Prevalence and risk factors of coliform-associated mastitis and antibiotic resistance of coliforms from lactating dairy cows in North West Cameroon.

BACKGROUND: Coliform bacteria are major causative agents of bovine mastitis, a disease that has devastating effect on dairy animal health and milk production. This cross-sectional study, carried out in the North West region of Cameroon, sought to determine the prevalence of bovine mastitis, coliforms associated with bovine mastitis, risk factors for infection and the antibiotic resistance pattern of coliform bacterial isolates.
MATERIALS AND METHODS: A total of 1608 udder quarters were sampled from 411 cows using a questionnaire, clinical examination, California Mastitis Test and milk culture. Primary isolation of coliform bacteria was done on MacConkey agar while identification of coliforms employed Gram-staining and biochemical testing. Each coliform bacterial isolate was challenged with 11 antibiotics using the Kirby-Bauer disc diffusion method.
RESULTS: The prevalence of mastitis was 53.0% (218/411) and 33.1% (532/1608) at the cow- and quarter-levels respectively. Overall, 21.9% (90/411) cows and 8.2% (132/1608) udder quarters showed coliform mastitis. Escherichia coli was isolated in 7.0% of mastitis milk, and other coliforms isolated were Enterobacter cloacae (12.6%), Klebsiella pneumoniae (2.4%), Enterobacter sakazakii (1.1%), Klebsiella oxytoca (0.8%), Citrobacter freudii (0.4%), Serratia ficaria (0.4%) and Serratia liquefaciens (0.2%). Lactation stage, breed, history of mastitis and moist/muddy faeces contaminated environment were significantly associated (P-value < 0.05) with coliform mastitis. Coliform isolates (99.0%; 203/205) were resistant to at least one antibiotic tested. Amoxicillin had the highest resistance (88.8%) while norfloxacin had the least resistance (3.4%). Multidrug resistance was exhibited by 52.7% (108/205) of the isolates in a proportion of 27.8% Enterobacter cloacae, 10.7% E. coli, 6.3% Klebsiella pneumoniae, 2.9% Enterobacter sakazakii, 2.0% Klebsiella oxytoca, 1.0% Citrobacter freundii, 1.0% Serratia ficaria, 0.5% Serratia liquefaciens and 0.5% Serratia odorifera.
CONCLUSION: Results indicate a need to educate these dairy farmers about mastitis (particularly subclinical), proper hygiene methods in milking and the public health implications of consuming contaminated raw milk.

1. Introduction

Bovine mastitis, an inflammation of the cow’s mammary gland, is the most common and most costly dairy cow disease worldwide [1, 2]. It adversely affects the business of milk production with substantial financial losses [3, 4] stemming from decreased quantity and quality of milk produced [5].

The majority of mastitis cases are of bacterial origin, with coliform bacteria being among the few predominant causative pathogens [6, 7]. Coliforms frequently implicated are Escherichia coli (E. coli), Enterobacter species, Klebsiella species, Serratia species and Citrobacter species [8, 9] with E. coli responsible for more than 80.0% of coliform mastitis cases [7, 10].

Coliform bacteria inhabit the gastrointestinal tract of most animals. These microorganisms are ubiquitous in the cow’s environment and cannot be easily eradicated even in well-managed dairy herds [11]. They mostly infect the mammary gland primarily through the exposure of the teat end to moisture, mud and faecal material, and exposure may repeatedly occur [6]. Coliform bacteria do not have a predilection for the mammary gland, thus are opportunistic pathogens. After infection with coliform bacteria, manifestations can be clinical, subclinical, or the bacteria may remain dormant. The severity of clinical signs ranges from a mild or moderate type of disease predominantly localized around the udder, to a more severe systemic illness, due to bacteraemia and septicaemia which may be fatal [8, 12]. About 60.0–70.0% of systemic clinical signs are associated with coliform infections [8]. Chronic coliform infections also occur and may be subclinical but typically elicit recurrent clinical episodes [13].

Most coliform infections are self-limiting, but if not cleared by the cow’s immune system, they may lead to unlimited growth of the coliform bacteria in the udder, making the use of antibiotic therapy, one of the essential methods to control the infection from advancing to complications [14, 15]. However, the report of antibiotic resistance of mastitis coliform bacterial isolates is a cause for concern [10, 16–18]. The frequent treatment failure, increased severity of the disease, and the ability of the bacteria to spread and transfer resistant phenotype to human populations via unpasteurized milk is a growing concern [19]. Monitoring antibiotic resistance in coliform bacteria is, therefore, important for public health reasons.

In Cameroon, cattle constitute the primary source of fresh milk, with 17.9% of households operating on dairy farming [20]. Similar to other sub-Saharan countries, milk production in Cameroon is dominated mostly by small-scale farmers [21] who get their income and nutrition from milk and milk products. Thus, dairying contributes significantly to alleviating poverty and reducing malnutrition, particularly in rural and peri-urban areas and serves as a source of income for the small-scale farmers who are primarily women [22]. Therefore, coliform mastitis can have huge economic impact on these small-scale famers who may not withstand the enormous financial losses associated with the disease. Moreover, apart from economic consequences associated with mastitis, there are also public health implications that have been linked to the handling and consumption of unpasteurized coliform-contaminated milk and/or milk products by humans [8, 23]. Milk could be contaminated by the Shiga toxigenic E. coli strain, which is risky due to their great zoonotic importance [24].

So far, there is limited information about coliform bacteria in bovine mastitis cases in the North West Region of Cameroon. To the best of our knowledge, the prevalence, risk factors and antibiotic resistance profile of coliform bacteria associated with bovine mastitis cases have not been investigated in our study area. Therefore, this study sought to determine the overall prevalence of bovine mastitis, isolate, identify and establish the prevalence of coliforms associated with bovine mastitis, particularly E. coli, elucidate risk factors for infection and determine antibiotic resistance pattern of coliform isolates from cow milk. The results of this study will provide important epidemiological data on bovine coliform-associated mastitis.

2. Materials and methods

2.1 Ethical considerations

The use of animals in the study was approved by the North West Regional Delegation of Livestock, Fisheries and Animal Industries. The objectives of this study were explained orally to dairy farmers, and their consent was obtained to participate in the study. Questionnaire responses about the dairy cows were given voluntarily, and farmers were permitted to withdraw their consent at any time.

2.2 Study area

The study was carried out in all seven administrative divisions in the North West region of Cameroon (Fig 1). These administrative divisions are: Boyo, Bui, Donga-Mantung, Menchum, Mezam, Momo and Ngoketunjia. The North West region is situated in the western highlands of Cameroon. It occupies a surface area of about 17, 300km2 between latitudes 5° 20’ and 7° 15’ N and longitudes 9° 30’ and 11° 15’ E at an altitude of 300 to 3000m above sea level. The region is characterized by two distinct seasons: a dry season from mid-October to mid-March and a rainy season from mid-March to mid-October, an average rainfall of about 2400mm and a daily average temperature of 23°C with a range between 15°C and 32°C [25]. Its agro-climatic conditions favour cattle rearing, thereby making the region one of the most important cattle production areas in Cameroon and one of Cameroon’s most favourable milk production areas [26].

Fig 1

Map of the study area showing sample collection sites.

2.3 Study design and population

A cross-sectional study was carried out to address the objectives of this study. Cattle herds were selected randomly based on accessibility to the farm and farmers’ willingness to participate. Data were collected from all lactating cows in each selected herd except those cows that had received antibiotics within the past 15 days.

The study included lactating cows used for dairy purposes (including cows raised for both milk and beef). The cows were reared under three husbandry systems; the extensive system where cows are left to wander and graze during the day but are enclosed at night, the semi-intensive system where cows graze freely on pasture but receive supplementary feeds, particularly during milking and are enclosed at night and the intensive system where cows remain confined and are catered for.

The cow breeds included in the study were: local African breeds consisting of Gudali, red Fulani, white Fulani and Boran breeds; pure exotic breeds consisting only Holstein-Friesian; and cross-breeds consisting of exotic (Holstein, Jersey, Simmental, Charolais and Brahman) and local species.

2.4 Determination of sample size

In the absence of documented evidence of a similar study in this area, the sample size was calculated from the formula (n = Z2 * P(1-P)/d2) recommended by Thrusfield [27], with a 95% confidence interval, at 5% desired absolute precision and expected prevalence of 50%. Where n = sample size, Z = α value of 95% confidence interval = 1.96, P = expected prevalence in population-based on previous studies, d = desired absolute error or precision = 5%. Hence, the expected minimum number of lactating dairy cows to be included in the study was 384.

2.5 Data collection

Data were collected from March to November 2019. Basic information on cow and herd management practices was collected using a semi-structured pretested questionnaire. Specifically, data captured in the questionnaire included cow information (such as age, breed, parity, stage of lactation and previous history of mastitis), herd size, husbandry system, method of milking, number of times cow was milked per day, cleanliness of cow environment and floor type. A trained veterinarian examined each cow for signs of clinical mastitis, which included evidence of pain, swelling, and milk changes such as the presence of clots, change of colour and consistency of milk, fever and body weakness [9]. The blindness of teats was also recorded.

2.6 California mastitis test

The California Mastitis Test (CMT) (ImmuCell® CMT, Portland) was performed for each quarter milk sample as a screening test for inflammation, particularly subclinical mastitis, as previously described [28, 29]. Briefly, two millilitres of quarter fore-milk and an equal amount of CMT reagent were collected into corresponding shallow wells of the CMT plastic paddle. A gentle circular motion was applied to the mixture in a horizontal plane for 15 seconds, and the result was scored as N (negative), T (trace), 1 (weakly positive), 2 (distinct positive) and 3 (strongly positive) based on thickening or gel formation. A quarter was positive if it had a score of ≥ 1, and a cow was considered positive when at least one quarter had a positive CMT score.

2.7 Milk sample collection for bacteriology

Quarter milk was collected aseptically from the teat into a sterile tube according to the procedure described by National Mastitis Council (NMC) [30]. Milk collection was done early in the morning before milking. The veterinarian’s hands were washed, disinfected and a new pair of gloves was worn before milk collection from each cow. The cow’s udder, especially teat, was thoroughly washed with clean running water and dried with disposable paper towels. Later, the teats were disinfected with cotton wool soaked in 70% ethanol and allowed to dry. A ball of separate cotton wool was used for each teat. The first few streams of milk were discarded, and approximately 10mL of milk were directly stripped from teats into pre-labelled screw-capped sterile plastic tubes. The samples were transported at +4°C in a cool box and stored at -20°C in the Laboratory for Emerging Infectious Diseases, University of Buea, for analysis.

2.7.1 Isolation and identification of coliforms

Each quarter milk sample was cultured for bacterial isolation using standard procedures [30]. An aliquot of 10μl of milk sample was inoculated by streaking on MacConkey agar (Liofilchem Diagnostic, Italy). Each frozen milk sample was thawed only once at room temperature and mixed vigorously. Unless otherwise stated, all incubations were done at 37°C for 24–48h. Colonial morphology, lactose fermentation and Gram reaction aided identification. Gram-negative bacteria were subcultured on eosin methylene blue for presumptive identification of E. coli. Presumptive colonies were subcultured on nutrient agar to get pure cultures. All Gram-negative bacterial isolates were subjected to the Analytical Profile index for Enterobacteriaceae (API 20E) (Biomerieux, 2002, France) testing following manufacturer’s instructions supported with oxidase testing using oxidase strips (Sigma, Switzerland) for confirmatory identification of coliforms.

2.7.2 Antibiotic susceptibility testing by disc diffusion technique

In vitro antibiotic susceptibility testing was done by Kirby-Bauer disc diffusion method on Mueller-Hinton agar (Liofilchem Diagnostic Srl, Italy) according to the Clinical and Laboratory Standards Institute [31]. Each of the coliform bacterial isolates was challenged with commonly used antibiotics in animal and human health. A panel of 11 antibiotics (Oxoid, England) was used and included: beta-lactams [ampicillin (10μg), amoxicillin (10μg), cephalothin (30μg)]; aminoglycosides [gentamicin (10μg), streptomycin (10μg)]; tetracyclines [tetracycline (30μg)]; quinolones [norfloxacin (5μg), nalidixic acid (30μg)]; folate inhibitors [cotrimoxazole (25μg)]; phenicols [chloramphenicol (10μg)] and macrolides [erythromycin (30μg)]. The turbidity of each bacterial inoculum was adjusted to that of 0.5 McFarland standard and then inoculated on Mueller-Hinton agar. The antibiotic discs were applied gently on the agar using sterile forceps, and plates were incubated at 35°C for 18h. Growth inhibition diameter was measured in millimetres and the data were interpreted using Clinical and Laboratory Standards Institute criteria [31] to classify isolates as susceptible, intermediate or resistant (Table 1). Isolates that were resistant to at least one antibiotic in three or more classes were termed multidrug-resistant [32].

Table 1

Antibiotic susceptibility test interpretative criteria and cut-off values for coliforms [31].

Antibiotic	Symbol	Disc potency	Interpretative categories and zone diameter breakpoints
			Susceptible	Intermediate	Resistant
Amoxicillin	AMX	10μg	≥21	14–20	≤13
Ampicillin	AMP	10μg	≥17	14–16	≤13
Cephalothin	KF	30μg	≥18	15–17	≤14
Gentamicin	CN	10μg	≥15	13–14	≤12
Streptomycin	STR	10μg	≥15	12–14	≤11
Tetracycline	TET	30μg	≥19	15–18	≤14
Nalidixic acid	NAL	30μg	≥19	14–18	≤13
Norfloxacin	NOR	5μg	≥17	13–16	≤12
Cotrimoxazole	COT	1.25/23.75 μg, (25μg)	≥16	11–15	≤10
Chloramphenicol	CHL	10μg	≥18	13–17	≤12
Erythromycin	ERY	30μg	≥23	14–22	≤13

2.8 Data analysis

The data generated from the field, laboratory analyses and questionnaire survey were entered into Microsoft Excel spreadsheets 2010 and was analyzed using MINITAB version 17 statistical package. Prevalence was calculated as a percentage value of the proportion of positive cases against total number sampled. The association between the explanatory/dependent and response/independent variables was analyzed using the chi-square test. Multivariate logistic regression analysis was employed to analyze the relative strength of the different risk factors on coliform-associated mastitis. The independent variables included in the model were those that showed statistical significance (P < 0.05) in the initial chi-square test. The model was assessed for goodness-of-fit using the Hosmer-Lemeshow test. Statistical differences were considered significant when P < 0.05.

3. Results

3.1 Characteristics of herds and cow population sampled

A total of 123 herds with a mean herd size of 13 (range: 1–102 cattle) were sampled, comprising 47.2% (58/123) that practised intensive farming, 5.7% (7/123) that practiced semi-intensive farming and 47.2% (58/123) that practised extensive farming. Milking was done manually in all herds, twice a day in 65 (52.8%) and once a day in the remaining 58 (47.2%). In 52.8% (65/123) of the herds (which included cows raised under the intensive and semi-intensive farming system), the udder was washed with clean running water before milking while 47.2% (58/123) of the herds (which included cows raised under the extensive farming system) did not clean the udder before milking. Gloves were not worn before milking in any of the herds and the teats were neither dipped into any disinfectant nor cleaned after milking. Four hundred and eleven lactating cows with a mean age of 6.4 years (age range 2–15 years), mean lactation number of 3 calves (range 1–11 calves) and mean lactation length of 5.4 months (range 1–12 months) were sampled, and a majority (22.4%) was local breeds

3.2 Clinical examination and CMT results

Upon examining the quarters of the 411 cows, it was observed that 36 out of 1644 quarters (2.2%) were nonfunctional (blind), and 5.8% of the 1608 functional quarters (within 33 cows) displayed clinical signs characterized by watery milk or clots, flakes or blood and swelling or sore udder/teat, with only two of these cows presenting additional signs of fever, depression and lack of appetite.

The remaining 1512 functional quarters without clinical signs had varied CMT scores (Fig 2). All the 96 quarters with evidence of clinical mastitis were positive by CMT.

Fig 2

Clinical examination and CMT results for functional quarters.

3.3 Prevalence of mastitis and coliform-associated mastitis

Table 2 depicts the prevalence of mastitis and coliform-associated mastitis in North West Cameroon. Out of the 411 cows examined, 53.0% (218/411) cows and 33.1% (532/1608) quarters had evidence of mastitis. The prevalence of subclinical mastitis was significantly higher compared to clinical mastitis (P = 0.000). The prevalence of coliform-associated mastitis at cow-level was 21.9% (90/411) and 8.2% (132/1608) at quarter-level. The proportion of coliform bacteria associated with mastitis at cow-level was higher (45.45%, 15/33) in clinical mastitis cases compared to subclinical cases (40.5%; 75/185). Conversely, the proportion at the quarter-level was higher (26.0%; 114/436) in subclinical cases than in clinical cases (18.8%; 18/96). However, the difference at both levels was not statistically significant. Among the herds visited, 78.9% (97/123) had at least a cow positive for mastitis.

Table 2

Prevalence of mastitis and coliform-associated mastitis in North West Cameroon.

Level	Number examined	Number positive for mastitis (%)		Total (%)	Number positive for coliform-associated mastitis (%)		Total (%)
		Clinical	Subclinical		Clinical	Subclinical
Cow	411	33 (8.0)*	185 (45.0)*	218 (53.0) a	15 (3.7)	75 (18.2)	90 (21.9) b
Quarter	1608	96 (6.0)#	436 (27.1)#	532 (33.1) c	18 (1.1)	114 (7.1)	132 (8.2) d

95% confidence interval: 53.0 ± 4.8%

95% confidence interval: 33.1 ± 2.3%

95% confidence interval: 21.9 ± 4.0%

95% confidence interval: 8.2 ± 1.3%

*(X2 = 144.233, P = 0.000)

(X2 = 260.363, P = 0.000

3.4 Occurrence of coliform bacteria in milk

The occurrence of coliform bacteria in all 1608 quarter milk samples was 12.8% (205/1608), and in mastitis samples, the occurrence was 24.8% (132/532). Even though coliform bacteria were isolated from non-mastitis milk samples, their isolation from mastitis samples was significantly (P = 0.000) higher at cow-level (41.3%; 90/218) and quarter-level (24.6%; 132/532) compared with non-mastitis samples (Table 3). The most frequently isolated coliform bacteria in mastitis quarters were Enterobacter cloacae (12.6%) and Escherichia coli (7.0%) (Table 4).

Table 3

Coliform bacterial isolation in mastitis and non-mastitis cows.

Clinical state	Cow-level			Quarter-level
	Number examined	Number of coliform positive (%)	P-value	Number examined	Number of coliform positive (%)	P-value
Mastitis	218	90 (41.3)	0.000	532	132 (24.8)	0.000
No mastitis	193	38 (19.7)		1076	73 (6.8)
Total	411	128 (31.1)		1608	205 (12.8)

Table 4

Occurrence of coliform bacterial isolates in quarter milk samples.

Mastitis state	Number of milk samples cultured	Number of coliform bacterial—culture positive quarter milk samples (%)									Total number of coliform positive quarters (%)
		Citrobacter freundii	Enterobacter cloacae	Enterobacter sakazakii	Escherichia coli	Klebsiella oxytoca	Klebsiella pneumoniae	Serratia ficaria	Serratia liquefaciens	Serratia odorifera
Clinical	96	0 (0.0)	7 (7.3)	1 (1.0)	8 (8.3)	1 (1.0)	1 (1.0)	0 (0.0)	0 (0.0)	0 (0.0)	18 (18.8)
Subclinical	436	2 (0.5)	60 (13.8)	5 (1.2)	29 (6.7)	3 (0.7)	12 (2.8)	2 (0.5)	1 (0.2)	0 (0.0)	114 (26.1)
Total mastitis	532	2 (0.4)	67 (12.6)	6 (1.1)	37 (7.0)	4 (0.8)	13 (2.4)	2 (0.4)	1 (0.2)	0 (0.0)	132 (24.8)
No mastitis	1076	0 (0.0)	60 (5.6)	4 (0.4)	0 (0.0)	6 (0.5)	2 (0.2)	0 (0.0)	0 (0.0)	1 (0.1)	73 (6.8)
Total	1608	2 (0.1)	127 (7.9)	10 (0.6)	37 (2.3)	10 (0.6)	15 (0.9)	2 (0.1)	1 (0.1)	1 (0.1)	205 (12.8)

Among the 90 coliform-associated mastitis cows, a majority (62.2%, 56/90) had only one of its quarters infected, while only 1.1% (1/90) cow had all four quarters infected (Fig 3). Among the 27 coliform-associated mastitis cows with two quarters infected, 23 of the cows had all two quarters infected with the same coliform bacteria while the remaining 4 cows had different coliform bacteria. For the 6 coliform-associated mastitis cows that had three quarters infected, 4 cows had all three quarters infected with the same coliform bacteria while the remaining 2 had two different coliforms. In the one coliform-associated mastitis cow, the same coliform bacteria infected all four of its quarters.

Fig 3

Number of quarters infected for coliform-associated mastitis cows.

3.5 Coliform-associated mastitis risk factors

Among the risk factors investigated in this study, chi-square analysis revealed that the cow-level prevalence of coliform mastitis was significantly (P < 0.05) associated with lactation stage, breed, history of mastitis and moist/muddy faeces contaminated environment, as shown in Table 5. Multivariate logistic regression analysis of risk factors significantly associated with coliform mastitis prevalence revealed that all the variables entered remained significant predictors of coliform mastitis (P < 0.05) (Table 6). The Hosmer-Lemeshow Goodness-of-Fit test suggested that the model fitted the data (X 2 = 9.44, P = 0.150).

Table 5

Risk factors associated with the prevalence of coliform mastitis.

Risk factor	Category	Number of cows examined	Number of positive cows	Coliform mastitis Prevalence (%)	Chi square	P-value
Age (years)	2–5	161	31	19.25	1.639	0.441
	> 5–9	203	46	22.66
	> 9	47	13	27.66
Parity	1	73	10	13.70	5.620	0.060
	2	124	24	19.35
	≥ 3	214	56	26.17
Lactation stage	Early (≤ 2 months)	112	39	34.82
	Mid (3–6 months)	110	13	11.82	17.827	0.000*
	Late (> 6 months)	189	38	20.11
Breed	Local	250	41	16.40	11.872	0.003*
	Pure exotic (Holstein)	92	26	28.26
	Exotic and local cross	69	23	33.33
History of mastitis	No	356	70	19.66	7.769	0.005*
	Yes	55	20	35.36
Husbandry system	Intensive	79	19	24.05	0.677	0.713
	Semi-intensive	35	6	17.14
	Extensive	297	65	21.89
Moist/muddy faeces contaminated environment	No	62	5	8.06	8.169	0.004*
	Yes	349	85	24.36
Floor type	Concrete	45	11	24.44	0.192	0.662
	Earth	366	79	21.58
Herd size	<5 cattle	74	19	25.7	0.809	0.667
	5–10 cattle	11	2	18.2
	>10 cattle	326	69	21.2

*Statistically significant variables (P < 0.05)

Table 6

Multivariate analysis of risk factors associated with coliform mastitis.

Risk factor	Category	Number of positive cows (%)	COR (95% CI)	AOR (95% CI)	P-value
Lactation stage	Early (≤ 2 months)	39 (34.82)	3.9863 (1.9849, 8.0057)	4.3289 (2.1004, 8.9220)	0.000*
	Mid (3–6 months)	13 (11.82)	1	1
	Late (> 6 months)	38 (20.11)	1.8903 (0.9581, 3.7295)	2.3747 (1.1787, 4.7845)
Breed	Local	41 (16.40)	1	1	0.022*
	Holstein–Friesian	26 (28.26)	2.0081 (1.1426, 3.5295)	1.2766 (0.6748, 2.4152)
	Exotic–local cross	23 (33.33)	2.5488 (1.3958, 4.6543)	2.5050 (1.3190, 4.7573)
History of mastitis	No	70 (19.66)	1	1	0.044*
	Yes	20 (35.36)	2.3347 (1.2706, 4.2899)	2.0493 (1.0289, 4.0819)
Moist / muddy faeces contaminated environment	No	5 (8.06)	1	1	0.000*
	Yes	85 (24.36)	4.8280 (1.8866, 12.3553)	5.8657 (2.2152, 15.5317)

COR, Crude odds ratio; AOR, Adjusted odds ratio; 1, Reference

*, Significant variables (P < 0.05)

3.6 Antibiotic resistance of coliform bacterial isolates

Out of the 205 coliform isolates, 203 (99.0%) exhibited resistance to at least one antibiotic tested, with 10 (4.9%) of the isolates exhibiting resistance to eight antibiotics. The highest resistance was against amoxicillin (88.8%), followed by cephalothin (75.1%) and erythromycin (61.0%), whereas the least resistance was observed with norfloxacin (3.4%), followed by gentamicin (10.2%), nalidixic acid (13.8%) and cotrimoxazole (19.0%) (Table 7).

Table 7

Coliform bacterial isolates resistant to each antibiotic tested.

Antibiotic disc (potency)			Number of resistant isolates (%)
	Citrobacter freudii (n = 2)	Enterobacter cloacae (n = 127)	Enterobacter sakazakii (n = 10)	Escherichiacoli (n = 37)	Klebsiella pneumoniae (n = 15)	Klebsiella oxytoca (n = 10)	Serratia ficaria (n = 2)	Serratia liquefaciens (n = 1)	Serratia odorifera (n = 1)	Total (n = 205)
KF (30μg)	1 (50.0)	107 (84.3)	10 (100.0)	27 (73.0)	4 (26.7)	1 (10.0)	2 (100.0)	1 (100.0)	1 (100.0)	154 (75.1)
AMX (10μg)	2 (100.0)	116 (91.3)	10 (100.0)	26 (70.3)	15 (100.0)	9 (90.0)	2 (100.0)	1(100.0)	1(100.0)	182 (88.8)
AMP (10μg)	2 (100.0)	66 (52.0)	4 (40.0)	19 (51.4)	14 (93.3)	8 (80.0)	2 (100.0)	1 (100.0)	1 (100.0)	117 (57.1)
NOR (5 μg)	0 (0.0)	2 (1.6)	1 (10.0)	4 (10.8)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	0 (0.0)	7 (3.4)
NAL (30μg)	0 (0.0)	19 (15.0)	1 (10.0)	7 (18.9)	0 (0.0)	1 (10.0)	0 (0.0)	0 (0.0)	0 (0.0)	28 (13.8)
GEN (10μg)	0 (0.0)	18 (14.2)	0 (0.0)	1 (2.7)	0 (0.0)	0 (0.0)	1 (50.0)	1 (100.0)	0 (100.0)	21 (10.2)
STR (10μg)	0 (0.0)	29 (22.8)	0 (0.0)	18 (48.6)	11 (73.3)	1 (10.0)	1 (50.0)	0 (0.0)	1 (100.0)	61 (29.8)
TET (30μg)	2 (100.0)	27 (21.3)	3 (30.0)	12 (32.4)	7 (46.7)	0 (0.0)	2 (100.0)	1 (100.0)	1 (100.0)	55 (26.8)
CHL (10μg)	1 (50.0)	47 (37.0)	4 (40.0)	7 (18.9)	4 (26.7)	0 (0.0)	2 (100.0)	0 (0.0)	0 (0.0)	65 (31.7)
ERY (30μg)	1 (50.0)	65 (51.2)	5 (50.0)	25 (67.6)	15 (100.0)	10 (10.0)	2 (100.0)	1 (100.0)	1 (100.0)	125 (61.0)
COT (25μg)	0 (0.0)	16 (12.6)	0 (0.0)	13 (35.1)	5 (33.3)	1 (10.0)	2 (100.0)	1 (100.0)	1 (100.0)	39 (19.0)

KF, Cephalothin,; AMX, Amoxicillin; AMP, Ampicillin; NOR, Norfloxacin; NAL, Nalidixic acid; GEN, Gentamicin; STR, Streptomycin; TET, Tetracycline; CHL, Chloramphenicol; ERY, Erythromycin; COT, Cotrimoxazole; μg, micrograms

Among the coliform isolates tested, 15.1% (31/205) and 31.2% (64/205) were resistant to one and two antibiotic classes, respectively. Resistance to three or more classes, multidrug resistance (MDR), was exhibited by 52.7% (108/205) isolates (in the proportion of: 27.8% (57/205) Enterobacter cloacae, 10.7% (22/205) E. coli, 6.3% (13/205) Klebsiella pneumoniae, 2.9% (6/205) Enterobacter sakazakii, 2.0% (4/205) Klebsiella oxytoca, 1.0% (2/205) Citrobacter freundii, 1.0% (2/205) Serratia ficaria, 0.5% (1/205) Serratia liquefaciens and 0.5% (1/205) Serratia odorifera) (Fig 4). All isolates (100.0%) of Citrobacter freundii and Serratia spp., 68.0% Klebsiella spp., 59.5% E. coli and 46.0% Enterobacter spp. were MDR.

Fig 4

Number of resistant coliform bacterial isolates against number of antibiotic classes.

4. Discussion

This is the first report of the presence and prevalence of bovine mastitis (a worldwide dairy animal disease associated with significant economic losses) in the North West region of Cameroon. The California mastitis test (CMT) is an on-farm screening test in which the degree of gel formation is scored to estimate somatic cell count in milk samples and thus detect mastitis cases. The CMT is most helpful in detecting subclinical mastitis but serves little use in detecting clinical mastitis, although accurate [33]. This is because the presence of clinical signs of mastitis establishes a diagnosis of clinical mastitis. Bacterial culture remains the gold standard for confirming mastitis caused by microorganisms [34].

According to this cross-sectional study, the overall prevalence of mastitis among lactating dairy cows as determined by CMT and clinical examination was 53.0%. This finding is relatively lower compared to reports in other parts of the country [35] and elsewhere in Africa [36, 37]. However, our finding is relatively higher compared with reports from other countries in Africa [38-40] and out of Africa [41]. This variability in the prevalence of mastitis in different reports could suggest the complexity of the disease. According to Radostits et al. [9], the prevalence of bovine mastitis is expected to vary from place to place with the interaction of several factors, including herd management, environment and cow-related factors.

As expected, the prevalence of subclinical mastitis was higher (45.0%) than that of clinical mastitis (8.0%). Similar reports of subclinical mastitis dominance over clinical mastitis have been reported in several studies [35, 37, 40]. According to Sori et al. [23], subclinical mastitis was higher than clinical mastitis because, in most cases of infection, the cow mounts defence mechanisms in the udder, which reduces the severity of the disease. Another reason is the unawareness of farmers about subclinical cases of mastitis since symptoms are not evident [42], such that it is not diagnosed early and treated. Hence, it is important to educate farmers particularly about subclinical mastitis.

The prevalence of coliform-associated mastitis among cows in this study was 21.9% and this was higher than 8.8% in Nigeria [43] and 7.2% in Rwanda [44]. The high prevalence may indicate contamination from soil and faecal matter. Coliform bacteria originate from the cow’s environment, such as faecal material, contaminated bedding, water and cow body sites [8]. They are generally acquired from the environment between milking through the teat canal into the udder when teat-ends contact an environmental site contaminated with coliform organisms [45]. Therefore, improving hygiene and reducing exposure of teat ends to environmental contamination is of paramount importance.

In this study, coliform bacteria were isolated in 12.8% of all quarter milk samples, which is relatively lower compared with 14.4% reported by Byarugaba et al. [46] in Uganda. The occurrence of coliform bacteria in all mastitis quarters was 24.8%, and this was lower compared to 66.0% in Tanzania [22] as well as 31.9% in Jordan [47]. The coliform bacteria isolated from mastitis milk samples were Enterobacter cloacae (12.6%), Escherichia coli (7.0%), Klebsiella pneumoniae (2.4%), Enterobacter sakazakii (1.1%), Klebsiella oxytoca (0.8%), Citrobacter freudii (0.4%), Serratia ficaria (0.4%), Serratia liquefaciens (0.2%), and this corroborates Hogan and Smith [8], who reported that these genera of coliform bacteria were frequently isolated from bovine mastitis cases. In their studies, Ahmed and Shimamoto [48] in Egypt and Kateete et al. [18] in Kampala-Uganda isolated these coliform genera, with Escherichia coli being the predominant coliform. Ngwa et al. [35] in Adamawa—Cameroon, reported E. coli as the predominant among coliforms associated with mastitis,. Makolo et al. [43] and Mbuk et al. [49] in Nigeria reported Klebsiella spp. as the predominant coliform. The differences in the relative occurrence of coliform bacteria could be due to differences in bacterial load of the various coliforms in the various environmental sources.

Mastitis is a complex disease influenced by several factors [9] and identification of risk factors in an area is important for the design of control programs [50]. Among the risk factors assessed, this study revealed a significant association (P < 0.05) of coliform mastitis prevalence with lactation stage, breed, clinical history and moist/muddy faeces contaminated environment.

The association of coliform mastitis with lactation stage was reported in previous studies [51, 52]. Cows in early lactation were four times more likely to have coliform mastitis than cows in mid-lactation. This could be due to impaired immune function in early lactation related to the stress of producing a high amount of milk [53]. It could also be due to the absence of a dry cow therapy regime. During the dry period, pathogens penetrate the teat canal from the cow environment, multiply, and this can be carried over to the post parturient period and ultimately cause mastitis [54].

The present finding of an association of coliform mastitis with a history of mastitis was in harmony with other reports [55-57]. Cows with a history of mastitis were two times more likely to have coliform mastitis than those with no history. The current result may imply that the treatment of cows for mastitis may not be effective in eradicating the pathogens [57]. It could also be due to repeated challenges of the mammary tissues with coliforms coupled with other stress factors resulting in more significant risks of re-infection from the environment [54].

As revealed in this study, Oliveira et al. [57] and Taponen et al. [58] also reported that breed was a significant risk factor of coliform mastitis. Exotic-local cross breeds and Holstein-Friesian breeds were 2.5 times and 1.3 times likely to have coliform mastitis than the local African breeds. The occurrence of mastitis is generally higher in high milk-yielding cows than low-yielding cows [50, 59] because the high-yielding cows may be associated with a looser teat canal and milk leaking tendency, which predispose the udder to coliform bacterial invasion via the teat opening [57].

Moisture, mud and manure present in the environment of the animals are primary sources of exposure for environmental mastitis pathogens [21], which agreed with this study as cows in moist/muddy faeces contaminated environments were 5.9 times more likely to have coliform mastitis than those in a clean environment.

Although this study did not show age and parity as significantly associated with coliform mastitis in cows, Hogan and Smith [8] reported increase susceptibility to coliform mastitis with an increase in age and parity. It was observed that farmers in this study did not keep records, so data on age and parity may not be accurate, particularly in cases where cows were brought from elsewhere into a herd.

In vitro, antibiotic susceptibility testing was performed on coliform isolates from both mastitis positive and negative quarters. Coliform bacterial isolates (88.8%) exhibited resistance against amoxicillin, although Mbuk et al. [49] reported no resistance. In our study, 75.1% and 57.1% of coliform isolates were resistant to cephalothin and ampicillin, respectively; Kateete et al. [18] reported a lower resistance of 33.0% for cephalothin and higher resistance of 71.0% for ampicillin in Uganda. Worldwide, the β-lactam class of antibiotics is the most commonly used in human and veterinary medicine [60, 61]. This may explain the reports of high resistance of coliform bacteria against them. This study found that 61.0% isolates were resistant to erythromycin; Mbuk et al. [49] and Makolo et al. [43] had more than 75.0% resistance. The use of chloramphenicol in food-producing animals in many countries has been prohibited to avoid the danger of resistance in human medicine [62]. Resistance against chloramphenicol was exhibited by 31.7% of coliform isolates in this study similar to studies elsewhere [43, 49].

This study revealed that norfloxacin (3.4%), gentamicin (10.2%), nalidixic acid (13.8%) and cotrimoxazole (19.0%) were the antibiotics with the least resistance by coliforms. Similar results of low resistance of coliform isolates to these antibiotics have been reported elsewhere [43, 49, 63]. The least resistance by norfloxacin in our study agrees with Wenz et al. [64] who recommended fluoroquinolones as the drugs of choice for mastitis caused by Gram-negative rods.

Generally, although coliform bacteria exhibited low resistance to some of the antibiotics tested and could be recommended as a drug of choice for coliform-associated mastitis, the pattern of resistance differed with specific coliform genera. According to World Health Organization reports [65], the resistance of Escherichia coli to fluoroquinolones is pervasive in a range of 0.0–98.0% in Africa. For example, in this study, resistance against nalidixic acid and norfloxacin was 18.9% and 10.8% for E. coli strains, respectively (Table 7). Resistance to gentamicin and cotrimoxazole was exhibited by 50.0% and 100.0% of Serratia spp. respectively. Thus, it is important to isolate the coliform mastitis pathogen and perform an antibiotic susceptibility test, if possible, before any antibiotic therapy.

Though the choice of antibiotic for mastitis treatment depended on the veterinarian in the study area, it was observed that the two most commonly used drugs for mastitis treatment were penicillin-streptomycin and tetracyclines. This study revealed that Klebsiella pneumoniae (46.7%), E. coli (32.4%), Enterobacter sakazakii (30.0%) and Enterobacter cloacae (21.3%) isolates were resistant to tetracycline. Mbuk et al. [49] reported more than 50.0% resistance to tetracycline by Klebsiella spp. and Enterobacter spp.. Makolo et al. [43] reported 100.0% resistance of E. coli isolates to tetracycline. Resistance to streptomycin was exhibited by 73.3% Klebsiella pneumoniae, 48.6% E. coli, 22.8% Enterobacter cloacae and 10.0% Klebsiella oxytoca. Resistance of coliforms to streptomycin conforms to the report by Makolo et al. [43], who revealed 100.0% resistance exhibited by Klebsiella pneumoniae and E. coli isolates each. Thus, it is not advisable to use these antibiotics to treat coliform-associated bovine mastitis without performing an antibiotic susceptibility test.

Analysis of MDR among coliform bacterial isolates revealed 52.7% (108/205) in the proportion of Enterobacter spp. (63 isolates, 30.7%), E. coli (22 isolates, 10.7%), Klebsiella spp. (17 isolates, 8.3%), Serratia spp. (4 isolates, 2.0%) and Citrobacter freundii (2 isolates, 1.0%). Similarly, Ahmed and Shimamoto [48] in Egypt reported 27.8% MDR among coliforms in the proportion of Klebsiella spp. (14 isolates, 12.6%), Enterobacter spp. (8 isolates, 7.1%), E. coli (5 isolates, 4.5%), Citrobacter freundii (3 isolates, 2.7%) and Serratia sp. (1 isolate, 0.9%). The presence of MDR coliform bacteria in milk is a serious cause for concern, particularly isolates exhibiting resistance to as many as seven classes of antibiotics like the two Enterobacter cloacae isolates in this study. The great differences observed in the antibiotic resistance of coliform bacteria from different studies indicate the importance of antibiotic susceptibility tests and periodic surveillance of the antibiotic susceptibilities associated with mastitis.

5. Conclusion

Based on the data obtained in this study area, the prevalence of mastitis among lactating cows was 53.0%, comprising 45.0% (185/411) subclinical and 8.0% (33/411) clinical cases. The prevalence of coliform-associated mastitis at the cow-level was 21.9% (90/411). Coliform bacteria were isolated from 12.8% (205/1608) of all quarter milk samples while 24.8% (132/532) of all mastitis quarters were positive for coliforms: Enterobacter cloacae (12.6%), Escherichia coli (7.0%), Klebsiella pneumoniae (2.4%), Enterobacter sakazakii (1.1%), Klebsiella oxytoca (0.8%), Citrobacter freundii (0.4%), Serratia ficaria (0.4%) and Serratia liquefaciens (0.2%). Prevalence of coliform mastitis was significantly (P < 0.05) associated with lactation stage, cow breed, history of mastitis and moist/muddy faeces contaminated environment. Amoxicillin had the least activity against coliform bacteria, and norfloxacin was the most active antibiotic. MDR was observed in 52.7% (108/205) of the coliform isolates.

Cross-sectional studies like this can only give associations but not causality, so an experimental study in this area will elucidate these associations as actual causes of coliform mastitis. However, the presence of mastitis, particularly coliform-associated mastitis, warrants the application of good hygiene practices in the milking process and the cow’s environment. Raising awareness of mastitis (especially subclinical mastitis) and the non-misuse of antibiotics among farmers through extension services and restricting consumption of unpasteurized milk is vital in improving cow and public health. Antibiotic resistance monitoring should also be implemented.

9 Dec 2021

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Reviewer #1: Review of:

Prevalence, Risk Factors and Antibiotic Resistance of Coliforms from Lactating Dairy Mastitis Cows in North West Cameroon

Comments:

L 1 The title of this manuscript is misleading. Clinical and subclinical infections of the mammary glands differ; whereas milk from cows with clinical mastitis is not approved for sale or consumption, but cows with subclinical mastitis may have their milk sold or consumed, depending on the Somatic Cell Count, which differs among countries. For example, in the EU, the SCC of milk for sale cannot exceed 400,000 over a 3-month period in herds that must be sampled at least once monthly. A more accurate title would simply delete the word “Mastitis” and discuss it in appropriate context in the manuscript.

L30 California Mastitis Test instead of California mastitis Test

L35 It is important here to define what is meant by the term “mastitis”. In this study, cows with active coliform organisms in their milk were classified as having mastitis, regardless of their SCC. Some of these cows would have met the European standards for salable milk. This needs to be clarified.

L37 It would be more correct to say that these cows had Coliform infections, rather than saying Coliform mastitis.

L81-82 This statement is incorrect. If only a few Coliform organisms were detected in milk from a quarter, then this would not be considered mastitis. It would be more correct to say that about 70-80% of clinical mastitis cases are associated with Coliform infections.

L88 There are several methods to treat Coliform infections instead of using antibiotics. For example, frequent milking to expel the infected milk, sometimes with use of oxytocin, can eliminate the infection without antibiotics.

L91-93 Transmission through the food chain can be reduced greatly by pasteurization of the milk.

L93-94 Monitoring resistance has no effect on control of mastitis unless cows with resistant Coliforms are culled.

L115 See this similar paper from Cameroon: https://www.heraldopenaccess.us/openaccess/bacterial-pathogens-involved-in-bovine-mastitis-and-their-antibiotic-resistance-patterns-in-the-adamawa-region-of-cameroon This paper may be from a different region and it differs in some respects from the current paper, but it clearly shows the causes of clinical mastitis in Cameroon.

L136 Although this is an important area in Cameroon, it is a tiny part of the country, Africa, and the world. What makes this study unique and of interest to scientists worldwide?

L186 Ideally, the veterinarian should have changed gloves between each teat, not just each cow.

L223 Please report the precise statistical models used for these analyses.

L232 Describe how the cows’ teats were cleaned before milking and whether their teats were dipped with disinfectants after milking. Also, did the milkers wear gloves and change gloves between cows? If none of these procedures were followed, then the levels of infection would be greater than if recommended milking procedures were used. Was the milking procedure recorded for each farm?

L234 What was the average lactation number for the cows? Among these herds, what was the average stage of lactation at sampling?

L252 ….13 out of 1644 quarters (2.2%) were….

L253-254 ….were blind, and 5.8% of the 1608 functional quarters (within 33 cows) displayed clinical signs characterized by watery milk or clots, flakes or blood.

L261 This entire section and tables are not useful or informative. There were too few cows per Division to be meaningful. The information may be useful for local farmers, but it is not useful for the scientific community at large.

L280 The term “subclinical mastitis” is not clearly defined by NMC or other organizations. Somatic cell count of milk that is acceptable for processing for human consumption differs among countries and even among processors within countries. Clearly, cows with extended periods of SCC above 100,000 produce less milk, but these cows are not classified as having mastitis unless the milk is abnormal or the udder is swollen, produces bloody milk or milk clots. It is more meaningful to look at the average SCC for the herd, but in this study, herd sizes are small, and a single cow can affect the average for the entire herd.

L280-onward Number of cows in each region is too small to be meaningful to the scientific community. This is useful information for the regions, but it is not useful for dairy farms elsewhere, because regions may differ in rates of infection.

L310 This section is useful and should be retained.

L350 This section is useful and should be retained.

L382 This section can be condensed to key points without making excessive comparisons among different countries. Also, include reports from dairy regions outside of Africa.

Reviewer #2: The paper describes investigations into the prevalence of Enterobacterales in milk samples from dairy cows in the North-West of Cameroon. As such this is a useful undertaking although the selective investigation of coliforms while neglecting other mastitis causing bacteria seems an odd approach. The very high prevalence of coliforms identified and the odd pattern with Enterobacter cloacae as the most frequent species shed doubt on the quality of sampling. Can the authors be sure, that their results truely reflect the situation in the udder and not (additional) environmental contamination of the samples with bacteria from the teat or udder surface or the surroundings? Interestingly, the authors never found a combination of different bacteria in the samples, which is a common issue in mastitis bacteriology. The authors should report, if they found the same bacteria in the udder quarters when several quarters were affected. Likewise they might discuss if certain bacteria were also clustered within herd, i.e. occuring in several cows in the same herd.

The authors spent a lot of room in the paper to the description of regional differences without providing evidence for the potential reasons for the differences. Including region in the statistical analysis might have helped to elucidate potential associations. For the international reader regions without a more detailed characterization of the differences between the regions are not useful.

The sample size calculation was done for independent entities. However, the authors report that they investigated 411 cows in only 123 farms. This means their sampling unit cow was not independent but clustered in farms. This is seemingly not refllected in the statistical analysis and -which is worse- neither included in the descriptive analysis nor in the discussion. An analysis of results also including herd size would be adequate.

Freezing milk samples prior to analysis usually is associated with a reduction in the number of coliform bacteria while it is not an issue with staphylococci. Therefore the high prevalence of coliforms is even more astonishing.

The antimicrobial test panel includes penicillin. This is not useful as coliforms are intrinsically resistant to penicillin. Therefore penicillin needs to be removed from the analysis and the analysis needs to be re-done. This also applies to the analysis of MDR as it is not valid if it includes penicillin.

When presenting the results of disk diffusion the authors need to present the used cut-off values. Ideally they would even provide the inhibition zone diameters.

Table 1 can be omitted as the information is included in table 8.

Tables 2 to 4 can be combined

Results in table 5 are odd as the number of cows is lower than in the other tables.

Only one decimal should be presented throughout.

In the discussion section excessive repetition of results should be avoided. Likewise comparison with other studies, while relevant should be condensed. Can the condition e.g. in Ethiopia or Tansania be compared with Cameroon?

A higher prevalence of subclinical as compared to clinical mastitis does not have to be elaborated as this is textbook knowledge.

The discussion on AMR cannot be evaluated as the results are not valid.

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Submitted filename: Coliform mastitis paper Cameroon.docx

Click here for additional data file.

9 Feb 2022

Point-by-point response letter

Dear Editor,

We appreciate your comments and those of the reviewers. We hope we have provided satisfactory responses/amendments to all the comments. The Point-by-point responses/ amendments are presented below.

Editor’s comments

Editor: We invite you to submit a revised version of the manuscript that addresses the points raised during the review process

Response: We have submitted a revised version of the manuscript addressing the points raised during the review process. Thank you for this opportunity. Hope we have addressed the points satisfactorily.

…………………………………………………………………………………………………

Editor: I will ask you to revise the writing of legends which, as the manuscript stands, are not informative at all.

Response: We have revised the writing of legends to make them more informative for all Tables except Table 6. We have also revised the legends for all Figures.

Table 1 (see L223-224….. New Table

Table 2 (see L280-281) ….. New Table

Table 3 (see L294)

Table 4 (see L298)

Table 5 (see L328)

Table 6 (see L333)

Table 7 (see L348)

Fig. 1 (see L137)

Fig. 2 (see L261)

Fig. 3 (see L310)

Fig. 4 (see L361-362)

…………………………………………………………………………………………………

Editor: I also encourage you to take into account the comment on the suitability of chosen antibiotics for coliforms.

Response: We have removed penicillin from the antibiotics panel and re-analyzed the results of the antibiotic susceptibility testing.

…………………………………………………………………………………………………

Editor: There seems to be a miscalculation in the number of coliform positive quarters (131 in the text and tables while, from figure 3, the calculated number is 55+(28*2)+(6*3)+(1*4)=133)

Response: We have done the correction (see Fig 3– L310). Actually, the number of coliform-associated mastitis quarters is 132. Thank you very much.

…………………………………………………………………………………………………

Editor: Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

Response: We have submitted all items requested above.

…………………………………………………………………………………………………

Reviewer #1 comments

Reviewer #1: L1 The title of this manuscript is misleading. Clinical and subclinical infections of the mammary glands differ; whereas milk from cows with clinical mastitis is not approved for sale or consumption, but cows with subclinical mastitis may have their milk sold or consumed, depending on the Somatic Cell Count, which differs among countries. For example, in the EU, the SCC of milk for sale cannot exceed 400,000 over a 3-month period in herds that must be sampled at least once monthly. A more accurate title would simply delete the word “Mastitis” and discuss it in appropriate context in the manuscript.

Response: The title has been revised. See L1-2

…………………………………………………………………………………………………

Reviewer #1: L30 California Mastitis Test instead of California mastitis Test

Response: This has been corrected. See L30

…………………………………………………………………………………………………

Reviewer #1: L35 It is important here to define what is meant by the term “mastitis”. In this study, cows with active coliform organisms in their milk were classified as having mastitis, regardless of their SCC. Some of these cows would have met the European standards for salable milk. This needs to be clarified.

Response: This is not true! In this study, we did not classify ALL cows with active coliform organisms in their milk as having mastitis. Mastitis was detected by clinical examination and the California Mastitis Test (CMT) – and we considered CMT scores ≥1 (which give SCC estimates ≥ 400,000 cells/ml) as positive (See L173). In fact, we identified 73 quarters (within 38 cows) that had active coliform organisms but were not classified as mastitic (see Table 3 – Line 294).

…………………………………………………………………………………………………

Reviewer #1: L37 It would be more correct to say that these cows had Coliform infections, rather than saying Coliform mastitis.

Response: We maintain coliform mastitis (coliform-associated mastitis) because, in this study, we detected mastitis by clinical examination and the California Mastitis Test (CMT). Mastitis positive cows or udder quarters that had coliform bacteria isolated from their milk were considered positive for coliform mastitis. While a total of 205/1608 (12.8%) quarter milk samples had active coliform bacteria, only 132 of these samples were from mastitic cows. Hence, coliform infection is not synonymous to coliform mastitis.

…………………………………………………………………………………………………Reviewer #1: L81-82 This statement is incorrect. If only a few Coliform organisms were detected in milk from a quarter, then this would not be considered mastitis. It would be more correct to say that about 70-80% of clinical mastitis cases are associated with Coliform infections.

Response: You are very right! The whole sentence has been deleted. …………………………………………………………………………………………………

Reviewer #1: L88 There are several methods to treat Coliform infections instead of using antibiotics. For example, frequent milking to expel the infected milk, sometimes with use of oxytocin, can eliminate the infection without antibiotics.

Response: This is true. We have revised the sentence to indicate that antibiotic therapy is not the only method to treat coliform infections (See L87-90)

…………………………………………………………………………………………………

Reviewer #1: L91-93 Transmission through the food chain can be reduced greatly by pasteurization of the milk.

Response: True. For clarity, we have rephrased the sentence by replacing food chain with unpasteurized milk (See L91-93)

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Reviewer #1: L93-94 Monitoring resistance has no effect on control of mastitis unless cows with resistant Coliforms are culled.

Response: We think monitoring antibiotic resistance has an effect on the control of mastitis even when cows with resistant coliforms are not culled. To substantiate, we quote this “Therefore, continuous monitoring of antimicrobial resistance (AMR) and application of AMR mitigation measures are required to control their spread to humans, animals, and the environment” (Abdi et al. 2021; https://doi.org/10.3390/ani11010131). However, for clarity, we have revised this sentence. See L94-95

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Reviewer #1: L115 See this similar paper from Cameroon: https://www.heraldopenaccess.us/openaccess/bacterial-pathogens-involved-in-bovine-mastitis-and-their-antibiotic-resistance-patterns-in-the-adamawa-region-of-cameroon. This paper may be from a different region and it differs in some respects from the current paper, but it clearly shows the causes of clinical mastitis in Cameroon.

Response: We have read the paper! Yes, this paper identifies 14 bacterial species as causes of mastitis (clinical and subclinical) in Cameroon. Similarly, in our study, we have identified nine coliform bacterial species including two species (Escherichia coli and Klebsiella pneumoniae reported in the previous study) and seven species reported only in our study. This means that our study has expanded the repertoire of bacterial isolates associated with mastitis in Cameroon. Hence, the results of this study have provided important epidemiological data on coliform-associated mastitis.

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Reviewer #1: L136 Although this is an important area in Cameroon, it is a tiny part of the country, Africa, and the world. What makes this study unique and of interest to scientists worldwide?

Response: This study is unique, in that, it has expanded knowledge on the epidemiology of mastitis in Cameroon in the following ways:

�  It has expanded the repertoire of bacterial isolates associated with mastitis in Cameroon. Bacterial isolates associated with mastitis may vary from herd to herd as well as within and between countries. Hence, the prompt identification and understanding of the diversity of the pathogens associated with mastitis is essential for effective control (Pascu et al., 2022; https://doi.org/10.3390/antibiotics11010057).

�  It has identified some risk factors of coliform-associated mastitis.

�  It has reported multidrug resistance of coliform bacteria in milk

Although our study area is a tiny part of the country, Africa, and the world, it has contributed data to the body of knowledge of bovine mastitis in Cameroon. Such knowledge is of interest to scientists worldwide seeking information from Cameroon on such a topic, which as at now is scanty in literature. Thus, the uniqueness and interest of this study from this tiny area to scientists worldwide cannot be overemphasized.

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Reviewer #1: L186 Ideally, the veterinarian should have changed gloves between each teat, not just each cow.

Response: We have noted this remarked and thank you very much. However, the veterinarian disinfected gloves with 70% alcohol between each teat.

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Reviewer #1: L223 Please report the precise statistical models used for these analyses.

Response: We have reported the precise statistical models used for these analyses (See L226)

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Reviewer #1: L232 Describe how the cows’ teats were cleaned before milking and whether their teats were dipped with disinfectants after milking. Also, did the milkers wear gloves and change gloves between cows? If none of these procedures were followed, then the levels of infection would be greater than if recommended milking procedures were used. Was the milking procedure recorded for each farm?

Response: Yes, we observed the milking procedures in the farms and these procedures have been reported (See L243-248).

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Reviewer #1: L234 What was the average lactation number for the cows? Among these herds, what was the average stage of lactation at sampling?

Response: We have included these details (See L249-250).

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Reviewer #1: L252 ….13 out of 1644 quarters (2.2%) were….

Response: We have effected this correction (See L253-254)

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Reviewer #1: L253-254 ….were blind, and 5.8% of the 1608 functional quarters (within 33 cows) displayed clinical signs characterized by watery milk or clots, flakes or blood.

Response: We have effected this correction (See L254- 255)

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Reviewer #1: L261 This entire section and tables are not useful or informative. There were too few cows per Division to be meaningful. The information may be useful for local farmers, but it is not useful for the scientific community at large.

Response: Okay. We have modified this entire section to capture prevalence in the region (i.e, the North West region) without the divisions. See L263.

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Reviewer #1: L280 The term “subclinical mastitis” is not clearly defined by NMC or other organizations. Somatic cell count of milk that is acceptable for processing for human consumption differs among countries and even among processors within countries. Clearly, cows with extended periods of SCC above 100,000 produce less milk, but these cows are not classified as having mastitis unless the milk is abnormal or the udder is swollen, produces bloody milk or milk clots. It is more meaningful to look at the average SCC for the herd, but in this study, herd sizes are small, and a single cow can affect the average for the entire herd.

Response: This remark is noted! A CMT score of ≥1 is expected to have an SCC of ≥400,000 according to the CMT kit we used. Since this was a cross-sectional study, SCC over an extended period could not be determined.

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Reviewer #1: L280-onward Number of cows in each region is too small to be meaningful to the scientific community. This is useful information for the regions, but it is not useful for dairy farms elsewhere, because regions may differ in rates of infection.

Response: We have removed the results of the divisions from this section and we have provided the results of the prevalence of coliform-associated mastitis in the North West region in Table 2 (See L280-281)

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Reviewer #1: L310 This section is useful and should be retained.

Response: Okay.

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Reviewer #1: L350 This section is useful and should be retained.

Response: Okay.

Reviewer #1: L382 This section can be condensed to key points without making excessive comparisons among different countries. Also, include reports from dairy regions outside of Africa.

Response: We have revised this section (see L365). Thank you very much.

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Reviewer #2 comments

Reviewer #2: The paper describes investigations into the prevalence of Enterobacterales in milk samples from dairy cows in the North-West of Cameroon. As such this is a useful undertaking although the selective investigation of coliforms while neglecting other mastitis causing bacteria seems an odd approach. The very high prevalence of coliforms identified and the odd pattern with Enterobacter cloacae as the most frequent species shed doubt on the quality of sampling. Can the authors be sure, that their results truly reflect the situation in the udder and not (additional) environmental contamination of the samples with bacteria from the teat or udder surface or the surroundings? Interestingly, the authors never found a combination of different bacteria in the samples, which is a common issue in mastitis bacteriology. The authors should report, if they found the same bacteria in the udder quarters when several quarters were affected. Likewise they might discuss if certain bacteria were also clustered within herd, i.e. occurring in several cows in the same herd.

Response:

- We followed aseptic measures during sample collection (See L183). So we are certain the results truly reflect the situation in the udder and not from the outside of the cow.

- We have reported the coliform bacteria type isolated from mastitis udder quarters when several quarters were affected (See L297-302). However, the same bacteria were identified in the udder quarters when several quarters were affected in majority of the cows (see L302 – 307).

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Reviewer #2: The authors spent a lot of room in the paper to the description of regional differences without providing evidence for the potential reasons for the differences. Including region in the statistical analysis might have helped to elucidate potential associations. For the international reader regions without a more detailed characterization of the differences between the regions are not useful.

Response: Thank you for this enlightenment. We have removed the results for the divisions since Reviewer 1 raised the same issue and requested we do not report. We have, therefore, reported only the overall prevalence for the North West region (see Table 2, L280-281).

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Reviewer #2: The sample size calculation was done for independent entities. However, the authors report that they investigated 411 cows in only 123 farms. This means their sampling unit cow was not independent but clustered in farms. This is seemingly not reflected in the statistical analysis and -which is worse- neither included in the descriptive analysis nor in the discussion. An analysis of results also including herd size would be adequate.

Response: Yes, in this study cows were considered independent entities. We have now added herd size in the analysis of the results (See Table 5, L328)

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Reviewer #2: Freezing milk samples prior to analysis usually is associated with a reduction in the number of coliform bacteria while it is not an issue with staphylococci. Therefore the high prevalence of coliforms is even more astonishing.

Response: True, freezing milk samples before analysis is usually associated with a reduction in the number of coliform bacteria. However, we did not investigate the number of coliform bacteria in this study to be able to notice the effect of freezing.

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Reviewer #2: The antimicrobial test panel includes penicillin. This is not useful as coliforms are intrinsically resistant to penicillin. Therefore penicillin needs to be removed from the analysis and the analysis needs to be re-done. This also applies to the analysis of MDR as it is not valid if it includes penicillin.

Response: We have removed penicillin from the antimicrobial test panel and the analysis has been re-done.

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Reviewer #2: When presenting the results of disk diffusion the authors need to present the used cut-off values. Ideally they would even provide the inhibition zone diameters.

Response: We have provided a table of zone diameter breakpoints of the various antibiotics against coliforms (See Table 1, L223-224)

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Reviewer #2: Table 1 can be omitted as the information is included in table 8.

Response: This has been done.

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Reviewer #2: Tables 2 to 4 can be combined

Response: We have combined the tables as one. But we revised the table to capture results of mastitis prevalence in the North West region without divisional prevalence. See L263.

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Reviewer #2: Results in table 5 are odd as the number of cows is lower than in the other tables.

Response: Table 5 has been deleted but the information has been maintained in text. See L368-273.

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Reviewer #2: Only one decimal should be presented throughout.

Response: This has been done. Thank you very much.

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Reviewer #2: In the discussion section excessive repetition of results should be avoided. Likewise comparison with other studies, while relevant should be condensed. Can the condition e.g. in Ethiopia or Tanzania be compared with Cameroon?

Response: We have revised the discussion section, making sure there is no excessive repetition.

Ethiopia, for example, has one of the largest livestock populations in Africa and modern dairy farming is beginning to flourish in this country in both urban and peri-urban areas of the major towns. Several studies have been conducted in various parts of the country on bovine mastitis leading to increased knowledge on prevalence of mastitis, the microbial diversity and risk factors associated with disease development. Cameroon has a comparatively smaller livestock population and very scanty literature on the occurrence of bovine mastitis.

However, these countries are in sub-Saharan Africa and the conditions in these countries may be more similar compared to countries elsewhere. Yet, the prevalence of bovine mastitis in these countries is expected to vary due to the interaction of several factors, including herd management, environment and cow-related factors.

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Reviewer #2: A higher prevalence of subclinical as compared to clinical mastitis does not have to be elaborated as this is textbook knowledge.

Response: In most sub-Saharan countries including Cameroon, subclinical mastitis has received little or no attention and efforts are focused on the treatment of clinical cases while high productive and economic losses come from subclinical mastitis. Therefore, highlighting this is very important for public awareness to guide mitigation. In fact, several different studies point out that subclinical mastitis is more economically important than clinical mastitis due to the fact that subclinical mastitis is more difficult to diagnose and therefore usually persists longer in the herds, causing production losses.

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Reviewer #2: The discussion on AMR cannot be evaluated as the results are not valid.

Response: The results are now valid because we have removed penicillin from the panel of antibiotics.

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Submitted filename: Response to Reviewers.docx

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13 Apr 2022

24 Apr 2022

Point-by-point response letter

Dear Editor,

We appreciate your comments and those of the reviewers. We hope we have provided satisfactory responses/amendments to all the comments. The Point-by-point responses/ amendments are presented below.

Editor’s comments

Editor: We invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Response: We have submitted a revised version of the manuscript addressing the points raised during the review process. Thank you for this opportunity. Hope we have addressed the points satisfactorily.

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Editor: Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.

• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.

• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

Response: We have submitted all items requested above.

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Editor: Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Response: We have reviewed the reference list to ensure it is complete and correct.

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Editor: While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/.

Response: We have uploaded figure files to the PACE digital diagnostic tool to ensure they meet PLOS requirements.

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Reviewer #3 comments

Reviewer #3: I only have minor comments for this version.

Lines 477-490: when it comes to the discussion of percent resistant isolates, I think the authors should be much more cautious in their statements. What is the meaning of 100% resistant isolates for a particular antibiotic when only one or two strains of a species have been isolated? Not much. If read as such, one could take as a general message that all Serratia spp. or Citrobacter freundii isolated from milk are resistant to tetracycline. Drawing such a conclusion from 1 or 2 isolates is really misleading. I think the authors should somehow nuance their remarks and focus on species for which they have at least 10 isolates.

Response: We have revised this. Serratia spp. and Citrobacter freundii with 4 and 2 isolates respectively have been excluded in the discussion. See L477-488

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Reviewer #3: Line 475: the statement "Thus, it is important to isolate the coliform mastitis pathogen and perform an antibiotic susceptibility test before any antibiotic therapy." is not always realistic when it comes to application on the field. Very often, when a clinical mastitis case is detected, antibiotics are used without any prior identification of the pathogen. I am not saying it is not important to follow antibiotic resistance of mastitis pathogens, I'm rather implying that it not easily feasible in the field and that it is more a question for surveillance authorities. This statement should be somehow modified.

Response: Thank you, this statement has been revised. See L475-476

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Submitted filename: Response to Reviewers.docx

Click here for additional data file.

26 Apr 2022

Prevalence and Risk Factors of Coliform-Associated Mastitis and Antibiotic Resistance of Coliforms from Lactating Dairy Cows in North West Cameroon

PONE-D-21-33571R2

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15 Jul 2022

PONE-D-21-33571R2

Prevalence and Risk Factors of Coliform-Associated Mastitis and Antibiotic Resistance of Coliforms from Lactating Dairy Cows in North West Cameroon

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