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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 17  |  Issue : 1  |  Page : 43-46

Bacteriological profile of gram-negative organisms from cases of septicemia and their antibiotic sensitivity pattern with special reference to beta-lactamase and carbapenemase production


1 Department of Microbiology, JNMC, Wardha, Maharashtra, India
2 Department of Second Year MBBS Student, JNMC, Wardha, Maharashtra, India

Date of Submission01-Oct-2021
Date of Decision06-Nov-2021
Date of Acceptance25-Dec-2021
Date of Web Publication25-Jul-2022

Correspondence Address:
Dr. Shital Moreshwarrao Mahajan
Department of Microbiology, JNMC, Sawangi, Wardha, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jdmimsu.jdmimsu_359_21

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  Abstract 


Background: Blood stream infections are amongst the most important causes of morbidity and mortality throughout the world. Septicemia is a serious bloodstream infection and a biggest challenge for the clinicians in selection of appropriate antimicrobial agents. Presence of intravascular catheters, immunosuppression and use of cytotoxic drugs are some precipitating factors for antibiotic resistance which is a global problem. Reports of multi-resistant bacteria causing septicemia in developing countries are increasing. Extended-spectrum beta-lactamases (ESBL) and Carbapenemase-producing Enterobacteriaceae (CRE) has caused a major public health concern. Aim and Objectives: To determine the bacteriological profile of septicemia and their antibiotic susceptibility profile with special references to β-lactamase and carbapemase productionin gram negative bacilli. Material and Method: During the 3 month period of study total 100 blood samples from suspected cases of septicaemia are collected. Isolates were processed and identified by standard protocols. Detection of extended spectrum β-lactamase (ESBL), inducible AmpC and MBL (Metallo β-lactamases) was done by using screening test and then confirmatory tests. Results: Out of 100 samples 68 gram negative organisms were isolated. Maximum growth was of Pseudomonas aeruginosa i.e. 34 , followed by Klebsiella pnemoniae i.e. 19 and Acinetobacter sps and E.coli. From all isolates 4.4% were ESBL, Metallo –beta-lactamases (MBL) production were seen in 5.9 %. 40% and 10.5% were MBL-E.coli and MBL-Klebseilla pnuemoniae respectively. Conclusion: Many of the isolates from cases of septicaemia were resistant to most of the recently used antibiotics. So, Detection of the etiological agents and their antibiotic sensitivity will definitely help in effective prophylactic measures, prompt and accurate diagnosis of septicaemia. Simple methods like disc potentiation test for ESBL, disk antagonism test for inducible AmpC producers and Combined disk potentiation test for MBL production are relatively inexpensive and less cumbersome, so it can also be performed as a part of routine sensitivity testing. High degree of antibiotic resistance is an alarming sign for development of antibiotic policies and protocols for treatment of septicaemia.

Keywords: Extended-spectrum beta-lactamase, Gram-negative bacilli, inducible AmpC, metallo beta-lactamases


How to cite this article:
Mahajan SM, Khushwah S. Bacteriological profile of gram-negative organisms from cases of septicemia and their antibiotic sensitivity pattern with special reference to beta-lactamase and carbapenemase production. J Datta Meghe Inst Med Sci Univ 2022;17:43-6

How to cite this URL:
Mahajan SM, Khushwah S. Bacteriological profile of gram-negative organisms from cases of septicemia and their antibiotic sensitivity pattern with special reference to beta-lactamase and carbapenemase production. J Datta Meghe Inst Med Sci Univ [serial online] 2022 [cited 2022 Aug 16];17:43-6. Available from: http://www.journaldmims.com/text.asp?2022/17/1/43/352235




  Introduction Top


Bloodstream infections are one of the most important causes of morbidity and mortality worldwide.[1] Continuous or transient presence of microorganisms within the bloodstream is known as bacteremia, while its dissemination throughout the body with evidence of systemic responses toward microorganisms with variable severity is septicemia.[2]

Septicemia is a serious bloodstream infection and a biggest challenge for clinicians. Many organisms such as  Escherichia More Details coli, Pseudomonas aeruginosa, Klebsiella species,  Neisseria More Details meningitidis, Haemophilus influenzae, and Gram-positive such as coagulase-negative staphylococci, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, and Enterococcus faecium can cause septicemia.[1]

Many precipitating factors such as presence of intravascular catheters, immunosuppression, and use of cytotoxic drugs are associated with cases of septicemia, especially in patients from intensive care unit setting.[2] Identification of the offending pathogen by bacteriological cultures and detection of sensitivity pattern are the mainstay of diagnosis and treatment of bloodstream infections.[2] Antibiotic resistance is a global concern. Reports of multiresistant bacteria causing septicemia in developing countries are increasing.[3] Extended-spectrum beta-lactamases (ESBLs) and carbapenemase-producing Enterobacteriaceae have caused a major public health concern.[4] Gram-negative bacilli-producing carbapenemase can cause serious outbreaks of nosocomial infections.[4] The wide availability of over-the-counter antibiotics and the inappropriate use of broad-spectrum antibiotics in the community may explain this situation.[3]

Microorganisms implicated in septicemia have developed increased drug resistance to commonly used antibiotics, thus making treatment extremely difficult.[5] Thus, the knowledge of both the common pathogens causing septicemia and their antimicrobial susceptibility is essential to select appropriate antimicrobial treatment. However, antimicrobial susceptibility patterns of the organisms may vary geographically and temporally, depending on local pathogens and the commonly used antibiotics.[6]

Hence, the present study was conducted to document the bacteriological profile of septicemia in adults as well as in neonates and their antibiotic susceptibility profile for planning strategy for the management of septicemia.


  Material and Methods Top


The present study was carried out in the department of microbiology at a tertiary care hospital from June 2019 to August 2019. A total of 100 blood samples were collected with all aseptic precaution and proceed for blood culture.[7] The isolates from the blood sample were identified by standard procedures from blood agar and MacConkey agar after incubation at 37°C for 18–24 h.[8] Cultures were labeled as negative if there was no growth after 1 week of incubation. Antimicrobial susceptibility of all bacterial isolates was carried out by Kirby–Bauer disk diffusion technique using Muller-Hinton agar (MHA) poured to a depth of 4 mm in a flat-bottomed 9 cm  Petri dish More Details as per the CLSI 2019 guidelines.[9]

Inoculum

The inoculum was prepared from the primary culture plate, by touching the tops of 3–5 colonies and suspended in peptone water. The turbidity was adjusted to 0.5 McFarland standards.[9]

The methods of antibiotic resistance detection are as follows:

Detection of extended-spectrum beta-lactamase

ESBL production was tested in all the isolates by initial screening test and then phenotypic confirmatory test (PCT) as per the CLSI 2019 guidelines.[9]

Initial screening test

This test was performed on MHA by disk diffusion method using ceftazidime (30 μg) disk. The lawn culture of 0.5 McFarland inoculum of the test strain will be exposed to a disk of ceftazidime. After incubation at 37°C for 16–18 h, the zone diameter ≤22 mm indicates ESBL production.[9]

Phenotypic confirmatory test

The lawn culture of 0.5 McFarland inoculum of the test strain will be done on MHA. Disks of ceftazidime (30 μg) and ceftazidime-clavulanic acid (30/10 μg) were used. After incubation at 37°C for 16–18 h, an increase of ≥5 mm in a zone diameter of ceftazidime-clavulanic acid versus its zone when tested alone confirms ESBL production.[9]

AmpC production

Screening test

It was done by using cefoxitin disk (30 μg) and the zone diameter ≤18 mm was suspected as AmpC producer.[10]

Ceftazidime-imipenem antagonism test

Ceftazidime (30 μg) and imipenem (10 μg) disks were placed 20 mm apart for the detection of inducible AmpC. Blunting of ceftazidime zone of inhibition adjacent to imipenem disk is confirmed for inducible AmpC production.[11]

Carbapenemase and metallo beta-lactamase production

Initial screening test

Lawn culture of 0.5 McFarland inoculum of the test strain was exposed to disk of imipenem (10 μg) and zone diameter around 16–21 mm indicates carbapenemase production.[9]

Combined disk test (disk potentiation test)

This test was for the detection of MBL production. Disk of imipenem (10 μg) and imipenem-EDTA (10/750 μg) was used. The difference of ≥7 mm in the zones of two disks indicated MBL production.[12]


  Observations and Results Top


A total of 100 blood samples from suspected cases of septicemia were collected and processed in the present study.

[Table 1] shows that males (55%) were more affected than females (45%). Furthermore, from [Table 1], it is observed that maximum cases were reported from age group of <1 year, i.e., 27%. Out of 100 samples, 68 Gram-negative organisms were isolated. [Table 2] shows that maximum growth was of P. aeruginosa, i.e., 34, followed by Klebsiella pneumoniae, i.e., 19, and Acinetobacter sp. and E. coli.
Table 1: Age- and gender-wise distribution (n=100)

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Table 2: Distribution of organisms in septicemia (n=100)

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[Table 3] shows antibiotic sensitivity testing in various Gram-negative organisms. In case of P. aeruginosa, out of all strains, 50% were sensitive to Imipenem, 17% to colistin, 20% to ceftazidime, 64% to ciprofloxacin, 14% to cotrimoxazole, 79% to piperacillin-tazobactam, 50% to piperacillin, and 8% to gentamicin. All strains were resistant to tetracycline. In case of K. pneumoniae, 10% were sensitive to Imipenem, 10% to ceftazidime, 42% to ciprofloxacin, and 31% to cotrimoxazole. In case of E. coli, all strains were resistant to Imipenem, piperacillin-tazobactam, piperacillin, and tetracycline and 20% were sensitive to ceftazidime and ciprofloxacin.
Table 3: Antibiotic sensitivity pattern in Gram-negative organisms

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[Table 4] shows that 4.4% from all Gram-negative organisms show ESBL production. Among these all strains, 15.8% were K. pneumoniae. MBL production was seen in 5.9%. 40% and 10.5% were MBL–E. coli and MBL–K. pneumoniae, respectively. Inducible AmpC production was 17.6% mainly detected in P. aeruginosa, i.e., 35.3%.
Table 4: β-lactamases production in Gram-negative organisms (n=68)

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  Discussion Top


Maximum cases were reported in males (55%) compared to females (45%) as shown in [Table 1]. Maximum isolates were from the age group of <1 year, i.e., 27%, followed by 11–20 years and 21–30 years; P value is significant. Male:female ratio is 1.2:1 which is similar to Ansari et al.,[6] i.e., 61.4% were males and 38.6% were females and male-to-female ratio is 1.59:1.

[Table 2] shows that in Gram-negative organisms, 34% were P. aeruginosa, 19% were K. pneumoniae, 10% were Acinetobacter sp., and 5% were E. coli, which is similar to Dagnew et al.[1] In the present study, from all isolates, 10% were Acinetobacter sp., which is similar to De et al.[13] in 2013 that was 9.18%.

[Table 3] shows that various organisms are sensitive to various antibiotics. In case of P. aeruginosa, out of all strains, 50% were sensitive to IPM, 17% to colistin, 20% to ceftazidime, 64% to ciprofloxacin, 14% to cotrimoxazole, 79% to piperacillin-tazobactam, 50% to piperacillin, and 8% to gentamicin. All strains were resistant to tetracycline. In case of K. pneumoniae, 10% were sensitive to IPM, 10% to ceftazidime, 42% to ciprofloxacin, and 31% to cotrimoxazole. In case of E. coli, all strains were resistant to IPM, piperacillin-tazobactam, piperacillin, and tetracycline and 20% were sensitive to ceftazidime and ciprofloxacin.

Galhotra et al.[14] reported that Gram-negative isolates showed resistance to ciprofloxacin, cephalosporin, cotrimoxazole, and aminoglycosides, but all were sensitive to carbapenem. Alqasim et al.[15] in 2018 observed that among all antibiotics tested in this study, imipenem was the most active agent as all E. coli isolates were imipenem-susceptible. Out of the 100 E. coli isolates, 92% were resistant to ampicillin, 55% to amoxicillin-clavulanic acid, and 12% to gentamicin. The resistance rates for ceftazidime, cefoxitin, tetracycline, and trimethoprim-sulfamethoxazole were 29%, 13%, 49%, and 54%, respectively.

[Table 4] shows that ESBL production was detected in 4.4% of Gram-negative organisms mainly in K. pneumoniae, i.e., 15.8%, which is similar to the study by McDanel et al.[16] in 2017, i.e., 28%. MBL production was detected in 5.9%; among them, 40% were E. coli and 10.5% were K. pneumoniae. From all isolates of Gram-negative organisms, mainly P. aeruginosa (35.3%) were inducible AmpC producers, i.e., carbapenemase producer, which is similar to the results of Ejikeugwu. et al.[17]


  Conclusion Top


In the present study, various causative agents of septicemia in adults and neonates include Acinetobacter sp., E. coli, K. pneumoniae, and P. aeruginosa. Resistance to commonly used antibiotics is mainly reported and it may be because of random use of antibiotics. Detection of the etiological agents and their antibiotic sensitivity will help clinicians in effective prophylactic measures, prompt and accurate diagnoses, and subsequent administration of targeted therapy to curb the excessive burden of the disease. Hence, it is useful to collaborate all the cost-effective methods for the detection of antibiotic resistance in commonly isolated organisms with routine antibiotic sensitivity testing for proper management of septicemia.

Acknowledgment

I am thankful to all the technical staff of the department of microbiology.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Dagnew M, Yismaw G, Gizachew M, Gadisa A, Abebe T, Tadesse T, et al. Bacterial profile and antimicrobial susceptibility pattern in septicemia suspected patients attending Gondar University Hospital, Northwest Ethiopia. BMC Res Notes 2013;6:283.  Back to cited text no. 1
    
2.
Mohanty A, Singh TS, Kabi A, Gupta P, Gupta P, Kumar P. Bacteriological profile and antibiotic sensitivity pattern of hospital acquired septicemia in a tertiary care hospital in northeast India. Asian J Pharm Clin Res 2017;10:186-9.  Back to cited text no. 2
    
3.
Jyothi P, Basavaraj MC, Basavaraj PV. Bacteriological profile of neonatal septicemia and antibiotic susceptibility pattern of the isolates. J Nat Sci Biol Med 2013;4:306-9.  Back to cited text no. 3
    
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Carmeli Y, Akova M, Cornaglia G, Daikos GL, Garau J, Harbarth S, et al. Controlling the spread of carbapenemase-producing Gram-negatives: Therapeutic approach and infection control. Clin Microbiol Infect 2010;16:102-11.  Back to cited text no. 4
    
5.
Motara F, Ballot DE, Perovic O. Epidemiology of neonatal sepsis at Johannesburg Hospital. South Afr J Epidemiol Infect 2005;20:90-3.  Back to cited text no. 5
    
6.
Ansari S, Nepal HP, Gautam R, Shrestha S, Neopane P, Chapagain ML. Neonatal septicemia in Nepal: Early-onset versus late-onset. Int J Pediatr 2015;2015:379806.  Back to cited text no. 6
    
7.
Collee JG, Marr W. Culture of bacteria. In: Collee JG, Marmion BP, Simmons A, editors. Mackie and McCartney Practical Medical Microbiology. 14th ed. New York: Churchill-Livingstone; 1996. p. 113-29.  Back to cited text no. 7
    
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Winn WC, Allen SD, Janda WM, Koneman EW, Procop GW, Woods GL, et al. Introduction to microbiology. In: Koneman's Color Atlas and Textbook of Diagnostic Microbiology. 6th ed. Philadelphia: Lippincott Williams & Wilkins; 2006. p. 67-110.  Back to cited text no. 8
    
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CLSI. Performance Standards for Antimicrobial Susceptibility Testing. CLSI Supplement M100. 29th ed., Vol. 39. Wayne, PA: Clinical and Laboratory Standards Institute; 2019.  Back to cited text no. 9
    
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Gupta G, Tak V, Mathur P. Detection of AmpC β lactamases in gram-negative bacteria. J Lab Physicians 2014;6:1-6.  Back to cited text no. 10
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11.
Cantarelli VV, Inamine E, Brodt TC, Secchi C, Cavalcante BC, Pereira Fde S. Utility of the ceftazidime-imipenem antagonism test (CIAT) to detect and confirm the presence of inducible AmpC beta-lactamases among Enterobacteriaceae. Braz J Infect Dis 2007;11:237-9.  Back to cited text no. 11
    
12.
Picão RC, Andrade SS, Nicoletti AG, Campana EH, Moraes GC, Mendes RE, et al. Metallo-beta-lactamase detection: Comparative evaluation of double-disk synergy versus combined disk tests for IMP-, GIM-, SIM-, SPM-, or VIM-producing isolates. J Clin Microbiol 2008;46:2028-37.  Back to cited text no. 12
    
13.
De AS, Rathi MR, Mathur MM. Mortality audit of neonatal sepsis secondary to Acinetobacter. J Glob Infect Dis 2013;5:3-7.  Back to cited text no. 13
    
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Galhotra S, Gupta V, Bains HS, Chhina D. Clinico-bacteriological profile of neonatal septicemia in a tertiary care hospital. J Mahatma Gandhi Inst Med Sci 2015;20:148.  Back to cited text no. 14
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Alqasim A, Abu Jaffal A, Alyousef AA. Prevalence of multidrug resistance and extended-spectrum β-lactamase carriage of clinical uropathogenic Escherichia coli isolates in Riyadh, Saudi Arabia. Int J Microbiol 2018;2018:3026851.  Back to cited text no. 15
    
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McDanel J, Schweizer M, Crabb V, Nelson R, Samore M, Khader K, et al. Incidence of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Klebsiella infections in the United States: A systematic literature review. Infect Control Hosp Epidemiol 2017;38:1209-15.  Back to cited text no. 16
    
17.
Ejikeugwu C, Esimone C, Iroha I, Okonkwo EC, Gugu T, Oli AN, et al. Detection of metallo-βlactamase (MBL) among carbapenem-resistant Gram-negative bacteria from rectal swabs of cow and cloacae swabs of poultry birds. Ann Med Health Sci Res 2017;7:51-6.  Back to cited text no. 17
    



 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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