In the last few decades, co-trimoxazole (SXT), an antibacterial combination of trimethoprim and sulfamethoxazole, has been used for treatment of upper respiratory tract infection due to Haemophilus influenzae. The usage of this antibiotic has become less important due to emergence of SXT-resistant strains worldwide. Most reports associate SXT resistance to the presence of variants of dihydrofolate reductase (DHFR) dfrA genes which are responsible for trimethoprim resistance; while the sulfamethoxazole (SMX) resistance are due to sulfonamide (SUL) genes sul1 and sul2 and/or mutation in the chromosomal (folP) gene encoding dihydropteroate synthetase (DHPS). This study aims to detect and analyse the genes that are involved in SXT resistance in H. influenzae strains that were isolated in Malaysia. Primers targeting for variants of dfrA, fol and sul genes were used to amplify the genes in nine SXT-resistant strains. The products of amplification were sequenced and multiple alignments of the assembled sequences of the local strains were compared to the sequences of other H. influenzae strains in the Genbank. Of the five variants of the dhfA genes, dfrA1 was detected in three out of the nine strains. In contrast to intermediate strains, at least one variant of folP genes was detected in the resistant strains. Multiple nucleotide alignment of this gene revealed that strain H152 was genetically different from the others due to a 15-bp nucleotide insert in folP gene. The sequence of the insert was similar to the insert in folP of H. influenzae strain A12, a strain isolated in United Kingdom. None of the strains had sul1 gene but sul2 gene was detected in four strains. Preliminary study on the limited number of samples shows that the TMP resistance was attributed to mainly to dfrA1 and the SMX was due to folP genes. Presence of sul2 in addition to folP in seven strains apparently had increased their level of resistance. A strain that lacked sul1 or sul2 gene, its resistance to sulfonamide was attributed to a 15-bp DNA insert in the folP gene.
Leaves of Andrographis paniculata and Orthosiphon stamineus were extracted with water, ethanol, methanol and chloroform to assess their potential as antibacterial and antioxidant agents. High performance liquid chromatography analysis showed that the methanolic extracts of A. paniculata and O. stamineus leaves gave the highest amounts of andrographolide and rosmarinic acid, respectively. These leaf extracts exhibited antimicrobial and antioxidant activities and, at the highest concentration tested (200 mg/mL), showed greater inhibitory effects against the Gram positive bacteria Bacillus cereus and Staphylococcus aureus than 10% acetic acid. Andrographis paniculata and O. stamineus methanolic and ethanolic leaf extracts also showed the strongest antioxidant activity as compared with the other extracts tested. The bioactive compounds present in these leaf extracts have the potential to be developed into natural antibacterial and antioxidant agents that may have applications in animal and human health.
Prior to the implementation of Haemophilus influenzae type b vaccination worldwide, H. influenzae has been one of the main causative agents of community acquired pneumonia and meningitis in children. Due to the lack of information on the characteristics of the H. influenzae isolates that have previously been collected in Malaysia, the H. influenzae were assessed of their microbial susceptibility to commonly used antibiotics. Emphasis was made on strains that were resistance to co-trimoxazole (SXT) and their mode of transfer of the antibiotic resistance determinants were examined. A collection of 34 H. influenzae isolates was serotyped and antimicrobial susceptibility tests were performed to 11 antibiotics. To the isolates that were found to be resistant to co-trimoxazole, minimum inhibition concentration (MIC) to SXT was performed using Etest while agar dilution method was used to measure the individual MICs of trimethoprim (TMP) and sulfamethoxazole (SUL). These isolates were also examined for presence of plasmid by PCR and isolation method. Conjugal transfers of SXT-resistant genes to SXT-susceptible hosts were performed to determine their rate of transfer. Result showed that 20.6% of the total number of isolates was serotype B while the remaining was non-typeable. Antimicrobial susceptibility profile of all the isolates revealed that 58.8% was resistant to at least one antibiotic. Majority of these isolates were equally resistant to ampicillin and tetracycline (29.4% each), followed by resistance to SXT (26.5%). From nine isolates that were found to be SXT-resistant, five contained plasmid/s. Conjugal transfer experiment showed that these five isolates with plasmid transferred SXT-resistance determinants at a higher frequency than those without. From these observations, it is postulated that plasmid is not involved in the transfer of SXT-resistance genes but presence of plasmid facilitates their transfer. The information obtained from this study provides some basic knowledge on the antimicrobial susceptibility pattern of the H. influenzae isolates and their mode of transfer of SXT-resistance genes.
Ciprofloxacin, a quinolone with good intracellular penetration may possibly be used for treatment of melioidosis caused by Burkholderia pseudomallei, but problems with resistance may be encountered. Amino acid substitutions in gyrA/gyrB have given rise to fluoroquinolone resistance in various microorganisms. Using published primers for gyrA and gyrB, PCR was performed on 11 isolates of B. pseudomallei with varying degrees of sensitivity to ciprofloxacin, followed by DNA sequencing to detect possible mutations. Results showed an absence of any point mutation in either gene. Local isolates have yet to develop full resistance to ciprofloxacin and probably other mechanisms of resistance may have been involved in the decreased sensitivity to ciprofloxacin.
An understanding of common pathogens and their antibiotic sensitivity patterns is critical for proper management of sepsis in Emergency Department (ED). The goal of the study was to identify common organisms isolated from blood cultures of patients attended to ED and their antimicrobial susceptibility. Beginning from 2002, all cases of positive blood culture collected by the ED, Hospital Universiti Sains Malaysia (HUSM) were recorded and analysed. Over the period of eight years, we documented 995 cases of positive blood cultures. Of these samples, 549 (55.2%) were Gram-negative bacteria; 419 (42.1%) were Gram-positive bacteria; 10 (1.0%) were anaerobic organisms; 10 (1.0%) were fungus; and 7 (0.7%) cases were mixed organisms. Gram-negative bacteria were observed to develop more resistance to antimicrobial agents, especially those commonly used in an outpatient setting with less than 80% sensitivity to ampicillin, cotrimoxazole and ciprofloxacin. By contrast, there has been no marked change in the sensitivity trends of Gram-positive bacteria over the same period. In conclusion, ED physicians are more equipped to initiate empirical antimicrobial therapy especially when dealing with possibility of Gram-negative sepsis.
Study site: Emergency department, Hospital Universiti Sains Malaysia (HUSM), Kelantan, Malaysia
Nosocomial blood stream infection (or nosocomial bacteremia) is a common problem in hospitals worldwide, including Malaysia. A three-year prospective cohort study (October 2003-March 2007) of the incidences, risk factors, and patterns of the microorganisms causing bacteremia was conducted using a validated surveillance form in three intensive care units (ICUs) in Malaysia. Center for Disease Control criteria were used to diagnose bacteremia. Patients were monitored from admission until the end point of study, which was the first detection of bacteremia in the blood in each patient. The frequency of occurrence of bacteremia with clinical symptoms was 10.7% (n=23). Bacteremia was observed to occur within a mean length of stay of 10 days in ICU. The rate of device-related infection was 10.4% per device utilization days with a device utilization rate of 95.9%/1000 patient days. The total number of patient days was 2309 and the period of device utilization was 2211 days. The common bacteria detected were extended-spectrum beta-lactamases (ESBLs) Klebsiella pneumoniae (n=6); Pseudomonas aeruginosa (n=6); Acinetobacter species (n=5); Methicillin-resistant Staphylococcus aureus (MRSA)(n=3); and (non- ESBL) Klebsiella pneumoniae (n=2). Multivariable analysis using Cox Proportional Hazard Model showed that the predictors for developing bacteremia were cancer, MRSA carriage, duration of central venous catheter (CVC) infusion, frequency change of CVC, and the administration of hydrocortisone drugs. These results indicate that a combination of nursing and medical interventions as well as patients' severity of illness could lead to bacteremia in ICU. Strategic implementation of quality assurance measures in ICUs could help to control this problem.
Methicillin-resistant Staphylococcus aureus (MRSA) is known to cause nosocomial infections and is now becoming an emerging problem in veterinary medicine. The objective of the study was to determine the presence of MRSA in 100 cats and dogs sampled between November 2007 and April 2008 at the University Veterinary Hospital, Universiti Putra Malaysia. MRSA was detected in 8% of pets sampled. Ten percent (5/50) and 6% (3/50) of the isolates were from dogs and cats, respectively. All MRSA isolates possessed the mecA gene and were found to be resistant to at least three antimicrobials with a minimum of Oxacillin MIC of 8 μg/mL. One isolate (CT04) had an extremely high MIC of >256 μg/mL. The MLST type ST59 found in this study have been reported earlier from Singapore and other countries as a strain from animal and community-associated MRSA respectively. Pulsed-field gel electrophoresis revealed five pulsotypes. Two isolates from cats (CT27 and CT33) and three isolates from dogs (DG16, DG20, and DG49) were respectively assigned to pulsotypes B and D. The study suggests that cats and dogs in Malaysia are potential reservoirs for MRSA.
Mesosomes of Staphylococcus aureus ATCC 25923 treated with antibiotics were examined morphologically under the electron microscope. The Transmission Electron Microscope Rapid Method was used to eliminate the artifacts due to sample processing. Mesosomes were seen in all the antibiotic treated bacteria and not in the control group. The main factor that contributes to the formation of mesosomes in the bacteria was the mode of action of the antibiotics. The continuous cytoplasmic membrane with infolding (mesosomes) as in the S. aureus ATCC 25923 is therefore confirmed as a definite pattern of membrane organization in gram positive bacteria assaulted by amikacin, gentamicin, ciprofloxacin, vancomycin and oxacillin antibiotics. Our preliminary results show oxacillin and vancomycin treated bacteria seemed to have deeper and more mesosomes than those treated with amikacin, gentamicin and ciprofloxacin. Further research is needed to ascertain whether the deep invagination and the number of mesosomes formed is associated with the types of antibiotic used.
Medicinal plants have many traditional claims including the treatment of ailments of infectious origin. In the evaluation of traditional claims, scientific research is important. The objective of the study was to determine the presence of antibacterial activity in the crude extracts of some of the commonly used medicinal plants in Malaysia, Andrographis paniculata, Vitex negundo, Morinda citrifolia, Piper sarmentosum, and Centella asiatica. In this preliminary investigation, the leaves were used and the crude extracts were subjected to screening against five strains of bacteria species, Methicillin Resistant Staphylococcus aureus (MRSA), Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli, using standard protocol of Disc Diffusion Method (DDM). The antibacterial activities were assessed by the presence or absence of inhibition zones and MIC values. M. citrifolia, P. sarmentosum and C. asiatica methanol extract and A. paniculata (water extract) have potential antibacterial activities to both gram positive S. aureus and Methicillin Resistant S. aureus (MRSA). None of the five plant extracts tested showed antibacterial activities to gram negative E. coli and K. pneumoniae, except for A. paniculata and P. sarmentosum which showed activity towards P. aeruginosa. A. paniculata being the most potent at MIC of 2 g/disc. This finding forms a basis for further studies on screening of local medicinal plant extracts for antibacteria properties.
Urinary Tract Infections (UTIs) consider as the most common infections worldwide, with higher risk in patients experienced Acute Appendicitis (AA). The purpose of this study was, to investigate the bacterial profile of UTIs in patients with non-ruptured AA postsurgically, and to assess age- and gender-related links of all AA cases in Karak region, Jordan. Urine samples obtained from 46 cases (32 male and 14 female) aged between 16-70 years were diagnosed as non-ruptured AA, following with isolation and characterization of isolated bacteria. Out of 46 AA cases, uropathogens isolated from 25 (54.3%) UTI cases. Out of these isolates; 42 (73.7%) were gram-negative isolates and 15 (26.3%) were gram-positive bacteria. The percentage of isolates were E. coli (26.3%), Enterobacter species (21%), Enterococcus faecalis and Klebsiella pneumoniea (10.5%) for each, Streptococcus saprophytics and Pseudomonas aeruginosa (7%) for each, Yersinia spp. and S. milleri (8.8%). Out of UTI cases, 20 cases (80%) possessed mixed culture, each of them had at least one of Enterobacterial species. i.e. Enterobacter spp. or E. coli or both. More precisely, out of all these positivecases, 2 cases had pure gram positive-bacterial infection (8%), while pure gram negative bacterial infection comprised 48% of them and the rest (44%) were mixed (gram-negative and gram-positive) bacterial infection. Moreover, study revealed a high prevalence rate of AA cases 24 (52.2%) in the ages of 16-22 years, then declining the rate with increasing the age, reaching the lowest rate (4.3%) in ages of 60-70. In addition to age factor, the males significantly more susceptible to AA cases than females by 2.2-fold. Antibiotic sensitivity test revealed high resistance capability of E. coli to the most used antibiotics except for nitrofurantoin. Bacterial isolates showing sensitivity against ciprofloxacin, trimethoprim/sulfamethoxazole, amoxicillin-Clavulanic acid and nitrofurantoin, with a superiority for the first two. Results demonstrate high prevalence rate of UTIs in patients with AA. For avoiding, the needless use of antibiotics through sticking to our accountability as healthcare provisioner to pursuit the antimicrobial management.
Bacterial mediated Silver nanoparticles is considered as an emerging Ecofriendly approach to eradicate human pathogens. This paper aims to provide the biological approach for the synthesis of silver nanoparticles from indigenously isolated bacteria. This study will be beneficial to control the nosocomial infections triggered by MRSA (Methicillin-resistant Staphylococcus aureus). The current study is the extracellular synthesis of silver nanoparticles by using the cell free filtrate of bacterial strains isolated from the soil. The optimization study was also carried out to obtain the maximum production of silver nanoparticles. Nanoparticles were confirmed and characterized by UV-Vis spectroscopy and Transmission Electron Microscopy (TEM) having the plasmon resonance peak between 420-450nm with 10-60nm in size range and most were spherical in shape. Synthesized silver nanoparticles showed a potential antibacterial activity against MRSA (Methicillin Resistant Staphylococcus aureus) in-vitro study. This is the green approach for the production of AgNPs, as there was no previous work done on the synthesis of silver nanoparticles by bacteria in this region of Southern Punjab, Pakistan and these nanoparticles can be used to treat nosocomial infection. These silver nanoparticles can be used in effective disease management as antimicrobial agent.
Nosocomial infection caused by Acinetobacter baumannii is common among immunocompromised patients. Treatment strategy is limited due to rapid resistance development and lack of novel antibiotic. Colistin has been the last line therapy with good in vitro activity against infections caused by multi-drug resistance A. baumannii. However, pharmacological updates are required to support dosing optimisation. This study aimed to determine the time-kill kinetic and resistance development after antibiotic exposure as well as post-antibiotic effect of colistin at different static concentrations in in vitro A. baumannii system. The static in vitro time-kill and post-antibiotic effect experiments were conducted against two clinical isolates as well as one reference isolate ATCC 19606. Time-kill and postantibiotic effect were studied at colistin concentrations ranging from 0.25MIC to 16.0MIC and 0.5MIC to 4.0MIC, respectively. Post-exposure resistance development was examined in time-kill study. Killing activity and post-antibiotic effect were in a concentration-dependent manner. However, delayed killing activity indicates colistin tolerance. Development of resistance after exposure was not detected except for the ATCC 19606 strain. Dosing suggestion based on the observations include administration of supplemental dose 3 MIU at 12 hours after loading dose, administration of maintenance dose 9 MIU in two divided doses and application of extended interval in renal adjustment dose. However, the information is applicable for non-colistin-heteroresistance A. baumannii with colistin MIC < 1.0 mg/L. As for heteroresistance and strain with colistin MIC > 1.0 mg/L, combination therapy would be the more appropriate treatment strategy.
Some of Vibrio species is well known as pathogenic bacteria in aquaculture and the marine industry. Its infection is able to generate a massive outbreak and affect the fish population, especially for net caged fish such as seabass. This study was conducted to investigate the prevalence of Vibrio spp. isolated from seabass (Lates calcarifer) in Sri Tujuh Lagoon, Tumpat, Kelantan. Then, to determine the antibiotic resistance in Vibrio isolates. Polymerase chain reaction (PCR) was used to detect Vibrio species using specific primer VR169 and VR744 with estimation base pair size band, 597 bp and further identified by sequencing. On the other hand, antibiotic susceptibility tests were continued by using 13 types of antibiotics; kanamycin (K30), chloramphenicol (C30), neomycin (N10), ampicillin (AMP10), nitrofurantoin (F300), tetracycline (TE30), streptomycin (S10), norfloxacin (NOR10), ciprofloxacin (CIP5), nalidixic acid (NA30), gentamicin (CN10), doxycycline (DO30) and sulfamethoxazole (SXT100). As a result, 14 Vibrio isolates were identified, including Vibrio fluvialis (n=6), Vibrio parahaemolyticus (n=3), Vibrio harveyi (n=2) and each isolate for Vibrio vulnificus, Vibrio alginolyticus and Vibrio spp. The results showed that all isolates were sensitive to most antibiotics except ampicillin, neomycin and streptomycin. The MAR index value was ranging from 0 to 0.31. This study demonstrates the prevalence of Vibrio spp. in seabass and the report on multidrug resistance strains that could be of concern to the fish farmers. In addition, data from this study can be further used in fish disease management plans.
Antimicrobial resistance (AMR) is a global health crisis. Despite the drug discovery efforts, AMR is increasing, and discoveries are nearly nil. It is thus critical to design new strategies. Probiotics are tapped as alternatives to antibiotics for the treatment of gut-associated diseases. Lactobacillus species, common in food products, can inhibit the growth of gut pathogens. Here, we demonstrate the antimicrobial activities of Lactobacillus species - Lactobacillus paracasei, Lactobacillus casei, and Lactobacillus delbrueckii subsp. bulgaricus are enhanced when cocultured with Salmonella enterica subsp. enterica serovar Typhimurium. Cell-free culture supernatants (CFCS) from cocultures of Lactobacillus spp. and Salmonella enterica serovar Typhimurium more potently inhibit pathogen growth than their monoculture counterparts. Interestingly, we discovered that Salmonella enterica serovar Typhimurium could enhance the production of antimicrobials from Lactobacillus spp., most evident in L. delbrueckii subsp. bulgaricus. Also, L. delbrueckii subsp. bulgaricus CFCS upregulates key Salmonella virulence genes, hilA and sipA. Whether this increases Salmonella's pathogenicity in vivo or reduces pathogen fitness and growth inhibition in vitro warrants further investigation. We propose that these probiotic isolates may be utilized for innovative natural food processing and preservation strategies to control Salmonella food contaminations. Importantly, our findings that Salmonella elicits an enhanced antimicrobial activity from Lactobacillus spp. provide evidence of a pathogen-mediated elicitation of antimicrobial production. Therefore, extending this phenomenon to other microbial interactions may help augment the strategies for drug discovery.
The present study was conducted to investigate the antimicrobial potential of essential oils of Curcuma longa and Syzygium aromaticum against multidrug-resistant pathogenic bacteria. Four identified bacterial isolates including Methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii were selected and their antibiotic sensitivity was checked by disc diffusion assay. C. longa and S. aromaticum were subjected to steam distillation to obtain their essential oils. The crude essential oils were fractioned by employing column chromatography. Crude essential oils and their fractions were evaluated for their antibacterial activity by agar well diffusion assay and minimum inhibitory concentrations were calculated. All the selected bacterial isolates showed resistance to three or more than three antibiotic groups and were declared as multidrugresistant (MDRs). Crude essential oils of C. longa and S. aromaticum exhibited antimicrobial activity against all selected isolates but S. aromaticum activity was better than the C. longa with a maximum 19.3±1.50 mm zone of inhibition against A. baumannii at 1.04 µL/mL MIC. GC/MS analysis revealed the abundance of components including eugenol, eugenyl acetate, b- caryophyllene, and a- Humulene in both crude oil and fractions of S. aromaticum. While the main components of C. longa essential oil were Ar-tumerone, a-tumerone, b- Tumerone, I-Phellandrene, a-zingibirene, b- sesquiphellandrene, and p- Cymene. This study highlights that plant-based essential oils could be a promising alternative to antibiotics for which pathogens have developed resistance. C. longa and S. aromaticum carry compounds that have antimicrobial potential against multiple drug-resistant bacteria including MRSA. E. coli, K. pneumoniae and A. baumannii.
The primary goal of this research was to elucidate the novel influence of Brachiaria decumbens supplementation on broiler chicken growth performance, nutritional digestibility, cecal microbiota, intestinal histomorphology, carcass characteristics, and meat quality. A total of 300 male day-old Ross 308 broiler chickens were randomly subjected to six different treatment groups having five replicates per treatment with 10 birds in each replicate. In treatment 1, broiler chickens were fed commercial diets with no added additives; in treatment 2, broiler chickens were offered commercial diets containing 100 mg/kg of the antibiotic oxytetracycline. However, in treatments 3, 4, 5, and 6, broiler chickens received similar commercial diets supplemented with 25 mg/kg, 50 mg/kg, 75 mg/kg, and 100 mg/kg of B. decumbens ground leaf powder, respectively, without antibiotics. Throughout the 42-day trial, the body weight gain and total feed intake for each replicate were recorded every week to determine the growth performance. Then, on 21th and 42nd day, ten broilers from each treatment (two in each replicate) were randomly selected and slaughtered to assess the digestibility of nutrients, histomorphology of the small intestine, the population of the cecal microbiota, carcass characteristics, as well as quality of both breast and drumstick muscle. There were differences (p
Prevalence, distribution and antibiotic resistance of Arcobacter spp. were investigated in cattle, goats, floor and treated water samples in this study. The prevalence of Arcobacter in adult and young was recorded as 8/110 (7.27%) and 4/83 (4.81%), respectively, which showed insignificant difference (P = 0.3503) in detection rates between adult and young cattle. A total of 33.33% of the floor samples and 11.11% of the treated water samples analysed were determined as positive for Arcobacter. Among the species isolated, over all, A. butzleri (45%) was the most frequently detected species, followed by A. skirrowii (5%). A. butzleri was isolated from adult cattle, floor and water samples at the rates of 75.0%, 33.4% and 50%, respectively. Co-colonization of species was not uncommon, and 50% of the samples were carrying more than one Arcobacter species. Only 12.5% sample from cattle (adult) was detected positive for only A. skirrowii. All samples from young animals, floor and water contained mixed isolates. None of the samples from goat farm was found to be carrying Arcobacter species. On profiling of antimicrobial resistance patterns, it was found that only one A. butzleri isolate (3.7%) was sensitive to all nine antibiotics tested. A. butzleri was found highly resistant to ampicillin (55.6%), followed by cefotaxime (33.4%) and ciprofloxacin (33.4%). Overall, 20% of the isolates showed multidrug resistance (resistant ≥4 antibiotics). Gentamicin and enrofloxacin can be used as drugs of choice for the treatment for Arcobacter infections.
The prevalence of resistance to rifampicin and fusidic acid among Malaysian strains of methicillin-resistant Staphylococcus aureus (MRSA) is increasing. This study aimed to determine the mechanisms of rifampicin and fusidic acid resistance and the genetic diversity of MRSA strains from a Malaysian tertiary hospital.