Antimicrobial-resistant bacterial populations and ... · obtained from inpatients vs outpatients....

Antimicrobial-resistant bacterial populations and antimicrobial resistance genes obtained from environments impacted by livestock and municipal waste.

Terry Arthur Meat Safety & Quality Research Unit U.S. Meat Animal Research Center

Research Team • Dr. Getahun Agga

• Dr. John Schmidt

• Dr. Lisa Durso

• Dr. Dayna Harhay

• Frank Reno

• Julie Dyer

• Trent Ahlers

• Kerry Brader

• Lawnie Luedtke

• Alberto Alvarado

• Bruce Jasch

• Greg Smith

Participating feedlots, swine operations, and municipal wastewater treatment facilities

USMARC Antimicrobial Resistance Research in Animal Agriculture

• What is the contribution of antimicrobial use in food animals to compromised human health outcomes due to resistance?

– Exposure

• Food

• Environment

• How can antimicrobial resistance in animal agriculture be mitigated?

Antimicrobial resistance came out of necessity and inevitability

• Most of the antibiotics we use clinically can be traced to bacterial origins – Aminoglycosides, b-lactams, glycopeptides, lipopeptides,

macrolides, rifampins, and tetracylines.

• Resistance was/is necessary – Antibiotic-producing bacteria need protection against self

destruction

– “Producer hypothesis” states that resistance spread by co-opting self immunity systems from antibiotic producers

• Resistance is inevitable – Microorganisms have been battling and surviving for millions of

years. They will continue to do so.

Multiple paths to achieve the same resistance Tetracycline efflux proteins are membrane-associated proteins that recognize and export tetracycline from the cell and are found in both Gram-positive and Gram-negative bacteria. - 22 different tetracycline efflux proteins, grouped according to sequence similarity: Group 1 are Tet(A), Tet(B), Tet(C), Tet(D), Tet(E), Tet(G), Tet(H), Tet(J), Tet(Z) and Tet(30); Group 2 are Tet(K) and Tet(L); Group 3 are Otr(B) and Tcr(3); Group 4 is TetA(P); Group 5 is Tet(V). In addition, there are the efflux proteins Tet(31), Tet(33), Tet(V), Tet(Y), Tet(34), and Tet(35). Ribosomal protection proteins are cytoplasmic proteins that display homology with the elongation factors EF-Tu and EF-G. Protection proteins bind the ribosome, causing an alteration in ribosomal conformation that prevents tetracycline from binding. - 10 ribosomal protection proteins: Tet(M), Tet(O), Tet(S), Tet(W), Tet(32), Tet(36), Tet(Q), Tet(T), Otr(A), and TetB(P). Tetracycline modification proteins include the enzymes Tet(37) and Tet(X), both of which inactivate tetracycline. In addition, there are the tetracycline resistance proteins Tet(U) and Otr(C).

Cantas et al. 2013. Front. Microbiol. 4:96.

Soil microbes

What counters antibiotic resistance in nature?

Chait et al. Nat. Chem. Biol. 2012 8:2-5

Antimicrobial resistance of Gram-negative bacilli isolates from inpatients and outpatients at Yaounde Central Hospital, Cameroon. Int J Infect Dis. 2004 Piéboji JG, et al. Our analysis suggests that the rate of resistance in nosocomial pathogens to a variety of antimicrobials, such as amoxicillin, amoxicillin/clavulanate, cefazolin, cefoxitin, cefotaxime and gentamicin, commonly used to treat nosocomial infections, is significantly higher in the hospital setting than the outpatient setting.

A comparison of inpatient versus outpatient resistance patterns of pediatric urinary tract infection. J Urol. 2014 Saperston KN, et al. Uropathogen resistance rates of several antibiotics are higher for urinary specimens obtained from inpatients vs outpatients.

During a September 2013 press conference to release a report on antibiotics, CDC director Thomas Frieden, MD, said, "Right now, the most acute problem is in hospitals. And the most resistant organisms in hospitals are emerging in those settings because of poor antimicrobial stewardship among humans." - CDC Telebriefing on today's drug-resistant

health threats, Sept. 16, 2013

Antibiotic Resistance by Source: human-cattle-swine

Objective: to compare, using identical methods, ARB and antimicrobial resistance genes from environments associated with municipal sewage treatment plant effluent, cattle feedlot runoff ponds, swine waste lagoons, and environments with minimal direct fecal impact (a municipal lake and a relict prairie).


3 municipal wastewater treatment plants

2 low impact environments

3 swine waste lagoons

3 feedlot runoff catchment ponds

Sampled summer and winter

4 samples each for solid and liquid-sediment slurry

USMARC Antimicrobial Research Initiative Approach

• Culture – Detect and quantify antimicrobial-resistant bacterial populations – Very sensitive – Isolates for characterization – Restricted to specific bacteria-resistance combinations

• Molecular – qPCR can detect and quantify antimicrobial resistance genes (ARG) – Large numbers of targets are manageable – Alive/dead? – Was the ARG in an organism of interest? – Moderate sensitivity – limited to number of primers/probes

• Metagenomic – Not limited to specific targets – Detection only – Low sensitivity? – Bioinformatics issues


Individual samples (n=174): Culture – enumeration and prevalence of specific resistant populations

3rd-generation cephalosporin-resistant (3GCr) E. coli

trimethoprim-sulfamethoxazole-resistant (COTr) E. coli 3GCr Salmonella enterica nalidixic acid-resistant (NALr) Salmonella erythromycin-resistant (ERYr) Enterococcus spp.

Pooled samples (n=44):

Molecular – PCR for 84 specific resistance genes

Environment

Organism Cattle Municipal Swine Low-

impact

E. coli 93.8a 100a 93.8a 93.8a

3GCr E. coli 79.2a 93.4a 72.9a 18.8b

COTr E. coli 81.3a 100a 81.3a 9.4b

Environment

Cattle Municipal Swine

Liquid matrix(log

CFU/ml)

n 24 24 24

E. coli 2.8a 2.7a 3.6a

3GCr E. coli 0.7a 0.9a 1.2a

COTr E. coli 1.3a,b 0.9b 2.0a

Solid matrix (log

CFU/g)

n 24 22 24

E. coli 2.4a 3.4a 1.5a

3GCr E. coli 0.13a 1.4a -0.5a

COTr E. coli 0.3a 1.9a -0.06a

Culture Analysis of antimicrobial resistant bacterial populations

E. coli

cattle (n = 48), municipal (n = 46), swine (n = 48), and low impact (n= 32) samples

Environment

Organism Cattle Municipa

l Swine

Low-

impact

Salmonella 52.1a 65.7a 39.6a 3.1b

3GCr Salmonella 35.4a 14.7a,b 2.1b 0b

NALr Salmonella 0a 4.3a 0a 0a

Salmonella


Environment

Organism Cattle Municipa

l Swine

Low-

impact

Enterococcus 100a 100a 100a 96.9a

ERYr Enterococcus 100a 84.9a 91.7a 18.8b

Environment

Cattle Municipal Swine

Liquid matrix(log

CFU/ml)

n 24 24 24

Enterococcus 3.1a 2.1a 3.1a

ERYr Enterococcus 2.7a 0.4b 2.4a

Solid matrix (log

CFU/g)

n 24 22 24

Enterococcus 2.9a,b 3.3a 1.9b

ERYr Enterococcus 2.1a 2.0a 0.8a

Enterococcus species


Cattle Municipal Swine Low

impact

Prevalence (%)

E. coli 93.8a 100 93.8a 93.8a

3GCr E. coli 79.2a 93.4a 72.9a 18.8b

COTr E. coli 81.3a 100 79.2a 9.4b

Enterococcus 100 100 100 96.9

ERYr

Enterococcus 100 84.9a 91.7a 18.8b

Bacterial count (mean log10)

E. coli 2.9a 3.32a 2.9a 0.34b

Enterococcus 3.3a 2.9a 2.9a 0.7b

Is it selection or enrichment?

Effects of manure on the abundances of culturable soil bacteria.

Nikolina Udikovic-Kolic et al. PNAS 2014;111:15202-15207

©2014 by National Academy of Sciences

The baseline level of culturable bacteria resistant to cephalothin was substantially higher in untreated soil than in manure (7.4% vs. 0.67%).

Qiagen qPCR array consisted of 84 resistance genes • aminoglycoside resistance (n=6) • class A beta-lactamases (22) • class B beta-lactamases (9) • class C beta-lactamases (11) • class D beta-lactamases (13) • fluoroquinolone resistance (10) • macrolide-lincosamide-streptograminB (MLSB) resistance (6) • multidrug efflux pumps (2) • tetracycline efflux pumps (2) • vancomycin resistance (2) • Staphylococcus aureus beta-lactam resistance (mecA) genes - MRSA

Molecular Analysis of antimicrobial resistance genes

44 pooled samples • cattle (n = 12), municipal (n = 12), swine (n = 12) and, low impact (n= 8)

Molecular Analysis of antimicrobial resistance genes

• 61 out of 84 unique antimicrobial resistance genes were detected from 41 of the 44-pooled samples

• 3 out of 8 low impact environment-pooled samples were negative for any of the resistance genes targeted

Box plot showing median distribution of antimicrobial resistance genes detected per pooled sample.

Antibiotic resistance classes Genes Cattle Municipal Swine Low-

impact

Class A beta-lactamases BES-1 4

CTX-M-1 Group 5

CTX-M-9 Group 2

GES 12 2

IMI & NMC-A 1

KPC 3

Per-1 group 2

Per-2 group 1 2 1

SFO-1 1 1

SHV 6

SHV(156D) 1

SHV(156G) 2 8 2

SHV(238G240E) 1 7 1

SHV(238S240K) 1 1

TLA-1 1 8

VEB 5 10 4 Class B beta-lactamases IMP-2 group 1

IMP-5 group 2 1

VIM-1 group 2

Antimicrobial Resistance Genes by Resistance Class

Pooled samples: cattle (n = 12), municipal (n = 12), swine (n = 12) and, low impact (n= 8)


impact

Class C beta-lactamases ACC-3 1

ACT 5/7 group 1 2 1

ACT-1 group 4 1 1

CMY-10 Group 7

DHA 1

FOX 2

LAT 4

MIR 3 1

MOX 9

Class D beta-lactamases OXA-10 Group 6 12 10

OXA-18 1

OXA-2 Group 11 12 11

OXA-23 Group 1

OXA-24 Group 4

OXA-48 Group 1

OXA-50 Group 1

OXA-51 Group 1

OXA-55 1

OXA-58 Group 4 5




impact

Fluoroquinolone resistance AAC(6)-Ib-cr 6 12 11

QnrA 1

QnrB-1 group 3

QnrB-4 group 1

QnrB-5 group 3

QnrB-8 group 1

QnrC 1

QnrD 1 1

QnrS 10

Macrolide resistance ereB 9 2 3

ermA 12 3 11

ermB 12 12 12 1

ermC 12 3 12

mefA 12 12 12 3

msrA 2

Multidrug resistance efflux pump oprm 1

Tetracycline resistance tetA 12 12 11 1

tetB 11 3 9




impact

Aminoglycoside resistance aacC1 3 12 1 1

aacC2 7 6 5

aacC4 5 1 11

aadA1 12 12 12

aphA6 3 6



* The numbers of class A beta-lactamase, class C beta-lactamase, and fluoroquinolone resistance genes detected were significantly higher (P < 0.05) in municipal samples than in cattle runoff or swine lagoon samples.

Venn diagram showing the number of specific genes identified by sample source.

Principal coordinate analysis showing the clustering of resistance genes by livestock, municipal and low impact environmental samples. Antimicrobial resistance genes were detected among cattle (n=12), low impact environment (n=8), municipal (n=12) and swine (n=12) pooled samples. Data points are colored as follows: green= cattle, red= low impact environment, black= municipal and blue = swine.

low impact

swine municipal

cattle


impact

Class A beta-lactamases GES 12 2

IMI & NMC-A 1

KPC 3

Class B beta-lactamases IMP-2 group 1

IMP-5 group 2 1

VIM-1 group 2

Class D beta-lactamases OXA-23 Group 1

OXA-24 Group 4

OXA-48 Group 1

OXA-51 Group 1

OXA-58 Group 4 5

Carbapenemase genes present in pooled samples


Isolate characterization

Ciprofloxacin Nalidixic acid Cefoxitin Ceftiofur

Cattle

E. coli (92) 2.2% 10.9% 25.0% 46.7%

Salmonella (46) 0.0% 0.0% 47.8% 47.8%

Municipal

E. coli (100) 35.0% 53.0% 12.0% 43.0%

Salmonella (40) 0.0% 5.0% 22.5% 22.5%

Swine

E. coli (101) 4.0% 4.0% 22.8% 39.6%

Salmonella (18) 0.0% 0.0% 5.6% 0.0%

Low impact

E. coli (25) 0.0% 0.0% 40.0% 32.0%

Cephalosporin and quinolone resistance in E. coli and Salmonella

Isolates from all plating media

n Ciprofloxacin Nalidixic acid Cefoxitin Ceftiofur Cattle

E. coli 24 0 4.2% 8.3% 8.3% Salmonella 27 0 0 11.1% 11.1%

Municipal E. coli 24 0 20.8% 4.2% 0.0%

Salmonella 30 0 0 3.3% 3.3% Swine

E. coli 24 0 0 20.8% 25.0% Salmonella 18 0 0 5.6% 0.0%

Low impact E. coli 16 0 0 18.8% 12.5%

Cephalosporin and quinolone resistance in E. coli and Salmonella

Media without targeted antibiotics

Co-resistance with 3GCr E. coli Ciprofloxacin resistance

Class R S Total

Cattle 31 31

Low impact 6 6

Municipal 22 15 37

Swine 1 29 30

Total 23 81 104 Ciprofloxacin resistance for ceftiofur resistant E. coli

Nalidixic acid resistance

Class R S Total

Cattle 2 29 31

Low impact 6 6

Municipal 26 11 37

Swine 1 29 30

Total 29 75 104 Nalidixic acid resistance for ceftiofur resistant E. coli

Trimethoprim-Sulfamethoxazole

Class R S Total

Cattle 3 28 31

Low impact 6 6

Municipal 27 10 37

Swine 30 30

Total 30 74 104

Trimethoprim/sulfamethoxazole resistance for ceftiofur resistant E. coli

Gentamicin resistance

Class R S Total

Cattle 31 31

Low impact 6 6

Municipal 14 22 37

Swine 30 30

Total 14 89 104

Gentamicin resistance for ceftiofur resistant E. coli

Prevalence of Extraintestinal Pathogenic E. coli Virulence Factors

Antimicrobial resistance in E. coli isolated from the low impact environments

Resistance phenotypes # of isolates Source AmApF 1 lake

AmApAzF 1 lake AzCSuTSxT 1 lake ApCSSuSxT 1 lake

AmApAzFCfAxCKSSuT 1 prairie AmApFCfAxSSuT 2 prairie AmApFCfAx 5 lake/prairie

Pansusceptable 13 lake/prairie 25

amoxicillin-clavulanic acid (Am), ampicillin (AP), azithromycin (Az), cefoxitin (F), ceftiofur (Cf), ceftriaxone (Ax), chloramphenicol (C), kanamycin (K), streptomycin (S), sulfisoxazole (Su), tetracycline (T), trimethoprim/sulphamethoxazole (SxT)

CONCLUSIONS

• The prevalences and concentrations of AMR E. coli, enterococci, and Salmonella were similar among the livestock and municipal sample sources.

• Highest diversity and relative abundance of antimicrobial resistance genes in municipal environments.

• Multivariate analysis of the distribution of antimicrobial resistance genes showed distinct clustering of samples with livestock (cattle and swine), low impact environment and municipal samples forming three separate clusters.

• Multidrug resistant isolates and multiple antimicrobial resistance genes can be found in environments with little to no direct impact by human or livestock waste.

• Enrichment of the native microbial flora may have as much impact on the ARB population and antibiotic selection.

• Isolation of resistant organisms is dependent on the media used for isolation.

ANTIMICROBIAL RESISTANCE: IF YOU LOOK FOR IT YOU WILL PROBABLY FIND IT

AMR Projects Recently Completed or Ongoing

• AMR in beef cow herds – No long term effects of antimicrobial treatments on AMR

• Effect of mass treatment with chlortet on AMR to 3GC – 25% on nontreated animals had to be pulled for treatment with a

“more critical” antibiotic

– Overall, no difference in AMR between treated and control groups

• Comparison of AMR detected in fecal samples from conventional cattle production vs cattle raised without antbiotics.

• Effect of antibiotic treatment on Salmonella colonization of bovine peripheral lymph nodes following a salmonellosis outbreak in fed cattle

[email protected] 402-762-4227

US Meat Animal Research Center

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Antimicrobial-resistant bacterial populations and ... · obtained from inpatients vs outpatients....

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