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.

Microbial Genomics: Innovative Targets and Mechanisms.

Multidrug resistance (MDR) has become an increasing threat to global health because bacteria can develop resistance to antibiotics over time. Scientists worldwide are searching for new approaches that go beyond traditional antibiotic discovery and development pipelines. Advances in genomics, however, opened up an unexplored therapeutic opportunity for the discovery of new antibacterial agents. Genomic approaches have been used to discover several novel antibiotics that target critical processes for bacterial growth and survival, including histidine kinases (HKs), LpxC, FabI, peptide deformylase (PDF), and aminoacyl-tRNA synthetases (AaRS). In this review, we will discuss the use of microbial genomics in the search for innovative and promising drug targets as well as the mechanisms of action for novel antimicrobial agents. We will also discuss future directions on how the utilization of the microbial genomics approach could improve the odds of antibiotic development having a more successful outcome.

A Landscape View of the Female Genital Tract Microbiome in Healthy Controls and Women With Reproductive Health Conditions Associated With Ectopic Pregnancy.

Disruption of the female genital microbiome is associated with several pregnancy complications, including miscarriage, preterm onset of labour, and tubal pregnancy. Ectopic pregnancy is a known cause of maternal morbidity and mortality, but early diagnosis and treatment of ectopic pregnancy remain a challenge. Despite growing established associations between genital microbiome and female reproductive health, few studies have specifically focused on its link with ectopic pregnancy. Therefore, the current review aims to provide a comprehensive account of the female genital microbiome in healthy and fertile women compared to those in ectopic pregnancy and its associated risk factors. The microbial diversity from various sites of the female genital tract was explored for a reliable proxy of female reproductive health in sequencing-based ectopic pregnancy research. Our report confirmed the predominance of Lactobacillus in the vagina and the cervix among healthy women. The relative abundance decreased in the vaginal and cervical microbiome in the disease state. In contrast, there were inconsistent findings on the uterine microbiome across studies. Additionally, we explore a spectrum of opportunities to enhance our understanding of the female genital tract microbiome and reproductive conditions. In conclusion, this study identifies gaps within the field and emphasises the need for visionary solutions in metagenomic tools for the early detection of ectopic pregnancy and other gynaecological diseases.

MgrB Mutations and Altered Cell Permeability in Colistin Resistance in Klebsiella pneumoniae.

There has been a resurgence in the clinical use of polymyxin antibiotics such as colistin due to the limited treatment options for infections caused by carbapenem-resistant Enterobacterales (CRE). However, this last-resort antibiotic is currently confronted with challenges which include the emergence of chromosomal and plasmid-borne colistin resistance. Colistin resistance in Klebsiella pneumoniae is commonly caused by the mutations in the chromosomal gene mgrB. MgrB spans the inner membrane and negatively regulates PhoP phosphorylation, which is essential for bacterial outer membrane lipid biosynthesis. The present review intends to draw attention to the role of mgrB chromosomal mutations in membrane permeability in K. pneumoniae that confer colistin resistance. With growing concern regarding the global emergence of colistin resistance, deciphering physical changes of the resistant membrane mediated by mgrB inactivation may provide new insights for the discovery of novel antimicrobials that are highly effective at membrane penetration, in addition to finding out how this can help in alleviating the resistance situation.

Anti- and Pro-Oxidant Properties of Essential Oils against Antimicrobial Resistance.

The rapid evolution of antimicrobial resistance (AMR) has remained a major public health issue, reducing the efficacy of antibiotics and increasing the difficulty of treating infections. The discovery of novel antimicrobial agents is urgently needed to overcome the challenges created by AMR. Natural products such as plant extracts and essential oils (EOs) have been viewed as potential candidates to combat AMR due to their complex chemistry that carries inherent pro-oxidant and antioxidant properties. EOs and their constituents that hold pro-oxidant properties can induce oxidative stress by producing reactive oxygen species (ROS), leading to biological damage in target cells. In contrast, the antioxidant properties scavenge free radicals through offsetting ROS. Both pro-oxidant and antioxidant activities in EOs represent a promising strategy to tackle AMR. Thus, this review aimed to discuss how pro-oxidants and antioxidants in EOs may contribute to the mitigation of AMR and provided a detailed description of the challenges and limitations of utilizing them as a means to combat AMR.

Host-Bacterial Interactions: Outcomes of Antimicrobial Peptide Applications.

The bacterial membrane is part of a secretion system which plays an integral role to secrete proteins responsible for cell viability and pathogenicity; pathogenic bacteria, for example, secrete virulence factors and other membrane-associated proteins to invade the host cells through various types of secretion systems (Type I to Type IX). The bacterial membrane can also mediate microbial communities' communication through quorum sensing (QS), by secreting auto-stimulants to coordinate gene expression. QS plays an important role in regulating various physiological processes, including bacterial biofilm formation while providing increased virulence, subsequently leading to antimicrobial resistance. Multi-drug resistant (MDR) bacteria have emerged as a threat to global health, and various strategies targeting QS and biofilm formation have been explored by researchers worldwide. Since the bacterial secretion systems play such a crucial role in host-bacterial interactions, this review intends to outline current understanding of bacterial membrane systems, which may provide new insights for designing approaches aimed at antimicrobials discovery. Various mechanisms pertaining interaction of the bacterial membrane with host cells and antimicrobial agents will be highlighted, as well as the evolution of bacterial membranes in evasion of antimicrobial agents. Finally, the use of antimicrobial peptides (AMPs) as a cellular device for bacterial secretion systems will be discussed as emerging potential candidates for the treatment of multidrug resistance infections.

Overview of the distribution of Burkholderia pseudomallei sequence types and the Emergence of sequence type 1342 in Malaysia.

Burkholderia pseudomallei, a Gram-negative bacterial pathogen that causes melioidosis, is of public health importance in endemic areas including Malaysia. An investigation of the molecular epidemiology links of B. pseudomallei would contribute to better understanding of the clonal relationships, transmission dynamics and evolutionary change. Multilocus sequence typing (MLST) of 45 clinical B. pseudomallei isolates collected from sporadic melioidosis cases in Malaysia was performed. In addition, a total of 449 B. pseudomallei Malaysian strains submitted to the MLST database from 1964 until 2019 were included in the temporal analysis to determine the endemic sequence types (STs), emergence and re-emergence of ST(s). In addition, strain-specific distribution was evaluated using BURST tool. Genotyping of 45 clinical strains was resolved into 12 STs, and the majority were affiliated with ST46 (n = 11) and ST1342 (n = 7). Concomitantly, ST46 was the most prevalent ST in Malaysia, which was first reported in 1964. All the Malaysian B. pseudomallei strains were resolved into 76 different STs with 36 of them uniquely present only in Malaysia. ST1342 was most closely related to ST1034, in which both STs were unique to Malaysia and first isolated from soil samples in Pahang, a state in Malaysia. The present study revealed a high diversity of B. pseudomallei in Malaysia. Localized evolution giving rise to the emergence of new STs was observed, suggesting that host and environmental factors play a crucial role in the evolutionary changes in B. pseudomallei.

Combinatorial Antimicrobial Efficacy and Mechanism of Linalool Against Clinically Relevant Klebsiella pneumoniae.

Antibiotic-adjuvant combinatory therapy serves as a viable treatment option in addressing antibiotic resistance in the clinical setting. This study was carried out to assess and characterize the adjuvant potential and mode of action of linalool against carbapenemase-producing Klebsiella pneumoniae (KPC-KP). Linalool exhibited bactericidal activity alone (11,250 µg/ml) and in combination with meropenem (5,625 µg/ml). Comparative proteomic analysis showed significant reduction in the number of cytoplasmic and membrane proteins, indicating membrane damage in linalool-treated KPC-KP cells. Upregulation of oxidative stress regulator proteins and downregulation of oxidative stress-sensitive proteins indicated oxidative stress. Zeta potential measurement and outer membrane permeability assay revealed that linalool increases the bacterial surface charge as well as the membrane permeability. Intracellular leakage of nucleic acid and proteins was detected upon linalool treatment. Scanning and transmission electron microscopies further revealed the breakage of bacterial membrane and loss of intracellular materials. Linalool induced oxidative stress by generating reactive oxygen species (ROS) which initiates lipid peroxidation, leading to damage of the bacterial membrane. This leads to intracellular leakage, eventually killing the KPC-KP cells. Our study demonstrated that linalool possesses great potential in future clinical applications as an adjuvant along with existing antibiotics attributed to their ability in disrupting the bacterial membrane by inducing oxidative stress. This facilitates the uptake of antibiotics into the bacterial cells, enhancing bacterial killing.

Neonatal intensive care unit (NICU) exposures exert a sustained influence on the progression of gut microbiota and metabolome in the first year of life.

Emerging evidence has shown a link between the perturbations and development of the gut microbiota in infants with their immediate and long-term health. To better understand the assembly of the gut microbiota in preterm infants, faecal samples were longitudinally collected from the preterm (n = 19) and term (n = 20) infants from birth until month 12. 16S rRNA gene sequencing (n = 141) and metabolomics profiling (n = 141) using nuclear magnetic resonance spectroscopy identified significant differences between groups in various time points. A panel of amino acid metabolites and central metabolism intermediates significantly correlated with the relative abundances of 8 species of bacteria were identified in the preterm group. In contrast, faecal metabolites of term infants had significantly higher levels of metabolites which are commonly found in milk such as fucose and ß-hydroxybutyrate. We demonstrated that the early-life factors such as gestational age, birth weight and NICU exposures, exerted a sustained effect to the dynamics of gut microbial composition and metabolism of the neonates up to one year of age. Thus, our findings suggest that intervention at this early time could provide 'metabolic rescue' to preterm infants from aberrant initial gut microbial colonisation and succession.

An Overview of the Genetic Variations of the SARS-CoV-2 Genomes Isolated in Southeast Asian Countries.

Monitoring the mutation dynamics of human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical in understanding its infectivity, virulence and pathogenicity for development of a vaccine. In an "age of mobility," the pandemic highlights the importance and vulnerability of regionalization and labor market interdependence in Southeast Asia. We intend to characterize the genetic variability of viral populations within the region to provide preliminary information for regional surveillance in the future. By analyzing 142 complete genomes from South East Asian (SEA) countries, we identified three central variants distinguished by nucleotide and amino acid changes.

First detection of Klebsiella quasipneumoniae producing OXA-181 carbapenemase in Malaysia.

The emergence of carbapenemase-producing Enterobacteriaceae has become a major global concern. OXA-48-like carbapenemase gene and its variants have been increasingly reported worldwide. This study reported the first OXA-181-producing Klebsiella quasipneumoniae isolate in Malaysia. This bacterium was isolated from blood specimen of a three-year-old boy with Alagille syndrome who had liver biopsy on October 2016. He had undergone liver transplant in India ten months previously. The genotypic and phenotypic characteristics of the strain were elucidated in this study. To our best knowledge, this is the first report of OXA-181-producing K. quasipneumoniae in Malaysia.

Transcriptomic analysis of multi-drug resistant Escherichia coli K-12 strain in response to Lavandula angustifolia essential oil.

To better understand the synergistic antibacterial activity between piperacillin and Lavandula angustifolia essential oil (LEO) against multidrug-resistant Escherichia coli, we performed microarray transcriptomic analysis of LEO when used alone and in combination with piperacillin against the non-treated control. In total, 90 genes were differentially expressed after the combination of LEO and piperacillin treatment. Among the up-regulated genes, nfsB, nemA, fruA, nfsB, nemA are known to control microbial metabolism and nitrotoluene degradation, which were observed only in the LEO-piperacillin combinatory treatment. Four candidate genes from the microarray result, srIA, srID, waaR and nfsB, were validated by qRT-PCR as these genes showed differential expression consistently in the two methods. Biochemical pathway analysis showed that there was upregulation of genes involved in several biological processes including fructose and mannose metabolism, phosphotransferase system (PTS), lipopolysaccharide biosynthesis and nitrotoluene degradation. Genes involved in microbial metabolism in diverse environments were found both up- and down-regulated in LEO-piperacillin combinatory treatment. Our study provides new information concerning the transcriptional changes that occur during the LEO and piperacillin interaction against the multidrug-resistant bacteria and contributes to unravel the mechanisms underlying this synergism.

Whole genome analysis of multidrug-resistant Citrobacter freundii B9-C2 isolated from preterm neonate's stool in the first week.

BACKGROUND: Resistance to colistin, the last line therapy for infections caused by multidrug-resistant Gram-negative bacteria, represents a major public health threat. Citrobacter freundii B9-C2 which was isolated from the stool of preterm neonate on the first week of life, displayed resistance to almost all major antibiotics, including colistin. Through whole genome sequencing (WGS), we characterised the genome features that underline the antibiotic-resistance phenotype of this isolate. METHODS: Genome of C. freundii B9-C2 was sequenced on an Illumina MiSeq platform. The assembled genome was annotated and deposited into GenBank under the accession number CP027849. RESULTS: Multiple antimicrobial resistance genes including blaCMY-66 were identified. Further, the presence of 15 antibiotic efflux pump-encoding resistance genes, including crp, baeR, hns, patA, emrB, msbA, acrA, acrB, emrR, mdtC, mdtB, mdtG, kdpE, mdfA and msrB, were detected and likely to account for the observed cephalosporins, carbapenems, aminoglycosides and monobactams resistance in C. freundii B9-C2. The isolate also presented unique virulence genes related to biofilm formation, motility and iron uptake. The genome was compared to publicly available genomes and it was closely related to strains with environmental origins. CONCLUSION: To the best of our knowledge, this is the first report of intestinal carriage of colistin-resistant C. freundii from the stool of a neonate in Malaysia. Using genomic analysis, we have contributed to the understanding of the potential mechanism of resistance and the phylogenetic relationship of the isolates with draft genomes available in the public domain.

Genomic Insights into Two Colistin-Resistant Klebsiella pneumoniae Strains Isolated from the Stool of Preterm Neonate During the First Week of Life.

Background: Klebsiella pneumoniae is a major opportunistic pathogen frequently associated with nosocomial infections, and often poses a major threat to immunocompromised patients. In our previous study, two K. pneumoniae (K36 and B13), which displayed resistance to almost all major antibiotics, including colistin, were isolated. Both isolates were not associated with infection and isolated from the stools of two preterm neonates admitted to the neonatal intensive care unit (NICU) during their first week of life. Materials and Methods: In this study, whole genome sequencing was performed on these two clinical multidrug resistant K. pneumoniae. We aimed to determine the genetic factors that underline the antibiotic-resistance phenotypes of these isolates. Results: The strains harbored blaSHV-27, blaSHV-71, and oqxAB genes conferring resistance to cephalosporins, carbapenems, and fluoroquinolones, respectively, but not harboring any known plasmid-borne colistin resistance determinants such as mcr-1. However, genome analysis discovered interruption of mgrB gene by insertion sequences gaining insight into the development of colistin resistance. Conclusion: The observed finding that points to a scenario of potential gut-associated resistance genes to Gram negative (K. pneumoniae) host in the NICU environment warrants attention and further investigation.

Diversity of endocervical microbiota associated with genital Chlamydia trachomatis infection and infertility among women visiting obstetrics and gynecology clinics in Malaysia.

The cervical microbiota constitutes an important protective barrier against the invasion of pathogenic microorganisms. A disruption of microbiota within the cervical milieu has been suggested to be a driving factor of sexually transmitted infections. These include Chlamydia trachomatis which frequently causes serious reproductive sequelae such as infertility in women. In this study, we profiled the cervical microbial composition of a population of 70 reproductive-age Malaysian women; among which 40 (57.1%) were diagnosed with genital C. trachomatis infection, and 30 (42.8%) without C. trachomatis infection. Our findings showed a distinct compositional difference between the cervical microbiota of C. trachomatis-infected subjects and subjects without C. trachomatis infection. Specifically, significant elevations of mostly strict and facultative anaerobes such as Streptococcus, Megasphaera, Prevotella, and Veillonella in the cervical microbiota of C. trachomatis-positive women were detected. The results from the current study highlights an interaction of C. trachomatis with the environmental microbiome in the endocervical region.

The emergence of carbapenem resistant Klebsiella pneumoniae in Malaysia: correlation between microbiological trends with host characteristics and clinical factors.

BACKGROUND: Carbapenem resistant Enterobacteriaceae is a growing concern worldwide including Malaysia. The emergence of this pathogen is worrying because carbapenem is one of the 'last-line' antibiotics. The main objective of this study was to determine the prevalence of genetic mechanisms and clinical risk factors of carbapenem resistant Klebsiella pneumoniae (K. pneumoniae) in Malaysia. METHODS: In this study, seventeen carbapenem resistant K. pneumoniae strains isolated from a tertiary teaching hospital in 2013 were studied. Minimal inhibitory concentration (MIC) of the bacterial strains was determined and genes associated with carbapenemases and extended-spectrum-beta-lactamases (ESBLs) were sequenced and compared with the closest representatives published in public domains. All strains were also sub-typed using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Statistical analyses were performed to determine the correlation between risk factors for acquiring carbapenem resistant K. pneumoniae and in-hospital mortality. RESULTS: The predominant carbapenemase was blaOXA-48, detected in 12 strains (70.59%). Other carbapenemases detected in this study were blaKPC-2, blaIMP-8, blaNMC-A and blaNDM-1. Nine different pulsotypes were identified and nine strains which were affiliated with ST101, the predominant sequence type had similar PFGE patterns (similarity index of 85%). Based on univariate statistical analysis, resistance to imipenem and usage of mechanical ventilation showed a statistically significant effect separately to in-hospital mortality. CONCLUSION: The diverse genetic mechanisms harbored by these carbapenem resistant K. pneumoniae facilitates its spread and complicates its detection. Thus, correlation between microbiological trends with host characteristics and clinical factors will provide a better insight of rational treatment strategies and pathogen control.

Intestinal carriage of multidrug-resistant gram-negative bacteria in preterm-infants during hospitalization in neonatal intensive care unit (NICU).

The prevalence and antibiotic susceptibility of intestinal carriage of Gram-negative bacteria among preterm infants admitted to the neonatal intensive care unit (NICU) in a tertiary teaching hospital in Malaysia were determined. A total of 34 stool specimens were obtained from preterm infants upon admission and once weekly up to two weeks during hospitalization. The presumptive colonies of Escherichia coli and Klebsiella pneumoniae were selected for identification, antibiotic susceptibility testing, and subtyping by using pulsed-field gel electrophoresis (PFGE). Out of 76 Gram-negative isolates, highest resistance was detected for amoxicillin/clavulanate (30.8%, n = 16), ceftriaxone (42.3%, n = 22), ceftazidime (28.8%, n = 15), cefoxitin (28.8%, n = 15), aztreonam (36.5%, n = 19), and polymyxin B (23.1%, n = 12). Three colistin resistant K. pneumoniae have also been detected based on E-test analysis. Thirty-nine isolates of K. pneumoniae and 20 isolates of E. coli were resistant to more than three antimicrobial classes and were categorized as multidrug resistant (MDR). PFGE analysis revealed a higher diversity in pulsotypes for K. pneumoniae (18 pulsotypes) in comparison to E. coli (four pulsotypes). In addition, a total of fifteen pulsotypes was observed from 39 MDR K. pneumoniae. The risk factors for antibiotic resistance were assessed using random forest analysis. Gender was found to be the most important predictor for colistin resistant while length, OFC, and delivery mode were showing greater predictive power in the polymyxin B resistance. This study revealed worrying prevalence rates of intestinal carriage of multidrug-resistant K. pneumoniae and E. coli of hospitalized preterm infants in Malaysia, particularly high resistance to polymyxins.

Antibacterial Mode of Action of Cinnamomum verum Bark Essential Oil, Alone and in Combination with Piperacillin, Against a Multi-Drug-Resistant Escherichia coli Strain.

This study aimed to investigate the mechanism of action of the cinnamon bark essential oil (CB), when used singly and also in combination with piperacillin, for its antimicrobial and synergistic activity against beta-lactamase TEM-1 plasmid-conferred Escherichia coli J53 R1. Viable count of this combination showed a complete killing profile at 20 h and further confirmed its synergistic effect by reducing the bacteria cell numbers. Analysis on the stability of treated cultures for cell membrane permeability by CB when tested against sodium dodecyl sulfate revealed that the bacterial cell membrane was disrupted by the essential oils. Scanning electron microscopy observation and bacterial surface charge measurement also revealed that CB causes irreversible membrane damage and reduces the bacterial surface charge. In addition, bioluminescence expression of Escherichia coli [pSB1075] and E. coli [pSB401] by CB showed reduction, indicating the possibility of the presence of quorum sensing (QS) inhibitors. Gas-chromatography and mass spectrometry of the essential oil of Cinnamomum verum showed that trans-cinnamaldehyde (72.81%), benzyl alcohol (12.5%), and eugenol (6.57%) were the major components in the essential oil. From this study, CB has the potential to reverse E. coli J53 R1 resistance to piperacillin through two pathways; modification in the permeability of the outer membrane or bacterial QS inhibition.

Essential oils, a new horizon in combating bacterial antibiotic resistance.

For many years, the battle between humans and the multitudes of infection and disease causing pathogens continues. Emerging at the battlefield as some of the most significant challenges to human health are bacterial resistance and its rapid rise. These have become a major concern in global public health invigorating the need for new antimicrobial compounds. A rational approach to deal with antibiotic resistance problems requires detailed knowledge of the different biological and non-biological factors that affect the rate and extent of resistance development. Combination therapy combining conventional antibiotics and essential oils is currently blooming and represents a potential area for future investigations. This new generation of phytopharmaceuticals may shed light on the development of new pharmacological regimes in combating antibiotic resistance. This review consolidated and described the observed synergistic outcome between essential oils and antibiotics, and highlighted the possibilities of essential oils as the potential resistance modifying agent.