The role of hatcheries, hatching eggs and one day old chicks in dissemination of beta lactam antibiotic resistance Pseudomonas aeruginosa (ESBL)

DISSCUSION

In the present study, a total of 55 Pseudomonas isolates (12.2%) were recovered from 450 collected samples collected from infertile egg, dead in shell embryos, baby chicks with omphalitis and hatcheries environment. P. aeruginosa was isolated from young chickens with high mortalities and late dead in shell embryos (Kebede 2010).

The isolation rate of P. aeruginosa was (12%) from dead in shell embryo. Nearly similar results were recorded by Rezk (2010) who isolated P. aeruginosa from dead in shell embryo with total recovery rate (15.9% ). Higher recovery rates were recorded in a previous study carried out by Mohamed (2004), Elsayed et al. (2016); Shahat et al. (2019) who isolated P.aeruginosa from dead in shell embryo with incidence rates of 52%, 21%and 19%respectively. On the other hand, lower incidence of P. aeruginosa 1.2% was mentioned by Amer et al.( 2017) .

In the present study, the incidence of P. aeruginosa in baby chicks was 15%. Similarly, Mohamed (2004) and  Farghal et al. (2017) isolated P.aeruginosa from one-day old chicks with a percentage of 17.6% and13% respectively. Higher result were obtained by Shahat et al. (2019) who isolated P.aeruginosa from young chicks with incidence rates of 42%. However, lower isolation rates 2.5% and 2.83% were reported by Abd El-Tawab et al.(2014); Amer et al. (2017). The isolation rate from hatcheries environment (6.7%) were similar to findings of Eraky et al. (2020) who isolated P. aeruginosa with an incidence  of 8% from hatcheries .

Beta-lactams are a significant antibiotic group used to control and prevent bacterial infections in humans and animals (Hawkey and Jones 2009). Their uses have always been followed by the development of resistance, most commonly caused by beta-lactamases (Bush and Bradford 2016) . ESBLs vary in their hydrolysis of cephalosporins. Therefore, resistance to at least one of them should be considered as positive in the screening test (Drieux et al. 2008; CLSI 2014). Twenty five out of 55 screened Pseudomonas isolates were found to be resistant to one or more of the screening agents, with an overall incidence of 45.45% (25/55) and were designated as (suspect ESBL producers). Incidence of resistance to ceftazidime , cefotaxime, ceftriaxone, and aztreonam was found to be 45.4 5% (25/55), 41.82 (23/55), 29.1% (16/55), and34.55% (19/55), respectively. In a study on antimicrobial sensitivity of 39 P. aeruginosa strains isolated from broilers infections in Egypt , resistance to ceftriaxone, cefotaxime and aztreonam were 74.5%,70.2% and 44.7% respectively .On the other hand, all isolates were sensitive for ceftazidime (Ismail 2016).

Since ESBLs are inhibited by clavulanic acid/sulbactam, tests that use these inhibitors to reverse ESBL resistance were used commonly for phenotypic confirmation of ESBL production (Drieux et al. 2008, CLSI 2014). Of the 25 ‘suspect ESBL producers’, ESBL production was confirmed in 8 isolates by double disk diffusion test. These results were conceded with Ismail (2016)  who reported that ESBLs was confirmed in 6/47 out of the investigated P. aeruginosa isolated from chickens. We were unable to confirm ESBL production in the remaining 17 isolates. These results might be due to concurrent production of other resistance mechanism like ACBLs and metallo beta-lactamases (Al-Ouqaili 2019).

The obtained result revealed that oprL gene was amplified in all the eight studied strains .These results drew near the other previous investigations Hassan ( 2013; Shahat et al. (2019). It considers a specific marker for molecular detection of P.aeruginosa and encodes a protein in the inner and outer membranes, which is essential for the invasion of epithelial cells (De Vos et al. 1997) .There is a strong relationship between the detection of oprL and phenotypic antibiotic resistance due to its implication in efflux transport systems affecting cell permeability (Qin et al. 2003, Lavenir et al. 2007). Also, Exotoxin A (toxA) was detected in all studied strains. This result came in accordance with those recorded by Elsayed et al. (2016), Shahat et al. (2019), Eraky et al. (2020). Exotoxin A (toxA), is the most toxic virulence factor detected in this organism (Dong et al. 2015). It inhibits protein biosynthesis, it has a necrotizing activity on tissues causing cell death and contributes to the colonization process (Michalska and Wolf 2015).

Resistance to -lactams in Gram-negative bacteria is primarily mediated by -lactamases. Many types of -lactamases have been described, but TEM-, SHV, OXA, CMY, and CTX-M-type -lactamases are the most common in Gram-negative bacteria (Bradford 2001). Regarding to the occurrence of Beta-lactamase genes in P. aeruginosa isolates, the obtained result revealed that- blaSHV and blaTEM genes were detected in (8/8) and (7/8) of studied isolates respectively. whereas none of the isolates harbored, blaOXA and blaCTX-M genes. Similarly, previous studies detected TEM-and SHV in P. aeruginosa (Elhariri et al. 2017 Abd El-Tawab et al. 2018).

ESBL P. aeruginosa was found to be resistant to ampicillin, chloramphenicol and trimethoprim/ sulphamethoxazole, tetracycline and streptomycin. While, it was found to be sensitive to Norfloxacin, Ciprofloxacin and colistin sulphate. These results came in accordance with Abd El-Tawab et al. (2014), Farghal et al. (2017), Shahat et al. (2019). Our results detected high multi resistance among isolates which may be due to the production of hydrolytic enzymes and the acquisition of resistance mechanisms by P. aeruginosa strains (Rostamzadeh et al. 2016).

Our experimental study was conducted to confirm pathogenicity of isolated virulent field strain of ESBL producing P. aeruginosa and to assess the efficacy of norfloxacin in controlling infection. Chicks in experimentally infected non treated group showed sleepy appearance, closed eyes, diarrhea. While the gross lesions revealed congestion of all internal organs, petechial hemorrhages on liver and spleen, pericarditis, and enteritis. Our results were parallel to the results of (Mohamed (2004) Eraky et al. (2020). Mortality rate was 43.3% in infected untreated group .Higher mortality rate (70%) were recorded by Eraky et al. (2020).

On the other hand, groups treated with norfloxacin in the drinking water at dose level of 10mg/Kg live body weight led to a great reduction in clinical signs, PM lesions, the mortality rates (20%), decrease in the number of pseudomonas positive birds and lowered the reisolation rate from different organs. Our results is similar to that reported by Badr et al.( 2006); Fatma (2013). Norfloxacin therapy has high therapeutic concentration in serum and tissues after the oral administration (Goossens et al. 1985)and accompanied with a prolonged excretion period, so the duration of the antibacterial activity was effectively prolongs (Avoire et al. 1990 Pecquet et al. 1990) and this explains the significant reduction in the reisolation rate among norfloxacin treated group.

The histopathological examination of lung in infected groups in first week showed congested pulmonary blood vessel with edema and hyperplasia of the lining epithelium of secondary bronchiole similar finding reported by Bakheet and Torra (2020) who reported that lung chick showed interstitial pneumonia with multiple thrombi in pulmonary blood vessels and hyperplasia in bronchial epithelium. At 2^nd week perivascular fibrosis with leucocytic cells infiltration and congestion of pulmonary blood vessels, these finding were supported by Shukla Satish (2016) Mowafy  and El Oksh (2017).The liver in infected group showed multiple areas of coagulative necrosis, focal replacement by erythrocytes and edema in 1^st week .while in 2^nd week, showed vacuolar degeneration of hepatocytes and dilated sinusoids .  Joh et al. (2005) reported that the prominent microscopic lesions in liver of the broilers infected with P. aeruginosa were multiple foci of coagulative necrosis with heterophilic infiltration. Also, Eraky et al. (2020) stated that the liver of chicks experimentally infected with pseudomonas  had dilated hepatic sinusoids, vacuolar degeneration of many hepatocytes, ,mononuclear inflammatory cell infiltration and heterophil. van Delden (2004) reported that elastin protein of connective tissues and its degradation was carried out by Las B enzyme which is an important enzyme of elastolytic activity that produced by P. aeruginosa. That explains the destruction in alveolar wall and blood vessel wall which led to hemorrhagic pneumonia, and destruction of hepatic sinusoids which led to hemorrhage (wide patches of accumulated red blood cells) in hepatic tissue.

The histopathological finding in the heart after 1^stweek from infection in infected group were edema, degenerated myocardial muscles, fibrinopurulent pericarditis. At2^nd week the cardiac muscles showed focal proliferation of connective fibrous tissues with diffuse interstitial infiltration with mononuclear cells, some heterophiles and edema. These findings were in accordance with a previous reported by Joh et al. (2005) who found  the serosal surface was covered with fibrino-purulent exudates, diffuse fibrinous exudates with bacterial colonization in the pericardium. Most histopathological changes of heart and liver were inflammatory in nature that come in agree with Timurkaan et al. (2008) who found inflammatory reaction through heart especially in young birds. Inflammatory cells infiltration mainly lymphocytes and heterophils in the different organs might be due to the antigenic stimulation of pseudomonas (Coles 1986 Hafez et al. 1987)

.

The histopathological finding in the intestine in infected group showed destruction of superficial villus, edema with inflammatory cells infiltration. Similarly Badr et al. (2006) reported that intestine in chicks infected with pseudomonas  showed epithelial hyperplasia of mucosa with cystic formation of goblet cells  .

The histopathological lesions in lung, heart, liver and intestine in infected treated group were less severe as compared to infected groups. fluoroquinolone therapy has the advantages of low minimal inhibitory concentration (MIC) and high therapeutic concentration in serum, tissues and feces after the oral administration (Goossens et al. 1985) accompanied with a prolonged excretion period which may persist for several days after therapy cessation, effectively prolongs the duration of the antibacterial activity (Pecquet et al. 1990). These results was matched with Badr et al. (2006) who reported that treatment of quail experimentally infected with pseudomonas with fluoroquinolone (Ciprofloxacin, Enrofloxacin, and Ofloxacin) reduced severity of lesions found that Liver of ciprofloxacin treated quail showing mild degenerative changes.

Conclusion

This study established the presence of ESBL-P. aeruginosa in infertile egg ,dead in shell embryo and baby chicks . These alarms the public health impact as one day old chicks may serve as vector for transmission of ESBL-P. aeruginosa to human. In addition, P. aeruginosa isolates showed marked resistance to several antibiotics that commonly used in the poultry production in Egypt. Hence, more attention should be paid to risk of bacterial contamination and the possible related effect on hatchability and health of produced one day old chicks. Subsequently, it will reduce the spread of antibiotic resistance to drugs of clinical interest.

The role of hatcheries, hatching eggs and one day old chicks in dissemination of beta lactam antibiotic resistance Pseudomonas aeruginosa (ESBL)

 Ghada Allam Abd EL –Dayem ^*; Ahmed H. Ramadan^** and Hanaa S. Ali ^***

^* Poultry Diseases Department, Animal Health Research Institute (AHRI) (Mansoura Branch), Agriculture Research Center (ARC), Dokki, Giza, Egypt.

^** Bacteriology Department, Animal Health Research Institute (AHRI) (Mansoura Branch), Agriculture Research Center (ARC), Dokki, Giza, Egypt.

^*** Pathology Department, Animal Health Research Institute (AHRI) (Mansoura Branch), Agriculture Research Center (ARC), Dokki, Giza, Egypt.

INTRODUCTION

Hatcheries are vulnerable to being reservoirs of infectious agents because of their central position within the poultry production chain (Wales and Davies 2020). The problem usually starts with contaminated eggs which are incubated under optimal condition for microbiological growth. The rate of embryonic death and vitality of newly hatched chicks are influenced by the extent of bacterial contamination in poultry hatcheries (Gehan 2009, Amer et al. 2017). Various bacterial pathogens include Escherichia coli, Salmonella species, Pseudomonas species Proteus species, and Enterobacter species, have been isolated from dead in shell embryos, baby chicks with omphalitis and hatcheries (Elsayed et al. 2016, Amer et al. 2017, Bakheet et al. 2017).

P.aeruginosa is a profiteer pathogen leads to respiratory infections, septicemia and high mortality of embryos and newly hatched chicks (Dinev et al. 2013 Farghal et al. 2017). Infection of yolk sac by pseudomonas is enhanced by its ability to degrade of other pathogen (Elsayed et al. 2016). Death of the chicks exposed to P. aeruginosa often due to Combinations of several bacteria multiplying in the yolk sac (Sarma 1985) 

P. aeruginosa declares plenty of virulence factors, which share in its toxicity and pathogenicity (Fadhil et al. 2016) .These include enterotoxins, exocytotoxins, and toxins produced by protein secretion systems, as a result of expression of certain virulence operons. Consequently, many of these virulence have been implemented in infection, septicemia, and fatal condition (McCarty et al. 2012; Tayabali et al. 2015) 

The introduction of extended-spectrum cephalosporins (ESCs) improved the treatment options in both veterinary and human medicine (Pfeifer et al. 2010) .Extended spectrum cephalosporins are one of the most important antibiotics in treatment of infections produced by P. aeruginosa isolates. However, resistance to these antimicrobials , mainly conferred by extended-spectrum β-lactamases (ESBL) and Amp C. β-lactamases, has recently gained importance and is considered as an emerging public health concern (Reich et al. 2013) .One-day-old chicks are shown to be a major risk factor for the introduction of ESBL/AmpC-producing bacteria in the broiler production chain (Dierikx et al. 2013) 

One of the main trouble characters of P. aeruginosa is a minor susceptibility to a lot of types of antimicrobials, making it a very hard pathogen to eliminate (Balasubramanian et al. 2013, Khattab et al. 2015) There are several mechanisms of resistance to antimicrobial agents ranging from efflux pump to mobile genetic elements and hydrolyzing enzymes have been described in clinical isolates of P. aeruginosa (Mesaros et al. 2007, Odumosu et al. 2013) .Unfortunately, it can expand their antibiotic resistance by mutations or by acquiring resistance through horizontal gene transfer causing therapeutic failures in humans (Cholley et al. 2010). Various class A ESBLs, such as TEM-, and SHV-, -type ESBLs, and class D ESBLs such as OXA-type ESBLs have been identified in P. aeruginosa (Nordmann and Guibert 1998, Livermore 2002). Therefore, this study was designed to investigate the role of hatcheries, hatching eggs and baby chicks in dissemination of ESBL producing pseudomonas. Moreover, characterization of their susceptibility to antimicrobial agents and evaluate their pathogenicity for baby chicks.

MATERIAL AND METHODS

Samples collection:

A total of 450 samples were collected from different hatcheries and farms in Dakahlia governorate. Out of them, 50 samples from the yolk of infertile egg,150 samples from internal organs (heart, liver, and yolk) of dead in shell embryo, 220samples from ( heart, liver, and yolk ) of diseased and freshly dead chicks (1-7 day) t hat  most of them shown omphalitis and 30 samples from hatcheries environment ( walls and equipments ).

Isolation and Biochemical identification of pseudomonas spp.:

Samples were collected in sterile plastic containers, kept in ice box and transported as soon as possible to the laboratory. Isolation of Pseudomonas isolates was done according to Shukla and Mishra (2015) .The samples were aerobically inoculated into nutrient broth for 24h at 37°C. A loop-full of inoculated broth were streaked onto MacConkey agar and Pseudomonas agar base media and incubated aerobically for 24h at 37°C. The non-lactose fermented colonies were randomly selected and sub-cultured onto nutrient agar plates to observe the pigmentation. Identification of suspected colonies was carried out through Gram staining, biochemical reactions and sugar fermentation.

Preliminary determination of ESBL producing Pseudomonas:

Pseudomonas isolates were subjected to antibiotic sensitivity testing by disc diffusion method on Mueller Hinton agar (Bayer et al. 1966). Inhibition zone diameters were interpreted according to Clinical and Laboratory Standards Institute (CLSI) guidelines. CLSI recommends an initial ‘screening test’ to detect resistance against one or more indicator substrates followed by ‘confirmatory test’ using one or more of the indicator substrates in combination with a betalactamase inhibitor, looking for synergy effects. All isolates were screened for resistance against four antimicrobial agents: cefotaxime (CTX, 30 µg), ceftazidime (CAZ, 30 µg), ceftriaxone (CTR, 30 µg) and aztreonam (AT, 30 µg). Resistance to at least one of the indicator antibiotics was considered as ‘positive’ screening test (Drieux et al. 2008; CLSI 2014).

Phenotypic confirmation of ESBL resistance:

 Isolates that were found positive in ‘screening test’ were subjected to ‘confirmatory test’ by Modified Double Disc Synergy Test (MDDST) by antibiotic discs of amoxicillin /clavulanic acid (30 µg) was placed at the center of the MH agar plate, and antibiotic discs containing cefotaxime (30 µg) and ceftazidime (30 µg) were placed at a distance of 15 mm (centre to centre) from the central disk, amoxycillin/clavulanic acid (30 µg). The plate was incubated at 37°C for 18-24 h. Any distortion or increase in the zone towards the disc of amoxicillin-clavulanate was considered as positive for the ESBL production (Paterson and Bonomo 2005) .

Antimicrobial susceptibility test:

Antibiotic susceptibility tests were performed for ESBL producing P. aeruginosa isolates by using the standard disc diffusion method (Kirby–Bauer) on Mueller–Hinton agar plates. Different antimicrobials were used such as Tetracycline (30 µg), chloramphenicol (30 μg), Ampicillin (10 μg), trimethoprim/ sulphamethoxazole (1.25-23.75µg) , Streptomycin (10 μg), colistin sulphate (10 μg) , Ciprofloxacin (5 μg), and Norfloxacin (10 μg). The interpretation of the measured zone was done according to CLSI (2014).

DNA extraction: DNA extraction from samples was performed using the QIAamp DNA Mini kit (Qiagen, Germany, GmbH) with modifications from the manufacturer’s recommendations. Briefly, 200 µl of the sample suspension was incubated with 10 µl of proteinase K and 200 µl of lysis buffer at 56^OC for 10 min. After incubation, 200 µl of 100% ethanol was added to the lysate. The sample was then washed and centrifuged following the manufacturer’s recommendations. Nucleic acid was eluted with 100 µl of elution buffer provided in the kit.

Oligonucleotide Primer. Primers used were supplied from Metabion (Germany) are listed in table (1).

PCR amplification. For PCR, primers were utilized in a 25- µl reaction containing 12.5 µl of EmeraldAmp Max PCR Master Mix (Takara, Japan), 1 µl of each primer of 20 pmolconcentration, 4.5 µl of water, and 6 µl of DNA template. The reaction was performed in an applied biosystem 2720 thermal cycler.

Analysis of the PCR Products.

 The products of PCR were separated by electrophoresis on 1.5% agarose gel (Applichem, Germany, GmbH) in 1x TBE buffer at room temperature using gradients of 5V/cm. For gel analysis, 20 µl of the PCR products were loaded in each gel slot. Generuler 100 bp ladder (Fermentas, Thermo Scientific, Germany) was used to determine the fragment sizes. The gel was photographed by a gel documentation system (Alpha Innotech, Biometra) and the data was analyzed through computer software.