Introduction: Antibiotic resistance is an important issue that may worsen the clinical evolution of high risks patients. The increasing use of colistin for the treatment of carbapenem-resistant Acinetobacter baumannii infections has led to the emergence of colistin resistance. Since A. baumannii is an important nosocomial pathogen and is responsible for severe infections, the use of antimicrobial combinations has been strongly recommended. Materials & Methods: This study aims to evaluate the in vitro effect of colistin/rifampicin and colistin/meropenem combinations against colistin-resistant A. baumannii isolates using two rapid E-test methods. Results: Synergy was observed with colistin in combination with rifampicin against all the isolates. Otherwise, colistin/meropenem combination showed indifference by E-test cross formation and synergy dependent by meropenem concentration by the E-test gradient. Discussion and conclusion: Nowadays A. baumannii is one of the most important emerging, opportunistic pathogens that can affect critical patients. Colistin, the main antibiotic effective against such bacterium, when associated with rifampicin shows an enhanced activity‎.

Keywords:  Acinetobacter baumannii, antibiotic resistance, antimicrobial stewardship, antimicrobial susceptibility test, colistin-resistance, E-test.

Introduction

Antibiotic resistance is a global emergency [33], so in recent years WHO and the EU have released the “Global action plan on antimicrobial resistance” and the “Council conclusions on the next steps under a One Health approach to combat antimicrobial resistance”, respectively, to attempt to limit its effects. [1-4] Medical device exhibits the advancement of the medical field and conjointly providing a larger contribution on lives. [34] Antibiotic resistance development has been a major issue for managing infections both in community and hospital settings. [35]

According to these guidelines, research is currently aimed worldwide to diminish the spreading of bacterial resistant strains and to isolate and produce new, effective antibiotics. [5, 6]

However, many studies are currently aimed to define the usefulness and the antibacterial activity of essential oils and probiotics or synbiotics used as antibiotic substitutes or supportive antimicrobial therapy. [7-13]

Acinetobacter baumannii is ubiquitous and can be found as colonizer of the skin, respiratory and digestive tract in both patients and healthcare professionals. It is an obliged aerobic Gram-negative coccobacillus, can survive in the environment for long periods, even up to 30 days, and may act as an opportunistic pathogen able to develop multidrug resistance against the major antibiotic classes including carbapenem. Microbes are also becoming resistant to available antimicrobial drugs, thus requiring alternative medications. [36] In recent years it emerged as one of the leading nosocomial pathogens, particularly in severely ill patients and, in the last decade, carbapenem-resistance associated with resistance to the other routinely tested antibiotics has become common for the majority of A. baumannii strains. [14, 15] The impressive number of acquired mechanisms of resistance makes it difficult to select an appropriate antimicrobial agent and leads to the renewed clinical use of colistin which is increasingly used as the last line of defense despite its nephrotoxicity and neurotoxicity.  [16, 17] More recently, resistance to colistin has been reported in A. baumannii clinical isolates probably because of its use in monotherapy, restricting further treatment options. [18]

The rapid emergence of resistance to colistin has been attributed to the high frequency of heteroresistant isolates which are rapidly selected as dominant resistant populations during colistin treatment. [19] Also, monotherapy is less effective than combination therapy, despite in vitro tests revealed drug susceptibility; thus, antimicrobial combinations are explored as potential therapeutic options. [20] Antimicrobial Stewardship Program has been an essential program in hospitals. One of its objectives is to prevent the emergence of antimicrobial resistance. [32] Bacterial infections that had been willingly cured by AM are lasting longer due to resistance emergence and this will lead to intense morbidity and mortality. [37]

Previous in vitro studies have investigated the benefits of different combination therapies against colistin-susceptible A. baumannii, especially the activity of imipenem, tigecycline, rifampicin and colistin alone or in combination. [21, 22]

A combination of antimicrobial agents, with a synergistic effect in vitro, has been associated with a better clinical outcome. Therefore, the clinicians need it to be performed synergist tests but both checkerboard and time-kill kinetic tests are time-consuming and unsuitable for daily laboratory routine.

To be clinically useful, a synergistic test should be rapid and easy to perform. [23] To study antibiotic synergy, E-test strips have been used in two different ways: in a cross formation and Mueller Hinton agar supplemented with an antibiotic. [24, 25]

This study aimed to evaluate in vitro the activity of colistin in combination with rifampicin or meropenem against colistin-resistant A. baumannii strains using two different E-test methods for the detection of synergy.

 

Materials and Methods

Twenty colistin-resistant Acinetobacter calcoaceticus baumannii complex strains collected at Policlinico University Hospital, Bari, Italy, were selected for the study. The strains were identified by biochemical profiling using the Vitek2 system (bioMerieux, France) and confirmed as A. baumannii by MALDI-TOF assay (bioMerieux, France). Antibiotic susceptibility was assessed using the GN202card of the Vitek2 system and the results were interpreted according to the current EUCAST clinical breakpoints. Colistin resistance was confirmed by broth microdilution (Merlin). [26]

To test synergy, MICs of colistin, meropenem, and rifampicin were determined by E-test. Briefly, each strain was matched to a 0.5 McFarland turbidity standard and inoculated in Mueller-Hinton agar plates. E-test strips (Liofilchem) were placed on the inoculated agar plates and incubated overnight at 35°C degrees in ambient air. 

E-test cross formation

Each isolate was matched to a 0.5 McFarland standard and streaked on Mueller-Hinton agar plates. The E-test strips of COL/MER and COL/RIF were placed on Mueller-Hinton agar in a cross formation, with a 90° angle at the intersection between the scales at their respective value of MICs. The plates were incubated at 35-37 °C overnight and the zones of inhibition were read as the value where the eclipse intersected the scale on E-test strips. The nature of drug interaction (synergy, indifference or antagonism) was determined based on the calculated fractional inhibitory concentration (FIC) index as described for the checkerboard method.

E-test agar dilution

Mueller Hinton agar, supplemented with rifampicin or meropenem were prepared at a final concentration of 32, 16, 8, 4, 2 µg/ml, respectively. Plates were inoculated with the same bacterial suspension used for E-test cross formation, E-test strips of colistin were applied and MICs of colistin were read after 24 hours of incubation at 35-37 °C.

 

Results

All A. baumannii strains were resistant to colistin (MIC₅₀ and MIC₉₀ > 16µg/ml), ciprofloxacin, gentamicin, imipenem, meropenem, and trimethoprim/sulfamethoxazole. 

Table 1 shows MICs obtained for COL and RIF alone and MICs obtained for the respective antibiotic combination. The concordance among both methods of synergy testing was observed for all isolates tested. In fact, with the E-test cross formation, the FIC index ranging from 0.18 to 0.5 (FICI <0.5 was considered synergistic) while with E-test gradient dilution MICs of COL decreased below the susceptibility breakpoint (MIC <2 µg/ml).

 

Table 1: Comparison of results by E-test cross formation and E-test gradient dilution for synergistic activity of colistin (COL) and rifampicin (RIF) combination. (-) indicates no growth

	E-test Cross formation					E-test Gradient dilution with RIF (ug/ml)
	MICs (ug/ml)					COL MIC (ug/ml)
Strains.	COL alone	COL col+rif	RIF alone	RIF col+rif	FICI	32	16	8	4	2
1	256	48	32	8	0.43	0.25	0.38	0.5	0.75	1
2	256	32	32	8	0.37	0.5	0.5	0.75	0.75	0.75
3	256	48	32	6	0.36	0.38	0.5	0.75	1	1
4	256	48	32	3	0.27	0.5	1	1	2	2
5	256	48	32	8	0.43	0.5	0.75	1	1	1.5
6	256	32	32	8	0.37	0.38	0.5	0.5	0.75	1
7	256	24	32	8	0.34	0.25	0.12	0.125	0.12	0.09
8	256	24	32	8	0.34	0.38	0.38	0.5	0.75	1
9	256	48	32	6	0.36	0.38	0.5	0.25	0.75	0.75
10	256	6	32	8	0.27	0.38	0.38	0.38	0.5	1
11	8	1.5	32	12	0.55	0.25	0.38	0.5	0.5	0.25
12	256	16	32	3	0.15	0.38	0.75	0.75	0.75	0.75
13	256	16	2	0.5	0.31	-	-	-	-	-
14	256	48	2	0.5	0.45	-	-	-	-	0.75
15	12	2	6	0.75	0.28	-	-	-	-	0.25
16	6	1.5	32	6	0.43	0.38	0.38	0.75	0.75	0.25
17	6	2	32	16	0.8	0.38	0.5	0.75	0.75	1.5
18	6	2	32	6	0.5	0.25	0.25	0.5	0.5	0.5
19	12	3	32	4	0.37	0.25	0.25	0.19	0.38	0.5
20	12	3	32	12	0.62	0.38	0.38	0.38	0.5	0.75

 

The results of the COL and MER combination are shown in table 2. With E-test cross formation, the FIC index ranged from 0.21 to 1.75. In detail, for 4 strains the antimicrobial combination was a synergistic while for all the other was indifferent. With E-test gradient dilution, the results showed synergistic activity-dependent to meropenem concentration in the medium.

 

Table 2. Comparison of results by E-test cross formation and E-test agar dilution for synergistic activity of colistin (COL) and rifampicin (RIF) combination. (-) indicates no growth

	E-test Cross formation					E-test Gradient dilution with MER (ug/ml)
	MIC (ug/ml)					COL MIC (ug/ml)
Strains	COL alone	COL col+mer	MER alone	MER Mer+col	FICI	32	16	8	4	2
1	256	128	12	6	1	-	-	1	6	16
2	256	128	32	8	0.7	-	1	1.5	3	3
3	256	32	32	6	0.3	-	1.5	3	6	256
4	256	64	32	8	0.5	-	16	256	256	256
5	256	128	32	16	1	-	1.5	4	256	256
6	256	128	32	8	0.7	-	1.5	1.5	2	2
7	256	128	32	16	1	-	0.38	1.5	2	4
8	256	128	32	12	0.8	-	0.75	1.5	4	4
9	256	256	16	6	1.3	-	-	3	12	12
10	256	128	32	12	0.8	-	1.5	8	256	256
11	8	4	32	16	1	-	1	1.5	2	2
12	256	24	32	4	0.2	-	1	1.5	6	4
13	256	128	32	12	0.8	-	-	1	4	4
14	256	96	32	8	0.6	-	0.75	1.5	1.5	2
15	12	6	32	8	0.7	0.5	0.75	1.5	1.5	1.5
16	6	6	32	32	2
17	6	3	32	16	1	-	0.75	1.5	3	3
18	6	1.5	32	6	0.4	-	1.5	1.5	2	1.5
19	12	6	32	6	0.6	-	2	3	16	8
20	12	6	32	16	1	-	0.38	0.75	1	0.75

 

At last, MICs of colistin decreased below to susceptibility breakpoint only in presence of meropenem concentration of 32-16 µg/ml.

 

Discussion

A. baumannii, commonly known for causing ventilator-associated pneumonia (VAP), bloodstream infections and sepsis, especially in critically ill hosts, is becoming difficult to treat because of the limited options available. [27]

The therapy of carbapenem-resistant strains often requires the use of colistin, but alarming resistance rates are rising and are slowly becoming a routine phenotype for this organism. [28, 29]

Colistin-resistant A.baumannii occurred among patients who received colistin to treat infection due to A. baumannii carbapenem-resistant but colistin susceptible (Nuovo). The colistin resistance is strongly associated with Lipid A modification by the addition of phosphoethanolamine. [19, 30]

Performing synergy tests may be helpful for the choice of combination therapy, especially in care units where patients are critically ill and where resistant strains are more common and difficult to treat. Currently, in patients with MDR gram-negative pneumonia, the colistin combination therapy turned out to be more successful than colistin monotherapy. [31, 32]

Then, a reliable in vitro test that could accurately predict the synergy of antibiotic combinations, simple to perform and reproducible would be an important contribution for its use in clinical settings.

We assessed in vitro activity of colistin in combination with meropenem and rifampicin against 20 colistin-resistant A. baumannii strains, using two rapid E-test methods. Our data showed that colistin/rifampicin combination presents high synergistic activity in vitro with both methods for all the tested strains; on the other hand, for colistin/meropenem combination there was no concordance of results. With E-test agar dilution the synergistic activity correlated to the concentration of meropenem and it was synergistic at 32-16 µg/ml. However, this study demonstrates that the colistin/rifampicin combination is effective against colistin-resistant strains while the colistin/meropenem combination may be synergistic only in the presence of an adequate concentration of meropenem.

Conclusions

Antimicrobial resistance is an increasingly serious global health problem for both humans and animals, which limits or renders treatment options less effective while decreasing the quality of life and increasing the cost of healthcare. In the EU, every year, are an estimated 25,000 deaths for infections caused by antimicrobial-resistant bacteria.

At this rate, it is assumed that by 2050 the number of people who died prematurely worldwide due to antimicrobial resistance will be 300 million.

Nowadays A. baumannii is one of the most important emerging, opportunistic pathogens that can affect critical patients. It requires complex antibiotic therapy, including colistin, the effect of which can be enhanced by associating rifampicin as demonstrated by our results.

Conflict of interests: The authors declare no conflicts of interest.

Funding: No grants or private funds were available for this paper.

References

1. Aflatoonian M R, Alizade H, Jajarmi M, Ghanbarpour R, Shamsaddini Bafti M, Askari A, Adib N, Khatami M. Phylotyping of Antibiotic Resistant, Shiga Toxin-Producing and Atypical Enteropathogenic Escherichia Coli Strains Isolated from Ovine and Caprine Carcasses in Iran. J. Biochem. Tech. 2018; Special Issue (2): 41-48.
2. Evans CD, Lewis JWS. Collaborative Antimicrobial Stewardship in the Health Department. Infect Dis Clin North Am. 2020; 34(1):145-160. DOI:10.1016/j.idc.2019.10.002
3. Ronga L, Abbasciano A, Calia C, Mosca A, Battista M, Sparapano E, et al. Trends in the antibiotic resistance of S. aureus clinical isolates: a 4 years retrospective study in a teaching hospital in South Italy. Infez Med. 2019; 27(3):266-27
4. World Health Organization. Global action plan on antimicrobial resistance. 2015 https://apps.who.int/iris/bitstream/handle/10665/193736/9789241509763_eng.pdf?sequence=1 (Last access: Feb. 15, 2020)
5. European Union. Council conclusions on the next steps towards making the EU a best practice region in combatting antimicrobial resistance. 2019 https://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:52019XG0625(01)&from=EN (Last access: Feb. 15, 2020).
6. Taramian S, Mahfoozi L, Soodmand M. Antibiotic Resistance Trend of Uropathogens in a Tertiary Teaching Hospital in Rasht, Iran, a Longitudinal Study. Int. J. Pharm. Phytopharm. Res. 2019; 9(1): 98-102.
7. Randhawa M A, Bawadekji A, Al Ali M, Oueslati M H, Jamith B W. Antimicrobial Effects of Methanolic Extract of Salvia Officinalis L, Including MRSA and Multidrug Resistant Acinetobacter Baumannii. Int. J. Pharm. Phytopharm. Res. 2018; 8(4): 1-5.
8. Hernando-Amado S, Coque TM, Baquero F, Martínez JL. Defining and combating antibiotic resistance from One Health and Global Health perspectives. Nat Microbiol. 2019; 4(9):1432-1442. doi: 10.1038/s41564-019-0503-9
9. Dalfino L, Puntillo F, Mosca A, Monno R, Spada ML, Coppolecchia S, et al. High-dose, extended-interval colistin administration in critically ill patients: is this the right dosing strategy? A preliminary study. Clin Infect Dis. 2012; 54(12):1720-6. DOI: 10.1093/cid/cis286.
10. Yu Z, Tang J, Khare T, Kumar V. The alarming antimicrobial resistance in ESKAPEE pathogens: Can essential oils come to the rescue? Fitoterapia. 2020; 140:104433. DOI: 1016/j.fitote.2019.104433
11. Man, A., Santacroce, L., Jacob, R., Mare, A., Man, L. Antimicrobial activity of six essential oils against a group of human pathogens: A comparative study. Pathogens 2019; 8(1):15 DOI: 10.3390/pathogens8010015
12. Valdivieso-Ugarte M, Gomez-Llorente C, Plaza-Díaz J, Gil Á. Antimicrobial, Antioxidant, and Immunomodulatory Properties of Essential Oils: A Systematic Review. Nutrients. 2019; 11(11). PII: E2786. DOI: 10.3390/nu11112786
13. Santacroce L, Charitos IA, Bottalico L. A successful history: probiotics and their potential as antimicrobials. Expert Rev Anti Infect Ther. 2019; 17(8):635-645. DOI: 10.1080/14787210.2019.1645597
14. Peters VBM, van de Steeg E, van Bilsen J, Meijerink M. Mechanisms and immunomodulatory properties of pre- and probiotics. Benef Microbes. 2019 Apr 19;10(3):225-236. DOI: 10.3920/BM2018.0066
15. Ballini, A., Santacroce, L., Cantore, S., Bottalico, L., Dipalma, G., De Vito, D., Saini, R., Inchingolo, F. Probiotics improve urogenital health in women. Open Access Maced J Med Sci 2018; 6(10):1-6. DOI: 10.3889/oamjms.2018.406
16. Ballini A, Santacroce L, Cantore S, Bottalico L, Dipalma G, Topi S, et al. Probiotics Efficacy on Oxidative Stress Values in Inflammatory Bowel Disease: A Randomized Double-Blinded Placebo-Controlled Pilot Study. Endocr Metab Immune Disord Drug Targets. 2019;19(3):373-381. DOI:10.2174/1871530319666181221150352.
17. Abdulsahib, W K, Fadhil O Q, Abood S J. Antimicrobial susceptibility pattern isolated from different clinical samples in Baghdad hospitals. J. Adv. Pharm. Edu. Res. 2020; 10(1): 51-59.
18. Maragakis LL, Perl TM. Acinetobacter baumannii: epidemiology, antimicrobial resistance, and treatment options. Clin Infect Dis, 2008; 46:1254-1263
19. Pormohammad A, Mehdinejadiani K, Gholizadeh P, Nasiri MJ, Mohtavinejad N, Dadashi M, et al. Global prevalence of colistin resistance in clinical isolates of Acinetobacter baumannii: A systematic review and meta-analysis. Microb Pathog. 2020; 139:103887. DOI: 10.1016/j.micpath.20103887
20. Fishbain J, Peleg AY. Treatment of Acinetobacter baumannii. Clin Infect Dis 2010; 51:79-84.
21. Gajdács M, Burián K, Terhes G. Resistance Levels and Epidemiology of Non-Fermenting Gram-Negative Bacteria in Urinary Tract Infections of Inpatients and Outpatients (RENFUTI): A 10-Year Epidemiological Snapshot. Antibiotics (Basel). 2019; 8(3). PII: E143. DOI: 10.3390/antibiotics8030143
22. Boral B, Unaldi Ö, Ergin A, Durmaz R, Eser ÖK; Acinetobacter Study Group. A prospective multicenter study on the evaluation of antimicrobial resistance and molecular epidemiology of multidrug-resistant Acinetobacter baumannii infections in intensive care units with clinical and environmental features. Ann Clin Microbiol Antimicrob. 2019; 18(1):19. DOI: 10.1186/s12941-019-0319-8
23. Quareshi ZA, Hittle LE, O’Hara JA, Rivera Syed A, Shields RK, Pasculle AW, et al. Colistin-resistant Acinetobacter baumannii: beyond carbapenem resistance. Clin Infect Dis 2015; 60:1295-1303.
24. Parchem NL, Bauer KA, Cook CH, Mangino JE, Jones CD, Porter K, et al. Colistin combination therapy improves microbiologic cure in critically ill patients with multidrug-resistant gram-negative pneumonia. Eur J Clin Microbiol Infect Dis 2016; 35:1433-1439.
25. Yoon J, Urban C, Terzian C, Mariano N, Rahal J J. In vitro double and triple synergistic activities of polymyxin B, imipenem, and rifampicin against multidrug-resistant Acinetobacter baumannii. Antimicrob Agents Chemother 2004; 48: 753-57.
26. Evrilia S R, Muhtadi A, Barliana M I, Winarni R. An evaluation of Ceftriaxone use in the antimicrobial stewardship program for surgical patients at a Hospital in Bandung. J. Adv. Pharm. Edu. Res. 2019; 9(1): 53-56.
27. Wareham DW, Bean DC. In Vitro Activities of Polymyxin B, Imipenem, and Rifampin against Multidrug-Resistant Acinetobacter baumannii. Antimicrob Agents Chemother 2006; 50:825–826.
28. Muthusamy D, Sudhishna A, Boppe A. In vitro activities of polymyxins and rifampicin against carbapenem-resistant Acinetobacter baumannii at a tertiary care hospital from South India. J. Clin and Diagnostic Research 2016; 10:15-18.
29. Mohammadi M, Khayat H, Sayehmiri K, Soroush S, Sayehmiri F, Delfani S, et al. Synergistic Effect of Colistin and Rifampin Against Multidrug-Resistant Acinetobacter baumannii: A Systematic Review and Meta-Analysis. Open Microbiol J. 2017; 11:63-71. DOI: 10.2174/1874285801711010063. eCollection 2017
30. Bonapace CR, White RL, Friedrich LV, Bosso JA. Evaluation of antibiotic synergy against Acinetobacter baumannii: a comparison with Etest, time-kill, and checkerboard methods. Diagnostic microbiology and Infectious Disease 2000; 38:43-50.
31. Tagliaferri E, Tascini C, Leonildi A, Ciullo I, Amadori F, Menichetti F. Easy synergism for colistin-resistant KPC-producing Klebsiella pneumonia: the E-test with supplemented agar. Clin Microbiol Infect 2015; 21: e7-e8.
32. http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Breakpoint_tables/v_9.0_Breakpoint_Tables.pdf (Las access: Feb. 15, 2020)
33. Falagas ME, Kasiakou SK. Colistin: the revival of polymyxins for the management of multidrug-resistant gram-negative bacterial infections. Clin Infect Dis 2005; 40(9):1333–1341.
34. Lesho E, Yoon EJ, McGann P, Snesrud E, Kwak Y, Milillo M, et al. Emergence of colistin-resistance in extremely drug-resistant Acinetobacter baumannii containing a novel pmrCAB operon during colistin therapy of wound infections. J Infect Dis 2013; 208:1142–51. 15.
35. Lopez-Rojas R, McConnell MJ, Jimenez-Mejias ME, Dominguez Herrera J, Fernandez-Cuenca F, Pachon J. Colistin resistance in a clinical Acinetobacter baumannii strain appearing after colistin treatment: effect on virulence and bacterial fitness. Antimicrob Agents Chemother 2013; 57:4587–9.
36. Trebosc V, Gartenmann S, Tötzl M, Lucchini V, Schellhorn B, Pieren M, et al. Dissecting Colistin Resistance Mechanisms in Extensively Drug-Resistant Acinetobacter baumannii Clinical Isolates. mBio. 2019;10(4):e01083-19. DOI: 10.1128/mBio.01083-19.
37. Beceiro, A., Llobet, E., Aranda, J., Bengoechea, J. A., Doumith, M., Hornsey, et al. Phosphoethanolamine modification of lipid A in colistin-resistant variants of Acinetobacter baumannii mediated by the pmrAB two-component regulatory system. Antimicrob Agents Chemother 2011; 55:3370–9.