Microbiology (2015), 161, 2310–2315
DOI 10.1099/mic.0.000183
TEM-1-encoding small plasmids impose dissimilar
fitness costs on Haemophilus influenzae and
Haemophilus parainfluenzae
Annette Søndergaard, Marianne Lund and Niels Nørskov-Lauritsen
Correspondence
Department of Clinical Microbiology, Aarhus University Hospital, Aarhus N, Denmark
Niels Nørskov-Lauritsen
[email protected]
Received 1 June 2015
Accepted 16 September 2015
Only two beta-lactamases, TEM-1 and ROB-1, have been observed in Haemophilus influenzae,
while four different TEM but no ROB enzymes have been found in Haemophilus parainfluenzae.
In order to investigate the mechanisms behind the dissemination of small beta-lactamaseencoding plasmids in H. influenzae and H. parainfluenzae, we assessed the fitness cost of
three TEM-1- (pPN223, pA1209, pA1606), one TEM-15- (pSF3) and one ROB-1-bearing
(pB1000) plasmid when expressed in either bacterial species. All plasmids were stable in
H. influenzae and H. parainfluenzae except pB1000, which showed on average (sample mean)
76 % curing in H. parainfluenzae after 5 days of subculture. Competition assays between
isogenic strains with and without plasmid showed no competitive disadvantage of pPN223 and
pA1606 in H. influenzae, or of pA1209 in H. parainfluenzae. In contrast, pSF3 and pB1000
were associated with significant competitive disadvantages in both species. Some of the
competitive disadvantages may be related to differences in plasmid copy number and mRNA
expression of the beta-lactamase genes, as revealed by quantitative PCR analysis. In
conclusion, plasmids encoding TEM beta-lactamases isolated from H. influenzae and H.
parainfluenzae can be stably transferred between species. The fast curing of pB1000 in H.
parainfluenzae observed in this study correlates to the fact that ROB-1 has never been reported
for this species. TEM-1-encoding plasmids are associated with the lowest level of fitness cost,
but different TEM-1 plasmids confer different levels of fitness cost on the two hosts.
INTRODUCTION
Resistance to beta-lactam antimicrobial agents in Haemophilus influenzae is commonly mediated by production of
beta-lactamases. Only two beta-lactamases, TEM-1 and
ROB-1, have been described for H. influenzae, with TEM1 being by far the more prevalent (95 %) of the two (Farrell
et al., 2005). Four different TEM beta-lactamases (TEM-1,
TEM-15, TEM-34 and TEM-182) have been documented
in H. parainfluenzae (Tristram et al., 2008; Garcı́a-Cobos
et al., 2013).
In H. influenzae, TEM-1 is primarily encoded on large integrative and conjugative elements (Leaves et al., 2000), and
less commonly (14–17 %) on small plasmids (Søndergaard
et al., 2012; Tristram et al., 2012; Fleury et al., 2014). Six
different blaTEM-1-bearing plasmids of 4–6 kb have been
characterized (Søndergaard et al., 2012; Tristram et al.,
Abbreviations: AMP, ampicillin; ESBL, extended spectrum betalactamase; sBHI, Brain Heart infusion broth supplemented with NAD
and haemin.
One supplementary table is available with the online Supplementary
Material.
2310
2012). These plasmids can be separated into three
groups: the two-gene plasmids encode blaTEM-1 and rep,
the three-gene plasmids also encode mob/pre, and the
four-gene plasmids also encode tnpR. The two-gene
group is only represented by pPN223 (4304 bp), which
also has a distinct backbone sequence. The three-gene
group encompasses pLFS5 and pA1209 (5142 bp), while
the four-gene group encompasses pLFH49, pLFH64
and pA1606 (5646 bp). Transcription of TEM-1 is regulated by two different promoters, namely Pa/Pb (pPN223
and pLFH49), and Prpt (pLFS5, pA1209, pLFH64 and
pA1606).
The plasmid encoding ROB-1 in H. influenzae is designated
pB1000 and is 4.6 kb in size (San Millan et al., 2010).
pB1000 has been detected in other Pasteurellaceae species
of animal origin (San Millan et al., 2007, 2009, 2010; Tristram et al., 2010), but not in H. parainfluenzae. In H. parainfluenzae blaTEM-15 and blaTEM-34 have been found on
small plasmids (3.7 kb and 5.5 kb, respectively), whereas
blaTEM-1 and blaTEM-182 have been described on large integrative and conjugative elements (Tristram et al., 2008;
Garcı́a-Cobos et al., 2013). Plasmid pSF3 encoding
TEM-15 (an extended spectrum beta-lactamase – ESBL)
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Printed in Great Britain
Fitness cost of small beta-lactamase plasmids
has not been fully sequenced, but TEM-15 is under control
of the Prpt promoter. A plasmid bearing TEM-34 was not
available for this investigation; the reported plasmid
(p72322) is identical to pLFH64, except for the single
amino acid substitution in TEM that renders the beta-lactamase resistant to clavulanic acid (Garcı́a-Cobos et al., 2013).
H. influenzae and H. parainfluenzae were routinely cultured in Brain
Heart Infusion broth (Oxoid), supplemented with XV supplement
(HTM supplement; Oxoid) (sBHI), and on chocolate agar plates
(Statens Serum Institut). Inoculated agar plates were incubated at
37 uC in an atmosphere containing 5 % CO2. Broth cultures were
incubated in ambient air at 37 uC with shaking (130 r.p.m.).
pB1000 has a competitive disadvantage of approximately
9 % per ten generations relative to the parental strain
when transformed into H. influenzae strain Rd (San
Millan et al., 2010). Fitness cost analyses of blaTEM-bearing
plasmids have not, to our knowledge, been performed.
To aid our understanding of the epidemiology of small
beta-lactamase-bearing plasmids, we assessed stability
and fitness cost, and performed expression analysis after
introduction of TEM-1-, TEM-15- and ROB-1-bearing
plasmids into recipient strains of H. influenzae and
H. parainfluenzae.
Plasmid extraction and transformation. Plasmid DNA was
extracted using a QIAprep Spin Miniprep kit (Qiagen) according to
the manufacturer’s instructions.
Electrocompetent cells were prepared from H. influenzae strain Rd
and H. parainfluenzae strain HK 23 and transformed with purified
plasmid DNA using electroporation as described elsewhere (Ubukata
et al., 2001). Transformants were selected on chocolate agar supplemented with 4 mg ampicillin (AMP) ml21. Electroporated controls
with no added DNA were plated on selective agar to assess the
selective quality of the agar. Selected colonies from each transformation experiments were subcultured twice on AMP-containing
agar. Production of beta-lactamase was confirmed using nitrocefin
dry slides (BBL DrySlide Nitrocefin; Becton, Dickinson and
Company).
Growth kinetics, plasmid curing and competition experiments.
METHODS
Bacterial strains, culture conditions and susceptibility testing.
The characteristics of the strains and plasmids used in this study are
listed in Table 1.
Ampicillin-susceptible H. influenzae strain Rd (Fleischmann et al.,
1995) and H. parainfluenzae strain HK 23 (CCUG 49489) (Hedegaard
et al., 2001) were used as transformation recipients. Small beta-lactamase bearing plasmids were isolated from strains of H. influenzae
(pPN223, pA1209 and pA1606) (Søndergaard et al., 2012) or
H. parainfluenzae (pSF3) (Tristram et al., 2008). Plasmid pB1000 was
kindly donated by Professor Bruno Gonzales-Zorn (San Millan et al.,
2010). The blaTEM-1 plasmids used in this study represent each of the
three TEM-1-bearing small plasmid types hitherto characterized in
H. influenzae.
The fitness cost of each plasmid was assessed by growth kinetics,
plasmid curing and competition between recipient strain and transformant. A saline suspension with an optical density of 1.0 McFarland
was prepared for each strain or transformant and used for the simultaneous execution of the three assays. The assays were repeated on
four separate occasions.
For measurement of growth kinetics, the saline suspensions were
diluted 1000 times in sBHI, and 200 ml was added to wells in triplicate
in a 96-well flat bottomed plate. Absorbance at 620 nm was measured
every 10 min for 24 h on a microplate spectrophotometer (Thermo
Scientific Multiskan GO Microplate Spectrophotometer) set on 37 uC
and 1 min of shaking every other minute.
To determine the stability of the plasmids, saline suspensions of
H. influenzae strain Rd and H. parainfluenzae strain HK 23
Table 1. Strains and plasmids used in this study
Strain or plasmid
Strains
Rd KW20
HK23 (CCUG 49489)
Rd/pPN223
Rd/pA1209
Rd/pA1606
Rd/pSF3
Rd/pB1000
HK23/pPN223
HK23/pA1209
HK23/pA1606
HK23/pSF3
HK23/pB1000
Plasmids
pPN223
pA1209
pA1606
pSF3
pB1000
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Description and characteristics
Reference
Amps H. influenzae recipient strain
Amps H. parainfluenzae recipient strain
Rd KW20 transformed with pPN223
Rd KW20 transformed with pA1209
Rd KW20 transformed with pA1606
Rd KW20 transformed with pSF3
Rd KW20 transformed with pB1000
HK23 transformed with pPN223
HK23 transformed with pA1209
HK23 transformed with pA1606
HK23 transformed with pSF3
HK23 transformed with pB1000
Fleischmann et al. (1995)
Hedegaard et al. (2001)
Søndergaard et al. (2012)
Søndergaard et al. (2012)
Søndergaard et al. (2012)
This study
This study
This study
This study
This study
This study
This study
rep, blaTEM-1; isolated from H. influenzae
rep, blaTEM-1, mob/pre; isolated from H. influenzae
rep, blaTEM-1, mob/pre, tnpR; isolated from H. influenzae
rep, blaTEM-15; isolated from H. parainfluenzae
mobA, mobB, mobC, blaROB-1; isolated from H. influenzae
Søndergaard et al. (2012)
Søndergaard et al. (2012)
Søndergaard et al. (2012)
Tristram et al (2008)
San Millan et al. (2010)
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2311
A. Søndergaard and others
transformed with each plasmid were diluted 1000 times in sBHI and
incubated at 37 uC with continuous shaking (130 r.p.m.). Every 24 h
for 5 days, the suspension was diluted 1000 times in fresh antibioticfree sBHI, and aliquots were plated on nonselective chocolate agar.
The proportion of colonies expressing beta-lactamase was assessed by
replica plating of 50 colonies on chocolate agar plates containing 4 mg
AMP ml21.
For competition experiments, 5 ml of the saline suspension of
H. influenzae strain Rd or H. parainfluenzae strain HK 23 were mixed
with equal amounts of the same strain transformed with the plasmid
in question in 5 ml of antibiotic-free sBHI. The mixture was incubated at 37 uC with continuous shaking (130 r.p.m.). Every 24 h for
5 days, a total of 5 ml of the mixture were diluted 1000 times in fresh
sBHI, and aliquots were plated on nonselective chocolate agar. The
proportion of colonies expressing beta-lactamase was assessed by
replica plating of 100 colonies on chocolate agar plates containing
4 mg AMP ml21.
Plasmid copy number and beta-lactamase expression. Strains
were grown in sBHI to an OD600 of 0.2–0.3 at 37 uC and 130 r.p.m.
Five millilitres of suspension was centrifuged and the pellet was
resuspended in 1 ml RNAprotect Bacterial reagent (Qiagen) and kept
at 220 uC until further use. RNA and DNA were extracted from the
suspension using a Magna Pure Compact instrument using a MagnaPure Compact Nucleic Acid Isolation kit (Large Volumen)
(Roche). The samples were split into an RNA sample for preparation
of cDNA, and a DNA sample for assessment of the plasmid copy
number. Residual DNA was degraded from the RNA sample using a
Turbo DNA free kit (Ambion/Life Technologies) and 50 % more
DNase than recommended by the manufacturer in a Veriti 96-well
Thermal Cycler (Applied Biosystems) for 30 min at 37 uC, and 5 min
at 95 uC. cDNA was prepared with TaqMan Reverse Transcription
reagents (Life Technologies) in a Veriti 96-well Thermo Cycler for
10 min at 25 uC, 30 min at 48 uC and 5 min at 95 uC. The cDNA and
purified DNA were mixed with primers, probes, and TaqMan Fast
Advanced Master Mix (Life Technologies) and quantitative PCR was
run in triplicates in a LightCycler 480 (Roche). RNA reverse transcriptase served as a negative control. Relative gene expression and
plasmid copy number were quantified with LightCycler Relative
Quantification software (Roche Applied Science) and normalized
to the single-copy housekeeping genes adk, fucK and mdh
(H. influenzae), or adk, mdh and recA (H. parainfluenzae). The PCRs
were designed to have similar amplification efficiency (primers and
probes are listed in Table S1, available in the online Supplementary
Material).
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Fitness cost of small beta-lactamase-encoding
plasmids in H. influenzae and H. parainfluenzae
None of the plasmids appeared to depress the growth rate
of H. influenzae strain Rd (Fig. 1a). For H. parainfluenzae
strain HK 23, plasmids pA1209 and pPN223 did not
affect the growth kinetics, while plasmids pA1606, pSF3
and pB1000 reduced both the growth rate and the maximal
cell density at stationary phase assessed after 24 h (Fig. 1b).
Competition assays between recipient and transformant
can reveal subtle disadvantages that are not readily disclosed by simple growth curve measurements (Andersson &
Hughes, 2010). Equal numbers of bacteria with and without plasmid were mixed and subcultured every 24 h for
5 days, and the proportion of bacteria carrying the plasmid
at each time point was monitored. Fig. 2 shows a significant
cost imposed on both H. influenzae and H. parainfluenzae
by TEM-15 (pSF3)- and ROB-1 (pB1000)-bearing plasmids: after only 24 h, H. parainfluenzae strain HK 23 harbouring plasmids pB1000 or pSF3 was almost completely
outnumbered by the parental strain without plasmid,
while the effects on H. influenzae were more protracted
(Fig. 2g–j). The competitive disadvantage of plasmid
pSF3 on H. parainfluenzae strain HK 23 may partly explain
why TEM-15, an ESBL, has only sporadically been reported
in this species (Tristram et al. 2008). However, it is a matter
of concern that the fitness cost of this replicon is lower in
the more pathogenic H. influenzae (Fig. 2g) than in its
theoretical native host (Fig. 2h). The competitive disadvantage of plasmid pB1000 on H. influenzae strain Rd found in
this study is in agreement with that reported by San Millan
et al. (2010). In contrast, the three TEM-1-bearing plasmids were differentially tolerated by the two bacterial
hosts: pPN223 and pA1606 did not impose a significant
fitness cost to H. influenzae strain Rd, and pA1209 did
not impose a significant fitness cost to H. parainfluenzae
strain HK 23, while the reverse combinations resulted in
significant disadvantages (Fig. 2a–f). The initial fitness
(b)
Rd
Rd/pPN223
Rd/pA1209
Rd/pA1606
Rd/pB1000
Rd/pSF3
OD620
OD620
(a)
RESULTS AND DISCUSSION
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
HK23
HK23/pPN223
HK23/pA1209
HK23/pA1606
HK23/pB1000
HK23/pSF3
0 1 2 3 4 5 6 7 8 9 101112131415161718192021222324
0 1 2 3 4 5 6 7 8 9 101112131415161718192021222324
Time (h)
Time (h)
Fig. 1. 24 h growth curves of (a) H. influenzae strain Rd isogenic transformants, and (b) H. parainfluenzae HK 23 isogenic
transformants. The curves represent the mean of three independent experiments.
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Microbiology 161
Fitness cost of small beta-lactamase plasmids
Relative percentage
Relative percentage
24
48
72
Time (h)
96
Rd/pSF3
Rd
24
48
72
Time (h)
96
120
48
72
Time (h)
96
24
48
72
96
24
48
72
Time (h)
96
48
72
Time (h)
96
120
HK23/pA1606
HK23
0
120
24
100
90
80
70
60
50
40
30
20
10
0
120
HK23/pSF3
HK23
Rd/pA1209
Rd
0
(i)
0
100
90
80
70
60
50
40
30
20
10
0
120
Rd/pA1606
Rd
0
100
90
80
70
60
50
40
30
20
10
0
24
(f)
100
90
80
70
60
50
40
30
20
10
0
120
(h)
100
90
80
70
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
0
120
HK23/pA1209
HK
0
(j)
96
(e)
100
90
80
70
60
50
40
30
20
10
0
0
(g)
48
72
Time (h)
Relative percentage
Relative percentage
(d)
24
HK23/pPN223
HK23
Relative percentage
0
(c)
100
90
80
70
60
50
40
30
20
10
0
Relative percentage
Relative percentage
Rd/pPN223
Rd
Relative percentage
(b)
100
90
80
70
60
50
40
30
20
10
0
Relative percentage
Relative percentage
(a)
100
90
80
70
60
50
40
30
20
10
0
24
48
72
Time
Time(h)
(h)
96
120
Rd/pB1000
Rd
0
24
48
72
Time (h)
96
120
HK23/pB1000
HK23
0
24
48
72
Time (h)
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96
120
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A. Søndergaard and others
Fig. 2. Competition profiles of H. influenzae strain Rd isogenic transformants and H. parainfluenzae strain HK 23 isogenic
transformants transformed with the beta-lactamase-encoding plasmid pPN223 (a, b), pA1209 (c, d), pA1606 (e, f), pSF3
(g, h) or pB1000 (i, j). The graphs represent the sample mean of four independent experiments with the standard deviation
represented by error bars.
cost imposed by pA1606 on H. influenzae was not detectable after 5 days (Fig. 2e). We did not sequence the plasmids at the end of the experiment, and mutations leading
to a decrease in fitness cost cannot be excluded. However,
the effect was similar in four separate experiments. Another
possible mechanism of amelioration of fitness cost of newly
introduced plasmids is reduction of plasmid copy number
(San Millan et al., 2015).
colonies) of the HK 23/pB1000 strains had lost pB1000.
pB1000 has been detected in several Pasteurellaceae species
(San Millan et al., 2007, 2009, 2010; Tristram et al., 2010),
but not in H. parainfluenzae. The imposed fitness cost and
the relatively fast curing of pB1000 may explain the absence
of ROB-1 in H. parainfluenzae.
Plasmid copy number and beta-lactamase
expression in relation to fitness cost of small
beta-lactamase-encoding plasmids
A recent study of H. influenzae AMP-resistance in Sweden
found that 16 % of 278 beta-lactamase expressing isolates
carried TEM-1 on small plasmids. Of the 44 small TEM-1bearing plasmids, four were pPN223, 19 were similar to
A1209, while 21 were similar to pA1606 (Fleury et al.,
2014). The low fitness cost of pA1606 could be implicated
in the high prevalence of this plasmid type, while the low
prevalence of pPN223 could be related to its lack of the
mob/pre mobilization gene. However, our fitness cost analysis does not give any clues to the high prevalence of
pA1209 in H. influenzae reported in that study.
Synthesis of plasmid copies, mRNA and encoded proteins
impose an energetic burden, which may lead to a fitness
cost for the recipient cell (Lenski, 1998). Therefore, plasmid copy number and beta-lactamase mRNA expression
were assessed for the five plasmids in H. influenzae strain
Rd and H. parainfluenzae strain HK 23.
Levels of beta-lactamase mRNA (normalized to the
expression of three housekeeping genes) are presented in
Fig. 3(a). The results show that TEM-15 and ROB-1 were
expressed at a higher level than TEM-1 in both species.
Variation in blaTEM promoters has previously been
reported and could affect TEM expression (Tristram
et al., 2007, 2012). Plasmids pA1606 and pA1209 carry
the Prpt promoter, whereas pPN223 carries the Pa/Pb
promoter. Variation in promoter sequences did not affect
transcription levels of TEM-1 (Fig. 3a).
The plasmid curing assay measures the stability of plasmids
within the host cells. This was performed by subculturing
the transformants in non-selective broth every 24 h for
5 days and assessing the proportion of cells that had lost
the plasmid and become susceptible to AMP. Curing was
only observed for H. parainfluenzae strain HK 23 harbouring the ROB-1-encoding plasmid pB1000. After 5 days,
76+ 12 % (N53; 31–44 colonies out of 50 replicate plate
(a)
(b)
2
1
HK23_pB1000
HK23_pSF3
HK23_pA1606
HK23_pA1209
0
HK23_pPN223
HK23_pB1000
HK23_pSF3
HK23_pA1606
HK23_pA1209
HK23_pPN223
Rd_pB1000
Rd_pSF3
Rd_pA1606
Rd_pA1209
Rd_pPN223
0
3
Rd_pB1000
20
4
Rd_pSF3
40
5
Rd_pA1606
60
6
Rd_pA1209
80
7
Rd_pPN223
Beta-lactamase DNA copies
Beta-lactamase mRNA
100
Fig. 3. Quantitative PCR analysis of beta-lactamase mRNA and DNA in isogenic transformants of H. influenzae strain Rd and
H. parainfluenzae strain HK 23. Data are normalized to housekeeping genes adk, mdh and fucK (H. influenzae) or adk, mdh
and recA (H. parainfluenzae). (a) Average mRNA expression of the various beta-lactamases (arbitrary units); (b) Average
plasmid copy number per cell. Each bar represents the mean quantitative PCR results of three (H. influenzae) or four
(H. parainfluenzae) individual plasmid-transformants with the standard deviation represented by error bars.
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Microbiology 161
Fitness cost of small beta-lactamase plasmids
Plasmid quantification (normalized to single-copy housekeeping genes) showed a generally lower plasmid copy
number per cell in H. parainfluenzae strain HK 23 compared to H. influenzae strain Rd (Fig. 3b). The plasmid
copy number of pA1209 was much lower in both species,
while pA1606, pSF3 and pB1000 were present in the highest
copy numbers per cell (Fig. 3b).
Hedegaard, J., Okkels, H., Bruun, B., Kilian, M., Mortensen, K. K. &
Nørskov-Lauritsen, N. (2001). Phylogeny of the genus Haemophilus
as determined by comparison of partial infB sequences. Microbiology 147, 2599–2609.
Leaves, N. I., Dimopoulou, I., Hayes, I., Kerridge, S., Falla, T., Secka,
O., Adegbola, R. A., Slack, M. P., Peto, T. E. & Crook, D. W. (2000).
Epidemiological studies of large resistance plasmids in Haemophilus.
J Antimicrob Chemother 45, 599–604.
High beta-lactamase expression in transformants harbouring pSF3 and pB1000 could contribute to the increased
fitness cost observed for these plasmids. However, the level
of mRNA expression and plasmid copy number of the
three TEM-1-encoding plasmids did not reflect the different
profiles of fitness cost observed for the two bacterial hosts.
Lenski, R. E. (1998). Bacterial evolution and the cost of antibiotic
In conclusion, we show that small plasmids encoding TEM
beta-lactamases isolated from H. influenzae and H. parainfluenzae can be stably transferred to recipient strains of the
other species. TEM-1-encoding plasmids are associated
with the lowest level of fitness cost in both species, but
different TEM-1 plasmids confer different burden on the
two hosts. The results are corroborated by the predominance of the TEM-1 beta-lactamase in H. influenzae,
while more studies on the prevalence and type of small
resistance plasmids in H. parainfluenzae are needed.
San Millan, A., Escudero, J. A., Gutierrez, B., Hidalgo, L., Garcia, N.,
Llagostera, M., Dominguez, L. & Gonzalez-Zorn, B. (2009).
resistance. Int Microbiol 1, 265–270.
San Millan, A., Escudero, J. A., Catalan, A., Nieto, S., Farelo, F.,
Gibert, M., Moreno, M. A., Dominguez, L. & Gonzalez-Zorn, B.
(2007). Beta-lactam resistance in Haemophilus parasuis is mediated
by plasmid pB1000 bearing blaROB-1. Antimicrob Agents Chemother
51, 2260–2264.
Multiresistance in Pasteurella multocida is mediated by coexistence
of small plasmids. Antimicrob Agents Chemother 53, 3399–3404.
San Millan, A., Garcia-Cobos, S., Escudero, J. A., Hidalgo, L.,
Gutierrez, B., Carrilero, L., Campos, J. & Gonzalez-Zorn, B. (2010).
Haemophilus influenzae clinical isolates with plasmid pB1000
bearing blaROB-1: fitness cost and interspecies dissemination.
Antimicrob Agents Chemother 54, 1506–1511.
San Millan, A., Santos-Lopez, A., Ortega-Huedo, R., Bernabe-Balas, C.,
Kennedy, S. P. & Gonzalez-Zorn, B. (2015). Small plasmid-mediated
antibiotic resistance in Haemophilus influenzae is enhanced by
increases in plasmid copy number and bacterial fitness. Antimicrob
Agents Chemother 59, 3335–3341.
ACKNOWLEDGEMENTS
Søndergaard, A., San Millan, A., Santos-Lopez, A., Nielsen, S. M.,
Gonzalez-Zorn, B. & Nørskov-Lauritsen, N. (2012). Molecular
Line Nymann Penstoft is acknowledged for excellent technical assistance with quantitative PCR.
organization of small plasmids bearing blaTEM-1 and conferring
resistance to b-lactams in Haemophilus influenzae. Antimicrob
Agents Chemother 56, 4958–4960.
Tristram, S., Jacobs, M. R. & Appelbaum, P. C. (2007). Antimicrobial
resistance in Haemophilus influenzae. Clin Microbiol Rev 20, 368–389.
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