Download Distribution and molecular characterization of

Journal of Antimicrobial Chemotherapy (2008) 61, 488– 497
doi:10.1093/jac/dkm539
Advance Access publication 28 January 2008
Distribution and molecular characterization of tetracycline resistance
in Laribacter hongkongensis
Susanna K. P. Lau1,2,3, Gilman K. M. Wong3, Maria W. S. Li3, Patrick C. Y. Woo1,2,3†
and Kwok-yung Yuen1,2,3*†
1
State Key Laboratory of Emerging Infectious Diseases, The University of Hong Kong, Hong Kong; 2Research
Centre of Infection and Immunology, The University of Hong Kong, Hong Kong; 3Department of Microbiology,
The University of Hong Kong, Hong Kong
Received 11 October 2007; returned 3 December 2007; revised 12 December 2007; accepted 13 December 2007
Objectives: Laribacter hongkongensis is a newly discovered bacterium associated with gastroenteritis
and found in freshwater fish. Although isolates resistant to tetracycline have been described, their
resistance mechanisms have not been studied.
Patients and methods: We describe the distribution and molecular characterization of tetracycline
resistance in 48 L. hongkongensis isolates from humans and fish.
Results: Three human isolates and one fish isolate were resistant to tetracycline (MIC 128 mg/L) and
doxycycline (MIC 8– 16 mg/L) and had reduced susceptibility to minocycline (MIC 1 –4 mg/L). A 3566 bp
gene cluster, which contains tetR and tetA, was cloned from one of the tetracycline-resistant strains,
HLHK5. While the flanking regions and 30 end of the tetA of HLHK5 were identical to the corresponding
regions of a tetC island in Chlamydia suis, the tetA gene was almost identical to that of transposon
Tn1721 and plasmids of Gram-negative bacteria, suggesting that the tetA/tetR of HLHK5 may have
arisen from illegitimate recombination. PCR and DNA sequencing showed the presence of tetA in the
other three tetracycline-resistant L. hongkongensis strains. Sequencing and characterization of a
15 665 bp plasmid, pHLHK22, from strain HLHK22 revealed the presence of a similar tetA/tetR gene
cluster. This novel plasmid also confers tetracycline resistance when transformed to Escherichia coli
and other L. hongkongensis isolates.
Conclusions: Horizontal transfer of genes, especially through Tn1721 and related plasmids, is likely an
important mechanism for acquisition and dissemination of tetracycline resistance in L. hongkongensis. The present study is the first report on identification of tetA genes in bacteria of the Neisseriaceae
family.
Keywords: freshwater fish, gastroenteritis, tetA gene, tetR gene
Introduction
As a result of the widespread use of tetracyclines for treatment
of various infections in humans, animals and plants, and as
growth promoters in animal feeds in some countries, there has
been a dramatic increase in tetracycline-resistant bacteria.1 There
are two main mechanisms of tetracycline resistance, namely
drug efflux and ribosomal protection, with tetracycline efflux
genes mostly found on mobile plasmids or transposon systems,
accounting for their widespread dissemination.2 – 5
Laribacter hongkongensis is a newly discovered Gramnegative, seagull-shaped rod that belongs to the Neisseriaceae
family of b-proteobacteria. After its first isolation from the
blood and empyema thoracis of a patient with alcoholic liver cirrhosis, the bacterium was subsequently found to be associated
with community-acquired gastroenteritis and traveller’s diarrhoea.6 – 9 L. hongkongensis is likely to be globally distributed,
as travel histories from patients suggested that it is present on at
least four continents, including Asia, Europe, Africa and Central
America.7,9,10 Although the causative role of L. hongkongensis
.....................................................................................................................................................................................................................................................................................................................................................................................................................................
*Correspondence address. State Key Laboratory of Emerging Infectious Diseases, Department of Microbiology, The University of Hong
Kong, University Pathology Building, Queen Mary Hospital, Hong Kong. Tel: þ852-28554892; Fax: þ852-28551241;
E-mail: [email protected]
†
These authors contributed equally to this article.
.....................................................................................................................................................................................................................................................................................................................................................................................................................................
488
# The Author 2008. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
For Permissions, please e-mail: [email protected]
Tetracycline resistance in Laribacter hongkongensis
in gastroenteritis is yet to be established,11 these data provide
strong evidence that the bacterium is a potential diarrhoeal
pathogen that warrants further investigations. Freshwater fish is
likely a reservoir for L. hongkongensis, with the highest recovery
rates from intestines of grass carp (60%) and bighead carp
(53%).9,12,13 Recently, the bacterium has also been isolated from
drinking water reservoirs in Hong Kong.14
L. hongkongensis has been found to carry resistance to multiple antibiotics. It is generally resistant to b-lactams, including
broad-spectrum penicillins and cephalosporins, but susceptible
to carbapenems, quinolones and aminoglycosides.6,7,9 We have
recently reported the cloning and characterization of a novel
class C b-lactamase gene from clinical isolates of L. hongkongensis.15 From our previous studies, 10% to 20% of L. hongkongensis isolates were also tetracycline resistant. However, the
molecular mechanism of tetracycline resistance in L. hongkongensis remains unknown. In this study, we describe the phenotypic and molecular characterization of tetracycline resistance in
L. hongkongensis isolated from humans and fish. A novel tetracycline resistance plasmid from one of the tetracycline-resistant
isolates was also characterized. The assembled 15 665 bp
sequence was found to contain the tetA/tetR gene cluster, supporting that gene exchange through plasmids is likely responsible for acquisition and dissemination of tetracycline resistance
in L. hongkongensis.
Materials and methods
Bacterial strains and microbiological methods
Bacterial strains and plasmids used for cloning experiments in this
study are listed in Table 1. The identification of all L. hongkongensis
isolates was confirmed phenotypically by standard conventional
biochemical methods and genotypically by 16S rRNA gene
sequencing.9
Antimicrobial susceptibility testing
MICs of tetracycline, doxycycline and minocycline were determined
using the macrodilution broth method for non-fastidious, aerobic
bacteria according to CLSI guidelines, using Mueller –Hinton broth
(Becton –Dickinson, Cockeysville, USA) with inoculum of 5 105 cfu/mL incubated at 358C for 20 h in ambient air.16 Control
strains Staphylococcus aureus ATCC 29213 and/or Escherichia coli
ATCC 25922 were included with each run.
Molecular characterization of tetracycline resistance in
L. hongkongensis strain HLHK5
A genomic DNA library from an L. hongkongensis strain, HLHK5,
was constructed as described in our previous publications.15,17,18 E.
coli DH5a cells were infected with pBK-CMV phagemid vector,
yielding pBK-CMV plasmid with the cloned inserts in E. coli
DH5a cells. Antibiotic-resistant clones were selected on Luria–
Bertani (LB) plates containing 50 mg/L kanamycin and 10 mg/L
tetracycline. Recombinant plasmid DNA was prepared with High
Pure Plasmid Isolation Kit (Roche, Mannheim, Germany) from
1 mL LB broth cultures incubated with 50 mg/L kanamycin and
10 mg/L tetracycline overnight at 378C. Sizes of inserted fragments
were estimated according to lAvaII digest DNA marker (MBI
Fermentas, Hanover, USA).
Cloned DNA fragments in pBK-CMV were sequenced using
vector primers of pBK-CMV (T3 and T7) and synthetic primers
(LPW806 50 -ATTCCGAGCATGAGTGCC-30 , LPW807 50 -CGAGC
AACGCCCGTCGAA-30 , LPW901 50 -GTGATAAAGAATCCGC
GC-30 , LPW1015 50 -ATGTCCACCAACTTATCA-30 , LPW1032
50 -CGAGTGAACCAGATCGCGC-30 , LPW1033 50 -GGGCATGAC
CGTCGTCGC-30 and LPW1414 50 -GGCACTCATGCTCGGA
AT-30 ) designed from the sequencing data of the first round of the
sequencing reaction (Figure 1). Bidirectional DNA sequencing was
performed with an ABI automatic sequencer (Perkin-Elmer,
Norwalk, CT, USA) according to the manufacturers’ instructions.
The DNA sequence was analysed by BLAST search with the
National Center for Biotechnology Information server at the
National Library of Medicine (http://www.ncbi.nlm.nih.gov)
(Bethesda, MD, USA). The searches were performed at both the
protein and DNA levels.
Distribution of tetA in L. hongkongensis and phylogenetic
analysis
Genomic DNA of the 24 human and 24 fish isolates of L. hongkongensis
(HKU1, HLHK2–24 and FLHK1–24) was extracted as described
Table 1. Bacterial strains and plasmids used in cloning experiments in this study
Relevant genotype or phenotypea
Source or reference
þ
F2v80dlacZDM15 D(lacZYA-argF) U169 recA1 endA1 hsdR17(r2
k , mk )
phoA supE44 l- thi-1 gyrA96 relA1
Tcr Ampr
Tcr Ampr
Invitrogen, Carlsbad, CA, USA
cloning vector; neomycin and kanamycin resistant
pBK-CMV with 2810 bp Sau3A-digested Tcr genomic fragment from
L. hongkongensis HLHK5 in pBK-CMV
pBK-CMV with 2764 bp Sau3A-digested Tcr genomic fragment from
L. hongkongensis HLHK5 in pBK-CMV
plasmid extracted from L. hongkongensis HLHK22, tetracycline resistant
Stratagene, La Jolla, CA, USA
this report
Strain or plasmid
Strains
E. coli DH5-a
L. hongkongensis HLHK5
L. hongkongensis HLHK22
Plasmids
pBK-CMV phagemid
pLTA1
pLTA2
pHLHK22
a
Tcr, tetracycline resistance; Ampr, ampicillin resistance.
489
9
9
this report
this report
Lau et al.
Figure 1. Genetic organization of the tetracycline resistance gene cluster in L. hongkongensis strain HLHK5 depicted by combined nucleotide sequences of
the inserts of two recombinant plasmids pLTA1 and pLTA2. Primers LPW806, LPW807, LPW901, LPW1015, LPW1032, LPW1033 and LPW1414 were
used for sequencing. Transcriptional direction is shown by arrows on the ORF boxes. The striped regions displayed 100% nucleotide identities to the
corresponding regions in a tetC island in C. suis. The meshed region had one nucleotide difference to the corresponding regions in the tetA system in
transposon Tn1721, S. enterica plasmid 6/9, Salmonella Typhimurium plasmid pU302L, E. coli plasmid pC15-1a, S. enterica plasmid pFPTB1, S. sonnei
plasmids pSS4 and pSSTAV, A. punctata plasmid pFBAOT6, Gram-negative bacteria, A. salmonicida plasmid pRAS1 and E. coli plasmid pAPEC-O2-R. The
vertical arrows indicate the boundaries of possible recombination sites from positions 1419– 1422 and 2910–2921, respectively. The shaded areas indicate the
corresponding regions homologous to C. suis and Tn1721.
previously.6 The prevalence of the cloned tetA gene of L. hongkongensis was studied by PCR using laboratory-designed primers (LPW806
and LPW901). For isolates positive for tetA gene by PCR, the complete
gene sequences were determined by additional PCR and sequencing
reactions using primers LPW806, LPW1015, LPW1032, LPW1033,
LPW1414 and LPW1697 50 -TCAGCGATCGGCTCGTTG-30 . The
deduced protein sequences of all tetA genes were compared with
known sequences in the GenBank by multiple sequence alignment
using the CLUSTAL W program.19 The phylogenetic relationships of
tetA genes of L. hongkongensis to the corresponding related genes were
made using the neighbour-joining method with ClustalX 1.83. The predictions of transmembrane regions were carried out with PRED-TM2
at http://biophysics.biol.uoa.gr/PRED-TMR2/.20
Sequencing and in silico analysis of tetracycline resistance
plasmid, pHLHK22, from strain HLHK22
Extraction of the plasmid, pHLHK22, from L. hongkongensis strain
HLHK22 was performed using the High Pure Plasmid Isolation Kit
(Roche Applied Science) according to the manufacturer’s instructions. The DNA sequence of pHLHK22 was determined using
primer walking with primers designed from the tetA gene sequence
of the plasmid and additional primers designed from the subsequent
rounds of the sequencing reactions. The nucleotide and deduced
amino acid sequences of the open reading frames (ORFs) of
pHLHK22 were compared with sequences in the GenBank. Protein
family analysis was performed using PFAM and InterProScan.21
Direct and inverted repeats were determined using dotmatcher
(EMBOSS-GUI). Phylogenetic tree construction was performed by
using the neighbour-joining method with GrowTree software
(Genetics Computer Group, Madison, WI, USA).
Determination of copy number of pHLHK22
The copy number of pHLHK22 was determined according to a published protocol.22 Overnight culture of HLHK22 was inoculated into
brain heart infusion (BHI) broth. When the culture reached a turbidity of 0.6 –1.0 at OD600, 1 mL of the culture was used for plasmid
DNA extraction using the High Pure Plasmid Isolation Kit (Roche
Applied Science), and the number of bacteria was determined by
back titration. The concentration of plasmid DNA was calculated by
measuring the absorbance of the plasmid DNA solution at 260 nm.
A plasmid of known copy number ( pBR322 in E. coli DH5a) was
used as the control. The experiment was performed three times and
the copy number of pHLHK22 was calculated using the following
formula:
No. of plasmids/bacterium ¼ ð6:02 1023
plasmids/mass of 1 plasmid) (mass of plasmid DNA in 1 mL of bacterial culture/no. of bacteria
in 1 mL of bacterial culture)
Segregational plasmid stability studies
The plasmid stability of pHLHK22 was determined according to a
published protocol.23 A single colony of HLHK22 containing
490
Tetracycline resistance in Laribacter hongkongensis
pHLHK22 was inoculated into BHI broth. Cells in the late exponential growth phase (12 h after inoculation) were diluted 1000-fold and
the procedure was repeated four times. After these subcultures, bacterial cells were plated on BHI agar plates. Ten colonies were
picked and the presence of pHLHK22 was determined by plasmid
DNA extraction.
Transformation experiments
Transformation of pHLHK22 was performed to assess its transformation efficiency and the potential transfer of tetracycline resistance from L. hongkongensis HLHK22 to tetracycline-susceptible
L. hongkongensis isolates. Transformation was performed by electroporation, using 1 mg of plasmids, according to standard protocol.24
Transformants were selected on LB agar plates with 10 mg/L tetracycline. The presence of plasmids in colonies of L. hongkongensis was
determined using the High Pure Plasmid Isolation Kit (Roche Applied
Science) and agarose gel electrophoresis as described earlier.
Conjugation experiments
Conjugation experiments using a streptomycin-resistant E. coli
strain HB101 and a spectinomycin-resistant L. hongkongensis strain
as recipients were performed by the broth mating and filter mating
method as described previously.25,26 Transconjugants were selected
on LB agar plates containing streptomycin (50 mg/L) plus tetracycline (80 mg/L) for E. coli strain HB101 and spectinomycin
(50 mg/L) plus tetracycline (80 mg/L) for the L. hongkongensis
strain.
Nucleotide sequence accession numbers
The tetA gene sequence data of strain HLHK5, tetALHK-5, and the
plasmid sequence of pHLHK22 have been lodged within the
GenBank sequence database under accession nos. AY903253 and
EF679779, respectively.
Results
Antibiotic susceptibility
The MICs of tetracycline, doxycycline and minocycline for the
48 isolates of L. hongkongensis (HKU1, HLHK2 – 24 and
FLHK1 – 24) are shown in Table 2. Among the 48 isolates, 4
were resistant to tetracycline (MIC 128 mg/L) and doxycycline
(MIC 8 –16 mg/L). These four isolates also had reduced susceptibility to minocycline (MIC 1– 4 mg/L). These four isolates
were isolated from Hong Kong, with three (HLHK5, HLHK16
and HLHK22) being human isolates and one (FLHK15) being a
fish isolate from a mud carp (Cirrhina molitorella). The other
44 isolates were susceptible to tetracycline (MIC 0.0625 – 2 mg/
L), doxycycline (MIC 0.0625 – 1 mg/L) and minocycline (MIC
0.0625 –0.5 mg/L).
were obtained. Sequencing of the inserts of the recombinant
plasmids revealed that all inserts contained two divergently
oriented ORFs, the first 636 bp encoding a protein of 211 amino
acids and the second 1266 bp encoding a protein of 421 amino
acids (Figure 1). The deduced amino acid sequence of the first
ORF had 92.8% amino acid identities with TetR of Chlamydia
suis (GenBank accession no. AAR96033.1) and 85.8% amino
acid identities with TetR of IncN plasmid R46 (GenBank accession no. NP_511232), plasmid pB3 (GenBank accession no.
YP_133931), E. coli plasmid pSC101 (GenBank accession no.
RPECYS) and Salmonella Typhimurium (GenBank accession
no. NP_958732). The deduced amino acid sequence of the
second ORF had 98.1% amino acid identities with TetA in
Salmonella enterica plasmid 6/9 (GenBank accession no.
CAF31521) and 97.6% amino acid identities with TetA in
Shigella sonnei plasmids (GenBank accession nos. AAN06707
and AAM22221). The deduced TetA protein possessed 12 transmembrane domains.
Sequencing of one of the recombinant plasmids, pLTA1,
revealed that upstream to the tetR gene of HLHK5, there are two
divergently transcribed ORFs. One incomplete ORF encodes a
putative IS1341 element transposase (GenBank accession no.
AAR96031) and the other ORF encodes a putative IS200
element transposase (GenBank accession no. AAR96032) from
C. suis. Both ORFs had 100% nucleotide identities to the
IS1341 and IS200 element transposases of C. suis (Figure 1).
Further analysis of the combined 3566 bp sequence from the
inserts of two recombinant plasmids, pLTA1 and pLTA2,
showed that the 50 1421 bp region and the 30 656 bp region had
100% nucleotide identities with the corresponding regions of a
genomic island containing a tetC system in C. suis; while the
region in between possessed only one nucleotide difference to
the corresponding regions of tetA systems of Gram-negative bacteria transposon Tn1721 (GenBank accession no. S87924),
S. enterica plasmid 6/9 (GenBank accession no. AJ628353),
Salmonella Typhimurium plasmid pU302L (GenBank accession
no. AY333434), E. coli plasmid pC15-1a (GenBank accession
no. AY458016), S. enterica plasmid pFPTB1 (GenBank accession no. AJ634602), S. sonnei plasmid pSS4 (GenBank accession no. AF534183), S. sonnei plasmid pSSTAV (GenBank
accession no. AF502943), Aeromonas punctata plasmid
pFBAOT6 (GenBank accession no. CR376602), Aeromonas salmonicida plasmid pRAS1 (GenBank accession no. AJ517790)
and E. coli plasmid pAPEC-O2-R (GenBank accession no.
AY214164) (Figure 1). This suggests possible recombination
sites between positions 1419 and 1422 and between positions
2910 and 2921, respectively.
E. coli DH5a cells harbouring the recombinant plasmid
pLTA2, containing the complete tetA and tetR genes, exhibited
increased levels of resistance to tetracycline compared with the
wild-type strain, indicating that the insert fragment in pLTA2
conferred tetracycline resistance (Table 1).
Distribution of tetA gene in L. hongkongensis
Molecular characterization of tetracycline resistance
in L. hongkongensis strain HLHK5
One of the four tetracycline-resistant strains, HLHK5, was
selected for identification of tetracycline resistance determinants.
Eight tetracycline- and kanamycin-resistant E. coli DH5a clones
harbouring the recombinant plasmids, named pLTA1 to pLTA8,
PCR for the tetA gene of L. hongkongensis showed bands at
908 bp in the 3, of the 24, human isolates that are resistant to
tetracycline (HLHK5, HLHK16 and HLHK22) and the only 1,
of the 24, fish isolate that is resistant to tetracycline (FLHK15).
Sequencing of the tetA genes of the four isolates showed that
the tetA gene of HLHK5 is identical to that obtained from the
491
Lau et al.
Table 2. MICs of tetracycline, doxycycline and minocycline and results of PCR amplification of the tetA gene
MIC (mg/L)b
Isolate/straina
L. hongkongensis
HKU1
HLHK2
HLHK3
HLHK4
HLHK5
HLHK6
HLHK7
HLHK8
HLHK9
HLHK10
HLHK11
HLHK12
HLHK13
HLHK14
HLHK15
HLHK16
HLHK17
HLHK18
HLHK19
HLHK20
HLHK21
HLHK22
HLHK23
HLHK24
FLHK1
FLHK2
FLHK3
FLHK4
FLHK5
FLHK6
FLHK7
FLHK8
FLHK9
FLHK10
FLHK11
FLHK12
FLHK13
FLHK14
FLHK15
FLHK16
FLHK17
FLHK18
FLHK19
FLHK20
FLHK21
FLHK22
FLHK23
FLHK24
E. coli DH5a
wild-type
with plasmid pLTA2
a
tetracycline
doxycycline
minocycline
tetA gene
0.25
0.5
0.25
0.25
128
0.5
0.125
0.5
0.5
0.5
0.5
0.5
0.25
0.5
0.5
128
2
0.5
0.25
1
0.5
128
0.5
1
0.0625
0.25
0.5
0.5
1
0.5
2
0.5
0.25
0.25
0.25
0.25
0.5
0.5
128
0.5
0.25
0.5
0.5
0.5
2
0.5
0.5
0.5
0.25
0.5
1
0.25
16
0.25
0.5
0.5
0.25
0.5
0.125
0.25
0.25
0.125
0.25
8
0.25
0.25
0.25
0.25
0.25
8
0.25
0.25
0.5
0.25
0.125
0.125
0.5
0.125
0.5
0.125
0.0625
0.125
0.125
0.0625
0.0625
0.0625
16
0.125
0.125
0.125
0.125
0.25
0.5
0.25
0.25
0.125
0.125
0.125
0.125
0.125
2
0.125
0.125
0.25
0.125
0.125
0.0625
0.125
0.125
0.0625
0.125
1
0.125
0.0625
0.25
0.125
0.125
2
0.0625
0.125
0.0625
0.25
0.0625
0.25
0.25
0.125
0.25
0.125
0.0625
0.125
0.125
0.0625
0.0625
0.125
4
0.25
0.125
0.125
0.125
0.125
0.5
0.25
0.25
0.125
2
2
2
2
þ
2
2
2
2
2
2
2
2
2
2
þ
2
2
2
2
2
þ
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
þ
2
2
2
2
2
2
2
2
2
4
256
2
32
1
4
2
þ
HKU1 and all HLHK isolates were isolated from humans, while all FLHK isolates were isolated from freshwater fish.
MIC cut-offs for tetracycline, doxycycline and minocycline: susceptible, 4 mg/L; intermediately resistant, 8 mg/L; and resistant, 16 mg/L.
b
492
Tetracycline resistance in Laribacter hongkongensis
E. coli DH5a clones and showed 97.7% to 98.2% nucleotide
and 97.1% to 98.1% amino acid identities to those of the other
three isolates (Figure 2). The tetA genes of strains HLHK22 and
FLHK15 were identical to that of Tn1721, indicating that the
proposed recombination event in strain HLHK5 has not
occurred. The tetA gene of strain HLHK16 displayed high similarities to that of uncultured bacterium plasmid pRSB101,
E. coli plasmid pFL129, Pseudomonas aeruginosa plasmid RP4
and Acinetobacter baumannii, than to Tn1721.
Sequencing and characterization of pHLHK22
During our study on the plasmid profiles of various strains of
L. hongkongensis, it was found that HLHK5 and HLHK22 possessed potential plasmids of size around 300 and 16 kb, respectively.27 Since attempts to extract the plasmid from HLHK5 were
unsuccessful, which could be related to its large size, further
characterization was performed on the plasmid from HLHK22
to determine if the tetA is plasmid-encoded and confers tetracycline resistance. The plasmid, pHLHK22, extracted from
L. hongkongensis HLHK22 consists of 15 665 bp. The overall
G þ C content is 61.1%. The copy number (mean + SD) of
pHLHK22 is 1.09 + 0.05. The plasmid is stable after four passages (240 generations) in the absence of selection. The
present plasmid is likely a theta, class A, replicative plasmid, as
it contains a predicted origin of replication with AT-rich region,
a DnaA box with five 16 bp direct repeats, and a putative replicative protein, repA, most homologous to those of other theta
replicative plasmids, such as IncW plasmid pSa in Shigella flexneri.28 Moreover, it shares all the nine positions of the consensus sequence, TT(A/T)TNCACA (TTTTCCACA in pHLHK22),
in the DnaA boxes observed in other classical examples of class
A plasmids of this group, such as plasmid F, pSC101, P1, R1
and R6K.29,30 The plasmid has 12 ORFs with nine genes
encoded in the sense direction and the other three in the antisense direction (Table 3 and Figure 3). These 12 ORFs include
both tetR and tetA genes which are divergently oriented. The
tetA gene of pHLHK22 possessed 100% nucleotide identity to
that of the parent strain HLHK22, suggesting that the tetA gene
of HLHK22 was plasmid encoded. The tetR possessed 79%
nucleotide identity to that of strain HLHK5 and was identical to
a tetR identified in a plasmid of E. coli.
Figure 2. Phylogenetic tree showing the relationships of the nucleotide sequence of tetA of L. hongkongensis to related tetA genes. The tree was constructed
by using the neighbour-joining method and bootstrap values calculated from 1000 trees. The scale bar indicates the estimated number of substitutions per
1000 nucleotides using the Kimura two-parameter correction. Names and accession numbers are given as cited in the GenBank database.
493
Table 3. ORFs of the L. hongkongensis plasmid pHLHK22 having significant database matches
Characteristics of ORFs
gene
name
start – end
(base)
Best match to known sequences in public databases
no. of
bases
number of
amino acids
frame
organism
description
794 –1411
1727–2122
2053–3327
3331–4008
4516–5082
5211–5558
618
396
1275
678
567
348
205
131
424
225
188
115
22
þ2
23
þ1
23
21
Nitrosomonas eutropha C71
Psychrobacter cryohalolentis K5
E. coli
E. coli
A. punctata
Acidovorax sp. JS42
mobA
6159–7643
1485
494
21
mobB
7643–7969
327
108
22
ORF4
9533–11 230
1698
565
þ2
ORF5
11 261 –11 632
372
123
þ2
ORF6
repA
12 349 –12 912
14 415 –15 644
564
1230
187
409
23
–1
Xanthomonas axonopodis pv.
glycines
Xanthomonas axonopodis pv.
glycines
Acidovorax avenae subsp.
citrulli AAC00-1
Agrobacterium tumefaciens str.
C58
P. aeruginosa
Xanthomonas campestris pv.
vesicatoria
E
value
Percentage amino
acid identity
ATPase of MinD/ParA family
putative acetyltransferase
tetracycline efflux protein
tetracycline repressor protein
DNA recombinase
addiction module toxin, RelE/
StbE
probable MobA
ZP_00671102
ABE75113
ABF71536
ABF71535
YP_067856
EAT54475
6e269
9e225
0
4e2123
2e2122
2e247
85%
34%
100%
100%
100%
87%
AAX12225
7e2125
35%
probable MobB
AAX12226
1e220
48%
TrbL/VirB6 plasmid conjugal
transfer protein
conjugal transfer protein TrbJ
EAT96946
2e253
30%
AAL46273
5e26
16%
KfrA protein
putative replication protein A
AAP22622
CAJ19910
5e210
8e2168
26%
76%
Lau et al.
494
ORF1
ORF2
tetA
tetR
ORF3
relE
GenBank
accession no.
Tetracycline resistance in Laribacter hongkongensis
Figure 3. Organization and replication region of pHLHK22. The thin arrows indicate the five 16 bp direct repeats of the putative origin of replication.
ORF1 of pHLHK22 encodes a putative ATPase involved in
plasmid partitioning belonging to the MinD/ParA family (PFAM
accession no. PF01656), with significant homology to a similar
protein in the plasmid pHLHK8 from another L. hongkongensis
strain HLHK8 we described previously.31 ORF2 encodes a putative acetyltransferase (PFAM accession no. PF00583). ORF3
encodes a putative recombinase of the resolvase/invertase family
(PFAM accession no. PF00239). Multiple alignments with the
amino acid sequences of closely related resolvases revealed the
presence of highly conserved motifs and amino acid residues
that are functionally important as determined by X-ray crystallography.32,33 RelE encodes a putative addiction module toxin
(PFAM accession no. PF05016) of the RelE –RelB toxin –antitoxin system, which regulates bacterial cell growth and stabilizes
plasmids by inhibiting translation of susceptible cells. mobA and
mobB encode relaxase/mobilization proteins belonging to the set
of genes within mobilization regions of small mobilizable plasmids in bacteria. These small mobilizable plasmids, though not
conjugative (self-transmissible), can be efficiently mobilized in
the presence of additional conjugative functions by additional,
self-transmissible plasmids. The presence of both mobA and
mobB in pHLHK22 suggests that the present plasmid may be
transmissible during the conjugal transfer of self-transmissible
plasmids. ORF4 encodes a putative conjugal transfer protein
(PFAM accession no. PF04610). ORFs 5 and 6 encode hypothetical proteins with low homologies to conjugal transfer protein
TrbJ and partition protein KfrA, respectively.
Transformation
Colonies [median 2.64 105 (range 1.40 103 – 6.67 103) per
mg of plasmid DNA], with plasmids recoverable, were observed
in 8 (50%) out of 16 L. hongkongensis strains when they were
transformed with pHLHK22. Colonies (2.18 105 per mg of
plasmid DNA), with plasmids recoverable, were observed in
E. coli DH5a when transformed with pHLHK22.
Conjugation
pHLHK22 was neither transferred by conjugation to E. coli
HB101 nor a L. hongkongensis isolate, suggesting that the
plasmid may not be self-transmissible, which is in line with the
presence of mobA and mobB. Further studies are required to
determine if it can be co-transferred during conjugation of selftransmissible plasmids.
Discussion
The present study is the first report on identification of tetA
genes in bacteria of the Neisseriaceae family. Among 24 epidemiologically unrelated L. hongkongensis isolates from humans
and 24 isolates from fish, 3 (12.5%) human and 1 (4.2%) fish
isolates were found to be resistant to tetracycline and doxycycline and had reduced susceptibility to minocycline. By
sequence analysis of the inserts of recombinant plasmids, a gene
cluster containing a tetA and a tetR gene from one
tetracycline-resistant human isolate, strain HLHK5, was identified. The two genes were divergently oriented with overlapping
promoters, typical of the tetracycline-regulated efflux systems in
Gram-negative bacteria, with the TetR protein acting as a repressor that is inactivated by tetracyclines through conformational
changes. From phylogenetic analysis, the tetA gene of L. hongkongensis is closely related to other known tetA genes
(Figure 2). The deduced TetA protein also possessed 12 transmembrane domains characteristic of other TetA proteins. The
presence of a similar tetA gene in all the 4 tetracycline-resistant
isolates, the absence of tetA gene in the 44 tetracyclinesusceptible isolates and the expression of tetracycline resistance
phenotype when cloned in E. coli suggest that this tetA gene is
responsible for tetracycline resistance in L. hongkongensis.
Furthermore, resistance to tetracycline and doxycycline with
reduced susceptibility to minocycline is also the typical
495
Lau et al.
antibiotic susceptibility profile of tetA-mediated tetracycline
resistance.34 The role of tetA in conferring tetracycline resistance
in L. hongkongensis was further proven by sequencing and
characterization of a 15 665 bp plasmid from one of the
tetracycline-resistant strains, HLHK22, which demonstrated that
the tetA/tetR of HLHK22 was plasmid-encoded and confers
tetracycline resistance when the plasmid was transformed to
eight L. hongkongensis strains and E. coli DH5-a. Although
most isolates of Gram-negative bacteria carry only a single type
of tet gene, more than one tetracycline resistance gene has been
identified in some bacteria.1,3,35 Further studies, including tetracycline uptake and efflux, and mutagenesis studies, can be performed to ascertain the role of the present tetracycline resistance
determinant in mediating tetracycline resistance.
The tetA and tetR gene cluster of L. hongkongensis strain
HLHK5 is likely a mosaic gene cluster that has originated from
recombination between phylogenetically different bacteria.
Mosaic antibiotic resistance genes have been well reported in
bacteria, the most notable example being pbp genes in
Streptococcus pneumoniae.36 In L. hongkongensis strain
HLHK5, the homology between its tetA island and that of
Gram-negative bacteria transposon Tn1721 and plasmids, and
between its 30 and flanking regions and those of a tetC system in
C. suis indicates that an illegitimate recombination event may
have occurred to produce the tetracycline resistance determinant
in HLHK5. Previous studies have shown that illegitimate recombination can occur with short stretches of homologous DNA in
bacteria.37 Such recombination events have also been proposed
in the generation of tetracycline resistance determinants in
Shigella.3 In the present gene cluster, there are also short
stretches of homologous nucleotides at the potential sites of
recombination. These short stretches of homologous nucleotides
may have allowed recombination between the sequences from
Tn1721 or other plasmids and C. suis after acquisition of genes
by horizontal transfer.
The high degree of homologies between the tetA genes of
L. hongkongensis and other known tetA genes indicates that horizontal gene transfer is an important mechanism for acquisition
and dissemination of tetracycline resistance in L. hongkongensis.
While the tetA gene of strain HLHK5 likely originates from
recombination between sequences from S. enterica or related
plasmids and C. suis, sequence analysis of the tetA genes of the
other three strains did not reveal such recombination event.
Nevertheless, the tetA genes of strains HLHK22 and FLHK15,
being identical in their nucleotide sequences, were also likely to
have been acquired from transposons or plasmids of other
Gram-negative bacteria, but without recombination. In contrast,
the tetA gene of strain HLHK16 appears to have a different
origin, as it is less similar to Tn1721. Our results suggest that
horizontal transfer of tetracycline resistance islands between
L. hongkongensis and other Gram-negative bacteria was frequent
with occasional recombination events. L. hongkongensis often
harbours plasmids of various sizes, with more than one plasmid
in some strains.31 Since sequencing of three plasmids from three
different strains revealed no selective markers previously,
E. coli – L. hongkongensis shuttle vectors have been constructed.27,31,38 The present plasmid, pHLHK22, being transferable to other L. hongkongensis strains and E. coli, conferring
tetracycline resistance, may serve as the backbone of new shuttle
vectors for L. hongkongensis.
The presence of tetA sequences closely related to the
Gram-negative bacteria transposon Tn1721 in three isolates of
L. hongkongensis supports that this transposon or related plasmids
are important for the dissemination of tetracycline resistance in
L. hongkongensis, as in the case of Aeromonas, another bacterium commonly found in fish populations.39,40 Since tetracyclines
are one of the most commonly used antibiotics in aquaculture to
control infections,41,42 further investigations are required to
assess its significance in the amplification and dissemination of
these transposons or plasmids in related bacterial populations.
Funding
This work is partly supported by the University Research Grant
Council grant (HKU7531/05M); University Development Fund,
HKU Special Research Achievement Award, The Croucher
Senior Medical Research Fellowship 2006 – 07, and Outstanding
Young Researcher Award, The University of Hong Kong; and
the Research Fund for the Control of Infectious Diseases of the
Health, Welfare and Food Bureau of the Hong Kong SAR
Government.
Transparency declarations
None to declare.
References
1. Chopra I, Roberts M. Tetracycline antibiotics: mode of action,
applications, molecular biology, and epidemiology of bacterial resistance. Microbiol Mol Biol Rev 2001; 65: 232–60.
2. Roberts MC. Update on acquired tetracycline resistance genes.
FEMS Microbiol Lett 2005; 245: 195–203.
3. Hartman AB, EssietII, Isenbarger DW et al. Epidemiology of
tetracycline resistance determinants in Shigella spp. and enteroinvasive Escherichia coli: characterization and dissemination of tet(A)-1.
J Clin Microbiol 2003; 41: 1023–32.
4. Kehrenberg C, Catry B, Haesebrouck F et al. tet(L)-mediated
tetracycline resistance in bovine Mannheimia and Pasteurella isolates.
J Antimicrob Chemother 2005; 56: 403–6.
5. Pratt A, Korolik V. Tetracycline resistance of Australian
Campylobacter jejuni and Campylobacter coli isolates. J Antimicrob
Chemother 2005; 55: 452–60.
6. Yuen KY, Woo PC, Teng JL et al. Laribacter hongkongensis
gen. nov., sp. nov., a novel Gram-negative bacterium isolated from a
cirrhotic patient with bacteremia and empyema. J Clin Microbiol 2001;
39: 4227–32.
7. Woo PC, Kuhnert P, Burnens AP et al. Laribacter hongkongensis:
a potential cause of infectious diarrhea. Diagn Microbiol Infect Dis 2003;
47: 551–6.
8. Lau SK, Woo PC, Hui WT et al. Use of cefoperazone
MacConkey agar for selective isolation of Laribacter hongkongensis.
J Clin Microbiol 2003; 41: 4839–41.
9. Woo PC, Lau SK, Teng JL et al. Association of Laribacter
hongkongensis in community-acquired human gastroenteritis with
travel and with eating fish: a multicentre case–control study. Lancet
2004; 363: 1941–7.
496
Tetracycline resistance in Laribacter hongkongensis
10. Ni XP, Ren SH, Sun JR et al. Laribacter hongkongensis isolated
from a community-acquired gastroenteritis in Hangzhou City. J Clin
Microbiol 2007; 45: 255– 6.
11. Farmer JJ, 3rd, Gangarosa RE, Gangarosa EJ. Does
Laribacter hongkongensis cause diarrhoea, or does diarrhoea ‘cause’
L hongkongensis? Lancet 2004; 363: 1923 – 4.
12. Lau SK, Woo PC, Fan RY et al. Seasonal and tissue distribution
of Laribacter hongkongensis, a novel bacterium associated with gastroenteritis, in retail freshwater fish in Hong Kong. Int J Food Microbiol
2007; 113: 62–6.
13. Teng JL, Woo PC, Ma SS et al. Ecoepidemiology of Laribacter
hongkongensis, a novel bacterium associated with gastroenteritis.
J Clin Microbiol 2005; 43: 919–22.
14. Lau SKP, Woo PCY, Fan RYY et al. Isolation of Laribacter
hongkongensis, a novel bacterium associated with gastroenteritis, from
drinking water reservoirs in Hong Kong. J Appl Microbiol 2007; 103:
507–15.
15. Lau SK, Ho PL, Li MW et al. Cloning and characterization of a
chromosomal class C b-lactamase and its regulatory gene in
Laribacter hongkongensis. Antimicrob Agents Chemother 2005; 49:
1957–64.
16. Clinical and Laboratory Standards Institute. Methods for Dilution
Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—
Seventh Edition: Approved Standard M7-A7. CLSI, Wayne, PA, USA,
2006.
17. Woo PC, Leung PK, Tsoi HW et al. Cloning and characterisation of
malE in Burkholderia pseudomallei. J Med Microbiol 2001; 50: 330–8.
18. Woo PC, Leung PK, Wong SS et al. groEL encodes a highly
antigenic protein in Burkholderia pseudomallei. Clin Diagn Lab
Immunol 2001; 8: 832–6.
19. Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving
the sensitivity of progressive multiple sequence alignment through
sequence weighting, position-specific gap penalties and weight matrix
choice. Nucleic Acids Res 1994; 22: 4673– 80.
20. Pasquier C, Hamodrakas SJ. An hierarchical artificial neural
network system for the classification of transmembrane proteins.
Protein Eng 1999; 12: 631– 4.
21. Bateman A, Coin L, Durbin R et al. The Pfam protein families
database. Nucleic Acids Res 2004; 32: D138– 41.
22. Smith MA, Bidochka MJ. Bacterial fitness and plasmid loss: the
importance of culture conditions and plasmid size. Can J Microbiol
1998; 44: 351–5.
23. Inui M, Roh JH, Zahn K et al. Sequence analysis of the cryptic
plasmid pMG101 from Rhodopseudomonas palustris and construction
of stable cloning vectors. Appl Environ Microbiol 2000; 66: 54–63.
24. Sambrook J, Russell DW. Molecular Cloning: A Laboratory
Manual. Cold Spring Harbor, 3rd edn. New York, USA: Cold Spring
Harbor Laboratory Press, 2001.
25. Bonnet R, Sampaio JL, Labia R et al. A novel CTX-M
b-lactamase (CTX-M-8) in cefotaxime-resistant Enterobacteriaceae
isolated in Brazil. Antimicrob Agents Chemother 2000; 44: 1936–42.
26. Duan RS, Sit TH, Wong SS et al. Escherichia coli producing
CTX-M b-lactamases in food animals in Hong Kong. Microb Drug
Resist 2006; 12: 145–8.
27. Woo PCY, Ma SSL, Teng JLL et al. Plasmid profile and construction of a small shuttle vector in Laribacter hongkongensis.
Biotechnol Lett 2007; 29: 1575–82.
28. Okumura MS, Kado CI. The region essential for efficient autonomous replication of pSa in Escherichia coli. Mol Gen Genet 1992;
235: 55 –63.
29. Messer W. The bacterial replication initiator DnaA. DnaA and
oriC, the bacterial mode to initiate DNA replication. FEMS Microbiol
Rev 2002; 26: 355–74.
30. Qin X, Hartung JS. Construction of a shuttle vector and transformation of Xylella fastidiosa with plasmid DNA. Curr Microbiol 2001;
43: 158–62.
31. Woo PC, Ma SS, Teng JL et al. Construction of an inducible
expression shuttle vector for Laribacter hongkongensis, a novel bacterium associated with gastroenteritis. FEMS Microbiol Lett 2005; 252:
57 –65.
32. Grindley NGF. Resolvase-mediated site-specific recombination.
Nucl Acids Mol Biol 1994; 8: 236–67.
33. Yang W, Steitz TA. Crystal structure of the site-specific recombinase gamma delta resolvase complexed with a 34 bp cleavage site.
Cell 1995; 82: 193–207.
34. Miranda CD, Kehrenberg C, Ulep C et al. Diversity of tetracycline resistance genes in bacteria from Chilean salmon farms.
Antimicrob Agents Chemother 2003; 47: 883–8.
35. Wilkerson C, Samadpour M, van Kirk N et al. Antibiotic resistance and distribution of tetracycline resistance genes in Escherichia
coli O157:H7 isolates from humans and bovines. Antimicrob Agents
Chemother 2004; 48: 1066–7.
36. Sibold C, Henrichsen J, Konig A et al. Mosaic pbpX genes of
major clones of penicillin-resistant Streptococcus pneumoniae have
evolved from pbpX genes of a penicillin-sensitive Streptococcus oralis.
Mol Microbiol 1994; 12: 1013–23.
37. de Vries J, Wackernagel W. Integration of foreign DNA during
natural transformation of Acinetobacter sp. by homology-facilitated
illegitimate recombination. Proc Natl Acad Sci USA 2002; 99: 2094–9.
38. Woo PCY, Teng JLL, Ma SSL et al. Characterization of a novel
cryptic plasmid, pHLHK26, in Laribacter hongkongensis. New Microbiol
2007; 30: 139–46.
39. Furushita M, Shiba T, Maeda T et al. Similarity of tetracycline
resistance genes isolated from fish farm bacteria to those from clinical
isolates. Appl Environ Microbiol 2003; 69: 5336–42.
40. Rhodes G, Huys G, Swings J et al. Distribution of oxytetracycline resistance plasmids between aeromonads in hospital and aquaculture environments: implication of Tn1721 in dissemination of the
tetracycline resistance determinant Tet A. Appl Environ Microbiol 2000;
66: 3883–90.
41. Lalumera GM, Calamari D, Galli P et al. Preliminary investigation
on the environmental occurrence and effects of antibiotics used in
aquaculture in Italy. Chemosphere 2004; 54: 661–8.
42. Rigos G, Nengas I, Alexis M et al. Potential drug (oxytetracycline and oxolinic acid) pollution from Mediterranean sparid fish farms.
Aquat Toxicol 2004; 69: 281–8.
497