CO2 and its role in reducing infection

Carbon Dioxide and its Place in Fighting Surgical Site Infections and Antimicrobial Resistance. Where it all started.

• Carbon dioxide has been used as a means of preserving liquid and solid foodstuff since the 1930s, owing to its inhibitory effect on bacterial growth. (Dixon and Kell., 1989)  (Haas et al., 1989) (Wei et al., 1991).  The first observations made on the effect of carbon dioxide on retarding bacterial growth were made in the late 1800s. (Frankel., 1889) (Gill & Tan., 1979)

• The scientific community became more interested in the microbiology of meat during the 1950s when meat products began to be shipped for longer distances due to the wide spread growth of the supermarket trade. (Nychas et al.,2008) Atmospheres of up to 100% CO2 have been shown to inhibit the growth of meat spoilage bacteria. The shelf life of meat can be extended by an increase in the concentration of carbon dioxide in the storage atmosphere (Stanbridge and Davies., 1998). 

• The packaging conditions and the gaseous composition of the atmosphere surrounding the meat greatly influence the composition of spoilage flora. (Borch et al., 1996) (Sechi et al., 2014) (Rossaint et al., 2015). Carbon dioxide is included for its inhibitory effect and to retard the growth of organisms produced by aerobic spoilage. (Adams and Moss., 1995) ( Gill 2003). 
• The bacteriostatic effect of carbon dioxide has been subject to much debate over the years. Originally it was thought that the effect was a result of oxygen displacement, or intracellular acidification. However, both of these theories have been discounted. (Daniels et al., 1984) 

Carbon dioxide in the clinical setting

• CO2 is widely used for laparoscopic colorectal surgery (LCS) and endoscopic vein harvesting procedures. When the LCS procedures are compared to open colorectal surgery (OCS) they have been shown to develop 40% less surgical site infections (SSI). (Aimag et al., 2011)  (Richards et al., 2003)

• Richards et al analysed results from 54,504 inpatient cholecystectomy procedures undertaken between 1992 and 1999. During this period the use of the laparoscopic technique, as opposed to open cholecystectomy, increased from 59% in 1992 to 79% in 1999. The laparoscopic technique is less invasive, requires shorter hospitalisation and is associated with faster recovery rates when compared to open cholecystectomy. Carbon dioxide is the most widely used gas during laparoscopic procedures. The risk of surgical site infections was lower in patients undergoing the laparoscopic technique, although infecting organisms were found to be similar. Inhibition of bacterial growth has been reported for the pathogen Staphylococcus Aureus.(Persson et al., 2004) In this study SSI data was collected for 342 patients, of which 116 had undergone laparoscopic technique with the remaining 226 undergoing an open procedure. The risk of SSI was significantly lower for laparoscopic cholecystectomy.

• A study of 122 patients (LCS 43, OCS 79), who underwent colorectal resections over a 12 month period in the UK, looked at the difference in infection rate between the laparoscopic and open procedures. The patients’ demographic and operative case-mix were similar for both groups. Infection rates for LCS were 7% whilst OCS infection rates were 25%. This translates to LCS infection rates being 72% lower than OCS over that period. (Howard et al., 2010) CO2 is used as the distension gas.

•  Further studies have found low infection rates in laparoscopic procedures, an example being a study of 670 patients in Japan, which found that rates of surgical site infections for laparoscopic surgery for colon cancer were 4%. (Nakamura et al., 2016).

• It is thought that the lower rates of SSIs in LCS compared with OCS procedures may be due to the bacteriostatic effect of carbon dioxide. (Persson et al., 2004) This study investigated the effect of CO2 on staphylococcus aureus at body temperature and at the difference between having S. aureus inoculated on blood agar and then exposed to either; anaerobic gas (5% CO2, 10% H20 and 85% N2), air or 100% CO2, at 37°C and over a 24hr period. The number of  S. aureus on blood agar was 100 times lower for the CO2 plate than the anaerobic one, and 1000 times lower for CO2 than the plate exposed to air. 

• "the number of bacteria was 100 times lower with CO2 than with air after 4 h and 1,000 times lower after 8 h" Persson et al., 2004

• "It is possible that CO2 inhibits not only bacteria but also immunological active cells, such as macrophages in a wound." Persson et al., 2004

• In broth there were fewer bacteria with CO2 than with air. After 2h the number of bacteria had increased with the air but not with the CO2. After 8h, the optical density measurement with air had increased from 0 to 1.2; the CO2 saw an increase to 0.01. 

• Staphylococcus Aureus accounts for 21% of SSIs detected by the Surgical Site Infection Surveillance Team in the United Kingdom (Public Health England, 2018) 

• The report analyses the rate of surgical site infections (SSIs) for 134,119 procedures including 105,771 from mandatory orthopaedic surveillance and 28,348 from 13 other voluntary surveillance categories. Surveillance is carried out yearly in the UK from April to the following March. A total of 201 hospitals reported data on varying procedures in 2017/18. 1,338 surgical site infections were detected during the inpatient stay or on readmission following the initial operation. It is interesting to note the difference between coronary artery bypass graft (CABG) procedures and those referred to as ‘Cardiac (non-CABG)’ procedures. Rates for SSIs after Coronary Artery Bypass Grafts (CABG) are more than double that of the Cardiac, non-CABG procedures ( these are predominantly heart valve surgery procedures). Both of these procedures are performed in the same theatres, by the same surgical teams. Both procedures entail patients undergoing sternotomy, therefore surgical wound sizes are very comparable. In the UK carbon dioxide is routinely used during heart valve surgery, however, it is not commonly used during CABG procedures. The rate of infection (inpatient only) for CABG procedures was reported at 2.12% of the 29,335 operations reported on since April 2013 , compared with 0.87% of the 16,706 other cardiac procedures. For in-patient and re-admission rates we see an increase to 3.5% of CABG procedures and 1.3% for other cardiac procedures. (Public Health England, 2018) This report highlights that in the UK you are more than twice as likely to acquire an SSI from a CABG procedure than you are from other, cardiac procedures. The main difference being that in the non CABG procedures CO2 is usually insufflated into the thoracic cavity before the surgical site is closed up. 

• A review on the effects of carbon dioxide on environmental microbes (Yu and Chen., 2019) further supports the finding that carbon dioxide has a bacteriostatic effect. The anti-microbial potential of using high pressure CO2against Gram-positive and Gram-negative bacteria was investigated using Pseudomonas aeruginosa and Bacillus subtilis. The survival ratio of both bacteria was reduced by seven orders of magnitude (Spilimbergo et al., 2002). High-pressure CO2 treatment is considered a promising sterilization technology because it will cause bacterial inactivation (Zhang et Al., 2006). Oceanic CO2 has been found to influence a deep sea isolate (Vibrio alginolyticus) by significantly suppressing the bacterial growth under 10.4mM CO2.  By using a CO2 injection system, Borrero- Santiago et al (2016) simulated a carbon dioxide leakage from a stable sub-seabed. They observed inhibited growth and a reduction in total cell number, that was concurrent with a lowering of the pH. This suggests that the higher concentrations of CO2 experienced by the microbes caused a reduction in population. 

• Anti-microbial resistance (AMR) is the ability of an organism to withstand treatment from antimicrobials such as antibiotics. Antibiotic resistance causes particular problems for blood stream infections (BSIs), such as gonorrhoea and tuberculosis (TB). The total number of antibiotic-resistant BSIs has increased by 35% from 2013 to 2017. (Public Health England ESPAUR Report, 2018) 
• It is widely known that a higher proportion of gram negative bacteria are resistant to antibiotics than gram positive bacteria. This is due to the outer membrane (OM) present in gram negative bacteria. The OM is impermeable except to the necessary nutrients which traverse the wall by diffusion through protein channels know as porins.
• A study into the effect of carbon dioxide on the growth of a common, gram negative bacteria, Pseudomonas fluroescens, found that carbon dioxide inhibits cell respiration. The author also looked at the difference between the inhibitory effect found when the bacteria was grown in the presence of CO2 as opposed to being grown in air. The inhibitory effect was the same whether cells had been grown in air or in the presence of CO2. (Gill Tan., 1979) This indicates that adaptive enzyme synthesis does not occur in response to CO2, further suggesting the benefits of using CO2 to inhibit bacterial growth.

• Enterobacteriaceae were responsible for 53% of SSIs in large bowel surgery from April 2016 to March 2017 (Public Health England 2018) .They were also responsible for 29% of SSIs in CABG surgery. Would insufflation of CO2 during CABG procedures lower the infection rate?

• The latest public health England report has found that the common pathogens found in SSIs include; enterobacteriaceae, pseudomonas, staphylococcus, MRSA and MSSA. Enterobacteriaceae made up the largest proportion of causative organisms in 2017/18 across all surgical categories. They accounted for 11.1% of knee replacement SSIs and 53% of SSIs from large bowel surgery. Overall the rate of SSIs attributed to Enterobacteriaceae averaged at 30.8% across all categories recorded in 2017/18/ The prevalence of enterobacteriaceae increased by 8% from 2016/17 to 2017/18, however the greatest relative increase in the proportion of causative organisms was for coagulase-negative staphylococci. This a gram positive bacteria strain, also known as CoNS.

High-pressure CO2 has been known to increase membrane permeability by introducing pores in the cell wall, therefore destroying the integrity of the cellular structure (Garcia-Gonzalez et al., 2010; Hong and Pyun., 2001) The damaging effect of pressurized CO2 on cell membranes has been demonstrated as having a direct relationship with inactivation of E.coli (Yao etal., 2014).Progressively longer exposure to CO2 has been linked with morphological changes (Wu et al, 2016).  Biophysical effects of supercrticial CO2 will lead to a significant perturbation of membrane architecture in E.coli because it can dramatically decrease the phosphatidyglycerol (PG) membrane lipid which plays an important role in membrane stability (Tamburini etal., 2014).

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