cronobacter research and surveillance in malaysia

Cronobacter research and surveillance in Malaysia

Norrakiah Abdullah Sani

Food Science ProgrammeSchool of Chemical Sciences and Food Technology

National University of Malaysia (UKM) Bangi, Selangor, Malaysia

[email protected]

UKM started operating from Lembah Pantai, Kuala Lumpur in 1970, and in October 1977, it moved to its present location in Bangi, an attractive and green valley area of 1,096 hectares.

UKM also has a branch campus in Kuala Lumpur and a teaching hospital in Cheras known as UKM Medical Centre (UKMMC).

List of Faculties1. Allied Health Sciences

2. Dentistry

3. Economics and Business

4. Education

5. Engineering and Built Environment

6. Graduate School of Business

7. Information Science and Technology

8. Islamic Studies

9. Law

10. Medicine

11. Pharmacy

12. Science and Technology

13. Social Sciences and Humanities

http://www.fskb.ukm.my/

http://www.ukm.my/fperg

http://www.ukm.my/fep

http://www.ukm.my/fpendidikan

http://www.ukm.my/jurutera

http://www.ukm.my/jurutera

http://gsb.ukm.my/

http://www.ftsm.ukm.my/

http://www.ukm.my/fpi

http://www.ukm.my/fuu

http://www.ukmmc.ukm.my/

http://www.ukm.my/farmasi/

http://www.ukm.my/fst

http://www.fssk.ukm.my/

Faculty of Science and Technology

1. School of Applied Physics

2. School of Biosciences and Biotechnology

3. School of Chemical Sciences and Food Technology

4. School of Mathematical Sciences

5. School of Environmental and Natural Resource Sciences

Cronobacter research and surveillance in Malaysia

Isolation, growth and survival characteristics of Cronobacter sakazakii and C. muytjensii in powdered infant formula (PIF) in Malaysia (2005-2007).

Detection of C. sakazakii and other Enterobacteriaceae in carton milk (2005-2006).

International survey for Cronobacter and related organisms in infant foods and formulas(2008) – poster P12.

C. sakazakii, Enterobacteriaceae and aerobic plate count in raw and pasteurized milk (2007-2008) – poster 13.

On-going

C. sakazakii and Enterobacteriaceae in drinking water (2008-2009).

Enterobacteriaceae, C.sakazakii and microbial population in PIF in the market (2008-2009).

Microbiological quality of infant cereal in the local markets (2008-2009).

Pending approval from MOH Ethical Committee

Identification of Cronobacter strains in PIF, water, milk preparation apparatus and environment in NICU of several hospitals (2009-2011).

Isolation, growth and survival characteristics of Cronobacter sakazakiiand C. muytjensii in PIF in Malaysia

Masomeh Ghassema, Uma Priya Kupusamyb, NorizanJaafarb, Abdul Salam Babjia, Stephen J. Forsythec, Norrakiah Abdullah Sani a

a Food Science Programme, School of Chemical Sciences and Food Technology, UniversitiKebangsaan Malaysia, 43600, Bangi, Selangor, Malaysia

b Chemistry Department of Malaysia, Jalan Sultan, 46661, Petaling Jaya, Selangor, Malaysia

c School of science and technology, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, UK

Isolation, growth and survival characteristics of Cronobacter sakazakiiand C. muytjensii in PIF in Malaysia

Cronobacter incidence in PIF obtained from supermarket, nurseries and hospitals.

ID presumptive Cronobacter strains using biochemical tests and molecular assays such as SYBR Green RT-PCR and 16S rDNA sequencing.

Growth and survival characteristics of Cronobacter strains in laboratory medium and reconstituted PIF at various temperatures.

Isolation of Cronobacter from PIF

91 PIF samples examined for Cronobacter and other Enterobacteriaceae.

◦ 75 PIF samples bought/ obtained from 7 different companies in the Malaysian market.

◦ 10 PIF samples collected from local nurseries.

◦ 6 PIF samples from two local hospitals in Hulu Langat District, Malaysia.

Identification of Cronobacter isolates

Presumptive Cronobacter colonies appeared entirely blue-green after 24h incubation on DFI agar, sub-cultured on Nutrient agar.

Biochemical identification systems :

- API20E (bioMérieux France)

- MicrogenTM GN-ID identification system

(GNA+ GNB, Microgen Bioproducts Ltd., UK)

Non-blue-green colonies also identified w/ biochemical kits.

C. muytjensii strain ATCC 51329 was used as the positive control organism.

◦ This was formerly the ATCC Preceptrol™ strain for the quality control of ‘Enterobacter sakazakii’ prior to the taxonomic revision.

DNA Extraction

12 presumptive Cronobacter blue-green colonies strains isolated from PIF and were identified using RT-PCR and 16S rDNA sequencing.

Resulting bacterial pellets subjected to DNA extraction by MasterPureTM Complete DNA and RNA Purifiction Kit (EPICENTRE, Madison,Wisconsin, U.S).

Identification of Cronobacter isolates with RT-PCR using SYBR Green The FailSafe™ Real-Time PCR Capillary Pre-Mix Selection Kit

(EPICENTRE, Madison,Wisconsin, U.S) was used for PCR amplification.

Cronobacter specific primers used (Liu et al., 2006) were:

- Forward (F) primer: 5'-TAT AGG TTG TCT GCG AAA GCG-3'

- Reverse (R) primer: 5'-GTCTTCGTGCTGCGAGTTTG-3'.

The master mix (20 µL) for PCR amplification were added to 10 µL of FailSafe RT-PCR Capillary 2X PreMix (C4) which include SYBR Green I (Molecular Probes, Inc., Eugene, Oregon).

Identification of Cronobacter isolates with RT-PCR using SYBR Green, continued

PCR was carried out in a Roche thermal cycler (Roche LightCycler 4559, Hoffmann-La Roche Inc., Basel, Switzerland)

Identification of Cronobacterisolates with 16S rDNA sequencing

All presumptive blue-green isolates obtained from 10 of 91 PIF samples (11%) identified with 16S rDNA sequencing using MicroSeq® 500 16S rDNA Bacterial Identification kit (Table 1).

Primers used for PCR amplification (Liu et al., 2006) were:

- Forward: 5'-TAT AGG TTG TCT GCG AAA GCG-3'

- Reverse: 5'-GTCTTCGTGCTGCGAGTTTG-3‘

Identification of Cronobacter isolates with 16S rDNA sequencing, continued..

PCR amplification done in a PCR thermal cycler (Gene Amp® PCR

Systems 2700, Applied Biosystems, USA) using the following thermal-cycling conditions:

◦ Initial incubation at 95°C for 10 min, 3–step cycling program for 30 cycles: denaturing at 95°C for 30 sec, annealing the primers at 60°C for 30 sec and 72°C for 45 sec for further primer extension.

◦ Final extension performed at 72°C for 10 min and cooled at 4°C. PCR products analyzed on 1.5% agarose gel w/ ethidium bromide staining.

◦ The gel was photographed in a gel visualizer (Alpha imager TM 2200, Alpha innotech, CA) under UV light.

◦ A Cleanup step was done to purify single-or doubled-stranded DNA fragments from PCR and other enzymatic reactions by using QIAquick PCR Purification Kits.

Identification of Cronobacter isolates with 16S rDNA sequencing, continued

Sequencing of PCR products carried out in thermal cycler using the following thermal-cycling conditions:

◦ 3–step cycling program, performed for 25 cycles, melting at 96°C for 10 sec, annealing the primers at 55°C for 15 sec and 60°C for 4 min for extension

◦ Final step of cooling at 4°C.

◦ After sequencing, DNA samples purified with CENTRI-SEP column (Applied Biosystems, USA)

◦ Resulting DNA sequences analyzed in the sequencer (3130 xl Genetic Analyzer, Applied Biosystems, USA) and compared to the library of National Centre for Biotechnology Information (NCBI) using BLASTn program.

Bacterial strains and growth conditions

Four strains:

◦ 3 strains of Cronobacter (UKMMGG1, UKMMGF1, and UKMMGH1) isolated from PIF.

◦ C. muytjensii (ATCC 51329).

Four growth conditions:

◦ 4 oC (refrigeration temp)

◦ 10oC (slightly abusive temp)

◦ 25oC (room temp in Malaysia)

◦ 37oC (optimum temp for growth)

◦ 45 and 50oC (maximum growth temps)

Population calculated as Log10 cfu/mL and plotted against incubation time (h) in different temperatures separately.

Data were analyzed by the Gompertz equation using ComBase statistical software package to obtain both generation and specific growth rate constant.

Generation time and specific growth rate constant results subjected to ANOVA using SPSS 14 (SPSS Inc., 2005) in order to determine significant statistical differences between growth (PIF and TSB) and plating (VRBGA and DFI) media and among Cronobacter strains.

Bacterial strains and growth conditions, continued

Bacterial strains and thermal resistance, continued

Five temperatures and time intervals:

◦ 52 and 54°C: 0, 10, 20, 30 & 35 min;

◦ 56°C: 0, 5, 12, 15 & 20 min;

◦ 58°C: 0, 5, 8, 10 & 12 min;

◦ 60°C: 0, 2, 5, 7 & 10 min

1 mL aliquots of each heating menstruum was serially diluted in MRD and plated on TSA plates with 1% sodium pyruvate, and incubated at 37°C for 24-48h using the surface drop method.

Bacterial strains and thermal resistance, continued

Experiments on two different days were performed for all strains at each temperature.

Themotolerance parameters (D and Z-values) were estimated using standard regression analysis based on log linear models.

D-values subjected to ANOVA using SPSS 14, to determine significant statistical differences among strains or temperatures.

RESULTS

Isolation of Cronobacter

From 116 isolates obtained from PIF, 12 isolates appeared entirely blue-green on DFI agar (10%).

Four of Cronobacter positive isolates were from PIF samples collected from nurseries and hospitals (3%).

Eight of Cronobacter positive isolates were from PIF samples in the market (7%).

TABLE 1

Differences in identification of presumptive Cronobacter spp. (E. sakazakii) isolates using biochemical and

16S rDNA profiling

Presumptive

Cronobacter

isolates

API20E biochemical

analysis

Microgen GN-ID

analysis (GNA+ GNB)

16S rDNA

sequencing

analysis

Identification % Profile Significant

Taxon

Identification % Octal

Code

Significant Taxon Significant Taxon

1 51.1 3305173 E. sakazakii 99.96 76021621 Salmonella group 1 E. sakazakii

2 Unacceptable 3326572 99.91 27470365 E. sakazakii E. sakazakii

3 95.1 3305573 E. cloacae 53.87 27460365 E. sakazakii E. sakazakii


5 77.6 3707573 E. sakazakii 53.87 27460365 E. sakazakii E. sakazakii






11 95.1 3305573 E. cloacae 53.87 27460365 E. sakazakii E. sakazakii


ATCC strain

5132999.9 3345373 E. sakazakii 96.82 27663325 E. sakazakii E. sakazakii

Biochemical Identification

Four strains had high % identification of E. sakazakiiusing Microgen GN-ID which was unacceptable using API20E profile.

Using API20E profile, 2 presumptive isolates identified as E. cloacae had high % identification as E.sakazakii using Microgen GN-ID.

One strain was confirmed as Salmonella with Microgen GN-ID but was identified as E. sakazakiiusing API20E profile.

TABLE 2.Enterobacteriaceae isolated from 91 PIF

Presumptive isolates

API20EAnalysis

Identification%

Significant Taxon

MicrogenAnalysis

Identification%

Percentage of isolates from IFM

1 87 E. cloacae 99.02 40

2 91.1 E. aerogenes 94.21 4.3

3 94.4 E. agglomerans 97.27 3.4

4 95.2 E. gergoviae 98.00 6

5 99 Escherichia coli 96.09 2.6

6 96.2 Citrobacter freundii 99.65 4.3

7 92 Hafnia alivei 92.01 2.6

8 99 Klebsiella pneumonia 97.27 6

9 94.5 Pantoea spp. 99.15 1.7

10 89.3 Salmonella group 1 95.08 6.6

11 94.3 Serratia ficaria 99.14 3.4

12 94.3 S. liquefaciens 89.65 7.8

13 89.9 Shigella spp. 94.32 1.7

14 92.2 X. maltophilia 83.87 1.7

*Total no. of Enterobacteriaceae isolated from PIF samples were 116.

RT-PCR Identification All 12 presumptive Cronobacter blue-green isolates from PIF and

C. muytjensii type strain ATCC 51329 confirmed using SYBR Green RT-PCR.

Fig. 1 (a). Lightcycler melting peaks analysis of five Cronobacter isolates from PIF and C. muytjensii type strain ATCC 51329

Fig. 1(b). LightCycler melting peaks analysis of seven Cronobacter isolates from PIF and C. muytjensii type strain ATCC 51329

RT-PCR Identification, continued

As analysis of melting temperature (Tm) on SYBR Green RT-PCR in Fig.1 (a) and (b) show, a Tm value of 86°C was observed for the positive control culture (ATCC 51329) and all 12 strains have the same melting peak at 86 ± 1°C and confirmed as E. sakazakii.

Fig. 2.

Light Cycle

Quantification

Analysis of Five (a)

and Seven (b)

Cronobacter

Isolates from PIF

and C. muytjensii

Type Strain ATCC

51329



The Ct value for each sample is proportional to the log of the initial amount of target DNA copies.

All 12 isolates also have Ct value of 15 to 18 cycles as shown in Fig 2 (a) and (b).

Growth Range of Cronobacter

Generation time & specific growth rate constant of three Cronobacter isolates and C. muytjensii type strain ATCC 51329 in PIF and TSB with four combinations of growth and plating media:

- TSB: VRBGA

- TSB: DFI

- PIF: VRBGA

- PIF: DFI

at 10, 25, 37 and 45°C were evaluated using ComBase software

Fig. 3. Growth curves of three C. sakazakiistrains, UKMMGF1 ( ), UKMMGG1 ( ), and UKMMGH1 ( ) and C. muytjensii ATCC 51329 ( ), grown and recovered in TSB: VRBGA (a) and PIF: DFI (b) at 10, 25, 37 and 45oC

0

2

4

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12

0 20 40 60 80 100 120 140 160

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12

0 20 40 60 80 100 120 140 160

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12

0 2 4 8 10 12 14 16 180

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0 2 4 8 10 12 14 16 18

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0 2 4 6 7 8 10

Time (Hr)

Log

10

nu

mb

er

of

cfu

/mL

of

Cro

no

ba

cter

stra

ins

(a) (b)

10oC

25oC

45oC

37oC

StrainTemp( C)

Generation time (h)

PIF TSB

DFI VRBGA DFI VRBGA

ATCC51329

10 2.49 0.05 3.19 0.01 3.23 0.09 3.27 1.48

25 0.41 0.01 0.45 0.01 0.47 0.01 0.63 0.01

37 0.27 0.03 0.28 0.05 0.28 0.06 0.30 0.02

45 0.26 0.01 0.27 0.01 0.27 0.06 0.28 0.04

UKMMGF1b10 3.10 0.01 3.60 0.30 3.87 0.17 3.87 0.39

25 0.51 0.01 0.52 0.02 0.62 0.13 0.64 0.03

37 0.28 0.06 0.29 0.05 0.29 0.05 0.30 0.01

45 0.27 0.05 0.28 0.01 0.29 0.04 0.30 0.02

UKMMGG1 10 4.98 0.54 5.02 0.31 5.14 0.82 5.20 0.20

25 0.49 0.02 0.51 0.02 0.60 0.05 0.63 0.02

37 0.28 0.02 0.29 0.01 0.31 0.03 0.32 0.01

45 0.27 0.18 0.27 0.01 0.27 0.01 0.28 0.01

UKMMGH1 10 3.17 0.02 3.59 0.02 3.61 0.06 3.64 0.01

25 0.52 0.01 0.57 0.05 0.63 0.05 0.65 0.10

37 0.32 0.03 0.33 0.01 0.36 0.02 0.36 0.01

45 0.26 0.12 0.27 0.01 0.28 0.09 0.29 0.07

= S

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TABLE 3. Generation times of four Cronobacter strains in PIF and TSB at various temperatures

StrainTemperature

( C)

Specific growth rate (h)-1

PIF TSB

DFI VRBGA DFI VRBGA

ATCC51329

10 0.28 0.005 0.22 0.001 0.21 0.006 0.21 0.101

25 1.67 0.018 1.53 0.016 1.48 0.038 1.10 0.018

37 2.55 0.312 2.47 0.444 2.43 0.471 2.33 0.218

45 2.66 0.104 2.6 0.022 2.53 0.470 2.43 0.307

UKMMGF1b10 0.22 0.001 0.19 0.015 0.18 0.008 0.18 0.018

25 1.36 0.001 1.32 0.062 1.12 0.224 1.08 0.056

37 2.45 0.444 2.42 0.413 2.36 0.348 2.32 0.016

45 2.58 0.405 2.47 0.002 2.35 0.319 2.27 0.175

UKMMGG1 10 0.14 0.018 0.14 0.009 0.13 0.027 0.13 0.005

25 1.42 0.056 1.35 0.060 1.16 0.131 1.09 0.045

37 2.5 0.172 2.36 0.009 2.23 0.232 2.14 0.016

45 2.55 1.406 2.53 0.123 2.53 0.0667 2.44 0.077

UKMMGH1 10 0.22 0.002 0.19 0.001 0.19 0.003 0.19 0.001

25 1.34 0.014 1.22 0.106 1.10 0.137 1.07 0.008

37 2.14 0.149 2.08 0.030 1.94 0.149 1.91 0.080

45 2.61 0.820 2.53 0.070 2.52 0.514 2.40 0.083

TABLE 4. Specific growth rate constant of four Cronobacter strains in PIF and TSB at various temperatures

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Growth Range of Cronobacter, continued

Mean generation times for four Cronobacter strains were 3.64, 0.5, 0.29 and 0.27 h in PIF and 3.98, 0.61, 0.31 and 0.28 min in TSB at 10, 25, 37 and 45°C, respectively.

No statistically significant differences found in generation time and specific growth rate constant among strains and growth media at 10, 25, 37 and 45°C (P> 0.05).

These comparisons also suggest that other three food isolates in PIF and TSB grow approximately at the same rate as ATCC 51329 at various temperatures.

D-value (min)

Temp (oC)

Strains52 54 56 58 60 Z-value (oC)

ATCC 51329 42.92 1.96 19.57 0.27 4.64 0.11 3.03 0.06 1.92 0.02 5.71 0.08

UKMMGF1 34.6 0.34 18.79 0.18 4.56 0.05 2.98 0.02 1.88 0.01 6.01 0.01

UKMMGG1 33.22 0.47 18.21 0.12 4.52 0.06 2.99 0.01 1.89 0.04 6.11 0.06

UKMMGH1 38.31 1.24 19.01 0.18 4.53 0.01 2.98 0.01 1.86 0.01 5.83 0.03

TABLE 5. D and Z-values of four strains of Cronobacter in PIF

= Standard deviation based on two replication (n=2)

Thermal Resistance of Cronobacter

D-values ranged from 42.92 min at 52°C for type strain ATCC 51329 to 1.86 min at 60°C for Cronobacter UKMMGH1.

D-values were significantly different at 52, 54 and 56°C comparing to 60°C (P< 0.05), but there were no significant differences in D-values at 58°C and 60°C (P> 0.05).

Z-values for Cronobacter isolated strains were higher than C. muytjensii type strain ATCC 51329 (5.71°C) which were in the same range (4-6°C) as reported by Tomlins and Ordal (1976) for most none-spore forming bacteria.

Discussion During investigation of feeding process in the NICU at

two local hospitals, it was found that powdered PIF was reconstituted and kept in small plastic bottles and then distributed into feeding tubes.

During handling and feeding infants, plastic bottles were opened and also were kept at room temperature of 25oC.

Presence of this organism in the reconstituted PIF could be due to post-processing contamination of the products.

This study showed that SYBR Green RT-PCR assay is an important tool for rapid identification of Cronobacter strains isolated from PIF.

Discussion

At 4°C, in TSB and PIF and for all four strains, the concentration of Cronobacter remained at the initial inoculum levels (103 cfu/mL) and did not multiply or decline with time.

These confirm the importance of proper refrigeration temperatures after reconstitution of PIF to ensure that this organism does not grow.

By increasing the temperature to 50°C, none of the food isolated Cronobacter strains and C. muytjensii type strain ATCC 51329 grew either in TSB or PIF.

Discussion

Cronobacter does not grow at proper refrigeration temperature of 4°C, but it can grow at slightly abusive temperature of 10°C (3.64h).

At room temperature of 25°C, the organism has a generation time of 29.92 min in reconstituted PIF.

Due to the exponential nature of bacterial growth, the risk of Cronobacter infection will also increase exponentially once the organism comes out of the lag period.

D and Z-values and thermal resistance of Cronobacter reported to be higher comparing to other members of Enterobacteriaceae.

Conclusions

Cronobacter was present in PIF with the incidence of 11%.

Other Enterobactreiaceae associated with NEC, the most common gastrointestinal emergency in the newborns, such as E. coli, K. pneumoniae, E. cloacae and Salmonella spp. were also present in IFM samples.

Conclusions

Importance must be given to proper preparation and storage of reconstituted PIF with respect to the growth of Cronobacter.

The ability of Cronobacter to multiply very quickly during holding time at room temperature (25°C) increases the risk of Cronobacter infection.

To reduce this risk, reconstituted PIF must be immediately used or kept below 5°C.

Acknowledgements

The Ministry of Health of Malaysia for the grant STGL-036-2005.

Dr. J. Baranyi and team of IFR UK, for their assistance in ComBase statistical software.

Sri Harminda P Hartantyo (Mian) for part assistance to the power point preparation.

TERIMA KASIHThank you for your attention

cronobacter research and surveillance in malaysia

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