Prevalence and Antimicrobial Susceptibility Patterns of
Bacteria from Milkmen and Cows with Clinical Mastitis in
and around Kampala, Uganda
David Patrick Kateete1*, Usuf Kabugo1,2, Hannington Baluku1, Luke Nyakarahuka2, Samuel Kyobe1,
Moses Okee1, Christine Florence Najjuka1, Moses Lutaakome Joloba1
1 Department of Medical Microbiology, School of Biomedical Sciences, Makerere University College of Health Sciences, Kampala, Uganda, 2 College of Veterinary Medicine
and Biosecurity, Makerere University, Kampala, Uganda
Abstract
Background: Identification of pathogens associated with bovine mastitis is helpful in treatment and management decisions.
However, such data from sub-Saharan Africa is scarce. Here we describe the distribution and antimicrobial susceptibility
patterns of bacteria from cows with clinical mastitis in Kampala, Uganda. Due to high concern of zoonotic infections, isolates
from milkmen are also described.
Methodology/Principal Findings: Ninety seven milk samples from cows with clinical mastitis and 31 nasal swabs from
milkmen were collected (one sample per cow/human). Fifty eight (60%) Gram-positive isolates namely Staphylococci (21),
Enterococci (16), Streptococci (13), Lactococci (5), Micrococci (2) and Arcanobacteria (1) were detected in cows; only one
grew Staphylococcus aureus. Furthermore, 24 (25%) coliforms namely Escherichia coli (12), Klebsiella oxytoca (5), Proteus
vulgaris (2), Serratia (2), Citrobacter (1), Cedecea (1) and Leclercia (1) were identified. From humans, 24 Gram-positive bacteria
grew, of which 11 were Staphylococci (35%) including four Staphylococcus aureus. Upon susceptibility testing, methicillinresistant coagulase-negative staphylococci (CoNS) were prevalent; 57%, 12/21 in cows and 64%, 7/11 in humans. However,
methicillin-resistant Staphylococcus aureus was not detected. Furthermore, methicillin and vancomycin resistant CoNS were
detected in cows (Staphylococcus hominis, Staphylococcus lugdunensis) and humans (Staphylococcus scuiri). Also,
vancomycin and daptomycin resistant Enterococci (Enterococcus faecalis and Enterococcus faecium, respectively) were
detected in cows. Coliforms were less resistant with three pan-susceptible isolates. However, multidrug resistant Klebsiella,
Proteus, Serratia, Cedecea, and Citrobacter were detected. Lastly, similar species grew from human and bovine samples but
on genotyping, the isolates were found to be different. Interestingly, human and bovine Staphylococcus aureus were
genetically similar (spa-CC435, spa-type t645 corresponding to ST121) but with different susceptibility patterns.
Conclusions/Significance: CoNS, Enterococci, Streptococci, and Escherichia coli are the predominant pathogens associated
with clinical bovine-mastitis in Kampala, Uganda. Multidrug resistant bacteria are also prevalent. While similar species
occurred in humans and cows, transmission was not detected.
Citation: Kateete DP, Kabugo U, Baluku H, Nyakarahuka L, Kyobe S, et al. (2013) Prevalence and Antimicrobial Susceptibility Patterns of Bacteria from Milkmen
and Cows with Clinical Mastitis in and around Kampala, Uganda. PLoS ONE 8(5): e63413. doi:10.1371/journal.pone.0063413
Editor: Stephen V. Gordon, University College Dublin, Ireland
Received September 18, 2012; Accepted April 4, 2013; Published May 7, 2013
Copyright: ß 2013 Kateete et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The Phoenix 100 ID/AST BDexpert system was purchased with support from the Swedish International Development Cooperation (Sida) through
Makerere University School of Graduate Studies. Activities in the Laboratories of the Department of Medical Microbiology have been funded by the National
Institutes of Health (Grant #s RO3 AI062849 and RO1 AI075637). The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: davidkateete@gmail.com
Bovine mastitis manifests either as subclinical, in which there’s
no visible symptom, or clinical, in which visible symptoms do
occur, varying from mild (flakes in milk, slight swelling of infected
quarter) to severe (abnormal milk secretions, hot swollen quarter/
udder, fever, rapid pulse, loss of appetite, depression and death)
[1].
Subclinical mastitis is relatively well documented in Uganda and
reports indicate that poor management as well as antimicrobial
resistance aggravate the condition [2,3]. While these important
studies demonstrate a growing problem of mastitis, there’s scanty
data on clinical mastitis in this country. Although subclinical
mastitis is economically more important to the dairy industry,
Introduction
Bovine mastitis is the inflammation of the mammary gland often
due to microorganisms that invade the udder, multiply and
produce toxins that are harmful to the mammary tissue [1].
Mastitis is a global problem responsible for huge financial losses to
dairy industries and economies at large due to poor milk quality,
reduced milk yield and increased expenditure on treatment and
sometimes death due to the disease itself or through culling of
affected cows [1]. In Uganda, the situation is no better in that
farmers incur heavy costs due to chemotherapy and reduced milk
production [2,3].
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Clinical Bovine Mastitis in Kampala, Uganda
samples were from cases reported by farmers for veterinary care.
Due to the high concern of zoonotic infections, nasal swabs were
simultaneously collected from milkmen to compare isolate profiles.
most farmers in Uganda are ignorant of it (due to concealed
symptoms) [2,3] but are aware of clinical mastitis, probably due to
the apparent symptoms which they perceive as an imminent threat
to cows. Besides, clinical mastitis is also of considerable importance
in that it causes both animal suffering and economic loss [4].
The effective control of mastitis heavily relies on husbandry and
management practices [1]; however, the identification of associated pathogens may be helpful in treatment and in making sound
management decisions [5,6]. Indeed, the probability of cure is
highly influenced by the characteristics of the pathogen involved,
implying that the identification of pathogens considerably
improves mastitis treatment protocols [6].
Bacteria causing clinical mastitis may be contagious or
environmental in origin [1] and for this the disease is categorized
as contagious or environmental. The bacteria associated with
either form in industrialized settings are well described [5,6,7]. It is
documented that contagious mastitis is caused by Staphylococcus
aureus, Streptococcus agalactiae, and Streptococcus dysgalactiae [5,6], and
the udder is the primary reservoir of contagious pathogens. The
mode of spread is from the infected quarter(s) to other quarters
and cows primarily at milking time. On the other hand,
environmental mastitis can be caused by coliforms (Escherichia coli,
Klebsiella pneumoniae, Klebsiella oxytoca and Enterobacter aerogenes);
environmental Streptococci (Streptococcus uberis, Streptococcus bovis
and Streptococcus dysgalactiae); and Enterococci (Enterococcus faecium
and Enterococcus faecalis). The environment of the cow is the primary
source of infection [1].
The above classification notwithstanding, it is now recognized
that the distinction between contagious and environmental mastitis
is not always clear and some bacteria can have contagious and
environmental modes of transmission. As such, surveillance data
has revealed changes in mastitis isolate profiles, which, among
other factors, are also influenced by setting [5,6,7,8,9]. This again
emphasizes the need for periodic evaluation of bacteria associated
with mastitis. Indeed, until recently coagulase negative staphylococci (CoNS) were considered to be less virulent and mainly
associated with subclinical mastitis. Yet, several studies in Europe
and North America now reveal that CoNS can cause clinical
mastitis [5,6,7,9].
Furthermore, with the global increase in antimicrobial resistance and zoonotic diseases, it has become important to
periodically determine profiles and antimicrobial susceptibility
patterns of pathogens associated with bovine mastitis. Indeed, the
problem of antimicrobial resistance has been blamed in part on
the heavy usage of antimicrobials in livestock production.
Antimicrobials are routinely used for therapeutic treatment of
disease, at sub-therapeutic concentrations to prevent disease
(prophylaxis) and for growth promotion [10,11]. For instance, in
Finland, cattle were reported to be the most treated animal species
[12] in which clinical mastitis was the most common indication for
antimicrobial treatment followed by subclinical mastitis [12].
Clearly, to elucidate mechanisms underlying the alarming
global trends in antimicrobial resistance, careful characterization
of antimicrobial resistance patterns among bacteria from food
animals particularly cattle is paramount. This requires use of
reliable methods in obtaining data on the bacterial distribution
and defining the profiles of species involved [9,13,14]. Moreover,
such data is also useful for infection control and in the
development of guidelines for appropriate antimicrobial usage in
Veterinary Medicine [5,6,15] [16].
Through conventional procedures and automated microbial
identification system, here we describe the distribution and
antimicrobial susceptibility patterns of bacteria associated with
clinical bovine mastitis in and around Kampala, Uganda. Bovine
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Results
Over the period of 1 year (February 2010 through March 2011),
97 bovine milk samples from cows with clinical mastitis in
Kampala were studied for the distribution of bacterial species and
antimicrobial susceptibility patterns. One sample per animal was
collected representing a total of 97 cows that were sampled. Most
cows belonged to exotic cattle breeds (Holstein Friesian, Jersey,
Guernsey; 52%, 50/97) or their crosses with indigenous cattle
(43%, 42/97); five (5%, 5/97) belonged to local breeds (East
African Zebu and Ankole).
Bovine samples were from a total of 34 farm units; 16 dairy
farms (50 samples), 17 zero-grazing units (35 samples) and one
communal grazing unit (12 samples), Table S1. Most exotic and
cross-breed cows were under organized farm units (dairy farms or
zero-grazing) while the indigenous cows were under communal
grazing. However, 15 cross-breeds were under the communal
grazing scheme (Table S1).
Identification of Bacteria
Following initial culturing and determination of Gram staining
properties, pure cultures were grown from single colonies, and
isolates were confirmed to species level through conventional
procedures and the Phoenix 100 ID/AST automated system
[17,18,19,20]. Due to controversy over the efficiency of this system
in identification of Gram-negative bacteria [18,21], presumptive
Gram negatives were identified through conventional methods
before subjecting to Phoenix 100 ID/AST. Generally there was
agreement between the Phoenix 100 ID/AST system and
conventional methods in the identification of common Gram
negatives (Escherichia coli, Klebsiella and Proteus vulgaris). However,
isolates of rare organisms, Gram-positive and Gram-negative alike
(Lactococci, Micrococci, Arcanobacteria, Cedecea, Serratia,
Citrobacter and Leclercia), were identified with Phoenix 100
ID/AST.
Bacterial Distribution in Milk (Bovine Samples)
Bacteria grew from 82 milk samples (85%, 82/97) of which 49
(51%, 49/97) grew pure cultures. Twenty two (23%, 22/97)
samples had mixed cultures but with a predominant colony type
which was pursued for further analysis. Eleven (11%, 11/97)
samples had mixed growth, from which pure cultures and selection
for further analysis depended on medical/veterinary importance
judged from morphological features of cells/colonies. Ultimately,
one isolate per sample was considered in further analyses.
There was no growth in 11 samples (11%, 11/97) while three
(3%, 3/97) were contaminated (at the site of collection) hence
discarded; one sample grew Candida albicans and was not included
in analyses. The bovine samples with no growth and those
contaminated on-site were mostly from cows under the communal
grazing scheme. This may reflect difficulty encountered in
sampling these animals (e.g. lack of restraint facilities to facilitate
cleaning of the udder and sampling). Nevertheless, mastitis of viral
origin, mycoplasma or un-cultivatable bacterial species may also
be responsible for the negative cultures.
i) Gram-positive bacterial species. There were 58 isolates
of Gram-positive bacteria (58/82, 71%), of which only one was
Staphylococcus aureus (1/58, 2%) while 20 were coagulase negative
Staphylococci (CoNS), (20/58, 34%). CoNS were identified as
Staphylococcus hycus (4), Staphylococcus saprophyticus (4), Staphylococcus
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Clinical Bovine Mastitis in Kampala, Uganda
xylosus (3), Staphylococcus sciuri (2), Staphylococcus epidermidis (1),
Staphylococcus haemolyticus (1), Staphylococcus hominis (1), Staphylococcus
lugdunensis (1), Staphylococcus gallinarum (1), Staphylococcus pasteuri (1)
and Staphylococcus intermedius (1).
Enterococci were 16 (16/58, 28%) identified as Enterococcus
faecium (5), Enterococcus hirae (4), Enterococcus faecalis (3), Enterococcus
gallinarum (2), Enterococcus durans (1) and Enterococcus raffinosus (1).
Streptococci were 13 (13/58, 22%), identified as Streptococcus bovis
II (5), Streptococcus acidominimus (3), Streptococcus uberis (3), Streptococcus
angionosus (1) and Streptococcus group C/G (1). Additionally,
Lactococci, Micrococci and Arcanobacteria were detected in
eight samples, speciated as; Lactococcus lactis species lactis (4/58,
7%); Lactococcus garvieae (1/58, 2%); Micrococcus lylae (2/58, 3%), and
Arcanobacterium pyogenes (1/58, 2%).
ii) Gram-negative bacteria. Twenty four (24/82, 29%)
coliforms were identified half of which were Escherichia coli (12/24,
50%). The others were Klebsiella oxytoca (5), Proteus vulgaris (2),
Serratia marcescens (2), Cedecea davisae (1), Citrobacter freundii (1) and
Leclercia adecarboxylata (1).
Thus, in Kampala and surrounding areas, CoNS, Enterococci,
Streptococci and Escherichia coli are the predominant bacteria
associated with clinical mastitis. The isolate profiles are summarized in Table S1.
tetracycline (7/21, 33%) and trimethoprim-sulfamethoxazole (6/
21, 29%).
Moreover, there were two vancomycin resistant staphylococci
(Staphylococcus hominis and Staphylococcus lugdunensis) which were also
MRS (i.e. methicillin-resistant-vancomycin-resistant staphylococci,
MR-VRS). Of note, while resistance to rifampicin was low (2/21,
10%), the two MR-VRS isolates were the ones resistant to this
drug, Table S2.
ii) Antimicrobial resistance among Staphylococci from
milkmen. All the 11 staphylococci from humans were susceptible
(11/11, 100%) to daptomycin, rifampicin, muprocin, moxifloxacin, linezolid and gentamicin. Thus, the difference in pansusceptibility between bovine and human isolates was ciprofloxacin to which three human-isolates were resistant (while all from
cows were susceptible) and rifampicin, to which two bovine MRVRS were resistant (while all from humans were susceptible),
Tables S1 and S2.
Similar to the antimicrobial susceptibility patterns of bovine
isolates, all the 11 (11/11, 100%) human isolates were resistant to
ampicillin and penicillin G, and were also found to be ‘‘betalactamase’’ producers. Again, the four human isolates of Staphylococcus aureus were susceptible to cefoxitine and oxacillin implying
they were MSSA. However, the human MSSA were also resistant
to trimethoprim-sulfamethoxazole and tetracycline. Furthermore,
as with bovine isolates, human CoNS were substantially resistant
to cefoxitin (7/11, 64%) and oxacillin (7/11, 64%) implying that
they were also MRS. Also, resistance to amoxicillin-clavulanate
(7/11, 64%) and tetracycline (7/11, 64%) was substantial.
Furthermore, three MRS (Staphylococcus scuiri) resistant to
vancomycin were detected in humans, Tables S1 and S2.
While the species distribution between humans and cows was
similar (i.e. MRS in milkmen -Staphylococcus sciuiri, Staphylococcus
saprophyticus, Staphylococcus xylosus and Staphylococcus intermedius were
also detected in cows), the antimicrobial resistance patterns
differed. Furthermore, while all the vancomycin resistant staphylococci (VRS) from humans and bovines were MRS, the species
were different (i.e. VRS from cows were Staphylococcus hominis and
Staphylococcus lugdunensis while the one from milkmen was Staphylococcus sciuiri). Overall, the staphylococcal species from bovine
samples that were not detected in milkmen were Staphylococcus
hycus, Staphylococcus lugdunensis and Staphylococcus gallinarum.
Figure 1 graphically depicts the antimicrobial resistance among
staphylococci in cows and milkmen. Details for the susceptibility
patterns of each isolate are provided in Table S2.
iii) Antimicrobial resistance among Enterococci from
cows. All Enterococci (16/16, 100%) from bovine samples were
susceptible to ampicillin. However, resistance to tetracycline (5/
16, 31%), vancomycin (3/16, 19%), teicoplanin (13%, 2/16),
erythromycin (3/16, 19%), daptomycin (1/16, 6%), and ciprofloxacin (1/16, 6%) was noted, but relatively low, Table 1.
Of concern was the detection of isolates resistant to vancomycin
and daptomycin (Enterococcus faecalis and Enterococcus gallinarum,
respectively), Table 1. Since these drugs are crucial in the
treatment of infections due to intractable pathogens, detection of
such isolates in milk is risky to consumers in case it is consumed
raw.
iv) Antimicrobial resistance among Enterococci from
milkmen. All enterococci (8/8, 100%) from humans were
susceptible to ampicillin, daptomycin, teicoplanin, vancomycin
and moxifloxacin while resistance to erythromycin was also low
(13%, 1/8), Table 1.
Lastly for the Gram-positives, Streptococci, Lactococci, Micrococci and Arcanobacteria were also identified to species level but
Bacterial Distribution in Nasal Swabs (Humans)
Thirty one nasal swabs from milkmen grew 24 (24/31, 77%)
bacterial isolates with no growth occurring in seven (7/31, 23%);
Gram-negative organisms were not detected (not surprising since
nares are not conducive for their growth).
Eleven Staphylococci (11/31, 35%) were detected in humans of
which four were Staphylococcus aureus. This implies that the nasal
carriage of Staphylococcus aureus in milkmen was 13% (4/31), lower
than that reported in hospital settings in Uganda [22]. Additionally, similar species of CoNS to those detected in cows were
identified; Staphylococcus scuiri (3), Staphylococcus saprophyticus (2),
Staphylococcus xylosus (1) and Staphylococcus intermedius (1). Importantly, these CoNS were detected in milkmen working on the same
farms where similar CoNS were detected in cows, Table S1.
Also detected in humans were eight Enterococci (8/31, 26%)
identified as Enterococcus faecium (4), Enterococcus faecalis (2) and
Enterococcus hirae (2). Additionally, two isolates were detected for
each of Streptococcus pneumoniae and Streptococcus bovis II (group D),
while one was identified for Lactococcus lactis species lactis.
Overall, the bacterial species detected in milkmen were similar
to those identified in bovine samples, Table S1. While this alluded
to a possibility of transmission between humans and cows, largely,
genotyping data did not support this notion (see ‘Genotyping’
below).
Antimicrobial Resistance Patterns
There were high levels of antimicrobial resistance among
isolates from cows and milkmen;
i) Antimicrobial resistance among Staphylococci from
cows. All Staphylococci (21/21, 100%) from bovine samples were
susceptible to daptomycin, ciprofloxacin, mupirocin, moxifloxacin,
linezolid and gentamicin. However, all the isolates (21/21 100%)
were resistant to ampicillin and penicillin G; expectedly, all were
found to be ‘‘beta-lactamase’’ producers. Of note, the sole isolate
of Staphylococcus aureus from cows was susceptible to cefoxitin and
oxacillin implying it was methicillin susceptible S. aureus (MSSA).
However, most isolates of CoNS were resistant to cefoxitin (12/21,
57%) and oxacillin (12/21, 57%), implying they were methicillin
resistant Staphylococci (MRS). Furthermore, Staphylococci were
substantially resistant to amoxicillin-clavulanate (11/21, 52%),
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Clinical Bovine Mastitis in Kampala, Uganda
Figure 1. Antimicrobial resistance among staphylococci from cows (panel A) and milkmen (panel B). Details in Tables S1 and S2.
doi:10.1371/journal.pone.0063413.g001
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Clinical Bovine Mastitis in Kampala, Uganda
between humans and livestock. Staphylococcus aureus was genotyped
with spa typing while multi-locus sequence typing (MLST) was
performed for the two Enterococci that were resistant to
vancomycin and daptomycin. However, owing to the diversity of
the species involved and paucity of genotyping methods, as well as
cost implications, RAPD genotyping was employed for the other
isolates.
Following MLST, the vancomycin resistant Enterococcus faecalis
was found to be unique with an allelic profile of 1 (gdh); 1 (gyd); 3
(pstS); 7 (gki); 21 (aroE); 1 (xpt); 5 (yqil); close to E. faecalis ST447
[allelic profile of 1 (gdh); 7 (gyd); 3 (pstS); 7 (gki); 6 (aroE); 1 (xpt); 5
(yqil)]. This strain was submitted to the MLST database to assign
the sequence type. However, the daptomycin resistant isolate of
Enterococcus faecium was found to be un-typable.
Overall, while similar bacterial species were detected in human
and bovine samples, and often on the same farm (Table S1), the
genotyped isolates displayed distinct patterns, Figure 2. Thus,
transmission between milkmen and cows was not detected (at least
for these isolates).
Interestingly however, isolates of Staphylococcus aureus were
genetically similar; all strains, human and bovine alike, belonged
to the same lineage, spa type t645 (spa-CC435, ST121) implying
genetic relatedness, Table 3. Moreover, the bovine and human
isolates were detected in samples from the same farm, Table 3.
However, as described above, the drug susceptibility data for the
bovine isolate was different from that of human isolates in that the
latter were resistant to trimethoprim-sulfermethaxole (SXT) and
tetracycline (in addition to ampicillin and penicillin G to which the
bovine isolate was resistant). In an attempt to account for this
difference, we performed plasmid profiling and indeed identified
differences among bovine and human isolates. All isolates
possessed an approx. 20 kb plasmid; however, the human isolates
were found to possess three additional smaller plasmids (approx. 5,
4 and 3 kb, respectively) that were missing in the bovine isolate.
Since antimicrobial resistance genes including those encoding
SXT and tetracycline resistance are plasmid-encoded, the
difference in susceptibility patterns may be attributed to the
acquisition of plasmids by the human isolates.
Nevertheless, the different DST patterns negate the possibility of
transmission in spite of the isolates being genetically similar.
Table 1. Antimicrobial resistance patterns among
Enterococci.
Isolates from cows (n = 16)
Species/isolate
Antimicrobial resistance pattern
E. faecium
ERY
E. faecium
DAP-ERY
Comment
DRE
E. faecium
E. faecium
E. faecium
ERY
E. faecalis
ERY-CIP-TET
E. faecalis
ERY-TET
E. faecalis
TEI-VAN
E. hirae
ERY-TET
VRE
E. hirae
E. hirae
TET
E. hirae
ERY
E. gallinarum
TEI-VAN
VRE
E. gallinarum
TEI-VAN
VRE
E. durans
TET
E. raffinosus
ERY-TET
Isolates from humans (n = 8)
E. faecalis
ERY-TET
E. faecalis
ERY-CIP-TET
E. faecium
TET
E. faecium
E. faecium
ERY
E. faecium
E. hirae
ERY
E. hirae
TET
DAP, Daptomycin; TEI, Teicoplanin; VAN; Vancomycin; ERY, Erythromycin; CIP,
Ciprofloxacin; TET, tetracycline.
In boldface type are isolates that were found to be resistant to daptomycin and
vancomycin, respectively (i.e., DRE, daptomycin resistant enterococcus, and
VRE, vancomycin resistant enterococcus).
doi:10.1371/journal.pone.0063413.t001
Highlight on Management Practices
For an insight into the management practices among the farm
units where samples were collected, a formal survey focusing on
veterinary care and milking practices was conducted using an
interview administered questionnaire;
the susceptibility patterns for these organisms are not included in
the Phoenix 100 AST panels hence are not reported.
v) Antimicrobial resistance among Gram-negative
isolates. All coliforms were susceptible to amikacin, gentamicin,
imipenem, meropenem, ceftazidime, ciprofloxacin and levofloxacin, Table 2. However, resistance to ampicillin was high (17/24,
71%) while it was moderate for cephalothin (8/24, 33%),
trimethoprim-sulfamethoxazole (8/24, 33%), cefuroxime (6/24,
25%) and amoxicillin-clavulanate (5/24, 21%). Also, resistance to
nitrofurontoin (4/24, 17%), colstin (4/24, 17%), cefoxitin (1/24,
4%), ertapenem (1/24, 4%), cefepime (1/24, 4%), aztreonam (1/
24, 4%) and piperacillin (1/24, 4%) was low. Although coliforms
were the least resistant with three pan-susceptible isolates,
multidrug resistant (MDR) isolates (Klebsiella oxytoca, Proteus vulgaris,
Serratia marcescenes, Cedecea davisae, and Citrobacter freundii) were
detected, Table 2.
Veterinary Care and Antimicrobial Usage
Farmers with dairy farms and zero grazing units reported that
they relied on veterinarians for veterinary services whenever they
encountered clinical mastitis. However, farmers practicing communal grazing relied on milkmen and herdsmen to treat mastitis
and involved veterinarians only when they encountered difficulty.
Intramammary infusions with ampicillin or tetracycline were
frequently used by most farmers. Also occasionally used were antiinflammatories such as calvasone, predinisolone, and dexamethathone.
Furthermore, most farmers reported poor response to treatment
particularly with ampicillin-based intramammary infusions (which
may be explained by the high proportion of isolates resistant to this
drug found in this study). Indeed, MRS (Staphylococcus hycus) isolates
were recovered in four cases where farmers reported poor
response to treatment. The intramammary infusions used to treat
these cows contained penicillin to which all staphylococci were
Genetic Relatedness among Human and Bovine Isolates
The similar bacterial species that were detected in milkmen and
cows (Staphylococci; Enterococcus; Streptococcus; Micrococcus)
were genotyped to determine relatedness and possible transmission
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Table 2. Antimicrobial resistance patterns among coliforms (n = 24).
Species
Antimicrobial resistance pattern
Comment
MDR
Citrobacter freundii
AMP-AMO-CEF-CEP-CFU
Escherichia coli
CEP
Escherichia coli
CEP
Escherichia coli
CEP
Escherichia coli
AMP-AMO-SXT-CEP
Escherichia coli
–
Escherichia coli
AMP-CEP
Pan-susceptible
Escherichia coli
AMP-SXT-CEP
Escherichia coli
AMP-SXT-CEP
Escherichia coli
AMP-SXT
Escherichia coli
CEP
Escherichia coli
AMP-AMO-CEP
Escherichia coli
AMP
Klebsiella oxytoca
SXT
Klebsiella oxytoca
–
Pan-susceptible
Klebsiella oxytoca
SXT-CEF-CEP-CFU-CFP-AZT-PIP
MDR
Klebsiella oxytoca
SXT
Klebsiella oxytoca
SXT-CEF-CEP-CFU-CFP-AZT-PIP
MDR
Leclercia adecarboxylata
–
Pan-susceptible
MDR
Proteus vulgaris
AMP-SXT-COL-CEP-CFU-NTR
Proteus vulgaris
AMP-NTR
Serratia marcescenes
AMP-AMO-COL-CEP-CFU-NTR
MDR
Serratia marcescenes
AMP-AMO-COL-CEF-CEP-CFU-NTR
MDR
Cedecea davisae
AMP-AMO-COL-CEF-CFT-CEP-CFU-NTR-ERT
MDR
AMP, Ampicillin; AMO; Amoxicillin-Clavulanate; SXT, trimethopprim-sulfamethoxazole; COL, Colistin; IMP, imipenem; CEF, Cefoxitine; CFT, Cefotaxim; CEP, Cephalothin;
CFU, Cefuroxime; CFP, Cefepime; AZT, Aztreonam; ERY, Erythromycin; NTR, Nitrofurantoin; PIP, Piperacillin-Tazobactum; ERT, Ertapenem.
In boldface type are isolates found to be multi-drug resistant (MDR).
doi:10.1371/journal.pone.0063413.t002
system. Staphylococci, Enterococci and Streptococci from milkmen and livestock were identified to species level, as well as rare
organisms such as Micrococcus, Arcanobacteria, Cedecea, Serratia, Citrobacter and Leclercia. Overall, CoNS, Enterococci,
Streptococci and Escherichia coli were the predominant bacteria
associated with clinical mastitis in Kampala. These organisms are
notorious agents of mastitis globally particularly in Europe [8,23]
and Asia [24] [25].
While further studies may be required, one can assume that in
Kampala, environmental clinical mastitis, for which coliforms are
most incriminated [1], is prevalent and may surpass the contagious
form of disease. This may not be surprising given the low levels of
hygiene and inappropriate husbandry practices encountered in
this study. Environmental mastitis usually reflects poor management practices [1], as previously reported [3]. Nevertheless (and
given the ambiguous understanding of mastitis disease forms),
contagious mastitis, also usually due to poor management practices
particularly at milking [1], could as well have contributed to the
high prevalence of environmental pathogens detected.
Subclinical mastitis has been studied before in Uganda [2,3];
however, there’s scanty data on clinical mastitis. Therefore, any
comparison with previous studies in Kampala and Uganda in
general is with respect to subclinical mastitis. In a previous study
[3], penicillin and oxacillin resistance was reported to be 86.8%
and 29.7%, respectively, while in the current study it was 100%
and 57%, respectively [3]. Furthermore, resistance to tetracycline
resistant. Following drug susceptibility testing (DST), gentamicinbased infusions were advised and a good response was reported
(i.e. cows were cured of clinical mastitis). However, the recommended withdraw period was not observed in that farmers
continued to consume or sell the milk from cows on treatment.
Milking Practices and Udder Hygiene
Milking machines are rare in this setting and most farmers rely
on hand-milking. Nevertheless, the milking technique employed
by milkmen was poor (i.e. pulling teats instead of squeezing them).
For dairy farms, there was no specific order of milking cows with
respect to health status (e.g. milking healthy cows before sick ones).
Teat dipping was practiced only on one farm. Furthermore,
communal grazing-farmers used the same individual (a herdsman)
for milking cows from different herds as he gathered cattle for
grazing.
Taken together and considering the isolate profiles described,
clinical mastitis in this setting is mostly environmental [1].
Discussion
In this study, we have employed contemporary bacterial
identification procedures to describe the bacterial species associated with clinical mastitis in Kampala, Uganda. Isolates which
previous studies in Uganda could not identify [2,3] have been
elucidated through the use of the Phoenix 100 ID/AST automated
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Clinical Bovine Mastitis in Kampala, Uganda
Figure 2. Distinct patterns among staphylococci (panel A), enterococci (panel B) and streptococci (panel C) following RAPD
genotyping. One isolate per lane.
doi:10.1371/journal.pone.0063413.g002
Furthermore, in a recent report on subclinical mastitis in periurban Kampala [2], infection with CoNS (54.7%) and Streptococci (16.2%) was found to be the most common bacteriological
outcome [2]. Six of the nine (67%) CoNS and four of the eight
(50%) Staphylococcus aureus were positive for penicillinase production. Although substantially high, this contrasted with the absolute
(100%) beta-lactamase production among Staphylococci in the
current study; the disagreement could be attributed to differences
in the previous study was higher than what we have reported (86%
vs. 33%). It is postulated that penicillin and tetracycline resistance
is exacerbated by the frequent usage by farmers of intramammary
infusions with those drugs [3]. Also in the previous study resistance
to gentamicin was reported albeit low while it was not detected in
the current study. The low gentamicin resistance in Uganda has
been attributed to the high cost of this drug which prohibits its
usage by farmers [3], in the end slowing emergence of resistance.
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Clinical Bovine Mastitis in Kampala, Uganda
Table 3. Staphylococcus aureus from cows (n = 1) and humans (n = 4) with similar Spa type, t645.
Date of collection
Isolate
number
23-Feb-2010
Ky9c
Cow (milk)
Katale (Farm A1)
RRSSSSSSSSSSSS
14:44:13:12:17:23:18:17
t645
ST-121
23-Feb-2010
Ky17n
Human (nares)
Katale (Farm A1)
RRRRSSSSSSSSSS
14:44:13:12:17:23:18:17
t645
ST-121
3-Jul-2010
Ky2n
Human (nares)
Kisubi (Farm A9)
RRRRSSSSSSSSSS
14:44:13:12:17:23:18:17
t645
ST-121
10-Dec-2010
Ky6n
Human (nares)
Entebbe (Farm B1)
RRRRSSSSSSSSSS
14:44:13:12:17:23:18:17
t645
ST-121
4-Mar-2011
105n
Human (nares)
Wakiso (Farm A12)
RRRRSSSSSSSSSS
14:44:13:12:17:23:18:17
t645
ST-121
Source
DSTa
Location
Spa repeat
Spa type
ST
a
Drug susceptibility testing. R, Resistant, S, susceptible, with respect to drugs in the following order: Ampicillin; Penicillin G; Trimethopprim-sulfamethoxazole;
Tetracycline; Cefoxitine; Oxacillin; Amoxicillin-Clavulanate; Teicoplanin; Vancomycin; Clindamycin; Erythromycin; Nitrofurantoin; Rifampicin; Ciprofloxacin.
All S. aureus were methicillin susceptible (MSSA).
doi:10.1371/journal.pone.0063413.t003
In an Algerian study the majority of bacteria from cows with
subclinical mastitis were CoNS [32]; another similar finding is that
Lactococcus lactis species lactis was also isolated [32]. However, there
was higher susceptibility of the isolates to antimicrobials including
penicillin, contrary to the findings in this study. While one may
point to differences in enforcement of regulations on antimicrobial
usage between Uganda and Algeria, MDR-CoNS are prevalent in
Nordic countries notable for sound antimicrobial regulations [33].
Meanwhile in the Sudan, Staphylococci also dominated isolates
recovered from cows with clinical and subclinical mastitis [34].
Interestingly, Arcanobacterium pyogenes, an emerging etiological agent
for bovine mastitis [35], was also identified in the Sudanese study.
Overall, the bacterial distribution in Africa appears similar but
with some important exceptions. For instance, there are differences in antimicrobial susceptibility patterns between isolates
reported in our study and those from Algeria; the isolate
distribution also differs between our study and the Sudanese (i.e.
Corynebacteria, Brucella, Pseudomonas and Aerococcus were
detected in Sudan but not in the current study).
in methodology. Interestingly however, the prevalence of Staphylococcus aureus (an organism highly associated with bovine mastitis)
was very low in both studies (i.e. of the 450 quarter samples in the
former study, Staphylococcus aureus grew only in eight while CoNS
grew in 246) [2].
Transmission of Bacteria between Milkmen and Cows
was not Detected
While transmission of bacterial species between humans and
livestock is increasingly being detected in farm workers in Europe
and much of the industrialized world [26], there’s so far no report
to indicate the same occurs in sub-Saharan Africa. Moreover,
methicillin resistant Staphylococcus aureus (MRSA), a common
finding in livestock workers [5], was not detected in this study.
Of concern however was the detection in cows and milkmen of
high levels of MDR bacteria of the same species implying that
transmission is possible. For most species however, transmission
was not detected in that the human and bovine isolates displayed
unrelated DST and RAPD patterns, implying that they were
indeed different.
However, the exception was Staphylococcus aureus for which a
bovine isolate presented a similar spa type to that of humans’.
Interestingly, the bovine and a human isolates were collected on
the same farm. Yet, the different DST patterns among these
isolates negate occurrence of transmission between milkmen and
cows.
Resistance genes in Staphylococcus aureus are often plasmidencoded and disseminate through Staphylococcus aureus populations
by horizontal gene transfer (HGT) mechanisms leading to strains
that are more resistant [27,28,29]. Thus, it’s possible that the
plasmids detected in the human isolates were acquired through
HGT and encode resistance to SXT and tetracycline. Of note, the
identified strain belonged to a lineage that occurs worldwide [30],
spa Type t645 (spa-CC435, ST121), and it was also the most
predominant lineage among Staphylococcus aureus causing surgical
site infections [31] at Mulago hospital, a national referral hospital
in Kampala. To date, there’re five strains of lineage t645 in the
Ridom database [http://spa.ridom.de/spa-t645.shtml] associated
with infection.
Europe
Given the contrast in animal husbandry practices and in
enforcement of antimicrobial usage between Uganda and Europe,
this discussion only serves to highlight global trends in isolate
profiles and antimicrobial resistance patterns without accounting
for differences or similarities.
In Europe, there are varying reports both in the distribution and
antimicrobial susceptibility patterns of bacteria causing mastitis.
For instance in Finland, CoNS dominated isolates from cows with
clinical mastitis in which symptoms were most severe in cows with
Staphylococcus hycus infection [33]. Interestingly, in the current
study, Staphylococcus hycus was also among the most prevalent
among the CoNS. Meanwhile in Estonia, the main bacterial
pathogens associated with clinical mastitis were Streptococcus uberis
and Escherichia coli [36] while subclinical mastitis was caused
mainly by Staphylococcus aureus and CoNS. Similar to our findings,
antimicrobial resistance was prevalent in Estonia, especially
penicillin resistance among Staphylococcus aureus and CoNS. In
Switzerland, high prevalence of MRS was found in livestock
production facilities [37] and in addition to beta-lactam resistance,
most strains were resistant to other non-beta-lactam antibiotics
[37]. Yet in Sweden, Staphylococcus aureus and CoNS are frequently
associated with subclinical mastitis but antimicrobial resistance is
very low [21].
Of note, human-CoNS species tend to be MDR yet their
counterpart, Staphylococcus aureus, is less prone to developing multiresistance to antimicrobials particularly in the Nordic countries
Situation in the Rest of Africa
Generally there’s little data on bovine mastitis from subSaharan Africa. Nevertheless, we highlight our findings in light of
countries where mastitis has been documented irrespective of
disease form. Since climate and management practices markedly
differ between countries, we only compare isolate profiles without
accounting for differences.
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Clinical Bovine Mastitis in Kampala, Uganda
[15] [16]. Also, CoNS species from bovines in Europe are most of
the time reported to be susceptible to antimicrobials [9,13,14], in
contrast with CoNS in this study. Differences in animal
husbandry, management practices as well as enforcement of
antimicrobial regulations could account for this. In veterinary
medicine, CoNS have become a problem and are currently
incriminated as causes in several episodes of clinical mastitis.
Setting
This study was conducted within farming units of Kampala and
surrounding areas including the adjoining districts of Wakiso,
Mukono, Mpigi, Luwero, Kamuli, Kayunga and Mityana [39].
Definition of Clinical Mastitis
A textbook definition of clinical mastitis was considered [1]; a
cow with visible signs of mastitis, either, mild (flakes or clots in
milk, slight swelling of infected quarter) or severe (abnormal
secretion, hot, swollen quarter or udder, fever, rapid pulse, loss of
appetite, dehydration and depression) [1]. As expected, cows with
severe signs were more common (since most were cases reported
by farmers for veterinary care). Cows were clinically re-examined
by field veterinarians to confirm symptoms prior to sample
collection.
Limitations
There’re some shortcomings in this report. First, the study was
based on mastitis cases from Kampala reported by farmers and
represents only those who could afford veterinary care. Thus, these
findings are not generalizable to the entire city or country. Also,
some animals were on medication and this could have affected
recovery of bacterial isolates. Additionally, most milkmen didn’t
consent limiting the human-sample size.
Secondly, whilst utmost care was taken to minimize contamination through strict adherence to standardized sampling
procedures, it is possible that some isolates could have been
contaminants from the cows’ environment given the ubiquity of
bacteria on cows. Nevertheless, the observed improvement in cure
rates among stubborn cases following DST implies that contamination was really minimal. Also, there was no bacterial growth in
several samples, bovine and human alike. Moreover, even in
settings with developed dairy industries, bacterial species previously thought to be commensals or contaminants are now
documented causes of clinical mastitis [13,14]. It is increasingly
becoming clear that there may be no difference between microbes
formerly considered pathogenic vs. the nonpathogenic ones [http://
www.einstein.yu.edu/uploadedFiles/casadevall/10_Casadevall_
Pirofski_09.pdf].
Collection of Milk Samples
Information on clinical mastitis cases was obtained from field
veterinarians who informed research assistants through telephone
calls and a farm visit was arranged. Milk samples were collected
consecutively from affected quarter(s) using sterile 50 ml centrifuge
tubes (Fisher Scientific, Leicestershire, UK). To minimize
contamination, we strictly adhered to the mastitis sample
collection protocol described by Dr. J.W. Schroeder, North
Dakota State University [www.ag.ndsu.edu/pubs/ansci/dairy/
as1129.pdf] [1]. Briefly, centrifuge tubes were labeled and forms
filled prior to each farm visit. At the farm, hands were washed with
soapy water while teats were washed with 70% ethanol and dried
individually with clean paper towels. Two squirts of milk were
discarded from the teat before dipping in a germicidal teat dip
(which contained 0.64% Sodium Chlorite) for 30 sec of contact
time. After wiping off the teat dip with an individual clean towel,
the teat end was thoroughly scrubbed with a cotton swab soaked in
70% ethanol. A clean swab was used for each teat. Then, a
centrifuge tube was opened under the teat and held at an angle so
that foreign material could not fall into the opening; nothing was
allowed to come in contact with the mouth of the tube. Approx.
5 ml of milk was collected from each infected quarter, and the
container was closed before removing it from beneath the teats.
During farm visits, samples were stored briefly in an ice-cold box
and promptly transported to the bacteriology laboratory for
culture.
Conclusions
Bovine clinical mastitis mainly due to CoNS, Enterococci,
Streptococci and Escherichia coli is prevalent in Kampala, Uganda.
Multidrug resistant bacteria notably coagulase negative Staphylococci and coliforms other than Escherichia coli (Klebsiella, Proteus,
Serratia, Citrobacter and Cedecea) are also prevalent. Of concern was
the detection of vancomycin and daptomycin resistant Enterococci
in cows, as well as methicillin and vancomycin resistant
staphylococci both in milkmen and cows. While the potential for
transmission of bacteria between humans and livestock occurs, it
was not detected in this study given the different genotypic and
susceptibility patterns exhibited by the isolates. Further studies are
required to ascertain this.
Human Samples
Nasal samples (swabs) were simultaneously collected from
milkmen who gave written informed consent, and similarly
transported in a separate ice-cold box to the bacteriology
laboratory.
Materials and Methods
Ethics Statement
Questionnaire
Written informed consent was sought from all the milkmen who
participated and those who did not consent were excluded.
Additionally, the study protocol and consent procedure were
approved by the Uganda National Council of Science and
Technology (UNCST) (reference # NS 371). The UNCST
registers and clears all research intended to be carried out in
Uganda and in so doing, it reviews the research protocols for their
scientific merit, safety and ethical appropriateness prior to issuing
permits for conducting studies. The research permit is granted at a
national level to facilitate the carrying out of research within the
country. All research in Uganda is registered and approved by the
UNCST [38].
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A formal survey with an interviewer administered questionnaire
was conducted to collect data on location, herd size, farming
system, clinical symptoms, breed, parity, age, milk-yield, stage of
lactation, treatment record and antimicrobial usage. This survey
was conducted among farm owners and had a high response rate
(100%).
Bacterial Cultures
Initially, samples were cultured on blood agar or on tryptic soy
agar (TSA) (for samples with no growth on blood agar plates).
Plates were incubated at 37uC for 24h. Further processing
followed the laboratory’s standard operating procedures for
identifying Gram-positive and Gram-negative bacteria.
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Clinical Bovine Mastitis in Kampala, Uganda
For AST, the Phoenix AST Indicator Solution was added to the
AST broth tubes and mixed by inversion. The AST side of the
combination panel contains 84 wells with dried antimicrobial
panels and one growth control well [17]. One free-falling drop of
the AST indicator was added to the AST broth tube [17], and
30 ml of the standardized ID broth suspension was transferred to
the AST broth and incubated up to 16 hours at 35 uC. Samples
were read automatically at the instrument’s set parameters.
Quality control and maintenance were performed according to
the manufacturer’s
recommendations [17]. Staphylococcus
aureus
TM
TM
ATCC 29213 and Enterococcus faecalis ATCC 29212 were
included in the ID and AST Panels for quality control.
Staphylococci were presumptively identified with a previously
described protocol that involves sequel testing of catalase positive
isolates with tube coagulase, Mannitol salt agar and DNase tests
[40]. Staphylococcus epidermidis was confirmed through culturing
CoNS isolates on TSA with 20 mg/ml of novobiocin (SigmaAldrich, St. Louis, MO, USA). Enterococci were presumptively
identified on the basis of catalase-negative, Gram-positive cocci
growing in the presence of 40% bile (bile-esculin agar, Difco,
Detroit, USA) and on 6.5% NaCl in brain heart infusion (BHI)
agar (Oxoid, London, UK) [41]. To distinguish Streptococci from
Enterococci, growth in BHI broth with 6.5% NaCl was employed
in which case Streptococci did not grow while Enterococci grew.
To isolate Gram-negative bacteria, a sample was plated on
TSA, blood and MacConkey agar, and incubated overnight at
37uC for 24h. Pure cultures were obtained by re-streaking single
colonies from MacConkey plates on TSA and incubating at 37uC
for 24h. Morphological features of isolates on TSA, blood and
MacConkey agar were examined prior to a series of biochemical
tests for identification of Escherichia coli, Proteus and Klebsiella species.
Tests involved sugar fermentation (sucrose, glucose, lactose, triple
sugar iron, mannitol); motility (Sulphur Indole & Motility test on
‘SIM’ medium); gas production; oxidase; and utilization of citrate
and urea [42].
Genotyping
To determine genetic relatedness and whether transmission of
bacteria occurs between humans and livestock, genotyping was
performed on isolates of the same species that were detected in
milkmen and cows.
i) Staphylococcus aureus. The x-region of Staphylococcus
aureus spa gene (0.2 kb to 0.4 kb) was amplified by PCR with the
method established before [43] using primers 1095F, 59-AGACGATCCTTCGGTGAG-39,
and
1517R,
59-CAGCAGTAGTGCCGTTTG-39. The PCR conditions were as follows:
94 uC for 5 min, followed by 31 cycles each consisting of 94 uC,
30 sec; 53 uC, 30 sec; 72 uC, 1 min and a final extension at 72 uC
for 10 min. The PCR products were purified with the QIAquick
PCR purification kit (Qiagen, Hilden, Germany) as per the
manufacturer’s instructions, and both strands sequenced (ACGT,
Wheeling, IL, USA) using the same primers. To obtain spa types,
the sequences were submitted to a free spaTyper data base
(http://fortinbras.us/cgi-bin/spaTyper/spaTyper.pl) and lineages
matching to query sequences determined. The data was also
submitted to the Ridom Spa server (http://spa.ridom.de/) for
comparison.
ii) CoNS, Enterococci, Lactococci and Streptococci. The
bacterial species belonging to the above genera were genotyped
with random amplification of polymorphic DNA (RAPD) typing
according to Reinoso et al, 2004 [44], with minor modifications.
The primer sequence used was 59-ACGCAGGCAC-39, under the
conditions: 94uC, 4 min, followed by 40 cycles of 94uC, 1 min,
36uC, 1 min and 72uC, 2 min, with a final amplification step at
72uC for 10 min. Amplicons were analyzed by agarose gel
electrophoresis at 90V for 5 hours on a 1% agarose gel. Images
were captured with a bioimager and analyzed with the
BioNumerics software v. 5 (Applied Maths NV, Sint-MartensLatem, Belgium).
iii) Daptomycin and vancomycin resistant Enterococci.
Since daptomycin and vancomycin are important drugs in the
treatment of microbial infections, the two Enterococci resistant to
these drugs (E. faecium and E. faecalis, respectively) were typed with
multi locus sequence typing (MLST) to ascertain their sequence
types. The primers used are summarized in Table S3 and were
obtained from [http://www.mlst.net/databases/default.asp].
For Enterococcus faecium the following conditions were used; PCR
reactions were performed in 50 ml mixture each containing 25 mL
HotStar Taq Master Mix (Qiagen), 40 pmol of each primer, and
milli-Q water to a final volume of 50 mL. One ml of crude DNA
prep was used as template for amplifications. The PCR
programme comprised of an initial denaturation at 95uC for
15 min, 35 cycles of 30 s at 94uC, 30 s at 50uC, and 30 s at 72uC,
followed by 5 min 72uC. The PCR products were purified with
the Qiaquick PCR purification kit following the manufacturer’s
instructions, and sequenced at ACGT (Wheeling, IL, USA) with
both the forward and reverse primers. Sequence chromatograms
Confirmation of Isolates to Species Level and
Antimicrobial Susceptibility Testing
To confirm the isolates to species level and their antimicrobial
susceptibility patterns, we employed the ‘Phoenix Automated
Microbiology System’ (Phoenix 100 ID/AST system) from Becton
and Dickson (Franklin Lakes, NJ, USA) [19]. This system has
combination testing panels that include: a) identification (ID) side
with dried substrates for bacterial identification; b) an antimicrobial susceptibility testing (AST) side with varying concentrations of
antimicrobial agents; and c) growth and fluorescent controls at
appropriate well locations.
The ID portion of the Phoenix panels utilizes a series of
conventional, chromogenic, and fluorogenic biochemical tests to
determine the identification of the organism. Acid production is
indicated by a change in the phenol red indicator when an isolate
is able to utilize a carbohydrate substrate. Chromogenic substrates
produce a yellow color upon enzymatic hydrolysis of either pnitrophenyl or p-nitroanilide compounds. Enzymatic hydrolysis of
fluorogenic substrates results in the release of a fluorescent
coumarin derivative. Organisms that utilize a specific carbon
source reduce the resazurin-based indicator. In addition, there are
other tests that detect the ability of an organism to hydrolyze,
degrade, reduce, or otherwise utilize a substrate.
Specimen processing and Gram staining procedure was
performed according to the manufacturer’s guidelines [19]. Then,
Phoenix panels were inoculated with a standardized inoculum
according to the manufacturer’s guidelines; occasionally, minor
modifications were done as described elsewhere [17,18,19].
Briefly, after determining the Gram staining properties of the
isolates, nonselective media (blood agar or TSA) was used to
prepare fresh pure cultures for isolate ID and AST [19]. Isolates
were inoculated into appropriate ID/AST combination panels
(PhoenixTM PMIC/ID for Gram-positive and PhoenixTM NMIC/
ID for Gram-negative isolates) that were loaded into the instrument and incubated at 35uC, according to the manufacturer’s
guidelines. The ID broth was inoculated with bacterial colonies
adjusted to a 0.5 McFarland standard. The suspension was poured
into the ID side of the Phoenix panel after an aliquot (30 ml) was
removed and saved for AST.
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Clinical Bovine Mastitis in Kampala, Uganda
were analyzed with BioEdit software and submitted to the MLST
database [http://www.mlst.net/databases/default.asp] for sequence
types.
For Enterococcus faecalis the following conditions were used; initial
denaturation at 94uC for 5 min; 30 cycles at 94uC for 30 s, 52uC
for 30 s and 72uC for 1 min; and extension at 72uC for 7 min.
Reactions were performed in 10 ml volumes with Custom master
mix (ThermoFisher, Surry, UK) and Taq polymerase (ThermoFisher, Surry, UK). The PCR products were purified as described
above for E. faecium, sequenced and analyzed similarly.
Nitrofurantoin; Piperacillin-Tazobactum; Ertapenem NA: Not
applicable DRE: Daptomycin resistant enterococcus VRE:
Vancomycin resistant enterococcus MSSA: Methicillin susceptible
Staphylococcus aureus MRS: Methicillin resistant Staphylococcus MRVRS: Methicillin-resistant, vancomycin-resistant Staphylococcus
DST: Drug susceptibility testing *Zero grazing is an approach
to animal management in which families contain livestock in an
enclosed, shaded area and carry fodder and water to them instead
of letting them wander in the open where they are more likely to
catch diseases or damage the environment [http://www.heifer.
org.za/faq/what_is_zero_grazing].
(XLS)
Data Analysis
The data was analyzed with STATA SE software version 11.2
(STATA Corp LP, College station TX 77849, USA). A P-value of
,0.05 was considered statistically significant.
The gel images for RAPD genotyping data were analyzed with
the Bionumerix software (Applied Maths NV, Sint-MartensLatem, Belgium). The spa and MSLT sequences were analyzed
with the BioEdit software and submitted to online databases
[http://spa.ridom.de/] and [http://www.mlst.net/databases/
default.asp], respectively, to obtain lineages.
Table S2 Antimicrobial resistance patterns of each
staphylococcal isolate.
(PDF)
Table S3 Primers for genotyping daptomycin and
vancomycin resistant enterococci.
(PDF)
Acknowledgments
We are highly indebted to the farmers, veterinarians, animal husbandry
officers, and milkmen for participating in this study. We also thank Mr.
Edgar Kigozi, Medical and Molecular Laboratories, MakCHS, for
extracting chromosomal and plasmid DNA from the bacteria and for
performing plasmid profiling.
Supporting Information
Isolate profiles (bovine and human samples).
R, resistant; S, susceptible, with respect to: Staphylococci:
Ampicillin; Penicillin G; Trimethopprim-sulfamethoxazole; Tetracycline; Cefoxitine; Oxacillin; Amoxicillin-Clavulanate; Teicoplanin; Vancomycin; Clindamycin; Erythromycin; Nitrofurantoin;
Rifampicin Enterococci: Daptomycin; Teicoplanin; Vancomycin; Erythromycin; Ciprofloxacin; Tetracycline. Gram-negatives: Ampicillin; Amoxicillin-Clavulanate; Trimethopprim-sulfamethoxazole; Colistin; Imipenem; Cefoxitine; Cefotaxim;
Cephalothin; Cefuroxime; Cefepime; Aztreonam; Erythromycin;
Table S1
Author Contributions
Conceived and designed the experiments: DPK. Performed the experiments: DPK UK HB MO SK. Analyzed the data: DPK UK MO CFN
LN. Contributed reagents/materials/analysis tools: DPK MLJ. Wrote the
paper: DPK.
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