Effect of inoculum density on susceptibility of Plesiomonas shigelloides to cephalosporins

Irith Wiegand* and Sonja Burak

Pharmaceutical Microbiology, University of Bonn, Meckenheimer Allee 168, 53115 Bonn, Germany


    Abstract
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objectives: Resistance of Plesiomonas shigelloides to cephalosporins at higher cell densities has been reported. We investigated whether these inoculum effects are due to the production of ß-lactamases.

Methods: ß-Lactamase production of five P. shigelloides strains was characterized by activity tests, SDS–PAGE and isoelectric focusing. For all strains, MIC values of different cephalosporins were determined by microdilution methodology using inocula of 1 x 105 cfu/mL and 1 x 106 cfu/mL. Subsequently, the morphology of cells was determined by light microscopy. For one isolate, kill kinetics of cefpodoxime were determined using batch cultures with the lower and higher inocula.

Results: Four of five P. shigelloides strains were shown to be ß-lactamase-positive, producing different amounts of constitutively expressed non-inducible enzymes. Inoculum effects for cephalosporin susceptibility were observed for all strains. Examination of cells revealed a very strong filamentation, with filament sizes ranging from 100 µm up to 2 mm. The kill kinetics with cefpodoxime showed similar killing capacities of the antibiotic at both inoculum sizes.

Conclusions: The reported resistance of P. shigelloides to cephalosporins at higher cell densities is not due to an inoculum-dependent regulation of ß-lactamases, but can be explained by the formation of extensive filaments.

Keywords: filamentation , ß-lactamases , inoculum effect


    Introduction
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 Acknowledgements
 References
 
Plesiomonas shigelloides is a ubiquitous Gram-negative, rod-shaped bacterium. It can be isolated from aquatic environments, the gut of several animals and from human faeces.1 It is regarded as a pathogen causing mainly gastrointestinal complications2,3 that are usually mild and self-limiting. Treatment with antibiotics is seldom required.4 However, extraintestinal infections have also been reported. Most frequently described are cases of septicaemia and meningitis, occurring mainly in patients with underlying health disorders and in immunocompromised patients. High mortality rates have been reported.5 For these severe infections, appropriate antibiotic therapy is necessary. Therefore, detailed knowledge about resistance mechanisms and antibiotic susceptibility of P. shigelloides is needed. P. shigelloides has been described as an organism showing pronounced inoculum effects in susceptibility testing with cephalosporins using microdilution methods.6 The effect of the inoculum density on susceptibility to ß-lactam antibiotics is frequently attributed to the cumulative activity of ß-lactamases at higher inocula.79 In one study, chromosomally encoded, non-inducible ß-lactamases were detected in 50% of the tested clinical and environmental P. shigelloides strains.10 All Plesiomonadae tested in another study showed high MIC values of several cephalosporins at the inoculum 1 x 107 cfu/mL, and a mechanism of ß-lactamase regulation was assumed to be the basis for this finding.11

The aim of this study was to investigate whether the inoculum effects with cephalosporins in P. shigelloides are due to the production of ß-lactamases. The effects of cephalosporins were determined for a strain with no detectable ß-lactamase production and for ß-lactamase-positive isolates producing different amounts of ß-lactamase.


    Material and methods
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 Acknowledgements
 References
 
Bacterial isolates

Five strains of P. shigelloides were examined. P6, P12 and P15 were originally isolated from humans, whereas P. shigelloides ATCC 14029 is a canine isolate and P. shigelloides P2 was found in aquarium sludge. The countries of isolation were the USA, Germany and Sweden.6 Escherichia coli ATCC 25922 was used as a comparative strain.

Antibiotic susceptibility testing

Susceptibility testing for all bacterial isolates was performed by a microdilution procedure in cation-adjusted Mueller–Hinton broth (Difco, Detroit, MI, USA) using inocula of 1 x 105 cfu/mL and 1 x 106 cfu/mL. Inocula were prepared using colonies from overnight agar plates (Mueller–Hinton agar; Difco). Inoculum sizes were adjusted photometrically to 1 x 108 cfu/mL (OD560 0.12–0.13), diluted, and samples were withdrawn for determination of the viable cell count. MIC values were determined visually after inoculation of microtitre plates containing the dehydrated ß-lactam antibiotics cefpodoxime, cefixime, ceftibuten, cefotaxime, ceftazidime, cefuroxime, ceftriaxone and cefotiam (Merlin-Diagnostika, Bornheim, Germany) with 100 µL of the appropriate bacterial suspension and incubation for 20 h at 37°C. MICs were also determined in the presence of 4 mg/L clavulanic acid (SmithKline Beecham Pharmaceuticals, Worthing, UK).

Cell morphology

The morphology of cells was determined by phase contrast microscopy using a Leica DM LB microscope (Leica Microsystems, Wetzlar, Germany). Morphological images were captured using a Leica DC 300F digital camera (Leica Microsystems) with 400-fold magnification.

Characterization of ß-lactamases

Cells were grown to an optical density at 600 nm (OD600) of 1.0 and then harvested by centrifugation at 4°C. The pellet was washed with 0.1 M KH2PO4/Na2HPO4 buffer (pH 7.0). Cells were resuspended in the same buffer and frozen overnight. Sonication on ice yielded the cell extract for ß-lactamase characterization. The ß-lactamase crude extracts were used for activity tests, SDS–PAGE and isoelectric focusing. The protein content of each sample was determined by the method of Lowry et al.,12 with bovine serum albumin (Serva, Heidelberg, Germany) used as a standard. ß-Lactamase activity was quantified spectrophotometrically according to Peter et al.13 by measuring the change in absorbance for two substrates. The following concentrations and wavelengths were used: 50 µM nitrocefin (Oxoid, Basingstoke, UK) at 485 nm and 100 µM cefotiam (Grünenthal, Stolberg, Germany) at 280 nm. Inducibility of the ß-lactamases was tested by adding 0.125 mg/L imipenem to exponentially growing cultures and determining the ß-lactamase activity compared with control cultures without inducer. The molecular weight of the ß-lactamases was estimated by separation of the proteins by SDS–PAGE in 13% acrylamide gels. Isoelectric focusing was performed in polyacrylamide gels with a pH range of 3.0–10.0 (Bio-Rad Lab., Munich, Germany). ß-Lactamase bands in the gels were visualized by staining with 1 mM nitrocefin solution.

Kill kinetics

For P. shigelloides strain P2, kill kinetics with 0.125 mg/L cefpodoxime (Sankyo Co., Tokyo, Japan) were determined twice. Batch cultures with inocula of 1 x 105 cfu/mL and 1 x 106 cfu/mL were incubated at 37°C and agitated at 90 rpm. Bacterial counts were performed by diluting the samples serially (1:10) in 0.9% NaCl solution, then plating a 0.05 mL aliquot on agar. Plates were read after colonies had formed following incubation at 37°C.


    Results
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 Acknowledgements
 References
 
All five strains were tested for the possession of ß-lactamases. ß-Lactamase bands could be detected in SDS–polyacrylamide gels and in isoelectric focusing gels for the strains ATCC 14029, P2, P6 and P12. In both gel types, crude protein extracts of these strains produced a single ß-lactamase band (not shown). The molecular weights of the ß-lactamases of the respective strains were determined to be 29, 29, 31 and 30 kDa. These data point to class A ß-lactamases. The enzymes differed in their pI values. Isoelectric points of the enzymes produced by strains ATCC 14029 and P2 were at pH 5.3 and pH 4.9, respectively. Strains P6 and P12 produced ß-lactamases with respective pIs of 5.0 and 5.2. No ß-lactamase band could be detected for protein extracts of strain P15 on either type of gel (Table 1).


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Table 1. Characterization of ß-lactamases of P. shigelloides strains used in the study

 
The determination of the specific ß-lactamase activity using nitrocefin as substrate revealed differences up to a factor of 10 in the amount of enzyme (Table 1). Induction with imipenem did not lead to an increase in specific ß-lactamase activity for strains ATCC 14029, P2, P6 and P12. For strain P15, it was not possible to detect any hydrolysis of nitrocefin. ß-Lactamase crude extracts of all strains showed no activity against cefotiam.

Uniform MIC values of the tested cephalosporins were determined for the five Plesiomonas isolates (Table 2). The strains were highly susceptible to the different antibiotics using an inoculum size of 1 x 105 cfu/mL. With a cell count of 1 x 106 cfu/mL, the MICs rose several dilution steps. For ceftibuten and ceftazidime, high increases of at least seven to 10 dilution steps could be determined. For cefpodoxime, cefixime, cefotaxime, cefotiam, cefuroxime and ceftriaxone the MIC values increased at least three to six dilution steps. A higher number cannot be excluded as concentrations below 0.03 mg/L were not tested.


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Table 2. P. shigelloides and E. coli MIC values (mg/L) using different inocula

 
For the ß-lactamase-positive strains ATCC 14029, P2, P6 and P12 the same increases in MIC values were seen irrespective of the amount of ß-lactamase produced. Furthermore, isolate P15 (with no detectable ß-lactamase activity) showed equivalent susceptibilities to the different cephalosporins in comparison with the ß-lactamase-producing strains. The increase in cephalosporin MICs with the higher inoculum seen with all tested P. shigelloides strains was not influenced by the presence of the ß-lactamase inhibitor clavulanic acid (data not shown).

The control strain E. coli ATCC 25922 showed no inoculum effects in susceptibility to the tested cephalosporins using the inoculum sizes of 1 x 105 and 1 x 106 cfu/mL.

The effects of cefixime, ceftibuten and cefpodoxime on the cell morphology of P. shigelloides strains ATCC 14029, P2, P15 and E. coli ATCC 25922 were examined. Phase-contrast microscopy of P. shigelloides cells from the wells of the MIC microtitre plates with visible turbidity revealed filament formation in all wells for all strains. Filament sizes ranged in length from 100 µm up to 2 mm (Figure 1). Examination of E. coli cells after incubation with cefpodoxime revealed only rods and short filaments (Figure 1); for cefixime and ceftibuten, filaments with a length up to 100 µm could be observed.



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Figure 1. Morphology of P. shigelloides P2, P15 and E. coli ATCC 25922. P. shigelloidesP2 cells without antibiotic (a), and after incubation with cefpodoxime (0.125 mg/L) for 20 h (b); P. shigelloides P15 cells without antibiotic (c), and after incubation with cefpodoxime (0.125 mg/L) for 20 h (d); E. coli ATCC 25922 cells without antibiotic (e), and after incubation with cefpodoxime (0.125 mg/L) for 20 h (f). Bar, 5 µm.

 
Phase contrast microscopy of P. shigelloides P2 revealed that cefotaxime, ceftazidime, cefuroxime, ceftriaxone and cefotiam also induced large filaments.

Reculturing of P. shigelloides P2 cells (initial cell count of 1 x 106 cfu/mL) from microtitre plate cavities revealed that in wells with clearly visible spots the viable cell count was reduced below the inoculum (Figure 2). Therefore, the visible growth observed in the microtitre cavities cannot be correlated with an increase in the cell number.



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Figure 2. Reculturing of P. shigelloides strain P2 (initial inoculum 1 x 106 cfu/mL) from MIC microtitre plate cavities. Row a, microtitre plate used for reculturing; row b, respective cefpodoxime concentrations (mg/L); row c, number of viable cells.

 
Kill kinetics were obtained with 0.125 mg/L cefpodoxime for strain P. shigelloides P2 using inocula of 1 x 105 and 1 x 106 cfu/mL, with the concentration of cefpodoxime being two or more dilution steps above the MIC determined with 1 x 105 cfu/mL, and three dilution steps below the MIC determined with 1 x106 cfu/mL. Growth controls with an initial cell count of 1 x 105 and 1 x 106 cfu/mL showed a lag phase of 1.5 h and reached stationary phase with 1 x 109 cfu/mL after 6 and 8 h, respectively. Similar killing capacity of cefpodoxime for both inocula was observed with a complete elimination of viable cells after 8–10 h (Figure 3). The optical density of the antibiotic-treated culture with an inoculum of 1 x 105 cfu/mL was below the detection limit during the experiment. The optical density of the antibiotic-treated culture with an inoculum of 1 x 106 cfu/mL increased during the first 4 h (Figure 3). The formation of long filaments was observed microscopically during this period. From 4–12 h the optical density decreased to a value where it remained stable over the next 12 h. The examined samples showed a breakdown of the long filaments to shorter fragments. Both cultures showed no visible turbidity after 24 h. The result for the batch culture with the inoculum of 1 x 106 cfu/mL seemed contrary to the results of the MIC determination, where—using the same inoculum size and cefpodoxime concentration—a visible bacterial mass was observed and very long filamentous cells were seen under the microscope. Therefore, this experiment was repeated without shaking the batch culture, in order to adapt the conditions to those of the MIC determination. Here, a dense mat of cells at the bottom of the flask was obtained after 24 h of incubation with 0.125 mg/L cefpodoxime. Phase-contrast microscopy revealed again very long, filamentous cells.



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Figure 3. Kill kinetics of 0.125 mg/L cefpodoxime for strain P. shigelloides P2 with initial cell count of 1 x 105 cfu/mL (filled triangles) and 1 x 106 cfu/mL (filled squares). Changes in the optical density (OD 600 nm) after treatment of P. shigelloides P2 (initial cell count of 1 x 106 cfu/mL) with 0.125 mg/L cefpodoxime are also included (open circles).

 

    Discussion
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 Acknowledgements
 References
 
Only four of five P. shigelloides strains examined produce a detectable ß-lactamase. Avison et al.10 had previously found enzymes with a variety of pI values (5.2–5.4, 5.9 and 6.7). In this study, we detected, in addition, enzymes with pI values of 4.9 and 5.0. The results of the characterization of the enzymes are in accordance with the previous observation that the ß-lactamases in P. shigelloides form a heterogeneous group of enzymes. The differences in the molecular weight of the ß-lactamases presented here also add to this conclusion.

MIC values determined were in accordance with the previously published results6,11 and show the characteristic inoculum-dependency pattern. It has been suggested that the production of ß-lactamases and inoculum-dependent regulation of the enzymes might be the reason for these findings. However, we showed that the characteristic pattern can also be found in a strain with no detectable ß-lactamase activity. Furthermore, the increased MIC values with the higher inoculum are not influenced by the presence of a ß-lactamase inhibitor. In addition, we were not able to determine any hydrolysis of cefotiam using crude ß-lactamase extracts, but did observe an increase in MIC values of this antibiotic using different inocula. From these results, we conclude that the dependency of cephalosporin MIC values on the inoculum size is not due to the production of the ß-lactamase.

Inoculum effects in Gram-negative bacilli are not only attributable to the production of ß-lactamases, but are also observed for antibiotics leading to the formation of filamentous cells.14 Formation of filaments is observed after treatment with a variety of ß-lactams, and in particular with aminothiazolyl cephalosporins.15 An inoculum dependency of MIC values of ceftazidime, cefoperazone, ceftriaxone and cefotaxime using E. coli, Salmonella typhimurium and Klebsiella pneumoniae at cell densities of 5 x 105 and 5 x 107 cfu/mL has been described by Eng et al.16 This was determined to be due to the increase in optical density caused by formation of filamentous cells. Using lower varying inoculum sizes (1 x 105 and 1 x 106 cfu/mL) of E. coli ATCC 25922, we could not see an increase in MIC values of the tested cephalosporins, although we could observe filamentous cells. Therefore, for E. coli the effect of filamentation and the resulting increase in cell mass was negligible for MIC determination using these cell numbers.

However, the situation for P. shigelloides was different. The extent of filamentation after cephalosporin exposure was outstanding. With a normal cell size of 2 µm, the cell length increased 1000-fold, as filaments up to 2 mm long were observed for cells taken from microtitre plate cavities prepared with the inoculum of 1 x 106 cfu/mL.

Filamentation of E. coli cells after treatment with cephalosporins that primarily target PBP3 is a well known phenomenon and has been described for a variety of ß-lactams e.g. ceftibuten,16,17 cefoperazone18 and for the oxyimino-cephalosporins ceftazidime19 and cefotaxime.20 In general, all oxyimino-cephalosporins show a high binding affinity to PBP3 due to the oxyimino-group at the C-7 position.21 PBP3 is needed for the formation of the septum during cell division.22 Cells without a functional PBP3 are able to elongate but fail to divide. Multichromosomal filaments are formed.23 Filaments induced by ß-lactams may recover when transferred into antibiotic-free medium. Furthermore, filaments may have normal cell functions. It has been shown that motility and chemotaxis is possible for cefalexin-induced E. coli filaments with a length up to 50 µm.24 Filamentation is seen for ß-lactams with highest affinity to PBP3 at sub-MIC concentrations as they bind to their preferential target, whereas at higher concentrations a bactericidal effect is observed as other PBPs are also affected.25

The PBP profile of P. shigelloides is not known. The formation of filaments is most likely due to the preferential inhibition of a PBP3-like penicillin-binding protein responsible for septum formation. Using a starting inoculum of 1 x 106 cfu/mL of P. shigelloides cells, filamentation is seen at concentrations above the MIC determined with an inoculum at 1 x 105 cfu/mL. Thus, one has to keep in mind that with the higher inoculum a 10-fold higher number of the preferential target penicillin-binding protein is present. The concentration to reach other PBPs therefore needs to be higher. The kill kinetics of cefpodoxime and the subculturing of cells from cefpodoxime microtitre MIC plates, however, reveal that even with a higher inoculum a pronounced killing of viable cells at filamentation-inducing concentrations can be observed. Furthermore, filaments were extremely sensitive to mechanical stress, since in the stirred batch culture, cells did not elongate to more than 100 µm. The following fragmentation correlated with the decrease in optical density. Those fragments were not viable when transferred to antibiotic-free medium, as a concomitant increase in viable cell count was not observed.

The reported resistance of P. shigelloides to cephalosporins at higher cell densities has to be reconsidered. Over a wide concentration range, cephalosporins seem not to inhibit the lateral growth of the individual P. shigelloides cell. The enormous increase in cell mass for the single P. shigelloides cell explains the clearly visible bacterial spots at the bottom of the microtitre plate. The minimal inhibitory concentration is defined as the lowest concentration that completely inhibits growth of the organism in microdilution wells, as detected by the unaided eye.26 Bacterial growth is defined as an increase in the number of cells that can be measured as an increase in microbial mass.27 In this respect, the MIC values determined for the higher inoculum are invalid, as the enormous increase in microbial mass is not due to an increase in cell numbers.

The aim of in vitro susceptibility testing is to provide information to prescribers on the choice of appropriate antibiotic therapy.28 The results presented in this study lead to the conclusion that the tested P. shigelloides strains are susceptible to cephalosporins. The bacterial response, however, might not be clinically favourable in septic cases where antibiotic therapy is clearly indicated. Antibiotic-induced, especially ß-lactam-induced, release of endotoxin due to filament formation with subsequent cell lysis, has been intensively studied in vitro.2931 It has been shown that there is a correlation between increase in microbial mass due to aberrant morphologies and endotoxin release.32 Endotoxin-related detrimental effects for the patient are suspected.15 However, the in vivo differences between antibiotics with high or low endotoxin release potential are under debate.33,34 Furthermore, it is questionable whether the extensive filamentation of P. shigelloides cells seen in vitro would also occur in vivo at the site of infection. Fatality of extraintestinal Plesiomonas infections cannot be correlated with cephalosporin therapy.3537 However, clinicians should be cautious when using filament-inducing ß-lactam antibiotics until more clinical data are available on the benefit of treatment of P. shigelloides infections with cephalosporins.


    Acknowledgements
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 Acknowledgements
 References
 
The provision of strains by Dr Ingo Stock is gratefully acknowledged. Part of this work was presented at the 13th ECCMID 2003, abstract P463.


    Footnotes
 
* Corresponding author. Tel: +49-22-873-5347; Fax: +49-22-873-5267; Email: wiegand{at}uni-bonn.de


    References
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Articles by Wiegand, I.
Articles by Burak, S.
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Articles by Wiegand, I.
Articles by Burak, S.