An IgM dot-immunobinding assay (IgM-DIA) was developed for the diagnosis of scrub typhus infection. The whole cell antigens of Karp, Kato and Gilliam strains of Rickettsia tsutsugamushi were immobilized onto nitrocellulose paper and reacted with patients sera. The presence of IgM R. tsutsugamushi specific antibody in the patient sera could be detected by the observation of a visible brown dot on the nitrocellulose paper. The IgM-DIA has a sensitivity of 90.4% and specificity of 81.4% as compared to the indirect immunoperoxidase test. The IgM-DIA is rapid, simple, cost-effective, does not require microscope or incubator. It is recommended as a rapid screening test for the diagnosis of scrub typhus infection in the field or rural area within the hyperendemic region.
The infection of Cryptococcus neoformans is acquired through the inhalation of desiccated yeast cells and basidiospores originated from the environment, particularly from bird's droppings and decaying wood. Three environmental strains of C. neoformans originated from bird droppings (H4, S48B and S68B) and C. neoformans reference clinical strain (H99) were used for intranasal infection in C57BL/6 mice. We showed that the H99 strain demonstrated higher virulence compared to H4, S48B and S68B strains. To examine if gene expression contributed to the different degree of virulence among these strains, a genome-wide microarray study was performed to inspect the transcriptomic profiles of all four strains. Our results revealed that out of 7,419 genes (22,257 probes) examined, 65 genes were significantly up-or down-regulated in H99 versus H4, S48B and S68B strains. The up-regulated genes in H99 strain include Hydroxymethylglutaryl-CoA synthase (MVA1), Mitochondrial matrix factor 1 (MMF1), Bud-site-selection protein 8 (BUD8), High affinity glucose transporter 3 (SNF3) and Rho GTPase-activating protein 2 (RGA2). Pathway annotation using DAVID bioinformatics resource showed that metal ion binding and sugar transmembrane transporter activity pathways were highly expressed in the H99 strain. We suggest that the genes and pathways identified may possibly play crucial roles in the fungal pathogenesis.
(1) Background: The antibiotic resistance (ABR) rates are escalating to seriously high levels worldwide. This study was conducted to determine physicians' perspectives on factors influencing ABR in Vietnam. (2) Methods: Focus group discussion (FGD) was conducted through in-depth interviews on ABR perspectives with 5-6 physicians from different geographical locations and hospitals in Vietnam between March and June 2020. The research questions were focused on three main themes of (a) knowledge deficiency on ABR and hospital-acquired infection, (b) antibiotic prescribing practice among clinicians in the healthcare setting, and (c) regulations and hospital policies on antibiotic use. The descriptive analysis was performed using QRS NVivo software. (3) Results: A total of six FGDs were conducted among 34 physicians (18 males, 16 females) aged 26-53 years old from six public and six private hospitals in Vietnam. Most of the participants were attending physicians (85.3%) and had 5-10 years of experience in surgical wards (55.9%). For theme (a), a majority of participants agreed that they had adequate information updates on how ABR develops in their clinical setting; and were well aware of hospital-acquired infections. For theme (b), the participants agreed that WHO guidelines and Vietnam national guidelines were two important reference documents in guiding physicians in antibiotic use. For theme (c), the FGD study revealed awareness of ABR, hospital antibiotic policies, and procedures for administrators on antibiotic use that were updated and complied with. (4) Conclusions: While different levels of control measures against ABR are ongoing in Vietnam, several weaknesses in the current antibiotic prescribing strategies in the hospital and clinical setting management policies have been identified in the healthcare system. The research findings will be helpful for policymakers to have better plans of action against ABR in Vietnam.
Colony morphology variation is a characteristic of Burkholderia pseudomallei primary clinical isolates, associated with variations in expression of virulence factors. Here, we performed comparative investigations on adhesion, invasion, plaque-forming abilities and protein profiles of B. pseudomallei wild-type (WT) and a small colony variant (SCV). The percentage of SCV adherence to A549 cells was significantly higher (2.73%) than WT (1.91%). In contrast, WT was significantly more efficient (0.63%) than SCV (0.31%) in invasiveness and in inducing cellular damage. Using 2-DE and MALDI TOF/TOF, 263 and 258 protein spots were detected in WT and SCV, respectively. Comparatively, 49 proteins were differentially expressed in SCV when compared with WT. Of these, 31 proteins were up-regulated, namely, nucleoside diphosphate kinase (Ndk), phosphoglycerate kinase (Pgk), thioredoxin (TrxA), putative ferritin DPS-family DNA-binding protein (DPS) and oxidoreductase (AhpC) that are known to be involved in adhesion, intracellular survival and persistence. However, among the 18 down-regulated proteins, enolase (Eno), elongation factor (EF-Tu) and universal stress-related proteins were associated with invasion and virulence. Differences observed in these protein profiles provide ample clues to their association with the morphotypic and phenotypic characteristics of colony variants, providing additional insights into the potential association of B. pseudomallei colony morphotypes with disease pathogenesis.
Carbapenems are the primary choice of treatment for severe Pseudomonas aeruginosa infection. However, the emergence of carbapenem resistance due to the production of metallo-β-lactamases (MBLs) is of global concern. In this study, 90 imipenem- (IPM- or IP-) resistant P. aeruginosa (IRPA) isolates, including 32 previously tested positive and genotyped for MBL genes by PCR, were subjected to double-disk synergy test (DDST), combined disk test (CDT), and imipenem/imipenem-inhibitor (IP/IPI) E-test to evaluate their MBLs detection capability. All three methods were shown to have a sensitivity of 100%. However, DDST was the most specific of the three (96.6%), followed by IP/IPI E-test interpreted based on the single criteria of IP/IPI ≥8 as positive (62.1%), and CDT was the least specific (43.1%). Based on the data from this evaluation, we propose that only IRPA with IP MIC >16 μg/mL and IP/IPI ≥8 by IP/IPI E-test should be taken as positive for MBL activity. With the new dual interpretation criteria, the MBL IP/IPI E-test was shown to achieve 100% sensitivity as well as specificity for the IRPA in this study. Therefore, the IP/IPI E-test is a viable alternative phenotypic assay to detect MBL production in IRPA in our population in circumstances where PCR detection is not a feasible option.
Differences in expression of potential virulence and survival genes were associated with B. pseudomallei colony morphology variants. Microarray was used to investigate B. pseudomallei transcriptome alterations among the wild type and small colony variant (SCV) pre- and post-exposed to A549 cells. SCV pre- and post-exposed have lower metabolic requirements and consume lesser energy than the wild type pre- and post-exposed to A549. However, both the wild type and SCV limit their metabolic activities post- infection of A549 cells and this is indicated by the down-regulation of genes implicated in the metabolism of amino acids, carbohydrate, lipid, and other amino acids. Many well-known virulence and survival factors, including T3SS, fimbriae, capsular polysaccharides and stress response were up-regulated in both the wild type and SCV pre- and post-exposed to A549 cells. Microarray analysis demonstrated essential differences in bacterial response associated with virulence and survival pre- and post-exposed to A549 cells.
In this study, 90 non-replicate imipenem-resistant Pseudomonas aeruginosa (IRPA) Malaysian isolates collected between October 2005 and March 2008 were subjected to a screening test for detection of the integron and the gene cassette. Class 1 integrons were detected in 54 IRPA clinical isolates, whilst three isolates contained class 2 integrons. Analysis of the gene cassettes associated with the class 1 integrons showed the detection of accC1 in isolates carrying bla(IMP-7) and aacA7 in isolates carrying bla(VIM-2). aadA6 was detected in two isolates carrying bla(IMP-4). Using random amplification of polymorphic DNA analysis, 14 PCR fingerprint patterns were generated from the 32 isolates carrying metallo-β-lactamase (MBL) genes (35.5 %), whilst 20 patterns were generated from the 58 non-MBL gene isolates (64.4 %). Based on the differences in the fingerprinting patterns, two clusters (A and B) were identified among the MBL-producing isolates. Cluster A comprised 18 isolates (56 %) carrying the bla(VIM) gene, whereas cluster B comprised 14 (44 %) isolates carrying the bla(IMP) gene. The non-MBL isolates were divided into clusters C and D. Cluster C comprised 22 non-MBL isolates harbouring class 1 integrons, whilst cluster D consisted of three isolates carrying class 2 integrons. These findings suggest that the class 1 integron is widespread among P. aeruginosa isolated in Malaysia and that characterization of cassette arrays of integrons will be a useful epidemiological tool to study the evolution of multidrug resistance and the dissemination of antibiotic resistance genes.
Burkholderia pseudomallei primary diagnostic cultures demonstrate colony morphology variation associated with expression of virulence and adaptation proteins. This study aims to examine the ability of B. pseudomallei colony variants (wild type [WT] and small colony variant [SCV]) to survive and replicate intracellularly in A549 cells and to identify the alterations in the protein expression of these variants, post-exposure to the A549 cells. Intracellular survival and cytotoxicity assays were performed followed by proteomics analysis using two-dimensional gel electrophoresis. B. pseudomallei SCV survive longer than the WT. During post-exposure, among 259 and 260 protein spots of SCV and WT, respectively, 19 were differentially expressed. Among SCV post-exposure up-regulated proteins, glyceraldehyde 3-phosphate dehydrogenase, fructose-bisphosphate aldolase (CbbA) and betaine aldehyde dehydrogenase were associated with adhesion and virulence. Among the down-regulated proteins, enolase (Eno) is implicated in adhesion and virulence. Additionally, post-exposure expression profiles of both variants were compared with pre-exposure. In WT pre- vs post-exposure, 36 proteins were differentially expressed. Of the up-regulated proteins, translocator protein, Eno, nucleoside diphosphate kinase (Ndk), ferritin Dps-family DNA binding protein and peptidyl-prolyl cis-trans isomerase B were implicated in invasion and virulence. In SCV pre- vs post-exposure, 27 proteins were differentially expressed. Among the up-regulated proteins, flagellin, Eno, CbbA, Ndk and phenylacetate-coenzyme A ligase have similarly been implicated in adhesion, invasion. Protein profiles differences post-exposure provide insights into association between morphotypic and phenotypic characteristics of colony variants, strengthening the role of B. pseudomallei morphotypes in pathogenesis of melioidosis.
In H. pylori infection, antibiotic-resistance is one of the most common causes of treatment failure. Bacterial metabolic activities, such as energy production, bacterial growth, cell wall construction, and cell-cell communication, all play important roles in antimicrobial resistance mechanisms. Identification of microbial metabolites may result in the discovery of novel antimicrobial therapeutic targets and treatments. The purpose of this work is to assess H. pylori metabolomic reprogramming in order to reveal the underlying mechanisms associated with the development of clarithromycin resistance. Previously, four H. pylori isolates were induced to become resistant to clarithromycin in vitro by incrementally increasing the concentrations of clarithromycin. Bacterial metabolites were extracted using the Bligh and Dyer technique and analyzed using metabolomic fingerprinting based on Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry (LC-Q-ToF-MS). The data was processed and analyzed using the MassHunter Qualitative Analysis and Mass Profiler Professional software. In parental sensitivity (S), breakpoint isolates (B), and induced resistance isolates (R) H. pylori isolates, 982 metabolites were found. Furthermore, based on accurate mass, isotope ratios, abundances, and spacing, 292 metabolites matched the metabolites in the Agilent METLIN precise Mass-Personal Metabolite Database and Library (AM-PCDL). Several metabolites associated with bacterial virulence, pathogenicity, survival, and proliferation (L-leucine, Pyridoxone [Vitamine B6], D-Mannitol, Sphingolipids, Indoleacrylic acid, Dulcitol, and D-Proline) were found to be elevated in generated resistant H. pylori isolates when compared to parental sensitive isolates. The elevated metabolites could be part of antibiotics resistance mechanisms. Understanding the fundamental metabolome changes in the course of progressing from clarithromycin-sensitive to breakpoint to resistant in H. pylori clinical isolates may be a promising strategy for discovering novel alternatives therapeutic targets.
The molecular types and genetic heterogeneity of Cryptococcus neoformans and C. gattii clinical isolates in Malaysia were determined in this study. Of 44 C. neoformans collected between 1980 and 2003, 42 (95.5%) were molecular type VNI, 2 (4.5%) were molecular type VNII. Of 17 C.gattii isolates, 13 (76.5%) were molecular type VGI, and 4 (23.5%) were molecular type VGII. A difference was noted when comparing the molecular types of cryptococcal isolates in the earlier and recent cases of cryptococcosis. While both molecular types VNI and VGI were equally predominant in the earlier cases of cryptococcosis, VNI was the most predominant molecular type isolated from the recent cases. VNII was a new molecular type, isolated from 5.1% of the recent cases. All the bird dropping isolates were molecular type VNI. The genetic heterogeneity of the two predominant molecular types, i.e., VNI, VGI clinical isolates and bird dropping isolates of C. neoformans were further determined by polymerase chain reaction (PCR) fingerprinting method, using (GTG)5 as single primer. Two clusters of cryptococcal isolates were distinguished at 68.5% of similarity, with cluster I consisting of VNI isolates and cluster II consisting of VGI isolates. Each cluster was further subdivided into three subtypes at >/=80% of similarity. Fourteen bird dropping isolates were grouped into a subtype within VN1, sharing 82.7% of similarity with the clinical isolates. A higher degree of similarities, ranging from 93.4-97.6% was noted between 3 bird dropping isolates with the clinical isolates in another subtype. This study demonstrated the existence of various molecular types of C. neoformans isolates in Malaysia and the genetic heterogeneity within the predominant molecular types. The study also provides evidence for genetic relatedness of clinical isolates with bird dropping isolates in the environment.
Ninety-three Malaysian extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae isolates were investigated for ciprofloxacin resistance. Two mismatch amplification mutation (MAMA) assays were developed and used to facilitate rapid detection of gyrA and parC mutations. The isolates were also screened for plasmid-mediated quinolone resistance (PMQR) genes including aac(6')-Ib-cr, qepA, and qnr. Ciprofloxacin resistance (MICs 4- ≥ 32 μ g/mL) was noted in 34 (37%) isolates, of which 33 isolates had multiple mutations either in gyrA alone (n = 1) or in both gyrA and parC regions (n = 32). aac(6')-Ib-cr was the most common PMQR gene detected in this study (n = 61), followed by qnrB and qnrS (n = 55 and 1, resp.). Low-level ciprofloxacin resistance (MICs 1-2 μ g/mL) was noted in 40 (43%) isolates carrying qnrB accompanied by either aac(6')-Ib-cr (n = 34) or a single gyrA 83 mutation (n = 6). Ciprofloxacin resistance was significantly associated with the presence of multiple mutations in gyrA and parC regions. While the isolates harbouring gyrA and/or parC alteration were distributed into 11 PFGE clusters, no specific clusters were associated with isolates carrying PMQR genes. The high prevalence of ciprofloxacin resistance amongst the Malaysian ESBL-producing K. pneumoniae isolates suggests the need for more effective infection control measures to limit the spread of these resistant organisms in the hospital.
The ciprofloxacin resistance of Klebsiella (K.) pneumoniae is mediated primarily through alterations in type II topoisomerase (gyrA) gene and plasmid-mediated quinolone resistance-conferring genes (qnr). This study aimed to define the prevalence of plasmid-mediated quinolone resistance-conferring genes (qnr) and type II topoisomerase (gyrA) alterations of a population of ciprofloxacin-resistant (n = 21), intermediate (n = 8), and sensitive (n = 18) K. pneumoniae isolates obtained from a teaching hospital at Kuala Lumpur, Malaysia.
The presence of Rickettsia felis, Bartonella henselae and B. clarridgeiae in 209 fleas (Ctenocephalides felis) obtained from domestic cats and dogs in several locations in Malaysia was investigated in this study. Using a polymerase chain reaction specific for the citrate synthase (gltA) and 17-kD antigenic protein (17kD) genes of rickettsiae, we detected R. felis DNA in 6 (2.9%) fleas. For detection of bartonellae, amplification of the heme-binding protein (pap31) and riboflavin synthase (ribC) genes identified B. henselae and B. clarridgeiae DNA in 24 (11.5%) and 40 (19.1%) fleas, respectively. The DNA of B. henselae and B. clarridgeiae was detected in 10 (4.8%) fleas. Two B. henselae genogroups (Marseille and Houston-1) were detected in this study; genogroup Marseille (genotype Fizz) was found more often in the fleas. The findings in this study suggest fleas as potential vectors of rickettsioses and cat-scratch disease in this country.
Little data are available on the prevalence and transmission of vector-borne diseases in stray dogs in Peninsular Malaysia. This study was designed to determine the occurrence of vector-borne pathogens in Malaysian stray dogs using serological and molecular approaches. In total, 48 dog blood samples were subjected to serological analysis using SNAP 4Dx kit (IDEXX Laboratories, Westbrook, ME). The presence of Ehrlichia and Anaplasma DNA in the dog blood samples and Rhipicephalus sanguineus (Latreille) ticks was detected using nested polymerase chain reaction assays. Positive serological findings against Ehrlichia canis and Anaplasma phagocytophilum were obtained in 17 (39.5%) and four (9.3%) of 43 dog samples, respectively. None of the dog blood samples were positive for Borrelia burgdorferi and Dirofilaria immitis. DNA of E. canis and A. phagocytophilum was detected in 12 (25.5%) and two (4.3%) of 47 dog blood samples, and 17 (51.5%) and one (3.0%) of 33 R. sanguineus ticks, respectively. Additionally, DNA of Ehrlichia spp. closely related to Ehrlichia chaffeensis was detected in two (6.1%) R. sanguineus ticks. This study highlights the prevalence of anaplasmosis and ehrlichiosis in dogs in Malaysia. Due to the zoonotic potential of Ehrlichia and Anaplasma spp., appropriate measures should be instituted for prevention and control of vector-borne diseases in dogs.
Infections caused by multidrug resistant Klebsiella pneumoniae have been increasingly reported in many parts of the world. A total of 93 Malaysian multidrug resistant K. pneumoniae isolated from patients attending to University of Malaya Medical Center, Kuala Lumpur, Malaysia from 2010-2012 were investigated for antibiotic resistance determinants including extended-spectrum beta-lactamases (ESBLs), aminoglycoside and trimethoprim/sulfamethoxazole resistance genes and plasmid replicons. CTX-M-15 (91.3%) was the predominant ESBL gene detected in this study. aacC2 gene (67.7%) was the most common gene detected in aminoglycoside-resistant isolates. Trimethoprim/sulfamethoxazole resistance (90.3%) was attributed to the presence of sul1 (53.8%) and dfrA (59.1%) genes in the isolates. Multiple plasmid replicons (1-4) were detected in 95.7% of the isolates. FIIK was the dominant replicon detected together with 13 other types of plasmid replicons. Conjugative plasmids (1-3 plasmids of ~3-100 kb) were obtained from 27 of 43 K. pneumoniae isolates. An ESBL gene (either CTX-M-15, CTX-M-3 or SHV-12) was detected from each transconjugant. Co-detection with at least one of other antibiotic resistance determinants [sul1, dfrA, aacC2, aac(6')-Ib, aac(6')-Ib-cr and qnrB] was noted in most conjugative plasmids. The transconjugants were resistant to multiple antibiotics including β-lactams, gentamicin and cotrimoxazole, but not ciprofloxacin. This is the first study describing the characterization of plasmids circulating in Malaysian multidrug resistant K. pneumoniae isolates. The results of this study suggest the diffusion of highly diverse plasmids with multiple antibiotic resistance determinants among the Malaysian isolates. Effective infection control measures and antibiotic stewardship programs should be adopted to limit the spread of the multidrug resistant bacteria in healthcare settings.
Bartonellosis is an emerging zoonotic infection responsible for a variety of clinical syndromes in humans and animals. Members of the genus Bartonella exhibit high degrees of genetic diversity and ecologic plasticity. The infection is usually transmitted to animals and humans through blood-feeding arthropod vectors such as fleas, lice, ticks and sandflies. This study was conducted to investigate the prevalence of Bartonella species in 184 beef cattle, 40 dairy cattle, 40 sheep and 40 goats in eight animal farms across Peninsular Malaysia. Bartonella-specific PCR assays and sequence analysis of partial fragments of the citrate synthase gene were used for detection and identification of B. bovis. Isolation of B. bovis was attempted from PCR-positive blood samples. Molecular heterogeneity of the isolates was investigated based on sequence analysis of gltA, ITS, rpoB genes, ERIC-PCR, as well as using an established multilocus sequence typing (MLST) method. The carriage rate of B. bovis in ticks was also determined in this study.
Amblyomma ticks parasitize a wide range of animals in tropical regions. This study describes the identification of Amblyomma ticks from wild snakes in Malaysia and the detection of potential human pathogens such as Rickettsia, Anaplasma, Ehrlichia and bartonellae in the ticks.