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ORIGINAL ARTICLE
Year : 2023 | Volume
: 14
| Issue : 1 | Page : 31-36
Bacteriological profile and antibiotic susceptibility patterns of Gram-negative bacilli isolated from lower respiratory tract infections
Divya Atray, S Sheethal
Department of Microbiology, Geetanjali Medical College and Hospital, Udaipur, Rajasthan, India
Correspondence Address:
Dr. S Sheethal
Department of Microbiology, Geetanjali Medical College and Hospital, Hiran Magri Extension, Manwakhera, NH-8 Bypass, Near Eklingpura Chouraha, Udaipur - 313 001, Rajasthan
India
Source of Support: None, Conflict of Interest: None
DOI: 10.4103/injms.injms_93_22
Background: Lower respiratory tract infections (LRTIs) are one of the most frequent infections seen in humans. Bacterial etiology of these infections is one of the major causes of morbidity and mortality. The emergence of drug resistance among bacteria is increasing throughout the world causing more severe infections because of their continuous mutation and multidrug resistance nature. Objectives: This study was focused on obtaining a comprehensive insight into the Gram-negative bacillary profile of LRTIs, their prevalence, and their antibiotic susceptibility patterns. Materials and Methods: The study was conducted for a duration of 6 months. Samples were obtained from patients of all ages and both sexes presenting with symptomatology suggestive of LRTIs. Following conventional culture, the isolated organisms were identified by various preliminary identification methods and biochemical tests. Antimicrobial sensitivity testing of Gram-negative isolates was performed by standard methods as recommended by CLSI 2019. Results: Out of the 1724 LRT specimens evaluated, 307 (17.80%) were culture positive. Our study showed that Gram-negative bacilli are the predominant cause (97.70%) of LRTIs with Klebsiella pneumoniae (42%) as the major pathogen followed by Escherichia coli (31.66%), Pseudomonas aeruginosa (25.33%), and Acinetobacter baumannii (1%). Extended-spectrum beta-lactamase production was detected in 3.33% and metallo-beta-lactamase in 2% of the total GNB pathogens. Conclusion: For effective management of LRTIs, an ultimate and detailed bacteriological diagnosis along with antimicrobial susceptibility testing is required to overcome the global problem of antibiotic resistance.
Keywords: Extended-spectrum beta-lactamase, lower respiratory tract infections, metallo-beta-lactamase, multidrug resistance, respiratory infections
One of the most frequently reported infections in humans is the infection of the respiratory tract.[1] Traditionally, the respiratory tract is divided into the upper respiratory tract and the lower respiratory tract[2] and the infections of these regions are designated simply as respiratory tract infections (RTIs) to generalize them or are called upper RTIs and lower respiratory tract infections (LRTIs), respectively. Of these infections, LRTIs are among the chief infective causes of morbidity and mortality[3] directly resulting in about 7 million deaths annually in the world.[4] LRTI is not a single disease but a group of specific infections such as acute bronchitis, pneumonia, and exacerbations of chronic lung disease each with diverse epidemiology, pathogenesis, symptomatology, and consequence.[3]
Hospitalized patients are particularly at risk for developing LRTIs, particularly those patients in intensive care units in comparison to patients infected in the community. The most common bacterial agents causing LRTIs include the Gram-positive cocci Streptococcus pneumoniae, Gram-negative bacilli (GNB) Haemophilus influenzae, Klebsiella pneumoniae, Escherichia coli, Pseudomonas spp., Acinetobacter spp., and other nonfermenting GNB. Multidrug-resistant (MDR) pathogens such as methicillin-resistant Staphylococcus aureus and MDR Enterobacteriaceae likely cause infections in patients with risk factors such as longer stay in the ICU.[3] Prophylactic management of respiratory infections with multiple antibiotics before the availability of culture reports can be presumed to be the cause of histrionic upsurge in antimicrobial resistance among the respiratory pathogens. This is a matter of global alarm.[5] Hence, the present study was conducted to investigate the various bacterial etiologies of LRTIs in our institution and to update the current antibiotic susceptibility patterns of these pathogens.
Materials and MethodsAll lower respiratory samples received in the laboratory during the study period from patients of all age groups and both sexes attending the outpatient departments (OPDs) and admitted in inpatient departments (IPDs) provisionally diagnosed to be suffering from LRTIs were included. This was a prospective study conducted between November 2019 and April 2020 in the Department of Microbiology.
Bacterial isolates from repeat cultures of previously recruited patients, bacterial isolates identified as commensals or contaminants, and specimens yielding mixed bacterial growth were excluded from the study.
The received samples were cultured on commercially available chocolate agar (CA) and 5% sheep blood agar (BA) along with MacConkey agar (MA) plates prepared using dehydrated media obtained from HiMedia Laboratories Pvt. Ltd., Mumbai, India, according to manufacturer's instructions. The CA and BA plates were incubated in a carbon dioxide (CO2) incubator (10% CO2) at 37°C for 24 h while MA plates were incubated at 37°C for 24 h in an aerobic atmosphere. Bacterial growth was identified using morphological characteristics, Gram staining, and biochemical tests following standard microbiological procedures. Following this, all the isolates were tested for antibiotic susceptibility to a set panel of drugs by modified Kirby–Bauer disc diffusion method in compliance with CLSI 2019 guidelines on Mueller–Hinton agar (MHA) plates. MHA plates were prepared from dehydrated media obtained from HiMedia Laboratories Pvt. Ltd., Mumbai, India, according to the manufacturer's instructions; the antibiotic discs were also commercially obtained from HiMedia Laboratories Pvt. Ltd., Mumbai, India.
Extended-spectrum beta-lactamase (ESBL) enzyme production and metallo-beta-lactamase (MBL) enzyme production in GNB were screened and confirmed as per CLSI guidelines.[6]
All GNB isolates were screened for ESBL enzyme production by Kirby–Bauer disk diffusion method by observing for susceptibility to cefotaxime (30 μg), ceftriaxone (30 μg), and ceftazidime (30 μg) discs. The confirmation of ESBL among screen-positive isolates was done by combined disc test in which ceftazidime (30 μg) alone and ceftazidime (30 μg) in combination with clavulanic acid (10 μg) were used. A zone size of more than 5 mm in the combination disc when compared to the single disc was taken as a confirmed ESBL-producing organismAll GNB isolates were screened for MBL enzyme production by testing their susceptibility to imipenem (10 μg) and meropenem (10 μg) antibiotic discs using Kirby–Bauer disk diffusion method. Resistant isolates were further subjected to phenotypic confirmation of MBL production using imipenem (10 μg) and imipenem (10 μg) + ethylenediaminetetraacetic acid (750 μg) discs. A zone size of more than 7 mm in the combination disc when compared to the single disc was taken as a confirmed MBL-producing organism. ResultsDuring the study period, a total of 1724 samples were collected from patients clinically diagnosed as having LRTIs. The total bacterial isolates were 307 (307/1724, 17.8%), among which 300 (300/307, 97.7%) were GNB, 4 (4/307, 1.3%) were Gram-positive cocci, and 3 (3/307, 0.9%) were identified as belonging to Candida spp. GNB were isolated from a majority of patients from IPD, 90% (270/300) in comparison to only 10% (30/300) from OPD. The culture positivity among the males was higher at 78% (234/300), whereas in females, it was 22% (66/300). The predominant age group was >61 years of age in both the sexes, 42.3% (127/300). The next common age groups to be affected by LRTIs were between 41–50 years (14.6% 44/300) and 31–40 years (12.6% 38/300) of age. The distribution of GNB obtained from various respiratory specimens is depicted in [Table 1].
Among the 300 GNB isolates, Klebsiella pneumoniae were the highest isolated (126/300, 42%), followed by Escherichia coli (95/300, 31.66%), Pseudomonas aeruginosa (76/300, 25.33%), and Acinetobacter baumannii (3/300, 1%), as depicted in [Figure 1]. The antibiotic susceptibility patterns of the isolated GNB and distribution of production of beta-lactamases, i.e., ESBL and MBL produced by them are shown in [Table 2] and [Table 3], respectively.
Figure 1: Distribution of Gram-negative bacterial isolates from lower respiratory tract infections Discussion LRTIs are an important cause of morbidity and mortality among humans affecting all age groups worldwide and demonstrate a range of symptoms and signs, varying in severity. In recent years, there has been a dramatic increase in antibiotic resistance among respiratory pathogens due to various mechanisms. As a consequence, bacterial infections of the lower respiratory tract have become a major cause of death. With emerging drug resistance of organisms to commonly used antibiotics, it is imperative to study their recent trends for effective management of these cases.[7] This study was undertaken to identify the most common GNB responsible for LRTIs, determine their antibiotic susceptibility patterns, and confirm the production of ESBLs and MBLs as a mechanism of resistance in our center.
A total of 1724 respiratory samples were processed in the laboratory during the study duration. Normal flora/no growth was reported in 82.19% of samples and 17.8% grew pathogens on culture. The predominant group of pathogenic organisms isolated was the GNB (17.4%) as supported by studies of Ahmed et al.[8] and Vishwanath et al.[9] who isolated 17.03% and 18.30% of GNB, respectively.
The most common pathogen isolated from patients suffering from LRTIs was Klebsiella pneumoniae (42%). Multiple studies have shown a wide range between 31.1% and 59.7% in which Klebsiella pneumoniae were isolated between 2013 and 2018.[10],[11],[12],[13] This pathogen is not spread through the air but through person-to-person contact (from patient to patient via the contaminated hands of health-care personnel, or other persons) or, less commonly, by contamination of the environment.[14] The other pathogens were Escherichia coli (31.66%), Pseudomonas aeruginosa (25.33%), and Acinetobacter baumannii (1%). Infections caused by E. coli result from: (a) hematogenous dissemination from either the gastrointestinal or urinary tracts and (b) aspiration from the pharynx.[15] On the other hand, infections caused by nonfermenters are commonly considered opportunistic infections and these bacteria are constantly finding new ways to avoid the effects of the antibiotics used to treat the infections they cause. Such pathogens can cause infections in patients who are on ventilators, patients with indwelling devices such as catheters, and patients with wounds from surgery or burns.[16],[17]
The etiological agents of LRTIs and their susceptibility patterns vary from area to area.[10] Hospital antibiograms are mandatory to guide empirical antimicrobial therapy and are an important component of detecting and monitoring trends in antimicrobial resistance. Reliable statistics on antibiotic resistance are mandatory to control resistant pathogens.
Slightly more than half the isolates of K. pneumoniae and E. coli showed good susceptibility to amoxicillin + clavulanic acid. Ahmed et al.'s[8] study reported the susceptibilities of these Enterobacteriaceae as low as 8.3% and 31.2%, respectively. Similar findings were also reported in the study of Goel et al.[18] in which K. pneumoniae was 100% resistant to this drug and E. coli showed 91.7% resistance. Testing for this drug was not performed for the nonfermenters as this drug is intrinsically resistant in them. Another combination drug ampicillin + sulbactam also showed good susceptibility in about half of the isolates. These findings of ours were similar to Bajpai et al.[19] who also reported sensitivity to this drug for K. pneumoniae and E. coli at 53.4% and 47.7%, respectively. Although P. aeruginosa is intrinsically resistant to this drug combination as well, A. baumannii is not. However, the A. baumannii isolates of our study showed resistance to this combination drug. The number of isolates was so small that this conclusion is not conclusive. The preferred beta-lactam + beta-lactam inhibitor combination preferred for treating Gram-negative bacteria is piperacillin + tazobactam. The sensitivity pattern to this combination revealed that all the four GNB groups isolated are highly sensitive. This highly misused drug in many centers has started showing resistance. One example is the study conducted by Ahmed et al.[8] who noted a sensitivity of only 16.7% for A. baumannii, 3.6% for E. coli, 18.2% for K. pneumoniae, and 1.9% for P. aeruginosa.
A higher resistance percentage for third generation than fourth generation of cephalosporins was observed. In a comparable study by Regha and Sulekha,[5] the GNB isolates showed high resistance to all generations of cephalosporins. These findings of our study can be directly extrapolated to mean that many of these isolates on the basis of the resistance shown to cephalosporins were considered screen positive for ESBL production. Treating GNB-caused LRTIs with cephalosporins empirically is not smart anymore with the emerging resistance patterns.
Generally, sensitivity to amikacin was favorable in our study for E. coli, K. pneumoniae, and P. aeruginosa. However, for A. baumannii, since the number of isolates was only three, it showed a common resistance pattern at 66.6%. There are two reports on similar ends of sensitivity towards amikacin. Saha et al.[13] observed 65.78% sensitivity among MDR isolates. On the other hand, Bajpai et al.[19] demonstrated 28.95% of resistance. Both rates are alarming. The causes behind the emergence of such organisms have been a matter of speculation.[19] Antipseudomonal aminoglycosides netilmicin and tobramycin were the most susceptible in our tested group of drugs. However, other studies by Bajpai et al.[19] and Goel et al.[18] have shown resistance even for these drugs.
Levofloxacin demonstrated a higher sensitivity percentage in comparison to ciprofloxacin except for P. aeruginosa where ciprofloxacin was found to have a comparatively higher sensitivity. Goel et al.[18] found higher sensitivity to ciprofloxacin for majority of isolates of E. coli, K. pneumoniae, and A. baumannii which showed 100%, 95.5%, and 89.7% sensitivity, respectively. Levofloxacin is a better drug in treating LRTIs than ciprofloxacin because being an oral drug with good compliance it is well absorbed, with moderate to excellent bioavailability.[20]
In the treatment of LRTIs, trimethoprim is the active component and its combination with sulfamethoxazole was postulated to decrease the emergence of resistance. Yet, bacterial resistance has steadily increased due to the widespread use of co-trimoxazole against most serious infections.[21] Many isolates of our study and isolates in the study by Elumalai et al.[4] echo these patterns, K. pneumoniae (64.5%), E. coli (88.2%), and A. baumannii (89.2%).
Carbapenems are frequently used as a last choice in treating serious infections such as LRTIs caused by GNB.[19] On those lines, carbapenems showed a higher sensitivity for all the isolates in the studies conducted by Bajpai et al.,[19] Regha and Sulekha,[5] and Goel et al.[18] In the study of Regha and Sulekha,[5] imipenem was the most active antibiotic against the GNBs. Similarly, our study also recorded good sensitivity zones to the carbapenem drugs imipenem and meropenem.
Almost three-quarters of all antibiotic consumptions are for RTIs.[22] Beta-lactams remain a cornerstone for antimicrobial chemotherapy of a large number of bacterial infections, but their efficacy has been increasingly opposed by dissemination of acquired resistance determinants among pathogenic bacteria. (Rossolini et al. presented in the 2005 15th European Congress of Clinical Microbiology and Infectious Diseases held in Copenhagen, Denmark, titled "Metallo-beta-lactamases: a last frontier for beta-lactams?") Nonfermenters are innately resistant to many antibiotics and are known to produce extended-spectrum β-lactamases and metallo-β-lactamases.[23] ESBL production was confirmed in 24.66% of isolates and MBL production in 14.33% of isolates.
Although the number of isolates of K. pneumoniae in our study was the highest, ESBL production was higher in E. coli, 8.42% (8 out of 95), than in K. pneumoniae, 1.5% (2 out of 126). In the study of Vishwanath et al.,[9] ESBL production in E. coli and K. pneumoniae was 70.6% and 65.9%, respectively, which was higher as compared to our study. Confirmed MBL production was seen among E. coli, 2.10% (2 out of 95); P. aeruginosa, 3.94% (3 out of 76); and K. pneumoniae, 0.79% (1 out of 126) isolates. Mishra et al.[2] and Pokhrel et al.[24] reported 0%, 3.3%, 0% and 0%, 2.4%, 4.2% of MBL-producing E. coli, P. aeruginosa, and K. pneumoniae, respectively.
The overall prevalence of ESBL and MBL among GNB in our study was 3.33% and 2%, respectively. ESBL production was 33% in the study carried out by Amutha et al.[25] and MBL production was detected to be 3.2%. ESBL production in their study was higher than observed in our study.
The treatment of infections caused by ESBL-producing organisms is carbapenems. However, carbapenem-resistant Enterobacteriaceae (CRE) have been increasingly reported worldwide.[26] Isolates which are CRE are often MDR and spread them to other groups of bacteria including the nonfermenters. Hence, colistin was re-introduced due to lack of discovery of new antibiotics. Colistin (polymyxin E) and polymyxin B are considered to be the most active in vitro agents against CRE.[3],[27] In our study, all the isolates exhibited 100% susceptibility toward colistin/polymyxin B. Being surrogate markers, susceptibility to one can be applied to the other. Minocycline, a tetracycline is another such broad-spectrum antibiotic effective against clinically resistant important pathogens, has excellent pharmacokinetic properties, and limited toxicity when used in serious infections.[28],[29] Our study showed 100% susceptibility to minocycline in all four isolate groups. Tigecycline is a glycylcycline antibiotic which has in vitro activity against Gram-negative bacteria including drug-resistant bacteria.[3] As Pseudomonas aeruginosa is intrinsically resistant to tigecycline, this antibiotic was not tested in those isolates but was tested in the other three organisms which showed 100% susceptibility.
Rapid diagnosis of the causative agents of RTIs is crucial in reducing morbidity and avoiding excessive and inappropriate antibiotic use which promotes the development of antimicrobial resistance.[30] With the findings of this study, we emphasize the importance of identifying the bacterial profile of LRTIs and determine the antimicrobial resistance pattern of the etiological agents. This will not only guide the clinician in administering appropriate antibiotic therapy to the patients but also help monitor the changing trends of these infections.[25]
ConclusionThis study has revealed GNB as major pathogens causing LRTIs with Klebsiella pneumoniae as the predominant pathogen followed by Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter baumannii. Antibiotic resistance among lower respiratory bacterial pathogens is alarming although ESBL and MBL production was reported at only 3.33% and 2%, respectively. While empirical therapy can be started with piperacillin + tazobactam or fourth-generation cephalosporins, treatment should be changed as per the culture and sensitivity reports procured from the microbiology laboratory after testing for resistance mechanisms in GNB. The last line of treatment for MDR pathogens resistant to carbapenems by mechanism of MBL production is colistin, tigecycline, and minocycline. If care is not taken to reduce MDRs and practice judicious antibiotic usage we may end up in a preantibiotic era once more.
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