Institut für Medizinische Mikrobiologie und Immunologie, Pharmazeutische Mikrobiologie, University of Bonn, Meckenheimer Allee 168, D-53115 Bonn, Germany
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Abstract |
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Introduction |
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Despite their occurrence in clinical specimens, little is known about the antimicrobial susceptibilities and particularly the natural antibiotic susceptibilities and resistances of Plesiomonas. Moreover, there is some confusion in the literature about the resistance of P. shigelloides to certain ß-lactam antibiotics like penicillins.1 There is strong evidence that this confusion has to be attributed to different conditions in susceptibility testing. In a previous study we showed that different media and inocula dramatically influenced the MICs for plesiomonadae of numerous ß-lactam antibiotics.13
The aim of the present paper was to create a database of the natural susceptibility of P. shigelloides strains originating from different sources to a wide range of antibiotics. To investigate whether and to what extent MICs of non- ß-lactams were dependent on the medium and inoculum, 10 representative strains were tested with these antibiotics using different media and inocula.
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Materials and methods |
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Seventy-four plesiomonadae isolated from humans (n = 50), water (n = 22) and animals (n = 2) were examined. An overview on the origin of the strains tested is shown in Table 1. Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853 served as controls for antibiotic susceptibility testing and were derived from the German culture collection of microorganisms and cell cultures in Braunschweig (DSMZ).
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The identification of the strains was confirmed using a commercial identification system for Enterobacteriaceae and several other Gram-negative bacteria (Micronaut-E, Merlin-Diagnostika, Bornheim, Germany). This identification system includes biochemical key reactions for Enterobacteriaceae and related taxa of clinical significance (including P. shigelloides). The inoculum for the identification tests was prepared from overnight cultures on IsoSensitest agar (Oxoid, Basingstoke, UK) in physiological saline and was 106 cfu/mL. The incubation time was 22 h, the incubation temperature was 37°C.
Antibiotics and antibiotic susceptibility testing
For all the strains, antibiotic susceptibility was determined by a microdilution procedure in cation-adjusted Mueller Hinton broth (CAMHB; Difco Laboratories, Detroit, MI, USA) according to the NCCLS standard, using a slightly increased inoculum of 1 x 106 cfu/mL. This inoculum is conceivable under normal testing conditions and was chosen to clarify the resistance potential of Plesiomonas. MICs were read visually after inoculation of antibioticcontaining microtitre plates (Merlin-Diagnostika) with 100 µL of the appropriate bacterial suspensions, and incubation for 22 h at 37°C. The MIC was defined as the lowest antibiotic concentration that inhibited visible growth. Ten representative plesiomonadae of clinical (n = 4), aquatic (n = 4) and mammalian origin (n = 2) were tested to all non-ß-lactam antibiotics using CAMHB, MuellerHinton broth (Oxoid) and IsoSensitest broth (Oxoid), and four different inocula (1 x 104, 1 x 105, 1 x 106 and 1 x 107 cfu/mL). For preparation of the inocula, 1820 h cultures grown on sheep blood agar (Oxoid) were used. An inoculum of 1 x 109 cfu/mL was adjusted in physiological saline and diluted to yield the final inoculum. Viable cell counting was undertaken to verify the prepared inocula (three-fold determinations). All susceptibility trials were carried out in duplicate. The antibiotics were kindly provided to Merlin-Diagnostika by the manufacturers.
Evaluation of natural antibiotic susceptibility
Plotting the MIC of a particular antibiotic for one species against the number of strains found with the respective MIC usually results in a bimodal distribution. Generally, one peak with relatively low MICs represents the natural population and one peak with higher MICs represents the strains with acquired (secondary) resistance. This is not valid for plesiomonadae and some ß-lactams.13 Analysis of the MIC distribution for all strains of one species of each antibiotic permitted determination of the biological thresholds, which limit the natural population at high MICs but not those strains with secondary resistance. Whether the MICs for the natural population were above or below the breakpoints of the standards, which assess the clinical susceptibility, was investigated. When the natural population was susceptible or intermediate according to the cited standard, it was described as naturally susceptible or naturally intermediate, respectively. When the natural population was clinically resistant, it was described as naturally (intrinsically) resistant. The method has been described in detail previously.1416 In the present study, breakpoints according to the NCCLS17 valid for Enterobacteriaceae, P. aeruginosa and other non-Enterobacteriaceae, Neisseria gonorrhoeae and staphylococci were applied. For antibiotics for which NCCLS clinical assessment criteria do not exist, breakpoints according to German,18 French19 or Swedish20 standards were employed. Breakpoints for apramycin, ribostamycin and lividomycin A were used as published recently.21
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Results |
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The MIC data of all antibiotics were reproducible for E. coli ATCC 25922 and P. aeruginosa ATCC 27853. The MICs for these strains were within the control limits for susceptibility testing according to NCCLS criteria (data not shown).
Dependency on medium and inoculum
Testing of non-ß-lactam antibiotics with different inocula generally did not influence the MICs, using inocula of 1 x 104, 1 x 105 or 1 x 106 cfu/mL. Using an inoculum of 1 x 107 cfu/mL, some plesiomonadae (and also E. coli ATCC 25922 and P. aeruginosa ATCC 27853) showed higher MICs for several unrelated antibiotics, such as the quinolones and antifolates, but the decrease in susceptibility was generally low (two or three MIC dilution steps). These dependencies on the inoculum were obtained in all media applied. MICs achieved with the two Mueller Hinton media and IsoSensitest broth were nearly identical. However, the MICs of tetracyclines and macrolides in IsoSensitest broth were two dilution steps higher than in MuellerHinton media (data not shown).
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Discussion |
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Concerning ß-lactam antibiotics, it was shown that P. shigelloides was naturally resistant to benzylpenicillin, oxacillin, amoxicillin, acylaminopenicillins (piperacillin, mezlocillin, azlocillin) and ticarcillin. It is most likely that discrepancies in the extent of resistance to these agents seen in several studies have to be attributed to the extreme inoculum dependency of the appropriate MICs, as shown recently.13 It is most remarkable that the two susceptibility patterns to several cephalosporins, piperacillin/tazobactam and aztreonam, which were the same as in our previous study,13 were also found for all strains with the susceptibility pattern of decreased MICs (pattern 1). Using an inoculum of 1 x 107 cfu/mL, all plesiomonadae, including one strain being susceptible to acylaminopenicillins and most susceptible to benzylpenicillin (see Table 2), showed the susceptibility pattern of increased MICs (pattern 2, data not shown). The phenotypic data of the present and our previous study13 indicate the presence of a novel mechanism of ß-lactamase expression and the presence of ß-lactamase(s) in all plesiomonadae. Resistance to amoxicillin and ticarcillin but susceptibility to co-amoxiclav might be due to chromosomally encoded class A enzymes, probably similar to those that can be found in Klebsiella spp. In a recent study, Avison et al.28 examined the production of ß-lactamases in 20 Plesiomonas strains and found three different enzymes in 10 of these strains, i.e. a group 2c carbenicillinase, a group 2d oxacillinase and a group 2a enzyme. Each of the 10 ß-lactamase-positive strains contained one of these enzymes. Because all the strains tested showed a similar antibiogramm it might be possible that failure to detect ß-lactamases in all the strains is due to the individual state of the bacterial culture during the ß-lactamase preparation. It should be noted that although all plesiomonadae examined so far are capable of showing resistance to certain penicillins and expressing two cephalosporin susceptibility patterns, there is a high strain-to-strain variation, as far as inoculum and medium dependency is concerned.13 From the data of the present and our previous study,13 there is evidence that ß-lactamase expression in plesiomonadae might be connected with quorum sensing.c29 Generally, during this process bacterial signals act at high cell density due to accumulations above a threshold level and mediate changes in gene expression.29 Quorum sensing can regulate a variety of cellular events and it was shown that the expression of the chromosomally encoded 2-N-acetyltransferase involved in the acetylation of peptidoglycan of Providencia stuartii was strongly influenced by its cell density.30 Interestingly, within the Enterobacteriaceae, Providencia and Proteus spp. might represent the closest neighbours of Plesiomonas31 and it was found that Providencia strains susceptible to cephalosporins with an inoculum of 5 x 105 cfu/mL were resistant to all cephalosporins with an inoculum of 5 x 106 cfu/mL.14
In contrast to ß-lactam antibiotics, there were no or only minor inoculum and medium dependencies seen in the susceptibility testing of non-ß-lactams. This gives further support to the idea that specific ß-lactamases rather than other mechanisms are responsible for the ß-lactam susceptibility patterns described. The natural resistance of P. shigelloides to most macrolides, lincosamides, streptogramins, glycopeptides and fusidic acid is likely to be attributed to the Plesiomonas outer membrane, which might prevent the entry into the cell of most of these antibiotics. The natural resistance patterns observed to these non-ß-lactams are a common feature of many Gram-negative organisms and can be found in nearly all Enterobacteriaceae but are not a general feature of Vibrionaceae. For example, Vibrio cholerae is highly susceptible to erythromycin32 and this macrolide is used successfully in the treatment of cholera and some other infections due to Vibrio spp.33 Thus, it is likely that the Plesiomonas cell envelope resembles the Enterobacteriaceae cell wall rather than that of several Vibrio spp. This is in agreement with some data on the composition of the Plesiomonas cell envelope, which contains lipid A and, in contrast to species of Vibrionaceae, the enterobacterial common antigen.1
The results of the present and our previous study13 show that P. shigelloides is naturally resistant to several antibiotics. Because the organism has the natural potential for resistance to a wide range of ß-lactams under conceivable testing conditions it might be useful to describe Plesiomonas as naturally resistant to a variety of ß-lactams, i.e. all penicillins but also some cephalosporins, like cefoperazone, ceftazidime and cefepime, and to renounce the use of these ß-lactams in the treatment of severe Plesiomonas infections. It should be discussed whether individual breakpoints for Plesiomonas should be included in the standards of antimicrobial susceptibility testing. The extreme inoculum dependency seen for numerous ß-lactams might give insights into a new mechanism of ß-lactamase expression. Several studies on the ß-lactamase expression of P. shigelloides have already started. Although mechanisms other than ß-lactamases cannot be excluded it is unlikely that they play an important part in the susceptibility to ß-lactams, according to the data from the present and our previous study.13
The data described represent a database about the natural susceptibility of P. shigelloides strains to a wide range of antimicrobial agents, which can be used for the validation of susceptibility test results of these unusual proteobacteria.
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Acknowledgements |
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Notes |
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References |
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Received 8 March 2001; returned 3 August 2001; revised 17 August 2001; accepted 22 August 2001