Multiple-locus variable-number tandem-repeat analysis (MLVA) genotyping of human Brucella isolates in Malaysia.

BACKGROUND: Brucellosis is one of the most common zoonotic diseases worldwide. It can cause acute febrile illness in human and is a major health problem. Studies in human brucellosis in Malaysia is limited and so far no genotyping studies has been done on Brucella isolates. The aim of the study was to determine the genetic diversity among Brucella species isolated from human brucellosis, obtained over a 6-year period (2009-2014). METHODS: In this study, the genotypic characteristics of 43 human Brucella melitensis isolates were analysed using multiple-locus variable-number tandem-repeat analysis (MLVA) which consisted of eight minisatellite loci (panel 1) and eight microsatellite loci; panels 2A (3 microsatellite loci) and panel 2B (5 microsatellite loci). Two human Brucella suis isolates were also investigated using the MLVA assay. RESULTS: Using panel 1 (MLVA8), two genotypes namely genotype 43 and 44 were obtained from the 43 B. melitensis isolates. Using the combination of panels 1 and 2A loci (MLVA11), two genotypes were obtained while using the complete panels 1, 2A and 2B, nine genotypes were obtained. The polymorphisms in using the complete panels (MLVA16) were observed in three loci from panel 2B, which showed a diversity index higher than 0.17. All B. melitensis isolates were closely related to the East Mediterranean group. For B. suis isolates, only genotype 6 and genotype 33 were obtained using panel 1 and MLVA11 respectively. CONCLUSION: In conclusion, the results of the present study showed a low genetic diversity among B. melitensis and B. suis isolates from human patients. Based on the MLVA16 assay, B. melitensis belonging to the East Mediterranean group is responsible for the vast majority of Brucella infections in our Malaysian patients. To our knowledge, this is the first genotyping study of human Brucella isolates in Malaysia.

A simple approach to obtain comparable Shigella sonnei MLVA results across laboratories.

Multilocus variable-number tandem repeat analysis (MLVA) is a promising subtyping tool to complement pulsed-field gel electrophoresis for discriminating closely related strains of some monomorphic organisms, including Shigella sonnei, which is one of the major foodborne pathogens. However, MLVA results are usually difficult to compare directly between laboratories, impeding the application of MLVA as a subtyping tool for disease surveillance and investigation of common outbreaks across regions or countries. It has long been a big challenge in seeking an approach that can be implemented to obtain comparable MLVA results across laboratories. By implementing a panel of calibration strains in each participating laboratory for data normalization, the MLVA results of 20 test strains were comparable even though some analytical conditions were different among the laboratories. This approach is simple, protocol independent, and easy to implement in every laboratory, and a small calibration set is sufficient to generate mathematical equations for accurate copy number conversion.

Active degradation-nitrification microbial assemblages in the hypoxic zone in a subtropical estuary.

In 2017 summer, we observed widespread bottom hypoxia at the lower estuary of the Pearl River estuary (PRE). Our previous study noticed that AOA and bacteria were highly abundant and clustered within the hypoxia zone. Moreover, nitrification and respiration rates were also evidently higher in these hypoxic waters. These observations prompt us to investigate whether these two oxygen-consuming microorganisms have symbiotic relationships and whether specific groups consistently coexist and form ecological-meaningful associations. In this study, we use network analysis to investigate the presence and active communities (DNA-RNA) based on bacterial and AOA communities sequencing (inferred from the 16S rRNA and amoA gene, respectively) to gain more insight into ecological-meaningful associations. We observed a highly diverse and active bacterial community in the hypoxia zone. The RNA networks were more modulized than the corresponding DNA networks, indicating that the active communities were better parsed into functional microbial assemblages. The network topology revealed that Gammaproteobacteria, Bacteroidetes (Flavobacteriales), Alphaproteobacteria (Rhodobacterales and Rhodospirillales), Marinimicrobia, Cyanobacteria (Synechococcales), and AOA sublineages were module hubs and connectors, indicating that they were the keystone taxa of the microbial communities. The hub-subnetwork further showed robust co-occurrence between Gammaproteobacteria, Bacteroidetes (Flavobacteriales), Alphaproteobacteria (Rhodobacterales and Rhodospirillales), Marinimicrobia with AOA sublineages, and Nitrospinae (presumably NOB) reflecting the formation of Degradation-Nitrification (sequential oxidation of Organic matter degradation to ammonia, then nitrate) microbial assemblage in the hypoxia zone. The subnetworks revealed AOA ecotype-specific modularization and niche partitioning of different AOA sublineages. Interestingly, the recurring co-occurrence of nitrifiers assemblage in the RNA subnetworks (SCM1-like-II (AOA) and Nitrospinae OTUs (NOB) suggests an active interaction via nitrite exchange. The Degradation-Nitrification microbial assemblage may contribute substantially to the oxygen consumption in the hypoxia formation in PRE. Our results provide new insight into the functional microbial assemblages, which is worth further investigation on their ecological implication in estuarine waters.

Characterization of Shigella sonnei in Malaysia, an increasingly prevalent etiologic agent of local shigellosis cases.

BACKGROUND: Shigellosis is a major public health concern worldwide, especially in developing countries. It is an acute intestinal infection caused by bacteria of the genus Shigella, with a minimum infective dose as low as 10-100 bacterial cells. Increasing prevalence of Shigella sonnei as the etiologic agent of shigellosis in Malaysia has been reported. As there is limited information on the genetic background of S. sonnei in Malaysia, this study aimed to characterize Malaysian S. sonnei and to evaluate the prospect of using multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) for subtyping of local S. sonnei. METHODS: Forty non-repeat clinical strains of S. sonnei isolated during the years 1997-2000, and 2007-2009 were studied. The strains were isolated from stools of patients in different hospitals from different regions in Malaysia. These epidemiologically unrelated strains were characterized using biotyping, antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE) and MLVA. RESULTS: The two biotypes identified in this study were biotype a (n = 29, 73%) and biotype g (n = 11, 27%). All the 40 strains were sensitive to kanamycin, ceftriaxone and ciprofloxacin. Highest resistance rate was observed for streptomycin (67.5%), followed by tetracycline (40%) and trimethoprim-sulfamethoxazole (37.5%). All the S. sonnei biotype g strains had a core resistance type of streptomycin - trimethoprim-sulfamethoxazole - tetracycline whereas the 29 biotype a strains were subtyped into eight resistotypes. All the strains were equally distinguishable by PFGE and MLVA. Overall, PFGE analysis indicated that S. sonnei biotype a strains were genetically more diverse than biotype g strains. Cluster analysis by MLVA was better in grouping the strains according to biotypes, was reflective of the epidemiological information and was equally discriminative as PFGE. CONCLUSIONS: The S. sonnei strains circulating in Malaysia throughout the period studied were derived from different clones given their heterogeneous nature. MLVA based on seven selected VNTR loci was rapid, reproducible and highly discriminative and therefore may complement PFGE for routine subtyping of S. sonnei.

Genetic and Ecological Diversity of Escherichia coli and Cryptic Escherichia Clades in Subtropical Aquatic Environments.

Escherichia coli not only inhabit the large intestines of human and warm-blooded animals but could also persist in the external environment. However, current knowledge was largely based on host-associated strains. Moreover, cryptic Escherichia clades that were often misidentified as E. coli by conventional diagnostic methods were discovered. Failure to distinguish them from E. coli sensu stricto could lead to inaccurate conclusions about the population genetics of E. coli. Based on seven housekeeping genes, we determine the genetic and ecological diversity of E. coli and cryptic clades as they occupy aquatic habitats with different characteristics and human impact levels in subtropical Hong Kong. Contrary to previous reports, clade II was the most abundant cryptic lineage co-isolated with E. coli, being especially abundant in relatively pristine subtropical aquatic environments. The phylogenetically distinct cryptic clades and E. coli showed limited recombination and significant genetic divergence. Analyses indicated that these clade II strains were ecologically differentiated from typical E. coli; some may even represent novel environmental Escherichia clades that were closely related to the original clade II strains of fecal origins. E. coli of diverse origins exhibited clonality amidst divergent genotypes STs, echoing other studies in that recombination in housekeeping genes was insufficient to disrupt phylogenetic signals of the largely clonal E. coli. Notably, environmental E. coli were less diverse than fecal isolates despite contributing many new alleles and STs. Finally, we demonstrated that human activities influenced the distribution of E. coli and clade II in a small aquatic continuum. Moving from relatively pristine sites toward areas with higher human disturbance, the abundance of clade II isolates and new E. coli genotypes reduces, while E. coli bearing class I integrons and belonging to CCs of public health concern accumulates. Altogether, this work revealed the new genetic diversity of E. coli and cryptic clades embedded in selected subtropical aquatic habitats, especially relatively pristine sites, which will aid a more thorough understanding of the extent of their genetic and functional variations in relation to diverse habitats with varied conditions.

Genetic Variation and Preliminary Indications of Divergent Niche Adaptation in Cryptic Clade II of Escherichia.

The evolution, habitat, and lifestyle of the cryptic clade II of Escherichia, which were first recovered at low frequency from non-human hosts and later from external environments, were poorly understood. Here, the genomes of selected strains were analyzed for preliminary indications of ecological differentiation within their population. We adopted the delta bitscore metrics to detect functional divergence of their orthologous genes and trained a random forest classifier to differentiate the genomes according to habitats (gastrointestinal vs external environment). Model was built with inclusion of other Escherichia genomes previously demonstrated to have exhibited genomic traits of adaptation to one of the habitats. Overall, gene degradation was more prominent in the gastrointestinal strains. The trained model correctly classified the genomes, identifying a set of predictor genes that were informative of habitat association. Functional divergence in many of these genes were reflective of ecological divergence. Accuracy of the trained model was confirmed by its correct prediction of the habitats of an independent set of strains with known habitat association. In summary, the cryptic clade II of Escherichia displayed genomic signatures that are consistent with divergent adaptation to gastrointestinal and external environments.

Draft Genome Sequences of 16 Strains of Escherichia Cryptic Clade II Isolated from Intertidal Sediment in Hong Kong.

The genus Escherichia includes several cryptic clades. Among them, the members of cryptic clade II have rarely been found, and their genome sequences remain largely uninvestigated. Here, we report the draft genome sequences of 16 strains of Escherichia cryptic clade II that were isolated from intertidal sediment in Hong Kong.

Prevalence and characterization of multidrug-resistant and extended-spectrum beta-lactamase-producing Escherichia coli from pediatric wards of a Malaysian hospital.

The emergence of Escherichia coli resistant to extended-spectrum cephalosporins (ESCs) is of concern as ESC is often used to treat infections by Gram-negative bacteria. One-hundred and ten E. coli strains isolated in 2009-2010 from children warded in a Malaysian tertiary hospital were analyzed for their antibiograms, carriage of extended-spectrum beta-lactamase (ESBL) and AmpC genes, possible inclusion of the beta-lactamase genes on an integron platform, and their genetic relatedness. All E. coli strains were sensitive to carbapenems. About 46% of strains were multidrug resistant (MDR; i.e., resistant to ≥3 antibiotic classes) and almost half (45%) were nonsusceptible to ESCs. Among the MDR strains, high resistance rates were observed for ampicillin (98%), tetracycline (75%), and trimethoprim/sulfamethoxazole (73%). Out of 110 strains, bla(TEM-1) (49.1%), bla(CTX-M) (11.8%), and bla(CMY-2) (6.4%) were detected. Twenty-one strains were ESBL producers. CTX-M-15 was the predominant CTX-M variant found and this is the first report of a CTX-M-27-producing E. coli strain from Malaysia. Majority (3.1%) of the strains harbored class 1 integron-encoded integrases with a predominance of aadA and dfr genes within the integron variable region. No gene cassette encoding ESBL genes was found and integrons were not significantly associated with ESBL or non-ESBL producers. Possible clonal expansion was observed for few CTX-M-15-positive strains but the O25-ST131 E. coli clone known to harbor CTX-M-15 was not detected while CMY-2-positive strains were genetically diverse.