High genetic diversity among Pseudomonas aeruginosa and Acinetobacter spp . isolated in a public hospital in Brazil

In Brazil and other regions of the world, Pseudomonas aeruginosa and Acinetobacter spp. have emerged as important agents of nosocomial infection and are commonly involved in outbreaks. The main objective of the present study was to evaluate the genetic relationship among P. aeruginosa and Acinetobacter spp. isolated from patients in a public university hospital in northwestern Paraná, Brazil, and report their antimicrobial resistance profile. A total of 75 P. aeruginosa and 94 Acinetobacter spp. isolates were phenotypically identified and tested for antibiotic susceptibility using automated methodology. Polymyxin B was tested by disk diffusion for P. aeruginosa. Metallo-b-lactamase (MBL) was detected using a disk approximation test. Genotyping was performed using enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR). Approximately 55% of the P. aeruginosa isolates and 92% of the Acinetobacter spp. isolates were multiresistant, but none were MBL-producers. ERIC-PCR revealed the presence of small clusters of carbapenem-resistant Acinetobacter spp., most likely OXA-type carbapenemase producers. Furthermore, high genetic diversity in P. aeruginosa and Acinetobacter spp. clinical isolates was observed, suggesting that cross-transmission is not very frequent in the studied hospital.


INTRODUCTION
Pseudomonas aeruginosa and Acinetobacter spp.have been the cause of nosocomial outbreaks worldwide (Cortes et al., 2009;Hosoglu et al., 2011), including in Brazil (Brito et al., 2003;Prates et al., 2011), where multidrug resistance is common among these isolates (Rossi, 2011).Carbapenems often represent the only effective treatment.However, carbapenem-resistant isolates have frequently been detected, leading clinicians to resume the use of older classes of antibiotics, such as polymyxins (Giamarellou, 2010).The production of carbapenemase enzymes has been an important mechanism of resistance in these bacteria.Metallo-β-lactamases (MBLs), enzymes able to hydrolyze all β-lactam antibiotics with the exception of monobactams, are particularly common in P. aeruginosa, and the genes that encode them are carried by highly mobile elements that play an important role in hospital environments (Cornaglia, Giamarellou, Rossolini, 2011).
The main objective of the present study was to describe the genetic relationship among P. aeruginosa and Acinetobacter spp.clinical isolates using ERIC-PCR and report their resistance profiles against antimicrobial agents that are routinely used in a public university hospital in northwestern Paraná, Brazil.

MATERIAL AND METHODS
Seventy-five P. aeruginosa and 94 Acinetobacter spp.isolates from various clinical specimens from patients admitted to a public hospital in northwestern Paraná, Brazil, between January 2007 and July 2009 were studied.Only one isolate from each patient was selected and stored at -80 ºC in the Laboratory of Medical Bacteriology, Department of Clinical Analysis and Biomedicine, State University of Maringá.No personal data were retrieved from the patients, so privacy could be assured and legal transgressions concerning human research could be avoided (Resolution 196/96 Brazil National Health Council,Health Ministry).The present study was approved by the Regulatory Commission of Academic Activities and Voluntary Services of the studied hospital.
Phenotypic identification and antimicrobial susceptibility testing (AST) were performed using an AUTO-SCAN-4 automated system (Siemens Microscan, Deerfield, IL, USA), with the exception of polymyxin B for P. aeruginosa, which was tested by disk diffusion.The data were interpreted according to the criteria of the Clinical and Laboratory Standards Institute (CLSI;2007-2009).

RESULTS
The studied institution is a small-size public hospital with three intensive care units (ICUs): adult, pediatric, and neonatal.P. aeruginosa and Acinetobacter spp.were isolated more frequently in male patients (62.7% and 59.6%, respectively), especially in patients older than 60 years (54.7% and 39.4%, respectively) and in ICUs (41.4% and 71.3%, respectively).P. aeruginosa isolates were detected at a higher frequency in urine samples, whereas Acinetobacter spp.isolates were mainly recovered from tracheal aspirate.
The resistance rate in P. aeruginosa isolates was less than 30% for most of the b-lactam antibiotics, and 54.6% were multiresistant (i.e., resistant to three or more antimicrobial classes; Magiorakos et al., 2012).All of the isolates were polymyxin B-susceptible.For Acinetobacter spp.isolates, the b-lactam resistance rate was greater than 90% to cephalosporins and greater than 50% to the tested carbapenems (Table I).Multiresistance was observed in 91.5%.None of the P. aeruginosa (n = 39) and Acinetobacter spp.(n = 88) isolates with total or intermediate resistance to ceftazidime were positive for MBL.
The ERIC-PCR applied to P. aeruginosa and Acinetobacter spp.clinical isolates showed a banding pattern with sizes that rang, from approximately 120 pb to 1200 pb and 120 pb and 1900 pb, respectively.
Considering a Dice correlation coefficient ≥ 0.95, 72 ERIC-PCR patterns were obtained in 75 P. aeruginosa clinical isolates.Seventy isolates (93.3%) were orphans, and the remaining five (6.7%) were included in two clusters comprising two (cluster A) and three (cluster B) isolates (Figure 1).
Two isolates that belonged to cluster A showed identical antimicrobial resistance profiles and were isolated from patients in different clinical units within a time interval of approximately 2 months.Isolates that belonged to cluster B, two with 100% similarity, had equal resistance profiles and were isolated from patients in an adult ICU within a time interval of only 4 days.The third isolate that belonged to cluster B, despite having the same resistance profile, was isolated 13 days later in a patient admitted to the pediatric unit (Table II).
A total of 70 Acinetobacter spp.isolates (74.5%) showed orphan ERIC-PCR patterns, and the remaining 24 (25.5 %) were included in 10 clusters (C-L) comprising two isolates each, with the exception of clusters D and H with three isolates each (Figure 2).
The several small clusters found in Acinetobacter spp.were generally detected in the same unit or units with displacement of patients, such as the adult ICU and medical clinic.Exceptions were observed in clusters D and J, which were composed of isolates from the adult and pediatric ICUs.Acinetobacter clusters included isolates with similar resistance profiles, with some differences related to aminoglycosides and carbapenems.Notably, 60% of the small clusters were carbapenems-resistant Acinetobacter spp.(Table II).

DISCUSSION
In the present study, the ERIC-PCR assessment and antimicrobial resistance profiles of P. aeruginosa and Acinetobacter spp.isolates from inpatients in a small-size teaching hospital in northwestern Paraná showed a high percentage of multiresistance and genetic diversity.Worrying rates of ceftazidime and carbapenems resistance were observed in P. aeruginosa isolates.However, they were smaller than those observed in isolates from other university hospitals in Brazil, where resistance to these antimicrobials was near or greater than 50% (Zavascki, Cruz, Goldani, 2004;Rocha et al., 2008).
In recent years, MBL production has represented an important mechanism of resistance to most b-lactam antibiotics, including carbapenems, especially in P. aeruginosa (Kunz, Brook, 2010).However, despite a resistance rate of just over 25% detected for carbapenems, none of the P. aeruginosa isolates tested were positive for MBL, demonstrating the probable participation of other resistance mechanisms.The isolation rates of MBL-producing P. aeruginosa have widely varied among Brazilian hospitals, with incidences ranging from 3.1% (Wirth et al., 2009) to 35% (Gonçalves et al., 2009).According to Rossi (2011), São Paulo metallo-β-lactamase (SPM) is the most prevalent MBL in Brazilian isolates.For Acinetobacter spp.isolated in the present study, the observed resistance rates of > 90% for third-generation cephalosporins and nearly 55% for carbapenems may be responsible for the difficulty treating these cases, which has been reported in other studies (Towner, 2009;Kunz, Brook, 2010).MBL was not detected in isolates from this genus, which corroborates other studies that found a low contribution of this enzyme to the increasing rates of resistance in Acinetobacter spp.(Mostachio et al., 2009;Kunz, Brook, 2010).OXA-type b-lactamases have shown an important role in carbapenem-resistant Acinetobacter spp.(Poirel, Naas, Nordmann, 2010).In Acinetobacter spp.isolates from Brazilian hospitals, carbapenem resistance is mostly related to b-lactamase OXA-23 (Gales et al., 2012).However, a recent study found a high prevalence of OXA-143 in these bacterial genera (Mostachio et al., 2012).Therefore, the small clusters of carbapenem-resistant Acinetobacter spp.isolated in this study may have been OXA-type carbapenemase producers.
Although clonal spread is commonly found in hospitals worldwide (Scott and Pitt, 2004;Cortes et al., 2009), including Brazil (Stheling, Leite, Silveira, 2010), the present study detected high genetic diversity, especially among P. aeruginosa isolates.Only two (2.7%) of the 75 isolates showed identical ERIC-PCR patterns (100% similarity), and five (6.7%) had ≥ 95% similarity.However, according to the phenotypic features and demographic conditions, the cross-spread of small clusters of P. aeruginosa and Acinetobacter spp., including carbapenem-resistant isolates, among patients in the hospital, was evident.Importantly, however, the findings of Saalfeld et al. (2009) should be considered, in which high genetic similarity was found among clinical and environmental Acinetobacter spp.isolates in an adult ICU in the same hospital from January to July 2008.These authors suggested that an endemic situation existed by observing the genotypic similarity of isolates using a Dice correlation coefficient > 90%.

CONCLUSION
Using ERIC-PCR, the present study found high genetic diversity among P. aeruginosa and Acinetobacter spp.isolates, suggesting that cross-contamination is not very frequent in the studied hospital.Because of the known applicability of ERIC-PCR, its good discriminatory power and reproducibility allowed an understanding of the epidemiology of these bacteria in the studied hospital environment.

FIGURE 2 .
FIGURE 2. Dendrogram and DNA-banding profiles that represent the genetic relationship among Acinetobacter spp.isolated from clinical specimens of inpatients in a public hospital in northwestern Paraná, Brazil.

TABLE I -
Resistance profiles of Pseudomonas aeruginosa and Acinetobacter spp.isolates from inpatients in a public hospital in northwestern Paraná, Brazil NT, not tested; a Tested isolates = 46; b Tested isolates = 76.FIGURE 1. Dendrogram and DNA-banding profiles that represent the genetic relationship among Pseudomonas aeruginosa isolated from clinical specimens of inpatients in a public hospital in northwestern Paraná, Brazil.