Abstract

Nosocomial infections have been recognized as one of the most critical problems in hospitalization, particularly in critical care units. As these infections prolong hospitalization, require extensive diagnostics and treatment, leads to excessive cost. The emergence of multidrug resistant pathogens has become a threat in critically ill, immuno-compromised patients due to the extensive use of antimicrobial. The most common types of nosocomial infections are pneumonia, urinary tract infections, meningitis, wound, soft tissue, surgical site infections and blood stream infections. These infections can be life threatening, capable of making of therapeutic options exceedingly difficult and limits the critical care settings. Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp are most common nosocomial pathogens. Among all nosocomial species multidrug resistance (MDR) A.baumannii is most pathogenic microorganism. Here a review on A. Baumannii in relation to nosocomial infection is conducted. This review includes risk factors, diagnosis modalities, pathogenesis, MDR properties, mechanism of MDR and treatment of A. Baumannii.

1. Introduction

Acinetobacter baumannii is gram negative, coccobacillus which is ubiquitous in hospital environments. In 1911, Martinus Willem Beijerinck, a Dutch Microbiologist discovered a gram negative, oxidase negative, non-fermentative organism named Micrococcus calcoaceticus from soil on a calcium acetate mineral medium, later on known to be in genus Acinetobacter. ^{1, 2} In 1968 Baumann et al., published comprehensive study on different organisms comprising a single genus, and then the genus Acinetobacter was widely accepted. ³ In late 1970s, Acinetobacter began to be recognized as a nosocomial pathogen. ⁴ In 1986, Bouvet and Grimont used DNA-DNA hybridization methods to propose 4 new species of Acinetobacter, including Acinetobacter baumannii. According to Wong et al., Acinetobacter baumannii can persist longer on dry surfaces under nutrient limiting conditions. They have observed that under such conditions the cell wall becomes thicker making it more persistence. This results into rapid transmission in hospitals and other natural environmental. ⁵ Certain outbreaks studies have revealed that few colonies of strains of Acinetobacter baumannii can remain viable for certain months to years on solid surfaces especially nosocomial surfaces. ⁶ Acinetobacter baumannii are known as “Iraqibacter” as it was present in wound infections in soldiers returned from Iraq and Afghanistan. A. baumannii has gained attention in last few years because of its multidrug resistance properties which make its potential nosocomial pathogen. ¹ Here a comprehensive review is prepared to summarise current information and development related to A. baumannii. It has given special emphasized on infections, risk factors, pathogenesis, genetic aspects for virulence and mechanism of antibiotic resistance.

2. Prevalence of A. baumannii in Various Diseases

As described earlier, A. baumannii infections has more prevalence in the patients who are hospitalized due to critical illness, who has serious underlying diseases, who were subjected to invasive and surgical procedures and who were treated with broad spectrum antibiotics for longer duration. ⁷ It is seen that most of A. baumannii infections involve fluid rich organ systems such as urinary tract, respiratory tract, and peritoneal cavity. The most common infections are pneumonia, urinary tract infection, meningitis, and trauma.

A. baumannii can cause hospital acquired or community acquired pneumonia. In hospitalized acquired pneumonia A. baumannii can be easily isolated from upper respiratory tract but it becomes difficult to differentiate between upper airway colonization from true pneumonia. Study has shown that A. baumannii is the second most common etiologic agent among all gram negative bacteria. ⁸ Only in US surveillance studies have shown that 5 to 10 % of cases from ICUs have A. baumannii infection as patients have longer ICU stay. ⁹ Study has shown that hospital acquired pneumonia due to A. baumannii in ICU has a frequency of 3-5% with mortality rate of 30-75%. ¹⁶ Whereas community acquired pneumonia has higher prevalence in Australia and Asia especially in rainy season. Patients with alcohol abuse, smoking, diabetes, and COPD are more prone to infection as compared to person with healthy habits. This is characterized by fulminant clinical condition and bloodstream infection. It has a mortality rate of 40% to 60%. ¹⁰ The major source of infection for community acquired pneumonia is throat carriage which can be spread up to 10% of community residents. ¹¹

Bacteraemia is a condition where blood stream gets infection with bacteria. In majority of cases the origin of bacteraemia is not known. In US Surveillance studies conducted during 1995-2002, it was observed that out of total bacteraemia episodes, around 1.3% of all nosocomial bloodstream infections are because of A. baumannii and it was the 10^th most common etiologic agent for bacteraemia. It was seen that in patients who are suffering from bacteraemia and hospitalized in ICU 34% - 43% mortality rate whereas patients of outside ICU have only 16% mortality rate. It was also seen that, A. baumannii has initiated more ICU- acquired bloodstream infections as compared to non-ICU ward infections. ^{11, 12} When A. baumannii gets associated with catheter of the patient then it will lead to urinary tract infection. However, the rate of infection is very less having rate of less than 2% of all ICU patients. In case of patients who are outside ICU, infection of A. baumannii is extremely rare. ^{11, 13} Meningitis is a clinical condition where swelling is seen in protective membrane of brain and spinal cord. There are various causes for meningitis including injuries, cancer, certain drug, and infection. Recent studies have shown that there is in increase in incidence of nosocomial meningitis due to A. Baumannii. It contributes to 10% of total nosocomial infection caused by gram negative bacteria with high mortality rate of around 70%. ¹⁴

A. baumannii is also found present in the wound of military population. It was predominant in the wounds of militants returned from Afghanistan or Iraq. Around 32.5% cased were having A. baumannii infection with open tibial fractures. Not only in military population, but also in common population A. baumannii can be seen in wound or skin infection. However, the rate of infection is 2.1% only. ^{7, 15, 16}

Few reports have also suggested that A. baumannii may cause endocarditis at some extent, especially in the conditions where prosthetic valves are involved. A particular case report of bloody diarrhea was reported in an infant due to Acinetobacter haemolyticus strain. Some reports revealed that Acinetobacter spp. may cause keratitis, peritonitis, endophthalmitis related to eye or contact lens surgery. ¹⁷

Infection with multidrug resistance (MDR) is seen with the patients who were hospitalized for longer period, especially in ICU. These patients were treated with third generation antibiotics for longer duration which make the microorganisms resistance against them. Studies have shown that the risk factors for MDR strains infection increases many folds in the patient who have undergone surgeries and exposed to infected patients. Treatment of such kind of infection is exceedingly difficult and leads to higher mortality rate.

3. Risk Factors for A. baumannii Infections

Risk factors for the A. baumannii are varying depending on the type of infection. The below table (Table 1) provides complete information about the risk factors associated with type of infection.

4. Pathogenesis and Virulence Factors of A. baumannii

In recent years, several virulence factors responsible for the pathogenicity in A. baumannii have been identified using phenotypic and genomic approaches. ¹⁸ Major factors include porins, exopolysaccharide, lipopolysaccharides, phospholipase, outer membrane vesicles, protein secretion systems, penicillin binding proteins etc.

Porins are made up of protein located on the surface of cell. They are known to play a variety of roles in maintaining cellular structural integrity, bacterial conjugation, antibiotic resistance, and pore formation. In A. baumannii, Omp A is one the most abundant porins present in outer membrane. Omp A plays a significant role in adherence and invasion. It also induced apoptosis by releasing cytochrome c and complement resistance. ^{18, 19} Omp A also regulates biogenesis of outer membrane vesicles and facilitates surface motility and biofilm formation. ^{20, 21} Other porins namely Omp 33-36 are responsible for cytotoxicity action through water passage channel. One study it was also shown that Omp33-36 induces apoptosis in connective tissues and immune cells by modulating autophagy in human cells. Studies have shown that deletion of the Omp 33-36 gene reduces adherence and invasion of human lung epithelial cells. ^{22, 23, 24, 25} In addition to porins, capsular exopolysaccharides and lipoproteins can also contribute to pathogenicity. Study has shown that the K locus has a conserved gene cluster in the patients having A. baumannii which determine the production of capsular polysaccharides responsible for pathogenicity. ²⁶ A study has revealed that antibiotics induce hyper-production of capsular polysaccharides, which increases resistance against host complement and increase its virulence. ²⁷ In a study carried out by Liou et al., has demonstrated that the presence of BfmS as virulence factor plays an important role in biofilm formation, adherence to eukaryotic cells, and resistance to human serum. ²⁸ Lipopolysaccharides are biomolecules made up of lipid A moiety, an oligosaccharide core and O antigen. They are immuno-reactive molecules which plays vital role in virulence and induce production of TNF and IL-8 from macrophages. ²⁹ Phospholipases are the enzymes responsible for phospholipids metabolism. These might be virulence factors in many bacteria. They are broadly classified into three classes such as A, C and D. Among the three, PL-C and PL-D function as virulence factors in A. baumannii. In a study conducted by Camarena et al. in 2010, they have shown that inactivation of two genes associate with phospholipase namely A1S_0043 and A1S_2055 lead reduction in cytotoxicity. ²⁹ Sthal et al., in their study has revealed that the virulence in A. baumannii strain ATCC 19606 is mediated by concreted action of three PLDs. ³⁰ In addition to LPS and porins, outer membrane vesicles (OMVs) also play vital role on pathogenesis. These OMVs are composed of LPS, periplasmic proteins, phospholipids, DNA/RNA. These molecules are secreted by outer membranes of gram-negative bacteria having 20-200 nm diameters. OMVs facilitate the pathogen to interact with the host cell without close contact. A. baumannii possess several OMVs belong to phospholipases and proteases, which are responsible for virulence. ^{31, 32} Previous studies have revealed that the presence of outer membrane vesicles can function as an acellular vaccine in A. baumannii strains. ³³ A. baumannii also known to have type II protein secretion system (T2SS) which is a multi-protein complex having similar structure as type IV pili system. ³⁴ T2SS is composed of pseudo pilus, ATPase, inner-membrane assembly and outer-membrane dodameric complex. The whole assembly is responsible for export of effector proteins. ^{34, 35} These proteins are responsible for virulence factor to invade into the host cells. T6SS is another protein system which was first identified in Pseudomonas aeruginosa and Vibrio cholera. T6SS is composed of accessory factors and structural proteins and has a spike like structure to penetrate the target cell. ³⁵ Type 5 secretory system of A. baumannii was found to mediate the biofilm formation and adherence to extracellular matrix which enhances the virulence efficiency. ³⁶ Not only proteins but sometime siderophore are also secreted from cells. Siderophores are low molecular weight, iron chelating compound. It is responsible for transportation of iron across the membranes. These siderosphores can modulate the host cellular pathways and add on virulence. Studies have confirmed that A. baumannii contains siderosphores as well as acinetobactin. Acinetobactin is a special class of siderosphore having catecholate and hydroxamate function groups, which can lead to impaired biosynthesis leading cell damage. In addition to all the above factors some other factors were also known to contribute to virulence characteristic. These factors as penicillin binding protein (PBP7/8), CipA, Sur A1, Tuf, etc. ^{37, 38} Below table summarize all major virulence determinant possessed by A. baumannii. (Table 2)

5. Mechanism of Antibiotic Resistance

Antibiotic resistance properties of A. baumannii make it more fatal pathogen as compared to other common pathogens. These include various enzymes, structural modifications, and genetic alterations. Among all the enzymes β-lactamase is a key enzyme which provides protection against penicillin, cephamycin, cephalosporins, and carbapenems. All these antibiotics are commonly known as β-lactam antibiotics as they contain β- lactam ring having four carbons. β-lactamase hydrolyse this structure and destroy all the antibiotics resulted into antibiotic resistance. Even it was observed that certain species of A. baumannii can resist carbapenems, a strong antibiotic used for MDR microorganisms. Beta metallo-beta lactamases and a class of D OXA of A. baumannii are known to destroy the drug and provide the resistance. A. baumannii was also known to alter the structure of porin and other proteins. Because of this modification antibiotics cannot enter the cells and not able to destroy the pathogen. Study has shown that A. baumannii gain resistance against colistin though alteration of membrane permeability only. Efflux pump present in the pathogen is responsible for throwing out the antibiotics out of the cell. There are four type of efflux pumps in A. baumannii which are major facilitator superfamily (MFS), resistance nodulation cell division (RND), small multidrug resistance (SMR) and multidrug and toxic compound extrusion (MATE). AdeABC gene was found responsible for this efflux activity. In addition to AdeABC gene, two other genes namely gyr A and par C were also found associate with antibiotic resistance. gyr A is a unit of DNA gyrase and par C is a subunit of topoisomerase IV. Point mutation in these two genes alters the membrane binding efficiency which makes them resistance against quinolones. More details about resistance due to enzymes, gene and efflux are summarised in the Table 3. ^{1, 39, 40, 41}

6. A. baumannii Infected Patient Management

As mentioned earlier there are several sites of infection. Based on the site of infections various diagnostic modalities are applied for detection of infection and patient managemenet. In case of ventilator associated pneumonia, chest radiography, sputum analysis, PCR, oxygen saturation, hemodynamic studies and acute phase reactants are determined. In cathater associated urinary tract infections, urine analysis and culture test are more preferred. In case of blood stream associated infection blood investigation, blood culture and ECG are preferred. In case of surgical site infections blood tests such as CRP, FBC, blood culture and swad culture are more preferred. Not limited to these tests, many other tests are also suggestive of A. bauminnii infection depending on the site of infection. ^{10, 13, 17, 18, 76}

7. Treatment

After the discovery of antibiotics in the early 19^th century, treatment of most of the infectious diseases was carried out using antibiotic. Excessive use of these antibiotics results into genesis of antibiotic resistance microorganisms which are not easily controlled by regular antibiotics. In case of Acinetobacter infections initially ampicillin, gentamicin and nalidixic acid ,either as mono or combination were highly used, and it was effective also at that time. But after few years, strains of Acinetobacter became resistance against these antibiotics. Hence, infection disease society of America has considered this specie as red alert pathogen. ¹¹ This high resistance capacity against broad spectrum antibiotic leads to discover the new molecules and re-evaluation of old molecules for efficacy. This is being achieved by applying knowledge of pharmacodynamic and pharmacokinetic. ⁷⁷ Few such antibiotics are now developed which are still effective against A. baumannii infection. Carbapenems like imipenem and meropenem are effective to certain geological location where strains as still sensitive against these molecules. According to a study, Latin America has 40% resistance, Europe & North America has 13% - 15% resistance, Singapore has 50% resistance, India has 85 % resistance and Pakistan has 62% - 100% resistance against these drugs. Hence these drugs are not suitable for these areas. For the other location they can be used. Sulbactam is a beta lactamase inhibitor which has intrinsic bactericidal activity against Acinetobacter, mediated by penicillin binding proteins. Like the carbapenems, susceptibility of these drugs on A. baumannii varies from various geographic locations. In Germany, France, and Spain sulbactam is used either as a single agent or as combination with ampicillin or cefoperazone. Studies have shown that combination of sulbactam with penicillin is more effective against A. baumannii as compare to sulbactam alone. ^{78, 79} Study have shown that meningitis, pneumonia, urinary tract infections caused by MDR A. baumannii can be treated with sulbactam with up to 67.5% healing rate. ⁸⁰ Tigecycline, a derivative of minocycline is universally used for treating community acquired pneumonia, skin infections, bacteraemia, UTIs by MDR A. baumannii. Jung et al., has conducted a study where critical patient who was suffering from ventilator associated pneumonia caused by A. baumannii was treated with tigecycline. This drug was proven effective against MDR A. baumannii. ^{81, 82} In 2005 Sader et al., has reported the very first case of tigecycline resistance. Studies have shown that strains from Israel have 66% resistance, India has 57.6%, Greece has 74.2% and Saudi Arabia has 56% resistance. ^{83, 84, 85} Colistin, is another broad-spectrum antibiotic which distracts the cell membrane resulting in the loss of integrity causes cell death. It was highly used between 1960s – 1970s, but due to neuro and nephrotoxicity it was discontinued. ⁸⁶ Studies have shown that colistin has 11% - 76% nephrotoxicity. ⁸⁶ Colistin and/or polymyxin E was last alternate for treating A. baumannii infections. ⁸⁷ In addition to polymyxin E other molecules like tigecycline, sulbactam and trimethoprim is also used in combination with colistin. Resistance to colistin was first time reported in 1999, in the Czech Republic. ⁸⁸ Daadmi et al., has reported 1.8% resistance to colistin in Saudi Arebia, Maspi et al., has reported 48.8% resistance in Iran ^{89, 90} and Gupta et al have reported 53.1% resistance. ⁹¹

8. Conclusion

Acinetobacter spp. are among one of the most potential nosocomial microorganisms across the globe. It is known to acquire extended resistance to most antimicrobial agents rapidly. They have potential to survive in hospital environments for a longer duration and therefore can result in nosocomial outbreaks. In view of this, and to control the spread of this organism, suitable safety programmes/strategies should be implemented by healthcare facilities. Research should also be focused on the development of new antibiotics which have better efficacy and lower resistance. At present the best way to avoid spreading MDR A. baumannii infection is to maintain cleanliness of the hospital and use antibiotic as per prescription and minimize antibiotic misuse/overuse. Colistin can be used as a last alternate for treatment of A. baumannii infection. Selection of antibiotic for treatment should be selected wisely keeping the geographical location in mind and the local antibiotic susceptibility profiles.

References