Abstract
In Eastern Africa, where 60 % of the world camel population is held, there is a long tradition
of preparing fermented camel milk known as suusac. The fermentation is spontaneous and
results in a product whose quality varies greatly, may be risky and even dangerous for
consumer health due to unpredictable microbial inhabitants. These risk factors arise not only
from unhygienic handling of camel milk but also from zoonotic bacteria usually attributed to
producing animal mastitis. Misuse of common antibiotics in treatment of camels could also
push bacteria to develop mechanisms to evade the inhibitory power of antibotics.
There is now a major change in consumer choice with shift towards good hygienic quality
camel milk and products and also readiness to pay more for the better quality. This calls for
development of formal camel dairy to address this value addition potential and also reduce
health risks leading to improved camel milk commercialisation.
It is therefore important to understand the predominant microorganisms in raw milk and
suusac and to ascertain their potential impact safety and quality of raw camel milk and
suusac. The diversity of pathogens and yeasts in raw and spontaneously fermented camel
milk in Kenya and Somalia was studied using phenotypic and molecular techniques.
Potential pathogenic microorganisms were studied for their virulence and antibiotic
resistance profiles. Technological properties of predominant lactic acid bacteria were studied
as prerequisite to the introduction of an adapted starter culture for suusac fermentation.
A total of 235 presumptive staphylococci isolated from 105 camel milk and related samples
from five locations in Kenya and two locations in Somalia were identified and characterized
phenotypically and genotypically. PCR amplification of the genes encoding antiphagocytic
XIX
capsular polysaccharides cap5 and cap8, and staphylococcal enterotoxins SEA to SEE and
SEG to SEJ was also carried out. Secondly, the antibiotic resistance patterns of 47
Staphylococcus aureus isolates was studied using microdilution assays to determine minimal
inhibitory concentrations and disc diffusion tests. Genotyping was then done using
microarray hybridization and confirmation of antibiotic resistance genes by PCR.
Escherichia coli, Klebsiella pneumoniae Pneumomia and Enterobacter cloacae which have
been implicated worldwide as producers of Extended Spectrum β-lactamases (ESBLs) were
the predominant Enterobacteriaceae in raw milk and suusac. Antibiotic resistance risk posed
by these bacterial pathogens was characterized for 95 isolates both phenotypically and
genotypically. Escherichia coli isolates were also evaluated for presence of virulence factors.
The prevalence and epidemiology of E. coli O157 and non-O157 shigatoxigenic E. coli
(STEC) along the raw and fermented camel milk marketing chain was studied in 70 samples.
Serotypes and associated virulence factors in the isolated strains were also determined.
Various selective media and immunomagnetic separation were used followed by multiplex
PCR for virulence genes stx1, stx2 and eae and for positive samples a second multiplex PCR
to type for the serotypes O157, O113 and O111. PCR-RFLP of the fliC gene also carried out
on the O157, O113 and O111STEC to elucidate the epidemiology of the serotypes.
Yeasts were identified using combination of both phenotypic and genotypic techniques.
Identification was done using API 20C AUX followed by Restriction Fragment Length
Polymorphism (RFLP) of intergenic spacers ITS1 and ITS2 using restriction endonucleases
HhaI, HinfI and HaeIII. RAPD was performed with (GTG)5, (GAC)5, (GACA)4
microsatellite primers and M13 core sequence (5'-GAG GGT GGC GGT TCT-3'). Sequence
XX
analysis of either ITS1 and ITS2 or the 26S rRNA encoding gene was performed on selected
isolates.
Finally, 95 lactic acid bacteria (LAB) consisting of Lb. helveticus, Lb. fermentum, Lc. lactis
subsp. Lactis and Str. thermophilus isolated from various camel milk and associated sources
from main camel milk production points in Kenya and Somalia were studied for acidification
and metabolic properties. Str. infantarius which is a putative human pathogen was also
studied due to its predominance amongst presumed streptococci isolated to ascertain its role
in the spontaneous fermentation. Initial screening was carried out in a microtiter assay and
LAB were then selected for fermentation in batch culture experiments using a CINAC
system which allowed continuous follow-up of pH changes during fermentation in camel
milk during incubation at 30, 35 and 40oC for 36 h. Parameters including maximal
acidification rate (Vmax), time and pH at which Vmax occurred (tmax and pHmax), lag phase (ta),
time during which the acidification rate was equal to or higher than Vmax/2 (dt50), time to
reach pH 5.0 (tpH5), final pH (pHF) and time to reach final pH (tpHF) were calculated.
Metabolites of sugars, lactose, glucose and galactose and flavour compounds, citrate, acetate,
acetaldehyde, diacetyl and ethanol, were also quantified using High Performance Liquid
Chromatography (HPLC). L(+)-/D(-)-lactic acid production was studied using an enzymatic
assay. Potential cultures were selected based on the number of desirable acidification kinetic
values for fast acidification and also flavor metabolites when compared to the other cultures.
Presumptive staphylococci increased along the market chain. There were 146 (62 %)
confirmed staphylococci isolates of which, 66 (45 %) were Staphylococcus aureus.
Coagulase positive staphylococci were predominant in raw camel milk directly obtained
from the camel (25 %), at the market level (23 %) and fermented milk (suusac) (21 %). S.
XXI
epidermidis accounted for 29 % of coagulase negative Staphyolococci (CNS) studied. The
remaining CNS were distributed among S. simulans (18 %), S. saprophyticus (11 %), S.
haemolyticus (2 %), S. hyicus (2 %), S. xylosus (2 %), S. lentus (1 %), S. carnosus (1 %) and
S. microti (1 %). Aerococcus viridans (1 %), Macrococcus caseolyticus (1 %) and M.
nishinomiyaensis (1 %) were also identified. The gene cap5 encoding antiphagocytic
capsular polysaccharide was observed for 9 (14 %) and cap8 for 16 (24 %) of the isolates.
Enterotoxin genes were observed in 47 % of the isolates with sej in 34 %, seb in 6 %, sed in
5 % and seg in 3 % of the isolates. Amongst the species enterotoxin genes were detected in
90 %, 65 %, 38 % and 22 % of the S. simulans, S. epidermidis, S. sapropyticus and S. aureus
respectively. Rep-PCR genotyping revealed diversity of the isolates though with close
similarities irrespective of the level along the market chain and sampling location indicating
ubiquity of the isolates in primary and secondary environments.
There were 11 (23 %), 12 (26 %), 5 (11 %), 6 (13 %), 3 (6 %) and 18 (38 %) isolates
resistant to ampicillin, gentamicin, streptomycin, tetracycline, trimethoprim, fusidic acid
respectively. Amongst the multi-resistant isolates 2 were resistant to 2 antibiotics, 7 to 3
antibiotics and 6 to 4 or more antibiotics. Based on microarray, all 3 isolates tested were
positive for the β-lactamase resistant genes (blaZ), tetracycline resistance with gene tet38 and
the Panton-Valentine leukocidin gene pvl. Additionally, 2 isolates harboured streptomycin
resistance gene and tetracycline resistance through the gene tet(K). PCR targeting these
genes was performed for all isolates and 6 were positive for tetK, 9 for blaZ and 2 isolates
harboured both tet(K) and blaZ genes.
Enterobacteriaceae were not detected at milking and first collection point but were present at
104- 106 CFU/ml in final market raw camel milk and 103-107 CFU/ml in suusac. The
XXII
Enterobacteriaceae belonged to 14 species and 10 genera. Predominant isolates were
Escherichia coli 1 (47), Klebsiella pneumoniae Pneumoniae (35) and Enterobacter sakazakii
(18). Salmonella arizonae, Serratia odorifera 1, Enterobacter cloacae and Escherichia coli 1
occurred at mean cell counts greater than 8 log cfu/ml. Enterotoxin genes stx1 and stx2 were
not detected in any of the E. coli isolates with only one isolate with sequence coding for
intimin (eae) was detected.
Of the isolates, 61 (63 %) were resistant to ampicillin, of which 46 (48 %) were E. coli, 45
(46%) K. pneumoniae Pneumoniae and 16 (7%) E. cloacae. ESBLs were not phenotypically
detected in any of the isolates by double disc diffusion test. However, PCR revealed
prevalence of blaSHV, blaCTX-M-3-like and blaCTX-M-14-like genes in 37 (60 %), 25 (40 %) and
11 (18 %) of the isolates respectively. K. pneumoniae Pneumoniae not only harbored
majority of the genes (74 % of K. pneumoniae Pneumoniae), but a strain of K. pneumoniae
Pneumoniae possessed all 3 genes and 13 harbored both blaSHV and blaCTX-M-3-like genes.
Thirty six percent of the isolates harbored either single or combinations of factors stx1, stx2
and eae with 78 % being stx1 positive, 18.6 % eae positive, 3.9 % stx1 and stx2 positive and
0.78 % stx2 and eae positive. Prevalence of isolates positive for the virulence factors stx1,
stx2 and eae increased from 32.6 % at herd level to 34.2 % in first collection point and 44.3
% in the final market. Though highest percentage of presumptive E. coli isolates (57 %) were
isolated using EMB agar while the rest were from CHROMagar (23 %) and CT-SMAC (21
%), amongst the isolates harboring virulence genes, 100 %, 12 % and 39 % were isolated
from CT-SMAC, EMB agar and CHROMagar respectively. Serotypes O157, O111 and O113
represented 94 %, 2 % and 4 % of the STEC respectively. Thirty nine different restriction
XXIII
endonuclease digestion profiles were revealed by the RFLP of the flic gene with O157 having
29 profiles bearing 7 clusters with common profiles.
There were low numbers of yeasts in milk at herd level but 4.4±1.4 log cfu/ml and 5 ±1.5 log
cfu/ml at the first collection point and final market respectively. Counts of up to 7.5 ± 2.5 log
cfu/ml were found in laboratory fermented suusac. Amongst the identified isolates, API
enabled the identification of 80 (47%) and resulted in either incorrect identification or
inability to identify the others. RFLP, RAPD and sequence analysis enabled complete
identification to species and some differentiation at strain level with RAPD allowing more
discrimination within species. There were 21 yeast species belonging to the genera
Rhodotorula, Cryptococcus, Candida, Saccharomyces, Trichosporon, Geotrichum and
Issatchenkia. The most frequently isolated yeasts were Saccharomyces cerevisiae (19 % of
the identified isolates), Candida inconspicua (12 %), Trichosporon mucoides (11 %),
Candida famata (11 %), Rhodotorula mucilaginosa (8 %), Candida lusitaniae (6 %),
Cryptococcus laurentii (5 %), Cryptococcus albidus (5 %), Candida guilliermondii (5 %),
and Trichosporon cutaneum (5 %). Lowest viable counts for the identified yeasts were 2.4
cfu/ml for C. tropicalis and highest were 7.6 cfu/ml (range; 2.4-8.5 cfu/ml) for C. famata and
8.0 cfu/ml (range; 2.6-8.5 cfu/ml) for C. guilliermondii.
When LAB were ranked in the increasing order of mean pH from the microtiter acidification
assay, the strains were Str. infantarius (5.32 ±0.36), Lb. helveticus (5.33±0.09), Lc. lactis
subsp. lactis (5.5±0.49), Lb. fermentum (5.67±0.53) and Str. thermophilus (5.7±0.15). Lc.
lactis subsp. lactis had a short ta (145 min), tmax (424.5 min) tpH5 (535 min) and tpHF (30h),
and low pHF (4.10) during incubation at 30 oC. Moreover, at 35oC, the Vmax increased from
0.00531 to 0.006805 pH units min-1, tmax decreased from 424 to 271 min and tpHF decreased
XXIV
from 30.75 to 19.00 min. Even though Str. thermophilus 150A3.1 showed short ta (183 min),
tmax (378 min) and low pHF (4.4) at 30oC, at 35oC Str. thermophilus was in the grouped with
slowest acidifiers. Str. thermophilus 150A3.1 however, produced the highest quantity of
galactose (3.8 g/l) and glucose (3.56 g/l) at 37oC. Str. thermophilus 221A11.3 performed
better at 35 than 30oC with increased Vmax (from 0.00169- 0.0063 pH units min-1) together
with shorter ta ( from 279 to 82.5 min) and tpHF (from 43 to 19.0 h). Str. thermophilus
221A11.3 also produced highest formate (2072 mg/l) at 35oC. Lb. helveticus showed second
highest Vmax (0.00937 pH units min-1), had a low pHmax (5.17), short tpH5 (705 min) and low
pHF (4.23) at 30oC. Lb. helveticus also produced highest amounts of acetaldehyde (106.7
mg/l at 37oC), ethanol (1590 mg/l at 37oC) and acetate (1540 mg/l at 35oC). However highest
quantities of D(-) lactate (1.338 g/l at 37oC) were also produced by Lb. helveticus. Mixed
cultures Lc. Lactis subsp. lactis + Str. thermophilus 150A3.1, acidifed at highest overall Vmax
(0.07999 pH units min-1) and Lc. lactis subsp. lactis + Lb. helveticus to lowest pHF (3.9). By
combining Lb. helveticus with either of Str. thermophilus 221A 11.3 and Str. thermophilus
150A3.1, they produced lactate at highest (7.62 g/l) and second highest (7 g/l) quantities
respectively at 35oC. Lb. helveticus + Str. thermophilus 221A 11.3 also reached pH 4.1
within 33.5 h at 30oC. Even though Str. infantarius had short relatively short tmax at 30oC, at
35oC it reached a lower pHmax (5.29) and acidified at a longer dt50 (1202 min) when
compared with other strains. The range in time it took to reach maximum acidification for all
the strains or their combinations was was however high (9-1204 min at 40oC incubation).
The high prevalence of toxin-producing staphylococci requires consideration for food
hygiene and safety especially regarding the aspect that the milk is consumed raw or as
fermented raw milk.
XXV
Controlled antibiotic therapy in Kenyan and Somali camels should be introduced to prevent
the increase of antibiotic resistant bacteria. Pastoralists should also be instructed on the use
for rapid detection mastitis tests and the best single antibiotic or their combinations that
minimize development of resistances.
Molecular methods remain the most reliable methods of choice for identification of ESBLproducing
enterobacteria isolates. The finding of a high diversity of enterobacteria in camel
milk especially at market level and suusac calls for measures to improve handling of camel
milk.
The higher prevalence of STEC in camel milk than in cases with milk from other species
indicates that this milk could be an important vehicle for transmission of STEC to humans.
Possibility of a continuous contamination with different STEC strains then distribution of
these strains during handling and storage of milk at this point was also revealed. This calls
for interventions on hygienic factors and animal health at all levels in the marketing chain.
A combination of phenotypic and molecular methods for proper yeast identification is
recommended. The high diversity and numbers of yeasts indicate their role in the
fermentation of camel milk and potential for inclusion as starter cultures.
In order to further select and adapt the starter cultures, sensory analysis, antibiotic
susceptibility tests and also survival of the cultures under different preservation methods
should be studied in order to optimize their application. The robust nature of Str. infantarius
calls for challenge tests including Str. infantarius as a contaminant to further select starter
combinations that would show competitive advantage over this predominant but potentially
pathogenic bacterium.