Am. J. Trop. Med. Hyg., 110(2), 2024, pp.

283–290
doi:10.4269/ajtmh.23-0199
Copyright © 2024 American Society of Tropical Medicine and Hygiene

Assessment of Antibiotic Resistance among Clinical Isolates of Enterobacteriaceae in Nepal

Ajaya Basnet,1,2* Mahendra Raj Shrestha,3 Basanta Tamang,2 Nayanum Pokhrel,4 Rajendra Maharjan,3 Junu Richhinbung Rai,5
Shrijana Bista,6 Shila Shrestha,1 and Shiba Kumar Rai7
1
Department of Medical Microbiology, Shi-Gan International College of Science and Technology, Tribhuvan University, Kathmandu, Nepal;
2
Department of Microbiology, Nepal Armed Police Force Hospital, Kathmandu, Nepal; 3Department of Clinical Laboratory, Nepal Armed Police
Force Hospital, Kathmandu, Nepal; 4Research Section, Nepal Health Research Council, Kathmandu, Nepal; 5Department of Microbiology,
Maharajgunj Medical Campus, Tribhuvan University Teaching Hospital, Institute of Medicine, Kathmandu, Nepal; 6Central Department of
Microbiology, Tribhuvan University, Kathmandu, Nepal; 7Department of Research and Microbiology, Nepal Medical College and Teaching
Hospital, Kathmandu, Nepal

Abstract. Clinicians face a global challenge treating infections caused by Enterobacteriaceae because of the high
rate of antibiotic resistance. This cross-sectional study from the Nepal Armed Police Force Hospital, Kathmandu, Nepal,
characterized resistance patterns in Enterobacteriaceae across different antimicrobial classes and assessed incidences
of multidrug-resistant (MDR) and extensively drug-resistant (XDR) infections. Enterobacteriaceae from clinical samples
were isolated on blood and MacConkey agar, except for urine samples on cysteine lactose electrolyte–deficient agar. To
determine antimicrobial susceptibility patterns, including MDR and XDR, the Kirby-Bauer disc diffusion method was
used. Statistics were performed using SPSS, v. 17.0. Members of the family were identified in 14.5% (95% CI: 16.2–
12.8%) of the total samples (N 5 1,617), primarily in urine (54.7%, 128/234), blood (19.7%, 46/234), and sputum (15.0%,
35/234). Escherichia coli (n 5 118, 44.2%) was the most predominant bacteria, followed by Citrobacter freundii (n 5 81,
30.3%). As much as 95.6% (392/410) of the isolates were penicillin-resistant, whereas only 36.2% (290/801) were
carbapenem-resistant. A total of 96 (36.0%) MDR and 98 (36.7%) XDR Enterobacteriaceae were identified. Proteus mira-
bilis (44.4%, 8/18) predominated MDR cultures, whereas C. freundii (53.1%, 43/81) predominated XDR cultures.
Multidrug resistant (38.4%, 71/154) and XDR Enterobacteriaceae (22.7%, 35/154) were chiefly uropathogens. Fluoro-
quinolone resistance rates in non-MDR, MDR, and XDR isolates were 19.9%, 63.2%, and 96.2%, respectively, whereas
cephalosporin resistance rates were 28.6%, 72.9%, and 95.4% and penicillin resistance rates were 67.0%, 97.4%, and
98.0%. One-seventh of patients visiting the hospital were found to be infected with Enterobacteriaceae, and of these
patients, at least one-fourth were infected with MDR strains.

INTRODUCTION specifically addressing incidents of XDR pathogens, particu-

Once-effective drugs that held promise in the 1940s for
eradicating infectious diseases around the world are no lon-
ger effective, as antimicrobial resistance (AMR) is progres-
sively becoming more common.1,2 At present, AMR is the
most pressing healthcare issue, as it correlates with longer
hospital stays, increased mortality, and greater healthcare
costs.3,4
Gram-negative bacteria, particularly those from the family
Enterobacteriaceae, can be resistant to antibiotics not only
phenotypically (viz., biofilms) but also genotypically (i.e.,
mutations or plasmid-encoded antibiotic-resistant genes),5
which can encompass multidrug-resistant (MDR)–associ-
ated infections (nonsusceptible to at least one agent in three
or more antimicrobial categories) as well as extensively
drug-resistant (XDR)–associated infections (nonsusceptible
to at least one agent in all but only one or two antimicrobial
categories).6 An analysis of more than 200 countries showed
that AMR contributed to an estimated 1.27 million deaths in
2019,7 which was approximately twice the number of deaths
from AIDS (0.65 million).8 As well, the WHO highlights the
report of the United Nations Ad hoc Interagency Coordinat-
ing Group on AMR, which estimates that around 10 million
people worldwide will die of drug-resistant infections by
2050.9
Although there is ample literature documenting incidents
of MDR pathogens, there is a noticeable dearth of literature
* Address correspondence to Ajaya Basnet, Department of Medical
Microbiology, Shi-Gan International College of Science and
Technology, Tribhuvan University, Shankha marg, Kathmandu 44600,
Nepal. E-mail: xlcprk@gmail.com
larly in low- and middle-income countries such as Nepal.
Therefore, this study aimed to determine the prevalence of
MDR and XDR strains within the Enterobacteriaceae family,
using clinical samples collected from a tertiary care hospital
in Nepal.
MATERIALS AND METHODS
Study design and population. A cross-sectional study
was conducted between September 2021 and February
2022, focusing on members of the family Enterobacteriaceae
isolated from clinical samples subjected to microbiological
culture and antimicrobial sensitivity testing at the Depart-
ment of Microbiology, Nepal Armed Police Force Hospital in
Kathmandu, Nepal.
Data collection. The Institutional Review Committee of the
Shi-Gan Health Foundation, Kathmandu, Nepal, approved
this study (Reference No.: 20790103). Informed consent was
obtained from adult participants (from parents or legal guar-
dians in the case of minors). The data collection sheet served
to document sample details and their corresponding microbi-
ological findings, which were subsequently entered into
Microsoft Excel version 10.0.
Inclusion and exclusion criteria. Only samples that met
the acceptance criteria (properly labeled or documented
samples, samples in sterile or leakproof containers, and
others) for microbiological culture analysis were included in
this study. The study excluded urine cultures that had three
or more bacteria, repeated samples with similar bacteria
from the same patient, and bacterial strains that were not
Enterobacteriaceae.

283
284 BASNET AND OTHERS

Laboratory analysis. meropenem 10 mg, ertapenem 10 mg), and others (chloram-

Sample processing and bacteriological analysis. Clinical
samples, including blood, central venous catheter tips, urine,
sputum, endotracheal suction, pleural fluid, and pus and
wound swabs, were processed for microbiological culture
analysis. Aseptically drawn blood samples were inoculated
into a biphasic brain-heart infusion medium and incubated
aerobically for 24 hours at 37 C. Other than urine samples,
which were streaked on cysteine-lactose electrolyte-
deficient agar, other samples were streaked on blood agar
and MacConkey agar. The streaked culture plates were
incubated aerobically for 24 hours at 37 C. Using standard
microbiological procedures, the samples were processed in
a biosafety cabinet class IIA/IIB as infectious.
Before the samples were reported as sterile, body fluids
and blood culture samples were reincubated for another
24 hours (up to 48 hours) and 48 hours (up to 72 hours),
respectively. Colony growth on agar plates was investigated
macroscopically, followed by Gram staining and biochemical
tests to identify the pathogens. A quantitative enumeration
of colony-forming units (CFUs) was performed for uropatho-
gens, with 104 CFUs/mL considered insignificant growth,
104 to 105 CFUs/mL doubtful growth (suggesting a repeat
specimen), and $ 105 CFUs/mL significant growth. A man-
ual of clinical microbiology was used to identify, isolate, and
characterize Enterobacteriaceae.10
Antimicrobial susceptibility testing. Antimicrobial suscepti-
bility testing of the identified isolate was performed on
Mueller Hinton agar (MHA) using the Kirby-Bauer disc
diffusion method, following the Clinical and Laboratory Stan-
dards Institute (CLSI; 30th edition) guidelines.11 For culture
and antimicrobial susceptibility testing, Escherichia coli
(America Type Culture Collection 25922) was used as a ref-
erence strain.
To begin, a sterile nutrient broth was inoculated with three
to five colonies from each isolate and incubated at 37 C until
it reached an optimal log phase. The suspension was stan-
dardized to maintain a 0.5 McFarland turbidity standard. The
standardized culture was swabbed onto the MHA plate and
left to dry and diffuse for about 5 minutes. The MHA plate
was inoculated with different commercially available antibi-
otic discs such as fluoroquinolones (ciprofloxacin 5 mg,
ofloxacin 5 mg, levofloxacin 5 mg), aminoglycosides (genta-
micin 10 mg, amikacin 30 mg), penicillins (ampicillin 10 mg,
carbenicillin 100 mg, amoxicillin-clavulanate 30 mg), cepha-
losporins (cefotaxime 30 mg, ceftriaxone 30 mg, ceftazidime
30 mg, cefepime 30 mg), carbapenems (imipenem 10 mg,
phenicol 30 mg and cotrimoxazole 25 mg). After being admin-
istered with six antibiotic discs, the 90 mm–diameter petri
plate was incubated at 37 C for 24 hours. Afterward, the
diameter of the susceptibility zone was measured with the
help of a Vernier caliper. The result was interpreted and
recorded as susceptible (S), intermediate (I), or resistant (R)
based on the CLSI guidelines.11
Definition of MDR and XDR. Multidrug-resistant was
defined as acquired nonsusceptibility to at least one agent in
three or more antimicrobial categories and XDR as nonsus-
ceptibility to at least one agent in all antimicrobial categories
except two or fewer (i.e., bacterial isolates remained suscep-
tible to only one or two categories).6
Statistical analysis. Statistical Package for Social Sci-
ence v. 17.0 (SPSS Inc., Chicago, IL) was used to analyze
the data. Study variables were presented as absolute num-
bers (n) and percentages (%). The x2 test was used to ana-
lyze the association between variables. A P value , 0.05
indicated a significant difference.
RESULTS
Bacterial strains. Among the total clinical samples
(N 5 1,617), 234 (14.5%) tested culture-positive for Enterobac-
teriaceae. Enterobacteriaceae (n 5 267) were frequently iso-
lated from urine (54.7%, 128/234) samples, followed by blood
(19.7%, 46/234) and sputum (15.0%, 35/234) samples. Three
blood samples, four sputum samples, and 26 urine samples
exhibited polymicrobial growth. Enterobacteriaceae chiefly
comprised E. coli (n 5 118, 44.2%), Citrobacter freundii
(n 5 81, 30.3%), and Klebsiella pneumoniae (n 5 39, 14.6%). A
majority of E. coli (91.5%, 108/118) (P , 0.001) were isolated from
urine samples, whereas C. freundii (38.3%, 31/81) (P , 0.001)
and K. pneumoniae (48.7%, 19/39) (P , 0.001) were isolated
from blood and sputum samples, respectively (Table 1).
Incidence of antimicrobial resistances in Entero-
bacteriaceae. Members of the family Enterobacteriaceae
exhibited a resistance rate of 95.6% (392/410) to penicillins,
71.6% (765/1068) to cephalosporins, and 65.2% (522/801)
to fluoroquinolones. Carbapenems (36.2%, 290/801), how-
ever, appeared to be the least resistant antimicrobial cate-
gory among Enterobacteriaceae (Table 2).
Antimicrobial susceptibility pattern among the Entero-
bacteriaceae isolates. The AMR profiles of Enterobacteria-
ceae were assessed for 17 antibiotics (Table 3). Carbenicillin
(n 5 81, 100%) (P 5 0.077), ceftazidime (n 5 70, 86.4%)

TABLE 1
Isolation of Enterobacteriaceae from different clinical samples
K. pneumoniae P. mirabilis S. Typhi Enterobacter
E. coli (n 5 118) C. freundii (n 5 81) (n 5 39) (n 5 18) (n 5 7) spp. (n 5 4)

Enterobacteriaceae n (%) P value n (%) P value n (%) P value n (%) P value n (%) P value n (%) P value

Urine (n 5 128) 108 (91.5) < 0.001 24 (29.6) < 0.001 11 (28.2) < 0.001 11 (61.1) 0.760 0 (0) – 0 (0) –
Blood (n 5 46) 3 (2.5) < 0.001 31 (38.3) < 0.001 3 (7.7) 0.063 3 (16.7) 0.848 7 (100) < 0.001 2 (50) 0.099
CVC tip (n 5 1) 1 (0.9) 0.26 0 (0) – 0 (0) – 0 (0) – 0 (0) – 0 (0) –
Sputum (n 5 35) 3 (2.5) < 0.001 12 (14.8) 0.949 19 (48.7) < 0.001 3 (16.7) 0.798 0 (0) – 2 (50) 0.043
Et secretion (n 5 12) 0 (0) – 9 (11.1) 0.001 3 (7.7) 0.297 0 (0) – 0 (0) – 0 (0) –
Pleural fluid (n 5 1) 0 (0) – 1 (1.2) 0.129 0 (0) – 0 (0) – 0 (0) – 0 (0) –
Pus (n 5 9) 3 (2.5) 0.357 4 (4.9) 0.498 2 (5.1) 0.623 1 (5.6) 0.675 0 (0) – 0 (0) –
Wound (n 5 2) 0 (0) – 0 (0) – 1 (2.6) 0.015 0 (0) – 0 (0) – 0 (0) –
CVC tip 5 central venous catheter tip; C. freundii 5 Citrobacter freundii; E. coli 5 Escherichia coli; Et secretion 5 endotracheal secretion; K. pneumoniae 5 Klebsiella pneumoniae; P. mirabilis 5
Proteus mirabilis; S. Typhi 5 Salmonella Typhi. Boldface P values are statistically significant (P , 0.05).
 ANTIBIOTIC RESISTANCE IN ENTEROBACTERIACEAE 285

TABLE 2
Antimicrobial resistance profiles of overall Enterobacteriaceae isolates
Resistant

Antibiotics Susceptible, n (%) Intermediate, n (%) n % Cumulative %

Fluoroquinolones
Ciprofloxacin (n 5 267) 49 (18.4) 33 (12.3) 185 69.3
Ofloxacin (n 5 267) 91 (34.1) 14 (5.2) 162 60.7 65.2
Levofloxacin (n 5 267) 60 (22.5) 32 (12.0) 175 65.5
Aminoglycosides
Gentamicin (n 5 267) 146 (54.7) 25 (9.3) 96 36.0
Amikacin (n 5 267) 91 (34.1) 78 (29.2) 98 36.7 36.3
Penicillins
Ampicillin (n 5 143) 6 (4.2) 5 (3.5) 132 92.3
Carbenicillin (n 5 267) 5 (1.9) 2 (0.7) 260 97.4 95.6
Cephalosporins
Cefotaxime (n 5 267) 44 (16.5) 23 (8.6) 200 74.9
Ceftriaxone (n 5 267) 60 (22.5) 21 (7.9) 186 69.7
Ceftazidime (n 5 267) 28 (10.5) 31 (11.6) 208 77.9 71.6
Cefepime (n 5 267) 44 (16.5) 52 (19.5) 171 64.0
Carbapenems
Imipenem (n 5 267) 86 (32.2) 58 (21.7) 123 46.1
Meropenem (n 5 267) 174 (65.2) 8 (3.0) 85 31.8 36.2
Ertapenem (n 5 267) 155 (58.1) 30 (11.2) 82 30.7
Others
Chloramphenicol (n 5 267) 114 (42.7) 42 (15.7) 111 41.6 –
Cotrimoxazole (n 5 267) 93 (34.8) 4 (1.5) 170 63.7 –
Amoxicillin-clavulanate (n 5 143) 77 (53.8) 12 (8.4) 54 37.8 –

(P 5 0.027), and imipenem (n 5 52, 64.2%) (P , 0.001) were
most resistant to C. freundii, whereas gentamicin (n 5 43,
53.1%) (P , 0.001), meropenem (n 5 42, 51.9%)
(P , 0.001), and chloramphenicol (n 5 41, 50.6%)
(P 5 0.048) were moderately resistant. Escherichia coli
showed resistance rates of 39.0% (46/118) (P 5 0.102) to
amoxicillin-clavulanate, 31.4% (37/118) (P , 0.001) to imi-
penem, 28.8% (34/118) (P , 0.001) to chloramphenicol,
21.2% (25/118) (P , 0.001) to gentamicin, and 15.3%
(18/118) (P , 0.001) to meropenem. Enterobacter spp.
showed 100% (4/4) resistance to carbenicillin, cefotaxime,
ceftriaxone, and ceftazidime. Likewise, K. pneumoniae
exhibited 100% resistance to carbenicillin (P 5 0.267)
and was highly resistant to ceftazidime (n 5 31, 79.5%)
(P 5 0.796) and ciprofloxacin (n 5 28, 71.8%) (P 5 0.713).
There was moderate resistance (48.7%, 19/39) to both mero-
penem (P 5 0.014) and ertapenem (P 5 0.008) in K. pneumo-
niae, but a higher resistance (64.1%, 25/39) to imipenem
(P 5 0.014). Susceptibility to ertapenem varied greatly across
different bacteria strains, ranging from a remarkable 100% for
Salmonella Typhi to a substantial 86.4% for E. coli and a lower
48.2% for C. freundii (Table 3).
Antimicrobial resistance based on isolation of Entero-
bacteriaceae from clinical samples. Resistance rates
among Enterobacteriaceae isolated from clinical samples
differed significantly (Table 4). A resistance rate of . 70%
was observed among uropathogens to ampicillin (110/154)
(P 5 0.897) and ceftazidime (111/154) (P 5 0.007). Blood-
stream pathogens were 100% (49/49) resistant to
amoxicillin-clavulanate (P 5 0.956), 93.38% (46/49) to peni-
cillins (P 5 0.001), and 85.7% (43/49) to aminoglycosides
(P 5 0.007). The resistance rates of Enterobacteriaceae iso-
lated from respiratory samples surpassed 80% for ceftazi-
dime, 70% for cefotaxime, and 60% for ciprofloxacin
(Table 4).
Incidences of non-MDR, MDR, and XDR among the
Enterobacteriaceae. Incidence of MDR and XDR strains in
Enterobacteriaceae is shown in Figure 1. Out of 267 isolates,
96 (36.0%) were MDR and 98 (36.7%) were XDR. Proteus
mirabilis (44.4%, 8/18) (P 5 0.043) and E. coli (44.1%,
52/118) (P 5 0.014) were the predominant MDR isolates.
However, K. pneumoniae (53.8%, 21/39) (P 5 0.036), C.
freundii (53.1%, 43/81) (P , 0.001), and E. coli (21.2%,
25/118) (P , 0.001) were the predominant XDR isolates.
An equal incidence of both MDR and XDR was found in
Enterobacter spp. (2/4, 50.0%) (Figure 1).
Incidence of isolation of MDR and XDR Entero-
bacteriaceae from clinical samples. A total of 71 (38.4%,
71/154) (P , 0.001) MDR Enterobacteriaceae and 35
(22.72%, 35/154) (P , 0.001) XDR Enterobacteriaceae were
detected in urine samples. Multidrug-resistant Enterobacter-
iaceae accounted for 14.3% (7/49) (P , 0.001) of the blood-
stream and 28.2% (11/39) (P 5 0.275) of the respiratory
isolates, whereas XDR Enterobacteriaceae made up 22.7%
(32/49) (P , 0.001) and 51.3% (20/39) (P 5 0.036), respec-
tively (Figure 2).
Incidence of AMR among non-MDR, MDR, and XDR
Enterobacteriaceae. The fluoroquinolones’ resistance rate
in non-MDR Enterobacteriaceae was 19.9%, whereas in
MDR and XDR Enterobacteriaceae it was 63.2% and
96.2%, respectively. There were 28.6% and 67.0% resis-
tance rates for cephalosporins and penicillins in non-MDR
Enterobacteriaceae, respectively, whereas they were 72.9%
and 97.4% in MDR Enterobacteriaceae and 95.4% and
99.0% in XDR Enterobacteriaceae. Multidrug-resistant
Enterobacteriaceae had resistance rates of 95.8%, 70.8%,
70.8%, and 68.8% for carbenicillin, ciprofloxacin, ceftriax-
one, and cotrimoxazole, respectively, whereas XDR Entero-
bacteriaceae had rates of 100%, 99.0%, 96.9%, and 96.9%
(Figure 3).
286 BASNET AND OTHERS

TABLE 3
Antimicrobial resistance and its significant association among the members of the family Enterobacteriaceae
E. coli (n 5 118) C. freundii (n 5 81) K. pneumoniae (n 5 39) P. mirabilis (n 5 18) S. Typhi (n 5 7) Enterobacter spp. (n 5 4)

Antibiotics n (%) P value n (%) P value n (%) P value n (%) P value n (%) P value n (%) P value

Fluoroquinolones
Ciprofloxacin 82 (69.5) 0.949 57 (70.4) 0.8 28 (71.8) 0.713 9 (50) 0.066 6 (85.7) 0.34 3 (75) 0.803
Ofloxacin 73 (61.9) 0.723 50 (61.7) 0.816 22 (56.4) 0.555 9 (50) 0.337 6 (85.7) 0.169 2 (50) 0.66
Levofloxacin 78 (66.1) 0.864 54 (66.7) 0.799 26 (66.7) 0.873 9 (50) 0.151 6 (85.7) 0.255 2 (50) 0.51
Aminoglycosides
Gentamicin 25 (21.2) < 0.001 43 (53.1) < 0.001 19 (48.7) 0.072 6 (33.3) 0.81 1 (14.3) 0.226 2 (50) 0.555
Amikacin 23 (19.5) < 0.001 43 (53.1) < 0.001 24 (61.5) < 0.001 5 (27.8) 0.416 1 (14.3) 0.212 2 (50) 0.578
Penicillins
Ampicillin 110 (93.2) 0.374 –* – –* – 16 (88.9) 0.56 6 (85.7) 0.501 –* –
Carbenicillin 116 (98.3) 0.399 81 (100) 0.077 39 (100) 0.267 14 (77.8) < 0.001 6 (85.7) 0.05 4 (100) 0.741
Cephalosporins
Cefotaxime 88 (74.6) 0.912 67 (82.7) 0.052 29 (74.4) 0.932 6 (33.3) < 0.001 6 (85.7) 0.504 4 (100) 0.243
Ceftriaxone 81 (68.6) 0.747 63 (77.8) 0.057 29 (74.4) 0.49 7 (38.9) 0.003 2 (28.6) 0.017 4 (100) 0.184
Ceftazidime 93 (78.8) 0.75 70 (86.4) 0.027 31 (79.5) 0.796 7 (38.9) < 0.001 3 (42.9) 0.024 4 (100) 0.283
Cefepime 70 (59.3) 0.152 61 (75.3) 0.011 28 (71.5) 0.275 7 (38.9) 0.021 2 (28.6) 0.047 3 (75) 0.645
Carbapenems
Meropenem 18 (15.3) < 0.001 42 (51.9) < 0.001 19 (48.7) 0.014 3 (16.7) 0.153 1 (14.3) 0.312 2 (50) 0.432
Imipenem 37 (31.4) < 0.001 52 (64.2) < 0.001 25 (64.1) 0.014 5 (27.8) 0.107 1 (14.3) 0.087 3 (75) 0.242
Ertapenem 16 (13.6) < 0.001 42 (51.9) < 0.001 19 (48.7) 0.008 3 (16.7) 0.181 0 (0) – 2 (50) 0.399
Others
Amoxicillin-clavulanate 46 (39.0) 0.102 –* – –* – 7 (38.9) 0.684 1 (14.3) 0.113 –* –
Chloramphenicol 34 (28.8) < 0.001 41 (50.6) 0.048 19 (48.7) 0.327 14 (77.8) 0.001 1 (14.3) 0.138 2 (50) 0.73
Cotrimoxazole 66 (55.9) 0.019 58 (71.6) 0.075 27 (69.2) 0.435 16 (88.9) 0.021 1 (14.3) 0.006 2 (50) 0.567
C. freundii 5 Citrobacter freundii; E. coli 5 Escherichia coli; K. pneumoniae 5 Klebsiella pneumoniae; P. mirabilis 5 Proteus mirabilis; S. Typhi 5 Salmonella Typhi. Boldface P values are
statistically significant (P , 0.05).
* Intrinsic resistance.

DISCUSSION across geographical regions and because few studies have

Resistance of pathogens to a wide range of antimicrobial
agents has become a major public health concern as the
development of new drugs has not kept pace.3 Unless infec-
tions with a drug-resistant microorganism are detected and
their incidence is known, it becomes difficult to implement
strategies to prevent drug resistance in healthcare settings.3
Because genetic variation may alter resistance patterns
examined XDR-associated Enterobacteriaceae infections,
this study examined resistance patterns in clinical isolates of
Enterobacteriaceae and estimated MDR and XDR incidence
rates.
The WHO has identified carbapenem-resistant Enterobac-
teriaceae and non-fermenters as critical or priority number
one in its list of priority pathogens for research, discovery,
and development of new antibiotics.12 In the present study,

TABLE 4
Incidence of isolation of drug-resistant Enterobacteriaceae from the clinical samples
Urine (n 5 154) Blood (n 5 49) Sputum (n 5 39) Et secretion (n 5 12) Pus (n 5 10)

Antibiotics n (%) P value n (%) P value n (%) P value n (%) P value n (%) P value

Fluoroquinolones
Ciprofloxacin (n 5 183) 100 (64.9) 0.072 42 (85.7) 0.006 25 (64.1) 0.447 8 (66.7) 0.84 8 (80) 0.454
Ofloxacin (n 5 160) 88 (57.1) 0.168 39 (79.6) 0.003 22 (56.4) 0.555 4 (33.3) 0.047 7 (70) 0.538
Levofloxacin (n 5 173) 95 (61.7) 0.122 40 (81.6) 0.009 24 (61.5) 0.569 7 (58.3) 0.591 7 (70) 0.762
Aminoglycosides
Gentamicin (n 5 95) 34 (22.1) < 0.001 29 (59.2) < 0.001 22 (56.4) 0.004 5 (41.7) 0.673 5 (50) 0.345
Amikacin (n 5 97) 38 (24.7) < 0.001 29 (59.2) < 0.001 21 (53.9) 0.016 7 (58.3) 0.112 2 (20) 0.264
Penicillins
Ampicillin (n 5 131) 110 (71.4) 0.897 12 (24.5) 1 5 (12.8) 0.399 0 (0) – 4 (40) 0.558
Carbenicillin (n 5 257) 148 (96.1) 0.128 48 (98.0) 0.778 39 (100) 0.267 12 (100) 0.561 10 (100) 0.597
Cephalosporins
Cefotaxime (n 5 198) 103 (66.9) < 0.001 46 (94.0) 0.001 32 (82.1) 0.265 9 (75) 0.994 8 (80) 0.705
Ceftriaxone (n 5 184) 97 (63.0) 0.006 42 (85.7) 0.007 30 (76.9) 0.286 7 (58.3) 0.382 8 (80) 0.469
Ceftazidime (n 5 205) 111 (72.1) 0.007 43 (87.8) 0.066 32 (82.1) 0.499 10 (83.3) 0.643 9 (90) 0.347
Carbapenems
Imipenem (n 5 121) 52 (33.8) < 0.001 35 (71.4) < 0.001 23 (59.0) 0.08 7 (58.3) 0.383 4 (40) 0.695
Meropenem (n 5 83) 27 (17.5) < 0.001 30 (61.2) < 0.001 17 (43.6) 0.088 5 (41.7) 0.454 4 (40) 0.572
Ertapenem (n 5 80) 25 (16.2) < 0.001 29 (59.2) < 0.001 17 (43.6) 0.059 5 (41.7) 0.4 4 (40) 0.516
Others
Chloramphenicol (n 5 109) 53 (34.4) 0.006 27 (55.1) 0.033 20 (51.3) 0.183 4 (33.3) 0.553 5 (50) 0.582
Cotrimoxazole (n 5 168) 90 (58.4) 0.038 36 (73.5) 0.114 28 (71.8) 0.254 7 (58.3) 0.694 7 (70) 0.671
Amoxicillin clavulanate (n 5 97) 44 (28.6) 0.665 49 (100) 0.956 2 (5.1) 0.819 0 (0) – 2 (20) 0.609
Et secretion 5 endotracheal secretion. Boldface P values are statistically significant (P , 0.05).
 ANTIBIOTIC RESISTANCE IN ENTEROBACTERIACEAE 287

FIGURE 1. Incidences of non-MDR, MDR, and XDR among the isolates of Enterobacteriaceae. AMR 5 antimicrobial resistance; C. freundii 5
Citrobacter freundii; E. coli 5 Escherichia coli; K. pneumoniae 5 Klebsiella pneumoniae; MDR 5 multidrug resistant; P. mirabilis 5 Proteus
mirabilis; S. Typhi 5 Salmonella Typhi; XDR 5 extensively drug resistant. *Significant association (P , 0.05) with MDR and^significant asso-
ciation (P , 0.05) with XDR.

Enterobacteriaceae were mainly responsible for urinary tract
infections (54.7%), followed by respiratory tract (20.51%)
and bloodstream (19.66%) infections, with E. coli (44.19%),
C. freundii (30.34%), and K. pneumoniae (14.61%) being the
most common pathogens. The findings of Folgori et al.13
also unveiled that Enterobacteriaceae were responsible for
the smallest number of bloodstream infections (2%). Our
result contradicts the finding of previously published studies,
which identified Klebsiella spp. as the most prevalent
FIGURE 2. Isolation of multidrug-resistant and extensively drug-
resistant Enterobacteriaceae from clinical samples. CVC tip 5 central
venous catheter tip; Et secretion 5 endotracheal secretion; MDR 5
multidrug resistant; XDR 5 extensively drug resistant. *Significant
association (P , 0.05) with XDR and ^significant association (P ,
0.05) with MDR.
Enterobacteriaceae in clinical samples.4,14 Similarly, Gajda
cs
et al.15 detected K. pneumoniae as the predominant Entero-
bacteriaceae, followed by E. coli and Enterobacter cloacae.
Because nearly half of the total culture-positive samples
were urine samples, E. coli—commonest pathogen for hos-
pital- and community-acquired urinary tract infections—was
more commonly isolated in this study.16
In this study, carbenicillin (2.62%), ampicillin (7.69%), cef-
tazidime (22.10%), ciprofloxacin (30.71%), and cotrimoxazole
(36.33%) had the lowest antimicrobial effect among Entero-
bacteriaceae, whereas ertapenem (69.29%) had the greatest
antimicrobial activity. It is in line with a study by Adhikari
et al.,17 who reported an increase in AMR in Enterobacteria-
ceae against ampicillin (76.7%), ceftazidime (51.5%), ceftriax-
one (51.0%), cotrimoxazole (48.7%), and ciprofloxacin
(43.9%). The Enterobacteriaceae in this study have a similar
(, 37%) cumulative resistance to both aminoglycosides and
carbapenems. Similar resistance was observed to aminogly-
cosides (5.8–48.4%) and carbapenems (0–29.6%) in an ear-
lier study by Adhikari et al.17 We use these reserve drugs as a
last resort when treating MDR bacterial infections, resulting in
very minimal spread of plasmid-encoded drug-resistant
genes and therefore least resistance to these antibiotics.
In this study, K. pneumoniae (P . 0.05), C. freundii (P . 0.05),
and Enterobacter spp. (P . 0.05) showed 100% resistance to
carbenicillin, unlike E. coli (98.31%) (P . 0.05) and P. mirabilis
(77.78%) (P , 0.05). Higher ceftazidime resistance (. 78%)
was observed among E. coli (P . 0.05), C. freundii (P , 0.05),
and K. pneumoniae (P . 0.05), compared with P. mirabilis
(38.89%) (P , 0.05) and S. Typhi (42.86%) (P , 0.05).
Studies in Nepal also reported higher rates of penicillin
288 BASNET AND OTHERS
FIGURE 3. Incidence of antimicrobial resistance among non-MDR, MDR, and XDR Enterobacteriaceae. AMGs 5 aminoglycosides; AMR 5 anti-
microbial resistance; CBPs 5 carbapenems; CEPHs 5 cephalosporins; FQs 5 fluoroquinolones; MDR 5 multidrug resistant; PI 5 penicillin;
XDR 5 extensively drug resistant.
resistance (74.9–100%) in Klebsiella spp. and E. coli, but
moderate rates of cephalosporin resistance (9.0–67.1%) in
S. Typhi, Klebsiella spp., and Citrobacter spp.17,18 A possi-
ble cause of resistance to b-lactam antibiotics is the
plasmidic spread of b-lactamases, such as extended-
spectrum b-lactamases, metallo-b-lactamases, and AmpC
b-lactamases, in Enterobacteriaceae, in hospitals, and in
the community.19 In this study, the antimicrobial activity of
meropenem and ertapenem against E. coli (P , 0.05) and
P. mirabilis (P . 0.05) was three times as effective as either
of the antibiotics for Citrobacter spp. and K. pneumoniae.
The higher carbapenem resistance among K. pneumoniae
in this study aligns with the findings of a Korean study,
where 64–80% of K. pneumoniae was found to be carba-
penem-resistant.20 Various carbapenemase enzymes,
including blaNDM, blaVIM, blaSPM, blaIMP, and blaKPC, could
contribute to such resistance.21,22
Although the prevalence of MDR (36.0%) in the current
study was comparable to that of Shrestha et al.18 (30.0%)
and Mahto et al.22 (30.2%), the prevalence of XDR (36.7%)
was higher than both of the studies (10% and 23%, respec-
tively). Studies from across the world have reported varying
rates of MDR (6.6–67.0%) and XDR (7.0–50%) in Enterobac-
teriaceae.3,23–26 Drug-resistant strains in the present study
may have resulted from over-the-counter antibiotic sales
and prescriptions of higher-generation antibiotics by general
practitioners. In this study, Enterobacter spp. (50.0%) were
the predominant MDR Enterobacteriaceae, followed by
P. mirabilis (44.4%) and E. coli (44.1%). On the other hand,
C. freundii (53.1%) was the most common XDR Enterobac-
teriaceae, followed by Enterobacter spp. (50.0%). In con-
trast, Aly and Balkhy27 identified E. coli and K. pneumoniae
as the most common MDR Enterobacteriaceae. Such
resistance will lead to treatment failure and an increased
mortality rate. This was evidenced in a study conducted by
Alkofide et al,28 which reported an alarming 84% fatality
rate among critically ill patients infected with MDR and
XDR Enterobacteriaceae.
This study found that MDR Enterobacteriaceae (34.8%)
(P . 0.05) were more likely to be isolated from urine sam-
ples, whereas XDR Enterobacteriaceae (51.3%) (P , 0.05)
were likely to be isolated from respiratory samples. Com-
pared with non-MDR and MDR Enterobacteriaceae, the XDR
Enterobacteriaceae showed higher rates of fluoroquinolone,
cephalosporin, and penicillin resistance. Uropathogens
showed higher resistance to ceftazidime (72.08%) (P , 0.05)
and ciprofloxacin (64.94%) (P . 0.05), but bloodstream
pathogens to cefotaxime (93.88%) (P , 0.05) and levofloxa-
cin (81.63) (P , 0.05) and respiratory pathogens to carbeni-
cillin (100%) (P . 0.05) and cefotaxime (. 80%) (P . 0.05).
These findings nonetheless mark the initial endeavor to
establish a link between AMR and clinical samples with
Enterobacteriaceae. Therefore, the findings of this study
cannot be directly compared with the existing literature.
However, they will serve as a crucial basis for further similar
research.
Recent studies have shown that difficult-to-treat resis-
tance (DTR), which distinguishes between antibiotic
strengths (higher efficacy) and weaknesses (lower toxicity),
has greater significance in identifying pathogens associated
with higher mortality risks, unlike MDR and XDR.29 The
detection of antibiotic-resistant pathogens must therefore
consider both classical resistance classifications and DTR
classifications. In addition, antimicrobial stewardship pro-
grams and prevention of overuse and over-the-counter sales
of antibiotics may help reduce AMR.3,25
The study had several limitations. First, this study was
conducted in a single tertiary care center, so the findings
 ANTIBIOTIC RESISTANCE IN ENTEROBACTERIACEAE
cannot be generalized to the entire nation or worldwide, as they
could overestimate or underestimate prevalence/incidence.
Second, because of financial limitations, the study was unable
to perform molecular analyses to identify antibiotic resistance
genes. Furthermore, it is important to consider that resistance
patterns might vary over an extended period of time, given the
limited duration of the study.
CONCLUSION
Escherichia coli, C. freundii, and K. pneumoniae caused
more than four-fifths of Enterobacteriaceae infections. Carba-
penems proved to be the most potent antibiotics among the
members of the family Enterobacteriaceae, whereas penicil-
lins proved to be the least effective antibiotics. Uropathogens
exhibited high resistance to ceftazidime, but bloodstream
and respiratory pathogens to carbenicillin. Extensively drug-
resistant infections associated with Enterobacteriaceae were
more prevalent than MDR infections. Multidrug-resistant and
XDR Enterobacteriaceae were frequently isolated from urine
samples and exhibited the highest resistance to penicillins.
To reduce AMR worldwide, clinical microbiology laboratories
must therefore actively detect, closely monitor, and report
MDR, or even XDR, bacterial strains.
Received April 1, 2023. Accepted for publication October 9, 2023.
Published online January 2, 2024.
Acknowledgment: The American Society of Tropical Medicine and
Hygiene (ASTMH) assisted with publication expenses.
Authors’ addresses: Ajaya Basnet, Department of Medical Microbiol-
ogy, Shi-Gan International College of Science and Technology, Trib-
huvan University, Kathmandu, Nepal, and Department of Microbiol-
ogy, Nepal Armed Police Force Hospital, Kathmandu, Nepal, E-mail:
xlcprk@gmail.com. Mahendra Raj Shrestha and Rajendra Maharjan,
Department of Clinical Laboratory, Nepal Armed Police Force
Hospital, Kathmandu, Nepal, E-mails: mahendrapath@gmail.com
and ryan_m10@hotmail.com. Basanta Tamang, Department of
Microbiology, Nepal Armed Police Force Hospital, Kathmandu,
Nepal, E-mail: tamangbasanta222@gmail.com. Nayanum Pokhrel,
Research Section, Nepal Health Research Council, Kathmandu,
Nepal, E-mail: nayanumpr@gmail.com. Junu Richhinbung Rai,
Department of Microbiology, Maharajgunj Medical Campus,
Tribhuvan University Teaching Hospital, Institute of Medicine,
Kathmandu, Nepal, E-mail: junuri15@gmail.com. Shrijana Bista,
Central Department of Microbiology, Tribhuvan University,
Kathmandu, Nepal, E-mail: shrijanabista800@gmail.com. Shila
Shrestha, Department of Medical Microbiology, Shi-Gan Interna-
tional College of Science and Technology, Tribhuvan University,
Kathmandu, Nepal, E-mail: shila.sth9@gmail.com. Shiba Kumar Rai,
Department of Research and Microbiology, Nepal Medical College
and Teaching Hospital, Kathmandu, Nepal, E-mail: drshibakrai@
gmail.com.
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