Understanding Pseudomonas Aeruginosa Resistant Pathogen Strains

Pseudomonas aeruginosa is a bacterium that has developed multi-drug resistance in many health care facilities. Resistance has arisen due to a low permeable outer cell membrane and the presence of efflux- pumping mechanisms. It is a problem particularly in patients with cystic fibrosis or burn wounds. Infection if not treated can lead to systemic sepsis which can result in organ failure and death. Currently research is being done to see how to develop an antibiotic that will effectively eliminate the bacterium. Previous research has indicated that preventing the development of a biofilm might help in increasing permeability and increasing access of antibiotics into the cell. The use siderophores conjugated with an antibiotic is also being researched as another possible treatment method.

In the last several decades antibiotic resistant microbes have increased in the severity of resistance, especially in health care facilities. Resistant bacteria have become a greater threat because microbes have become not only resistant, but have developed multi-drug resistance. This makes treatment more difficult because it becomes harder to find a remedy that is able to effectively kill the bacteria without also harming uninfected cells. Gram negative Pseudomonas aeruginosa resistant strains are one particular type of bacteria that is becoming a growing problem in hospitals and is a big contributor to nosocomial infections (1). Specifically the bacteria can be found in those with cystic fibrosis and burn wound patients. This resistance can be acquired when exposed to antibiotics, multi-drug resistance to the widely used imipenem and profloxacin specifically, has been observed (2,3). In general these resistance strains of P. aeruginosa do not instantly arise in patients, over time resistance emerges and then becomes more prominent due to certain risk factors that increase the probability of acquiring a resistant strain. Some of these risk factors are long hospital stays which can lead to cross-transmission, the use of broad spectra antibiotics, or exposure to P. aeruginosa that is either susceptible, or resistant to antimicrobials.

P. aeruginosa infection is a concern because it can spread throughout the body and cause a systemic inflammatory response, multiple organ failure and death (6). It is hypothesized that the sepsis is caused when P. aeruginosa sheds syndecan 1. Syndecan 1 is a proteoglycan that is attached to the cell surface, when it is cleaved from the cell surface proteoglycan ectodomains are released which affects the immune systems defensive ability (6). In healthy individuals syndecan 1 is shed in low undetectable levels. In burn wounds though the level dramatically increases, it is this increase that is suspected to be the problem.
Mice were used as subjects to study the effects of high levels of syndecan 1 shedding. Some of the mice had mutations that prevented them from shedding syndecan 1. These syndecan 1 shedding negative mice were compared with syndecan 1 shedding positive wild type mice in response to the virulence of P. aeruginosa. The syndecan 1 shedding negative mice were found to be less susceptible to infection. This did not inhibit the growth of the bacterium, but fewer syndecan 1 negative mice became infected. Those that did become infected did not develop sepsis. The infection did not spread beyond the primary infection site (6).
The research done indicates sydecan 1 shedding does play a role in P.aeruginosa infection and sepsis. Syndecan 1 shedding negative mice displayed overall a decrease in mortality, infection and sepsis than compared with the wild type syndecan 1 shedding positive mice (6). The decrease of virulence of P. aeruginosa may be because syndecan 1 shedding is involved in perivascular cuffing (6). Without a blood supply infection can easily flourish because the body’s immune system is unable to transport the needed immune system mediators to the burn wound. The infection can grow and eventually can lead to systemic sepsis. If syndecan 1 shedding can be prevented though, the blood supply is still in contact with the immune system and the body’s own immune system can fight against the infection. An understanding of the role of syndecan 1 shedding and its relationship with P. aeruginosa sepsis leads to the possibility of using syndecan 1 shedding neutralizing agents as a treatment for serious burn wounds (6).
In Brazil a study was done on P.aeruginosa found in burn patients. Resistance to ciprofloxacin and imipenem or both arose in 41% of the patients participating in the study. Imipenem, ciprofloxacin or both have been administered to most of the patients for 10 or more days. The resistant strains were isolated as soon as 10 days (3). When the isolated strains were studied and molecular typed it was found that the susceptible and resistant strains for each patient were not related (3). Since the two strains were not related it could be hypothesized that the resistance did not occur due to the bacteria instantly developing its own new mechanisms. It is suspected that resistance emerged due to random mutation and this mutation was then passed to other cells and spread from patient to patient. Each strain in a patient did not develop resistance, but acquired it from another strain. Microbials help select for the resistant cells by eliminating all susceptible organisms until only the mutated strain remains (3).In P. aeruginosa resistance may be partly due to the impermeability of the outer membrane and multi-drug efflux mechanisms (4).
P. aeruginosa is capable of producing a biofilm. A biofilm is secreted by certain bacteria and is composed of an exopolysaccharide matrix. This biofilm can act as a barrier against antimicrobial agents. It can also block out components of a host’s own immune system. This leaves the host defenseless against the bacterium. A biofilm can also protect “persistor cells” that can later repopulate the biofilm leading to chronic infections (5). P. aeruginosa was isolated from a wound and grown in vitro in order to determine a specific time line in forming a mature biofilm. The bacterium was grown in trypic soy broth with cover slips present in the broth. These slips were removed at specific hour intervals starting at three hours and ending at 24 hours. The cover slips where then dyed and observed using light microscopy.
A biofilm first started to form around five hours after inoculation. After 10 hours a biofilm has formed a dense coating around the bacterium. Once a biofilm has completely formed disruption of the film is much more difficult, therefore the best time to prevent a biofilm and weaken resistance is during the early stages before the bacterium has the opportunity to fully develop a protective barrier(5). This experiment was useful in understanding biofilms and in explaining some of factors of P.aeruginosa impermeability. The experiment though does not address the issue of what treatments specifically would be effective against the bacterium. The experiment focuses on when the best time is for treatment. Further experiments could be done to see what types of microbials work the best and if there are any other factors that contribute to the cell membrane impermeability.
Currently the use of siderophores conjugated with antibiotics is being researched as a method of overcoming the obstacle of low permeability of P. aeruginosa. The siderophore pyoverdin is used to pick up the scarce Fe +2 in the environment and transports it though the outer cell membrane into the periplasmic space (7). The “Trojan Horse” strategy is based on the idea if an antibiotic molecule can be bound to a siderophore the bacterium will allow the sideriphore in along with the undetected antibiotic. Several examples of this naturally occurring have been found, such as albomycins, ferrimycins, and salimycins (7). To test this theory antibiotic conjugates were first successfully produced. These conjugates where then tested to see how well they function as an iron chelator and as an antibiotic. The results were slightly varied depending on the specific structure of the conjugates, and the siderophore that was specifically used. Some antibiotic pyoverdin conjugates were able to function both as chelators and as an antibiotic, but were only affective against P. aeruginosa strains that were able to use the original parent siderophores from which the conjugates were derived from (7). Catecholate conjugates showed different results of iron absorption in vitro and in vivo due to methylation effects and tautomerization. These conjugates though did show a drastic increase of antibiotic activity regardless of the conditions (7).
The Trojan Horse strategy may by successful after further research has been done. Experimenting with different siderophore-antibiotic conjugates have shown the bacterium is able to use the conjugates as it would an unmodified siderophore and the attached antibiotics were able to enter the cell by using the cell’s natural pathway and were affective in varying degrees. Further research is needed though, a conjugate that successfully works both as a chelator and an antibiotic would bring the most effective treatment. Two of the siderophores of P. aeruginosa that are known are infective. To use the Trojan Horse strategy the “perfect conjugate” needs to be produced. A conjugate that is able to be utilized by a cell as a chelator, function as an antibiotic, and is non-infectious (7).

P. aeruginosa is only one particular bacterium that has become a problem of nosocomial infections. Many other resistant bacteria have arisen in health care facilities, as well as other public areas. The growing resistance can partly be attributed to the frequent use of microbials. This alone though it not the cause of the resistance. Antimicrobials select for resistant microbes and facilitate the growth of the resistant colonies. Resistance itself though comes from a cell’s ability to actively export substances, and a low permeable membrane such as the use of biofilm. Another factor that contributes to the spread in hospitals is the occurrence of cross-transmission. Resistant strains are easily passed from one patient to another.
To reduce the presence and or spread of resistant P. aeruginosa preventative measures should be reinforced. Some of these preventative measures are prudent use of broad spectra antimicrobials, and practicing infection control procedures has an effect on slowing the spread between individuals. Continuing research on how P. aeruginosa and other resistant bacteria have developed resistance and learning what mechanisms are used will help in future research. Further extensive research of how resistance occurs will help in producing an effective antibiotic that will be able to bypass the mechanisms that are currently understood and eventually even those that are not.

1.Akcay NM, Altoparlak U, Erol S, et al. The Risk Factors for Acquisition of Imipenem-Resistant Pseudomonas aeruginosa in the Burn Unit. Burn. 2005;31: 870-873.

2. Andremont A, Belloc S, Karabinis A, et al. Acquisition of Multidrug-Resistant Pseudomonas aeruginosa in Patients in Intensive Care Units: role of antibiotics with
antipseudomonal activity. Clinical Infection Diseases. March 2004;38:670-677.

3. Ferreira ACB, Gobara S, Costa S, Sauaia N, et al. Emergence of Resistance in Pseudomonas aeruginosa and Acinetobacter species after the use of antimicrobials for burned patients. Infection Control and Hospital Epidemiology. Oct. 2004; 25(10):868-872.

4. Alborzi A, Farshad S, Japoni A, et al. Susceptibility patterns and cross-resistance of antibiotics against Pseudomonas aeruginosa isolated from burn patients in the South of Iran. Burns. October 2005;32:343-347.

5.Cassaniga BS, Davis S, Harrison-Belestra C, et al. A wound-isolated Pseudomonas aeruginosa grows a biofilm in vitro within 10 hours and is visualized by light microscopy. Dermatol Surg. 2003;29:631-635.

6. Hamood A, Haynes A, Griswold JA, et al. Syndecan 1 Shedding Contributes to Pseudomonas aeruginosa Sepsis. Infection and Immunity. December 2005;73(12):7914-7921.

7. Budzikiewicz H. Siderophore-Antibiotic Conjugates Used as Trojan Horses Against Pseudomonas aeruginosa. Current Topics in Medicinal Chemistry. 2002;1:73-82.