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Article

Isolation of Pathogenic Bacteria from Dairy Cow Mastitis and Correlation of Biofilm Formation and Drug Resistance of Klebsiella pneumoniae in Jiangsu, China

by
Wendi Cao
1,2,
Yi Xu
3,
Yicai Huang
2 and
Tianle Xu
1,2,4,*
1
Joint International Research Laboratory of Agriculture and Agri-Product Safety, Ministry of Education of China, Yangzhou University, Yangzhou 225009, China
2
College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China
3
College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China
4
International Joint Research Laboratory in Universities of Jiangsu Province of China for Domestic Animal Germplasm Resources and Genetic Improvement, Yangzhou 225009, China
*
Author to whom correspondence should be addressed.
Agriculture 2023, 13(10), 1984; https://0-doi-org.brum.beds.ac.uk/10.3390/agriculture13101984
Submission received: 14 September 2023 / Revised: 7 October 2023 / Accepted: 11 October 2023 / Published: 12 October 2023
(This article belongs to the Special Issue Pathology and Veterinary Diagnostics of Farming Animals)
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Abstract

:
According to recent investigations, the proportion of mastitis caused by environmental pathogens, such as K. pneumoniae, has increased. In this research, the epidemiology of pathogens in milk samples collected from four farms in the Jiangsu Province was carried out. The results show that 16 pathogens were detected in 186 positive milk samples. It was found that K. pneumoniae had the lowest sensitivity to penicillin (0%) and amoxicillin (4%) compared to its sensitivity to gentamicin (92%) and piperacillin (89%). A total of eight ESBL-producing strains were detected. Crystal violet staining showed that 46 of the 68 isolates of K. pneumoniae had strong biofilm-forming ability, which was related to the tetracycline resistance phenotype (p < 0.05). The detection rate of the ESBL-resistant gene (blaSHV) reached 100%. The results show that resistance genes blaVIM, blaOXA-10, and blaTEM were correlated with drug-resistance phenotypes to varying degrees. The present study indicates the prevalence of bovine mastitis-derived pathogens in part of Jiangsu Province and reveals the distribution of β-lactam resistance genes and the strong biofilm-forming ability of K. pneumoniae and its relationship with tetracycline resistance. This study provided theoretical support and guidance for rational drug use and disease prevention and control on farms.

1. Introduction

As a common disease in the dairy industry, bovine mastitis has a great impact on the economy of dairy farming, which can lead to large production losses [1]. According to the symptoms and milk changes, bovine mastitis is divided into two types: clinical and subclinical mastitis. Although there are no obvious clinical symptoms of recessive mastitis, it increases the number of somatic cells and bacteria in milk and changes the physiological properties of milk. The occurrence of mastitis in cows is caused by many pathogenic factors, such as immune deficiency, pathogenic infection, environmental stress, etc. [2]. The pathogenic bacteria that cause mastitis in bovines include contagious and environmental pathogenic bacteria. The contagious pathogenic bacteria mainly include Streptococcus, Staphylococcus, and Enterobacter, while the environmental pathogenic bacteria mainly include Acinetobacter, Pseudomonas, Klebsiella, and so on [3]. Currently, cross-infection by complex bacteria is a difficult problem in the treatment of mastitis.
Gram-negative bacteria such as E. coli and K. pneumoniae account for a large proportion of environmental pathogenic infections [4]. Compared with E. coli, acute mastitis caused by K. pneumoniae has more serious clinical symptoms [5]. Research shows that clinical mastitis caused by K. pneumoniae does not respond well to antimicrobial therapy, resulting in prolonged infection and significantly decreased milk production [6]. Meanwhile, against the background of excessive antibiotic use, a large number of drug-resistant strains have gradually emerged, which brings great challenges to prevention and control. Studies have shown that the drug-resistance phenotypes of K. pneumoniae from bovine mastitis in different regions are quite diverse. Luo et al. [7] found that K. pneumoniae from cow mastitis at four large-scale farms in the Hunan Province was sensitive to gentamicin and ciprofloxacin and showed strong resistance to penicillin, tetracycline, erythromycin, and vancomycin. Gao et al. [8] isolated K. pneumoniae from large-scale farms in 24 provinces and autonomous regions of China that were resistant to cefepime (5.7%), ciprofloxacin (4.7%), ceftazidime (4.2%), meropenem (2.8%), ertapenem (0.5%), and other drugs. Under the influence of various factors, such as medication, environment, feeding, and management, K. pneumoniae can show higher resistance to antibiotics through its own evolution and adaptation to different environments. Following the extensive use of antibacterial drugs, K. pneumoniae carries antimicrobial drug-decomposing enzymes. There are many resistance genes encoding antibacterial drug-decomposing enzymes, including those encoding extended-spectrum β-lactamases (ESBL: SHV, TEM, CTX-M, etc.) [9] and carbapenemases (KPC, VIM, OXA, etc.) [10].
K. pneumoniae has extremely strong film-forming ability. Ashwath et al. found that 97.1% of 70 strains of multidrug-resistant K. pneumoniae isolated from hospitals formed biofilms [11]. Bacterial biofilms will colonize on medical devices and increase drug resistance, which brings difficulties to the treatment of K. pneumoniae [12]. Moreover, biofilm production will regulate the phagocytosis of macrophages and the expression of cytokines, which can inhibit the immune response of macrophage cells and increase the expression of cytokines [13]. In the present study, an epidemiological investigation of pathogenic bacteria of bovine mastitis was carried out at farms in the Jiangsu Province in order to uncover the epidemic trends and characteristics of K. pneumoniae. Because it carries drug-resistant genes and is biofilm-forming, it is expected to effectively orient the rational use of drugs on farms, which is helpful to accurately prevent and control bovine mastitis caused by this organism.

2. Materials and Methods

2.1. Isolation of Bacteria from Milk Samples

In the current research, 208 clinical mastitis milk samples were collected from four farms in different areas of Jiangsu Province from March to August 2022. According to the recommendations of the National Mastitis Commission, the California Mastitis Test (CMT) was used for testing [14]. To prevent contamination during sampling, the first stream of milk was discarded, and the teats were subsequently exposed to iodine tincture for 30 s and dried using an individual towel. In total, 20~30 mL of milk samples were collected in the sterilized 50 mL tube after labeling and cryopreservation. Milk samples (80 μL) were cultured in 5% fresh sheep blood agar at 37 °C for 18~24 h. According to the size, morphology, color, hemolysis, and transparency of the colonies, the typical colonies were selected and cultured at 37 °C with 200 rpm overnight. The bacterial suspensions of selected colonies were streaked and passaged on Luria-Bertain agar, and the cultured colonies were inoculated in a liquid medium and incubated aerobically at 37 °C for 24 h. The genomic DNA of suspected pathogens was isolated using a DNA extraction kit (Tiangen, Beijing, China). The products of the PCR reaction were purified, and 16S rDNA pyrophosphate was sequenced. The sequences were then aligned using SnapGene (GSL Biotech, Chicago, IL, USA) [15]. Finally, the NCBI database was used for Blast alignment of the PCR product sequences for species identification. The identified strains were stored in a cryopreservation solution containing 15% glycerol at −80 °C. K. pneumoniae was taken for analysis and identification of antimicrobial susceptibility, resistance gene detection, and biofilm-forming capacity. All of the experimental protocols were performed in accordance with the approved guidelines and regulations by the Animal Experiment Committee of Yangzhou University (YZU202302-021).

2.2. Determination of Antimicrobial Susceptibility

The Kirby–Bauer (K–B) disk was applied on Mueller–Hinton agar with 0.5 McFarland units of cultured bacteria to detect the antibacterial effect of each antibiotic [16]. Tetracyclines (tetracycline (TE, 30 μg)), macrolides (azithromycin (AZM, 15 μg)), aminoglycosides (streptomycin (SM, 10 μg)), gentamicin (GM, 10 μg), β-lactams (cefalotin (CEF, 30 μg)), penicillin (PEN, 10 μg), amoxicillin (AMX, 20 μg), piperacillin (PIP, 100 μg), ceftazidime (CAZ, 30 μg), ceftazidime/clavulanic acid (TED, 30–10 μg), cefotaxime (CTX, 30 μg), and cefotaxime/clavulanic acid (CD, 30–10 μg) were selected. According to the Clinical & Laboratory Standards Institute [16] standard manual, the drug sensitivity of K. pneumoniae isolates was determined. The K. pneumoniae ATCC 4352 was taken as a standard reference, and the test results were recorded as sensitive or resistant. When there was no zone diameter interpretation standard, the instructions of the antibacterial disc from the manufacturer were considered.
The identification of ESBL isolates was determined by the K–B disk diffusion method according to the susceptibility of ceftazidime and cefotaxime. If the inhibition zone of ceftazidime was ≤22 mm or the inhibition zone of cefotaxime was ≤27 mm, the strain was initially determined to produce ESBL K. pneumoniae. In order to further determine whether the isolates produced ESBLs, the difference in the diameter of the inhibition zone of ceftazidime/clavulanic acid in ceftazidime, cefotaxime, and cefotaxime/clavulanic acid was calculated. If the difference in the diameter of the inhibition zone of any one of the compound preparation discs in the single drug disc was ≥5 mm, the strain was identified as producing ESBL K. pneumoniae.

2.3. Detection of Drug-Resistance Genes

In this study, PCR was used to detect drug-resistance genes of the isolated K. pneumoniae. A total of 9 kinds of Carbapenem resistance genes, including blaKPC-2, blaNDM-1, blaVIM, blaOXA-48, and blaOXA-10 and ESBL resistance genes blaTEM, blaSHV, blaVEB, and blaPER were detected [17,18,19]. Primers used in the current study has been listed in Table S1. The amplification conditions were as follows: 95 °C pre-denaturation 3 min; then 35 cycles of denaturation at 94 °C for 30 s; annealing at 55 °C for 30 s, extension at 72 °C for 1 min; and final extension at 72 °C for 8 min. The amplified PCR products were verified on a 1% agarose gel and sequenced (Tsingke, Beijing, China). The presence of the target genes was defined by recognizing the visible bands on the gel electropherogram.

2.4. Biofilm Formation Determination

The biofilm-forming ability of 68 K. pneumoniae isolates was determined using a 24-well plate and cultured in the incubator at 37 °C for 48 h. Each isolate was set up with three replicates, and a negative control group containing only broth was set up accordingly. Crystal violet staining was used to stain the biofilm. The bacterial suspension in the suction hole was washed with phosphate buffer solution and dried at room temperature for 15 min. The biofilm was fixed with methanol solution and stained with 1% crystal violet dye solution. After washing the dye solution with sterile ultrapure water, the crystal violet bound to the biofilm was dissolved with 30% acetic acid solution, and the absorbance was measured at 590 nm using a spectrophotometer (Tecan, Spark, Switzerland). Set the average value of the negative control group as OD0, the critical value ODC = OD0 + 3SD (standard deviation), then the relative value SI = OD/ODC, SI is divided into three grades [20]: 0 < SI ≤ 1.5, biofilm formation ability is defined weak or no biofilm formation ability; 1.5 < SI < 2.5 is defined as medium biofilm formation ability; SI ≥ 2.5 is defined as strong biofilm-formation ability.

2.5. Statistical Analysis

A chi-square test (χ2) was used to compare the statistical significance between the groups. The difference was significant when p < 0.05; p < 0.01 indicated that the difference was extremely significant. Pearson correlation coefficient (r) is a non-parametric index reflecting the degree of correlation between the two variables, and its value is between −1 and 1. With the help of Spss26.0 software, the chi-square test and Pearson correlation coefficient were used to analyze the correlation between biofilm and drug-resistance genes and drug-resistance phenotypes.

3. Results

3.1. Identification of Bacteria in Milk Samples Derived from Mastitis-Infected Dairy Cows at Various Farms

Pathogenic bacteria were isolated and cultured from 208 clinical milk samples from cows with mastitis. The results show that pathogenic bacteria were positively detected in 186 milk samples with a detection rate of 89.42%. The highest detection rate of pathogenic bacteria in milk samples was 94.32% at farm A, while the lowest detection rate was 84.62% at farm C (Figure 1). The total number of bacterial species was a total of 438 isolates of 16 pathogenic bacteria that were detected in milk samples, among which the majority were environmental pathogenic bacteria. K. pneumoniae (15.35%) accounted for the highest proportion, followed by E. coli (13.70%), Pseudomonas aeruginosa (12.33%), and Streptococcus agalactiae (10.5%) compared to the other detected bacteria. Most of the milk samples collected from the four farms were infected by the first two pathogens. As compared with farm A (42.31%), farm B (44.23%), and farm D (40.38%), farm C (51.92%) accounted for the highest proportion of milk samples in which both pathogens were isolated. The detection rate of K. pneumoniae at farms A, B, and D was 21.82%, 14.95%, and 12.93%, respectively, while E. coli (14.29%) had the highest detection rate at farm C (Table 1).

3.2. Research on Biofilm and Drug-Resistance Characteristics

In this study, 68 strains of K. pneumoniae isolates showed different levels of resistance to four types (eight kinds) of antibiotics. The results show that K. pneumoniae had the lowest sensitivity to penicillin (0%), followed by amoxicillin (4%), while it had a strong sensitivity to cefalotin (88%) and piperacillin (89%). It had the strongest sensitivity to the aminoglycoside gentamicin (93%), and it had a lower sensitivity to streptomycin (51%) than gentamicin (93%) (Figure 2). The K. pneumoniae isolates also showed high sensitivity to tetracycline (72%). The ESBL determination test was performed on 68 K. pneumoniae isolates. Eight ESBL-producing strains were detected from the four farms, with detection rates of 12.5% (3/24), 12.5% (2/16), 15% (2/13), and 7% (1/15) (Figure 3). The results of crystal violet staining showed that 46 of the 68 K. pneumoniae isolates had strong biofilm-forming ability, 17 had moderate biofilm-forming ability, and 5 had weak or no biofilm-forming ability. The results of analyzing the correlation between biofilm and drug-resistance phenotype showed a significant correlation between biofilm-forming ability and the tetracycline resistance phenotype (p < 0.05). Among the strains with strong biofilm-forming ability, there were more strains sensitive to tetracycline, gentamicin, azithromycin, piperacillin, and cefalotin than strains that were resistant to those drugs (Table 2).

3.3. Analysis of Drug-Resistance Genotype of K. pneumoniae

In the present study, we selected 68 strains of K. pneumoniae for the detection of nine drug-resistance genes. The results showed that five drug-resistance genes were detected (Table 3); the positive detection rate of the ESBL drug-resistance gene blaSHV was as high as 100%, while the detection rate of blaTEM was 64.7%. The positive detection rates of blaNDM-1, blaVIM, and blaOXA-10 in carbapenem-resistant genes were lower than 50%. Furthermore, 31 strains of K. pneumoniae carried two drug-resistance genes simultaneously, most of which (19 isolates) carried ESBL drug-resistance genes blaSHV and blaTEM. As shown in Table 4, K. pneumoniae isolated carrying carbapenem-resistant gene blaVIM were significantly positively correlated with azithromycin resistance (R = 0.296). The resistance gene blaOXA-10 was significantly positively correlated with resistance to tetracycline (R = 0.287) and streptomycin (R = 0.250) and was significantly negatively correlated with resistance to azithromycin (R = −0.279) and amoxicillin (R = −0.242). However, the ESBL-resistant gene blaTEM was positively correlated with resistance to two β-lactam antibiotics: cefathiophene (R = 0.270) and piperacillin (R = 0.250).

4. Discussion

Bovine mastitis is a common disease in the dairy industry and is induced by multiple causes. The main pathogens involved in mastitis are often differentially prevalent in different regions. It is crucial to investigate the dominant pathogens in each region in order to prevent and treat mastitis in bovines. In this study, 208 milk samples from cows with clinical mastitis collected from four farms in parts of the Jiangsu Province were isolated for identification of bacterial pathogens. The results show that the detection rate of pathogenic bacteria was as high as 84.92%, indicating that bacteria are important pathogens causing mastitis in dairy cows. The infection pattern of mastitis pathogens in bovines on the experimental farm is complex, and there were more milk samples with two pathogens isolated at the same time, which is consistent with previously reported results [21]. It was found that the number of milk samples containing two pathogens was the highest in northern Jiangsu, even for the increasing infections with 3–4 pathogens, indicating that there is a serious mixed infection of pathogens in bovines in this area. In this study, K. pneumoniae, E. coli, Pseudomonas aeruginosa, and Streptococcus agalactiae were widely prevalent in the sampling areas, which was different from the results reported in another study in Lanzhou, China [22]. This may be caused by differences between regions, sampling seasons, feeding management styles, and drug treatments.
Antibiotics are currently widely used to treat bacterial infections, but the abuse of antibiotics leads to the emergence of drug-resistant bacteria and poses a certain threat to global public health [23]. The results show that K. pneumoniae displayed different degrees of resistance to four types of antibiotics, especially to penicillin and amoxicillin among the β-lactams, which was consistent with the results of Sun et al. [24]. This indicates that the treatment at the farms may have been based on β-lactam application. β-lactam drugs are commonly used to treat bacterial infections caused by Enterobacteriaceae [25]. However, extensive use of this drug has accelerated the emergence of drug-resistant bacteria, especially ESBL-producing strains. The results of this study indicate that although the detection rate of ESBL-producing strains at each farm was low, it is still imperative to pay attention to environmental hygiene and prevent the spread of ESBL-producing strains between adjacent farms.
It is known that the formation of biofilm enhances the drug resistance of K. pneumoniae to a certain extent [26]. Many factors contribute to K. pneumoniae producing biofilms, such as quorum sensing, capsule, and fimbriae [27]. Due to the production of biofilm, K. pneumoniae is more likely to colonize the respiratory tract, gastrointestinal tract, and urethra [28]. The results of this study show that the biofilm-forming ability of K. pneumoniae was related to the resistance phenotype of tetracycline, which was different from the results of Türkel [29] and Mirzaei [30]. This may be related to the resistance mechanism of tetracycline and the state of K. pneumoniae biofilm. Tetracycline can be used against both Gram-negative and Gram-positive bacteria. Its mechanism of action is mainly to target 30S prokaryotic ribose subunits, thereby preventing interactions with aminoacyl t-RNA and ribosomes and inhibiting peptide chain extension and protein synthesis [31]. The biofilm produced by planktonic bacteria will first form a protein film on the surface of the object, and the bacteria are still partially sensitive to antibiotics [32]. It is indicated that the biofilm-forming ability of K. pneumoniae is strong, but its colonizing ability may be weak, resulting in enhanced sensitivity to tetracycline.
Drug-resistance genes carried by plasmids can be spread between bacteria through the mechanisms of conjugation, transformation, or transduction. Moreover, K. pneumoniae can be used as a key host and an important transmission carrier of drug-resistance genes, which is one of the main reasons for its increased multidrug resistance [33]. Five carbapenemase-resistant genes and four ESBL-resistant genes were selected for PCR detection in this study, and the results show that five genes were detected, among which the ESBL-resistant genes blaSHV and blaTEM had the highest detection rates. This is in accordance with the results of the drug-resistant genes in mastitis-derived K. pneumoniae isolates from cows on farms in China, as reported by Maierhaba [34]. A total of 189 subtypes of the blaSHV gene have been reported [35]. Among them, blaSHV-11 is located on the chromosome of K. pneumoniae, which is a kind of ESBL gene naturally carried by K. pneumoniae [36]. Most blaSHV subtypes are located on plasmids, which easily spread among K. pneumoniae with other drug-resistance genes [37]. blaTEM is also a common ESBL-resistant gene with many sub-genotypes. BlaSHV and blaTEM genes can hydrolyze penicillins, cephalosporins, and other drugs and mediate the resistance of K. pneumoniae carrying the gene to various β-lactam drugs [38]. The correlation results show that the blaTEM gene was significantly positively correlated with resistance to cefalotin (0.270) and piperacillin (0.250), indicating that the resistance of K. pneumoniae isolates to these drugs may be mediated mainly by the blaTEM gene in these regions.
K. pneumoniae acquires carbapenemase-resistant genes by horizontal or vertical transmission with the help of mobile element plasmids, which enhances resistance to β-lactam drugs. According to the Ambler classification, carbapenemase-resistant genes can generally be divided into three categories: A, B, and D. Among them, the blaKPC gene is the most common in class A enzymes. Studies have shown that most of the ST 11 K. pneumoniae strains prevalent in China carry the blaKPC-2 gene [39]. However, the blaKPC-2 gene was not detected in the K. pneumoniae isolates in the current study, indicating that the gene has not spread in those areas of Jiangsu Province. There were blaNDM, blaVIM, blaIMP, and other resistance genes in class B metalloenzymes. The blaNDM-1 and blaVIM genes were detected in the test, with detection rates of 36.8% and 17.6%, respectively. These results are inconsistent with the results of K. pneumoniae-carrying genes at the farm in Hubei reported by Wu [40]. This might be attributed to the different geographical locations, large differences in the molecular characteristics of K. pneumoniae at various farms, and different medications. Class D enzymes are mainly OXA type. Studies have shown that K. pneumoniae carrying both blaOXA-48 and blaNDM genes can produce super-drug-resistant strains [41]. At present, the detection rate of the blaOXA-48 gene in China is low [42]; the results show that this gene was not detected in K. pneumoniae isolates, but 13 (19.1%) of K. pneumoniae isolates carried blaOXA-10 gene. The chi-square test and Spearman correlation coefficient results indicate that there were different levels of correlation between carbapenemase-resistant genes and some antibiotics. There was a significant positive correlation between blaVIM and azithromycin (0.296). BlaOXA-10 was significantly positively correlated with tetracycline (0.287) and streptomycin (0.250) and significantly negatively correlated with azithromycin (−0.279) and amoxicillin (−0.242), indicating that carbapenemase-resistant genes may coexist with other antimicrobial-resistant genes on the plasmid, and jointly mediate K. pneumoniae resistance to multiple drugs. The specific resistance mechanism remains to be further studied.

5. Conclusions

The detection rate of pathogenic bacteria in the mammary glands of dairy cows in part of Jiangsu was high, and there were frequent cases of mixed bacterial infection. There were many environmental pathogenic bacteria, and the detection rate of K. pneumoniae was the highest. In all, 68 strains of K. pneumoniae showed different sensitivity to different types of antimicrobial drugs but had strong resistance to β-lactam drugs. In addition, the detection rate of β-lactamase genes was high, and K. pneumoniae biofilm formation was strong and correlated with the tetracycline resistance phenotype. This study provides a basis for understanding the prevalence of mastitis pathogens in lactating cows and the rational use of antimicrobials for the treatment of K. pneumoniae-induced mastitis.

Supplementary Materials

The following supporting information can be downloaded at: https://0-www-mdpi-com.brum.beds.ac.uk/article/10.3390/agriculture13101984/s1, Table S1 Primers used for the PCR experiment.

Author Contributions

Conceptualization, W.C. and T.X.; methodology, W.C.; validation, Y.H. and Y.X.; formal analysis, W.C.; investigation, W.C.; resources, T.X.; data curation, W.C.; writing—original draft preparation, W.C.; writing—review and editing, T.X.; project administration, T.X.; funding acquisition, T.X. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the National Natural Science Foundation of China (32102731); The Open Project Program of Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou University (JILAR-KF202204); The Open Project Program of International Joint Research Laboratory in Universities of Jiangsu Province of China for Domestic Animal Germplasm Resources and Genetic Improvement: (IJRLD-KF202214).

Institutional Review Board Statement

The manuscript does not contain experiments using animals or human studies.

Data Availability Statement

Data are available on request from the authors.

Acknowledgments

We appreciate the support of the milk sample provisions from the dairy farms located in the 4 correlated farms.

Conflicts of Interest

The authors declare no conflict of interest.

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Agriculture 13 01984 g001
Figure 1. The detection rate of pathogenic bacteria and the infection pattern of pathogenic bacteria in each farm. The blue bar in the chart represents one kind of pathogenic bacteria infection; orange bar indicates two types; gray bar represents three types; yellow bar represents four types; the line indicates the detection rate of pathogenic bacteria in each farm.
Figure 1. The detection rate of pathogenic bacteria and the infection pattern of pathogenic bacteria in each farm. The blue bar in the chart represents one kind of pathogenic bacteria infection; orange bar indicates two types; gray bar represents three types; yellow bar represents four types; the line indicates the detection rate of pathogenic bacteria in each farm.
Agriculture 13 01984 g001
Agriculture 13 01984 g002
Figure 2. Drug-resistance phenotype of K. pneumoniae. Using 4 kinds of 8 antibiotics to analyze the drug sensitivity of K. pneumoniae. Blue, gray, and orange bar charts represent drug resistance levels with resistant (R), sensitive (S), and intermediate (I), respectively.
Figure 2. Drug-resistance phenotype of K. pneumoniae. Using 4 kinds of 8 antibiotics to analyze the drug sensitivity of K. pneumoniae. Blue, gray, and orange bar charts represent drug resistance levels with resistant (R), sensitive (S), and intermediate (I), respectively.
Agriculture 13 01984 g002
Agriculture 13 01984 g003
Figure 3. The detection rate of ESBLs in each farm.
Figure 3. The detection rate of ESBLs in each farm.
Agriculture 13 01984 g003
Table 1. Distribution of detected pathogens in each farm.
Table 1. Distribution of detected pathogens in each farm.
Pathogenic BacteriaFarm AFarm BFarm CFarm D
Detection Rate/%Detection Rate/%Detection Rate/%Detection Rate/%
Escherichia coli20 (18.18)13 (12.15)15 (14.29)12 (10.34)
Klebsiella Pneumoniae24 (21.82)16 (14.95)13 (12.38)15 (12.93)
Pseudomonas aeruginosa12 (10.91)15 (14.02)14 (13.33)13 (11.21)
Enterobacter cloacae9 (8.18)5 (4.67)3 (2.86)5 (4.31)
A.calcoaceticus2 (1.82)3 (2.80)4 (3.81)3 (2.59)
Megasphaera Rogosa--6 (5.71)3 (2.59)
Shigella Castellani-4 (3.74)--
Staphylococcus aureus8 (7.27)9 (8.41)9 (8.57)12 (10.34)
Staphylococcus sciuri3 (2.73)6 (5.61)5 (4.76)6 (5.71)
Staphylococcus chromogenes2 (1.82)5 (4.67)3 (2.869 (7.76)
Streptococcus agalactiae10 (9.09)13 (12.15)11 (10.48)12 (10.34)
Enterococcusfaecalis5 (4.55)2 (1.87)7 (6.67)6 (5.17)
Streptococcus dysgalactiae5 (4.55)6 (5.61)7 (6.67)6 (5.17)
Bacillus cereus3 (2.73)6 (5.61)3 (2.86)4 (3.45)
Bacillus licheniformis4 (3.64)2 (1.87)-6 (5.17)
Lactococcus garvieae--1 (0.95)-
Others3 (2.73)2 (1.87)4 (3.81)4 (3.45)
Table 2. Correlation analysis between film-forming ability and drug-resistance phenotype.
Table 2. Correlation analysis between film-forming ability and drug-resistance phenotype.
Antimicrobial Biofilm Forming Abilityχ2p
StrongModerateWeak
TER83014.9970.005
I313
S35132
GMR3108.460.932
I100
S42165
SR16747.7420.102
I221
S2880
AZMR4005.1430.273
I652
S36123
PR421334.9300.085
I442
S000
AMXR311142.6080.723
I1261
S300
PIPR4210.6770.713
I004
S42150
CEFR5210.3620.834
I000
S41154
TE, tetracycline; S, streptomycin; GM, gentamicin; AZM, azithromycin; CEF, cefalotin; P, penicillin; AMX, amoxicillin; PIP, piperacillin.
Table 3. Detection results of drug-resistance genes of K. pneumoniae isolates.
Table 3. Detection results of drug-resistance genes of K. pneumoniae isolates.
TypesDrug-Resistance GeneIsolates (No.)Positive Rates (%)
CarbapenemblaNDM-12536.8
blaVIM1217.6
blaOXA-101319.1
blaKPC-200
blaOXA-4800
ESBLsblaSHV68100
blaTEM4464.7
blaVEB00
blaPER00
Table 4. Correlation between drug-resistance genes and drug resistance.
Table 4. Correlation between drug-resistance genes and drug resistance.
AntimicrobialDrug-Resistance Genes
blaNDM-1blaVIMblaOXA-10blaTEM
TE0.1480.0670.287 *0.113
S0.2150.0860.250 *−0.111
GM0.1310.1610.0040.092
AZM−0.0670.296 *−0.279 *0.199
CEF0.006−0.0490.1710.270 *
P0.028−0.192−0.202−0.218
AMX−0.1640.004−0.242 *−0.006
PIP0.043−0.1570.2050.250 *
TE, tetracycline; S, streptomycin; GM, gentamicin; AZM, azithromycin; CEF, cefalotin; P, penicillin; AMX, amoxicillin; PIP, piperacillin; * showed a significant correlation (p < 0.05).
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Cao, W.; Xu, Y.; Huang, Y.; Xu, T. Isolation of Pathogenic Bacteria from Dairy Cow Mastitis and Correlation of Biofilm Formation and Drug Resistance of Klebsiella pneumoniae in Jiangsu, China. Agriculture 2023, 13, 1984. https://0-doi-org.brum.beds.ac.uk/10.3390/agriculture13101984

AMA Style

Cao W, Xu Y, Huang Y, Xu T. Isolation of Pathogenic Bacteria from Dairy Cow Mastitis and Correlation of Biofilm Formation and Drug Resistance of Klebsiella pneumoniae in Jiangsu, China. Agriculture. 2023; 13(10):1984. https://0-doi-org.brum.beds.ac.uk/10.3390/agriculture13101984

Chicago/Turabian Style

Cao, Wendi, Yi Xu, Yicai Huang, and Tianle Xu. 2023. "Isolation of Pathogenic Bacteria from Dairy Cow Mastitis and Correlation of Biofilm Formation and Drug Resistance of Klebsiella pneumoniae in Jiangsu, China" Agriculture 13, no. 10: 1984. https://0-doi-org.brum.beds.ac.uk/10.3390/agriculture13101984

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