UMYU Journal of Microbiology Research

E-ISSN: 2814 – 1822; P-ISSN: 2616 – 0668

ORIGINAL RESEARCH ARTICLE

Characterization of Mammaliicoccus sciuri and Mannitol-Fermenting Staphylococci from Small Ruminants and Chickens in the Federal Capital Territory, Nigeria

Martha Echioda-Ogbole^1, James Agbo Ameh ¹, Samuel Mailafia ¹, Olatunde Hamza Olabode¹, Bridget Maria Jessica Adah¹, Ikoojo Charity Ikwe-Agada¹, Odey E Odey¹and Edet E. Udo²

¹Department of Veterinary Microbiology, Faculty of Veterinary Medicine, University of Abuja, Nigeria.

²Department of Microbiology, College of Medicine, Kuwait University, Kuwait

* Correspondence: echioda.martha@uniabuja.edu.ng,

Abstract

Mammalicoccus and Staphylococcus species are Gram-positive bacteria found on the skin and mucous membranes of some mammals. This study aimed to identify, determine the species distribution, and assess the antimicrobial profile of mannitol-fermenting non- S. aureus species from chickens, sheep, and goats in the Federal Capital Territory, Nigeria. Forty-seven isolates from chickens (n = 15), sheep (n = 18), and goats (n = 14) were screened using the Staph Latex Kit (Labmann, UK) and identified using the Analytical Profile Index Kit. Antimicrobial susceptibility testing (AST) was performed using disk diffusion and E-test methods. Mammalicoccus sciuri, Staphylococcus intermedius, and Staphylococcus xylosus were the three species identified. Mammaliccoccus sciuri was found to be the most predominant species with 79% (n=37) prevalence, followed by Staphylococcus intermedius with 15% (n = 7), while S. xylosus was the least common with 4.25% prevalence. The AST results showed complete sensitivity of all isolates (100 %) to cefoxitin, penicillin, gentamicin, kanamycin, rifampicin, spectinomycin, and ciprofloxacin. Isolates were, however, resistant to trimethoprim (48.93%), tetracycline (15%), erythromycin (9%), and amikacin (4%). Ten percent of the isolates exhibited multidrug resistance. This study documents a high occurrence of Mammalicoccus sciuri in small ruminants and chickens. Periodic AST should be conducted to determine the level of antimicrobial use in food animals and to facilitate effective monitoring and reporting of AMR in animals.

Key words: Mammaliicoccus, Staphylococcus, chickens, sheep. Goats, antimicrobial resistance

INTRODUCTION

Mammaliicoccus is a recently reassigned genus under the family Staphylococcocea (Madhaiyan et al., 2020). The novel genus was formally referred to as the Staphylococcus sciuri group, including S. vitulinus, S. lentus, S. fleurettii, and S. stepanovicii (Adesoji et al., 2024). Mammalicoccus and Staphylococcus are commensal bacteria found on the skin and mucous membranes of humans and animals (de Moura et al., 2023). Although both species are considered to be of low virulence, they are increasingly becoming pathogenic and are reported to be implicated in life-threatening animal and human infections (Bora, 2018). They have established themselves as important pathogens, exhibiting increasing trends towards antibiotic resistance (May et al., 2014).

Mammaliicoccus sciuri is the most common species of the genus Mammaliicoccus and has been reported to cause mastitis and metritis in cattle (Schnitt et al.,2021), septicemia in poultry and wild animals such as tigers, buffaloes, and elephants (Singh et al., 2024). Staphylococci are a diverse group of bacteria that can cause a wide spectrum of diseases in mammals, resulting in an increased healthcare burden (Frey et al., 2013). Aside S. aureus, which is reported to be mostly incriminated in cases of mastitis and skin infections, other species of staphylococci such as S. epidermitis, S. intermedius, S. hyicus, and S. delphini have been reported to cause mastitis and pyoderma in cows, horses, pigs, goats, and dogs (Frey et al., 2013). S. xylosus and S. gallinarum have been reported to be associated with endophthalmitis and pyelonephritis in chickens (Vela et al., 2012). These infections are sometimes very difficult to treat because staphylococci rapidly develop resistance to many antimicrobial agents to which they were once susceptible to (Echioda-Ogboleet al., 2025). The development of resistance to antimicrobial agents by Staphylococci are due to a number of mechanisms, including the production of beta-lactamase enzymes which hydrolyses the amid bond of beta-lactam antibiotics thereby inactivating the drug (Torimiro and Olusayo, 2017), acquisition of chromosomal mecA gene carried on staphylococcal cassette chromosoomemec (SCCmec) which encodes a low- affinity penicillin binding proteins (PBP2a), associated with methicillin and oxacillin resistance (Zhu et al ., 2017). Mammaliicoccus sciuri is a known reservoir for the mecA gene and a potential carrier of other antimicrobial and virulence genes to S. aureus (de Moura et al., 2023).

Mannitol fermentation is a species signature of S. aureus, use in low-resource settings along with the tube coagulase test (TCT) for the identification of the organism. Although these traditional test methods have been reported to identify S. aureus (Echioda-Ogbole et al., 2018), their performance and results are subjective and vary from setting to setting (Bello and Qahtani, 2006). Studies have shown that some coagulase-negative staphylococci (CoNS) species can ferment mannitol and produce golden-yellow colonies typical of S. aureus due to the high salt content of the Mannitol salt agar (Thakur et al., 2017). There is a dearth of information on mannitol-fermenting non- S. aureus species in the study area. Hence, this study was undertaken to identify Staphylococci species other than S. aureus from chickens, sheep, and goats in the Federal Capital Territory, Nigeria.

MATERIALS AND METHODS

Sample collection

Simple random sampling was used to collect 684 samples from chickens, sheep, and goats from livestock markets in the six Area Councils of the FCT. The samples consist of 228 nasal swab samples from sheep and goats each, and 228 trachea swabs from chickens. A sterile swab stick was inserted into the nostrils of sheep and goats, and the trachea of chickens, and then gently rubbed against the mucosal surface for approximately 4 to 5 seconds. Individual swab sticks were placed in sterile cryo-vials containing Mueller-Hinton broth (Oxoid, UK) supplemented with 6.5% Sodium Chloride (NaCl), appropriately labeled, and placed in a flask containing icepacks. They were then immediately transported to the Department of Veterinary Microbiology laboratory, University of Abuja, for processing.

Bacteria isolation and identification

All bacteriological culture media (Oxoid, Basingstoke, Hampshire, UK) were prepared according to manufacturers' instructions. Each sample was analyzed individually by inoculating it into 5ml of Muller Hinton broth and incubating at 37°C for 24 hours. A loopful of about 10-μl of the inoculum from Mueller Hinton broth (Oxoid, England) was streaked onto prepared plates of Mannitol salt agar (Oxoid, Basingstoke, Hampshire, UK) and incubated at 37^°C for 24 hours. Following incubation, all cultured plates were examined for colonial morphology and pigmentation. Smooth, shiny, convex, distinct golden yellow colonies were picked with a sterile wire loop and transferred onto nutrient agar plates incubated at 37^°C for 24 hours to obtain a pure culture. The pure isolates were inoculated on nutrient agar slants, incubated at 37^°C for 24 hours, and then stored in the refrigerator at 4^°C for further characterization.

Biochemical identification of isolates

Preliminary identification of the isolates was done based on Gram reaction (microscopic appearance) and biochemical characterization based on catalase, oxidase, and coagulase tests as described by Cheesbrough (2016). Isolates were further screened using the Staph Latex Kit (Labmann, UK).

Identification of mannitol-fermenting Staphylococcus species

The Analytic Profile Index (API) Kit, a standardized system utilizing miniaturized biochemical tests and a specially adapted database, was employed for the identification of Staphylococcus, Micrococcus, and Kocuria. The test strip consists of 20 microtubes containing dehydrated substrates including D-glucose, D-fructose, mannose, maltose, lactose, D-trhalose, mannitol, xylitol, melibiose, nitates, Vogesproskauer, raffinose, xylose, saccharase, methyl-α-D-glucopyranoside, N-acetyl-glucosamine, Arginine DiHydrolase, and Urease. Forty-seven mannitol-fermenting non-S.aureus isolateswere characterizedusing the API Kit (BioMerieux, Marcy-I’Etoile, France).

Antimicrobial susceptibility testing

Antimicrobial susceptibility testing was performed using the disk diffusion method of Kirby-Bauer with some modifications and in accordance with the guidelines of the Clinical Laboratory Standards Institute (CLSI, 2018).The following antibiotic disks with their corresponding concentration were tested: Cefoxitin (30 μg), Penicillin (10 units), Gentamicin (10 μg), Amikacin (30μg), Erythromycin (15 μg), Clindamycin (2 μg), Chloramphenicol (30 μg), Tetracycline (30 μg), Trimethoprim (25μg), Rifampicin (5 μg), Spectinomycin 100μg, Fusidic acid (10 μg), Ciprofloxacin (5 μg), Kanamycin (30 μg).Minimum inhibitory concentrations (MICs) for Vancomycin, Teicoplanin, Linezolid, and Tigecycline were determined using E-test strips (BioMerieux, Marcy-I’Etoile, France) according to the manufacturer’s instructions. The results were interpreted according to Clinical and Laboratory Standards Institute guidelines (CLSI, 2020).

RESULTS

Forty-seven mannitol-positive isolates were identified as S. sciuri (M. sciuri), S. intermedius, and S. xylosus. Of the three species, M. sciuri was found to be the most predominant with 79% prevalence (n = 37), followed by S. intermedius with 15% (n = 7), while S. xylosus was the least common with 4.25% prevalence (Table 1).

Table 1: Prevalence of mannitol-fermenting non-S. aureus species and their percentage identity range

Key: %= percentage, S. index= similarity index

Of the 37 M. sciuri species, 10 (27%) were recovered from chickens and goats each, while 17 (46%) were from sheep. S. xylosus was isolated only from chickens (n = 2), giving a prevalence of 4.3%, while the distribution of S. intermedius in chickens, sheep, and goats are 29% (n = 2), 43% (n = 3) and 29% (n = 2), respectively.

The Antimicrobial susceptibility testing results showed complete sensitivity of all isolates (100 %) to cefoxitin, penicillin, gentamicin, kanamycin, rifampicin, spectinomycin, and ciprofloxacin. Isolates were, however, resistant to trimethoprim (48.93%), tetracycline (15%), erythromycin (9%), amikacin (4%), with 2% resistance to chloramphenicol and mupirocin (Table 2).

Table 2: Antimicrobial susceptibility of mannitol-fermenting Staphylococcus species from food animals in FCT

KEYS: % = percentage positive, µg = microgram

Eight (8) resistance phenotypes were observed among the other Staphylococci isolates, and 5 (10.63%) of the isolates exhibited multidrug resistance. Three (20%) of the multidrug-resistant isolates were from chickens, while 2 (11.11%) were from sheep. None of the isolates from goats were multidrug resistant (Table 3).

Table 3: Antimicrobial resistance profiles of mannitol-fermenting Staphylococcus species from food animals in FCT

KEYS: * = multidrug resistant, Em = erythromycin, TET = tetracycline, TMP = Trimethoprim, C = chloramphenicol, MUPH = high level mupirocin resistance, AMC = amikacin

DISCUSSION

In this study, Mammaliicoccus sciuri was found to be the most predominant mannitol-fermenting non-S. aureus specie identified, followed by S. intermedius, while S. xylosus was the least common. All three strains were detected in chickens, while two species (S. sciuri and S. intermedius) were detected in sheep and goats, as S. xylosus was not detected in either sheep or goats in this study. This study differs from that of Mamza et al. (2020) in northeastern Nigeria, who reported S. sciuri in sheep and goats with a lower prevalence of 3.6 and 7.2%, respectively. In other part of the world, both S. sciuri and S. xylosus have been reported from different food animals such as chickens (Nemeghaire et al., 2014), sheep (Wesołowska et al., 2023) and goats (Egyir et al.,2022). Staphylococci species other than S. aureus (SOSA), such as S. xylosus, S. intermedius, S. cohnii, S. epidermidis, S. hyicus, S. lentus, S. haemolyticus, and S. lugdunensis are reported more in pigs in Nigeria than in small ruminants and chickens (Lawal et al., 2021).

Although M. sciuri is a primary animal bacterial species, its clinical relevance is increasing due to its pathogenic potential, and it has been reported to be associated with fatal infections in animals (Sands et al., 2022) and in humans (Jesumirhewe et al., 2024). S. intermediusis a coagulase positive Staphylococcus frequently misidentified as S. aureus and has been isolated from cases of dog bites in humans(Wang et al., 2013). All three staphylococcal species isolated in this study have been reported to be part of the normal floral of the skin and mucous membranes of different animal species, but with serious zoonotic potentials (Chen et al., 2017). A recent study by Battalia et al. (2023) involving genomic analysis of S. xylosus identified a number of loci in the organism with similar homology to known virulence factors of S. aureus, which shows that S. xylosus has more pathogenic potential than M. sciuri.

The AST results in this study showed complete sensitivity of all the isolates to cefoxitin, penicillin, gentamicin, kanamycin, rifampicin, spectinomycin, ciprofloxacin, vancomycin, teicoplanin, linezolid, and tigecycline. This finding is similar to that reported by Zhou et al. (2017), who reported high susceptibility of staphylococcal isolates from goats in China. The high susceptibility of isolates in this study could be attributed to the fact that the chickens, sheep and goats sampled in this study are home grown, owned by small holder farmers who may not have used antimicrobials, and thus, shows that the animals have not been exposed to cefoxitin, penicillin, gentamicin, kanamycin, rifampicin, spectinomycin, ciprofloxacin, vancomycin, teicoplanin, linezolid and tigecycline antibiotics. However, 48 % of the isolates were found to be resistant to trimethoprim, tetracycline resistance was also seen in 15% of the isolates, while erythromycin and amikacin resistance were observed in 9 % and 4% of the isolates, respectively. In this study,10.63% of the isolates exhibited multidrug resistance; three of the multidrug-resistant isolates were from chickens, while 2 were from sheep. None of the isolates from goats were multidrug-resistant. Resistance to these agents could be attributed to misuse of the antimicrobial agents in the study area or due to environmental exposure (Panyako et al, 2022). Clinical isolates of M. sciuri have been previously reported to be multidrug-resistant, carrying antimicrobial resistance genes against major classes of antibiotics such as oxazolidones, phenicols, and aminoglycosides (Li et al., 2016).

CONCLUSION

In conclusion, this study has shown that M. sciuri, S. intermedius, and S. xylosus are predominant mannitol-fermenters found in the upper respiratory tracts of small ruminants and chickens. Mamaliicoccussciuri was found to be most common with 79 % prevalence, followed by S. intermedius with 15 % prevalence, while S. xylosus was the least common with 4.2 % prevalence.

FUNDING

This research work was supported by the Institutional Based Research Grant (TETF/DR&D/CE/UNI/ABUJA/IBR2021/VOL.1).

CONFLICT OF INTEREST

All authors hereby declare that they do not have any conflict of interest.

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