Indian Journal of Animal Research

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Indian Journal of Animal Research, volume 57 issue 10 (october 2023) : 1375-1379

Phylogenetic Group, Biofilm Formation and Drug Resistance of Escherichia coli Isolated from Goose and Fish in Henan, China

XiaoLing Zhang1, Siyuan Jia2, LuYue Cui2, Hong Yang2, Li Yi2,*
1College of Animal Science and Technology, Henan University of Science and Technology, Luoyang, China.
2College of Life Science, Luoyang Normal University, Luoyang, China.
Cite article:- Zhang XiaoLing, Jia Siyuan, Cui LuYue, Yang Hong, Yi Li (2023). Phylogenetic Group, Biofilm Formation and Drug Resistance of Escherichia coli Isolated from Goose and Fish in Henan, China . Indian Journal of Animal Research. 57(10): 1375-1379. doi: 10.18805/IJAR.BF-1604.
Background: Escherichia coli (E. coli) is a pathogen that sickens both humans and animals. There are relatively few reports on the correlation between drug resistance and biofilm formation ability of E.coli isolated from goose and fish in water environment of China. This study investigated the genetic typing, antibiotic sensitivity and biofilm formation ability of E. coli strains isolated from goose and fish.

Methods: The phylogenetic clustering, drug resistance and biofilm formation ability of E. coli were tested by triple PCR, crystal violet microplate and Microdilution method. 

Result: The results showed the prevalence of group B2 and D in fish-derived E.coli accounted for 59% and 12%, goose-derived E. coli accounted for 48% and 9%, respectively. Some strains could form biofilm and biofilm formation ability was associated with the drug resistance. The MIC values of strains growing in biofilm were 2-16 times higher than those of corresponding planktonic bacteria. The antibiotic resistance among biofilm-forming isolates was significantly higher than that strains unable to form biofilm (p<0.05). 
Escherichia coli (E.coli) is a pathogen that sickens both humans and animals, often causing pneumonia, meningitis and sepsis in humans. Geese infected with E.coli show septicemia, balloon inflammation and granuloma and other lesions (Yeh et al., 2017; Yu et al., 2018). Fish infected with E.coli mainly show ulcers and congestion in the viscera (Assefa et al., 2019). At present, the prevention and treatment of E.coli disease is mainly based on antibiotics. However, with the continuous use of antibiotics, E.coli has developed strong drug resistance. Because the infection is difficult to cure and the spread of drug resistance, E.coli disease has become one of the most prevalent and widespread diseases in the world, which also poses a serious threat to human health (Geurtsen et al., 2022).

Bacterial biofilms are bacterial congregate membrane-like substance formed by bacteria and their exocytic secretions, which can help bacteria resist various harsh environments. Numerous studies have shown that the biofilm condition of bacteria is more resistant to drug and more able to evade the attack by the immune system than the planktonic conditionÿand this is one of the most important causes of bacterial infections (Yi et al., 2019). There are relatively few reports on the correlation between drug resistance and biofilm formation ability of E.coli from goose and fish in water environment of China. Therefore, in this study, the clinical isolates of E.coli from goose and fish were used as the research objects to explore the relationship between biofilm formation and drug resistance, in order to provide scientific basis for the prevention and control of E.coli disease from the perspective of anti-biofilm.
The experiment was conducted from January 2019 to August 2021 at College of Life Science, Luoyang Normal University.
 
Bacterial strains
 
There were 100 strains of goose-derived E.coli isolates and fish-derived E.coli isolated respectively, which were isolated in four different cities of Luoyang, Xinyang, Shangqiu and Zhengzhou in Henan province. The quality control strain was E.coli (ATCC25922). The strains were grown in Luria-Bertani broth (LB).
 
Reagent
 
Norfloxacin (NOR), Florfenicol (Nuflo), Gentamicin (GEN), Spectinomycin  (SPT), Tilmicosin (TILM), Tylosin (TYL), Chlortetracycline (CTC), Tetracycline (TE), Amoxicillin (AMX), Doxycycline (DOX), Enrofloxacin (ENR) and Trimethoprim (TMP) were purchased from Soleibao Technology Co., LTD.
 
Phylogenetic group identification of E. coli
 
Through the reference’s methods (Clermont et al., 2000), three pairs of primers used for phylogenetic grouping of E. coli, chuA, yiaA and TSPE4.C2, were designed (Table 1). Genomic DNA was extracted from the isolates and E. coli was identified by multiplex PCR.

Table 1: Primers used for phylogenetic group of E. coli.


 
Biofilm formation ability test
 
The crystal violet microplate method was used for determination (Li et al., 2021; Liu et al., 2020). The critical point ODc value for judging whether the biofilm can be formed is 2 times the absorbance (OD) value of the negative control well (Wang et al., 2016; Yi et al., 2020).
 
Drug sensitivity test
 
Antibacterial activity was detected by using the microdilution method according to the CLSI standards (Andrews, 2001; Bhatia and Sharma, 2015). Among them, one strain was randomly selected from the biofilm-positive strains for analyzed the drug sensitivity of biofilm and planktonic state. The final drug concentration of wells 1-10 was 128, 64, 32, 16, 8, 4, 2, 1, 0.5 and 0.25 μg/ml. The 11th well and the 12th well were used as medium control and bacterial liquid control respectively. In addition, according to the results of drug susceptibility tests, the antimicrobial resistance spectrum of biofilm-positive strains and biofilm-negative strains was statistically analyzed
Bacterial clustering
 
The results showed fish-derived E. coli isolates were classified into phylogenetic four groups: group A (8%), group B1 (21%), group B2 (59%), group D (12%). The goose-derived isolates were classified into phylogenetic four groups: group A (10%), group B1 (33%), group B2 (48%), group D (9%). The fish-derived and goose-derived E.coli isolates were mainly B2+D.

Phylogenetic clustering is an important method for typing E.coli. According to previous reports, phylogenetic clustering of E.coli can be divided into four groups: A, B1, B2 and D, among which B2 and D are considered to be the main pathogenic groups (Javed et al., 2021; Lee et al., 2016). Pathogenic strains belonging to group B2 and, to a lesser extent, group D, are known to carry more virulence factor genes than strains of groups A and group B1 (Nowrouzian et al., 2005). More specifically, the B2 phylogenetic group of E. coli includes important pathogens such as extraintestinal pathogenic, adherent-invasive and uropathogenic strains (Deshpande et al., 2015). Zhu Ge ​et al. (2014) discovered avian pathogenic E. coli and human extraintestinal infection of large intestine Most of the bacilli belong to group B2 (Zhu Ge​ et al., 2014). In this study, it was found that the E. coli isolated from goose and fish were dominated by group B2 and D. These data provide a helpful reference about the ecological distribution and genetic evolution of E. coli in the area. The results were consistent with the results of dominant evolutionary grouping of avian pathogenic E.coli reported by Wang et al. (2026) (Wang et al., 2016). However, it was different from the research results of other researchers (Higgins et al., 2007; Kuczkowski et al., 2016), which may be due to the different animal sources, sampling areas and feeding environment.
 
Biofilm formation ability
 
The results of this study showed that some strains of E.coli isolated from goose and fish could form biofilm. Among the fish-derived E.coli isolates, 69% strains had biofilm formation ability, including 36% with strong ability, 33% with weak ability and 31% without biofilm formation ability. Among the goose-derived E.coli isolates, 60% strains had biofilm formation ability, including 42% with strong ability, 18% with weak ability and 40% without biofilm formation ability. This indicates that the biofilm formation rate of E. coli from goose and fish in Henan, China is high. This may be due to the long-term use of low concentrations of antibiotics in feeding (Chakraborty et al., 2020; Li et al., 2021). Earlier, other workers also reported biofilm production by Ecoli from chicken, pig, duck and chicken products (Li et al., 2021; Wang et al., 2011; Wang et al. 2016). However, no report could be traced in literature on the biofilm forming ability and drug resistance by E.coli isolated from goose and fish.
 
Antibiotic sensitivity
 
The results of the drug sensitivity test of E. coli isolated from fish were shown in Table 2.

Table 2: Relationship between phylogenetic group and drug sensitivity of E. coli from fish.



All isolates showed a high resistance rate (84% - 100%). The results of the drug sensitivity test of E. coli isolated from goose were shown in Table 3.

Table 3: Relationship between phylogenetic group and drug sensitivity of E. coli from geese.



All isolates also showed a high resistance rate (48% -100%). The drug-resistance rate of the phylogenetic groups B2 and D was higher than the resistance rate of the phylogenetic groups A and B1. This study found that E. coli isolated from fish and goose had high multiple drug resistance rates. The reason may be related to different breeding environments. From the point of view of the aquaculture environment when collecting samples, the aquaculture density of fish is highly concentrated and in order to ensure the water quality of fish farming, appropriate amount of fungicides will be regularly put into the water. Long-term use of large amounts of antibacterial drugs can easily lead to high resistance rate of E. coli from fish. In the breeding process of geese, some antibacterial drugs will be used regularly, which will increase the resistance of E. coli.

At present, most of the drug susceptibility test objects are planktonic bacteria. In this study, the MIC values against planktonic and biofilm E.coli were detected and the results showed that the MIC values would increase with the formation of biofilm. Compared with planktonic bacteria, the MIC values of tetracycline, aureomycin, enrofloxacin, doxycycline, TMP trimethoprim, norfloxacin and gentamicin against biofilm fish-derived E.coli were increased by 2, 8, 8, 4, 4, 16 and 2 times, respectively. The MIC values of tetracycline, aureomycin, enrofloxacin, doxycycline, TMP trimethoprim and norfloxacin against biofilm goose-derived E. coli were increased by 2, 8, 4, 2, 8 and 2 times, respectively (Table 4).

Table 4: MIC value of antibiotics to different types of E. coli strains (μg/ml).



Similarly, Significant differences were observed between MICs of planktonic cells and MICs of UPEC biofilms, indicating a higher level of bacterial tolerance in biofilm form (Rafaque et al., 2020). (Wang et al. 2020) found that the E. coli minimum biofilm eradication concentrations were generally two times higher than the planktonic minimum inhibitory concentrations (Wang et al., 2020). They believed that MIC would significantly increase with the formation of biofilm, which further indicated that the formation of biofilm could increase the drug resistance of bacterial strains. Therefore, biofilm is of great significance for the prevention and treatment of clinical E.coli. It is necessary to evaluate the bacterial status of infection during clinical medication. Especially when the clinical treatment effect is poor, drug resistance and biofilm factors need to be considered.
 
Correlation analysis of biofilm formation ability and drug resistance
 
Table 5 indicated that the isolates showed multidrug-resistance (MDR) (100%).

Table 5: Resistance spectrum of biofilm-positive strains and biofilm-negative strains to antibiotics.



Among fish-derived E.coli isolates, 90 strains were resistant to more than 8 drugs and the biofilm-positive strains accounted for 69% (62/90) and the biofilm-negative strains accounted for 31% (28/90). Among goose-derived E.coli isolates, 91 strains were resistant to more than 8 drugs and the biofilm-positive strains accounted for 59% (54/91) and the biofilm-negative strains accounted for 41% (37/91). The antibiotic resistance of biofilm-forming E. coli isolates was found to be significantly higher than that of strains unable to form biofilm (p<0.05). The results of this study showed the number of drug resistance of biofilm-positive strains was higher than that of biofilm-negative strains, further proving that the biofilm-forming ability of bacteria is closely related to drug resistance. Similarly, Qian et al., (2022) revealing that the populations that exhibited more robust biofilm formation likely contained larger proportions of extensively drug-resistant (XDR) isolates. Dumaru et al., (2019) found that there was strong association between the MDR-status and biofilm-production in gut bacteria (Dumaru et al., 2019). Katongole et al., (2020) demonstrated a high prevalence of biofilm-forming Uropathogenic E. coli strains that are highly associated with the MDR phenotype (Katongole et al., 2020).
The biofilm formation ability was associated with the drug resistance. In this study, phylogenetic clustering detection, biofilm formation ability and drug resistance of E. coli from goose and fish were studied to provide reference for the pathogenic mechanism and prevention and control of E.coli from goose and fish and to lay a foundation for the subsequent research on the drug resistance mechanism of E. coli.
This work was supported by the Henan Provincial Science and Technology Research Project (232102110095).
None

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