Prevalence of multidrug-resistant Acinetobacter baumannii and Pseudomonas aeruginosa in an Italian hospital

Journal of Infection and Public Health (2013) 6, 179 185 Prevalence of multidrug-resistant Acinetobacter baumannii and Pseudomonas aeruginosa in an Italian hospital M.A. De Francesco, G. Ravizzola, L. Peroni, C. Bonfanti, N. Manca 1 Institute of Microbiology, University of Brescia, Italy Received 3 August 2012; received in revised form 30 November 2012; accepted 30 November 2012 KEYWORDS P. aeruginosa; A. baumannii; Antibiotic resistance; Infection; Antibiotics Summary The severity and extent of disease caused by multidrug-resistant organisms (MDROs) varies by the population(s) affected and the institution(s) at which these organisms are found; therefore, preventing and controlling MDROs are extremely important. A retrospective study of patients who were infected with Acinetobacter baumannii or Pseudomonas aeruginosa was performed at the Spedali Civili Hospital in Brescia, Italy, from 2007 to 2010. A total of 167 (0.52%) A. baumannii isolates and 2797 P. aeruginosa (8.7%) isolates were identified among 31,850 isolates. Amikacin and colistin were the most active agents against A. baumannii strains. Multidrug resistance (MDR) was observed in 57 isolates (54%). Most MDR isolates (42 out of 57, 73%) were resistant to four classes of antibiotics. P. aeruginosa was recovered more frequently from the respiratory tract, followed by the skin/soft tissue, urine and blood. Colistin, amikacin and piperacillin/tazobactam were active against 100%, 86% and 75% of P. aeruginosa isolates, respectively. A total of 20% (n = 316) of P. aeruginosa isolates were MDR. In summary, A. baumannii was more rare than P. aeruginosa but was more commonly MDR. Epidemiological data will help to implement better infection control strategies, and developing a local antibiogram database will improve the knowledge of antimicrobial resistance patterns in our region. 2013 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved. Corresponding author at: Institute of Microbiology, P. le Spedali Civili, 1, 25123 Brescia, Italy. Tel.: +39 030 3995860; fax: +39 030 3996071. E-mail address: defrance@med.unibs.it (M.A. De Francesco). 1 The paper is dedicated to the memory of the late Professor Nino Manca. 1876-0341/$ see front matter 2013 King Saud Bin Abdulaziz University for Health Sciences. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jiph.2012.11.006

180 M.A. De Francesco et al. Introduction Nosocomial infections are one of the most common complications of hospitalization and lead to increased morbidity and mortality [1,2]. These infections prolong hospitalization, require more extensive diagnostics and treatment and are associated with additional costs [3,4]. Infection with multidrug-resistant pathogens can also complicate treatment. Antibiotic resistance is a daunting phenomenon with a growing impact on patient safety, particularly in ICUs [5]. Critically ill patients are prone to colonization and infection by antibiotic-resistant bacteria because of the frequent exposure of these patients to antibiotics and the presence of multiple, often invasive, devices. This dangerous array of risk factors drives a vicious cycle of increased infection incidence, increased need for broadspectrum antibiotics, reduced antimicrobial efficacy and increased selection of antibiotic resistance. Multidrug-resistant organisms (MDROs) are resistant to one or more classes of antimicrobial agents, such as -lactams (penicillins, cephalosporins, monobactams and carbapenems), fluoroquinolones and aminoglycosides. During the past several decades, a shift in the MDR dilemma from gram-positive to gram-negative bacteria has been noted, which is in part due to the small number of new antimicrobial agents that are active against resistant gram-negative strains [6]. Gramnegative pathogens that have acquired epidemiological importance among nosocomial infections include Acinetobacter baumannii and Pseudomonas aeruginosa. A. baumannii is a cause of outbreaks in hospitals [7,8], and the MDR patterns observed among isolates often leave carbapenems as the only effective treatment for severe infections [8]. However, carbapenem-resistant A. baumannii is emerging worldwide and has been observed in different countries [7,9 11]. There are limited therapeutic options for infections caused by these isolates. P. aeruginosa is also a common gram-negative nosocomial pathogen. This organism is an important cause of hospital-acquired pneumonia and urinary tract, wound and bloodstream infections [12]. Infections caused by this pathogen are often difficult to treat because of the multidrug-resistant nature of this bacterial species, and P. aeruginosa strains are often carbapenem resistant, which can severely limit the available therapeutic choices [13]. The purposes of this study were the following: (a) to determine the prevalences of A. baumannii and P. aeruginosa in patients with nosocomial infections at Brescia s main hospital; and (b) to analyze the antimicrobial susceptibility patterns of these two microorganisms determined as part of an internal laboratory surveillance study from 2007 to 2010. Methods Bacterial isolates A retrospective study of all A. baumannii and P. aeruginosa isolates from different clinical specimens collected from patients with nosocomial infections and processed by the microbiology laboratory between 2007 and 2010 was conducted at the Spedali Civili Hospital in Brescia, Italy. Spedali Civili is a major hospital with an average of approximately 47,000 hospitalizations annually. Infections were considered nosocomial if they first appeared 48 h after admission. Infections that were likely to have been acquired before hospital admission were not considered nosocomial. Blood, urine, tracheal aspirate, bronchi alveolar lavage, sputum, purulent wound, skin ulcer and catheter tip samples collected from patients admitted to all units (ICU and other departments) were eligible. Duplicate isolates were excluded. Clinical specimens were plated onto blood agar and MacConkey agar and incubated overnight at 37 C. After incubation for 24 h at 37 C, the organisms were identified using the VitekTM system (biomerieux, Marcy-l Etoile, France). Antimicrobial susceptibility Antimicrobial susceptibility was assessed using the Vitek2 system (biomerieux) and the AST-GN24 card according to the manufacturer s instructions. The results obtained after a maximum of 15 h of incubation were analyzed and interpreted by AES 4.02 software. The MICs determined by the system identified the microorganism as susceptible, intermediate or resistant according to the criteria published by the CLSI [14]. Antibiotic resistance was categorized into five groups: (1) resistance to extended-spectrum penicillins (piperacillin/tazobactam), (2) resistance to cephalosporins (ceftazidime), (3) resistance to carbapenems (imipenem), (4) resistance to aminoglycosides (amikacin) and (5) resistance to quinolones (ciprofloxacin). The breakpoints for these antimicrobials were as follows: amikacin, S 16 and R 32; ceftazidime, S 8 and R 16; imipenem,

Multidrug-resistant A. baumannii and P. aeruginosa 181 Table 1 Prevalence of A. baumannii strains obtained during the study period and their distributions in different clinical samples. A. baumannii Total = 23 a Total = 21 Total = 48 Total = 75 No. of infections/colonizations b 21 17 36 46 A. baumannii No (%) No (%) No (%) No (%) Type of infected/colonized specimen Respiratory tract 8 (38) 6 (35.3) 18 (50) 24 (52.2) Skin/soft tissue 7 (33.3) 7 (41.2) 10 (27.8) 15 (32.6) Blood 1 (4.8) 3 (17.6) 4 (11.1) 0 Urine 3 (14.3) 0 2 (5.5) 3 (6.5) Other clinical specimens 2 (9.6) 1 (5.9) 2 (5.5) 4 (8.7) a Total strains, including duplicates. b All clinical specimens positive for A. baumannii;. S 4 and R 16; ciprofloxacin, S 1 and R 4 (all for both strains); and piperacillin/tazobactam, S 16/4 and R 64/4 for A. baumannii strains and S 64/4 and R 128/4 for P. aeruginosa strains. Colistin susceptibility was tested using the Kirby Bauer disk diffusion with 10 g colistin disks (OXOID S.p.A., Milan, Italy). The inhibition zone diameters were interpreted according to the CLSI guidelines for colistin (resistant 10 mm and susceptible 11 mm). The strains were defined as R0 (absence of resistance to all five classes of antibiotics), R1 (resistant to one class of antibiotics), R2 (resistant to two classes of antibiotics), R3 (resistant to three classes of antibiotics), R4 (resistant to four classes of antibiotics) and R5 (resistant to five classes of antibiotics). In agreement with previous reports, the term multidrug resistance was used to describe resistance to three or more classes of antimicrobial agents [15,16]. Statistical analysis The Fisher exact test and the chi-square test for linear trend analysis were used to evaluate both the differences in the distributions of the isolates and the trend in the susceptibility rate. A p value of <0.05 was considered significant. Results A. baumannii Among the 31,850 isolates collected from hospitalized patients over the 4-year study period, 167 (0.52%) were identified as A. baumannii. Specimens were collected from the respiratory tract (46.6%; n = 56), skin/soft tissue (32.5%; n = 39), blood (6.6%; n = 8), urine (6.6%; n = 8) and other locations (7.5%; n =9) (Table 1). Increases in the percentages of strains isolated from the respiratory tract and skin/soft tissue were observed in 2009 and 2010. More A. baumannii isolates than P. aeruginosa isolates were resistant to the tested antibiotics (Table 2). The percentage of susceptible A. baumannii strains was 82% for amikacin, approximately 20% for imipenem and piperacillin/tazobactam and 16% for ceftazidime and ciprofloxacin. All strains were susceptible to colistin. Fifty-seven isolates (54%) were MDR (Table 3). Three isolates were resistant to three classes of antibiotics, and most MDR isolates (42 out of 57, 73%) were resistant to four classes of antibiotics. Eleven isolates were resistant to all five classes of antimicrobials. A significant increase (p < 0.05) in the percentage of R5 isolates was observed in 2010. P. aeruginosa P. aeruginosa was identified in 2797 isolates (8.8%) of the 31,850 isolates collected from hospitalized patients during the study period. Like A. baumannii, most P. aeruginosa strains were recovered from the respiratory tract (42.8%; n = 790), followed by skin/soft tissue (26%; n = 480), urine (13.5%; n = 349), other clinical specimens (11%; n = 204) and blood cultures (6.5%; n = 121) (Table 4). However, a significant decrease (p < 0.01) in the percentage of P. aeruginosa strains isolated from the respiratory tract over time was observed;

182 M.A. De Francesco et al. Table 2 Antimicrobial susceptibility patterns of 167 A. baumannii and 2797 P. aeruginosa strains collected from 2007 to 2010. Antimicrobial agents A. baumannii P. aeruginosa CLSI breakpoint interpretation CLSI breakpoint interpretation %S %I %R %S %I %R Amikacin 82 0 18 86 0 14 Ceftazidime 16 0 84 69 0 31 Ciprofloxacin 16 0 84 58 0 42 Imipenem 24 0 76 64 0 36 Piperacillin/tazobactam 19 0 81 75 0 25 %S = percent susceptible; %I = percent intermediate; %R = percent resistant. Table 3 Trends in the antimicrobial resistance patterns of A. baumannii isolates tested from 2007 to 2010. n =21 n =17 n =31 n =36 R0 11 (52.4) 8 (47) 11 (35.5) 10 (27.8) R1 1 (4.8) 2 (11.8) 1 (3.2) 1 (2.8) R2 0 1 (5.9) 1 (3.2) 1 (2.8) R3 a 0 0 2 (6.5) 1 (2.8) R4 a 9 (42.8) 5 (29.4) 15 (48.4) 14 (38.8) R5 a 0 1 (5.9) 1 (3.2) 9 (25) * a Multidrug-resistant isolates. * p < 0.05. ** p < 0.01. Table 4 Prevalence of P. aeruginosa strains obtained during the study period and their distribution in different clinical samples. P. aeruginosa Total = 575 a Total = 732 Total = 798 Total = 692 b No. of infections/colonizations 362 482 534 466 P. aeruginosa No (%) No (%) No (%) No (%) Type of infected/colonized specimen Respiratory tract 198 (54.7) 197 (41) ** 216 (40) ** 179 (38) ** Skin/soft tissue 79 (21.8) 154 (32) ** 133 (25) 114 (24.5) Blood 14 (3.9) 32 (6.6) 44 (8.2) ** 31 (6.6) Urine 65 (18) 52 (11) ** 71 (13.3) 61 (13.1) Other clinical specimens 6 (1.6) 47(9.7) ** 70 (13) ** 81 (17) ** a Total strains, including duplicates. b All clinical specimens positive for P. aeruginosa. * p < 0.05. ** p < 0.01.

Multidrug-resistant A. baumannii and P. aeruginosa 183 Table 5 2010. Trends in the antimicrobial resistance patterns of Pseudomonas aeruginosa isolates tested from 2007 to n = 328 n = 402 n = 452 n = 392 R0 141 (43) 179 (44.5) 214 (47.3) 163 (41.6) R1 63 (19.2) 87 (21.7) 82 (18.1) 89 (22.7) R2 60 (18.3) 62 (15.4) 69 (15.3) 49 (12.5) R3 a 31 (9.5) 29 (7.2) 23 (5.1) * 42 (10.7) ** R4 a 26 (7.9) 37 (9.2) 49 (10.8) 33 (8.4) R5 a 7 (2.1) 8 (2) 15 (3.4) 16 (4.1) a Multidrug-resistant isolates. * p < 0.05. ** p < 0.01. in contrast, a significant increase was observed in the recovery of P. aeruginosa from different clinical specimens (p < 0.01). Colistin (100% susceptible), amikacin (85%) and piperacillin/tazobactam (75%) had the highest susceptibility rates among the antimicrobial agents tested. The susceptibility rates for carbapenems and cephalosporins ranged from 64% for imipenem to 69% for ceftazidime. Lower activity was observed for ciprofloxacin (58%). Of the 1574 isolates from infected/colonized patients, 20% (n = 316) were MDR (Table 5). Most isolates were resistant to four classes of antibiotics (145 out of 316, 45.8%), followed by three classes of antibiotics (125 out of 316, 39.5%) and all five classes of antimicrobial agents (46 out of 316, 14.5%). A significant decrease (p < 0.05) in the percentage of R3 strains was observed from 2007 to 2009, followed by a significant increase (p < 0.01) from 2009 to 2010. Discussion The data collected as part of an internal laboratory surveillance program were used in the present study to draw conclusions about the occurrence of epidemiologically important pathogens responsible for nosocomial infections. Despite the increased rates of A. baumannii infection reported worldwide and the findings of the clonal expansion of these microorganisms in clinical settings, particularly ICUs [7,8,17], A. baumannii represented only 0.52% of all organisms isolated over the 4-year study period. The most serious current problem regarding the treatment of Acinetobacter infections is acquired MDR, resulting in the availability of few effective antimicrobial agents. MDR can be to the result of the presence of a single resistance determinant that confers resistance to more than one class of antimicrobial agent, such as MDR pumps. MDR can also be due to the presence of multiple resistance determinants [18]. Some strains of A. baumannii that are resistant to all antibiotics have been detected [19,20]. A surveillance study in Korea in 2009 [21] showed that the resistance rates of Acinetobacter were very high: 67% of isolates were resistant to fluoroquinolones, 48% to amikacin, 66% to ceftazidime and 51% to imipenem. This resistance trend was largely similar to that observed in a study conducted by The Surveillance Network (TSN) in the U.S. [22]. In our study, we observed more frequent resistance to imipenem (80%), ceftazidime and ciprofloxacin (84%), whereas resistance to amikacin was less frequent (18%). Fifty-seven isolates (54%) were MDR, with an increase in the percentage of strains resistant to all five classes of antibiotics. Nosocomial strains of P. aeruginosa are frequently resistant to a broad range of antibiotics. The prevalence of nosocomial strains of P. aeruginosa appears to be increasing worldwide, especially as a cause of ventilator-associated pneumonia; their prevalence in high-risk populations, such as patients with severe burn injuries, is also increasing [23]. Pseudomonas is most commonly isolated from the respiratory tract, followed by wounds, urine and blood [19]. This finding is in agreement with our results. Pseudomonas is intrinsically resistant to most antibiotics. Antimicrobial resistance develops under selective pressure, and multiple mechanisms are responsible: the (hyper-) production of enzymes, such as beta-lactamases and DNA gyrases; active efflux pumps; and permeability changes [19]. Our results show that colistin, amikacin and piperacillin/tazobactam were the most active antipseudomonal antimicrobials evaluated, a result

184 M.A. De Francesco et al. that is corroborated by the findings of other studies [12]. Additionally, the limited activity of ciprofloxacin could be due to the overuse of this antimicrobial agent in clinical settings. In this study, we found that 316 strains (20%) were MDR. The majority of isolates were resistant to four classes of antibiotics (145 out of 316, 45.8%). Among MDR P. aeruginosa isolates, colistin showed good activity and could therefore be a useful alternative treatment; however, this agent is has considerable toxicity [15,24]. The ongoing surveillance of these microorganisms is important to help direct antimicrobial therapy and monitor the emergence of potentially drug-resistant strains in Brescia, Italy. Conflict of interest None declared. Funding No funding sources. Ethical approval Not required. References [1] Geffers C, Sohr D, Gastmeier P. Mortality attributable to hospital-acquired infections among surgical patients. 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