| Clinical Microbiology | Research Article

Evaluation of blaOXA-48-like point mutation carbapenemase-

producing Enterobacterales in Prapokklao Hospital, Thailand
Sirijan Santajit,1,2 Witawat Tunyong,3 Thida Kong-Ngoen,3 Weewan Arsheewa,4 Woranich Hinthong,5,6 Pornpan Pumirat,3 Nitat
Sookrung,7,8 Nitaya Indrawattana7,8

AUTHOR AFFILIATIONS See affiliation list on p. 11.

ABSTRACT Carbapenemase-producing Enterobacterales (CPE) isolates increasingly carry

oxacillinase-48 (OXA-48)-like enzymes encoded by blaOXA-48-like, which can confer high
levels of carbapenem resistance. This aims to determine the prevalence of CPE and
genetic variation among blaOXA-48-like-carrying isolates recovered from Prapokklao
Hospital in Chanthaburi Province, Thailand in 2016–2017. In total, 122 carbapenem-
resistant Enterobacterales (CRE) isolates were recovered from clinical samples. CRE
were evaluated using standard biochemical tests and MIC test strips. Carbapenemase
production was assessed through the modified Hodge test (MHT), modified carbape­
nem inactivation method (mCIM), and EDTA-modified carbapenem inactivation method
(eCIM). Detection of blaOXA-48-like mutations was conducted via PCR and confirmed
by Sanger sequencing. Among these CRE isolates, 72 (59.02%), 44 (36.07%), 3 (2.46%),
and 3 (2.46%) were Klebsiella pneumoniae, Escherichia coli, Enterobacter aerogenes, and
Enterobacter cloacae, respectively. The MHT identified carbapenemase production in
108 isolates (88.52%). Based on the mCIM, 81 isolates (66.39%) were carbapenemase
producers. Seventy-three isolates (59.84%) were eCIM-positive, indicating metallo-β-lac­
tamase production. Three distinct genetic variants of the blaOXA-48-like gene were

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identified among the isolates, including the wild-type and two point mutation types
harboring the mutations E168Q and S171A (mutation type 1) and E168Q, S171A, and
R214S (mutation type 2). Multiple-sequence alignment and in silico analysis revealed
variation of R214 located in the β5–β6 loop. This study identified blaOXA-48-like point
mutation groups and carbapenemase production, predominantly metallo-β-lactamases,
among CRE isolates, especially K. pneumoniae and E. coli. These findings highlight the
importance of implementing stringent infection control measures and active antimicro­
bial resistance surveillance to combat the spread of difficult-to-treat, metallo-β-lacta­
mase-producing CRE in healthcare settings.

IMPORTANCE In this study, we aimed to investigate genetic variation and CPE

among blaOXA-48-like carrying isolates recovered from Prapokklao Hospital, Chanthaburi
Editor John Osei Sekyere, University of Pretoria,
Province, Thailand, during 2016–2017. A total of 122 carbapenem-resistant Enterobacter­ Pretoria, Gauteng, South Africa
ales (CRE) were recovered from clinical samples in Prapokklao Hospital. All CRE samples
Address correspondence to Nitaya Indrawattana,
were confirmed by standard biochemical tests and minimum inhibitory concentration [email protected].
(MIC) test strips (E-test). The carbapenemase production was determined using the
The authors declare no conflict of interest.
modified Hodge test (MHT), the modified carbapenem inactivation method (mCIM),
and EDTA-CIM (eCIM). Three single mutations (E168Q, S171A, and R214S) were character­ See the funding table on p. 11.
ized in this study. This mutation might reflect the hydrolysis of the modified β-lactam Received 23 January 2024
spectrum, especially carbapenem, by OXA-48-like. Our report provides evidence of the Accepted 2 September 2024
blaOXA-48-like point mutation and carbapenemase-producing phenotype of CRE detected Published 17 October 2024

in this healthcare setting. Effective control measures and active surveillance of drug Copyright © 2024 Santajit et al. This is an open-
resistance in nosocomial pathogens are crucial for controlling diseases associated with access article distributed under the terms of the
Creative Commons Attribution 4.0 International
difficult-to-treat bacteria.
license.

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KEYWORDS antibiotic resistance, blaOXA-48-like, CPE, carbapenemases, eCIM, modified

hodge test, mCIM

T he global spread of carbapenemase-producing Enterobacterales (CPE) has contrib­

uted to the increased prevalence of carbapenem resistance in recent decades,
representing a danger to public health (1). Carbapenem-resistant Enterobacterales
(CRE) comprises a substantial order of Gram-negative bacteria that are non-suscepti­
ble (intermediate or resistant) to at least one carbapenem (ertapenem, meropenem,
doripenem, or imipenem) (2). The most concerning carbapenem-resistant organisms
(CROs) include Escherichia coli, Klebsiella pneumoniae, and Enterobacter spp (3). These
bacteria can cause serious conditions such as urinary tract infections, sepsis, diarrhea,
and systemic infections. Drug-resistant strains of these organisms have been documen­
ted in multiple regions (4, 5). CRE isolates are particularly alarming because of their
involvement in various infections that result in elevated mortality rates and their
potential to disseminate carbapenem resistance through mobile genetic elements (4,
6). Strains carrying plasmid-encoded resistance genes efficiently transfer these genes to
other species.
The emergence of carbapenem resistance among Enterobacterales primarily arises
from the production of carbapenem-hydrolyzing β-lactamases, specifically carbapene­
mases belonging to classes A (KPC), B [metallo-β-lactamases (MBLs); e.g., New Delhi
metallo-β-lactamase (NDM), Verona integron‐encoded metallo-β-lactamases (VIM), and
Imipenemase (IMP)], and D [oxacillinase-48 (OXA-48)-like and its variants] (7). Class A
and D enzymes employ a hydrolytic mechanism centered around serine, whereas class B
enzymes are MBLs characterized by the presence of zinc in their active sites (8). Currently,
more than 1,000 β-lactamase variants have been documented, and their abundance
continues to steadily increase (9, 10). Since the early 2000s, there have been global
reports of CPE outbreaks, and CPE has become established in some countries (11). In
2018, data submitted to the European Antimicrobial Resistance Surveillance Network
revealed that 0.1% of E. coli isolates and 7.5% of K. pneumoniae isolates from invasive
infections were resistant to carbapenem (12). In Europe, the incidence of CPE, including
both infections and colonization, has gradually increased since 2009. Although most

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cases involve patients with recent travel or hospitalization abroad, there is a growing
number of locally acquired cases, often linked to OXA-48-like CPE (13, 14).
The key mechanism of resistance to CRE is the acquisition of carbapenemases, a
group of β-lactamases. These enzymes can be classified into classes A (e.g., KPC), B
[metallo-β-lactamases (MBLs); e.g., NDM, VIM, IMP), and D (OXA-48-like and variants).
Class D carbapenemases, such as OXA-48-like, play a significant role in the emergence of
resistance to last-resort β-lactam antibiotics. OXA-48-like β-lactamase confers resistance
to carbapenems and can lead to life-threatening infections due to limited treatment
options. Class D carbapenemases play a significant role in the emergence of resistance
to β-lactam antibiotics, especially those regarded as treatment options of last resort (15).
A critical clinically significant carbapenemase is the class D β-lactamase OXA-48-like,
which poses a considerable threat to public health because it confers resistance to the
last-line antibiotics carbapenems, resulting in a range of life-threatening infections and
their rapid global dissemination (16, 17).
OXA-48-like β-lactamase was discovered in 2004 in a K. pneumoniae isolate from
Istanbul, Turkey. Subsequently, it spread quickly across Europe, the Middle East, and
the Mediterranean region, posing a growing threat (18). Several OXA-48-like variations
have been reported globally since OXA-48-like was first identified (16, 19). Although
OXA-48-like effectively hydrolyzes penicillins, its ability to hydrolyze carbapenems is
limited, and it exhibits minimal activity against extended-spectrum cephalosporins. The
recent identification of several OXA-48-like variants indicates their wide distribution.
A four-amino acid deletion in the β5–β6 loop exhibits expanded-spectrum cephalo­
sporin hydrolytic activity but no ability to hydrolyze carbapenem (20). However, when
combined with other resistance mechanisms, such as compromised outer membrane

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permeability and extended-spectrum β-lactamase (ESBL) production, the resistance level

to these antibiotics in OXA-48-like-producing bacteria can be significantly higher. This
situation can be highly lethal because treatment options for serious infections caused
by these bacteria are severely restricted (21). For infection control and prevention,
a CPE screening test should be performed (22, 23). According to a European study,
intrahospital and interhospital transmission of CPE occurs more frequently within nations
than between nations (24). To halt the spread of CPE, hospital-level and nationwide
surveillance is required.
Although OXA-48-like variants have been reported globally, there is a scarcity of
information regarding the sequence types disseminated in Thailand. This study aimed
to examine the prevalence of CPE and blaOXA-48-like variant genes/point mutations
at Prapokklao Hospital in Chanthaburi Province, Thailand. Understanding the local
epidemiology and molecular characteristics of CPE is crucial for implementing effec-
tive infection control measures and antimicrobial resistance surveillance to combat the
spread of these difficult-to-treat pathogens in healthcare settings.

MATERIALS AND METHODS

CRE study design and bacterial confirmation
Carbapenem-resistant Enterobacterales (CRE) was defined as isolates non-susceptible
(intermediate or resistant) to at least one carbapenem antibiotic (meropenem or
ertapenem) based on minimum inhibitory concentrations (MICs) determined by E-test
according to the manufacturer’s instructions (Liofilchem, Roseto degli Abruzzi, Italy) (1, 2,
22).
This cross-sectional study estimated the blaOXA-48-like gene among bacterial isolates
recovered at Prapokklao Hospital between 2016 and 2017.
A total of 282 non-duplicate CRE isolates were identified by colony morphology,
Gram staining, and biochemical tests including oxidase, triple sugar iron utilization,
ornithine decarboxylase, indole production, motility, and citrate utilization tests (12).

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The blaOXA-48-like gene was amplified and further subjected to DNA sequencing to
investigate sequence mutations, and analyzed in silico (20). Carbapenemase production
was investigated in blaOXA-48-like-positive samples. A graphical abstract summarizing this
study is presented in Fig. 1.

Detection of blaOXA-48-like, DNA sequencing, and multiple sequence align­

ment
Bacterial genomic DNA was isolated from bacteria grown overnight at 37°C in 3 mL of
Luria–Bertani (LB) broth. Bacterial cultures were centrifuged for 1 min at 12,000 rpm, and
the supernatant was removed. Crude DNA extracts were obtained by suspending the
pellet in 300 µL of distilled water and boiling at 95°C for 10 min, followed by centrifuga­
tion at 12,000 rpm for 5 min. The supernatant containing DNA was transferred to a new
Eppendorf tube, and the DNA quantity was measured using a NanoDrop-1000 spectro­
photometer (Thermo Fisher Scientific, Waltham, MA, USA).
PCR targeting the blaOXA-48-like gene was used as the standard to assess the perform­
ance of genotypic tests. Therefore, PCR amplification to detect blaOXA-48-like was
performed on a PCR cycler (Bio-Rad, Hercules, CA, USA). The sequence containing the
β5–β6 loop, which comprises the catalytic part of the OXA-48-like enzyme against β-
lactams, was amplified using the specific primer pair OXA-48-F (forward primer; 5′-TTGGT
GGCATCGATTATCGG-3′) and OXA-48-R (reverse primer; 5′-GAGCACTTCTTTTGTGATGGC
-3′) (25). The amplification mixture (25 µL) contained Taq DNA polymerase, 10 × Taq
buffer, 25 mM MgCl2, 10 mM dNTPs, 10 mM forward and reverse primer solutions, 1.25
units/L (Thermo Fisher Scientific), and 100 ng/L DNA template. PCR was performed with
initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for
30 s, annealing at 56°C for 30 s, and extension at 72°C for 30 s, with a final extension at

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FIG 1 Graphical abstract of the study. Bacterial culture and confirmation of CRE from the stock solutions. Phenotypic assays for carbapenemase production and
the detection of OXA-48-like point mutations were performed.

72°C for 7 min. The PCR products were analyzed by electrophoresis on 1.5% agarose gels
in 0.5 × Tris-borate-EDTA buffer. The gels were stained with SafeView FireRed (Applied
Biological Materials, Richmond, BC, Canada), and the PCR products were visualized with
UV light using the ChemiDoc MP imaging system (Bio-Rad).
Clinical K. pneumonia isolates harboring blaOXA-48-like from a previous study were

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used as positive controls (26). The blaOXA-48-like amplicons (743 bp) were purified
and subjected to Sanger sequencing. The nucleotide sequences were examined using
software available on the National Center for Biotechnology Information website
(http://www.ncbi.nlm.nih.gov). Multiple sequence alignments were generated using CLC
Sequence Viewer Version 8 (27). The phylogenetic tree of the blaOXA-48-like variant/point
mutations was constructed using MEGA11 software (28) and the neighbor-joining
method (29).

Modified Hodge test

The MHT was performed on CRE isolates according to the (30) guidelines to detect the
presence of carbapenemase (30). The following medium and carbapenem antibiotics
were used: 10 µg ertapenem disks (Oxoid, Thermo Fisher Scientific), 10 µg meropenem
disks (Oxoid), and Mueller–Hinton agar (MHA; Difco, Detroit, MI, USA). A 0.5 McFarland
standard suspension of E. coli ATCC 25922, the MHT carbapenem susceptible indicator
organism, was suspended in Mueller–Hinton broth (Difco) after being grown overnight.
An undiluted suspension or a 1:10 diluted suspension was plated onto MHA in
accordance with the CLSI disk diffusion procedure (30). Each of the ertapenem or
meropenem disks was placed in the middle of the plate, and the test isolates were
thoroughly streaked using a 10 µL disposable loop from the edge of the central disk
to the plate periphery. Each plate was tested with up to four isolates. After overnight
incubation at 35°C in ambient air, the indicator organism inhibition zone was carefully
observed for enhanced growth around the test organism streak (a cloverleaf-type
indentation) that appeared after the test organism grew at the intersection of the
streak. Two indentation levels, measuring 2 and 3 mm, were employed as positive cutoff

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values. The enhanced growth indicates positive carbapenemase production, whereas no

enhanced growth indicates negative carbapenemase production.

Modified carbapenem inactivation method and EDTA-modified carbapenem

inactivation method
The goal of the mCIM and eCIM is to assess the potential of the tested bacteria to
produce carbapenemases. The mCIM and eCIM involve incubating the bacteria with a
meropenem disk and subsequently assessing whether the antibiotic is inactivated. The
use of EDTA in the eCIM method enhances the detection of MBLs, which comprise a
class of carbapenemases that are inhibited by chelating agents such as EDTA. Briefly, a
1 µL loopful of bacteria was resuspended in a 2-mL tube containing tryptic soy broth
(TSB). Then, in a separate tube containing 2 mL of TSB supplemented with 5 mM EDTA
(achieved by adding 20 µL of 0.5 mM EDTA to 2 mL of TSB), another 1 µL loopful of
bacteria was resuspended. Then, a meropenem disk was placed in each tube, and these
tubes were incubated at 35°C for 4 h. After this incubation period, the disks were placed
on MHA plates that had just been inoculated with a 0.5 McFarland suspension of E. coli
ATCC 25922 strains that were susceptible to carbapenem.
The reaction plates were incubated at 35°C for 20 h, and the results of the mCIM
and eCIM were interpreted in accordance with the CLSI 2020 (30) criteria. The mCIM
results were interpreted as positive if the zone was 6–15 in size, intermediate (defined as
positive) when pinpoint colonies were found inside a 16–18 mm zone, and negative if
the zone was 19 mm or larger. If the eCIM zone size was at least 5 mm larger than that
measured in the mCIM, the isolate was indicated as positive for metallo-carbapenemase
production, whereas if the increase in zone size was less than 4 mm, the isolate was
classified as negative. E. coli ATCC 25922 (carbapenemase positive) and K. pneumoniae
strains (carbapenemase negative, blaKPC positive, and blaNDM positive) from a previous
study (26) were used as internal controls for the mCIM and eCIM tests in compliance with
CLSI criteria. In addition, the MHT, mCIM, and eCIM were independently repeated by two
different investigators to determine the repeatability of the results.

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In silico studies: homology modeling and superimposition
Mutations within the OXA-48-like enzymes of representative CPE isolates were first
identified by sequencing, followed by sequence alignment with reference sequence
(accession number NG_049762.1:101–898) to determine the specific single nucleotide
polymorphisms (SNPs). The roles of these mutations were then established using
predictive homology modeling-based 3D structure analysis and protein superimposition
of carbapenems within the binding pocket (β5–β6 loop). Structural investigation of
OXA-48-like wild-type (WT) and mutation groups was performed using SWISS-MODEL
(http://swissmodel.expasy.org) (31). The quality of the generated homology models of
OXA-48-like enzymes was then evaluated using Ramachandran plots (32). Superimposi­
tion to compare OXA-48-like mutation groups with the WT complexed with meropenem
(PDB ID: 6ZRP) was performed using PyMol software (PyMol Molecular Graphics System,
Version 2 edu, Schrodinger, LLC) (33). The CLICK server (http://mspc.bii.a-star.edu.sg/
click), which is a topology-independent tool, compared the superimposed 3D structures
without a scoring function measuring structural similarity (34). Discovery Studio 3.5 was
used to analyze and visualize all models.

RESULTS
Phenotypic detection of CPE
The phenotypic analysis employed the MHT, mCIM, and eCIM to investigate carba­
penemase-producing strains according to the CLSI guidelines for 2020. Out of 122
isolates, 108 (88.52%) CRE isolates were MHT positive, including 62 K. pneumoniae
isolates (50.82%), 41 E. coli isolates (33.61%), 3 E. aerogenes isolates (2.46%), and 2
E. cloacae isolates (1.64%). The mCIM and eCIM results indicated that 93 isolates

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TABLE 1 Phenotypic detection of CRE using the MHT, mCIM, and eCIM and evaluation of OXA-48-like mutation groupsa

Bacterial Carbapenemase production Evaluation of OXA-48-like mutation

pathogens groups
MHT mCIM eCIM No. of OXA-48-like type mutations (%)
Ertapenem Meropenem
No. of No. of positive No. of No. of positive No. of serine No. of MBLs E168Q S171A R214S
negative samples negative samples carbapenemase positive
samples samples positive

E. coli 11 (9.02%) 33 (27.05%) 3 (2.46%) 41 (33.61%) 3 (2.46%) 36 (29.51%) 41 (33.61%) 41 (33.61%) 34 (27.87%)
(n = 44)
K. pneumoniae 11 (9.02%) 61 (50.00%) 10 (8.20%) 62 (50.82%) 20 (16.39%) 29 (23.77%) 68 (55.74%) 68 (55.74%) 67 (54.92%)
(n = 72)
E. aerogenes 0 (0%) 3 (2.46%) 0 (0%) 3 (2.46%) 0 (0%) 3 (2.46%) 3 (2.46%) 3 (2.46%) 3 (2.46%)
(n = 3)
E. cloacae 1 (0.82%) 2 (1.64%) 1 (0.82%) 2 (1.64%) 0 (0%) 2 (1.64%) 3 (2.46%) 3 (2.46%) 3 (2.46%)
(n = 3)
Total 23 (18.85%) 99 (81.15%) 14 (11.48%) 108 (88.52%) 23 (18.85%) 70 (57.38%) 114 (93.44%) 114 (93.44%) 106 (86.89%)
(n = 122)
a
mCIM, modified carbapenem inactivation method; MHT, modified Hodge test; eCIM, EDTA-modified carbapenem inactivation method; n, number 254 of isolates; No.,
number; MBLs, metallo-β-lactamases.

produced carbapenemase (76.23%). Of these, 23 isolates produced serine carbapene­

mase (18.85%), including 3 E. coli isolates (2.46%) and 20 K. pneumoniae isolates (16.39%).
In all, 70 isolates (81.15%) were metallo-β-lactamases producers, including 36 E. coli
isolates (29.51%), 29 K. pneumoniae isolates (23.77%), 3 E. aerogenes isolates (2.46%), and
2 E. cloacae isolates (1.64%, Table 1). A representative result is presented in Fig. 2.

Analysis of the β5–β6 loop in OXA-48-like

Genomic bacteria amplified the β5–β6 loop, which comprises the catalytic part of

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the OXA-48-like enzyme against β-lactams (Fig. S1). PCR amplicons were subjected to
nucleotide sequencing (Fig. S2).
Three-point mutations were identified in OXA48-like, including E168Q in 114 isolates
(93.44%), S171A in 114 isolates (93.44%), and R214S in 106 isolates (86.89%). In this
study, the enzyme mutations were classified into three types, namely WT [seven isolates

FIG 2 Screening and identification of carbapenemase-producing strains using the MHT, mCIM, and eCIM. (A) Representative results of the MHT. Isolates 1, 2, 7,
and 8 were MHT positive. (B) Representative results of the mCIM and eCIM. Thirteen isolates were mCIM positive and eCIM negative, whereas 24 isolates were
mCIM negative and eCIM negative. n, negative; p, positive.

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FIG 3 (A) Alignment of the amino acid sequence for OXA-48-like point mutations. The β5–β6 loop is framed in black for class D lactamases. Numbers were
assigned using an OXA-48-like sequence. (B) Cladogram of the amino acid alignment of different OXA-48-like enzymes using the ClustalW2 sequence alignment
program. Comparison of the deduced amino acid sequences among the OXA-48-like genes of the representative pattern of CRE isolates (wild type, V1, and V2).

(5.74%)], mutation 1 [V1 (E168Q/S171A), eight isolates (6.56%)], and mutation 2 [V2
(E168Q/S171A/R214S), 107 isolates (87.70%)]. The multiple sequence alignment and

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phylogenetic tree of the OXA-48-like mutation groups are depicted in Fig. 3A.

Computerized simulation of mutations within the β5–β6 loop of OXA-48-like

Homology modeling of the OXA-48-like enzyme produced reliable Ramachandran plots.
The percentages of WT, V1, and V2 residues in the most favored regions were 97.92%,
0.71%, and 0.00%, respectively. The percentages of WT, V1, and V2 residues in rotamer
outliers were 98.13%, 0.71%, and 0.00%, respectively, whereas those in Ramachandran
outlier regions were 98.34%, 0.71%, and 0.00%, respectively (Fig. S1B).
The in silico study was performed using pairwise structure superposition, which was
performed using the CLICK server. The percent coverages of the overlapping structures
between the modeled WT and V1, as well as the modeled WT and V2, indicated almost
perfect superpositions of the key binding site residues of each pair (Table S1). Amino acid
substitution in OXA-48-like located in β5–β6 might enhance the catalytic activity of the
enzyme for meropenem. The results of superimposition and the surface illustration of
the OXA-48-like mutation groups are presented in Fig. 4.

DISCUSSION
Currently, as carbapenems represent the primary treatments for infections caused by
multidrug-resistant pathogens, it is imperative to employ effective treatment options,
especially for Enterobacterales. However, the inappropriate use of antibiotics has led to
the emergence of carbapenem resistance through carbapenemase production, including
class D OXA-48 in Gram-negative bacteria, which has become a significant concern
in clinical practice. Monitoring of carbapenemases both in genotypic and phenotypic
manner is often neglected due to the low MICs of carbapenems and also requires

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FIG 4 (A) Superposition of OXA-48-like WT (magenta), V1 (green), and V2 (cyan), which are magnified in the upper part
of the figure. (B) Surface illustration of the OXA-48-WT, OXA-48- V1, and OXA-48-V2 models with the meropenem-binding
site. Mutated amino acids (E168, yellow; S171, blue; R214, red) are shown in stick representation. The meropenem ligand is
depicted in gray.

another strategy in addition to routine susceptibility testing (35, 36). In this study, we
investigated blaOXA-48-like variants/point mutations in CPE isolates and provided a new
context for laboratory detection and therapeutic management. These variants or point
mutations pose a significant risk to patients with infections attributed to these isolates
(37). To the best of our knowledge, few official reports have described CPE isolates
carrying variants or point mutations OXA-48-like in our country (38–40).
This study demonstrated a high prevalence of carbapenem resistance among
Enterobacterales isolates, with K. pneumoniae and E. coli being the predominant species

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exhibiting carbapenem resistance. This finding aligns with previous global and regional
reports identifying K. pneumoniae and E. coli as the major plasmid-mediated species
harboring the carbapenem-resistant blaOXA-48-like gene among CPE isolates, along with
Enterobacter spp. (41, 42). A sizeable proportion of isolates (81.15%) were identified as
metallo-β-lactamase (MBL) producers, including E. coli (29.51%), K. pneumoniae (23.77%),
E. aerogenes (2.46%), and E. cloacae (1.64%). The high prevalence of MBL-producing
Enterobacterales observed in our study is concerning and reflects the global challenge
posed by these organisms, which are associated with limited treatment options and
potential for rapid dissemination. The diverse in vitro sensitivity profiles observed for
carbapenems and the classification of isolates as resistant or indeterminate to imipenem
and meropenem can be attributed to the multidrug resistance conferred by the synthesis
of extended-spectrum β-lactamases (ESBLs) and the presence of various carbapenema­
ses. Accurate detection of carbapenemase production is crucial for guiding appropriate
antimicrobial therapy and implementing effective infection control measures (43).
The MHT, recommended by CLSI for detecting CPE, is a simple and widely imple­
mented phenotypic method in clinical laboratories. However, while it has a high
sensitivity for detecting class A (KPC) and class D (OXA-48) carbapenemases, the MHT
does not reliably identify MBL producers (44). The mCIM and eCIM employed in our
study effectively identified 76.23% of isolates as carbapenemase producers, including
both serine carbapenemases (18.85%) and the substantial proportion of MBL produc­
ers mentioned earlier. These phenotypic methods serve as valuable screening tools
for carbapenemase production, complementing molecular techniques that provide
definitive identification of specific carbapenemase variants/point mutations. The mCIM
and eCIM serve as valuable screening tools for carbapenemase production, which is
crucial for guiding appropriate antimicrobial therapy and implementing infection control
measures (45–47).
Molecular studies enabled the definitive identification of OXA-48-like CPE in our
setting. Given the current global emergence of this enzyme group, with OXA-48 and
OXA-163 being the most prevalent, it is crucial to characterize the specific variants/point
mutations circulating. The 11 OXA-48-like mutation groups identified to date exhibit
varying hydrolytic activities against carbapenems, geographical distributions, associa­

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tions with particular bacterial species, and resistance mechanisms (47).
In our study, we identified two distinct OXA-48-like point mutations harboring
mutations found in OXA-181 (E168Q and S171A) and OXA-232 (R214S). The OXA-181
with E168Q and S171A mutations was previously reported in K. pneumoniae isolates from
India. Similarly, the R214S mutation found in the OXA-232 was initially identified in a K.
pneumoniae isolate recovered from a patient transferred from India to Mauritius in France
(48).
The R214S substitution is located in the β5-β6 loop region, which is known to
significantly influence the hydrolytic nature of class D β-lactamases (49). Modifications
in this loop can alter the hydrolytic profile, potentially converting non-carbapenemases
into carbapenemases, or vice versa (50). Such mutations may enable advantageous
contacts with specific substrates or alleviate steric hindrance for substrate binding,
leading to changes in the β-lactam spectrum of hydrolysis (51). The β5-β6 loop of
OXA-48-like enzymes inhibits the binding of expanded-spectrum cephalosporins, while
its effect on the rate of carbapenem turnover is variable—it may either decrease
carbapenem turnover by hindering substrate binding or potentially increase carbape­
nem turnover by facilitating catalytic efficiency and promoting substrate interactions,
depending on the specific structural and biochemical properties of the enzyme (49).
The identification of these OXA-48-like variants/point mutations in our setting,
particularly their associations with K. pneumoniae isolates and potential links to the
Indian subcontinent, highlights the need for continued molecular surveillance and
epidemiological investigations. Importantly, mutations or deletions within the β5-β6
loop can potentially alter these effects. For instance, certain modifications in this region
have been observed to enhance the enzyme ability to hydrolyze expanded-spectrum

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Research Article Microbiology Spectrum

cephalosporins or increase the rate of carbapenem hydrolysis. Conversely, other changes

may diminish or abolish the enzyme carbapenemase activity. Therefore, the β5-β6 loop
acts as a critical structural determinant of OXA-48-like enzymes substrate specificity
and catalytic efficiency, particularly regarding their interactions with expanded-spectrum
cephalosporins and carbapenems.
This study reveals that out of 114 bacterial isolates identified with three-point
mutations in OXA48-like genes, only 108 tested positive for carbapenemase production
using the MHT. This situation highlights an interesting observation. While all isolates
with three-point mutations in OXA48-like were expected to exhibit carbapenemase
activity, not all of them tested positive in the MHT. Several factors could contribute to
this discrepancy, such as the sensitivity of detection methods, the presence of non-func­
tional mutations, and other mechanisms of carbapenem resistance. Overall, the finding
that not all isolates with three-point mutations in OXA48-like tested positive in the
MHT underscores the importance of using multiple detection methods and considering
various factors that may influence test results when assessing carbapenemase produc­
tion in clinical isolates (7, 8, 24).
Monitoring mutations in this region can provide insights into the potential impact
on antimicrobial resistance profiles and guide appropriate therapeutic strategies. Clonal
molecular research is necessary to further elucidate the origin, spread, and molecular
epidemiology of OXA-48-like CPE infections in our region and globally.
Antibiotic stewardship is critical to prevent the emergence of bacterial resistance.
The appropriate use of antimicrobial control systems in clinical settings reduces the
need for carbapenems, thereby reducing the rate of carbapenem resistance. This
approach has also displayed an association with a decline in the number of CRE
isolates. Nevertheless, further analysis of the data is essential to ascertain the full impact
of manipulating prescriptions for CRE strains (52). Addressing antimicrobial resistance
necessitates adopting the “one health” approach and recognizing the interconnected­
ness of humans, animals, and the environment as potential sources of CPE infections.
In this context, ongoing education and vigilant surveillance of CPE are imperative for
preventing and controlling its transmission in healthcare facilities. This effort should
engage all healthcare professionals in both direct and indirect contact with patients.

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In addition, it is advisable to implement programs focusing on research and epide­
miological surveillance, enhancing the involvement of clinical laboratories through
the development of flowcharts and the characterization of CPE. Surveillance culture
tailored to local epidemiology should be used as a tool for informed decision-making
based on the generated data (53). Our study had multiple limitations, indicating the
need for a prospective evaluation with a larger sample size. The small number of
cases is attributed to the novelty of emerging infections by CPE carrying blaOXA-48-like
variants/point mutations in our facility. Unfortunately, prospective analyses were not
conducted because of constraints posed by our limited laboratory resources.
Our findings suggest a high prevalence of CPE based on phenotypic detection. In
addition, we investigated OXA-48-like enzyme mutations. Clarification of the mecha­
nisms responsible for carbapenem resistance in Enterobacterales has significant clinical
implications, and research, including in silico studies, is needed to facilitate the develop­
ment of preventive strategies and personalized antibiotic therapies. Considering the
implications for infection control practices, antimicrobial stewardship, and public health
interventions, ongoing surveillance is necessary to monitor changes in OXA-48-like CPE
prevalence and characteristics over time.

ACKNOWLEDGMENTS

This research was funded by the National Research Council of Thailand (NRCT) and
Mahidol University (N42A660376). We would like to thank the Center for Scientific and
Technological Equipment Walailak University, School of Allied Health Sciences, Walai­
lak University, and Department of Microbiology and Immunology, Faculty of Tropical
Medicine, Mahidol University, for providing the required laboratory instruments.

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Research Article Microbiology Spectrum

N.I., S.S., and P.P. conceived the study and designed the experiments. S.S., P.P., W.T., and
T.K.-N. performed the experiments. N.I., S.S., P.P., W.H., and N.S. analyzed and interpreted
the data. N.I., N.S., and W.A. contributed to the acquisition of reagents, materials, analysis
tools, and data. N.I., S.S., and P.P. wrote the manuscript.

AUTHOR AFFILIATIONS
1
Department of Medical Technology, School of Allied Health Sciences, Walailak
University, Nakhon Si, Thammarat, Thailand
2
Research Center in Tropical Pathobiology, Walailak University, Nakhon Si, Thammarat,
Thailand
3
Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol
University, Bangkok, Thailand
4
Department of Microbiology, Prapokklao Hospital, Chanthaburi, Thailand
5
Princess Srisavangavadhana College of Medicine, Chulabhorn Royal Academy, Bangkok,
Thailand
6
Department of Infection Biology, Faculty of Infectious and Tropical Diseases, London
School of Hygiene and Tropical Medicine, London, United Kingdom
7
Siriraj Center of Research Excellence in Allergy and Immunology, Faculty of Medicine
Siriraj Hospital, Mahidol University, Bangkok, Thailand
8
Biomedical Research Incubator Unit, Research Department, Faculty of Medicine Siriraj
Hospital, Bangkok, Thailand.

AUTHOR ORCIDs

Sirijan Santajit http://orcid.org/0000-0002-1273-2618

Nitaya Indrawattana http://orcid.org/0000-0003-1571-528X

FUNDING

Funder Grant(s) Author(s)

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National Research Council of Thailand (NRCT) N42A660376 Nitaya Indrawattana

AUTHOR CONTRIBUTIONS

Sirijan Santajit, Conceptualization, Data curation, Formal analysis, Investigation,

Methodology, Validation, Visualization, Writing – original draft, Writing – review and
editing | Witawat Tunyong, Data curation, Formal analysis, Methodology | Thida
Kong-Ngoen, Formal analysis, Methodology | Weewan Arsheewa, Resources | Wora­
nich Hinthong, Formal analysis, Software | Pornpan Pumirat, Conceptualization, Formal
analysis, Methodology, Writing – original draft, Writing – review and editing | Nitat
Sookrung, Formal analysis, Resources, Software | Nitaya Indrawattana, Conceptualization,
Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project
administration, Resources, Software, Supervision, Validation, Visualization, Writing –
original draft, Writing – review and editing

DATA AVAILABILITY

The data supporting the findings of this study, including the genomic sequences
and homology modeling results, are publicly available. The blaOXA-48-like amplicon
sequences have been deposited in the National Center for Biotechnology Information
(NCBI) under accession number NG_049762.1. The structural data for the OXA-48-like
wild-type enzyme complexed with meropenem is available in the Protein Data Bank
(PDB) under accession ID 6ZRP. The predictive homology modeling and superimposi­
tion analyses were conducted using SWISS-MODEL (https://swissmodel.expasy.org) and
PyMOL Molecular Graphics System, Version 2.0 (Schrodinger, LLC). The 3D structures

Month XXXX Volume 0 Issue 0 10.1128/spectrum.00198-24 11

Research Article Microbiology Spectrum

generated in this study were evaluated using Ramachandran plots and further analyzed
with the CLICK server (http://mspc.bii.a-star.edu.sg/click) for structural comparison. For
any additional data requests or further inquiries, please contact the corresponding
author.

ETHICS APPROVAL

Clinical bacterial isolates kept in bacterial stock were recovered and used in this study.
Ethical approval for the study was obtained from the Ethics Committee in Human
Research Walailak University (approval no. WU-EC-AL-3-226-66).

ADDITIONAL FILES

The following material is available online.

Supplemental Material

Supplemental material (Spectrum00198-24-S0001.pdf). Fig. S1 and S2; Table S1.

Supplemental data (Spectrum00198-24-S0002.pdf). Nucleotide sequence alignment.

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