Tareq M. Osaili

International survey of Cronobacter sakazakii and other Cronobacter spp. in follow up formulas and infant foods

A coordinated survey for Cronobacter and related organisms in powdered infant formula, follow up formula and infant foods was undertaken by 8 laboratories in 7 countries in recognition of and in response to the data needs identified in an FAO/WHO call for data in order to develop global risk management guidance for these products. The products (domestic and imported) were purchased from the local market and were categorised according to their principle ingredients. A total of 290 products were analysed using a standardised procedure of pre-enrichment in 225 ml Buffered Peptone Water (BPW), followed by enrichment in Enterobacteriaceae Enrichment (EE) broth, plating on the chromogenic Cronobacter Druggan-Forsythe-Iversen (DFI) agar and presumptive identification with ID 32 E. Presumptive Cronobacter isolates were identified using 16S rRNA gene sequence analysis. Aerobic plate counts (APC) of the products were also determined on nutrient agar. Fourteen samples had APC>10(5) cfu/g, 3 of which contained probiotic cultures. C. sakazakii was isolated from 27 products; 3/91 (3%) follow up formulas (as defined by Codex Alimentarius Commission), and 24/199 (12%) infant foods and drinks. Hence C. sakazakii was less prevalent in follow up formula than other foods given to infants over the same age range. A range of other bacteria were also isolated from follow up formulas, including Acinetobacter baumannii, Enterobacter cloacae, Klebsiella pneumoniae, Citrobacter freundii, and Serratia ficaria. There was significant variation in the reconstitution instructions for follow up formulas. These included using water at temperatures which would enable bacterial growth. Additionally, the definition of follow up formula varied between countries.

Effect of desiccation, starvation, heat, and cold stresses on the thermal resistance of Enterobacter sakazakii in rehydrated infant milk formula

Enterobacter sakazakii is an opportunistic foodborne pathogen that has been isolated from powdered infant milk formula. This study determined the effect of desiccation, starvation, heat and cold stresses on the thermal inactivation of E. sakazakii in rehydrated infant milk formula (RIMF). Stressed cells were mixed with RIMF at 52, 54, 56, and 58 degrees C for various time periods. The D- and z-values were determined by using linear regression analysis. D-values for unstressed E. sakazakii at 52, 54, 56, and 58 degrees C were 15.33, 4.53, 2, and 0.53 min, respectively. Desiccation and heat stresses, but not starvation or cold stress, caused significant (P < 0.05) reduction in D-values. The z-values of desiccated, starved, heat stressed, and cold stressed E. sakazakii were not significantly different from unstressed cells (4.22 degrees C). Thermal resistance of E. sakazakii in RIMF is affected by the environmental stresses; that is, desiccation and heat stresses that may surround the bacterium prior to the contamination of infant formula. The results of this study may be of use to regulatory agencies, infant milk producers, and infant caregivers to design heating processes to eliminate E. sakazakii that may be present in infant milk formula.

Thermal inactivation studies of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes in ready-to-eat chicken-fried beef patties.

Thermal inactivation studies were used to determine the D- and z-values of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes in ready-to-eat chicken-fried beef patties. Inoculated meat was packaged in sterile bags, which were immersed in a circulated water bath and held at 55, 57.5, 60, 62.5, 65, 67.5, and 70 degrees C for different lengths of time. D- and z-values were determined with a linear regression model. Average D-values at temperatures 55 to 70 degrees C were 27.62 to 0.04 min for E. coli 0157:H7, 67.68 to 0.22 min for Salmonella, and 81.37 to 0.31 min for L. monocytogenes. The z-values were 5.2 degrees C for E. coli O157:H7, 6.0 degrees C for Salmonella, and 6.1 degrees C for L. monocytogenes. The results of this study can be used by food processors to validate their processes and help eliminate pathogenic bacteria associated with chicken-fried beef products.

Impact of environmental stress desiccation, acidity, alkalinity, heat or cold on antibiotic susceptibility of Cronobacter sakazakii.

Cronobacter sakazakii is an emerging foodborne pathogen that has been implicated in severe forms of meningitis, septicemia or necrotizing colitis in pre-term neonates. Although illness outbreaks (primarily associated with powdered infant formula, PIF) caused by this pathogen are rare, the case-fatality rate may reach 50%. Successful treatment of C. sakazakii infection is reliant upon clinical use of antibiotics (AB) such as ampicillin. Recent reports showed increased resistance of C. sakazakii to broad-spectrum antibiotics. The objective of this study was to evaluate the effect of extreme pH (3.5 for 30 min or 11.25 for 5 min), cold (4°C for 24h), heat (55°C for 5 min), and desiccation (cells were dried at 40°C for 2h and held at 21°C for 4 d) stresses on susceptibility of five isolated strains of C. sakazakii to streptomycin, gentamicin, kanamycin, neomycin, tetracycline, doxycycline, tilmicosin, florfenicol, ampicillin, amoxicillin, vancomycin, ciprofloxacin and enrofloxacin. All unstressed strains of C. sakazakii were sensitive to streptomycin, gentamycin, kanamycin, ciprofloxacin, enrofloxacin, ampicillin and amoxicillin, but were moderately resistant or resistant to the rest. Exposing cells to alkaline or acidic stress did not change their sensitivity toward streptomycin, gentamycin, kanamycin or ciprofloxacin, but their resistance toward the other AB was increased. Cells stressed by desiccation showed increased sensitivity toward streptomycin, gentamicin, kanamycin, ciprofloxacin, enrofloxacin, ampicillin and doxycycline, but showed resistance toward the others. Cold-stressed cells were more sensitive to streptomycin, gentamicin, kanamycin, and ciprofloxacin compared with heat-stressed cells, but both heat and cold-stressed cells showed increased resistance toward all the other AB. Results obtained will help in understanding the effect of environmental stresses during processing on C. sakazakii susceptibility to AB.

Heat resistance of Cronobacter species (Enterobacter sakazakii) in milk and special feeding formula.

Aim:  To determine D‐ and z‐values of Cronobacter species (Enterobacter sakazakii) in different reconstituted milk and special feeding formula and the effect of reconstitution of powdered milk and special feeding formula with hot water on the survival of the micro‐organism.

Influence of desiccation on the sensitivity of Cronobacter spp. to lactoferrin or nisin in broth and powdered infant formula

Although outbreaks caused by Cronobacter spp. (Enterobacter sakazakii) are rare, infections by this organism have a case-fatality rate which may reach 80%. Powdered infant milk formula (PIMF) is considered a major source for human infection with Cronobacter spp. The organism has the capability to survive in dry environments for long periods (approximately 2 years). Current interest in the use of natural antimicrobials including lactoferrin (LF) and nisin has developed because of the desire for preservative-free food products. The objective of the present study was to evaluate the antimicrobial activity of bovine LF or nisin against undesiccated and desiccated Cronobacter spp. cells in 0.2% peptone water (PW) and reconstituted PIMF at different temperatures. In 0.2% PW, 2.5 mg/ml LF was able to inactivate 4 log(10) CFU/ml of undesiccated cells of Cronobacter spp. in 4 h at 37 degrees C but at lower temperatures, higher concentrations of LF as well as longer exposure were needed to achieve the same effect as at 37 degrees C. Similarly, the effect of nisin against undesiccated cells of Cronobacter spp. was concentration and temperature dependent in 0.2% PW. It was found that 1500 IU/ml caused a 4 log(10) CFU/ml reduction of undesiccated cells of Cronobacter spp. at 21 degrees C and 37 degrees C. Desiccated Cronobacter spp. cells in 0.2% PW were more sensitive to LF action than were undesiccated cells. A 4 log(10) CFU/ml reduction was obtained with 2.5 mg/ml LF after 1 h at 21 and 37 degrees C or 8 h at 10 degrees C. In contrast, desiccated cells of Cronobacter spp. were more resistant to nisin. Furthermore, neither LF nor nisin had detectable antimicrobial activity against desiccated or undesiccated Cronobacter spp. in reconstituted PIFM. Heating at 55 degrees C for 5 min with nisin in reconstituted PIFM did not enhance the antimicrobial activity of nisin. Unexpectedly, nisin appeared to protect Cronobacter spp. from the damaging effects of heat treatment. The reduced antimicrobial activity of LF and nisin in reconstituted PIMF was potentially explained by the higher concentration of Ca(2+), Mg(2+) and Fe(3+) in the latter.

Effects of osmotic pressure, acid, or cold stresses on antibiotic susceptibility of Listeria monocytogenes

Prevalence of antibiotic resistance of Listeria monocytogenes isolated from a variety of foods has increased in many countries. L. monocytogenes has many physiological adaptations that enable survival under a wide range of environmental stresses. The objective of this study was to evaluate effects of osmotic (2, 4, 6, 12% NaC), pH (6, 5.5, 5.0) and cold (4 °C) stresses on susceptibility of three isolates of L. monocytogenes towards different antibiotics. The minimal inhibitory concentrations (MICs) of tested antibiotics against unstressed (control), stressed or post-stressed L. monocytogenes isolates (an ATCC strain and a meat and dairy isolate) were determined using the broth microdilution method. Unstressed cells of L. monocytogenes were sensitive to all tested antibiotics. In general, when L. monocytogenes cells were exposed to salt, cold and pH stresses, their antibiotic resistance increased as salt concentration increased to 6 or 12%, as pH was reduced to pH 5 or as temperature was decreased to 10 °C. Results showed that both meat and dairy isolates were more resistant than the ATCC reference strain. Use of sub-lethal stresses in food preservation systems may stimulate antibiotic resistance responses in L. monocytogenes strains.

Effects of Extended Dry Storage of Powdered Infant Milk Formula on Susceptibility of Enterobacter sakazakii to Hot Water and Ionizing Radiation

Infant milk formula has been identified as a potential source of Enterobacter sakazakii, which has been implicated in neonatal meningitis and necrotizing enterocolitis. This study was undertaken to determine whether the length of E. sakazakii storage in powdered infant milk formula (PIMF) affected the ability of the pathogen to survive subsequent reconstitution of the powder with hot water or treatment with gamma radiation. Five E. sakazakii strains were mixed individually with PIMF and kept for up to 12 months at 25 degrees C. After storage PIMF was reconstituted with water at 60 to 100 degrees C or was exposed to < or = 5 kGy of gamma radiation. Without any treatment secondary to drying, E. sakazakii counts decreased < 1 log/g after 1 month but decreased about 4 log/g during storage for 8 to 12 months. Dry storage decreased thermal resistance but increased resistance of E. sakazakii to ionizing radiation in PIMF. Reconstitution of contaminated powder with water at 70 degrees C after 1 month of dry storage reduced E. sakazakii viability slightly, > 2 log/g, and after powder was stored for 12 months all E. sakazakii strains were eliminated. In contrast, desiccation substantially increased the resistance of E. sakazakii strains to ionizing radiation. Although the D-value for E. sakazakii IMF1 following overnight storage in PIMF was 0.98 kGy, > 4 kGy was required to kill 1.5 log/g of the same strain that had survived 12 months in dry PIMF. Results suggested that low-dose irradiation will more effectively eliminate E. sakazakii from PIMF if the treatment is applied shortly after PIMF manufacture.

Detergent and Sanitizer Stresses Decrease the Thermal Resistance of Enterobacter sakazakii in Infant Milk Formula

This study determined the effect of acid, alkaline, chlorine, and ethanol stresses on the thermal inactivation of Enterobacter sakazakii in infant milk formula. Unstressed or stressed cells were mixed with reconstituted powdered infant milk formula (PIMF) at temperatures between 52 and 58 degrees C for various time periods or mixed with PIMF prior to reconstitution with hot water between 50 and 100 degrees C. D- and z-values were determined using liner regression analysis. In general, detergent and sanitizer stresses decreased the thermal resistance of E. sakazakii in infant milk formula. The results of this study may be of use to regulatory agencies, manufacturers, and infant caregivers to design heating processes to eliminate E. sakazakii.

Efficacy of the thin agar layer method for the recovery of stressed Cronobacter spp. (Enterobacter sakazakii).

Cronobacter spp. (Enterobacter sakazakii) are emerging opportunistic pathogens for all age groups, and are of particular concern when it comes to infants. Prior to contaminating food, the organism may be exposed to a variety of stresses, leading to a generation of sublethally injured cells that may not be detected by selective media unless a protracted recovery period is included in the isolation procedure. This study evaluated the efficacy of the thin agar layer (TAL) method for the recovery of Cronobacter cells that had been exposed to various stress conditions. Five strains of C. sakazakii and C. muytjensii were exposed to starvation, heat, cold, acid, alkaline, chlorine, or ethanol, with or without further exposure to desiccation stress. The recovery of the stressed cells was determined on tryptone soy agar (TSA; nonselective control medium), violet red bile glucose agar (VRBGA; selective agar), Druggan-Forsythe-Iversen (DFI; selective agar), and TAL media (viz., VRBGA overlaid with TSA, and DFI overlaid with TSA). Regardless of stress type, there were no significant differences among the recoveries of stressed desiccated Cronobacter spp. cultures on TSA, DFI+TSA, and VRBGA+TSA, but there was significantly less recovery on VRBGA. The recovery of prestressed desiccated Cronobacter spp. on DFI+TSA was similar to that on TSA, whereas the recovery on VRBGA+TSA was lower. DFI+TSA performed better than VRBGA+TSA did in differentiating Cronobacter spp. within mixed bacterial cultures. The results of this study suggest the use of the TAL method DFI+TSA as an improved method for the direct recovery of stressed Cronobacter spp.