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 Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 17  |  Issue : 1  |  Page : 25-29

Study of gram-positive isolates from cases of septicemia and their antibiotic sensitivity pattern with special reference to methicillin-resistant Staphylococcus aureus


Department of Microbiology, JNMC, Wardha, Maharashtra, India

Date of Submission17-Jan-2022
Date of Decision28-Feb-2022
Date of Acceptance16-Mar-2022
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_210_22

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  Abstract 


Background: Infections of the bloodstream are a leading source of illness and mortality in all types of communities. Bacteremia is the presence of bacteria in the bloodstream for long or short periods. Aim and Objectives: Dissemination of the bacteria throughout the body with evidence of systemic responses toward microorganisms is septicemia. Many organisms including Gram positive such as Coagulase negative staphylococci, Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, and Enterococcus faecium can cause septicemia. Many precipitating factors such as presence of intravenous catheters, immunocompromised state, and use of cytotoxic drugs may lead to increase in cases of septicemia. Isolation of the offending pathogens and knowledge about sensitivity and resistance pattern of the isolates remain the mainstay of the diagnosis. This study was conducted to cite the bacteriological etiology of septicemia in adults as well as in neonates and to decide the strategy for the cure of septicemia cases along with their antibiotic susceptibility profile. Materials and Methods: Duration of this study was 3 months. In this duration, 100 blood samples from suspected cases of septicemia were processed. Results: Gram positive organisms isolated from the specimens were collected and identified by standard protocols. Antibiotic sensitivity and detection of methicillin resistant Staphylococcus aureus were done by using Kirby Bauer disc diffusion method. Conclusion: Various causative agents were isolated from blood samples. Some of them are resistant to the drugs that are commonly used for the treatment of septicemia. Hence, isolation of the etiological agent along with the detection of its antibiogram pattern is important for early diagnosis and treatment of cases of septicemia.

Keywords: Bacteremia, inducible clindamycin resistance, methicillin-resistant Staphylococcus aureus, septicemia


How to cite this article:
Mahajan SM. Study of gram-positive isolates from cases of septicemia and their antibiotic sensitivity pattern with special reference to methicillin-resistant Staphylococcus aureus. J Datta Meghe Inst Med Sci Univ 2022;17:25-9

How to cite this URL:
Mahajan SM. Study of gram-positive isolates from cases of septicemia and their antibiotic sensitivity pattern with special reference to methicillin-resistant Staphylococcus aureus. J Datta Meghe Inst Med Sci Univ [serial online] 2022 [cited 2022 Aug 16];17:25-9. Available from: http://www.journaldmims.com/text.asp?2022/17/1/25/352217




  Introduction Top


Infections of the bloodstream are a leading source of illness and mortality in all types of communities.[1] Bacteremia is the presence of bacteria in the bloodstream for long or short periods. Septicemia is the spread of bacteria throughout the body with indications of systemic reactions to microorganisms.[2]

Septicemia is a serious bloodstream infection. In underdeveloped countries, the rise in cases of septicemia poses major challenge for a clinician to treat and manage the causative etiology. Nowadays, these cases have become more complicated due to the development of multidrug resistance in the causative agent, which is the mainstay of treatment for septicemia.[3] Septicemia can be caused by a variety of species, including Gram-positive bacteria such coagulase-negative staphylococci (CONS), Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, and Enterococcus faecium.[1]

Many precipitating factors such as presence of intravenous devices, immunocompromised state, and use of cytotoxic drugs are associated with rise in cases of septicemia, especially in critical patients from critical units. Isolation of the offending pathogens by culture on different media and report about sensitivity pattern of the isolates remains the mainstay of diagnosis and treatment of septicemia.[2] Neonatal septicemia is a leading cause of neonatal impairment and death. Septicemia in newborns is defined as a widespread bacterial infection confirmed by a positive blood culture within the first 4 weeks of life.[4] According to the World Health Organization, out of 5 million neonatal deaths a year, 98% are occurring in underdeveloped countries.[5]

Septicemia is becoming more common due to multidrug-resistant bacteria all over the world. The widespread availability of over-the-counter medicines and the widespread usage of broad-spectrum antibiotics in the population may explain the multidrug resistance problem.

Septicemia-causing microbes have gained enhanced multidrug resistance to commonly prescribed medicines, making therapy extremely challenging.[6] Thus, having all knowledge regarding the most frequent etiological agents that cause septicemia, as well as their antimicrobial susceptibility patterns, is critical for selecting suitable antimicrobial therapy. These pathogens vary geographically in their antimicrobial susceptibility patterns and are temporally dependent on the commonly used antibiotics and the local pathogens.[7]

As a result, this study was carried out to determine the microbiological etiology of septicemia in adults and neonates, as well as to create a strategy and correct approach for septicemia care based on their antibiotic susceptibility profile.


  Materials and Methods Top


From June to August 2019, the current investigation was conducted in a tertiary care hospital's department of microbiology.

Sample collection

Blood culture

With a disposable needle and syringe, 2 ml (in neonates) and 5 ml (in adults) of blood were extracted and inoculated into blood culture bottles containing 20 ml and 50 ml of brain–heart infusion broth, respectively. The blood and broth were gently mixed, and the bottles were transferred to the laboratory and incubated aerobically at 37°C. Then, use 5% sheep blood agar (BA) and MacConkey agar (MA) plates to subculture. Gram staining, colony features, and biochemical properties were used to identify the isolates. If there was no growth after 1 week of incubation, the culture was labeled as negative. According to the CLSI 2019 standards, all bacterial isolates were tested for antimicrobial susceptibility using the Kirby-Bauer disc diffusion technique.[8],[9]

Processing of the specimen

Standard microbiological techniques were used to identify Enterobacteriaceae isolates by examining their shape, colony features, and biochemical responses.[10]

Culture

BA and MA were used to inoculate the samples. The plates were incubated for 18–24 h at 37°C overnight.[11]

Identification of isolates

Standard microbiological procedures were used to identify the isolates, which included looking at colony features, morphology, and biochemical reactions.

  • Colony characteristics were investigated including size, shape, elevation, margin, surface, opacity, consistency, medium change, and pigment production[11]
  • Morphology: A Gram stain was applied to the fixed smear as stated[12]
  • Biochemical reactions: Catalase, oxidase, coagulase, carbohydrate fermentation (lactose, glucose, mannitol, and sucrose), indole, methyl red, citrate utilization, urease generation, H2S production test, and other biochemical reactions were carried out according to conventional protocols. All biochemical tests included appropriate positive and negative controls.[10]


Biochemical reaction

Identification of genus Staphylococcus


  Catalase test Top


The catalase test is the most popular way to tell the difference between Staphylococcaceae and Streptococcaceae bacteria. Catalase is present in Staphylococcus and Micrococcus, but not in Stomatococcus, which has a mild response.[13]

Identification of species Staphylococcus aureus


  Coagulase test Top



  Slide coagulase test Top


Principle

The bound coagulase is determined by slide coagulase (clumping factor). When floating in plasma, the clumping factor attaches to the bacterial cell wall, and fibrin threads develop between the bacterial cells, causing them to clump into visible aggregates (fibrinogen).[14]

Reagent

Human plasma with EDTA.

Quality control

The coagulability of the plasma was determined by adding one drop of 5% calcium chloride to 0.5 ml plasma. Within 10–15 s, a clot should form.

  • Positive control – S. aureus
  • Negative control – Staphylococcus epidermidis.


Interpretation

Appearance of coarse clumps within 10–15 s was considered positive test.

Staphylococcus lugdunensis may also give slide coagulase test positive. If no clumping was noticed after 2 min, the test was declared negative. All of the isolates were then subjected to a tube coagulase test.


  Antimicrobial susceptibility testing Top


Each isolate was subjected to antimicrobial susceptibility test as per the CLSI 2019 guidelines[9] by Kirby-Bauer disk diffusion technique.[15]


  Kirby-Bauer disk diffusion technique Top


Medium

Muller-Hinton agar (MHA) was put into a flat-bottomed 9 cm  Petri dish More Details to a depth of 4 mm (25 ml).

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 set to McFarland standards of 0.5.

Antibiotic disks

Commercially available (HiMedia Lab, Mumbai) disks of 6 mm diameter with recommended potencies were used. Disks used were as follows:

  • Piperacillin (100 μg)
  • Piperacillin-tazobactam (100/10 μg)
  • Cefoxitin (30 μg)
  • Ceftazidime (30 μg)
  • Ceftazidime-clavulanic acid (30/10 μg)
  • Imipenem (10 μg)
  • Imipenem-EDTA (10/750 μg)Gentamicin (10 μg)
  • Amikacin (30 μg)
  • Netilmicin (30 μg)
  • Ciprofloxacin (5 μg).


Reading and interpretation

The antibiotic disc was used to measure the diameters of the zone of inhibition. Interpretation, i.e., susceptible, intermediate, and resistant, was done with reference to the CLSI 2019 guidelines.[9]

Quality control

Each batch of MHA and antibiotic disks were tested by using  Escherichia More Details coli ATCC 25922 control strains.


  Methicillin-resistant Staphylococcus aureus detection Top



  Cefoxitin disc diffusion testing Top


A cefoxitin disc diffusion test was performed on all S. aureus isolates using a 30 g disc. The isolate was produced as a 0.5 McFarland standard suspension and then cultured on MHA plate. Zone diameters were measured after a 24-h incubation period at 37°C. A resistance zone diameter of <21 mm was observed, whereas a sensitive zone diameter of more than 22 mm was reported.[9]


  Inducible clindamycin resistance: D-zone test Top


The inducible clindamycin resistance of S. aureus isolates was investigated using a double-disc diffusion test (D-zone test). On the MHA plate inoculated with the test organism, a clindamycin disc (2 g) was placed 15 mm apart from an erythromycin disc (15 g). The strain S. aureus ATCC 25923 was used as a control. After 16–18 h of incubation at 35°C, the plates were examined.[9]


  Reading and interpretation Top


Positive D-zone test, i.e., existence of inducible clindamycin resistance, was demonstrated by flattening the zone (D shaped) of the clindamycin disc toward the side facing the erythromycin disc. Growth of the organism <14 mm zone of inhibition was taken as constitutive clindamycin resistance. The MS phenotype was defined as staphylococcal isolates that were resistant to erythromycin (zone size 13 mm) but sensitive to clindamycin (zone size 21 mm) and gave a circular zone of inhibition around clindamycin.


  Observations and Results Top


In the present study, a total of 32 isolates isolated from clinically suspected cases of septicemia were included.

[Table 1] shows the maximum growth of S. aureus, i.e., 16. Rest of the organisms isolated were CONS and Enterococcus sp.
Table 1: Distribution of organisms in septicemia (n=32)

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[Table 2] shows that all isolates are sensitive to vancomycin and linezolid. In the present study, out of 16 isolates of S. aureus, 75% were sensitive to clindamycin followed by erythromycin (62.5%) and cefoxitin (50%).
Table 2: Antibiotic sensitivity pattern of Gram-positive organisms

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[Table 3] shows that 29.6% of S. aureus were MRSA by cefoxitin disk diffusion test and 14.8% were CONS which are methicillin resistant. 3.7% of S. aureus and 7.4% of CONS showed inducible clindamycin resistant.
Table 3: Methicillin and inducible clindamycin resistance in staphylococci (n=27)

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  Discussion Top


In the present study, a total of 32 isolates isolated from clinically suspected cases of septicemia were included. A total of 32 blood samples were collected from patients and processed in the microbiology department.

Distribution of organisms in septicemia

From [Table 1], Gram-positive organisms, 16% were S. aureus, 11% were CONS, and 5% were Enterococcus sp.

According to Muley et al.[16] reported in 2015, the most common Gram-positive pathogen was S. aureus (22.9%).

Antibiotic sensitivity pattern of Gram-positive organisms

[Table 2] demonstrates that all Gram-positive organisms were susceptible to vancomycin and linezolid, except 12 (75%) S. aureus, 5 (45.5%) CONS, and 1 (20%) Enterococcus sp., which were clindamycin resistant. Cefoxitin sensitivity was found in 50% of S. aureus and 36.4% of CONS. It indicates that these isolates were sensitive to methicillin. 62.5% of S. aureus and 27.3% of CONS were resistant to erythromycin.

Galhotra et al.[17] in 2015 discovered that all Gram-positive isolates were susceptible to vancomycin and linezolid. This finding is similar to the finding in the present study. Prabhu et al.[18] in 2011 observed that 54 (28.42%) of S. aureus were erythromycin resistant which is also a similar finding to the present study. The incidence of methicillin resistance is discussed in [Table 3].

Methicillin and inducible clindamycin resistance in staphylococci

[Table 3] shows that 44.4% were resistant to methicillin and 11.1% were inducible clindamycin resistant. In methicillin-resistant Gram-positive cocci, 29.6% were S aureus and 14.8% were CONS. In inducible clindamycin resistance, 3.7% were S. aureus and 7.4% were CONS.

Kumari et al.[19] in 2008 also reported that the prevalence of MRSA was 26.14%. Galhotra et al.[17] in 2015 observed in their study that from all staphylococcal isolates, 50% were MRSA and 60% were methicillin-resistant CONS (MR-CONS).

According to Ciraj et al.,[20] 26 (17.3%) of the 150 S. aureus strains were MRSA and 124 (82.6%) were methicillin-sensitive S. aureus (MSSA). There were 90 methicillin-sensitive CONS and four MR-CONS among the CONS.

Muley et al.[16] reported 18.1% of the Staphylococcus isolates were MRSA. Deotale et al.[21] in 2010 observed that inducible clindamycin resistance was 14.5% in all isolates of S. aureus isolated from clinical samples by using D-test.

In 2011, Prabhu et al.[18] in 2011 found that 20 (37.52%) of all S. aureus isolates were positive for inducible clindamycin resistance using the D-test.


  Conclusion Top


The present study identified various causative agents of septicemia in adults and neonates such as CONS and S. aureus.

  • Along with the etiological agents, antibiotic sensitivity and various resistance patterns are demonstrated
  • The detection of etiological agents and their antibiotic sensitivity would undoubtedly aid ineffectual preventative efforts, timely and accurate diagnoses, and subsequent delivery of tailored medication to reduce the disease's excessive burden
  • In the present study, MRSA, MR-CONS, and inducible clindamycin resistance in Gram-positive organisms are seen.
  • Antibiotic resistance is a concerning sign for the creation of antibiotic policies and procedures for septicemia therapy
  • The pathogen pattern of septicemia is changing time to time and over regions so that regular re-evolution of various etiological agents is necessary for an early and accurate diagnosis and correct treatment
  • More epidemiological and clinical research is needed to track changes in the bacteria that cause newborn sepsis in the future.


Acknowledgment

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

Financial support and sponsorship

The project was funded by ICMR.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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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
    
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Mohanty A, Singh ST, Kabi A, Gupta P, Gupta P, Kumar P. Bacteriological profile and antibiotic sensitivity pattern of hospital acquired septicaemia in a tertiary care hospital in north east India. Asian J Pharm Clin Res 2017;10: 186-9.  Back to cited text no. 2
    
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Alam MS, Pillai PK, Kapur P, Pillai KK. Resistant patterns of bacteria isolated from bloodstream infections at a university hospital in Delhi. J Pharm Bioallied Sci 2011;3:525-30.  Back to cited text no. 3
    
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Agnihotri N, Kaistha N, Gupta V. Antimicrobial susceptibility of isolates from neonatal septicemia. Jpn J Infect Dis 2004;57:273-5.  Back to cited text no. 4
    
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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. 5
    
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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. 8
    
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CLSI. Performance Standards for Antimicrobial Susceptibility Testing. 29th ed. CLSI Supplement M100. Vol. 39. Wayne, PA: Clinical and Laboratory Standards Institute; 2019.  Back to cited text no. 9
    
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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. 10
    
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Collee JG, Duguid JP, Fraser AG, Marmion BP, Simmons A. Laboratory strategy in the diagnosis of infective syndromes. In: Collee JG, Fraser AG, Marmion BP, Simmons A, editors. Mackie and McCartney Practical Medical Microbiology. 14th ed. Delhi: Churchill Livingstone; 2007. p. 53-94.  Back to cited text no. 11
    
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Burke V. Notes on the gram stain with description of a new method. J Bacteriol 1922;7:159-82.  Back to cited text no. 12
    
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Collee JG, Miles RS, Watt B. Tests for the identification of bacteria. In: Collee JG, Fraser AG, Marmion BP, Simmons A, editors. Mackie and McCartney Practical Medical Microbiology. 14th ed. Delhi: Churchill Livingstone; 2007. p. 131-49.  Back to cited text no. 13
    
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Williams RE, harper GJ. Determination of coagulase and alpha hemolysin production by staphylococci. Br J Experimental Pathol 1946;27:72-81.  Back to cited text no. 14
    
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Bauer AW, Kirby WM, Sherris JC, Turck M. Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 1966;45:493-6.  Back to cited text no. 15
    
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Muley VA, Ghadage DP, Bhore AV. Bacteriological profile of neonatal septicemia in a tertiary care hospital from western India. J Glob Infect Dis 2015;7:75-7.  Back to cited text no. 16
    
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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. 17
  [Full text]  
18.
Prabhu K, Rao S, Rao V. Inducible clindamycin resistance in Staphylococcus aureus isolated from clinical samples. J Lab Physicians 2011;3:25-7.  Back to cited text no. 18
[PUBMED]  [Full text]  
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Kumari N, Mohapatra TM, Sing YI. Prevalence of methicillin resistant Staphylococcus aureus (MRSA) in tertiary – Care hospital in eastern Nepal. J Nepal Med Assoc 2008;47:53-6.  Back to cited text no. 19
    
20.
Ciraj AM, Vinod P, Sreejith G, Rajani K. Inducible clindamycin resistance among clinical isolates of Staphylococci. Indian J Pathol Microbiol 2009;52:49-51.  Back to cited text no. 20
[PUBMED]  [Full text]  
21.
Deotale V, Mendiratta DK, Raut U, Narang P. Inducible clindamycin resistance in Staphylococcus aureus isolated from clinical samples. Indian J Med Microbiol 2010;28:124-6.  Back to cited text no. 21
[PUBMED]  [Full text]  



 
 
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  In this article
Abstract
Introduction
Materials and Me...
Catalase test
Coagulase test
Slide coagulase test
Antimicrobial su...
Kirby-Bauer disk...
Methicillin-resi...
Cefoxitin disc d...
Inducible clinda...
Reading and inte...
Observations and...
Discussion
Conclusion
References
Article Tables

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