Scottish Salmonella Reference Laboratory, Stobhill Hospital, 133 Balornock Road, Glasgow G21 3UW, UK
Received 27 October 2004; returned 2 February 2005; revised 18 March 2005; accepted 20 April 2005
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Abstract |
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Methods: Etest strips were used to determine the ciprofloxacin MICs for nalidixic acid-resistant salmonellae isolated from humans during 19932003.
Results: The percentage of nalidixic acid-resistant salmonellae isolated from human infection in Scotland has increased from 1.4% in 1993, to 8.8% in 1998 and 21.3% in 2003. Of 493 strains of nalidixic acid-resistant Salmonella tested, a single isolate of Salmonella Typhimurium RDNC was resistant to ciprofloxacin at the NCCLS breakpoint of 4 mg/L. Eleven isolates were resistant at the Scottish Salmonella Reference Laboratory breakpoint of 0.5 mg/L, while 466 isolates (94.5%) had reduced susceptibility with MICs of 0.125 mg/L and <0.5 mg/L.
Conclusions: These results show that very few non-typhoidal isolates of Salmonella in Scotland that are resistant to nalidixic acid are also resistant to ciprofloxacin at the NCCLS recommended breakpoint of 4 mg/L. The majority of isolates examined, however, do show reduced susceptibility at 0.125 mg/L. This confirms the necessity for continuing antimicrobial surveillance of resistance to this important antibiotic.
Keywords: resistance surveillance , fluoroquinolones , Etest
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Introduction |
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Many European countries have also noted increased resistance to quinolones. In 1996 in England and Wales, 12% of S. Typhimurium isolates were resistant to ciprofloxacin at 0.125 mg/L, a 12-fold increase since 1994.13 Recent figures from Denmark show that the incidence of nalidixic acid-resistant zoonotic salmonella infections rose from 0.8% in 1995 to 8.5% in 2000.14
The USA is also monitoring the emergence of quinolone-resistant Salmonella. In 1996, 0.4% of non-Typhi Salmonella isolates had decreased susceptibility (MIC 0.25 mg/L) to ciprofloxacin. In 2001, that figure had risen to 1% with 0.2% of these classed as resistant to ciprofloxacin.15
Quinolone and fluoroquinolone antimicrobials inhibit tertiary supercoiling of bacterial DNA, primarily by inhibiting the action of DNA gyrase, a Type II topoisomerase, which consists of two GyrA and two GyrB subunits encoded by gyrA and gyrB, respectively.
A single point mutation in gyrA between amino acids 67 and 106 (known as the quinolone resistance-determining region or QRDR) can give rise to nalidixic acid (a first-generation quinolone) resistance among isolates of Salmonella. This resistance is usually accompanied by a reduction in the susceptibility of these isolates to ciprofloxacin.16,17
All Salmonella isolates of human, veterinary and environmental origin received by the Scottish Salmonella Reference Laboratory (SSRL) are tested for susceptibility against a panel of 14 antibiotics, including nalidixic acid (40 mg/L) and ciprofloxacin (0.125 mg/L and 0.5 mg/L) using a validated in-house breakpoint method. Data from these tests indicated an increase in the incidence of reduced susceptibility to quinolones similar to that observed in other countries.
This study was undertaken to fully characterize the levels of resistance to ciprofloxacin observed in non-Typhoidal Salmonella serotypes in Scotland over the 10 year period 19932003. Accurate MIC data are essential to allow the assessment of any trend towards reduced susceptibility to ciprofloxacin in nalidixic acid-resistant clinical isolates.
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Materials and methods |
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All Salmonella isolates used in this study were taken from the SSRL culture collection of strains isolated in Scotland.
Selection was based on the following criteria: (i) human origin; (ii) nalidixic acid-resistant by breakpoint method; (iii) received by SSRL during 1993, 1998 and 2003. Quality control strains and duplicate samples were excluded and only a single isolate from epidemiologically related outbreaks was included.
In total, 493 isolates of Salmonella were included in the study (Tables 1 and 2).
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Isolates were stored on Dorset Egg slopes at room temperature.
Media
Isolates were inoculated onto cysteine lactose electrolyte deficient (CLED) agar (DM110; Mast Diagnostics, Merseyside, England) plates and incubated overnight at 37°C. For susceptibility tests, MacConkey agar (CM507; Oxoid, Hampshire, England), diagnostic sensitivity agar (CM261; Oxoid) and tryptone water (tryptone, L42 oxoid, 1% w/v; sodium chloride, 301325Q, VWR International Ltd, Leicester, England; distilled water, pH 7.4) were used. Nalidixic acid was purchased from SigmaAldrich (N-4382), Dorset, England. For Etests, MuellerHinton agar plates were purchased from E & O Laboratories, Bonnybridge, Scotland.
Susceptibility testing by breakpoint
A single colony from an overnight MacConkey agar plate was resuspended in 3 mL of tryptone water and incubated at 37°C for 4 h; 100 µL was then inoculated into 3 mL of sterile water, 0.5 mL of which was transferred into a well in a perspex block. Diagnostic sensitivity agar plates containing known concentrations of antibiotic (for nalidixic acid, 40 mg/L) were inoculated with the bacterial dilutions from the perspex block using a Lidwell multipoint inoculator. The plates were allowed to dry and were incubated overnight at 37°C.
Isolates were deemed susceptible if no growth was observed on the antibiotic plate, resistant if there was confluent growth on the antibiotic plate and intermediate if growth was detectable but visibly less than on the control plate (no antibiotic added to the agar).
SSRL routinely uses two breakpoints of 0.125 and 0.5 mg/L for ciprofloxacin susceptibility testing due to the possible clinical significance of this important antimicrobial.
Susceptibility testing by Etest
Ciprofloxacin Etest strips (AB Biodisk, Solna, Sweden) were purchased from Biostat, Stockport, UK (product code 51000868) and stored at 20°C.
Colonies taken from a fresh overnight CLED plate were resuspended in 3 mL of sterile 0.85% saline to a turbidity of 0.5 McFarland units and inoculated onto MuellerHinton agar plates using a cotton wool swab and a rotary plater. Etest strips were placed onto the plates, which were then incubated at 37°C for 18 h. MIC values were determined according to the manufacturer's instructions.
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Results |
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In 1998, the number of nalidixic acid-resistant isolates had risen to 186 (8.8%) from a total of 2109. The MIC of ciprofloxacin was measured by Etest and 173 (93%) were found to have reduced susceptibility. Two of the 173 isolates, both isolates of S. Typhimurium DT104, had high-level resistance with MICs of 0.5 mg/L (Table 1).
Of the 1254 isolates received in 2003, 267 (21.3%) were nalidixic acid-resistant, 260 (97.4%) of which had reduced susceptibility to ciprofloxacin. Only four isolates of Salmonella were found to have MICs >0.5 mg/L in 2003: S. San-diego, 0.75 mg/L; S. Virchow and S. Haifa both 1 mg/L; S. Typhimurium DT RDNC, 12 mg/L (Table 1). The distribution of the MICs of ciprofloxacin for nalidixic acid-resistant Salmonella isolates received by the SSRL in 1993, 1998 and 2003 is summarized in Figure 1.
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Of the 21 different serotypes identified, S. Enteritidis, S. Hadar, S. Typhimurium and S. Virchow were the four main contributors to the levels of nalidixic acid resistance seen in the 493 isolates investigated. S. Enteritidis was the most common, accounting for 72% of all isolates, followed by S. Hadar, S. Typhimurium and S. Virchow, which accounted for 8.9, 8.3 and 5.7% of the total, respectively. Levels of resistance to ciprofloxacin were also very high among these serotypes with 100% of S. Hadar, 95.5% of S. Enteritidis, 92.8% of S. Virchow and 82.9% of S. Typhimurium all resistant at 0.125 mg/L.
S. Enteritidis showed the greatest increase in resistance to nalidixic acid of any individual serotype over the 3 years studied. This was almost entirely due to S. Enteritidis phage type (PT) 1. In 1993, SSRL received 59 isolates of S. Enteritidis PT1 none of which were resistant to nalidixic acid. By 2003, 145 (72.9%) from a total of 199 were resistant at 40 mg/L. (Table 3). Cross-resistance to ciprofloxacin at 0.125 mg/L mirrored this trend. In 1998, 74 (39.8%) of the 186 isolates studied were isolates of S. Enteritidis PT1, 70 (94.6%) of which were resistant to low-level ciprofloxacin. In 2003, 145 (54.3%) of 267 isolates were S. Enteritidis PT1, 140 (96.6%) being cross-resistant to ciprofloxacin at 0.125 mg/L. Over 50% of the S. Enteritidis PT1 isolates received by SSRL in 1998 were isolated from patients with a history of foreign travel (as indicated by epidemiological information submitted with the isolate), however, this had decreased in 2003 to 30%. This apparent decrease may be misleading in that epidemiological information is rarely complete at the time of submission of the isolate to SSRL and therefore the number of patients with a history of foreign travel may be higher than it first appears.
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Discussion |
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In 1993, 47.5% of the isolates found to have reduced susceptibility to ciprofloxacin were S. Enteritidis PT4, which until recently was the most commonly isolated phage type from human Salmonella infections in Scotland. By 2003, this had decreased to only 8.8%. However, low-level ciprofloxacin resistance in S. Enteritidis PT1 had increased from zero in 1993 to 52% in 2003, mirroring the increased incidence of this phage type. This may be a result of expansion of a single nalidixic acid-resistant clone of S. Enteritidis PT1, as analysis of these isolates with PFGE all generate identical profiles (A. Murray and D. J. Brown, unpublished data). However, the increasing occurrence of quinolone resistance in S. Enteritidis isolates belonging to several different phage types indicates that the overall increase in quinolone resistance in this serotype is not due to the clonal expansion of a single resistant strain (Table 4).
In 1993, SSRL received a single isolate of PT3, fully susceptible to both nalidixic acid and ciprofloxacin. However, in 2003, 11 from a total of 12 PT3 isolates were resistant to both nalidixic acid and 0.125 mg/L ciprofloxacin, of which 36% were isolated from people returning from foreign travel. Greater than 50% of 74 S. Enteritidis PT1 isolates were of foreign origin in 1998. This decreased to 30% of 145 S. Enteritidis PT1 isolates in 2003. Nevertheless, resistance to ciprofloxacin is increasingly being seen in isolates of foreign origin. Scientists in Finland have found that among foreign Salmonella isolates from travellers returning from Southeast Asia, ciprofloxacin resistance increased from 3.9 to 23%.20 A study in Denmark also found that quinolone resistance in isolates from patients with a known history of foreign travel was 8.9% compared with 2.4% of domestically acquired infections.14
No isolates of PT1 or PT4 had high-level resistance to ciprofloxacin. This appears to be true for most of the isolates investigated, since only 11 (2.2%) had MICs of 0.5 mg/L. Of six isolates of Salmonella found to have MICs >0.5 mg/L, two were isolated in 1993 and four were isolated in 2003. Resistance to nalidixic acid (a first-generation quinolone) in isolates of Salmonella is regarded as an indicator of decreased susceptibility to ciprofloxacin21,22 and is therefore often used as a screening tool. Recent reports have shown that high-level ciprofloxacin resistance is often a result of the sequential acquisition of mutations in a number of genes, namely gyrA, parC and parE23 and less frequently in gyrB.24
In order to avoid this sequence of events, it has been proposed that when administering quinolones for treatment of Salmonella infections, dosing strategies be based on a concentration that has been named the mutant prevention concentration (MPC) and not on the MIC, which can allow the selection of antibiotic-resistant mutants. The MPC is defined as no colony recovery when 1010 cells are applied to agar plates.25 A recent study has proposed MPC values of ciprofloxacin for strains of S. Enteritidis and S. Typhimurium.26
The occurrence of high-level fluoroquinolone resistance (ciprofloxacin 4 mg/L) in non-typhoidal Salmonella isolates remains rare worldwide but there have been increasing numbers of reports in recent years.6,2729 Although relatively few isolates have tested resistant to high concentrations of ciprofloxacin in Scotland, we think there can be no doubt that the trend towards high-level resistance experienced in other countries will occur here too. The results of this study suggest it is imperative that surveillance of Salmonella isolates be continued at the breakpoint level of 0.125 mg/L, and we would suggest that the NCCLS reconsiders the recommended resistance breakpoint of 4 mg/L. We would also recommend that any resistance detected at or above the 0.125 mg/L breakpoint be confirmed by the MIC method. This is of particular importance when dealing with cases of typhoid fever or extra-intestinal sources.
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Acknowledgements |
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