Pseudomonas aeruginosa is an important causative agent of nosocomial infections. This bacterium is considered an opportunistic pathogen that affects the health of immunocompromised individuals such as those with diabetes, cancer, cystic fibrosis, advanced HIV infections (acquired immunodeficiency syndrome, AIDS), severely burn patients and those that underwent major surgeries.1

As pathogen, Pseudomonas aeruginosa is of increasing clinical importance because of their innate resistance to multiple agents and their ability to develop high-level multidrug resistance (MDR) due to the presence of several virulence factors encoded in its genome (<-- -->Poole, 2016). Several studies have shown that this innate resistance is directly associated to the expression of bacterial efflux pumps and porins such as the Resistance-Nodulation-cell-Division (RND)-type efflux pump, MexAB-OprM, MexC–MexD–OprJ, MexE–MexF–OprN, and MexXY-OprM.2 These important RND-type efflux pumps are constitutively expressed in wild-type cells and are responsible for the intrinsic resistance to most antimicrobial agents.3–7

The MexAB-OprM system was initially characterized from an isolate of P. aeruginosa cultured under iron-depleted conditions in the presence of the iron chelator 2,2’- dipyridyl (Dipy).8,9 Under this condition, protein levels for the MexAB-OprM members were increased, suggesting that the efflux pump plays an essential role in its survival under iron deficiency conditions8,10,11 (Oglesby-Sherrouse et al., 2009)<-- -->.

The MexAB-OprM system includes the proteins MexA, MexB, OprM, and MexR. MexB is the molecular subunit that selectively recognizes specific antibiotics and mediates their expulsion; MexA functions as the interconnecting bridge between OprM and MexB. OprM is a lipoprotein localized in the outer membrane whose role is mediating the final expulsion of the antibiotic across the bacterial outer membrane. MexR acts as repressor of the MexAB-OprM system and autoregulates MexR.12

In addition to the MexAB-OprM efflux pump system, Pseudomonas aeruginosa produce small molecules known as siderophores, which are iron-chelating molecules that contributes to their virulence; siderophores have also been connected to mechanisms of antibiotic resistance (<-- -->Djapgne et al., 2014). Under iron-limiting conditions, P. aeruginosa produces two main siderophores, pyoverdine and pyochelin, which scavenge iron from host proteins, contributing to its virulence.13–15 The ferri-pyoverdine complex uptake is carried out by TonB-dependent receptors with the help of the transporter FpvB,16 resulting in its internalization into the periplasm.17 In addition to its molecular function, the pyoverdine receptor genes have also been used to genotype several strains of P. aeruginosa.18

Another important resistance mechanism in Gram-negative bacteria such as P. aeruginosa is the production of β-lactamases, hydrolytic enzymes involved in the degradation of antibiotics (β-lactams). Among this group, the extended-spectrum β-lactamases (ESBLs) represent an important sub-class of enzymes that confer resistance to oxyimino-cephalosporins, such as cefotaxime (CTX), ceftriaxone, and ceftazidime (CAZ), and the monobactam aztreonam (ATM). Because of this enhanced resistance phenotype against some of the most effective antibiotics, strains carrying ESBLs genes can become a serious clinical problem.19

Several genes coding β-lactamases have been identified at low frequency in P. aeruginosa.20 blaTEM-1 was the first plasmid-encoded β-lactamase gene isolated from E. coli; this isolate came from a blood culture of a patient named Temoniera in Greece, hence the acronym TEM.21 blaTEM-1-like β-lactamases have also been found in other species of the family Enterobacteriaceae and other bacterial species such as Pseudomonas aeruginosa, Haemophilus influenzae, and Neisseria gonorrhoeae.22 Among the ESBL sub-class, SulfHydryl Variable-2 (SHV-2) ESBLs were first identified in a German strain of Klebsiella ozaenae; this novel enzyme is capable of hydrolyzing cefotaxime and to a lesser extent ceftazidime.23 Several other studies have reported the existence of non-blaTEM and non-blaSHV hydrolytic enzymes that do not share the evolutionary and functional origin of typical EBSLs.24 These non-blaTEM and non-blaSHV plasmid-encoded β-lactamases and cefotaximases (blaCTX-M) are able to hydrolyze cephalothin and cefotaxime, and to lesser extend benzylpenicillin and ceftazidime.23 Therefore, the characterization of β-lactamases from drug-resistant P. aeruginosa can greatly contribute to the understanding of mechanisms of molecular pathogenesis, antibiotic resistance and virulence in bacteria<-- -->.25,26

To better understand the intrinsic virulence of circulating strains of Pseudomonas aeruginosa from Panamá, we surveyed and characterized the antibiotic resistance profiles and the prevalence of virulence factors in 51 clinical isolates of P. aeruginosa obtained from children admitted to Hospital del Niño-Panamá.

Pseudomonas aeruginosa is a Gram-negative bacterium with a capacity to produce a wide range of virulence factors which makes it a MDR pathogen for which there are limited therapeutic options. Therefore, the understanding of resistance mechanisms can facilitate the development of new therapeutics.

In this study, we tested the resistance pattern of P. aeruginosa using antibiotics of the following groups: aminoglycosides (amikacin and gentamicin), fluoroquinolones (ciprofloxacin), other β-lactams (cephalosporins, ampicillins) (ceftazidime, cefepime, cefotaxime, piperacillin, ampicillin), pyrimethamine (trimethoprim), nitrofuran (Nitrofurantoin) and carbapenems (meropenem and imipenem).

In Panama, the Central Reference Public Health Laboratory (LCRSP) of the Gorgas Memorial Institute for Health Studies (ICGES) reviewed the National Antibiotic Resistance Database from 2007 until 2013.30 For the purpose of our study, we chose the Gram-negative bacterium P. aeruginosa because of its medical importance and innate ability to develop antibiotic resistance. In our study, this bacterium was shown to be more susceptible to two aminoglycosides gentamicin (100%) and amikacin (98%), fluoroquinolones (ciprofloxacin, 96%) and two third generation cephalosporins, ceftazidime (88%) and cefepime (94%), as shown in Fig. 1. By contrast, the isolates tested in this study showed increased resistance to antibiotics in the following categories: (i) penicillins (ampicillin 69%, piperacillin 22%); (ii) pyrimethamines (trimethoprim 65%); (iii) nitrofurans (nitrofurantoin 63%), and (iv) third-generation cephalosporin cefotaxime (53%), in contrast to previous reports.30 Resistance values for carbapenems oscillated between 25 and 33% for meropenem and imipenem, respectively.

In the United States, the National Healthcare Safety Network reported that Pseudomonas aeruginosa is the sixth most common nosocomial pathogen and the second most common in ventilator-associated pneumonia in hospitals.31 Nguyen et al.31 reported 9.7% resistance rate to at least one aminoglycoside and 19.3% to at least one carbapenem compared to 2% and 25-33%, respectively, in this study. The reported resistance rate for cephalosporins was 10.3% (cefepime or ceftazidime) compared to 6%, 12% and 53% for cefepime, ceftazidime and cefotaxime, respectively, in this study. Resistance for one fluoroquinolone was reported in 21.6% of isolates compared to 4% in our study. Overall, the MDR index was reported to be 14.2% (previous study), much lower than the 59% observed in the present study. The difference between the two studies could be explained by the presence of intrinsic genetic differences between circulating isolates, which is corroborated by the presence of various resistance mechanisms and/or genes that contribute to virulence and resistance to various drugs representatives of seven antibiotic classes.

The MexAB-OprM efflux system is responsible for the development of resistance to a number of antimicrobials including quinolones, macrolides, tetracyclines, lincomycin, chloramphenicol, novobiocin, and β-lactams.3,6,7,32,33 In our study we detected the presence of genes of the MexABR-OprM efflux system under iron-limiting conditions, as evidenced by the growth of P. aeruginosa under these conditions (CAA medium) and the presence of amplicons for mexA, mexB and mexR by multiplexed standard PCR (Fig. 2A). These results are in line with two previous reports where all components were encoded in the genome and induced by the iron-limiting conditions and their detections indicate that they confer or allow the development of resistance to different antibiotics.12,33–36 The functional expression of each system components and its individual contribution to the resistance phenotype measured is the topic of a follow up study.

Given that iron is key for the development of antibiotic resistance in Pseudomonas aeruginosa, in this bacterium the mechanism of iron uptake is mediated by siderophores, pyocheline and pyoverdine, making them all crucial for the establishment of infection processes.15,37–39 Several studies have shown that increasing concentrations of iron decreased resistance to antibiotics.40,41 Under iron-repleted conditions, P. aeruginosa shows enhanced resistance to the antibiotics tigecycline and tobramycin; the production of pyoverdine was shown to be an important factor for resistance against tigecycline.41,42 In the present study, all 51 clinical isolates were pyoverdine producers (Table 1) and were PCR-positive for all pyoverdine receptor genes, being fpvAI-type the most prevalent type (44%), followed by fpvAIII-type (31%) and fpvAII-type (31%) (Fig. 2B). Our results agree with a previous study where the clinical isolates studied showed a similar prevalence43 meaning that production of pyoverdine increases virulence of the bacteria.38 A previous study associated P. aeruginosa antibiotic resistance with the presence of fpvA genes; the reported prevalence for the receptor genes fpvAI, fpvAII, and fpvAIII were 30%, 20%, and 23%, respectively.44 Recently, a whole genome sequence of two MDR strains revealed as well that gene encoding putative proteins for iron uptake and other virultent factors were detected in both strains. The two siderophores pyochelin and pyoverdine were observed in both MDR strains and their corresponding receptors genes.45 These results agree with those found in this study for all MDR.

Pyoverdine pigment production is ubiquitous amongst β-lactamase-producing strains of P. aeruginosa. Because both pyoverdine and β-lactamase production are necessary for the acquisition of drug resistance capabilities,25,26 we decided to screen our isolates for the presence of ESBLs genes such as blaTEM, blaCTX-M and blaSHV using PCR. Twenty-two strains were PCR positive for the presence of the ESBL blaTEM1-like genes (43%) (Fig. 2C); no PCR products were obtained for blaCTX-M and blaSHV genes. All blaTEM1-positive isolates were highly similar to the blaTEM1 gene of Pseudomonas aeruginosa strain SJP2. An earlier study in P. aeruginosa by Girlich et al.46 reported a 0% prevalence of CTX-M-like and SHV-like genes, and the presence of a gene that coded for a β-lactamaseVEB-1 (blaTEM-like) inserted in an integron; these results are in line with the present study. By contrast, a study by reported a similar prevalence for blaTEM-like genes (26.7%), as well as low prevalences for low-frequency β-lactamases such as blaCTX (17.3%) and blaSHV (10.7%).47 Similarly, a study by Bokaeian et al.48 reported prevalences of 30% for blaTEM-like, 6.6% for blaVHS-like and 0 % for blaCTX-like β-lactamases. Furthermore, certain strains of Pseudomonas and Acinobacter spp. exhibit profound differences in the prevalence of β-lactamase-like genes; in these strains the most prevalent type is blaVHS-like (75%), followed by blaCTX-like (57.5%) and finally blaTEM (15%).23 Together the presence of β-lactamases and the MexAB-OprM efflux pump system greatly contributes to resistance against many classes of β-lactams in P. aeruginosa.7 Our initial screening provided important clues regarding the potential pathogenicity and geographic occurrence of circulating nosocomial strains in Panama.

Different approaches have been used to fight infections caused by P. aeruginosa, from the design of new drugs to metagenomics projects aimed at leading to the discovery of new antibiotics; however, despite such advances, together they have been of limited success41 due to the diversity of resistance mechanisms that allows the bacterium to evade a wide variety of antibiotics. New therapeutic strategies have been developed, i.e. by combining iron-chelators with antibiotics, given the preponderant physiological role of iron in the physiology of this pathogen.42,49–52 The virulence factors identified in our study provides valuable information regarding the prevalence of resistance genes and their potential impact on treatments that exploit the unique physiology of the pathogen. More importantly, this information can be used to better guide future control strategies, understand the evolution and dissemination of strains hoping to prevent the development of resistance in Pseudomonas aeruginosa.