Antibiotic resistance, putative virulence factors and curli fimbrination among Cronobacter species

Antibiotic resistance, putative virulence factors and curli fimbrination among Cronobacter species

Microbial Pathogenesis 136 (2019) 103665 Contents lists available at ScienceDirect Microbial Pathogenesis journal homepage: www.elsevier.com/locate/...

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Microbial Pathogenesis 136 (2019) 103665

Contents lists available at ScienceDirect

Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath

Antibiotic resistance, putative virulence factors and curli fimbrination among Cronobacter species

T

O.A. Odeyemia,b,1, N. Abdullah Sanib,∗ a

Aquaculture Microbiology Laboratory, Ecology and Biodiversity Centre, Institute for Marine and Antarctic Studies, University of Tasmania, Launceston, Australia Food Safety, Security and Quality Research Group, Centre for Biotechnology and Functional Food, Faculty of Science and Technology, National University of Malaysia (UKM), Bangi, 43600, Selangor, Malaysia b

A R T I C LE I N FO

A B S T R A C T

Keywords: Virulence factor Biofilm formation Antibiotic resistance Cronobacter sakazakii

This study aimed to investigate antibiotic resistance and putative virulence factors among Cronobacter sakazakii isolated from powdered infant formula and other sources. The following 9 cultures (CR1-9) were collected from our culture collection: C. sakazakii and 3 Cronobacter species: C. sakazakii ATCC® 29544™, C. muytjensii ATCC® 51329™, C. turicensis E866 were used in this study. Isolates were subjected to antibiotic susceptibility and the following virulence factors (protease, DNase, haemolysin, gelatinase, motility and biofilm formation) using phenotypic methods. All the bacteria were able to form biofilm on agar at 37 °C and were resistant to ampicillin, erythromycin, fosfomycin and sulphamethoxazole. It was observed from this study that tested strains formed weak and strong biofilm with violet dry and rough (rdar), brown dry and rough (bdar), red mucoid and smooth (rmas) colony morphotypes on Congo red agar. Rdar expresses curli and fimbriae, while bdar expresses curli. Both biofilm colony morphotypes are commonly found in Enterobacteriaceae including Salmonella species. This study also reveals a new colony morphotypes in Cronobacter species. Conclusively, there was correlation between putative virulence factors and antibiotic resistance among the tested bacteria. Further study on virulence and antibiotic resistance genes is hereby encouraged.

1. Introduction

from clinical specimens, food (powder infant formula, ready-to-eat foods), beverages, water, meat, vegetables and cheese [6,7]. The incidence and detection of Cronobacter in these samples and household contamination increased the potential risks for infections in immunecompromised adults as stated by Baumgartner et al. [8]. Powdered infant formula (PIF) is so far the only food source that has been epidemiologically linked to outbreaks caused byCronobacter. In developed countries, a lot of research on Cronobacter has been focused on the potential presence of these pathogens in PIF raw materials [6], processing facilities [9,10], and final products [11]. In 2004, Iversen and co-workers reported the ability of Cronobacter spp. to form biofilm on various surfaces like glass, stainless steel, silicon, latex and polycarbonate [12]. It was observed that attachment of the cells appears rapidly to plastic (a hydrophobic material) and hydrophilic materials [13]. Nutrient availability and temperature of the growth medium are important factors influencing biofilm formation [14]. It was reported that over 75% of Cronobacter spp. produced biofilm in infant formula milk while less than 20% produced biofilm in diluted tryptone soy broth when both are used as growth medium [15]. More so,

The bacterial genus Cronobacter was formerly known as Enterobacter sakazakii, and was first defined as a new genus in 2007 [1]. It is a member of the Enterobacteriaceae family and is closely related to the Enterobacter and Citrobacter genera. In recent years, the Cronobacter genus has undergone a number of revisions and currently contains 10 species [2,3]. The following were the formally recognized species: C. sakazakii, C. muytjensii, C. dublinensis, C. universalis, C. turicensis, C. condimenti, C. malonaticus, C. helveticus, C. pulveris and C. zurichensis including former bacterial species Enterobacter sakazakii, E. helveticus, E. pulveris and E. turicensis [4], making it difficult to ascertain the specific Cronobacter species that were reported prior to 2007 scientific publications. Holy and Forsythe [4] classified Cronobacter spp. into two major groups: Group 1 comprising of C. sakazakii and C. malonaticus which are mostly from clinical samples and Group 2 comprising of C. turicensis and C. universalis with less reported frequency. Cronobacter spp. are ubiquitous bacteria as described by Cawthorn et al. [5] and ElSharoud et al. [6] due to the fact that the pathogen has been isolated



Corresponding author. E-mail address: [email protected] (N. Abdullah Sani). 1 Current address: Research Performance and Analysis, Research Division, University of Tasmania, Australia. https://doi.org/10.1016/j.micpath.2019.103665 Received 30 June 2017; Received in revised form 9 August 2019; Accepted 9 August 2019 Available online 09 August 2019 0882-4010/ © 2019 Elsevier Ltd. All rights reserved.

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CRITERION™, Hardy Diagnostics, USA) and 8 g/L Congo red (C6767 Sigma-Aldrich, USA) were used. However, Congo red was prepared separately, and the constituents were autoclaved before adding to sterile BHA, sucrose and agar constituents Spot inoculated CRA plates were incubated at 37 °C for 24 h. Plates were then observed for dry crystalline black colonies for slime production and red colonies indicating non-slime production [20]. Thereafter, colony morphology or multicellular morphology (also known as curli fimbriae expression) was observed and recorded. This test was performed in triplicate for each isolate.

temperature influences biofilm formation. Kim et al. [14] reported that no biofilm was formed by Cronobacter at 12 °C in any of the growth media that were studied. Biochemical substances such as D-galactose, heteropolysaccharide, D-glucose, glucuronic acid, D-fucose and D-mannose enhanced biofilm formation among Cronobacter spp [16]. These biochemical substances hence increase resistance of these bacteria to antibiotic, detergents and other environmental stresses. Kim et al. [17] reported that most disinfectants used in food service kitchen, hospitals and day care centres are not effective enough to eliminate bacterial cells trapped within these organic matrices, hence, biofilm formation on equipment and in hospital environments increases the risk of infections in infants and immune - compromised adults. Formation of biofilm in infant formula milk has been observed after 24 h on enteral feeding tubes increasing the risk for neonatal infection, as enteral feeding tubes can remain in situ for several days at normal body temperature while administering nutrients to the infants at 2–3 h intervals [18]. There is limited study of this pathogen in this part of the world. Therefore, studies on theincidence, virulence and antibiotic resistance of Cronobacter spp. are imperative. This study therefore aimed to investigate antibiotic resistance, putative virulence factors and multicellular colony morphology among Cronobacter sakazakii previously isolated in Malaysia and compared to reference strains.

2.4. DNase assay All the bacterial isolates were investigated for the presence of nuclease enzyme using DNase test agar (263220, BD Difco™, USA) prepared according to manufacturer's instruction. Isolates were then be streaked on the solidified medium and incubated as at 37 °C for 24 h. Plates were observed while positive with clear coloration zone were noted. 2.5. Proteolytic assay Presence of protease enzymes among bacteria was carried out using modified method of Dada et al. [21]. Briefly, skim milk agar was prepared with the following composition (skim milk powder 10 g/L – Sunlac, New Zealand; yeast extract 1 g/L (C7001 CRITERION™, Hardy Diagnostics, USA) and bacteriological grade agar 15 g/L (C5001 CRITERION™, Hardy Diagnostics, USA). Other components were firstly prepared and autoclaved at 121 °C for 15 min and allowed to cool to 55 °C before adding sterilely prepared skim milk solution. Both mixtures were swirled gently and allowed to mix thoroughly before pouring into sterile Petri dishes. Bacteria were spot inoculated into the plates and incubated at 37 °C. Plates were observed after 24 h of incubation for possible clear zones.

2. Materials and methods 2.1. Sources and preparation of strains The strains used in this study have been isolated from PIF, milk preparation counter, water bath and bottle rack in a previous study. A total of 12 stock cultures (3 typed cultures from University College Dublin, Ireland) and 9 cultures previously isolated from PIF sold in Malaysia, milk preparation counter, water bath and bottle rack in neonatal intensive care units (NICU) from two hospitals. A bead from each stock culture was placed into sterile Tryptone soya broth – TSB (CM 0129, Oxoid™, UK) prepared according to manufacturer's instruction and incubated at 37 °C for 24 h. Thereafter, 20 μL of the broth was transferred into 10 mL sterile Cronobacter screening broth - CSB (CM 1121, Oxoid™, UK) and incubated for 24 h. A loopful inoculum of each culture was streaked on freshly prepared Enterobacter Sakazakii Isolation agar – ESIA (CF 3706, Biolife, Italy) and Brilliance Enterobacter Sakazakii agar - DFI (CM 1055, Oxoid™, UK). Colonies with blue green coloration on ESIA agar were further streaked on Tryptone Soya agar – TSA (CM 0131, Oxoid™, UK). Yellow colonies were selected and further purified on ESIA agar. Stock cultures with yellow coloration in CSB broth, blue green colonies on ESIA, DFI and yellow on TSA were selected as Cronobacter species and used to prepare fresh stock cultures for subsequent experiment.

2.6. Haemolytic assay The haemolytic activity of the Cronobacter species was assessed on blood agar plates prepared as using blood agar base (CM 0271, Oxoid™, UK), while 3% v/v defibrinated sheep blood (SR0051, Oxoid™, UK) was added to the sterile blood agar base. Freshly prepared cultures of Cronobacter spp. from TSA plates were inoculated on plates and observed for haemolysis zone around colonies after incubation for 24 h at 37 °C [21]. 2.7. Gelatinase assay Adapted method of Dada et al. [21] was used to screen isolates for the presence of gelatinase enzyme. Briefly, Brain-Heart Infusion agar (CM 0375, Oxoid™, UK) was supplemented with 10 g/L Bacto™ peptone (S71604, BD Difco™, USA) and 30 g/L gelatine bacteriological (LP0008, Oxoid™, UK). Plates were incubated overnight at 37 °C and observed for clear zone around the colonies indicating hydrolysis of gelatine.

2.2. Standardization of inocula Standardization of inocula prior to biofilm experiment was done as follows: A loopful of each culture was spread on freshly prepared TSA and incubated at 37 °C for 24 h before sub culturing at 37 °C for 24 h in 10 mL TSB. One millilitre (1 mL) of each culture was serially diluted in sterile Buffered Peptone Water – BPW (CM 0509, Oxoid™, UK) to 108 dilution factors to give 108 colony forming units (per millilitre (CFU/ mL). However, cells were also enumerated on agar as follows: 20 μL of culture from dilution factor 6 was streaked on TSA and counted.

2.8. Motility test Modified method of Kalai Chelvam et al. [22] was used for this test. The compositions of the agar (100 mL) used are as follow: Swarm agar (1% w/v tryptone (LP0043, Oxoid™, UK), 0.5% w/v NaCl (106404, Merck Germany) and 0.6% w/v bacteriological grade agar (C5001 CRITERION™, Hardy Diagnostics, USA) and swim agar (1% w/v tryptone, 0.5% w/v NaCl and 0.25% w/v bacteriological grade agar. All the components were added and autoclaved at 121 °C for 15 min before pouring into Petri dishes and allowed to solidify. Therafter, 20 μL of Cronobacter species was then spot inoculated at the centre of the plates before incubating at 37 °C for 48 h. Plates with bacterial growth

2.3. Biofilm formation and multicellular morphology on Congo red agar Qualitatively, all Cronobacter species were screened for biofilm formation using modified Congo Red Agar (CRA) method previously described by and Hassan et al. [19]. Briefly, 37 g/L Brain Heart Infusion Broth (CMO 375, Oxoid™, UK), 50 g/L of Sucrose (C7021 CRITERION™ Hardy Diagnostics, USA), 10 g/L of bacteriological grade agar (C5001 2

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spreading from the spot of inoculation to the edge of the plate were noted as positive and measured in millimetre (mm).

Table 1 List of Cronobacter species used in this study. Code

Species

Sources

CR1

C. sakazakii

CR2

C. sakazakii

CR3 CR4 CR5 CR6 CR7 CR8 CR9 CR10 CR11 CR12

C. C. C. C. C. C. C. C. C. C.

Milk preparation counter, NICU, P Hospital Milk preparation counter, NICU, P Hospital Ward Water Bath, NICU, P Hospital Bottle rack, NICU, S Hospital Powdered infant formula milk Powdered infant formula milk Powdered infant formula milk Powdered infant formula milk Raw milk American Type of Culture Collection University College Dublin American Type of Culture Collection

2.9. Antibiotics susceptibility Antibiotic resistance and susceptibility of bacteria used for biofilm assay was tested against 10 antibiotics using disc diffusion method. Each isolate from standardized inoculum (108 CFU/mL) was aseptically streaked on Brain Heart Infusion agar - BHIA (CM 0375, Oxoid™, UK) using sterile swab. Ten (10) Oxoid made antibiotic discs were then placed on the surface of the solidified agar and allowed to diffuse into the agar for 20 min before incubating at 37 °C for 24 h. All the experiments were carried out in duplicate. Multidrug resistance (resistance to more than 3 antibiotics tested) according to Oteo et al. [23] was noted. The size of the zone of inhibition was measured in millimetre (mm) after 24 h and interpreted as breakpoints for antibiotics according to the National Committee for Clinical Laboratory Standards (CLSI 2006) sensitive (S) – inhibition zone ≥ 18 mm, intermediate (I) – inhibition zone 13–17 mm and resistance (R) – inhibition zone < 13 mm [24].

sakazakii sakazakii sakazakii sakazakii sakazakii sakazakii sakazakii sakazakii ATCC® 29544™ turicensis E866 muytjensii ATCC® 51329™

virulence factors. CR 2 showed the least (3) virulence factors. Hierarchical clustering analysis of putative virulence factors revealed 2 clusters with 2 sub clusters each. Cluster 1 consist of sub cluster 1 with 6 of the isolates (CR 3, CR 4, CR 6, CR 9, CR 10 and CR 12) while sub cluster 2 consist of CR 1. This clustering was based on presence and absence of hemolysis among the isolates. Cluster 2 consist of isolate CR 2 as only member of sub cluster 1 while sub cluster 2 consist of 3 species C. sakazakii and C. turicensis E866. The ubiquitous nature of this species of bacteria revealed that there could be different mechanisms used to survive within the environment and potential host(s). It was initially thought Cronobacter spp. are limited to powdered infant formula. However, studies have shown that other sources can serve as natural reservoir and routes of transmission for Cronobacter species [28] According to Jaradat et al. [28], Cronobacter spp. are opportunistic pathogens mostly implicated in newborn and infant infections. According to Cruz-Cordova et al. [29], all species of Cronobacter have been associated with human infections except C. condiment. Since not all Cronobacter spp. are linked to infantile infections and other human infections, it is assumed that there can be variation of virulence among the strains [29]. They cause meningitis, necrotizing enterocolitis and bacteremia as a result of the presence of virulence factors which are believed to be caused by various factors [30]. Virulence factors are gene mediated abilities of bacteria to cause diseases in host after successful invasion [31]. These properties include cell surface proteins used for purpose of host attachment there has been increase in Cronobacter research worldwide, yet, the exact mechanism of pathogenesis of the genus of bacteria remain unknown [28]. Production of enterotoxin in bacteria has been a major virulence factor that marks the pathogenicity. Pagotto et al. [32] firstly described presence of putative enterotoxin activity in four out of 18 Cronobacter isolates that were tested using suckling mice assay. They further tested filtrates of the isolates on cultured mammalian cells and observed that one of the isolates were toxic to both Vero and Y-1 cells. In 2007, Raghav and Aggarwal [33] purified and characterize enterotoxin from Cronobacter isolates Similarly, Yang et al. [34] in their showed that filtrates from two out of eight strains studied were toxic to Vero cells. Researchers also found haemolysin genes (hly) although no activity has been found [35,36]. In this current study, gelatinase, DNase and biofilm formation were found in all the tested Cronobacter. None of the isolates including reference strains possessed protease enzyme unlike the studies of Pagotto et al. [32] and Kothary et al. [37] that found proteolytic enzymes that lyse cells and create tissue damage at the site of infection in mice. In a recent study, putative virulence factors were identified in Cronobacter [38]. Biofilm formation among bacteria has been ascribed as virulence factor. This is as a result of the fact that biofilm helps bacteria to survive in harsh and unusual environment. Bacteria cells mostly occur either as non-adherent single cell state or as adhesive multicellular state mostly referred to as biofilm. Formation of biofilm enables

2.10. Antibiotic resistance and virulence factors hierarchical clustering analysis Resistance pattern similarities among Cronobacter species considered in this study was performed using paleontological statistics software package for education and data analysis (PAST) tool by unweighted average linkage (UPGMA) two-way Ward's method clustering. This was carried out to determine phenotypic antibiotic resistance relatedness of the isolated bacteria by constructing dendrogram adapted from Adeleke and Omafuvbe [25] using results of antibiotic resistance test of the 10 antibiotics tested coded as 0 for susceptibility and 1 for resistance [26]. Similar method was used for the following tested putative virulence factors: biofilm formation, DNase, protease, gelatinase, motility and haemolysis. 2.11. Multi antibiotic resistance (MAR) index Multi antibiotics resistance index of the isolates was investigated following modified method of Odeyemi et al. [27]. MAR = x/y where x is the number of resistant antibiotics and y is the total number of antibiotics tested. MAR index value > 0.2 indicates existence of isolate(s) from high – risk contaminated sources involving frequent antibiotics usage however, values ≤ 0.2 show bacteria isolates are from source with less use of antibiotics. 3. Results and discussion 3.1. Virulence factors and multicellular morphology Cronobacter species used in this study and their sources are listed in Table 1. In this study, the following phenotypic virulence biomarkers were examined among -the isolates: biofilm formation, DNase enzyme, motility, protease, gelatinase and haemolysin. It was obserted that all the tested C. sakazakii were able to form biofilm on agar as seen in Fig. 1. Isolates CR 3, CR 4 and CR 9 were weak biofilm producers with rough dry colony surfaces (rdar). Isolates CR 6, CR 7, CR 8 and CR 11 were strong biofilm producer with brown rough dry colony surfaces (bdar). Isolate CR 10 formed weak biofilm with red mucoid and smooth colony surface (rmsc). Isolates CR 2 formed weak biofilm with smooth colony surface while CR 1 and CR 5 formed strong biofilm with smooth colony surface. They all elicited DNase and gelatinase enzymes and are motile except CR 2 that was non-motile. Only CR 1 showed presence of all the 6 virulence factors tested as shown in Fig. 2. Five C. sakazakii strains (CR 3, CR 4, CR 6, CR 9, CR 10) and C. muytjensii ATCC®51329™ (CR 12) exhibited 5 virulence factors respectively while 3 isolates (CR 5, CR 7, CR 8) and C. turicensis (CR 11) showed presence of only 4 3

Microbial Pathogenesis 136 (2019) 103665

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Fig. 1. Biofilm formation and curli fimbrination among Cronobacter species on Congo red agar. A. (CR 3, CR 4 and CR 9) weak biofilm producers with rough dry colony surfaces; B. (CR 6 and CR 7) stronger biofilm producer with rough dry colony surfaces; C. (CR 2 and C. sakazakii ATCC® 25944™) weak biofilm with smooth colony surfaces; D. (CR 1, CR 5) strong biofilm producers with smooth colony surfaces; E. (CR 8 and CR 11) strong biofilm producers with rough dry colony surfaces; F. CR 10 weak biofilm producer with red mucoid and smooth colony surface and G. Control non-biofilm former.

swarming involve movement on solid surface or agar. Motility is required by bacteria to invade host cell. All the bacteria tested were positive for motility except CR 2. Both motility and production of exopolysaccharides (EPS) are required in biofilms formation. Exopolysaccharides (EPS) components such as cellulose, curli fimbriae, O antigen capsule, colonic is required for biofilm formation [42]. According to Kalai Chelvam et al. [22] fimbriae or curli are important for attachment of bacteria to surfaces and gives a signal for initiation of microcolony formation. Recent indicated that cellulose and flagella also found in other Enterobacteriaceae are important in C. sakazakii biofilm formation [43]. In this study, variation of colony morphology on solid agar was observed among the isolates and similarity in the multicellular colony morphology of Cronobacter and other member of the family Enterobacteriaceae. Most study on multicellular colony morphology studied are mainly on other enteric bacteria species especially Escherichia coli and Salmonella. It was revealed in a study in 2006 that persistence of Salmonella to desiccation was due to the presence of rdar morphotypes which is a multicellular phenotype characterized by fimbria- and cellulose-mediated colony pattern formation [44]. Each of the isolates used in this study showed either rdar or bdar morphology. A

bacteria to evade and circumvent host immune system thereby enabling bacteria to resist antibiotics and disinfectants [39]. Du et al. [40] stated that only little scientific information is available regarding Cronobacter biofilm formation. It was observed that attachment of the cells appears rapidly to plastic (a hydrophobic material) and hydrophilic materials [13]. Nutrient availability and temperature of the growth medium are important factors influencing biofilm formation [41]. It was reported that over 75% of Cronobacter spp. produced biofilm in infant formula milk while less than 20% produced biofilm in diluted tryptone soy broth when both are used as growth medium [15]. The presence of all the screened virulence factors in CR 1 and more than one in all the other isolates calls for public health concern. Since the isolation of these bacteria occurred from neonatal intensive care unit (NICU), precautions must be taken to avoid cross contamination of the PIF milk fed to neonates. Although no outbreak of Cronobacter infection has been reported in Malaysia, this study serves to unravel the capability of these bacteria in clinical environment. Bacterial motility and biofilm formation have been attributed to pathogenicity of bacteria [22]. Swimming and swarming are both used by motile bacteria to migrate. Bacterial swimming involve movement in broth or semi-solid media while 4

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O.A. Odeyemi and N. Abdullah Sani

Fig. 2. Dendrogram based on putative virulence factors among Cronobacter species using Jacaard Two-way similarity, Paired group cluster method and UPGMA for clustering generated based on similarity level of 95%.

new morphology was observed in reference strain CR 10. It showed red mucoid and smooth colony surface (rmsc). Fig. 3. Dendrogram based on antibiotic resistance among Cronobacter species using Jacaard Two-way similarity, Paired group cluster method and UPGMA for clustering generated based on similarity level of 95%.

3.2. Antibiotic resistance Resistance and susceptibility to commercial antibiotics was investigated among the isolates. The 10 antibiotics tested, and their concentrations are listed in Table 2. All the tested Cronobacter species were resistant to sulphamethoxazole, ampicillin, fosfomycin and erythromycin respectively while all the isolates were susceptible to ciprofloxacin, nalixidic acid and tetracycline as shown in Figs. 3 and 4. Only 3 (CR 1, CR 8 and CR 10) where susceptible to kanamycin while others were resistant to it. The following isolates were resistant to rifampicin: CR 2, CR 6, CR 7, CR 9, CR 11 and CR 12. Both CR 11 and CR 12 were resistant to gentamicin. Others were susceptible to the antibiotic. Hierarchical clustering analysis of antibiotic resistance of the isolates based on resistance and susceptibility to kanamycin revealed two clusters 1 and 2. Cluster 1 consisted of 2 sub clusters. Sub cluster 1 comprised of C. turicensis E866 and C. muytjensii ATCC® 51329™. Sub cluster 2 comprised of CR 2, CR 6, CR 7 and CR 9 respectively. Isolates in cluster 1 were resistant to tested antibiotics compared with isolates in

cluster 2 which consisted of 3 sub clusters. Sub cluster 1 of cluster 2 consisted of 3 isolates (CR 3, CR 4 and CR 5). Sub cluster 2 consisted only of CR 10, while sub cluster 3 consisted of CR 1 and CR 8. All the isolates including reference strains showed high multi antibiotic resistance index (MAR) as shown in Table 3. Four C. sakazakii and 2 reference strains have MAR index of 0.6 while 5 had 0.5 MAR index. The least MAR index was observed in CR 1. Development of resistance to antibiotics among bacterial pathogens is of global concern posing danger to public health. Initially, infections caused by Cronobacter are treated with ampicillin in combination with gentamycin or chloramphenicol [45]. High level of antibiotic resistance was observed among C. sakazakii tested in this study. This was similar to recent study of antibiotic resistance among C. sakazakii isolated from domestic kitchens in United States [46]. Over 76% of the isolates were resistant to penicillin, ciprofloxacin (57.1%), tetracycline (66.6%), and nalidixic acid (47.6%). However, none of the isolates was resistant to gentamicin unlike this study where 2 isolates were resistant to gentamicin. Similarly, C. sakazakii isolated from ingredients of infant foods in Korea were resistant to ampicillin but susceptible to nalidixic acid, tetracycline, gentamicin, ciprofloxacin, chloramphenicol, ampicillin and kanamycin [47]. Al-Nabulsi et al. [48] showed stressed C. sakazakii streptomycin, gentamicin, kanamycin and ciprofloxacin are effective against both stressed and unstressed C. sakazakii cells were sensitive to gentamycin, kanamycin, streptomycin, ciprofloxacin, ampicillin and amoxicillin but resistant to neomycin, doxycycline, ampicillin, amoxicillin and vancomycin. This revealed that stressed such as desiccation, low nutrient and others can impact antibiotic resistance on bacteria.

Table 2 List of antibiotics tested. S/No

Antibiotics

Concentration

Company

1 2 3 4 5 6 7 8 9 10

Ampicillin (Amp) Gentamicin (Cn) Ciprofloxacin (Cip) Tetracycline (Te) Sulphamethoxazole (Rl) Rifampicin (Rd) Fosfomycin (Fos) Nalidixic acid (Na) Kanamycin (K) Erythromycin (E)

10 μg 10 μg 5 μg 30 μg 25 μg 5 μg 50 μg 30 μg 35 μg 15 μg

Oxoid™, Oxoid™, Oxoid™, Oxoid™, Oxoid™, Oxoid™, Oxoid™, Oxoid™, Oxoid™, Oxoid™,

UK UK UK UK UK UK UK UK UK UK

5

Microbial Pathogenesis 136 (2019) 103665

O.A. Odeyemi and N. Abdullah Sani

Fig. 4. The percentage of Cronobacter species resistant to antibiotics tested in this study. Table 3 Antibiotics resistance profiling (Antibiogram) of Cronobacter species. Code

Species

Antibiogram

MAR index

CR CR CR CR CR CR CR CR CR CR CR CR

C. C. C. C. C. C. C. C. C. C. C. C.

Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp Amp

0.4 0.6 0.5 0.5 0.5 0.6 0.6 0.5 0.6 0.5 0.6 0.6

1 2 3 4 5 6 7 8 9 10 11 12

sakazakii sakazakii sakazakii sakazakii sakazakii sakazakii sakazakii sakazakii sakazakii sakazakii ATCC® 29544™ turicensis E866 muytjensii ATCC® 51329™

E E E E E E E E E E E E

Fos Fos Fos Fos Fos Fos Fos Fos Fos Fos Fos Fos

Rd Rd K Rl K Rl K Rl K Rl Rd K Rl Rd K Rl Rd Rl Rd K Rl K Rl Rd K Rl Rd K Rl

[3]

[4] [5]

[6]

[7]

[8]

4. Conclusion

[9]

In conclusion, we investigated antibiotic resistance, putative virulence factors and multicellular colony morphology among C. sakazakii isolates recovered from raw milk, powdered infant formula milk and milk preparation environment. This study reveals that C. sakazakii are capable of forming biofilm, phenotypically express virulence factors and show resistance to commercial antibiotics. Although, this was a phenotypic study of virulence factors among the isolates and the result obtained gave insight into morphological characteristics of C. sakazakii isolated in Malaysia however, further study on genetic study of virulence and antibiotic resistance genes is hereby encouraged.

[10]

[11]

[12] [13]

[14]

Acknowledgement [15]

This research was supported by the UKM research grant (DIP-2014007) and the Ministry of Education grant (FRGS/1/2014/STWN03/ UKM/02/1).

[16]

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