Aerobic vs. Anaerobic Blood Culture

The early detection of bacteremia through blood cultures is not just a diagnostic tool but a life-saving measure. It underscores the crucial role healthcare professionals play in identifying organisms within the blood stream that may lead to sepsis or septic shock. This has a significant impact on patient outcomes.

Standard laboratory practice for blood cultures uses a vented bottle for aerobes and an unvented bottle for anaerobes. Bacteremia can result from activities such as ordinary vigorous toothbrushing and severe infections. However, not all bacteremia leads to sepsis and bacteremia caused by toothbrushing poses very low risk.

Sepsis, a life-threatening condition, is a severe whole-body response to bacteremia or another infection. Symptoms, including fever, weakness, tachycardia, rapid breathing, and elevated white blood cell counts characterize it. If sepsis progresses, it can lead to septic shock, a condition where dangerously low blood pressure occurs, causing the internal organs to lack blood flow. Septic shock occurs when sepsis causes dangerously low blood pressure. As a result, the internal organs lack blood flow and may fail.

At least 90% of bacteria that cause bloodstream infections grow in aerobic bottles. This fact has sparked an ongoing debate about the relevance of routinely using anaerobic bottles in both sets when obtaining blood cultures. However, most recent studies advocate using aerobic and anaerobic bottles for blood culture to optimize the overall detection of bloodstream infections, a practice that directly impacts patient care.¹

This review provides a comprehensive understanding of aerobic and anaerobic blood cultures. It highlights the differences between aerobic and anaerobic bacteria and their clinical significance in diagnosing bloodstream infections. It will enlighten individuals interested in microbiology and infectious diseases and equip them with valuable knowledge.

Aerobic and Anaerobic Bacteria

Aerobic bacteria dominate the vast microbial world and are primarily symbiotic. They often live on, around, or in us, benefiting us. After consuming oxygen, these aerobic bacteria release carbon dioxide, akin to human respiration, reassuring us about their role in our ecosystem.

Fascinatingly, anaerobic bacteria have evolved a unique survival strategy. They have adapted to low or no-oxygen environments, surviving by primarily adopting a parasitic lifestyle and feeding off their host. This remarkable adaptation, necessitated by their less permeable cell walls, which limit their access to external resources, is a testament to their resilience. Clostridium difficile, which can cause severe diarrhea, is an example of an anaerobic bacterium. Unlike aerobic bacteria, anaerobic bacteria can survive and grow in environments without oxygen, underscoring their adaptability.

Some organisms can live with or without oxygen and are excellent hybrid-like adapters. For example, Escherichia coli, a common cause of urinary tract infections, is a facultative anaerobic bacterium that requires oxygen to grow and survive in most instances but can survive in low—or no-oxygen environments. A facultative anaerobic organism can produce energy by aerobic respiration if oxygen is present but switch to fermentation if oxygen is absent.

Aerobic and anaerobic bacteria are part of the normal flora of human skin and mucosal membranes and are not just microorganisms. They are fascinating entities that can maintain our health or cause serious infections. Understanding their roles is a captivating journey that underscores the importance of diagnosing bloodstream infections.

Aerobic and Anaerobic Blood Cultures 

The two bottles, aerobic and anaerobic, are considered to be part of a set of blood cultures. Contamination with skin flora usually occurs when one commensal skin organism appears in only one of the two sets. It is important to note that some organisms do not grow well in blood cultures and require different, specialized techniques. 

Blood cultures are drawn using venipuncture with an antiseptic skin and bottle technique. When followed correctly, this method ensures the accuracy and reliability of the results. While antibiotic therapy may cause false negative blood culture results because it inhibits the growth of microbes, the overall protocol is designed to provide the most accurate diagnosis possible. 

Facultative anaerobic organisms, capable of cellular respiration in oxygen-rich and oxygen-poor environments, are often found in blood cultures. Examples of facultative anaerobes include Escherichia coli, Pseudomonas aeroginosa, Staphylococcus spp., Listeria spp., Salmonella, Shewanella oneidensis, and Yersinia pestis. Obligate aerobic bacteria live only in aerobic environments, cannot survive without oxygen, and are less common in blood cultures. 

If microorganisms are detected in blood culture, a gram stain test is performed using a primary crystal violet stain to a heat-fixed bacterial culture smear. Gram staining distinguishes the bacteria as gram-positive or gram-negative and provides information about their shape and identity. The gram stain also identifies yeast or fungi.

Gram staining differentiates bacteria by the characteristics of their cell walls, not whether they are aerobic or anaerobic. Bacteria are classified as rod-shaped (bacilli), spherical (cocci), or spiral-shaped (spirochetes). It should be noted that not all bacteria can be classified as gram-positive or gram-negative. Some bacteria are gram-variable or gram-indeterminate. 

Most blood cultures take 24 to 48 hours for sufficient growth to occur. During the 1980s, gram-positive organisms became more common than gram-negative organisms in causing bacteremia.³

Most Common Organisms Found In Positive Blood Cultures

The most common microbes identified in blood cultures are: 

• Staphylococcus aureus 
  • A gram-positive spherically shaped aerobic bacterium. 
  • Part of the body’s microbiota, including the range of organisms that may be commensal, mutualistic, or pathogenic.  
  • It is coagulase-positive, producing coagulase and converting hydrogen peroxide to water and oxygen. 
  • Usually found in blood cultures as a result of a respiratory infection 
  • One of the leading organisms causing antibiotic resistance is methicillin-resistant organisms. 

• Escherichia coli and other members of the family Enterobacteriaceae 
  • A gram-negative, rod-shaped, coliform, facultative anaerobic bacterium. 
  • Usually found in blood cultures due to a lower intestine infection or fecal contamination.
  • Enterobacteriaceae are gram-negative bacteria that include Salmonella, Klebsiella, Shigella, Enterobacter, and Citrobacter. 

• Enterococcus species 
  • These lactic acid bacteria are gram-positive cocci occurring primarily in pairs (diplococci).
  • Facultative anaerobes 

• Pseudomonas aeruginosa 
  • Encapsulated, gram-negative, rod-shaped
  • Aerobic-facultatively anaerobic bacteria 
  • Considered a multidrug-resistant pathogen of severe illnesses, including hospital-acquired (nosocomial) infections such as ventilator-associated pneumonia and various sepsis syndromes.
  • Considered by the World Health Organization as one of the greatest threats to humans because of its antibiotic resistance.² 

• Coagulase-negative staphylococcus 
  • Unable to produce coagulase, an enzyme that causes blood clot formation.
  • Facultative anaerobes from the order of Bacillales.
  • An example is Staphylococcus epidermidis, which is commensal to the skin but can cause severe infections in central venous catheters or immunocompromised patients. 

• Candida albicans 
  • Opportunistic pathogenic yeast
  • Common in the human gut flora
  • It can cause severe infections in immunocompromised patients.

Obligate anaerobic bacteria are found in about 0.5 to 13% of all positive blood cultures.4 These pathogens are causative agents in multiorgan failure and can be responsible for severe, life-threatening infections. They are commonly found in bacteremia as a result of bite wounds and gas gangrene. Anaerobic bacteremia may occur in advanced-age patients and severe immunocompromised states. Mortality is high, ranging from 25% to 44%.⁵ 

Common anaerobes found in bacteremia include: 

• Cutibacterium spp. (formerly known as Propionibacterium) 
  • Part of the pathogenesis of inflammatory acne 
  • Cause of opportunistic infections involving skin and soft tissue, endocarditis device infections, and implant-associated infections associated with biofilm formation in orthopedics, neurosurgery, and ophthalmology. 

• Bacteroides spp. 
  • Components of the bacterial florae of mucous membranes and a common cause of endogenous infections 
  • Responsible for infections in the central nervous system, head, neck, chest, abdomen, pelvis, skin, and soft tissues. 
  • Part of the gut flora, but they are consummate opportunistic pathogens. 
  • Fastidious organisms that make them difficult to isolate and are often overlooked because of their slow growth.
  • Possess immunomodulatory effects that affect the body’s energy balance. 

• Parabacteroides and Bacteroides spp.
  • Part of the digestive system’s microbiota.
  • Responsible for intra-abdominal, vaginal, pilonidal, perianal, and brain abscesses and soft tissue infections. 

• Clostridium spp.
  • These gram-positive bacteria are the causative agents of botulism and tetanus.
  • Clostidioides difficile is a significant cause of diarrhea in patients who have already been on antibiotics such as clindamycin. 

• Fusobacterium spp.
  • These gram-negative bacteria are generally found in the oral cavity, gastrointestinal tract, and female genital tract.
  • It may cause serious, opportunistic infections such as tooth abscesses, periodontal disease, skin ulcers, Lemierre’s syndrome, Crohn’s disease, and Ludwig’s angina.

Differences Between Aerobic and Anaerobic Blood Cultures

Blood cultures are incubated at body temperature to promote the growth of the microorganisms, typically for up to five days, although most pathogens are discovered within the first 48 hours. Slower-growing organisms are usually suspected of cardiovascular infections such as endocarditis. Aerobic pathogens typically cause sepsis, endocarditis, and catheter-related infections.

Manual blood culture systems may be more subjective. The bottles are examined for cloudiness, gas production, frank microbial colony growth, or a change in color from blood digestion, called hemolysis. To increase sensitivity, samples from the bottles are inoculated onto agar plates or subcultured to determine whether there are signs of positive growth.

Automated systems consist of sensors that measure the levels of gases inside the bottle, particularly carbon dioxide. Improvements in blood cultures have led to using the lysis-centrifuge method to detect fungi, mycobacteria, and Legionella. The process involves collecting blood in a tube with a lysis agent to destroy red and white blood cells and then spinning the blood in a centrifuge. Other methods have been developed for rapid identification using genetic techniques such as polymerase chain reaction (PCR) and microassays.

If detected on blood culture, anaerobic bacteria are usually responsible for severe infections. These include intra-abdominal abscesses, pelvic inflammatory disease (PID), dental or head and neck infections, and aspiration pneumonia. Typically, anaerobic infections are caused by a disruption of the mucosal surface and entry of the anaerobic bacteria with deep tissue invasion. Timely identification of anaerobic bacteremia is vitally essential to begin empiric therapy since many of these cases progress to local abscesses, sepsis, and even septic shock. 

Blood Culture Collection and Transport

Diagnosing bloodstream infections depends on clinical and technical factors in collecting and transporting blood culture tubes. Efficient, accurate, and rapid diagnosis of bacteremia and fungemia leads to the prompt administration of the most appropriate, targeted antibiotic therapy, preferably within 24- 48 hours. The treatment goals include optimal clinical outcomes, reducing mortality and costs, and mitigating the development of antibiotic resistance.

The preclinical process of obtaining blood cultures includes: 

• Timing and preparation for blood culture collection.
  • Blood cultures should be collected as soon as possible and before other lab tests.
  • The procedure should be performed carefully under standardized, sterile conditions using peripheral venipuncture, and maintaining optimal aseptic no-touch technique throughout the entire procedure.
  • Aerobic bottle for aerobic bacteria.
  • Anaerobic bottle for anaerobic bacteria.
  • Mask both the patient and health care provider. 

• Skin asepsis.
  • 2% chlorhexidine and IPA (or povidone-iodine) in 70% isopropyl alcohol.
  • Disinfect 6-7 cm skin area for 30 seconds- let dry for at least 30 seconds. Povidone iodine takes longer to dry. 

• Blood volume is the most critical parameter for the detection of organisms.
  • A blood culture set includes an aerobic and an anaerobic bottle of 16-20 mL total. Two sets consist of 4 bottles, each with 8-10 mL of blood for most bottle manufacturers. Two sets should always be drawn from two different sites.

• Sampling method and staff safety.
  • The stopper of each blood culture bottle is not sterile and must be disinfected before inoculation with blood for 15 seconds with IPA and should be covered with a sterile cover to maintain asepsis.
  • Divert and sequester 1mL of the first aliquot of blood. Use of ISDDs have been shown to reduce blood culture contamination rates to less than 1%.
  • The aerobic bottles should be filled first.
  • Blood samples such as CBC, chemistry, or other studies should be filled after blood cultures. 

• Medium to be used.
  • Both aerobic and anaerobic bottles should be used.
  • Fungal media is optional. Most laboratories don’t use mycosis media since most cases of candidemia can be obtained using aerobic media if volume is sufficient. 

• Time to blood culture transportation.
  • Prompt transport time ensures optimal recovery of microorganisms. Within two hours is optimal, and four hours is the maximum acceptable limit.
  • If available, blood culture bottles should be placed in satellite incubators, and no refrigeration should be used. 

• Quality assurance and quality management.
  • Mark all necessary information on the vials, including patient data, number of samples, sampling site, time, and date.
  • Indicate execution of blood cultures in the patient’s medical record.

Interpreting Blood Culture Results

Bloodstream infection remains a worldwide concern. Positive blood cultures indicate the presence of bacteria in the bloodstream, but the patient’s clinical assessment and additional laboratory testing determines if sepsis or septic shock is present. The identified type of bacteria provides valuable clinical guidance for appropriate antibiotic therapy.

he American Society for Microbiology (ASM) and the Clinical Laboratory Standards Institute (CLSI) have recommended that an overall blood culture contamination rate should not exceed 3%.⁶ Despite this recommendation, approximately 40% of positive blood cultures are false positives due to contamination. The ASM and Infectious Diseases Society of America (IDSA) 2024 Guidelines state that blood cultures contaminated with skin microbiota during collection are common, but laboratories should be able to achieve contamination rates below 3% with target rates of 1% when best practices are used.⁴

Conclusion

• Blood cultures are vital diagnostic tools for identifying pathogens responsible for patient infections.
• Blood cultures should be obtained before beginning antibiotic therapy.
• Accurately identifying pathogens improves clinical management, decreases costs, and reduces antibiotic resistance.
• Understanding the differences between aerobic and anaerobic blood cultures helps provide correct practice in the pre-analytic stage of blood culture collection.
• Proper collection protocols, transport, and handling of blood culture bottles are essential.
• Antibiotic stewardship and infection prevention personnel should learn and understand the differences between aerobic, anaerobic, and fungal blood culture media, why reducing blood culture contamination risks is critical, and provide proper patient data tracking.

References

¹Lafaurie, M., d’Anglejan, E., Donay, J. L., Glotz, D., Sarfati, E., Mimoun, M., Legrand, M., Oksenhendler, E., Bagot, M., Valade, S., Bercot, B., & Molina, J. M. (2020). Utility of anaerobic bottles for the diagnosis of bloodstream infections. BMC infectious diseases, 20(1), 142. https://doi.org/10.1186/s12879-020-4854-x 

²Spagnolo, Anna Maria; Sartini, Marina; Cristina, Maria Luisa. Pseudomonas aeruginosa in the healthcare facility setting. Reviews in Medical Microbiology 32(3):p 169-175, July 2021. | DOI: 10.1097/MRM.0000000000000271   

³Mahon, C.R., Lehman, D.C., Manuselis, G. (2014). Textbook of Diagnostic Microbiology (5th ed.). New York: Saunders. Pp.865-6. 

⁴Márió Gajdács, Marianna Ábrók, Andrea Lázár, Gabriella Terhes, Edit Urbán, Anaerobic blood culture positivity at a University Hospital in Hungary: A 5-year comparative retrospective study, Anaerobe, Volume 63, 2020, 102200, ISSN 1075-9964, https://doi.org/10.1016/j.anaerobe.2020.102200. 

⁵O. Opota, A. Croxatto, G. Prod’hom, G. Greub, Blood culture-based diagnosis of bacteraemia: state of the art, Clinical Microbiology and Infection, Volume 21, Issue 4, 2015, Pages 313-322, ISSN 1198-743X, https://doi.org/10.1016/j.cmi.2015.01.003. 

⁶Doern, G. V., Carroll, K. C., Diekema, D. J., Garey, K. W., Rupp, M. E., Weinstein, M. P., & Sexton, D. J. (2019). Practical Guidance for Clinical Microbiology Laboratories: A Comprehensive Update on the Problem of Blood Culture Contamination and a Discussion of Methods for Addressing the Problem. Clinical microbiology reviews, 33(1), e00009-19. https://doi.org/10.1128/CMR.00009-19