• LY333328;
  • vancomycin-resistant enterococci;
  • inoculum effect


  1. Top of page
  2. Abstract
  6. Acknowledgments
  7. References

Objectives: To determine the in vitro activity and inoculum effect of LY333328, a semisynthetic glycopeptide, against vancomycin-susceptible and vancomycin-resistant enterococcal isolates.

Methods: One hundred and seventy-six enterococcal isolates (117 vancomycin-susceptible, 29 VanA-type and 30 VanC-type isolates) obtained from surveillance cultures of 139 intensive care unit patients were studied by the standard agar dilution method. Vancomycin resistance determinants were characterized by PCR.

Results: The activity of LY333328 was comparable (MIC range, 0.1–2 mg/L) to those of vancomycin (0.1–4 mg/L) and teicoplanin (0.06–1 mg/L) for vancomycin-susceptible isolates. LY333328 was more active (0.1–8 mg/L) than vancomycin (256 to >1024 mg/L) and teicoplanin (32–512 mg/L) against VanA-type isolates, and similar (0.2–1 mg/L) to teicoplanin (0.1–0.5 mg/L) against VanC-type isolates. The MIC distribution of LY333328 displayed a narrower range than that of vancomycin, with no clear distinction between susceptible and resistant populations. The increment in the inoculum size, from 104 to 106 CFU/spot, of susceptible isolates increased the MIC values of LY333328, vancomycin and teicoplanin by factors of 11.4, 1.6 and 3.8, respectively. The corresponding factors for LY333328 for VanA-type and VanC-type isolates were 3.5 and 6.4, respectively.

Conclusions: LY333328 displays an excellent in vitro activity against vancomycin-susceptible and -resistant enterococci. Nevertheless, the inoculum size used in susceptibility tests should be carefully controlled.


  1. Top of page
  2. Abstract
  6. Acknowledgments
  7. References

Although enterococcal infections are not usually considered to be associated with high mortality rates, the dramatic increase of multiresistant enterococcal strains has spurred interest in this pathogen [1]. During the last few years, glycopeptide-resistant enterococci (GRE) have emerged as a major public-health concern, particularly in intensive care units (ICUs) in institutions in the USA [2]. Progress has been made in defining risk factors for colonization or infection by these strains [3], and now several guidelines for the control and prevention of infections due to GRE have been proposed [4], although precise treatment protocols remain to be defined. Penicillin G or aminopenicillins, alone or combined with an aminoglycoside, remain the therapy of choice for infections due to enterococci. The increased resistance to penicillins and high-level resistance to aminoglycosides observed in GRE have prompted a review of this regimen, as no effective antimicrobial therapy is available against many GRE [5]. Various combinations of cell wall-active antimicrobials, including carbapenems and glycopeptides, have been successfully assayed in animal models [2, 3]. Oral novobiocin and bacitracin have been used to suppress GRE fecal carriage, and a new streptogramin combination, quinupristin/dalfopristin, has been evaluated [5, 6]. Lastly, recently developed glycopeptides have been shown to be active in vitro against GRE [7–10].

LY333328 is a semisynthetic glycopeptide derived from the N-alkylation of LY264826, a naturally occurring glycopeptide with the same core structure as vancomycin [9]. Early reports have shown excellent antimicrobial activity of LY333328 against Gram-positive organisms, including staphylococci, streptococci and enterococci [8–11]. The objective of this study was to compare the in vitro activity of LY333328 against a collection of enterococcal isolates obtained from surveillance cultures derived from ICU patients. Organisms tested included both vancomycin-susceptible and-resistant enterococci. The inoculum effect in conventional media used for susceptibility testing was also investigated.


  1. Top of page
  2. Abstract
  6. Acknowledgments
  7. References

The investigation was performed with enterococcal isolates recovered from surveillance specimens of 139 long-term intubated patients admitted to the ICU at a 400-bed teaching hospital during the period 1994–96. A hundred and sixty enterococcal isolates were obtained from rectal swabs and 16 isolates from nares, oropharynx and bronchial aspirates. Fifty-nine were vancomycin resistant and 117 vancomycin susceptible. Isolates were obtained by plating specimens on a commercial selective azide agar (m-Enterococcus agar, DIFCO, Detroit, Mi, USA) and additionally on selective azide agar supplemented with 6 mg/L of vancomycin. For identification of enterococci to the species level, the biochemical scheme of Facklam and Collins, and the criteria for motility and pigment production were used [12]. Pulsed-field gel electrophoresis [13] was performed to avoid redundancy of repeated vancomycin-resistant isolates. When available, a single vancomycin-susceptible isolate per patient was also included for comparative purposes.

LY333328 and vancomycin were obtained from Lilly Research Laboratories (Indianapolis, Ind., USA). The other antibiotics were obtained from the following manufacturers: Hoechst-Marion Roussel, Barcelona, Spain (teicoplanin); SmithKline Beecham, Madrid, Spain (ampicillin); and Merck Sharp & Dohme, Madrid, Spain (imipenem). MICs were determined by standard agar dilution method according to NCCLS criteria with an inoculum size of 104 CFU/spot using Mueller-Hinton agar (Oxoid, Basingstoke, UK) [14]. For comparative purposes, MICs were also determined for 45 selected enterococcal isolates using standard broth microdilution [14].

All isolates with MIC values ≥4 mg/L of vancomycin were genetically typed by PCR to detect the presence of vanA, vanB, vanC1 and vanC2 genes. DNA extraction was performed with the Instagene Matrix commercial kit (BioRad, Hercules, Ca, USA) according to the manufacturer's instructions. Primers, reaction mixtures and PCR conditions followed published studies for the detection of vanA [15], vanB [16], vanC1 [17] and vanC2 [18] genes. Enterococcus faecium U2A1 [19], E. faecalis SF-299 (provided by George M. Eliopoulos), E. gallinarum U2A3 [19] and E. casseliflavus RYC46E (this study) were used as positive controls for the detection of vanA, vanB, vanC1 and vanC2 genes, respectively. E. faecalis ATCC 29212 was used as negative control.

β-Lactamase production was assayed for all enterococcal isolates by incubating 50 μL of an overnight enterococcal culture with 50 μL of nitrocefin 100 mM at 35°C for 2 h.

The inoculum size effect on MIC values (103, 104, 105 and 106 CFU/spot) was studied in Mueller-Hinton agar with 45 selected isolates displaying different levels of susceptibility to vancomycin and LY333328. In addition, the inoculum effect (103, 104, 105 and 106 CFU/mL) was also assayed by microdilution. Control strains were also included in the inoculum effect study.


  1. Top of page
  2. Abstract
  6. Acknowledgments
  7. References

The glycopeptide-resistance genotype characteristics of the enterococcal isolates described in this study are listed in Table 1. Of the 176 isolates, 29 were positive for vanA PCR; 28 isolates contained the vanC1 gene and two isolates contained the vanC2 gene. No VanB-type enterococcal isolates were isolated during the surveillance period. In Europe, in contrast to the USA, this situation is not unusual, as VanB isolates from surveillance cultures are much less common than VanA isolates [20–22]. Moreover, in Spain the incidence of VanB isolates is particularly low [23]. It is of interest to note that, although E. faecium represents the majority of GRE isolates recovered in the US [3, 16], this species represents the minority of our VanA-type isolates (34.5%). On the other hand, in our study, 90.9% of the isolates were recovered from fecal samples. The epidemiologic relevance of these isolates derives from the suggestion that intestine is the reservoir of clinically relevant enterococcal isolates. Several studies have demonstrated that previous gastrointestinal colonization is a risk factor for infection with GRE [21, 24].

Table 1.  Activity of LY333328 against 176 enterocococcal isolates compared with those of two other glycopeptides and two β-lactam agents
 MIC (mg/L)
Organisms (no. tested)VangenotypeAntimicrobialRange50%90%Mode
  1. aNo vanA, vanB, vanC1 or vanC2 determinants amplified by PCR assays.

  2. bTwo E. durans and one E. avium.

 faecalis (103)a vancomycin0.1–4222
E. faecium (11)aLY3333280.1–10.510.5
Enterococcus spp.LY3333280.5–1
(3b)a vancomycin2
E. faecalis (19)vanALY3333282–8484
  vancomycin256 to > 1024>1024>1024>1024
E.faecium (10)vanALY3333280.1–40.540.2
  vancomycin512 to >10241024>10241024
E. gallinarum (28)vanCLY3333280.2–
E. casseliflavus (2)vanCLY3333280.5–1

LY333328 inhibits the same step in peptidoglycan biosynthesis as vancomycin [8, 25]. It is highly active against Gram-positive organisms with MIC90 values 10–100 times lower than that of vancomycin [7, 10]. Previous studies have demonstrated that LY333328 displays good inhibitory activity against both vancomycin-susceptible and -resistant Enterococcus isolates [7–11, 26] with a bactericidal concentration-dependent effect [11]. Table 1 shows the activity of LY333328, vancomycin, teicoplanin and two β-lactam antibiotics against ICU isolates. Only one isolate of E. faecium, susceptible to vancomycin, displayed ampicillin MICs higher than 16 mg/L. None of the enterococcal isolates produced β-lactamase. Table 2 shows the comparative activity of LY333328 against control strains used in PCR experiments and susceptibility testing.

Table 2.  Comparative in vitro activity of LY333328 against quality control and reference strains
  MIC range (mg/L)a
StrainGlycopeptide resistance phenotypeVancomycinTeicoplaninLY333328
  1. aTwelve determinations for each strain.

Enterococcus faecalis ATCC 29212 2–40.2–0.50.2–0.5
E.faecium U2A1VanA1024 to > 1024128–5121–4
E. faecalis SF-299VanB16–320.06–0.20.2–1
E. gallinarum U2A3VanC14–80.2–0.50.2–1
E. casseliflavus RYC46EVanC24–80.2–0.50.2–0.5

Considering all enterococcal isolates tested, LY333328 displayed a high intrinsic activity, with MIC50/MIC90 of 0.5/2 mg/L. The corresponding values for vancomycin and teicoplanin were 2/1024 and 0.2/128 mg/L, respectively. Against glycopeptide-susceptible enterococcal isolates, the modal MIC values for LY333328, vancomycin and teicoplanin were 0.5, 2 and 0.2 mg/L, respectively. Vancomycin and LY333328 tend to present a better intrinsic activity against E. faecium than against E. faecalis, considering both vancomycin-resistant and -susceptible isolates. This trend is in agreement with the analysis of susceptibility data of vancomycin-susceptible strains reported in other publications by Jones et al [26] and could be confirmed from the data on vancomycin-susceptible enterococci in a recent European survey [27]. Moreover, results from Fraise et al [7] show the higher activity of glycopeptides, including LY333328, against E. faecium vancomycin-resistant isolates.

VanA-type E. faecalis and E. faecium isolates were inhibited by a concentration of LY333328 at least 256-and 64-fold lower than that required for inhibition with vancomycin or teicoplanin, respectively (Table 1). The modal MIC values of LY333328 for E. gallinarum and E. casseliflavus were similar to those of teicoplanin (0.2 mg/L), but 40-fold lower than those of vancomycin (8 mg/L). The MIC50/MIC90 of LY333328 against VanC-type isolates was similar to that of teicoplanin.

Figure 1 shows MIC distribution of all enterococcal isolates tested, vancomycin susceptible and those with vanA and vanC determinants. Of note is the fact that vancomycin displayed a wide range of MIC distributions with clearly defined susceptible and VanA-type resistant populations, while LY333328 MIC values had a narrower range, without a clear gap between susceptible and VanA-type resistant populations (Figure 1). This effect demonstrates the higher intrinsic activity of LY333328 compound against resistant isolates, which could be related to a secondary mechanism of inhibition [25]. Our results obtained with vancomycin-susceptible isolates and with E. gallinarum and E. casseliflavus (VanC type) are similar to those of previously reported investigations [9–11]. In contrast, LY333328 MIC values observed for VanA-type isolates were slightly higher than those previously described [9, 10]. A concentration of 4 mg/L of LY333328 inhibited 78.9% and 100% of highly vancomycin-resistant E. faecalis and E. faecium isolates, respectively. Twenty per cent (four of 19 isolates) of vancomycin-resistant E. faecalis isolates showed LY333328 MIC values of 8 mg/L, which were repeatedly obtained in different agar dilution assays with an inoculum of 104 CFU/spot. To our knowledge, the highest MIC value encountered for LY333328 in vancomycin-resistant enterococci was 4 mg/L [26]. As agar dilution may field higher values for LY333328, we also investigated by a broth microdilution method (using an inoculum of 105 CFU/mL) those E. faecalis isolates displaying 8 mg/L of LY333328. Using a microdilution methodology, LY333328 MIC values were 4 mg/L. Moreover, 45 selected isolates with different levels of susceptibility to glycopeptides yielded LY333328 MIC values by microdilution 1-2-fold dilution lower than those previously obtained by agar dilution. Nevertheless, the general form of MIC distribution obtained using microdilution-based methodologies did not differ from that of the agar dilution.


Figure 1. Distribution of vancomycin, teicoplanin and LY333328 MIC values for vancomycin-susceptible and -resistant (VanA-type and VanC-type) enterococcal isolates determined using an agar dilution method according to NCCLS guidelines [14].

Previous in vitro studies with a vancomycin-resistant E. faecium and a methicillin-resistant Staphylococcus aureus strain have demonstrated that the bactericidal activity of LY333328 is affected by a large inoculum (107–108 CFU/mL), although a slight increase in drug concentration seems to compensate easily for this effect [28]. Our studies with 44 enterococcal isolates (14 vancomycin-susceptible, 21 VanA-type and nine VanC-type isolates) clearly demonstrate that LY333328 MIC values were higher when the inoculum size was increased, both by agar and by microdilution methods. By agar dilution, an increment in the inoculum size, from 104 to 106 CFU/spot, in vancomycin-susceptible isolates increased MIC values of LY333328, vancomycin and teicoplanin by factors of 11.4, 1.6 and 3.8, respectively. The MIC values of LY333328 increased by factors of 3.4 for VanA-type and 6.4 for VanC-type isolates. By microdilution, the increment in the inoculum size, from 104 to 106 CFU/mL, in susceptible, VanA-type and VanC-type isolates increased MIC values of LY333328 by factors of 5.4, 3.6 and 1.1, respectively (Figure 2). Moreover, for the reference VanB-type strain an incoculum effect was also observed for LY333328, with an increased factor of 5. This inoculum effect has not been previously reported.


Figure 2. Comparative activity of vancomycin, teicoplanin and LY333328 against 14 vancomycin-susceptible and 30 vancomycin-resistant (21 VanA type, nine VanC type) enterococcal isolates with different inoculum sizes (expressed as folddilution change of MIC values).

In summary, LY333328 shows high intrinsic activity against enterococci susceptible and resistant to currently available glycopeptides. This excellent in vitro activity and the bactericidal activity described by other authors [11] support the potential use of this new glycopeptide for the treatment of enterococcal infections, particularly in ICU patients, where resistant isolates are increasingly encountered. Our data suggest that the inoculum effect, an important factor in high-density infections, should be carefully controlled in microbiological susceptibility studies with LY333328.


  1. Top of page
  2. Abstract
  6. Acknowledgments
  7. References

This study was partially presented at the 37th Inter-science Conference on Antimicrobial Agents and Chemotherapy, Toronto, 28 September to 1 October 1997 (abstract F-4). This study was supported by a grant from Lilly of Spain. We are grateful to Carmen Torres and Jesús Blázquez for providing PCR technology, Begoña Sánchez del Saz for her technical assistance, and George M. Eliopoulos for providing the SF-299 control strain.


  1. Top of page
  2. Abstract
  6. Acknowledgments
  7. References
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