Antimicrobial Susceptibility Testing Guidelines Research Articles

Investigation of Escherichia coli isolates from pigs and humans for colistin resistance in Lao PDR- a cross-sectional study

BackgroundIn Laos, colistin is not currently registered for use in humans. This One Health study aimed to estimate the prevalence of meat-producing pigs carrying colistin-resistant Escherichia coli, and investigate if E. coli causing invasive human infections were colistin-resistant. MethodsBetween September 2022 and March 2023, rectal swabs were collected from 895 pigs from abattoirs in 9/17 Lao provinces. Pig rectal swabs and stored E. coli isolates from human blood cultures, submitted to Mahosot Hospital Microbiology laboratory between 2005 and 2022, were screened for colistin resistance on selective chromogenic agar with organism identification confirmed using MALDI-TOF MS. Suspected colistin-resistant isolates underwent colistin susceptibility testing by broth microdilution following European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. Isolates with MIC values of ≥2 μg/ml were tested for plasmid-mediated colistin resistance genes (mcr-1, mcr-2, and mcr-3) by multiplex SYBR Green PCR. ResultsA total of 15/620 (2.41%) invasive human E. coli isolates were phenotypically colistin-resistant by broth microdilution (MIC values 4 to 8 μg/ml). The earliest isolate was from 2015 in a patient from Phongsaly province in Northern Laos. A total of 582/895 (65.02%) pig rectal swab samples contained colistin-resistant E. coli. The detected colistin resistance genes were predominantly mcr-1 (57.8%, 346/598), followed by mcr-3 (20.23%,121/598), and 22.24% (133/598) were found to co-harbour mcr-1 and mcr-3. Among the 15 human isolates with colistin MIC values of ≥4 μg/ml, 12/15 were mcr-1. ConclusionsWe found that colistin resistant E. coli is causing invasive infection in humans in Laos despite the fact it is not available for human use. Use in animals seems to be widespread, confirmed by high carriage rates of colistin-resistant E. coli in pigs. It is probable that food-producing animals are the source of colistin-resistant E. coli bloodstream infection in Laos, although these have been infrequent to date. This is a serious public health concern in the region that needs to be addressed by appropriate enforceable legislation.

Fosfomycin susceptibility testing and resistance mechanisms in Enterobacterales in South Africa.

Fosfomycin treatment of urinary tract infections is increasingly attractive due to escalating antibiotic resistance rates among urinary pathogens. Standard antibiotic susceptibility testing methods perform poorly for fosfomycin as there is poor correlation between susceptibility results and clinical outcomes in urinary pathogens other than Escherichia coli. We evaluated the performance of fosfomycin susceptibility testing in E. coli and Klebsiella pneumoniae to determine whether fosfomycin susceptibility is associated with molecular resistance mechanisms. Forty-six each of E. coli and K. pneumoniae clinical isolates were obtained from a tertiary hospital in South Africa, from 01 June 2017 to 31 January 2018. Agar dilution, disk diffusion, and gradient diffusion were performed and interpreted using the Clinical Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing guidelines. Molecular resistance mechanisms were identified by whole genome sequence analysis. Disk diffusion and gradient diffusion were accurate alternatives for fosfomycin susceptibility testing in E. coli (98% categorical agreement), but not in K. pneumoniae (47% categorical agreement). All E. coli isolates contained at least one resistance mechanism, but only one isolate with a fosA gene was resistant. In K. pneumoniae, 63% (29/46) and 70% (32/46) of isolates were susceptible to fosfomycin, using Clinical Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing breakpoints, respectively, despite all isolates containing a fosA gene and a uhpT mutation. A better understanding of fosfomycin susceptibility and improved antibiotic susceptibility testing tools could improve diagnostic capability and clinical guidelines for fosfomycin treatment of urinary tract infections. This study highlights the importance of adhering to interpretive guidelines when performing antimicrobial susceptibility testing and the need for simplified, accurate and standardised susceptibility testing methodology and interpretation for fosfomycin in Enterobacterales organisms.

Antimicrobial Susceptibility and Characterization of Extended-Spectrum β-Lactamase-Producing Escherichia coli Isolated from Stools of Primary Healthcare Patients in Ethiopia.

Antimicrobial resistance of Escherichia coli is a growing problem in both developed and developing countries. This study aimed to investigate the phenotypic antimicrobial resistance of E. coli isolates (n = 260) isolated from the stool specimen of patients attending public health facilities in Addis Ababa and Hossana. This study also aimed to characterize phenotypically confirmed extended-spectrum beta-lactamase (ESBL)-producing E. coli isolates (n = 22) using whole-genome sequencing. Resistance to 18 different antimicrobials was assessed using the disc diffusion method according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. The highest resistance rate among the E. coli isolates was found for ampicillin (52.7%), followed by trimethoprim-sulfamethoxazole (29.6%). Of all isolates, 50 (19.2%) were multidrug-resistant and 22 (8.5%) were ESBL producers. ESBL genes were detected in 94.7% of the sequenced E. coli isolates, and multiple β-lactamase genes were detected in 57.9% of the isolates. The predominant ESBL gene identified was blaCTX-M-15 (78.9%). The blaTEM-1B gene was detected in combination with other ESBL genes in 57.9% of the isolates, while only one of the sequenced isolates contained the blaTEM-1B gene alone. The blaCTX-M-3 gene was detected in three isolates. The genes blaCTX-M-15 and blaTEM-1B as well as blaCTX-M-15 and blaTEM-169 were confirmed to coexist in 52.6% and 10.5% of the sequenced E. coli isolates, respectively. In addition, blaOXA-1 was identified together with blaCTX-M-15 and blaTEM-1B in one isolate, and in one isolate, blaTEM-169 together with blaCTX-M-15 and blaTEM-1B was found. The results obtained show that measures need to be taken to reduce the spread of drug resistance and ensure the long-term use of available antimicrobials.

In vitro susceptibility to fosfomycin in clinical and environmental extended-spectrum beta-lactamase producing and/or ciprofloxacin-non-susceptible Escherichia coli isolates.

Extended-spectrum beta-lactamase producing and ciprofloxacin-non-susceptible Escherichia coli are clinical and environmental issues. We evaluated the susceptibility profile of fosfomycin in non-susceptible E. coli isolated from urine and the environment. We measured the activity of fosfomycin against 319 and 36 E. coli strains from urine and environmental isolates, respectively, collected from rivers. Fosfomycin resistance profiles were investigated using the minimal inhibitory concentration (MIC), according to the Clinical and Laboratory Standards Institute (CLSI) and the European Committee for Antimicrobial Susceptibility Testing (EUCAST) guidelines. Antibiotic susceptibility testing revealed that 5% and 6.6% of urine samples were non-susceptible to fosfomycin according to CLSI and EUCAST guidelines, respectively. The fosfomycin MIC50/90 was 0.5/4 mg/L. Of the 36 E. coli isolates from river water, 11.1% and 13,8% were non-susceptible to fosfomycin according to CLSI and EUCAST, respectively (range ≤0.25 ≥512 mg/L). All the isolates with MIC ≥512 mg/L for fosfomycin showed the fosA3 gene. Fosfomycin resistance was more frequent in the environment than in clinical samples.

Characterization of Ceftriaxone-Resistant Haemophilus influenzae Among Korean Children.

Haemophilus influenzae is a frequently encountered pathogen responsible for respiratory tract infections in children. Following the detection of ceftriaxone-resistant H. influenzae at our institution, we aimed to investigate the resistance mechanisms of ceftriaxone in H. influenzae, with a particular focus on alterations in penicillin-binding protein 3 (PBP3) and β-lactamase production. Among H. influenzae isolates collected at Asan Medical Center Children's Hospital from March 2014 to April 2019, ceftriaxone-resistant strains by the disk-diffusion test were included. Ceftriaxone minimum inhibitory concentrations (MICs) were determined using the E-test according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. The presence of β-lactamase was assessed through cefinase test and TEM-1/ROB-1 polymerase chain reaction (PCR). PBP3 alterations were explored via ftsI gene sequencing. Out of the 68 collected strains, 21 exhibited resistance to ceftriaxone in disk diffusion tests. Two strains were excluded due to failed subculture. Among 19 ceftriaxone-resistant H. influenzae isolates, eighteen were non-typeable H. influenzae, and twelve were positive for TEM-1 PCR. Isolates were classified into groups II (harboring only N526K, n = 3), III (N526K+S385T, n = 2), III+ (S385T+L389F+N526K, n = 11), and III-like+ (S385T+L389F+R517H, n = 3) according to the PBP3 alteration pattern. With a median ceftriaxone MIC of 0.190 mg/L (range, 0.008-0.750), the median ceftriaxone MIC was the highest in group III-like+ (0.250 mg/L), followed by groups III+ (0.190 mg/L), III (0.158 mg/L), and II (0.012 mg/L). All three strains belonging to group II, which did not harbor the S385T substitution, had ceftriaxone MICs of ≤ 0.125 mg/L. The emergence of ceftriaxone-resistant H. influenzae with ceftriaxone MIC values of up to 0.75 mg/L was observed even in children in South Korea, with most associated with S385T and L389F substitutions. The N526K mutation alone does not significantly impact ceftriaxone resistance. Further large-scale studies are essential to investigate changes in antibiotic resistance patterns and factors influencing antibiotic resistance in H. influenzae isolated from pediatric patients in Korea.

Vibrio species: development of EUCAST susceptibility testing methods and MIC and zone diameter distributions on which to determine clinical breakpoints.

Most human infections caused by Vibrio spp. do not warrant antimicrobial treatment but in severe cases, targeted antimicrobial treatment can be lifesaving. For Vibrio spp., standardized antimicrobial susceptibility testing (AST) guidelines with EUCAST methodology are lacking. In this study, we aimed to produce data suitable for EUCAST to establish clinical MIC breakpoints and zone diameter correlates for Vibrio spp. An intercontinental collection (N = 524) comprising five important Vibrio spp. (V. alginolyticus, V. cholerae, V. fluvialis, V. parahaemolyticus and V. vulnificus) was organized. All isolates were subjected to broth microdilution (BMD) against 11 antimicrobial agents according to ISO 20776-1 using unsupplemented Mueller-Hinton broth on freeze-dried Sensititre panels (Thermo Scientific, UK), and most isolates (n = 371) were also tested with disc diffusion according to EUCAST methodology for non-fastidious organisms. Aggregated results were used to generate MIC and zone diameter distributions and to prepare graphs of MIC-zone diameter correlation. Based on these results, the EUCAST Steering Committee determined clinical susceptible (S) and resistant (R) MIC (mg/L) breakpoints (S≤/R>) for the five Vibrio spp. for piperacillin/tazobactam (1/1), cefotaxime (0.25/0.25), ceftazidime (1/1), meropenem (0.5/0.5), ciprofloxacin (0.25/0.25), levofloxacin (0.25/0.25), azithromycin (4/4), doxycycline (0.5/0.5) and trimethoprim/sulfamethoxazole (0.25/0.25). The corresponding zone diameter breakpoints were identified. We demonstrated the validity of using standard BMD and EUCAST disc diffusion methodology for AST of five Vibrio spp., and generated suitable data to allow EUCAST to determine clinical MIC and zone diameter breakpoints for five pathogenic Vibrio spp., including both non-toxigenic and toxigenic V. cholerae.

Phenotypic and genomic characterization of Pseudomonas aeruginosa isolates recovered from catheter-associated urinary tract infections in an Egyptian hospital.

Catheter-associated urinary tract infections (CAUTIs) represent one of the major healthcare-associated infections, and Pseudomonas aeruginosa is a common Gram-negative bacterium associated with catheter infections in Egyptian clinical settings. The present study describes the phenotypic and genotypic characteristics of 31 P. aeruginosa isolates recovered from CAUTIs in an Egyptian hospital over a 3 month period. Genomes of isolates were of good quality and were confirmed to be P. aeruginosa by comparison to the type strain (average nucleotide identity, phylogenetic analysis). Clonal diversity among the isolates was determined; eight different sequence types were found (STs 244, 357, 381, 621, 773, 1430, 1667 and 3765), of which ST357 and ST773 are considered to be high-risk clones. Antimicrobial resistance (AMR) testing according to European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines showed that the isolates were highly resistant to quinolones [ciprofloxacin (12/31, 38.7 %) and levofloxacin (9/31, 29 %) followed by tobramycin (10/31, 32.5 %)] and cephalosporins (7/31, 22.5 %). Genotypic analysis of resistance determinants predicted all isolates to encode a range of AMR genes, including those conferring resistance to aminoglycosides, β-lactamases, fluoroquinolones, fosfomycin, sulfonamides, tetracyclines and chloramphenicol. One isolate was found to carry a 422 938 bp pBT2436-like megaplasmid encoding OXA-520, the first report from Egypt of this emerging family of clinically important mobile genetic elements. All isolates were able to form biofilms and were predicted to encode virulence genes associated with adherence, antimicrobial activity, anti-phagocytosis, phospholipase enzymes, iron uptake, proteases, secretion systems and toxins. The present study shows how phenotypic analysis alongside genomic analysis may help us understand the AMR and virulence profiles of P. aeruginosa contributing to CAUTIs in Egypt.

A Prospective, Observational and Comparative Study of In-Vitro Susceptibility of Isepamicin against Clinical Isolates from Various Clinical Sources (Triple I Study - Isepamicin in India)

Objectives: This study aims to investigate the in-vitro susceptibility of clinical isolates against Isepamicin and compared it with Gentamicin and Amikacin.
 Patients and Methods: In this multicentre prospective study, clinical specimens of patients were collected from three different regions of India. Clinical isolates from urine, intra-abdominal, broncho-alveolar lavage, endotracheal secretion, and sterile blood were included. The E-test was used to quantify the minimal inhibitory concentration (MIC) for Isepamicin, Gentamicin, and Amikacin. The percentages of bacterial isolates were categorized as susceptible, intermediate, and resistant according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines as per Comité de l'Antibiogramme de la Société Française de Microbiologie (CA-SFM) recommendation.
 Results: A total of 8 different bacterial isolates were collected from 150 clinical samples obtained from 50 patients. Respiratory (63 [42%]) and urine (44 [29.3%]) specimens were the most common sources for bacterial strains. The most identified bacterial isolates were K. pneumoniae (40 [26.6%]) and P. aeruginosa (38 [25%]). Isepamicin was found to be highly effective in urine samples and showed excellent sensitivity against E. coli (93.3%), followed by P. aeruginosa (57.9%) and K. pneumoniae (55.0%). Antimicrobial sensitivity was highest for Isepamicin (60/108 [56%]) at MIC≤1 mg/L and was most effective against Gram-negative bacterial isolates from the intensive care units (ICUs).
 Conclusions: Isepamicin could treat E. coli infections and could be an effective therapy in the treatment of urinary tract infections (UTIs). Moreover, it could also be used as an alternative to Gentamicin and Amikacin against resistant cases.
 Keywords: Aminoglycosides, Amikacin, E. coli, Gentamicin, Gram-negative bacteria, Isepamicin.

Analysis of cumulative antibiogram reports in intensive care units at an Egyptian University Hospital

BackgroundInstitutions must have access to antibiograms to monitor changes in antimicrobial resistance and direct empirical antibiotic therapy. The first facility-specific cumulative antibiogram was launched in the ICU in 2019. Consequently, many antibiogram-operation-related actions have been adopted in the institution based on reported data. This study aimed to analyze the cumulative antibiogram reports for multiple intensive care units (ICUs) for 2020, and compare the antimicrobial susceptibility testing (AST) patterns between the 2019 and 2020 years in an academic medical center. MethodsThis cross-sectional study was performed of routine bacterial culture and AST data extracted from a laboratory information system in a 2252-bed capacity hospital. Only the first diagnostic isolate of a given species per patient per year was included in the study. Interpretation and reporting were done in accordance with the applicable Clinical and Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing guidelines. ResultsOf the 46,791 clinical isolates, the Gram-negative bacilli isolation rate witnessed a significant increase: 35,670 isolates in 2020 versus. 33,652 isolates in 2019. Klebsiella pneumoniae showed a statistically significant increase, mainly in pediatric, emergency, and cardiothoracic ICUs (p < 0.001). Neonatal and pediatric ICUs showed statistically significant increases in Pseudomonas aeruginosa and Proteus mirabilis isolates (p < 0.001). A statistically significant decrease was noted in the prevalence of Acinetobacter, Escherichia coli, Burkholderia cepacia, and Enterobacter cloacae. The sensitivities of K. pneumoniae and E. coli to imipenem and tigecycline significantly improved (p < 0.001). The sensitivity to colistin was significantly decreased (p < 0.001). The sensitivity of P. aeruginosa isolates to colistin and carbapenems was improved (p < 0.001). We reported a statistically significant decrease in all Gram-positive cocci (11,121 in 2020 versus. 11,528 in 2019). Staphylococcus aureus showed a statistically significant increase (p < 0.001), particularly in the medical ICU. ConclusionThe high susceptibility rates of Enterobacteriaceae toward colistin and tigecycline, should be cautiously considered in empiric therapy while looking for alternatives. The majority of isolates of Gram-positive cocci were coagulase negative staphylococci (CONS), we still need to confirm whether they are true pathogens or commensals before considering anti-staphylococcal agents in the empirical therapy. We underscored some corrective actions that might have improved the susceptibility rates, such as antibiotic cycling

Molecular Detection of Extended Spectrum Beta-Lactamase Gene in Clinical Isolates of Klebsiella Species in Jos North Central of Nigeria

Aims: The aim of the study was to determine the antimicrobial susceptibility of Klebsiella species, prevalence of Extended Spectrum Beta-Lactamases (ESBLs) and the gene coding ESBL production in Klebsiella Species obtained from clinical specimens of patients attending Plateau State Specialist and Jos University Teaching Hospitals both in Jos North Central of Nigeria.&#x0D; Study Design: This was hospital based study conducted in the Department of Medical Microbiology unit of Plateau State Specialist and Jos University Teaching Hospital (JUTH) during the periods of January to April 2020.&#x0D; Methodology: Out of 156 non-motile, oxidase negative gram negative organisms collected, 137 isolates were identified to be Klebsiella species using oxoid Microbact 24E kit for Enterobacteriaceae identification and Microsoft for bacteria identification. Antimicrobial Susceptibility Testing (AST) and ESBL screening was done by Kirby-Bauer disk diffusion method using oxoid Single disk antibiotics which include Ceftazidime (30µg), Cefotaxime (30µg), Imipenem (10µg), Ciprofloxacin (5µg), Gentamicin (10µg), Ampicillin (10 µg), Aztreonam (30 µg), and Azithromycin (15 µg) on Mueller Hinton Agar (oxoid).Break point was determined by CLSI AST guidelines. The isolates that showed resistance or reduced susceptibility to Ceftazidime and Cefotaxime are suspected to be ESBL positive Klebsiella. Phenotypic confirmation of ESBL positive isolates was carried out using Double Disk Synergy Test (DDST) with Ceftazidime 30µg (oxoid), Cefotaxime 30µg (oxoid) and Amoxicillin Clavulanic acid 20/10ug (Oxoid).Genotypic detection of ESBL gene was done using Polymerase Chain Reaction (PCR).&#x0D; Results: Antimicrobial Susceptibility Test showed Imipenem to be highly sensitive to ESBL positive Klebsiella pneumonia with 100% sensitivity. Gentamicin, Ampicillin, and Azithromycin showed reduced susceptibility. Genotypic results showed presence of bla TEM, bla SHV genes at 868bp and 972bp band size with no occurrence of bla CTX-M gene. There was multiple occurrence of gene in some of the isolates tested while some recorded no ESBL gene. The ESBL prevalence of 15.3% was recorded in this study.&#x0D; Conclusions: The prevalence of 15.3% recorded in this study is low compared to other studies from some other states within the country. Efforts should be put in place to ensure non-proliferation of this ESBL Klebsiella strain as it creates not only a therapeutic challenge but health problem.

The First Report of Chryseobacterium gleum Caused Ventriculoperitoneal-shunt Infection in a Child.

To The Editors: Ventriculoperitoneal (VP) shunt is one of the most widely used and effective methods in the treatment of hydrocephalus in childhood. VP shunt infections have been observed in approximately 3.2%–25%, and Chryseobacterium spp. is a rare cause of VP shunt infections.1,2 Herein, we report the first VP shunt infection caused by Chryseobacterium gleum. A 12-month-old female (corrected age 8 months) born via spontaneous vaginal delivery, weighed 700 g at 25 weeks of gestation, was admitted to our emergency department with a fever with a maximum peak temperature of 39°C, projectile vomiting, and decreased oral intake. Her history included long-term ventilation in the neonatal intensive care unit with a diagnosis of moderate bronchopulmonary dysplasia. At 4 months of age, she developed intraventricular hemorrhage and progressive hydrocephalus, and a VP shunt was inserted. She had several episodes of VP shunt infections caused by S. epidermidis and S. aureus. At admission, the patient was febrile, tachycardic (heart rate, 160/min) and tachypneic (respiratory rate of 48/min). Physical examination showed weakness, lethargy and sleepiness. Her total Glasgow Coma Scale score was 12 (E3V4M5). Laboratory tests were as follows: white blood cell (WBC) count: 12.5 × 103/μL (neutrophil 37% and lymphocyte 54%), C-reactive protein: 20 mg/L, procalcitonin: 0.09 μg/L. The cerebrospinal fluid (CSF) WBC count was 120/mm3, protein 107 mg/dL and glucose 42 mg/dL. With the preliminary diagnoses of sepsis and shunt infection, intravenous (IV) cefotaxime (300 mg/kg/day) and IV vancomycin (60 mg/kg/day) was initiated empirically. Peripheral/catheter blood cultures and CSF samples were inoculated into aerobic and anaerobic culture bottles (BacT/ALERT, BioMérieux, Durham, USA). The patient underwent VP shunt externalization. The blood and urine cultures were negative during the patient’s follow-up. Chryseobacterium gleum was isolated in the CSF, shunt and ventricular catheter tip cultures on the 3rd day of treatment. The organism had high-level minimum inhibitory concentration (MIC) levels for meropenem (MIC = 32 μg/mL), imipenem (MIC = 32 μg/mL) and piperacillin-tazobactam (MIC = 250 μg/mL). MIC level of ciprofloxacin was 0.125 μg/mL and trimethoprim-sulfamethoxazole was 0.5 μg/mL. Cefotaxime and vancomycin were switched to IV trimethoprim/sulphamethoxazole (5 mg/kg every 6 hours) and ciprofloxacin (10 mg/kg every 8 hours). On the 5th day of the combined therapy, the patient’s CSF sample was evaluated and showed a WBC count of 30/mm3, glucose 42 mg/dL and protein 360 mg/ dL, and the CSF culture was sterile. Antibiotics were continued for 21 days. The patient was clinically stable, and no adverse effects or seizures were observed during or after the treatment. The extra ventricular drainage was removed, the VP shunt was inserted following 2 consecutive negative CSF cultures, and the patient was discharged. Chryseobacterium species are common in nature but are not found in human flora. Most cases are nosocomial and are frequently associated with immunosuppression or implantable devices. Chryseobacterum gleum is typically a low-virulence organism that can cause serious infections. It has been reported that Chryseobacterum gleum can cause infections such as central-line-associated bloodstream infection, community-acquired pneumonia and urinary tract infection in adults.3–5 Unlike previous reports, Chryseobacterium was isolated in the cerebrospinal fluid in our study. Chryseobacterum gleum commonly colonizes mechanical devices, causing device-associated infections, and can form biofilm. According to this study, VP shunt may be a risk factor for Chryseobacterium gleum infections. This report emphasizes the significance of Chryseobacterium species as a causative agent of a wide range of infections, and VP shunts can pose a risk for this agent. Data on the management of Chryseobacterium is limited. Antimicrobial susceptibility testing guidelines for Chryseobacterium infections should be developed for more effective and accurate treatment.

Genetic determinants of macrolide and tetracycline resistance in penicillin non-susceptible Streptococcus pneumoniae isolates from people living with HIV in Dar es Salaam, Tanzania

BackgroundOver one million yearly deaths are attributable to Streptococcus pneumoniae and people living with HIV are particularly vulnerable. Emerging penicillin non-susceptible Streptococcus pneumoniae (PNSP) challenges therapy of pneumococcal disease. The aim of this study was to determine the mechanisms of antibiotic resistance among PNSP isolates by next generation sequencing.MethodsWe assessed 26 PNSP isolates obtained from the nasopharynx from 537 healthy human immunodeficiency virus (HIV) infected adults in Dar es Salaam, Tanzania, participating in the randomized clinical trial CoTrimResist (ClinicalTrials.gov identifier: NCT03087890, registered on 23rd March, 2017). Next generation whole genome sequencing on the Illumina platform was used to identify mechanisms of resistance to antibiotics among PNSP.ResultsFifty percent (13/26) of PNSP were resistant to erythromycin, of these 54% (7/13) and 46% (6/13) had MLSB phenotype and M phenotype respectively. All erythromycin resistant PNSP carried macrolide resistance genes; six isolates had mef(A)-msr(D), five isolates had both erm(B) and mef(A)-msr(D) while two isolates carried erm(B) alone. Isolates harboring the erm(B) gene had increased MIC (> 256 µg/mL) towards macrolides, compared to isolates without erm(B) gene (MIC 4-12 µg/mL) p < 0.001. Using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines, the prevalence of azithromycin resistance was overestimated compared to genetic correlates. Tetracycline resistance was detected in 13/26 (50%) of PNSP and all the 13 isolates harbored the tet(M) gene. All isolates carrying the tet(M) gene and 11/13 isolates with macrolide resistance genes were associated with the mobile genetic element Tn6009 transposon family. Of 26 PNSP isolates, serotype 3 was the most common (6/26), and sequence type ST271 accounted for 15% (4/26). Serotypes 3 and 19 displayed high-level macrolide resistance and frequently carried both macrolide and tetracycline resistance genes.ConclusionThe erm(B) and mef(A)-msr(D) were common genes conferring resistance to MLSB in PNSP. Resistance to tetracycline was conferred by the tet(M) gene. Resistance genes were associated with the Tn6009 transposon.

Prevalence of multidrug-resistant and extensively drug-resistant phenotypes of Gram-negative bacilli isolated in clinical specimens at Centre Hospitalo-Universitaire Ibn Rochd, Morocco.

antimicrobial resistance in gram-negative bacilli is one of the major concerns in public health. We aimed to evaluate gram-negative bacilli epidemiology, antimicrobial profiles, and the resistance´s mechanism for Enterobacteriaceae isolated from specimens of hospitalized patients in wards of University Hospital Center Ibn Rochd of Casablanca, Morocco. a prospective study of the patient's specimens, collected from December 2016 to 31st March 2017. Isolation and identification were performed using conventional biochemical tests. According to the European Committee on Antimicrobial Susceptibility Testing guidelines, antibiotic susceptibility was determined. Polymerase Chain Reaction (PCR) was used to detect β-lactamase and carabapenemase genes: CTX-M, SHV, TEM, OXA-48, NDM, and VIM among the Enterobacteriaceae. according to inclusion criteria, 38 Enterobacteriaceae, 25 Acinetobacter baumannii (A. baumannii), and 10 Pseudomonas aeruginosa (P. aeruginosa) were included during the study period; these bacteria were mainly responsible for bacteremia. Fifty-five percent of enterobacteria were extended-spectrum β-lactamase (ESBL), 42% EBSL and carbapenemase, and 3% carbapenemase, with high coresistances. Eighty-four percent of A. baumannii were XDR. All P. aeruginosa were MDR; amikacin showed the best activity (70% susceptibility). The genotypic approach revealed the presence of blaCTX-M, blaSHV, blaTEM in 68%, 22%, and 11% respectively. Of the 22 carbapenemase-producers, 41% were blaOXA-48 and 18% blaNDM; none had blaVIM. Furthermore, various genes coexistence were detected: blaCTX-M+blaOXA-48; blaCTX-M+blaNDM; blaCTX-M+blaSHV+blaOXA-48; and blaSHV+blaOXA-48. findings revealed highly resistance rate among isolates. This raises the need to control antibiotics and regular screening to identify dynamics promoting resistance. Thus, we recommend developing antimicrobial stewardship programs and improving hygiene systems to prevent the nosocomial spreading of these phenotypes in our center.

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

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

Modified Drug-Susceptibility Testing and Screening Culture Agar for Colistin-Susceptible Enterobacteriaceae Isolates Harboring a Mobilized Colistin Resistance Gene mcr-9.

Three isolates of the Enterobacter cloacae complex harboring mcr-9, a member of the colistin resistance mcr gene family encoded on plasmids, were susceptible to colistin, with MICs of 0.125 to 0.5 μg/mL in standard broth microdilution (BMD) tests using cation-adjusted Mueller-Hinton broth (CA-MHB) in accordance with European Committee on Antimicrobial Susceptibility Testing guidelines. In contrast, their MICs for colistin were significantly higher (4 to 128 μg/mL) when BMD tests were performed using brain-heart infusion (BHI) medium, Luria-Bertani (LB) broth, tryptic soy broth (TSB), or CA-MHB supplemented with casein, tryptonen or peptone. Colistin significantly induced mcr-9 expression in a dose-dependent manner when these mcr-9-positive isolates were cultured in BHI or CA-MHB supplemented with peptone/casein. Pretreatment of mcr-9-positive isolates and Escherichia coli DH5α harboring mcr-9 with colistin significantly increased their survival rates against LL-37, a human antimicrobial peptide. Electrospray ionization time-of-flight mass spectrometry analysis showed that a lipid A moiety of lipopolysaccharide was partially modified by phosphoethanolamine in E. coli DH5α harboring mcr-9 when treated with colistin. Of 93 clinical isolates of Enterobacteriaceae, only the mcr-9-positive isolates showed MICs to colistin that were at least 32 times higher in BHI than in CA-MHB. These mcr-9-positive isolates grew on a modified BHI agar, MCR9-JU, containing 3 μg/mL colistin. These results suggest that the BMD method using BHI is useful when performed together with the BMD method using CA-MHB to detect mcr-9-positive isolates and that MCR9-JU agar is useful in screening for Enterobacteriaceae isolates harboring mcr-9 and other colistin-resistant isolates.

To Be or Not to Be Resistant: CLSI Versus EUCAST Guidelines for Staphylococcus lugdunensis

Abstract Introduction/Objective Staphylococcus lugdunensis is a coagulase-negative Staphyloccocus, but infections can be as severe as S. aureus. Performing antimicrobial susceptibility testing (AST) is paramount to appropriate treatment. Recently, the College of American Pathologists is requiring clinical labs to implement updated guidelines. We evaluated how the Clinical and Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines would impact our hospital’s resistance rates for S. lugdunensis. Methods/Case Report Cultures with S. lugdunensis isolated between 04/2018 to 02/2022 were included. Minimal inhibitory concentrations (MICs) (Vitek2 and Microscan Walkaway) reported (clindamycin, doxycycline, daptomycin, linezolid, and vancomycin) were interpreted using CLSI (M100Ed32) and EUCAST (v12.0) guidelines. The range of MICs (95% Confidence Interval) was calculated. Results (if a Case Study enter NA) A total of 125 isolates from 120 patients from specimen types including wound (49%), tissue (42%), blood (6%), and body fluid (3%) were identified. Five patients had multiple cultures of S. lugdunensis, four from two wound/tissue sites and one with blood and biopsy positive. For clindamycin (0.7-1.3 ug/mL), EUCAST guidelines determined two more isolates (from blood and wound, MIC=0.5 ug/mL) as resistant. Most drastically, the rate of resistance for doxycycline (0.6-1.0 ug/mL) was 11% higher per EUCAST guidelines (susceptible: 89% EUCAST vs 100% CLSI). The difference was seen in isolates &amp;gt; 2 ug/mL, all isolated from wound and tissue. Regardless of the guidelines used, 100% of the isolates were susceptible to daptomycin (0.4-0.5 ug/mL), linezolid (1.2-1.4 ug/mL), and vancomycin (0.6-0.7 ug/mL); 23% were resistant to oxacillin (1.7-2.0 ug/mL). Conclusion EUCAST guidelines would have reported more resistant doxycycline and clindamycin isolates at our institution. To ensure that clinical teams are utilizing AST results to provide appropriate care, a team effort consisting of the microbiology lab, infectious diseases physicians, and antibiotic stewardship committee is needed to determine which guidelines should be followed.

Antimicrobial Activity of Ceftazidime-Avibactam and Comparators Against Fluoroquinolone-Resistant Klebsiella pneumoniae Collected Globally from Antimicrobial Testing Leadership and Surveillance: 2018-2019.

This study assessed the in vitro antimicrobial activity of ceftazidime-avibactam (CAZ-AVI) and a panel of comparator agents, including aztreonam, cefepime, ceftazidime, meropenem, imipenem, colistin, piperacillin-tazobactam, and tigecycline against isolates of fluoroquinolone-resistant (FQ-R) Klebsiella pneumoniae collected in 2018 and 2019 from the Antimicrobial Testing Leadership and Surveillance (ATLAS) program. Susceptibility and minimum inhibitory concentration were determined using broth microdilution for all antimicrobial agents by a central reference laboratory according to the Clinical and Laboratory Standards Institute guidelines and European Committee on Antimicrobial Susceptibility Testing guidelines. Of all the K. pneumoniae isolates (n = 10,906), 44.1% (4,814/10,906) were FQ-R. Of these, 71.3% (3,432/4,814) were extended-spectrum β-lactamase (ESBL)-positive, and 10.4% (499/4,814) were CAZ-AVI-resistant. CAZ-AVI showed high susceptibility (>87%) against all the FQ-R K. pneumoniae isolates. However, metallo- β-lactamase-positive isolates showed low susceptibility (3.8%; 18/470) to CAZ-AVI. Among the different geographical regions, CAZ-AVI showed the highest activity against isolates collected from North America (98.2%, 216/220) and lowest against those collected from Asia Pacific (APAC) (81.7%; 882/1,079). Among comparator agents, carbapenems showed a relatively lower susceptibility (<71.5%), while only tigecycline and colistin were active (>85%) across all isolates. In conclusion, CAZ-AVI may be a potential treatment option for FQ-R K. pneumoniae isolates. However, increasing CAZ-AVI resistance among ESBL-positive and metallo-β-lactamase-positive isolates and in isolates from APAC warrants continuous surveillance.

Ceftazidime-avibactam activity against Gram-negative respiratory isolates collected between 2018 and 2019

The objective of this study was to assess the in vitro activity of ceftazidime-avibactam (CAZ-AVI) and a panel of comparator agents against isolates of Enterobacter spp., Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa collected in 2018 and 2019 by different centres worldwide from patients with respiratory tract infections. Susceptibility and minimum inhibitory concentration (MIC) of all organisms were determined using broth microdilution methodology for CAZ-AVI, and a panel of comparator antimicrobial agents by a central reference laboratory according to Clinical and Laboratory Standards Institute guidelines and European Committee on Antimicrobial Susceptibility Testing guidelines. CAZ-AVI demonstrated potent antimicrobial activity against isolates of Enterobacter spp. (97.6% susceptibility, MIC90, 1 µg/ml), E. coli (98.5% susceptibility, MIC90, 0.25 µg/ml), K. pneumoniae (94.7% susceptibility, MIC90 2 µg/ml), and P. aeruginosa (91.2% susceptibility, MIC90 8 µg/ml). CAZ-AVI was also active (susceptibility >85%) against MDR isolates for all organisms except P. aeruginosa. Only a small proportion (<10%) of all isolates of Enterobacter spp. and E. coli were identified as XDR compared to isolates of K. pneumoniae and P. aeruginosa isolates (>20%). Susceptibility to CAZ-AVI was high (>70%) among XDR isolates of Enterobacter spp., K. pneumoniae, and E. coli, compared to P. aeruginosa (<70%). Among the comparator agents, only colistin showed consistent activity across all the organisms (>85%). Gram-negative respiratory isolates collected in 2018-2019 showed high susceptibility to CAZ-AVI; CAZ-AVI represents a potential treatment option against infection caused by these organisms.

First Belgian Report of Ertapenem Resistance in an ST11 Klebsiella Pneumoniae Strain Isolated from a Dog Carrying blaSCO-1 and blaDHA-1 Combined with Permeability Defects.

Klebsiella pneumoniae of sequence type (ST) 11 is a hyper-epidemic nosocomial clone, which is spreading worldwide among humans and emerging in pets. This is the first report, to the best of our knowledge, of multidrug-resistant (MDR) K. pneumoniae ST11 carrying blaSCO-1 and blaDHA-1, isolated from a four-month-old dog in Belgium. Antimicrobial susceptibility testing (AST) of the isolate, performed via broth microdilution following the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines, revealed resistance to eight different classes of antimicrobials, including carbapenems, in particular ertapenem, third-generation cephalosporins and fluoroquinolones. A hybrid approach, combining long- and short-read sequencing, was employed for in silico plasmid characterization, multi-locus sequence typing (MLST) and the identification and localization of antimicrobial resistance (AMR) and virulence-associated genes. Three plasmids were reconstructed from the whole-genome sequence (WGS) data: the conjugative IncFIB(K), the non-mobilizable IncR and the mobilizable but unconjugative ColRNAI. The IncFIB(K) plasmid carried the blaSCO-1 gene, whereas IncR carried blaDHA-1, both alongside several other antimicrobial resistance genes (ARGs). No virulence genes could be detected. Here, we suggest that the resistance to ertapenem associated with susceptibility to imipenem and meropenem in K. pneumoniae could be related to the presence of blaSCO-1 and blaDHA-1, combined with permeability defects caused by point mutations in an outer membrane porin (OmpK37). The presence of the blaSCO-1 gene on a conjugative IncFIB(K) plasmid is worrisome as it can increase the risk of transmission to humans, to animals and to the environment.

Evaluating the Role of Wastewaters as Reservoirs of Antibiotic-Resistant ESKAPEE Bacteria Using Phenotypic and Molecular Methods.

IntroductionWastewaters carrying thousands of human specimens from the community and representing the diversity of antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs) directly from the community mirror the extent of AR spread in the community and environment. This study aimed to investigate the occurrence and distribution of antibiotic-resistant ESKAPEE bacteria in the community versus clinical settings through monitoring nonclinical and clinical wastewaters.MethodologySeven wastewater samples were collected from different environmental sources. Isolates were obtained on general and selective media, biochemically characterized and antimicrobial-susceptibility tests performed by disk diffusion against 13 antibiotics according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines using MastDisc disk cartridges, and 16S rRNA metagenomic analysis was performed for two water samples.ResultsOf 43 isolates, all representatives of the ESKAPEE group were recovered from clinical wastewaters, but Gram-positive cocci were not obtained from nonclinical wastewaters. The most predominant isolate was Pseudomonas aeruginosa (n=15; 33%), followed by Escherichia coli (n=9; 20%). Complete (100%) resistance to eleven of the tested antibiotics was observed, with only a few isolates being susceptible to clarithromycin, amikacin, and gentamicin. The lowest (79%) resistance rate was observed for linezolid. The multiple antibiotic resistance (MAR) index was calculated, and the resistance phenotype was independent of the wastewater source, indicated by x2 (P=0.766). Metagenomic analysis replicated the results, as Pseudomonas spp., Acinetobacter spp., and Escherichia spp. were found to be predominant. The integrase gene (IntI1) was also amplified in Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa.ConclusionWastewaters are significant carriers of drug-resistant ESKAPEE bacteria and play an important role in their dissemination. This study endorses the periodic surveillance of water systems to evaluate the presence and burden of antibiotic-resistant pathogens.