Acinetobacter baumannii has emerged as one of the most problematic nosocomial pathogens to eradicate using available antimicrobial agents, and its occurrence has increased especially in patients admitted to intensive care units (ICUs). In a recent Italian survey, this microorganism ranked third among the most relevant pathogens in ICUs and was one of the most resistant microrganisms to all families of antibiotics 1. The reasons for this emergence in nosocomial settings can be found among: i) its particular characteristics of being a colonizer of multiple body sites of hospitalized patients; ii) its ability to survive for a long time on inanimate surfaces; and iii) its tendency to be intrinsically resistant and to acquire new resistance determinants.

The clinical role of A. baumannii has been very controversial: it was considered a colonizer for a long time but recently, due to the report of severe illness with high associated mortality 2 and occurrence of some severe infections in bacteriemic patients, has been considered – by some clinicians – an indicator of severity, leading to the hypothesis that some changes can take place in this strain i.e. the acquisition of some virulence factors.

Treatment of Acinetobacter spp. infections has been limited to a few broad-spectrum agents including carbapenems, fluoroquinolones, ceftazidime, trimethoprim/sulfamethoxazole, sulbactam, amikacin and some tetracyclines. As resistance to carbapenems and other alternative drugs has emerged 34567, often conferring a multi-drug-resistance (MDR) phenotype to this microrganism, the popularity of an old class of drugs, i.e. the polymyxin class of agents, has increased. However, colistin-resistant A. baumannii has been recently reported 89, and in spite of documented clinical success, colistin demonstrated dose-dependent nephrotoxicity 10.

In this context, it is mandatory to look at the activity of new antimicrobial agents and numerous reports have indicated that tigecycline, a novel compound belonging to a new class of antimicrobial agents – glycylcyclines -, displays inhibitory activity against Acinetobacter spp. 11121314. This study sought to determine the in vitro activity of tigecycline and comparators against Italian carbapenem resistant and susceptible A. baumannii strains, isolated throughout Italy during a two-year surveillance study 1. The presence of carbapenem-resistance genes was also investigated. These data can add new information to the in vitro activity of the most used antibiotics against this sample of epidemiologically representative Italian isolates.

The 107 strains included in the study were collected from 9 centres in Italy, mainly located in the Central-South area of the country. Lungs and blood were the most common sources of the isolates (41.6% of all isolated came from lower respiratory tract infections, and 20.3% were isolated from blood cultures). In this survey, A. baumannii was also isolated from urinary catheters, complicated skin and skin structure (both 13.8%), and intra-abdominal infections (2.7%).

The results of the in vitro susceptibility testing, expressed as distribution, MIC₅₀, MIC₉₀ and percentage of resistance are presented in Table 1. The vast majority of strains of A. baumannii showed a multi-drug resistant phenotype (MDR), in particular, 53 strains were simultaneously resistant to three antibiotics, namely fluoroquinolones, ceftazidime and aminoglycosides or imipenem, while 44 strains were resistant to four antibiotics, the above three including carbapenems. 17% of strains were resistant also to ampicillin/sulbactam. More than 90% of strains were simultaneously resistant to ciprofloxacin, trimethoprim/sulphametoxazole, aztreonam, and ceftazidime; around 70% of strains were also resistant to gentamicin, amikacin, and tetracycline, while resistance to carbapenems was 50% for imipenem and 59% for meropenem. Among the tetracyclines, the unimodal distribution of susceptibility of tigecycline was comparable with that of doxycycline (93 and 94% susceptible, respectively). In our sample of strains colistin was active (106 out of 107 strains were susceptible). One strain was resistant to this drug, with a MIC of 16 mg/L.

The molecular characterization of the carbapenem-resistance gene content of the 58 isolates that were resistant to meropenem is shown in Table 2. The bla_OXA51 and bla_OXA58-like, were the most diffused genes: 43 strains carried both these determinants and 14 strains carried the bla_OXA51-like gene. In our sample of carbapenem-resistant strains, only one carried the bla_oxa23-like together with the bla_oxa51 gene; all strains were negative for MBL (bla_VIM and bla_IMP) enzymes (table 2). Six out of the 14 strains carrying the bla_OXA-51-like gene, demonstrated a susceptible or intermediate level of resistance to imipenem while they were resistant to meropenem, suggesting that other mechanisms can be responsible for carbapenem resistance in these strains. In the 43 strains carrying the bla_OXA51 and bla_OXA58-like genes there is an association between the presence of the OXA-58 enzyme and resistance to carbapenems.

A preliminary observation on the level of resistance to carbapenems demonstrated that meropenem is a more hydrolysable substrate with respect to imipenem, and in all cases, tigecycline remained active in all carbapenem-resistance-strains.

Time-kill studies with increasing concentrations of tigecycline above the MIC, demonstrated that this antibiotic has a maximal killing effect near the MIC, without any increased rate at multiples of the MIC (Table 3). Its bacteriostatic activity was similar in all strains tested irrespective of their resistance to other antibiotics such as carbapenems.

Acinetobacter baumannii is one of the most important nosocomial pathogens, able to cause severe infections, occurring principally in immunosuppressed patients, in patients with serious underlying diseases, or subjected to invasive procedures and treatment with broad-spectrum antibiotics 19. This microorganism is also able to spread in high-risk wards, causing outbreaks. Nevertheless, until recently, carbapenems represented a viable choice for treating infections caused by this organism due to a diffused sensitivity to both imipenem and meropenem. In Italy, until 2000, very few strains were resistant 20, and only few genotypes appeared to occur in multiple hospitals causing outbreaks 212223242526. The recent Italian survey previously cited 1 demonstrated that A. baumannii was the third pathogen for severity in high-risk wards, frequently derived from nosocomial pneumoniae (VAP) or bacteremia. In our experience, nosocomial isolates of this microorganism were also co-pathogens in cSSSI and a few strains also came from IAIs. Approximately half of the 107 single isolates were MDR to three or four classes of antibiotics, including carbapenems, while they were almost fully susceptible to tigecycline, doxycycline and colistin. In our results, tigecycline is active, with a MIC₉₀ of 2 mg/L against 93% of strains with maximum killing at the MIC concentrations.

The activity of tigecycline in our Italian isolates is comparable with that of many other national and global studies 12272829, including the worldwide program T.E.S.T.(Tigecycline Evaluation and Surveillance Trial, 12 that reported a MIC₉₀ value of 1 mg/L for a large number of A. baumannii isolates (4353) worldwide. The same study reported the isolation of 14.8% imipenem-resistant strains. Our data are also in line with the results obtained in other European studies 3031 in which tigecycline demonstrated its activity with MIC₉₀ of 2 mg/L against strains showing a multiple resistance phenotype.

A. baumannii isolates resistant to various classes of antibiotics are emerging worldwide, and the recent resistance or reduced susceptibility to carbapenems, is considered a serious clinical problem due to the role of first choice therapy that these drugs have had until now.

Numerous mechanisms, including decreased permeability, efflux pump overexpression, and carbapenemase production, can be responsible for the resistance to carbapenems 73233. Class B (IMP and VIM enzymes) and D (oxacillinases) beta-lactamases are the most important group of enzymes able to hydrolyze carbapenems: although MBLs are not the predominant enzymes in A. baumannii, several IMP-enzymes have been described in various countries 34353637 while VIM-1, first identified in Italy in a strain of P. aeruginosa 38, is now reported in Korea 39, in Poland 40 and, as sporadic isolates, in many other parts of the world. Most worrisome are the carbapenemase-hydrolyzing oxacillinase (OXA) clusters that have been identified in A. baumannii in nine major subgroups 74142, including the most diffused throughout the world OXA-23, OXA-24, OXA-51, OXA-58 enzymes and their variants 43. The significant contribution of these enzymes to carbapenem resistance in A. baumannii has been emphasized, particularly when they are accompanied by IS_Aba1 and IS_Aba3 in the naturally occurring plasmid 44, due to the fact that these sequences can provide the promoter required for expression of linked antibiotic-resistance genes. The OXA-51/69-like beta-lactamases are, instead, "naturally occurring" chromosomal enzymes in this species, isolated from four continents, and their expression varies according to the presence of IS_Aba1 as previously discussed 45. All these enzymes have been found in different isolates from various countries 2446474849. In our strains, OXA-51 alone or associated with other enzymes was found. As recently suggested 32, these enzymes are very poor carbapenemases and may be natural chromosomally encoded in the vast majority of A. baumannii isolates, regardless of their susceptibility or resistance to carbapenems. OXA-58 enzymes are the most diffused oxacillinases in 43 strains of A. baumannii. As recently demonstrated, these enzymes may contribute to carbapenem resistance 3245, although it cannot be excluded that other mechanisms may additionally affect the activity of these drugs.

The recent emergence of high-resistance rates to tigecycline in multiple clones of MDR strains is also cause for concern. Various Authors 505152 have reported a few cases of high tigecycline-resistance in nosocomial isolates: the involvement of an overexpression of the AdeABC multidrug efflux pump in the decreased susceptibility of tigecycline in these strains was postulated 5253. To date, tigecycline has been approved for the treatment of complicated intra-abdominal and complicated skin and skin structure infections; the well documented activity of this drug against A. baumannii isolates and the high concentration reached by this drug in alveolar cells (77.5 fold higher than serum)29, have indicated its use in infections sustained by these MDR microorganisms 54, despite the lack of approved clinical indications and criteria for in vitro susceptibility testing. In this situation, due to the importance that tigecycline can have in the treatment of MDR A. baumannii infections, it is absolutely mandatory to monitor isolates for evidence of acquired microbiological resistance, requiring further investigations to understand better all the possible mechanisms underlying this phenomenon.

Our results have shown that the MDR resistance phenotype is very diffused in A. baumannii isolated from severe infections in Italy. We have documented that almost half of the strains are also resistant to carbapenems and, as published by other Authors 55, also colistin has not escaped development of resistance: in fact, we isolated one strain resistant to this drug. Carbapenem-resistance in these strains is due to the presence of three different oxacillinases and the most diffused hydrolytic enzymes are the OXA-51/OXA-58 enzymes, carried in 43 strains. Tigecycline shows a potent antibacterial activity with a unimodal distribution of MIC and a MIC₉₀ of 2 mg/L; the drug has indeed a bacteriostatic effect at the MIC concentration, and its activity is not influenced by the other mechanisms of resistance carried in these microorgansims, including the resistance to carbapenems. Furthermore recent studies demonstrated that tigecycline achieved maximal killing near the MIC in this species and concentration escalation studies demonstrated that this drug needs to approach concentrations higher than those currently achieved in the bloodstream to adequately treat A. baumannii infections 13.

These in vitro results, obtained in a representative sample of Italian isolates – all from documented severe infections – need to be confirmed by the clinical efficacy of this drug from Phase 3 clinical trials regarding the treatment of nosocomial pneumonia and other infections sustained by this microrganism. For the moment, the evaluation of the microbiological and pharmacological profile of tigecycline in each patient and the careful assessment of susceptibility can assist physicians in deciding on tigecycline use.

GN and SS received funding from Wyeth – Italy.

MML participated in the study design, interpretation of the results, and co-drafted the manuscript; TG participated in the study design, co-performing MIC and killing curves, analysis and interpretation of data; GF participated in the study design, co-performing DNA extractions and PCR experiments; NMV and ND participated in design and provided the strains for the study; CA, FG and GN participated in design, in the coordination of the study and in the revision of the manuscript; SS participated in the design the study, interpretation of the data, co-drafted the manuscript and participated in the final revision