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Letter
Isolation of Enterobacter
sakazakii from Midgut of Stomoxys calcitrans
Joanne V. Hamilton,* Michael J. Lehane,† and Henk R. Braig†
*University of Wales, Aberystwyth, Ceredigion, UK; and †University of
Wales, Bangor, Gwynedd, UK
To the Editor: Enterobacter sakazakii, a gram-negative
rod-shaped bacterium, is an emerging foodborne pathogen that can cause
meningitis, sepsis, or necrotizing enterocolitis in newborns, particularly
affecting premature or other immunocompromised infants. Although an environmental
reservoir and mode of transmission for E. sakazakii has not been
clearly identified, a growing number of reports suggest that powdered
milk–based infant formulas can be a vehicle for infection. We report the
isolation of E. sakazakii from the guts of larvae of the stable
fly, Stomoxys calcitrans, demonstrating an environmental reservoir
for E. sakazakii and raising the possibility that environmental
contamination by insects may be important in the spread of this opportunistic
organism.
The first two cases of neonatal meningitis caused by E. sakazakii
were reported in the United Kingdom in 1961; both infants died (1).
Subsequently, cases of meningitis, septicemia, necrotizing enterocolitis,
brain abscesses, cerebral infarctions and dermoid cysts of the posterior
fossa caused by E. sakazakii have been reported worldwide.
Although most documented cases involve infants, reports also describe
adult infections. Adults with E. sakazakii infections usually have
serious underlying diseases or malignancies, but adult meningitis caused
by E. sakazakii infection has never been reported. Concern is growing
regarding antibiotic resistance in E. sakazakii. Recently, an E.
sakazakii wound infection in an adult patient has been reported, which
was resistant to multiple antibiotics and required prolonged treatment
with broad-spectrum antibiotics (2).
Illness and death associated with E. sakazakii infection in neonates
vary considerably, and mortality rates as high as 80% have been recorded
(3). Implicated infection sources include incubators,
the birth canal, a blender in a milk kitchen where powdered infant formula
was reconstituted and hospital environments (E. sakazakii growth
on an agar plate in a ward specifically under infection control against
multidrug-resistant Staphylococcus aureus [MRSA]). The presence
of E. sakazakii in powdered infant formula has been reported often.
An investigation of Enterobacteriaceae found in powdered substitutes for
breast milk from 35 countries indicated that 52.2% of the 141 samples
tested contained members of this family. E. agglomerans, E. cloacae,
E. sakazakii, and Klebsiella pneumoniae were the most frequently
isolated organisms (4). More recently, E. sakazakii
has been detected in ultra–heat-treated milk, spoiled tofu, lettuce, and
traditional fermented bread (khamir), and in beer mugs rinsed mechanically
or in open vats. Although more vehicles for transmission are being identified,
the environmental reservoir has remained elusive. A study in 1990 did
not isolate this organism from samples of surface water, mud, soil, rotting
wood, bird dung, grain, rodents, domestic animals, cattle, or raw cow's
milk (5). We report the isolation of E. sakazakii
from the gut of larvae of the blood-sucking insect Stomoxys calcitrans.
S. calcitrans were reared in a laboratory culture (originally
isolated from farms in North Wales, U.K.) as previously described (6).
Larvae were removed from culture medium, washed in sterile isotonic saline
solution, and surface sterilized by immersion in 100% ethanol. Larval
guts were dissected aseptically and placed individually onto standard
LB agar plates. Plates were incubated at 24°C overnight and were examined
for the presence of bacteria the next day. Subcultures of each different
colony type were streaked onto fresh LB plates until pure colonies were
obtained.
A sample was taken from each pure colony and an attempt was made to identify
the bacteria by using standard polymerase chain reaction (PCR) amplification
of the genes encoding bacterial 16S rRNA (7). We identified
E. sakazakii, Providencia stuartii, Erwinia carotovora,
Micrococcus luteus, and Serratia marcescens. The E. sakazakii
colony had the typical yellow pigmentation and both colony morphologic
features reported for this bacterium (3).
The growing reports of E. sakazakii infections and its implied
role as an emerging foodborne pathogen are of concern to clinicians, the
food industry, and consumers. The high mortality rate and severity of
this infection in infants, coupled with the lack of ecologic information
about this organism, have fueled much debate. The voluntary product recall
of a powdered infant formula by its manufacturer in 2001, after E.
sakazakii was found in the finished product (8),
exemplifies the need for stringent process control and the use of aseptic
techniques during preparation, handling, storage, and use of reconstituted
infant milk. The problem of foodborne transmission is further exacerbated
by the high tolerance to heat of E. sakazakii (3).
The overall risk for infection may depend on several factors, including
numbers of bacteria present in the product, handling after preparation,
and underlying patient characteristics.
We have identified the larval gut of the stable fly, Stomoxys calcitrans,
as an environmental reservoir for E. sakazakii. Stable flies have
a worldwide distribution; they are blood feeders that primarily feed
on cattle, horses, dogs, pigs, and humans, but they will also take a blood
meal from reptiles and birds. Stomoxys spp. may be found wherever
cattle, pigs, or horses are kept, and more specifically, S. calcitrans
is common in cowsheds, making contamination of milk a possibility.
The geographic distribution of S. calcitrans correlates well with
the incidence of E. sakazakii infections. Both S. calcitrans
and E. sakazakii infections have been reported from Denmark, the
United States, Israel, the United Kingdom, and Germany. Additionally,
Stomoxys spp. have a worldwide distribution and are likely present
in all countries reporting E. sakazakii infection.
A recent study has isolated E. sakazakii from the guts of a laboratory
colony of Mexican fruit flies, Anastrepha ludens (9).
Furthermore, a technical report from a pest exterminating company in the
United States records the presence of Enterobacter sakazakii in
the house fly (Musca domestica), although the exact location of
the bacterium on or in the fly is unclear. Insects are thus a likely major
environmental reservoir of E. sakazakii. This conclusion suggests
that in addition to stringent control measures during manufacturing and
use of foodstuffs, reducing arthropod presence in hospital and manufacturing
environments could result in a substantial reduction in the transmission
of E. sakazakii. New control methods will likely need to be developed
because some evidence suggests that insect traps that electrocute could
play a role in the spread of infectious disease agents such as bacteria
and viruses (10).
Acknowledgments
This study was supported
by grants from BBSRC and The Wellcome Trust.
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