Journal of Antimicrobial Chemotherapy (1999) 43, 47-54
© 1999 The British Society for Antimicrobial Chemotherapy
Amoxycillin tolerance in Helicobacter pylori
a VA Medical Center and Baylor College of Medicine, Houston, TX 77030, USA; b Institute of Internal Medicine and c Institute of Hygiene and Preventive Medicine, University of Sassari, Italy
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Abstract |
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Resistance to amoxycillin in Helicobacter pylori has only recently been reported. To demonstrate the existence of resistance, and to test for the presence of tolerance, 17 amoxycillin-resistant strains of H. pylori, first isolated in Sardinia (Italy) and the USA, were studied. Four amoxycillin-sensitive strains were used as controls. Primary isolates of all test strains exhibited amoxycillin resistance; ß-lactamase activity was not detected. Amoxycillin resistance was lost after storage of strains at -80°C but could be rescued by plating these strains on to amoxycillin gradient plates. MICs and MBCs from rescued isolates ranged from 0.5 to 32 mg/L and from 32 to >1024 mg/L, respectively. MBC/MIC ratios
32 are
characteristic of antibiotic tolerance. The ratios of MBC/MIC of amoxycillin ranged from 32 to
>1024 for the test strains, indicating that these strains were tolerant to the antibiotic.
Amoxycillin resistance does occur in H. pylori. Amoxycillin susceptibility testing of H. pylori isolates in patients who fail therapy should include determination of the MBC
to detect tolerance. |
Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Helicobacter pylori is implicated in the aetiology of duodenal and gastric ulcer disease. 1,2 A large number of studies have demonstrated that successful treatment of H. pylori infection results in healing of gastritis and cure of peptic ulcer disease. 3,4, 5 Oral triple-therapy schedules using metronidazole or tinidazole have proved highly effective in the treatment of H. pylori infection, 5 but as resistance to metronidazole becomes more prevalent, metronidazole is increasingly being replaced by amoxycillin in triple therapy. Initially, cure of H. pylori infections was achieved in up to 90% of treated patients, with simple and well- tolerated treatment schedules combining medium- or high-dose omeprazole with amoxycillin. 6,7 More recently, the effectiveness of amoxycillin-containing therapies has varied widely, and inexplicably, ranging from 0 to 90%4,8 ,9 ,10 and enthusiasm for dual therapy has consequently declined.
Amoxycillin-resistant isolates of H. pylori have recently been identified in patient
samples in Italy, the USA and Canada.
11,12 The group of
patients from Italy in whom amoxycillin-resistant H. pylori were found were
participants in a clinical trial that included omeprazole and amoxycillin. The presence of
amoxycillin resistance was associated with a marked reduction in efficacy of treatment.
13 During attempts to
confirm and characterize H. pylori amoxycillin resistance, it was observed that freezing H. pylori strains at -80°C resulted in loss of the amoxycillin resistance
phenotype. A similar phenomenon has been described previously with other bacteria exhibiting
tolerance to ß-lactam antibiotics.14,15,16,17Tolerance is said to be exhibited by bacteria when there is a significant difference
between inhibitory and cidal concentrations.
18,19 Usually it is
accepted that a bacterial strain shows tolerance to a ß-lactam antibiotic if the MBC/MIC
ratio is 32,20
,21,22 but some
authors have suggested ratios ranging from 8 to 100.
18,23 Although
antibiotic tolerance is less well understood than other mechanisms of resistance to
ß-lactams, it can be exhibited by a number of clinically relevant Gram-negative and
Gram-positive pathogenic bacteria, including Haemophilus influenzae,
24 Legionella pneumophila,
25 Streptococcus pneumoniae
26and Staphylococcus aureus.
14
Loss of tolerance by staphylococci 14 and streptococci 15,16 has been described after storage in broth at 20°C, 4°C and -70°C. The resistance phenotype can be restored by consecutive transfers on to penicillin gradient-agar plates. 16,27 Tolerance has been proposed as one possible explanation for the poor response of some bacterial infections to penicillin therapy.28,29,30,31,32
The aims of this study were to demonstrate the existence of amoxycillin-resistant H. pylori, to attempt to rescue amoxycillin resistance in strains that had lost the resistance phenotype after freezing and to test whether H. pylori resistance to amoxycillin corresponds to the phenomenon of tolerance to ß-lactam antibiotics.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Isolation and identification of bacterial strains
Amoxycillin-resistant H. pylori strains were isolated from three patients from Florida and California with peptic ulcer disease. Fourteen amoxycillin-resistant strains were isolated from dyspeptic patients from Sardinia, Italy. The strains were stored at -80°C for 5–10 months, in cysteine–Albimi broth containing 20% glycerol, until processed. 33 All strains were frozen at the same rate by placing the bacterial stock vials directly at -80°C. For subculture, frozen cultures were thawed rapidly (in <1 min) at room temperature.
Antibiotic susceptibility testing with Etest
Before and after storage at -80°C, the MICs of amoxycillin were determined by the Etest method (AB Biodisk, Uppsala, Sweden). Mueller–Hinton agar plates (150 mm) containing 5% sheep blood (BBL, Cockeysville, MD, USA) were covered with each suspended inoculum to produce a lawn of bacterial growth. Etest results were read after incubation at 37°C for 72 h in an atmosphere with 99% relative humidity and 12% CO2. MICs were determined by noting the point of intersection of the elliptical zone of inhibition with the graded Etest strip. Isolates were considered resistant to amoxycillin when the MIC was .8 mg/L, the susceptibility breakpoint recommended in guidelines for fastidious organisms. 34
ß-Lactamase assay
The chromogenic cephalosporin method was used to test the production of ß-lactamase. Cefinase discs (Becton– Dickinson Microbiology Systems, Cockeysville, MD, USA) impregnated with nitrocefin, a chromogenic cephalosporin, were moistened with a drop of sterile distilled water. Several well-isolated colonies from the agar plate were selected and smeared over the surface of the disc. The presence of ß-lactamase was indicated by a change in the colour of the disc.35 Activity was considered absent if no colour change had occurred after 2 h of incubation at room temperature.
Use of amoxycillin gradient plates
A modification of the method described by Kim & Anthony 27 was used to restore amoxycillin resistance. Stock solutions of amoxycillin (Sigma Chemical Co., St Louis, MO, USA) were prepared at 2000 mg/L by addition of autoclaved double-distilled water. The stock solutions were freshly prepared and sterilized by passage through 0.22 µm filters. Gradient plates were prepared with brain heart infusion agar (BHI agar) (Difco Laboratories, Detroit, MI, USA). After autoclaving, the medium was cooled to 50°C and 10 mL of sterile horse serum (HyClone Labs, Logan, UT, USA) and amoxycillin solution were added to prepare the bottom layer of the gradient plate. Plates were prepared with amoxycillin concentrations ranging from 0.5 to 32 mg/L. The top layer was prepared by adding 10 mL of sterile horse serum to BHI agar. The bottom layer was poured into sterile Petri dishes (150 mm) placed approximately at a 30° angle; after this layer had solidified, the top agar layer was added.
Frozen strains that were resistant to amoxycillin before storage, and strains that were sensitive to amoxycillin, were subcultured on BHI agar plates containing 7% defibrinated horse blood (BHIB agar plates) (Cocalico Biologicals, Reamstown, PA, USA) and incubated for 3–5 days at 37°C in 12% CO2 and 95–99% relative humidity. A loopful (c. 10 µL) of bacterial growth was inoculated on to the gradient plates by streaking the isolate from the zone of lowest to the zone of highest antibiotic concentration. The plates were monitored for the presence of bacterial growth initially after 48 h and then daily for up to 7 days. When growth was noted, bacteria from the half of the plate with highest antibiotic concentration were streaked on to a gradient plate containing the next concentration of amoxycillin. Usually the passages were done every 3–7 days on to the next higher gradient concentration.
MIC determination by the agar dilution method
H. pylori from amoxycillin gradient plates were subcultured on to amoxycillin-free BHIB agar plates and incubated for 3–4 days to obtain abundant bacterial growth. Bacterial suspensions corresponding to a McFarland No. 1 standard (3 x 108 cfu/mL) were prepared in phosphate-buffered saline. A final inoculum of 3 x 10 7 cells was plated on BHIB agar plates containing serial doubling dilutions of amoxycillin. The plates were incubated at 37°C in 12% CO2 and 95–99% relative humidity, and examined after 72 h. The MIC was defined as the lowest antibiotic concentration preventing visible growth. All tests were performed in duplicate.
MBC determinations
MBCs were determined using a method similar to that described by Goodwin et al.
36 Serial doubling dilutions of amoxycillin (from 1024
mg/L to 0.016 mg/L) were made in BHI broth containing 0.25% yeast extract and 10% horse
serum. An initial inoculum of 3 x 107 cfu was suspended in serial doubling
dilutions of amoxycillin in BHI broth,
resulting in a final inoculum of 1 3 10
6 cfu/mL. The bottles were incubated at 37°C, with an atmosphere containing
12% CO2
and 95–99% humidity. After 72 h of incubation, 10 mL of cell suspension was cultured
on BHIB agar amoxycillin-free plates and incubated for 72 h. The MBC was defined as the
lowest concentration killing 99.9% of the original inoculum. All tests were performed in
duplicate.
Killing curves
Killing curves were constructed using two amoxycillin-resistant strains (SS47 and SS260) after being passaged on amoxycillin gradient plates, one amoxycillin-sensitive strain (127JC) and a strain that was resistant before freezing, tested without passage on gradient plates (SS211). An initial inoculum of 3 x107 cfu was suspended in each amoxycillin dilution in BHI broth, resulting in a final inoculum of 1 x 106 cfu/mL. Bacterial killing was determined by exposing the strains to the amoxycillin concentration corresponding to the MIC and MBC of each particular strain, except for strain 127JC, which was tested at 2 x MBC, i.e. SS47 was exposed to 16 mg/L (MIC) and 1024 mg/L (MBC); SS260 to 32 mg/L (MIC) and 1024 mg/L (MBC); SS 211 to 0.32 mg/L (MIC) and 8 mg/L (MBC); and 127JC to 0.032 mg/L (MIC) and 0.064 mg/L (2 3 MBC). Ten-microlitre aliquots were taken after 0, 12, 19, 24, 36, 44, 74, 81 and 87 h of incubation at 37°C in an atmosphere containing 12% CO2 and 95–99% humidity, and plated on to BHIB agar plates. Colony counts were performed after incubation for 72 h, as previously described. 14,37 Antibiotic-free controls were also tested for strains SS260 and 127JC.
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Results |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Identification of amoxycillin-resistant H. pylori
Typical Etest results for amoxycillin-sensitive strains ranged from <0.016 to 0.094 mg/L (Table I). The MIC of amoxycillin that denotes resistance for H. pylori has not been defined. NCCLS guidelines recommend that fastidious organisms should be considered resistant to amoxycillin if MICs are >8 mg/L. 34 Fourteen H. pylori isolates from Italy, identified from subjects in a clinical trial of amoxycillin plus omeprazole, showed pre-treatment amoxycillin resistance with MICs >256 mg/L when tested by the Etest method ( Table I). Similarly, all the strains isolated from patients in the USA had initial MICs >256 mg/L (Table I). The amoxycillin-resistant H. pylori strains were tested for the production of ß-lactamase by the chromogenic cephalosporin method and were negative. After the initial few passages, usually less than three, strains were stored at -80°C in cysteine broth as previously described. 33
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Effect of freezing on H. pylori amoxycillin resistance phenotype and rescue of amoxycillin resistance
To confirm the occurrence of amoxycillin resistance, and to characterize further the putative underlying mechanisms of resistance, H. pylori was cultured from frozen stocks and MICs were determined after storage. After freezing, the resistant strains demonstrated MICs ranging from <0.016 to 0.047 mg/L, well within the range of amoxycillin-susceptible strains (Table I). Several studies have shown that penicillin-tolerant bacterial strains such as group A streptococci, may lose their penicillin resistance phenotype after storage, and that resistance can be restored by consecutive transfers on to penicillin gradient agar plates. 15,27 Using an adaptation of the gradient agar plate method previously described, 27 the amoxycillin resistance phenotype of H. pylori could be rescued. Strains were rescued to different levels of amoxycillin resistance (1.0, 2.0, 4.0, 7.0, 16 and 32 mg/L) by consecutively passaging them on plates containing progressively higher concentrations of amoxycillin (Table II). Strains rescued to each amoxycillin concentration were used for Etest, MIC and MBC testing. Three strains were rescued to the extent that they were resistant to 1 mg/L of amoxycillin, two were rescued to 2 mg/L, two were rescued to 4 mg/L, one to 7 mg/L, one to 16 mg/L and two strains to 32 mg/L of amoxycillin (Table II). There was a good agreement between the results of Etest and agar dilution MIC determinations (Table II). The MIC values correlated well with the concentration in the gradient plate at which the strains were taken for MIC determinations. For example, strain SS47 and SS158 were grown at 16 mg/L and 7.0 mg/L, and had MICs of 16 mg/L and 8.0 mg/L, respectively (Table II).
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MBC and MBC/MIC determinations
Penicillin tolerance is identified by an MBC/MIC ratio 32.
20 At MICs of >32 mg/L, the amoxycillin concentrations
required to demonstrate an MBC of 32 x MIC are excessively high (>1024 mg/L),
precluding a uniform antibiotic solution in the culture medium. Therefore, to demostrate
amoxycillin tolerance, the MBC/MIC ratios were determined only with strains rescued up to 32
mg/L. However, H. pylori strains have been rescued up to resistance to 64 and 128
mg/L
amoxycillin. Eleven resistant H. pylori strains and four sensitive strains were tested,
after being passaged on amoxycillin gradient plates (Table II); strain
SS211 was a resistant strain tested directly from the frozen stock, without passage on
amoxycillin
gradient plates. All strains were tested at amoxycillin concentrations up to 1024 mg/L. The
MBCs for amoxycillin-resistant strains ranged from 32 to >1024 mg/L, while those for
sensitive strains ranged from 0.032 to 0.125 mg/L (Table II). Strain
SS211 had a low MIC (0.32 mg/L) but a relatively high MBC (8 mg/L).
The MBC/MIC ratios for the amoxycillin-resistant strains, tested after serial passage on gradient
plates, ranged from 32 to 1024 and differed markedly from the ratios observed in
amoxycillin-sensitive strains (
3.9), confirming the presence of antibiotic tolerance in the
resistant strains (Table II and Figure 1).
|
) and MBC
(
) values of H. pylori strains.
MICs and MBCs were tested after resistance to amoxycillin was partially rescued by serial
passages of H. pylori on amoxycillin-containing gradient plates, except strain SS211.
Note that the gap between the MIC and MBC for tolerant strains is wider than that of sensitive
strains.Killing curves
The use of time–kill curves has been proposed as the most reliable method for detecting antibiotic tolerance, 17,34 with tolerance being defined as a rate of killing lower than that in non-tolerant organisms. Two amoxycillin-tolerant H. pyloristrains (SS260 and SS47) were tested, as well as a sensitive strain (127JC) and SS211 (see above)(Figure 2). Each strain was tested at both its MIC and MBC. The pattern of bacterial killing for the strains exhibiting amoxycillin tolerance was different from that in the sensitive control. Tolerant strains (SS260 and SS47) showed a low rate of killing during the initial 19–36 h, in contrast to the rapid initial killing observed in the control strain, 127JC (Figure 2). The amoxycillin-resistant and tolerant strain SS260, and the sensitive strain 127JC, were tested in the absence of antibiotic and no significant bacterial killing was observed. Strain SS211, which had an MIC of 0.32 mg/L but an MBC of 8 mg/L, had an intermediate pattern, with a lower rate of death when tested at the MIC than at the MBC.
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) mg/L; SS47 (b) was incubated
with amoxycillin at
concentrations of 16 (
) or 1024 (
) and the sensitive strain 127JC (c, |
Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Failure of therapy for H. pylori infection has been related to poor compliance with the treatment regimen and with the development of antibiotic resistance. 4 The antibiotics most widely used in the treatment of H. pylori infection include metronidazole, clarithromycin, amoxycillin and tetracycline. Antimicrobial resistance to metronidazole and clarithromycin is increasing and may eventually undermine the effectiveness of regimens that use these drugs. Amoxycillin resistance has not been described and it was thought that amoxycillin would have a long period of usefulness for the treatment of this infection. The frequency of amoxycillin-resistant H. pylori in the community is unknown. Attempts to produce amoxycillin-resistant H. pylori strains in the laboratory have failed. 38,39
The loss of the amoxycillin resistance phenotype upon freezing makes the identification,
confirmation and study of amoxycillin resistance in H. pylori difficult. The molecular
mechanisms responsible for this behaviour are unknown, but the same phenomenon has been
described in a number of other bacterial species.14,15,16 As with other
species, the antibiotic resistance phenotype of strains lost after freezing can be restored by
passaging the strain on antibiotic-containing gradient plates.
16,27 Only strains that
were initially resistant demonstrated the resistance phenotype in amoxycillin-containing gradient
plates, with resistance of 32 mg/L (the highest value tested). The amoxycillin-resistant
strains all had a large gap between their MIC and MBC, with MBC/MIC ratios ranging from 32
to >1024, indicating development of tolerance to amoxycillin.
The term drug tolerance was coined by Tomasz et al. 17 and was first applied to Staphylococcus aureus by Best et al.to describe an isolate that was inhibited, but not killed, by oxacillin. 40 Sabath et al. described the isolation of tolerant strains of staphylococci from deep-seated sites of infection, with an unsuspectedly high frequency. 20 Subsequent studies implicated tolerance in the failure of therapy of clinical infections treated with antibiotics that inhibit cell wall synthesis.29,30,31,32 41,42 Our results with H. pylori show that the MBC/MIC ratios meet the criteria for H. pylori tolerance to amoxycillin.
Amoxycillin tolerance was detected in H. pylori strains after rescue of amoxycillin resistance using gradient plates. The process was repeated using a previously resistant strain, SS211, taken directly from frozen stocks. This strain's behaviour was phenotypically intermediate, failing to achieve the arbitrary classification of tolerance (MBC/ MIC >32). Amoxycillin-resistant strains that were not tolerant to the antibiotic were not identified. The fact that the amoxycillin resistance phenotype could be restored, but could not be induced in previously sensitive strains, suggests a possible genetic basis for amoxycillin tolerance in H. pylori, rather than an epiphenomenon related to in-vitro passage on amoxycillin plates. To characterize the amoxycillin tolerance phenotype further, time–kill experiments were performed. Killing curves of amoxycillin- resistant and -tolerant strains are typical in that they demonstrated a low rate of killing during the first 19–36 h ( Figure 2). In contrast, a sensitive strain showed a steady decrease in bacterial counts over time, with a rapid decline in the number of viable cells in the first 24 h.
Our results suggest that penicillin tolerance may be responsible, at least in part, for the failure of amoxycillin-containing treatment regimes for H. pylori in Italy, and possibly in other regions of the world. The failure to identify amoxycillin-resistant H. pylori prior to this study may be related to the instability of this phenotype. All the H. pylori strains described here had MIC values (Etest) >256 mg/L when tested before freezing. It is conceivable that this level of resistance results from selective pressure in vivo, related to amoxycillin therapy. Our results suggest that, when amoxycillin is used and there is a high rate of treatment failure, the presence of amoxycillin resistance and tolerance should be considered. The microbiological study of isolates from patients in whom therapy fails, or whose disease recurs, should include both an MIC and an MBC determination. The MBC values may more accurately characterize H. pylori resistance to amoxycillin. Our study also indicates that it is important to determine the MIC and MBC for H. pylori strains during the initial steps of culture, to avoid losing the resistance phenotype after freezing. Resistance of all commonly used antibiotics to treat H. pylori infection has now been described, indicating the need for the development of new treatment strategies.
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Acknowledgments |
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This work was supported by the Department of Veterans Affairs and by the generous support of Hilda Schwartz. Part of this study was presented as an oral communication in: Digestive Disease Week, May 10–16, 1997, Washington, DC (see Gastroenterology (1997), 112, abstract A105).
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Notes |
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* Corresponding author. Room 3A353 (111D), Department of Medicine, VA Medical Center, 2002 Holcombe Boulevard, Houston, TX 77030, USA. Tel: +1-713-794-7280; Fax: +1-713-790-1040; E-mail: asepulv{at}bcm.tmc.edu
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