Pyocyanin (PCN) is a blue–green pigment mainly produced by Pseudomonas aeruginosa. It has antimicrobial effects on a variety of organisms. Ampicillin (AMP) and cefotaxime (CTX) are widely used against bacterial pathogens as important antibacterial agents. The effect of PCN on the antibacterial action of AMP and CTX has been studied. PCN from P. aeruginosa was extracted by chloroform method. Two PCN concentrations (2 and 4 µg/ml) were combined separately with a variety of AMP and CTX concentrations. Change in the antibacterial activity of AMP and CTX against 14 isolated pathogenic bacteria after combination has been investigated. The antibacterial activity of AMP decreased against 7 bacterial isolates when mixed with 4 µg/ml of PCN, while this activity increased against the majority of bacteria after mixing with 2 µg/ml. CTX activity was also reduced against 12 bacterial isolates by 4 µg/ml of PCN, while it was not generally affected by 2 µg/ml. in conclusion; PCN had variable effects on the antibacterial activity of AMP and CTX, which varied mainly between decreased activity and absence of effects. A low concentration of PCN was more effective in increasing the action of AMP, but had no effect on the action of CTX.
Research Article
Potential effects of pyocyanin on the antibacterial action of ampicillin and cefotaxime against human pathogenic bacteria
https://doi.org/10.21203/rs.3.rs-2074872/v1
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ampicillin
cefotaxime
antimicrobial
bacteria
pyocyanin
Pyocyanin (PCN) is a blue–green colored pigment produced by various microorganisms as a phenazine derivative (Özyürek Gür and Bilkay, 2016; O’Malley, Reszka and Britigan, 2004). P. aeruginosa is the most common PCN-producing bacterium (Özyürek et al., 2016; Karpagam Sudhakar and Lakshmipathy, 2013), which acts as one of its virulence factors (Rodŕguez-Rojas et al., 2009). The antimicrobial action of PCN is demonstrated by numerous studies against the growth of a number of bacterial and fungal species (El-Shouny and Hamza, 2011; Gharieb et al., 2013). As an antibacterial agent, PCN is more effective on Gram-positive bacteria than on Gram-negative bacteria (El-Shouny et al., 2011; Gharieb et al., 2013). Staphylococcus aureus, Bacillus licheniformis, and Bacillus subtilis, as Gram-positive bacteria, were found to be more susceptible to PCN than Gram-negative bacteria like Escherichia coli (El-Shouny et al., 2011). Ampicillin, an antibiotic derived from penicillin by the addition of an amino group to the benzylpenicillin molecule, is broadly active against bacteria by inhibiting microbial cell wall synthesis (Raynor, 1997). It inhibits 50–60% of clinical Gram-negative isolates (Shibl, 1994). Resistance to ampicillin has recently increased in coliform bacteria (Raynor, 1997).
Cefotaxime is a third generation cephalosporin with broad-spectrum antibacterial activity against both aerobic and anaerobic Gram-positive and Gram-negative bacteria (Carmine et al., 1983). It has the capability of penetrating the bacterial cell wall with high β-lactamase stability and has a high affinity with important target enzymes (Shrinner et al., 1980). It is also more suitable for use than ampicillin or the first and second generation of cephalosporins because of its activity against multidrug-resistant Enterobacteriaceae (Carmine et al., 1983).
The effect of PCN on the antibacterial action of ampicillin and cefotaxime has been investigated against a variety of human pathogenic bacteria.
Microorganism isolation
Pseudomonas aeruginosa strain MH382164 registered in GenBank was used as a producer of PCN. Other strains of bacteria were isolated from patients suffering from a variety of diseases upon admission to Al-Kafeel Hospital in Karbala, Iraq, in December 2017. A number of samples, including urine, skin swabs, stool and oral swabs, were collected to isolate the bacteria. Bacterial samples were cultivated on Nutrient agar, Blood agar and MacConkey agar and incubated at 37°C for 24 hours. The morphological characteristics of cultured bacteria were diagnosed as a first step followed by the application of API-20 (BioMérieux, France). Enterobacteriaceae members were diagnosed by API-20E medium, Staphylococcus species by API-20 Staph medium, and Streptococcus species byAPI-20 Strep medium. A total of 14 strains of bacteria were diagnosed consisting of 8 strains of Gram negative (Acinetobacter baumannii, Citrobacter freundii, E. coli, Enterobacter cloacae, Klebsiella pneumoniae, Proteus mirabilis, Salmonella species, and Shigella species) and 6 strains of Gram positive (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae and Enterococcus faecalis).
Pyocyanin (PCN) production
P. aeruginosa was grown in 150 ml of sterilized Luria-Bertani medium (LB) prepared by dissolving 10 g of peptone, 10 g of NaCl and 5 g of yeast extract in 1 liter of D.W. and incubated at 37°C for 1–5 days. Turning media to green is a good indicator for PCN production.
Extraction of PCN
PCN in medium broth culture LB was purified from growing bacteria by filtering through the filter paper as a first step and then centrifuged at 6000 RPM for 10 min. The supernatant was further filtered with a 0.2 µm microfilter to obtain bacterial-free PCN. Chloroform was added to extract PCN from liquid media at a ratio of 1:1. The brown top layer was thrown away, while the second layer with the blue color was retained to obtain PCN. The process was repeated 2–3 times, after which the extracted PCN was dried in an oven at 50°C to evaporate the chloroform. The concentration of PCN was determined by measuring the absorption of the PCN solution by a spectrophotometer at 520 nm in accordance with the following formula (El-Fouly et al., 2015):
Concentration of PCN (µg/ml) = O.D520× 17.072.
where:
O.D520: optical density at 520 nm
Effects of PCN on the activity of antibacterial agents
The effect of PCN on the antibacterial activity of ampicillin (AMP) and cefotaxime (CTX) (Strides Pharma science KRSG, Karnataka, India) against isolated bacteria was measured as minimum inhibitory concentration (MIC) using a CLSI dilution method (M07-A10) (2015). Isolated bacteria were sub-cultured in Mueller-Hinton Broth (MHB) (Himedia, India) for 24 h at 35°C. The PCN was used in two concentrations (2 and 4 µg/ml) to mix with different concentrations of each antibacterial agent. Each concentration of PCN was mixed with 5 concentrations of AMP (0.125, 2,4,6, and 8µg/ml) or CTX (0.625, 10,15,20, and 25 µg/ml). A stock solution of antibacterial agent was prepared by the following formula:
where the potency of ampicillin is 10 µg/ml and that of cefotaxime is 30 µg/ml.
An inoculum from each bacterial isolate was prepared with 1–2×108 cfu/ml after adjusting with a standard 0.5 McFarland solution. A plastic microdilution plate (96 well) was used to determine the MIC value for a combination. A concentration of 100 µl of each standardized bacterial count in MHB was added in one well, followed by a concentration of 100 µl of each combination. Multiple controls were used in a microdilution plate, including MHB with or without bacteria, PCN, AMP or CTX alone, a mixture of PCN with a bacteria-free antibacterial agent and distilled water. A sheet of aluminum was used to cover the inoculated plate to prevent drying of the contents and contamination during incubation periods. The results were visually read to determine whether bacterial growth was present or absent.
The antibacterial activity of ampicillin and cefotaxime was measured after mixing with two PCN concentrations. The effect of 4 µg/ml PCN on AMP antibacterial activity increased its MIC for seven bacterial isolates, including six Gram-negative bacteria and one Gram positive (S. aureus). Meanwhile, the concentration of 2 µg/ml of PCN increased only the ampicillin resistance of A. baumannii by increasing the MIC value to more than 8 µg/ml (Table 1). An increase in AMP activity for isolated bacteria was very clear when mixed with 2 µg/ml PCN, while such action was shown only in S. pyogenes after mixing at 4 µg/ml. However, no changed in the antibacterial action of AMP after combination with 4 µg/ml of PCN was shown on five bacterial isolates, including four strains of Gram positive and one of Gram negative (A. baumannii), while ampicillin with 2 µg/ml PCN had no such effect (Table 1).
The MIC value of CTX for twelve bacterial isolates was increased in the presence of 4 µg/ml PCN, while only four showed an elevated MIC at 2 µg/ml PCN. Meanwhile, the value of MIC of CTX that combined with 4 µg/ml against A. baumannii and Shigella spp. as well as that combined with 2 µg/ml against nine isolates of bacteria revealed no change in this value compared with CTX alone (Table 2). A decrease in antibacterial activity of CTX was observed only against E. faecalis after combination with 2 µg/ml of PCN (Table 2).
The PCN is a blue to green soluble pigment mainly produced by P. aeruginosa, a Gram negative, motile, and rod bacteria (El-Fouly et al., 2015). It is immediately secreted in solid culture media leading to the blue color, whereas in liquid media it appears with a dark green color (Gahlout et al., 2017; Priyaja, 2012). The main function of PCN to P. aeruginosa is elimination of other competing microorganisms (Mathew et al., 2011). Gram-positive bacteria are considered to be more susceptible to antimicrobial activity of PCN compared to the Gram-negative group (El-Shouny et al., 2011; Gharieb et al., 2013; Jameel et al., 2017). This was noted when Staphylococcus aureus showed lower MIC value (20 µg/ml) of PCN than E. coli (50 µg/ml) (El-Fouly et al., 2015).
Combining PCN with other chemical agents may increase or decrease the antimicrobial effect of each of them relative to when they were used alone. Silver nanoparticles with PCN showed synergic antimicrobial effects on three bacterial species (Staphylococcus aureus, E. coli, and Pseudomonas aeruginosa) and one yeast (Candida albicans) (Nowroozi and Rashnonejad, 2012). The antibacterial activity of the PCN on Staphylococcus aureus increased after being combined in equal volume with bacteriocin (50 µl/50 µl) at 72 h of contact, while action against E. coli decreased after mixing 30 µl of PCN with 70 µl of bacteriocin within 24 h of contact (Markraphael et al., 2017).
In this study, the role of the PCN in the activity of two antibacterial agents (ampicillin and cefotaxime) was examined in isolated bacteria. The combination of ampicillin with 4 µg/ml PCN decreased ampicillin activity against most isolated bacteria through an increase in the ampicillin MIC value. Reduced antibacterial action of ampicillin in combination with different compounds was also mentioned in other studies. The bactericidal activity of ampicillin for group B Streptococcal meningitis was limited by chloramphenicol (Weeks and Baker, 1981). This could be related to the fact that chloramphenicol acts as a bacteriostatic agent, while ampicillin is a bactericide agent and combining both antibiotics will reduce the activity of the other (Ocampo et al., 2014). The water alcohol extract from various parts of the plant Passiflora cincinnata showed antagonistic effects with ampicillin on Staphylococcus aureus and E. coli (Siebra et al., 2018). Furthermore, certain types of Agrimonia eupatoria L. extracts showed antagonistic effects on ampicillin activity when tested for E. coli (Muruzović et al., 2017). Although Streptococcus pyogenes is resistant to ampicillin (Evans et al., 1994), this study revealed that it is more sensitive to a combination of ampicillin and high concentrations of PCN.
The inhibitory effect of PCN on ampicillin activity was significantly reduced following a reduction in PCN concentration to 2 µg/ml. Thus, a low concentration of PCN may be considered useful to improve the antibacterial effect of ampicillin. This type of combination will provide a promising indication for the use of ampicillin against resistance bacteria. Acinetobacter baumannii showed high resistance to combining low concentration of PCN with ampicillin. The bacterium is known to be multi-drug resistant to most antibiotics, including ampicillin (Ibrahim et al., 2000). This resistance capacity of this bacterium is related to its content of broad-spectrum beta-lactamase (BSEL) genes (Jayaseelan and Dharmarai, 2014).
As noted with ampicillin when combined with PCN, the antibacterial activity of cefotaxime was also inhibited against some bacterial isolates. Meanwhile, there was no PCN activity on the MIC value of cefotaxime for most other species when used at high or low concentrations. The antibacterial effect of cefotaxime has been shown to decrease in combination with other agents. Chloramphenicol as a bacterial agent reduced cefotaxime activity in 26 clinical isolates of Gram-negative rods, group B streptococci, and Staphylococcus aureus (Asmar and Dajani, 1988). Cefotaxime activity against 24 Propionibacterium acne strains also decreased as a result of the antagonistic action of linezolide, ciprofloxacin or clindamycin (Mory et al., 2005). Aminoglycosides may also have inhibitory effects on the action of cefotaxime, but such results have not been clear (Carmine et al., 1983).
On the other hand, PCN showed no effect on antibiotic activity, as indicated by some bacterial strains in which they were not affected by a combination of PCN with ampicillin or cefotaxime compared with any of these antibiotics alone. This finding was quite clear when combining a low concentration of PCN with cefotaxime. Matrine agent showed no effect on the antibacterial activity of cefotaxime against Klebsiella pneumoniae in comparison with the synergistic effects of baicaleine and clavulanic acid (Ibrahim et al., 2000).Combination of ampicillin with the water extract of Agrimonia eupatoria L. exhibited no antibacterial effects on P. aeruginosa (Muruzović et al., 2017).
The main mechanism for the action of the PCN on organisms depends mainly on altering the normal transport of electrons in the respiratory chain and the formation of oxygen-free radicals (Ran and Lau, 2003). The PCN also has the ability to pass through the cell membrane of the organism and reacts with NADH and NADPH leading to its conversion to reduce form (O’Malley et al., 2004). Thus, PCN will provide any agent which combined with an increased ability to kill a target organism. Conversely, the decline in antibacterial activity of agents combined with the PCN may be related to the antagonistic effects between these components. However, the stability of antibacterial activity of any agent after combining with PCN is most likely related to competition in antibacterial action between these combatants when the action of one will impede the action of another.
In conclusion; PCN had variable effects on the antibacterial activity of ampicillin and cefotaxism, which ranged mainly from decreased activity to no effects. A low concentration of PCN was most effective in enhancing the action of ampicillin without effect on cefotaxime.
Funding: There is no funding for this research
Conflict of interesting: The authors state that there is no conflict of interest.
Availability of data and material: Not applicable
Code availability: Not applicable
Author contributions: Conceptualization: A.A.A.; Methodology: J.K.M.; Formal analysis and investigation: A.A.A.; Writing: A.A.A.; Resource: J.K.M: Supervision: A.A.A.
Consent to participate: Not applicable
Consent for publication: Not applicable
Declaration of patient consent: All of the involved subjects were volunteers and signed a consent form.
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Table (1): Minimal inhibitory concentration (MIC) of ampicillin combined with pyocyanin
|
Bacterial isolate |
MIC (µg/ml) |
|||||
|
PCN (2 µg/ml) |
PCN (4 µg/ml) |
|||||
|
AMP
|
PCN |
PCN and AMP |
AMP
|
PCN |
PCN and AMP |
|
|
S. aureus |
6 |
- |
0.125 |
6 |
4 |
8 |
|
S. epidermidis |
6 |
- |
0.125 |
6 |
4 |
6 |
|
S. pneumoniae |
6 |
- |
0.125 |
6 |
4 |
6 |
|
S. pyogenes |
8 |
- |
0.125 |
8 |
4 |
4 |
|
S. agalactiae |
8 |
- |
0.125 |
8 |
4 |
8 |
|
A. baumannii |
8 |
- |
> 8 |
8 |
4 |
8 |
|
C. freundii |
4 |
- |
0.125 |
4 |
4 |
6 |
|
E. faecalis |
6 |
- |
0.125 |
6 |
4 |
6 |
|
E. cloacae |
8 |
- |
0.125 |
8 |
4 |
> 8 |
|
E. coli |
4 |
- |
0.125 |
4 |
4 |
> 8 |
|
K. pneumoniae |
6 |
- |
0.125 |
6 |
4 |
8 |
|
P. mirabilis |
6 |
- |
0.125 |
4 |
4 |
8 |
|
Salmonella spp. |
6 |
- |
0.125 |
4 |
4 |
8 |
|
Shigella spp. |
6 |
- |
0.125 |
4 |
4 |
8 |
(-): no inhibition
PCN; pyocyanin, AMP: Ampicillin
Table (2): Minimum inhibitory concentration (MIC) of cefotaxime combined with pyocyanin
|
Bacterial isolate |
MIC (µg/ml) |
|||||
|
PCN (2 µg/ml) |
PCN (4 µg/ml) |
|||||
|
CTX
|
PCN |
PCN and CTX |
CTX
|
PCN |
PCN and CTX |
|
|
S. aureus |
10 |
- |
15 |
10 |
4 |
25 |
|
S. epidermidis |
15 |
- |
15 |
15 |
4 |
25 |
|
S. pneumoniae |
10 |
- |
10 |
15 |
4 |
25 |
|
S. pyogenes |
20 |
- |
20 |
20 |
4 |
25 |
|
S. agalactiae |
10 |
- |
10 |
15 |
4 |
25 |
|
A. baumannii |
20 |
- |
> 25 |
20 |
4 |
20 |
|
C. freundii |
10 |
- |
15 |
10 |
4 |
25 |
|
E. faecalis |
15 |
- |
10 |
15 |
4 |
20 |
|
E. cloacae |
15 |
- |
15 |
15 |
4 |
20 |
|
E. coli |
10 |
- |
15 |
10 |
4 |
25 |
|
K. pneumoniae |
15 |
- |
15 |
10 |
4 |
25 |
|
P. mirabilis |
15 |
- |
15 |
15 |
4 |
25 |
|
Salmonella spp. |
15 |
- |
15 |
15 |
4 |
20 |
|
Shigella spp. |
15 |
- |
15 |
20 |
4 |
20 |
(-): no inhibition
PCN; pyocyanin, CTX: cefotaxime
No competing interests reported.