Pseudomonas aeruginosa is a Gram-negative bacterium, which is categorized as an opportunistic pathogen due to its ability to cause infections mainly in immunocompromised patients. It is a ubiquitous microorganism, metabolically versatile, which is able to adapt to many environments (Gilligan, 1995; Lyczak et al., 2000). An important virulent characteristic is the formation of biofilms and its natural multiresistance to a wide range of antibiotics and disinfectants (Drenkard and Ausubel, 2002; Wolska and Szweda, 2009; Poole, 2011; Rybtke et al., 2015). This microorganism has been associated with nosocomial infections and outbreaks in Intensive Care Units (ICU) for adults, children and neonates (Thuong et al., 2003; Agodi et al., 2007; Zhang et al., 2012). It is a microorganism with the capacity to colonize different surfaces and in hospitals this is common in the colonization of humid sources, such as air conditioning units, sink faucets, and medical equipment (automatic ventilators and humidifiers; Agodi et al., 2007; Kerr and Snelling, 2009).

Approximately, a 30% of the general population carries P. aeruginosa on their skin and in their mucosa and intestine (Thuong et al., 2003; Agodi et al., 2007). This bacterium is associated with chronic recurrent infections in patients with cystic fibrosis and it represents a high mortality in children with underlying conditions such as hemato-oncology diseases, cardiovascular surgeries, extended hospitalization in the ICU, gastrointestinal malformations, and prematurity (Fergie et al., 1994; Zhang et al., 2012). Some reports have shown that the incidence of bacteremia due to P. aeruginosa falls between 0.09 and 3.8 cases per 1,000 patients with a greater frequency in boys (Grisaru-Soen et al., 2000) with underlying conditions, such as hemato-oncological diseases. Nonetheless, P. aeruginosa also causes infections, such as ear infections or skin infections in healthy people exposed to poorly chlorinated water in swimming pools or tubs for hydromassage (Mena and Gerba, 2009; Rybtke et al., 2015).

The genome of P. aeruginosa is highly variable due to the insertion of different mobile elements, such as genomic and pathogenic islands that contribute to chromosomal organization and genetic content thereby providing versatility to the bacteria that allows for better adaptation to different niches (Shen et al., 2006; Wiehlmann et al., 2007). Horizontal gene transfer (HGT) is a major force in bacterial evolution conferring a great variability between the species (Jolley and Maiden, 2010; Darmon and Leach, 2014).

The majority of the studies related to P. aeruginosa and pediatric cohorts have been performed in patients with cystic fibrosis (Cf; Kus et al., 2004; Kidd et al., 2015), which have genetic and phenotypic characteristic well-studied. The distribution of pilin alleles amongst CF human isolates belong to pilin group I. Biofilm production is thought to be a hallmark of chronic colonization of the CF lung; P. aeruginosa “hypermutators” can be isolated from 37 to 54% of the patients with chronic CF infections. MutS, a critical component of the mismatch repair system, is commonly lost in hypermutator strains, resulting in elevated mutation rates. P. aeruginosa hypermutator strains isolated from chronically infected patients are often more resistant to antibiotics, possess a mucoid phenotype with small-colony variants on culture medium, and lose both the lipopolysaccharide (LPS) O-antigen and motility (Deretic et al., 1994; Mahenthiralingam et al., 1994; Govan and Deretic, 1996; Häußler et al., 1999, 2003; Oliver et al., 2000; Leone et al., 2008; Chung et al., 2012; Kidd et al., 2012; Rybtke et al., 2015). Published data show the importance of P. aeruginosa as a cause of bacteremia in patients who develop neutropenia following chemotherapy and the bacterium has been associated with nosocomial infections (Pronovost et al., 2006). However, there is little published data on the genetic characteristics and the susceptibility patterns of P. aeruginosa strains isolated from blood samples from children who developed bacteremia and/or neutropenia following chemotherapy (Oliver et al., 2015; Peña et al., 2015). In the present study, we characterized a collection of P. aeruginosa strains isolated from the blood of 60 children with a background of underlying conditions that developed bacteremia and neutropenia post-chemotherapy in a highly specialized hospital in Mexico City.

We carried out the characterization of 60 P. aeruginosa isolates. All isolates showed biochemical patterns of P. aeruginosa (data not shown). The isolates were isolated from children, of whom 65% were female and 35% male. The median age of patients at the time of P. aeruginosa bacteremia diagnosis was 5.5 years with a range of 1 month to 14 years and 8 months. All patients admitted to hospital had underlying disease. The most common underlying diseases were hematological and oncological disease (52%) including acute lymphoblastic leukemia, non-Hodgkin lymphoma, solid tumor, hemophagocytic syndrome, histiocytosis and aplastic anemia, and prematurity (24%), other underlying diseases were gastrointestinal malformations, congenital cardiopathy, Wiscott-Aldrich disease, Dandy Walker syndrome, Chiari's malformation, and nephrogenic and diabetes insipidus (Figure 2). Medical records were unavailable for eight patients.

In 28 patients, bacteremia was related to a central catheter. Bacteremia was present in 21 children after chemotherapy treatment, with neutropenia and fever developing. The overall case fatality associated with P. aeruginosa bacteremia was 13.3% (8 of 60), who developed septic shock and multi-organ failure (Table 1).

European epidemiological surveillance programs show that P. aeruginosa is one of the most frequently isolated Gram-negative microorganisms from patients admitted to ICU (Pujol and Limón, 2013). The most important risk factors leading to development of nosocomial infections associated with P. aeruginosa in patients are a long period of hospitalization, the existence of a serious pre-existing condition and exposure to invasive procedures (Fergie et al., 1994; Yetkin et al., 2006; Yang et al., 2011). P. aeruginosa-associated infections have a high mortality rate due to the presence of virulence factors in the bacterium, innate and acquired multidrug resistance, and immune impairment of the host (Fergie et al., 1994; Lyczak et al., 2000; Corona-Nakamura et al., 2001; Thuong et al., 2003; Poole, 2011). Different studies have shown that P. aeruginosa is generally acquired from the hospital environment, person-to-person contact, indirect transmission via contaminated hands, through contaminated respiratory care equipment, catheters, irrigating solutions, and from the use diluted antiseptics and cleaning solutions (Corona-Nakamura et al., 2001; Thuong et al., 2003; Yetkin et al., 2006). Generally, P. aeruginosa outbreaks in hospitals are associated with clonally-related strains and through cross transmission in immunocompromised patients with underlying diseases, such as those with malignancies, burns, and prematurity (Agodi et al., 2007; Zhang et al., 2012; Cies et al., 2015). Although P. aeruginosa is considered an opportunistic pathogen, it has several virulence factors. These are encoded on plasmids or chromosomal genes, such as lasB (encoding for elastase), toxA (exotoxin-A), pilA (type fimbrial precursor type IV pilin), plcH (hemolytic phospholipase C precursor), phzA1 (phenazine biosynthesis protein), toxR (positive transcriptional regulator of toxA transcription), lecA (lectin; Wick et al., 1990; Walker et al., 1995; Rumbaugh et al., 1999; Woods, 2004; Shen et al., 2006; Wolska and Szweda, 2009; Morita et al., 2015), and four type III effectors: ExoU (phospholipase A2), ExoY (adenylate cyclase), ExoS (ADP-ribosylates numerous proteins, including members of the Ras protein family) and ExoT (a type III cytotoxin that functions as an anti-internalization factor with an N-terminal RhoGAP domain and a C-terminal ADP-ribosyltransferase domain; Sun and Barbieri, 2003; Jia et al., 2006; Cisz et al., 2008; Sun et al., 2012). These last two effectors are closely related to each other and participate in inhibiting phagocytic cells (neutrophil and macrophage function) and in bacterial uptake by epithelial cells (Engel and Balachandran, 2009). The characterization of strains from our current study showed the presence of all virulence genes in 100% of the strains, indicating that these genes are present in the structural integrity of the bacterial chromosome. Contrary to report by other authors (Feltman et al., 2001; Engel and Balachandran, 2009; Oliver et al., 2015; Peña et al., 2015), almost all our strains presented the exoU gene. Determination of the TTSS genotype showed that a high percentage was exoS+/exoU+ genotype. However, not all strains present the exoT gene. The presence of both exoS and exoU genes has been associated with acute infection in humans, such as bacteremia, and correlates with a worse outcome in clinical infections, a higher bacterial burden and a greater risk of death in mechanically ventilated patients (Feltman et al., 2001; Engel and Balachandran, 2009; Peña et al., 2015). While it is true that the results of the present study confirm this important relationship, between the exoS+/exoU+ genotype and bacteremia, the infections were resolved via antimicrobial treatment, probably due to low resistance of most of our strains to fluoroquinolones, aminoglycosides and ceftazidime, a third generation cephalosporin indicated for the treatment of patients who develop fever associated with neutropenia. On the other hand, registered mortalities in our study were reduced. Analysis of the results showed that there was no association between the exoS+/exoU+ genotype and risk of death.

The characterization of TFP alleles in our strains show diversity in TFP alleles to a greater extent than previously documented (Kus et al., 2004). A novel TFP allele was detected on the transposase accessory gene located between tRNA and pilA, which has been documented in other Pseudomonas species. A novel group of the pilA_VI gene with low sequence identity in its 3′ end to other pilA groups of different P. aeruginosa strains was also detected. The analysis of PilA_VI amino acid sequence showed a homology between 31.4 and 42.6% with respect to other groups of PilA (Figure S1). Although, we did not have experimental evidence of the PilA expression, the amino acids sequence is highly homologous in its first 127 aa to other sequences of PilA available in the databases, as it is showed in Figure S1. Using the Swiss-model (homology modeling) software, we obtained a virtual protein structure 100% homologous to P. aeruginosa fimbrial protein in its first 125 amino acids (image not shown), although from 126 to 142 aa, no homology was found with respect to the C-terminal region of the same PilA protein. Interestingly, we observed the lack of the two-cysteine residues within of its disulfide-bonded loop (DSL) region, which are involved in the disulfide bond formation contributing to the pilin assembly into fibers and its adhesive capacity (Harvey et al., 2009). The detection of this novel allele supports the notion of horizontal transfer of genes and recombination within homologous regions between bacteria and the incorporation of novel DNA into one of the hypervariable regions of the P. aeruginosa chromosome. The singularity of the accessory gene (transposase) and pilA_VI gene confirmed Kus's observations that each pilin type is stringently associated with a specific accessory gene. Additionally, the presence of the transposase gene immediately adjacent to the tRNA^thr gene confirms that a mechanism of bacteriophage-mediated transduction was involved in the generation of the new TFP allele. This is not surprising, since it is known that tRNA genes are hotspots for bacteriophage integration, where we detected greater genetic variability, as seen in the lack of characterization of three of our strains. The characterization of our strains isolated from the blood of children with bacteremia showed the predominance of two TFP alleles (group I and II). In her study, Kus reported a similar percentage of group I pilins within environmental strains and pediatric CF isolates, while in other human isolates, there appears to be an approximately equal distribution of strains within pilin groups I, II, and III (Kus et al., 2004). The characterization of different populations of P. aeruginosa isolated from different sources is required in order to determine if there is a correlation between the pilA allele and a specificity niche.

In addition to virulence genes, the bacterium acquired foreign DNA in combinations of specific blocks of genes that contributed to virulence and/or adaptation to specific niches. These strain-specific segments of the genome are found in limited chromosomal locations, referred to as genomic islands (GEIs), which are acquired by HGT (Ou et al., 2006; Boyd et al., 2008; Juhas et al., 2009). Depending on their functions, they encode for pathogenicity, symbiosis, fitness, metabolic, or resistance traits (Hacker and Kaper, 2000; Dobrindt et al., 2004; Juhas et al., 2009). A large number of GEIs in the P. aeruginosa chromosome have been described, but these GEIs are found in varying numbers in some strains and not in others (Schmidt et al., 1996; Liang et al., 2001; Larbig et al., 2002). In the present study, all the strains isolated from children diagnosed with bacteremia possessed the PAPI-2 Island and more than half of them had PAPI-1 with both islands presenting a mosaic structure. Most of the PAPI-2 genes are related to mobility functions, including integrase genes, transposase genes, one pseudogene, and portions of insertion sequences in addition to the presence of seven ORFs that correspond to hypothetical proteins of unknown function (He et al., 2004). Interestingly, in the right end of PAPI-2 there are two genes that correspond to the exoU gene and its chaperone spcU (He et al., 2004). As mentioned previously, exoU encodes a type III effector (ExoU) that plays an important role in pathogenesis. ExoU is a potent cytotoxin with phospholipase A2 activity, which has been associated with the development of septic shock in an animal model (Kurahashi et al., 1999). The presence of exoU in almost all our strains isolated from blood samples taken from patients with bacteremia corroborates the data previously reported by Kurahashi. The presence of exoU on PAPI-2 defines this island as a pathogenicity island and it is very likely that the expression of ExoU for P. aeruginosa strains facilitates the spread through tissues favoring the arrival of bacteria to bloodstream (He et al., 2004; Kulasekara et al., 2006). On the other hand, PAPI-1 genes are involved in adhesion and/or motility, although, the majority of their genes encode for hypothetical proteins, making this island unique (He et al., 2004; Qiu et al., 2006; Carter et al., 2010; Harrison et al., 2010). This island contains two pairs of two-component regulatory systems, which through mutational analysis have been shown to affect plant and mammalian pathogenesis (He et al., 2004). In addition to all genes being involved in type 4 fimbrial assembly and function in the pil chromosomal region, the PAPI-I Island has a set of genes (pilL, pilN, pilO, pilQ, pilR, pilS, pilT, pilV, and pilM) involved in type IVb pilus biogenesis that contributes to adherence onto synthetic surfaces, such as catheters (Giltner et al., 2011), which provide an entrance to the circulatory system.

The presence of both PAPI-I and PAPI-2 islands in more than half of our strains show that these islands are contributing to P. aeruginosa virulence, promoting colonization on catheter surfaces and skin injury with the induction of proinflammatory mediators, and passing the bacteria into the blood system benefitting the survival and fitness of the bacterium. The antimicrobial resistance profiles showed that more than half of our strains isolated from children had a moderate resistance profile, which may provide some explanation as to the low mortality rate reported in our study. The number of MDR and XDR strains detected is still low in the present study (Pediatric Hospital), nevertheless, continuous epidemiological surveillance is necessary to monitor MDR and XDR strain presence considering the continuous admission of patients to different hospital services and horizontal genes transfer from hospital microbiota to patient's native microbiota. Analysis of chromosomal profiles and MLST of the strains showed great genetic variability among our population, indicating that there is no clonal relationship. However, it is striking that in a cluster of nine strains, six were isolated from urine and all nine share the ST309, TFP allele (group II), and have the XDR (six strains) or MDR (two strains) phenotype. There are reports of P. aeruginosa high-risk clones circulating in hospitals worldwide, which present specific genetic characteristics (ST111, ST235, and ST175) linked to MDR or XDR phenotype (Cabot et al., 2012; Mulet et al., 2013; Witney et al., 2014; Oliver et al., 2015; Peña et al., 2015). The increasing prevalence of these clones complicates the clinical landscape, limiting therapeutic options and having significant impact on morbidity and mortality. The presence in our population of strains ST309 linked to the MDR or XDR phenotype, make them a potential high-risk clone, which was not documented as such, previously. However, it is important to highlight that in our study, this clone was not associated with any of the mortality cases. Studies in other populations and hospitals settings are recommended in order to determine the presence of ST309 as part of a potential high risk clone, its distribution throughout hospitals in Mexico and its importance to the severity of the clinical outcome.

Based on the overall analysis of the results obtained in this study, we found that the strains of P. aeruginosa causing bacteremia in each child harbored exoU and exoS. This results are support for the Berthelot's observations (Berthelot et al., 2003), who characterized genetically and phenotypically 92 P. aeruginosa strains isolated from blood: where, they identified four groups of strains (TTSS types) according to level of type III protein secretion and kinetics of cytotoxicity. Additionally, they made the detection of exoU and exoS genes by real-time PCR. They found a strong correlation among exoU+ and exoS+ genotype and TTSS phenotype. They concluded that the most of the bacteremic strains (80%) were strongly cytotoxic for macrophages and that the ExoU-secreting isolates killed the phagocytes more rapidly. Based on Berthelot's study, we can deduce that our strains being cytotoxic. It is likely that exoU was acquired through horizontal transfer of PAPI-2 from one strains to other. It also appears likely that the patients were carrying the majority of the strains prior to hospital admission and immunosuppression caused by underlying disease favored the multiplication of microorganisms and adherence to catheter surfaces. The presence of exoU on PAPI-2 island gives bacteria the ability to disseminate into the circulation and produce bacteremia, and in some cases the development of septic shock (Engel and Balachandran, 2009).

We identified a reduce number of exogenous β-lactamases among strains, with KPC β-lactamase being the most frequent. However, the presence of a potential high-risk clone, ST309 with a MDR or XDR phenotype, circulating throughout of our hospital could create a serious health problem.

Multiresistant bacteria serve as hosts for the multiple genetic elements (genes, integrons, transposons, and plasmids) that confer their antibiotic resistance phenotypes. This important characteristic allows to the bacteria to be a “successful” bacterial strain, which is an extremely effective vehicle for the dissemination of any genetic element (s) for at least two reasons: (a) all of the hosted resistance elements are transmitted vertically (i.e., from mother to daughter cells) by virtue of the strain's spread and its increasing prevalence and (b) a successful strain has multiple opportunities to act as a donor and to transfer its resistance elements horizontally to other strains, species or genera (Maatallah et al., 2011; Woodford et al., 2011). So that, the identification of a successful multiresistant strain or clone should receive prompt attention to avoid HGT of antimicrobial resistance into bacterial populations, and its dissemination to different hospitals and different regions.

Additionally a high-risk clone should have important characteristics: (a) to be pathogenic (to have virulence factors); (b) to have a resistance profile to at least three groups of antibiotics (extensive drug resistance) and (c) to be present in different places (Woodford et al., 2011). ST309 strains have been documented in France, Australia, Malaysia and even Brazil, which have been isolated from water and some clinical samples such as bronchial lavage, blood, and urinary tract (P. aeruginosa PubMLST website).

Despite the fact that the relative contributions of endogenous and exogenous sources to P. aeruginosa acquisition are not well-established. At this moment, we can assume that in our study hospital, P. aeruginosa infections are not the result of epidemic outbreaks, since the strains associated with infection were highly variable and they were not acquired in the hospital setting.

To conclude, genetic and phenotypic characterization of 60 isolates of P. aeruginosa associated with blood infections in children admitted to a highly specialized hospital in Mexico, showed that the infections were caused by strains with great diversity in their accessory genome. In the majority of the cases, there was no cross-infection between patients associated with a single clone. The P. aeruginosa strains isolated from blood and involved in bacteremia were TFP allele group I and II, and cytotoxic (exoU+ and exoS+). The results support the idea that the presence of PAPI-I and PAPI-2 in the strains contributed to greater virulence, which is associated with better adherence and dissemination into the bloodstream leading to an increased risk of septicemia. We identified the presence of ST309 strains isolated from urinary tract, which possess virulence genes, an extensive drug resistance phenotype, important characteristics that would contribute to make them a potential high-risk clone.

RM, conceived and designed the experiment, wrote the paper. GM and CR critical review of the article and review the article for intellectual content. RM, GD, and AC, Final approval of the version to be published. JM, CR, performed the experiments of Antimicrobial susceptibility and made the analysis of results. GD, made all genetic analysis corresponding to MLST, Type III Secretion System Genotype and Analyzed in general all results. LE and CR, Designed the primers and performed the experiments for the characterization of the β-lactamase genes and MLST and determined TFSS genotype. DI performed the experiments for PFGE, virulence genes and GEIs determination.

This research was funded by DGAPA-PAPIIT Grant number IN212513 from Universidad Nacional Autónoma de México. The funder has no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript.