Antibacterial Resistance and Bacterial Load in Milk Exposed to Dog Saliva in Sudan

Received 27 Jul, 2024

Accepted 24 Sep, 2024

Published 31 Dec, 2024

Background and Objective: Dogs saliva contains several types of bacteria, some of which are pathogenic to humans. Dogs contribute to many zoonotic diseases that may be transmitted by saliva. Dog bites and dog scratches are common sources of diseases among humans, particularly in children who use to play with dogs. This study aimed to identify and characterize bacteria present in dog saliva, quantify bacterial load in milk contaminated by dog saliva and assess bacterial susceptibility to twelve antibiotics. Materials and Methods: Two experiments were conducted: The first involved 150 swabs of police dog saliva in Bori and the second used 150 milk samples mixed with dog saliva from various areas. Bacteria were cultured on multiple media and tested against several antibiotics, with inhibition zones measured to determine sensitivity, intermediate response, or resistance. Results: Five bacterial species were identified: Staphylococcus spp., Streptococcus spp., Micrococcus spp., Gemella morbillorum and Bacillus spp. Staphylococcus aureus was the predominant species. Co-trimoxazole showed the highest effectiveness, with 81.7% sensitivity, followed by Ciprofloxacin at 73.3%. The bacterial load increased with extended licking, with the highest counts in Gandahar and the lowest in Kafori. Conclusion: This study concludes that Staphylococcus aureus, is predominant in dog saliva, with bacterial load increasing as milk licking recurs. Co-Trimoxazole and Ciprofloxacin are the preferred treatments.

INTRODUCTION

The interaction between humans and dogs is widespread, with dogs serving multiple roles, from pets to service animals. This close relationship poses potential health risks due to the transmission of pathogens via saliva. Previous studies have identified a diverse array of bacterial species in dog saliva. Key studies, such as those by Toth et al.¹ and Veir and Lappin², have documented the presence of Staphylococcus spp., Streptococcus spp., Pasteurella spp. and various Gram-negative bacteria. These studies highlight the potential for pathogenic bacteria to be present in dog saliva, which can pose a risk if transmitted to humans. Research by Johler et al.³ indicated that food items contaminated with dog saliva could harbor high levels of bacteria, leading to food-borne illnesses. This is particularly concerning when considering dairy products, such as milk, which can provide an ideal growth medium for bacteria.

Staphylococcus species, particularly Staphylococcus aureus and Staphylococcus intermedius, have been extensively studied due to their ability to cause a range of infections in humans and animals. Studies by Grudlewska-Buda et al.⁴ and Cuny et al.⁵ have shown that these bacteria can be resistant to multiple antibiotics, posing significant challenges in clinical settings. The presence of bacteria in dog saliva and their subsequent transfer to food items has been a topic of research, especially concerning the safety of consuming food contaminated by dog saliva. This study aims to isolate and characterize bacteria from dog saliva, quantify bacterial load in milk contaminated by dog saliva and assess the antibiotic susceptibility of isolated bacteria.

MATERIALS AND METHODS

Study area: Conducted across various locations (Tuti Island, Gandahar, EL-Thawrah, EL-Salha, Kafori, EL-Shuhada and Bori) in Khartoum from November, 2018 to November, 2021.

Study design: A descriptive cross-sectional study was conducted over three years, analyzing samples for bacterial contamination and antibiotic susceptibility.

Microbiological cultures and bacterial isolates
Sample collection: One hundred and fifty swabs from police dogs’ saliva were collected at the police dog center in Bori. One hundred and fifty milk samples were mixed with dog saliva from domestic dogs in areas such as Elthawrah, Tuti Island, Gandahar, Elsalha, Kafori and Elshuhada.

Primary culture: Samples were cultured on blood agar and nutrient agar under aerobic conditions. Visual and microscopic examination for bacterial growth and characteristics^6-8.

Bacterial identification and count: Bacteria identified using conventional bacteriological methods and Colony-Forming Unit (CFU) counts⁹.

Identification by biochemical tests⁷
For gram-positive bacteria:

•	Catalase test: A culture sample was placed on a slide with a drop of 3% hydrogen peroxide10. The presence of gas bubbles indicated a positive result

•	Coagulase test:

	•	Slide test: A bacterial suspension in normal saline was mixed with undiluted human plasma11. Clumping within 5-10 sec indicated a positive result
	•	Tube test: A diluted plasma sample was mixed with a broth culture and incubated at 37°C overnight12. Clot formation indicated a positive result

•	Voges-Proskauer test: This test differentiates between Staphylococcus aureus and Staphylococcus intermedius. The broth media was inoculated, incubated and then treated with alpha-naphthol and KOH10. The development of a red color indicated a positive result
•	Staining techniques13

Differentiation between bacteria: Novobiocin (5 μg/disk), basitracin (10 International Units) and optochin (5 μg/disk) disks were obtained from Sigma-Aldrich (Merck) and were used to differentiate bacterial species and observe hemolysis in blood agar¹⁴. The presence or absence of spores and colony pigmentation were also noted.

Table 1:

Standard zone of inhibition to different antibiotics

		Zone of inhibition (diameter in mm)
Antibiotic disk	Disk potency	Resistant	Intermediate	Sensitive
Linezolid (LZ)	30 mcg	20 or less	-	21 or more
Ciprofloxacin (CIP)	5 mcg	15 or less	16-20	21 or more
Roxithromycin (RF)	15 mcg	9 or less	Oct-20	21 or more
Ampicillin (AS)	10 mcg	11 or less	Dec-14	15 or more
Cefotaxime (CF)	30 mcg	14 or less	15-22	23 or more
CoTrimexothazol (BA)	25 mcg	10 or less	Nov-15	16 or more
Tetracycline (TE)	30 mcg	14 or less	15-18	19 or more
Cephalexin (PR)	30 mcg	14 or less	15-17	18 or more
Cloxacillin (CX)	5 mcg	10 or less	11-Dec	13 or more
Gentamicin (GM)	10 mcg	12 r less	13-14	15 or more
Levofloxacin (LE)	5 mcg	13 or less	14-16	17 or more
Lincomycin (LM)	2 mcg	14 or less	15-20	21 or more
Source: Company of Axium Laboratories, Paharganj, New Delhi

Table 2:

Percentages of gender of dogs from all areas

Gender	Frequency	Percentage
Male	162	54
Female	138	46
Total	300	100

Antimicrobial susceptibitity: Antibiotic susceptibility tested against twelve antibiotics, including Ampicillin, Co-Trimoxazole, Cephalexin, Tetracycline, Cefotaxime, Ciprofloxacin, Levofloxacin, Gentamycin, Lincomycin, Linezoled, Roxithromycin and Cloxacillin. Pure cultures from nutrient agar were inoculated into nutrient broth and incubated until light turbidity appeared. The suspension was spread on Mueller-Hinton agar and antibiotic discs were placed on the medium. The plates were incubated at 37°C for 18-24 hrs and the zones of inhibition were measured to determine sensitivity or resistance¹⁵. The standard zone of inhibition to different antibiotics was shown in Table 1. The antibiotic disks and their corresponding zones of inhibition were used to classify bacteria as resistant, intermediate or sensitive based on a standard interpretative chart.

RESULTS

In the study samples were collected from male and female dogs of different ages, the males had the highest percentage as shown in Table 2.

One hundred and fifty milk mixed with saliva samples were collected from different areas including Tuti Island, Elshuhada, Elthawrah, Kafori, Gandahar and Elsalha and 150 swab samples were collected from the saliva of police dogs from Bori in Khartoum state shown in Table 3-9.

Staphylococcus aureus had the highest percentage in all areas. In Tuti Island, S. aureus had the highest percentage. In Gandahar, S. aureus and B. lentus were the highest percentages. In Elthawrah and Elsalha, S. aureus and S. intermedius were the highest percentages. In Kafori, S. aureus and B. badius were the highest percentages. In Elshuhada,S. aureus and S. sciuri were the highest percentages. In the Police Dogs Center in Bori, S. aureus and S. intermedius had the highest percentage. Staphylococcus aureus was the highest percentage (Table 3-10).

The count of CFU bacteria due to licking milk three times was carried out for studied bacteria (Table 11). The highest load of bacteria was in samples which were collected from Gandahar (lick one was 7.0×10⁶, lick two was 8.5×10⁶ and lick three was 9.2×10⁶), while the lowest load of bacteria was in samples which were collected from Kafori (lick one was 1.2×10⁶, lick two was 2.3×10⁶ and lick three was 3.4×10⁶).

Table 3:

Types of bacteria isolated from milk mixed with saliva of dogs at Tuti Island

No.	Gram stain	Catalase	Coagulase	VP test	Motility	Species
1	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
2	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
3	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
4	G+ve Rods	+ve	-ve	-ve	Motile	Bacillus
5	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
6	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
7	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
8	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
9	G+ve cocci	+ve	-ve	-ve	Non motile	Micrococcus
10	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
11	G+ve cocci	+ve	-ve	+ve	Non motile	Micrococcus
12	G+ve cocci	+ve	-ve	+ve	Non motile	Micrococcus
13	G+ve cocci	-ve	-ve	-ve	Non motile	Streptococcus
14	G+ve Rods	+ve	-ve	+ve	Motile	Bacillus
15	G+ve cocci	-ve	-ve	-ve	Non motile	Streptococcus
16	G+ve Rods	+ve	-ve	-ve	Motile	Bacillus
17	G+ve Rods	+ve	-ve	-ve	Motile	Bacillus
18	G+ve cocci	-ve	-ve	-ve	Non motile	Streptococcus
19	G+ve Rods	+ve	-ve	-ve	Motile	Bacillus
20	G+ve Rods	+ve	-ve	-ve	Motile	Bacillus
VP: Voges Proskauer

Table 4:

Types of bacteria isolated from milk mixed with saliva of dogs at Gandahar

No.	Gram stain	Catalase	Coagulase	VP test	Motility	Species
21	G+ve Rods	+ve	-ve	-ve	Motile	Bacillus
22	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
23	G+ve Rods	+ve	-ve	-ve	Motile	Bacillus
24	G+ve Rods	+ve	-ve	-ve	Motile	Bacillus
25	G+ve cocci	-ve	-ve	-ve	Non motile	Streptococcus
26	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
27	G+ve Rods	+ve	-ve	+ve	Motile	Bacillus
28	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
29	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
30	G+ve Rods	+ve	-ve	+ve	Motile	Bacillus
31	G+ve cocci	-ve	-ve	-ve	Non motile	Streptococcus
32	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
33	G+ve cocci	-ve	-ve	-ve	Non motile	Streptococcus
34	G+ve cocci	-ve	-ve	-ve	Non motile	Streptococcus
35	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
36	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
37	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
38	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
39	G+ve cocci	-ve	-ve	-ve	Non motile	Streptococcus
40	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
41	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
42	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
43	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
44	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
45	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
46	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
47	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
48	G+ve Rods	+ve	-ve	-ve	Motile	Bacillus
49	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
50	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
51	G+ve cocci	-ve	-ve	-ve	Non motile	Streptococcus
52	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
53	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
54	G+ve cocci	+ve	-ve	-ve	Non motile	Micrococcus
55	G+ve cocci	+ve	-ve	-ve	Non motile	Micrococcus
56	G+ve cocci	-ve	-ve	-ve	Non motile	Gemella morbillorum
57	G+ve cocci	-ve	-ve	-ve	Non motile	Gemella morbillorum
58	G+ve cocci	+ve	+ve	-ve	Non motile	Micrococcus
59	G+ve cocci	-ve	-ve	-ve	Non motile	Gemella morbillorum
60	G+ve cocci	+ve	-ve	-ve	Non motile	Micrococcus

Table 5:

Types of bacteria isolated from milk mixed with saliva of dogs at Elthawrah

No.	Gram stain	Catalase	Coagulase	VP test	Motility	Species
61	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
62	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
63	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
64	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
65	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
66	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
67	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
68	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
69	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
70	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
71	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
72	G+ve cocci	+ve	-ve	-ve	Non motile	Micrococcus
73	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
74	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
75	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
76	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
77	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
78	G+ve cocci	+ve	-ve	-ve	Non motile	Streptococcus
79	G+ve cocci	+ve	-ve	-ve	Non motile	Streptococcus
80	G+ve cocci	+ve	-ve	-ve	Non motile	Micrococcus
81	G+ve cocci	-ve	-ve	-ve	Non motile	Streptococcus

Table 6:

Types of bacteria isolated from milk mixed with saliva of dogs at Elsalha

No.	Gram stain	Catalase	Coagulase	VP test	Motility	Species
82	G+ve cocci	+ve	-ve	+ve	Non motile	Micrococcus
83	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
84	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
85	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
87	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
88	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
89	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
90	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
91	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
92	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
93	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
94	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
95	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
96	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
97	G+ve cocci	+ve	-ve	-ve	Non motile	Micrococcus
98	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
99	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
100	G+ve cocci	-ve	-ve	-ve	Non motile	Streptococcus
101	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
102	G+ve cocci	-ve	-ve	-ve	Non motile	Streptococcus
103	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
104	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
105	G+ve Rods	+ve	-ve	-ve	Motile	Bacillus
106	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
107	G+ve cocci	+ve	-ve	+ve	Non motile	Micrococcus
108	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
109	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
110	G+ve Rods	+ve	-ve	-ve	Motile	Bacillus
111	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
112	G+ve Rods	+ve	-ve	-ve	Motile	Bacillus
113	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
114	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
115	G+ve cocci	+ve	-ve	-ve	Non motile	Micrococcus
116	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus

Table 7:

Types of bacteria isolated from milk mixed with saliva of dogs at Kafori

No.	Gram stain	Catalase	Coagulase	VP test	Motility	Species
117	G+ve cocci	+ve	-ve	+ve	Non motile	Micrococcus
118	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
119	G+ve Rods	+ve	-ve	-ve	Motile	Bacillus
120	G+ve Rods	+ve	-ve	-ve	Motile	Bacillus
121	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
122	G+ve cocci	-ve	-ve	-ve	Non motile	Streptococcus
123	G+ve Rods	+ve	-ve	-ve	Motile	Bacillus
124	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
125	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
126	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
127	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
128	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
129	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
130	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
131	G+ve Rods	+ve	-ve	-ve	Motile	Bacillus

Table 8:

Types of bacteria isolated from milk mixed with saliva of dogs at Elshuhada

No.	Gram stain	Catalase	Coagulase	VP test	Motility	Species
132	G+ve cocci	+ve	+ve	+ve	Non motile	Micrococcus
133	G+ve cocci	+ve	+ve	+ve	Non motile	Micrococcus
134	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
135	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
136	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
137	G+ve cocci	-ve	-ve	-ve	Non motile	Streptococcus
138	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
139	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
140	G+ve cocci	-ve	-ve	-ve	Non motile	Streptococcus
141	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
142	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
143	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
144	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
145	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
146	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
147	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
148	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
149	G+ve cocci	-ve	-ve	-ve	Non motile	Streptococcus
150	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus

Table 9:

Types of bacteria isolated from saliva of dogs at Bori

No.	Gram stain	Catalase	Coagulase	VP test	Motility	Species
1	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
2	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
3	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
4	G+ve cocci	+ve	+ve	+ve	Non motile	Micrococcus
5	G+ve Rods	+ve	-ve	-ve	Motile	Bacilllus
6	G+ve Rods	+ve	-ve	+ve	Motile	Bacilllus
7	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
8	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
9	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
10	G+ve cocci	+ve	+ve	+ve	Non motile	Micrococcus
11	G+ve Rods	+ve	-ve	-ve	Motile	Bacilllus
12	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
13	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
14	G+ve Rods	+ve	-ve	+ve	Motile	Bacilllus
15	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
16	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
17	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
18	G+ve Rods	+ve	+ve	-ve	Motile	Bacilllus
19	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
20	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
21	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
22	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
23	G+ve Rods	+ve	+ve	+ve	Motile	Bacilllus
24	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
25	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
26	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
27	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
28	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
29	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
30	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
31	G+ve Rods	+ve	+ve	-ve	Motile	Bacilllus
32	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
33	G+ve Rods	+ve	-ve	-ve	Motile	Bacilllus
34	G+ve Rods	+ve	-ve	-ve	Motile	Bacilllus
35	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
36	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
37	G+ve cocci	+ve	+ve	-ve	Non motile	Micrococcus
38	G+ve Rods	+ve	-ve	+ve	Motile	Bacilllus
39	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
40	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
41	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
42	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
43	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
44	G+ve Rods	+ve	-ve	-ve	Motile	Bacilllus
45	G+ve Rods	+ve	-ve	-ve	Motile	Bacilllus
46	G+ve Rods	+ve	-ve	-ve	Motile	Bacilllus
47	G+ve Rods	+ve	-ve	+ve	Motile	Bacilllus
48	G+ve Rods	+ve	-ve	+ve	Motile	Bacilllus
49	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
50	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
51	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
52	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
53	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
54	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
55	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
56	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
57	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
58	G+ve cocci	+ve	-ve	-ve	Non motile	Micrococcus
59	G+ve cocci	+ve	-ve	+ve	Non motile	Micrococcus
60	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
61	G+ve cocci	+ve	-ve	+ve	Non motile	Micrococcus
62	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
63	G+ve cocci	-ve	-ve	-ve	Non motile	Streptococcus
64	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
65	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
66	G+ve cocci	+ve	-ve	-ve	Non motile	Micrococcus
67	G+ve Rods	+ve	-ve	-ve	Motile	Bacilllus
68	G+ve Rods	+ve	-ve	-ve	Motile	Bacilllus
69	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
70	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
71	G+ve Rods	+ve	-ve	-ve	Motile	Bacilllus
72	G+ve Rods	+ve	-ve	-ve	Motile	Bacilllus
73	G+ve Rods	+ve	+ve	+ve	Motile	Bacilllus
74	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
75	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
76	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
77	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
78	G+ve cocci	-ve	-ve	-ve	Non motile	Streptococcus
79	G+ve cocci	-ve	-ve	-ve	Non motile	Streptococcus
80	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
81	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
82	G+ve Rods	+ve	-ve	+ve	Motile	Bacilllus
83	G+ve Rods	+ve	-ve	-ve	Motile	Bacilllus
84	G+ve Rods	+ve	-ve	-ve	Motile	Bacilllus
85	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
86	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
87	G+ve cocci	-ve	-ve	-ve	Non motile	Streptococcus
88	G+ve cocci	-ve	-ve	-ve	Non motile	Streptococcus
89	G+ve cocci	-ve	-ve	-ve	Non motile	Streptococcus
90	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
91	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
92	G+ve cocci	-ve	-ve	-ve	Non motile	Streptococcus
93	G+ve cocci	-ve	-ve	-ve	Non motile	Streptococcus
94	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
95	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
96	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
97	G+ve cocci	-ve	-ve	-ve	Non motile	Streptococcus
98	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
99	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
100	G+ve cocci	-ve	-ve	-ve	Non motile	Streptococcus
101	G+ve cocci	-ve	-ve	-ve	Non motile	Streptococcus
102	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
103	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
104	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
105	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
106	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
107	G+ve cocci	-ve	-ve	-ve	Non motile	Streptococcus
108	G+ve cocci	-ve	-ve	-ve	Non motile	Streptococcus
109	G+ve cocci	-ve	-ve	-ve	Non motile	Streptococcus
110	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
111	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
112	G+ve cocci	-ve	+ve	-ve	Non motile	Streptococcus
113	G+ve cocci	-ve	+ve	-ve	Non motile	Streptococcus
114	G+ve cocci	-ve	+ve	-ve	Non motile	Streptococcus
115	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
116	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
117	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
118	G+ve cocci	-ve	+ve	-ve	Non motile	Streptococcus
119	G+ve cocci	-ve	+ve	-ve	Non motile	Streptococcus
120	G+ve cocci	-ve	+ve	-ve	Non motile	Streptococcus
121	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
122	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
123	G+ve cocci	-ve	+ve	-ve	Non motile	Streptococcus
124	G+ve cocci	-ve	+ve	-ve	Non motile	Streptococcus
125	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
126	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
127	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
128	G+ve cocci	-ve	+ve	-ve	Non motile	Streptococcus
129	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
130	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
131	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
132	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus
133	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
134	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
135	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
136	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
137	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
138	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
139	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
140	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
141	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
142	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
143	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
144	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
145	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
146	G+ve Rods	+ve	-ve	+ve	Motille	Bacilllus
147	G+ve Rods	+ve	-ve	+ve	Motille	Bacilllus
148	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
149	G+ve cocci	+ve	+ve	-ve	Non motile	Staphylococcus
150	G+ve cocci	+ve	+ve	+ve	Non motile	Staphylococcus

Table 10:

Percentage of bacterial species isolated from milk mixed with saliva from different samples

Bacterial species	Tuti Island (20)	Bori (150)	Gandahar (40)	EL-Shuhada (19)	EL-Thawrah (21)	EL-Salha (34)	Kafori (15)
Staphylococcus intermedius	1 (5%)	18 (12%)	4 (10%)	1 (5.26%)	5 (23.81%)	5 (14.71%)	2 (17.33%)
Staphylococcus aureus	5 (25%)	47 (31.33%)	11 (27.5%)	5 (26.32%)	9 (42.86%)	14 (41.18%)	7 (46.67%)
Staphylococcus epidermidis	1 (5%)	11 (7.33%)	3 (7.5%)	2 (10.53%)	0	3 (8.82%)	0
Staphylococcus sciuri	1 (5%)	5 (3.33%)	3 (7.5%)	4 (21.05%)	2 (9.52%)	3 (8.82%)	0
Staphylococcus caseolyticus	0	7 (4.67%)	3 (7.5%)	2 (10.53%)	0	0	00
Streptococcus pneumoniae	3 (15%)	10 (6.67%)	2 (5%)	1 (5.26%)	1 (4.76%)	1 (2.94%)	1 (6.67%)
Streptococcus pyogenes	0	7 (4.67%)	2 (5%)	2 (10.53%)	2 (9.52%)	1 (2.94%)	00
Streptococcus sanguis	0	5 (3.33%)	2 (10%)	0	0	0
Bacillus badius	2 (10%)	9 (6%)	1 (2.5%)	0	0	2 (5.88%)	3 (20%)
Bacillus subtilis	2 (10%)	9 (6%)	0	0	0	0	0
Bacillus lentus	2 (10%)	7 (4.67%)	5 (12.5%)	0	0	1 (2.94%)	1 (6.67%)
Micrococcus agilis	2 (10%)	1 (0.67%)	1 (2.5%)	1 (5.26%)	1 (4.76%)	0	0
Micrococcus kristinae	1 (5%)	2 (1.33%)	3 (7.5%)	0	1 (4.76%)	0	1 (6.67%)
M. mucilaginosus	0	4 (2.67%)	0	0	0	1 (2.94%)	0
Micrococcus lylae	0	1 (0.67%)	0	1 (5.26%)	0	3 (8.82%)	0
Gemella morbillorum	0	7 (4.67%)	3 (7.5%)	0	0	0	0

Novobiocin, Basitracin and Optochin were used to differentiate between some bacteria shown in Table 12, VP test and the position of spore was used to differentiate between Bacillus spp. as shown in Table 13. In species of M. kristinae the pigmentation of colonies was yellow colour, M. mucilaginosus had no pigmentation in colonies, M. agilis appeared red colour, while M. lylae showed cream colour in the surface of colonies, presented in Table 14.

Various bacterial species were tested against various antibiotics as shown in Table 15 Co-Trimexothazol showed the best antibiotic against different species of bacteria as 245 isolates were sensitive (81.7%) from the total isolates. Ciprofloxacin followed Co-Trimexothazol in a percentage of sensitivity, 220 isolates were sensitive to Ciprofloxacin (73.3%) from the total isolates. While Cloxacillin showed the lowest percentage against different bacteria as 14 isolates were sensitive (4.7%) from the total isolates.

Table 11:

Number of the CFU/mL from milk after licking for three times

Sample No.	Lick one (CFU/mL)	Lick two (CFU/mL)	Lick three (CFU/mL)
Tuti Island
1	3.6×106	5.0×106	6.0×106
2	3.5×106	6.0×106	7.5×106
3	4.0×106	5.8×106	7.0×106
4	4.9×106	7.3×106	8.0×106
5	6.0×106	8.0×106	8.7×106
6	4.0×106	6.0×106	7.5×106
7	2.5×106	3.8×106	5.0×106
8	1.5×106	2.9×106	3.7×106
9	1.4×106	2.1×106	3.0×106
10	2.2×106	3.1×106	4.4×106
11	1.8×106	2.5×106	3.4×106
12	2.6×106	3.2×106	4.8×106
13	3.8×106	5.6×106	6.4x106
14	2.6×106	3.2×106	4.0×106
15	2.9×106	3.7×106	4.6×106
16	3.8×106	5.1×106	6.0×106
17	1.7×106	3.0×106	4.5×106
18	4.9×106	6.4×106	7.2×106
19	1.6×106	2.5×106	3.5×106
20	2.2×106	3.7×106	4.0×106
Gandahar
21	3.2×106	5.0×106	6.3×106
22	5.5×106	7.3×106	8.0×106
23	2.4×106	4.0×106	5.9×106
24	3.3×106	5.9×106	7.2×106
25	4.0×106	5.3×106	6.0×106
26	7.0×106	8.5×106	9.2×106
27	3.3×106	4.0×106	5.5×106
28	2.3×106	3.9×106	5.6×106
29	3.8×106	5.0×106	7.5×106
30	1.5×106	3.0×106	4.9×106
31	2.4×106	3.7×106	4.5×106
32	1.9×106	2.7×106	5.7×106
33	2.6×106	4.0×106	6.2×106
34	2.0×106	3.1×106	4.9×106
35	1.7×106	3.0×106	4.3×106
36	3.7×106	6.8×106	9.0×106
37	3.8×106	5.0×106	6.7×106
38	2.1×106	3.2×106	5.0×106
39	4.0×106	5.6×106	7.2×106
40	3.1×106	4.4×106	6.4×106
41	2.9×106	3.9×106	5.0×106
42	3.3×106	4.7×106	6.0×106
43	2.9×106	3.8×106	5.0×106
44	3.6×106	5.6×106	7.4×106
45	2.0×106	3.3×106	4.0×106
46	1.4×106	2.3×106	3.6×106
47	1.3×106	2.5×106	4.1×106
48	2.0×106	2.9×106	4.0×106
49	2.9×106	4.8×106	7.0×106
50	2.6×106	3.5×106	4.8×106
51	2.4×106	3.5×106	6.0×106
52	1.5×106	2.2×106	4.0×106
53	3.7×106	5.2×106	7.0×106
54	3.8×106	5.0×106	6.0×106
55	4.0×106	5.5×106	6.0×106
56	1.9×106	2.3×106	3.3×106
57	2.9×106	4.0×106	5.5×106
58	2.3×106	3.0×106	4.1×106
59	2.0×106	2.9×106	3.4×106
60	2.9×106	4.0×106	6.0×106
ELthawrah
61	3.5×106	4.2×106	6.6×106
62	5.0×106	6.3×106	7.5×106
63	1.7×106	2.5×106	4.0×106
64	3.0×106	4.5×106	6.0×106
65	2.1×106	3.0×106	5.4×106
66	3.7×106	4.9×106	6.0×106
67	1.5×106	2.3×106	3.8×106
68	3.1×106	4.0×106	5.2×106
69	2.7×106	3.3×106	4.5×106
70	2.2×106	3.9×106	5.0×106
71	4.0×106	5.3×106	6.9×106
72	1.4×106	2.5×106	3.7×106
73	3.1×106	4.8×106	5.9×106
74	1.5×106	2.9×106	3.5×106
75	3.7×106	4.5×106	6.0×106
76	2.8×106	4.9×106	6.8×106
77	2.7×106	5.5×106	7.0×106
78	2.4×106	3.6×106	5.0×106
79	1.3×106	2.1×106	3.0×106
80	3.0×106	4.5×106	5.7×106
81	3.7×106	4.9×106	6.0×106
ELsalha
82	4.0×106	5.5×106	7.0×106
83	1.9×106	2.8×106	4.1×106
84	3.5×106	4.3×106	6.0×106
85	1.2×106	2.2×106	3.2×106
86	3.6×106	5.6×106	6.7×106
87	2.9×106	3.5×106	4.2×106
88	1.3×106	2.1×106	3.2×106
89	3.8×106	5.3×106	6.3×106
90	1.4×106	2.8×106	4.0×106
91	3.9×106	5.1×106	6.0×106
92	4.9×106	5.6×106	7.0×106
93	1.6×106	2.3×106	4.7×106
94	4.7×106	6.2×106	7.3×106
95	5.0×106	6.6×106	7.5×106
96	1.5×106	2.5×106	3.8×106
97	2.1×106	3.0×106	4.2×106
98	1.8×106	2.7×106	3.9×106
99	2.5×106	3.6×106	5.0×106
100	3.4×106	4.5x106	5.8×106
101	2.3×106	3.1×106	4.0×106
102	2.6×106	3.0×106	4.2×106
103	1.7×106	2.3×106	3.1×106
104	2.5×106	3.1×106	4.0×106
105	5.3×106	6.2×106	7.0×106
106	3.0×106	4.1×106	5.4×106
107	1.3×106	2.2×106	3.5×106
108	2.6×106	3.9×106	5.0×106
109	5.0×106	6.3×106	7.1×106
110	1.6×106	2.7×106	3.5×106
111	2.0×106	3.5×106	4.2×106
112	3.5×106	4.3×106	5.5×106
113	2.2×106	3.0×106	4.7×106
114	1.9×106	2.7×106	3.2×106
115	2.4×106	3.5×106	5.1×106
Kafori
116	1.2×106	2.3×106	3.4×106
117	3.3×106	4.0×106	5.3×106
118	5.1×106	5.9×106	6.2×106
120	5.0×106	6.6×106	7.3×106
121	2.1×106	3.4×106	5.0×106
122	1.6×106	2.5×106	3.4×106
123	1.8×106	2.8×106	3.6×106
124	2.0×106	3.8×106	5.0×106
125	3.3×106	4.0×106	5.5×106
126	1.7×106	2.9×106	3.8×106
127	2.6×106	3.2×106	4.5×106
128	5.0×106	6.2×106	7.3×106
129	3.3×106	4.0×106	5.5×106
130	2.3×106	3.2×106	4.0×106
131	2.5×106	3.3×106	4.3×106
ELshuhada
132	4.5×106	5.2×106	6.1×106
133	1.7×106	2.9×106	3.8×106
134	3.1×106	4.0×106	5.2×106
135	2.4×106	3.5×106	4.8×106
136	1.9×106	2.8×106	3.6×106
137	3.2×106	4.5×106	5.9×106
138	2.5×106	3.7×106	5.1×106
139	4.4×106	5.4×106	6.7×106
140	2.4×106	3.5×106	5.0×106
141	2.1×106	3.2×106	4.3×106
142	3.5×106	4.3×106	5.5×106
143	3.2×106	4.0×106	5.3×106
144	2.2×106	3.5×106	4.5×106
145	4.0×106	5.2×106	6.6×106
146	6.1×106	7.3×106	8.2×106
147	7.0×106	8.5×106	9.2×106
148	2.7×106	3.5×106	4.4×106
149	4.2×106	5.3×106	6.1×106
150	1.8×106	2.6×106	3.3×106

Table 12:

Some antibiotics used to differentiate some bacterial species and observe appearing of haemolysis

Genus	Novobiocin	Basitracin	Optochin	Haemolysis
Staphylococcus caseolyticus	S	-	-	-
Staphylococcus sciuri	R	-	-	-
Streptococcus pyogenes	-	S	R	Beta haemolysis
Streptococcus pneumoniae	-	S	S	Alpha haemolysis
S: Sensitive and R: Resistant

Table 13:

VP test, position and shape of spore used to differentiate some species of Bacilli

Genus	VP test	Position and shape of spore
Bacillus badius	-ve	Central and oval
Bacillus subtilis	+ve	Central and oval
Bacillus lentus	-ve	Terminal and oval

Table 14:

Genus of Micrococcus spp showed different pigmentation on the surface of colonies

Genus	Pigmentation of colonies
M. kristinnae	Yellow
M. mucilaginosus	-
Megachile agilis	Red
Micrococcus lylae	Cream

Table 15:

Sensitivity percentage of antibiotic disk against various bacterial species

Antimicrobial agent	Resistant	Intermediate	Sensitive
Ampicillin (10 μg)	80 (26.6%)	20 (6.7%)	200 (66.7%)
Co-Trimexothazol (25 μg)	34 (11.3%)	21 (7.0%)	245 (81.7%)
Cephalexin (30 μg)	55 (18.3%)	37 (12.3%)	208 (69.3%)
Tetracycline (30 μg)	55 (18.3%)	53 (17.7%)	192 (64%)
Cefotaxime ( (30 μg)	38 (12.7%)	95 (31.7%)	167 (55.7%)
Ciprofloxacin (5 μg)	22 (7.3%)	58 (19.3%)	220 (73.3%)
Levofloxacin (5 μg)	38 (12.7%)	45 (15%)	217 (72.3%)
Gentamycin (10 μg)	62 (20.7%)	42 (14%)	196 (65.3%)
Lincomycin (2 μg)	89 (29.7%)	78 (26%)	133 (44.3%)
Linezoled (30 μg)	114 (38%)	7 (2.3%)	179 (59.7%)
Roxithromycin (15 μg)	76 (25.3%)	105 (35%)	119 (39.7%)
Cloxacillin (5 μg)	281 (93.7%)	5 (1.7%)	14 (4.7%)

DISCUSSION

This study’s findings aligned with previous literature that highlights the presence of Gram-positive bacteria, particularly Staphylococcus aureus, in canine oral flora. Similar studies have identified S. aureus as a significant bacterium in dog saliva, emphasizing its potential as a source of contamination in various environments, including food sources¹⁶. The identification of Co-Trimoxazole and Ciprofloxacin as effective treatments for these bacterial isolates also resonates with existing research, which suggests that these antibiotics are generally effective against Gram-positive bacteria, including S. aureus¹⁷.

The results obtained in this study reveal that a significant amount of bacteria, more than 1×10⁵ CFU/mL, are introduced into sterile milk licked by a dog three times. Furthermore, the types of bacteria introduced can be harmful to human health. This study evaluated all isolated bacterial genera against different antibiotics. Staphylococcus spp. and Streptococcus spp. were isolated from dog saliva, aligned with findings by Georges and Adesiyun¹⁸, who recorded that dog bites to children between the ages of 8-12 in Trinidad presented risk factors and preventive strategies for reducing injuries. Most studies on dog injuries derive from hospital data, primarily from emergency departments, with the most common injury sites being the face, head and neck, especially in children under five years old. Bacteria such as Staphylococcus spp. Streptococcus spp. and Pasteurella multocida were isolated from dog bite wounds.

This study isolated S. aureus from dog saliva, similar to findings by Robertson et al.¹⁹, who reported dogs as main reservoirs of many infective stages of parasites transmissible to humans and other domestic animals. There is evidence of resistant organism transfer between animals and people, highlighting dogs' public health significance due to their close companionship with humans. In the United States, up to 43 million (36.5%) households own a dog. Saliva, a biological fluid, offers advantages as a diagnostic medium over blood due to non-invasive, simple collection and repeated sampling without discomfort to the patient. In human medicine, saliva is gaining attention as an alternative to blood analysis. In dogs, analytes like C-reactive protein, cortisol, alpha-amylase, adenosine deaminase or muscle enzymes have been measured in saliva²⁰.

This study detected various bacterial species among dogs kept as pets in Khartoum State. Samples were collected from milk mixed with dog saliva from Tuti Island, Elshuhada, Elthawrah, Kafori, Gandahar and Elsalha and direct saliva samples using sterile cotton swabs from Bori (police dogs). All isolated bacteria were Gram-positive, with some capable of causing numerous human diseases. The genus Staphylococcus exhibited the highest percentage, with 181 isolates (60.33%) of the total isolation. This genus displayed different shapes, colors and sizes. Five Staphylococcus species were fully identified: Staphylococcus aureus, Staphylococcus intermedius, Staphylococcus sciuri, Staphylococcus epidermidis and Staphylococcus caseolyticus. Staphylococcus intermedius was isolated from 18 samples of licked milk. Known to be carried in dog saliva, Staphylococcus intermedius poses a serious health hazard for dog owners and those consuming contaminated food. This finding aligned with Kempker et al.²¹, who isolated Staphylococcus intermedius from a woman with a history of endoscopic pituitary edema resection presenting with foul-smelling nasal discharge. Staphylococcus intermedius is a potential human pathogen.

The results of this study, which isolated Staphylococcus intermedius from dog saliva, were substantiated by Kikuchi et al.²², who reported a case of a 37 year-old female with Staphylococcus intermedius in a surgical wound infection. A month later, a case of mastoid cavity infection due to ear licking by a dog was reported. These cases emphasize the danger of consuming food contaminated by dog saliva carrying Staphylococcus intermedius. The association of human infection with Staphylococcus intermedius may be attributed to its status as normal dog skin flora²³. Dog and cat bites are primary sources of bacterial diseases, some fatal²⁴. Maurelli et al.²⁵ demonstrated that bodily contact significantly contributes to infection occurrence.

The genus Micrococcus showed 24 isolates (8%) from the total number, with a high percentage isolated from Bori. This study isolated Micrococcus spp. from dog saliva, substantiated by Abrahamian and Goldstein²⁶, who isolated Micrococcus spp., from dog bites. The genus Streptococcus showed 40 isolates (13.33%) from the total number, with a high percentage isolated from Bori. In this study, Streptococcus pneumoniae, Streptococcus sanguis and Streptococcus pyogenes isolation aligned with Fredrick et al.²⁶.

Three Bacillus species were isolated in this study: Bacillus lentus, B. badius and B. subtilis, comprising 43 isolates (14.33%) of the total. This genus showed different shapes, colors and sizes, with some having mucoid colonies. The study found M. mucilaginosus sensitivity to various antibiotics, similar to von Eiff et al.²⁷, who found M. mucilaginosus sensitivity in 63 isolates. Leyden et al.²⁸ counted aerobic bacteria colonies from dogs’ moist areas, such as the axillae or toe web spaces, reaching 10⁷ bacteria per cm², while dry areas like the forearm or trunk harbored fewer bacteria per cm², substantiating²⁸. Anaerobic bacteria are present on human skin, with colony counts up to 10⁶ bacteria per cm^{2 29}. Ribeiro et al.³⁰ reported that Strep pyogenes has significant importance as an opportunistic animal pathogen causing a variety of purulent infections. These infections pose a huge economic problem in livestock breeding, it is considred to be a part of the biota of skin and mucous membrane of the upper respiratory and urogenital tracts of animals.

According to Moosavy et al.³¹, most animal bites to humans in the Saudi Arabia involve snakes, dogs, cats, rodents and foxes. Their study also identified Actinobacteria in dog saliva, mirroring the findings of Wilhem et al.³², who detected Actinobacteria in the oral cavities of dogs. Additionally, Harvey³³ documented the isolation of Gram-positive bacteria from the mouths of dogs in various studies, highlighting that the prevalence of periodontal disease increases with age, affecting around 80% of dogs. The most common treatment for periodontal disease in dogs is scaling, which usually requires general anesthesia.

The study revealed a significant bacterial load, exceeding 1×10⁵ CFU/mL, in the milk mixed with dog saliva. The isolated bacteria included Staphylococcus aureus, Staphylococcus intermedius and various Streptococcus species, which are known to cause infections in humans. The antimicrobial susceptibility testing showed that many of the isolated bacteria were resistant to multiple antibiotics.

CONCLUSION

The study concludes that saliva, particularly from dogs, contains pathogenic bacteria like S. aureus, making it a significant source of infection. The bacterial count in milk increased with repeated licking, highlighting the risk of pathogen transmission through saliva in food and drinks. Co-trimoxazole emerged as the most effective antibiotic against these bacteria, followed by Ciprofloxacin, while Fungistatin was recommended for preventing fungal contamination. Future recommendations include more extensive surveillance of antibiotic-resistant bacteria, public education on the risks of dog saliva and the implementation of preventive hygiene measures to reduce bacterial transmission.

SIGNIFICANCE STATEMENT

This study underscores the importance of understanding the microbial composition of dog saliva and its implications for public health. The isolation and characterization of bacteria from milk mixed with dog saliva reveal significant bacterial contamination and antimicrobial resistance, posing potential health risks. Continuous research and public health initiatives are crucial to address these concerns and safeguard human health.

REFERENCES

1. Tóth, A.G., I. Tóth, B. Rózsa, A. Dubecz and Á.V. Patai et al., 2022. Canine saliva as a possible source of antimicrobial resistance genes. Antibiotics, 11.
2. Veir, J.K. and M.R. Lappin, 2010. Molecular diagnostic assays for infectious diseases in cats. Vet. Clin. North Am. Small Anim. Pract., 40: 1189-1200.
3. Johler, S., D. Weder, C. Bridy, M.C. Huguenin, L. Robert, J. Hummerjohann and R. Stephan, 2015. Outbreak of staphylococcal food poisoning among children and staff at a Swiss boarding school due to soft cheese made from raw milk. J. Dairy Sci., 98: 2944-2948.
4. Grudlewska-Buda, K., J. Bauza-Kaszewska, N. Wiktorczyk-Kapischke, A. Budzyńska, E. Gospodarek-Komkowska and K. Skowron, 2023. Antibiotic resistance in selected emerging bacterial foodborne pathogens-An issue of concern? Antibiotics, 12.
5. Cuny, C., A. Friedrich, S. Kozytska, F. Layer and U. Nübel et al., 2010. Emergence of methicillin-resistant Staphylococcus aureus (MRSA) in different animal species. Int. J. Med. Microbiol., 300: 109-117.
6. Rammell, C.G., 1962. Inhibition by citrate of the growth of coagulase-positive staphylococci. J. Bacteriol., 84: 1123-1124.
7. Feltham, R.K.A. and G.I. Barrow, 2003. Cowan and Steel's Manual for the Identification of Medical Bacteria. 3rd Edn., Cambridge University Press, Cambridge, England, ISBN: 9780521543286, Pages: 331.
8. Tittsler, R.P. and L.A. Sandholzer, 1936. The use of semi-solid agar for the detection of bacterial motility. J. Bacteriol., 31: 575-580.
9. Koch, A.L., 2004. Growth Measurement. In: Methods for General and Molecular Microbiology, Reddy, C.A., T.J. Beveridge, J.A. Breznak, G.A. Marzluf, T.M. Schmidt and L.R. Snyder (Eds.), ASM Press, Washington, DC, United States, ISBN: 9781683671619, pp: 172-199.
10. Clark, W.M. and H.A. Lubs, 1915. The differentiation of bacteria of the Colonaerogenes family by the use of indicators. J. Infect. Dis., 17: 160-173.
11. Dickson, J.I. and R.R. Marples, 1986. Coagulase production by strains of Staphylococcus aureus of differing resistance characters: A comparison of two traditional methods with a latex agglutination system detecting both clumping factor and protein A. J. Clin. Pathol., 39: 371-375.
12. Grov, A., B. Myklestad and P. Oeding, 1964. Immunochemical studies on antigen preparations from Staphylococcus aureus: 1. Isolation and chemical characterization of antigen A. Acta Pathologica Microbiol. Scand., 61: 588-596.
13. Preston, N.W. and A. Morrell, 1962. Reproducible results with the gram stain. J. Pathol. Bacteriol., 84: 241-243.
14. Murray, P.R., K. Rosenthal and M.A. Pfaller, 2020. Medical Microbiology E-Book. 9th Edn., Elsevier Health Sciences, Amsterdam, Netherlands, ISBN: 9780323674508, Pages: 872.
15. Balouiri, M., M. Sadiki and S.K. Ibnsouda, 2016. Methods for in vitro evaluating antimicrobial activity: A review. J. Pharm. Anal., 6: 71-79.
16. Rubin, J.E., K.R. Ball and M. Chirino-Trejo, 2011. Antimicrobial susceptibility of Staphylococcus aureus and Staphylococcus pseudintermedius isolated from various animals. Can. Vet. J., 52: 153-157.
17. Moellering Jr., R.C., 1998. Antibiotic resistance: Lessons for the future. Clin. Infect. Dis., 27: S135-S140.
18. Georges, K. and A. Adesiyun, 2008. An investigation into the prevalence of dog bites to primary school children in Trinidad. BMC Public Health, 8.
19. Robertson, I.D., P.J. Irwin, A.J. Lymbery and R.C.A. Thompson, 2000. The role of companion animals in the emergence of parasitic zoonoses. Int. J. Parasitol., 30: 1369-1377.
20. Pfaffe, T., J. Cooper-White, P. Beyerlein, K. Kostner and C. Punyadeera, 2011. Diagnostic potential of saliva: Current state and future applications. Clin. Chem., 57: 675-687.
21. Kempker, R., M. Eaton, D. Mangalat and T. Kongphet-Tran, 2009. Beware of the pet dog: A case of Staphylococcus intermedius infection. Am. J. Med. Sci., 338: 425-427.
22. Kikuchi, K., C. Piao, I. Itoda, H. Hidai and H. Yamaura et al., 2004. Molecular confirmation of transmission route of Staphylococcus intermedius in mastoid cavity infection from dog saliva. J. Infect. Chemother., 10: 46-48.
23. Wang, N., A.M. Neilan and M. Klompas, 2013. Staphylococcus intermedius infections: Case report and literature review. Infect. Dis. Rep., 5.
24. Orton, D.W. and W.H. Fulcher, 1984. Pasteurella multocida: Bilateral septic knee joint prostheses from a distant cat bite. Ann. Emerg. Med., 13: 1065-1067.
25. Maurelli, M.P., A. Santaniello, A. Fioretti, G. Cringoli, L. Rinaldi and L.F. Menna, 2019. The presence of Toxocara eggs on dog’s fur as potential zoonotic risk in animal-assisted interventions: A systematic review. Animals, 9.
26. Abrahamian, F.M. and E.J.C. Goldstein, 2011. Microbiology of animal bite wound infections. Clin. Microbiol. Rev., 24: 231-246.
27. von Eiff, C., M. Herrmann and G. Peters, 1995. Antimicrobial susceptibilities of Stomatococcus mucilaginosus and of Micrococcus spp. Antimicrob. Agents Chemother., 39: 268-270.
28. Leydcn, J.J., K.J. McGinley, K.M. Nordstrom and G.F. Webster, 1987. Skin microflora. J. Invest. Dermatol., 88: 65-72.
29. Fredricks, D.N., 2001. Microbial ecology of human skin in health and disease. J. Invest. Dermatol. Symp. Proc., 6: 167-169.
30. Ribeiro, M.G., R.M. Risseti, C.A.D. Bolaños, K.A. Caffaro and A.C.B. de Morais et al., 2015. Trueperella pyogenes multispecies infections in domestic animals: A retrospective study of 144 cases (2002 to 2012). Vet. Q., 35: 82-87.
31. Moosavy, S.H., M. Shahi, J. Rafinejad, S. Zare, A. Madani and S. Navidpour, 2016. Epidemiological aspect of scorpion sting in Bandar Abbas, Iran, during 2009-2011. Electron. Physician, 8: 2286-2290.
32. Wilhem, S., M. Kovalik and C. Favrot, 2011. Breed-associated phenotypes in canine atopic dermatitis. Vet. Dermatol., 22: 143-149.
33. Harvey, C.E., 2005. Management of periodontal disease: Understanding the options. Vet. Clin. North Am.: Small Anim. Pract., 35: 819-836.