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Fulltext Horizontal gene transfer: a vehicle for the dissemination of resistance and virulence determinants during colonization and disease

Asinamai Athliamai Bitrus, Siti Khairani Bejo, Sarah Othman, Zunita Zakaria

The Pertanika Journal of Scholarly Research Reviews, 2017;3(3):51-60.
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The successful in vivo horizontal transfer of mobile genetic elements carrying resistance and virulence determinants have contributed immensely to a global dissemination of virulent and multi-drug resistant pathogens. In addition, the pathogenesis of MRSA infection is enhanced via initial colonization of the skin through the component of the microbial surface antigen recognizing adhesive matrix molecules and by their ability to evade host immune response. Furthermore, it was also observed that the genetic diversity of pathogenic MRSA is due to its’ ability to rapidly acquire resistance and virulence determinants. A characteristic feature that made it one of the most important nosocomial pathogen worldwide. Similarly, the expression of virulence gene in MRSA has been observed to be regulated by the accessory gene regulator system (agr). These system is made up of a series of genes whose product build up quorum-sensing regulatory mechanisms that is growth dependent. In addition, at a certain growth stage, the agr systems triggers a pronounced changes in the expression of genes called the quorum sensing. The findings of this review affirms the importance of horizontal gene transfer in the dissemination of resistance and virulence determinants and as well as the genetic diversity of MRSA.
Fulltext Molecular epidemiology: a valuable tool for determination of emerging and clonality of methicillin resistant Staphylococcus aureus (MRSA)

Bitrus, Asinamai Athliamai, Zunita Zakaria, Siti Khairani-bejo, Abdul-nasir Tijjan, Sarah Othman

The Pertanika Journal of Scholarly Research Reviews, 2016;2(2):121-134.
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Methicillin-resistant Staphylococcus aureus (MRSA) is one of the leading nosocomial
pathogen that is also emerging as a zoonotic pathogen. In this review, it was observed that rapid
emergence of new MRSA clones at a higher frequency has ushered in a new knowledge on the clonality
and epidemic potentials of MRSA. Secondly, the success of treatment and management of MRSA
infection is threatened by the diversity in the clonal types. This is because different clones harbours
different antibiotics resistance characteristics and as such respond differently to treatment. Furthermore,
clonal replacement of hospital-acquired MRSA with community -acquired MRSA has also been
observed. Thirdly, the transmission of MRSA even though previously thought to be exclusively within
the hospital setting through hand contact and nasal colonization has now spread to the community and in
addition human to animal and animal to human transmission has also been observed. Similarly, pet
owners, veterinarians and farmers have been described as high-risked group with potentials of becoming
reservoirs of MRSA. Furthermore, the adoption of hand hygiene in healthcare setting have to a great
extent reduced the incidence of MRSA in the hospital. And lastly, the advent of molecular typing such as
Pulsed Field Gel Electrophoresis (PFGE), Multi Locus Sequence Typing (MLST), Staphylococcal protein
A typing (Spa typing) and Double Locus Sequence Typing (DLST) have proven to be a useful tool in
providing valuable information on the evolution and clonal diversity of MRSA. These in turn help
researchers to answer some pertinent questions on the epidemiology of MRSA.
Fulltext Molecular detection, identification and differentiation of Burkholderia pseudomallei

Rabiu Muhammad, Aliyu, Mohamed Mustapha, Noordin, Zunita Zakaria, Siti Khairani-bejo

The Pertanika Journal of Scholarly Research Reviews, 2016;2(2):69-79.
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Burkholderia pseudomallei are Gram negative highly pathogenic bacteria of humans and
animals causing a multisystemic disease called melioidosis. They have recently gained a lot of interest
from the research community and public health organisations because of their great potential to be
used as an agent of bioterrorism. This has made the search for simple, rapid, accurate and the most
definitive means of their detection, identification and discrimination very critical and necessary. This
article aimed to review the molecular techniques used for detection, identification and differentiation
of B. pseudomallei. Although, culture and isolation techniques maintained their usefulness in
confirming cases of melioidosis, their time limitation (can take up to a week for confirming diagnosis)
leads to the search for rapid and simple techniques. Consequently, serology-based tests have been
developed which are both faster and less sophisticated. However, the presence of high background
titre levels and cross-reaction with other organisms make it less reliable. Thus, efforts have been
directed to explore rapid and accurate molecular techniques and resulting in the development and
validation of various PCR-based identification techniques targeting either single or multiple genes.
Although requiring some level of instrumentation and expertise, PCR-based techniques have been
reported to be very useful in diagnosis of melioidosis. We recommend the 16S rRNA PCR (especially
augmented with other molecular methods such as gene sequencing and analysis) and MLST
techniques for timely detection, identification and differentiation of B. pseudomallei for routine
diagnosis and epidemiological studies respectively.
Fulltext Determining the efficacy of CRISPR-Cas9 mechanisms on the uppP Gene of Klebsiella pneumonia using Lysozyme assay

Mohd Nasharudin Razak, Raha Abdul Rahim, Abdul Rahman Omar, Zunita Zakaria, Nurulfiza Mat Isa

Malaysian Journal of Medicine and Health Sciences, 2020;16(4):226-233.
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Introduction: Multidrug resistance bacteria is alarming worldwide. A lot of research were done and are ongoing to search for the best, convenient and economically affordable ways to fight them. With the latest genome editing tool; Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology, this research was performed to develop a novel strategy to genetically modify the genome and inhibit the growth of Klebsiella pneumoniae (UPM ESBLKP1), an Extended Spectrum Beta Lactamases (ESBL) organism. Methods: A CRISPR-Cas9 vector was construct- ed together with guide RNAs designed specifically for the targeted uppP gene, a gene responsible for bacterial cell growth and protection. Results: The growth and cell wall integrity of the modified Klebsiella pneumoniae (UPM ESBLKP1) were significantly inhibited and reduced, respectively. Interestingly, wild type Klebsiella pneumoniae showed a normal growth curve while modified strains showed a faster doubling rate when supplemented with Luria-Bertani media. In contrast, slower growth rate of modified strain was observed in the M9 minimal media. This explained the higher doubling rate of mutants on nutrient rich medium earlier is being related to gene recovery. They grew slowly in the minimal media as they were adapting to a new environment while recovering the uppP gene and surviving, proving the success of its gene modification. Conclusion: The developed CRISPR-gRNA system was able to modify the targeted Klebsiella pneumoniae gene hence providing an opportunity to develop a new drug for Klebsiella pneumoniae infection as an alternative to antibiotics.
Fulltext New sites of localisation of Pasteurella multocida B:2 in buffalo surviving experimental haemorrhagic septicaemia

Annas S, Zamri-Saad M, Jesse FF, Zunita Z

BMC Vet Res, 2014;10:88.
PMID: 24721163 DOI: 10.1186/1746-6148-10-88

Haemorrhagic septicaemia (HS) is an acute septicaemic disease of buffalo and cattle caused by Pasteurella multocida B:2 and E:2. Field outbreaks of HS are known to result in localisation of bacteria in the tonsils of surviving buffalo, confirming that animals can become carriers and the role of respiratory tract in the transmission of the disease. This report describes additional sites of localisation of P. multocida B:2 in surviving buffalo following experimental induction of HS.
ESBL Producing E. coli in Chickens and Poultry Farms Environment in Selangor, Malaysia: A Cross-Sectional Study on Their Occurrence and Associated Risk Factors With Environment and Public Health Importance

Aliyu AB, Jalila A, Saleha AA, Zunita Z

Zoonoses Public Health, 2024 Dec;71(8):962-971.
PMID: 39289890 DOI: 10.1111/zph.13179

INTRODUCTION: Extended-spectrum β-lactamase-producing Escherichia coli (ESBL-EC) are widespread multidrug-resistant zoonotic bacteria that threaten animal production, food safety and antimicrobial therapy worldwide including Malaysia. Poultry has been reported as one of the pathways for human exposure to ESBL-EC. There has been little research on the occurrence of ESBL-EC within the Malaysian poultry food chain. Hence, the objectives of the study were to determine the occurrence of ESBL-EC in chickens and to identify the potential risk factors associated with their occurrence in poultry farms.
METHODS: A cross-sectional study was conducted on 400 samples, consisting of 240 cloacal swabs from chickens and 160 from poultry farms environments in eight districts in Selangor, Malaysia using culture and disk combination methods and multiple polymerase chain reaction assays. In the determination of possible factors associated with the presence of ESBL-EC at poultry farms, a questionnaire was used to obtain the information and data.
RESULTS: The findings demonstrated the wide distribution of ESBL-EC in all the farms with an overall occurrence of 37.2%. Farms in Gombak, Klang and Hulu Selangor had the highest occurrence rates at 62%, 50% and 50%, respectively, followed by farms in Petaling 38%, Sepang at 34%, Kuala Langat at 26% and Kuala Selangor at 24%, and the lowest was in Hulu Langat 14%. Among the study samples, chickens had the highest occurrence rate at 45.4%, followed by chicken house floors at 40% and flies at 30%, while feed and water samples at 17.5% and 12.5%, respectively. The present study indicated the high occurrence and wide dissemination of ESBL-EC in chickens and poultry farms environment. The ESBL-EC occurrence was associated with several factors including imprudent use of antibiotics, poor husbandry, management and biosecurity practices at the farms.
CONCLUSIONS: Our study showed the presence and spread of ESBL EC among chickens in the farms and their environment; this may lead to being spread to outside of farm environment by flies, vermins, flying birds, litter and farm wastes and possibly to humans upon contact with the contaminated environment and by poultry meat. Thus, the findings of the study can assist to serve as a piece of useful information to veterinary authority in designing evidence-based mitigation strategies for the control of ESBL-EC in poultry farms.
Staphylococcal cassette chromosome mec (SCCmec) and characterization of the attachment site (attB) of methicillin resistant Staphylococcus aureus (MRSA) and methicillin susceptible Staphylococcus aureus (MSSA) isolates

Bitrus AA, Zunita Z, Khairani-Bejo S, Othman S, Ahmad Nadzir NA

Microb Pathog, 2018 Oct;123:323-329.
PMID: 30053600 DOI: 10.1016/j.micpath.2018.07.033

This study was designed to screen for SCCmec types and to characterize the attachment site (attB) and universal insertion site (orfX) of SCCmec in a collection of 27 isolates (n = 11) methicillin resistant S. aureus and (n = 16) methicillin susceptible S. aureus isolates in Malaysia. Screening of SCCmec types and characterization of the attachment site was carried out using PCR amplification and Sanger's sequencing method. The result showed that a large proportion of the MRSA isolates carried SCCmec type III 7/11 (63%). Three isolates 3/11 (27%) and 1/11 (9.0%) carried SCCmec type II and IVd respectively. Amplification of the universal insertion site of the SCCmec (orfX) and attachment site (attB) showed that all 16 S. aureus isolates were positive for the orfX gene, while only 7 were positive for the attB gene. Phylogenetic diversity showed that the isolates clustered around strains with features similar to a community acquired MRSA. In conclusion, a high carriage rate of SCCmec type III was observed. The result also showed that all the S. aureus isolates have the orfX structure; however, not all isolates possesses the attB site on the 3' end of the orfX region.
Occurrence of Campylobacter species from broiler chickens and chicken meat in Malaysia

Sinulingga TS, Aziz SA, Bitrus AA, Zunita Z, Abu J

Trop Anim Health Prod, 2020 Jan;52(1):151-157.
PMID: 31273582 DOI: 10.1007/s11250-019-01995-y

Campylobacter is reported as a major cause of foodborne illness worldwide. Consumption of contaminated chicken meat is considered a significant risk factor of Campylobacter infection in humans. This study investigated the occurrence of non-Campylobacter jejuni-Campylobacter coli, in broiler chickens (n = 210) and chicken meat (n = 109). The samples were collected from seven broiler chicken farms (n = 210 cloacal swabs), 11 markets (n = 84 chicken meat), and 5 supermarkets (n = 25 chicken meat) located in different districts of Selangor State. Campylobacter were isolated from cloacal swabs using the Cape Town Protocol and from meat samples using the method of Duffy et al. (2007) with some modifications for Campylobacter isolations which were reported effective in the isolation of non-C. jejuni-C. coli Campylobacter species. The isolates were identified by Gram staining for cellular morphology, wet mount for motility and biochemical tests. Confirmation of presumed Campylobacter isolates was carried out using multiplex PCR (mPCR). One hundred seven (107/210) or 50.9% and twenty-nine (29/109) or 26.6% of chickens and chicken meat samples respectively were positive for Campylobacter species. Among the Campylobacter isolates from chickens, C. jejuni was the most predominantly isolated species (69.5%), followed by C. coli (16.2%). Campylobacter fetus and C. upsaliensis were the non-C. jejuni-C. coli Campylobacter species isolated in this study, at 9.3% and 2.5% respectively. Overall, the findings indicated broiler chickens were colonized not only by the common Campylobacter species but also by other Campylobacter species. We found the Cape Town Protocol useful to detect the occurrence of non-C. jejuni-C. coli isolates in chickens.
Fulltext In vitro transfer of methicillin resistance determinants mecA from methicillin resistant Staphylococcus aureus (MRSA) to methicillin susceptible Staphylococcus aureus (MSSA)

Bitrus AA, Zunita Z, Bejo SK, Othman S, Nadzir NA

BMC Microbiol, 2017 04 04;17(1):83.
PMID: 28376716 DOI: 10.1186/s12866-017-0994-6

BACKGROUND: Staphylococcus aureus more than any other human pathogen is a better model for the study of the adaptive evolution of bacterial resistance to antibiotics, as it has demonstrated a remarkable ability in its response to new antibiotics. This study was designed to investigate the in vitro transfer of mecA gene from methicillin resistant S. aureus to methicillin susceptible S. aureus.
RESULT: The recipient transconjugants were resistant to erythromycin, cefpodoxime and were mecA positive. PCR amplification of mecA after mix culture plating on Luria Bertani agar containing 100 μg/mL showed that 75% of the donor and 58.3% of the recipient transconjugants were mecA positive. Additionally, 61.5% of both the donor cells and recipient transconjugants were mecA positive, while 46.2% and 41.75% of both donor and recipient transconjugants were mecA positive on LB agar containing 50 μg/mL and 30 μg/mL respectively.
CONCLUSION: In this study, the direction of transfer of phenotypic resistance as well as mecA was observed to have occurred from the donor to the recipient strains. This study affirmed the importance of horizontal transfer events in the dissemination of antibiotics resistance among different strains of MRSA.