Clinical and Molecular Epidemiology of Multidrug-Resistant P. aeruginosa Carrying aac(6')-Ib-cr, qnrS1 and blaSPM Genes in Brazil.

We described a comprehensive analysis of the molecular epidemiology of multidrug-resistant (MDR) P. aeruginosa. Molecular analysis included typing by Pulsed Field Gel Electrophoresis, identification of genes of interest through PCR-based assays and sequencing of target genes. Case-control study was conducted to better understand the prognostic of patients and the impact of inappropriate therapy in patients with bacteremia, as well as the risk factors of MDR infections. We observed a high rate of MDR isolates (40.7%), and 51.0% of them was independently associated with inappropriate antibiotic therapy. Bacteremia was detected in 66.9% of patients, and prolonged hospital stay was expressive in those resistant to fluoroquinolone. Plasmid-mediated quinolone resistance genes (PMQR), qnrS1 and aac(6')Ib-cr, were detected in two different nosocomial isolates (5.3%), and the aac(6')-Ib7 variant was detected at a high frequency (87.5%) in those negative to PMQR. The presence of mutations in gyrA and parC genes was observed in 100% and 85% of selected isolates, respectively. Isolates harboring PMQR genes or mutations in gyrA and parC were not closely related, except in those containing SPM (São Paulo metallo-β-lactamase) clone. In addition, there is no study published in Brazil to date reporting the presence of Pseudomonas aeruginosa isolates harboring both qnrS1 and aac(6')Ib-cr genes, with alarming frequency of patients with inappropriate therapy.

Introduction

Currently, the increasing incidence of MDR P. aeruginosa is a global problem as a consequence of the ability of this microorganism to develop resistance to almost all antibiotics that are available for treatment, either by mutations present in chromosomal gene, or by horizontal gene transfer [1,2]. In Brazil, this problem is aggravated because of either the very high density of the antimicrobials use, especially β-lactams (including carbapenems) and fluoroquinolones [3,4] and the lack of affirmative action’s for prevention of these infections [5,6].

Carbapenems are a therapeutic choice for the treatment of severe infections caused by P. aeruginosa. However, its use has been threatened mainly by the increased incidence of isolates resistant to this class of antibiotics [7-9]. Among the carbapenem resistance mechanisms, there is the presence of carbapenemases, which are a heterogeneous group of β-lactamases comprising Classes A (penicillinases), B (metalloenzymes) and D (oxacillinases), with the ability to hydrolyze imipenem and meropenem in addition to other penicillins and cephalosporins [10]. The genes encoding the carbapenemases are usually located in plasmids, which significantly increase the risk of their dissemination [11,12]. The carbapenem resistance in P. aeruginosa infection occurs mainly by metallo-β-lactamase (MBL), in addition to the appearance of other β-lactamases with carbapenemase activity, including KPC (Klebsiella pneumoniae carbapenemase) and OXA-carbapenemase [7,9,13,14].

In recent years, the production of MBL by P. aeruginosa isolates has assumed epidemiological importance and is associated with high mortality rates [15]. MBLs confer resistance to carbapenems, and are generally encoded by mobile elements facilitating the spread of antibiotic resistance [16-18]. The blaSPM-1 gene has been spread in Brazilian regions by the persistent MDR P. aeruginosa, and studies have reported concerns about its worldwide spread and potential pandemic [7, 18–20]. The increased prevalence of infections and colonization by P. aeruginosa São Paulo metallo-β-lactamases (SPM)-producers suggests that the gene blaSPM-1 settled successfully in plasmids and is associated with high risk of formation of clones, which can facilitate its global spread [21, 22].

Beyond the potent in vitro anti-pseudomonal activity, the use of fluoroquinolone antibiotics has spread widely in the past decade, and its usage popularity has also facilitated the emergence of resistant and MDR strains [23]. The resistance to fluoroquinolones is mainly due to (i) the point mutations in quinolone resistance determining regions (QRDR) in the DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE) genes, (ii) the presence of plasmid-mediated quinolone resistance (PMQR) determinants, and/or (iii) the decreased uptake of the drug due to the loss of a membrane-bound porin and/or drug extrusion via efflux pumps [24]. The co-existence of mutations in genes encoding for type-II topoisomerase and PMQR is often found together in microorganisms of the Enterobacteriaceae family, and the presence of PMQR determinants may promote QRDR mutations, increasing the resistance to fluoroquinolone [25, 26]. Although PMQR genes, such as qnrA, qnrB, qnrC, qnrD, qnrS, qepA and aac(6’)-Ib-cr, have been increasingly reported in bacterial pathogens within the Enterobactericeae family, they have not been detected frequently in P. aeruginosa isolates [24, 27]. To the best of our knowledge, as far as we know, this is the first description of the presence of PMQR genes in P. aeruginosa isolates in Brazil.

In addition, only a few studies have examined the consequences of resistance to antibiotics and the impact of inappropriate therapy on the outcome of patients with MDR P. aeruginosa infections. Thus, this study was performed to evaluate this factor, and to identify the risk factors in patients with MDR P. aeruginosa infections. In addition, we also aimed to determine: (i) the presence of metallo-β-lactamases genes in carbapenem-resistant isolates; (ii) the co-occurrence of PMQR determinants and altered gyrA and parC genes in fluoroquinolone-resistant isolates; and (iii) the pattern of clonal spread of P. aeruginosa in the hospital environment.

Materials and Methods

Study design and data collection

Active surveillance was conducted from May 2009 to December 2012 and from April to October 2014 for the detection of patients with P. aeruginosa infections resistant to carbapenems and fluoroquinolones at Uberlândia University Hospital (Brazil). In total, 242 episodes of P. aeruginosa infections obtained from 236 patients were included in the study. From this surveillance, two case-control studies were conducted: (i) to determine the risk factors associated with MDR P. aeruginosa infections, and (ii) to determine the risk factors associated with antimicrobial resistance and treatment outcome in patients with P. aeruginosa bacteremia. In both studies, only the first episode of each infection was considered. The demographic, clinical and epidemiological data of the patients were also obtained through review of medical records, following the model of National Healthcare Safety Network (NHSN).

Definitions

According to the Centers for Disease Control and Prevention (CDC), bacteremia was defined as the presence of viable bacteria in the blood, documented by a positive blood culture result [28]. The isolates were considered to be nosocomial if the infection occurred >48 h after admission and no clinical evidence of infection on admission existed [29]. The criteria used for defining MDR phenotype was: non-susceptible to ≥1 agent in ≥3 antimicrobial categories [30]. Previous antibiotic use was considered when the patient received therapy with any antibiotic for at least 72 h over a period of 30 days prior to the microbiological infection diagnosis [31]. The antimicrobial therapy was considered to be appropriate if the initial antibiotics, which were administered within 24 h of acquisition of a blood culture sample, included at least one antibiotic that was active in vitro [32]. The 30-day mortality was considered as the number of deaths of patients with infections during hospitalization that occurred within 30 days of the diagnosis of infection [33], and the 5-day mortality, also known as early mortality, was considered as the number of deaths within 5 days of hospitalization [34]. Hospital stays were considered prolonged if they reached or exceeded 45 days [35]. It was considered MIC50 and MIC90 represent the concentration of antimicrobial agent (μg/mL) that inhibited 50% and 90%, respectively, of the isolates tested [36].

Clinical microbiological and antibiotic resistant profile

Microbial identification and antimicrobial susceptibility tests were performed on a VITEK II system (bioMérieux, Brazil) for the following antimicrobials: aminoglycoside (gentamicin, amikacin), carbapenems (imipenem, meropenem), cephalosporin (cefepime), fluoroquinolone (ciprofloxacin) and penicillin plus β-lactamase inhibitors (piperacillin-tazobactam). Quality-control protocols were used according to the standards of the Clinical and Laboratory Standard Institute [37, 38]. The isolates with intermediate susceptibility were considered as resistant.

The minimum inhibitory concentration (MIC) and the confirmation test of resistance to imipenem (≥8 μg/mL) were performed by the E-test® method, according to the manufacturer’s guidelines (AB Biodisk, Sweden) [38]. In addition, resistance to ciprofloxacin was confirmed by broth microdilution method according to Capuano [39] with modifications, and the interpretations also were made according to CLSI [38], considering resistance to ciprofloxacin ≥4 μg/mL.

Characterization of strains harboring MBL and PMQR genes

Forty clinical P. aeruginosa fluoroquinolone-resistant isolates were selected, being obtained from 39 patients, with various clinical infections (urinary infection, pneumonia, wound infection, otitis, and bloodstream infection).

DNA extraction was performed using a PureYield™ Plasmid Miniprep System (Promega, Brazil). Amplification of the MBL and PMQR markers (blaIMP, blaVIM, blaSPM, blaGIM, blaSIM, qnrA, qnrB, qnrC, qnrD, qnrS, qepA and aac(6’)-Ib-cr) were performed using primers listed in S1 Table (available in the Supporting Information). The reaction mixture (25 μL) contained 1.0 μL DNA template (10 ng), 12.5 μL GoTaq® Green Master Mix (Promega) and 0.5 μL of each primer. Amplifications were performed in Mastercycler Personal (Eppendorf) using the following program: initial denaturation at 95°C for 2 min followed by 30 cycles of 30 seconds at 95°C, 1 min at annealing temperature (54°C for blaIMP, blaVIM, blaSPM, blaGIM and blaSIM; 51°C for qnrA, qnrB, qnrC, qnrS and qnrD; 52°C for qepA and aac(6’)-Ib-cr), 1 min at 72°C and a final extension steap of 5 min at 72°C. Multiplex PCR was performed for genotypic characterization of different MBL (blaIMP, blaVIM, blaSPM, blaGIM and blaSIM) and PMQR (qnrA, qnrB, qnrC, and qnrS) genes; and, for the others genes individual PCRs were carried out (qnrD, qepA and aac(6’)-Ib-cr). The amplified PCR products were visualized by electrophoresis in 1.5% agarose gel by the photo documentation System L-Pix EX (Loccus Biotechnology, Brazil).

Sequencing of PMQR genes and QRDRs mutations

The PCR products of target regions for QRDR mutations (gyrA and parC) and PMQR genes (qnrS and aac(6’)-Ib) were sequenced (primers listed in S1 Table), using an automatic sequencer ABI-PRISM 3100 Genetic Analyzer (Applied Biosystems, USA). Sequences were edited using SeqMan Pro alignment was carried out with MegaAlign, both of Lasergene package version 10 (DNAStar, USA) and deposited at GenBank (http://www.ncbi.nlm.nih.gov/genbank/) [accessions numbers: KT962252 (qnrS), KT987419 (aac(6’)-Ib-cr) and KT987420 (aac(6’)-Ib)].

Pulsed-Field Gel Electrophoresis (PFGE)

Isolates were typed according to the protocols described by Galetti [40] with modifications, following digestion of genomic DNA with SpeI restriction enzyme (Promega). DNA fragments were separated on 1% (w/v) agarose gels in 0.5x TBE [Tris–borate–ethylene diamine tetra-acetic acid (EDTA)] buffer using a CHEF DRIII apparatus (Bio-Rad, USA) with 6 V/cm, pulsed from 5 s to 40 s, for 21 h at 12°C. Gels were stained with ethidium bromide and photographed under ultraviolet light. Computer-assisted analysis was performed using BioNumerics 5.01 software (Applied Maths, Belgium). Comparison of the banding patterns was accomplished by the unweighted pair-group method with arithmetic averages (UPGMA) using the Dice similarity coefficient.

Statistical analysis

The Chi-square or Fisher’s exact test was used to compare discrete variables. The comparison of two quantitative variables was made using the Mann–Whitney test for nonparametric variables and the Student t test for parametric variables. Two-sided tests were used for all analyses. Multivariate analysis was performed using multiple logistic regression and the values were included when significance was <0.05 in univariate analysis. To determine inappropriate therapy for mortality within 30 days of hospitalization, a multiple logistic regression model was used to control for the effects of confounding variables. All p-value <0.05 was considered statistically significant. The epidemiological data were analyzed through the programs Graph Pad Prism® 5.0 (La Jolla, USA) and BioEstat 5.0 (Tefé, Brazil).

Ethical considerations

The data and the samples analyzed in the present study were obtained in accordance with the norms and approved by the Federal University of Uberlandia Ethics Committee (UFU), through license number 36601814.7.0000.5152. For this study, samples were collected at the Microbiology Laboratory of the Clinical Hospital, with no contact to the patient and with the permission of the Hospital. Moreover, this study was retrospective and there are no patient identification when performed data collection, so the ethics committee dismissed the informed consent term and clarified.

Results

From May 2009 to December 2012 and from April to October 2014, a total of 236 non-repetitive patients with P. aeruginosa infections at the University Hospital were included in the study. The univariate analysis and independent risk factors associated with MDR P. aeruginosa infections are summarized in Table 1. According to antimicrobial susceptibility testing results, MDR P. aeruginosa infections occurred in 40.7% of the cases. Data from these patients (MDR) were compared with a sensitive P. aeruginosa infections group (non-MDR). In the whole series, prior exposure to carbapenems and inappropriate therapy as well as the co-morbidity condition (diabetes mellitus) were significant in the univariate analysis by MDR P. aeruginosa infections. The results of multivariate analyses showed that factors independently associated with MDR P. aeruginosa were patients who received inappropriate therapy. The Kaplan–Meier cumulative survival estimates (Fig 1) for patients with inappropriate versus appropriate therapy showed that the first group had a lower probability of survival than the group that received appropriate therapy (P = 0.0047). The 30-day mortality rate of the first group was 55.3%, whereas that of the second group was 34.4%. Furthermore, of the total mortality, 5-day mortality rate was 40.6% (54/133) independently of the therapy received.

Table 1

Univariate and multivariate analyses, mortality and independent risk factors associated with multidrug-resistant P. aeruginosa infections.

Risk factor/ characteristics	Total	MDR1	non-MDR	Univariate		Multivariate
	n = 236 (%)	n = 96 (%)	n = 140 (%)	OR2 (CI3 95%)	P value	OR (CI 95%)	P value
Age [mean]; ±SD4	52.7; ±22.9	56.5; ±18.3	50.6; ±25.1	-	0.2111	-	-
Male	166 (70.3)	65 (67.7)	101 (72.1)	0.81 (0.46–1.42)	0.4638	-	-
Female	70 (29.7)	31 (32.3)	39 (27.8)	1.23 (0.70–2.17)	0.4638	-	-
Length of hospital stay days [mean]; ±SD	55.0; ±69.7	54.7; ±46.5	53.3; ±80.9	-	0.0669	-	-
Invasive procedures	210 (88.9)	86 (89.6)	123 (87.9)	1.19 (0.52–2.72)	0.6824	-	-
Mechanical ventilation	126 (53.4)	58 (60.4)	68 (48.6)	1.62 (0.95–2.74)	0.0731	-	-
Tracheostomy	120 (50.8)	56 (58.3)	64 (45.7)	1.66 (0.98–2.81)	0.0568	-	-
Urinary catheter	163 (69.1)	71 (73.9)	92 (65.7)	1.48 (0.83–2.63)	0.1783	-	-
Central venous catheter	192 (81.4)	77 (80.2)	115 (82.1)	0.88 (0.45–1.71)	0.7078	-	-
Surgical drain	41 (17.4)	17 (17.7)	24 (17.1)	1.04 (0.52–2.06)	0.9103	-	-
Enteral probes/gastric nutrition	172 (72.9)	71 (73.9)	101 (72.1)	1.10 (0.61–1.97)	0.7580	-	-
Haemodialysis	71 (30.1)	29 (30.2)	42 (30.0)	1.01 (0.57–1.78)	0.9727	-	-
Co-morbidity conditions	146 (61.9)	57 (59.4)	89 (63.6)	0.84 (0.49–1.43)	0.5144	-	-
Heart failure	60 (25.4)	27 (28.1)	33 (23.6)	1.27 (0.70–2.29)	0.4300	-	-
Neoplasia	36 (15.2)	9 (9.4)	27 (19.3)	0.43 (0.19–0.97)	0.0375	-	-
Diabetes mellitus	40 (16.9)	22 (22.9)	18 (12.9)	2.01 (1.01–4.00)	0.0430*	1.9907 (0.97–4.09)	0.0608
Chronic renal failure	66 (28.0)	28 (29.2)	38 (27.1)	1.10 (0.62–1.97)	0.7337	-	-
Previous use of antimicrobial	195 (82.6)	82 (85.4)	113 (80.7)	1.40 (0.69–2.83)	0.3490	-	-
Carbapenems	119 (50.4)	56 (58.3)	63 (45.0)	1.71 (1.01–2.89)	0.0442*	0.8928 (0.51–1.55)	0.6873
Fluoroquinolone	51 (21.6)	24 (25.0)	27 (19.3)	1.41 (0.76–2.64)	0.2753	-	-
Cephalosporin (3 and 4th generation)	161 (68.2)	65 (67.7)	96 (68.6)	0.96 (0.55–1.68)	0.8888	-	-
Inappropriate therapy	85 (36.0)	49 (51.0)	36 (25.7)	3.01 (1.73–5.23)	< 0.0001*	3.0169 (1.72–5.31)	0.0001*
Mortality
Total mortality	133 (56.4)	54 (56.2)	79 (56.4)	0.99 (0.59–1.68)	0.9783	-	-
30-day mortality	99 (41.9)	42 (43.7)	57 (40.7)	1.133 (0.67–1.92)	0.6425	-	-
5-day mortality	54 (22.9)	25 (26.0)	28 (20.0)	1.408 (0.76–2.61)	0.2746	-	-

1Multidrug-resistant;

2Odds ratio;

3Confidence interval;

4Standard deviation.

*P ≤ 0.05 –statistically significant for risk factor.

Fig 1

Survival curve using the Kaplan–Meier method for patients who received appropriate antimicrobial therapy compared with those who received inappropriate therapy.

P ≤ 0.05 –statistically significant.

Antimicrobial therapy and clinical outcome of patients with or without bacteremia caused by P. aeruginosa were evaluated and it can be observed that patients with bacteremia caused by isolates resistant to carbapenems, had a high 5-day mortality rate. Moreover, the time of hospital stay was significantly higher for the MDR and fluoroquinolone-resistant groups when compared with susceptible group, and the latter was also independently associated in the multivariate analyses (Table 2).

Table 2

Antimicrobial therapy and clinical outcome of patients with bacteremia caused by P. aeruginosa resistant to carbapenems, fluoroquinolones and multiresistant.

	Patients with bacteremia	Patients without bacteremia	Univariate
	n = 158 (%)	n = 78 (%)	P value	OR1 (CI2 95%)
Carbapenem resistant isolates3
Total	70 (100.0)	39 (100.0)	-	-
5-day mortality	22 (31.4)	5 (12.8)	0.0310*	3.117 (1.073–9.051)
30-day mortality	36 (51.4)	14 (35.9)	0.1188	1.891 (0.8453–4.229)
Inappropriate therapy	32 (45.7)	24 (61.5)	0.1131	0.5263 (0.2368–1.170)
Prolonged hospital stay5	40 (57.1)	15 (38.5)	0.0615	2.133 (0.9582–4.749)
Fluoroquinolone resistant isolates4
Total	67 (100.0)	31 (100.0)	-	-
5-day mortality	21 (31.3)	4 (12.9)	0.0796	3.082 (0.9561–9.932)
30-day mortality	33 (49.2)	9 (29.0)	0.0600	2.373 (0.9534–5.904)
Inappropriate therapy	30 (44.8)	21 (67.7)	0.0343	0.3861 (0.1579–0.9440)
Prolonged hospital stay6	38 (56.7)	7 (22.6)	0.0016*	4.493 (1.701–11.86)
Multidrug-resistant isolates
Total	67 (100.0)	29 (100.0)	-	-
5-day mortality	21 (31.3)	4 (13.8)	0.0820	2.853 (0.8809–9.241)
30-day mortality	32 (47.8)	11 (37.9)	0.3738	1.496 (0.6141–3.645)
Inappropriate therapy	30 (44.8)	19 (65.5)	0.0620	0.4267 (0.1727–1.055)
Prolonged hospital stay	39 (58.2)	7 (24.1)	0.0022*	4.378 (1.644–11.66)

1Odds ratio;

2Confidence interval;

3Imipenem and/or meropenem;

4Ciprofloxacin and/or norfloxacin;

5Length of stay ≥ 45 days;

6Significant risk factor by multivariate analyses P = 0.0088*, OR = 3.8151 (CI = 1.40–10.38); *P ≤ 0.05 –statistically significant.

The antimicrobial resistance analysis of the 242 P. aeruginosa isolates is presented in Table 3. The resistance to carbapenem and fluoroquinolones were the most frequents, 45.9% and 42.6% respectively, followed by resistance to aminoglycosides (38.4%), cephalosporin (3rd and 4th generation) (34.3%) and piperacilin/tazobactam (24.8%). No isolate was resistant to polymyxin.

Table 3

Pseudomonas aeruginosa antimicrobial resistance and sensitivity profiles to the main antimicrobials of isolates from 242 episodes of hospital infections.

Antimicrobials	Resistant		Sensitive
	Isolates	Rate (%)	Isolates	Rate (%)
Cephalosporin (3rd and 4th generation)	83	34.3	159	65.7
Carbapenem	111	45.9	131	54.1
Aminoglycoside	93	38.4	149	61.6
Polymyxin	0	0	242	100.0
Piperacilin/Tazobactam	60	24.8	182	75.2
Fluoroquinolone	103	42.6	139	57.4

Table 4 summarizes the characterization of forty P. aeruginosa isolates that were resistant to fluoroquinolones (100%) and/or resistant to carbapenems (80%), with 87.5% (35/40) of nosocomial origin and 12.5% (5/40) classified as community acquired. The isolates were recovered from clinical specimens as blood (52.5%; 21/40), tracheal secretion (20.0%; 8/40), urine (15.0%; 6/40), tissue fragment (10.0%; 4/40) and otitis (2.5%; 1/40). All patients with bloodstream infection used a central venous catheter, and 50% of patients with urinary infection had a urinary catheter. Furthermore, 86.7% (13/15) of the patients with pneumonia had ventilator-associated pneumonia (VAP). Antimicrobial susceptibility results were analyzed and 87.5% (35/40) isolates was characterized as MDR. The resistance rates to carbapenem, cefepime, piperacillin/tazobactam, and aminoglycoside were 80% (32/40), 72.5% (29/40), 57.5% (23/40) and 55% (22/40), respectively (data not shown). Moreover, 40% community isolates were characterized as MDR. The minimum inhibitory concentrations of imipenem and ciprofloxacin to inhibit 90% of 38 P. aeruginosa isolates selected were ≥32 and 64 μg/mL, respectively.

Table 4

Characterization of P. aeruginosa isolates resistant to fluoroquinolones and/or carbapenems with regard to the minimum inhibitory concentration, blaSPM, blaVIM, qnrS, aac(6´)-Ib-cr and aac(6´)-Ib genes, quinolone resistance determining region mutations and the PFGE patterns.

Characteristics	Positive/analyzed (%)
Phenotype
Multiresistant	35/40 (87.5)
Non-multiresistant	05/40 (12.5)
Metallo-β-lactamase
SPM	05/32 (15.6)
VIM	02/32 (06.2)
PMQR/aac(6′)-Ib1
qnrS1	01/38 (02.6)
aac(6′)-Ib-cr	01/38 (02.6)
aac(6′)-Ib7	28/32 (02.6)
QRDR2 mutations
gyrA: Thr83Ile	20/20 (100.0)
parC: Ser87Leu	16/20 (80.0)
parC: Glu91Lys	01/20 (05.0)
gyrA + parC: Thr83Ile + Ser87Leu	16/20 (80.0)
gyrA + parC: Thr83Ile + Glu91Lys	01/20 (05.0)
PFGE3 patterns
A	03/21 (14.3)
B	02/21 (09.5)
C	02/21 (09.5)
D-Q4	01/21 (04.8)
Predominant clones
A, B and C	07/21 (33,3)
Antimicrobial	MIC5 (μg/mL)
Imipenem
MIC50	≥32
MIC90	≥32
Ciprofloxacin
MIC50	16
MIC90	64

1Plasmid-mediated quinolone resistance or aminoglycoside 6′-N-acetyltransferase type Ib;

2Quinolone-resistance determining region;

3Pulsed field gel electrophoresis;

4each clone;

Minimum inhibitory concentration (n = 38). Of the total isolates, 12.5% are of Community origin (5/40).

Multiplex PCR to MBL was conducted only for carbapenem-resistant P. aeruginosa (32/40) from nosocomial and community infections, with 85.7% (30/35) and 40.0% (2/5), respectively, and seven isolates showed amplifications consistent with MBL genes indentified as blaSPM-1 and blaVIM types.

The frequency of isolates that presented PMQR genes (qnrS and aac(6’)-Ib-cr) was 5.3% (2/38). The only MDR isolate of nosocomial origin that presented the qnrS gene was recovered from lung, with the following MIC for ciprofloxacin and imipenem, 64 μg/mL and ≥32 μg/mL, respectively. This isolate did not show to present the MBL genes tested (blaIMP, blaVIM, blaSPM, blaGIM and blaSIM). Overall, 85% (34/40) of the isolates harboured aac(6’)-Ib gene, and was recovered from blood (38.2%; 13/34), tracheal aspirate (32.4%; 11/34), urine (17.6%; 6/34) and tissue fragment (11.8%; 4/34). The sequencing results showed that only one nosocomial isolate, recovered from tissue fragment and showed non-MDR profile, presented the cr variant (aac(6’)-Ib-cr gene), with a MIC of 64 μg/mL for ciprofloxacin. Besides that, 87.5% (28/32) harboured the aac(6’)-Ib variant. None of the isolates harboured qnrA, qnrB, qnrC, qnrD, and qepA genes. Moreover, among PMQR-positive isolates none had community origin.

Of the 20 P. aeruginosa isolates evaluated for QRDR mutations in gyrA and parC (Thre83Ile; Ser87Leu; Glu91Lys), none presented concomitant PMQR determinants (Table 4).

A total of 17 PFGE patterns (A-Q) of P. aeruginosa were observed among the 21 isolates analyzed, comprising 3 main clones: A (14.3%; 3/21), B (9.5%; 2/21), C (9.5%; 2/21) (Table 4 and Fig 2). The pulsotype A had two subtypes (A and A1), and they belonged to isolates that contained the blaSPM gene, while B and C had only one.

Fig 2

UPGMA dendrogram of PFGE profiles of 21 clinical P. aeruginosa isolates used in this study using the Dice coefficient under 1% tolerance and 1% optimization.

A similarity coefficient of 80% was chosen for cluster definition. 1Plasmid-mediated quinolone resistance determinants or aminoglycoside 6′-N-acetyltransferase type Ib; 2Metallo-β-lactamase; 3negative; 4not tested.

Discussion

Recent studies have shown that multi-drug resistance and several virulence determinants are key factors that contribute to the global spread of P. aeruginosa in hospitals [41-43]. The present study evaluated the risk factors for the development of infections caused by MDR P. aeruginosa, as well as those associated with antimicrobial resistance and treatment outcome in patients with bacteremia. The independent risk factors for development of MDR P. aeruginosa infections include prior use of antibiotics (carbapenems, fluoroquinolones, broad-spectrum cephalosporins, and aminoglycosides), being bedridden or in the intensive care unit, prolonged hospital stay, P. aeruginosa infection or colonization within a previous period of one year, malignant disease, mechanical ventilation, and history of chronic obstructive pulmonary disease [44, 45]. Data of univariate analysis from this study have corroborated some of these risk factors. However, only inappropriate therapy was a risk predictor independent associated for developing MDR P. aeruginosa infections, but it must be considered as such within the epidemiological context [46]. Some studies provide evidences of a general view that the development of MDR can be caused by treatment that is inappropriate, incorrect and widespread use of carbapenems [3, 46]. The pressure of carbapenems use has contributed to an explosive increase of KPC (carbapenemase-producing K. pneumoniae), which has been responsible for 70.9% of K. pneumoniae infections in the hospital of this study (data not shown).

Among patients with bacteremia (158 patients), 44.3% had isolates resistant to carbapenems, 42.4% resistant to fluoroquinolones and 42.4% with a multidrug-resistant profile. The association of bacteremia with antimicrobial resistant isolates is common, but few studies have addressed this problem systematically [47-49]. In hospitalized patients, the association between bacteremia and fluoroquinolone-resistant P. aeruginosa was observed in our study with a high frequency of patients remained hospitalized longer (56.7%), independently associated with bacteremia. Similar frequencies were also observed for those patients with MDR isolates (58.2%). In the carbapenem-resistant group, was observed a higher early mortality rate (5 days) that was statistically significant with those that had bacteremia infections. These aspects have often been observed in developing countries like Brazil [48, 50, 51], where the macro and micro-regional differences in relation to the hospitals are extremely significant, as well as the characteristics of the hospitalized population [29, 52]. Besides, the lack of the microbiology laboratories and human and financial resources, a result of poor implementation of control practices for prevention of nosocomial infections, favors the intra and inter-hospital transmission of resistant pathogens that exhibit adaptation to the environment [6, 52]. Another aspect that must be considered is that the antimicrobial use is commonly abusive, empirical and often less judicious [6, 52]. A study performed by Dantas [53] in the same university hospital of this study, showed that the density of antibiotic use was much higher when compared with hospitals with similar size in other countries, allowing MDR strains such as Gram-negative non-fermentative bacteria to emerge and spread quickly [54].

In addition to the multi-drug resistance, special attention was given in this study to P. aeruginosa resistant to carbapenems, considering the significant increase of this resistance in Latin America and widespread of different clones associated with the production enzymes of the MBL type [55-58]. The predominant MBL-encoding gene in Brazil is blaSPM-1, which has been disseminated by the MDR P. aeruginosa clone SP/ST277, considered a high-risk clone [7, 18, 59, 60]. The blaSPM-1 gene was first detected in São Paulo, and later in others cities in Brazil, moreover several studies have shown its global spread and pandemic potential, causing important morbidity and mortality in hospital infections [18, 43, 60, 61]. In our study, we observed a high frequency of SPM among the isolates, especially those belonging to clone A. In addition, two isolates harboring blaVIM were detected. An increase in the rates of P. aeruginosa isolates containing the gene blaVIM has been also observed in others hospitals in Brazil [62, 63]. The frequent data among different types of metallo-β-lactamase in Brazil, and not in other countries, especially those developed, suggest the spread of specific clones and the knowledge of these facts may contribute to improving the multidrug resistance scenario.

Besides the carbapenem resistance, resistance to fluoroquinolones has become an increasing problem, so far, only a few studies have investigated the occurrence of PMQR in P. aeruginosa [27, 29, 64, 65]. PMQR is an important phenomenon that is being disseminated worldwide and the most relevant PMQR genes to date are the aac(6’)-Ib-cr, qnr and genes encoding efflux pumps such as qepA [24]. Surprisingly, the results from this study demonstrated for the first time the presence of PMQR genes in clinical isolates of P. aeruginosa in Brazil (5.3%), as well as a very significant high frequency not shown in other studies of the aac(6’)-Ib variant. The PMQR rate in our study was higher than that some reported in the literature [27, 65]. According to the study reported by Jiang et al. [27], the frequency of clinical isolates carrying the aac(6’)-Ib-cr gene was 1.9% (2/106 isolates), and the total rate of PMQR determinants was 3,8% among P. aeruginosa isolates, while in the Yang and colleagues [65] study, only one in 256 P. aeruginosa isolates (0.4%) showed a PMQR gene. The higher detection frequency of these genes in our study reflects most likely the complexity of epidemiology and resistance mechanisms associated with P. aeruginosa in developing countries. The co-existence of different PMQR genes in the same clinical isolate was not observed throughout this study, although it has been reported by Jiang et al. [27].

Regarding the target site mutations in QRDR of fluoroquinolone resistant P. aeruginosa, we observed mutations consistent with those published previously in all isolates tested [66-68]. Of total, 20 Pseudomonas aeruginosa of nosocomial origin were evaluated for mutations in gyrA and parC, and none of them presented PMQR genes. However, according to literature evidences, the chromosomal QRDR mutations in gyrA and parC genes are crucial to fluoroquinolone resistance, and their association with PMQR determinants may have an additional role that contributes to resistance to fluoroquinolones [26].

Another interesting observation from this study was the identification of a mixed wound infection by P. aeruginosa and E. coli in the patient who presented P. aeruginosa harboring the PMQR gene aac(6’)-Ib-cr. In a previous evaluation of E. coli infections in the same hospital, 71.4% had aac(6’)-Ib-cr gene (data not shown). The epidemiology of P. aeruginosa in this hospital proved to be complex and can be explained by its involvement in the propagation through transference of plasmids to different species as well as by clonal spread.

PFGE results, in this study, suggested that P. aeruginosa isolates harboring PMQR genes or mutations in gyrA and parC were not closely related, except in those containing SPM clone, wherein in the literature showing results with a similarity clonal among isolates of MDR P. aeruginosa [69].

In conclusion, our results confirm previous findings regarding the dissemination of SPM-type clones in Brazil, and contribute whit additional evidence that may indicate that inappropriate therapy may be a crucial factor to the emergence of MDR isolates, besides being related to worse prognosis. Additionally, this study demonstrates for the first time in Brazil the presence of the PMQR determinants in P. aeruginosa, spreading in the hospital. Future follow-up surveillance studies of molecular epidemiology in Brazilian hospitals have crucial importance to infection-control practices and reduce the effects of these infections on hospital patients.

Primer nucleotide sequences and amplicon sizes of the PCR carried out for the detection and/or sequencing of antimicrobial resistance genes in this study.

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