Investigation of Extended Spectrum Β-Lactamases (ESBL)-Producing Enterobacteriaceae and Cronobacter Spp in Infant Formulas and Cereal-Based Foods for Children

Yıl 2017, Sayı: 1, 19 - 32, 26.04.2017

Pınar Cava Gümüş İsmail Hakkı Tekiner Burcu Çakmak Zeynep Tacer Caba Haydar Özpınar

Öz

Aim: The
extended spectrum β-lactamase (ESBL) producing Enterobacteriaceae have attracted attention due to their ability of
delaying or cancelling antimicrobial therapy. The foods for infants and
children may occasionally contain pathogens such as ESBL-producing Enterobacteriaceae, including Cronobacter spp., because of
inappropriate production, storage and handling conditions. This situation leads
to a greatest risk of infection to infants and children through their consumption.
The objective of this study was to investigate the occurrence of ESBL-producing
Enterobacteriaceae, including Cronobacter spp., from a total of 115
samples of various foods from different brands for infants and children sold in
Istanbul, Turkey (20 locally produced infant formula, 20 imported infant
formula, 20 starch, 20 rice flour, 20 semolina, and 15 milk powder).

Methods: For isolation of Enterobacteriaceae ISO 21528-2:2004 was
followed. The isolated colonies were identified by Mass Spectrometer. The identified
colonies were then exposed to pre-enrichment at 35-370C for 18-24
hours, the suspension was transferred to a Chromogenic ESBL selective media,
and allowed for an incubation at 35-370C for 18-24 hours again. The
presumptive ESBL producers were subjected to ESBL screening test using a
combination of ceftazidime, cefotaxime, and cefpodoxime ± clavulanic acid.
Finally, MIC values for ESBL confirmation were conducted by Merlin Micronaut-S
beta-lactamase VII kit, the readings were automatically evaluated by Sifin
Software integrated with a spectrometer. All procedures were performed
according to the Guidelines of CLSI (2013).

Results: Of 18 ESBL suspected isolates (7 P. agglomerans, 1 E. aerogenes, 3 E.  cloacae/asburiae, 2 K. oxytoca, 2 L. adecarboxylata,
and 3 C. sakazakii), 5 isolates (3 P. agglomerans, 1 E. cloacae/ asburiae, and 1 E.
aerogenes) were positive for ESBL-production, whereas none of C. sakazakii isolates was
ESBL-producers.

Discussion and Conclusion: Some of the
analysed infant formulas and cereal-based foods for children (locally produced
infant formulas, rice flour, and starch) were determined to be posing a serious
health risk for the infants and children due to contamination by ESBL-producing
Enterobacteriaceae, except for C. sakazakii isolates.

Anahtar Kelimeler

Cereal-based food, Children, Cronobacter spp., Enterobacteriaceae, ESBL, Infant formula

Bebek Mamaları ve Tahıl Bazlı Çocuk Gıdalarında Genişlemiş Spektrumlu Β-Laktamazlar (GSBL) Üreten Enterobacteriaceae ve Cronobacter Spp. Araştırılması

Öz

Amaç: Genişlemiş spektrumlu β-laktamaz
(GSBL) üreten Enterobacteriaceae türleri
antimikrobiyal tedavileri başarısız kıldıkları için artan şekilde ilgi
görmektedirler. Yenidoğan ve çocuk gıdaları uygunsuz üretim, depolama ve
hazırlama koşulları sebebiyle GSBL-üreten Enterobacteriaceae
suşları, özellikle Cronobacter
türleri dahil olmak üzere, patojen mikroorganizmalar içerebilmektedir. Bu tür
gıdaların tüketilmesi yenidoğan ve çocuk sağlığı açısından son derece tehlikeli
enfeksiyonlara yol açmaktadır. Bu çalışmada İstanbul ilinde satışa sunulan
farklı markalara ait toplam 115 örnekte (20 yerli üretim bebek maması, 20 ithal
bebek maması, 20 nişasta, 20 pirinç unu, 20 irmik ve 15 süt tozu) GSBL-üreten Enterobacteriaceae ve Cronobacter türlerinin varlıklarının
araştırılması amaçlanmıştır.

Yöntem: Örneklerde Enterobacteriaceae türlerinin izolasyonu ISO 21528-2:2004
talimatları takip edilerek yapılmıştır. İzole edilen Enterobacteriaceae türleri Kütle Spektrometresi ile
tiplendirilmiştir. Tiplendirmesi tamamlanmış izolatlar ön zenginleştirme amaçlı
35-370C/18-24 saat inkübe edildikten sonra, kromojenik GSBL seçici
besiyerine transfer edilmiş ve tekrar 35-370C/18-24 saat inkübasyona
bırakılmıştır. İnkübasyon sonunda gelişen GSBL şüpheli koloniler seftazidim,
sefotaksim ve sefpodoksim ± klavulanik asit içeren antibiyotik diskler
kullanılarak incelenmiştir. İnceleme sonucu güçlü şüpheli GSBL pozitif olduğu
belirlenen kolonilerin MİK değeri tespiti Merlin Micronaut-S beta-lactamase VII
kiti kullanılarak yapılmış, MİK bulguları spektrometre tarafından ölçülmüş ve
Sifin yazılımı ile otomatik olarak değerlendirilmiştir. GSBL tarama ve MİK
tespiti CLSI (2013) talimatları takip edilerek yapılmıştır.

Bulgular: Mikrobiyolojik İnceleme sonucu elde
edilen toplam 18 adet GSBL şüpheli izolat arasında (7 adet P.  agglomerans, 1 adet E. aerogenes, 3 adet E. 
cloacae/asburiae, 2 adet K.
oxytoca, 2 adet L. adecarboxylata
ve 3 adet C. sakazakii), 5 adet
izolatın (3 adet P. agglomerans, 1
adet E. cloacae/ asburiae ve 1 adet E. aerogenes) GSBL pozitif oldukları,
diğer taraftan C. sakazakii
izolatların GSBL üretmedikleri tespit edilmiştir.

Tartışma ve Sonuç: Yerli üretim
bebek mamaları, pirinç unu ve nişasta örneklerinin, C. sakazakii izolatları hariç, GSBL-üreten Enterobacteriaceae türleri içerdikleri, bu sebeple bebek ve çocuk
sağlığı açısından ciddi sağlık riski oluşturdukları görülmüştür.

Anahtar Kelimeler

Tahıl bazlı gıda, Çocuk, Cronobacter spp., Enterobacteriaceae, GSBL, Bebek maması

Kaynakça

• Kim SA, Oh SW, Lee YM, Imm JY, Hwang IG, Kang DH, Rhee MS. Microbial contamination of food products consumed by infants and babies in Korea. Lett Appl Microbiol. 2011;53: 532–538.
• Öndeş N, Özpinar H. Occurrence of ESBL-Producing Enterobacteriaceae in Cubed Beef Samples. Kafkas Univ Vet Fak Derg. 2016; 22(1): 79-83.
• Tekiner İH, Özpinar H. Occurrence and characteristics of extended spectrum beta-lactamases-producing Enterobacteriaceae from foods of animal origin. Braz J Microbiol. 2016; 47(2): 444-51.
• Shaker R, Osaili T, Al-Omary W, Jaradat Z, Al-Zuby M. Isolation of Enterobacter sakazakii and other Enterobacter spp. from food and food production environments. Food Control. 2007;18: 1241-1245.
• Holy O, Forstythe S. Cronobacter spp. as emerging causes of healthcare-associated infection. J Hosp Infect. 2014;86: 169-177.
• Li Y, Chen Q, Zhao J, Jiang H, Lu F, Bie X, Lu Z. Isolation, identification and antimicrobial resistance of Cronobacter spp. isolated from various foods in China. Food Control. 2014; 37:109-114.
• Caubillia-Barron J, Hurrel E, Townsend S, Cheetham P, Loc-Carrillo C, Fayet O. Genotypic and phenotypic analysis of Enterobacter sakazakii strains from an outbreak resulting fatalities in a neonatal intensive care unit in France. J Clin Microbiol. 2007;45(12):3979-3985.
• Himelright I, Harris E, Lorch V, Anderson M, Jomes T, Craig A. Enterobacter sakazakii infections associated with the use of powdered infant formula-Tennessee, 2001. J Am Med Assoc. 2002;287(17): 2204-2205.
• FAO/WHO (2004) Enterobacter sakazakii and other microorganisms in powdered infant formula: Meeting report. Microbiological Risk Assessment Series No. 6, Rome, Italy.
• FAO/WHO (2006) Enterobacter sakazakii and Salmonella in powdered infant formula: Meeting report. Microbiological Risk Assessment Series No. 10, Rome, Italy
• Oliphant CM, Eroschenko K. Antibiotic Resistance, Part 2: Gram-negative Pathogens. J Nurse Practitioners. 2015;11: 79-86.
• Shakil S, Akram M, Ali SM, Khan AU. Acquisition of extended-spectrum b-lactamase producing Escherichia coli strains in male and female infants admitted to a neonatal intensive care unit: molecular epidemiology and analysis of risk factors. J Med Microbiol. 2010; 59: 948–954.
• Tham J, Mats W, Eva M, Inga O. Prevalence of extended-spectrum beta-lactamase-producing bacteria in food. Infect Drug Resist. 2012; 5: 143-147.
• Stewardson AJ, Renzi G, Maury N, Vaudaux C, Brossier C, Fritsch E, Pitet D, Heck M, Zwaluw KV, Reuland A, Laar T, Snelders E, Vandenbroucke-Grauls C, Kluytmans J, Edder P, Schrenzel J, Harbarth S. Extended-Spectrum β-Lactamase–Producing Enterobacteriaceae in Hospital Food: A Risk Assessment. Infect Cont Hosp Ep. 2014;35(4): 375-383.
• Wiklund S, Hallberg U, Kahlmeter G, Tammelin A. Living with extended-spectrum beta-lactamase: a qualitative study of patient experiences. Am J Infec Control. 2013; 41: 723-727.
• Chon JW, Song KY, Kim SY, Hyeon JY, Seo KH. Isolation and characterization of Cronobacter from desiccated foods in Korea. J Food Sci. 2012;77(7): 354-358.
• Lee YD, Park JH, Chang H. Detection, antibiotic susceptibility and biofilm formation of Cronobacter spp. from various food from Korea. Food Control. 2012;24(1-2): 225-230.
• Molly C, Cagney C, O’Brien S, Iversen C, Fanning S, Duffy G. Surveillance and characterisation by pulsed-field gel electrophoresis of Cronobacter spp. in farming and domestic environments, food production animals and retails foods. Int J Food Microbiol. 2009;136 (2): 198-203.
• Kandhai MC, Heuvelink AE, Reij MW, Beumer RR, Dijk R, Van Tilburg JJHC, Van Schothorst M, Gorris LGM. A study into the occurrence of Cronobacter spp. in the Netherlands between 2001 and 2005. Food Control. 2010;21: 1127–1136.
• Ben Said L, Jouini A, Klibi N, Dziri R, Alonso CA, Boudabous A, Ben Slama K, Torres C. Detection of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in vegetables, soil and water of the farm environment in Tunisia. Int J Food Microbiol. 2015; 203: 86–92.
• Anon. (2004). International Standart ISO 21528-2:2004 (E) Microbiology of food and animal feeding stuffs - Horizontal methods for the detection and enumeration of Enterobacteriaceae.
• Anon. (2013). Clinical and laboratory standards institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Third Informational Supplement. M100-S23.
• Ergün F, Ergün Ö. Ülkemizde tüketime sunulan yerli ve ithal bebek mamalarının genel mikrobiyolojik kriterleri ve bazı patojenlerin varlığı yönünden incelenmesi. İstanbul Üniv. Vet. Fak. Derg. 1999;25: 57-64.
• Çetinkaya E, Josept S, Ayhan K, Forsythe SJ. Comparison methods for the microbiological identification and profiling of Cronobacter species from ingredients used in preparation of infant formula. Mol Cell Probe. 2012;27(1): 60-64.
• Kropp KA, Lucid A, Carroll J, Belgrudov V, Kelly WB, Templeton K, Smith C, Dickinson P, O’Driscoll A, Ghazal P, Sleator RD. Draft Genome Sequence of a Pantoea sp. Isolated from a Preterm Neonatal Blood Sepsis Patient. Genome Announ. 2014;2(5): e00904-14.
• Mardaneh J, Dallal MMS. Isolation, identification and antimicrobial susceptibility of Pantoea (Enterobacter) agglomerans isolated from consumed powdered infant formula milk (PIF) in NICU ward: First report from Iran. Iran J Microb. 2013;5(3): 263-267.
• Meropol SB, Stange KC, Jacobs MR, Weiss JK, Bajaksouzian S, Bonomo RA. Bacterial Colonization and Antibiotic Resistance in a Prospective Cohort of Newborn Infants During the First Year of Life. Open Forum Infect Dis. 2016; 3(4): ofw221.4.