Hygienic Studies on Biofilms in Drinking Water Systems in Poultry Farms: Isolation, Molecular Identification, and Antibiotic Sensitivity

| In poultry production, drinking water must be free from pathogens that pose a risk for infection. Biofilms are significant contributors to water contamination with pathogens, and aid in the genetic exchange among bacterial populations that cause antibiotic resistance. The study included four-layer chicken houses receiving the same water source: A growing pullet house with iron water pipes and three production layer houses with polyvinyl chloride (PVC) water pipes. Biofilm samples were collected, during February 2020, by swabbing the inner surfaces of drinking water pipes. The heterotrophic bacterial counts were determined. Afterwards, colonies were purified and molecularly identified using the 16S rRNA by PCR test. A total of 31 antimicrobials were used for antibiotic sensitivity testing of the bacterial isolates. In the PVC pipes, more bacterial densities were found than in the iron pipes (2×10 19 and 2×10 12 colony forming units/ml, respectively). Pseudomonas , Enterococcus , Staphylococcus , and Sphingopyxis were identified from iron pipes, while Acinetobacter , Pseudomonas , and Bacillus were confirmed from PVC pipes. Multidrug resistance to at least three antibiotic groups was identified in 67% of the isolates. Staphylococcus , Enterococci , Sphingopyxis , Bacillus , and Acinetobacter were found to be originated from water sources highly contaminated with antibiotics overuse. While all Pseudomonas strains originated from water environments free from antibiotics contamination. In terms of bacterial density and antibiotic resistance patterns, biofilms possess a significant role in harboring and disseminating pathogenic strains leading to production problems in poultry. So, programs for the prevention and control of biofilm buildup in poultry drinking systems are required.

Water is an essential nutrient for poultry. Biofilm could induce blockage of the water system and, subsequently, impair the adequacy of water flow as well as the flock performance (Fairchild and Ritz, 2009). The resistance of organisms in biofilm increases toward disinfection and medication, so the biofilm becomes a repository for the continuous dissemination of bacteria within the water flow to form biofilms in other parts of the pipelines and spreading pathogenic bacteria along the drinking line. In many cases, the aesthetic properties of drinking water are negatively affected by biofilm organisms. When biofilms are formed on surfaces of iron pipes, corrosion of iron pipes can occur, which lead to metal particles being detached into drinking water (Camper et al., 1998;September et al., 2007;Hoiby et al., 2010).
In the past few decades, extensive use of antibiotics for medication and promoting growth in veterinary sector has caused an upsurge in the emergence of multidrug-resistant (MDR) bacteria (Page and Gautier, 2012;Boeckel et al., 2015). Horizontal transfer of antibiotic resistance genes (ARGs) between antibiotic-resistant bacteria (ARBs) and environmental bacteria was found to be facilitated in water environments (Martinez, 2012;Labella et al., 2021). Resistance genes could be maintained and spread by those environmental resistant bacteria (Taylor et al., 2011). Thus, veterinary and public health risks may arise from acquiring ARGs through consuming drinking water obtained from such environments (Wellington et al., 2013). The implication of biofilm formation and the occurrence of multiple antibiotic-resistant bacteria have become a critical problem for veterinary and public health (Oliveira et al., 2010).
Multidrug-resistant bacteria are prevalent in poultry, poultry products, carcasses, litter, and fecal matter. Many studies reported the evolution of antibiotic-resistant bacteria in the poultry production sector, such as Escherichia coli (Tadesse et al., 2012), Campylobacter (Richter et al., 2015), and Staphylococcus (Bortolaia et al., 2016). A study on 337 strains of Salmonella Pullorum from China revealed that resistance to cefamandole, trimethoprim, and co-trimoxazole was higher for biofilm-forming bacteria when compared to the non-biofilm ones (Gong et al., 2013). A positive correlation was observed between antibiotic resistance and the biofilm-producing capability of the bacteria (Zhang et al., 2017). Vancomycin-resistant Enterococcus faecium was isolated from farm animals and surface water (Iversen et al., 2002), as well as, from several human hosts (Novais et al., 2006). Biofilm lining the drinking water system has mixtures of bacteria, nutrients, and antimicrobial agents (Hirsch et al., 1999), which could help resistant bacteria and gene transfer within the bacterial population. Bacterial biofilms showed resistance to several types of antibiotics either by mutations or the acquisition of foreign DNA (Hoiby et al. 2010). The control of biofilms will, therefore, improve bird health and minimize antibiotic treatments (Linden, 2014).
The current study aimed to isolate and identify biofilm bacteria found in the drinking water systems of layer chicken farms. Moreover, the study examined the effectiveness of different antibiotics in combating isolated bacteria inside poultry production facilities as an integral part of overall prevention and control measures.

bird Houses
The present study was conducted at a shaver chicken layer farm in Ayyat (Giza), Egypt, in February 2020. The farm included one growing pullet house (15 weeks age; 7500 birds/house) and three egg-producing layer houses (69 weeks age; 30,000 birds/house). Pullets were raised in the growing house for 15 weeks before being transferred to the production houses. Growing pullet house was equipped with iron water pipelines with a diameter of one inch and bell drinkers. All three production houses were equipped with Polyvinyl chloride (PVC) water pipelines (1-inch diameter) and automatic drinking systems with drinking nipples. Houses were all closed with fully controlled environments. No treatment or sanitization was performed on underground water before it was used for birds. The average daily water consumption rates were 1300 and 7000 liters/house for growing and production houses, respectively. Drinking water was supplemented with vitamin E and selenium 4-weeks before sampling. The performance of both the growing and production houses was suboptimal throughout the production cycle (Tables 1-2).

samplinG of biofilms from water pipelines
A total of eight biofilm samples were collected from the water systems of the four-layer chicken houses. Two samples were collected from the iron pipelines of the growing house (Biofilm A), and six samples were collected from the PVC pipelines of the three production houses (Biofilm B). Sterile cotton swabs, moistened with sterile normal saline, were used to scrape biofilm from the inner surface of the pipelines. Swabs were collected in sterile conical flasks and transferred to the laboratory in an icebox within four hours before being examined (Baird et al., 2017;Lin et al., 2015). Change % a -6.7 -8.3 -5.8 -7.6 -6.4 -6.4 -7.6 -6.7 -7.2 -6.8 -6.8 -7.2 -6.8 -7.2 -6.9 a Percentage of change between the actual and target body weights.

HeterotropHic count and isolation of biofilm bacteria
The heterotrophic bacterial count of biofilm samples was done following the standard protocols of the American Public Health Association's (Baird et al., 2017). Briefly, 1-ml from the collected samples were serially diluted in 9 ml sterile saline solution and then 0.1 ml from each dilution were spread on plate count agar plates and incubated for 24 hours at 37°C to allow bacterial growth.
For bacterial isolation, microbial enrichment was performed by inoculating 1ml from each biofilm sample into a nutrient broth medium with the following ingredients (g/l): 3, Beef Extract; 5, Peptone and 5, NaCl, and then incubated at 37°C for 24 hours with shaking at 150 rpm (Gehring et al., 2012). Bacterial isolation and purification were done by streaking on nutrient agar plates (Sanders, 2012). Suspected colonies were picked up and transferred to sterile nutrient agar plates to check purity. Then purified molecular identification and sequencinG of biofilm bacteria All DNA of bacterial isolates were extracted using Quick-DNA™ Fungal/Bacterial Microprep Kit (Zymo research #D6007) according to the manufacturer's protocol. For molecular identification of bacterial isolates, ribosomal 16S rRNA genes were amplified using the universal bacterial primers NVZ-1 (forward; 5′-GCGGATCCGCGGC-CGCTGCAGAGTTTGATCCTGGCTCAG-3′) and NVZ-2 (reverse; 5′-GGCTCGAGCGGCCGCCCGG-GTTACCTTGTTACGACTT-3′) (Lopez et al., 2006). The protocol for 16S rRNA genes amplification and sequencing were performed by Sigma Scientific Services Co. (http://sigmaeg-co.com/) (Tables 3 -4). PCR products clean up were performed using GeneJET™ PCR Purification Kit (Thermo K0701), according to the manufacturer's instructions.  The amplified 16S rRNA fragments of bacterial isolates were sequenced at GATC Company using ABI 3730xl DNA sequencer by using forward and reverse primers. The NCBI BLAST website (https://blast.ncbi.nlm.nih. gov/Blast.cgi) was used to get the most closely similar 16S rRNA gene sequences.
For all biofilm isolates, the Multiple Antibiotic Resistance Index (MARI) was estimated. A value above 0.2 indicates that the bacteria came from a potentially contaminated water source where antibiotics are commonly applied.
Rates smaller than or equal to 0.2 refer to bacteria from water environments where antibiotic use is rare or nonexistent (Titilawo et al., 2015;Labella et al., 2021).
MARI= the number of antibiotics the bacterial isolate was resistant to ÷ the total number of antibiotics the isolate was tested against.

bird performance
The growing pullet house displayed lower performance than what should be in the correspondent ages. Throughout the growing period, the average body weights were suboptimal. The differences from optimal weights ranged from -5.8% to -8.3% (Table 1). Furthermore, the production houses showed suboptimal egg production % throughout the production cycle. The change % from the optimal egg production levels ranged from -66.7% to -10.4% (Table 2).

antibiotic sensitivity of biofilm isolates
Table (7) displays bacterial isolates' sensitivity to antibiotics. In the iron water pipes (sample A), P. aeruginosa showed susceptibility to all antibiotics tested, while Staph. saprophyticus, E. faecalis, E. casseliflavus, and S. terrae showed varied resistance patterns. The Staphylococcus saprophyticus strain was resistant to cefpodoxime, penicillin, erythromycin, clindamycin, and doxycycline. Both E. faecalis and E. casseliflavus showed resistance to erythromycin and doxycycline. Additionally, we noticed rifampin resistance in E. faecalis, and gentamicin resistance in E. casseliflavus. Sphingopyxis terrae showed resistance to amikacin, doripenem, ceftriaxone, cefpodoxime, cefotaxime, cefepime, nalidixic acid, aztreonam, trimethoprim-sulfamethoxazole, and doxycycline.
In biofilm isolates of PVC water pipes (sample B), one P. aeruginosa isolate (MW193120) was sensitive to all tested antibiotics, while the other three isolates (P. aeruginosa-MW035344, Bacillus luti, and Acinetobacter kookii) displayed different levels of resistance. The B. luti strain was resistant to ceftazidime, cefpodoxime, cefepime, penicillin, and rifampin. The P. aeruginosa strain (MW035344) was only resistant to norfloxacin. The Acinetobacter kookii strain was resistant to ceftriaxone, cefepime, and trimethoprim-sulfamethoxazole (Table 7).
Sixty-seven percent of the isolates showed resistance to at least three antibiotic groups (Table 7). Multidrug resistance was primarily observed in Staph. saprophyticus, E. faecalis, E. casseliflavus, and S. terrae isolated from iron pipes biofilms (sample A), and Bacillus luti, and Acinetobacter kooki isolated from PVC pipes biofilms (sample B).
The MARI index was used to trace the source of antibiotic resistance of the isolates. Using the Krumperman (1983) criteria, the isolated strains of Staph. saprophyticus, E. faecalis, E. casseliflavus, and S. terrae isolated from iron pipes, as well as Bacillus luti, and Acinetobacter kookii isolated from PVC pipes originated from highly contaminated water sources, involving frequent use of antibiotics. Nevertheless, all isolates of P. aeruginosa strains from iron and PVC pipes originated from water environments free from antibiotics contamination (Table 8).

DIScuSSIon
Biofilms samples were collected from drinking water pipes of 4-layer houses to characterize the bacterial population and test their antimicrobial resistance. Physical examination of biofilm samples revealed that they were slimy. This finding is consistent with (Cunha et al., 2019), who stated that biofilm slime was a pseudo-capsule formed by bacteria, especially staphylococci. Moreover, the biofilm layer found on the inner surface of the PVC pipes was more abundant and blackish than the layer formed within the iron pipes. These findings agreed with what was stated by (Cerrato et al., 2006) that water in PVC pipes showed more manganese levels and black colour than detected in water flowed in iron pipes. According to Cerrato et al. (2006), the scale layer of the PVC pipe was composed of white and brown layers, with manganese making up approximately 6% of the brown layer. Furthermore, Lehtola et al. (2004) mentioned in their study that PVC pipe was used as a cost-efficient alternative to iron pipe. On the other hand, PVC could release phosphorous and biodegradable compounds that enhance biofilm formation and microbial regrowth.
In the current study, the PVC pipe showed a total microbial count higher than the iron pipe (Tables 5-6). The PVC pipes were constructed in the production layer houses of 69-weeks of age, while the iron pipes were in the growing pullet house of 15-weeks of age. Biofilms are highly hydrated structures that protect bacteria from desiccation and antibacterial agents. The formation of biofilm may be attributed to the time factor and the lack of concurrent water sanitization, as untreated groundwater was used in all layer houses. Drinking water system hydrodynamics

Journal of Animal Health and Production
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December 2021 | Volume 9 | Issue 4 | Page 449 (flow rate, velocity, turbulence, and shear stress) vary from growing to production houses. Hydrodynamics affect the exchange of trace nutrients, disinfectants, oxygen, heat, and microorganisms inside the pipe system (Fish et al., 2016). Douterelo et al. (2013) stated that the biofilm community will vary according to water system hydrodynamics. Cowle et al., 2020 stated that lower water flows assisted the attachment and propagation of biofilm bacterial biomass. While higher water flows weakened the biofilm attachment and hindered the development of biofilm.
Nine bacterial isolates were detected in water pipes biofilms, five isolates from iron pipes (Staphylococcus saprophyticus, Enterococcus faecalis, Enterococcus casseliflavus, Pseudomonas aeruginosa, and Sphingopyxis terrae) and four isolates from PVC pipes (two Pseudomonas aeruginosa isolates, Bacillus luti, and Acinetobacter kookii). Drinking water can transmit various pathogenic bacteria that affect layer hen performance (do Amaral, 2004). Groundwater was considered a crucial problem in poultry production because it is vulnerable to contamination from sewage water and other possible sources (Cloete et al., 2003). A previous study detected indicative bacteria of fecal pollution in three different types of water samples: creek, drain, and artesian well, confirming the contamination of underground and superficial water resources (do Amaral et al., 1994).
Prior studies revealed that staphylococci and enterococci are common microbiota in the intestinal tract of chickens but are also opportunistic pathogens that cause diseases in poultry (Rosenstein and Gotz, 2012;Nowakiewicz et al., 2017;Syed et al., 2020). In previous research, staphylococci were isolated from eggs of layer chickens which were contaminated from the environment or the faeces of the birds (Fahim et al., 2021). Recently, Staphylococcus sap-rophyticus was identified as an emerging foodborne uropathogenic bacterium developing resistance to antibiotics (Sommers et al., 2017). Staphylococcus spp. has been known to have the biofilm-forming ability due to its microbial surface components recognizing adhesive matrix molecules (MSCRAMM) and biofilm formation genes that help in better host colonization (Culotti and Packman, 2015;Maes et al., 2019). Enterococci are resistant to unfavorable environmental conditions and have a considerable impact on soil and water contamination. Furthermore, enterococci can acquire antimicrobial resistance and virulence determinants (Nowakiewicz et al., 2017). All enterococci strains can form a biofilm, as reported by Woźniak-Biel, et al. (2019). As recommended by the European Council directive (ECD), that no enterococci should be spotted in drinking waters (ECD, 1998).

Pseudomonas aeruginosa, Sphingopyxis terrae, Bacillus and
Acinetobacter bacteria are widely spread in environments and can be easily detected in soil, freshwater, and could endure intense environmental conditions. Pseudomonas aeruginosa and Sphingopyxis terrae are strong biofilm producers could co-exist and interact with a wide range of bacteria (del Mar Cendra and Torrents, 2021;Labella et al., 2021;Sharma et al., 2020). Pseudomonas has biofilm-forming capabilities, besides the ability to support the attachment of other pathogenic bacteria like Campylobacter. In the current study, Pseudomonas isolates represented one-third the numbers of isolates, and these results are in agreement with those of (Maes et al., 2019) who regarded Pseudomonas as the most abundant isolates in drinking water systems of broiler houses. Sphingopyxis is a gram-negative bacterium that was isolated from the biofilm of iron pipe, and this agrees with the results of (Lee et al., 2010). Sphingopyxis possesses the ability of biofilm formation as a secondary

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December 2021 | Volume 9 | Issue 4 | Page 450 colonizer (Douterelo et al., 2014). Sphingomonas was reported to be especially abundant in biofilms developed at low water flows, which agreed with our findings (Cowle et al., 2020). Bacillus and Acinetobacter bacteria has high capacity to adhere to surfaces and forms biofilm (Ebrahimi et al., 2021;Rajitha et al., 2021). Acinetobacter is gram-negative bacteria exhibiting a strong biofilm-forming ability (Maes et al., 2019). Acinetobacter was previously isolated from diseased chickens (Liu et al., 2016); besides it possesses public health importance, as it was isolated from wild animals and humans, therefore it needs further investigation (Wilharm et al., 2018;Wareth et al., 2019). Gram-positive Bacillus spp. was isolated from the biofilm of the PVC pipe (Maes et al., 2019). Bacillus can induce corrosion in the iron pipe which represents economic loss and the need to change the pipe (Makris et al., 2014). Bacillus cereus is a common contaminant of poultry feed and lead to severe diarrhea and malnutrition. Bacillus was isolated from hemorrhagic lung infected chicken (Zuo et al., 2020).
Results of the antibiotic sensitivity revealed Staphylococcus resistance to penicillin, doxycycline, cefpodoxime, clindamycin, and erythromycin antibiotics. Additionally, Staphylococcus showed intermediate resistance to norfloxacin. These results agreed with (Bakheet et al., 2018), who proved the resistance of 90% of Staphylococcus isolates to penicillin. Also, Onaolapo et al. (2017) reported Staphylococcus resistance to doxycycline. Intermediate resistance of staphylococcal isolate to norfloxacin agreed with Farghaly et al. (2015), who reported intermediate resistance to norfloxacin in 7.1% of poults staphylococcal isolates. In Egypt, tetracycline and erythromycin are used frequently by veterinarians to treat staphylococcal infections, as well as other bacterial infections. Hence, these traditional antibiotics may not remain able to control staphylococcal infections, as previously reported in Belgium (Nemati et al., 2008).
Two Enterococcus isolates were identified in the biofilm of the iron pipe, Enterococcus faecalis and Enterococcus casseliflavus. Both enterococci strains showed resistance to doxycycline and sensitivity to vancomycin, and this agreed with (Stępień-Pyśniak et al., 2016). Enterococci showed sensitivity to teicoplanin, ciprofloxacin, and resistance to erythromycin. These results agree with findings reported by (Van den Bogaard et al., 2002). Enterococcus showed sensitivity toward ampicillin and nitrofurantoin. On contrary, da Costa et al. (2007) observed ampicillin and nitrofurantoin resistance in 36.2% and 1.2% of enterococci isolates from broiler feed; respectively.
One Pseudomonas isolate was found in the biofilm of iron pipe and two isolates were detected in the biofilm of the PVC pipe. Pseudomonas isolates were tested for antibi-otic sensitivity, and they displayed sensitivity to most of the tested antibiotics. However, one showed resistance to norfloxacin and intermediate sensitivity to ofloxacin and aztreonam. These results are not agreeable with Kebede (2010) and Isichei-Ukah et al. (2018). Variation in antibiotic sensitivity results might be due to the misuse of antibiotics in the field, and the physicochemical properties of the cell wall, besides the antibiotic inhibiting enzymes (Koncicki et al., 1988).
One Acinetobacter isolate was identified from the biofilm of the PVC pipe. Acinetobacter displayed resistance to cefepime, ceftriaxone, and trimethoprim-sulfamethoxazole. While it showed intermediate resistance to ceftazidime and susceptibility to other tested antibiotics. These results showed differences from what was reported by (Kittinger et al., 2018). They reported the highest resistance to cefotaxime and low resistance to cefepime. Results agreed with Van Looveren et al. (2004) and Labella et al. (2021) who mentioned that the majority of Acinetobacter strains exhibited resistance toward cephalosporins.
The Sphingopyxis terrae isolate from iron pipes was resistant to 66.7% of cephalosporins and intermediately resistant to the remaining 33.3% (Table 7). Sphingopyxis was commonly resistant to cephalosporins, according to Labella et al. (2021). In addition, it showed resistance to nalidixic acid and trimethoprim-sulfamethoxazole. These results agreed with Vaz-Moreira et al. (2011) who reported fluoroquinolone and sulfonamide resistances as the second most widespread in Sphingomonadaceae, following beta-lactam resistance. Bacillus luti isolated from the PVC biofilm exhibited resistance to cephalosporins, penicillin, and intermediate resistance to erythromycin. These results agreed with Labella et al. (2021) who reported Bacillus resistance to ceftazidime, cefipime, and erythromycin.
All isolates except Pseudomonas (6 out of 9 -66.7%) exhibited multiple resistance to antibiotics belonging to three or more groups. The percentage represented two times the prevalence (37.2%) reported by Labella et al. (2021). In addition, they found a significant relationship between the multiple antibiotic resistance index (MARI) and the capacity of bacteria to produce biofilm. In the current study, MARI highlighted contamination of the water environment with bacteria originated from antibiotic-rich sources. Hence, most of these bacteria were suggested to be recycled from the poultry farm wastewater and then leaked to underground water. Therefore, control measures should be targeted to the well, including the appropriate design and location of the well besides maintenance to protect the well from any contamination. Proper cleaning and disinfection of the drinking water system between flocks and regular water testing and treatment are crucial to protect

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concluSIon
Multiple factors contributed to the formation of biofilm inside water pipelines used in layer chicken farms. Various surface materials have an impact on biofilm formation, composition, and bacterial population. Polyvinyl chloride (PVC) pipes enhanced the build-up of biofilm when compared to iron pipes. Additionally, water flow, water sanitization routine, and duration of production affected the propagation of bacteria and the development of biofilms inside the water systems. Various bacteria could grow and multiply within biofilm formed inside drinking systems, some of which represented a veterinary health concern and could affect the performance, production, and health of birds, like Pseudomonas aeruginosa and Enterococcus spp. The bacterial population identified from biofilms inside iron and PVC water pipes in the poultry farm differed from each other. Many of the isolated bacteria from the biofilms represented potential hazards to poultry health and performance. The biofilm environment increases pathogenic bacteria's antimicrobial resistance. Interestingly, the antibiotic resistance profile of these bacteria varied from one isolate to another. Doxycycline, trimethoprim, and erythromycin, which were frequently used in commercial poultry farms, showed inefficacy against multiple tested isolates as Enterococcus spp. and Staphylococcus spp. that have risk impacts on poultry health. Hence, control programs of monitoring, testing, cleaning, and disinfection are a must for combating the biofilm build-up within drinking systems. The responsible use of antibiotics became essential beside the longterm policies to ban the use of antibiotics in food-producing animals and poultry. Otherwise, consumers of poultry eggs and meat, as well as public health are at risk.