Acinetobacter baumannii in suspected bacterial infections: Association between multidrug resistance, virulence genes, & biofilm production

Acinetobacter baumannii is a non-fermentative, oxidase-negative, Gram-negative coccobacillus1. It is a predominant pathogen involved in hospital-associated infections, causing ventilator-associated pneumonia (VAP), septicaemia, urinary tract infection and meningitis2. It can survive and thrive under diverse conditions of pH, temperature and dry and humid environments and form biofilms on several objects, such as medical devices involving ventilators, catheters and respirometers3. These significantly acquire resistance to broad-spectrum antibiotics. The spread of carbapenem-resistant A. baumannii (CRAB) is emerging as a global problem and is causing life-threatening infections due to the lack of therapeutic alternatives4. Several mechanisms of resistance to many classes of antibiotics are known to exist in A. baumannii; among which the presence of Ambler’s class D OXA-type oxacillinase genes and class B metallo-β-lactamase genes is predominant.

The virulence factor genes such as outer membrane protein A (OmpA) and biofilm-associated protein (Bap) in A. baumannii are associated with adhesions, biofilm production and the development of antibiotic resistance5. The biofilm state is considered more resistant to the host defence and is metabolically less vigorous, consequently more resistant to antibiotics6. The study also stated that the relationship between the formation of biofilm and antibiotic resistance is controversial1. So far, there are scanty data available on this highly drug-resistant bacterium from Sikkim, the northeastern part of India. Therefore, the present study aimed to investigate the occurrence of A. baumannii in clinical specimens of suspected bacterial infections and further to see the association of biofilm production with multidrug-resistance and virulence factor genes in A. baumannii isolates.

Material & Methods

Study design: This cross-sectional hospital-based study was carried out at the department of Microbiology, Sikkim Manipal Institute of Medical Sciences (SMIMS), which is associated with its own 500-bed Central Referral Hospital located in Gangtok, Sikkim after procuring clearance from the Institutional Ethics Committee. All clinical specimens received for the bacterial culture in the department of Microbiology, Central Referral Hospital, Gangtok, Sikkim, were included for the isolation of A. baumannii. Specimens were collected from individuals admitted and those visiting the outpatient department (OPD) of the Central Referral Hospital with suspected bacterial infections having fever as a major symptom and, with or without other symptoms as per the different organ systems involved. After isolation and identification, those confirmed A. baumannii were further used for the study. The repeat samples from the same individuals showing repeated growth of A. baumannii were excluded from the study. A total of 18,400 different clinical specimens were processed over a period of two years (September 2018-August 2020) from individuals attending the various units in this hospital.

Isolation and identification of Acinetobacter baumannii: All clinical specimens received for bacterial culture and sensitivity were inoculated on MacConkey agar (HiMedia, Mumbai) and HiCrome Acinetobacter agar (HiMedia) media for the isolation of A. baumannii. All non-repetitive isolates of A. baumannii were incorporated in this study. The identification of the isolates as an Acinetobacter species was performed based on the characteristics of the colonies, Gram staining and biochemical characteristics by the VITEK-2 System (bioMerieux, Marcy I’Etoile, France) using Gram-negative identification cards (bioMerieux).

Molecular detection using real-time PCR: All isolates were confirmed at the species level as A. baumannii by real-time PCR (qPCR) with the detection of the bla_OXA-51-like gene. PCR was carried out in a 25 μl reaction volume with 3 μl of extracted DNA as described by Turton et al7. The PCR conditions were 94°C for three minutes, followed by 35 cycles at 94°C for 45 sec, 57°C for 45 sec, 72°C for one minute and a final extension at 72°C for five minutes8.

Antibiotic susceptibility test: The antibiotic susceptibility of the isolates was determined by the VITEK-2 system (bioMerieux, Marcy I’Etoile, France) and reported according to the CLSI 2018 guidelines8. Antibiotics, including piperacillin/tazobactam, ceftriaxone, cefoperazone/sulbactam, cefepime, imipenem, meropenem, gentamicin, ciprofloxacin, tigecycline, colistin and trimethoprim/sulphamethoxazole (bioMerieux) were used. A. baumannii resistant to three or more (≥3) classes of antibiotics tested was considered multidrug-resistant (MDR-A. baumannii).

Biofilm production test: All A. baumannii isolates were tested for the production of biofilm using a microtitre plate assay. The isolates were grown overnight in Trypticase Soy Broth (HiMedia) with 0.25 per cent glucose at 37°C. The overnight growth was adjusted to 0.5 McFarland turbidity using the same broth and further diluted in a ratio of 1:40 in the supplemented medium. A 200 μl aliquot of cell suspension was inoculated in sterile 96-well polystyrene microtitre plates. After 24 h of incubation, the wells were washed thrice with 200 μl PBS (phosphate-buffered saline) dried and stained with one per cent crystal violet (HiMedia). The stained crystal violet was solubilized in 200 μl of ethanol–acetone (80:20 v/v) for five min, and the optical density at 620 nm (OD 620) was read using a microplate reader. Every assay was carried out in triplicate, and the average optical density was considered according to that reported by Badave and Kulkarni9 with modification. The following values were allotted for the determination of biofilm: non-biofilm producers: OD 620<0.0898, weak biofilm producers: 0.0898>OD 620<0.179, medium biofilm producers: 0.179>OD 620<0.2688 and strong biofilm producers: OD 620>0.2688. The cut-off value of 0.0898 was obtained after calculating the mean OD (0.0318) and the standard deviation of the empty microtitre plate stained by the method mentioned above. The cut-off value was taken as three standard deviations above the mean OD.

Detection of carbapenem-resistant genes and virulence factor genes: The bacterial DNA of A. baumannii was extracted from the overnight broth culture in Tryptic Soy Broth using the Bacterial DNA mini spin preparation kit (Helini Biomolecules, Chennai). Among the carbapenem-resistant genes, the genes encoding class D oxacillinase (bla_OXA-23-like, bla_OXA-24-like and bla_OXA-58-like), class B metallo-β-lactamases (bla_IMP-1 and bla_VIM-1) and the presence of ISAba1 the insertion sequence were assessed using real-time PCR for carbapenem-resistant clinical A. baumannii isolates. Of the virulence factor genes, the presence of either Bap or OmpA was assessed. The primer details are mentioned in Supplementary Table10-14.

Statistical analysis: All data were recorded, and segregated using the software, SPSS version 19 (IBM Corp., Chicago, IL, USA). The baseline data for this study were expressed as a percentage. The Z-test was used to compare variables such as the MDR rates between the intensive care unit (ICU) and other units of the hospital and the Chi-square test for biofilm formation between MDR and non-MDR isolates and the association between MDR and virulence genes. P<0.05 was considered significant.

Results

Isolation of A. baumannii: A total of 307 A. baumannii were isolated from 18,400 clinical specimens cultured. These isolates were obtained from 60.26 per cent male to 39.73 per cent female individuals between the ages of 10 days and 90 yr (Table I). Site sampling analyses showed that the majority of A. baumannii isolates were from endotracheal tube specimens [138 (44.95%)], followed by sputum [60 (19.54%)], pus [53 (17.26%)], urine [23 (7.49%)] and blood [18 (5.86%)], and <2 per cent were from body fluids, catheter-tip specimens and urogenital specimens (Table II). The majority of isolates were from specimens of the lower respiratory tract, and none were obtained from throat swabs and tissue. A. baumannii isolated from ICUs, other wards and OPDs added upto 142 (46.25%), 152 (49.51%) and 13 (4.23%), respectively (Table III).

Antibiotic susceptibility test: The susceptibility to antibiotics of 307 A. baumannii is shown in the Figure. The resistance obtained against most of the tested antibiotics was penicillin/tazobactam (81.43%), ceftriaxone (80.13%), cefepime (79.15%), imipenem (76.22%), meropenem (75.24%), gentamicin (72.31%), ciprofloxacin (74.26%) and trimethoprim/sulphamethoxazole (75.24%). However, the resistance rates for tigecycline and colistin were 2.93 per cent and 8.46 per cent, respectively. The overall MDR isolates were 242 (78.82%), and segregation was 92.25 per cent in ICUs and 71.05 per cent in the other units of the hospital. The MDR was high in endotracheal tube specimens (96.37%), followed by pus (88.67%). The MDR distribution according to specimens and different units of the hospital is displayed in Table III.

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Figure

Antibiotic susceptibility pattern of Acinetobacter baumannii isolates (n = 307).

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Biofilm forming ability: The biofilm formation test revealed that out of 307 A. baumannii isolates, 205 (66.78%) were biofilm producers, and 102 (33.22%) were non-biofilm producers. The strong, medium and weak biofilm producers were 7 (2.28%), 32 (10.42%) and 166 (54.08%), respectively. The biofilm producers and their MDR rates are represented in Table IV.

Virulence factor genes: Of the 307 A. baumannii isolates, the virulence factor gene OmpA was present in 281 (91.53%), and Bap was detected in 98 (31.92%) isolates.

Carbapenem-resistant genes: Among the 307 A. baumannii, the results for the 100 randomly selected CRAB isolates assessed for the various carbapenem-resistant genes showed that among the class D oxacillinase, the bla_OXA-23-like gene was predominantly detected in 96 (96%) isolates, the bla_OXA-58-like gene in 6 (6%) isolates and none of the isolates harboured the bla_OXA-24-like gene. For the class B metallo-β-lactamases, the bla_IMP-1 gene and the bla_VIM-1 gene were detected in 4 (4%) and 8 (8%) isolates, respectively. The insertion sequence ISAba1 was detected in 76 (76%) CRAB strains. Interestingly, some of the isolates were seen co-harbouring more than one carbapenem resistance gene.

Discussion

The present study demonstrates an isolation rate of 1.66 per cent of A. baumannii in several clinical specimens. This finding is, however, lower than that reported imperviously studies, showing 6.94 per cent and 4.21 per cent isolation rates15,16. 64.49 per cent of the A. baumannii were significantly isolated from bronchopulmonary specimens (endotracheal tube: 44.95% and sputum: 19.54%). The concordant data with greater isolation of 62.1 per cent, 44.7 per cent and 39 per cent from bronchopulmonary specimens have been previously reported16,17. The highest isolation from lower respiratory specimens may be due to the colonization of bacteria promoted by the endotracheal tube and the survival of bacteria in the endotracheal tube due to the formation of biofilm that increases the possibility of VAP18. Biofilms in the endotracheal tube are an important virulence mechanism and promote the persistence of the pathogen resulting in a treatment failure since bacteria in the form of biofilms are less susceptible to the host’s immune defences, less metabolically active and consequently, increasingly resistant to antibiotics19. The occurrence of VAP enhances the risk of mortality in critically ill individuals when it is caused predominantly by MDR bacteria6.

The susceptibility pattern to antibiotics showed high resistance to almost all classes, including carbapenems. Carbapenems are generally considered drugs of preference for the MDR-A. baumannii isolates; however, high rates of carbapenem-resistant isolates have been detected in recent decades. The mortality rate for the most frequent CRAB infections (mostly hospital-associated pneumonia and bloodstream infections) has reached 60 per cent20. A review study reported that the mortality rate of infected individuals with CRAB doubled compared to strains susceptible to carbapenem21. CRAB has topped the WHO list of risks to human health and in the recent decades, has emerged as the predominant pathogen causing hospital-associated infections22. This could be due to the influence of unsuitable antimicrobial therapy and the survival rate of CRAB infections. The mortality rate of 86.1 per cent in individuals receiving inappropriate antimicrobial therapy, which was substantially higher than 33.7 per cent of those receiving proper antimicrobial therapy, was noted23. The new but last alternatives are colistin and tigecycline for the treatment of A. baumannii infection, which is also slowly rising with resistant isolates24.

The present study demonstrated an MDR rate of 78.82 per cent in the entire hospital, which, when segregated, is 92.25 per cent of the ICUs and 71.05 per cent of other units. The inappropriate overuse of broad-spectrum antibiotics, one of the main reasons, has led to a rise in the prevalence of MDR-A. baumannii in the ICU. Large surveillance studies by Vincent et al25 reported that around 70 per cent of ICU in patients were given antibiotics either for therapy or prophylaxis. Another factor supporting the prevalence of MDRAB in the ICU is that this organism exhibits and acquires drug resistance in a short period. A 10 yr study from Poland reported Acinetobacter spp. as the predominant ICU bacteria with 87 per cent extensively drug resistant and a rapid rise in resistance to carbapenems over the decade26. Therefore, this is an opportune time to undertake more comprehensive studies on A. baumannii drug resistantce particularly in Sikkim and other parts of India to provide not only baseline information but also preventive strategies that could be implemented for proper clinical management in the future.

The biofilm formation in bacteria is considered a system of self-defence, as it withstands and resists various stresses1. The biofilm production showed 66.78 per cent positivity, with most isolates being 54.07 per cent weak, 10.42 per cent medium and only 2.28 per cent strong biofilm producers, and most of these isolates were associated with device-related infections. Among 198 A. baumannii isolates from the lower respiratory tract, some also showed a similar pattern of biofilm production, with 96 (48.48%) weak, 25 (12.62%) medium and 4 (2.02%) strong biofilm producers. Out of 205 biofilm producers, 83.9 per cent are MDR isolates, and among 242 total MDR isolates, 172 (71.07%) are biofilm producers, indicating a positive association between biofilm formation and MDR A. baumannii (P<0.05). 100 per cent, 78.2 per cent and 84.33 per cent of strong, medium and weak biofilm producers were MDR, respectively. To support antibiotic resistance in biofilm producers, a study by Yang et al27 reported the various contributing factors that may play an important role, such as bacterial aggregation that impairs antibiotic diffusion in biofilm, increased expression of exopolymeric substances during biofilm formation and changes in genotypic and phenotypic features due to physiological and stress heterogenicity of biofilm-forming cells. In contrast, another study1 reported no relation between the biofilm formation by A. baumannii and antibiotic resistance, and no notable differences in the pattern of antibiotic resistance between different groups of biofilm formation1.

The presence of different virulence factor genes responsible for adherence, biofilm formation and antibiotic resistance is already known, however, the extent to which these promote the biofilm formation is not clearly understood. We observed the presence of OmpA in 281 (91.53%) A. baumannii isolates, and all (205) biofilm-producing isolates were positive for the OmpA gene which is known to play a role in host cell adhesion, antibiotic resistance and biofilm formation. The OmpA expression enhances antibiotic resistance by decreasing the rate of diffusion of β-lactam antibiotics, and OmpA may combine with the efflux pump to trigger out the antibacterial compounds from the bacterial periplasm, leading to resistance development28. In our study, of the 242 MDR A. baumannii, OmpA was detected in 221 (91.32%) isolates. This indicates a positive association between the development of the MDR phenotype and the presence of OmpA (P<0.05). A. baumannii lacking the OmpA was found to be less virulent to human respiratory tract epithelium due to reduced adherence to the host cell, and its over-expression is a vital risk factor causing high morbidity and even mortality in the case of A. baumannii, causing bacteraemia and nosocomial pneumonia5.

The Bap is another factor responsible for being a highly divergent protein exposed to the surface and is essential for the adherence to bronchial cells to maintain the structural integrity and the formation of water channels within the biofilm29. Our study detected Bap in 98 (31.92%) A. baumannii isolates; of which MDR was seen in 89 (90.81%), indicating a positive association (P<0.05). The presence of Bap is associated with the formation of matured biofilm in various medical devices; however, our findings revealed that of the 205 biofilm-producing strains, Bap was only found in 92 (44.87%) isolates. Interestingly, the six Bap-producing A. baumannii isolates were negative for biofilm, and among 113 (36.8%) biofilm-producing strains, Bap was not detected, which indicates that biofilm formation was not regulated by a single factor and may be multifactorial30.

The carbapenemase-encoding genes revealed that the production of class D oxacillinase is predominant amongst clinical A. baumannii isolates. The bla_OXA-23-like gene was detected in 96 per cent of isolates. The presence of the bla_OXA-23-like gene is considered one of the potential virulence markers and a significant cause of carbapenem resistance. This result is worrisome because it could cause outbreaks of CRAB infections. The outbreak caused by A. baumannii producing the bla_OXA-23-like gene has been reported worldwide and is a dominant resistant determinant in the Asian subcontinent31. The other oxacillinase and metallo-β-lactamases detected is also responsible for the dissemination of resistant A. baumannii strains. The insertion sequence ISAba1, when present upstream of the carbapenem-hydrolyzing β-lactamases, may increase the production of β-lactamases, thereby increasing the spread of CRAB strains. Seventy-six per cent prevalence of the ISAba1 insertion sequence was seen in our study. The mechanism of resistance to carbapenem caused by the overexpression of bla_OXA-like genes due to the presence of upstream insertion promoter sequences such as ISAba1 is more prevalent32.

There were some limitations in this study. First, the participants’ clinical history, hospitalization details and outcome data were not included in the study; hence, the relationship between MDR isolates and their association with drug consumption could not be analyzed. Second, the other virulence factor genes and carbapenem resistance genes that are also associated with biofilm formation and the development of antibiotic resistance were not included in this study.

Overall, the results of this study showed high isolation rate of A. baumannii from lower respiratory tract specimens. The bacterial colonization and biofilm formation in the endotracheal tube probably favour lower respiratory tract infections. The MDR rate in biofilm-producing A. baumannii isolates was significantly higher compared to biofilm-negative isolates. The high existence of MDR A. baumannii and its positive association with biofilm formation indicate nosocomial distribution and a threat to hospital settings. This study also suggests that biofilm formation is a process regulated by multiple factors. The high prevalence of carbapenem resistance genes such as the bla_OXA-23-like gene and the insertion sequence ISAba1, as well as the coexistence of various carbapenem resistance genes in a single isolate, is of concern given their ability to cause outbreaks in hospital settings. A need for strict antimicrobial stewardship is emphasized and more comprehensive molecular surveillance of various genes implicated in biofilm-specific resistance mechanisms is suggested. Understanding these mechanisms in detail can provide better facilities for the treatment and prevention of CRAB infections.