Genotypic Diversity, Antibiotic Resistance, and Virulence Phenotypes of Stenotrophomonas maltophilia Clinical Isolates from a Thai University Hospital Setting.

Stenotrophomonas maltophilia is a multidrug-resistant organism that is emerging as an important opportunistic pathogen. Despite this, information on the epidemiology and characteristics of this bacterium, especially in Thailand, is rarely found. This study aimed to determine the demographic, genotypic, and phenotypic characteristics of S. maltophilia isolates from Maharaj Nakorn Chiang Mai Hospital, Thailand. A total of 200 S. maltophilia isolates were collected from four types of clinical specimens from 2015 to 2016 and most of the isolates were from sputum. In terms of clinical characteristics, male and aged patients were more susceptible to an S. maltophilia infection. The majority of included patients had underlying diseases and were hospitalized with associated invasive procedures. The antimicrobial resistance profiles of S. maltophilia isolates showed the highest frequency of resistance to ceftazidime and the lower frequency of resistance to chloramphenicol, levofloxacin, trimethoprim/sulfamethoxazole (TMP/SMX), and no resistance to minocycline. The predominant antibiotic resistance genes among the 200 isolates were the smeF gene (91.5%), followed by blaL1 and blaL2 genes (43% and 10%), respectively. Other antibiotic resistance genes detected were floR (8.5%), intI1 (7%), sul1 (6%), mfsA (4%) and sul2 (2%). Most S. maltophilia isolates could produce biofilm and could swim in a semisolid medium, however, none of the isolates could swarm. All isolates were positive for hemolysin production, whereas 91.5% and 22.5% of isolates could release protease and lipase enzymes, respectively. In MLST analysis, a high degree of genetic diversity was observed among the 200 S. maltophilia isolates. One hundred and forty-one sequence types (STs), including 130 novel STs, were identified and categorized into six different clonal complex groups. The differences in drug resistance patterns and genetic profiles exhibited various phenotypes of biofilm formation, motility, toxin, and enzymes production which support this bacterium in its virulence and pathogenicity. This study reviewed the characteristics of genotypes and phenotypes of S. maltophilia from Thailand which is necessary for the control and prevention of S. maltophilia local spreading.

Characterisation of Salmonella enterica clones carrying mcr-1 plasmids in meat products and patients in Northern Thailand using long read sequencing.

Salmonella spp. is an important foodborne pathogen associated with consumption of contaminated food, especially food of livestock origin. Antimicrobial resistance (AMR) in Salmonella has been reported globally and increasing AMR in food production is a major public health issue worldwide. The objective of this study was to describe the genetic relatedness among Salmonella enterica isolates, which displayed identical DNA fingerprint profiles. Ten S. enterica isolates were selected from meat and human cases with an identical rep-PCR profile of serovars Rissen (n = 4), Weltevreden (n = 4), and Stanley (n = 2). We used long-read whole genome sequencing (WGS) on the MinION sequencing platform to type isolates and investigate in silico the presence of specific AMR genes. Antimicrobial susceptibility testing was tested by disk diffusion and gradient diffusion method to corroborate the AMR phenotype. Multidrug resistance and resistance to more than one antimicrobial agent were observed in eight and nine isolates, respectively. Resistance to colistin with an accompanying mcr-1 gene was observed among the Salmonella isolates. The analysis of core genome and whole genome MLST revealed that the Salmonella from meat and human salmonellosis were genetically related. Hence, it could be concluded that meat is one of the important sources for Salmonella infection in human.

Characterization of two siderophores produced by Bacillus megaterium: A preliminary investigation into their potential as therapeutic agents.

BACKGROUND: Microorganisms produce siderophores in order to scavenge iron from the environment and this study focuses on the characterization of the two siderophores secreted by Bacillus megaterium. The general biological properties and pharmacokinetics following oral application of these compounds are reported. METHODS: Under optimized culture conditions, the siderophores were harvested, purified by chromatography and identified using LC-MS and NMR. Two dihydroxamate siderophores were isolated, schizokinen (MW = 420) and schizokinen imide (MW = 402). RESULTS: Both compounds demonstrate strong antioxidant activity and were found to be relatively nontoxic to both human hepatocellular carcinoma (Huh7) and peripheral blood mononuclear cells. The siderophores possess a strong affinity for iron(III) and decrease the levels of the labile iron pool (LIP) in iron-loaded cells in a concentration-dependent manner. Schizokinen, was detected as both the free siderophore and the iron complex in the plasma and urine of rats after oral gavage. CONCLUSIONS: However, the bioavailability was low and thus schizokinen, like deferoxamine, has no potential as an orally active iron chelator for the treatment of systemic iron overload. GENERAL SIGNIFICANCE: By virtue of the high affinity of schizokinen for tribasic metals, this siderophore does have considerable potential for the chelation of gallium(III) and the development of clinical diagnostic reagents.

Core genome sequence analysis to characterize Salmonella enterica serovar Rissen ST469 from a swine production chain.

Salmonella enterica subsp. enterica serotype Rissen is the predominant serotype found in Thai pork production and can be transmitted to humans through contamination of the food chain. This study was conducted to investigate the genetic relationships between serovar Rissen isolates from all levels of the pork production chain and evaluate the ability of the in silico antimicrobial resistance (AMR) genotypes to predict the phenotype of serovar Rissen. A total of 38 serovar Rissen isolates were tested against eight antibiotic agents by a disk diffusion method and the whole genomes of all isolates were sequenced to detect AMR genetic elements using the ResFinder database.A total of 86.84% of the isolates were resistant to tetracycline, followed by ampicillin (78.96%) and sulfonamide-trimethoprim (71.05%). Resistance to more than one antimicrobial agent was observed in 78.95% of the isolates, with the most common pattern showing resistance to ampicillin, chloramphenicol, streptomycin, sulfonamide-trimethoprim, and tetracycline. The results of genotypic AMR indicated that 89.47% of the isolates carried tet(A), 84.22% carried blaTEM-1B, 78.95% carried sul3, and 78.95% carried dfrA12. The genotypic prediction of phenotypic resistance resulted in a mean sensitivity of 97.45% and specificity of 75.48%. Analysis by core genome multilocus sequence typing (cgMLST) demonstrated that the Salmonella isolates from various sources and different locations shared many of the same core genome loci. This implies that serovar Rissen has infected every stage of the pork production process and that contamination can occur in every part of the production chain.

Quantification and rep-PCR characterization of Salmonella spp. in retail meats and hospital patients in Northern Thailand.

Human salmonellosis is a major public health problem worldwide. Infections can pass to humans by contact with contaminated substances in the food chain. This study aimed to determine the prevalence and contamination levels of Salmonella isolated from pork, chicken and beef sold in different types of retail stores in Chiang Mai and Lamphun provinces and to investigate the genetic relatedness among Salmonella isolates in food chains in that area. A total of 360 meat samples from supermarkets, mini-grocery stores and fresh markets were obtained. Salmonella Rissen and S. Weltevreden were found in all meat sample types and in human cases. The overall prevalence of Salmonella in the chicken, pork and beef samples was 34.17%, 32.50% and 3.33%, respectively. Quantitatively, Salmonella contamination was highest in pork (1.24 log10 MPN/g), followed by chicken (1.08 log10 MPN/g), and beef (0.75 log10 MPN/g). The highest frequency of Salmonella contamination was found at the fresh markets (85.71%), whereas the highest quantity of contamination level was from mini-grocery stores (1.27 log10 MPN/g). The rep-PCR analysis results revealed that some of the Salmonella from meat samples and human cases were identical clones.

Lipid-modified azurin of Neisseria meningitidis is a copper protein localized on the outer membrane surface and not regulated by FNR.

The laz gene of Neisseria meningitidis is predicted to encode a lipid-modified azurin (Laz). Laz is very similar to azurin, a periplasmic protein, which belongs to the copper-containing proteins in the cupredoxin superfamily. In other bacteria, azurin is an electron donor to nitrite reductase, an important enzyme in the denitrifying process. It is not known whether Laz could function as an electron transfer protein in this important pathogen. Laz protein was heterologously expressed in Escherichia coli and purified. Electrospray mass spectrometry indicated that the Laz protein contains one copper ion. Laz was shown to be redox-active in the presence of its redox center copper ion. When oxidized, Laz exhibits an intense blue colour and absorbs visible light around 626 nm. The absorption is lost when exposed to diethyldithiocarbamate, a copper chelating agent. Polyclonal antibodies were raised against purified Laz for detecting expression of Laz under different growth conditions and to determine the orientation of Laz on the outer membrane. The expression of Laz under microaerobic and microaerobic denitrifying conditions was slightly higher than that under aerobic conditions. However, the expression of Laz was similar between the wild type strain and an fnr mutant, suggesting that Fumarate/Nitrate reduction regulator (FNR) does not regulate the expression of Laz despite the presence of a partial FNR box upstream of the laz gene. We propose that some Laz protein is exposed on the outer membrane surface of N. meningitidis as the αLaz antibodies can increase killing by complement in a capsule deficient N. meningitidis strain, in a dose-dependent fashion.

Cytochrome c-based domain modularity governs genus-level diversification of electron transfer to dissimilatory nitrite reduction.

The genus Neisseria contains two pathogenic species (N. meningitidis and N. gonorrhoeae) in addition to a number of commensal species that primarily colonize mucosal surfaces in man. Within the genus, there is considerable diversity and apparent redundancy in the components involved in respiration. Here, we identify a unique c-type cytochrome (cN ) that is broadly distributed among commensal Neisseria, but absent in the pathogenic species. Specifically, cN supports nitrite reduction in N. gonorrhoeae strains lacking the cytochromes c5 and CcoP established to be critical to NirK nitrite reductase activity. The c-type cytochrome domain of cN shares high sequence identity with those localized c-terminally in c5 and CcoP and all three domains were shown to donate electrons directly to NirK. Thus, we identify three distinct but paralogous proteins that donate electrons to NirK. We also demonstrate functionality for a N. weaverii NirK variant with a C-terminal c-type heme extension. Taken together, modular domain distribution and gene rearrangement events related to these respiratory electron carriers within Neisseria are concordant with major transitions in the macroevolutionary history of the genus. This work emphasizes the importance of denitrification as a selectable trait that may influence speciation and adaptive diversification within this largely host-restricted bacterial genus.

Tied down: tethering redox proteins to the outer membrane in Neisseria and other genera.

Typically, the redox proteins of respiratory chains in Gram-negative bacteria are localized in the cytoplasmic membrane or in the periplasm. An alternative arrangement appears to be widespread within the betaproteobacterial genus Neisseria, wherein several redox proteins are covalently associated with the outer membrane. In the present paper, we discuss the structural properties of these outer membrane redox proteins and the functional consequences of this attachment. Several tethered outer membrane redox proteins of Neisseria contain a weakly conserved repeated structure between the covalent tether and the redox protein globular domain that should enable the redox cofactor-containing domain to extend from the outer membrane, across the periplasm and towards the inner membrane. It is argued that the constraints imposed on the movement and orientation of the globular domains by these tethers favours the formation of electron-transfer complexes for entropic reasons. The attachment to the outer membrane may also affect the exposure of the host to redox proteins with a moonlighting function in the host-microbe interaction, thus affecting the host response to Neisseria infection. We identify putative outer membrane redox proteins from a number of other bacterial genera outside Neisseria, and suggest that this organizational arrangement may be more common than previously recognized.

Roles of c-type cytochromes in respiration in Neisseria meningitidis.

Three c-type cytochromes were identified in Neisseria meningitidis, based on predictions from genome sequences, that were hypothesized to be involved in electron transport to terminal electron acceptor reductases for oxygen (the cytochrome cbb(3) oxidase) and nitrite (the nitrite reductase, AniA). Mutants were generated by allelic exchange with disrupted copies of the genes encoding these cytochromes and the phenotypes of the resultant mutants analysed. It was found that cytochrome c(5) is required for in vivo nitrite reductase activity, whereas cytochromes c(x) and c(4) are both required for efficient growth using oxygen as an electron acceptor. Mutants in c(x), c(4), and c(x)+c(4) have a decreased capacity to reduce oxygen, but there is a background oxygen-reduction activity, indicating that there may be other routes for electron transfer from the cytochrome bc(1) complex to the cytochrome cbb(3) oxidase, whereas cytochrome c(5) appears to be the sole route of electrons to the nitrite reductase in N. meningitidis. Interestingly, cytochrome c(x) is highly similar to a domain of copper nitrite reductases from various proteobacteria, whereas cytochrome c(5) has high identity with a domain of the cytochrome cbb(3) oxidase of Neisseria gonorrhoeae, yet these two proteins function in oxygen respiration and nitrite respiration, respectively. This highlights a limitation of predicting protein function from similarity to known proteins, i.e. very closely related protein domains in different organisms can have different redox partners.