Emergence of Multi-Drug Resistant Pseudomonas aeruginosa in Broiler Chicks

Pseudomonas aeruginosa is an opportunistic pathogenic bacterium responsible for serious problems in poultry farms and is one of the most relevant pathogens causing human opportunistic infections. Poultry has been suggested to be a reservoir for antibiotic resistance bacteria that may aggravate the problem of pseudomonas infection. The present work was applied to investigate the drug resistance among P. aeruginosa isolated from chicks in different poultry farms and its hazard to human health. A total of 460 broiler chicks constituted 46 private farms were examined for the presence of P. aeruginosa infection. Samples were collected from internal organs of broiler chicks subjected to bacteriological examination and identification. Thirty two P. aeruginosa isolates were recovered from 183 broiler chicks (39.78%) were positive for isolation of P. aeruginosa constituted 32 positive farms with a prevalence of (69.57%). Antimicrobial drug assay was applied against 14 different antimicrobial agents constituted 10 antibiotic genera. The majority of the isolates were sensitive to 3rd generation quinolones (levofloxacin, Enrofloxacin and Danofloxacin) in incidences 81.25%, 59.375% and 46.875% respectively. The sensitivity to Aminoglycosides (Gentamycin and Tobramycin) ranged from 37.5% to 43.75% while Polymyxins showed 34.375%. The least sensitivity was towards Phenicoles (Florfenicol) and Tetracyclines (Doxycycline), 9.375% for each. Antibiotic resistant pattern of the isolated P. aeruginosa revealed that all the isolates were multidrug resistant with MARindices for most isolates was determined to be > 0.6 indicating the misuse of antibiotics in poultry farms. P. aeruginosa isolates showed complete resistance towards cefotaxime, cefradine, nalidixic acid, and spectinomycin (100% for each) with high resistance rates among sulfamethxazole/ trimethoprim, amoxicillin (96.875, 93.75, respectively), doxycycline and florfenicol (90.625 for each), followed by colistine sulphate, gentamycin, tobramycin, danofloxacin, and enrofloxacin with percentages of 68.75, 62.5, 56.25 53.1 3, 40.6% respectively, which all posing a significant threat to public health. In conclusion poultry farms should take strict measures to improve the management of animal nutrition and production hygiene to overcome possible sources of pseudomonas infection. The misuse of antibiotics leads to the development of resistant bacteria that may transfer from poultry to humans. Strict supervision and enforcement of laws to control antibiotic usage in food chain within established safe levels must be done.


Introduction
Pseudomonas aeruginosa is Gram-negative, aerobic, motile, non-capsulated and non-spore forming bacteria. The organisms are ubiquitous, often associated with soil, water and humid environments [1]. P. aeruginosa is the most predominant pseudomonas species causing mortality among birds specially chickens. P. aeruginosa in chickens is associated with respiratory manifestations, diarrhea and septicemia [2]. The organism is ubiquitous, often associated with soil, water, and humid environments. Generally, it is considered to be an opportunistic organism that produces respiratory infections, septicemia and other forms when introduced into tissues of susceptible birds with greatest losses in very young birds [3]. P. aeruginosa is the most common pseudomonad causing infections. Virulent strains can cause diarrhea, dehydration, dyspnea, septicemia and death to newly hatched chicks. The infection may occur through skin wounds or contaminated vaccines, egg dipping or egg inoculation or through contamination of needles used for injection, infection can also spread from infected to susceptible flocks on the same premises under conditions. P. aeruginosa is capable of infecting/colonizing a wide range of ecological niches, including aquatic and soil habitats, animals and plants [4]. P. aeruginosa, is a ubiquitous microorganism, is one of the most relevant pathogens causing human opportunistic infections [5]. Also, P. aeruginosa is one of the most frequent and severe causes of acute nosocomial infections, particularly affecting immunocompromised patients or those admitted to the Intensive Care Unit (ICU) [6]. Likewise, P. aeruginosa is the most frequent and severe driver of chronic respiratory infections in patients suffering from cystic fibrosis (CF) or other chronic underlying diseases [7].
On the other hand, Resistance to antimicrobials is a global public health concern that is impacted by both human and non-human usage. Antimicrobial resistance is an emerging concern to public health, and food-producing animals are known to be a potential source for transmission of resistant bacteria to humans. Recently, the importance of poultry as a source of foodborne diseases and antimicrobial-resistant organisms was highlighted [8,9,10]. With the global increase in antibiotic resistance, there is the need for all countries to preserve the effectiveness of essential antibiotics, especially those that are of critical importance [11,12] for human health. The present work was applied to investigate the drug resistant among P. aeruginosa isolated from chicks as food animal and its hazard to human health.

Sample Collection
A total of 460 broiler chicks (320 freshly dead and 140 living ailing ( were collected from 46 poultry farms from different governorates in Egypt (Giza, Dakahlia, Kaliobia, Monofia, Kena and Aswan). Samples were taken from liver, heart, lung, yolk and bone marrow were collected under complete aseptic conditions and brought to the bacteriology unit of poultry diseases department-Animal Health Research Institute, Dokki, Giza and submitted to bacteriological examination.

Isolation of Pseudomonas
Isolation and identification of pseudomonas from collected samples was done according to Koneman et al., 1997 andQuinn et al., 2011 [13, 14]. In brief, A loopful from tested samples were directly taken and inoculated into nutrient broth then a loopful from the previously incubated tubes was streaked on to the surface of nutrient agar, pseudomonas cetrimide ager and MacConkey agar, and incubated at 37°C for 24 hours. Then a loop full of inoculated nutrient broth was streaked on to pseudomonas cetrimide ager (selective media), nutrient agar and MacConkey agar, incubated aerobically for 24hrs at 37°C. The suspected colonies were identified biochemically according to [14].

Antimicrobial Drug Assay
Antimicrobial in-vitro susceptibility testing of the isolated pseudomonas strains against various chemotherapeutics was screened for susceptibility against 14 antibiotics by disc diffusion method and the interpretation was assigned as sensitive, intermediate and resistant performed according to the recommendations of The Clinical and Laboratory Standard Institute [15]. The antibiotics used for the susceptibility testing are classified by the WHO according to importance to human medicine [16,17] (table 1)

Determination of Multi-drug Resistance Index (MDRI)
MDR index (MDRI) of individual isolates was calculated by dividing the number of antibiotics to which the isolate was resistant by the total number of antibiotics to which the isolate was exposed [18]. Isolates with MDRI values of more than 0.2 were considered highly resistant.

Prevalence of Isolation of P. aeruginosa
In this investigation, the examination of 460 chicks collected from 46 private farms (10 chick per farm) revealed that 32 farms (69.57%) were positive for pseudomonas isolation from which 183 chicks (39.78%) were positive for isolation of P. aeruginosa (table 2).

Antibiogram of the Isolated P. aeruginosa
Sensitivity testing of the isolated P. aeruginosa revealed that most of the isolates were sensitive to Levofloxacin (81.25%) followed by Enrofloxacin (59.375%), Danofloxacin (46.875%) and Tobramycin (43.75%) while, sensitivity to Gentamycin and Colistin sulphate were 37.5%, 34.375% respectively and the least sensitivity was towards Florfenicol and Doxycycline (9.375% for each), table 3.

Multi-drug Resistance Index (MDRI)
The investigation of antibiotic resistant of the isolated P. aeruginosa revealed that all the isolates were multidrug resistant to at least 1 agent in at least 5 antimicrobial categories used recording a multidrug resistant index ranged of 0.4 to 0.9 (table 4).

Drug Resistant P. aeruginosa in Accordance to Public Health Hazard
Studying Antibiotic resistance to different antibiotics among P. aeruginosa isolated from broiler chicks in accordance to their importance to human health (

Discussion
Pseudomonas is a good example of environment associated infection and may cause a serious problem in poultry farms.
Birds at any age may be infected; young birds are most susceptible. Severely stressed or immunodeficient birds and concurrent infections with viruses and other bacteria enhance susceptibility to Pseudomonas infection.
In this investigation, a high prevalence of pseudomonas positive farm (69.57%) with high incidence of pseudomonas isolation from which the examined chicks (39.78%) was detected which constitute a hazard for both poultry and public health These records are much higher than that reported by Saif-Edin, 1983 [19] who isolated the same organism with an incidence of 21.6% at kena Governorate. Mohamed, 1996 [20] found that 13 strains of P. aeruginosa isolated from 150 baby chicks were collected from twenty broiler and balady flocks from different localities at Sharkia province during two years of investigation. One hundred were in diseased conditions while the remaining was freshly dead, the incidence was 8.7%. Mohamed, 2004 [21] and Hebat Allah, 2004, [22] isolated P. aeruginosa from baby chicks at Assiut governorate in percentage of 17.6% for both. Hassan, 2013 [23] examined 150 samples from Hubbard and Ross broiler chickens (130 samples from diseased chickens and 20 samples from apparently normal chickens) and 50 samples from one-dayold chicks (40 samples from diseased chicks and 10 from apparently normal chicks) and Indicated that 38 and 4 isolates of P. aeruginosa were isolated from samples of chickens and chicks, with an incidence of 25.3% and 10% respectively. The elevation in the incidence of isolation indicated increased environmental pollution and decreased in the biosecurity programs applied in poultry farms as most developing countries as Egypt suffering from the release of pharmaceutical waste containing active pharmaceutical compounds from antibiotic manufacturing plants, into the rivers or the environment, which constituted a focus of infection with resistant organisms posing a significant threat to public health [24,25].
On the other hands, Sensitivity testing of the isolated P. aeruginosa revealed that most isolates were mostly sensitive to 3rd generation quinolones (levofloxacin, Enrofloxacin and Danofloxacin) in incidences (81.25%) to (59.375%), (46.875%) respectively while complete resistant to 1st generation quinolones (Nalidixic acid). Also, the sensitivity to Aminoglycosides (Gentamycin and Tobramycin) ranged from 37.5% to 43.75% while Polymyxins showed 34.375%. The least sensitivity was towards Phenicoles (Florfenicol) and Tetracyclines (Doxycycline), 9.375% for each Antibiotic sensitivity of the isolated P. aeruginosa was also detected by many authors. Kurkure et al., 2001 [26] stated that P. aeruginosa isolated from broiler were sensitive to gentamycin and ciprofloxacin in a percentage 88.57% and 62.85% respectively. Abd El-Tawab et al., 2014 [27] reported that P. aeruginosa isolates were sensitive to colistin sulphate and norfloxacin. Also, Tawakol et al., 2018 [28] reported that P. aeruginosa isolates were highly sensitive to ciprofloxacin, colistin sulphate, norfloxacin and gentamycin with percentages of 90%, 90%, 70% and 65% respectively while Doxycycline, penicillin, ceftazidime and streptomycin showed resistance with a percentages of 75%, 65%, 60% and 50% respectively. The most worrisome characteristic of P. aeruginosa is its low antibiotic susceptibility, which is attributable to low permeability of the bacterial cellular envelopes and action of multidrug efflux pumps. In addition to this intrinsic resistance, P. aeruginosa can get resistance by mutation either in chromosomally encoded genes or by the horizontal gene transfers of antibiotic resistance determinants [29,30].
On studying of antibiotic resistant pattern of the isolated P. aeruginosa the results showed that all the isolates were multidrug resistant to at least 1 agent in at least 5 antimicrobial categories used recording a multidrug resistant index ranged of 0.4 to 0.9 (table 4).
Unfortunately, rates of antibiotic resistance in P.
aeruginosa are increasing worldwide [29,31]. However, in the majority of the published studies, multidrug resistance was defined as resistance to at least three drugs from a variety of antibiotic classes, mainly aminoglycosides, antipseudomonal penicillins, cephalosporins, carbapenems and fluoroquinolones [32]. In Egypt, there is a lack of information on the degree of antimicrobial usage in poultry, medications through growth promoters included in their feed and among curative or preventive medicines. Another important element that has been overlooked in developing countries is the release of environmental pollution through pharmaceutical waste containing active pharmaceutical compounds from antibiotic manufacturing plants, into the rivers or the environment, contributes to the selection of antibiotic resistant organisms posing a significant threat to public health [24,25]. Pseudomonas aeruginosa is also an opportunistic pathogen in human [31]. Antimicrobials used in poultry production are employed for therapeutic and nontherapeutic purposes [33,34]. Many of these antibiotics employed in poultry production also serve as essential medicines for use in humans in many countries [33].
On the other hand, Investigating the drug-resistance among P. aeruginosa isolated from broiler chicks in accordance to their importance to human health (table 5), revealed complete resistance towards 3 rd generation Cephalosporins and 1st generation quinolones (Nalidixic acid) followed by amoxicillin, colistine sulphate and 3rd generation quinolones which all classified according to WHO as a critical Important antibiotic for human use (Category I). Also, complete resistance was recorded among 1st generation cephalosporines, (cefradine) which classified as highly important antibiotics (Category II). A high resistance rates among Sulfonamid & Trimethoprim, Tetracyclines Phenicoles, Aminoglycosides and Complete resistance was found in all isolates towards the aminocyclitols spectinomycin which included as an important drug for human medicine (Category III).
Many of these antibiotics that employed in animal production also serve as essential medicines for use in humans in many countries [33,35]. The misuse of antibiotics in food-animal production is one of the most important factors contributing to the global surge and spread in antibiotic resistance. [36,37,38]. With the global increase in antibiotic resistance, there is the need for all countries to preserve the effectiveness of essential antibiotics, especially those that are of critical importance [12,39]. Also, careful use of antibiotics and the establishment of scientific monitoring systems are the best way to limit the adverse effects of the misapplication of antibiotics and to ensure the safety of animal-derived food and environment [40]. On the other hand, more researches for the development of new efficient and safe antibiotic alternatives with studying the effects of combined use of antibiotics and their alternatives for maintaining a healthy agricultural economy and preservation of potent antibiotics for efficacious therapy in human.