Characterization of Enterococci- and ESBL-Producing Escherichia coli Isolated from Milk of Bovides with Mastitis in Egypt
Abstract
:1. Introduction
2. Materials and Methods
2.1. Sample Collection and Cultivation
2.2. Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS)
2.3. Antibiotic Susceptibility Testing using Broth Microdilution
2.4. Antibiotic Susceptibility Testing using the VITEK 2 System
2.5. DNA Extraction and Detection of Resistance-Associated Genes
2.6. GenoSerotyping, Detection of Antibiotic Resistance and Virulence-Associated Genes of E. coli Isolates by Microarray Analysis
3. Results
3.1. Bacterial Isolation and Identification by MALDI-TOF MS
3.2. Antimicrobial Susceptibility Profiles of Enterococcus Isolates
3.3. Antimicrobial Susceptibility Profiles of Escherichia coli Isolates
3.4. Detection of Resistance-Associated Genes in Enterococcus Isolates
3.5. Detection of Resistance-Associated Genes in Coliform Bacteria
3.6. GenoSerotyping and Analysis of Escherichia coli Isolates by Microarray Investigation
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Antibiotic | Target Gene | Primer Sequences (5′–3′) | Expected Amplicon Size (bp) | Reference |
---|---|---|---|---|
Methicillin/ oxacillin | mecA | F: TCC AGA TTA CAA CTT CAC CAG G R: CCA CTT CAT ATC TTG TAA CG | 161 | [15] |
mecB | F: TTA ACA TAT ACA CCC GCT TG R: TAA AGT TCA TTA GGC ACC TCC | 2263 | [16] | |
mecC | AL3: TCA AAT TGA GTT TTT CCA TTA TCA AL4: AAC TTG GTT ATT CAA AGA TGA CGA | 1931 | [16] | |
Penicillin | blaZ | F: AAG AGA TTT GCC TAT GCT TC R: GCT TGA CCA CTT TTA TCA GC | 517 | [17] |
Vancomycin | vanA | F: ATG AAT AGA ATA AAA GTT GCA ATA R: CCC CTT TAA CGC TAA TAC GAT CAA | 1030 | [18] |
vanB | F: AAG CTA TGC AAG AAG CCA TG R: CCG ACA AAA TCA TCC TC | 536 | [18] | |
vanC1 | F: GGA ATC AAG GAA ACC TC R: CTT CCG CCA TCA TAG CT | 822 | [19] | |
Erythromycin | erm(B) | F: GAA AAG GTA CTC AAC CAA ATA R: AGT AAC GGT ACT TAA ATT GTT TAC | 639 | [20] |
erm(A) | F: TAT CTT ATC GTT GAG AAG GGA TT R: CTA CAC TTG GCT TAG GAT GAA A | 138 | [15] | |
erm(C) | F: CTT CTT GAT CAC GAT AAT TTC C R: ATC TTT TAG CAA ACC CGT ATTC | 189 | [15] | |
Macrolide | msrC | F: AAG GAA TCC TTC TCT CTC CG R: GTA AAC AAA ATC GTT CCC G | 342 | [21] |
Tetracycline | tetK | F: TCG ATA GGA ACA GCA GTA R: CAG CAG ATC CTA CTC CTT | 169 | [22] |
tetL | F: TCG TTA GCG TGC TGT CAT R: GTA TCC CAC CAA TGT AGC CG | 267 | [22] | |
tetM | F: GTG GAC AAA GGT ACA ACG AG R: CGG TAA AGT TCG TCA CAC AC | 406 | [22] | |
tetO | F: AACTTA GGC ATT CTG GCT CAC R: TCC CAC TGT TCC ATA TCG TCA | 515 | [22] | |
Aminoglycoside | aac6-aph2 | F: CCA AGA GCA ATA AGG GCA TA R: CAC TAT CAT AAC CAC TAC CG | 219 | [23] |
aac-aphD | F: TAA TCC AAG AGC AAT AAG GGC R: GCC ACA CTA TCA TAA CCA CTA | 227 | [15] | |
Linezolid, chloramphenicol | optrA | F: AGG TGG TCA GCG AAC TCA R: ATC AAC TGT TCC CAT TCA | 1400 | [24] |
Oxazolidinone | cfr | F: TGA AGT ATA AAG CAG GTT GGG AGT CA R: ACC ATA TAA TTG ACC ACA AGC AGC | 400 | [24] |
Lincosamide | lnuD | F: ACG GAG GGA TCA CAT GGT AA R: TCT CTC GCA TAA TAA CCT TAC GTC | 475 | [25] |
lnuA | F: GGT GGC TGG GGG GTA GAT GTA TTA ACT GG R: GCT CTC TTT GAA ATA CAT GGT ATT TTT CGA TC | 323 | [26] | |
Colistin | mcr-1 | F: AGT CCG TTT GTT CTT GTG GC R: AGA TCC TTG GTC TCG GCT TG | 320 | [27] |
mcr-2 | F: CAA GTG TGT TGG TCG CAG TT R: TCT AGC CCG ACA AGC ATA CC | 715 | [27] | |
mcr-3 | F: AAA TAA AAA TTGTTC CGC TTA TG R: AAT GGA GAT CCC CGT TTT T | 929 | [27] | |
mcr-4 | F: TCA CTT TCA TCA CTG CGT TG R: TTG GTC CAT GAC TAC CAA TG | 1116 | [27] | |
mcr-5 | F: ATG CGG TTG TCT GCA TTT ATC R: TCA TTG TGG TTG TCC TTT TCT G | 1644 | [28] |
Origin of Milk | Number of Milk Samples | Enterococcus faecalis | Enterococcus casseliflavus | Enterococcus hirae | Escherichia coli | Enterobacter cloacae | ||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
n | % | n | % | n | % | n | % | n | % | |||
Clinical mastitis | Cattle | 22 | 5 | 22.7 | 1 | 4.5 | 1 | 4.5 | 2 | 9.1 | 0 | 0.0 |
Buffalo | 10 | 3 | 30.0 | 0 | 0.0 | 1 | 10.0 | 1 | 10.0 | 1 | 10.0 | |
Subclinical mastitis | Cattle | 5 | 3 | 60.0 | 1 | 20.0 | 0 | 0.0 | 2 | 40.0 | 0 | 0.0 |
Buffalo | 13 | 2 | 15.4 | 0 | 0.0 | 0 | 0.0 | 2 | 15.4 | 0 | 0.0 | |
Total | 50 | 13 | 26.0 | 2 | 4.0 | 2 | 4.0 | 7 | 14.0 | 1 | 2.0 |
Antibiotic | Class | Enterococcus faecalis (n = 13) | Other Enterococcus Species (n = 4) | ||||||
---|---|---|---|---|---|---|---|---|---|
S | I | R | Resistance Rate (%) | S | I | R | Resistance Rate (%) | ||
Ampicillin | β-Lactam | 8 | 0 | 5 | 38.4 | 2 | 0 | 2 | 50.0 |
Cefoxitin | β-Lactam; cephamycin | 1 | 0 | 12 | 92.3 | 0 | 0 | 4 | 100 |
Ceftaroline | Cephalosporin 5th generation | 1 | 0 | 12 | 92.3 | 1 | 2 | 1 | 25.0 |
Clindamycin | Lincosamide | 0 | 0 | 13 | 100 | 0 | 0 | 4 | 100 |
Daptomycin | Cyclic lipopeptide | 0 | 0 | 13 | 100 | 0 | 0 | 4 | 100 |
Erythromycin | Macrolide | 0 | 1 | 12 | 92.3 | 0 | 0 | 4 | 100 |
Erythromycin/ clindamycin | 0 | 0 | 13 | 100 | 0 | 0 | 4 | 100 | |
Fosfomycin | Epoxide antibiotic | 1 | 0 | 12 | 92.3 | 0 | 0 | 4 | 100 |
Fusidic acid | Steroide antibiotic | 1 | 0 | 12 | 92.3 | 0 | 0 | 4 | 100 |
Gentamicin | Aminoglysides | 0 | 0 | 13 | 100 | 0 | 0 | 4 | 100 |
Gentamicin high level | Aminoglysides | 1 | 0 | 12 | 92.3 | 0 | 1 | 3 | 75.0 |
Linezolid | Oxazolidinone | 1 | 0 | 12 | 92.3 | 0 | 1 | 3 | 75.0 |
Moxifloxacin | Fluorchinolone 4th generation | 1 | 0 | 12 | 92.3 | 0 | 0 | 4 | 100 |
Mupirocin | 1 | 2 | 10 | 76.9 | 0 | 2 | 2 | 50.0 | |
Oxacillin | beta-Lactam | 2 | 0 | 11 | 84.6 | 0 | 0 | 4 | 100 |
Penicillin G | beta-Lactam | 1 | 5 | 7 | 53.8 | 1 | 2 | 1 | 25.0 |
Rifampicin | Ansamycine | 0 | 0 | 13 | 100 | 0 | 0 | 4 | 100 |
Synercid | Streptogramine | 0 | 0 | 13 | 100 | 0 | 2 | 2 | 50.0 |
Teicoplanin | Glycopeptide | 3 | 0 | 10 | 76.9 | 3 | 0 | 1 | 25 |
Tigecycline | Glycylcycline | 2 | 0 | 11 | 84.6 | 1 | 0 | 3 | 75.0 |
Trimethoprim/ sulphamethoxazole | Dihdrofolatreductase/ Sulfonamide | 0 | 0 | 13 | 100 | 0 | 0 | 4 | 100 |
Vancomycin | Glycopeptide | 2 | 1 | 10 | 76.9 | 2 | 1 | 1 | 25.0 |
Isolate | Phenotypic Antimicrobial Resistance | Detected Resistance-Associated Genes |
---|---|---|
19CS0095-1 | PEN, STR, CAZ, CIP, LEV, GEN, AMK, TET, ERY, CMP, RAM, T/S | erm(B), tetK |
19CS0065 | PEN, STR, AMC, CAZ, IMP, ERY, CMP, RAM, T/S | erm(B) |
19CS0080-1 | PEN, STR, AMC, CAZ, IMP, CIP, LEV, GEN, TET, ERY, CMP, RAM, T/S | tetL, tetK |
19CS0092-1 | PEN, STR, AMC, CAZ, IMP, CIP, LEV, GEN, AMK, TET, ERY, CMP, RAM, T/S | erm(B), msrC, tetL |
19CS0078-1 | PEN, STR, CIP, LEV, GEN, AMK, TET, ERY, CMP, RAM, T/S | erm(B), aac6-aph2, tetL |
19CS0069 | PEN, STR, CAZ, CIP, LEV, GEN, AMK, TET, ERY, CMP, AM, T/S | msrC |
19CS0098-1 | PEN, STR, AMP, CAZ, CIP, LEV, GEN, TET, ERY, CMP, RAM, T/S | erm(B), tetL |
Isolate | PIP | TZP | CTX | CAZ | FEB | ATM | IMP | MEM | AMK | GEN | TOB | CIP | MXF | TGC | FOS | CT | T/S | |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
19CS0095-1 | R | I | R | R | R | R | S | S | S | R | R | R | R | S | S | S | R | ESBL |
19CS0065 | R | I | R | R | R | R | S | S | S | S | S | S | S | S | S | S | R | ESBL |
19CS0080-1 | R | I | R | R | R | R | S | S | S | R | S | S | S | S | S | S | R | ESBL |
19CS0092-1 | R | I | R | R | R | R | S | S | S | R | R | S | R | S | R | S | R | ESBL |
19CS0078-1 | R | I | R | R | R | R | S | S | S | R | R | R | R | R | R | R | R | ESBL |
19CS0069 | R | I | R | R | R | R | S | S | S | R | R | S | R | S | R | S | R | ESBL |
19CS0098-1 | R | I | R | R | R | R | S | S | S | R | R | R | R | S | S | S | R | ESBL |
Enterococcus faecalis (n = 13) | Other Enterococcus Species (n = 4) | ||||
---|---|---|---|---|---|
Positive (n) | % | Positive (n) | % | ||
Vancomycin resistance genes | vanA | 7 | 53.8 | 1 | 25.0 |
vanB | 0 | 0.0 | 3 | 75.0 | |
vanC1 | 0 | 0.0 | 1 | 25.0 | |
Erythromycin resistance genes | erm(A) | 1 | 7.7 | 0 | 0.0 |
erm(B) | 13 | 100 | 4 | 100 | |
erm(C) | 0 | 0.0 | 0 | 0.0 | |
Penicillin resistance gene | blaZ | 11 | 84.6 | 1 | 25.0 |
Linezolide resistance genes | optrA | 2 | 15.4 | 0 | 0.0 |
cfr | 0 | 0.0 | 0 | 0.0 | |
Macrolide resistance gene | msrC | 3 | 23.1 | 0 | 0.0 |
Aminoglycoside resistance genes | aac-aphD | 13 | 100 | 3 | 75.0 |
Tetracycline resistance genes | tetK | 0 | 0.0 | 0 | 0.0 |
tetM | 11 | 84.6 | 1 | 25.0 | |
tetL | 13 | 100 | 4 | 100 | |
tetO | 0 | 0.0 | 0 | 0.0 | |
Lincosamide resistance genes | lnuA | 2 | 15.4 | 0 | 0.0 |
lnuD | 1 | 7.7 | 2 | 50.0 |
19CS0065 | 19CS0069 | 19CS0078-1 | 19CS0080-1 | 19CS0092-1 | 19CS0095-1 | 19CS0098-1 | |
---|---|---|---|---|---|---|---|
Escherichia coli | + | + | + | + | + | + | + |
dnaE a | + | + | + | + | + | + | + |
gada | + | + | + | + | + | + | + |
gapA a | - | + | - | + | + | + | + |
ihfA a | + | + | + | + | + | + | + |
rrsa | + | + | + | + | + | + | + |
O-serotyping | 08 | 086 | - | - | 086 | - | O157 |
H-serotyping | H11 | H12 | H21 | H11 | H12 | H16 | H21 |
lpfA b | - | - | + | - | - | - | + |
tshc | - | + | - | - | + | - | - |
astA d | - | + | + | - | + | - | + |
cbad | - | + | - | - | + | - | - |
celB d | - | + | - | - | + | - | - |
cmad | - | + | + | + | + | + | + |
hemL e | + | + | + | + | + | + | + |
intl1/2 e | + | + | + | + | + | + | + |
iroN e | - | - | - | - | - | + | - |
isse | - | - | - | - | - | + | - |
blaCTX-M1,M15 f | - | + | - | - | + | - | - |
blaTEM f | - | + | + | - | + | + | + |
blaCTX-M9 f | + | - | + | + | - | + | + |
Aminoglycosides g | aadA1, aadA4 | aadA1, aphA, strA, strB | aadA1, aphA, strA | aadA1, aadA4, aphA | aadA1, aphA, strA, strB | strA, strB | aadA1, aphA, strA |
Chloramphenicol g | cmlA1 | cmlA1, floR, catA1 | cmlA1, floR | cmlA1, floR | cmlA1, floR, catA1 | floR | cmlA1, floR |
Macrolides g | - | mphA | - | - | mphA | mphA | - |
Quinolones g | - | qnrA1, qnrS | - | - | - | - | qnrA1, qnrS |
Tetracycline g | - | tetA | tetA | - | - | tetA | - |
Sulphonamides g | sul3 | sul1, sul2, sul3 | sul1, sul3 | sul3 | sul3 | sul2 | sul3 |
Trimethoprim g | dfrA17 | dfrA1 | dfrA12 | dfrA17 | dfrA1, dfrA14 | dfrA12 | dfrA12, dfrA14 |
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Ahmed, W.; Neubauer, H.; Tomaso, H.; El Hofy, F.I.; Monecke, S.; Abd El-Tawab, A.A.; Hotzel, H. Characterization of Enterococci- and ESBL-Producing Escherichia coli Isolated from Milk of Bovides with Mastitis in Egypt. Pathogens 2021, 10, 97. https://doi.org/10.3390/pathogens10020097
Ahmed W, Neubauer H, Tomaso H, El Hofy FI, Monecke S, Abd El-Tawab AA, Hotzel H. Characterization of Enterococci- and ESBL-Producing Escherichia coli Isolated from Milk of Bovides with Mastitis in Egypt. Pathogens. 2021; 10(2):97. https://doi.org/10.3390/pathogens10020097
Chicago/Turabian StyleAhmed, Wedad, Heinrich Neubauer, Herbert Tomaso, Fatma Ibrahim El Hofy, Stefan Monecke, Ashraf Awad Abd El-Tawab, and Helmut Hotzel. 2021. "Characterization of Enterococci- and ESBL-Producing Escherichia coli Isolated from Milk of Bovides with Mastitis in Egypt" Pathogens 10, no. 2: 97. https://doi.org/10.3390/pathogens10020097
APA StyleAhmed, W., Neubauer, H., Tomaso, H., El Hofy, F. I., Monecke, S., Abd El-Tawab, A. A., & Hotzel, H. (2021). Characterization of Enterococci- and ESBL-Producing Escherichia coli Isolated from Milk of Bovides with Mastitis in Egypt. Pathogens, 10(2), 97. https://doi.org/10.3390/pathogens10020097