Serotyping of sub-Saharan Africa Salmonella strains isolated from poultry feces using multiplex PCR and whole genome sequencing.

BACKGROUND: Salmonella enterica remains a leading cause of food-borne diseases worldwide. Serotype information is important in food safety and public health activities to reduce the burden of salmonellosis. In the current study, two methods were used to determine serotypes of 111 strains of Salmonella isolated from poultry feces in Burkina Faso. First, Salmonella Multiplex Assay for Rapid Typing (SMART) Polymerase Chain Reaction (PCR) was used to determine the serovars of the S. enterica isolates. Second, serovar prediction based on whole genome sequencing (WGS) data was performed using SeqSero 2.0. RESULTS: Among the 111 Salmonella isolates, serotypes for 17 (15.31%) isolates were identified based on comparison to a panel of representative SMART codes previously determined for the 50 most common serovars in the United States. Forty-four (44) new SMART codes were developed for common and uncommon serotypes. A total of 105 (94.59%) isolates were serotyped using SeqSero 2.0 for serovar prediction based on WGS data. CONCLUSION: We determined that SeqSero 2.0 was more comprehensive for identifying Salmonella serotypes from Burkina Faso than SMART PCR.

Genome Analysis of Multidrug-Resistant Escherichia coli Isolated from Poultry in Nigeria.

Escherichia coli is one of the most common commensal bacteria of the gastrointestinal tract of humans and warm-blooded animals. Contaminated poultry can lead to disease outbreaks in consumers causing massive economic losses in the poultry industry. Additionally, commensal E. coli can harbor antibiotic resistance genes that can be transferred to other bacteria, including pathogens, in a colonized human host. In a previous study on antimicrobial resistance of E. coli from food animals from Nigeria, multidrug-resistant E. coli were detected. Three of those isolates were selected for further study using whole-genome sequencing due to the extensive drug resistance exhibited. All of the isolates carried the extended-spectrum ß-lactamase (ESBL) genes, blaCTX-M15 and blaTEM-1, whereas one isolate harbored an additional ESBL, blaOXA-1. All of the tetracycline-resistant isolates carried tet(A). The genes aac3-IIa and aacA4, conferring resistance to aminoglycosides, were identified in an E. coli isolate resistant to gentamicin and tobramycin. In two E. coli isolates, dfrA14, qnrS1, and sulII, were detected conferring resistance to trimethoprim, fluoroquinolones, and sulfonamides, respectively. The third isolate carried dfrA17, no fluoroquinolone resistance gene, an additional sulI gene, and a chloramphenicol resistance gene, catB3. Mutations in candidate genes conferring resistance to fosfomycin and fluoroquinolones were also detected. Several efflux systems were detected in all the E. coli isolates and virulence-associated genes related to serum resistance, motility, and adhesion. E. coli and non-E. coli origin prophages were also identified in the isolates. The results underline the higher resolution power of whole-genome sequencing for investigation of antimicrobial resistance, virulence, and phage in E. coli.

Whole-genome sequence of Chryseobacterium oranimense, a colistin-resistant bacterium isolated from a cystic fibrosis patient in France.

For the first time, we report the whole-genome sequence analysis of Chryseobacterium oranimense G311, a multidrug-resistant bacterium, from a cystic fibrosis patient in France, including resistance to colistin. Whole-genome sequencing of C. oranimense G311 was performed using Ion Torrent PGM, and RAST, the EMBL-EBI server, and the Antibiotic Resistance Gene-ANNOTation (ARG-ANNOT) database were used for annotation of all genes, including antibiotic resistance (AR) genes. General features of the C. oranimense G311 draft genome were compared to the other available genomes of Chryseobacterium gleum and Chryseobacterium sp. strain CF314. C. oranimense G311 was found to be resistant to all ß-lactams, including imipenem, and to colistin. The genome size of C. oranimense G311 is 4,457,049 bp in length, with 37.70% GC content. We found 27 AR genes in the genome, including ß-lactamase genes which showed little similarity to the known ß-lactamase genes and could likely be novel. We found the type I polyketide synthase operon followed by a zeaxanthin glycosyltransferase gene in the genome, which could impart the yellow pigmentation of the isolate. We located the O-antigen biosynthesis cluster, and we also discovered a novel capsular polysaccharide biosynthesis cluster. We also found known mutations in the orthologs of the pmrA (E8D), pmrB (L208F and P360Q), and lpxA (G68D) genes. We speculate that the presence of the capsular cluster and mutations in these genes could explain the resistance of this bacterium to colistin. We demonstrate that whole-genome sequencing was successfully applied to decipher the resistome of a multidrug resistance bacterium associated with cystic fibrosis patients.

Loss of LPS is involved in the virulence and resistance to colistin of colistin-resistant Acinetobacter nosocomialis mutants selected in vitro.

OBJECTIVES: Acinetobacter nosocomialis has increasingly been reported as an opportunistic pathogen causing nosocomial infections. Although it is more susceptible to all antimicrobial agents than Acinetobacter baumannii, MDR clinical isolates have also been described. In addition, several studies have shown a high percentage of resistance to colistin. Therefore, in the present study we investigated the mechanism of resistance to colistin in this microorganism. METHODS: Colistin-resistant strains were selected from the original colistin-susceptible A. nosocomialis strain following multi-step mutant selection. Comparative genomic and proteomic analyses of both colistin-susceptible and colistin-resistant A. nosocomialis strains were performed. In addition, virulence was investigated using the Caenorhabditis elegans assay. RESULTS: The colistin-resistant mutants selected showed a lower resistance profile for other types of antibacterial agents together with a significant decrease in virulence. The LT50 (i.e. time required to kill 50% of the nematodes) for the colistin-susceptible strain (WT) was 7 days compared with 9 days for the colistin-resistant strain (256) (P < 0.0001). In the genomic studies, several mutations were observed in the lpxD genes, leading to the loss of LPS in the colistin-resistant strains. The proteomic studies showed several up- and down-regulated proteins that may be involved in colistin resistance or in a decrease in the resistance profile for several antibiotics. CONCLUSIONS: This study shows that the mechanism of resistance to colistin by A. nosocomialis is mainly associated with the loss of LPS due to mutations in the lpxD gene, although changes in the expression of some proteins cannot be ruled out. In addition, the acquisition of colistin resistance is related to a decrease in virulence.

Abiotic stress induces change in Cinnamoyl CoA Reductase (CCR) protein abundance and lignin deposition in developing seedlings of Leucaena leucocephala.

Aboitic stress such as drought and salinity are class of major threats, which plants undergo through their lifetime. Lignin deposition is one of the responses to such abiotic stresses. The gene encoding Cinnamoyl CoA Reductase (CCR) is a key gene for lignin biosynthesis, which has been shown to be over-expressed under stress conditions. In the present study, developing seedlings of Leucaena leucocephala (Vernacular name: Subabul, White popinac) were treated with 1 % mannitol and 200 mM NaCl to mimic drought and salinity stress conditions, respectively. Enzyme linked immunosorbant assay (ELISA) based expression pattern of CCR protein was monitored coupled with Phlorogucinol/HCl activity staining of lignin in transverse sections of developing L. leucocephala seedlings under stress. Our result suggests a differential lignification pattern in developing root and stem under stress conditions. Increase in lignification was observed in mannitol treated stems and corresponding CCR protein accumulation was also higher than control and salt stress treated samples. On the contrary CCR protein was lower in NaCl treated stems and corresponding lignin deposition was also low. Developing root tissue showed a high level of CCR content and lignin deposition than stem samples under all conditions tested. Overall result suggested that lignin accumulation was not affected much in case of developing root however developing stems were significantly affected under drought and salinity stress condition.

NDM-5 carbapenemase-encoding gene in multidrug-resistant clinical isolates of Escherichia coli from Algeria.

Here, we report the first autochthonous cases of infections caused by blaNDM-5 New Delhi metallo-ß-lactamase-producing Escherichia coli strains recovered from urine and blood specimens of three patients from Algeria between January 2012 and February 2013. The three isolates belong to sequence type 2659 and they coexpress blaCTX-M-15 with the blaTEM-1 and blaaadA2 genes.

Emergence of VIM-2 and IMP-15 carbapenemases and inactivation of oprD gene in carbapenem-resistant Pseudomonas aeruginosa clinical isolates from Lebanon.

We report here the emergence of VIM-2 and IMP-15 carbapenemases in a series of clinical isolates of carbapenem-resistant Pseudomonas aeruginosa in Lebanon. We also describe the disruption of the oprD gene by either mutations or insertion sequence (IS) elements ISPa1328 and ISPre2 isoform. Our study reemphasizes a rapid dissemination of the VIM-2 carbapenemase-encoding gene in clinical isolates of P. aeruginosa in the Mediterranean basin.

ARG-ANNOT, a new bioinformatic tool to discover antibiotic resistance genes in bacterial genomes.

ARG-ANNOT (Antibiotic Resistance Gene-ANNOTation) is a new bioinformatic tool that was created to detect existing and putative new antibiotic resistance (AR) genes in bacterial genomes. ARG-ANNOT uses a local BLAST program in Bio-Edit software that allows the user to analyze sequences without a Web interface. All AR genetic determinants were collected from published works and online resources; nucleotide and protein sequences were retrieved from the NCBI GenBank database. After building a database that includes 1,689 antibiotic resistance genes, the software was tested in a blind manner using 100 random sequences selected from the database to verify that the sensitivity and specificity were at 100% even when partial sequences were queried. Notably, BLAST analysis results obtained using the rmtF gene sequence (a new aminoglycoside-modifying enzyme gene sequence that is not included in the database) as a query revealed that the tool was able to link this sequence to short sequences (17 to 40 bp) found in other genes of the rmt family with significant E values. Finally, the analysis of 178 Acinetobacter baumannii and 20 Staphylococcus aureus genomes allowed the detection of a significantly higher number of AR genes than the Resfinder gene analyzer and 11 point mutations in target genes known to be associated with AR. The average time for the analysis of a genome was 3.35 ± 0.13 min. We have created a concise database for BLAST using a Bio-Edit interface that can detect AR genetic determinants in bacterial genomes and can rapidly and easily discover putative new AR genetic determinants.

A Genetic Locus in Elizabethkingia anophelis Associated with Elevated Vancomycin Resistance and Multiple Antibiotic Reduced Susceptibility.

The Gram-negative Elizabethkingia express multiple antibiotic resistance and cause severe opportunistic infections. Vancomycin is commonly used to treat Gram-positive infections and has also been used to treat Elizabethkingia infections, even though Gram-negative organisms possess a vancomycin permeability barrier. Elizabethkingia anophelis appeared relatively vancomycin-susceptible and challenge with this drug led to morphological changes indicating cell lysis. In stark contrast, vancomycin growth challenge revealed that E. anophelis populations refractory to vancomycin emerged. In addition, E. anophelis vancomycin-selected mutants arose at high frequencies and demonstrated elevated vancomycin resistance and reduced susceptibility to other antimicrobials. All mutants possessed a SNP in a gene (vsr1 = vancomycin-susceptibility regulator 1) encoding a PadR family transcriptional regulator located in the putative operon vsr1-ORF551, which is conserved in other Elizabethkingia spp as well. This is the first report linking a padR homologue (vsr1) to antimicrobial resistance in a Gram-negative organism. We provide evidence to support that vsr1 acts as a negative regulator of vsr1-ORF551 and that vsr1-ORF551 upregulation is observed in vancomycin-selected mutants. Vancomycin-selected mutants also demonstrated reduced cell length indicating that cell wall synthesis is affected. ORF551 is a membrane-spanning protein with a small phage shock protein conserved domain. We hypothesize that since vancomycin-resistance is a function of membrane permeability in Gram-negative organisms, it is likely that the antimicrobial resistance mechanism in the vancomycin-selected mutants involves altered drug permeability.

Genome analysis of Salmonella enterica serovar enteritis strains isolated from poultry and humans in Burkina Faso.

Whole-genome sequencing (WGS) was used to characterize four Salmonella enterica Enteritidis isolates from poultry (n=2) and human (n=2) from Ouagadougou, Burkina Faso. Antimicrobial resistance genes, chromosomal mutations, and mobile genetic elements were identified by analysis of WGS data using sequence homology.

Characterization of three Francisella tularensis genomes from Oklahoma, USA.

Francisella tularensis , the causative agent for tularaemia, is a Tier 1 select agent, and a pan-species pathogen of global significance due to its zoonotic potential. Consistent genome characterization of the pathogen is essential to identify novel genes, virulence factors, antimicrobial resistance genes, for studying phylogenetics and other features of interest. This study was conducted to understand the genetic variations among genomes of F. tularensis isolated from two felines and one human source. Pan-genome analysis revealed that 97.7 % of genes were part of the core genome. All three F. tularensis isolates were assigned to sequence type A based on single nucleotide polymorphisms (SNPs) in sdhA. Most of the virulence genes were part of the core genome. An antibiotic resistance gene coding for class A beta-lactamase was detected in all three isolates. Phylogenetic analysis showed that these isolates clustered with other isolates reported from Central and South-Central USA. Assessment of large sets of the F. tularensis genome sequences is essential in understanding pathogen dynamics, geographical distribution and potential zoonotic implications.

Transcriptomic and Metabolomic Analysis of a Fusidic Acid-Selected fusA Mutant of Staphylococcus aureus.

Physiological experimentation, transcriptomics, and metabolomics were engaged to compare a fusidic acid-resistant Staphylococcus aureus mutant SH10001st-2 to its parent strain SH1000. SH10001st-2 harbored a mutation (H457Y) in the gene fusA which encodes the fusidic acid target, elongation factor G, as well as mutations in a putative phage gene of unknown function. SH10001st-2 grew slower than SH1000 at three temperatures and had reduced coagulase activity, two indicators of the fitness penalty reported for fusA-mediated fusidic acid- resistance in the absence of compensatory mutations. Despite the difference in growth rates, the levels of O2 consumption and CO2 production were comparable. Transcriptomic profiling revealed 326 genes were upregulated and 287 were downregulated in SH10001st-2 compared to SH1000. Cell envelope and transport and binding protein genes were the predominant functional categories of both upregulated and downregulated genes in SH10001st-2. Genes of virulence regulators, notably the agr and kdp systems, were highly upregulated as were genes encoding capsule production. Contrary to what is expected of mid-exponential phase cells, genes encoding secreted virulence factors were generally upregulated while those for adhesion-associated virulence factors were downregulated in SH10001st-2. Metabolomic analysis showed an overall increase in metabolite pools in SH10001st-2 compared to SH1000, mostly for amino acids and sugars. Slowed growth and metabolite accumulation may be byproducts of fusA mutation-mediated protein synthesis impairment, but the overall results indicate that SH10001st-2 is compensating for the H457Y fitness penalty by repurposing its virulence machinery, in conjunction with increasing metabolite uptake capacity, in order to increase nutrient acquisition.

Resistance Genes, Plasmids, Multilocus Sequence Typing (MLST), and Phenotypic Resistance of Non-Typhoidal Salmonella (NTS) Isolated from Slaughtered Chickens in Burkina Faso.

The emergence of antimicrobial-resistant bacteria in developing countries increases risks to the health of both such countries' residents and the global community due to international travel. It is consequently necessary to investigate antimicrobial-resistant pathogens in countries such as Burkina Faso, where surveillance data are not available. To study the epidemiology of antibiotic resistance in Salmonella, 102 Salmonella strains isolated from slaughtered chickens were subjected to whole-genome sequencing (WGS) to obtain information on antimicrobial resistance (AMR) genes and other genetic factors. Twenty-two different serotypes were identified using WGS, the most prevalent of which were Hato (28/102, 27.5%) and Derby (23/102, 22.5%). All strains analyzed possessed at least one and up to nine AMR genes, with the most prevalent being the non-functional aac(6')-Iaa gene, followed by aph(6)-Id. Multi-drug resistance was found genotypically in 36.2% of the isolates for different classes of antibiotics, such as fosfomycin and ß-lactams, among others. Plasmids were identified in 43.1% of isolates (44/102), and 25 plasmids were confirmed to carry AMR genes. The results show that chicken can be considered as a reservoir of antibiotic-resistant Salmonella strains. Due to the prevalence of these drug-resistant pathogens and the potential for foodborne illnesses, poultry processing and cooking should be performed with attention to prescribed safe handling methods to avoid cross-contamination with chicken products.

Apt (Adenine Phosphoribosyltransferase) Mutation in Laboratory-Selected Vancomycin-Intermediate Staphylococcus aureus.

Comparative genomic sequencing of laboratory-derived vancomycin-intermediate Staphylococcusaureus (VISA) (MM66-3 and MM66-4) revealed unique mutations in both MM66-3 (in apt and ssaA6), and MM66-4 (in apt and walK), compared to hetero-VISA parent strain MM66. Transcriptional profiling revealed that both MM66 VISA shared 79 upregulated genes and eight downregulated genes. Of these, 30.4% of the upregulated genes were associated with the cell envelope, whereas 75% of the downregulated genes were associated with virulence. In concordance with mutations and transcriptome alterations, both VISA strains demonstrated reduced autolysis, reduced growth in the presence of salt and reduced virulence factor activity. In addition to mutations in genes linked to cell wall metabolism (ssaA6 and walK), the same mutation in apt which encodes adenine phosphoribosyltransferase, was confirmed in both MM66 VISA. Apt plays a role in both adenine metabolism and accumulation and both MM66 VISA grew better than MM66 in the presence of adenine or 2-fluoroadenine indicating a reduction in the accumulation of these growth inhibiting compounds in the VISA strains. MM66 apt mutants isolated via 2-fluoroadenine selection also demonstrated reduced susceptibility to the cell wall lytic dye Congo red and vancomycin. Finding that apt mutations contribute to reduced vancomycin susceptibility once again suggests a role for altered purine metabolism in a VISA mechanism.

Genomic Analysis of Multidrug-Resistant Escherichia coli from Surface Water in Northeast Georgia, United States: Presence of an ST131 Epidemic Strain Containing blaCTX-M-15 on a Phage-Like Plasmid.

Surface water is suspected of playing a role in the development and spread of antimicrobial-resistant (AR) bacteria, including human pathogens. In our previous study, 496 Escherichia coli isolates were recovered from water samples collected over a 2-year period from the Upper Oconee watershed, Athens, GA, United States, of which 34 (6.9%) were AR isolates. Of these, six isolates were selected based on their multidrug resistance (MDR) phenotypes, the presence of mobile genetic elements, and their pathogenic potential and were subjected to whole-genome sequence (WGS) analysis to enhance our understanding of environmental MDR E. coli isolates. This study is the first report on genomic characterization of MDR E. coli from environmental water in the United States through a WGS approach. The sequences of the six MDR E. coli isolates were analyzed and the locations of their AR genes were identified. One of the E. coli isolates was an ST131 epidemic strain, which also produced an extended-spectrum ß-lactamase encoded by the blaCTX-M-15 gene, carried on a plasmid that is a member of a very rarely reported family of phage-like plasmids. This is the first time an in-depth sequence analysis has been done on a blaCTX-M-15- containing phage-like plasmid, the presence of which suggests a new emerging mechanism of AR gene transmission.

Carriage and Gene Content Variability of the pESI-Like Plasmid Associated with Salmonella Infantis Recently Established in United States Poultry Production.

Salmonella Infantis carrying extended spectrum ß-lactamase blaCTX-M-65 on a pESI-like megaplasmid has recently emerged in United States poultry. In order to determine the carriage rate and gene content variability of this plasmid in U.S. Salmonella Infantis, whole genome sequences of Salmonella isolates from humans and animals in the U.S. and internationally containing the pESI-like plasmid were analyzed. The U.S. Department of Agriculture Food Safety and Inspection Service (FSIS) identified 654 product sampling isolates containing pESI-like plasmids through hazard analysis and critical control point (HACCP) verification testing in 2017 and 2018. The Centers for Disease Control and Prevention identified 55 isolates with pESI-like plasmids in 2016-2018 through the National Antimicrobial Resistance Monitoring System. Approximately 49% of pESI-like plasmids from FSIS verification isolates and 71% from CDC NARMS contained blaCTX-M-65. Pan-plasmid genome analysis was also performed. All plasmids contained traN and more than 95% contained 172 other conserved genes; 61% contained blaCTX-M-65. In a hierarchical clustering analysis, some plasmids from U.S. animal sources clustered together and some plasmids from South America clustered together, possibly indicating multiple plasmid lineages. However, most plasmids contained similar genes regardless of origin. Carriage of the pESI-like plasmid in U.S. appears to be limited to Salmonella Infantis and carriage rates increased from 2017 to 2018.

Comparison of Antimicrobial Resistance and Pan-Genome of Clinical and Non-Clinical Enterococcus cecorum from Poultry Using Whole-Genome Sequencing.

Enterococcus cecorum is an emerging avian pathogen, particularly in chickens, but can be found in both diseased (clinical) and healthy (non-clinical) poultry. To better define differences between E. cecorum from the two groups, whole-genome sequencing (WGS) was used to identify and compare antimicrobial resistance genes as well as the pan-genome among the isolates. Eighteen strains selected from our previous study were subjected to WGS using Illumina MiSeq and comparatively analyzed. Assembled contigs were analyzed for resistance genes using ARG-ANNOT. Resistance to erythromycin was mediated by ermB, ermG, and mefA, in clinical isolates and ermB and mefA, in non-clinical isolates. Lincomycin resistance genes were identified as linB, lnuB, lnuC, and lnuD with lnuD found only in non-clinical E. cecorum; however, lnuB and linB were found in only one clinical isolate. For both groups of isolates, kanamycin resistance was mediated by aph3-III, while tetracycline resistance was conferred by tetM, tetO, and tetL. No mutations or known resistance genes were found for isolates resistant to either linezolid or chloramphenicol, suggesting possible new mechanisms of resistance to these drugs. A comparison of WGS results confirmed that non-clinical isolates contained more resistance genes than clinical isolates. The pan-genome of clinical and non-clinical isolates resulted in 3651 and 4950 gene families, respectively, whereas the core gene sets were comprised of 1559 and 1534 gene families in clinical and non-clinical isolates, respectively. Unique genes were found more frequently in non-clinical isolates than clinical. Phylogenetic analysis of the isolates and all the available complete and draft genomes showed no correlation between healthy and diseased poultry. Additional genomic comparison is required to elucidate genetic factors in E. cecorum that contribute to disease in poultry.

Circulation of emerging NDM-5-producing Escherichia coli among humans and dogs in Egypt.

The emergence of NDM-producing Escherichia coli has considerably threatened human and animal health worldwide. This study describes for the first time in Egypt, the draft genome sequences of emerging NDM-5-producing E. coli from humans and dogs, and investigates genetic relatedness between isolates from both sources. Two E. coli from human urine and seven from environmental clinical samples of dogs exhibited resistance to carbapenems and harbouring blaNDM were subjected to Illumina Miseq whole-genome sequencing (WGS). Assembly and analysis of the reads were performed to identify resistance genes, multilocus sequence types (MLST), plasmid replicon types (Inc) and insertion sequences (IS) of the blaNDM region; core genome MLST (cgMLST) analysis was also performed. Two different NDM alleles were identified; blaNDM-5 in E. coli HR119 from the urine of a healthy person and environmental samples of dogs, and blaNDM-1 in E. coli HR135 from a human patient's urine. Multiple mobilizable resistance genes to different antimicrobial classes were identified except the colistin resistance gene, mcr. E. coli isolates from humans and dogs were assigned to different sequence types (STs). Using cgMLST, dog isolates clustered together with only 1-2 allellic differences; however, human E. coli showed 1,978 different allelles compared with dog isolates. Plasmidfinder results indicated the presence of an IncX3 replicon in blaNDM-5 -producing E. coli; however, blaNDM-1 was linked to IncCoIKP3. Notably, the NDM region (3 Kb) in all isolates from humans and dogs was highly similar with variable flanking sequences that represented different IS elements. This study reports the first emergence of NDM-5-producing E. coli from dogs in Egypt that shared some genetic features with human isolates and could be considered potential public health threats.

Improved method of in vitro regeneration in Leucaena leucocephala - a leguminous pulpwood tree species.

Leucaena leucocephala is a fast growing multipurpose legume tree used for forage, leaf manure, paper and pulp. Lignin in Leucaena pulp adversely influences the quality of paper produced. Developing transgenic Leucaena with altered lignin by genetic engineering demands an optimized regeneration system. The present study deals with optimization of regeneration system for L. leucocephala cv. K636. Multiple shoot induction from the cotyledonary nodes of L. leucocephala was studied in response to cytokinins, thidiazuron (TDZ) and N(6)-benzyladenine (BA) supplemented in half strength MS (½-MS) medium and also their effect on in vitro rooting of the regenerated shoots. Multiple shoots were induced from cotyledonary nodes at varied frequencies depending on the type and concentration of cytokinin used in the medium. TDZ was found to induce more number of shoots per explant than BA, with a maximum of 7 shoots at an optimum concentration of 0.23 µM. Further increase in TDZ concentration resulted in reduced shoot length and fasciation of the shoots. Liquid pulse treatment of the explants with TDZ did not improve the shoot production further but improved the subsequent rooting of the shoots that regenerated. Regenerated shoots successfully rooted on ½-MS medium supplemented with 0.54 µM α-naphthaleneacetic acid (NAA). Rooted shoots of Leucaena were transferred to coco-peat and hardened plantlets showed ≥ 90 % establishment in the green house.