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ORIGINAL ARTICLE
Year : 2016  |  Volume : 29  |  Issue : 2  |  Page : 349-353

Biofilm formation by blood stream staphylococcal isolates from febrile neutropenic cancer patients


1 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Oncology, Faculty of Medicine, Menoufia University, Menoufia, Egypt

Date of Submission15-Oct-2014
Date of Acceptance18-Dec-2014
Date of Web Publication18-Oct-2016

Correspondence Address:
Hadeel M Shalaby
Clinical Pathology Department, Faculty of Medicine, Menoufia University, 80 Sharaf El-Din Street, Quesna, Menoufia, 32631
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.192435

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  Abstract 

Objectives:
The aim of this study was to define the main causative organisms of bacteremia in febrile neutropenic patients and to study the prevalence of biofilm forming capability of staphylococci in blood isolates of these patients.
Background:
Blood stream infections remain the main cause of morbidity and death in patients undergoing treatment for cancer. Researchers showed that the first step of staphylococcal infection is the attachment to surfaces of various materials and biofilm formation. Biofilms render the cells less accessible to the defense system of the host, thus impairing the action of antibiotics. Therefore, methods identifying strains with a capacity for biofilm formation are necessary to develop effective strategies for biofilm control and improve patient care.
Patients and methods:
The study included 185 participants divided into two groups; group I included 155 cancer patients and group II included 30 healthy participants as control. The participants were subjected to the following: medical history, blood cultures, antimicrobial susceptibilities of the staphylococcal isolates, Congo red agar (CRA) test, and microtiter plate (MTP) test.
Results:
Positive blood culture represented 33% of cases; of them, gram-positive bacteria represented 78.5% (40 cases staphylococci): 17 cases were of Staphylococcus aureus and 23 cases were of Staphylococcus epidermidis. Biofilm-positive strains of S. aureus were 4/17 (23.6%) when assessed with CRA and 6/17 (35.3%) when assessed with MTP. Biofilm-positive strains of S. epidermidis were 7/23 (30.4%) when assessed with CRA and 10/23 (43.5%) when assessed with MTP. Most strains that form biofilm were more resistant to most antibiotics compared with other strains.
Conclusion:
There is an association between biofilm production with persistent infection and antibiotic therapy failure. The MTP is the test of choice in detecting biofilm formation among staphylococci.

Keywords: biofilm, febrile neutropenia, staphylococci


How to cite this article:
El-Hendi AA, Abo El-Fotouh MA, El-Shalakany AH, Shalaby HM. Biofilm formation by blood stream staphylococcal isolates from febrile neutropenic cancer patients. Menoufia Med J 2016;29:349-53

How to cite this URL:
El-Hendi AA, Abo El-Fotouh MA, El-Shalakany AH, Shalaby HM. Biofilm formation by blood stream staphylococcal isolates from febrile neutropenic cancer patients. Menoufia Med J [serial online] 2016 [cited 2019 Dec 10];29:349-53. Available from: http://www.mmj.eg.net/text.asp?2016/29/2/349/192435


  Introduction Top


Patients with malignancies receiving intensive chemotherapy have an increased risk of development of neutropenia and febrile episodes [1]. Despite major advances in prevention and treatment, febrile neutropenia (FN) remains one of the most serious complications of cancer chemotherapy [2]. Over the past decade, there has been a considerable change in the spectrum and antibiotic susceptibility patterns of pathogens causing infection in FN patients. Knowledge of locally prevalent pathogens and their sensitivities is essential as it helps to guide antimicrobial therapy in neutropenic patients [3]. There was increase in the incidence of gram-positive pathogens in the 1980s and 1990s. Thereafter, the most common bacterial etiologic agent isolated from blood cultures in most centers was reported to be coagulase-negative staphylococci (CoNS) [1]. Biofilm is defined as the accumulation of microorganisms and their extracellular products to form a highly structured bacterial community on a surface [4]. Bacterial biofilm has long been considered a virulence factor contributing to infections associated with various medical devices and causing nosocomial infection [5]. The presence of biofilm in CoNS represents a significant bacterial virulence factor [4]. Moreover, a number of Staphylococcus aureus infections are associated with the formation of biofilms [6].

We aimed to study the prevalence of biofilm forming capability of staphylococci in blood isolates of FN cancer patients and to determine the antimicrobial susceptibility of these isolates.


  Patients and Methods Top


This study was carried out in the Clinical Pathology Department and the Oncology Department, Faculty of Medicine, Menoufia University. The study included 185 participants divided into two groups. Group I included 155 FN cancer patients (96 male and 59 female patients), and their ages ranged between 21 and 65 years. Group II included 30 healthy volunteers from healthcare members at the Oncology Department as a control. All participants were subjected to the following: full medical history and complete physical examination, blood cultures to diagnose bacteremia, identification of type of bacteria, antimicrobial susceptibilities of the staphylococcal isolates to different antimicrobial agents, and phenotypic characterization of biofilm formation by the Congo red test and the microtiter plate (MTP) test.

  1. Sampling: A total volume of 8–10 ml of peripheral blood was aseptically collected from cancer patients and inoculated into blood culture bottles.
  2. Laboratory methods: Positive blood culture bottles were subcultured on blood agar and MacConkey's agar, and then were incubated aerobically at 37°C for 24 h. Staphylococcal strains were identified using gram stain, catalase test to differentiate them from streptococci, and finally DNAase agar and mannitol agar to distinguish S. aureus from CoNS.
  3. Antimicrobial susceptibilities of staphylococcal isolates to the following antibiotics were ascertained: cefepime, ceftriaxone, ciprofloxacin, levofloxacin, cefoxitin, erythromycin, azithromycin, clindamycin, amoxicillin clavulanate, ampicillin sulbactam, oxacillin, vancomycin, methicillin, meropenem, and imipenem.
  4. Phenotypic detection of the biofilm was carried out using the following tests:


  1. Congo red agar (CRA) test: Plates were inoculated with test organism and incubated at 37°C for 24–48 h aerobically. On CRA, biofilm-producing strains formed black colonies with a dry crystalline consistency, whereas biofilm nonproducing strains formed pink colonies [5].
  2. MTP: Isolates from fresh agar plates were inoculated in 10 ml trypticase soy broth with 1% glucose and incubated for 24 h at 37°C and then diluted (one in 100) with fresh medium. Individual wells of sterile, polystyrene, flat-bottom tissue culture plates were filled with 0.2 ml aliquots of the diluted cultures and only broth served as control to check sterility and nonspecific binding of media. The tissue culture plates were incubated for 24 h at 37°C. After incubation, the content of each well was gently removed by tapping the plates. The wells were washed four times with 0.2 ml of PBS (pH 7.2) to remove free-floating planktonic bacteria; wells were dried for 1 h at 60°C and then 1% solution of crystal violet was added to each well (this dye stains the cells but not the polystyrene plates). The plates were incubated at room temperature for 15 min and rinsed thoroughly and repeatedly with water. Adherent cells, which usually formed biofilm on all side wells, were uniformly stained with crystal violet. The optical density of the wells was measured at 570 nm using micro ELISA auto reader [7].



  Results Top


In the present study, bacteremia represented 33% of cases (51 cases). Gram-positive bacteremia represented 78.5% with 40 staphylococcal isolates. Of them, 17 (33.3%) were S. aureus strains and 23 (45.1%) were Staphylococcus epidermidis strains. Gram-negative bacteremia represented 21.5% with 11 isolates; of them, four (7.8%) were Klebsiella spp., four (7.8%) were Proteus spp., two (3.9%) were  Escherichia More Details coli, and one (1.9%) was Enterobacter spp. ([Table 1]). Biofilm-positive strains of S. aureus were 4/17 (23.6%) when assessed with CRA and 6/17 (35.3) when assessed with MTP ([Table 2]). Biofilm-positive strains of S. epidermidis were 7/23 (30.4%) when assessed with CRA and 10/23 (43.5) when assessed with MTP ([Table 3]). There was no significant difference between the two methods as regards biofilm detection. Antimicrobial susceptibilities of staphylococcal isolates showed that strains that form biofilm were more resistant to cefepime, ciprofloxacin, ceftriaxone, oxacillin, methicillin, imipenem, and meropenem compared with other strains that do not form biofilm ([Table 4]).
Table 1: Isolated organisms of bacteremia in cancer patients

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Table 2: Comparison between Congo red agar and microtiter plate in detecting biofilm in Staphylococcus aureus

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Table 3: Comparison between Congo red agar and microtiter plate in detecting biofilm in Staphylococcus epidermidis

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Table 4: The relation between biofilm formation and antimicrobial resistance pattern

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Comparing the patients and controls as regards biofilm formation, there was statistically significant difference between the two groups on using CRA or MTP for detecting biofilm formation.


  Discussion Top


In the present study, bacteremia in FN cancer patients represented 33% of cases. According to the global reports, the prevalence of bacteremia in patients with cancer ranged between 5.7 and 44% [8].

Mildly higher results were obtained by El Mahallawy et al. [9]. They performed a prospective cohort study on pediatric cancer patients at National Cancer Institute of Cairo that revealed a 46% positive blood culture for bacteremia [9].

However, Seif El-Din et al. [0] reported higher results in their study in which bacteremia reached 54.7%. These higher results may be due to strict inclusion criteria of the patients, in which they excluded patients who had received empirical antibiotics therapy [0].

In the present study, we found that bacterial strains isolated from blood cultures from febrile cancer patients had the following distribution: 21.5% of the strains were gram-negative organisms and 78.5% of the strains were gram positive.

This is in accordance with other studies, which reported that gram-positive organisms account for 60–70% of bacteremias in cancer patients [Locus et al. [1], Hughes et al. [2], Marie et al. [3], and Hsin et al. [4].

This high percentage of gram-positive bacteremia presented by these workers was attributed to the factors that possibly contribute to the shift in gram-positive isolates, such as increased use of indwelling central venous catheters, fluoroquinolone prophylaxis, and high-dose chemotherapy inducing oral mucositis [15],[16].

In contrast, El Mahallawy et al. [9] stated another distribution in which gram-negative organisms constitute the higher percentages. They suggested this high percentage to be most likely derived from endogenous sources such as the gastrointestinal tract. Similar reports have been published by Seif El-Din et al. [0].

In the present study, the gram-positive cocci were distributed as follows: 33% were S. aureus and 45.1% were S. epidermidis. This pattern was in accordance with that reported by Ramphal [7], Kim et al. [8], Ashour and El-Sharif [9], and Eslaminezhad et al. [0]. CoNS were the predominant etiological pathogens of bacteremia. Similar results have been reported by Seif El-Din et al. [0].

In the present study, we assayed isolated staphylococcal strains for qualitative biofilm-forming ability by means of the CRA test: 23.6% strains of S. aureus and 30.4% strains of S. epidermidis had biofilm-forming ability. Similar results were reported by De Silva et al. [1], who reported that only 25% of the tested CoNS were biofilm positive by CRA.

However, some authors found different results. Arciola et al. [5] found that biofilm formation among staphylococci isolated from catheter-associated infection was found in 61% of S. aureus and 49% of S. epidermidis isolates. El Mahallawy et al. [2] also assayed the staphylococcal blood isolates of FN pediatric cancer patients at the NCI, Cairo, and reported that 60% of S. aureus and 24.4% of CoNS were CRA positive. Similar results have been reported by Seif El-Din et al. [0].

In the present study, we performed quantitative detection of biofilm using MTP, which showed that 35.3% of S. aureus strains were biofilm forming and 43.5% strains of S. epidermidis were biofilm forming.

This was similar to the results reported by De Silva et al. [1], who reported 42% of S. epidermidis isolated from bacteremia in neonatal ICU to be positive biofilm producer using MTP.

However, Seif El-Din et al. [0] reported higher results concerning S. aureus, in which 57% were biofilm forming.

The most important advantage of the MTP assay in addition to the phenotypic biofilm production information presented by CRA is the ability of this method to differentiate between weak and strong biofilm producers. This reflects the severity of the condition and so may help in the determination of suitable line of management [3].

In the present study, isolates forming biofilm were more resistant to broad spectrum antibiotics. This supports the fact that biofilm adds to the virulence profile of staphylococcalstrains isolated from blood stream infection. The results were consistent with those of Seif El-Din et al. [0], De Silva et al. [1], and El Mahallawy et al. [2].


  Conclusion Top


The results obtained by this study confirm that MTP is the recommended test for screening of biofilm formation among staphylococci. In infection caused by biofilm producing staphylococci, the differentiation with respect to biofilm phenotype by MTP might help to modify antibiotic therapy.

Conflicts of interest

There are no conflicts of interest.[23]

 
  References Top

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Freifeld AG, Bow EJ, Sepkowitz KA, Boeckh MJ, Ito JI, Mullen CA, et al. Clinical practice guideline for the use of antimicrobial agents in neutropenic patients with cancer: 2010 update by the Infectious Diseases Society of America. Clin Infect Dis 2011; 52:56–93.  Back to cited text no. 1
    
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Boles B, Horswill A. Staphylococcal biofilm disassembly. Trends Microbiol 2011; 19:449–455.  Back to cited text no. 6
    
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Hsin P, Hsueh P, Chin Y. Bacteremia in haematological and oncological children with febrile neutropenia: experience in tertially medical center in Taiwan, J Microbiol Immunol Infect 2003; 36:197–202.  Back to cited text no. 14
    
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Paganini H, Staffolani V, Zubizarreta P. Viridans streptococci bacteraemia in children with fever and neutropenia: a case–control study of predisposing factors. Eur J Cancer 2003; 39:1284–1289.  Back to cited text no. 15
    
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21.
De Silva G, Kantzanou M, Justice A, Massey R, Wilkinson A, Day N, Peacock S. The ica operon and biofilm production in coagulase-negative staphylococci associated with carriage and disease in a Neonatal Intensive Care Unit. J Clin Microbiol 2002; 40:382–388.  Back to cited text no. 21
    
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El Mahallawy H, Loutfy S, El-Wakil M, Abd El-Aal, Morcos H. Clinical implications of icaA and icaD genes in coagulase negative Staphylococci and S. aureus bacteremia in febrile neutropenic pediatric cancer patients. Pediatr Blood Cancer 2009; 52:824–828.  Back to cited text no. 22
    
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    Tables

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



 

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