absence of microorganisms
E. italicus group: italicus and camelliae. They have low biochemical activity in comparison with the
15.2.1 Material required for analysis Obligatory
• Bolton Broth
• Modified Charcoal Cefoperazone Deoxycholate Agar (mCCDA)
• Columbia Blood Agar (CBA)
• Brucella Broth
• Oxidase Kovacs Reagent
• Microaerobic gas-generating kits
• Laboratory incubator set to 37 ± 1°C
• Laboratory incubator set to 41.5 ± 1°C
• Laboratory incubator set to 25 ± 1°C Optional
• Mueller Hinton Blood Agar
• 3% Hydrogen Peroxide
• Nalidixic acid (30 μg) discs
• Cephalothin (30 μg) discs
• Sodium Hippurate Solution
• Ninhydrin Solution
• Indoxyl acetate discs (2.5 to 5.0 mg)
15.2.2 Procedure
A general flowchart for the detection of thermotolerant Campylobacter in foods using the presence/absence method ISO 10272-1:2006 is shown in Figure 15.1.
a) Enrichment: Following the procedures described in Chapter 2, homogenize 25 g or 25 ml of sample with 225 ml of Bolton Broth. Incubate at 37 ± 1°C/4–6 h, then at 41.5 ± 1°C/44 ± 4 h, in a microaerobic atmosphere (oxygen content of 10 ± 3%, carbon dioxide 5 ± 3%, optional hydrogen ≤10%, with the balance nitrogen).
Note a.1) The sample quantity of analytical unit can vary, as long as the dilution 1:10 is kept.
Note a.2) The appropriate microaerobic atmosphere can be obtained using commercially available gas-generating kits, following precisely the manu-facturers’ instructions, particularly those relating to the volume of the jar and the capacity of the gas-generating kit. Alternatively, the jar may be filled with an appropriate gas mixture prior to incubation. As an alternative to incubation in a microaerobic atmosphere, the enrichment broth can be incubated in screw capped bottles or flasks filled with enrichment broth, leaving a
headspace of less than 2 cm, and tightly closing the caps.
b) Selective-differential plating: Using the culture obtained in the Bolton Broth, inoculate the surface of the selective isolation medium, Modified Charcoal Cefoperazone Deoxycholate Agar (mCCDA). Proceed in the same manner with a second Campylobacter selective isolation medium, chosen by the laboratory. Incubate the m-CCDA plates at 41.5 ± 1°C/44 ± 4 h in a microaero-bic atmosphere. Incubate the second isolation medium plates according to the manufacturers’
instructions.
Note b.1) ISO 10272-1:2006 recommends a second isolation medium based on a principle different from mCCD agar. Examples of isolation media to be used are Skirrow Agar, Karmali Agar and Preston Agar.
After the incubation period, examine the plates for typical colonies of Campylobacter. The typi-cal colonies on mCCDA are grayish, often with a metallic sheen, with a tendency to spread. Other forms of colonies may occur. Follow the manufac-turers’ instructions to select typical colonies on the second isolation medium.
c) Confirmation: For confirmation, take at least one typical colony from each plate and a further four colonies if the first is negative. Streak each colony onto a Columbia Blood Agar (CBA) plate and incubate the plates in a microaerobic atmosphere at 41.5 ± 1°C/24–48 h. Use the pure cultures obtained on CBA for examination of morphology, motility, microaerobic growth at 25°C, aerobic growth at 41.5°C and presence of oxidase.
c.1) Morphology and motility: From the CBA plate suspend the culture into 1 ml of Brucella Broth and examine for morphology and motility using a microscope. Cultures showing curved bacilli with a spiraling
“corkscrew” motility should be retained for the confirmatory tests below.
c.2) Growth at 25°C (microaerobic): Inoculate the culture from the CBA plate onto the sur-face of a new CBA plate. Incubate the plate at 25 ± 1°C/44 ± 4 h in a microaerobic atmos-phere and examine for growth of Campylo-bacter colonies.
c.3) Growth at 41.5°C (aerobic): Inoculate the culture from the CBA plate onto the surface of a new CBA plate. Incubate the plate at 41.5 ± 1°C/44 ± 4 h in an aerobic atmosphere
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37±1 C/4-6h in microaerobic atmosphere
and after at 41.5±1 C/44±4h in microaerobic atmosphere
o
o
Second isolation medium chosen by the laboratory in microaerobic atmosphere
(incubation according to manufacturers’ instructions) Modified Charcoal Cefoperazone
Desoxycolate Agar (m-CCDA) 41.5±1 C/44±4h in microaerobic atmosphere
o
Columbia Blood Agar (CBA) in microaerobic atmosphere 41.5±1 C/24 - 48ho
Typical colonies (5) (grayish often with a metallic sheen)
CONFIRMATION
Brucella Broth (emulsify)
MORPHOLOGY AND MOTILITY GROWTH AT 25°C MICROAEROBIC (-)
GROWTH AT 41.5 AEROBIC (-)
°C
CBA 25±1 C/44±4h in microaerobic atmosphere
o
CBA 41.5±1 C/44±4h in aerobioc atmosphere
o
(curved bacilli with a spiraling cork screw motility
Homogenization
OXIDASE TEST (+) Columbia Blood Agar (CBA)
in microaerobic atmosphere 41.5±1 C/24 - 48ho Typical colonies (5)
Wet mount slide
Campylobacter spp.
Presence in 25g Confirmed culture(s)
Figure 15.1 Scheme of analysis for detection of thermotolerant Campylobacter in foods using the presence/absence method ISO 10272-1:2006.
and examine for growth of Campylobacter colonies.
c.4) Oxidase test: Using a platinum/iridium loop or glass rod, take a portion of a well-isolated colony from each individual CBA plate and streak it onto a filter paper moistened with the Oxidase Kovacs Reagent. The appearance of a mauve, violet or deep blue color within 10 s indicates a positive reaction. If a com-mercially available oxidase test kit is used, fol-low the manufacturer’s instructions.
Consider as Campylobacter spp. the cul-tures exhibiting the following characteristics:
small curved bacilli with a spiraling “cork-screw” motility, microaerobic growth at 25°C negative, aerobic growth at 41.5°C negative, oxidase positive.
d) Species identification (optional): Among the Campylobacter spp. growing at 41.5°C, the most frequently encountered species are Campylo-bacter jejuni and CampyloCampylo-bacter coli. Other spe-cies have, however, been described (Campylobacter lari, Campylobacter upsaliensis and some oth-ers). If necessary, the following tests permit their differentiation.
d.1) Catalase test: Deposit a loop of the culture from the CBA plate into a drop of 3% Hydro-gen Peroxide on a clean microscope slide. The test is positive if bubbles appear within 30 s.
Positive control: Staphylococcus aureus NCTC 8532, negative control: Enterococcus faecalis NCTC 775.
d.2) Sensitivity to nalidixic acid and to cepha-lothin: From the culture on CBA plate pre-pare a suspension in Brucella Broth (density
0.5 on the McFarland scale). Dilute this suspension 1/10 with the same broth and flood the surface of a Mueller Hinton Blood Agar plate with the suspension. After 5 min drain off excess suspension, dry the plates (drying cabinet at 37°C/10 min) and place a disc of nalidixic acid and a disc of cephalothin on the surface of the medium. Incubate the plates at 37 ± 1°C/22 ± 2 h in a microaero-bic atmosphere (without inverting). Cultures showing visible growth in contact with the disc are classified as resistant. Cultures show-ing inhibition halo (any size) around the discs are classified as susceptible.
d.3) Hippurate hydrolysis: From the culture on CBA plate prepare a suspension in 0.4 ml of Sodium Hippurate Solution and incubate at 37°C/2 h in a water bath or at 37°C/4 h in an incubator. Add 0.2 ml of Ninhydrin Solution and incubate at 37°C/10 min (water bath or incubator) without shaking. Development of a dark violet color indicates a positive reac-tion. A pale violet color or no color change indicates a negative reaction. Positive control:
Campylobacter jejuni NCTC 11351, negative control: Campylobacter coli NCTC 11366.
d.4) Indoxyl acetate hydrolysis: From the cul-ture on CBA plate place a loopful on an Indoxyl Acetate disc and add a drop of sterile distilled water. A color change to dark blue within 5–10 min indicates a positive reaction (indoxyl acetate hydrolysis). No color change indicates a negative reaction. Positive control:
Campylobacter jejuni NCTC 11351, negative control: Campylobacter lari NCTC 11352.
e) Interpretation of the results: Consider as Campy-lobacter spp. the cultures showing the following
Table 15.2 Characteristics of Campylobacter species growing at 41.5°C.
Test C. jejuni C. coli C. lari C. upsaliensis
Catalase + + + – or slight
Nalidixic acid Sa Sa R/Sb S
Cephalothin R R R S
Hydrolysis of hippurate + – – –
Indoxyl acetate + + – +
+ = positive, – = negative, S = sensitive, R = resistant.
a An increase in the resistance to nalidixic acid of C. jejuni and C. coli strains has been shown.
b Both sensitive and resistant C. lari strains exist.
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characteristics: morphology of small curved bacilli, spiralling “corkscrew” motility, negative growth at 25°C (microaerobic), negative growth at 45°C (aerobic), oxidase test positive.
Campylobacter species growing at 41.5°C may be identified at a species level according to Table 15.2.
15.3 References
Gupta, A., Nelson, J.M., Barrett, T.J. Tauxe R.V., Rossiter, S.P., Friedman, C.R., Joyce, K.W., Smith, K.E., Jones, T.F., Hawkins, M.A., Shiferaw, B., Beebe, J.L., Vugia, D.J., Rabatsky-Ehr, T., Benson, J.A., Root, T.P., Angulo, F.J. & NARMS Working Group (2004) Antimicrobial resistance among Campylobacter strains, United States, 1997–2001. Emerging Infectious Diseases, 10(6):1102–1109.
ICMSF (International Commission on Microbiological Specifica-tions for Foods) (1996) Microorganisms in Foods 5. Microbiologi-cal Specifications of Food pathogens. London, Blackie Academic &
Professional.
ICMSF (International Commission on Microbiological Specifica-tions for Foods) (2002) Microorganisms in Foods 7. Microbiological Testing in Food Safety Management. New York, Kluwer Academic/
Plenum Publishers.
International Organization for Standardization (2006) ISO 10272-1:2006. Microbiology of food and animal feeding stuffs – Horizontal method for the detection and enumeration of Campylobacter – Part 1: Detection Method. Geneva, ISO.
On, S.L.W. & Holmes, B. (1991) Reproducibility of tolerance tests that are useful in the identification of campylobacteria. Journal of Clinical Microbiology, 29, 1758–1788.
On, S.L.W. & Holmes, B. (1992) Assessment of enzyme detection tests useful in identification of campylobacteria. Journal of Clini-cal Microbiology, 30, 746–749.
On, S.L.W. & Holmes, B. (1995) Classification and identification of campylobacters, helicobacters and allied taxa by numerical analysis of phenotypic characters. Systematic and Applied Micro-biology, 18, 374–390.
Pearson, A.D. & Healing, T.D. (1992) The surveillance and control of campylobacter infection. Communicable Disease Review, 2(12), R133-R139.
Stanley, K. & Jones, K. (2003) Cattle and sheep farms as reservoir of Campylobacter. Journal of Applied Microbiology, 94, 104S–113S.
Tauxe, R.V., Hargrett-Bean, N. & Patton, C.M. (1998) Campylo-bacter isolates in the United States, 1982–1986. Morbidity and Mortality Weekly Report, 37(SS-2), 1–13.
Vandamme, P., Dewhirst, F.E., Paster, B.J., On, S.L.W. (2005a) Family I. Campylobacteraceae. In: Brenner, D.J., Krieg, N.R. &
Staley, J.T. (eds), Bergey’s Manual of Systematic Bacteriology. 2nd edition. Volume 2, Part C. New York, Springer. pp. 1145–1146.
Vandamme, P., Dewhirst, F.E., Paster, B.J. & On, S.L.W (2005b) Genus I. Campylobacter. In: Brenner, D.J., Krieg, N.R. & Staley, J.T. (eds), Bergey’s Manual of Systematic Bacteriology. 2nd edition.
Volume 2, Part C. Springer, New York, 2005. pp. 1147–1160.
WHO (World Health Organization). (2000) Campylobacter Fact Sheet No 255. [Online] Available from: http://www.who.int/
mediacentre/factsheets/fs255/en/ [Accessed 1st November 2011].
16 Cronobacter
16.1 Introduction
Cronobacter ( Enterobacter sakazakii) causes a foodborne disease classified by the International Commission on Microbiological Specifications for Foods (ICMSF, 2002) in Risk Group IB: “diseases of severe hazard for restricted population; life threatening or resulting in substantial chronic sequelae or presenting effects of long duration”. The FAO/WHO (Food and Agriculture Organization/World Health Organization) expert meet-ings have identified all infants (less than 12 months of age) as the population at particular risk for Cronobacter infections. Among this group, those at greatest risk are neonates (less than 28 days), particularly preterm, low-birth-weight (less than 2500 g), and immunocompro-mised infants, and those less than two months of age.
Infants of HIV-positive mothers are also at risk, because they may specifically require infant formula and may be more susceptible to infection (Codex Alimentarius/
CAC/RCP 66, 2008 revision 1 2009).
16.1.1 Taxonomy
The information below is from Iversen et al. (2007) and Iversen et al. (2008).
Cronobacter is a member of the family Enterobac-tericeae and until 1980 was designated as a yellow pig-mented variant of Enterobacter cloacae. In 1980 it was defined as the new species [Enterobacter sakazakii] by Farmer et al (1980). DNA-DNA hybridization gave no clear generic assignment for [E. sakazakii] as it was shown to be 53–54% related to species in two different genera, Enterobacter and Citrobacter. However the spe-cies was placed in Enterobacter as it appeared phenotypi-cally and genotypiphenotypi-cally closer to E. cloacae than to C.
freundii, the type species of these genera.
Iversen et al. (2007) proposed the subdivision of the species [E. sakazakii] in new species allocated in the new
genus Cronobacter. Iversen et al. (2008) reported officially the taxonomy of the new genus and the new species.
16.1.1.1 Cronobacter Iversen et al. 2008, gen. nov.
The members of the genus Cronobacter are Gram-negative rods, oxidase-negative, catalase-positive, and facultative anaerobic. Generally motile, they reduce nitrate, utilize citrate, hydrolyse aesculin and arginine and test posi-tive for L-ornithine decarboxylation. Acid is produced from D-glucose, sucrose, raffinose, melibiose, cellobi-ose, D-mannitol, D-manncellobi-ose, L-rhamncellobi-ose, L-arabincellobi-ose, D-xylose, trehalose, galacturonate and maltose. Generally positive for acetoin production (Voges-Proskauer test) and negative for the methyl red test, indicating 2,3-bu-tanediol rather than mixed acid fermentation. Negative reactions include hydrogen sulphide (H2S) production, urea hydrolysis, lysine decarboxylation and β-D-glucuro-nidase activity. The production of indole is variable.
The temperature range for growth is between 6°C and 45°C (in brain heart infusion broth) and the pH range is between pH 5.0 and 10.0, with no growth below pH 4.5.
Able to grow in up to 7% (w/v) NaCl but not in 10%.
The differentiation among the new genus Cronobacter and some other genera of the Enterobacteriaceae family is shown in the Table 16.1. The differentiation among the new Cronobacter species is shown in the Table 16.2.
16.1.2 Epidemiology
The information below is from Codex Alimentarius/
CAC/RCP 66 (2008 revision 1 2009).
According to the FAO/WHO (2008) experts all the six species of Cronobacter should be considered patho-genic. The incidence of infections in infants is low but have been documented as sporadic cases or as outbreaks with severe consequences.
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Table 16.1 Biochemical characteristics used to differentiate the new genus Cronobacter from some other genera of the Enterobacteriaceae family (Iversen et al., 2007).
Species α−Glc VP ADH ODC SAC RAF CEL ARA CIT MR ADO SOR LDC H2S
Cronobacter spp. + + + + + + + + + − − − − −
Buttiauxella agrestis v − − + − + + + + + − − − −
Citrobacter koseri − − v + v − + + + + + + − −
Citrobacter freundii − − v − v v v + v + − + − +
Edwardsiella tarda − − − + − − − − − + − − + +
Enterobacter aerogenes − + − + + + + + + − + + + −
Enterobacter asburiae − − v + + v + + + + − + − −
Enterobacter cancerogenus − + + + − − + + + − − − − −
Enterobacter cloacae − + + + + + + + + − v + − −
Enterobacter georgoviae − + − + + + + + + − − − + −
Enterobacter hormaechei − + v + + − + + + v − − − −
Enterobacter pyrinus v v − + + − + + − v − − + −
Enterobacter helveticus + − − − − − + + − + − − − −
Enterobacter turicensis + − − − − − + + − + − − − −
Escherichia coli − − v v v v − + − + − + (+) −
Hafnia alvei (−) (+) − + − − (−) + − v − − + −
Klebsiella pneumoniae (−) + − − + + + + + − + + + −
Kluyvera spp. v − − + + + + + (+) + − v v −
Leclercia adecarboxylata − − − − v v + + − + + − − −
Morganella morganii − − − + − − − − − + − − − (−)
Pantoea spp. − v − − v v v + v v − v − −
Proteus vulgaris + − − v (+) − − − v v − − − +
Providencia spp. − − − − v − − − v + v − − v
Rahnella aquatilis − − v − + + + + (−) − − + − −
Raoultella terrigena − + − (−) + + + + v v + + + −
Salmonella spp. − − v (+) − − v (+) v + − v (+) v
Serratia marcescens v + − + + − − − + (−) v + + −
Yersinia enterocolitica − − − + + − v + − + − + − −
4-NP-α-Glc = metabolism of 4-NP-α-glucoside; VP = Voges-Proskauer; ADH = arginine dihydrolase; ODC = ornithine decarboxylase;
SAC = acid from sucrose; RAF = acid from raffinose; CEL = acid from cellobiose; ARA = acid from arabinose; CIT = use of citrate as sole carbon source (Simmons); ADO = acid from adonitol; SOR = acid from sorbitol; LDC = lysine decarboxylase; MR = methly red test; H2S = production of hydrogen sulphide.
+ = 90–100% positive; (+) = 80–90% positive; v = 20–80% positive; (–) = 10–20% positive; – = less than 10% positive.
Manifestations may occur as meningitis and bacterae-mia, with variable fatality rates which may be as high as 50 percent (reported in at least one outbreak). Surviv-ing infants have sequelae such as retardation and other neurological conditions.
Outbreaks of Cronobacter species infections have been linked to powdered formulae, especially cases occurred in neonatal intensive care setting. Low number of Cronobacter cells is present in a proportion of powdered formulae. The microorganism has also been detected in other types of food, but only powdered formulae has been linked to outbreaks of disease. For infants at great-est risk in neonatal intensive care settings, commercially
sterile liquid infant formula should be used. If a non-commercially sterile feeding option is chosen, an effec-tive decontamination procedure should be used.