Methods
Clinical isolates of catalase negative gram-positive coccoid or coccobacillary pairs and chains with α- or γ-hemolysis in our institute during 1997–2004 were subject to an identification aid by API 20 STREP (bioMérieux, Inc., Lyon, France), following the instruction manual. Those identified as
Leuconostoc by API 20 STREP were re-examined by the same kit and by API 50 CHL (bioMérieux, Inc., Lyon, France) according to the instruction manuals, by
Leuconostoc conventional phenotypic assays, by
Leuconostoc- and
Lactobacillus-specific PCR's, and by 16S rDNA sequence analysis as previously described [
4]. The 800-bp 16S rDNA fragment corresponds to Escherichia coli positions 10 to 806. The sequencing results were compared with those available in the GenBank, using BLASTN. Criteria for our conventional phenotypic assays for
Leuconostoc are catalase-negative gram-positive coccoid or coccobacillary bacteria evaluated after growth in thioglycolate broth at 35°C for 24–48 hours [
5], vancomycin MIC ≥ 256 μg/ml by Etest (AB BIODISK, Solna, Sweden), CO2 production from glucose in de Man, Sharp, Rogosa (MRS) broth (Difco, Detroit, MI, USA) with Durham tubes and negative pyrrolidonyl arylamidase (PYR), leucine arylamidase (LAP), and arginine dihydrolase (ADH) [
6].
Leuconostoc-specific PCR was performed on all isolates as described [
7], with slight primer modifications, as stated below. These modifications were to make the primer sequences most complementary and specific to
Leuconostoc strains in GenBank. Forward and reverse primer sequences were 5'-CACAGCGAAAGGTGCTTGCAC-3' and 5'-GATCCATCTCTAGGTGACGCC-3', respectively. To further assess accuracy of the API 20 STREP kit, additional catalase-negative gram-positive coccoid or coccobacillary isolates during 2005–2006 with vancomycin MIC ≥ 256 μg/ml were also evaluated by the same phenotypic and genotypic assays (isolates 31–38 in Table
1). Our gold standard for
Leuconostoc identification is that the organisms fulfill both conventional phenotypic criteria and either or both of the genotypic assays (PCR and 16S rDNA sequence analysis). As we suspected that some isolates might have been
Lactobacillus, also a lactic acid bacteria with overlapping phenotypes,
Lactobacillus-specific PCR was also performed on all isolates as described [
8]. PCR using universal primers targeting bacterial 16S rRNA conserved sequences was also performed to ensure template quality. The forward primer Y1 corresponds to positions 20 to 43 in the
E. coli 16S rRNA sequence and the reverse primer Y2 corresponds to
E. coli positions 361 to 338 [
9]; this protocol gave positive results for all isolates in the study.
Leuconostoc mesenteroides ATCC 8293,
Pediococcus pentosaceus ATCC 33316,
Lactobacillus pentosus ATCC 8041 and
Lactobacillus plantarum ATCC 14917 served as controls for all assays. This study has been approved by The Institutional Review Board of The Faculty of Medicine, Chulalongkorn University.
Table 1
Comparison of various identification methods for 4 ATCC reference strains of Lactobacillus, Pediococcus, Leuconostoc, and 26 catalase-negative, gram-positive clinical isolates from 1997–2004 (numbers 1–26) initially identified as Leuconostoc by API 20 STREP or 7 isolates from 2005–2006 expressing high levels of vancomycin resistance (numbers 31–33 and 35–38).
Lactobacillus plantarum ATCC 14917* |
Enterococcus avium (63.2) |
Lactobacillus plantarum (99.9) | B | -/- | +/+ | - | -/- | R | +ve Lactobacillus
|
Lactobacillus plantarum (100) |
Lactobacillus pentosus ATCC 8041* |
Leuconostoc (81.1) |
Lactococcus lactis ssp lactis 1 (82.5) | B | -/- | +/+ | + | -/- | R | +ve Lactobacillus
| N/A |
Pediococcus pentosaceus ATCC 33316* |
Leuconostoc (39.3) |
Lactobacillus pentosus (84.3) | C | +/+ | +/+ | - | -/- | R | Neg |
Pediococcus pentosaceus (99.4) |
Leuconostoc mesenteroides ATCC 8293 |
Leuconostoc (96.8) |
Leuconostoc mesenteroides ssp mesenteroides/dextranicum 1 (95.7) | Cb | -/- | -/- | + | -/- | R | +ve Leuconostoc
|
Leuconostoc mesenteroides (99.3) |
1-pus* |
Streptococcus suis biotype I (85.6) |
Lactobacillus acidophilus (97.4) | C-Ch | -/+#
| +/-#
| - | -/- | S | Neg |
Streptococcus suis (100) |
2-blood* |
Leuconostoc (99.8) |
Lactococcus lactis ssp lactis 1 (90.5) | Cb | +/+ | -/- | + | -/- | R | Neg | N/A |
3-corneal discharge |
Leuconostoc (97.9) |
Lactobacillus brevis 3 (98.8) | C-Ch | -/- | -/- | + | -/- | R | +ve Leuconostoc
| N/A |
4-ascitic fluid |
Leuconostoc (99.9) |
Leuconostoc mesenteroides spp mesenteroides/dextranicum 2 (99.9) | C | -/- | -/- | + | -/- | R | +ve Leuconostoc
|
Leuconostoc lactis or garlicum (99.5) |
5-ascitic fluid* |
Leuconostoc (93.6) |
Lactobacillus acidophilus 1 (85.1) | Cb | +/+ | -/- | + | -/- | R | Neg | N/A |
6-blood |
Leuconostoc (95.4) |
Leuconostoc lactis (96.0) | Cb | -/- | -/- | + | -/- | R | +ve Leuconostoc
|
Leuconostoc lactis or garlicum (99.6) |
7-blood |
Leuconostoc (68.5) |
Leuconostoc lactis (92.0) | Cb | -/- | -/- | + | -/- | R | Neg | N/A |
8-blood* |
Streptococcus mitis 1 (81.1) |
Lactobacillus acidophilus (92.1) | Cb | -/- | +/-#
| - | -/- | S | Neg | N/A |
9-blood* |
Leuconostoc (97.2) |
Leuconostoc mesenteroides spp cremoris (99.9) | Cb | +/+ | -/- | + | -/- | R | Neg |
Weissella cibaria (100) |
10-blood* |
Abiotrophia adiacens (46.9) Aerococcus viridans 2 (27.5) |
Lactobacillus delbrueckii spp delbrueckii (78.1) | Cb | -/- | -/- | - | -/- | S | Neg | N/A |
11-blood* |
Leuconostoc (92.2) |
Lactobacillus acidophilus (72.5) | C-Ch | -/- | -/+#
| - | -/- | S | Neg | N/A |
12-ascitic fluid* |
Lactococcus lactis spp cremoris (47.2) Leuconostoc (45.1) |
Lactobacillus salivarius (99.9) | C-Ch | -/- | -/- | - | -/- | R | +ve Lactobacillus
| N/A |
13-blood* |
Streptococcus sanguis (49.2) other streptococci (48.5) |
Leuconostoc mesenteroides spp cremoris (98.7) | Cb | -/- | +/-#
| - | -/- | S | Neg | N/A |
14-blood* |
Leuconostoc (39.0) Lactococcus lactis ssp cremoris (37.9) |
Lactobacillus acidophilus 1 (88.2) | C | -/- | -/+#
| - | -/- | S | Neg |
Streptococcus pasteurianus (99.9) |
15-blood* |
Streptococcus bovis (64.8) |
Leuconostoc lactis (87.9) | Cb | -/- | +/-#
| - | -/- | R | Neg |
Weissella confusa (99.9) |
16-blood* |
Enterococcus faecium (98.7) |
Lactobacillus plantarum 1 (98.6) | Cb | +/+ | +/-#
| - | +/-#
| S | Neg |
Enterococcus faecium (99.9) |
17-blood* |
Aerococcus viridans (62.6) |
Lactobacillus delbrueckii spp delbruekii (95.5) | Cb | -/- | -/- | - | -/- | S | Neg |
Actinomyces odontolyticus (98.9) |
18-brain abscess* |
Streptococcus constellatus (99.9) |
Lactobacillus acidophilus (82.1) | C-Ch | +/+ | +/-#
| + | -/- | S | Neg |
Streptococcus anginosus or constellatus (99.7) |
19-blood* |
Streptococcus bovis biotype II (64.8) |
Lactobacillus acidophilus 1 (98.1) | C-Ch | -/- | +/-#
| + | -/- | S | Neg |
Streptococcus constellatus (99.7) |
20-blood* |
Leuconostoc (92.2) |
Weissella viridescens (99.8) | C | -/+#
| -/+#
| - | -/- | R | Neg |
Weissella viridescens (99.9) |
21-lung swab* |
Leuconostoc (99.9) |
Lactobacillus acidophilus (78.4) | Cb | +/+ | -/- | + | -/- | R | Neg |
Weissella cibaria (100) |
22-bone* |
Leuconostoc (99.6) |
Lactobacillus coprophilus (96.9) | Cb | +/+ | -/- | + | -/- | R | Neg |
Weissella confusa (99.61) |
23-NR* |
Leuconostoc (98.8) |
Lactobacillus coprophilus (96.9) | Cb | +/+ | -/+#
| - | -/+#
| R | Neg |
Weissella confusa (100) |
24-NR* |
Aerococcus viridans (48.5) Lactococcus lactis ssp cremoris (41.6) |
Leuconostoc mesenteroides spp cremoris (94.7) | C-Ch | -/+#
| +/-#
| - | -/- | S | Neg |
Streptococcus anginosus (99.9) |
25-pus* |
Streptococcus constellatus (99.9) |
Lactobacillus delbrueckii (80.4) | C-Ch | +/+ | +/-#
| - | -/- | S | Neg |
Streptococcus constellatus (99.7) |
26-duodenal* content |
Leuconostoc (89.9) |
Lactobacillus salivarius (99.9) | Cb | -/- | +/-#
| - | -/- | R | +ve Lactobacillus
|
Lactobacillus salivarius (100) |
31-urine* |
Leuconostoc (99.7) |
Lactobacillus acidophilus 1 (49.5) | Cb | +/+ | -/- | + | -/- | R | Neg |
Weissella cibaria (99.5) |
32-gastric content |
Leuconostoc (99.4) |
Leuconostoc lactis (95.3) | C-Ch | -/- | -/- | + | -/- | R | +ve Leuconostoc
|
Leuconostoc garlicum or lactis (99.2) |
33-tissue biopsy |
Leuconostoc (99.9) |
Leuconostoc mesenteroides spp mesenteroides/dextranicum2 (99.9) | C-Ch | -/- | -/- | + | -/- | R | +ve Leuconostoc
|
Leuconostoc garlicum or lactis (99.6) |
35-ascitic fluid* |
Leuconostoc (65.8) |
Pediococcus pentosaceus 1 (63.6) | C | -/- | +/+ | - | -/- | R | neg |
Pediococcus stilesii (91.5) |
36-blood |
Leuconostoc (92.8) |
Lactobacillus collinoides or fermentum 1 (98.3) | Cb | -/- | -/- | + | -/- | R | neg |
Weissella confusa (99.9) |
37-pleural fluid* |
Leuconostoc (69.2) |
Lactobacillus salivarius (99.9) | Cb | -/- | +/+ | - | -/- | R | neg | N/A |
38-tissue biopsy* |
Leuconostoc (82.7) |
Pediococcus pentosaceus (99.9) | C | +/+ | +/+ | - | -/- | R | neg |
Pediococcus pentosaceus (98.2) |
Results
Our clinical bacteriology laboratory has a busy service, serving a 1,500-bed university hospital. Out of several thousands of gram-positive bacteria isolated during 1997–2004, 26 catalase-negative gram-positive isolates (isolates 1–26) were initially identified as Leuconostoc by API 20 STREP. 7 catalase-negative gram-positive strains with vancomycin MIC ≥ 256 μg/ml were isolated during 2005–2006 (isolates 31–33 and 35–38). Thus, 33 clinical isolates entered the study. As 16S rDNA sequencing analysis was performed after the other tests, the results were not complete. Some isolates could not be retrieved, and some could not be amplified.
11 isolates of isolates 1–26 were reproducibly identified by API 20 STREP as
Leuconostoc (Table
1). Only 3 of the 11 isolates, however, were confirmed by both genotypic and all defined phenotypic criteria (Table
1). 7 catalase-negative gram-positive isolates with vancomycin MIC ≥ 256 μg/ml (isolates 31–33 and 35–38) were all identified as
Leuconostoc by API 20 STREP, only 2 of which were confirmed genotypically. API 20 STREP identified
Lactobacillus pentosus ATCC 8041 and
Pediococcus pentosaceus ATCC 33316 as
Leuconostoc with 81.1% and 39.3% identity, respectively. Regarding all 31 non-leuconostoc strains (including reference strains and clinical isolates), API 20 STREP identified 10 of them as
Leuconostoc with over 90% identity. 16S rDNA sequencing data were available in 7 of the 10 isolates and all were closely-related
Weissella spp. 6 isolates were read as
Leuconostoc with 50–90% identity. Two of these were
Lactobacillus pentosus ATCC 8041 and
Lactobacillus salivarius (isolate 26) and two were
Pediococcus (isolates 35 and 38). API 50 CHL identified almost all
Leuconostoc correctly, at least to the genus level, except for isolate 3. The kit, however, identified
Pediococcus pentosaceus ATCC 33316, 7 out of 8
Weissella spp., all 6 streptococci, and 1
Enterococcus as either
Lactobacillus or
Leuconostoc. Of all 37 standard and clinical strains, 14 demonstrated at least one discrepant biochemical test results between API 20 STREP and manual phenotypic assays (Table
1). All these belonged to non-leuconostoc isolates and do not affect conventional phenotypic interpretation of these isolates as non-leuconostoc. Identity percentages given by API 20 STREP and API 50 CHL had poor correlations with PCR or phenotypes.
With regard to the 7 isolates of catalase-negative gram-positive bacteria with high-level vancomycin resistance which were all identified as Leuconostoc by API 20 STREP, only 2 of them were confirmed as such by genotypic assays. API 50 CHL correctly identified both isolates as Leuconostoc, one of which was correct at the species level.
Discussion
Commercial diagnostics have been widely used in bacteriology laboratories to identify common organisms, such as
Streptococcus, to the species level, or to identify unusual gram-positive organisms in clinical specimens, e.g.
Aerococcus,
Lactobacillus, and
Leuconostoc, among others. Studies illustrating inaccurate identification of various gram-positive pathogens have been published [
3,
10‐
14]. In this study, our purpose is to raise an awareness that
Leuconostoc, an emerging human pathogen, can be overdiagnosed by certain commercial diagnostics.
Occasional discrepant results among the same biochemical tests obtained from API 20 STREP and from manual conventional assays are not unexpected, as incomplete agreement of various automated and manual systems have been reported [
15‐
17]. Reproducibility of API 20 STREP for
Leuconostoc identification is only moderate in our study. Previous studies have shown higher consistency of bacterial identification by commercial diagnostics [
18,
19]. Clinical isolates in our study were initially identified during the time spanning from 1997–2006 and thus repeated API 20 STREP testing was done months or years thereafter. Our lower reproducibility could be, at least partly, due to loss or change in some characteristics by repeated subculture [
20,
21].
All 6 standard and clinical
Leuconostoc strains were correctly identified by API 20 STREP and 5 by API 50 CHL, at least at the genus level. On the contrary, specificity of
Leuconostoc identification by these API kits were only moderate at best. As evidenced by 16S rDNA sequence analysis, most of the isolates misidentified as
Leuconostoc by API systems were in the genus
Lactobacillus and
Weissella, which are closely-related bacteria, followed by
Pediococcus, also one of the lactic acid bacteria [
6]. API 20 STREP failed to identify these isolates obviously because
Lactobacillus and
Weissella are not listed in the Identification Table [
22]. Even some strains of streptococci and enterococci initially were identified as Leuconostoc.
Streptococcus constellatus isolate 19 was misidentified at the species level. API 50 CHL misidentified most
Weissella in this study obviously because only one species,
Weissella viridescens, is included in the Identification Table of the kit.
Our conventional phenotypic criteria correlated well with
Leuconostoc-specific PCR and 16S rDNA sequence analysis in almost all isolates, except for isolates 7 and 36 (Table
1). Isolate 7 was phenotypically compatible with
Leuconostoc but negative by PCR. This could be closely-related bacteria which are certain lactobacilli such as
L. sanfrancisco, or
L. fructosus, or
Weissella [
23], or some rare
Leuconostoc not detected by our PCR protocol.
Weisella is a recently-described genus found in a variety of foods. Some of its members used to be
Leuconostoc paramesenteroides and heterofermentative lactobacilli. Reliability of the conventional phenotypic criteria in this study is evidenced by the fact that only 1 of the 8
Weissella isolates and none of the 2
Lactobacillus isolates (one identified by 16S rDNA sequencing and both by PCR) was misidentified as
Leuconostoc.
The importance of accurate identification of
Leuconostoc also needs to be emphasized in the clinical arena. Case reports based on incomplete and/or inappropriate phenotypic criteria with or without assistance of commercial diagnostics are subject to potential errors [
24‐
27], given the fact that
Leuconostoc and related bacteria possess overlapping phenotypes. Flawed clinical reports include an incorrect argument that heterofermentative
Lactobacillus must hydrolyze arginine [
25], while in fact
L. sanfrancisco and
L. fructosus do not [
23], and labeling the organism as
Leuconostoc even though the organism was LAP positive [
26].
Two major limitations of API 20 STREP are noted. Firstly, the test contains
Leuconostoc in its list, while some other medically-important lactic acid bacteria with overlapping phenotypes such as
Lactobacillus,
Weissella and
Pediococcus, are not included. It is of note, however, that, according to the manufacturer,
Leuconostoc is a multiple taxon of
Leuconostoc and
Lactobacillus and if a strain is identified as
Leuconostoc, a note "POSSIBILITY OF
Lactobacillus spp" is included in the report. Considering
Leuconostoc as a multiple taxon of
Leuconostoc and
Lactobacillus by the manufacturer is not very practical, as
Leuconostoc and
Lactobacillus are distinct bacteria, microbiologically and clinically. Given that human infections by these lactic acid bacteria are emerging, these organisms could obviously be misidentified as
Leuconostoc by API 20 STREP, potentially contributing to cumulative incorrect reporting in medical literature and incorrect understanding of its clinical spectra and epidemiology. Secondly, while clinical isolates of
Leuconostoc, as a rule, are LAP and ADH negative, the test lists
Leuconostoc as 70% LAP and 10% ADH positive [
22]. Appropriate modifications of the kit criteria for
Leuconostoc would significantly enhance its accuracy. For clinical laboratories, we propose that all catalase-negative gram-positive coccoid or coccobacillary bacteria with high level of vancomycin resistance (MIC ≥ 256 μg/ml) be tested with the manual phenotypic assays listed in Table
1: Gram's staining of the isolate grown in thioglycolate broth, arginine dihydrolase (ADH), leucine arylamidase (LAP), gas production in MRS broth, and pyrrolidonyl arylamidase (PYR) test. Users of this method are to accept that, even though more accurate than the current API systems, these conventional assays could still occasionally misidentify certain lactobacilli and
Weissella as
Leuconostoc. This practical guide should minimize avoidable inaccurate identification of this emerging pathogen.
Acknowledgements
The authors thank Dr. Somboon Tanasupawat, Department of Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand, for ATCC bacteria used in the study, Dr. Nattachai Srisawat, Department of Medicine, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand, for assistance with clinical data collection, Ms. Sivanee Joybai, Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand for technical assistance, all during March and April 2004, Chulalongkorn Medical Research Center and Faculty of Medicine OPR 11 Special Laboratories for core molecular facilities throughout the study during 2004–2007. This study was supported by Ratchadapiseksompotch Pilot Study Fund from Faculty of Medicine, Chulalongkorn University and Development Grant for New Faculty/Researchers, Chulalongkorn University in 2004. During the study, Dr. Kulwichit was supported by Faculty Fund, Faculty of Medicine (2004–2007); Research Scholar Support Fund, King Ananda Mahidol Foundation (2004–2005); and Research Scholar Fund, Thailand Research Fund (2004–2005 and 2006–2007).
This work was completed at the Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand, and was presented, in part, at The 44th Interscience Conference on Antimicrobial Agents and Chemotherapy (ICAAC), Washington, D.C., October 30–November 2, 2004
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
WK conceived of and designed the study, participated in its coordination, analyzed data, and drafted the manuscript. SN participated in coordination of the study, carried out experiments on API systems and conventional assays of bacteria, and assisted with PCR assays. TC performed 16S rDNA sequence analysis. SK carried out all PCR assays. CU performed susceptibility tests. AC helped design and coordinated the study. All authors read and approved the final manuscript.