Lactamase Genes Research Articles

Predominance of multi-drug resistant extended spectrum β lactamase producing bacteria from marine fishes

The present study aimed to determine the extended spectrum beta lactamase (ESBL) genes in the bacteria from fresh exportable fish samples collected along South east coast of India, Chennai. ESBL genes are the base for the antibiotic resistance in pathogens and it transmitted from one to other species. Totally 2670 isolates were isolated from 293 fish samples which belong to 31 species dominated by Aeromonas, Klebsiella, Serratia, Leclerica, Proteus, Enterobacter, Acinetobacter, Haemophilus, Escherichia, Shigella sp. Out of 2670 isolates, 1958 isolates have multi drug resistant capacity with ESBL genes of blaCTX, blaSHV, blaTEM and blaAmpC and 712 isolates are not detected ESBL genes. The present study revealed that, the contamination of fresh fish sample with pathogenic bacteria resistant to multiple antibiotics can incriminate seafood as a potential carrier and accentuate an immediate need to prevent environmental infectivity and distribution. Further, hygienic facilitated markets should be developed with ensured quality of seafood.

Bacterial genome sequencing tracks the housefly-associated dispersal of fluoroquinolone- and cephalosporin-resistant Escherichia coli from a pig farm.

The regular use of antimicrobials in livestock production selects for antimicrobial resistance. The potential impact of this practice on human health needs to be studied in more detail, including the role of the environment for the persistence and transmission of antimicrobial-resistant bacteria. During an investigation of a pig farm and its surroundings in Brandenburg, Germany, we detected abundant cephalosporin- and fluoroquinolone-resistant Escherichia coli in pig faeces, sedimented dust, and house flies (Musca domestica). Genome sequencing of E. coli isolates revealed large phylogenetic diversity and plasmid-borne extended-spectrum beta lactamase (ESBL) genes CTX-M-1 in multiple strains. [Correction added on 28 February 2023, after first online publication: In the preceding sentence, 'and TEM-1' was previously included but has been deleted in this version.] Close genomic relationships indicated frequent transmission of antimicrobial-resistant E. coli between pigs from different herds and across buildings of the farm and suggested dust and flies as vectors for dissemination of faecal pathogens. Strikingly, we repeatedly recovered E. coli from flies collected up to 2km away from the source, whose genome sequences were identical or closely related to those from pig faeces isolates, indicating the fly-associated transport of diverse ESBL-producing E. coli from the pig farm into urban habitation areas. The observed proximity of contaminated flies to human households poses a risk of transmission of antimicrobial-resistant enteric pathogens from livestock to man.

English

Fresh fruits are popular sources of healthy diets with low energy density. Since they are consumed raw, it may act as a source of foodborne disease and a reservoir for antibiotic-resistant organisms. This study aimed to determine microbial prevalence among the fruits sold around hospital along with antimicrobial profiles. Thirty-five different types of fruits were bought from around Dhaka Medical College and Hospital (DMCH) and analyzed for the presence of bacteria. Antibiotic sensitivity, detection of ESBL, AmpC β-lactamase, and MBL positive strains were done by standard methods followed by PCR to detect ESBL, AmpC β-lactamase and MBL genes. Twenty-seven different organisms were isolated: Klebsiella spp. (33.33%), Citrobacter spp. (29.64%), Enterobacter spp. (22.22%), Escherichia coli (11.11%) and Staphylococcus aureus (3.70%). Among them, 48.15% were resistant to different antibiotics. Only one organism (Citrobacter spp.) produced ESBL phenotypically (7.69%). Two (15.38%) were positive for AmpC β-lactamase and one of these (Enterobacter spp.) possessed SHV and CTX-M15A genes by PCR. Imipenem resistance was 84.62% of the antibiotic-resistant organisms and 10 (90.91%) were phenotypically MBL positive. By PCR, one Enterobacter spp. had MBL encoding gene OXA-48.  Fresh fruits, contaminated with pathogens, might be a source of resistant organisms' transmission and contribute to public health issues.   Key words: Antibiogram, bacteria, Bangladesh, fresh fruits, fruit venders around hospital.

Antimicrobial susceptibility pattern of extended-spectrum beta-lactamase-producing uropathogens in aminu Kano teaching hospital, Northwestern Nigeria.

UTI is the most common nosocomial infection among hospitalized patients. Emerging increasing resistance has been observed among uropathogens of the family enterobacterales. Extended spectrum beta lactamase genes encode for multidrug resistance and unrestricted use of antibiotics in hospitals provides an environment for spread of infections with limited treatment options. This necessitates therapies based on culture and antimicrobial sensitivity to improve patients' outcomes We aimed to determine susceptibility pattern of ESBL uropathogens among hospitalized patients in Aminu Kano Teaching Hospital, Kano. Three hundred and eighty-nine urine samples were obtained from in-patients with UTI between April 2020 and April 2021. Five samples were rejected and remaining analyzed. Susceptibility testing was done by modified Kirby Bauer technique. Clinical Laboratory Standards Institute guidelines 2019 (CLSI 2019) and the European Committee on Antimicrobial Susceptibility testing guidelines version 2 (EUCAST version 2) were used for screening and confirmation of ESBL production respectively. Of the 384-urine processed, 105 (27.3%) were gram negatives and 81 were Enterobacterales, Isolation rates were E. coli-55.6%, K. pneumoniae-29.6%, Citrobacter spp.-12.3%, P. mirabilis-1.2% and Morganella spp.-1.2%. Among Enterobacterales, 32 (39.5%) were ESBL producers. Prevalence of ESBL were 62.5% for Escherichia coli, 28.1% for Klebsiella pneumoniae and 9.4% for Citrobacter species. Susceptibility showed that ESBL-producing Isolates were highly susceptible to amikacin (96.9%). Resistance to other antibiotics varied from 3.1% to 100%. We recommend strengthening laboratory capacity, antibiotics stewardship and Infection control to prevent spread of resistant pathogens including ESBLs.

Klebsiella infections in a pediatric intensive care unit: incidence, antimicrobial susceptibility, and resistance genes

BackgroundInfections with multidrug-resistant K. pneumoniae is associated with high morbidity and mortality especially among critically ill patients. This was the main principle to conduct a detailed study about this organism, its resistance pattern, and type of its resistance genesSubjects and methodsA cross-sectional study was carried out in a pediatric intensive care unit on patients with age range from 1 month to 12 years over a period of 1 year with positive K. pneumoniae using standard microbiological culture and antibiogram sensitivity testing. All collected samples were processed using multiplex PCR technique to identify the most relevant resistant genes.ResultsForty-four patients had 54 positive cultures for K. pneumoniae, out of which 17 patients (38.6%) passed away. The most prevalent-resistant gene was New Delhi metallo-beta lactamase (NDM) gene (65.4%) followed by cefotaximase (CTX-M) gene (57.7%). Extensively drug-resistant K. pneumoniae was detected in (15.9%) of the results and was proved to be independent risk factor increasing mortality odds 139 folds.ConclusionThe evolution of resistance of Klebsiella pneumoniae was proved to be associated with a high mortality rate. Continuous widespread surveillance of Klebsiella pathogen focusing on identification of resistance genes and antibiotic resistance pattern is highly recommended.

Analysis of Extended Spectrum Beta Lactamase (ESBL) Genes of Non-Invasive ESBL Enterobacterales in Southeast Austria in 2017.

Extended spectrum beta lactamases producing Enterobacteriaceae are a major player in the antibiotic resistance challenge. In general, the situation regarding antibiotic resistance in Austria is very good compared to many other countries. Perhaps this is why there is a lack of data on the distribution of ESBL genes in the clinical setting. The aim of this study was to collect data on ESBL genes from a larger sample of human non-invasive clinical isolates from one region in Austria. In total, 468 isolates from different sample materials isolated at the Medical University of Graz from 2017 were examined. The most frequent organisms were Escherichia coli and Klebsiella pneumoniae. Among the enzymes produced, CTX-M-15 was clearly dominant, exotic ESBLs were only represented by three Proteus mirabilis isolates harboring genes for VEB-6 and one P. mirabilis for CTX-M-2, respectively. Compared to other countries, the results are in line with the expectations. The data help to better classify the many studies from the non-clinical field in Austria and to shift the focus slightly away from the exotic results and sample sites.

Genetic Relationship between Salmonella Isolates Recovered from Calves and Broilers Chickens in Kafr El-Sheikh City Using ERIC PCR.

A prevalent bacterial intestinal infection with severe economic damage is salmonellosis. Our study was carried out to diagnose Salmonella from chickens and calves, to determine its resistance to antimicrobials' phenotypic and genotypic characterization of integrons and β lactamase genes in the multidrug resistance of different Salmonella serotypes, and to detect the genetic relationship between Salmonella isolates collected from different origins using an ERIC PCR. In total, 200 samples from diseased chicken and diarrheic calves were obtained from 50 various farms from Kafr El-sheikh, Egypt. Salmonella poultry isolates were characterized as S. Typhimurium (3/8), S. Enteritidis (3/8), and S. Kentucky (2/8), but Salmonella isolates from cattle were S. Enteritidis (1/2) and S. Kentucky (1/2). When antibiotic susceptibility testing was completed on all of the isolates, it showed that there was multidrug resistance present (MDR). A PCR was applied for identifying the accompanying class 1 integrons and ESBLs from MDR Salmonella isolates (two isolates of S. Kentucky were divided as one from calf and one from poultry). Our results detected blaTEM and class 1 integron, but were negative for bla IMP, bla VIM, and bla SHV. An ERIC PCR was conducted for understanding the clonal relation between various β-lactamase-producing MDR Salmonella isolates. The same four previously mentioned isolates were also tested. The two isolates of S. Enteritidis isolated from poultry and calves had 100% similarity despite indicating that there were interactions between broilers and calves living on the same farm that caused infection from the same Salmonella strains, while the other two isolates of S. Kentucky showed only 33% serovarities.

High prevalence of resistance to third-generation cephalosporins detected among clinical isolates from sentinel healthcare facilities in Lagos, Nigeria

BackgroundAntimicrobial resistance (AMR) in bacterial pathogens is a worldwide concern that demands immediate attention. Most information on AMR originates from high-income countries and little is known about the burden in Africa, particularly Nigeria. Using four sentinel sites (General hospitals) in Lagos State, this study sought to estimate the burden of AMR.MethodsThis is a hospital-based surveillance using secondary health care centres. Four sites were randomly selected and included in the study. Clinical isolates were collected over a period of 6 months for each site from August 2020 to March 2021. All isolates were characterised and analysed for resistance to 15 antibiotics using the Kirby-Baur method. Multiplex PCR assay was used for the detection of Extended spectrum beta lactamase genes. Data analysis was done using SPSS version 27.0.ResultsFour hundred and ninety-nine (499) patients consented and participated in this study, consisting of 412 (82.6%) females and 87 (17.4%) males. The mean age ± SD of the participants was 33.9 ± 13.8 with a range of 1–89 years. The majority (90.8%) of the participants were outpatients. Two hundred and thirty-two (232) isolates were obtained from 219 samples, comprising of 120 (51.7%) Gram positive and 112 (48.3%) Gram negative organisms. Key bacterial pathogens isolated from this study included Staphylococcus aureus (22.8%), Escherichia coli (16.4%), Staphylococcus spp. (15.9%), Enterococcus spp. (7.3%) and Klebsiella pneumoniae (6.5%). There was high prevalence of multi-drug resistance (79.3%) among the isolates with 73.6% of Staphylococcus aureus phenotypically resistant to methicillin and 70% possessed the MecA gene. 76.5% of Enterococcus spp. isolated were Vancomycin resistant. Overall, resistance to Cephalosporins was most frequently/commonly observed (Cefotaxime 87.5%).ConclusionA high incidence of AMR was identified in clinical bacteria isolates from selected general hospitals in Lagos State, highlighting the necessity for the implementation of national action plans to limit the prevalence of AMR. Surveillance via collection of isolates has a lot of promise, especially in resource-limited environments.

Molecular epidemiology and antimicrobial susceptibility of Pseudomonas spp. and Acinetobacter spp. from clinical samples at Jimma medical center, Ethiopia.

IntroductionPseudomonas aeruginosa (P. aeruginosa) and Acinetobacter baumannii (A. baumannii) can cause difficult-to-treat infections. We characterized molecular epidemiology of ceftazidime-resistant P. aeruginosa and carbapenem-resistant A. baumannii at a tertiary hospital in Ethiopia.Materials and methodsNon-fermenting gram-negative bacilli (n = 80) isolated from admitted patients were subjected for species identification by MALDI-TOF. Pseudomonas species resistant to ceftazidime or meropenem, and Acinetobacter species resistant to meropenem, or imipenem were selected for whole genome sequencing. DNA extracted with EZ1 Advanced XL instrument (Qiagen, Hilden, Germany) was sequenced on Illumina (HiSeq2500) using libraries prepared by NEXTRA-kits (Illumina). Raw reads were assembled using SPAdes 3.13.0, and assembled genomes were used to query databases for resistome profile and sequence types.ResultAmong Pseudomonas species isolated, 31.7% (13/41), and 7.3% (3/41) were non-susceptible to ceftazidime, and meropenem, respectively. Carbapenem-resistance was 56.4% (22/39) among Acinetobacter species. Moreover, 92% (12/13) of Pseudomonas species non-susceptible to ceftazidime and/or meropenem, and 89.4% (17/19) of Acinetobacter species encoded multiple resistance genes for at least three classes of antimicrobials. The prevalent β - lactamase genes were blaOXA–486 (53.8%, 7/13), blaCTX–M–15 (23.0%, 3/13) among Pseudomonas, and blaGES–11 (57.8%, 11/19) among Acinetobacter. The blaOXA–51-like β - lactamase, blaOXA–69 (63.1%, 12/19) was the most prevalent carbapenemase gene among Acinetobacter isolates. Single isolates from both P. aeruginosa, and A. baumannii were detected with the blaNDM–1. Sequence type (ST)1 A. baumannii and ST274 P. aeruginosa were the prevalent sequence types. A cgMLST analysis of the ST1 A. baumannii isolates showed that they were closely related and belonged to the international clonal complex one (ICC1). Similarly, ST274 P. aeruginosa isolates were clonally related.ConclusionThe prevalence of MDR isolates of Pseudomonas and Acinetobacter spp. was high. A. baumannii isolates were clonally spreading in the admission wards at the hospital. Emergence of blaNDM–1 in the intensive care, and surgical wards of the hospital is a severe threat that requires urgent intervention.

Investigation of blaIMP-1, blaVIM-1, blaOXA-48 and blaNDM-1 carbapenemase encoding genes among MBL-producing Pseudomonas aeruginosa

Pseudomonas aeruginosa (P. aeruginosa) places among major opportunistic nosocomial pathogen which has developed extensive drug resistance. Due to uncontrolled consumption of antibiotics, multidrug-resistant (MDR) P. aeruginosa species are increasingly isolated from various settings of hospitals globally. This research aimed to determine the genes reproducing blaOXA-48, blaIMP-1, blaVIM-1 and blaNDM-1 metallo beta lactamase (MBL) genes from MDR P. aeruginosa isolates. Herein, the isolates of 200 P. aeruginosa have been obtained from microbiology laboratory of burn ward. The antibiotic susceptibility profile was using Disc Diffusion Method (DDM and in compliance with CLSI (Clinical and Laboratory Standards Institute) advice. Study of MBL-bearing strains and the existence of encoding genes was specified by means of polymerase chain reaction. In addition, pulsed field gel electrophoresis (PFGE) is performed for typing. In this study, 124 (62%) were extended spectrum β-lactamase producer and 51 (25.5%) were MBL producers. Moreover, 148 (74%) isolates were MDR-P. aeruginosa. Additionally, 42 (21%), 21 (10%), 10 (5%) and 2 (1%) isolates carried the blaIMP-1, blaOXA-48, blaVIM-1 and blaNDM-1 genes, respectively. The PFGE showed no genetic relationships among isolates. The study observed high rate of MDR P. aeruginosa in hospital settings, though not being outbreak, which nearly half of them carried carbapenemase enzymes. Therefore, the proper control of related infections and appropriate prescription and consumption of antibiotics is essential.

Extended Spectrum Beta-Lactamase (ESBL) Resistant genes in Salmonella species Isolated from Ready-to-eat Foods and Food Handlers in Port Harcourt Metropolis, Nigeria

Aim: To assess the Extended Spectrum Beta-Lactamase (ESBL) resistant genes in Salmonella spp isolated from ready-to-eat foods and food handlers in Port Harcourt Metropolis, Nigeria.
 Study Design: A cross-sectional study.
 Place and Duration of Study: This study was conducted in selected places in Port Harcourt, between November 2019 and June 2021.
 Methodology: A total of 350 food specimens and 230 food handlers were collected and the social demographic information were collected using a questionnaire survey. The samples were analysed for contamination with Salmonella species using conventional and molecular methods. Salmonella species were isolated from samples using Salmonella-Shigella agar (SSA), Xylose-lysine desoxycholate agar (XLD), MacConkey agar (MA), Blood agar (BA) after pre-enrichment and enrichment method has been done using peptone water broth and Selenite F. Salmonella Chromogenic medium (SCM) was also used to confirm the isolate. Antibiotic susceptibility patterns of the Salmonella isolates were determined using Kirby Bauer disk diffusion method. The presence of Extended Spectrum Beta Lactamase genes (CTX-M, SHV, and TEM) in the Salmonella isolates were screened using double disc synergy method and also investigated using Polymerase chain reaction (PCR) technique.
 Results: Results showed that of the 11 Salmonella species isolated from the food handlers, 7(63.6%) harboured extended spectrum beta-lactamase (ESBL) genes while 18 (64.3%) out of 28 Salmonella isolated from street vended food sample were positive for ESBL genes. However, there was no significant difference in the proportion of ESBL producers observed in the street vended food and those in food handlers’ sample (P= 0.964) PCR result revealed the presence of genes encoding for TEM, SHV and CTX-M.
 Conclusion: Street vended foods such as white rice/stew, jollof rice, rice/beans stew, porridge beans, beans/stew, moi moi, abacha, and roasted plantain in Port Harcourt harbour Salmonella species that possess the following Extended Spectrum Beta Lactamase (ESBL) genes as CTX-M, SHV and TEM. This should receive particular attention, as the presence of extended spectrum beta-lactamase genes isolated, indicate public health hazard and gives a warning signal for the possible occurrence of food borne diseases and multi-drug resistance.

Detection of virulent genetic markers like bla tem, bla imp1 of pathogenic e. coli strain isolated from Iraqi frozen chicken meat

This research was conducted at the Department of Biology, College of Science faculty for Women, University of Kufa, Iraq. From August 2020 to April 2021, one hundred twenty samples (frozen chicken) were collected from various brands (15) and origins (5) from various locations in AL-Najaf Province. The goal of this study was to look at the incidence of Escherichia coli with extended-spectrum -lactamase genes (blaTEM, bla IMP1) in chicken meats in Iraq. The samples were obtained aseptically in a sterile plastic container then transferred to the laboratory inside an icebox for bacteriological investigation. The results revealed that Escherichia coli had the highest percentage(33, 27%)out of 120 of bacterial species isolated from frozen chicken meat then Enterobacter clocace ssp dissolvens(30, 25%) then different species as Klebsiella pneumonia ssp pneumonia( 21,18%), Klebsiella oxytoca (10,8%), Staphylococcus lantus (5,4%), Enterococcus faecalis (5,4%), Enterococcus gallinarum ( 3,3%), Enterobacter kobi (4,3%), Leclercia adecarboxylata(2,2%), Kluyvera cryocrescens (3,2.5%), Vagococcus fluvialis(2 ,2%), Enterobacter hormaechei (1,1%), Enterobacter ludwigii(1,1%). Recognition of resistance genes by PCR test yielded a negative outcome to bla IMP1 identification in all Escherichia coli isolates, while 8 (24.2 percent) of isolates were positive for blaTEM gene of Escherichia coli out of 33 examined isolates.

Dispersal of antibiotic resistance genes in an agricultural influenced multi-branch river network

Rivers in agricultural regions serve as an important sink for livestock and poultry farm runoff, fertilizer runoff, and country living sewage, which could bring antibiotic resistance genes (ARGs) contaminations. However, the diversity and distribution of ARGs has not been well documented in the agricultural influenced river. Here, the diversity of ARGs, and their relationship with biochemical factors were determined in the surface water in an agricultural region of the Jialing River and its five rural branches. The 218 unique ARGs encoding resistance to eight major antibiotic classes have been detected using high-throughput quantitative PCR. The branches of the river had a remarkably higher abundance of ARGs than the mainstream. The aminoglycoside, beta_Lactamase, MLSB, and Multidrug resistance genes were significantly enriched in the branches compared to the mainstream. Compared with the mainstream, the ARGs profiles in the branches showed obvious higher spatial variability. Significant correlation between ARGs profiles and bacterial community structures were observed, and network analysis further showed that the ARGs were associated with their potential hosts, such as Ottowia and Novosphingobium. Redundancy discrimination analysis revealed that Cu content has a significant contribution to the increase of ARGs in the river. The microbial diversity index was negatively correlated with the abundance of the ARGs. These results provide evidence for the enrichment of ARGs in the agricultural influenced river and branches due to the joint influence of chemical and microbial variables.

Report on carbapenemase-producing rare sequence types of Escherichia coli and Enterobacter hormaechei

Introduction and Aim: Carbapenem Resistant Enterobacteriaceae (CRE) have emerged at an alarming rate. Multi locus sequence typing (MLST) is an important parameter for identifying drug resistant organisms. The present study was carried out for elucidating the mechanisms of CRE and MLSTs associated with CRE. Materials and Methods: CRE (n=14) were obtained from various clinical samples and subjected to Rapidec Carba NP (CNP) test and multiplex polymerase chain reaction (M-PCR) and five isolates proceeded for whole genome sequencing (WGS). ?-lactamase (bla) genes were analysed using Resfinder and CARD tool. Bioinformatics tools: mlplasmids, plasmid finder, mobile element finder, and Center for Genomic Epidemiology (CGE) toolbox were used. Results: All isolates (n=14) were positive for CNP and bla genes using M-PCR. Isolates (J21, J22, J23, J27) were identified as Escherichia coli while (J34) was Enterobacter hormaechei. MLST showed E. coli isolates (J21& J22) as ST648; E. coli (J23) was ST940; E. coli (J27) was ST 2851, and E. hormaechei (J34) was closest to ST1325. Genes blaTEM, blaNDM & blaampC were found to be present in all isolates; blaCTX-M was present in all E. coli isolates but not in E. hormaechei. blaOXA was present in E. coli (J23) and in E. hormaechei (J34); while ESBL blaSFO-1 in E. hormaechei (J34). Conclusion: ESBLs (blaTEM & blaCTXM) and metallo beta-lactamase -MBL (blaNDM) cause carbapenem resistance in rare sequence types of E. coli while; ESBL (blaSFO-1) and MBL (blaNDM) cause carbapenem resistance in E. hormaechei.

Genetic Characterization of Pathogenic Escherichia Coli Isolated from Smallholding Dairy Buffaloes, their Products, and Some Environmental Sources

This study was conducted to evaluate the potential existence of pathogenicEscherichia coli in smallholding dairy buffaloes, their products as well as in some environmental sources. In total, 150 samples including an equal sample size (n = 25) of raw milk, Kareish cheese, water, feed, udder and rectal swabs were examined. Phenotypic and genotypic procedures were used to characterize the bacterial population. Twenty-eight isolates were confirmed as E. coli from the examined samples (18.66%). The recovered isolates were categorized as Enterohemorrhagic E. coli (n=11),Enteropathogenic E. coli(n=9), Enterotoxigenic E. coli(n= 5)and Enteroinvasive E. coli (n=3). All isolates were resistant to erythromycin followed by oxacillin (89.28%), and nalidixic acid (67.85%), while 96% of the recovered strains displayed high sensitivity to imipenem. A great proportion of the recovered E. coli (67.9%) exhibited multidrug resistance. The majority of isolates (92.9%) harbored stx1either alone or in association with stx2 which present in 60.7% (17/28) while eaeA genes were detected in 50% among the recovered isolates in association with stx1 or combined with stx2 (n =11). Twelve isolates (42.86%) expressed all the examined beta lactamase encoding genes; while only 25% of the isolates harbored all tested beta lactamase genes in common with stx1, stx2 and eaeA genes.The results herein indicated that the pathogenicE. coli could be present in the examined samples which pose a relevant threat and negative impact on animal as well as consumer health. The study emphasizes the necessity of developing long-term strategies to assure dairy food safety.

An inducible transposon mutagenesis approach for the intracellular human pathogen Chlamydia trachomatis

Background: Chlamydia trachomatis is a prolific human pathogen that can cause serious long-term conditions if left untreated. Recent developments in Chlamydia genetics have opened the door to conducting targeted and random mutagenesis experiments to identify gene function. In the present study, an inducible transposon mutagenesis approach was developed for C. trachomatis using a self-replicating vector to deliver the transposon-transposase cassette - a significant step towards our ultimate aim of achieving saturation mutagenesis of the Chlamydia genome. Methods: The low transformation efficiency of C. trachomatis necessitated the design of a self-replicating vector carrying the transposon mutagenesis cassette (i.e. the Himar-1 transposon containing the beta lactamase gene as well as a hyperactive transposase gene under inducible control of the tet promoter system with the addition of a riboswitch). Chlamydia transformed with this vector (pSW2-RiboA-C9Q) were induced at 24 hours post-infection. Through dual control of transcription and translation, basal expression of transposase was tightly regulated to stabilise the plasmid prior to transposition. Results: Here we present the preliminary sequencing results of transposon mutant pools of both C. trachomatis biovars, using two plasmid-free representatives: urogenital strain C. trachomatis SWFP- and the lymphogranuloma venereum isolate L2(25667R). DNA sequencing libraries were generated and analysed using Oxford Nanopore Technologies’ MinION technology. This enabled ‘proof of concept’ for the methods as an initial low-throughput screen of mutant libraries; the next step is to employ high throughput sequencing to assess saturation mutagenesis. Conclusions: This significant advance provides an efficient method for assaying C. trachomatis gene function and will enable the identification of the essential gene set of C. trachomatis. In the long-term, the methods described herein will add to the growing knowledge of chlamydial infection biology leading to the discovery of novel drug or vaccine targets.

An inducible transposon mutagenesis approach for the intracellular human pathogen Chlamydia trachomatis.

Background: Chlamydia trachomatis is a prolific human pathogen that can cause serious long-term conditions if left untreated. Recent developments in Chlamydia genetics have opened the door to conducting targeted and random mutagenesis experiments to identify gene function. In the present study, an inducible transposon mutagenesis approach was developed for C. trachomatis using a self-replicating vector to deliver the transposon-transposase cassette - a significant step towards our ultimate aim of achieving saturation mutagenesis of the Chlamydia genome. Methods: The low transformation efficiency of C. trachomatis necessitated the design of a self-replicating vector carrying the transposon mutagenesis cassette (i.e. the Himar-1 transposon containing the beta lactamase gene as well as a hyperactive transposase gene under inducible control of the tet promoter system with the addition of a riboswitch). Chlamydia transformed with this vector (pSW2-RiboA-C9Q) were induced at 24 hours post-infection. Through dual control of transcription and translation, basal expression of transposase was tightly regulated to stabilise the plasmid prior to transposition. Results: Here we present the preliminary sequencing results of transposon mutant pools of both C. trachomatis biovars, using two plasmid-free representatives: urogenital strain C. trachomatis SWFP- and the lymphogranuloma venereum isolate L2(25667R). DNA sequencing libraries were generated and analysed using Oxford Nanopore Technologies' MinION technology. This enabled 'proof of concept' for the methods as an initial low-throughput screen of mutant libraries; the next step is to employ high throughput sequencing to assess saturation mutagenesis. Conclusions: This significant advance provides an efficient method for assaying C. trachomatis gene function and will enable the identification of the essential gene set of C. trachomatis. In the long-term, the methods described herein will add to the growing knowledge of chlamydial infection biology leading to the discovery of novel drug or vaccine targets.

Evaluation of antibiotic resistance patterns in clinical isolates of Klebsiella pneumoniae in Bangladesh

The treatment of K. pneumoniae infections has become more difficult due to the emergence of multidrug-resistant strains. This study aimed to evaluate antimicrobial resistance patterns and detect different types of beta-lactamase genes among MDR and non-MDR Klebsiella pneumoniae in various clinical samples. A total of 150 Klebsiella pneumoniae were identified from different clinical samples by conventional microbiological procedures. Antibiotic sensitivity was detected by the Kirby-Bauer disc diffusion method. Extended-spectrum beta-lactamase, Metallo-β-lactamase, and carbapenemase were phenotypically detected by double-disk synergy test, combined disk assay, and modified Hodge test. PCR also detected different β lactamase genes and virulence genes. The majority of Klebsiella pneumoniae were multi-drug resistant (82%). The bacteria were found resistant to most β-lactam antibiotics, aminoglycosides, ciprofloxacin, cotrimoxazole, carbapenem, piperacillin, and tazobactam. However, only about 0.7% colistin resistance was observed. A double-disk synergy test was performed among the isolates for phenotypic detection of ESBL producing Klebsiella pneumoniae. Only 16.6% of isolates were found to be ESBL producers. Among β-lactamase genes, NDM (23.34%), OXA-48(8%), and KPC (7.3%) were detected. Other prevalent β lactamase genes VIM and IMP were not detected. In summary, the prevalence of MDR Klebsiella pneumoniae is high in Bangladesh, which may complicate the treatment of hospitalized patients.

Distribution of blaTEM Gene among Escherichia Coli Strains Isolated from Different Clinical Samples in Erbil City

Background: Emerging antibiotic resistance and Extended-Spectrum Beta-Lactamase (ESBL) producing Escherichia coli causing different nosocomial infections are rapidly increasing at alarming levels and it poses a major health burden in the 21st century globally. Objective: The aim of this study is to determine the distribution of blaTEM gene ESBLproducing E. coli from clinical specimens in different hospitals in Erbil city. Methods: A total of 200 samples were collected from (sputum, urine, wound) from patients attended public hospitals in Erbil city. The isolation and identification of Escherichia coliand antimicrobial susceptibilitywere performed by using Vitek 2 compact -lactamase production in E.coli was confirmed by using both Double disk diffusion and Standard disk diffusion techniques. Moreover, PCR technique was used for genotypic detection of an ESBL gene blaTEMaccording to the standard protocol. Results: Out of 200 samples 60(30%) of E. coli isolates and the highest rate of E. coli isolates were obtained from the urine samples (20%) and the lowest number was isolated from sputum specimens (2.5%). The ESBL-producing Escherichia coli isolates were detected using double disk synergy test (76.7%) in comparison to standard disk diffusion test (80%). Genotypic screening results confirmed that all ESBL-producing E. coli isolates (66.7%) were carried blaTEM gene (700 bp) in clinical specimens (50% urine, 13.33% wound and 3.33% sputum). All ESBL-positive E. coli isolates showed high rates of susceptibility to Carbapenems antibiotic group including Imipenem (83.3%), Meropenem (81.7%), and Ertapenem (80.0%). Conclusions: -lactamase gene in ESBL-producing E. coli observed in this study for the first time is considered as alarming because there is a limited treatment options remained for infections. Attempts to reduce the dissemination of multi-drug resistant E. coli through compliance with strict hospital infection control and prevention standards are imperative. Findings of this study may help clinicians selecting appropriate antimicrobial therapy in patients with different infections caused by ESBLproducing E. coli.

English

Antimicrobial surveillance and identifying the genetic basis of antimicrobial resistance provide important information to optimize patient care. The present study is an analytical cross sectional study aimed to determine the prevalence of multidrug resistant (MDR), extensively drug resistant (XDR), pan drug resistant (PDR) and extended-spectrum ?-lactamases genes among Gram-negative bacteria isolated in Saudi Arabia.  A total number of 386 non-duplicate Gram-negative isolates were collected. Identification and susceptibility testing were done using automation system (BD Phoenix™). The extracted DNAs were subjected to multiplex polymerase chain reaction (PCR). The results showed that only 15 (3.9%) of isolates were fully susceptible, the overall prevalence of XDR, MDR, PDR was 129 (33.4%), 113 (29.3%) and 48(12.4%) respectively. High resistant rate was observed against the antibiotic agents of cephalosporins class 79.3% followed by the agents of penicillins class 69.4%. The most dominant resistant gene was bla SHV which was detected in 106/386 (27.5%) isolates, followed by bla CTX-M 90/386 (23.3%). Bla CTX-M showed significant relation with all used antibiotic except ampicillin/clavulanic acid, aztreonam, cefoxtin, and meropene. The isolates which showed frequent resistant genes were: Klebsiella pneumoniae 90/124 (72.6%), A. baumanni 37/67 (55.2%), and P. mirabilis 24/44 (54.5%). These findings underscore the need for optimization of current therapies and prevention of the spread of these organisms.   Key word: Multidrug resistant (MDR), extensively drug resistant (XDR), pan drug resistant (PDR), extended spectrum beta lactamase (ESBL), bla SHV, bla TEM, bla CTX-M.