Migrating Type 2 Dendritic Cells Prime Mucosal Th17 Cells Specific to Small Intestinal Commensal Bacteria.

Dendritic cells (DCs) are professional APCs equipped with MHC-restricted Ags, costimulations, and cytokines that effectively prime and differentiate naive T cells into distinct functional subsets. The immune signals that DCs carry reflect the route of Ag uptake and the innate stimuli they received. In the mucosal tissues, owing to the great variety of foreign Ags and inflammatory cues, DCs are predominantly activated and migratory. In the small intestine, CD4 Th17 cells are abundant and have been shown to be regulated by DCs and macrophages. Using a mouse commensal bacteria experimental model, we identified that the early priming step of commensal-driven Th17 cells is controlled by bona fide Zbtb46-expressing DCs. CCR7-dependent migration of type 2 DCs (DC2s) from the small intestine to the mesenteric lymph nodes (MLNs) is essential for the activation of naive CD4 T cells. The migratory DC2 population in the MLNs is almost exclusively Esam+ cells. Single-cell RNA sequencing highlighted the abundance of costimulatory markers (CD40 and OX40) and chemokines (Ccl22 and Cxcl16) on MLN migratory DCs. Further resolution of MLN migratory DC2s revealed that the Th17-polarizing cytokine IL-6 colocalizes with DC2s expressing CD40, Ccl17, and Ccl22. Thus, early Th17 cell differentiation is initiated by a small subset of migratory DC2s in the gut-draining lymph nodes.

Evaluation of methicillin-resistant Staphylococcus aureus (MRSA) bacteremia: Epidemiology, clinical characteristics, and outcomes in the older patients in a tertiary teaching hospital in Malaysia.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) bacteremia is a major concern in the global healthcare system. However, data from Asian regions dealing with the singularity of this infection in older persons is lacking. We aimed to identify the differences in the clinical characteristics and outcomes of MRSA bacteremia between adults aged 18-64 and ≥ 65 years. METHODS: A retrospective study cohort was conducted at the University Malaya Medical Centre (UMMC) on cases of MRSA bacteremia from 2012 to 2016. Patient demographic and clinical data were collected for risk factors analyses. RESULTS: New cases of MRSA bacteremia showed a trend of increase from 0.12 to 100 admissions in 2012 to 0.17 per 100 admissions in 2016 but a drop was observed in 2014 (0.07 per 100 admissions). Out of the 275 patients with MRSA bacteremia, 139 (50.5%) patients were aged ≥ 65 years old. Co-morbidities and severity at presentation were significantly higher among older adults, including diabetes mellitus (p = 0.035), hypertension (p = 0.001), and ischemic heart disease (p < 0.001), as well as higher Charlson Comorbidity Index (p < 0.001) and Pitt bacteremia scores (p = 0.016). Central line-associated bloodstream infections were more common among younger patients (37.5% vs. 17.3% in older patients, p < 0.001), while skin and soft tissue infections are more frequent among older adults (20.9% vs. 10.3% in younger patients, p = 0.016). All-cause mortality and in-hospital mortality were significantly higher in older patients (82.7% and 56.1% vs. 63.2% and 28.7% in younger patients, p < 0.001). Multivariate analysis revealed age ≥ 65 years (adjusted odds ratio: 3.36; 95% confidence interval: 1.24-9.13), Pitt score ≥ 3 (2.15; 1.54-3.01), hospital (6.12; 1.81-20.72) and healthcare (3.19; 1.30-7.81) acquisition of MRSA, indwelling urinary catheters (5.43; 1.39-21.23), inappropriate targeted treatment (8.08; 1.15-56.86), lack of infectious disease team consultation (2.90; 1.04-8.11) and hypoalbuminemia (3.31; 1.25-8.79), were significant risk factors for 30-day mortality. CONCLUSION: Older patients' risk of mortality from MRSA bacteremia was three times higher than younger patients. Our data will contribute to developing and validating a robust scoring system for risk-stratifying patients to achieve better management and improved clinical outcomes.

The Microtubule-Associated Protein Lis1 Regulates T Lymphocyte Homeostasis and Differentiation.

The microtubule-associated protein lissencephaly 1 (Lis1) is a key regulator of cell division during stem cell renewal and differentiation. In this study, we examined the role of Lis1 in T lymphocyte homeostasis and fate diversification in response to microbial infection. T cell-specific deletion of Lis1 resulted in depletion of the peripheral CD4(+) and CD8(+) T lymphocyte pool owing to a loss of homeostatic, cytokine-induced proliferation. In contrast, cognate Ag-triggered proliferation was much less affected, enabling Lis1-deficient CD8(+) T cells to differentiate into terminal effector cells in response to microbial infection. Strikingly, however, the specification of Lis1-deficient long-lived memory CD8(+) T lymphocytes was impaired due, in part, to an apparent failure to differentiate appropriately to IL-15. Taken together, these findings suggest that Lis1 plays an important role in T cell homeostasis and the generation of memory T lymphocytes.

Costimulation Endows Immunotherapeutic CD8 T Cells with IL-36 Responsiveness during Aerobic Glycolysis.

CD134- and CD137-primed CD8 T cells mount powerful effector responses upon recall, but even without recall these dual-costimulated T cells respond to signal 3 cytokines such as IL-12. We searched for alternative signal 3 receptor pathways and found the IL-1 family member IL-36R. Although IL-36 alone did not stimulate effector CD8 T cells, in combination with IL-12, or more surprisingly IL-2, it induced striking and rapid TCR-independent IFN-γ synthesis. To understand how signal 3 responses functioned in dual-costimulated T cells we showed that IL-2 induced IL-36R gene expression in a JAK/STAT-dependent manner. These data help delineate a sequential stimulation process where IL-2 conditioning must precede IL-36 for IFN-γ synthesis. Importantly, this responsive state was transient and functioned only in effector T cells capable of aerobic glycolysis. Specifically, as the effector T cells metabolized glucose and consumed O2, they also retained potential to respond through IL-36R. This suggests that T cells use innate receptor pathways such as the IL-36R/axis when programmed for aerobic glycolysis. To explore a function for IL-36R in vivo, we showed that dual costimulation therapy reduced B16 melanoma tumor growth while increasing IL-36R gene expression. In summary, cytokine therapy to eliminate tumors may target effector T cells, even outside of TCR specificity, as long as the effectors are in the correct metabolic state.

Algal chloroplast produced camelid VH H antitoxins are capable of neutralizing botulinum neurotoxin.

We have produced three antitoxins consisting of the variable domains of camelid heavy chain-only antibodies (VH H) by expressing the genes in the chloroplast of green algae. These antitoxins accumulate as soluble proteins capable of binding and neutralizing botulinum neurotoxin. Furthermore, they accumulate at up to 5% total soluble protein, sufficient expression to easily produce these antitoxins at scale from algae. The genes for the three different antitoxins were transformed into Chlamydomonas reinhardtii chloroplasts and their products purified from algae lysates and assayed for in vitro biological activity using toxin protection assays. The produced antibody domains bind to botulinum neurotoxin serotype A (BoNT/A) with similar affinities as camelid antibodies produced in Escherichia coli, and they are similarly able to protect primary rat neurons from intoxication by BoNT/A. Furthermore, the camelid antibodies were produced in algae without the use of solubilization tags commonly employed in E. coli. These camelid antibody domains are potent antigen-binding proteins and the heterodimer fusion protein containing two VH H domains was capable of neutralizing BoNT/A at near equimolar concentrations with the toxin. Intact antibody domains were detected in the gastrointestinal (GI) tract of mice treated orally with antitoxin-producing microalgae. These findings support the use of orally delivered antitoxins produced in green algae as a novel treatment for botulism.

Presensitizing with a Toll-like receptor 3 ligand impairs CD8 T-cell effector differentiation and IL-33 responsiveness.

The synthetic double-stranded RNA poly(I:C) is commonly used as an adjuvant to boost CD8 T-cell function; however, polyinosinic:polycytidylic acid [poly(I:C)] can also suppress autoimmune disease. The mechanism by which a single adjuvant achieves two distinct immunoregulatory roles is unknown. Although it is clear that coadministration of poly(I:C) with antigen elicits strong adjuvant effects in mice, we found that poly(I:C) injection before antigen substantially reduced antigen-dependent CD8 T-cell responses. Notably, CD8 T cells sensitized in poly(I:C)-pretreated mice failed to fully up-regulate IL-33R (ST2), which led to impaired T-cell receptor-independent responses to IL-33. In contrast, nonsensitized effector CD8 T cells responded robustly to IL-33 using a two-signal cytokine mechanism. During an acute lung response to Staphylococcus aureus enterotoxin, peripheral injection of poly(I:C) manifested a suppressive process by inhibiting the differentiation of both antigen- and IL-33-responsive CD8 effectors systemically. These findings highlight that early exposure to double-stranded RNA reverses its role as an adjuvant and, importantly, prevents IL-33R up-regulation on CD8 effector T cells to dampen inflammation.

A multicentre study to determine the in vitro efficacy of flomoxef against extended-spectrum beta-lactamase producing Escherichia coli in Malaysia.

Background: The high burden of extended-spectrum beta-lactamase-producing (ESBL)-producing Enterobacterales worldwide, especially in the densely populated South East Asia poses a significant threat to the global transmission of antibiotic resistance. Molecular surveillance of ESBL-producing pathogens in this region is vital for understanding the local epidemiology, informing treatment choices, and addressing the regional and global implications of antibiotic resistance. Methods: Therefore, an inventory surveillance of the ESBL-Escherichia coli (ESBL-EC) isolates responsible for infections in Malaysian hospitals was conducted. Additionally, the in vitro efficacy of flomoxef and other established antibiotics against ESBL-EC was evaluated. Results: A total of 127 non-repetitive ESBL-EC strains isolated from clinical samples were collected during a multicentre study performed in five representative Malaysian hospitals. Of all the isolates, 33.9% were isolated from surgical site infections and 85.8% were hospital-acquired infections. High rates of resistance to cefotaxime (100%), cefepime (100%), aztreonam (100%) and trimethoprim-sulfamethoxazole (100%) were observed based on the broth microdilution test. Carbapenems remained the most effective antibiotics against the ESBL-EC, followed by flomoxef. Antibiotic resistance genes were identified by PCR. The blaCTX-M-1 was the most prevalent ESBL gene, with 28 isolates (22%) harbouring blaCTX-M-1 only, 27 isolates (21.3%) co-harbouring blaCTX-M-1 and blaTEM, and ten isolates (7.9%) co-harbouring blaCTX-M-1, blaTEM and blaSHV. A generalised linear model showed significant antibacterial activity of imipenem against different types of infection. Besides carbapenems, this study also demonstrated a satisfactory antibacterial activity of flomoxef (81.9%) on ESBL-EC, regardless of the types of ESBL genes.

Genetic fine structure of a Salmonella enterica serovar Typhi strain associated with the 2005 outbreak of typhoid fever in Kelantan, Malaysia.

Among enteric pathogens, Salmonella enterica serovar Typhi is responsible for the largest number of food-borne outbreaks and fatalities. The ability of the pathogen to cause systemic infection for extended durations leads to a high cost of disease control. Chronic carriers play important roles in the evolution of Salmonella Typhi; therefore, identification and in-depth characterization of isolates from clinical cases and carriers, especially those from zones of endemicity where the pathogen has not been extensively studied, are necessary. Here, we describe the genome sequence of the highly virulent Salmonella Typhi strain BL196/05 isolated during the outbreak of typhoid in Kelantan, Malaysia, in 2005. The whole-genome sequence and comparative genomics of this strain should enable us to understand the virulence mechanisms and evolutionary dynamics of this pathogen in Malaysia and elsewhere.

Genotypic and phenotypic characterization of Escherichia coli isolated from indigenous individuals in Malaysia.

Objectives: The occurrence of asymptomatic verocytotoxin (VT)-producing Escherichia coli (VTEC) infections among humans in recent years is posing a high risk to public health. Thus, the role of asymptomatic human carriers as a source of dissemination should not be underestimated. This study aimed to elucidate the phenotypic and genotypic characteristics of E. coli in the stool samples collected from indigenous individuals in Malaysia. Materials and Methods: E. coli strains (n=108) were isolated from stool samples obtained from 41 indigenous individuals. All strains were subjected to Repetitive Extragenic Palindromic-Polymerase Chain Reaction (REP-PCR) typing and confirmation of VTEC variants. Non-duplicate strains were selected based on REP-PCR profiles and further subjected to antimicrobial susceptibility test (AST). The genotypic and phenotypic characteristics of the strains were then correlated with the demographic data of the subjects. Results: A total of 66 REP-PCR profiles grouped in 53 clusters (F=85%) were obtained. Four genetically distinct strains were confirmed as VTEC (eaeA-positive). The predominant resistance was against ampicillin (34.2%), followed by trimethoprim-sulfamethoxazole (32.9%), ampicillin-sulbactam (5.5%), and ciprofloxacin (1.4%). All isolates were sensitive to amoxicillin-clavulanate, cefuroxime, ceftriaxone, imipenem, and meropenem. Conclusion: Genetically diverse E. coli and VTEC strains were found to colonize the intestines of the indigenous populations. This study is important for the prospective surveillance of E. coli among the indigenous individuals in Malaysia, especially in asymptomatic VTEC infection and antimicrobial resistance phenomenon.

Molecular characterization of Vibrio cholerae O1 El Tor strains in Malaysia revealed genetically diverse variant lineages.

Vibrio cholerae O1 El Tor variants have been the major causative agents of cholera worldwide since their emergence in the 2000s. Cholera remains endemic in some regions in Malaysia. Therefore, we aimed to investigate the genetic characteristics of the V. cholerae O1 El Tor strains associated with outbreaks and sporadic cases to elucidate the molecular evolution among the strains circulating in this region. A total of 45 V. cholerae O1 El Tor strains isolated between 1991 and 2011 were examined. All strains were subjected to phenotypic characterization, and molecular characterization including detection of virulence genes and CTX prophage (CTXΦ) by Polymerase Chain Reaction (PCR) and genotyping by Pulsed-Field Gel Electrophoresis (PFGE). All strains were phenotypically confirmed as El Tor biotype and were mostly Ogawa serotype (96%). Antimicrobial susceptibility testing showed that the outbreak strains isolated in 1991 (Sabah) and 2009 (Terengganu) were all multidrug-resistant while the sporadic strains were resistant to erythromycin and furazolidone only. All strains (n = 45) were resistant to erythromycin. The virulence genes ctxA, ctxB, ompW, rfb, rtxC, tcpA, tcpI, rstR, zot and hlyA were present in all strains. The outbreak strains isolated in 1991 harboured El Tor cholera toxin gene (ctxB3) while sporadic strains from 2004 to 2011 harboured classical ctxB1. Four distinctive CTXΦ arrays were identified among the El Tor variants, one of which co-occurred with El Tor strains during the 2009 outbreak in Terengganu. PFGE analysis revealed that a genetically diverse El Tor variants population persisted in Sabah. The co-existence of multiple El Tor variants together with the prototypic El Tor strains suggested a multiclonal emergence of V. cholerae O1 El Tor variants in this region.

The oxazolidinone derivative locostatin induces cytokine appeasement.

Damaging inflammation arising from autoimmune pathology and septic responses results in severe cases of disease. In both instances, anti-inflammatory compounds are used to limit the excessive or deregulated cytokine responses. We used a model of robust T cell stimulation to identify new proteins involved in triggering a cytokine storm. A comparative proteomic mining approach revealed the differential mapping of Raf kinase inhibitory protein after T cell recall in vivo. Treatment with locostatin, an Raf kinase inhibitory protein inhibitor, induced T cell anergy by blocking cytokine production after Ag recall. This was associated with a reduction in Erk phosphorylation. Importantly, in vivo treatment with locostatin profoundly inhibited TNF-alpha production upon triggering the Ag-specific T cells. This effect was not limited to a murine model because locostatin efficiently inhibited cytokine secretion by human lymphocytes. Therefore, locostatin should be a useful therapeutic to control inflammation, sepsis, and autoimmune diseases.

Molecular Chaperone GRP94/GP96 in Cancers: Oncogenesis and Therapeutic Target.

During tumor development and progression, intrinsic and extrinsic factors trigger endoplasmic reticulum (ER) stress and the unfolded protein response, resulting in the increased expression of molecular chaperones to cope with the stress and maintain tumor cell survival. Heat shock protein (HSP) GRP94, also known as GP96, is an ER paralog of HSP90 and has been shown to promote survival signaling during tumor-induced stress and modulate the immune response through its multiple clients, including TLRs, integrins, LRP6, GARP, IGF, and HER2. Clinically, elevated expression of GRP94 correlates with an aggressive phenotype and poor clinical outcome in a variety of cancers. Thus, GRP94 is a potential molecular marker and therapeutic target in malignancies. In this review, we will undergo deep molecular profiling of GRP94 in tumor development and summarize the individual roles of GRP94 in common cancers, including breast cancer, colon cancer, lung cancer, liver cancer, multiple myeloma, and others. Finally, we will briefly review the therapeutic potential of selectively targeting GRP94 for the treatment of cancers.

Genomic analysis revealed a novel genotype of methicillin-susceptible Staphylococcus aureus isolated from a fatal sepsis case in dengue patient.

Staphylococcus aureus (S. aureus) is an opportunistic pathogen capable of causing serious health implications in susceptible individuals once it invades the host's protective barriers. Methicillin-susceptible S. aureus (MSSA) often receives lesser attention although it has been frequently associated with serious infections in human. We aim to investigate the genomic features of a highly virulent yet pan susceptible MSSA strain (coded as HS-MSSA) which caused concurrent bacteraemia in a dengue patient, ultimately resulted in sepsis death of the patient. Whole genome sequence analysis was performed. The draft genome of HS-MSSA is approximately 2.78 Mb (GC content = 32.7%) comprising of 2637 predicted coding sequences. In silico genotyping of the HS-MSSA strain revealed a novel combined genotype (t091/ST2990). The HS-MSSA carries a SaPIn1-like pathogenicity island that harbours the staphylococcal enterotoxin and enterotoxin-like genes (sec3 and selL). The strain-specific ß-lactamase (blaZ)-bearing plasmid region was identified in HS-MSSA. Core genome phylogeny showed that the HS-MSSA strain shared a common ancestry with the European MRSA clone. We report herein the genomic features of an MSSA lineage with novel genotype previously not reported elsewhere.

Type 2 dendritic cells mediate control of cytotoxic T cell resistant tumors.

Type 2 DCs (DC2s) comprise the majority of conventional DCs within most tumors; however, little is known about their ability to initiate and sustain antitumor immunity, as most studies have focused on antigen cross-presenting DC1s. Here, we report that DC2 infiltration identified by analysis of multiple human cancer data sets showed a significant correlation with survival across multiple human cancers, with the benefit being seen in tumors resistant to cytotoxic T cell control. Characterization of DC subtype infiltration into an immunotherapy-resistant model of breast cancer revealed that impairment of DC1s through 2 unique models resulted in enhanced DC2 functionality and improved tumor control. BATF3 deficiency depleted intratumoral DC1s, which led to increased DC2 lymph node migration and CD4+ T cell activation. Enhancing DC2 stimulatory potential by genetic deletion of Hsp90b1 (encoding molecular chaperon GP96) led to a similar enhancement of T cell immunity and improved survival in a spontaneous breast cancer model. These data highlight the therapeutic and prognostic potential of DC2s within checkpoint blockade-resistant tumors.

Investigation of biofilm formation in methicillin-resistant Staphylococcus aureus associated with bacteraemia in a tertiary hospital.

Biofilm formation is an important physiological process in Staphylococcus aureus (S. aureus) that can cause infections in humans. In this study, the ability of 36 methicillin-resistant S. aureus (MRSA) clinical isolates to form biofilm was studied based on genotypic and phenotypic approaches. These isolates were genotyped based on the microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) and biofilm-associated genes (icaAD) via polymerase chain reactions. Phenotyping was performed based on the determination of the strength of biofilm formation of MRSA isolates in vitro. The most prevalent MSCRAMMs and biofilm-associated genes were clfA, eno, and icaD, followed by clfB. The fnbB (38.9%) and ebpS (11.1%) occurred less frequently among the MRSA isolates, while bbp and fnbA genes were absent from all isolates. The MRSA isolates were mostly moderate to strong biofilm formers, despite the heterogeneity of the MSCRAMM profiles. MRSA isolates from different infection sources (primary, catheter-related bloodstream, or secondary infections) were capable of forming strong biofilms. However, persistent bacteraemia was observed only in 19.4% of the MRSA-infected individuals. This study suggested that persistent MRSA bacteraemia in patients might not be associated with the biofilm-forming ability of the isolates.

One-step differential detection of Salmonella enterica serovar Typhi, serovar Paratyphi A and other Salmonella spp. by using a quadruplex real-time PCR assay.

Diseases caused by typhoidal and non-typhoidal Salmonella remain a considerable threat to both developed and developing countries. Based on the clinical symptoms and serological tests, it is sometimes difficult to differentiate the Salmonella enterica serovar Paratyphi A (S. enterica serovar Paratyphi A) from serovar Typhi (S. enterica serovar Typhi). In this study, we developed a quadruplex real-time polymerase chain reaction (PCR) assay with an internal amplification control (IAC), to simultaneously differentiate S. enterica serovar Paratyphi A from serovar Typhi and to detect other Salmonella serovars which cause salmonellosis in humans. This assay was evaluated on 155 salmonellae and non-salmonellae strains and demonstrated 100% specificity in species differentiation. Inclusion of an IAC did not affect the efficiency of the assay. Further evaluation using a blind test on spiked stool, blood and food specimens showed that the detection limit was at 103 -104 CFU/mL (or g) and a high PCR efficiency with different targets (R2 > 0.99), except for S. enterica serovar Paratyphi A in blood. This assay has been applied to clinical specimens to detect the causative agents of gastrointestinal infections and has successfully identified 6 salmonellosis patients from the 50 diarrhoea patients. The quadruplex real-time PCR developed in this study could enhance the detection and differentiation of salmonellae. This assay could be applied to stools, blood and food based on the notable performance in the simulation tests and field evaluation.

Genetic mechanisms and correlated risk factors of antimicrobial-resistant ESKAPEE pathogens isolated in a tertiary hospital in Malaysia.

BACKGROUND: Knowledge on the epidemiology, genotypic and phenotypic features of antimicrobial-resistant (AMR) ESKAPEE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., and Escherichia coli) and their association with hospital-acquired infections (HAIs) are limited in Malaysia. Therefore, we evaluated the AMR features and resistance mechanisms of the ESKAPEE pathogens collected in a tertiary hospital located in the capital of Malaysia. METHODS: A total of 378 AMR-ESKAPEE strains were obtained based on convenience sampling over a nine-month study period (2019-2020). All strains were subjected to disk diffusion and broth microdilution assays to determine the antimicrobial susceptibility profiles. Polymerase chain reaction (PCR) and DNA sequence analyses were performed to determine the AMR genes profiles of the non-susceptible strains. Chi-square test and logistic regression analyses were used to correlate the AMR profiles and clinical data to determine the risk factors associated with HAIs. RESULTS: High rates of multidrug resistance (MDR) were observed in A. baumannii, K. pneumoniae, E. coli, and S. aureus (69-89%). All organisms except E. coli were frequently associated with HAIs (61-94%). Non-susceptibility to the last-resort drugs vancomycin (in Enterococcus spp. and S. aureus), carbapenems (in A. baumannii, P. aeruginosa, and Enterobacteriaceae), and colistin (in Enterobacteriaceae) were observed. Both A. baumannii and K. pneumoniae harbored a wide array of extended-spectrum ß-lactamase genes (blaTEM, blaSHV, blaCTX-M, blaOXA). Metallo-ß-lactamase genes (blaVEB, blaVIM, blaNDM) were detected in carbapenem-resistant strains, at a higher frequency compared to other local reports. We detected two novel mutations in the quinolone-resistant determining region of the gyrA in fluoroquinolone-resistant E. coli (Leu-102-Ala; Gly-105-Val). Microbial resistance to ampicillin, methicillin, and cephalosporins was identified as important risk factors associated with HAIs in the hospital. CONCLUSION: Overall, our findings may provide valuable insight into the microbial resistance pattern and the risk factors of ESKAPEE-associated HAIs in a tertiary hospital located in central Peninsular Malaysia. The data obtained in this study may contribute to informing better hospital infection control in this region.

Genomic comparison and phenotypic profiling of small colony variants of Burkholderia pseudomallei.

Burkholderia pseudomallei (B. pseudomallei) is an intracellular pathogen that causes melioidosis, a life-threatening infection in humans. The bacterium is able to form small colony variants (SCVs) as part of the adaptive features in response to environmental stress. In this study, we characterize the genomic characteristics, antimicrobial resistance (AMR), and metabolic phenotypes of B. pseudomallei SCV and wild type (WT) strains. Whole-genome sequence analysis was performed to characterize the genomic features of two SCVs (CS and OS) and their respective parental WT strains (CB and OB). Phylogenetic relationship between the four draft genomes in this study and 19 publicly available genomes from various countries was determined. The four draft genomes showed a close phylogenetic relationship with other genomes from Southeast Asia. Broth microdilution and phenotype microarray were conducted to determine the AMR profiles and metabolic features (carbon utilization, osmolytes sensitivity, and pH conditions) of all strains. The SCV strains exhibited identical AMR phenotype with their parental WT strains. A limited number of AMR-conferring genes were identified in the B. pseudomallei genomes. The SCVs and their respective parental WT strains generally shared similar carbon-utilization profiles, except for D,L-carnitine (CS), g-hydroxybutyric acid (OS), and succinamic acid (OS) which were utilized by the SCVs only. No difference was observed in the osmolytes sensitivity of all strains. In comparison, WT strains were more resistant to alkaline condition, while SCVs showed variable growth responses at higher acidity. Overall, the genomes of the colony morphology variants of B. pseudomallei were largely identical, and the phenotypic variations observed among the different morphotypes were strain-specific.

ß-Lactam Resistance in Upper Respiratory Tract Pathogens Isolated from a Tertiary Hospital in Malaysia.

The rise of antimicrobial resistance (AMR) among clinically important bacteria, including respiratory pathogens, is a growing concern for public health worldwide. Common causative bacteria for upper respiratory tract infections (URTIs) include Streptococcus pneumoniae and Haemophilus influenzae, and sometimes Staphylococcus aureus. We assessed the ß-lactam resistant trends and mechanisms of 150 URTI strains isolated in a tertiary care hospital in Kuala Lumpur Malaysia. High rates of non-susceptibility to penicillin G (38%), amoxicillin-clavulanate (48%), imipenem (60%), and meropenem (56%) were observed in S. pneumoniae. Frequent mutations at STMK and SRNVP motifs in PBP1a (41%), SSNT motif in PBP2b (32%), and STMK and LKSG motifs in PBP2x (41%) were observed in S. pneumoniae. H. influenzae remained highly susceptible to most ß-lactams, except for ampicillin. Approximately half of the ampicillin non-susceptible H. influenzae harboured PBP3 mutations (56%) and only blaTEM was detected in the ampicillin-resistant strains (47%). Methicillin-susceptible S. aureus (MSSA) strains were mostly resistant to penicillin G (92%), with at least two-fold higher median minimum inhibitory concentrations (MIC) for all penicillin antibiotics (except ticarcillin) compared to S. pneumoniae and H. influenzae. Almost all URTI strains (88-100%) were susceptible to cefcapene and flomoxef. Overall, ß-lactam antibiotics except penicillins remained largely effective against URTI pathogens in this region.

In Vitro Efficacy of Flomoxef against Extended-Spectrum Beta-Lactamase-Producing Escherichia coli and Klebsiella pneumoniae Associated with Urinary Tract Infections in Malaysia.

The increasing prevalence of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae has greatly affected the clinical efficacy of ß-lactam antibiotics in the management of urinary tract infections (UTIs). The limited treatment options have resulted in the increased use of carbapenem. However, flomoxef could be a potential carbapenem-sparing strategy for UTIs caused by ESBL-producers. Here, we compared the in vitro susceptibility of UTI-associated ESBL-producers to flomoxef and established ß-lactam antibiotics. Fifty Escherichia coli and Klebsiella pneumoniae strains isolated from urine samples were subjected to broth microdilution assay, and the presence of ESBL genes was detected by polymerase chain reactions. High rates of resistance to amoxicillin-clavulanate (76-80%), ticarcillin-clavulanate (58-76%), and piperacillin-tazobactam (48-50%) were observed, indicated by high minimum inhibitory concentration (MIC) values (32 µg/mL to 128 µg/mL) for both species. The ESBL genes blaCTX-M and blaTEM were detected in both E. coli (58% and 54%, respectively) and K. pneumoniae (88% and 74%, respectively), whereas blaSHV was found only in K. pneumoniae (94%). Carbapenems remained as the most effective antibiotics against ESBL-producing E. coli and K. pneumoniae associated with UTIs, followed by flomoxef and cephamycins. In conclusion, flomoxef may be a potential alternative to carbapenem for UTIs caused by ESBL-producers in Malaysia.