Nalidixic acid-resistant strains of Salmonella showing decreased susceptibility to fluoroquinolones in the mid-west region of the Republic of Ireland

Rachel Gorman and Catherine C. Adley*

Microbiology Laboratory, Department of Chemical and Environmental Sciences, University of Limerick, Ireland

Keywords: Salmonella, nalidixic acid, fluoroquinolones

Sir,

Salmonellosis is an important public health problem worldwide. To date 2435 serotypes of salmonella have been identified. As salmonella has been implicated in a number of foodborne outbreaks in the Republic of Ireland, its prevention, surveillance and control has become a public health priority.1 Salmonella infection is primarily associated with gastroenteritis. This illness poses a more serious health risk to sensitive populations in the community such as the elderly, young and the immunocompromised, where hospitalization may be required. In such cases effective antimicrobial treatment is essential, and the fluoroquinolones are a very important class of antibiotic in the treatment of salmonellosis. Very few published data are available on fluoroquinolone antimicrobial susceptibility testing of salmonella in the Republic of Ireland. Three previous reports in particular have demonstrated fluoroquinolone susceptibility testing of salmonella isolates from 1996,2 1997–19983 and 2000.1 We now provide an update on the occurrence of reduced susceptibility to nalidixic acid and five fluoroquinolone agents in salmonella isolates from humans and food animals in the mid-west region of the Republic of Ireland between mid-2000 and 2002.

Our study examined a total of 195 salmonella isolates from human (51), food (eight) and veterinary (136) sources in the mid-west region of the Republic of Ireland. In our laboratory these salmonella isolates were tested against nalidixic acid (30 µg), ciprofloxacin (5 µg), ofloxacin (5 µg), pefloxacin (5 µg), norfloxacin (10 µg) and enrofloxacin (5 µg) (Oxoid), in accordance with NCCLS guidelines for antimicrobial disc susceptibility testing.4 The ciprofloxacin MIC was determined by broth macrodilution in IsoSensitest broth (Oxoid) with an inoculum of 105 cfu/mL.5 Escherichia coli ATCC 25922 was included on each test occasion and all results were within recommended limits, indicating the validity of our test procedures.

Nalidixic acid resistance was observed in 2.6% (5/195) of salmonella isolates; these resistant strains were Salmonella enteritidis (two human isolates), Salmonella typhimurium (two bovine isolates) and Salmonella dublin (one bovine isolate). One porcine strain of Salmonella derby expressed intermediate resistance to nalidixic acid (Table 1). Overall, 3.9% (2/51) of human salmonella strains and 2.2% (3/136) of animal salmonella strains were nalidixic acid resistant.


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Table 1.  Summary of disc diffusion zone diameters for nalidixic acid and five fluoroquinolones, and ciprofloxacin MICs
 
The ciprofloxacin-resistant breakpoint has been categorized into low level CpL (MIC 0.25–1.0 mg/L) and high level CpH (MIC > 1 mg/L).6 In our study the MICs of ciprofloxacin were >10-fold lower than those categorized as CpL. Although our ciprofloxacin MICs were quite low (0.01–0.04 mg/L), disc diffusion results did show reduced zone diameter measurements for strains of salmonella expressing nalidixic acid resistance. For example, in S. enteritidis nalidixic acid-susceptible strains, a ciprofloxacin 5 µg disc showed a zone diameter of 32 mm. In contrast, S. enteritidis nalidixic acid-resistant strains showed a zone diameter of 25 mm, suggesting reduced susceptibilities to the fluoroquinolones. Similar differences in zone diameter can also be seen for S. dublin and S. typhimurium (Table 1). This is of particular concern considering that ciprofloxacin and ofloxacin are the antibiotics of choice at the Mid-West Regional Hospital for first-line defence against salmonellosis.

Cormican et al.2 reported the antimicrobial susceptibility testing of 181 Salmonella enterica clinical isolates obtained in 1996. Nalidixic acid susceptibility testing was not performed; however, all strains were found to be susceptible to a ciprofloxacin 5 µg disc, which corresponds to an MIC breakpoint of 1 mg/L according to NCCLS criteria.4 A report by Foley et al.1 included 665 S. enterica isolates from human clinical and food laboratories countrywide in 2000. This report did not examine fluoroquinolone susceptibility; however, nalidixic acid resistance was tested for, and was found in 8.6% of isolates.

In a study of 122 human and animal S. typhimurium DT104 isolates collected between 1997 and 1998 in Ireland, Murphy et al.3 found that 5.6% (6/108) expressed nalidixic acid resistance. Of particular interest to our report is that four of these six nalidixic acid-resistant strains were obtained from the same regional veterinary laboratory in the mid-west region as the strains in our study. In this study, ciprofloxacin MICs for nalidixic acid-resistant salmonella were 0.01–0.04 mg/L, 10-fold less than that observed by Murphy et al.,3 who note that the low numbers of nalidixic acid-resistant isolates indicate the potential for resistance to the fluoroquinolones in the future. In comparison with the Murphy et al.3 study, where all clinical strains were nalidixic acid susceptible, 4% of clinical strains in our study expressed nalidixic acid resistance.

In contrast to data from England and Wales where ciprofloxacin resistance has been reported,6 we did not observe any ciprofloxacin resistance. Threlfall et al.6 showed that when ciprofloxacin resistance is categorized into low level CpL and high level CpH, the occurrence of CpL between 1996 and 1999 has increased, and note that CpH resistance has been associated with foreign travel.

It has been suggested previously that increased incidences of resistance to the fluoroquinolones in zoonotic salmonella are associated with their use in animals.6 Future monitoring of fluoroquinolone usage in animal and human medicine, as well as foreign travel, must also be emphasized. The common finding of most antimicrobial susceptibility reports is the need for support for continued monitoring and surveillance of antimicrobial resistance. Our report further emphasizes this need at both a regional and national level in the Republic of Ireland.

Acknowledgements

We would like to acknowledge the assistance afforded by the personnel of the Public Health Laboratory and the Microbiology Laboratory at the Mid-West Regional Hospital, Limerick, and the personnel at the Regional Veterinary Laboratory, Knockalisheen, Limerick. This work is funded by an Irish American Partnership Fellowship and Strategic Research grant (no. ST99/043).


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Table 2.  Zone size criteria (mm) for quinolones and equivalent MIC breakpoint for ciprofloxacin (mg/L) according to NCCLS standards4
 
Footnotes

* Corresponding author. Tel: +353-61-202646; Fax: +353-61-202568; E-mail: catherine.adley{at}ul.ie Back

References

1 . Foley, B., Cormican, M., Fitzgerald, M. & McKeown, P. (2001). Epidemiology of Salmonella infections in Ireland, 2000. Epi-Insight 2, 2–3.

2 . Cormican, M., Butler, C., Morris, D., Corbett-Feeney, G. & Flynn, J. (1998). Antibiotic resistance amongst Salmonella enterica species isolated in the Republic of Ireland. Journal of Antimicrobial Chemotherapy 42, 116–8.[Free Full Text]

3 . Murphy, T. M., McNamara, E., Hill, M., Rooney, N., Barry, J., Egan, J. et al. (2001). Epidemiological studies of human and animal Salmonella Typhimurium DT104 and DT104b isolates in Ireland. Epidemiology and Infection 126, 3–9.[ISI][Medline]

4 . National Committee for Clinical Laboratory Standards. (1997). Performance Standards for Antimicrobial Disk Susceptibility Tests—Sixth Edition: Approved Standard M2-A6. NCCLS, Wayne, PA, USA.

5 . Andrews, J. M. (2001). Determination of minimum inhibitory concentrations. Journal of Antimicrobial Chemotherapy 48, Suppl. S1, 5–16.[Abstract/Free Full Text]

6 . Threlfall, E. J., Ward, L. R., Skinner, J. A. & Graham, A. (2000). Antimicrobial drug resistance in non-typhoidal salmonellas from humans in England and Wales in 1999: decrease in multiple resistance in Salmonella enterica serotypes Typhimurium, Virchow, and Hadar. Microbial Drug Resistance 6, 319–25.[ISI][Medline]