Study of Hospital Acquired Methicillin-Resistant Staphylococcus aureus in a Tertiary Care Hospital of Bihar

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Introduction

Staphylococcus aureus (S. aureus), a very common pathogen in clinical practice, causes a broad spectrum of diseases ranging from minor skin infections, osteomyelitis, food poisoning to pneumonia, toxic shock syndrome, wound infections, and bacteremia [1]. Methicillin-Resistant Staphylococcus aureus (MRSA) are strains of Staphylococcus aureus that are resistant to methicillin and a large group of Beta-lactam antibiotics which include penicillin and the cephalosporins [2]. MRSA emerged as a nosocomial pathogen in the early 1960s and resulted in many sporadic and large outbreaks in different hospitals throughout the world. By the 1970s MRSA was endemic in many hospitals but in the 1990s it unexpectedly spread rapidly in communities to become Community Associated MRSA (CA-MRSA) [3,4]. An MRSA isolate is classified as ‘Hospital-Acquired MRSA’ (HA-MRSA) if it is isolated from a patient who has given a positive culture taken more than 48 hours after hospital admission and treated in the inpatient setting [5]. Bacteremia, surgical wound infections, and pneumonia are the most common nosocomial infections caused by MRSA [6]. There has been a consistent increase in the prevalence of MRSA infections throughout the world, especially in hospital settings as shown in data by various studies and antimicrobial surveillance programs including the National Nosocomial Infections Surveillance System (NNISS) which reported an escalation of MRSA infections among large hospitals in the United States from 4% in the 1980s to 50% in the late 1990s [7,8]. The problem of the MRSA infection is augmented by its ability to cause cross infections, colonization, and resistance to multiple classes of commonly used antimicrobials [1,9]. Transmission of MRSA stains can occur by various routes including the hands of healthcare workers, contaminated surfaces, and instruments or even it may be airborne [10,11].

Compared with MSSA (Methicillin Susceptible Staphylococcus aureus) strains, infections caused by MRSA strains are associated with higher morbidity, mortality, and health care burden [12].

Studies conducted by various authors have found that various risk factors are significantly associated with acquisition and infection by MRSA and those risk factors can vary in different regions and health care setups [13,14].

Taking into consideration the fact that MRSA has become one of the most important pathogens in hospital and community settings. This study was undertaken to determine the prevalence and associated risk factors for MRSA infections which will be crucial to formulate infection control and prevention policies and to determine the antimicrobial susceptibility pattern of MRSA isolates which will guide the clinicians to construct proper antimicrobial therapy for such infections and reduce antibiotic misuse and its associated adverse effects including antimicrobial resistance.

Materials and Methods

After approval of the Institute Ethical Committee (Memo No: IGIMS/2014/260 Acad.), this cross-sectional study was conducted in the Department of Microbiology, I.G.I.M.S, Patna, Bihar, from February 2014 to November 2015.

Various clinical samples like pus; swabs (from wounds, eye, ear, throat, and urethra); body fluids (Ascitic fluid, Pleural fluid, Peritoneal fluid, Bile, Liver aspirates, CSF); tissue samples, sputum, CV lines, catheter tips, shunts, ET Tubes, and urine, from patients only after 48 hours of admission in various departments, were included in this study.

Staphylococcus aureus was identified using the standard microbiological methods and MRSA by cefoxitin disc (30 μg) and agar screening media. Antimicrobial susceptibility tests were done on Mueller-Hinton agar with commercially available antibiotic discs (Hi-Media, Mumbai) according to CLSI 2014 guidelines (Figure 1-3) [15]. Isolates screened as MRSA were further tested for identification and antimicrobial susceptibility testing using VITEK-2 Compact (Biomérieux, USA).

Figure 1. Slide coagulase test

Figure 2. Tube coagulase test

Figure 3. Antibiotic susceptibility test in MHA (K-B disc diffusion)

Based on previous studies by various authors prevalence of HA-MRSA was assumed as 25% and with a precision of 10%, the sample size was estimated as a minimum of 73 [16]. Repeat samples from the same patient were not included in the study.

Analysis of Risk Factors

Demographic, laboratory, and clinical data of all the patients who were admitted for more than 48 hours and culture yields Staphylococcus aureus, was divided into two groups-patients with MRSA isolates (HA-MRSA) and patients with MSSA isolates (HA-MSSA). All the variables were recorded for both these 2 groups for statistical analysis.

Statistical Analysis

All the data were plotted in Microsoft Excel Sheet (v.2007). Descriptive statistics were used to describe characteristics of HA-MRSA and HA-MSSA cases. Associations between categorical variables were tested using Chi-square tests.

All statistical analyses were performed using MedCalc (v.19.1.3) and a p-value of ≤ 0.05 was considered statistically significant.

Results

A total of 14182 clinical samples were received in the Microbiology laboratory from February 2014 to November 2015, out of which 102 hospital-acquired Staphylococcus aureus isolates were taken for further study. Out of the 102 Staphylococcus isolates, 27 (26.5%) were identified as MRSA strains and 75 (73.5%) were MSSA strains (Figure 4).

Of the 102 patients who had the Staphylococcal infection, 56 (54.9%) patients were male, and 46 (45.1%) were female with a male: female ratio of 1.2. Likewise, the majority of the patients with MRSA isolates were also male, 14/27 (51.85%), and male to female ratio for MRSA isolates, was 14:13 or approximately 1.1:1. In the case of patients with MSSA isolates, it was also the male patients who form the majority i.e. 42/75 (56%), and the male-to-female ratio was 42:33 or approximately 1.3:1 (Figure 1).

Figure 4. Prevalence of HA-MRSA and HA-MSSA isolates

The majority of the patients from whom Staphylococcus aureus was isolated were in the age group of 0-40 years, 65/102 (63.7%). In the case of patients with isolation of HA-MRSA strains majority of the patients were of 0-40 years of age, 18/27 (66.7%) which was also seen in patients with isolation of HA-MSSA strains, 47/75 (62.7%) (Table 1, and Table 2).

The majority of the 102 strains of Staphylococcus aureus were isolated from pus 38.2% (39/102) followed by blood 23.5% (24/102) and urine 16.7% (17/102). Likewise, 11 isolates (40.74%) of HA-MRSA strains were isolated from pus followed by 7 isolates (25.93%) from blood and 4 isolates (14.8%) from urine samples. In the case of HA-MSSA isolates, the majority of them were obtained from pus 37.3% (28/75) followed by blood 22.7% (17/75) and urine 17.3% (13/75) (Table 4).

The majority of the HA-MSSA isolates showed resistance to Penicillin (98.7%) followed by Ampicillin (94.7%) and Nalidixic acid (72%). All the isolates were sensitive to Vancomycin and Linezolid followed by Co-trimoxazole (97.3%), Chloramphenicol (89.3%), and Amikacin (84%).

All the strains of HA-MRSA were resistant to Penicillin (100%), Ampicillin (100%) followed by Nalidixic acid (96.3%), Erythromycin (92.6%), and Gentamicin (85.2%). All the isolates were sensitive to Vancomycin and Linezolid (100%) followed by Co-trimoxazole (77.8%) and Amikacin (70.4%).

For urinary isolates, Nitrofurantoin showed 50% and 23.1% resistance for HA-MRSA and HA-MSSA isolates (Table 5).

Several risk factors were analyzed for association with HA-MRSA infections and nine of them were found to be statistically significant (p ≤ 0.05) and those risk factors are the length of stay in hospital or ICUs, burn injury, severe trauma, and presence of open wounds, catheterization, use of mechanical ventilators, use of antibiotics before admission and more than two previous hospital admissions (Table 6).

Discussion

The prevalence of HA-MRSA in this study was 26.5% (27/102) which shows the significant role of MRSA as a nosocomial pathogen. Studies done by various authors have proved the importance of MRSA in a hospital environment. A pilot program of MRSA surveillance conducted in various centers across India in 1996 found 26.65% of Staphylococcal strains to be MRSA in Mumbai, 47.1% in Bangalore, and 42.5% in New Delhi [16]. Verma S, et al. in 2000, reported that the prevalence of MRSA in their center was as low as 12% in 1992 increasing to 80.89% in 1999 [17]. Other authors have reported diverse findings as far as isolation of MRSA is concerned viz. 54.85% from Uttar Pradesh (Anupurba, et al.), 31.1% from Tamil Nadu (Rajaduraipandi K, et al.), 44% from New Delhi (Tyagi A, et al.), and 38.14% from Sikkim (Tsering DC, et al.) [18-20].

Our finding corresponds to findings by other authors from different states of India where all have seen that MRSA has a significant prevalence in healthcare-associated infections.

In this study, patients from age groups consisting of pediatric and young adults contribute to the majority of cases (65, >63%) which is seen in the case of both HA-MRSA (66.7%) and HA-MSSA (62.7%) isolates. Studies conducted by Chen CJ, et al., and Eshwara VK, et al. have shown a similar age group consisting of children and young adults are the major contributors (>57%) of Staphylococcal cases including MRSA isolates [21,22]. A study conducted by Waness A, et al. however reported higher infection rates of MRSA in persons above 65 years of age [23]. This finding is quite different from the present study in which only 14 (13.7%) patients are there in the age group of 61 and above with isolation of HA-MRSA and HA-MSSA isolates.

In this study, there was a male predominance in cases of both HA-MRSA (M:F=1.1:1) and HA-MSSA (M:F=1.3:1) isolates. This may be due to the fact like differences in MRSA carriage in males as compared to females due to differences in hand hygiene and daily activities. A study conducted by Winkler J, et al. showed that female hormones like estrogen may have some role in limiting colonization, and in rural settings; females less readily seek medical care for their ailments [24]. A study conducted by Humphreys H, et al. showed that there is a male predominance (OR>1.3) in staphylococcal infections including MRSA infections which is in harmony with our study [25].

Most of the Staphylococcal isolates (74/102, 72.5%) which include both HA-MRSA and HA-MSSA were isolated from Pediatrics, Medicine, Surgery, and Orthopedics departments and ICUs corresponding to those departments. A study conducted by Severin, et al. reported a high prevalence of MRSA carriage in patients admitted in surgical wards [26]. A study conducted by Arora S, et al. also showed a similar finding where Orthopedics (75/250), Pediatrics (56/250), Medicine (44/250), Surgery (26/250), and Gynecology department (27/250) contributes to the majority of MRSA isolates [27]. The above studies including the current study established the fact that strict MRSA infection control practices are needed especially in those departments and surveillance for MRSA carriage may be a crucial step towards infection control and prevention.

Most of the Staphylococcal isolates (HA-MRSA and HA-MSSA) were isolated from pus, blood, and urine samples (80/102, 79.5%). Anupurva S, et al. reported maximum isolation (200/381, i.e. 52.5%) from pus and wound swabs [18]. Rajaduraipandi, et al. also reported that the majority of the MRSA isolates (>62%) were from blood and pus samples [19]. The findings of their studies correspond to our findings and emphasize the fact if the primary Gram stain from such samples showed the presence of Gram-positive cocci in clusters, then further tests for identification of MRSA should be carried out.

The antimicrobial susceptibility pattern of MRSA isolates showed that they are resistant to multiple classes of antibiotics and many of those isolates are resistant to more than three different classes of antibiotics which makes them important Multi-Drug Resistant Organisms (MDRO) regarding hospital-associated infections [28]. In this study, a very high resistance (>95%) is seen against beta-lactam antibiotics like Penicillin and Ampicillin and shouldn’t be used empirically whenever hospital-acquired staphylococcal infections are suspected. In this study, we have also found a statistically significant difference (p ≤ 0.05) among sensitivity shown by various antibiotics like Erythromycin, Clindamycin, Gentamicin, and Tetracyclin among HA-MRSA and HA-MSSA isolates. This infers that those antibiotics may show a good sensitivity when used against HA-MSSA isolates but may be ineffective when used against HAMRSA isolates and thus they should be only used after antimicrobial susceptibility testing. Vancomycin and Linezolid provide the best option against HA-MRSA and HA-MSSA isolates (100% sensitivity).

Anupurba S, et al. reported in a study that 80% of their MRSA strains were resistant to antibiotics like cephalexin, ciprofloxacin, penicillin, co-trimoxazole, gentamycin, erythromycin, and tetracycline [18]. A study conducted by Arora S, et al. showed that 73% of the MRSA strains were multidrug-resistant [27]. Although Vancomycin is still the empirical treatment of choice in severe MRSA infections in less severe conditions oral antibiotics should be preferably used depending on the antimicrobial susceptibility testing [29,30].

In this study, authors have analyzed various risk factors that may be associated with HA-MRSA infections in comparison to HA-MSSA infections based on previous studies conducted by various authors [13,14]. The authors have found that some of the risk factors were statistically significant (p ≤ 0.05) among patients with MRSA isolates. Length of stay (>1 week) in hospital wards and ICUs, burn injury, severe trauma and open wounds, use of broad-spectrum antibiotics at the time or before admission to hospital, catheterization, patients on mechanical ventilators, and patients having more than 2 previous admissions in hospitals are important risk factors for MRSA infections than MSSA infections.

A study conducted by Harinstein L, et al. showed that many risk factors like catheterization and more than 2 previous hospitalizations are associated with the conversion of MRSA colonization into MRSA infections [31].

A study conducted by Yao Z, et al. showed Length of Stay (LOS) in the hospital and the use of mechanical ventilation as important risk factors [32].

Another study by Yamakawa K. et al. showed that four risk factors which include intubation, presence of open wounds, antimicrobial therapy, and use of steroids if occurred within 24 hours of ICU admission are associated with MRSA infections [33].

Current study results are in harmony with findings of those above-mentioned authors whereas, in some risk factors like diabetes mellitus and liver cirrhosis, we don’t find statistical significance as compared to work done by Wu HP, et al. [34].

The knowledge of various risk factors may help in identifying susceptible patients for MRSA colonization or infections and active surveillance of those patients along with health care workers attending those patients may play a crucial role in controlling HA-MRSA infections and prevent associated mortality, morbidity, and economic burden of health care [35,36].

Conclusion

According to this study, MRSA, with its high prevalence, continues to be an important pathogen in Bihar. These organisms are multidrug-resistant and selecting proper antimicrobials for empirical therapy should be based on the current antimicrobial susceptibility pattern, the severity of the infection, and other associated risk factors of the patient. Active surveillance of patients based on their risk profile for colonization of MRSA may play an important role in the control and prevention of MRSA spread and related infections which will, in turn, reduce the associated mortality, morbidity, and cost of health care.

Limitations of the Study

In this study, due to a lack of molecular methods and related infrastructure, authors were unable to do molecular typing of different MRSA strains and to work out their spread among the hospital environment. There may be other risk factors that need to be evaluated in further studies.

Declarations

Conflicts of Interest

The authors declared no potential conflicts of interest concerning the research, authorship, and/or publication of this article

Authors Funding

The financial and infrastructure support was provided by the Dept. of Microbiology, I.G.I.M.S, Patna as per directions by the Institutional Ethical Committee. No external funding was utilized in this study.

Author’s Contributions

Dr. Suprakash Das reporting microbiologist and preparation of manuscript. Dr. Keshav Kumar Bimal collected and analyzed the data. Dr. Namrata Kumari assisted in paper writing and statistical analysis. Dr. Shailesh Kumar assisted in drafting the manuscript and critical revision.

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