Qnr Prevalence in <i>Escherichia coli</i> Isolates from Wastewater from Livestock Farms in Hamedan, Iran

the emergence of antibiotic resistance in E. coli strains has led to a variety of clinical illnesses in both humans and animals, with consequences for human health and the environment. this cross-sectional study was conducted to determine the risk of increased antibiotic resistance by calculating the multiple antibiotic resistance (MAR) index of E. coli in Port Harcourt, Rivers State, Nigeria. A higher MAR index close to 1 was interpreted as having a higher level of antibiotic resistance, while a lower MAR index close to 0 is an indicative of little to no resistance. In this study, a total of 200 samples from clinical (urine and stool) and non-clinical sources (soil and poultry) were randomly collected following ethical approval. Escherichia coli was isolated on Eosin Methylene Blue agar and identified using standard biochemical tests. The antimicrobial susceptibility profile was determined using the disk diffusion methodology with Mueller-Hinton agar according to the instructions of the Clinical and Laboratory Standards Institute (CLSI). Escherichia coli isolates were amplified in this investigation using a 16s rRNA gene-based polymerase chain reaction (PCR) technique. for both 73% clinical (n=100) and 35% non-clinical (n=100) isolates, amoxicillin/clavulanic acid showed the highest rate of resistance with 82.2% (60 of 73) and 100% (35 of 35) respectively, whereas nitrofurantoin showed the lowest rate of resistance at 1.4% (1 of 73) clinical and no resistance was found for the nonclinical E. coli isolates. The study presumptively finds a higher resistance to 4 drug classes, with 42%, and the lowest of 1% to 6 drug classes of antibiotics. Additionally, MAR indices > 0.2 were observed in this study, which indicates excessive use of antibiotics. MAR index of 0.4 was the most frequent, with a 25% prevalence. The results of this investigation show that a MAR index of 0.4 suggests widespread antibiotic resistance, with 25% of the bacterial isolates from both sources exhibiting resistance to the tested antibiotics. A MAR index > 0.2 in this study indicates that 14.8% of the E. coli isolates were ineffective against the tested antibiotics. The bacteria isolates in this study have developed resistance to multiple antibiotics if the MAR index is greater than 0.2, which is an indication of overuse or inappropriate antibiotic use. The genotypic test verified 82.9% non-clinical E. coli (found in environmental soil and poultry samples) and 82.2% clinical E. coli (found in patient urine and stool samples). In this study, E. coli isolates were genotypically identified using the 16s rRNA gene. the high levels of MAR indices in E. coli as presented in this study could be indicative of antibiotic treatment failures in both clinical and nonclinical settings, which can serve as a potential reservoir of drug-resistant E. coli that are harmful and therefore require continuous monitoring.

Locked Nucleic Acids Can Enhance the Analytical Performance of Quantitative Methylation-Specific Polymerase Chain Reaction

Urinary tract infections (UTIs) impose significant challenges among patients infected with multidrug-resistant Escherichia coli (E. coli). The emergence of the extended-spectrum beta-lactamase (ESBL) strains complicates the treatment and exacerbates life-threatening infections. The comprehensive analysis of the resistance mechanisms combined with identifying the relevant genes amend the implications encountered with the devastating effects generated by the pathogenic microorganisms. This study sought about the prevalence of ESBL-producing E. coli in UTIs patients in Jordan, focusing on the prevalence of blaOXA and blaCTX-M resistance genes. A total of 500 urine samples were compiled from patients diagnosed with UTIs between November 2023 and May 2024. The samples were collected from five governmental hospitals distributed in three districts in Jordan (North, Middle, and South). Urine samples were cultured in the microbiology lab and the percentage of E. coli isolates was explored. Later on, isolates were tested for ESBL production using the double disk synergy test. Thus, molecular detection of the blaOXA and blaCTX-M genes was performed using polymerase chain reaction (PCR) assays. Statistical analysis was conducted to assess the prognostic correlation between the prevalence of resistance genes and variables such as location, age, and gender. In the current study, we investigated the prevalence of ESBL-producing E. coli in UTIs across Jordanian hospitals, focusing on the prevalence of the blaOXA and blaCTX-M genes with the correlation with the epidemiological factors. Our findings revealed an overall prevalence of 18% for ESBL- E. coli isolates, with the highest prevalence in the Northern governorates exhibiting 50% (OR = 1.67, 95% CI: 1.103-2.519). Females showed a higher prevalence of the blaOXA gene (62.29%) compared to males (37.7%) (p = 0.008). Moreover, the blaCTX-M gene was universally present in all samples with a prevalence of 98%. The multinomial regression analysis has revealed a significant correlation (p = 0.001) between location on the distribution of the blaOXA gene among the study population. The findings of the current study highlight alarming rates for the prevalence of ESBL-producing E. coli in UTIs across Jordan in general and in the northern districts as specific. Emphasizing the crucial need for effective surveillance, antibiotic stewardship, and infection control strategies. The significant regional variations in resistance rates suggest that localized factors, such as antibiotic misuse and environmental influences combined with the widespread of blaCTX-M and blaOXA genes point to the need for targeted antibiotic resistance surveillance in Jordan.

Background: Escherichia coli is known to inhabit the gastrointestinal tract of poultry and other animals. E. coli infection can cause major economic losses in poultry production. The development of resistance to the commonly used antimicrobials in the treatment of such infection can be a setback in the poultry sector. Objective: This study was carried out to determine the antibiotic resistance profile of E. coli isolated from chickens in Imo State, Nigeria. Materials and Methods: Twelve poultry farms were selected across the 3 senatorial zones of the state using purposive random sampling. A total of 120 cloacal samples were collected from chickens using sterile swabs. The samples were streaked onto MacConkey agar (MCA) and incubated at 37o C overnight. Pink colonies on the MCA were streaked onto eosin-methylene blue agar, incubated for 24 hours at 37o C, and confirmed by indole, methyl red, Voges-Proskauer, and citrate (IMVIC) tests. Antimicrobial susceptibility test was carried out using disc diffusion technique, and the inhibition zone diameter was measured and recorded as resistant or susceptible. Results: Twenty isolates out of the 120 samples were identified as E. coli. Twenty isolates were highly resistant to 5 antibiotics out of 10 antibiotics used. Antibiotic resistance patterns were as follows: amoxicillin (AMP) (95%), cephalothin (CEP) (90%), nalidixic acid (NA) (85%), trimethoprim (SXT) (85%), cefoperazone (PEF) (80%), ampicillin (AMP) (70%) ofloxacin (OFX) (15%), ciprofloxacin (CIP) (10%), gentamicin (CN) (10%), and streptomycin (S) (10%). Sixteen resistance patterns were recorded with AMC-SXT-AMP-CEP-NA-PEF being the most prevalent. Conclusion: This study shows that multi-drug resistant E. coli strains are present in poultry farms in Imo State.

Validation of Roche LightCycler Epstein-Barr Virus Quantification Reagents in a Clinical Laboratory Setting

Real-Time PCR: An Essential Guide

This study investigated the seasonal prevalence and the antimicrobial susceptibility of Campylobacter jejuni and Campylobacter coli in 264 samples of chicken meat. The samples encompassed wings (n=88), breasts (n=79) and thighs (n=97) and were purchased from different butcheries and markets in Elazig province, in Eastern Turkey, between December 2009 and November 2010. The meat samples were tested for Campylobacter presence and the collected isolates were identified as Campylobacter jejuni and Campylobacter coli using polymerase chain reaction (PCR). Resistance rates to 7 antimicrobials were investigated by the disk diffusion method. Campylobacter jejuni was found at a higher prevalence (41.7%) than C. coli (14.4%); C. jejuni was isolated most frequently from breast samples (53.2%) than from thighs (40.2%) and wings (32.9%) samples. The prevalence of C. jejuni and C. coli peaked during the Summer (June-August), with the highest peak occurring in July (77.3%). The lowest prevalence (30%) was detected in February. The prevalence in the Summer (June-August) was significantly higher (71.2%) than the one reported during the Winter (December-February) (39.4%, P< 0.05). The highest resistance rate among C. jejuni isolates was observed to tetracycline (38.2%), nalidixic acid (29.1%), and ciprofloxacin (24.5%). Campylobacter coli also showed a high resistance to these antibiotics, although in slightly different proportions: tetracycline (42.1%), ciprofloxacin (31.6%), and nalidixic acid (26.3%). None of the C. jejuni or C. coli isolates was resistant to gentamicin.

Studies on the microorganisms associated with spoilt carrots obtained from Ose Market, Onitsha, Nigeria were carried out using standard cultural techniques. Nutrient agar, sabouraud dextrose agar and Eosin methylene blue agar were the growth media for the isolation of the heterotrophic bacteria, fungi and coliforms. The bacteria were identified as Serratia marcescens, Escherichia coli and Corynebacterium bovis while the fungi was identified on the basis of their colonial and microscopic characteristics as Penicillium digitatum, Rhizopus stolonifer, Aspergillus niger and Alternaria alternata. Escherichia coli was predominantly isolated among the bacterial isolates (50%) while Aspergillus niger occurred most frequently than the other fungal species (40%). These organisms may have been introduced to the carrots during growth, harvesting, handling, storage and distribution. The presence of the organisms is a public health risk because of the diseases known to be caused by them. It is therefore imperative that adequate hygienic practices must be put in place during the storage and handling of carrots. Spoilt carrots must also not be consumed as they contain a teaming population of bacteria and fungi, some of which are pathogenic to humans.

Transmission of Escherichia Coli O157 by the formit route is under reported since interest in E. Coli O157 is usually arisen during outbreak situations which are mostly linked to consumption of food of bovine origin (Armstrong et al., 1996; Fukushima et al., 1999 and Allison et al., 2000). In December 2005 while working on the isolation of E. Coli O157 from cattle herds in Zaria, Kaduna State, a 1– year old child of a member of the team came down with a mild watery diarrhea. He was not presented to any Physician, since he was observed to be alert, active and with a normal appetite. However, stool specimens were collected from the child on days 2 and 3 of the diarrhea, (which was self limiting on day 3). Stool specimens were also collected from all the five members of his family. All items that the boy had contact with including a laboratory coat, bunch of keys and shoes were swabbed. Finally samples of all the boy's food and drinks were taken. Microbiological, Serological and Polymerase Chain Reaction (PCR) Profiling Assays. l the samples were cultured on Sorbitol - MacConkey (SMAC) agar, after enrichment using Modified Tryptone Soya Broth (MTSB) supplemented with Novobiocin (Oxoid Ltd, Basingstoke, England). All colourless colonies on SMAC agar from the samples were biochemically confirmed as E. coli by the methods described by (Cowan, 1981) and serologically as E. Coli O157:NM using the Remel Wellcolex E. coli O157:H7 kit (Remel Europe Ltd, Kent UK). Shigatoxin profiling was done using the VTEC-RPLA kit (Oxoid Ltd, Basingstoke England). Antimicrobial resistance profiling was carried out by the method of Bauer et al. (1966) using 16 antimicrobial agents: Ampicillin (AMP10), Ceftazidime (CAZ30), Cefuroxime (CXM30), Cephazolin (KZ30), ciprofloxacin (CIP5), compound Sulphonamides (S3300), gentamicin (CN10), Kanamycin (K30), Chloramphenicol (C30), Nalidixic acid (NA30), Neomycin (N10), Norfloxacin (NOR2), streptomycin (S10), Sulphamethoxazole (RL25), Co-trimoxazole (SXT25) and tetracycline (TE30) (Oxoid Ltd, Basingstoke, England). The assays were controlled using a control strain of E. Coli O157:H7 (ATCC 43895). Total DNA was isolated from 1 ml of brain heart infusion (BHI) broth culture grown overnight based on the method of Silhavy et al (1984). The Eae primer (Operon Ltd, Dusseldorf, Germany) was used (Germani et al., 1997). The PCR mix consisted of 2.50&#956l 10 x buffer, 1.50&#956l MgCl2, 0.50 dNTPs, eae-f 0.40&#956l, Taq polymerase (Ampli Taq Gold, Applied Biosystems, California, U.S.A.) 0.50&#956l, and 4.0&#956l of template DNA. The volume of the mix was adjusted to 25&#956l by addition of 15.2&#956l of nuclease free sterile water. DNA amplification was carried out in a Gene Amp PCR System 9700 Thermocycler (Applied Biosystems, California, U.S.A.) using an initial denaturation step at 94oC for 5 minutes, followed by 30 cycles of amplification with denaturation step at 94oC for 1 minute, annealing at 53oC for 1 minute, and extension at 72oC for 10 minutes and cooled to 4oC. Keywords : Escherichia Coli O157: NM, Formit, Transmission Nigerian Veterinary Journal Vol. 28 (3) 2007 pp. 53-55

Background: The prevalence of Klebsiella pneumoniae in surgical site infections (SSIs) has increased recently. This study aimed to evaluate the antimicrobial resistance patterns and biofilm formation capacity of K. pneumoniae strains isolated from SSIs. Methods: A total of 63 K. pneumoniae isolates were obtained from patients with SSIs. Antimicrobial susceptibility testing was determined using the Kirby-Bauer method. Molecular analyses were performed to confirm the presence of virulence and antibiotic resistance genes. Biofilm formation was determined using a semiquantified microtiter plate assay, and optical density measurements were used to classify the isolates into weak, moderate, and strong biofilm producers. Biofilm structure was observed using field-emission scanning electron microscopy. Statistical analyses were performed using SPSS software version 16 (SPSS Inc., Chicago, IL, USA), and data were analyzed and presented in terms of frequency and percentage. Results: The frequencies of fimH, mrkD, mrkA, wcaG, and magA were 98.4%, 96.8%, 77.7%, 61.9%, and 7.9%, respectively. The highest rates of antibiotic resistance were observed for cefazolin, cefuroxime, and piperacillin/tazobactam and 98.4% of the isolates were resistant to at least one antibiotic agent. The most prevalent resistance genes were blaSHV (42.8%), blaCTX-M (31.7%), blaTEM (28.5%), and blaOXA48 (22.2%). All the tested isolates were able to produce biofilms, and 76.2% were classified as strong biofilm producers. Conclusions: Klebsiella pneumoniae is one of the common pathogens in SSIs, and due to its antibiotic resistance and the presence of multiple virulence factors, proper controlling strategies need to be carried out.

Background: Plasmid-mediated quinolone resistance (PMQR) determinants are commonly characterized in Klebsiella pneumoniae isolates worldwide and complicate the treatment of infections caused by this bacterium. Objectives: This study aimed to investigate the prevalence of PMQR determinants and molecular typing of blood isolates of K. pneumoniae in Milad hospital in Tehran, Iran, within 2018 - 2019. Methods: A total of 100 K. pneumoniae isolates were tested for susceptibility to quinolones using the disk diffusion method. The minimum inhibitory concentrations (MICs) of ciprofloxacin (CIP) and levofloxacin (LEV) were determined using the microdilution broth method. The PMQR determinants were detected by polymerase chain reaction (PCR) assay, and the genetic relationship between the isolates was assessed using enterobacterial repetitive intergenic consensus (ERIC)-PCR. Results: The resistance rates of the isolates to LEV, CIP, nalidixic acid, and norfloxacin were determined to be 62, 46, 29, and 23%, respectively. Eighty-one isolates were resistant to at least one tested quinolone. A high-level CIP and LEV resistance (MIC &gt; 32 mg/L) was observed in 15 (18.51%) and 36 (44.44%) isolates, respectively. The PMQR genes were detected in 71 (87.65%) isolates. The oqxAB, qnrS, qnrD, qnrB, aac(6')-Ib-cr, qnrA, qepA, and qnrC genes were detected in 71 (87.65%), 30 (37%), 25 (30.8%), 24 (29.6%), 18 (22.2%), 17 (21%), 17 (21%), and 8 (9.9%) isolates, respectively. The ERIC-PCR revealed 64 genotypes among quinolone-resistant isolates. Conclusions: The high prevalence of PMQR genes observed in this study is a significant concern for public health since they can contribute to the spread of fluoroquinolone resistance among clinical isolates. The ERIC-PCR revealed high heterogeneity among the studied isolates, indicating that they emerged from different sources.

An SYBR Green real-time polymerase chain reaction (PCR) assay was developed for Arcobacter detection in food and wastewater samples. The assay was applied to 36 chicken and 33 wastewater samples, and the results were compared with those obtained for conventional PCR, multiplex PCR, and culture isolation. Isolates were identified by multiplex PCR and restriction fragment length polymorphism analysis of PCR-amplified DNA fragment, and typed by randomly amplified polymorphic DNA. Arcobacter sp. was detected in 25 of the 26 chicken carcasses (96%) and in 4 of the 10 liver samples (40%) by real-time PCR. Twenty-five chicken samples were positive also by conventional PCR, but in most of them the detection was only possible after 48-h enrichment. Arcobacter butzleri was the most frequently detected species. Twenty-four Arcobacter isolates were obtained from chicken samples, where A. butzleri is the only identified species. All the wastewater samples (100%) were positive for Arcobacter sp. by real-time PCR without enrichment. A. butzleri and Arcobacter cryaerophilus were detected by multiplex PCR. Fifteen samples were found to be positive by culture. Thirty-six isolates were obtained; all of them were identified as A. butzleri by multiplex PCR. However, by PCR-restriction fragment length polymorphism, 34 were identified as A. butzleri, 1 as A. cryaerophilus, and another 1 as Arcobacter skirrowii. A great genetic heterogeneity was observed by randomly amplified polymorphic DNA-PCR profiling. The real-time PCR assay developed in this work showed better detection levels than conventional PCR, together with shorter times of testing samples. Therefore, it could be used as a rapid and accurate instrument for monitoring Arcobacter contamination levels in food and water samples.

A Novel Semiquantitative Fluorescence-Based Multiplex Polymerase Chain Reaction Assay for Rapid Simultaneous Detection of Bacterial and Parasitic Pathogens from Blood

Objective: Pseudomonas aeruginosa is among the most common nosocomial opportunistic pathogens capable of acquiring multi-drug resistance. Recent data on antibiotic resistance in Northern Cyprus is limited. We aimed evaluate antibiotic susceptibility profiles of P. aeruginosa strains isolated from clinical samples and contribute to the update of antibiotic use policies in the country. Methods: Total of 504 P. aeruginosa strains sent to Microbiology Laboratory from various clinics from September 2016 to October 2019 were retrospectively analyzed. Clinical samples were cultured on %5 sheep blood agar and eosin-methylene blue agar. BD Phoenix™ 100 (Becton Dickinson Co., Sparks, Maryland, USA) automated bacterial identification system and conventional antimicrobial susceptibility testing was used. Antimicrobial susceptibility was determined in accordance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. The distribution of P. aeruginosa isolates according to hospital services, sample types, admission types, age group, and antibiotic resistance rates were investigated. Results: P. aeruginosa was mostly isolated from urine (37.3%), deep tracheal aspirate (17.9%) and abscess/wound material (17.9%) samples. Strains were most commonly obtained from the intensive care clinics (35.9%). Infections were observed mainly in &gt;65 age group (49.6%) and in-patients (70.8%) group. Antibiotic resistance rates were detected as 47.7% for aztreonam, 32% for cefepime, 26.3% for ceftazidime, 26.4% for imipenem, 25.6% for ciprofloxacin, 24.6% for meropenem, 19.7% for piperacillin-tazobactam, 13.5% for gentamicin, 12.2% for colistin and 5.3% for amikacin. Multi-drug resistant P. aeruginosa rate was 19.2%. When compared with 2010-2014, a substantial increase in rates of cefepime, ceftazidime, ciprofloxacin, piperacillin-tazobactam and carbapenem resistance was detected within the last three years. Conclusions: In this study, the highest resistance rates were detected against aztreonam and cefepime, and the highest susceptibility was observed for colistin and amikacin. These results may be used for empirical treatments, establishment of rational antibiotic use policies and prevention of emerging antibiotic resistance problem in Cyprus.

Background: Urinary tract infection (UTI) is one of the most common human bacterial infections. The frequent use of antibiotics is associated with increased resistance of many bacterial agents to the treatment.Materials and Methods: A total of 9456 urine specimens were collected from patients referring to Pol-e Dokhtar health centers. All urine samples were inoculated on blood agar plates (BAP) and eosin methylene blue (EMB) agar. Those specimens positive for selected pathogen were finally identified. The antimicrobial susceptibility and resistance patterns of the UTI pathogens to common antibiotics were determined using disk diffusion method according to Clinical and Laboratory Standards Institute (CLSI) guidelines.Results: Out of 9456 investigated urine samples, 1036 samples (10.95%) were positive, of which 823 samples (79.4%) belonged to female subjects and 213 samples (20.6%) belonged to males. The most common identified urine tract pathogens were Escherichia coli (69.4%), Staphylococcus epidermidis (15.15%) and Staphylococcus aureus (7.05%). Also, co-trimoxazole (sulfamethoxazole/trimethoprim) displayed the highest antibiotic resistance (58.1%) and nitrofurantoin (90.1%) and gentamicin (88%) were shown to be the most frequent cause of sensitivity.Conclusion: The findings of our study demonstrate that E. coli is the most common UTI pathogen. Given the high antibiotic resistance of co-trimoxazole, it is recommended to avoid indiscriminate consumption of this antibiotic and perform antibiogram tests for the treatment of UTIs.