British Journal of Biomedical Science

ISSN: 0967-4845 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/tbbs20

Biomedical Science in Brief

M. Lotfy, G. Badra, W. Burham, F.Q. Alenzi, J.F. Bermejo-Martin, D. Bernardo,

Marta Dominguez-Gil, Ana Alonso, M.C. Garcia-Arevalo, M. Pino, Ortiz
De Lejarazu, J.M. Eiros, J. Ardura, A.J. Leön, J.A. Garrote, S. Resino, A.
Blanco-Quirös, A. Muñoz-Fernández, E. Arranz, V.K. Gopinath, M. Musa,
A.R. Samsudin, W. Sosroseno, M. Shigematsu, Y. Harada, T. Sekizuka, O.
Murayama, S. Takamiya, B.C. Millar, J.E. Moore, M. Matsuda, S. Elshibly, J. Xu,
B.C. Millar, C. Armstrong & J.E. Moore

To cite this article: M. Lotfy, G. Badra, W. Burham, F.Q. Alenzi, J.F. Bermejo-Martin, D. Bernardo,
Marta Dominguez-Gil, Ana Alonso, M.C. Garcia-Arevalo, M. Pino, Ortiz De Lejarazu, J.M. Eiros,
J. Ardura, A.J. Leön, J.A. Garrote, S. Resino, A. Blanco-Quirös, A. Muñoz-Fernández, E. Arranz,
V.K. Gopinath, M. Musa, A.R. Samsudin, W. Sosroseno, M. Shigematsu, Y. Harada, T. Sekizuka,
O. Murayama, S. Takamiya, B.C. Millar, J.E. Moore, M. Matsuda, S. Elshibly, J. Xu, B.C. Millar,
C. Armstrong & J.E. Moore (2006) Biomedical Science in Brief, British Journal of Biomedical
Science, 63:4, 171-184, DOI: 10.1080/09674845.2006.11732742

To link to this article: http://dx.doi.org/10.1080/09674845.2006.11732742

Published online: 23 May 2016.

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Combined use of honey, bee propolis and
myrrh in healing a deep, infected wound in
a patient with diabetes mellitus
M. LOTFY*, G. BADRA†, W. BURHAM‡ and F. Q. ALENZI§
*
Biomedical Research Unit, Molecular and Cellular Biology Department, Genetic
Engineering and Biotechnology Research Institute, Minufiya University, Sadat City;
†
Department of Hepatology and Internal Medicine, National Liver Institute,
Minufiya University, Minufiya; ‡Department of Surgery, Faculty of Medicine,
Mansoura University, Mansoura, Egypt; §Department of Medical Laboratories,
College of Applied Medical Sciences, King Faisal University, Dammam, Saudi Arabia
Diabetes mellitus is a group of diseases characterised by high
levels of blood glucose resulting from defects in insulin
production, insulin action, or both. Diabetes can be
associated with serious complications including diabetic foot
disease. Diabetic foot disease is estimated to affect
Downloaded by [University of Exeter] at 14:11 31 May 2016
15% of people with diabetes.1
Wound healing is a process that involves inflammation,
proliferation/regeneration and finally remodeling. The
normal orderly pattern is disrupted in chronic non-healing
wounds, which are characterised by decreased levels of
growth factors and increased protease activity. Wound
healing is affected by serum albumin, tissue oxygenation,
infection, hyperglycaemia, cytokines and proteases.2
A marker of non-healing wounds may be the prolonged
presence of extracellular matrix molecules in the dermis.3
Other markers and potential mediators include increased
levels of transforming growth factor (TGF)-β3,4 proteolytic
factors such as matrix metalloproteinases,5 and the absence
of IGF-I.6
Wound care includes a variety of approaches to enhance
healing, with treatment of infection, vascular reconstruction,
achieving adequate glycaemic control, removal of pressure,
and ongoing wound debridement being important aspects
of this care.2
A deep wound with tissue loss in the right foot of a
65-year-old male patient with diabetes mellitus was treated
by a standard protocol that included strict control of blood
sugar level. In addition, an antibiotic regimen was included
to combat anaerobic and aerobic infection. Also, a paste
consisting of myrrh, bee propolis and honey (MPH) was
applied to the wound. Following treatment, the wound
settled and healed well (Fig. 1).
The patient presented to the out-patient clinic of Minufiya
University Hospital (MUH) with possible osteomyelitis.
A foot wound showed severe oedema and the patient was
unable to bear weight on the foot. On examination, the
patient had a large abscess beneath the skin of the foot.
An incision (3 cm) was made, pus was drained and the
wound was cleaned. The patient returned home with
antibiotic (gatifloxacin; 400 mg twice a day for five days) and
an anti-inflammatory agent.
X-ray examination showed no bony abnormality and
inflammation was prominent around the smallest toe.
Correspondence to: Dr Mahmoud Lotfy
Molecular and Cellular Biology Department, Genetic Engineering and Biotechnology
Research Institute, Minufiya University, Sadat City, PO 79, Minufiya, Egypt
Email: mlotfy2000@yahoo.com
BIOMEDICAL SCIENCE IN BRIEF 171
Fig. 1. The foot wound showing the healing process during treatment
with the MPH paste.
Two days later, the patient’s foot was re-examined and
debridement of the wound was performed to remove dead
skin and necrotic tissue inside the opened cavity.
At all times during treatment, blood sugar level was
controlled (in the range 150–170 mg/dL) using insulin. The
patient was kept on metronidazole (1500 mg/day) and
combined amoxicillin with clavulanic acid (1500 mg/day) for
10 days. Thereafter, ciprofloxacin (1500 mg/day) was used
instead of the combined amoxicillin with clavulanic acid.
Pentoxifylline, vitacid calcium (vitamin C and calcium
carbonate) and vitazinc (vitamins A and E plus zinc) were
added to the treatment regimen to aid vascularity and
healing. From the beginning of treatment until the deep
wound healed, the patient was maintained on an oral dose
of bee propolis (400 mg/day). Erythrocyte sedimentation rate
(a good indicator of treatment efficacy) was 125 mm prior to
treatment and dropped to 65 mm after two weeks and
25 mm after four weeks, where it stabilised.
The most significant results were obtained during the use
of the MPH paste (800 mg bee propolis, 50 g myrrh, mixed
together in honey). The paste was prepared every three days
and stored in a refrigerator. Wound cleaning was performed
daily using standard methods in addition to the MPH paste
to fill the wound cavity. The effectiveness of the paste in
keeping the wound clean was indicated by a complete
absence of pus and cellular exudate. After four weeks the
wound had healed well and the patient returned to work.
Poor wound healing in people with diabetes is well
recognised.7 However, there is little information about many
aspects of foot care in people with diabetes, including
wound healing.8 The American Diabetes Association 9
suggests a range of predisposing factors to explain poor
healing of wounds in people with diabetes, including
abnormal cellular and/or inflammatory pathways,
peripheral neuropathy and vascular disease and/or tissue
hypoxia. Abnormal cellular function, particularly in
fibroblasts and neutrophils, has been found in people with
diabetes. In vitro, hyperglycaemia may be toxic to these
cellular elements, while in vivo it may result in a greater
susceptibility to infection.
Modest differences in the function of neutrophils,
macrophages and fibroblasts associated with hyperglycaemia
BRITISH JOURNAL OF BIOMEDICAL SCIENCE 2006 63 (4)
 172 BIOMEDICAL SCIENCE IN BRIEF
have been postulated, but these have not been
demonstrated conclusively in vivo. Advanced glycosylation
end products accumulate in diabetes as a result of
hyperglycaemia, leading to the non-enzymatic glycosylation
of collagen.10 This process results in the production of
abnormal collagen, which is highly inflexible and prone to
breakdown, particularly over pressure areas.11
In this report, a paste is described that keeps a wound
clean, which is especially important in cases that involve
tissue loss. The MPH paste contains safe and effective
components that have prominent antimicrobial activity.
Myrrh is an oleogum resin obtained from the stem of the
plant Commiphora molmol. It is a safe, natural flavouring
substance approved by the US Food and Drug
Administration.12,13 In experimental studies on Swiss albino
mice, myrrh from C. molmol exhibited no mutagenicity and
proved to be a potent cytotoxic drug against Ehrlich solid
tumour cells. The antitumour potential of C. molmol was
comparable with that of the standard cytotoxic drug
cyclophosphamide. 14 Studies with animal and human
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models demonstrate antischistosomal and other
antiparasitic activity for myrrh and have found it to be safe
and effective.15,16
Myrrh has considerable antimicrobial activity and is
used in a variety of diseases.17 It has antibacterial and
antifungal activity against standard pathogenic strains of
Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa
and Candida albicans.18 In addition, it has an antidiabetic
effect, especially in non-insulin-dependent diabetes
mellitus (NIDDM). 19 Moreover, Myrrh has found
pharmacological application in the reduction of cholesterol
and triglycerides.20
Propolis is a resinous substance collected from trees by the
bee Apis mellifera, which uses it as a building and insulating
material in the hive. It is known that propolis has
antimicrobial, antioxidative, anti-ulcer and antitumour,
anti-inflammatory, hypotensive and immune stimulatory
activities.21
Antimicrobial activity has been observed against
S. aureus,22,23 Streptococcus pyogenes,24 Gram-positive and
Gram-negative bacterial species and Candida species,25,26
S. mutans,27 anaerobic bacteria in the human oral cavity,28
salmonellas,29 and other microorganisms including
mycobacteria.30 Antibacterial activity of propolis against
Staphylococcus aureus is higher when extracts are prepared in
60–80% ethanol.22 In vitro synergy between propolis and
antimicrobial drugs has been investigated, 31,32 and
preparations combining propolis with antibiotic and
antifungal agents are of potential medical interest.26
Honey is an ancient remedy that has regained popularity
as an alternative treatment for antibiotic-resistant bacteria.
Both honey and sugar pastes are considered useful as topical
antimicrobial agents, mainly because of their high
osmolarity and the ability to minimise water availability to
bacteria.33 Although the dilution of honey by wound fluid is
likely to reduce the efficacy of its osmotic effect, the slow and
sustained production of hydrogen peroxide by some types
of honey (e.g., manuka honey) is capable of maintaining
an antimicrobial effect at a concentration approximately
1000-fold higher than that used commonly in antiseptic
solutions (i.e., 3%).33 Also, certain components of manuka
honey (e.g., flavonoids and aromatic acids) demonstrate
antimicrobial properties.33
BRITISH JOURNAL OF BIOMEDICAL SCIENCE 2006 63 (4)
Honey is also an effective wound deodorant, an effect
attributed to the presence of glucose, which is metabolised
by bacteria in preference to proteinaceous necrotic tissue,
resulting in the production of lactic acid and not the
malodorous compounds generated by protein degradation.33
In addition, the observed benefits of honey in infected
wounds may be attributed to the high glucose content and
low pH, both of which stimulate macrophages.34
In the present case study, application of MPH resulted in a
clean and odour-free wound, which healed well. However,
the results of this single case need to be confirmation in
a study of a larger number of patients. In the meantime,
use of the MPH paste would appear to reduce the cost of
deep wound treatment and improve the outcome in the
patients affected.
References
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1997; 22: 511–23.
2 Bloomgarden ZT. Diabetes complications. Diabetes Care 2004;
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3 Loots MA, Lamme EN, Zeegelaar J, Mekkes JR, Bos JD,
Middelkoop E. Differences in cellular infiltrate and extracellular
matrix of chronic diabetic and venous ulcers versus acute
wounds. J Invest Dermatol 1998; 111: 850–7.
4 Jude EB, Blakytny R, Bulmer J, Boulton AJ, Ferguson MW.
Transforming growth factor-beta 1, 2, 3 and receptor type I and
II in diabetic foot ulcers. Diabet Med 2002; 19: 440–7.
5 Lobmann R, Ambrosch A, Schultz G, Waldmann K, Schiweck S,
Lehnert H. Expression of matrix-metalloproteinases and their
inhibitors in the wounds of diabetic and non-diabetic patients.
Diabetologia 2002; 45: 1011–6.
6 Blakytny R, Jude EB, Martin Gibson J, Boulton AJ, Ferguson MW.
Lack of insulin-like growth factor 1 (IGF1) in the basal
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J Pathol 2000; 190: 589–94.
7 Bouter KP, Storm AJ, de Groot RRM et al. The diabetic foot in
Dutch hospitals: epidemiological features and clinical outcome.
Eur J Med 1993; 2: 215–8.
8 Pecoraro RE, Ahroni JH, Bpyko EJ et al. Chronology and
extremities of tissue repair in diabetic lower-extremity ulcers.
Diabetes 1991; 40: 1305–13.
9 American Diabetes Association. Consensus development
conference on diabetic foot wound care. Diabetes Care 1991; 22:
1354–60.
10 McInnes A. Guide to the assessment and management
of diabetic foot wounds. The Diabetic Foot 200; 4 (Suppl 1):
SI–II.
11 lkes RS, Wolfe JHN. The diabetic foot. BMJ 1991; 303:
1053–5.
12 Hall BL, Oser BL. Recent progress in the consideration of
flavoring ingredients under the food additive amendment. 3.
GRAF substances. Food Technol 1965; 19: 151–97.
13 Ford RA, Api AM, Letizia CS. Monographs on fragrance to raw
materials. Food Chem Toxicol 1992; 30 (Suppl): 91S–92S.
14 Al Harbi MM, Qureshi S, Ahmed MM, Rafatulla S, Shah AH.
Effect of Commiphora molmol (oleogum resin) on the cytological
and biochemical changes induced by cyclophosphamide in
mice. Am J Chin Med 1994; 22: 77–82.
15 Botros S, Sayed H, El-Dusoki H et al. Efficacy of Mirazid in
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infected school children and households. Am J Trop Med Hyg
2005; 72 (2): 119–23.
16 Sheir Z, Nasr AA, Massoud A et al. A safe, effective, herbal
antischistosomal therapy derived from myrrh. Am J Trop Med
Hyg 2001; 65: 700–4.
17 El Ashry ES, Rashed N, Salama OM, Saleh A. Components,
therapeutic value and uses of myrrh. Pharmazie 2003; 58 (3):
163–8.
18 Dolara P, Corte B, Ghelardini C et al. A. Local anaesthetic,
antibacterial and antifungal properties of sesquiterpenes from
myrrh. Planta Med 2000; 66 (4): 356–8.
19 Al-Awadi F, Fatania H, Shamte U. The effect of a plant mixture
extract on liver gluconeogenesis in streptozotocin induced
diabetic rats. Diabetes Res 1991; 18 (4):163–8.
20 Michie CA, Cooper E. Frankincense and myrrh as remedies in
children. J R Soc Med. 1991; 84: 602–5.
21 Lotfy M. Biological activity of bee propolis in health and disease.
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22 Fernandes Júnior A, Balestrin ECC, Cunha MLRS. Anti-
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23 Fernandes Júnior A, Leomil L, Fernandes AAH, Sforcin JM.
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L. and Brazilian stingless bees. J Venom Anim Toxins 2001;
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24 Bosio K, Avanzini C, D’avolio A, Ozimo O, Savoia D. In vitro
activity of propolis against Streptococcus pyogenes. Lett Appl
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25 Drago I, Mombelli B, De Vecchi E, Fassina MC, Tocalli L,
Gismondo MR. In vitro antimicrobial activity of propolis dry
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antimicrobial activity of propolis and synergism between
propolis and antimicrobial drugs. Microbiol Res 2003; 158:
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27 Koo H, Rosalen PL, Cury JA, Park YK, Bowen WH. Effects of
compounds found in propolis on Streptococcus mutans growth
and on glucosiltransferase activity. Antimicrob Agents Chemother
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28 Santos FA, Bastos EMA, Uzeda B, Carvalho MAR, Farias ESA,
Braga FC. Antibacterial activity of Brazilian propolis and
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29 Orsi RO, Sforcin JM, Rall VLM, Funari SRC, Barbosa L,
Fernandes Júnior A. Susceptibility profile of Salmonella against
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Brazil. J Venom Anim Toxins 2005; 11: 109–16.
30 Banskota AH, Tezuka Y, Kadota S. Recent progress in
pharmacological research of propolis. Phytother Res 2001; 15:
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31 Scheller S, Dworniczak S, Waldemar KK, Rajca M, Tomczik A,
Shani J. Synergism between ethanolic extract of propolis (EEP)
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32 Fernandes Júnior A, Balestrin EC, Betoni JEC, Orsi RO, Cunha
MLRS, Montelli AC. Propolis: anti-Staphylococcus aureus activity
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BIOMEDICAL SCIENCE IN BRIEF 173
Interleukin (IL)-1β, IL-6 and IL-8 in nasal
secretions: a common role for innate
immunity in viral bronchial infection in
infants?
J. F. BERMEJO-MARTIN*, D. BERNARDO*, MARTA
DOMINGUEZ-GIL†, ANA ALONSO‡, M. C. GARCIA-AREVALO*,
M. PINO‡, R. ORTIZ DE LEJARAZU†, J. Mª EIROS†, J. ARDURA‡,
A. J. LEÓN*, J.A. GARROTE*, S. RESINO§, A. BLANCO-
QUIRÓS*, Mª. A. MUÑOZ-FERNÁNDEZ§ and E. ARRANZ*
*
Mucosal Immunity Laboratory, Pediatrics Department. Institute of Molecular
Biology and Genetics; †Virology Laboratory, Hospital Clínico Universitario;
‡
Pediatrics Deparment. Hospital Clínico Universitario, Valladolid; and
§
Immunomolecular Biology Laboratory. HGU Gregorio Marañón, Madrid, Spain
There is growing evidence to support a role for the immune
response in lower respiratory tract infection (LRTI) due to
viruses in children.1,2 Respiratory syncytial virus (RSV) is the
most frequent aetiological agent of LRTI, but other
respiratory viruses (human metapneumovirus [hMPV],
parainfluenza virus types 1, 2 and 3, influenza virus B,
adenovirus types 1, 2 and 5, and mycoplasmas) can produce
symptoms indistinguishable from those elicited by RSV.
Currently, however, there is controversy over the ability of
the different viral agents to induce pro-inflammatory
cytokines. Differences in the locally secreted cytokine profile
in nasal washes between RSV and metapneumovirus
infections have been described, as have similarities between
RSV and influenza.3 Additionally, immune function in
neonates differs from that in adults.4
At birth, the immune system is not fully mature. Neonatal
antigen-presenting cells tend to be deficient in interleukin
(IL)-12. Furthermore, T cells secreting interferon (IFN)-γ
(Th1 cells) are more likely to undergo apoptosis after antigen
exposure. This might explain in part the Th2-skewed
immunity in newborns. Neonatal immune cells appear
unable to provide strong responses because neonatal
antigen-presenting cells fail to up-regulate major
histocompatibility complex (MHC) class II and co-
stimulatory molecules. Antibody levels and classes are also
different in early life to those found in adulthood.
Neonatal antibody is characterised by increasing levels of
maternal immunoglobulin during the last trimester of
pregnancy, which is replaced during the first year of life
progressively by neonatal IgM, then IgG and finally IgA
production. These special features of the neonatal immune
system mean that young children are much more
susceptible to infectious disease; however, with ageing,
mortality due to infection decreases rapidly such that by the
age of 10 years it is reduced 10- to 100-fold.4
Greater understanding of the immune response in LRTI is
important for better understanding of the physiopathology
of the condition, which may contribute to the development
Correspondence to: J. F. Bermejo
Mucosal Immunity Laboratory, Pediatrics and Immunology Department,
Facultad de Medicina, Valladolid, Spain
Email: bermejo@ped.uva.es
BRITISH JOURNAL OF BIOMEDICAL SCIENCE 2006 63 (4)
 174 BIOMEDICAL SCIENCE IN BRIEF

Table 1. Comparison of cytokine levels. Cytokine values are expressed as median and interquartile range.

Group 1 (A) Group 2 (B) Controls for Controls for P(a) P(b) P(c)
(n=8) (n=14) Group 1 (C) Group 2 (D)
(n=6) (n=11)
IL-6 502 (1033) 135 (296) 19 (33) 28 (34) 0.008* 0.001* 0.110
IL-8 8749 (8765) 3565 (3895) 540 (737) 913 (1479) 0.001* 0.001* 0.029*
IL-10 215 (457) 105 (206) 20 (114) 34 (64) 0.228 0.051 0.330
IL-2 33 (137) 16 (109) 12 (32) 12 (48) 0.414 0.687 0.714
IFNγ 36 (71) 14 (55) 3 (12) 14 (46) 0.345 0.536 0.868
IL-12p70 119 (283) 87 (114) 42 (108) 36 (200) 0.108 0.202 0.365
TNF-α 257 (295) 100 (266) 78 (198) 27 (154) 0.282 0.222 0.330
IL-1β 851 (5036) 704 (937) 104 (83) 124 (295) 0.013 *
0.001 *
0.664
IL-5 33 (63) 35 (75) 14 (36) 10 (17) 0.345 0.120 0.714
IL-4 18 (137) 40 (94) 22 (46) 18 (26) 0.573 0.095 0.664

P : Difference between groups (significance). Table 2. Characteristics of patients in Groups 1 and 2.

P(a): Difference between (A) and (C). Values are expressed as median and interquartile range
Downloaded by [University of Exeter] at 14:11 31 May 2016

P(b): Difference between (B) and (D). (except Gender and Prematurity).
P(c): Difference between (A) and (B).
*
P<0.05.
Group 1 Group 2 P value
of better treatment strategies. This is particular important in (n=8) (n=14)
severe cases of LRTI in young children. Age 1.5 (2.2) 7 (5.25) 0.005*
This study examines the cytokine profile in nasal washes (months)
from children under the age of one year, diagnosed with Gender 3/5 8/6 0.440
severe LRTI that showed bronchial involvement and clinical (male/female)
findings of viral respiratory disease. A control group Severity 4 (2.1) 3.25 (3.75) 0.330
showing no respiratory disease is used for comparison. (M-WCAS score)
The patient group comprised children up to the age of 12 Days 6.5 (3.5) 4 (3.5) 0.188
months who needed hospitalisation in the paediatric in hospital
department of the Hospital Clínico Universitario, Valladolid, Weight 4510 (2655) 7200 (2877) 0.008*
Spain, from February to May 2005, all of whom had signs of (grams)
viral bronchial illness (tachypnoea, prolonged expiratory
C reactive protein 4.2 (8.9) 8.95 (37.85) 0.297
time, wheezing, rales, chest retractions, dyspnoea of sudden (mg/dL)
appearance, fever).5 Those children who showed acute and
Prematurity 6/2 9/5 0.604
severe presentation were recruited for the study (n=22).
(term/premature)
The decision to hospitalise was made independently by the
attending physicians on the basis of clinical findings alone P : Difference between groups (significance). *
P<0.05.
(e.g., respiratory distress requiring oxygen therapy, poor
feeding with signs of dehydration and/or apnoea). Evaluation microfugue (Eppendorf), cytokine levels were determined
of clinical severity was performed at admission following the in the supernatants using the FlowCytomix multiplex
modified Wood`s Clinical Asthma Score (M-WCAS).5 human Th1/Th2 10plex kit (Bender) and five-colour flow
Those children under one year old with no respiratory or cytometry (Cytomics, Beckman Coulter).
inflammatory pathology admitted during the observation Differences in infant characteristics and levels of cytokines
period were included in the study as age-matched controls. were analysed for significance by a non-parametric test
In all cases, parent or other legal permission was requested (Mann-Whitney). Children with clinical signs of viral
prior to sample extraction and this was recorded in the bronchial infection who were RSV-positive (Group 1, n=8,
patient’s history. age median [IQR]: 1.5 [2.2] months) were compared with an
Nasopharyngeal aspirate samples were obtained within 24 age-matched control group (n=6, age median [IQR]: 2 [1]
hours of admission to hospital by gently flushing the infants’ months). The differences in ages between the RSV-positive
nostrils with 4 mL sterile saline solution. Secretions were group and the control group were not statistically significant
divided into aliquots, snap frozen immediately in dry ice by the Mann-Whitney test (P=0.852).
and then stored at –70˚C until tested. The group of children with clinical signs of viral bronchial
Viral presence was diagnosed in infected infants and infection but negative for the screened viruses (Group 2,
excluded in controls on nasopharyngeal aspirates by direct n=14, age median [IQR]: 7 [5.2] months) was also compared
immunofluorescence staining (Imagen, Dako, Denmark) for with an age-matched control group (n=11, age median
RSV, adenovirus, parainfluenza types 1, 2 and 3, and [IQR]: 4 [4] months). Once again, the differences in ages
influenza A and B after viral culture on MDCK, LLCMK2, between Group 2 and the control group were not statistically
A549 and Hep2 cells. significant by the Mann-Whitney test (P=0.536). The χ2 test
After centrifugation of nasal samples at 5000 rpm in a was used to compare the effects of prematurity and gender.

BRITISH JOURNAL OF BIOMEDICAL SCIENCE 2006 63 (4)


Respiratory syncytial virus was isolated in eight children,
while 14 children were negative for all the viruses screened.
A common pattern in the profile of nasal cytokine secretion
in young children suffering from severe LRTI was apparent.
When the children in Group 1 were compared with its age-
matched control group, IL-1β, IL-8 and IL-6 levels were
higher in the former (P=0.008, P=0.001 and P=0.013,
respectively; Table 1). When similar comparison was made
between Group 2 and its age-matched control group, IL-1β,
IL-8 and IL-6 levels were also higher in the former (P=0.001,
Table 1). In addition, when Groups 1 and 2 were compared,
IL-8 levels were significantly higher in Group 1 patients
(P=0.029, Table 1). As shown in Table 2, differences in the M-
WCAS score for severity between Groups 1 and 2 were not
statistically significant; thus, differences between cytokine
levels in these groups could not be attributed to differences
in clinical severity.
IL-1β, IL-8, and IL-6 are produced during the very early
stages of infection.6 IL-1β stimulates almost all local and
systemic inflammatory responses. IL-6, which can be
Downloaded by [University of Exeter] at 14:11 31 May 2016
induced by IL-1β, is pyrogenic, induces the liberation of
acute-phase reactants by the liver, and, in turn, switches off
pro-inflammatory cytokine production. While IL-1β
mediates the initial adhesive reaction of neutrophils to the
endothelium, IL-8 appears to be essential for the directed
migration of leucocytes into infected tissue. Thus, it is not
unexpected that these cytokines play an important role
independently of the causative viral agent in children under
the age of one year (with an immature immune response
and underdeveloped specific responses).
Laham et al. previously reported lower pro-inflammatory
cytokine production in human metapneumovirus infection
than in RSV and influenza infections.3 On the basis of these
differences, and given that these viruses lead to identical
clinical manifestations, they concluded that innate
inflammation is not critical for the induction of respiratory
symptoms in viral respiratory diseases.
However, Laham’s study compared only infected patients,
and it is important to include non-respiratory pathology
control groups in any study of cytokine secretion profiles. In
the present study, despite the finding of higher levels of IL-
8 in Group 1, the inflammation-related mediators (IL-1β, IL8,
IL6) were clearly elevated in Groups 1 and 2, compared with
their respective age-matched controls. Both groups showed
clinical signs of viral infection, despite the fact that those in
Group 2 were negative for the viruses screened. However,
the virus culture methods employed are less sensitive than
are polymerase chain reaction (PCR)-based methods.
The small number of patients studied here, together with
the number of parameters measured, means that larger
studies are required to limit the possible role of type 1 and
type 2 statistical errors, and such studies are currently
underway. Nevertheless, the immature immunological
status of the studied children supports a possible role for
these innate factors of immunity in RSV-mediated disease.
Comparison of cytokine profiles between patients with mild
and severe disease is also needed.
Why some children present with severe disease while
others do not remains unexplained. However, genetics
seems to explain, at least in part, the different clinical
pictures (i.e., the presence of mutations in the Toll-like
receptor family).7 This particular aspect deserves further
study.
BIOMEDICAL SCIENCE IN BRIEF 175
Finally, IL-1β and IL6, together with the chemokine IL-8,
have an important role in mediating viral clearance;
however, they may also mediate immune-mediated
pathogenesis, leading to exacerbation of the inflammatory
response in the bronchial tree. Some authors have proposed
immunomodulatory approaches to treat the inflammatory
component in LRTI caused by RSV.8,9
On the basis of the results presented here, this approach
deserves further study, not only in RSV infection but also in
those patients with non-identified viral agents who show
clinical criteria of viral LRTI (at least in children less than a
year old).
In conclusion, the high levels of IL-1β, IL-6 and IL-8 in
nasal aspirates reveal the important role played by innate
immunity in bronchial viral disease in young children. This
is independent of the causative viral agent
This study received financial support from the Nutriben Prize of the
Asociación Española de Pediatría and from Fondo de Investigaciones
Sanitarias (project PI050358). Dr Bermejo-Martin was supported
by FIS, CD05/00153. David Bernardo received a grant from the
Ministerio de Educación y Ciencia (programa FPU). Alberto León
received a grant from Junta de Castilla y León (O.C. 14/11/03-O.R.
26/01/0). J. A. Garrote and S. Resino received a grant from Fondo de
Investigaciones Sanitarias. E. Arranz was supported by the
Programa Ramón y Cajal. The authors acknowledge the cooperation
of the nursing team in the Infants’ Section of Hospital Clínico
Universitario de Valladolid, who kindly performed the nasal washes.
Also, thanks are due to Mrs. Ana Sanz for her assistance.
References
1 McNamara P, Flanagan BF, Selby AM, Hart CA, Smyth RL. Pro-
and anti-inflammatory responses in respiratory syncytial virus
bronchiolitis. Eur Respir J 2004; 23:106–12.
2 Openshaw PJ. Antiviral immune responses and lung
inflammation after respiratory syncytial virus infection. Proc Am
Thorac Soc 2005; 2 (2): 121–5.
3. Laham FR, Israele V, Casellas JM et al. Differential production of
inflammatory cytokines in primary infection with human
metapneumovirus and with other common respiratory viruses
of infancy. J Infect Dis 2004; 189: 47–56.
4 Openshaw PJ, Yamaguchi Y, Tregoning JS. Childhood infections,
the developing immune system, and the origins of asthma.
J Allergy Clin Immunol 2004; 114: 1275–7.
5 Martinon-Torres F, Rodriguez-Nunez A, Martinon-Sanchez JM.
Heliox therapy in infants with acute bronchiolitis. Pediatrics
2002; 109: 68–73.
6 Van Reeth K, Van Gucht S, Pensaert M. In vivo studies on
cytokine involvement during acute viral respiratory disease of
swine: troublesome but rewarding. Vet Immunol Immunopathol
2002; 87 (3–4): 161–8.
7 Tal G, Mandelberg A, Dalal I et al. Association between common
Toll-like receptor 4 mutations and severe respiratory syncytial
virus disease. J Infect Dis 2004; 189: 2057–63.
8 Sheeran P, Jafri H, Carubelli C et al. Elevated cytokine
concentrations in the nasopharyngeal and tracheal secretions of
children with respiratory syncytial virus disease. Pediatr Infect
Dis J 1999; 18 (2): 115–22.
9 Rutigliano JA, Graham BS. Prolonged production of TNF-alpha
exacerbates illness during respiratory syncytial virus infection. J
Immunol 2004; 173: 3408–17.
BRITISH JOURNAL OF BIOMEDICAL SCIENCE 2006 63 (4)
 176 BIOMEDICAL SCIENCE IN BRIEF
Role of interleukin-1β and tumour necrosis
factor-α on hydroxyapatite-induced
phagocytosis by murine macrophages
(RAW264.7 cells)
V. K. GOPINATH*, M MUSA†, A. R. SAMSUDIN*
and W. SOSROSENO*†
*
School of Dental Sciences and †Department of Immunology,
School of Medical Sciences, Universiti Sains Malaysia, 16150 Kota Bharu, Malaysia
Hydroxyapatite (HA) is used in bone reconstruction in
dental and orthopaedic surgery. Characteristic features of
HA such as bioactivity, osteoconduction, osteoinduction and
the fact that it is similar in composition to bone mineral are
factors that favour its use in regeneration of hard tissues.1
However, when HA is implanted in tissue, monocytes
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/macrophages are attracted to the implant site.2 Analysis of
tissue from animal models3 and humans demonstrate the
phagocytosis of HA particles.4
The most common causes of failure in implants is aseptic
loosening, which is mostly triggered by wear particles that
activate macrophages to produce cytokines such as
interleukin (IL)-1 and IL-6, thereby initiating bone
resorption around the implant.5 It has been shown that both
IL-1β and tumour necrosis factor-α (TNFα) are identified in
the periprosthetic tissues of patients who undergo revision
surgery for total joint replacement,6 indicating that these
cytokines play a crucial role in inflammation and osteolysis.
Indeed, implant materials such as titanium and alumina
ceramic particles stimulate the production of pro-
inflammatory cytokines by murine or human macrophages
in vitro.7,8
Recently, this group showed that murine macrophage
phagocytic activity induced by HA is under the regulation of
inducible nitric oxide synthase (iNOS).9 Therefore, this study
aims to evaluate the effect of HA-induced phagocytosis on
IL-1β and TNFα production and to demonstrate whether or
not murine macrophages (RAW264.7 cells) use these
cytokines in an autocrine fashion.
Hydroxyapatite powder (3.5–8 µm particle size) was
suspended in sterile saline.10 Latex beads (3 µm; Sigma,
St. Louis) were used as a control. The HA particles were
stained with crystal violet prior to use in the phagocytic
assay.
Hydroxyapatite-induced phagocytosis by RAW264.7 cells
was assessed as previously described.9 Briefly, RAW 264.7
cells, obtained from ATCC, were cultured in Dulbecco’s
modified Eagle’s medium (supplemented with 1%
penicillin/streptomycin) and 10% heat-inactivated fetal calf
serum in a humidified atmosphere of 5% CO2. Five million
cells were incubated with 1 x 105 HA particles or latex beads
in culture medium (1 mL) in a sterile tube for 7, 15, 30 and 60
min. At each time point, culture supernatants were
harvested and IL-1β and TNFα levels were determined by
Correspondence to: Dr. V. K. Gopinath
School of Dental Sciences, Universiti Sains Malaysia,
16150 Kota Bharu, Malaysia
Email: gopinath@kb.usm.my
BRITISH JOURNAL OF BIOMEDICAL SCIENCE 2006 63 (4)
IL-1b (pg/mL
Minutes
Fig. 1. Effect of HA and latex bead particles on the release of IL-1β
by RAW264.7 cells at various incubation times. Untreated cell
culture supernatants were used as negative controls. The bars
represent standard deviation.
an enzyme-linked immunosorbent assay (ELISA) kit (Bender
MedSystems, Vienna, Austria) according to the
manufacturer ’s instructions. Unstimulated cell culture
supernatants were used as a control. All materials used in
the cell culture process were obtained from Sigma.
RAW264.7 cells were stimulated with HA or latex beads in
the presence of various concentrations of anti-murine IL-1β
or TNFα antibody (Sigma). Phagocytic index (PI) was
assessed at 60 min. The number of phagocytosed cells per
100 cells was assessed by light microscopy (Leica, Germany).
The PI was calculated by counting the number of engulfed
particles per macrophage, as described previously.11 All
experiments were carried out in triplicate.
Data analysis included paired t-test at different time
points and one–way analysis of variance followed by
Fisher’s least squared difference in the experiments where
neutralising IL-1β and TNFα antibodies were used with
activated cells (SPSS, Chicago, IL).
IL-1β and TNFα were detected at 30 and 60 min in
unstimulated RAW264.7 cell cultures (Figs. 1 and 2).
Following activation of the cells, IL-1β concentration was
higher in cultures treated with latex beads than with HA
(P<0.05; Fig. 1). Murine macrophages activated with HA and
latex bead particles produced significantly higher levels
of IL-1β when compared to unstimulated controls at 15,
30 and 60 min (P<0.05). Levels of IL-1β were not detected at
7 min following activation with HA or latex beads.
Examining the trend within groups, IL-1β production
increased at 7–15 and 15–30 min (P<0.05) but did not
increase significantly at 30–60 min following activation with
HA or latex beads.
TNFα levels in the cultures treated with HA were higher
than in those treated with latex beads (P<0.05; Fig. 2).
Examining the trend within groups, TNFα production
showed significant increases at 7–15 ,7–30 and 7–60 min
(P<0.05); however, the HA-treated group also showed
significance at 15–60 min (P<0.05) but did not increase
significantly at 15–30 and 30–60 min. In the latex bead-
treated group, TNFα production did not increase
significantly at 15–30, 15–60 or 30–60 min.
Pretreatment of cells with anti-murine IL-1β antibody

TNF-α (pg/mL)
Minutes
Fig. 2. Effect of HA and latex bead particles on the release of TNFα
by RAW264.7 cells at various incubation times. Untreated cell
culture supernatants were used as negative controls. The bars
Downloaded by [University of Exeter] at 14:11 31 May 2016
represent standard deviation.
resulted in lower phagocytic activity induced by HA and
latex beads (P<0.05; Fig. 3). Similarly, HA- and latex bead-
induced phagocytosis by RAW264.7 cells was significantly
reduced when the cells were coated with anti-murine TNFα
(P<0.05; Fig. 4).
The present study shows that murine macrophages
upon ingestion of HA and latex beads secreted IL-1β and
TNFα. Latex beads used as a control in the present study are
know to activate macrophages to produce cytokines as
observed in previous in vitro studies where latex beads
activated J774.2 cells to produce TNF-α12 and human
monocyte to secrete IL-1β and TNFα.13 Unstimulated
RAW 264.7 cells that produced IL-1β and TNFα at 30 and
60 minutes were used as a negative control in the present
study. This indicated spontaneous activation of the
macrophages during isolation and culture; this is in
correlation with previous studies using RAW 264.7 cells.14
The present study shows that HA and latex beads
stimulate RAW264.7 cells to produce TNFα in a time-
PI
Anti-TNF-α (ng/mL)
Fig. 4. RAW264.7 cells stimulated with HA or latex bead in
the presence of anti-murine TNFα. The PI was assessed at
60 min by calculating the number of ingested particles per
macrophage.
BIOMEDICAL SCIENCE IN BRIEF 177
PI
Anti-IL-1β (µg/mL)
Fig. 3. RAW264.7 cells stimulated with HA or latex beads in the
presence of anti-murine IL-1β. The PI was assessed at 60 min by
calculating the number of ingested particles per macrophage. The
bars represent standard deviation.
dependent manner. These results are not unexpected as
macrophages during phagocytosis are known to produce
cytokines.15 Similarly, murine macrophages (ANA-1 cells)
and human monocytes (THP-1 cells) synthesise TNFα and
IL-1β when the cells come into contact with implant
materials such as titanium and HA.7,8,16,17 This suggests that
implant materials activate macrophages to produce pro-
inflammatory cytokines.
While TNFα produced by HA-stimulated RAW264.7 cells
was detected after 7-min incubation, IL-1β production by the
same cell cultures was observed later at 15 min, suggesting
that TNFα may be produced earlier than IL-1β during
HA-induced macrophage phagocytosis; however, the exact
mechanism to explain these findings remains unclear.
Previous reports have shown that TNFα messenger RNA
(mRNA) expression in human macrophages responding to
titanium is detected earlier than is IL-1β gene expression.18
TNF-α is also known to induce production of IL-1β.19
Therefore, it can be assumed that HA stimulates RAW264.7
cells to produce TNFα initially, which then in turn initiates
production of IL-1β; however, this remains to be confirmed.
It should also be noted that IL-1β production by latex
bead-activated cells was higher than that by HA-stimulated
cells. However, HA-activated macrophages secreted
significantly higher amounts of TNFα than did latex bead-
activated cells. This might be due to the physical structure of
the particle, as study has shown that fewer titanium particles
than polyethylene particles are required to stimulate peak
production of TNFα and IL-1β.20
The present study also demonstrated that decreased
phagocytosis of HA and latex beads was observed when the
cells were incubated with HA or latex beads in the presence
of anti-murine TNFα or murine IL-1β antibody. This
suggests that TNFα and IL-1β produced by HA-stimulated
RAW264.7 cells may be required to induce phagocytosis.
Macrophages are the main source of both cytokines,
which are required for cell activation and function.19,21
Defective phagocytosis, as seen in TNFα- and IL-1β-deficient
mice,22,23 highlights the importance of the role of TNFα and
IL-1β in murine macrophage phagocytosis induced by HA.
Extrapolation of the results from the present study to
BRITISH JOURNAL OF BIOMEDICAL SCIENCE 2006 63 (4)
 178 BIOMEDICAL SCIENCE IN BRIEF
humans remains speculative; however, they confirm
previous reports that show HA-stimulated human
macrophages produce TNFα and IL-1β,16,17 which suggests
that HA-stimulated RAW264.7 cells are a good model of HA-
induced human macrophage responses. Furthermore, when
used as an implant in humans, HA activates macrophages,24
and analysis of periprosthetic tissues from patients with
implants has identified cytokines such as TNFα and IL-1β.6
Thus, it seems plausible that macrophages are activated
immediately after implantation with HA and then
subsequently produce TNFα and IL-1β. This may enhance
macrophage infiltration and function and induce pro-
inflammatory responses to surrounding implanted
materials, as is seen in other pathophysiological disease
processes.25
In summary, the results of the present study show that HA
stimulates murine macrophages (RAW264.7 cells) to produce
IL-1β and TNFα; however, addition of neutralising
antibodies to IL-1β or TNFα result in reduction of particle-
induced phagocytosis. This suggests that HA-induced
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phagocytic activity by murine macrophages is dependent on
the presence of IL-1β and TNFα.
This research was supported by an e-IRPA category EA grant (No.
06-02-05-00029 EAR) from the Malaysian Government. The
hydroxyapatite was kindly prepared by staff of the School of
Material and Mineral Resources Engineering, Universiti Sains
Malaysia, Malaysia.
References
1 LeGeros RZ. Properties of osteoconductive biomaterials:
calcium phosphates. Clin Orthop Relat Res 2002; 395: 81–98.
2 Heymann D, Pradal G, Benahmed M. Cellular mechanisms of
calcium phosphate ceramic degradation. Histol Histopathol 1999;
14: 871–7.
3 Rahbek O, Kold S, Bendix K, Overgaard S, Soballe K. No effect
of hydroxyapatite particles in phagocytosable sizes on implant
fixation: an experimental study in dogs. J Biomed Mater Res A
2005; 73: 150–7.
4 Bloebaum RD, Beeks D, Dorr LD, Savory CG, DuPont JA,
Hofmann AA. Complications with hydroxyapatite particulate
separation in total hip arthroplasty. Clin Orthop Relat Res 1994;
298: 19–26.
5 Jiranek WA, Machado M, Jasty M et al. Production of cytokines
around loosened cemented acetabular components. Analysis
with immunohistochemical techniques and in situ
hybridization. J Bone Joint Surg Am 1993; 75: 863–79.
6 Catelas I, Campbell PA, Dorey F, Frausto A, Mills BG, Amstutz
HC. Semi-quantitative analysis of cytokines in MM THR tissues
and their relationship to metal particles. Biomaterials 2003; 24:
4785–97.
7 Soloviev A, Schwarz EM, Kuprash DV et al. The role of p105
protein in NFkappaB activation in ANA-1 murine macrophages
following stimulation with titanium particles. J Orthop Res 2002;
20: 714–22.
8 Yagil-Kelmer E, Kazmier P, Rahaman MN, Bal BS, Tessman RK,
Estes DM. Comparison of the response of primary human blood
monocytes and the U937 human monocytic cell line to two
different sizes of alumina ceramic particles. J Orthop Res 2004; 22:
832–8.
9 Gopinath VK, Musa M, Samsudin AR, Lalitha P, Sosroseno W.
BRITISH JOURNAL OF BIOMEDICAL SCIENCE 2006 63 (4)
Role of nitric oxide in hydroxyapatite-induced phagocytosis by
murine macrophage cell line (RAW264.7). Arch Oral Biol 2006;
51: 339–44.
10 Nordsletten L, Hogasen AK, Konttinen YT, Santavirta S,
Aspenberg P, Aasen AO. Human monocytes stimulation by
particles of hydroxyapatite, silicon carbide and diamond:
in vitro studies of new prosthesis coatings. Biomaterials 1996; 17:
1521–7.
11 Barid I, Nawawi S, Sosroseno W. Effects of parachlorophenol
and camphorated parachlorophenol on the phagocytic activities
of a murine macrophage cell line (RAW264.7). J Endod 2002; 28:
806–10.
12 Olivier CR, Hindie J-L, Duval M, Bomila-Koradjim G, Nagel
MD. Uptake of polystyrene bead bearing functional groups by
macrophages and fibroblasts. Colloids Surf B Biointerfaces 2004;
33: 23–31.
13 Harada Y, Wang JT, Doppalapudi VA et al. Differential effects of
different forms of hydroxyapatite and hydroxyapatite/
tricalcium phosphate particulates on human monocyte/
macrophages in vitro. J Biomed Mater Res 1996; 31: 19–26.
14 Huang SL, Cheng WL, Lee CT, Huang HC, Chan CC.
Contribution of endotoxin in macrophage cytokine response to
ambient particles in vitro. J Toxicol Environ Health 2002; 65:
1261–72.
15 Underhill DM, Ozinsky A. Phagocytosis of microbes: complexity
in action. Annu Rev Immunol 2002; 20: 825–52.
16 Laquerriere P, Grandjean-Laquerriere A, Guenounou M,
Laurent-Maquin D, Frayssinet P, Nardin M. Correlation between
sintering temperature of hydroxyapatite particles and the
production of inflammatory cytokines by human monocytes.
Colloids Surf B Biointerfaces 2003; 30: 207–13.
17 Grandjean-Laquerriere A, Laquerriere P, Guenounou M,
Laurent-Maquin D, Phillips TM. Importance of the surface area
ratio on cytokine production by human monocytes in vitro
induced by various hydroxyapatite particles. Biomaterials 2005;
26: 2361–9.
18 Garrigues GE, Cho DR, Rubash HE, Goldring SR, Herndon JH,
Shanbhag AS. Gene expression clustering using self-organizing
maps: analysis of the macrophage response to particulate
biomaterials. Biomaterials 2005; 26: 2933–45.
19 Aggarwal BB, Samanta A, Feldman M. TNF-alpha. In:
Oppenheim JJ, Feldman M, eds. Cytokines Reference.
Amsterdam/New York: Elsevier/Academic Press, 2000: 413–34.
20 Rader CP, Sterner T, Jakob F, Schutze N, Eulert J. Cytokine
response of human macrophage-like cells after contact with
polyethylene and pure titanium particles. J Arthroplasty 1999; 14:
840–8.
21 Dinarello CA. Interleukin-1. Cytokine Growth Factor Rev 1997; 8:
253–65.
22 Fantuzzi G, Sacco S, Ghezzi P, Dinarello CA. Physiological and
cytokine responses in IL-1 beta-deficient mice after zymosan-
induced inflammation. Am J Physiol 1997; 273: 400–6.
23 Marino MW, Dunn A, Grail D et al. Characterization of tumor
necrosis factor-deficient mice. Proc Natl Acad Sci USA 1997; 94:
8093–8.
24 Chatelet JC, Setiey L. Long-term bone behavior in total primary
hip arthroplasty with a fully hydroxyapatite-coated femoral
stem: a continuous series of 120 cases with twelve years follow-
up (in French). Rev Chir Orthop Reparatrice Appar Mot 2004; 90:
628–35.
25 Esch T, Stefano G. Proinflammation: a common denominator or
initiator of different pathophysiological disease processes. Med
Sci Monit 2002; 8: 1–9.
 BIOMEDICAL SCIENCE IN BRIEF 179

Genetic heterogeneity of the cytolethal Table 1. Isolates of C. lari analysed in the present study.

distending toxin B (cdtB) gene locus among

isolates of Campylobacter lari Isolate Source Country Accession
number
UN C. lari JCM2530T Seagull Japan AB 266778
M. SHIGEMATSU*, Y. HARADA*, T. SEKIZUKA*, O. MURAYAMA*, UN C. lari 28 Mussel N. Ireland AB 266779
S. TAKAMIYA†, B. C. MILLAR‡, J. E. MOORE‡ and M. MATSUDA*
*
UN C. lari 48 Mussel N. Ireland AB 266780
Laboratory of Molecular Biology, Graduate School of Environmental Health
Sciences, Azabu University, Fuchinobe 1-17-71, Sagamihara 229-8501; †Department UN C. lari 84C-1 Human N. Ireland AB 266781
of Molecular and Cellular Parasitology, Juntendo University School of Medicine, UN C. lari 84C-2 Human N. Ireland AB 266782
Bunkyoku Hongo 2-1-1, Tokyo 113-8421, Japan; and ‡Department of Bacteriology, UN C. lari 99 Sea water N. Ireland AB 266783
Northern Ireland Public Health Laboratory, Belfast City Hospital, Belfast BT9 7AD, UN C. lari 170 Seagull Japan AB 266784
Northern Ireland, UK
UN C. lari 264 Mussel N. Ireland AB 266785
UN C. lari 274 Mussel N. Ireland AB 266786
Thermophilic Campylobacter species, primarily C. jejuni and C. UN C. lari 288 Black-tailed gull Japan AB 266787
coli, are curved Gram-negative bacteria that are the UN C. lari 293 Seagull Japan AB 266788
recognised cause of acute bacterial diarrhoea around the UN C. lari 296 Human Canada AB 266789
world.1,2 C. lari is a relatively recently discovered thermophilic UN C. lari 298 Human Canada AB 266790
Downloaded by [University of Exeter] at 14:11 31 May 2016

Campylobacter species first isolated from mammalian and UN C. lari 299 Human USA AB 266791
avian species, particularly seagulls of the genus Larus.1,3 C. lari
UN C. lari 381 Mussel N. Ireland AB 266792
has also been shown to be a cause of clinical infection.4–7
An atypical group of isolates of urease-positive UN C. lari 2316A3 NA NA AB 266793
thermophilic campylobacters (UPTC) was first isolated from UPTC CF89-12 River water Japan AB 266794
the natural environment in England in 1985.8 Thereafter, UPTC CF89-14 River water Japan AB 266795
these organisms were described as a biovar or variant of UPTC NCTC12892 River water England AB 266796
C. lari.9,10 Subsequent reports described four human isolates UPTC NCTC12893 River water England AB 266797
in France.9,11 Some additional isolates of UPTC have also been
UPTC A1 Seagull N. Ireland AB 266798
reported in Ireland,12–14 in The Netherlands15 and in Japan.16,17
The possible association of UPTC with human disease UPTC A3 Seagull N. Ireland AB 266799
remains unclear. Two representative taxa, namely urease- UPTC 89049 Human France AB 266800
negative (UN) C. lari and UPTC, occur within the species of UPTC 92251 Human France AB 266801
C. lari.18 NA: not available.
Although several Campylobacter species’ cytotoxins have
been identified,19,20 only the cytolethal distending toxin
(CDT) has been characterised in detail.21,22 The cdt genes of A schematic representation of the cdtB gene and its
C. jejuni have been cloned and characterised by Pickett et al.21 genetic loci for C. lari RM2100 (GenBank Accession No.
However, in relation to the cdt genes, no reports have yet AAFK00000000)23 including the locations of a primer pair for
appeared for C. lari. the cdtB (JCB common up and JCB common down [Asakura
Therefore, the aim of the present study is to clone, primer])24 employed in the present study for PCR
sequence and analyse the cdtB gene of C. lari isolates and amplification is shown in Figure 1. This primer pair was
compare the sequences obtained with those of other designed to generate a product of approximately 700 bp
thermophilic campylobacters. (equivalent to a 90% segment of the cdtB structural gene of
Twenty-four isolates of C. lari (UN C. lari [n=16] and UPTC C. lari RM2100, AAFK00000000) of the cdtB gene with
[n=8]) were used in the present study (Table 1), together C. jejuni, C. coli and C. fetus isolates.24 The polymerase chain
with three reference strains (JCM2530T, NCTC12892 and reaction (PCR) was performed in 50-µL reaction volumes, for
NCTC12893). The test organisms were isolated from 30 cycles at 94˚C for 30 sec, 55˚C for 30 sec, 72˚C for 45 sec,
different sources in several countries. The organisms were followed by a final extension of 72˚C for 5 min.
cultured on blood agar containing defibrinated horse blood Amplified PCR products were separated by 1.0% (w/v)
(Nippon Bio-Test, Tokyo, Japan) and supplemented with agarose gel electrophoresis in 0.5x TBE at 100 V and detected
campylobacter-selective medium (Nissui, Tokyo, Japan), by ethidium bromide staining. PCR products amplified by
under microaerophilic conditions at 37˚C for two days. the constructed primer pair for the partial cdtB gene were
Template DNA was prepared by boiling in water at 95˚C purified using a QIA quick PCR purification kit (Qiagen, CA,
for five minutes. The PCR mixture contained 10 mmol/L USA) and inserted in the pGEM-T vector using the pGEM-T
Tris-HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl2, Easy Vector System (Promega, Tokyo, Japan).
400 µmol each dNTP, 1 µmol each primer, and 1 unit of Thermus Sequencing of the cloned cdtB gene fragment was
aquaticus (Taq) DNA polymerase (Takara Bio, Shiga, Japan). performed (Hitachi DNA autosequencers SQ-5500L and SQ-
5500EL) after a dideoxy nucleotide sequencing reaction,
Correspondence to: Dr. Motoo Matsuda using a Thermo Sequenase premixed cycle sequencing kit
Laboratory of Molecular Biology, School of Environmental Health Sciences, (Amersham Pharmacia Biotech, Tokyo, Japan). Sequence
Azabu University, Fuchinobe 1-17-71, Sagamihara 229-8501, Japan analysis of the PCR amplicons was carried out using the
Email: matsuda@azabu-u.ac.jp GENETYX-MAC (version 9) computer software.

BRITISH JOURNAL OF BIOMEDICAL SCIENCE 2006 63 (4)

 180 BIOMEDICAL SCIENCE IN BRIEF
Fig. 1. Schematic representation of the cdtB genes of C. lari,
including A) the locations of a primer pair for the amplification of the
cdtB gene fragment of C. lari and B) nucleotide sequences of the
primer pair.24
Nucleotide sequences of approximately 720 bp of the
Downloaded by [University of Exeter] at 14:11 31 May 2016
partial cdtB gene fragments from 24 C. lari isolates were
compared to each other and to accessible sequence data of
other thermophilic campylobacters (C. lari RM2100,
AAFK00000000; C. jejuni RM1221, CP000025; C. coli RM2228,
AAFL00000000), employing CLUSTAL W software (1.7
program),25 which was incorporated in DDBJ. A
phylogenetic tree was constructed by the unweighted pair
group method, using arithmetic means analysis (UPGMA)
available on the GENETYX-MAC program (version 9.0).
In the present study, the primer pair (JCB common up and
down, Asakura primer) 24 amplified PCR products of
approximately 720 bp in length with all 24 isolates of C. lari
(UN C. lari [n=16] and UPTC [n=8]) (data not shown). For
PCR cloning of the partial cdtB fragments, PCR products
were purified and inserted in the pGEM-T vector using the
TA cloning procedure. The nucleotide and deduced amino
acid sequence data of the partial and possible open reading
frame (ORF) of the cdtB gene fragments cloned and
sequenced from 24 isolates of C. lari are accessible in the
DDBJ/EMBL/GenBank nucleotide sequence database (Table 1).
The nucleotide sequences of the partial cdtB gene
fragments (approximately 720 bp) of 16 UN C. lari isolates
showed 81–100% similarity to each other. Those of eight
UPTC isolates showed 74.7–99.6% similarity to each other. In
addition, those of the cdtB gene fragments showed
74.7–100 % similarity among 16 UN C. lari isolates and eight
UPTC isolates. Moreover, the nucleotide sequences of the
cdtB gene fragments of 25 C. lari isolates, including C. lari
RM2100, showed 63.9–88.0% sequence similarity to those of
C. jejuni RM1221 and C. coli RM2228.
When the deduced amino acid sequences of the partial
and possible ORFs of the cdtB fragments of 24 C. lari isolates
were determined, the partial and possible ORFs were
estimated to consist of 241–242 amino acids. The deduced
amino acid sequence alignments were also examined for
partial and possible ORFs of the cdtB fragments of 24 C. lari
isolates, as well as those of C. lari RM2100, C. jejuni RM1221
and C. coli RM2228. The partial and possible ORFs of the cdtB
gene fragments from 25 C. lari isolates, including C. lari
RM2100, showed 78.2–99.6 % amino acid sequence similarity
to each other, and 66.6–78.5% amino acid sequence similarity
to those of the ORFs of C. jejuni RM1221 and C. coli RM2228.
Thus, cdtB had a high sequence heterogeneity in both
BRITISH JOURNAL OF BIOMEDICAL SCIENCE 2006 63 (4)
nucleotide and deduced amino acid sequences with the
24 C. lari isolates examined. An extremely high sequence
variability of the cdtB was also demonstrated between the
24 C. lari isolates and the other two thermophilic
campylobacters (C. jejuni and C. coli).
In relation to the cdt gene in campylobacters, Martinez et al.
found the cdt gene variant, a putative shortened cdtB gene
fragment in two of the 100 C. jejuni isolates derived from
several sources (humans and animals) and countries, by a
multiplex PCR procedure. Although, they observed several
point mutations throughout the remaining cdtA, -B and -C
sequences, it has become very clear that cdt genes from
C. jejuni are highly homologous.26
In the present study, the partial cdtB gene (approximately
720 bp), similar in length in 24 isolates of C. lari, was
examined using a constructed PCR primer in silico (Asakura
primer). Thus, essentially all the C. lari isolates employed in
the present study had the cdtB gene. However, high genetic
heterogeneity of 74.7–100% nucleotide sequence similarity
of the partial cdtB gene fragment was identified among all
24 isolates of C. lari by cloning and sequencing procedures.
This is the first demonstration of the genetic heterogeneity
of cdt genes among C. lari isolates by cloning and sequencing
procedures.
A dendrogram showing phylogenetic relationships was
constructed using UPGMA, based on the nucleotide
sequence information of the partial cdtB gene fragments
amplified from 24 C. lari isolates (Fig. 2) and demonstrated
hypervariability among the cdtB genes of 24 C. lari isolates.
This implies that the C. lari organisms examined are
genetically variable, at least based on the cdtB gene
information obtained, and separate from the other two
thermophilic campylobacters, C. jejuni and C. coli.
Previously, this group demonstrated genetic
hypervariability of 31 isolates of UPTC, as determined by
multilocus enzyme electrophoresis typing (MLEE).14 The
present results for the cdtB gene from eight UPTC isolates is
consistent with the results of the genetic hypervariability
obtained by MLEE.
UPTC A1
UPTC A3
UPTC 92251
UN C. lari 381
UPTC 89049
UN C. lari 274
UN C. lari 84C-2
UN C. lari 288
UN C. lari 299
UN C. lari 84C-1
UN C. lari 48
UN C. lari 2316A3
UN C. lari RM2100
UN C. lari 264
UN C. lari 296
UN C. lari 28
UN C. lari JCM2530T
UN C. lari 298
UN C. lari 170
UN C. lari 293
UPTC CF89-12
UPTC CF89-14
UN C. lari 99
UPTC NCTC12892
UPTC NCTC12893
C. coli RM2228
C. jejuni RM1221
0.5 0.1 0.1824
Fig. 2. Phylogenetic tree based on sequence similarity data of the
cdtB gene fragments from 24 C. lari isolates examined and
thermophilic isolates of C. lari RM2100, C. jejuni RM1221 and
C. coli RM2228. Values in the figure represent evolutionary distances.

References
1 Benjamin J, Leaper S, Owen R J, Skirrow MB. Description of
Campylobacter laridis, a new species comprising the nalidixic
acid-resistant thermophilic Campylobacter (NARTC) group. Curr
Microbiol 1983; 8: 231–8.
2 Blaser MJ, Taylor DN, Feldman RA. Epidemiology of
Campylobacter jejuni infections. Epidemiol Rev 1983; 5: 157–76.
3 Skirrow MB, Benjamin J. ‘1001’ Campylobacters: cultural
characteristics of intestinal campylobacters from man and
animals. J Hyg (Camb) 1980; 85: 427–42.
4 Nachamkin I, Stowell C, Skalina D, Jones AM, Hoop RM,
Smibert RM. Campylobacter laridis causing bacteremia in an
immunosuppressed patient. Ann Intern Med 1984; 101: 55–7.
5 Simor A E, Wilcox L. Enteritis associated with Campylobacter
laridis. J Clin Microbiol 1987; 25: 10–2.
6 Martinot M, Jaulhac B, Moog R et al. Campylobacter lari
bacteremia. Clin Microbiol Infect 2001; 7: 96–7.
7 Werno AM, Klena JD, Shaw GM, Murdoch DR. Fatal case of
Campylobacter lari prosthetic joint infection and bacteremia in an
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immunocompetent patient. J Clin Microbiol 2002; 40: 1053–5.
8 Bolton FJ, Holt AV, Hutchinson DN. Urease-positive
thermophilic campylobacters. Lancet 1985; i: 1217–8.
9 Mégraud F, Chevrier D, Desplaces N, Sedallian A, Guesdon JL.
Urease-positive thermophilic Campylobacter (Campylobacter
laridis variant) isolated from an appendix and from human
feces. J Clin Microbiol 1988; 26: 1050–1.
10 Owen RJ, Costas M, Sloss L, Bolton FJ. Numerical analysis of
electrophoretic protein patterns of Campylobacter laridis and
allied thermophilic campylobacters from the natural
environment. J Appl Bacteriol 1988; 65: 69–78.
11 Bezian MC, Ribou G, Barberis-Giletti C, Megraud F. Isolation of
a urease-positive thermophilic variant of Campylobacter lari from
a patient with urinary tract infection. Eur J Clin Microbiol Infect
Dis 1990; 9: 895–7.
12 Wilson IG, Moore JE. Presence of Salmonella spp. and
Campylobacter spp. in shellfish. Epidemiol Infect 1996; 116: 147–53.
13 Kaneko A, Matsuda M, Miyajima M, Moore JE, Murphy PG.
Urease-positive thermophilic strains of Campylobacter isolated
from seagulls (Larus spp.). Lett Appl Microbiol 1999; 29: 7–9.
14 Matsuda M, Kaneko A, Stanley T et al. Characterization of
urease-positive thermophilic Campylobacter subspecies by
multilocus enzyme electrophoresis typing. Appl Environ
Microbiol 2003; 69: 3308–10.
15 Endtz HP, Vliegenthart JS et al. Genotypic diversity of
Campylobacter lari isolated from mussels and oysters in The
Netherlands. Int J Food Microbiol 1997; 34: 79–88.
16 Matsuda M, Kaneko A, Fukuyama M et al. First finding of
urease-positive thermophilic strains of Campylobacter in river
water in the Far East, namely, in Japan, and their phenotypic
and genotypic characterization. J Appl Bacteriol 1996; 81: 608–12.
17 Matsuda M, Shibuya T, Itoh Y et al. First isolation of urease-
positive thermophilic Campylobacter (UPTC) from crows (Coruvs
levaillantii) in Japan. Int J Hyg Environ Health 2002; 205: 321–4.
18 Matsuda M, Moore JE. Urease-positive thermophilic
Campylobacter species. Appl Environ Microbiol 2004; 70: 4415–8.
19 Johnson WM, Lior H. A new heat-labile cytolethal distending
toxin (CLDT) produced by Campylobacter spp. Microb Pathog
1988; 4: 115–26.
20 Schulze F, Hanel I, Borrmann E. Formation of cytotoxins by
enteric Campylobacter in humans and animals. Zentralbl Bakteriol
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21 Pickett CL, Pesci EC, Cottle DL, Russell G, Erdem AN, Zeytin H.
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Prevalence of cytolethal distending toxin production in
Campylobacter jejuni and relatedness of Campylobacter spp. cdtB
genes. Infect Immun 1996; 64: 2070–8.
22 Pickett CL, Whitehouse CA. The cytolethal distending toxin
family. Trends Microbiol 1999; 7: 292–7.
23 Fouts DE, Mongodin EF, Mandrell RE et al. Major structural
differences and novel potential virulence mechanisms from the
genomes of multiple Campylobacter species. PLoS Biol. 2005; 3:
e15. 0072–85. http://biology.plosjournals.org/
24 Asakura M, Yoshida E, Taguchi M, Kobayashi K, Yamazaki S.
Poster presentation at the 77th annual meeting of the Japanese
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2004; 59: 283.
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the sensitivity of progressive multiple sequence alignment
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and weight matrix choice. Nucleic Acids Res 1994; 212: 4673–80.
26 Martinez I, Mateo E, Churruca E, Girbau C, Alonso R,
Fernandez-Astorga A. Detection of cdtA, cdtB and cdtC genes in
Campylobacter jejuni by multiplex PCR. Int J Med Microbiol 2006;
296: 45–8.
Molecular diagnosis of native mitral valve
endocarditis due to Corynebacterium
striatum
S. ELSHIBLY*, J. XU†, B. C. MILLAR†, C. ARMSTRONG*
and J. E. MOORE†
*
Department of Microbiology, Craigavon Area Hospital, Craigavon, Co. Armagh;
and †Northern Ireland Public Health Laboratory, Department of Bacteriology,
Belfast City Hospital, Lisburn Road, Belfast, Northern Ireland
Corynebacterium striatum is a relatively rare causal agent of
infective endocarditis (IE). Its association with IE is
complicated by its relatively slow growth, its role as a
possible contaminant in such cases, and the phenotypic
difficulty in laboratory identification.
The present study reports a case of culture-positive
endocarditis due to C. striatum in a 77-year-old woman who
showed no risk factors for endocarditis. To date, there have
been 12 reports of endocarditis due to C. striatum, which are
discussed and summarised.
Currently, use of molecular microbiological methods has
been limited, but such techniques have been adopted in
several clinical microbiology laboratories. Molecular
methods of identification using the polymerase chain
reaction (PCR) and sequencing of 16S ribosomal DNA
(rDNA) from the causal agent isolated by blood culture may
be very useful in the identification of causal agents in
culture-positive endocarditis, which prove difficult to
identify using a conventional approach.
The 77-year-old patient presented to hospital with a
chronic three-month history of weight loss, fatigue and
arthralgia. A history was taken in accordance with the
Correspondence to: Dr. B. Cherie Millar
Northern Ireland Public Health Laboratory,
Belfast City Hospital, Belfast BT9 7AD, Northern Ireland, UK
Email: bcmillar@niphl.dnet.co.uk
BRITISH JOURNAL OF BIOMEDICAL SCIENCE 2006 63 (4)
 182 BIOMEDICAL SCIENCE IN BRIEF
questionnaire criteria for endocarditis, as described
previously by Paturel et al.1 The patient had no risk factors
for IE nor any underlying disease. She was febrile, anaemic
and demonstrated septic embolisation. On auscultation,
she had a new cardiac murmur, which was shown to
originate from leakage from her native mitral valve. In
addition, the presence of a vegetation on this valve was
demonstrated.
On presentation, she had an elevated C-reactive protein
(CRP) of 244.7 mg/L. This rose to 255.5 mg/L and then
decreased to 46.4 mg/L nine days after commencement of
intravenous antibiotics. Three sets of blood cultures were
taken, all of which grew a Gram-positive rod provisionally
identified by conventional techniques as a Corynebacterium
sp. Antibiotic treatment was continued for a further seven
weeks and the patient was discharged without any signs or
symptoms of IE.
Phenotypic identification of the Gram-positive organism,
isolated from three sets of blood cultures (isolate identifier:
CAHE68720) was performed using the API Corynebacterium
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system (bioMérieux, Las Halles, France). It gave the profile
3140345 and an identification of Brevibacterium sp. (47%).
A repeat test gave the API profile 3100305, yielding
Corynebacterium group G (55.6%). The organism was
sensitive to vancomycin, teicoplanin, gentamicin and
rifampicin by standard in vitro antibiotic disc diffusion
susceptibility assay.
Given the relatively poor phenotypic identification
obtained, the isolate was forwarded for molecular
identification through PCR amplification and direct
sequencing of a large but partial region of the 16S rRNA
gene, corresponding to the base position of approximately
811–1374 of Escherichia coli ATCC 25922 16S rRNA (GenBank
accession number: X80724). All DNA isolation procedures
were carried out in accordance with the DNA contamination
management guidelines of Millar et al.2 and in a Class II
biological safety cabinet (MicroFlow, England). This was
situated in a room separated from that used to set up nucleic
acid amplification reaction mixes, and also from the ‘post-
PCR’ room, in order to minimise contamination and the
possibility of false-positive results.
Bacterial DNA was extracted from the isolate using the
Roche high-purity PCR template preparation kit (Roche,
England) in accordance with the manufacturer ’s
instructions. Extracted DNA was transferred to a clean tube
and stored at –80˚C prior to PCR. For each batch of
extractions, a negative extraction control containing all
reagents other than the isolate was performed. Reaction
mixes (50 µL) were set up as follows: 10 mmol/L Tris–HCl
(pH 8.3), 50 mmol/L KCl, 2.5 mmol/L MgCl2 200µmol/L (each)
dATP, dCTP, dGTP and dTTP, 1.25 units Thermus aquaticus
(Taq) DNA polymerase (Amplitaq, Perkin Elmer), 0.2 µmol/L
appropriate ‘broad range’ primers (PSL3 [forward] 5’ -AGG
ATT AGA TAC CCT GGT AGT CCA-3’ and P13P4 [reverse]
5’ - AGG CCC GGG AAC GTA TTC AC -3’) and 4 µL DNA
template.
Prior to PCR cycling, sealed tubes containing DNA
template and all PCR reagents were introduced to the
thermal cycler at 96˚C to avoid non-specific annealing during
the initial ramp stage. The reaction mixtures were subjected
to the following thermal cycling parameters in a Perkin Elmer
Cetus 2400 thermocycler: 96˚C for 3 min followed by 40
cycles at 96˚C for 1 min, 55˚C for 1 min, 72˚C for 1 min, and
BRITISH JOURNAL OF BIOMEDICAL SCIENCE 2006 63 (4)
a final extension at 72˚C for 10 min. During each run,
molecular-grade water was included randomly as several
negative controls and DNA templates from Staphylococcus
aureus were included as a positive control, as appropriate.
Following amplification, samples (15 µL) were removed
from each reaction mixture and examined by electrophoresis
(80V, 45 min) in gels composed of 2% (w/v) agarose (Gibco,
UK) in TAE buffer (40 mmol/L Tris, 20 mmol/L acetic acid,
1 mmol/L EDTA [pH 8.3]), and then stained with ethidium
bromide (5 µg/100 mL).
Gels were visualised under ultraviolet (UV) illumination
using a gel image analysis system (UVP Products, England)
and all images were archived as digital graphic (.bmp) files.
Amplicons chosen for automated sequencing were purified
using a QIAquick PCR purification kit (Qiagen, UK) and
eluted in Tris–HCl (10 mmol/L [pH 8.5]) prior to sequencing,
in order to remove dNTPs, polymerases, salts and primers.
The amplicon was sequenced on the ALF II Express
automated sequencer using the primer PSL which was
labelled with Cy-5 fluorescent dye and used in conjunction
with the Sequenase fluorescence-labelled primer cycle
sequencing kit (Thermo, Amersham, UK).
The resulting sequence obtained (570 bp) was compared
with those stored in the GenBank data system using FASTA
alignment software (www.ebi.ac.uk), and deposited in
GenBank (accession number DQ018338). On BLAST analysis
in combination with previously reported criteria used for
interpretation of partial 16S rRNA gene sequences,5 the
sequence gave a 100% identification for C. striatum
(GenBank accession number AY008302) followed by
C. xerosis X81906 (99% identity) and an uncultured
Rhodococcus sp. (98% identity).
At present, there are approximately 73 recognised species
in the genus Corynebacterium, including C. striatum. This
species was first described by Chester in 1901 as Bacterium
striatum, and then by Eberson in 1918. 6 Several
Corynebacterium spp. have been described as causal agents of
IE.7 Over the past 15 years, however, there have only been 10
cases of C. striatum endocarditis. In a review of the 12 cases
in the literature, seven patients were male. The median age
was 62.6 years for females (age range: 46–72 years) and 59
years for males (age range: 24–76 years), as shown in Table 1.
Native valves have been most frequently infected (three
mitral, three aortic, one pulmonary and one tricuspid);
however, there have been three cases of prosthetic valve
endocarditis (two aortic, one mitral). In total to date, two
deaths have been attributed to C. striatum endocarditis, with
aortic valve involvement (Table 18–17).
The current case highlights the important role of
molecular methods in the correct identification of the causal
agent, namely C. striatum. Identification of the genus
Corynebacterium and differentiation of species in the genus is
usually based on differential biochemical tests.18 However, in
most diagnostic laboratories, the API system is employed for
the rapid and convenient speciation of isolates in this genus.
Previously, Funke et al.,19 although concluding that the API
Coryne system is a useful tool for identifying the diverse
group of coryneform bacteria encountered in the routine
clinical laboratory, noted that additional tests were required
when using version 2.0 of the kit instead of version 1.0, in
order to identify strains completely. In the case reported
here, the API Coryne system was not able to identify the
isolate correctly, even to the genus level, yielding the close
 BIOMEDICAL SCIENCE IN BRIEF 183

Table 1. A review of the literature describing cases of Corynebacterium striatum endocarditis previously reported.

Patient Predisposing Echocardiographic Valve Antibiotic therapy Surgery Survival Ref

(age, sex) condition for IE findings
72, F RF in childhood. Mitral TOE: inconclusive due to NK Vancomycin/rifampicin None Yes (8)
valvotomy and prosthetic what was believed to be
mitral valve replacement calcified vegetations from
30 y later. Culture-negative previous episode of
IE, 18 m previously culture-negative IE
61, F RF in childhood Partially calcified vegetation Mitral Vancomycin/gentamicin None Yes (8)
on mitral valve
50, M* Previous mycotic aneurysm TOE: 15 mm vegetation on Aortic Antibiotic therapy unsuccessful. Aortic Yes (9)
for which he underwent aortic valve visible before i. Penicillin/gentamicin valve
surgical treatment. and after antibiotic therapy ii. Vancomycin/netilmicin replaced
iii Vancomycin/gentamicin/
doxycycline
62, F Diastolic murmur after TOE: No evidence of Bioprosthetic Vancomycin None Yes (10)
surgery for a bioprosthetic vegetation or abscess aortic
aortic valve replacement formation valve
a few years previously
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72, F Aortic valve replacement TTE indicated a 15 mm Periprosthetic i.Vancomycin/gentamicin, None No (11)
with a metallic prosthesis vegetation on the mitral aortic ii benzyl penicillin
valve; however, this was valve
not confirmed at
post mortem
68, M Mitral valve regurgitation TTE: moderate left Mitral i.Vancomycin, None Yes (12)
(2 y) ventricular dysfunction, ii. Penicillin
mitral valve regurgitation.
TOE: mitral regurgitation
and vegetation on atrial
aspect of anterior
mitral valve leaflet
73, M Pacemaker 6 y previously. TTE: normal Tricuspid i. Vancomycin Removal of Yes (13)
Batteries replaced and TOE: vegetation on old ii. Oral co-trimoxazole/ old electrode
electrode cut, although electrode wire which rifampin wire via
distal part left in place. remained after previous iii. Vancomycin due to jugular vein
Draining sinus tract removal penicillin resistance
TOE after surgical removal
of wire. Vegetation visible
on tricuspid valve
24, M Ventriculoatrial shunt TTE/TOE: 10 mm vegetation Pulmonary i. Amoxicillin/netilmicin Bed sore treated Yes (14)
on pulmonary valve close ii. Amoxicillin (source of infection)
to distal extremity of iii Amoxicillin, Endovascular
ventriculoatrial shunt catheter; netilmicin/teicoplanin process attempted
TTE after 7 w therapy: iv. Oral amoxicillin to remove catheter
vegetation unchanged; in pulmonary
TOE 20 m later: no vegetations artery (failed)
54, M None TTE: moderate aortic Aortic i. Ampicillin/vancomycin Aortic valve Yes (7)
insufficiency following detection ii. Ampicillin + gentamicin replacement
of new murmur. iii. Vancomycin alone
TTE/TOE: ruptured aortic (due to rash)
tricuspid valve cusp with severe
aortic insufficiency
76, M† No history of Echo: aortic/tricuspid Aortic Parenteral ampicillin/ Died prior to No (15)
heart disease insufficiency gentamicin planned emergency
surgery to replace
aortic valve
46, F Unknown Unknown Unknown Initial treatment failure None Yes (16)
with linezolid.
Switched to daptomycin/
rifampicin
68, M Aortic and mitral TTE: 10 mm vegetation on Prosthetic Vancomycin due to antibiotic None Yes (17)
prosthetic valves prosthetic mitral valve mitral valve pan-resistance
RF: rheumatic fever; IE: infective endocarditis; TTE: transthoracic echocardiography; TOE: transoesphageal echocardiography; NK: not known.
*
Polymicrobial, multiresistant infective endocarditis due to Staphylococcus epidermidis and Corynebacterium striatum.
†
Causative organism most closely resembled Corynebacterium striatum.

BRITISH JOURNAL OF BIOMEDICAL SCIENCE 2006 63 (4)

 184 BIOMEDICAL SCIENCE IN BRIEF
phylogenetic neighbour Brevibacterium sp. (47%) on first
testing and eventually Corynebacterium group G (55.6%) on
subsequent testing.
Where conventional blood culture yields a viable culture,
it is important to be able to identify it reliably to aid patient
management, in particular antimicrobial susceptibility and
the prescribing of an appropriate regimen of anti-infective
chemotherapy, and for epidemiological reporting of
aetiological agents of IE. Molecular methods may be
introduced as part of a polyphasic approach to aid the
identification of a culture-positive case, but are not required
in the primary diagnostic setting.
Conventional phenotypic characterisation of cultures can
be problematic (e.g., speciation of enterococci and coagulase-
negative staphylococci) and molecular identification
primarily by sequence analysis allows these organisms to be
speciated in such cases. Additionally, occasional
employment of these molecular methods may help to
quality assure the correct phenotypic identification of
cultures from patients with IE.
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Although primary diagnostic laboratories may be able to
detect and isolate a significant organism from the blood
cultures of patients with IE, these laboratories may have
problems with the correct and reliable identification of such
isolates. This may be due to the fact that the cultures are
unusual in that they are not observed frequently in the
clinical laboratory, either in the context of IE or other
infections (e.g., Weissella confusa20) or where the organism is
new (e.g., Streptococcus nov. sp.21).
Where a new organism is identified by 16S rRNA
sequence-based methods, further work is required to fully
characterise and describe the new taxon, whether a new
species or indeed a new genus. Generally, this necessitates
the forwarding of such isolates to a specialist taxonomy
laboratory, as even those primary diagnostic laboratories
with specialist molecular ability are often not suited for an
in-depth analysis of the taxonomic positioning of such
isolates. Limitations of routine identification systems
(e.g., API, Vitek, etc.) in primary diagnostic laboratories
create problems in identifying difficult-to-identify
organisms reliably, and employment of 16S rRNA gene
sequencing has proved reliable in such cases.22
In conclusion, this report demonstrates the value of
molecular methods in the correct identification of
phenotypically difficult-to-identify causal agents of IE.
Furthermore, it represents the first instance in which PCR
and direct sequencing has been employed to aid the
identification of C. striatum endocarditis. Diagnostic clinical
laboratories should be familiar with the existence of such
techniques and have a mechanism in place to permit the
referral of such organisms to a specialist/reference laboratory
with experience in these molecular techniques. 5 19
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20 Flaherty JD. Levett PN, Dewhirst FE, Troe TE, Warren JR,
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21 Woo PC, Tam DM, Leung KW et al. Streptococcus sinensis sp. nov.,
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