The Role of Flies in Spreading the Extended-Spectrum β-lactamase Gene from Cattle

Abstract

The spreading of antimicrobial-resistant bacteria and genes from food-producing animals to humans has been a subject of increasing concern. To clarify the role of flies in spreading the extended-spectrum β-lactamase (ESBL) gene from food-producing animals to humans, we isolated and characterized a third-generation cephalosporin-resistant Escherichia coli strain from flies and cattle feces from a cattle barn. Cephalosporin-resistant strains were isolated from 14.3% (13/91) of houseflies, 10.3% (7/68) of false stable flies, and 7.5% (7/93) of cattle feces. Twenty-seven cephalosporin-resistant strains were tested for the presence of antimicrobial resistance genes. Of the 27 samples, 22 isolates from 11 houseflies, 5 false stable flies, and 6 cattle feces samples harbored the bla_CTX-M-15 gene. All bla_CTX-M-15-harboring isolates belonged to phylogenetic group D and the ST38 clonal group. Analysis of pulsed-field gel electrophoresis showed that these isolates were divided into two clusters, indicating that flies carried several of the same clones that were detected in cattle feces. All bla_CTX-M-15 gene-harboring plasmids were transferable and were members of incompatibility group FIB. These results suggest that transferable plasmids encoding ESBL were prevalent among flies and cattle. As vectors, flies may play an important role in spreading ESBL-producing bacteria from food-producing animals to humans.

Introduction

The emergence and spread of antimicrobial resistance has become a major global public health concern. A component of this problem is the spreading of the plasmid-encoded extended-spectrum β-lactamase (ESBL) gene, which can confer resistance to third-generation cephalosporins.²⁹ Third-generation cephalosporins are clinically important antimicrobials in human and veterinary medicine. In recent years, the most prevalent type of ESBLs has been CTX-M.²⁹ In particular, CTX-M-15, a member of the CTX-M-1 group, is the most widely disseminated ESBL globally.^16,29

Since the report of Swann et al.³, the spreading of antimicrobial-resistant bacteria and genes from food-producing animals to humans has been investigated. Nationwide surveillance studies have indicated that antimicrobial resistances have been harbored in food-producing animals.^10,24,33 However, the connection between antimicrobial resistance in bacterial isolates from food-producing animals and clinical isolates of humans is uncertain, because the ecology of these bacteria and their genes in the agricultural and urban environment is not well understood.^1,13,32

Antimicrobial-resistant bacteria are released into the environment via feces from food-producing animals and sanitary insects, including flies that feed on feces.¹ Flies move freely between food-producing animals and humans owing to their strong flight capabilities,¹¹ possessing a great potential for dissemination of antimicrobial resistance from food-producing animals to humans.¹² In addition, recent reports have shown that fly guts provide a suitable environment for the horizontal transfer of conjugative plasmids among Escherichia coli strains.^2,27 Therefore, fly-mediated dissemination of antimicrobial resistance from food-producing animals to humans is drawing increasing attention.

The purpose of this study was to clarify the role of the flies in the spread of ESBL-producing bacteria from food-producing animals to humans. We identified and characterized a third-generation cephalosporin-resistant E. coli isolated from flies and cattle feces from a cattle barn.

Materials and Methods

Sample collection

All fecal and fly samples were collected from a cattle barn during August–October 2010 in Ebetsu City (Hokkaido prefecture, Japan). A total of 231 flies were collected using a sweep net. Flies were placed individually in sterile 50-ml Falcon tubes for laboratory processing. The fly species, including 91 houseflies (Musca domestica), 68 false stable flies (Muscina stabulans), and 72 stable flies (Stomoxys calcitrans), were morphologically identified by a stereomicroscope. A total of 93 samples of cattle feces were collected from the same cattle barn.

Bacterial isolation

Individual flies were surface sterilized with sodium hypochlorite and ethanol as previously described.²⁰ The samples were then washed thrice with sterile distilled water and homogenized in potassium-buffered saline. The homogenized flies and cattle feces were inoculated into deoxycholate-hydrogen sulfate-lactose (DHL) agar medium (Nissui Pharmaceutical, Tokyo, Japan) or DHL agar medium supplemented with 2 μg/ml cefpodoxime (DHL-C; Daiichi-Sankyo, Tokyo, Japan). The isolation agar media were incubated at 37°C overnight. The isolate that was identified as E. coli by colony morphology and API 20E tests (Sysmex, Kobe, Japan) was selected.

Antimicrobial susceptibility testing

We performed minimal inhibitory concentration (MIC) determinations using the broth microdilution method with an Eiken frozen plate (Eiken Chemistry, Tokyo, Japan) according to the Clinical Laboratory Standards Institute (CLSI) guidelines.⁷ The following antimicrobial agents were tested: ampicillin (Sigma-Aldrich, St. Louis, MO), cefazolin (Sigma-Aldrich), cefpodoxime (Daiichi-Sankyo), streptomycin, kanamycin, gentamicin, tetracycline, chloramphenicol, nalidixic acid, ciprofloxacin, trimethoprim, fosfomycin, and colistin (Sigma-Aldrich). The resistance breakpoints were defined for the antimicrobials in accordance with CLSI guidelines.⁷ We obtained breakpoints for colistin, which were not defined by the CLSI guidelines, from a report on the Japanese Veterinary Antimicrobial Resistance Monitoring system.²⁵ In this study, we defined the breakpoint for streptomycin as 64 μg/ml by taking into consideration the midpoint between the peaks of each MIC distribution. E. coli ATCC25922 was used as a quality-control strain. The cefpodoxime-resistant isolates (MIC ≥8 μg/ml) were selected for further tests.

Characterization of resistance genes

The DNA from the cefpodoxime-resistant isolates was extracted from cultures with an InstaGene Matrix (Bio-Rad Laboratories, Tokyo, Japan). The presence of genes encoding bla_TEM, bla_SHV, bla_OXA, bla_CTX-M, bla_ACC, bla_FOX, bla_MOX, bla_DHA, bla_CIT, and bla_EBC was determined by multiplex PCR as previously described.⁹ For the CTX-M-1 group, an additional PCR procedure was performed using external primers as previously described,²³ and all amplicons were subsequently sequenced. The tetA and aac(6′)-Ib-cr genes were screened by PCR.^26,31

Pulsed-field gel electrophoresis and phylogenetic grouping

The bla_CTX-M-1 _group-harboring isolates were typed by pulsed-field gel electrophoresis (PFGE) analysis according to the Pulse Net CDC protocol.⁴ Genomic DNA in each agarose plug was digested with XbaI (Takara Bio, Shiga, Japan). The PFGE procedure was performed using the CHEF-DRIII system (Bio-Rad Laboratories) under the following conditions: switch time, 2.2–54.2 seconds; running time, 18 hours; included angle, 120°; voltage, 6 V/cm; and temperature, 14°C. The PFGE profiles were analyzed using the BioNumerics program (Applied Maths, Sint-Martens-Latem, Belgium). Similarity and diversity were assessed by applying the Dice coefficient. Cluster analysis was performed using the unweighted pair group method with arithmetic means (UPGMA; position tolerance of 0.4% and optimization of 0.5%).

E. coli phylogenetic groupings (A, B1, B2, and D) were determined using a multiplex PCR method that was used for the chuA and yjaA genes and tspE4.C2 fragment, as previously described by Clermont et al.⁶

Sequence-type determination

Multilocus sequence typing (MLST) was performed as previously described.³⁴ Gene amplification and sequencing were performed using the primers specified on the E. coli MLST Web site (http://mlst.ucc.ie/mlst/dbs/Ecoli). Allelic profile and sequence type (ST) were determined as per the scheme on the E. coli MLST Web site.

Transferability of the bla_{CTX-M-1 group} genes and plasmid characterization

Transferability was tested by performing previously described broth-mating assays with slight modifications.³⁶ In brief, the recipients used were rifampicin-resistant E. coli K12 ML4909 strains, with a mating temperature set at 30°C. Transconjugants were selected on Mueller–Hinton agar supplemented with 50 μg/ml rifampicin (Sigma-Aldrich) and 32 μg/ml cefpodoxime. All bla_CTX-M-1 _group-carrying plasmids were replicon typed by using the PCR-based replicon-typing method, as previously described.¹⁴ The plasmid DNA was purified from the parental strains and transconjugants by using a modified alkaline lysis method,¹⁵ and the plasmid sizes were estimated using the BAC-Tracker supercoiled DNA ladder (Epicentre Biotechnologies, Madison, WI). Southern hybridization was subsequently performed. The plasmids were transferred using downward capillary transfer to Hybond-N+ nylon membrane (GE Healthcare, Chalfont St Giles, United Kingdom), and the membrane was treated according to standard procedures.³⁰ DNA probe labeling, hybridization, and detection were performed using the digoxigenin (DIG)-PCR and DIG Nucleic Acid Detection Kits (Roche Diagnostics, Basel, Switzerland) according to the manufacturer's instructions. DNA probes were constructed with the β-lactamase gene (bla_CTX-M15) and replicon FIB gene-purified PCR products.

Results

Isolation of E. coli from flies and cattle feces

By using DHL agar, E. coli was isolated from 42.9% (39/91) of houseflies, 67.6% (46/68) of false stable flies, and 66.7% (62/93) of cattle feces samples (Table 1). Cefpodoxime-resistant E. coli was isolated from only two houseflies (2.2%). By using DHL-C agar, cefpodoxime-resistant E. coli was isolated from 12.1% (11/91) of houseflies, 10.3% (7/68) of false stable flies, and 7.5% (7/93) of cattle feces samples. No E. coli were obtained from the 72 stable flies. In total, 27 cefpodoxime-resistant strains were isolated from 13 houseflies (14.3%; 2 from DHL and 11 from DHL-C), 7 false stable flies (10.3%), and 7 cattle feces samples (7.5%; Table 2).

Table 1.

Isolation Rate of Escherichia coli from Flies and Cattle Feces

	Isolation rate (%)
Origin	DHL	DHL containing cefpodoxime ^a
Housefly (Musca domestica)	39/91 (42.9%)	12/91 (13.2%)
False stable fly (Muscina stabulans)	46/68 (67.6%)	7/68 (10.3%)
Stable fly (Stomoxys calcitrans)	0/72 (0%)	0/72 (0%)
Cattle feces	62/93 (66.7%)	7/93 (7.5%)

DHL agar medium supplemented with 2 μg/ml cefpodoxime.

DHL, deoxycholate-hydrogen sulfate-lactose.

Table 2.

Antimicrobial Resistance of Escherichia coli Isolates

		Housefly		False stable fly		Cattle feces
Antimicrobial agents	Break point (μg/ml)	DHL (n=39)	DHL containing cefpodoxime (n=12)^a	DHL (n=46)	DHL-containing cefpodoxime (n=7)^a	DHL (n=62)	DHL containing cefpodoxime (n=7)^a
Ampicillin	32^b	3 (7.7%)	12 (100%)	1 (2.2%)	7 (100%)	6 (9.6%)	7 (100%)
Cefazolin	4^b	2 (5.1%)	11 (91.7%)	0	7 (100%)	3 (4.8%)	7 (100%)
Cefpodoxime	8^b	2 (5.1%)	11 (91.7%)	0	7 (100%)	0	7 (100%)
Streptomycin	64^c	6 (15.4%)	11 (91.7%)	9 (19.6%)	6 (85.7%)	7 (11.3%)	7 (100%)
Kanamycin	64^b	1 (2.6%)	2 (16.7%)	0	1 (14.3%)	8 (12.9%)	1 (14.3%)
Gentamicin	16^b	0	0	1 (2.2%)	0	0	0
Tetracycline	16^b	8 (20.5%)	12 (100%)	11 (23.9%)	7 (100%)	28 (45.2%)	7 (100%)
Chloramphenicol	32^b	0	0	0	1 (14.3%)	1 (1.6%)	0
Nalidixic acid	32^b	0	1 (8.3%)	1 (2.2%)	0	0	0
Ciprofloxacin	4^b	0	0	0	0	0	0
Trimethoprim	16^b	2 (5.1%)	10 (83.3%)	0	6 (85.7%)	1 (1.6%)	7 (100%)
Fosfomycin	256^b	0	0	0	0	0	0
Colistin	16^d	0	0	0	0	0	0

DHL agar medium supplemented with 2 μg/ml cefpodoxime.

The value was a CLSI breakpoint.

The value was set as the midpoint between the peaks of each MIC distribution.

The value was a JVARM breakpoint.

JVARM, Japanese Veterinary Antimicrobial Resistance Monitoring Program; CLSI, Clinical Laboratory Standards Institute; MIC, minimum inhibitory concentration.

Antimicrobial resistance

The proportions of antimicrobial-resistant E. coli obtained using DHL and DHL-C are shown in Table 2. Among E. coli isolates from houseflies and false stable flies grown in DHL agar, a high percentage of isolates with resistance to tetracycline (20.5% and 23.9%, respectively) and streptomycin (15.4% and 19.6%, respectively) were observed. Among E. coli isolates from cattle feces, however, isolates with resistance to tetracycline (45.2%), kanamycin (12.9%), and streptomycin (11.3%) were observed.

By using DHL-C agar, a high percentage of isolates with resistance to ampicillin, cefazolin, and cefpodoxime were observed among E. coli isolates from all origins. In addition, most of these isolates were resistant to streptomycin, tetracycline, and trimethoprim.

Antimicrobial-resistant genes

Twenty-seven cefpodoxime-resistant isolates were tested for the presence of antimicrobial-resistant genes. Twenty-two isolates from 11 houseflies, 5 false stable flies, and 6 cattle feces samples harbored the bla_CTX-M-1 _group genes. Sequencing of the resulting PCR products demonstrated identity with bla_CTX-M-15 genes. All isolates harbored the bla_CTX-M-15 genes containing bla_TEM and tetA. Three additional cefpodoxime-resistant isolates (from one housefly and two false stable flies) harbored the bla_CTX-M2 _group genes. The β-lactamase genes were not detected in the remaining two cefpodoxime-resistant isolates (from one housefly and one cattle feces sample).

No bla_SHV, bla_OXA, bla_ACC, bla_FOX, bla_MOX, bla_DHA, bla_CIT, bla_EBC, or aac(6′)-Ib-cr genes were detected in any of the cefpodoxime-resistant isolates.

PFGE analysis of bla_CTX-M-15-carrying E. coli

The genetic relationships, based on PFGE results, among the 22 bla_CTX-M-15-carrying isolates are shown in Figure 1. Analysis using the UPGMA resulted in the classification of the genes into two clusters with an 80% similarity level (Fig. 1). The first cluster contained 12 isolates derived from 4 houseflies, 4 false stable flies, and 4 cattle feces samples. The second cluster contained 10 isolates derived from 7 houseflies, 1 false stable fly, and 2 cattle feces samples.

FIG. 1.

Pulsed-field gel electrophoresis analysis showing percent similarities of 22 isolates carrying bla_CTX-M-15 genes. DNA samples for pulsed-field gel electrophoresis analysis were digested with XbaI.

Phylogenetic characterization revealed that all 22 isolates harbored the bla_CTX-M-15 genes belonging to group D. All 22 bla_CTX-M-15-harboring isolates were subjected to seven-locus MLST and exhibited the same combination of alleles across the seven sequenced loci, corresponding to an established ST, ST38.

bla_CTX-M-15-carrying plasmid characterization

Transconjugants derived from all 22 isolates harboring the bla_CTX-M-15 genes were established. Southern hybridization analysis revealed that the length of the plasmid harboring the bla_CTX-M-15 genes in all isolates and transconjugants of this study was approximately 120 kbp; further, all belonged to incompatibility group FIB.

Discussion

The current study using DHL-C showed that cefpodoxime-resistant E. coli were isolated from 11 houseflies (12.1%), 7 false stable flies (10.3%), and 7 cattle feces (7.5%). By contrast, we were able to detect cefpodoxime-resistant E. coli isolates from only two houseflies (2.2%) in non-selective DHL medium. These results suggest that cefpodoxime-resistant isolates were not predominant in the cattle barn, despite their wide distribution.

E. coli was detected in housefly and false stable fly gut, but not in stable fly gut. Both houseflies and false stable flies feed on cattle feces, while stable flies are blood-feeding insects. An analysis of the results of PFGE and MLST in bla_CTX-M-15-harboring E. coli showed that houseflies and false stable flies carried several of the same clones that were detected in cattle feces. Chakrabati et al. reported that the prevalence of antimicrobial-resistant enterococci in houseflies decreased with increasing distance from the cattle feedlot.⁵ These results suggest that the source of ESBL-producing E. coli in housefly and false stable fly gut was the cattle feces from the cattle barn.

In this study, we observed closely related isolates harboring the bla_CTX-M-15-carrying incompatibility group FIB plasmids in houseflies, false stable flies, and cattle feces samples. This is an interesting finding, because ESBL-producing E. coli clones have rarely been reported to show clonal dissemination. Furthermore, the incompatibility group I1 plasmid has been the most commonly observed vector that is subject to clonal dissemination, especially in animals.³⁵ It has been reported that once ESBL-producing plasmids are acquired, clonal dissemination is another likely mechanism for the perpetuation of resistance.¹⁸ These results suggest that the bla_CTX-M-15-carrying incompatibility group FIB plasmid had invaded the cattle barn and that clonal dissemination subsequently occurred between flies and cattle. However, further studies are required to clarify the invasion routes of the antimicrobial-resistant-carrying plasmids.

The bla_CTX-M-15-carrying plasmids in E. coli isolated from flies were observed for the first time in this study. Furthermore, bla_CTX-M-15-carrying plasmids from cattle were observed for the first time in Japan. bla_CTX-M-15-carrying plasmids, particularly found in B2-O25b:H4-ST131 E. coli, have been spreading globally.^16,27 The bla_CTX-M-15 genes are commonly found in large plasmids that often carry other antimicrobial-resistant genes, including bla_TEM-1, tetA, bla_OXA-1, and aac(6′)-lb-cr, classifying most of them as members of incompatibility group F.⁸ In this study, however, bla_CTX-M-15-harboring E. coli belong to phylogenetic group D and ST38, not B2-O25b:H4-ST131. In France, the bla_CTX-M-15-carrying plasmids from cattle were derived from non-ST131 E. coli isolates that were highly similar to those found in ST131 E. coli isolates in humans.²¹ bla_CTX-M-15-carrying plasmids from human clinical isolates derived from ST131 E. coli have recently been reported in Japan,^16,22 although the molecular structure of the plasmids has not been examined. Therefore, the bla_CTX-M-15-carrying plasmids derived from non-ST131 E. coli in this study and those from ST131 E. coli isolates in humans were not comparable. In future studies, we will carefully monitor the ESBL-producing bacteria in food-producing animals, flies, and humans isolates.

The bla_CTX-M-15-carrying plasmid in fly gut can be transmitted to other bacteria by conjugative transfer. Previous reports have shown that housefly gut provides a suitable environment for the horizontal transfer of conjugative plasmids among enterococci.² Flies often contain pathogenic bacteria such as Shigella sp., Vibrio cholera, E. coli O157:H7, Staphylococcus aureus, and Salmonella sp.¹¹ These results suggest that ESBL genes derived from cattle feces are capable of transfer to pathogenic bacteria in fly gut.

In addition to cattle barns, flies are also observed in pig pens and hen houses,^12,19 and ESBL-producing bacteria have been observed in swine and poultry feces.¹⁷ ESBL genes derived from swine and poultry feces may also be capable of transferring to the pathogenic bacteria in fly gut. Although most flies do not travel a distance greater than two miles, certain individual flies can travel as far as 20 miles.¹² It has been suggested that flies carry several bacterial pathogens of humans from hospitals into neighboring communities and vice versa.²⁸ Therefore, flies could transfer antimicrobial-resistant bacteria from farms into the urban area. To prevent the transmission of ESBL-producing bacteria from food-producing animals to humans, pest control in the rearing environment of food-producing animals would be most effective. Multi-farm and urban area studies are now in progress in order to confirm this hypothesis.

In conclusion, flies are both vectors and amplifiers in the transmission of ESBL-producing bacteria from food-producing animals to humans. Ensuring good practices and hygiene around calving areas is important for reducing the dissemination of ESBL-producing bacteria.

Footnotes

Acknowledgments

This work was supported by MEXT KAKENHI Grant Number 24590754. The authors thank Dr. Hitoshi Sasaki of Rakuno Gakuen University for his technical advice.

Disclosure Statement

No conflicts of interest exist.

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The Role of Flies in Spreading the Extended-Spectrum β-lactamase Gene from Cattle

Abstract

Introduction

Materials and Methods

Sample collection

Bacterial isolation

Antimicrobial susceptibility testing

Characterization of resistance genes

Pulsed-field gel electrophoresis and phylogenetic grouping

Sequence-type determination

Transferability of the blaCTX-M-1 group genes and plasmid characterization

Results

Isolation of E. coli from flies and cattle feces

Antimicrobial resistance

Antimicrobial-resistant genes

PFGE analysis of blaCTX-M-15-carrying E. coli

blaCTX-M-15-carrying plasmid characterization

Discussion

Footnotes

Acknowledgments

Disclosure Statement

References

Transferability of the bla_{CTX-M-1 group} genes and plasmid characterization

PFGE analysis of bla_CTX-M-15-carrying E. coli

bla_CTX-M-15-carrying plasmid characterization