Isolation, molecular typing and antibiotic sensitivity profiling of enteric bacterial pathogen from chicken eggs

doi:10.21203/rs.3.rs-1549539/v1

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Isolation, molecular typing and antibiotic sensitivity profiling of enteric bacterial pathogen from chicken eggs

https://doi.org/10.21203/rs.3.rs-1549539/v1

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Food is a basic need of life; nevertheless its contamination due to pathogenic bacteria, especially antibiotic-resistant bacteria (ARB) in the poultry world, is a big threat to public health. In this study, enteric bacteria were isolated from chicken eggs and their antibiotic resistance against a panel of 18 different antibiotics belonging to 09 different antibiotic classes was determined. A total of 300 egg samples (i.e. 150 egg shells and 150 egg interiors) were collected and evaluated for bacterial growth. It was observed that 46% of the samples (n = 138) were positive for the growth of bacteria, while 54% of the samples (n = 162) did not show any bacterial growth in specified culture conditions. From those growth positive samples, three genera of Salmonella (from both the egg shell and the egg interior) and a genus of Enterobacter species, i.e. Enterobacter spp. (only from egg shells), were isolated using enrichment techniques and identified on the basis of cultural, morphological, biochemical, and 16S rRNA gene sequencing characteristics. The antibiotic sensitivity profiling results revealed that 50% of the isolated Enterobacter species were only resistant to amoxycillin, azomax (azithromycin), and fosfomycin, while 100% resistant to cephalothin, cefuroxime sodium, erythromycin, and vancomycin. On the other hand, 50% of the isolated species of Salmonella were resistant to amoxycillin, urixin, fosfomycin, and amoxycillin, whereas all Salmonella spp. were completely resistant to cephalothin, cefuroxime sodium, erythromycin, vancomycin, moxifloxacin, and azomax (azithromycin). The findings of the current study would increase the awareness of ordinary people to understand and overcome the associated risks associated with the utilization of contaminated eggs or other food products.

Antibiotics resistant bacteria (ARB)

Poultry eggs

Enterobacter

Salmonella

Biochemical reactions

16S rRNA

A balanced diet mainly contains the proteins and essential nutrients that play vital role in the foundation of well-adjusted food generally obtained from two major sources, i.e. animals and plants. In Pakistan, the beef, mutton, milk, chicken and eggs are major sources of protein (Maqbool et al. 2005). Chicken eggs are highly rich in high-quality proteins, carbohydrates, easily digestible fats, minerals and valuable vitamins. The chicken eggs are most versatile food (Matt et al. 2009) examination of the eggshell, membranes, albumen and yolk. At the same time because of its nutritional importance, an egg provides an excellent environment for bacterial growth and development (Stepien-Pysniak 2010). The bacterial penetration the information regarding penetration of bacteria after inoculation is based on, into chicken eggs is accomplished by three main mechanisms: adherence to eggshell and then penetration through eggshell pores, cracks survival in albumen and multiplication and growth in yolk (Al-Bahry et al. 2012).

A variety of bacteria, specifically the enteric bacteria inhabit both the interior and exterior of the chicken eggs. All the enteric bacteria belonging to the family Enterobacteriaceae are bacilli, facultative aerobe, gram-negative and non-spore former. The Enterobacteriaceae family has the following genera including Salmonella, Shigella, Klebsiella, Citrobacter, Edwardsiella, Enterobacter, Morganella, Serratia, Hafnia, Proteus, Erwinia, Providencia, Escherichia, Yersinia (Ateba and Setona 2011). Since last 60 years, antibiotics have played most important role by saving lives of millions of people. However, the antimicrobial drug resistance is the worldwide problem in developing as well as in developed countries (Lestari et al. 2012). In recent years, the resistance to the antibiotics by pathogenic bacteria developed a threat. It leads us to predict that we are returned to the time before the antibiotic’s discovery. Now, the most possible choice would be the introduction of different antimicrobial products to cope with the alarming situation; which would further be used in the experiments to eradicate the diseases caused by different microbial species (Hleba et al. 2020). The wide use of antimicrobial agents throughout the world is accepted as main factor of risk for the increase in antimicrobial resistance to antibiotics, which leads to the emergence of resistant bacterial species as well as resistance genes in community of fauna and human beings (Lukášová and Šustáčková 2003).

Bacteria are normally more susceptible species and become resistant to antimicrobial agents by the mutation or by gaining the new drug resistant genes. Some of the bacterial species are inherently resistant to specific antibiotics, whereas sensitive to others. Change in any of the prerequisites might render a resistance to drugs by bacteria which were sensitive earlier (Lukášová and Šustáčková 2003; Yehia et al. 2013). The data revealed that isolated Enterobacteriaceae and non –Enterobacteriaceae from poultry intestine have the patterns of antimicrobial resistance. The early scientists have determined the zone of inhibitions (in mm) of the antibiotics by using standard interpretative chart on the test microbial species. Enterobacter aerogenes, Klebsiella pneumoniae sp., Leclerciaadecarboxylata, Enterobacter cloacae, Kluyvera spp. exhibited the highest resistance of 94.11%, 88.23%, 57.82% against the tested panel of antibiotics. Two bacterial species, Aeromonashydrophill aand Cedecealapagei, revealed 47.07% of resistance against tried, whereas, the Salmonella arizonae, Escherishia coli, as well as Klebsiella oxycota were found very less resistant, 35.29, 41.17 and 29.41%, respectively (Yehia et al. 2013).

In this study, the enteric bacteria were isolated using enrichment technique and identified on the basis of cultural, morphological, biochemical and 16S rRNA gene sequencing characteristics from chicken eggs (i.e. egg shells and egg interiors). And the antibiotic resistance against panel of 18 different antibiotics belonging to 09 different antibiotic classes was determined.

2.1 Sample collection

Total of 300 samples of poultry eggs were collected from different general stores of city Khairpur. The eggs were examined for the occurrence of enteric bacteria on eggs' shell and eggs' interior content (150 eggshells and 150 egg interior). Five eggs from different trays of each store were randomly collected in sterilized polythene bags and were kept under refrigerated conditions until transportation and processing of the samples.

2.2 Media Enrichment

To reduce the chance of inappropriate results, the hen's egg samples, a combination of three and four enrichments of the pre-enrichment plating on selective media was performed. The lactose broth was used as enrichment medium for the isolation of enteric bacteria. Homogenized suspension of each sample (10 mL) was moved to 90 mL of lactose broth and was incubated at 37°C for 24 to 48 hours. Tubes showing turbidity of enteric bacterial colonies were6 selected for primary isolation.

2.3 Isolation of enteric bacteria

The sampling from the surface of intact egg shell was carried out by using swab technique as described by (Jamshidi et al. 2010). Briefly, a sterile cotton swab was dipped in sterile blubbered peptone water to swab the entire surface area of the egg shell. In order to collect material of intact eggs, the eggs were surface sterilized by immersion in 70% alcohol for 2 minutes and then air dried in a sterile chamber for 10 minutes. Sterile eggs were cracked with a sterile knife. Each eggs contents were thoroughly mixed and mixed egg content (10 mL) was inoculated into a 90 mL of lactose broth medium for the enrichment. The inoculated medium was then incubated at 37°C for 24 hours. After enrichment, the culture was transferred and streaked onto Salmonella-Shigella (SS) agar and Bismuth Sulfate (BS) agar then incubated at 37°C for 24 to 48h. The plates were observed for typical colonies on the plate and two colonies were randomly selected, purified and subjected to examination of the basic biochemistry.

2.4 Identification and characterization

3.5.1 Preliminary identification

All the bacterial isolates were exposed for their morpho-microscopic characteristics. And then, the assenting identification of the bacterial isolates was carried through biochemical tests (Cheesbrough 1991).

3.5.2 Molecular identification using 16SrRNA sequencing

For the molecular identification, the selected bacterial isolates were sent to Macrogen Inc., Seol, Korea, for commercial amplification and sequencing of 16S rRNA gene. The purified PCR products of approximately 1,400 bp were sequenced by using universal amplification (27F/1492R) and sequencing (518F/800R) primers. Both the obtained nucleotide sequences amplified using forward and reverse primers were then assembled using online CAP3 Sequence Assembly Program (http://doua.prabi.fr/software/cap3) in order to get a single consensus sequence and that sequence was used for phylogeny re-construction. Phylogeny re-construction analyses of the consensus sequences of each isolate was performed for scrutiny of percentage similarities index against nucleotide sequence databases available at National Center for Biotechnology Information (NCBI) website using Basic Local Alignment Search Tool (BLAST) program. The sequence FASTA files of closely related organisms from NCBI database were retrieved and the used for phylogenetic correlation analyses. The trees showing the evolutionary history and evolutionary distances, as inferred by neighbor-joining (Saitou and Nei 1987) and maximum composite likelihood method, respectively, were re-constructed using online phylogentic tree reconstruction utility available after BLAST analysis at NCBI website.

2.5 Antibiotic sensitivity profiling

The antimicrobial susceptibility of the pure bacterial isolates was tested using Kirby-Bauer’s disc diffusion method according to the standard protocols and guidelines of CLSI (Clinical and Laboratory Standards Institute) (NCCLS, 2007). A panel of eighteen different antibiotics was selected for antibiotic sensitivity profiling of the selected bacteria isolated from egg samples. The antibiotics panel comprised of one each from sulfonamide, glycopeptide, pipemidic acid and phosphonic antibiotics; two each from β-lactams, aminoglycosides and macrolides; three from cephalosporins and four from fluoroquinolones group of antibiotics. The brand names, abbreviations and potencies of each antibiotic is presented in Table S1.

2.6 Statistical analysis

The statistical analysis of the data was carried out using Microsoft Excel (2010) and Statistix software (Version 8.1). All the experiments were carried out in triplicates and the data represented in this thesis are mean of three replicates ± standard deviation. The statistical analysis included analysis of variance (ANOVA) and least significant difference (LSD) all-pairwise comparison tests.

3.1 Prevalence of enteric bacteria on egg shell and in egg interior

A total of 300 egg samples, i.e. 150 egg shells and 150 egg interiors were collected and evaluated for the bacterial growth on nutrient agar and lactose broth, respectively. It was observed that 46% of the samples (n = 138) were found to be positive for growth of bacteria, while 54% of the samples (n = 162) did not show any bacterial growth in specified culture conditions. Moreover, 82% (n = 123) of the total 150 egg shell samples were found to be positive for growth of bacteria, while 27 samples (18%) did not show any bacterial growth (Fig. 1A-B). It was also observed that the bacterial prevalence was very low in egg interior given that only 10% of the samples (n = 15) were found to be positive for growth of bacteria, while 90% of the egg interior samples (n = 135) did not show any bacterial growth (Fig. 1C-D).

3.2 Preliminary identification of the selective isolates

The egg samples showing positive growth on nutrient agar were chosen for the isolation of enteric bacteria. Then, four dissimilar type of bacterial isolates i.e. ES1, ES2, ES4, and ES5 founded on their characteristics like colonial color, size, shape, margin and appearing isolated and pure cultures were founded. Table S2 shows colonial characteristics of isolates. It was observed that the colonial characteristics of ES1 isolate were smooth, shiny, low convex colonies with entire edges. Similarly, isolates ES2, ES4 and ES5 shared parallel colonial features like smooth, circular, low convex, translucent colonies having size in the range of 2–3 mm in diameter. Afterwards, the isolates were preserved as agar slants for short term preservation and glycerol broth (15–20%) stocks for long term preservation of bacterial strains at − 20°C.

Following the pure culturing of each bacterial isolate, the isolates were further subjected to preliminary identification through biochemical and microscopic examination (Table S3). The microscopic examination for the morphological features was based on Gram staining, cell wall staining, capsule staining and spore staining. The microscopic examination of the isolates revealed that all the four isolates, ES1, ES2, ES4 and ES5 were Gram negative, non-sporulated, non-capsulated and motile bacteria, whereas they somehow differed in their biochemical profiles. The biochemical characterization of the bacterial isolates included all essential tests like IMViC test, TSI, sugar tests (glucose, maltose, lactose and sucrose), nitrate reduction test, oxidase test. The biochemical characteristic of the ES1 showed a positive test for voges proskauer, citrate, catalase, glucose, sucrose and triple sugar iron tests, while the remaining tests were negative. Thus, on the basis of morphological and biochemical characteristics ES1 isolate was tentatively identified as Enterobacter spp. On the other hand, the biochemical tests for bacterial isolates ES2, ES4 and ES5 were found to be similar, i.e. Methyl red, glucose, maltose, catalase, nitrate reduction test and triple sugar iron tests were positive and the remaining tests were found to be negative signifying the isolates to be species of the genus Salmonella (Table S3). Therefore, on the basis of microscopic and biochemical tests, the bacterial strains were tentatively identified ES1 isolate as Enterobacter spp., whereas, ES2, ES4 and ES5 as Salmonella spp.

3.3 16s rRNA gene sequence analysis

The phylogenetic correlation analysis of ES1 isolate revealed that the isolates shared 99% similarity with the strains of Enterobacter sp. (GenBank: MW131452) as shown in Fig. 2, thus, the strain was named as Enterobacter sp. ES1. On the other hand, the bacterial isolates ES2, ES4 and ES5 showed the maximum similarity (99% and 100%, respectively) with the strains of genus Salmonella. The phylogenetic and similarity analyses of all three isolates showed close relatedness to Salmonella enterica (GenBank: MW165860, MW165863, MW165867), and the isolates were named as Salmonella enterica strains ES2, ES4 and ES5, respectively.

3.4 Antibiotic sensitivity profiling of enteric bacterial isolates

The antibiotic sensitivity profile of the bacterial isolate ES1 is shown in Fig. 3a. It was found that the ES1 isolate was sensitive to 66.67% of the tested antibiotics including ceftriaxone (27mm), sulphamethoxazole (32.6mm), amikacin (26.97mm), urixin (26.83mm), meropenem (27.37mm), ciprofloxacin (38.70mm), sparfloxacin (31.03mm), gentamicin (24.27mm), moxifloxacin (26.37mm), enoxacin (31.00mm), azomax (23.03mm) and piperacillin-tazobactam (27.57mm). Contrastingly, the ES1 has developed resistance to almost 33.33% of the antibiotics used in this study, which included cefuroxime sodium, erythromycin, cephalothin, amoxycillin, vancomycin and fosfomycin (Fig. 5).

On the other hand, Fig. 3b shows the antibiotic sensitivity profile of ES2. It was observed that the strain ES2 also showed a significant sensitivity to ceftriaxone (28.5mm), sulphamethoxazole (33.3mm), amikacin (25.77mm), urixin (25.77mm), meropenem (25.97mm), ciprofloxacin (29.6mm), sparfloxacin (32.33mm), gentamicin (25.93mm), moxifloxacin (26.67mm), enoxacin (30.33mm), and piperacillin-tazobactam (23.27mm), while resistance to cefuroxime sodium, erythromycin, cephalothin, vancomycin. Contrary to ES1 isolate, the ES2 isolate was found sensitive to amoxycillin (19mm) and fosfomycin (17.33mm), while resistant to azomax. In general, the ES2 isolate showed a resistance against 28% (i.e. against 5 out of 18) of the antibiotics tested, whereas it showed sensitivity almost 78% antibiotics Fig. 5.

However, Fig. 4a shows the antibiotic sensitivity profile of ES4 isolate, and its percentage resistance. It was observed that majority of the antibiotics were effective and the bacterial isolate ES4 has shown sensitivity towards them. These included ceftriaxone (33.27mm), amikacin (32.3mm), urixin (18.27mm), meropenem (34.3mm), ciprofloxacin (25.27mm), sparfloxacin (29.5mm), gentamicin (30.23mm), fosfomycin (14.8mm), enoxacin (24.03mm), amoxycillin (21.93mm), and piperacillin-tazobactam (29.27mm). Contrarily, the ES4 displayed a complete resistance towards cefuroxime sodium, erythromycin, cephalothin, vancomycin, sulphamethoxazole, moxifloxacin and azomax Fig. 5. Generally, ES4 isolate showed sensitivity against 61.11% of the antibiotics used in current study, while it has developed resistance towards 38.88% of the antibiotics tested. Similarly, the antibiotic sensitivity profile of the ES5 is shown in Fig. 4b.The isolate ES5 displayed sensitivity towards 61% of the antibiotics while complete resistance against 39% of the antibiotics tested in present study. The isolate ES5 was sensitive to a group of antibiotics including ceftriaxone (31.23mm), amikacin (20.27mm), urixin (16.07mm), meropenem (29.93mm), ciprofloxacin (24.03mm), sparfloxacin (25.7mm), gentamicin (29.3mm), fosfomycin (16.27mm), enoxacin (25.7mm), amoxycillin (26mm), and piperacillintazobactam (27.22mm). On the other hand, ES5 isolate was highly resistant against cefuroxime sodium, erythromycin, cephalothin, amoxycillin, vancomycin, sulphamethoxazole, moxifloxacin and azomax (Fig. 5).

Table 1 shows the average zone of inhibitions for each antibiotic against selected bacterial isolates followed by alphabetical letters indicating their homogenous group at 95% probability (P < 0.05). The values followed by same alphabetical letter were considered to have a non-significant difference as compared to other members of that group. It is noteworthy that higher F-values (F > 90) and lowest P-values (P < 0.01) denoted the significant differences among the treatments.

Table 1 LSD All-Pairwise comparisons test of bacterial isolates by antibiotics

Antibiotics	Average Zone of Inhibition (mm)[1]
Antibiotics	*Enterobacter* spp. ES1	*Salmonella enterica* ES2	*Salmonella enterica* ES4	*Salmonella enterica* ES5
CE	27.00CD[2]	28.50BCD	33.87AB	31.23A
SUL	32.60B	33.30A	0.00F	0.00F
AMI	26.93CD	22.10FG	32.30ABC	20.27D
URI	26.83CD	25.77DEF	18.27E	16.07E
CEF	0.00F	0.00I	0.00F	0.00F
ME	25.37CDE	25.97CDE	34.30A	29.93A
ER	0.00F	0.00I	0.00F	0.00F
CEP	0.00F	0.00I	0.00F	0.00F
AM	0.00F	19.00GH	21.93D	26.00BC
VAN	0.00F	0.00I	0.00F	0.00F
CIP	38.70A	29.60ABC	25.27D	24.03C
SPX	31.03B	32.33A	29.50C	25.70BC
GM	24.27DE	25.93CDE	30.23BC	29.30A
FOS	0.00F	17.33H	14.80E	16.27E
MXF	26.37CD	26.67BCDE	0.00F	0.00F
EN	31.00B	30.33AB	24.03D	25.70BC
AZM	23.03E	0.00I	0.00F	0.00F
TZP	27.57C	23.27EF	29.27C	28.22AB
Grand Mean	18.93	18.89	16.32	15.15
CV	9.58	12.06	13.45	12.55
Standard Error for Comparison	1.48	1.86	1.79	1.55
Critical Value for Comparison	3	3.77	3.64	3.15
F-value	184	93.5	127	142
P-value[3]	0.000001	0.00002	0.000001	0.000001

[1] Average Zone of inhibition of three replicate experiments

[2] The alphabetical letters following each value of inhibitory zone indicate the least significant differences at 95% probability. The values in each column followed by same alphabetical letter were considered as non-significantly different from each other (P>0.05).

[3] P-value at 95% probability (i.e. Alpha=0.05)

3.5 Group-wise antibiotic sensitivity profile of the bacterial isolates

Selected antibiotics belonging to different classes of antibiotics were tested to determine the antibiotic sensitivity profile of the bacterial isolates. Generally, there were significant differences in activities of the antibiotics of even the same group. Therefore, the antibiotic sensitivity data was statistically interpreted in order to find out that which members of an antibiotic group are still effective or have become in-effective due to development of resistance in the bacterial isolates. The study was also carried out to suggest the most effective antibiotics regime to treat zoonotic infections caused by Enterobacter or Salmonella species.

3.5.1 β-lactam and Cephalosporin

The antibiotic sensitivity of the four isolates tested against 06 antimicrobial agents of β-lactam and cephalosporin antibiotics is shown in Fig. 6A. All the isolates were susceptible to β-lactam group of antibiotics except ES1, which showed complete resistance towards amoxicillin. Similarly, ES2 and ES5 did not show significant difference (P > 0.05) in inhibition zones for β-lactam group of antibiotics, however ES1 and ES4 displayed significant differences (P < 0.05) among the treatments. Contrarily, all the four isolates showed complete resistance to cephalothin and cefuroxime sodium, whereas there was significant sensitivity (P < 0.01) of ceftriaxone against all the four bacterial isolates.

3.5.2 Sulfonamide, Aminoglycoside and Macrolide

The bacterial isolates ES1, ES2, ES4 and ES5 were tested for antimicrobial sensitivity against one sulfonamide (Sulphamethoxazole), two aminoglycoside (amikacin and gentamicin) and two macrolide (erythromycin and azomax) antibiotics as shown in Fig. 6B. In general, all the four bacterial isolates were sensitive to Amikacin and gentamicin antibiotics, whereas the isolates ES1 and ES2 also showed significant and highest sensitivity (P < 0.01) to sulphamethoxazole. Contrarily, both the i.e. ES4 and ES5, were completely resistant to sulphamethoxazole. In addition, all the four bacterial isolates were resistant to an erythromycin, while only ES1 showed moderate sensitivity towards azomax, the macrolide antibiotics.

3.5.3 Fluoroquinolones

The antimicrobial activity of four fluoroquinolones antibiotics, i.e. enoxacin, ciprofloxacin, sparfloxacin and moxifloxacin as shown in Fig. 6C. Although, all the isolates were sensitive to enoxacin, ciprofloxacin and sparfloxacin antibiotics, however both the isolates, i.e. ES4 and ES5, displayed complete resistance against moxifloxacin antibiotic. Contrarily, the isolates ES1 and ES2 showed significant sensitivity (P < 0.05) to moxifloxacin. Figure 6C also signified that among the tested fluoroquinolones, sparfloxacin was rated as most significant treatment against all the bacterial isolates.

3.5.4 Glycopeptide, Pipemidic acid and Phosphonic

The antibiotic sensitivity of the four isolates against 03 antimicrobial agents i.e. vancomycin, urixin and fosfomycin, the representatives of glycopeptide, pipemidic acid and phosphonic antibiotics, respectively, is shown in Fig. 6D. All isolates were significantly sensitive (P < 0.01) to urixin (Pipemidic acid) antibiotic, while the isolates were resistant to the glycopeptide, vancomycin. In addition, only ES1 isolates was found to be resistant to fosfomycin whereas the other three isolates displayed significant sensitivity (P < 0.05) to fosfomycin (the phosphonic antibiotic).

The hen’s egg is more proteinase food supplement and rich in nutrients. Hens are used as food purpose and hatching of chicks too. Contrary to this whole egg provides a brilliant environment for the growth of bacterial microflora, plus pathogenic bacteria (Stepien-Pysniak 2010). In present study, a total of 300 egg samples, i.e. 150 egg shells and 150 egg interiors were collected and evaluated for the bacterial growth on nutrient agar and lactose broth, respectively (Fig. 1). In general, there was significant difference in positive vs. negative samples being highest prevalence (82%) of pathogenic bacteria on egg shells and very low bacterial prevalence in egg interior (10%, n = 15). On the other hand, approximately 90% of the egg interior samples (n = 135) did not show any bacterial growth. The egg albumen may possesses some antimicrobial defense mechanisms, such as its organization in the aluminous sac and the viscosity of its protein (Al-Jaff 2005). The penetrability of some pathogens through the shell, light brown fertile and brown infertile eggs was subjected to microbiological analyses (Al-Jaff 2005). Among all, four different types of bacterial isolates i.e., Enterobacter spp. and Salmonella enterica based on their morpho-microscopic and molecular characteristics were identified. Microbiologist also isolated Salmonella bacteria and many other microorganisms from the shell of hen’s eggs (Stepien-Pysniak 2010).

In a pilot study conducted in Grenada with 160 table eggs, majority of the bacterial isolates belong to Enterobacteriaceae group (Sabarinath et al. 2009). The results of research showed that incidence of Salmonella spp. in chicken egg from the general shop in Khulna city is higher than the normal ratio (Hasan et al. 2009). More over incidence of Salmonella spp. in broken eggs is relatively very high than the fresh eggs (Hasan et al. 2009). The shell probably receives microorganisms when passing through the cloaca and during the time until the egg is used (Al-Jaff 2005). The fact that eggs can be contaminated or infected through the shell or transovarially, makes them a probable cause of pathogens contributing in the etiology of foodborne illnesses in humans (Stepien-Pysniak 2010). It has been observed in both the US and the UK union in common conduction of infection caused by S. enteritidis in human (Zou et al. 2012). These species possessed great resistance power against the action of antibiotics and retain their growth even in the presence of drugs. These microorganisms develop resistance to antibiotics under the mechanisms such as choice, change, phage transduction, and conveyance while microbial resistance can either be intrinsic in the organism or developed through the environment (Chinedum 2005).

In this study, the antibiotic sensitivity of the four isolates was tested against 06 antimicrobial agents of β-lactam and cephalosporin antibiotics. All the bacterial isolates were susceptible to β-lactam group of antibiotics except Enterobacter sp. ES1, which showed complete resistance towards Amoxicillin. Similarly, S. enterica ES2 also showed complete resistance towards Amoxicillin. Clinically, E. cloacae and E. hormaechei are the maximum collective species in the genus Enterobacter. For 10 years, the two species have been increasingly considered as causative agent for the infections in hospitalized patients. They are characterized by resistance to broad-spectrum β-lactam agents by producing extended-spectrum β-lactamase and inducible chromosome-encoded cephalosporinase (Davin-Regli et al. 1997). Resistance of Enterobacter species to extended-spectrum cephalosporin is identified to be interceded by hyper manufacture of chromosomal AmpC β-lactamases (Davin-Regli et al. 1997). S. enterica ES5 did not show significant difference in inhibition zones for β-lactam group of antibiotics, however, Enterobacter sp. ES1 and S. enterica ES4 displayed significant differences among the treatments. Contrarily, all the four isolates showed complete resistance to cephalothin and cefuroxime sodium, whereas there was significant sensitivity of ceftriaxone against all the four bacterial isolate (Learn-Han et al. 2009). Learn- Han et al. (2009) also reported the order of resistant profile of S. enterica against ceftriaxone, cefuroxime, ampicillin, cephalothin and cefotaxime to be 14%, 12%, 10%, 9% and 7%, respectively (Learn-Han et al. 2009). In present study, the antimicrobial activity of four fluoroquinolone antibiotics, i.e. enoxacin, ciprofloxacin, sparfloxacin and moxifloxacin were examined. Although, all the isolates were sensitive to enoxacin, ciprofloxacin and sparfloxacin antibiotics, fluoroquinolones are often the treatment of choice in the cases of life-threatening Salmonellosis due to multidrug-resistant strains (Giraud et al. 2000).

Fluoroquinolones and the third-generation cephalosporin’s are recommended for use in areas where there are resistant organisms (Chiu et al. 2002). However, the S. enterica isolates, i.e. S. enterica ES4 and S. enterica ES5, displayed complete resistance against moxifloxacin antibiotic. While fluoroquinolone used animals that is failed to produce any resistance (Chiu et al. 2002). However, Enterobacter species showed significant sensitivity to moxifloxacin, also signified that among the tested fuoroquinolones, sparfloxacin was rated as most significant treatment against all the bacterial isolates. Fluoroquinolone is used for treatment of invasive gastrointestinal infections across the world (Aarestrup et al. 2003). Moreover, the bacterial isolates Enterobacter sp. ES1, S. enterica ES2, S. enterica ES4 and S. enterica ES5 were tested for antimicrobial sensitivity against one sulfonamide (sulphamethoxazole), two aminoglycoside (amikacin and gentamicin) and two macrolide (erythromycin and azomax) antibiotics. In general, all the four bacterial isolates were sensitive to amikacin and gentamicin antibiotics, whereas the Enterobacter sp. ES1 and S. enterica ES2 also showed significant and highest sensitivity to sulphamethoxazole. Contrarily, both the S. enterica isolates, i.e. ES4 and ES5, were completely resistant to sulphamethoxazole. S. enterica serotype typhimurium is frequent agents of bacterial enteritis that shows great resistance against multiple drugs, especially sulfonamides (Tosini et al. 1998). However, all the four bacterial isolates were resistant erythromycin, while only Enterobacter sp. ES1 showed moderate sensitivity towards Azomax, the macrolide antibiotics.

Antibiotics showed a great effective result against the gram’s positive bacteria, on other hand gram’s negative bacteria posed great resistance against these drugs (Poole 2005). While, the antibiotic sensitivity of the four isolates against three antimicrobial agents, i.e. vancomycin, urixin and fosfomycin, the representatives of glycopeptide, pipemidic acid and phosphonic antibiotics, respectively. All isolates were significantly sensitive to urixin (pipemidic acid) antibiotic, while the isolates were resistant to the glycopeptide, vancomycin. The increase of resistance to vancomycin new classes of antibiotics, revealed active against MRSA, such as linezolid and daptomycin, is irritating (Ge et al. 2008). In addition, only Enterobacter sp. ES1 isolates was found to be resistant to Fosfomycin, whereas, the other three isolates displayed significant sensitivity to fosfomycin (the phosphonic antibiotic). Clinical effectiveness of an old antibiotic, fosfomycin, high effective activity against multidrug-resistant gram-negative bacilli belonging to the family Enterobacteriaceae (Wachino et al. 2010). Unfortunately, resistance develops rapidly when fosfomycin is used as monotherapy, therefore, combinations with other antimicrobials are preferred in clinical practice for the treatment of serious infections (Souli et al. 2011).

In this study, four bacterial isolates were isolated from collected egg shells and egg interiors and were identified. The results revealed the prevalence of Salmonella enterica in both the egg shell and the egg interior samples, while Enterobacter strains were only prevalent on egg shell. The antibiotic sensitivity profile of the bacterial isolates revealed that Enterobacter species had developed complete resistance to cephalothin, cefuroxime sodium, erythromycin, and vancomycin, whereas Salmonella species, in addition to these antibiotics, also showed complete resistance to sulphamethoxazole, azomax and moxifloxacin. On the other hand, 50% of both the genera had also developed resistance to amoxycillin, azomax, and fosfomycin. Overall, all the four isolates had developed resistance against 30–40% of the antibiotics tested. Fortunately, some antibiotics like meropenem, piperacillin-tazobactam, ceftriaxone, amikacin, gentamycin, enoxacin, ciprofloxacin, and sparfloxacin are still working and revealed 100% sensitivity against these pathogenic bacteria. Therefore, the current study would be helpful to raise awareness in both of the producers and consumers of the eggs and related food products. This would also be helpful to improve the quality of food and in turn would be beneficial for control and curing of zoonotic infections caused by food borne enteric bacterial pathogens.

Declaration of interest: none

Acknowledgement: The authors are extremely thankful for the support of the Higher Education Commission (HEC), Government of Pakistan for funding through NRPU Projects (ID: 6236).

Aarestrup FM, Wiuff C, Mølbak K, Threlfall EJ (2003) Is it time to change fluoroquinolone breakpoints for Salmonella spp.? Antimicrobial Agents and Chemotherapy 47:827–829
Al-Bahry S, Mahmoud I, Al-Musharafi S, Al-Ali M (2012) Penetration of spoilage and food poisoning bacteria into fresh chicken egg: a public health concern. Global Journal of Bio-Science and Biotechnology 1:33–39
Al-Jaff BM (2005) The risk of bacterial contamination in hen eggs of Sulaimani poultries. Journal of Zankoy Sulaimani 8:63–71
Ateba CN, Setona T (2011) Isolation of enteric bacterial pathogens from raw mince meat in Mafikeng, North-West Province, South Africa. Life Science Journal 8
Cheesbrough M (1991) Mannual of tropical diseases
Chinedum IE (2005) Microbial resistance to antibiotics. African journal of Biotechnology 4
Chiu C-H et al. (2002) The emergence in Taiwan of fluoroquinolone resistance in Salmonella enterica serotype Choleraesuis. New England Journal of Medicine 346:413–419
Davin-Regli A et al. (1997) A nosocomial outbreak due to Enterobacter cloacae strains with the E. hormaechei genotype in patients treated with fluoroquinolones. Journal of clinical microbiology 35:1008–1010
Ge Y, Biek D, Talbot GH, Sahm DF (2008) In vitro profiling of ceftaroline against a collection of recent bacterial clinical isolates from across the United States. Antimicrobial agents and chemotherapy 52:3398–3407
Giraud E, Cloeckaert A, Kerboeuf D, Chaslus-Dancla E (2000) Evidence for active efflux as the primary mechanism of resistance to ciprofloxacin in Salmonella enterica serovar Typhimurium. Antimicrobial agents and chemotherapy 44:1223–1228
Hasan MN, Ara N, Mamun S, Rahman MM, Rahman MH (2009) Prevalence of Salmonella spp. in chicken egg from Khulna city. Journal of Innovation and Development Strategy 3:1–6
Hleba L, Kačániová M, Pochop J, Lejková J, Čuboň J, Kunová S (2020) Antibiotic resistance of Enterobacteriaceae genera and Salmonella spp., Salmonella enterica ser. typhimurium and enteritidis isolated from milk, cheese and other dairy products from conventional farm in Slovakia. Journal of microbiology, biotechnology and food sciences 9:1–20
Jamshidi A, Kalidari GA, Hedayati M (2010) Isolation and identification of Salmonella Enteritidis and Salmonella Typhimurium from the eggs of retail stores in Mashhad, Iran using conventional culture method and multiplex PCR assay. Journal of food safety 30:558–568
Learn-Han L et al. (2009) Molecular characterization and antimicrobial resistance profiling of Salmonella enterica subsp. enterica isolated from ‘Selom’(Oenanthe stolonifera). International Food Research Journal 16:191–202
Lestari ES, Severin JA, Verbrugh HA (2012) Antimicrobial resistance among pathogenic bacteria in Southeast Asia. Southeast Asian Journal of Tropical Medicine & Public Health 43:385–422
Lukášová J, Šustáčková A (2003) Enterococci and antibiotic resistance. Acta Veterinaria Brno 72:315–323
Maqbool A, Bakhsh K, Hassan I, Chattha MWA, Ahmad AS (2005) Marketing of commercial poultry in Faisalabad city (Pakistan). Journal of Agriculture and Social Sciences 1:327–331
Matt D, Veromann E, Luik A (2009) Effect of housing systems on biochemical composition of chicken eggs. Agronomy research 7:662–667
Poole K (2005) Efflux-mediated antimicrobial resistance. Journal of Antimicrobial Chemotherapy 56:20–51
Sabarinath A, Guillaume V, Guillaume B, Mathew V, DeAllie C, Sharma RN (2009) Bacterial contamination of commercial chicken eggs in Grenada, West Indies. West indian veterinary journal 9:4–7
Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular biology and evolution 4:406–425
Souli M et al. (2011) In vitro interactions of antimicrobial combinations with fosfomycin against KPC-2-producing Klebsiella pneumoniae and protection of resistance development. Antimicrobial agents and chemotherapy 55:2395–2397
Stepien-Pysniak D (2010) Occurrence of Gram-negative bacteria in hens' eggs depending on their source and storage conditions. Polish journal of veterinary sciences 13:507
Tosini F et al. (1998) Class 1 integron-borne multiple-antibiotic resistance carried by IncFI and IncL/M plasmids in Salmonella enterica serotype Typhimurium. Antimicrobial Agents and Chemotherapy 42:3053–3058
Wachino J-i, Yamane K, Suzuki S, Kimura K, Arakawa Y (2010) Prevalence of fosfomycin resistance among CTX-M-producing Escherichia coli clinical isolates in Japan and identification of novel plasmid-mediated fosfomycin-modifying enzymes. Antimicrobial agents and chemotherapy 54:3061–3064
Yehia HM, Riyadh K, Arabia S (2013) Antimicrobial resistance patterns of Enterobacteriaceae and non–Enterobacteriaceae isolated from poultry intestinal. Life Sci. J 10:3438–3446
Zou M, Keelara S, Thakur S (2012) Molecular characterization of Salmonella enterica serotype Enteritidis isolates from humans by antimicrobial resistance, virulence genes, and pulsed-field gel electrophoresis. Foodborne Pathogens and Disease 9:232–238

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Isolation, molecular typing and antibiotic sensitivity profiling of enteric bacterial pathogen from chicken eggs

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Sample collection

2.2 Media Enrichment

2.3 Isolation of enteric bacteria

2.4 Identification and characterization

3.5.1 Preliminary identification

3.5.2 Molecular identification using 16SrRNA sequencing

2.5 Antibiotic sensitivity profiling

2.6 Statistical analysis

3. Results

3.1 Prevalence of enteric bacteria on egg shell and in egg interior

3.2 Preliminary identification of the selective isolates

3.3 16s rRNA gene sequence analysis

3.4 Antibiotic sensitivity profiling of enteric bacterial isolates

3.5 Group-wise antibiotic sensitivity profile of the bacterial isolates

3.5.1 β-lactam and Cephalosporin

3.5.2 Sulfonamide, Aminoglycoside and Macrolide

3.5.3 Fluoroquinolones

3.5.4 Glycopeptide, Pipemidic acid and Phosphonic

4. Discussion

5. Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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