Pakistan Jurnal f Nutritin 9 (7): 659-663, 2010 ISSN 1680-5194 Asian Netwrk fr Scientific Infrmatin, 2010 Islatin f Enterbacter sakazakii frm Pwdered Fds Lcally Cnsumed in Nigeria B.O. Aigbekaen and C.E. Oshma Department f Micrbilgy, University f Benin, P.M.B. 1154, Benin City, Ed State, Nigeria Abstract: The presence Enterbacter sakazakii was investigated frm pwdered fd samples using enrichment prcedure f Enterbacteriaceae Enrichment Brth (EEB), Vilet Red Bile Glucse Agar (VRBGA) and Tryptic Sy Agar (TSA). A ttal f 140 fd samples were tested. E. sakazakii was islated frm 20/70 pwdered infant fds, 13/50 pwdered milk and 5/20 milk-based prducts, making a ttal f 27.1% prevalence. The islates were tested fr their susceptibility t 15 antibitics. The results shwed that streptmycin was the mst effective (94.7%), fllwed by flxacin (92.1%), levflxacin (84.2%), ciprflxacin (79.0%), peflxacin (79.0%) and gentamycin (65.8%). The rganism was resistant t rifampicin and amxicillin. Key wrds: Fd, Enterbacter sakazakii, antibitic, streptmycin, infant milk INTRODUCTION Enterbacter sakazakii is a gram negative, facultative anaerbic, straight rd-shaped bacterium. It belngs t the family Enterbacteriaceae and genus Enterbacter that cntains a number f species including E. agglmerans, E. clacae, E. aergenes and E. gergviae. It is a clifrm having dimensins f 3 µm in length and 1 µm in width. The cells are mtile by peritrichus flagella and d nt frm spres (Farmer et al., 1980; Farmer and Kelly, 1992). Enterbacter sakazakii is an pprtunistic human pathgen that has been implicated in severe frms f septicemia (Lai, 2001), necrtizing enterclitis (Van Acker et al., 2001) and meningitis (Bar-Oz et al., 2001), especially in nenates with mrtality rate varying frm 40-80% (Muytjens et al., 1988). The Internatinal Cmmissin fr Fds, due t the seriusness f pathlgies with E. sakazakii has ranked the rganism as severe hazard fr restricted ppulatins, life threatening r substantial chrnic sequelae r lng lasting (ICMSF, 2002). E. sakazakii has been islated frm a wide range f fds including ultra high-temperature treated milk (UHT milk), cheese, meat, vegetables, grains, srghum seeds, rice seeds, herbs, spices, fermented bread, fermented beverage, tfu and sur tea (Gassem, 2002; Leclercq et al., 2002; Iversen and Frsythe, 2003). Despite this, studies have cnfirmed the cnnectin between nenatal E. sakazakii infectin and infant milk frmulas (Muytjens et al., 1988; Biering et al., 1989; Simmns et al., 1989; Nazarwec-White and Farber, 1997b; Van Acker et al., 2001). The US Fd and Drug Administratin (FDA, 2002) has issued an alert t health care prfessinals abut the risk assciated with E. sakazakii infectins amng nenates fed with milk-based infant frmula. The alert stated that a majr cntributin t the avidance f E. sakazakii infectins in premature babies and nenates is the preventin f cntaminatin f infant milk frmula during prductin and bttle preparatin. Several utbreaks have ccurred in nenatal intensive care units as a result f infectins by the rganism: in May/ June 1994 in France where 13 nenates were infected and three died, June/ July 1998 in Belgium where 12 nenates develped necrtizing enterclitis and tw twin brthers died and a mre serius ne in Tennessee in 2001 (CDC, 2002). These utbreaks were traced t cntaminated pwdered infant frmula by the rganism (Van Acker et al., 2001). But mst recently in 2008, a ttal f five babies were lst in New Mexic due t E. sakazakii infectin (CDC, 2009). These have alerted the US Centers fr Disease Preventin and Cntrl (CDC) n the cnsumptin f pwdered infant frmula, and have brught the rganism t limelight. E. sakazakii is an emerging fd brne pathgen that had been discvered in the develped cuntries. It is likely that there is a significant under-reprting f infectins in all cuntries. The absence f reprts is prbably due t a lack f awareness f the prblem rather than an absence f illness. Since infant frmula is widely used, the presence f E. sakazakii in infant frmula and its ptential effects in infants culd as well be a significant public health prblem in mst cuntries. This study prvides an infrmatin n the determinatin n the incidence f E. sakazakii in pwdered infant fds and antibitic susceptibility pattern f the rganism. Crrespnding Authr: B.O. Aigbekaen, Department f Micrbilgy, University f Benin, P.M.B. 1154, Benin City, Ed State, Nigeria 659
MATERIALS AND METHODS Cllectin f samples: A ttal f 140 different cmmercial fd samples frm different manufacturers were purchased frm retail stres acrss Benin City, Nigeria. The samples cmpsed f 70 pwdered infant fds (recmmended fr frm birth t ne year ld infants), 50 pwdered milk and 20 milk-based prducts. The samples were labeled apprpriately and apprved by Natinal Agency fr Fd and Drug Administratin and Cntrl (NAFDAC). Detectin and Islatin f Enterbacter sakazakii: The methd f Fd and Drug Administratin (2002) fr the islatin f the pathgen was used. Fr the preenrichment step, 10 g f each pwder was aseptically disslved in 50 ml f sterile water pre-warmed t 40 C, t make a slutin in the 60ml screw-capped bttles. Frm the bttle, 10-fld dilutin was made using sterile test tubes. The test tubes were screwed. The tubes were incubated at 36 C fr 24 h in an incubatr. Fr the enrichment step, 90 ml f Enterbacteriaceae Enrichment Brth (EEB) was prepared int the 100 ml screw-capped bttles. The incubated tubes (10 ml each) were aseptically inculated int the bttles cntaining 90ml f EEB. A bttle was left uninculated t act as a cntrl. The bttles were incubated at 36 C fr 24 h in an incubatr. The selectin step was carried ut by aseptically streaked a lp full f the incubated bttles int a duplicate plates f Vilet red bile glucse agar. The plates were then incubated at 36 C fr 24 h in an incubatr. Frm the cultured plates, purple clnies were picked using sterile inculating lp and subcultured int Tryptic Sy Agar (TSA) plates in duplicates by streaking. The plates were incubated at 25 C fr 72 h in an incubatr. The islated clnies were further detected by bichemical methds. Antibitic susceptibility test: Antibitic susceptibility test was perfrmed using Kirby-Bauer disc diffusin methd. The medium used fr this test is Tryptic sy agar. Prepared plates cntaining the medium were dried in ht air ven fr abut five minutes. A sterile inculating lp was used t pick islated clnies and emulsified int 5 ml f sterile nutrient brth. After prper hmgenizatin, the brth was pured int the agar surface t make a lawn f grwth. The surface f the agar plate was allwed t dry fr 5 min. A sterile frceps was used t place the antibitic discs unt the inculated plates. The antibitic discs were in tw sets and each set was used fr islatin. The antibitics cntained in the discs were: streptmycin (30 µg), nrflxacin (10 µg), chlramphenicl (30 µg), ciprflxacin (10 µg), levflxacin (20 µg), gentamycin (10 µg), rifampicin (20 µg), amxicillin (20 µg), flxacin (10 µg), peflxacin (10 µg), augmentin (30 µg), cephalexin (10 µg), nalidixic acid (30 µg), trimethprimsulfamethxazle (30 µg), ampicillin (30 µg). The plates were incubated at 37 C fr 24 h in an incubatr. After incubatin, the diameter f the zne f inhibitin was measured in millimetres using a ruler. All results were recrded apprpriately and interpreted using the Natinal Cmmittee fr Clinical Labratry Standards (NCCLS) interpretatin chart (NCCLS, 2002). RESULTS A ttal f 140 fd samples were tested fr the presence f E. sakazakii, 38 samples were fund psitive. The psitive strains f E. sakazakii frmed yellw clnies n Tryptic sy agar after 48 h f incubatin at 25 C and satisfied the bichemical screening. Table 1 indicates the number f E. sakazakii islates btained frm each fd brand. The rganism was detected in all the fd brands except in Burnvita. Amng the pwdered infant fds, SMA was the mst prevalent, while Friscrem and Nutrend were the least prevalent fr the rganism. Peak chc had the highest prevalence amng the milk-based prducts, with Burnvita had zer prevalence. The result in Table 2 shwed that 38(27.1%) f the ttal 140 pwdered fds were psitive fr E. sakazakii, with pwdered infant fds having the highest frequency f 33.9%, fllwed by pwdered milk (32.7%) and milkbased prducts (31.4%) having the least. On testing the 38 islates frm the samples using 15 antibitics in antibitic susceptibility discs, almst all the islates were sensitive t streptmycin (94.7%), fllwed by flxacin (92.1%), levflxacin (84.2%), ciprflxacin (79.0%), peflxacin (79.0%) and gentamycin (65.8%) in the decreasing rder (Table 3). Cephalexin (42.1%), nrflxacin (31.6%), amxicillinclavulanate (31.6%) was prly sensitive; while nalidixic acid (10.5%), trimethprim-sulfamethxazle (10.5%), chlramphenicl (7.9%), ampicillin (5.3%) were resistant. But rifampicin (0%) and amxicillin (0%) were strngly resistant. DISCUSSION Enterbacter sakazakii is an emerging fd-brne pathgen that had been linked with infantile meningitis, septicemia and necrtizing clitis transmitted thrugh the cnsumptin f cntaminated pwdered infant fds and ther milk prducts (Lai, 2001; Van Acker et al., 2001; Bar-Oz et al., 2001). This study was set up t investigate the incidence f this rganism in pwdered fds and its prducts. E. sakazakii was islated frm the three fd typespwdered infant fd, pwdered milk and milk-based prducts, in 27.1% f samples. Amng the three fd types, pwdered infant fd had the highest frequency. 660
Table 1: Enterbacter sakazakii islated frm pwdered fds N. f SP (+) with SN (-) with Fd samples/brand samples E. sakazakii E. sakazakii Pwdered infant fd SMA 10 4 6 NAN 10 3 7 Peak 123 10 3 7 Nutrend 10 2 8 Sya 10 3 7 Cerelac 10 3 7 Friscrem 10 2 8 Pwdered milk Peak 10 3 7 Cwbell 10 3 7 Nunu 8 2 6 Cast 8 1 7 Blue bat 7 2 5 Jag 7 2 5 Milk-based prduct Mil 4 1 3 Ovaltine 4 1 3 Peak chc 4 2 2 Cwbell chc 4 1 3 Burnvita 4 0 4 SP = Samples Psitive (+) with E. sakazakii SN = Samples Negative (-) with E. sakazakii Table 2: Ttal (percentage) cntaminatin f the sample type Ttal N. (%) f sample Sample type number cntaminated Pwdered infant fd 70 20 (33.9) Pwdered milk 50 3 (32.7) Milk-based prduct 20 5 (31.4) Ttal 140 38 (27.1) Pak. J. Nutr., 9 (7): 659-663, 2010 These results crrelated with the wrks f (Muytjens et al., 1988; Biering et al., 1989; Simmns et al., 1989; Nriega et al., 1990; Nazarwec-White and Farber, 1997b; Iversen and Frsythe, 2003; Shaker et al., 2007), wh had fund a direct relatinship between infant frmula and E. sakazakii. Muytjens et al. (1988) tested 141 samples f pwdered infant milk frmula manufactured in different cuntries. They fund that E. sakazakii and ther Enterbacteriaceae were islated frm 14.1 and 52.2% f the ttal samples respectively. Nazarwec-White and Farber (1997b) surveyed the presence f E. sakazakii in 120 dried infant milk samples (five manufacturers) btained frm Canadian retail market and reprted that the prevalence f this bacterium ranged between 0 and 12% f the samples/manufacturer. Iversen and Frsythe (2004) islated E. sakazakii frm 24% f 82 pwdered infant milk frmulas. Many studies have fcused n the infant frmula as the main surce f this serius pathgen (Pstupa and Aldva, 1984; Muytjens et al., 1988; Nazarwec-White and Farber, 1997b; Van Acker et al., 2001; Blck et al., Enterbacter sakazakii like ther Enterbacter species 2002). Despite the fact that frmulas are expsed t have acquired resistance by inactivating brad spectrum heat treatment during prcessing, E. sakazakii was still islated frm these prducts. Pst-prcessing cntaminatin f the infant frmula frm fd prductin envirnments may be respnsible fr the presence f this pathgen in infant frmula since standard pasteurizatin practices are effective fr the inactivatin f the rganism (Iversen et al., 2004). Nazarwec-White and Farber (1997a) stated that E. sakazakii can gain access t the pwder frm the envirnment r frm the additin f the ingredients at the pwder stage, especially using the Dry-mix prcess f prductin. Iversen and Frsythe (2003) reprted that the presence f E. sakazakii in pwdered infant milk frmula depends n the prcess cnditins and nature f the prduct. Hwever, the prevalence f the rganism fllwing the drying stage and survival in pwdered fds fr a lng time may be due (in part) t the rganism s ability t resist desiccatin and smtic stress (Arku et al., 2008). Unlike cmmercially available ready-t-feed liquid infant frmula, which is sterile, pwdered infant frmula (including dried bvine milk and milk prducts) is nt a sterile prduct (FDA, 2002). Pwdered infant frmula has been knwn t be cntaminated, n ccasin, with bacterial pathgens, including Bacillus species, Clstridium species, Staphylcccus species and Enterbacteriaceae, ntably Crnbacter (Frsythe, 2005). Therefre, hygienic measures and practices must be used during the manufacture f frmula t minimize entry f cntaminants int the prcess. Fr the case f the milk-based prduct, the rganism was nt detected in Burnvita, E. sakazakii culd nt be ruled frm being a pssible pathgen f this prduct, putting int cnsideratin that each sample represented itself nly. This result is unusual as the rganism has been islated frm a wide range f fds including chclate, spices, rice seeds, cereals, ptat flur and pasta (Shaker et al., 2007). Als, it culd that the rganism is unequally distributed in the sample r its presence escaped detectin (Muytjens et al., 1988). Frm the results btained frm the antibitic sensitivity test (Table 3), streptmycin (94.7%) seems t be a better drug fr treatment. Others include Oflxacin (92.1%), levflxacin (79.0%), peflxacin (79.0%) and gentamycin (65.8%). Streptmycin and gentamycin are aminglycsides; while Oflxacin, levflxacin, ciprflxacin and peflxacin are quinlnes. These results were cnsistent with the findings by Stck and Wiedemann (2002) that E. sakazakii is susceptible t tetracyclines, aminglycsides, quinlnes, antiflates and chlramphenicl. Aminglycsides and quinlnes inhibit prtein synthesis and DNA replicatin respectively in the rganism (McKane and Kandel, 1996). beta-lactam antibitics due t the prductin f betalactamases (Drudy et al., 2006). Other mechanisms 661
Table 3: Number (percentage) f E. sakazakii islates sensitive t antibitics N. f Antibitics islates --------------------------------------------------------------------------------------------------------------------------------------------- Sample brands tested S NB CH CPX LEV RD CN AML SMA 4 4(100) 1(25) 0(0) 3(75) 3(75) 0(0) 3(75) 0(0) NAN 3 3(100) 1(33.3) 1(33.3) 3(100) 3(100) 0(0) 2(66.7) 0(0) Peak 123 3 3(100) 0(0) 0(0) 2(66.7) 3(100) 0(0) 2(66.7) 0(0) Nutrend 2 2(100) 1(50) 1(50) 2(100) 2(100) 0(0) 1(50) 0(0) Sya 3 2(66.7) 1(33.3) 0(0) 1(33.3) 3(100) 0(0) 1(33.3) 0(0) Cerelac 3 2(66.7) 2(66.7) 0(0) 1(33.3) 2(66.7) 0(0) 2(66.7) 0(0) Friscrem 2 2(100) 1(50) 0(0) 1(50) 1(50) 0(0) 2(100) 0(0) Peak 3 3(100) 1(33.3) 0(0) 3(100) 1(33.3) 0(0) 1(33.3) 0(0) Cwbell 3 3(100) 1(33.3) 0(0) 2(66.7) 3(100) 0(0) 3(100) 0(0) Nunu 2 2(100) 0(0) 0(0) 2(100) 1(50) 0(0) 2(100) 0(0) Cast 1 1(100) 1(100) 1(100) 1(100) 1(100) 0(0) 0(0) 0(0) Blue bat 2 2(100) 1(50) 0(0) 2(100) 2(100) 0(0) 2(100) 0(0) Jag 2 2(100) 0(0) 0(0) 2(100) 2(100) 0(0) 1(50) 0(0) MBP 5 5(100) 1(20) 0(0) 5(100) 5(100) 0(0) 3(60) 0(0) Ttal (%) 38 94.7 31.6 7.9 79.0 84.2 0.0 65.8 0.0 N. f Antibitics islates ------------------------------------------------------------------------------------------------------------------------------------------- Sample brands tested OFX PEF AU CEP NA SXT PN SMA 4 4(100) 4(100) 1(25) 1(25) 0(0) 1(25) 0(0) NAN 3 3(100) 2(66.7) 1(33.3) 1(33.3) 0(0) 0(0) 0(0) Peak 123 3 3(100) 1(33.3) 2(66.7) 0(0) 0(0) 0(0) 1(33.3) Nutrend 2 2(100) 1(50) 1(50) 1(50) 0(0) 0(0) 0(0) Sya 3 3(100) 3(100) 1(33.3) 2(66.7) 1(33.3) 0(0) 1(33.3) Cerelac 3 2(66.7) 2(66.7) 0(0) 1(33.3) 0(0) 0(0) 0(0) Friscrem 2 2(100) 2(100) 0(0) 2(100) 0(0) 1(50) 0(0) Peak 3 2(66.7) 2(66.7) 0(0) 2(66.7) 1(33.3) 1(33.3) 0(0) Cwbell 3 3(100) 2(66.7) 0(0) 2(66.7) 0(0) 0(0) 0(0) Nunu 2 2(100) 1(50) 0(0) 1(50) 1(50) 0(0) 0(0) Cast 1 1(00) 1(100) 0(0) 0(0) 0(0) 1(100) 0(0) Blue bat 2 2(100) 2(100) 1(50) 1(50) 0(0) 0(0) 0(0) Jag 2 2(100) 2(100) 1(50) 1(50) 0(0) 0(0) 0(0) MBP 5 4(80) 5(100) 4(80) 1(20) 1(20) 0(0) 0(0) Ttal (%) 38 92.1 79.0 31.6 42.1 10.5 10.5 5.3 S = Streptmycin, CN = Nrflxacin, CH = Chlramphenicl, CPX = Ciprflxacin, LEV = Levflxacin, RD = Rifampicin, CN = Gentamycin, AML = Amxicillin, OFX = Oflxacin, PEF = Peflxacin, AU = Amxicillin-clavulanate, CEP = Cephalexin, NA = Nalidixic Acid, SXT = Trimethprim-sulfamethxazle, PN = Ampicillin. NZI = N Zne f Inhibitin include: decreased cell permeability, active efflux, mdificatin f drug receptr site, synthesis f resistant metablic pathway and acquisitin f plasmids and transpsns (Cha et al., 2007). These culd be the reasns fr the strng resistance against cephalexin (42.1%), amxicillin-clavulanate (31.6%), ampicillin (5.3%) and amxicillin (0%) as bserved in this research. Other studies have als shwn rifampicin, macrlides and fusidic acid t be resistant (Stck and Wiedemann, 2002). Cnclusin: E. sakazakii is an emerging pathgen, ften transmitted thrugh the cnsumptin f pwdered infant fds and its prducts. It is respnsible fr series f infectins with ptential fatal utcmes in infants. The findings frm this study shwed that E. sakazakii was detected in pwdered infant fds, pwdered milk and milk-based prducts. The rganism was fund t be resistant t sme antibitics; the quinlnes, in cmbinatin with an aminglycsides wuld be a better chice drug fr treatment f infectins caused by this rganism. REFERENCES Arku, B., N. Mullane, E. Fx, S. Fanning and K. Jrdan, 2008. Enterbacter sakazakii survives spray drying. Int. J. Dairy Technl., 61: 102-108. Bar-Oz, B., A. Preminger, O. Peleg, C. Blck and I. Arad, 2001. Enterbacter sakazakii infectin in the newbrn. Acta Paedia., 90: 356-358. Biering, G., S. Karlssn, N. Clark, N.C., Karlssn, K.E. Jnsdttir, P. Ludvigssn and O. Steingrimssn, 1989. Three cases f nenatal meningitis caused by Enterbacter sakazakii in pwdered milk. J. Clin. Micrbil., 27: 2054-2056. Blck, C., O. Peleg, N. Minster, B. Bar-Oz, A. Simhn, I. Arad and M. Shapir, 2002. Cluster f nenatal infectins in Jerusalem due t unusual bichemical variant f Enterbacter sakazakii. Eur. J. Clin. Micrbil. Infect. Dis., 21: 613-616. 662
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