The Emergence of Multidrug-Resistant Gram-Positive Bloodstream Infections in India—A Single Center Prospective Cohort Study

Abstract

Introduction: Gram-positive bloodstream infections (BSIs) are an emerging health concern, especially in resource-limited settings. There is a paucity of data regarding the antimicrobial resistance (AMR) pattern of Gram-positive BSIs. The rise in multidrug-resistant infections further convoluted antibiotic selection. We aimed to assess the incidence, clinical and microbiological profile, antimicrobial resistance (AMR) and outcome in Gram-positive BSIs. Methods: This was a single-center prospective study conducted at a tertiary care hospital in Western India. All patients (age ≥18 years) with culture-proven Gram-positive BSIs were included. Data were collected on all patients’ demography, risk factors, AMR and clinical outcome. Results: A total of 210 clinically significant isolates were grown from July 2020 to December 2021. The incidence of Gram-positive BSIs was 29% (n=61); 55.9% of cases were healthcare-associated, while 44.1% were community-acquired. Coagulase-negative staphylococci (CoNS) were the major isolates (36.1%), followed by Enterococcus spp. (27.9%), methicillin-susceptible Staphylococcus aureus (MSSA) (18%) and methicillin-resistant Staphylococcus aureus (MRSA) (14.7%). The proportion of vancomycin and teicoplanin-resistant CoNS isolates was 13.6% and 19%. Among Enterococcus isolates, the proportion of vancomycin-resistant enterococci (VRE) and linezolid-resistant enterococci (LRE) were 11.8% and 5.9%. The overall mortality in Gram-positive BSIs was 42.6%. Older age, MRSA infection, septic shock, and high NLR were significantly associated with mortality. On the Cox regression model, age ≥65 years (HR: 2.5; 95%CI: 1.1-5.8; p=0.024) and MRSA infection (HR: 3.6; 95%CI: 1.5-8.5; p=0.021) were found as independent predictors of 30-day mortality. Conclusions: This study found substantial mortality with Gram-positive BSIs, especially MRSA infections. Moreover, the emergence of VRE and LRE is also alarming. Active surveillance of AMR and evaluation of mortality predictors may help overcome the therapeutic challenges in managing BSIs.

Introduction

Bloodstream infection is the presence of viable microorganisms in the bloodstream that 1elicit the inflammatory response characterized by altered clinical, laboratory or hemodynamic parameters [1]. The threat of bloodstream infections is looming with ever-increasing incidence (122-215 cases/100000 population) and an estimated crude mortality rate of 8-17% [2,3,4]. Primary BSIs are bacteremia with an unidentified nidus of infection, or where the infection is directly introduced by contaminated medical devices. In secondary BSIs, there is evidence of another host tissue invasion, other than blood, by the same organism. Gram-positive organisms are the most common etiology causing catheter-related BSIs, the majority being coagulase-negative staphylococci (CoNS), followed by enterococci and Staphylococcus aureus. The attributable mortality rate is around 12-25% in these Gram-positive BSIs [5].

In the past two decades, the rising incidence of Gram-positive BSIs (especially S. aureus) has been a significant cause of concern. Another worrisome trend is the rise in MRSA BSIs incidence compared to MSSA, which is also contributing to the high mortality [6]. The situation is no different in the Indian subcontinent, with an ever-increasing burden of MRSA and VRE (vancomycin-resistant Enterococcus) infections [7]. Another worry is the lack of reliable markers for the early detection of Gram-positive BSIs, with procalcitonin and NLR (neutrophil to lymphocyte ratio) usually favoring Gram-negative BSIs. There is a paucity of data on Gram-positive BSIs regarding the aforementioned factors; this study aims to assess the clinical and microbiological profile and antibiotic resistance patterns (AMR) of Gram-positive BSIs.

Methods

Data collection

This prospective observational study was carried out at a tertiary care center in Western Rajasthan, India, from July 2020 to December 2021. All patients (age ≥18 years) with blood culture positivity for Gram-positive bacteria and clinical evidence of sepsis were included. Those with isolation of multiple organisms from blood culture were excluded. BSIs were classified as healthcare-associated and community-acquired based on their clinical presentation. The study was approved by our Institutional Ethical Committee (reference no. AIIMS/IEC/2020/3175). Gram-positive BSIs were defined by the presence of viable Gram-positive bacterial microorganisms in the bloodstream (documented through positivity of one or more blood cultures) that have elicited an inflammatory response translated into an alteration of clinical, laboratory, and hemodynamic parameters [1]. For CoNS, positivity of at least two blood cultures for the same organism was considered significant.

The following clinical data were recorded: demographic parameters (age, gender, date of hospitalization), clinical symptoms, risk factors for BSIs (central venous catheters, mechanical ventilation), comorbidities (diabetes, hypertension, COVID-19, malignancy), complications (septic shock, acute kidney injury, multiorgan dysfunction, etc.), identifiable primary source and outcome. Laboratory parameters and inflammatory markers, including C-reactive protein (CRP) and procalcitonin, were also collected. On admission, a quick sequential organ failure assessment (q-SOFA) score was calculated for each patient. According to the CLSI guideline, bloodstream infections were classified into community-acquired and healthcare-associated. Septic shock was defined as persisting hypotension requiring vasopressors to maintain MAP ≥65 mmHg and having a serum lactate level >2 mmol/L despite adequate volume resuscitation.

Identification of bloodstream infections and antimicrobial susceptibility patterns

Blood cultures were collected aseptically for all suspected patients with sepsis at presentation. Each blood culture BACTEC (Becton Dickinson, USA) bottle was inoculated with eight to ten mL of blood, taken aseptically, loaded onto the automated system, and incubated for five days minimum. When the blood culture was flagged positive, the culture samples were subjected to Gram stain and cultured onto blood agar and mannitol salt agar. Only Gram stain-positive blood cultures were included in the study. After 16-18 hours of incubation at 37 °C, the cultures were read for colonies’ growth, and the Gram stain and coagulase tests were performed for further workup. Antibiotic susceptibility testing was done by Kirby Bauer’s disk diffusion method. Zone inhibition diameters were interpreted as sensitive (S), intermediate (I) and resistant (R). The criteria for the zone of inhibition of different antibiotics were set according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [8]. A multidrug-resistant organism (MDRO) was defined as acquired non-susceptibility to at least one agent in three or more antimicrobial categories [9].

Statistical analysis

Data analysis was done using SPSS software, version 20.0 (IBM Corp, USA). Mean ± standard deviation (SD) were calculated for continuous variables. Categorical data and median were represented as percentages or frequencies. The independent student t-test was used for normally distributed data, and the Mann-Whitney U test for skewed data. Analysis of categorical variables was done by the Fisher exact test. Univariate Cox regression analysis was performed to determine the various clinical predictors of mortality. Statistically significant variables on univariate analysis (p<0.1) were selected for multivariate Cox regression analysis. The results of Cox regression are presented as hazard ratio (HR) and their 95% confidence intervals (CIs). Log-rank test was used to compare survival probability in different groups like MRSA vs non-MRSA infections. P<0.05 was considered statistically significant.

Results

During the study period from July 2020 to December 2021, 223 isolates were grown. After removing contaminants (n=13, 5.8%), 210 isolates were considered clinically significant. Those with multiple isolates (≥2 isolates in the same or subsequent culture) were excluded from the analysis. Overall, the incidence of Gram-positive bloodstream infections was 29% (61/210) in the study period (18 months).

Demographic and clinical characteristics

The demographic and clinical characteristics of all patients are summarized in

Table 1. Demographic, clinical and laboratory data of Gram-positive bloodstream infections.

Data are presented as median (IQR), mean±SD, or n (%). p values were calculated by χ² test or t-test, as appropriate. MSSA – methicillin-susceptible Staphylococcus aureus; MRSA – methicillin-resistant Staphylococcus aureus; CoNS – coagulase-negative staphylococci; qSOFA – quick Sequential Organ Failure Assessment; NLR – neutrophil-lymphocyte ratio; CRP – C reactive protein; AST – aspartate aminotransferase; ALT – alanine aminotransferase.

. The mean age of all patients with Gram-positive BSIs was 55 years, with more than two-thirds being male patients. Around 47.5% of the patients (n=29) were from the intensive care unit (ICU).

Among all Gram-positive BSIs isolates, the most common species were CoNS (36%), followed by Staphylococcus aureus (33%) and Enterococcus (28%). Among S. aureus isolates, the proportion of methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) were 18% and 15%, respectively. Moreover, 56% of BSIs were healthcare-associated, and 44% were community-acquired infections. CoNS and Enterococcus were the commonest etiology for healthcare-associated BSIs (38% each). However, S. aureus was the most common etiology for community-acquired BSIs (44%). In addition, among all MRSA BSIs, the majority (66.6%, 6/9) were community-acquired (CA-MRSA). Diabetes (28%) and hypertension (25%) were the most common comorbidities (Table 1). Overall, 15% of the patients had been admitted with COVID-19 pneumonia and, later on during same hospitalization course, developed Gram-positive BSIs. Central venous catheter and mechanical ventilation were the major risk factors (38% and 33%, respectively). Pneumonia was the most common source identified in 28.3% of cases. Skin/soft tissue infection, meningitis, infective endocarditis and urinary tract infections were seen in 11.7%, 6.6%, 4.9%, and 4.9% of the patients, respectively. In 42% of the cases, the source could not be identified. Septic shock was documented in 35% of cases, multiple organ dysfunction syndrome (MODS) was seen in 25% of cases and acute respiratory distress syndrome (ARDS) in 21.6% of cases.

Antimicrobial susceptibility and outcomes

We collected data on the pattern of antimicrobial resistance to various commonly used antibiotics, for pathogens involved in Gram-positive BSIs. The antibiogram for different microorganisms is shown in

Table 2. Antibiotic resistance pattern of Gram-positive microorganisms isolated from patients with bloodstream infections.

CoNS – coagulase-negative staphylococci; MRSA – methicillin-resistant Staphylococcus aureus; MSSA – methicillin-susceptible Staphylococcus aureus; S. pneumoniae – Streptococcus pneumoniae.

. Among CoNS, vancomycin and teicoplanin resistance was seen in 13.6% and 19% of the isolates. None of the CoNS isolates was found resistant to linezolid. Most enterococcal isolates were resistant to penicillin and ampicillin (Table 2). In this study, the percentages for vancomycin-resistant enterococci (VRE) and linezolid-resistant enterococci (LRE) were 11.8% and 5.9%. None of the S. aureus isolates was resistant to glycopeptide antibiotics (vancomycin, teicoplanin and daptomycin) – Table 2.

The overall mortality rate in Gram-positive BSIs was 42.6% (n=26). We analyzed the various clinical determinants for mortality in Gram-positive BSIs. Age ≥ 65 years, septic shock on presentation, MRSA infection, and NLR>10 were found to be associated with high mortality (

Table 3. Univariate and multivariate regression analysis showing various predictors of mortality in patients with Gram-positive bloodstream infections.

HR – hazard ratio; MRSA  – methicillin-resistant Staphylococcus aureus; qSOFA – quick Sequential Organ Failure Assessment; NLR – neutrophil-lymphocyte ratio.

). On multivariate Cox regression analysis, older age (≥65yrs) (HR: 3.6, 95%CI: 1.5-8.5; p=0.02; Figure 1) and MRSA infection (HR: 2.5, 95%CI: 1.1-5.8, p=0.02; Figure 2) were identified as independent predictors of 30-days mortality. In this study, we did not find any association between qSOFA score, CRP and procalcitonin levels and mortality.

Discussion

Data are scarce regarding Gram-positive BSIs, with most literature focusing on Gram-negative BSIs. This study aimed to analyze the incidence and associated outcomes of Gram-positive bloodstream infections in our tertiary care institute. In this study, the incidence of Gram-positive BSIs was 29%, comparable to other studies. Literature reveals heterogenicity in the incidence of Gram-positive BSIs, which varies from 22% to 59% depending on the study population characteristics, blood culture methods, and interpretation of culture reports [10,11,12]. CoNS were the most common etiology, which was explained by the high proportion of healthcare-associated infections with indwelling devices in this study population. Staphylococcus aureus is now emerging as the leading cause of Gram-positive BSIs [12], surpassing Gram-negative BSIs as the most common cause according to recent bloodstream infection surveillance studies [4,5]. In this study, most patients were elderly, which explained the high incidence of S. aureus bacteremia. Previous studies have also highlighted the age-dependent increase in the incidence of S. aureus BSIs [13]. The MRSA incidence was 45% in this study, similar to a result of a large multicenter study from India (41%) [14]. Another important observation was the high incidence of CA-MRSA in this study (around two-thirds of cases). CA-MRSA usually causes skin and soft tissue infections and pneumonia; however, in this study, only two patients had this clinical presentation. We did not evaluate the patients for staphylococcal cassette chromosome mec (SCCmec), an important characteristic of CA-MRSA infections (type IV and V).

In this study, among all CoNS isolates, vancomycin resistance was significantly high (14%) compared to previous studies [15], which is alarming. Notably, previous reports have also documented heterogeneous vancomycin resistance (vancomycin-intermediate subpopulation) in CoNS isolates [16]. Owing to the inadvertent use of vancomycin and selection pressure, the prevalence of vancomycin-resistant CoNS could further propagate. Among Enterococcus isolates, we found a high incidence of VRE (11.8%), consistent with a recent study from a South Indian tertiary center (19%) [17]. Moreover, the incidence of LRE was alarmingly high (6%), which was not seen in previously published reports (<1%) [18]. Previous antibiotic exposure, person-to-person transmission, and environmental contaminants are the various factors contributing to drug-resistant enterococcal infection.

This study found a significant mortality rate in Gram-positive BSIs (43%), which was high compared to the previous reports (5% to 13%) [19,20]. This disparity in mortality rate could be due to various risk factors, such as the difference in age groups, a high proportion of ICU patients, healthcare-associated infections, comorbidities, MDR infections and source of bacteremia. Interestingly, mortality due to MRSA BSIs was 88.8% (8/9 cases). This could be attributed to several factors, one of them being the elderly population. A report by Van Hal et al. described that older patients had twice the risk of dying from Staphylococcus aureus bacteraemia [21]. Except for one, all patients with CA-MRSA BSIs died. Although we did not identify Panton-Valentine leukocidin gene (PVL) positivity in our study cohort, this could have contributed to high mortality compared to non-toxin-producing strains, especially in patients with necrotizing pneumonia. Irrational use of antibiotics, particularly in MRSA BSIs, is also an important factor causing high mortality; 6 out of 9 patients in this group received irrational antibiotics, and 5 (55.5%) succumbed to infection. Studies also showed improved outcomes in staphylococcal infections where infectious disease consultation was taken [22]. Another critical factor contributing to the poor outcome was represented by comorbidities, which were present in nearly 50% of the patients. COVID-19 was found to be an important predisposing factor in this study. Furthermore, the mortality rate was 80% in these patients, consistent with previous reports of high mortality in COVID-19 pneumonia [23]. Around one-third of patients in this study had septic shock on admission, contributing to increased mortality. Septic shock is usually more associated with Gram-negative bacteremia. However, when it is present, it was found to be an independent predictor of mortality [24]. Procalcitonin levels can be the early indicator of bacteremia (especially Gram-negative BSIs); however, this study did not find any correlation between procalcitonin levels and mortality. This result was consistent with previous reports, which showed the poor response of procalcitonin levels in predicting Gram-positive BSIs [25]. Similarly, we did not find any correlation of NLR with mortality, although some earlier reports advocate the role of high NLR in predicting poor outcomes in Gram-positive BSIs [26].

This study has some important limitations which need to be highlighted. First, this was a single-center study with a relatively small sample size, so the results should be interpreted carefully. Second, we did not collect data regarding the follow-up culture positivity, which is also an important determinant of outcome. Lastly, we did not perform the molecular characterization of isolates and epidemiological typing (like SCC mec genotyping and PVL analysis in S. aureus bacteremia), so we do not know to what extent the high mortality is attributed to these factors.

Conclusions

This study reiterates the emerging threat of Gram-positive bloodstream infections, which carry substantial mortality, especially with MRSA infections. The preponderance of CA-MRSA is also a looming threat along with MDR enterococcal isolates (VRE and LRE strains). Active surveillance of AMR, judicious use of antibiotics and infection control practices are paramount to curtail this problem. Further studies are warranted to elucidate new prognostic markers for Gram-positive BSIs, as procalcitonin levels and NLR were not prudent in these settings.