Development and Validation of an Efficient Multiplex PCR Assay for Simultaneous Detection of Six Common Foodborne Pathogens and Hygiene Indicators.

Microbial contamination in foods could lead to illnesses and substantial losses in both food industry and public health sectors. Rapid detection of microbial hazards (i.e., pathogens, hygiene indicator microorganisms) can accelerate surveillance and diagnostic processes reducing transmission and minimizing undesirable consequences. This study developed a multiplex PCR (m-PCR) for the detection of six common foodborne pathogens and hygiene indicators using specific primers for uidA of Escherichia coli, stx2 of Escherichia coli O157:H7, invA of Salmonella spp., int of Shigella spp., ntrA of Klebsiella pneumoniae, and ail of Yersinia enterocolitica and Yersinia pseudotuberculosis. Sensitivity of the m-PCR was 100 fg or ∼20 bacterial cells. Each primer set amplified only the targeted strain, and specificity was demonstrated by lack of nonspecific bands with DNA from 12 other bacterial strains. Following ISO 16140-2:2016, the relative limit of detection of the m-PCR was comparable to that of the gold-standard method; however, the processing time was five times faster. The m-PCR was applied to detect the six pathogens in 100 natural samples (50 pork meat and 50 local fermented food samples) and compared to results of the gold-standard method. Positive cultures for Klebsiella, Salmonella, and E. coli were 66%, 82%, and 88%, respectively, of meat samples and 78%, 26%, and 56%, respectively, of fermented food samples. Escherichia coli O157:H7, Shigella, and Yersinia were not detected in any of the samples by both standard and m-PCR methods. The developed m-PCR assay showed comparable results with the traditional culture technique proving its rapid and reliable detection of six foodborne pathogens and hygiene indicators in food.

Halogenated trimethoprim derivatives as multidrug-resistant Staphylococcus aureus therapeutics.

Incorporation of halogen atoms to drug molecule has been shown to improve its properties such as enhanced in membrane permeability and increased hydrophobic interactions to its target. To investigate the effect of halogen substitutions on the antibacterial activity of trimethoprim (TMP), we synthesized a series of halogen substituted TMP and tested for their antibacterial activities against global predominant methicillin resistant Staphylococcus aureus (MRSA) strains. Structure-activity relationship analysis suggested a trend in potency that correlated with the ability of the halogen atom to facilitate in hydrophobic interaction to saDHFR. The most potent derivative, iodinated trimethoprim (TMP-I), inhibited pathogenic bacterial growth with MIC as low as 1.25 µg/mL while the clinically used TMP derivative, diaveridine, showed resistance. Similar to TMP, synergistic studies indicated that TMP-I functioned synergistically with sulfamethoxazole. The simplicity in the synthesis from an inexpensive starting material, vanillin, highlighted the potential of TMP-I as antibacterial agent for MRSA infections.

Listeria monocytogenes MerR-Like Regulator NmlRlm: Its Transcriptome and Role in Stress Response.

NmlR, a negative transcription regulator in the MerR family, is involved in oxidative and nitrosative stress response in Neisseria gonorrhoeae and Haemophilus influenzae. In this study, the objective was to characterize the role and the regulon of NmlR in the foodborne Listeria monocytogenes. An L. monocytogenes nmlR null mutant strain was constructed. Transcriptomes of strain 10403S wild type (WT) and ΔnmlRlm strains grown to the stationary phase were determined by mRNA sequencing. Differential expression analyses revealed 74 genes with altered expression levels (>9-fold difference), comprising 46 negatively and 28 positively regulated genes. Twenty-four NmlRlm-dependent genes overlap with the members of previously identified regulons of HrcA, a negative regulator of heat response in L. monocytogenes, and of alternative sigma factor σ(H). Phenotypic characterization revealed that the ΔnmlRlm strain survived significantly less than the WT under acid stress (pH 2.5 for 1 h) and oxidative stress (3% hydrogen peroxide for 1 h). In addition, nmlRlm deletion also resulted in a significant decrease (p < 0.0005) of cell length and enhanced intracellular growth in a differentiated macrophage-like U937 cell line during entry into stationary phase. These findings indicate that NmlRlm is not only involved in oxidative stress response but also contributes to other characteristics such as acid tolerance and intracellular growth, either through direct regulation or co-regulation with other regulators such as HrcA and σ(H).

Molecular characterization of Thai Salmonella enterica serotype typhimurium and serotype 4,5,12:i:- reveals distinct genetic deletion patterns.

In order to better understand the relationship between Salmonella serotypes Typhimurium and its monophasic variant 4,5,12:i:- found in Thailand, a total of 138 isolates from various sources were characterized using different molecular subtyping methods (i.e., pulsed-field gel electrophoresis [PFGE] and polymerase chain reaction [PCR]) and antibiotic resistance (AbR) patterns. PFGE revealed 52 distinct band patterns among these isolates, 3 of which were shared between the two serotypes. PCR characterization of genomic deletion patterns reveals that Thai S. 4,5,12:i:- isolates contain a distinct deletion pattern in the fljAB region, which can be used as a specific genetic marker for primary identification of S. 4,5,12:i:- sources. AbR study shows that, among 50 representative serotype-confirmed strains, 48.28% (14/29) of Salmonella Typhimurium and 90.48% (19/21) of 4,5,12:i:- isolates are multidrug-resistant Salmonella as they are resistant to at least 3 antimicrobial categories. The AmpST pattern for resistance to ampicillin, streptomycin, and tetracycline was found in high proportions of Salmonella Typhimurium (10 of 29) and S. 4,5,12:i:- (15 of 21) isolates.

Four New Sequence Types and Molecular Characteristics of Multidrug-Resistant Escherichia coli Strains from Foods in Thailand.

The presence of antibiotic-resistant Escherichia coli in food is a serious and persistent problem worldwide. In this study, 68 E. coli strains isolated from Thai food samples were characterized. Based on antibiotic susceptibility assays, 31 of these isolates (45.59%) showed multiple antibiotic resistance (MAR) index values > 0.2, indicating high exposure to antibiotics. Among these, strain CM24E showed the highest resistance (it was resistant to ten antibiotics, including colistin and imipenem). Based on genome sequencing, we identified four isolates (namely, CF25E, EF37E, NM10E1, and SF50E) with novel Achtman-scheme multi-locus sequence types (STs) (ST14859, ST14866, ST14753, and ST14869, respectively). Clermont phylogrouping was used to subtype the 68 researched isolates into five Clermont types, mainly A (51.47%) and B1 (41.18%). The blaEC gene was found only in Clermont type A, while the blaEC-13 gene was predominant in Clermont type B1. A correlation between genotypes and phenotypes was found only in Clermont type B1, which showed a strong positive correlation between the presence of an afa operon and yersiniabactin-producing gene clusters with the colistin resistance phenotype. Strain SM47E1, of Clermont type B2, carried the highest number of predicted virulence genes. In summary, this study demonstrates the pressing problems posed by the prevalence and potential transmission of antimicrobial resistance and virulence genes in the food matrix.

Enhancing effect of natural adjuvant, panduratin A, on antibacterial activity of colistin against multidrug-resistant Acinetobacter baumannii.

Colistin- and carbapenem-resistant Acinetobacter baumannii is a serious multidrug resistant (MDR) bacterium in clinical settings. Discovery of new antibacterial drugs against MDR is facing multiple challenges in drug development. Combination of known antibiotics with a robust adjuvant might be an alternative effective strategy for MDR treatment. In the study herein, we report an antibiotic adjuvant activity of a natural compound panduratin A from fingerroot (Boesenbergia rotunda) as a potent adjuvant to colistin. The present study investigated the antibiotic adjuvant effect of panduratin A against 10 colistin- and carbapenem-resistant A. baumannii. Antibacterial activities were tested by broth microdilution method. Biofilm assay was used to determine the efficacy of panduratin A in biofilm formation inhibition on two representative strains Aci46 and Aci44. Genomic and transcriptomic analyses of colistin- and carbapenem-resistant A. baumannii strains were used to identify potential resistance and tolerance mechanism in the bacteria. Panduratin A-colistin combination showed an increased effect on antibacterial in the A. baumannii. However, panduratin A did not improve the antibacterial activity of imipenem. In addition, panduratin A improves anti-biofilm activity of colistin against Aci44 and Aci46, the colistin- and carbapenem-resistant A. baumannii. Panduratin A markedly enhances bactericidal and anti-biofilm activity of colistin against colistin- resistant A. baumannii. Based on genome comparisons, single nucleotide polymorphism (SNP) patterns in six genes encoding biofilm and lipid A biosynthesis were shared in Aci44 and Aci46. In Aci44, we identified a partial sequence of pmrB encoding a polymyxin resistant component PmrB, whereas a full length of pmrB was observed in Aci46. RNA-seq analyses of Aci44 revealed that panduratin A-colistin combination induced expression of ribosomal proteins and oxidative stress response proteins, whereas iron transporter and MFS-type transporter systems were suppressed. Panduratin A-colistin combination could promote intracellular reactive oxygen species (ROS) accumulation could lead to the cidal effect on colistin-resistant A. baumannii. Combination of panduratin A and colistin showed a significant increase in colistin efficacy against colistin- resistant A. baumannii in comparison of colistin alone. Genomic comparison between Aci44 and Aci46 showed mutations and SNPs that might affect different phenotypes. Additionally, based on RNA-Seq, panduratin A-colistin combination could lead to ROS production and accumulation. These findings confirmed the potency of panduratin as colistin adjuvant against multidrug resistant A. baumannii.

A novel variant in Salmonella genomic island 1 of multidrug-resistant Salmonella enterica serovar Kentucky ST198.

Salmonella enterica serovar Kentucky ST198 is a major health threat due to its resistance to ciprofloxacin and several other drugs, including third-generation cephalosporins. Many drug-resistant genes have been identified in the Salmonella genomic island 1 variant K (SGI1-K). In this study, we investigated the antimicrobial resistance (AMR) profile and genotypic relatedness of two isolates of ciprofloxacin-resistant (CIPR) S. Kentucky ST198 from poultry in Northeastern Thailand. We successfully assembled the complete genomes of both isolates, namely SSSE-01 and SSSE-03, using hybrid de novo assembly of both short- and long-read sequence data. The complete genomes revealed their highly similar genomic structures and a novel variant of SGI1-K underlying multidrug-resistant (MDR) patterns, including the presence of blaTEM-1b, which confers resistance to beta-lactams, including cephalosporins and lnu(F) which confers resistance to lincomycin and other lincosamides. In addition, the chromosomal mutations in the quinolone resistance-determining region (QRDR) were found at positions 83 (Ser83Phe) and 87 (Asp87Asn) of GyrA and at positions 57 (Thr57Ser) and 80 (Ser80Ile) of ParC suggesting high resistance to ciprofloxacin. We also compared SSSE-01 and SSSE-03 with publicly available complete genome data and revealed significant variations in SGI1-K genetic structures and variable relationships to antibiotic resistance. In comparison to the other isolates, SGI1-K of SSSE-01 and SSSE-03 had a relatively large deletion in the backbone, spanning from S011 (traG∆) to S027 (resG), and the inversion of the IS26-S044∆-yidY segment. Their MDR region was characterized by the inversion of a large segment, including the mer operon and the relocation of IntI1 and several resistance genes downstream of the IS26-S044∆-yidY segment. These structural changes were likely mediated by the recombination of IS26. The findings broaden our understanding of the possible evolution pathway of SGI1-K in fostering drug resistance, which may provide opportunities to control these MDR strains.IMPORTANCEThe emergence of ciprofloxacin-resistant (CIPR) Salmonella Kentucky ST198 globally has raised significant concerns. This study focuses on two poultry isolates from Thailand, revealing a distinct Salmonella genomic island 1 variant K (SGI1-K) genetic structure. Remarkably, multiple antibiotic resistance genes (ARGs) were identified within the SGI1-K as well as other locations in the chromosome, but not in plasmids. Comparing the SGI1-K genetic structures among global and even within-country isolates unveiled substantial variations. Intriguingly, certain isolates lacked ARGs within the SGI1-K, while others had ARGs relocated outside. The presence of chromosomal extended-spectrum ß-lactamase (ESBL) genes and lincosamide resistance, lnu(F), gene, could potentially inform the choices of the treatment of CIPRS. Kentucky ST198 infections in humans. This study highlights the importance of understanding the diverse genetic structures of SGI1-K and emphasizes the role of animals and humans in the emergence of antimicrobial resistance.

Evidence of international transmission of mobile colistin resistant monophasic Salmonella Typhimurium ST34.

S. 4,[5],12:i:-, a monophasic variant of S. enterica serovar Typhimurium, is an important multidrug resistant serovar. Strains of colistin-resistant S. 4,[5],12:i:- have been reported in several countries with patients occasionally had recent histories of travels to Southeast Asia. In the study herein, we investigated the genomes of S. 4,[5],12:i:- carrying mobile colistin resistance (mcr) gene in Thailand. Three isolates of mcr-3.1 carrying S. 4,[5],12:i:- in Thailand were sequenced by both Illumina and Oxford Nanopore platforms and we analyzed the sequences together with the whole genome sequences of other mcr-3 carrying S. 4,[5],12:i:- isolates available in the NCBI Pathogen Detection database. Three hundred sixty-nine core genome SNVs were identified from 27 isolates, compared to the S. Typhimurium LT2 reference genome. A maximum-likelihood phylogenetic tree was constructed and revealed that the samples could be divided into three clades, which correlated with the profiles of fljAB-hin deletions and plasmids. A couple of isolates from Denmark had the genetic profiles similar to Thai isolates, and were from the patients who had traveled to Thailand. Complete genome assembly of the three isolates revealed the insertion of a copy of IS26 at the same site near iroB, suggesting that the insertion was an initial step for the deletions of fljAB-hin regions, the hallmark of the 4,[5],12:i:- serovar. Six types of plasmid replicons were identified with the majority being IncA/C. The coexistence of mcr-3.1 and blaCTX-M-55 was found in both hybrid-assembled IncA/C plasmids but not in IncHI2 plasmid. This study revealed possible transmission links between colistin resistant S. 4,[5],12:i:- isolates found in Thailand and Denmark and confirmed the important role of plasmids in transferring multidrug resistance.

Functional and genomic characterization of a novel probiotic Lactobacillus johnsonii KD1 against shrimp WSSV infection.

White Spot syndrome virus (WSSV) causes rapid shrimp mortality and production loss worldwide. This study demonstrates potential use of Lactobacillus johnsonii KD1 as an anti-WSSV agent for post larva shrimp cultivation and explores some potential mechanisms behind the anti-WSSV properties. Treatment of Penaeus vannamei shrimps with L. johnsonii KD1 prior to oral challenge with WSSV-infected tissues showed a significantly reduced mortality. In addition, WSSV copy numbers were not detected and shrimp immune genes were upregulated. Genomic analysis of L. johnsonii KD1 based on Illumina and Nanopore platforms revealed a 1.87 Mb chromosome and one 15.4 Kb plasmid. Only one antimicrobial resistance gene (ermB) in the chromosome was identified. Phylogenetic analysis comparing L. johnsonii KD1 to other L. johnsonii isolates revealed that L. johnsonii KD1 is closely related to L. johnsonii GHZ10a isolated from wild pigs. Interestingly, L. johnsonii KD1 contains isolate-specific genes such as genes involved in a type I restriction-modification system and CAZymes belonging to the GT8 family. Furthermore, genes coding for probiotic survival and potential antimicrobial/anti-viral metabolites such as a homolog of the bacteriocin helveticin-J were found. Protein-protein docking modelling suggests the helveticin-J homolog may be able to block VP28-PmRab7 interactions and interrupt WSSV infection.

Incidence, genetic diversity, and antimicrobial resistance profiles of Vibrio parahaemolyticus in seafood in Bangkok and eastern Thailand.

Background: Emergence of Vibrio parahaemolyticus pandemic strain O3:K6 was first documented in 1996. Since then it has been accounted for large outbreaks of diarrhea globally. In Thailand, prior studies on pandemic and non-pandemic V. parahaemolyticus had mostly been done in the south. The incidence and molecular characterization of pandemic and non-pandemic strains in other parts of Thailand have not been fully characterized. This study examined the incidence of V. parahaemolyticus in seafood samples purchased in Bangkok and collected in eastern Thailand and characterized V. parahaemolyticus isolates. Potential virulence genes, VPaI-7, T3SS2, and biofilm were examined. Antimicrobial resistance (AMR) profiles and AMR genes (ARGs) were determined. Methods: V. parahaemolyticus was isolated from 190 marketed and farmed seafood samples by a culture method and confirmed by polymerase chain reaction (PCR). The incidence of pandemic and non-pandemic V. parahaemolyticus and VPaI-7, T3SS2, and biofilm genes was examined by PCR. AMR profiles were verified by a broth microdilution technique. The presence of ARGs was verified by genome analysis. V. parahaemolyticus characterization was done by multilocus sequence typing (MLST). A phylogenomic tree was built from nucleotide sequences by UBCG2.0 and RAxML softwares. Results: All 50 V. parahaemolyticus isolates including 21 pathogenic and 29 non-pathogenic strains from 190 samples had the toxRS/old sequence, indicating non-pandemic strains. All isolates had biofilm genes (VP0950, VP0952, and VP0962). None carried T3SS2 genes (VP1346 and VP1367), while VPaI-7 gene (VP1321) was seen in two isolates. Antimicrobial susceptibility profiles obtained from 36 V. parahaemolyticus isolates revealed high frequency of resistance to colistin (100%, 36/36) and ampicillin (83%, 30/36), but susceptibility to amoxicillin/clavulanic acid and piperacillin/tazobactam (100%, 36/36). Multidrug resistance (MDR) was seen in 11 isolates (31%, 11/36). Genome analysis revealed ARGs including blaCARB (100%, 36/36), tet(34) (83%, 30/36), tet(35) (42%, 15/36), qnrC (6%, 2/36), dfrA6 (3%, 1/36), and blaCTX-M-55 (3%, 1/36). Phylogenomic and MLST analyses classified 36 V. parahaemolyticus isolates into 5 clades, with 12 known and 13 novel sequence types (STs), suggesting high genetic variation among the isolates. Conclusions: Although none V. parahaemolyticus strains isolated from seafood samples purchased in Bangkok and collected in eastern Thailand were pandemic strains, around one third of isolates were MDR V. parahaemolyticus strains. The presence of resistance genes of the first-line antibiotics for V. parahaemolyticus infection raises a major concern for clinical treatment outcome since these resistance genes could be highly expressed under suitable circumstances.

Roles of Alternative Sigma Factors in Invasion and Growth Characteristics of Listeria monocytogenes 10403S Into Human Epithelial Colorectal Adenocarcinoma Caco-2 Cell.

Listeria monocytogenes is a Gram-positive facultative intracellular bacterium with a broad host range. With its housekeeping sigma factor and four alternative ones (namely SigB, SigC, SigH, and SigL), L. monocytogenes can express genes in response to changing environments. However, the roles of these sigma factors in intracellular survival are still unclear. The objectives of this study were to characterize the role of each alternative σ factor on L. monocytogenes invasion and growth inside human epithelial colorectal adenocarcinoma Caco-2 cells. We used L. monocytogenes 10403S wild type and its 15 alternative sigma factor deletion mutants at a multiplicity of infection of 100 and 1 in invasion and intracellular growth assays in the Caco-2 cells, respectively. At 1.5, 2, 4, 6, 8, 10, and 12 h post-infection, Caco-2 cells were lysed, and intracellular L. monocytogenes were enumerated on brain-heart infusion agar. Colony-forming and growth rates were compared among strains. The results from phenotypic characterization confirmed that (i) SigB is the key factor for L. monocytogenes invasion and (ii) having only SigA (ΔsigBCHL strain) is sufficient to invade and multiply in the host cell at similar levels as the wild type. Our previous study suggested the negative role of SigL in bile stress response. In this study, we have shown that additional deletion of the rpoN (or sigL) gene to ΔsigB, ΔsigC, or ΔsigH could restore the impaired invasion efficiencies of the single mutant, suggesting the absence of SigL could enhance host invasion. Therefore, we further investigated the role of SigL during extracellular and intracellular life cycles. Using RNA sequencing, we identified 118 and 16 SigL-dependent genes during the extracellular and intracellular life cycles, respectively. The sigL gene itself was induced by fivefolds prior to the invasion, and 5.3 folds during Caco-2 infection, further suggesting the role of SigL in intracellular growth.

Nephroprotective potential of Panduratin A against colistin-induced renal injury via attenuating mitochondrial dysfunction and cell apoptosis.

Colistin is a last-resort polypeptide antibiotic widely used to treat against multidrug-resistant Gram-negative bacterial infections. However, this treatment is associated with nephrotoxicity. The aim of this study was to examine the potential protective effect of panduratin A, a bioactive compound of Boesenbergia rotunda, on colistin-induced nephrotoxicity in both in vivo and in vitro models. Intraperitoneal injection of 15 mg/kg colistin for 7 days markedly promoted renal tubular degeneration, increased blood urea nitrogen (BUN) levels, and upregulated the expression of renal injury biomarker and apoptosis proteins. In addition, treatment with colistin increased oxidative stress and apoptosis in mice kidney tissues. Interestingly, these defects were attenuated when co-administered of colistin with panduratin A (2.5 or 25 mg/kg). The underlying mechanisms of panduratin A attenuating colistin toxicity was investigated in human renal proximal tubular cells (RPTEC/TERT1). The mechanisms by which colistin-triggered cytotoxicity was determined by analysis of cell death, reactive oxygen species (ROS) levels, mitochondria function as well as the expression of proteins related to apoptosis pathway. Colistin treatment (200 µg/ml) significantly increased cell apoptosis, elevated ROS production, reduced mitochondrial membrane potential, and decreased anti-apoptotic protein (Bcl-2) expression. These effects were notably suppressed by co-treatment with panduratin A (5 µM). Collectively, panduratin A exerts as a novel nephroprotective agent to protect against colistin-induced renal injury by attenuating mitochondrial damage and renal cell apoptosis.

Differential Interaction between Invasive Thai Group B Streptococcus Sequence Type 283 and Caco-2 Cells.

The emergence in Southeast Asia of invasive group B Streptococcus (GBS) infections in adults by sequence type (ST) 283 is suggested to be associated with fish consumption. Genotyping of 55 GBS clinical isolates revealed that 33/44 invasive isolates belonged to ST283/capsular polysaccharide type (CPS) III. This included 15/16 isolates recovered from younger adults aged 16-36 years. Seven ST283/CPSIII isolates from the blood, cerebrospinal fluid, or joint fluid were selected by the patient's age at random to perform interaction studies with intestinal epithelial Caco-2 monolayers. The invasion efficiency profiles from this study classified these isolates into two groups; a higher invasion efficiency group 1 recovered from patients aged between 23 and 36 years, and a lower invasion efficiency group 2 recovered from the elderly and neonate. Intracellular survival tests revealed that only group 1 members could survive inside Caco-2 cells up to 32 h without replication. Additionally, all isolates tested were able to traverse across polarized Caco-2 monolayers. However, the timing of translocation varied among the isolates. These results indicated the potential of GBS invasion via the gastrointestinal tract and showed phenotypic variations in invasiveness, intracellular survival, and translocation efficiency between genetically closely related ST283 isolates infecting young adults and those infecting the elderly.

Transcriptomic and phenotypic analyses identify coregulated, overlapping regulons among PrfA, CtsR, HrcA, and the alternative sigma factors sigmaB, sigmaC, sigmaH, and sigmaL in Listeria monocytogenes.

A set of seven Listeria monocytogenes 10403S mutant strains, each bearing an in-frame null mutation in a gene encoding a key regulatory protein, was used to characterize transcriptional networks in L. monocytogenes; the seven regulatory proteins addressed include all four L. monocytogenes alternative sigma factors (σ(B), σ(C), σ(H), and σ(L)), the virulence gene regulator PrfA, and the heat shock-related negative regulators CtsR and HrcA. Whole-genome microarray analyses, used to identify regulons for each of these 7 transcriptional regulators, showed considerable overlap among regulons. Among 188 genes controlled by more than one regulator, 176 were coregulated by σ(B), including 92 genes regulated by both σ(B) and σ(H) (with 18 of these genes coregulated by σ(B), σ(H), and at least one additional regulator) and 31 genes regulated by both σ(B) and σ(L) (with 10 of these genes coregulated by σ(B), σ(L), and at least one additional regulator). Comparative phenotypic characterization measuring acid resistance, heat resistance, intracellular growth in J774 cells, invasion into Caco-2 epithelial cells, and virulence in the guinea pig model indicated contributions of (i) σ(B) to acid resistance, (ii) CtsR to heat resistance, and (iii) PrfA, σ(B), and CtsR to virulence-associated characteristics. Loss of the remaining transcriptional regulators (i.e., sigH, sigL, or sigC) resulted in limited phenotypic consequences associated with stress survival and virulence. Identification of overlaps among the regulons provides strong evidence supporting the existence of complex regulatory networks that appear to provide the cell with regulatory redundancies, along with the ability to fine-tune gene expression in response to rapidly changing environmental conditions.

Listeria monocytogenes 10403S Alternative Sigma-54 Factor σL Has a Negative Role on Survival Ability Under Bile Exposure.

Listeria monocytogenes is a Gram-positive bacterium causing listeriosis in animals and humans. To initiate a foodborne infection, L. monocytogenes has to pass through the host gastrointestinal tract (GIT). In this study, we evaluated survival abilities of L. monocytogenes 10403S wild type (WT) and its isogenic mutants in alternative sigma (σ) factor genes (i.e., sigB, sigC, sigH, and sigL) under simulated gastric, duodenal, and bile fluids. Within 10min of exposures, only bile fluid was able to significantly reduce survival ability of L. monocytogenes WT by 2 logs CFU/ml. Loss of sigL showed the greatest bile resistance among 16 strains tested, p<0.0001, (i.e., WT, four single alternative σ factor mutants, six double mutants, four triple mutants, and one quadruple mutant). To further investigate the role of σL in bile response, RNA-seq was conducted to compare the transcriptional profiles among L. monocytogenes 10403S ΔBCH triple mutant (lacking sigB, sigC, and sigH genes; expressing housekeeping σA and σL) and ΔBCHL quadruple mutant (lacking all alternative sigma factor genes; expressing only σA) strains under BHI and 1% bile conditions. A total of 216 and 176 differentially expressed genes (DEGs) were identified in BHI and bile, respectively. We confirmed that mpt operon was shown to be strongly activated by σL. Interestingly, more than 80% of DEGs were found to be negatively regulated in the presence of σL. This includes PrfA regulon and its mediated genes (i.e., hly, hpt, inlB, clpP, clpE, groL, and inlC) which were downregulated in response to bile in the presence of σL. This result suggests the potential negative role of σL on bile survival, and the roles of σL and σB might be in a seesaw model prior to host cell invasion.

Colistin and Carbapenem-Resistant Acinetobacter baumannii Aci46 in Thailand: Genome Analysis and Antibiotic Resistance Profiling.

Resistance to the last-line antibiotics against invasive Gram-negative bacterial infection is a rising concern in public health. Multidrug resistant (MDR) Acinetobacter baumannii Aci46 can resist colistin and carbapenems with a minimum inhibitory concentration of 512 µg/mL as determined by microdilution method and shows no zone of inhibition by disk diffusion method. These phenotypic characteristics prompted us to further investigate the genotypic characteristics of Aci46. Next generation sequencing was applied in this study to obtain whole genome data. We determined that Aci46 belongs to Pasture ST2 and is phylogenetically clustered with international clone (IC) II as the predominant strain in Thailand. Interestingly, Aci46 is identical to Oxford ST1962 that previously has never been isolated in Thailand. Two plasmids were identified (pAci46a and pAci46b), neither of which harbors any antibiotic resistance genes but pAci46a carries a conjugational system (type 4 secretion system or T4SS). Comparative genomics with other polymyxin and carbapenem-resistant A. baumannii strains (AC30 and R14) identified shared features such as CzcCBA, encoding a cobalt/zinc/cadmium efflux RND transporter, as well as a drug transporter with a possible role in colistin and/or carbapenem resistance in A. baumannii. Single nucleotide polymorphism (SNP) analyses against MDR ACICU strain showed three novel mutations i.e., Glu229Asp, Pro200Leu, and Ala138Thr, in the polymyxin resistance component, PmrB. Overall, this study focused on Aci46 whole genome data analysis, its correlation with antibiotic resistance phenotypes, and the presence of potential virulence associated factors.

Alternative σ Factors Regulate Overlapping as Well as Distinct Stress Response and Metabolic Functions in Listeria monocytogenes under Stationary Phase Stress Condition.

Listeria monocytogenes can regulate and fine-tune gene expression, to adapt to diverse stress conditions encountered during foodborne transmission. To further understand the contributions of alternative sigma (σ) factors to the regulation of L. monocytogenes gene expression, RNA-Seq was performed on L. monocytogenes strain 10403S and five isogenic mutants (four strains bearing in-frame null mutations in three out of four alternative σ factor genes, ΔCHL, ΔBHL, ΔBCL, and ΔBCH, and one strain bearing null mutations in all four genes, ΔBCHL), grown to stationary phase. Our data showed that 184, 35, 34, and 20 genes were positively regulated by σB, σL, σH, and σC (posterior probability > 0.9 and Fold Change (FC) > 5.0), respectively. Moreover, σB-dependent genes showed the highest FC (based on comparisons between the ΔCHL and the ΔBCHL strain), with 44 genes showing an FC > 100; only four σL-dependent, and no σH- or σC-dependent genes showed FC >100. While σB-regulated genes identified in this study are involved in stress-associated functions and metabolic pathways, σL appears to largely regulate genes involved in a few specific metabolic pathways, including positive regulation of operons encoding phosphoenolpyruvate (PEP)-dependent phosphotransferase systems (PTSs). Overall, our data show that (i) σB and σL directly and indirectly regulate genes involved in several energy metabolism-related functions; (ii) alternative σ factors are involved in complex regulatory networks and appear to have epistatic effects in stationary phase cells; and (iii) σB regulates multiple stress response pathways, while σL and σH positively regulate a smaller number of specific pathways.

Sequence Analyses and Phenotypic Characterization Revealed Multidrug Resistant Gene Insertions in the Genomic Region Encompassing Phase 2 Flagellin Encoding fljAB Genes in Monophasic Variant Salmonella enterica Serovar 4,5,12:i:- Isolates From Various Sources in Thailand.

Salmonella enterica serovar 4,5,12:i:- (S. 4,5,12:i:-), a monophasic variant of Salmonella Typhimurium (STm) lacking the phase 2 flagellin encoding genes fljAB, has become increasingly prevalent worldwide. The increasing trends in multidrug resistant (MDR) S. 4,5,12:i:- prevalence also pose an important global health threat. Though many reports have characterized phenotypic and genotypic drug resistance of this serovar, few studies have characterized antimicrobial resistance of this serovar in Thailand. In this study, 108 S. 4,5,12:i:- isolates from various sources in Thailand and four international S. 4,5,12:i:- isolates were screened using polymerase chain reaction (PCR) to detect the presence of five target regions which are associated with antimicrobial resistant (AMR) genes, in the genomic region that contained fljAB genes in STm. We determined AMR phenotypes of all isolates by Kirby-Bauer disk diffusion method. Whole genome sequencing (WGS) was performed on 53 representative isolates (based on differences in the pulsed filed gel electrophoresis profiles, the sources of isolate, and the PCR and AMR patterns) to characterize the genetic basis of AMR phenotype and to identify the location of AMR determinants. Based on PCR screening, nine PCR profiles showing distinct deletion patterns of the five target regions have been observed. Approximately 76% of isolates (or 85 of 112 isolates), all of which were Thai isolates, contained five target regions inserted between STM2759 and iroB gene. A total of 21 phenotypic AMR patterns were identified with the predominant AmpST resistant phenotype [i.e., 84% (or 94 of 112) tested positive for resistance to ampicillin, streptomycin, and tetracycline], and 89% (or 100 of 112) were found to be MDR (defined here as resistant to at least three classes of tested antimicrobials). Using WGS data, a total of 24 genotypic AMR determinants belonging to seven different antimicrobial groups were found. AMR determinants (i.e., blaTEM - 1 , strB-A, sul2, and tetB, conferring resistance to ampicillin, streptomycin, sulfonamides, and tetracycline, respectively) were found to be inserted in a region typically occupied by the phase 2 flagellin encoding genes in STm. These resistant genes were flanked by a number of insertion sequences (IS), and co-localized with mercury tolerance genes. Our findings identify AMR genes, possibly associated with multiple IS26 copies, in the genetic region between STM2759 and iroB genes replacing phase 2 flagellin encoding fljAB genes in Thai S. 4,5,12:i:- isolates.

Modulation of stress and virulence in Listeria monocytogenes.

Listeria monocytogenes can respond rapidly to changing environmental conditions, as illustrated by its ability to transition from a saprophyte to an orally transmitted facultative intracellular pathogen. Differential associations between various alternative sigma factors and a core RNA polymerase provide a transcriptional mechanism for regulating bacterial gene expression that is crucial for survival in rapidly changing conditions. Alternative sigma factors are key components of complex L. monocytogenes regulatory networks that include multiple transcriptional regulators of stress-response and virulence genes, regulation of genes encoding other regulators, and regulation of small RNAs. In this article, the contributions of various sigma factors to L. monocytogenes stress response and virulence are described.

Listeria monocytogenes σA Is Sufficient to Survive Gallbladder Bile Exposure.

Listeria monocytogenes is a foodborne Gram-positive bacterium causing listeriosis in both animals and humans. It can persist and grow in various environments including conditions countered during saprophytic or intra-host lifestyles. Sigma (σ) subunit of RNA polymerase is a transcriptional factor responsible for guiding the core RNA polymerase and initiating gene expression under normal growth or physiological changes. In L. monocytogenes, there is one housekeeping sigma factor, σA, and four alternative sigma factors σB, σC, σH, and σL. Generally, σA directs expression of genes required for normal growth while alternative σ factors alter gene expression in response to specific conditions (e.g., stress). In this study, we aimed to determine the exclusive role of σA in L. monocytogenes by comparing a wild type strain with its isogenic mutant lacking genes encoding all alternative sigma factors (i.e., sigB, sigC, sigH, and sigL). We further investigated their survival abilities in 6% porcine bile (pH 8.2) mimicking gallbladder bile and their transcriptomics profiles in rich medium (i.e., BHI) and 1% porcine bile. Surprisingly, the results showed that survival abilities of wild type and ΔsigBΔsigCΔsigHΔsigL (or ΔsigBCHL) quadruple mutant strains in 6% bile were similar suggesting a compensatory role for σA. RNA-seq results revealed that bile stimulon of L. monocytogenes wild type contained 66 genes (43 and 23 genes were up- and down-regulated, respectively); however, only 29 genes (five up- and 24 down-regulated by bile) were differentially expressed in ΔsigBCHL. We have shown that bile exposure mediates increased transcription levels of dlt and ilv operons and decreased transcription levels of prfA and heat shock genes in wild type. Furthermore, we identified σA-dependent bile inducible genes that are involved in phosphotransferase systems, chaperones, and transporter systems; these genes appear to contribute to L. monocytogenes cellular homeostasis. As a result, σA seemingly plays a compensatory role in the absence of alternative sigma factors under bile exposure. Our data support that the bile stimulon is prone to facilitate resistance to bile prior to initiated infection.