Biofilm Formation and Colistin Susceptibility of Acinetobacter baumannii Isolated from Korean Nosocomial Samples

Abstract

Biofilm formation, a virulence factor of Acinetobacter baumannii, is associated with long-term survival in hospital environments and provides resistance to antibiotics. Standard tests for antibiotic susceptibility involve analyzing bacteria in the planktonic state. However, the biofilm formation ability can influence antibiotic susceptibility. Therefore, here, the biofilm formation ability of A. baumannii clinical isolates from Korea was investigated and the susceptibility of biofilm and planktonic bacteria to colistin was compared. Of the 100 clinical isolates examined, 77% exhibited enhanced biofilm formation capacity relative to a standard A. baumannii strain (ATCC 19606). Differences between the minimal inhibitory concentrations and minimal biofilm-inhibitory concentrations of colistin were significantly greater in the group of A. baumannii that exhibited enhanced biofilm formation than the group that exhibited less ability for biofilm formation. Thus, the ability to form a biofilm may affect antibiotic susceptibility and clinical failure, even when the dose administered is in the susceptible range.

Introduction

Acinetobacter species are ubiquitous, nonlactose fermenting gram-negative coccobacilli and have emerged as a major cause of hospital-acquired infections, especially ventilator-associated pneumonia in Asia and Europe.⁸ Nosocomial infections caused by Acinetobacter baumannii have been associated with an 8–40% increased risk in mortality. Moreover, a substantial increase in the resistance to all major classes of antibiotics by A. baumannii has been observed,³⁰ further complicating the treatment of these infections.

Biofilm formation is one of the virulence factors of A. baumannii associated with long-term survival in the environment and enhances resistance to antibiotics.¹⁸ A. baumannii can survive on fingertips, glass, plastics, and other environmental surfaces, even dry surfaces. Also, biofilm formation contributes to the high level of resistance to desiccation and disinfection, facilitating the survival of bacteria in a hospital setting.⁷ Moreover, the ability to form a biofilm facilitates contact with susceptible patients, leading to outbreaks of medical device-related infections and ventilator-associated pneumonia.^4,15

Despite the high rate of treatment failure, colistin is the last option for treating multidrug-resistant A. baumannii infections. However, colistin has a relatively low intrinsic efficacy, suboptimal lung penetration, and the risk of significant renal toxicity.⁹

The minimal inhibitory concentration (MIC) is the lowest concentration of an antimicrobial that can inhibit the growth of a microorganism and is commonly used to determine the efficacy of an antimicrobial agent. However, the MIC represents the sensitivity of the bacteria in their planktonic state and does not reflect the susceptibility of cells in a biofilm state. Organisms that cause device-related and other chronic infections that grow in biofilms in or on these devices are extremely difficult to treat due to the intrinsic resistance provided by the biofilm lifestyle to numerous antimicrobial agents and products of the immune system.⁵ Under these circumstances, MICs do not correlate with treatment outcomes or antimicrobial efficacies. Generally, bacteria require much higher concentrations of antibiotics to inhibit their growth in vivo than in vitro. For some antibiotics, the concentration required to kill bacteria in a biofilm may be 1,000-fold greater than that required to kill planktonic bacteria of the same strain.²⁴

Only a few studies have reported on biofilm formation of A. baumannii clinical isolates and antimicrobial susceptibility of the biofilm state.^15,29 Therefore, we investigated the biofilm formation ability of A. baumannii clinical isolates and the difference in MICs of colistin for both planktonic and biofilm cells.

Materials and Methods

Bacterial strains

A total of 100 A. baumannii isolates was obtained between July and December 2011 from the Keimyung University Dongsan Medical Center, Daegu, Korea. Most isolates (90%) originated from sputum. The isolates were identified using conventional biochemical methods, and 16S rRNA sequencing. A. baumannii ATCC 19606 (ATCC, Manassas, VA) was used as a positive control for the biofilm formation assay and Escherichia coli DH-5α (ATCC) strain was used as a negative control. A. baumannii ATCC 19606, E. coli DH-5α, and the 100 clinical isolates of A. baumannii were maintained on Muller–Hinton (MH) agar at 35°C.

Antibiotics and experimental compounds

Colistin sulfate was obtained from Sigma-Aldrich (St. Louis, MO). A colistin solution was prepared (5.12 mg/ml) and frozen until used. A resazurin (Sigma-Aldrich) solution was prepared in phosphate-buffered saline (PBS) at a concentration of 0.02 mg/ml. When resazurin enters the bacterial cell, it is reduced to resorufin, resulting in a change in the blue color and production of very bright red fluorescence. Thus, resazurin can be used to measure bacterial viability.²

Biofilm formation assay

Biofilm formation was assayed by crystal violet staining, as described previously.²⁵ Fresh bacterial suspensions were prepared from overnight cultures and adjusted to an optical density (OD₆₀₀) of 0.1. The bacterial suspensions (100 μl) were inoculated into individual wells of a 96-well plate and incubated at 35°C for 24 hr. After an overnight incubation, the plates were gently washed twice with 200 μl of PBS, air-dried, and stained with 0.1% crystal violet (100 μl) for 15 min at room temperature. Plates were gently washed twice with PBS, the stain was solubilized with 99% ethanol, and the OD₅₇₀ of the supernatant was measured using a Victor 3 microplate reader (PerkinElmer, Waltham, MA). Biofilm formation was defined as the optical density of solubilized crystal violet in 99% ethanol that was higher than the average optical density of A. baumannii ATCC 19606. All experiments were performed in triplicate. Each plate included the following controls: media alone, A. baumannii ATCC 19606 (positive control), and E. coli DH-5α (negative control).

Colistin susceptibility assay

Twenty-eight isolates were selected to compare the colistin susceptibility between planktonic and biofilm states; 14 isolates were selected from samples producing more biofilm than A. baumannii ATCC 19606 and 14 isolates were selected from those producing less biofilm than ATCC 19606.

Planktonic susceptibility test (microdilution test)

MICs were determined using the broth microdilution test according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI, M100-S22, January 2012). To standardize the inoculum density for a susceptibility test, a barium sulfate (BaSO₄) turbidity standard equivalent to a 0.5 McFarland standard was used.

The inoculated microdilution plates were incubated at 35°C for 18–20 hr. After the incubation, 10 μl of resazurin was added and the plates were shaken and incubated at 35°C for 1 hr. After incubation with the resazurin, visual MICs were recorded. Planktonic MICs were defined as the lowest drug concentrations that prevented a change from blue color at 60 min after the addition of resazurin.

Biofilm susceptibility test

The microplate resazurin biofilm susceptibility test was used to determine the susceptibility of biofilms to colistin.²⁶ Isolated colonies from MH agar plates incubated for 18–22 hr were used to prepare inocula. Assays were performed in flat-bottom, polystyrene tissue-culture microtiter plates containing 5×10⁵ CFU/ml in cation-adjusted MH broth with final well volumes of 100 μl. Plates were incubated at 35°C without shaking. After 24 hr, 50 μl was removed from all experimental and control wells, and 50 μl of the appropriate drug dilution was added. Twofold dilutions of colistin in cation-adjusted MH broth were prepared external to the plates, diluted from 256 to 0.125 μg/ml. The plates were incubated at 35°C for 24 hr without shaking. After 24 hr, 10 μl resazurin was added to the wells and the plates were shaken gently and incubated for 1 hr. After 1 hr of incubation with the resazurin, visual minimal biofilm inhibition concentrations (MBICs) were recorded. MBICs were defined as the lowest drug concentrations that prevented a change from a blue color after 60 min of incubation with resazurin.

Statistical analyses

Data management and statistical analyses were performed with SPSS software version 21.0 (SPSS, Chicago, IL). For the comparison of the differences in MICs and MBICs, the concentration was changed with 2 logarithms. The differences of MICs and MBICs were compared with Student's t-test. A p-value <0.05 was considered statistically significant.

Results

Biofilm formation by A. baumannii clinical isolates

The ability of A. baumannii ATCC 19606 to form a biofilm has been established.^10,31 Biofilm formation ability was defined as an optical density of solubilized crystal violet in ethanol that was higher than the average optical density of A. baumannii ATCC 19606. The mean optical density of A. baumannii ATCC 19606 was 0.637. Seventy-seven out of 100 strains (77%) produced more biofilm than A. baumannii ATCC 19606. The interquartile range of the optical density for the biofilm mass was 0.588–1.11, and the average was 0.899 (Fig. 1).

FIG. 1.

The biofilm mass of Acinetobacter baumannii clinical isolates. To measure the relative amount of biofilm, crystal violet-stained biofilms were solubilized with ethanol (99%) for 5 min. The solubilized crystal violet was measured at 570 nm using a Victor 3 microplate reader. (A) The optical density of the solubilized crystal violet from the biofilm staining. The average optical density of the standard strain of A. baumannii ATCC 19606 was 0.637 (dotted line). (B) The interquartile range of the optical density for the biofilm mass was 0.588–1.11, and the average was 0.899.

Colistin susceptibility of planktonic and biofilm cells

Colistin susceptibility was assayed for both planktonic and biofilm states. Each experiment was performed with 28 isolates. Fourteen isolates were selected from tested isolates producing more biofilm than A. baumannii ATCC 19606 and 14 isolates from those producing less biofilm than the ATCC 19606 strain. The MIC values for colistin were determined using a broth microdilution assay. Among the 28 tested isolates, 24 isolates were susceptible to colistin in the planktonic state (Table 1). The MICs were ≤2 μg/ml. The MICs of the other isolates were 4 μg/ml (three strains) and 32 μg/ml (one strain).

Table 1.

MICs and MBICs of Colistin Against A. baumannii

Low level of biofilm^a (n=14)			High level of biofilm^b (n=14)
Biofilm mass (OD)	MIC (μg/ml)	MBIC (μg/ml)	Biofilm mass (OD)	MIC (μg/ml)	MBIC (μg/ml)
0.125	1	8	1.285	1	256
0.139	2	64	1.330	1	256
0.150	1	8	1.333	0.5	64
0.157	2	32	1.442	2	256
0.181	1	8	1.444	0.5	16
0.200	1	32	1.855	1	256
0.228	1	64	1.943	2	64
0.292	32	256	2.085	2	32
0.294	1	64	2.133	2	256
0.294	1	8	2.242	1	64
0.324	4	8	2.321	0.5	32
0.325	2	8	2.332	4	256
0.334	2	32	2.475	1	64
0.365	4	128	2.519	2	256

Clinical isolates of A. baumannii producing less biofilm than ATCC 19606 (n=14).

Clinical isolates of A. baumannii producing more biofilm than ATCC 19606 (n=14).

MBIC, minimal biofilm inhibition concentration; MIC, minimal inhibitory concentration.

When biofilms of the same strains were tested, MBICs were at least eightfold higher relative to the planktonic MICs (Table 1). These data were in accordance with previous reports that indicated an increase in drug resistance due to biofilm formation when compared to a planktonic state.²⁴ The median MIC of the 28 isolates was 1 μg/ml and the median MBIC of the 28 isolates was 64 μg/ml. The median MIC and MBIC of high biofilm-producing strains versus the low biofilm-producing strains were 1 and 160 versus 1.5 and 32.

The MICs and the MBICs were changed with 2 logarithms for the t-test (Table 2). The average of the difference between MBICs and MICs was about a twofold increase in high biofilm-producing strains (Table 2). This suggested that strains that produce more biofilm required a higher antibiotic concentration for the inhibition of bacterial growth.

Table 2.

Comparison of Differences Between MICs and MBICs

	Low level of biofilm (n=14)	High level of biofilm (n=14)	p-Value
Average of differences^a	3.786	6.429	<0.001

Difference=log₂ (MBIC)−log₂ (MIC).

Discussion

In the present study, biofilm production varied extensively among the strains and 77% of the A. baumannii clinical isolates exhibited a greater capacity for biofilm formation than A. baumannii ATCC 19606. This is a relatively higher rate than that observed in other studies. Only a few reports have described the biofilm formation ability of A. baumannii clinical isolates. In the United States, 55% of A. baumannii strains isolated from wounds were reported to be biofilm-forming strains, with a median biofilm mass of 0.125±0.061.²⁹ A study from Spain reported that 63% of A. baumannii isolates formed a biofilm.²⁸ In Korea, it was previously reported that all 23 strains of multidrug-resistant A. baumannii formed biofilm.¹⁸ The higher rate of biofilm-forming strains may be one of the reasons for the recent increase in the incidence of nosocomial A. baumannii infections such as the catheter-associated urinary tract infection, bacteremia, and nosocomial pneumonia in Korea.¹⁷

A bacterial biofilm is a surface-associated community arranged in a tertiary structure and is often encased in an extracellular matrix composed of macromolecule(s) such as carbohydrates, nucleic acids, and/or proteins.³ Biofilm formation is medically relevant as a contributing factor to the chronic nature of infections and the inherent resistance to antimicrobial therapy. In ventilator-associated pneumonia, the formation of a biofilm on the surface of the endotracheal tube is an almost universal phenomenon and has been related to pathogenesis.²⁷ A. baumannii and Pseudomonas aeruginosa are the most prevalent bacteria that form a biofilm on endotracheal tubes. It has been reported that there is a microbial link between airway colonization, biofilm formation, and ventilator-associated pneumonia development.¹² Bacterial survival on endotracheal tubes through biofilm formation serves as a pathogenic mechanism for microbial persistence and impaired response to antimicrobial therapy.

The treatment of A. baumannii infections has become a challenge for clinicians. These infections have become a real threat to international public health due to the rapid spread of antimicrobial resistance and the slow development of novel antimicrobials.⁶ Furthermore, the genetic potential of multidrug-resistant A. baumannii to carry and transfer diverse antibiotic resistance determinants poses a major threat in hospitals.²³ This contributes largely to the severity and lethality of infections, as well as the high rate of chronic infections, as the organism is able to persist and survive for a long period of time, even after treatment with disinfectants and antibiotics.

Biofilm-related infections are 10–1,000 times more resistant to the effects of antimicrobial agents.²⁰ Indeed, many nosocomial pathogens exist predominantly in a biofilm state within the tissue or on the surface of medical devices, and such infections are extremely difficult to treat. The MIC has been used as a gold standard for the determination of antimicrobial efficacy. It is generally understood that antimicrobials that are ineffective in preventing growth will also be clinically ineffective. However, an organism that is sensitive in vitro may not be sensitive in vivo, as in the case of colistin in A. baumannii infections. Many reasons for colistin failure have been reported, such as a low intrinsic efficacy, suboptimal lung penetration, and the risk for significant renal toxicity.^14,19

In this study, the median MICs of isolates in the planktonic state were 1 μg/ml. This suggests that most isolates were susceptible to colistin when in the planktonic state. However, when in the biofilm state, the median MBIC was 64 μg/ml. Thus, A. baumannii biofilms require a much higher concentration of colistin to kill A. baumannii, which is growing in a biofilm.

On an average, there was about a twofold increase in the MBICs relative to the MICs in strains producing a large amount of biofilm. These findings suggest that the strains producing more biofilm required a higher antibiotic concentration for inhibition. This might influence the failure of colistin, despite the administration of a dose within the range of susceptibility. Unfortunately, we do not have clinical data to confirm if what we observed in vitro was applicable to patient outcomes.

Consistent with these results, previous studies have shown that the concentration of colistin required to eradicate a multidrug-resistant P. aeruginosa biofilm was 4- to 10-fold higher than the MICs of planktonic cells.²¹ It was suggested that further studies are needed for other biofilm-forming bacteria.

In the present study, a large proportion of clinical isolates of A. baumannii from the Dongsan Medical Center (Daegu, Korea) was found to produce biofilm. The previous study reported that the ability to form biofilm is higher for A. baumannii strains corresponding to International clone (IC)-II such as sequence type (ST) 2, ST 25, and ST 78.¹¹ There are few studies about the epidemic clones in Asian countries, including Korea. Some studies about clonal diversity of Korean isolates showed IC-II was most prevalent.^13,16 It seems to need further experiments about the virulence-related traits like biofilm-forming ability of the epidemic strain assigned to distinct genotypes.

The strains that produced more biofilm required higher concentrations of colistin to inhibit bacterial growth in the biofilm state. These results suggest that biofilm formation is an important factor influencing colistin susceptibility of A. baumannii in vivo. In the near future, the routine determination of the biofilm inhibition concentration may be needed in the treatment of medical device-related A. baumannii infections and ventilator-associated A. baumannii pneumonia, especially if the device cannot be removed. The association with biofilm formation may help in the determination of the dose of colistin and predicting clinical outcomes.

Apart from biofilm, another important mechanism against colistin resistance is gene mutation. There are two gene mutations associated with colistin resistance. One is the total inactivation of the lipopolysaccharide (LPS) through mutations in LPS-producing genes (lpxA, lpxC, and lpxD).²² The second mechanism is modification of lipid A components of LPS through mutations in the pmrA and pmrB genes encoding two-component signaling protein.¹ We need additional experiments about the gene mutations to confirm the biofilm effect on colistin susceptibility.

These results suggest the need for further investigation involving a large number of multicentric A. baumannii isolates collected from Korea. Additional research is needed on possible links between biofilm formation and nosocomial infection such as application of MBIC to patient care and outcomes, as well as methods for the control of biofilm-related infections.

Footnotes

Acknowledgments

The authors thank Hui-Jung Jung, Hyejin Park, and Yun yi Yang for their support and technical assistance in experiments.

Disclosure Statement

The authors declare that they have no competing interests.

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