The Journal

The Journal ofofInfectious

InfectiousDiseases
Diseases
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N T AARR TT II CC L E

Host and Pathogen Biomarkers for Severe Pseudomonas

aeruginosa Infections
Carlos Juan,1 Carmen Peña,2 and Antonio Oliver1
1
Servicio de Microbiología and Unidad de Investigación, Hospital Universitario Son Espases, Instituto de Investigación Sanitaria de Palma, Palma de Mallorca, and 2Servicio de Medicina Interna,
Hospital Virgen de los Lirios, Alcoy, Spain

Pseudomonas aeruginosa is among the leading causes of severe nosocomial infections, particularly affecting critically ill and immu-
nocompromised patients. Here we review the current knowledge on the factors underlying the outcome of P. aeruginosa nosocomial
infections, including aspects related to the pathogen, the host, and treatment. Intestinal colonization and previous use of antibiotics
are key risk factors for P. aeruginosa infections, whereas underlying disease, source of infection, and severity of acute presentation are

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key host factors modulating outcome; delayed adequate antimicrobial therapy is also independently associated with increased mor-
tality. Among pathogen-related factors influencing the outcome of P. aeruginosa infections, antibiotic resistance, and particularly
multidrug-resistant profiles, is certainly of paramount relevance, given its obvious effect on the chances of appropriate empirical
therapy. However, the direct impact of antibiotic resistance in the severity and outcomes of P. aeruginosa infections is not yet
well established. The interplay between antibiotic resistance, virulence, and the concerning international high-risk clones (such as
ST111, ST175, and ST235) still needs to be further analyzed. On the other hand, differential presence or expression of virulence
factors has been shown to significantly impact disease severity and mortality. The likely more deeply studied P. aeruginosa virulence
determinant is the type III secretion system (T3SS); the production of T3SS cytotoxins, and particularly ExoU, has been well estab-
lished to determine a worse outcome both in respiratory and bloodstream infections. Other relevant pathogen-related biomarkers of
severe infections include the involvement of specific clones or O-antigen serotypes, the presence of certain horizontally acquired
genomic islands, or the expression of other virulence traits, such as the elastase. Finally, recent data suggest that host genetic factors
may also modulate the severity of P. aeruginosa infections.
Keywords. Pseudomonas aeruginosa; virulence; biomarkers; antibiotic resistance; high-risk clones; nosocomial infections.

Pseudomonas aeruginosa, a ubiquitous microorganism, is one
of the most relevant pathogens causing human opportunistic
infections [1]. P. aeruginosa is a leading cause of severe nosoco-
mial infections, particularly in critically ill and immunocom-
promised patients [2, 3], and is difficult to treat because of
its limited susceptibility to antimicrobial agents [4] and the
frequent emergence of antibiotic resistance during therapy [5].
Indeed, P. aeruginosa is the top pathogen causing ventilator-
associated pneumonia and burn wound infections and is
a major cause of nosocomial bacteremia, with a very high
(>30%) associated mortality rate [2, 3, 6]. Community-acquired
P. aeruginosa infections in immunocompetent patients are
overall less prevalent, although the pathogen is a frequent
cause of otitis externa (swimmer’s ear) and hot tub folliculitis.
Likewise, P. aeruginosa is the most frequent and severe driver of
chronic respiratory infections in patients with cystic fibrosis or
other chronic underlying diseases, such as bronchiectasis and
chronic obstructive pulmonary disease [1].
Correspondence: A. Oliver, Servicio de Microbiología, Hospital Son Espases, Ctra. Valldemossa
79, 07010 Palma de Mallorca, Spain (antonio.oliver@ssib.es).
The of Infectious Diseases®® 2017;215(S1):S44–51
Infectious Diseases
Journal of
© The Author 2016.7 Published by Oxford University Press for the Infectious Diseases Society
of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.
DOI: 10.1093/infdis/jiw299
The versatility and adaptability conferred by the large pro-
portion (>8%) of regulatory genes encoded in its large genome
(>6 mega base pairs), its remarkable repertoire of virulence de-
terminants, and its outstanding capacity to evade the activity of
antimicrobial treatments makes P. aeruginosa one of the most
feared bacterial pathogens [1, 7].
In addition to the pathogen, the outcome of the infections is
modulated by factors related to the treatment and the host. It
has been well documented that decreased antibiotic effectiveness
or delayed initiation of therapy may contribute to adverse results
[8]. Likewise, the severity of the underlying disease may be syner-
gistic with infection due to resistant organisms. Severity of acute
illness presentation is associated with a higher mortality [2] and
might be determined not only by P. aeruginosa virulence, but
also by the interaction between the bacteria and innate immunity
host response, as well as by the complex combinations of genetic
polymorphisms modulating human susceptibility to infections.
Therefore, here we review the current knowledge on the potential
factors underlying the outcome of P. aeruginosa infections, includ-
ing the aspects related to the pathogen, the host, and treatment.
THE MULTIPLE FACES OF P. AERUGINOSA
ANTIMICROBIAL RESISTANCE
P. aeruginosa is genetically equipped with outstanding intrinsic
antibiotic resistance machinery [7, 9, 10]. The production of an

S44  • JID 2017:215 (Suppl 1) •  Juan et al Biomarkers for P. aeruginosa Infections • JID • S1

inducible AmpC cephalosporinase, the constitutive (MexAB-
OprM) or inducible (MexXY) expression of efflux pumps,
and the reduced permeability of its outer membrane are likely
the mechanisms having a greater impact on the basal lower sus-
ceptibility of P. aeruginosa to antibiotics, compared with other
gram-negative pathogens.
In addition to its remarkable intrinsic resistance, P. aeruginosa
shows an extraordinary capacity for further developing resistance
to all available antibiotics through the selection of mutations in
chromosomal genes. The main mechanisms for developing resis-
tance to the antipseudomonal penicillins, cephalosporins, and
monobactams, occurring in >20% of P. aeruginosa clinical iso-
lates, is the selection of mutations in peptidoglycan-recycling
genes (ampD, dacB, and ampR) leading to the constitutive over-
expression of AmpC [11–13]. Moreover, specific mutations
in ampC have been recently shown to increase the resistance
of P. aeruginosa to antipseudomonal cephalosporins [14].
Likewise, mutational inactivation of the porin OprD confers re-
sistance to imipenem and reduced susceptibility to meropenem
[15]. In fact, the prevalence of imipenem-resistant isolates fre-
quently exceeds 20%, and nearly all of them are OprD deficient
[11]. The mutational overexpression of one of at least 4 of the
P. aeruginosa efflux pumps also plays a major role in acquired
resistance [11, 16, 17].
In addition to efflux pump overexpression, fluoroquinolone
resistance in P. aeruginosa frequently results from target muta-
tions in topoisomerases, including DNA gyrase (GyrA/GyrB)
and type IV topoisomerases (ParC/ParE) [18]. Overall, fluoro-
quinolone resistance rates vary geographically but range from
30% to 40% in many countries. On the other hand, the preva-
lence of polymyxin ( polymyxin B and colistin) resistance is still
very low (<5%) but has been increasing in the past years owing
to the frequent use of these last-resource antibiotics for the
treatment of multidrug-resistant (MDR) and extensively
drug-resistant (XDR) strains [19]. Polymyxin resistance most
frequently results from the modification of the lipopolysaccha-
ride (LPS), caused by the addition of a 4-amino-4-deoxy-L-
arabinose moiety in the lipid A structure [20]. The underlying
mutations are frequently tracked to the PmrAB or PhoPQ 2-
component regulators, which lead to the activation of the
arnBCADTEF operon [20]. More-recent studies revealed that
mutations in the ParRS 2-component regulator, in addition
to conferring colistin resistance due to the activation of the
arnBCADTEF operon, lead to a MDR profile caused by the
overexpression of MexXY and the repression of OprD [21].
In addition to frequent mutation-driven resistance, the
increasing prevalence of horizontally acquired resistance in
P. aeruginosa is a growing threat. Indeed, the prevalence of
the most concerning acquired β-lactamases, particularly class
B carbapenemases (metallo-β-lactamases), including VIM,
IMP. and NDM enzymes, is increasing worldwide although
with uneven distribution, ranging from <1% to close to 50%,
S2 • JID • Juan et al
depending on the hospital and geographic area [22]. These con-
cerning resistance elements are strongly linked to the interna-
tional high-risk clones, particularly ST235 [23].
Regardless of the involved mechanisms, the prevalence of
MDR/XDR P. aeruginosa is certainly on the rise worldwide. Al-
though important geographical differences exist, and consensus
definitions (resistance to at least three of eight defined antibiotic
classes) have not been reached until very recently [19], the prev-
alence of MDR P. aeruginosa has increased in the past decade
and currently ranges from 15% to 30% in many areas [24–26].
Moreover, an important proportion of MDR strains meet the
criteria of XDR (resistance to all but 1 or 2 of the 8 classes), lim-
iting further our therapeutic arsenal. For instance, a recent large
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multicenter study of P. aeruginosa bloodstream infections per-
formed in Spain revealed that 28% of the isolates were MDR and
that, of these, 52% (15% of all isolates) met the XDR criteria,
with most being only susceptible to polymyxins, amikacin,
and/or the new β-lactam–β-lactamase inhibitor combinations
ceftolozane-tazobactam and ceftazidime-avibactam (unpub-
lished data).
THE VERSATILE VIRULENCE REPERTOIRE OF
P. AERUGINOSA
Even as an opportunistic pathogen of environmental origin,
P. aeruginosa has a notable number of virulence determinants
and genes required for establishing infections, which are encod-
ed in the core of its large genome and therefore shared by most
of the strains, independently of whether they are isolated from
the environment or from infected plants or humans. Neverthe-
less, particular P. aeruginosa strains may contain additional ge-
nomic islands, acquired by horizontal gene transfer and
representing 10% or even more of the genome [27], which
may include pathogenicity islands that further increase their
virulence potential, such as PAPI-1 and PAPI-2 described in
the hypervirulent strain PA14 [28, 29]. Moreover, up to 12
P. aeruginosa genomic islands (PAGIs) have been described
so far, and many of them have been demonstrated to play a
major role in virulence [30–32]. The expression of P. aeruginosa
virulence determinants is tightly controlled by multiple cell-to-
cell communication signaling systems [33], as well as by
multiple 2-component response regulators that frequently
interconnect antibiotic resistance and virulence [34]. Supple-
mentary Table 1 shows a summary of P. aeruginosa genes
involved in virulence, as well as the type of existing evidence
(in vitro, animal model, or epidemiological).
P. aeruginosa is able to secret a large number of exoproducts
that interact with specific host targets. Up to 6 types of secretion
systems, designated type I (T1SS) to type VI (T6SS), have been
identified [35]. Among them, the T3SS is of paramount rele-
vance for P. aeruginosa virulence. There are 4 known T3SS ef-
fector cytotoxins: ExoT, ExoY, ExoS, and ExoU. While exoT and
exoY are detected in the vast majority of strains, exoS and exoU
Biomarkers for P. aeruginosa Infections  •  JID 2017:215 (Suppl 1) • S45
are nearly mutually exclusive. The presence of exoS is found in
58%–72% of the isolates and is typically associated with an in-
vasive phenotype, such as that seen in PAO1 or PAK. On the
other hand, exoU is less frequent (in 28%–42% of isolates)
but associated with a highly cytotoxic phenotype, such as that
seen in PA14 or PA103 strains [36].
INTERNATIONAL HIGH-RISK P. AERUGINOSA
CLONES
The global success of bacterial pathogens is expected to be de-
termined by a complex interplay between pathogenicity, epide-
micity, and antibiotic resistance [37]. The fitness cost of
antibiotic resistance mechanisms [38], the existence of regulato-
ry networks interconnecting resistance and virulence [34, 39],
and natural genetic engineering linking antibiotic resistance de-
terminants and clonal success through genetic capitalism [40]
are thought to be the main elements of this intricate equation
[41]. For decades, multiple reports have warned of the occur-
rence of epidemic outbreaks caused by MDR/XDR strains with-
in the hospital environment. More recently, concerning reports
have provided evidence of the existence of MDR/XDR global
clones disseminated in several hospitals worldwide that have
been denominated high-risk clones [23, 42].
Typically, molecular epidemiology surveys of P. aeruginosa
isolates recovered from nosocomial infections, as well as sur-
veys of environmental isolates and those from patients with
cystic fibrosis, reveal a remarkable clonal diversity, with most
isolates represented by single genotypes. However, further
analyses show that this is the case for antibiotic-susceptible
strains but not for MDR and, particularly, XDR strains. For ex-
ample, a recent multicenter study of P. aeruginosa bloodstream
infections performed in Spain revealed that nearly all of each
susceptible isolate were represented by single genotypes,
while clonal diversity was much lower among MDR and, spe-
cially, XDR strains [7, 8]. In fact, 73 of 81 XDR isolates (90%)
belonged to just 3 clones, corresponding to the 3 major inter-
national MDR/XDR high-risk clones: ST111, ST175, and
ST235 [24].
The interplay between virulence and antibiotic resistance is
also a major point to consider: antibiotic resistance mechanisms
may (1) determine a biological cost compromising bacterial vir-
ulence, (2) have a positive or negative direct effect on virulence,
and (3) be statistically associated to certain virulence traits [43–
46]. One such example is the hypervirulent exoU+ T3SS geno-
type, which is linked to specific clonal lineages, such as the
MDR high-risk clone ST235. Moreover, a recent study showed
that the 3 major high-risk clones are associated with a defined
set of biological markers that, surprisingly, included defective
motility and pigment production, as well as reduced in vitro fit-
ness [47]. On the other hand, high-risk clones showed increased
biofilm formation and spontaneous mutant frequencies.
Thus, the biological markers of P. aeruginosa high-risk clones
S46 • JID 2017:215 (Suppl 1)  •  Juan et al
appeared to show certain similarities to those typically resulting
from adaptation to chronic infections [47].
RISK FACTORS AND HOST MARKERS FOR SEVERE
P. AERUGINOSA INFECTIONS
Although colonization usually precedes P. aeruginosa infec-
tions, the exact source and mode of transmission are often
unclear because of its ubiquitous presence in the hospital envi-
ronment. As an endogenous source of endemic or epidemic
infection with gram-negative bacilli, the intestinal reservoir is
central to the epidemiology of P. aeruginosa because prior rectal
colonization is typically present in most patients developing in-
fections. In a recent prospective study, we assessed the temporal
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changes and the dynamic characteristics of intestinal coloniza-
tion by non-MDR, MDR, and XDR P. aeruginosa [48]. Intesti-
nal colonization occurred earlier for non-MDR P. aeruginosa
(Supplementary Figure 1), but the use of fluoroquinolones
and carbapenems (including antipseudomonal carbapenems
but also ertapenem) promoted digestive tract colonization
with MDR/XDR strains [48]. In the same prospective study,
by means of serial active surveillance and genotypic studies,
we demonstrated that the risk of developing P. aeruginosa clin-
ical infection among colonized patients is 15 times the risk
among noncolonized patients (Supplementary Figure 2), and
genotyping revealed a high concordance between surveillance
and clinical samples [49]. Thus, the study demonstrated that in-
testinal colonization is a key requirement for the development
of P. aeruginosa infections.
In addition to pathogen-related factors (see below), poor out-
comes of P. aeruginosa infections have been associated with fac-
tors related to host, such as the presence of underlying disease
[50, 51], the source of infection [2, 3, 52], and/or the severity of
acute presentation [2, 3, 51, 52]. Table 1 summarizes the risk fac-
tors associated with mortality of P. aeruginosa bloodstream in-
fections as documented in a recent large multicenter prospective
study [53].
Regarding antibiotic treatment, several studies demonstrated
that inappropriate initial antimicrobial therapy [3, 52] was inde-
pendently associated with increased mortality. Thus, the presence
of resistant strains reduces treatment options and enhances the
risk of inadequate empirical therapy. Several authors recommend-
ed empirical combination therapy in patients with known or sus-
pected P. aeruginosa infection as a means of decreasing the
likelihood of administering inadequate empirical antimicrobial
therapy; however, the limited number of drugs available increases
the complexity of management. Moreover, a recent prospective
study [54] with P. aeruginosa bloodstream infections was unable
to demonstrate a significant association between adequate combi-
nation antibiotic therapy and survival (Supplementary Figure 3),
even in a subgroup of patients with septic shock.
The severity of acute illness presentation is known to be as-
sociated with a higher mortality rate [2], and it is possible that
Biomarkers for P. aeruginosa Infections • JID • S3
Table 1. Crude Analysis of Variables Impacting Mortality in sepsis is dominated by a hyperinflammatory state mediated by
Pseudomonas aeruginosa Bloodstream Infections a systemic production of cytokines (interleukin 1, interleukin 6,
and tumor necrosis factor α), which are required for adequate
Covariate Survived Died HR (95% CI) P Value
host defense against infection. Occurring almost simultane-
Subjects 449 (71) 183 (29) . . .
ously, the production of antiinflammatory cytokines (eg, inter-
Carbapenem 94 (21) 51 (28) 1.3 (.9–1.8) .11
resistance leukin 10) reduces local and systemic toxicity and serves to
Age, y, median 65.5 (54.5–76.5) 71.5 (60.5–79.5) 1.1 (1.0–1.0) .002 balance the inflammatory state. However, an excessive inflam-
(range)
Male sex 311 (69) 122 (67) 1.1 (.8–1.5) .60
matory response may progress to sepsis and, ultimately, to
Nosocomial/ 424 (94) 175 (96) 1.1 (.5–2.2) .80 multiple organ dysfunction syndrome; on the other hand, an
healthcare excessive antiinflammatory response may hamper the resolu-
acquisition
Charlson index, 2 (1–4) 3 (2–5) 1.1 (1.1–1.2) <.001 tion of the infection [55]. Several researchers have shown that
median (IQR) the levels of both proinflammatory and antiinflammatory
High-risk 207 (46) 134 (73) 2.7 (2.0–3.8) <.001
bacteremia
cytokines may be correlated with illness severity and that

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Origin of bacteremia unbalanced inflammatory response is a strong predictor of
Urinary tract 142 (32) 28 (15) 0.4 (.3–.7) <.001 mortality [56].
Vascular 100 (22) 21 (11) 0.5 (.3–.8) .002 In the case of P. aeruginosa infection, much influence on dis-
catheter
Unknown 99 (22) 71 (39) 2.0 (1.5–2.7) <.001
ease outcome has been mainly attributed to different bacterial
Respiratory 34 (7.5) 39 (21) 2.5 (1.8–3.6) <.001 phenotypes. However, host genotype is an important determi-
tract nant factor in human host susceptibility to major diseases, in-
Abdominal/ 54 (12) 10 (5.5) 0.5 (.2–.9) .02
biliar cluding infection [57]. There is increasing evidence that some
Soft tissue 11 (2.5) 9 (5.5) 1.6 (.8–3.1) .17 interpopulation and interindividual differences in the attack
Others 9 (2) 5 (3) 1.3 (.5–3.1) .60 rate and prognosis of specific infectious organisms are due to
Clinical presentation inherited genetic variants and, for the most part, to multicom-
Pitt score ≥ 2 139 (30) 132 (72) 4.5 (3.3–6.3) <.001
plex genetic traits ( polygenetic traits) [57, 58]. An efficient ex-
Shock/MODS 68 (15) 92 (50) 4.2 (3.1–5.6) <.001
initial perimental method to dissect complex genetic traits still needs
Shock/MODS 38 (9) 97 (56) 7.7 (5.7–10.4) <.001 to be established, but recently, a new community resource—
at 48 h
Collaborative Cross mice—has been implemented as a common
Adequate 290 (70) 106 (64) 0.8 (.6–1.1) .19
empirical platform for mammalian complex genetic traits. This innovative
antimicrobial
model system can potentially reproduce the variable responses
Adequate 241 (57) 97 (67) 1.5 (1.1–2.1) .02
treatment in of disease severity observed in humans during a specific infec-
first 24 h
tion and has demonstrated that host genetic factors determine a
Additional therapy 166 (37) 33 (18) 0.4 (.3–.6) <.001
differential response to P. aeruginosa airway infection [59].
Data are no. or no. (%) of patients, unless otherwise indicated, and are from [53].
Abbreviations: CI, confidence interval; HR, hazard ratio; IQR, interquartile; MODS, multiorgan PATHOGEN PHENOTYPIC AND GENETIC MARKERS
dysfunction syndrome.
FOR SEVERE P. AERUGINOSA INFECTIONS

Among pathogen-related factors influencing the outcome of

the different presentation is determined not only by the
P. aeruginosa virulence factors produced, but also by the inter-
action between the bacteria and host’s innate immune response,
as well as by the complex combinations and variations of host
genes.
Innate immune cells recognize microorganisms through
sensing of common microbial structures known as pathogen-
associated molecular patterns (PAMPs). Receptors on the sur-
face and in the cytoplasm of immune and nonimmune cells,
known as pattern recognition receptors (PRRs) and nucleo-
tide-binding oligomerization domain-like (NOD-like) recep-
tors, recognize and attach to PAMPs. Activation of NOD-like
receptors through PAMPs leads to the formation of a multipro-
tein complex called the inflammasome, which contributes to the
production of proinflammatory cytokines. The early phase of
P. aeruginosa infections, antibiotic resistance, and particularly
MDR/XDR profiles, is certainly of paramount relevance. In-
deed, delayed adequate antimicrobial treatment is one of the
main risk factors for mortality during acute P. aeruginosa nos-
ocomial infections, and resistance to first line antibiotics, such
as carbapenems, as well as MDR/XDR profiles, obviously in-
crease the chances for it to occur. Likewise, second-line antimi-
crobial agents, such as colistin, might not be as effective or safe
as the first-line treatments. Thus, it is perhaps not surprising
that carbapenem resistance and MDR/XDR profiles are well-
documented risk factors for mortality in P. aeruginosa (blood-
stream) infections [2, 3, 51, 53, 60]. However, the real impact
of antibiotic resistance in the severity and outcomes of
P. aeruginosa infections, besides the effect on appropriate ther-
apy chances, is not so well established. It is known that antimi-
crobial resistance may frequently determine a fitness cost, thus

S4 • JID • Juan et al Biomarkers for P. aeruginosa Infections  •  JID 2017:215 (Suppl 1) • S47

reducing virulence and consequently disease severity [61].
However, this effect may vary significantly from one resistance
mechanism to another, and it may depend as well on the spe-
cific genetic context of the involved strains. Although it is the
exception rather the rule, some resistance mechanisms, such
as the inactivation of the carbapenem porin OprD, have been
found to enhance in vivo fitness and pathogenesis [46].
Likewise, MDR/XDR international high-risk clones have been
found to show a reduced in vitro fitness and to be defective
in some virulence determinants, such as bacterial motility or
pigment production [47]. Moreover, recent in vivo data using
a mouse sepsis model support these finding [62]. There are
also some clinical data supporting potentially reduced pathoge-
nicity in resistant strains. The analysis of the mortality dynam-
ics in a large cohort of P. aeruginosa bloodstream infections
revealed that, while carbapenem resistance (Supplementary
Figure 4) and MDR profiles (Figure 1) were associated with
late (30-day) mortality, the excess of mortality explained by an-
timicrobial resistance was not evident during the first 2 days
after onset of bacteremia, and an adjusted analysis with Cox re-
gression showed a significant interaction between antimicrobial
Figure 1. Probability estimate for 30-day mortality of Pseudomonas aeruginosa bacteremia according to resistance phenotype. MDR, multidrug resistant. Reproduced with
permission from Peña et al [24].
S48 • JID 2017:215 (Suppl 1)  •  Juan et al
resistance and underlying disease: the risk fell steadily as the
number of comorbidities rose [53]. Indeed, a large number of
deaths occurred within the first 24–72 hours after the diagnosis
of P. aeruginosa infection and were mainly caused by non-MDR
P. aeruginosa [24, 53, 54].
In addition to antimicrobial resistance, bacterial virulence is
certainly a major driver of disease severity and outcome. As
commented above, there are a large number of P. aeruginosa in-
trinsic virulence factors that are fully required for establishing
infections. Moreover, the differential presence or expression of
some of these virulence factors may determine major interstrain
variability in virulence and, thus, potentially have a major im-
pact on disease severity and mortality. Among P. aeruginosa vir-
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ulence determinants, the T3SS is likely the one more deeply
studied. A number of previous studies have evaluated the im-
pact of T3SS in the outcome of P. aeruginosa respiratory or
bloodstream infection [63–67]. Most of them have evaluated
whether the involved strains secreted (as evidenced by immuno-
blotting) T3SS cytotoxins in vitro and concluded that strains
that produced at least one of them, designated “secretors,”
were associated with a worse outcome. Additionally, recent
Biomarkers for P. aeruginosa Infections • JID • S5
data showed that the exoU+ genotype was associated with high-
er probability of developing pneumonia in patients with cul-
tures of respiratory specimens positive for P. aeruginosa [63].
In recently published work, we asked whether the T3SS geno-
type could be used as prognostic marker [24]. For this purpose,
we analyzed a large multicenter prospective cohort of individu-
als with P. aeruginosa bacteremia. Multivariate analysis showed
for the first time that the exoU+ genotype is independently as-
sociated with an increased risk of early (within first 5 days) mor-
tality due to P. aeruginosa bloodstream infections (Figure 2). In
contrast, late mortality was not influenced by T3SS genotype
but was significantly higher among patients infected by MDR
strains (Figure 1).
We also investigated the potential association between T3SS
genotypes and resistance profiles [24]. Interestingly, we found
that the exoU+ genotype was significantly more frequent
among non-MDR strains. However, a closer analysis revealed
that the exoU+ genotype was positively linked to the moderately
resistant phenotype and negatively linked to the XDR pheno-
type. Since clonal diversity was extremely high among the mod-
erately resistant phenotype isolates, the excess of exoU+ strains
Figure 2. Probability estimate for 5-day mortality of Pseudomonas aeruginosa bacteremia, according to type III secretion system exoU genotype. Reproduced with permission
from Peña et al [24].
S6 • JID • Juan et al
is apparently not a consequence of the presence of specific
clones. Overall, the exoU+ genotype was detected in 21% of sus-
ceptible isolates but up to 34% of moderately resistant isolates.
Regarding individual antibiotics among moderately resistant
isolates, the prevalence of the exoU+ genotype was highest
among carbapenem-resistant isolates (48% for imipenem), fol-
lowed by fluoroquinolone-resistant isolates (32% for ciprofloxa-
cin) and cephalosporin-resistant isolates (31% for ceftazidime);
in contrast, the exoU+ genotype was particularly infrequent
among aminoglycoside-resistant isolates (6% for gentamicin).
Thus, our results supported and expanded recent findings sug-
gesting an association between the T3SS and fluoroquinolone
resistance [63, 68–70], which is a major step forward for under-
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standing the interplay between resistance and virulence.
On the other hand, the negative association between the
exoU+ genotype and XDR phenotypes was determined by the
widespread international high-risk clones ST175 and ST111
[47, 71, 72], which were all exoU−/exoS+. It should be noted,
however, that there is a third international high-risk clone,
the exoU+/exoS− ST235, that, although very infrequent in
our series, has caused numerous outbreaks worldwide and is
Biomarkers for P. aeruginosa Infections  •  JID 2017:215 (Suppl 1) • S49
associated with a particularly poor outcome [73, 74]. Thus, a
high prevalence of this high-risk clone could have yielded a sig-
nificantly higher impact of MDR/XDR strains on the mortality
rate. These results indicate that, for analyzing the impact of
XDR strains on the outcome of infections, the specific clonal
types, and particularly their T3SS genotype, should be consid-
ered. Likewise, a recent study revealed that P. aeruginosa isolates
belonging to some O antigen serotypes ( particularly O1) were
epidemiologically associated with a worse prognosis than other
serotypes (such as O2) [75]. Thus, correlation between sero-
types, clonal lineages, and T3SS genotypes should be further an-
alyzed. Besides the T3SS genotype, a recent study has revealed
that the presence of a novel 124–kilobase pair genomic island
(PAGI-12) was epidemiologically associated with a significantly
higher mortality rate among individuals with ventilator-associ-
ated pneumonia. Finally, recent work has suggested that elastase
activity might be a marker of disease severity and increased risk
of mortality among individuals with P. aeruginosa bacteremia
[76].
FUTURE PERSPECTIVES
While MDR/XDR profiles and delayed effective therapy are
well-documented risk factors for mortality among individuals
infected with P. aeruginosa, the direct impact of antibiotic resis-
tance on the severity and outcomes of P. aeruginosa infections
should to be further explored. Likewise, the impact of interna-
tional high-risk clones on the pathogenesis of P. aeruginosa in-
fection needs to be addressed. Moreover, assessment of the
differential presence or expression of virulence factors should
be further explored as useful prognostic markers of severe
P. aeruginosa infections. Finally, host biomarkers and genetic
factors modulating the severity of P. aeruginosa infections
need to be characterized.
Supplementary Data
Supplementary materials are available at http://jid.oxfordjournals.org.
Consisting of data provided by the author to benefit the reader, the posted
materials are not copyedited and are the sole responsibility of the author, so
questions or comments should be addressed to the author.
Notes
Financial support. This work was supported by the Ministerio de
Economía y Competitividad of Spain, Instituto de Salud Carlos III, cofi-
nanced by European Regional Development Fund A way to achieve Europe,
through the Spanish Network for the Research in Infectious Diseases
(RD12/0015 and RD16/0016 and grants PI11/00164, PI12/00103,
SAF2012-38539, PI15/00088, and PI15/02212); and the European Union,
through the 11th Call of the Innovative Medicines Initiative (grant COM-
BACTE-MAGNET to A. O.)
Potential conflicts of interest. All authors: No reported conflicts. All
authors have submitted the ICMJE Form for Disclosure of Potential Con-
flicts of Interest. Conflicts that the editors consider relevant to the content
of the manuscript have been disclosed.
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