ß-Lactamase expression in Plesiomonas shigelloides

Matthew B. Avison*, Peter M. Bennett and Timothy R. Walsh

Bristol Centre for Antimicrobial Research and Evaluation (BCARE), Department of Pathology and Microbiology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have examined the production of ß-lactamases by 11 clinical and nine environmental isolates of Plesiomonas shigelloides from Czechoslovakia, the Czech Republic and Cuba. Ten isolates (50%) expressed detectable amounts of a chromosomally encoded, non-inducible ß-lactamase, though all isolates showed a broadly similar resistance profile: low-level resistance to ampicillin and higher-level resistance to carbenicillin. All strains were susceptible to cephalosporins and meropenem. Three clinical isolates expressed a ß-lactamase similar to a class 2c carbenicillinase, with a pI of 5.2 and three expressed an enzyme similar to a class 2d oxacillinase, with a pI of 5.3. The environmental isolates produced a variety of penicillinases, indicating that there is a reservoir of heterogeneous ß-lactamase genes in this species.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Plesiomonas shigelloides has been reported as the causative agent for rare outbreaks of diarrhoeal disease in humans.1 Increasing numbers of other types of infection that are attributable to the bacterium have also been reported.2 Several virulence-associated factors have been described in isolates of P. shigelloides,2 but most have the potential to cause infection even in the absence of such virulence factors. Immunosuppressed individuals are more susceptible and many patients infected extra-intestinally die.2 Resistance to antimicrobial drugs has been reported in this species, with resistance to aminoglycosides and penicillins being most common.3,4

The aim of this study was to determine the prevalence and type of ß-lactamases in clinical and environmental isolates of P. shigelloides. The strains examined were isolated over a period of some 30 years from 1964 in Czechoslovakia, the Czech Republic and Cuba. Given the historical context of these isolates, it was of particular interest to see if the ß- lactamases produced by isolates collected at specific times, or from specific geographical regions showed similarity.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Bacterial strains

Eleven clinical and nine environmental strains of P. shigelloides were isolated in Cuba, Czechoslovakia and the Czech Republic. The identity of each was confirmed using API 20NE test strips (bioMérieux, La Balme les Grottes, France). All isolates gave the same API profile, so are likely to be the same species. The origin, year of isolation and serovar of each isolate are recorded in Table IGo.


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Table I. The P. shigelloides isolates used and MICs of various ß-lactams against them
 
Materials

Unless otherwise stated, media used were nutrient broth and nutrient agar (Oxoid, Basingstoke, UK). ß-Lactams used were nitrocefin, clavulinic acid, BRL 42715, ampicillin and carbenicillin (SmithKline Beecham, Worthing, UK); oxacillin, cephalothin, cephaloridine and ceftazidime (Sigma Chemical Co., St Louis, MO, USA); piperacillin (Lederle, Carolina, Puerto Rico) and meropenem (Zeneca Pharmaceuticals, Macclesfield, UK). All other reagents were from Sigma or BDH (Poole, UK).

Susceptibility tests

Antibiotic susceptibility tests were performed as described previously.5 The MIC of the test ß-lactam was defined as the lowest concentration of the antibiotic that prevented growth after incubation at 37°C for 18 h. BSAC breakpoints were used to define resistance to particular antibiotics.6

Induction, preparation and assay of ß-lactamases

Bacterial strains were cultured, induction was attempted and cells were pelleted as described previously.5 Following resuspension of the pellet in 1 mL of extraction buffer, the cells were disrupted in a Ribolyser (Hybaid, Teddington, UK) in tubes containing silica beads (Hybaid Blue matrix), with a single 30 s burst (amplitude 6). Cell debris was pelleted by centrifugation (10 min, 4°C, 15000g) and the supernatant was used directly as a source of ß-lactamase.

Hydrolysis of ß-lactam antibiotics was examined as described previously.5 The protein concentration of each bacterial extract was determined using the Bio-Rad protein assay reagent (Bio-Rad, München, Germany) according to the manufacturer's instructions. One unit of ß-lactamase activity is defined as that required to hydrolyse 1 µmol/min of substrate at 25°C. Specific activity is therefore defined as the number of units/mg of protein in the assay.

Isoelectric focusing

Ten micrograms of total protein of the bacterial extracts (as used above) was loaded on to a precast isoelectric focusing gel (Novex, San Diego, CA, USA) with a pH gradient of pH 10.0 to pH 3.0. The pH gradient was maintained using pH 10.0 cathode and pH 3.0 anode buffers and the gel was resolved according to the manufacturer's instructions. ß-Lactamases were visualized following incubation of the gel for 5 min in a solution of 50 mM nitrocefin (made up in 50 mM MOPS, pH 7.0). Chromogenic isoelectric focusing standards (Bio-Rad, pI 10.0 to 3.0) were run in parallel to allow the estimation of the pI of each ß- lactamase detected.

Selection of derepressed mutants

An overnight culture in nutrient broth of the isolate to be tested was produced; 200 µL of this culture was spread on to a nutrient agar plate containing ampicillin (100 mg/L) or piperacillin (10 mg/L). Plates were incubated for up to 36 h at 37°C.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
All 20 P. shigelloides isolates showed low-level resistance to ampicillin (MIC range 4–32 mg/L, mode 16 mg/L) and higher-level resistance to carbenicillin (MIC range 32–256 mg/L, mode 64 mg/L). Isolates were susceptible to piperacillin, meropenem and all of the cephalosporins tested (Table IGo).

Nine of the 20 isolates (45%) expressed a ß-lactamase that was readily detected using isoelectric focusing gel electrophoresis and a further isolate expressed a very low level of enzyme activity (Table IIGo). All of these 10 isolates produced a single ß-lactamase band (not shown); three having ß-lactamases with a pI of 5.2, three with a pI of 5.3, two with a pI of 5.4 and one each with a pI of 5.9 and 6.7 (Table IIGo).


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Table II. Characterization of ß-lactamases from 10 isolates of P. shigelloides: substrate profiles and pIs
 
The substrate profiles of the various ß-lactamases were determined: four isolates expressed a group 2c carbenicillinase, four expressed a group 2d oxacillinase and two expressed a group 2a enzyme (Table IIGo). The serine ß- lactamase inhibitors BRL 42715 and clavulinic acid both reduced the ampicillinase activity in all of the extracts to undetectable levels (Table IIGo).

ß-Lactamase expression in the 20 P. shigelloides isolates was not increased in the presence of 0.25, 0.5 or 1 x MIC concentrations of ampicillin or carbenicillin, as set out in Materials and methods (data not shown). In addition, we were unable to isolate mutants derepressed for ß-lactamase synthesis from any of the isolates with selection on ampicillin at 100 mg/L or piperacillin at 10 mg/L (data not shown).


    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
All of the P. shigelloides isolates showed a resistance profile similar to that previously reported for other P. shigelloides isolates.5 No correlation between the MICs of ß-lactams for an isolate and its production of a ß-lactamase was found, so it is likely that the isolates that do not express a ß-lactamase have some other mechanism of resistance to penicillins.7 Indeed, we found evidence of expression of a single ß-lactamase in only 10 of the 20 isolates (50%). It is possible that the other isolates have ß-lactamase genes but, if so, they are expressed at levels below the sensitivity of our assay.

The ß-lactamases detected were acidic (as determined by isoelectric focusing), with 80% of enzymes having pIs of <5.5. Their substrate profiles indicate that they are not known acidic group 2b ß-lactamases like TEM or SHV.8 Specifically, the three clinical isolates from Santiago, Cuba each expressed a class 2d oxacillinase with a pI of 5.2, and the three clinical isolates from Prague, Czech Republic, expressed a class 2c carbenicillinase with a pI of 5.3. We could find no other published examples of ß-lactamases with similar pIs and substrate profiles to either type of enzyme,8 so it is likely that the ß-lactamases in these two groups of isolates are previously unknown. The environmental isolates of P. shigelloides, obtained from water, sewage and animal samples in the Czech Republic, displayed a wide range of ß-lactamases, in terms of both their pIs and their substrate profiles (Table IIGo). It appears from this study, therefore, that a diverse pool of ß-lactamases is present in P. shigelloides in the environment. It is interesting to note that some of the eight isolates that express enzymes that hydrolyse carbenicillin, piperacillin and oxacillin to varying degrees were isolated in the mid-1960s, before any of these drugs had been developed. This and the wide distribution of ß-lactamases in the environment indicate that acquisition of these ß-lactamase genes has not arisen simply from the selective pressure of ß-lactam therapy.

The genus Plesiomonas has been reported to be most similar to the genus Aeromonas.9 Organisms of this genus express three co-inducible chromosomal ß-lactamases, including an oxacillinase.5 However, ß-lactamase activities could not be induced in any of the P. shigelloides isolates (data not shown), even in isolates that expressed no detectable basal levels of ß-lactamase activity. This finding, together with the fact that we could not isolate ß-lactamase hyperproducers (indicative of genetic derepression) from any of the P. shigelloides strains (data not shown), leads us to conclude that ß-lactamase expression in this species is not inducible.


    Acknowledgments
 
We thank Dr E. Aldová for providing the P. shigelloides isolates and G. E. Aymes for technical assistance. This work was supported by grants from the Wellcome Trust and the British Society for Antimicrobial Chemotherapy.


    Notes
 
* Corresponding author. Tel: +44-117-9287541; Fax: +44-117-9287896; E-mail: matthewb.avison{at}bris.ac.uk Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Soweid, A. M. & Clarkston, W. K. (1995). Plesiomonas shigelloides: an unusual cause of diarrhoea. American Journal of Gastroenterology 90, 2235–6.[ISI][Medline]

2 . Abbott, S. L., Kokka, R. P. & Janda, J. M. (1991). Laboratory investigations on the low pathogenic potential of Plesiomonas shigelloides. Journal of Clinical Microbiology 29, 148–53.[ISI][Medline]

3 . Reinhardt, J. F. & George, W. L. (1985). Comparative in vitro activities of selected antimicrobial agents against Aeromonas species and Plesiomonas shigelloides. Antimicrobial Agents and Chemotherapy 27, 643–5.[ISI][Medline]

4 . Clark, R. B., Lister, P. D., Arneson-Rotert, L. & Janda, J. M. (1990). In vitro susceptibilities of Plesiomonas shigelloides to 24 antibiotics and antibiotic–ß-lactamase-inhibitor combinations. Antimicrobial Agents and Chemotherapy 34, 159–60.[ISI][Medline]

5 . Walsh, T. R., Payne, D. J., MacGowan, A. P. & Bennett, P. M. (1995). A clinical isolate of Aeromonas sobria with three chromosomally mediated inducible ß-lactamases: a cephalosporinase, a penicillinase and a third enzyme displaying carbapenemase activity. Journal of Antimicrobial Chemotherapy 35, 271–9.[Abstract]

6 . Philips, I., Andrews, J., Bint, A. J., Bridson, E., Brown, D. F. J., Cooke, E. M. et al. (1988). Breakpoints in in-vitro antibiotic sensitivity testing. Report by a working party of the British Society for Antimicrobial Chemotherapy. Journal of Antimicrobial Chemotherapy 21, 701–10.[ISI][Medline]

7 . Hawkey, P. M. (1998). The origins and molecular basis of antibiotic resistance. British Medical Journal 317, 657–60.[Free Full Text]

8 . Bush, K., Jacoby, G. A. & Medeiros, A. A. (1995). A functional classification scheme for ß-lactamases and its correlation with molecular structure. Antimicrobial Agents and Chemotherapy 39, 1211–33.[Free Full Text]

9 . Schubert, R. H. W. (1984). Genus IV. Plesiomonas Habs and Schubert 1962, 324AL. In Bergey's Manual of Systematic Bacteriology, vol. 1, (Krieg, N. R. & Holt, J. G., Eds), pp. 548–50. Williams & Wilkins, Baltimore, MD.

Received 25 October 1999; returned 23 January 2000; revised 28 January 2000; accepted 6 February 2000