BioScience Trends. 2016; 10(1):14-21.14 Nosocomial infection and its molecular mechanisms of antibiotic resistance Jufeng Xia1, Jianjun Gao2,*, Wei… [610340]

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BioScience Trends. 2016; 10(1):14-21.14
Nosocomial infection and its molecular mechanisms of antibiotic
resistance
Jufeng Xia1, Jianjun Gao2,*, Wei Tang1
1 Hepato-Biliary-Pancreatic Surgery Division, Department of Surgery, Graduate School of Medicine, The University of Tokyo, Tokyo,
Japan;
2 Department of Pharmacology, School of Pharmaceutical Sciences, Qingdao University, Qingdao, Shandong Province, China.
1. Introduction
Nosocomial infection, also known as hospital-acquired
infection, is a kind of infection, which is contracted
from the environment or staff of a healthcare facility
(1). It can be spread in various hospital environments,
including nursing homes, wards, operating rooms,
or other clinical settings. Infection happens in the
clinical setting through a large number of pathways. In
addition to contaminated equipment, bedding articles,
or aerosols, staff also can spread infection ( 2). An
epidemiological investigation implemented by WHO
in fifty five hospitals of fourteen countries from four WHO Regions (Europe, Eastern Mediterranean, South-
East Asia and Western Pacific) revealed an average of
8.7% of hospital patients had a nosocomial infection.
At any time, over 1.4 million people worldwide suffer
from infectious complications acquired in hospitals ( 3).
The morbidities of nosocomial infection were reported
from hospitals in the European, Eastern Mediterranean,
South-East Asia and Western Pacific as 7.7, 11.8, 10.0,
and 9.0% respectively ( 4). Nosocomial infections
could lead to functional disability and mental stress of
patients. In addition, nosocomial infections are also one
of the leading causes of death ( 5).
In hospitals, patients are exposed to a diversity
of microbes. Many different bacteria, viruses, fungi
and parasites may lead to nosocomial infections ( 6).
Most recently hospital-acquired infections are caused
by common bacteria that usually lead to no or milder
disease compared to in-patients such as, Staphylococcus
aureus , enterococci , Pseudomonas spp . and
Enterobacteriaceae (7). After being infected, patients
commonly receive antibiotics. Through selection and Summary Nosocomial infection is a kind of infection, which is spread in various hospital environments,
and leads to many serious diseases ( e.g. pneumonia, urinary tract infection, gastroenteritis,
and puerperal fever), and causes higher mortality than community-acquired infection.
Bacteria are predominant among all the nosocomial infection-associated pathogens, thus
a large number of antibiotics, such as aminoglycosides, penicillins, cephalosporins, and
carbapenems, are adopted in clinical treatment. However, in recent years antibiotic resistance
quickly spreads worldwide and causes a critical threat to public health. The predominant
bacteria include Methicillin-resistant Staphylococcus aureus , Pseudomonas aeruginosa ,
Klebsiella pneumoniae , Escherichia coli , and Acinetobacter baumannii . In these bacteria,
resistance emerged from antibiotic resistant genes and many of those can be exchanged
between bacteria. With technical advances, molecular mechanisms of resistance have been
gradually unveiled. In this review, recent advances in knowledge about mechanisms by
which ( i) bacteria hydrolyze antibiotics ( e.g. extended spectrum β-lactamases, ( ii) AmpC
β-lactamases, carbapenemases), ( iii) avoid antibiotic targeting ( e.g. mutated vanA and mecA
genes), ( iv) prevent antibiotic permeation ( e.g. porin deficiency), or ( v) excrete intracellular
antibiotics ( e.g. active efflux pump) are summarized.
Keywords: Hospital-acquired infection, mutations, PBP2a, SCC mec, OprD, MexEF-OprN.DOI: 10.5582/bst.2016.01020 Review
Released online in J-STAGE as advance publication February
11, 2016.
*Address correspondence to:
Dr. Jianjun Gao, Department of Pharmacology, School of
Pharmaceutical Sciences, Qingdao University, Qingdao 266021,
Shandong Province, China.
E-mail: gaojj@qdu.edu.cn

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BioScience Trends. 2016; 10(1):14-21.exchange of genetic resistant elements, antibiotics boost
the emergence of multi-drug resistant strains. Bacteria
which are sensitive to the antibiotics are suppressed or
killed, while resistant strains survive and may become
endemic and burst out in the hospital ( 8,9). Based on
previous research, the major mechanisms of antibiotic
resistance include extended spectrum β-lactamases (ESBLs), AmpC β-lactamases, carbapenemases,
staphylococcal cassette chromosome mec (SCC mec),
VanA ligase, porin deficiency, and active efflux pumps
(Figure 1). The above mechanisms will be introduced in
the following sections.
2. Hydrolyzing antibiotics I: ESBLs
The emergence of third-generation cephalosporins in
clinical treatment in the early 1980s was reported as
a significant breakthrough to antagonize β-lactamase-
mediated antibiotic resistance. Soon after, the first
research into plasmid-encoded-lactamases which are
able to hydrolyze extended-spectrum cephalosporins
was reported in 1983 ( 10). The genes, which encode
β-lactamases much similar to SHV-1, TEM-1, and
TEM-2, exhibited mutations of single nucleotides and
were soon discovered to have the ability to acquire
resistance to extended-spectrum cephalosporins (Table
1) (11,12 ). For now,various ESBLs contained in Gram-
negative bacteria such as E. coli , K. pneumoniae , A.
baumannii and P . aeruginosa have proved to be capable
of resistance to most of β-lactam antibiotics. Because
ESBLs-producing bacteria are able to hydrolyze a large
number of β-lactam antibiotics, the utility of those
antibiotics for infections caused by such bacteria is
reduced. Moreover, the plasmids containing the genes
that encode ESBLs usually also contain genes that
cause resistance to aminoglycosides and trimethioprim/
sulfamethoxazole. There have been more and more
reports of plasmid-induced attenuation in susceptibility
to aminoglycosides, often being associated with plasmid-15
Figure 1. Molecular mechanisms of antibiotic resistance
Table 1. Resistance-related β-lactamases
β-lactamases
Extended spectrum
β-lactamases
(ESBLs)
AmpC
CarbapenemaseFamilies
TEM family and SHV family
CTX-M family
XOA family
Others (PER-1, VEB family,
GES family, IBC-2, BES-1,
IBC-1, SFO-1, and TLA-1)
FOX family, CMY family, LAT
family, DHA family, and MOX
family
IMP family and VIM family
KPC family
OXA familyTargets
Targets of broad-spectrumβ-lactamases(Am
inopenicillins,benzylpenicillin,carboxypen
icillins, narrow-spectrum-cephalosporins),
oxyimino-cephalosporins,monobactam
Targets of broad-spectrumβ-lactamases,
cefepime
Same as above
Same as TEM family and SHV family
Targets of broad-spectrumβ-lactamases,
cephamycins
Targets of broad-spectrumβ-lactamases,cep
hamycins,carbapenems
Same as above
Same as aboveSusceptibility
(to clavulanic acid)
++++
++++
+
++++
0
0
+++
+Classes
A
A
D
A
C
B
A
D Ref.
(19-27 )
(28-30 )
(21,31,32 )
(33, 34 )
(35-40 )
(41-44 )
(41-44 )
(41,42,45 )

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BioScience Trends. 2016; 10(1):14-21.16
2.5. Other members of ESBLs
Other members of ESBLs are rare and have been
discovered predominantly in P . aeruginosa and in some
small areas: PER-1 was found in France, Italy, and
Turkey; VEB family was found Southeast Asia; and GES
family and IBC-2 were found in South Africa, France,
and Greece ( 33). A part of these ESBLs were discovered
in Enterobacteriaceae as well, but other rare ESBLs,
such as BES-1, IBC-1, SFO-1, and TLA-1, have been
discovered only in Enterobacteriaceae (34).
3. Hydrolyzing antibiotics II: AmpC β-lactamases
AmpC β-lactamases, which are usually induced by
β-lactams, are expressed in many Gram-negative
bacteria. Mutations in relevant genes lead to increasing
expression levels and promote the emergence of
cephalosporin resistance in Enterobacter cloacae
(35). The AmpC β-lactamases in E. coli are present
at a low expression level and the AmpC-encoded
gene is deficient in the chromosome of klebsiella and
salmonella strains. However, plasmid-expressed AmpC
β-lactamases can endow those bacteria with similar
resistance as Enterobacter cloacae mentioned above.
Until recently, more than twenty AmpC β-lactamases
have been found expressed by plasmids ( 36).
As shown in Figure 1, The ampC-related regulatory
pathway includes the following three elements: ( i)
AmpG which is a permease on the inner membrane;
(ii) AmpD which is an amidase in cytoplasm; and
(iii) AmpR, a transcription factor, is a member of
the LysR family, a group of regulatory proteins ( 37).
These three elements are necessary for expression of
AmpC β-lactamases in both Enterobacteriaceae and
P . aeruginosa (38). In the regular process of cell wall
recycling, 1,6-anhydromuropeptides are disassembled
from the cell wall and transferred into the cytoplasm
by AmpG permease. The 1,6-anhydromuropeptides
are cut by AmpD protein to produce tripeptides, which
are subsequently transformed into UDP-MurNAc-
pentapeptides. UDPMurNAc-pentapeptides couple
with AmpR proteins combining in the intergenic region
between ampR and ampC , and generating a structure
that inhibits activation of ampC . Low expression levels
of AmpC are generated, and the β-lactamase localizes to
the periplasmic space. When β-lactams, such as cefoxitin
and imipenem, permeate the outer membrane of bacteria,
they reach the periplasmic space, and combine with
target penicillin binding proteins (PBPs). The amount
of 1,6-anhydromuropeptides increases, and AmpD is
unable to efficiently deal with the high levels of cell wall
pieces. The anhydro-MurNAc-peptides substitute UDP-
MurNAc-pentapeptides binding to AmpR, leading to a
structural change of the enzyme. AmpR is changed into
a role of transcriptional promoter, AmpC is produced at
higher levels, and concentration of AmpC rises in the induced cephalosporin resistance ( 13,14 ). Even when
plasmid-mediated attenuation of susceptibility of
quinolone y is absent, there also is an obvious correlation
between quinolone resistance and ESBLs production
(15-17 ). The reason for such correlation is not yet
understood.
2.1. TEM family
Mutations of single nucleotides at many sites in
genes coding TEM-1β-lactamases can be achieved
in the laboratory with complete activity ( 18). Those
mutations, which change the ESBLs phenotype
transform the configuration of the active site of the
enzyme, and allow interaction between active site and
oxyimino-β-lactams ( 18-20 ). Exposing the active site
to β-lactam substrates also leads to susceptibility of the
ESBLs to β-lactamase inhibitors, such as clavulanic
acid. More than one hundred and thirty members of the
TEM family are now recognized, and their diversity
supplies a useful pathway to trace the transmission of
individual resistance genes ( 21,22 ).
2.2. SHV family
SHV-1 coincides in 68% of its amino acids sequence
with TEM-1 and shares its molecular structure (23).
Like the TEM family, members of the SHV family have
certain amino acid mutations at the active site. More
than fifty members of the SHV family recently have
been identified through unique combinations of amino
acid replacements ( 24). The SHV family recently has
been shown in surveys of resistant strains in Europe
and America ( 25,26 ). SHV-5 and SHV-12 are prevalent
among the members of the SHV family ( 27).
2.3. CTX-M family
Another family of ESBLs not a member of the TEM
or SHV families was named CTX-M to emphasize
its greater activity against cefotaxime compared to
ceftazidime. More than forty members of CTX-M
are currently known ( 28). Belying their name, some
hydrolyze ceftazidime more rapidly than they do
cefotaxime. CTX-M-14, CTX-M-3, and CTX-M-2 are
the most widespread ( 29,30 ).
2.4. OXA family
Twelve members of the OXA family have recently
been discovered (21). They were found mainly in P.
aeruginosa in clinical samples from France and Turkey
(31). Major members of the OXA family are relatively
resistant to clavulanic acid-induced inhibition. Some
of them have resistance mainly to ceftazidime, but
OXA-17 shows stronger resistance to cefotaxime and
cefepime than to ceftazidime ( 32).

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BioScience Trends. 2016; 10(1):14-21.17
periplasmic space. When the concentration of β-lactam
decreases below its "alarm level" the amount of anhydro-
MurNAc-peptides in cytoplasm also decreases, and
AmpD's ability to efficiently cut these peptides is
restored. In another case, mutations of nucleotides in
genes leads to deficiency of AmpD or down-regulates
expression of ampD damage in the process of wall
fragment recycling and leads to increased concentration
of anhydro-MurNAc-peptides in the cytoplasm. As a
result, the combining of anhydro-MurNAc-peptides
to AmpR makes AmpR "locked" in a structure as
transcriptional activator of ampC, and produces high
levels of AmpC β-lactamases ( 37,39,40 ).
4. Hydrolyzing antibiotics III: Carbapenemases
Carbapenemases are a kind of β-lactamase with various
hydrolytic abilities. They have been identified to have
ability to damage penicillins, cephalosporins, and
carbapenems. Bacteria generating the carbapenemases,
which resist these antibiotics by breaking β-lactams,
frequently lead to serious nosocomial infections.
Carbapenemases belong to the A, B, and D molecular
class of β-lactamases ( 41). Class A and D β-lactamases
have a mechanism of serine-based hydrolysis, while
class B β-lactamases are metallo-β-lactamases which
have zinc in their active site ( 42). The carbapenemases
group of class A contains members of the KPC, NMC,
IMI, SME, and GES families. Among these families,
the KPC carbapenemases are the most predominant,
usually existing on plasmids in Klebsiella pneumoniae
(43,44 ). The carbapenemases group of class D contains
OXA β-lactamases usually found in Acinetobacter
baumannii . The metallo-β-lactamases were first found
in Pseudomonas aeruginosa strains, but at present,
there is an increasing worldwide emergency of this
class of β-lactamases in the Enterobacteriaceae (45).
5. Avoid antibiotics targeting I: mecA mutation
SCC mec is a mobile genetic element of Staphylococcus
bacterial strains. This genetic sequence contains the
mecA gene, which codes for resistant proteins to the
antibiotic methicillin, and is the only known way for
Staphylococcus species to spread the gene in the wild
by horizontal gene transfer. mecA leads to resistance
to methicillin and other β-lactam antibiotics. After
mecA is absorbed into bacteria, it is inserted into the S.
aureus chromosome ( 46,47 ). mecA produces penicillin-
binding protein 2a (PBP2a), which is much different
from former penicillin-binding proteins because
mutations have changed its conformation to make it
hard to bind methicillin or other β-lactam antibiotics to
its active site. Thus, PBP2a can continuously promote
the transpeptidation required for peptidoglycan cross-
linking to perform cell wall synthesis in the presence
of antibiotics. As a result of the incapability of PBP2a to combine with β-lactam moieties, activation of mecA
promotes resistance to all other β-lactam antibiotics
including methicillin ( 48). mecA is controlled by
regulatory genes mecI and mecR1 . MecI often combines
with the mecA promoter and plays an inhibitor role
(49). In the existence of β-lactam antibiotics, MecR1
promotes a signaling transduction pathway that
causes activation of mecA (50). This activation is
initiated by MecR1-induced cleavage of MecI, which
decreases MecI inhibition. mecA is also regulated by
two co-repressors BlaI and BlaR1. blaI and blaR1 are
homologous to mecI and mecR1 , respectively, and
usually play a role as regulators of blaZ which leads to
penicillin resistance ( 51,52 ). The nucleotide sequences
recognized by MecI and BlaI are the same, thus BlaI
can also combine with the mecA operator to inhibit
activation of mecA (53).
6. Avoid antibiotics targeting II: vanA mutation
Glycopeptides repress cell wall synthesis in Gram-
positive bacteria by combining with the C-terminal
D-Ala-D-Ala of the pentapeptide precursors of
peptidoglycan, further blocking the reactions of
transglycosylation and transpeptidation ( 54). Recently,
glycopeptide-resistant enterococci have spread
throughout the whole world. So far, seven types of
resistant elements (VanA, -B, -C, -D, -E, -G, and -L) in
enterococci have been discovered and they have seven
corresponding operons ( vanA , -B, -C, -D, -E, -G, and
-L) which play roles of synthesis of a novel combining
site (peptidoglycan precursors terminating in D-Ala-
D-lactate in VanA, -B, and -D type or D-Ala-D-serine
in VanC, -E, -G, and -L type) leading to a decreased
affinity to glycopeptides and substitution of the normal
precursors ending in D-Ala-D-Ala ( 55-57 ).
A two-component regulatory system VanR-
VanS controls vancomycin resistance in vancomycin-
resistant enterococci (VRE) and vancomycin-
resistant Staphylococcus aureus (VRSA) ( 58). VanS
is a membrane-related sensor for vancomycin which
regulates the phosphorylation of VanR. VanR is a
transcriptional activator of the operon which encodes
VanH, VanA and VanX. VanH is a dehydrogenase
which converts pyruvate to D-Lac, and VanA is a
ligase which combines D-Ala and D-Lac by creating
an ester bond between them. Vancomycin can only
combine with D-Ala- D-Ala but not to D-Ala-D-Lac,
and thus vancomycin resistance appears. VanX is a
dipeptidase which cleaves the normal peptidoglycan
component D-Ala-D-Ala that prevents it from
leading to vancomycin sensitivity. VanY is a D,D-
carboxypeptidase that cuts the end D-Ala residue of
the peptidoglycan if substitution of D-Ala-D-Ala by
VanX is not thorough. Thus, D-Ala-D-Lac substitutes
for the normal D-Ala-D-Ala in peptidoglycan synthesis
resulting in vancomycin resistance ( 59-61 ).

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BioScience Trends. 2016; 10(1):14-21.18
7. Prevent antibiotics permeation: oprD mutation and
porin deficiency
The outer membrane of Gram-negative bacteria has a
semi-permeable barricade which decreases the import
of antibiotics, and the outer membrane of P . aeruginosa
is only 8% as permeable as that of Escherichia coli
(62). However, for surviving, P . aeruginosa has to
allow import of nutrients through the outer membrane,
and this is achieved through a system of water-filled
protein channels named porins. DNA sequencing of the
P . aeruginosa genome has recognized one hundred and
thirty known or supposed outer membrane proteins,
with sixty four of these outer membrane proteins
classified into three families of porins ( 62). These
porins play a significant physiological role in the
transport of sugars, amino acids, and phosphates, and
so on ( 63,64 ). Some hydrophilic antibiotics, such as
β-lactams, aminoglycosides, tetracyclines, and some
fluoroquinolones, have been shown to pass through the
outer membrane porins ( 65-68 ). Thus, deficiency of
porins can diminish susceptibility of P . aeruginosa to
some antibiotics.
OprD porin-mediated resistance contains mechanisms
that down-regulate the transcriptional level of the oprD
gene and/or mutations which replace the translational
production of a normal porin. At the transcriptional level
of oprD , disturbing mechanisms contain ( i) breakdown
of the oprD promoter, ( ii) terminating the transcription of
oprD prematurely, ( iii) co-regulation with trace metal ion
resistance, ( iv) salicylate-induced decrease, and ( v) down-
regulated transcriptional expression by coregulation
with the active efflux pump encoded by mexEF-oprN.
The oprD promoter breakdown appears as a result of
deletions or insertions in the upstream region of oprD.
It was reported that a deletion containing the putative
promoter and initiation codon blocked transcription of
oprD (69-72 ). Based on previous research, IS1394 and
an ISPa16-like insertion element have been proposed as
an upstream region of the oprD in imipenem-resistant
strains of P . aeruginosa showing down-regulated oprD
expression ( 73,74 ).
8. Excreted intracellular antibiotics: mexEF-oprN
and actived efflux pump
On the one hand, the deficiency of porins such as
OprD is an effective obstacle for antibiotic import into
the cell, on the other hand, a decrease in antibiotic
concentration can also be realized via export through
membrane-located efflux pumps. Efflux pumps have
been classified into five superfamilies ( 75,76 ). The
superfamilies contain ( i) the ATP-binding cassette
(ABC) superfamily, ( ii) the small multidrug resistance
superfamily, ( iii) the major facilitator superfamily, ( iv)
the resistance-nodulation-division (RND) superfamily,
and ( v) the multidrug and toxic compound extrusion superfamily.
One of the most important regulatory mechianisms
is the coincident overproduction of the MexEF-OprN
efflux pump and downregulation/upregulation of
the OprD porin ( 77). In wild P . aeruginosa , MexT is
silenced owing to either the existence of repressing
mutations or the deficiency of a secondary effecter
(78). As a result, expression of mexEF-oprN stays at
a low level, and expression of oprD stays at a basal
level providing a proper amount of OprD in the outer
membrane sufficient for normal cellular intake ( 79). In
nfxC-type mutants, MexT becomes active via a mutation
in mexT . The activated MexT protein up-regulates
transcription of mexEF-oprN causing overexpression
of the efflux operon and overproduction of the MexEF-
OprN efflux pump. At the same time, MexT down-
regulates oprD at the transcriptional and translational
levels, leading to a decreased amount of OprD (80). On
the other hand, loss of MexS, a supposed oxidoreductase/
dehydrogenase, has been thought to lead to formation
of secondary metabolites which may serve as effecters
for MexT ( 77). These effecters could combine with
MexT, change the structure of the regulatory protein,
and alter MexT into an activating situation. As a result,
MexT can up-regulate the expression of mexEF-oprN
and down-regulate the expression of oprD , similar to
the mechanism mentioned above. There also is a third
mechanism. Loss of the universal regulatory protein
MvaT is also associated with the positive regulation of
the mexEF-oprN operon ( 81). The mechanism of MvaT-
related regulation has not been discovered, but it works
independent of MexT and MexS. In contrast to the
MexT- and MexS-related regulatory mechanisms, loss
of MvaT leads to a positive regulation of both mexEF-
oprN and oprD expression.
9. Conclusion
The capacity of bacteria to evolve resistance to
antibiotics has long been realized, but our knowledge
about the tremendous variety of molecular mechanisms
has been enriched enormously most recently. Technology
advances in genomics, proteomics, and structural biology
have analyzed many of the molecular mechanisms
promoting resistance and will continuously provide more
and more intensive explanations. Based on these newest
discoveries, the development of novel antibiotics, which
can resist or grant knowledge of resistance mechanisms
will be accelerated. For speeding up development of new
antibiotics, academic institutions and pharmaceutical
companies should make joint efforts in the future.
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(Received January 3, 2016; Revised January 31, 2016;
Accepted February 4, 2016)