Prevalence and antibiogram of multidrug resistant Staphylococcus haemolyticus from various clinical samples in a tertiary care hospital in Hyderabad, India

Abstract

Background: 

Staphylococcus haemolyticus (S. haemolyticus) is one of the important organisms in hospital-acquired infections. This strain comprises many enzymes, cytolysins, and surface substances which contribute to its virulence. We in the present study tried to evaluate the prevalence of methicillin-resistant S. haemolyticus causing nosocomial infections in a Tertiary Care Hospital.

Methods: 

A total of n = 95 non-repeated S. hemolyticus were isolated in the Department of microbiology out of n = 2116 various clinical specimen present. The strains collected were initially identified by colony morphology on Nutrient agar, Sheep blood agar, Chocolate agar, Uri Chrom agar, gram staining, catalase, coagulase (both slide coagulase and tube coagulase), mannitol fermentation, Amino acid decarboxylation.

Results: 

n = 95 (4.5%) samples of n = 2116 samples were positive for S. haemolyticus. Most positive cultures of S. haemolyticus were from pus samples n = 40 out of n = 95. The susceptibility pattern revealed none were susceptible to penicillin and 94% susceptibility was found with Linezolid and 100% susceptibility to Nitrofurantoin. The vancomycin Minimum Inhibitory Concentration (MIC) results showed 71.57% (68/95) strains were susceptible; out of these 12 (12.63%) were showing MIC < 1 μg/mL, n = 56 isolates (58.94%) were showing MIC value less than n = 2 μg/mL, and none of them were less than MIC 0.5 μg/mL.

Conclusion: 

In this study, variable antimicrobial resistance susceptibilities were shown by S. haemolyticus isolates. Some clinical situations may require a single strain of S. haemolyticus to be identified up to the species level with their antibiogram. Critical and timely detection of drug-resistant S. haemolyticus in hospital settings will be a helpful guide in the management and prevent further proliferation of drug resistance.

INTRODUCTION

Staphylococcus haemolyticus (S. haemolyticus) is a skin commensal but it has gained attention in recent times as an emerging pathogen of nosocomial infections.[¹] S. haemolyticus is a gram-positive, non-motile, non-spore-forming, facultative anaerobic bacteria that grows on a wide range of substrates such as glucose, maltose, sucrose, and trehalose. It tests negative for coagulase, DNase, ornithine decarboxylase, phosphatase, urease, and oxidase. S. haemolyticus is an emerging pathogen of nosocomial infections, and the most frequently isolated staphylococcal (previously termed as coagulase-negative staphylococci [CoNS]) species alongside Staphylococcus epidermidis.[²] S. haemolyticus is the second most frequently isolated amongst other Staphylococcus spp in hospital-acquired infections often associated with the insertion of medical devices. S. haemolyticus becomes a crucial factor in nosocomial infections caused by multi-resistant staphylococci.[³] Studies on the prevalence of nasal colonization with methicillin-resistant S. haemolyticus amongst health care are minimal. S. haemolyticus possesses a thick cell wall (60-80 nm). Like the other gram-positive cocci, the cell wall is composed of peptidoglycan, teichoic acids, and proteins. L-lysine is the di-amino acid attached at position 3 of the peptide subunit and a glycine-rich interpeptide bridge present in the peptidoglycan is a characteristic feature of this microbe.[³] The two predominant cross-bridges found across the peptidoglycan layer are COOH-Gly-Gly-Ser-Gly-Gly-NH₂ and COOH-Ala-Gly-Ser-Gly-Gly-NH₂. Changes that may take place as a consequence of mutations in the cross bridging can play a role in the development of glycopeptide resistance. S. haemolyticus is a significant cause of bacteremia associated with vascular catheters. S. haemolyticus can colonize central venous catheters. Common infections include valve endocarditis, septicemia, peritonitis, and urinary tract infections. S. haemolyticus also colonizes medical devices such as prosthetic valves, Cerebrospinal Fluid (CSF) shunts, orthopedic prosthesis, and intravascular and urinary catheters induced during surgical interventions.[⁴] In recent years, S. haemolyticus demonstrates the highest level of antimicrobial resistance among other Staphylococci. Well elucidated mechanisms of antibiotic resistance include the horizontal gene transfer, also referred to as lateral gene transfer, in which antibiotic-resistant genes are transferred from one bacterial species to another via conjugation, transformation, transposons, or plasmids.[⁵] The increasing number of CoNS in hospital-acquired infections is closely related to their antimicrobial resistance and the ability to survive in a hospital environment. With this background, we in the present study aimed

1. To evaluate the prevalence of (MRS) Staphylococcus haemolyticus causing infections in tertiary care hospitals.
2. To identify the patterns of antibiotic susceptibility of Staphylococcus haemolyticus among various clinical samples.

SUBJECTS AND METHODS

This cross-sectional study was conducted in the Department of Microbiology, Mediciti Institute of Medical Sciences Hospital, Hyderabad. Ethical committee approval was obtained from the Institutional Ethical Committee. Informed consent was taken from all the patients. Isolation and identification of S. haemolyticus were performed. A total of n = 95 non-repeated S. haemolyticus were isolated in the Department of Microbiology from n = 2116 various clinical specimen. The strain collected was initially identified by colony morphology (Nutrient agar, Sheep blood agar, Chocolate agar, Uri Chrom agar), gram staining, catalase, Coagulase (both slide coagulase and tube coagulase), mannitol fermentation, Amino acid decarboxylation Novobiocin susceptibility, acetoin, urease production. Gram stain all the exudative samples, a preliminary gram stain was done, most of the clinical specimens showed gram-positive cocci in singles and clusters. Samples were inoculated on sheep blood agar, Chrom Agar, for CSF Chocolate agar, urine – CLED and Uri Chrom plates were incubated at a temperature from 35°C to 37°C, after 18-24 hours plates were observed for the findings. Colony morphology moderate to large-sized, low convex, circular, smooth, pale white opaque colonies with beta hemolysis were observed on blood agar. Chrom agar showed pale white opaque smooth colonies [Figure 1].

Figure 1: Showing the colonies of Staphylococcus haemolyticus on agar plates

Positive findings: Catalase – positive, Slide and tube coagulase-negative, Blood agar shows beta hemolysis Novobiocin sensitive, Ornithine not decarboxylated, Urease negative, Mannose was not fermented, Acetoin produced. Antibiotic susceptibility was performed by both Conventional disc diffusion and automated– identification and susceptibility by walkaway 96s using Positive Breakpoint Combo 29 panel. In the conventional method, the antibiotic susceptibility of test strains was performed on Mueller Hinton agar using the Kirby Bauer disc diffusion method as per Clinical and Laboratory Standard Institute (CLSI) guidelines.[⁶] The commonly used antibiotics are Penicillin 10 U, Amoxicillin 20 μg, Erythromycin 15 μg, Ciprofloxacin 5 μg, Gentamycin 10 μg, Clindamycin 2 μg, Chloramphenicol 30 μg, Cefoxitin 30 μg, Sulfamethoxazole/trimethoprim (1.25/23.75 μg), Linezolid 30 μg, and Tetracycline 30 μg. In automated– identification and susceptibility by walkaway 96s (fully automated ID system) using Positive Breakpoint Combo 29 panel. Antibiotics tested are Amoxicillin/K Clavulanate (4/2 μg/mL), Ampicillin (2-8 μg/mL), Ampicillin/Sulbactam (8/4-16/8 μg/mL), Cefazolin (4-16 μg/mL), Cefoxitin Screen (4 μg/mL), Chloramphenicol (8-16 μg/mL), Ciprofloxacin (1-2 μg/mL), Clindamycin (0.25-4 μg/mL), Erythromycin (0.25-4 μg/mL), Gentamicin (1-8 μg/mL), Inducible Clindamycin Test (4/0.5 μg/mL), Levofloxacin (0.5-4 μg/mL), Linezolid (0.5-4 μg/mL), Moxifloxacin (0.5-4 μg/mL), Nitrofurantoin (32-64 μg/mL), Oxacillin (0.25-2 μg/mL), Penicillin (0.03-8 μg/mL), Synercid (0.25-2 μg/mL), Tetracycline (1-8 μg/mL), Trimethoprim/Sulfamethoxazole (0.5/9.5-2/38 μg/mL), Vancomycin (0.25-16 μg/mL) [Table 1]. No significant differences were found between the conventional and automated identifications and susceptibility. S. haemolyticus isolates were found to be resistant to at least three classes of antibiotics, which were categorized as multidrug resistance (MDR).[⁷]

Table 1: Showing the Vancomycin Mic Susceptibility Pattern

RESULTS

Out of n = 95 S. haemolyticus, 41.66%(40/95) were obtained from pus, 19.44% (18/95) were obtained from wound swabs, 13.88% (13/95) were obtained from ulcer swabs, 16.66% (15/95) were obtained from tissue and CSF, peritoneal fluid and urine 2.77% (3/95) from each sample were isolated [Figure 2].

Figure 2: Showing the isolation of staphylococcus haemolyticus from various specimens

Maximum strains of S. haemolyticus were isolated from pus and minimum in CSF, urine, and peritoneal fluid. No strains were isolated from blood culture. Among Staphylococci species isolated, 49.5% were S. aureus, 20.8% were S. epidermidis, and 29.7% were S. haemolyticus. The common species isolated was S. aureus followed by S. haemolyticus and S. epidermidis amongst important clinical specimens. The results of the antibiotic susceptibility test are shown in Figure 3.

Figure 3: Antibiotic susceptibility pattern of staphylococcus haemolyticus isolates

Among n = 95 Staph haemolyticus the susceptibility percentage of S. haemolyticus is minimally observed in Penicillin (0%), Ampicillin 5.20%, followed by Gentamycin 21.05%, and Erythromycin 25%, maximum susceptibility is seen in Nitrofurantoin (100%), and linezolid (94%), followed by vancomycin (72%) 29.47% of strains were susceptible to Methicillin by cefoxitin screen test showing higher percentage (70.53%) of methicillin-resistant S. haemolyticus in our study. Vancomycin susceptibility pattern of n = 95 isolates. Vancomycin MIC tested was 0.25 μg/mL, 0.5 μg/mL, 1 μg/mL, 2 μg/mL, 4 μg/mL, 8 μg/mL and 16 μg/mL. Out of n = 95 S. haemolyticus isolates, 71.57% (68/95) strains were susceptible out of these 12 (12.63%) were showing MIC < 1, n = 56 isolates (58.94%) were showing MIC value less than n = 2, none of them were less than mic 0.5.

DISCUSSION

In the current study, we found that n = 95 (4.5%) samples of n = 2116 samples were positive for S. haemolyticus. Most positive cultures of S. haemolyticus were from pus samples n = 40 out of (n = 95). The susceptibility pattern revealed none were susceptible to penicillin and 94% susceptibility was found with Linezolid and 100% susceptibility to Nitrofurantoin. The vancomycin MIC results showed that 71.57% (68/95) strains were susceptible out of these 12 (12.63%) were showing MIC < 1 μg/mL, n = 56 isolates (58.94%) were showing MIC value less than n = 2 μg/mL, none of them were less than mic 0.5 μg/mL. S. haemolyticus is an important nosocomial pathogen and approximately 90% of coagulase-negative staphylococci strains isolated from clinical specimens with chronic diseases.[^8,9] In this study, n = 40 (41.66%) pus samples were found to be positive for S. haemolyticus. Singh et al.,[¹⁰] in a similar study in North India, found S. haemolyticus (47.8%) as the most common isolate found in various clinical samples. The variability in the appearance of S. haemolyticus from different samples may be due to greater colonization of CoNS in the skin as compared to the mucous membrane. S. haemolyticus and S. epidermis frequently colonize the moist body surfaces such as the perineal area, inguinal area, and axillae. Although they are natural colonizers of skin but have adapted to become nosocomial pathogens because of their ability to produce biofilm and produce resistance to antibiotics. In this study, S. haemolyticus were found to be completely resistant to penicillin and 5.2% sensitivity to ampicillin. S. haemolyticus also showed lower sensitivity to other non-beta lactam antibiotics such as ciprofloxacin 27.77% sensitive and Gentamycin 21.05%. Singh et al.[¹⁰] have shown a higher resistance of 84% and lower sensitivity of S. haemolyticus to ciprofloxacin. Similarly, for gentamicin, 84% resistance was seen. Dziri et al.[¹¹] in Tunisian hospital found that Staphylococcus hemolyticus, Staphylococcus epidermidis, and Staphylococcus saprophyticus were detected species in methicillin-resistant CoNS in environmental samples. Among CoNS, Staphylococcus hemolyticus is the second most documented cause of infections like wound infection, bacteremia, prosthetic valve endocarditis, and urinary tract infection. S. haemolyticus isolates are ranked as the most antibiotic-resistant species among the CoNS.[¹²] Barros et al.[¹³] found n = 64 clinical isolates of S. hemolyticus and Polymerase Chain Reaction (PCR) of the mecA gene, 87% were found to be methicillin-resistant. Approximately 55% harbored staphylococcal cassette chromosome mec element (SCCmec) type V and only one SCCmec type IV. Shah et al.[¹⁴] in Pakistan found 40.1% of isolates of CoNS were methicillin-resistant. Most of them were susceptible to Vancomycin, Linezolid, Chloramphenicol, Minocycline, and Rifampicin. Vancomycin is an anti-staphylococcal antibiotic to which resistance is rarely seen. However, cases of CoNS resistance to vancomycin are now appearing and most of them have developed the biofilms Raad et al.[¹⁵] This makes the microorganisms extremely resistant to antibiotics and the therapeutic failure of the drug. Hence, drugs like Linezolid may be needed in such cases. Another possible way to decrease resistance is the modification of dose regimens' use of beta-lactamase inhibitors or maybe using agents from a different class Sharma et al.,[¹⁶] Kitti et al.,[¹⁷] investigating methicillin-Resistant Staphylococci Clinical isolates from a Tertiary Hospital in Northern Thailand found more than 70% of MRSA and MR-CoNS were resistant to cefoxitin, penicillin, oxacillin, erythromycin, clindamycin, gentamicin, and ciprofloxacin. CoNS identification and susceptibility must be carefully checked to avoid treatment failures and unnecessary use of other antibiotics may also be avoided.

CONCLUSION

In conclusion, antibiotic resistance is a growing threat in India. The indiscriminate use of antibiotics is one of the important reasons for this development. In this study, variable antimicrobial resistance susceptibilities were shown by S. hemolyticus isolates. Some clinical situations may require a single strain of S. haemolyticus that may have to be identified up to the species level with their antibiogram. Critical and timely detection of drug-resistant S. haemolyticus in hospital settings will be helpful in guiding its management and preventing further proliferation of drug resistance.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

Acknowledgements

Authors wish to thank the Department of Microbiology, Mediciti Institute of Medical Sciences Hospital, Hyderabad for their assistance during the conduction of the study.