Detection of multidrug-resistance Klebsiella pneumoniae isolated from oropharyngeal swab in broiler chickens

Klebsiella pneumoniae is an important pathogen that causes infection in the respiratory system of animals and humans. Livestock and poultry therapy for bacterial infections, such as K. pneumoniae, further increases the risk of microbial resistance to antibiotics and impacts human health globally. This study aimed to detect multidrug-resistant K. pneumoniae in the oropharyngeal swab of broiler chickens. The Samples were collected from 100 broilers in closed-house and open-house system cages (50 samples of each). Identification conducted by fenotipic and genotypic characterization. Isolates characterized are followed by antibiotic sensitivity testing using Gentamicin, Ciprofloxacin, Chloramphenicol, Erythromycin, Clindamycin, and Tetracycline. The study successfully identified 5 Klebsiella pneumoniae isolates. Two isolates (4%) were isolated from closed-house system cages, and 3 isolates (6%) were isolated from open-house system cages. Two isolates from closed-house system cages have shown resistance to tested antibiotics except Chloramphenicol, while the 3 isolates from open-house farms still showed sensitivity to Ciprofloxacin (66%) and Chloramphenicol (33%). According to the results, it can be concluded that multidrug-resistant Klebsiella pneumoniae can be isolated from closed-house and open-housed system cages with different prevalence rates.


Introduction
Poultry is an important industrial sector in Indonesia.The poultry sector has contributed to satisfying the public's need for animal protein by producing meat and eggs.The biggest challenge faced in the poultry industry is infection by pathogenic bacteria that infect the digestive and respiratory systems.The Productivity rate on a chicken farm is primarily determined by farm management [1].The housing system was designed to comfort chickens and protect them from the weather effects (hot and cold), infectious agents, and the influence of other animals and humans.To obtain optimal protection, the poultry industry has begun to develop closed-house system cages to control farm management [2].
Klebsiella pneumoniae belongs to the Enterobacteriaceae family, a facultative anaerobic bacterium, rod-shaped, Gram-negative, non-motile, and produces a capsul.Bacterial cells measuring 0.3-1.0μm in diameter and 0.6-6.0μm in length show mucoid colonies [3].K. pneumoniae typically colonizes the mucosal surfaces (oropharynx and gastrointestinal) of humans and mammals, and widely distributed in the environment (air, plants, insects, and soil).The pathogen was reported to cause liver abscesses, respiratory and urinary tract infections, septicemia, and other diseases [4].K. pneumoniae was integrated into three subspecies, K. pneumoniae subsp.pneumoniae, K. pneumoniae subsp.ozaenae and K. pneumoniae subsp.rhinoscleromatis [5].More than 130 serotypes K. pneumoniae have been identified and 77 serotypes have been recognized as reference strains based on serological reactivity tests [6,7].
K. pneumoniae has been reported as an opportunistic pathogen [8], that can induce severe infection, especially in immunosuppressed hosts [9].K. pneumoniae is one of the most important infectious agents in the poultry industry.Infection in the chicken respiratory tract causes pneumonia and contributes to mortality or decreased production [10,11], mainly in chicks.Therapeutic failures usually produce mortality rates due to the emergence of antibiotic-resistant strains.Resistance in bacteria was brought out by inappropriate administration of antibiotics, which encourages pathogens to mutate and develop antibiotic resistance.The existence of multidrug-resistant strains negatively impacts the sustainability of the poultry industry and human health.Therefore, the research was conducted to explore the existence of multidrug-resistant K. pneumoniae in broiler farms.

Ethical approval
The use of animal experiments and procedures in this study were approved by the animal ethics committee of the Institute for Research and Community Service, Bogor Agricultural University, with approval number: 144/KEH/SKE/VII/2019.

Isolation and identification
One hundred samples were collected from 20-day-old broiler chickens in closed-house and open-house systems (50 samples each) in Aceh Besar district, Indonesia.Samples were collected from oropharyngeal chickens using sterile cotton swabs and cultured in BHIB (Oxoid).Oropharyngeal samples were inoculated into BHIB medium and incubated for 24 hours at 37°C.The bacterial suspension on BHIB media was then cultured on Eosin Methylene Blue agar (Oxoid) and MacConkey agar (Oxoid) medium, and incubated in a candle jar for 24 hours at 37°C.Phenotypic characteristics were identified through colony morphology, Gram staining, catalase test, carbohydrate fermentation (sucrose, mannitol, lactose, and glucose), IMVIC test (indole, methyl red, Voges-Proskauer, indole motility, and citrate), TSIA, and urease.
The phenotypically identified isolates were further confirmed genotypically based on rpoB gene (RNA polymerase β subunit) by using a specific primer pair of F-CAACGGTGTGGTTACTGACG and R-TCTACGAAGTGGCCGTTTTC. Nucleic acids of 5 isolates were extracted by using the boiling method.Amplification carried out in a Bio-Rad thermal cycler.PCR conditions were as follows: Initial activation at 95°C for 3 min, followed by 30 cycles at 95 °C for 30 s, 55 °C for 90 s, and 72 °C for 60 s, and a final elongation at 72 °C for 10 min.The amplification products were separated in 1.5% agarose gel electrophoresis for 30 min at 120 V and visualized using a UV transilluminator.

Antimicrobial susceptibility testing
Antibiotic-resistant evaluation of 5 isolates confirmed K. pneumoniae was conducted according to the Clinical and Laboratory Standards Institute (CLSI) guidelines.The Kirby-Bauer disc diffusion test carried out antibiotic susceptibility testing.Briefly, the inoculum prepared in saline to the density of a McFarland 0.5, and spread onto Mueller-Hinton Agar (Oxoid).Antibiotic disks were placed onto the plates and incubated at 37°C overnight.Each sample was tested against six antibiotics (Oxoid): Gentamicin (10μg), Ciprofloxacin (5μg), Chloramphenicol (30μg), Erythromycin (15μg), Clindamycin (2μg), and Tetracycline (30μg).The Zone of inhibition was interpreted and classified as sensible, resistant and intermediate.

Fenotipic characterization
The bacterial culture onto the media obtained 5 isolates suspected of being K. pneumoniae.K. pneumoniae colonies on MacConkey agar medium showed pink colonies, mucoid, and 3-5mm in size.On EMB agar media, K. pneumoniae colonies showed red-purplish and mucoid (Fig. 1).Based on Gram staining, the bacteria classified as Gram-negative, non-spore-forming bacilli, and surrounded by a thick capsule.

Prevalence of Klebsiella pneumoniae in broiler chickens
The bacterial identification based on phenotypic and molecular characteristics was found in 5 bacterial isolates.Two isolates were found from chickens in closed-house system cages and 3 from chickens in openhouse system cages (Table 1).

Antimicrobial resistance
The antimicrobial resistance test showed extensive drug resistance (XDR) to tetracycline, Erythromycin, Gentamicin, Clindamycin, and Ciprofloxacin.Two isolates from chickens in closed-housed system cages showed sensitive reactions to Chloramphenicol but were resistant to the other's antibiotic.Otherwise, one isolate from an open-housed system cage is still sensitive to Ciprofloxacin, and two isolates are sensitive to Chloramphenicol (Table 2).K. pneumoniae infection in chickens leads to an interesting topic for further research regarding the risk of zoonoses and antibiotic resistance.Bacterial diseases were usually treated with antimicrobial agents.However, excessive and inappropriate use of antibiotics in livestock and poultry has increased bacteria resistance against antimicrobial agents [12,13].Emerged of MDR K. pneumoniae MDR is a serious worldwide phenomenon.In this study, antibiotic resistance in K. pneumoniae isolates reached 66% to 100%.
Almost all isolates from closed-housed system cages resisted almost all antibiotics except chloramphenicol.On the other hand, 3 isolates from open-housed system cages showed 100% resistance to tetracycline, erythromycin, gentamicin, and clindamycin, while chloramphenicol and ciprofloxacin showed 33% -66% resistance.The high level of antibiotic resistance in broiler chickens can occur from the uncontrolled use of antibiotics as Antibiotic Growth Promotors (AGP).Feed in broiler farming is the main component, the poultry feed that contains antibiotics was the main cause of resistance development [14].In addition, resistant strains in livestock and poultry can appear when the host is exposed to resistant strains in the environment [15].Antibiotic Resistance causes K. pneumoniae to be identified as an important disease in public health because of the difficulty in selecting therapeutic agents [16].The bacterial resistance to antibiotics was closely related to the success rate of infectious disease therapy and give impact on health, welfare and economy.Infectious diseases caused by resistant bacteria result in prolonged illness, increased risk of death and longer therapy [17].The World Health Organization (WHO) declared K. pneumoniae as one of the pathogens that most require new types of antibiotics worldwide [18].The potential risk of spreading bacteria from animals to humans and the emergence of multidrug resistance bacteria increased world attention associated with the health issue [12].The resistance of K. pneumoniae can be influenced by the capability of bacteria to produce thick capsules.Polysaccharide capsules play a role in increasing the virulence and pathogenicity of pathogens.The capsule mediated the pathogen to evade the phagocytosis system by polymorphonuclear cells and macrophages [19], and protect the bacteria against drying [20].Capsule and biofilm formation facilitate K. pneumoniae survival through the bactericidal activity of antimicrobial peptides, serum and complement, and phagocytic activity [21].Furthermore, [22) reported an inhibition phagocytosis to K. pneumoniae, indicated by rarely present of macrophage in the lung areas with encapsulated bacteria.
Moreover, the resistance properties of K. pneumoniae are also encoded in plasmid genes and are the main source of resistance genes [23,24].The emergence of hypervirulent K. pneumoniae strains and increasing resistance to antimicrobials expands the host targets and organs susceptible to infection, including in healthy and immunocompetent individuals [24,25].In addition, the emergence of multi-drug resistant K. pneumoniae also increases the risk of spreading resistance genes in the environment [23,24].
Acute respiratory infections are the main cause of mortality and morbidity due to disease.Early diagnosis is very important to be efficient in diagnosis time and avoid inappropriate use of antibiotics [25].Prolonged use of antibiotics in treating respiratory diseases and as growth promoters will cause additional problems in the form of the emergence of strains that are resistant to antibiotics.

Conclussion
Multidrug-resistant K. pneumoniae can be isolated from oropharyngeal broiler chickens in closed-house and open-house system cages.Two isolates (4%) was found from closed-house system cages, and 3 isolates (6%) from open-house system cages.Two isolates from closed-house have shown resistance to tested antibiotics except Chloramphenicol, while the 3 isolates from open-house farms still showed sensitivity to Ciprofloxacin (66%) and Chloramphenicol (33%).

Table 1 .
Prevalence of Klebsiella pneumoniae based on the source of samples.

Table 2 .
Antimicrobial resistance patterns of K. pneumoniae from broiler