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Phylogenetic Background, biofilm and antibiogram profile of E.coli Isolated

from Raw Milk Soled in The Local Markets at Al-Nasiriyah City-Iraq

Article in International Journal of Pharmaceutical Sciences · January 2020

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Research Article

Phylogenetic Background, biofilm and antibiogram

profile of E.coli Isolated from Raw Milk Soled in The
Local Markets at Al-Nasiriyah City- Iraq
MUSLIM DHAHR MUSA1,RABAB SHAKER SALMAN2,ABBAS DAKHEEL MUTAR3
1
.

ABSTRACT
Current study was aimed to evaluate the potential risk of raw milk consumption that sold at local market to
publichealth. Toachieve this mission the presence of E.coli was investigated as hygienic, quality indicator of raw
milk also,phylogrouping, biofilm production ability and antibiogram was employed. Thus 54 raw milk samples
were collected from two levels (markets and farms) at Al –Nasiriyah city south of Iraq, in period from January
to March 2018. Traditional cultural and biochemical methods were used to isolate and identify E.coli, while
molecular method ( Cleremont method) was used to determine phylogenetic groups. E.coli was detected in 15
samples (27.8%), recovery percentage was significantly higher (40.7%) at market than farm (14.8%).
Phylogenetic analysis revealed that all isolated strains were belong to two phylogroups;A (53.4%) and B2
(46.6%). According to the source of isolation phylogroup B2 was found only at market level, while phylogroup
A was found in both market and farmlevel. This study found that most strains of phylogroup B2 (83.4%) were
strong biofilm producer and harbor a potential antibiotics resistance to commonly used antibiotics, as
determine in this study the poor hygienic practice of entire process beginning from milking through
transporting to selling at market was responsible of contamination of milk with pathogenic strains that belong
to phylogroup B2 which is mostly probably come from human source .In conclusion the raw milk constitute a
serious public health threat to people who consumed raw milk
Keywords: E.coli , Raw milk, Phylogenetic grouping, Antibiogram, Biofilm, Iraq

INTRODUCTION

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 Isolation & Identification of E.coli

Biofilm Production Assay

MATERIALS AND METHODS

Samples Collection

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ANTIBIOGRAM ANALYSIS

STATISTICAL ANALYSIS

PHYLOGENETIC ANALYSIS

RESULTS

Table 1: The recovery percentages of E.colifrom raw milk at two isolation levels

Isolation level No. of samples Positive Recovery % P-value

Market level 27 11 40.7% ≥0.05
Farm level 27 4 14.8
Total 54 15 27.8
P-value=

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 Fig.1: characteristic morphology (Metalic – Fig.2: hemolytic activity of E.coli on Blood agar
Sheen ) of E.coli on EMB agar plate

Table 2: distribution of E.colistrains according to phylogenetic groups

M1 2345678

Phylogenetic group A Phylogenetic group B2

Fig.3: Agarose gel Electrophoresis( 2%) of amplified three genetic markers (chuAand
yjaATSPE4.C2 ), after 45 minutes at 105 V,stained with Ethidium bromide and transilluminated by
gel-documentation. M: ladder(2000bp), 1,4,5,6,7,8:YjaA 2,3 (YjaAandChuA)

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 Table 3: distribution of E.colistrains to produce biofilm according to phylogenetic groups

Biofilm formation ability

P-Value
Phylogen
etic Non-producer Weak Moderate Strong
groups (Mean±SD of (Mean±SD of (Mean±S (Mean±SD of
OD) OD) D of OD) OD) P > 0.05
A 3 3 1 1
B2 0 1 1 5(83.4%)
3 4 2 6
Total
(0.014±0.003) (0.029±0.002) (0.13) (0.234±0.051
OD of Negative control=0.03

Table 4: Antibiogram analysis of E.coliphylogroups isolated from raw milk

DISCUSSION

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REFERENCES
1. Uddin A, Motazzim-ul-haque H, Noor R.
Isolation and Identification of Pathogenic
Escherichia coli , Klebsiella spp . and
Staphylococcus spp . in Raw Milk Samples
1179| International Journal of Pharmaceutical Research | Oct - Dec 2019 | Vol 11 | Supplementary Issue 1
Collected from Different Areas of Dhaka City ,
Bangladesh. Stamford J Microbiol. 2011;1(1):19-23.
2. FDA e Food and Drug Administration. Grade
“A” pasteurized milk ordinance. Public Heal Serv
FDA.
3. Yohannes G. Isolation, Identification and
Antimicrobial Susceptibility Testing of
Escherichia coli Isolated from Selected Dairy
Farms in and Around Mekelle, Tigray, Ethiopia. J
Vet Sci Technol. 2018;9(2). doi:10.4172/2157-
7579.1000518
4. Verraes C, Claeys W, Cardoen S, et al. A review
of the microbiological hazards of raw milk from
animal species other than cows. Int Dairy J.
2014;39(1):121-130.
doi:10.1016/j.idairyj.2014.05.010
5. Tadesse HA, Gidey NB, Workelule K, et al.
Antimicrobial Resistance Profile of E . coli
Isolated from Raw Cow Milk and Fresh Fruit
Juice in Mekelle , Tigray , Ethiopia. Vet Med Int.
2018:1-7.
6. Bali OS, Lajnef R, Felfoul I, Attia H, Ayadi MA.
Detection of Escherichia coli in unpasteurized
raw Milk ISSN 2249-8516 Original Article
Detection of Escherichia coli in unpasteurized
raw Milk. Int J Agric Food Sci. 2013;3(2):53-55.
7. RICHARD K. ROBINSON. DAIRY
MICROBIOLOGY HANDBOOKe. THIRD EDIT. A
JOHN WILEY & SONS, INC; 2002.
8. Claeys WL, Cardoen S, Daube G, et al. Raw or
heated cow milk consumption: Review of risks
and benefits. Food Control. 2013;31(1):251-262.
doi:10.1016/j.foodcont.2012.09.035
9. P. K. Virpari, J. B. Nayak, M. N. Brahmbhatt and
HCT. Study on isolation, molecular detection of
virulence gene and antibiotic sensitivity pattern
of Escherichia coli isolated from milk andmilk
products. Vet World. 2013;6(8):541– 545.
10. Soomro AH, Arain MA, Khaskheli M, Bhutto B.
Isolation of Escherichia Coli from Raw Milk and
Milk Products in Relation to Public Health Sold
under Market Conditions at Tandojam , Pakistan
Isolation of Escherichia Coli from Raw Milk and
Milk Products in Relation to Public Health Sold
under Market Con. Pakistan J Nutr.
2002;1(3):151-153. doi:10.3923/pjn.2002.151.152
11. Liebert MA, Smith JL, Fratamico PM, Gunther
NW. Extraintestinal Pathogenic Escherichia coli.
Foodborne Pathog Dis. 2007;4(2).
doi:10.1089/fpd.2007.0087
12. ORSKOV, F. and O. Escherichia coli serotyping
and disease in man and animals. Can J Microbiol.
1992;(38):699-704.
13. Bonacorsi P, Clermont O, Bingen E. Rapid and
Simple Determination of the Escherichia coli
Phylogenetic Group. 2000;66(10):4555-4558.
14. OLIVIER CLERMONT, STE´PHANE
BONACORSI AEB. Rapid and Simple
Determination of the Escherichia coli
Phylogenetic Group. Appl Environ Microbiol.
View publication stats
2000;66(10):4555-4558.
15. Carlos C, Pires MM, Stoppe NC, et al.
Escherichia coli phylogenetic group
determination and its application in the
identification of the major animal source of fecal
contamination. BMC Microbiol. 2010;10(June).
doi:10.1186/1471-2180-10-161
16. M. Abdallah, C. Benoliel, D. Drider, P. Dhulster
N-EC. Biofilm formation and persistence on
abiotic surfaces in the context of food and
medical environments. Arch Microbiol.
2014;196(7):453–472.
17. Ombarak RA, Hinenoya A, Awasthi SP, et al.
Prevalence and pathogenic potential of
Escherichia coli isolates from raw milk and raw
milk cheese in Egypt. Int J Food Microbiol.
2016;221:69-76.
doi:10.1016/j.ijfoodmicro.2016.01.009
18. Igbinosa IH, Igbinosa EO, Okoh AI. Detection of
antibiotic resistance , virulence gene
determinants and biofilm formation in
Aeromonas species isolated from cattle. Env Sci
Pollut Res. 2015. doi:10.1007/s11356-015-4934-4
19. Basson A, Flemming LA CH. Evaluation of
adherence, hydrophobicity, aggregation
characteristics and biofilm development of
Flavobacterium johnsoniae-like isolates from
South African aquaculture systems. Microb Ecol.
2007;55:1-14.
20. CLSI. 28th Edition M100 Performance Standards
for Antimicrobial Susceptibility Testing This.; 2018.
doi:10.1007/978-3-662-48986-4_300418
21. Abbas BA, Ghadban MK, Alghanim AM.
Microbial evaluation of milk and milk products
during a past two decades, in Basrah Southern
Iraq: A review. Annu Res Rev Biol. 2017;14(2):1-8.
doi:10.9734/ARRB/2017/34855
22. De Campos ACLP, Punõ-Sarmiento JJ, Medeiros
LP, et al. Virulence Genes and Antimicrobial
Resistance in Escherichia coli from Cheese Made
from Unpasteurized Milk in Brazil. Foodborne
Pathog Dis. 2018;15(2):94-100.
doi:10.1089/fpd.2017.2345
23. Amin WF, Ahmed EH, Embarak MS. Molecular
Detection of Enterotoxigenic E . coli in raw Milk
and Milk Products. Int J Curr Microbiol Appl Sci.
2017;6(11):856-864.
24. Kabir SML. Molecular detection and
characterization of Escherichia coli isolated from
raw milk sold in different markets of Bangladesh.
Bangl J Vet Med. 2016;14(2):271-275.
25. Garbaj, A.M., Awad, E.M., Azwai, S.M., Abolghait
SK, Naas, H.T., Moawad, A.A., Gammoudi, F.T.,
Barbieri I and, Eldaghayes IM.
Enterohemorrhagic Escherichia coli O157 in
milk and dairy products from Libya: Isolation and
molecular identification by partial sequencing of
16S rDNA. Vet World. 2016;9(11):1184-1189.
1180| International Journal of Pharmaceutical Research | Oct - Dec 2019 | Vol 11 | Supplementary Issue 1
26. Picard B, Garcia JS, Gouriou S, Duriez P, Brahimi
N BE, Al E. The link between phylogeny and
virulence in Escherichia coli extraintestinal
infection. Infect Immun. 1999;67:546-553.
27. Zhang D, Zhang Z, Huang C, et al. The
phylogenetic group, antimicrobial susceptibility,
and virulence genes of Escherichia coli from
clinical bovine mastitis. J Dairy Sci.
2018;101(1):572-580. doi:10.3168/jds.2017-
13159
28. Müştak HK, Günaydin E, Kaya İB, et al. Phylo-
typing of clinical Escherichia coli isolates
originating from bovine mastitis and canine
pyometra and urinary tract infection by means
of quadruplex PCR. Vet Q. 2015;35(4):194-199.
doi:10.1080/01652176.2015.1068963
29. Milanov D, Prunić B, Velhner M, Todorović D,
Polaček V. Investigation of biofilm formation and
phylogenetic typing of Escherichia coli strains
isolated from milk of cows with mastitis. Acta Vet
Brno. 2015;65(2):202-216. doi:10.1515/acve-
2015-0017
30. Carlos C, Pires MM, Stoppe NC, et al.
Escherichia coli phylogenetic group determination
and its application in the identification of the
major animal source of fecal contamination. BMC
Microbiol. 2010;10:161. doi:10.1186/1471-2180-
10-161
31. Gordon DM CA. The distribution and genetic
structure of Escherichia coli in Australian
vertebrates: host and geographic effects.
Microbiology. 149:3575-3586.
32. Chakraborty A, Saralaya V, Adhikari P, Shenoy S,
Baliga S, Hegde A. Characterization of
Escherichia coli Phylogenetic Groups Associated
with Extraintestinal Infections in South Indian
Population. Ann Med Health Sci Res.
2015;5(4):241-246. doi:10.4103/2141-
9248.160192
33. And B formation on biotic, Escherichia abiotic
surfaces in the presence of antimicrobials by,
Mastitis coli I from cases of bovine. Silva, V. O.,
L. O. Soares, A. Silva Junior, H. C. Mantovani, Y.
F. Chang, and M. A. Moreira. Appl Env Microbiol.
2014;80:6136–6145.
34. B.R. Paneto, R.P. Schocken-Iturrino2, C.
Macedo, E. Santo JMM. Occurrence of toxigenic
Escherichia coli in raw milk cheese in Brazil.
2007:508-512.
35. Alharbi NS, Khaled JM, Kadaikunnan S, Alobaidi
AS, Sharafaddin AH. Prevalence of Escherichia
coli strains resistance to antibiotics in wound
infections and raw milk Saudi Journal of
Biological Sciences Prevalence of Escherichia coli
strains resistance to antibiotics in wound
infections and raw milk. Saudi J Biol Sci.
2018;(November). doi:10.1016/j.sjbs.2018.11.016