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Clinica Chimica Acta 451 (2015) 7177

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Clinica Chimica Acta


journal homepage: www.elsevier.com/locate/clinchim

Neonatal infections due to multi-resistant strains: Epidemiology, current


treatment, emerging therapeutic approaches and prevention
Chryssoula Tzialla , Alessandro Borghesi, Margherita Pozzi, Mauro Stronati
Neonatal Intensive Care Unit, Fondazione IRRCS Policlinico San Matteo, Pavia, Italy

a r t i c l e

i n f o

Article history:
Received 22 September 2014
Received in revised form 19 February 2015
Accepted 22 February 2015
Available online 4 March 2015
Keywords:
Antibiotics
Antimicrobial treatment
Multidrug-resistance
Neonate
Neonatal sepsis

a b s t r a c t
Severe infections represent the main cause of neonatal mortality accounting for more than one million neonatal
deaths worldwide every year. Antibiotics are the most commonly prescribed medications in neonatal intensive
care units. The benets of antibiotic therapy when indicated are clearly enormous, but the continued and widespread use of antibiotics has generated over the years a strong selective pressure on microorganisms, favoring the
emergence of resistant strains. Health agencies worldwide are galvanizing attention toward antibiotic resistance
in gram-positive and gram-negative bacteria. Infections in neonatal units due to multidrug and extensively
multidrug resistant bacteria are rising and are already seriously challenging antibiotic treatment options.
While there is a growing choice of agents against multi-resistant gram-positive bacteria, new options for
multi-resistant gram-negative bacteria in the clinical practice have decreased signicantly in the last 20 years
making the treatment of infections caused by multidrug-resistant pathogens challenging mostly in neonates.
Treatment options are currently limited and will be some years before any new treatment for neonates become
available for clinical use, if ever.
The aim of the review is to highlight the current knowledge on antibiotic resistance in the neonatal population,
the possible therapeutic choices, and the prevention strategies to adopt in order to reduce the emergency and
spread of resistant strains.
2015 Elsevier B.V. All rights reserved.

1. Introduction
Neonatal sepsis represents the main cause of neonatal mortality accounting for more than one million neonatal deaths worldwide every
year, and antibiotics are the most commonly prescribed medications
in neonatal intensive care units (NICU) [1,2]. The development of antibiotic resistance (resistance of a microorganism to an antimicrobial drug
Abbreviations: ASP, antimicrobial stewardship programs; CA-MRSA, communityassociated MRSA; CDC, Centers for Disease Control and Prevention of Atlanta; CONS,
coagulase-negative Staphylococcus; CPE, carbapenemase-producing Enterobacteriaceae;
CSF, cerebrospinal uid; ECDC, European Centre for Disease Prevention and Control; EMA,
European Medicines Agency; EOS, early-onset sepsis; ESBL, extended spectrum lactamase; FDA, the Food and Drug Administration; GBS, group B streptococcus; GNB,
gram-negative bacteria; GPB, gram-positive bacteria; HA-MRSA, hospital-associated
MRSA; IDSA, Infectious Diseases Society of America; KCP, Klebsiella pneumoniae
carbapenemase; KCP-Kp, KCP K. pneumonia; LOS, late-onset sepsis; MALDI-TOF MS,
matrix-assisted laser desorption ionization time-of ight mass spectrometry; MRSA,
Methicillin-resistant Staphylococcus aureus; MDR, multi-drug resistant; NICU, Neonatal intensive care unit; PCR, polymerase chain reaction; PVL, Panton-Valentine leukocidin;
VISA, vancomycin-intermediate S. aureus; VLBW, very low birth weight infants; VRE,
vancomycin-resistant enterococci; VRSA, vancomycin-resistant S. aureus; WHO, World
Health Organization; XDR, extensively drug-resistant.
Corresponding author at: Neonatal Intensive Care Unit, Fondazione IRCCS Policlinico
San Matteo, Piazzale Golgi 19, 27100 Pavia, Italy. Tel.: +39 0382 502704.
E-mail address: c.tzialla@smatteo.pv.it (C. Tzialla).

http://dx.doi.org/10.1016/j.cca.2015.02.038
0009-8981/ 2015 Elsevier B.V. All rights reserved.

that was originally effective for treatment of infections caused by it),


on the other hand, is rising among many microorganisms in healthcare
settings as well as in community and is associated with increased morbidity and mortality [3]. The risks of antibiotic resistance have been recognized as a nations' security matter at the sixty-sixth World Health
Assembly on 2013 [4] and the Institute of Medicine suggests that the
control and reduction of infections caused by antibiotic-resistant pathogens is one of the most important issues that medical community must
approach [5].
Data from the Centers for Disease Control and Prevention of Atlanta
(CDC) report 2 million cases of infection with resistant bacteria in the
United States (US) every year with at least 23,000 deaths as direct result
and the cost to the US health system has been estimated to be $2134
billion dollars yearly [6]. European Medicines Agency (EMA) and
European Centre for Disease Prevention and Control (ECDC) estimates
that 25,000 deaths per year are direct consequence of a multidrug resistance infection [7] and World Health Organization (WHO) report 3.7% of
new cases worldwide [3]. A national survey of infectious diseases specialists conducted on 2011 by the Emerging Infectious Network report
that more than 60% of specialists had seen an untreatable infection
due to a pan-resistant pathogen within the prior year [8].
Recently the most common resistant bacteria have been reported as
the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus,

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Klebsiella pneumonia, Acinetobacter baumanii, Pseudomonas aeruginosa


and Enterobacter species) to highlight that they escape the effects of
antibacterial drugs [9].
2. Epidemiology of resistant pathogens in the NICU
Neonatal sepsis are distinct in early-onset (EOS) due to vertical
transmission and late-onset (LOS) usually hospital acquired. Even if
the risk to develop an infection due to resistant pathogens is usually related with late-onset nosocomial infections, since hospitalized neonates
are frequently exposed to wide-broad antibiotics, there are potential
risks also around early-onset infections.
EOS is most often caused by group B streptococcus (GBS) (43%),
followed by Escherichia coli (15.529%) [10]. LOS is caused by Grampositive bacteria (GPB) (7081%), most often coagulase-negative
Staphylococcus (CONS) (4245%), followed by S. aureus (1013%).
Gram-negative bacteria (GNB) cause 19%25% of all LOS in the NICU
and they are associated with greater mortality (1936%). Of Gramnegative isolates Enterobacteriacae (910%) were the most common
followed by E. coli (78%), Pseudomonas spp (23%) and other organisms
(14%) [11,12].
2.1. Gram positive bacteria
GBS remains sensitive to penicillin, ampicillin, and the rst generation cephalosporins. However there have been described cases of increase in the MIC of penicillin and ampicillin for some strains. The
proportion of GBS strains showing in vitro resistance to clindamycin
or erythromycin has increased over the last 20 years. In studies published between 2006 and 2009, the prevalence of resistance among invasive strains of GBS isolates in the United States ranges from 25 to
32% for erythromycin and 1320% for clindamycin [13].
Dual resistance of isolates to both drugs was also very high, with
94.3% of clindamycin-resistant isolates being also resistant to erythromycin and 71.5% of erythromycin-resistant isolates exhibiting coresistance to clindamycin [13,14].
Enterococci are uncommon pathogens in neonatal settings, even
though, ampicillin-resistant, and more recently, an increasing rate of infections due to VRE has been described in children and neonates
[1517]. In particular in the US the rate of vancomycin resistance
among Enetrococcus faecium isolates is estimated around 60% [9].
Most hospital acquired CONS have widespread resistance to
many usually prescribed antibiotics on the neonatal units, including
penicillin, synthetic penicillins and gentamicin. In addition they
could be multidrug-resistant, for example resistant to gentamicin, rifampin, erythromycin, and clindamycin [1618].
Methicillin-resistant S. aureus (MRSA) has become a frequent source
of infection affecting premature and critically ill neonates in NICUs with
a wide variability of infection rates from institution to institution. Data
from different studies in NICU population report, despite variations in
prevalence measurements, a rate of colonized or infected neonates
with MRSA between 0.6% and 8.4% [19]. The epidemiology of MRSA is
changing from being exclusively a hospital acquired pathogen to a pathogen with widespread distribution in the community [20,21]. This
MRSA strains are genotypically and phenotypically distinct [19]. Recently a vertical transmission of MRSA from mother to infants has been described. Similarly, the dominant MRSA clones in the NICU have been
changing from hospital-associated (HA) to community-associated
(CA) clones [16,1921].
By denition MRSA is resistant to methicillin and all -lactam antibiotics. HA strains are more often resistant to multiple types of antibiotics;
by contrast CA strains are often susceptible to different non--lactam
antibiotics [19]. CA-MRSA strains are more often associated with expression of Panton-Valentine leukocidin (PVL) that cause the production of cytoxins [20].

Even though an increase in vancomycin MIC values, within the susceptible range, has been registered among isolates of MRSA, CONS or
S. aureus strains vancomycin-intermediate or vancomycin-resistant
haven't been documented in the NICU population [16,17].
2.2. Gram negative bacteria
Gram-negative bacteria are often resistant to at least one class of antibiotics usually used in health care settings, and bacteria multi- or
extensively-resistant to conventional antibiotics are frequently isolated.
Pan-resistant pathogens are rarely isolated in the NICUs [16,22] and the
most frequent resistance has been described against piperacillintazobactam, ceftazidime, and/or gentamicin [17].
Gentamicin resistance (antibiotic widely used for empiric therapy in
NICU due to aminoglycoside-converting enzymes) is increasing among
Enterobacteriaceae. Since the enzyme that confers resistance to gentamicin may not include other agents of antimicrobial category the
gentamicin-resistant bacteria could be still susceptible to tobramycin
or amikacin [22].
The emergence of ESBL-producing Enterobacteriaceae resistant
to penicillins and cephalosporins, and often to other antibiotic classes
(i.e. uoroquinolones and aminoglycosides) has become a problem
threatening health. ESBL-producing bacteria are found frequently in the
community meaning that they are possible causes of an EOS due to a vertical transmission [16,17,22]. E. coli and K. pneumonia are probable to acquire ESBLs, even if these enzymes are also noted in other species [16].
Even more threatening, is the increasing in the community as well as
in the hospital of carbapenemase-producing Enterobacteriaceae (CPE)
that are no susceptible to carbapenems. The European antimicrobial
resistance surveillance network report increasing percentages of
K. pneumoniae carbapenemase (KPC)-producing isolates between
2005 and 2010 in Europe even if these bacteria are not yet common in
neonatal population [9,16,17,22,23].
Failure to start early treatment with antibiotics active against resistant strains is linked with poorer prognosis [16,17].
A recent study conducted by Giuffr et al. [24] in a NICU shows the
colonization by KCP-K. pneumoniae (KCP-Kp), in 10 out of 54 neonates
admitted in the NICU in the period between September 18 and November 14, 2012, without occurs however, cases of infection. According to
the authors this is the rst report of a KCP-Kp colonization outbreak in
a NICU.
Rates of infection due to resistant P. aeruginosa and MDR
Acinetobacter spp continue to increase in US and globally. P. aeruginosa
is intrinsically resistant to many commonly used antibiotics. Resistance
to both quinolones and carbapenems is increasing among P. aeruginosa
isolates and recent reports document resistance to polymyxins [9].
However, resistance to multiple antibiotics is rare in neonates. Some
strains of MDR Acinetobacter baumannii exhibit discordant resistance
to carbapenems, being susceptible to impenem but resistant to
meropenem and doripenem due to increased expression of naturally
occurring carbapenemases [17].
3. Antibiotic use and emergence of resistant strains
The extensive and indiscriminate use of antibiotics over several decades in hospitals, outpatient regime in the territory and outside the
health care settings, to the farms to promote growth in food-producing
animals, has generated over the years a strong selective pressure on microorganisms, favoring the emergence of resistant strains [25].
The development of resistance is a normal evolutionary process for
microorganisms, but it is accelerated by the selective pressure exerted
by widespread use of antibacterial drugs.
Already in his Nobel Prize speech in 1945, Alexander Fleming, who
discovered penicillin, warned that bacteria could become resistant to
these remarkable drugs and that it was appropriate to reduce the use
of penicillin to slow the development of resistance [26].

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The ECDC, drawing up a list of European countries for daily use of antibiotics in descending order, identies Italy as one of the countries
where consumption is higher on average 2630 daily doses per 1000 inhabitants between 2008 and 2010, compared with 1115 daily doses per
1000 inhabitants consumed in the same period in the Netherlands,
which is the most virtuous of the league [5].
The CDC reports that, in general, more than half of hospitalized patients receive at least one antibiotic during hospitalization, and of
these, about two-thirds receive broad-spectrum antibiotics [6].
In particular nearly all very low birth weight infants (VLBW) admitted
to NICUs receive empirical antibiotic treatment in the rst days of life, in
spite the evidence of sterile cultures and low incidence of cultureproven bacterial sepsis in this population [1,10]. Indeed more than 95%
of infant admitted to the NICU receive empiric antibiotics, but only 1%
to 5% have positive initial blood cultures [27].
Several studies show that widespread and inappropriate use of antibiotics is very common in NICUs.
In a national prevalence study conducted for the Pediatric Prevention
Network on 29 NICUs [28] in the United States, 43.3% of all patients
were receiving antimicrobials on the probing date; 22.7% were receiving
one agent, 58.5% two agents, 15.5% three, 2.9% four and 0.4% ve. The
most common agents were gentamicin (22.3%), ampicillin (20.4%),
vancomycin (10.9%), cefotaxime (6.6%), and tobramycin (3.1%).
Patel et al. [29], in a retrospective observational study of two hundred neonates admitted in 4 NICUs revealed that, in 323 antibiotic
courses, 35% of neonates received at least 1 inappropriate course due
to, more commonly, continuation of antibiotics than to initiation of
therapy (39% vs 4% respectively, P b 0.001). Vancomycin was the most
commonly used drug with 895 antibiotic-days, 284 of which were considered inappropriate. Carbapenems were the agents most frequently
used inappropriately (43% of antibiotic-days).
Of 6956 VLBW neonates included in a study of the National Institute
of Child Health and Human Development National Research Network, 56%
received at least one course of antibiotic treatment initiated after the
third day of postnatal life, even if culture-proven sepsis was diagnosed
in only 21% of all infants [30].
A recent review shows that even if screening for EOS were undertaken in approximately 1012% of all babies, only 3.2% of screened babies
had any microbiological or clinical and laboratory evidence of bacterial
infection, and they were all treated with antibiotics empirically for at
least two days [31].
A number of in vitro and in vivo studies have shown that, although
short courses of carbapanems and third-generation cephalosporins
cover a broad spectrum of bacteria, their prolonged and intensive use selects resistant bacteria. Overuse of third-generation cephalosporins favors
the emergence of extended spectrum -lactamase (ESBL)-producing
strains of GNB in NICUs [32].
de Man et al. [33] in order to investigate the effects of empiric antibiotic therapy on the selection of resistant bacterial strains, evaluated
436 infants admitted to 2 NICUs within the same hospital subjected to
two different regimes of empirical antibiotic therapy based on the spectrum of activity (narrow-spectrum vs broad-spectrum) and subsequently exchanged after 6 months. The investigators demonstrated
that the relative risk for colonization with resistant strains per 1000 patients at risk was 18-fold higher in the broad-spectrum regimen group
(penicillin or ucloxacillin plus tobramycin) than in the narrowspectrum regimen group (amoxicillin plus cefotaxime).
In another study, investigators reported a signicant reduction of colonization from 32% to 10.8% and of nosocomial infections from 18 to 2
cases per year caused by multi-resistant bacteria (oxacillin-resistant
S. aureus and GNB resistant either to aminoglycosides or to thirdgeneration cephalosporins), after institution of educational measures
and restriction in the use of third-generation cephalosporins [34].
In a prospective surveillance study conducted by Mammina et al.
[35] between 2003 and 2004, 55.2% of admitted neonates resulted
colonized with Gram-negative multidrug-resistant (MDR) bacteria.

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Ampicillin-sulbactam and gentamicin were the most frequently used


antibiotics with mean time of administration of 9.2 and 6.8 days, respectively. Administration days of both antimicrobials were not different
between neonates colonized with MDR bacteria, colonized with susceptible bacteria and not colonized. Nevertheless, the total exposure to antimicrobial drugs was signicantly higher in the group of neonates
colonized with MDR bacteria than in neonates colonized with susceptible bacteria or not colonized (8.0 days vs 5.5 vs 2.3; P b 0.1).
A recent study conducted by Tsai et al. describes 1106 episodes of
bacteremia in 8 years in their NICU: 35.5% were caused by GNB, and
18.6% by multi-resistant strains; the most frequent mechanism of
resistance was the production of ESBL (67.1%), especially from
K. pneumoniae (59.6%). More importantly, in the multivariate analysis
the authors identify as independent risk factors for the emergence of resistance the exposure to third-generation cephalosporins (P b 0.001),
and carbapenems (P = 0.017) [36].
In recent study conducted by Isodis et al. on 253 neonates hospitalized in NICU screened for vancomycin-resistant enterococci (VRE), 101
(39.9%) were colonized during a biphasic outbreak in a period from June
to December 2008. Multivariate analysis identied administration of
antimicrobial therapy for LOS (p = 0.008) and hospitalization during
the rst month of the outbreak (p = 0.002) as independent risk factors
for VRE colonization [15].

4. Treatment of infections due to resistant bacteria


Antibiotic therapy in neonatal infections with resistant strains is a
challenging matter since adequate treatment could be based on agents
with limited neonatal experience.

4.1. Treatment of infections with antibiotic-resistant GPB


Glycopeptide antibiotics remain adequate as treatment of most
staphylococcal infections [28,29] with vancomycin having a number of
advantages over teicoplanin. According to the American Academy of
Pediatrics' Committee on Infectious Diseases for serious infections due
to MRSA and CONS vancomycin is the drug of choice [37].
However in case of gram-positive infections unresponsive or in
case of patient who has recently received several courses of vancomycin
and could therefore harbor vancomycin resistant strains linezolid
has been the most used in neonatology [17,19] or trimethoprimsulfamethoxazole with or without gentamicin [37], even if the use of
daptomycin has been described in few reports in case of persistent
staphylococcal bacteraemia in neonates [38,39]. The doses given in neonates have reported to be higher and shorter interval than those recommended for adults without adverse events [38].
Infections due to multi-drug resistant HA-MRSA strains should be
treated with a combination of vancomycin plus gentamicin and/or rifampin, but alternatives such as trimethoprim-sulfamethoxazole, linezolid,
quinupristin-dalfopristin, uoroquinolones or tigecycline could also be
considered pending results of susceptibility tests [19,37]. The use of
quinupristin-dalfopristin is not approved by the Food and Drug Administration (FDA) in children younger than 16 years, and uoroquinolones
and tigecycline are not approved for use in children younger than
18 years but may be used in special circumstances after careful assessment of risk and benets [37]. Life-threatening CA-MRSA strain infections should be treated with a combination of vancomycin plus
gentamicin and/or rifampin [19]; in case of PVL-producing CA-MRSA
combinations of linezolid, clinadamycin and/or rifampicin are recommended along with intravenous immunoglobulin [17].
The combination of vancomycin plus rifampin is recommended for
the treatment of meningitis from MRSA since rifampicin has a good cerebrospinal uid (CSF) penetration and has also been reported successful for the treatment of persistent CONS bacteraemia [17].

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Vancomycin is also the cornerstone for the treatment of penicillinresistant pneumococci infections combined with cefotaxime because
the majority of this bacteria show an intermediate resistance [17].
Several novel antibiotics active against gram-positive bacteria are
currently in diverse phases of development and undergoing clinical trials. In particular it has been reported advanced-generation cephalosporins like ceftaroline and ceftobiprole with antimicrobial activity against
multidrug-resistant staphylococci (including MRSA, vancomycinintermediate S. aureus [VISA], hetero-resistant VISA, and vancomycinresistant S. aureus VRSA) [4043], and lipoglycopeptides agents with
promising activity against multidrug-resistant gram-positive pathogens
like oritavancin and dalbavancin and telavancin [44,45]. All three of
these agents are promising alternatives for the treatment of complicated skin and soft-tissue infections in adults but there are no data on
their pharmacokinetics in neonates [17,44,45]. Other new agents
(omadacycline and tedizolid) as well as revisited older agents
(fosfomycin and fusidic acid) appear promising but require further
study for their potential role [46].
4.2. Treatment of infections with antibiotic-resistant GNB
GNB are often resistant to at least one class of antibiotic that is used as
standard, and may be from time to time resistant to all-rst-line antibiotics. The development of new antibiotics active against resistant GNB
has not progressed in parallel with increasing rates of resistance. Indeed
over the past 30 years only few new classes of antibiotics have been introduced and moreover pharmaceutical industry has reduced drug development programs leading to serious restriction of treatment options for
MDR-GNB in the future. This scenario of limited therapeutic options has
prompted renewed interest in older and more toxic antimicrobials [17].
In recent review Gray et al. [22] summarized the best antimicrobial
treatments for serious infections with antibiotic-resistant Gramnegative bacteria in neonates as follows:
1) Aminoglycoside-resistant GNB: in case of pan-aminoglycoside resistant bacteria alternative antibiotics include cefotaxime, piperacillintazobactam and carbapenems. It would be better to avoid the use of
piperacillin-tazobactam in case of suspected or conrmed meningitis because it is not sure that it can provide effective protection
against beta-lactamase enzymes in CSF.
2) ESBL-producing GNB: Carbapenems have high stability to hydrolysis
by ESBLs and penetrate easily porins. Based in this activity carbapenems are the cornerstone of treatment and should normally be used
as monotherapy. Meropenem is the most widely used carbapenem
in neonates. Doripenen is a new carbapenem with higher activity
against P. aeruginosa but with limited experience and with pharmacokinetic studies ongoing in neonates [47].
Recent publications in adults and children highlight the utility of
ertapenem, particularly in urinary tract infections but they are limited clinical data evaluating his role in invasive ESBL infections [48].
Other new carbapenems in different phases of clinical trials in adults
are biapenem, panipenem, tomopenem and FSI-1686 [49].
3) Carbapenem-resistant GNB: nonsusceptibility to carbapenems coexists with resistance to other classes of antibiotic like aminoglycosides
and uoroquinolones and optimal treatment is not established. The
use of ciprooxacine in neonatal life-threatening infections is supported from some information provided from current literature
and because it offers a good CFS penetration. For lower-level of resistance higher doses and/or continuous infusions of carbapenems plus
another active agent such as an aminoglycoside, uroquinolone or
colistin could be used but no trials conrm this treatment. Data on
prolonged infusion of doripenem exists in adult population but
there is limited published experience on its use in patients aged
b 18 years [48]. For higher-level of resistance colistin could be considered. Recent clinical reports have demonstrated a more favorable
tolerability and safety prole of colistin compared with reports from

several decades ago with nephrotoxity and neurotoxity varied in different studies from 3.5% to 22% and from 0% to 4% of children, respectively [50,51]. Colistin is largely experienced in the neonatal
population, but it must be kept in mind that is not effective against
Proteus and Serratia.
In case of MDR P. aeruginosa strains colistin is the drug of last resort,
although when MICs are elevated to all available -lactams,
prolonging the infusion of meropenem with the addition of an aminoglycoside, uroquinolone, or colistin can be considered.
Some carbapenem-resistant and MDR strains of A. baumanii remain
susceptible to some aminoglycoside, like tobramycin. In adults, intravenous tobramycin has been reported to be effective for treating
infections with susceptible strains but there is little experience in neonates. However colistin is the most reliable agent in vitro [29 17] although it has been used with variable success for treatment of
pneumonia, bacteremia and meningitis [48].
4) Extensively drug-resistant (XDR) GNB: last-resort treatments include chloramphenicole and trimethoprim or co-trimoxazolo even
if are lacking data on their efcacy in serious neonatal infections.
Fosfomycin is active against extensively drug-resistant Enterobacteriaceae, A. baumannii and P. aeruginosa with the ability to achieve adequate concentrations in urine, plasma, broncholaveolar lavage uid
and CSF with iv formulations. Resistance to fosfomycin can be developed rapidly when used as monotherapy; therefore, even if clinical
data are limited, most experts recommend to combine fosfomycin
with other antibiotic classes. Currently there is limited experience
of using fosfomycinin neonates.
Tigecycline, active against hard to treat pathogens like many
multidrug-resistant GPB e GNB, is inactive against P. aeruginosa [52],
and should be considered in patients infected with A. baumannii
when non other options are available [48]. Due to the possible effects
on the bone growth in children and because it determines permanent
teeth discoloration and enamel hypoplasia in children aged b8 years,
tigecycline use in neonates could only be justied in extreme cases.
Unfortunately, to date we cannot identify new candidate compounds
in late-stage development for the treatment of MDR Enterobacteriaceae.
The Infectious Diseases Society of America (IDSA) in a recent update
[53] has identied 7 parenteral drugs in phase 2 or 3 of clinical development for treatment of infections caused by MDR GNB: 4 -lactam plus
-lactamase inhibitor (ceftolozane/tazobactam, ceftazidime/avibactam,
ceftaroline/avibactam and MK-7655/imipenem) products that act by
inhibiting the -lactamases so the partner antibiotic can interfere with
cell wall synthesis; 2 protein synthesis inhibitors (plazomicin a next generation neoglycoside and eravacycline a broad-spectrum uorocycline
antibiotic that binds to bacterial ribosomes) and one peptide mimetic
(brilacidin a peptide mimetic that disrupts bacterial membranes). Unfortunately, none of the 7 drugs demonstrates activity against the entire
spectrum of clinically relevant GNB resistance. In addition the authors
report that some promising compounds like carbavance a -lactam/lactamase inhibitor, BAL30072 a siderophore sufactam and GSK052 a
novel tRNA synthesis inhibitor are in phase 1 development. Other
newer potent compounds such as carbapenemase inhibitors [54] and
novel polymyxin lipopeptides [55] against MDR and XDR gram-negative
microorganisms still necessitate the evaluation of clinical trials.
In the absence of an evidence-based strategy for treating multidrugresistant Enterobacteriaceae, Gray et al. [34] recommend that the microbiological response to treatment must be controlled trough repeat
cultures.

5. Reduction of therapeutic arsenal against multi-resistant germs


In parallel with the emergence of resistant microorganisms and consequent reduction of the therapeutic means available to combat bacterial infections, the available arsenal is gradually reducing in part

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because of the lack of investment by the pharmaceutical industry in the


discovery of new antibiotic molecules.
Indeed the number of new antibacterials that make it through the
complete development process and ultimately receive FDA approval
has precipitously decreased over the past 25 years. According to data
from the CDC and IDSA, in the early 80s, namely between 1980 and
1984, the FDA has approved 19 new molecular entities; in the years
19851989, 11 new molecules, as well as in the periods 19901994
and 19951999. The number of new molecular entities approved
was reduced to only 4 in the years 20002004 in the 3 years from
2005 to 2009; and only one new antibiotic has been approved in
the period 20102012 [6,53]. Moreover only two classes of synthetic
antibiotics were developed in the past 50 years: uoroquinolones
and oxazolidinones (linezolid) [56].
The discovery of new antibiotics must face a number of challenges
that make the development of new antibiotic drugs more difcult compared to other non-antibiotic drugs. These have been summarized in a
recent review by Lewis et al. [56].
First, the poor penetration of antibiotics in prokaryotic cells requires
the delivery of higher amounts of a compound on average 23 times
higher concentrations of typical drugs acting against targets in eukaryotic cells which, in turn, increases the risk of toxicity and narrows the
therapeutic range.
In addition, specically targeting GNB is even more challenging, as
not only the inner membrane restricts the penetration of hydrophilic
substances, but also the outer membrane further reduces the number
of compounds that may be effective, and the multidrug-resistant
pumps extrude any compounds that leak in through the outer
membrane.
Even when these pharmacodynamics-related issues are resolved,
the development of a new drug needs to face the pharmacokineticsrelated issues; indeed, the search for molecules with physicochemical
properties to improve the likelihood of bioavailability (e.g. by applying
the Lipinski's rules) may not match with the need of a compound with
physicochemical properties that improve penetration into prokaryotes.
Once discovered, the compound must be tested in clinical trials;
however, the identication and recruitment of patients infected with
multi-resistant bacteria may be difcult, as most infections are caused
by pathogens susceptible to the available compounds.
Finally, there is modest return on investment on antibiotic development compared to other drugs. Indeed, antibiotic therapy is typically
short-term, lasting only some days, while drugs such as antihypertensives, anti-inammatories, medications to lower blood cholesterol and
diabetes medications, used for years or decades or lifelong, are much
more protable; and, in any case, resistance to the new antibiotics will
eventually develop, limiting their use and the prots that it produces.
This has led, in recent decades, the withdrawal of several large pharmaceutical companies from antibacterial research and development
compromising the infrastructure for discovering and developing antibiotic molecules [53]. The picture that emerges is of a world in which the
arsenal to ght the microorganisms is always poorer.
The IDSA recognizing the need for new drugs continues to propose
legislative, regulatory and funding solutions to solve the problem of
the antibiotic pipeline. They aim to create trough the 10 '20 Initiative campaign launched in 2010 a sustainable global antibacterial
drug research and development enterprise with the power to develop
10 new safe and efcacious systemically administrated antibiotics by
2020 [53].

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The strategies to slow the development and spread of MDR bacteria


are focusing on the following aims to [36,8]:
1) improve the knowledge of MDR bacteria and antibiotic use through
networks that undertake surveillance at national and international
levels and developing more effective early warning systems to improve health security
2) conserve and steward the effectiveness of existing treatments
through improving infection prevention and control in health-care
settings, development of educational programs to facilitate optimal
use of antibiotics and reducing the antimicrobial use in animal husbandry.
3) stimulate the development of new antibiotics, alternative treatments and preventive strategies (including new forms of immunotherapy and new vaccinations)
6.1. Antibiotic stewardship policies
It is necessary that the prescription of antibiotics is strictly regulated.
The alarm that physicians and patient overuse antibiotics was rst
raised by Sir Alexander Flemming as early as 1945. Since then different
antimicrobial stewardship programs (ASP) have been developed with
the aim to reduce unnecessary therapies. Nevertheless, only in 2007,
the Infectious Diseases Society of America, together with other professional organizations, published guidelines in order to implement multidisciplinary ASP [57].
Recently the CDC of Atlanta published a paper (available on: http://
www.cdc.gov/media/releases/2014/p0304-poor-antibiotic-prescribing.
html) that encourages the adoption, in every hospital, of an ASP to provide clinicians with the information and tools they need to make the
right decision. Every stewardship program, according CDC, must include
7 components: 1) Dedicated human, nancial, and technology resources,
2) A physician or other leader responsible for overall outcomes, 3) A pharmacist leader focused on prescribing, 4) An action to improve prescribing,
such as requiring reassessment of prescriptions after 48 h for drug choice,
dose, and duration, 5) Monitoring of prescribing and resistance patterns,
6) Regular reporting of resistance information to clinicians and 7) Education about resistance and judicious prescribing. Moreover CDC suggests
that the elements of the ASP should be checked through a checklist,
which serves to determine that all key elements and actions for an optimal antibiotic prescription are present, and to guide physicians in prescribing in order to restrict the overuse of antibiotics in hospitals [58,59].
In neonatal settings CDC recommendations are also valid and recently, several authors suggested different strategies that might be helpful in
a NICU that include implementation of systems for surveillance of
bloodstream infections, education of practitioners concerning the development of resistance, use of narrow spectrum empirical antibiotic
policy and stop of empirical treatment or documented justication for
continuation when blood cultures are negative, use of narrowest
spectrum antibiotics for a proven infection, formulary restriction and
pre-authorization requirements for selected antimicrobial agents like
cephalosporins, meropenem, vancomycin and teicoplanin [60,31].
Patel et al. [61] suggest that although specic guidelines for neonates
are often lacking, antibiotic stewardship principles like those proposed
by the Get Smart for Health Care Campaign of the CDC can be applied
to the NICU along with the development of an interdisciplinary antimicrobial stewardship team and metrics to measure successful implementation of ASP.
6.2. Infection prevention and control

6. Prevention of multi-resistant germs emergence and spread


Since antibiotic resistance cannot be eradicated, different strategies
have been proposed from several world health organizations to manage
to limit the threat to, and minimize the impact on, human and animal
health.

A way to slow spread of MDR bacteria is to reduce the number of infections. Whenever an infection is prevented, one less person may be
prescribed drugs. Infections can be prevented by vaccines, hygiene, sanitation as well as by isolation during infectivity [62,63]. Consistent hand
sanitation before and after interaction with patient or his environment

76

C. Tzialla et al. / Clinica Chimica Acta 451 (2015) 7177

is the milestone of prevention [16,18]. Despite recent dissemination of


clear hand hygiene guidelines from the WHO and several studies that
conrm the connection between inadequate hand hygiene and HA
infection, compliance among health givers, remains low [18]. Rigorous
efforts to improve hand washing rates are critical and must continue
[8,16]. Robotic and automated disinfection technologies are available
for deployment in healthcare settings but clinical validation to assess
their real efcacy is needed [8,63].
6.3. Discovery of new antibiotics and development of innovative treatments
Most of the potential bacterial targets for antibiotics are still unexploited. It is assumed that there are approximately 200 conserved essential proteins in bacteria, but the current antibiotics only hit few
targets or pathways [56]. Future efforts should focus on the discovery
of compounds directed against these new targets and a better alignment
of economic and regulatory approaches to antibiotic development could
facilitate antibiotic research.
The development of new treatments or preventive strategies could
help to reduce the use of antibiotics. Spellberg et al. [63] suggest possible therapies with reduced potential to drive resistance that may in the
future be introduced into clinical practice. The author suggest immunebased therapies like infusion of monoclonal antibodies and white cells
that kill microbes or biologic agents that don't kill bacteria but capable
of altering the ability of bacteria to trigger inammation and cause infection and drugs that can modulate host-microbe interactions without
attacking directly microbial targets like moderation of host inammation (agonists or antagonists of cytokines) and limitation of microbial
growth by sequestration of host nutrients or probiotics administration
that compete with microbial growth. Such treatments could limit the
pathogenicity of microorganisms without exercising at the same time
a selective pressure that induces resistance
6.4. Development of rapid diagnostic tests and discovery of biomarkers to
enable appropriate antibiotic use
Advances in molecular microbiology have provided new assays focused on rapid identication of pathogens and genetic markers related
to antimicrobial resistance. These technologies are based on a variety
of established and new applications to detect nucleic acids and/or proteins in body uids and swabs. The advantages include the ability to tailor antimicrobial therapy to a pathogen's susceptibilities avoiding
antibiotic misuse and overuse.
In particular nucleic acid-based amplication technologies have
evolved from traditional and real time polymerase chain reaction
(PCR) to newer methods and the older molecular assays developed as
single-analyte tests have been moved toward multiplexed cartridgebased systems. Matrix-assisted laser desorption ionization time-of ight
mass spectrometry (MALDI-TOF MS) platforms can be used routinely
for the rapid identication of bacteria, fungi, mycobacteria and parasites
and the time of rst result is approximately 1020 min. Preliminary
studies demonstrate that MALDI-TOF MS can also detect certain types
of antibiotic resistance mechanisms [64].
Although methods like real-time PCR have high sensitivity and specicity, molecular assays still do not have sufcient sensitivity to replace
microbiological cultures [65].
Furthermore research is ongoing to identify new biomarkers [66] for
bacterial infection potentially useful in clinical practice. Metabolomics a
method based on the study of the complete set of metabolites contained
in a biological sample could be able to identify a small number of metabolites for identication of septic newborns [66,67].
7. Conclusions
Antibiotic resistance is a public health concern worldwide. The global burden of antimicrobial resistance is rising and is associated with

increased morbidity and mortality in clinical and community setting. Infections in neonatal units due to multidrug and extensively multidrug
resistant bacteria are also rising and are already seriously challenging
antibiotic treatment options. Treatment of neonates infected with
these pathogens is largely based on case reports and on experience of
treating other age groups.
The late-stage clinical development pipeline for antibacterials remains unacceptably lean. Although some important molecules are currently in diverse phases of development and undergoing clinical trials
for treatment of infections due to resistant GPB, very few drugs have
reached advanced stages of development for infection due to MDR
gram-negative bacilli.
At this point it is essential to preserve the efcacy of existing drugs
through measures to minimize the development and spread of resistance to them, while efforts to develop new treatment options proceed.
Antibiotic stewardship policies and improving infection prevention and
control on international, national and local level are the major recommended approaches. Moreover development of rapid, reliable, accurate,
simple tests that will reduce time to diagnosis and identify resistant
strains could improve the quality of care and patients outcome. Thorough understanding of resistance mechanism and innovation in new
drugs and vaccines is necessary.

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