Background & objectives: Linezolid (LZD) is increasingly being used in tuberculosis (TB) treatment. However, LZD resistance has already been reported, which is highly alarming, given its critical ...therapeutic role. This study was aimed to phenotypically and genotypically assess LZD resistance in Mycobacterium tuberculosis (MTB) isolates at a laboratory in a tertiary care centre in Mumbai, India.
Methods: A sample of 32 consecutive LZD-resistant MTB isolates identified by liquid culture susceptibility testing was subjected to whole-genome sequencing (WGS) on the Illumina NextSeq platform. Sequences were analyzed using BioNumerics software to predict resistance for 12 antibiotics within 15 min.
Results: Sixty eight of the 2179 isolates tested for LZD resistance by MGIT-based susceptibility testing (June 2015 to June 2016) were LZD-resistant. Thirty two consecutive LZD-resistant isolates were analyzed by WGS to screen for known mutations conferring LZD resistance. WGS of 32 phenotypically LZD-resistant isolates showed that C154R in the rplC gene and G2814T in the rrl gene were the major resistance determinants.
Interpretation & conclusions: LZD resistance poses an important risk to the success of treatment regimens, especially those designed for resistant isolates; such regimens are extensively used in India. As LZD-containing regimens increase in prominence, it is important to support clinical decision-making with an improved understanding of the common mutations conferring LZD resistance and their frequency in different settings.
The influence of turning and environmental contamination on six spontaneous cocoa bean heap fermentations performed in Ghana was studied through a multiphasic approach, encompassing both ...microbiological (culture-dependent and culture-independent techniques) and metabolite target analyses. A sensory analysis of chocolate made from the fermented, dried beans was performed as well. Only four clusters were found among the isolates of acetic acid bacteria (AAB) identified: Acetobacter pasteurianus, Acetobacter ghanensis, Acetobacter senegalensis, and a potential new Acetobacter lovaniensis-like species. Two main clusters were identified among the lactic acid bacteria (LAB) isolated, namely, Lactobacillus plantarum and Lactobacillus fermentum. No differences in biodiversity of LAB and AAB were seen for fermentations carried out at the farm and factory sites, indicating the cocoa pod surfaces and not the general environment as the main inoculum for spontaneous cocoa bean heap fermentation. Turning of the heaps enhanced aeration and increased the relative population size of AAB and the production of acetic acid. This in turn gave a more sour taste to chocolate made from these beans. Bitterness was reduced through losses of polyphenols and alkaloids upon fermentation and cocoa bean processing.
Recent studies portend a rising global spread and adaptation of human- or healthcare-associated pathogens. Here, we analyse an international collection of the emerging, multidrug-resistant, ...opportunistic pathogen Stenotrophomonas maltophilia from 22 countries to infer population structure and clonality at a global level. We show that the S. maltophilia complex is divided into 23 monophyletic lineages, most of which harbour strains of all degrees of human virulence. Lineage Sm6 comprises the highest rate of human-associated strains, linked to key virulence and resistance genes. Transmission analysis identifies potential outbreak events of genetically closely related strains isolated within days or weeks in the same hospitals.
At present, there is much variability between MALDI-TOF MS methodology for the characterization of bacteria through differences in e.g., sample preparation methods, matrix solutions, organic ...solvents, acquisition methods and data analysis methods. After evaluation of the existing methods, a standard protocol was developed to generate MALDI-TOF mass spectra obtained from a collection of reference strains belonging to the genera
Leuconostoc,
Fructobacillus and
Lactococcus. Bacterial cells were harvested after 24
h of growth at 28
°C on the media MRS or TSA. Mass spectra were generated, using the CHCA matrix combined with a 50:48:2 acetonitrile:water:trifluoroacetic acid matrix solution, and analyzed by the cell smear method and the cell extract method. After a data preprocessing step, the resulting high quality data set was used for PCA, distance calculation and multi-dimensional scaling. Using these analyses, species-specific information in the MALDI-TOF mass spectra could be demonstrated. As a next step, the spectra, as well as the binary character set derived from these spectra, were successfully used for species identification within the genera
Leuconostoc,
Fructobacillus, and
Lactococcus. Using MALDI-TOF MS identification libraries for
Leuconostoc and
Fructobacillus strains, 84% of the MALDI-TOF mass spectra were correctly identified at the species level. Similarly, the same analysis strategy within the genus
Lactococcus resulted in 94% correct identifications, taking species and subspecies levels into consideration. Finally, two machine learning techniques were evaluated as alternative species identification tools. The two techniques, support vector machines and random forests, resulted in accuracies between 94% and 98% for the identification of
Leuconostoc and
Fructobacillus species, respectively.
Spontaneous organic cocoa bean box fermentations were carried out on two different farms in Brazil. Physical parameters, microbial growth, bacterial species diversity mainly lactic acid bacteria ...(LAB) and acetic acid bacteria (AAB), and metabolite kinetics were monitored, and chocolates were produced from the fermented dry cocoa beans. The main end-products of the catabolism of the pulp substrates (glucose, fructose, and citric acid) by yeasts, LAB, and AAB were ethanol, lactic acid, mannitol, and/or acetic acid. Lactobacillus fermentum and Acetobacter pasteurianus were the predominating bacterial species of the fermentations as revealed through (GTG)5-PCR fingerprinting of isolates and PCR-DGGE of 16S rRNA gene PCR amplicons of DNA directly extracted from fermentation samples. Fructobacillus pseudoficulneus, Lactobacillus plantarum, and Acetobacter senegalensis were among the prevailing species during the initial phase of the fermentations. Also, three novel LAB species were found. This study emphasized the possible participation of Enterobacteriaceae in the cocoa bean fermentation process. Tatumella ptyseos and Tatumella citrea were the prevailing enterobacterial species in the beginning of the fermentations as revealed by 16S rRNA gene-PCR-DGGE. Finally, it turned out that control over a restricted bacterial species diversity during fermentation through an ideal post-harvest handling of the cocoa beans will allow the production of high-quality cocoa and chocolates produced thereof, independent of the fermentation method or farm.
► Organic cocoa fermentations in Brazil are dominated by restricted bacterial species diversity. ► Lb. fermentum, and A. pasteurianus prevail in organic cocoa fermentations in Brazil. ► Three novel LAB species were isolated from Brazilian cocoa bean box fermentations.
Two lactic acid bacteria, strains 257(T) and 252, were isolated from traditional heap fermentations of Ghanaian cocoa beans. 16S rRNA gene sequence analysis of these strains allocated them to the ...genus Weissella, showing 99.5 % 16S rRNA gene sequence similarity towards Weissella ghanensis LMG 24286(T). Whole-cell protein electrophoresis, fluorescent amplified fragment length polymorphism fingerprinting of whole genomes and biochemical tests confirmed their unique taxonomic position. DNA-DNA hybridization experiments towards their nearest phylogenetic neighbour demonstrated that the two strains represent a novel species, for which we propose the name Weissella fabaria sp. nov., with strain 257(T) (=LMG 24289(T) =DSM 21416(T)) as the type strain. Additional sequence analysis using pheS gene sequences proved useful for identification of all Weissella-Leuconostoc-Oenococcus species and for the recognition of the novel species.
The increasing number of infections caused by
resistant to clinically important antibiotics is a global concern for human and animal health. High overall levels of extended-spectrum beta-lactamase ...(ESBL)-producing and ciprofloxacin-resistant (ciproR)
in livestock are reported in Belgium. This cross-sectional study aimed to genotypically characterize and trace ESBL-and ciproR-
of Belgian food-producing animals.
A total of 798 fecal samples were collected in a stratified-random sampling design from Belgian broilers and sows. Consequently, 77 ESBL-
and 84 ciproR-
were sequenced using Illumina MiSeq. Minimum inhibitory concentration (MIC) for fluoroquinolones and cephalosporins were determined. Molecular
typing, resistance and virulence gene determination, and plasmid identification was performed. Scaffolds harboring ESBL or plasmid-mediated quinolone resistance (PMQR) genes were analyzed to detect mobile genetic elements (MGEs) and plasmid origins. Core genome allelic distances were used to determine genetic relationships among isolates.
A variety of
sequence types (ST) (
= 63), resistance genes and virulence profiles was detected. ST10 was the most frequently encountered ST (8.1%,
= 13). The pandemic multidrug-resistant clone ST131 was not detected. Most farms harbored more than one ESBL type, with
(41.6% of ESBL-
) being the most prevalent and
(
= 3) being the least prevalent. PMQR genes (15.5%,
= 13) played a limited role in the occurrence of ciproR-
. More importantly, sequential acquisition of mutations in quinolone resistance-determining regions (QRDR) of
and
led to increasing MICs for fluoroquinolones. GyrA S83L, D87N and ParC S80I mutations were strongly associated with high-level fluoroquinolone resistance. Genetically related isolates identified within the farms or among different farms highlight transmission of resistant
or the presence of a common reservoir. IncI1-I(alpha) replicon type plasmids carried different ESBL genes (
,
and
). In addition, the detection of plasmid replicons with associated insertion sequence (IS) elements and ESBL/PMQR genes in different farms and among several STs (e.g., IncI1-I(alpha)/IncX3) underline that plasmid transmission could be another important contributor to transmission of resistance in these farms.
Our findings reveal a multifaceted narrative of transmission pathways. These findings could be relevant in understanding and battling the problem of antibiotic resistance in farms.
In this study, the diversity of the native lactic acid bacteria (LAB) population in nem chua, a popular traditional Vietnamese uncooked fermented meat, was described using a combination of ...culture-dependent and culture-independent methods. A total of two hundred seventy-three LAB isolates were subjected to a polyphasic identification approach combining (GTG)5-PCR fingerprinting and phenylalanyl-tRNA synthase α subunit (pheS) and RNA polymerase α subunit (rpoA) gene sequence analysis. LAB associated with nem chua were identified as Lactobacillus pentosus (21%), Lactobacillus plantarum (29.7%), Lactobacillus brevis (5%), Lactobacillus paracasei (0.4%), Lactobacillus fermentum (0.7%), Lactobacillus acidipiscis (0.4%), Lactobacillus farciminis (23%), Lactobacillus rossiae (0.4%), Lactobacillus fuchuensis (0.7%), Lactobacillus namurensis (0.4%), Lactococcus lactis (0.4%), Leuconostoc citreum (9.5%), Leuconostoc fallax (1%), Pediococcus acidilactici (1%), Pediococcus pentosaceus (4%), Pediococcus stilesii (1%), Weissella cibaria (0.7%) and Weissella paramesenteroides (0.7%). Furthermore, PCR-DGGE was also applied as a culture-independent method in this study. Results indicated the presence of species of which no isolates were recovered, i.e. Lactobacillus helveticus/crispatus, Lactococcus garvieae and Vagococcus sp. Conversely, not all isolated bacteria were detected by PCR-DGGE. Principal component and discriminant analysis disclosed correlations between the different production locations and certain isolated LAB species and strains and/or DGGE bands suggesting possible influences of locally prevailing production practices on the nem chua LAB microbiota.
► High degree of LAB diversity (a total of 21 different LAB species) in nem chua ► Specific species or strains could be associated with different production practices. ► Combining culture-dependent and -independent methods is favorable. ► First step in better understanding interrelation LAB composition and nem chua ► Perspectives for developing new starter cultures for the fermentation of nem chua
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