often linked to its capacity to form biofilms, which enhance its resistance to antibiotics and immune system clearance, making treatment challenging (Fredheim EG et al. 2009). Additionally, its genetic plasticity allows for the horizontal transfer of resistance genes, posing a risk of amplifying resistance across bacterial populations (Sharma 2020). The growing prevalence of multidrug-resistant S. haemolyticus highlights the urgent need for studies that address its epidemiology and resistance mechanisms, particularly in regions like India, where antibiotic misuse is widespread (da Costa PM et al. 2013).

Antibiotic resistance in India has become an escalating public health concern, driven by factors such as over-the-counter availability of antibiotics, inadequate infection control practices, and widespread misuse (Laxminarayan R et al. 2016). While substantial research has focused on hospital-acquired pathogens, there is a paucity of data on the antibiotic resistance profiles of pathogens isolated from non-hospital settings like educational environments (Kumari H et al. 2020). A comparative analysis of S. haemolyticus isolates from these distinct environments could provide insights into the ecological niches of this pathogen and the potential for inter-environmental transmission of resistant strains.

This study aims to investigate and compare the antibiotic susceptibility patterns of S. haemolyticus recovered from frequently hand-touched surfaces in hospital and UBE environments in central India. By identifying resistance trends and potential reservoirs of multidrug-resistant S. haemolyticus, the findings could inform targeted interventions to control the spread of resistant pathogens in both healthcare and community settings.

Materials and Methods

Sampling from frequently hand-touched surfaces of UBEs and hospital settings

Samples were collected from frequently hand-touched surfaces at various public places in UBEs and various hospital settings in Vidarbha, Maharashtra. A total of 200 isolates were tested, 100 from each environment (urban and hospital). The sampling was carried out in 0.9% sterile saline along with a swab, which was used for the sampling and kept in microcentrifuge tubes. After sampling, the bottles are kept in a cool condition and the samples are transferred to the laboratory in 2 hours.

Isolation of staphylococci species

To isolate staphylococci species from the sampled environment, a selective medium of mannitol salt agar (MSA – MH118-500G) has been used. The contaminated swab was used to inoculate the sterile media via the spread plate technique and incubated at 37 ° C for 24-48 hours. The isolated colonies were further maintained on an MSA plate under the same conditions.

Screening of staphylococci species

The colonies were screened for the potential staphylococci characteristics, including performing the conventional method such as gram staining, catalase, and coagulase tests, and the identification of the selected isolates at the species level using the VITEK 2 (BioMerieux) (Pincus DH et al. 2010).

Antibiotic susceptibility testing

Initially, ampicillin (10 µg) and methicillin (10 µg) antibiotics were checked for resistance and only those strains which are positive for the resistance were taken forward to investigate other antibiotics such as oxacillin (1 µg), gentamicin (10 µg), amoxicillin (10 µg), mupirocin (20 µg), erythromycin (15 µg), cefoxitin (30 µg), fusidic acid (10 µg), cefepime (30 µg), penicillin G (1 unit) and piperacillin (100 µg) (HI media, India). Antibiotic sensitivity test carried out on Muller Hinton Agar (MHA – M173-500G). A bacterial load of 0.5 McFarland standard was made with 24 hrs old culture maintained on nutrient broth. The lawn was prepared, and further antibiotic discs were placed on MHA plates and incubated for 24 hrs at 37ºC. After incubation, the growth of inhibition for antibiotic resistance was noted with the standard scale of the Clinical Laboratory Standards Institute (CLSI 2018) Table 1.

Statistical analysis

In this study, the per-drug sensitivity of UBEs and hospital-based CoNS were statistically analyzed using an unpaired T-test with P<0.05 to determine significance via GraphPad Prism software.

Table 1: Antibiotic susceptibility profiling of S. haemolyticus recovered from frequently touched surfaces of UBE and hospital settings and their zone of inhibition in mm.

S- Sensitive, R- Resistant, I- Intermediate. AMP (Ampicillin):- S – >29, R – <28, MET (Methicillin):- S – >18, R – <17, OXA (Oxacillin):- S – >18, R– <17, AMX (Amoxicillin):- S – >28, R – <28, FOX (Cefoxitin):- S – >25, R – < 25, FEN (Cefepime):- S – >24, R – <24, FUA (Fusidic acid):- S – >24, R– <24, MUP (Mupirocin):- S – >37, R – <31, GEN (Gentamicin):- S – >15, R – <12, ERN (Erythromycin):- S – >23, R – <13, PEN (Penicillin G):- S – >29, R – <28, PIP (Piperacillin):- S – >22, R – < 22

Results and Discussion

Surveying staphylococci species in urban built environments and hospital settings

Staphylococci colonies were grown on Mannitol Salt Agar (MSA) plates. Microbial loads varied significantly across locations. In UBE bus stops had an average colony count of 36 (range: 0-153), ATMs averaged 29 colonies (range: 3-58), while cafes showed a lower mean of 6.8 colonies (range: 0-28). Gardens and automobiles displayed typical counts of 13 and 54 colonies, respectively. In hospitals, washbasins had an average of 8.2 colonies (range: 0-23), lifts averaged 2 colonies (range: 0-8), and wheelchairs, doors, and tables had mean counts of 48, 86, and 49, respectively, reflecting substantial variability (Figure a). Circular colonies with yellow and pink hues on MSA agar indicated the presence of S. aureus and coagulase-negative staphylococci (CoNS) species, which are gram-positive, catalase-positive, and exhibit variable coagulase responses (Figure b).

Screening of staphylococci species

All the colonies grown in MSA are gram-positive and catalase-positive and exhibit variable coagulase responses (both positive and negative).

Antimicrobial susceptibility analysis

Antibiotic-resistant staphylococci isolates from urban and hospital contexts demonstrated considerable disparities in resistance patterns. In UBEs and hospital settings, 51% and 33% were shown antibiotic-resistant staphylococci respectively. This represented a significant 18% difference between both environments, demonstrating that antibiotic resistance is more prevalent in metropolitan constructed environments (Figure c).

Figure a: No. of colonies recovered from various frequently touched surfaces of (a)UBEs (b) Hospital settings.

Figure b: Results of Gram stain, catalase and coagulase tests.

Figure c: Antibiotic susceptibility test of isolates recovered from different directions of: (A) urban built environments and (B) hospital settings towards various antibiotics used in this study.