Comparative Analysis of Antimicrobial Resistance,Integrons,and Virulence Genes Among Extended-Spectrum β-Lactamase-Positive Laribacter hongkongensis from Edible Frogs and Freshwater Fish

Abstract

Aquatic animals are now recognized to be major hosts of potentially pathogenic Laribacter hongkongensis. A comparative study was carried out among extended-spectrum β-lactamase (ESBL)-producing L. hongkongensis isolated from frogs (47 isolates) and fish (41 isolates) to examine phenotypic and genotypic antimicrobial resistance profiles, integrons, virulence factors, and genetic relatedness. Isolates from frogs showed a higher incidence of antibiotic resistance compared with those from fish for most of the antimicrobials tested, especially trimethoprim–sulfamethoxazole, tetracycline, ciprofloxacin, levofloxacin, and streptomycin. Multidrug-resistant strains were also found more frequently among frog isolates (5.44 traits on average) than among fish isolates (3.29 traits). In frog isolates, class 1 integrons and the resistance genes sul1, sul2, tetA, tetR, and aac(6′)-Ib-cr showed a clearly higher incidence compared with isolates from fish. In contrast, bla_TEM-1 was higher in fish isolates than in frog isolates. Correlation analysis showed that sul1, sul2, tetA, and tetR were significantly associated with class 1 integrons in frog isolates. The correlations indicated a potential co-selection risk of bacterial resistance to antibiotics. In addition, the distribution of three virulence-associated determinants for the type IV bundle-forming pili gene (bfpA), ferric aerobactin receptor gene (iucD), and iron-responsive element gene (ireA) was markedly higher in strains isolated from frogs than in those isolated from fish. No obvious genetic relatedness was observed between both populations. The large differences found in the incidence of antibiotic resistance, integrons along with the multiple resistance genes, virulence factors, and genetic fingerprints determined by pulsed-field gel electrophoresis suggest a high degree of antibiotic resistance and pathogenicity potential of ESBL-producing L. hongkongensis from isolates found in frogs.

Introduction

Laribacter hongkongensis (L. hongkongensis) is a gram-negative foodborne organism that is associated with gastroenteritis and diarrhea in humans.^1,2 Since its first discovery in the stool of six patients with diarrhea in Hong Kong,³ this bacterium has been discovered in the gut of diverse aquatic animals and drinking water reservoirs to date.^4–6 Freshwater fish and Chinese tiger frogs have been recognized to be the primary hosts for L. hongkongensis and the sources of human infection.^7,8 Interestingly, previous studies have revealed that frog isolates exhibited higher genetic diversity than those of fish and showed a significant genetic correlation and similar resistance phenotypes with humans,^4,9,10 indicating that some frog L. hongkongensis isolates might be more virulent and adapted to humans than others.

Antimicrobial resistance in pathogenic bacteria has been a major threat to medical practice and public health, and genes that mediate resistance are rapidly evolving and diversifying.¹¹ The prevalence of extended-spectrum β-lactamase (ESBL)-producing gastroenteritis-causing pathogens in food-producing animals and edible animal products is also increasing worldwide.¹² ESBL producers, particularly those with resistance to other antimicrobial agents, such as fluoroquinolones, aminoglycosides, and sulfonamides, are often associated with treatment failures.¹³

Integrons are genetic elements that harbor various antibiotic resistance genes and are frequently associated with the development of multidrug resistance in gram-negative bacteria.¹⁴ Class 1 integrons are the most prevalent and have been found to be the primary contributors to the dissemination of antimicrobial-resistant genes.¹⁵

Importantly, the presence of various virulence-associated genes in ESBL producers may increase the pathogenicity and complicate the therapeutic strategy.¹⁶ The complete genome analysis of an isolate from one patient indicates that the virulence of L. hongkongensis may be multifactorial.¹⁷ Virulence strategies involved in autoaggregation, biofilm formation, invasion, and other virulence-encoding genes, such as collagenases, cytotoxins, hemolysins, RTX toxins, lipopolysaccharides, patatin-like proteins, and phospholipase A1, have also been identified in L. hongkongensis.¹⁷ Nevertheless, information regarding the virulence profile of L. hongkongensis is still limited to date.

Recently, we found an alarmingly high rate of ESBL L. hongkongensis isolates in food animals randomly collected from Shenzhen (75.8% in frogs; 89.1% in fish). Shenzhen is located in the southern portion of Guangdong Province, which is the largest center for aquaculture in China. More than 70% of the freshwater fish for sale in Hong Kong are imported from southern China.⁶ Therefore, understanding the prevalence of antimicrobial resistance and the pathogenicity of ESBL-producing L. hongkongensis from aquatic animals is critical from the public health perspectives.

In this study, we compared the distribution of integrons, resistance genes, antimicrobial resistance profiles, and virulence-associated genes among ESBL-producing L. hongkongensis strains from frogs and fish and specifically analyzed the relationship between antimicrobial resistance and integrons.

Materials and Methods

Bacterial strains

A total of 108 L. hongkongensis isolates were selected for this study, including 62 from Chinese tiger frogs and 46 from grass carps. These strains were randomly isolated from five local markets in the city of Shenzhen, China, from April 2015 to October 2015. All the isolates were identified by using a 16S rRNA gene-based PCR assay in combination with a series of biochemical tests.¹⁸ The biochemical tests of putative L. hongkongensis isolates indicated that the isolates reacted with arginine, catalase, cytochrome oxidase, dihydrolase, and urease and were only slightly positive for sugar oxidation–fermentation. The details of the isolations are summarized in Supplementary Table S1.

Phenotypic detection of ESBL production and antimicrobial resistance

The susceptibility of L. hongkongensis to 25 antimicrobial agents, including ampicillin (AMP), piperacillin (PRL), piperacillin–tazobactam (TZP), cefazolin (CFA), cephalothin (CEP), cefoperazone (CFP), cefotaxime (CTX), ceftriaxone (CRO), ceftazidime (CAZ), cefoxitin (FOX), cefuroxime (CXM), cefepime (CEF), aztreonam (ATM), imipenem (IPM), amikacin (AMK), gentamicin (GEN), streptomycin (STR), tetracycline (TET), ciprofloxacin (CIP), levofloxacin (LEV), trimethoprim–sulfamethoxazole (SXT), chloramphenicol (CHL), erythromycin (ERY), rifampicin (RIF), and polymyxin (PB), was tested using the disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines for Enterobacteriaceae (except for erythromycin and rifampicin) and Staphylococcus spp. (for erythromycin and rifampicin).¹⁹

After first screening for the phenotypic identification of ESBL producers by the disk diffusion method using cefotaxime, ceftazidime, ceftriaxone, and aztreonam, the isolates with reduced susceptibility to any of these drugs were screened again by the double-disk synergy test for further phenotypic confirmation.¹⁹ The test was recorded as positive when the zone of inhibition of cefotaxime plus clavulanic acid or ceftazidime plus clavulanic acid was ≥5 mm larger than their respective single disks. Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 25923, and Klebsiella pneumoniae ATCC 700603 were used as controls. Multidrug resistance (MDR) was defined as the resistance to three or more different antibiotic classes.

Detection of antimicrobial resistance genes, class 1 integrons, and gene cassettes

The total DNA of 88 strains was extracted by using a rapid boiling method. The genes encoding TEM, CTX-M-1, CTX-M-9, SHV, and OXA-1 type β-lactamases were screened by PCR.²⁰ Additionally, all isolates were subjected to the detection of other genes that confer resistance to sulfonamide (sul1, sul2, and sul3), tetracycline (tetA, tetB, tetD, tetE, tetK, tetL, tetR, and tetX), and fluoroquinolones [qnrA, qnrB, qnrC, qnrD, qnrS, qepA, and aac(6’)-Ib-cr].^21–25 PCR amplification was performed to screen for the presence of class 1 integrons; the variable regions of the class 1 integrons were amplified and further sequenced according to a previous report.²⁶

Virulence genotyping

Each strain was analyzed for the presence of 10 virulence genes (Table 3), including toxin genes (hlyA, stx1, and stx2), genes encoding protectins (traT and iss), siderophore-related genes (iucD and ireA), and genes associated with bacterial adhesion (fimH, bfpA, and eaeA), as described elsewhere.^27,28

Pulsed-field gel electrophoresis analysis

Genetic relatedness among the ESBL-producing strains was assessed by the use of pulsed-field gel electrophoresis (PFGE) after the digestion of DNA with XbaI and following the procedures as previously documented.⁴ Salmonella enterica serovar Braenderup H9812 was used as the molecular size marker in the PFGE experiment.²⁹ The PFGE profiles were analyzed using BioNumerics software (Version 7.6; Applied Math, Austin, TX).

Statistical analysis

Statistical analysis and the comparison of proportions were performed using chi-square test or Fisher's exact test (SPSS software, version 20.0). p-Values of <0.05 were considered significant.

Results

Occurrence of ESBL-producing L. hongkongensis isolates and antimicrobial resistance

Of the 108 L. hongkongensis isolates tested, 47/62 (75.8%) frog and 41/46 (89.1%) fish isolates were confirmed as ESBL producers. All the 88 ESBL-producing L. hongkongensis isolates were characterized for their phenotypes of antimicrobial resistance to 25 commonly used antimicrobials by the disk diffusion method. The comparison of resistance to the different antimicrobials among the L. hongkongensis isolates from frogs and fish is presented in Fig. 1.

FIG. 1.

Antimicrobial susceptibility of the 88 ESBL-producing Laribacter hongkongensis strains. AMP, ampicillin; PRL, piperacillin; TZP, piperacillin–tazobactam; CFA, cefazolin; CEP, cephalothin; CFP, cefoperazone; CTX, cefotaxime; CRO, ceftriaxone; CAZ, ceftazidime; FOX, cefoxitin; CXM, cefuroxime; CEF, cefepime; ATM, aztreonam; IPM, imipenem; AMK, amikacin; GEN, gentamicin; STR, streptomycin; TET, tetracycline; CIP, ciprofloxacin; LEV, levofloxacin; SXT, trimethoprim–sulfamethoxazole; CHL, chloramphenicol; ERY, erythromycin; RIF, rifampicin; PB, polymyxin; ESBL, extended-spectrum β-lactamase. Frog group represents resistance rate from frogs higher than that of fish; fish group represents resistance rate from fish higher than that of frogs.

The frog isolates were resistant to 19 of the 25 antimicrobials tested to different degrees, with a percentage of resistance to cephalosporins (CTX, CRO, CEP, CFP, CAZ, CFZ, and FOX), rifampicin, tetracycline, and sulfonamides of more than 50%. However, fish isolates exhibited resistance to 13 of the 25 tested antimicrobial agents, with a percentage of resistance to cephalosporins (CTX, CRO, CEF, CFP, CAZ, and CFZ) and rifampicin of more than 50%.

The low incidence of antimicrobial resistance to cefepime, chloramphenicol, erythromycin, piperacillin, and streptomycin was observed in both frog and fish isolates. Notably, the resistant rates of five antibiotics (trimethoprim–sulfamethoxazole, tetracycline, ciprofloxacin, levofloxacin, and streptomycin) were significantly higher for the isolates from frogs than for the isolates from fish (p < 0.05). In contrast, only two antibiotics (aztreonam and cefoperazone) showed significantly higher rates of resistance for the isolates from fish than for the isolates from frogs.

Of the 88 strains, multiresistance strains were also more frequent among the frog isolates than among the fish isolates (74.5% vs. 53.7%, p < 0.05). Remarkably, multiresistance frog isolates showed a higher average number of antibiotic resistance traits (5.44 traits) than did fish isolates with an average of 3.29 traits per strain. All 35 multiresistance frog isolates were grouped into 33 different phenotypes, and the most frequent multiresistance profile was CFP/CEP/CFZ/CTX/CAZ/CRO/FOX/RIF/TET/SXT, whereas there were 15 different multiresistance patterns observed among all 22 MDR isolates. The dominant multiresistance profile was AMP/CFP/CEP/CFZ/CTX/CAZ/CRO/FOX/RIF.

Distribution of resistance genes

A total of 23 genes conferring resistance against antimicrobials belonging to four antimicrobial families (beta-lactams, sulfonamides, tetracycline, and fluoroquinolones) were chosen to determine their distributions in frog and fish ESBL-producing L. hongkongensis isolates (Table 1). PCR and sequencing revealed that all frog and fish strains were positive for at least one of the main β-lactamase gene groups. Of these, bla_CTX-M-9 was predominant, being present in 40 (85.1%) of the frog isolates and 40 (97.6%) of the fish isolates. The bla_TEM-1 gene was positive in more fish isolates (87.8%) than in frog isolates (63.8%). One fish isolate but no frog isolate was bla_SHV-1 positive, and this isolate simultaneously contained bla_CTX-M-9 and bla_TEM-1. However, none of the isolates contained the bla_CTX-M-1 and bla_OXA1 genes.

Table 1.

Distribution of Resistance Genes and Integrons Among Extended-Spectrum β-Lactamase-Producing Laribacter hongkongensis Isolates from Frogs and Fish

Variable	No. (%) of isolates			p
Variable	Total (n = 88)	Frog (n = 47)	Fish (n = 41)	p
Beta-lactams
TEM-1	66 (75.0)	30 (63.8)	36 (87.8)	0.013
CTX-M-9	80 (90.9)	40 (85.1)	40 (97.6)	0.063
SHV-1	1 (1.1)	0 (0)	1 (2.4)	0.466
Tetracycline
tetA	23 (26.1)	19 (40.4)	4 (9.8)	0.001
tetR	24 (27.3)	20 (42.6)	4 (9.8)	0.001
Sulfonamides
sul1	30 (34.1)	26 (55.3)	4 (9.8)	0.000
sul2	19 (21.6)	19 (40.4)	0 (0)	0.000
Fluoroquinolones
aac(6′)-Ib-cr	9 (10.2)	8 (17.0)	1 (2.4)	0.033
Integrons
class 1	26 (29.5)	20 (42.6)	6 (14.6)	0.004

Boldface data indicate statistical significance.

Of the three sulfonamide resistance genes tested, sul3 was not detected in any of the isolates tested. The sul1 gene was detected more frequently in the frog isolates (55.3%) than in the fish isolates (9.8%). Nineteen frog isolates were positive for the sul2 gene, whereas none of the fish isolates possessed the sul2 gene. Seventeen frog isolates coharbored the sul1 and sul2 genes.

Among the various tetracycline resistance genes screened, only tetA and tetR were found in both groups of isolates. The two genes had a similarly high prevalence among the frog isolates (tetA in 40.4% and tetR in 42.6%, respectively) but showed a lower incidence among the fish isolates (tetA in 9.8% and tetR in 9.8%, respectively). The tetA/tetR gene clusters were found in 18 frog isolates and 4 fish isolates, respectively.

Of the seven fluoroquinolone resistance genes detected, only aac(6′)-Ib-cr was found in eight isolates from frogs and one isolate from fish.

Characterization of integrons and their relationship with antibiotic resistance

The prevalence of class 1 integrons in the ESBL-producing strains from frogs and fish was analyzed by PCR targeted to the intI1 gene. Of the 88 isolates, 26 (25.9%) contained a class 1 integron. The proportion of class 1 integron-positive strains was higher in the frog isolates than in the fish isolates (42.6% vs. 14.6%, p = 0.004, Table 1).

The variable region of the class 1 integron-positive isolates was amplified using primers 5′-CS/3′-CS. All the 20 class 1 integron-positive frog isolates and 2 of the 6 class 1 integron-positive fish strains were positive for the variable region, with the inserted gene cassette sizes varying from 0.8 to 2.0 kb, whereas the variable region was not amplified from the remaining 4 class 1 integron-positive fish strains. Five different gene cassettes that confer resistance to trimethoprim (dfrA1 and dfrA27), streptomycin or spectinomycin (aadA1), kanamycin and gentamicin (aacA4), and rifampicin (arr3) were detected among the 20 frog isolates. The different cassette combinations are described in Table 2. In comparison, only one kind of gene cassette (dfrA1) was found in the two of integron-positive fish isolates.

Table 2.

Characterization of Extended-Spectrum β-Lactamase-Producing Strains Harboring Class 1 Integron from Frogs and Fish

Source	No. of isolates with this type of integron	Gene cassettes arrays	Resistance phenotype (no. of isolates)		sul genotype (no. of isolates)			tet genotype (no. of isolates)
Source	No. of isolates with this type of integron	Gene cassettes arrays	SXT	TET	sul1	sul2	sul1+sul2	tetA	tetR	tetA+tetR
Frog
	11	dfrA1-orfC	11	9	1	1	8	1	0	8
	3	dfrA1-aadA1	3	1	2	0	1	0	0	1
	3	dfrA1-arr3	3	3	0	0	3	0	0	3
	2	dfrA1-aacA4	2	1	0	0	2	0	0	1
	1	dfrA27-arr3	1	1	0	0	1	0	0	1
Total	20		20	15	3	1	15	1	0	14
Fish
	2	dfrA1-orfC	0	0	0	0	0	0	0	0
	4	Unknown	0	0	1	0	0	0	0	0
Total	6		0	0	1	0	0	0	0	0

SXT, trimethoprim–sulfamethoxazole; TET, tetracycline.

A statistically significant (p < 0.05) association was detected between the incidence of antimicrobial resistance (genes) and class 1 integrons among the collection of frog ESBL-producing L. hongkongensis isolates. As expected, all the frog isolates harboring class 1 integrons showed an MDR phenotype. The resistance to trimethoprim–sulfamethoxazole and tetracycline was significantly associated with the presence of integrons. Moreover, the associations between the class 1 integrons and the resistance genes sul1, sul2, tetA, and tetR were clear, as described in Table 2.

Virulence genotyping and combination patterns

Table 3 summarizes the incidence of various virulence genes among the ESBL-producing L. hongkongensis isolates. In total, genes that encode virulence determinants related to tissue invasion, such as protectins (traT and iss) and siderophore-related proteins (ireA), as well as adhesin-related proteins (fimH and bfpA), were commonly found among the strains we analyzed, whereas the toxin genes, such as stx1 and stx2, were not detected in any of the strains. Only a small number of the strains harbored the hlyA gene. Similarly, iucD and eaeA were present in fewer than 10% of the isolates.

The two most prevalent virulence genes from both frog and fish isolates were fimH and traT, which ranged from 93.6% to 100%, followed by iss, which ranged from 53.2% to 73.2%. The hlyA and eaeA genes were observed among 2 and 1 frog isolates, respectively, whereas no fish isolate harbored either gene. Additionally, a significantly higher prevalence of three virulence genes (bfpA, ireA, and iucD) was observed among the frog isolates than among the fish isolates.

Table 3.

Distribution of Virulence-Associated Genes Between Frog and Fish Extended-Spectrum β-Lactamase-Producing Laribacter hongkongensis Isolates

Category	Genes	Description/function	No. (%) of strains			p
Category	Genes	Description/function	Total (n = 88)	Frog (n = 47)	Fish (n = 41)	p
Adhesins	fimH	d-Mannose-specific adhesin, type 1 fimbria	88 (100)	47 (100)	41 (100)	1.000
	bfpA	Type IV bundle-forming pili	26 (29.5)	19 (40.4)	7 (17.1)	0.020
	eaeA	Outer membrane protein intimin	1 (1.1)	1 (2.1)	0 (0)	1.000
Toxins	hlyA	α-Hemolysin	2 (2.3)	2 (4.3)	0 (0)	0.497
	stx1	Shiga toxin I	0 (0)	0 (0)	0 (0)	—
	stx2	Shiga toxin II	0 (0)	0 (0)	0 (0)	—
Siderophores	iucD	Ferric aerobactin receptor (iron uptake, transport)	7 (8.0)	6 (12.8)	1 (2.4)	0.028
	ireA	Iron-responsive element, a siderophore receptor	21 (23.9)	16 (34.0)	5 (12.2)	0.023
Protectins	traT	Surface exclusion, serum survival	84 (95.5)	44 (93.6)	40 (97.6)	0.620
	iss	Serum survival gene	55 (62.5)	25 (53.2)	30 (73.2)	0.077

Boldface data indicate statistical significance.

Multiple virulence genes were common and varied between the two sources in this study. All ESBL-producing L. hongkongensis strains but one fish isolate showed the presence of ≥2 of the virulence genes we tested. Twenty virulence gene profiles of the 10 putative virulence genes examined were observed, as shown in Supplementary Table S2. Among the frog strains, 18 distinct combination patterns were found, and each included only a few strains. The four most frequent patterns (n ≥ 5) fimH-traT-iss, fimH-traT-iss-bfpA, fimH-traT-bfpA, and fimH-traT-ireA were present in 10, 7, 7, and 6 isolates, respectively. The fish strains exhibited 8 different profiles, and 30 strains belonged to either the fimH-traT-iss (25 isolates) profile or the fimH-traT (5 isolates) profile. In total, these two major profiles accounted for 73.2% of the fish strains.

Pulsed-field gel electrophoresis

Figure 2 shows a dendrogram with the fingerprinting patterns obtained by PFGE of the 88 ESBL-producing L. hongkongensis strains analyzed. The PFGE revealed five clusters (clusters A, B, C, D, and E) with similarities over 65.0%. Clusters D and E included 28 strains that all originated from fish. The remaining 13 fish strains were divided into clusters A (5 strains) and B (8 strains), which also included 7 and 27 frog isolates, respectively. Cluster C grouped 13 strains all from frogs that were mostly positive for class 1 integrons harboring dfrA1 gene cassettes, as well as sul and tet genes. In addition, some identical profiles were observed in clusters B (five sets), C (two sets), D (one set), and E (two sets).

FIG. 2.

PFGE analysis of the 88 ESBL-producing L. hongkongensis strains. ^aGC, grass carp; TF, raising tiger frog. ^bAMP, ampicillin; PRL, piperacillin; TZP, piperacillin–tazobactam; CFA, cefazolin; CEP, cephalothin; CFP, cefoperazone; CTX, cefotaxime; CRO, ceftriaxone; CAZ, ceftazidime; FOX, cefoxitin; CEF, cefepime; ATM, aztreonam;; STR, streptomycin; TET, tetracycline; CIP, ciprofloxacin; LEV, levofloxacin; SXT, trimethoprim-sulfamethoxazole; CHL, chloramphenicol; ERY, erythromycin; RIF, rifampicin.

Discussion

There is the potential for L. hongkongensis strains from aquatic animals to affect humans either by direct transmission or by horizontal exchange of mobile genetic elements harboring various resistance and virulence genes. In this study, ESBL-producing L. hongkongensis isolates from freshwater frogs and fish were characterized for the distributions of antibiotic resistance phenotypes and genotypes, integron profiles, and virulence potentials to obtain insight into the putative risk for human health.

Our study demonstrates that nearly all the ESBL-producing L. hongkongensis isolates exhibited resistance to the 25 antibiotics with different levels. For seven antibiotics (CTX, CRO, CEF, CFP, CAZ, CFZ, and RIF), the resistance rates were more than 50%, indicating that the strains from frogs and fish had a high resistance to antibiotics. This result probably reflects the selective pressures in the aquatic environment exerted by the use of various classes of antibiotics in the prevention and treatment of disease in aquatic animals.³⁰ Not surprisingly, all strains were still susceptible to several antimicrobial agents for which low resistance rates were also reported worldwide,^31,32 such as piperacillin–tazobactam, cefuroxime, imipenem, amikacin, gentamicin, and polymyxin B. These antimicrobial agents may be the preferred choice to treat infections.

Interestingly, frog ESBL-producing strains showed a significantly higher prevalence of resistance for several antibiotics tested and multi-drug resistance than did fish isolates. In addition, the average number of resistance traits per strain was also much higher, and more complex multiresistance profiles were observed in the frog isolates. These results indicate that the frog ESBL producers have acquired a wide range of resistance to antibiotics and MDR compared with those from fish. The highly different resistant patterns found between these two hosts are most likely due to the differences in their aquaculture environments and antibiotic treatment strategies in their rearing practices. Thus, it is necessary to explore the pathogenic features of different hosts, which could help in developing preventative strategies for reducing the incidence of infection.

In the present study, the bla_CTX-M-9 gene was the most prevalent in all the ESBL-producing strains. This is consistent with E. coli isolates from Chinese animal,^33–35 in which the CTX-M-9 group is also the major type and is detected more frequently than the bla_TEM-1 and bla_SHV genes. A significantly higher incidence of the bla_TEM-1 gene was observed among the fish isolates than among the frog isolates. TEM-1, although a non-ESBL enzyme, is not uncommonly found among ESBL producers since several TEM-1 variants could also confer ESBL properties.³⁶

Although the high prevalence of various β-lactamase genes was observed among strains in this study, it is possible that other mechanisms are involved in resistance to β-lactams. A previous study showed that the chromosomal AmpC gene of L. hongkongensis was generally resistant to most β-lactams except the carbapenems,⁷ and the AmpC genes were also identified in all strains in the present study (data not shown). Therefore, we should not exclude the possible role of chromosomal AmpC gene of L. hongkongensis in resistance to β-lactams. The exact mechanism of resistance to β-lactams remains to be determined.

This study revealed that 40.4% of frog and 9.8% of fish ESBL-producing strains were positive for the tetA gene, whereas other tet genes were not detected in both sources. Previous studies have also suggested such a prevalence of tetracycline resistance in isolates of human and fish origin.³⁷ These tetA genes were presented not only among tetracycline-resistant isolates but also in the six sulfonamide- and tetracycline-susceptible isolates. Additionally, 95.7% of the tetA-positive strains also concurrently contained the tetR gene, which is a tetracycline repressor gene that regulates the expression of tet genes.³⁸

The sul1 and sul2 genes were detected in 55.3% and 40.4%, respectively, of the frog ESBL producers. Seventeen strains were positive for both of the two genes encoding sulfonamide resistance, whereas four sulfonamide-susceptible fish strains were sul1-positive. This trend was also observed in other reports that indicated that sul1 is the most common mechanism of resistance to sulfonamides, followed by the sul2 gene, which seems to have increased in some clinical isolates of E. coli.^23,39–41 The existence of several sulfonamide-resistant genes could be the consequence of the constant pressure generated by sulfonamides and other commonly used antibiotics.⁴²

In the present study, except for the eight frog and one fish ESBL producers that harbored the aac(6′)-Ib-cr gene, none of the strains was found to be positive for any of the acquired quinolone resistance (AQR) genes, such as qnrA, qnrB, qnrC, qnrD, qnrS, and qepA. Similar to our observations, a previous study also reported that only the aac(6′)-Ib-cr gene was discovered in resistant L. hongkongensis strains.⁴³ Some strains exhibited resistance toward quinolones, although they did not harbor any of the AQR determinants. Such resistance could be conferred via chromosomal mutations in quinolone resistance-determining regions.^44,45

Resistance genes can be carried on integrons, which facilitate their transmission among various bacterial pathogens.⁴⁶ Class 1 integrons are the most common in bacteria isolated from different environments, and they are generally regarded as effective markers of multidrug resistance.¹⁴ Our study showed that ∼42.6% of the frog ESBL-producing isolates tested contained a class 1 integron, and five different gene cassettes were confirmed in these integron-positive strains. The high occurrence of dfrA and aadA gene cassettes, which confer resistance to sulfamethoxazole and trimethoprim and frequent association with the prevalence of the sul genes, has also been reported by several previous studies.^47,48 These gene cassettes might contribute to sulfonamide resistance among the frog ESBL producers in this study.

In contrast, fish ESBL-producing strains were distinguished by a significantly lower prevalence (14.6%) of class 1 integrons than the frog strains. Only two of the integron-positive strains possessed dfrA gene cassettes. This might suggest that the frequencies of the exchange of antibiotic-resistant gene cassettes in ESBL-producing L. hongkongensis could differ between frogs and fish. A greater diversity of gene cassette types identified in frog strains may raise a major public health concern.

Remarkably, some interesting gene associations were observed between the presence of class 1 integrons and the resistance genes sul1, sul2, tetA, and tetR among the frog ESBL-producing strains. Similar to other studies, the sul1 gene was always associated with class 1 integrons.^42,49 In contrast, sul2 was often detected on a broad range of host plasmids and had no known connection to integrons as previously reported.⁵⁰ Other resistance genes, including tetA and tetR, did not present as gene cassettes on integrons in this study and are mostly transferred in association with plasmids.⁵¹ It is highly likely that these plasmids could also potentially mobilize the integrons harboring other resistance genes. Further studies should be performed to investigate the prevalence of plasmids and the role in the transfer of these resistance genes among the L. hongkongensis strains.

Virulent bacterial pathogens have posed a severe challenge for modern medicine, as these strains harboring virulence genes are often highly resistant to antibiotics and very difficult to treat.⁵² As a potential gastroenteritis-causing bacterium, L. hongkongensis must possess certain genetic determinants to survive in multiple hosts.⁵³ According to the results from our study, most ESBL-producing L. hongkongensis isolates possessed some of the virulence markers commonly known to be related to extraintestinal pathogenic E. coli (ExPEC) strains or/and enteropathogenic E. coli (EPEC) strains.²⁸ This finding cannot be compared, as the incidence of virulence factors of L. hongkongensis in ESBL producers is rarely reported.

The comparison of the two source strains demonstrated a greater variety of virulence genes among the frog ESBL-producing strains, and the average number of virulence factor traits were higher among the frog strains (3.40) than among the fish strains (3.02), suggesting a high virulence potential of the frog strains. The significant differences in the distribution of the three virulence genes represented by bfpA, iucD, and ireA were also found between the frog and fish ESBL-producing strains. The bfpA gene encodes for the production of type IV bundle-forming pili, which can be involved in localized adherence, and is commonly found in EPEC strains.⁵⁴ The iucD gene encodes a ferric aerobactin receptor and is involved in iron uptake and transport.²⁷ Similarly, the ireA gene encodes an iron-responsive element that contributes to iron acquisition.⁵⁵

The diversity of virulence genes present in ESBL-producing strains from the two hosts may be a result of their adaption to given environments, which stimulate the genes to recombine, exchange, and disseminate.^56,57 Nevertheless, a common trend was observed for the genes fimH and traT, which were found in almost all the frog and fish ESBL producers, suggesting that these two traits were probably acquired early during the evolution of L. hongkongensis and therefore now present in nearly all members of the bacterium.

Twenty combination patterns of virulence genes were found among the 88 ESBL-producing strains in this study. Approximately 93.6% of the frog strains and 85.4% of the fish strains contained 3 or more of the 10 virulence genes that we detected, which imply that multiple factors must be involved in the complicated process of L. hongkongensis pathogenesis.

To investigate the virulence gene profiles of L. hongkongensis from different hosts, we also analyzed the genome sequence of two clinical isolates, the L. hongkongensis HLGZ1 (CP022115.1) and HLHK9 (CP001154.1),^10,17 to search the 10 virulent factors investigated in this study. For L. hongkongensis HLGZ1, we only confirmed the existence of traT and fimH genes. However, none of the 10 genes were present in L. hongkongensis HLHK9. Nevertheless, no clear relatedness was observed among the two human strains and aquatic animal strains with respect to their virulence profiles. Further studies are in need for a better understanding the virulence status of L. hongkongensis that infect humans, with more clinical isolates.

The PFGE analysis showed that the majority of the fish isolates (68.3%) were grouped together in clusters D and E without isolates from frogs and showed an overall similarity coefficient higher than 65.0%, indicating a high degree of genetic relatedness among these isolates. Similarly, close genetic relatedness was observed in cluster D, including 13 isolates solely from frogs. Eleven of these isolates carried class 1 integrons containing the dfrA gene cassette, as well as some resistance genes (sul1, sul2, tetA, and tetR). However, most of the frog isolates were found in clusters A and B with the other fish isolates, suggesting the presence of genetic interrelationship between the two sources. In addition, some sets of identical profiles were observed in the same host or different hosts, which may imply the existence of a successful clone among different animals.

Conclusion

Our study highlights the multiple factors involved in the antimicrobial resistance and pathogenesis of ESBL-producing L. hongkongensis isolates from two major hosts associated with human infection. Our results reveal that the frog isolates have acquired a wide range of antibiotic resistance (genes), integrons, virulence factors, and genetic fingerprints compared with the fish isolates, which might pose a threat to public health. The results also suggest a possible separation of host-adapted populations of ESBL-producing L. hongkongensis from these two sources.

Footnotes

Authors' Contributions

J.H. and Z.L. conceived and designed the study. M.F., H.G., and Y.H. collected the isolates. Ling W., L.F., and Li W. performed the experiments. Ling W., L.F., and Z.L. analyzed the data. Ling W. and L.F. wrote the article. J.H., Z.L., and Q.C. revised the article.

Acknowledgment

This work was supported by the National Nature Science Foundation of China (Grant No. 81373052).

Disclosure Statement

The authors declare that they have no conflicts of interest.

Supplementary Material

References

Teng

J.L.

, Woo

P.C.

, Ma

S.S.

, Sit

T.H.

, Ng

L.T.

, Hui

W.T.

, Lau

S.K.

, and Yuen

K.Y.

. 2005. Ecoepidemiology of Laribacter hongkongensis, a novel bacterium associated with gastroenteritis. J. Clin. Microbiol. 43:919–922.

Woo

P.C.

, Lau

S.K.

, Teng

J.L.

, and Yuen

K.Y.

. 2005. Current status and future directions for Laribacter hongkongensis, a novel bacterium associated with gastroenteritis and traveller's diarrhoea. Curr. Opin. Infect. Dis. 18:413–419.

Woo

P.C.

, Kuhnert

, Burnens

A.P.

, Teng

J.L.

, Lau

S.K.

, Que

T.L.

, Yau

H.H.

, and Yuen

K.Y.

. 2003. Laribacter hongkongensis: a potential cause of infectious diarrhea. Diagn. Microbiol. Infect. Dis. 47:551–556.

Feng

J.L.

, Hu

, Lin

J.Y.

, Liu

, Chowdhury

, Zhang

, Li

J.D.

, Shi

, Yamasaki

, and Chen

. 2012. The prevalence, antimicrobial resistance and PFGE profiles of Laribacter hongkongensis in retail freshwater fish and edible frogs of southern China. Food Microbiol. 32:118–123.

Lau

S.K.

, Woo

P.C.

, Fan

R.Y.

, Lee

R.C.

, Teng

J.L.

, and Yuen

K.Y.

. 2007. Seasonal and tissue distribution of Laribacter hongkongensis, a novel bacterium associated with gastroenteritis, in retail freshwater fish in Hong Kong. Int. J. Food Microbiol. 113:62–66.

Lau

S.K.

, Woo

P.C.

, Fan

R.Y.

, Ma

S.S.

, Hui

W.T.

, Au

S.Y.

, Chan

L.L.

, Chan

J.Y.

, Lau

A.T.

, Leung

K.Y.

, Pun

T.C.

, She

H.H.

, Wong

C.Y.

, Wong

L.L.

, and Yuen

K.Y.

. 2007. Isolation of Laribacter hongkongensis, a novel bacterium associated with gastroenteritis, from drinking water reservoirs in Hong Kong. J. Appl. Microbiol. 103:507–515.

Lau

S.K.

, Ho

P.L.

, Li

M.W.

, Tsoi

H.W.

, Yung

R.W.

, Woo

P.C.

, and Yuen

K.Y.

. 2005. Cloning and characterization of a chromosomal class C beta-lactamase and its regulatory gene in Laribacter hongkongensis. Antimicrob. Agents Chemother. 49:1957–1964.

Lau

S.K.

, Lee

L.C.

, Fan

R.Y.

, Teng

J.L.

, Tse

C.W.

, Woo

P.C.

, and Yuen

K.Y.

. 2009. Isolation of Laribacter hongkongensis, a novel bacterium associated with gastroenteritis, from Chinese tiger frog. Int. J. Food Microbiol. 129:78–82.

Wang

, Zhu

, Liu

, Zheng

, Feng

, Chen

, Yu

, Jiang

, and Hu

. 2017. Multi-locus sequence typing of Laribacter hongkongensis isolates from freshwater animals, environment and diarrhea patients in southern China. Int. J. Food Microbiol. 245:98–104.

10.

H.K.

, Chen

J.H.

, Yang

, Li

A.R.

, Su

D.H.

, Lin

Y.P.

, and Chen

D.Q.

. 2017. Emergence and genomic analysis of MDR Laribacter hongkongensis strain HLGZ1 from Guangzhou, China. J. Antimicrob. Chemother. 73:643–647.

11.

Cantas

, Shah

S.Q.

, Cavaco

L.M.

, Manaia

C.M.

, Walsh

, Popowska

, Garelick

, Burgmann

, and Sorum

. 2013. A brief multi-disciplinary review on antimicrobial resistance in medicine and its linkage to the global environmental microbiota. Front. Microbiol. 4:96.

12.

Raja

K.M.

, and Ghosh

A.R.

. 2014. Molecular insight of putative pathogenicity markers with ESBL genes and lipopolysaccharide in Laribacter hongkongensis. Appl. Biochem. Biotechnol. 174:1935–1944.

13.

Coque

T.M.

, Baquero

, and Canton

. 2008. Increasing prevalence of ESBL-producing Enterobacteriaceae in Europe. Euro. Surveill. 13:, pii: 19044.

14.

Cambray

, Guerout

A.M.

, and Mazel

. 2010. Integrons. Annu. Rev. Genet. 44:141–166.

15.

Chen

, Zheng

, Yu

, Huang

, Zheng

, Zhang

, Guan

, Zhuang

, Chen

, and Topp

. 2011. Class 1 integrons, selected virulence genes, and antibiotic resistance in Escherichia coli isolates from the Minjiang River, Fujian Province, China. Appl. Environ. Microbiol. 77:148–155.

16.

Dutta

T.K.

, Warjri

, Roychoudhury

, Lalzampuia

, Samanta

, Joardar

S.N.

, Bandyopadhyay

, and Chandra

. 2013. Extended-spectrum-beta-lactamase-producing Escherichia coli isolate possessing the Shiga toxin gene (stx1) belonging to the O64 serogroup associated with human disease in India. J. Clin. Microbiol. 51:2008–2009.

17.

Woo

P.C.

, Lau

S.K.

, Tse

, Teng

J.L.

, Curreem

S.O.

, Tsang

A.K.

, Fan

R.Y.

, Wong

G.K.

, Huang

, Loman

N.J.

, Snyder

L.A.

, Cai

J.J.

, Huang

J.D.

, Mak

, Pallen

M.J.

, Lok

, and Yuen

K.Y.

. 2009. The complete genome and of proteome Laribacter hongkongensis reveal potential mechanisms for adaptations to different temperatures and habitats. PLoS Genet. 5:e1000416.

18.

Feng

J.L.

, Yan

, Chowdhury

, Neogi

S.B.

, Yamasaki

, Shi

, Hu

, and Chen

. 2011. Identification and characterization of integron-associated antibiotic resistant Laribacter hongkongensis isolated from aquatic products in China. Int. J. Food Microbiol. 144:337–341.

19.

CLSI. 2014. Performance Standards for Antimicrobial Susceptibility Testing; 24th Informational Supplement, M100-S24. CLSI, Wayne, PA.

20.

Costa

, Poeta

, Saenz

, Vinue

, Rojo-Bezares

, Jouini

, Zarazaga

, Rodrigues

, and Torres

. 2006. Detection of Escherichia coli harbouring extended-spectrum beta-lactamases of the CTX-M, TEM and SHV classes in faecal samples of wild animals in Portugal. J. Antimicrob. Chemother. 58:1311–1312.

21.

L.K.

, Martin

, Alfa

, and Mulvey

. 2001. Multiplex PCR for the detection of tetracycline resistant genes. Mol. Cell Probes. 15:209–215.

22.

Moller

T.S.

, Overgaard

, Nielsen

S.S.

, Bortolaia

, Sommer

M.O.

, Guardabassi

, and Olsen

J.E.

. 2016. Relation between tetR and tetA expression in tetracycline resistant Escherichia coli. BMC Microbiol. 16:39.

23.

Kerrn

M.B.

, Klemmensen

, Frimodt-Moller

, and Espersen

. 2002. Susceptibility of Danish Escherichia coli strains isolated from urinary tract infections and bacteraemia, and distribution of sul genes conferring sulphonamide resistance. J. Antimicrob. Chemother. 50:513–516.

24.

Kim

H.B.

, Park

C.H.

, Kim

C.J.

, Kim

E.C.

, Jacoby

G.A.

, and Hooper

D.C.

. 2009. Prevalence of plasmid-mediated quinolone resistance determinants over a 9-year period. Antimicrob. Agents Chemother. 53:639–645.

25.

Cavaco

L.M.

, Hasman

, Xia

, and Aarestrup

F.M.

. 2009. qnrD, a novel gene conferring transferable quinolone resistance in Salmonella enterica serovar Kentucky and Bovismorbificans strains of human origin. Antimicrob. Agents Chemother. 53:603–608.

26.

, Shi

, Yang

, Xiao

, Li

, and Yamasaki

. 2006. Analysis of integrons in clinical isolates of Escherichia coli in China during the last six years. FEMS Microbiol. Lett. 254:75–80.

27.

Gao

, Jia

, Wang

, Xiong

, Gao

, and Liu

. 2015. The avian pathogenic Escherichia coli O2 strain E058 carrying the defined aerobactin-defective iucD or iucDiutA mutation is less virulent in the chicken. Infect. Genet. Evol. 30:267–277.

28.

Chapman

T.A.

, Wu

X.Y.

, Barchia

, Bettelheim

K.A.

, Driesen

, Trott

, Wilson

, and Chin

J.J.

. 2006. Comparison of virulence gene profiles of Escherichia coli strains isolated from healthy and diarrheic swine. Appl. Environ. Microbiol. 72:4782–4795.

29.

Hunter

S.B.

, Vauterin

, Lambert-Fair

M.A.

, Van Duyne

M.S.

, Kubota

, Graves

, Wrigley

, Barrett

, and Ribot

. 2005. Establishment of a universal size standard strain for use with the PulseNet standardized pulsed-field gel electrophoresis protocols: converting the national databases to the new size standard. J. Clin. Microbiol. 43:1045–1050.

30.

Jiang

H.X.

, Tang

, Liu

Y.H.

, Zhang

X.H.

, Zeng

Z.L.

, Xu

, and Hawkey

P.M.

. 2012. Prevalence and characteristics of beta-lactamase and plasmid-mediated quinolone resistance genes in Escherichia coli isolated from farmed fish in China. J. Antimicrob. Chemother. 67:2350–2353.

31.

Grami

, Mansour

, Dahmen

, Mehri

, Haenni

, Aouni

, and Madec

J.Y.

. 2013. The blaCTX-M-1 IncI1/ST3 plasmid is dominant in chickens and pets in Tunisia. J. Antimicrob. Chemother. 68:2950–2952.

32.

Araujo

, Silva

A.T.

, Tacao

, Patinha

, Alves

, and Henriques

. 2017. Characterization of antibiotic resistant and pathogenic Escherichia coli in irrigation water and vegetables in household farms. Int. J. Food Microbiol. 257:192–200.

33.

P.L.

, Chow

K.H.

, Lai

E.L.

, Lo

W.U.

, Yeung

M.K.

, Chan

P.Y.

, and Yuen

K.Y.

. 2011. Extensive dissemination of CTX-M-producing Escherichia coli with multidrug resistance to ‘critically important’ antibiotics among food animals in Hong Kong, 2008–2010. J. Antimicrob. Chemother. 66:765–768.

34.

Sun

, Zeng

, Chen

, Ma

, He

, Liu

, Deng

, Lei

, Zhao

, and Liu

J.H.

. 2010. High prevalence of bla(CTX-M) extended-spectrum beta-lactamase genes in Escherichia coli isolates from pets and emergence of CTX-M-64 in China. Clin. Microbiol. Infect. 16:1475–1481.

35.

Zheng

, Zeng

, Chen

, Liu

, Yao

, Deng

, Chen

, Lv

, Zhuo

, Chen

, Liu

J.H.

. 2012. Prevalence and characterisation of CTX-M beta-lactamases amongst Escherichia coli isolates from healthy food animals in China. Int. J. Antimicrob. Agents, 39:305–310.

36.

Diwan

, Chandran

S.P.

, Tamhankar

A.J.

, Stalsby Lundborg

, and Macaden

. 2012. Identification of extended-spectrum beta-lactamase and quinolone resistance genes in Escherichia coli isolated from hospital wastewater from central India. J. Antimicrob. Chemother. 67:857–859.

37.

Lau

S.K.

, Wong

G.K.

, Li

M.W.

, Woo

P.C.

, and Yuen

K.Y.

. 2008. Distribution and molecular characterization of tetracycline resistance in Laribacter hongkongensis. J. Antimicrob. Chemother. 61:488–497.

38.

Shi

, Zhang

, Tian

, Yang

, and Zhang

. 2018. Characteristics of ARG-carrying plasmidome in the cultivable microbial community from wastewater treatment system under high oxytetracycline concentration. Appl. Microbiol. Biotechnol. 102:1847–1858.

39.

Frank

, Gautier

, Talarmin

, Bercion

, and Arlet

. 2007. Characterization of sulphonamide resistance genes and class 1 integron gene cassettes in Enterobacteriaceae, Central African Republic (CAR). J. Antimicrob. Chemother. 59:742–745.

40.

Hammerum

A.M.

, Sandvang

, Andersen

S.R.

, Seyfarth

A.M.

, Porsbo

L.J.

, Frimodt-Moller

, and Heuer

O.E.

. 2006. Detection of sul1, sul2 and sul3 in sulphonamide resistant Escherichia coli isolates obtained from healthy humans, pork and pigs in Denmark. Int. J. Food Microbiol. 106:235–237.

41.

Trobos

, Jakobsen

, Olsen

K.E.

, Frimodt-Moller

, Hammerum

A.M.

, Pedersen

, Agerso

, Porsbo

L.J.

, and Olsen

J.E.

. 2008. Prevalence of sulphonamide resistance and class 1 integron genes in Escherichia coli isolates obtained from broilers, broiler meat, healthy humans and urinary infections in Denmark. Int. J. Antimicrob. Agents, 32:367–369.

42.

Antunes

, Machado

, Sousa

J.C.

, and Peixe

. 2005. Dissemination of sulfonamide resistance genes (sul1, sul2, and sul3) in Portuguese Salmonella enterica strains and relation with integrons. Antimicrob. Agents Chemother. 49:836–839.

43.

Chen

D.Q.

, Yang

, Luo

Y.T.

, Mao

M.J.

, Lin

Y.P.

, and Wu

A.W.

. 2013. Prevalence and characterization of quinolone resistance in Laribacter hongkongensis from grass carp and Chinese tiger frog. J. Med. Microbiol. 62:1559–1564.

44.

Nam

Y.S.

, Cho

S.Y.

, Yang

H.Y.

, Park

K.S.

, Jang

J.H.

, Kim

Y.T.

, Jeong

J.W.

, Suh

J.T.

, and Lee

H.J.

. 2013. Investigation of mutation distribution in DNA gyrase and topoisomerase IV genes in ciprofloxacin-non-susceptible Enterobacteriaceae isolated from blood cultures in a tertiary care university hospital in South Korea, 2005–2010. Int. J. Antimicrob. Agents, 41:126–129.

45.

Abbassi

M.S.

, Ruiz

, Saenz

, Mechergui

, Ben Hassen

, and Torres

. 2010. Genetic background of quinolone resistance in CTX-M-15-producing Klebsiella pneumonia and Escherichia coli strains in Tunisia. J. Chemother. 22:66–67.

46.

Sajjad

, Holley

M.P.

, Labbate

, Stokes

H.W.

, and Gillings

M.R.

. 2011. Preclinical class 1 integron with a complete Tn402-like transposition module. Appl. Environ. Microbiol. 77:335–337.

47.

Bean

D.C.

, Livermore

D.M.

, Papa

, and Hall

L.M.

. 2005. Resistance among Escherichia coli to sulphonamides and other antimicrobials now little used in man. J. Antimicrob. Chemother. 56:962–964.

48.

Sunde

, and Norstrom

. 2005. The genetic background for streptomycin resistance in Escherichia coli influences the distribution of MICs. J. Antimicrob. Chemother. 56:87–90.

49.

Paulsen

I.T.

, Littlejohn

T.G.

, Radstrom

, Sundstrom

, Skold

, Swedberg

, and Skurray

R.A.

. 1993. The 3' conserved segment of integrons contains a gene associated with multidrug resistance to antiseptics and disinfectants. Antimicrob. Agents Chemother. 37:761–768.

50.

Enne

V.I.

, Livermore

D.M.

, Stephens

, and Hall

L.M.

. 2001. Persistence of sulphonamide resistance in Escherichia coli in the UK despite national prescribing restriction. Lancet. 357:1325–1328.

51.

Jacobs

, and Chenia

H.Y.

. 2007. Characterization of integrons and tetracycline resistance determinants in Aeromonas spp. isolated from South African aquaculture systems. Int. J. Food Microbiol. 114:295–306.

52.

Beceiro

, Tomas

, and Bou

. 2013. Antimicrobial resistance and virulence: a successful or deleterious association in the bacterial world? Clin. Microbiol. Rev. 26:185–230.

53.

Raja

M.K.

, and Ghosh

A.R.

. 2014. Laribacter hongkongensis: an emerging pathogen of infectious diarrhea. Folia Microbiol (Praha). 59:341–347.

54.

Martinez-Laguna

, Calva

, and Puente

J.L.

. 1999. Autoactivation and environmental regulation of bfpT expression, the gene coding for the transcriptional activator of bfpA in enteropathogenic Escherichia coli. Mol. Microbiol. 33:153–166.

55.

Russo

T.A.

, Carlino

U.B.

, and Johnson

J.R.

. 2001. Identification of a new iron-regulated virulence gene, ireA, in an extraintestinal pathogenic isolate of Escherichia coli. Infect. Immun. 69:6209–6216.

56.

Scaletsky

I.C.

, Aranda

K.R.

, Souza

T.B.

, and Silva

N.P.

. 2010. Adherence factors in atypical enteropathogenic Escherichia coli strains expressing the localized adherence-like pattern in HEp-2 cells. J. Clin. Microbiol. 48:302–306.

57.

Rogers

E.A.

, Das

, and Ton-That

. 2011. Adhesion by pathogenic corynebacteria. Adv. Exp. Med. Biol. 715:91–103.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.20 MB

0.03 MB