The study determined the prevalence and genetic profiles of ESBL-producing uropathogens among members of the Enterobacteriaceae family at Specialist Hospital Sokoto, Nigeria. A total of 64 Gram-negative uropathogens were isolated from 365 urine samples, with E. coli and Salmonella arizonae being most prevalent. The isolates showed high resistance to cotrimoxazole, nalidixic acid, ciprofloxacin and norfloxacin. 64.1% of isolates were multidrug resistant. ESBL production was detected in 23.4% of isolates. PCR analysis showed 73.3% of ESBL producers contained the blaCTX-M gene and 26.7
Candidemia in HIV-positive patients in Dschang District Hospital (West Region...Claude Nangwat
Candidemia has been identified as a public health problem in HIV-infected patients. The evaluation of CD4 count, transaminases and blood glucose, are being used as a means to monitor the health of HIV-infected patients, without excluding the diagnosis of candidemia and other opportunistic infections. In order to contribute in improving the care of HIV-infected patients attending Dschang District Hospital and later on, in other hospitals in Cameroon, we conducted from June to September 2014 a cross-sectional study, with general objective; to determine the association between candidemia and selected biochemical and haematological parameter changes in HIV-infected patients, as a possible indicator in monitoring HIV disease progression.
To do this, blood samples were collected from HIV-infected patients assigned to the UPEC of Dschang District Hospital for follow up, and haemogram report, CD4 counts, ALAT level, ASAT level, and glucose level in blood were evaluated by cytometric and spectrophotometric assays. Candida species were isolated from some blood samples, and then identified using CHROMagar Candida culture medium. The broth microdilution method was afterwards used to test the susceptibility of the fungal isolates vis-a-vis three conventional antifungal agents.
Mycological analysis of blood samples showed that eight (08) patients had candidemia, a prevalence of 6.11%. Eight (08) isolates were obtained from these eight (08) candidemic HIV-infected patients; this consisted of 4(50%) Candida albicans, 3(37.5%) Candida parapsilosis and 1(12.5%) Candida glabrata. All these isolates were resistant (MICs ranged from 2 to >256 µg/mL) to the antifungals used, that is, ketoconazole, amphotericin B and nystatin.
A significant correlation was found between candidemia and white blood cell count, with a correlation coefficient of r = 0.240 (p < 0.05). Based on the results obtained, the systematic diagnosis of candidemia should be performed in patients infected with HIV in Cameroon in order to improve on their care.
Key words: Candidemia, HIV, biochemical parameters, hematological parameters, Antifungals activities.
Candidemia in HIV-positive patients in Dschang District Hospital (West Region...Claude Nangwat
Candidemia has been identified as a public health problem in HIV-infected patients. The evaluation of CD4 count, transaminases and blood glucose, are being used as a means to monitor the health of HIV-infected patients, without excluding the diagnosis of candidemia and other opportunistic infections. In order to contribute in improving the care of HIV-infected patients attending Dschang District Hospital and later on, in other hospitals in Cameroon, we conducted from June to September 2014 a cross-sectional study, with general objective; to determine the association between candidemia and selected biochemical and haematological parameter changes in HIV-infected patients, as a possible indicator in monitoring HIV disease progression.
To do this, blood samples were collected from HIV-infected patients assigned to the UPEC of Dschang District Hospital for follow up, and haemogram report, CD4 counts, ALAT level, ASAT level, and glucose level in blood were evaluated by cytometric and spectrophotometric assays. Candida species were isolated from some blood samples, and then identified using CHROMagar Candida culture medium. The broth microdilution method was afterwards used to test the susceptibility of the fungal isolates vis-a-vis three conventional antifungal agents.
Mycological analysis of blood samples showed that eight (08) patients had candidemia, a prevalence of 6.11%. Eight (08) isolates were obtained from these eight (08) candidemic HIV-infected patients; this consisted of 4(50%) Candida albicans, 3(37.5%) Candida parapsilosis and 1(12.5%) Candida glabrata. All these isolates were resistant (MICs ranged from 2 to >256 µg/mL) to the antifungals used, that is, ketoconazole, amphotericin B and nystatin.
A significant correlation was found between candidemia and white blood cell count, with a correlation coefficient of r = 0.240 (p < 0.05). Based on the results obtained, the systematic diagnosis of candidemia should be performed in patients infected with HIV in Cameroon in order to improve on their care.
Key words: Candidemia, HIV, biochemical parameters, hematological parameters, Antifungals activities.
— We performed analysis of the biological properties of African swine fever virus (ASF) isolate Odintsovo 02/14. Domestic pigs were inoculated with 50 (low) or 5000 (high) hemadsorbing doses (HAD) of the virus via intranasal (IN) or intramuscular (IM) routes, to investigate the pathogenesis of ASF virus Odintsovo 02/14 isolate. Our results indicated that filtered 10% spleen suspension of ASFV isolate Odintsovo 02/14 induced an acute disease in pigs, resulting in 100% mortality rate. For cultural viral suspension (3rd passage), produced in a PBM cells mortality rate was 85.7%. We also present an analysis of the complete genome of African swine fever virus (ASF) Odintsovo 02/14 isolate. It is 189 333 nucleotide long and contains more than 160 open reading frames (ORFs). Complete nucleotide sequence of the genome of Odintsovo 02/14 isolate was obtained using pyrosequencing method and used to determine differences between the nucleotide sequences in the genomes of Odintsovo 02/14 and Georgia 01/2007. The genome of ASF virus Odintsovo 02/14 contains substitutions, insertions and deletions in genes encoding structural, membrane, and regulatory proteins, DNA reparation enzymes, host immune response evasion proteins, and MGF genes. The intergenic region I73R/I329L of Odintsovo 02/14 isolate contains 10-nucleotide long tandem repeat sequence, missing in Georgia 01/2007. Keywords— African swine fever (ASF), experimental challenge, complete genome sequencing, intergenic region, tandem repeats.
Single Cell Insights: Studying Environmental Microbial Communities Cell by CellQIAGEN
Dissecting complex microbial communities has incredible potential in the quest to decipher the world around us and find new sources of enzymes, antibiotics and other drugs. The challenge that remains is how to gain a deeper understanding of the roles and interactions of individual microbes. Single cell sequencing has emerged as an innovative investigational approach that provides a view of the cell-by-cell community by separating out individual microbes prior to sequencing. This issue of Single Cell Insights reviews peer-reviewed journal articles that describe applications and methodologies for single cell sequencing of environmental microbial communities.
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
All manuscripts are subject to rapid peer review. Those of high quality (not previously published and not under consideration for publication in another journal) will be published without delay.
Next generation sequencing for Identification and Characterization of plant v...Malyaj R Prajapati
Plant viruses have always been a challenge to plant growth and crop production in several parts of the world. Virus can be transmitted by vegetative propagation, fungi, nematodes, aphids, leaf hoppers, plant hoppers, beetles, white flies, and so forth. Viruses symptoms vary with the infecting virus and the infected part which includes leaf spots, leaf blights, root rots, fruit rots, fruit spots, wilt, dieback and decline. It is causing economic losses by reducing crop quality, quantity and nutritional value. Thus, their reliable detection is of a crucial importance for plant protection. While the adoption of molecular techniques such as RT-PCR has increased the speed and accuracy of virus diagnostics, such techniques only allow the detection of known viruses, i.e., each test is specific to one or a small number of related viruses. Therefore, unknown virus can be missed and testing can be slow and expensive if molecular tests are unavailable. NGS technology is one of the most popular tools for virus diagnostics. It is highly efficient, rapid diagnostics tools, and low-cost high-throughput and deep RNA sequencing. Due to the capacity to target multiple unique signature loci of virus in an infected plant metagenome and also useful for discovery of new virus and new hosts. It is including virus genome sequencing, discovery and detection, ecology and epidemiology, replication and transcription. By using deep RNA-seq requires fast and robust bioinformatics methods to enable host sequence removal and virus classification. Future developments in this area, including the use of bioinformatics tools for identification and characterization of multiple plant virus and analysis of diversity of plant viruses.
Resistance to antimicrobials by pathogenic microorganisms has raised serious global clinical concerns in recent times. The present study aimed at detection of β-lactam resistance genes in S. aureus isolates from women with symptomatic and asymptomatic cases of urinary tract infections in Nasarawa state, Nigeria. A total of 200 non-repetitive midstream urinal samples were analysed and 50 (29%) bacterial isolates were identified as S. aureus. The susceptibility profile of the bacterial isolates to tested antibiotics was Nitrofurantoin (74.1%), Gentamicin (72.4%), Ciprofloxacin (65.5%), Ofloxacin (56.9), Augmentin (36.2%), Cotrimozazole (29.3%), Ampicillin (27.6%), Erythromycin (25.8%), Ceftazidine (20.7%) and Cefurozime (10.3%). Thirteen bacterial isolates were found to be resistant to all β-lactam antibiotics tested, out of which 7 were confirmed β-lactamase producers using the acidometric and iodometric methods. The detection of β-lactamase genes (blaZ, blaI and blaR1) was carried out and only five of the isolates were found to be expressing the blaI genes. This research finding suggests that β-lactam resistance by S. aureus may not be dependent only on the blaZ, blaI and blaR1 genes.
Comparative Study of the Prevalence and Antibiogram of Bacterial Isolates fro...iosrjce
The study compared the prevalence and antibiogram of bacterial isolates from the urinary and
genital tracts of pregnant women attending ante-natal clinics in Imo State. Urine and High vaginal swab (HVS)
samples were collected from across the three geopolitical zones of Imo State (Owerri, Orlu and Okigwe).
Federal Medical Centre (FMC) Owerri, Imo State University Teaching Hospital (IMSUTH) Orlu and General
Hospital Okigwe (GHO) were used as focal points. A total of 1197 samples were obtained from women and
used. Infection was significantly more with the urine samples than the HVS samples (P < 0.05) while
polymicrobial growth was more observed with the HVS samples. Escherichia coli was the predominantly
isolated organism (38.3%) from the urine samples while Staphylococcus aureus (29.1%) was the predominant
bacterial isolates in HVS. Other commonly isolated bacterial species include; Enterococcus faecalis and
Staphylococcus epidermidis, Klebsiella pneumoniae, Proteus mirabilis and Bacteriodes were solely isolated
from urine while Lactobacillus was solely isolated from HVS. Overall antibiogram showed ciprofloxacin to be
the most effective antibiotic followed by nalidixic acid and pefloxac in for both specimens. Generally, multidrug
resistance was more in urine isolates (55.7%) than vaginal isolates (53.6%) with many showing the same
resistance patterns. The rate of multi/drug resistance in both samples is high (>50%) and worrisome. These call
for routine HVS as well as urine culture to be carried out on all antenatal women to ensure holistic antenatal care/ management.
— We performed analysis of the biological properties of African swine fever virus (ASF) isolate Odintsovo 02/14. Domestic pigs were inoculated with 50 (low) or 5000 (high) hemadsorbing doses (HAD) of the virus via intranasal (IN) or intramuscular (IM) routes, to investigate the pathogenesis of ASF virus Odintsovo 02/14 isolate. Our results indicated that filtered 10% spleen suspension of ASFV isolate Odintsovo 02/14 induced an acute disease in pigs, resulting in 100% mortality rate. For cultural viral suspension (3rd passage), produced in a PBM cells mortality rate was 85.7%. We also present an analysis of the complete genome of African swine fever virus (ASF) Odintsovo 02/14 isolate. It is 189 333 nucleotide long and contains more than 160 open reading frames (ORFs). Complete nucleotide sequence of the genome of Odintsovo 02/14 isolate was obtained using pyrosequencing method and used to determine differences between the nucleotide sequences in the genomes of Odintsovo 02/14 and Georgia 01/2007. The genome of ASF virus Odintsovo 02/14 contains substitutions, insertions and deletions in genes encoding structural, membrane, and regulatory proteins, DNA reparation enzymes, host immune response evasion proteins, and MGF genes. The intergenic region I73R/I329L of Odintsovo 02/14 isolate contains 10-nucleotide long tandem repeat sequence, missing in Georgia 01/2007. Keywords— African swine fever (ASF), experimental challenge, complete genome sequencing, intergenic region, tandem repeats.
Single Cell Insights: Studying Environmental Microbial Communities Cell by CellQIAGEN
Dissecting complex microbial communities has incredible potential in the quest to decipher the world around us and find new sources of enzymes, antibiotics and other drugs. The challenge that remains is how to gain a deeper understanding of the roles and interactions of individual microbes. Single cell sequencing has emerged as an innovative investigational approach that provides a view of the cell-by-cell community by separating out individual microbes prior to sequencing. This issue of Single Cell Insights reviews peer-reviewed journal articles that describe applications and methodologies for single cell sequencing of environmental microbial communities.
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
All manuscripts are subject to rapid peer review. Those of high quality (not previously published and not under consideration for publication in another journal) will be published without delay.
Next generation sequencing for Identification and Characterization of plant v...Malyaj R Prajapati
Plant viruses have always been a challenge to plant growth and crop production in several parts of the world. Virus can be transmitted by vegetative propagation, fungi, nematodes, aphids, leaf hoppers, plant hoppers, beetles, white flies, and so forth. Viruses symptoms vary with the infecting virus and the infected part which includes leaf spots, leaf blights, root rots, fruit rots, fruit spots, wilt, dieback and decline. It is causing economic losses by reducing crop quality, quantity and nutritional value. Thus, their reliable detection is of a crucial importance for plant protection. While the adoption of molecular techniques such as RT-PCR has increased the speed and accuracy of virus diagnostics, such techniques only allow the detection of known viruses, i.e., each test is specific to one or a small number of related viruses. Therefore, unknown virus can be missed and testing can be slow and expensive if molecular tests are unavailable. NGS technology is one of the most popular tools for virus diagnostics. It is highly efficient, rapid diagnostics tools, and low-cost high-throughput and deep RNA sequencing. Due to the capacity to target multiple unique signature loci of virus in an infected plant metagenome and also useful for discovery of new virus and new hosts. It is including virus genome sequencing, discovery and detection, ecology and epidemiology, replication and transcription. By using deep RNA-seq requires fast and robust bioinformatics methods to enable host sequence removal and virus classification. Future developments in this area, including the use of bioinformatics tools for identification and characterization of multiple plant virus and analysis of diversity of plant viruses.
Resistance to antimicrobials by pathogenic microorganisms has raised serious global clinical concerns in recent times. The present study aimed at detection of β-lactam resistance genes in S. aureus isolates from women with symptomatic and asymptomatic cases of urinary tract infections in Nasarawa state, Nigeria. A total of 200 non-repetitive midstream urinal samples were analysed and 50 (29%) bacterial isolates were identified as S. aureus. The susceptibility profile of the bacterial isolates to tested antibiotics was Nitrofurantoin (74.1%), Gentamicin (72.4%), Ciprofloxacin (65.5%), Ofloxacin (56.9), Augmentin (36.2%), Cotrimozazole (29.3%), Ampicillin (27.6%), Erythromycin (25.8%), Ceftazidine (20.7%) and Cefurozime (10.3%). Thirteen bacterial isolates were found to be resistant to all β-lactam antibiotics tested, out of which 7 were confirmed β-lactamase producers using the acidometric and iodometric methods. The detection of β-lactamase genes (blaZ, blaI and blaR1) was carried out and only five of the isolates were found to be expressing the blaI genes. This research finding suggests that β-lactam resistance by S. aureus may not be dependent only on the blaZ, blaI and blaR1 genes.
Comparative Study of the Prevalence and Antibiogram of Bacterial Isolates fro...iosrjce
The study compared the prevalence and antibiogram of bacterial isolates from the urinary and
genital tracts of pregnant women attending ante-natal clinics in Imo State. Urine and High vaginal swab (HVS)
samples were collected from across the three geopolitical zones of Imo State (Owerri, Orlu and Okigwe).
Federal Medical Centre (FMC) Owerri, Imo State University Teaching Hospital (IMSUTH) Orlu and General
Hospital Okigwe (GHO) were used as focal points. A total of 1197 samples were obtained from women and
used. Infection was significantly more with the urine samples than the HVS samples (P < 0.05) while
polymicrobial growth was more observed with the HVS samples. Escherichia coli was the predominantly
isolated organism (38.3%) from the urine samples while Staphylococcus aureus (29.1%) was the predominant
bacterial isolates in HVS. Other commonly isolated bacterial species include; Enterococcus faecalis and
Staphylococcus epidermidis, Klebsiella pneumoniae, Proteus mirabilis and Bacteriodes were solely isolated
from urine while Lactobacillus was solely isolated from HVS. Overall antibiogram showed ciprofloxacin to be
the most effective antibiotic followed by nalidixic acid and pefloxac in for both specimens. Generally, multidrug
resistance was more in urine isolates (55.7%) than vaginal isolates (53.6%) with many showing the same
resistance patterns. The rate of multi/drug resistance in both samples is high (>50%) and worrisome. These call
for routine HVS as well as urine culture to be carried out on all antenatal women to ensure holistic antenatal care/ management.
Methicillin-resistant Staphylococcus aureus (MRSA) infections have been recognized for decades as hospital acquired MRSA (HA-MRSA). Nowadays, MRSA is also recognized as a worldwide emerging community-associated pathogen. Community associated- MRSA (CA-MRSA) has been shown to be more virulent with a high degree of severity of disease when compared to HA-MRSA.
SALMONELLA ARIZOANE: AN UNCOMMON UROPATHOGEN?Nuhu Tanko
Salmonella arizonae is usually an uncommon uropathogen from many studies. But from this study, it was the second most prevalent uropathogen after E.coli.
The prevalence of extended spectrum beta-lactamases (ESBLs) among Escherichia...Open Access Research Paper
The prevalence of extended spectrum β-lactamases among 246 clinical isolates from Abia State University teaching Hospital patients was investigated. The isolates were made up of 134 Escherichia coli and 112 Klebsiella species. Antimicrobial susceptibility of the isolates was determined by the disc diffusion method. ESBL phenotypes were determined by the double disc synergy method using ceftazidime, cefotaxime, ceftriaxone and co-amoxiclav. Out of the 246 isolates, 125 (50.8%) were ESBL producers, made up of 62(50.8%) E. coli and 63 (50.4%) Klebsiella isolates. Seventeen (54.8%) of the ESBL producing E. coli isolates were from in-patients while 45 (47.9%) were from out-patients. For the ESBL positive Klebsiella spp., 14 (45.2%) and 49 (52.1%) were from in-patients and out-patients respectively. ESBL producing isolates were also found to be more prevalent among the female patients (72.8%) than among the male patients (27.2%). The isolates also expressed high rates of resistance to other classes of antibiotics tested. However, Amikacin was found to have excellent performance against the urinary isolates tested and therefore is recommended for the treatment of infections caused by Escherichia coli and Klebsiella species. This study shows high prevalence of ESBL producing E. coli and Klebsiella isolates clinical samples of patients attending the Abia State University Teaching Hospital Aba, Abia State Nigeria.
Background: The widespread use of antibiotics has resulted in emergence of community-acquired antibiotic resistance among uropathogens in outpatient’s population. This constitutes an impediment in the management of urinary tract infection (UTI) in both community and hospital settings. Objective: The aim of this study was to determine the current antibiotic resistance trends, extended spectrum beta-lactamase (ESBL) production and plasmid profile of uropathogens from outpatients. Methods: A total of 370 mid-stream urine samples were collected and cultured by standard methods. Isolated uropathogens were identified using appropriate biochemical methods. The modified Kirby Bauer disk method was used for antibiotic susceptibility test. The ESBL-producing uropathogens were identified and their plasmid DNA extraction and curing were carried out by standard methods. Results: About 35.7% and 32.7% of uropathogens were multi-drug resistant and ESBL-producing respectively. There was higher prevalence of ESBL-production among isolates from female patients (62.5%) when compared to that from male patients (37.5%). The isolated uropathogens were most resistant to Cefotaxime, and most sensitive to Imipenem. Resistance to antibiotics by ESBL-producing uropathogens was found to be plasmid-mediated. Conclusion: Community acquired Uropathogens from outpatients were multidrug resistant due to ESBL production localized on plasmids, a probable cause of treatment failures experienced in Uyo.
A study of antibiotic resistance of Extended-Spectrum Beta-Lactamases produci...Premier Publishers
Background: Extended-Spectrum Beta-Lactamases - producing Enterobacteriaceae are common in hospitals. This study aims to describe the antibiotic resistance of these bacteria and their associated demographic and clinical factors. Methods: It was a prospective study of 73 isolates of Extended-Spectrum Beta-Lactamases - producing Enterobacteriaceae for a period of six months from July to December 2019 in the laboratory of Befelatanana. Results: This study showed 73 (6.3%) isolates of Extended-Spectrum Beta-Lactamases- producing Enterobacteriaceae, represented by 25 (34.2%) isolates of Klebsiella spp, 24 (32.9%) isolates of Escherichia coli, 22 (30.1%) isolates of Enterobacter spp and 2 (2.7%) isolates of Proteus spp. The antibiotic resistance of these bacteria varied from 0% to 100% for all of the antibiotics tested. Resistance to aminoglycosides ranged from 0% (amikacin) to 69.9% (gentamycin). Resistance to quinolones ranged from 43.8% (levofloxacin) to 76.7% (nalidixic acid). Similarly, 60 (82.2%) isolates were resistant to cotrimoxazole and 25 (34.2%) isolates to chloramphenicol. Patients under 20 years (57.1%) (p=0.03), men (52.2%)(p=0.11; NS), patients with respiratory samples (83.3%)(p=0.004), with pus (61.9%)(p=0.02) and hospitalized in surgery and intensive care units (68.4%)(p=0.0009) were the most affected by these enterobacteria. Conclusion: Extended-Spectrum Beta-Lactamases - producing Enterobacteriaceae are responsible for severe infections and the majorities are multi-resistant bacteria.
Keywords: Beta-lactamase, Enterobacteriaceae, antibiotic resistance, amikacin, imipenem.
Emergence of ESBL worldwide has become a threat to successful treatment of noocomial infections. This deals with detection and treatment of ESBL infetions.
A total number of 74 coagulase negative Staphylococci were isolated from orthopaedic patients in Ahmadu Bello University Teaching Hospital, Zaria, Nigeria. They were further characterized into various Staphylococci species using API STAPH identification kit: Staph xylosus (31.1%), Staph lentus (10.8%), Staph hominis (10.8%), Staph cohnii cohnii (5.4%), Staph epidermidis (4.1%) others were Staph cohnii ureal., Staph hyicus, Staph lugdunensis (2.7% each) Staph caprae , Staph capitis, Staph haemolyticus, Staph scuiri, Staph chromogenes and Staph warneri (1.4% each). Microcossus spp was 8.2% while 13.5% isolates were undetermined. Kirby Baurer disk method was used for the antibiotics susceptibility test, the result showed gentamicin and ciprofloxacin to be most active (96.6%), followed by vancomycin (93.1) and pefloxacin (87.9). The isolates were resistant to ampicillin (96.6), amoxicillin clavulanic acid (65.5%), clindamycin 41.4%). The aim of this study is to classify the coagulase negative Staphylococci isolates into species and to determine their antibiotic susceptibility
Nuhu et al_Poster NAPA2016 correction and observation
1. MOLECULAR CHARACTERIZATION OF EXTENDED SPECTRUM
BETA-LACTAMASE-PRODUCING GRAM-NEGATIVE
UROPATHOGENS AT SPECIALIST HOSPITAL SOKOTO, NIGERIA
*T. Nuhu1, R.O. Bolaji2, H.Y. Ungo1, A. Olowookere1 and B.O. Olayinka2
1Department of Pharmaceutics and Pharmaceutical Microbiology, Usmanu Danfodiyo University, Sokoto. (nuhu.tanko@yahoo.com)
2Department of Pharmaceutics and Pharmaceutical Microbiology, Ahmadu Bello University, Zaria. (busayoolayinka@yahoo.com)
Background: Extended Spectrum Beta-Lactamase (ESBL)-producing Enterobacteriaceae are fast becoming the leading cause of antibiotic resistant infection.
Objective of the study: The study determined the prevalence and genetic profiles of ESBL-producing uropathogens among members of the family Enterobacteriaceae at
Specialist Hospital Sokoto.
Methodology: A total of 365 mid-stream urine samples collected over a period of 4 months were studied. Isolates were identified using Microgen Identification Kit, GN-
ID. Susceptibility testing was performed using the modified Kirby Bauer method, and results were interpreted according to Clinical Laboratory Standard Institute (CLSI),
2012. ESBL production was detected by Double Disc Synergy Test. Molecular characterization of the ESBL positive isolates were confirmed by PCR method.
Results: A total of 64 Gram negative uropathogens were isolated from 365 urine samples. The isolates were made up of E. coli (29.7%), Salmonella arizonae (23.4%),
Klebsiella oxytoca (10.9%), Klebsiella pnuemoniae (4.7%), Enterobacter gergoviae (9.4%), Enterobacter aerogenes (1.6%), Citrobacter freundii (6.3%), Serratia
marscense (6.3%), Proteus mirabilis (1.6%), Edwardsiella tarda (1.6%), Acinetobacter iwoffii (1.6%), Burkholderia pseudomallei (1.6%), and Pseudomonas aeruginosa
(1.6%). The antibiotic susceptibility profile of the isolates to seven (7) commonly prescribed antibiotics used in the treatment of UTI - associated infections showed that
the isolates were resistant to cotrimoxazole (71.9%), nalidixic acid (67.2%), ciprofloxacin (54.7%), norfloxacin (53.1%), gentamicin (50%), amoxicillin/clavulanic acid
(48.4%) and nitrofurantoin (29.7%). Majority of the isolates were multidrug resistant (64.1%), 15.6% were extended drug resistant (XDR), 4.7% were pandrug resistant
(PDR), while 15.6% do not fall into any of the classification. The PCR genomic DNA of the 15 MDR-ESBL-producing isolates showed that 73.3% were CTX-M, while 26.7%
were OXA.
Conclusion: The prevalence of ESBL in this study was 23.4%. Molecular characterization of the selected MDR-ESBL - producing isolates showed that they harboured the
blaCTX-M gene (73.3%) and blaOXA gene (26.7%).
Keywords: Molecular characterization, ESBL, Uropathogens, Sokoto
INTRODUCTION
Urinary Tract Infections (UTIs) has become the most common hospital-acquired infection, accounting for as many as 35% of nosocomial infections, and it is the second most
common cause of bacteremia in hospitalized patients . It is one of the most common bacterial diseases worldwide that is characterized by a wide range of symptoms from mild
irritative voiding to bacteremia, sepsis, or even death . Hawkey (2008), stated that “since the introduction of antibiotics in the 1950s, there has been a steady and significant
increase in the number of resistant strains of bacteria”. The most important draw-back in the treatment of UTIs is the development of antimicrobial resistance. The improper and
uncontrolled use of many antibiotics resulted in the occurrence of antimicrobial resistance, which became a major health problem worldwide.
Antimicrobial resistance among pathogenic bacteria is increasing worldwide especially against beta-lactam drugs, due to the development of beta-lactamase enzymes (NNIS
System, 2003) which destroy the beta-lactam ring of these antibiotics, thus preventing the action of penicillin binding proteins (PBPs) (Livermore, 1995). In the battle against beta-
lactamase mediated bacterial resistance, development of third generation cephalosporins in the early 1980s was a major breakthrough. But soon, a new plasmid encoded beta-
lactamase capable of hydrolyzing the extended spectrum cephalosporins was reported (Knothe et al., 1983; Jacoby and Medeiros, 1991).
Beta-lactamase producing Enterobacteriaceae has become a global problem . The incidence and type of beta-lactamase enzyme varies in different geographical locations. The
ability of bacteria to produce extended-spectrum beta-lactamases (ESBLs) that hydrolyze penicillins, cephalosporins, and monobactams has resulted in intractable infections and
serious consequences for infection control (Marchandin et al., 1999; Philippon et al., 1989).
A commonly used working definition is that, ESBLs are beta-lactamases capable of conferring bacterial resistance to the penicillins; first, second and third generation
cephalosporins; and aztreonam (but not the cephamycins or carbapenems) by hydrolysis of these antibiotics, and which are inhibited by beta-lactamase inhibitors such as
clavulanic acid (Paterson and Bonomo, 2005).
ESBL producing bacteria especially E. coli and K. pneumoniae have emerged as serious pathogens both in hospital and community acquired UTIs (Venkatadri et al., 2014). The
study sought to determine the prevalence of ESBL-producing uropathogens and characterize the ESBL genes.
MATERIALS AND METHODS
Sample Collection:Early morning, mid-stream clean catch urine samples were collected by patients in a sterile disposable containers with screw caps. Prior to urine collection,
patients were counseled on how to collect urine sample by observing all aseptic conditions to avoid contamination.
Isolation and Culturing of Urine Samples:Urine samples were inoculated on CLED Agar using calibrated wire loop. Samples with counts up to and greater than 105cfu/ml were
counted microscopically and considered positive for further analysis.
Antibiotic Susceptibility Testing (AST):The antibiotic susceptibility of the isolates was determined against seven (7) commonly prescribed antibiotics in Specialist Hospital Sokoto
using the modified Kirby Bauer disc agar diffusion (CLSI, 2012).
Screening and Phenotypic Confirmatory Test for ESBL Production: Gram-negative isolates were screened for ESBL production. The double disc synergy test (DDST) was
adopted to confirm for the presence of ESBLs.
Molecular Characterization of Resistant Isolates: Genomic DNA extraction was carried out using Zymo Research Quick-gDNATM MiniPrep. To ascertain that genomic DNA was
actually extracted, the eluent was subjected to agarose gel electrophoresis. Amplification of resistant DNA fragments was carried out using Dream TaqTM DNA polymerase, which is
an enhanced Taq DNA Polymerase optimized for high throughput PCR applications. A 2% agarose gel was used to resolve the PCR genomic DNA fragments with their primers .
CONCLUSIONS
E. coli and Salmonella arizonae were the most prevalent Gram-negative isolates. Gram-negative uropathogens
were generally resistant to Cotrimoxazole (73.3%), Nalidixic acid (66.7%), Norfloxacin (53.5%), and
Ciprofloxacin (50.5%). Multidrug resistant isolates were 64.1%, extended drug resistant (15.6%), pandrug
resistant (4.7%) and 15.6% did not fall into the category of classification. All the ESBL producers were MDR,
and their MARI range from 0.3 to 1.0.ESBL production was 23.4% among the Gram-negative isolates. The
incidence of bla CTX-M and bla OXA gene were reported with 26.7% producing (bla CTX-M and bla OXA), 73.3%
produced bla CTX-M only, and 26.7% produced bla OXA only.
29.7
23.410.9
4.7
9.4
1.6
6.3
6.3
1.6
1.6
1.6 1.6
1.6
E. coli
Sal arizonae
Kleb oxytoca
Kleb pneumoniae
E. gergoviae
E. aerogenes
C.freundii
S. marscence
P. mirabilis
E. tarda
A. iwoffii
B. pseudomallei
Ps aeruginosa
Fig. 2: Percentage distribution of Gram-negative isolates from urine.
0
10
20
30
40
50
60
70
80
PercentageSusceptibility
Antibiotics
Sensitive
Resistant
Fig.2: Percentage susceptibility of Gram-negative uropathogens
Fig. 3:Genomic DNA PCR analysis of CTX-M gene in
ESBL isolates
Fig.4: Genomic DNA PCR analysis of OXA gene in ESBL isolates
14th NATIONAL CONFERENCE AND SCIENTIFIC MEETING, NIGERIA ASSOCIATION OF PHARMACISTS IN ACADEMIA. FACULTY OF PHARMACY, OLABISI ONABANJO UNIVERSITY , SAGAMU, OGUN STATE, NIGERIA. 11TH – 15TH JULY, 2016
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