E-test Method Research Articles

Pannonibacter anstelovis sp. nov. Isolated from Two Cases of Bloodstream Infections in Paediatric Patients.

This study describes two cases of bacteraemia sustained by a new putative Pannonibacter species isolated at the U.O.C. of Microbiology and Virology of the Policlinico of Bari (Bari, Italy) from the blood cultures of two patients admitted to the Paediatric Oncohaematology Unit. Pannonibacter spp. is an environmental Gram-negative bacterium not commonly associated with nosocomial infections. Species identification was performed using Sanger sequencing of the 16S rRNA gene and Whole-Genome Sequencing (WGS) for both strains. Genomic analyses for the two isolates, BLAST similarity search, and phylogeny for the 16S rDNA sequences lead to an assignment to the species Pannonibacter phragmitetus. However, by performing ANIb, ANIm, tetranucleotide correlation, and DNA-DNA digital hybridization, analyses of the two draft genomes showed that they were very different from those of the species P. phragmitetus. MALDI-TOF analysis, assessment of antimicrobial susceptibility by E-test method, and Analytical Profile Index (API) tests were also performed. This result highlights how environmental bacterial species can easily adapt to the human host and, especially in nosocomial environments, also gain pathogenic potential through antimicrobial resistance.

Role of MIC levels and 23S rRNA mutation sites to clarithromycin in 14-day clarithromycin bismuth quadruple therapy for Helicobacter pylori eradication: A prospective trial in Beijing

BackgroundRising clarithromycin resistance undermines Helicobacter pylori (H. pylori) treatment efficacy. We aimed to determine clarithromycin's minimum inhibitory concentration (MIC) levels and identify specific mutation sites in the 23S ribosomal subunit (23S rRNA) that predict treatment outcomes in a 14-day regimen of clarithromycin bismuth quadruple therapy (amoxicillin 1g, clarithromycin 500 mg, rabeprazole 10 mg, and colloidal bismuth pectin 200 mg). Materials and methodsWe included adult H. pylori patients who hadn't previously undergone clarithromycin-based treatment, either as initial or rescue therapy. Exclusions were made for penicillin allergy, recent use of related medications, severe illnesses, or inability to cooperate. Patients underwent a 14-day clarithromycin bismuth quadruple therapy. Gastric mucosa specimens were obtained during endoscopy before eradication. MIC against amoxicillin and clarithromycin was determined using the E-test method. The receiver operating characteristic (ROC) curve helped to find the optimal clarithromycin resistance MIC breakpoint. Genetic sequences of H. pylori 23S rRNA were identified through Sanger Sequencing. (ChiCTR2200061476) ResultsOut of 196 patients recruited, 92 met the inclusion criteria for the per-protocol (PP) population. The overall intention-to-treat (ITT) eradication rate was 80.00 % (84/105), while the modified intention-to-treat (MITT) and PP eradication rates were 90.32 % (84/93) and 91.30 % (84/92) respectively. No amoxicillin resistance was observed, but clarithromycin resistance rates were 36.19 % (38/105), 35.48 % (33/93), and 34.78 % (33/92) in the ITT, MITT, and PP populations respectively. Compared with the traditional clarithromycin resistance breakpoint of 0.25 μg/mL, a MIC threshold of 12 μg/mL predicted better eradication. Among 173 mutations on 152 sites in the 23S rRNA gene, only the 2143A > G mutation could predict eradication outcomes (p < 0.000). ConclusionsInterpretation of elevated MIC values is crucial in susceptibility testing, rather than a binary "susceptible" or "resistant" classification. The 2143A > G mutation has limited specificity in predicting eradication outcomes, necessitating further investigation into additional mutation sites associated with clarithromycin resistance.

Clinical distribution of carbapenem genotypes and resistance to ceftazidime-avibactam in Enterobacteriaceae bacteria.

Bacterial resistance is a major threat to public health worldwide. To gain an understanding of the clinical infection distribution, drug resistance information, and genotype of CRE in Dongguan, China, as well as the resistance of relevant genotypes to CAZ-AVI, this research aims to improve drug resistance monitoring information in Dongguan and provide a reliable basis for the clinical control and treatment of CRE infection. VITEK-2 Compact automatic analyzer was utilized to identify 516 strains of CRE collected from January 2017 to June 2023. To determine drug sensitivity, the K-B method, E-test, and MIC methods were used. From June 2022 to June 2023, 80 CRE strains were selected, and GeneXpert Carba-R was used to detect and identify the genotype of the carbapenemase present in the collected CRE strains. An in-depth analysis was conducted on the CAZ-AVI in vitro drug sensitivity activity of various genotypes of CRE, and the results were statistically evaluated using SPSS 23.0 and WHONET 5.6 software. This study identified 516 CRE strains, with the majority (70.16%) being K.pneumoniae, followed by E.coli (18.99%). Respiratory specimens had highest detection rate with 53.77% identified, whereas urine specimens had the second highest detection rate with 17.99%. From June 2022 to June 2023, 95% of the strains tested using the CRE GeneXpert Carba-R assay possessed carbapenemase genes, of which 32.5% were blaNDM strains and 61.25% blaKPC strains. The results showed that CRE strains containing blaKPC had a significantly higher rate of resistance to amikacin, cefepime, and aztreonam than those harboring blaNDM. The CRE strains isolated from Dongguan region demonstrated a high resistance rate to various antibiotics used in clinical practice but a low resistance rate to tigecycline. These strains produce Class A serine carbapenemases and Class B metals β-lactamases, with the majority of them carrying blaNDM and blaKPC. Notably, CRE strains with blaKPC and blaNDM had significantly lower resistance rates to tigecycline. CAZ-AVI showed a good sensitivity rate with no resistance to CRE strains carrying blaKPC. Therefore, CAZ-AVI and tigecycline should be used as a guide for rational use of antibiotics in clinical practice to effectively treat CRE.

Determination of Virulence Factors and Antibiotic Resistance Profile of E. coli O157:H7 in some Marine and Freshwater Fish from Samsun, Türkiye

In this study, Escherichia coli O157:H7 contamination was investigated in Engraulis encrasicolus, Merlangius euxmus, Trachurus mediterraneus, Mullus barbutus, Tinca tinca, Cyprinus carpio and Oncorchynhus mykiss (total of 300 samples). The presence of certain virulence genes such as shiga toxin 1 (stx1), shiga toxin 2 (stx2), intimin (eaeA), and hemolysin (hlyA) was determined. The E-test method was employed to determine the antibiotic resistance profile and minimum inhibitory concentration (MIC) values of the isolates. Out of a total 141 freshwater fish samples, E. coli O157:H7 was detected in 5 of them (3.5%). In 159 marine fish, 2 of them (1.25%) were E. coli O157 positive. In terms of virulence characteristics, the E. coli O157:H7 isolate obtained from the Oncorhynchus mykiss sample had the stx1, eaeA, and hlyA genes. Both eaeA and hlyA genes were determined as positive in the E. coli O157 isolate identified from the Merlangius euxmus sample. Resistance to tetracycline, cefotaxime, cefepime, ciprofloxacin, gentamicin, ertapenem and cefoxitin was demonstrated in both samples. In this study, the presence of E. coli O157 in fish, detection of virulence genes and antibiotic resistance profile indicate the potential risk of infection from contaminated seafood.

Antibiotic Resistance of Streptococcus spp. Isolated from the Root Surface of Extracted Teeth to Penicillin V and Clindamycin Using E-Test

Background: Antibiotics are used to treat and prevent oral infections. Clindamycin and penicillin are widely used drugs in this field. Poor use of antibacterial agents causes the emergence of antibiotic-resistant bacteria. Objectives: The present study examined and compared the frequency distribution of resistance of streptococci isolated from the root surface of extracted teeth to penicillin V and clindamycin. This study was conducted in 2021 in Yazd, Iran. Methods: This was an analytical cross-sectional study performed in vitro. The samples were taken from the root surfaces of 50 teeth immediately after extraction and transferred to the laboratory in a transfer medium. After cultivation, isolation, and identification, the antibiotic resistance of isolates was measured by the E-test method. The data were analyzed using SPSS software (version 23). Results: Streptococci spp. were isolated from 46 samples (92%) without environmental contamination. The minimum inhibitory concentration (MIC) 50 and MIC 90 for penicillin V were 0.5 μg/mL and 1 μg/mL, respectively, and for clindamycin, 0.125 μg/mL and 0.19 μg/mL, respectively, which was significantly (P = 0.0001) lower for clindamycin. Intermediate sensitivity to penicillin V was 95.6%, and resistance to clindamycin was 21.8%. Conclusions: It seems that except in limited cases where there is resistance to clindamycin, this antibiotic is a more effective drug to control the bacteria in the mouth. However, in some cases, aerobic isolates showed intermediate sensitivity to penicillin V; instead, resistance to clindamycin was observed.

Colistin monotherapy or combination for the treatment of bloodstream infection caused by Klebsiella pneumoniae: a systematic review and meta-analysis.

Bloodstream infection of Klebsiella pneumoniae (BSI-KP) were associated with increased mortality. Klebsiella pneumoniae was tested to susceptible to colistin by E-test and broth microdilution method in clinical laboratory. This study aimed to assess the efficacy of colistin versus tigecycline, carbapenem monotherapy and combination in the treatment of BSI-KP. Electronic databases such as PubMed, Web of Science and Embase were searched. The last search was in November 24th, 2022, addressing the colistin, carbapenems and tigecycline monotherapy and combination treatments in patients with BSI-KP. The primary outcomes were 30-day or 28-day mortality. OR where available with 95% CI were pooled in random-effects meta-analysis. Following the outlined search strategy, a total of 658 articles were identified from the initial database searching. Six studies, 17 comparisons were included. However, they all were observational design, lacking high-quality randomized controlled trials (RCTs). Moderate or low-quality evidences suggested that colistin monotherapy was associated with an OR = 1.35 (95% CI = 0.62-2.97, P = 0.45, Tau2 = 0.00, I2 = 0%) compared with tigecycline monotherapy, OR = 0.81 (95% CI = 0.27-2.45, P = 0.71, Tau2 = 0.00, I2 = 0%) compared with carbapenem monotherapy. Compared with combination with tigecycline or carbapenem, Colistin monotherapy resulted in OR of 3.07 (95% CI = 1.34-7.04, P = 0.008, Tau2 = 0.00, I2 = 0%) and 0.98 (95%CI = 0.29-3.31, P = 0.98, Tau2 = 0.00, I2 = 0% ), respectively. Colistin, carbapenem and tigecycline monotherapy showed similar treatment effects in patients who suffered from BSI-KP. Compared with colistin monotherapy, colistin combined tigecycline therapy might play the synergism effects. retrospectively registered.

Neisseria gonorrhoeae antimicrobial resistance patterns and associated risk factors in women of childbearing potential in northwestern Ethiopia

BackgroundsNeisseria gonorrhoeae causes gonorrhea and poses public health problems, including antimicrobial resistance. Current data on gonorrhea in prenatal participants in the study area are required. Thus, we aimed to identify gonorrhea prevalence, antimicrobial resistance, and risk factors among antenatal care clinic visitors in northwestern Ethiopia.MethodsA cross-sectional study was conducted from March to August 2022 at the University of Gondar Comprehensive Specialized Hospital. We recruited 278 study participants using convenient sampling techniques. Sociodemographic, clinical and behavioral risk factors were recorded using pre-tested questionnaires. Endocervical swabs were collected by a physician, transported to the microbiology laboratory, immediately inoculated into modified Thayer-Martin medium, and it was incubated at 37 °C for 24–48 hours. Gram staining and biochemical tests were used to identify the organism. AMR testing was performed using disc diffusion and E-test methods. Data were entered in EPI-info version 7 and exported and analyzed in SPSS version 26. A p-value ≤0.05 was considered as statistically significant. Results were presented in words, tables and figure.ResultsOf 278 subjects enrolled, majority (44.6%) were 26–35 years, with a mean age of 29.9 (SD = ±7.2) years, 69.4% were urban residents, and 70.5% were married. Twenty-one (7.6%) participants had gonorrhea. Overall antimicrobial resistance ranged from 19 to 100%. High resistant to tetracycline (100%) and penicillin (85.7%) were observed by both tests. Ciprofloxacin resistance was 52.4% by disc diffusion and 85.7% by E-test. By E-test, all isolates were sensitive to ceftriaxone, cefixime, azithromycin and spectinomycin; however, 7 (33.3%), 9 (42.9%), 9 (42.9%) and 5 (23.8%) isolates showed resistant to these antibiotics with disk method. Prevalence of beta-lactamase producing Neisseria gonorrhoeae was 85.7%. Alcohol consumption (p = 0.032), condom-free sexual practice (p = 0.010), multiple sexual partners (p < 0.001), pelvic pain (p = 0.018), and dysuria (p = 0.021) revealed increased risk of infection.ConclusionsCompared with many previous studies in Ethiopia, we found high prevalence, antimicrobial resistance, and beta-lactamase-positive isolates. Multiple sexual partners, alcohol consumption, not using condom, pelvic pain and dysuria were predictors of this infection. Continuous large-scale monitoring of pathogen is essential for its prevention and control.

In vitro evaluation of tigecycline synergy testing with nine antimicrobial agents against Enterobacter cloacae clinical strains.

Enterobacterales (especially carbapenem-resistant) are considered an urgent threat to public health. The available antibiotic therapy is limited due to the increase of multidrug-resistant (MDR) strains. Tigecycline, a minocycline derivative, has emerged as a potential key agent in the treatment of MDR isolates. The aim of the study was to evaluate the synergistic effect of tigecycline in combination with nine antimicrobial agents-ceftazidime/avibactam, colistin, ertapenem, gentamicin, imipenem, levofloxacin, meropenem/vaborbactam, polymyxin B, and rifampicin. Eighty clinical Enterobacter cloacae strains were obtained from patients of two University Hospitals in Bialystok, Poland. The E-test method was used to determine synergistic interactions. Among all combinations, synergy was reported in 61% of cases, addition in 32%, and indifference in 7%. The highest synergy rates were observed in tigecycline combinations with: ceftazidime/avibactam (60/80; 75%), imipenem (60/80; 75%), polymyxin B (55/80; 68.75%) and rifampicin (55/80; 68.75%), while the lowest synergy rate was noted in tigecycline-levofloxacin (26/80; 32.5%). The tigecycline-gentamicin showed the highest rate of indifference; antagonism, was not observed in any combination. In conclusion, tigecycline appears more suitable for use in combination therapy rather than as monotherapy and can be effectively paired with various antimicrobial agents against MDR E. cloacae. Further research will be necessary to confirm these results.

Characterization of Verona Integron-Encoded Metallo-β-Lactamase-Type Carbapenemase-Producing Escherichia coli Isolates Collected over a 16-Year Period in Bolzano (Northern Italy).

Multidrug-resistant Escherichia coli, particularly carbapenemase producers, are a major source of concern. This study aims to investigate the long-term epidemiology of Verona integron-encoded metallo-β-lactamase (VIM)-producing E. coli in the health district of Bolzano, Northern Italy, by examining the phenotypic and genotypic characteristics of 26 isolates obtained during 2005-2020. Isolates were identified with matrix-assisted laser desorption/ionization time-of-flight, susceptibility testing was by Vitek 2, Sensititre, and Etest; carbapenemase activity was confirmed by Etest and Carbapenemase Inactivation Method (CIM) test; and the VIM-antigen was identified by the NG-Test CARBA 5. Genome sequencing was performed on an Illumina MiSeq platform. Carbapenem minimum inhibitory concentrations varied across methodologies, and overall category agreement between phenotypic methods was low. All 23 sequenced isolates contained blaVIM-1. Eleven (47.8%) isolates belonged to the clonal lineage ST131, with fimH30 being the most common subclone. In Bolzano ST131-fimH30 was present as early as 2005. While the ST131 clonal lineage predominated for the first 10 years, various clonal lineages were present, especially in subsequent years, indicating the concurrent circulation of multiple clonal lineages. Future efforts should focus on the implementation of surveillance methods, including genomic analysis, as well as the use of updated infection control strategies and antibiotic stewardship programs to prevent the spread of these carbapenem-resistant strains.

A comparative evaluation of colistin Minimum Inhibitory Concentration determination by reference broth microdilution with other commonly used phenotypic methods in Multidrug-Resistant Gram-negative bacilli

Background: over the past decade, the dependency on colistin as a last resort antibiotic has driven the global emergence of colistin resistance among many bacterial species. This study comparatively evaluated the colistin Minimum Inhibitory Concentration (MIC) by various phenotypic methods, including the reference method of Broth Microdilution (BMD), other approved methods of Colistin Broth Disk Elution (CBDE), and Colistin Agar Test (CAT) and widely available method of Epsilometer Test (E-test) among Multidrug Resistant (MDR) Gram-negative bacteria. Methods: ninety Gram-negative bacterial isolates that were resistant to three or more classes of drugs (MDR) were included in the study. All the MDR isolates were subjected to colistin susceptibility determination by BMD, CBDE, CAT, and E-test. Results: amongst 1118 samples, 90 (8.05%) samples yielded MDR Gram-negative bacilli. All the MDR Gram-negative isolates were colistin intermediate by all four methods of phenotypic colistin susceptibility. Three Acinetobacter baumannii and two Klebsiella pneumoniae isolates that had MIC of 2 μg/mL by BMD, displayed MIC of &lt;1 μg/mL by CBDE and CAT. Three isolates (2 Citrobacter koseri and 1 Enterobacter spp.) showed higher MIC by the E-strip method in comparison to BMD. Conclusions: our study holds significance, as there is a paucity of data comparing the four phenotypic methods for colistin MIC determination; BMD is the most reliable, gold standard method, but it is labor-intensive and requires technical expertise. In the present study, CBDE and CAT methods showed good concordance with BMD, and are easy to perform with limited logistics. Thus, they can be used as an alternative to BMD. We found that even though the Etest method was less accurate, it can still be used with caution to exclude the possibility of colistin resistance.

Prevalence of carbapenemases among Gram-negative bacteria in Tunisia: first report of KPC-2 producing Acinetobacter baumannii.

The rapid evolution of the antibacterial resistance problem worldwide, including the Mediterranean countries, constitutes a real threat to public health. This study aims to characterize carbapenemase encoding genes among Gram-negative bacteria collected from some Tunisian hospitals. Twenty-two clinical carbapenem-resistant Gram-negative bacteria were recovered, and identified by the matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) method. Antibiotic resistance was tested by disk diffusion method on Muller-Hinton Agar. The minimum inhibitory concentration (MIC) for imipenem was revealed by the E-test method. Carbapenemase encoding genes were screened by polymerase chain reaction (PCR). Genetic relatedness was performed by the pulsed field gel electrophoresis (PFGE) method. Our isolates, identified as K. pneumoniae (n = 7), P. mirabilis (n = 1), A. baumannii (n = 13), and P. aeruginosa (n = 1), presented high MIC values for imipenem. Enterobacerales were resistant to carbapenems due to OXA-48 production. Only, four K. pneumoniae harbored the blaNDM-1 gene. VIM-2 production was detected in P. aeruginosa. However, OXA-23 production was observed in A. baumannii isolates, one of which co-produced the KPC-2 enzyme that was identified for the first time in Tunisia in this species. A high genetic diversity was demonstrated by pulsed-field gel electrophoresis in K. pneumoniae and A. baumannii after XbaI and ApaI digestion respectively. Our findings highlight the spread of various unrelated clones of carbapenemase-producers in some Tunisian hospitals as well as the spread of several carbapenemase types.

The Causative Agents and Antibiotic Susceptibility Patterns of Healthcare-Associated Urinary Tract Infections (HAUTIs) in Isfahan, Iran: The Impact on Empiric Therapy

Background: Healthcare-associated urinary tract infections (HAUTIs) are the most prevalent healthcare-associated infection worldwide. They can lead to major problems in hospitalized patients, including the increase in the length of the hospital stay, the cost of the hospitalization, and the inappropriate administration of antibiotics. Objectives: We aim to investigate the prevalence and antibacterial sensitivity of various bacteria that cause HAUTIs. Methods: This cross-sectional study was conducted in three referral hospitals in Isfahan, Iran. Urine specimens were taken from urinary catheters of the patients with a suspected diagnosis of HA-UTI. Bacteria were identified by conventional methods, and antimicrobial sensitivity testing was performed by disk diffusion and E-test methods according to standard guidelines. Data was extracted from WHONET software, and further statistical analysis was performed by SPSS v.21. Results: Among 283 HAUTIs, Escherichia coli was the most prevalent pathogen (33.2%), followed by Enterococcus spp. (25.9%), Klebsiella pneumonia (16.6%), Pseudomonas aeruginosa (8.8%), and Acinetobacter baumannii (8.8%). Escherichia coli was more common in females. The most active antibiotics against Gram-negative isolates were nitrofurantoin (69.8%), carbapenems (69.7%), and aminoglycosides (68.3%), and the most effective antibiotics against Gram-positive isolates were linezolid (100%) and nitrofurantoin (74.5%). Conclusions: This study showed that nitrofurantoin, carbapenems, and aminoglycosides are the most effective antibiotics against Gram-negative causes of HAUTIs. Also, linezolid and nitrofurantoin demonstrated significant activity against Gram-positive isolates. As a result, for the empiric treatment of nosocomial UTI in the area under study, judicious use of the mentioned antibiotics is recommended.

In-vitro Susceptibility Testing Methods for Ceftazidime-avibactam against Carbapenem-resistant Enterobacterales: Comparison with Reference Broth Microdilution Method.

β-lactam antibiotics, mainly cephalosporins, and carbapenems, have been the mainstay of treatment for infections caused by Enterobacterales. However, their role in treating clinical infections has become limited because of the increase in resistance. There is a need to have cost-effective and rapid methods for antimicrobial susceptibility testing methods for newer antibiotics like ceftazidime-avibactam against carbapenem-resistant Enterobacterales (CRE), which can be applied in routine clinical microbiology laboratories. With this aim, the present study was conducted to compare the disk diffusion and gradient diffusion, i.e., the E-test method with the reference broth microdilution (BMD) method for in-vitro testing of ceftazidime-avibactam against CRE. A total of 111 CRE isolates from various clinical samples were included. Conventional PCR (Polymerase Chain Reaction) was done for the detection of genes encoding carbapenemases and to see their expression, modified carbapenem inactivation method (mCIM) along with EDTA (Ethylenediaminetetraacetic acid) carbapenem inactivation method (eCIM) was done. 42.3% (47/111) isolates were resistant to ceftazidime-avibactam by the standard broth microdilution method; however, 45.9% (51/111) were resistant by both disk diffusion and E-test. In 5.4% of isolates (similar in both methods), microbroth dilution method results did not match with E-strip and disk diffusion. Very major errors (VME) by both disk diffusion and E-test were found in 2.1% (1/47), and major errors (ME) were found in 7.8% (5/64) isolates (similar isolates in both methods). The overall categorical agreement (CA) rate was 94.6% for both E-test and disk diffusion, and the essential agreement (EA) rate was 90.1% (100/111) for E-test. 98% (109/111) of CRE harbored carbapenemase genes either singly (30.3%) or in combination with others (69.7%). In conclusion, for CRE, E-test and the disk diffusion method for ceftazidimeavibactam depicted an acceptable performance as an alternative to the reference broth microdilution method.

Microbiological Profiles and Inflammatory Biomarkers of Bacteremia in Children in a Teaching Hospital in Kuwait: An 8-Year Retrospective Study

Objectives: The objectives of this study were to determine the bacterial profiles and prevalence of antibiotic resistance patterns of bacteria causing bacteremia in febrile children and to compare levels of inflammatory markers between children with and without bacteremia in Kuwait from 2015 to 2022. Materials and Methods: Isolates from all episodes of significant bacteremia (n = 96) during the study period were recorded and evaluated. Microorganisms were identified using standard microbiological methods. Antimicrobial susceptibility testing was carried out using the VITEK2 system and Etest method. Extended-spectrum β-lactamase (ESBL) production by Enterobacterales was detected by the double-disk diffusion method and VITEK2 system. Patient age, gender, and inflammatory markers were collected at admission and compared between patients with and without bacteremia. Results: A majority of the patients were infants (37, 40%) and newborns (13, 14%). The main ports of entry were the lower respiratory tract, the genitourinary tract, and the gastrointestinal tract. Streptococcus pneumoniae was the most common pathogen (16, 16.7%) followed by Escherichia coli (12, 12.5%), Staphylococcus aureus (10, 10.4%), and Streptococcus agalactiae (9, 9.4%). High rates of resistance to ampicillin, cefuroxime, ciprofloxacin, and trimethoprim-sulfamethoxazole were observed among the Enterobacterales. The prevalence of ESBL-producing E. coli and K. pneumoniae were 45% and 29%, respectively. The prevalence of methicillin-resistant S. aureus was 30%. Patients with bacteremia had significantly higher white blood cell (WBC) counts, absolute neutrophil count (ANC), C-reactive protein (CRP), and neutrophil-lymphocyte ratio (NLR). Conclusion: Continuous surveillance of the prevalence and antimicrobial susceptibility patterns of blood isolates is imperative for the formulation of antibiotic policy. WBC, ANC, CRP, and NLR could be valuable indicators of bacteremia in febrile children.

Clostridioides difficile from Fecally Contaminated Environmental Sources: Resistance and Genetic Relatedness from a Molecular Epidemiological Perspective.

Clostridioides difficile is the most important pathogen causing antimicrobial-associated diarrhea and has recently been recognized as a cause of community-associated C. difficile infection (CA-CDI). This study aimed to characterize virulence factors, antimicrobial resistance (AMR), ribotype (RT) distribution and genetic relationship of C. difficile isolates from diverse fecally contaminated environmental sources. C. difficile isolates were recovered from different environmental samples in Northern Germany. Antimicrobial susceptibility testing was determined by E-test or disk diffusion method. Toxin genes (tcdA and tcdB), genes coding for binary toxins (cdtAB) and ribotyping were determined by PCR. Furthermore, 166 isolates were subjected to whole genome sequencing (WGS) for core genome multi-locus sequence typing (cgMLST) and extraction of AMR and virulence-encoding genes. Eighty-nine percent (148/166) of isolates were toxigenic, and 51% (76/148) were positive for cdtAB. Eighteen isolates (11%) were non-toxigenic. Thirty distinct RTs were identified. The most common RTs were RT127, RT126, RT001, RT078, and RT014. MLST identified 32 different sequence types (ST). The dominant STs were ST11, followed by ST2, ST3, and ST109. All isolates were susceptible to vancomycin and metronidazole and displayed a variable rate of resistance to moxifloxacin (14%), clarithromycin (26%) and rifampicin (2%). AMR genes, such as gyrA/B, blaCDD-1/2, aph(3')-llla-sat-4-ant(6)-la cassette, ermB, tet(M), tet(40), and tetA/B(P), conferring resistance toward fluoroquinolone, beta-lactam, aminoglycoside, macrolide and tetracycline antimicrobials, were found in 166, 137, 29, 32, 21, 72, 17, and 9 isolates, respectively. Eleven "hypervirulent" RT078 strains were detected, and several isolates belonged to RTs (i.e., RT127, RT126, RT023, RT017, RT001, RT014, RT020, and RT106) associated with CA-CDI, indicating possible transmission between humans and environmental sources pointing out to a zoonotic potential.

The Influence of Medium Composition on EUCAST and Etest Antifungal Susceptibility Testing.

There is an ongoing effort to optimize and revise antifungal susceptibility testing (AFST) methods due to the rising number of fungal infections and drug-resistant fungi. The rising antifungal resistance within Candida and Aspergillus species, which are common contributors to invasive fungal infections (IFIs), is a cause for concern, prompting an expanding integration of in vitro AFST to guide clinical decisions. To improve the relevance of in vitro AFST results to therapy outcomes, influential factors should be taken into account. The tested medium is one of several factors that could affect the results of AFST. The present study evaluated the effect of two complex media (Sabouraud dextrose and Columbia) versus the standard defined medium (RPMI 1640) on the AFST results of amphotericin B, posaconazole, and voriconazole against Candida spp. and Aspergillus spp. representatives, utilizing the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Etest methods. Overall, Candida species exhibited higher variability in minimum inhibitory concentration (MIC) across different media (more than three log2 dilutions) comparing to Aspergillus spp., while quality control isolates showed consistency regardless of tested media, antifungals, and methods. When comparing tested methods, MIC variation was mostly detected using EUCAST than it was using Etest.

In Vitro Susceptibility of Clinical Isolates to Ceftriaxone Alone and Ceftriaxone in Combination With Sulbactam or Tazobactam: A Comparative Study of Broad-Spectrum β-Lactam Antibiotics in India.

Background This study was designed to evaluate the current in vitro susceptibility of clinical isolates to broad-spectrum β-lactam antibiotics. Methodology Bacterial isolates, cultured from 180 non-repetitive clinical samples between April and November 2022 at three hospitals in India, were used to evaluate the minimum inhibitory concentration (MIC) of broad-spectrum β-lactam antibiotics using the Epsilometer test (E-test) method. Test antibiotics were ceftriaxone and ceftriaxone in combination with β-lactamase inhibitors (BLIs) sulbactam and tazobactam. Comparator antibiotics included amoxicillin + BLI clavulanic acid, piperacillin + tazobactam, cefotaxime, and cefepime. The MIC values obtained were used to assess the susceptibility of the isolates and to compute the efficacy ratios (ERs) of the antibiotics. Results Among the 180 clinical isolates, ~89% were gram-negative bacteria, the most prevalent ones being Escherichia coli and Klebsiella pneumoniae. Of the gram-negative isolates, ~37% were susceptible/intermediately susceptible to ceftriaxone, and ~29% were susceptible to ceftriaxone + BLIs. The test antibiotics had ER >10 against 85%-95% E. coli isolates, whereas comparator antibiotics had ER >10 against 31%-68% isolates. The differences between the test antibiotics and piperacillin + tazobactam or cefotaxime were statistically significant. Ceftriaxone, ceftriaxone + sulbactam, and ceftriaxone + tazobactam had ER >10 against 78%, 100%, and 90% of K. pneumoniae isolates, while the corresponding percentages for cefotaxime, piperacillin + tazobactam, and cefepime were 100%, 64%, and 80%, respectively. The difference between ceftriaxone + BLIs and piperacillin + tazobactam was statistically significant. Ceftriaxone + BLIs had ER >10 against all E. coli isolates producing extended-spectrum β-lactamases (ESBLs); the percentage of isolates was significantly higher than that for piperacillin + tazobactam. Ceftriaxone + tazobactam had ER >10 against all ESBL-producing K. pneumoniae isolates; ceftriaxone and ceftriaxone + sulbactam had ER ranging 6-10. Conclusions Ceftriaxone and ceftriaxone in combination with sulbactam and tazobactam are promising antibiotics to explore against prevalent infectious microorganisms such as E. coli and K. pneumoniae. Ceftriaxone + tazobactam also holds promise against ESBL-producing variants.

Detection of colistin resistance via four methods among Gram-negative bacteria

Objective: We aimed to find the most accurate and robust method for the detection of colistin resistance among Gram-negative bacteria by comparing the performance of four methods. Methods: Colistin resistantance was determine by disk diffusion (DD), Etest, colistin broth disk elution (CBDE) and compared with standard broth microdilution (BMD) method. Results: An exploratory study was conducted in a tertiary hospital in Jaipur, India, from November 2021 to November 2022. Of the 384 isolates tested, 41 (10.7%) were resistant against colistin by BMD and CBDE testing. Compared with BMD method, the categorical agreement of DD and Etest methods were 65.9% and 78.1%, respectively, and the rates of very major error were 34.1% and 21.9%, respectively. Conclusions: Due to the high frequency of very major error, the use of DD and Etest are not recommended by Clinical laboratory and standards institute (CLSI) for colistin susceptibility testing while CBDE is the recommended method which yields a categorical agreement of 100% with BMD method. The CBDE method can be used in routine laboratories for the detection of colistin resistance.

Detection of Methicillin-resistant Staphylococcus aureus (MRSA) resistant to vancomycin and linezolid in bulk tank milk by E-test method

Staphylococcus aureus is considered a serious threat to public health, besides is one of the most common causes of subclinical mastitis in dairy cows. Therefore, especially methicillin-resistant Staphylococcus aureus (MRSA) is among the most critical reasons for antibiotic treatment. Monitoring the antibiotic resistance of MRSA from livestock animals and foods is of great significance. The E-test method is a suitable option for detecting antibiotics' minimum inhibition concentration (MIC) value because it is an easy and fast assay to perform. This study aimed to detect vancomycin, teicoplanin, and linezolid resistance of milk-borne 34 MRSA isolates with subclinical mastitis by the E-test method to determine MIC values. The study determined that 8.8% of MRSA isolates isolated from milk with subclinical mastitis were also resistant to vancomycin and 11.7% to linezolid, while none of the isolates were determined to be resistant to teicoplanin, according to EUCAST. The data obtained from the study reveal that the efficacy of vancomycin and linezolid antibiotics, which are commonly used against MRSA infections, are in danger. Especially before MRSA treatment, MIC values of antibiotics should be determined, and appropriate antibiotics should be used in effective doses.

Looking for Stenotrophomonas maltophilia treatment: in vitro activity of ceftazidime/avibactam alone and in combination with aztreonam

During the Sars-Cov-2 pandemic, Stenotrophomonas maltophilia (S.maltophilia) secondary pulmonary infections have increased, especially in critically ill patients, highlighting the need for new therapeutic options. Trimethoprim-sulfamethoxazole (SXT) is the treatment of choice but the increase of resistant strains or adverse drug reactions limited its clinical use. Recently ceftazidime/avibactam (CZA) has been approved for the treatment of multi drug resistant (MDR) Gram-negative bacteria infections, including hospital acquired pneumonia. The aim of this study was to evaluate the in vitro activity of ceftazidime/avibactam (CZA) alone and in combination with aztreonam (ATM) against S. maltophilia clinical isolates by E-test method. Susceptibility of SXT and levofloxacin (LEV) was also investigated. Our results showed 22% of resistance to CZA, 2% to SXT and 26% to LEV. CZA in combination with ATM demonstrated synergistic activity against 86% of the strains, including all those resistant to CZA. The combination of CZA with ATM provides a new therapeutic option for the treatment of severe respiratory infections in critically ill patients.