Blood stream infection or Septicemia is a potential life threatening condition where the bacteria, its toxins and other infectious agents enters into the blood and evokes a severe inflammatory response. Sepsis is most commonly caused by aerobic bacteria. On response to infection in the blood, body triggers the immune system and cause damage to tissues and organs, which leads to shock, multiorgan failure. Blood stream infections can be primary or secondary from any other organ’s primary focus of infection like endocarditis, pneumonia, UTI, meningitis and others Sepsis is one of the most frequent causes of death worldwide, but there are challenges in collecting reliable data at the population level [1]. From data published in 2020, there were 48.9 million cases and 11 million sepsis-related deaths worldwide, representing 20% of all global deaths [2]. Almost half (20 million) of all estimated sepsis cases worldwide occurred in children under 5 years of age. Mortality rate and incidence rate of sepsis vary from region to region, highest burden of sepsis in low middle income countries and sub-saharan Africa [3]. Sepsis can affect any age group, but people who are older, very young, pregnant and people with co morbidities like cardiovascular disease, diabetes, and kidney disease. Prolonged hospitalization, use of central lines, catheter and ventilator support for management of the patient could lead to nosocomial blood stream infections. Several chronic diseases, sociodemographic factors, poor access to health care systems and quality of care are associated with the occurrence of sepsis and its case fatality rate [4]. Blood culture is the best approach to identify the pathogen in blood stream infections. The antimicrobial resistance is spreading drastically worldwide, extended spectrum beta lactamases Enterobacteriaceae, methicillin resistant Staphylococcus aureus, metallo beta lactamase non fermenters are being observed in community and hospital settings [5]. Regular monitoring of antimicrobial susceptibility pattern of blood stream infections is primarily important to fight against the infections. Firstly, the strict antimicrobial stewardship programme with the distribution of hospital antibiogram on a determined frequency, secondly, strong and dedicated laboratory tests with the provision of automated antimicrobial sensitivity testing and rapid diagnostic tests for MDR and finally, clinician compliance to laboratory directed therapy will help to face the real challenges in the management of sepsis. AIM: To study the pathogens associated with blood stream infections and the antibiotic sensitivity testing OBJECTIVES: 1. To assess the pathogens in blood stream infections 2. To know the most common pathogens isolated from blood stream infections 3. To determine the antibiotic susceptibility pattern of isolates.

A Retrospective cohort study was conducted on patients diagnosed to have septicemia at the department of Microbiology of Government General Hospital/Medical College, Nandyal, Andhra Pradesh. A total of 466 patients attended various clinical departments with clinical manifestations of septicemia were included in this study during the period of one year (January 2024 to December 2024). Ethical committee approved this study. Informed consent was taken from all the patients included in this study. Inclusion criteria: 1. Patients presenting clinical manifestations of sepsis 2. Patients of all ages and both sexes Exclusion criteria: 1. Hospital acquired blood stream infections 2. Isolate which is considered as a contaminant or colonizer after evaluation of clinical, laboratory and radiological findings. Study Procedure: Under aseptic precautions blood samples were collected from a peripheral vein and 10-20 ml and 3-5 ml of blood was inoculated immediately into labeled adult and pediatric blood culture bottles of BD Company. Two bacTalert blood culture bottles per each patient were preferable to increase the pathogen detection rate. These were loaded into BD BACTEC automatic blood monitoring system. Positively flagged Blood Cultures were further processed via direct Gram’s stain and inoculation onto culture media. Culture media routinely used for processing for blood bottles were Nutrient agar, 5% sheep blood agar, Macconkey agar. After inoculation of the sample, these culture media plates were incubated at 370C for 24 hours. The colony characteristics observations, its gram stain and biochemical reactions were performed as per standard precautions [6]. Antimicrobial Susceptibility Testing: Antimicrobial susceptibility of isolates was determined using the Modified Kirby-Bauer disk diffusion method. The results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [7]. AST is done by modified Kirby bauer disc diffusion method on Mueller Hinton agar based on CLSI guidelines. The quality check done with the quality control strains. – Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853 and S.aureus ATCC 25923. Antibiotic disks used for Gram positive organisms testing were penicillin (10U), amoxyclav (30 µg), amikacin (30 µg), ciprofloxacin (5 µg), erythromycin (5µg), clindamycin (2µg), cotrimoxazole (1.25 µg/23.75 µg), cefoxitin (30 µg), linezolid (30 µg), vancomycin (30µg) and teicoplanin (30µg). Gram negative isolates antibiotics were: ampicillin, amoxyclav (30 µg), cefotaxime (30 µg), cefipime (30 µg), Cefotaxime+clavulanic acid (30/10 µg), levofloxacin (5 µg), ertapenem (10 µg), meropenem (10 µg), amikacin (30 µg), tigecycline (15 µg) and colistin (50 µg). Non fermenter isolates antibiotics were: ceftazidime (30 µg), piperacillin+tazobactum (100/10 µg), Cefotaxime+clavulanic acid (30/10 µg), cefaperazone+sulbactum (75/30 µg), minocycline (30 µg), levofloxacin (5 µg), meropenem (10 µg), tobramycin (10 µg), gentamicin (10 µg) and colistin (50 µg). Standard Quality Control strains were used as a part of testing. Multi Drug testing was done for all strains isolated according to CLSI guidelines. Statistical Analysis: The data was collected from laboratory culture registers and the results were obtained in the Microsoft Excel and statistically analysed by calculating the numbers and percentages of all descriptive variables.

Clinical specimens of 466 were evaluated in this study. Out of 466 blood culture tests, 178 (38.1%) yielded growth in culture media. Out of these blood samples, S.aureus (17.7%) were predominantly noted followed by Pseudomonas species (38.1%), Candida (14.5%, E.coli (13.02%), Coagulase Negative Staphylococci (CoNS) (11.4%), Klebsiella (10.4%) Fig 1. Gram negative isolates antibiotic susceptibility pattern shows around 50% were sensitive to cefotaxime, cefipime, amoxyclav, Cefotaxime+clavulanic acid, around 90% isolates were sensitive to Amikacin, Meropenem, Ertapenem, Levofloxacin and almost all the isolates were sensitive to tetracycline, colistin and tigecycline. Isolates of Salmonella and Citrobacter were very few, those were sensitive strains. Table 1. Antibiotic susceptibility pattern of Enterobacteriaceae Antibiotics Esch.coli (n=25) Klebsiellaspp .(n=20) Salmonella (n=2) Citrobacter (n=1) Ampicillin 4 (16%) IR 1(50%) IR Amoxyclav 12 (48%) 10 (50%) 2(100%) 1 (100%) Cefotaxime 12 (48%) 10 (50%) 2(100%) 1 (100%) Cefepime 15 (60%) 10 (50%) 2(100%) 1 (100%) Cefotaxime+clavulanic acid 16 (64%) 13 (65%) 2(100%) 1 (100%) Ertapenem 22 (88%) 16 (80%) 2(100%) 1 (100%) Meropenem 23 (92%) 17 (85%) 2(100%) 1 (100%) Amikacin 25 (100%) 18 (90%) 2(100%) 1 (100%) Levofloxacin 18 (72%) 16 (80%) 2(100%) 1 (100%) Tetracycline 25 (100%) 18 (90%) 2(100%) 1 (100%) Colistin 25 (100%) 20 (100%) 2 (100%) 1 (100%) Tigecycline 25 (100%) 20 (100%) 2 (100%) 1 (100%) Gram positive pathogens from CAP patients showed about 60% of isolates sensitive to erythromycin. Around 80% of isolates were sensitive to cotrimoxazole, ciprofloxacin, clindamycin, and meropenem. Among Staphylococcus aureus isolates 60% (15 out of 25) were sensitive to cefoxitin, signifying methicillin sensitive Staphylococcus aureus. Table 2. Antibiotic susceptibility pattern of Gram Positive isolates Antibiotics S.aureus (n=25) CONS (n=22) Enterococci spp (n=7) Streptococcus spp (n=10) Penicillin 6 (24%) 6 (27.2%) 5 (70.1%) 8 (80%) Amoxyclav 15 (60%) 13 (59.09%) 5 (70.1%) 8 (80%) Cefoxitin 15 (60%) 14 (63.6%) IR 8 (80%) Erythromycin 12 (48%) 12 (54.5%) 4 (57.1%) 7 (70%) Cotrimoxazole 16 (64%) 14 (63.6%) IR 7 (70%) Ciprofloxacin 18 (72%) 14 (63.6%) 6 (85.7%) 10 (100%) Amikacin 25 (100%) 22 (100%) 7 (100%) 10 (100%) Clindamycin 16 (64%) 15 (68.1%) IR 7 (70%) Meropenem 22 (88%) 20 (90.9%) 7 (100%) 10 (100%) Linezolid 25 (100%) 22 (100%) 7 (100%) 10 (100%) Teicoplanin 25 (100%) 22 (100%) 7 (100%) 10 (100%) Vancomycin 25 (100%) 22 (100%) 7 (100%) 10 (100%) Pseudomonas spp were <60% sensitive to all the first line antibiotics like: ceftazidime, ceftriaxone, levofloxacin, cefotaxime+clavulanic acid, piperacillin+tazobactum, cefoperazone+sulbactam, 70-80% sensitive to second line antibiotic aminoglycosides – levofloxacin, meropenem and 90-100% sensitive to gentamicin, tetracycline, tobramycin and colistin. Other non-fermenters showed 70-80% susceptibility to levofloxacin, tetracycline, gentamicin, tobramycin, meropenem. 55% of isolates were sensitive to ceftazidime, cefotaxime+clavulanic acid, piperacillin+tazobactum, cefoperazone+sulbactam (Table 3). Table 3. Antibiotic susceptibility pattern of Non fermenters Gram Negative bacilli Organism CAZ CTR LE TE COT G PIT TOB CFS CXA MRP CL Pseudomonas (n=32) 14 (43.7%) 13 (40.6%) 24 (75%) 28 (87.5%) IR 30 (93.7%) 18 (56.2%) 28 (87.5%) 18 (56.2%) 17 (53.1%) 25 (78.1%) 32 (100%) Acinetobacter (n=4) 0 (0%) 0 (0%) 3(75%) 4(100%) 2(50%) 4(100%) 2(50%) 3(75%) 2(50%) 0 (0%) 3(75%) 4(100%) NF GNB (n=7%) 4(57.1%) 2(28.5%) 5(71.4%) 5(71.4%) 4(57.1%) 5(71.4%) 4(57.1%) 5(71.4%) 4(57.1%) 4(28.5%) 5(71.4%) 7(100%)

Bloodstream infections (BSI) are infectious diseases defined by the presence of viable bacterial or fungal microorganisms in the bloodstream (later demonstrated by the positivity of one or more blood cultures) that elicit or have elicited an inflammatory response characterized by the alteration of clinical, laboratory and hemodynamic parameters. A blood culture test measures and analyzes the growth of germs that can cause infections, such as bacteria or fungi. Blood culture is mandatory to perform in sepsis because it helps to start the accurate antibiotic therapy of those patients in critical situation. The present study is aimed to find all the most possible pathogens responsible for blood stream infections. Though the sensitivity rate is less in blood cultures and even there is a chance to get contaminants during the sample collection, this study will help clinicians to think about the empirical therapy in blood stream infections. 38.1% (178/466) were blood culture positive in septicemia patients. Out of these blood samples, S.aureus (17.7%) were predominantly noted followed by Pseudomonas species (38.1%), Candida (14.5%, E.coli (13.02%), Coagulase Negative Staphylococci (CONS) (11.4%), Klebsiella (10.4%). Remaining blood pathogens (Streptococcus pyogenes (1.0%), Salmonella spp (1.04%), Streptococcus pneumoniae (4.1%), Acinetobacter (2.08%), NFGNB (3.6%), Enterococci (3.6%), Citrobacter (0.5%) were below 10% in culture isolation. In similar to this as study from South India observed 108 (38.71%) isolates were Gram positive cocci while 171(61.29%) were Gram negative bacilli. Five fungal isolates. Staphylococcus aureus was the most common isolate followed by Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Acinetobacter and Enterococcus species. 24.37% of all isolates were MDR [8]. A study from North India on blood culture samples showed out of 565 positive cultures, 447 (79.1%) showed bacterial growth; Gram positive were 306 (54.2%) and Gram negative were 141 (24.9%). Candida species were isolated from 118 (20.9%) of positive samples. The most frequently identified Gram-positive bacteria were Coagulase-negative staphylococci 208 (67.9%) and the most common Gram-negative isolates were Acinetobacter species 89 (63.1%) [9]. Mehta M et al [10] noted among the 567 qualifying samples, Pseudomonas aeruginosa (19.75%), Escherichia coli (15.17%), Klebsiella pneumoniae (14.99%), and Salmonella enterica serovar Typhi (12.87%) were the most frequently isolated Gram-negative bacteria other than Citrobacter, Acinetobacter, Proteus, and Enterobacter spp. collectively accounting for 80.96% of the isolates. Staphylococus aureus (13.86%) and Enterococcus feacalis (2.35%) were most frequently isolated Gram-positive bacteria other than other Streptococcus and Staphylococcus spp. collectively accounting for 18% of the isolates. Nishanthy M et al [11] among these culture growth organisms, 82 were Gram positive (55.4%) isolates and 74 were Gram negative (50%). The most common isolate was Coagulase negative Staphylococcus trailed by Escherichia coli and Staphylococcus aureus, Acinetobacter species and Pseudomonas aeruguinosa, Klebsiella pneumoniae and Enterococcus species. CONS are usually skin commensals and have the capability of causing opportunistic infections especially in hospitalized patients and in those patients underwent procedure or intervention or surgery. CONS reporting from microbiology department should do after thorough evaluation of microbial growth, other laboratory findings like CRP, WBC, procalcitonin and the patient condition as they can be commensal or colonizer. Improper sample collection is the most important which should keep in mind. Finally, the wrong reporting as a pathogen may mislead the clinician in arriving at final diagnosis. Anjum et al [8] reported 12.9% of CONS among gram positive isolates, Vasudeva et al [12] reported 30% of GPC isolates to be CONS and Banik et al [13] reported 23.31% CONS. Gram negative isolates antibiotic susceptibility pattern shows around 50% were sensitive to cefotaxime, cefipime, amoxyclav, Cefotaxime+clavulanic acid, around 90% isolates were sensitive to Amikacin, Meropenem, Ertapenem, Levofloxacin and almost all the isolates were sensitive to tetracycline, colistin and tigecycline. This is in similar to the study done by Shrestha S et al [14]. Nazir A et al [9] reported vancomycin and linezolid for gram positive isolates and colistin and tigecycline for gram negative isolates as the most sensitive drugs. Anjum et al [8] stated Piperacillin-Tazobactam and Gentamicin had good sensitivities against GNB. Mehta M et al [10] observed 82.66% of isolates were sensitive to cefoperazone+sulbactum, 76.61% of amikacin and 65.17% ciprofloxacin sensitivity among gram negative bacteria. Nishanthy M et al [11] documented that all the Gram negative isolates were susceptible to Carbapenems, with susceptibility rate of 97% for E. coli, 93% for Acinetobacter, 93% for Pseudomonas aeruginosa and almost all these isolates showed 100% susceptibility to Colistin and Polymyxin-B. Gram positive pathogens from CAP patients showed about 60% of isolates sensitive to erythromycin. Around 80% of isolates were sensitive to cotrimoxazole, ciprofloxacin, clindamycin, and meropenem. Good sensitivity with vancomycin, linezolid, teicoplanin, also reported same by other studies [15-17]. Among Staphylococcus aureus isolates 60% (16 out of 21) were sensitive to cefoxitin, signifying Methicillin sensitive Staphylococcus aureus. In line with this study Anjum et al [8] and Banik et al [13] observed the similar percentage of MRSA in their localities, percentage being 39.6% and 42% respectively. Anjum et al [8] noted CONS were highly susceptible to doxycycline, Ciprofloxacin, Linezolid and Vancomycin while penicillin, azithromycin, erythromycin, and clindamycin showed poor response. Enterococcal isolates were high susceptibility to linezolid, vancomycin, ciprofloxacin and high level gentamicin. Mehta M et al [10] noted among Gram-positive isolates, vancomycin (100%), ciprofloxacin (89.74%) showed the highest activity against Staphylococcus spp. Nishanthy M et al [11] noted linezolid, vancomycin and clindamycin were highly susceptible to majority of the Gram positive isolates. Meshram P et al [18] did a study on blood stream infections in Central India reported higher percentage of Methicillin Resistant Staphylococcus aureus which was 75%. Vancomycin, linezolid, teicoplanin, doxycycline, sometimes macrolides and flouoroquinolones are good option to treat MRSA infections, but the patient condition and the culture report need to be evaluated before choosing these drugs to avoid emergence of antimicrobial resistance. Non fermenter Gram negative bacilli were moderate sensitive to all the first line antibiotics like: ceftazidime, ceftriaxone, levofloxacin, cefotaxime+clavulanic acid, piperacillin+tazobactum, cefoperazone+sulbactam, and good sensitivity towards aminoglycosides, levofloxacin, meropenem, gentamicin, tetracycline, tobramycin and colistin. Anjum et al [8] noted the isolates were moderately resistant to Aztreonam and ceftazidime and showing good susceptibility (>80%) to Aminoglycosides, fluoroquinolones (i.e. levofloxacin), carbapenems and Piperacillin-tazobactam. Acinetobacter species were sensitive to Meropenem (93%), piperacillin-tazobactam (100%) and 4th generation cephalosporins (84%). One isolate of Carbapenem-Ampicillin Sulbactam Resistant (CASR) Acinetobacter baumannii was isolated. The epidemiology and pathogen profile of BSIs vary between regions [19]. Overall, isolation of gram positive and gram negative pathogens varies depending on the regulations framed by government on infection prevention, lifestyle of community, their practices and antibiotic policy. Gram positive isolates expressed poor sensitivity to erythromycin, clindamycin, amoxyclav cefoxitin, and cotrimoxazole. Gram negative isolates were moderate sensitive to amoyxclav, ceftriaxone, cefipime, beta lactam and beta lactamase inhibitors. Non fermenters were moderately sensitive to ceftazidime, ceftriaxone, cotrimoxazole, piperacillin+tazobactum cefoperazone+sulbactum cefotaxime+clavulanic acid. Thinking about the moderate susceptibility pattern of isolates, clinicians may start high end antibiotics like polymyxin, colistin which may pose pan drug resistant, this will ultimately spoil the antibiotics pipeline and cause the emergence of antimicrobial resistance drastically worldwide [20]. The combination of antibiotic therapy by keeping vision on synergistic effect along with the evaluation of empiric therapy in the hospital, central line associated blood stream infections surveillance and microbiological cultures will help to recovery of infected patient and also slow the phase of emergence of antimicrobial resistance [21].

In this study among gram negative isolates Pseudomonas, Escherichia coli and Klebsiella were predominant pathogens, whereas among gram positive isolates S.aureus and CONS were majority isolates and Candida is the third most common pathogen responsible for Septicemia. Beta lactam and beta lactamase inhibitors, fluoroquinolones, cotrimoxazole yielded good result in good percentage of studied population, whereas third line antibiotics showed high sensitivity. Patient with septicemia is a medical emergency as the virulence factors of pathogens quickly imbalance the immune system, coagulation abnormalities and impairs tissue oxygenation. Judicial usage of antibiotics by clinicians, provision of hospital anitbiogram at least annually by microbiologist and adherence to antimicrobial stewardship policy will help to manage blood poisoning patients effectively.

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