International Journal of Microbiology
Volume 2016 (2016), Article ID 3103672, 7 pages
http://dx.doi.org/10.1155/2016/3103672

Research Article

IncF Plasmids Are Commonly Carried by Antibiotic Resistant Escherichia coli Isolated from Drinking Water Sources in Northern Tanzania

Beatus Lyimo,¹ Joram Buza,¹ Murugan Subbiah,² Sylivester Temba,¹ Honest Kipasika,¹ Woutrina Smith,³ and Douglas R. Call1,2

¹Nelson Mandela African Institution of Science and Technology, 447 Arusha, Tanzania
²Paul G. Allen School for Global Animal Health, Washington State University, Pullman, WA 99164, USA
³One Health Institute, School of Veterinary Medicine, University of California, Davis, CA 95616, USA

Received 6 November 2015; Accepted 29 February 2016

Academic Editor: Sisinthy Shivaji

Copyright © 2016 Beatus Lyimo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract.

The aim of this study was to identify the replicon types of plasmids, conjugation efficiencies, and the complement of antibiotic resistance genes for a panel of multidrug resistant E. coli isolates from surface waters in northern Tanzania. Standard membrane filtration was used to isolate and uidA PCR was used to confirm the identity of strains as E. coli. Antibiotic susceptibility was determined by breakpoint assay and plasmid conjugation was determined by filter-mating experiments. PCR and sequencing were used to identify resistance genes and PCR-based replicon typing was used to determine plasmid types. Filter mating experiments indicated conjugation efficiencies ranged from 10⁻¹ to 10⁻⁷. Over 80% of the donor cells successfully passed their resistance traits and eleven different replicon types were detected (IncI1, FIC, P, FIIA, A/C, FIB, FIA, H12, K/B B/O, and N). IncF plasmids were most commonly detected (49% of isolates), followed by types IncI1 and IncA/C. Detection of these public health-relevant conjugative plasmids and antibiotic resistant traits in Tanzanian water suggests the possible pollution of these water sources from human, livestock, and wild animal wastes and also shows the potential of these water sources in the maintenance and transmission of these resistance traits between environments, animals, and people.

1. Introduction

Increased mortality and morbidity due to antibiotic treatment failure make antimicrobial resistance (AMR) one of the 21st century’s major global public health challenges [1]. Overuse and misuse of antibiotics are considered major reasons for the emergence of resistant bacteria in many low-income countries [2, 3]. Antibiotic resistance has been documented for enteric bacteria from various water sources and these water sources could facilitate dissemination of resistant bacteria to a wider community of people and animals [4]. This is particularly true for low-income countries like Tanzania where water sources are frequently shared between animals and people [5, 6]. For example, a report from Kenya reported a high prevalence of antibiotic resistance E. coli from water and fish in Lake Victoria [ampicillin (64%), tetracycline (76%), and cotrimoxazole (80%)] [7] where untreated water is consumed routinely.

Tanzanian hospitals have reported a high proportion (80%–90%) of clinical E. coli isolates that are resistant to antibiotics such as ampicillin, cotrimoxazole, tetracycline, gentamicin, and amoxicillin/clavulanic acid. These bacteria infect people within a healthcare system where, in most cases, there are no laboratory diagnostics to guide antibiotic treatment [8–10]. Another study reported a high number of antibiotic resistant E. coli, possessing resistance to cephalosporins, from free-range buffalo, zebra, and wildebeest [11]. These animals were located in mixed grazing areas with potential contact with people and livestock. Contaminated water was suspected as the source of resistant bacteria found in these wild animals [11]. Contaminated water most likely plays a role in the dissemination of antibiotic resistant bacteria and the probability of transmission likely increases when people and animals use that water.

Antimicrobial resistance (AMR) genes are transferred to other bacteria, sometimes at the species level, by horizontal gene transfer (transduction, transformation, and conjugation). Plasmid-mediated horizontal transfer of multidrug resistance between different bacteria is a major concern because this contributes to the evolution and emergence of antibiotic resistant bacteria in the environment [12, 13].

For the last decade, polymerase chain reaction-based replicon typing (PBRT) has been used to identify major plasmid types found in Enterobacteriaceae, including incompatibility (Inc) groups (HI2, HI1, I1-γ, X, L/M, N, FIA, FIB, FIC, W, Y, P, A/C, T, K, and B/O) [14, 15]. Plasmids belonging to group IncF frequently harbor that is often associated with , , and aac(6 ′)-Ib-cr resistance genes [16]. Replicon groups IncA/C and l1 are frequently associated with Enterobacteriaceae and harbor multiple resistance genes including resistance for extended-spectrum cephalosporins and carbapenems [17–19].

In northern Tanzania, surface water such as rivers and ponds is often shared between animals and people on daily basis. Consequently, these water sources become polluted with human and animal excreta and might harbor antibiotic resistant enteric bacteria. Consumption of water containing these bacteria is likely to increase the risk that antibiotic resistant and pathogenic bacteria will be transmitted. Nevertheless, to date, no studies have been conducted in Tanzania to determine if drinking water represents a risk factor for transmission of antibiotic resistant bacteria to people and animals. The objective of this study was to characterize the replicon types of plasmids that harbor drug resistant traits, their conjugation efficiencies, and the complement of antibiotic resistance genes for a panel of multidrug resistant E. coli isolates that were obtained from drinking water sources in northern Tanzania.

2. Methods

2.1. Study Design

Convenience sampling was used to collect water samples between March and August 2014. Each source was visited twice and one sample was collected from each source per visit (in Tanzania, March is the rainy season and August is during dry season). Sample locations included the Kilimanjaro Region (Moshi Municipal, Moshi Rural, and Hai Districts), the Arusha Region (Arusha City, Arumeru, Longido, and Monduli Districts), and the Manyara Region (Simanjiro and Babati Districts). A convenience approach was used to select sampling sites with appropriate permission from local authorities. Water samples from ponds were collected from localities near Maasai villages. All sites, including streams, may have been impacted by people and wildlife. We also collected opportunistic samples from taps and wells.

2.2. Isolation and Identification of E. coli

Water samples were collected in 500 mL sterile bottles and were transported in cooler boxes with ice packs to the laboratory for processing within 6 h of collection. Out of 500 mL, 100 mL water samples were analyzed using a standard membrane filtration technique with minor modifications [20]. Following filtration, each filter membrane was placed on a chromogenic selective agar plate (HiCrome E. coli Agar, HiMedia Laboratories Prt. Ltd., Mumbai, India). The agar plates were initially incubated at 37°C for 4 h, followed by incubation for 16–22 h at 44°C. Plates produced 1–200 CFU of which individual colonies were subcultured to ensure purity for further characterization. The identity of E. coli isolates was confirmed using a PCR genotyping test that detects the presence of the uidA gene [21].

Antibiotic break point assays were used to determine the resistance profile of each E. coli isolate against a panel of important antibiotics. MacConkey (MAC) (Thermo Oxoid Remel) agar plates with each antibiotic at their CLSI recommended minimum inhibitory concentrations [22] (Table 1) were used to perform the break point assays [23]. E. coli strains K-12 (negative control; susceptible to all antibiotics tested) and H4H E. coli (positive control; resistant to all antibiotics tested) were used as reference strains for antibiotic susceptibility testing.

Table 1: Antibiotic concentration tested against E. coli from surface waters in northern Tanzania.

Name
Code
Source
Concentration (µg/mL)

Amoxicillin/clavulanate potassium
Amx/Clv
MP Biomedicals, Illkirch, France
32/16

Ampicillin
Amp
Fisher Scientific, Fair Lawn, New Jersey
32

Ceftazidime
Ceftaz
Sigma-Aldrich
16

Chloramphenicol
Chlo
Sigma-Aldrich
32

Ciprofloxacin
Cip
Sigma-Aldrich
4

Kanamycin
Kan
Sigma-Aldrich
64

Streptomycin
Str
Sigma-Aldrich
16

Sulfamethoxazole
Sul
MP Biomedicals
512

Tetracycline
Tet
GTS, San Diego, CA
16

Trimethoprim
Tri
MP Biomedicals
16

2.3. Plasmid Characterization

A set of 31 E. coli isolates that were susceptible to nalidixic acid and resistant to more than 2 antibiotics tested were chosen for this study. Filter-mating experiments were performed to determine the conjugation rates of plasmids with the nalidixic acid susceptible MDR (wild-type) E. coli isolates as donors and a plasmid-free recipient strain (E. coli K-12, nalidixic acid resistant, ) as recipient as described earlier [23, 24] with minor modifications. Briefly, single colonies of E. coli K-12 and potential donor strains were grown separately overnight in LB medium (Luria-Bertani medium; Difco™ LB Broth Lennox, Sparks, MD, USA) at 37°C. Equal quantities (10 μL) of overnight cultures of donor and recipient strains were added on top of a nitrocellulose (~1 cm²) membrane overlaid on LB agar with no antibiotics. After 24 h of incubation at 37°C, cells from the membrane were suspended in 500 μL of sterile phosphate-buffered saline (PBS, pH 7.0) and spread onto LB agar plates containing 32 μg/mL nalidixic acid (Sigma-Aldrich) and another antibiotic to which the donor cells were resistant (Table 2). Colonies that grew on these selective agar plates were considered transconjugants. The conjugation efficiency of plasmid was calculated by dividing the number of transconjugants by the number of donor cells. Transconjugants were screened for their donor’s antibiotic resistance phenotypes and presence of tet(A), tet(B), , , and genes [25, 26]. CTX-M grouping (group 1, group 2, and group 9) was further evaluated for all CTX-M positive isolates. All CTX-M E. coli isolates were positive for CTX-M group 1 and the PCR products were subsequently sequenced by Functional Bioscience (Madison, WI). Sequencher (ver 5.0) software was used to process sequence traces, and the final sequences were analyzed with CLC Genomic Workbench 7.0.2 (CLC Bio Aarhus, Denmark) and compared with the reported sequences from GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi).

Table 2: Sample collection sites, phenotypes of donor and transconjugants, conjugation efficiency, resistance genes, and associated plasmid replicons.

District
Resistance phenotypes of donors
Abx used for selection
Resistance genes in transconjugants
Conjugation efficiency
Replicon type in transconjugants
Resistance phenotypes of transconjugants

Arusha urban
StrSulTri
Str
ND
—
I1
Str

StrTri
Str
ND
—
FIC
Str

Moshi urban
AmpStrSulTetTri
Tet
tet(B), TEM-1
5.8 × 10⁻³
ND
AmpStrSulTetTri

AmpStrSulTetTri
Amp
tet(A)
3.83 × 10⁻³
P, FIIA
AmpStrSulTetTri

AmpStrSulTet
Amp
tet(A)
9.88 × 10⁻¹
ND
AmpStrSulTet

SulTetTri
Tet
tet(A)
9.93 × 10⁻¹
ND
SulTetTri

StrTetTri
Tet
tet(B)
2.00 × 10⁻⁷
ND
StrTetTri

AmpStr
Amp
ND
2.7 × 10⁻⁴
FIIA
Amp

Moshi rural
AmpStrSulTetTri
Amp
tet(A), TEM-1
8.33 × 10⁻⁶
A/C, P, FIB
AmpStrSulTetTri

AmpSulTetTri
Tet
tet(A)
7.86 × 10⁻²
FIA, FIB
AmpSulTetTri

AmpStrSulTetTri
Tet
tet(A)
2.05 × 10⁻⁷
A/C
AmpStrSulTetTri

Hai rural
AmpStrSulTetTri
Amp
ND
7.49 × 10⁻³
ND
Amp

AmpStrSulTet
Amp
ND
—
ND
Amp

Simanjiro rural
AmpCeftazKanStrSulTetTri
Amp

6.5 × 10⁻²
N, FIB, FIA
AmpStrSulTetTri

AmpCeftazChloStrSulTetTri
Amp

—
N, H12
AmpCeftazStrSul

AmpStrTetTri
Tet
tet(B)
1.08 × 10⁻⁴
ND
AmpStrSulTet

AmpSulTetTri
Amp
ND
6.25 × 10⁻⁴
ND
AmpSul

TetTri
Tet
ND
—
ND
Tet

Monduli rural
AmpCeftazChloKanStrSulTetTri
Tet
tet(A),
9.26 × 10⁻²
H12, K/B
AmpCeftazChloKanStrSulTetTri

AmpStrSulTetTri
Amp
tet(A)
3.19 × 10⁻⁴
ND
AmpSulTet

AmpStrSulTet
Amp
ND
1.07 × 10⁻²
I1, FIB
AmpSul

AmpSulTet
Amp
ND
7.91 × 10⁻³
B/O
AmpSulTet

AmpSul
Amp
ND
2.45 × 10⁻²
ND
AmpSul

Longido rural
AmpStrSulTetTri
Amp
ND
5.31 × 10⁻³
B/O, FIC
AmpSul

AmpStrSulTri
Amp
ND
8.93 × 10⁻⁴
FIC
AmpSul

AmpSulTetTri
Tet
ND
8.99 × 10⁻⁵
FIC, A/C, FIIA
AmpSulTet

AmpStrTet
Amp
ND
1.33 × 10⁻⁴
I1, K/B
AmpStrTet

Arumeru periurban
AmpChloKanSulTetTri
Amp
tet(B),
8.95 × 10⁻²
I1, FIB, FIA, K/B
AmpKanStrSulTetTri

AmpChloSulTetTri
Amp
ND
1.66 × 10⁻²
ND
AmpSul

AmpStrSulTetTri
Amp
ND
5 × 10⁻³
ND
Amp

AmpSulTetTri
Amp

8.33 × 10⁻¹
ND
AmpSulTetTri

Abx, antibiotic; Amp, ampicillin; Ceftaz, ceftazidime; Cip, ciprofloxacin; Chlo, chloramphenicol; Kan, kanamycin; Str, streptomycin; Sul, sulfamethoxazole; Tet, tetracycline; Trm, trimethoprim; ND, not detected.

PCR-based replicon typing was used with genomic DNA of the transconjugants using the methods described by Johnson et al. [15]. Briefly, pellets from 1 mL of overnight culture were resuspended with 200 μL of nanopure water and placed in a heating block at 100°C for 10 min. The lysed suspension was cooled to room temperature and centrifuged briefly to pellet debris. Supernatant was transferred to a new vial and stored at −80°C until ready for testing against 18 different sets of primers [15] that were grouped into three multiplex primer panels [15]. The following PCR conditions were used: 5 min at 94°C, 30 cycles of 30 s at 94°C, 30 s at 60°C, and 90 s at 72°C, and a final extension of 5 min at 72°C. The amplified PCR products were visualized using 1.5% Tris-acetate-EDTA agarose gel containing 0.2 μg/mL ethidium bromide alongside a 1 kb ladder (Gene ruler 1 Kb, Life Technologies).

3. Results

Thirty-one MDR isolates were selected and used as donors to test if the resistance determinants were transferrable to recipient E. coli isolates by conjugation. Of these, antibiotic resistance traits were successfully transferred by 25 isolates with conjugation efficiencies ranging from 10⁻¹ to 10⁻⁷ (Table 2). IncF plasmids were attributable to the highest conjugation efficiency 1.8 × 10⁻¹. Importantly, over 80% of the donor cells successfully passed a “penta-resistant” phenotype that included resistance to ampicillin, streptomycin, sulfamethoxazole, tetracycline, and trimethoprim. PCR testing of transconjugants showed that tet(A) was most commonly associated with conjugative plasmids (33%) followed by (24%), tet(B) (17%), (8%), and (0%).

A total of 11 replicon types were detected among the 31 MDR isolates (Table 3). IncF replicon types (IncF IA, IB, IC, and IIA) were predominant (49%) and were mainly associated with E. coli isolates that were resistant to ampicillin, streptomycin, sulfonamide, tetracycline, and trimethoprim. Replicon types IncX, IncW, IncL/M, IncY, IncHI1, IncT, and Inc K were not detected. Replicon types N, H12, FIB, and FIA were associated with and resistance to ampicillin, ceftazidime, streptomycin, sulfamethoxazole, tetracycline, and trimethoprim. After conjugation, one recipient was positive for four different replicons (I1, FIB, FIA, and K/B).

Table 3: Frequency of plasmid replicon types detected from E. coli isolates () from surface waters in northern Tanzania.

Replicon type
Number of isolates
Percent of isolates

FIB
5
16%

FIC
4
13%

I1
4
13%

FIA
3
10%

FIIA
3
10%

A/C
3
10%

K/B
3
10%

P
2
6%

HI2
2
6%

B/O
2
6%

N
2
6%

The total percentage sums to 106 because some isolates were positive for more than one replicon type.

4. Discussion

Plasmid-mediated horizontal transfer of multidrug resistance traits plays a key role in the dissemination of antimicrobial resistance around the world [27]. Our study shows that E. coli isolated from Tanzanian water sources harbor multiple plasmids belonging to major plasmid replicon types such as IncF, A/C, l1, and N. Most of these plasmids were associated with transfer of antibiotic resistance traits via conjugation with rates that varied between 10⁻¹ and 10⁻⁷ using filter-mating assays. Moreover, these plasmids harbor multiple antibiotic resistance genes that are associated with plasmid replicon types such as IncF, A/C, N, l1, H12, and B/O. Studies show that plasmid-mediated horizontal gene transfer occurs within and between E. coli and Pseudomonas isolated from sewage and lake water [13]. Given the presence of resistant E. coli in biologically contaminated water from Tanzania, it is likely that their presence contributes to the long-term persistence of resistance traits in people and animals who share these water resources.

Among the 11 replicon types found, IncF group plasmids were detected more frequently than other tested groups. This is in accordance with previous studies where IncF plasmids were found predominantly in E. coli from clinical samples (rectal samples, gastric aspirate samples, and vaginal sample) [28, 29] and in E. coli from people (feces and UTI patients) and poultry (fecal swab) [15]. IncFIB was the most frequently detected (16%) replicon type, similar to what has been reported for E. coli isolates collected from fecal samples of healthy people and cattle in Nigeria [30]. The overall proportion of IncF-positive E. coli was 49%, which is lower than that observed in Germany (71%) [31] but higher than that observed in E. coli isolates from fecal samples of healthy people and food animals in Switzerland (45%) [27]. IncF type plasmids have a “narrow” host range although they are well adapted to E. coli and are frequently associated with the presence of tet(A), , and [31, 32]. In this study, plasmid type IncF was associated with tet(A), , and .

The CTX-M-15 β-lactamases are disseminated worldwide and are usually located in the conjugative plasmids [33]. Detection of this trait in E. coli isolated from water sources is a public health concern because CTX-M-15 β-lactamases are commonly associated with urinary tract infections [30]. Another study from a hospital in Tanzania found CTX-M-15 in Klebsiella pneumoniae that can be associated with neonatal sepsis [34]. In this study, detection of CTX-M-15 in E. coli from water suggests a possible contamination of human, livestock, and wild animal excreta and thus consumption of this untreated water is clearly a potential risk for transmission back to people [35].

The IncI1 plasmid type was the second most prevalent replicon type (13%) and these plasmids also harbor multiple resistance genes. E. coli can reportedly maintain IncI1 plasmids without antibiotic selection pressure and with little or no apparent fitness cost to the host bacterium [36]. Importantly, it is also a conjugative plasmid commonly detected in E. coli recovered from humans and animals with the conjugation efficiencies ranging between 10⁻² and 10⁻⁷ [15, 16, 28, 34]. The IncI1 plasmid carrying and has been associated with the recent 2011 outbreak of E. coli O104 in Germany [37]. In addition, bacteria carrying lncl1 plasmids were responsible for community and hospital acquired infections [38, 39].

IncA/C plasmids are typically larger (~150 kb) than others with lower conjugation efficiencies [24, 40, 41]. About 10% of E. coli isolates from our water samples harbored IncA/C plasmids and these were associated with resistance to ampicillin, streptomycin, sulfonamide, tetracycline, and trimethoprim. Importantly, IncA/C plasmids can harbor a large number of antimicrobial resistance genes and the broad-host spectrum coupled with an ability to spread via conjugation transfer within bacteria communities means that they can transfer an arsenal of resistance traits to pathogens of people and animals [40, 42, 43]. Isolation of E. coli with IncA/C from environmental water samples that are consumed by humans and animals on daily basis is a major public health concern. The replicon typing methods have some pitfalls including the obvious inability to detect unknown replicons [15]. For example, 14 isolates in the current study transferred their resistance phenotypes to the recipients’ cells but no plasmid replicon was detected. Detection of these public health important conjugative plasmids and antibiotic resistant traits in Tanzanian water suggests the possible pollution of these water sources from human, livestock, and wild animal wastes and also shows the potential of these water sources in the maintenance and transmission of these resistance traits between environment, animals, and people. Therefore, appropriate intervention strategies should be identified and implemented to reduce the water pollution.

Competing Interests

No competing financial interests exist.

Acknowledgments

Lisa Orfe, Lisa Jones, Deogratius Mshanga, Samson Lyimo, and Lidia Munuo provided technical assistance. This work was funded in part by the COSTECH through the Nelson Mandela Africa Institution of Science and Technology, the Paul G. Allen School for Global Animal Health, and the National Science Foundation (DEB1216040).

References

G8 Science Ministers: G8 Science Ministers Statement, Department for Business, Innovation and Skills, London, UK, 2013, https://www.gov.uk/government/publications/g8-science-ministers-statement-london-12-june-2013.

T. T. Lina, B. K. Khajanchi, I. J. Azmi et al., “Phenotypic and molecular characterization of extended-spectrum beta-lactamase-producing Escherichia coli in Bangladesh,” PLoS ONE, vol. 9, no. 10, Article ID e108735, 2014.

P. M. Hawkey and A. M. Jones, “The changing epidemiology of resistance,” Journal of Antimicrobial Chemotherapy, vol. 64, supplement 1, pp. i3–i10, 2009.

G. C. A. Amos, P. M. Hawkey, W. H. Gaze, and E. M. Wellington, “Waste water effluent contributes to the dissemination of CTX-M-15 in the natural environment,” Journal of Antimicrobial Chemotherapy, vol. 69, no. 7, pp. 1785–1791, 2014.

F. Baquero, J.-L. Martínez, and R. Cantón, “Antibiotics and antibiotic resistance in water environments,” Current Opinion in Biotechnology, vol. 19, no. 3, pp. 260–265, 2008.

M. W. Jenkins, S. Tiwari, M. Lorente, C. M. Gichaba, and S. Wuertz, “Identifying human and livestock sources of fecal contamination in Kenya with host-specific Bacteroidales assays,” Water Research, vol. 43, no. 19, pp. 4956–4966, 2009.

J. H. O. Onyuka, R. Kakai, D. M. Onyango et al., “Prevalence and antimicrobial susceptibility patterns of enteric bacteria isolated from water and fish in lake Victoria basin of western Kenya,” International Journal of Biological, Biomolecular, Agricultural, Food and Biotechnological Engineering, vol. 5, pp. 762–769, 2011.

S. E. Mshana, M. Matee, and M. Rweyemamu, “Antimicrobial resistance in human and animal pathogens in Zambia, Democratic Republic of Congo, Mozambique and Tanzania: an urgent need of a sustainable surveillance system,” Annals of Clinical Microbiology and Antimicrobials, vol. 12, article 28, 2013.

A. Christopher, S. E. Mshana, B. R. Kidenya, A. Hokororo, and D. Morona, “Bacteremia and resistant gram-negative pathogens among under-fives in Tanzania,” Italian Journal of Pediatrics, vol. 39, no. 1, article 27, 2013.

E. Nelson, J. Kayega, J. Seni et al., “Evaluation of existence and transmission of extended spectrum beta lactamase producing bacteria from post-delivery women to neonates at Bugando Medical Center, Mwanza-Tanzania,” BMC Research Notes, vol. 7, no. 1, article 279, 2014.

A. A. S. Katakweba, K. S. Møller, J. Muumba et al., “Antimicrobial resistance in faecal samples from buffalo, wildebeest and zebra grazing together with and without cattle in Tanzania,” Journal of Applied Microbiology, vol. 118, no. 4, pp. 966–975, 2015.

S. Chaturvedi, R. Chandra, and V. Rai, “Multiple antibiotic resistance patterns of rhizospheric bacteria isolated from Phragmites australis growing in constructed wetland for distillery effluent treatment,” Journal of Environmental Biology, vol. 29, no. 1, pp. 117–124, 2008.

M. R. Shakibaie, K. A. Jalilzadeh, and S. M. Yamakanamardi, “Horizontal transfer of antibiotic resistance genes among gram negative bacteria in sewage and lake water and influence of some physico-chemical parameters of water on conjugation process,” Journal of Environmental Biology, vol. 30, no. 1, pp. 45–49, 2009.

A. Carattoli, A. Bertini, L. Villa, V. Falbo, K. L. Hopkins, and E. J. Threlfall, “Identification of plasmids by PCR-based replicon typing,” Journal of Microbiological Methods, vol. 63, no. 3, pp. 219–228, 2005.

T. J. Johnson, Y. M. Wannemuehler, S. J. Johnson et al., “Plasmid replicon typing of commensal and pathogenic Escherichia coli isolates,” Applied and Environmental Microbiology, vol. 73, no. 6, pp. 1976–1983, 2007.

A. Carattoli, “Resistance plasmid families in Enterobacteriaceae,” Antimicrobial Agents and Chemotherapy, vol. 53, no. 6, pp. 2227–2238, 2009.

P. M. Gann, J. Hang, R. J. Clifford et al., “Complete sequence of a novel 178-kilobase plasmid carrying in a Providencia stuartii strain isolated in Afghanistan,” Antimicrobial Agents and Chemotherapy, vol. 56, no. 4, pp. 1673–1679, 2012.

T. Sekizuka, M. Matsui, K. Yamane et al., “Complete sequencing of the -positive IncA/C plasmid from Escherichia coli ST38 isolate suggests a possible origin from plant pathogens,” PLoS ONE, vol. 6, no. 9, Article ID e25334, 2011.

T. L. Poole, T. S. Edrington, D. M. Brichta-Harhay, A. Carattoli, R. C. Anderson, and D. J. Nisbet, “Conjugative transferability of the A/C plasmids from Salmonella enterica isolates that possess or lack blaCMY in the A/C plasmid backbone,” Foodborne Pathogens and Disease, vol. 6, no. 10, pp. 1185–1194, 2009.

Environmental Protection Agency (EPA), “Method 1603: Escherichia coli (E. coli) in water by membrane filtration using modified membrane-Thermotolerant Escherichia coli Agar (Modified mTEC),” 2002

S. Iqbal, J. Robinson, D. Deere, J. R. Saunders, C. Edwards, and J. Porter, “Efficiency of the polymerase chain reaction amplification of the uid gene for detection of Escherichia coli in contaminated water,” Letters in Applied Microbiology, vol. 24, no. 6, pp. 498–502, 1997.

Clinical and Laboratory Standards Institute, “Performance standards for antimicrobial susceptibility testing; twenty-fourth informational supplement,” CLSI M100-S24, Clinical and Laboratory Standards Institute (CLSI), 2014.

A. T. Adesoji, A. A. Ogunjobi, I. O. Olatoye, and D. R. Douglas, “Prevalence of tetracycline resistance genes among multi-drug resistant bacteria from selected water distribution systems in southwestern Nigeria,” Annals of Clinical Microbiology and Antimicrobials, vol. 14, no. 1, article 35, 18 pages, 2015.

M. Subbiah, E. M. Top, D. H. Shah, and D. R. Call, “Selection pressure required for long-term persistence of blaCMY-2-positive IncA/C plasmids,” Applied and Environmental Microbiology, vol. 77, no. 13, pp. 4486–4493, 2011.

C. Eckert, V. Gautier, and G. Arlet, “DNA sequence analysis of the genetic environment of various blaCTX-M genes,” Journal of Antimicrobial Chemotherapy, vol. 57, no. 1, pp. 14–23, 2006.

M. Saladin, V. T. B. Cao, T. Lambert et al., “Diversity of CTX-M β-lactamases and their promoter regions from Enterobacteriaceae isolated in three Parisian hospitals,” FEMS Microbiology Letters, vol. 209, no. 2, pp. 161–168, 2002.

J. Wang, R. Stephan, M. Karczmarczyk, Q. Yan, H. Hächler, and S. Fanning, “Molecular characterization of blaESBL-harboring conjugative plasmids identified in multi-drug resistant Escherichia coli isolated from food-producing animals and healthy humans,” Frontiers in Microbiology, vol. 4, article 188, 2013.

G. Marcadé, C. Deschamps, A. Boyd et al., “Replicon typing of plasmids in Escherichia coli producing extended-spectrum β-lactamases,” Journal of Antimicrobial Chemotherapy, vol. 63, no. 1, pp. 67–71, 2009.

C. Branger, O. Zamfir, S. Geoffroy et al., “Genetic background of Escherichia coli and extended-spectrum β-lactamase type,” Emerging Infectious Diseases, vol. 11, no. 1, pp. 54–61, 2005.

C. Inwezerua, N. Mendonça, V. Calhau, S. Domingues, O. E. Adeleke, and G. J. Da Silva, “Occurrence of extended-spectrum beta-lactamases in human and bovine isolates of Escherichia coli from Oyo state, Nigeria,” Journal of Infection in Developing Countries, vol. 8, no. 6, pp. 774–779, 2014.

S. E. Mshana, C. Imirzalioglu, H. Hossain, T. Hain, E. Domann, and T. Chakraborty, “Conjugative IncFI plasmids carrying CTX-M-15 among Escherichia coli ESBL producing isolates at a University hospital in Germany,” BMC Infectious Diseases, vol. 9, article 97, 2009.

E. Fidelma Boyd, C. W. Hill, S. M. Rich, and D. L. Hard, “Mosaic structure of plasmids from natural populations of Escherichia coli,” Genetics, vol. 143, no. 3, pp. 1091–1100, 1996.

G. Peirano and J. D. D. Pitout, “Molecular epidemiology of Escherichia coli producing CTX-M β-lactamases: the worldwide emergence of clone ST131 O25:H4,” International Journal of Antimicrobial Agents, vol. 35, no. 4, pp. 316–321, 2010.

S. E. Mshana, T. Hain, E. Domann, E. F. Lyamuya, T. Chakraborty, and C. Imirzalioglu, “Predominance of Klebsiella pneumoniae ST14 carrying CTX-M-15 causing neonatal sepsis in Tanzania,” BMC Infectious Diseases, vol. 13, no. 1, article 466, 2013.

K. Grenet, D. Guillemot, V. Jarlier et al., “Antibacterial resistance, wayampis amerindians, French Guyana,” Emerging Infectious Diseases, vol. 10, no. 6, pp. 1150–1153, 2004.

E. A. J. Fischer, C. M. Dierikx, A. van Essen-Zandbergen et al., “The IncI1 plasmid carrying the gene persists in in vitro culture of a Escherichia coli strain from broilers,” BMC Microbiology, vol. 14, article 77, 2014.

A. Mellmann, D. Harmsen, C. Cummings et al., “Prospective genomic characterization of the German enterohemorrhagic Escherichia coli O104:H4 outbreak by rapid next generation sequencing technology,” PLoS ONE, vol. 6, no. 7, Article ID e22751, 2011.

K. L. Hopkins, E. Liebana, L. Villa, M. Batchelor, E. J. Threlfall, and A. Carattoli, “Replicon typing of plasmids carrying CTX-M or CMY β-lactamases circulating among Salmonella and Escherichia coli isolates,” Antimicrobial Agents and Chemotherapy, vol. 50, no. 9, pp. 3203–3206, 2006.

Â. Novais, R. Cantón, R. Moreira, L. Peixe, F. Baquero, and T. M. Coque, “Emergence and dissemination of Enterobacteriaceae isolates producing CTX-M-1-like enzymes in spain are associated with IncFII (CTX-M-15) and broad-host-range (CTX-M-1, -3, and -32) plasmids,” Antimicrobial Agents and Chemotherapy, vol. 51, no. 2, pp. 796–799, 2007.

R. L. Lindsey, P. J. Fedorka-Cray, J. G. Frye, and R. J. Meinersmann, “Inc A/C plasmids are prevalent in multidrug-resistant Salmonella enterica isolates,” Applied and Environmental Microbiology, vol. 75, no. 7, pp. 1908–1915, 2009.

M. Wiesner, M. Fernández-Mora, M. A. Cevallos et al., “Conjugative transfer of an IncA/C plasmid-borne gene through genetic re-arrangements with an IncX1 plasmid,” BMC Microbiology, vol. 13, article 264, 17 pages, 2013.

T. J. Welch, W. F. Fricke, P. F. McDermott et al., “Multiple antimicrobial resistance in plague: an emerging public health risk,” PLoS ONE, vol. 2, no. 3, article e309, 2007.

T. J. Johnson and K. S. Lang, “IncA/C plasmids: an emerging threat to human and animal health?” Mobile Genetic Elements, vol. 2, no. 1, pp. 55–58, 2012.