Biofilm Formation by Acinetobacter Spp. in association with Antibiotic Resistance in clinical samples Obtained from Tertiary Care Hospital

 

Rachna Chaturvedi^1*, Preeti Chandra¹, Vineeta Mittal²

¹Amity Institute of Biotechnology, Amity University, Uttar Pradesh Lucknow-226028

²Ram Manohar Lohia Institute of Medical Sciences, Lucknow

 

ABSTRACT:

Acinetobacter spp., is an opportunistic, gram negative and non-fermenter organism, has occurred as one of the maximum bothersome pathogens for health care organisations worldwide. This organism generally targets the most susceptible hospitalized patients causing pneumonia mainly in patients on mechanical ventilation in ICU, having urinary tract infection, bloodstream infections, skin infections etc. It accounts for about 10% of all nosocomial infections. Its clinical significance over last 15 years has been forced by its amazing capacity to get resistant elements, making it one of the antagonistic organisms in the current antibiotic era. Most of Acinetobacter strains are resistant to all known antibiotics have been reported and they are acting in interaction with this emerging resistance profile, with this ability. Acinetobacter isolatesare able to survive for long periods throughout the hospital environment.Itrecurrently causes infections associated with medical devices, e.g. vascular catheters, cerebrospinal fluid shunts, urinary catheter or an endotracheal tube. Microbial biofilms are considered as virulence factors and is a well-known pathogenic mechanism in such infections. The ability to form biofilms on biotic or abiotic surfaces is a common trait among Acinetobacter strains. Therefore, the present study was undertaken to study antibiotic resistance patterns and association the resistance profile with bio-film production in various clinical isolates of Acinetobacter spp. A total of 35 isolates of Acinetobacter species were obtained for study from various clinical specimens like pus, urine, sputum, blood culture specimens from the patients admitted in hospital. Isolates obtained from different medical specimens were managed and confirmed by conservative microbiological procedures. The isolates of Acinetobacter are identified on the basis of certain biochemical test like catalase positive, oxidase negative, urease negative, nitrate negative, indole negative and non-motility test. A total of 35 isolates were subjected to susceptibility testing by disc diffusion method according to CLSI guidelines for 14 clinically relevant antibiotics. Screening for biofilm production was done quantitatively by micro titre plate assay method. In the present study, 75 % isolates were showing antibiotic resistance and 22.8% isolates produced bio film along with antibiotic resistance. Biofilm formers isolates showed high resistance to imipenem, cephotaxime, ceftazidime, tobramycin, ciprofloxacin as compared to non-biofilm formers. We selected to study the biofilm formation and antibiotic resistance of clinical isolates of Acinetobacter species to understand its ability to persist in the hospital environment to cause outbreaks.

 

 

 

 

INTRODUCTION:

Acinetobacter spp are nonfermentative gram negative bacilli, has emerged as one of the most troublesome pathogens for health care institutions globally, related to outbreaks of nosocomial infections¹. This organism commonly targets the most vulnerable hospitalized patients causing pneumonia particularly in patients on mechanical ventilation, urinary tract infection, bloodstream infections, skin infections etc. It is frequently isolated from respiratory tract through which it contaminates installed catheters. This will eventually lead to fever, pneumonia, bacteremia, urinary tract infections, septicaemia and meningitis among critically ill patients in ICU^{2, 3, 4,5} It accounts for about 10% of all nosocomial infections^6,7 Its clinical significance over last 15 years has been propelled by its remarkable ability to acquire resistant determinants, making it one of the organisms threatening pathogen in the current antibiotic era. Acinetobacter strains resistant to all known wide range of antibiotics as cephalosporin, penicillins, carbapenems, fluoroquinolones and aminoglycosides. This activity has been reported and acting in synergy with this emerging resistance profile is the ability of Acinetobacter to survive for long periods throughout the hospital environment^7,8. As Acenetobacter spp. is responsible for the majority of nosocomial infections. There are different virulence factors, amongst them the most important one is the ability to produce biofilms and their survival in hospital environment which is related to their high degree of antibiotic resistance^{9,10,11,12,13}. Biofilm formation on invasive devices by strains of Acinetobacter spp. is considered to be an important virulence factor for such infections. The biofilm formation plays a crucial role in survival of bacteria under stressed environmental conditions. Biofilms are complex mixtures of microbes which are predominantly attached to hard surfaces. They are often enclosed by thick polysaccharide layer which makes them resistant to antibiotics and thus very hard to eliminate^13,14. The ability to produce biofilms grounds bacteria to tolerate comparatively tough situations. Bacteria binding to non-natural planes in hospitals encourage long-lasting strength and forbearance for waterless surroundings and use of several metabolic resources. These structures make abolition of biofilm-associated bacteria practically impossible from hospital environments^3,12,15,16. On the other hand, sensitivity and resistance to different antibiotics as well as microbial digestion due to formation of biofilm will be decreased. This is attributable to deficiency of nourishment in the biofilm deepness. Slower metabolism and antibiotic resistance lead to bacterial distribution which can generate a rapid critical situation¹⁷. The infection is challenging to eliminate as Acinetobacter spp. growing in biofilm are resistant to most of the antimicrobials so restraining therapeutic selections. Biofilm formation on surfaces and countenance of multidrug resistance always favours propagation of Acinetobacter spp. in hospital setting.This may increase the occurrence of nosocomial infections triggered by bacteria, particularly in patients in intensive care and persons in burn units and patients under surgical treatment^9,10,11.Acinetobacter recurrently causes infections accompanying to medical devices, e.g. vascular catheters, cerebrospinal fluid shunts or Foleys catheter. Biofilm formation is a renowned pathogenic mechanism in such type of infections. The potential of Acinetobacter to produce biofilm may describe its extraordinary antibiotic resistance and existence in the hospital environment^8,18. Therefore the present study was undertaken to detect biofilm formation and its correlation between biofilm formation and the antibiotic resistance in the in the clinical isolates of Acenetobacter spp. isolated from tertiary care hospital.

 

MATERIALS AND METHODS:

The present study was conducted in the Department of Microbiology at Dr. Ram Manohar Lohia Institute of Medical Sciences Lucknow. A total of 1125 clinical samples were received in the Department of Microbiology for bacterial culture and sensitivity from patients were included in the present study. Samples had collected from several clinical sources (blood, urine, pus, sputum of hospital unit) and from different environments as from ICU, surgical ward and gastro ward.

 

Isolation and Identification:

Isolation is done for different samples by using different type of media. Specimens were found to be placed in brain-heart infusion broth medium and then transferred to microbiology lab for culture. Isolates were plated using standard laboratory methods including growth on blood agar (non-haemolytic), MacConkey agar medium (Merck, Germany) at 37°C showing a pink to light purple appearance and identified by Gram stain (Gram negative coccobacilli), catalase test (positive), oxidase test (negative), oxidative-fermentative test for glucose (producing acid from glucose in oxidative state), sulphide test indole test motility test, methyl red-Voges-Proskauer test, citrate utilization test, urease test, triple sugar iron test (non-fermentative or alkaline/alkaline).

 

Antibiotic sensitivity test:

Antibiotic sensitivity test for Acinetobacter spp. was done using most common and therapeutically important antibiotics by Kirby Bauer disk diffusion method on Mueller Hinton agar. The antibiotics used in present study were as Ampicillin sulbactam (As), Cefoperazone-sulbactam (Cfs), Ceftriaxone (Ctx), Ceftazidime (Caz), Gentamycin (G), Chloramphenicol (Cmp), Ciprofloxacin (Cip), Imipenem (Imp), Amikacin (An), Tetracycline (Tet), Tobramycin (Tob), Doripenem (Dori), Doxycycline (Do), Levofloxacin (Levo). Interpretation of antimicrobial susceptibility testing by disc diffusion test was done as per Clinical Laboratory and Standard Institute (CLSI) Guidelines^{19, 20}.

 

Biofilm formation:

The quantitative biofilm detection method was performed by micro titre plate method as determined by Christensen et al is generally considered to be the gold-standard technique for bio-film detection^21,22.Test organisms were inoculated in 10 ml TSB and were incubated at 37ºC for 24hours. The overnight growth was diluted in a ratio of 1:40 in TSB-0.25% glucose Each well of a 96-well flat-bottomed polystyrene tissue culture plate (3 wells for each isolate) was filled with 20 mL of the overnight culture (equivalent to 0.5McFarland standard). Sterile TSB medium (180 mL) was further added into each well. The biofilm producing isolates of Acenetobacter spp. was used as positive control. Wells inoculated with sterile broth were considered as negative control. The plates were protected with a lid and incubated aerobically for 24 h at 37°C. After incubation, the content of each well was removed and the wells were carefully washed, three times, with 0.2 mL of phosphate buffer saline (pH 7.2) in order to remove free floating plank tonic bacteria. Wells were filled with 200 µl of 99% of methanol to fix adherent bacteria for 15 min. The plates were emptied and desiccated in overturned position. Desiccated plates were stained with 0.2 ml of 2% crystal violet for 7 min and this is followed by washing with distilled water to take away extra stain. After the plates were entirely waterless, 160 µl of 33% glacial acetic acid was further added to the wells to take out additional stain. The wells were washed another time in 200 microliter of ethanol-acetone (80:20 v/v) to solubilise crystal violet. Micro plate reader was used to determine the optical density at 620 nm (OD 620).Agreeing with the absorbance values, the test bacteria were classified: as none (-), weak (+), moderate (++) and strong (+++) believer cells. The cut-off absorbance value (ODc) was calculated as three standard deviations above the mean OD of the negative control. Tests were performed in triplicates and results were averaged.

 

RESULTS:

In the present study total 1125 samples were processed for the isolation, identification of Acinetobacter spp.Out of 1125 samples, 412 (36.6%) were found to be Gram-negative bacteria. Of total Gram-negative bacteria 210 (51%) were found as non-fermenter. Out of these total non-fermenter bacteria, Acinetobacterspp. were 35 (16.6%). Out of total 1125 samples, 35 (3.1%) isolates of Acinetobacter spp. were isolated and were answerable for infections in hospital admitted patients. In this study the isolates of Acinetobacter were isolated from different types of samples i.e. from Blood samples 12 (34.2%) followed by from urine sample 10 (28.5%), from pus 8 (22.8%), and from sputum 5 (14.2%) (Table1) and isolated from different types of hospital environment, i.e.18 (51.4%) isolates were from ICU, 10(28.5%) from surgical ward and 7 (20%) from gastro ward (Table 2). This Study also reveals that Acinetobacterinfection were common in patients in age group >50 years as 22 (62.8%)) followed by >30 years 13 [37.1%])and in male patients 21 (60%) followed by female patients 14 (40%).

 

In the present study different types of common antibiotics showed resistance and susceptibility against isolates of Acinetobacter spp. (Table 3). All the antibiotics used in present study, have deviations in their resistance and susceptibility patters against Acinetobacter isolates Maximum resistance was recorded for Imipenem (80%), followed by Ceftazidime (71.4%), Tobramycine (68.5%), Chloramphenicol (65.7 %), Doripenem (60%), Ciprofloxacin (57.1%), Tetracycline (54.2 %), Ceftriaxone (51.4%), Doxycycline 16(48.5%), Levofloxacin 15(45.7%), Cefoperazone 13 (40.0 %), Ampicillin 8 (25.7%), Amikacin (11.4 %) and Gentamycin (8.5%)] (Fig 1)

 

In this study out of 35 isolates 20(57.1%) isolates are positive for biofilm and 15 (42.8 %) are biofilm negative. Out of 20 biofilm producers, 12 (60%) are moderate biofilm producers and 8 (22.8%) are strong biofilm producers (Fig 2 and image 1).Comparison of antibiotic resistance results among biofilm producers and biofilm non producers has been shown in Table 4. Maximum resistance by biofilm producers was shown to Imipenem (89.7%) followed by Piperacillin (90.0%), followed by Ceftazidime (85.0 %), Tobramycin and Chloramphenicol (80.0%) and Doripenem (75.0%).

 

Table 1: Percentage of Acinetobacter isolates from different Sources

 

Table 2: Percentage of isolates from different environment.

 

Table 3: Antibiotic resistance pattern of Acinetobacter species

Antibiotics	No. of Resistance(n=35)	% of Resistance
Imipenem	28	80.0%
Ceftazidime	25	71.4%
Tobramycin	24	68.5%
Chloramphenicol	23	65.7%
Doripenem	21	60.0%
Ciprofloxacin	20	57.1%
Tetracycline	19	54.2%
Ceftriaxone	18	51.4%
Doxycycline	17	48.5%
Levofloxacin	16	45.7%
Cefoperazone sulbactam	14	40.0%
Ampilicin sulbactam	9	25.7%
Amikacin	4	11.4 %
Gentamycin	3	8.5%

 

 

Fig 1 Antibiotic resistance pattern (%) of Acenetobacter isolates

 

Table 4:Antibiotic resistance in biofilm positive and biofilm negative isolates

Antibiotics	No. of resistance isolates (n=35)	Biofilm positive resistance isolates(n=20)	Biofilm negative resistance isolates(n=15)
Imipenem	28 (80.0%)	18 (90.0%)	10 (66.6%)
Ceftazidime	25 (71.4%)	17 (85.0%)	08 (53.3%)
Tobramycin	24 (68.5%)	16 (80.0%)	08 (53.3%)
Chloramphenicol	23 (65.7%)	16 (80.0%)	07 (46.6%)
Doripenem	21 (60.0%)	15 (75.0%)	06 (40.0 %)

 

 

Fig 2 Biofilm formation by Acinetobacter isolates     

 

Fig 3 Biofilm producers and Non biofilm producers

 

Fig 4 Ratio of strong, moderate and non-biofilm producers 

 

 

Fig 5 Antibiotic resistance in biofilm positive and biofilm negative Acinetobacter isolates

 

DISCUSSION:

Acenetobacter spp. is an opportunistic pathogen and one of the main causes of nosocomial infections during the past few de-cades²³. Out of all the “unusual” pathogens till this time these isolates plays a major role in colonization and nosocomial infections including bacteraemia, urinary tract infections and secondary meningitis but their major role is as agents of nosocomial pneumonia, particularly in patients confined to intensive care units (ICU’s). Such infections are often particularly problematic the clinician because of widespread resistance of the pathogen to a large number of antibiotics²⁴. In the present study out of 35 isolates, 28 (80%) were resistant to Imipenem. and drugs like Amikacin and Gentamycin have been reported to be susceptible. In contrary to our results Mostofi et al²⁵. reported that clinical strains of Acenetobacter spp. Were resistant to Gentamicin (61%). High Imipenem resistance has been reported by other studies however drugs like amikacin, Ceftazidime, Quinolone have been reported effective in several other studies.^{26, 27}..In the present study out of 35 isolates 20(57.1%) isolates (08strong and 12 moderate) were biofilm producers by Microtitre plate method. This is in acconcordance with the study of Rao SR et al in which 62% isolates of Acinetobacter were biofilm producers²⁸. The results of standard microtiter plate using a qualitative approach is also almost in agreement with Kaleem's study. In his study, 63% of the bacteria produced biofilm and the rest were not able to do so¹⁷ This study was also comparable with the other study in which 63% isolates were biofilm producers²⁸. The present study reveals that Maximum resistance by biofilm producers was against to Imipenem (90.0%) followed by ceftazidime (85.0 %), Tobramycin and Chloramphenicol (80.0%) and Doripenem (75.0%) that is also evident from earlier studies that biofilm producing isolates of Acinetobacter spp. were resistant to Imipenem (89.7%), Amikacin in (79.4%), Ciprofloxacin (76.9%), and Aztreonam (74.3%)²⁹. Similar results were also reported in other studies in which biofilm positiveAcinetobacterspp. were resistance to Ceftazidime (95%), Cefepime (95%), Aztreonam (85%), Ciprofloxacin (85%), Amikacin (80%), Imipenem (65%) and pipercillin+Tazobactum (40%)¹¹. Analyses of the results of this study also indicate that there is a significant correlation between power of biofilm formation and antibiotic resistance; considering that, more than 90% of the bacteria with the ability to form biofilm were MDR. This result is in accordance with Hassan et al.¹⁷

 

It has been observed that normally, two properties are frequently associated with biofilm producers, as the increase in the synthesis of exopolysaccharide and the improvement of antibiotic resistance³⁰ Beside the, mechanism of antibiotic resistance such as production of chromosomally encoded β-lactamases, efflux pumps and mutations in the molecules of antibiotic target, also contribute to the survival of bacteria in biofilms³¹. In our study biofilm producers isolates have high levels of resistance (>75%) to Imipenem, Ceftazidime, Tobramycin Chloramphenicol and Doripenim is similar to other studies which show high levels of resistance (> 75%) to Imipenem, Ciprofloxacin, Cefotaxime, Amikacin and Aztreonam in India. ^{11, 27} Our results were with the finding of up to 90% resistance to both Imipenem and Ceftazidime It is evident from our study that high levels of antibiotic resistance was found in biofilm positive and less antibiotic resistance in biofilm negative hence, were able to observe an association between biofilm formation and resistance to most of the test antibiotics. However, with the high levels of antibiotic resistance, there was significant association between biofilm production and resistance to Imipenem, Ceftazidime, Tobramycin Chloramphenicol and Doripenim in biofilm producers and nonbiofilm producers was observed³². Nucleo et al.³³ showed that sub-minimum inhibitory concentrations of Imipenem induced biofilm formation in a clinical isolate of Acinetobacter in vitro.

 

CONCLUSION:

Acinetobacterspp. with speedy multiplication are coming out of unlimited resistance to even to the newer antibiotics. They more resistant to antibiotics in comparison to other gram-negative organisms. Due to easy continued existence in the hospital environment, they are most effective to cause nosocomial infections. The Combination of antibiotic resistance and biofilm forming ability of these isolates plays a decisive role in their in-vitro and in-vivo survival. With our Study it is demonstrated the ability of the clinical isolates of Acinetobacter spp to produce biofilm and resistance to commonly used antibiotics such as Cephalosporin, Aminoglycosides, Quinolone, Carbapenem was observed among biofilm producers Therefore our study concludes that there is a positive correlation between biofilm formation and multiple drug resistance in Acenetobacter spp. This trend of multidrug resistance among Acinetobacter spp. is a matter of concern Gentamycin and Amikacin may be the only effective therapeutic agent in the study. Therefore there is a need for alternate method of treatment for the infections caused by multidrug resistant Acenetobacter spp. Combination therapy can be an effective option. So a greater understanding of the antibiogram of Acenetobacter spp. will help in development of effective treatment. More research activities such as other methods of biofilm detection and further molecular support are needed to be carried out against these types of multidrug resistance microorganisms which are an upcoming crisis in the medical field.

 

REFERENCES:

1.      Fournier PE, Richet H. The epidemiology and control of Acinetobacter baumannii in health care facilities. Clin Infect Dis. 2006; 42:692–9. [PubMed]

2.      Jawad A, Heritage J, Snelling AM, Gascoyne-Binzi DM, Hawkey PM. Influence of relative humidity and suspending menstrua on survival of Acinetobacter spp. on dry surfaces. J Clin Microbiol. 1996; 34:2881–7. [PMC free article [Pub Med]

3.      Akers K, Chaney C, Barsoumian A, Beckius M, Zera W, Yu X, et al. Aminoglycoside resistance and susceptibility testing errors in Acinetobacter baumannii-calcoaceticus complex. J Clin Microbiol 2010; 48(4): 1132-8.

4.      Aliakbarzade K, Farajnia S, Karimi Nik A, Zarei F, Tanomand A. Prevalence of aminoglycoside resistance genes in Acinetobacter baumannii isolates. Jundishapur J Microbiol 2014; 7(10): e11924.

5.      Ardebili A, Lari AR, Talebi M. Correlation of ciprofloxacin resistance with the AdeABC efflux system in Acinetobacter baumannii clinical isolates. Ann Lab Med 2014; 34(6): 433-8.

6.      Karlowsky JA, Draghi DC, Jones ME, Thornsberry C, Friedland IR, Sahm D. Surveillance for antimicrobial susceptibility among clinical isolates of Pseudomonas aeruginosa and Acinetobacter baumannii from hospitalized patients in the United States, 1998–2001. Antimicrob Agents Chemother. 2003; 47:1681–88. [PMC free article] [PubMed]

7.      Guillou Joly ML. Clinical impact and pathogenicity of Acinetobacter. Clin Microbiol Infect. 2005; 11:868–73. [PubMed]

8.      Peleg AY, Seifert H, Paterson DL. Acinetobacter baumannii: emergence of a Successful pathogen. Clin Microbiol Rev. 2008; 21:538–82. [PMC free article] [PubMed]

9.      Abdi-Ali A, Hendiani S, Mohammadi P, Gharavi S. Assessment of biofilm formation and resistance to imipenem and ciprofloxacin among clinical isolates of Acinetobacter baumannii in Tehran. Jundishapur J Microbiol 2014; 7(1): e8606.

10.   de Breij A, Gaddy J, van der Meer J, Koning R, Koster A, van den Broek P, et al. CsuA/BABCDE-dependent pili are not involved in the adherence of Acinetobacter baumannii ATCC 19606T to human airway epithelial cells and their inflammatory response. Res Microbiol 2009; 160: 213-8.

11.   Dheepa M, Rashme VL, Appalaraju B. Comparison of biofilm production and multiple drug resistance in clinical isolates of Acinetobacter baumanii from a tertiary care hospital in South India. Int J Pharm Biomed Sci 2011; 2(4): 103-7.

12.   Espinal P, Martí S, Vila J. Effect of biofilm formation on the survival of Acinetobacter baumannii on dry surfacees. J Hosp Infect 2012; 80(1): 56-60.

13.   M'hamedi I, Hassaine H, Bellifa S, Lachachi M, Kara Terk I, Djeribi R. Biofilm formation by Acinetobacter baumannii isolated from medical devices at the intensive care unit from medical 532 Ebrahim Babapour et al./Asian Pac J Trop Biomed 2016; 6(6): 528–533 devices at the intensive care unit of the University Hospital of Tlemcen (Algeria). Afr J Microbiol Res 2014; 8(3): 270-6.

14.   Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME, Giske CG, et al. Multidrug-resistant, extensively drug-resistant and pan drug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect 2012; 18(3): 268-81

15.   Freeman DJ, Falkiner FR, Keane CT. New method for detecting slime production by coagulase negative staphylococci. J Clin Pathol 1989; 42: 872-4.

16.   Houang ET, Sormunen RT, Lai L, Chan CY, Leong AS. Effect of desiccation on the ultrastructural appearances of Acinetobacter baumannii and Acinetobacter lwoffii. J Clin Pathol 1998; 51(10): 786-8.

17.   Hassan A, Usman J, Kaleem F, Omair M, Khalid A, Iqbal M. Evaluation of different detection methods of biofilm formation in the clinical isolates. Braz J Infect Dis 2011; 15(4): 305-11

18.   Rodriguez BJ, Marti S, Soto S, Fernandez CF, Cisneros JM, Pachon J. Biofilm formation in Acinetobacter baumannii: associated features and clinical implications. Clin Microbiol Infect. 2008; 14(3):276–78. [PubMed]

19.   Bauer AW, Kirby WM, Sherris JC, Turuk M. Antimicrobial susceptibility testing by standardized single disc method. Am J Clin Pathol. 1966; 45(4):493–96. [PubMed]

20.   Clinical and Laboratory Standards Institute. Performance Standards for Antimicrobial Susceptibility testing; Twenty third Informational Supplement. CLSI document M100-S23.Wayne, PA:Clinical and Laboratory Standards Institute; 2013.

21.   Toledo AA, Valle J, Solano C, Arrizubieta MJ, Cucarella C, Lamata M, et al. The enterococcal surface protein, Esp, involved in Enterococcus faecalis biofilm formation. Appl Environ Microbiol. 2001; 6:4538–45. [PMC free article] [PubMed]

22.   Christensen GD, Simpson WA, Younger JJ, Baddour LM, Barrett FF, Melton DM, et al. Adherence of coagulase-negative staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices. J Clin Microbiol. 1985; 22(6):996–1006. [PMC free article] [PubMed]

23.   Constantiniu S, Romaniuc A, Iancu LS, Filimon R, Taras¸i I. Cul-tural and biochemical characteristics of Acinetobacter spp. strainsisolated from hospital units. J Prev Med 2004; 12: 35-42.

24.   BergoneBerezin E, Towner KJ. Acinetobacter spp. as Nosocomial Pathogens: Microbiological, Clinical, and Epidemiological Features. Clin Microbiol Rev. 1996; 9:148–65. [PMC free article] [PubMed]

25.   Mostofi S, Mirnejad R, Masjedian F. Multi-drug resistance inAcinetobacterbaumannii strains isolated from the clinical speci-mens of three hospitals in Tehran-Iran. Afr J Microbiol Res 2011; 5: 3579-982.

26.   Vijaya BS, Govindan R, Preeti P, Kurt S, Daniel T, Prapas P, et al. Genetic relatedness and molecular characterization of multidrug resistant Acinetobacter baumannii isolated in central Ohio, USA. Ann Clin Microbial Antimicrob. 2009; 8:21.

27.    Beck SCM, Jarvis WR, Brook JH, Culver DH, Potts A, Gay E, et al. Epidemic bacteremia due to Acinetobacter baumannii in five intensive care units. Am J Epidemiol. 1990; 132(4):723-33.

28.   .Rao SR, R Uma K, Singh SP, Shashikala P, Kanungo R, Jayachandran S, Prashanth K. Correlation between biofilm production and multiple drug resistance in imipenem resistant clinical isolates of Acinetobacter baumannii. Indian J Med Microbio. 2008; 26(4):333-7.

29.   Rodroguez BJ, Marti S, Soto S, Fernandez CF, Cisneros JM, Pachon J, et al. Bio film formation in Acinetobacter baumannii: associated features and clinical implications. Clin Microbiol Infect. 2008; 14:276-8.

30.   Bala M, Gupte S, Aggarwal P, Kaur M, Manhas A. Biofilm producing multidrug resistant Acinetobacter species from a tertiary care hospital: a therapeutic challenge. Int J Res Med Sci 2016; 4:3024-6.

31.   Costerton JW, Stewart PS, Greenberg EP. Bacterial biofilms: a common cause of persistent infections. Science. 1999; 284:1318–22. [PubMed]

32.   Hoiby N, Bjarnsholt T, Givskov M, Molin S, Ciofu O. Antibiotic resistance of bacterial biofilms. Int J Antimicrob Agents. 2010; 35(4):322-32. [DOI] [PubMed]

33.   Nucleo E, Steffanoni L, Fugazza G, Migliavacca R, Giacobone E, Navarra A, et al. Growth in glucose-based medium and exposure to subinhibitory concentrations of imipenem induce biofilm formation in a multidrug-resistant clinical isolate of Acinetobacter baumannii. BMC Microbiol. 2009; 9:270. [DOI] [PubMed]

 

 

 

 

 

Accepted on 21.05.2019           © RJPT All right reserved

Research J. Pharm. and Tech 2019; 12(8): 3737-3742.