Keywords
gram-negative isolates - antibiotic susceptibility - bacterial profile - biliary tract
infections - clinical profile
Introduction
Bile is a sterile secretion produced by the liver and stored in the gallbladder. It
helps remove microorganisms entering the biliary tract by its flushing action. Bactibilia
occurs either due to the presence of stones in the gallbladder or biliary tract.[1]
[2] Microorganisms from the duodenum contents may enter the gallbladder due to biliary
tract obstruction leading to septicemia through the portal venous channel.[3] Eventually, the bacteria presumably translocate into the circulation causing a systemic
infection. Acute cholangitis spans a continuous clinical spectrum and can progress
from a local biliary infection to advanced disease with sepsis and multiple organ
dysfunction syndrome.[2] According to recent studies, biliary tract infections (BTIs) are associated with
mortality rates of 9 to 12%.[4] BTIs predominantly have bacterial etiology with gram-negative aerobic flora being
the most prevalent. The most commonly isolated aerobic pathogens are Escherichia coli, Klebsiella spp., Pseudomonas spp., Enterococcus spp., and Salmonella spp.[2] Candida may be isolated in very few of the cases. Additionally, 4 to 5% of BTIs
are caused by more than one organism (polymicrobial).[5]
[6]
In the past few years, although there has been no change in the bacteriological profile
of organisms responsible for BTIs, there has been a significant change in the drug
susceptibility pattern of these organisms. These infections with increased frequency
of extended-spectrum β-lactamase (ESBL) and carbapenemase-producing Enterobacteriaceae
(CPE) organisms lead to redundancy of currently used empiric antibiotic therapy.[7]
[8] Therefore, choosing an antibiotic regimen remains challenging for clinicians.
Therefore, this study aimed to know the microbiological profile and current antibiotic
susceptibility patterns of isolates from patients with BTIs admitted to a teaching
hospital in North India.
Material and Methods
This retrospective cross-sectional study was conducted in the department of microbiology
in a tertiary care hospital. This study was carried out over 1 year from March 2022
to Feb 2023.
Sample Collection
Bile samples were collected by intraprocedural extraction through endoscopic retrograde
cholangiopancreatography (ERCP) or during surgery procedures. In case of a complicated
or perforated gallbladder, bile was collected from the perihepatic space under aseptic
conditions. Depending on the clinical presentation, empirical antibiotics were started
before sample collection on the basis of the hospital antibiotic policy. Bile samples
were received in the microbiology laboratory for culture and antibiotic sensitivity
testing.
Sample Processing
All the bile samples received in the microbiology laboratory were inoculated in blood
culture bottles for enrichment and were processed in an automated BACTEC or BacT/ALERT
system. Once a blood culture bottle flagged positive, a Gram stain was done from the
broth in the bottle to look for any organism. Simultaneously all the positive bottles
were subcultured on blood agar and MacConkey agar plates. The plates were incubated
at 37°C for 18 to 24 hours. Identification of isolated organisms and antimicrobial
susceptibility was done by the VITEK 2 system.[9] Characterization of isolates was done as multidrug resistant (MDR), extensively
drug resistant (XDR), and pan drug resistant (PDR). MDR was defined as acquired nonsusceptibility
to at least one agent in three or more antimicrobial categories. XDR was defined as
nonsusceptibility to at least one agent in all but two or fewer antimicrobial categories
(i.e., bacterial isolates remain susceptible to only one or two categories), and PDR
was defined as nonsusceptibility to all agents in all antimicrobial categories.[10] Demographic, clinical, and histopathological details of the patients with positive
bile culture were included. In addition, positive bile cultures were correlated with
blood cultures received concurrently from the respective patient.
Statistical Analysis
Statistical analysis was performed by using the chi-squared test or Fisher's exact
test as appropriate and probability levels <0.05 by the two-tailed test were considered
statistically significant.
Result
A total of 226 bile samples were received in the microbiology laboratory for culture
and sensitivity, 123 (54.4%) samples from male patients and 103 (45.6%) samples from
female patients. Out of these, 136 (60.2%) samples were found to be culture positive.
Culture positivity was similar among female (63/103 [61.2%]) and male (73/123 [59.3%])
patients. The majority of patients in both the genders were ≥40 years. Hypertension
(33.1%) was the most common underlying disease followed by diabetes (28.7%) and cardiovascular
disease (8.1%). Common manifestations presented by patients were abdominal pain (77.89%),
jaundice (42.24%), and fever (41.52%). Cholangitis (acute inflammation and infection
of the biliary duct system) was present in 28 (20.6%) patients. Out of 136 cholecystitis
patients, 53 (39%) patients were clinically diagnosed with cholelithiasis or choledocholithiasis,
35 (25.7%) had malignancy,12 (8.8%) patients had bile duct injury, and 36 (26.4%)
patients had concurrent perihepatic collection, pancreatitis, and ascites. These patients
were referred patients with prior intervention ([Table 1]). Histopathological examination revealed adenocarcinoma as the most common finding
in malignant cases.
Table 1
Demographic and clinical characteristics in patients with biliary tract infections
(n = 136)
|
Clinical characteristics
|
Number (%)
|
|
Male (
n
= 73)
|
|
< 40 y
|
9 (6.6)
|
|
> 40 y
|
64 (47)
|
|
Females (
n
= 63)
|
|
< 40 y
|
19 (13.9)
|
|
> 40 y
|
44 (32.5)
|
|
Primary disease
|
|
Benign
|
101 (74.3)
|
|
Malignant
|
35 (25.7)
|
|
Underlying diseases
|
|
Hypertension
|
45 (33.1)
|
|
Diabetes
|
39 (28.7)
|
|
Cardiovascular diseases
|
11 (8.1)
|
|
Manifestation
|
|
Abdominal pain
|
97 (71.3)
|
|
Jaundice
|
33 (24.3)
|
|
Fever
|
21 (15.4)
|
|
Route of bile collection
|
|
Endoscopic
|
75 (55.1
|
|
During surgery
|
61 (44.9)
|
A total of 142 isolates were obtained from 136 patients with bile culture positive,
as 6 (15.3%) of the samples yielded more than one organism. There was no definite
pattern observed in polymicrobial infection. Among 142 isolated organisms, 120 (84.5%)
were gram-negative bacilli, 19 (13.3%) were gram-positive cocci, and 3 (2.1%) were
Candida spp. Escherichia coli was predominantly found in 33% bile samples followed by Klebsiella spp. (19%) &Pseudomonas spp. (18%). Among Gram positive, Enterococcus spp. was the most common isolate followed by Streptococcus and coagulase-negative Staphylococcus. Besides, a few isolates of Acinetobacter spp., Salmonella spp., and Sphingomonas paucimobilis were also obtained ([Fig. 1]).
Fig. 1 Percent distribution of microorganisms isolated from bile specimens (n = 142).
Further, a correlation between the age and gender of the patient with the type of
isolates/pathogen from bile was also made. No significant association was seen between
age and isolates from bile, whereas Salmonella spp. isolates were significantly high in patients younger than 40 years (p < 0.05). While comparing the gender with isolated bacteria, although the distribution
of bacterial isolates was variable in both genders, it was not statistically significant
except in the case of Enterococcus (p < 0.05). In the case of BTI with Enterococcus, males were significantly higher than females.
Specimens for blood culture samples were received from 40 of the patients with positive
bile cultures. Out of these, only nine samples were found to be positive. However,
out of these nine patients, seven blood samples showed the same organism as had been
isolated from bile.
The antibiotic susceptibility profile of gram-negative isolates showed that tigecycline
and aminoglycosides were the most effective drugs. E. coli showed 93.6 and 95.7%sensitivity to both tigecycline and amikacin. Other antibiotics
that were found to be effective were gentamicin, imipenem, and cefoperazone sulbactam.
The isolates were less susceptible to cephalosporins and amoxicillin clavulanate combination.
However, Pseudomonas spp. and Klebsiella spp. showed very high resistance against cephalosporins and carbapenems ([Fig. 2]). Among the gram-negative isolates, approximately 70% of the isolates were MDR with
approximately 80 and 75% being the ESBLs and carbapenemase producers, respectively.
Fig. 2 Antibiotic susceptibility profile of predominant gram-negative isolates.
Gram-positive isolates (Enterococcus) were 100% susceptible to linezolid, whereas 25 and 16.7% of the isolates were resistant
to vancomycin and teicoplanin, respectively ([Fig. 3]).
Fig. 3 Antibiotic susceptibility profile of Enterococcus spp. (n = 12).
Discussion
In the present study, 136/226 (60.2%) bile samples received were found to be culture
positive. Our results were in accordance with that of Shenoy et al[11] who reported a culture positivity of 56% bile samples. On the other hand, some authors
have reported considerably lower bile culture positivity (23.6–26.5%).[12]
[13] This can be ascribed to the fact that it being a tertiary care setup most of the
patients are referral patients. Additionally, with state of art in the house microbiology
laboratory, the isolation rate is possibly better.
BTIs such as acute or chronic cholecystitis and cholangitis are more common in patients
presenting with biliary tract obstruction due to cholelithiasis, choledocholithiasis,
or malignancy.[14] We also observed that 39% of patients had a history of stones in the biliary tract
and another 25.7% had malignancy.
Gender is one of the most important independent risk factors for cholelithiasis. In
the present study, bile culture positivity was more in females as compared to the
males, which can be correlated with the high incidence of cholelithiasis in females,
although this difference was not found to be significant. Female predominance was
observed in the literature.[11]
[14]
[15] In the present study, 80.2% of the patients with positive bile culture belong to
the elder age groups (>40 years). Similar findings have been reported in other studies
as well.[14]
[16]
In the present study, hypertension, diabetes, and cardiovascular diseases were the
most common underlying diseases. Abdominal pain, jaundice, fever, and loss of appetite
were common manifestations. Similar observations have also been reported in few other
studies.[11]
[17]
In most cases, bile infections are caused by a single organism, but in a few cases
mixed flora can be obtained. The mixed growth may be due to some antibiotic-resistant
bacteria along with food habits and environmental factors. In our study, monomicrobial
growth was seen in 95.6% of samples, while polymicrobial (two organisms) growths were
detected in only 4.4% of the cases. Our results were in concordance with a study done
by Farhana et al,[18] where a single organism was predominantly isolated from bile. Gram-negative isolates
were the leading (84.5%) cause of BTIs, with a lesser number (13.3%) of gram-positive
isolates, because gram-negative isolates constitute the majority of gut flora.[11] Among the gram-negative flora, consistent with previous studies, E. coli was the predominant isolate followed by Klebsiella spp. and Pseudomonas spp., in the present study. Among gram-positive flora, Enterococcus spp. remained the most common isolate as has been observed in previous studies as
well.[15]
[16] However few other studies have reported a swift increase in the incidence of Enterococci in BTI, which may be due to the use of empirical antibiotic therapy against gram-negative
bacteria.[19]
[20]
A high-level resistance against all the gram-negative isolates has been observed against
β -lactam drugs including penicillin, cephalosporins, and β-lactamase inhibitor combinations.
This may be correlated with the high incidence of ESBL producers among these gram-negative
isolates. However, the susceptibility profile of carbapenems is variable (25–50%)
in these isolates; E. coli is less resistant to carbapenems as compared to K. pneumoniae and Pseudomonas. Additionally, tigecycline and aminoglycosides were relatively effective against
gram-negative isolates. Susceptibility to cephalosporins against gram-negative isolates
was reported to be higher in comparison with aminoglycosides in a few studies, while
Shahi et al observed higher resistance to cephalosporins, which corroborates with
the present study.[21]
Among the gram-positive isolates, vancomycin, teicoplanin, and linezolid were the
most effective drugs showing good sensitivity, which was also found in the study by
Sharma et al.[22]
Only 22.5% of the patients with positive bile culture had a positive blood culture,
and 77.7% of these patients had septicemia with the same organism as had been isolated
from bile. Blood culture is comparatively easy to collect for isolation of etiological
agents, but studies have revealed that more than 50% of patients with bactobilia were
negative for blood culture,[23] probably because of lesser bacterial load in the blood of these patients.
There are no published guidelines for antibiotic treatment in bile duct infections.
Therefore, this study helps determine the responsible pathogens and local antibiotic
susceptibility pattern for successful treatment. The data will help in the selection
of empiric antibiotics depending on the individual case. The limitations of the study
include not taking data of antibiotic use before sample collection. As our hospital
is a referral center, most of the patients were already on antibiotics, which can
defer the actual picture of flora and antibiogram.
This study emphasizes on the flora and antibiotic profile of BTIs and enabling the
clinicians to choose appropriate regimes.
In conclusion, the predominant flora was of the gram-negative organisms, exhibiting
a heightened resistance to cephalosporins and carbapenems. As a result, the treatment
of BTIs has become increasingly difficult. Urgent and efficacious treatment is imperative
to mitigate the perils associated with such infections. Consequently, a comprehensive
assessment of bacterial infection incidence and resistance patterns in individuals
with BTI is instrumental in enabling health care professionals to make informed decisions
in selecting the most appropriate antibiotic treatment.
