Phenotypic Methods For Detection Research Articles

Prevalence, Molecular Characterization and Antibiogram Pattern of Livestock Associated Methicillin Resistant Staphylococcus aureus in Livebird Markets Gusau, Zamfara State, Nigeria

Study’s Excerpt The prevalence and antibiotic resistance patterns of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) in poultry markets within the Gusau metropolis was examined. Phenotypic and molecular (PCR) detection methods were combined to identify the LA-MRSA isolates. Multidrug resistance (MDR) phenotypes were observed in 72.2% of the isolates. Full Abstract Poultry farms have been identified as hotspots for the emergence and spread of antibiotic-resistant bacteria, especially livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA). In this study, a total of 320 swab samples were randomly collected from fecal litter, Hands of poultry handlers, Soil, Feed, and Water from five different poultry markets in the Gusau metropolis. The LA-MRSA isolates involved were detected culturally and identified phenotypically by the use of PCR assay. The pooled prevalence of the LAMRSA isolates in the samples was 22(100%). The chi-square test was used to compare the prevalence of LAMRSA among the samples collected at 95% confidence interval and 0.05 level of significance. There is no statistically significant difference in the prevalence of LAMRSA among the samples. Findings from the antibiogram study conducted revealed that all the 22(100%) LAMRSA isolates were resistant to Penicillin antibiotics, with resistance to other antibiotics following the order: Nitrofurantoin, 17(77.3%), Sulphamethoxazole, 13(59.1%), Chloramphenicol 6(27.28%), Ciprofloxacin 5(22.7%) and Erythromycin 4(18.2%). A total of 14 multidrug resistant phenotypes were identified from the 22 LAMRSA isolates with 16(72.2%) demonstrating cross resistant to ≥3 of the six different classes of antibiotics used. The MDR rate in soil samples was the highest (27.3%) (p<0.05) compared to other MDR isolates from the other sources. Of all the isolates, 22(100%) demonstrated a MAR Index ranging from 0.2-0.8. These are disturbing trends with significant risks to humans and animals. Suggesting a potential for inoculation of self and transmission to others, particularly through those who sell and make daily contact with live birds. Therefore, the government should awaken the consciousness of poultry farmers/handlers by way of sensitization on the need to improve water quality and practice routine personal and environmental hygiene as it relates to poultry management, with a focus on antibiotic stewardship within the one health framework so as to lower endemicity and equally break the chain of transmission of LA- MRSA across the farm to fork continuum.

Phenotypic and genotypic perspectives on detection methods for bacterial antimicrobial resistance in a One Health context: research progress and prospects.

The widespread spread of bacterial antimicrobial resistance (AMR) and multidrug-resistant bacteria poses a significant threat to global public health. Traditional methods for detecting bacterial AMR are simple, reproducible, and intuitive, requiring long time incubation and high labor intensity. To quickly identify and detect bacterial AMR is urgent for clinical treatment to reduce mortality rate, and many new methods and technologies were required to be developed. This review summarizes the current phenotypic and genotypic detection methods for bacterial AMR. Phenotypic detection methods mainly include antimicrobial susceptibility tests, while genotypic detection methods have higher sensitivity and specificity and can detect known or even unknown drug resistance genes. However, most of the current tests are either genotypic or phenotypic and rarely combined. Combining the advantages of phenotypic and genotypic methods, combined with the joint application of multiple rapid detection methods may be the trend for future AMR testing. Driven by rapid diagnostic technology, big data analysis, and artificial intelligence, detection methods of bacterial AMR are expected to constantly develop and innovate. Adopting rational detection methods and scientific data analysis can better address the challenges of bacterial AMR and ensure human health and social well-being.

Antibiotic Resistance Profiles of Extended-Spectrum β-Lactamase (ESBL)- and Metallo-β-Lactamase (MBL)-Producing Klebsiella pneumoniae Isolates From Diabetic Foot Ulcers: Implications for Treatment Strategies.

Background Diabetic foot ulcers (DFUs) are prevalent complications of diabetes mellitus, often leading to severe infections and adverse clinical outcomes. Klebsiella pneumoniae, a gram-negative bacterium, has emerged as a significant causative agent in DFU infections, raising concerns due to its increasing antibiotic resistance, particularly in extended-spectrum β-lactamase (ESBL) and metallo-β-lactamase (MBL) production. Aim This study aimed to comprehensively assess the prevalence, antibiotic resistance profiles, and clinical correlates of ESBL- and MBL-producing K. pneumoniae isolates specifically derived from DFUs. Methods A cross-sectional observational study was conducted at Krishna Vishwa Vidyapeeth from January 2023 to June 2023, involving 126 patients diagnosed with DFUs. Clinical and demographic data were collected, and wound swabs underwent microbiological analysis. Phenotypic detection methods were employed to identify ESBL and MBL production, followed by standardized antibiotic susceptibility testing. Results Among the 126 isolates tested, 36 (28.6%) were identified as ESBL-producing and 21 (16.7%) as MBL-producing strains. ESBL-producing isolates exhibited high resistance rates to antibiotics such as ampicillin (92.3%), amoxicillin-acid (84.6%), and cephalosporins, including ceftriaxone (76.9%), and cefepime (73.8%). MBL-producing isolates demonstrated even broader resistance profiles, including resistance to fluoroquinolones (ciprofloxacin, 60.0%; levofloxacin, 57.1%), aminoglycosides (gentamicin, 42.9%), and carbapenems (meropenem, 38.1%; imipenem, 35.7%). Conclusion This study identifies a significant prevalence of ESBL- and MBL-producing K. pneumoniae in DFUs, showcasing high antibiotic resistance rates. Comorbidities correlate significantly with the presence of resistant isolates, necessitating treatment strategies for effective management.

Detection of Carbapenemase Production in Klebsiella pneumoniae Isolates by Rapid Carbapenemase Detection Method in an OXA-48 Endemic Region

Background: Carbapenem resistance is one of the major global public health threats caused by the overuse or misuse of carbapenems, which are often the last resort in treating multidrug-resistant infections. Today, timely detection of carbapenemase-producing organisms is critical for infection control. Therefore, rapid and accurate detection of carbapenemases using a cost-effective method with high sensitivity and specificity is essential to guide appropriate treatment and prevent further spread. Objectives: This study aimed to evaluate the sensitivity of the newly developed rapid carbapenemase detection method (rCDM) with isolates whose resistance genes were determined by molecular methods and to compare it with the modified carbapenem inactivation method (mCIM), whose diagnostic performance characteristics are well-defined. Methods: A total of 130 Klebsiella pneumoniae isolates (65 carbapenem-susceptible, 65 carbapenem-resistant) from various clinical specimens were included in the study. Identification and antibiotic susceptibility testing of the isolates was performed using the BD Phoenix™ automated system. Phenotypic carbapenemase detection methods, mCIM and rCDM, were studied for all carbapenem-sensitive and carbapenem-resistant isolates with known resistance genes. Results: Among carbapenem-resistant K. pneumoniae isolates, blaOXA-48 was the most frequently detected resistance gene (78%). Using PCR results as the reference method, the sensitivity of rCDM was 100%. When rCDM and mCIM results were compared, both phenotypic methods were 100% compatible, with no statistically significant difference observed. Conclusions: Rapid carbapenemase detection method is a rapid and reliable phenotypic carbapenemase detection method with high sensitivity. Its ease of use and interpretation make it suitable for use in laboratories with limited resources. Additionally, its excellent performance in detecting common carbapenemase types and high consistency with mCIM support its utility in various laboratory settings without requiring additional materials or technical infrastructure.

Complete Genome Sequence of a Klebsiella pneumoniae Strain Carrying Novel Variant blaKPC-203, Cross-Resistant to Ceftazidime/Avibactam and Cefiderocol, but Susceptible to Carbapenems, Isolated in Italy, 2023.

Klebsiella pneumoniae is a concerning pathogen, responsible for hospital-associated outbreaks. Multi drug resistant (MDR) strains are especially hard to treat. We conducted whole-genome sequencing on a MDR K. pneumoniae strain in order to identify genomic features potentially linked to its phenotype. DNA sequencing was performed on the Illumina iSeq 100 platform. Genome assembly was carried out with SPAdes. The genome was annotated with RASTtk. Typing was performed with MLST and Kaptive. Antibiotic resistance genes were detected with AMRFinderPlus and Abricate, and further verified with BLAST. The strain exhibited resistance to ceftazidime/avibactam and cefiderocol, but remained susceptible to carbapenems. The strain belonged to sequence type ST101, serotype O1:K17. The analysis of antibiotic resistance genes indicated that the strain carried a novel KPC variant, designated as KPC-203, featuring a EL deletion at amino acid position 166-167, within the Ω-loop, and a nine-amino-acid insertion (LAVYTRAPM) at position 259. Sequence alterations were found in porin genes ompK35 and ompK36. Unlike molecular testing, which was able to detect the KPC-203 variant, all phenotypic carbapenemase detection methods achieved negative results. KPC-203, a novel KPC variant, showed a sequence modification in a cephalosporin resistance-associated hotspot. Interestingly, such alterations typically correlate with the restoration of carbapenem susceptibility. We hypothesize that KPC-203 likely led to resistance to ceftazidime/avibactam and cefiderocol, while maintaining susceptibility to carbapenems.

A comparative evaluation of five phenotypic methods for identification of carbapenemase-producing Enterobacteriaceae: a modified carbapenemase detection test.

Rapid detection of carbapenemase-producing Enterobacteriaceae (CPE) is urgently needed to prevent their spread in healthcare settings. Here, we have evaluated the performance of the phenotypic methods for detection of carbapenemase production directly from bacterial cultures. A total of 99 clinical and rectal Enterobacteriaceae isolates were included (81 carrying known carbapenemase-encoding genes and 18 without carbapenemase production). All isolates were subjected to the five phenotypic tests including in-house Carba NP (iCarba NP), modified-Carba NP, E-Test MBL, modified Hodge test (MHT), and commercial combination disk test. Test results were read at different time points for iCarba NP and modified-Carba (1 min, 5 min, 15 min, 1 h and 2 h). The sensitivity and specificity of the iCarba NP were 78.87% and 100%, respectively, whereas those of the modified-Carba NP test were 95.06% and 94.44%, respectively. False-negative results were detected in four OXA-48 isolates with the use of modified-Carba NP, whereas one non-carbapenemase isolate had false-positive results. The sensitivity/specificity was 91.30%/100% and 80.25%/83.33% for the E-Test MBL and MHT, respectively. The sensitivity and specificity of the aminophenylboronic acid synergy test were 100% and 97.94%, respectively, whereas those of the dipicolinic acid synergy test were 82.61% and 96.23%, respectively. Rapid, simple, and reliable methods are needed for laboratory detection of CPE isolates to improve the detection and surveillance of these clinically relevant pathogens in an epidemiological context. We conclude that the modified-Carba NP test can be one of the reliable tests for the prediction of carbapenemase-producing bacteria.IMPORTANCEThe emergence of carbapenem resistance among Gram-negative bacteria is a serious global health threat. Here, we investigate the performance of the five phenotypic assays against carbapenemase-producing and carbapenemase-non-producing Enterobacteriaceae. Accurate and rapid detection of CPE isolates is critically required for clinical management and treatment of infections caused by these organisms. Among the five evaluated phenotypic tests, the mCNP test presented the highest sensitivity (95.06%) and, therefore, can be considered the best test to be used as a screening phenotypic methodology.

Prevalence of Extended-spectrum Beta-lactamase (ESBL), Metallo Beta-lactamase (MBL) and Carbapenemase Producing Acinetobacter Species Isolated from Various Clinical Samples in Tertiary Care Hospital

Acinetobacter is an important nosocomial pathogen causing health care associated infections. It is highly antibiotic resistant gram-negative bacilli. The study was done to determine the prevalence of Acinetobacter species isolated from various clinical samples with their antibiotic susceptibility pattern. To determine the antimicrobial susceptibility pattern of the isolated Acinetobacter species and also the multidrug resistant mechanisms by phenotypic characterization. The retrospective study was carried out in patients diagnosed with Acinetobacter infection in the Microbiology Department, Krishna Institute of Medical Sciences, Krishna Vishwa Vidyapeeth, Deemed To Be University, Karad, a tertiary care hospital, including the clinical departments, during the period of two years from November 2020 till November 2022. Organism identification, biochemical test, antibiotic resistance pattern and phenotypic tests such as ESBL, MBL and Carbapenemase production were performed as per the Clinical and Laboratory Standards Institute (CLSI). The Modified Hodge test (MHT) was performed for Carbapenemase production detection in Acinetobacter species. A total of 150 Acinetobacter species were isolated from clinical samples. Acinetobacter baumannii was the most prevalent species 138 (92%), followed by Acinetobacter lwoffii 10 (7%) and Acinetobacter hemolyticus 2 (1%). The isolates showed highest resistance to Ampicillin 130 (87%) and sensitive to Colistin 113 (75.3%). Most of the isolates of of Acinetobacter baumannii showed maximum ESBL 21(14%) and MBL 75 (93%) production. Modified Hodge test showed positive results in Acinetobacter baumannii, only 11 (7%). The study showed that Acinetobacter baumannii was the most prevalent species showing drug resistance by phenotypic detection methods. At present Acinetobacter is a frequent pathogen in hospital acquired infections in critically ill patients admitted to ICU. The isolates of Acinetobacter species in our study showed resistant to most commonly used antibiotics. The study showed that ESBL production in Acinetobacter was 22 (15%) and MBL 80 (53%). Most Acinetobacter isolates were Multi Drug Resistant (MDR). MDR Acinetobacter is widely increasing due to inappropriate use of antibiotics in healthcare hospital. In our study, detection of carbapenemase by Modified Hodge test was positive in 11 (7%) isolates.

Evaluation of Phenotypic Tests for Carbapenemase Detection in Enterobacteriaceae in Tunisia.

Introduction: Resistance to carbapenems in Enterobacteriaceae is a challenge for public health. Carbapenemase production is the leading mechanism. This work aims to evaluate four phenotypic methods for carbapenemase detection in comparison with a molecular method. Materials and Methods: Thirty-seven nonrepeating Enterobacteriaceae strains with decreased susceptibility to ertapenem were included. Imipenem MIC, Modified Hodge Test (MHT), Neo-Rapid Carb Kit® and KPC, MBL, and OXA-48 Confirm Kit® were performed. Isolates were tested for blaOXA-48, blaNDM, and blaVIM genes by end-point polymerase chain reaction. The results of the molecular study were used as a reference test to determine the performances of the phenotypic tests. Results: Imipenem resistance does not seem to be a good marker for carbapenemase production with a sensitivity of 54% (95% CI: 38-71). MHT showed 82% sensitivity (95% CI: 65-91). Overall, the enzymatic test showed the best performances for carbapenemase detection with 100% sensitivity (95% CI: 89-100) and the best turnaround time. The characterization of carbapenemases classes by the combined discs test demonstrated 88% overall sensitivity (95% CI: 72-95). Conclusion: The results of this study support the combination of the enzymatic and the combined disc tests for carbapenemase detection in Enterobacteria.

Comparison of two invitro phenotypic methods (Tissue Culture plate and Congo Red Agar) for Detection of Biofilm Formation by Enterococci

Comparison of two invitro phenotypic methods (Tissue Culture plate and Congo Red Agar) for Detection of Biofilm Formation by Enterococci

BIOFILM FORMATION AND VIRULENCE FACTORS DISTRIBUTION AMONG CLINICAL ISOLATES OF Pseudomonas aeruginosa

Introduction: Pseudomonas aeruginosa is an opportunistic microorganism frequently isolated in the hospital environment and has been classified by the World Health Organization as a critical pathogen due to its resistance to antibiotics and its varied virulence factors. Objective: To evaluate biofilm formation and the expression of virulence factors of 30 clinical isolates and 2 standard strains of P. aeruginosa. In addition, this work aims to analyze the distribution of virulence factors in the different categories of biofilm formation. Methodology: Biofilm formation assays were performed in polystyrene microplate with crystal violet coloring and the strains were classified in groups. Other virulence factors were studied with specific phenotypic detection methods. Results: All P. aeruginosa were able to form biofilms, with 46.9% classified as strong formers, 25% as moderate and 28.12% as weak biofilm formers. Most isolates expressed the virulence factors analyzed and among them, pyoverdine was the most prevalent, followed by the production of lipases B, hemolysin, and protease. Lipases A showed the lowest incidence. Conclusion: Although no significant correlation was detected between biofilm formation of P. aeruginosa clinical isolates and virulence factors, it was observed that in the strong formers group there was expression of alkaline protease and lipases B in all strains. The results suggest that the pathogenicity of P. aeruginosa is a multifactorial phenomenon and that virulence factors, although present in most isolates, occur independently of the biofilm formation category of the studied strains.

Assessment of four in vitro phenotypic biofilm detection methods in relation to antimicrobial resistance in aerobic clinical bacterial isolates.

The lack of standardized methods for detecting biofilms continues to pose a challenge to microbiological diagnostics since biofilm-mediated infections induce persistent and recurrent infections in humans that often defy treatment with common antibiotics. This study aimed to evaluate diagnostic parameters of four in vitro phenotypic biofilm detection assays in relation to antimicrobial resistance in aerobic clinical bacterial isolates. In this cross-sectional study, bacterial strains from clinical samples were isolated and identified following the standard microbiological guidelines. The antibiotic resistance profile was assessed through the Kirby-Bauer disc diffusion method. Biofilm formation was detected by gold standard tissue culture plate method (TCPM), tube method (TM), Congo red agar (CRA), and modified Congo red agar (MCRA). Statistical analyses were performed using SPSS version 17.0, with a significant association considered at p<0.05. Among the total isolates (n = 226), TCPM detected 140 (61.95%) biofilm producers, with CoNS (9/9) (p<0.001) as the predominant biofilm former. When compared to TCPM, TM (n = 119) (p<0.001) showed 90.8% sensitivity and 70.1% specificity, CRA (n = 88) (p = 0.123) showed 68.2% sensitivity and 42% specificity, and MCRA (n = 86) (p = 0.442) showed 65.1% sensitivity and 40% specificity. Juxtaposed to CRA, colonies formed on MCRA developed more intense black pigmentation from 24 to 96 hours. There were 77 multi-drug-resistant (MDR)-biofilm formers and 39 extensively drug-resistant (XDR)-biofilm formers, with 100% resistance to ampicillin and ceftazidime, respectively. It is suggested that TM be used for biofilm detection, after TCPM. Unlike MCRA, black pigmentation in colonies formed on CRA declined with time. MDR- and XDR-biofilm formers were frequent among the clinical isolates.

Phenotypic Detection of Carbapenemase Production in Gram Negative Bacilli from Clinical Isolates in a Tertiary Care Hospital in Telangana

Antimicrobial resistance (AMR) in Gram negative bacteria (GNB) has become a critical health concern across the globe. Unveiling of β-lactamase, extended spectrum β-lactamase (ESBL) and AmpC β-lactamase producing bacteria has led to the development of multidrug-resistant organisms (MDRO’s). Carbapenems are considered to be very effective in morbid infections caused by MDRO’s. Now, the upsurge of carbapenem resistance among GNB is an issue of concern as these infections are very difficult to treat. Rapid and reliable methods to detect these CPO’s in all Microbiology laboratories is essential to streamline the antimicrobial therapy. Accordingly, this study is conducted to determine the enormity of CPO’s among clinical isolates by various phenotypic tests along with differentiation of serine β-lactamases from metallo-β-lactamases. This is a Prospective Cross-sectional study meticulously planned &amp; conducted for a period of one year. Among the 76 suspected Carbapenemase Producing Organisms (CPO’s), 42% were Klebsiella spp. followed by Escherichia coli (25%), Pseudomonas aeruginosa (24%), Citrobacter spp. (5%) and Proteus spp. (4%). Out of the total isolates 82% of the isolates were confirmed as CPO’s by Carba NP test, whereas 93% by mCIM test. 53% of the total isolates tested were Serine-β-lactamase producers and 41% were Metallo-β-lactamase producers. In conclusion, Carba NP test and mCIM in conjunction with eCIM test could be considered as reliable phenotypic diagnostic methods for carbapenemase detection to guide the clinicians for initiating antibiotic therapy.

Phenotypic methods for detection of metallo-β-lactamase (MBL) production in multidrug resistant gram negative bacilli – comparative study

Introduction: One of the most common mechanism of resistance of bacterial pathogens to β-lactam antibiotics is production of β-lactamase, there are different types of Beta lactamases, which are expressed by drug resistant gram-negative bacteria. Carbapenemases (Metallo beta lactamases/MBL) are the β-lactamases with the widest spectrum of activity. Early detection of MBL-producing organisms is crucial to establish appropriate antimicrobial therapy and to prevent their interhospital and intrahospital dissemination. Several phenotypic methods are available for the detection of MBL producing bacteria. As there is no standardized method present study was done to screen MDR gram negative bacilli isolated from clinical samples for MBL-production by a low cost, convenient and sensitive procedure. Methods: All non-duplicate MDR gram negative isolates obtained from various clinical samples were screened for carbapenam resistance. All carbapenam resistant bacteria were screened for production of MBL by 3 phenotypic tests (Double disc synergy, combined disk test, Hodge test). The results were compared and analyzed on the basis of results obtained by E test. Results: During Study Period, 988 non duplicate gram negative bacilli were isolated, 70.64% (698) were multi drug resistant. Amongst Total number of MDR Isolates to carbapenam resistance was seen in 62(9.28%). These 62 isolates that were resistant to carbapenam were tested for MBL production. 54 (87%) of these 62 isolates showed MBL production by disc potentiation test whereas 41 isolates (66%) gave positive result by DDST. By Modified Hodge test, out of 62, 48 isolates (77.4%) were MBL positive. Compared to E- test, the Sensitivity Specificity and Accuracy for Disc potentiation test was 90%,100% and 90.32%, for Modified Hodge test was 80%,100% and 80.6% and for Double disc synergy test was 68.3%,100% and 69.3%. Conclusion: In our study, in comparison to MBL E test, disc potentiation test is more sensitive than double disc synergy test and Modified Hodge test for detection of MBL phenotypically

Treatment outcomes with benzylpenicillin and non-benzylpenicillin antibiotics, and the performance of the penicillin zone-edge test versus molecular detection of blaZ in penicillin-susceptible Staphylococcus aureus (PSSA) bacteraemia.

The blaZ gene encodes penicillinase, which inactivates penicillin. As there were reports on suboptimal sensitivity for the penicillin zone-edge test, a phenotypic method for blaZ detection, we investigated treatment outcomes in patients with penicillin-susceptible Staphylococcus aureus (PSSA) bacteraemia (phenotypically negative for penicillinase), subjecting isolates to molecular testing for blaZ retrospectively. A retrospective cohort study was conducted on 121 patients with a first episode of PSSA bacteraemia from 1 January 2012 to 31 October 2015 at Tan Tock Seng Hospital (TTSH), Singapore. Patients were grouped into IV benzylpenicillin and non-benzylpenicillin groups. The primary outcome was overall treatment failure, defined as either 30 day all-cause mortality and/or 90 day relapse. The penicillin (P10) zone-edge test was repeated on archived PSSA isolates, concurrently with penicillin MIC determination via gradient diffusion and PCR for blaZ. Among 121 patients, 57 patients (47.1%) received IV benzylpenicillin as the predominant antibiotic. There was no significant difference in overall treatment failure between treatment with the benzylpenicillin [7/57 (12.3%)] versus non-benzylpenicillin groups [12/64 (18.8%)] (P = 0.33) or cloxacillin/cefazolin [6/37 (16.2%)] (P = 0.59). For 112 PSSA isolates available for testing, repeat penicillin zone-edge testing was negative for penicillinase production, corroborating previous results. A single PSSA isolate with a negative penicillin zone-edge test was found to be positive for blaZ. We found no differences in overall treatment failure between patients with PSSA bacteraemia treated with benzylpenicillin, anti-staphylococcal β-lactams cefazolin/cloxacillin and other antimicrobials, when using the penicillin zone-edge test as the phenotypic method for blaZ screening.

The optimized carbapenem inactivation method for objective and accurate detection of carbapenemase-producing Acinetobacter baumannii.

The modified carbapenem inactivation method (mCIM) recommended by the Clinical and Laboratory Standards Institute is not applicable for detecting carbapenemases in Acinetobacter baumannii. Four currently reported phenotypic detection methods, namely, the modified Hodge test, the mCIM, the adjusted mCIM, and the simplified carbapenem inactivation method (sCIM), did not perform well in our 90 clinical A. baumannii isolates. Thus, the minimal inhibitory concentrations (MICs) of carbapenems and the existence and expression of carbapenemase-encoding genes were detected to explain the results. According to the E-test, which was more accurate than the VITEK 2 system, 80.0 and 41.1% were resistant to imipenem (IPM) and meropenem (MEM), respectively, and 14.4 and 53.3% exhibited intermediate resistance, respectively. Five β-lactamase genes were found, of which blaOXA-51-like, blaTEM, and blaOXA-23-like were detected more frequently in 85 non-susceptible strains. The expression of blaOXA-23-like was positively correlated with the MIC values of IPM and MEM. Therefore, an improved approach based on the mCIM, designated the optimized CIM (oCIM), was developed in this study to detect carbapenemases more accurately and reproducibly. The condition was improved by evaluating the factors of A. baumannii inoculum, incubation broth volume, and MEM disk incubation time. Obvious high sensitivity (92.94%) and specificity (100.00%) were obtained using the oCIM, which was cost-effective and reproducible in routine laboratory work.

ASSESSMENT OF ANTIBIOTICS RESISTANCE AND BIOFILM PRODUCTION AMONG BACTERIAL SPECIES ISOLATED FROM CONTACT LENSES

Contact lenses (CLs) wearing has been increased globally during recent decades, which is one of the main risk factors for developing several ocular infections. Resistant CLs bacterial infections are mainly due to the CLs contamination by bacteria producing biofilm. This study was aimed to assessment of antibiotics resistance and biofilm production among bacterial species isolated from contact lenses in Mukalla city, Hadhramout, Yemen. This cross-sectional study was carried out on 298 participants women during a period from October 2022 to January 2023. The CLs swab samples were collected, then inoculated onto culture media and incubated aerobically at 37°C for 24 hrs. The bacterial isolates were identified by conventional bacteriological methods of cultural characteristics, Gram staining and biochemical test. Antibiotics susceptibility testing was performed by disc diffusion method. Bacterial biofilm production on CLs was detected by tube method (TM) and Congo red agar (CRA) method. The prevalence of CLs bacterial infection was 54.4%. Enterobacter spp. 37.1%, followed by Escherichia coli 28.4%, Pseudomonas aeruginosa 11.7%, Klebsiella pneumoniae 6.8% were the most common Gram-negative isolated from CLs. Staphylococcus epidermides 3.7% and other coagulase negative staphylococci (CoNS) 12.3% were the most common species of Gram-positive bacteria isolated from CLs. The CRA method was found to be effective phenotypic screening method for detection of biofilm production of bacterial isolates from CLs. Prevalence of antibiotics resistance and multi-drug resistance (MDR) biofilm producing strains was found. In conclusion, there is a high prevalence of CLs use by females in Mukalla city, Hadhramout especially for cosmetic purposes. CLs infection due to improper care practices leads to eye complications. Increasing awareness is crucial to avoid identified risk factors for ocular infection.

The Role of Efflux Pumps in Antibiotic Resistance of Gram Negative Rods

Antibiotic resistance is an important public health problem today, causing increased morbidity and mortality. Resistance to antibiotics in bacteria can develop by various mechanisms such as a change in the target site of the drug, a change in the outer membrane permeability, enzymatic defusing of the drug and efflux of the antimicrobial compound. Some bacteria have the potential to develop resistance to more than one drug by using several mechanisms together. One of the important resistance mechanisms of bacteria is active efflux pumps (EPs). EPs are pump proteins found in all cell types, located in the cell membrane. They are responsible for the excretion of various intracellular and extracellular substances (antibiotics, etc.) out of the cell. There is much research on various antimicrobials that cause antibiotic resistance in Gram negative rods, but studies on EPs are relatively few. Due to the concern that antibiotics will be insufficient in the treatment of diseases, a good understanding of EPs and the discovery of new EP inhibitors will shed light on the future of humanity. In this review, the structure of bacterial EPs in Gram negative bacteria, the role of EPs in multidrug resistance, the importance of EP inhibitors in the fight against antibiotic resistance and the phenotypic and genotypic detection methods of EPs are discussed.

MCC-Net: A class attention-enhanced multi-scale model for internal structure segmentation of rice seedling stem

Internal structural parameters of rice seedling stems are of great significance for rice growth detection, rice selection, breeding, and damage examination. Aiming at the problems of non-repeatability and low detection accuracy in the existing plant internal structure phenotypic traits detection methods, this paper presents a non-destructive segmentation method for examining the internal structure of rice seedling stems based on deep learning. We use a standard X-ray CT imaging technology to obtain non-destructive tomographic images of rice seedling stems and then design a class attention-enhanced multi-scale segmentation model (MCC-Net), where UNet is used as the backbone network. Specifically, the proposed MCC-Net mainly consists of three core components: multi-scale convolutional block (MCB), coordinate spatial attention (CSA) module, and class attention enhancement (CAE) module. MCB is the main component of the encoder to improve the feature extraction ability of the model for regions of different sizes in the internal structure. CSA is embedded into the UNet skip connections to enhance the expression of effective features and automatically locate the regions with different structures of rice seedling stems. CAE is designed to calculate the dependencies between image pixels and categories, which can enhance the feature expression from the perspective of categories and correct the category errors in the segmentation results. The experimental results show that MIOU, average dice coefficient and average precision of our proposed MCC-Net model on the self-built rice seedling stem CT image dataset are 92.56%, 96.33% and 96.59% respectively. Compared with several state of the art models, the proposed model achieves better segmentation performance on the rice seedling stem CT image dataset.

Genetic and Phenotypic Characterization of the First Canadian Case of Ambler Class A Carbapenemase FRI-8

This study investigated the mechanism of carbapenem resistance in an Enterobacter cloacae complex positive by the modified carbapenem inactivation method (mCIM) but negative by the Rosco Neo-Rapid Carb Kit, β CARBA, and conventional PCR for common carbapenemase genes (KPC, NDM, OXA-48, IMP, VIM, GES, and IMI/NMC). Using whole genome sequencing (WGS) data we confirmed the identification of Enterobacter asburiae (ST1639) and the presence of blaFRI-8 located on a 148kb IncFII(Yp) plasmid. This is the first occurrence of a clinical isolate harboring the FRI-8 carbapenemase and the second occurrence of FRI in Canada. This study highlights the need to use both WGS and phenotypic screening methods for detection of carbapenemase-producing strains if we consider the growing diversity of carbapenemases.

BETA-LACTAMASES: PHENOTYPIC IDENTIFICATION METHODS

Today, β-lactam antibiotics are the most widely used. This is due to their low toxicity, wide spectrum of action and variety of drugs. Therefore, the bacterial resistance factor in the form of β-lactamase production is of particular interest. Bacteriological laboratories have classical methods for detecting β-lactamases. However, the combination of bacterial production of enzymes of different classes, together with the overexpression of some of them, causes difficulties in recognizing the production of extended-spectrum enzymes, which is critical for making clinical decisions. Currently known phenotypic detection methods for β-lactamases and extended-spectrum carbapenemases, when performed correctly, allow precise elucidation of the mechanism of resistance. However, these methods are very labor-intensive, difficult to implement and require highly qualified staff of bacteriological laboratories. Perhaps methods based on molecular diagnostic approaches, due to automation and standardization, high accuracy and specificity in identifying β-lactamase genes, reducing research time, and the ability to identify uncultivable or slowly growing microorganisms, could simplify the determination of mechanisms of antibiotic resistance in the future.