Research Note. MS : Received 25 May 2011/Accepted 15 August 2011 ABSTRACT

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1 2177 Journal of Food Protection, Vol. 74, No. 12, 2011, Pages doi: / x.jfp Copyright G, International Association for Food Protection Research Note Two Cases of Hemorrhagic Diarrhea Caused by Cronobacter sakazakii in Hospitalized Nursing Infants Associated with the Consumption of Powdered Infant Formula J. PARRA FLORES, 1,2 S. ARVIZU MEDRANO, 1 J. SILVA SÁNCHEZ, 3 AND E. FERNÁNDEZ-ESCARTÍN 1 * 1 Departamento de Investigación y Posgrado en Alimentos, Facultad de Química, Universidad Autónoma de Querétaro, Querétaro, Querétaro, 76010, México; 2 Facultad de Ciencias de la Salud y de los Alimentos, Universidad del Bio Bio, Chillán, Ñuble, Chile; and 3 Departamento de Diagnóstico Epidemiológico, Instituto Nacional de Salud Pública, Cuernavaca, Morelos, México MS : Received 25 May 2011/Accepted 15 August 2011 ABSTRACT Two cases of acute gastroenteritis occurred in 5-month-old infants hospitalized in a mother-and-child hospital in Queretaro, Mexico, on 24 January C. sakazakii was recovered from the powdered infant formula (PIF), rehydrated PIF (R-PIF) fed to infants, and their fecal samples. The microorganism was present at levels of 0.33 most probable number (MPN)/g and 24 MPN/ ml in PIF and R-PIF, respectively. The total ingested dose for the day before the onset of the diarrheic syndrome ranged between 2,160 and 3,600 MPN/ml. All strains of C. sakazakii recovered from the three sources (R-PIF, PIF, and fecal matter) showed identical biotypes, adhesion and invasiveness factors, and pulsed-field gel electrophoresis profiles. No deaths were observed. Salmonella, Shigella, and enterotoxigenic Escherichia coli were not found in food or fecal samples. Cronobacter sakazakii is considered a particularly aggressive emerging opportunistic pathogen in nursing patients. A large number of infection incidents occur in hospital environments (nosocomial), resulting in frequent outbreaks (2, 3, 4). The clinical picture developed by victims consists mainly of meningitis, septicemia or necrotizing enterocolitis in infants (22, 25), though diarrhea, urinary tract infections, and sepsis (11) have also been observed. The fatality rate can be as high as 80% (17). The methodology for the phenotypic identification of clinical and environmental isolates from cases has evolved from the culture characteristics, pigment production, and biochemical tests from when the bacterium was recognized as a coliform bacterium belonging to the species Enterobacter cloacae in More recently, culture and molecular techniques led first to the creation of the species Enterobacter sakazakii and, subsequently, to the genus Cronobacter (8, 14, 15). The minimum infective dose for Cronobacter spp. is unknown, but levels of and 0.92 most probable number (MPN)/g (7) have been detected in the powdered infant formula (PIF) used in rehydrated PIF (R-PIF) fed to affected infants. This work describes an outbreak of bloody diarrhea in two infants on the same day, associated with the consumption of prepared R-PIF at a hospital. It also includes the characterization of C. sakazakii strains recovered from PIF, R-PIF, and fecal matter according to biochemical phenotype, antibiotic resistance profile, virulence factors, and molecular genotype. * Author for correspondence. Tel: z52 (442) , Ext 5581; Fax: z(52) ; efescart@uaq.mx. MATERIALS AND METHODS Clinical cases. In the course of a prospective study at a maternity hospital to investigate the incidence of Cronobacter and Escherichia coli in food and environmental samples at its lactarium (milk kitchen), we became aware of two episodes of sporadic acute bloody diarrhea in infants. Case 1. Case 1 was a male infant 4 months of age and born after a full term of pregnancy with a weight of 2,620 g, who was hospitalized with severe psychomotor damage. He developed sepsis, epilepsies, and grade II reflux. Gastrostomy was performed, and he was treated with cefotaxime and vancomycin for 2 weeks. After 24 h of consuming the R-PIF (90 ml every 4 h), he presented with a bloody diarrheic syndrome that was resolved after treatment with cefotaxime for 7 days. Case 2. Case 2 was a female infant born by caesarean section with a weight of 2,500 g. At 4 months, the baby presented with a subarachnoid hemorrhage associated with an automobile accident. A month later, she was readmitted to hospital with pneumonia symptoms and treated with clindamycin and amikacin. She was fed with R-PIF (150 ml every 4 h) by orogastric tube, and on the fifth day developed bloody diarrhea with mucus (which coincides with case 1). She remained with the described symptoms for 72 h, while on the antimicrobial treatment. The bacteriological examination found an absence of Salmonella, Shigella, and enterotoxigenic E. coli in the fecal material of both infants. Sampling. One year prior to the incident, 21 samples of PIF had been collected, 10 for premature infants and 11 for children aged 0 to 6 months. For 29 samples of R-PIF, 11 were for premature infants and 18 for infants aged from 0 to 6 months. In

2 2178 PARRA FLORES ET AL. J. Food Prot., Vol. 74, No. 12 addition, 155 and 17 environmental and fecal samples were collected, respectively. PIF samples were collected in the manufacturer s cans, and R- PIF milk in the same bottles used to feed the infants. A bottle of R- PIF for each day was routinely kept under refrigeration for analysis in the event of an infectious case occurring. Environmental samples were collected in sterile Whirl-Pak bags (Nasco, Ft. Atkinson, WI), and fecal matter was taken directly from the soiled diapers. All samples were transported in containers at 4 to 7uC and analyzed within 3 h. Isolation from PIF and R-PIF. As recommended by the Food and Agriculture Organization in 2002 (9), R-PIF from the lactarium was preenriched by incubating at 35uC for 24 h. For PIF, 25-g amounts were suspended in 178 ml of sterile distilled water and preenriched. In both cases, the enrichment took place in Mossel broth (BD Difco, Sparks, MD) at a 1/10 dilution and with incubation at 35uC for 24 h. The colonies were isolated on violet red bile agar plates (Bioxon, BD, Lomas de Chapultepec, Mexico) with 1% glucose and methylene blue eosin agar (Bioxon, BD). They were purified in trypticase soy agar and transferred to chromogenic Enterobacter sakazakii agar (DFI formulation, code CM1055, Oxoid, Basingstoke, England). Stool specimens. Portions of bloody mucus obtained directly from the soiled diapers were enriched in Mossel broth and incubated at 35uC for 24 h. Additionally, the mucus was streaked directly onto violet red bile agar 1% glucose plates and MacConkey agar (Merck KGaA, Darmstadt, Germany) plates and incubated at 35uC for 24 h. Suspicious colonies were isolated using the scheme as described for PIF and R-PIF. Identification of Cronobacter spp. Cronobacter sp. identification was confirmed in samples and strains from PIF, R-PIF, and fecal matter by using PCR techniques with the primers Esakf and Esakr as described by Lehner et al. (18). The control strain was C. sakazakii ATCC Identification of C. sakazakii. Characterization of C. sakazakii was based on pigment production, a-glycosidase activity, and biochemical tests using the BIOLOG system (Biolog, Inc., Hayward, CA). Finally, the pathogen was identified by PCR amplification of the rpob region with primers Csakf and Csakr as previously described by Stoop et al. (23). C. sakazakii ATCC and ATCC were used as control strains. Quantification of C. sakazakii. Quantification of C. sakazakii in PIF and R-PIF samples was carried out by the MPN technique (19). The presence of C. sakazakii was confirmed in each tube using PCR as previously described. Antibiotic susceptibility. Bacterial susceptibility to antibiotics was determined by the disk diffusion method using guidelines established by Bauer et al. (1) and recommended by the Clinical and Laboratory Standards Institute (5), using commercial antibiotic discs. A total of 12 antibiotic discs (Bio-Rad, Hercules, CA), with ampicillin (10 mg), amikacin (30 mg), levofloxacin (5 mg), cephalothin (30 mg), cefotaxime (30 mg), ceftriaxone (30 mg), chloramphenicol (30 mg), gentamicin (10 mg), netilmicin (30 mg), nitrofurantoin (300 mg), cefepime (30 mg), and trimethoprimsulfamethoxazole (25 mg), were employed. Characterization of the resistance or susceptibility profile of the isolates was performed by measuring inhibitory zones and then comparing these against the interpretative chart to determine the sensitivity of the isolates to the antibiotics. E. coli ATCC was included as a reference strain. Virulence test. Strains of C. sakazakii were evaluated in triplicates for adhesion and invasiveness factors using HEp-2 cells (20) at the Bowel Bacteriology Laboratory of the Federico Gomez Children s Hospital of Mexico. Enteroaggregative E. coli O42 and enteropathogenic E. coli 1198 were used as positive controls, while E. coli K-12 HB101 was used as a negative control. PFGE. Molecular typing was performed by pulsed-field gel electrophoresis (PFGE). Whole-cell DNA from C. sakazakii was obtained as previously described by Kaufmann et al. (16). Chromosomal DNA was digested with XbaI endonuclease (Gibco BRL, Gaithersburg, MD) and separated in 1% agarose gels (pulsed-field certified, Pronadisa, Madrid, Spain) with the GenePath system (Bio-Rad). Electrophoresis was performed at 6 V/cm for 23 h with an initial pulse time of 1 s and a final pulse of 30 s. Gels were stained with ethidium bromide and photographed. Interpretation of the images was done according to the criteria of Tenover et al. (24), and analysis with the Gel Compar II system (Applied Maths NV, Sint-Martens-Latem, Belgium ). RESULTS AND DISCUSSION The prospective work underway on the microbiology of milk and the environmental factors of the lactarium, as well as the availability of daily information on the presence of any acute infectious process among hospitalized infants, gave us a unique opportunity to study the factors that had contributed to the two incidents of diarrhea. Accumulated data prior to these events showed that the incidence of C. sakazakii in the PIF from the lactarium was 23%. According to Friedemann (11), the main source of C. sakazakii in neonatal infections has been contaminated PIF, both for single cases and outbreaks. The contamination level of Cronobacter spp. in R-PIF up to the time of the incident was low, with a mean of 0.72 MPN/ml and a range of to 2.3 MPN/ml in 7 samples. In four independent PIF samples, the levels were between and 0.25 MPN/g, with a mean of MPN/g. C. sakazakii was detected only in one PIF sample, with a concentration of MPN/g. The stored R-PIF (24 h), which was analyzed on the day when the two cases of diarrhea occurred, yielded 24 MPN/ml of C. sakazakii. The PIF used for the preparation of R-PIF fed to the infants contained 0.33 MPN/g. We determined that the water temperature used in the rehydration of the PIF was 45uC and not 70uC as recommended by World Health Organization (26). A recent report by Forsythe (10) concludes that the inactivation provided by the use of 70uC as the rehydration temperature would eliminate any intrinsic cells of C. sakazakii that may be present in the PIF. Occasionally, the presence of Cronobacter spp. (later identified as C. sakazakii) was detected in environmental samples from the lactarium, such as the access door handles, refrigerator doors, the wash basin, and the air conditioning ventilation grid. However, none of the isolated environmental strains of C. sakazakii showed similarity to the genotype of strains recovered from the milk or fecal matter. An ongoing possibility is the multiplication of surviving pathogens from the PIF, which could have survived the rehydration process as described by Van Acker et al. (25) and Nazarowec-White and Farber

3 J. Food Prot., Vol. 74, No. 12 HEMORRHAGIC DIARRHEA CAUSED BY C. SAKAZAKII 2179 FIGURE 1. Dendrogram of 23 C. sakazakii strains isolated from different sources. Blocks A and B group strains isolated during the outbreak. Blocks C and D correspond to strains isolated from the same sources before or 4 months after the outbreak. (21). Despite the available quantitative data on the presence of C. sakazakii, it is not easy to establish the number of bacteria causing the diarrheic syndrome that may have been ingested. Exposure to the pathogen did not occur on only a single occasion, as is common when foodborne outbreaks occur. However, what was possible to estimate is that in the first exposure (approximately 24 h prior to the day of the start of the symptoms), the infants consumed around 24 cells/ml of milk, giving total exposure doses of 2,160 and 3,600 CFU of C. sakazakii. This dose was repeated on four occasions, although the R-PIF was refrigerated. The quantification of the bacterium in the refrigerated R-PIF approaches the minimal infective dose of 1,000 CFU estimated by Iversen et al. (13), although it differs from that of 10,000 CFU suggested by the Food and Agriculture Organization (9). Thus, the total population of C. sakazakii that was consumed by the affected infants was approximately between 8,000 and 14,000 total cells within the 12-h period required for the complete use of the batch of prepared R-PIF. Gurtler and Beuchat (12) found that C. sakazakii can survive for at least 72 h but does not grow at 4uC in different types of R-PIF, but can grow slowly at 8uC. Subsequently, the monitoring of the temperature of a sample of milk held under similar conditions and practices (as described by the lactarium personnel) yielded an initial high temperature of 40uC before being placed in the refrigerator. After 180 min of refrigeration, the R-PIF reached a temperature of 8uC, and during storage (up to the 12 h programmed for its consumption), it ranged between 7.2 and 9.0uC. Apparently the opportunities for an increase in the population of C. sakazakii in the R-PIF were of minimal significance. In order to identify the genetic relatedness of the C. sakazakii isolates, PFGE was performed. The genotypes of 12 strains isolated from PIF, R-PIF, and fecal samples during the two cases in this incident and 11 strains from similar samples taken before and after the incident were determined. PFGE identified two main groups, A and B. Group A included nine isolates of C. sakazakii from PIF (8705), R-PIF (8721, 8708, 8716, and 8724), and two fecal samples from the affected infants (8706 and 8709). In addition, two isolates from PIF (8720 and 8715) had the same genotype,

4 2180 PARRA FLORES ET AL. J. Food Prot., Vol. 74, No. 12 TABLE 1. Antibiotic resistance profiles for strains of C. sakazakii involved in the outbreak according to origin and clonal group Strain Source Clonal group Antibiotic resistance profile a 8721 R-PIF A CF, NF 8705 PIF A CF, AM, NF 8708 R-PIF A CF, NF 8716 R-PIF A CF, NF 8724 R-PIF A CF, AM, NF 8706 Case 1 fecal matter A AM, NF 8709 Case 2 fecal matter A CF, AM, NF 8700 Case 1 fecal matter B CF, CTX, AM, CRO, NET, GE, CL 8719 Case 1 fecal matter B CF, CTX, AM, CRO, NET, GE, CL 8702 Case 2 fecal matter B CF, FEP, CL 8701 Case 2 fecal matter B CF, CTX, AM, CRO, NF, CL 8703 Case 2 fecal matter B CF, AM a CF, cephalothin; NF, nitrofurantoin; AM, ampicillin; CTX, cefotaxime; CRO, ceftriaxone; NET, netilmicin; GE, gentamicin; CL, chloramphenicol; FEP, cefepime. but these were isolated later. Group B included five isolates from the two affected infants (8700, 8719, 8701, 8702, and 8703) (Fig. 1). These results indicate that there were two genotypically different C. sakazakii strains implicated in the outbreak at the hospital. All C. sakazakii strains obtained from the outbreak adhered to and were invasive for HEp-2 cells. Antibiotic resistance tests were conducted on 12 strains of C. sakazakii. Two fecal strains in case 1 (8700 and 8719) showed a similar antimicrobial resistance profile for seven antibiotics. Except for one strain (8706), the remaining isolates were resistant to cephalothin (Table 1). The bloody diarrhea syndrome present in the two infants was relatively benign if we consider the reported lethality of meningitis and septicemia in cases of C. sakazakii reported in the literature (9, 22, 25). The epidemiology of neonatal C. sakazakii infections has recorded some incidents of diarrhea, urinary tract infections, and conjunctivitis (11). The diarrhea in this incident was resolved in 24 to 48 h, which could be associated with the administration of antibiotics to both infants. Apparently incidents similar to the one described in this communication are not frequent at the hospital. These cases are the only ones reported and affected two individuals in the course of the 16-month study. The incident that we studied clearly meets the generally accepted definition of an outbreak (6). In 34 reports of hospital infection gathered from 17 countries, the most frequent number of affected infants was 1 or 2, with rare extreme cases of 10 and 12 patients (11). Three issues deserve attention in this incident: (i) the fact that the milk rehydration temperature (40uC approximately) was well below that recommended by the World Health Organization (70uC), (ii) PIF as a primary contamination source of C. sakazakii during R-PIF preparation, and (iii) the detection of C. sakazakii in the environmental samples of the lactarium. 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