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 Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 7  |  Issue : 1  |  Page : 56-60

Antimicrobial resistance pattern of bacterial isolates and genetic resistance determinants of carbapenemase producers in bloodstream infections


Department of Microbiology, Institute of Medical Sciences and SUM Hospital, Bhubaneswar, Odisha, India

Date of Submission23-Feb-2019
Date of Decision22-Nov-2019
Date of Acceptance20-Jan-2020
Date of Web Publication19-Jun-2020

Correspondence Address:
Swati Jain
142, Doctors Enclave, Campus.3, Institute of Medical Sciences and SUM Hospital, Kalinga Nagar, Bhubaneswar - 751 003, Odisha
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/cjhr.cjhr_22_19

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  Abstract 


Introduction: Bloodstream infection (BSI) continues to be a significant cause of disease and death in hospitalized patients worldwide. These are among the most common healthcare-associated infections with a mortality rate of 20%–50%. The emergence of multidrug resistance among the organisms causing BSI is of great concern. Objectives: The study was undertaken in a medical intensive care unit of a tertiary care hospital in Eastern India to evaluate the spectrum of pathogens causing BSI, their antimicrobial resistance patterns and carbapenemase enzyme production using polymerase chain reaction (PCR). Materials and Methods: A total of 550 blood samples from clinically suspected cases of BSIs were studied from July 2016 to June 2018. Blood samples were inoculated and incubated in BacT/ALERT (BioMerieux) system. Identification and antibiotic susceptibility testing was conducted in Vitek-2 (BioMerieux) as per the Clinical Laboratory Standards Institute guidelines. Methicillin-resistant Staphylococcus aureus (MRSA) and β-lactamase production were also noted in the Vitek-2 method. All Gram-negative isolates were studied for carbapenemase production by the genotypic method using PCR. Results: Of 550 samples, 116 samples yielded the growth of various bacterial isolates. Of these, 54 (47%) were Gram-positive organisms and 62 (53%) were Gram-negative organisms. S. aureus was the most common organism isolated followed by Klebsiella pneumoniae. MRSA was observed in 66.7% of S. aureus isolates. Among Gram-negative isolates, 43.5% were found to be β-lactamase producers and 66.1% were carbapenemase producers. Most of the carbapenemase producers were encoded by the OXA gene (58%). Conclusion: This study emphasizes the importance of antibiotic policy and its stringent application, which will eventually help us to control the menacing rise in antimicrobial resistance.

Keywords: Bloodstream infection, carbapenemase, methicillin-resistant Staphylococcus aureus, OXA gene, β-lactamase


How to cite this article:
Jena L, Swain B, Jain S. Antimicrobial resistance pattern of bacterial isolates and genetic resistance determinants of carbapenemase producers in bloodstream infections. CHRISMED J Health Res 2020;7:56-60

How to cite this URL:
Jena L, Swain B, Jain S. Antimicrobial resistance pattern of bacterial isolates and genetic resistance determinants of carbapenemase producers in bloodstream infections. CHRISMED J Health Res [serial online] 2020 [cited 2020 Jul 16];7:56-60. Available from: http://www.cjhr.org/text.asp?2020/7/1/56/286883




  Introduction Top


Bloodstream infection (BSI) continues to be a significant cause of disease and death in hospitalized patients worldwide despite advances in therapy and supportive care.[1] They are among the most common healthcare-associated infections with a mortality rate of 20%–50%.[2] Multiple invasive therapeutic and diagnostic procedures in intensive care units (ICUs) that are carried out on an extremely vulnerable group of the population who have an increased risk of becoming infected makes them the epicenter of these infections. Physiologic stress of infections accompanied by the increasingly added burden of multidrug-resistant organisms (MDROs) hinders therapy of these infections, with consequential adverse clinical and economic outcomes.[3] The greatest threat is to treat infections caused by β-lactamase producing Gram-negative bacteria. Among the β-lactamases, the most feared are the carbapenemases, especially metallobeta-lactamases because of their ability to hydrolyze all β-lactams, including the carbapenems. These enzymes are often associated with laboratory reports of false susceptibility to carbapenems as their presence does not always produce a resistant phenotype on conventional disc diffusion method or automated susceptibility testing methods.[4] Hence, special tests are required to detect the resistance mechanisms involved as the isolate may be reported falsely susceptible while harboring carbapenemase enzyme, resulting in treatment failure and dissemination of the resistant isolates.[5] Therefore, genotypic detection of carbapenemase genes is the gold standard, although it only detects a prespecified set of known genes. In the ICU, the causative agents of BSIs implicated are Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterococcus faecalis, Acinetobacter baumannii, and Candida spp.[6],[7] Hence, knowledge of local pathogens and susceptibility patterns is essential to start prompt and appropriate empirical therapy and also to formulate and update antibiotic policy.


  Materials and Methods Top


The present study was conducted prospectively in the department of Microbiology, IMS and SUM Hospital, over a period of 2 years extending from July 2016 to June 2018 with approval from the hospital ethical committee. A total of 550 venous blood samples from adult patients were collected following all the aseptic precautions and before administration of antibiotics from the clinically suspected cases of septicemia admitted in medical ICU (MICU). The blood was inoculated into the BacT/ALERT (BioMerieux) blood culture bottles and was incubated at 37°C in BacT/ALERT 3D system, fully automated blood culture system for the detection of aerobic growth in blood samples. The blood samples were incubated for a maximum period of 7 days, and if there was no growth, the result was read as negative. Culture bottles which were flagged positive in the system were subcultured on blood agar and MacConkey agar medium. The identification of growth on the plates was done based on colony morphology and Gram stain. Based on Gram reactivity, 0.5% McFarland suspension from the colonies was prepared which was then subjected to identification and antimicrobial susceptibility testing on VITEK 2 (BioMerieux) as per the Clinical Laboratory Standards Institute guidelines and manufacturer's instructions. Methicillin-resistant S. aureus (MRSA) and β-lactamase production were also noted in the Vitek-2 method in addition to the screening by conventional disc diffusion methods. All the Gram-negative bacilli isolates were studied for carbapenemase production by the genotypic method using polymerase chain reaction (PCR). Genomic DNA of each bacterial isolate was extracted using the HipurA™ Bacterial Genomic DNA purification kit (Himedia) according to the manufacturer's instruction. Predesigned primers with specific oligonucleotide sequences were used to detect the resistance genes for the study, i.e., blaIMP, blaNDM, blaOXA, blaKPC, and blaVIM as reported earlier by Poirel et al. were used. After amplification by PCR, the amplicons were detected by the development of specific bands on gel electrophoresis [Table 1] and [Figure 1].[8]
Table 1: List of primers for the detection of bla IMP, bla VIM, bla KPC, bla OXA, and bla NDM genes

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Figure 1: Banding pattern of NDM, OXA, IMP, and VIM genes through agarose gel electrophoresis (lane-M: 100 bp ladder, Himedia, Mumbai, India) (a) NDM gene: Presence of 621 bp band indicates resistance genes. Presence of band seen in lane 5, 6, 14, 16, and 17 (b) OXA gene: Presence of 438 bp band indicates resistant genes. Presence of band seen in lane 3, 4, 6, 8, and 9 (c) IMP gene: Presence of 232 bp band indicates resistant genes. Presence of band seen in lane 8, 9, 10, 14, 15, 18, and 19 (d) VIM gene: Presence of 390 bp band indicates resistant genes. Presence of band seen in lane 1, 2, 8, 16, and 18

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Due to nonavailabilty of known strains with specific resistance genes, we have only utilized few known extended-spectrum β-lactamases (ESBL)-negative E. coli and K. pneumoniae as negative controls.


  Results Top


A total of 550 clinically suspected cases of BSI admitted in MICU over a period of 2 years were included in the study.

Of 550 samples, 134 were flagged positively in BacT/ALERT automated blood culture method. Of 134 samples, 116 samples (86.5%) yielded growth of bacteria, 7 samples (5.25%) showed fungal growth and 11 samples (8.25%) did not grow on the culture plate. Among the positive bacterial culture (116), maximum number of patients were in the age group of >50 years (70%) and 64% isolates were recovered from male patients. Most of the cases (75%) were associated with comorbid conditions such as diabetes, hypertension, chronic obstructive pulmonary disease, chronic kidney disease, and malignancy.

Of 116 bacterial isolates recovered, 54 (47%) were Gram-positive organisms and 62 (53%) were Gram-negative organisms. Among Gram-positive organisms, S. aureus (24/54, 44.5%) was the most common organism isolated followed by Coagulase-negative Staphylococcus (16/54, 29.6%) [Table 2].
Table 2: Distribution of Gram-positive isolates (n=54)

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Among Gram-negative organisms, K. pneumoniae (18/62, 29%) was the frequently isolated organism followed by E. coli (14/62, 22.6%) [Table 3].
Table 3: Distribution of Gram-negative isolates (n=62)

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Of 62 Gram-negative bacteria, 37 isolates belonged to the family Enterobacteriaceae and 25 were nonfermenting Gram-negative bacteria.

Gram-positive organisms showed the following susceptibility pattern (MIC by Vitek-2): Linezolid (87%), vancomycin (81.5%), tigecycline (70%), gentamicin (42.6%), amikacin, piperacillin-tazobactam and teicoplanin (37%), netilmicin (31.5%), ofloxacin and imipenem (27.8%), levofloxacin (24%), lincomycin (18.5%), meropenem, cefoperazone-sulbactam and cefepime-tazobactam (14.8%), ampicillin (13%) and clarithromycin (11%). From 24 S. aureus isolates, 16 (66.7%) were MRSA.

Gram-negative organisms belonging to the family Enterobacteriaceae showed the following susceptibility pattern: Colistin (75.7%), tigecycline and meropenem (48.6%), amikacin (43.2%), imipenem, cefoperazone-sulbactam and piperacillin-tazobactam (40.5%), gentamicin (27%), cotrimoxazole (21.6%), ciprofloxacin (19%), amoxicillin-clavulanic acid (16.2%), netilmicin (13.5%), tobramicin (10.8%), cefuroxime (8.1%), cefixime (5.4%), and levofloxacin and ofloxacin (2.7%).

Nonfermenting Gram-negative bacteria showed the following susceptibility pattern: Colistin and meropenem (40%), levofloxacin (32%), ceftazidime and tigecycline (28%), doripenem (20%), imipenem (16%), amikacin (12%), cefoperazone-sulbactam and ofloxacin (8%), and amoxicillin-clavulanic acid (4%).

From 62 Gram-negative isolates, 27 (43.5%) were found to be β-lactamase producers. 2 of 2 (100%) Citrobacter, 12 of 14 (85.7%) E. coli and 13 of 18 (72.2%) Klebsiella were β-lactamase producers.

Of 62 Gram-negative isolates, 41 (66.1%) were found to be carbapenemase producers by the genotypic method [Figure 2].
Figure 2: Distribution of carbapenemase-producing isolates

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Most of the carbapenemase producers were encoded by the OXA gene (58%) followed by the NDM gene (43.3%), IMP gene (22.6%), and VIM gene (17.7%). No KPC genes were detected in our study [Table 4].
Table 4: Distribution of carbapenemase genes in Gram-negative isolates

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The carbapenemase-producing strains were mostly susceptible to colistin (53%) followed by amikacin (26.8%) and tigecycline (21.9%).

The mortality rate was found to be 38% (44 of 116 cases), 51 of 116 cases (44%) got recovered, and 21 cases (18%) left against medical advice.


  Discussion Top


BSIs are a major cause of morbidity and mortality among patients in ICUs. The cause of infection in ICU is multifactorial, and consequences depend on pathogens associated, source of infection in ICU, underlying risk factors, timely intervention, and appropriate treatment received.

In the present study, majority of the patients admitted in MICU with clinical signs of sepsis were in the age group of >50 years. This result is well correlated with the study of Prowle et al. and van Gestel et al.[9],[10] This can be due to the vulnerability of the extremes of the age groups.

In this study, the prevalence rate of BSI was found to be 21% (116 from 550 cases), which is comparable to other studies done in India.[11],[12] In contrast, the high prevalence rate of 42% was reported by Ramana et al.[13] Prevalence rate variation may be due to factors such as geographic allocations, patient type, timing, and number of blood cultures or differences in the blood culture system.

Although our study showed the predominance of Gram-negative bacteria (53%) which is well in accordance with a study by Vijaya Devi et al.,[14] the most common organism isolated was S. aureus (24/116) followed by K. pneumoniae (18/116), CoNS (16/116), and E. coli (14/116). Other organisms isolated were A. baumannii, Enterococcus species, P. aeruginosa, Burkholderia cepacia, Streptococcus species, Citrobacter freundii, Enterobacter aerogenes, P. mirabilis, and Serratia marcescens. The similar distribution of microorganisms was noted by Sultana et al.[15] Although CoNS can be a skin colonizer, it is now a well-described pathogen associated with central venous lines.

Our study showed the susceptibility of Gram-positive organisms to be the most toward linezolid (87%), vancomycin (81.5%), tigecycline (70%) and the least toward meropenem, cefoperazone-sulbactam and cefepime-tazobactam (14.8%), ampicillin (13%), and clarithromycin (11%). Similar findings were also reported in a study conducted by Orsini et al.[1]

Gram-negative isolates of our study were most susceptible to colistin and tigecycline and were least susceptible to levofloxacin, ofloxacin, cefuroxime, cefixime, and amoxicillin-clavulanic acid. Similar to our study, Fayyaz et al. have also reported higher resistance to third-generation cephalosporins and quinolones.[16]

In our study, 16 of 24 S. aureus isolates were MRSA (66.7%), which is in concordance to the findings of a study done in Maharashtra.[11]

In the present study 27 of 62 Gram-negative isolates were found to be β-lactamase producers. Twelve of 14 (85.7%) E. coli, 13 of 18 (72.2%) K. pneumoniae, and 2 of 2 (100%) Citrobacter were β-lactamase producers. A similar study carried out by Agrawal et al. showed E. coli as the most common isolate (50.7%) producing β-lactamase.[17]

In our study, 41 of 62 Gram-negative isolates were found to be carbapenemase producers. Eleven of 12 (91.6%) A. baumannii, 10 of 14 (71%) E. coli, 5 of 7 (71%) Pseudomonas, 9 of 18 (50%) K. pneumoniae, 4 of 6 (66.4%) Burkholderia and 1 of 1 (100%) Proteus were carbapenemase producers. Among the carbapenemase producers, OXA gene was the most prevalent one (58%) followed by the NDM gene (43.3%), IMP gene (22.6%) and VIM gene (17.7%). No KPC genes were detected in our study. Similar finding showing OXA gene to be the most common carbapenemase-producing gene was reported by Jeong et al.[18] In contrast, Dwivedi et al. reported 42.8% as the prevalence rate of carbapenamase production among Gram-negative organisms causing BSI and IMP (58.3%) to be the most prevalent gene for MBL producers.[19]

The limitation of our study is that we have not used positive controls due to the nonavailability of well-characterized strains; though, we have utilized few known ESBL-negative E. coli and K. pneumoniae as negative controls.


  Conclusion Top


This study has given us the knowledge about BSI and its incidence in MICU of our hospital; the most common organisms causing BSI and their antibiotic susceptibility pattern. Staphylococcus was the most common organism isolated followed by K. pneumoniae. Gram-positive organisms were most susceptible to vancomycin, linezolid, and tigecycline. Gram-negative organisms were most susceptible to colistin and tigecycline. The presence of MDROs in our study calls for intensive infection control practices and continuous antibiotic surveillance. Formulation of hospital antibiotic policy and its strict implementation is the need of the hour. It will certainly help the health-care providers to control the rising antimicrobial resistance, which is a serious problem these days.

Acknowledgment

We are grateful to S“O”A University for their constant support and encouragement.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Sultana Q, Ansari H, Ansari WM. Bacteriological profile and antimicrobial susceptibility patterns of organisms responsible for blood stream infections. Indian J Microbiol Res 2016;3:113-7.  Back to cited text no. 15
    
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