| Mastitis is a common disease in dairy cows and is a significant factor affecting milk production and quality.It can also cause irreversible damage to the cow’s body and severely restrict the development of animal husbandry.Pseudomonas aeruginosa is a conditionally pathogenic bacterium that is shared by both humans and animals,and has a high detection rate in mastitis,often causing persistent chronic infections.Therefore,this study isolated and identified P.aeruginosa from clinical milk samples collected from some areas in northern Jiangsu,China.The strains were then subjected to MLST typing,and their drug resistance phenotypes,β-lactamase genes,virulence genes,and biofilm were detected,and the correlation between biofilm formation,drug resistance,and virulence genes was explored.The aim of this study was to understand the distribution of drug resistance and virulence genes of P.aeruginosa in dairy cow mastitis in northern Jiangsu and the relationship between biofilm formation ability of the strains,in order to provide a basis for the prevention and treatment of P.aeruginosa infection in production.Using the traditional combination of bacterial culture and 16S rDNA identification,486 strains of bacteria were isolated from 187 clinical mastitis milk samples,of which 63 strains were Pseudomonas aeruginosa,with a detection rate of 12.96%,and the bacterial phylogenetic tree showed that 63 strains of Pseudomonas aeruginosa had strong homology..The MLST typing of the 63 strains of Pseudomonas aeruginosa was performed by PCR amplification of the housekeeping gene.A total of 16 ST types were obtained,with 5 major ST types accounting for 65%of the strains.By comparing the bacterial phylogenetic tree with the typing results,most of the strains with the same ST type were located on the same branch of the evolutionary tree,further proving their high homology.The drug susceptibility of Pseudomonas aeruginosa to 10 antibiotics includingβ-lactams,aminoglycosides,fluoroquinolones,and tetracyclines was determined using the KB disk diffusion method.The overall resistance of 63 P.aeruginosa strains was not strong,with a 100%resistance rate to penicillin.The strains showed strong resistance to the aminoglycoside antibiotics streptomycin and gentamicin,with resistance rates of 69.84%and 49.21%,respectively.The resistance rates to ciprofloxacin,imipenem,meropenem,and cefotaxime were all 0,with meropenem and cefotaxime showing the strongest antibacterial effects,with sensitivity rates of 100%.Additionally,only 7 ESBL-producing strains were identified,accounting for 11.11%of the total strains.There were differences in the antibiotic resistance phenotypes of the 5 major ST types,with strains that were intermediate to gentamicin only appearing in the ST641 type,and strains resistant to piperacillin only appearing in the ST277 and ST450 types.The overall resistance rates of different ST types were also significantly different,and the resistance phenotypes of strains with the same ST type were not completely consistent,indicating that the resistance of strains to antibiotics is influenced by their own genetic adaptability.The test results of β-lactamase genes showed that all 63 strains of Pseudomonas aeruginosa tested positive for β-lactamase genes,and a total of six types ofβ-lactamase genes were detected,with no detection of the PER gene.The SHV gene had the highest detection rate at 96.83%,followed by the GES gene at 41.27%,while the detection rates of VEB,CTX-M-1,and OXA genes were very low.There were 11β-lactamase genotypes in the 63 strains of Pseudomonas aeruginosa,with 30 strains having a single β-lactamase genotype,and 29 of those strains being SHV type strains.Although the detection rate of β-lactamase genes in Pseudomonas aeruginosa was relatively high in this study,the susceptibility test showed a very low resistance toβ-lactam antibiotics,which may be related to the less frequent use of β-lactam antibiotics in the dairy farms of northern Jiangsu.In this study,15 virulence genes were detected by PCR amplification,and the results showed that the overall detection rate of virulence genes in the 63 strains of Pseudomonas aeruginosa was relatively high,with detection rates of over 90%for 9 virulence genes.There were 58 strains carrying 11 or more virulence genes,accounting for 92.06%of the total,and the most frequently detected virulence gene combination was the 14 virulence genes,which were found in 19 strains,accounting for 30.16%of the total.In addition,7 strains of Pseudomonas aeruginosa carried all 15 virulence genes.With the exception of ciprofloxacin-resistant strains and a lower number of piperacillin-resistant strains,the frequencies of six virulence genes,exoU,pyo,toxA,lasA,lasR,and rhlL,in resistant strains were mostly lower than those in sensitive strains,indicating a correlation between drug resistance phenotype and virulence genes.Strains with higher drug resistance tended to carry fewer virulence genes,indicating weaker virulence.The results of the biofilm assay showe that 63 strains of Pseudomonas aeruginosa,there were relatively few strains with strong biofilm formation ability,with only 12 strains accounting for 19.05%of the total,while the highest number of strains had moderate biofilm formation ability,with 27 strains accounting for 42.86%of the total.In this study,strains with strong biofilm formation ability had a lower detection rate of virulence genes exoU and toxA,with P<0.05,indicating a significant difference.There were a relatively high proportion of strains with strong biofilm formation ability among strains without the virulence genes pyo,lasR,and rhlL,but the difference was not significant.In addition,the average number of virulence genes carried by the 63 strains of P.aeruginosa was inversely proportional to their biofilm formation ability,i.e.,strains with strong biofilm formation ability carried fewer virulence genes.Furthermore,this study found that drug-resistant strains were more likely to form strong biofilms,but the difference was not significant.In this study,we compared the number of bacteria growing in the state of plankton and the state of biofilm to test whether biofilm could enhance the resistance of Pseudomonas aeruginosa to gentamicin.The results showed that the number of Pseudomonas aeruginosa grown in the biofilm state was significantly higher than that in the planktonic state,and there were differences among strains with different biofilm formation abilities,with strains that had strong biofilm formation abilities showing greater resistance to gentamicin.Therefore,biofilms can enhance the antibiotic resistance of Pseudomonas aeruginosa,and the formation of biofilms may be related to the number of virulence genes carried.In summary,this experiment isolated and identified the clinical mammary source of Pseudomonas aeruginosa in dairy cows in some areas of northern Jiangsu,and analyzed the correlation between virulence genes,drug resistance characteristics and biofilm formation ability of the isolates,so as to provide experimental support for the prevention and treatment of Pseudomonas aeruginosa infection in production. |
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