Similarities between restriction endonuclease digestion profiles were check details analyzed by using Unweighted Pair Group Method with Arithmetic Mean (UPGMA) of BioNumerics
software (Applied Maths, Kortrijk, Belgium). Multi-locus sequence typing and phylogenetic analysis The MLST scheme available at http://www.pasteur.fr/Eltanexor mw recherche/genopole/PF8/mlst/Lmono.html was used. The nucleotide sequences of internal fragment of the following genes, acbZ (ABC transporter), bglA (beta-glucosidase), cat (catalase), dapE (succinyl diaminopimelate desuccinylase), dat (D-amino acid aminotransferase), ldh (L-lactate dehydrogenase), and lhkA (histidine kinase), were obtained by PCR using published primers (Table 1) with the exception of primers for lhkA. A new pair of primers for lhkA (lhkAF 5′-GTTTTCCCAGTCACGACGTTGTATTATCAAAGCAAGTAGATG-3′ and lhkAR 5′-TTGTGAGCGGATAACAATTTCTTTCACTTTTTGGAATAATAT-3′) were designed to amplify the lhkA gene from the isolates which had no amplification products when the published primers were used. A 50-μl reaction was composed as follows: 5.0 μl of 10 × pfu buffer with 1.5 mM MgCl2, 125 μM each of deoxynucleoside triphosphate mix, 0.2 μM forward and reverse primers, 0.5U of pfu DNA polymerase, and 2U of rTaq DNA polymerase.
The PCR amplification conditions were as follow: 94°C for 4 min and 30 cycles of 94°C for 30 s, 52°C for 30s, and 72°C for 2 min, followed by one cycle of 72°C for Bafilomycin A1 order 10 min and hold triclocarban indefinitely at 4°C. The purified PCR products were sent for sequencing commercially. For each isolate, the allele combination at the 7 loci defines
an allelic profile or sequence type (ST). Minimum spanning tree (MST) analysis was used to infer relationships among the isolates and was done using BioNumerics (Applied Maths, Belgium). Neighbor-joining tree of the seven concatenated housekeeping gene sequences was constructed using MEGA 4.0 [30]. A clonal complex (CC) is defined based on eBURST algorithm with member STs differing by only one of the 7 MLST genes [23]. Results Serotyping The 212 isolates used in this study were typed into seven of the 13 known serotypes: 1/2a, 1/2b, 1/2c, 3a, 3b, 4b and 4c. The most frequent serotypes are 1/2c, 1/2a and 1/2b with a frequency of 36.8%, 33.5% and 19.8% respectively. The remaining 4 serotypes account for only 9.9% of the total isolates. Pulsed-field gel electrophoresis PFGE analysis divided the 212 isolates into 61 pulse types (PTs). PTGX6A16.0004 was predominant and accounts for 26.5% of the isolates, followed by GX6A16.0011 (17 isolates), and GX6A16.0009 (13 isolates). Thirty two PTs (52.5%) were represented by only a single isolate. A UPGMA dendrogram was constructed for the 61 PTs based on presence or absence of bands. The PTs are divided into 3 clusters. Cluster I contained all serotype 1/2c isolates, the majority of serotype 1/2a isolates. Cluster II contained all serotype 4b and 1/2b isolates and the remaining serotype 1/2a isolates.