Characterization of Escherichia coli Isolates from Healthy Food Handlers in Hospital

Abstract

Recent studies have reported that Escherichia coli in fecal samples of healthy humans could also serve as important reservoirs of drug-resistant bacteria. Limited data are available for E. coli–resistant profiles of healthy food handlers in hospitals who provide food service to inpatients and hospital staffs. E. coli isolates were recovered from hospital healthy food handlers, and one random selected isolate from each food handler was subjected to antimicrobial susceptibility testing, phylogenetic typing, and screening for antimicrobial-resistant mechanisms by polymerase chain reaction amplification. Ciprofloxacin-resistant isolates were further characterized by mutation analysis in the quinolone resistance determining regions (QRDRs) of GyrA and ParC. And extended-spectrum β-lactamase (ESBL) producing isolates were screened for bla_CTX-M by polymerase chain reaction amplification and DNA sequence analysis. In total, more than 50% (47/92) of E. coli isolates from healthy food handlers showed multidrug-resistant profiles and 50% (46/92) isolates carried intI. Resistance prevalence of the B2 phylogenetic group was significantly lower than that of the non-B2 groups for all tested antimicrobials (p < 0.05) except chloramphenicol and tetracycline. Seven isolates of phylogenetic group A (n = 3) and D (n = 4) produced ESBL, and 12 isolates of phylogenetic group A (n = 5), B2 (n = 2), and D (n = 5) were resistant to ciprofloxacin. Transferable quinolone resistance determinants were identified in four isolates. Point mutations in QRDRs of GyrA or ParC were identified among 59 out of 62 E. coli isolates showing decreased susceptibility or resistance to ciprofloxacin. Genes encoding CTX-M enzyme were identified in seven ESBL-producing isolates. The preponderance in hospital food handlers of multidrug-resistant E. coli makes it important to introduce control measures such as improved biosecurity to ensure that they do not pass through the food service and limit inpatient therapeutic options.

Introduction

Antimicrobial resistance has become a major public health concern worldwide because of clinical infection treatment heavily relying on antimicrobials and the quick emergence and dissemination of various resistant mechanisms.²³. Since resistant mechanisms could not only transfer vertically through cell replication, but also transfer horizontally to other organisms (such as conjugation, phage transduction, or transposition), the reservoir identification of antimicrobial resistance determinants has become a key step in efficient prevention program development. As one of the most common intestinal bacteria, Escherichia coli has been reported as important reservoirs of transposable elements encoding antimicrobial resistance.^2,13 In clinics, E. coli is one of the most commonly isolated microorganisms in clinical specimens and multidrug-resistant isolates are frequently reported. Recent studies have reported that E. coli in fecal samples of healthy humans could also serve as important reservoirs of drug-resistant bacteria.^25,28 As a special group of healthy humans, the healthy food handlers in hospitals should be investigated for the distribution of multidrug-resistant isolates.

The objective of our study was to determine the drug resistance prevalence of random selected fecal E. coli isolates of healthy food handlers in a hospital and analyze the associated resistance genes and the phylogenetic groups of these isolates.

Materials and Methods

Sample collection and E. coli isolation

During a health examination from June to July in 2009, solid formed stool samples were collected from 103 healthy food handlers who had not taken antibiotics in 3 months before sample collection and worked in dining halls of a military hospital in Beijing. All individuals were given informed consent for participation in this study. This military hospital has approximately 2,000 patient beds, 4,000 physicians and nurses, and 3,000 other routine workers. The sampled food handlers were comprised of 43 men and 60 women (age range: 18 to 55 years). For E. coli isolation, a loopful of stool samples was directly streaked on Statens Serum Institut (SSI) enteric agar (Statens Serum Institut, Denmark). From each sample, one red single colony was selected and further confirmed by the API 20E test (BioMérieux, Beijing, China). All confirmed E. coli isolates were kept in brain heart infusion broth with 50% glycerol at −70°C freezer for further study.

Antimicrobial susceptibility testing

The minimal inhibitory concentrations (MICs) of 10 antimicrobials were determined via broth microdilution method, including ampicillin, cefepime, ceftazidime, cefotaxime, chloramphenicol, ciprofloxacin, gentamicin, nalidixic acid, tetracycline, and trimethoprim-sulfamethoxazole. All susceptibility results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) interpretive standards.⁶ Isolates with MIC to ceftriaxone or ceftazidime higher than 1 mg/L were further screened for extended-spectrum β-lactamase (ESBL) production by determination of synergy between 0.25 and 128 mg/L ceftazidime or cefotaxime and 4 mg/L clavulanic acid. Isolates showing a ≥8-fold concentration decrease in an MIC for either ceftazidime or cefotaxime tested in combination with clavulanic acid versus its MIC tested alone were considered as producing ESBL. All ESBL-producing isolates were counted as resistant to cefepime, ceftazime, and cefotaxime as recommended by the CLSI standards.⁶ E. coli ATCC 25922, E. coli ATCC 35218, and K. pneumoniae ATCC 700603 were used as quality control organisms in antimicrobial susceptibility experiments.

Polymerase chain reaction amplification and DNA sequence analysis

Phylogenetic analysis of E. coli isolates was determined through the presence or absence of chuA, yjaA, and TspE4.C2 as previously described.⁵ The quinolone resistance determining regions (QRDRs) of gyrA and parC in nalidixic or ciprofloxacin-resistant E. coli isolates were amplified by polymerase chain reaction (PCR).¹⁰ All 92 isolates were screened by PCR targeting the class 1 integrase encoding gene intI1¹⁵ and transferable quinolone resistance determinants, including qnrA, qnrB, qnrS, aac-(6′)-Ib, and qepA.^22,24,30 A multiplex PCR method was applied to screen for bla_CTX-M in ESBL producing isolates,²⁹ and the sequences of bla_CTX-M like genes were analyzed as previously described.⁹ All PCR products were either directly sequenced or cloned into pMD18-T plasmid (Takara Biotechnology Cooperation, Dalian, China) for sequence analysis at Takara Biotechnology Cooperation. The sequences obtained were analyzed by Sequencher 4.6 software (Gene Codes Corporation, Ann Arbor, MI). The search for homologous sequences was performed by using the BLASTN program at the U.S. National Center for Biotechnology Information Web site (www.ncbi.nlm.nih.gov/BLAST/). A new nucleotide bla_CTX-M-like sequence identified in this article has been submitted to the National Center for Biotechnology Information Data Libraries (GenBank) and the Lahey Clinic (www.lahey.org/Studies/). The predicted amino acid sequences of GyrA and ParC were analyzed for amino acid changes by comparison with wild-type GyrA (GenBank accession number NP_416734) and ParC (GenBank accession number NP_417491) of E. coli K-12 (GenBank accession number NC_000913). The qnrB and qnrS allele numbers were designated based on the qnr gene nomenclature.

Results

E. coli isolation and phylogenetic analysis

In total, 92 E. coli isolates were recovered from 103 healthy food handlers. Eleven isolates belonged to phylogenetic group A, 44 isolates belonged to group B2, 37 isolates belonged to group D, and no isolates belonged group B1.

Antimicrobial susceptibility of E. coli isolates

Among 92 E. coli isolates, 17 (18.5%) isolates were susceptible to all tested antimicrobials according to the CLSI interpretive standards. Resistance to tetracycline was the most common (62/92, 67.4%), followed by nalidixic acid (60/92, 65.2%), trimethoprim-sulfamethoxazole (46/92, 50%), and ampicillin (44/92, 47.8%). Resistance prevalence in the B2 phylogenetic group was significantly lower than that in the non-B2 groups for all tested antimicrobials (p < 0.05) except chloramphenicol and tetracycline. Seven isolates (7.6%) of phylogenetic group A (n = 3) and D (n = 4) produced ESBLs and 13 isolates (14.0%) of phylogenetic group A (n = 5), B2 (n = 3) and D (n = 5) were resistant to ciprofloxacin (Table 1).

Table 1.

Resistance Phenotypes of Escherichia coli Isolates Recovered from Stool Samples of Healthy Foodhandlers in Beijing, China

	No. of resistant isolates
Antimicrobial agents	MIC, mg/L	A group (N = 11)	B2 group (N = 44)	D group (N = 37)	Total (N = 92)
Ampicillin	≥32	9 (81.8%)	15 (34.1%)	20 (54.1%)	44 (47.8%)
Cefepime	≥32	3 (27.3%)	0 (0%)	4 (10.8%)	7 (7.6%)
Ceftazidime	≥32	3 (27.3%)	0 (0%)	4 (10.8%)	7 (7.6%)
Ceftriaxone	≥64	3 (27.3%)	0 (0%)	4 (10.8%)	7 (7.6%)
Nalidixic acid	≥32	11 (100%)	24 (54.5%)	25 (67.6%)	60 (65.2%)
Ciprofloxacin	≥4	5 (45.5%)	3 (6.8%)	5 (13.5%)	13 (14.1%)
Chloramphenicol	≥32	2 (18.2%)	2 (4.5%)	4 (10.8%)	8 (8.7%)
Gentamicin	≥16	4 (36.4%)	5 (11.4%)	10 (27.0%)	19 (20.7%)
Trimethoprim–sulfamethoxazole	≥4/76	9 (81.8%)	17 (38.6%)	20 (54.1%)	46 (50%)
Tetracycline	≥16	10 (90.9%)	27 (61.4%)	25 (67.6%)	62 (67.4%)

MIC, minimal inhibitory concentration.

Among 92 E. coli isolates, 47 isolates showed multidrug-resistant profiles (resistant to more than three categories of antimicrobials), including 9 isolates of phylogenetic group A, 17 isolates of group B2, and 21 isolates of group D. Multidrug-resistant profiles AMP-GEN-NAL-SXT-TET (n = 11) and Amp-Nal-Tet (n = 10) were the most common. All ciprofloxacin-resistant isolates were multidrug resistant. Five ESBLs producing isolates of group A (n = 3) and D (n = 2) showed resistance to all tested non-β-lactam antimicrobials (Table 2).

Table 2.

Multidrug-Resistant Profiles of Escherichia coli Isolates Recovered from Stool Samples of Healthy Foodhandlers in Beijing, China

	No. of resistant isolates
Resistant profiles	A (N = 11)	B2 (N = 44)	D (N = 37)	Total
AMP-CAZ-CHL-CIP-CTX-FEP-GEN-NAL-SXT-TET	3	0	2	5
AMP-CAZ-CTX-FEP-NAL-SXT-TET	0	0	2	2
AMP-CHL-CIP-GEN-NAL-SXT-TET	1	0	0	1
AMP-CHL-CIP-NAL-SXT	1	1	0	2
AMP-CHL-CIP-NAL-SXT-TET	0	0	2	2
AMP-CHL-NAL-STX-TET	0	0	1	1
AMP-CIP-GEN-NAL-SXT-TET	0	0	1	1
AMP-GEN-NAL-SXT-TET	0	6	6	12
AMP-GEN-SXT	0	0	1	1
AMP-NAL-SXT-TET	2	0	2	4
AMP-NAL-TET	2	6	2	10
CHL-NAL-SXT-TET	0	1	0	1
CIP-NAL-SXT-TET	0	1	0	1
NAL-SXT-TET	0	2	2	4
Total	9	17	21	47

AMP, ampicillin; CAZ, ceftazidime; CHL, chloramphenicol; CIP, ciprofloxacin; CTX, cefotaxime; FEP, cefepime; GEN, gentamicin; NAL, nalidixic acid; TET, tetracycline; SXT, trimethoprim/sulfamethoxazole.

Antimicrobial-resistant mechanisms analysis

Gene intI1 was identified in 46 isolates of phylogenetic group A (N = 9), B2 (N = 17) and D (N = 20). Of these 46 isolates, 39 isolates were multidrug resistant. The percentages of resistance detected were as the followings (% among intI1-positive/% among intI1-negative isolates): ampicillin (76/20), cefepime (15/0), ceftazidime (15/0), cefotaxime(15/0), chloramphenicol (13/0), gentamicin (41/0), nalidixic acid (80/50), ciprofloxacin (28/0), tetracycline (87/46), and trimethoprim-sulfamethoxazole (85/13). The resistance prevalence of all tested antimicrobials between intI1-positive and intI1-negative isolates was highly significant (p < 0.01). After PCR screening and sequence analysis, transferable quinolone resistance determinants were identified in 4 out of 92 E. coli isolates as qepA1 in 2 isolates and qnrS1 or qnrB6 in another 2 isolates. Point mutations in QRDRs of gyrA or parC were identified among 59 out of 62 E. coli isolates with decreased susceptibility (≥0.06 mg/L) or resistance to ciprofloxacin. A single GyrA mutation was found in 32 isolates (S83L 29 isolates, D87Y 2 isolates, and S83A 1 isolate) and double GyrA mutations were found in 13 isolates (S83F and D87N 12 isolates, S83F, and D87I 1 isolate). Twelve ciprofloxacin-resistant isolates accumulated three point mutations as GyrA (S83L and D87N) and ParC (S80I). One ciprofloxacin-resistant E. coli isolate harbored four point mutations: GyrA (S83L, D87N) and ParC (S80I, E84G). Genes encoding CTX-M enzyme were identified in seven ESBL producing isolates, including bla_CTX-M-14 (n = 5), bla_CTX-M-79 (n = 1), and bla_CTX-M-14-like gene (n = 1), which has two new point mutations (G830A and R277H) compared to bla_CTX-M-14. This new bla_CTX-M-14-like gene has been assigned accession numbers in the GenBank and the Lahey Clinic as the following: HQ913565 (CTX-M-106) (Table 3).

Table 3.

Characteristics of Extended-Spectrum β-Lactamase Producing and Ciprofloxacin-Resistant Escherichia coli Isolates Recovered from Stool Samples of Healthy Foodhandlers in Beijing, China

Strain no.	Antibiotic-resistance profiles	NAL MIC	CIP MIC	Phylogenetic groups	bla_CTX-M	QRDR mutations	PMQR	IntI1
E49	AMP-CAZ-CTX-FEP-NAL-SXT-TET	32	0.125	D	CTX-M-106	D87Y		P
E51	AMP-CAZ-CTX-FEP-NAL-SXT-TET	32	0.125	D	CTX-M14	D87Y		P
E125	AMP-CAZ-CHL-CIP-CTX-FEP-GEN-NAL-SXT-TET	>512	>4	D	CTX-M14	GyrA (S83L, D87N), ParC (S80I, E84G)		P
E35	AMP-CAZ-CHL-CIP-CTX-FEP-GEN-NAL-SXT-TET	>512	>4	D	CTX-M79	GyrA (S83L, D87N), ParC (S80I)	qepA1	P
E40	AMP-CAZ-CHL-CIP-CTX-FEP-GEN-NAL-SXT-TET	>512	>4	A	CTX-M14	GyrA (S83L, D87N), ParC (S80I)		P
E50	AMP-CAZ-CHL-CIP-CTX-FEP-GEN-NAL-SXT-TET	>512	>4	A	CTX-M14	GyrA (S83L, D87N), ParC (S80I)		P
E64	AMP-CAZ-CHL-CIP-CTX-FEP-GEN-NAL-SXT-TET	>512	>4	A	CTX-M14	GyrA (S83L, D87N), ParC (S80I)		P
E193	AMP-CHL-NAL-STX-TET	128	0.25	D		NO	qepA1	P
E227	-	16	0.25	D		NO	qnrB6	n
E32	AMP-CHL-CIP-NAL-SXT-TET	>512	>4	D		GyrA (S83L, D87N), ParC (S80I)		P
E96	AMP-CIP-GEN-NAL-SXT-TET	>512	>4	D		GyrA (S83L, D87N), ParC (S80I)		P
E395	AMP-CHL-CIP-NAL-SXT-TET	>512	>4	D		GyrA (S83L, D87N), ParC (S80I)		P
E52	AMP-CHL-CIP-NAL-SXT	>512	>4	A		GyrA (S83L, D87N), ParC (S80I)		P
E459	AMP-CHL-CIP-GEN-NAL-SXT-TET	>512	>4	A		GyrA (S83L, D87N), ParC (S80I)		P
E55	AMP-CHL-CIP-NAL-SXT	>512	>4	B2		GyrA (S83L, D87N), ParC (S80I)	qnrS1	P
E376	AMP-CIP-NAL-SXT-TET	>512	>4	B2		GyrA (S83L, D87N), ParC (S80I)		P
E394	CIP-NAL-SXT-TET	>512	>4	B2		GyrA (S83L, D87N), ParC (S80I)		P

QRDR, quinolone resistance determining regions; PMQR, plasmid-mediated quinolone-resistant mechanisms; p, positive; n, negative.

Discussion

Since different E. coli clones exist in the intestinal tract,¹ isolation method is an important factor affecting the types of E. coli isolates recovered from fecal samples. In this study, bacteria in fecal samples were streaked on a nonselective indicator medium (SSI) that facilitated the recovery of dominant E. coli isolates in each sample. As a result, more than 50% (47/92) of the isolates were multidrug resistant and 50% (46/92) of the isolates harbored integrase I that were much higher than the prevalence in other studies of healthy humans.^8,25,27 If appropriate antimicrobials were added in the isolation media as in a previous study,¹⁹ higher prevalence of resistant isolates should be expected. These data illustrated that fecal bacteria from healthy hospital food handlers could serve as important reservoirs of multidrug-resistant isolates. The following reasons might be the causes of high prevalence of resistant bacteria among the food handlers in this study. First, hospital is a place consuming large volume of various antimicrobials and colonized by various multidrug-resistant bacteria in its environment, especially a large hospital in this study. Healthy humans working in this environment, such as food handlers might have more chances to be colonized by drug-resistant bacteria than people in the community. Second, food handlers who processed large volume of raw animal food products each day were exposed to high risks of multidrug-resistant E. coli colonization of animal sources. Plenty of studies have shown that food-producing animals were important reservoirs of multidrug-resistant bacteria, and these isolates could transmit to humans through food contact.^17,18,20 Third, the population exposure to antimicrobial use was increasing sharply in the last 10 years worldwide. Especially in clinics, multidrug-resistant bacteria have become more and more popular among nosocomial infections.^3,26 It is interesting to conduct a case–control study to investigate the prevalence of resistant isolates among healthy community residents and cooks in the community and hospitals.

As we know, fluoroquinolones and third-generation cephalosprins were two popular empirical options to treat severe gastrointestinal infections caused by pathogenic bacteria.¹¹ In this study, 7 E. coli isolates were found to carry ESBLs encoding genes, 13 isolates were resistant to ciprofloxacin and 4 isolates harbored plasmid-mediated quinolone-resistant determinants. Since ESBLs encoding genes and plasmid-mediated quinolone-resistant determinants were usually carried by conjugatable plasmids,^19,31 these resistant genes might transmit to other pathogens, such as Salmonella, pathogenic E. coli during a severe infection treatment. The colonization of E. coli isolates carrying these resistant genes among hospital food handlers may endanger the inpatients through food service whose immune system is not as strong as the healthy humans in the community. After sequence analysis, isolates carrying different bla_CTX-M and plasmid mediated quinolone-resistant determinants were found which illustrated these genes were introduced into these food handlers at various times and then disseminated among the population by cross-transmission. Especially, bla_CTX-M-14 was also identified among these food handlers, which has also been identified as the dominant ESBL-encoding gene from both the human clinical and animal samples in China.^17,21 It is necessary to monitor the prevalence and sources of these resistant bacteria and develop proper prophylaxis programs.

Among 92 E. coli isolates in this study, no phylogenetic group B1 isolates were identified. Studies have reported similar phylogenic group distribution patterns among healthy humans, which has also been discussed in a previous report.⁴ This substantial under-representation of the B1 type might be related to the characteristics of this population or could also simply be a reflection of the enormous overall diversity in the E. coli species pool. Besides the lack of B1 type isolates, drug-resistant isolates were significantly more prevalent in the non-B2 groups than in the B2 phylogenetic group (p < 0.05). Previous study has found similar trend that isolates from the B2 phylogenetic group appeared to be the least resistant to antibiotics.¹⁴ These findings indicated a close relationship of E. coli resistance prevalence and phylogenetic groups.

Integrons play an important role in antibiotic resistance determinants dissemination because they are able to capture, integrate, and express gene cassettes encoding antibiotic resistance.¹² Integrase is the core enzyme to catalyze the site-specific recombination, which is a good indictor for the presence of integron.⁷ In this study, a high prevalence of intI (46/92) was detected in fecal E. coli of healthy humans, which illustrated that food handlers could be an important reservoir of integrons. Higher resistance prevalence to all tested antimicrobials was observed among integron-positive isolates with respect to integron-negative isolates (p < 0.01). This fact could be explained by the presence of resistance genes in the conserved or variable region of integrons (as is the case for genes associated with sulfamethoxazole, trimethoprim resistance) or by the inclusion of resistance genes in the same mobile elements that carried integrons.¹⁶ Especially, 100% correlation of ciprofloxacin or third-generation cephalosprin resistance to the identification of int1 further emphasized the transfer of integron-carrying elements played a dominant role in the development of multidrug resistance in Enterobacteriaceae. Besides these, highly significant difference was observed for the prevalence of intI between phylogenetic group B2 and non-B2 group, which further supported the lower prevalence of antimicrobial-resistant isolates in B2 group.

In summary, E. coli isolates from healthy hospital food handlers were shown to harbor antimicrobial resistance genes, especially resistance to third-generation cephalosporins or ciprofloxacin. The preponderance in hospital food handlers of multidrug-resistant E. coli makes it important to introduce control measures such as improved biosecurity to ensure that they do not pass through the food service and limit inpatient therapeutic options.

Funding

This research was supported by grant (2009BADB9B01) from the Ministry of Science and Technology and grant (30972487) from the National Natural Science Foundation of China.

Disclosure Statement

No competing financial interests exist.

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