Multilocus sequence analysis of the genus Citrobacter and description of Citrobacter pasteurii sp. nov.

Abstract

Multilocus sequence analysis of the genus Citrobacter and description of Citrobacter pasteurii sp. nov. Species of the genus Citrobacter are considered to be inhabitants of human and other animal guts and can be found in varied environmental habitats (Borenshtein & Schauer, 2006). In humans, several species of the genus Citrobacter are regarded as opportunistic pathogens and have been associated with various infections, including urinary tract infections, wound infections, pneumonia, abscesses, septicaemia, meningitis, endocarditis and diarrhoea (Lipsky et al., 1980;Doran, 1999;Mohanty et al., 2007;Samonis et al., 2009;Vaz Marecos et al., 2012;Chowdhry & Cohen, 2012). Citrobacter rodentium can cause colitis and transmissible colonic hyperplasia in mice (Luperchio & Schauer, 2001) and uses molecular mechanisms of pathogenesis similar to those observed for enteropathogenic and enterohaemorrhagic strains of the species Escherichia coli causing diarrhoea and haemolytic uraemic syndrome in humans (Petty et al., 2011). At the time of writing, the genus Citrobacter comprises 11 species: Citrobacter freundii (the type species of the genus), Citrobacter amalonaticus, Citrobacter braakii, Citrobacter farmeri, Citrobacter gillenii, Citrobacter koseri, Citrobacter murliniae, C. rodentium, Citrobacter sedlakii, Citrobacter werkmanii and Citrobacter youngae (Brenner et al., 1993;Frederiksen, 2005). Since the large DNA hybridization study of Brenner and colleagues (Brenner et al., 1993), only three novel species have been described: C. rodentium (Schauer et al., 1995), C. gillenii and C. murliniae (Brenner et al., 1999). Identification of isolates of species of the genus Citrobacter can be tentatively achieved using biochemical profiles or matrixassisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF) (Richter et al., 2013; Kolinská et al., 2014). Phylogenetic relationships based on 16S rRNA sequences distinguished three groups (Warren et al., 2000): group I (C. freundii, C. youngae, C. braakii, C. werkmanii, C. gillenii and C. murliniae); group II (C. amalonaticus, C. farmeri, C. sedlakii and C. rodentium); and group III (C. koseri). 16S rRNA gene sequence variation provides limitedAbbreviations: ANI, average nucleotide identity; MALDI-TOF, matrixassisted laser desorption/ionization time-of-flight mass spectroscopy; MLSA, multilocus sequence analysis.The GenBank/EMBL/DDBJ accession numbers for the gene sequences of the type strains of species of the genus Citrobacter resulting from this study are KM515966 to KM515975, KP728117 and KP057683 for rrs, KM515976 to KM515985 for fusA, KM516006 to KM516012 for rpoB, KM515996 to KM516005 for pyrG, and KM515986 to KM5159 95 for leuS.The annotated genomic sequence of strain CIP 55.13

Phylogeny and Comparative Genomics Unveil Independent Diversification Trajectories of qnrB and Genetic Platforms within Particular Citrobacter Species

“…Individual nucleotide sequences of genes included in the MLSA scheme of the Citrobacter genus (rpoB, pyrG, fusA, and leuS) (6) and recN were aligned and the average rates of similarity calculated using MEGA version 5.2.2 (http://www.megasoftware.net/) (10). The leuS gene sequences from Clermont et al were included in this analysis (6).…”

“…The leuS gene sequences from Clermont et al were included in this analysis (6). Similarity scores of the leuS and recN genes were calculated and individual phylogenetic trees were constructed in MEGA using the neighbor-joining (NJ) method (11), and genetic distances were calculated using the Kimura two-parameter model (12) in the case of nucleotide sequences and using the Jones-Taylor-Thornton (JTT) model (13) for LeuS and RecN protein sequences.…”

“…Isolates included in this study were identified by biochemical methods (7), mass spectrometry (MALDI-TOF MS; Bruker Daltonik, Germany), and sequencing of 16S rRNA (8), leuS (leucine tRNA synthetase) (6), and recN (DNA repair protein) genes. PCR amplification and further sequencing of recN genes were performed by using primers recN-Fw (5=-ATTGCCATTGATGCTC TCGG-3=) and recN-Rv (5=-ANCGAGTCGGCCTGATCGT-3=) to amplify a 637-bp internal fragment and the following amplification conditions: one cycle of 3 min at 95°C; 35 cycles of 1 min at 95°C, 1 min at 56°C, and 1 min at 72°C; and 1 cycle of 1 min at 72°C.…”

“…Recently, Clermont et al described a multilocus sequence analysis (MLSA) based on partial sequences of rpoB (␤ subunit of RNA polymerase gene), pyrG (CTP synthetase gene), fusA (protein synthesis elongation factor-G gene), and leuS (leucine tRNA synthetase gene) that allowed the discrimination of the 12 recognized Citrobacter species, namely, Citrobacter freundii, C. amalonaticus, C. braakii, C. farmeri, C. gillenii, C. koseri, C. murliniae, C. rodentium, C. sedlakii, C. werkmanii, C. youngae, and C. pasteurii (6).…”

“…In fact, most of the methods conventionally used to identify Citrobacter spp. (biochemical or phenotypic features, matrix-assisted laser desorption ionization-time of flight mass spectrometry [MALDI-TOF MS], or 16S rRNA gene sequencing) (6)(7)(8) have low discriminatory power, hindering the accurate discrimination of these species.…”