Diagnostic Microbiology and Infectious Disease 95 (2019) 1–4

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Diagnostic Microbiology and Infectious Disease

journal homepage: www.elsevier.com/locate/diagmicrobio

Bacteriology

A selective culture medium for screening linezolid-resistant gram-

positive bacteria
Patrice Nordmann a,b,c,d,⁎, Angel Rodríguez-Villodres a,e,f, Laurent Poirel a,b,c
a
Medical and Molecular Microbiology Unit, Department of Medicine, Faculty of Science, University of Fribourg, Fribourg, Switzerland
b
INSERM European Unit (IAME, France), University of Fribourg
c
Swiss National Reference Center for Emerging Antibiotic Resistance (NARA), Fribourg, Switzerland
d
Institute for Microbiology, University of Lausanne and University Hospital Centre, Lausanne, Switzerland
e
Clinical Unit of Infectious Diseases, Microbiology and Preventive Medicine, University Hospital Virgen del Rocío, Seville, Spain
f
Institute of Biomedicine of Seville (IBiS), University Hospital Virgen del Rocío/CSIC/University of Seville, Seville, Spain

a r t i c l e i n f o a b s t r a c t

Article history: The SuperLinezolid medium was developed for screening resistance to linezolid (LZD) in Gram-positive bacteria
Received 25 January 2019 (Staphylococcus spp., Enterococcus spp.). It was evaluated using LZD-susceptible (n = 20) and LZD-resistant (n =
Received in revised form 14 March 2019 17) Gram-positive isolates. The sensitivity was found to be 82% at 24 h (3 out of 17 isolates being missed), and
Accepted 16 March 2019
reached 100% at 48 h. At 48 h, a single LZD-susceptible isolate grew (specificity 95%). By testing stools spiked
Available online 21 March 2019
with LZD-resistant Gram-positive strains, an excellent performance of the medium was observed, with a lowest
Keywords:
detection limit ranging from 101 to 102 CFU/ml. Overall, this medium is accurate for detection of LZD-resistant
Linezolid Gram-positive isolates after 24 h of culture.
Antibiotic © 2019 Published by Elsevier Inc.
Screening
Enterococci
Staphylococci

Oxazolidinone antibiotics such as linezolid (LZD) are increasingly In addition, acquired transferable LZD resistance genes have been re-
used as a consequence of an increased rate of multidrug-resistant ported, namely the cfr, cfr(B), cfr(C), optrA, and poxtA genes (Sadowy,
Gram-positive pathogens (Leach et al., 2011). Considering the wide dif- 2018). The cfr gene encodes a methylase modifying the C-8 position of
fusion of methicillin-resistant Staphylococcus aureus and Staphylococcus A2503 residue in the 23S rRNA methylases (Kehrenberg et al., 2005;
epidermidis isolates worldwide (Purrello et al., 2016), as well as the Schwarz et al., 2000), and confers resistance not only to LZD but also
wide diffusion of vancomycin-resistant Enterococcus faecium and to phenicols, lincosamides, pleuromutilins, and streptogramins (so
Enterococcus faecalis (Zahedi Bialvaei et al., 2017), there is a significant called PhLOPSA resistance phenotype), but noticeably spares the novel
need to rely on the use of LZD to treat infections caused by those oxazolidinone tedizolid (Long et al., 2006). This gene has been identified
multidrug-resistant isolates (Cattoir and Giard, 2014). Therefore, it not in S. aureus, enterococci, Streptococcus suis, and Bacillus spp., but also in
surprising that occurrence of LZD-resistant isolates is now increasingly several Gram-negative bacteria such as Escherichia coli and Proteus
reported, including in S. aureus, S. epidermidis, E. faecium, and vulgaris. The cfr(B) gene sharing 72% nucleotide identity with cfr was
E. faecalis (Bi et al., 2017; Bourgeois-Nicolaos et al., 2014; Gu et al., found in S. aureus, also conferring a PhLOPSA resistance phenotype
2013). Consequently, accurate and rapid identification of LZD-resistant (Marín et al., 2015). The cfr(C) gene encoding a protein sharing ca.
isolates is needed. 55% amino acid identity with Cfr(A) and Cfr(B) was found in
The main mechanism of resistance to LZD in Gram-positive bacteria Campylobacter spp. (Tang et al., 2017).
corresponds to a specific mutation (G2576 T) in the 23S rRNA gene, In addition to those Cfr-like 23S rRNA methylases, another acquired
preventing the binding of the drug to its target, i.e. the ribosome resistance trait to LZD has been reported, being OptrA (Wang et al.,
(Sadowy et al., 2018). Some other mutations have been reported in 2015). It belongs to the ABC-F family of ATP-binding proteins, and has
the 23S rRNA gene, leading to modifications of the L3, L4, and L22 ribo- been characterized as a ribosomal protection protein. The correspond-
somal proteins, but they are much less frequent (Bi et al., 2017; Gu et al., ing gene was first identified in E. faecalis, and later in Staphylococcus
2013; Pfaller et al., 2017a, 2017b). sciuri and Streptococcus gallotycus (Sharkey et al., 2016).
Finally, another ribosomal protection protein, PoxtA, was recently
⁎ Corresponding author. Tel.: +41-26-300-9583. identified from a human methicillin-resistant S. aureus (MRSA) clinical
E-mail address: patrice.nordmann@unifr.ch (P. Nordmann). isolate isolate, and further identified in E. faecalis and E. faecium of

https://doi.org/10.1016/j.diagmicrobio.2019.03.006
0732-8893/© 2019 Published by Elsevier Inc.
2 P. Nordmann et al. / Diagnostic Microbiology and Infectious Disease 95 (2019) 1–4

Table 1
Preparation of the SuperLinezolid medium.

Compounds Stock solution Quantity or volume to adda Final concentration

BHI agar powder - 14.8 g 3.7%

Distilled water - 400 ml
Linezolid 2 mg/ml 300 μl 1.5 μg/ml
Aztreonam 10 mg/ml 80 μl 2 μg/ml
Colistin sulfate 15 mg/ml in water in glass tubes 400 μl 15 μg/ml
Amphotericin B 20 mg/ml in D(+)-glucose 10% 100 μl 5 μg/ml
a
The volume of 400 ml of SuperLinezolid medium was for twenty plates.

animal origin (Antonelli et al., 2018). That resistance mechanism con- with species being intrinsically resistant or heteroresistant to colistin
fers reduced susceptibility not only to oxazolidinone, but also to tetracy- such as Proteus spp., Serratia spp., Hafnia spp., or Enterobacter cloacae)
clines and phenicols. (Poirel et al., 2017), aztreonam was added at a concentration of 2 μg/ml.
Taking in account the potential clinical threat represented by a diffu- While addition of aztreonam contributed to inhibit growth of Gram-
sion of LZD-resistant strains, our aim was to develop a selective culture negative bacteria (unless they produced broad-spectrum ß-lactamases),
medium for screening of LZD-resistant bacteria both among human and it did not modify the growth of the Gram-positive bacteria tested,
animal isolates. including LZD-susceptible and -resistant isolates. Amphotericin B
(Bristol-Myers-Squibb, Rueil-Malmaison, France) was added as an anti-
1. Material and methods fungi molecule at a final concentration of 5 μg/ml. Cultures were
incubated at 37°C during 18 h. When no growth was observed after
1.1. Preparation of the SuperLinezolid medium 24 h, the incubation period was extended to 48 h to definitely assess
that no growth actually occurred.
The necessity to prevent contamination by Gram negatives and fungi The instructions for the preparation of SuperLinezolid medium are
was taken in account for the development of this medium. Based on our indicated in Table 1. The stock solutions may be kept at −20°C. For pre-
own experience of developing screening media (Nordmann et al., 2012, paring the SuperLinezolid medium, the diluted powder of BHI was
2016), the optimal medium retained was based on the Brain Heart Infu- autoclaved at 121°C for 15 min. After cooling this medium for one
sion (BHI) medium (ref 3,564,014; Bio-Rad, Cressier, Switzerland). This hour at 56°C, the antibiotic stock solutions were added (Table 1). Once
medium was rich enough to enhance the growth of the Gram-positive poured, the plates were stored at 4°C and protected from direct light ex-
isolates we tested. posure. The SuperLinezolid plates were kept one month and no major
To determine the optimal concentrations of each compound of the effect on its performances was noticed over time (data not shown).
SuperLinezolid medium, a series of different preliminary tests was (See Fig. 1.)
performed, using six linezolid-resistant and two linezolid-susceptible A total of thirty-seven isolates of various Gram-positive species
isolates, including Staphylococci and Enterococci strains. Using an inocu- (Enteroccocus faecium, Enterococcus faecalis, Enterococcus casseliflavus,
lum with an optical density of 0.5 Mac Farland (inoculum of ~10 8 CFU/ Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus
ml), a 1000-fold dilution of the strains to be tested was made in normal capitis) recovered in France were tested to evaluate the performance
saline solution and a 100-μl volume was plated onto the SuperLinezolid of the SuperLinezolid medium. Seventeen of those isolates were resis-
medium. To quantify the viable bacteria in each dilution, BHI agar me- tant to LZD, and twenty isolates were susceptible. In addition, a total
dium was inoculated concomitantly with 100 μl of suspension and incu- of eleven Gram-negative isolates were tested, including Enterobacteria-
bated overnight at 37°C. A range of concentrations varying from 0.25, ceae (n = 9), Pseudomonas aeruginosa (n = 1), and Acinetobacter
0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4 mg/L was tested, and the 1.5 mg/L concen- baumannii (n = 1) isolates. All those isolates were clonally-unrelated
tration was retained since it allowed to obtain the optimal sensitivity (data not shown).
and specificity values. Colistin sulfate (tablets, MAST DIAGNOSTICS, The lowest limit of detection with the SuperLinezolid medium was de-
Merseyside, UK) was added in the medium at a final concentration of termined for all the tested strains. Using an inoculum with an optical den-
15 μg/ml to prevent the growth of colistin- susceptible Gram-negative sity of 0.5 McFarland standard (inoculum of ∼108 CFU/ml), serial 10-fold
isolates. In addition, considering that growth of colistin-resistant dilutions of the isolates were made in normal saline, and 100-μl portions
Gram-negative strains might be a source of contamination (particularly were plated onto the SuperLinezolid medium. To quantify the viable

Fig. 1. LZD-resistant S. epidermidis (A), and E. faecium (B) growing onto the SuperLinezolid medium.
 P. Nordmann et al. / Diagnostic Microbiology and Infectious Disease 95 (2019) 1–4 3

Table 2 dilution on the screening medium. The sensitivity and specificity were
Lowest limits of detection of the SuperLinezolid medium for linezolid-resistant isolates. determined using the same cut-off value set at ≥10 3 CFU/mL
Strains MIC of linezolid (mg/L) Growth on the At 48 h (Nordmann et al., 2016).
SuperLinezolid MICs of LZD were determined using the broth microdilution method
medium in Mueller-Hinton broth, as recommended by the CLSI (Clinical and
At 24 h
Laboratory Standards Institute, 2018). For each strain, an inoculum cor-
E. faecium CNR-15-307 32 + + responding to 5 × 10 5 CFU/ml was distributed in the 96-well tray
S. aureus 2015S421 8 + +
(Sarstedt, Nümbrecht, Germany). The evaluation of the selective me-
S. aureus ST2015–1386 32 + +
S. capitis ST 2015–2014 N256 + + dium was performed in triplicate.
S. epidermidis ST2014–0603 8 + + According to the CLSI and EUCAST breakpoints (www.eucast.org/
S. epidermidis A N256 + + clinical_breakpoints/), Staphylococcus spp. and Enterococcus spp. iso-
S. epidermidis 2 N256 + + lates with MIC values of LZD ≤ 4 mg/L are categorized as susceptible,
S. epidermidis 22 N256 + +
whereas those with MIC values ≥8 mg/L are categorized as resistant.
S. epidermidis ST N256 + +
2015–1734
S. epidermidis ST N256 + + 2. Results
2015–1739
S. epidermidis LESC 256 + +
Overall, all LZD-resistant Gram-positive isolates tested grew on the
S. epidermidis HM-1 16 − +
S. epidermidis HM-2 16 − + SuperLinezolid medium after 24 h, except three LZD-resistant
S. epidermidis HM-3 256 + + S. epidermidis isolates (HM-1, HM-2, and HM-6) that did not grow
S. epidermidis HM-4 32 + + after 24 h of incubation (even at a concentration of 10 3 CFU/ml), but
S. epidermidis HM-5 256 + + grew after 48 h (Table 2). As expected, no growth was observed for
S. epidermidis HM-6 8 − +
the 11 Gram-negative isolates tested.
E. faecalis ATCC 29212 2 − −
E. faecalis N95 1 − − The sensitivity was found to be 82% at 24 h (3 out of 17 isolates being
E. faecalis N89 1 − − missed), and reached 100% at 48 h. At 48 h, a single LZD-susceptible iso-
E. faecalis 2953 2 − − late grew (specificity 95%).
E. faecalis 2094 2 − −
For all the LZD-susceptible isolates, the lowest limit of detection was
E. faecalis 2146 2 − −
E. faecalis 2147 2 − − found above the cut-off value of 103 CFU/ml, being in fact ≥1 × 106 CFU/
E. casseliflavus 2149 2 − − ml (Table 2). By contrast, all the LZD-resistant isolates grew on the
S. aureus ATCC 29213 2 − − SuperLinezolid medium in 24 h and the lowest limit of detection was
S. aureus C1013 1 − − below the cut-off value. All LZD-susceptible Gram-positive did not
S. aureus C1014 1 − −
growth after 24 h of incubation when the inoculum was up to
S. aureus 2954 2 − −
S. aureus 2092 2 − − 10 3 CFU/ml; small colonies were obtained only for a single isolate
S. aureus 2973 2 − + (S. aureus 2973) after 48 h of incubation when using this same inocu-
S. aureus 3108 2 − − lum. Therefore, a specificity of 100% was observed after a 24-h culture.
S. aureus 2959 2 − −
Finally, no Gram-negative isolate grew after either 24 h or 48 h of
S. aureus 2732 1 − −
S. epidermidis N30 1 − −
incubation.
S. epidermidis N79 1 − − The spiked stools containing LZD-resistant strains grew with a low-
S. epidermidis 2145 2 − − est detection limit ranging from 10 1 to 102 CFU/ml (Table 3), thus nicely
corresponding to the required sensitivity. In addition, no false-positive
isolate was recovered, thus showing an excellent specificity.
bacteria in each dilution, BHI agar was inoculated concomitantly with
100 μl of suspension and was incubated overnight at 37°C. The number 3. Discussion
of viable colonies was counted after 24 h of culture at 37°C. The sensitivity
and specificity cut-off values were set at 1 × 103 CFU/ml i.e., a limit value We developed here a selective medium allowing screening and de-
of 1 × 103 CFU/ml and above was considered as « not efficiently detected » tection of Gram-positive bacteria exhibiting resistance to LZD. Consider-
(Schwarz et al., 2000). ing that LZD-resistant isolates are currently emerging in different parts
Spiked stools were also tested using this selective culture medium, of the world, this selective medium may be used to perform prospective
done in triplicate. Spiked fecal samples were made by adding 100 μl of screening, and epidemiological surveys. Some surveys had used media
each strain dilution to 900 μl of fecal suspension that was obtained by supplemented with LZD for selection of LZD-resistant isolates but nei-
suspending 5 g of freshly pooled feces from three healthy volunteers ther systematic evaluation nor development of such media had been
in 50 ml of distilled water, as done previously (Nordmann et al., performed (Bourgeois-Nicolaos et al., 2014; Lode et al., 2001).
2016). A non-spiked fecal suspension was used as negative control. The SuperLinezolid medium may detect all clinically-relevant bacte-
The lowest detection limit was determined by plating 100 μl of each rial species exhibiting resistance to LZD, regardless of the resistance

Table 3
Lowest limits of detection of the SuperLinezolid medium for a series of linezolid-resistant isolates in spiked stools.⁎

Strains Species Genotype MIC of linezolid (μg/ml) Lowest limit of detection

2015S421 S. aureus Cfr+ 8 101

CNR E. faecium CfrC + OptrA+ 32 101
ST2015–2014 S. capitis Mutations RNA T/2319/C; G/2576/T N256 102
ST2014–0603 S. epidermidis Mutations RNA T/2504/A; C/2534/T 8 101
LESC S. epidermidis ND (cfr-; optrA-) 256 101
HM-2 S. epidermidis ND (cfr-; optrA-) 16 101⁎
HM-3 S. epidermidis Cfr+ 256 101
HM-6 S. epidermidis Cfr+ 8 102
⁎ Only after 48 h growth.
4 P. Nordmann et al. / Diagnostic Microbiology and Infectious Disease 95 (2019) 1–4

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