Antimicrobial Resistance in Coagulase-Positive Staphylococci Isolated from Various Animals in Bosnia and Herzegovina

Abstract

The objective of this study was to determine and compare the antimicrobial resistance profiles of clinical Staphylococcus pseudintermedius (n = 90) and Staphylococcus aureus (n = 61) isolates from different animal species in Bosnia and Herzegovina. Among S. pseudintermedius isolates from dogs (n = 86), resistance to penicillin (87.2%) was most common followed by amoxicillin (76.7%) and amoxicillin/clavulanic acid (67.4%). A total of 25 isolates were found to be resistant to oxacillin of which 21 were mecA-positive and multidrug resistant (MDR). The most frequent MDR pattern was penicillins-lincosamides-cephalosporins-macrolides-fluoroquinolones. Among the 61 methicillin-susceptible S. pseudintermedius isolates, 18 were MDR (penicillins-lincosamides-macrolides). Most of S. aureus isolates were resistant to penicillin (50.8%), followed by amoxicillin (41%) and amoxicillin/clavulanic acid (31%). Resistance to cefoxitin was detected in only two isolates. All S. aureus isolates were mecA and mecC-negative. MDR was observed in six S. aureus isolates of which five were from cattle (penicillins-lincosamides-fluoroquinolones). Isolates from red foxes (Vulpes vulpes) were sensitive to most of the antimicrobials tested. The high number of methicillin-resistant S. pseudintermedius and MDR isolates in dogs exposed in this study underlines the urgent need for establishment of national antimicrobial resistance surveillance program in animals in the country, as well as for the surveillance of veterinary antimicrobial consumption.

Introduction

Staphylococcus aureus and Staphylococcus pseudintermedius occur as commensals and opportunistic pathogens in animals and humans. Apart from being one of the most common bacterial pathogens in humans, S. aureus also affects companion and production animals, and consequently has caused financial losses worldwide.^1,2 S. pseudintermedius is the most frequently isolated bacterial pathogen from clinical canine specimens, mainly associated with skin, wound, ear, and urinary tract infections.³ Less commonly, this Staphylococcus sp. has been found in other animals, such as cats, horses, and cattle, as well as in humans.⁴ The increased detection of methicillin-resistant Staphylococcus pseudintermedius (MRSP) and methicillin-resistant Staphylococcus aureus (MRSA) has become a major concern for animal and public health.^5,6 The transmission of MRSA can occur between animals and humans and vice versa, whereas MRSP is not considered to be a major zoonotic pathogen.^7,8 However, both MRSP and MRSA are resistant to a broad range of antimicrobials, which limits therapeutic options of infections and complicates their management.^1,3 MRSP isolates from dogs showed resistance to the major classes of antibiotics used in veterinary medicine. Aside from β-lactams, resistance was observed to aminoglycosides, fluoroquinolones, macrolides, lincosamides, trimethoprim, tetracycline, and chloramphenicol.^3,9 In addition to multidrug-resistant (MDR) MRSP isolates, which have been reported to reach up to 97.8%,⁵ increased MDR methicillin-susceptible Staphylococcus pseudintermedius (MSSP) is a growing concern.^10–13 Although many countries have established antimicrobial resistance surveillance programs⁷ recommended by World Organization for Animal Health,¹⁴ surveillance and monitoring of antimicrobial resistance in animals in Bosnia and Herzegovina (B&H) are still lacking. Considering that resistance rates vary depending on the geographic region, local investigations are required.^13,15 So far, there has been only one reported case of MRSA in animals in B&H, isolated from a household canary bird (Serinus canaria domestica).¹⁶ Apart from a canary bird, there have been no reported antimicrobial susceptibility patterns of S. pseudintermedius and S. aureus from animals in B&H. The objective of this study was to determine and compare the antimicrobial resistance profiles (particularly methicillin resistance and MDR) of clinical S. pseudintermedius and S. aureus isolates from different animal species in B&H.

Materials and Methods

Bacterial isolates

Clinical isolates of S. pseudintermedius (n = 86) and S. aureus (n = 59) from various animals (Table 1) with different clinical conditions, collected in the period 2012–2019 at the bacteriology laboratory of the Veterinary faculty-University of Sarajevo were used in this study. Isolates of S. pseudintermedius (n = 4) and S. aureus (n = 2) obtained from the lungs of red foxes (Vulpes vulpes) during a regular hunting season were also tested. All isolates were identified by standard bacteriological testing, including hemolysis and colony morphology, Gram-stain, the catalase test, coagulase production, maltose fermentation, O-nitrophenyl-β-d-galactopyranoside test (β-galactosidase) and sensitivity to polymyxin B.^17,18 PCR assays were performed as described previously with minor modifications.¹⁹ In brief, for DNA extraction, several colonies of overnight cultures were resuspended in 100 μL of Tris-EDTA (TE) buffer (10mM Tris-HCl, 1mM ethylenediaminetetraacetic acid, pH 8.0), heated at 100°C for 15 minutes and cooled. After centrifugation at 16,100 × g for 3 minutes, 5 μL of the supernatant was used as template for PCR. PCR mixes were made up in 25 μL volumes containing 5 μL extracted DNA, 12.5 μL Taq PCR Master Mix (Qiagen), and 0.2 μM of each primer (Eurofins, MWG, Operon) specific for S. aureus (au-F3, 5′-TCGCTTGCTATGATTGTGG-3′; au-nucR, 5′-GCCAATGTTCTACCATAGC-3′) or for S. pseudintermedius (pse-F2, 5′-TRGGCAGTAGGATTCGTTAA-3′; pse-R5, 5′-CTTTTGTGCTYCMTTTTGG-3′). Analysis was performed in a thermocycler (Applied Biosystems) with an initial DNA denaturation at 95°C for 2 minutes, followed by 30 cycles of 95°C for 30 seconds, 50°C for 35 seconds, 72°C for 1 minute, and with a final extension at 72°C for 2 minutes. The PCR products and 100–1,000 bp DNA size marker (Bio-Rad) were resolved by electrophoresis on 1.5% agarose gel at 70 V for 45 minutes, stained with ethidium bromide, and examined under a transilluminator (VilberLourmat).

Table 1.

Description of the Isolates Used in This Study

Animal species	Clinical site of isolation (No. of isolates)	Staphylococcus spp. (No. of isolates)
Dog	Skin (34), nasal mucose (14), ear (10), wound (8), eye (6), vagina (6), pharynx (4), urine (3), milk (1)	Staphylococcus pseudintermedius (86)
Red fox (Vulpes vulpes)	Lungs (6)	S. pseudintermedius (4)
Red fox (Vulpes vulpes)	Lungs (6)	Staphylococcus aureus (2)
Cat	Skin (2), ear (1)	S. aureus (3)
Parrot	Skin (2)	S. aureus (2)
Cattle	Milk (41), nasal mucose (2)	S. aureus (43)
Sheep	Lungs (4)	S. aureus (4)
Goat	Milk (6), lungs (1)	S. aureus (7)
Total number	151	S. pseudintermedius (90)
Total number	151	S. aureus (61)

Antimicrobial susceptibility testing

The susceptibilities of all staphylococci against nine antimicrobial agents were tested using the disk diffusion method according to the Clinical Laboratory Standard Institute (CLSI) guidelines.²⁰ The following antimicrobials were used: amoxicillin (25 μg), amoxicillin/clavulanic acid (20 + 10 μg), cephalothin (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), erythromycin (15 μg), clindamycin (2 μg), and penicillin G (1 IU) (Condalab). The inhibition zone diameters were interpreted according to the CLSI guidelines.²⁰ Isolates that showed resistance to three or more antimicrobial classes were defined as MDR.^21,22

Phenotypic detection of methicillin resistance in the isolates was assessed using resistance to cefoxitin (30 μg) for S. aureus and to oxacillin (2 μg) for S. pseudintermedius, according to CLSI disk diffusion method and the interpretative criteria.²⁰ In addition, PCR assays specific for detection of mecA and mecC (mecA_LGA251) (methicillin resistance) genes were performed as described previously.²³

Results

Among S. pseudintermedius isolates from dogs (n = 86), resistance to penicillin (87.2%) was most common followed by amoxicillin (75.6%) and amoxicillin/clavulanic acid (67.4%). Of these isolates, 39 (45.3%) were MDR. A total of 25 isolates (29%) were found to be resistant to oxacillin of which 21 (84%) were mecA-positive and MDR (Table 2). The most frequent MDR pattern in oxacillin-resistant isolates was penicillins-lincosamides-cephalosporins-macrolides-fluoroquinolones (15/21; 71.4%). The 21 mecA gene positive S. pseudintermedius isolates were detected in the samples of skin (7/34; 20.6%), nasal mucose (5/14; 35.7%), wound (4/8; 50%), urine (3/3; 100%), and pharynx (2/4; 50%). The majority of MRSP isolates were susceptible to chloramphenicol (18/21; 85.7%); two isolates were sensitive to ciprofloxacin (2/21; 9.5%), and only one isolate to cephalothin (1/21; 4.7%). None of the isolates harbored mecC gene. Among the 61 MSSP isolates, 18 (29.5%) were MDR (Table 2). The most common MDR profile was penicillins-lincosamides-macrolides.

Table 2.

Antimicrobial Resistance in Staphylococcus pseudintermedius Isolates (n = 86) from Dogs

Antimicrobial agent	MSSP (n = 61), n (%) resistant	MRSP (n = 25), n (%) resistant	Total (n = 86), n (%) resistant
β-Lactams
Penicillin	50 (82)	25 (100)	75 (87)
Amoxicillin	41 (67.2)	25 (100)	65 (75.6)
Amoxicillin/clavulanic acid	33 (54)	25 (100)	58 (67.4)
Cephalothin	1 (1.6)	20 (80)	21 (24.4)
Non β-lactams
Clindamycin	27 (44.3)	21 (84)	48 (55.8)
Erythromycin	24 (39.3)	20 (80)	44 (51.2)
Ciprofloxacin	1 (1.6)	19 (76)	20 (23.3)
Chloramphenicol	10 (16.4)	3 (12)	13 (15.1)
MDR	18 (29.5)	21 (84)	29 (33.7)

MDR, multidrug resistant; MRSP, methicillin-resistant Staphylococcus pseudintermedius; MSSP, methicillin-susceptible Staphylococcus pseudintermedius.

Most of S. aureus isolates were resistant to penicillin (50.8%), followed by amoxicillin (41%) and amoxicillin/clavulanic acid (31%). Resistance to cefoxitin was detected in only two isolates (3.3%, cattle n = 1, cat n = 1) (Table 3). All S. aureus isolates were mecA and mecC-negative. MDR was observed in six S. aureus isolates (6/61; 9.8%). MDR isolates from cattle (5/43; 11.6%) showed resistance to penicillins, lincosamides, and fluoroquinolones. MDR pattern in S. aureus from a goat (1/7; 14.3%) was penicillins-macrolides-lincosamides.

Table 3.

Antimicrobial Resistance in Staphylococcus aureus Isolates (n = 59) from Food Producing and Companion Animals

Antimicrobial agent	Cattle (n = 43)				Goats (n = 7)					Sheep (n = 4)					Cats (n = 3)					Parrots (n = 2)
Antimicrobial agent	S	I		R	S	I		R		S	I		R		S	I		R		S	I		R
	n (%)				n (%)					n (%)					n (%)					n (%)
AM	24 (55.8)	0	19 (44.2)		5 (71.4)		1 (14.3)		1 (14.3)	4 (100)		0		0	1 (33.3)		0		2 (66.7)	0		0		2 (100)
AMC	26 (60.5)	0	17 (39.5)		7 (100)		0		0	4 (100)		0		0	2 (66.7)		0		1 (33.3)	1 (50)		0		1 (50)
CEPH	42 (97.7)	0	1 (2.3)		7 (100)		0		0	4 (100)		0		0	2 (66.7)		1 (33.3)		0	2 (100)		0		0
CHL	38 (88.4)	2 (4.6)	3 (7)		7 (100)		0		0	4 (100)		0		0	3 (100)		0		0	1 (50)		0		1 (50)
CIP	22 (51.1)	11 (25.6)	10 (23.3)		7 (100)		0		0	4 (100)		0		0	2 (66.7)		0		1 (33.3)	1 (50)		0		1 (50)
CLI	11 (25.6)	23 (53.4)	9 (21)		1 (14.3)		5 (71.4)		1 (14.3)	1 (25)		3 (75)		0	0		2 (66.7)		1 (33.3)	1 (50)		1 (50)		0
E	11 (25.6)	31 (72.1)	1 (2.3)		2 (28.6)		0		5 (71.4)	3 (75)		1 (25)		0	1 (33.3)		1 (33.3)		1 (33.3)	0		2 (100)		0
FOX	42 (97.7)	0	1 (2.3)		7 (100)		0		0	4 (100)		0		0	2 (66.7)		0		1 (33.3)	2 (100)		0		0
P	18 (42)	0	25 (58)		7 (100)		0		0	4 (100)		0		0	0		0		3 (100)	0		0		2 (100)

AM, amoxicillin; AMC, amoxicillin/clavulanic acid; CEPH, cephalothin; CHL, chloramphenicol; CIP, ciprofloxacin; CLI, clindamycin; E, erythromycin; FOX, cefoxitin; I, intermediate; P, penicillin G; R, resistant; S, sensitive.

Isolates from red foxes were sensitive to most of the antimicrobials tested (Table 4).

Table 4.

Antimicrobial Resistance in Staphylococcus spp. Isolates from the Lungs of Red Foxes (Vulpes vulpes)

Antimicrobial agent	Staphylococcus pseudintermedius (n = 4)			Staphylococcus aureus (n = 2)
	S	I	R	S	I	R
	n (%)			n (%)
AM	3 (75)	0	1 (25)	1 (50)	0	1 (50)
AMC	4 (100)	0	0	2 (100)	0	0
CEPH	4 (100)	0	0	2 (100)	0	0
CHL	3 (75)	1 (25)	0	2 (100)	0	0
CIP	3 (75)	1 (25)	0	2 (100)	0	0
CLI	0	3 (75)	1 (25)	1 (50)	1 (50)	0
E	4 (100)	0	0	1 (50)	1 (50)	0
OX/FOX	4 (100)	0	0	2 (100)	0	0
P	3 (75)	0	1 (25)	1 (50)	0	1 (50)

OX, oxacillin.

Discussion

The prevalence of MRSP varies between different regions^7,9,24 and in some countries reached up to 66.5 and 73%.^10,25 MRSP were more frequently reported in Europe (70.6%) than in other parts of the world.²⁶ A high rate (24.4%) of MRSP isolates in this study is most likely due to uncontrolled use of antimicrobials in the country. The frequency of MRSP isolates is consistent with those recently reported in Spain and Romania (27%).^10,13 However, it was much higher than those in other European countries, such as the Netherlands (7%),²⁷ neighboring Croatia (7.5%),²⁸ Finland (14%),²⁹ and France (16.9).¹² Lower antimicrobial resistance in some countries could be due to more strict monitoring of antimicrobial usage in companion animals when compared with others.¹⁰

Although all oxacillin-resistant isolates were found to be resistant to penicillin and amoxicillin, mecA gene was not determined for four of them. Moreover, all oxacillin-resistant isolates were also mecC-negative. Apart from mec genes, β-lactam resistance in staphylococci can be associated to the presence of blaZ- or blaARL genes. In addition, staphylococci can carry either mecA or blaZ gene, or both.^9,11,30 MRSP isolates from different geographic regions can have dissimilar mecA responses to oxacillin, showing slow, moderate, or robust response regarding oxacillin-induced mecA expression.³¹ The hyperproduction of β-lactamases or penicillin-binding proteins (PBPs) alternatively produced by another type of antibiotic resistance gene or point mutations in PBP genes, as observed in borderline oxacillin-resistant S. aureus, could be related to phenotypic methicillin resistance in the absence of the mecA gene.^32,33

This study revealed penicillin, amoxicillin, and amoxicillin/clavulanic acid resistance in both, S. pseudintermedius and S. aureus, more frequently detected in canine S. pseudintermedius isolates. This finding correlates with the common use of these antimicrobials in B&H, regardless that penicillin is not routinely used for treatment of staphylococcal infections in dogs due to widespread resistance.³⁴ Amoxicillin and amoxicillin/clavulanic acid are among the most often used antimicrobials for dogs in many European countries.³⁵ A high rate of resistance to amoxicillin in this study is not surprising considering that this antimicrobial is the most commonly used drug in B&H for therapy of various conditions in dogs. The percentage of S. pseudintermedius (67.4%) resistant to amoxicillin/clavulanic acid was higher than previously reported in Croatia (7.5%), Portugal (7.6%), Romania (14.3%), and the United Kingdom (47.4%).^10,11,28 This finding corresponds to the frequent use of this antibiotic in dogs in B&H. Canine pyoderma, respiratory infections, as well as some other conditions in dogs are commonly treated with this agent.^5,36,37 In addition, the MSSP isolates from dogs also showed resistance to clindamycin (44.3%) and erythromycin (39.3%), whereas in MRSP isolates the resistance rates were even higher (100% and 95.2%, respectively), which is in agreement with previous studies.^9,26 Increased prevalence of MRSP has resulted in substantial use of clindamycin in treatment of canine skin infections,³⁸ despite that the earliest studies demonstrated very high resistance to this antimicrobial.³⁹ Similar resistance rates to clindamycin and erythromycin could be explained by the resistance mechanism. The methylase gene ermB is responsible for MLS (macrolide, lincosamide, and streptogramin B) resistance in S. pseudintermedius. The expression of this gene can be inducible or constitutive; however, the isolates with constitutive resistance were more frequently detected.^9,38 Clindamycin and erythromycin are unavailable for use in animals in B&H, which indicates constitutive resistance of the isolates. Therefore, the effectiveness of these antimicrobials to treat MSSP and MRSP infections is doubtful.

In contrast, most of the canine isolates were sensitive to chloramphenicol (81.4%). This antimicrobial is not available for parenteral use in animals in B&H and thus, is less commonly used in small animal practice when compared with some other countries. Opposite to our results, previously reported prevalence rate of chloramphenicol resistance among MRSP isolates worldwide was 43.1%,²⁶ whereas in Europe and North America was 57.3%.⁹ MRSP isolates from Europe were often found to be resistant to chloramphenicol, whereas isolates from North America were frequently susceptible to this antimicrobial.^3,9 However, chloramphenicol resistance rates also vary among European countries,^10,28,29 most likely due to frequency of use of this antimicrobial and regional prescribing recommendations. Some countries reported an increase in the resistance to chloramphenicol over time.^11,40 Most of MRSP isolates in this study were recovered from skin infections. Although MRSP can be found in different body sites, it is commonly associated with canine pyoderma,³ and it was isolated in up to 59% of the cases.⁵ The prevalence of MRSP in canine pyoderma was found to be 66.5% in Japan.²⁵ In addition, MRSP is frequently isolated from wound infections.^9,27,41 High rates of MRSP and MDR S. pseudintermedius from urinary tract infections in canines, as well as temporal increase in MDR resistance in MRSP isolates have raised concerns.⁵ The frequent detection of MDR in MSSP (29.5%) and MRSP isolates (100%) in this study is consistent with those from previous studies.^10–13 MDR pattern (β-lactams/lincosamides/macrolides) in MSSP isolates was similar to that in MRSP isolates (β-lactams/lincosamides/macrolides/fluoroquinolones). However, the main difference between them was resistance to cephalothin and ciprofloxacin. The percentage of MSSP isolates susceptible to cephalothin and ciprofloxacin was high (88.5%), which corresponds with the fact that these antimicrobials are rarely used in small practice in our country. On the contrary, almost all mecA-positive isolates exhibited resistance to cephalothin and ciprofloxacin. Cephalothin resistance was not unexpected as mecA is responsible for β-lactams resistance of MRSP, including cephalosporins,¹ which are frequently used in dogs in Europe.³⁵ These drugs are the most commonly prescribed antimicrobials for off-label indications in the European Union (EU).⁴² High ciprofloxacin resistance (87.4%) was observed in MRSP isolates from Europe and North America.⁹ An increase in the resistance rates for fluoroquinolones has been reported in several European countries,^11–13 which correlates with routine use of fluoroquinolones in small animal practice.³⁵ The high prevalence of MDR probably reflects worldwide dissemination of the sequence type 71 (ST71), which is also the most frequently identified clone in Europe. The ST71 clone is typically MDR and considered to be more fluoroquinolone-resistant than other STs.^13,34,43However, new clones have emerged, including the ST496 clone in France, which is resistant to all veterinary-licensed antibiotics.⁴⁰ Considering the variations in antimicrobial resistance among the different MRSP lineages and that therapy for the infections dependent on the clonal type involved,²⁶ further studies should include identification of MRSP clones associated with canine infections in B&H.

Among S. aureus isolates from food producing animals, resistance to penicillin was frequently detected in cattle isolates (25/43; 58%), mainly recovered from milk samples (95%). In B&H, various infections in cattle are commonly treated with this antibiotic. Despite the high percentage of penicillin resistance in S. aureus from bovine mastitis (36%), penicillins are the second most used antimicrobial class in food-producing animals in the EU.^44,45 In this study, the highest sensitivity was observed for cephalothin and cefoxitin (97.7%), followed by chloramphenicol (88.4%). All three antibiotics are unavailable for use in B&H and chloramphenicol is not authorized for use in food-producing animals.⁴⁶ Very high susceptibility to first-generation cephalosporins was also reported for S. aureus in other countries in Europe.⁴⁵ Prevalence of MRSA in dairy milk is considered to be low in Europe⁴⁷ and varies between 0% and 13.8%.⁴⁴

This study detected only one S. aureus isolate resistant to cefoxitin and to all antimicrobials except ciprofloxacin. However, mecA and mecC genes were not detected. Similar to our results, of 192 S. aureus isolates collected from cases of clinical mastitis in dairy cows across Europe, only five (2.6%) were resistant to oxacillin, whereas two were negative for mecA.⁴⁷ Nearly half of S. aureus isolates from cattle, and all from sheep and goats showed sensitivity to ciprofloxacin that is rarely used in B&H. Since fluoroquinolones are critically important antimicrobial agents, they should not be used as a first-line treatment and without prior antimicrobial susceptibility testing.⁴⁸ Only one S. aureus from companion animals was resistant to cefoxitin, but negative for mecA and mecC genes. This isolate was recovered from skin infection of a cat and showed resistance to penicillin, amoxicillin, amoxicillin/clavulanic acid, and ciprofloxacin.

To the best of our knowledge, this is the first study reporting the antimicrobial resistance profiles of S. pseudintermedius and S. aureus isolated from the lungs of red foxes. In foxes, S. pseudintermedius was more frequently identified when compared with S. aureus.^49,50 S. pseudintermedius was isolated from rectal swabs of foxes,⁴⁹ but it was also associated with meningoencephalitis and nephritis in these canine.^51,52 These observations support that foxes, same as dogs, are the natural host of S. pseudintermedius that may act as canine commensal and opportunistic pathogen.⁴ In our study, the fox isolates generally displayed susceptibility to the antimicrobials. Resistance to penicillin and amoxicillin was observed for two isolates and no MRSP or MRSA were identified. In the U.K. study, all isolates from the foxes were coagulase-negative, resistant to methicillin, and mecA was detected in 89% of isolates.⁵³ Regarding S. aureus in foxes, most of isolates were found to be MSSA and so far, only one mecC-MRSA was identified.^50,54 Previous studies also reported a high level of sensitivity of S. pseudintermedius from foxes.^51,52,55 The findings in this and previous studies suggest that the foxes were not directly exposed to clinical antimicrobial agents. However, wild animals can acquire antimicrobial-resistant bacteria through contact with humans, animals, and the environment and, therefore, may serve as reservoirs of antibiotic resistance.⁵⁶

Conclusion

The high number of MRSP and MDR isolates in dogs exposed in this study underlines the urgent need for establishment of national antimicrobial resistance surveillance program in animals in B&H, as well as for the surveillance of veterinary antimicrobial consumption. The antimicrobial patterns of S. aureus observed in food producing animals, along with the uncontrolled movement of animals and the relative absence of disease control measures imply a requirement to monitor antimicrobial susceptibility patterns in staphylococci to ensure appropriate antimicrobial treatment and to prevent antimicrobial resistance.

Footnotes

Disclosure Statement

The authors declare that no competing financial interests exist.

Funding Information

This study was partly supported by project (Grant No. 11-05-14-20526-1/16) “Methicillin-resistant and multi-resistant coagulase-positive staphylococci isolated from veterinary clinical samples in the Sarajevo Canton” of the Ministry of Education, Science and Youth of Sarajevo Canton, Bosnia and Herzegovina.

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