Isolation of Enterobacter sakazakii from Midgut of Stomoxys calcitrans.

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 1991; 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 1991 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 bloodsucking 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[degrees]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 carotova, Micrococcus Luteus, and Serratia marcescens. The E. sukazakii 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 mad 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 era 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 sonic evidence suggests that insect traps that electrocute could play a role in the spread of infectious disease agents such as bacteria and viruses (10).

Joanne V. Hamilton, * Michael J, Lehane, ([dagger]) and Henk R. Braig ([dagger])

* University of Wales, Aberystwyth, Ceredigion, UK; and ([dagger]) University of Wales, Bangor, Gwynedd, UK

Acknowledgments

This study was supported by grants from BBSRC and The Wellcome Trust.

References

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(8.) Himelright I, Harris E, Lorch V, Anderson M, Jones T, Craig A, et al. Enterobacter sakazakii infections associated with the use of powdered infant formula--Tennessee, 2001 (Reported from MMWR 2002;51:297-300). JAMA 2002;287: 2204-5.

(9.) Kuzina LV, Peloquin JJ, Vacek DC, Millet TA. Isolation and identification of bacteria associated with adult laboratory Mexican fruit flies. Anastrepha ludens (Diptera: Tephritidae). Curr Microbiol 2001;42: 290-4.

(10.) Urban JE, Broce A. Killing of flies in electrocuting insect traps releases bacteria and viruses. Curr Microbiol 2000;41:267-70.

Address for correspondence: Jeanne Hamilton. Institute of Biological Sciences. University of Wales, Aberystlwyth, Ceredigion. SY23 3DA. UK; tax: [phone]; email: [e-mail]