Phenotypic Study on the Bacterial Isolates from Equine with Respiratory Disorders regarding Antimicrobial Drug Resistance Текст научной статьи по специальности «Биологические науки»

2021, Scienceline Publication

Worlds Veterinary Journal

World Vet J, 11(1): 98-109, March 25, 2021

DOI: https://dx.doi.org/10.54203/scil.2021 .wvj 14

Phenotypic Study on the Bacterial Isolates from Equine with Respiratory Disorders regarding Antimicrobial Drug Resistance

M. Fawzy Nehal1, M. Osman Kamelia2, N. F. Azza 1, R. A. Abd Elmawgoud Shaimaa1, S.A. El Shafii Soumaya1*, M. A. Shahein1 and E. M. Ibraheem1

'Animal Health Research Institute; Equine Bacterial Diseases Unit, Giza, Egypt 2Faculty of Veterinary Medicine, Cairo University, Egypt

"•"Corresponding author's Email: [email protected]; : 0000-0003-1071-8377

ABSTRACT

Upper respiratory tract infection and pneumonia in foals are primarily caused by a bacterial infection. Gramnegative bacteria are commonly found in neonatal pneumonia although gram-positive and mixed infections could be accompanied. The current study aimed to detect the different pathogens causing respiratory disorders in the equine, describe the antimicrobial resistance in these pathogens, and determine the types of antimicrobial isolates. A total of 203 different samples were collected from 42 horse foals, 5 adult horses, and 4 donkey foals from June 2019 to April 2020. All samples were subjected to bacteriology analysis and isolated bacteria were analyzed using susceptibility test for different antibacterial agents. The findings indicated that 38 (74.5%) animals were positive for the isolation of bacteria causing respiratory disorders. The most predominant isolates were Klebsiella pneumoniae subsp. Pneumoniae followed by Staphylococcus aureus, Streptococcus equi, Pseudomonas aeruginosa, Streptococcus zooepidemicus, Proteus mirabilis, Rhodococcus equi, Stenotrophomonas maltophilia, and Streptococcus mitis. Stenotrophomonas maltophilia is isolated from all organs, including the lungs. All K. pneumoniae isolates were sensitive to lomefloxacin, cefotaxime, meropenem, enrofloxacin, neomycin, and chloramphenicol. The Pseudomonas aerugenosa (P. aeruginosa) is sensitive to aztreonam and 20% of isolates sensitive to Piperacillin-tazobactam. All Proteus mirabilis were sensitive to ampicillin-sulbactam, piperacillin-tazobactam, and cefoperazone. Stenotrophomonas maltophilia was only sensitive to oxytetracycline and lomefloxacin. Staphylococcus aureus was susceptible to Piperacillin-tazobactam (50%), 25% to lomefloxacin; Streptococcus equi were sensitive to vancomycin 33.3% while 16.7% to erythromycin and doxycycline, Streptococcus zooepidemicus (100%) were sensitive to cefotaxime, meropenem, and doxycycline. All isolates of Enterococcus species were sensitive to penicillin, piperacillin-tazobactam, and lomefloxacin. Moreover, Rhodococcus equi (one isolate) was only sensitive to clarithromycin. The antimicrobial susceptibility test illustrated the presence of multidrug-resistant and pan-drug resistant isolates which proved the indiscriminate and extensive use of antibiotics. In conclusion, resistance monitoring data and risk assessment identified several direct and/or indirect predisposing factors to be potentially associated with MDR development in the equine health sector of Egypt. The predisposing factors may be attributed to insufficient veterinary healthcare, monitoring, and regulatory services, in addition to the intervention of animal health service providers, and/ or farmers' lack of knowledge about drugs. The misuse and overuse of antibiotics have led to the evolution of antibiotic-resistant bacteria in equine in Egypt.

Keywords: Antimicrobial agents, Klebsiella pneumoniae, Streptococcus zooepidemicus. INTRODUCTION

Substantial morbidity and mortality in foals are commonly due to lower and upper respiratory tract infections that is attributed to the interactions between innate immunologic factors and management risk factors (Galvin and Corley 2010). Neonatal pneumonia is commonly caused by Gram-negative bacteria, although Gram-positive and mixed infections do occur. The development of pneumonia can be complex in the foal as it can be caused by multiple organisms-viruses, bacteria, and even internal parasites (Leguillette et al., 2002).

Pneumonia in foals is primarily caused by a bacterial infection and among all isolates, Streptococcus zooepidemicus and Rhodococcus equi are the most important Gram-positive bacteria. These organisms can be obtained from pure culture or a pleurimicrobial infection. Several other aerobic bacterial species may also occur, including, Actinobacillus spp, Bordetella bronchiseptica, Escherichia coli, Klebsiella pneumoniae, Pasteurella spp, Pseudomonas spp, Salmonella spp., and Staphylococcus spp. (Welsh, 1984). Klebsiella spp. is concerned as a common cause of bacterial pneumonia in horses, but few reports describe the clinical presentation and disease progression (Estell et al., 2016). Strangles is a highly contagious disease caused by the abscess-forming bacteria Streptococcus equi, mainly foals, and horses of any age can also be infected. It seems to cause severe and economically important respiratory disease in horses (Erol et al., 2012; Rush, 2014). One Health (OH) is a vital conceptualization when the intervention that occurs

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between humans, animals, and the environment is considered. The horses' footprint on the well-being of the environment and humans forces the incorporation of the horse in any roadmap to achieve OH (Lonker et al., 2020). Antimicrobial resistance in equine medicine has received relatively limited attention which encourages individuals to indulge in this endeavor to throw light on the situation of microbial resistance in the bacterial community allocated in the respiratory tract of equines.

The aim of this study was to detect the rate of different pathogens causing respiratory disorders in equine and describe the rate of antimicrobial resistance in pathogens, and to determine the type of antimicrobial isolates.

MATERIALS AND METHODS

A database search was performed of submissions to Equine Bacterial Diseases Unit (EBDU) within time interval June 2019 to April 2020 for the bacterial culture of samples from foals, adults, and donkeys (Table 1). Samples were enriched on buffer peptone water and incubated at 37C for 18-24 hours. The enriched samples were cultured on duplicated plates blood agar and staph strep media with strep supplement and 5% sheep blood (UK standard, 2014a). Also, the enriched samples were cultured on mannitol media or Baird Barker media, and plates incubated at 37°C for 24 hours (UK standard, 2014b), and on Tinsdale media at 37°C for 24-72 hours aerobically (UK standard, 2014c). Small Colonies showed p hemolysis or a hemolysis, which were examined for catalase test and oxidase test, golden yellow on mannitol or black colonies with hallow zone on Baird Barker. Non-hemolytic colonies and Tinsdale agar showed small dark brown colonies. Furthermore, enrichment samples were cultured on blood agar, MacConkey agar (UK standard, 2014d), and pseudomonas agar (CN media, UK standard, 2015). All suspected colonies were further biochemical identified using S.R.O. GP24 and S.R.O. GN24 kits (diagnostics.S.R.O.TM).

Susceptibility test for different isolates against antimicrobial agents

The type, symbol, and concentration of antimicrobial agent used were illustrated in supplementary Table 2. Each culture was cultured onto a non-inhibitory agar medium. After incubation at 35°C overnight, four or five well-isolated colonies were selected and transferred to Mueller-Hinton broth and vortex thoroughly, incubated the broth at 35°C, and then adjusted the turbidity (0.5 McFarland standard tube). The procedure continued by using a sterile cotton swab, dipping into the suspension, and culturing over the entire surface of the medium, and rotating the plate approximately 60 degrees after each application. This procedure was repeated three times to ensure an even distribution of the inoculum, CLSI, 2012). The antimicrobial discs were applied to the plates and incubated at 35°C for 16 to 18 hours. The diameter of the zones of complete inhibition was measured. Interpretation of results was recorded according to CLSI (2017). Pareto chart was used to demonstrate the contribution of each type of bacteria in respiratory infections. It was conducted using QI Macros software that has been loaded to the startup directory of Microsoft Office Excel 2013.

Table 1. Type and numbers of samples collected from Table 2. List of antimicrobial disks used for antibiotic private farms in Egypt from June 2019 to April 2020_ sensitivity test_

Total Number of Serial Antimicrobial agents Symbol Concentration (MR )

Period of collection Type of animals Type of samples

number of each type of 1 Penicillin P 10

animals samples 2 Oxacillin OX 1

3 Ampicillin AMP 10

Nasal swabs

Foals 9 9 4 Ampicillin-sulbactam SAM 20

(20 days- Feces 1 5 Ampicillin-clavulanic acid AMC 30

3years) 12 Internal 81 6 Piperacillin-tazobactam TZP 110

6-12/2019 organs* Nasal swabs 7 8 Cephalexin Cephradine CL CE 30 30

Feces Internal 9 Cefotaxime CTX 30

Adults 2 14 10 11 Cefoperazone Cefquinome CFP CEQ 75 30

Subtotal organs* 12 Meropenem MEM 10

23 105 13 Aztreonam ATM 300

Nasal swabs 10 14 Clarithromycin CLR 10

Foals (20 10 feces 10 15 Erythromycin E 15

days- 16 Oxytetracycline OT 30

3years 11 Internal 52 17 Chloramphenicol C 30

1-4/2020 organs* 18 Norfloxacin NO 10

Nasal swabs - 19 Ofloxacin OFX 5

Adults 3 feces - 20 Lomefloxacin LOM 10

Internal 18 21 kanamycin K 30

organs* 22 Novobiocin NV 30

Subtotal 24 90 23 Streptomycin S 10

24 Neomycin N 10

3/2020 Donkey's 4 Nasal swabs 4 25 Amikacin AK 30

foal Feces 4 26 Linezolid LZD 30

27 Clindamycin DA 2

Subtotal 4 8 28 Vancomycin VA 30

Total 51 203 30 Amoxicillin- clavulanic acid Amox-clav 30

*Internal organs: Lung, trachea, liver, spleen, heart, kidney, and Intestine 31 Doxycycline D 30

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RESULTS AND DISCUSSION

Recently, an obvious growing interest in equine breeding and industry in Egypt has been observed, which has a great impact on the healthcare of horses as a whole, and particularly on their respiratory infection.

Pneumonia in equine is most frequently caused by Gram-positive bacteria which may be accompanied by Gramnegative (Estell et al., 2016). Out of 51 horses (203 samples), 38 (74.5%) animals were positive for isolation of bacteria causing respiratory disorders. The rates of different bacteria isolated from different samples in foals and adults are illustrated in Table 3. As can be seen, 36 isolates were obtained (17.7%) which was less than the obtained of Toombs-Ruane et al. (2015, 63%). These different results may be attributed to the different environmental or climatic conditions. About 23.8% of the microorganisms were isolated from samples collected during the period June 2019 to December 2019 and 12.2% collected during the period January 2020 to April 2020. Samples of diseased donkey's foals showed no bacteria. The decrease in the isolation rate may be attributed to slight care of the hygienic management (Saastamoinen et al., 2015).

Klebsiella pneumoniae (K. pneumoniae) showed the highest rate of isolation regarding internal organs (26.3%, Table 4), followed by Staphylococcus aureus (S. aureus), Streptococcus equi subsp. Equi (S. equi subsp. Equi), and Pseudomonas aeruginosa (P. aeruginosa) (10.5%, 4.5%, and 3.8% respectively). Also, Proteus mirabilis (P. mirabilis) and Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) were isolated at the same rate of 2.3%. Nasal samples of foals showed one isolate S. aureus and one isolate of Rhodococcus equi (R. equi) isolated from fecal samples. In adult horses, only Enterococcus species isolated from internal organs had a rate of 6.2% (Table 5). Klebsiella spp are a common cause of bacterial pneumonia but cases are not well-described in the literature, as Estell et al. (2016) stated that mixed infection (polymicrobic infection) is more common in older foals, in which S. zooepidemicus is the most predominant, followed by Actinobacillus suis, and Pasteurella spp. The obtained results of E. coli, Klebsiella pneumoniae were on the contrary with Wood et al. (2005) who found that S. zooepidemicus and S. pneumoniae are the most common ones followed by Actinobacillus, Pasteurella, and Mycoplasma equirhinis.

Stenotrophomonas maltophilia (S. maltophilia) is isolated from all organs, including the lung, for the first time in Egypt . Recently, S. maltophilia is being recorded as a human nosocomial infection causing pneumonia with increasing incidence and has not previously been associated with lower airway disease in the horse. However, Winther et al. (2009) reported the clinical findings, laboratory diagnosis, and response to treatment of seven cases of respiratory infection with S. maltophilia in horses.

Table 6 and Figures 1 and 2 showed the rate of single and mixed infection in dead animals, where 5 animals showed mixed infection with K. pneumoniae and S. aureus (13.1%), also S. aureus with Ps. aeruginosa was a mixed infection in 7.9% of cases. The K. pneumoniae indicated the highest rate of single infection (26.3%). Stenotrophomonas maltophilia (2.6%) as it isolated from all organs, including lung, is isolated in Egypt for the first time. These obtained results were in agreement with those reported by Wilson (2001).

Antimicrobial agent's action occurs by interrupting specific metabolic functions within bacterial cells. There are four primary targets for antimicrobial action, including disruption of cell wall synthesis, inhibition of DNA/RNA synthesis, inhibition of protein biosynthesis, or interference with a crucial metabolic pathway (Roberts, 2005). There has been a scarcity in the studies investigating the antimicrobial resistance profile in the bacteria that have been isolated from the respiratory tract of horses (Johns and Adams, 2015; Älvarez-Narvaez et al., 2020; Lönker et al., 2020).

The K. pneumoniae, isolates were sensitive to lomefloxacin, cefotaxime, meropenem, enrofloxacin, neomycin, and chloramphenicol (15.4%, 13.3%, 13.3%, 6.7%, 6.7%, and 6.7%, respectively, Table 7). Fluoroquinolones are predominantly active against Gram-negative aerobes, including Enterobacteriaceae and Pseudomonas aeruginosa, against Mycoplasma spp., Rickettsia spp., and Ehrlichia spp. They have limited Gram-positive coverage, except for many Staphylococcus spp. (Haggett and Wilson, 2008). Enrofloxacin is the only fluoroquinolone presently in clinical use in horses. Although different doses have been reported in the literature for other fluoroquinolones, there is a lack of reliable data (Bousquet-Melou et al., 2002; Davis et al., 2006; Fernandez-Varon et al., 2006).

The P. aeruginosa is sensitive to aztreonam (100%) and 20% of isolates sensitive to Piperacillin-tazobactam. The monobactams do not have any activity against Gram-positives or anaerobic bacteria. However, they are highly effective against certain Gram-negative bacteria, especially the enteric Gram-negative rods and can be used for Pseudomonas aeruginosa (Chirality, 2012). All Proteus mirabilis (3 isolates) were sensitive to ampicillin-sulbactam, piperacillin-tazobactam, and cefoperazone (100%). Only, 33.3% of isolates were sensitive to enrofloxacin, Stenotrophomonas maltophilia (one isolate) was sensitive to oxytetracycline (Table 7). These results were in accordance with O'Hara et al. (2000) and Deredjian et al. (2016). As can be seen in Table 7, S. aureus was susceptible to Piperacillin-tazobactam (50%) and 25% to lomefloxacin. It was recorded that the bactericidal activity of piperacillin/tazobactam was noticed 1 hour after drug administration for S. aureus, E. coli, and P. aeruginosa (Lemmen et al., 2004). Moreovr, it was found that S. equi (causing strangles) was sensitive to doxycycline and erythromycin (16.7%). S. zooepidemicus was sensitive to cefotaxime, meropenem, and doxycycline (100%), which supported the findings of Lemmen et al. (2004).

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R. equi (one isolate) was sensitive only to Clarithromycin (Table 7). Pneumonia caused by R. equi is a major health problem for equine industries on a worldwide basis. A combination of macrolide with rifampin is recommended for the treatment of infection caused by R. equi (according to the in-vitro activity) when there are no highly effective preventatives (Gigue're et al., 2011). Heatmap analysis showed the intensity of antibiotic resistance of different isolates based on the percentage of resistance (Figure 3). Each row indicates the type of isolate and each column represents the type of antimicrobial agents most of which showed 100% resistance. The phenotypic resistance pattern, prevalence, and diversity of the four Gram-ve bacteria species K. pneumoniae, P. aeruginosa, P. mirabilis, and Stenotrophomonas maltophilia isolates are recorded in Table 8. They were tested for their resistance phenotypic profile against 25 antibiotics representing 9 classes. They were resistant to the 15 antibiotics. Moreover, the five Gram-positive bacteria species isolated from the respiratory tract (S. aureus, S. equi, and S. zooepidemicus), feces Enterococcus spp., and one isolate of R. equi were tested for their phenotypic resistance patterns against 27 antibiotics representing 11 classes (Table 9).

This diversity of Gram-negative bacteria and Gram-positive bacteria isolated from the respiratory tract reflect the capacity of AMR revealed the indiscriminate and extensive use of antibiotics which has led to the emergence and extent spread of resistant pathogenic bacteria (Wolska et al., 2012; Garza-Cervantes et al., 2020). Highly resistant Gramnegative bacteria were Pseudomonas aeruginosa and Klebsiella pneumoniae have become very difficult to treat pathogens (Boucher et al., 2009) and are, therefore, considered as the ESKAPE pathogens (Pendleton et al., 2013), including some Gram-positive bacteria, such as Staphylococcus, S.equi, and S.zooepidemicus as well as Enterococcus species (Coates et al., 2002; Smith and Romesberg, 2007; Hegreness et al., 2008).

In the present study, 11 isolates of K. pneumoneae were typed as multidrug resistance (MDR) and 4 isolates were pan-drug resistance (PDR), all isolates of P. aeruginosa were PDR while all isolates of P. mirabilis and S. maltophilia were MDR (Table 10). While all isolates of Gram-positive isolates were PDR except the two isolates of S. zooepidemicus which were MDR (Table 11). Antibiotic resistance (El Zowalaty et al., 2015; Magiorakos et al., 2012) is classified into MDR which is not susceptible to at least one representative from each of three categories of selected antimicrobial compound families (El Zowalaty et al., 2015; Fodor et al., 2020). Extreme or extensively drug-resistant (XDR) is not susceptible to at least a single representative of all but very few categories of antimicrobial compound families. The PDR is not susceptible to any of the tested or empirical representatives of all known antimicrobial compound families (El Zowalaty et al., 2015).

The MDR and PDR isolates are inconsistent in medical literature, disqualifying reliable comparison of data. In order to reach a standardized definition, we applied the multidrug resistance definition from human medicine (Magiorakos et al., 2012). This adaption was limited by the unattainability of certain susceptibility results and differing antimicrobial agents in human and veterinary medicine. Therefore, the establishment of a standard definition of MDR bacteria in veterinary medicine should be supported.

Table 3. Rate of different bacteria isolated from different samples collected from private equine farms during the period from June 2019 to April 2020

Results

Period of sample collected Number of animals Age of horses Type of samples Total Number of samples Number of positive samples Number of negative samples % of positive results

Foals Internal organs 81 18 63 22.2

21 (20 days- 3 Nasal 9 0 9 0

6-12-2019 2 years) Adults over 3 years Fecal Internal organs Nasal Fecal 1 14 0 0 1 6 0 0 0 8 0 0 0 42.9 0 0

Subtotal 23 105 25 80 23.8%

Internal organs 52 11 41 21.2

11 Foals nasal 10 0 10 0

1-4-2020 Fecal 10 0 10

3 Adults Internal organs Nasal Fecal 18 0 18 0

Subtotal 14 90 11 73 12.2%

Internal organs 0 0 0 0

Donkey's foal Nasal 4 0 4 0

Fecal 4 0 4 0

1-4/2020 4 Internal organs 0 0 0 0

Adult donkeys Nasal 0 0 0 0

Fecal 0 0 0 0

Subtotal 8 0 8

Total 50 - - 203 36 161 17.7%

* Percentage calculated according to total number of each type of samples

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Table 4. Number and type of different bacteria isolated from different samples of foals during the period from June 2019 to April 2020.

Type of samples Number of samples Type of isolated bacteria Number of isolated organisms Percentage*

Nasal swabs 23 Staph. aureus 1 4.3

Fecal swabs 15 Rhodococcus equi 1 6.7

Stenotrophomonas maltophilia 1 0.8

Staph. aureus 8 6.01

Streptococcus. zooepidemicus 3 2.3

Internal organs 133 Streptococcus equi subsp. equi 6 4.5

Streptococcus mitis 1 0.8

Pseudomonas aeruginosa 5 3.8

Klebsiella pneumoniae 15 11.2

Proteus mirabilis 3 2.3

Total 171 - 44 61.9

* Percentage calculated according to the total number of samples

Table 5. Number of different bacteria isolated from different samples in adult horses' equine during the period from June 2019 to April 2020.

Type of samples Number of samples Type of isolated bacteria Number of isolated organisms Percentage*

Internal organs 32 Enterococcus spp. 2 6.2%

Total 32 - 2 6.2%

* Percentage calculated according to the total number of samples

Table 6. Rate of isolated bacteria among infected horses during the period from June 2019 to April 2020.

Type of bacteria Type of positive organs Number of isolates in IO of foals Number of isolates in Fecal swab Number of isolates in Nasal swab Number of isolates in IO of adults Number of positive animals Rate of bacterial isolates*

Rhodococcus equi - 0 1 0 0 1 2.6%

Klebsiella pneumoniae All organs 10 0 0 0 10 26.3%

Staphylococcus aureus All organs 3 0 1 0 4 10.5%

Klebsiella pneumoniae + Staphylococcus aureus Lung + trachea 5 0 0 0 5 13.1%

Streptococcus equi subsp. equi Lung + trachea 3 0 0 0 3 7.9%

Streptococcus equi +Pseudomonas aeruginosa All organs 3 0 0 0 3 7.9%

Streptococcus zooepidemicus Lung 3 0 0 0 3 7.9%

Pseudomonas aeruginosa All organs 2 0 0 0 2

Streptococcus mitis Lung, liver, spleen 1 0 0 0 1 2.6%

Stenotrophomonas maltophilia All organs 1 0 0 0 1 2.6%

Proteus mirabilis All organs 3 0 0 0 3 7.9%

Enterococcus species All organs 0 0 0 2 2 5.3%

Total 34 1 1 2 38 89.5

* Rate of bacterial isolates was calculated according to the total Number of animals (38), IO: Internal organs

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Table 7. Susceptibility antimicrobial agents for different bacterial isolates.

Antimicrobial agents Gram negative bacteria Gram negative

Klebsiella. pneumoniae Pseudomonas aeruginosa Proteus mirabitis Stenotroph omon as maltophiüa Staphylococcus aureus Streptococcus equi Streptococcus zooepidemicus Enterococcus species Rhodococcus equi

No. 1 % No. 1 % No. 1 % No. 1 % No. 1 % No. 1 % No. 1 % No. 1 % No. 1 %

ß-lactam

Penicillins

Penicillin 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 100 0 0

Oxacillin - 0 0 0 0 0 0 0 0 0 0 - - 0 0 0 0

Ampicillin 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

p-Lactam/p-Lactamase Inhibitor Combinations

Ampicillin-sulbactam 0 0 0 0 3 100 0 0 0 0 0 0 0 0 0 0 0 0

Ampicillin-clavulanic acid 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Piperacillin-tazobactam 0 0 1 20 3 100 0 0 4 50 - - 0 0 2 100 0 0

Cephems

Cephalexin 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Cephradine 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Cefotaxime 0 0 0 0 0 0 0 0 0 0 0 0 3 100 0 0 0 0

Cefoperazone 2/15 13.3 0 0 3 100 0 0 0 0 0 0 - - 0 0 0 0

Cefquinome 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Monobactam

Meropenem 2/15 13.3 0 0 0 0 0 0 0 0 0 0 3 100 0 0 0 0

Aztreonam 0 0 5 100 0 0 0 0 - - - - - - 0 0 0 0

Non ß-lactam Macrolides

Clarithromycin 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 100

Erythromycin 0 0 0 0 0 0 0 0 0 0 1 16.7 0 0 0 0 0 0

Tetracyclines

Oxytetracycline 0 0 0 0 0 0 1 100 0 0 0 0 0 0 0 0 0 0

Doxycycline - - - - - - - 0 0 1 16.7 3 100 0 0 0 0

Fluoroquinolones

Norfloxacin 0 0 0 0 1 33.3 0 0 0 0 0 0 0 0 0 0 0 0

Ofloxacin 0 0 0 0 0 0 0 0 1 12.5 0 0 0 0 0 0 0 0

Lomefloxacin 2/13 15.4 0 0 0 0 0 0 2 25 0 0 0 0 2 100 0 0

Enrofloxacin 1/15 6.7 0 0 1 33.3 0 0 /- - - - - - - - 0 0

Aminoglycosides

Kanamycin 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Phenicols

chloramphenicol 1/15 6.7

Novobiocin 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Streptomycin 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Neomycin 1/15 6.7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Amikacin 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Oxazolidinones

Linezolid - - - - - - - 0 0 0 0 0 0 0 0 0 0

Lincosamides

Clindamycin - - - - - - - 0 0 0 0 0 0 0 0 0 0

Glycopeptides

Vancomycin - - - - - - - - 0 0 2 33.3 0 0 0 0 0 0

(-): Not applied

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To cite this papery Nehal MF, Osman KM, Azza NF, Shaimaa RAA, Soumaya SAS, Shahein MA and Ibraheem EM (2021). Phenotypic Study on the Bacterial Isolates from Equine with Respiratory Disorders regarding Antimicrobial Drug Resistance. World Vet. J., 11 (1): 98-109.

Table 8. Phenotypic resistance pattern of Gram-negative bacteria isolated from all samples.

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Pseudomonas aeruginosa

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Pseudomonas aeruginosa

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Proteus mirabilis

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Nehal MF, Osman KM, Azza NF, Shaimaa RAA, Soumaya SAS, Shahein MA and Ibraheem EM (2021). Phenotypic Study on the Bacterial Isolates from Equine with Respiratory Disorders regarding Antimicrobial Drug Resistance. World Vet. J., 11

(1): 98-109.

Table 9. phenotypic resistance pattern of Gram-positive bacteria isolated from different samples

Bacterial isolates ß-lactam ß-Lactam/ß-Lactamase Inhibitor Combinations Cephems Monobactam Macrolides Tetracyclines Phenicols Fluoroquino-lones Aminoglycosides Oxazolidinone Lincosamide Glycopeptide

S nilli •s e Ph

Penicillin Oxacillin Ampicillin Ampicillin-sulbactam Ampicillin -clavulanic acid Piperacillin-tazobactam Cephalexin Cephradine Cefotaxime Cefoperazone Cefquinome Meropenem Clarithromycin Erythromycin Oxytetracycline Doxycycline Chloramphenicol Norfloxacin Ofloxacin Lomefloxacin kanamycin Novobiocin Streptomycin Neomycin Amikacin Linezolid Clindamycin Vancomycin

Staphylococcus aureus R R R R R S R R R R R R R R R R R S S R R R R R R R R

Staphylococcus aureus R R R R R S R R R R R R R R R R R R S R R R R R R R R

Staphylococcus aureus R R R R R S R R R R R R R R R R R R R R R R R R R

Staphylococcus aureus R R R R R S R R R R R R R R R R R R R R R R R R R

Staphylococcus aureus R R R R R R R R R R R R R R R R R R R R R R R R R

Staphylococcus aureus R R R R R R R R R R R R R R R R R R R R R R R R R

Staphylococcus aureus R R R R R R R R R R R R R R R R R R R R R R R R R

Staphylococcus aureus R R R R R R R R R R R R R R R R R R R R R R R R R

Streptococcus equi R R R R R R R R R R S R R R S R R R R R R R R R S R S

Streptococcus equi R R R R R R R R R R R R R R R R R R R R R R R R R R S

Streptococcus equi R R R R R R R R R R R R R R R R R R R R R R R R R R R

Streptococcus equi R R R R R R R R R R R R R R R R R R R R R R R R R R R

Streptococcus equi R R R R R R R R R R R R R R R R R R R R R R R R R R R

Streptococcus equi R R R R R R R R R R R R R R R R R R R R R R R R R R R

Streptococcus zooepidemicus R R R R R R S R S R R R S R R R R R R R R R

Streptococcus zooepidemicus R R R R R R S R S R R R S R R R R R R R R R

Streptococcus zooepidemicus R R R R R R S R S R R R S R R I R R R R R R

Enterococcus species S R R R R S R R R R R R R R R R R R S R R R R R R R R

Enterococcus species S R R R R S R R R R R R R R R R R R S R R R R R R R R

Rhodococcus equi R R R R R R R R R R R R S R R R R R R R R R R R R R R R

R: Resistant, S.: Sensitive, I: intermediate

—105

^oOS39EhS3SapSr: Nehal MF, Osman KM, Azza NF, Shaimaa RAA, Soumaya SAS, Shahein MA and Ibraheem EM (2021). Phenotypic Study on the Bacterial Isolates from Equine with Respiratory Disorders regarding Antimicrobial Drug Resistance. World Vet. J., 11 (1): 98-109.

Table 10. Multidrug resistance profiles of the Gram negative bacterial species isolated from respiratory tract of equines

Number of Number of resistant resistant AB AB classes Antibiotics Number of isolates Type of resistance Total number of Isolates (n = 24)

9 5 P, AMP, S, AMC, TZP, CL, CTX, CEQ, CLR 1 MDR

12 6 P, AMP, AMC, TZP, CL, CTX, CEQ, C, ENR, NV, S, N 1 MDR

16 8 P, AMP, AMC, TZP, CL, CTX, CEQ, MEM, ATM, CLR, C, LOM, ENR, NV, S, N 1 MDR

16 8 P, AMP, AMC, TZP, CL, CE, CEP CEQ, MEM, ATM, CLR, C, LOM, ENR, NV, N 1 MDR

18 8 SAM, TZP, CL, CE, CTX, CEP, CEQ, MEM, ATM, CLR, E, OT, C, NO, OFX, NV, S, N 1 MDR 15 (K.pneumoniae)

20 8 SAM, AMC, TZP, CL, CE, CTX, CEP, CEQ, MEM, ATM, CLR, E, OT, C, NO, OFX, ENR, NV, S, N 2 MDR

20 8 SAM, AMC, TZP, CL, CE, CTX, CEP, CEQ, MEM, ATM, CLR, E, OT, C, NO, OFX, ENR, NV, S, N 2 MDR

22 8 SAM, AMC, TZP, CL, CE, CTX, CEP, CEQ, MEM, ATM, CLR, E, OT, C, NO, OFX, LOM, ENR, NV, S, N, AK 2 MDR

23 9 AMP, SAM, AMC, TZP, CL, CE, CTX, CEP, CEQ, MEM, ATM, CLR, E, OT, C, NO, OFX, LOM, ENR, NV, S, N, AK 4 PDR

23 9 P, OXA, AMP, SAM, AMC, TZP, CL, CE, CTX, CEP, CEQ, MEM, CLR, OT, C, NO, OFX, LOM, K, NV, S, N, AK 2 PDR

22 9 P, OXA, AMP, SAM, AMC, TZP, CL, CE, CTX, CEP, CEQ, MEM, CLR, OT, C, NO, OFX, K, NV, S, N, AK 2 PDR 5 (P. aeruginosa)

21 9 P, OXA, AMP, SAM, AMC, CL, CE, CTX, CEP, CEQ, MEM, CLR, OT, C, NO, OFX, K, NV, S, N, AK 1 PDR

17 8 P, OXA, AMP, AMC, CE, CTX, CEQ, MEM, ATM, CLR, OT, C, K, NV, S, N, AK 1 MDR

3 (P. mirabilis)

16 8 P, OXA, AMP, AMC, CTX, CEQ, MEM, ATM, CLR, OT, C, K, NV, S, N, AK 2 MDR

6 8 SAM, AMC, CE, CEQ, MEM, ATM 1 MDR 1 S. maltophilia)

P: Penicillin, OXA: Oxacillin, Amp: Ampicillin, SAM: Ampicillin-sulbactam, AMC: Ampicillin -clavulanic acid, PRL: Piperacillin-tazobactam, CFX: Cephalexin, CE: Cephradine, CTX: Cefotaxime, CPZ: Cefoperazone, CEQ: Cefquinome, MEM: Meropenem, ATM: Aztreonam, CLR: Clarithromycin, OXT: Oxytetracycline, C: Chloramphenicol, NOR: Norfloxacin, OFX: Ofloxacin, LOM: Lomefloxacin, ENR: Enrofloxacin, K: kanamycin, NO: Novobiocin, S: Streptomycin, N: Neomycin, AK: Amikacin, MDR: Multidrug resistant, PDR: Pan-drug resistant, n: Number, AB: Antibiotic.

Table 11. Multidrug resistance profiles of the Gram +ve bacteria species isolated from respiratory tract and feces of equines

Number of resistant AB Number of resistant Antibiotics AB classes Number of isolates Type of AMR Number of isolates (n = 19)

24 11 P, OXA, Amp, SAM, AMC, CFX, CE, CPZ, TZP, CEQ, MEM, CLR, OXT, NOR, K, NV, DO, NO, S, N, AK, DA, VA, LZD 1 PDR

25 11 P, OXA, Amp, SAM, AMC, CFX, CE, CPZ, TZP, CEQ, MEM, CLR, OXT, NOR, OFX, K, NV, DO, NO, S, N, AK, DA, VA, LZD 1 PDR

25 11 P, OXA, Amp, SAM, AMC, CFX, CPZ, TZP, CEQ, MEM, CLR, E, OXT, NOR, OFX, LOM, NV, DO, NO, S, N, AK, DA, VA, LZD 3 PDR 8 (S. aureus)

25 11 P, OXA, Amp, SAM, AMC, CFX, CPZ, TZP, CEQ, MEM, CLR, OXT, NOR, OFX, LOM, K, NV, DO, NO, S, N, AK, DA, VA, LZD 2 PDR

26 11 P, OXA, Amp, SAM, AMC, CFX, CPZ, TZP, CEQ, MEM, CLR, E, OXT, NOR, OFX, LOM, K, NV, DO, NO, S, N, AK, DA, VA, LZD 1 PDR

23 8 P, OXA, AMP, SAM, AMC, CE, CTX, KF, CEP, CEQ, CLR, E, OTX, C, NOR, OFX, LOM, K, NV, S, N, AK, DA 1 MDR

26 11 P, OXA, AMP, SAM, AMC, CE, CTX, KF, CEP, CEQ, MEM, CLR, E, OTX, DO, C, NOR, OFX, LOM, K, NV, S, N, AK, LNZ, DA 1 PDR 6 (S. equi Equi)

27 11 P, OXA, AMP, SAM, AMC, CE, CTX, KF, CEP, CEQ, MEM, CLR, E, OTX, DO, C, NOR, OFX, LOM, K, NV, S, N, AK, LNZ, DA, VA 4 PDR

18 19 9 9 P, AMP, SAM, AMC, CE, CF, CEQ, CLR, E, OTX, NOR, OFX, K, NV, S, AK, DA, VA P, AMP, SAM, AMC, CE, CF, CEQ, CLR, E, OTX, NOR, OFX, LOM, K, NV, S, AK, DA, VA 1 2 MDR MDR 3 S. Zooepidemicus)

24 11 OXA, AMP, SAM, AMC, CE, CTX, KF, CEP, CEQ, MEM, ATM, CLR, E, OTX, DO, NOR, OFX, K, NV, N, AK, LNZ, DA, VA 2 PDR 2 (Enterococcus)

26 11 P, OXA, AMP, SAM, AMC, CE, CTX, KF, CEP, CEQ, MEM, E, OTX, DO, C, NOR, OFX, LOM, K, NV, S, N, AK, LNZ, DA, VA 1 PDR 1 R. equi

P: Penicillin, OXA: Oxacillin, Amp: Ampicillin, SAM: Ampicillin-sulbactam, AMC: Ampicillin-clavulanic acid, PRL: Piperacillin-tazobactam, CFX: Cephalexin, CE: Cephradine, CTX: Cefotaxime, CPZ: Cefoperazone, CEQ: Cefquinome, MEM: Meropenem, CLR: Clarithromycin, E: Erythromycin, OXT: Oxytetracycline, DO: Doxycycline, NOR: Norfloxacin, OFX: Ofloxacin, LOM: Lomefloxacin, K: kanamycin, NO: Novobiocin, S: Streptomycin, N: Neomycin, AK: Amikacin, LIN: Linezolid, DA: Clindamycin, VA: Vancomycin, MDR: Multidrug resistant, PDR: Pan-drug resistant, n: number, AB: Antibiotic

106

intenral organs of foals 0__0 BR.equi , ^— HK.pneumonea | □ S.aureus 1 \ HK.pneumonea + S.aureus A 2 vôk \ \ H S.equisubsp. Equi S. equi +Ps. areuginosa u S.zooepidemicus 1 ^ ^^^^^^^ e Ps.aeruginosa £ S.mitis H Stenotrophomonas maltopliilia y / / ^^^^^^ ^ P.mirablis u Enterococcus sppcies 3 / 1

Figure 1. Number and type of isolates in internal organs of dead foals

Figure 2. Pareto chart showing the rate of participation of different bacteria in respiratory infections in equine

Antibiotic classes

? S F & 1 I

f

ff t ulllf S fgfSili-l I 1 IlilS I III. Ill3"3

P S 3 ff * ?S F " - " B s ~

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P. aeruginosa P. mirabilis

S. zooepidemicus Enterococcus sp

IS Hi-! ■ J ■ m

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100% of isolates was resistant 85%-85% of isolates was resistant 70% -84% of isolates was resistant

0% resistance of isolates was resistant | 33%-67% of isolates was resistant Not applied

Figure 3. Heat map analysis showed the intensity of resistance of different isolates against different antimicrobial classes

107

CONCLUSION

Stenotrophomonas maltophilia isolated from all organs, including the lung, is one of the first reports of isolation in Egypt. High rates of recorded antimicrobial resistance towards commonly used antibiotics emphasize the importance of individual bacteriological and antimicrobial susceptibility testing to guide antimicrobial therapy. The routine application of antimicrobials in the livestock industry has a dual effect, one acts as an advantage (beneficial for the health and productivity of the animal) while the other is considered as an important disadvantage with a global concern that is the significant evolution of different pathogenic bacterial strains having multidrug resistance (MDR) properties. In the present study, resistance monitoring data and risk assessment identified several direct and/or indirect predisposing factors to be potentially associated with MDR development in the equine health sector of Egypt. Affecting factors are inadequate veterinary healthcare, observing and controlling services, enhancing animal health knowledge among facility providers, and filling farmers' knowledge gap on drugs, and MDR which have resulted in the misuse and overuse of antibiotics leading to the evolution of antibiotic-resistant bacteria in equine in Egypt. Execution of extensive MDR, PDR, and XDR surveillance in equine and awareness programs for farmers along with the strengthening of the capacity of General Veterinary Services are recommended for effective containment of MDR emergence and spreading in the equine health sector in Egypt.

DECLARATION

Competing interests

Authors declare no conflict of interest.

Authors' contributions

Soumaya, S. A. El Shafii was responsible for project administration and validation. Nehal, M. Fawzy, Soumaya, S. A. El Shafii, Azza, N. F. and Shaimaa, R. A. Abd Elmawgoud cooperated in conceptualization, formal analysis, investigation, methodology, and writing the original draft. Kamelia, M. Osman, Momtaz A. Shahin, and Essam Ibrahim were helpful in data curation, writing, reviewing, and editing. All authors reviewed and approved the last edition of article for publishing in the present journal.

Acknowledgments

This paper is based upon work supported by Science, Technology & Innovation Funding Authority (STDF) under the grant (Research Support Grant (STDF - RSG) / Capacity Building Grants).

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